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Journal of the Italian Society of Anatomic Pathology
and Diagnostic Cytopathology,
Italian Division of the International Academy of Pathology
Periodico bimestrale – POSTE ITALIANE SPA - Spedizione in Abbonamento Postale - D.L. 353/2003 conv. in L. 27/02/2004 n° 46 art. 1, comma 1, DCB PISA
Aut. Trib. di Genova n. 75 del 22/06/1949
Vol. 106 September 2014
Società Italiana di Anatomia Patologica e Citopatologia Diagnostica,
Divisione Italiana della International Academy of Pathology
Journal of the Italian Society of Anatomic Pathology and
Diagnostic Cytopathology
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Journal of the Italian Society of Anatomic Pathology
and Diagnostic Cytopathology,
Editor-in-Chief
Marco Chilosi, Verona
Associate Editor
Roberto Fiocca, Genova
Managing Editor
Roberto Bandelloni, Genova
Scientific Board
R. Alaggio, Padova
G. Angeli, Vercelli
M. Barbareschi, Trento
C.A. Beltrami, Udine
G. Bevilacqua, Pisa
M. Bisceglia, S. Giovanni R.
A. Bondi, Bologna
F. Bonetti, Verona
C. Bordi, Parma
A.M. Buccoliero, Firenze
G.P. Bulfamante, Milano
G. Bussolati, Torino
A. Cavazza, Reggio Emilia
G. Cenacchi, Bologna
P. Ceppa, Genova
C. Clemente, Milano
M. Colecchia, Milano
G. Collina, Bologna
P. Cossu-Rocca, Sassari
P. Dalla Palma, Trento
G. De Rosa, Napoli
A.P. Dei Tos, Treviso
L. Di Bonito, Trieste
C. Doglioni, Milano
V. Eusebi, Bologna
G. Faa, Cagliari
F. Facchetti, Brescia
G. Fadda, Roma
G. Fornaciari, Pisa
M.P. Foschini, Bologna
F. Fraggetta, Catania
E. Fulcheri, Genova
P. Gallo, Roma
F. Giangaspero, Roma
W.F. Grigioni, Bologna
G. Inghirami, Torino
L. Leoncini, Siena
M. Lestani, Arzignano
G. Magro, Catania
A. Maiorana, Modena
E. Maiorano, Bari
T. Marafioti, Londra
A. Marchetti, Chieti
D. Massi, Firenze
M. Melato, Trieste
F. Menestrina, Verona
G. Monga, Novara
R. Montironi, Ancona
B. Murer, Mestre
V. Ninfo, Padova
M. Papotti, Torino
M. Paulli, Pavia
G. Pelosi, Milano
G. Pettinato, Napoli
S. Pileri, Bologna
R. Pisa, Roma
M.R. Raspollini, Firenze
L. Resta, Bari
G. Rindi, Parma
M. Risio, Torino
A. Rizzo, Palermo
J. Rosai, Milano
G. Rossi, Modena
L. Ruco, Roma
M. Rugge, Padova
M. Santucci, Firenze
A. Scarpa, Verona
A. Sidoni, Perugia
G. Stanta, Trieste
G. Tallini, Bologna
G. Thiene, Padova
P. Tosi, Siena
M. Truini, Genova
V. Villanacci, Brescia
G. Zamboni, Verona
G.F. Zannoni, Roma
Editorial Secretariat
G. Martignoni, Verona
M. Brunelli, Verona
Società Italiana di Anatomia Patologica e Citopatologia Diagnostica,
Divisione Italiana della International Academy of Pathology
Governing Board
SIAPEC-IAP
President:
G. De Rosa, Napoli
President Elect:
M. Truini, Genova
General Secretary:
L. Molinaro, Torino
Past President:
C. Clemente, Milano
Members:
M. Basciu, Monza
A. Bondi, Bologna
G. Botti, Napoli
F. Crivelli, Busto Arsizio
A. Fassina, Padova
R. Fiocca, Genova
A.M. Florena, Palermo
A. Marchetti, Chieti
D. Massi, Firenze
G. Mazzoleni, Bolzano
L. Resta, Bari
G. Tinacci, Firenze
Associate Members
Representative:
T. Zanin, Genova
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Vol. 106 September 2014
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Sommario
Relazioni ................................................................................................................................... pag. 93
Comunicazioni orali .....................................................................................................................» 153
Poster............................................................................................................................................» 235
Indice degli Autori .......................................................................................................................» 309
RELAZIONI
PATHOLOGICA 2014;106:93-152
Giovedì, 23 ottobre 2014
Sala Donatello – 09.30-11.30
Lesioni neoplastiche rare in ginecologia
Moderatori: Giovanni Negri (Bolzano), Laura Viberti (Torino)
Le alterazioni morfologiche dell’endometriosi.
Relazioni con il cancro
A. Pesci
Anatomia Patologica Ospedale Sacro Cuore Don Calabria, Negrar
Verona
L’endometriosi è una patologia cronica benigna caratterizzata
dalla presenza di tessuto endometriale al di fuori della cavità
uterina. Colpisce circa il 10-12% delle donne in età fertile e la
percentuale sale al 25-30% nelle donne con problemi di fertilità
e fino al 40-70% nella categoria di donne con dolore cronico.
La patogenesi è multifattoriale, legata a fattori genetici, ormonali ed infiammatori. Quello che si conosce al momento è
che l’endometrio eutopico di donne con endometriosi produce
fattori di crescita e citochine che gli permettono di attecchire
e proliferare in un microambiente, favorevole, che non è il
proprio.
Non esiste una classificazione patologica dell’endometriosi
ma viene mutuata quella chirurgico-laparoscopica che distingue l’endometriosi:
Di superficie: focolai presenti sul peritoneo pelvico;
Endometrioma o cisti ovarica;
Endometriosi profonda (deep infiltrating endometriosis DIE):
massa solida costituita da tessuto endometriale e fibroleiomuscolare che infiltra il peritoneo per > 5 mm e che coinvolge i
legamenti utero-sacrale, retto-sigma, vagina e vescica.
La relazione esistente tra endometriosi e cancro è studiata da
molti anni.
Sicuramente esistono delle similitudini tra endometriosi e
cancro perché l’endometriosi infiltra e distorce l’anatomia
degli organi, può localizzarsi ai linfonodi e diffondere oltre la
cavità pelvica localizzandosi nell’alto addome e più raramente
a polmoni e fegato.
È stata dimostrata da diversi studi l’associazione tra endometriosi e ben definiti istotipi di carcinomi ovarici, adenocarcinoma endometrioide (EOC) e carcinoma a cellule chiare
(CCC), sulla base della similitudine morfologica tra queste
neoplasie ed il cancro, la presenza di focolai di endometriosi
nello stesso ovaio e/o per la continuità tra focolai di endometriosi e cancro.
Studi condotti su larghe cohorti hanno dimostrato che sebbene
l’endometriosi sia una patologia benigna il rischio di sviluppare carcinoma ovarico è aumentato di circa 1,6%. Valore che
aumenta in rapporto all’età della paziente e quindi alla presenza di endometriosi da lungo tempo. Nella nostra casitica l’incidenza di cancro associato all’endometriosi rispecchia quello
riportato in letteratura per quello che riguarda l’endometriosi
ovarica (1,6%), mentre lo sviluppo di cancro in endometriosi
extraovarica, nello specifico intestinale è dello 0,6%.
Studi molecolari stanno dimostrando la presenza di alterazioni
di geni soppressori tumorali quali ARID1A, PTEN e PIK3 in
un’alta percentuale di EOC e CCC e nei focolai di endometriosi contigui alla neoplasia.
Le lesioni che precedono lo sviluppo del cancro sono meno
definite.
Il concetto di endometriosi atipica è molto dibattuto e la gestione clinica di questi casi molto controversa. Con endometriosi atipica ci si riferisce a due situazioni:
1. Iperplasia ghiandolare complessa tipica e/o atipica come
quella che insorge nell’utero, molto infrequente e dovuta ad
una situazione di iperestrogenismo.
2. Atipia su “epitelio piatto”, atipia citologica che viene
definita di grado lieve e di verosimile origine reattiva o
severa (lesione precancerosa) quando presenta nuclei ipertrofici, ipercromatici o chiarificati, aspetti squamoidi del
citoplasma ed affollamento o stratificazione cellulare con la
presenza di piccole proiezionib papillari.
Quando l’endometriosi atipica severa si associa a metaplasia
mucinosa è ritenuta una lesione correlata allo sviluppo di
neoplasia siero-mucinosa di tipo borderline. Tipicamente tale
neoplasia è caratterizzata da papille edematose, con un ricco
infiltrato infiammatorio di tipo granulocitario neutrofilo e
rivestite da un epitelio con aspetti di tipo sieroso, mucinosoendocervicale, ossifilo/squamoide o null type.Queste lesioni
si associano tipicamente ad endometriosi, si presentano come
cisti uni o multiloculate, possono essere bilaterali e si sviluppano in donne giovani. In uno studio condotto in collaborazione con l’Anatomia Patologica del Gemelli di Roma (Prof. GF
Zannoni) abbiamo dimostrato come queste lesioni esprimano
marcatori di tipo mulleriano essendo diffusamente ed intensamente positive per i Recettori degli Estrogeni e Progesterone,
CA125 e PAX8 mentre risultano WT1 negativi.Negli ultimi
anni si è infine descritta un’interessante lesione che colpisce
i focolai di endometriosi a carico dell’appendice vermiforme e che può creare problemi di diagnosi differenziale con
neoplasie primitive dell’appendice. In questa regione focolai
di endometriosi possono determinare iperplasia delle fibre
leiomuscolari della parete con la creazione di mucoceli e lo
stravaso di laghi di muco oppure andare incontro a fenomeni
di metaplasia mucinosa dell’epitelio endometriosico e processi iperplastici che mimano neoplasie mucinose appendicolari
di basso grado.
Ovarian clear carcinoma
G.F. Zannoni
Anatomia Patologica, Policlinico A. Gemelli, Università Cattolica del
Sacro Cuore, Roma
Ovarian clear cell carcinoma constitutes approximately 5-6%
of all ovarian malignancies in the world, while in Japan, the
prevalence is as high as 15-25%. It has also been recognized
to show an association with endometriosis, high degree of
recurrence, and resistance to chemotherapy, thus resulting
in poor prognosis. Clear cell carcinomas, included in type 1
carcinomas, exhibit morphological, molecular and clinical
features that do not entirely resemble either type I or type
II tumors. Indeed, these tumors are typically high-grade,
whereas other type I are low-grade, and the frequency in
clear cell carcinoma of KRAS, BRAF and TP53 mutations
94
is generally lower than type 1 tumours.. In particular, some
authors reported mutations of KRAS from 4.7%, to 26% of
ovarian clear cell carcinomas. On the other hand, other authors failed to observe KRAS mutations in a large cohort of
ovarian clear cell carcinomas. In a recent paper published on
Wirkov Arch we observed a KRAS gene mutations in 13.6%
of ovarian clear cell carcinomas which appears significantly
lower compared to other ovarian carcinoma subtypes, especially mucinous tumors. On the other hand, the frequency of
KRAS mutation, documented in our study, is similar to those
reported for ovarian endometrioid carcinomas (7%), thus supporting the hypothesis of a potential common origin of these
two tumor histotypes. Recent developments in the molecular
biology of clear cell carcinoma have identified multiple signaling pathways associated with development and progression
of this cancer and support the hypothesis that it has a distinct
gene expression profile relative to other histotypes. Specifically, the expression profile signature of clear cell carcinomas
appears to centre around oxidative stress, hypoxia, and cytokine related pathways.
Overall, however, the pathogenesis of clear cell carcinomas
still remains understudied and unknown. Results obtained show
that clear cell carcinoma has unique clinical characteristics and
immunohistochemical features different from other epithelial
ovarian cancers, suggesting that it should be studied separately
in order to establish new treatment molecular targeted strategies, thus improving survival in patients with advanced disease.
Two years ago in a paper published on IJGP we compared ovarian clear cell carcinoma with Type 1 and type 2.
With respect to clinicopathological features, we demonstrated
that, in keeping with previous reported results, clear cell carcinomas are characterized by unilaterality, capsular integrity
and low stage at diagnosis, while bilaterality, capsular infiltration and advanced stage characterize type I and type II tumors
(with incidence lower in type I compared to type II). Therefore, similarities between clear cell and type I carcinomas are
limited to patient’s age, tumor dimension, and incidence of
lymph node and extranodal metastases, while only patient’s
age and incidence of extranodal metastases are comparable
between clear cell and type II groups. Assessment of immunohistochemical parameters also shows that while clear
cell carcinomas typically exhibit low Ki67 and p53 levels,
as expected, their expression increase from type I to type II
carcinomas. The low mitotic activity observed in clear cell
carcinomas could account for the higher incidence of capsular
integrity and early stage disease in this histotype when compared to the two main categories family.
An interesting observation is that hormone receptor profile
differs significantly among the three populations examined,
with clear cell carcinomas showing unique features distinguishing them from both type I and II tumors. Indeed, the
majority of them were negative for ER_ and PR, while showing ER_ positivity mostly confined to the nucleus, with no
cytoplasmic staining (80% of cases). On the other hand, both
type I and II tumors were generally positive for ER_ and PR
(although PR positivity was lower in type II when compared
to type I), and commonly showed ER_ immunoreaction in
both nuclear and cytoplasmic compartments. Our results are
consistent with previous literature data demonstrating that the
expression of ER_ and PR is significantly lower in clear cell
carcinomas when compared to other histotypes..
Only limited data are available on the expression of HER2/
Neu in epithelial ovarian cancer, where positivity varies
between 1.8% and 76%, with different methods of detection
(IHC, FISH, CISH and ELISA among the others), and differ-
CONGRESSO ANNUALE DI ANATOMIA PATOLOGICA SIAPEC-IAP 2014
ent cut-off value. Literature data in epithelial ovarian cancer
also report an association between HER2/Neu overexpression
and advanced stage, poor histological grade, higher recurrence
frequency and shorter survival time, as well as with lower
sensitivity to platinum-based chemotherapy. Results from
our study show that HER2/Neu is significantly up-regulated
in clear cell carcinomas compared to type I and II tumors, in
keeping with findings from a large population study, showing
that, as compared to all other subtypes, clear cell carcinomas
have significantly higher levels (19%)..The higher incidence
of HER2/neu positivity in clear cell carcinomas raises the
possibility that patients with this histotype might be candidates for trials evaluating anti-HER2 MAbs (trastuzumab,
Herceptin), in combination with standard chemotherapy.
These data show that clear cell carcinomas have unique
clinical characteristics and immunohistochemical features
different from other epithelial ovarian cancers.. A diseasebased approach might be more appropriate in the surgical and
therapeutic management of this histotype, characterized by a
poor prognosis in advanced stages and relatively resistant to
standard carboplatin-paclitaxel chemotherapy.
References
1
Gilks CB, Bell, DA, Longrace TA, et al. “Clear cell tumours” in WHO
Classification of Tumours of female reproductive organs, Editors Kurman RJ, Carcangiu ML, Herrington CS, Young R, WHO press 2014
2
Zannoni GF, Morassi F, Prisco MG, et al. Clinicopathologic and
immunohistochemical features of ovarian clear cell carcinomas in
comparison with type I and type II tumors. Int J Gynecol Pathol
2012;31:507-16.
3
Zannoni GF, Improta G, Chiarello G, et al. Mutational status of KRAS,
NRAS, and BRAF in primary clear cell ovarian carcinoma. Virchows
Arch 2014;465:193-8.
I tumori ovarici della granulosa: di tipo adulto
e giovanile
M. De Nictolis
Paper not received
Il carcinoma tubarico
M.L. Carcangiu
Direttore S.C. Anatomia Patologica 1 Fondazione IRCCS Istituto Nazionale dei Tumori
Il carcinoma primitivo della tuba è estremamente raro con
una stimata incidenza di 0.41/100 000 donne. Questo è anche
dovuto alla rigidità dei criteri usati per stabilire la origine
tubarica di una neoplasia. Questi infatti prevedono che il volume maggiore del tumore si trovi nella tuba, che la crescita
neoplastica sia intraluminale e che l’interessamento ovarico
sia minimo. Neoplasie che non soddisfano questi requisiti
sono state classificate come carcinomi tubo-ovarici.
Il tipo istologico più comune è il sieroso, seguito dall’endometrioide e dal carcinoma a cellule chiare che è molto raro. La
localizzazione nella regione fimbrica e l’invasione della parete
tubarica sono fattori prognostici sfavorevoli.
La diffusione extra organo del carcinoma tubarico è sempre
stata considerata sovraponibile a quella del carcinoma ovarico, seppure con una, mai confermata, maggiore incidenza di
metastasi a distanza.
L’ultimo decennio ha visto un rinnovato interesse nella patologia ovarica e tubarica
con il focalizzarsi degli studi principalmente sulle basi morfologiche e molecolari del carcinoma sieroso ovarico e delle
RELAZIONI
lesioni tubariche intra-epiteliali non–invasive. Il carcinoma
sieroso è ora considerato comprendere due entità con diversa
patogenesi, morfologia e comportamento clinico: carcinoma
sieroso di basso grado e carcinoma sieroso di alto grado.
Mentre tradizionalmente è stato assunto che la maggior parte
dei carcinomi sierosi inclusi quelli di alto grado originasse dal
mesotelio che riveste la superficie ovarica, dopo essere questo
andato incontro a cambi metaplastici, una origine tubarica del
carcinoma sieroso di alto grado ovarico e peritoneale è stata
recentemente proposta. Numerose osservazioni morfologiche
e molecolari sembrano suffragare questa ipotesi che ha preso
spunto da studi su ooforectomie profilattiche in donne con
mutazioni BRCA nelle quali focolai di atipia epiteliale, carcinomi in situ e carcinomi invasivi microscopici, sono stati
trovati in oltre il 50% delle tube esaminate, ma non nelle ovaie
e specificatamente non nell’epitelio superficiale ovarico. È
stato quindi teorizzato che carcinomi tradizionalmente classificati come primitivi ovarici o primitivi peritoneali, in realtà
derivino da lesioni pre-neoplastiche tubariche che, essendo
preferenzialmente localizzate nella regione fimbrica, hanno la
capacità di impiantarsi direttamente nell’ovaio, probabilmente
nelle aree della superficie ovarica dove è avvenuta l’ovulazione. L’accesso alla cavità peritoneale che la porzione fimbrica
della tuba ha per ragioni anatomiche puo’ inoltre spiegare il
ruolo di lesioni pre-neoplastiche tubariche nell’origine dei
carcinomi primitivi peritoneali.
Appare di conseguenza evidente la inadeguatezza e inutilità
degli sforzi atti ad attribuire una precisa sede di origine ad un
carcinoma sieroso a diffusione prevalentemente peritoneale/
pelvica.
Il carcinoma sieroso tubarico farebbe quindi parte con il carcinoma ovarico e quello peritoneale del cosiddetta “neoplasia
sierosa pelvica”.
Bibliografia
Alvarado-Cabrero I, Navani SS, Young RH, et al. Tumors of the fimbriated end of the fallopian tube: a clinicopathologic analysis of 20 cases,
including nine carcinomas. Int J Gynecol Pathol 1997;16:189-96.
Navani SS, Alvarado-Cabrero I, Young RH, et al. Endometrioid carcinoma of the fallopian tube: a clinicopathologic analysis of 26 cases.
Gynecol Oncol 1996;63:371-8.
Alvarado-Cabrero I, Young RH, Vamvakas EC, et al. Carcinoma of the
fallopian tube: a clinicopathological study of 105 cases with observations on staging and prognostic factors. Gynecol Oncol 1999;72:36779.
Carcangiu ML, Peissel B, Pasini B, et al. Incidental carcinomas in prophylactic specimens in BRCA1 and BRCA2 germ-line mutation carriers, with emphasis on fallopian tube lesions: report of 6 cases and
review of the literature. Am J Surg Pathol 2006;30:1222-30. Medeiros F, Muto MG, Lee Y, et al. The tubal fimbria is a preferred
site for early adenocarcinoma in women with familial ovarian cancer
syndrome. Am J Surg Pathol 2006;30:230-6.
Jarboe E, Folkins A, Nucci MR, et al. Serous carcinogenesis in the
fallopian tube: a descriptive classification. Int J Gynecol Pathol
2008;27:1-9.
Perets R, Wyant GA, Muto KW, et al. Transformation of the fallopian
tube secretory epithelium leads to high-grade serous ovarian cancer
in Brca; Tp53; Pten models. Cancer Cell 2013;24:751-65.
Tumori borderline mucinosi dell’ovaio
S. Losito
S.C. Anatomia Patologica e Citopatologia, Istituto Nazionale Tumori
IRCCS Fondazione G. Pascale, Napoli
Finora, i tumori mucinosi borderline dell’ovaio venivano
classicamente distinti in due tipi: Intestinale ed Endocervicale, con notevoli differenze epidemiologiche, cliniche ed
istopatologiche.
95
Una delle più importanti novità della recente classificazione WHO dei Tumori degli Organi Riproduttivi Femminili,
pubblicata nel corso del 2014, è proprio l’introduzione, tra i
Tumori Epiteliali Ovarici, del Tumore Sieromucinoso, come
istotipo distinto nelle rispettive forme benigne, borderline e
maligne.
Permane, come requisito per la definizione di Tumore Borderline, l’assenza di “ovvia infiltrazione stromale”, ma compare,
per la definizione di istotipo Sieromucinoso, con i sinomini
Tumore Mucinoso Borderline Mulleriano e Tumore Mulleriano Atipico Proliferativo, la presenza di due o più tipi cellulari
mulleriani in ragione di > 10% dell’epitelio; i tipi più frequenti di epitelio sono il sieroso e il mucinoso endocervicale, ma
anche l’endometrioide, il cellule chiare, il transizionale e lo
squamoso possono essere rappresentati.
Clinicamente, il Tumore Sieromucinoso Borderline è sovrapponibile al Tumore Sieroso Borderline, per l’elevata incidenza di bilateralità (40%) e per la possibilità non trascurabile
di essere diagnosticato in stadio avanzato, per una discreta
incidenza (20%) di impianti epiteliali e desmoplastici non
invasivi; dal punto di vista istogenetico, il Tumore Sieromucinoso Borderline sembra essere correlato all’endometriosi,
per l’elevata incidenza di questa patologia (1/3 dei casi), per
l’immunofenotipo (WT1-, ER+, PgR+, Vim+) e per l’elevata
presenza di mutazione di ARID1A, riscontrata nei carcinomi
endometrioidi e a cellule chiare.
I Tumori Mucinosi Borderline di tipo Intestinale sono molto
più frequenti (30-50% dei Tumori Borderline in Europa e
Nord America-70% in Asia), unilaterali, di grosse dimensioni, pluriloculati, rivestiti da epitelio di tipo gastrointestinale,
spesso simile all’epitelio foveolare gastrico, come suggerisce
la frequente positività immunistochimica per Claudina 18 e
per MUC5.
Fondamentale è l’estensivo campionamento di entrambi i tipi
di neoplasia, per la frequente coesistenza, nella stessa lesione,
di aree benigne, borderline e maligne e per il riconoscimento
di fattori di rischio morfologici quali: presenza di focolai di
carcinoma intraepiteliale, di tumore borderline microinvasivo
e di carcinoma microinvasivo o invasivo, oltre che di eventuali noduli murali, descritti come noduli reattivi sarcoma –like,
sarcomi, carcinomi anaplastici e leiomiomi.
Nelle forme intestinali, alla progressione morfologica tra
benigno e maligno corrisponde la presenza di un’identica mutazione di KRAS come evento precoce nella trasformazione
neoplastica. Questo istotipo è praticamente sempre diagnosticato al I stadio, essendo nella quasi totalità dei casi le forme
bilaterali e/o avanzate di natura metastatica.
Bibliografia
Kurman RJ, Carcangiu ML, Herrington CS, et al. WHO Classification of
Tumors of Female Reproductive Organs 2014.
Halimi SA, Maeda D, Shinozaki-Ushiku A, et al. Claudin-18 overexpression in intestinal-type mucinous borderline tumour of the ovary.
Histopathology 2013;63:534-44.
Wu CH, Mao TL, Vang R, et al. Endocervical-type mucinous borderline
tumors are related to endometrioid tumors based on mutation and
loss of expression of ARID1A. Int J Gynecol Pathol 2012;31:297-303.
Yasunaga M, Ohishi Y, Oda Y, et al. Immunohistochemical characterization of mullerian mucinous borderline tumors: possible histogenetic
link with serous borderline tumors and low-grade endometrioid tumors. Hum Pathol 2009;40:965-74.
Vang R, Gown AM, Barry TS, et al. Ovarian atypical proliferative
(borderline) mucinous tumors: gastrointestinal and seromucinous
(endocervical-like) types are immunophenotypically distinctive. Int J
Gynecol Pathol 2006;25:83-9.
96
Patologia rara e non neoplastica in ginecologia
Moderatori: Mauro Biancalani (Empoli), Augusto Giannini (Prato)
Endometrial carcinoma associated with lynch
syndrome
C. Riva, L. Cimetti, I. Carnevali, F. Sessa, M.G. Tibiletti
Department of Surgical and Morphological Sciences, University of
Insubria-Varese
U.O.Anatomia Patologica, Ospedale di Circolo-Varese
One hundred years ago, when the pathologist AS Whartin
first described the “cancer family G” that was later proven
to be a Lynch Syndrome (LS) family, the major types of
tumors found were uterine carcinomas in females and gastric
cancers in males. The tumor spectrum of LS has evolved over
the time to include colorectal cancers as the most frequent,
followed by neoplasms of the uterus, ovary , stomach, small
intestine, hepatobiliary tract, urinary tract, brain and skin. LS
is an autosomal dominant condition characterized by germline
mutations of DNA mismatch repair (MMR) genes, including
hMSH2, hMSH6, hMLH1 and hPMS2. The MMR system
repairs DNA replications errors and it plays an important
role in DNA replication accuracy. Functional inactivation of
MMR genes by mutation or epigenetic mechanisms leads to
microsatellite instability (MSI). MSI can be present in tumors
from LS patients but it is also observed in up to 25% of various sporadic tumors including endometrial carcinomas.
Endometrial carcinoma in the most common extracolonic
tumor in LS patients: in women the lifetime risk of colorectal
cancer is over 30%, but more than 40% to 50% will develop
endometrial carcinoma.
The frequency of germline MMR gene mutations among unselected patients with endometrial carcinoma has been found to
be approximately 2%, while in patients younger than 50 years
the incidence is increased up to 9%. Moreover, early-onset gynaecological cancer frequently represents the first malignancy
in LS. Identification of these patients is important to propose
surveillance strategies as they show increased risk for synchronous and/or metachronous colorectal cancer and other tumors.
After the initial and revised Amsterdam criteria (1991 and
1998), the Bethesda guidelines (2003) were introduced to
broaden testing recommendations and to identify a greater
proportion of affected individuals, including two subsets of
gynecologic cancer populations, endometrial cancer patients
under 45y and those with multiple LS-related neoplasms.
Detection of LS cases is currently achieved through somatic
genetic tests, immunohistochemistry (IHC) for MMR proteins, microsatellite instability (MSI) assay, and germinal
genetic tests, mutational analysis of MMR genes.
In summary LS- related uterine neoplasms often show an
early onset and they frequently represent the “sentinel neoplasia”. LS-associated tumors are more often endometrial or
isthmic endometrioid carcinomas, sometimes showing tumor
heterogeneity, ambiguous features and/or dedifferentiated
components; abundancy of tumor-infiltrating lymphocytes
and of peritumoral lymphocytes are frequently observed. All
these clinopathological features suggest to perform IHC for
MMR proteins and MSI assay as first step. In patients with
loss of MMR proteins and MSI, genetic counseling and mutational analysis should be then considered.
References
Karamurzin Y, Rutgers KL. DNA Mismatch Repair Deficiency in endometrial carcinoma. Int J Gynecol Pathol 2009;28:239-52.
Garg K, Soslow RA. Endometrial carcinoma in women aged 40 years and
younger. Arch Pathol Lab Med 2014;138:335-42.
Rabban JT, Calkins SM, Karnezis AN, et al. Association of tumor morphology with mismatch-repair protein status in older endometrial
cancer patients. Am J Surg Pathol 2014;38:793-800.
Le metaplasie endometriali: un dubbio
diagnostico
G. Negri
Paper not received
Chronic endometritis: an unknown lesion
L. Resta, M.G. Fiore, R. Rossi, A. Marzullo, E. Cicinelli*
Dipartimento dell’Emergenza e dei Trapianti d’Organo. Sezione di
Anatomia Patologica. * Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Ostetricia e Ginecologia 3, Università degli
Studi di Bari
Chronic endometritis represents one of the most controversial
matters in gynecologic pathology. As a matter of fact, its rate
remarkably differs in case-reports and this affects diagnostic
uncertainties involving both the clinician and the pathologist.
From the clinical point of view chronic endometritis can
be very insidious, with few or no symptoms, only revealed
during investigations due to unexplained infertility. In other
cases it can be misdiagnosed as a dysfunctional metrorrhagia,
since a non-coordinated maturation of endometrial glands
and stroma during menstrual cycle is often associated to the
inflammatory condition: the delicate endometrial maturation
sequence is definitely affected by the presence of germs as
well as by the cellular and humoral reaction of the body. Evolution of the inflammation is modified by cyclic changes of a
mucosa whose main features are exfoliation, restoration and
maturation within a few weeks.
A pathologist can be strongly puzzled when diagnosing an endometritis. For quite some time, pathologists have considered
the presence of an inflammatory infiltration with plasma cells
in it as the only unequivocal sign of endometritis 1.
Recently 2, plasma cells have been observed even when
the physiological or pathologic conditions were not strictly
inflammation related: asymptomatic women suffering from
urinary infections, myomas, polyps, IUD, proliferative alterations in cases of anovulation, or patients treated with progesterone. All these factors tend to make the pathologist’s work
even harder in diagnosing the endometritis.
Moreover, we cannot exclude an endometrial infection if,
besides infiltration, there are factors such as an aggressive
behaviour against glands, the presence of an exudate inside
gland lumina, or the formation of granulomas.
Another element supporting the diagnosis of endometritis is
the “spindle cell” alteration in the endometrial stroma, firstly
described by Kurman and Mazur 3. This alteration had never
been considered fundamental for a correct diagnosis, but from
RELAZIONI
our experience, we can say that this stromal change was present in 70.9% of patients suffering from endometritis. It is difficult to explain how this alteration takes place. As these cells
have a particular aspect, we may suppose that the reason why
they turn into spindle cells is due to myofibroblasts present in
many inflammatory foci. Another possibility to explain this
change is either to consider a former alteration in the cells of
endometrial stroma, or to suppose the activation of blast cells.
An important sign, peculiar to chronic endometritis, is the
presence of micropolyps that can be observed during hysteroscopy 4-6.
Micropolyps have different histological aspects 5: in most
cases, they were excrescences formed by a vascular loop
surrounded by inflammatory cells. Another sign is the localized oedematous swelling, with enhancement of the mucosal
surface.
Considering that there is no statistically meaningful correlation between the type and severity of inflammation and the
presence of micropolyps, we can suppose that the latter is a
distinct phenomenon, more related to inflammatory haemodynamics phenomena.
The frequency of micropolyps observed during hysteroscopy
is lower than their presence on histological preparations. This
depends on the small size of these polyps that may be wasted
because of a non-orthogonal embedding of the specimen.
Our study shows the following results:
a) Micropolypoid vegetations are formed in the endometrium;
b) they are a macroscopic evidence of a phlogosis-related process;
c) they can be useful markers for the histologist.
As for the correlation between the morphologic picture of
endometritis and the microbiological exam, paying attention
to 438 cases, where there was a diagnosis of suspect endometritis, 302 cases tested positive to it and 32 negative. We can
consider this result a good one since the correlation concerned
334 cases out of 438, that is to say 76.26%.
In 86 cases the histological exam showed inflammatory infiltration though the cultural exam was negative; we can suppose that
we might have been dealing with different germs we did not test.
In rare cases (n = 18) where the cultural exam was positive
and the histological one negative, we can either suspect a
faulty sampling of the endometrium, or a low pathogenicity of
the germs found. There is no relation between a particular type
of inflammatory infiltration and an etiological factor.
In the past, we tried to identify some peculiar aspects of particularly dangerous phlogoses such as Chlamydia and Ureaplasms: besides that, we dealt with a giant cellular granuloma-
97
tous infiltration typical in tuberculosis, not so rare in female
genitals even today.
Both our experience and the latest investigations highlighted
the heterogeneity of morphologic pictures in phlogoses,
caused by these germs. Therefore, how should we consider
the morphologic pictures we observed? A first consideration
concerns the frequency of this pathology (438 cases out of
2190), with few or no symptoms, disguised as hypofertility or
dysfunctional metrorrhagia.
A fundamental question has to be considered: can we speak of
a clinically relevant inflammatory lesion every time we come
across inflammatory infiltration? This question is frequently
debated in the literature. Undoubtedly, not all cases of histological “endometritis” have the same importance. In cases
when there is the cultural evidence (sometimes supported
by serologic and/or molecular examinations with negative
results), or there are symptoms compatible with an inflammatory condition (abnormal bleeding, troubles with other pelvic
organs, infertility or hypofertility) the suspect for endometritis
is reliable and true. From our experience, an infiltration showing aggressive behaviour against glands, an inflammatory cell
gathering inside glands (a sign of their destruction) or a more
structured infiltration are also relevant.
In conclusion, our investigation has highlighted how in some patients in a fertile age having different symptoms, there is hysteroscopic, histological and microbiological proof of an inflammatory condition. Micropolyps can be considered both histological
and hysteroscopic markers as well as the spindle cell alteration in
stromal elements. None of these markers has an absolute value,
but when an infiltration shows the previously mentioned characteristics, endometritis becomes a reasonable diagnosis.
References
1
Kiviat NB, et al. Endometrial histopathology in patients with cultureproved upper genital tract infection and laparoscopically diagnosed
acute salpingitis. Am J Surg Pathol 1990;14:167-75.
2
Heatley MK. The significance of plasma cells in the endometrium. J
Obstet Gynaecol. 2005;25:711-2.
3
Kurman RJ, Mazur MT. Benign Diseases of the Endometrium. In:
Bleustein’s Pathology of the Female Genital Tract. Kurman RJ (Ed.)
Sprinter Verlag eds. Fourth Edition NewYork 1996; 371.
4
Cicinelli E, et al. Detection of chronic endometritis at fluid hysteroscopy. J Minim Invasive Gynecol 2005;12:514-8.
5
Cicinelli E, et al. Endometrial micropolyps at fluid hysteroscopy suggest
the existence of chronic endometritis. Hum Reprod 2005;20:1386-9.
6
Cicinelli E, et al. Chronic endometritis: correlation among hysteroscopic, histologic, and bacteriologic findings in a prospective trial with
2190 consecutive office hysteroscopies. Fertil Steril 2008;89:677-84.
7
Resta L, et al. Histology of micro polyps in chronic endometritis. Histopathology 2012;60:670-4.
98
Sala Giotto – 09.30-11.30
Le indicazioni del consensus di Vancouver sul carcinoma renale
nella realtà italiana
Moderatori: Maurizio Colecchia (Milano), Rodolfo Montironi (Ancona)
Classificazione
G. Martignoni
Dipartimento di Patologia e Diagnostica, Università di Verona
La classificazione dei tumori renali e in particolare quella
delle neoplasie a cellule renali è andata incontro a significativi
aggiornamenti nel corso degli ultimi trent’anni basantisi su
una miglior comprensione dei caratteri morfologici, immunoistochimici, citogenetici e molecolari delle stesse.
Due importanti consensus conferences tenutesi a Heidelberg
e a Rochester nel 1996 e nel 1997 hanno fornito indicazioni
fondamentali per la classificazione dei tumori renali dell’Organizzazione Mondiale della Sanità (OMS-WHO) redatta nel
dicembre del 2002 a Lione ed edita nel 2004. In quest’ultima
ciascuna singola entità è stata descritta non solo con le classiche caratteristiche patologiche, quali l’aspetto macroscopico
e microscopico, ma anche con quelle genetiche. Inoltre alcune neoplasie sono state inquadrate nosograficamente come
benigne, adenoma tubulo-papillare, oncocitoma e adenoma
metanefrico, e il carcinoma sarcomatoide è stato riconosciuto
piuttosto che come entità a sé stante come estremo grado di
dedifferenziazione comune a tutti gli istotipi,.
Nel corso dell’ultima decade alcuni nuovi tumori sono state
identificati e pertanto la Società Internazionale di Patologia Urologica (ISUP) ha organizzato una nuova consensus
conference tenutasi nel marzo del 2012 a Vancouver al fine
di suggerire raccomandazioni riguardo le nuove neoplasie a
cellule renali e le entità emergenti nonché nuovi concetti e
chiarificazioni sui tumori renali già presenti nella classificazione OMS-WHO attualmente in uso. Il metodo utilizzato è
consistito in una meticolosa revisione della letteratura, in un
survey inviato e compilato prima della consensus conference
e infine in una discussione plenaria conclusasi con un voto da
parte dei delegati nel corso della stessa.
Le nuove neoplasie a cellule renali proposte dall’ISUP a
Vancouver sono state il carcinoma a cellule renali (RCC)
tubulo cistico, il RCC associato a malattia cistica acquisita,
il RCC a cellule chiare (tubulo-)papillare, il RCC t(6;11) con
conseguente ridenominazione dell’intero gruppo dei RCC
con traslocazione come “MiT Family Translocation RCC”
e il RCC associato alla sindrome ereditaria caratterizzata da
leiomiomatosi e RCC.
Il RCC tubulocistico è radiologicamente identificato come
cisti complessa, tipo 3-4 secondo il sistema classificativo di
Bosniak, in soggetti di sesso maschile con età media di 60 anni, macroscopicamente si presenta ben circoscritto con aspetto
traslucido e multicistico e risulta microscopicamente composto da tubuli di dimensioni variabili con rivestimento unifilare
costituito da cellule con citoplasma eosinofilo e nucleo rotondo e nucleolato. Questi elementi sono usualmente racemasi
positivi e presentano trisomia per i cromosomi 7 e 17 come il
RCC papillare. Il RCC associato a malattia cistica a cquisita è
l’istotipo che insorge con maggior frequenza nei reni terminali
ed è composto da voluminosi elementi cellulari eosinofili che
talora presentano aggregazione di tipo cribriforme; in esso si
identificano cristalli di ossalato. Le cellule neoplastiche sono
positive per racemasi e mostrano svariate alterazioni cromosomiche di tipo numerico. Il RCC a cellule chiare (tubulo-)
papillare è una neoplasia originariamente descritta come quella precedente nei reni terminali, ma successivamente descritta
anche in parenchima renale normale con frequenza pari a circa
il 3% delle neoplasie a cellule renali. Essa è composta da elementi cellulari con citoplasma chiarificato aggregantisi in papillare e tubuli che presentano una peculiare distribuzione dei
nuclei che ricorda quella di un endometrio in fase secretiva.
Questi tumori sono risultati costantemente positivi per citocheratina 7 e negativi per racemasi e geneticamente non appaiono presentare alcuna alterazione riguardante i cromosomi 3,
7 e 17. Il RCC t(6;11), come gli altri RCC con traslocazione,
sono stati inizialmente descritti in soggetti in età pediatrica e
giovanile, ma in seguito sono stati riportati anche in pazienti
adulti. Presentano peculiari aspetti morfologici quali la formazione di strutture tipo pseudorosetta e una costante positività
per HMB45 e catepsina K. Tutti queste entità presentano nella
stragrande maggioranza dei casi un comportamento non aggressivo e in particolare nessun caso di RCC a cellule chiare
(tubulo-) papillare ha metastatizzato né recidivato. Il RCC
associata alla sindrome ereditaria caratterizzata da leiomiomatosi e RCC è un tumore renale renale composto da elementi
cellulari con ampio citoplasma eosinofilo e nucleo circondato
da alone chiaro, dotato di voluminoso nucleolo, formanti
strutture papillari. Questa neoplasia insorge nella sindrome
suddetta e mostra mutazioni germline nel gene codificante per
l’enzima fumarato idratasi localizzato sul cromosoma 1q42. I
casi di questa neoplasia descritti in letteratura hanno presentato comportamento aggressivo.
Nel corso della consensus conference sono stati inseriti nel
gruppo delle entità emergenti il RCC follicolare “thyroidlike”, il RCC associato a mutazione della succinico deidrogenasi B e il RCC con traslocazione ALK e sono stati proposti
alcuni nuovi concetti riguardo il RCC a cellule chiare e in
particolare riguardo la sua variante multicistica, il RCC papillare, il carcinoma cromofobo e i tumori oncocitici “ibridi” , il
RCC dei dotti collettori, il RCC midollare, il RCC mucinoso,
tubulare e a cellule fusate, l’angiomiolipoma e in particolare
quello epitelio ide, il nefroma cistico e il tumore misto epitelio-stromale e il sarcoma sinoviale primitivo del rene.
Infine nella consensus conference si sono trattati e sono state
formulate raccomandazioni riguardo il campionamento e la
stadiazione, il grading e altri parametri prognostici e infine
utilizzo e prospettive di biomarcatori per la diagnosi e la prognosi del RCC.
Bibliografia
Srigley JR, Delahunt B, Eble JN, et al The International Society of Urological Pathology (ISUP) Vancouver Classification of Renal Neoplasia. Am J Surg Pathol 201;37:1469-89.
Delahunt B, Cheville JC, Martignoni G, et al The International Soci-
RELAZIONI
ety of Urological Pathology (ISUP) Grading System for Renal Cell
Carcinoma and Other Prognostic Parameters. Am J Surg Pathol
2013;37:1490-504.
Trpkov K, Grignon DJ, Bonsib SM, et al Handling and Staging of Renal
Cell Carcinoma: The International Society of Urological Pathology
Consensus (ISUP) Conference Recommendations. Am J Surg Pathol
2013;37:1505-17.
Tan PH, Cheng L, Rioux-Leclercq N, et al. Renal Tumors: Diagnostic
and Prognostic Biomarkers. Am J Surg Pathol 2013;37:1518-31.
Fattori Prognostici nel carcinoma del rene
M. Scarpelli
Anatomia Patologica, Università Politecnica delle Marche, Ancona
Il carcinoma renale è una malattia eterogenea con una prognosi variabile in rapporto a diversi fattori. I fattori prognostici
sono legati alle caratteristiche del tumore come definite dalla
classificazione TNM corrente, a parametri istologici e a fattori
clinici.
Fra i parametri istologici di valore prognostico discussi nella
Consensus Conference sulle Neoplasie Renali organizzata
dall’ISUP (International Society of Urological Pathology) a
Vancouver nel 2012 sono stati considerati rilevanti: istotipo,
grado, differenziazione sarcomatoide e rabdoide e necrosi
tumorale.
Per quanto riguarda l’istotipo è stato raggiunto un accordo sul
fatto che il carcinoma papillare ed il cromofobo abbiano una
prognosi migliore rispetto al carcinoma a cellule chiare e che
il carcinoma dei dotti collettori abbia una prognosi infausta.
Per le nuove entità considerate nella stessa Consensus Conference meritevoli di essere incluse nella prossima classificazione WHO sono disponibili al momento solo dati incompleti su
piccole casistiche.
Per quanto riguarda il grado si è raggiunto un accordo sul fatto
che debba essere riportato il grado più alto visibile nel tumore
mentre non si è raggiunto un accordo sulla estensione minima
dell’area da prendere in considerazione ai fini del grading. Si
è inoltre raggiunto un accordo sul fatto che il grado dovrebbe
essere applicato al carcinoma a cellule chiare e al carcinoma
papillare ma non al carcinoma cromofobo.
È stato raggiunto un accordo sul fatto che per questi due
istotipi la assegnazione del grado dovrebbe essere basata
esclusivamente sulla prominenza nucleolare per i gradi da 1 a
3 mentre i tumori di grado 4 dovrebbero essere definiti dalla
presenza di estremo polimorfismo nucleare, cellule giganti e/o
differenziazione sarcomatoide o rabdoide.
Sulla base della prominenza nucleolare è stato proposto un
nuovo sistema di grading.
Per quanto riguarda la necrosi si è raggiunto un accordo sul
fatto che la presenza o assenza di necrosi dovrebbe essere
inclusa di routine nel referto istopatologico e che la valutazione deve essere basata sia sulla osservazione macroscopica
che su quella microscopica. Per il carcinoma a cellule chiare
la componente di necrosi dovrebbe essere riportata come
percentuale.
Campionamento e stadiazione
M.R. Raspollini
Istologia e Diagnostica Molecolare Azienda Ospedaliera Universitaria Careggi, Firenze
La conferenza di consenso della Società Internazionale di
Patologia Urologica che si è svolta a Vancouver nel 2012 ha
non solo rivisto la classificazione delle neoplasie renali, ma
99
ha focalizzato la propria attenzione su aspetti tecnici della
diagnostica tra cui il campionamento e la stadiazione. Queste
raccomandazioni sono il frutto di un accordo di maggioranza
basato su una survey online dei membri della Società modulata da un panel di esperti internazionali. Le raccomandazioni
pubblicate sulla rivista American Journal Surgical Pathology
nel 2013 prevedono i seguenti importanti aspetti: 1) inchiostrare il margine chirurgico (sia per nefrectomia radicali che
per le parziali); 2) misurare il tumore lungo l’asse maggiore,
escludendo dalla misurazione l’eventuale presenza di trombo
cavale o della vena renale; 3) eseguire campionamento di
un blocchetto per centimetro di tumore, con un minimo di
tre blocchetti e, nel caso di tumori multipli, includere almeno un minimo di cinque tumori di dimensioni maggiori; 4)
determinare l’invasione del grasso perirenale eseguendo il
campionamento con sezioni multiple perpendicolari al tumore; 5) determinare l’invasione del grasso del seno renale e
l’aderenza-invasione della vena cava; 6) campionare il parenchima renale macroscopicamente non coinvolto dal tumore;
7) campionare la ghiandola surrenale dopo aver valutato il
suo eventuale coinvolgimento per contiguità all’esame macroscopico per differenziarlo, in caso di invasione vascolare,
da metastasi; 8) valutare i linfonodi e l’area ilare in caso di
nefrectomia radicale.
La stadiazione dei tumori è critica nella stratificazione dei
pazienti e del loro trattamento. Un importante passo è determinare le dimensioni del tumore, l’infiltrazione del tumore
nel grasso perirenale, e del seno, e la sua estensione vascolare.
Particolare attenzione va fatta sull’esame macroscopico: a
questo riguardo il patologo deve essere estremamente familiare con l’anatomia renale, sia per il campionamento di per sé,
che per l’accurata descrizione del reperto. Viene dato particolare risalto alla valutazione dei margini rispetto la vena renale,
e all’invasione sia del grasso perirenale che di quello del seno
renale. Quest’ultimo, inizialmente riconosciuto solo per la
stadiazione del nefroblastoma, è ritenuto ora importante anche
per la valutazione del carcinoma renale a cellule chiare, rappresentando l’invasione un fattore prognostico estremamente
negativo. Viene perciò raccomandato che nell’incertezza di
una presenza d’invasione del seno, si campionino almeno
tre blocchi dell’interfaccia tumore-seno. Un altro importante
aspetto per la stadiazione è il campionamento del margine della vena renale per valutare al microscopio l’eventuale aderenza
e/o invasione. Infine, è essenziale campionare il parenchima
renale distante dal tumore per evidenziare eventuali patologie
glomerulari, tubulo-interstiziali, o vascolari associate. La VII
edizione del sistema di stadiazione TNM del 2010 ha introdotto diverse modifiche che necessitano di questi accorgimenti
al momento del campionamento. Sicuramente l’adesione a
queste recenti raccomandazioni permetterà una stadiazione
più aderente all’attuale sistema TNM, ma anche un più facile e
attendibile confronto di risultati fra casistiche dando maggiore
spessore ai risultati dei conseguenti trattamenti.
Bibliografia
Bonsib SM. Renal veins and venous extension in clear cell renal carcinoma. Mod Pathol 2007;20:44-53.
Delahunt B, Cheville JC, Martignoni G, et al. The International Society
of Urological Pathology (ISUP) Grading System for Renal Carcinoma
and other prognostic parameters. Am J Surg Pathol 2013;37:1490504.
Srigley JR, Delahunt B, Eble JN, et al. The International Society of Urological Pathology (ISUP) Vancouver Classification of Renal Neoplasia. Am J Surg Pathol 2013;37:1469-89.
Tan PH, Cheng L, Rioux-Leclercq N, et al. Renal Tumors. Diagnostic and
Prognostic Biomarkers. Am J Surg Pathol 2013;37:1518-31.
Trpkov K, Grignon DJ, Bonsib SM, et al. Handling and staging of renal
100
cell carcinoma. The International Society of Urological Pathology
Consensus (ISUP) Conference Recommendations. Am J Surg Pathol
2013;37:1505-17.
Biomarkers in renal cell carcinoma
M. Brunelli, G. Martignoni
Department of Pathology and Diagnostics, University of Verona, Italy
The International Society of Urological Pathology convened a
consensus meeting on renal cell carcinoma, to address issues
relating to renal neoplasia. The role of immunophenotypical
and cytogenetic biomarkers in the diagnosis and assessment of
prognosis of renal tumors has been addressed.
At phenotypical level, a panel of antibodies has been applied
to the assessment of renal tumors, and these include cytokeratins, vimentin, a-methylacyl CoA racemase (AMACR),
carbonic anhydrase IX (CAIX), Pax 2, Pax 8, RCC marker,
CD10, E-cadherin, kidney-specific cadherin, parvalbumin,
claudin-7, claudin-8, S100A1, CD82, CD117, TFE3, TFEB,
thrombomodulin, uroplakin III, p63, and S100P.
Clear cell RCC is usually positive for vimentin, keratin,
EMA, CD10, Pax 2, RCC marker, and CAIX and negative
for kidney-specific cadherin and parvalbumin. CD10 and Pax
2 upregulation is due to VHL inactivation in clear cell RCC;
CAIX is also consistently.
Papillary RCC type 1 is positive for vimentin, keratins, CK7,
AMACR, and RCC marker, and negative for CD117, kidneyspecific cadherin, and parvalbumin. Papillary RCC type 2 has
variable staining patterns.
Chromophobe RCC shows diffuse reactivity for E-cadherin,
kidney-specific cadherin, parvalbumin, CD117, EMA, broadspectrum keratins, and CK7 and no expression of vimentin,
CAIX, and AMACR.
Collecting duct carcinoma is often positive for EMA, CK7,
high–molecular weight keratin, Pax 2, and Pax 8 and negative
for CD10 and CK20.
Renal oncocytoma usually express parvalbumin, S100A1 and
stains few cells for CK7.
There was consensus that 5 entities should be recognized as
new distinct epithelial tumors within the classification system:
tubulocystic renal cell carcinoma (RCC), acquired cystic disease-associated RCC, clear cell (tubulo) papillary RCC (cyto-
keratin 7++, HMWCK+), the MiT family translocation RCCs
(in particular t(6;11) RCC) (cathpesin-k++, melanocytic
markers ++), and hereditary leiomyomatosis RCC syndromeassociated RCC. In addition, there are 3 rare carcinomas that
were considered as emerging or provisional new entities:
thyroid-like follicular RCC; succinate dehydrogenase B deficiency-associated RCC (succinate dehydrogenase B++); and
ALK translocation RCC (break-apart ALK probe+).
Questions with consensus online responses immunohistochemistry is occasionally used for histologic subtyping and
used before diagnosing unclassified RCC. At molecular level,
FISH is the most commonly used molecular platform. TFE3
and TFEB analysis (immunohistochemistry and/or FISH)
should be requested when RCC is diagnosed in a patient under
30 years of age, and/or when the morphologic appearances are
suggestive. Pax 2 and/or Pax 8 are the most useful markers in
confirming a renal primary.
At cytogenetic molecular level, clear cell RCC shows chromosome 3p aberrations, most commonly loss of 3p and mutations
of the VHL gene, whereas papillary RCC shows a variety of
abnormalities, most often trisomies of chromosomes 7 and 17.
Chromophobe RCC is known to harbor multiple numerical
losses of chromosomes 1, 2, 6, 10, and 17, whereas for collecting duct carcinomas there are many numerical and structural
aberrations. Translocation RCCs are defined by translocations involving chromosome Xp11.2, resulting in TFE3 gene
fusions. Another variant of translocation-associated RCC is
characterized by fusion of the TFEB gene on chromosome 6p
to the alpha gene on 11q12, which leads to expression of the
TFEB protein.
Currently, the majority of patients with advanced clear cell
RCC receive vascular endothelial growth factor (VEGF)
pathway antagonists as first-line therapy for metastatic disease. Analysis of VHL mutation status and that of plasma
CAIX, VEGF, sVEGFR2, tissue inhibitor of metalloproteinase 1 (TIMP-1), and Ras p21. Other targeted agents being
pursued in advanced clear cell RCC include selective mTOR
inhibitors.
References
Srigley JR, Delahunt B, Eble JN, et al.; ISUP Renal Tumor Panel. The
International Society of Urological Pathology (ISUP) Vancouver
Classification of Renal Neoplasia. Am J Surg Pathol 2013;37:1469-89.
Sala Giotto – 15:30-17.00
Attualità e controversie in patologia prostatica
Moderatori: Marco Carini (Firenze), Paolo Viacava (Lido di Camaiore)
Aspetti attuali e controversi dello score di
Gleason
M. Colecchia
Fondazione IRCCS Istituto dei Tumori di Milano
Ad oggi il metodo più affidabile in nostro possesso per valutare il rischio relativo al potenziale biologico di aggressività
nel carcinoma della prostata consiste nel gradare la neoplasia
secondo il Gleason score modificato nell’ISUP grading del
2005 1. Quando esso è utilizzato in modo accurato, ad esempio
esaminando i campioni di prostatectomia radicale, costituisce
un potente strumento per predire le metastasi ed il rischio di
morte. Il sistema di Gleason definisce cinque patterns morfo-
logici. Studi recenti hanno modificato il Gleason score eliminando praticamente dall’uso comune i pattern 1 e 2 2 lasciando
i patterns 3, 4 e 5 quali patterns principali del carcinoma.
Il Gleason score è la somma dei patterns prevalente e secondario di Gleason nella prostatectomia radicale e del pattern prevalente e del più elevato nei campioni bioptici. Ad esempio un
carcinoma score di Gleason 3+4=7 presenta pattern prevalente
3 ma ha anche una componente 4. Tumori che presentano solo
pattern 3 saranno diagnosticati con Gleason score 3+3=6: queste neoplasie di basso grado generalmente non metastatizzano
3
, mentre i carcinomi con patterns 4 e 5 sono responsabili di
elevata morbidità e mortalità cancro-specifica 4.
Ancora meritevole di studi clinico-patologici è la controversa
101
RELAZIONI
questione se la neoplasia prostatica di Gleason score 3+3=6
meriti o meno di essere definita carcinoma 5. In merito alla
proposta di un novello sistema di gradazione prognostica si
cercherà un consenso promosso da un panel di esperti della
International Society of Uropathology, che si terrà a Chicago
nel Novembre 2014.
Bibliografia
1
Epstein JI, Allsbrook WC Jr, Amin MB, et al.; ISUP Grading Committee.The 2005 International Society of Urological Pathology (ISUP)
Consensus Conference on Gleason Grading of Prostatic Carcinoma.
Am J Surg Pathol 2005;29:1228-42.
2
Berney DM. Low Gleason score prostatic adenocarcinomas are no
longer viable entities. Histopathology 2007;50:683-90.
3
Ross HM, Kryvenko ON, Cowan JE, et al. Do adenocarcinomas of the
prostate with Gleason score (GS) ≤6 have the potential to metastasize
to lymph nodes? Am J Surg Pathol 2012;36:1346-52.
4
Eggener SE, Scardino PT, Walsh PC, et al. Predicting 15-year prostate cancer specific mortality after radical prostatectomy. J Urol
2011;185:869-75.
5
Pierorazio PM, Walsh PC, Partin AW, et al. Prognostic Gleason grade
grouping: data based on the modified Gleason scoring system. BJU Int
2013;111:753-60.
Prostate changes related to therapy
R. Montironi
Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71,
I-60126 Torrette, Ancona, Italy
The number of newly diagnosed cases of prostate cancer
(PCa) has doubled in the last few years. Factors that have
contributed to this rise include the aging of the population,
coupled with growing awareness of the importance of early
detection by measurement of serum prostate-specific antigen
(PSA) levels and digital rectal examination. Many of these
prostate cancers, whose most likely precursor is prostatic intraepithelial neoplasia, are curable. In the hope of improving
long-term disease-free survival, urologists are now performing radical prostatectomy (RP) increasingly often in men
with PCa that is localized upon presentation and with a life
expectancy of more than 10 years. Radiation and hormone and
therapy have also been used in PCa. Other ablative therapies,
such as cryotherapy, and emerging focal therapies, including
high-intensity focused ultrasound, photodynamic therapy, and
interstitial laser thermotherapy, may have morphological effects on prostate tissue.
Morphological effects are often identified in needle biopsies
and surgical specimens. A range of histological changes are
seen in the non-neoplastic prostate and in the pre-neoplastic
and neoplastic areas. Other ablative therapies, including cryotherapy, and emerging focal therapies, such as high-intensity
focused ultrasound, photodynamic therapy, and interstitial
laser thermotherapy, may induce changes on the prostate. As
new compounds are developed for prostate cancer treatment,
it is important to document their effects on benign and neoplastic prostate tissue.
Over the past few of years, the introduction of new agents
with different mechanisms of action and favorable safety
profiles has led to an improvement in overall survival and
quality of life for men with metastatic castration resistant
prostate cancer. The impetus for new drug development has
followed important molecular genetic discoveries based on
the possibility that prostate cancer could be considered not a
clinically heterogeneous disease but a collection of homogeneous subtypes identifiable by molecular criteria, and perhaps
vulnerable to targeted therapies. In particular, our knowledge
of the molecular circuitry of tumor invasion, metastases and
treatment resistance, has become more refined. The number of
new targets, potentially useful, has grown exponentially. This
is reflected in the vast array of diverse targeted agents that are
currently being evaluated in human trials. In the future years,
challenge will be to identify the agents that target validated
pathways, and refining their role in the natural history of the
disease. As new therapies are developed for prostate cancer,
it is important to document their effects on benign and malignant prostate tissue and to understand possible implications
for traditional prognostic factors.
Carcinoma intraduttale della prostata
A. Lopez-Beltran
Cordoba University Medical School and Champaplimaud Clinical
Center
Although histological and molecular evidence points to the
fact that IDC-P is a late-stage intraductal spread of existing,
high-grade invasive prostate cancer (PCa), there are a few
cases of IDC-P without an associated invasive component.
Such cases suggest that IDC-P, in some instances, may act as
a precursor lesion in the HGPIN pathway of cancer or possibly as a separate de novo pathway.
Histological features
Cohen et al. were the first to describe three histological patterns for IDC-P.
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cells, approximately 2 cell-layers thick, that span the lumen in the absence of stromal support. In some cases, the
trabeculae fail to span the entire lumen and form a focal
papillary pattern.
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punched-out round spaces comprising greater than half of
the luminal space. Small foci of comedonecrosis can be
found occasionally in this subtype.
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of cells with frequent foci of central comedonecrosis. Positive Ki-67 nuclear staining was confined to the perimeter
compartment in types A and B but was found in both the
central and perimeter compartments in type C.
These different patterns of IDC-P correlated with the frequency of high-grade (Gleason 4/5) invasive carcinomas in
a stepwise fashion, with type C associated with the highest
volume and highest grade tumors. In this regard, types A and
B could be regarded as low-grade and medium grade patterns,
respectively.
Recently, Guo and Epstein described four subtypes of IDCP, including solid, dense cribriform, loose cribriform, and
micropapillary. They proposed diagnostic criteria of IDC-P
in prostate biopsy as a proliferation of malignant prostatic
glandular cells filling ducts and acini, with some basal cell
preservation. The first tier criterion is a solid or dense
cribriform pattern. If the first tier criterion is not present, a
diagnosis of IDC-P can be made if loose cribriform or micropapillary pattern is identified with one of the following: 1)
prominent nuclear pleomorphism (nuclear size greater than
6× normal) or 2) non-focal comedonecrosis (>1 duct showing comedonecrosis).
This IDC-P subtyping is currently considered as the international standard.
The low grade spectrum of IDC-P overlaps with HGPIN.
Even though IDC-P with low grade morphology does not
meet the diagnostic criteria for IDC-P in prostate biopsies,
102
it should not be simply dismissed as HGPIN. In lesions that
cannot be confidently classified, the term “atypical intraductal
proliferations” has been used [7].
Molecular features
There is molecular evidence that IDC-P and HGPIN represent
distinct entities. Dawkins et al. studied twenty radical prostatectomy specimens with IDC-P for loss of heterozygosity
(LOH) at known loci on chromosomes strongly associated
with prostate cancer and compared the results of HGPIN with
invasive Gleason grade 3 and grade 4 carcinomas. Loss of
heterozygosity was detected in 60% of IDC-P, 29% of Gleason grade 4 cancers, 9% of HGPIN, and no Gleason grade 3
cancers. Based on these findings, the authors concluded that
IDC-P is a distinct pathologic process from HGPIN.
Bettendorf et al. analyzed HGPIN, IDC-P, and invasive PCa
for LOH of TP53 and RB1 (tumor suppressor genes) and used
comparative genomic hybridization on HGPIN and IDC-P
to further characterize the two types of lesions. LOH of both
TP53 and RB1 were found frequently in IDC-P (52%) and
tumor tissue in extraprostatic extension (44%), and rarely in
HGPIN (19%) and benign prostatic tissue (17%). Comparative genomic hybridization showed that all of the HGPIN lesions lacked chromosomal imbalances in contrast to IDC-P
where 8/11 cases demonstrated chromosomal imbalances.
Han et al. analyzed E-twenty six (ETS) gene aberrations, the
most common of which in prostate cancer is the TMPRSS2ERG fusion, using a break-apart fluorescence in-situ hybridization assay to help establish distinguishing features between
HGPIN and IDC-P. ERG gene rearrangement was found in
75% of the cases of IDC-P and in none (0/16) of the cases of
HGPIN, and the ERG gene status was concordant between
IDC-P and adjacent invasive PCa. Recently, it has been shown
that immunohistochemical stains using antibodies against
ERG correlate strongly with the status of the ERG gene fusion, potentially making it an attractive marker for IDC-P
versus HGPIN. Other researchers have investigated deletions
involving the PTEN locus in IDC-P versus HGPIN. Loss of
PTEN, a tumor suppressor, occurs in up to 70% of invasive
prostatic carcinomas and is uncommon in HGPIN. Using immunohistochemistry, cytoplasmic loss of PTEN was identified in 84% of IDC-P and was not identified in any cases of
HGPIN (0/39).
There is some evidence that IDPC may be regulated by
estrogens and their receptors. Mosquera et al. have identified five morphological features of PCa associated with
TMPRSS2-ERG gene fusion, including blue-tinged mucin,
cribriform growth pattern, macronucleoli, signet-ring cell
features, and IDPC. In a subsequent study, the same group of
authors have identified an 87 gene expression signature for
TMPRSS2- ERG tumours that was associated with oestrogen
receptor (ER) signalling pathways. TMPRSS2- ERG expres-
sion was found to be increased by ER agonist (estrogens) and
decreased by ERβ agonists. In fact, IDPC and related Gleason
pattern 4/5 PCa may express ER and the oestrogen inducible
progesterone receptor (PR), indicating that these tumours are
regulated by estrogens and could be targeted accordingly.
Conclusions
IDC-P, a proliferation of prostate adenocarcinoma cells
distending and completely spanning the lumen of normal
prostatic ducts and acini, is strongly associated with invasive,
high-grade prostate cancers. This is especially true on needle
core biopsies where only IDC-P is found without invasive
carcinoma, leading some authors to advocate for definitive
treatment or immediate rebiopsy. Genetic studies indicate that
IDC-P represents intraductal spread of invasive carcinoma,
rather than a precursor lesion.
References
Watts K, Li J, Magi-Galluzzi C, Zhou M. Incidence and clinicopathological characteristics of intraductal carcinoma detected in prostate
biopsies: a prospective cohort study. Histopathology 2013;63:574-9.
Risbridger GP, Taylor RA, Clouston D, Sliwinski A, et al. Patient-derived Xenografts reveal that intraductal carcinoma of the prostate is a
prominent pathology in BRCA2 mutation carriers with prostate cancer
and correlates with poor prognosis. European Urology (in Press)
Cohen RJ, McNeal JE, Baillie T. Patterns of differentiation and proliferation in intraductal carcinoma of the prostate: significance for cancer
progression. Prostate 2000;43:11-9.
Guo CC, Epstein JI. Intraductal carcinoma of the prostate on needle
biopsy: histologic features and clinical significance. Mod Pathol
2006;19:1528-35.
Shah RB, Zhou M. Atypical cribriform lesions of the prostate: clinical
significance, differential diagnosis and current concept of intraductal
carcinoma of the prostate. Adv Anat Pathol 2012;19:270-8.
Dawkins HJ, Sellner LN, Turbett GR, et al. Distinction between intraductal carcinoma of the prostate (IDC-P), high-grade dysplasia (PIN),
and invasive prostatic adenocarcinoma, using molecular markers of
cancer progression. Prostate 2000;44:265-70.
Bettendorf O, Schmidt H, Staebler A, et al. Chromosomal imbalances,
loss of heterozygosity, and immunohistochemical expression of TP53,
RB1, and PTEN in intraductal cancer, intraepithelial neoplasia, and
invasive adenocarcinoma of the prostate. Genes Chromosomes Cancer
2008;47:565-72.
Han B, Suleman K, Wang L, et al. ETS gene aberrations in atypical
cribriform lesions of the prostate: implications for the distinction between intraductal carcinoma of the prostate and cribriform high-grade
prostatic intraepithelial neoplasia. Am J Surg Pathol 2010;34:478-85.
Lotan TL, Gumuskaya B, Rahimi H, et al. Cytoplasmic PTEN protein loss
distinguishes intraductal carcinoma of the prostate from high-grade
prostatic intraepithelial neoplasia. Mod Pathol 2013;26:587-603.
Mosquera JM, Perner S, Demichelis F, et al. Morphological features of
TMPRSS2-ERG gene fusion prostate cancer. J Pathol 2007;212:91101.
Montironi R, Scarpelli M, Cheng L, et al. Do not misinterpret intraductal
carcinoma of the prostate as high-grade prostatic intraepithelial neoplasia! Eur Urol 2012;62:518-22.
Bonkhoff H, Wheeler TM, van der Kwast TH, et al. Intraductal carcinoma of the prostate: precursor or aggressive phenotype of prostate
cancer? Prostate 2013;73:442-8.
103
RELAZIONI
Neoplasie della vescica e problematiche di diagnosi differenziale
Moderatori: Antonio Lopez Beltran (Cordoba), Maria Rosaria Raspollini (Firenze)
Varietà istologiche
E. Bollito
Paper not received
Istopatologia del carcinoma vescicale pT1
C. Patriarca
Anatomia Patologica St Anna, Como
Una corretta stadiazione del materiale proveniente da TUR
rappresenta un prerequisito necessario alle scelte dell’Urologo, quando si è di fronte a un carcinoma della vescica.
Riguardo una più precisa definizione del carcinoma infiltrante
il connettivo sottoepiteliale (T1), sarebbe bene fornire una
qualche stima della profondità di invasione, benché non vi sia
un’indicazione in tal senso da parte di WHO/ISUP. Le ragioni
per cui è conveniente “stimare” l’invasione del connettivo
sono due:
1. Scelta dell’opzione cistectomia in caso di fallimento del
BCG.
2. Scelta dell’opzione cistectomia in caso di primo episodio
ma con malattia T1 profonda e multifocale.
Certo, l’ideale sarebbe poter eseguire delle resezioni en bloc
delle neoplasie vescicali.
Proviamo a mettere per un momento a tacere le obiezioni che
una simile proposta talora solleva (le dimensioni eccessive
della neoplasia, la scelta di un resettore idoneo, la sede talvolta impervia della neoformazione, il rischio di perforazione
della parete, etc) e immaginiamo di inviare al patologo con
questa modalità le neoformazioni vegetanti della vescica 1;
diciamo per prudenza quelle inferiori ai 2 centimetri, dopo
tutto un numero non esiguo di casi.
Siccome la base muscolare della resezione en bloc tende
a flettersi verso l’asse della vegetazione, il patologo potrà
fissare il pezzo “a fresco” su una superficie di sughero, se la
lesione non è troppo piccola; inoltre, sempre se la neoplasia
non è troppo piccola, potrà anche segnare i margini della resezione con inchiostro di china. In ogni caso il patologo e il
tecnico di istologia maneggeranno materiale vescicale con una
caratteristica inaspettata: l’orientabilità.
Ciò che il patologo fino ad oggi ha indicato in un referto
di TURB è: l’istotipo della lesione, il grading, l’eventuale
infiltrazione con una stima approssimativa nel caso di una neoplasia T1, e l’embolizzazione neoplastica quando presente.
Vediamo ora ciò che il patologo potrebbe dire di più, nel caso
di una resezione unica e orientabile della neoplasia:
r VOB TUJNB NBDSPTDPQJDB EFMMB EJNFOTJPOF EFM UVNPSF DIF
sappiamo essere un fattore prognostico.
r 6OB WBMVUB[JPOF JTUPMPHJDB EFJ NBSHJOJ MBUFSBMJ EFMMB SFTFzione, anche se non sempre secondo la letteratura 2. Poiché
la recidiva precoce è un importante fattore prognostico
anche questo dato può rivelarsi prezioso.
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profondità di infiltrazione 3. L’inattuabilità della suddivisione in T1a e T1b nella maggior parte dei casi con le TURB
convenzionali è esperienza comune.
r 6OBWBMVUB[JPOFJONJMMJNFUSJEFMMBQSPGPOEJUÆEJJOGJMUSB[JPne. L. Cheng 4 ha fornito un dato di valutazione micrometrica dello spessore di infiltrazione, stabilendo in 1,5 mm una
soglia significativa per il PFS. In talune aree anatomiche
della vescica, come il trigono e la parete anteriore, un simile spessore di infiltrazione è tuttavia spesso superiore allo
spessore della lamina propria 5 e fa supporre uno sconfinamento in T2 della neoplasia, non sempre documentabile nel
caso di TURB convenzionale, composta com’è da singoli
frammenti profondi tutto-tumore.
r 6OBWBMVUB[JPOFJONJMMJNFUSJEFMMBEJTUBO[BEBMNBSHJOFEJ
resezione muscolare profondo.
r 6OB RVBOUJGJDB[JPOF NJHMJPSF EFMMFWFOUVBMF FUFSPHFOFJUÆ
della neoplasia.
È evidente come un simile approccio eliminerà la necessità
di reTURB per ora in via teorica e in ogni caso solo dopo
studi controllati, ma è fin d’ora chiaro come le resezioni en
bloc riducano il ben noto rischio di seeding e reimpianto del
tumore uroteliale.
In definitiva la questione, vista dalla parte del patologo, riveste caratteristiche simili a quelle di altre “biopsie” che la
surgical pathology tratta da tempo come “pezzi operatori”,
quali i polipi intestinali cancerizzati o i coni cervicali uterini;
vista dalla parte dell’uropatologo si riassume in una richiesta
di “pari dignità” per le neoplasie uroteliali papillari.
Nell’attesa che questo approccio si faccia strada, sono sopraggiunte alcune novità dalla letteratura, come il substaging sec
van Rhijn 6. A tal proposito verranno esposti i dati preliminari
di uno studio retrospettivo multicentrico (St. Anna, Como,
San Raffaele e INT Milano e Humanitas Rozzano).
Sullo sfondo restano tuttavia i problemi interpretativi quotidiani relativi ai pattern di infiltrazione del carcinoma della
vescica, sia del tipo convenzionale che delle varianti, responsabili sia di overstaging che di understaging. In particolare,
in assenza di caratteristiche di infiltrazione chiara (differenziazione paradossa, sgocciolamento, reazione infiammatoria
stromale) anche il carcinoma uroteliale convenzionale può
sollevare problemi interpretativi. Inoltre, bisogna considerare
le diverse entità e varianti/istotipi, che possono essere presentate come pattern ghiandolare (adenocarcinoma, adenocarcinoma a cellule chiare, adenoma nefrogenico, adenocarcinoma
uracale, mullerianosi/ carcinoma micro cistico etc.), pattern
a singole cellule (carcinoma plasmacitoide, adenocarcinoma
a cellule ad anello con castone etc), pattern a cellule fusate
(carcinoma sarcomatoide, sarcomi, tumore miofibroblastico
infiammatorio), e pattern a nidi/piccoli nidi (nested carcinoma, carcinoma micro papillare). Verranno esposti alcuni dei
quadri istologici più tipici e problematici.
Bibliografia
1
Naselli A, Introini C, Germinale F, et al. En bloc transurethral resection of bladder lesions: a trick to retrieve specimens up to 4.5 cm. BJU
Int 2012;6:960-3. Int J Urol 2010;17:708-14.
2
Ukai R, Hashimoto K, Iwasa T, et al. Transurethral resection in one
piece (TURBO) is an accurate tool for pathological staging of bladder
tumor. Int J Urol 2010;8:708-14.
3
Angulo JC, Lopez JI, Grignon DJ, et al. Muscolaris mucosae differentiates two populations with different prognosis in stage T1 bladder
cancer. Urology 1995;45:47-53.
104
4
5
6
Cheng L, Neumann RM, Weaver AL, et al. Predicting cencer progression in patients with stage T1 bladder carcinoma. J Clin Oncol
1999;17:3182-7.
Paner GP, Ro JY, Wojcik EM, et al. Further characterization of the
muscle layers and lamina propria of the urinary bladder by systematic
histologic mapping: implications for pathologic staging of invasive
urothelial carcinoma. Am J Surg Pathol 2007;9:1420-9.
Bas WG van Rhijn et al. A New and Highly Prognostic System to
Discern T1 Bladder Cancer Substage. Eur Urol 2012;378-81.
Problematiche istopatologiche dei tumori
vescicali muscolo invasivi
M. Fiorentino
Paper not received
Sala Botticelli – 10.30-12.30
Malattie autoimmuni del fegato
Moderatori: Massimo Rugge (Padova), Luca Saragoni (Forlì)
Autoimmune hepatitis
M. Guido
Dipartimento di Medicina, DIMED Sezione di Anatomia Patologica
Università degli Studi di Padova
Autoimmune hepatitis (AIH) is an immune-mediated liver
disorder characterized by female preponderance, elevated
transaminase and immunoglobulin G levels, seropositivity for
autoantibodies and interface hepatitis. Presentation is highly
variable and AIH should be considered in the differential
diagnosis of any increase in liver enzyme levels.
Since transaminases and IgG levels do not reflect the extent of
histological inflammatory activity, or the presence or absence
of cirrhosis, liver biopsy is mandatory both to confirm the
diagnosis and to evaluate the severity of liver damage. Some
hepatologists require liver biopsy before withdrawal of treatment, though histological resolution does not assure stable
remission.
Interface hepatitis is the hallmark of AIH, though it is not
exclusively observed in this condition. It is characterised by
a lymphoplasmacytic infiltrate crossing the limiting plate and
invading the liver parenchyma. Although plasma cells are
usually abundant at the interface and throughout the lobule,
they are not a constant feature of the disease: their absence
does not exclude AIH nor does their presence exclude other
diseases. The hepatic lobules show inflammatory cell infiltrate, and in severe cases, there may be diffuse hepatocyte
injury associated with hepatocyte swelling and “lobular disarray.” Apoptotic bodies may be numerous. Confluent hepatocyte necrosis may be seen, particularly in newly diagnosed,
untreated patients. When lobular hepatocyte injury is extensive, regenerative features with bile ductular proliferation
or hepatocyte rosetting are usually seen. Centrilobular, perivenular, necrosis is a recognized feature of AIH. This pattern
of injury may be predominant, with relative portal sparing in
up to 18% of patients. The inflammatory lesions usually spare
the biliary tree, but up to 12% of biopsies may show duct
destruction or lymphocytic infiltration of bile duct epithelium
without ductopenia. Diffuse “florid” inflammatory changes
of the bile ducts should rise the suspicion of an overlap with
sclerosing cholangitis, which occurs more frequently in the
paediatric setting. In progressed AIH, fibrosis develops with
formation of portal-portal and portal-central bridges which
results in cirrhosis, often with severe necroinflammatory activity. Histological changes of AIH are not pathognomonic,
but in the appropriate clinical setting, the diagnosis is not difficult. The major differential diagnosis includes other forms
of chronic hepatitis, including viral hepatitis, which can be
excluded by serologic testing. Autoantibodies are commonly
found in chronic hepatitis C, but true overlap with AIH is very
rare. Close correlation with clinical data is warranted in such
cases. Drug-induced hepatitis may be indistinguishable from
AIH, and a careful history is required to exclude medications,
herbal products, and nutritional supplements. Drugs or other
toxins can also induce AIH with a serological profile resembling either type 1 AIH or type 2 AIH. AIH may overlap with
PBC clinically and the distinction is important because the
treatment differs.
Malattie biliari
G. Faa
Paper not received
Fegato e autoimmunità
A. Floreani
Dipartimento di Scienze Chirurgiche, Oncologiche e Gastroenterologiche, Università di Padova
Le principali malattie autoimmuni del fegato sono: l’epatite
autoimmune, la cirrosi biliare primitiva (CBP) e la colangite
sclerosante primitiva (CSP).
Epatite autoimmune: è una malattia rara, caratterizzata dalla
preponderanza del sesso femminile, da ipergammaglobulinemia, dalla positività per autoanticorpi non organo-specifici,
dall’associazione con HLA DR3 e DR4 e da una buona risposta alla terapia immunosoppressiva. Si riconoscono diversi
sottotipi: tipo 1 caratterizzato da anticorpi antinucleo (ANA)
e/o antimuscolo liscio (SMA). Il tipo 2 è caratterizzato da
anticorpi microsomiali anti fegato e rene (LKM). Il tipo 3 è
caratterizzato dalla positività per SLA (soluble liver antigen),
ma è una forma estremamente rara. La forma di tipo 1 interessa sia l’età giovanile che l’età geriatrica. Recentemente 127
pazienti del Nord Inghilterra sono stati arruolati in uno studio
retrospettivo e sono stati valutati con lo scoring system internazionale. 130 avevano una diagnosi di epatite autoimmune
definita (score >15), 21 probabile (score tra 10 e 15), mentre
3 (2.5%) avevano uno score <10. Il sintomo di presentazione
più importante era l’ittero in 52 pazienti (50.5%) e astenia +
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RELAZIONI
artralgie in 24 (23.3%). In 27 pazienti (26.2%) era già presente
cirrosi alla presentazione.
CBP: è una malattia colestatica ad eziologia sconosciuta che
tipicamente interessa le donne di media età. È caratterizzata
dalla distruzione dei dotti biliari intra-epatici interlobulari,
da infiammazione granulomatosa portale, e dall’associazione
con condizioni autoimmuni extraepatiche quali: s. di Sjogren,
artrite reumatoide, patologia tiroidea, ecc. È una malattia
eterogenea; accanto alla forma classica caratterizzata dalla
positività degli anticorpi antimitocondrio (AMA) e dalla
sindrome colestatica, si riconosce una variante chiamata in
passato colangite autoimmune, ma meglio inquadrabile come CBP AMA negativa e una forma chiamata sindrome da
overlap tra CBP ed epatite autoimmune. La CBP AMA negativa è caratterizzata da biopsia epatica compatibile con CBP,
negatività per AMA, positività per ANA con ERCP Normale
e buona risposta biochimica al trattamento con prednisolone
ed azatioprina. Numerosi studi hanno dimostrato che la CBP
AMA negativa e la CBP classica sono sovrapponilbili per
presentazionee decorso clinico, quadro istologico, anomala
espressione di autoantigeni mitocondriali sul polo apicale
delle cellule biliari. Si definisce sindrome da overlap CBP/
epatite cronica autoimmune una patologia caratterizzata dalla
coesistenza dei criteri per la diagnosi probabile o definitiva di
epatite autoimmune e dei criteri diagnostici per CBP. Questa
sindrome è un’entità rara in Italia. Nella nostra esperienza è
stata osservata in 2/170 pazienti con CBP. Il gruppo di studio
internazionale per l’epatite autoimmune ha raccomandato di
inquadrare questi pazienti come CBP e non come varianti
dell’epatite autoimmune.
CSP: è una sindrome colestatica ad eziologia ignota, associata nel 50-70% dei casi a malattie infiammatorie croniche
intestinali (nei 2/3 dei casi a colite ulcerosa). È una patologia
cronica, caratterizzata dalla distruzione infiammatoria e distruzione obliterativa dei dotti epatici intra e/o extraepatici,
lentamente progressiva, a decorso oscillante e con esito in
cirrosi biliare. L’età mediana di presentazione è 35 anni. Il 3040% dei pazienti è asintomatico al momento della diagnosi;
l’incidenza di colangiocarcinoma varia dal 6 al 36% dei casi.
In età pediatrica può essere estremamente eterogenea; si riconoscono infatti: una forma neonatale, la forma autoimmune
che è analoga alla forma dell’adulto overlap/epatite autoimmune, la forma associata a malattie infiammatorie intestinali,
la forma associata a istiocitosi X, la forma associata ad immunodeficienze. Nell’adulto accanto alla forma classica esiste la
variante overlap CSP/epatite autoimmune che è presente nel
15-20% dei casi e risponde abbastanza bene alla terapia immunosoppressiva associata all’acido ursodesossicolico.
Anatomia patologica e molecolare
Moderatori: Roberto Fiocca (Genova), Luca Messerini (Firenze)
Lesioni preneoplastiche del colon-retto
M. Risio
Paper not received
Colorectal carcinoma: from the genetic
landscape to the clinical management of the
biospecimen
M. Fassan
ARC-NET Research Centre, University of Verona, Italy
An increasing understanding of both the phenotypic alterations and the molecular mechanisms occurring during colorectal carcinogenesis has characterized the last two decades. In
particular, the discovery of the key mutated/altered genes in
colorectal cancers (CRCs) has proven useful for the design of
a new generation of targeted therapies for clinical intervention.
As a result, multidisciplinary diagnostic approaches integrating clinical evaluation, histology and molecular profiling is
the current practice for CRC patients’ stratification according
to the most appropriate therapeutic regimens. In this setting,
the role of surgical pathologists has become central for clinical decision-making. Routine practice in surgical pathology
laboratories has also changed accordingly. The structuring
of adequate molecular pathology platforms and the upstream
control of the multistep process leading from tumor sampling
to molecular analysis have proved to be crucial. The upcoming step is the standardization of all these procedures and of
pathology reports; this will finally lead to a next-generation
management of CRC patients.
Grading e Staging dei GEP-NET: evidenze e punti
critici
G. Rindi
Istituto di Anatomia Patologica, Università Cattolica del Sacro Cuore
UCSC – Policlinico A. Gemelli - Roma, Italy
I tumori neuroendocrini (NET) del tratto gastroenteropancreatico (GEP) sono un gruppo di lesioni epiteliali neoplastiche
originalmente note come “carcinoidi” e caratterizzate dall’espressione di marcatori neuroendocrini 1. I NET sono descritti
in tutti gli organi del corpo sebbene quelli del tratto GEP ne
rappresentino la frazione a maggiore incidenza nella popolazione 2. In base alle indicazioni fornite dell’organizzazione
mondiale della sanità (WHO 2010) 3 4 i NET sono raccolti
sotto la definizione ombrello di “neoplasia”, comprendente
lesioni a grado istologico basso, intermedio ed alto. Cellule
tumorali con un fenotipo significativamente simile alle loro
controparti normali compongono i carcinoidi/NETs, a rifletterne la controparte normale. La complessità del sistema
neuroendocrino diffuso del tratto GEP con 14 tipi cellulari
differenti 5 è riflessa dai nove tipi di carcinoide/NETs con
diversa composizione cellulare ivi osservati 1. L’attuale
classificazione WHO 2010 introduce i due nuovi strumenti
classificativi del grading (G1-G3) e dello staging TNM
(Tumor, Node, Metastasis) come proposto dalla Società
Europea dei Tumori Neuroendocrini (ENETS) 6 7. Il sistema
di grading definisce tre categorie (G1-G3) in base alla conta
mitotica (G1 <2, G2 2-20 e G3 >20 mitosi per 10 campi ad
alto ingrandimento/2mm2 in aree ad alta densità mitotica) ed
all’indice di proliferazione per Ki67 (G1 ≤2%, G2 3-20% e
G3>20% con anticorpo MIB1 in % di 500-2000 cellule tumo-
106
rali in aree ad elevata densità di marcatura). Si identificano
tre classi WHO, la classe 1 WHO, definita tumore neuroendocrino NET G1; la classe 2 WHO, NET G2 e la classe 3
WHO definita carcinoma neuroendocrino (NEC), per definizione G3. Il grading ha solidamente dimostrato la sua significatività tecnica e, soprattutto, la sua efficacia prognostica in
diverse serie tumorali pubblicate da gruppi indipendenti 8-22.
Resta tuttavia dibattuta l’efficacia del grading per la stratificazione terapeutica di lesioni G3 23-25. Il TNM WHO 2010 è
sostanzialmente sovrapponibile allo schema classificativo per
organo proposto dall’ENETS 6 7. Tuttavia la WHO 2010 ne
limita l’applicazione solo ai NET G1-G2 (“carcinoidi”) e sono presenti differenze significative per il pancreas (lo stesso
schema indicato per l’adenocarcinoma) e per l’appendice (T
esclusivamente dimensionale). Mentre per il pancreas evidenze comparative recenti sostengono una maggior efficacia
del sistema di staging proposto da ENETS 21 resta aperta la
definizione dello staging ottimale per l’appendice. Le linee
guida oncologiche correnti per il trattamento dei pazienti con
neoplasie neuroendocrine si basano largamente sugli schemi
classificativi di grading e staging della WHO 2010 per la
scelta di specifiche opzioni terapeutiche.
Bibliografia
1
Rindi G and Wiedenmann B. Neuroendocrine neoplasm of the gut and
pancreas: new insights. Nat Rev Endocrinol 2012;8:54-64.
2
Yao JC, Hassan M, Phan A, et al. One hundred years after “carcinoid”: epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States. J Clin Oncol 2008,26:306372.
3
Bosman F, Carneiro F. World Health Organization Classification of
Tumours, Pathology and Genetics of Tumours of the Digestive System.
Lyon: IARC Press, 2010.
4
Rindi G, Arnold R, Capella C, et al. 2010 Nomenclature and classification of digestive neuroendocrine tumours. In: World Health
Organization Classification of Tumours, Pathology and Genetics of
Tumours of the Digestive System. Eds F Bosman & F Carneiro. Lyon:
IARC Press, pp 10-12.
5
Rindi G, Leiter AB, Kopin AS, et al. The normal endocrine cell of
the gut: changing concepts and new evidences. Ann N Y Acad Sci
2004;1014:1-12.
6
Rindi G, Kloppel G, Alhman H, et al. TNM staging of foregut (neuro)
endocrine tumors: a consensus proposal including a grading system.
Virchows Arch 2006;449:395-401.
7
Rindi G, Kloppel G, Couvelard A, et al. TNM staging of midgut and
hindgut (neuro) endocrine tumors: a consensus proposal including a
grading system. Virchows Arch 2007;451:757-62.
8
Fischer L, Kleeff J, Esposito I, et al. Clinical outcome and long-term
survival in 118 consecutive patients with neuroendocrine tumours of
the pancreas. Br J Surg 2008;95:627-35.
9
Pape UF, Jann H, Muller-Nordhorn J, et al. Prognostic relevance of
10
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14
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17
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21
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24
25
a novel TNM classification system for upper gastroenteropancreatic
neuroendocrine tumors. Cancer 2008;113:256-65.
La Rosa S, Klersy C, Uccella S, et al. Improved histologic and clinicopathologic criteria for prognostic evaluation of pancreatic endocrine
tumors. Hum Pathol 2009;40:30-40.
Zerbi A, Falconi M, Rindi G, et al. Clinicopathological features of
pancreatic endocrine tumors: a prospective multicenter study in Italy
of 297 sporadic cases. Am J Gastroenterol 2010;105:1421-9.
Couvelard A, Deschamps L, Ravaud P, et al. Heterogeneity of tumor
prognostic markers: a reproducibility study applied to liver metastases
of pancreatic endocrine tumors. Mod Pathol 2009;22:273-81.
Yang Z, Tang LH, Klimstra DS. Effect of tumor heterogeneity on
the assessment of Ki67 labeling index in well-differentiated neuroendocrine tumors metastatic to the liver: implications for prognostic
stratification. Am J Surg Pathol 2011;35:853-60.
Landerholm K, Zar N, Andersson RE, et al. Survival and prognostic
factors in patients with small bowel carcinoid tumour. Br J Surg
2011;98:1617-24.
Jann H, Roll S, Couvelard A, et al. Neuroendocrine tumors of midgut
and hindgut origin: Tumor-node-metastasis classification determines
clinical outcome. Cancer 2011;117: 3332-41.
Strosberg JR, Cheema A, Weber J, et al. Prognostic validity of a novel
American Joint Committee on Cancer Staging Classification for pancreatic neuroendocrine tumors. J Clin Oncol 2011;29:3044-9.
La Rosa S, Inzani F, Vanoli A, et al. Histologic characterization and
improved prognostic evaluation of 209 gastric neuroendocrine neoplasms. Hum Pathol 2011;42:1373-84.
Norlen O, Stalberg P, Oberg K, et al. Long-term results of surgery for
small intestinal neuroendocrine tumors at a tertiary referral center.
World J Surg 2012;36:1419-31.
Dhall D, Mertens R, Bresee C, et al. Ki-67 proliferative index predicts
progression-free survival of patients with well-differentiated ileal neuroendocrine tumors. Hum Pathol 2012;43:489-45.
Goodell PP, Krasinskas AM, Davison JM, et al. Comparison of
methods for proliferative index analysis for grading pancreatic welldifferentiated neuroendocrine tumors. American Journal of Clinical
Pathology 2012;137:576-82.
Rindi G, Falconi M, Klersy C, et al. TNM staging of neoplasms of the
endocrine pancreas: results from a large international cohort study. J
Natl Cancer Inst 2012;104:764-77.
McCall CM, Shi C, Cornish TC, et al. Grading of well-differentiated
pancreatic neuroendocrine tumors is improved by the inclusion of
both Ki67 proliferative index and mitotic rate. Am J Surg Pathol
2013;37:1671-7.
Sorbye H, Welin S, Langer SW, et al. Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3): The NORDIC NEC
study. Ann Oncol 2013;24:152-60.
Scoazec JY, Couvelard A, Monges G, et al. Well-differentiated
grade 3 digestive neuroendocrine tumors: myth or reality? The
PRONET study group. ed/’, eds. 2012. ASCO Annual Meeting.
2012.
Velayoudom-Cephise FL, Duvillard P, Foucan L, et al. Are G3
ENETS neuroendocrine neoplasms heterogeneous? Endocr Relat
Cancer 2013;20:649-57.
Sala Caravaggio – 09.30-11.30
Update in patologia pleuro-polmonare neoplastica
Moderatori: Bruno Murer (Venezia), Oscar Nappi (Napoli)
Problemi diagnostici nei tumori neuroendocrini
del polmone
M. Papotti, M. Volante
Università di Torino e AOU San Luigi, Orbassano, Torino
I tumori neuroendocrini (NET) del polmone appartengono
ad uno spettro di lesioni classificate nell’attuale schema
dell’OMS 1 in base alla combinazione tra aspetti strutturali
(crescita organoide rispetto a diffusa) ed aspetti citologici (dimensione cellulare, atipia, indice mitotico, necrosi cellulare).
L’OMS prevede solo criteri diagnostici su base morfologica.
Il carcinoide tipico (TC) rappresenta meno dell’1% dei tumori
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RELAZIONI
polmonari ed ha una classica crescita organoide (trabecolare,
acinare) di cellule poligonali con minime atipie. La necrosi è
sempre assente e la conta mitotica è inferiore a <2/10 campi
ad alto ingrandimento, high power fields (HPF). Tumori con
caratteristiche simili ma dimensioni inferiori a < 5 mm sono
invece definiti tumorlets. Il carcinoide atipico (AC) è una
neoplasia estremamente rara, correlata al fumo di sigaretta,
in genere di basso grado, ma spesso associata a metastasi linfonodali ed anche a distanza. Il quadro morfologico è sovrapponibile a quello del TC, ma la necrosi può essere presente
(solitamente focale) e/o la conta mitotica è compresa fra 2 e
10/10 HPF.
Il carcinoma neuroendocrino (NE) a grandi cellule, large cell
NE carcinoma (LCNEC) è in parte simile al AC, ma è costituito da grandi cellule con atipie ed ampio nucleolo, in presenza
di estesa necrosi ed un alto indice mitotico (>10/10 HPF).
Nella classificazione OMS del 2004, il LCNEC è inserito nel
gruppo dei carcinomi di grandi cellule, peraltro costituito da
neoplasie non-NE, e va distinto dagli altri sottotipi in base
alla morfologia ed il profilo immunofenotipico (espressione
di cromogranina A e sinaptofisina associata ad assenza di
citocheratine di alto peso molecolare).
Il carcinoma di piccole cellule, small cell lung carcinoma
(SCLC) è la più comune neoplasia polmonare NE ed è classicamente costituito da piccole cellule con scarso o minimo
citoplasma e piccoli nuclei con cromatina finemente dispersa
o talora condensata. Questi aspetti microscopici pongono una
diagnosi differenziale attenta con forme poco differenziate
(e basaloidi) del carcinoma squamoso del polmone. Infine,
“carcinomi NE combinati” vengono raramente osservati nel
polmone, quale risultato di una coesistenza tra componente di
SCLC o LCNEC con campi morfologicamente riconoscibili
di adenocarcinoma o carcinoma squamoso. Invece, la focale
differenziazione NE in adenocarcinomi (o più raramente
carcinomi squamosi) convenzionali non è considerata in
questo contesto, in quanto il significato clinico e l’impatto
prognostico non sono del tutto definiti 2. Il corretto inquadramento classificativo dei NET primitivi del polmone non può
prescindere da un’accurata diagnostica differenziale non
solo con altre forme di carcinomi polmonari, ma anche con
localizzazioni metastatiche al polmone di vari tipi di tumori.
Tra questi sono compresi vari tumori di piccole cellule (da
distinguere dal SCLC), neoplasie indifferenziate pleomorfe
(da distinguere dal LCNEC) ed anche altri tumori a fenotipo
NE con possibili localizzazioni polmonari (NET gastroenteropancreatici, carcinoma midollare della tiroide, paraganglioma,
feocromocitoma, carcinoma di Merkel, etc). Le principali difficoltà diagnostiche derivano dall’analisi di piccole biopsie o
materiali citologici, in cui la quantità di cellule diagnostiche
o la qualità a seguito di artefatti tecnici possono impedire un
corretto giudizio. Mentre non è difficile definire la natura NE,
grazie alla determinazione di marker NE specifici (compresa
la assenza di citocheratine di alto peso molecolare in tutti i
tumori NE di qualunque sede 3), meno attendibile può risultare la definizione dell’istotipo, in particolare la distinzione
su biopsia tra carcinoidi (basso grado) e carcinomi NE (alto
grado). Al riguardo, i marker NE non sono di assoluta utilità
ed il marcatore più utile si è rivelato il Ki67, che ha una distribuzione nucleare e appare positivo, se presente, anche in
aree di artefatti da schiacciamento, così frequenti in biopsie
bronchiali di carcinomi NE 4. Nella diagnostica dei NET polmonari non è ufficialmente ammesso il ricorso all’indice di
proliferazione definito dal Ki67, che invece è uno dei parametri richiesto per il grading dei NET gastroenteropancreatici.
Nella pratica, il Ki67 viene frequentemente valutato ed è stato
anche estesamente studiato in letteratura, con il riscontro di
valori medi di proliferazione nei 4 sottotipi di NET polmonari
in parte sovrapponibili a quelli osservati in altri organi (da
una meta-analisi di circa 1800 casi pubblicati, il TC ha valori
medi di circa 1.5%, l’AC di 7%, LCNEC del 50% e SCLC del
60%) [si veda rassegna di Pelosi 2014]. Un cutoff del 4% è
risultato utile per stratificare pazienti con significative differenze di sopravvivenza libera da malattia e assenza di recidive.
Tale valore è analogo a quello usato nei NET addominali per
la distinzione fra tumori di grado 1 e 2. In un recente studio
5
, è stata dimostrata l’efficacia dell’uso combinato del Ki67
con i due parametri “ufficiali” per la classificazione dei NET
polmonari (conta mitotica e necrosi). Adattando i valori di
cutoff per il Ki67 al 4% e 25% (diversi da quelli in uso nei
NET addominali), si è osservato che era possibile ottenere un
sistema di grading a tre gruppi con prognosi più omogenea e
significative differenze di sopravvivenza, attraverso il riscontro di almeno 2 su 3 dei parametri sopra menzionati (mitosi,
necrosi e ki67). A parte il ruolo prognostico dell’indice Ki67,
altri parametri morfologici e biomolecolari non sono correntemente in uso per la caratterizzazione dei NET polmonari.
L’invasività del tumore e la sua diffusione (ad es metastasi
linfonodali locoregionali) sono incorporate nello stadio di
malattia (che in parte ripercorre quello del TNM per i tumori
broncopolmonari), ma l’interessamento linfonodale non è in
assoluto un fattore sfavorevole, osservandosi anche in casi
di TC, associati ad una lunga sopravvivenza (oltre 30 anni).
Il profilo molecolare dei NET polmonari è stato studiato in
anni recenti, ma non sono emerse applicazioni pratiche per la
pratica quotidiana. L’assetto genetico dei carcinoidi rispetto
ai carcinomi NE di alto grado è stato trovato differente da un
gruppo 6 con conseguente critica alla esistenza di uno continuum di lesioni da AC a LCNEC. Mutazioni di TP53 sono quasi
ristrette ai carcinomi NE di alto grado, così come le mutazioni
del gene MEN1 sono più caratteristiche dei tumori carcinoidi.
Infine, un problema emergente nella diagnostica dei NET, in
analogia con quella di altri tumori, è la necessità di determinare la presenza di marcatori predittivi di risposta a specifiche
terapie mirate. Nei NET polmonari sono stati studiati diversi
bersagli, quali i recettori per la somatostatina, molecole che
regolano l’angiogenesi (recettori per VEGF) e le molecole
regolatorie la trasduzione del segnale intracellulare (ad es via
del mTor) 7. Recentemente, sono stati studiati oncogeni quali
il MET, la cui amplificazione può rappresentare un bersaglio
per inibitori specifici dell’oncogene stesso 8. In questo scenario, ancora parecchia strada resta da compiere, per una migliore
stratificazione dei casi, che pur appartenendo alla stessa categoria diagnostica, possono seguire comportamenti biologici
assai divergenti, soprattutto nel campo del AC. Quest’ultimo,
a fronte di un aspetto istologico blando e di basso grado, può
presentare, anche all’esordio, metastasi loco regionali, ma
anche a distanza, comprese localizzazioni cerebrali o ossee.
Queste richiedono un costante follow up durante terapie mirate, chemioterapiche o biologiche. Per ora, solo l’indice di
Ki67 consente previsioni prognostiche, ma mancano valori di
cutoff validati in questo gruppo di neoplasie.
Bibliografia
1
Travis 2004.
2
Rekhtman 2010.
3
Sturm 2003.
4
Pelosi 2005
5
Rindi 2014.
6
Swarts 2012.
7
Righi 2010.
8
Song 2010.
108
Alterazioni molecolari dei carcinomi a grandi
cellule del polmone
G. Pelosi
Paper not received
Lung Adenocarcinoma Heterogeneity
R. Franco, F. Zito Marino
SC Anatomia Patologica e Citopatologia Istituto dei tumori “Fondazione G. Pascale”
Lung Adenocarcinoma (ADC) is the most frequently diagnosed histological type of primary lung cancer accounting for
almost half of all lung cancers. ADC is a very heterogeneous
tumor not only for the clinical and radiologic presentation
but also for histological and molecular features. Histologic
intratumoral heterogeneity in ADCs is considered as both a
frequent minor heterogeneity due to the occurrence of several
growth patterns, such as lepidic, acinar, papillary, micropapillary and solid patterns in mixed adenocarcinomas (mADCs)
and an unusual major heterogeneity as in the adenosquamous
lung carcinoma (AdSqLCs).
Recently, major revisions have been made to the classification
and grading of ADCs, leading to an improvement of tumors
diagnosis with a subsequent increase in patient survival. Thus,
the 2011 IASLC/ATS/ERS classification of lung adenocacinomas has introduced some significant modifications to the
2004 World Health Organization (WHO) ADCs classification. In particular, the comprehensive histologic subtyping of
the different patterns of mADCs by a percentage of 5% increments is recommended, in order to determine the predominant pattern. In addition Sica et al. have proposed a grading
system based upon the comprehensive histologic subtyping
of mADCs, providing a grading score based on the main two
growth patterns. This pattern-based scoring system has proved
to improve both mADCs subclassification and better prognostic stratification.
Although molecular characterization through the definition
of genetic mutations, such as EGFR and ALK mutations, is
crucial for lung cancer patients treatment, however few data
are reported in the literature about molecular features of the
mADCs/AdSqLCs. In the literature, the main histologic subtypes ascribed to EGFR mutations are lepidic or mixed subtype with lepidic component; papillary and micropapillary; on
the other hand ALK-R are described in many ADC subtypes,
including lepidic, papillary, micropapillary and acinar. The
high rate of morphological heterogeneity in ADCs could reflect a heterogeneous molecular profiling.
References
Cadioli A, Rossi G, Costantini M, et al. Lung cancer histologic and immunohistochemical heterogeneity in the era of molecular therapies:
analysis of 172 consecutive surgically resected, entirely sampled pulmonary carcinomas. Am J Surg Pathol 2014;38:502-9.
Travis WD, Brambilla E, Noguchi M, et al. International association for
the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 2011;6:244-85.
Yoshizawa A, Motoi N, Riely GJ, et al. Impact of proposed IASLC/ATS/
ERS classification of lung adenocarcinoma: prognostic subgroups
andimplications for further revision of staging based on analysis of
514 stage I cases. Mod Pathol 2011;24:653-64.
Sica G, Yoshizawa A, Sima CS, et al. A grading system of lung adenocarcinomas based on histologic pattern is predictive of disease recurrence in stage I tumors. Am J Surg Pathol 2010;34:1155-62.
Motoi N, Szoke J, Riely GJ, et al. Lung adenocarcinoma: modification of
the 2004 WHO mixed subtype to include the major histologic subtype
suggests correlations between papillary and micropapillary adenocarcinoma subtypes, EGFR mutations and gene expression analysis. Am
J Surg Pathol 2008;32:810-27.
Il Mesotelioma ignorante
G. Rossi, M.C. Mengoli, A. Cavazza*
Anatomia Patologica, Azienda Policlinico di Modena; * Unità Operativa di Anatomia Patologica, Azienda Arcispedale S. Maria Nuova /
IRCCS, Reggio Emilia
La diagnosi di mesotelioma pleurico (considerando una serie
consecutiva personale di 100 casi) non presenta generalmente
grandi difficoltà per il patologo quando si presenta nella sua
forma classica, ovverosia con un franco ispessimento pleurico
diffuso associato a dolore toracico, versamento consensuale
recidivante e nella variante istologica epitelioide. Fortunatamente, questo accade nell’80-90% circa di tutti i mesoteliomi
pleurici.
In un 10% circa dei casi, tuttavia, il mesotelioma non sembra
seguire le regole scritte nei testi di oncologia o di anatomia
patologica e può manifestarsi con una sintomatologia fuorviante, come localizzazione metastatica in sedi particolarmente inusuali, piuttosto che con morfologia non convenzionale o
con espressione anomala dei marcatori immunoistochimici.
In tutti questi casi, la diagnosi può rivelarsi particolarmente
difficoltosa. La conoscenza di un’esposizione lavorativa o
non-lavorativa alle fibre di asbesto è certamente importante
nel porre il sospetto di mesotelioma, così come il dato epidemiologico che prevede un incremento dell’incidenza di questa
neoplasia in Europa nei prossimi 2-3 decenni.
È probabile quindi che un patologo si confronti con un mesotelioma in situazioni decisamente non convenzionali, quando
meno se lo aspetta, ma deve sempre tenere in considerazione
la remota possibilità che, ad esempio, una blanda proliferazione a pattern “lepidico” nel polmone di un paziente anziano
che si presenta con pneumotorace o interstiziopatia polmonare
possa in realtà essere un mesotelioma. Un nodulo cutaneo, una
massa dei tessuti molli, una metastasi linfonodale, un addome
acuto sono tutte possibili presentazioni di mesotelioma.
Una proliferazione neoplastica “ad anello con castone”, a
cellule chiare, a piccole cellule, con aree mixoidi, con stroma
linfo-istiocitoide rappresentano tutte possibili varianti morfologiche inusuali di un mesotelioma.
Una valutazione errata dell’espressione di alcuni marcatori
mesoteliali (calretinina fortemente espressa ma solamente a
livello citoplasmatica, WT-1 citoplasmatico) o l’aberrante
reattività di un mesotelioma per marcatori considerati nonmesoteliali (es. TTF-1 o CDX2) possono condurre ad una
diagnosi sbagliata, soprattutto se il patologo non è esperto di
patologia toracica o quando non si tengano in considerazione
i dati clinico-anamnestici e radiologici.
In conclusione, la diagnosi di mesotelioma può essere estremamente semplice nei casi classici (“carcinomas looks like
carcinoma and mesotheliomas looks like mesotheliomas,”
– Thomas Colby, 117th Semi-Annual Seminar “Pulmonary
pathology”, 5 Dicembre 2004, Hyatt Regency, San Francisco,
CA USA), mentre richiede sempre un’attenta valutazione
globale dei dati clinico-anamnestici e radiologici insieme ai
reperti immuno-morfologici tutte le volte che la neoplasia
non rispetta le regole convenzionali. L’immunoistochimica
ha generalmente un ruolo di conferma. Infine, da un punto di
vista medico-legale e per tutelare i diritti dei pazienti esposti
ad asbesto e delle loro famiglie, una diagnosi descrittiva di
“non esclusione” di mesotelioma appare del tutto ragionevole
109
RELAZIONI
di fronte a neoplasie maligne indifferenziate che rimangano
non meglio classificabili anche dopo uno scrupoloso esame
morfologico ed immunofenotipico.
Bibliografia
Pisani RJ, Colby TV, Williams DE. Malignant mesothelioma of the
pleura. Mayo Clin Proc 1988;63:1234-44.
Attanoos RL, Dojcinov SD, Webb R, et al. Anti-mesothelial markers in
sarcomatoid mesothelioma and other spindle cell neoplasms. Histopathology 2000;37:224-31.
van Zandwijk N, Clarke C, Henderson D, et al. Guidelines for the diagnosis and treatment of malignant pleural mesothelioma. J Thorac Dis
2013;5:E254-E307.
Larsen BT, Klein JR, Hornychová H, et al. Diffuse intrapulmonary malignant mesothelioma masquerading as interstitial lung disease: a distinctive variant of mesothelioma. Am J Surg Pathol 2013;37:1555-64.
Husain AN, Colby T, Ordonez N, et al. Guidelines for pathologic diagnosis of malignant mesothelioma: 2012 update of the consensus
statement from the International Mesothelioma Interest Group. Arch
Pathol Lab Med 2013;137:647-67.
Cavazza A, Pasquinelli G, Agostini L, et al. Foamy cell mesothelioma.
Histopathology 2002;41:369-71.
Ordóñez NG. Mesothelioma with clear cell features: an ultrastructural and immunohistochemical study of 20 cases. Hum Pathol
2005;36:465-73.
Ordóñez NG. Mesothelioma with signet-ring cell features: report of 23
cases. Mod Pathol 2013;26:370-84
Khalidi HS, Medeiros LJ, Battifora H. Lymphohistiocytoid mesothelioma.
An often misdiagnosed variant of sarcomatoid malignant mesothelioma. Am J Clin Pathol 2000;113:649-54.
Rossi G, Cavazza A, Turrini E, et al. Exclusive intrapulmonary lepidic
growth of a malignant pleural mesothelioma presenting with pneumothorax and involving the peritoneum. Int J Surg Pathol 2006;14:234-7.
Maiorana A, Giusti F, Cesinaro AM, et al. Cutaneous metastases as the
first manifestation of pleural malignant mesothelioma. J Am Acad
Dermatol 2006;54:363-5.
Cavazza A, Rossi G, Agostini L, et al. Small-cell mesothelioma of the
pleura: description of a case. Pathologica 2002;94:247-52.
Ordóñez NG. Mesotheliomas with small cell features: report of eight
cases. Mod Pathol 2012;25:689-98.
Update in patologia polmonare non-neoplastica
Moderatori: Mattia Barbareschi (Trento), Marco Chilosi (Verona)
I granulomi nel polmone
Eziologie proteiformi dietro la medesima
maschera: il danno polmonare fibrosante
G. Rossi
Paper not received
Ruolo della biopsia trans bronchiale nella
diagnosi di interstiziopatie polmonari
P. Graziano
Paper not received
A. Cavazza
Paper not received
Sala Brunelleschi – 09.30-12.30
Quanto e come sono preparati i patologi in Citologia?
Moderatori: Pietro Gallo (Roma), Domenico Ientile (Palermo)
Esigenze dell’utenza
C. Gentili
Paper not received
La preparazione dei citotecnici e la
preparazione post-universitaria
M.R. Giovagnoli
Dipartimento di Medicina Molecolare, Università “La Sapienza”, Roma
La formazione dei tecnici in ambito citopatologico ha visto
vari fasi legate ai differenti quadri normativi che si sono susseguiti nel tempo. Dai Corsi biennali per tecnici di colpo citologia, ai diplomi triennali per tecnici di laboratorio, nei quali
al terzo anno era previsto un indirizzo di citologia e citogenetica, fino alla unificazione dell’ insegnamento universitario a
livello europeo che introducendo la laurea triennale ha di fatto
chiuso gli spazi, seppur limitati, che erano stati fino ad allora
utilizzati per la formazione di base in citopatologia.
A questo l’accademia ha risposto proponendo dei master dedicati, sicuramente validi, ma con il limite dovuto al fatto che
a tali modelli formativi non poteva essere riconosciuta una
funzione professionalizzante.
D’altra parte le Regioni, perlomeno alcune, hanno proposto
dei corsi di durata diversa che sono rimasti però episodici e
non hanno permesso una formazione omogenea di personale
dedicato alla citologia.
Le Società Scientifiche, dal canto loro, non sono riuscite a
promuovere una strategia formativa a lungo raggio, limitandosi a corsi di aggiornamento estremamente validi per chi già
lavorava nel campo ma non forieri di nuove forze e per lo più
attivati dalla buona volontà di singoli patologi particolarmente
sensibili al problema. Né, dette società, si sono poste il proble-
110
ma o hanno avuto la forza di indicare e proporre al legislatore
degli standard qualitativi minimali per lo svolgimento di tale
delicata attività professionale.
Attualmente con il differente quadro lavorativo che si è venuto a creare con il passaggio allo screening basato sull’ HPV
quale test primario, c’è stata una “riconversione” di alcuni
citopatologi alle attività di laboratorio biomolecolare, mentre
una richiesta di nuovi professionisti si è sicuramente ridotta,
almeno a livello pubblico.
C’è però da considerare che l’attività di diagnostica citopatologica continua ad essere rilevante nel settore extraginecologico, dove anzi l’affinarsi delle tecniche endoscopiche e la
necessità di avere materiale di qualità per le analisi molecolari
indirizzate alla cura personalizzata del paziente aprono nuovi
orizzonti, e, d’altra parte, l’accrescersi delle richieste diagnostiche sempre di più pesa su di un personale di Anatomia
Patologica ogni giorno più scarso.
Si aprono quindi nuove necessità formative che potrebbero essere
indirizzate ad un personale tecnico di elevato livello quale quello
che si è venuto formando negli ultimi decenni, ma che ancora una
volta non trovano una risposta istituzionale o almeno concordata
da parte di tutti i possibili attori del sistema: Università , Regioni,
Società Scientifiche, rappresentanti di categoria.
Il futuro della citologia in Italia ed in Europa
A. Fassina
Paper not received
Cervical cancer and screening in Nigeria
R.O. Egejuru
Paper not received
Nuove problematiche in citologia tiroidea
Moderatori: Luca Molinaro (Torino), Vania Vezzosi (Firenze)
New issues in thyroid cytology: Italian
classification and reporting system for thyroid
cytology 2014
F. Nardi
Università La Sapienza, Rome
The 2014 italian consensus for the classification and reporting
of thyroid cytology has been published on line on may 2014
on the Journal of Endocrinological Investigation 1. This document is an update of the former 2007 consensus statement 2
and is accomplished with a review of the literature from 2007
until 2013. Its main purpose is to provide the Italian cytopathologists and clinicians with a reliable tool for their clinical
practice. The present classification is comparable with the
major international reporting systems 3-5, following the recommendations of the 2009 European Federation of Cytology
Societies 6. The 2014 Italian Consensus confirms the previous
5-tiered reporting scheme with increasing expected risk of
malignancy and suggested clinical actions for each category,
but includes a sub-classification of the “non diagnostic” category for cystic lesions and the separation of the “indeterminate” category into two subclasses with an expected different
risk of malignancy.
The main differences in respect to the former Italian classification concern the TIR 1 and TIR 3 categories.
In the TIR 1 category are introduced 3 changes:
A sample with significant cytologic atypia that raises the suspicion of malignancy does not require a minimum number of
follicular cells to be included into a suspicious category
Samples obtained from a cystic lesion that are characterized
by erythrocytes, cellular debris and hemosiderin-laden macrophages without abundant colloid with a cellularity that does
not meet the requirements for adequacy should be subclassified as TIR 1C (Cystic)
US-guided core needle biopsy may be considered in case of
repeatedly non diagnostic FNA samples.
The TIR 3 category has been subdivided in two subcategories
at different expected risk of malignancy on the basis of ar-
chitectural and cytological alterations and of the background
component:
TIR 3A, low-risk indeterminate lesion (LRIL), the suggested
clinical action is a follow-up. During the follow-up repeat
FNA is recommended.
TIR 3B, high-risk indeterminate lesion (HRIL), whose patients should be preferentially addressed to surgery.
The subdivision of the TIR 3 category in two subcategories
TIR 3A and TIR 3B with different risk of malignancy and
different clinical actions is similar, but not identical and
with different terminology, to the American 3 4 and English
5
classifications. The subdivision has the purpose to reduce
the number of patients to address to surgery. A prospective,
multicenter trial will be planned to validate the clinical effectiveness of this classification.
Table I summarize the new italian classification with diagnostic categories, expected risk of malignancy and suggested
clinical actions.
References
1
Nardi F, Basolo F, Crescenzi A, et al. Italian Consensus for the classification and reporting of thyroid cytology. J Endocrinol Invest 2014
May 1 (ahead of publication)
2
Fadda G, Basolo F, Bondi A, et al. Cytological classification of thyroid nodules. Proposal of the SIAPEC-IAP Italian consensus Working
Group. Pathologica 2010;102:405-408
3
Baloch ZW, Li Volsi VA, Asa SL, et al. Diagnostic terminology and
morphologic criteria for cytologic diagnosis of thyroid lesions: a synopsis of the National Cancer Institute Thyroid Fine-needle Aspiration
State of the Science Conference. Diagn Cytopahol 2008;36:425-37.
4
Ali SZ, Cibas ES. The Bethesda system for reporting thyroid cytopathology: definitions, criteria and explanatory notes. Springer New
York 2010.
5
Cross PA, Chandra A, Giles T, et al. Guidance on the reporting of
thyroid cytology specimens. Htpp://www.rcpath.org/resources/pdf/
g089guidance on the reporting of thyroid cytology final.pdf, 2009.
6
Kocjan G, Cochand-Priollet B, de Agustin PP, et al. Diagnostic terminology for reperting thyroid fine needle aspiration cytology: European
Federation of Cytology Societies thyroid working party symposium.
Lisbon 2009. Cytopathology 201021.86-92
111
RELAZIONI
Tab. I. Italian Thyroid Cytology Classification System 2014.
Code
TIR1
TIR1C
Diagnostic category
Non diagnostic
Non diagnostic - Cystic
Expected Risk of malignancy (%)
Not defined
Low (variable on the basis of clinical
findings)
<3
Suggested Actions
Repeat US-guided FNA after at least one month
Evaluate the clinical setting and/or repeat FNA
TIR2
Non malignant
TIR3A
Low-risk indeterminate
lesion (LRIL)
< 10 °
Repeat FNA/clinical follow-up
TIR3B
High-risk indeterminate
lesion (HRIL)
15-30°
Surgery
TIR4
Suspicious of malignancy 60-80
Surgery (consider frozen section)
TIR5
Malignant
Surgery. In selected cases additional diagnostic
procedures
> 95
Follow-up
° Expected rate of malignancy for the TIR3 subcategories are mainly founded on clinical experience and are only partially based on the evidence of
published data.
New issues in thyroid cytology:
the controversial category of indeterminate
for malignancy (TIR 3)
G. Fadda
MD, MIAC – Head, Cytopathology Section – Division of Anatomic
Pathology and Histology – Università Cattolica, “Agostino Gemelli”
School of Medicine and Hospital
The “Indeterminate for Malignancy” cytological group of
thyroid lesions still remains a controversial subject in the classification of fine needle aspiration (FNA) specimens. If the
surgical removal of a nodule diagnosed as indeterminate has
been suggested, as many as 70% of patients would undergo
an unnecessary operation. Based on the Bethesda System for
Reporting Thyroid Cytopathology (BSRTC), the Atypia of
undetermined significance/Follicular lesion of undetermined
significance (AUS/FLUS) category has a 5-15% risk of malignancy which increases in the groups of Follicular Neoplasm/
Suspicious for Follicular neoplasm (FN/SFN) and Hurthle cell
neoplasm (HCN) to 15-30%. The recent update of the Italian
reporting system for thyroid classification SIAPEC-AIT 2014
has introduced, on the basis of architectural and cytological
atypia, two new subclasses among the TIR3 category: TIR3A
and TIR3B, with the purpose of decreasing the amount of unnecessary surgeries.
The TIR3A subgroup, low-risk indeterminate lesion (LRIL),
is comparable to the AUS/FLUS category of BSRTC and the
Thy3a class of the British Thyroid Association (BTA) and
bears an estimated maximum risk of malignancy of 10%. The
latter subgroup is characterized by increased cellularity with
scattered microfollicular structures (<60% of the whole cellularity) and exclusively mild cytologic atypia.
The TIR3B subgroup is similar to the FN/SFN category of
BSRTC and the Thy3f category of BTA identifies high-risk
indeterminate lesions (HRIL) with an expected risk of malignancy of 15-30% higher than the TIR3A group. It is characterized by high cellularity in a predominant microfollicular/
trabecular arrangement (>60% of the cell component), with
focal cytologic atypia (mostly moderate, rarely severe) suggestive for a “follicular neoplasm” (FN). Samples composed
almost exclusively of Hurthle cells (“Hurthle cell neoplasm”)
are included in the TIR 3B subcategory.
For all these reasons is recommended a clinical and sonographic follow-up for TIR3A lesions and the repeat of the
FNA within six months, while surgery is the most appropriate
indication for TIR3B nodules. However, the surgical or conservative management of these novel subcategories depends
upon both the cytological and clinical data and requires the
consultation between the cytopathologist and the clinician.
References
Nardi F, Basolo F, Crescenzi A, et al. Italian Consensus for the classification and reporting of thyroid cytology. J Endocrinol Invest, 2014 May
1 (ahead of publication)
Baloch ZW, Li Volsi VA, Asa SL, et al. Diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions: a synopsis
of the National Cancer Institute Thyroid Fine-needle Aspiration State
of the Science Conference. Diagn Cytopahol 2008;36:425-37.
Ali SZ, Cibas ES. The Bethesda system for reporting thyroid cytopathology: definitions, criteria and explanatory notes. Springer New York
2010.
Cross PA, Chandra A, Giles T et al. Guidance on the reporting of thyroid
cytology specimens. Htpp://www.rcpath.org/resources/pdf/g089guidance on the reporting of thyroid cytology final.pdf, 2009.
Bongiovanni M, Spitale A, Faquin WC, et al. The Bethesda System
for Reporting Thyroid Cytopathology: A Meta-Analysis. Acta Cytol
2012;56:333-9.
Vanderlaan PA, Marqusee E, Krane JF. Usefulness of Diagnostic Qualifiers for Thyroid. Fine-Needle Aspirations With Atypia of Undetermined
Significance. Am J Clin Pathol 2011;136:572-577
New issues in thyroid cytology: Lo spazio delle
nuove tecnologie
E.D. Rossi
MD, PhD MIAC1 Division of Anatomic Pathology and Histology Università Cattolica del Sacro Cuore, “Agostino Gemelli” School of
Medicine, Rome Italy
Thyroid nodules are a worldwide finding in the general population, taking into account both non-neoplastic and neoplastic
entities, so the distinction between benign and malignant
neoplasms is controversial. Fine-needle aspiration cytology
(FNAC) represents the most important and, generally, primary
diagnostic tool for evaluating thyroid lesions. Although its
high diagnostic accuracy and sensitivity, the morphological
evaluation is not always sufficient. In fact 10-30% of FNAC
samples were given an “indeterminate” diagnosis due to a
112
cellular follicular proliferation, a “grey zone category” that
causes a high number of unnecessary thyroidectomies with
additional morbidity and higher healthcare costs. Its optimization represents a challenging request with the goal of
ruling out a malignant outcome that would require surgical
treatment. The major issue is represented by the difficulties
in identifying the follicular variant of papillary thyroid carcinoma (FVPC) due to the vague nuclear features in cytological
samples that often fall short of a malignant diagnosis. Hence,
several recent studies have encouraged the application of
ancillary techniques (including immunocytochemistry-ICC
and molecular analyses). Rarely does the detection of positive
ICC result in more than a suggestion of malignancy, while the
high specificity of molecular detections is a strong indicator
of cancer
Immunohistochemistry (IIC) has been introduced in the
routine pathology practice since the beginning of the 1970s.
Firstly it has been traditionally used on thyroid pathology for
the identification of cell origin (such as thyroglobulin, calcitonin or parathyroid hormone) in tumors arising either in the
gland or outside it. Secondly the introduction of the markers
of malignancy, which may distinguish malignant from benign
lesions irrespective the histological features of carcinomatous
evolution (especially capsular or vascular invasion), has represented a pillar of the morphologic diagnostics of thyroid
cancer. In this latter perspective, numerous immunocytochemical (ICC) stains have been examined as potential markers of malignancy in thyroid FNA. A significant challenge to
this modality is the overlap in ICC marker expression between
cases of indeterminate nodules and well-differentiated malignancies.
HBME-1(Hector Battifora mesothelial antigen) and Galectin-3 have reached the highest specificity and sensitivity in
discriminating between benign and malignant lesions mainly
when used in panels. There has been an association between
galectin-3 and thyroid carcinoma, with a positive predictive value ranging from 78%-100%. A retrospective study
estimated a 71% decrease in unnecessary surgeries; however
6% of cancers would have been missed. An important point
is the threshold used for making a positive interpretation has
markedly varied from >10% of cells staining to >50%. Also of
note, there is no accepted standard methodology with regard
to testing. Reports vary from use of formalin-fixed paraffin
embedded material, conventional smears, or liquid-based
preparations. Neverthless it is essential to underline that none
of the immunomarkers studied have shown a diagnostic accuracy sufficient for using them as single antibody characteristic
of malignancy so that the use of an immunopanel made up of
two o more immunomarkers is strongly encouraged by several
authors.
Among the outstanding variety of markers studied and used to
identify malignant lesions, a special note should be addressed
in including HBME-1, CK19, and to a lesser extent CD44.
In two different experiences Rossi et al and Fadda et al
highlighted the application of an immunocytochemical panel
consisting of HBME-1 and Galectin-3 as the best choice to
discriminate between low and high risk of malignancy in
follicular proliferations, showing a 81% overall diagnostic
accuracy, which increased to 92% when a concordant positive
panel was applied.
CD44 has shown great sensitivity for papillary carcinoma
(97%), although relative poor specificity with atypical / benign cases showing greater than 20% positive expression.
On the opposite hand, the role of CD56 as a marker for
identifying follicular epithelial cells of the normal thyroid is
emerging mainly on histological samples even though it may
be also carried out on cytological specimens. Recent papers
show 98.6% sensitivity and 95.8% specificity in distinguishing PTC from other follicular benign thyroid lesions
A recent meta-analysis of 95 studies of ICC markers in
thyroid FNA identified CK19, HMBE-1, and galectin-3 as
showing sufficient sensitivity and specificity to serve as a cost
effective means to improve diagnostic accuracy. The National
Cancer Institute (NCI), however, did not include ICC testing
among its recommendations for ancillary testing of indeterminate thyroid FNA.
Eventually, utilization of a panel of antibodies may be the
best method of choice for ICC markers. Similar to molecular
panels with numerous markers, this improves the relative sensitivity and specificity.
Recent scientific data have shown that molecular alterations
of specific pathways play a pivotal role in some types of thyroid cancer and, importantly, arise early in the tumorigenic
process, justifying the use of these as markers of malignancy.
In particular, papillary thyroid carcinoma-PTC, the most
common thyroid malignancy, may carry BRAF, RET/PTC or
N-RAS mutations. Taken together these evidences, this growing literature regarding ancillary testing is being reflected in
the revised management guidelines for patients with thyroid
nodules and differentiated thyroid cancer, recently published
by the American Thyroid Association. They suggest utilization of a molecular panel (including BRAF, RAS, RET/PTC
and PAX8-PPAR_g) for patients with indeterminate FNA
cytology as a possible aid in guiding their management. Recent experiences published by Rossi et al revealed that in the
group of suspicious for malignancy (SM), the detection of a
BRAF mutation has a 100% histological correlation with a
diagnosis of thyroid carcinoma (p=0.0353) and also significantly matched with three aggressive parameters chosen such
as lymph-node metastases (p<0.0001), extra capsular invasion (p=0.03) and multi-focality (p=0.0003), so that a more
aggressive surgical treatment is appropriate for these cases.
Furthermore, the BRAF V600E mutation has been highly associated with PTC and more often with the aggressive variants
(e.g. tall cell variant and columnar variant) with much lower
rate of BRAF mutations in FVPCs.
Although BRAF mutation has been typically performed using
DNA-based techniques, the recent introduction of monoclonal
V600E antibody (clone VE1) is likely to be an alternative
strategy to detect this mutation in thyroid lesions on cytology.
Two different papers from Zimmerman et al and Rossi et al
showed good sensitivity and specificity ranging from 82% to
86% on cytological samples.
Although its limitation probably due to the limited series published, VE1 antibody represents a feasible first-line approach
for evaluating BRAF mutation and might be a valid tool in
selecting cases for molecular analysis. Rossi et al highlighted
a statistical significance between molecular and VE1 positivity (p<0.0001) in PTC. Nevertheless, in their experience
BRAF mutation was more accurate in identifying VE1 negative cases.
Eventually, the use of molecular markers, either alone or as a
panel, will continue to expand and influence management of
thyroid nodules. While no single test may be able to provide
adequate positive and negative predictive value, an expanded
panel of existing tests, or the addition of novel markers, may
make this an attainable goal in the future.
ICC stains are used with a broad range of testing methodologies, specimen preparations, and diagnostic thresholds. While
there is utility for a select panel of markers, this appears more
113
RELAZIONI
applicable to individual institutions as a “home-brew” form of
testing. Variability of specimen preparation and the subjectivity/thresholds of interpretation make standardized widespread
use of ICC markers less likely.
References
Rossi ED et al. Cancer Cytopathol 2005.
Cochand Priollet B et Thyroid 2011; 21: 1067-73.
Fadda G et al Eur J Endocrin 2011; 165: 447-453.
Rossi ED et al Eur J Endocrin 2013; 168: 853-9.
Nikiforova MN et al. Thyroid 2009; 19:1351-61.
Rossi ED et al. Cancer Cytopathol 2014 Mar 17doi.
Clinical actions
F. Orlandi, I. Messuti, F.Arecco
Department of Oncology, University of Turin
The new Italian consensus for the classification and reporting
of thyroid cytology has been recently published on the Journal
of Endocrinological Investigation, aiming to update the previous consensus according to the indications of the European
Federation of Cytology Societies and, above all, to reduce the
rate of non diagnostic and indeterminate reports. The most
significant novelties concern TIR1 and TIR3 categories.
TIR1 category includes non diagnostic samples (inadequate
and/or non-representative). FNA repetition is recommended,
at least 1 month later. Core needle biopsy has to be considered
in case of repeatedly non diagnostic samples. In this category
THYc has been added. It includes cystic lesions with an esti-
mate high probability of benignity. Cytological report has to
be integrated with clinical and US examinations in order to
exclude the cystic form of DTC.
TIR2 category includes non malignant/benign lesions. FNA
repetition is recommended in case of nodule growth or structural changes, and in patients who are undergoing ablative
treatments.
The TIR3 category (indeterminate) has been divided in TIR3a
and TIR3b. TIR3a includes lesions with low probability of
malignancy (<10%), suggesting a conservative approach and
a periodic follow up. During the follow-up FNA repetition
is recommended. TIR3b includes lesions with higher risk of
malignancy (15-30%), recommending surgery.
In TIR 4 (suspicious of malignancy) and TIR5 (malignant)
categories surgery is strongly recommended. The extent of
surgery should be evaluated on the basis of the cytology report
and of the clinical and US setting.
This classification is aimed to give cytopathologists and clinicians a reliable and useful tool in the clinical practice, correlating the morphological features with an increasing risk of
malignancy in order to define an appropriate clinical management. A prospective and controlled study is needed to validate
the clinical effectiveness of this classification.
Diagnosi molecolare del nodulo tiroideo
citologicamente indeterminato: oltre BRAF
F. Basolo
Paper not received
Sala Michelangelo – 09.30-11.30
Lesioni melanocitarie della cute
Moderatori: Claudio Clemente (Milano), Gaetano De Rosa (Napoli)
Microscopia confocale nelle neoplasie
pigmentate
G. Pellacani
Clinica Dermatologica, Università di Modena e Reggio Emilia
La Microscopia Laser Confocale a riflettenza rappresenta una
innovativa metodica di indagine non invasiva della cute a risoluzione quasi-istologica, fornendo immagini di sezioni orizzontali dell’epidermide e del derma superficiale con una risoluzione laterale di circa 1 μm. La sua principale applicazione
è la dermato-oncologia, in quanto ha permesso di migliorare
l’accuratezza diagnostica, di definire i margini chirurgici e
valutare l’efficacia di trattamenti topici conservativi di tumori
cutanei. Risulta utile nella diagnosi differenziale di lesioni
melanocitarie ed epiteliali benigne e maligne. In particolare
nella diagnosi differenziale di lesioni melanocitarie sono stati
descritti aspetti cito-architetturali specifici di melanoma che
consentono di porre una diagnosi più accurata rispetto alla
sola dermoscopia, quali la presenza di cellule pagetoide e di
cellule atipiche alla giunzione, il disordine citoarchitetturale
alla giunzione e la presenza di nidi disomogenei. D’altra parte
la presenza di papille di forma regolare e circondate da anelli
di cellule riflettenti e l’assenza di cellule atipiche consente di
porre con elevata sicurezza diagnosi di lesione melanocitaria
benigna. Rispetto alle metodiche attualmente in uso quale la
dermoscopia, la microscopia confocale permette una maggiore specificità diagnostica, offrendo la possibilità di evitare
escissioni non necessarie di lesioni benigne, con il mantenimento della stessa sensibilità offerta dalla dermoscopia.
Pertanto, la microscopia confocale si dimostra un’utile ausilio
diagnostico nell’oncologia dermatologica.
Melanocitosi dermiche
C. Clemente
Servizio di Anatomia Patologica e Citopatologia Casa di Cura San Pio X,
Milano, Servizio di Anatomia Patologica e Citodiagnostica, I.R.C.C.S. Policlinico San Donato, Gruppo San Donato, San Donato Milanese, Milano
Il nevo blu e le sue varianti (cellulato, atipico e combinato) insieme al nevo blu desmoplastico, al nevo penetrante profondo,
alla macchia Mongolica, al nevo di Ota e di Ito, costituiscono
un gruppo di lesioni definito da Elder 1 come “melanocitosi
cutanee” dermiche. Queste lesioni sono caratterizzate da una
proliferazione di melanociti dendritici, solitamente pigmentati, o epitelioidi situati nel derma papillare e nel derma reticolare e da una marcata e diffusa positività immunoistochimica
con l’anticorpo anti-HMB45.
114
Il nevo blu è un tumore melanocitico benigno in cui la profondità del pigmento melanico, presente nelle cellule neviche nel
derma profondo, è responsabile del caratteristico colore bluastro della lesione da cui prende il nome. Il nevo blu interessa
prevalentemente i giovani, con uguale frequenza in entrambi i
sessi. Le sedi più frequenti sono il dorso delle mani e dei piedi
e i glutei. Le lesioni possono anche essere localizzate al tronco
e al cuoio capelluto. Sono segnalati casi in sede extracutanea:
cavità orale 2, utero 3 4, vagina 5, funicolo spermatico 6, prostata
7 8
, un caso è stato descritto in un teratoma maturo ovarico
cistico 9 e nell’albero bronchiale 10. Clinicamente, i nevi blu
presentano un colore che varia dal blu, al blu-grigio o al blu
dorato. Il nevo blu è caratterizzato da tre aspetti morfologici
principali: 1) proliferazione di melanociti dendritici nel derma
reticolare, 2) presenza di melanofagi, talora numerosi e 3) reazione stromale fibrogenetica. I melanociti dendritici mostrano
forma fusiforme, allungata tipica, con estensioni citoplasmatiche sottili e prominenti, granuli intracitoplasmatici di pigmento melanico che a volte oscurano il nucleo. Il pigmento è
di colore marrone scuro e finemente granulare che differenzia
la cellula del nevo blu da un macrofago in cui il pigmento è
denso, grossolano e spesso si sovrappone e oscura il nucleo.
Nel nevo blu, il nucleo è ovale con una sottile membrana nucleare e il nucleolo spesso poco appariscente. Solo raramente
è presente pleomorfismo. La modalità di crescita è a cellule
singole o in piccoli fasci, disposti tra le fibre di collagene del
derma con aspetto a volte serpiginoso senza frammentazione
delle fibre collagene. La reazione stromale è spesso responsabile dell’apparente scarsa cellularità della lesione, che ad ingrandimento panoramico, può simulare un istiocitoma fibroso
benigno dermico. Spesso si può osservare una tendenza delle
cellule neviche a disporsi intorno agli annessi cutanei o lungo
i vasi o i nervi. La fibrogenesi mostra vari gradi, da lieve ad
intensa, fino alla formazione di fasci densi che danno l’aspetto
desmoplastico della lesione o a volte simulare aspetti neuroidi. Quando la sclerosi è prevalente e la neoplasia assume un
aspetto a lente biconvessa, con margini ben definiti rispetto al
derma, spesso la neoplasia viene identificata con il termine di
nevo desmoplastico. Effettivamente la differenza e una netta
separazione tra nevo desmoplastico e nevo blu con marcata fibrogenesi forse non esiste. Nel nevo blu, frammisti ai melanociti spesso sono presenti melanofagi con citoplasma pieno di
grossolani granuli di pigmento melanico. Le mitosi sono rare
e tipiche ma solitamente sono assenti. La presenza di una zona
libera dalla proliferazione di melanocitici, immediatamente
sotto l’epidermide (Grenz zone) è caratteristica. L’epidermide
può talvolta presentare iperplasia e iperpigmentazione dei
melanociti basali con caratteri molto simili a ciò che si osserva
in un istiocitoma fibroso benigno dermico. Spesso è possibile
osservare nel nevo blu focolai multipli di proliferazione. Una
variante del nevo blu è il nevo blu combinato quando è associato ad un altro nevo, spesso acquisito ma anche congenito,
a un nevo di Spitz, a un nevo penetrante profondo o ad altri
nevi (acrale, desmoplastico, ecc.). La diagnosi differenziale di
nevo blu solitamente non presenta difficoltà soprattutto nella
forma tipica di proliferazione a cellule dendritiche pigmentate
a focolai multipli.
Il nevo di Ota e di Ito presentano caratteri clinici e sede tipica e molto raramente vengono sottoposti a biopsia a scopo
diagnostico. I caratteri morfologici delle lesioni sono analoghi
a quelli di un nevo blu con minore cellularità e maggiore
sclerosi.
La macchia mongolica è una lesione congenita che ha una
colorazione simile al nevo blu ed è localizzata quasi esclusivamente in sede sacrale. È una lesione piatta e di scarsa
consistenza, pianeggiante, a differenza dei nevi blu che di
solito sono più consistenti e leggermente rilevati. In diagnosi
differenziale devono essere presi in considerazione l’istiocitoma fibroso benigno dermico, soprattutto nella variante con
depositi di emosiderina tipo emangioma sclerosante; anche
un tatuaggio può talora simulare la macchia mongolica o il
nevo blu.
Il nevo blu cellulato è un caratteristico tumore melanocitico
frequentemente localizzato in sede coccigea, sacrale o nella
zona dei glutei. Presenta una marcata cellularità che coinvolge non solo il derma papillare e reticolare, ma, spesso anche
l’ipoderma con un caratteristico aspetto a forma di battacchio
di campana. Il nevo blu cellulato presenta frequentemente una
crescita multinodulare e differenti aspetti possono essere identificati: 1) fascicolato, 2) alveolare, 3) neuro-nevoide oltre ad
una caratteristica struttura bifasica. La variante mista bifasica
(fascicolata e alveolare) è la più frequente ed è composta da
grandi noduli di cellule fusiformi mescolati con nidi di cellule
epitelioidi, talora risultanti dalla sezione trasversale di fasci
cellulari di cellule fusate. Questi noduli sono separati tra loro
da zone di fibrosi più o meno estese con melanofagi e caratteristiche cellule tipo nevo blu. Le mitosi sono rare e possono
essere osservate nei noduli con un certo pleomorfismo. La
variante alveolare presenta nidi rotondeggianti, con bordi netti, spesso apigmentati o meno pigmentati delle aree fusate o
neuroidi; i nuclei sono vescicolosi con nucleoli poco evidenti;
il citoplasma spesso è chiaro e talora anche abbondante. I
nidi sono circondati da melanociti dendritici pigmentati e
melanofagi mescolati con scarse fibre collagene. Lo stroma
può occasionalmente presentarsi edematoso. Nella variante
neuro-nevoide il differenziamento cellulare presenta caratteri
schwanniani e le cellule fusate tendono ad aggregarsi in fasci
che simulano il caratteristico aspetto dei tumori dei nervi
periferici; collagene e macrofagi possono essere abbondanti.
A volte i blu cellulati possono essere così sorprendentemente
proliferanti anche con ulcerazione da simulare un melanoma
nodulare. Tuttavia, la presenza di cellule dendritiche con melanofagi in uno stroma fibroso tra le isole nodulari di cellule
più epitelioidi con citoplasma chiaro e l’aspetto bifasico con
alternanza di aree pigmentate e apigmentate sono di grande
aiuto nel differenziare il nevo blu cellulato dal melanoma
nodulare. In generale i nuclei sono simili con scarso polimorfismo e talora si possono osservare figure mitotiche, ma
le mitosi non sono atipiche e soprattutto non sono numerose.
Alcuni nevi congeniti ed acquisiti possono evolvere per maturazione/regressione a noduli fibrosi per la presenza di marcata
desmoplasia. Il riconoscimento del nevo con desmoplasia è
importante per distinguere queste neoplasie da altre lesioni
cutanee benigne come le fibrosi/fibromatosi, l’ istiocitoma
fibroso, il neurofibroma dermico ma anche e soprattutto dal
melanoma desmoplastico. La presenza di desmoplasia in
fenomeni nevo può essere correlata a regressione o a un processo reattivo. La proliferazione del nevo è, di solito, centrata
nel derma papillare, ma può anche essere estesa al derma reticolare mentre l’attività giunzionale spesso è minima o assente.
L’epidermide sovrastante può presentare iperplasia pseudoepiteliomatosa, acantosi e irregolare ipercheratosi. Le cellule
neviche sono disposte singolarmente o in piccoli gruppi, nidi
o cordoni e cellule fusiformi, in prevalenza cellule di tipo C e
di tipo A B epitelioidi sono meno frequenti o rare, ma quando
presenti sono ben evidenti. A volte possono essere presenti
cellule giganti bizzarre con aspetti simil-gangliari. Non è raro
il ritrovamento di inclusioni intranucleari causate da invaginazioni del citoplasma al nucleo. Mitosi sono rare e mitosi
atipiche sono assenti. Il pigmento melanico è irregolarmente
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RELAZIONI
distribuito in granuli di diverse dimensioni e quantitativamente variabile. Rari sono i melanofagi. Lo stroma è costituito da
fasci di collagene eosinofili di solito molto più spessi di quelli
normalmente presenti nel derma papillare. Lo stroma spesso
circonda e isola le singole cellule o i nidi.
Il nevo penetrante profondo è un nevo acquisito benigno
che spesso ha un colore che sfuma dal marrone a blu ad una
colorazione nerasttra. Necrosi, ulcerazione o e significativa attività mitotica sono assenti. La neoplasia è caratteristicamente
ben circoscritta ma profondamente penetrante e di solito incuneata nel derma profondo in corrispondenza di un annesso
cutaneo o lungo di esso. La base, più ampia, della lesione si
trova verso l’epidermide e l’apice o gli apici sono nel derma
profondo o anche nel tessuto adiposo sottocutaneo, con un
caratteristico aspetto a triangolo rovesciato. La componente
nevica più superficiale è costituita da nidi di cellule neviche,
circondati da macrofagi intensamente pigmentati, che di solito
sono simili ai nodi presenti nel derma profondo. Tale aspetto
è molto caratteristico e rappresenta l’unica eccezione alla
regola presente in tutti i nevi della progressiva maturazione
dalla superficie alla profondità della lesione. La maggior parte
delle lesioni hanno un focale o più raramente diffuso infiltrato
linfoide reattivo. Le cellule del nevo penetrante profondo sono
S100 e HMB 45 positive; l’ immunoreattività con siero anti
p16 può essere variabile con isolate cellule positive. Anche
il nevo penetrante profondo si pone in diagnosi differenziale
con un melanoma nodulare, tuttavia il melanoma presenta nidi
con aspetti clonali di cellule atipiche, necrosi, mitosi e infiltrato linfoide a focolai. I melanomi mostrano una architettura
simil-trapezoidale con base ampia in profondità e porzione più
ristretta in superfice, al contrario del nevo penetrante profondo che si estende in profondità a cuneo e mostra base superficiale più ampia. Inoltre i melanomi tendono ad espandersi
lateralmente oltre la porzione verticale dermica con aspetti
asimmetrici, mentre i nevo penetrante profondo si estende
nel derma lungo i fasci neuro-vascolari. Inoltre, il melanoma
mostra molto più pleomorfismo e nidi policlonali.
Bibliografia
1
Elder, 1991.
2
Lovas, 1983.
3
Patel, 1985.
4
Tobon, 1977.
5
Heim, 1993.
6
Rodriguez, 1968.
7
Nogueras-Gimeno, 1993.
8
Jao, 1971.
9
Tsang, 1993.
10
Ferrara, 1995.
Lesioni melanocitarie lentigginose
C. Urso
Anatomia Patologica Ospedale S. M. Annunziata Azienda Sanitaria
di Firenze
Le lesioni melanocitiche lentigginose costituiscono un insieme disomogeneo di lesioni cutanee, benigne e maligne, clinicamente classificabili come macchie e accomunate istologicamente da un aumentato numero di melanociti singolarmente
disposti lungo una giunzione dermo-epidermica modificata,
con allungamento delle creste (pattern lentigginoso). Il gruppo
può comprendere, oltre alla lentigo simplex, sulla quale è definito il modello, la lentigo labiale, la lentigo solare, le melanosi
genitali, il nevus spilus, il nevo persistente/recidivante, il nevo
lentigginoso, il nevo acrale, la lentigo maligna e il melanoma
acrale lentigginoso. Infine, negli ultimi decenni, sono state
oggetto di attenzione alcune lesioni melanocitiche di soggetti
in età adulto-avanzata, grandi, discromiche a margini frastagliati, istologicamente anch’esse caratterizzate da pattern
lentigginoso. Tali lesioni, inizialmente definite come nevi
lentigginosi displastici dell’età senile 1, successivamente sono
state indicate come nevi pigmentati lentigginosi con atipia 2 e
come nevi lentigginosi atipici dell’età senile 3, sottolineando
il loro possibile ruolo di precursori del melanoma, in particolare di uno specifico tipo di melanoma in situ, simile alla
lentigo maligna, ma in aree non fotoesposte, lentigo maligna
nevoide, ma anche di un tipo di melanoma invasivo, melanoma a piccole cellule 3. Questo gruppo di lesioni lentigginose
atipiche dell’età senile sono state in seguito “riscoperte” e
reinterpretate, con attenzione particolare al segmento più
atipico dello spettro e l’introduzione del termine di melanoma
lentigginoso 4, la cui diagnosi differenziale con il nevo lentigginoso e il nevo lentigginoso atipico rimane comunque non
facile. A complicare ulteriormente il gruppo, infine, si è inoltre recentemente aggiunto una lesione denominata melanoma
a nidi dell’età senile e considerata evoluzione del melanoma
lentigginoso 5.
References
1
Kossard S. Lentiginous dysplastic naevi in the elderly: a potential precursor for malignant melanoma. Australas J Dermatol 1991;32:27-37.
2
Blessig K. Benign atypcal naevi diagnostic difficulties and continued
controversy. Histopathology 1999;34:189-98.
3
Kossard S. Atypical lentiginous junctional naevi of the elderly and
melanoma. Australas J Dermatol 2002;43:93-101.
4
King R, Page RN, Googe PB, et al. Lentiginous melanoma: a histologic pattern of melanoma to be distinguished from lentiginous nevus.
Mod Pathol 2005;18:1397-401.
5
Pennacchia I, Garcovich, Gasbarra R, et al. Morphological and molecular characteristics of nested melanoma of the elderly (evolved
lentiginous melanoma). Virchows Arch 2012;461:433-9.
Melanomi che simulano sarcomi
M.C. Montesco
Paper not recevid
Melanomi mucosali
G. Botti
Direttore Dipartimento di Patologia Diagnostica e di Laboratorio
Istituto Nazionale Tumori IRCCS Fondazione G.Pascale Napoli Napoli
Il melanoma mucosale (MM) rappresenta un raro (solo l’1.4%
di tutti i melanomi) sottotipo di melanoma con caratteristiche
cliniche, biologiche ed epidemiologiche, differenti da quelle
dei melanomi cutanei: I) differente incidenza (MM 2.2 casi
per milione/anno vs 153.5 casi per milione/anno melanomi
cutanei), II) trend di incidenza stabile in confronto a quello dei
cutanei che è in progressivo aumento, III) i fattori di rischio
per l’insorgenza dei MM ad oggi non sono ancora noti, IV) le
informazioni disponibili in letteratura riguardano popolazioni
non caucasiche.
La notevole aggressività dei MM e, quindi, la loro prognosi
particolarmente sfavorevole, rende sempre più impellente una
dettagliata caratterizzazione di queste lesioni tumorali.
Ad oggi non esiste un sistema di stadiazione collegialmente
condiviso e generalmente si ricorre a stadiazioni in uso, spesso eterogenee, nei vari distretti interessati, in cui la neoplasia
è presente. L’istituzione di un sistema di stadiazione riproducibile e condiviso, per singola sede anatomica mucosale,
potrebbe costituire pertanto un obiettivo primario al fine di
116
consentire un ulteriore supporto all’ approccio prognostico/
terapeutico.
Studi in letteratura forniscono dati esigui e frammentati a causa
e della rarità della patologia e della mancanza di studi multicentrici; appare quindi necessario definire parametri oggettivi e
condivisibili, morfologici e molecolari, dei più comuni sottotipi
di MM non viscerali (testa-collo, anorettale e vulvovaginale).
Le mutazioni più frequenti nell’ambito dei melanomi interessano principalmente tre oncogeni: BRAF, c-KIT e NRAS. Nei
melanomi cutanei sono molto frequenti (40-60%) le mutazioni
del gene BRAF (serina/treonina chinasi) raramente descritte nei
MM (0-10%). Le mutazioni di NRAS coinvolgono il 5-14%
dei MM (vs 26% dei cutanei), mentre più frequenti risultano le
mutazioni e/o amplificazione del gene c-KIT (recettore tirosin
chinasi) riscontrate nel 39% dei casi.
Una attenta caratterizzazione delle alterazioni genetiche dei
MM, inoltre, consentirebbe una strategia terapeutica mirata,
considerando che gli attuali approcci terapeutici loco-regionali
e sistemici appaiono inadeguati e non condivisi. Queste sono
le basi per la proposta di uno studio multicentrico SIAPEC
IAP/GISD – IMI che ha come obiettivo principale quello di
formulare delle linee guida generali e condivise che forniscano
un utile strumento clinico diagnostico e prognostico per i MM.
Il progetto si articola in 5 fasi principali: I) Arruolamento dei
casi (melanomi mucosali non oculari) mediante revisione e
conferma morfologica ed immunoistochimica dei casi arruolati,
con relative informazioni cliniche e follow-up; contemporanea
realizzazione di un data base unico di pazienti con melanoma
mucosale non oculare provenienti dai diversi centri arruolati; II)
Identificazione dei criteri clinico/patologici (sede, dimensioni,
% necrosi, indice di proliferazione, indice mitotico, espressione
di BCl2, focalità, sistema di stadiazione condiviso per singola
sede anatomica mucosale, etc) condivisibili, di possibile impatto
prognostico; caratterizzazione del profilo molecolare (pathways
di NRAS, BRAF e c-KIT) effettuabile presso il proprio centro
o in altro centro disponibile. III) Elaborazione statistica uni/
multivariata dei dati registrati. IV) Standardizzazione del workup clinico diagnostico, con chirurghi specialisti e oncologi. V)
Produzione risultati e formulazione di linee guida condivise
anche con i Clinici (IMI-SIAPEC e AIOM).
I risultati attesi consistono nell’implementazione della casistica dei MM non oculari, con la creazione di un data base
virtuale mediante il quale potrebbe essere possibile una standardizzazione dei parametri morfo-molecolari dei MM, ad
impatto prognostico-predittivo.
Bibliografia
Aulmann S, Sinn HP, Penzel R, et al. Comparison of molecular abnormalities in vulvar and vaginal melanomas. Mod Pathol. 2014 Mar; 7
Beadling C, Jacobson-Dunlop E, Hodi FS, et al. KIT gene mutations and
copy number in melanoma subtypes. Clin Cancer Res 2008;14:6821-8.
Carlos A Torres-Cabala, Wei-Lien Wang, Jonathan Trent, et al. Correlation between KIT expression and KIT mutation in melanoma: a study
of 173 cases with emphasis on the acral-lentiginous/ mucosal type.
Mod Pathol 2009;22:1446-56.
Curtin JA, Busam K, Pinkel D, et al. Somatic activation of KIT in distinct
subtypes of melanoma. J Clin Oncol 2006;24:4340-6.
Grimaldi AM, Cassidy PB, Leachmann S, et al. Novel approaches in melanoma prevention and therapy. Cancer Treat Res 2014;159:443-55.
Gru AA, Becker N, Dehner LP, et al. Mucosal melanoma: correlation
of clinicopathologic, prognostic, and molecular features. Melanoma
Res. 2014;24:360-70.
Jakob JA, Bassett RL Jr, Ng CS, et al. NRAS mutation status is
an independent prognostic factor in metastatic melanoma. Cancer
2012;118:4014-23.
Keller DS, Thomay AA, Gaughan J, et al. Outcomes in patients with
mucosal melanomas. J Surg Oncol 2013;108:516-20.
Poynter JN, Elder JT, Fullen DR, et al. BRAF and NRAS mutations in
melanoma and melanocytic nevi. Melanoma Res 2006;16:267-73.
Postow MA, Hamid O, Carvajal RD. Mucosal melanoma: pathogenesis,
clinical behavior, and management. Curr Oncol Rep 2012;14:441-8.
Maldonado JL, Fridlyand J, Patel H, et al. Determinants of BRAF mutations in primary melanomas. J Natl Cancer Inst 2003;95:1878-90.
Mihajlovic M, Vlajkovic S, Jovanovic P, et al. Primary mucosal melanomas: a comprehensive review. Int J Clin Exp Pathol 2012;5:739-53.
Tacastacas JD, Bray J, Cohen YK, et al. Update on primary mucosal
melanoma. J Am Acad Dermatol. 2014;71:366-75.
Woodman SE, Davies MA. Targeting KIT in melanoma: a paradigm of
molecular medicine and targeted therapeutics. Biochem Pharmacol
2010;80:568-74.
Zebary A, Jangard M, Omholt K, et al. KIT, NRAS and BRAF mutations
in sinonasal mucosal melanoma: a study of 56 cases. Br J Cancer
2013;109:559-64.
Patologia molecolare e terapie target
nel melanoma
M. Barberis
Paper non received
Venerdì, 24 ottobre 2014
Le linee guida per la valutazione di HER2 nella mammella
Moderatori: Simonetta Bianchi (Firenze), Anna Sapino (Torino)
Linee guida ASCO/CAP 2013 per la valutazione
HER2
Le diverse linee guida per la valutazione dello
stato del gene HER2 a confronto: limiti e vantaggi
A. Sapino
Paper non received
C. Marchiò
Dipartimento di Scienze Mediche, Università degli Studi di Torino
I carcinomi della mammella con espressione equivoca (score
2+) della proteina HER2 rappresentano fino al 16% di tutti i
carcinomi di nuova diagnosi e vanno sottoposti a test di ibridizzazione in situ (in situ hybridization, ISH) per la valutazione dello stato del gene HER21. I risultati ISH dipendono dalla
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RELAZIONI
scelta di linee guida disponibili e possono essere influenzati
dalla presenza dell’eterogeneità dell’amplificazione di HER2
come anche dalla presenza del fenomeno di gain/amplificazione del centromero del cromosoma 17 (CEP17)2. Recentemente è stato proposto l’utilizzo di metodiche alternative
alla ISH, soprattutto nel tentativo di risolvere quei casi che
risultano equivoci anche al test ISH, tuttavia le recenti linee
guida americane (ASCO/CAP2013)3 sottolineano come dati a
tale riguardo siano ancora scarsi. Abbiamo recentemente condotto un’analisi di performance delle linee guida ASCO/CAP
(versione 20074 e versione 20133) e delle raccomandazione
FDA/EMA5,6 su una casistica di 957 carcinomi della mammella con score 2+. Su un sottogruppo di 122 casi abbiamo
anche valutato se un test addizionale PCR-based (“Multiplex
Ligation-dependent Probe Amplification”, MLPA) potesse
essere di aiuto nel risolvere casi di dubbia interpretazione7.
I nostri risultati dimostrano che i carcinomi della mammella
con espressione equivoca di HER2 sono in larga parte carcinomi estrogeno positivi con alto indice proliferativo (media di
Ki67=23.8%). In tale popolazione il gain del CEP17 si riscontra in circa il 27.4%, mentre l’amplificazione è decisamente
meno frequente (0.7%). Il tasso di amplificazione di HER2
varia significativamente a seconda delle raccomandazioni
utilizzate (dal 15% con ratio ASCO/CAP 2007 al 29.5% con
numero di copie FDA/EMA) e in generale è maggiore quando
viene utilizzato il numero di copie di HER2 come singolo
parametro. Secondo l’algoritmo introdotto dalle recenti linee
guida americane i carcinomi ISH positivi sono circa il 20%.
I carcinomi che presentano stato equivoco di HER2 anche dopo
ISH (12% circa) sono più frequentemente estrogeno-positivi
e presentano un tasso di proliferazione maggiore rispetto ai
carcinomi HER2 non amplificati, indicando una aggressività
biologica maggiore. Il test MLPA applicato a tale sottogruppo
ha identificato in larga parte una assenza di amplificazione di
HER2, tuttavia un bassa concordanza di tale metodica con i dati
ISH è stata riscontrata per la categoria dei carcinomi HER2 amplificati, a causa della presenza di amplificazione eterogenea.
Tale osservazione pone pertanto un caveat all’applicazione di
tali metodiche come esame di scelta alternativo alla ISH.
Bibliografia
1
Sapino A, Goia M, Recupero D, et al. Current Challenges for HER2
Testing in Diagnostic Pathology: State of the Art and Controversial
Issues. Front Oncol 2013;3:129.
2
Marchiò C, Lambros MB, Gugliotta P, et al. Does chromosome 17
centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis. J
Pathol 2009;219:16-24.
3
Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists
clinical practice guideline update. J Clin Oncol 2013;31:3997-4013.
4
Wolff AC, Hammond ME, Schwartz JN, et al. American Society of
Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in
breast cancer. J Clin Oncol 2007;25:118-45.
5
http://www.ema.europa.eu/ema/
6
http://www.fda.gov/Drugs/default.htm
7
Sapino A, Maletta F, Verdun di Cantogno L, et al. Gene status in HER2
Equivocal Breast Carcinomas: impact of distinct recommendations and
contribution of a PCR-based method. The Oncologist 2014, in press.
I risultati sullo studio di concordanza diagnostica del gruppo GIPAM
Moderatori: Simonetta Bianchi (Firenze), Anna Sapino (Torino)
Studio della concordanza diagnostica su core
biopsy
A. Rizzo
Coordinatore breast unit ULSS 8 U.O. Anatomia Patologica Ospedale
S. Giacomo, Castelfranco Veneto (Tv)
Il cancro della mammella rappresenta uno dei più frequenti
tumori nella popolazione femminile. Gli screening sistematici basati sulla mammografia biennale hanno ridotto significativamente la mortalità. Tuttavia, tali benefici possono
essere raggiunti soltanto se siano garantiti standard qualitativi
elevati di tutte le attività sanitarie del percorso diagnosticoterapeutico 1. Un’accurata diagnosi patologica comprensiva
delle informazioni prognostiche e predittive di risposta alla
terapia è indispensabile e cruciale nel corretto management
delle pazienti 2. La consapevolezza della necessità di aumentare l’appropriatezza della diagnosi istologica di lesioni
mammarie su core biopsy e la valutazione della qualità del
materiale diagnostico esaminato appaiono come aspetti indispensabili e propedeutici anche alla successiva valutazione
dei fattori prognostico- predittivi di risposta terapeutica. Vista
l’introduzione sempre più frequente di terapie neoadiuvanti
nel trattamento dei tumori mammari, appare necessaria una
puntuale caratterizzazione classificativa delle lesioni osservate. Come sottolineato dalle nuove linee guida ASCO per la valutazione di HER 2, alcuni istotipi particolari (ad esempio, il
carcinoma micropapillare) possono presentare delle positività
di membrana di tipo incompleto (baso-laterale) che risultano
talora mostrare amplificazione genica alla successiva indagine
in FISH/SISH: ciò consente il recupero di casi HER2 positivi
che altrimenti non verrebbero trattati.
Obiettivo chiave di un programma di qualità è il continuo
confronto tra i patologi. Programmi di garanzia della qualità
sulla diagnosi patologica sono attivi soltanto in pochi paesi
europei. Le aree su cui sono attivi tali programmi sono relative alla diagnosi istopatologica e alla diagnosi dei fattori
prognostico-predittivi. In Italia, esistono alcune realtà regionali che hanno sviluppato attività in tal senso, tra cui l’Emilia
Romagna, il Piemonte, la Toscana, il Trentino Alto Adige e il
Veneto. Un CQ nazionale è attivo per l’analisi del gene HER2
mediante FISH/SISH ed è coordinato dalla SIAPEC. Gli
schemi di controllo di qualità della diagnosi istocitopatologica attivi in Europa operano tradizionalmente su set di vetrini
di lesioni mammarie: lo schema del Regno Unito (NHSBSP
EQA) ad esempio prevede la circolazione annuale di set di
70 vetrini tra i vari centri. L’uso dei vetrini virtuali può rappresentare una valida alternativa negli schemi di controllo di
qualità 3. L’utilizzo del vetrino virtuale nella fase formativa a
distanza consente il confronto su un preparato unico per tutti i
patologi, consentendo di eliminare le inevitabili differenze di
colorazione (ad esempio: HER 2 2+) nel caso in cui fosse necessario ricorrere a vetrini consecutivi da spedire alle singole
118
unità operative. Ciò consente una valutazione dei risultati più
obiettiva e può costituire il primo nucleo di un futuro archivio
didattico online, eventualmente utilizzabile per successivi
eventi FAD e/o come materiale didattico per giovani patologi
in formazione. Per tali motivi, il GIPAM (Gruppo Italiano Patologia Mammaria) ha organizzato un confronto diagnostico
interistituzionale su 42 vetrini virtuali ottenuti da preparati
istologici di core biopsy” convenzionale (CB) o “core biopsy
vacuum-assisted” (VABB) di patologia mammaria, su 7 casi
di carcinoma della mammella immunocolorati per HER 2 e
su 6 casi per Ki-67, nell’ottica di un programma di garanzia e
miglioramento continuo della qualità. Le risposte istologiche
delle CB , una per centro, sono state refertate secondo le categorie diagnostiche B1-B5 sec. Linee Guida Europee 2006.
Scopo di questo set di vetrini è valutare la concordanza diagnostica su casi di cui è noto il riscontro istologico definitivo
per i casi di malignità o dubbi ed un opportuno follow-up per
le lesioni diagnosticate come benigne. Come gold standard è
stato considerata la diagnosi di maggioranza 4.
Tutti i casi discordanti saranno presentati durante l’evento
residenziale per il confronto e la discussione tra i partecipanti.
L’ analisi statistica sarà anche condotta separatamente tra
centri che leggono abitualmente core biopsy/VABB e centri
che refertano solo occasionalmente microistologia in quanto
osservano di routine citologia agoaspirativa. I casi di carcinomi mammari immunocolorati per HER 2 con vari pattern
di immunoespressione (0, 1+, 2+ amplificato alla FISH, 2+
non amplificato alla FISH, 3+) sono stati refertati (una diagnosi per centro), secondo il documento di consenso AIOM
SIAPEC 2010. Sui casi di carcinomi mammari immunocolorati per Ki-67 con varie percentuali di positività (alta e bassa
proliferazione), l’analisi statistica sarà effettuata utilizzando
come cut off il 14% e il 20% di cellule neoplastiche immunoespresse.
Una prima fase a distanza è avvenuta tramite accesso al sito
dell’ULSS di Rovigo (https://servizi.azisanrovigo.it/) per la
lettura di tutti i vetrini da parte dei singoli patologi.
Hanno aderito al progetto circa 50 U.O. di Anatomia Patologica ospedaliere ed universitarie del territorio nazionale. Hanno
partecipato all’evento anche più patologi afferenti alla stessa
unità operativa, formulando in tali casi una diagnosi finale “di
consenso ” del centro che è stata registrata su appositi tabulati
e successivamente inviata ai coordinatori nazionali dell’iniziativa. Dopo l’arrivo delle risposte da parte dei vari centri,
sono stati messi online i corrispettivi vetrini virtuali dei campioni operatori agganciati a ciascun caso di core/VABB che
sia stato sottoposto ad intervento chirurgico o le relative indagini immunoistochimiche: tutto ciò per consentire di giungere
alla discussione plenaria con maggiore consapevolezza delle
proprie risposte.
I vetrini rimarranno online anche dopo l’evento residenziale,
per quanti volessero confrontare le proprie valutazioni dopo
la discussione collegiale. La presentazione dei risultati e la
discussione sui singoli casi nella giornata residenziale sarà
effettuata dopo aver reso anonimi i risultati dei singoli centri
partecipanti.
Bibliografia
1
Perry N, Broeders M, de Wolf C, et al. European Guidelines for
quality assurance in breast cancer screening and diagnosis. Fourth
Edition, European Commission 2006.
2
Wolff AC et al. American Society of Clinical Oncology; College of
American Pathologists. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human
epidermal growth factor receptor 2 testing in breast cancer. J Clin
Oncol 2007;25:118-45.
3
Leong FJ, Graham AK, Schwarzmann P, et al. Clinical trial of telepathology as an alternative modality in breast histopathology quality
assurance. Telemed J 2000;6:373-7.
4
Ellis I. Quality Assurance Guidelines for Breast Pathology Services
(second edition). Sheffield NHS Breast Cancer Screening Programme
July 2011. ISBN 978-1-84463-072-1 (NHSBSP publication n° 02).
Discussione risultati HER2
I. Castellano
Paper not received
Discussione risultati Ki67
F. Pietribiasi
Paper not received
Telepatologia
Moderatori: Daniela Massi (Firenze), Roberto Mencarelli (Verona)
Controllo di qualità su vetrino virtuale: una
storia nuova
A. Rizzo
Paper not received
La diagnostica su vetrino virtuale: prospettive
attuali
L. Molinaro
Paper not received
Telepatologia Oltrefrontiera: esperienze
Africane
A.M. Ferrari
Associazione Patologi oltre Frontiera Servizio di Anatomia Patologica Casa di Cura San Pio x
La “tele patologia” è l’acquisizione e la trasmissione di immagini di campioni istologici e/o citologici da una sede remota
ad un’altra sede remota , utilizzata per diagnosi, consulto o
per insegnamento.
Nel 2005 l’associazione APOF, Patologi oltre Frontiera, ha
119
RELAZIONI
iniziato un progetto in Zambia all’ospedale Mtendere di Chirundu, per costruire ed organizzare un reparto di Anatomia
Patologica.
Due membri dello staff locale sono stati istruiti per un periodo di 8 mesi da alcuni membri dell’associazione e, alla
fine dell’addestramento, essi sono stati in grado di allestire
sia preparati istologici che citologici, oltre che leggere strisci
convenzionali da Pap test. È stato creato inoltre un sito internet apposito dove tutti i casi pervenuti all’ospedale vengono
registrati e dove vengono inserite, tramite uno scanner ed un
programma particolare, le immagini di tutti i casi istologici e
dei casi citologici sospetti o positivi.
Dal 2007 il sistema è pienamente attivo e da allora sono stati
svolti anche controlli di qualità con l’invio dei preparati isto e
citologici direttamente in Italia.
Da allora il sistema è stato utilizzato o sta per essere utilizzato
anche in altri progetti dell’associazione, offrendo un utile servizio di diagnosi a quegli ospedali dei paesi in via di sviluppo
dove sono presenti reparti chirurgici, servizi di endoscopia
o di aspirazione con ago sottile, ma non patologi in grado di
fornire diagnosi accurate ed in tempi accettabili.
La lettura dei pap test da parte di tecnici addestrati opportunamente e di un servizio di tele patologia come quello attuato
dall’associazione APOF permette inoltre uno screening efficace e a basso costo in quei paesi con un’alta incidenza di
carcinoma cervicale.
A national networking system: possible
scenarios
A. Gasparetto
Azienda ULSS 18 Rovigo Local Healthcare Authority
Introduction
Speaking about digital pathology networks it is possible to
meet different kinds of applications and IT infrastructures or
configurations. Educational purposes, quality control studies,
scientific studies, qualified second opinion, routine diagnosis,
for each kind of network it is possible to underline the respective peculiarities and implementation difficulties. The way in
which a project is developed and the final objectives must be
properly analyzed for a successful implementation.
Methods
Analyzing the different kinds of telepathology networks
configurations it is possible to represent the following architectures. For educational purposes usually a single center is
equipped with one or more slide scanners used for digitizing
the glass slides and for publishing them into a dedicated web
portal; in it students or other users can view specific cases or
test their skills. Differently for qualified 2nd opinion, different
labs, typically those which want to receive the opinion, are
equipped with one or more slide scanners for digitizing the
glass slides and for publishing them into a single web portal
that manage the opinion request, the counseling report and,
in some cases, online meetings. For quality control studies,
usually, the glass slides are digitized by one or more centers
and then collected and published into a web portal; in it the
pathologists can insert the diagnosis related to each case, for
the following data analysis and discrepancies evaluation.
Finally, a digital pathology network for the routine cases
management must be configured for handling all glass slides
digitized from one or more centers and must tackle the integration problem related to technical (systems integration) and
organizational aspects (processes integration).
Monitoring the different kinds of experiences born in Italy we
can find a lot of projects with a regional or national anatomic
pathology labs involvement but, unfortunately, coordinated at
local level (hospitals, Universities, University hospitals, …)
or, in some rare cases, at regional level. Consequently each
institution develops its own system, a lot of times without the
possibility of sharing images with other centers because the
related standards are not completely defined, increasing, at the
same time, the heterogeneity of the context.
Recent scientific articles about the diagnostic reliability of
telepathology, report many experiences that indicate it is
possible to successfully use digital pathology for diagnostic
interpretation 1,2,3. Unfortunately at the moment in Italy do not
exist technical and legal guidelines for using digital pathology
systems for diagnostic purposes; furthermore generally, there
are no certifications for using telepathology in diagnostic area.
Results
Each kind of aforementioned network of digital pathology
needs of specific technical and organizational prerequisites.
The complexity of these elements increase with the number of
different systems and institutions involved, as well as with the
necessity to introduce a new technology within consolidated
processes.
Even if the solution of the technical challenges seems to be
the first objective, only solving the organizational and legal
aspects it will be possible to build and effectively use a large
scale digital pathology network.
Conclusions
In order that digital pathology become a tool regularly used
by pathologists during their work, there must be a national
step by step strategy and coordination that can solve all the
open issues, helping to find the right standards for archiving
and sharing images and joining together all professional skills
necessarily involved as ICT, clinical engineering, forensic
medicine, privacy experts for creating the rules for using this
new technology properly.
References
1
Thorstenson S, Molin J, Lundström C. Implementation of large-scale
routine diagnostics using whole slide imaging in Sweden: Digital pathology experiences 2006-2013. J Pathol Inform 2014;5:14.
2
Bauer TW, Slaw RJ. Validating Whole-Slide Imaging for Consultation
Diagnoses in Surgical Pathology. Arch Pathol Lab Med 2014 May 19.
3
Pantanowitz L, Sinard JH, Henricks WH, et al. Validating whole slide
imaging for diagnostic purposes in pathology: guideline from the
College of American Pathologists Pathology and Laboratory Quality
Center. Arch Pathol Lab Med. 2013;137:1710-22.
From Venice to Paris: what’s new in digital
pathology
V. Della Mea
Dept. of Mathematics and Computer Science, University of Udine,
Italy
Introduction
With the 2014 edition, the traditional series of the European
Congress on Telepathology and International Congress on
Virtual Microscopy changed its name to European Congress
on Digital Pathology. This has been proposed to reflect recent
developments in the field and also a novel, integrated view
of the many applications of digital technology to pathology.
Being held every two years, the time span between congresses
permits to appreciate novel findings that usually are presented
during the scientific session, as well as commercial products
displayed at the booths.
120
In the 12th edition, held in Paris in June 2014 1, participation
was slightly above the previous Venice congress 2, and as
usual was worldwide more than European, with attendants
coming from 29 different countries.
The main themes that could be recognised this year were:
the digital image as practical tool for the modern pathologist;
the issue of storing digital slides with concrete solutions;
the traditional topic of quantitative image analysis;
and finally, discussions about the possible new role of the
pathologist with the availability of novel tools, towards the
so-called integrative pathology.
The digital pathologist
Regarding the theme 1, some proof is appearing that working
on digital images might provide better quality vs. the usual
microscope view. This regards in particular quantitative analyses that, when done on digital images (even not automatically), can benefit in terms of precision and documentation,
like mitotic count. Digital images also allow to better exploit
traditional sampling techniques like those described by morphometry and stereology.
However, in order to use digital images in practice, a workstation able to replicate the ergonomically optimal performance
of microscopes is needed. Different efforts have been presented, including the Leeds Virtual Microscope that is similar to
the microscope in terms of speed, view field, and resolution.
Storage of digital slides
Until now, techniques for efficient storage of digital slides
have been somewhat underconsidered because until few time
ago digital slides were mostly a research tool. However, if
they become a common tool in pathology practice, the issue
of the extremely large and continously growing amount of
memory needed arises, together with the bandwidth needs for
their distribution.
Fortunately, in the last years the concept of cloud computing
started to become common, and applications to digital slides
begin to appear. At the congress, three vendors were advertising
cloud-based storage solutions specifically targeted at pathology.
Another issue related to storage (and transmission) is technical standards. Like in Venice, the Congress hosted pre-meetings of DICOM and IHE, which discussed currently available
standards for digital slides and also for Anatomic Pathology
Information Systems.
Quantitative image analysis
Image analysis still remains one crucial topic at the congress.
While many advancements have been made, methods for
recognising tumor areas inside a specimen, quantifying immunohistochemistry, detecting rare events including mitoses
are important research subjects.
Integrative pathology
Finally, totally new methods are being introduced for analysing specimens that overlap with specialties adjacent to pathology and in particular with laboratory medicine.
As an example, new no-stain imaging systems have been presented that base their image generation on chemical properties
acquired by means of spectroscopy, driving to images that do
not need staining to be acquired. Since images are only one
possible output of those systems, one question is whether is
the pathologist the right person to deal with them.
This from one side represent a possible source of problems
for the traditional pathologist, part of which expertise could
become no more useful because substituted by other methods,
but on the other side the pathologist capacity of dealing with
morphological aspects may provide novel duties for him/her.
In fact, the final round table presented the importance of the
integration approach for solving the complex problem of predictive pathology – a view in which the pathologist becomes
the professional able to integrate the various perspectives
provided by novel techniques.
References
1
Proceedings of the 12th European Congress on Digital Pathology,
Paris, June 2014, Diagn Pathol, in press.
2
Proceedings of the 11th European congress on telepathology and 5th
international congress on virtual microscopy. Diagn Pathol. 2012 Jun
6;8 Suppl 1:S1-S45.
Patologia ultrastrutturale
Moderatori: Giovanna Cenacchi (Bologna), Leonardo Resta (Bari)
“Nanomics” in surgical pathology
L. Resta, R. Rossi, M.G. Fiore, T. Montrone, D. Piscitelli
Anatomia Patologica, Università degli Studi di Bari
The years 1950-70 are thought to be the years of largest
changes of the electron microscopy (EM) in the future of
surgical pathology: a novel plan for the study of subcellular
architecture and intercellular matrix has been revealed to
the investigator. In this period most of usual methods of
ultrastructure were performed. In the years 1970-2000 the
electron microscopy were utilized in the renal pathology,
neuromuscular diagnosis, liver and skin diseases, diagnosis
of undifferentiated neoplasms. Moreover, the electron microscope was a constant and particular instrument in several
research field.
Since 1990 the introduction and large utilization in surgical
pathology of a series of new technologies (immunohistochem-
istry, molecular biology, genetic techniques) obscured the use
of the EM in the routine histopathology. The ultrastructural
procedure is considered a difficult field, a very expansive and
time consuming technology.
But in special diagnostic fields, the EM is today an irreplaceable technological procedure. We can summarized four special conditions:
Evaluation of subcellular structures non otherwise evidenced,
the architecture of which induces a several defect of the function. Examples of this occurrence are the diagnosis of neuromuscular diseases and the conformation of the cilia. The study
of such type of diseases requests a long time of experience, in
order to avoid grossly confusion, as confirmed by the series
referred in the literature 1.
Evaluation of damaged structures, not otherwise evidenced
by histochemical or immunohistochemical procedures. The
main field is represented by the renal pathology. A not negligible amount of renal pathology is diagnosed only with the
RELAZIONI
use of EM: Alport’ syndrome, minimal change GN, unusual
deposits of different nature, etc. 2.
Diagnosis of undifferentiated neoplasms with an equivocal
immunohistochemical and/or molecular expression. Today,
we have a very high confidence in these new technologies.
But, the neoplastic cell may lose some natural antigen and
produce atypical ones, and our research is arbitrarily confused. Also the genetic mutations expected are not present in
all the tumors: an accurate ultrastructural observation may be
useful in some cases. In other tumors, as the salivary tumors
or the metastases of unknown origin, the ME reveals the possibility of shape mutations of pluripotential cells 3.
Research of parasites, viruses, bacteria, and other substances
not evidenced with common diagnostic procedures. An affected
structure may be slightly modified in the form observed at light
microscope, but it may hide an insidious phantom. We can
confirm a Whipple’ disease, protein crystals, metal poisoning 4.
The EM offers today a significant help to surgical pathology. In a situation of spending review, this technology is not
more expansive as respect to other procedures, but it strongly
depends on the clinical users with which is essential a continuous collaboration. On the other hand, the possibility of a
diffuse and modern formation of a new generation of pathologists in the ultrastructural diagnosis is of great importance for
the future of this discipline. As in other fields of diagnostic
pathology, the lack of specialists in EM may promote its acquisition by other professionals.
In a systematic and complete form of diagnostic pathology the
EM may be flanked to other more diffused “omics” and it may
become the “nanomic” of surgical pathology, or, as proposed
by MG Farqhuar (2009) ‘In the age of harvesting the cell genoma and proteoma, we should also not forget to pay attention
to harvesting the cell “structurome”’ 5.
References
1
Simoneau T, et al. Impact of cilia ultrastructural examination on
the diagnosis of primary ciliary dyskinesia. Pediatr Dev Pathol
2013;16:321-6.
2
Liapis H. Electron microscopy in kidney research: seeing is believing.
Ultrastruct Pathol 2013;37:340-5.
3
Resta L, et al. Role of electron microscopy in diagnosis of parotid
tumours. Acta Otorhinolaryngol Ital 2005;25:150-2.
4
Principi M, et al. A mysterious case of gastroparesis: could the secret
be found in a drink? Inflamm Allergy Drug Targets 2013;12:187-9.
5
Farquhar MG: Foreword. in Pavelka M & Roth J: Functional Ultrastructure. Atlas of tissue Biology and Pathology. 2nd Edition. Springer
Wien New York Ed. 2010.
Ultrastructural diagnosis in renal pathology:
evidences in special cases
T. Montrone, L. Resta, M.G. Fiore, R. Rossi, D. Piscitelli
Anatomia Patologica, Università degli Studi di Bari
The electron microscopy is a fundamental step in the diagnosis of kidney biopses and ultrastructural aspects have a
different importance for definitive diagnosis.
The main patterns used for diagnosis are cellular architecture,
type and site of eventual deposits, structure of GBM and mesangium, changes of subcellular structures.
The role of EM changed in the last 40 years, after the introduction in the routine of new instruments for diagnosis (immunofluorescence, immunohistochemistry); today it is very
useful both for diagnosis in nephrotic syndrome, to classify
glomerular diseases and to identify new categories, and to
monitor therapy and evolution of some conditions.
121
We performed electron microscopy in about 170 kidney biopsies in a period of 4 years, and sometimes it was essential
or helpful for diagnosis, in other times its contribute was not
necessary.
In a lot of cases (23) (13,70%) it was no possible to do a
certain diagnosis because clinical and histological dates were
not sufficient, or because ultrastructural alterations were completely nonspecific; in these case we did only a descriptive
report.
12 cases (7,15%) were non diagnostic due to processing
artifacts or sampling errors, and 3% were ultrastructurally
completely normal.
In 12,5% of diagnosis it was essential. In MCD (5 cases),
when we can observe effacement of podocytes, in absence
of other alterations, and it is the only way to distinguish this
from early stages of membranous nephropathy; in membraneproliferative EM help to differentiate type I (3 case) and II
(1 cases). In Alport (11 cases) syndrome is basic the “basket weaving” aspect, with reduplication and splitting of the
lamina densa of GBM, and also in non-amyloidotic fibrillary
glomerulophathy the aspects and diameter of fibrils can be
observed only with ultrastructural examination.
In 20,85% was helpful: in FSGS (16 cases) EM is useful to
distinguish primary from secondary forms, and it is very important to stage the disease; in amyloidosis it confirms in the
presence of specific type of fibrils.
In IgA nephropathy (17 cases) presence of immunodeposits
typically in mesangial and paramesangial areas is an important complementary aspect for diagnosis.
EM permits to distinguish membranous glomerulopathy from
other diseases with the same clinical manifestations.
The EM results not useful in the remnant cases and so it could
be not requested.
Our experience reveals that in some circumstances the ultrastructural approach arises as a sudden and unexpected event
and it permits to do a diagnosis.
In the literature EM is essential in a percentage of cases from
10% to 25%, and useful/confirmatory in the 5% to 61%.
Case 1. A 18 year old female shows clinically a nephrotic
syndrome. Renal biopsy shows tubular atrophy and interstitial
fibrosis in about one half of the sample. Small foci of foam
cells are present in the interstitium. In glomeruli we notice a
pattern of mesangial expansion with a minimal and segmental
hypercellularity.
The GBM is irregularly expanded and the capillary lumina are
compressed and sometime obliterated by eosinophilic PAS
positive, material.
The immunofluorescence does not reveal any deposits.
A diagnosis of “unclassified” nephropathy is performed. At
ultrastructural examination, we note an irregular thickness
of the GBM, with a double contour pattern, rarefaction of
subendothelial space and diffuse deposition of electrondense
fibrillary substance in the extracellular matrix. The fibers
measure 30-40 nm in diameter, with a periodicity of 450560nm different from normal type of collagen III. These
fibrils are similar to collagen but are atypical, for shape,
sometimes they are curved, bent, curled or spiral-shaped, and
arrangement. The diagnosis of “Collagen III Glomerulopathy
“ is made. This rare condition must be distinguished from
type I of membrane-proliferative glomerulopathy, which is
characterized by an important mesangial hypercellularity and
by subendothelial immunedeposits of IgG and C3. Another
similar aspect is observed in nail-patella syndrome, in which
collagen fibers are in the lamina densa of GMB, with a “motheaten” appearance but are unremarkable ultrastructurally,
122
furthermore the disease is genetically determined and it is
associated with bone and nail alterations.
The Collagen III Glomerulopathy is a very rare disease (50
cases in literature), it affects patients of all the ages, with onset
in childhood or in late adulthood.
It usually is sporadic (adult forms) and familial cases seem to
have a autosomal recessive pattern of inheritance with a variable gene penetrance.
The pathogenesis is not clear: probably IL-4 stimulates mesangial cells and/or podocytes to synthesize collagen III, or
probably there is an altered degradation of this type of protein,
with involvement of other sites (reported association with
perisinusoidal fibrosis).
For confirmation of the diagnosis we can use levels of type
III procollagen peptide in serum and urine, and positivity of
collagen III at IHC/IF in kidney biopsy.
There is no treatment for this disease, and prognosis is variable: it develops into a ESRD in 3-10 years.
Case 2. A 70 year old man, with a nephrotic syndrome.
Kidney biopsy shows large and hypocellular glomeruli. The
mesangial matrix is replaced by a large amount of a hyaline
eosinophilic, PAS positive, silver stain negative material. Immunofluorescence excludes immunodeposits. The diagnosis
of “unclassified glomerulopathy” is made.
The EM examination of a frozen renal fragment reveals the
presence of a large amount of interstitial regular fibers measuring 12-15 nm in diameter, organized in straights bundels
rather regular. The diagnosis of “fibronectin glomerulopathy”
is performed. This rare disease shows an ultrastructural pattern similar to amyloidosis, but the amyloidotic fibers are less
large (8-10 nm) and hazardously disposed, On the contrary,
in the “immunotactoid glomerulopathy” we observe larger
microtubules (10-90 nm), with a central cavitation disposed
in a fascicular arrangement.
This condition is very rare and it exhibits an autosomal dominant pattern of inheritance or it could be sporadic. Probably it is
caused by an altered catabolism of fibronectin, and mutations in
the FN1 gene (2q34 cromosome) are associated with this disease.
The diagnosis is made on kidney biopsy and it is confirmed by
positivity for fibronectin at IHC/IF.
The disease develops slowly in ESRD (10-15 years) and
there are no specific treatments all forms of renal replacement
therapy have been used with good success for patients with
end-stage renal disease, but FNG can recur in a transplanted
kidney and the risk of recurrence is uncertain.
Case 3. A 14 year old female, affected by acute renal failure,
with low level of complement factors in the serum. Kidney
biopsy shows aspects suggestive for a membrano-proliferative
glomerulopathy. The immunofluorescence reveals the presence of glomerular C3 deposits, without antibodies. The diagnosis is: “suspected for C3 nephropathy”.
At ultrastructural examination, the GBM shows an irregular thickening with presence of electrondense “hump-like” material, mainly
in the subepithelial spaces, but also in the mesangium and in the
membranes. The diagnosis of “C3 nephropathy” is confirmed.
This is a rare and complex disease in which we can distinguish
3 sub-types, identified with the ultrastructural examination:
1. Dense deposit disease (DDD) with intramembranous ribbon-like deposits;
2. C3GN with discontinuous, mesangial, subendothelial and
subepithelial non ribbon-like deposits;
3. CFHR5 with mesangial, subendothelial, and occasional
subepithelial, hump-like deposits.
It affects mainly young people and it is often familiar ( with
autosomal dominant genetic inheritance).
Etiopathogenesis is not clear, probably the disease is caused
by a dysregulation of complement alternative pathway.
Isolated deposits of C3 without immunoglobulins in kidney
biopsies, and low levels of this factor in serum are important
for diagnosis. It develops an ESRD in 10-15 years.
The present demonstration point the importance of electron
microscopy in the definition of glomerular disease, especially in
the rare form, in which the differential diagnosis is very difficult.
The use of a morphometric system is important in the definition of the exact nature of fine structure.
References
1
Mokhtar GA, Jallalah SM. Role of electron microscopy in evaluation
of native kidney biopsy: a retrospective study of 273 cases. Iran Kidney Dis 2011;5:314-9.
2
Sihem Darouich S, Goucha RL, Jaafoura MH, et al. Value of electron
microscopy in the diagnosis of glomerular diseases. Ultrastructu
Pathol 2010;34:49-61.
3
Haas M. A reevaluation of routine electron microscopy in the examination of native renal biopsies. J AM Soc Nephrol 1997;8:70-6.
4
Yasuda T, Imai H, Nakamoto Y, et al. Collagenofibrotic glomerulopathy; A systemic disease. Am J Kidney Dis1999;33:123-7.
5
Cohen AH. Collagen Type III Glomerulopathies. Adv Chronic Kidney
Dis 2012;19:101-6.
6
Otsuka Y, Takeda A, et al. A recurrent fibronectin glomerulopathy in a renal transplant patient: a case report. K Clin Transplant
2012;26(Suppl 24):58-63.
7
Patro KC, Jha R, Sahay M, et al. Collagenofibrotic glomerulopathyCase report with review of literature. Indian Journal of Nephrology
2011;21:52-5.
8
Fujigaki Y, Kimura M, Yamashita F et al. An isolated case with
predominant glomerular fibronectin deposition associated with fibril
formation. Nephrol Dial Transplant 1997:12: 2717-22.
9
Castelletti F, Donadelli R, Banterla F, et al. Mutations in FN1 cause
glomerulopathy with fibronectin deposits. Proc Natl Acad Sci USA
2008:105:2538-43.
10
Barbour TD, Pickering MC, Cook HT. Recent insights into C3 glomerulopathy. Nephrol Dial Transplant 2013;28:1685-93.
Update sul ruolo della microscopia elettronica
nella diagnosi dei tumori dei tessuti molli
A. Franchi
Paper not received
Myofibrillar myopathies: an integrated
diagnosis
V. Papa1, R. Salaroli1, L. Badiali De Giorgi2, R. Rinaldi3, C.
Graziano4, G. Ricci5, F. Paoloni6, M. Fanin6, G. Siciliano5, C.
Angelini6, G. Cenacchi1
Department Biomedical Neuromotor Sciences, Alma Mater University Bologna; 2UO Pathology, 3UO Neurology, 4UO Medical Genetics
S.Orsola-Malpighi Hospital, Bologna; 5Department Clinical Experimental Medicine, Pisa University; 6Department Neurosciences, Padua University and IRCCS S. Camillo, Venice, Italy
1
Myofibrillar myopathies (MFMs) are often overlooked disorders because of their clinical and genetic heterogeneity.
They are rare inherited or sporadic progressive neuromuscular disorders involving distal muscles: cardiomyopathy and
peripheral neuropathy can be associated. A small proportion
of MFM patients carries disease-associated mutations in the
desmin, alphaB-crystallin, myotilin, filamin C, BAG3, titin,
FHL1 and ZASP genes 1, otherwise others share candidate
chromosomal loci without identified gene. So far, no clinical
diagnostic criteria have been established. MFMs diagnosis is
based on characteristic morphological features in the muscle
biopsy as myophatic such as myofibrillar structural changes
RELAZIONI
and abnormal sarcoplasmic protein aggregates. Immunohistochemical analysis reveals positive reactions in affected
fibers against Z-disc and related proteins but cannot identifies
different MFM subtypes 2. Electron microscopy reveals clues
to the mutated gene pointing out ultrastructural differences
between subgroups 3, but only genetic analysis remains, by
now, the only way to a definite diagnosis. Nevertheless,
MFM morphological features may overlap with those of other
neuromuscular disorders showing protein aggregates, (e.g,
Inclusion Body Myopath,IBM and Limb Girdle Muscular
Dystrophies 1A and 1F) 4. Therefore, differential diagnosis
could be very complex. Trying to identify a morphological
marker we studied muscle biopsies from MFM and sporadic
IBM patients with histological, enzyme histochemical, ultrastructural and immunohistochemical techniques using above
all antibodies towards desmin, myotilin and Major Histocompatibility Complex class I (MHC-I). This study has identified
similarities and dissimilarities between MFM and sIBM cases
and has given more insights into the effects of protein aggregation within muscle fibres. Progress in molecular studies of
MFMs in combination with classical morphological and ultrastructural approaches leads to improvements in diagnostic and
opens up prospects for the development of subtype-specific
prevention and therapy 1.
References
1
Olivé M, et al. Myofibrillar myopathies: new developments. Curr Opin
Neurol 2013;26:527-35.
2
Claeys KG, et al. Differential involvement of sarcomeric proteins in
myofibrillar myopathies: a morphological and immunohistochemical
study. Acta Neuropathol 2009;117:293-307.
3
Claeys KG. Electron Microscopy in myofibrillar myopathies reveals
clues to the mutated gene. Neuromuscul Disord 2008;18:656-66.
4
Schroder R. Protein aggregate myopathies: the many faces of an expanding disease group. Acta Neuropathol 2013;125:1-2.
Application of the future in nanomics
G. Cenacchi
Dipartimento di Scienze Biomediche e Neuromotorie, Alma Mater
Università di Bologna
The suffix -ome as used in molecular biology refers to a totality of some sort; it is an example of a “neo-suffix” formed by
abstraction from various Greek terms in -ωμα, a sequence that
does not form an identifiable suffix in Greek. Data integration
is now a very commonly used notion in life sciences research.
However, there is still no unified definition of data integration, nor taxonomy for data-integration methodologies despite
some recent efforts on this topic. Omics sciences (i.e genomics, metabolomics, proteomics, and pharmacogenomics) have
just started to contribute here by generating gene, protein
expression, metabolite data at global level and large scale. The
explosion of the omes and omics terms reflects 1) reinforced
holistic point of view in understanding life, 2) expansion and
differentiation of relatively simple chunk of life into various
encapsulated biological domains, and 3) technical advancement in computer science to integrate complex biological
data through bioinformatics. The neologism “nanomics” could
indicate the discipline applied to nanometer-sized both biological and non-biological structures. Nanobiotechnology is a
new focus in technological science: it plays a key role in the
creation of nanodevices , including liposomes, nanoparticles
and polymeric nanoassemblies, such as polymeric micelles and
vesicles for the analysis of living systems on a molecular level.
Moreover, nanomedicine allows for improved understanding
of human life while using the knowledge on human organism
123
at a molecular level. The use of nanotechnological approaches
and nanomaterials opens new prospects for the creation of
drugs and systems for their directed transport. The application
of nanotechnology can provide significant advantages for the
diagnosis, treatment and management of diseases. Nano-scaled
devices are a promising platform for specific detection of pathological targets, facilitating the analysis of biological tissues in
real-time, while improving the diagnostic approaches and the
efficacy of therapies. In this way, the ability of nanodevices
to specifically target pathological events not only can enhance
therapeutic efficacies, while lessening side effects and reducing costs, but also can provide a useful approach for studying the nano-pathophysiology of diseased tissues Inorganic
nanoparticles have also been used for constructing multifunctional nanodevices with precise control of their size and shape.
These nanoparticles present unique electronic, optical, magnetic and mechanical properties, which have been exploited
in a wide range of biomedical applications, including biosensing, imaging, tissue engineering, heating (hyperthermia), and
targeted drug and gene delivery. Inorganic nanoparticles can
also provide a framework for incorporating various functions,
as their surface can be readily modified with lipids, polymers
or biomolecules, thus, improving their biocompatibility and
performance at the biological interface. Nevertheless, toxicity
concerns and slow excretion of inorganic nanoparticles must
be deeply investigated and the electron microscopy likely represents the key nano-tool in this new nanomics era. Electron
microscopy is also strategic in the study of biological nanoparticles suche as microvesicles and exosomes. Microvesicles,
ranging from 0.1 to 2.0 μm, are defined as intact, submicron,
phospholipid-rich vesicles that are released from the cell
membrane of a diverse range of cells upon activation, with for
example growth factors or cytokines and/or during apoptosis.
Exosomes denote a family of nanoparticles with a diameter in
the range of 30–120 nm that are secreted by most cell types of
the body. They arise within endosomal compartments called
multivesicular bodies, which bud internally to form intraluminal vesicles. On fusion with the plasma membrane, multivesicular bodies release their contents into the extracellular fluid,
at which point the vesicles are referred to as exosomes. These
data suggested that exosomes were used by the cells as a major
route of excretion, which would allow them to dispose of any
unused or harmful RNA and proteins, in case no appropriate
lysosomal degradation systems were available. Exosomes are
now regarded as a distinct cellular entity specifically capable
of carrying cargos like RNA, proteins, lipids etc. to be shared
between cells. Exosomes are now thought to play key roles in
cell-to-cell communication, antigen presentation, and in the
pathogenesis of retroviral infections and prion diseases such
as novel means of delivering therapeutics lipids, polymers or
diagnostic biomolecules.
References
López E, Madero L, López-Pascual J, et al. Clinical proteomics and
OMICS clues useful in translational medicine research. Proteome Sci
2012;10:35.
Yameen B, Choi WI, Vilos C, et al. Insight into nanoparticle cellular
uptake and intracellular targeting. J Control Release 2014 Jun 28. pii:
S0168-3659(14)00447-7.
Oliveira ON Jr, Iost RM, Siqueira JR Jr, et al. Nanomaterials for Diagnosis: Challenges and Applications in Smart Devices Based on
Molecular Recognition. ACS Appl Mater Interfaces 2014 Jul 9. [Epub
ahead of print).
Srinivasan A, Rastogi A, Ayyavoo V, et al. Nanotechnology-based approaches for the development of diagnostics, therapeutics, and vaccines. Monoclon Antib Immunodiagn Immunother 2014;33:186-91.
Johnsen KB, Gudbergsson JM, Skov MN, et al. A comprehensive over-
124
view of exosomes as drug delivery vehicles - Endogenous nanocarriers
for targeted cancer therapy. Biochim Biophys Acta 2014;1846:75-87.
Inal JM, Kosgodage U, Azam S, et al. Blood/plasma secretome and microvesicles. Biochim Biophys Acta 2013;1834:2317-25.
Kastelowitz N, Yin H. Exosomes and microvesicles: identification and
targeting by particle size and lipid chemical probes. Chembiochem
2014;15:923-8.
Sala Botticelli - 08.30 – 12.30
Qualità e gestione del rischio in anatomia patologica:Problemi e soluzioni
pratiche – Parte I
Moderatori: Roberto Riccardo Pietro Giardini (Milano), Giuseppe Santeusanio (Roma)
L’errore nella determinazione dei biomarcatori:
cause, conseguenze e soluzioni
M. Chilosi
Paper not received
L’Anatomia Patologica come verifica di qualità
nel processo clinico-assistenziale
G. Santeusanio
Paper not received
Audit in Anatomia Patologica: Dalla teoria alla
pratica
C. Della Rocca
Dipartimento di Scienze e biotecnologie medico-chirurgiche “Sapienza” Università di Roma, Polo Pontino – Sede di Latina
Per Audit si intende un esame sistematico e indipendente
finalizzato a verificare che le attività svolte per la qualità e
i risultati ottenuti siano congruenti con quanto stabilito, ed
efficaci per il raggiungimento degli obiettivi. Si distingue un
Audit di Sistema, che riguarda un’intera organizzazione o una
sua parte, che ha un modello di riferimento e mira a posizionare l’organizzazione rispetto a tale modello di riferimento, e un
Audit di Processo o di Prodotto, che riguarda un processo o un
prodotto, che ha un modello di riferimento (standard) e verifica la congruità con tale standard tramite l’analisi di indicatori.
L’Audit clinico è un tipico Audit di processo o prodotto in
cui l’oggetto è la pratica clinica e i suoi risultati, ha una forte
connotazione tecnico professionale e sfrutta la valutazione tra
pari; si tratta nei fatti di “una iniziativa condotta da clinici che
cerca di migliorare la qualità lavorativa complessa e gli outcome dell’assistenza attraverso una revisione tra pari strutturata
per mezzo della quale i clinici esaminano la propria attività e
i propri risultati in confronto a standard espliciti e la modificano se necessario”. I contesti in cui trova applicazione l’Audit
in campo medico sono la valutazione sistematica della qualità
dell’assistenza erogata rispetto allo standard di riferimento
(monitoraggio sistematico) e la valutazione di problematiche/
criticità individuate, occasionale, alla ricerca della non conformità, palese o non, ai fini del miglioramento del processo/prodotto (miglioramento continuo delle prestazioni). L’Anatomia
Patologica, come è noto, è una realtà lavorativa complessa
nella quale la diagnosi di malattia tramite studio di alterazioni
morfologiche di cellule e tessuti si raggiunge tramite una sequenza articolata di attività manuali, tecniche e di atti medici
che hanno l’obiettivo della formulazione di un referto in un
ambito di applicazione vasto, caratterizzato, di sovente, da un
gran numero di prestazioni richieste. È evidente che in tale
realtà lo strumento dell’Audit è indispensabile non solo per la
verifica e il miglioramento della qualità del servizio erogato e
per la risoluzione di problemi/criticità, ma anche come forma
di “difesa” dell’attività del servizio in ordine a situazioni di
rischio che si generano altrove e ricadono sulle attività del
servizio stesso. In questo senso è indispensabile pervenire a
standard e indicatori di processo e risultato condivisi, a livello
almeno nazionale, nella nostra disciplina per evitare che altre
“autorità sovraordinate” (Aziende, Regioni, ecc) possano
proporne liberamente di propri, senza la necessaria competenza. È evidente il ruolo che la SIAPEC deve rivestire in tale
ambito stante anche la considerazione incontrovertibile che la
figura dell’anatomo-patologo è sempre centrale anche nelle
azioni di Audit clinico a livello Dipartimentale/Aziendale, per
l’importanza del suo apporto allo sviluppo dell’iter diagnostico terapeutico dell’assistito e la sua frequente funzione di
controllo di qualità ultimo (in sede autoptica) delle prestazioni
assistenziali erogate.
Standardizzazione delle procedure:
problemi e soluzioni
R. Giardini
IRCCS Istituto Auxologico Italiano, Anatomia Patologica, Milano
L’Anatomia Patologica sta sperimentando in questi ultimi
tempi svariati modelli di cambiamento, sia in ambito organizzativo che in ambito professionale, che, in misura variabile
da realtà a realtà. hanno iniziato a modificare, ed incideranno
sempre più profondamente in un futuro non troppo lontano, il
modo di praticare la specialità: svariate tecniche innovative
in immunocitochimica, l’espansione della patologia molecolare e l’estesa informatizzazione da un lato, la disponibilità
di sofistica tecnologie di automatizzazione e di controllo del
tracciato operativo che conduce alla diagnosi stanno portando
all’acquisizione di nuove ed eccitanti informazioni nel campo
della pratica anatomopatologica. Questo fenomeno, tuttavia,
se da un lato incrementa le finalità dell’informazione diagnostica, aggiungendovi dati di tipo prognostico e predittivo di risposta alla terapia, dall’altro impone la necessità di verificare,
attraverso meccanismi di convalida basati sull’evidenza, che
le informazioni prodotte siano “di qualità”. Se l’applicazione
sempre più estensiva della tecnologia esige, ogni giorno di
più, che anche il patologo debba sforzarsi di abbandonare
l’eminence based medicine, in pratica le situazioni contingenti
ed i problemi quotidiani rendono, tuttavia, difficile l’applicazione sistematica di pratiche evidence based. Una prima scon-
125
RELAZIONI
tata risposta a questa esigenza è quella di rivolgersi a manuali
di procedure ed a linee guida, che nascono come strumento
che ha l’obiettivo di permettere all’operatore di fare scelte
“informate”, basandosi sull’analisi delle prove scientifiche e
sulla valutazione dei rischi e dei benefici di qualsiasi azione.
Di più, nel tempo, manuali di procedure e linee guida si sono
rivelati uno strumento di aggiornamento per i professionisti,
di educazione ed informazione per i pazienti e di riferimento
esterno con cui si rende possibile una verifica di quel che il
professionista è in grado di produrre. A questo punto non ci
dovrebbero più essere problemi nello standardizzare le proprie
procedure, nell’uniformarle all’evidenza, ad esempio nella
descrizione macroscopica di un pezzo operatorio, per evitare
verbosità e per minimizzare il rischio di omettere informazioni importanti. Ma, se si vuole approfondirne l’analisi, non è
quasi mai così facile. Occorre considerare che il vantaggio più
grande dell’anatomia patologica come specialità medica è che
la biologia di una malattia rimane per la gran parte costante,
risultando in un vasto corpo di conoscenze che potrebbe
anche non cambiare mai. All’opposto, la nostra conoscenza
della malattia è rapidamente in espansione e sia le evidenze
sia le modalità del trasferimento di quelle conoscenze nella
pratica quotidiana possono essere molteplici ed alla fine
creare sconcerto e portare a risultati inferiori alle aspettative.
Nella standardizzazione delle proprie procedure operative non
basta fare riferimento ad evidenze della letteratura, occorre
valutare la loro applicazione nella situazione particolare, ed
agire di conseguenza. Per standardizzare una procedura occorre quindi per prima cosa tener presente tutti i fattori che
possono influenzare, ad esempio in una diagnosi di neoplasia,
la possibilità di porre una diagnosi corretta di tumore: fattori
non controllabili come i fattori di rischio del paziente (sintomi, biochimica, stadio clinico, precedenti diagnosi, etc) e
fattori controllabili: ad esempio, nel carcinoma della prostata,
quelli controllabili dall’urologo, come il numero delle agobiopsie effettuate, il metodo bioptico random o ecoguidato,
la localizzazione delle biopsie, la qualità delle stesse, legata
all’esperienza dell’operatore ed alla disponibilità logistica;
quelli controllabili dal patologo (esperienza e professionalità
dei tecnici di laboratorio nel processare e tagliare i frustoli,
il numero dei frustoli inclusi per biocassetta, il numero delle
sezioni per campione, etc) e quelli controllabili dalla collaborazione di entrambi: valga per tutti il riscontro, per quest’ultimo punto, che la percentuale di diagnosi positiva aumenta da
24% a 31% se le biopsie vengono adagiate nelle biocassette
direttamente al momento dell’atto bioptico, rispetto al lasciarle fluttuare nelle provette per la consegna al laboratorio.
Una procedura standard può avere successo e quindi portare
ad un miglioramento della qualità diagnostica solo se viene
costruita anche rispetto alla realtà lavorativa in cui dovrebbe
operare: costruire una procedura con standard troppo elevati
rispetto alla reale situazione delle risorse di apparecchiature,
di personale, di logistica significa votare quella procedura al
suo più o meno precoce abbandono, con tutti problemi che ne
conseguono (ad esempio, per rimanere nel campo della patologia prostatica, non attivare una collaborazione con urologo,
implementare una procedura di taglio delle agobiopsie o di
campionamento dei pezzi operatori da prostatectomia senza
prima aver organizzato con la direzione strategica attrezzature
e personale). Standardizzare una procedura non deve avere
il significato di un polveroso ingranaggio che appesantisce
la macchina diagnostica, ma deve essere l’occasione per ripensare la nostra organizzazione ed adeguarla allo standard
che dobbiamo raggiungere, ad esempio richiedendo adeguate
risorse: solo così si sarà capaci di risolvere i problemi che
una procedura standard può portare nell’impatto con le nostre
abitudini lavorative.
La richiesta di preparati citoistologici e delle
inclusioni in paraffina per consulenza / seconda
opinione: problemi e soluzioni
A. Fabiano
Paper not received
Responsabilità professionale medica: l’evoluzione della giurisprudenza
Moderatore: Ilaria Pagni (Firenze)
Responsabilità sanitaria e risarcimento tra
danno alla salute e perdita di chance
La giurisprudenza della Corte dei Conti sulla
responsabilità sanitaria
G. Travaglino
Paper not received
S. Auriemma
Paper not received
Accertamento della responsabilità medica:
aspetti processuali
A. Didone
Paper not received
126
Sala Botticelli –15.30-17.30
Profili di responsabilità per malpractice sanitaria
Moderatori: Filippo Crivelli (Milano), Fabio Maria Vecchio (Roma)
Politiche per la gestione del rischio in Sanità
A. Ghirardini
Paper not received
Citato in giudizio per una diagnosi errata:
tipologie e caratteristiche dell’errore
dell’anatomopatologo
E. De Dominicis1, S. Fratoni 2, G. Santeusanio 3
Medicina Legale, Dipartimento di Medicina Sperimentale e Chirurgia, Università degli Studi di Roma “Tor Vergata”, Roma; 2 UOC di
Anatomia e Istologia Patologica, Azienda USL Roma C, Roma; 3 Dipartimento di Medicina Sperimentale e Chirurgia, Università degli
Studi di Roma “Tor Vergata”, Roma; UOC di Anatomia e Istologia
Patologica, Azienda USL Roma C, Roma
1
Nel corso dell’ultimo decennio, il tema dell’errore nelle
attività mediche ha avuto un crescente interesse, soprattutto
per quanto riguarda l’approccio che dovrebbe essere seguito
nell’impostare le azioni correttive, in grado di minimizzare
l’impatto del fenomeno.
In questi ultimi anni, si è assistito ad un forte incremento
delle controversie giudiziarie per casi di responsabilità civile
e penale avviati da pazienti che si ritenevano insoddisfatti o
danneggiati dall’operato dei medici, dentro e fuori dal Servizio Sanitario Nazionale.
Con tutta l’attenzione concentrata sul tema dell’errore in medicina vale la pena chiedersi: (i) quanti errori si verifichino in
anatomia patologica, (ii) quali siano le cause, (iii) cosa si intenda per errore diagnostico, (iiii) quali errori possano provocare la
richiesta di risarcimento per danni all’anatomopatologo e (iiiii)
quali possano essere le conseguenze dell’errore diagnostico.
Il tasso di errore in Anatomia Patologica, considerando varie casistiche e metodi di rilievo 1-11, varia da 0,26% a 6,8%
(studi prospettici), da 0,8% a 8,8% (studi retrospettivi), da
1,0% a 45,0% (revisione inter-dipartimentale), da 0,08% a
4,7% (revisioni intra-dipartimentale), da 0,2% a 2,5% (esame
intraoperatorio), da 0,12% a 6,8% (referti corretti, Q-Pobes,
CAP). I tassi di errori clinicamente significativi variano dallo
0,08% (11) al 24% (7).
Secondo Meyer e coll. 12 e Zarbo e coll. 13 le cause d’errore
in Anatomia Patologica sono riconducibili a: errata interpretazione, errata identificazione (paziente, campione biologico,
contenitore, biocassetta/inclusione in paraffina, vetrino istologico/citologico), campione insufficiente e referto incompleto.
Per errore diagnostico in anatomia patologica si intende “una
diagnosi che non rappresenta la vera natura della malattia”
ossia “una diagnosi che viene contraddetta dall’esito clinico”.
L’errore diagnostico in anatomia patologica può essere secondario a errori che si verificano nel servizio clinico o durante
il trasporto, a errori che si verificano nel servizio di anatomia
patologica e a errori che si verificano dopo che il referto sia
stato inviato al clinico richiedente.
Errori che si verificano al di fuori del servizio di anatomia
patologica
Questo tipo di errore causa spesso, con effetto “a cascata”,
errori maggiori nelle fasi successive delle attività del servizio
di anatomia patologica. Si tratta di errori che si verificano nel
servizio clinico. Tali errori comprendono:
la fornitura di un campione inadeguato per la diagnosi;
la fornitura di un campione in un fissativo non idoneo o addirittura senza fissativo o in un contenitore non idoneo;
la etichettatura di un campione in maniera errata o illeggibile
(identificazione inesatta per quanto concerne il paziente, la
sede della lesione o la lateralità);
una richiesta con informazioni cliniche inadeguate, sbagliate
o addirittura assenti.
Si possono verificare anche errori nella fase di trasporto.
Questi errori comprendono la perdita del campione e la distruzione del campione (per congelamento o surriscaldamento
durante il trasporto).
Errori che si verificano nel servizio di anatomia patologica
Si tratta di errori che si verificano nel servizio di Anatomia
Patologica e comprendono:
Errori che si verificano nella fase di ricezione e registrazione del campione biologico comprendono gli errori di
etichettatura che originano dalla non corretta registrazione ed
accettazione del campione biologico da parte del personale
tecnico (assegnazione di un campione al paziente sbagliato,
errata identificazione del sito di origine del campione, errata
identificazione della lateralità del campione, assegnazione del
campione al clinico / reparto sbagliato).
Errori che si verificano nella sala prelievi comprendono
descrizioni macroscopiche e campionamenti incompleti o
inesatti, scambio di campioni, errata etichettatura delle biocassette, contaminazione.
Errori che si verificano nel laboratorio di istologia comprendono errori nella processazione, errori nella fase della inclusione in paraffina, errori nella fase del taglio, errori nella etichettatura dei vetrini, scambio di vetrini quando si allestiscono più campioni contemporaneamente e contaminazione (al
momento della inclusione in paraffina, al momento del taglio
della inclusione e allestimento delle sezioni, al momento della
colorazione). Errori durante l’esame intraoperatorio possono
derivare da errori tecnici di allestimento, taglio e colorazione
del campione e dallo scambio di vetrini o campioni quando si
allestiscono più campioni contemporaneamente.
Errori che si verificano durante la lettura del preparato
istologico al microscopio comprendono scambio di vetrini
(assegnare la diagnosi al paziente sbagliato), errori causati da
problemi cognitivi, referti mal formulati, referti incompleti,
referti incomprensibili e referti con errori tipografici.
Errori che si verificano al di fuori del servizio di anatomia
patologica dopo la refertazione
Dopo la lettura al microscopio i risultai vengono comunicati
al clinico richiedente tramite il referto. In questa fase gli errori
si riferiscono alla consegna del referto al clinico. Tali errori
comprendono la consegna di referti al clinico / reparto / ambulatorio sbagliato, l’inserimento del referto nella cartella clinica
sbagliata, il non inserimento del referto nella cartella clinica e
la non consegna del referto al paziente.
Gli errori in medicina possono essere errori di trattamento
terapeutico od errori diagnostici. In Anatomia Patologica gli
errori sono errori diagnostici, che esitano in eventi avversi
127
RELAZIONI
o per diagnosi sbagliate o per diagnosi differite tardive, con
conseguenti interventi terapeutici sbagliati omissivi, ovvero
con interventi necessari tardivi.
La richiesta di risarcimento per danni all’anatomopatologo può essere avanzata per errore nella formulazione della
diagnosi istopatologica, errore nella formulazione di fattori
prognostici - predittivi e per ritardo o mancata comunicazione
del referto al clinico richiedente.
Le conseguenze di tali errori possono essere varie: (i) ritardo
della diagnosi e conseguente ritardo della terapia, quando una
lesione “neoplastica” viene diagnosticata come “non neoplastica”; (ii) interventi chirurgici non necessari (asportazione di
un organo o parte di organo), trattamenti inutili come chemioterapia e radioterapia quando una lesione “non neoplastica”
viene diagnosticata come “neoplastica”; (iii) controlli bioptici
non necessari (aumentata sorveglianza endoscopica per la
diagnosi di displasia su esofago di Barrett); (iiii) recidiva (es.
un margine di resezione positivo per neoplasia che non è stato
segnalato sul referto istopatologico); (iiiii) perdita della vita.
Zarbo e coll. 13 e Raab e coll. 14 classificano i gradi di severità
dell’errore in base alla ricaduta clinica, come variabili “da
nessun impatto” sulla cura del paziente, a gradi diversi di morbilità, fino a “morbilità di grado elevato” e/o “morte”. I gradi
di severità dell’errore sono stati classificati come:
Nessun danno: Errore che non ha provocato danno al paziente
(ad esempio un errore di tipografia che non ha avuto alcuna
incidenza sulla gestione del paziente)
Danno mancato: Errore scoperto prima che si traducesse in
evento dannoso (ad esempio un errore che è stato rilevato in una
conferenza clinico-patologica prima di una eventuale terapia)
Evento dannoso: Un errore che ha provocato un danno al paziente, stratificato come di seguito:
Grado 1: Errore che ha procurato un maggiore controllo o
procedure bioptiche (ad esempio, aumentata sorveglianza
endoscopica per la diagnosi di displasia su esofago di Barrett);
Grado 2: Errore che ha determinato un’ulteriore procedura
chirurgica invasiva (ad esempio, la resezione di un quadrante
della mammella) o il ritardo nel trattamento per un periodo
inferiore a 6 mesi;
Grado 3: Errore che ha determinato un trattamento inutile
(per esempio, chemioterapia o radioterapia), una procedura
chirurgica definitiva o ritardo nel trattamento per un periodo
superiore a 6 mesi;
Grado 4: Errore che ha determinato perdita della vita
Sulla scorta delle suddette indicazioni e classificazioni, sarebbe auspicabile uno studio / raccolta dati, di tipo retrospettivo e
soprattutto prospettico, da parte dei servizi di AP su territorio
nazionale, mediata e centralizzata da un gruppo di lavoro dedicato SIAPEC, che monitorizzi gli errori in AP, li classifichi,
ne attualizzi i dati, con lo scopo di creare un database regionale di errori e controversie giudiziarie in anatomia patologica, con raccomandazioni, linee guida e procedure al fine di
conoscere e gestire le situazioni che più frequentemente sono
a rischio di errori e di richieste di risarcimento del danno agli
anatomopatologi.
La proposizione conclusiva della pur breve analisi classificatoria è l’auspicio ad una più approfondita ricerca sul tema
attualissimo dell’errore dell’anatomo-patologo potrebbe collegarsi al titolo – provocatorio? – di questo scritto, che intende
prospettare la vastità e la complessità del sistema in cui può
nascere un referto errato, con seguito di citazione in giudizio.
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Profili penali della responsabilità per
malpractice dell’anatomopatologo
V.Cirese
Studio Legale Cirese
I grandi progressi compiuti dalla scienza e dalla professione
medica, soprattutto nella seconda metà del ventesimo secolo,
si sono caratterizzati per la possibilità, enormemente accresciuta, di diagnosticare e curare molte malattie, con complete
guarigioni senza esiti, ovvero con menomazioni residue accettabili. Questi successi sono quasi quotidianamente vantati,
attraverso i mezzi di comunicazione di massa, e questa forma
di “promozione” ha indubbie ricadute positive sul prestigio
della medicina e dei medici, con rilevanti risvolti economici
per un certo numero di essi, per le industrie che producono
farmaci e strumenti, per la sanità privata, etc..
Naturalmente, esistono categorie di malattie a decorso inevitabilmente cronico – come, ad esempio, molte patologie
vascolari, neurologiche, psichiatriche, reumatiche e parte di
quelle oncologiche – nell’ambito delle quali i progressi, e
quindi i successi, sono limitati. Queste malattie fanno ancora
parte delle delusioni accettate dai pazienti e dai loro congiunti, perché non precedute da speranze infondate. È invece sul
versante assai vasto delle malattie, per le quali sono in qualche
misura giustificate delle attese ottimistiche, che la reazione di
rifiuto dell’insuccesso è proporzionale alla gravità dell’evento
negativo, valga per tutti l’esempio del parto.
L’eccessiva e irrealistica esigenza di informazione può trasformare il rapporto medico-paziente in un rapporto contrattuale,
non dissimile da quelli commerciali, con una sorta di “capitolato” sottoscritto dal medico e dal paziente all’inizio della
prestazione sanitaria. Questa esasperazione – che è l’eccesso
128
opposto della frequente, eccessiva reticenza o sbrigatività dei
medici nell’informare il paziente e chiedergli il suo consenso
– è una delle facce della professione che, da parte dell’opinione pubblica e della magistratura, si tende progressivamente a
connotare, per una irrealistica obbligazione di risultato e non
soltanto di mezzi. Il sistema dei DRG non ha certo migliorato
il problema del tempo a disposizione per instaurare un corretto
rapporto medico-paziente. Né si possono nascondere le carenze del servizio sanitario pubblico, in cui il soffocamento della
lottizzazione partitica ha avuto conseguenze negative sia sulle
risorse umane (troppo spesso l’apparato pubblico è stato utilizzato come trampolino per carriere politiche o mediche), non
rispondenti ai canoni della singola preparazione o bravura, sia
sulle strutture realizzate con logiche distanti dall’efficienza e
dal migliora-mento del servizio. Al contrario, il singolo medico responsabile si è trovato (e si trova) stretto nella morsa di
chi, nel caso di palesi carenze di strutture o di disservizi, non
può fare a meno di prestare comunque la propria opera per
non incorrere nel reato di omissione di atti d’ufficio, essendo
pubblico ufficiale o incaricato di pubblico servizio. Tuttavia,
accettando di prestare la propria opera in un contesto carente,
si espone ugualmente a responsabilità, potendo rispondere
quantomeno per colpa (imprudenza), per aver accettato l’eventualità del verificarsi di un rischio.
A ciò si aggiunga che spesso il contenzioso nei confronti
del singolo è strumentale ad ottenere l’intervento in giudizio
dell’ente (quale responsabile civile) da parte del danneggiato
o della pubblica accusa. Esiste poi la possibilità di rivalsa
dell’Azienda, nel caso che il professionista sia stato riconosciuto colpevole con sentenza passata in giudicato per dolo e
colpa grave. È negata invece la possibilità al singolo medico,
che sia stato imputato, di citare nel giudizio penale l’ente, perché risponda di quelle omissioni che, al verificarsi dell’evento
infausto, hanno determinato l’instaurarsi del processo.
Diverso, invece, sarebbe, se l’ente rispondesse per proprio
conto delle proprie carenze, sussistendo una normativa di tutela della sicurezza nella pratica medica relativa alla struttura,
ad esempio, sulla stregua della disciplina antinfortunistica di
matrice comunitaria, adottata per le imprese, disciplina che è
autonoma e concorrente e che anticipa la soglia di punibilità al
verificarsi del rischio, indipendente-mente dall’evento dannoso.
Un certo orientamento giurisprudenziale – rafforzato talvolta
dalla inconsapevole partecipazione emotiva della magistratura
alle aspettative del paziente – inquadrato in un contesto legislativo locale e comunitario inadeguato quando non mancante
e il compattarsi di frange o categorie sociali in organizzazioni
a difesa dei diritti della collettività completano il novero delle
ragioni di un incremento nella conflittualità medico/paziente.
A ben vedere, i magistrati – responsabili delle evoluzioni
giurisprudenziali – costituiscono un’interfaccia solo apparentemente asettica ed equidistante tra i “contendenti”, mentre
in realtà essi, nella inscindibile veste di giudici, ma anche di
membri della società che li chiama a giudicare, non possono
non essere partecipi degli stessi sentimenti che animano la comunità intera nei confronti dei medici e della Medicina. Sono
sentimenti in cui oggi domina l’attesa, spesso esagerata e tramutata in pretesa di risultati favorevoli non sempre possibili.
L’inevitabile, quanto forse inconsapevole, partecipazione dei
magistrati al sentimento collettivo e la loro meditata adesione
all’attuale orientamento dottrinale e giurisprudenziale di massima tutela del paziente, spiega, in buona sostanza, il progressivo abbandono di un orientamento generale più comprensivo
nei confronti dei medici, dovuto forse anche al tradizionale
“rispetto” nei confronti della categoria e anche alla millenaria
accettazione dell’ineluttabilità della malattia e della morte.
Conseguentemente, si è avuto il passaggio progressivo a quella giurisprudenza severa, non di rado eccessiva.
Il fenomeno del maggior rigore degli indirizzi giurisprudenziali non è limitato all’Italia, ma come già evidenziato è comune a Stati altrettanto sviluppati. Ad esempio, in Germania e
Svizzera, sempre più spesso con l’inversione dell’onere della
prova, è il medico a dover dimostrare di non aver causato
danni al paziente o di non essere stato inadempiente nello
svolgimento delle sue funzioni. In queste due nazioni, come
anche in Belgio, Francia, Lussemburgo, Spagna, è in crescita
il numero dei contenziosi (nel Regno Unito è addirittura triplicato negli ultimi cinque anni), così come l’entità media del
risarcimento.
Il quadro appena delineato è quello di un settore vasto, potente, ma assolutamente sfilacciato: un gigante scoordinato,
un agglomerato disorganico e vulnerabile capace soltanto di
difendersi con mezzi spuntati e perversi, come la cosiddetta
“medicina difensiva”. La “medicina difensiva” è da considerarsi un fenomeno sostanzialmente nuovo, direttamente
conseguente all’incremento patologico del contenzioso giudiziario nei confronti dei medici. A prima vista, essa non si
discosta, nei connotati comportamentali che la caratterizzano,
dalla tipica condotta professionale che si avvale di scelte
operative in discreta misura opzionali, cioè di scelte caso per
caso dei mezzi diagnostici e, soprattutto, terapeutici, ritenuti
più opportuni e utili al paziente. La differenza essenziale consiste nei motivi delle scelte che, nella “medicina difensiva”,
non sono dettate dall’interesse primario del paziente, bensì
dall’obiettivo del medico di prevenire denunce giudiziarie. La
“medicina difensiva” è ormai una realtà, un frutto perverso e
preoccupante del contenzioso giudiziario, che assilla la classe
medica e turba la regolare evoluzione della pratica professionale, che ha invece l’obbligo di convogliare gli strumenti
forniti dal progresso scientifico e tecnologico entro i binari dei
propri millenari principi deontologici: i quali pongono al centro dell’opera del medico l’esclusivo interesse del paziente.
L’analisi della mutata realtà socio-sanitaria mostra come
luogo privilegiato per il verificarsi di episodi di “medical
malpractice” una struttura organizzata dove l’attività sanitaria viene tradotta in servizio; tuttavia, raramente, nella
ricostruzione giudiziaria della vicenda, viene preso in esame
un disservizio o una disfunzione organizzativa della struttura
sanitaria, e si continua ad aprire indagini, sempre e comunque
sulla colpa professionale dell’operatore sanitario (o dell’équipe, ossia dei soggetti-persone fisiche che operano nella
struttura), anche in quei casi in cui è palese il disservizio o la
carenza organizzativa.
Invero la struttura a mezzo del legale rappresentante e non
il medico ha l’obbligo di garantire la sicurezza delle cure
predisponendo la dovuta organizzazione di mezzi e di persone, potendosi spesso rinvenire anche collegamenti causali
tra organizzazione deficitaria e danni al paziente per inosservanza degli standard di sicurezza imposti dalla legge (colpa
specifica) o da un generale dovere di diligenza, prudenza,
perizia (colpa generica).
L’amministrazione dell’ente deve predisporre meccanismi di
controllo dei rischi per i pazienti, ciò configurando un preciso obbligo giuridico di attivarsi con la previsione in caso di
violazione, di sanzioni almeno nell’ambito del diritto punitivo
amministrativo. Attualmente manca nel S.S.N. un sistema
concordato e condiviso (nazionale-regionale-locale) di monitoraggio-gestione del rischio clinico e manca la previsione
normativa dell’obbligo in capo alle aziende ospedaliere di istituire unità di gestione del rischio clinico, per individuare i
rischi, eventi avversi e fonti di responsabilità.
RELAZIONI
In caso di carenze strutturali, organizzative, mancato
approntamento del sistema di monitoraggio del rischio
ed eventi avversi, occorre prevedere una responsabilità
autonoma in capo ai legali rappresentanti delle strutture
(o loro preposti), con precisi obblighi e sanzioni.
Solo un sistema sanitario che contempli diversi profili di
responsabilità, che includa un vero e proprio sistema
(nazionale/regionale/locale) di gestione del rischio clinico
e preveda obblighi e sanzioni in caso di inadempimento,
consentirà più ampie garanzie di sicurezza per i pazienti.
Il “sistema” per essere tale, deve raccordare le Unità di rischio clinico a livello aziendale (obbligatorie) con Agenzie
di raccordo a livello regionale e un Osservatorio Nazionale
(presso il Ministero della Salute).
Al monitoraggio dei rischi e degli eventi avversi nelle strutture sanitarie, deve seguire la raccolta e la fruizione dei dati a
livello regionale e nazionale che consentirà di emettere direttive e raccomandazioni per aver modelli più omogenei e più
efficienti di erogazioni delle cure nelle realtà locali, evitando
il più possibile penalizzanti disparità di trattamento dei pazienti, relativamente a livelli di qualità delle cure e sicurezza
delle prestazioni erogate e assicurando la piena collaborazione
con le società scientifiche per emettere raccomandazioni ed
elevare lo standard della prevenzione dei rischi e della corretta
prestazione.
Alla luce delle premesse, che precedono, appare inconfutabile che il problema di fondo, in tema di colpa professionale
sanitaria, consista nel ritrovare una soluzione che contemperi
sia l’esigenza di tutelare adeguatamente il bene della vita
e della salute del paziente, sia quella di assicurare una
valutazione della condotta del medico confacente alla
complessità dell’attività svolta, posto che la giurisprudenza,
originariamente e per un lungo lasso di tempo in posizione di
benevolenza e indulgente considerazione verso i medici, sia
stata progressivamente sostituita da indirizzi severi.
Non vi è dubbio che gli orientamenti dominanti, recentemente, si siano dimostrati più rigorosi anche nella valutazione
della colpa medica in ambito anatomo-patologico, in considerazione del peso giuridico di un errore professionale in un
settore così specializzato e tuttavia così ampio, nonché per
la maggior severità di valutazione dell’errore commesso dallo
specialista (a fronte delle specifiche capacità tecniche che
l’acquisizione del titolo specialistico comporta, legittimanti
maggiori aspettative).
Il primo comma dell’art. 3 della “legge Balduzzi”:
gradualità della colpa e prospettive medico
legali
E. De Dominicis, G. Santeusanio*
Università di Roma “Tor Vergata”, Roma; * Università di Roma “Tor
Vergata”, UOC Anatomia e Istologia Patologica, Azienda USL Roma
C, Roma
In virtù della legge Balduzzi, introdotta con il decreto legge 13
settembre 2012 n. 158 1, convertito, con modificazioni, nella
legge 8 novembre n. 189 2, agli operatori del diritto penale –
giudici, avvocati e medici legali – non resta che dimostrare
che il fatto illecito, causativo di danno alla salute della persona
per malpractice sanitaria, non può non avere che i connotati
della colpa lieve ossia il grado della colpa media; e, quindi,
reclamare l’esenzione della responsabilità penale, ai sensi
dell’articolo 3 del predetto decreto del 2012.
E, tuttavia, la linea di demarcazione tra la colpa normale e la
colpa professionale ovverossia tra la culpa levis e la culpa lata
129
non è facile da ritrovare, siccome tutte le possibili differenziazioni sembrano messe in crisi da due norme del codice civile.
Innanzitutto da quel secondo comma dell’articolo 1176, che
recita: “nell’adempimento delle obbligazioni inerenti l’esercizio di un’attività professionale, la diligenza deve valutarsi con
riguardo alla natura dell’attività esercitata”.
L’altra norma civilistica, che integra la nozione di colpa grave, è offerta dall’articolo 2236, il quale stabilisce che “se la
prestazione implica la soluzione di problemi tecnici di speciale difficoltà, il prestatore d’opera non risponde dei danni, se
non in caso di dolo o colpa grave”.
Lasciamo da parte il dolo, si pone allora il problema di definire la colpa grave e di differenziarla dalla colpa lieve, almeno
in via astratta e generale.
Ed infatti, è proprio il predetto articolo 2236 del codice civile
che, nel fare riferimento esplicito ad attività professionali di
elevata complessità, pone l’esigenza di ricavare la nozione di
colpa grave dalla colpa professionale e di rapportarla alla natura della concreta prestazione ed alla particolare complessità
di essa.
Può dirsi subito che per le prestazioni professionali di carattere tecnico-scientifico, come quelle mediche ai sensi degli
articoli 1176 e 2236 c.c., non sembra esserci molto spazio
per la colpa lieve ma solo per la colpa grave. Una colpa lieve
in se è irrilevante: non solo perché è facilmente coperta da
polizza di assicurazione ma perché, come proclama il decreto
Balduzzi, può costituire una condizione di non punibilità sul
piano penale. È la colpa grave quella che scatena le giuste
preoccupazioni dei medici 3.
Peraltro, è noto agli studiosi della materia che ai sensi dell’articolo 43 del codice penale, l’illecito penale colposo viene
caratterizzato da due proposizioni esplicative della “colpa
generica” e della “colpa specifica”.
Si afferma nella norma che il delitto è colposo quando l’evento, anche se previsto, non è voluto e si verifica a causa di
negligenza, imprudenza ed imperizia (colpa generica) ovvero
per inosservanza di leggi, regolamenti, ordini e discipline
(colpa specifica).
Ed ancorché i reati colposi contro la persona siano molteplici
– fatta ovviamente esclusione per le contravvenzioni ove la
colpa costituisce la regola base – appare opportuno rammentare che il codice penale prevede, poi, nei dettagli le fattispecie
tipiche dell’omicidio colposo (art. 589 c.p.), delle lesioni personali colpose (art. 590 c.p.) cui devono aggiungersi le altre
ipotesi specifiche di lesioni alla persona, disciplinate dall’articolo 582 c.p.; e, specificatamente quelle lievi, se la malattia
ha durata non superiore a 20 giorni e quelle gravissime, se la
malattia ha durata permanente, ai sensi dell’articolo 583 c.p.
Si ricordi che con la legge n. 24 del 1963 venne riformato
l’articolo 582 c.p. e scomparvero le cosiddette lesioni gravi
che erano quelle con prognosi non superiore a 40 giorni.
Orbene, sia nell’omicidio colposo sia nei reati per lesioni
colpose l’evento è disvoluto e si verifica per una delle cause
previste dal suindicato articolo 43 c.p.; nel senso, cioè, che
l’evento lesivo è contro l’intenzione ancorché sia prevedibile
ed, altresì, evitabile (ipotesi di colpa cosciente).
Orbene, se questa breve rassegna introduttiva viene calata
nella problematica della responsabilità medica e si affronta,
altresì, il tema della esenzione della responsabilità penale, in
virtù dell’articolo 3 del decreto Balduzzi, emerge, per altri
profili, un dato di grande importanza scientifica e pratica: la
colpa civilistica attribuibile al medico dagli articoli 1176 e
2236 c.c. è esclusivamente colpa grave; cioè colpa non solo
cosciente, ma colpa in concreto, contraria alle linee guida ed
alle buone regole della professione sanitaria che si ritengo-
130
no esigibili nel caso di una infermità oggetto di trattamento
medico-chirurgico in ambito ospedaliero.
Inserendo il quadrante della responsabilità per colpa grave
dentro il sistema penale emerge che il tasso di colpevolezza
(colpa lieve, grave, cosciente od incosciente) attiene alla misura della punibilità del reo e, quindi, all’applicazione della
pena in relazione al particolare comportamento dell’imputato,
come può dedursi dall’articolo 133 c.p. e dall’articolo 61 n.
3 c.p.
In altri termini, nel processo penale il grado o la gravità della
colpa è funzionale alla erogazione della pena e alla valutazione dell’elemento soggettivo del reato.
Mentre nel diritto civile il medico andrebbe esente da responsabilità se non ha agito con colpa grave, nel diritto penale la
stessa colpa, se lieve, opererebbe come scriminante soggettiva
e, quindi, come causa di esclusione della responsabilità penale.
C’è, pertanto, una asincronia tra sistema civile e sistema penale, ancorché il problema della colposità dell’illecito sanitario
sia unitario.
Tutti gli studi sull’illecito colposo, riguardanti la responsabilità medica, concordano nell’affermare che non esiste un
unico modello di colpa, ma esso va rapportato alla fattispecie
illecita ed al rapporto di causalità tra prestazione medica ed
evento lesivo. La colpa in concreto, richiamando comportamenti negligenti ossia prestazioni connotate da imperizia
od imprudenza è quella esigibile in concreto e riferibile alla
complessità della malattia del paziente e, quindi, a situazioni
extrapenali in se flessibili.
La disposizione affermativa dell’esonero della responsabilità medica in sede penale delimita la scriminante soggettiva
all’ipotesi della colpa lieve, fermo restante il rispetto delle
linee giuda, accreditate dal mondo scientifico ed omologate
presso l’autorità ministeriale; e delle buone pratiche medicochirurgiche generalmente accettate dalla comunità scientifica.
Orbene, la proposizione contenuta nel decreto legge n. 158
del 2012 faceva esplicito riferimento agli articoli 1176 e
2236 c.c., mentre nella legge di conversione il quadro è stato
cambiato.
Secondo alcuni si è trattato di un travisamento del concetto di
colpa medica nella fase di conversione del decreto Balduzzi,
siccome è noto che la gravità della colpa in sede penale incide
sulla misura della sanzione, ai sensi dell’articolo 61 n. 3 c.p. e
dell’articolo 133 c.p. Secondo altri autori si è voluto affidare
al giudice un ambito evolutivo e creativo più ampio ed elastico nell’ambito del cosiddetto diritto vivente.
In passato non sono mancate nella giurisprudenza della Corte
di Cassazione aperture verso soluzioni empiriche ed evolutive, a condizione che fossero comunque rispettate le cosiddette
linee giuda (Cass. N. 4391 del 2011).
Di recente la IV Sezione penale della Corte di Cassazione ha
dichiarato l’abolitio criminis per colpa lieve del medico, ai
sensi dell’articolo 3 della legge Balduzzi n. 189 del 2012, con
motivazioni prudenziali e di notevole spessore creativo.
Ora c’è da chiedersi se queste aperture della giurisprudenza
di legittimità preludano ad un indirizzo consolidato sulla responsabilità medica, in modo da affermare la gradualità della
colpa nelle fattispecie penali, ancorché caratterizzate sul piano
positivo da un unico grado di colpa.
Si tratterà di riconoscere se la colpa professionale possa integrare una fattispecie penale il cui precetto sia in bianco ed
ove l’integrazione offerta dal contributo della medicina legale
possa rappresentare una sorta di colpa “multigraduale”; cioè,
una colpa media od in concreto che sia l’unica esigibile nella
particolare circostanza in cui si è verificata.
Il nodo da sciogliere sta, quindi, nell’offrire una visione dina-
mica della colpa lieve i cui confini con la colpa grave dovrebbero essere individuati in concreto, rappresentando una colpa
medica unitaria concreta e non astratta e, comunque, vicina
alla verità storica dei fatti illeciti posti in contestazione.
Il criterio rilevante sul piano dell’esonero della responsabilità penale per illecito colposo risiede, a nostro avviso, nella
rappresentazione di una colpa unitaria e “multigraduale”, ove
la misura della gravità emerga dall’analisi della fattispecie in
concreto e dal riscontro del comportamento effettivamente
tenuto dal medico.
Ed, invero, se si volesse passare in rassegna la più recente giurisprudenza della Cassazione in tema di responsabilità medica
verrà ad emersione un dato, forse inaspettato, ma assolutamente oggettivo: che è impossibile una definizione teoretica
della colpa specifica del medico. Occorrerà, perciò, partire dal
concetto di colpa media o multigraduale per costruire quella
fattispecie di esonero della responsabilità penale disciplinata
dal decreto Balduzzi.
Del resto, se si legge l’ordinanza della Corte Costituzionale
n. 295 del 2 dicembre 2013, che ha dichiarato la manifesta
inammissibilità della questione di legittimità costituzionale
dell’articolo 3 del decreto legge 13 settembre 2012 n. 158 si
vedrà che la Corte, in qualità di giudice delle leggi e della loro
conformità alla Costituzione, ha respinto la problematica devolutagli dal Tribunale di Milano non per motivi sostanziali,
bensì per questioni formali. Infatti, la Corte ha denunciato il
vizio della mancanza di rilevanza della questione, così come
enunciata dal giudice a quo, reputandola insussistente. Così
facendo la Corte Costituzionale non ha deciso sulla questione
della non manifesta infondatezza e, quindi, sull’eventuale
contrasto dell’articolo 3 della legge Balduzzi con varie norme
e principi costituzionali, tra cui il principio di tassatività della
norma penale, la finalità rieducativa della pena, la violazione
del principio di ragionevolezza e la disparità di trattamento tra
operatori sanitari.
La Corte Costituzionale non ha, cioè, potuto sciogliere la
contraddizione tra l’abolitio criminis per colpa lieve, come
introdotta dall’articolo 3 del decreto Balduzzi, ed il presunto
contrasto con i principi di ragionevolezza e di tassatività della
fattispecie penale, siccome è noto che non esiste una definizione astratta del concetto di colpa grave, così come non esiste
una esposizione teorica della colpa lieve.
Inoltre, il ruolo giocato dalle linee giuda e dalle buone pratiche accreditate dalla comunità scientifica accresce l’imprecisione e l’indeterminatezza perché esse contengono soltanto
regole di perizia, senza fare alcun cenno ai comportamenti che
potrebbero ritenersi, in via astratta, negligenti od infondati 4.
Peraltro, come è stato opportunamente osservato, il decreto
Balduzzi sembra avere spostato l’ambito di analisi dal tema
dell’intensità della colpa lieve o grave, quale misura per graduare la sanzione ex articolo 133 c.p., alla linea di discrimine
tra esistenza o inesistenza del reato colposo.
La colpa penale assumerebbe, quindi, una duplice configurazione: di strumento di valutazione della pena da erogare e,
nei casi di osservanza delle linee guida e delle buone pratiche
accreditate, di causa di non punibilità dell’illecito colposo.
Quindi, è possibile affermare che la disposizione abolitiva
della responsabilità penale ha molto valorizzato la portata e
l’applicazione delle linee giuda accreditate dalla comunità
scientifica.
Ed, invero, prima del decreto Balduzzi la buona pratica clinica non costituiva il valore scriminante che oggi gli viene
riconosciuto.
Secondo la Corte di Cassazione (sezioni penali) la norma civilistica indicata all’articolo 2236 c.c. può, dunque, essere presa
131
RELAZIONI
in considerazione anche nel processo penale quando ricorrono
le condizioni per affermare che il medico si sia trovato ad affrontare prestazioni professionali in condizioni di emergenza
e con problemi di particolare difficoltà tecnica.
Si tratterebbe, dunque, dell’applicazione nel processo penale di una norma extrapenale perché ricorrono le condizioni
dell’integrazione tipiche della cosiddetta norma penale in
bianco (cfr. Corte Costituzionale sentenza n. 166 del 28 novembre 1973; Corte di Cassazione Sez. IV penale n. 39592
del 21 giugno 2007; Cassazione Sez. IV penale n. 16328
del 5 aprile 2011; Cassazione Sez. IV penale n. 4391 del 22
novembre 2011: amplius Cassazione Sez. IV penale n. 16237
del 29 gennaio 2013).
Si può dire che si riscontra nella giurisprudenza penale di
legittimità un importante filone di indirizzo orientato ad applicare nel processo l’articolo 2236 c.c., a condizione che ricorrano, de facto, quelle condizioni di imprevedibilità ed inevitabilità specifiche dell’emergenza medica e che sussistano tutti
gli altri presupposti che tendano a legittimare l’applicazione
della scriminante prevista dal decreto Balduzzi.
Il concetto di colpa lieve va, quindi, calato nel processo e
nei suoi complessi profili scientifici, che solo un’autorevole
consulenza medico legale è in grado di svelare e porre in
evidenza 5.
Proprio la più recente giurisprudenza della Cassazione penale
ha posto in nuova luce le linee giuda accreditate ed omologate,
rapportandole alla teoria dell’esigibilità e della colpa in concreto nel giudizio di responsabilità penale.
Ad esempio, le ragioni dell’urgenza, le situazioni complessive
della struttura ospedaliera, lo sciopero del personale ed altre
concrete condizioni di emergenza potrebbero essere tali da far
emergere la colpa lieve quale discriminante soggettiva negli
illeciti colposi.
Si può aggiungere che l’immotivato distacco dalle linee guida
potrebbe, altresì, determinare l’emersione della colpa grave.
Conclusivamente può dirsi che se il medico ha rispettato con
diligenza e competenza quelle linee può sostenersi che negli
illeciti colposi possa emergere solo la colpa lieve che costituisce, ora, esimente o condizione di non punibilità nel campo
penale.
Bibliografia
1
Gazzetta Ufficiale n. 214 13 settembre 2012.
2
Gazzetta Ufficiale n. 263 10 novembre 2012.
3
Bona M. La responsabilità medica civile e penale dopo il decreto
Balduzzi. Maggioli Editore 2013.
4
Genovese U, Mariotti P, Zoja R. Le linee guida e la responsabilità
sanitaria. Maggioli Editore 2013.
5
Introna F. Metodologia medico legale nella valutazione della responsabilità medica per colpa. Riv It Med Leg 1996; 3: 1295-98.
sia, sia nella formulazione di una proposta per la risoluzione
della stessa”1.
La modifica introdotta dal legislatore nel 2013, con il c.d. decreto del fare (d.l. 69/2013)2, ha però sostituito l’ultimo periodo “sia nella formulazione di una proposta per la risoluzione
della stessa”, con “anche con formulazione di una proposta
per la risoluzione della stessa”, spostando così l’accento dalla
fase facilitativa dei rapporti alla fase valutativa di essi3.
Appare evidente che, oggi, la mediazione è un istituto finalizzato alla composizione di possibili controversie, nel quale è
decisivo il ruolo del terzo compositore4 che deve determinare,
con la sua autorevolezza, l’incontro delle volontà delle parti
litiganti, producendo la sintesi autodeterminata della vertenza
ed evitando che essa divenga una lite giudiziaria5.
Questi, infatti, deve essere inteso come terzo neutrale, che non
porta soluzioni, ma si propone come un elemento in presenza
del quale si modifica una relazione, in quanto lavora per facilitare la comunicazione6.
Il ricorso a tale strumento è stato reso obbligatorio, rappresentando condizione di procedibilità della domanda giudiziale,
nelle materie del condominio, dei diritti reali, delle divisioni,
delle successioni ereditarie, dei patti di famiglia, delle locazioni, del comodato, degli affitti di azienda, del risarcimento
del danno derivante da responsabilità medica e sanitaria, della
diffamazione con il mezzo della stampa o con altro mezzo di
pubblicità, dei contratti assicurativi, bancari e finanziari.
La scelta delle materie è stata ancorata, a mio avviso7 erroneamente, a dati statistici (il numero dei giudizi pendenti),
piuttosto che alla effettiva necessità di un passaggio razionalizzatore; di ciò è prova anche il fatto che non sono state
ricomprese tutte le ipotesi di responsabilità professionale, ma
solo quelle a carattere sanitario8.
A ben vedere, nella prima stesura era ricompresa la sola
responsabilità medica e l’estensione dell’applicazione della
norma anche alle professioni sanitarie si è avuta solo con il
d.l. 69/2013, con il quale si è preso atto dell’evoluzione giurisprudenziale in materia e della sostanziale equiparazione,
sotto il profilo della responsabilità nei confronti del paziente,
fra la prestazione del medico e quella della struttura sanitaria9,
sul presupposto che il rapporto che si instaura fra paziente e
1
2
3
4
L’istituto della mediazione civile nella
responsabilità medica
P. Sandulli
Professore presso l’Università degli studi di Teramo
Posizione del tema
Con il termine mediazione, secondo la definizione fornita
nell’art. 1 dal d.lgs. n. 28/2010, reso in attuazione dell’art. 60
della legge 18.6.2009, n. 69 in materia di mediazione finalizzata alla conciliazione delle controversie civili e commerciali
(pubblicato sulla G.u. n. 53 del 5.3.2010), si intende “l’attività, comunque denominata, svolta da un terzo imparziale e
finalizzata ad assistere due o più soggetti sia nella ricerca di
un accordo amichevole per la composizione di una controver-
5
6
7
8
9
In merito alla normativa dettata con il d.lgs. n. 280/201, vedi P. Sandulli, La mediazione finalizzata alla conciliazione delle controversie
civili e commerciali, in A. Didone (a cura di), Il processo civile competitivo, Milano, 2010, 979 ss.
Resasi necessaria a seguito della decisone della Corte costituzionale n.
272 del 6 dicembre 2012, in La Mediazione, n. 1/2013, 31.
Vedi, in merito, G. Raiti, La media-conciliazione dopo il decreto del
fare, in N. L. C. C. 2014, 245.
C. Punzi, voce Conciliazione e tentativo di conciliazione, in Enc. Dir.,
aggiornamento IV, Milano 2000, 327 ss.
V. P. Calamandrei, Il concetto di lite nel pensiero di F. Carnelutti, in
Opere giuridiche, Napoli 1965, vol. I, 217.
V. Trombini, Responsabilità medica e mediazione nelle controversie,
in Consumatori, Diritti e Mercato n. 11/2011.
P. Sandulli, Le alternative al giudizio e l’economia di mercato, alla
luce della sentenza n. 272, del 2012, della Corte costituzionale, in La
mediazione, 1/2013, 13.
Dai dati in possesso dell’ANIA (associazione nazionale fra le imprese
assicuratrici) nel 2011 risultano inviate circa 31.500 denunce nei confronti di medici e strutture sanitarie, con una media di 85 esposti al
giorno.
Le Sezioni Unite della Suprema Corte di Cassazione, con sentenza 11
gennaio 2008, n. 577 (in Giust. civ. 2009, 11, 2532) hanno ribadito che
“per quanto concerne la responsabilità della struttura sanitaria nei confronti del paziente è irrilevante che si tratti di una casa di cura privata o di
un ospedale pubblico in quanto sostanzialmente equivalenti sono a livello
normativo gli obblighi dei due tipi di strutture verso il fruitore dei servizi.
132
struttura sanitaria deve qualificarsi alla stregua di un contratto
d’opera professionale10.
Le origini della mediazione in campo medico.
Ma le origini del sistema alternativo di risoluzione delle controversie in campo medico risalgono certamente ad un’epoca
molto più lontana: già nel 1946, il d. lgs. n. 233, all’art 3, demandava la funzione di conciliatore all’Ordine professionale,
prescrivendo che: “ Al Consiglio direttivo di ciascun Ordine
e Collegio spettano le seguenti attribuzioni:1) compilare e
tenere l’albo dell’Ordine e del Collegio e pubblicarlo al principio di ogni anno; b) vigilare alla conservazione del decoro
e della indipendenza dell’Ordine e del Collegio; c) designare
i rappresentanti dell’Ordine o Collegio presso commissioni,
enti ed organizzazioni di carattere provinciale o comunale; d)
promuovere e favorire tutte le iniziative intese a facilitare il
progresso culturale degli iscritti; e) dare il proprio concorso
alle autorità locali nello studio e nell’attuazione dei provvedimenti che comunque possono interessare l’Ordine od il
Collegio; f) esercitare il potere disciplinare nei confronti dei
sanitari liberi professionisti inscritti nell’albo, salvo in ogni
caso, le altre disposizioni di ordine disciplinare e punitivo
contenute nelle leggi e nei regolamenti in vigore; g) interporsi, se richiesto, nelle controversie fra sanitario e sanitario, o
fra sanitario e persona o enti a favore dei quali il sanitario
abbia prestato o presti la propria opera professionale, per
ragioni di spese, di onorari e per altre questioni inerenti
all’esercizio professionale, procurando la conciliazione della
vertenza e, in caso di non riuscito accordo, dando il suo parere sulle controversie stesse”.
L’operatività della norma, tuttavia, ha trovato scarsa attuazione, nonostante il crescente numero di azioni giudiziarie intentante nei confronti della classe medica, tanto che negli anni
successivi le singole Regioni si erano attivate per l’istituzione
di appositi sistemi di gestione stragiudiziale del contenzioso.
Tra tutte, si ricorda come nel Veneto, con legge regionale n.
31/2009, era stata istituita una Commissione conciliativa, con
il compito di “comporre in via stragiudiziale le controversie
per danni da responsabilità civile derivanti da prestazioni
sanitarie erogate dalle aziende ULSS ed ospedaliere, nonché
dalle strutture private provvisoriamente accreditate”, con il
compito di decidere “in tutti i casi in cui un paziente o i suoi
aventi causa ritengano che vi sia stato un danno causato da
un errore nella diagnosi o nella terapia ovvero dall’omessa
o irregolare informazione, qualora obbligatoria per legge”.
Sulla legittimità della norma si era pronunciata anche la Corte
costituzionale11, che ne aveva decretato la “correttezza”, sottolineando come fosse finalizzata a ridurre il fenomeno della
medicina difensiva che nuoce alla qualità della prestazione
sanitaria e ponendo luce sul fatto che “… la predisposizione di
servizi volti a facilitare la conciliazione stragiudiziale in materia di danni da responsabilità sanitaria, mediante l’offerta
di un supporto tecnico-giuridico è stata ed è oggetto di altre
leggi regionali: legge della Regione Abruzzo 9 maggio 1990,
n. 65 (Integrazione della legge regionale n. 20 del 2 aprile
1985, recante «Norme di salvaguardia dei diritti dell’utente
dei servizi delle U.L.S.S.»); art. 65-quater della legge della
Regione Lombardia 7 gennaio 1986, n. 1 (Riorganizzazione e
10
11
Secondo la dottrina si riversa in ipotesi di obbligazione plurisoggettiva
“ex latere debitoris” inquadrabile nella categoria delle “obbligazioni
soggettivamente complesse con prestazione indivisibile ad attuazione
congiunta” (in quanto una esecuzione parziaria o non simultanea sarebbe “inutiliter data” non essendo in grado di realizzare l’interesse del
creditore).
Corte cost., 15 maggio 2010, n. 178, in Giust. civ. 2011, 7-8, 1668.
programmazione dei servizi socio-assistenziali della Regione
Lombardia), abrogata dall’art. 28, comma 1, lettera a), della
legge della Regione Lombardia 12 marzo 2008, n. 3 (Governo
della rete degli interventi e dei servizi alla persona in ambito
sociale e sociosanitario); art. 4-bis della legge della Provincia di Bolzano 5 marzo 2001, n. 7 (Riordinamento del Servizio Sanitario provinciale). Anche enti pubblici diversi dalle
Regioni hanno emanato normative in materia: si veda, ad
esempio, l’Ordine provinciale di Roma dei medici chirurghi e
degli odontoiatri, che ha istituito l’ufficio “Accordia”, quale
sportello conciliativo delle controversie derivanti dai rapporti
tra medici e pazienti”.
Con l’intervento del decreto legislativo n. 28/2010, il preventivo ricorso alle forme di ADR (alternative dispute resolution)
è stato esteso a livello nazionale, allo scopo di deflazionare
l’ingente contenzioso nascente intorno alla responsabilità
del medico e della struttura sanitaria, ed ad esso è seguito il
decreto legge n. 158/2012, convertito in legge 189/2012, (c.d.
Decreto Balduzzi), che ha previsto una serie di cautele per
limitare il ricorso ai giudici nei confronti dei medici.
In particolare, agendo sul piano del diritto penale, è stata
introdotta12 una scriminante per i medici che provino di aver
12
D. l.. 158/2012, art. 3: “Responsabilità professionale dell’esercente
le professioni sanitarie: 1. (L’esercente la professione sanitaria che
nello svolgimento della propria attività si attiene a linee guida e buone
pratiche accreditate dalla comunita’ scientifica non risponde penalmente per colpa lieve. In tali casi resta comunque fermo l’obbligo di
cui all’articolo 2043 del codice civile. Il giudice, anche nella determinazione del risarcimento del danno, tiene debitamente conto della
condotta di cui al primo periodo.)
2. Con decreto del Presidente della Repubblica, adottato ai sensi
dell’articolo 17, comma 1, della legge 23 agosto 1988, n. 400, (da
emanare entro il 30 giugno 2013), su proposta del Ministro della
salute, di concerto con i Ministri dello sviluppo economico e dell’economia e delle finanze, sentite l’Associazione nazionale fra le imprese
assicuratrici (ANIA), (la Federazione nazionale degli ordini dei medici
chirurghi e degli odontoiatri, nonché) le Federazioni nazionali degli
ordini e dei collegi delle professioni sanitarie e le organizzazioni
sindacali maggiormente rappresentative delle categorie professionali
interessate, anche in attuazione dell’articolo 3, comma 5, lettera e), del
decreto-legge 13 agosto 2011, n. 138, convertito, con modificazioni,
dalla legge 14 settembre 2011, n. 148, al fine di agevolare l’accesso
alla copertura assicurativa agli esercenti le professioni sanitarie, sono
disciplinati le procedure e i requisiti minimi e uniformi per l’idoneita’
dei relativi contratti, in conformità ai seguenti criteri: a) determinare i
casi nei quali, sulla base di definite categorie di rischio professionale,
prevedere l’obbligo, in capo ad un fondo appositamente costituito, di
garantire idonea copertura assicurativa agli esercenti le professioni
sanitarie. Il fondo viene finanziato dal contributo dei professionisti
che ne facciano espressa richiesta (in misura definita in sede di
contrattazione collettiva,) e da un ulteriore contributo a carico delle
imprese autorizzate all’esercizio dell’assicurazione per danni derivanti
dall’attività medico-professionale, determinato in misura percentuale
ai premi incassati nel precedente esercizio, comunque non superiore al
4 per cento del premio stesso, con provvedimento adottato dal Ministro
dello sviluppo economico, di concerto con il Ministro della salute e il
Ministro dell’economia e delle finanze, sentite (la Federazione nazionale degli ordini dei medici chirurghi e degli odontoiatri, nonché) le
Federazioni nazionali degli ordini e dei collegi delle professioni sanitarie; b) determinare il soggetto gestore del Fondo di cui alla lettera a) e
le sue competenze senza nuovi o maggiori oneri a carico della finanza
pubblica; c) prevedere che i contratti di assicurazione debbano essere
stipulati anche in base a condizioni che dispongano alla scadenza la
variazione in aumento o in diminuzione del premio in relazione al
verificarsi o meno di sinistri e subordinare comunque la disdetta della
polizza alla reiterazione di una condotta colposa da parte del sanitario
(accertata con sentenza definitiva).
3. Il danno biologico conseguente all’attività dell’esercente della
professione sanitaria è risarcito sulla base delle tabelle di cui agli
articoli 138 e 139 del decreto legislativo 7 settembre 2005, n. 209,
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RELAZIONI
arrecato danno al paziente con “colpa lieve”, purchè si siano
attenuti alle linee guida codificate nell’ambito di convegni e
seminari a carattere internazionale. In conseguenza, a seguito
della riforma Balduzzi, i medici possono essere ritenuti penalmente responsabili solo se hanno agito con dolo o colpa grave.
In ottica civilistica poco è cambiato, dato il richiamo all’art.
2043 c.c. e, sul punto, si segnala una delle prime pronunce di
merito13 che ha fatto chiarezza sui presupposti della responsabilità civile del professionista sanitario, dopo la normativa
Balduzzi.
Nella sentenza si legge, infatti, che se “nulla è cambiato con
riferimento alle strutture sanitarie, i cui profili di responsabilità rimangono quelli delineati da SS.UU. 577/2008, l’art.
3 del decreto citato è invece intervenuto sulla responsabilità
dei soggetti esercenti professioni sanitarie, affermando che
costoro, in caso si siano attenuti alle linee guida e alle buone
pratiche accreditate dalla comunità scientifica, non rispondono penalmente per colpa lieve; in tali casi rimane fermo
l’obbligo di cui all’art. 2043 c.c., ma il giudice, anche nella
determinazione del risarcimento del danno, tiene debitamente
conto della condotta di cui al primo comma.”
L’unico obbligo, quindi, espressamente previsto sarebbe quello di risarcire il danno provocato.
Il procedimento di mediazione e i suoi effetti sul successivo
giudizio.
Il ricorso al procedimento di mediazione, nella pratica, riduce
la durata del conflitto14 e, certamente, ne limita i costi, tentando anche un riavvicinamento delle parti: ciò è tanto vero che
se ne prevede una durata massima di tre mesi, con la fissazione di un primo incontro non oltre i 30 giorni dal deposito
della domanda. Al detto incontro le parti potranno stabilire se
continuare o meno nella procedura di mediazione.
Se non vi sono i presupposti per proseguire, la mediazione
sarà chiusa senza costi per i partecipanti (fatta eccezione per
le spese di avvio, pari a 40 euro più IVA).
Pur essendo stata resa obbligatoria l’assistenza di un legale
– per meglio garantire le parti rispetto a comportamenti e
valutazioni che potrebbero compromettere, anche in maniera
rilevante, la loro posizione nel successivo giudizio di merito -,
sono state reintrodotte le norme relative al gratuito patrocinio
13
14
eventualmente integrate con la procedura di cui al comma 1 del predetto articolo 138 e sulla base dei criteri di cui ai citati articoli, per tener
conto delle fattispecie da esse non previste, afferenti all’attività di cui
al presente articolo.
4. Per i contenuti e le procedure inerenti ai contratti assicurativi per i
rischi derivanti dall’esercizio dell’attività professionale resa nell’ambito del Servizio sanitario nazionale o in rapporto di convenzione, il
decreto di cui al comma 2 viene adottato sentita altresi’ la Conferenza
permanente per i rapporti tra lo Stato, le regioni e le province autonome di Trento e di Bolzano. Resta comunque esclusa a carico degli
enti del Servizio sanitario nazionale ogni copertura assicurativa della
responsabilita’ civile ulteriore rispetto a quella prevista, per il relativo
personale, dalla normativa contrattuale vigente.
5. Gli albi dei consulenti tecnici d’ufficio di cui all’articolo 13 del
regio decreto 18 dicembre 1941, n. 1368, recante disposizioni di attuazione del codice di procedura civile, devono essere aggiornati con
cadenza almeno quinquennale, al fine di garantire, oltre a quella medico legale, una idonea e qualificata rappresentanza di esperti delle discipline specialistiche dell’area sanitaria anche con il coinvolgimento
delle societa’ scientifiche, (tra i quali scegliere per la nomina tenendo
conto della disciplina interessata nel procedimento).
6. Dall’applicazione del presente articolo non (devono derivare) nuovi
o maggiori oneri a carico della finanza pubblica.
Trib. Cremona, I sez., 19 settembre 2013.
Cass. civ. sez. un., 22 luglio 2013, n. 17781, in Giust. civ. Mass.,
1/2013, 742.
e stabilito che in caso di mancata presentazione della controparte, senza giustificato motivo, il giudice, nella successiva
causa, potrà condannarla15 al versamento in favore dell’Erario
di una somma di importo corrispondente al contributo unificato, applicando la sanzione prevista all’art. 8, comma 5, del
d.lg. n. 28 del 2010.
Detto comportamento, inoltre, potrà essere valutato come utile
argomento di prova all’interno del processo, in virtù di quanto
disposto dall’art. 116, secondo comma c.p.c., consentendo
così l’ingresso di un’ ulteriore ipotesi di prova atipica nel
processo civile
È stato, poi, introdotto anche un criterio di competenza territoriale che mira ad impedire, per esempio, ad un’impresa, di
chiamare in mediazione un consumatore presso un organismo
la cui sede sia molto lontana dalla sua residenza, per rendergli
difficile o impossibile la partecipazione: oggi, infatti, è competente per territorio solo l’Organismo che si trovi nel luogo
del giudice territorialmente competente per la controversia.
Ove, poi, le parti raggiungano l’accordo, non dovranno più
rivolgersi anche al Tribunale per renderlo esecutivo, ma è sufficiente che i legali ne attestino il rispetto dell’art. 474 c.p.c.,
ovvero che esso non è contrario all’ordine pubblico (cioè non
contiene pattuizioni contrastanti con i principi fondamentali
dell’ordinamento) o a norme imperative.
Sono, inoltre, stati introdotti alcuni benefici fiscali, quali
l’esenzione dall’imposta di registro fino al limite di euro
50.000,00 per i verbali di accordo e il credito di imposta per
le parti che hanno sostenuto le spese di mediazione.
Va, infine, precisato che l’istituto ha carattere sperimentale e
ne è stata prevista una prima durata di soli 4 anni.
Conclusioni
Il limite di durata imposto, appare, tuttavia, uno dei lati più
negativi16 delle recenti modifiche introdotte dal d.l. 69/2013
che, se, da una parte, hanno avuto l’indubbio merito di aver
chiarito l’importanza della mediazione finalizzata alla conciliazione, avendone enfatizzato il ruolo, dall’altra, hanno
rallentato il cammino di consapevolezza circa l’utilità del ricorso a procedure alternative ai giudizi, nell’ottica del bonario
componimento delle vertenze.
Ponendo un limite di durata si è, infatti, rallentato il progredire
della cultura della pacificazione che deve portare a considerare le ADR come utili istituti finalizzati al perseguimento del
diritto vantato e non come agevole rimedio ad un processo
troppo lungo ed articolato, finalizzato esclusivamente a smaltire gli arretrati dei giudici.
Le stesse compagnie di assicurazione, peraltro, dovrebbero
incentivarne il ricorso, sia per il notevole risparmio economico che ne trarrebbero, grazie ai costi di gestione del conflitto
certamente inferiori rispetto a quelli di un processo ordinario,
che per la riduzione dei tempi di definizione della pratica,
con diminuzione del carico di lavoro anche per gli uffici di
liquidazione dei sinistri.
Il Legislatore, tuttavia, non pare aver attentamente valutato
questo aspetto, non avendo fatto menzione del ruolo svolto
dalle compagnie: nel silenzio della norma, dunque, non sembra neppure possibile ipotizzare una estensione dell’istituto
del c.d. “intervento in causa di terzo”, di cui agli artt. 106 e ss.
c.p.c., all’interno del procedimento di mediazione.
15
16
Tra le prime pronunce in materia si segnale Trib. Termini Imerese, 9
maggio 2012.
Per una più approfondita disamina delle modifiche apportate
all’istituto, si veda P. Sandulli, Processo civile. Lavori in corso, Roma,
2013, 289 ss.
134
Al contrario, ad avviso di chi scrive, la presenza – obbligatoria - dell’assicuratore renderebbe certamente più efficace
l’istituto, vieppiù ove fosse reso “certo” anche il pagamento
degli eventuali indennizzi, attraverso l’introduzione di un rigido sistema di vincoli e scadenze tassative. Da ciò potrebbero
trarre giovamento anche gli assicurati, ove fosse ipotizzata
una riduzione dei premi, o, comunque, il mancato aumento, in
caso di esito positivo della mediazione.
A ben vedere, sarebbe opportuno addirittura inserire apposite
clausole di mediazione all’interno dei contratti, vincolando
così le parti, sin dalla sottoscrizione, a ricorrere al detto procedimento prima di procedere a qualsivoglia azione giudiziaria.
L’istituto in esame, infatti, dovrebbe rappresentare un’opportunità per il cittadino e non un mero obbligo procedimentale,
laddove finalizzato ad eliminare le criticità nel rapporto
medico paziente che, spesso, sono l’unica causa dell’ingente
ricorso al contenzioso in materia sanitaria.
È auspicabile, pertanto, che dalla diffusione di questo istituto
possano giovarsi i rapporti medico – paziente che per il futuro
potranno tornare ad essere impostati su di una reciproca fiducia e non su basi di diffidenza.
Certamente, per ottenere ciò è necessario che vi siano organismi di mediazione specializzati in materia17, che possano anche avvalersi di consulenti tecnici nelle ipotesi di malpractice,
al fine di favorire una soluzione più meditata ed equa, nella
ricerca di un giusto procedimento18 di media conciliazione, in
applicazione dei principi garantiti dalla Carta costituzionale.
Ciò perché, si ricorda, lo scopo istituzionale degli organismi
di mediazione dovrebbe essere quello di facilitare, con la
loro autorevolezza, i rapporti tra le parti, predisponendole a
recuperare un clima di ritrovata fiducia, unico nel quale può
crescere la cultura scientifica del Paese.
17
18
Una delle più rilevanti carenze della attuale normativa è
costituita dalla poca attenzione dedicata agli organismi di
mediazione che, invece, dovrebbero essere qualificati in
virtù delle specifiche specializzazioni in quanto non tutti,
in questo settore, possono fare tutto.
F. P. Luiso, Il modello italiano di mediazione. Il “giusto”
procedimento di mediazione (contraddittorio, riservatezza,
difesa, proposta), in Giur. It., Gennaio 2012, 213-217
Sessione patologia testa/collo
Moderatori: Maria Pia Foschini (Bologna), Eugenio Maiorano (Bari)
Hot topics nella diagnostica dei carcinomi naso/
sinusali
A. Franchi
Paper not received
La diagnosi delle lesioni HPV correlate nel
distretto testa/collo
S. Staibano
Paper not received
Utilità della FISH in patologia testa/collo
T. Dorji, V. Grazioli
Centro Diagnostico Italiano, Milano
Squamous cell carcinoma of the head&neck (HNSCC) is the
sixth most common cancer worldwide and represents 90%
of all the oral cavity cancers. This cancer is characterized by
high mortality, high morbidity, and despite advances in surgery and chemotherapy, there has been little improvement in
survival rates over the past 4 decades 1-4. The most important
risk factor for HNSCC are tobacco use, alcohol consumption
and high-risk type of human papilloma virus (HR-HPVs) 5 6.
Patients with oropharynx cancers associate with HR-HPV has
relatively good prognosis 7.
The biological pathway of HNSCC is usually a multistep progression characterized by progressive accumulation of genetic
abnormalities, and mutation of genes on chromosomes induced by geno-toxic agents including aromatic hydrocarbons,
nitrosamines of tobacco smoke and viral particles such as
human papilloma virus 8, may finally lead to various schemes
of chromosomal rearrangements as well as to patterns of unbalanced chromosomal aberrations. Frequent amplifications
on chromosomes 1q, 3q, 8q, 10q, 11q13, 15q and 22q and
deletions on chromosomes 3p, 9p, 13q, 14q, 17p, 18q and 21q
were detected in HNSCC by cytogenetic and comparative
genomic hybridization technique 9-12.
In the era of molecular targeted therapy against the cancer,
hematoxilin&eosin (H&E) and immunohistochemistry (IHC)
remains the cornerstone in the current cancer diagnosis but
molecular genetic testing like PCR, RT-PCR, fluorescence
in situ hybridization (FISH), DNA sequencing, CGH, nucleic
acid microarray and cytogenetic is firmly becoming an imperative component in the daily practice of contemporary
surgical pathologist for better treatment of patients.
Since the introduction of FISH in late 1980s many variations of the procedure have been developed over the time
and today this method has become an important tool to visualize numerical and structural chromosomal aberrations in
interphase nuclei of cytological and histological specimens.
In consideration of the fact that the development and progression of cancer are associated with or driven by chromosomal
abnormalities, FISH has become an important tool for routine
analysis of chromosomal aberrations for diagnosis, prognosis
and predictive purpose.
The combination of molecular cytogenetic methods, including
comparative genomic hybridization, spectral karyotyping and
fluorescence in situ hybridization has allowed detailed characterization and quantification of 3q26 (harbouring hTREC
gene) in squamous cell carcinoma of mucosal origin at different anatomic sites like cervix, lung, head and neck and
oesophagus 13-15.
135
RELAZIONI
FISH analysis employing 3q26 probe in precursor lesions
and invasive squamous carcinoma of the uterine cervix and
larynx showed that the gain of hTERC signals increased with
increasing severity of dysplasia 16 17. This could represent the
activation of telomerase which can lead to cellular immortalization, prevent from apoptosis and potentially promote
tumorogenesis, and finally encodes the RNA template for
adding the repeat sequence of telomeres to the end of DNA.
We evaluated the gain of 3q26 applying a dual-colour fluorescence in situ hybridization in precursor lesions of the oral
cavity in order to assess the utility of 3q26 gain in the clinical practice that would anticipate the diagnosis and help the
clinician to treat the patient with minimally invasive surgery.
30 patients (16 female and 14 males with age range 60-63
years) with oral precursor lesions were enrolled for this study
and long-term follow-up information were available (ranging
from 5 to 10 years). Recently modified Ljubljana classification for grading the squamous intraepithelial lesions of the
larynx was adopted for grading the precursor lesions 18.
For Fluorescence in situ hybridization, slides were prepared
according to our standard laboratory FISH protocol and were
analysed using the Ikonoscope®, an automated system developed specifically for counting the probe signals. A cell with
three or more 3q26 signal was considered abnormal regardless
of the signal numbers of CEP 7 (used as a control probe). For
positive results with gain of hTERC gene, the percentage of
nuclei with combination of the entire abnormal signal pattern
should be more than the cut-off value. Cut-off vale as 7,28%
was adopted as described by Liu Y et al. 17 19 20 21.
A gain of 3q26 was observed in 10 cases, where the histological
diagnosis was HG-SIL (five cases), CIS (one case) and LG-SIL
(four cases). Subsequently, eight patients developed an invasive
OSCC, one LG-SIL progressed to HG-SIL and only one case
showed no evidence of disease after 167 months.
FISH analysis in the remaining 20 cases did not show gain of
3q26. Histological diagnosis was HG-SIL in 3 cases and LGSIL in 17 cases. Only one case developed an OSCC after 16
months of follow-up period and the remaining 19 showed no
evidence of disease progression after a mean follow-up period
of 90 months.
Therefore, gain of 3q26 showed a sensitivity of 90% while the
specificity reached 95%.
Despite the limited number of cases enrolled in our study,
our data emphasizes the desirability and the concrete possibility to evaluate the gain of 3q26 as an early diagnostic
and perhaps predictive marker in precursor lesions of the oral
cavity. A good correlation that we obtained in the transition
from SILs to OSCC suggests that we might be able to stratify
the precursor lesions of the oral cavity into high risk and low
risk categories. Analysis of 3q26 in conjunction with other
cytogenetic markers like EGFR and HER2 would provide
the clinicians with better armamentarium for the treatment of
cancer patient in the era of targeted molecular therapy. After
more than a century, the dream of Paul Ehrlich’s “magic
bullet” against the cancer in the form of targeted molecular
therapy becomes true.
References
1
Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002. CA
Cancer J Clin 2005;55:74-108. 2
Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence,
mortality, and prevalence across five continents: defining priorities to
reduce cancer disparities in different geographic regions of the world.
J Clin Oncol 2006;2414:2137-50. 3
Boyle P, Levin B, editors. World Cancer Report 2008. WHO Press;
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Feller L, Lemmer J. Oral Squamous Cell Carcinoma: Epidemiology,
Clinical Presentation and Treatment. Journal of Cancer Therapy
2012;3,:263-8.
Hashibe M, Brennan P, Benhamou S, et al. Alcohol drinking in
never users of tobacco, cigarette smoking in never drinkers, and the
risk of head and neck cancer: pooled analysis in the International
Head and Neck Cancer Epidemiology Consortium. J Natl Cancer
Inst 2007;99:777–89.
Kreimer AR, Clifford GM, Boyle P, et al. Human papillomavirus types
in head and neck squamous cell carcinomas worldwide: a systematic
review. Cancer Epidemiol Biomarkers Pre 2005;14:467-75.
Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 2010;363:2435.
Petti S. Lifestyle Risk Factors for Oral Cancer. Oral Oncology
2009;45:340-50.
Carey TE, Van Dyke DL, Worsham MJ. Nonrandom chromosome aberrations and clonal populations in head and neck cancer. Anticancer
Res 1993;13:2561-8.
Van Dyke DL, Worsham MJ, Beninger MS, et al. Recurrent cytogenetic abnormalities in squamous cell carcinoma of the head and neck
region. Genes Chromosomes Cancer 1994;9:192-206.
Sidransky D. Molecular markers in cancer: can we make better predictions? Int J Cancer 1995;64:1-2.
Bockmuhl U, Schwendel A, Dietel M, et al. Distinct patterns of chromosomal alterations in high- and low-grade head and neck squamous
cell carcinomas. Cancer Res 1996;56:5325-9.
Kokalj Vokač N, Cizmarević B, Zagorac A, et al. An evaluation
of SOX2 and hTERC gene amplifications as screening markers in
oral and oropharyngeal squamous cell carcinomas. Mol Cytogenet
2014;7:5.
Singh B, Gogineni SK, Sacks PG, et al. Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement
of 3q amplification.Cancer Res 2001;61:4506-13.
Heselmeyer K, Macville M, Schrock E, et al. Advanced-staged cervical carcinomas are defined by a recurrent pattern of chromosomal
aberrations revealing high genetic instability and a consistent gain of
chromosome arm 3q. Genes Chromosomes Cancer 1997;19:233-40.
Heselmeyer K, Schrock E, du Manoir S, et al. Gain of chromosome 3q
defines the transition from severe dysplasia to invasive carcinoma of
the uterine cervix. Proc Natl Acad Sci USA 1996;93:479-84.
Liu Y, Dong XL, Tian C, et al. Human telomerase RNA component
(hTERC) gene amplification detected by FISH in precancerous lesions
and carcinoma of the larynx. Diagn Pathol 2012;7:34.
Gale N, Blagus R, El-Mofty SK, et al. Evaluation of a new grading
system for laryngeal squamous intraepithelial lesions-a proposed unified classification. Histopathology 2014.
Tu Z, Zhang A, Wu R, et al. Genomic amplification of the human telomerase RNA gene for differential diagnosis of cervical disorders. Cancer Genet Cytogenet 2009;191:10-6. Jiang J, Wei LH, Li YL, et al. Detection of TERC amplification in
cervical epithelial cells for the diagnosis of high-grade cervical lesions and invasive cancer: a multicenter study in China. J Mol Diagn
2010;12:808-17. Xiang L, Yang H, Li J, et al. Different amplification patterns of
the human telomerase RNA gene in invasive cervical carcinomas
and cervical intraepithelial neoplasia grade III. Diagn Cytopathol
2012;40:849-55. Correspondingly,6.4% was established as the standard cut-off
value for interpreting the testing results from all study centers.
Tumori delle ghiandole salivari analoghi
alla mammella
M.P. Foschini
Dipartimento di Scienze Biomediche e Neuromotorie, Università di
Bologna, Anatomia Patologica “M. Malpighi”, Ospedale Bellaria,
Bologna
Ghiandole salivari e mammella sono ghiandole esocrine
con analoga struttura e possono essere sede di neoplasie simili. Sono noti i carcinomi della mammella che hanno aspetto
136
analogo alle ghiandole salivari, quali il carcinoma adenoidocistico, il carcinoma acinico e altri.
Analogamente nelle ghiandole salivari possiamo avere carcinomi che più frequentemente vediamo nella mammella.
La forma più nota è il carcinoma duttale. Si tratta di un
carcinoma che presenta aspetto sovrapponibile al carcinoma
duttale della mammella. Può presentare una forma in situ ed
una forma infiltrante e può presentare vario grado di aggressività anche se, nelle ghiandole salivari prevalgono le forme
ad alto grado.
Il carcinoma duttale colpisce più frequentemente soggetti di
età superiore ai 50 anni, di sesso maschile. La parotide è la
sede più colpita, anche se lo troviamo anche a carico della
sottomandibolare e delle ghiandole salivari minori intraorali.
A livello istologico si riconoscono una componente intraduttale ed una componente invasiva. Applicare i marcatori di
mioepitelio e di cellule basali è utile per differenziare le due
componenti.
Il carcinoma duttale delle ghiandole salivari può presentare
vari aspetti istologici, come la forma sarcomatoide, la forma
micropapillare e mucoide.
A livello immunoistochimico il carcinoma duttale delle ghiandole salivari esprime marcatori di epitelio, quali citocheratine
a basso peso molecolare. Inoltre risulta spesso positivo per
recettore per Androgeno e per HER2.
Recentemente è stato descritto il carcinoma secretorio delle
ghiandole salivari, caratterizzato morfologicamente da aspetto
analogo alla forma mammaria. All’esame immunoistochimico è positivo per proteina S-100, mammaglobina, e vi-
mentina. Inoltre presenta una traslocazione (12;15) (p13;q25)
che determina un gene di fusione ETV6-NTRK3. Le stesse
modificazioni genetiche si riscontrano nella forma mammaria.
L’aspetto morfologico del carcinoma secretorio delle ghiandole salivari è in genere blando, e il comportamento clinico a
bassa aggressività. Recentemente sono stati descritti tre casi
di carcinoma secretorio delle ghiandole salivari con aspetti di
trasformazione in alto grado.
Bibliografia
Connor A, Perez-Ordoñez B, Shago M, et al. Mammary analog secretory
carcinoma of salivary gland origin with the ETV6 gene rearrangement
by FISH: expanded morphologic and immunohistochemical spectrum
of a recently described entity. Am J Surg Pathol 2012;36:27-34.
Simpson RH. Salivary duct carcinoma: new developments-morphological
variants including pure in situ high grade lesions; proposed molecular
classification. Head Neck Pathol 2013;7(Suppl 1):S48-58.
Simpson RH, Prasad AR, Lewis JE, et al. Mucin-rich variant of salivary
duct carcinoma: a clinicopathologic and immunohistochemical study
of four cases. Am J Surg Pathol 2003;27:1070-9.
Skalova A. Mammary analogue secretory carcinoma of salivary gland
origin: an update and expanded morphologic and immunohistochemical spectrum of recently described entity. Head Neck Pathol
2013;7(Suppl 1):S30-6.
Skálová A, Vanecek T, Sima R, et al. Mammary analogue secretory carcinoma of salivary glands, containing the ETV6-NTRK3 fusion gene:
a hitherto undescribed salivary gland tumor entity. Am J Surg Pathol
2010;34:599-608.
Skálová A, Vanecek T, Majewska H, et al. Mammary analogue secretory
carcinoma of salivary glands with high-grade transformation: report
of 3 cases with the ETV6-NTRK3 gene fusion and analysis of TP53,
β-catenin, EGFR, and CCND1 genes. Am J Surg Pathol 2014;38:23-33.
Neuropatologia: Istopatologia dell’epilessia non neoplastica
Moderatori: Anna Maria Buccoliero (Firenze), Felice Giangaspero (Roma)
Le aspettative del clinico
F. Giordano, R. Guerrini
Paper not received
Istopatologia delle displasie focali corticali
E. Aronica
Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, The Netherlands
Le displasie corticali focali (FCD) rappresentano malformazioni cerebrali focali frequentemente diagnosticate o sospettate in pazienti sottoposti a trattamento chirurgico per
epilessia intrattabile. La disponibilità di campioni cerebrali
clinicamente ben caratterizzati ha fornito un’opportunità
unica per definire meglio le caratteristiche neuropatologiche, neurochimiche e molecolari delle FCD. Dopo la prima
descrizione delle caratteristiche neuropatologiche delle FCD
fornita da Taylor nel 1971, sono stati proposti diversi sistemi
di classificazione per le FCD. Tuttavia, dal momento che lo
spettro morfologico delle FCD è ampio, lo sviluppo di una
classificazione unitaria e completa rappresenta una grande
sfida e richiede aggiornamenti continui. Una task force della
Commissione per i Metodi Diagnostici della Lega Internazionale contro l’Epilessia (ILAE) ha recentemente generato una
nuova classificazione dei diversi sottotipi di FCD sulla base
delle caratteristiche istopatologiche. La classificazione della
ILAE rappresenta la base per nuovi studi clinici per definire
meglio le caratteristiche cliniche, elettrofisiologiche e neuroradiologiche specifiche, ma può anche guidare studi futuri
che dovranno contribuire a sviluppare nuove teorie relative
alla patogenesi molecolare e epilettogenicità delle diverse
varianti di FCD. I meccanismi cellulari e molecolari alla base
delle FCD sono ancora poco chiari e modelli animali che
riproducono con precisione le caratteristiche istopatologiche
specifiche delle varianti di FCD non sono ancora disponibili.
Tuttavia, recentemente stanno emergendo nuove ipotesi sulla
patogenesi molecolare della FCD tipo II.
I recenti progressi nella classificazione neuropatologica delle
FCD saranno presentati e le evidenze neuropatologiche e di
ricerca di base saranno discusse, evidenziando il coinvolgimento di meccanismi patogenetici convergenti, che possono
creare la base per nuove strategie terapeutiche in pazienti
con FCD o altre malformazioni cerebrali patogeneticamente
correlate.
News dal WHO
F. Giangaspero
Paper not received
137
RELAZIONI
Venerdì, 24 Ottobre 2014
Controlli di qualità
Analisi del controllo di qualità di RAS
e prospettive future
N. Normanno
Cell Biology and Biotherapy Unit, Istituto Nazionale per lo Studio e
la Cura dei Tumori “Fondazione Giovanni Pascale” - IRCCS, Naples,
Italy
L’analisi delle mutazioni degli esoni 2, 3 e 4 dei geni KRAS
ed NRAS è indispensabile per una corretta programmazione
terapeutica nei pazienti con carcinoma del colon retto metastatico. Per assicurare una adeguata qualità delle analisi
di patologia molecolare sul territorio nazionale, la Società
Italiana di Anatomia Patologica e Citopatologia (SIAPEC) e
l’Associazione Italiana di Oncologia Medica (AIOM) hanno
organizzato un programma di controllo di qualità per l’analisi
mutazionale dei geni RAS e del gene BRAF, che riveste un
importante ruolo prognostico nel carcinoma del colon retto.
Il programma ha visto la partecipazione di 88 centri, di cui
4 da altre nazioni. Ai centri partecipanti sono stati inviati 10
campioni da analizzare in un periodo massimo di tre settimane
con le metodiche impiegate nella routine clinica. I laboratori
che non hanno superato il primo round hanno avuto comunque
la possibilità di partecipare ad un secondo round. La metodica
più utilizzata dai centri partecipanti è stato il pirosequenzia-
mento, che per la prima volta nel contesto dei programmi di
controllo di qualità organizzati da AIOM e SIAPEC risulta
essere più frequente del sequenziamento di Sanger. I risultati
del I round del controllo di qualità sono stati molto preoccupanti: 9 laboratori (10,2%) non hanno sottomesso i risultati
nel periodo consentito e, soprattutto, 23 centri (26,2%) hanno
commesso un errore grave di genotipizzazione (falso positivo
o falso negativo), determinando pertanto un tasso di insuccesso del primo round del 36,4%. Molti dei laboratori che hanno
fallito il primo round hanno comunque superato il secondo
round che è stato condotto con metodologia identica al primo
round. Infatti, al termine del secondo round, solo 9/88 centri
non hanno superato il controllo di qualità (10,25). I risultati
estremamente negativi del primo round richiedono tuttavia
una profonda riflessione e l’attivazione di una strategia che
consenta un miglioramento della attuale organizzazione, attraverso la costruzione di reti, percorsi e modelli organizzativi
condivisi su base nazionale nonché di controlli di outcome da
affiancare ai programmi di controllo di qualità.
Focus sul carcinoma colon-rettale metastatico
D. Santini
Paper not received
Patologia Molecolare
Moderatori: Massimo Barberis (Milano), Francesca Castiglione (Firenze)
Il ruolo di EGFR nel CA Polmonare
Citologia e Biomarcatori
A. Marchetti
Paper not received
G.C. Troncone
Paper not received
ALK: Immunoistochimica o Biologia Molecolare?
Nuove Metodologie
G. Fontanini
Paper not received
F. Buttitta
Paper not received
138
Patologia Molecolare
Moderatori: Lorenzo Antonuzzo (Firenze), Giancarlo Troncone (Napoli)
Ruolo di RAS nel carcinoma colon-rettale
L. Antonuzzo
Paper not received
Gist: future prospettive diagnostiche
A. Scarpa
Paper not received
BRAF nel melanoma: come e quando
M. Barberis
Paper not received
Il sistema RAS nel carcinoma colon rettale
G. Giuffrè
Department of Human Pathology “Gaetano Barresi”, University of
Messina
Colorectal cancer (CRC) is the third most frequently diagnosed type of cancer and approximately 40% of CRC patients
develop metastatic cancer. In this advanced stage of disease,
surgery is often not practicable and chemotherapy is usually
suggested for the treatment of metastatic CRC (mCRC) patients. The recent introduction of monoclonal antibody drugs
targeting epidermal growth factor receptors (EGFR), such as
cetuximab and panitumumab, in combination to chemotherapy
regimens has widened treatment options in mCRC. However,
not all patients can benefit from these agents; in fact, patients
whose tumours carry an activating mutations in exon 2 of the
gene KRAS are much less likely to respond to antibodies that
block EGFR signalling. These findings led to a requirement
for KRAS mutational testing of mCRC and restrict the use of
anti-EGFR antibodies to patients with a wild-type (WT) KRAS
exon 2 status. More recently, prospective-retrospective studies underlined that additional mutations in RAS family genes,
namely in exons 2, 3 and 4 of KRAS and NRAS, predicted a
lack of response to anti-EGFR therapy. Based on these data,
regulatory authorities in North America and Europe updated
their guidelines and in particular the EMEA requires testing
of exons 2, 3 and 4 of KRAS and NRAS for use of cetuximab
and panitumumab.
In RAS mutation testing the pathologist plays a central role; in
fact, he is responsible to select a sufficient quantity of invasive
tumour cells needed for analysis from different histopathological samples. Either biopsy or resection specimen tissue can
be used for RAS testing though use of resection tissue is preferable. Macrodissection or microdissection techniques should
be utilized to enrich the sample of tumour cells, especially
in case with relevant intra-tumour heterogeneity. However,
the amount of tumour area required for testing depend on the
RAS assay utilized; in any case, the minimum neoplastic cell
content tested should be at least two times the assay’s limit of
detection. A variety of assays are available for RAS mutation
testing, with different performances regarding sensitivity and
specificity. These techniques fall into two main categories,
screening technologies that are able to detect all RAS mutations, and targeted technologies that detect only specific RAS
mutations with a higher sensitivity. In the former group are
comprised direct sequencing, pyrosequencing, high resolution
melting analysis, while in the latter one allele-specific Real
Time PCR and Strip Assay. However RAS analysis should
include at least KRAS codons 12, 13, 59, 61, 117 and 146 and
NRAS codons 12, 13, 59 and 61 in which harbour the most
frequent somatic mutation. In the future, the comprehensive
integrated analysis of whole EGFR signalling pathways will
enable us to identify most of the mCRC patients who are
unlikely to respond to anti-EGFR therapies. Prospectically
the use of emerging technologies, such as next-generation sequencing allowing to screen simultaneously multiple somatic
mutations in multiple genes in a single test run, might represent an optimal solution for rapid selection of mCRC patient
to address to anti-EGFR therapy.
Ruolo dei Mirna: prospettive future
S. Bosari, V. Vaira
Università degli Studi di Milano, Dipartimento di Fisiopatologia
medico-chirurgica e dei Trapianti, Fondazione IRCCS Ca’ Granda,
Ospedale Maggiore Policlinico
Gli RNA non codificanti (ncRNA) sono una classe di molecole funzionalmente attive. Con la loro scoperta avvenuta nel
1993 1 si è dimostrato che un filamento di RNA non codificante per alcuna proteina può svolgere una funzione biologica
rilevante. All’interno di questa famiglia vi sono i microRNA
(miRNA), molecole di RNA a singolo filamento lunghe 1822 paia di basi. La loro funzione è di inibire la traduzione
degli RNA messaggeri (mRNA) a proteina attraverso un appaiamento non perfetto nella regione regolatoria del mRNA
denominata 3’UTR. Un singolo miRNA puo’ avere molteplici
mRNA targets, regolando interi processi biologici. Cosi come
i geni codificanti, i miRNA sono deregolati nelle patologie
umane inclusi i tumori 2. I miRNA possono essere iper- o ipo
- espressi, amplificati, deleti, o soggetti a regolazione epigenetica, e rappresentano una nuova classe di molecole “patologiche” e target terapeutici. Inoltre i miRNA sono molecole
conservate nell’evoluzione e tra specie diverse, permettendo
il loro studio in modelli animali di malattia.
Nel campo della Patologia Molecolare e dell’oncologia i
miRNA hanno assunto notevole importanza. Grazie alle loro
dimensioni ridotte ed alla incorporazione in microvescicole,
i miRNA sono stabili e più facilmente riscontrabili in tessuti
di archivio fissati in formalina o in fluidi corporei (miRNA
circolanti), rispetto agli RNA messaggeri. Questa caratteristica
fa sì che i miRNA possano diventare biomarcatori di patologie,
di prognosi o predittivi di risposta a trattamenti farmacologici.
Una recente meta-analisi di quarantatré studi di espressione
di miRNA in 20 tipologie diverse di neoplasie ha evidenziato
come aumentati livelli di miR-21 o ridotta espressione di let7, siano marcatori comuni di patologia neoplastica e di prognosi infausta. Nonostante i miRNA non siano ancora entrati
nella pratica clinica, molti studi hanno identificato dei miRNA
139
RELAZIONI
associati alla risposta a terapie. Ad esempio in pazienti affetti
da leucemia mieloide cronica e portatori del riarrangiamento
BCR-ABL i livelli del miR-451 sono inversamente correlati
all’espressione della proteina chimera 3. In pazienti affetti da
epatocarcinoma in stadio avanzato, elevati livelli epatici del
miR-425-3p predicono la risposta al trattamento con Sorafenib in termini di maggiore tempo alla progressione e maggiore
tempo di sopravvivenza senza progressione di malattia. La
valutazione differenziale dei livelli di espressione del miRNA
nel tessuto cirrotico rispetto a quello tumorale del paziente
può identificare i pazienti maggiormente responsivi al trattamento. Infine, anche i polimorfismi nelle regioni 3’UTR
dei target dei miRNA possono essere predittori di risposta a
farmaci. Il polimorfismo di un singolo nucleotide nel 3’UTR
di KRAS (LCS6) soggetto a regolazione da parte di let-7,
influenza la risposta a terapia anti-EGFR in pazienti affetti da
carcinoma colo-rettale metastatico 4.
Grazie alla presenza e stabilità dei miRNA nel siero o nel
plasma, pannelli di espressione dei miRNA circolanti sono
emersi come utili biomarcatori non invasivi di malattia 5. La
diagnosi precoce è sempre più importante, specialmente per
le patologie neoplastiche, dove l’intervento chirurgico è una
valida opzione terapeutica negli stadi non avanzati di malattia.
In questo contesto, si è evidenziato che la presenza a livello
serico di due microNA (miR-320 and miR-574-3p) ed un
ncRNA, l’RNU6-1, rappresenta un indicatore diagnostico di
glioblastoma 6. Nella patologia toracica, uno studio condotto
in soggetti sottoposti a screening per carcinoma del polmone
non a piccole cellule mediante tomografia computerizzata, ha
validato una signature di 24 miRNA circolanti come marcatore diagnostico accurato e specifico di malattia (87% e 81%,
rispettivamente). I pazienti individuati come ad alto rischio
dalla signature molecolare hanno mostrato anche una sopravvivenza ridotta, confermando il valore di questi biomarcatori
anche a livello prognostico oltre che diagnostico 7.
Recentemente, la somministrazione di molecole di miR-34a
sintetiche (MIRX34) coniugate a nanoparticelle liposomiali
è entrata in protocolli sperimentali terapeutici per pazienti
affetti da epatocarcinoma o da metastasi epatiche con diversa
primitività (Fase 1, trial NCT01829971). Il miR-34a non è
però stato il primo miRNA ad essere utilizzato come farmaco.
Il più avanzato trial clinico è rappresentato dalla sperimentazione sulla somministrazione di un antagonista del miR-122
(Miravirsen) per la terapia dell’epatite virale C 8.
I microRNA offrono quindi un’attraente alternativa ai marcatori correntemente utilizzati in clinica per la diagnosi precoce
di tumori, diagnosi differenziale, stadiazione prognostica
e scelta del regime terapeutico. La standardizzazione delle
procedure per una loro quantificazione accurata e univoca
consentirà la loro validazione ed il loro utilizzo in campo
clinico-assistenziale.
Bibliografia
1
Lee et al., Cell;75:843.
2
Hayes J et al., Trends in Mol Med 2014;20:460.
3
Scholl V et al., Leukemia Res 2012;36:119.
4
Chin LJ et al., Cancer Res 2008;68:8535.
5
Mitchell PS et al., PNAS 2008;105:10513.
6
Manterola et al, Neuro Oncol 2014;16:520.
7
Sozzi G et al., JCO 2014.
8
Janssen HLA et al NEJM 2013; 368.
Neoplasie mammarie e FISH
V. Vezzosi
Paper not received
Cellule tumorali circolanti prospettive future
M. Pazzagli, F. Salvianti e P. Pinzani
Dipartimento di Scienze Biomediche, Sperimentali e Cliniche “Mario
Serio”, Università di Firenze, Italia
È ben noto che cellule tumorali circolanti (CTC) sono presenti
nel sangue di pazienti affetti da tumori solidi, anche se il loro
significato biologico e la loro importanza da un punto di vista
clinico sono ancora argomento di dibattito 1.
Contrariamente a quanto si potrebbe pensare la disseminazione di cellule da parte della massa tumorale può essere anche
un evento precoce nella progressione neoplastica 2.
La presenza in circolo delle CTC è una condizione necessaria, anche se non sufficiente allo sviluppo di una metastasi,
dal momento che una parte di esse va incontro ad apoptosi;
inoltre il potenziale metastatico di una cellula è determinato
da un elevato numero di variabili che rendono il processo di
metastatizzazione estremamente complesso 1.
La presenza di cellule tumorali circolanti sembra avere un
significato prognostico in pazienti affetti da stadi avanzati
della patologia, come attestano studi che associano il numero
di CTC con la sopravvivenza e la progressione della malattia
nel cancro della mammella 3, del colon 4, e della prostata 5.
L’identificazione delle CTC in stadi precoci della malattia potrebbe permettere di stimare il rischio di sviluppare metastasi
e sembrano esserci risultati incoraggianti in tal senso, anche
se sono necessari studi su scala più ampia affinché questo
parametro possa contribuire in futuro alla gestione clinica dei
pazienti 6.
Una delle applicazioni più interessanti dello studio delle CTC
riguarda la possibilità di un loro impiego come “biopsia liquida” in quanto esse rappresentano un “surrogato” del tessuto
tumorale, che può essere ottenuto in maniera non invasiva,
ed il loro conteggio può essere usato a fini diagnostici, prognostici e di monitoraggio della malattia. Studi recenti hanno
mostrato che le CTC possono avere un genotipo diverso
dal tumore primario e possono essere caratterizzate da una
notevole eterogeneità intra ed inter individuale. Gli studi più
recenti confermano la necessità di una caratterizzazione molecolare delle CTC , anche a livello della singola cellula, al
fine di identificare marcatori specifici nei confronti dei quali
esistano terapie mirate.
Bibliografia
1
Mocellin et al., 2006.
2
Pantel et al., 2008.
3
Cristofanilli et al., 2004.
4
Cohen et al., 2004.
5
de Bono et al., 2008.
6
Pantel e Alix-Panabières, 2010.
140
Sala Michelangelo – 09.30-11.30
Sessione di ematopatologia
Moderatori: Simonetta Di Lollo (Firenze), Vito Franco (Palermo)
Diagnostica dei linfomi non-Hodgkin nel midollo
osseo
E. Sabattini
Paper not received
Aspetti diagnostici e biologici dei linfomi
primitivi del sistema nervoso centrale
M. Ponzoni
Paper not received
Is Burkitt lymphoma a polymicrobial disease?
Evidence from RNA Sequencing
M.R. Ambrosio1, F. Abate2, G. De Falco1, P.P. Piccaluga3, S.
Lazzi4, L. Mundo1, B.J. Rocca1, S. Gazaneo1, C. Bellan1, R.
Rabadan2, S.A. Pileri3, L. Leoncini1
Department of Medical Biotechnologies, University of Siena, Italy;
Department of Biomedical Informatics, Center for Computational
Biology and Bioinformatics Columbia University, New York, USA; 3
Department of Specialistic, Diagnostic and Experimental Medicine,
University of Bologna, Italy; 4 Section of Pathology, Azienda Ospedaliera Universitaria Senese, Italy
1
2
Introduction
Recent data provide insights into the emerging concepts
of polymicrobial disease pathogenesis for endemic Burkitt
lymphoma (eBL). In particular, the potential mechanisms by
which Plasmodium falciparum and Epstein-Barr virus (EBV)
infection could interact have been addressed. Plasmodium
falciparum impacts on immunity and viral persistence by exhaustion of EBV specific T-cell response and Toll-like receptor (TLR)-9 mediated reactivation of EBV latently infected
memory B-cells. EBV encodes several latent proteins essential for viral immortalization of B cells, but EBNA-1 protein
is the only EBV latent protein consistently expressed in eBL.
Other EBV latent and lytic transcripts are not consistently
present, although they have been occasionally reported in a
subset of eBL tumors. Therefore the underlying mechanism
linking EBV infection of B-cells to the emergence of malignancy remains undiscovered.
In addition, epidemiological studies suggest that malaria
and EBV alone cannot account for the eBL cases in high
risk regions. In fact, malaria and EBV are ubiquitous within
the lymphoma belt of Africa and, unless other cofactors are
involved, the tumour should be much more common than it
is. Arboviruses and plant tumor promoters are other possible
local cofactor.
Material and methods
To determine if previously unidentified pathogens are present
in BL and to understand their contribution in the pathogenesis
of this disease, we established a tissue sample cohort that could
be assayed for viral RNA. We used a subtractive algorithmic
approach by which the input reads were aligned to host refer-
ence with different alignment program (bowtie2, blastN and
Megablast) and a computational subtraction was applied at each
step. Resulting unmapped not-host reads were the input for
pathogens identification. Validations were carried out by PCR,
RT-qPCR and immunohistochemistry on the same cohort of
primary tumors and on an additional series of samples.
Results
6 of known herpes viruses have been identified in tumor
samples. The presence of Herpes virus 4 (EBV) in 100%
of the samples was a positive control. 7/20 cases presented
Human herpes virus (HHV) 5 (cytomegalovirus), 5/20 cases
presented HHV8 (Kaposi Sarcoma-associated herpes virus
- KSHV), 1/20 case presented Human T-lymphotropic virus
(HTLV) 1. These results were validated by PCR and immunohistochemistry on primary tumors which confirmed the
presence of these additional pathogens in the tumors microenvironment. We then deeply investigated the latency of EBV
in eBL and asked the question whether the virus could be
reactivated (lytic or lysogenic phase). Our findings revealed a
diversity of non-canonical latency associated gene expression
program with a subset of viral episomes initiating lytic reactivation as indicated by expression of genes corresponding to
lytic program (i.e. BZLF-1/ZEBRA and early BMRF-1/Ea-D,
BHRF-1/Ea-R) and the expression of LMP2a. These results
were validated by RT-qPCR and immunohistochemistry in
primary tumors samples.
Conclusions
Our data supports the view that BL is a polymicrobial disease
and that lytic EBV reactivation may also contribute to the
development of EBV-associated lymphomas. Taken together,
these findings suggest potential therapeutic strategies for
virus-associated cancer. In fact, to avoid the tumor’s evolutionary escape from the already available therapies, we need
to identify and target the multiple pathways dysregulating in
the neoplastic cells, creating a genetic bottleneck too narrow
for the tumor’s survive.
References
Rochford R, Cannon MJ, Moormann AM. Endemic Burkitt’s lymphoma:
a polymicrobial disease? Nature Reviews Microbiology 2005;3:182-7.
Gorres KL, Daigle D, Mohanram S, et al. Activation and repression of
Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus lytic
cycles by short- and medium-chain fatty acids. J Virol 2014 May 7
[Epub ahead of print].
Söderberg-Nauclér C, Peredo I, Stragliotto G. Valganciclovir in patients
with glioblastoma. N Engl J Med 2013;369:2066-7.
Leucemia acute, associazioni inusuali
S. Ascani
Paper not received
141
RELAZIONI
Neoplasie mieloproliferative, proposte di
aggiornamento della classificazione WHO
U. Gianelli
Servizio di Emopatologia, UOC di Anatomia Patologica, Università degli Studi di Milano, IRCCS Fondazione Ca’ Granda, Ospedale
Maggiore Policlinico, Milano
In accordo con la classificazione dell’Organizzazione Mondiale della Sanità (WHO), le principali entità che costituiscono
il capitolo delle neoplasie mieloproliferative (NPM) possono
essere distinte in due gruppi, sulla base della presenza o
dell’assenza del riarrangiamento BCR-ABL1. Nel gruppo
delle NMP BCR-ABL1 negative sono comprese, come è
noto, la Trombocitemia essenziale (TE), la Policitemia vera
(PV) e la Mileofibrosi primaria (MFP). Nel 2005, la scoperta
della mutazione JAK2V617F, che è identificabile in più del
90% dei casi di PV e nel 50-60% dei casi di TE e MFP, ha
rivoluzionato i criteri diagnostici di queste neoplasie, rappresentandone il primo marcatore genetico. La successiva
identificazione di ulteriori mutazioni a carico dell’esone 12
di JAK2 e della mutazione MPLW515K/L presenti rispettivamente in quasi tutti i casi di PV negativi per JAK2V617F e
nel 5% circa dei casi di TE e MFP ha suggerito la possibilità
di una classificazione molecolare delle NMP e ha consentito
di includere la presenza di tali mutazioni nei criteri diagnostici
delle NMP, nell’aggiornamento della classificazione WHO
(2008). Alla fine del 2013, due gruppi di ricerca distinti hanno
identificato una serie di mutazioni del gene della Calreticolina, presenti con elevato frequenza (70-80%) dei casi di TE e
MFO JAK2-negativi. Alla luce di tali riscontri e di altri dati
della letteratura, la frequenza dei casi di TE e MFP che mostrano mutazioni di JAK2, MPL, Calreticulina o che risultano
“tripli-negativi” è risultata rispettivamente pari a 55-60%,1525%, 4-8% e 9-16% a seconda delle casistiche. Sulla base di
tali evidenze, il comitato di revisione della classificazione
WHO ha proposto di modificare i criteri diagnostici della TE
e della MFP, includendo tra i criteri maggiori, la presenza
di mutazioni a carico dei suddetti geni. A questo proposito,
si deve comunque sottolineare il concetto che tali mutazioni
non sono specifiche di una o più NMP e che dunque l’analisi
morfologica del midollo osseo rappresenta ancora un criterio
diagnostico imprescindibile.
Un discorso a parte riguarda la revisione dei criteri diagnostici
della PV, che dal punto di vista molecolare si associa nella
quasi totalità dei casi alle mutazioni di JAK2. Recentemente,
Barbui T et al. hanno riportato un’ampia serie di pazienti
JAK2-positivi, con caratteristiche morfologiche a carico del
midollo osseo analoghe a quanto descritto per la PV e con livelli di emoglobina superiori alla norma, ma inferiori a quanto
richiesto dalla classificazione WHO per una diagnosi di PV
in fase policitemica. Per tali pazienti è stato introdotto il concetto di “masked-PV. Tali evidenze, unitamente a precedenti
descrizioni di altri gruppi, hanno suggerito nella proposta di
revisione della classificazione WHO, di ridurre il valore di
emoglobina e/o ematocrito richiesti come criterio maggiore
per la diagnosi di PV a 16.5 g/dl (49%) per gli uomini e a 16.0
g/dl (48%) per le donne.
Bibliografia
Tefferi A, et al. Leukemia 2014;28:1407-13.
Nangalia J, et al. New Eng J Med 2013;369:2391-405.
Klampfl T, et al. New Eng J Med 2013;369:2379-90
Barbui T, et al. Am J Hematol 2014;89:52.
Modifiche stromali midollari nelle neoplasie
mieloidi, dalla prognosi alla patogenesi
C. Tripodo
Paper not received
Sabato, 25 ottobre 2014
Sessione parti molli
Moderatori: Angelo Paolo Dei Tos (Treviso), Alessandro Franchi (Firenze)
The role of grading and myogenic
differentiation in retroperitoneal liposarcomas
P. Collini
Department of Diagnostic Pathology and Laboratory Medicine, Soft
Tissue and Bone Pathology, Histopathology and Pediatric Pathology
Unit - IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Well differentiated/dedifferentiated liposarcoma (WD/DDLS) is
the most frequent sarcoma histotype in retroperitoneum. In the
last 2013 WHO ‘Classification of Tumours of Soft Tissue and
Bone’, WDLS is defined as a locally aggressive mesenchymal
neoplasm composed either entirely or partly of a mature adipocytic proliferation showing significant variation in cell size and
at least focal nuclear atypia in both adipocytes and stromal cells.
DDLS is considered as a progression of WDLS, either in the primary or a recurrence, to a usually non-lipogenic sarcoma of variable histologic grade. Both WDLS and DDLS are characterized
by supernumerary ring and giant marker chromosomes containing amplified sequences originating from the 12q14-15 region.
MDM2 (12q15) is consistently amplified and overexpressed.
Dedifferentiation occurs in up to 10% of WDLS, being a timedependent phenomenon, more frequent in retroperitoneum
respect to limbs. Histologically DDLS is characterized by
an abrupt/gradual transition from WDLS to a non-lipogenic
sarcoma usually of high grade. Exceptionally the two components are intermingled. DDLS is characterized by local recurrence in at least 40% of cases, distant metastases in 15-20% of
cases, and overall mortality of about 30% at 5 years.
The application of a grading to the DD component has been
variably performed using a two-tiered system (low grade vs
high grade) or the FNCLCC system, with some modification
or not. Instead of ‘low-grade DDLS’, in 2007 Evans introduced the term of ‘cellular WDLS’ encompassing all WDLS
components more cellular than a sclerosing WDLS but with a
142
mitotic index lower than 5/10HPF. Grading of the DD component is a still debated issue.
The DD component usually features a UPS, a myxofibrosarcoma, or a fibrosarcoma. In 5-10% of cases it can also show
‘heterologous differentiation’ in the form of myogenic, osteochondrosarcomatous, or angiosarcomatous differentiation. This
evenience is usually considered not to affect clinical outcome.
Along these lines, a series of 144 consecutive cases of retroperitoneal WD/DDLS uniformly treated from 2002 and 2011
at our Institution was studied for the prognostic significance
of FNCLCC grading and myogenic differentiation. In this
series, FNCLCC and absence/presence of myogenic differentiation with or without a rhabdomyoblastic component
significantly predicted the outcome. As a consequence, they
should be factored in the treatment decision making and possibly used to stratify patients in clinical trials.
La nuova classificazione WHO dei tumori ossei
A. Franchi
Paper not received
Nuovi marcatori immunoistochimici per la
diagnosi delle neoplasie dei tessuti molli
A.P. Dei Tos
Paper not received
Approccio diagnostico ai tumori maligni delle
guaine nervose periferiche
M.C. Montesco
Paper not received
Biobanche
Moderatori: Mattia Barbareschi (Trento), Giorgio Stanta (Trieste)
Biobanking and BioMolecular Resorces
Research Infrastructure - European Consortium:
Challenges and opportunities for Italian
Biobanks
M.L. Lavitrano
Università Milano-Bicocca & BBMRI, Italy
Realizing the full promise of precision medicine, whose goal
is to provide the best available care for each individual, requires that researchers and health-care providers have access
to vary large sets of samples and health and disease-related
data linked to individual patients. This emerging challenge
can only be addressed by innovative solutions developed in
an international and interdisciplinary collaborative scientific
effort. The European Strategy Forum for Research Infrastructures Roadmap identified the need of an infrastructure for
biobanks and biomolecular resources with the aim of both
modernize the ways in which biomedical research is conducted and, over time, lead to dramatically improved patient
care. Biological resources are indeed the essential raw material for the advancement of biotechnology, human health, and
research and development in life sciences.
Biobanking and Biomolecular Resources Research Infrastructure (BBMRI) is one of the largest and remarkable RI
in Europe in the life-science sector, it gained the legal ERIC
(European Research Infrastructure Consortium) status in November and became operational in December 2014. Italy is
one of the funding Member State together with Austria, Belgium, Czech Republic, Estonia, Finland, France, Germany,
Greece, Malta, The Netherlands, Sweden. BBMRI-ERIC is
born with the aim of increasing the efficacy and excellence
of European bio-medical research by I) facilitating access
to quality-defined human health/disease-relevant biological
resources including associated data in an efficient, ethically
and legally compliant manner, ii) reducing the fragmentation
of the bio-medical research landscape through harmonization
of procedures, implementation of common standards and
fostering high-level collaboration. BBMRI-ERIC is a distributed Infrastructure with the headquarter (Central Executive
Management Office) located in Graz, Austria, and operational
sites in different Member States (National Nodes), providing services to biobanks and researchers and a new model
of public-private-partnership - Expert Centres - to improve
efficacy of biospecimen research for academia and industry.
BBMRI-IT is a distributed infrastructure including Biobanks
& Biological Resources Centres allocated all over Italy. They
include, among others 18 Universities, 23 IRCCS (Institute of
health care and research), 40 Hospitals, 8 patient associations
and about 90 Biobanks, Biological Resources Centres and
Collections. The main goals of BBMRI-IT are i) to take Italian
collections of biological resources, Biobanks and networks to
a new level of coordination and efficiency, ii) to supply new
common services for the community of the Italian biobanks, iii)
to provide better access for users from public and private sector,
iv) to contribute to the pan-European research infrastructure
BBMRI-ERIC. To reach these goals BBMRI.IT is developing
and implementing tools to improve interoperability of research
databases, to facilitate public/private partnership, to harmonize
SOPs, to implement quality management criteria and finally to
develop conditions to ensure sustainability of Biobanks.
Management of the Italian Biobank
infrastructure
B. Parodi
IRCCS AOU San Martino, IST Istituto Nazionale per la Ricerca sul
Cancro, Genova, Italy
Italian Biobanks and Biological Resources Centers are the
core of the Italian BBMRI infrastructure; they are mainly
disease-oriented (cancer, genetics, multi-specialististic) and
143
RELAZIONI
most of them take part in regional networks and national and
international thematic networks. The population biobanks operate within a thematic network that participates in BBMRILPC (Large Population Cohorts).
A case apart is represented by Archival tissues, which can be used
for research under particular conditions. The national network of
tissue archive is part of the European network Impactsnetwork.
To build the National infrastructure, following a first survey
of potentially interested bioresources, BBMRI Italy has submitted to biobanks an on-line self-assessment questionnaire.
More than 80 collections and biobanks have participated to
the survey, and the responses were evaluated by a special
commission, on the basis of the requirements for participation
in BBMRI-ERIC, as defined in the BBMRI-ERIC partner
charter: - policy on access to samples, - data protection, - informed consent - infrastructure - quality system. The curators
of biobanks were also contacted individually, and this has
allowed to have a more accurate picture of the national scene.
The parameters for the evaluation of the impact of bioresources in scintific research, as defined by the BRIF initiative, have
also been taken into account.
The vast majority of Italian biobanks has already demonstrated
to operate in accordance with the principles of BBMRI-ERIC,
and collections interested in participating in the network that
have not yet completed the process of quality assurance intend
to do so with the support of the National node.
Each biobank has now its own web page on the BBMRI
Italy website (www.BBMRI.IT), where contacts, details of
the available samples and the services offered to the scientific
community are available. Biobanks can currently be searched
in the database by name, responsible, type of samples, Region,
participation in thematic and regional networks. The website
has been developed in accordance with the requirements of
the European Infrastructure BBMRI-ERIC, in order to promote scientific collaboration among European bioresources.
The Common Service Quality of BBMRI Italy is now operational and it aims to: - Map and monitor biobanks, repositories
and biomolecular resources; - Harmonize and standardize
SOPs; - Develop criteria for accreditation and certification; Implement quality management criteria; - Improve interoperability; - Set up training in quality control; - Standardize and
set up proficiency testing; Promote a certification program.
Organizzazione a livello regionale e sostenibilità
delle biobanche
A. Zanobini
Paper not received
Accesso alle biobanche: ruolo dei comitati etici
e delle associazioni dei pazienti
S. Casati
PhD & MD in Bioetica clinica, Presidente Comitato Etico di Ateneo UNIMIB
Il biobanking è un processo che ha bisogno del coinvolgimento
attivo di tutti gli attori che lo rendono possibile, in particolare
dei cittadini che sono motore e destinatari del biobanking stessi. Mentre Nature intitolava un suo editoriale ormai famoso
“Biobanks need publicity”, si integrava la Raccomandazione
Europea sull’uso scientifico del materiali biologici umani con
un articolo dedicato all’“informazione pubblica”: senza conoscenza di base, comprensione dello scopo e dell’importanza
del biobanking per la ricerca e per una cura personalizzata i
cittadini e chi li rappresenta difficilmente possono interagire
costruttivamente e scegliere di agevolare il processo stesso.
Le associazioni dei pazienti e i comitati etici svolgono allora
una funzione determinante, enzimatica, nel promuovere un dialogo costante con i cittadini e con clinici, che sono a tutti gli
effetti gli interlocutori naturali per avviare una raccolta consapevole dei materiali, e nel monitorare a qualità del processo
stesso. È interessante approfondire come in alcuni contesti di
eccellenza, il Network Telethon delle biobanche genetiche e
il Network dei comitati etici, si sia riconosciuta la necessità
di fare “squadra” per implementare e sostenere il biobanking
stesso e si siano intrapresi dei programmi in formazione e di
biobanking di successo grazie alle Associazioni e al convenire
da parte dei comitati sull’urgenza di individuare dei requisiti
condivisi di buona pratica biobanking, che faciliterebbero sia
l’attività valutativa dei Comitati stessi che divulgativa e di
supporto verso i clinici.
Opportunità e vincoli nei rapporti tra biobanche
e partners industriali
M. Macilotti
Paper not received
Quality management and SOPs in frozen tissue
biobanks
M.G. Daidone, S. Veneroni, G. Pelosi
Department of Experimental Oncology, Fondazione IRCCS Istituto
Nazionale dei Tumori, Milan, Italy
Translational research is dependent on human sample collections and associated clinical and research data and currently
increased expectations concern the perceived opportunities
of personalized medicine combined with molecular targeting
of therapies 1-3. To reach such goals several factors should be
considered, which stem from clearly stated hypotheses and
statistically significant observations resulting from studies of
robust statistical design and adequate power in which patient
clinical outcome is analyzed as a function of biomarkers and
molecular alterations, considered singly and/or in association.
In recent years, guidelines for the design and reporting of biomarker studies have been adopted by several major journals 4.
To obtain robust and reliable results, the scientific community
must find ways to access large numbers of well-annotated
and high-quality samples in order to satisfy the needs of complex study designs in which innovative high-throughput approaches are challenged in the context of clinical studies 5.
Therefore, cooperation in correlative and/or integrated large
multi-center translational research projects appears a logical
solution 6. Sample exchangeability is currently a major topic
for biobanks: a plethora of tools, best practices and guidelines
have been released to assist the community in implementing
primarily technical and infrastructural measures 7-12.
Biobankers recognize the importance of implementing harmonization to allow exchangeability of samples and associated
data among institutes, since the human samples collected
today form the foundation of what can be analyzed in the future, sometimes after decades of storage. This is an important
strategy since sharing of samples between different medical
centers is foreseen to be the future standard for performing
medical translational research, while incompatibilities in
sample and associated data quality originating from different
144
institutes can potentially block the possibility for meaningful
sharing. Incompatible quality can introduce an institutionalbased bias that can confound the outcome of statistical analysis and may obscure interesting biologically and clinically
relevant findings 13.
To ensure a constant and equivalent sample quality, it is important to have comparable pre-analytical conditions for each
of the collected samples used together in one study. In biobanking, the pre-analytical phase can be divided in three distinct phases: pre-acquisition, acquisition and post-acquisition
phase. Biorepositories should establish standard operating
procedures (SOPs) and work instructions that begin within the
pre-analytical phase and cover everything until distribution
of the sample for use in a research project. Unfortunately, the
pre-acquisition phase cannot be entirely encapsulated by SOPs
and work instructions. Depending on the type of sample many
parameters can be identified that may influence the results. In
the case of tissue samples these include patient treatment (e.g.
type of intervention or operating procedures, clamping time,
cold ischemic time etc.); drug intake (e.g. use of anesthetics,
antibiotics, pre-treatment, pain treatment, concomitant disease
treatment) and even time to food intake. Although it may not
be possible to control these factors, such information can be
recorded and added to the sample dataset assuring that it can
later be considered to either case selection or interpretation and
correlations during the analysis of the results. The acquisition
and post-acquisition phase cover the rest of the pre-analytical
phase, which can be captured completely in biorepository
SOPs. To guarantee constant sample quality SOP’s need to
adhere at least to the minimal internationally accepted methods
and standards 11. Additionally, there are instruments like Biospecimen Reporting for Improved Study Quality (BRISQ) 14,
that is a checklist for the issues that need to be reported on the
sample characteristics for improving research reproducibility
and for recognition of well-collected and stored samples, whose
use is recommended during the review process of publications
for studies dealing with human samples.
To constantly guarantee the required high quality samples, it is
crucial that acquisition, preservation, storage and distribution
are organized within well-structured and sustainable biorepositories, with dedicated trained staff. These biorepositories need
alarm systems, back-up capacity, emergency power, sample
tracking software, internationally accepted conservation procedures, work instructions, staff education program, contingency
plans, quality assurance and quality control, research support,
knowledge on transport/shipment, regulatory procedures and
data handling complete with solutions concerning privacy issues. Ideally, proficiency testing, external quality assurance as
well as biobank certification or accreditation should be in place.
Implementing such measures will most likely be successful
and sustainable when samples are collected and stored within
well-organized institutional biorepositories (such as hospital
integrated biorepositories) where only few collecting points and
storage facilities are allowed.
Next to sample quality, poor study design might possibly be
the most important reason for study failure, as it can result in
worthless data or incomparable results 5. When designing a
large study, all of the involved disciplines in the translational
research cycle, including surgeons, pathologists, nurses, molecular biologists, biochemists, statisticians, epidemiologists
and bio-informaticians, can and must contribute to avoid
this type of failure. When tissues are involved, pathologists,
with the necessary knowledge on the underlying morphology
and tissue heterogeneity, are essential for adequate sampling
of the material, while when blood samples are considered,
clinical chemists can give valuable contributions. A multidisciplinary involvement needs to be carefully considered to
ascertain optimal cohort selection, unexpected bias and test
sensitivity.
Hospital-integrated biorepositories can only exist when they
are fully supported by all stakeholders in the hospital and embedded into the local infrastructure with approval of the board
of the institute as recommended for tissue banks. For example,
pathologists are stakeholders who have the responsibility for
the large diagnostic tissue biobanks archived in clinical hospitals, that can be used for research purposes under strict conditions 15. Since all stakeholders can play a critical role in translational research and since they can be directly involved in
the sample collection and storage process, the use of samples
in research projects must be well-managed. The stakeholder
roles must be respected in order to achieve optimal and successful collaborations between projects within the biorepository. Only this way, the hospital-integrated biorepositories can
become fully integrated into the local curative and diagnostic
pathways as part of the normal hospital infrastructure.
It is clear that there are many essential issues contributing
to high impact translational research on samples, which may
classified into three main categories: 1) high sample quality
and equivalent standards (technical requirements), 2) good
study design with appropriate stakeholder involvement and
3) adequate statistical power to address the posed hypothesis.
These three principles drive the potential study impact, and
increase the chances of the research impacting on innovation of patient care. Amongst the instruments of choice for
implementing and securing these three key factors one should
involve crucial elements such as synergistic cooperation in
networks by exploitation of win-win situations, dedicated biorepositories with dedicated personnel, access rules including
external access and exchangeability of samples. Apart from
the factors that can be influenced directly by the scientific
community, there are also important external factors to deal
with like the financial and the social environment.
References
1
Mendelsohn J, Ringborg U, Schilsky R. Personalized cancer medicine
- a strategy to counteract an increasing cancer challenge. Mol Oncol
2012;6:109-10.
2
Vucic EA, Thu KL, Robison K, et al. Translating cancer ‘omics’ to
improved outcomes. Genome Res 2012;22:188-95.
3
de Paoli P. Institutional shared resources and translational cancer
research. J Transl Med 2009;7:54.
4
McShane L, Altman D, Sauerbrei W, et al. For the Statistics Subcommittee of the NCI-EORTC Working Group on Cancer Diagnostics.
REporting recommendations for tumor MARKer prognostic studies
(REMARK). Nature Clinical Practice Oncology 2005;2:416-22.
5
Bacchetti P, Deeks S, McCune J. Breaking free of sample size
dogma to perform innovative translational research. Sci. Transl. Med
2011;3:87ps24.
6
Riegman P, Morente M, Betsou F, et al. and the Marble Arch International Working Group on Biobanking for Biomedical Research.
Biobanking for better healthcare. Mol Oncol 2008;2:213-22.
7
OECD (Ed.). OECD Best Practice Guidelines for Biological Resource
Centers – General Best Practice Guidelines for all BRCs. OECD,
Paris 2007; http://www.oecd.org/dataoecd/7/13/38777417.pdf. (Accessed July 2013).
8
OECD (Ed.). Guidelines on Human Biobanks and Genetic Research Databases. OECD, Paris 2009 http://www.oecd.org/dataoecd/41/47/44054609.pdf (Accessed July 2013).
9
International Society for Biological and Environmental Repositories
(ISBER). Best practices for repositories: collection, storage, retrieval
and distribution of biological materials for research. Biopreserv
Biobank 2012;10:79-161.
10
NCI Best Practices for Specimen Resources. NCI, Bethesda. 2007;
http://biospecimens.cancer.gov/global/pdfs/NCI_Best_Practices_060507.pdf (Accessed July 2013).
145
RELAZIONI
11
12
13
14
15
Caboux E, Plymoth A, Hainaut P. (Eds.). International Network of
Biological Resource Centres for Cancer Research: Recommendations
on Common minimal Technical Standards. IARC/WHO, Lyon 2007.
ABN (Austrelasian Biospecimen Network). Biorepository Protocols.
2007; http://www.abrn.net/pdf/ABN_SOPs_Review_Mar07_final.pdf
1. (Accessed July 2013).
Mosley JD, Keri RA. Intrinsic bias in breast cancer gene expression
data sets. BMC Cancer 2009;9:214-24.
Moore HM, Kelly AB, Jewell SD, et al. Biospecimen reporting for
improved study quality (BRISQ). J Proteome Res 2011;10:3429-38.
Bevilacqua G, Bosman F, Dassesse T, et al. The role of the pathologist in tissue banking: European Consensus Expert Group Report.
Virchows Arch 2010;456:449-54.
Quali opportunità dai tessuti congelati?
F. Buttitta
Paper not received
Ricerche cliniche retrospettive nei tessuti
d’archivio
G. Stanta
Università di Trieste, Dipartimento di Scienze Mediche
Si è tenuto recentemente a Graz un workshop in cui, da parte
delle maggiori organizzazioni europee che si occupano di ricerca clinica, si sono proposti nuovi canoni e modelli per la ricerca clinica retrospettiva, con particolare attenzione a quella
che comporta analisi molecolari nei tessuti fissati e inclusi.
C’è un grosso scetticismo nella letteratura mondiale da parte
di organizzazioni ufficiali sugli studi di ricerca clinica pubblicati per biomarcatori prognostici e predittivi in oncologia.
Si mette in evidenza come questi studi siano spesso contraddittori e che per ottenere l’applicazione clinica di un biomarcatore trascorre un periodo di quasi dieci anni. Le cause di
questa irriproducibilità degli studi sono state individuate sia a
livello di disegno dello studio, sia a livello di standardizzazione tecnica. Per il disegno dello studio si propongono nuovi
modelli che vengono sviluppati in modo simil-prospettivo, basati se possibile su popolazione. Per quanto riguarda, invece,
le soluzioni tecniche, viene consigliata un’accurata microdissezione dei tessuti, considerando anche il tipo di area funzionale e una standardizzazione delle metodiche utilizzate con
l’uso descritto già nel disegno dello studio. Dovrebbero essere
applicate SOPs e IQC, simili a quelle che dovrebbero essere
utilizzate in diagnostica. Sono stati presentati già quattro modelli di ricerca che si basano sull’esperienza di organizzazioni
internazionali, quali l’OECI per una validazione rapida dei
biomarcatori clinici, il modello EPAAC per un’analisi dei
risultati della terapia con lungo follow-up e modelli regionali
che si adattano a specifiche condizioni locali. A questi modelli, inoltre, si aggiunge un’organizzazione di rete europea,
proposta recentemente anche per i tessuti d’archivio, che può
essere organizzata per paesi partecipanti sul modello della
base delle biobanche BBMRI.
Aspetti pratici della diagnostica cardiovascolare, dall’esame macroscopico alla
formulazione del referto
Moderatori: Annalisa Angelini (Genova), Augusto Orlandi (Roma)
Patologia del miocardio
Patologia coronarica
O. Leone, M.L. Tardio
Paper not received
G. D’Amati, C. Giordano
Paper not received
Patologia valvolare
C. Basso, S. Rizzo
Paper not received
146
I possibili errori diagnostici in patologia pediatrica:
una “survival guide” per i patologi – I Parte
Moderatori: Emanuele D’Amore (Vicenza), Libero Lauriola (Roma)
Torsioni ovariche e tumori
Le appendiciti “con sorpresa”
R. Boldrini
A.M. Buccoliero
Servizio di Anatomia Patologica Ospedale Pediatrico Bambino
Gesù IRCCS, Roma
Anatomia Patologica AOU Meyer, Firenze
L’intervento in urgenza per addome acuto legato a torsioni
ovariche può configurare un dilemma sia per il chirurgo che
deve scegliere tempestivamente il comportamento più congruo da adottare che per il patologo pediatra che si trova a
dover effettuare una diagnosi su tessuto spesso compromesso
dall’infarcimento emorragico.
Nel 2010 Oltmann et al presenta una casistica di 114 casi
pediatrici (età media 10 anni) con reperto intra-operatorio di
torsione ovarica. I reperti patologici evidenziano come nel
40% dei casi si tratti di tessuto ovarico normale, nel 33% di
formazioni cistiche benigne, nel 23% di neoplasie benigne
(teratoma, cistoadenoma, dermoide, fibroma), nel 3,5% di neoplasie maligne (tumore sieroso borderline, tumore a cellule
della granulosa, disgerminoma).
Inoltre Oltmann, combinando la sua casistica con una revisione complessiva della letteratura, identifica 707 torsioni
ovariche e segnala come la percentuale di neoplasie maligne
scenda all’1,8%
L’esame di una casistica di 131 lesioni ovariche operate
nell’ultimo quinquennio nell’ospedale Pediatrico Bambino
Gesù ha evidenziato 42 torsioni che hanno mostrato percentuali del 26% di tessuto ovarico normale , del 60% di cisti
ovariche (2% ovaio policistico), del 4% di neoplasie benigne
riferibili a teratomi maturi , mentre non sono state osservate
neoplasie maligne. L’elevata percentuale di cisti ovariche
appare correlata all’invio alla Chirugia neonatale dell’Ospedale Bambini Gesù di pazienti in età neonatale con diagnosi
ecografica prenatale di torsione ovarica.
La casistica del Protocollo di centralizzazione tumori germinali ha consentito di osservare 4 neoplasie benigne (teratomi
maturi) ed 1 neoplasia maligna (disgerminoma) ad insorgenza
su ovaio torto. In paraticolare quest’ultimo caso è risultato
di difficoltoso inquadramento diagnostico , in relazione allo
stato di degenerazione del campione.
Dai dati della letteratura e dall’esperienza personale si desume
come l’incidenza di neoplasie maligne nei casi di torsione
ovarica pediatrici sia più bassa di quanto non si aspetti.
Tali dati supportano l’indicazione a trattamenti più conservativi (detorsione e sorveglianza post-operatoria) riservando
il reintervento solo in caso di neoplasie accertate istologicamente.
Bibliografia
Oltmann SC, Fischer A, Barber R, et al. Pediatric ovarian malignancy
presentino as ovarian torsion: incidence and relevance. J Pediatric
Surgery 2010;45;135-9.
L’addome acuto è una condizione morbosa addominale a
prognosi anche grave se non trattato prontamente. Nonostante
esso possa ricondursi a svariate patologie di organi sia addominali che extra addominali, l’appendicite acuta ne rappresenta la causa di gran lunga più’ frequente in particolare nei
bambini di età superiore ai due anni.
L’appendice può tuttavia essere colpita da una varietà di
patologie congenite ed acquisite comprese le neoplasie tutte
potenzialmente in grado di determinare una condizione di
addome acuto.
I tumori dell’appendice nel bambino sono rari. Più frequenti
sono i tumori endocrini che però più che l’età pediatrica prediligono soprattutto l’età giovanile con un picco di incidenza
intorno ai 30-40 anni.
Eccezionalmente l’intervento di appendicectomia porta alla
luce patologie assolutamente inaspettate nel bambino come
è il caso recentemente osservato di metastasi da adenocarcinoma gastrico in un preadolescente.
Unusual adamantinoma of the tibia:
metastasis at presentation with complete
sarcomatoid transformation and loss of
epithelial markers
A.Parafioriti, E. Armiraglio, P.A. Daolio*, S. Bastoni*, A.
Di Bernardo
Anatomia Patologica AO Istituto Ortopedico Gaetano Pini, Milano; *Chirurgia Ortopedica Oncologica, AO Istituto Ortopedico
Gaetano Pini, Milano
Adamantinoma is a rare, biphasic low grade malignancy of
long bones. The tumor is heterogeneous due to the variable
proportion of the epithelial and stromal components, and is
related to osteofibrous dysplasia with the development of
mesenchymal/to/epithelial transformation. We describe a case
of metastatic adamantinoma with sarcomatoid transformation and loss of epithelial differentiation. A 34 old female
presented with a lesion of the proximal diaphysis of the tibia
and a second lesion in the iuxtaosseous soft tissue of the distal
tibia. On histological examination of the surgical resection,
the former lesion had the morphology and the phenotype of a
classic adamantinoma with occasional small keratins negative
spindle cell foci suggestive of initial mesenchymal transformation, the latter was an overt sarcomatoid spindle cell
neoplasm mimicking synovial sarcoma, negative for epithelial
markers and SYT/SSX fusion transcript. The patient did not
undergo either relapse or metastasis in a 5 years follow/up
after surgery. Very few cases of sarcomatoid transformation
in adamantinoma have been reported, compatible with an
epithelial to mesenchymal dedifferentiation but just in only
147
RELAZIONI
one a complete loss of epithelial immunophenotype has been
described. To our knowledge this is the second such case and
furthermore the dedifferentiation took place mainly in a synchronous metastatic localization.
Le lesioni mesenchimali superficiali
R. Alaggio
Paper not received
I possibili errori diagnostici in patologia pediatrica:
una “survival guide” per i patologi – II Parte
Moderatori: Rita Alaggio (Padova), Anna Maria Buccoliero (Firenze)
Pediatric follicular lymphomas
E. D’Amore
Ospedale San Bortolo, Vicenza, Italy
Pediatric follicular lymphomas (PeFL) have been recognized
as a peculiar variant of follicular lymphomas (FL) in the 2008
WHO Classification of tumors of Haematopoietic and Lymphoid Tissues.
They are rare tumors accounting for less than 5% of Non
Hodgkin lymphomas in the Italian pediatric registry of the
AIEOP (Associazione Italiana di Emato Oncologia Pediatrica).
1-2% of the Non Hodgkin lymphomas and, compared with the
usual FL of the adulthood, they.
Compared with the usual FL of the adulthood, PeFL are more
frequently discovered at a lower stage, have little propencity
to relapse or progress, and rarely show an unfavorable evolution, even when associated with diffuse areas.
Morphologically PeFL have almost always a high histologic
grade and are difficult to differentiate from reactive follicular
hyperplasia, as they are mostly BCL2 negative and often have
a large number of tingible-body macrophages imparting a
starry sky pattern.
However recent studies have shown that PeFL can be dissected in several morphologic subtypes 1:
Adult type FL (Usual FL)
Cases similar to the usual type of FL seen in adult patients
are very rare in the pediatric age, but have been found in
young adults (under the age of 30). Usual FL they are tipically
formed of closely packed and monomorphic bcl2 positive follicles without a starry sky pattern, and they may have a low
histologic grade.
PeFL of the tonsils and the Waldeyer ring
This type of follicular lymphoma is characterized by large
follicles predominantly composed of centroblasts or blasts of
intermediate size which are variably positive for BCL2 protein and uniformly positive for MUM1.
About 50% of these tumors have IRF4 breaks by FISH, and
occasionally the partner gene for the translocation is the IGH,
but other yet unknown genes can be involved and could be
responsible for the upregulation of MUM1 expression.
PeFL involving lymph nodes
PeFL involving the lymph nodes are the most difficult to
differentiate from a follicular hyperplasia. They feature large
ill-defined follicles of irregular shape and with attenuated
cuffs of mantle cells, and they are predominantly composed
of monotonous small to medium sized blastoid cells with a
starry sky pattern. The tumor cells are usually negative for
both BCL2 and MUM1 proteins.
Most of these lymphomas do not carry the BCL2/IHG translocation of the adult follicular lymphomas or the IGH/IRF3
fusion characterizing the PeFL of the Waldeyer ring. Very
rare cases have been described to carry the cytogenetic abnormality t(14;16)(q32;q24) and the IGH / ETO2 translocation 2,
and it has been recently shown that a subgroup of cases with a
more aggressive behavior have recurrent loss of heterozygosity in 1p36 associated with TNFRSF14 mutations 3. However
for most PeFL involving lymph nodes the molecular events
are unknown and their pathogenesis in unclear.
PeFL of the testis (4cases)
Testicular PeFL usually preserve the overall testicular architecture and are formed of very small naked follicles infiltrating between seminiferous tubules. They are composed
predominantly of usual centroblasts and they are negative for
both BCL2 and MUM1.
The molecular abnormalites have not been studied extensively, but a few cases with BCL6 mutation or translocation
have been reported 4.
The treatment of pediatric follicular lymphomas is controversial and should be carefully evaluated for each individual
case. A series of 25 German patient have been treated according to NHL-BFM protocols for mature B-cell non-Hodgkin’s
lymphoma 5; however a more conservative approach not
including systemic chemotherapy has also been advocated
other authors 6.
References
1
Liu Q, et al. Follicular Lymphomas in Children and Young Adults.
A Comparison of the Pediatric Variant With Usual Follicular Lymphoma. AJSP 2013;37:333-43.
2
Salaverria I, et al. The CBFA2T3/ACSF3 Locus Is Recurrently Involved in IGH Chromosomal Translocation t(14;16)(q32;q24) in
Pediatric B-Cell Lymphoma with Germinal Center Phenotype. Genes
Chromosomes & Cancer 2012;51:338-43.
3
Martin-Guerrero I, et al. Recurrent loss of heterozygosity in 1p36 associated with TNFRSF14 mutations in IRF4 translocation negative
pediatric follicular lymphomas. Haematologica 2013;98:1237-41.
4
Pileri SA, et al. Primary follicular lymphoma of the testis in childhood:
an entity with peculiar clinical and molecular characteristics. J Clin
Pathol 2002;55:684-8.
5
Oschlies I, et al. Pediatric follicular lymphoma – a clinico-pathological study of a population-based series of patients treated within the
Non-Hodgkin’s Lymphoma - Berlin-Frankfurt-Münster (NHL-BFM)
multicenter trials. Haematologica 2010;95:253-9.
6
Atra A, et al. Conservative management of follicular non-Hodgkin’s
lymphoma in childhood. Br J Haematol 1998;103:220-3.
7
Lorsbach RB, et al. Clinicopathologic analysis of follicular lymphoma
occurring in children. Blood 2002;99:1959-64.
148
Timomi ed iperplasia timica
L. Lauriola
Paper not received
IgG4 related disease
B. Famengo, E.S.G. D’Amore
U.O di Anatomia Patologica, ULSS6-Vicenza
Recently, immunoglobulin G4 related disease (IgGRD), has
been recognized as a novel clinical entity, caracterized by
mass-forming lesions or diffuse infiltrative process, specific
histopathological findings associated with elevation of serum
IgG4 (not always) and response to steroid.
A clear description and nomenclature of this condition has
been achieved in the past 10 years.
In 2001 Hamano et al. 1 reported elevated serum IgG4 in
patients with sclerosing pancreatitis, also called autoimmune
pancreatitis, and, in 2002, infiltration of IgG4 plasma-cells in
tissue samples from the same group.
The observation that patients with autoimmune pancreatitis
have extra-pancreatic lesions rich in IgG4 plasma-cell led to
concept of IgG4-related disease.
There are many synonyms because IgG4 disease is a systemic
condition and can involve many organs, so the term IgG4related disease was proposed upon the second meeting of the
Umehara group 2 3 and this name is actually recommended.
Immunoglobulin G4 is the least common of the 4 subclasses
of IgG and accounts for less than 5% of the totally IgG in
healthy persons; IgG4 production is controlled primarily by
type 2 helper T cells.
The IgG4 molecule is assumed to play a role in certain
immune-mediated conditions, such as pemphingus, a subset
of idiopathic membranous glomerulonephritis and thrombotic
thrombocytopenic purpura, but these disorders are distinct
from IgG4RD.
IgG4RD can affect any organ or tissue, the lacrimal glands,
salivary glands and pancreas are the major affected organ.
Disorders as Mickuliz’s syndrome, Kuttner’s tumor, Riedel’s
thyroiditis are now considered to fall within this disease
spectrum.
Because multiorgan involvement may occur, the International
Symposium on IgG4-RD 4 proposed a nomenclature for individual organ manifestation, using IgG4-related as a modifier
(for example IgG4-related sialadenitis, IgG4-related sclerosing cholangitis).
The pancreatic manifestation of disease was termed type 1
autoimmune pancreatitis-type1 AIP.
Epidemiologic and Clinical Features
Few data exist on the global incidence and prevalence of
IgG4-RD, but recent date indicate that it is not a rare disease.
Almost all epidemiological studies come from Japan.
There is a male predominance, mostly patients are middle-age
and elderly.
IgG4-RD may occur in children.
There are 11 pediatric patients reported in literature with autoimmune pancreatitis (AIP) or likely AIP, most cases failed to
report IgG4 levels, so it is difficult to establish whether they
meet I the IgG4 related autoimmune pancreatitis criteria 5.
There are few report of probable IgG4-RD that describe orbital involvement in children 6.
Very rare pediatric cases of idiopathic retroperitoneal fibrosis
were reported as manifestation of IgG4-RD 7.
The relationship of IgG4-RD with a myofibroblastic inflam-
matory tumor (IMT) has been studied 8. IMT and IgG4-RD
are distinct diseases in spite of some morphologic similarities.
The clinical picture is heterogeneous, the disease may manifest as a tumefactive lesion or as diffuse infiltrative lesion,
with a single o multiple site involvement, syncronous or
metachronous.
Constitutional symptoms are uncommon, laboratory findings
are often inconspicous.
IgG4 levels cannot be used as a sole criterion for diagnosis or
exclusion of IgG4-RD.
Type 1 AIP is frequently associated (50-80% of cases) with
extrapancreatic lesions (biliary tract, kidney, salivary gland,
retroperitoneum, lymph node).
A significant proportion of cases of retroperitoneal fibrosis,
sclerosing mesenteritis, mediastinal fibrosis and periaortitis
probably represent a manifestation of IgG4 disease.
Localized or ic lymphadenopathy can be found in 80% of
patients with IgG4-RD.
Enlarged lymph node can be found in resected specimen (pancreas o submandibular gland), and the lymphadenopathy can
be asymptomatic.
When the lymphadenopaty is generalized, the differential
diagnosis with lymphoma, malignancy or multicentric Castleman disease is required.
In IgG4-RD the lymph node are generally less than 2 cm,
there is not fewer or constitutional symptoms, LDH levels is
not increased.
Treatment
Most IgG4-RD patients respond favourably to glucocorticoid
treatment, spontaneous remission do occur.
The pediatric patients reported in literature have a disease that
is similar, but not identical, to adult AIP type 1, with typical
good response to corticosteroids.
Recently the use of mycophenolate mofetil has been adovocated, also in pediatric patient with IgG4 related pancreatitis 5.
Histopathological features
In the appropriate clinical context, the histopathological and
immunohistochemical analysis is a key component in the diagnosis of IgG4-RD 9.
The three major histopathological features associated with
IgG4-RD are:
1) Dense lymphoplasmacytic infiltrate with lymphocytic infiltrate composed predominantly of T cells. Plasma cells are
an essential component. Reactive lymphoid follicle may be
observed. Eosinophils can be found.
2) Storiform-type fibrosis.
3) Obliterative phlebitis, the veins are obliterated by a dense
lymphoplasmacytic infiltrate. Elastin stains may be required
for identification.
Phlebitis without obliteration of the lumen are also consistent
with diagnosis.
Conversely, the finding of granulomas and a prominent neutrophilic infiltrate are inconsistent with diagnosis.
A pathological diagnosis of IgG4-RD requires the presence of
two of the three major histological features.
Variability of histological findings exists in different organ.
Morphologic features in common in the pancreas, salivary
gland and lacrimal gland are sclerosis of interlobular septa,
interstial infiltration of lymphocytes and plasma cells, atrophy
and loss of acini.
In pancreas are often more prominent stromal edema or myxoid changes and obliterative pheblitis.
In salivary and lacrimal glands reactive lymphoid follicle are
common, obliterative phlebitis often is not seen.
149
RELAZIONI
Histopathology of lymph nodes in IgG4-RD differs from the
usual findings and could be categorized in 3 pattern 10:
1) Type I Multicentric Castleman disease-like: the architecture is preserved, mixed of hyperplastic follicles and follicles
with regressive changes, many plasma cells in the interfollicular zone.
2) Type II Follicular hyperplasia: reactive follicular hyperplasia, interfollicular zone with lymphocites and plasma cells.
3) Type III Interfollicular expansion: marked distorsion of
the architecture, lymphoid follicles often atrophic, the interfollicular zone is expanded by immunoblasts, mature and
immature plasma cells, eosinophils.
Two additional patterns have been also reported:
r 1SPHSFTTJWFUSBOTGPSNBUJPOPGHFSNJOBMDFOUSFMJLF
r /PEBMJOGMBNNBUPSZQTFVEPUVNPSMJLF
Assessment of the IgG4 immunohistochemical stain
The diagnosis of the IgG4RD cannot be established in the
absence of an immunoistochemical stain for IgG4.
Elevation in the absolute number of IgG4+cells and IgG4+/
IgG+ ratio is a prerequisite for the diagnosis.
Cut-off for the number of IgG4 cells: Absolute number of
IgG4+ cells > 50/HPF is highly specific.
The appropriate cutoff may vary from organ to organ.
IgG4/IgG ratio: IgG4+/IgG+ plasmacell ratio > 40% is mandatory for histological diagnosis, in the presence of the other
typical findings of the disease.
Recommandations to IgG/IgG4 count are:
r *H(BOE*H(DFMMTDPVOUVTJOHUIFQSJOUFEQIPUPHSBQIT
of the same microscopic field at x40 objective lens or microscope.
r "WFSBHFOVNCFSPG*H(QMBTNBDFMMTJOUISFFYGJFMET
with the highest number of IgG4+ plasma cells and calculation of IgG4+-to IgG ratio
Diagnostic criteria for IgG4 related disease
The diagnosis of the IgG4RD required appropriate clinical
and histopathological findings with careful clinico-pathological correlation.
Organ-specific diagnostic criteria has been proposed.
The guidelines for the pathological diagnosis proposed in the
Consensus Statement on the Pathology of IgG4-related disease includes three category 9:
1)
Histologically
Highly
suggestive
2) Probable
histological
features
At least 2 major histologic features
IgG4 counts (variable from organ to organ)
IgG4+ /IgG+ cell ratio:>40%
The majority of cases show clinical and
serological findings typical for IgG4-RD. If
these are lacking: histopathology is the
defining feature.
Cases either lacking the full histological
spectrum and the immunohistochemical
profile of IgG4-related disease.
Organs where the concept of IgG4-related
disease is not completely established
(meningeal or cutaneous site)
Needle biopsies non-completely
representative
Cases with only a single histopathological
feature (typically a dense lymphoplasmacytic
infiltrate, and required numbers of IgG4+ cells)
Additional evidence required:
Serum IgG4 >135 mg/dl.
Radiology
Insufficient
Evidence
Outside the previous two categories
Diagnosis of IgG4-related disease not excluded
Potential reasons include: sampling artifact,
effects of previous therapy, progression to a
fibrotic stage.
These criteria may not apply to certain organ, as lymph node,
lung and oral mucosa.
The Japanese comprehensive diagnostic criteria for IgG4-RD
2
includes characteristics:
1. Clinical: characteristic diffuse/localized swelling or masses
in single or multiple organs.
2. Laboratoristic: elevated serum IgG4 concentrations(135
mg/dl).
3. Pathological.
(1) Marked lymphocyte and plasmacyte infiltration and fibrosis.
(2) Infiltration of IgG4+ plasma cells: ratio of IgG4+/IgG+ cells >40 %
and >10 IgG4+ plasma cells/HPF.
The diagnosis of IgG4-RD is:
Definite: if are present 1) + 2) + 3)
Probable: if are present 1) + 3)
Possible: if are present 1) + 2)
Pathological criteria should be strictly observed because the
presence of numerous IgG4 + plasma cells and a high IgG4/
IgG ratio has been reported in various inflammatory conditions (rheumatoid synovitis, inflammatory oral and skin
lesions) and in epithelial neoplastic lesions (a peritumoral
inflammatory response).
Discussion and conclusion
The term IgG4 related disease includes a wide variety of disease with similar histopathological findings.
The cause of the disease are still not clear (some abnormal
immunological mechanism are involved).
The diagnosis of IgG4 related disease should be seriously
considered when we see a lesion with morphologic features
of pseudolymphoma, inflammatory pseudotumor with many
plasma cells, unexplained abundance of mature plasma-cells
in extranodal sites.
The finding of concomitant or prior history of pancreatic,
lacrimal and salivary gland support the diagnosis.
In lymph node morphologic features as Castleman-like disease, atypical lymphoid hyperplasia rich in plasma cell or
angioimmunoblastic T cell lymphoma-like pattern with associated good general condition, are suggestive for IgG4 RD.
Strict clinical and pathological criteria are necessary in diagnosis of IgG4-RD.
IgG4 –RD may occur in children and the correct diagnosis
requires an accurate clinical history and laboratory studies.
References
1
Hamano H, Kawa S, Horiuchi A, et al. High serum IgG4 concentrations in patients with sclerosing pancreatitis. N Engl J Med
2001;344:732-8.
2
Umehara H, Okazaki K, Masaki Y, et al. A novel clinical entity, IgG4related disease (IgG4RD): general concept and details. Mod Rheumatol 2012;22:1-14.
3
Umehara H, Okazaki K, Masaki Y, et al. Comprehensive diagnostic
criteria for IgG4-related disease (IgG4-RD), 2011. Mod Rheumatol
2012;22:21-30.
4
Stone JH, Khosroshahi A, Deshpande V, et al. Recommendations for
the nomenclature of IgG4-related disease and its individual organ
system manifestations. Arthr Rheum 2012;64:3061-7.
5
Mannion M, Randy QC. Successful treatment of pediatric IgG4 related systemic disease with mycophenolate mofetil: case report and a
review of the pediatric autoimmune pancreatitis literature. Ped Rheum
2011;9:1.
150
6
7
8
9
10
Griepentrong GJ, Vickers RW, Karesh JW, et al. A clinicopathological
case study of two patients with pediatric orbital IgG4-related disease.
Orbit 2013;32:389-91.
Laco J, Podhola M, Kamaràdovà K, et al. Idiopathic vs secondary
retroperitoneal fibrosis: a clinicopathological study of 12 cases, with
emphasis to possible telationship to IgG4 related disease. Virchow
Arch 2013;463:721-30.
Saab St, Hornick JL, Fletcher CD, et al. IgG4 plasma cells in inflammatory myofibroblastic tumor: inflammatory marker or pathogenic
link? Mod Pathol 2011;24:606-12.
Deshpande V, Zen Y, Chan JK, et al. Consensus statement on the
pathology of IgG4-related disease. Mod Pathol 2012;25:1181-92.
Cheuk W, Chan JK. IgG4-related sclerosing disease: a critical appraisal of an evolving clinicopathologic entity. Adv Anat Pathol
2010;17:303-32.
Salivary gland tumors in pediatric age
P. Collini
Department of Diagnostic Pathology and Laboratory Medicine, Soft
Tissue and Bone Pathology, Histopathology and Pediatric Pathology
Unit - IRCCS Istituto Nazionale dei Tumori, Milan, Italy
The most frequent causes of enlargement of salivary gland in
children are inflammatory processes and benign cysts. The
most common tumors are vascular neoplasms, most common
in newborns and infants. Epithelial tumors are very rare, accounting for less than 3% of all head and neck tumors. About
5% of them occur in patients up to 18 years of age, being more
frequent after 10 years of age and extremely rare in newborns.
In our Institution, 52 cases have been retrieved from 1974 to
2004. There were 25 females and 27 males, with a median age
of 15 years (73% over 10 years of age). The most frequent
was polymorphous adenoma (71.2%) in the parotid gland,
followed by mucoepidermoid carcinoma (MEC) (23.1%) in
the parotid gland, acinic cell carcinoma (3.8%) and adenoid
cystic carcinoma (ACC) (1.9%). MEC represented 80% of
malignant tumors, being low-grade in the vast majority of
cases. This is in contrast with adult MEC, which are prevalently high-grade tumors. Overall, in children salivary gland
carcinomas usually are low-grade and have a good prognosis.
A similar series of 17 cases of our Institution from 2000 to
2012 gave similar results. Recently, a pathognomonic fusion
transcript has been identified in MEC (MECT1-MAML2), associated with lower grades and improved survival, suggesting
both diagnostic and prognostic roles for it.
Patologia del sistema nervoso enterico
P. Nozza
Paper not received
Pediatric esophagitis
G. Pennelli, F. Galuppini, R. Alaggio, M. Rugge
Specialist in Surgical Pathology University of Padova Department of
Medicine DIMED Surgical Pathology and Cytopathology Unit
Esophagitis occurs throughout the entire pediatric age. Most
esophageal biopsies are performed during the diagnostic
evaluation of children presenting with nonspecific symptoms such as vomiting, abdominal pain, failure to thrive and
dysphagia1. Gastroesophageal reflux (GER) is the most frequent cause of histologic esophagitis. An increased frequency or duration of those episodes may result in pathologic
effects on the esophageal mucosa or airways (gastroesophageal reflux disease – GERD). Approximately 10% of infant
population has an abnormal quantity of acid GER (tested
on pH probes) that results in signs or symptoms. GERD has
different clinical manifestation between infants and children.
While older children have similar symptoms to adults (heartburn, epigastric abdominal pain, dysphagia, regurgitation),
in infants more frequent are nonspecific irritability, apnea or
malnutrition2. The histological findings of GERD include i)
epithelial alteration (basal cell hyperplasia and spongiosis,
anysocitosis and increased in mitotic rate in basal layer) ii)
changes in lamina propria (elongation, increased number
and vascular dilatation of papillae) and iii) intraepithelial
inflammatory cell infiltrates (eosinophils, lymphocytes, neutrophils). None of the abovementioned alterations is specific
for GERD. Even in the presence of severe inflammation not
all tissue samples are equally involved, therefore, several
specimen biopsies may be necessary to establish the histologic diagnosis of esophagitis. The treatment of GERD
consists primarily with acid suppression thanks to proton
pump inhibitors (PPI) and H2 receptor antagonist. The most
important complications of reflux esophagitis are ulceration,
peptic strictures, secondary infections and Barrett’s esophagus (BE). Because it’s necessary a prolonged exposure to
severe gastroesophageal reflux to develop, BE rarely occurs
in children 1.
In symptomatic patients, who are not responsive to antireflux treatments, the most frequent disease is eosinophilic
esophagitis (EoE). The most recently update consensus recommendations 3 state that EoE is a chronic, immune/antigen
mediated esophageal disease. Clinically, it is characterized by
symptoms related to esophageal dysfunction. Pathologically,
at least one biopsy specimen must feature 15 eosinophils/hpf.
The disease is confined to the esophagus and other causes
of esophageal eosinophilia should be excluded. EoE should
remit with treatment of dietary exclusion, topical corticosteroids, or both. In addition to the eosinophilic-predominant
inflammation, other histologic features present in EoE include
aggregates of microabscesses, surface layering of eosinophils,
several eosinophils mixed with desquamated luminal debris,
extracellular eosinophil granules, basal cell hyperplasia and
lamina propria fibrosis. The effective incidence of EoE is
unknown, although the prevalence appears to be increasing
in pediatric and adult population. Although the pathophysiology of EoE is currently uncertain, evidence implicates food
and aeroallergen hypersensitivity in genetically predisposed
individual as contributory factors 4. Genome-wide expression analyses have isolated a remarkably conserved geneexpression profile irrespective of age and gender, suggesting
a genetic contribution. EoE has characteristics of mainly TH2
type immune response but also some TH1 cytokines, which
appear to strongly contribute to tissue fibrosis. Eosinophildegranulation products appear to play a central role in tissue
remodeling. EoE is frequently associated with extraintestinal
allergic symptoms (asthma, eczema and chronic rhinitis) and
recently some Authors have described an association between
EoE and celiac disease: the prevalence of EoE in subjects
with celiac disease was about 10-times higher than that of the
general population 5.
The most common causes of infectious esophagitis are Candida and herper simplex virus (HSV) 1. Most of these infections
occur in immunocompromised patients, although mucosal
damage due to physical or chemical causes may predispose
to opportunistic infection. The most common symptoms of
infectious esophagitis include odynophagia, dysphagia, chest
pain, regurgitation and vomiting. Pathologically, the inflammation infiltrate is mainly represented by neutrophilis. In
151
RELAZIONI
Candida infection, white plaques cover the mucosa and are
composed of an acute inflammatory exudate admixed with
necrotic debris, pseudohyphae and budding yeast. In Herpes
esophagitis, typical lesions are shallow ulcers, covered by a
non-specific acute inflammatory exudate. Biopsies obtained
from the edge of HSV-ulcer can feature characteristic viral
cytopathic effect in the squamous epithelium.
Less frequent etiologies of pediatric esophagitis are immunodeficient disorders, drug, ingestion of corrosive agents, radiation, esophageal strictures, systemic disorders with esophageal involvement and the rare “Sloughing esophagitis”.
Pediatric esophagitis are a complex disease with a variety of
nonspecific symptoms and a poor correlation between endoscopic and histologic findings. Therefore, adequate mucosal
biopsy sampling and thoroughly histological assessment are
essential for a correct diagnosis and hence design an appropriate therapeutic approach.
References
1
Ruchelli ED, Liacouras CA. Esophageal disorders in childhood. In:
Russo P, et al. Pathology of pediatric gastrointestinal and liver disease. Springer-Verlag Berlin Heidelberg 2014.
2
Lighdale JR, Gremse DA; Section on Gastroenterology, Hepatology
and Nutrition. Gastroesophageal reflux: management guidance for
pediatrician. Pediatrics 2013;131:e1684-95.
3
Liacouras CA, Furuta GT, Hirano I, et al. Eosinophilic esophagitis:
Updated consensus recommendations for children and adults. J Allergy Clin Immunol 2011;128:3-20 e6.
4
Raheen M, Leach ST, Day AS, et al. The pathophisiology of eosinophilic esophagitis. Front Pediatr 2014;2:41.
5
Pellicano R, De Angelis C, Ribaldone DG, et al. 2013 Update on celiac disease and eosinophilic esophagitis. Nutrients 2013;5:3329-36.
Sala Brunelleschi - 08.30-12.30
Carcinoma della mammella: dalla diagnostica alle tecniche per la definizione
del trattamento terapeutico
Moderatori: Tiziano Berlingieri (Firenze), Tiziano Zanin (Genova)
Approccio multidisciplinare alla diagnostica del
carcinoma della mammella
C. Cigala, A. Comi
A.O. San Paolo, Milano
Ogni anno nel nostro Paese, 5mila donne contraggono un tumore della mammella: l’incidenza è aumentata del 14% negli
ultimi 6 anni, con un picco del 29% circa tra i 25 e i 44 anni
d’età, e ad aggravare il quadro concorrono l’allungamento
della vita media e l’incremento dei fattori di rischio. Nell’ottobre 2006 il Parlamento Europeo ha votato la nuova Risoluzione che ribadisce quanto affermato in quella del 2003, invitando gli Stati membri a garantire entro il 2016 la creazione
a livello nazionale di Unità di Senologia, poiché è dimostrato
che il trattamento del tumore della mammella in centri multidisciplinari aumenta le possibilità di sopravvivenza e migliora
la qualità di vita. L’approccio al carcinoma mammario è
completamente cambiato, non solo rispetto a 30 anni fa, ma
per molti aspetti anche da cinque anni fa ad oggi. Richiede un
costante aggiornamento e la professionalità non di un singolo,
ma di un gruppo che deve lavorare in modo integrato, con un
continuo scambio di dati, informazioni, discussione e conclusioni diagnostiche e terapeutiche. Il modello ideale per far
convergere questi professionisti e consentire una presa in carico completa delle pazienti con carcinoma mammario, anche in
considerazione dell’incidenza epidemiologica estremamente
elevata della malattia, è rappresentato dalla creazione non
solo di percorsi ma anche di spazi dedicati e riservati, definiti
Breast Unit. Le indicazioni delle linee guida sono precise:
per essere una Breast Unit bisogna trattare almeno 150 nuovi
casi di cancro del seno primario ogni anno. Tutto il percorso
di cura deve avvenire nell’ambito di questo centro specializzato che deve seguire protocolli scritti condivisi da tutti gli
operatori, perché è ormai chiaro che la migliore garanzia per i
pazienti è quella di mettersi nelle mani di persone che abbiano
davvero dimestichezza con quanto devono combattere: è noto
che la variabilità nel trattamento di casi simili è all’origine
dei fallimenti terapeutici. La diagnostica anatomo-patologica
è divenuta negli ultimi anni sempre più un elemento inserito in un ambito anatomo-clinico multidisciplinare, di cui è
necessario comprendere tutti i risvolti per poter condurre al
meglio il test diagnostico-predittivo con il più alto grado di
efficacia ed efficienza. Da questo punto di vista, l’accuratezza
della diagnostica patologica non può più essere assicurata solo
dentro i termini del solo laboratorio, ma va garantita dall’interfacciamento di diverse attività extra-laboratorio e la vera
complessità sta nella loro integrazione. Proprio per questo il
laboratorio di Anatomia Patologica deve essere in grado di
fornire risultati affidabili e sicuri. Solo l’attenta valutazione
delle fasi metodologiche e tecniche, analitiche e preanalitiche,
nonchè la partecipazione a programmi di Controllo di Qualità
possono garantire uno standard di processo elevato.
Citologia della mammella
F. Pagano
Cytopatlogy assistant Ente Ospedaliero Polo Universitario Osp. L.
Sacco Milano
“Omnis cellula e cellula”, quindi anche una sola cellula
possiede, come tale, le caratteristiche di malignità di tutta la
neoplasia, e la precisa identificazione delle sue alterazioni
morfologiche rappresenta in effetti una valutazione diagnostica e questo è il fondamento della citologia.
Nonostante gli ampi progressi della ricerca biologica sui markers delle neoplasie maligne, la diagnostica pratica del cancro
è data da indagini istocitologiche, radiologiche ed endoscopiche, che come fattore comune hanno il riconoscimento di
caratteristiche morfologiche.
La citologia agoaspirativa mammaria è anch’essa basata sulla
morfologia e tende ad avere anch’essa lo stesso valore diagnostico dell’esame istopatologico
Una parte dei criteri istopatologico di malignità sono rappresentati da:
152
1) anaplasia delle cellule tumorali;
2) accrescimento invasivo tendente a sostituirsi nei tessuti
normali circostanti;
3) rapidità ed autonomia di accrescimento.
La citologia mammaria non potendo contare sulla struttura
istologica, dipende esclusivamente dal primo punto elencato
ma si è dimostrata una indagine non solo tecnicamente semplice e ripetibile, ma anche accurata nell’identificazione del
tumore in fase precoce o ripetitiva, utilizzando aghi da 25G o
27G, valutando il materiale in fase di prelievo e indirizzandolo nelle colorazioni successive sia routinarie che immunocitochimiche che di citogenetica.
In linea di massima per la citologia agoaspirativa mammaria
si ha bisogno di almeno tre operatori, il medico che deve
occuparsi di raccogliere un campione adeguato, un tecnico
citologo che deve allestire i preparati ed eseguire lo screenig
e il citopatolo responsabile delle conclusioni diagnostiche.
linee guida ASCO/CAP (Wolff et al, JCO 2013) nelle quali
vengono affrontati eridiscussi sia i parametri pre-analitici sia
quelli interpretativi. Nel testo vengonoribaditi l’importanza
dei tempi di fissazione, sia per i prelievi bioptici che per i
pezzioperatori, viene suggerito un algoritmo laboratoristico
e viene riproposta unarivisitazione della classificazione delle
lesioni 2+; viene inoltre introdotta una nuovaclasse di tumori
FISH positivi monosomici. Gli autori delle linee pongono
inoltrel’attenzione sulla classe dei tumori classificati come
negativi (0/1+) potenzialmenteamplificati. La nuova classificazione, condivisa dagli oncologi, permette una nuovastratificazione delle pazienti affette da carcinoma infiltrante della
mammella con unprobabile maggior reclutamento di pazienti
candidabili alla terapia personalizzata.
Linfonodo sentinella con OSNA-Test
C.M. Antonacci
Her2-neu: immunoistochimica e FISH nel
carcinoma mammario
P. Balzarini, M. Cadei
Sezione di Anatomia Patologica, Dipartimento di Medicina Molecolare eTraslazionale, Università dgeli Studi di Brescia, Spedali Civili
di Brescia
Nelle pazienti affette da carcinoma infiltrante della mammella, la tipizzazioneimmunoistochimica e molecolare del
proto-oncogene c-erbB-2 è mandatoria per iltrattamento terapeutico a target immunologico. L’approccio metodologico
consta diuna prima valutazione con metodo immunoistochimico che permette di stratificare lelesioni in 4 classi con
score progressivo da 0 (negativi) a 3+ (positivi). Secondo
leindicazioni AIFA le pazienti con marcatore immunoistochimico francamente positivo(3+) possono beneficiare del
trattamento con Trastuzumab mentre le pazienti diclasse
intermedia (2+) necessitano di una caratterizzazione molecolare al fine diidentificare la presenza della amplificazione del
gene che riqualifica le lesioni comecandidabili al trattamento
personalizzato. Recentemente sono state pubblicate lenuove
Anatomo Patologo presso Dipartimento di Laboratorio Unità di Anatomia Patologica, Ospedale Luigi Sacco, Milano
Il linfonodo sentinella è il primo linfonodo che riceve la linfa
del tumore mammario, è il linfonodo in cui è più probabile il
riscontro di metastasi, sicchè è un elemento essenziale nella
stadiazione e prognosi del tumore.
Il metodo OSNA ha il vantaggio a) di processare tutto il linfonodo, b) di ottimizzare il trattamento chirugico c) eliminare
errori di campionamento e taglio d) standardizzazione del
metodo, e) di ovviare alla mancanza frequente di personale
tecnico in Anatomia patologica.
OSNA (One Step Nucleic acid Amplification) è una determinazione quantitativa dell’mRNA della citocheratina 19
mediante amplificazione RT-LAMP
La indagine molecolare può dare questi risultati.
NEGATIVO: assenza di mRNA CK19
MICROMETASTASI: range tra 250- 5000 copie mRNA CK 19
MACROMETASTASI: copie > di 5000copie di mRNACK19
In Europa gli utilizzatori del metodo OSNA sono circa 190 distribuiti prevalentemente in Spagna, Francia, Italia. Inghilterra e
Germania In Italia i Centri utilizzatori OSNA sono > 32 centri.
COMUNICAZIONI ORALI
PATHOLOGICA 2014;106:153-234
Sala Donatello – 2° piano – ore 11,30-12,30
Patologia ginecologica
Moderatori: M. De Nictolis, G.F. Zannoni
Expression of MECA-79 and HECA-452 on
endometrial epithelium in infertile women with
chronic endometritis. An immunohistochemical
and ultrastructural study
G. Arborea, L. Resta1 R. Rossi1, F. Gaudio1, C. Nardelli2, R. Depalo2, E. Cicinelli3
1
DETO, sezione di Anatomia Patologica, Policlinico, Bari, Italia; 2Unità
PMA, Policlinico, Bari, Italia; 3Dipartimento di Ostetricia e Ginecologia,
Policlinico, Bari, Italia.
Background. In humans, the a so-called “window of implantation” in the midluteal phase represents the maximal receptivity of
the endometrium for embryo implantation.
The L-selectin adhesion system has been strongly proposed to
be one of the most important candidate pathway to mediate the
initial embryo-maternal interactions. In support, it was demonstrated that human blastocyst expresses L-selectin on its external
surface raising the possibility that these molecules may partecipate in the early stages of human blastocyst attachment.
Aim of the present study is to examinate molecular bases of these
initial interactions and to correlate the infertility with inflammatory condition (chronic endometritis).
Methods. We studied ten endometrial biopsies from fertile females in age beetween 29 and 45 years with chronic endometritis
and two endometrial biopsies from infertile females (31 and 36
years) subjected to hormone therapy. All of these biopsies were
obtained during midluteal phase, from day 19 to day 24. We
compared these samples with twenty endometrial biopsies of secretory phase from fertile females without inflammation.
Our samples were studied using several approaches, including immunostaining for light microscopy and immunogold for
trasmission electron microscopy (TEM) using MECA-79 and
HECA-452 antibody. Immunohistochemical expression of Lselectin was examinate and slides were score with semiquantitative scoring system as follows: distribution of staining – absent
(0), <10% (1), 10-30% (2), 30-50% (3), 50-70%(4), 70-100% (5)
plus strenght of staining – weak (1), moderate (2), or strong (3).
endometrial glandular and surface epithelium were scored.
Results. MECA-79 IHC-level of glandular and surface epithelium wasn’t significantly different between women with inflammation and cases control (6,85 vs 6,42) instead HECA-452 IHClevel was significantly lower for sterile females and women with
chronic endometritis versus cases control (5 vs 3,42). The results
of immunogold TEM illustrated the expression of MECA-79 and
HECA-452 in cell membrane of endometrial microvilli in the
midluteal phase and in the lumen secretum.
Conclusions. This is the first study which tried to correlate
chronic inflammation with L-Selectin ligands (MECA-79 and
HECA-452) expression in human endometrial surface during
window of implantation.
Thanks immunogold TEM we confirm subcellular localization of
MECA-79 and HECA-452 in the human microvilli.
Mutational status of KRAS, BRAF, NRAS and PIK3CA
in primary clear cell ovarian carcinoma
G. Chiarello1, G. Improta2, A. Pettinato3, M. Petrillo4, D. Damiani1, C. Mosseri1, G.F. Zannoni1, F. Fraggetta3
1
Dipartimento di Patologia dell’Università Cattolica del Sacro Cuore
Roma; 2 Laboratory of Clinical Research and Advanced Diagnostics, Ospedale IRCCS-CROB, Rionero in Vulture Potenza, Italia; 3 Dipartimento
di Patologia, Ospedale Cannizzaro, Catania, Italia; 4 G. Scambia Dipartimento di Ostetricia e Ginecologia, Università Cattolica del Sacro Cuore
Roma
Introduction. Ovarian clear cell carcinoma (OCCC) is a subtype
of epithelial ovarian cancer with characteristic biological features
and aggressive clinical behaviour. The histopathological classification of EOC has been recently revised, and currently, two broad
categories are distinguished1. Type I EOC (including low-grade
serous, mucinous, endometrioid, and clear cell carcinoma) are
confined to the ovary at diagnosis, grow slowly and are chemo
resistant. Type II EOC (high-grade serous, undifferentiated
carcinomas, carcinosarcomas) typically show poor histological differentiation, early extra-ovarian spread and respond well
to platinum-based chemotherapy2.Type I tumors often harbour
somatic mutations of genes encoding protein kinases including
KRAS, BRAF, PI3KCA, and ERRB2, along with other signalling molecules, such as CTNNB1 and PTEN. In contrast, Type
II tumors generally lack these mutations but are characterized by
chromosomal instability and high frequency of TP53 mutations3,4.
In this complex scenario, ovarian clear cell carcinoma (OCCC) is
considered an entity distinct from the above cited type I EOCs, due
to its specific biological behaviour. Recent findings have demonstrated that OCCCs typically show a higher frequency of PIK3CA
mutations, which suggests that aberrations in telomere biology
may play an important role in the pathogenesis of OCCC5. On the
other hand, KRAS and BRAF mutations have been recognized as
a frequent event in low grade serous ovarian cancer, but only few
data are currently available on the mutational status of these genes
in OCCCs. For these reasons, we designed a clinico-pathological
study with the aim to evaluate the incidence of KRAS, NRAS,
BRAF an d PIK3CA hot spot mutations in a consecutive single
Institution series of patients with OCCC.
Methods. Between December 2006 and June 2012, 22 patients
with a proven diagnosis of OCCC were admitted to our Institutions.
In all cases, final diagnosis was established according to FIGO
and WHO criteria. All women received complete surgical staging.
The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany)
was used for pyrosequencing analysis of KRAS, NRAS, BRAF
and PIK3CA hot spotregions on 2.5-μm sections of formalinfixed
paraffin-embedded tissue from primary OCCC. Table 3 shows the
primer sequence for exon 9 and exon 20 of PIK3CA gene.
Results. Genomic profiling was conducted on a consecutive
series of 22 women with OCCC. The clinicopathological characteristics of the study patients are summarized in Table 1. Pyrosequencing analysis of KRAS, NRAS, BRAF and PIK3CA hot
spot regions revealed the presence of mutations at codon 12 in
exon 2 of KRAS in 3 of 22 (14 %) cases The following mutations
were found: p.G12V(gly12 val12), p.G12A (gly12→ala12),
and p.G12S (gly12→cys12) (Table 3).Concerning PIK3CA we
found mutations at exon 9 (c.1633G>A; p.E545K) and exon
20 (c.1633G>A; p.E545K) in 6 of 22 cases (27,3%).We found
no mutations in the hot spotregions of NRAS (exons 2, 3, 4) or
BRAF (exon 15).The median age of women with a KRAS mutated OCCC was 74 years. These OCCC were unilateral FIGO stage
IA lesions in two cases associated with foci of endometriosis.
Conclusions. OCCC accounts for 5 to 13 % of all epithelial ovarian malignancies. From a clinical point of view, it usually occurs
154
at a younger age than high-grade serous EOC. It often presents as
a pelvic mass confined to the ovary, often arising in association
with foci of endometriosis 6. Moreover, the clinical behaviour
of OCCC is aggressive, with a low response rate to standard
platinum-based chemotherapy and poor prognosis compared to
high-grade serous EOC. We observed a KRAS gene mutation
in 14% of OCCCs which seems significantly lower than in other
ovarian carcinoma subtypes, especially mucinous tumors. This
frequency of KRAS mutation is similar to that reported for ovarian endometrioid carcinomas (7 %), which supports the hypothesis
of a common origin of these two tumor types2,7,8. Interestingly,
we found KRAS mutations only in codon 12, exon 2, but not in
codon 13, exon 2 nor in NRAS or BRAF. Interestingly, the commonly described p.G12D mutation was not found in our series,
which emphasizes the specific biological features of OCCCs.
Finally, we confirmed the absence of BRAF hot spot mutations
in OCCCs9, making BRAF alterations a very rare event in ovarian cancer. Kuo KT et al.10 analyzing a series of 97 cases of clear
cell ovarian carcinomas have demonstrated mutations of PIK3CA
in more than one third of cases (46%). Several studies in the literature show that among all epithelial ovarian carcinomas those
clear cell have the highest frequency of mutations of the PIK3CA.
Rahman M et al11 have demonstrated PIK3CA mutations in 16/56
cases (28.6%) of clear cell ovarian carcinomas in Japanese patients. In our study we found PIK3CA mutations in 27% (6/22)
equally distributed in exon 9 (p.545K) and 20(p.H1047R). The
type of mutations found are in line with the results of the Japanese and Taiwan series 10,11 suggesting pathogenesis and progression of ovarian clear cell carcinomas are common to different
ethnic groups. Considering the poor response to platinum-based
chemotherapy protocols, these studies suggest that the cascade
PI3KT-AKT-mTOR-HIF (phosphoinisitide 3-kinase, v-akt murine thymoma viral oncogene homolog, mammarian target of rapamycin, and hypoxia induced factor) could be target of specific
therapies 12. New therapeutic prospects may in fact derive from
these observations: there are new drug molecules such as GDC0941, NVP-BEZ235, PI-103 and SF-1126 that target PIK3. In the
future it may be useful to evaluate through clinical trials the efficacy of these drugs correlate them with clinicalpathological data.
Tab. III.
Aminoacido
N
GGT
Mutazione
GTT
gly12
val12
1
GGT
GCT
gly12
ala12
1
GGT
ACT
gly12
ser12
1
References
1
Kurman RJ, Shih IM. The Origin and Pathogenesis of Epithelial
Ovarian Cancer: A Proposed Unifying Theory. Am J Surg Pathol
2010;34:433–443.
2
Del Carmen M. et al. Clear cell carcinoma of the ovary: A review of
the literature. Gynecol Oncol126 (2012) 481–490.
3
Chan JK et al. Do clear cell carcinomas have poorer prognosis compared to other epithelial cell types? A study of 1411 clear cell ovarian.
Gynecol Oncol 2008:109; 370–376.
4
Rechsteiner M et al. TP53 mutations are common in all subtypes of
epithelial ovarian cancer and occur concomitantly with KRAS mutations in the mucinous type. Exp and Mol Pathol 95 235–241 2013.
5
Wu RC et al. Frequent somatic mutations of the telomerase reverse
transcriptase promoter in ovarian clear cell carcinoma but not
in other major types of gynaecological malignancy. J Pathol2014
232:473–81
6
Anglesio MS et al. Clear cell carcinoma of the ovary: a report from
the first ovarian clear cell symposium, June 24th, 2010. Gynecol Oncol2011; 121: 407-415
7
Kurman RJ,et al (2005) Serous borderline tumours of the ovary. Histopathology2005 47:310–315
8
Sang YT,et al. KRAS (but not BRAF) mutations in ovarian serous
borderline tumor are associated with recurrent low-grade serous
carcinoma. J Pathol2013 231:449–56
9
Rechsteiner Met al. TP53 mutations are common in all subtypes of
epithelial ovarian cancer and occur concomitantly with KRAS mutations in the mucinous type. 2013 Exp Mol Pathol 95: 235–241
10
Kuo KT et al Frequent activating mutations of PIK3CA in ovarian
clear cell carcinoma Am J Pathol 2009,174:1597–1601
11
Rahman M et al Clinicopathologic and biological analysis of PIK3CA
mutation in ovarian clear cell carcinoma Hum Pathol 43, 2197–2206
2012
12
Gemignani M et al Role of KRAS and BRAF gene mutations in mucinous ovarian carcinoma. Gynecol Oncol2003 90: 378–381
Penile metastasis from rectal carcinoma
A. D’Amuri1, C. Fasano1, G. Carluccio2, C. Gabrieli1, F. Floccari3, M. Filotico1
Tab. I.
Characteristic
All cases
Age, median (range), years
FIGO Stage
I-II
III-IV
Bilateral ovarian lesion
No
Yes
Dimension of ovarian lesion,
median (range), mm
Occurence of endometriotic foci
No
Yes
All cases Number (%)
22
50 (29-75)
U.O. Anatomia Patologica - Dipartimento dei Servizi Diagnostici, Azienda Ospedaliera “Card. G. Panico” Tricase, Tricase (LE), Italia; 2U.O.
Urologia - Dipartimento di Chirurgia, Azienda Ospedaliera “Card. G.
Panico” Tricase, Tricase (LE), Italia; 3U.O. Anatomia Patologica P.O.
“Sacro Cuore di Gesù” Gallipoli, ASL LECCE, Gallipoli (LE), Italia
16 (72.7)
6 (27.316
Introduction. Metastatic carcinoma of the penis as result of adenocarcinoma of the rectum was first described by Eberth in 1870.
It is a rare clinico-pathologic entity arising most frequently from
primary tumours in the genito-urinary tract and rectum. It is frequently associated with disseminated metastasis. The prognosis is
poor with most of the patients dying within one year. In literature
less than 300 penile metastasis were described; 50 of them are
originate from colorectal carcinoma. We report a case of adenocarcinoma of rectum with multiple penile metastasis.
Methods. A 68-year old-man with a history of rectum adenocarcinoma in March 2013 (pT3, pN1, pMx), received adjuvant
chemo-radiotherapy post-operatively. In April 2014 at clinical examination showed and indurated lesion palpable in the glans penis
beneath the covered preputial skin with multiple ulcerative hard
nodules along the penile urethra and right corpus cavernosa close
the root of penis. The patient underwent total penectomy based on
the clinical diagnosis of penile metastasis from rectal carcinoma.
Results. A histopathological examination of the resected specimen revealed an infiltration by moderately differentiated adenocarcinoma with abundant extracellular mucinous areas in the pre-
21 (95.5)
1 (4.5)
120 (20-180)
18(81.8)
4(18.2)
Tab. II.
Ex9 Fw
5’-BiotinGGGAAAATGACAAAGAACAGCT
Ex9 Rw
5’-ACCTGTGACTCCATAGAAAATCTT
Ex9 S1
5’-CAATAGAAAATCTTTCTCCT
Ex20 Fw
5-BiotinATTCGAAAGACCCTAGCCTTAGA
Ex20 Rw
5’-TGCTGTTTAATTGTGTGGAAGATC
Ex20 S1
5’-GTTGTCCAGCCACC
1
155
COMUNICAZIONI ORALI
putial, glans penis, corpus spongiosum and corpus cavernosum
consistent with his primary rectal tumour. Immunohistochemistry
of tumor cells demonstrated positive staining for cytokeratin 20
and CDX2 (useful marker of colorectal carcinoma) and negative
staining for cytokeratin 7, which supported our diagnosis because
the majority of rectal cancer does not stain for cytokeratin 7, but
instead does stain for cytokeratin 20, and also because urothelial
cells commonly shows positive staining for cytokeratin 7.
Conclusion. Tumors metastasizing to the penis are rare. This
event is believed to be due to abundant vascularity of the penis
and its proximity to pelvic organs and the metastasis mechanism
is thought to be secondary to venous retrograde flow because of
the intense pelvic, lumbar and penile vein communication, lymphatic and arterial dissemination, as well as contiguity. Twentytwo percent of all metastasis to the penis originate in the gastrointestinal tract, particularly in the sigmoid colon and rectum. Penile
metastasis tends to show a poor prognosis because the metastasis
to the penis, in most cases, tends to be part of widely disseminated disease. The majority of patients die within one year.
On the other hand, a weak WT1 immunostaining was recognized
in only 5 cases (11%), with the remaining 40 patients (89%) resulting negative for WT1 staining.
Only 1 patients (2,5%) showed extraovarian diffusion with a non
invasive seromucinous borderline ovarian tumors peritoneal implant. All the other cases (97,5%) were at FIGO STAGE I.
Conclusions. Seromucinous borderline ovarian tumors are characterized by a very favorable prognosis, and a strong association
with ovarian or pelvic mullerianosis.
The immunohistochemical profile of seromucinous borderline
tumors showing strong immunostaining for ER PR, CA125, and
PAX8, seems completely different from the profile of intestinaltype mucinous borderline ovarian tumors, which usually show a
negative immunostaining for ER, PR, CA125.
To our knowledge this is the first series in which the expression
of PAX8 was documented.
Fig. 1. IHC panel in seromucinous borderline tumors A) ER B) PAX-8
C) CA125 D) PR.
References
Park JC, Lee WH, Kang MK, et al. Priapism secondary to penile metastasis of rectal cancer. World J Gastroenterol 2009;15:4209-11.
Yildirim M, Coskun A, Purten M, et al. A clinical case of the penile
metastasis from the rectal carcinoma. Radiol Oncol 2010;44:121-3.
Gbenou Goris MC, Wahidy T, Llinares K, et al. Atypical phimosis secondary to a preputial metastasis from rectal carcinoma. Case Rep
Oncol 2011;4:542-6.
Kimura Y, Shida D, Nasu K, et al. Metachronous penile metastasis from
rectal cancer after total pelvic exenteration. World J Gastroenterol
2012;18:5476-8.
Seromucinous borderline ovarian tumors:
immunohistochemical profile and clinical
correlations
L. Santoro, G. Chiarello, P. Castelli*, D. Damiani, C. Mosseri, G.
Zamboni*, G.F. Zannoni, A. Pesci*
Fig. 2. ER variable expression
Istituto di Anatomia Patologica, Università Cattolica del Sacro Cuore,
Roma; * Istituto di Anatomia Patologica, Ospedale Sacro Cuore-Don Calabria, Negrar
Introduction. Seromucinous borderline ovarian tumors are very
rare entity, with only few cases series reported until now1-7. Here,
we describe a large consecutive series of Seromucinous borderline
ovarian tumors, investigating the immunohistochemical profile and
clinical correlations of this rare entity.
Methods. The electronic database of the Pathology Department of
the Catholic University of the Sacred Heart, of Rome, and of the
Hospital of the Sacred Heart, Negrar, Italy, were retrospectively reviewed in order to identify women with proven diagnosis of Seromucinous borderline ovarian tumor. Specimens were retrieved, and
a careful rejoined evaluation of each case was conducted. Immunohistochemistry was performed in order to evaluate the expression
levels of the following markers: ER, PR, PAX8, CA125, WT1.
Results. Forty-five cases of Seromucinous borderline ovarian
tumor diagnosed between January 2000 and March 2013 were
identified. Median age at the time of diagnosis was 37 (range 2157), and the median tumor size was 6 cm (range 2-16). The vast
majority of patients showed unilocular masses (89%), with only 4
cases presenting multilocular lesions.
All seromucinous borderline ovarian tumors showed a papillary
structure, with a diffuse inflammatory infiltration in the stroma,
and among the neoplastic cells. A variable percentage of serous,
squamous, endometrioid and undifferent cells was observed.
Mullerianotic foci were documented, consisting in 31% of endometriosis and 20% of endosalpingiosis.
All cases showed a strong immunostaining for ER, PR, PAX8,
and CA125 (Fig. 1). Interestingly we found variable ER positivity
in different cell type (Fig. 2).
References
Kurman RJ, Carcangiu ML, Herrington SC, et al. WHO histological classification of tumours of the ovary. In: Tumors of Female Reproductive
Organs. Lyon, France: IARC Press; 2014
Shappell HW, Riopel MA, Smith Sehdev AE, et al. Diagnostic criteria
and behavior of ovarian seromucinous (endocervical-type mucinous
and mixed cell-type) tumors: atypical proliferative (borderline) tumors, intraepithelial, microinvasive, and invasive carcinomas. Am J
Surg Pathol 2002;26:1529–41.
Rutgers JL, Scully RE. Ovarian mixed-epithelial papillary cystadenomas
of borderline malignancy of mullerian type: a clinicopathologic analysis. Cancer 1988;61:546–554.
Lee KR, Scully RE. Mucinous tumors of the ovary: a clinicopathologic
study of 196 borderline tumors (of intestinal type) and carcinomas,
156
including an evaluation of 11 cases with ‘‘pseudomyxoma peritonei.’’
Am J Surg Pathol 2000;24:1447–64.
Rodriguez IM, Irving JA, Prat J. Endocervical-like mucinous borderline
tumors of the ovary: a clinicopathologic analysis of 31 cases. Am J
Surg Pathol 2004;28:1311–8.
Lee KR, Nucci MR. Ovarian mucinous and mixed epithelial carcinomas
of mullerian (endocervical-like) type: a clinicopathologic analysis of
four cases of an uncommon variant associated with endometriosis. Int
J Gynecol Pathol 2002;22:42–51.
Masafumi yasunaga MD et al. Immunihistochemical characterization of
mullerian mucinous borderline tumors: possible histogenetic link with
serous borderline tumors and low-grade endometrioid tumors. Human
pathology 2009; 40:965-974
Ligneous cervicitis: a rare benign, condition that
mimics cervical adenocarcinoma. A report of two
cases
E. Pacella1, N. Piol1, L. Mastracci1, F. Grillo1, E. Fulcheri1,2, S.
Carinelli3, G.F. Zannoni4, VG. Vellone1
DISC-Anatomia Patologica, Università di Genova; 2 UOSD Centro di diagnostica e patologia feto-placentare, Istituto Giannina Gaslini, Genova;
3
Divisione di Anatomia Patologica, IEO, Milano; 4 Istituto di Anatomia
Patologica, Università Cattolica del S.Cuore, Roma
1
Introduction. Carcinoma of the cervix is still the second cause
of cancer related mortality in women aged 20 to 39 years old
in the USA. Tumors with glandular differentiation are steadily
rising in incidence and this represents a problem in diagnostic
pathology. The putative precursor lesion for cervical adenocarcinoma (ADK) is still debated and a common, widely recognized,
nomenclature does not exist.
Adenocarcinoma in situ (AIS, corresponding to HG-CGIN of
UK authors) is the recognized precursor of invasive ADKand
is related to high-risk HPV with usually strongly and diffusely
positive immunostain for p16. A lesser degree of certainty exists
for earlier lesions variously named as endocervical glandular
dysplasia (EGD or LG-CGIN) whose diagnostic criteria, relations
with HPV infection and evolution to invasive cancers are not well
defined and universally accepted.
Several pseudoneoplastic conditions may mimic neoplastic and
pre-neoplastic lesions such as microglandular hyperplasia, reactive/reparative atypia, endometriosis, tuboendometrioid metaplasia, endometriosis and mesonephric remnants.
Ligneous (wood-like) cervicitis is a chronic non-infectious,
pseudomembranous disease characterized by extensive fibrin
deposition in various organs. Although the most frequent site is
the conjunctiva, the female genital tract may also be affected with
reactive epithelial changes that may mimic malignancy. This rare
condition seems related to an inherited defect in plasminogen
activity and it is known to cause infertility.
Case report. We report two cases of ligneous inflammation of
the cervix in absence of any preceding conjunctival or systemic
symptoms.
Case 1: A 31 years old woman, para 1, gravida 2, presented
with post-coital bleeding. Colposcopy revealed granulation tissue on the cervix. She had a history of condilomata and HPV
infection in 2006 treated with diathermocoagulation; followup Pap smears were all negative. Histological examination of
cervical biopsies revealed a proliferation of mostly irregular
“back-to-back” glands, lined by a cuboidal or low columnar
epithelium with reduced muciparity and hyperchromatic or
vesiculated nuclei, occasionally with nucleoli and only rare
mitotic figures. These glands were embedded in an amorphous
eosinophilic material in which neutrophils and some lymphocytes were dispersed. Immunohistochemistry showed intense
but inconstant positivity to p16 and CEA and a proliferation
index of 60% (evaluated with Ki67 antibody). All these features were suspicious for cervical adenocarcinoma with rare,
clear and microglandular features. The patent underwent cone
biopsy showing the same, previously described, lesion with no
sign of stromal invasion and ambiguous HPV positivity. Concurrent HC2 test were positive for HR-HPV. The case was sent
in consultation with the final diagnosis of ligneous cervicitis.
Serum levels of plaminogen resulted decreased to 33% (range
80-120%) confirming the diagnosis.
Case 2: A 27 years old woman, para 3 gravida 4, suffered a
miscarriage and during dilation and curettage the gynaecologist
saw a friable and bleeding area of the cervix which was biopsied.
Histologically a chronic and active, erosive cervicitis with mature
and immature metaplasia was seen. There was a focal area with
microglandular pattern with little intervening stroma lined by a
cuboidal epithelium with loss of muciparity and slightly hypercromatic but relatively monomorphic nuclei with only occasional
mitotic figures. This glands were embedded in an amorphous
amyloid-like material. Neutrophils were present both around and
inside the glands. Immunostain for p16 and CEA revealed intense
but inconstant positivity. Proliferation index was 20% with Ki67
antibody. All these features completely overlapped those of the
previously reported case We concluded that the histomorphologic
findings were consistent with a diagnosis of atypical (probably
reactive) microglandular hyperplasia consistent with ligneous
cervicitis. The patients has been advised to test for HPV and
plasminogen defect.
Discussion. Only a few cases of ligneous inflammation of the
female genital tract have been reported to date. Patients are usually premenopausal and infertility is often reported. We present
two cases of ligneous cervicitis in young women with a recent
miscarriage, both with no ocular or systemic symptoms and
uneventful medical or family history. The clinical and histological aspects of ligneous cervicitis are suggestive for an aberrant
response to diverse inflammatory stimuli. It is believed that this
disease is triggered by injury with hemorrhage and accumulation
of fibrinogen and fibronectin.
This chronically recidivating inflammation may cause reparative/
reactive changes of the endocervical epithelium to such an extent
that it can simulate malignant transformation. P16 positivity and
a high proliferative index, as seen in both our cases, may further,
erroneously, suggest a carcinogenic mechanism. However the
positivity for p16, although strong, was not uniform in all areas
of the samples and no clear signs of HPV infection were observed
in epitheliamapart from the lesion.
These cases show the importance of correlating pathologic, colposcopic (pseudomembranes with granulation tissue) and laboratoristic (serum plasminogen levels) findings in the diagnosis
of ligneous cervicitis because of the rarity of the disease and the
heterogeneity at presentation.
Histologically it represents a diagnostic pitfall that can lead a
young patient to an unnecessary, demolitive surgery with possible
medico-legal consequences.
Abnormalities of glandular cells on first level
cytology: what do they really mean? The Genoa
experience
V.G. Vellone1, E. Cartesegna1, P. Calamaro1, F. Sarocchi1, L.
Abete1, L. Mastracci1, F. Grillo1, E. Fulcheri1,3
1
DISC-Anatomia Patologica, Universitˆ di Genova; 2 UOSD Centro di
diagnostica e patologia feto-placentare, Istituto Giannina Gaslini
Pap test is the leading and most successful procedure for screening and diagnosis of precursor lesions of cervical cancer. For
largely unknown reasons, cervical adenocarcinoma and its
precursor lesions are steadily increasing in Western Countries,
hence the presence of atypical cells with glandular phenotype on
first level cytology represents an emerging problem.
All patents with a previous, first level diagnosis of atypical
glandular cell (AGC), adenocarcinoma in situ (AIS) and invasive
adenocarcinoma (ADK) admitted to IRCCS San Martino-IST,
157
COMUNICAZIONI ORALI
Genoa between 2010 and 2013 were collected and compared with
the results of subsequent second level investigations (cytology,
colposcopy, HC2 test, histology).
A total of 163 patients included in our study reported a first
level diagnosis of AGC: 129 AGC with atypical endocervical
cells, 22 AGC with atypical endometrial cells and 12 AGC
NOS. AGC patients were further subdivided in: “AGC favor
reactive” (n=111), and “AGC/favor neoplastic” (n=52). The
majority of patients (84,2%) discovered the lesion during
the screening program and were asymptomatic. The group
“AGC/favor neoplastic” was significantly related to lesions of
endocervical phenotype (p=0,0067). In addition, about 1/3 of
all cases showed a concurrent squamous lesion on first level
cytology, including 24 cases of ASC-US/ASC-H and 26 cases
of L-SIL/H-SIL; the majority were AGC of endocervical type,
while just 2 cases of AGC of endometrial type were found in
association with L-SIL.
Second level investigations consisted of different diagnostic
procedures: colposcopy was the main implemented procedure in
136 cases; 36 cases were evaluated with all available examinations while in just 6 cases a mere repetition of the Pap smear was
performed.
Secondary evaluation of AGC cases lead to the identification of
5 cases of adenocarcinoma, 5 cases of endocervical dysplasia, 17
cases of CIN2-3 and 37 cases of CIN1. For 99 patients the second
level investigation revealed a benign condition or a normality
status.
“AGC/favoring neoplastic lesion” resulted more frequently associated with neoplastic and pre-neoplastic lesions, but without a
statistical significance.
Six patients were diagnosed with AIS on Pap test screening: in
four of these cases secondary evaluation revealed an invasive
adenocarcinoma (three of endocervical type, one of endometrial
type), and in the other two cases diagnosis of AIS was confirmed
on second level examination.
Three patients received a diagnosis of adenocarcinoma on first
level examination, all of them confirmed on secondary evaluation, resulting in two endometrial adenocarcinomas and one
endocervical adenocarcinoma.
In conclusion, “atypical glandular cell” diagnosis on first level
cytologic screening is an important red flag for the clinician:
although the majority of AGC reflected a wide variety of underlying benign conditions, further investigations in these patients
lead to the identification of a not negligible number of invasive
and pre-invasive carcinomas, also of squamous phenotype. Of
remarkable note, all cases of AIS and adenocarcinoma observed
in our study were confirmed on second level investigation, hence
supporting the opinion that these two diagnostic categories on
Pap test screening highly recommend a prompt and thorough
subsequent evaluation.
Sala Giotto – 2° piano – 08,30-09,30
Interesse generale
Moderatori: P. Bufo, G. MIkuz
The fabry nephropathy: the electronic microscopy
role in the diagnosis and in the follow up.
M. Cervasio1, E. Guadagno1, S. Campione1, G. Luongo1, R. Maffucci1, M.L. Del Basso De Caro1, A. Pisani2, M. D’Armiento1
Department of Adavanced Biomedical Sciences, Division of Pathology,
University of Naples “Federico II”, Naples, Italy; 2Department Public
Health, Division of Nephrology, University of Naples “Federico II”, Naples, Italy
1
Background. Fabry disease (FD) is a X-linked disorder in
which mutations of GLA gene result in deficiency of the enzyme
-galactosidase A ( -GalA) and subsequent progressive, intralysosomal deposition in cells of various tissues of undegraded
glycosphingolipid products, especially globotriaosylceramide,
resulting in end-organ manifestations. Later in life, FD results
in cardiomyopathy, cerebrovascular disease renal failure and
stroke. These later disease manifestations are non-specific and
also common in diabetes, arterial hypertension and atheromas; as
a consequence, FD is under-diagnosed and screening of high-risk
groups is important for case findings as it is pedigree analysis
of affected patients. To date the prevalence of classical FD has
been estimated at about 1:117000 births and 1:40000 males, but
in several screening studies the frequency was up to 1% of births.
Kidney involvement is a prominent feature of FD, characterized
by an insidious development, and that’s why it is recommended
screening of patients for FD when there is unexplained chronic
kidney disease (CKD) without reliable renal diagnosis (renal
phenotype variant) . It is recommended enzyme measurement
for -Gal A activity as a primary tool in males younger than 50
years, followed by confirmation with mutation analysis when
positive; it is suggested mutation analysis as a primary tool for
screening in females (after informed consent). Transplant Fabry
patients have higher mortality than regular transplant population.
Enzyme replacement therapy (ERT) has been shown to slow the
progression of FD nephropathy.
Material and methods. From 54 patients affected by FD, collected in the regional registry at Nephrology Division of the
University of Naples “Federico II”, of with diagnosis of FD, 15
cases aged between 14 and 65 years, 7 males and 8 females, were
selected from nephrologist for kidney biopsy. All patients were
affected by unexplained CKD and proteinuria. Three cores for
each patient were obtained; one core was fixed in Bouin solution, embedded in paraffin and stained with Haematoxylin-Eosin,
Jones’ silver stain, Pas and Red Sirius tricrome stains, for light
microscopy evaluation; one core was frozen for routine immunofluorescence and a third one was in glutaraldehyde fixed, Eponembedded for ultrastructural examination.
Results. Of the 15 analyzed cases (TAB.1), at light microscopy 7
cases showed classical FD findings: glomerular visceral epithelial
cells and tubular cells were prominently vacuolated; 3 cases were
characterized by progressive glomerulosclerosis and interstitial
fibrosis, 5 cases did not show any histological lesions, 1 case was
a IGA nephropathy. The immunofluorescence was usually negative. It was positive for IgA just in the IgA glomerulonephritis
and it was positive in the glomeruli with advanced lesions for
IgM and C3 (in the capillary walls and mesangial regions). The
ultrastructural examination showed abundant typical osmiophilic
lysosomal inclusion bodies in 13 cases and was negative in 2
female patients, with light symptoms, one with intronic mutation that reduce transcript level, the other with classic FD light
microscopy but negative ultrastructural examination and with a
missense mutation that reduce transcript level; further genetic
investigation revealed a mitochondrial pathology. Therefore the
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ultrastructural examination is the most sensitive test in the morphological diagnosis
Conclusions. Our data confirm that FD affected patients are a
heterogeneous population and it makes it difficult to study its
natural course and to conduct large scale clinical trials. Renal
core biopsy is not essential for diagnosis in the classical phenotype because FD is a multisystem disease; other less invasive
approaches are in use for diagnosis.
However, we propose it in the flowchart for screening for FD
in CKD patients. Reasons for that are: 1) diagnosis in unclear
case 2) diagnosis in subjects with only inexplicable proteinuria
or CKD without affected relatives to reveal de novo FD 3) to
reveal a superimposed kidney disease 4) to invalidate a suspect
of FD (some lesions are not totally specific for FD, for example
thesaurismosis or those reported during therapy inhibiting lysosomal enzymes. 5) it will be an excellent approach to assessment
of renal lesions before initiating ERT and to evaluate the effect
of ERT 6) to evaluation the female carriers for living donation.
Therefore electron microscopy must be performed whenever possible to avoid delays in eventual specific therapy.
Finally we raccomend the screening for FD in all patients (on
dialysis and not) with CKD without a clear diagnosis, even in the
case of negative family history as de novo mutations can occur.
The real prevalence should be derived from screening in the
healthy population at young age; however this approach remains
problematic for several reasons. For that is desirable a high quality registry of all treated and untreated patients.
References
W. Terryn, P. Cochat, R. Froissart et al. Fabry nephropathy: indications
for screening and guidance for diagnosis and treatment by the European renal best practice. Nephrol Dial Transplant (2013) 28: 505-517
I pathology cockpit diagnostic station: validation
according to College of American Pathologists
Pathology and Laboratory Quality Center
recommendation at the Hospital Trust and
University of Verona
M. Brunelli1, S. Beccari1, R. Colombari2, M. Chilosi1, M. Lunardi2, G. Martignoni1, A. Scarpa1, A. Eccher1
Department of Pathology and Diagnostics, University of Verona, Italy;
Fracastoro Hospital, San Bonifacio, ULSS 20, Veneto Region, Verona,
Italy.
1
2
Introduction. Validation of digital whole slide images is crucial to
ensure that diagnostic performance is at least equivalent to that of
glass slides and light microscopy. The College of American Pathologists Pathology and Laboratory Quality Center recently developed
recommendations for internal digital pathology system validation.
Following these guidelines we sought to validate the performance of
a digital approach for routine diagnosis by using an iPad and digital
control widescreen-assisted workstation through a pilot study.
Methods. 61 histopathological slides were scanned by ScanScope Digital Slides Scanner (Aperio, Vista, CA). Two independent
pathologists performed diagnosis on virtual slides in front of a
widescreen by using two computer devices (ImageScope viewing
software) located to different Health Institutions (Verona) connected by local network and a remote image server using an iPad
tablet (Aperio, Vista, CA), after uploading the Citrix receiver for
iPad. Quality indicators related to image characters and workflow of the e-health cockpit enterprise system were scored based
on subjective (high vs poor) perception.
Results. The whole glass slides encountered 10 liver, 10 renal,
10 gastric and 10 prostate biopsies, 5 excisional skin biopsies,
5 lymph-nodes, 6 immuno- and 5 special stains were available
for intra- and internet remote viewing. Scan times averaged two
minutes and 54 seconds per slide (standard deviation 2 minutes
34 seconds). Reliance on glass slide, image quality (resolution
and color fidelity), slide navigation time, simultaneous viewers
in geographically remote locations were considered of high
performance score. Side by side comparisons between diagnosis
performed on tissue glass slides versus widescreen were excellent
showing an almost perfect concordance.
Conclusions. We validated our institutional digital pathology system for routine diagnostic cases facing with whole slide images
in a cockpit enterprise digital system or iPad tablet. Computer
widescreens are best for diagnosing scanned glass slide in respect
to iPad. For urgent request, iPad may be used. Legal aspects have
to be soon faced with to permit the clinical use of this technology
in a manner that does not compromise patient care.
New subtypes of de novo renal cell carcinoma
after kidney transplantation according to the ISUP
2013 Vancouver Classification of Renal Neoplasia
M. Brunelli1, A. D’Errico-Grigioni2, L. Boschiero3, C. Ghimenton1, A. Caliò1, G. Martignoni1, A. Eccher1
Department of Pathology and Diagnostics, University of Verona, Italy; 2
Department of Specialized Medicine, Diagnostics and Experimental, University of Bologna, Italy; 3Transplant Kidney Surgery, AOUI, Verona, Italy.
1
Introduction. De novo renal cell carcinoma (RCCs) may arise in
kidney after transplantation and these tumours affect the survival
of patients who usually receives at time zero drugs for reducing
rejection of the transplant kidney, performing immunosoppression. A recent (2013) International Consensus was obtained in
Vancouver (ISUP Vancouver Classification of Renal Neoplasia)
in regard to the introduction on new 5 subtypes of renal cell
carcinoma. The incidence and impact of such a new subtypes of
tumours to survival after kidney transplantation is not known.
Methods. De novo RCCs after kidney transplantation were reviewed though a period of 30 years. Immunophenotypical analysis including CD13, CD10, CK7, AMACR, CAIX, GATA-3,
S100A1 and parvalbumin was assessed. ph mTOR and ph 70S6K
were also evaluated.
Results. Five clear cell, 5 papillary, 1 sarcomatoid NAS, and 1
unclassifiable RCCs were subtyped. We identified the subtype
“clear cell-papillary RCC” among news (1/13). One papillary
RCC showed clear cell changes and one oncocytic changes. Nine
staged pT1, three pT3, one pT3 with sarcomatoid features. One
died with metastases (lung) (clear cell G4) and two developed
recurrences (sarcomatoid, G3). All tumours revealed intense and
diffuse positivity for both ph mTOR and ph 70S6K molecules.
One clear cell and the low grade clear cell-papillary RCCs
showed a weaker staining for mTOR.
Conclusion. A new type of tumour called “clear cell-papillary”
was reported among de novo RCCs after kidney transplantation.
This tumour is reported to show a benign appearance although the
presence of clear neoplastic cells. The knowledge of such a presence is importance for the management and follow-up of patients
after kidney transplantation who develops de novo RCCs.
ST2L immunohistochemical expression in carotid
atherosclerotic plaque: a new marker of plaque
instability?
F. Buffelli, F. Ambrosi, G. Serio, A. Marzullo
Università degli Studi di Bari, DETO, Sezione di Anatomia Patologica.
Soluble ST2 (sST2), a novel biomarker predictive for heart disease, has recently been shown associated with the progression of
atherosclerotic disease in a mouse model. ST2, the receptor for the
Th2 associated cytokine interleukin (IL)-33, is a member of the
IL-1 receptor family that has recently gained significant interest in
the biomarker research field. ST2 complex is constituted by a tissue transmembrane component (ST2L) and a soluble form (sST2)
ST2L, expressed on the surface of many inflammatory cells is the
159
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receptor trough which signalling of IL-33 has been found to exert
a cardioprotective role.
The present study was designed to assess the immunohistochemical expression of the ST2 receptor (ST2L) in atherosclerotic
plaques of internal carotid arteries in patients with and without
cerebro-vascular symptoms. In addition, ST2L expression was
correlated to patient clinical data and atherosclerotic plaque
histological features, namely stenosis, inflammation, lipid core
dimension, angiogenesis, calcification.
34 cases (23 asymptomatic and 11 symptomatic) were recruited
for the study. All the clinical and morphological parameters examined were uniformly distributed between the two populations
with a mild predominance of a large lipid core and inflammation
in asymptomatic cases. ST2L expression resulted more expressed
with a membranous pattern in macrophages observed in carotid atherosclerotic plaques of symptomatic patients (63.6% vs
39.2%). Significantly ST2L was expressed in the endothelium of
new formed vessels at the shoulder region of the plaque, but not
(apart from few cases) in the endothelium covering the residual
lumen of the vessel. This data was also correlated with plaque
angiogenesis assessed as microvascular density and number of
mastcells detected by immunohistochemistry (tryptase).
In conclusion ST2L, as the soluble counterpart, could be a promising predictive factor for vulnerable plaque and its predictive
value seems to be more powerful than the traditional morphological factors. In addition ST2L positivity could be a valuable
marker of angiogenesis that is an essential step in the mechanism
of plaque formation.
Periostin: a new player in cardiac myxomas
A. Di Vito1, C. Mignogna2, G. Santise3, F Conforti2, V Zuccalà2,
L Maltese2, G Donato2
1
Department of Experimental and Clinical Medicine, University Magna
Græcia, Catanzaro; 2 Department of Health Sciences, Pathology Unit,
University Magna Græcia, Catanzar; 3 Department of Cardiac Surgery,
Sant’Anna Hospital, Catanzaro
Cardiac myxoma is the most common benign cardiac tumor generally localized in the left atrium. The majority of cardiac myxomas occurs sporadically while a relatively small proportion of
cases develops as a part of Carney complex syndrome. Histogenesis of cardiac myxomasis still not fully characterized, however
recent data suggest an origin from subendocardium precursors
which can differentiate in both endocardial and myocardial lines.
Myxoma cells are organized in rings, cords and nests showing
variable immunopositivity for both vascular markers CD31 and
CD34, as well as for Calretinin, which has been proved to be quite
useful in discriminating cardiac myxoma from mural thrombi and
papillary fibroelastomas. The extracellular matrix is organized
in an Alcian blue positive myxoidstroma, partially resistent to
predigestion with hyaluronidase, composed of variable amounts
of proteoglycans, elastin and collagen. Interestingly, vascular
channels seem to develop often from myxomatous structures.
We previously reported that the expression of some extracellular
matrix proteins (Tenascin c) and hyaluronic acid receptor (CD44)
on cell surface is involved in angiogenesis occurring during neoplastic transformation1. In addition, we confirmed the role of mast
cell-derived tryptase (detected in almost cases in left atrium) in
angiogenesis and in myxoma mesenchymal cells proliferation also forming pseudovascular structures2. Recent evidences suggest
a key role of Periostin, a matricellular protein, in the development
of endocardial cushions and atrioventricular junction as well as in
cardiac remodeling of post-infarction heart. According to recent
literature, Periostin action could cooperate with Tenascin-c3. In
this study, a series of 13 sporadic cases of cardiac myxoma were
examined by immunohistochemical analysis for the presence of
Tenascin-c and Periostin. It appears that Tenascin-C and Periostin
expression levels are interrelated, suggesting a synergic role in
development of cardiac myxomas. Immunohistochemical staining was also performed for the myocardiocyte marker CD117. In
all cases analyzed, cells were negative for CD117, confirming the
absence of myofibroblastic origin. In conclusion, the detection of
Periostin overespression in cardiac myxoma, combined with the
absence of CD117-positive cells strongly corroborate the idea
that cardiac myxoma cells originate from precursors of cushion
endocardium.
References
1
Donato G, Conforti F, Camastra C, Ammendola M, Donato A, Renzulli A (2014) The role of mast cell tryptases in cardiac myxoma:
Histogenesis and development of a challenging tumor. Oncol Lett
8(1):379-383.
2
Donato G, Conforti F, Zuccalˆ V, Russo E, Maltese L, Perrotta I,
Amorosi A (2009) Expression of tenascin-c and CD44 receptors in
cardiacmyxomas. Cardiovasc Pathol 18(3):173-7.
3
Kudo A (2011) Periostin in fibrillogenesis for tissue regeneration:
periostin actions inside and outside the cell. Cell Mol Life Sci
68(19):3201-7.
Diagnostic and forensis autopsy.
An old and new methodological approach
D. Bellis1, A. Giancarlo1, M. Fammartino2
SC Anatomia Patologica; 2 SC Direzione Sanitaria – P.O. Martini ASLTO1 Torino
1
Introduction. Until the 1960s the autopsy was essential to the
discovery and understanding of disease. In 1993 the Royal College of Pathologists published “The Autopsy and Audit”, and
recommended that at least 10% of hospital deaths were autopsied.
This approach was no longer considered acceptable by Guidelines
on Autopsy Practice (2002). Today hospital autopsy rates are falling and it has been questioned if the autopsy is still useful. This
is in part because of new medical diagnostic technology, but also
because clinicians may not seek autopsies because of potential
medico-legal consequences. Other factors implicated in the declining rate include inadequate communication of autopsy results
to clinicians and inadequate training of young pathologists.
Based on this background, we felt it was appropriate to study the
utility of the diagnostic and forensic autopsy in medical practice
today, organizing an educational course, together legal doctors
and sanitary direction, for medical, paramedical and technicians
of our hospital. The finalities of the course were to investigate
if autopsy is still useful and necessary in clinical practice and to
produce a guide line and PDTA for post-mortem diagnoses.
Material and methods. The course was organized by Surgical
Pathology Service of Martini Hospital, ASLTO1, Coroner Service ASLTO1 and Health Department of Martini Hospital. We
selected 5 docents: 2 surgical pathologists (Dr Donata Bellis, Dr
Giancarlo Abbona), 2 coroners (Dr Maurizio Cravello, Dr Moreno Bertoni) and the Health Department director Dr Marinella
Fammartino. Students were 30: internists, coroners, pathologists,
doctors of Health Department and technicians. The arguments
analyzed were: the History of autopsy, the diagnostic autopsy,
the medico-legal autopsy, the prenatal and neonatal autopsy, legal
practice guidelines and protocols and a summary report about
PDTA (Protocol to Diagnostic and therapeutic Assistant). During
the course were discussed representative cases of medico-legal,
pediatric and diagnostic autopsies to definite the causes of dead
and to draw their epicrisis (initial, intermediate and final death’s
causes). The methodology was based on magistral lectures and
cases discussion.
Results. This course provided major conscience of role of diagnostic and forensic autopsies in clinical practice; also emerged
the necessity of working together for an ethical and functional
system with more rationality of the resources. An intimate connection between clinicians, parents and legal doctors appeared
the more useful approach to formulate the PDTA. We found that
160
autopsy, including histology, is an accurate mean for determining
natural deaths causes as well as suddenly deaths with unknown
medical conditions, or criminally suspected deaths. The autopsy
also appeared to play an important role in public health, like
investigation of deaths in the workplace, in custody, as well as
sudden and unexpected infant’s deaths. We also investigated the
value of additional or alternative tests, such as toxicology, genetic
tests, mineralogy analyses and resonance imaging; there was the
evidence they help to support, but not to adequately replace the
conventional autopsy.
ANATOMIA PATOLOGICA P.O. MARTINI
PDTA RISCONTRO DIAGNOSTICO
In caso di:
· Morte inaspettata
· Sospetta malattia professionale
· Giunti Cadavere
· Morte in caso di sperimentazione terapeutica
· Morte intraoperatoria o in corso di esame strumentale
· Morte entro 48 ore dallintervento o da procedure diagnostiche invasive
· Morte di una gravida o di una puerpera entro 7 giorni dal
parto
· Morte in reparto psichiatrico
· Morte malformativa
· Morte da cause non sufficientemente chiare da permettere
la compilazione del certificato del decesso.
· Morte neonatale o morte fetale endouterina e infantile
improvvisa e vittime SIDS (sudden infant death syndrome)
· Morte da cause contagiose
È da richiedere il riscontro diagnostico per la identificazione
della/e causa/e di morte.
Il medico di reparto o di Pronto Soccorso richiede l’autorizzazione alla Direzione Sanitaria. Se non ci sono delle condizioni
che richiedono la segnalazione del decesso alla Procura della
Repubblica (causa di morte non naturale di tipo traumatica o
naturale per colpa) la Direzione Sanitaria richiederˆ al Servizio
di Anatomia Patologica di eseguire il riscontro diagnostico.
Il patologo incaricato, prima di eseguire il riscontro diagnostico, consulterˆ la cartella clinica e in caso di Giunto Cadavere
accerterˆ le condizioni precedenti la morte tramite consultazione dei parenti o di testimoni.
Il Patologo dopo aver eseguito il riscontro diagnostico
formulerˆ la prima diagnosi clinico morfologica e compilerˆ
le carte di morte e la scheda ISTAT. Se necessario eseguirˆ
dei campioni di tessuto biologico (secondo le linee guida
regionali, nazionali, internazionali) per l’esame istopatologico
(È possibile, a seconda dei casi, eseguire anche degli esami
cito-istologici tramite agoaspirazione o agobiopsia).
Il patologo redarrà il referto diagnostico conclusivo che
comprenderà la ricostruzione della storia clinica (anamnesi
familiare, fisiologica, patologica remota e prossima), la descrizione macroscopica e la diagnosi istologica dei tessuti/
organi campionati e la diagnosi conclusiva (Epicrisi).
Salvo casi complessi, che richiedono ulteriori indagini (immunoistochimica, molecolare, genetica, mineralogica) il referto
verrà inviato all’ufficio cartelle cliniche entro 30 giorni.
Il patologo sarà sempre disponibile per i parenti del defunto
(secondo la normativa vigente sulla privacy).
In caso di malattia professionale, malattia diffusiva farà
segnalazione alla Direzione Sanitaria secondo le normative
vigenti e le procedure aziendali.
Conclusion. These results confirmed those reported by J Roulson
and colleagues (2005) with a meta-analysis and review: “With an
increasing emphasis on audit and clinical governance, the audit
of diagnostic accuracy is essential”. Although alternatives to the
autopsy are being researched, the autopsy, including histology,
remains the most accurate means for determining the cause of
death. Autopsy results must be given to clinicians promptly and
reports must contain an adequate summary correlating the clinical
and autopsy findings.
References
J Roulson, E W Benbow, P S Hasleton: Discrepancies between clinical
and autopsy diagnosis and the value of post mortem histology; a metaanalysis and review. Histopathology 2005; 47: 551-559.
Sington JD, Cottrell BJ. Analysis of the sensitivity of death certificates
in 440 hospital deaths: a comparison with necropsy findings. J. Clin.
Pathol. 2002; 55: 499–502.
Roberts ISD, Benbow EW, Bisset R et al. Accuracy of magnetic resonance imaging in determining cause of sudden death in adults: comparison with conventional autopsy. Histopathology 2003; 42: 424–30.
Gas chromatographic detection of endocrinedisrupting chemicals in brain samples from SIUDS
and SIDS victims
T. Pusiol1, L. Roncati2, V. Termopoli3
1
Company of Provincial Health Services, Institute of Pathology, Santa
Maria del Carmine Hospital, Rovereto (TN), Italy; 2 Department of Diagnostic and Clinical Medicine and of Public Health, Section of Pathology,
University of Modena and Reggio Emilia, Modena (MO), Italy; 3 LC-MS
Laboratory, DiSTeVA, University of Urbino, Urbino (PU), Italy
Keywords. Gas Chromatography-Mass Spectrometry (GC-MS);
Endocrine-Disrupting Chemicals (EDCs); Sudden Intrauterine
Unexplained Death Syndrome (SIUDS); Sudden Infant Death
Syndrome (SIDS).
Introduction. Sudden intrauterine unexplained death syndrome
(SIUDS) and sudden infant death syndrome (SIDS) represent one
of the main open issue in the social and scientific setting of the
modern medicine and they are facets of a multifactorial problem.
The National Institute of Child Health and Human Development
(NICHD) strategic plan 2000 has stated that SIDS is a developmental disorder and it takes its origin from fetal development 1.
The neuropathological investigation plays an important role in the
analysis of these deaths. However, only a few studies in this field
have adequately examined the neurological substrates, although
even subtle congenital structural abnormalities of the autonomic
nervous system can determine dysfunctions in the control of vital
functions, leading to unexpected and sudden death 2-4. External
risk factors, such as maternal smoking, alcohol and drugs abuse,
environmental pollution, are known to be potential SIDS and
SIUDS contributors. In the last few years, researches on new
environmental risk factors for fetal and neonatal development
have been directed to the adverse effects of endocrine-disrupting
chemicals (EDCs). Some of these chemicals, such as organochlorine compounds, are well known for being harmful to the
endocrine system. They are able to interfere with important
biological processes in humans and other mammals. In relation
to their lipophilic properties, they are capable of crossing the
placenta and enter the fetal bloodstream, affecting the normal
development of the fetus and causing preterm birth, lower birth
weight, growth retardation, psychomotor and cognitive dysfunctions 5-7. As a result, many current efforts are focusing on the
study of transplacental transfer of EDCs and their determination
in cord blood, serum and maternal adipose tissues, to understand
better the in utero exposure dynamics 8. All the existing literature
clearly demonstrates the adverse effects of the prenatal exposure
to some persistent pollutants. Therefore, the assessment of possible fetal exposures, together with the determination of EDCs in
fetal and newborn brain tissues, is a critical element to establish a
potential correlation with these sudden death syndromes. Several
publications on the detection of polybrominated diphenyl ethers
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(PDBEs) and polychlorinated biphenyl (PCB) in human fetal
liver have been reported 9,10. However, up to now, informations
on the detection of these compounds in brain samples are completely missing. Therefore, the major aim of this study has been
the detection and the quantification of selected EDCs in brain
tissues, and their possible correlation with brain anomalies in
SIUDS and SIDS victims.
Methods. The study has included 24 cases of sudden perinatal
death, in whom a complete autopsy and analysis of the clinical
history did not establish any cause of death. More in detail, 16 SIUDS (age 25-41 gestational weeks) and 8 SIDS (age 2 hours -6.5
months), occurring in the Autonomus Province of Trento (Italy)
and in other neighboring Italian regions during a 3-year period
(2011-2013), have been studied. According to the guidelines provided by the Italian law n¡ 31 of 2 February 2006 ‘governing of the
diagnostic examination on victims of sudden infant death syndrome
and unexpected death of the fetus’ 11, brains of all the victims were
submitted to an in-depth anatomo-pathological examination, particularly of the autonomic nervous system. After removal two fresh
samples of cerebral cortex (0.5-1 cm3) for chemical investigation,
the brainstem and cerebellum, where the main vital centers are
situated (arousal control, sleep-wake transition, cardio-respiratory
input, upper digestive tract regulation, etc.), were standardly processed and embedded in paraffin. Transverse serial sections were
made at intervals of 30 mm. For each level, six/seven 5-µm-thick
sections were obtained, two of which were routinely stained for
histological examination, using alternately hematoxylin-eosin and
KlŸver-Barrera stains. The routine histological evaluation of the
brainstem was focused on: locus coeruleus, parafacial/facial complex, superior olive, retrotrapezoid nucleus, parabrachial/KšllikerFuse complex in the pons/mesencephalon; hypoglossal nucleus,
dorsal motor vagal nucleus, tractus solitarius and ambiguus nuclei,
pre-Bštzinger complex, inferior olive and arcuate nucleus in the
medulla oblongata; intermediolateral nucleus in the spinal cord. In
the cerebellum, the cortex layers (external granular layer, molecular layer, Purkinje cell layer and internal granular layer) and the
medullary deep nuclei (dentate nucleus, fastigial nuleus, globose
nucleus and emboliform nucleus) were examined. Portions of cerebral cortex of the victims were sent to LC-MS Laboratory of the
University of Urbino for chemical characterization. Each defrosted
brain sample was weighed (approximately 0.5 g) and homogenized
with 2 mL of n-hexane to obtain a dense reach supernatant. The
homogenized tissue was transferred into a solid phase extraction
161
(SPE) cartridge containing 500 mg of C18 sorbent, in order to retained most of the matrix impurities and to release the compounds
of interest with hexane. The SPE cartridge was conditioned with 4
mL of n-hexane, before purification step, and it was washed with
1 mL of n-hexane, followed by 1 mL of dichloromethane after elution step. The extraction method was developed and validated in
terms of accuracy, precision, limit of quantification (LOQ), limit
of detection (LOD) and linearity. Nine isotopically labeled internal
standards (ISTDs) were used for method validation. The validated
method was subsequently applied to human tissues from SIUDS
and SIDS victims. A mixture of 20 organochlorine compounds
(EPA CLP mix), chlorpyrifos, chlorfenvinfos, captan, boscalid and
bisphenol A were purchased from Sigma-Aldrich (Milan, Italy).
All solvents used (n-hexane and dichloromethane) were supplied
by Merk (Suprasolv, 99% purity, Merk, Germany). Stock solutions were prepared in n-hexane at 100 µg/mL concentration. A
standard mixture containing all compounds (25 specific EDCs and
9 ISTDs) was prepared by appropriate dilution and stored at 4¡C in
the dark. SPE cartridges HyperSep-C18 (500 mg/6 mL) were purchased by Thermo Scientific (Bellefonte, USA). The analyses on
the extracted samples were performed by an Agilent Technologies
gas chromatograph 6890N equipped with a single quadrupole mass
spectrometer 5975C TAD/MS, working in electron ionization. The
chromatographic separation was carried out using an HP-5MS
(Agilent J&W GC columns, Folsom, CA, USA), i.d. 30.0 m x 0.25
mm, containing 5% phenyl-methylsiloxane, with a phase thickness
of 0.25 µm. Carrier gas was helium at 1 mL/min (constant flow).
The GC oven temperatures were programmed as follows: 80¡C,
held for 1 min, ramped at 30¡C/min to 180¡C, ramped at 3¡C/
min to 225¡C, held for 4 min, ramped at 20¡C/min to 300¡C, held
for 4.08 min (total acquisition time: 25 min). Splittless sample
injection of 1 µL at 250¡C was selected. The transfer line and ion
source temperature were kept at 290¡C and 300¡C, respectively.
Results. In the brainstem the hypoplasia of one or more nuclei
of the raphe system (obscurus, pallidus, median, magnum, caudal
linear raphe nuclei) was the most frequent alteration, seen in 5
SIUDS and in 2 SIDS cases. In the same 5 SIUDS victims, the
raphe nuclei hypoplasia was associated to the hypoplasia of the
facial/parafacial complex in the caudal pons. Developmental
disorders of other nuclei was less common, such as the agenesis
of the arcuate nucleus (Fig. 1), found in 1 SIUDS and 2 SIDS,
the hypoplasia of the retrotrapezoid nucleus, found in 1 SIUDS
and 1 SIDS, and the hypoplasia of the olivary nuclei (the superior
Fig. 1. Medulla oblongata (insert): a well-preserved arcuate nucleus (red arrows) in an infant died for chest crush (A, KlŸver-Barrera staining, original
magnification, 4x) is compared to an arcuate nucleus agenesis (red circles), found in a SIDS victim (B, KlŸver-Barrera staining, original magnification 4x).
162
olivary nucleus in 1 SIUDS and 1 SIDS and the inferior olivary
nucleus in 1 SIUDS). Noteworthy were also the agenesis of the
intermediolateral nucleus in the spinal cord; the hypoplasia of the
Kὂlliker-Fuse complex, of the hypoglossal nucleus and of the solitary nucleus were seldom ascertained. At the level of cerebellum
interesting findings were the cerebellar cortex immaturity, with
regard to the age, and the hyperplasia of the Purkinje cell layer,
observed in 1 SIDS victim. Among the target analytes, some organochlorine pesticides (OCPs), that is α-chlordane, γ-chlordane,
heptachlor, p,p-DDE, p,p-DDT, and the two most common used
organophosphorus pesticides (OPPs), chlorpyrifos and chlorfenvinfos, have been found in 8 and 3 samples, respectively. It is
very interesting to point out the actual presence of two common
used organophosphate pesticides in certain samples. These nonpersistent compounds, also called contemporary-use pesticides,
are currently available in place of organochlorine compounds,
that have been banned since 1980s. Even though they are environmentally non persistent, their extensive use in pest control in
intensive agricultural areas, such as Northern Italy, expose population to continuous low concentrations. Fetuses, newborns and
pregnant women represent the most exposed subjects.
Discussion. From our histopathological results obtained analyzing a 24-case series of sudden perinatal death, it is emerged that
both unexplained fetal and infant deaths share common congenital anomalies of the central autonomic nervous system. Noteworthy in the brainstem of the unexplained fetal death is the frequent
association between the hypoplasia of the facial/parafacial complex and the hypoplasia of the raphe system. In particular, the
hypodevelopment of the parafacial nucleus, consisting of “preinspiratory” neurons with the main function of hierarchical modulation of the breathing circuitry, is an exclusive very frequent
finding in unexplained stillbirths 12. The detection of some EDCs
in 8 positive samples out of 24 fetal and neonatal brain specimens
confirms the possibility of such chemicals to pass from mother to
fetus and to be collected in brain tissue. This is consistent with
literature data that report the presence of these selected pollutants
in other human tissues 9,10. It is recommended to determine these
substances in autopsy findings to establish a possible correlation
with SIUDS and SIDS events. It is also interesting that 8 brain
samples have shown the presence in part-per-billion (ppb) levels
of several OCPs and 3 samples have evidenced the presence of
two OPPs, chlorpyrifos and chlorfenvinfos, the most common
pesticides used in apples cultivation. These findings are in accordance with the widespread presence of persistent contaminants in
intensive agricultural areas represented in Northern Italy.
References
1
U.S. National Institute of Child Health and Human Development
Strategic Plan. From Cells to Selves. U.S. Government Printing Office,
Washington DC, 2000, p. 13.
2
Rodriguez ML, McMillan K, Crandall LA, Minter ME, Grafe MR,
Poduri A, et al. Hippocampal asymmetry and sudden unexpected death
in infancy: a case report. Forensic Sci Med Pathol 2012;8:441-46.
3
Grafe MR, Kinney HC. Neuropathology associated with stillbirth.
Semin Perinatol 2002;26:83-88.
4
Sparks DL, Hunsaker JC. Neuropathology of sudden infant death
(syndrome). Childs Nerv Syst 2002;18:568–92.
5
Ranjit N, Siefert K, Padmanabhan V. Bisphenol A and disparities in
birth outocomes: a review and directions for future research. J Perinatol 2010;30:2-9.
6
Siddiqui MKJ, Srivastava S, Srivastava SP, Mehrota PK, Mathur N,
Tandon I. Persistent chlorinated pesticides and intra-uterine foetal
growth retardation: a possible association. Int Arch Occup Environ
Health 2003;76:75-80.
7
Eszenaki B, Rosas LG, Marks AR, Bradman A, Harley K, Holland N,
et al. Pesticide toxicity and the developing brain. Basic Clin Pharma
Toxicol 2008;102:228-36.
8
Porpora MG, Lucchini R, Abballe A, Ingelido AM, Valentini S, Fuggetta E, et al. Placental transfer of persistent organic pollutants: a
preliminary study on mother-newborn pairs. Int J Environ Res Public
Health 2013;10:669-711.
9
Doucet J, Tague B, Arnold DL, Cooke GM, Hayward S, Goodyer
CG. Persistent organic pollutant residues in human fetal liver and
placenta from Greater Montreal, Quebec: a longitudinal study from
1998 through 2006. Environ Health Perspect 2009;117:605−10.
10
Rawn D, Gaertner D, Sun W, Casey V, Curran I, Cooker G, et al.
Polybrominated diphenyl ethers (PBDEs) in Canadian human fetal
liver and placental tissues. Organohalogen Compd 2011;73:563−66.
11
Legge 2 Febbraio 2006, n¡ 31. Disciplina del riscontro diagnostico
sulle vittime della sindrome della morte improvvisa del lattante (SIDS)
e di morte inaspettata del feto. Gazzetta Ufficiale della Repubblica
Italiana, Serie generale, 2006, p. 4.
12
Lavezzi AM, Matturri L. Hypoplasia of the parafacial/facial complex:
a very frequent finding in sudden unexplained fetal death. Open Neurosci J 2008;2:1-5.
Sala Giotto – 2° piano – 14,30-15,30
Uropatologia
Moderatori: M.P. Gardiman, G. Martignoni
Tumor infiltrating mast cells depend on c-kit
for influencing prostate cancer histotype
specification
L. Bongiovanni1, E. Jachetti2, C. Guarnotta1, A. Gulino1, A. Rigoni2, S. Sangaletti2, M.P. Colombo2, C. Tripodo1
1
Department of Health Sciences, Human Pathology Section, University of
Palermo, Palermo; 2 Molecular Immunology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS “Istituto
Nazionale Tumori”, Milan.
Introduction. The role of mast cells (MCs) as promoters or
inhibitors of tumor development has been widely studied, but
data in prostate cancer are still not conclusive. In TRAMP mice,
which develop spontaneous prostatic cancers, tumor infiltrating
MCs have been demonstrated to be decisive for progression
of well differentiated (WD) prostatic adenocarcinomas while
dispensable for poorly differentiated (PD) adenocarcinomas
(1). It is well known that recruitment ad activation of MCs at
tumor sites are mediated by SCF interaction with its receptor
c-kit expressed on MCs (2). In this setting, MC targeting (treating TRAMP mice with the MC stabilizer Cromolin, or using
MC-deficient KitWsh-TRAMP mice) was observed to prevent
prostatic adenocarcinoma (AC) onset but, on the other hand,
it associated with a higher occurrence of highlyaggressive
neuroendocrine carcinomas (NECs) expressing high levels of ckit (1). The concomitant expression of c-kit on MCs, NE tumor
cells and the prostate stem cells (PSCs) compartment (3) rose
up the idea of a possible function of MCs as homeostatic regulators of PSCs (1,4), via competition for the same ligand (SCF),
and that the absence of MCs could deregulate ordinary PSCs
differentiation into luminal epithelial cell. On these bases, we
investigated the therapeutic effects of the tyrosine kinase inhibi-
COMUNICAZIONI ORALI
163
tor Imatinib on the stromal myeloid components (MCs) favoring
AC growth and on the tumor NE component.
Materials and methods. We used the transgenic adenocarcinoma
prostate mouse (TRAMP) model, in which -426/+28 fragment of
rat probasin (rPB) is gene fused to the early region of SV40 (large
and small T antigens), leading to androgen-dependent oncogene
expression and tumor development from PIN (8-16 weeks) towards
in situ AC (16-24 weeks) and poorly differentiated AC (25-30
weeks) (5). KitWsh-TRAMP mice were generated by crossing
TRAMP mice with MC-deficient KitWsh mice (6). Imatinib (50 mg/
kg) was inoculated intra peritoneum 5 times a week from 8 to 30
weeks of age. Human prostate cancer samples were gathered from
the archives of the Human Pathology Section of the University of
Palermo School of Medicine. Histopathologic and immunohistochemical (IHC) analyses were carried out on formalin-fixed and
paraffin-embedded specimens. MC distribution and density on
mouse tissues were estimated by staining slides with toluidine blue,
MCs were counted out of 5 high-power microscopic fields (400x),
and counts were expressed as average. Confocal microscopy
analysis was carried out on slides from OCT embedded prostates
stained with Draq5 and DAPI (blue), Ki67, cytokeratin 8 (Ck8),
p63, or Synaptophysin. Sections were analyzed with a Microradiance 2000 (Bio-Rad Laboratories) confocal microscope equipped
with Ar (488 nm), HeNe (543 nm), and red diode (638 nm) lasers.
Confocal images (512 Å~ 512 pixels) were obtained using a 20Å~,
0.5 NA Plan Fluor DIC or 60Å~, 1.4 NA oil immersion lens and
analyzed using ImageJ software.
Results. In our series 55% of TRAMP mice developed infiltrating
ACs and only a minor fraction (5,26%) developed NECs (figure
B). In contrast 66% of KitWsh-TRAMP mice analyzed did not develop ACs, but showed a high incidence of NECs (25%). Chronic
treatment of TRAMP with Imatinib led to a lower incidence of
ACs, with 44,4% of mice carrying in situ AC and without signs
of infiltrating AC. Moreover, Imatinib administration was associated with a high incidence of NECs (33,3%), which was
similar to that of KitWsh-TRAMP mice. Double IF analysis at an
early time-point in tumor development (16 weeks) showed a high
proliferation index (Ki-67+ cells) of basal cells (p63+) and of NE
cells (Synaptophysin+) in the prostates of KitWsh-TRAMP mice
compared to TRAMP mice, where instead the majority of proliferating cells were, as expected, of luminal cytotype (CK8+, Ki67+). These data suggest a marked propensity in KitWsh-TRAMP
mice to an early differentiation of PSCs into NE cells. Treatment
with Imatinib also influenced the intensity and composition of
inflammatory peritumoral infiltrates: except for MCs, we did
not find appreciable differences in the inflammatory infiltrates
of TRAMP and KitWsh-TRAMP mice, which usually showed
moderate mixed inflammatory infiltrates rich in neutrophils and
eosinophils. In contrast, Imatinib-treated mice showed poor peritumoral inflammation with a prevalence of lymphocytes. Evaluations of toluidine blue stained sections in untreated TRAMP mice
showed accumulation and degranulation of MCs preferentially
around PIN and WD ACs areas, while MCs were virtually absent
in NECs (Figure A). Imatinib treatment did not affect the average
MCs density nor their degranulation status. IHC analysis of c-kit
on mice samples showed an expected positivity on MCs and on
neoplastic cells of NECs. We also performed c-kit staining on
human prostatic ACs with different grade of malignancy and on
prostatic NEC. Normal prostatic epithelium showed the presence
of intraepithelial c-kit positive cells (PSCs or NE cells) in addition to rare stromal MCs. Besides stromal tumor infiltrating MCs,
c-kit analysis on well-differentiated ACs showed the presence of
scattered c-kit positive cells into the neoplastic glands. Poorly
differentiated carcinomas showed only the presence of stromal
positive MCs while we did not find positivity of neoplastic cells.
Human prostatic NEC showed a diffuse positivity for c-kit similar
to that of NEC developing in mice (Fig. 1).
Discussion. Expression of c-kit has been demonstrated on mouse
and human prostatic NECs by in vitro assays, IHC and gene
expression profiles, the latter showing that c-kit is part of NE
molecular signature (1). Oncogenic tyrosine kinases are ideal
druggable targets as inhibition of their catalytic activity resulted
in the induction of apoptosis or senescence of the neoplastic
cell (7), thus we used the tyrosine kinases inhibitor Imatinib
mesylate to affect c-kit pathway on both MCs and neoplastic NE
cells, to hamper the occurrence of AC and to protect, on the other
hand, against NEC onset. However, in our series, Imatinib-treated
mice developed aggressive NE tumors with similar frequency
and histological appearance of KitWsh-TRAMP mice, suggesting
that c-kit signaling pathway could be dispensable in prostatic NE
neoplasms or that NE neoplastic cells may develop mechanisms
of Imatinib resistance that deserve further investigation. Recent
studies also highlighted the essential contribution of the immune
system for Imatinib action (8). Imatinib has been demonstrated to
induce a cytotoxic CD8+ T immune response against the tumor
cells along with apoptosis of infiltrating regulatory T cells (Tregs)
Fig. 1. On the top, toluidine blue stained sections showing degranulated MCs around a WD AC in a TRAMP mouse (fig.1, 200x) and MCs
absence in a NEC of an Imatinib-treated mouse (fig.2, 200x). On the
bottom, IHC analysis for c-kit in human prostatic tumors showing
scattered stromal MCs infiltrating a PD AC (fig.3, 400x) and c-kit expression of neoplastic cells in NEC (fig.4, 200x). Insets, magnification
400x.
Fig. 2. Frequency of prostatic lesions occurred in TRAMP, KitWshTRAMP and Imatinib-treated TRAMP mice. Of note, all ACs arisen in
Imatinib-treated TRAMP mice were in situ ACs.
164
in a GIST mouse model (7). Other studies demonstrated a role
of NK cells (9) and of tumor associated macrophages (10) in the
Imatinib-driven antitumoral immune response. These findings are
in line with our evidence of predominantly lymphoid inflammatory infiltrates in Imatinib-treated TRAMP mice differing from
the mixed, neutrophil- and eosinophil-rich, infiltrates of TRAMP
and KitWsh-TRAMP mice. Since in previous studies Imatinib response has been correlated with the skewing of T cell infiltrates
towards cytotoxic effectors (i.e. positive TCD8+/Treg ratio) (7),
we could hypothesize that Imatinib failure in controlling NEC
could be at least in part due to an unfavorable immune infiltrate
polarization, which also deserves further investigations. In conclusion, our results reveal that within the microenvironment of
nascent prostate cancers elements with different sensitivity to
c-kit inhibition may be identified. Targeting of c-kit by Imatinib
treatment can alter the complex cross-talk between transforming
prostate cells and bystander mast cells towards suppression of
adenocarcinoma development and unpredicted fostering of highly
aggressive neuroendocrine cancers.
References
1
Pittoni P, Tripodo C, Piconese S et al. Mast cell targeting hampers
prostate adenocarcinoma development but promotes the occurrence
of highly malignant neuroendocrine cancers. Cancer Res. 2011 Sep
15;71(18): 5987-97.
2
Huang B, Lei Z, Zhang GM et al. SCF-mediated mast cell infiltration
and activation exacerbate the inflammation and immunosuppression
in tumor microenvironment. Blood. 2008 Aug 15;112(4):1269-79.
3
Leong KG, Wang BE, Johnson L, Gao WQ. Generation of a prostate
from a single adult stem cell. Nature 2008;456:804–8.
4
Pittoni P, Colombo MP. The dark side of mast cell-targeted therapy in
prostate cancer. Cancer Res. 2012 Feb 15;72(4):831-5.
5
Greenberg NM, DeMayo F, Finegold MJ et al. Prostate cancer in a
transgenic mouse. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):343943.
6
Grimbaldeston MA, Chen CC, Piliponsky AM et al. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo. Am J Pathol. 2005 Sep;167(3):83548.
7
Balachandran VP, Cavnar MJ, Zeng S et al. Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the
inhibition of Ido. Nat Med. 2011 Aug 28;17(9):1094-100.
8
Zitvogel L, Kroemer G. Anticancer effects of imatinib via immunostimulation. Nat Med. 2011 Sep 7;17(9): 1050-1.
9
#PSH$5FSNF.5BrFC+FUBMNovel mode of action of c-kit tyrosine
kinase inhibitors leading to NK celldependent antitumor effects. J Clin
Invest. 2004 Aug;114(3):379-88.
10
Cavnar MJ, Zeng S, Kim TS et al. KIT oncogene inhibition drives
intratumoral macrophage M2 polarization. J Exp Med. 2013 Dec
16;210(13):2873-86.
Overcome in part heterogeneity in clear cell renal
cell carcinoma: a second-look for chromosomes
9p and 14q for retargeting patients affected by
metastases
M. Brunelli, S. Knuutila, L. Cheng, S. Pedron, C. Ghimenton, M.
Chilosi, A. Scarpa, G. Martignoni
Introduction. Losses of chromosomes 9p and 14q are associated
with worse outcomes in patients affected by clear cell renal cell
carcinoma (RCC) and are helpful for prognostic risk stratification.
Both chromosomal loci harbor several hot spot molecular pathways
suitable for targeted therapies. Intratumor heterogeneity may foster
tumor adaptation and therapeutic failure. We sought to investigate
the presence of losses of the hot spots of chromosomal loci 9p and
14q in primary clear cell RCC and matched metastatic tissues.
Methods. CD10 and CD13 were performed on 7 cases of clear
cell RCC with hematogenous tissue metastases. Cytogenetic
FISH analysis was performed on primary and matched metastatic
tissues by using specific probes mapping the 9p and 14q loci.
Results. Loss of chromosome 9p was observed in 85% of both
primary clear cell RCCs and in matched metastases; 14% showed
discordance between primary and metastases showing gains. Loss
of chromosome 14q was observed in 58% of both primary and
matched metastases. Only 3/7 (42%) did show equal status of loss
of chromosome 14q.
Conclusion. Heterogeneity of cytogenetic status between metastatic
and primary clear cell RCCs is observed for loss of chromosome 14q
rather than chromosome 9p. The impact of chromosome 14q cytogenetic status, harboring the HIF1 driven gene for angiogenesis, may
drive efficacy of targeted inhibitors whereas loss of chromosome 9p,
harboring many hot spot genes, seems related to the metastatic behavior per se, without cytogenetic modulation. Re-profiling metastatic
tissue for re-targeting patients affected by metastatic RCC may be
proposed to overcome resistance to targeted inhibitors.
Cathepsin-k is espressed in a subset of castration
resistant prostate adenocarcinoma and
peritumoral stroma and may be considered as a
therapeutical target for patients at risk for bone
metastases
M. Brunelli, A. Caliò, A. Eccher, C. Ghimenton, G. Riva, M.
Chilosi, G. Martignoni
Introduction. Cathepsin-k plays a role in osteoclast-mediated
bone degradation and it may be also produced by cancer cells that
metastasize to bone. Cathepsin-k actively contributes to stromal
remodeling and invasion and is also expressed in the peritumoral
stroma of adenocarcinomas arising from different organs. Metastatic adenocarcinoma to the bone from patients affected by
castration-resistant prostate cancer is a topic of interest for new
suitable biomarkers predictive of responsiveness to chemo- or targeted therapies. We sought to investigate cathepsin-k in a cohort
of advanced castration-resistant prostate adenocarcinoma.
Methods. 16 patients affected by castration resistant prostate
adenocarcinoma were recruited with at least 5 years of followups. Cathepsin-k (Abcam; clone 3F9, 1:2000) was evaluated
immunophenotypically on formalin-fixed and paraffin embedded
tissue. High 2+ (>30% of cells), low 1+ (<30%) and zero (absence) were scored and distinguishing neoplastic cells and cells from
the peritumoral stroma. Low (1+) and absence (0) of scoring was
interpreted as negative and 2+ a positive evaluation.
Results. All prostate adenocarcinoma showed at least Gleason
score 8 at prostatectomy. Cathepsin-k was scored positive in 2/16
(12%) neoplastic tumour cells and in 5/16 (31%) peritumoral
stroma. The neuroendocrine and acinar subtypes of adenocarcinoma scoring positive also developed bone metastases after
respectively four and five years and subsequently died.
Conclusions. Patients affected by castration-resistant prostate adenocarcinoma may be tested for cathepsin-k molecule and patients
scoring high 2+ positivity may be candidate to promising targeted
therapeutical treatment such as odanacatib or in combination with
zoledronic acid. As cathepsin-k inhibitors are being developed for
cancer therapy, mainly for bone metastases, our study expands the
spectrum of cancer that potentially could be responsive.
Testing mTOR pathway active biomarkers in clear
cell renal cell carcinoma: strategy and methods
to be used to stratify patients for more likely
response to mTOR inhibitors
M. Brunelli, C. Fiorini, S. Pedron, S. Sanavio, C. Zampini, M.
Ficial, M. Chilosi, A. Scarpa, G. Martignoni
Department of Pathology and Diagnostics, University of Verona, Italy.
Introduction. mTOR inhibitors play an important role in the
targeted treatment of clear cell renal cell carcinoma, however no
COMUNICAZIONI ORALI
pre-selection of patients is available to predictive high efficacy
to targeted inhibitors in the metastatic setting. Different potential
mechanisms may also lead to resistance to mTOR inhibitors. Evidence of activation of predictive biomarkers by molecular testing
on formalin-fixed and paraffin embedded cancerous tissue blocks
does also depend to different technique used. The visualization
of active mTOR pathways has not been tested and compared on
the same cohort of patients by using simultaneously the most
common techniques such as immunohistochemistry, Western blot
and immunofluorescence analysis. We sought to investigate and
compare the capability of three major techniques in evidencing
active function of molecules related to mTOR pathways on renal
cell carcinoma.
Methods. 16 patients affected by clear cell renal cell carcinoma
were recruited. Immunohistochemichal (IHC), Western blot
(WB) and immunofluorescence (IF) were performed by using
the following molecules for active detection of mTOR pathways:
mTOR, ph mTOR, p70S6k, ph p70S6k polyclonal and monoclonal, ph S6 Rb and ph 4EBP1.
Results. No one molecules was simultaneously revealed by all three techniques. ph p70S6k monoclonal was detected by two (IHC
and IF) methods. The other molecules were detected by only one
technique as follows: IHC showed the sensitivity to detecting the
expression of ph mTOR (Ser 2448) and ph p70S6K (Thr389)
molecules, WB was the sensitive method to detect the expression
of p70-S6K molecules (Thr389), ph S6Rb and ph 4EBP1 ones
(Thr37/46) and the IF was sensitive in detecting the expression
of mTOR (Ser235/236).
Conclusions. 1) there is a significant difference in detecting
mTOR pathway’s active molecules by using three major techniques such as IHC, WB and IF analysis; 2) the methods to detect
active molecules of the mTOR pathways is important when justifying responsiveness or resistance to targeted drugs; 3) clinical
trials need an agreement for standard methods on tissue in order
to correlate mTOR biomarkers and clinical end-points.
Prognostic role of the Receptor Tyrosine Kinase
AXL in Renal Cell Carcinoma
F. Collina1, F. Morelli1, L. La Sala1, E. La Mantia1, S. Scala3, G.
Botti 1, S. Pignata2, R. Franco1
Pathology Unit, National Cancer Institute “Fondazione G. Pascale”,
Naples, Italy; 2 Urology Unit, National Cancer Institute “Fondazione G.
Pascale”, Naples, Italy; 3 Immunology Unit, National Cancer Institute
“Fondazione G. Pascale, Naples, Italy
1
Background. Renal Cell Carcinomas (RCC) is characterized
by a complex molecular biology and an unpredictable behavior.
The only curative treatment of RCC is surgery and, in cases of
metastatic RCC, systemic therapies such as immunotherapy are
performed. Since 2005, a series of tyrosine kinase inhibitors have
been approved by the FDA for the treatment of advanced stage
RCC. The Axl proto-oncogene is a tyrosine kinase receptor member of TAM family and is activated by growth arrest-specific
gene-6 (Gas 6) ligand. Little is known about the role that this
receptor plays in the RCC. It is known that Axl is overexpressed
in RCC cell lines and tissues,[1] that the Axl activation, in vitro,
induces cell growth and MAPK pathway activation.[2] Moreover,
Axl mRNA overexpression were strongly correlated with worse
prognosis.[3,4]
Aim. The aim of this work was to evaluate, in vivo, the protein
expression on a series of 166 RCC samples and its potential
association to clinicopathological features and outcome. Furthermore, Axl was correlated to the apoptotic index and to its downstream proteins expression (PI3K, pAkt, pNF-kB p65, pERK and
pSTAT3) and to the alterations present at gene level.
Methods. The samples were included in a prognostic Tissue
MicroArray and the proteins expression analysis was performed
by immunohistochemistry. To detect the apoptotic index, was
165
Fig. 1. Citoplasmic and membrane Axl expression (40x). a) Axl immunonegativity in clear cell RCC. Axl stains the stroma; b) Axl immunopositivity in clear cell RCC; c) Axl immunonegativity in Papillary RCC; d)
Axl immunopositivity in Papillary RCC.
Fig. 2. Low and high expression of Axl downstream proteins pERK,
PI3K, pAKT, pNF-kB p65 and pSTAT3. (40x)
166
Fig. 3. Low and high apoptotic index in RCC samples. (40x) The brown
staining identifies the apoptotic cells, whereas the non apoptotic cells
are stained with methyl green.
used the TUNEL method, instead the gene amplification analysis was conducted by FISH. All the statistical analysis were
carried out using the SPSS version 20.0 software (SPSS Inc.,
Chicago, USA). The association between Axl expression and the
clinicopathological parameters was conducted using the χ2 Test.
Progression free survival (PFS) and Overall survival (OAS) was
calculated using the Kaplan–Meier method.
Results and conclusions. The Axl expression directly correlated
with the T (p = 0.004), nuclear Furhman grade (p = 0.009) and
with PFS (p = 0.02). The association with receptor downstream
proteins showed that Axl correlated statistically with pERK (p =
0.006), PI3K (p= 0.001) and pNF-kB p65 (p = 0.018). Moreover,
there was a statistical trend between the Axl expression and
cytoplasmic pAKT expression (p = 0.06) and a trend of inverse
correlation with the apoptotic index, indicating the probable activation of proliferative and antiapoptotic pathways. FISH analysis
revealed 5 cases with gene amplification, 18 copy gains, 10 trisomic, and 100 diploid for Axl, but the statistical analysis showed
no correlation between the amplification and protein expression.
Therefore, Axl might be used as a prognostic factor in RCC and,
given that an Axl inhibitors series have already been developed
and that there are ongoing clinical trials, this receptor could be a
new target in RCC treatment.
Reference
1
Chung BI, Malkowicz SB, Nguyen TB et al. Expression of the
proto-oncogene Axl in renal cell carcinoma. DNA Cell Biol. 2003
Aug;22(8):533-40.
2
Gustafsson A, Bostrom AK, Ljungberg B et al. Gas6 and the Receptor
Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma. PLoS One
2009;4(10):e7575.
3
Gustafsson A, Martuszewska D, Johansson M, et al. Differential
expression of Axl and Gas6 in renal cell carcinoma reflecting tumor
advancement and survival. Clin Cancer Res 2009;15(14):4742-9.
4
Linger RM, Keating AK, Earp HS. Taking aim at Mer and Axl receptor tyrosine kinases as novel therapeutic targets in solid tumors.
Expert Opin Ther Targets. 2010 October; 14(10): 1073–1090.
Prolaris CCP score assessed on pre-operative
formalin-fixed paraffin-embedded biopsies
stratifies risk for prostate cancer
L. Daniele1, M. Barale2, D. Pacchioni1, L. Delsedime1, D. Galliano1, P. Gontero2, A. Sapino1
Dipartimento di Scienze Mediche, Università di Torino; 2 Dipartimento
di Scienze Chirurgiche - Divisione di Urologia, AOU Città della Salute e
della Scienza di Torino
1
Introduzione. Prostate cancer has a highly variable natural history and an accurate assessment of the tumour aggressiveness
based on clinical and pathological features can be challenging.
The cell cycle progression (CCP) score (Prolaris, Myriad Genetic
Laboratories, Inc.) has been proven to hold prognostic value in
predicting disease progression in various clinical settings by
utilizing biopsies, TURP and surgical specimens. The aims of
our study were: i) to evaluate the expression of the CCP score
in a series of pre-operative prostatic biopsies; ii) to correlate the
results of the molecular test with the immunohistochemical (IHC)
expression of know prognostic markers (Ki67, p53, ERG) and
with the clinico-pathological parameters of the lesions on radical
prostatectomies.
Metodi impiegati. We selected 57 preoperative prostatic biopsies
formalin-fixed and paraffin-embedded (FFPE) with a histological
diagnosis of adenocarcinoma and a minimal extension of the lesion of 0.5 mm. RNA was extracted and subjected to CCP score
analysis (assessment of quantitative expression of 31 cell cycle
progression genes and 15 housekeeping genes). Subsequently,
the CCP score was compared with the clinico-pathological parameters of the American Urological Association (AUA) and new
risk classes were obtained. Finally IHC analysis of Ki67, P53 and
ERG was performed and results compared to the CCP score.
Risultati. An adequate quantity of RNA for CCP score evaluation was obtained in 54/57 biopsies. The CCP score ranged from
1.8 and 1.7. By comparing the results with the AUA risk classes,
27% of the cases were categorized as more aggressive while 23%
was labelled as less aggressive. The proliferation index (Ki67)
ranged from 2% to 37%, p53 and ERG expression was detected in
46% and 50% of cases, respectively. There was a significant association between high Ki67 and a high prognostic risk as assessed
by the molecular test (p=0.0005). In addition, ERG positivity was
significantly associated with a low prognostic risk (p=0.002).
Finally, within the subgroup of cases that underwent radical
prostatectomy (52% of the whole cohort), 70% with a pathological staging higher than the clinical staging assessed properatively
showed a higher prognostic risk against 22% of the cases with the
same pathological staging (p=0.037).
Conclusioni. The CCP score is a molecular test feasible in very
small amount of FFPE tissues and is able to create new prognostic
risk classes for prostate cancer.
The evaluation of glomerulitis and interstitial
inflammation is clinically relevant in IgA
nephropathy
P. Viola1, G. Lattanzio2, L. Centurione1, P. Felaco1, T. D’Antuono2,
R. Di Pietro1, M. Bonomini1, F.B. Aiello1
Dipartimento di medicina sperimentale e scienze dell’invecchiamento,
Università “G. d’Annunzio” Chieti; 2 ASL2 Lanciano-Vasto-Chieti, UO
Anatomia Patologica, Ospedale “SS Annunziata” Chieti
1
Introduction. IgA Nephropathy (IgAN), the most common
form of glomerulonephritis in the world, is characterized by a
high variable course ranging from a totally benign condition to
progressive long-term renal failure. Approximately 25 to 30%
of patients reach end-stage renal disease (ESRD) after 10 years.
The variability in clinical course justifies the efforts to identify
histological and clinical features that predict the development of
renal failure.
The pathogenesis of IgAN is not completely clear, it is well
accepted, however, that an aberrant glycosylation of IgA is involved. Galactose-deficient IgA1 molecules are recognized by
anti-glycan (or anti-IgA1) IgG and the resultant immune complexes are too bulky to enter the space of Disse in the liver. The
IgA1-containing immune complexes escaping normal clearance
mechanisms reach the renal circulation, pass through the larger
fenestrae in the glomerular capillaries and overly the mesangium.
Their binding to CD89 expressed on the surface of mesangial
cells leads to the production of pro-inflammatory cytokines, recruitment of circulating cells, and inflammation.
Histologically the most common alteration in IgAN is mesangial
proliferation and matrix expansion, but this is not specific and
immunofluorescence studies are necessary for the diagnosis. A
predominant deposition of IgA is observed mainly within the
mesangial region, with focal paramesangial or subendothelial
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COMUNICAZIONI ORALI
extension. Ultrastructurally, electron dense deposits are present
predominantly within mesangial regions of glomeruli. Importantly, the biopsies meeting these criteria present with a variety
of histological patterns ranging from a minimal lesions to a diffuse proliferative glomerulonephritis. There are still grey zones
regarding the identification of patients with IgAN most likely to
progress. In addition, no causal therapy is available for IgAN and
its treatment is not well defined. Studies targeting histological
and immunohistochemical features in renal transplant patients
have demonstrated that the degree of the glomerulitis, which in
humoral rejection is significantly associated with the deposition
of the C4d fragment of the complement component is of prognostic value in terms of graft survival. We have therefore performed
a retrospective study investigating the histological and clinical
relevance of these additional markers in IgAN patients.
Methods. Glomerulitis was studied in 32 IgAN biopsies, classified according to the Oxford and to the 3-G classifications, and
in 12 Minimal Change Disease (MCD) biopsies (time interval
2003-2012). Follow up clinical data (mean follow up: 45.8 ±
19.1 months) were available for 21 IgAN patients. CD68 immunostaining was performed in all cases and the number of CD68
positive glomerular and interstitial macrofages was evaluated.
C4d immunostaining was performed in all cases, and 2 biopsies
of lupus nephritis served as positive controls. For evaluation of
glomerular C4d (Gl-C4d) staining biopsies showing C4d positive
endothelial cells in more than 50% of glomeruli were considered
GlC4d positive. Peritubular C4d (PTC-C4d) staining was considered positive when more than 50% of peritubular capillaries
showed C4d staining.
Results. No significant differences in age (35.59 + 11.69 vs 39.50
+ 10.81) and male/female ratio (21/11 vs 6/6) were observed
among the IgAN and the MCD groups. The number of glomerular
and interstitial macrofages in IgAN biopsies was significantly
higher than in MCD biopsies (P < 0.001 and P < 0.001, respectively).
The number of interstitial, but not that of glomerular macrophages positively correlated with the severity of the disease according both to the Oxford and to the 3-G classifications.
In IgAN patients with serum creatinine level < 1,3 mg/dl at the
time of biopsy a significantly lower number of
interstitial macrophages was observed (P < 0.006). In addition,
an inverse correlation was found between the number of interstitial macrophages and the Glomerular Filtration Rate (GFR) (r
= - 0.463, P < 0.008) at the time of biopsy. Importantly, similar
results were observed at the end of the follow up.
Gl-C4d or PTC-C4d deposition was not observed in IgAN or in
MCD biopsies, whereas Gl-C4d positivity
(diffuse and segmental), but not PTC-C4d positivity was present
in the 2 lupus nephritis biopsies.
Conclusions. Gl- or PTC-C4d positivity was not present in any
of the 32 IgAN or of the 12 MCD biopsies, while diffuse GLC4d positivity was observed in both cases of lupus nephritis.
This finding may be helpful in the differential diagnosis between
mesangial glomerulonephritis, in particular when histological and
immunofluorescence data overlap.
The inverse correlation between the number of interstitial macrophages and the GFR, and the positive
correlation with Oxford and 3-G classifications scoring suggest
that a high number of interstitial macrophages are indicative of a
more severe nephropathy.
References
Batal I, Lunz III JG, Aggarwal N, et al. A critical appraisal of methods to
grade transplant glomerulitis in renal allograft biopsies. Am J Transplant 2010;10:2442-52.
Papadimitriou JC, Drachenberg CB, Munivenkatappa R, et al.Glomerular
inflammation in renal allograft biopsies after the firstyear: cell types
and relationship with antibody-mediated rejection andgraft outcome.
Transplantation 2010;90:1478-85.
Magil AB. Infiltrating cell types in transplant glomerulitis: relationshipto
peritubular capillary C4d deposition. Am J Kidney Dis 2005;45:10849.
Aiello FB, Furian L, Della Barbera M, et al. Glomerulitis and endothelial
cell enlargement in C4d+ and C4d−acute rejections of renal transplant patients, Human Pathology (2012) 43, 2157–2166.
Regele H, Exner M, Watschinger B, et al. C4d deposition is associatedwith inferior kidney allograft outcome independently of cellularrejection. Nephrol Dial Transplant 2001;16:2058-66.
Manno C, Strippoli GFM, D’Altri C, et al. A Novel Simpler Histological
Classification for Renal Survival in IgA Nephropathy: A Retrospective
Study. Am J of Kid Dis, Vol 49, 6, 2007:763-775.
A Working Group of the International IgA Nephropathy Network and the
Renal Pathology Society. The Oxford classification of IgA nephropathy: pathology definitions, correlations, and reproducibility. Kidney
International (2009) 76, 546–556.
Sala Botticelli – 2° piano – 08,30-09,30
Apparato digerente - 1
Moderatori: F.P. D’Armiento, V. Villanacci
HER2 status in gastric and gastro-oesophageal
junction cancer: primitive versus metastatic
lesions
M. Amato, G. Perrone, D. Righi, C. Pellegrini, M. Pandolfi, R.
Coppola, A. Onetti Muda
Università Campus Bio-Medico di Roma, Laboratorio di Diagnostica molecolare e Citogenetica, UOC Anatomia Patologica.
Introduction. HER2 status assessment in histological samples
of gastric and gastro-oesophageal junction carcinoma is essential
to determine which patients might benefit from trastuzumab
therapy. Trastuzumab-based therapy is used to treat metastatic
disease, but HER2 amplification status is often evaluated in the
primary tumor. However, the exact relationship in terms of HER2
amplification status between bioptic and surgical resection specimens, or between primary and metastatic tumors has not been
fully clarified. Aim of our work was to evaluate the concordance
rate between primary gastric cancer and distant metastatic lesions
in terms of HER2 status.
Materials and methods. We evaluated 38 cases of gastric cancer
with distant metastatic disease, selected on the basis of availability of primary and metastatic tissue. Biopsy specimens from
primary tumor were also included in 25 cases. HER2 amplification status was assessed first by standard IHC according to the
HER2 scoring system for gastric cancer. Equivocal cases (IHC
2+ score) were evaluated by FISH using the SPEC HER2/CEN
17 Dual Color Probe Kit (Zytovision).
Results. HER2 amplification (IHC 3+ and IHC 2+/FISH+) was
168
observed in 9/38 (23,7%) specimens from metastatic lesions. On
the other hand, HER2 amplification was observed in 6/38 (15,8%)
primary tumors (surgical and/or biopsy specimens). Concordance
between primary and metastatic HER2 status was 91% (k-value
0,82). In particular, three patients with metastatic HER2 positive
lesions were found HER2 negative in the respective primary
cancers and one patient with primitive HER2 positive lesions
was found negative in metastatic disease. Moreover, HER2 status
was analyzed in 16 cases both on bioptic and surgical specimens;
concordance status was 83% (k-value 0,66): two HER2 positive
lesions on biopsy resulted negative on surgical specimens and one
HER2 negative biopsy resulted positive on respectively surgical
specimen. Interestingly, these three patients resulted HER2 positive on their metastatic disease.
Conclusions. In line with our previous data (G. Perrone ed al.,
Histopathology 61:1;134-5, 2012), results showed that there is a
good concordance rate in terms of HER2 status between primary
and metastatic lesions, as well as in biopsy and surgical removed
specimens. However, a higher rate of HER2 positive status was
found in metastatic lesions (23,7%) rather than in the related
primary cancer (15,8%). These results are similar to data recently
reported on breast cancer (Fabi A, et al. Clin Cancer Res. 2011)
and suggest that HER2+ distant metastasis may arise from a
small, undetectable HER2+ subclone within primary disease. In
conclusion, our data underline the importance of testing, whenever possible, HER2 status of metastatic sites in patients who are
candidates for trastuzumab-based therapy.
Microscopic Residual tumor after
pancreaticoduodenectomy for cancer: preliminary
results of a multicentric prospective randomized
trial
G. Perrone, D. Campani, P. Spaggiari, M. Amato, R. Coppola,
A. Onetti Muda
UOC Anatomia Patologica, Università Campus Bio-Medico di Roma
Introduction. Resection margin and lymph-node status after
Pancreatico-Duodenectomy (PD) for cancer are the most powerful prognostic factors. Recent studies showed that a standardized
pathological evaluation of the specimen increases the rate of
microscopic residual tumour (R1) and nodal metastases after PD
for cancer. We report the preliminary results of a multicentric
prospective randomized study that compared standardized Vs
non-standardized methods to evaluate the surgical specimen.
Material and methods. Surgical specimens after PD performed
for ampullary, biliary and pancreatic carcinoma were eligible for
inclusion. Four different pancreatic surgery Units took part into
the study: Campus Bio-Medico University of Rome; Humanitas
Institute of Milan (Surgeons: M. Montorsi, A. Zerbi); University
of Pisa (Surgeons: U. Boggi, F. Vistoli), University of Katowice
(Surgeon: S. Mrowiec). Eligible cases were randomly allocated
to receive two different methods of pathological evaluation:
GROUP A: the specimen was evaluated with the standardized
method (Verbeke CS et Al. in Br. J. Surg. 2006; 93; 1232–1237)
and GROUP B: the specimen was evaluated with the method
conventionally used by each centre. The primary endpoint of
the study was to verify if the standardized method determines a
significant variation of R1 rate. The secondary endpoints were the
evaluation of “T” and “N” status, number of examined blocks and
number of examined lymph-nodes in the two groups.
Results. From March 2013 until now, we randomly allocated 29
and 27 specimens to be managed with the standardized and the
conventional method respectively. At final histology: 13 cases
(23.2%) resulted ampullary cancer (group A: 9 patients, group B:
4 patients, p = n.s.); 8 cases (14.3%) resulted distal common bile
duct cancer (group A: 5 patients, group B: 3 patients, p = n.s.);
35 cases (62.5%) resulted pancreatic head ductal adenocarcinoma
(group A: 15 patients, group B: 20 patients, p = n.s.). Mean size
of the tumor was 2.59 cm (group A: 2.43 cm, group B: 2.77 cm,
p = n.s.). R1 resection rates were 58.6% (group A) and 40.7%
(group B) (p < 0.05). Average number of examined blocks was
44,4 (group A) and 23,4 (group B) (p < 0.05). Mean number of
examined lymphnodes was 34,8 (group A) and 21,9 (group B) (p
< 0.05). Metastatic lymphnodes were found in 72.4% (group A)
and in 63.0% (group B) (p < 0.05).
Discussion. This is the first prospective randomized study focusing the role of pathological evaluation of the specimen after PD
for cancer. Preliminary results of our study confirm that the use
of the standardized method increases the incidence of R1 resections, the number of examined lymph-nodes and the rate of nodal
metastases assuming that at least two third of PD for cancer can
not be considered “radical resections”.
Gastroesophageal reflux disease among pediatric
patients: difficulties on finding a suitable
histopathological score
S. Campione1, P. Quitadamo2, M. Giordano1, R. Ippolito1, E.
Miele2, A. Staiano2, F.P. D’armiento1, M. D’Armiento1
Department of Advanced Biomedical Sciences, Section of Pathology,
University of Naples Naples, Italy “Federico II”; 2 Department of Translational Medical Sciences, Section of Pediatrics, University of Naples
“Federico II”, Naples, Italy
1
Background. Gastroesophageal reflux disease (GERD) is a common disease clinically characterized by a constellation of symptoms and signs, most often regurgitation, vomiting, retrosternal
pain, heartburn and dysphagia. On the basis of endoscopy, pH
testing and pathology, different disorders are distinguished:
erosive esophagitis (EE), with endoscopy evident lesions; non
erosive reflux disease (NERD), either with histopathological or
esophageal pH alterations; functional heartburn (FH), without
any organic alteration. Distinctions between these groups is important for the assessing of therapy and follow up of the patients.
Histopathology is a validated tool in these patients’ differentiation. GERD is also common among pediatric patients. However,
pediatric diagnostic protocols are different. One of the difference
is based on the scanty help that comes from histopathological
analysis. The aim of our study was to try the enforceability of a
histopathological validated score among pediatric patients and to
match it with a validated clinical severity scoring system.
Material and methods. 84 children, aged between 2 and 17 years
were selected from physicians of the Pediatric clinics of the University of Naples Federico II. All children had symptoms and signs
suspected to be related with GERD and underwent careful clinical
examinations, completed the PSGQ clinical severity score and
esophageal endoscopy. During endoscopy, 4 biopsies were taken
from the distal esophagus. All biopsies were sampled in the same
container and fixed in formalin 4%. Care was taken at the moment
of paraffin embedding, so that biopsies could be well oriented for
histological examination. All sections were Haematoxylin-eosin
stained and observed by 2 blinded pathologists. Firstly, a different
possible diagnosis was excluded, such as infectious or eosinophilic
esophagitis. Secondly, the Yerian Fiocca score was applied. Attention was made on basal cell hyperplasia, papillae elongation, dilatation of intercellular spaces, presence of intraepithelial eosinophils
or neutrophils. A severity score for each point and a combined
severity score was applied according to the author’s instructions:
three different levels, from 0 to 2, where 2 is the most severe.
Results. Of the 84 analyzed patients, 6 had both clinical and histopathological data suggestive of eosinophilic esophagitis; 9 were
not suited for histopathological score evaluation; in 30 cases the
result was 0, in 34 was 1, in 5 was 2. Histopathological results
compared with clinical score are evidenced in picture. No statistical correlation between clinical severity score and histopathological score was found.
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COMUNICAZIONI ORALI
Hepatic Epitelioid Hemangioendothelioma
Diagnosis By Fine Needle Cytology And “Small
Biopsy” And Review Of The Literature
S. Campione1, M. Giordano1, V. Papacciuoli1, I. Cozzolino2, P.
Mainenti1, V. D’Alessandro2, M. D’Armiento1
Departments of Advanced Biomedical Sciences and of Public Health2,
Federico II University, Naples
1
Discussion. Histopathological analysis is relevant in assessing
GERD and in distinguishing it from FH among adult patients.
Moreover, it is necessary to exclude other causes of esophageal
diseases, such as infectious or systemic disease, that could present as GERD and mimic it. A histopathological severity score can
also be useful to address therapy and follow up. This has been
validated and is of current use among adult patients. Not the same
is considered among pediatric patients. Current guidelines in
diagnosis, therapy and follow up of pediatric patients admit that
histopathological analysis can be not helpful in pathology assessing. Our experience follows this trend, since we also did not find
any correlation between clinical severity and histopathological
findings. Different explanations should be found for these unsatisfactory results. Firstly, clinical diagnosis of GERD in pediatric
patients is different from adults: guidelines stress the relevance of
history and clinical examination in assessing the diagnosis. Probably most relevant complaints in children are related to functional
disorders, and this can explain the lack of evident histopathological alterations. Secondly, validated scores for adult patients must
not necessarily fit for pediatric patients. In the past some studies
have highlighted the role of different histopathological elementary lesion, but contrasting results have been produced. However,
investigation correlated with clinical severity must be continued
in order to find new useful histopathological elementary lesions.
Finally, histopathological assessment of pediatric patients is
harder: specimens are smaller and more difficult to orientate.
References
Yerian L., R. Fiocca, L. Mastracci et al. Refinement and Reproducibility
of Histologic Criteria for the Assessment of Microscopic Lesions in
Patients with Gastroesophageal Reflux Disease: the Esohisto Project
Dig Dis Sci 2011 Sep;56(9):2656-65.
Savarino E., P. Zentilin, L Mastracci. Microscopic esophagitis distinguishes patients with non-erosive reflux disease from those with functional heartburn. J Gastroenterol. 2013 Apr;48(4):473-82
Kleinman L., S. Nelson, S. Kothari-Talwar. Development and psychometric evaluation of 2 agestratified versions of the Pediatric GERD
Symptom and Quality of Life Questionnaire. J Pediatr Gastroenterol
Nutr. 2011 May;52(5):514-22
Vandenplas Y., Cd. Rudolph, C. Di Lorenzo. Pediatric gastroesophageal reflux clinical practice guidelines: joint recommendations of the
North American Society for Pediatric Gastroenterology, Hepatology,
and Nutrition (NASPGHAN) and the European Society for Pediatric
Gastroenterology, Hepatology, and Nutrition (ESPGHAN). J Pediatr
Gastroenterol Nutr. 2009 Oct;49(4):498-547
Introduction. Hepatic Epithelioid Hemangioendothelioma
(HEHE) is a rare vascular neoplasms with unpredictable malignant potential. Although US, TC and MR features are well
established, a correct preoperative diagnosis is most of the times
mandatory in order to choose the proper therapeutic actions.
Case report. The patient is a 54-year-old woman who presented
a persistent pain in the right hypochondrium and weight loss.
An abdominal ultrasound examination and, subsequent, CT scan
show several focal, confluent hepatic lesions. An ultrasoundguided fine-needle aspiration (US-FNA) is performed on largest
lesion located in segment IV-V. A Diff Quik smear is evaluated
on site for adequacy and preliminary diagnostic hypothesis.
The other smears are alcohol fixed and Papanicolaou stained.
A second pass is suspended in formalin for paraffin-embedded
cell block and immunocytochemical study. The antibodies for
CKAE1AE3, CK7, CK19, CD31, CD34 and pS-100 are used.
The smears show a moderate cellularity composed by cells
individually dispersed or arranged in small groups; the cells
mainly show an epithelioid aspect. The epithelioid cells have
a round/oval nuclei with abundant eosinophilic cytoplasm with
ill-defined borders; some cells show a tufted aspect. In the
background benign hepatocytes are observed. Immunostained
cell block sections show CD34 positivity and CKAE1AE3,
CK7, CK19 and pS-100 negativity. A cytological diagnosis of
vascular proliferation with epithelioid component is performed.
Afterwards, during a mini-laparotomy a increased liver size
with multiple nodules without signs of disease in the rest of
the abdomen is revealed and a hepatic “small biopsy” (SB) of
2,2x1,5x1,1cm is performed. Histological sections stained with
hematoxylin and eosin show a mesenchimal proliferation of
epithelioid and dendritic cells, with pleomorphic nuclei, partly
arranged in solid areas, partly in loose areas where vascular-like
spaces surrounded by a dense fibrous stroma are observed. Focally trapped liver tissue is recognized. The immunophenotype
of the lesion is CD34 and FVIII-RAg positive and CK7, Hepatocyte and CK20 negative. The hepatic epithelioid hemangioendothelioma diagnosis is performed. The patient is actually in
good general condition and waiting for hepatic transplantation;
repeated total CT scan do not show any signs of metastasis.
Discussion. HEHE is a very rare neoplasm of vascular origin
with non specific and variable clinical manifestation. The tumors
usually present as multiple nodular lesions involving both lobes
of the liver simulating a metastatic disease. A multidisciplinary
approach provided by FNA, SB and imaging (US, CT and MR),
is essential for a correct diagnostic approach and the most appropriate therapeutic choice.
References
Makhlouf HR, Ishak KG, Goodman ZD. Epithelioid hemangioendothelioma of the liver: a clinicopathologic study of 137 cases. Cancer.
1999; 85(3):562-82.
Jurczyk M, Zhu B, Laskin W, Lin X. Pitfalls in the diagnosis of hepatic
epithelioid hemangioendothelioma by FNA and needle core biopsy.
Diagn Cytopathol. 2014;42(6):516-20.
170
Differential diagnosis between bile duct adenoma
and intrahepatic cholangiocarcinoma: an
immunohistochemical algorithm
R. Cappellesso1, C. Mescoli1, P. Capelli2, C. Luchini2, F. Sivieri1,
S. Sarcognato1, M. Rugge1, M. Guido1
Department of Medicine, University of Padova, Padova, Italy; 2 Department of Pathology and Diagnostics, University of Verona, Verona, Italy
1
Background. The differential diagnosis between intrahepatic
cholangiocarcinoma (CC), an aggressive liver tumor, and bile
duct adenoma (BDA), an indolent lesion, is fundamental but
sometimes challenging, mainly in the biopsy context. Often,
morphological analysis alone is not sufficient to achieve a correct final diagnosis and must be sustained by clinical features.
Recently, several immunohistochemical markers, such as p53,
p16, and S100P, have been proposed as helpful tools to improve
the diagnostic performances. However, singularly these markers
show a low sensitivity and are not used in the routine. The aim of
this study was to evaluate whether the combination of these three
immunohistochemical markers may help to distinguish intrahepatic CC from BDA.
Materials and methods. Overall, the resected specimens of 30
intrahepatic CC and 21 BDA were retrieved from the archives
of the Pathology Units of the University of Padova and of the
University of Verona. Immunohistochemical analysis of p53,
p16, and S100P was performed in all cases. p53 was considered
positive when a moderate to strong nuclear immunoreaction was
observed, whereas positive p16 and S100P stain was defined by
the presence of cytoplasmic immunostaining in more than 5%
of tumor cells. An algorithm was built and its diagnostic performance analysed.
Results. Positive immunostaining for p53 was observed in 56.7%
of CC and in none of the BDA. S100P stained 23.3% of CC,
whereas it was totally absent in all BDA. p16 was positive in
73.3% of CC and in all BDA samples. When taken individually,
the markers showed low sensitivity (56.7%, 26.6%, and 23.3%,
for p53, p16, and p53, respectively), although the specificity was
excellent (100% for all). However, if considered sequentially (as
shown in figure 1), these markers allowed to distinguish 22 out
of 30 CC from the BDA, reaching a sensitivity of 73.3% and a
specificity of 100%, with a PPV of 100% and a NPV of 72.4%.
Fig. 1. The diagnostic algorithm allowed the correct identification of
22 out of 30 CC.
Conclusions. A diagnostic algorithm based on the combined
immunostaining of p53, p16, and S100P is helpful in differentiating intrahepatic CC from BDA. Further larger studies should be
performed to validate the proposed algorithm.
The clinical impact of HER2-status assessment in
gastric cancer
R. Cappellesso1*, M. Fassan1,2,3*, G. Mazzoleni4, E. D’Amore5,
M. Barbareschi6, M. Pizzi1, M. Guido1,7, M. Rugge1
Department of Medicine (DIMED), Surgical Pathology & Cytopathology Unit, University of Padua, Padua, Italy; 2 Istituto Oncologico Veneto
- IOV-IRCCS, Padua, Italy; 3 Comprehensive Cancer Center, The Ohio
State University, Columbus, OH, USA; 4 Service of Pathology, Regional
General Hospital, Bolzano, Italy; 5 Department of Pathology, San Bortolo
Hospital, Vicenza, Italy; 6 Unit of Surgical Pathology, Laboratory of Molecular Pathology, Trentino Biobank, S. Chiara Hospital, Trento, Italy; 7
Pathology Department ULSS15 Alta Padovana; Padua-Italy, Italy; * The
first two authors contributed equally
1
Background. Among Gastric (Gc) and Gastro-Esophageal (GEc)
adenocarcinomas, HER2-status discriminates patients who may
benefit from HER2-targeted therapy. Thus, the reliability of its
assessment is crucial. Currently, in most laboratories immunohistochemistry (IHC) represent the first diagnostic step for screening
cases with a score 0 or 1+, which are hence excluded from any
further evaluation. In cases showing an IHC score 2+, HER2 gene
amplification must be demonstrated by in situ hybridization (ISH)
to be assimilated to IHC score 3+ cases and treated. However,
IHC analysis is affected by pre-analytical variables (manly fixation) and by a frankly pathologist-dependent assessment with low
inter- and intra-observer reproducibility. Aim of this study was
to evaluate HER2 amplification prevalence and the consistence
its current detection protocol in a large series of gastric cancers.
Materials and methods. A multi-Institutional archival series of
1,040 radically treated gastric adenocarcinomas (824 Gc and 216
GEc) was retrieved. 720 synchronous lymph node metastases
were also considered. In total, 5,426 neoplastic tissue microarray
cores were analyzed. HER2-status was assessed by dual-color
chromogenic in situ hybridization (CISH) and by 2 different immunohistochemical (IHC) protocols (CB11 and 4B5).
Results. Overall, 114/1.040 (11%) cases were HER2-amplified
(in 6/114 cases, amplification only involved lymph node metastasis). As currently assessed, IHC excluded the large majority of
non-amplified cases but lacked the identification of about 10% of
HER2-amplified cases. In particular, the CB11 protocol showed
a score 0 in 4/114 (3.5%) and a score 1+ in 9/114 (7.9%) CISHpositive cases. The 4B5 was scored as 0 in 1/114 (0.9%) and 1+
in 6/114 (5.3%) HER2-amplified cases. Overall, CB11 presented
a sensitivity of 88.6%, a specificity of 96.9%, a PPV of 77.7%,
and a NPV of 98.6%, whereas for 4B5 they were 93.9%, 96.1%,
78.8%, and 99.2%, respectively. By lowering the threshold of eligibility for ISH analysis to score 1+, HER2-testing would reach
a more clinically acceptable sensitivity of 96.5% (CB11) and
99.1% (4B5). Accordingly with this novel system, about 20% of
all gastric cancer should be analyzed also by ISH.
Conclusions. In this large Gc/GEc series, the prevalence of
HER2 amplification was low (11%). Thinking to a Western world
context in which gastric cancer is a relatively rare tumor with a
poor prognosis, the opportunity to perform ISH analysis also to
score 1+ patients should be carefully considered.
171
COMUNICAZIONI ORALI
Sala Botticelli – 2° piano – 14,30-15,30
Apparato digerente - 2
Moderatori: G. Lanza, G. Zamboni
Poorly differentiated clusters (PDCs) grading
system predicts unfavorable prognosis in stage IIA
colorectal carcinomas
G. Branca1, L. Reggiani Bonetti2, A. Ieni1, G. Tuccari1, V. Barresi1
Dipartimento di Patologia Umana “G. Barresi”, Policlinico “G. Martino”, Messina, Italia; 2 Dipartimento Att. Integr. di Lab., Anat. Pat. e Med.
Leg., Policlinico Universitario di Modena, Modena, Italia
1
Introduction. The histological grade of colorectal carcinoma
(CRC) is generally assessed as codified by the World Health
Organization (WHO), by taking into account the percentage of
glandular component in the tumour. This grading system shows
a significant inter-observer variability, which may limit its prognostic value. Recently, a novel grading system based on the
counting of poorly differentiated clusters (PDC) in the tumour
stroma has been proposed; this grading system seems to have
higher inter-observer reproducibility and prognostic significance
than the WHO one.
According to the current (7th) edition of the UICC TNM (tumor,
node metastasis) Classification of Malignant Tumours staging
CRCs in stage II are tumours that spread into subserosa or into
non peritonealized pericolic or perirectal tissue (pT3), or tumours
that perforate visceral peritoneum and/or directly invade other
organs or structures (pT4), in the absence of regional lympho
nodes spread (N0) and of distant metastasis (M0). Stage II is
further subdivided in stage IIA, IIB and IIC, respectively, and the
5-year survival rate ranges from 75% to 95%. Due to its overall
good prognosis, controversies do exist on the benefits of adjuvant
5-fluorouracil based chemotherapy in stage II CRC. However a
percentage of these tumours do recur during the follow-up. Hence
there is the need to identify histopathological markers able to
predict the clinical course of these neoplasias in order to select
those tumours with higher probability to recur and which could
be submitted to chemotherapy. On this premise, the aim of the
present study was to assess the prognostic value of PDC grading
system in stage IIA CRC.
Materials and methods. Fifteen surgically resected TNM Stage
IIA (pT3N0Mx) CRCs, which occurred between 2002 and 2004
and which were characterized by disease progression/relapse
within five years from the initial diagnosis, were taken from the
Cancer Registry of the province of Modena, Italy, and included
in the present study. Then, a comparable number of 25 stage IIA
CRCs occurred in the same years at the Department of Human
Pathology of University of Messina, with no evidence of disease
relapse/progression were selected. Finally, the cohort of the
study included a total of 40 cases (age range: 48-90 years; mean
age: 69,55 years). None of the patients received chemotherapy
for their cancer. In all the cases, pathological staging had been
performed according to the TNM system. The maximum size in
centimeters had been noted for each tumour (range 1.8-12.0 cm;
median: 4 cm). According to their localization, 21 CRCs were
located in the right colon including caecum, ascending colon,
and transversum and 19 in the left colon, including descending
colon and sigma. All cases had been formalin-fixed paraffinembedded for the histological evaluation with haematoxylin and
eosin (H&E) stain. For each case, we revised the H&E-stained
slides and assessed the histological grade, according to the WHO
criteria, as well as lymphovascular invasion (LVI), tumour bud-
ding, perineural invasion, pattern of growth at the invasive margin of each tumour (infiltrative or pushing) and myenteric spread
(MS). LVI was established by using immunohistochemistry with
podoplanin (D2-40) as target antigen and recorded when at least
one tumour cell cluster was clearly visible inside a D2-40 positive vessel, as previously extensively reported. Tumour budding
was defined as isolated single cancer cells or clusters of cells
composed of less than five elements in the stroma of the actively
invasive margin of the tumour. After choosing one field where
the budding was the most intensive, a budding count was made
underx200 magnification, with a count of less than five foci considered negative and a count of five or more as positive, according to literature data. For each case, the count and the histological
grading based on PDC counting were assessed on haematoxylin
and eosin stained sections prepared from a longitudinal tissue
sample of the whole tumour including its deepest part. In each
specimen, PDC were quantified according to the procedure previously described. In detail, the whole tumour was firstly scanned
at a lower power magnification to identify the area with the
highest number of PDC. Then, the clusters were counted under
the microscopic field of a x20 objective lens (i.e., a microscopic
field with a major axis of 1 mm), by using a Zeiss microscope. At
least three sections per patient were examined and the maximum
number of PDC found in all sections was considered for grading.
More specifically, cancers with <5 PDC, 5 to 9 PDC, and ≥10
clusters were classified as grade 1 (G1), grade 2 (G2) and grade 3
(G3) respectively, as previously suggested. MS was assessed on
haematoxylin-eosin (H&E) slides as well as by the immunohistochemical detection of Auerbach’s plexus cells using the neural
marker S-100. CRC was considered to be positive for when a
horizontal cancer spread along the Auerbach’s plexus zone was
evident at invasive cancer front.
Chi-squared test was carried out to assess whether any significant
difference in the various clinico-pathological parameters (site,
size, WHO grade, tumour border configuration, tumour budding, peri-neural infiltration, LVI, PDC grade, PDC presence,
MS) was present between the recurring and not recurring cases.
Disease-free survival was assessed by the Kaplan-Meier method,
with the date of primary surgery as the entry data. The end point
was characterized as the length of survival to disease progression
(metastatic disease) of colorectal carcinoma. Follow-up ranged
between 9 and 162 months. The Mantel-Cox log-rank test was
applied to assess the strength of association between survival time
and each of the parameters (site, size, WHO grade, tumour border
configuration, tumour budding, peri-neural infiltration, LVI, PDC
grade, PDC presence, MS) as a single variable. A probability (P)
value less than 0, 05 was considered statistically significant. Data
were analysed using the SPSS package version 6.1.3 (SPSS Inc.,
Chicago, IL, USA).
Results. According to the WHO grading system, 2 CRCs (5%)
were well-differentiated (G1), 27 (67%) were moderately differentiated (G2), and 11 (28%) were poorly differentiated (G3).
When we used PDC grading system, 28 CRCs (70%) were G1,
6 (15%) were G2 and 6 (15%) were G3, respectively. LVI was
present in only 2 CRCs (5%). With reference to the pattern of
neoplastic growth, 26 CRCs (65%) showed an infiltrative invasive front, while 14 CRCs (35%) exhibited a pushing invasive
pattern. Furthermore, tumour budding was evident in 13 CRCs
(33%) out of 40, and perineural invasion in 6 (15%). Finally, only
172
10 CRCs (10%) revealed a MS. Chi-squared test showed that the
presence of PDC and mainly higher PDC grade were significantly
more frequent among the cases characterized by disease progression (P= 0.0036; P= 0.0219). No significant differences in the
other clinico-pathological parameters were found between the
two groups of CRCs. Univariate analysis revealed that presence
of PDC as well as PDC grading were significantly negative prognostic parameters associated with a shorter disease-free interval
in stage IIA CRC (P= 0.0011; P= 0.0003)
Conclusion. Our data demonstrated the relevant negative prognostic value of PDC grading assessment for Stage IIA (pT3N0Mx) CRCs. We suggest that this grading system may be used
as a predictor of stage IIA CRC clinical behavior. If our findings
are confirmed in larger cohorts of tumours, PDC grading might
represent a useful histological marker to select aggressive cases
which may benefit from adjuvant chemotherapy.
clinico-pathological; 7. Ki67 index: manual versus digital evaluation; 8. group G3 NET; 9. histology diagnostic report and 10.
multidisciplinary group of experts.
Conclusion. In this preliminary retrospective analysis, prospective and epidemiologic study, overall agreement between grade
and differentiation was good. In according to the literature G3
ENETS GEP could constitute a heterogeneous subgroup of NEN
as regards diagnosis, prognosis and treatment. Our preliminary
study representing a pioneering effort to provide the medical
community in our territory with basic information on a number
of patients with GEP-NETs.
GEP-NETs: a clinicopathologic analysis of 55 cases
Università Campus Bio-Medico di Roma, UOC Anatomia Patologica
A. D’Amuri1, F. Floccari2, S. Senatore2, S. Leo3, G. Crisman4
Background. Over the past 2 decades, endoscopic ultrasonography (EUS) coupled with fine-needle aspiration biopsy (FNA) has
become an effective tool to define suspected pancreatic lesions.
Also, the advent of neoadjuvant therapies for pancreatic cancer
has increased the need for unequivocal histological diagnosis.
However, the reported diagnostic accuracy of EUS-FNA in solid
pancreatic lesions ranges between 62% and 96%, with significant
variability in terms of sensitivity and specificity. In contrast to
breast and thyroid pathology, a standardized reporting system for
cytological examination of solid pancreatic lesions is lacking, and
this absence could account for such discrepancy. Here we report
the results of a large series of FNA solid pancreatic mass samples
classified in a standardized five-class diagnostic system , based
on literature data as well as on personal experience.
Methods. 487 EUS/FNAC samples of solid pancreatic mass were
reassessed using 5 clinically relevant pathologic categories:
PANC1: non diagnostic\inadequate (scant cellularity, smearing
artifacts, obscuring blood).
PANC2: negative for neoplasm (normal acinar and/or ductal
epithelium).
PANC3: atypical/inconclusive (mild to moderate cell atypia,
with a low suspicion of malignancy, often in an inflammatory
background).
PANC4: suspicious for carcinoma (strongly suggesting malignancy but cytological features are not sufficient in terms of quantity and/or quality for a definitive diagnosis).
PANC5: diagnostic of carcinoma (adenocarcinoma, metastatic
disease, neuroendocrine tumors).
Histological confirmation or clinical follow up (at least 6 months)
was available for 417 cases. Sensibility, specificity, accuracy,
positive and negative predictive value were calculated.
Results. Applying the above-mentioned classification, 251/487
FNA (51.5%) resulted as PANC5, 67/487 (13.7%) as PANC4,
42/487 (8.6%) as PANC3, 72/487 (14.7%) as PANC2 and 55/487
(11.3%) as PANC1. Considering the 417 cases with clinical
follow-up, a final diagnosis of carcinoma was obtained in 100%
of PANC5, 100% of PANC4, 50% of PANC3, 32.7% of PANC2,
67.7% of PANC1 cases. Overall false-positive and false-negative
rate was 0% and 8.3%. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 90.5%,
100%, 100%, 61%, 91.7% respectively.
Conclusions. EUS-FNA-based cytological examination of solid
pancreatic lesions is an accurate procedure for diagnosis and
planning of therapy. Atypical/inconclusive diagnosis (PANC3)
may have a detrimental effect on clinical management of patients.
In such cases, supplementary diagnostic techniques, such as tumor marker analysis, could be employed on the same samples in
order to enhance the EUS-FNA diagnostic yield. Of note, benign
FNA findings (PANC2) do not necessarily exclude the pres-
Anatomia Patologica P.O. “Sacro Cuore di Gesù” Gallipoli, ASL LECCE,
Gallipoli (LE), Italia; 3U.O. Oncologia - GELP UNIT, P.O. “Vito Fazzi”
Lecce, ASL LECCE, Lecce, Italia; 4U.O. Anatomia Patologica, Azienda
Ospedaliera di Desenzano del Garda, Desenzano del Garda (BS), Italia
1
Introduction. Gastroenteropancreatic neuroendocrine tumors
(GEP-NETs) are diagnostically challenging entities, as they comprehend a rare and heterogeneous group of epithelial neoplasm
with neuroendocrine differentiation that can arise in all organs
derived from the primordial gut. Histopathology allows the distinction of two categories: poorly differentiated aggressive carcinomas and well-differentiated neoplasm. In contrast to the 2000
WHO classification of NET in which morphologic differentiation
was the first criterion, the 2010 WHO classification of NET is
based mostly on histological grade. NET are now classification
into three main categories: NET G1 (mitotic count <2/10 HPF
and/or ≤2% Ki67 index), NET G2 (2-20/10 HPF and/or 3-20%),
and NEC of small or large cell type. While NET G1 and G2 are
well-differentiated tumors, NEC are considered poorly differentiated G3 tumors. We looked at the agreement between grade and
differentiation to determine whether all GEP-NET can be readily
classified according to the 2010 WHO classification and compared the histopathological criterion of the two WHO classification
(2000 vs 2010).
Methods. We performed a preliminary retrospective analysis,
prospective and epidemiologic study of all newly diagnosed
GEP-NETs at the Tricase (LE) and Gallipoli (LE) Hospitals
between 2005-2014 years. A research of the files of the Pathology
Unit of the two Hospitals was carried out to retrieve available
data on all newly diagnosed GEP-NETs cases with the two classifications (WHO 2000 and 2010). The investigators have indicate morphologic differentiation (according to WHO 2000) and
elements of histological grade (mitotic index, Ki67 proliferation
index), according to ENETS and WHO 2010.
Results. Fifty-five (55) GEP-NETs tumors were diagnosed in
ours institutions from January, 2005 to May, 2014. The most
common primary tumor site is the stomach (20 cases) followed
by colo-rectal (14 cases), small intestine (jejunum and ileum)
(11 cases), appendix (7 cases) and pancreas (3 cases). In according to the previous 2000 WHO classification and 2010 WHO
and ENETS classifications were subdivide and reclassified
in NET G1 (26 cases), NET G2 (16 cases) and NEC G3 (13
cases). Critical points discoveries have been: 1. discrepancies
between mitotic count and Ki67 proliferation index; 2. correct
grading in biopsy versus surgical specimens; 3. misdiagnosis;
4. intratumoral heterogeneity in proliferation rate; 5. appropriate
neuroendocrine immunohistochemical markers; 6. correlations
Definition of a cytological standardized reporting
system in EUS-FNA solid pancreatic masses
G. Deda, G. Perrone, D. Borzomati, F. Di Matteo, G. Nappo, R.
Coppola, A. Onetti Muda
173
COMUNICAZIONI ORALI
ence of pancreatic malignancy, and repeated sampling should be
considered whenever the presence of malignancy be suspected
clinically. In summary, a uniform reporting system for pancreatic FNA would facilitate communication among pathologists,
surgeons, radiologists and other health care providers, allowing
easy and reliable sharing of data from different laboratories for
collaborative studies.
Obliterative portal venopathy in subjects without
portal hypertension: an unknown planet
M. Guido1, S. Sarcognato1, A. Sonzogni2, M. Senzolo3, S. Fagiuoli4, M. Pizzi1, L. Giacomelli1, G. Colloredo5
Department of Medicine-DIMED, General Pathology & Cytopathology
Unit, University of Padova; Padova; 2 Pathology and 4 Gastroenterology
Units, Ospedali Riuniti, Bergamo; 3 Multivisceral Transplant Unit, Department of Surgery, Oncology and Gastroenterology, Padua University
Hospital, Padua; 5 Liver Unit, Seriate Hospital, Bergamo. Italy
1
Background and aims. Obliterative portal venopathy (OPV)
represents a spectrum of histological lesions traditionally associated to non-cirrhotic portal hypertension (NCPH). Recently, OPV
has been observed in the absence of clinical signs of portal hypertension and a proportion of the affected subjects subsequently
developed portal hypertension. Thus early recognition of OPV
by pathologists is advisable, which should prompt clinicians to
search for immune-mediate or prothrombotic conditions. Pathological diagnosis of OPV is usually easy in symptomatic subjects.
However, lesions may be often so subtle that the liver biopsy may
be read as being normal. Therefore, in the absence of a high index
of suspicion, the diagnosis of OPV might be underestimated. This
retrospective study aimed to assess the prevalence of OPV in
patients without clinical signs of portal hypertension (PH), who
underwent liver biopsy (LB) for long-lasting abnormal liver function tests (LFTs) of unknown etiology.
Methods. Four-hundred eighty-two consecutive LBs were blindly
reviewed in order to evaluate the presence of portal vessel abnormalities belonging to the spectrum of OPV: phlebosclerosis,
aberrant portal vessels (APVs), portal vessel fragmentation (PVF),
intrahepatic portal vein thrombosis (PVT). In positive biopsies,
other lesions usually reported in NCPH were also assessed (i.e.
portal fibrosis/inflammation, incomplete fibrous septa, sinusoidal
dilatation/fibrosis, abnormal central veins, lobular necrosis/inflammation, and nodular regenerative hyperplasia). For each case, the
histological features were matched with clinical/laboratory findings.
Results. OPV-related lesions were identified in 96/482 (19.9%)
patients (M/F ratio: 0.92; mean age 41±10.8 years) and were
mostly associated with mild LFTs abnormalities. Interval between first detection of LFTs abnormalities and LB ranged from
6 to 336 months (median= 48). Screening for serum autoantibodies turned positive in 49% of 88 tested patients, while 5.9% of
51 tested cases had a pro-thrombotic condition (factor V Leiden
mutation [n= 1], MGUS [n= 1], protein C alteration [n= 1]). No
cases were associated with HIV-infection or exposure to vascular
injury-inducing toxins. In 13.5% of cases, OPV was associated
with systemic and/or organ-specific immune disorders (Hashimoto’s thyroiditis [n=4], Basedow’s disease [n= 2], rheumatoid
arthritis [n= 1], celiac disease [n= 2], psoriasis [n= 1], Sjogren’s
disease [n= 1], inflammatory bowel disease [n= 1], multiple sclerosis [n= 1]). APVs and PVF were the most prevalent vascular
changes (96.9% and 74%), while phlebosclerosis and PVT were
only rarely reported (9.4% and 1.1%). Among the other evaluated
lesions, sinusoidal dilatation and portal fibrosis were most frequently documented (65.6% and 60.4%, respectively). Nodular
regenerative hyperplasia was detected in 9.4% of cases.
Conclusions. OPV changes are fairly common among patients
with unexplained LFT abnormalities, even in the absence of
PH. In the present series, OPV was frequently associated with
histological and/or clinical findings known to occur in NCPH,
suggesting that the patients may be in an early pre-clinical phase
of NCPH. This condition is probably largely underestimated in
the daily practice by both clinicians and pathologists.
Dilated intercellular spaces in eosinophilic
esophagitis
M. Salemme1, V. Villanacci2, M. Cadei3, M. Fuoti4, G. Gennati5,
A. Ravelli6
1
Deparment of Pathology – Spedali Civili, Brescia (Italy); 2 Deparment
of Pathology – Spedali Civili, Brescia (Italy); 3 Deparment of Pathology –
Spedali Civili, Brescia (Italy); 4 Gastroenterology and GI Endoscopy Unit,
University Department of Pediatrics, Children’s Hospital– Spedali Civili,
Brescia (Italy); 5 Gastroenterology and GI Endoscopy Unit, University
Department of Pediatrics, Children’s Hospital– Spedali Civili, Brescia
(Italy); 6 Gastroenterology and GI Endoscopy Unit, University Department of Pediatrics, Children’s Hospital– Spedali Civili, Brescia (Italy)
Introduction. Dilated intercellular spaces (DIS) in the esophageal epithelium can be induced by acid and reduced by proton
pump inhibitors (PPI), and are thus considered a marker of gastroesophageal reflux disease (GERD). Over the years, however,
DIS have also been reported in esophagitis unrelated to GERD.
Because DIS have never been formally measured in eosinophilic
esophagitis (EoE), we aimed at detecting and measuring DIS in
EoE before and after nutritional or pharmacological therapy.
Methods. In 22 children with EoE DIS were measured by morphometry and transmission electron microscopy (TEM), before
and after treatment with topical steroids (n=16) and/or exclusion
diet (n=13). Thirty children undergoing upper GI endoscopy with
biopsy for non-esophageal disorders acted as controls.
Results. In controls the mean (±SD) number of esophageal eosinophils was 0.91(±0.47) and the mean DIS values were 0.62(±0.08)
μm at morphometry and 0.33(±0.24) μm at TEM. In patients
with EoE, the mean (±SD) number of esophageal eosinophils
decreased from 31.8(±6.96) to 6.64(±5.01) (p<0.0001), and the
mean DIS values decreased from 2.26(±0.21) to 1.23(±0.20) μm
at morphometry (p<0.0001), and from 2.24(±0.28) to 0.98(±0.19)
μm at TEM (p<0.0001), respectively before and after treatment.
Conclusions. DIS are a prominent morphological feature of EoE,
where they can be significantly reduced by an appropriate non-PPI
therapy.
Clear cell adenocarcinoma of the prostate and
colon. An unusual case and a review of the
literature
S. Sina, C. Zanella, E. Baritono*, A. Remo*, A. Fioravanzo, E.
Manfrin, M. Chilosi
Università degli Studi di Verona-Ospedale di Borgo Roma;
Patologica Ospedale “Mater Salutis” Legnago, Verona
*
Anatomia
Primary clear cell adenocarcinoma of the colon is a rare entity.
Elderly men, particularly in their left sited colon, are more commonly affected than women. We report the case of a 78-yearold man who presented to Mater Salutis Hospital in Legnago,
Verona, with anemia, weight loss and associated symptoms of
bowel sub-occluding. Before the admission, during an urological
exploration due to a total PSA dosage of 30mg/ml, multiple prostate biopsies were performed and the diagnosis of acinar adenocarcinoma of the prostate with clear cell aspects was formulated.
A therapy with LH-RH analogues was proposed to the patient.
During the current admission an abdominal CT-scan confirmed
the presence of a lesion sub-occluding the lumen in the cecumascending colon. Serological CEA, CA19-9, a -fetoprotein were
within normal limits. A right hemicolectomy with ileo-anastomosis was performed. The postoperative course was regular.
The histological colon examination revealed a clear cell adenocarcinoma infiltrating the serosa and the radial margin. The tumor
grade was poorly differentiated with vascular, but no perineural
174
invasion. The cut margin was negative. Lymph node were negative 0/12. (T4 N0 Mx).
In the suspect of a possible metastasis from the prostate clear cell
adenocarcinoma, immunohistochemical stainings of the colonic
adenocarcinoma were performed, thus showing a diffuse positivity of neoplastic cells for cytokeratin 20, CEA, CDX2, E-Caderin,
but the negativity for cytokeratin 7, vimentin and PSA.
A skilful review of the prostate adenocarcinoma, added of immunohistochemical stainings positive for PSA and negative for
cytokeratin 20 and CDX2, confirmed the previously diagnosis of
a clear cell adenocarcinoma, with a prostate primitivity.
After surgery, our patient completed 8 cycles of chemotherapy
sec. Schema (Xelox day 1,8,22). CT-scan staging showed no
progression of the colonic disease.
Clinical data and immunohistochemistry are crucial for establishing the origin of the neoplastic cells. A positivity for cytokeratin
20 and CDX2 is highly specific and sensitive for the enteric
origin. In our case, the association of enteric and prostate immunohistochemical markers have been essential in solving a so
unusual case.
More research and information about clear cell adenocarcinoma
of the colon is needed, especially in regard to prognosis and in
order to provide the best treatment option.
Carcinosarcoma of the colon: report of a case
V. Terrinazzi1, P. Pretelli1, C. Caporalini1, M. Rotellini1, L. Novelli2, C.E.Comin1, L. Messerini1
Department of Experimental and Clinical Medicine, Section of Specialistic Surgery and Histopathologic and Molecular Diagnosis, University
of Florence, Florence, Italy 2Unit of Histopathology, Department of Biomedicine, Careggi Hospital, Florence, Italy.
1
Introduction. Carcinosarcoma is a rare malignant tumor. This
tumor occurs in various anatomic locations such as head and
neck, respiratory tract, female urogenital system and other sites.
Carcinosarcoma of the colon was first reported by Weider and
Zekan in 1986. Since then, less than 20 cases have been reported.
Carcinosarcoma is a biphasic tumor characterized by a mixed
composition of carcinomatous and sarcomatous areas. Carcinosarcoma has a tendency to distantly metastasize and shows dismal
prognosis. We report a case of carcinosarcoma of the colon with
an osteoid component.
Patient and methods. The patient was a 83 years old woman
with severe oppression in the lower abdomen and an increased
abdominal distention developed in a short time. Instrumental examinations revealed a large abdominal mass. The clinical suspicion was a gastrointestinal stromal tumor (GIST). During surgery,
intraoperative frozen sections of tumor samples revealed a poorly
differentiated malignancy. The patient underwent large bowel resection with a complete surgical resection of the neoplastic mass.
Macroscopically, the lesion measured 40x20x16 cm. and was
elastic and soft with multiple hemorrhagic areas, grossly infiltrating the surrounding adipose tissue. The tumor appeared to arise
from the left colonic wall. Representative sections of the mass
were fixed in 10% buffered formalin and embedded in paraffin.
Sections 4 µm thick were then stained with hematoxylin-eosin.
Immunohistochemical studies were performed on representative
sections. The following antibodies were applied: cytocheratins
(CK7, CAM5.2), desmin, smooth muscle actin, CD34, DOG1,
CD117, D2-40, CD34, synaptofisin and calretinin.
Results. Pathological examination of the resected specimen
showed a biphasic proliferation composed of spindle and polygonal cells. The spindle cells had round to ovoid nuclei with
coarse chromatin pattern, distinct nucleoli and were organized in
a fascicular pattern. The polygonal cells had large bizarre nuclei
and abundant eosinophilic glassy cytoplasm. Histologically, the
tumor showed anaplastic features and did not make any organoid
or glandular pattern. Solid areas built up of spindle cells with focal areas of osteoid differentiation were also present. The tumor
showed immunohistochemical positive stain for cytocheratins
(CK7, CAM5.2) in the polygonal cells and for desmin in the
spindle cells. The other markers were completely negative in both
polygonal and spindle cells.
Conclusion.On the basis of morphology and immunohistochemical results, the tumor was diagnosed as carcinosarcoma of the left
colon. The peculiarity of our case are the site of the tumor, the
size of the lesion (up to 40 cm in the largest diameter) and the
histological findings of a clear osteoid differentiation, which is
an extremely rare event in colonic carcinosarcoma. After eight
months from surgery, the patient was still alive without residual
tumor.
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Staroz F, Botton A, Potet F: Malignant tumors of the colon with two components (carcinosarcoma). Ann Pathol 1995; 15:457-458.
Roncaroli F, Montironi R, Feliciotti F: Sarcomatoid carcinoma of the
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Shan S, Kim DH, Harster G: Carcinosarcoma of the colon and spleen:
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p-CREB expression in human gliomas: a potential
marker to differentiate astrocytoma from
oligodendroglioma
V. Barresi1, S. Mondello2, G. Branca1, R. Thangavelu Soundara2,
E. Vitarelli1, G. Tuccari1
Department of Human Pathology “Gaetano Barresi”, University of Messina, Italy 2Department of Neurosciences, University of Messina, Italy
1
Introduction. Cyclic-AMP responsive element binding protein
(CREB) is a ubiquitous transcription factor. Following its activation by serine-threonine kinases and phosphorylation on ser133,
it may bind to cyclic-AMP response elements (CREs) sequences
present in the promoters of target genes. By the regulation of gene
transcription, phosphorylated CREB (p-CREB) plays important
roles in various cellular processes, including tumorigenesis and
cancer progression. Consistent with these effects, p-CREB is
over-expressed in several tumours, including prostate, breast,
lung and ovarian cancer and its expression correlates with higher
proliferation index and adverse prognosis in these cancers. pCREB seems also to have a role in gliomagenesis and high pCREB expression was demonstrated in vivo in human gliomas
from histological grade II to IV compared with normal brain
parenchyma. Due to its involvement in the oncogenesis and progression of gliomas, p-CREB has been identified as a potential
target for novel therapies against these tumours. However, gliomas have different biological behaviour according to their histological subtype. According to the World Health Organization
(WHO) Classification of central nervous system (CNS) tumours,
diffuse gliomas are subdivided into two main groups – astrocytomas and oligodendrogliomas- which represent separate clinical
and molecular entities showing different prognoses and treatment responses. For this reason, in the present study we aimed
to investigate whether p-CREB expression varies in gliomas
according to their type, astrocytic or oligodendroglial. Since the
pathologic criteria for classifying diffuse gliomas as astrocytoma
or oligodendroglioma are prone to significant subjectivity and
interobserver variability, we included in the study only oligodendrogliomas showing 1p/19q co-deletion, which represents
the molecular signature of these tumours, and only astrocytomas
lacking 1p/19q co-deletion.
Materials and methods. Fifty-three cases of formalin fixed
and paraffin embedded surgically resected gliomas, including
11 diffuse astrocytomas (DA) (WHO grade II), 9 anaplastic
astrocytomas (AA) (WHO grade III), 13 glioblastomas (GBL)
(WHO grade IV), 11 oligodendrogliomas (OD) (WHO grade II)
and 9 anaplastic oligodendrogliomas (AOD) (WHO grade III)
were submitted to the immunohistochemical procedures against
p-CREB. p-CREB immunoexpression was also evaluated in fragments of normal brain tissue present in some of the specimens
(5 cases), as well as in formalin fixed and paraffin embedded
samples of human normal brain parenchyma taken at autopsy
from a patient dead of myocardial infarction. p-CREB immunohistochemical expression was based on the presence of nuclear
staining. The intensity of p-CREB immunostaining was graded
on a scale of 0 to 3, with 0 indicating no staining, 1 indicating
weak staining, 2 indicating moderate staining, and 3 indicating
strong staining. The extent of positive immunoreactivity for pCREB (0 to 1; 1 = 100%) was calculated as the percentage of
cells that had nuclear staining for p-CREB. Finally, an intensity
distribution (ID) score was obtained for each specimen as the
product of immunostaining intensity (0 to 3) and immunoreactivity extent (0 to 1). p-CREB expression was also investigated
by Western blot analysis on paired frozen specimens of 3 OD, 3
AODs, 5 DAs and 5 GBLs.
In all the cases p53 overexpression and Ki-67 labelling index (LI)
were assessed by immunohistochemistry. 1p/19q codeletion had
been documented by FISH analysis in all of the ODs and AODs,
while all of the astrocytic tumours lacked this chromosomal
alteration. Unpaired Student’s t-tests and analysis of variance
(ANOVA) were carried out to assess whether any significant difference in p-CREB score was present among tumours of different
type (astroglial versus oligodendroglial derivation) or histological grade or on the basis of p53 over-expression. Spearmann test
was performed to investigate the statistical correlation between
p-CREB ID score and Ki-67 LI. A P value lower than 0.05 was
considered to be statistically significant.
Results. Nuclear p-CREB immuno-reactivity was found only in
the reactive astrocytes but not in the oligodendrocytes, neurons or
endothelial cells in the specimens of normal brain parenchyma. In
all of the neoplastic specimens, nuclear p-CREB immunostaining
was evidenced in the endothelial cells of the tumour vessels. The
neoplastic cells showed p-CREB immuno-expression in 41/53
cases. p-CREB was expressed in all the astrocytic tumors and
its xpression was higher in high grade than in low grade neoplasias by both immunohistochemistry (P=0.048) and western
blot (P=0.001). On the other hand, the large majority of oligodendrogliomas did not display any immuno-expression for this
protein and staining involving less than 10% of neoplatic cells
was evidenced in only 4 cases. Western blot results for p-CREB
were consistent with the immunohistochemistry study. Indeed,
marked p-CREB level was detected in low grade astrocytomas,
while considerable reduction was noticed in oligodendrogliomas
(P=0,0003). When all the cases were considered, regardless of
their grade and histotype, p-CREB and p53 expression were
significantly correlated (P<0,0001). In addition, a significant
positive correlation was present between p-CREB ID score and
Ki-67 LI (P= 0,0127).
Conclusions. We demonstrated for the first time that p-CREB is
differently expressed in human astrocytomas and oligodendrogliomas. If this peculiar finding is confirmed in future studies,
p-CREB expression may be used as a novel diagnostic tool to
differentiate astrocytomas and oligodendrogliomas. p-CREB expression in the vessels of gliomas, regardless of the tumour type,
is also an interesting finding which warrants further investigation
and it may represent a target for therapeutic strategies aimed at
interfering with angiogenesis in gliomas.
Glioblastomas with EGFR Amplification and
Overexpression are selectively responsive to
adjuvant Chemotherapy with Metronomic
Temozolomide through inhibition of NF-kB
transcriptional activity
M. Cominelli1,*, S. Grisanti2,*, L. Buttolo3, D. Medicina1, M.F.
Bonetti1, R. Galli4, F. Facchetti1, P.L. Poliani1, #
1
Pathology Unit, Department of Molecular and Translational Medicine,
University of Brescia, Italy; 2Medical Oncology and 3Neurosurgery Departments, Spedali Civili of Brescia, University of Brescia, Italy; 4Neural
Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells &
176
Gene Therapy, San Raffaele Scientific Institute, Milan; Italy. *These Authors equally contributed to the work presented; # Corresponding author
Keywords: Glioblastoma; Temozolomide; EGFR amplification
and overexpression; Metronomic Treatment.
Introduction. Temozolomide (TMZ) responsiveness in Glioblastoma (GBM) remains difficult to predict, mainly due to lack
of robust predictive biomarkers, other than MGMT promoter
methylation. Notably, within a group of newly diagnosed GBMs
(n=105) treated at our Institution by either Standard (SS; n=55)
or Metronomic (MS; n=50) adjuvant TMZ schedules, we identified Patients with significant long-term survival (≥35 months).
We thus investigated possible molecular profiles associated with
longer survival following TMZ treatment.
Methods. Histopathological and Molecular features, including
MGMT, EGFR/EGFRvIII, IDH1 and PTEN status, were correlated with either progression-free (PFS) and overall survival (OS).
Human-derived GBM Cancer Stem Cell (CSC) lines were used
to investigate in vitro molecular mechanism(s) correlated to TMZ
responsiveness. Surgically removed recurrences were studied to
investigate molecular changes occurring during therapy in vivo.
Results. Improved OS was observed in MS- compared to SStreated Patients (p=0.035). Importantly, long-term survivors were
highly represented within MS-treated cohort. We thus studied in
detail 70 Patients with homogeneous clinical-pathological features all receiving post-surgical standard concomitant radiochemotherapy followed by either SS (n=40) or MS (n=30) TMZ
schedules. No correlation was found between survival and gene
classifiers associated with different molecular GBM subtypes,
including YKL40, Met, p53, IDH1, Olig2, PDGFR. Conversely, robust correlation was found between EGFR amplification/overexpression and PFS and OS, but only in MS-treated
Patients (P<0.0005 by log-rank test; P=0.007 and P=0.001 by
Cox model respectively, that for OS remained significant after
Bonferroni correction; Pinteraction<0.0005). Notably, longterm survival following MS treatment was independent from
MGMT and EGFRvIII status, and was more pronounced in EGFR-overexpressing GBMs with PTEN loss (p<0.0005). Most
remarkably, in vitro findings confirmed selective dose- and
time-dependent decrease in survival of TMZ-treated EGFRpos
human-derived GBM-CSC lines, which occurred through inhibition of NF-κB transcriptional activity. In addition, reduction
of percentage of EGFR-amplified cells, along with significant
decrease of NF-κB/p65 expression (P<0.01), were observed in
specimens from recurrent MS-treated EGFR-overexpressing
GBMs. Finally, we cross-validated EGFR expression and TMZ
responsiveness in the original cohort of 105 Patients confirming that MS-treated EGFR-overexpressing GBMs strongly
correlate with improved OS (p<0.0005).
Conclusions. GBMs characterized by EGFR overexpression/amplification might strongly benefit from MS TMZ administration,
with additional advantage if carrying PTEN loss.
EZH2 expression in gliomas: a useful diagnostic
and prognostic marker
M. Cominelli1, M.F. Bonetti1, S. Pellegatta2, G. Finocchiaro2, F.
Facchetti1, P.L. Poliani1
Pathology Unit, Department of Molecular and Translational Medicine,
University of Brescia, Italy; 2Neurological Institute Besta, Milan, Italy
1
Introduction. Enhancer of Zeste 2 (EZH2) is a histone methyltransferase that belong to the Polycomb Repressive Complex-2
(PCR2). EZH2 over-expression has been associated with aggressive behaviour in different types of cancer, including breast and
prostate carcinomas. We have previously described that EZH2
in glioblastoma is highly expressed in the cancer stem-like component and correlate with tumour aggressiveness*. Aim of this
study is to provide a detailed analysis of EZH2 expression and
clinico-pathological correlations in a large cohort of gliomas.
Methods. We have evaluated EZH2 expression by immunohistochemistry in 296 gliomas of different histotype and WHO grade
(G), including 210 astrocytomas [G-I 30(14%), G-II 41(19%),
G-III 33(16%) G-IV 106(51%)], 59 oligodendrogliomas [G-II
26(44%), G-III 33(56%)] and 27 ependymomas [G-I 9(33%),
G-II(55%), G-III 3(11%)]. Gliobalstomas have been selected for
being primary glioblastomas, as assayed by clinico-pathological
parameters, including negativity to monoclonal antibody IMab-1,
specific for R132H-containing IDH1 mutation (IDH1-R132H),
the most frequent IDH1 mutation in secondary glioblastomas.
Clinico-pathological parameters (age, sex, radiological/histopathological features, proliferation index, tumor localization, surgery,
RT/CT treatment, Karnosky scale, RPA classes, MGMT status,
overall survival and time-to-progression) have been considered.
Results. Data show strong correlation between EZH2 expression
(both for intensity and percentage of positive cells) and tumor
progression and grade in all histotypes with the large majority
of high grade gliomas displaying strong EZH2 staining (68%).
Particularly, in Glioblastomas EZH2 expression strongly correlates with proliferation index (p<0.005), small cell component
(p<0.005), vascular proliferation (p<0.05) and, most importantly,
with lower survival, as stratified by surgical excision and treatment (p<0.005). Interestingly, preliminary MRI studies show a
correlation between cerebral blood volume (CBV) and intensity
of EZH2 expression. No significant association has been found
with MGMT status, p53 expression and EGFR expression/amplification.
Conclusions. Overall, data suggest that increased expression of
EZH2 takes place during glioma progression and have a valuable
diagnostic significance, particularly in the differential diagnosis
from grade II and grade III gliomas. Moreover, in glioblastomas
high levels of EZH2 expression (high intensity of expression and
>50% of positive cells) has a significant prognostic value identifying patients with lower survival.
References
Orzan F, Pellegatta S, Poliani L, Pisati F, Caldera V, Menghi F, Kapetis
D, Marras C, Schiffer D, Finocchiaro G. Enhancer of zeste 2 (ezh2)
is up-regulated in malignant gliomas and in glioma stem-like cells.
Neuropathol Appl Neurobiol. 2010
Primary leptomeningeal melanomatosis:
morphologic, immunophenotypic and molecular
analysis of two cases
G. Corradi, M. Ficial, G. Burato, S. Beccari, M. Chilosi, A.
Scarpa, C. Ghimenton
Dipartimento di Anatomia Patologica, AOUI Verona
Background. Primary leptomeningeal melanomatosis (PLM)
is a rare, aggressive variant of primary malignant melanoma
of the central nervous system that arises from melanocytes
within the leptomeninges. It occurs most frequently in adults,
but pediatric cases have also been reported. Clinical presentation includes seizures, signs of increased intracranial
pressure, psychiatric disturbances, cranial nerve palsies, and
spinal cord compression. The diagnosis of PLM requires the
exclusion of malignant melanoma in other sites. Because of
PLM poor prognosis, new therapeutic strategies are required.
Recent studies suggest that BRAF mutations are absent in
PLM. On the other hand, two pediatric cases showed NRAS
mutations in different studies.
We report two cases of PLM showing morphologic and immunophenotypic features and molecular expression of BRAF and
NRAS.
Methods. Case 1. A 38 years-old man was hospitalized for headache, sickness and signs of spinal cord compression. TC and MRI
revealed diffuse thickening of the leptomeninges, with abnormal
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COMUNICAZIONI ORALI
enhancement on the post-contrast images. Clinical hypothesis of
meningitis, chronic subarachnoid hemorrage or meningeal lymphoma were made. Analysis of cerebrospinal liquor revealed a
low glucose and high protein levels and absence of atypical cells.
With no clinical improvement with steroid therapy a frontal lobe
biopsy was performed with intraoperatory consultation. The patient died ten days after the biopsy and an autopsy was performed.
Case 2. A 4 years-old child underwent TC and MRI for headache
and signs of intracranial pressure. The images revealed a diffuse
thickening of the leptomeninges and, even in this case, a intraoperatory biopsy was made.
Results. In the first case, the brain and spinal leptomeninges were
diffusely thickened and black with extension of the neoplasia to
the optic nerves, Gasser ganglia and spinal roots on necroscopic
examination.
Both on intraoperatory biopsy and on autoptic samples, histological review revealed the presence of diffuse polygonal neoplastic
cells, often pigmented, with sparse mitoses and vesicular nuclei
containing prominent nucleoli. Immunophenotypic profile was
consistent with PML (S-100 +; HMB-45 +; Melan-A +; GFAP –;
Cytokeratins –). The pediatric case showed the same morphological and immunophenotypical pattern.
BRAF mutation was absent in both patients, while only the pediatric case showed NRAS alteration.
Discussion. In our cases, the morphological and immunophenotipical features of PLM were similar to those observed in
malignant melanoma of other sites, as previously reported. The
absence of BRAF mutation described in literature is confirmed
in our study. On the other hand, according to what reported in
literature, the presence of NRAS mutation suggests a role in the
pathogenesis of pediatric PLM with possible and future therapeutic implications.
Nuclear ß-catenin expression and stem cell
markers in medulloblastoma: correlation with
histological subtypes
E. Guadagno, A. Imperato*, G. Pettinato, M.L. Del Basso De Caro
Department of Advanced Biomedical Sciences-Pathology Section. University of Naples FedericoII, Italy; * Department of Neurosciences and Reproductive and Odontostomatological Sciences, Division of Neurosurgery,
University of Naples, Federico II
Introduction. Medulloblastoma generally affects patients in the
first two decades of life, accounting for about a fifth of all intracranial neoplasms of childhood. According to the 2007 WHO
classification of tumors of the Central Nervous System, it belongs
to primitive embryonal grade IV tumors.
Although the majority of them shows a classic histopathology,
there are other three variants (nodular/desmoplastic, with Extensive Nodularity, Large-Cell/Anaplastic), which have distinct
clinical, as well as microscopic characteristics.
An important molecular feature that is known to predict a good
prognosis in medulloblastoma is the finding of nuclear β-catenin
in neoplastic cells.
Not much is known about the expression of stem cell markers in
this tumor.
The aim of our study was to evaluate the expression in medulloblastoma of nuclear β-catenin and of CD133 and CD166, two
cancer stem cells markers whose reactivity has already been
explored in other neoplasms, and to correlate the results with different histological subtypes.
Material and methods. 14 cases of medulloblastoma were retrospectively analyzed to study the expression of β-catenin, CD133
and CD166 by immunohistochemistry.
Results. We observed nuclear positivity for β-catenin in 5 cases
and in particular, in one case among 5 nodular subtypes, in 3 cases among 7 classic subtypes and in the unique case with extensive
nodularity. As regards instead stem cell markers, we observed
positivity for CD166 in 50% of cases (5/7 classic variant and 2/5
nodular variant) and for CD133 in 2 cases of 14, respectively one
for the nodular and the classic subtype.
Conclusions. We observed a higher expression of β-catenin in
the classic variant and, even more important, a different pattern of
expression of two stem cell markers, with CD166 more frequently detected. This could mean that CD166 is more useful in identifying cerebral cancer stem cells and, therefore, in identifying
patients that will have more often chemo- and radio-resistance.
There wasn’t any significant difference in the frequency of
positivities of CD133 and CD166 between the nodular and classic subtype.
Multiple meningiomas: a case of death
E. Guadagno1, V. Natella1, M. Pieri2 , M.L. Del Basso De Caro1,
M. Gelsomina1
Dept of Advanced Biomedical Sciences University Federico II of Naples,
Italy. 1 Morphological and Functional Diagnostics, 2 Forensic Medicine
Background. Meningiomas are intracranial tumors originating
from arachnoidal cap cells. Multiple intracranial meningiomas
are rare and consist of two or more lesions occuring either simultaneously or sequentially in different locations.
Materials and methods. We describe a post-mortem case of
multiple meningothelial meningioma in a 43-year-old male found
death in his bedroom by his parents, apparently for unknown
causes. Close to the body hand-written notes, reporting his last
wills, and testament were found. Although the decedent lived
together with his parents, he conducted a completely isolated life.
No clinical symptoms were documented, but only diagnostic hypothesis of depressive syndrome and double personality disorders
(unmotivated aggressiveness and anger) referred by his parents.
Nothing in the patient’s history was suggestive of the presence
of a tumor.
Results. Systematic chemical-toxicological analyses were performed on blood, urine, bile, liver and brain samples in order to
investigate the presence of any drug and/or xenobiotic that could
be involved in the fatal hexitus: they were all negative. Sampling of brain areas showed multiple diffuse zones which, at the
microscopic examination, were characterized by marked edema
and fibrosis of the leptomeninges with dilated and congested
vessels, by lobulated or nested aggregates of epithelioid cells,
with angiomatous stroma, even with hyaline aspects. Psammoma
bodies were also present. These morphological features configure
the framework of meningothelial variant of meningioma, in the
multicentric form, considering the spread and jump localization
of the lesions.
Surprising autopsy diagnosis in unclear initial
situation.
A case of intravascular B cell lymphoma
V. Natella1, E. Guadagno1, M.L. Del Basso De Caro1, G. Luongo1, F. Policino2, G. Mansueto1
Department of Advanced Biomedical Sciences-Pathology Section. University of Naples Federico II, Italy; 2 Department of Advanced Biomedical
Sciences-Legal Medicine Section. University of Naples Federico II, Italy
1
Background. Intravascular large B cell lymphoma (IVLBCL) is
a rare extranodal non-Hodgkin lymphoma characterized by the
proliferation of malignant cells within the lumen of small vessels,
with a predilection for the central nervous system and the skin,
and with highly aggressive course. The clinical presentation of
IVLBCL is non-specific and may consist of change in neurological deficits, fever of unknown origin, cutaneous lesions, without
specific neuroimaging patterns.
Materials and methods. we described a case of a post-mortem
IVLBCL diagnosis in a 62-year-old female with non-specific
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clinical symptomatology, and without cutaneous lesions, nor
lymphnode swelling, no injuries of the skull at the autopsy external examination.
Results. microscopically the blood vessels of the leptomeninges,
cerebral cortex and white matter showed multifocal occlusion
due to large lymphoid cells with strong immunoreactivity for
B lymphocyte marker CD20 and a cohesive growth pattern. In
addition, multifocal haemorragic areas and signs of cortical and
medullary damage from hypoxia and ischemia. Intravascular
atypical lymphoid B-cells were observed also within the lumen
of the vessels in the liver, kidneys, node, adrenal gland, lung,
and heart. Therefore, the diagnosis was intravascular large B-cell
lymphoma (IVLBCL).
ß-catenin and stem cell markers (CD133 and CD166)
expression in craniopharyngiomas after radical
excision via an endoscopic endonasal approach:
neuropathological and immunoistochimical study
with clinical implications.
V. Varone1, M.L. Del Basso De Caro1, A. Somma1, E. Guadagno1, A. Di Somma2, L.M. Cavallo2, G. Pettinato1
Department of Advanced Biomedic Sciences, section of Pathology, University of Naples Federico II; 2 Department of Neurosciences, Reproductive and Odontostomatological Sciences, Division of Neurosurgery, University of Naples Federico II.
1
Introduction. Craniopharyngiomas (CPs) are a heterogeneous
group of benign but locally aggressive epithelial tumors arising
within the sellar region, along the path of the craniopharyngeal
duct and account for 1.2-4% of all intracranial tumors and 6-9%
of all pediatric brain tumors. CPs are composed of two subtypes,
adamantinomatous (adaCP) and papillary (papCP), differing
both in histopathological features as well as in frequency, clinical manifestation and prognosis. Histologically, papCPs show a
papillary architectural pattern of growth, with a well differentiated squamoid epithelium and pseudopapillae. This subtype is
infrequent, occurring almost exclusively in adults and shows a
favorable outcome. Conversely, the majority of CPs are adaCPs
originating in childhood. Microscopically they are characterized by anastomosing trabeculae of squamous epithelium with
a peripheral palisaded layer and more loosely arranged “stellate
reticulum”. This subtype is also associated with compact keratin
material (“ghost cells” or “wet keratin”), fibrosis, dystrophic
mineralization and cholesterol cleft formation (xanthogranulomatosus inflammation). Although adaCP is histologically benign
(WHO grade I) and does not metastatize, this tumor shows tendency to infiltrate the critical parasellar structures and to regrowth
or recur even after total surgical removal or after subtotal excision
with adjuvant irradiation (the rate of recurrence is 23 and 27%
respectively, 5 years after surgery). However, the pathogenesis of
CPs is still poorly understood and genetic and molecular features
currently support a distinct pathogenesis of the two subtypes:
papCP may arise from metaplastic transformation of mature epithelial cells of anterior pituitary gland, while adaCP may derive
from ectopic embryonic remnant cells of the craniopharingeal
duct and Rathke’s pouch. Moreover, previous studies have reported a role of the canonical Wnt signaling and β-catenin in the
pathobiology of adaCPs. β-catenin is a 92 kD protein normally
found in the cytoplasm of the cell in a submembranous location;
mutations of the -catenin gene (CTNNB1) result in nuclear accumulation of this protein and a constitutive activation of Wnt
signaling. Mutations in the β-catenin gene are found in around
70% of adaCPs and nuclear β-catenin is detected in up to 94%
of adaCPs, even in the absence of CTNB1 mutations, suggesting that other genetic or epigenetic events can activate the Wnt
signaling in adaCPs. The nuclear accumulation of β-catenin occur
only in small “whirl-like” clusters of neoplastic cells in adaCPs
but never in papCPs or in other tumors of the sellar region. These
clusters are often present at the invading edge of the tumor and
share some features with pituitary stem cells. Thus, the activation
of Wnt signaling and the aberrant nuclear expression of β-catenin
are striking evidences pointing towards the existence of cancer
stem cells in adaCP. The aim of the present study is to evaluate
-catenin and stem cell markers (CD133 and CD166) immunohistochemical expression in CPs. These data have been investigated
in few studies in the pertinent literature and could be strongly
associated with patient’s prognosis and outcome, explaining the
irradiation resistance and the capacity to recurrence of adaCPs.
Methods. We analyzed a collective of twenty craniopharyngiomas
samples, comprising 13 relapsed CP and 7 non relapsed CP; all
the specimens were obtained from the archives of the Department
of Advanced Biomedical Sciences, section of Pathology, University of Naples Federico II and were histopathologically classified
according the guidelines of the world health organization (2007
WHO). The group of relapsed patients included one case of papCP
and twelve cases of adaCP; this group consisted of three female
and ten male aged 4-69 years (mean age 43 years) at the time of
surgery; in eight cases the surgical removal was performed, for
both the primary tumor and the relapse, in the Department of Neurosurgery of the University of Naples Federico II. Therefore, we
also analyzed, for five relapsed cases, the corresponding primary
tumor samples (the other three primary CP sample were inadequate
for the present study). For the remaining five case of relapsed CP,
the primary tumor was removed elsewhere. The disease free interval ranged from six month to two years from the primary surgical
removal. The group of non relapsed patients included four papCPs
and three adaCPs, consisting of one female and six male, aged 2766 years (mean age 50 years) at the time of surgery. The follow-up
period ranged from six to eight years. All the patients included in
the study underwent radical excision via an endoscopic endonasal
approach. Haematoxylin and eosin stained slides were reviewed
and the histopathological diagnosis of CP with the proper subtype
was verified by two pathologists from the Department of Advanced
Biomedical Sciences, section of Pathology, University of Naples
Federico II. Immunohistochemical staining for beta-catenin and
for the cancer stem cell markers CD133 (Prominin1) and CD166
(ALCAM) was performed on sections from one representative
formalin-fixed paraffin embedded tissue block. Nuclear immunoreactivity for β-catenin was semiquantitatively evaluated on the basis
of staining intensity and proportion, according to the immunoreactivity score described by Liu et al (1): immunoreactivity score=
intensity score x proportion score. The intensity score was defined
as follows: 0, negative (only at the cell membrane); 1, weak; 2,
moderate; 3, strong. The proportion score was defined as follows:
0, negative; 1, ≤ 10% of positive cells; 2, 11-50%; 3, 5180%; 4,
>80%. The total score ranged from 0 to 12. Immunoreactivity was
divided into three groups on the final score: a total score of 0-2
defined a low level; a total score of 3-6 defined a moderate level
and a total score of >6 defined a high level. Immunoreactivity for
CD133 and CD166 was semiquantitatively evaluated on the basis
of staining intensity, distribution and proportion. The intensity
score was defined as follows: 0, negative; 1, weak; 2, moderate;
3, strong. The distribution was evaluated at the cell membrane or
in the cytoplasm; the proportion was evaluated as diffuse or focal.
The relationships between expression of these three immunohistochemical markers and various clinicopathological indicators and
clinical outcomes of the tumours were analysed.
Results. Among 7 non relapsed CPs, 6 cases (4 papCPs; 3
adaCPs) showed negative nuclear immunoreactivity for catenin;
only one case of non relapsed adaCP showed a low level of immunoreactivity (total score=2). Among 13 relapsed CPs, all the
12 cases of adaCPs showed mainly a moderate level of -catenin
immunoreactivity. The only case of relapsed papCP presented, as
expected, negative nuclear immunoreactivity for -catenin; this
case, maybe, represent a case of tumor regrowth rather than a
true relapse (6 month disease free interval). The distribution pat-
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COMUNICAZIONI ORALI
tern was, as expected, more prominent in whirl-like cell clusters.
CD133 and CD166 showed an increased expression in relapsed
CPs, expecially in adaCPs, rather then in non relapsed CPs; the
immunoreactivity was mainly weak/moderate, the pattern of
distribution was predominantly diffuse and at the cell membrane.
Moreover, the distribution pattern of both CD133 and CD166
strongly resembled that of cells showing nuclear β-catenin accumulation and, as expected, was more prominent in whirl-like
clusters. The five primary CP samples that we also analyzed, in
addition to the twenty CP samples, showed similar results to the
corresponding relapsed tumors, both for β-catenin and for cancer
stem cell markers.
Conclusion. Our results indicate the presence in adaCP, expe-
cially in relapsed tumors, of cell clusters characterized by nuclear
expression of β-catenin and by increased expression of the stem
cell markers CD133 (Promin 1) and CD166 (ALCAM).
Tumor stem cell-like cell clusters are known to be less sensitive
to radiotherapy, therefore, causing tumor recurrences. The potential impact of these special cell clusters may involve the future
CPs management, including postoperative additional treatment.
References
Liu LK et al Upregulation of vimentin and aberrant expression of
E-cadherin/β-catenin complex in oral squamous carcinoma: correlation with clinicopathological features and patient outcome. Mod.
Pathol. 2009; 23; 213-224
Sala Caravaggio – 2° piano – 14,30-15,30
Patologia polmonare
Moderatori: L. Novelli, G. Pelosi
Pulmonary calcifying fibrous tumor: a case report
C. Caporalini , P. Pretelli , M. Rotellini , V. Terrinazzi , L. Novelli2, A. Gonfiotti3, L. Messerini1, C.E. Comin1.
1
1
1
1
Department of Experimental and Clinical Medicine, Section of Specialistic Surgery and Histopathologic and Molecular Diagnosis, University
of Florence, Florence, Italy; 2 Unit of Histopathology, Department of Biomedicine, Careggi Hospital, Florence, Italy; 3General Thoracic Surgery,
Careggi Hospital, Florence, Italy.
1
Introduction. Calcifying fibrous tumor (CFT) is a rare, benign,
fibrous lesion typically found in subcutaneous and deep soft tissues of the extremities, trunk and neck of children, teenagers and
young adults.13,12 A few cases have been reported in the pleura
and mediastinum.4-10 We present the case of a man with a giant
CFT of the left lung, involving the main hilum and treated with a
left intrapericardial-pneumonectomy. This is the widest thoracic
CFT lesion reported and the second case of a pulmonary location
described in the literature.11
Patient and methods. A 31-year-old man was referred to our
hospital with the presumptive diagnosis of a wide neoplastic
lesion of the left lung, causing dyspnea and chest pain. The
computer tomography (CT) showed a large lesion of the lower
left lobe. The lesion was also adherent to the pericardium and descending aorta. At the left sided thoracotomy, a giant lesion at the
left pulmonary hilum, involving all its main structures (left main
bronchus, pulmonary artery and veins at the origin) was found.
Repeated frozen sections were done and showed spindle cells
without features of malignancy. The pleura was completely free
from disease. Since the dimension and the location of the tumor,
an intrapericardial-pneumonectomy was necessary. On gross examination, the lesion measured 14 x 10 x 8 cm, it was lobulated,
well circumsbribed and firm but unencapsulated. On cut surface it
appared white to tan to gray and the calcifications sometimes imparted a gritty texture. Representative sections of the mass were
fixed in formalin and embedded in paraffin. Conventional hematoxylin and eosin slides were done. Immunohistochemical studies
were performed on representative sections testing the following
antibodies: vimentin, factor XIII, cytokeratin AE1/AE3, EMA,
CD34, desmin, smooth muscle actin, CD99 and S100 protein.
Results. Conventional hematoxylin and eosin slides showed
a paucicellular proliferation of relatively small, incospicous,
mature fibrocytes set in a densely collagenous background. Dystrophic or psammomatous calcifications and a variable inflammatory infiltrate, composed chiefly of lymphocytes and plasma
cells, were present. Lymphoid aggregates with active germinal
centers were also observed. No atypical features were identified; there were no necrosis or mitoses. Immunohistochemically,
spindle-shaped cells were positive only for vimentin and factor
XIII, negative for cytokeratin, EMA, CD34, desmin, smooth
muscle actin, CD99 and S100 protein. The histologic findings
were compatible with a diagnosis of CFT.
Conclusions. CFT is a rare benign mesenchymal tumor commonly characterised histologically by hyalinized collagenous
fibrous tissue, with bland spindle cells, psammomatous or dystrophic calcifications and focal lymphoplasmacytic infiltrate. The
histogenesis and pathogenesis of the lesion remain unclear.6,13
CFT is usually located in the soft tissues of the extremities and
trunk; other locations are rare and anecdotally reported in the
mesentery, peritoneum, pleura, chest wall, and mediastinum.
The most important diagnostic aid is to bear this entity in mind
and focus the attention on the differential diagnosis. The most
important differential diagnosis of CFT includes other spindle
cell tumors or pseudo-tumor conditions. Dystrophic calcifications
and psammomatous bodies are specific to CFT which distinguish
the tumor from inflammatory pseudotumor. Absence of cellular
atypia, storiform pattern, frequent mitosis, and bizarre tumor
cells are the characteristics that distinguish CFT from soft tissue
sarcomas such as leiomyosarcoma, fibrosarcoma and malignant
fibrous histiocytoma. Since the well known tendency of CFT to
local recurrence, the proper treatment is a complete resection. In
our case a left pneumonectomy was performed. After 12-month
follow-up, the patient remains free of disease.
References
1
Fetsch JF, Montgomery EA, Meis JM. Calcifying fibrous pseudotumor. Am J Surg Pathol.1993;17(5):502-8.
2
Rosenthal NS, Abdul-Karim FW. Childhood fibrous tumor with psammoma bodies. Clinicopathologic features in two cases. Arch Pathol
Lab Med. 1988;112(8):798-800.
3
Jaiswal SS, Agrawal A, Sahai K, Nair SK. Large retroperitoneal calcifying fibrous tumor. Med J Armed Forces India. 2013;69(2):184-6.
4
Ağaçkıran Y, Fındık G, Aydoğdu K, Günay E, Günay S, Kaya S. An
extremely rare case of multiple calcifying tumor of the pleura. Tuberk
Toraks. 2012;60(4):385-8.
5
Giardino AA, Ramaiya NH, Shinagare AB, Jagannathan JP, Stachler
MD, Raut CP. Case report: Calcifying fibrous tumor presenting as an
asymptomatic pelvic mass. Indian J Radiol Imaging. 2011;21(4):3068.
6
Isaka M, Nakagawa K, Maniwa T, Saisho S, Ohde Y, Okumura
T, Kondo H, Nakajima T. Disseminated calcifying tumor of the
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8
9
10
11
12
13
Rev. 2013 Jun;39(4):328-39) and on protocols for the pathologic
reporting. Recommendations include the use immunohistochemical stains in the differentiation of epithelioid MM from various
metastatic carcinomas, and in the identification of sarcomatoid
MM. Immunohistochemical panels should contain both positive
and negative markers. The aim of this study was to verify the
reporting of MM and use of immunohistochemistry (IHC) in the
practice of pathology, taking advantage of a regional populationbased mesothelioma registry and a pathology-based mesothelioma archive.
Methods. The source of cases were 2 databases covering the
years 1980-2000 (pathology-based mesothelioma archive (PMA)
and 2001-2013 (mesothelioma registry of the Lazio region (Regional-Operative-Center, COR-Lazio). We reviewed pathology
reports and recorded the following elements: type of procedure
(thoracentesis/paracentesis, core biopsy, excisional biopsy, surgery, autopsy); site of the tumor (pleura, peritoneum, pericardium
and tunica vaginalis); type of specimen (histology, cytology);
histologic type [not otherwise specified (NOS), epithelial, sarcomatoid, mixed]. We analyzed IHC for each case, evaluating
positive mesothelioma markers, positive carcinoma markers and
additional markers, as typewritten out in full by the pathologist
on the final report and the clone of the antibody (if indicated).
We calculated the number of antibodies applied in the diagnostic
panel of epithelial MM, whether composed of only 2 antibodies
(1 mesothelial and 1 epithelial) or composed of 4 or more antibodies (2 or more mesothelial markers with 2 or more epithelial
markers) to analyze whether and how pathologists follow the
recommendations of the literature.
Results. Overall, in the 2 databases there are 1018 MM with a
pathology-based diagnosis (113-PMA; 905-COR); 88% with
histology (60% epithelial, 22% NOS, 9% sarcomatoid and 9%
mixed) on biopsy specimens (82%); 90% with IHC. The 85% are
MM of the pleura. We have preliminarily reviewed the patholo-
pleura: review of the literature and a case report with immunohistochemical study of its histogenesis. Gen Thorac Cardiovasc Surg.
2011;59(8):579-82.
Jiang K, Nie J, Wang J, Li J. Multiple calcifying fibrous pseudotumor
of the bilateral pleura. Jpn J Clin Oncol. 2011;41(1):130-3.
Suh JH, Shin OR, Kim YH. Multiple calcifying fibrous pseudotumor
of the pleura. J Thorac Oncol. 2008;3(11):1356-8.
Shibata K, Yuki D, Sakata K. Multiple calcifying fibrous pseudotumors disseminated in the pleura. Ann Thorac Surg. 2008;85(2):e3-5.
Jeong HS, Lee GK, Sung R, Ahn JH, Song HG. Calcifying fibrous
pseudotumor of mediastinum: a case report. J Korean Med Sci. 1997
Feb;12(1):58-62.
Soyer T, Ciftci AO, Gucer S, Orhan D, Senocak ME. Calcifying fibrous pseudotumor of lung: a previously unreported entity. J Pediatr
Surg.2004;39(11):1729-30.
Hoffmann H, Beaver ME, Maillard AA. Calcifying fibrous pseudotumor of the neck. Arch Pathol Lab Med. 2000;124(3):435-7.
Agaimy A, Bihl MP, Tornillo L, Wünsch PH, Hartmann A, Michal
M. Calcifying fibrous tumor of the stomach: clinicopathologic and
molecular study of seven cases with literature review and reappraisal
of histogenesis. Am J Surg Pathol. 2010;34(2):271-8.
Malignant mesothelioma: evaluation on pathology
reporting (1980-2013)
C. Carnovale Scalzo1, I. Cozzi1, G. Minelli2, E. Rullo1, E. Romeo3, L. Ancona3, F. Forastiere3, V. Ascoli1
Department of Radiological, Oncological and Anatomo-Pathological
Sciences, Sapienza University, Viale Regina Elena 324, 00161 Rome, Italy; 2 Unit of Statistics, National Institute of Public Health (ISS),Viale Regina Elena 299, 00161 Rome, Italy; 3 Department of Epidemiology, Lazio
Regional Health Service, Via di Santa Costanza 53, 00198 Rome, Italy
1
Introduction. Malignant mesothelioma (MM) is difficult to
diagnose. Several articles have independently focused on practical guidelines for the pathological diagnosis (Husain et al Arch
Pathol Lab Med- Vol 137, May 2013; Pinto et al Cancer Treat
Tab. I. Immunostains in 376 pleural MM
Positive mesothelioma markers
Calretinin
Cytokeratin 5/6
WT-1
HBME-1
Podoplanin
Thrombomodulin
Positive carcinoma markers
TTF-1
CEA
CD15
MOC-31
Ber-EP4
B72.3
Additional markers
Pancytokeratin #
Vimentin
Desmin
CD10
EMA
Actina (SMA)
Antibody clone reported in the report
1980-2000
41 epithelial
5 sarcomatoid
N°
% positive
N°
% positive
4
100
1
100
2
100
0
13
100
4
75
-
2001-2013
295 epithelial
35 sarcomatoid MM
N°
% positive
N°
% positive
283
97
33
56
158
92
17
47
78
90
5
80
107
91
16
0
15
100
4
31
12
100
-
18
15
11
3
5
6
36
0
1
1
-
0
0
-
221
184
66
20
43
7
2
2
9
10
12
0
13
13
4
6
1
0
0
0
0
0
13
15
12
-
100
93
100
-
5
3
1
-
100
100
114
58
32
4
63
8
74
99
84
6
100
97
12
25
33§
18
17
3
12
12
35
90
100
5
66
41
58
100
¤ or other cytokeratins including CAM 5.2 (CK 7/8), CK18, and CK7
0
-
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COMUNICAZIONI ORALI
Tab. II. Immunostains in 295 epithelial MM. Positive carcinoma markers vs Positive mesothelioma markers
additional markers
Positive
carcinoma
markers
1 marker
2 markers
more than 2 markers
Total
N°
N°
N°
N°
N¡
%
additional markers
4
1,4%
1
0,3%
1
0,3%
1
0,3%
7
2,4%
gic reports of 443 MM of the pleura: a) 75 epithelial (55% with
IHC) and 8 sarcomatoid (5 with IHC) for the period 1980-2000;
and b) 321 epithelial (92% with IHC) and 39 sarcomatoid (90%
with IHC) for the period 2001-2013. Table 1 shows the results of
IHC performed in 376 pleural MM. Table 2 shows a focus on the
295 epithelial MM with IHC, in which 76 pathologists were involved (43=1 diagnosis; 29=2-9 diagnoses; 5=14-69 diagnoses).
In 18% of cases the IHC panel consisted of 2 carcinoma markers
and 2 mesothelioma, whereas in 10% the panel was expanded to
more than 2 carcinoma markers and more than 2 mesothelioma
markers. 8.5% of MM was diagnosed using only 2 antibodies (1
mesothelial and 1 epithelial).
Conclusions. Our data reflect the changes that occurred in the
diagnosis of MM in the last three decade. In the more recent period analyzed, the use of IHC is above 90% in epithelial MM and
90% in sarcomatoid MM. As positive mesothelioma marker is
concerned, the use of calretinin is consolidated being performed
in 96% of epithelial MM in 2001-2013, after the commercial
availability of the antibody. As positive carcinoma marker, TTF1 and CEA are the most used antibodies. There is variability
both in the use of antibodies (up to 56 different types), and in the
number of diagnosis per pathologist [median 5 (SD±2,12); range
1-69], and in the number of IHC markers. There is a need of standardization of the pathologic reporting of MM.
Cyto-histological correlation for the
characterization of morphological patterns in lung
adenocarcinomas
P. Morbini1, M. Agozzino2, C. Tinelli3
Dipartimento di Medicina Molecolare, Unità di Anatomia Patologica,
Università di Pavia e IRCCS Policlinico S. Matteo, Pavia; 2 Laboratori
sperimentali di Ricerca, Area Trapiantologica, IRCCS Policlinico S. Matteo, Pavia; 3 Unità di Biometria e Biostatistica, IRCCS Policlinico S. Matteo, Pavia
1
Introduction. A new histopathological classification of lung
adenocarcinomas (ADK) has been proposed by an international
and multidisciplinary panel in 2011 (1) with the aim of providing
better correlation with biological data and prognostic and predictive factors as compared with the current classification included
in the 2004 WHO blue book on thoracic tumors. According to
the new classification, 5 morphological patterns are recognised
(acinar, papillary, micropapillary, solid and lepidic), while mucinous tumors are classified separately; the presence of any signet
ring or clear cell component is quantified independently of the
observed patterns. This classification proposal is intended also
as an instrument towards a more precise correlation of specific
histopatological features with emerging predictive molecular
markers associated with targeted treatments.
Positive mesothelioma markers
1 marker
2 markers
more than 2 markers
12
9
5
4,1%
3,1%
1,7%
25
30
20
8,5%
10,2%
6,8%
16
53
44
5,4%
18,0%
14,9%
3
29
42
1,0%
9,8%
14,2%
56
121
111
19,0%
41,0%
37,6%
As stated in the paper presenting the new ADK classification and
in a previous paper specifically dealing with lung cancer cytology
(2), the identification of histopatological patterns and percentage
quantification in ADK is limited to the analysis of a surgical lung
samples, while only histotype characterization (ADK vs squamous
or small cell caricnoma, or non-small cell favouring a specific
histotype) is required on biopsy or cytological samples (2). Given
that some ADK patterns are consistently associated with higher
biological aggressiveness and worse prognosis, and are considered “high grade”, namely solid, micropapillary (1), together with
recently characterized cribriform (3) and signet ring (4), and that
only a limited proportion of lung cancer patients are eligible for
surgery, we addressed the issue of investigating a possible correlation between features that can be recognised in small samples and
the codified histopathological patterns. We considered CT-guided
fine-needle aspiration (FNA) ADK samples because this type of
sample allows, in our experience, to evaluate several architectural and cytomorphological characters, as well as the presence of
diverse extracellular materials, and defined a series of recurrent
parameters. Of note FNA samples have also been shown to be adequate for molecular testing (5). The defined parameters were then
searched in a series of FNA samples, together with the sample cellularity, and the results were correlated with the histopathological
patterns observed in the corresponding surgical samples and with
the patient outcome. The final objective of our study was that of
characterizing in FNA samples features that significantly associate
with specific ADK patterns and/or worse outcome, and that could
increase in the future the prognostic role of FNA.
Materials and methods. Patients The study included 99 paired
consecutive lung FNA and surgical samples from 98 patients (61
males, mean age 67.34 SD 8.41), collected between August 2005
and July 2013, with a cytological and histopathological diagnosis
of pulmonary ADK. Patients that had undergone neoadjuvant
therapy before surgery and all other lung cancer histotypes or
secondarisms were excluded.
Samples FNA samples had been processed with formalin fixation
and paraffin inclusion (“cell block”); hematoxylin and eosinstained slides were used for revision. Surgical lobectomies had
been routinely processed for histopathological examination.
Cytological parameters As distinct architectural parameters we
defined the presence of papillary (a), micropapillary (b), solid
(c), acinar (d), cribriform (e) and chain-like (f) aggregates of
neoplastic cells and the presence of isolated neoplastic cells
(g); cytomorphological parameters distinguished tall columnar
(h) and cuboidal cells (i), goblet cells (l), signet ring cells (m),
and the presence of intracytoplasmic mucinous vacuoles in non
overtly muciparous cells (n). Necrosis (o), extracellular mucous
(p) and microcalcifications/psammoma bodies (q) were defined
as extracellular parameters.
182
Histopathological patterns. Surgical samples were classified according to the 2011 lung ADK classification (1). The presence of
any pattern (acinar, papillary, micropapillary, solid and lepidic)
was recorded with the percentage extension. Cribriform pattern
was added considering its recent association with worse prognosis (3). Moreover, the presence of a mucinous component was
recorded, distinguishing if it represented the totality or only a part
of the tumor. Finally, any clear cell or signet ring component was
separately recorded.
Histopathological case review. All samples underwent a double
blind analysis by two pathologists (PM and MA). All discordant
data were discussed and a final consensus was reached.
Statistical analysis Fisher exact test was used to analyse the association between each combination of cytological and histopatological parameters. Univariate analysis was used to investigate
the association of each parameter with the risk of cancer-related
death and disease recurrence. P values equal or less than 0.05
were considered as statistically significant. Histopathological patterns were analyzed in two distinct runs, one considering only the
predominant patter, and one considering their absolute presence
independently of their proportion in the tumor tissue.
Results. The following significant associations were found for
cytological features with the presence or absence of any histopathological patterns independently of the sample cellularity:
- papillae: solid (p 0.017), papillary (p 0.004)
- micropapillae: acinar (p: 0.014), micropapillary (p: 0.037
- solid: solid (p 0.000), cribriform (p.0.000), lepidic (p 0.002),
clear cell (p 0.015)
- acinar: solid (p 0.032), lepidic (p 0.024)
- cribriform: acinar (p: 0.029), cribriform (p 0.001)
- chains: acinar (p. 0.025), solid (p 0.000), lepidic (p 0.009)
- intracytoplasmic mucus: clear cells (p 0.29), cribriform (p: 0.020)
- goblet cells: micropapillary (p 0.008), lepidic (p 0.008), mucinous (p 0.000), signet ring (p 0.004)
- cuboid cells: acinar (p: 0.037), solid (p 0.017), lepidic (p 0.004)
- columnar cells: solid (p 0.026), lepidic (p 0.05), mucinous
(p 0.007), signet ring (p 0.002)
- signet ring cells: micropapillary (p 0.018), cribriform (p: 0.009),
mucinous (p 0.009), signet ring (p 0.001)
- extracytoplasmic mucus: acinar (p: 0.042), mucinous (p 0.007),
signet ring (p 0.004)
- necrosis: solid (p 0.013), cribriform (p: 0.013), lepidic (p 0.003),
clear cells (p 0.017)
- psammoma bodies: micropapillary (p 0.045)
The following significant associations were found for cytological
features with the predominant histopathological pattern independently of the sample cellularity and of minor components:
- papillae: acinar (p 0.035), papillary (p 0.000)
- solid: solid (p 0.003)
- acinar: solid (p 0.049)
- cribriform: acinar (p 0.32), cribriform (p 0.043)
- chains: solid (p 0.002)
- goblet cells: lepidic (p 0.050)
- columnar: solid (p: 0.004)
- necrosis: solid (p 0.043)
The risk of death was significantly associated, beside clinical
stage, with cribriform cytoarchitecture (p 0.023, HR 2.955, CI
1.157-7.55). Risk of disease recurrence was significantly associated with clinical stage, single cells (p 0.032, HR 0.432,
CI 2-0.931) and acinar cytoarchitecture (p 0.022, HR 2.462, CI
1.136-5.33).
Discussion. The finding of papillary, solid or micropapillary
cytoarchitectural features in FNA samples was significantly
associated with the presence of the corresponding histological
pattern as a component of the surgically resected tumor. Similarly, goblet cells, extracellular mucous pools, and signet ring
cells were strongly associated with the presence of a mucinous
component in the tumor. These cytological features were even
more strongly associated with the presence of signet ring cells
in the surgical sample. Finally, the presence of necrotic material
was strongly associated with a clear cell, solid or cribriform pattern, while psammoma bodies were indicative of a micropapillary
pattern. Non-consistent associations found between some cytohistological pairs (acinar-solid and reverse, papillary-solid and
reverse) can be justified on the basis of a recurrent, significant
association of the two histological pattern in the surgical samples,
where commonly more than 1 pattern is present (1).
While FNA cytological profile appears to be highly representative of the overall histological patterns that are observed in corresponding surgical samples, in particular for those patterns that are
recognized to have a negative prognostic impact, the cytological
analysis is less effective in identifying the predominant pattern
present in the surgical sample.
Considering that in most patients FNA are the only samples available for tumor diagnosis and characterization, a report providing
information of the presence of cytological features associated
with high risk histology could improve risk stratification for
patients with advanced stage tumors. Analyzing the correlation
183
COMUNICAZIONI ORALI
of cytological pattern with the molecular profile of the tumor
could further improve the diagnostic power of FNA cytological
analysis.
References
1
Travis WD, Brambilla E, Noguchi M, et al. International association
for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung
adenocarcinoma. J Thorac Oncol 2011;6:244-85.
2
Travis WD, Rekhtman N, Riley GJ, et al. Pathologic diagnosis of advanced lung cancer based on small biopsies and cytology: a paradigm
shift. J Thorac Oncol 2010;5:411-4.
3
Kadota K, Yeh YC, Sima CS, Rusch VW, et al. The cribriform pattern
identifies a subset of acinar predominant tumors with poor prognosis
in patients with stage I lung adenocarcinoma: a conceptual proposal
to classify cribriform predominant tumors as a distinct histologic subtype. Mod Pathol 2014; 27:690-700.
4
Ou SH, Ziogas A, Zell JA. Primary signet-ring carcinoma (SRC)
of the lung: a population-based epidemiologic study of 262 cases
with comparison to adenocarcinoma of the lung. J Thorac Oncol
2010;5:420-7
5
Stella GM, Scabini R, Inghilleri S, et al. EGFR and KRAS mutational
profiling in fresh non-small cell lung cancer (NSCLC) cells. J Cancer
Res Clin Oncol 2013;139:1327-35
Discussione
Well-differentiated mucinous adenocarcinoma
arising in pulmonary leiomyomatous hamartoma
P. Pretelli1, A. Cavazza2, G. Rossi3, M. Rotellini1, C. Caporalini1,
L. Novelli4, L. Messerini1, C.E. Comin1, K.O. Leslie5
Department of Experimental and Clinical Medicine, Section of Specialistic Surgery and Histopathologic and Molecular Diagnosis, University of
Florence, Florence, Italy; 2Department of Oncology, Division of Pathological Anatomy, S. Maria Nuova Hospital, Reggio Emilia, Italy; 3Section of
Pathology, Azienda Ospedaliero-Universitaria Policlinico, Modena, Italy;
4
Unit of Histopathology, Department of Biomedicine, Careggi Hospital,
Florence, Italy; 5Mayo Clinic, Scottsdale, Arizona, USA.
1
Introduction. Pulmonary leiomyomatous hamartomas (PLH) are
extremely rare lesions. The growth of excessive smooth muscle
in the lung parenchyma has been described in a wide range of
neoplastic and nonneoplastic conditions including such diverse
entities as lymphangioleiomyomatosis (LAM), smooth muscle
proliferation associated with HIV infection, benign metastasizing
leiomyoma from the uterus, primary and metastatic leiomyosarcoma, and even idiopathic pulmonary fibrosis. PLH are more
frequent in women than men (reported ratio 1.5:1) 1-2 Because
these lesions represent a tumorlike overgrowth of normal tissue,
leiomyomatous hamartoma is an appropriate term to designate
this particular entity.2 Cases of PLH, associated with other benign conditions (such as bronchogenic cyst or pneumothorax),
have been reported.1-2 To the best of our knowledge, no cases of
PLH associated with malignant lesions have been previously described. Difficulties may arise when hamartoma is combined with
a second neoplasm, a very rare event with a potentially significant
impact on prognosis.3 We report the first case of PLH associated
with a small focus of adenocarcinoma.
Patient and methods. A 73-year-old man was admitted to our
hospital with dyspnea. Chest X-ray film demonstrated a wellcircumscribed mass of 5.5 cm in diameter in the upper lobe of the
left lung. No enlarged mediastinal lymph nodes were observed.
The patient underwent surgery without a previous diagnosis.
Representative sections of the mass were fixed in 10% buffered
formalin and embedded in paraffin. Sections 4 µm thick were
then stained with hematoxylin-eosin. Immunohistochemical studies were performed on representative sections. The following
antibodies were used: smooth-muscle actin, desmin, myogenin,
cytokeratins CAM 5.2 and 7, TTF-1.
Results. Pathological examination showed a lesion composed
of spindle cells arranged in interlacing fascicles and connective
tissue. The cells showed eosinophilic, fibrillary cytoplasm and
blunt, elongated nuclei. No mitotic activity was observed. Respiratory epithelial-lined clefts were seen. The immunohistochemical profile showed positivity for smooth-muscle actin and for
desmin; whereas, cytokeratins and TTF-1 were negative. A focal
area (maximum diameter 0.7 cm) of well-differentiated mucinous
adenocarcinoma was observed. Therefore, complete sampling of
the lesion was performed and no further adenocarcinoma foci
were found. The neoplastic mucinous cells stained for CK7 and
were negative for TTF-1.
Conclusion. Pulmonary hamartoma (PH) combined with a second neoplasm is a very rare event.3-5 Both epithelial and mesenchymal tumors have been described within PH. In particular, the
first report concerns the association of a lipoma-like well-differentiated liposarcoma within a PH.4 As regards epithelial tumors,
the previously associations reported in the English literature
include a PH combined with a typical carcinoid/tumorlet, and 2
PH combined with salivary gland-type tumors.3,5 We herein have
reported the first case of PLH in association with a malignant
tumor, a well-differentiated mucinous adenocarcinoma.
References
1
Cavazza A, Rossi G, Paci M, et al. Primary pulmonary leiomyoma,
with clear cell features and with admixed epithelial spaces: report of
a case presenting with pneumotorax. Pathologica 2003; 95:108-114.
2
Nistal M, Hardisson D, Riestra ML. Multiple pulmonary leomyomatous hamartomas associated with a bronchogenic cyst in a man. Arch
Pathol Lab Med 2003; 127:194-196.
3
Cavazza A, Paci M, Rossi G. Pulmonary hamartoma associated with
typical carcinoid/tumorlet. Wirchows Arch 2006; 449:392-393.
4
Rossi G, Cavazza A, Valli R, et al. Atypical lipomatous tumour
(lipoma-like well-differentiated liposarcoma) arising in a pulmonary
hamartoma and clinically presenting with pneumothorax. Lung Cancer 2003; 39:103-106.
5
Pelosi G, Rodriguez J, Viale G, et al. Salivary gland-type tumors with
myoepithelial differentiation arising in pulmonary hamartoma. Am J
Surg Pathol 2006; 30:375-387.
The use of immunohistochemistry for the
identification of egfr mutations
M. Rotellini1, P. Pretelli1, C. Caporalini1, L. Novelli2, G. Baroni1,
L. Messerini1, C.E. Comin1
Department of Experimental and Clinical Medicine, Section of Specialistic Surgery and Histopathologic and Molecular Diagnosis, University of
Florence, Florence, Italy; 2 Unit of Histopathology, Department of Biomedicine, Careggi Hospital, Florence, Italy.
1
Introduction. In the last years there have been remarkable advances in the treatment of non-small-cell carcinoma (NSCLC),
with a clear departure from the historical management of advance lung cancer based upon a relatively simple dichotomy of
small-cell carcinoma (SCLC) or NSCLC.1 Infact, the historical perspective radically changed in 2004 with the discovery
of the epidermal growth factor receptor (EGFR) mutations
in patients with lung adenocarcinoma and their response to
EGFR tyrosine-kinase inhibitors (TKIs). ALK fusions were
later found to also be limited to adenocarcinoma and current
guidelines recommend initial EGFR testing and ALK testing
in patients with adenocarcinoma or mixed tumors with adenocarcinoma components.2 Lung adenocarcinoma is now considered a disease that could be largely classified into clinically
relevant molecular subsets by oncogenic driver mutations,
each with unique clinicopathologic features and potential opportunities for targeted therapies.3As emphasized by the new
184
classification of lung adenocarcinoma in 2011, the role of the
pathologist is central to achieve the goal represented not only
by a most accurate diagnosis, but also by a correct management of biological tissue in a way that immunohistochemical
and molecular studies can be performed to obtain predictive
and prognostic data that will lead to improvement in patients
outcomes.4 EGFR is a gene constituited by 24 exons, 6 of
which (exons 18-24) representing the tyrosine-kinase domain.
This peculiar site is responsible of gene activity and it is the
region where the relevant mutations are located. In fact, 8085% of activating gene mutations are represented by the deletion E746-A750 in exon 19 and by the point mutation L858R
in exon 21 (so called “hot spot mutations”). The constitutional
activation of EGFR by mutation promotes the development
and progression of malignancy because the gene is implicated in proliferation, angiogenesis, metastasis and apoptosis
inhibition.5 A variety of methods are used to detect EGFR
mutations, including Sanger sequencing, pyrosequencing, real
time PCR. In the last few years, rabbit monoclonal antibodies
against the two most common mutations (del E746-A750 and
L858R) were developed for detecting EGFR mutations. These
antibodies seem to have high specificity but low sensitivity.
Materials and methods. Our series was represented by 37 cases.
Specimens were obtained from surgical lung resection material
(20), biopsy specimens of primary pulmonary neoplasms (i.e. endobronchial biopsies or core biopsies, 6); biopsies from metastatic
sites (10), and one cell block. All cases were previously analyzed
with molecular techniques to identify the mutational status of
EGFR gene. All surgical lung resections was classified with the
criteria of the new classification proposed in 2011;4 pathological
stage were assessed by the indications of the last edition of the
American Joint Committe on Cancer (AJCC).8 Tissue sections
(4-µm thick) were deparaffinized in xylene and rehydrated using
a graded series of ethanol solutions. A heat-induced epitope retrival with Target Retrieval Solution (Dako, Carpinteria, CA, USA)
were performed. The primary antibodies included the rabbit
monoclonal antibody against human EGFR with delE746-A750
mutation (1:100 dilution, clone 6B6, Cell Signaling Technology,
Danvers, MA, USA) and the rabbit monoclonal antibody against
human EGFR L858R mutation (1:50 dilution, clone 43B2, Cell
Signaling Technology). The immunoreactions were detected using EnVision Plus system (Dako) and 3,3’-diaminobenzidine,
followed by counterstaining with hematoxylin. Positive and
negative controls included cases previously studied by molecular
analysis. Immunostaining for each mutation specific antibody
was evaluated by using a light microscope at different magnifications. Specificity, sensitivity, positive predictive value (PPV) and
negative predictive value (NPV) of each antibody were evaluated.
Results. In the molecular-based analyses, delE746-A750 was
detected in 18 cases, and L858R mutation in 19 cases. In the
immunohistochemistry-based analyses, a positive reaction was
detected in 14 cases for the del-specific antibody, and in 18 cases
for the L858R mutation. With the molecular-based results set as
gold standard, the specificity and sensitivity of the del-specific
antibody were 100% and 77.7% respectively, while the specificity and sensitivity of the L858R specific antibody were 100% and
94.8% respectively. Both specific antibodies showed a VPP of
100%, while VPN for del-specific antibody and L858R specific
antibody was 88.2% and 96.7% respectively.
Conclusion. Our results showed extremely high specificity
and lower sensitivity, in particular regarding the identification
of the delE746-A750. We demonstrated that immunohistochemistry could be used when dealing with small biopsy samples or scarce cytology when DNA content may be unsuitable
for molecular analysis. Another aspect, is the possibility to
use immunohistochemistry in determining EGFR status in tissues obtained from bone biopsies as decalcification processes
often result in DNA degradation hindering mutation detection.
Moreover, the immunohistochemical technique could be a
valid screening method.
References
1
Kerr KM. Personalized medicine for lung cancer: new challenges for
pathology. Histopathology, 2012; 60:531-546
2
Johnson DH, Schiller JH, Bunn PA. Recent clinical advantages in lung
cancer management. J ClinOncol 2014; 32:973-982.
3
Pan Y, Zhang Y, Li Y, et al. ALK, ROS1 and RET fusions in 1139
lung adenocarcinomas: A comprehensive study of common and fusion
pattern-specific clinicopathologic, histologic and cytologic features.
Lung Cancer 2014; 84:121-126.
4
Travis WD, Brambilla E, Noguchi M. International Association for
the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary classification of lung
adenocarcinoma. J ThoracOncol 2011; 6:244-285.
5
Yatabe Y. EGFR mutations and the terminal respiratory unit. Cancer
Metastasis Rev 2010; 29:23-36.
6
Roskoski R. Anaplastic lymphoma kinase (ALK): structure, oncogenic
activation, and pharmacological inhibition. Pharmacological Research
2013; 68:68-94.
7
Sasaki T, Rodig SJ, Chirieac LR, et al. The biology and treatment
of EML4-ALK non-small cell lung cancer. European J Cancer 2010;
46:1773-1780.
8
Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, editors. AJCC cancer staging manual (7thed.). New York, NY: Springer
2010.
185
COMUNICAZIONI ORALI
Lung metastasis from TTF-1 positive sigmoid
adenocarcinoma: pitfalls and management
A. Remo1, S. Sina2, E. Baritono1, S. Bonfante1, A. Fioravanzo2,
E. Manfrin2, M. Chilosi2
1
Anatomia Patologica Ospedale “Mater Salutis” Legnago, Verona; 2 ULSS 21
Ospedale di Legnago Verona Dipartimento di Anatomia Patologica
The lung is a frequent site of metastatic involvement, and in many
cases the differential diagnosis between a metastasis and a primary
carcinoma is a substantial question. TTF-1 is considered as a reliable marker for differential diagnosis in distinguishing primary
lung carcinoma and metastasis, especially when dealing with an
adenocarcinoma or a large-cell carcinoma. It was generally thought
that adenocarcinomas arising in the gastrointestinal tract do not express TTF-1. Recently, it has been reported that a small percentage
(1.8%-5.8%) of intestinal adenocarcinoma TTF-1 positive show
differences in sensitivity/specificity depending on the antibody
clones. We report a case of lung localization of a TTF-1 positive adenocarcinoma in a patient with a history of colon adenocarcinoma.
Based on the current results and previous reports, we propose the
following criteria for diagnosing lung metastasis from TTF-1 positive intestinal adenocarcinoma. 1) Clinical features and anamnestic
history are diagnostic milestones, and provide very important information as a prognostic parameter of primary carcinoma and the time
interval between the two localizations (primary and metastasis). 2)
The histologic features are compatible with an enteric differentiation. 3) TTF-1 must be tested in the primary carcinoma. 4) In lung
lesions, in association with TTF-1, it could be useful to test other
immunohistochemical markers such as CDX-2 and NapsinA. 5)
Testing other immunohistochemical or molecular markers in either
lesion is not very useful. Heterogeneity between primary and metastatic lesions has been reported in the literature. Application of the
above-mentioned criteria would simplify diagnosis of lung metastases from TTF-1 positive intestinal adenocarcinoma.
Sala Brunelleschi – 4° piano – 08,30-09,30
Osso e parti molli e Patologia pediatrica
ModeratorI: D. Moncini, G. Tinacci
Immunohistochimical expression of p16 in
pediatric lipoblastomas
R. Cappellesso1, E. D’Amore2, V. Guzzardo1, E. Vassarotto1, M.
Rugge1, R. Alaggio1
Department of Medicine (DIMED), Surgical Pathology & Cytopathology
Unit, University of Padua, Padua, Italy; 2 Department of Pathology, San
Bortolo Hospital, Vicenza, Italy
1
Background. Lipoblastoma is an uncommon benign adipocytic
lesion of the childhood morphologically encompassing all the
steps of adipocytic differentiation. It may show striking histologic
similarity to liposarcoma (myxoid [ML] or well differentiated
[WDL]). Recently, p16 positive immunostaining has been found
in about 30% of ML and in up to 89% of WDL and it has proved
to be useful in the differential diagnosis from lipoma, with higher
sensitivity and specificity than MDM2 and CDK4 immunohistochemisty. Indeed, p16 inhibits cell cycle progression by binding CDK4, whose gene is frequently amplified in WDL, as for
MDM2 gene. Conversely, lipoblastomas usually harbor PLAG1
gene amplification or rearrangement that is not linked to p16, thus
they are expected to be negative for p16 immunostaining. The
aim of this study was to investigate the morphologic spectrum of
lipoblastomas and the performances of p16 immunohistochemistry in differentiating liposarcomas from lipoblastomas.
Materials and methods. The study was carried out on surgical
specimens of 21 lipoblastomas (age range 0-9 yr, mean 2.7 yr),
16 liposarcomas (11 WDL and 5 ML; age range 57-65 yr, mean
60 yr), and 4 classic lipomas (age range 44-62 yr, mean 54yr) retrieved from the archives of the Pathology Unit of the University
of Padova. Immunohistochemical analysis of p16 was considered
positive when a strong nuclear immunoreaction was observed in
more than 10% of tumor cells, according to the criteria proposed
by Tway K et al. and revised by Gonzalez RS et al.
Results. Lipoblasts were evident in 15 lipoblastomas and were
embedded in variable amount of myxoid matrix in the majority
of cases. In 2 large congenital masses this was prominent and was
associated with a spindle cell component mimicking a sarcoma.
In 6 cases, lipoblasts were rare and mostly found along vascular
septa, thus simulating a WDL. p16 immunostaining was positive
in 2 lipoblastomas and in 12 out of 16 liposarcomas (8 WDL and
4 ML). All lipomas were negative. Overall, p16 showed a sensitivity of 75%, a specificity of 90%, a positive predictive value
(PPV) of 86%, and a negative predictive value (NPV) of 83% in
differentiating liposarcomas from lipoblastomas.
Conclusions. Lipoblastomas are morphologically heterogeneous
and seldom can simulate both WDL and ML (6/21 and 2/21 cases
in the present series, respectively). In adipocytic lesions, p16
immunohistochemical positivity is highly suggestive of liposarcoma and may be helpful in difficult cases for the differentiation
from lipoblastoma. Although promising, such finding should be
interpreted with caution since also two lipoblastomas resulted
positive. Further validation in larger series is needed.
Elastofibroma dorsi: focus on extracellularmatrix
C. Mignogna1, A. Di Vito*, F. Conforti1, V. Zuccalà1, L. Maltese1, G. Donato1
1
Department of Health Sciences,Pathology Unit, University Magna
Græcia,Catanzaro; 2 Department of Experimental and Clinical Medicine,
University Magna Græcia,Catanzaro
Elastofibroma dorsi is regarded as a reactive process of uncertain
origin and, in some degree as fibroblastic tumor-like lesion. It
results from a peculiar proliferation of fibroblastic or myofibroblastic cells, with accumulation of abnormal collagen and mainly
elastic fibers in the periscapular region. As far as accumulation
of elastic fibers in elastofibroma is concerned, it’s not clear if it
results from a degenerative process due to repeated mechanical
friction, or from an altered de novo synthesis. However, genetic
predisposition was suggested according to the chromosomal
alterations reported. In contrast to its peculiar clinical diagnosis,
pathogenesis of elastofibroma phenotypes has not yet characterized. In our study, we examined 11 cases of elastofibroma,
by using histochemical and immunohistochemical methods, in
order to highlight the distribution of specific extracellular matrix
proteins. Cases were tested with Alcian blue ph2.5 staining and
with a combined ialuronidase digestion procedure (ialuronidases
from bovine testis), and immunostochemically with Tenascin C
and Periostin. Immunohistochemical analysis display Periostin
and Tenascin C positivity both in vessel’s wall as well as in extracellular matrix in all cases examined. Such proteins are typical
186
of embryonic development whereas in adult tissue are detected
almost exclusively during neoplastic process. In addition, many
studies show as mechanical tension is able to stimulate expression and deposition of a wide range of matrix proteins (Elastin,
Tenascin-C, Periostin) in peripheral tissues1-2. Such evidences
strongly suggest that similar mechanisms can also occur in elastofibroma development. Overall, our results confirm the involvement of specific extracellular matrix proteins in elastofibroma
development. Especially, Periostin and Tenascin C expression in
blood vessels may indicate a key role in active neovascularization
occurring in elastofibromas.
References
Järvinen TA, Józsa L, Kannus P, Järvinen TL, Hurme T, Kvist M, PeltoHuikko M, Kalimo H, Järvinen M. (2002) Mechanical loading regulates the expression of tenascin-C in the myotendinous junction and
tendon but does not induce de novo synthesis in the skeletal muscle.
Journal of Cell Science 116(5)
Rosselli-Murai L.K., L.O. Almeida, C. Zagni, P. Galindo-Moreno, M.
Padial-Molina, S.L. Volk, M.J. Murai, H.F. Rios, C.H. Squarize, R.M.
Castilho (2013) Periostin responds to mechanical stress and tension
by activating the mTOR signaling pathway. Plos One 8(12):e83580.
Histosonographic characterization of
subcutaneous cavernous angiolipoma
T. Pusiol1, L. Roncati2, A. Maiorana2
1
Company of Provincial Health Services, Institute of Pathology, Santa
Maria del Carmine Hospital, Rovereto (TN), Italy;2 Department of Diagnostic and Clinical Medicine and of Public Health, Section of Pathology,
University of Modena and Reggio Emilia, Modena (MO), Italy.
Keywords. angiolipoma; subcutaneous angiolipoma; cavernous
angiolipoma; histology; ultrasound.
Introduction. Angiolipoma is a benign soft-tissue tumor which
combines the morphological features of lipoma with those of
hemangioma. The ratio of lipomatous and vascular components is
variable, reaching the latter the highest percentage in the cellular
Fig. 1. 3D ultrasound scan of subcutaneous cavernous angiolipoma:
a not homogenus sonographic appearance, due to vascular lacunae
filled by blood (black gaps), is noticeable.
variant of the tumor 1. It is important to distinguish this variant
because cellular angiolipoma can assume an infiltrative growth
with tendency to local recurrence after a borderline surgical excision. Angiolipoma tipically occurs in the subcutaneous tissue as
a single nodule, rarely more than 2 cm in diameter, or as multiple
nodules, especially in syndromic conditions, such as Cowden
and Birt-Hogg-Dube syndromes 2,3. The tumor can cause pain if
it compresses the adjacent peripheral nerves; the pain correlates
also with the degree of vascularity 4. About the two-thirds of all
angiolipomas appear in the forearm, followed by the upper arm
and trunk. It is seldom localized at the level of the internal organs.
The tumor is more common in males and its incidence is higher
in the second decade of life, while only sporadic cases in children
or in adults over the age of 50 years are described. Microscopically, the tumor consists of mature adypocites separated by a
branching network of small vessels, which often contain fibrin
thrombi 4,5. On the contrary, the term ‘vascular caverns’ have to
be refered to large vascular channels lined by flattened endothelium and engorged by blood, with a neoplastic or malformative
nature. Here, we describe the first case in literature of subcutaneous cavernous angiolipoma, delivering its first histosonographic
characterization.
Methods. A 75-year-old man was admitted to the hospital ward
for an ultrasound scan on the region of the left pectoralis major
muscle, where a painful enlarging subcutaneous swelling had
appeared about 6 months before. The echography revealed the
presence of an extrafascial discoid neoformation, measuring
2.32x2.27x0.56 cm. The lesion showed an inhomogeneous sonographic appearance, with ill-defined margins (Fig. 1). For this
reason a surgical excision was performed. Grossly, the lesion
appeared with a distinctive bluish color. The surgical specimen
was fixed in 10% neutral buffered formalin and then paraffin
embedded. The tissue sections were submitted to haematoxylin/
eosin staining, following the standard procedures.
Results. The tumor was histologically composed by a well-differentiated lipomatous component intermingled with large gaps,
Fig. 2. Histological picture of subcutaneous cavernous angiolipoma:
large vascular channels, haphazardly arranged, are engorged by
blood. They are lined by a single layer of endothelial cells (insert); the
vascular component is mingled with the fat one (haematoxylin/eosin,
original magnifications 4x and 20x).
187
COMUNICAZIONI ORALI
lined by vascular endothelium devoid of cytologic atypia. The extent of these gaps was such as to far exceed the vascular dilations
that can be found in classic angiolipoma. Therefore, a diagnosis
of subcutaneus cavernous angiolipoma was rendered (Fig. 2).
Discussion. For the first time in literature, we describe a case
of subcutaneous cavernous angiolipoma. This soft-tissue entity
should be reserved to subcutaneous angiolipoma with very large
vascular gaps, haphazardly arranged and engorged by blood.
The first and only description of a cavernous angiolipoma dates
back to 2008, when Farooq and collegues described its spinal occurence 6. The differential diagnosis of this new entity depends
on vascular density: the hypovascular tumor can be mistaken
with a common lipoma, while the hypervascular lesion should be
distinguished from cavernous hemangioma and related variants,
intramuscular hemangioma (also called infiltrating angiolipoma),
spindle cell hemangioma, arteriovenous and venous malformations and Kaposi’s sarcoma. Histopathogenetic theories about
subcutaneous cavernous angiolipoma include a possible differentiation from a pluripotent mesenchymal cell into both lipomatous
and angiomatous counterparts, due to inflammation or trauma,
and a progressive development from a congenital malformation
/ benign hamartoma. In support of the latter thesis, there is a
5-10% familial incidence of classic angiolipoma and the frequent
presence of many mast cells inside the tumor. Moreover, about
a third of all angiolipomas express androgen receptors and this
datum leads to suspect a hormonal influence in the development
of these subcutaneous tumors 7,8. Currently, there is no evidence
that subcutaneous cavernous angiolipoma can undergo malignant
transformation.
References
1
Hunt SJ, Santa Cruz DJ, Barr RJ. Cellular angiolipoma. Am J Surg
Pathol 1990;14:75-81.
2
Chung JY, Ramos-Caro FA, Beers B, Ford MJ, Flowers F. Multiple
lipomas, angiolipomas, and parathyroid adenomas in a patient with
Birt-Hogg-Dube syndrome. Int J Dermatol 1996;35:365-67.
3
Levitt J, Lutfi Ali SA, Sapadin A. Multiple subcutaneous angiolipomas associated with new-onset diabetes mellitus. Int J Dermatol
2002;41:783-85.
4
Dixon AY, McGregor DH, Lee SH. Angiolipomas. An ultrastructural
and clinicopathological study. Hum Pathol 1981;12:739-47.
5
Muthulakshmi R, Yesudian P, Kamalam A, Thambiah AS. Unusual
clinical presentation of angiolipoma. Int J Dermatol 1981;20:608.
6
Farooq MU, Samaraweera R, Heilman T, Chang HT. Spinal cavernous
angiolipoma. Arch Neurol 2008;65:981-82.
7
Jain R, Bitterman P, Lamzabi I, Reddy VB, Gattuso P. Androgen
receptor expression in vascular neoplasms of the breast. Appl Immunohistochem Mol Morphol 2014;22:132-35.
8
Cesinaro AM, Roncati L, Maiorana A. Estrogen receptor alpha
overexpression in multinucleate cell angiohistiocytoma: new insights
into the pathogenesis of a reactive process. Am J Dermatopathol
2010;32:655-59.
Fetus affected by Pena-Shokeir syndrome Type I
in monochorionic diamniotic twin pregnancy
Fig. 1. monochorionic diamniotic placenta.
L. Sollima , G. Calvisi , L. Melchiorri , G. Di Luigi , G. Carta ,
P. Leocata1
1
2
1
3
or decreased in utero movement. The fetal akinesia/hypokinesia
sequence (or Pena-Shokeir syndrome type I) is characterized
by multiple joint contractures, facial anomalies and pulmonary
hypoplasia. Whatever the cause, the common feature of this
sequence is decreased foetal activity. Failure of normal deglutition results in polyhydramnios, and a lack of movement of the
diaphragm and intercostal muscles leads to pulmonary hypoplasia. The lack of normal fetal movement also results in a short
umbilical cord and multiple joint contractures. Ulnar deviation
of the hands, rocker-bottom feet, camptodactyly, sparse dermal
ridges and absence of palmar flexion creases are the other components of the fetal akinesia sequence. The face is expressionless, with hypertelorism, telecanthus and poorly folded, small,
and posteriorly angulated ears, and the mouth is small with
micrognathia and high-arched palate. Cryptorchidism is often
observed The Pena-Shokeir syndrome is not a unitary entity but
is etiologically heterogeneous. One-half of cases are sporadic,
in approximately 50 % of published cases an autosomal recessive mode of inheritance were documented. Maternal myasthenia gravis has been diagnosed in some cases, and experiments
in animal models show that curarization of the mother induces
fetal akinesia. Plausible causes of fetal immobility should be
myogenic, neurogenic, ischemic/anoxic.
Materials and methods. We described an unusual case of a fetus affected by Pena Shokeir syndrome Type I, characterized by
pulmonary hypoplasia, distal joint contractures, cryptorchidism
and facial dysmorphism, in a monochorionic diamniotic twin
pregnancy. The second fetus was healthy. A 41-year-old woman,
terzi gravida nulliparous, was subjected to ultrasound during the
12th week of pregnancy showing polyhydramnios and reduction of movements in the uterus limited to one of the fetuses . A
chorionic villus sampling (CVS) was performed. Chromosome
analysis showed a normal 46+XY complement. At week 24 the
ultrasound demonstrated polyhydramnios and increased discrepancy between the movements of the two fetuses; furthermore
one of fetuses presented flexion of the arms and legs, twisting of
hands and feet and signs of pulmonary hypoplasia. At the 31th
week of pregnancy a fetoscopy with fetal cord clamping (selective reduction) and drainage of polyhydramnios was performed
and at the 32th week caesarean section was done. RESULTS.
The healthy baby weighed 1.5 kg and had an Apgar index of
7-9. The dysmorphic fetus presented severe thanatological alterations, short umbilical cord, arthrogryposis, ulnar deviation of
the hands, rocker-bottom feet, camptodactyly, facies amimica,
oedematous head, micrognathia, low-set ears. Assessment of
organs and viscera revealed hypoplastic lungs and cryptorchidism. Because colliquative necrosis of brain was impossible to
examine the brain and look for possible causes of the disease.
Microscopically scalp appeared edematous, iliopsoas muscle was
characterized by fibrosis with fatty infiltration and by moderate
3
Anatomia Patologica, Dipartimento di Medicina clinica, Sanità pubblica, Scienze della Vita e dell’Ambiente, Università degli Studi dell’Aquila,
L’Aquila, Italia; 2 U.O.C. Anatomia Patologica Ospedale Civile “San
Salvatore”, L’Aquila, Italia; 3 Ginecologia ed Ostetricia, Dipartimento
di Medicina clinica, Sanità pubblica, Scienze della Vita e dell’Ambiente,
Università degli Studi dell’Aquila, L’Aquila, Italia
1
Introduction. Pena-Shokeir syndrome is a rare, early lethal
disorder with an estimated incidence of 1:12,000. It was first
identified by Pena and Shokeir in 1974, although early descriptions resulted in the eponym, it has recently been suggested
that Pena-Shokeir is not a diagnosis or a specific syndrome but
rather a description of a phenotype produced by fetal akinesia
188
Fig. 2. Dysmorfic fetus and facies amimica with low-set ears.
Fig. 3. Rocker-bottom feet and camptodactyly.
References
Pena SD, Shokeir MH. Syndrome of camptodactyly, multiple ankyloses,
facial anomalies and pulmonary hypoplasia: a lethal condition. J Pediatr 1974; 85(3): 373-5.
Hall JG. Analysis of Pena-Shokeir phenotype. Am J Med Genet 1986;
25: 99-117.
Hall JG. Pena-Shokeir phenotype (fetal akinesia deformation sequence)
revisited. Birth Defects Res A Clin Mol Teratol 2009; 85:677-94.
Moessinger AC. Fetal akinesia deformation sequence. An animal model.
Pediatrics 1983; 72:857-63.
Paladini D et al. Pena-Shokeir phenotype with variable onset in three consecutive pregnancies. Ultrasound Obstet Gynecol 2001; 17: 163-165
Evans M et al. Fetal Reduction From Twins to a Singleton: A Reasonable
Consideration? Obstetrics & Gynecology 2004; 104: 102-109
Risk factors of Osteonecrosis
G. Luongo1, V. Natella1,V. Papacciuoli1, F. Carbone2, V. Franzese2, A. Bernasconi2, F. Sadile2, G. Mansueto1
Department of Advanced Biomedical sciences, University of Naples Federico II; 2 DAI di Chirurgie Specialistiche e Nefrologia U.O. di Ortopedia
e Traumatologia
1
Fig. 4. Hypoplastic lungs.
Fig. 5. Microscopic features of oedema of the scalp; fibrosis with fatty
infiltration of iliopsoas.
Fig. 6. Testis: atrophy of testicular tubules, fibrosis and hypertrophy
of Leydig-cells.
Background. Osteonecrosis, also known as avascular necrosis or
ischemic necrosis of the femoral head, is a pathologic process that
results from interruption of blood supply to the bone. The etiology of osteonecrosis is believed to be multifactorial and associated
in some cases with both a genetic predilection and exposure to
certain risk factors such as trauma, use of corticosteroids, excessive alcohol intake, smoking, hemoglobinopathies, coagulation
and myeloproliferative disorders.
Materials and methods. From 74 patients (47 males and 27
females, aged between 13 and 76 years, and a follow-up from
two months to 21 years) we selected 26 cases with follow-up
(18 with ipsilateral osteonecrosis and 8 with bilateral osteonecrosis).
The necrosis, fibrosis and bone remodeling were analyzed semiquantitatively (0 absent, 1 minimal, 2 low, 3 mildly, 4 high).
Conclusion. We identified three groups: the first group with unknown etiology and acute onset (short story); the second one with
insidious onset (long story) and worsening limitation of ROM;
and the third group characterized by hematological, neoplastic
and not. For each group we observed a specific morphological
pathological aspect.
Fatal alveolar-interstitial pneumonitis in a
case of Treacher Collins syndrome without
tracheoesophageal fistula
G. Luongo, V. Natella, P. Valeria, A. Marano, M. D’Armiento,
G. Mansueto
Department of Advanced Biomedical Sciences, University of Naples Federico II
variability in diameter of muscle fibers, lungs showed borderline
hypoplasia and testis showed atrophy of testicular tubules, fibrosis and hypertrophy of Leydig-cells. Conclusions. The normal
46+XY complement, without chromosomal alterations, allow to
exclude differential diagnoses like Cerebro Oculo Facio Skeletal
syndrome 1 (COFS1-Pena-Shokeir syndrome, Type II), Trisomy
18 and Lethal Congenital Contracture Syndrome (LCCS). The
macroscopic and microscopic features, the medical history, the
course of the disease and the phenotype of the fetus suggest the
diagnosis of Pena-Shokeir syndrome, Type I.
Background. Treacher Collins syndrome (TCS, OMIM 154500)
is a rare congenital birth disorder characterized by severe craniofacial defects. It occurs with an incidence of 1:50000 live births
with autosomal dominant inheritance in types 1 and 2 (TCS1,
TCS2) and autosomal recessive in type 3 (TCS3). The majority
of individuals with TCS are heterozygous for a mutation in the
TCOF1 gene, located in 5q32-q33.1. At present, there is no clear
genotype–phenotype correlation to discern between dominant
and recessive types, sporadic and familial mutations or to predict disease severity.Hypoplasia of the mandible and zygomatic
arches, abnormalities in shape, size and position of the auricles
often associated with atresia of the external auditory canal,
macrostomia, eyelid colobomas and downward slanting of the
palpebral fissures are the most common clinical features of TCS.
Conductive hearing loss, cleft palate, and dental malformations
are often present.
COMUNICAZIONI ORALI
Materials and methods. Came under our observation for autopsy an infant of one month and thirteen days died of cardiorespiratory arrest after arriving in the first aid unconscious and in
the absence of breathing and pulse.
He was born at thirty-eight weeks of pregnancy normally passed
as a result of planned caesarean section with an APGAR score of
1’:8, 5’:9 (BW 3440 gr, BL 50 cm, BHC 34,5).
At autopsy the body was 51 cm tall, weighed 3,3 kg and had a
diffuse bluish coloration of the nail bed as cyanosis. On physical
examination several craniofacial abnormalities were noted including mandibular hypoplasia, retrognathia, downward slanting of the
palpebral fissures (antimongoloid slant), an angioma of the internal
third of the upper right eyelid, bilaterally preauricular process (appendages), two bilateral preauricular breaks in the skin that end
up in a dead-end and agenic right external auditory canal. These
malformations were soon investigated at birth with a genetic counseling with which it was discovered that a mother’s cousins had
mandibular hypoplasia, the mother was born with bilateral ptosis,
greater on the left; and his maternal grandfather was affected by
unilateral hearing loss as well as his paternal grandmother.
Conclusion. The presence of structural abnormalities and the
familial history had allowed the diagnosis of Treacher Collins
syndrome a few days after birth.
At autopsy the internal examination showed that the pharyngeal walls were lined by mucous membrane with slight reddened
folds. Esophagus was normal shaped and without fistulas with
nearby organs. The laryngeal vestibule was characterized by
reddened, thickened and edematous mucosa with an appreciable
reduction in the lumen and thickening of the aryepiglottic folds.
The heart weighed 25 gr and appeared normal.
The lungs appear reddish widespread, showing increased size
and normal shape and reveal subcentimeter areas of bluish-red
color more at the level of the pulmonary fissures and areas of
parenchymal consolidation. No marked abnormalities, except for
congestion, were found in the other organs.
The corresponding microscopic findings of the narrowed laryngeal vestibule was of a mucosa lined by squamous epithelium
thickened and hyperplastic subtended by edematous and fibroticlike areas. Furthermore was observed the presence of hyperplastic
lymphoid tissue.
The lungs showed an alveolar structure partly preserved and partly
with an areas of septal fibrosis. In the alveolar spaces and interalveolar septa was observed the presence of multinucleated cells, erythrocytes extravasation, histiocytic elements and poor lymphmonocytic
component in the absence of a significant portion of granulocytes.
Post-mortem diagnosis was alveolar-interstitial pneumonitis in
a case of Treacher Collins syndrome with several craniofacial
abnormalities (mandibular hypoplasia, retrognathia, downward
slanting of the palpebral fissures, bilaterally preauricular process
(appendages), two bilateral preauricular breaks in the skin that
end up in a dead-end and agenic right external auditory canal) and
airway narrowing and respiratory compromise.
In our case it was just an acute respiratory failure that result
in in the infant’s death. The cause was a widespread infectiveinflammatory process of the airways with particular involvement
of the lung parenchyma (broncopneumonia of interstitial origin),
in the absence of tracheoesophageal fistula.
Alport syndrome: a patologist point of view
M. D’Armiento1, S. Campione1, F. Nuzzi3, E. Guadagno1, M. Balletta2, M. Cervasio1, C. Pecoraro3, G. De Rosa1.
Department of Advanced Biomedical Sciences, Section of Pathology, University of Naples “Federico II”, Naples, Italy; 2 Department of Public Health,
Section of Nephrology, University of Naples “Federico II”, Naples, Italy; 3
Pediatric Nephrology Unit, AORN Santobono-Pausilipon, Naples, Italy
1
Background. Alport syndrome (AS) is a hereditary, progressive,
hematuric nephropathy characterized by glomerular basement
189
membrane abnormalities, frequent hearing defects and ocular
anomalies. Hematuria is a common presenting sign of glomerular disease and is sometimes associated with kidney failure later in life. When isolated, in children and young adults,
microscopic hematuria can reveal an underlying genetic disorder, such as Alport syndrome or a thin basement membrane
nephropathy, but also other diseases, which often present with
isolated microscopic hematuria, among them hereditary angiopathy, aneurysms, and muscle cramps syndrome (HANAC),
IgA nephropathy, and CFHR5 nephropathy.
We focus on familial microscopic hematuria (MH) of glomerular origin, that can be expression of a heterogeneous group of
monogenic conditions involving several genes, some of which
remain unknown, and that could be associated with kidney
failure later in life. The disease is associated with mutations
in genes encoding the alpha3, alpha4, or alpha5 chains of type
IV collagen, COL4A3, or COL4A4 in the autosomal forms of
the disease, COL4A5 in the more frequent X-linked variety.
Pathologic studies play an important role in evaluating patients with AS besides genotyping. Difficulties still exist in
diagnosing AS, and misdiagnosis is a not-so-rare event, even
in adult patients evaluated with renal biopsy.
Ultrastructural changes in glomerular basement membrane
and frequent abnormal expression of type IV collagen chains
in renal and skin basement membranes are crucial elements
for the diagnosis of AS, determination of the mode of inheritance, and genetic counseling. Animal models have provided
invaluable tools to study the mechanisms that lead to progressive deterioration of the glomerular basement membrane
and ultimately to renal failure, and to evaluate benefits of
potential targeted therapies. However, Difficulties still exist
in diagnosing AS; Pathologic studies play an important role in
evaluating patients with AS besides genotyping.
Materials and methods. A retrospective evaluation of 470
pediatric native kidney biopsies (aged between 0,5 and
17 years) performed at “S.C. Nefrologia-Dialisi Pediatrica
AORN Santobono-Pausilipon” from 2000 to 2013 and from
3540 nephropathic babies was done by 2 renal pathologists.
Morphologic alterations were evaluated according to light
microscopy, immunofluorescence and electron microscopy
(EM) findings in order to assess the contribution of EM to
the final diagnosis. All cases suspected as AS were addressed
to genetic testing for important diagnostic and prognostic
information that are relevant to the patient and their family,
particularly when kidney transplantation is considered.
Results. Of 470 pediatric native kidney biopsies ultrastructural examination revealed 61 AS, 43 males and 18 females.
2 previously diagnosed mesangial proliferative glomerulonephritis (MsPGN), and 5 focal and segmental glomerulosclerosis (FSGS) were the most common misdiagnosis. FSGS
was the most frequent misdiagnosis in female X-linked AS
(fXLAS) patients, and MsPGN in male X-linked AS (mXLAS) patients; 3 IgA GN were also misdiagnosed. Previously
misdiagnosed mXLAS patients and autosomal recessive AS
(ARAS) were corrected after electronic microscopy, while
misdiagnosed fXLAS patients were corrected after a family
member diagnosed and after rechecking electronic microscopy and/or collagen-IV alpha-chains immunofluresence study
(COL-IF). The α5 COL-IF was not useful (1 case positive);
the expression of the α3 chain was diffuse in 5 cases and
segmental or absent in 18 cases. Diffuse staining for the α3
COL-IF is early positive prognostic marker and the patients
present a better prognosis (with null or truncating COL4A5
mutations).
Conclusions. The ultrastructural examination is the most
significant diagnostic tool in suspected AS diagnosis (97%, χ
2
6,2). COL-IF is an additional criterion for AS diagnosis: we
found that patients with less than 3 diagnostic criteria reached
190
have increased risk of misdiagnosis. The molecular genetics
emerges as a powerful tool for a definite diagnosis when all
the above conditions enter the differential diagnosis, while in
many at-risk related family members, a molecular diagnosis may
obviate the need for another renal biopsy. However, there are
still numbers of cases in which EM is essentially needed to reach
definitive diagnosis (also to suggest genetic analysis).
thy. J Am Soc Nephrol 24: 364–375, 2013.
Artuso R, Fallerini C, Dosa L, et al. Advances in Alport syndrome diagnosis using next-generation sequencing. European Journal of Human
Genetics (2012) 20, 50–57.
Liapis H, Foster K, Theodoropoulou E. Phenotype/genotype correlations
in the ultrastructure of monogenetic glomerular diseases. Ultrastruct
Pathol. 2004 Jul-Aug;28(4):181-97.
References
Savige J, Gregory M, Gross O, et al. Expert Guidelines for the Management of Alport. Syndrome and Thin Basement Membrane Nephropa-
Sala Brunelleschi – 4° piano – 14,30-15,30
Citologia
Moderatori: C. Gentili, G. Mazzoleni
Cytopathologists can reliably perform ultrasoundguided thyroid fine needle aspiration: a one-year
audit on 3715 consecutive aspirates
C. Bellevicine, E. Vigliar, U. Malapelle, P. Pisapia, R. Morrone,
A. Vetrani, G. Troncone
Department of Public Health, University of Naples Federico II, Pathology
Division
Objective. In our Pathology Department, fine needle aspiration
(FNA) of palpable thyroid nodules are performed by cytopathologists who ensure correct sample management and rapid on-site
evaluation (ROSE). Conversely, ultrasound (US)-guided FNA
have traditionally been taken by endocrinologists and radiologists
in outside clinics, where the presence of a cytopathologist is not
always feasible. To overcome this limitation, cytopathologists
have started to perform US-guided FNA by themselves. This
study retrospectively evaluates one-year of this novel practice.
Methods. A total of 2225 US-guided FNAs was performed in our
clinic by cytopathologists, whereas 1490 aspirates were taken by
a group of non-cytopathologists. Among those, 756 FNAs were
taken by a single experienced endocrinologist. The distribution
of the Bethesda classification categories was evaluated in each of
these groups. Results. FNAs performed by cytopathologists were
more often diagnostic and better prepared than those taken by
non-cytopathologists, including those taken by the experienced
endocrinologist (p<0.01) (Table 1). This latter operator yielded a
Tab. I. Bethesda categories distribution in US-guided FNAs taken by cytopathologists (CUS) and non-cytopathologists (N-CUS).
Bethesda
CUS FNAs (%)
Categories
Nondiagnostic
72 (3.2%)
AUS/FLUS
164 (7.3%)
Benign
1881 (84.5%)
FN/SFN
40 (1.7%)
Suspicious for
22 (0.9%)
Malignancy
Malignant
46 (2%)
Total
2225
* statistically significant.
N-CUS FNAs (%) p-value
385 (25.8%)
101 (6.7%)
919 (61.6%)
30 (2%)
20 (1.3%)
<0.01*
0.4
<0.01*
0.6
0.3
35 (2.3%)
1490
0.5
higher rate of suspicious and malignant FNAs, reflecting a more
appropriate clinical triage of worrisome nodules. Conclusion.
Although the endocrinologist’s evaluation is crucial to select
clinically relevant thyroid nodules, cytopathologists can reliably
perform US-guidance in addition to their traditional expertise in
sampling, specimen preparation and ROSE.
Blood-borne metastatic carcinomas to thyroid
gland: a fifteen cases experience on fine needle
cytology in ten years
I. Cozzolino1, A. Vetrani2
Specialist in Anatomic Pathology and 2 Professor of Anatomic Pathology
and Chief Cytology Service, Department of Public Health “Federico II”
University of Naples (IT), Cytology Service.
1
Introduction. The thyroid gland usually is considered to be an
infrequent site of blood-borne metastatic carcinoma (BMC). The
secondary thyroid cancer involvement may be for direct spread
from carcinomas arising in thyroid adjacent anatomical structures
or the metastasis may clinically manifest as a goiter by means of
the arrival of cancer cells by blood stream from a distant origin.
Purpose of this study is to present a fifteen BMC to thyroid gland
on Fine Needle Cytology (FNC) experience in ten years in one
institution which occurred precisely as goiter.
Materials and methods. From January 2003 to December 2013
in the out patients office of Cytology Service of the University of
Naples “Federico II” a total of 28,077 thyroid FNC, either with
and without ultrasonography (US) control, have been performed.
The clinical presentation, the microscopic patterns and metastases
incidence to the thyroid gland are presented. The BMC to thyroid gland incidence data is discussed also compared with other
thyroid malignancies also in the light of the specific literature.
Results. In this period (ten years) 513 (1,8%) out 28,077
thyroid FNC were classified malignant and 15 (2,9%) out 513
were BMC to thyroid gland. Therefore the BMC to thyroid
gland is 0.05% of all the our series. In all cases FNC has proved
to be useful addressing quickly diagnosis and indicating also
the site of origin.
Conclusion. In our experience and by the results of the literature, BMC to thyroid gland constitute certainly a rare event,
however, in a history of malignancy, it is appropriate that a
thyroid nodule must be suspected as metastatic until proven
otherwise.
COMUNICAZIONI ORALI
References
Nikirof YE, Biddinger PW, Thomposon LDR. Diagnostic pathology and
molecular genetics of the thyroid. Baltimore: Lippincott Williams &
Williams, 2009:348-356.
De Lellis RA, Lloyd RV, Heitz PU, Eng C. World Health Organization classification of Tumours. Tumours of Endocrine Organs. Lyon:
Chapman and Hall, 1993.
Papi G, Fadda G, Corsello SM, et al. Metastases to the thyroid gland:
prevalence, clinicopathological aspects and prognosis: a 10-year
Experience. Clinical Endocrinology. 2007;66:565–571.
Schmid KW, Hittmair A, Ofner C, Tötsch M, Ladurner D. Metastatic
tumors in fine needle aspiration biopsy of the thyroid. Acta Cytol.
1991; 35:722-724
Cervical Vaginal Screening: Cyto-hystologic
correlation with P16 test and Hr-Hpv Dna
G. Di Benedetto1, A. Santarsiere1, S. Esposito1, G. Carillo1, A. di
Iasi1, M. Manno1, L. Agozzino2
1
UOC of Pathological Anatomy – P.O. Aversa (Ce)- 2 -Second University
of Naples (SUN)
Background. Although the histological features of cervical intraepithelial neoplasia (CIN) are well understood, inconsistent use
and misinterpretation of such features may occur due to intra-and
interobserver variability (1,2). On the basis of morphology alone,
it can be difficult to distinguish between squamous metaplasia
coupled with hyperplasia (SMH) and CIN I, and between CIN I
and CIN II-III. Moreover, small biopsy size, tangential sectioning, thermal artifact, coexistent inflammatory or reactive lesions
and application of subjective criteria all increase the difficulty for
the diagnosis of CIN (3). Therefore, objective diagnostic methods
in addition to histology are required to accurately diagnose CIN
in histological specimens (4).
Human papillomavirus (HPV) plays an etiologic role in cervical carcinogenesis and is detectable in preinvasive and invasive
cervical epithelial neoplasms (5,6). The most common high-risk
HPV subtypes include types 16 and 18, which account for ~70%
of HPV species detected in cervical cancer. Low-risk types, 6 and
11, account for ~90% of HPV species present in genital warts.
Infections with low-risk types have been known to cause genital warts and low-grade cervical abnormalities. Most recently,
several studies have analyzed the diagnostic performance of a
novel p16/Ki-67 dual-stained cytology approach that combines
simultaneous p16 and Ki-67 detection in the same cell as a hallmark of cell-cycle deregulation (7) . P16 is a cyclin-dependent
kinase inhibitor that regulates the transition from G1 to S phase
of the cell cycle and normally functions as a tumor suppressor . Although p16 levels are reduced in a variety of malignant
tumors, this gene product has been shown to be upregulated (or
overexpressed) in the majority of high-grade cervical dysplasias
and carcinomas induced by high-risk HPV subtypes (8). p53, as
a tumor suppressor, is one of the major factors controlling cell
proliferation. As the ‘guardian of the genome’, it arrests the cell
cycle in response to DNA damage or directs the damaged cell to
an apoptotic pathway.
The aim of the present study was to investigate human papillomavirus (HPV) infection and p16INK4A, to evaluate their roles in
the pathological diagnosis and grading for cervical intraepithelial
neoplasia (CIN).
Materials and methods. The study was conducted on patients
from the A.S.L. of Caserta, who underwent cervical vaginal cytology from June 2012 to April 2013.
2001 Bethesda classification has been used and we have considered for our research the following categories : atypical squamous
cells of undetermined significance (ASC-US), low grade squamous intraepithelial lesion (LGSIL or LSIL), high grade squamous intraepithelial lesion (HGSIL or HSIL). Colposcopy was
performed and biopsies were taken if clinically indicated. Biopsy
confirmed CIN1+ was used as clinical end point for the study.
191
The detection of HPV DNA was identified with the INFORM
HPV III Family 16 Probe (B)(Ventana) that contains a cocktail
of HPV genomic probes in a formamide-based diluent. The intended targets are the common high-risk HPV genotypes found
to be associated with cervical neoplasia. The probe cocktail has
demonstrated affinity to the following genotypes: 16, 18, 31, 33,
35, 39, 45, 51, 52, 56, 58, and 66. The INFORM HPV II Family 6
Probe (B) (Ventana) contains a cocktail of HPV genomic probes
in a formamide-based diluent. The intended targets are the HPV
genotypes found commonly in condyloma and some early cervical intraepithelial lesions, which are not commonly associated
with cervical cancer For all tissue specimens, a parallel section
stained for p16 using the CINtec Histology Kit (Roche mtm laboratories) was separately evaluated regarding its staining pattern.
These data can not to be used to study the diagnostic accurancy
of cytology why the invitations for the women to perform cervical vaginal screening, did not respond to criteria, in according to
GISCI.
Results. Cytology and Hystology. We have performed 18962 pap
test of whose 240 Lsil, 24 Hsil, 194 Asc-Us, 4 Ag-Us, 3 Asc-H
and 92 biopsies with diagnosis of 54 CIN1, 8 CIN2, 9 CIN3 and
21 cervicites.
Precision. The precision to cytologic diagnosis (Predictive Positive Value) Hsil=100%, Lsil= 72%.
P16 and Hpv. Here, we studied the immunohistochemical expression of p16 in 92 dysplastic cervical lesions, 54 cervical intraepithelial neoplasms-grade 1 (CIN-I), 8 CIN-II, and 9 CIN-III.
In addition, we performed HPV detection and genotyping. We detected HR-HPV in 16/54 CIN-I, 8/8 CIN-II and 9/9 CIN-III cases.
P16(INK4a) immunoreactivity was observed in 6/16 CIN-I HRHPV-positive, 8/8 CIN-II HR-HPV-positive and 9/9 CIN-III
cases.
Conclusion. p16(INK4a) expression in dysplastic cervical lesions is related to high-risk human papillomavirus (HR-HPV)
infection. The immunohistochemical expression of this protein in
these lesions allows an increase in diagnostic reproducibility in
biopsies and the introduction of prognostic factors in low-grade
lesions could to be useful to divide CIN 1 without p16 positive
by CIN1 with p16 positive.
The E6 E7 proteomic study and interaction with p53 pRb would
be use to better study the evolution of carcinogenesis of cervix
carcinoma.
References
1
Martin CM and O’Leary JJ: Histology of cervical intraepithelial neoplasia and the role of biomarkers. Best Pract Res Clin Obstet Gynaecol 25: 605-615, 2011.
2.
Dascau V, Furau G, Furau C, Paiusan L, et al: Cervical intraepithelial
neoplasia in the ‘dr. Salvator vuia’ clinical obstetrics and gynecology
hospital - arad during the 2000-2009 period. Maedica (Buchar) 7:
138-142, 2012.
3
Walts AE, L Lechago J and Bose S: P16 and Ki67 immunostaining is
a useful adjunct in the assessment of biopsies for HPV-associated anal
intraepithelial neoplasia. Am J Surg Pathol 30: 795-801, 2006.
4
Hans Ikenberg, Christine Bergeron, Dietmar Schmidt, Henrik Griesser, Francisco Alameda, Claudio Angeloni, Johannes Bogers, Roger
Dachez, Karin Denton, Jalil Hariri, Thomas Keller, Magnus von
Knebel Doeberitz, Heinrich H. Neumann, Luis M. Puig-Tintore, Mario
Sideri, Susanne Rehm, Ruediger Ridder, for the PALMS Study Group
Screening for Cervical Cancer Precursors With p16/Ki-67 DualStained Cytology: Results of the PALMS Study. DOI:10.1093 October
4, 2013.
5
Monsonego J, Pintos J, Semaille C, Beumont M, et al: Human papillomavirus testing improves the accuracy of colposcopy in detection of
cervical intraepithelial neoplasia. Int J Gynecol Cancer 16: 591-598,
2006.
6
Massad LS, Einstein MH, Huh WK and Katki HA: 2012 updated
consensus guidelines for the management of abnormal cervical cancer
screening tests and cancer precursors. J Low Genit Tract Dis 17 (5
Suppl 1): S1-S27, 2013.
7
Tsoumpou I, Arbyn M, Kyrgiou M, Wentzensen N, et al: p16(INK4a)
192
8
immunostaining in cytological and histological specimens from the
uterine cervix: a systematic review and meta-analysis. Cancer Treat
Rev 35: 210-220, 2009.
Sano T, Oyama T, Kashiwabara K, Fukuda T and Nakajima T: Expression status of p16 protein is associated with human papillomavirus
oncogenic potential in cervical and genital lesions. Am J Pathol 153:
1741-1748, 1998.
Oral microhistology: an innovative technique for
a first level diagnosis in malignant and potentially
malignant oral lesions
A. Marsico2, E. Amodeo1, R. Navone1,
Dipartimento di Scienze Biomediche e Oncologia Umana dell’Università
di Torino (Sez. di Anatomia Patologica); 2 Servizio di anatomia Patologica
Ospedale Koelliker, Torino
1
Objectives. Squamous cell carcinoma of the oral cavity (OSCC),
although frequent (6th cause of cancer-related mortality worldwide), still has a low survival rate as diagnosis is often late and
there is a lack of simple and reproducible diagnostic tests able to
identify early stage precancerous potentially malignant lesions
(PMLs). These are clinically subdivided into classes I and II: the
former are manifestly clinically suspicious and the latter have
an apparently innocent appearance. To date, the diagnosis of
oral cavity OSCC and PMLs has been based exclusively on the
scalpel (surgical) biopsy. This is an invasive technique, limited to
a restricted area and of difficult application with multiple lesions
and, last but not least, generally only used for class I lesions.
Oral diagnostic cytology alone, whilst providing useful information (sensitivity is higher than the Pap test, while specificity is
similar), does not suffice for the diagnosis of OSSC and PMLs
(Navone R et al: The impact of liquid-based cytology for the
diagnosis of oral dysplasia and carcinoma. Cytopathology 2007;
18: 35660).
Methods. Recently, an original, less invasive sampling method
which does not generally require anaesthetic and uses a dermatological disposable curette, provided small epithelial fragment
from oral mucosa with results comparable to the scalpel biopsy
(Navone R et al: Oral Potentially Malignant Lesions: First Level
Microhistological Diagnosis from Tissue Fragments Sampled in
Liquid-Based Diagnostic Cytology. J Oral Pathol Med 2008, 37:
358-63). However, only experts in specialised centres did this
sampling. As the territorial (private practise) dentist is the first
to observe an apparently innocent lesion i.e. class II PMLs, after
a brief training period, a clinical trial was set up in collaboration
with them. Samples were obtained according to our instructions
with the curette technique by 50 dentists and treated for histological examination (paraffin inclusion, haematoxylin-eosin staining)
as routine small biopsies.
Results. Ten of the 150 samples were inadequate (6.6%),
131/140 negative (hyperkeratosis, parakeratosis or simple hyperplasia), 6/140 (4.3%) low-grade dysplasia (OIN 1), 2/140 (1.4%)
high-grade dysplasia (OIN 2-3) and 1/140 (0.7%) OSCC.
Our aforementioned study, done in a specialised centre, reported
a 3.6% rate of inadequate samples (6/164). Although inadequate
samples in this field trial are higher (6.6%, 10/150), it must be
considered that “inexpert persons” did sampling and results are
still very good as this is a first level test.
Conclusions. The sampling with the “curette technique” and
the use of “microhistology” may well be an effective firstlevel
method to distinguish those reactive, or inflammatory lesions
requiring only follow-up, from positive lesions (dysplasia and
OSCC) to be sent to the specialised second-level centres for
routine scalpel biopsy. Moreover, this method can use this
material also with flow cytometry to evaluate ploidy: the finding of aneuploidy allowed for the identification of lesions that
were at risk of evolution (Pentenero M et al: DNA aneuploidy
and dysplasia in oral potentially malignant disorders. Oral
Oncol 2009; 45: 887-90) and the selection of individuals who
required a stricter follow-up regime. Lastly, curette sampling,
which covered ample surface areas and/or multiple lesions,
led to a reduction in the number of patients that were required
to return for further investigations as well as the quantity of
surgical (scalpel) biopsies. Consequently, there is a positive
cost/benefit ratio for the hospital and less discomfort for patients. Therefore, the adoption of this technique will allow the
dentist, who is the first to see the preneoplastic and neoplastic
oral lesions, to manage even those apparently innocent lesions
(class II) of difficult definition and/or not yet considered for
biopsy, in the most appropriate manner.
Morphologic parameters are able to predict
BRAFV600E mutated malignancies on thyroid FNAC.
A new cytological insight
E.D. Rossi1, T. Bizzarro1, M. Martini, S. Capodimonti, G. Fadda1,
#
L.M. Larocca1, #F. Schmitt2,3
these authors shared senior authorship; 1 Division of Anatomic Pathology and Histology - Università Cattolica del Sacro Cuore, “Agostino
Gemelli” School of Medicine, Rome Italy; 2 Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal; 3 D-epartment of Laboratory Medicine and Pathobiology, Faculty of
Medicine, University of Toronto and Department of Pathology, University
Health Network, Toronto, Canada
#
Introduction. BRAFV600E mutation is the most common diagnostic/prognostic marker in papillary thyroid carcinoma (PTC).
Its evaluation is typically performed using DNA-based techniques
nonetheless few papers have recently proposed the morphological
prediction of BRAFV600E on thyroid histological PTCs. We investigated this morphological parameter in our cytological series.
Material and methods. We re-analyzed all the 72 cyto-histological samples diagnosed as “positive for malignancy-favoring
PTC” on FNAC in the period January 2012-December 2013. We
included 22 male and 50 female patients. The cytological cases
were processed with liquid based cytology (LBC). We performed
molecular and immunocytochemistry for VE1 BRAFV600E antibody.
Results. We reported 47 mutated and 25 wild type (WT) cases
with 100% cyto-histological concordance. The cytological evaluation revealed plump cells (abundant eosinophilic cytoplasm and
PTC nuclei) in all 47 mutated cases with only 6 focal (less than
20% cells) plump component. Furthermore, 5 out 25 WT cases
showed focal plump cells. A typical sickle nuclear shape was
referred only in the mutated cases. VE1 yielded 100% positivity
in all 24 mutated cases tested including 3 cases with focal plump
cells. Our diagnostic accuracy for these parameters resulted in
100% for sickle shape cells and 93% for plump cells.
Conclusions. We demonstrated that BRAF V600E mutation
might be predicted in cytological samples with good inter-observer agreement. While the detection of plump cells, mainly focal,
might be also observed in WT cases, the detection of sickle shape
nuclei provided the highest specificity as predictive mutational
parameter. These morphological features might be a valid tool in
selecting cases for molecular analysis.
References
Virk RK et al. Virchow Arch 2014; 464: 435-42
Finkelstein A et al. Histopathol 2012; 60: 1052-59
Rossi ED et al. Virchow Arch 2014 Jun 14. [Epub ahead of print]
193
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How to classify follicular adenoma with papillary
architecture diagnosed by thyroid FNA? Report of
a case with histological correlation
E. Vigliar1, V. Varone2, G. Pettinato2, C. Bellevicine1, G. Troncone1.
Department of Public Health, University of Naples “Federico II”, Napoli, Italy; 2 Department of Advanced Biomedical Science, University of
Naples “Federico II”, Napoli, Italy.
1
Introduction. Follicular adenoma with papillary architecture or
“ papillary adenoma” have been traditionally termed “papillary
hyperplastic nodules”, although they show complex papillary
growth and well-defined fibrous capsule, unlike the usual papillary hyperplastic areas in benign lesions. This case highlights the
possibility of classifying these lesions as follicular neoplasms
with papillary architecture even on cytological samples.
Case history. We report a case of a 45 year-old woman presenting with a 10 mm left lobe thyroid nodule, featuring hypoechoic
ultrasound (US) appearance. The material obtained by the first
pass of an US guided fine needle aspiration (FNA) was smeared
and Diff-Quik stained for rapid evaluation. The on-site impression was that of a high cellular smear featuring a striking
papillary pattern. To better evaluate these changes alcohol-fixed
Papanicolaou stained smears were also prepared. The presence
of a large number of three-dimensional cell clusters, displaying
papillary architecture and fibro-vascular cores, was confirmed.
In the background the colloid was scant. At higher magnification the thyrocytes were small or medium in size, with occasional microvacuolated clear cytoplasms, and with round and
regular nuclei. Since papillary thyroid carcinoma (PTC) nuclear
features were completely absent a diagnosis of suspicious for
follicular neoplasm with papillary architecture was rendered,
recommending surgical excision. The histology showed an
encapsulated lesion with extensive papillary architecture. The
nuclei were round and showed regular basal polarity, with little
crowding and overlapping. No PTC features (i.e. grooves, optically clear nuclei or intranuclear cytoplasmic inclusion) were
detected. There was no capsular or vascular invasion. CD56
immunostaining, a marker that it is usually loss in PTC, showed
a strong membranous positivity. Only a few cells were positive
after staining by the proliferating cell marker Ki-67. A final
diagnosis of follicular adenoma (FA) with papillary architecture
was rendered.
Discussion. Besides PTC, papillary architecture may be observed
in different benign thyroid lesions, thus that relying only on papillae recognition may led to misdiagnosis, mostly in FNAs. Multinodular goiter with degenerative changes, Hashimoto thyroiditis
with hyperplastic/reactive areas and FA may all display a papillary
pattern. However, the fibrovascular cores of true papillary fronds
lack in nodular goiter aspirates while in Hashimoto thyroiditis the
inflammatory background may aid the correct cytological diagnosis. The current case highlights the possibility that the cytological
diagnosis of a FA with papillary architecture is difficult. The scant
colloid background, the high cellularity and true papillary structures may be raise the suspect of malignancy. However, the careful
observation at higher magnification generally rule-out PTC with
ease; in fact its nuclear features lacks. As in present case, these
features can be better classified on FNA as “follicular neoplasm”
including a note on their papillary architecture. On histology, these
benign nodules have been traditionally termed “papillary hyperplastic nodules” although they show complex papillary growth,
unlike the usual papillary hyperplastic areas in benign nodules;
moreover, they harbor activating somatic mutations in the TSH
receptor or GNAS gene and they are now known to be clonal lesions. Hence, these lesions are best classified as “FA with papillary
architecture” or “ papillary adenoma”. As we have previously reported, the correct classification of lesion displaying true papillary
structures is even more difficult if the cells are mostly oncocytic.
In fact, encapsulated papillary oncocytic neoplasia (EPON) closely
mimics both on cytology and histology oncocytic PTC. As for FA
with papillary architecture, EPONs usually lack PTC nuclear features and a correct cytological diagnosis can be usually rendered
on careful morphological scrutiny. The diagnostic terminology to
classify benign neoplastic lesions showing a papillary pattern is
controversial. In fact, the Bethesda terminology does not provide
keys to classify this type of lesions. The most pertinent diagnostic
class to report our case was that of suspect for follicular neoplasm.
However, as matter of the fact the Bethesda terminology warns to
not include in this class papillary follicular patterned lesions. This
point should be taken into account in the future refinements of the
Bethesda system
Effectiveness analysis of an opportunistic
cytology-based screening program in Southern
Zambia
F. Maletta1, D. Balmativola1, E. Ferrario2, L. Viberti3-4, M. Barbero5, S. Guzzetti4-6
Department of Medical Sciences, University of Turin, Turin, Italy;
Mtendere Mission Hospital Chirundu, Zambia; 3 Casa di Cura Sedes
Sapientiae, Turin, Italy; 4Associazione Patologi Oltre Frontiera (APOF),
NGO; 5Department of Obstetrics and Gynecology, Ospedale Cardinal
Massaia, Asti, Italy; 6 Department of Pathology, Ospedale Martini - ASL
TO1, Turin, Italy
1
2
Introduction. In 2008, the Mtendere Mission Hospital (MMH)
in Chirundu, Zambia, launched a cytology-based opportunistic
cervical screening program in collaboration with the non-governmental organization Pathologists Beyond Borders Association
(Associazione Patologi Oltre Frontiera - APOF) and with the
Italian Society of Colposcopy (Società Italiana di Colposcopia
e Patologia Cervico-Vaginale - SICPCV). The adhesion of local
women was fostered by an awareness campaign organized by the
MMH and by the presence of an outpatients gynaecological clinic
with colposcopy and surgical wards available for treatments.
During the entire length of the screening program (2008-2013),
APOF and SICPCV oversaw the training plan for local health
staff to be employed in the screening program.
Aim of this study was to evaluate the performance of such screening program and to identify any corrective actions to be applied
for its improvement.
Methods. From 2008 to 2013, about three thousand women were
examined at the MMH; for each of them a Pap test (smear) was
performed. The smears were evaluated by two local cytoscreeners, who photographed selected microscopic fields of all abnormal cases with a digital camera. Images were sent via web for
a remote second opinion rendered by a group of Italian expert
pathologists. Negative cases were directly diagnosed by the cytoscreeners on site, with no second opinion.
Women with an abnormal smear (as confirmed by the expert
pathologists) and women with negative and/or unsatisfactory Pap
smear showing clinical signs of a cervical lesion were referred for
a second-level diagnostic procedure (colposcopy, biopsy).
Cytological Pap test and histological biopsies results were collected. Accuracy of the screening program was evaluated by
calculating sensitivity, specificity, positive predictive value
(PPV) and negative predictive value (NPV) of cytology towards
histology (considered as the gold standard) in two situations: 1)
negative versus positive for lesions of any grade: 2) negative +
low-grade lesions versus high grade lesions.
Results. Cytology: we collected data on a total of 2934 women,
each of them performing a Pap smear at the time of the first
examination. 2854 (97.3%) Pap smears were considered satisfactory for cytological evaluation; of these, 2458 (86.1%) were
diagnosed as negative, 230 (8.1%) as low-grade lesions (ASCUS, L-SIL) and 166 (5.8%) as high-grade lesions (ASC-H, AGC,
H-SIL, carcinoma).
194
Histology: in women undergoing a second-level examination, 152
biopsies were performed under colposcopic guidance: 14 (9.2%)
resulted negative, 45 (29.6%) were low-grade (CIN1) and 93
(61.2%) high-grade (CIN2, CIN3 and carcinoma) lesions.
Cyto-histological correlation: Of 14 negative biopsies, 5 (36%)
followed a negative Pap test, 6 (43%) had a previous diagnosis
of ASC-US/L-SIL and 3 (21%) a diagnosis of high-grade lesion
(ASC-H, AGC, H-SIL, carcinoma). Of 45 CIN1 biopsies, the
previous cytological diagnosis was: negative in 13 cases (29%),
positive for low-grade lesions in 21 cases (47%) and positive
for high-grade lesions in 11 cases (24%). Finally, of 93 CIN2+
cases, the preceding Pap test was negative in 16 (17%), positive
for low-grade lesion in 15 (16%), positive for high-grade lesion
in 61 (66%) and inadequate in 1 (<1%) patient.
We first assessed the accuracy of the screening program in discriminating women with no histological lesions from those with
positive histology (lesions of any grade). Thus, we observed a
sensitivity of 78.3%, a specificity of 35.7%, a PPV of 92.3%and
a NPV of 14.7%. We performed the same analysis to assess its
accuracy in discriminating negative and low-grade from high
grade lesions,and obtained a sensitivity of 66.3%, a specificity of
76.3%, a PPV of 81.3% and a NPV of 59.2%.
Conclusions. It is difficult to compare the available literature,
usually referring to large and well established cervical cancer
screening program in wealthy Countries, with data obtained
from the first 6 years of activity of the opportunistic screening
program performed at the MMH. Several major differences exist between these two situations in terms of target population,
adherence to follow-up and training and autonomy of local
cytoscreeners.
However, at least two considerations can be made: (i) the percentage of second-level referral is very low (only 30% of women with
positive Pap test underwent biopsy); (ii) in the effort to discriminate negative and low- grade from high-grade lesions, our data
showed a sensitivity of 66.7%, thus meaning that at least 1/3 of
high grade lesions were not properly recognized with cytology.
Since the main target of a screening program is the detection of
lesions in asymptomatic women (and to keep the number of false
negative cases as low as possible), future efforts should aim at
improving a proper recognition of the cytological changes worthy
of further investigation. Additional training programs for local
cytoscreeners should aim at improving their ability to point out
the exact criteria of adequacy of a Pap test and recognize lowgrade lesions.
In conclusion, the establishment of a cytology-based screening
program in such particular conditions highlights some limitations
that must be addressed and overcome in order to align its effectiveness to the levels reached in high-income Countries.
Sala Michelangelo – Piano inferiore – 08,30-09,30
Patologia testa collo - 1
Moderatori: M. Basciu, M. Truini
Correlation study of Fine-Needle Aspiration
biopsies in the head and neck region with
Histologic Diagnosis: accuracy, advantages and
pitfalls
L. Maccio, L. Losi, A.M. Cesinaro, G. Ficarra, A. Maiorana, S.
Corrado
Istituto di Anatomia Patologica, Università di Modena e Reggio Emilia,
Policlinico di Modena, Italia
Background. Fine-Needle Aspiration (FNA) is an important
adjuvant to the diagnosis of palpable lesions of the head and neck
region and its usefulness, safety and accuracy have been repeatedly proved. [1] The use of quick staining methods also allows a
rapid provisional diagnosis to be achieved in many cases, guiding
further management [2]. However, the role of FNA is still controversial owing to its diverse morphological patterns and overlapping features between benign and malignant lesions. [3-4] The
main objective of this study was to evaluated the effectiveness of
FNA’s performed at the Department of Pathology of Policlinico
di Modena on head and neck masses and to evaluate the advantages and pitfalls of FNA, as compared to histopathology.
Materials and methods. During a 10-year period (2004-2013),
699 patients who underwent with FNA’s for head and neck
masses were reviewed and correlated with clinical and/or histological findings. Smears stained with the Diff-Quick method
were used to provide rapid provisional diagnoses in all cases.
Specimens were subsequently fixed in 95% alcohol and stained
with Haematoxylin and Eosin. Cases was divided into 3 anatomic
sites (salivary glands, lymph nodes and soft tissues) and classified
according to the following diagnostic categories: non neoplastic
lesions, benign neoplasms, malignant neoplasms, unsatisfactory
and uncertain/borderline lesions. (Table 1) In 422 cases in wich
radical surgical procedures were performed, cytological and histological diagnoses were compared. Sensitivity, specificity, accuracy, true-positive rate, predictive positive value and efficiency
were calculated for all cases and for each anatomic site.
Results. Aspirates were obtained from 699 patients comprising 351 males and 348 females. The age range was 1-96 years
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COMUNICAZIONI ORALI
Tab. I. Cytological diagnostic categories of 699 cases.
Diagnosi FNA
Non neoplastic
lesion
Benign
neoplasm
Malignant
neoplasm
Uncertain/
borderline
lesion
Unsatisfactory
Total
Salivary
glands
106
(21%)
232
(45.8%)
40
(8%)
77
(15%)
Lymph
nodes
48
(25.9%)
74
(41.6%)
20
(11.2%)
Soft
tissue
3
(21.4%)
5
(35.8%)
1
(7%)
-
52
(10.2%)
507
(72.5%)
Total
157
(22.4%)
237
(34%)
115
(16.4%)
97
(13.9%)
36
(20.3%)
178
(25.4%)
5
(35.8%)
14
(2.1%)
93
(13.3%)
699
(100%)
Tab. II. Sensitivity, specificity, positive predictive value and negative predictive value of FNA.
CONCORDANCE
SENSIBILITY
SPECIFICITY
VPP*
VPN°
Salivary
glands
71.1%
88.6%
73.5%
91.2%
73.5%
Lymph
nodes
91.6%
94.7%
88.2%
94.7%
88.2%
Soft
tissue
100%
100%
100%
100%
100%
Total
87.5%
94.4%
87.2%
95.3%
87.2%
* VPP= positive predictive value ; °VPN=negative predictive value
Tab. III. Comparison between our experience and other studies in literature.
Flynn et
al 1990
SPECIFICITY
99%
SENSIBILITY
82%
VPP
98%
UNSATISTACTORY 5%
CONCORDANCE
91.4%
Fulcinti
et al 1997
100%
86.4%
100%
5.5%
93.6%
Nanda et
al 2011*
86.4%
84.6%
80.1%
-
Nostra
casistica
87.2%
94.4%
95.3%
13.3%
93.5 %
* VPP= positive predictive value
(mean 56,9). Salivary gland lesions constituted the main group
aspirated (72,5%), lymph nodes and soft tissues were involved
in 25,5% and 2% respectively. There were 157 (22,4%) cases
of non neoplastic lesions, 237 cases (34%) of benign lesions,
115 cases (16,4%) of malignant lesions, 93 cases (13,3%) of
unsatisfactory specimens and 97 cases (13,9%) of uncertain/
borderline lesions. In 422 patients cytological and histological
concordance was evidenced. Sensitivity, specificity, positive
predictive value and negative predictive value of FNA for each
categories were calculated. (Table 2) The malignant neoplasms
of salivary glands showed a critical accuracy of 56,2%, due to
the complex cytological interpretation of these types of lowgrade tumor, that usually show minimal cellular atypia. The rate
of unsatisfactory specimens depends in most cases by the presence of non palpable masses from which it is difficult to obtain
diagnostic material with the needle aspiration. In addition, in our
experience FNA was helpful in distinguishing benign from malignant lesions in order to direct clinicians to the correct management for the patient. In fact, high values of diagnostic accuracy
were observed in our study; especially the sensitivity was of
94,4%, much higher a value than reported in the literature.[4-5-6]
(Table 3)
Conclusion. Fine-Needle Aspiration is safe, easy to perform
and very useful as initial diagnostical investigation for head and
neck masses. The main role of this technique is to differentiate
malignant from benign lesions and a close cooperation between
pathologist and clinicians is essential to maximize the diagnostic
sensitivity, expecially in the selection of palpable masses.
References
1
Platt JC, Davidson D, Nelson CL, et al. Fine-needle aspiration
biopsy:An analysis of 89 head and neck cases. J Oral Maxilofac Surg
48:702, 1990
2
Roland NJ, Caslin AW, Turnbull LS, et al. Fine needle aspiration
cytology of salivary gland lesions reported immediately in a head and
neck clinic. J Laryngol Otol 1993; 107:1025-1028.
3
Kamal MM, Dani AA, Kotwal MN, et.al Aspiration cytology of salivary gland lesions advantages and pitfalls. Indian J Pathol Microbiol
1994; 37:281-287
4
Nanda KDS, Mehta A, Nanda J, Fine-needle aspiration cytology: a
reliable tool in the diagnosis of salivary gland lesions. J Oral Pathol
Med 2012; 41:106-112.
5
Flynn MB, Wolfson SE, Thomas S, et al. Fine needle aspiration biopsy in clinical management of head and neck tumors. J Surg Oncol
1990; 44:214
6
Fulciniti F, Califano F, Zupi A, et al. Accuracy of Fine Needle Aspiration Biopsy in head and neck tumors. J Oral Maxillofac Surg 1997;
55:1094-1097
Detection of predictive bio-markers in a
salivary duct carcinoma: a case report with
immunohistochemical and molecular study
R. De Cecio1, M. Cantile1, G. Scognamiglio1, F. Fulciniti1, F.
Longo2, F. Ionna2, G. Botti1, S. Losito1
Pathology Unit, Istituto Nazionale Tumori Fondazione “G. Pascale”, Naples, Italy; 2 Head and Neck Medical Oncology Unit, Istituto Nazionale
Tumori Fondazione “G. Pascale”, Naples, Italy
1
Introduction. Over the years, several molecular alterations associated with the pathogenesis and progression of the salivary
glands tumors have been well-characterized. Particularly, the
mutational status and/or aberrant expression of certain markers,
such as EGFR, HER2, cKIT, BRAF and AR, mainly identified
in some subgroups of salivary duct carcinomas (SDCs) highly
aggressive, currently represent molecular targets for new and
efficacious drugs routinely employed in the treatment of some
common cancers [1-4].
Methods. We describe the case of 52 years old woman with a
palpable mass in the left parotid, with cytological examination
suggestive for epithelial neoplasia likely metastasis by papillary
thyroid tumor.
Microscopically analysis revealed glandular parenchyma with
neoplastic proliferation characterized by intraductal component
and high-grade infiltrating component, consisting of small glands
and nests of tumor cells, immunohistochemically positive for
CK19, CEA, p53.
Final diagnosis was “High grade G3 ductal carcinoma of the salivary gland, with an outbreak of nodular adenosis”. Subsequently,
10 widespread lymph nodal metastases, associated to perineural
tissue and masseter infiltration, were diagnosed.
Finally, we decided to verify the presence of specific molecular
alterations already described in the literature, in particular: i)
EGFR and BRAF mutational status and ii) Androgen receptor
expression.
Results and conclusions.The sample was negative for EGFR
mutation and positive for BRAF V600E mutation, but showed
an high immunohistochemical expression of AR.
A rich literature is instead associated with the expression and
the role of AR in salivary glands tumors. A comparative study
between SDC and invasive ductal carcinoma of the breast (IDC)
showed high AR expression only in SDC, suggesting a main role
of AR in this tumor development and progression [4-6].
Concerning the therapeutic potential of anti-AR drugs several
studies report the benefits of anti-androgen therapy in salivary
196
Unit, Department of Biomedical, Experimental and Clinical Sciences,
University of Florence, Italy; 3 Pathology Department, Istanbul Medical
Faculty, Istanbul, Turkey, 4 Ophtalmology Unit, Careggi University Hospital, Florence, Italy
glands tumors histotypes. In one series of 10 patients with SDC
and over-expression of AR, 50% of them was enormously benefited from treatment with bicalutamide [6].
In conclusion we suggest that the expression analysis of these
biomarkers associated with histomorphological data, could then
provide the oncologist the opportunity to create a proper stratification of patients for customized therapies also for salivary gland
tumors.
References
1
Cantile M, Losito S, Longo F, Aquino G, Malzone G, Fulciniti F,
Caponigro F, Franco R, Ionna F, Botti G. Detection of Predictive
Markers for Therapeutic Stratification of Salivary Glands Tumors.
Curr Drug Targets. 2014 Jun 12.
2
Williams MD, Roberts DB, Kies MS, et al. Genetic and expression
analysis of HER-2 and EGFR genes in salivary duct carcinoma: empirical and therapeutic significance. Clin Cancer Res. 2010; 16:226674.
3
Nardi V, Sadow PM, Juric D, et al. Detection of novel actionable
genetic changes in salivary duct carcinoma helps direct patient treatment. Clin Cancer Res. 2013;19:480-90.
4
Nasser SM, Faquin WC, Dayal Y. Expression of androgen, estrogen,
and progesterone receptors in salivary gland tumors. Frequent expression of androgen receptor in a subset of malignant salivary gland
tumors. Am J Clin Pathol. 2003;119:801-6.
5
Jaspers HC, Verbist BM, Schoffelen R, et al. Androgen receptorpositive salivary duct carcinoma: a disease entity with promising new
treatment options. J Clin Oncol. 2011; 29:e473-6.
6
Moriki T, Ueta S, Takahashi T, et al. Salivary duct carcinoma: cytologic characteristics and application of androgen receptor immunostaining for diagnosis. Cancer. 2001;93:344-50.
SF3B1 mutation has prognostic significance in
uveal melanoma
E. Projetto1, L. Simi2, N. Buyukbabani3, C. Mazzini4, M. Puccioni4, V. Maio1, D. Massi1, P. Pinzani2
1
Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Italy, 2 Clinical Biochemistry
Background. Uveal melanoma (UM) is the most frequent primary tumour of the eye, causes severe visual morbidity and is
fatal in approximately 50% of patients.1 At molecular level, it is
clearly distinct from cutaneous melanoma and shows a different
pattern of driver mutations, including GNAQ, GNA11, BAP-1,
EIF1AX and SF3B1.2 In particular, SF3B1 is a component of the
spliceosome and is involved in splicing of the pre-mRNA.3 The
aim of the study was to determine the frequency of SF3B1 mutations in a series of primary uveal melanomas and to analyze the
SF3B1 mutational status in relation to clinico-pathological, other
molecular features and outcome.
Methods. We retrospectively collected 67 UM obtained by
enucleation surgery. Tissue samples were analyzed by means of
direct sequencing for mutations of SF3B1,GNAQ, GNA11 and
BAP-1 genes. The association of clinico-pathological features
with mutational status was assessed using the Student test for
continue and Chi square test for categorical variables. The survival rates were calculated using the Kaplan Meier method and
differences in survival were tested using the log rank test.
Results. SF3B1 mutations were identified in 13/67 (19.4%)
cases, including 9 (13.4%) R625H mutation and 4 (6%) R625C
mutation. Among SF3B1-mutated cases, 7 were males and 6 were
females, median age was
53.5 years. Among the SF3B1-mutated cases, 7 cases were also
associated with a Q209L mutation for GNA11 (2/4 R625C and
5/9 R625H). All SF3B1-mutated cases were GNAQ wild-type
and BAP1 (exon 4 and exon 7) wild-type. SF3B1 mutation was
significantly associated with younger age (p=0.048) and favorable outcome (p=0.028).
Conclusions. We herein show that SF3B1 is recurrently mutated
in UM, not in coincidence with BAP-1 mutations and rarely with
GNA11 mutations, and that SF3B1 mutation is associated with a
better prognosis. Future studies will focus on how these downstream molecular events may affect UM biology and clinical
management decisions in individual patients.
References
Virgili G, Gatta G, Ciccolallo L, et al. Incidence of uveal melanoma in
Europe. Ophthalmology 2007;114:2309–15.
Dono M, Angelini G, Cecconi M, et al. Mutation frequencies of GNAQ,
GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single
case of uveal melanoma. Br J Cancer 2014;110:1058-65.
Harbour JW, Roberson ED, Anbunathan H, et al. Recurrent mutations at
codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat Genet
2013;45:133-5.
Axl tyrosine kinase receptor relates with p-AKT
L. La Sala 1, F. Collina 1, S. Losito 1, M.G. Chiofalo 2, G. Botti 1,
R.M. Melillo 3, R. Franco1, L. Pezzullo2.
SC Anatomia Patologica e Citopatologia, INT Fondazione G. Pascale,
Napoli; 2 SSD Chirurgia OncologicaTiroide e Paratiroide, INT Fondazione G. Pascale, Napoli; 3 IEO del CNR, Napoli
1
Background. Thyroid cancer is the most common endocrine
disease, but its key oncogenic drivers remain undefined. The Axl
proto-oncogene is a receptor tyrosine kinase member of TAM
receptors family. Some reports indicated that Axl and its ligand
growth arrest-specific gene-6 (Gas6) might be involved in tumor
progression of several human cancers. Moreover, recently, it has
been shown that aberrant expression of Axl and Gas6 mRNA
are strongly correlated with the outcome of patients. The binding
of Gas6 to Axl induces the activation of the signaling pathways
MAPK/ERK and PI3K/Akt. Preliminary studies have shown an
aberrant expression of Axl and Gas6, in vitro, in thyroid cancer cell
197
COMUNICAZIONI ORALI
Fig. 1. Axl receptor pathway.
Fig. 3. AXL expression in papillary thyroid cancer. A) Low cytoplasmic
AXL immunopositivity (20x); B) Low cytoplasmic AXL immunopositivity
(40x); C) High cytoplasmic AXL immunopositivity (20x); D) High cytoplasmic AXL immunopositivity (40x). E) Membrane Axl expression in
thyroid cancer (20x); F) Membrane Axl expression in thyroid cancer
(40x)
Fig. 2. pAKT expression in papillary thyroid cancer. A) Low pAKT expression (20x); B) Low pAKT expression (40x); C) High nuclear pAKT
expression (20x); D) High nuclear pAKT expression (40x); E) High
nuclear and cytoplasmic pAKT expression (20x); F) High nuclear and
cytoplasmic pAKT expression (40x).
Fig. 4. Detection of V600E Braf mutation by Real Time PCR.
lines, and in vivo, on a small series of thyroid carcinomas. Furthermore, the phosphorylation of Akt, but not of ERK, was observed
upon Gas6 stimulation of thyroid cancer cells. The BRAF V600E
mutation (BRAF mutation) has been reported in 18% to 87% of
thyroid cancers and including in up to 69% of papillary thyroid
carcinomas (PTC). This mutation is thought to play an important
role in the pathogenesis of PTC through activation of the mitogenactivated protein kinase pathway, which affects the transcriptional
regulation and expression of various genes that are involved in cell
proliferation, survival, and cancer progression
Aim. In this work we wanted to investigate the role of Axl
expression on a series of 51 thyroid carcinomas, included in
a prognostic TMA, associating their expression to the clinical
pathological features. Moreover, we evaluated the V600E BRAF
mutation levels and the activation of MAPK and PI3K/Akt pathways downstream through the analysis of the pAKT and pERK
expression and the apoptotic index.
Methods. We used microarray technology comprising 51 thyroid
carcinomas. Axl, Gas6, pERK and pAKT expression were valued
by immunohistochemistry using monoclonal antibodies. The
V600E BRAF mutation was evalueted by Real time PCR. The
apoptotic index was evaluted by Tunel assay.
Results and conclusion. Axl positivity was observed mainly in
tumoral cells. Most of the samples displayed membrane positivity, but a small number of specimens showed cytosolic staining
into perinuclear vescicles (probably representing the Golgi apparatus), a typical pattern of overexpressed proteins. Axl showed
low expression in 23 cases out of 51 tumors (45%), a high expression in 28 of 51 cases (55%).
Statistical analysis showed that the high expression of Axl in
thyroid carcinomas correlates with the relapse (p = 0.022). The
expression of Axl in primary carcinomas correlates with the
expression of this receptor in the lymph node metastasis (p =
0.001) and with the expression of cytoplasmic pAKT (p = 0.029),
198
indicating the likely activation of this pathway downstream but
not with the expression of pErk. Tunel expression is inversely
correlated with nuclear (p=0.053) and cytoplasmic (p=0.088) expression of p-AKT. BRAF mutation was observed in 18 (35,2%)
of the 51 cases in this subset. Our data confirm the role of Axl
in the pathogenesis of thyroid tumors and highlight in particular
his prognostic role in thyroid tumors, being responsible for the
induction of the metastatic process, mainly through deregulation
of apoptosis via pAkt signaling. BRAF(V600E)-positive tumors
were at higher risk of developing more aggressive behavior.
References
Avilla E, Guarino V, Visciano C et al. Activation of TYRO3/AXL Tyrosine
Kinase Receptors in Thyroid Cancer. Cancer Res 2011;71:1792-1804.
Walts AE, Pao A, Sacks W, et al. BRAF genetic heterogeneity in
papillary thyroid carcinoma and its metastasis. Human Pathology
2014;45:935–941.
Expression of the ATM substrate CCDC6 protein predicts
Oral Squamous Cell Carcinomas response to Radiation
Therapy
F. Merolla1, G. Ilardi1, D. Russo1, S. Varricchio1, M. Mascolo1, G.
De Rosa1, S. Staibano1, A. Celetti2
Dipartimento di Scienze Biomediche Avanzate, Università degli studi di
Napoli “Federico II”; 2IEOS/CNR, Napoli
1
The CCDC6 (Coiled Coil Domain Containing 6) gene product,
formerly known as H4(D10S170), is a proapoptotic, ubiquitously expressed, 65 KDa nuclear and cytosolic phospho-protein,
lacking significant homology to known genes[1]. Ccdc6 gene
was first identified in chromosomal rearrangements involving
the RET gene, known as RET/PTC, as prevalent chromosomal
alteration in thyroid papillary carcinomas from patients with
radiation exposure history[2]. Previously unidentified kinase fusions including CCDC6-RET have recently been reported in lung
adenocarcinoma[3]. A 60 aminoacid fragment of the CCDC6
coiled coil domain, included in the RET/PTC rearrangement, has
been shown to be necessary for homodimerization, constitutive
activation and transforming ability of the oncoprotein. Merolla et
al reported the involvement of CCDC6 in ATM-mediated cellular
response to DNA damage, supporting the idea that CCDC6 action
impairment could play a role in carcinogenesis[4]. Moreover,
CCDC6 gene product involvement in DNA damage response has
been further supported by the findings of CCDC6PP4c interaction: CCDC6 has been found to inhibit the PP4c mediated dephosphorylation of gamma-H2AX. Following exposure to ionizing radiations, depletion of CCDC6 increases dephosphorylation
of gamma-H2AX, limiting the amount of the phosphor-histone
which then influences repair of DNA Double Strand Breaks[5].
With 42,440 new cases estimated in 2014, which accounts for
2.5% of new cases, oral squamous cell carcinomas (OSCC) is the
6th most common cancer in US; surgery and Radiation Therapy
(RT) represent the standards of care for early-stage and locally
advanced resectable lesions. The natural history of the disease
is variable and difficult to predict and it mainly depends on the
disease’s stage; mortality is substantially unchanged over the past
decades, in 2014 the estimates deaths in US are 8,390, OSCC in
fact accounts for 1.4% of all cancer deaths[6]. In OSCC tumors,
early recurrences sometimes appear following RT treatment.
Radioresistance is one of the main determinants of treatment
outcome in oral cancer, but its prediction is difficult, due to the
complexity of the molecular changes and the poor knowledge
of OSCC tumor’s biology. Relapses, usually associated to poor
prognosis, are often related to an intrinsic radio-resistance. Radioresistant OSCC tumors primarily show alterations in intracellular pathways involved in cell proliferation, apoptosis and DNA
repair[7]. In the present study, we investigated, by IHC, CCDC6
protein expression in a selected series of RT treated OSCC and
correlated its expression to tumor relapse and patients survival.
We performed our study on selected OSCC cases from the ar-
chives of the Pathology Section of the Advanced Biomedical
Sciences Department, University of Naples “Federico II”. The
studied population ranged from January 2000 to December 2013;
the mean patient’s age was 63,3 years old, ranging from 30 to
89. None of the samples showed a differentiation grade of G1,
29% were G2, 9.7% showed moderate to poor differentiation
grade, 61.3% were poorly differentiated. The mean follow-up
time was 30 months (median 26 months, range 1 to 98). By the
end of the follow-up 58% patients died of disease, 42% did not
show any adverse events. All the tumor samples were formalin
fixed and paraffin embedded. IHC studies were performed with a
polyclonal anti-CCDC6 antibody (AbCam 1:250 dilution). In our
study CCDC6 IHC expression significantly associated with RT
response, directly correlating with OSCC patients survival. Our
results let envisage a role for CCDC6 as a protein marker predictive of Radio-therapy response.
References
1
Celetti A, Cerrato A, Merolla F, Vitagliano D, Vecchio G. et al.
H4(D10S170), a gene frequently rearranged with RET in papillary thyroid carcinomas: functional characterization. Oncogene.
2004;13:109–121
2
Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R. et al.
PTC is a novel rearranged form of the RET proto-oncogene and is
frequently detected in vivo in human thyroid papillary carcinomas.
Cell. 1990;13:557–563
3
Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S. et al. RET, ROS1
and ALK fusions in lung cancer. Nat Med. 2012;13:378–381
4
Merolla F, Pentimalli F, Pacelli R, Vecchio G, Fusco A. et al. Involvement of H4(D10S170) protein in ATM-dependent response to DNA
damage. Oncogene. 2007;13:6167–6175
5
Merolla F, Luise C, Muller MT Pacelli R, Fusco A. et al. Loss of
CCDC6, the first identified RET partner gene, affects pH2AX S139
levels and accelerates mitotic entry upon DNA damage. PLos ONE.
2012;13(5)
6
Howlader N, Noone AM, Krapcho M, Garshell J, Neyman N, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Cho H, Mariotto A,
Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2011, National Cancer Institute. Bethesda, MD,
http://seer.cancer.gov/csr/1975_2011/
7
Perri F, Pacelli R, Della Vittoria Scarpati G, Cella L, Giuliano M,
Caponigro F, Pepe S. Radioresistance in head and neck squamous
cell carcinoma: Biological bases and therapeutic implications. Head
Neck. 2014 Jul 4.
DNA Methylation analysis by bisulfite next
generation sequencing to early detect oral
squamous cell carcinoma from oral scrapings
L. Morandi1, S. Asioli1, A. Tarsitano2, C. Marchetti2, D. Gissi3, V.
Monti1, L. Montebugnoli3, M.P. Foschini1
Department of Biomedical and Neuro Motor Sciences, Section of Anatomic Pathology “M.Malpighi” at Bellaria Hospital, University of Bologna, Bologna, Italy; 2 Unit of Maxillofacial Surgery, S. Orsola Hospital
Bologna, Italy; 3 Section of Oral Science, Department of Biomedical and
Neuro-Muscular Sciences, University of Bologna, Bologna, Italy
1
Introduction. Oral squamous cell carcinoma (OSCC) is the most
frequent neoplastic disease in head and neck region and it is commonly preceded by potentially malignant lesions. In addition
patients affected by OSCC can experience second primary OSCC,
with a frequency ranging between 17% and 30% after surgical
treatment. The development of non invasive, highly sensitive and
specific biomarkers to identify premalignant lesion, lesions that
will undergo malignant transformation and patients that will develop an OSCC during follow-up is urgently required. Clinical and
histological features of preneoplastic lesions are not able to provide
enough information to this purpose and many molecular approaches have been investigated to improve the current strategies for early
cancer detection, reliable prognosis and personalised therapy.
Methods. Oral scraping DNA from 8 healthy donors, 9 oral lichen planus (OLP), 3 proliferative verrucous leukoplakia (PVL),
COMUNICAZIONI ORALI
5 dysplasia and 8 OSCC were collected and bisulfite treated.
A set of differentially methylated promoter genes in OSCC
(GP1BB, ZAP70, p16, MGMT, CDH1, KIF1A, miR137, miR375)
were investigated by Next Generation Sequencing (BisulfiteNGS, 454 Roche). ReadSeqs in Fasta format were anayzed by
QuMA (http://quma.cdb.riken.jp/). The statistical significance
between lesions and normal epithelia from the same patient and
from a pool of healthy donors were evaluated with the MannWhitney U-test.
Results. GP1BB hypomethylation was detected in 6 out of 7
(85%) OSCC cases; ZAP70 and miR137 were found to be hypermethylated in 83% and 75% of OSCC cases respectively. The
same aberration pattern of methylation was found even in 100%
of premalignant lesions. OLP showed promoter hypermethylation
only for miR137 in 100% of cases. PVL revealed no aberrant
methylation pattern for all of the 8 markers.
Conclusion. In our preliminary results, Bisulfite-NGS analysis
of GP1BB, ZAP70 and miR137 promoters from oral scrapings
allows to discriminate premalignant and neoplastic lesions from
OLP, PVL and normal samples. Premalignant lesions share the
same epigenetic modifications of OSCC. These data confirm that
CpG methylation changes may play a role in oral cancer progression and that DNA methylation analyses may have significant
utility in wider studies that seek to derive biomarkers or potentially druggable targets to improve oral cancer outcomes.
Markers of squamocellular junction epithelium in
normal tonsils and oropharyngeal cancer:
an immunohistochemical study
P. Morbini, G. Capello, P. Alberizzi
UO Anatomia Patologica, Fondazione IRCCS Policlinico S. Matteo e Università di Pavia, Pavia
Introduction. HPV infection has recently been identified as the
causative agent of a subset of squamous cell carcinomas arising
in the oropharyngeal region with a specific tropism for tonsillar
epithelium (1). Oncogenic HPV infection in other areas of the
head and neck compartment on the other hand has been rarely
described. The factors influencing the selective susceptibility of
tonsillar epithelium to HPV-induced oncogenesis are far from
being elucidated (2). In the uterine cervix, however, Herfs et al.
(3) have recently identified a population of cuboidal cells located
at the squamo-columnar (SC) junction that show a unique gene
expression profile, different from adjacent ectocervical and endocervical transitional zone epithelium. Gene expression analysis
allowed to identify a five protein-signature that recognized SC
Fig. 1.
199
cells, and this signature was used to document the presence of
these cells in the early stages of cervical embryonic development,
in the adult SC junction, in HPV-infected cell lines and in all
analyzed HPV-associated cervical dysplasias and cancers. The
proteins that specifically recognized SC cells were cytokeratin (CK)7, anterior gradient (AGR)2, cluster differentiation
(CD)63, matrix metalloproteinase (MMP)7, and guanine deaminase (GDA). The Authors concluded that the profile of expression of the 5 markers allowed the recognition of an embryonic
cell population in the cervix susceptible to HPV infection and of
cancers originating from these cells, although the mechanisms by
which viral–host cell interactions lead to malignancy in the SC
junction population remain to be elucidated. Considering the lack
of information on factors regulating the susceptibility of tonsillar
epithelium to HPV infection and neoplastic transformation, the
present study was designed to analyze the expression profile of
the SC junction markers in normal tonsils and in oropharyngeal
carcinomas, correlating the expression profile with tumor HPV
status and the presence of a transcriptionally active (i.e. oncogenic) infection
Methods. Non neoplastic tonsils: we selected 24 normal tonsils
(NT) obtained at tonsillectomy performed for benign condition in
patients of different ages (range 3-82 years, all males) Oropharyngeal cancers (OPC): 41 consecutive OPC collected between January 2010 and and June 2013 were fully characterized for HPV
infection by means of HPV DNA in situ hybridization (ISH),
HPV DNA amplification and genotyping with SPF LiPA, HPV16
E6 gene amplification; viral transcriptional activity was assessed
indirectly with p16 immunostain, and directly with HR HPV E7
mRNA ISH (4,5). The mean age of patients was 63.68, 34 were
males. Twenty OPC showed viral mRNA and p16 expression
and were classified as HPV-associated, while 21 were negative,
despite 5 of them hosted HR HPV DNA. Unstained slides obtained from both NT and OPC were immunostained with rabbit
anti-GDA (Sigma-Aldrich), mouse anti-AGR2 clone EPR3278
(GeneTex Inc), mouse anti-CD63 cloneNK1/C3 (Abcam), mouse
anti-CK7 clone OV-TL 12/30 (Dako), and rabbit anti-MMP-7
(Neomarkers) antibodies. p16 immunostain was also performed
on NT samples. All reactions were performed on the immunostainer Benchmark XT (Ventana), using the Ultraview DAB v3
revelation system. Semiquantitative analysis was used to evaluate
the results of immunohistochemical reactions: in NT, expression
in cells lining tonsillar crypts was distinguished from surface
squamous epithelium; absence of staining was graded 0, the
presence of scattered isolated positive cells as grade 2, clustered
positive cells as grade 2, and diffuse immunostain of the crypt
200
epithelium as grade 3. OPC samples were scored according to the
H scoring system. Briefly, the highest staining intensity present
in tumor cells for each marker was given a score of 0 to 3. The
percentage of tumor cells staining at the highest intensity in the
sample was also estimated within 5% increments with respect to
the total tumor area. The H score was derived from the cross-product of the intensity score (0 to 3) and of the percentage of tumor
staining at the highest intensity (0% to 100%). An H score of >60
was defined as positive. Statistical analysis correlated the degree
of expression with patients age for NT, and with HPV status, tumor
site and morphology for OPC.
Results. In NT, expression of grade 2 or 3 in cells of the crypts
was found in 13 cases (54%) with AGR2 antibidy, in 18 (75%)
with CK7, in 24 (100%) with GDA, in 8 (33,3%) with CD63, and
in 6 (25%) with MMP7 (fig 1, upper row). Absence of expression
was not observed with AGR2, CK7 and GDA; 8 (33,3%) and 9
(37,5%) cases were negative respectively with CD63 and MMP7.
Only 1 case scored grade ≥2 for all 5 markers; none scored grade
≤1 for all markers. No correlation was observed between patient
age and degree of expression. Surface squamous epithelium
resulted constantly negative. p16 was strongly expressed on
crypt cells in all NT samples. In OPC positive results for AGR2
were found in 15 (36,5%), for CK7 in 12 (29,2%), for GDA in
16 (39%), for CD63 in 7 (17%), and for MMP7 in 21 (51,2%)
(fig.1, lower row). No OPC was positive for all markers; 6 were
completely negative.
As far as the correlation with HPV status was concerned, Fisher
exact test showed a significant association between AGR2 expression and transcriptionally active HPV infection, p16 expression, and the presence of HR HPV DNA; CK7 expression
was significantly associated with transcriptionally active HPV
infection, the presence of HR HPV DNA, and non-keratinizing
morphology.
Discussion. The epithelium of the tonsillar crypts expressed the
markers of SC junction indentified in cervical cells susceptible to
HPV infection, but with high variability in terms of proportion of
positive cells and of markers expressed in each single case. This
could be explained by a different degree of maturation of tonsillar
cells with respect to SC junction cells that are considered to be
embryonic remnants in the cervical mucosa. Although the expression of two markers (AGR2 and CK7) was significantly more
frequent in OPC hosting oncogenic HPV infection and in those
positive for HR HPV DNA; we were not able to replicate the observation of Herfs and al. in cervical intraepithelial and invasive
squamous cell cancers and adenocarcinomas, reporting a 100%
positivity for all tested markers (3). In particular, the association
of both significant markers with non-keratinizing OPC supports
the hypothesis that HPV selectively infects crypt epithelium,
while non-HPV associated conventional keratinizing carcinomas
originate from surface squamous epithelium (2), that was constantly negative for all markers in our study. Why HPV targets
tonsillar crypt epithelial cells is far to be clarified, however this
notion is potentially relevant for prevention and early diagnosis
of this emerging cancer affecting mostly young subjects. Further
studies are needed, possibly focusing on different cell populations
(surface vs deep reticular) lining the crypts
References
Jemal A, Simard EP, Dorell C, Noone AM, Markowitz LE, Kohler B, et
al. Annual Report to the Nation on the Status of Cancer, 1975-2009,
featuring the burden and trends in human papillomavirus (HPV)associated cancers and HPV vaccination coverage levels. J Natl Cancer
Inst 2013;105:175-201
Kim SH, Koo BS, Kang S, Park K, Kim H, Lee KR, et al. HPV integration
begins in the tonsillar crypt and leads to the alteration of p16, EGFR
and c-myc during tumor formation. Int J Cancer 2007;120:1418-25.
Herfs M, Yamamoto Y, Laury A, Wang X, Nucci MR, McLaughlinDrubin ME, et al. A discrete population of squamocolumnar junction
cells implicated in the pathogenesis of cervical cancer. Proc Natl Acad
Sci USA 2012;109:10516-21.
Ukpo OC, Flanagan JJ, Ma X, Luo Y, Thorstad WL, Lewis JS. High-risk
human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient
outcomes in oropharyngeal squamous cell carcinoma. Am J Surg
Pathol 2011;35:1343-50
Gao G, Chernock RD, Gay HA, Thorstad WL, Zhang TR, Wang H, et al.
A novel RT-PCR method for quantification of human papillomavirus
transcripts in archived tissues and its application in oropharyngeal
cancer prognosis. Int J Cancer 2013;132:882-90
CD25 and p-nfkb expression in the tumor
inflammatory microenvironment of the thyroid
cancer: a preliminary report
G. Pannone1, M. Mattoni1, A. Santoro2, S. Cagiano1, M.C. Pedicillo1, P. Bufo1
1
Department of Surgical Sciences, Section of Anatomic Pathology, University of Foggia, Foggia, Italy; 2 Department of Laboratory and Services,
Institute of Histopathology and Diagnostic Cytopathology, Fondazione di
Ricerca e Cura ‘Giovanni Paolo II’-UCSC, Campobasso, Italy
Introduction. A functional relationship between chronic inflammation and cancer has been envisaged long ago and it has
become evident that an inflammatory microenvironment is an
essential component of all tumors, including those for which a
direct causal relationship with inflammation is not yet proven.
Several reports indicate the presence of immune-inflammatory
cell infiltrate also in thyroid cancer. Thyroid cancer is the most
common endocrine malignancy and its incidence is increasing
worldwide. An association between Hashimoto’s thyroiditis
and thyroid cancer has been reported in about the 30%of the
cases and the increased incidence of carcinomas in patients
with thyroiditis suggests it might be a precancerous condition.
T regulatory lymphocytes (Tregs) and natural killer (NK) cells
play an important role in the dysfunction of the host immune
system in cancer patients. Tregs are an important subset of
T-cells that regulate T-cell activation. They play a major role
in peripheral tolerance to self-antigens. Indeed, upregulation
of Treg cells suppressed experimental autoimmune thyroiditis
in mice. Treg cells are characterized by constitutive expression of high levels of the alpha chain of the IL-2 receptor
(CD25), and their development is regulated by the FOXP3
gene. Interestingly, both FOXP3 and CD25 have been found
to be associated with autoimmune thyroid diseases. It is well
known that the promoter in the CD25 gene contains multiple
DNA regulatory elements that bind to key transcription factors, in particular nuclear factor-kappa B (NF-κB). NFκB is
a protein complex that controls the transcription of DNA.
The NF-κB pathway is critical to tumor formation since it not
only promotes cell survival via the expression of antiapoptotic proteins, but it also promotes the transcription of many
immune-related genes and proinflammatory cytokines that can
influence tumor progression through the mobilization of host
cells. J.P. Russel and colleagues showed that the expression
of RET/PTC oncogene in thyrocyte cell lines causes nuclear
translocation of NF-κB and the activation of proinflammatory
mediators.
Materials and methods. the aim of this study was to investigate
the expression of CD25 as well asNFkB and its phosphorylated
form in human thyroid cancer, Hashimotos’s thyroiditis and thyroid adenoma and to evaluate its clinical and prognostic significance. Protein expression was analyzed by immunohistochemistry in 80patients with thyroid cancer. The 80 cases of cancer
were also compared with 10cases of Hashimoto’s thyroiditis and
5 cases of thyroid adenoma.
Results. in our study,CD25 was mainly detected in the nucleus
of the inflammatory cells su chas in that of thyroid epithelial cells
(thyrocytes) and neoplastic cells. Protein staining was mainly
detected in the T lymphocytes of outermost zone of the lymphoid
follicles. Moreover, in all cancer alterations there was a more
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COMUNICAZIONI ORALI
evident level of p-NFkB than in normal peritumoral tissues.
Again, p-NFkBstaining was evident in neoplastic cells but it did
not appear in inflammatory cells. Protein staining was mainly
detected in the cytoplasm of the neoplastic cells, and only focal
nuclear signals were observed. Expression of CD25 and p-NFkB
correlated significantly with invasive phenotype (p<0,05).
Conclusions. Our study suggests an important role of CD25 and
p-NFkB in thyroid cancer progression. The expression of CD25
and p-NFkB may identify a cohort of tumors with aggressive
biological and clinical behavior. Therefore, our preliminary
results point out that the assessment of CD25 and p-NFkB may
be useful as prognostic factor in patients with thyroid cancer.
Dermopatologia - 1
Moderatori: C. Miracco, A.R. Palomba
Synchronous or metachronous occurrence of
cutaneous melanoma and lymphoproliferative
diseases: a report of two cases
A.M. Anniciello1*, M. Cerrone1*, L. Marra1, L. Benedetto2, G.
Botti1
Unit of Pathology, Istituto Nazionale per lo Studio e la cura dei tumori
“Fondazione G.Pascale” IRCCS, Naples, Italy; 2 Unit of Surgery “Melanoma - Soft Tissues – Head & Neck – Skin Cancers”, Istituto Nazionale
per lo Studio e la cura dei tumori “Fondazione G.Pascale” IRCCS, Naples,
Italy; * AM Anniciello and M Cerrone are equally contributed.
1
Introduction. Recent studies in the literature have shown an increased risk of 42% developing non-Hodgkin lymphoma (NHL)
in patients with cutaneous melanoma.
In particular, patients who have NHL history have a higher risk
developing a secondary neoplasm, often a skin cancer, mainly
as squamous cell carcinoma, basal cell carcinoma, Merkel cell
carcinoma and melanoma (1;2). Melanoma can also occur as a
secondary neoplasm, especially in immune-suppressed patients
for a solid organ transplant, viral infections and autoimmune
disorders. Melanoma induces immunosuppression but is also
associated with immune-deficiency (3).
It has been showed that the prognosis is worse if both diseases
coexist and in particular the OS at 5 years is 96.2% in patients
with primary MM compared to OS of 60.9% in patients with a
history of primary CLL (4).
Since 1995, Adami et al. emphasized the association between
lymph proliferative disorders and skin diseases suggesting that
UV radiation may play a role in the increased incidence of both
cancers (5)
Methods. Here we described two cases of melanoma patient with
a contemporary or later NHL diagnosis.
In the first case a 58-year-old man presented a skin lesion diagnosed as melanoma, with a thickness of Breslow of mm 0, 6 Clark
level III, with a low mitotic rate (0xmmq), staged as T1a N0 M0.
Three years later he developed several spleen deep and inaccessible nodules, with a final diagnosis of Non Hodgkin lymphoma
3b sec. WHO.
In the second case a 63 year old man, with an oncological history of Chronic Lymphocytic Leukemia (LLC), developed three
nodules two of which in the right pectoral region and one is right
axillary lymph node draining the dermatome region. The two dermal lesions were nodular melanoma metastases. At conventionally stained with hematoxylin and eosin (H&E) the lymph node
presented dual cell population with a central core of metastatic
epithelioid cells (positive immunostaining for S100 and MART1)
and a peripheral atypical lymphoid component.
Results and conclusions. Immunohistochemical phenotype of
case 1 is CD10 + weak, diffuse and intense CD 20+, CD3 -, bcl2-,
MUM1- with Ki67 40%. The atypical cells result negative to IHC
for melanoma markers (MART1-,S100-, HMB45-). The diagnosis was Non Hodgkin lymphoma 3b sec. WHO in a patient with a
Fig. 1. a) H- E staining in case 1, A) of fixed melanoma sample and B) NHL (20X). The formalin-fixed, paraffin-embedded (FFPE) tissue block specimens
were sectioned (3-μm thick), deparaffinized and rehydrated. Each section was stained with hematoxylin and eosin.
202
Fig. 2. IHC analysis of splenic neoplasm in Case 1 (20X): A) Moderate
expression of CD10 B) High expression of CD 20; negative immunostaining for CD3 (C) and Bcl2 (E). D) Positive immunostaining for Ki67
(40%). In the white box is represents fluorescence in situ hybridization
(FISH) patterns with Vysis LSI BCL2 Dual Color, Break Apart for nontraslocated signal.
Fig. 4. Immunohistochemistry in lymphomatous zone (20X): A) negative immunostaining for CD3. Positive expression of CD 23 (B) and
Bcl2 (C). D) low expression of Ki67. E) The area with Melanoma is positive for S100. F) the same zone is positive for V600E mutation in 15
exone of BRAF gene, shown with Real Time PCR-FRET Probe analysis
history of cutaneous melanoma. FISH analysis for the t(14;18)
(q32;q21) translocation was performed.
In the second case immunohistochemistry confirmed a diagnosis
of melanoma metastases in lymph nodes with coexistent NHL.
Patients treated with chemotherapy and radiation therapy for
the NHL have a higher risk of developing other cancers such
as thyroid and lung tumors and melanoma (6). In conclusion,
we suggest that during follow-up of one of the two tumors must
consider the possibility of occurrence of other diseases. Frequent
dermatological visits as well as the appropriate blood tests and
an early diagnosis can certainly improve the prognosis of these
“related” diseases.
Fig. 3. H&E-stained case 2 A and B) in the same nodal structure is present NHL and metastatic melanoma, respectively in the left and right portions
of the area (20X)
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COMUNICAZIONI ORALI
References
1
Brewer JD et al. Lymphoma-associated skin cancer:incidence, natural
history, and clinical management. Int J Dermatol. 2014 Mar;53(3):26774
2
Onajin O, Brewer JD. Skin cancer in patients with chronic lymphocytic
leukemia and non-Hodgkin lymphoma. Clin Adv Hematol Oncol. 2012
Sep;10(9):571-6.
3
Dunn GP et al. Cancer Immunoediting: Integrating Immunity’s Roles
in Cancer Suppression and Promotion. Nat Immunol. 2002; 991-8.
4
Brewer JD et al. Effects of chronic lymphocytic leukemia on the development and progression of malignant melanoma. Dermatol Surg 2010;
36: 368–376.
5
Adami J et al. Sunlight and non-Hodgkin’s lymphoma: a populationbased cohort study in Sweden. Int J Cancer. 1999 Mar 1;80(5):641-5.
6
Morton LM, Curtis RE et al. Second malignancy risks after nonHodgkin’s lymphoma and chronic lymphocytic leukemia: differences
by lymphoma subtype. J Clin Oncol. 2010 Nov 20;28(33):4935-44.
Italian patients in good health, without history of previous vaccination. They responded completely to topical steroid treatment;
in two patients steroid therapy was associated with antibiotics.
ALDY seems to be a relatively rare disease, given the small
number of cases reported so far despite its clinico-pathological
description more than a decade ago; on the other hand, it is reasonable to think that other genuine cases are still misdiagnosed
both clinically and histologically. The striking geographical
distribution of ALDY is an interesting topic of investigation to
uncover its causative agents.
Tab. I. Clinical data of 5 patients.
Gender
M
F
M
F
M
Annular lichenoid dermatitis (of youth): five new
cases of a poorly recognized entity
Age
39
38
35
19
13
Anat. Site
Flank/leg
Trunk
Flank
Trunk/armpit
Abdomen
N. of lesions
Multiple
1
1
Multiple
2
A.M. Cesinaro
Department of Anatomic Pathology, Azienda Ospedaliero-Universitaria
Policlinico, Modena, Italy
Introduction. Annular lichenoid dermatitis of youth (ALDY), an
entity described in children and young patients, is characterized
by a clinical presentation mimicking usually morphea, annular
erythema, vitiligo, or mycosis fungoides (MF), and by a peculiar
histology to be differentiate mostly from mycosis fungoides and
vitiligo (1). It has been observed more recently also in adult
patients (2).
Materials and methods. Five cases of ALDY were collected by
the author in the Department of Anatomic Pathology, Azienda
Ospedaliero-Universitaria of Modena (Italy). Clinical data are
listed in table 1. Biopsies were sent by dermatologists working in
public hospitals and private practice. A clinical diagnosis suspicious of morphea was provided in one case, together with clinical
picture. The other cases were sent with a description of the lesions
as annular/oval patches or plaques; one patient referred pruritus
in an early phase. Punch biopsies were routinely processed for
histologic examination. Immunohistochemical stains were performed, applying a panel of antibodies against CD3, CD4, and
CD8 (Ventana, CA, USA).
Results. By histology, all cases presented the typical features
described in the original paper by Annessi and coworkers: basket
woven slight hyperkeratosis, elongated rete ridges with basal
vacuolization and frequently squared apex, lichenoid lymphocytic infiltrate made of CD4+/CD8+ elements, with slight predominance of CD8 especially at the dermal-epidermal interface.
The follow-up was available in all patients but one, and ranged
from 4 to 32 months. The patients were treated with topical steroids (2 cases) and with antibiotics plus topical steroids (2 cases).
Complete resolution of the lesions was observed in the 4 patients
with available follow-up.
Discussion. Clinical appearance of ALDY can simulates a series
of dermatoses, such as morphea, vitiligo, annular erythema or
MF, whereas histopathological differential diagnosis comprehends mostly MF and the inflammatory phase of vitiligo. Among
the differential diagnoses, MF is the most important, all the more
since cases of genuine MF showing histological features reminiscent of ALDY have been observed.
If pathogenesis of ALDY relies probably on an immunological
process, analogously to other lichenoid dermatoses, the causative
agent is still uncertain. The large majority of cases reported in
the literature are patients living in the Mediterranean area, thus
indicating that peculiar local triggers could be responsible. Borrelia infection has been investigated with negative results; another
patient was reported to have developed the dermatosis after vaccination for Hepatitis B. The five cases herein reported belong to
References
Annessi G, Paradisi M, Angelo C, et al. Annular lichenoid dermatitis of
youth. J Am Acad Dermatol 2003; 49: 1029-36.
Cesinaro AM, Sighinolfi P, Greco A, et al. Annular lichenoid dermatitis
of youth ... and beyond: a series of 6 cases. Am J Dermatopathol 2009;
31: 263-7.
Wells’ Syndrome in childhood: a case report with
spontaneous resolution and a review of the
literature
G. Crisman1, A. D’Amuri2, I. Neri3, S.D. Infusino3, A. Patrizi3,
B. Passarini3
U.O. Anatomia Patologica, Azienda Ospedaliera Desenzano del Garda, Desenzano d.G. (BS), Italy; 2 U.O. Anatomia Patologica, Dipartimento dei Servizi Diagnostici, Az. Osped. “Card. G. Panico”, Tricase
(LE), Italy; 3 U.O. Dermatologia, Dipartimento di Medicina Specialistica, Sperimentale e Diagnostica, Università di Bologna, Bologna, Italy
1
Introduction. Originally described in 1971 by Wells’ as an
autoinflammatory, autoimmune, granulomatous dermatitis with
eosinophilia, Wells’ Syndrome (WS), also known as Eosinophilic
Cellulitis (EC), is still considered a challenging diagnosis in
childhood due to its variable clinical expression and the lack of
clear diagnostic criteria. Wells’ Syndrome (WS) or Eosinophilic
Cellulitis (EC) is clinically characterized by recurrent erythematous and edematous plaques, and by alopecia and scarring in case
of scalp involvement.1,2 However, an expanding spectrum of clinical presentation has been described in the last decades, including
nodular lesions, blisters, papulo-vescicular eruption, itchy escoriated inflammatory papules typically associated with urticarial
rash, deafness and, often, with relapsing fever. Lymphadenopathy
and arthralgias can also occur. Trunk and extremities represent
the most common site of onset, followed by the head and neck.
Mild to moderate eosinophilia and/or neutrophilia are seen approximately in 50% of patients. 3,4 Histologically, early lesions
(acute stage) are characterized by a diffuse and heavy infiltrate
of eosinophils in either superficial and deep dermis. Lymphocytes and plasma cells may be present. The epidermis may show
spongiosis. In the second (subacute) stage, due to the degeneration and degranulation of eosinophils, eosinophilic material and
nuclear debris are observed within collagen fibers thus leading to
so-called “flame figures”. Sometimes, histiocytes surrounding the
“flame figures” are observed. In the third (resolution) stage, less
eosinophils and histiocytes are observed, whereas multinucleated
giant cells forming granulomas are present. Vasculitis is usually
not observed. 5,6 Due to the variable expression and the lack of
clear diagnostic criteria, WS is still considered a challenging diagnosis in childhood and up to 31 cases have been reported so far.
204
Fig. 1. Clinical presentation of erythematous, edematous, painful
lesion 20x10 cm in-diameter of the dorsal face of the right thigh and
a smaller, similar lesion of the left thigh.
The author herein report on a case of a 6-year-old girl presented
with an erythematous, figurated lesions of the thighs.
Material and methods (case report). A healthy 6-year-old
girl presented with a 7 days history of an annular erythematous,
edematous, itchy plaque, 20x10 cm in-diameter of the dorsal
face of her right thigh and a smaller, similar lesion of the left
thigh (Fig. 1). In the previous two years, she suffered of febrile
convulsions. Her development since birth was otherwise uncomplicated and her family history was unremarkable. Six months
before our evaluation she has been hospitalized for the onset of
an figurate urticarial rash, without fever, on her right thigh which
subsequently worsened with the extension to the left thigh. In the
suspicion of an erysipela, she underwent to an amoxicilline and
clavulanic systemic therapy without improvement thus, a punch
skin biopsy was performed. Histological examination revealed
a deep dermal eosinophilic infiltrate with flame figures (Fig. 2).
PAS-stain (Periodic acid-Schiff) resulted negative for fungi. A
diagnosis of WS was made. The lesions evolves into violaceous
plaques and spontaneously healed over 4-6 weeks. Two recurrences were observed in the subsequent 6-month follow-up and
spontaneously resolved as well.
Results (discussion). First described by Wells in 1971 as “granulomatous dermatitis with eosinophilia”, and subsequently renamed “Wells’ syndrome” in 1979 by Spigel GT and Winkelmann
RK. WS represents an uncommon inflammatory dermatosis of
unknown aetiology in the majority of cases. 1,2 Over 200 cases of
WS have been published so far, and only 31 reported a childhood
onset. Of 31 reported cases with a childhood onset, with a mean
age of 5.7 years, ranging from birth to 14-year-old, a slight male
prevalence could be suggested (18M:13F, male to female ratio of
1,3:1). 3-13 A localized hypersensitivity reaction with an eosinophilic-induced cytotoxicity caused by several triggers, such as arthoropod bites, vaccines, bee stings, and infections (i.e., varicella,
molluscum contagiosum, mumps), and erysipelas, was proposed
as the major cause of WS in childhood. Laboratory exams mainly
highlight eosinophilia and/or neutrophilia in a mild to moderate
degree up to 50% of cases. Cutaneous lesions, when present, may
be useful in achieving a correct diagnosis by a simple punch biopsy and in starting an effective therapy to avoid irreversible organ
damage. Typically, in adults cellulitis-like plaques are the more
frequently cutaneous manifestations and WS has been reported in
association with several haematological disorders, such as polycitemia vera, non-Hodgkin’s lymphoma, chronic lymphocytic
leukaemia and chronic myeloid leukaemia. 8,9 In childhood,
figurate and annular erythematous, edematous plaques are the
most common clinical presentation thus, erythema annulare such
as annular urticaria, erythema multiforme or annular erythema
of infancy should be considered by the clinician as a possible
differential diagnosis. It should be noted that no association of
WS with haematological disorders has been reported. Biological
course reflects the great variability in clinical presentation. Thus,
a large spectrum of responses, from a rapid resolution to a chronic
disease with exacerbations and remissions with a mean duration
of at least 3 years in childhood and 5 years in adulthood, was
described. 8,9 By considering the great spectrum of symptoms, WS
could be difficult to suspect at the first visit. Histological features
may represent a diagnostic tool when clinical presentation and
anamnestic data resulted incomplete or conflicting. As a matter of
fact, a diffuse and heavy infiltrate of eosinophils in either superficial and deep dermis and, eventually, the presence of “flame figures” represent two diagnostic clues for histological descriptive
report of “eosinophilic cellulitis with or without flame figures”.
However, this histological presentation is not pathognomonic of
WS, since it could be observed in other conditions, such as parasitic infections, spider bites, herpes gestationis, follicular mucinosis
and Churg-Strauss vasculitis. Thus, a clinical and histological
correlation should be performed to achieve the correct diagnosis.
7,8,10
According to the literature, of 31 WS of the childhood, 8
patients spontaneously resolved without any treatments (even with
Fig. 2. Histological examination showed focal hyperpigmentation of a deep dermal eosinophilic (a, H&E, 10x) infiltration with flame figures in the
absence of vasculitis (b, H&E, 40x).
COMUNICAZIONI ORALI
some recurrences) as our patient, whereas 21 patients underwent to
topical steroids, antibiotics, antihistamines and antiviral therapies
gaining a resolution within 3 years from the time of diagnosis, often
with one or multiple recurrences. 3-13 Only in 2 cases the therapeutic
scheme was not provided 14.
Conclusions. In conclusion, we suggest the importance of obtaining a complete medical history of each patient, which should
include all kind of possible triggers, such as arthropod bites, bee
stings, infections, medications or vaccinations even though in
more than a half of previous reported childhood WS a specific
trigger could not be identified. We report on this case to underline
the importance of clinical and histological correlation, even if apparently conflicting, in the aim to achieve the correct diagnosis
and to schedule a correct therapeutic approach.
References
1
Wells GC. Recurrent granulomatous dermatitis with eosinophilia.
Trans St Johns Hosp Dermatol Soc. 1971; 57:46-56.
2
Spiegl GT, Winkelmann RK. Wells’ syndrome. Recurrent granulomatous dermatitis with eosinophilia. Arch Dermatol 1979; 115:611-13.
3
Caputo R, Marzano av, ezzosi P, et al. Wells syndrome in adults and
children. Arch Dermatol 2006; 142:1157-1161.
4
Gilliam AE, Bruckner AL, Howard RM et al. Bullous ‘‘Cellulitis’’
with Eosinophilia: case report and review of Wells’ syndrome in childhood. Pediatrics 2005;116:e149–e155.
5
Brehmer-Anderson E., Kaaman T., Skog E. et al. The histopathogenesis of the flame figures in Well’s syndrome based on five cases. Acta
Derm Venerol 1986; 66:213-219.
6
Wood C., Miller A.C., Jacobs A. et al. Eosinophilic infiltration with
flame figures. Am J Dermatopathol 1986; 8:186-193.
7
Davis MDP, Brown AC, Blackston RD et al. Familial eosinophilic
cellulitis, dysmorphic habitus, and mental retardation. J Am Acad
Dermatol. 1998; 38:919-928.
8
Aberer W, Konrad K, Wolff K. Wells’ syndrome is a distinctive disease entity and not a histologic diagnosis. J Am Acad Dermatol. 1988;
18(1 Pt 1):105-14.
9
Nakazato S, Fujita Y, Hamade Y. Wells’ syndrome associated
with chronic myeloid leukaemia. Acta Derm Venereol. 2013
May;93(3):375-6.
10
Shorr WF, Tausheck AL, Dickson KB, Melski JW. Eosinophilic Cellulitis (Wells’ Syndrome): histologic and clinical features in arthropod
bite reaction. J Am Acad Dermatol. 1984; 11:1043-9.
11
Kuwahara R, Randall MB, Eisner MG. Eosinophilic cellulitis in a
newborn. Pediatr Dermatol 2001; 18:89-90.
12
Powell JG, Ramsdell A, Rothman L. Eosinophilic cellulitis (Wells
Syndrome) in a pediatric patient: a case report and review of the
literature. Cutis 2012; 89(4):191-194.
13
Wood C, Miller AC, Jacobs A, Har R, Nickoloff BJ. Eosinophilic infiltration with flame figures. A distinctive tissue reaction seen in Wells’
syndromeand other diseases. Am J Dermatopathol 1986; 8:186 93.
Atrophoderma of Pasini and Pierini in a 10-yearold girl: a case of a spontaneous resolution and a
review of the literature
G. Crisman1, A. D’Amuri3, I. Neri2, A. Patrizi2, B. Passarini2
U.O. Anatomia Patologica, Azienda Ospedaliera Desenzano del Garda,
Desenzano d.G. (BS); 2 Dermatology, Department of Specialised, Experimental and Diagnosed Medicine, University of Bologna, Bologna, Italy;
3
U.O. Anatomia Patologica, Dipartimento dei Servizi Diagnostici, Az.
Osped. “Card. G. Panico”, Tricase (LE)
1
Introduction. Atrophoderma of Pasini and Pierini (APP) mainly
occurs in young women and presented with single or multiple
hyperpigmented or blue to violet in color, round or ovoid, localized atrophic plaques usually sited on the back and lumbosacral
region, but chest, arms and abdomen have been reported as well.
Only two congenital forms have been reported so far.
Material and methods (case report). A healthy 10-year-old
Caucasian girl presented to us with a multiple, asymptomatic,
hyperpigmented atrophic plaques on her left back since birth. Her
development since birth was uncomplicated and her family and
medical history was unremarkable and parents had noted the con-
205
genital plaques that gradually enlarged and acquired a dark pigmentation and a progressive depression since the age of 9-yearold. Physical examination revealed three large, ovoid in shape,
irregularly and slightly depressed, hyperpigmented plaques with
prominent vasculature on the left back. The overlying skin was
atrophic without induration and ranging in color from violaceous
to gray-brown .The lesions were well demarcated and movable.
(Fig. 1) A 4-mm punch skin biopsy has been performed and histological examination revealed a focal hyperpigmentation of the
epidermis and a moderate reduced dermal thickness with homogenized collagen fibers. (Fig. 2) Weigert stain revealed a normal
amount and distribution of elastic tissue fibers. Thus, a diagnosis
of atrophoderma of Pasini and Pierini was posed.
Results (discussion). First described by Pasini in 1923 and subsequently by Pierini and Vivoli in 1936, Atrophoderma of Pasini and
Pierini(APP) represents a rare, primary dermal, atrophic process
of benign clinical course and unknown aetiology, even though a
positive ANA and an association with a previous or concomitant
Borrelia burgorferi infection has been reported in a small number
of patients. APP usually occurs in the second or third decade (mean
age of onset of 30years), with a male to female ratio of 1:2 to 1:6.
It is still controversial whether it represents a variant of localized
scleroderma (morphea) or a distinct entity sui generis.(1-4).
Clinically, single or multiple plaques, with a bilateral and symmetrical distribution, characterized by a violaceous or graybrown colour, round or ovoid in shape are observed. The lower
back and lumbosacral region represent the most common site of
onset, even though some cases arisen on chest, arms and abdomen
have been reported as well. Unilateral involvement has also been
described (5).
Fig. 1. Clinical presentation of the three large, ovalar, irregularly and
slightly depressed, hyperpigmented plaques with prominent vasculature sited on the left back.
206
Fig. 2. Histological examination showed focal hyperpigmentation of
the epidermis and a moderate reduced dermal thickness with homogenized collagen fibers.
Histological examination of skin biopsy usually highlights unspecific changes: the epidermis may be atrophicor normal and it
oftenly shows an increased melanin hyperpigmentation; in early
phase, a mild, mainly perivascular and periadnexal lymphohistiocytic inflammatory infiltrate and slight homogenization of the
collagen bundles are observed, whereas in older lesions a mild to
moderate dermal atrophy as well as dermal sclerosis, with thickened and tightly packed collagen fibers are seen. The appendage
structures are usually normal. Weigert stain is commonly used to
highlights the elastic tissue fibers, which are reported to be normal in distribution in APP, even though in some cases a reduction
and a fragmentation has been also detected. (4) According to the
literature, only two congenital cases have been reported. (6-7) In
2006, Kim SK et al reported on a 2-year-old girl presented with
erythematous atrophic lesion on the right shoulder since birth.
Histological examination revealed a slightly homogenization of
the dermal collagen bundles and a severe reduction of dermal
thickness, whereas elastic tissue resulted normal in distribution.
Six years later, Handler MD et al described a large atrophic plaque
on the lower back of a Caucasian man since birth. Histologically,
a dermal atrophy with normal elastic fibers distribution have
been highlighted. In both cases, a favourable clinical course has
been reported and an etiopathogenetic mechanism has not been
suggested. In our case, clinical examination of the lesion and the
histological changes, characterized by a moderate reduced dermal
thickness with homogenized collagen fibres, led to the diagnosis
of a APP. Interestingly, all the three congenital cases reported a
unilateral presentation. (Table 1) In this context, it is worth noting
the possibility of an alteration in the composition of the extracellular matrices during the gestation period, since no trauma nor
infection have occurred in our patient and in the two congenital
cases reported. In 2000, Yokoyama Y at el., in the aim to confirm
APP as a different nosologic entity, measured the disaccharide
units of skin GlycosAminoGlycan (GAG) and in particular Dermatan Sulphate (DS – which is found to be predominant in the
dermis) in two patient affected by APP (16-year-old and 65-yearold respectively, none with congenital lesion) and one patient
with atrophic phase morphoea. An increased total GAG per
punch-biopsied skin and a decrease or unchanged amount of DS
per mg dry weight was noted in APP patients, whereas opposite
results where obtained from the morphoea of atrophic phase thus
suggesting a different pathogenetic mechanism. It is thus possible
to suggest the hypothesis that a mosaic distribution of regulatory
genes of GAG and DS in particular could be responsible of this
kind of congenital atrophodermic lesion (8).
Conclusions. In conclusion, this represents the third case of
documented congenital APP and we would like to underline that
this peculiar type of dermal atrophy should be considered in differential diagnosis of congenital dermal skin lesions.
References
1
Winkelmann RK. Localized cutaneous scleroderma. Semin Dermatol
1985; 4: 90-103.
2
Stoner MC, Dixon SL. Atrophoderma Pasini,Pierini. Arch Dermatol
1990; 126: 1641-44.
3
Saleh Z, Abbas O, Dahdah MJ et al. Atrophoderma of Pasini and
Pierini: a clinical and histopathological study. J Cutan Pathol 2008;
35: 1108-14. 4) Buechner SA, Rufli T. Atrophoderma Pasini-Pierini:
clinical and histopathological findings and antibodies to Borreli burgorferi in thirty-four patients. J Am Acad Dermatol 1994; 30, 441-446.
5
Iriondo M, Bloom RF, Neldner KH. Unilateral atrophoderma of Pasini and Pierini. Cutis 1987; 39: 69 70.
6
Kim SK, Rhee SH, Kim YC et al. Congenital atrophoderma of Pasini
and Pierini. J Corean Med Sci 2006; 21: 169-71.
7
Handler MZ, Alshaiji JM, Shiman MI, Elgart GW, Schachner LA.
Congenital idiopathic atrophoderma of Pasini and Pierini. Dermatol
Online J. 2012; 18(4):4.
8
Yokoyama Y, Akimoto S, Ishikawa O. Disaccharide analysis of skin
glycosaminoglycans in atrophoderma of Pasini and Pierini. Clin Exp
Dermatol 2000; 25(%):436-40.
Cytological atypia in basal cell carcinoma: clinical,
histological and cytological correlations
G. Crisman1, A. D’Amuri3, S.D. Infusino2, A. Patrizi2, B. Passarini2
U.O. Anatomia Patologica, Azienda Ospedaliera Desenzano del Garda,
Desenzano d.G. (BS), Italy; 2 U.O. Dermatologia, Dipartimento di Medicina Specialistica, Sperimentale e Diagnostica, Università di Bologna,
Bologna, Italy; 3 U.O. Anatomia Patologica, Dipartimento dei Servizi Diagnostici, Az. Osped. “Card. G. Panico”, Tricase (LE), Italy
1
Background. Basal Cell Carcinoma (BCC) represents the most
common Non-Melanoma Skin Cancer (NMSC). Histologically, a
monomorphous, small, uniform in shape and size, basaloid cells proliferation, with peripheral palisading, is observed. Up to now, cells
with large, hyperchromatic nuclei, multinucleated or vesiculated
cells, as well as atypical cells and bizarre mitoses have been noted.
Tab. I. General characteristic of the included studies.
References
Gender
Kim SK et al., 2006
Handler MZ et al., 2012
Neri I et al., 2014
Female
Age at time of
diagnosis
2 years old
Number
of lesions
1
Male
5 months
1
Female
10 years old
3
Clinical
presentation
Unilateral, slighty depressed,
erythematous atrophic plaque
Unilateral, atrophic plaque with
prominent vasculature
Unilateral, large, ovalar,
irregularly and slightly
depressed, hyperpigmented
plaques, with prominent
vasculature
Site
Right
shoulder
Lower right
back
Left back
COMUNICAZIONI ORALI
Material and methods. A literature search for peer-reviewed
and indexed publications from 1960 to 2014 using the PubMed,
Medline, Medline in Process, Embase and Cochrane search engines was performed by using the following key words: basal cell
carcinoma and epidemiology, basal cell carcinoma and atypia,
basal cell carcinoma and atypical nuclei, basal cell carcinoma and
mitoses, basal cell carcinoma and giant cells, basal cell carcinoma
and monster cells, basal cell carcinoma and clinical course. Two
dermatopathologists systematically and independently reviewed
the consecutive BCC specimens collected from January 2006
to December 2007 at the Dermatopathology Laboratory of the
Dermatology Unit (University of Bologna). The personal and
family history of patients, the clinical presentation, management
and follow-up of BCCs were retrospectively collected. Patients
with basal cell nevus syndrome, patients with incomplete clinical
histological and/or follow-up data and patients with a previous
diagnosis of BCC were excluded from the study. The reviewing
pathologists assigned a degree of atypia to each case using the
following criteria: i) grade 1 atypia: mild architectural alteration and/or mild cytological atypia ; ii) grade 2 atypia: moderate
architectural atypia and cytological atypia and/or high number
of mitoses whether typical or atypical; iii) grade 3 atypia: severe
cytological atypia with prompt hyperchromatic or multinucleated or vesiculated nuclei and/or bizarre mitoses and/or architectural alterations (Fig. 1a and 1b). The dermatopathologists were
blinded to the previous clinical diagnosis of the lesion, patients’
data and followup. Cases with discordant atypia classification
between the two reviewing dermatopathologists were reviewed
again in a consensus session in order to obtain a final diagnosis.
The authors subsequently matched the histological features with
the clinical data with the aim of verifying whether cellular and/or
architectural atypia of BCC were related with clinical presentation, patients’ gender or age, site of onset or clinical behaviour.
Results. 1149 consecutive BCC specimens from 1134 patients
were studied. The patients were 658 males and 476 females, with
a male to female ratio of 1.4 : 1, with a mean age of 69.1 years
(range 20-99 years) and a mean BCC size of 0.86 cm (range 0.2
– 4.1 cm). Almost half of the lesions arose on the face (540/1149:
207
47%), including the nose, forehead, eyelid, lip, and cheek,
whereas other sites such as the thorax, shoulders and back were
less common (15% and 14% respectively). Histological examination revealed in all cases a monomorphous, small basaloid cells
proliferation, with peripheral palisading, stromal retraction and
mucin production. Neoplastic nuclei are usually uniform in shape
and size, and occasionally some necrotic cells were detected.
Mitoses varied from 0 to 30 per 10-high-power field. Lesions
showing severe cytological atypia with prompt hyperchromatic
or multinucleated or vesiculated nuclei and/or bizarre mitoses
and/or architectural alterations were classified separately as grade
3 atypia, being rarer in percentage and more worrisome for the
pathologist. These atypical cells may be observed and recognized
also at low magnification and represented more than 50% of the
total cell population in the majority of cases. In our study, 104
BCC with grade 3 atypia were detected (104/1149: 9.1%).
Grade 3 atypia was more commonly observed in the elderly (mean age 75 years, range 43-97 years), and generally in BCCs greater in size with a mean diameter of 0.95cm (range 0.3-2.4cm);
the nose (29/104: 27.9%) represented the most common site of
onset, followed by another sun-exposed area, such as the face
(28/104: 26.9%), and the legs (9/104: 8.65%). During the 6 to 7
years of follow-up, more than a half (71 cases) of the lesions did
not recur (68.2%), 11 recurrences were recorded (10.6%), 11 new
solitary BCCs were detected (10.6%) and 11 new multiple BCCs
appeared (10.6%).
In 1964 Okun MR at al. reported 5 cases of BCC presenting with
giant cells and nuclear atypicality. None of the lesions showed
dyskeratosis or area of squamous differentiation 5. Five years
later, in 1969 Rupec M et al.
reported the presence of giant cells with multiple nuclei in 7 cases of BCC. 6 In both papers, the atypical cells were interspersed
among the “common” basaloid neolpastic cells and did not represent the major component of the lesion. Ochiai et al. reported a
case of BCC with giant cells in 1987 10 and in 1995, Garcia JA
et al reported a series of 19 cases of BCC with scattered large,
pleomorphic cells (so-called “pleomorphic basal cell carcinoma”)
analyzed with the image analysis. Garcia suggested the term
Fig. 1. a) Grade 3 atypia: severe cytological atypicality, visible at low magnification (H&E 4x); b) Grade 3 atypia: multinucleated cells with irregular,
large, hyperchromatic nuclei (H&E 20X).
208
“pleomorphic” BCC to describe a new (even though already
described) histological variant characterized by the presence of
mononucleated and multinucleated giant cells with bizarre nuclei.
However, all these aberrations referred to a small component of
the tumour cells and peripheral palisading and stromal retraction,
mucin production and apoptosis were conserved 11. Four years later, Cutlan RT et al. analyzed 12 cases of abnormal cells found in
BCC using immunohistochemistry (Ki-67, Bcl-2 and PCNA). All
aberrant cells stained positive for the tested makers in higher percentage than surrounding small neoplastic cells thus suggesting
a preserved capability of these monster cells of cell division 12.
Conclusions. In conclusion, BCC with pleomorphic giant cells
represents an uncommon histological variant of BCC. Also
referred to BCC with giant cells, BCC with monster cells and
pleomorphic BCC, over 40 cases of BCC with “atypical cells”
have been reported so far 5-12. These cells have been described
as mono-or multinucleated, irregular in shape and size, with prominent nuclei, 2 to 10 times larger than the surrounding tumour
cells, with hyperchromatic nuclei and phagocytic activity in some
cases. These cells could be easily recognized at low magnification and showed random distribution except in areas of squamous
differentiation. However, the exact nature and function of these
cells is still unknown. They have been reported to be a rare to
occasional finding, never representing more than 10% of the total
cell population. Even if a small sample of lesions were collected
in each study, Cutlan et al. suggested that the incidence of giant
cells should be around 2,5% 12. All authors suggested that these
atypicalities showed a biological potential similar to the conventional form of BCC 5-12. In contrast with previous reports, we have
demonstrated in a large case study that abnormal, pleomorphic
giant cells may represent more than 50% of the neoplastic cell
population and could comprise around 9% of routinely processed
BCC. The long term period of follow up of our patients leads us
to suggest with reasonable certainty that these alterations are not
related to an increased risk of local recurrences and/or metastatic
potential. Thus, we would like to underline how the presence of
these histological aberrancies may represent only an incidental
histological finding without any prognostic significance.
References
Silverberg E, Boring CC, Squires TS. Cancer statistics 1990. CA Cancer
J Clin 1990; 40:9-26.
Gallagher RP, Hill GB, Bajdik CD et al. Sunlight exposure, pigmentary
factors and risk of nonmelanocytic skin cancer. I. Basal cell Carcinoma. Arch Dermatol 1995; 131:157-163.
Gallagher RP, Ma B, McLean DI et al. Trends in basal cell carcinoma,
squamous cell carcinoma and melanoma of the skin . J Am Acad Dermatol 1990; 23:413-21.
Miller SJ. Biology of Basal cell carcinoma (Part 1). J Am Acad Dermatol
1991; 24:1-13.
Okun MR, Blumenthal G. Basal cell epithelioma with giant cells and
nuclear atypically. Arch Dermatol 1964; 89: 598-600.
Rupec M, Vakilzadeh F. Uber das Vorkommen von mehrkemigen Riesenzellen in Basaliomen. Arch Klein Exp Dermatol. 1969; 235:198.
Pinkus H, Mehregan AH. Basal cell epithelioma. A guide to dermatohistopathology (3rd. Ed), Appleton-Century-Crofts, New York 1981;
pp-459-461.
Ono T, Egawa K, Higo J et al. Basal cell epithelioma with giant tumourcells. J Dermatol 1985; 12:344-348.
Ochiai Tm Suzuki H, Morioka S. Basal cell epithelioma with giant tumourcells: light and electron microscopic study. J Cutan Pathol 1987;
14:242-247.
Elston DM, Bergfeld WF, Petroff N. Basal Cell Carcinoma with monster
cells. J Cutan Pathol 1993; 20:70-73.
Garcia JA, Cohen PR, Herzberg AJ et al. Pleomorphic basal cell carcinoma. J Am Acad Dermatol 1995; 32(5 Part I):740-746.
Cutlan RT, Maluf HM. Immunohistochemical characterization of pleomorphic giant cells in basal cell carcinoma. J Cutn Pathol 1999;
26:353-356.
Human Dirofilariasis Repens with unusual
localization: report of three new cases in Apulia
Region
A. D’Amuri1, F. Floccari2, C. Gabrieli1, C. Fasano1, M. Filotico1,
G. Crisman3
Gallipoli (LE), Italia; 3U.O. Anatomia Patologica, Azienda Ospedaliera di
Desenzano del Garda, Desenzano del Garda (BS), Italia
1
Introduction. Human zoonotic filariasis due to Dirofilaria repens is a world-wide infection accidentaly transmitted to man by
a zooanthropophilic bloodsucking mosquito belonging to the generae Culex. Dirofilariasis live in the subcutaneous tissue of their
hosts causing the formation of nodules which normally requires
surgical removal and an histological examination. Rare localizations have also been reported in deep tissues like lung, spermatic
cord and uterine round ligament. We reported three new cases of
human infections in rare sites for this parasitosis, diagnosed in
Italian patients living in Southern Italy (Apulia).
Methods. A 45-year-old Caucasian man revealed a left inguinoscrotal hernia; incidentally, a solid fatty mass within the funiculus
spermaticus of the left testis was discovered during hernioplastic
and an orchiectomy was performed for suspicion of malignancy.
A 51-year-old Caucasian man complained of swelling and a
painful mass in the inguinal area. Lymphoadenopathy due to
lymphadenitis of unknown etiology was suspected. A 69-yearold Caucasian man presented a subcutaneous abdominal swelling
mass. A clinical diagnosis of foreign body granuloma after skinpenetrating trauma was performed.
Results. In the first case histopathological examination demonstrated a severe granulomatous reaction with eosinophils around
cross section of a nematode identified as Dirofilaria repens. The
second patient at microscopy examination of the excised lymph
nodes and perinodal adipose tissue revealed a reactive nodal
hyperplasia with widespread adipose tissue. There was an inflammatory infiltrate rich in lymphocytes and eosinophils around
cross sections of a nematode identified as D. repens. In the last
case histology showed a subcutaneous tissue with a conspicuous
inflammatory infiltrate with a granulomatous reaction around
cross sections of a nematode identified as D. repens.
Conclusion. In both of our patients, histopathology revealed
cross sections of the nematode enclosed in the surgical excised nodules. There was an associated granulomatous reaction
with many eosinophils surrounding the nematode. The correct
identification of a D. repens nematode relied upon histological
examination of the microscopic features, including a thick laminated cuticle with external longitudinal ridges and the presence
of a well-developed circumferential musculature interrupted by
two lateral cords. Little has been reported about the benefits of
antifilarial medication, taken alone in association with surgical
therapy. The aim of this report is to better characterize dirofilariasis in endemic areas in order to increase awareness of this
entity and its changing geographical distribution. We hope to
provide histopathologists and parasitologists with new useful
data concerning the nematode.
References
Pampiglione S, Arlotta MR, Carlà TG, et al. Human dirofilariasis in
southern Italy. I. The Puglia Region. Pathologica 1994;86:528-32.
Pampiglione S, Rivasi F, Angeli G, et al. Dirofilariasis due Dirofilaria
repens in Italy, an emergent zoonosis: report of 60 new cases. Histopathology 2001;38:344-54.
Pampiglione S, Fioravanti ML, Piccolotti D, et al. Human dirofilariasis in
Italy: a new case in the spermatic cord. Parassitologia 2002;44:93-6.
D’Amuri A, Senatore SA, Carlà TG, et al. Cutaneous dirofilariasis resulting in orchiectomy. J Cutan Pathol 2012;39:304-5.
209
COMUNICAZIONI ORALI
Dermopatologia - 2
Moderatori: G. Botti, M.C. Montesco
Subcutaneous nodule of a tymic carcinoma in a 64
year old man
A. Di Lorito1, C. Marinelli2, G. De Luca1, L. Ranieri1, G. Lattanzio1
Medicine and Science of Aging, Santissima Annuziata Hospital, Chieti,
Italy; 2 Medicine and Science of Aging, G. Bernabeo, Ortona, Italy
1
Background. Thymic carcinoma is a rare tumor, accounting for
less than 0,5% of mediastinal malignancies. It usually spreads,
has a high risk of recurrence, and has a poor survival rate . Thymic carcinoma is divided into subtypes, depending on the types
of cells in which the cancer begin.
Distant metastasis have been reported in up to 7% of cases and
they usually involve lung, liver, bone and soft tissue.
The treatment is not standardized, although complete surgical removal gives the best length of survival. Tumor size and histology
are very important factors for the clinical outcome.
We described a patient with a thymic carcinoma diagnosed in a
subcutaneous nodule of the chest.
Methods. A 64-year-old man was admitted to our hospital for
cough and hemoftoe. Chest X ray showed an opacity in the mediastinum. The chest computed tomography (CT) scan revealed a
solid mass, in the anterior mediastinum, with lymphoadenopathy.
There was no evidence of the tumor elsewhere on abdominal CT
scans and bone scintigraphy.
A subcutaneous nodule, suspected for malignancy, was removed
and analyzed. The sample was made up of skin of 2,6x1 cm with
subcutaneous tissue of 2,7x2,6 cm inside of which a white nodule
with well defined borders has been found.
Results. The histological examination showed in the subcutaneous tissue irregular solid nests of infiltrating tumor. The solid
nests were made up of polygonal atypical cells with oval nuclei
and prominent nucleoli, with moderate amount of eosinophilic
cytoplasm. The nests were separated by broad bands of fibrous
tissue. Coagulation necrosis was absent but the tumor cell nests in
some areas were interspersed with typical lymphocytes.
A panel of different immunoistochemical markers has been
analyzed. The tumor cells were positive for CKAE1/AE3, CROMOGRANIN and CD117, negative for TTF1, CDX2, HMB45.
According to the morphology, immunohistochemical profile and
to the clinical data, the final diagnosis of well differentiated squamous cell thymic carcinoma has been made.
Conclusions. Well differentiated squamous cell thymic carcinoma is a rare neoplasm and it is considered a diagnostic challenge
to both the histopathologist and clinicians. Surgical removal with
radiotherapy is the treatment of choice and the most relevant
prognostic factors are the tumor size and the histological grade.
Intravascular proliferation of reactive atypical
CD30+ T-cells: a diagnostic pitfall
V. Natella, G. Luongo, A. Somma, G. Mansueto
Department of Advanced Biomedical Sciences University Federico II of
Naples, Italy
Background. CD30 is a trasmembrane glycoprotein member
of the tumor necrosis factor receptor superfamily, generally expressed in activated B and T cells. In the past CD30 expression
in cutaneous lymphocytic infiltrate was considered as a reliable
marker for primitive lymphoma (lymphomatoid papulosis, pri-
mary cutaneous anaplastic large T cell lymphoma, and large cell
transformation of mycosis fungoides) or for secondary involvement of systemic lymphoproliferative diseases.
Furthermore, in the past years, CD30 positive lymphoid atypical
cells have been detected in several reactive inflammatory condition (infections, drugs reactions, etc.).
Intravascular lymphoma is a rare, fatal, extranodal non-Hodgkin
lymphoma characterized by the proliferation of neoplastic cells
within the lumen of small vessels, and involve the skin in 40% of
patients. Usually IVL show a B-cell phenotype, only few cases
are of T-cell or NK-cell origin. In literature 2 cases of intravascular large T cell lymphoma CD30+ with an aggressive course
have been reported.
Therefore the presence of an atypical CD30+ T-cell proliferation
within blood vessels in a benign inflammatory conditions may
represent a potential diagnostic pitfall for intravascular lymphoma.
Methods. We present two cases of benign atypical intravascular CD30+ T-cell proliferation occurring in a 51-year-old man
with a clinical history of sinus pilonidalis, and in a 40-year-old
woman with an epidermal cyst in the mammary region. In both
cases the microscopic examination showed a diffuse, dermal,
mixed inflammatory infiltrate (lymphocytes, plasma cells, and
neutrophils) and an intravascular atypical lymphoid proliferation
resulting positive for CD3, CD4, CD30, and negative for CD20,
and ALK.
Although the histopathological features suggested an intravascular T-cell lymphoma CD30+, the evidence of normal laboratory
test, and the clinical history (unique lesion, absence of constitutional signs and symptoms) favoured the diagnosis of a reactive
condition.
Conclusion. These two cases demonstrate that reactive intravascular proliferation of atypical lymphoid cells CD30+ may mimic
an intravascular lymphoma and represent a potential pitfall for
pathologists. The nature of this intravascular growth pattern of
reactive lymphoid blasts is unclear; it is postulated that these cells
could be in transit within an area of severe chronic inflammation
Cutaneous macroglobulinosis as an initial
symptom of incipient Waldenstrom disease: a case
report
V. Natella, E. Mezza, A. Somma, G. Mansueto
Department of Advanced Biomedical Sciences University Federico II of
Naples, Italy
Background. Cutaneous macroglobulinosis is a rare skin manifestation of Waldenstrom macroglobulinemia (WM), consisting
in an isolated deposition of immunoglobulin M (IgM) in the
dermis.
Waldentrom macroglobulinemia is a lymphoproliferative disorder characterized by the production of monoclonal IgM by malignant B cells that proliferate in the bone marrow, lymphnodes, and
spleen. The clinical manifestations are due to neoplastic B-cell
infiltration (lymphnode enlargement, splenomegaly, and bone
marrow involvement with cytopenia) and to the overproduction
of IgM resulting in hyperviscosity syndrome. Cutaneous manifestations of WM are rare and classified into specific and nonspecific ones. Specific skin lesions consist of neoplastic infiltrates
of lymphoplasmacytic cells and deposits of monoclonal IgM.
210
Most patients presented with papules on the upper and lower
extremities. Nodules, plaques, or ulcerated lesions were also
reported, as well as involvement of the trunk, face, and scalp.
Methods. A healthy 83 year-old woman presented to the dermatologist for evaluation of asymptomatic erythematous papules and plaques on her face and legs. Biopsies of the lesions
showed a dermal vascular proliferation and an extravascular and
intravascular deposition of a pink homogeneous material. This
eosinophilic material was strongly PAS positive and negative
for amyloid stains (Congo red). Direct immunofluorescence
performed on the biopsies revelead a dermal and an intravascular
deposition of IgM. These findings were consistent with IgM- associated cutaneous macroglobulinosis.
Conclusion. Cutaneous macroglobulinosis is a rare condition that
should be considered in the histopathological differential diagnosis of eosinophilic dermal deposits, as it could represent an initial
symptom of incipient Waldenstrom disease.
Evaluation analysis of BRAF gene mutation
status of primary and metastatic melanomas
by immunohistochemistry and comparison with
molecular biology techniques
F. Pellegrino1, G. Giannini2, F. Buttitta3, G. Soda4
1
Istituto di Anatomia Patologica “Sapienza” Università di Roma;2 Dip.
Medicina Molecolare “Sapienza” Università di Roma; 3 Dipartimento di
Anatomia patologica, patologia clinica e medicina trasfusionale Ospedale SS. Annunziata Chieti; 4 Istituto di Anatomia Patologica “Sapienza”
Università di Roma
Background. In a large proportion of patients with metastatic
melanomas a specific mutation (V600E) in the BRAF gene has
been identified.
The current gold-standard for the detection of this mutation
involves the use of molecular techniques. Recently, a new monoclonal antibody (VE1) against mutant BRAF (V600E) has been
used to identify this specific mutation. Mutant BRAF protein has
become an important therapeutic target in patients with metastatic
melanomas.
Materials and methods. In this study we collected 100 melanoma samples (37 of metastatic melanoma and 63 of primary
cutaneous melanoma). DNA was extracted from formalin-fixed
and paraffin embedded sections and then analyzed by molecular
biology techniques to determine the mutation status of the codon
V600.
In 42 cases, PCR and direct sequencing have been used; in the remaining 58, Real-Time PCR cobas test 4800 has been performed.
An additional consecutive section of each sample has been tested
by immunohistochemistry (IHC) with the monoclonal antibody
VE1 “Spring-Bioscience” and the Detection Kit “Bond Polymer
Refine Red Detection”.
Results. The comparison of the results of BRAF gene mutation
status obtained by conventional molecular biology methods and
by IHC showed a significant correlation. The antibody has shown
a high sensitivity and specificity for detecting the presence of
BRAF V600E mutation.
Conclusions. Currently, the investigation of the BRAF gene
mutation status requires the use of molecular methods because
these are the only methods accepted by competent authorities
(AIFA, etc.) for therapy with drugs directed against the mutant
BRAF protein.
Molecular techniques are sensitive and reliable, but on the other
hand they are expensive, they require a significant amount of
biological material and they’re not always available in diagnostic
pathology laboratories.
Furthermore, molecular techniques are also time consuming.
In fact, a BRAF mutation status test may take a few days to be
performed.
On the contrary, IHC shows several potential advantages:
1) ability to perform the test within 24-48 hours after surgical
excision of the tumor;
2) very low economic impact;
3) minimum quantities of tissue needed;
4) widespread availability of IHC, in all diagnostic pathology
laboratories.
The only limit of IHC is the inability to detect BRAF mutations
other than V600E, whereas molecular methods are able to recognize all of them.
Our results suggest the use IHC as a first level screening test for the
detection of BRAF V600E mutations; negative cases would be then
tested with molecular techniques as a second level screening test, in
order to identify possible BRAF mutations other than V600E.
Our purpose is put forward in order to optimize costs and time
consuming, with an increase of benefits for patients
References
Capper D, Berghoff AS, Magerle M, et al Immunohistochemical testing
of BRAF V600E status in 1,120 tumor tissue samples of patients with
brain metastases. Acta Neuropathol (2012) 123:223-233.
Capper D, Preusser M, Habel A, ed al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol (2011) 122:11-19.
Long GV, Wilmott JS, et al. Immunohistochemistry is highly sensitive and
specific for the detection of V600E BRAF mutation in Melanoma. Am
J Surg Pathol 2013;37:61-65.
Marin C, Beauchet A, Capper D, et al. Detection of BRAF pV600E mutations in Melanoma by immunohistochemistry has a good interobserver
reproducibility. Arch Pathol Lab Med doi: 10.5858/arpa. 2013-0031OA.
Chen Q, Xia C, Deng Y, et al. Immunohistochemistry as a quick screening
method for clinical detection of BRAF (V600E) mutation in melanoma
patients. Tumor Biol. 2014 Jun;35(6):5727-33.
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Sala Donatello – 2° Piano – 08,30-09,30
Patologia mammaria - 1
Moderatori: S. Bianchi, S. Di Lollo
Metastatic tumors to the breast. Report of 4 cases
and review of the literature
V.Angione1, S. Bertozzi2, A.P. Londero3, S. Bernardi2, G. De
Maglio, A. De Pellegrin1, E. D’Alessandro1, S. Pizzolitto1
1
Dipartimento di Medicina di Laboratorio SOC Anatomia Patologica AOU
“SSMM della Misericorda”, Udine, Italy; 2 Dipartimento di Chirurgia
Generale, AOU “SSMM della Misericordia”, Udine, Italy; 3 Clinica Ostetrica e Ginecologica, AOU “SSMM della Misericordia”, Udine, Italy
Introduction. We report our experience on metastatic tumors to
the breast emphasizing their incidence, clinical and pathologic
features as documented in the last 6 years (2008-2013)
Methods. Case search was conducted through the archives of the
Department of Surgery and the electronic files of the Department
of Pathology between 2008-2013, retrieving the cases of metastatic tumors to the breast and the incidence thereof.
Results. We identified 4 patients who underwent breast surgery
for extramammary tumors. All the patients were women.
Case 1. The patient was 55 years old and had evidence of serous
carcinoma of ovary. She presented in January 2008 with recurrent mastitis and a breast core needle biopsy revealed lymphatic
spread by cancer cells which were positive for broad spectrum
keratins and Estrogen receptors. Progesterone receptors were
negative. In February 2008 she underwent hysterectomy and bilateral salpingo-oophorectomy for bilateral serous papillary carcinoma of ovary. In August 2008 right mastectomy and axillary
dissection was carried out for diffuse cancer spread of the mammary lymphatics and metastases to 21 out of 24 lymph nodes. After a fourth line chemotherapy, she died of intestinal subocclusion
and progressively physical wasting in November 2009.
Case 2. The patient was 68 years old and had history of neuroendocrine carcinoma of lung. In July 2004, a bronchial biopsy
was deemed consistent with atypical carcinoid (neuroendocrine
carcinoma, intermediate type). In October of the same year, the
left lower lobe was resected showing evidence of neuroendocrine
carcinoma of lung, intermediate type, pT1N1Mx. In January
2008 a metastatic tumor was seen in the thyroid, and in July
2008 a nodule was discovered in the superior sector of the left
breast: the mammary tumor measured 7 mm. A needle aspirate
yielded atypical cells suspicious for malignancy. In September, a
left quadrant biopsy plus a biopsy of a sentinel lymph node was
performed. Routine and immunohistochemical examination of
the breast tumor were deemed consistent with a neuroendocrine
carcinoma metastatic from primay in lung.
Case 3. The patient was 49 years old and had history of melanoma of toe. A core needle biopsy of right breast, performed at
a community hospital, revealed a papillary tumor suspicious of
papillary carcinoma. Wide biopsy of right inferior extern quadrant of breast was done in July 2009. The breast tumor showed a
metastatic, partially cystic melanoma. Tumor cells were negative
for broad spectrum keratins, estrogen and progesterone receptors,
and positive for S100 protein, HMB45 and Melan A. Follow up
at October 2013 is negative for tumor recurrence or metastatic
disease.
Case 4. The patient was 73 years old and had history of renal
cell carcinoma. In November 2011, a biopsy of a 1 cm nodule
of left superior internal quadrant of breast revealed a metastatic
carcinoma consistent with renal primary. In november 1999 radical left nephrectomy, plus partial pancreatectomy and omentec-
tomy had been performed for renal cell carcinoma, stage IV. In
August 2013, resection of omentum was carried out due to a 5 x
4 cm mass diagnosed as metastasis of renal cell carcinoma. The
patient is currently alive, symptom free with evidence of minimal
metastatic tumor.
Conclusion. In our series the incidence of metastatic tumors to
the breast is comparable to published data, with 4/1500 cases
(0.26% vs 0.2-1.3% of literature). The spectrum of tumors capable to metastatize to the mammary gland is likewise consistent
with available reports addressing the subject, namely melanoma,
renal carcinoma, neuroendocrine carcinoma, ovarian carcinoma.
We have documented only cases of metastases to the female
breast. Published cases of metastases to male breast, although
available, are very rare.
In one of our 4 cases, the breast metastasis was the first clinical
evidence of the neoplastic disease, whereas in case 3 the melanoma was initially neglected. The interval between the diagnosis
of the primary tumor and the breast metastasis varies although it
can be as long as 4 years (case of neuroendocrine carcinoma) or
12 years (renal cell carcinoma). Metastases to the breast from an
extramammary neoplasm usually indicates a disseminated metastatic disease and a poor prognosis, localized metastatic disease
may be encountered yet. In addition to evaluation of the proper
clinical context, metastatic tumor to the breast may be suspected
in cases of preponderant lymphatic breast infiltration, along to
appropriate immunohistochemical evaluation especially of apparently “triple negative” breast carcinoma.
Case 3: metastatic melanoma to breast
(mimicking papillary carcinoma)
Monosomy of chromosome 17 in breast
carcinoma. A 3:1 ratio at Her-2 testing should
be interpreted as Her-2 negative for gene
amplification
M. Brunelli, G. Bogina, L. Marcolini, G. Zamboni, E. Manfrin,
A. Caliò, G. Martignoni, F. Bonetti
Department of Pathology and Diagnostics, University of Verona, Italy;
Pathology, Sacro Cuore Hospital, Negrar, Verona, Italy
Introduction. Recent guidelines (ASCO/CAP 2013) addressed
and get guide to appropiate Her-2 scoring among common and
uncommon patterns of Her-2 values. Among minor abnormalities, monosomy of chromosome 17 may affect the interpretation
of Her-2/neu amplification gene testing and its prevalence range
from 1% up to 38% of breast carcinoma. The skewing of the ratio
due to single centromeric 17 probe may bring to false positive
evaluation of gene amplification.
Methods. 201 breast carcinoma were reviewed at genomic level
for Her-2/neu gene amplification. FISH analysis was performed
by double probes (Her-2 LSI/CEP17) (Abbott). Monosomy of
chromosome 17 was assessed when at least 70% of nuclei harbored single fluorescent centromeric signals. Ratio was initially
scored and subsequently corrected by monosomy. Her-2 immunotests (Hercept Test) was matched for all cases.
Results. 26 cases were amplified (13%). 20 cases scored 3+, six
scored 2+. Monosomy of chromosome 17 was revealed in 3/201
cases (1,5%). 1/26 amplified cases showed monosomy of chromosome 17 and scored 3+. Differently, two out of three cases
with monosomy revealed a 3:1 (LSI/CEP) count ratio, and all
212
these three cases notably scored 0 at immunotest. The three cases
were re-scored after serial sections ranging from 3 to 5, 10 and
15 μm. Monosomy due to biological reasons rather than nuclear
truncation was observed when cut-off set to 70% of nuclei.
Conclusions. 1) monosomy of chromosome 17 may be observed
in 2% of breast carcinoma; 2) the skewing of the ratio due to single centromeric 17 probe may bring to false positive evaluation of
gene amplification; 3) monosomy due to biological reasons rather
than nuclear truncation was observed when cut-off set to 70% of
nuclei; 4) when dealing with cases showing a 3:1 ratio (Her-2/
CEP) the interpretation should be “Her-2
Carcinosarcoma of the breast (metaplastic
carcinoma): a case report and short review
of the literaturere
negative or basal-like tumours). Carcinosarcoma of the breast is
an aggressive type of breast cancer with a worse prognosis than
classical breast carcinomas. Carcinosarcoma of the breast is rare
and there are a few numbers of published cases.
References
Wargotz Es, Norris HJ. Metaplastic carcinomas of the breast. III. Carcinosarcoma. Cancer 1989;64:1490-9.
Reis-Filho JS, Milanezi F, Steele D, et al. Metaplastic breast carcinomas
are basal-like tumours. Histopathology 2006;49:10-21.
Ilhan E, Vardar E, Ozkok G, et al. A Rare Tumour of the Breast: Carcinosarcoma. J Clin Med Res 2010;2:96-8.
Rungta S, Kleer CG. Metaplastic Carcinomas of the Breast: Diagnostic
Challenges an New Translational Insights. Arch Pathol Lab Med
2012;136:896-900.
A. D’Amuri1, C. Fasano1, C. Gabrieli1, F. Floccari2, M. Filotico1
Semiautomatic digital analysis of equivocal (2+)
IHC Her2 correlated with FISH test
Gallipoli (LE), Italia
T. Marcuzzo1, F. Giudici1, E. Ober2, D. Bonazza1-3, M. Pinamonti1-3, D. Bonifacio1, C. Rizzardi1-2, F. Zanconati1-2-3
1
Introduction. Metaplastic breast carcinoma (MBC) is a rare form
of breast cancer (comprising less than 1% of invasive carcinomas). Carcinosarcoma is a subgroup of MBC, and it is probably
the rarest of all primary malignancy of the breast (0,1% of all
cases). The cells of origin for breast carcinosarcoma are not yet
agreed upon fully, but many researchers feel that they are of myoepithelial origin. Breast carcinosarcomas may themselves present
in a variety of mixtures, such as homogeneously adenosquamous,
or heterogeneously epithelial (adenocarcinoma) and mesenchimal
(matrix, spindle cell and sarcomatous). In common practice now,
all breast carcinomas with obvious carcinomatous and sarcomatous features will tend to be referred to as “biphasic metaplastic
sarcomatoid carcinoma” of the breast.
Methods. A 37-year-old woman (married, having 2 children)
presented with a mass in her left breast which has grown rapidly
in 2 months. The clinical features, mammography and ultrasound
were suggestive of fibroadenoma. The surgical specimen was a
nodular circumscribed fragment, size 3 x 3 x 2,5 cm, grayishwhite ill-defined cut surface. On histopathological examination
revealed disorganization presenting area with epithelial and mesenchymal characteristics. Necrosis was also observed. Epithelial
areas with a ductal nature were determined to be of histologic
grade 3.
Results. The tumor cells were positive for p53, p63, CKAE1/
AE3 and EMA in carcinomatous areas, and vimentin in sarcomatous areas. All tumour cells were negative for estrogen and
progesteron receptors, HER-2 and Ki-67 proliferation index
was 60% positive. No metastasis was found in systemic radiological investigations. Left modified radical mastectomy was
performed. No residual tumor was found in the excision area
in the mastectomy specimen. Axillary lymph nodes (23) was
consistent with reactive hyperplasia. Having received 5 courses
chemotherapy, the case is now in her 29th month and under
follow-up with no disease.
Conclusion. Carcinosarcoma is a general term describing biphasic lesions simultaneously containing malignant epithelial and
malignant mesenchymal tissue components. The origin of breast
carcinosarcoma is controversial. Myoepithelial cells are believed
to originate from a single stem cell like spindle-cells. They have
also been reported to develop from existing cystosarcoma phyllodes, fibroadenoma and cystic backgrounds. The epithelial component in a tumor may be composed of undifferentiated carcinoma,
adenocarcinoma, in situ carcinoma, infiltrative ductal carcinoma
or squamous carcinoma. Mesenchymal components may contain
different elements ranging from undifferentiated mesenchymal
areas to fibroblastic, chondorblastic or osteoclastic areas. Hormone receptors and c-erb-B2 (HER-2) are usually negative (triple-
Dipartimento di Scienze Mediche, Chirurgiche e della Salute-Università
degli Studi di Trieste; 2 Azienda Ospedaliero-Universitaria Ospedali Riuniti di Trieste; 3 Scuola di Specializzazione in Anatomia Patologica Università di Udine -Sede Aggregata di Trieste
1
Introduction. The determination of HER2 status became routine
in processing breast cancer specimens for approving the use of
Herceptin in the patient’s therapy. The immunohistochemistry
evaluation is carried out by the pathologist using an optical
microscope, with a low magnification lens, making the results
subjective, even though it is regulated by strict specific guidelines [1]. In this study we used a semiautomatic digital image
analysis software, dedicated to the evaluation of the different
Her2 membrane patterns: the application of a standardized and
reproducible method could be useful to guarantee the repeatability and reliability of the evaluation.
Materials and methods. We select 46 Her2 immunohistochemical equivocal slides (CB11 clone, 1:300 dilution, Aczon), taken
from consecutive surgical specimens of breast cancer during
2013. These cases were submitted to fluorescence in situ hybridization test (PathVysion Dualcolor, Vysis). All the immunohistochemical slides were scanned using a digital microscope
(D-Sight Fluo Instrument) and analyzed using a specific software
(VISIA Imaging S.r.l). Two cases were valuated as unsuitable
for the digital analysis, while the 44 remaining cases were divided into two categories (Uniform and Heterogeneous) based
on the immunohistochemical pattern intensity distribution of the
slice. The division into these categories was made using a low
magnification lens (2X-4X), examining all the slice. Nine cases
presented an heterogeneous staining distribution, with areas of
different staining intensity, while the 35 remaining were uniform.
The heterogeneous cases were not included because they were
too few and can’t be combined with the uniform ones for their
different analysis procedure. The analysis made on the remaining
35 cases, determined on the membrane staining intensity and also
on their entirety, divided the cases in three categories as Negative
(1+), Equivocal (2+) and Positive (3+). The analysis was taken
on specific areas of the dimension of about 20 mm2, spending a
variable time from 90 to 120 minutes. We compared the results
with the ones obtained by the previous FISH test. Results. Of the
35 analyzed cases 21 (60%) results again Equivocal, needing a
more sensible test, as the demonstration of Her2 amplification
with ISH method. Of the remaining 14 cases, 10 were valuated
as Positive (3+), and 4 as Negative (1+), and each of them were
concordant with FISH test. Conclusion . In our experience the application of the digital analysis is of significant impact. In 40% of
the cases the analysis lead to a definitive diagnosis on a samples
defined as “equivocal”, in agreement with FISH. In pathologist’s
work routine the application of this semiautomatic analysis could
213
COMUNICAZIONI ORALI
define more safety each equivocal Her2 immunohistochemical
slide. There could decrease the application of more expensive
and time consuming FISH analysis. The small number of samples
selected in this study limits the substance of the evidenced data.
A future continuance of this study, including a large number of
clinical record, will lead to more certainty and statistical validation of the method.
References
1
Antonio C. Wolff, M. Elizabeth H. Hammond, David G. Hicks. Et
al: Recommendations for Human Epidermal Growth Factor Receptor
2 Testing in Breast Cancer: America Society of Clinical Oncology/
College of American pathologists Clinical Practice Guideline Update.
American Society of Clinical Oncology, 2013.
Clinical rilevance of her2 gene amplification
heterogeneity in breast cancer in light of updated
2013 asco/cap guideline
C. Marinelli1, A. Di Lorito2, P. Viola2, L. Ranieri2, G. De Luca2,
D. Angelucci1
Medicine and Science of Aging, G. Bernabeo, Ortona, Italy; 2 Medicine
and Science of Aging, Santissima Annuziata Hospital, Chieti, Italy
1
Background. The updated 2013 ASCO/CAP guideline recommendations for HER2 testing define HER2-positive breast cancer
with evidence of protein overexpression (IHC) or gene amplification (ISH) in >10% of tumor cells. If results are equivocal
(revised criteria) reflex testing should be performed using an
alternative assay (IHC or ISH).
Moreover many new paper have described HER2 heterogeneity
as a more frequent occurrence than previously thought.
Genomic heterogeneity refers to conditions where more than one
population of tumor cells exists within the same tumor as clones
of amplified and non-amplified tumor cells, diffuse intermingling
of amplified and non-amplified cells across the tumor, or as
isolated amplified cells in a predominantly non-amplified tumor.
Here we report an interesting heterogeneous case for HER2
gene amplification. The discrepancy between morphologicalmolecular features and clinical behavior is only apparent and lies
in HER2- heterogeneity.
Methods. Mammography of a 52-years-old woman revealed a
lower-external opacity 12 mm in size in the left breast. An ultrasound
guided biopsy was performed which showed an invasive ductal carcinoma coexisting with low grade ductal carcinoma in situ.
The patient underwent left breast segmentectomy with axillary
sentinel lymph node biopsy. Sentinel node was negative also
after immunohistochemical studies (pNO). Histological examination of surgical specimen confirmed the diagnosis of invasive
ductal carcinoma grade 1 sec. Elston&Ellis (tubules formation:
2; nuclear pleomorphism: 2; mitotic rate: 1) 12 mm in diameter
(pT1c). Margins were clear.
Immunohistochemically, the invasive tumor was positive (90%)
for estrogen receptor (ER) and negative for progesterone receptor
(PgR) and Ck5. The proliferation index was low (MIB1: 10%).
Results. HER2 protein expression resulted equivocal (2+) based
on circumferential membrane staining, imcomplete, moderate
within >80% of the invasive tumor cells. So a dual-probe CISH
was carried out on the same block.
Such as recommended the number of CEP17 and HER2 signals
were prior counted in 20 non-overlapping and contiguous invasive cancer cell nuclei in 2 tumor areas.
Due to the evidence of genetic heterogeneity additional cells
(>60) were counted. Scattered amplified cells (HER signals ≥6;
ratio HER2/CEP17 >2) were found in a predominantly nonamplified tumor (ratio <2).
Because the proportion of these amplified cells was estimated
<10% the ISH test resulted equivocal too. A reflex test was necessary. A different block of the tumor was subjected to immunohistochemical analysis (score 2+) and then to dual-probe CISH.
The molecular profile was the same of the first block with isolated (<10%) amplified cells in a overall non-amplified tumor.
The case was considered HER-negative and ineligible for targeted-therapy based on current data.
The final diagnosis was of a well differentiated invasive ductal
carcinoma (G1); pT1c; pN0 (sn) with favorable prognostic factors
(ER+; MIB1:10%).
Less than a year away the patient shows multiple bone metastases.
Assuming that the population could be present HER2 overexpression or gene amplification a bone biopsy was proposed but
the patient refused it.
Conclusions. The new HER2 testing guideline considers HER2negative a case with evidence of gene amplification <10%. It is
recommended to assign HER2 status according to overall mean
score; also reporting proportion of amplified cells. It is important to
underscore the evidence of HER2-heterogeneity and the coexisting
of different clones with a different biological potential. Although
the case will be considered HER2- negative and ineligible for targeted therapy based on the current data; however the patient would
benefit from subsequential re-testing of distant metastases.
Sala Donatello – 2° Piano – 14,30-15,30
Patologia mammaria - 2
Moderatori: F. Pietribiasi, A. Rizzo
EDX microanalysis of breast cancers: elemental
and phenotypic characterization and relationship
to radiodensity of microcalcifications
M. Scimeca1, N. Toschi2,4,5, C. Antonacci1, C.A. Pistolese3, E.
Giannini1, L. Scarano3, E. Bonanno1
1
Anatomic pathology Section, Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome; 2 Medical Physics Section,
Department of Biomedicine and Prevention, University of Rome “Tor
Vergata”, Rome; 3 Diagnostic Imaging Section, Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome,Italy;(4)
Department of Radiology, Athinoula A. Martinos Center for Biomedical
Imaging, Boston, MA, U.S.A.;(5) Harvard Medical School, Boston, MA,
U.S.A.
Background. While mammary microcalcifications have a crucial
role in breast cancer detection the processes that induce their
formation are unknown [1-3]. Also, recent studies have described
the occurrence of the epithelial–mesenchymal transition (EMT)
in breast cancer [4-5]. We investigated whether microcalcification features detectable through imaging (e.g. shape and density) contain information related to elemental composition and
morphological appearance. Moreover, to better define the phenomenon of microcalcifications, we hypothesized that epithelial
cells acquire mesenchymal characteristics and become capable of
producing breast microcalcifications.
Methods. The mammographic exam was performed by using
the digital mammograph (Senographe DS IS4-3 GE Medical
214
System). Images were used to extrapolate pixel-wise values of
density-related microcalcification features. Sixty breast sample
biopsies with microcalcifications underwent energy dispersive
X-ray microanalysis to examine the elemental composition of
the microcalcifications. Breast sample biopsies without microcalcifications were used as controls. The ultrastructural phenotype
of breast cells near to calcium deposits was also investigated to
verify EMT in relation to breast microcalcifications. The mesenchymal phenotype and tissue mineralization were studied by immunostaining for vimentin, BMP-2, 2-microglobulin, -catenin
and osteopontin (OPN).
Results. The complex formation of calcium hydroxyapatite
(HA) was significantly associated with malignant lesions
whereas calcium-oxalate (CO) is mainly reported in benign lesions. Notably, for the first time, we observed the presence of
magnesium-substituted hydroxyapatite (Mg-HAp), which was
frequently noted in breast cancer but never found in benign
lesions. The morphological aspect showed by microcalcifications made of Mg-HAp were classified as “casting type”,
whereas those made of HA were “powdery” and “crushed
stones” and those composed by CO were often “pop cornlike”. Pixel measured in correspondence to microcalcifications
revealed that Mg-HAp microcalcifications were significantly
more dense than those made of HA and CO. Morphological
studies demonstrated that epithelial cells with mesenchymal
characteristics were significantly increased in infiltrating
carcinomas with microcalcifications and in cells close to
microcalcifications with ultrastructural features typical of
osteoblasts. These data were strengthened by the rate of cells
expressing molecules typically involved during physiological
mineralization (i.e. BMP-2, OPN) that discriminated infiltrating carcinomas with microcalcifications from those without
microcalcifications.
Conclusions. We found significant differences in the elemental
composition of calcifications between benign and malignant lesions. Observations of cell phenotype led us to hypothesize that
under specific stimuli, mammary cells may acquire mesenchymal
characteristics transforming themselves into cells with an osteo-
Fig. 1. Breast cancer microcalcifications and mesenchymal phenotype.
blast-like phenotype, and are able to contribute to the production
of breast microcalcifications.
All together these preliminary data, if confirmed in a larger batch
of samples, could became a new challenge to improve diagnostic tools in the radiological and histological diagnosis of breast
carcinoma.
Breast carcinoma is frequently associated with microcalcifications. A) RX of a vacuum assisted breast biopsy specimens. The
picture showed the ROI identifying microcalcifications and relative pixel-wise values density-related (MED). B) H&E section
showed a microcalcification, inside a comedocarcinoma, made of
Mg-HAp. C) Transmission electron micrograph of a microcalcification made of Mg-HAp. D) Vimentin positive cells in a ductal
in situ comedocarcinoma in proximity of calcium deposits. E)
Immunogold labeling for vimentin: the ultrastructural analysis
demonstrated a diffuse positive reaction to vimentin antibody.
F) Double staining for pan-cytokeratin (brown stain) and vimentin (red cytoplasmic stain).The co-localization of both markers
(arrows) catch the transition epithelium-mesenchyma just as it
is occurring. G) B2-microglobulin signal in cells surrounding
calcium deposits.
References
1
Jemal A, SIiegel R, Ward E, Murray T, Xu J, Thun MJ: Cancer statistics, 2007. CA Cancer J Clin 2007, 57:43–66.
2
Ferranti C, Coopmans de Yoldi G, Biganzoli E, Bergonzi S, Mariani
L, Scaperrotta G, Marchesini M: Relationships between age, mammographic features and pathological tumour characteristics in nonpalpable breast cancer. Br J Radiol 2000, 73(871):698–705.
3
Gülsün M, Demirkazik FB, Ariyürek M: Evaluation of breast microcalcifications according to Breast Imaging Reporting and Data System
criteria and Le Gal’s classification. Eur J Radiol 2003, 47(3):227–231.
4
Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY,
Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak
K, Brisken C, Yang J, Weinberg RA: The epithelial-mesenchymal
transition generates cells with properties of stem cells. Cell 2008,
133(4):704–715.
5
Morel AP, Livre M, Thomas C, Hinkal G, Ansieau S, Puisieux A: Generation of breast cancer stem cells through epithelial-mesenchymal
transition. PLoS One 2008, 3(8):e2888.
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Comparison between invasive breast cancer with
extensive peritumoral vascular invasion and
inflammatory breast carcinoma.
A clinicopathologic study of 161 cases
S. Sina, S. Bonfante*, P. Parcesepe, A. Fioravanzo, A. Remo*,
E. Manfrin
Università degli Studi di Verona-Ospedale di Borgo Roma; * Anatomia Patologica Ospedale “Mater Salutis” Legnago, Verona
Objectives. Extensive peritumoral neoplastic lymphovascular
invasion (ePVI) is a marker of aggressiveness in invasive breast
carcinoma (BC).
Methods. We explored the impact of ePVI on different BC
subtypes. In a total of 2,116 BCs, 91 ePVI-BC, 70 inflammatory
breast carcinomas (IBCs) and 114 casual BCs as control group
(CG-BC) were recruited.
Results. Patients affected by ePVI-BC were younger, had larger
tumors, higher histologic grade, elevated Ki-67 score, Her2/neu
overexpressed and lymph node metastases compared with CG (P
< .001). Interestingly, only younger mean age at diagnosis differentiated patients with ePVI-BC from patients affected by IBC.
ePVI-BC showed a clinical outcome intermediate between the
prognoses of IBC and CG-BC.
Conclusions. Results suggest that ePVI-BC and IBC may share
some pathological processes providing a novel perspective on the
heterogeneity of BC. Epidemiologic data and molecular studies
on gene expression features are needed to rationally classify these
tumors into their identified subtypes.
p16 immunohistochemistry is helpful to increase
the diagnostic accuracy of low-grade and highgrade ovarian serous carcinomas
S. Sina, A. Fioravanzo, A. Remo*, E. Piazzola, P. Parcesepe, E.
Manfrin
Università degli Studi di Verona-Ospedale di Borgo Roma; * Anatomia Patologica Ospedale “Mater Salutis” Legnago, Verona
Introduction. Considering the new knowledge on the pathogenesis of serous carcinomas and the different therapeutic implications of a correct interpretation of the grading, we paid attention
to the evaluation of the predictive value of the immunohistochemical patterns of p53 and p16 in the discrimination between
low-grade serous carcinomas (LGSC) and high-grade serous
carcinomas (HGSC).
Materials and methods. All cases of ovarian tumor disease have
been extracted from the computer files of the Surgical Pathology
Unit of the G.B. Rossi Hospital, University of Verona, from 2003 to
the first quarter of 2013. The folder information of each patient was
built with the histological data, the results of immunohistochemistry, the medical and surgical treatments and the follow-up. Serous
ovarian tumors were selected and slides reviewed in order to update
the former diagnosis to the new diagnostic criteria as stated by the
fourth edition of the WHO Classification of Tumors of Female
Reproductive Organs. We paid specific attention to the cytological
grading and the mitotic activity to distinguish LGSC from HGSC.
The interpretation of the different p53 patterns was based on the
presence or absence of reaction and on the intensity of the nuclear
staining. According to the literature, p53 immunohistochemistry
was scored as: “absent” (less than 10% of tumor cell nuclei stain.
ing for p53), “wild-type” (focal and weak p53 expression in a
percentage between 10% and 60% of neoplastic cells) and “overexpressed” (more than 60% of tumor cell nuclei staining for p53).
Complete absence of p53 indicates a null mutation, whereas p53
overexpression is indicative of a missense mutation. These two
extreme staining patterns are combined as aberrant expression
of p53 (1). p16 immunohistochemistry was scored as “patchy”
(less than 90% of tumor cell nuclei staining for p16) and “block”
(more than 90% of tumor cells are positive for p16). A pattern of
p16 block stands for a pathologic protein contrary to a pattern of
p16 patchy (2).
Results. In our record of serous tumors there were 26 HGSC
and 4 LGSC. We observe that among the LGSC the combination
of wild-type p53 and patchy p16 is the most common of the six
combinations we can obtain crossing the three different patterns
of p53 and the two of p16 (Tab. I).
Invasive pleomorphic lobular carcinoma with HER2
overexpression
L. Sollima1, A. Chiominto2, A. Marinucci1, L. Melchiorri1, A. Dal
Mas2, P. Leocata1
Anatomia Patologica, Dipartimento di Medicina clinica, Sanità pubblica, Scienze della Vita e dell’Ambiente, Università degli Studi dell’Aquila,
L’Aquila, Italia; 2 U.O.C. Anatomia Patologica Ospedale Civile “San Salvatore”, L’Aquila, Italia
1
Introduction. Invasive pleomorphic lobular carcinoma (IPLC) has
been identified as a distinct histological entity from classic infiltrating lobular carcinoma (ILC) and is reported to be associated with
a more aggressive clinical behavior than classic ILC . It has been
reported to account for <1% of all epithelial malignancies in the
breast. Unlike the classic variant, the tumor cells of the pleomorphic
variant of ILC are larger, have abundant eosinophilic cytoplasm
with large pleomorphic hyperchromatic nuclei that show prominent
nucleoli which can be irregular in some cases, thereby making it difficult to differentiate from a high grade invasive ductal carcinoma.
Duct formation is distinctly absent. Its morphological features have
been well described by different authors but there are conflicting
data in literature concerning the presence of estrogen receptor (ER),
progesterone receptor (PR) and Her2/neu receptor.
Materials and methods. We report a case of invasive pleomorphic lobular carcinoma (IPLC) which strongly expresses estrogen receptor, progesterone receptor and HER-2. A 65-year-old
woman, without family history of breast, ovarian, uterine or colon
cancer, presented asymmetric density on mammogram. A vague
palpable mass was identified in the upper outer quadrant of the
right breast. No local adenopathy was appreciated. In February
2014 an ultrasound guided biopsy of the right breast mass was
performed which showed an invasive mammary carcinoma ER
and PR + in 90% of neoplastic cells, Ki 67 + in 5% of neoplastic
cells and HER-2 (score 3+) in 95% of neoplastic cells. One month
later the patient underwent right breast quadrantectomy with axillary sentinel lymph node biopsy.
Results. On gross exam a solid lesion, measuring 0,8 cm in maximal dimension, was identified. On microscopic exam the lesion
was an invasive carcinoma with highly atypical tumor cells infiltrating in a trabecular and cord like pattern. The tumor cells were
large with relatively abundant eosinophilic cytoplasm, enlarged
Tab. I. Immunohistochemical results Diagnosis
HGSC
LGSC
total
p53 overexpr. p53 overexpr.
p16 patchy
p16 block
0
5
0
0
0
5
p53 absent
p16 patchy
5
1
6
p53 absent
p16 block
10
0
10
p53 wild-type p53 wild-type
p16 patchy
p16 block
2
4
3
0
5
4
total
26
4
30
216
Fig. 1. Microscopic findings of IPLC.
Fig. 2. E-Cadherin.
Fig. 3. Estrogen recepror + 95%, Progesterone Receptor + 95%, Ki67
+ 10%, Herceptest 3+ 90%.
verse relationship between HER-2 and estrogen and progesterone
receptors using HER-2 testing and correlated HER-2 status with
histologic features in 3655 unselected invasive breast carcinomas.
When HER-2 status was analyzed according to the histologic
features of tumors, they found that HER-2 positivity virtually
was limited to invasive breast carcinoma of the ductal type,
while HER-2 positivity was observed only in the 0,8% of lobular
carcinomas. Among these, only the pleomorfic type was HER2 +
(score 3+) confirmed with FISH, like the present case. It would be
interesting to note if cytogenetics in such a case is different from
the more common hormone positive variants of PLC.
References
Yu J, Dabbs DJ, Shuai Y, Niemeier LA, Bhargava R. Classical-type
invasive lobular carcinoma with HER2 overexpression: clinical,
histologic, and hormone receptor characteristics. Am J Clin Pathol.
2011 Jul;136(1):88-97.
Lal P, Tan LK, Chen B. Correlation of HER-2 status with estrogen and
progesterone receptors and histologic features in 3,655 invasive
breast carcinomas. Am J Clin Pathol. 2005 Apr;123(4):541-6.
Butler D, Rosa M. Pleomorphic lobular carcinoma of the breast: a morphologically and clinically distinct variant of lobular carcinoma. Arch
Pathol Lab Med. 2013 Nov;137(11):1688-92.
Frolik D, Caduff R, Varga Z. Pleomorphic lobular carcinoma of the breast:
its cell kinetics, expression of oncogenes and tumour suppressor genes
compared with invasive ductal carcinomas and classical infiltrating
lobular carcinomas. Histopathology. 2001 Nov;39(5):503-13.
Manucha V, Khilko N, Reilly K, Zhang X. Invasive pleomorphic lobular
carcinoma, negative for ER, PR and Her/2neu--a case report. Int J
Clin Exp Pathol. 2011 Jan 30;4(2):200-5.
Breast pathology in ancient human remains.
An approach to mummified mammary gland by
modern investigation methods
L. Ventura1, R. Gaeta2, V. Giuffra2, C. Mercurio1, M.L. Pistoia3,
A. Ciccozzi3, M. Castagna4, G. Fornaciari2
U. O. C. di Anatomia Patologica, Ospedale San Salvatore, L’Aquila;
Divisione di Paleopatologia, Dipartimento di Ricerca Traslazionale e
delle Nuove Tecnologie in Medicina e Chirurgia, Università, Pisa; 3 U. O.
S. Dipartimentale di Diagnostica Senologica, Dipartimento di Diagnostica per Immagini, Ospedale San Salvatore, L’Aquila; 4 Dipartimento di
Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia,
Università, Pisa
1
2
nuclei with vesicular chromatin and prominent nucleoli. Mitotic
figures was 4 per 10 high power field. No lymphovascular invasion and metastasis of the sentinel node were seen. One block of
the tumor was subjected to immunohistochemical analysis. The
invasive tumor was E-cadherin negative and was diagnosed as
invasive pleomorfic lobular carcinoma (IPLC) The tumor cells
(95%) were positive for both estrogen recptor and progesterone
recptor, like the classic variant of lobular carcinoma. The IPLC
was strongly positive for Her2/neu (score 3+), unlike the most of
lobular carcinomas.
Conclusions. As mentioned above, conflicting data exist in literature concerning the presence of ERs and PRs. PLCIS is known
to be immunohistochemically positive for hormone receptors in
addition to a high proliferation rate and absence of HER2/neu
over expression/ amplification. In 2005 Lal et al studied the in-
Background. After death, fat hydrolysis often turns out in mammary glands collapse. The rate and degree of breast size reduction
is quite variable, resulting in a wide range of prominence in mummified female bodies. Sometimes breast appears unidentifiable,
whereas in other circumstances it remains easily recognizable,
permitting sex identification in about half of spontaneously mummified human remains.
As a matter of fact, breast is often observed in mummies but it is
investigated only occasionally, an issue in part explained by the
need for conservative purposes. To the best of our knowledge, paleopathological studies of the breast are extremely rare with only
one case of lactational changes and one case of fibroadenoma
described in literature. Histological investigation of mummified
mammary glands demonstrates that the epithelial component
does not survive postmortem alterations, differently from fibrous
tissue. For this reason, a carcinoma should be easily identifiable
in a mummified breast because of cancer-induced fibrosis, but
breast cancer has never been reported in mummies to date.
Aim of the present study is to gather the information obtained
applying modern investigation methods to the mummified breasts
of three women dating back to 15th, 16th, and 20th century, in
order to find out features of pathological significance and validate
the approach.
Methods. The first case is represented by a partially (50%) mummified female, dating back to XX century, found in the San Michele Arcangelo church in Sermoneta (Latium, central Italy). The
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COMUNICAZIONI ORALI
subject was 40-50 years of age at death and her body measured
148 cm, showing good preservation of superficial structures of
the inferior and left body regions, including her left breast (Fig.
1). This was entirely submitted to radiology, using a Senographe
Essential digital mammograph (GE Medical Systems). Samples
from the outer quadrants were rehydrated with Sandison solution,
and routinely processed to obtain histological sections stained
with hematoxylin-eosin, Masson’s trichrome, von Kossa, and red
alizarin stains.
The second case was that of Margherita Sozzini (dead in 1511).
In 1459 she had married Salimbene Capacci who became Rector
of the Hospital of Santa Maria della Scala in Siena (Tuscany, central Italy) 20 years later. She was entrusted the responsibility and
direction of the Institution’s female ward including management
of the foundlings, maintaining her functions after the death of her
husband. Her well-preserved, artificial mummy was found in the
Santissima Annunziata church of Santa Maria della Scala Hospital. The imaging studies were performed using a CT scanner GE
Sytec, Kv 120, MA 100, with slice thickness 10 mm, and field of
view 2 cm. The breasts, well-preserved with a recognizable nipple, were laterally descended (Fig. 2). Fragments of the left breast
were rehydrated using Sandison solution, and routinely processed
to obtain histological sections stained with hematoxylin-eosin.
The third case regarded Mary of Aragon (1503-1568), marquise
of Vasto, a noblewoman of the Italian Renaissance. Famed for her
beauty, Maria belonged to the intellectual and religious circles of
Ischia, whose leading figures were Giulia Gonzaga and Victoria
Colonna, a friend of Michelangelo. Her artificial mummy, part of
the famous series of the Aragoneses preserved in the Basilica of San
Domenico Maggiore in Naples, was studied and resulted affected
by obesity, syphilis, viral hepatitis, and a genital condyloma. Her
breasts, dried and collapsed into the chest, appeared to be well preserved. During autopsy, mammary glands were entirely sampled by
a “skin-sparing” technique and revealed remarkable dimensions: 12
x 11 x 1 cm for the left breast, 16 x 10 x 1 cm for the right one (Fig.
3). Both specimens were submitted to mammography, samples were
rehydrated with Sandison solution and routinely processed to obtain
histological sections stained with hematoxylin-eosin, Masson’s trichrome, periodic acid-Schiff, Van Gieson, and Von Kossa methods.
Results. Mammography performed in the mummy from Sermoneta showed diffuse microcalcifications of the outer breast
quadrants, quite similar to those observed in modern patients
affected by epithelial proliferative lesions (Fig. 4). Histology displayed collagen fibers diffusely colonized by fungal spores, and
scattered roundish structures with only focal calcium deposits,
von Kossa-and red alizarin-positive. Such findings could not be
certainly attributed to breast pathology or post-morten changes.
The mummy of Margherita Sozzini did not undergo mammography, and CT scout scans did not allow to detect significant alterations of the breast. Histology showed fibrous tissue with empty,
round spaces, possibly related to fat tissue. Given the age at death
of the subject, the picture can be due to fibrous changes.
In the mummy of Mary of Aragon mammography revealed
multiple, bilateral microcalcifications, mostly on the right breast
(Fig. 5). Microscopic examination highlighted a mixture of dense
fibrous tissue and empty spaces possibly related to adipose tissue.
Many fibrous walls of cysts were suggestive of breast fibrocystic changes. Single and grouped microcalcifications were also
evident, appearing as rounded basophilic granules, with a sligthly
eosinophil center (Fig. 6). In the upper outer quadrant of the
right breast, a roundish formation (0.8 cm in largest diameter) of
fibrous-hyaline connective suggested a fibroadenoma.
Fig. 1.
Fig. 3.
Fig. 2.
Fig. 4.
218
Fig. 5.
Conclusions. The breast has not been widely studied in mummified human remains, though it can be recognized in many ancient
bodies. To the best of our knowledge, this is the first series of
mummified mammary glands investigated by using modern technologies. Following this approach, we were able to find different
alterations. Fibrocystic change appeared a possible diagnosis in
two out of three subjects, whereas hyaline fibroadenoma could be
suspected in one case. No firm conclusion can be drawn about the
significance of microcalcifications, as they may be the result of a
pathologic process or related to post-mortal/taphonomic phenomena. Additional data on larger mummy series, as well as the resort
to compositional investigation methods (e. g., energy dispersive
X-ray analysis), may be of help in understanding morphological
details of ancient breasts.
Nevertheless, the investigation methods of modern senology
may allow an effective approach to ancient breast pathology,
disclosing good morphological details in mummified specimens.
Furthermore, the alterations induced by neoplastic diseases (fibrosis) should be easily identified in ancient breast by following
this approach.
References
Aufderheide AC. The scientific study of mummies. Cambridge University
Press, 2003; 492-493.
Zimmerman MR. Mummies of the arctic regions. In: Spindler K, Wilfing
H, Rastbichler-Zissernig E, zur Nedden D, Nothdurfter H (eds). Human mummies. Springer, Wien, 1996; 83-92.
Zimmerman MR, Aufderheide AC. The frozen family of Utqiagvik: the
autopsy findings. Arctic Anthrop 1984; 21: 53-63.
Zimmerman MR. Paleopathology in alaskan mummies. Amer Sci 1985;
73: 20-25.
Cockburn A, Cockburn E, Reyman TA. Mummies, disease and ancient
cultures. Cambridge University Press, 1980; 93-95.
Ventura L, Ciccozzi A, Pistoia L. Diagnostica “senologica” nei resti
umani antichi. Indagine mammografica su ghiandola mammaria
mummificata della serie di San Michele Arcangelo. In: Di Falco A (ed)
Chiesa di San Michele Arcangelo. Storia del restauro (2006-2013),
2013; 5: 135-136.
Ventura L, Mercurio C, Miranda G, Papola F. Antropologia e paleopatologia dei resti umani mummificati dell’Oratorio dei Battenti. In: Di
Falco A (ed), Chiesa di San Michele Arcangelo. Storia del restauro
(20062013), 2013; 5: 128-137.
Giuffra V, Naccarato AG, Caramella D, Fornaciari A, Marvelli S, Fornaciari G. The rector of the hospital and his wife: two artificial mummies
of the late 15th century from Siena (central Italy). In: Atoche P, Rodriguez C, Ramirez MA (eds), Mummies and science. World mummies
Research, 2008; 529-536.
Barraco D, Ciranni R, Caramella D, Fornaciari G. A case of mastopathy
in a 16th century italian mummy. XIIIth European Meeting of the Paleopathology Association, Chieti, 18-23, 2000. J Paleopathol 1999; 11:
Fig. 6.
Systems biology analysis reveals NFAT5 as novel
biomarker and master regulator of inflammatory
breast cancer invasiveness
A. Remo, P. Parcesepe, S. Sina, M. Pancione, M. Ceccarelli, E.
Manfrin
ULSS 21 Ospedale di Legnago Verona Dipartimento di Anatomia Patologica
Introduction. Inflammatory Breast Cancer (IBC) is the most
aggressive variant of breast cancer, however, there are no specific gene signatures and reliable biomarkers to improve IBC
classification into molecularly distinct subtype. We applied a
network-based strategy to gain insight into master regulators
(MRs) linked to IBC pathogenesis.
Methods. In-silico modeling and algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) on IBC gene
expression data (n=197) were employed. The DAVID and IPA
enrichment analyses of KEGG and canonical pathways, were
selected to identify novel Master Regulators (MRs) strongly connected with IBC disease condition. The expression pattern of the
top candidate genes were evaluated in two independent cohorts
of IBCs (n=41) and non-IBC (n=82) tissue microarrays by using
immunohistochemistry. The staining pattern of non-neoplastic
mammary epithelial cells, was used as a normal control.
Results. Using in-silico modeling of network-based strategy,
we identified three top enriched MRs (NFAT5, CTNNB1 and
MGA) strongly linked to IBC subtype. We show that NFAT5
expression is significantly higher in IBC than non-IBC subgroup
(70% vs 20%; p=0.0022). Accordingly, the majority of NFAT5positive IBC subtype displayed increased nuclear expression in
comparison with non-IBC specimens (89% vs 12% p<0.0001). In
IBC subtype, aberrant activation of NFAT5 can occur independently of WNT/ -catenin signaling suggesting that a substantial
portion of biological effects, is mediated by NFAT5 pathway.
Concomitant inactivation of NFAT5 or -catenin signaling was
strongly linked to non-IBC accounting for the best prognosis of
this subtype. Interestingly, MGA and NFAT5-regulated genes,
were significantly increased in IBC compared to non-IBC, (30%
vs 7%; 23% vs 0%; P<0.0001) reinforcing the central role of
NFAT5 in IBC pathogenesis.
Conclusions. We identified a novel transcription factor
NFAT5 that potentially might be developed into routine clinical practice as a putative biomarker of IBC subtype.
We provide evidence that NFAT-signaling pathway activation
could help to identify aggressive form of BC and potentially be a
guide to assignment of subtype-specific therapeutic agents.
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Sala Caravaggio – 2° Piano – 08,30-09,30
Patologia testa collo - 2
Moderatori: C. Giardina – M. Santucci
Cadherins alterations in fibro-osseous lesions of
the jaws. A tissue microarray study: a preliminary
report
G. Pannone1, A. Santoro1,2, M. Mattoni1, B. Pantaleo1
Department of Clinical and Experimental Medicine – Institute of Pathological Anatomy Section of Oral Pathology - University of Foggia- Italy;
2
Department of Laboratory and Services, Institute of Histopathology and
Diagnostic Cytopathology Fondazione di Ricerca e Cura ‘Giovanni Paolo
II’-UCSC, Campobasso, Italy
1
Introduction. Fibrous Dysplasia (FD), caused by GNAS mutation, and Hyperparathyroidism-Jaw tumor syndrome (HPT-JT),
due to HRPT2/CDC73 mutation are two disease belonging to the
group of Benign fibro-osseous lesions (BFOLs).
Despite the causal mutations have been already demonstrated, the
exact molecular mechanisms by which the gene mutations transfer pathological signals in bone precursor cells and differentiated
cells are not well understood.
Methods. Tissue Microarray (TMA) based immunohistochemical expression of cadherins (E-cadherin, N-cadherin, P-cadherin,
OB-cadherin) has been analyzed in formalin fixed, paraffin embedded samples from 9 patients, respectively affected by Fibrous
Dysplasia (n.8) and HPT-JT syndrome (n.1), and in 7 controls,
represented by reactive fibromixoid tissue, reactive mature fibrous tissue, normal bone with haematopoietic cells and remodelling bone surrounding developmental odontogenic cyst.
Results. We detected that loss of expression of OB-Cadherin in
osteoblast lineage in FD was accompanied by up-regulation of Ncadherin and P-cadherin, while E-cadherin showed a minor role
in these pathological processes.
Discussion. This study confirms the complete absence of Ecadherin and OB-cadherin but, on the other hand, shows that the
OB-cadherin loss is vicariate by the considerable increase of Ncadherin expression and by the expression of P-cadherin.
Conclusions. In conclusion, we retain that the evaluation of histological characteristics, together with a complete histochemical
and immunohistochemical analysis of key-molecules involved in
osteoblast differentiation, is essential for the diagnosis, classification as well as the appropriate clinical therapeutical
management of fibrous-osseous lesions.
A case of ectopic thymus within the parathyroid
gland
E. Guadagno, G. Pettinato,V. Varone
Department of Advanced Biomedical Sciences-Pathology Section. University of Naples FedericoII, Italy
Introduction. We describe the case of a 65 year old female who
underwent thyroidectomy for a papillary carcinoma. During the
surgery, a small nodule in the right bed of the gland was found
and its macroscopic aspect was that of a lipoma, according to the
surgeon.
Materials and methods. On gross examination, the thyroid gland
weighed 33 g and was composed of a right lobe measuring 5x3x1
cm, an istmus 1.5x1 cm and a left lobe 5.5x4x2 cm. The cut section revealed, in the left lobe, the presence of a white nodule that
measured 1.3 cm in maximum diameter. The small nodule of the
right bed of the thyroid measured 3.5x1.8 cm and its cut section
appeared lobulated and yellowish.
Results. Microscopically, the white nodule was a papillary carcinoma, as it was expected, but the yellowish nodule was composed
of rests of ectopic thymus with wide adipose involution, within the
parathyroid gland intimately admixed in a well encapsulated mass.
Conclusions. Thymic tissue can be found in ectopic locations,
due to a failure of the organ to migrate to its final destination
during embryonic development. Disturbances in the embryologic
development of the thymus can produce a series of congenital
anomalies that frequently represent an incidental finding in adult
life, but that can even give rise to thymomas.
In literature only one case 1 is described showing the same morphological aspects of our case. Therefore, it could represent a
specific pathological entity, that is still unknown.
References
1
van Hoeven KH,Brennan MF: ‘’Lipothymoadenoma of the parathyroid’’. Arch Pathol Lab Med: 1993; 117 (3): 312-4.
An immunohistochemical study of ERCC1
expression in Head and Neck squamous cell
carcinomas
D. Russo, G. Ilardi, M. Mascolo, S. Varricchio, M.L. Vecchione,
G. De Rosa, S. Staibano, F. Merolla
Dipartimento di Scienze Biomediche Avanzate, Università degli Studi di
Napoli “Federico II”, via S. Pansini, 5 80131 Napoli
Introduction. DNA damage, in the form of nucleotides modifications and DNA breaks, can result from endogenous and
exogenous sources: cellular metabolism, routine errors in DNA
replication and recombination as well as exposure to genotoxic
agents, such as ultraviolet light, oxidative stress and chemical
mutagens. Nucleotide Excision Repair (NER) removes a broad
spectrum of DNA damages, especially bulky DNA lesions (such
as cyclobutane pyrimidine dimers (CPDs)) induced by UV,
and those induced by chemical carcinogens like those found in
tobacco smoke (such as benzo(a)pyrene derivatives) and those
caused by common chemotherapeutics such as cisplatin. Tissues
of Head and Neck region are heavily exposed to carcinogens able
to induce neoplastic transformation via bulky DNA lesion formation, moreover multimodality treatment of squamous cell carcinoma of the head and neck (SCCHN) often involves radiotherapy
and cisplatin-based therapy. For these reasons NER pathway,
of which ERCC1 is a rate-limiting factor, is of great interest in
understanding Head and Neck cancers (HNC) onset and response
to therapy, since elevated activity of DNA repair mechanisms are
associated with cisplatin and possibly RT resistance.
Materials and methods. We have determined excision repair
cross-complementing group 1 (ERCC1) IHC expression in a
series of head and neck squamous cell carcinomas, treated with
surgery, from the archive of our pathology department and correlated it to several clinico-pathological features, in particular
with tumor aggressiveness and response to therapy. In order to
determine IHC epression of ERCC1 the following antibody was
used: XPF (H-300) sc28718 (Santa Cruz Biotechnology, 1:200)
Results and conclusion. ERCC1 overexpression, IHC determined, proved to directly correlate to treatment resistance and
consequently poor prognosis, envisaging a role for ERCC1 as a
negative predictive marker, useful to stratify patients risk in order
to better address them to a personalized therapeutic approach.
220
A rare case of coexistence of metastasis from
head and neck squamous cell carcinoma and
tuberculosis within a neck lymph node
M. Siano1, D. Caroppo1, M. Mascolo1, D. Russo1, G. Ilardi1, F.
Merolla1, L. Califano2, S. Staibano1
1
Dipartimento di Scienze Biomediche Avanzate, 2Dipartimento di Neuroscienze e Scienze riproduttive ed odontostomatologiche, Università degli
Studi di Napoli “Federico II”, Napoli
Introduction. Mycobacterium tuberculosis (MTB) infects one
third of the world’s population and is the second leading cause of
death from an infectious disease after HIV. Lymphadenitis is the
most frequent manifestation of extrapulmonary tuberculosis and
neck swelling is the commonest presentation. Although many
authors have described the association of tuberculosis and cancer,
coexistence of tuberculosis and metastatic carcinoma in lymph
nodes is a rare event. Here we present a case of laterocervical
TBC-lymphadenitis co-existing with a metastatic squamous cell
carcinoma of the head and neck region. A 86-year-old woman
noted, in September 2013, the appearance of a small lesion on the
left cheek mucosa. She was followed at the department of Oral
and Maxillofacial Surgery of the University of Naples “Federico
II”. The physical examination revealed the presence of an ulcerated lesion, 2 cm of diameter, lightly painful on palpation, with
hard/elastic consistence. The histological picture of an incisional
biopsy of the lesion, revealed a poorly differentiated, invasive
squamous cell carcinoma. The diagnosis was confirmed on the
whole excision of the lesion. Clinical examination, a few months
later, revealed the presence of a node swelling in the left laterocervical region of about 3 cm in diameter, firm, fix and painful on
palpation, which showed a rounded morphology at US, ranging
in size between 20 and 23 mm of maximum diameter. A wholebody PET showed tracer hyperaccumulation restricted to the
retromandibular and left parapharyngeal regions. A left laterocervical lymphadenectomy was then performed, and the sample was
sent to the institutional Pathology Unit.
Methods and materials. The surgical specimen was composed
of a salivary gland, twelve single lymph nodes and a single mass
of few fused lymph nodes (maximum diameter: 3 cm).
Results. At microscopic examination, the lymph-node mass
showed massive necrosis lined by granulomatous inflammation with palisading epithelioid-and Langhans’ giant histiocytes
(Figure 1), in close association with metastatic sheets from a
squamous cell carcinoma (Figure 2). The immunohistochemistry
clearly evidenced the tumor nests (pan-cytokeratin positive) and
the granulomatous reaction area (CD68 immunoreactive) (Figure
34). Basing on these results, the patient was carefully re-questioned about her previous pathological history, and an episode of
tuberculosis in the 60s finally emerged.
At present, the patient has been redirected to the department of
infectious diseases for further investigation.
Conclusion. An association between tuberculosis and cancer has
been frequently found in humans. Warthin reported two cases of
carcinoma and tuberculosis of mammary glands, in 1988 [1], and
Kaplan et al. examined the frequency of the coexistence between
different cancer types and tuberculosis in a retrospective study
[2]. Up to date, only a few cases of tuberculosis and metastatic
carcinoma in a single lymph node has been reported [3-4-5].
Here we describe an unexpected, rare case of node metastasis
from oral squamous cell carcinoma coexisting with an infective
granulomatous reaction due to mycobacterium tuberculosis.
Only few cases of coexisting squamous cell carcinoma metastasis and TBC-lymphadenitis, in the same lymph node, have been
described so far in the literature [6-7]; to our knowledge, our
case represents the first involving the oral cavity as the source
of the primary tumor. A case of OSCC with node metastasis
and TB infection has been reported by Wang at al, but in this
instance the two pathological entities were found in different
lymph nodes [8]. Cancer can be associated with a granulomatous response in the draining lymph node [9]; in particular,
squamous cell carcinoma can elicit this reaction, being tumor
keratins the reaction target [7]. Nevertheless, the possibility of
TBC-lymphadenitis should always be considered even in nonendemic areas, particularly in elderly oncology patients, due to
the cancer-induced immune suppression.
References
1
Warthin AS The coexistence of tuberculosis and carcinoma of the
mammary gland Am J Med Sci 1899, 118:25
2
Kaplan MH, Armstrong D and Rosen P Tuberculosis complicating
neoplastic disease: a review of 201 cases Cancer 1974, 33:850-858.
3
Pandey M, Abraham EK, K C, Rajan B. Tuberculosis and metastatic
carcinoma coexistence in axillary lymph node: A case report. World J
Surg Oncol. 2003 Apr 7;1(1):3.
4
Avninder SP, Saxena S. Infiltrating ductal carcinoma of the breast,
metastatic to axillary lymph nodes harboring primary tuberculous
lymphadenitis. Pathol Oncol Res. 2006;12(3):188-9.
5
Salemis NS, Razou A. Coexistence of breast cancer metastases and
tuberculosis in axillary lymph nodes--a rare association and review
of the literature. Southeast Asian J Trop Med Public Health. 2010
May;41(3):608-13.
6
Gheriani H, Hafidh M, Smyth D, O’Dwyer T. Coexistent cervical
tuberculosis and metastatic squamous cell carcinoma in a single
lymph node group: a diagnostic dilemma. Ear Nose Throat J. 2006
Jun;85(6):397-9.
7
Barwad A, Gowda KK, Dey P. Co-existent of tuberculosis and
squamous cell carcinoma in a lymph node diagnosed by fine needle
aspiration cytology. Cytopathology. 2012 Aug;23(4):276-7. doi:
10.1111/j.1365-2303.2011.00878.x. Epub 2011 May 25.
8
Wang WC, Chen JY, Chen YK, Lin LM. Tuberculosis of the head
and neck: a review of 20 cases. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod. 2009
9
Alka Bhatia, Yashwant Kumar, Anjali Solanki Kathpalia Granulomatous inflammation in lymph nodes draining cancer: A coincidence or a
significant association International Journal of Medicine and Medical
Sciences Vol.1(2) pp. 013-016, February, 2009.
Subglotic MALT-lymphoma of the larynx:
an unusual presentation of chronic cough
S. Sina, E. Baritono*, S. Bonfante*, A. Fioravanzo, P. Parcesepe,
E. Manfrin, A. Remo*
Università degli Studi di Verona-Ospedale di Borgo Roma;
Patologica Ospedale “Mater Salutis” Legnago, Verona
*
Anatomia
Background. Extranodal non-Hodgkin lymphomas limited to
the larynx are rare, accounting for less than 1% of all laryngeal
neoplasms. The most common site of development of primary
laryngeal lymphomas is the supraglottic region. In most cases,
the presenting symptoms are hoarseness, dysphagia, dyspnea, and
cervical lymphadenopathy. They consist mainly of non-Hodgkin
lymphoma, especially of diffuse large B-cell lymphoma and
mucosa-associated lymphoid tissue.
Case presentation. We report a case of a primary Extranodal
marginal zone of mucosa associated lymphoid tissue (“Malt
Lymphoma”) of the larynx in a 73-year-old non-smoker woman,
presented as chronic cough, unresponsiveness to oral corticosteroid. We present a detailed report of his clinical, histopathological
data as well as treatment options.
Conclusion. In patients with chronic cough, uncommon causes
should be considered when cough persists after evaluation for
common causes. If cough persists after the consideration of the
most common causes, CT scan and a bronchoscopic evaluation
are fundamental for the diagnosis of tumors of the upper and
lower respiratory tract.
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Isolated Langerhans cell histiocytosis of thyroid
gland associated with papillary thyroid carcinoma
and lymphocytic thyroiditis. Report of a case with
complete clinical, cytological, histological and
immunophenotipical features
V. Varone1, G. Ciancia1, C. Cipullo1, R. Maffucci1, A. Vetrani2,
G. Pettinato1, I. Cozzolino2
Departments of Advanced Biomedical Sciences, Histology Service 1 and
Public Health, Cytology Service 2, “Federico II” University of Naples (IT)
Introduction. Langerhans cell histiocytosis (LCH) is a rare disorder characterized by infiltration of the involved tissues by large
numbers of Langerhans cells. LCH can affect any organ of the
body, even particular sites as thyroid, above all in patients with
multifocal disease. Isolated thyroid involvement is extremely
rare, as well as the association with other thyroid disease. We
present a case of LCH of the thyroid gland associated with
papillary thyroid carcinoma (PTC) and lymphocytic thyroiditis,
reporting with the complete clinical, cytological, histological and
immunophenotipical features.
Methods. A 24-yr-old woman in autoimmune thyroiditis follow
up presented at ultrasound evaluation a hypoechoic nodule measuring 1.4 X 1 cm, involving the right lobe; the nodule was solid
with calcifications.
US-FNA of the thyroid, using a 23-gauge needle, was performed
in order to classify the lesion. The May-Grunwald-Giemsa
stained smears showed a highly cellularity represented by two
distinct cell populations. The first, which was predominant, consisted of loosely cohesive clusters and dissociated single cells
characterized by aboundant pale and eosinophilic cytoplasm
and irregular, often incised, nuclei. This population was interpreted as dendritic-histiocytic type. Conversely, the second cell
population, follicular type, consisted of few large monolayer or
three-dimensional sheets of cuboidal cells, with mild to moderate
nuclear abnormalities, some with atypical grooved and elongated
nuclei. Furthermore, the cells were mixed with a background
of various inflammatory cells (mainly lymphocytes and neutrophils) with numerous eosinophils. Calcifications were observed
too. The atypia observed in follicular population suggested only
a PTC diagnosis, as not all the required cytological criteria for
such a diagnosis were present. According to the SIAPEC consensus, the final diagnosis was suspicious for malignancy (Tir4)
with dendritic-histiocytic type population associated. The patient
underwent total thyroidectomy. Grossly, a firm intralobular, 1.2
cm diameter nodule was found in the right lobe. Histologically,
we observed the coexistence of a classical PTC, with areas of
squamous and solid differentiation, and an ill-defined area (4
mm) consisted of a dissociated cell population characterized by
large eosinophilic and finely vacuolated cytoplasm, bean-shaped
and folded nuclei; these cells, diagnosed presumptively as Langerhans cell, showed, as expected, strong immunoistochemical
positive reactivity with S-100 protein and CD1a. The Langerhans
cell were mixed with a background of neutrophils and numerous
eosinophils. The PTC showed immunoistochemical positive reactivity with CK19, TTF-1, Thyroglobulin and no reactivity with
CD56. Moreover, another area (5 mm) of LCH was observed in
the left lobe. The remaing gland was characterized by a diffuse
lymphocytic thyroiditis with well-developed lymphoid follicoles,
often with germinal centre and oxyphil metaplasia of glandular
epithelium (“Hashimoto-like” thyroiditis).
Results. On the basis of the histological and immunohistochemical features, a diagnosis of PTC associated with LCH and lymphocytic thyroiditis (“Hashimoto-like”) was made, confirming
the cytological suspicion. At CT control were not lymph node
swelling, hepatosplenomegaly and bone lesions and at clinical
examination were not found skin lesions.
Conclusion. LCH thyroid involvement is rare and the association
with PTC remains not frequent and unclear, as there have only
been a few reported cases and, only in a recent study, an increased
presence of CD1a-positive dendritic cells in PTC was showed.
However, a correct diagnosis is possible, both on cytological
and histological sample, based on some salient morphological
and phenotypical features. Surgical removal is the gold standard
treatment for isolated LCH; associated PTC may require further
treatment, such as iodine therapy.
References
IARC, 2004.
Patten DK, Wani Z, Tolley N. Solitary langerhans histiocytosis of the
thyroid gland: a case report and literature review. Head Neck Pathol.
2012;6(2):279-89.
Mucinous variant of follicular adenoma of the
thyroid gland: a rare and still unclear neoplasm
V. Varone, G. Ciancia, R. Maffucci, V. Papacciuoli, G. Pettinato
Departments of Advanced Biomedical Sciences, Histology Service, “Federico II” University of Naples (IT).
Introduction. Follicular adenoma with extracellular mucin deposition is a rare and still unclear entity, since several mechanism
have been proposed to explain the etiology of the mucin-producing thyroid neoplasms. Therefore, in these neoplasms, the large
amount of mucin presents an intracellular location, often with a
typical signet-ring cell appearance. We report a rare case of follicular neoplasm of the thyroid gland with extensive extracellular
mucin deposition, diagnosed as a mucinous variant of follicular
adenoma (sec WHO) and discuss data of most recent literature.
Methods. A 25-yr-old woman presented with a slowly growing
mass of the right lobe of the thyroid gland; the patient had no complaints and the hormonal evaluation revealed normal thyroid hormone profile with a highly elevated thyroglobulin levels; normal
calcitonin level. Ultrasound showed a isoechoic 21-mm-diameter
nodule at the lower pole of the right lobe, near the isthmus; the
nodule was solid, with cystic areas and peri- and intranodular
vascularisation.The patient underwent total thyroidectomy. The
resected specimen weighed 35 grams and measured 10x3.5x2 cm;
a well-circumscribed, round shaped nodule measuring 2.0x1.9
was observed at the lower pole of the right lobe near isthmus. On
the cut surface, the nodule was dark-brown and translucent. Histologically, the tumor showed mainly a microcystic and reticular
growth pattern, consisting of anastomosing trabeculae and cords
of neoplastic cells floating in a abundant extracellular basophilic
mucin pools. Follicular structure and microfollicles containing
colloid or mucinous material were rarely observed. The tumor
cells were cuboidal or polygonal and showed a moderate to
aboundant eosinophilic cytoplasm, vescicular nuclei with inconspicuous nucleoli; intracytoplasmatic vacuole with compression
and displacement of nuclei (“signet-ring cell” appearance) was
occasionaly found. No nuclear irregularities neither pseudoinclusion were found in the present case. The neoplastic cells showed
immunoistochemical positive reactivity with thyroglobulin and
thyroid transcription factor (TTF-1) whereas they were negative
for calcitonin. The tumor was circumscribed by a thick fibrous
capsule; neither capsular or vascular invasion was observed.
Results. On the basis of the histological and immunohistochemical features, a diagnosis of mucinous variant of follicular adenoma of the thyroid gland was made.
Conclusion. Mucin deposition can be rarely observed in primary
thyroid tumors, including follicular neoplasms, medullary carcinoma, poorly differentiated carcinomas, papillary carcinomas,
mucoepidermoid and primary mucinous carcinomas. Therefore,
the differential diagnosis should consider all these entities as
well as a possible metastasis to the thyroid from other primary
sites (particularly the salivary gland). The only two case reported
in literature as follicular adenoma with extensive extracellular
222
mucin deposition were characterized by atypical nuclear changes
(frequent nuclear inclusions) and mucin spillage into the capsule.
According to authors these features do not fully belong to the
mucinous variant of follicular adenoma reported in the WHO
classification. However, the biological behaviour, clinical course
and treatment of this neoplasm should be the same of follicular
neoplasm, determined on the basis of some critical histological
features (capsular and vascular invasion) rather than the presence
of mucin deposition.
References
IARC, 2004.
Kim NR, Cho HY, Pi–a-Oviedo S, De La Roza G, Lee YD, Ro JY. Follicular adenoma with extensive extracellular mucin deposition: report
on two cases. Clin Med Insights Case Rep. 2012;5:155-62.
Melanoma of the conjunctiva Nestin and Ghrelin
positive. An insight on new prognostic markers
P. Viola1, M. Pugliese2, C.E. Gallenga3, G. Lattanzio1, A. Marchetti1
1
Dept of Aging Medicine, G D’Annunzio University Chieti, Italy; 2 Dept.
Medical Oral Sciences and Biotechnology, Section of Otolaryngology, G
D’Annunzio University, Chieti Pescara, Italy; 3 Dept Medical Surgical
Communication/Behaviour, Section Ophthalmology, University of Ferrara, Italy
Introduction. Malignant melanoma of the conjunctiva is a rare
tumor that carries a significant mortality and local recurrence. It
can arise de novo or from clinically distinct precursors, including primary acquired melanosis (PAM) or nevi, those originating de novo are considered at higher risk. It’s very important to
recognize the precursor malignant lesions for an early diagnosis
and before spreading. Histological classification of conjunctival
melanomas is lacking and therefore skin melanoma classification is adopted. Treatment remains unclear because validated
protocols do not exist. However it requests a more aggressive
surgical approach in the advanced cases and a combination of
more conservative surgery with radiotherapy or cryotherapy in
the other cases, as well as patient annual assessments. The mutational analysis for BRAF could be useful in promoting biological
therapy if metastatic disease developed. There is evidence in
literature saying that the presence of nestin in the specimen is a
factor of poor prognosis and Ghrelin could be related with the
rapid growing of this tumor and its duplication time. We reported
a ninety-four year old woman referred for a very large epibulbar
conjunctival mass located at the left temporal limbus/fornix area
in which both markers were identified.
Material and metodhs. A ninety-four year old woman presented
with a seven-eight months history of a left epibulbar pigmented
mass at the temporal limbus. The lesion gradually increased in
size occupying the entire palpebral rim preventing the patient
to see and masking the bulbous. Previous medical history was
unremarkable; comorbidities at the time of presentation were
hypertension on treatment, gallbladder stones and a mild renal
failure. On clinical examination a round-shaped, deep brown
mass of hard-elastic consistency was noted on the globe of the
eye. At neck palpation two hard-elastic nodes in sub-mandibular
region were appreciated. Ultrasonography of the neck revealed
many reactive-inflammatory nodes with a maximum diameter
of 1,3 cm located in sub-mandibular and later-cervical regions,
without inner echoes of discontinuity or lower reflectivity nests.
Ultrasonography of the ocular bulbous (B-scan) revealed that the
neoplastic mass was adherent but not infiltrating the lateral rectus
muscle and the sclera; the muscle cone internal structures resulted
not affected. Fine needle aspiration cytology (FNAC) from the
mass was performed, which showed the presence of cells with
intracytoplasmatic pigment indicative of melanoma. Blood tests,
abdominal ultrasonography and thorax and bones Rx for searching any distant spread were negative. Because of the mass dimensions, appearing larger than two months before, the tumor was removed by using a wide surgical resection technique: exenteration
of the left eye. Histological examination confirmed the diagnosis
of melanoma and after the surgery, the patient underwent monitoring protocols, and total body follow up with unenhanced CT
scan, showed no evidence of metastatic disease after 36 months.
From a treatment point of view, BRAF mutations were examined
and for research purposes immunohistochemical stains for nestin
and ghrelin were performed.
Results. Macroscopically the left eye measured 5x4.5x2.4 cm
including conjunctiva, extra ocular muscle insertions, optic nerve
and ophthalmic artery. Upon sectioning a dark round lesion measuring 2.8x2.3 cm was noted. It involved the conjunctiva dislocating the iris, but it appeared completely excised. Histologically the
lesion has been reported has malignant melanoma composed of
atypical melanocytes involving more than one conjunctival quadrant but surgical margins were free of tumor (pt2c). Molecular
analysis for BRAF (codon 15) showed no evidence of mutation
to address the therapy. Immunohistochemistry confirmed the
melanocytic nature of the lesion (Melan-A+, HMB45+, S100+)
and interestingly staining for Nestin and Ghrelin was surprisingly
positive. Despite the age of the patient and fast growing rate of
this extralarge conjunctival melanoma, she did very well without
signs of metastasis for 36 months after orbital exenteration.
Conclusion. Malignant melanoma of the conjunctiva is a rare
tumor that carries a significant mortality and local recurrence.
Histological classification of conjunctival melanomas is lacking
and therefore skin melanoma classification is adopted. Treatment
remains unclear because validated protocols do not exist. From
a treatment point of view, detection of BRAF mutations in exon
15 could address to biological drugs (such as Vemurafenib) in
case of developing of metastatic disease. Nestin is an intermediate filament protein and marker for melanoma initiating cells; its
immunohistochemical expression has been correlated with tumor
progression and is associated with poor prognosis in conventional
melanomas. Since its expression was significantly associated with
reduced survival in multivariate analysis, it is thought to be an
aggressive melanoma feature. Nestin is also a neuronal stem cell/
progenitor cell marker of central nervous system development and
it has been shown to modulate cell proliferation in embryonic and
undifferentiated tissues. Hence the presence of mechanisms involving nestin in melanoma growth may indicate more dedifferentiated
tumor and raise the possibility for a marker of progression. In fact,
in contrast to benign melanocytic naevi, which are negative for nestin, strong positivity for nestin was seen in all 9 cases of malignant
melanoma by Ehrman and by Hanet in conjunctival melanomas.
Strong positive result for nestin is also well documented in our
case. Ghrelin (Ghr) is a 28-aminoacid peptide, natural ligand for
the growth hormone segretagogue receptor (GSHR1a) that induces
a potent release of growth hormone (GH), with other physiological
distinct functions. Widely expressed in different tissues, besides
its orexigenic activity, Ghr exerts several non-metabolic effects, as
myorelaxation on cardiac, skeletal and smooth muscle cells. At the
best of our knowledge, we do not found in the web literature any
previous report of Ghr in melanomas. In a previous works, we also
found and confirmed Ghr in eye layers and aqueous humour, but
also in choroidal melanoma and in this conjunctival case. These
data need to be confirmed by more researches, in order to explain
how both these growth factors (nestin and ghrelin) act in the melanomas development and growth and their significance for staging
and prognosis.
References
Shields CL. Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Tr. Am
Ophth Soc. 2000;98:471-492.
Pugliese M,Gallenga CE, Aharrh-Gnama A et al. XXLarge conjunctival
223
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melanoma B-RAF negative for codone mutation, proliferating under
corneal epithelium layer. III Orbitoplastic Course PS 5, Sept.2013,
Ravello Italy.
Brychtova S, Fluraskova M, Hlobilkova A and coll. Nestin expression in cutaneous melanomas and melanocytic nevi. J Clin Pathol.
2007;34:370-75.
Ladstein RG, Bachmann IM, Straume O and coll. Nestin expression
is associated with aggressive cutaneous melanoma of the nodular
type. Mod Pathol. 2013;sep 13.doi:10.1038/modpatol.2013.151.[Epub
ahead of print].
Lai S, Piras F, Spiga S and coll. Nestin and vimentin colocalization affects the subcellular location of glucocorticoid receptor in cutaneous
melanoma. Histopathology. 2013;62:487-98.
Florenes VA, Holm R, Myklebost O and coll. Expression of the neuro-
ectodermal intermediate filament nestin in human melanomas. Cancer
Res. 1994;54:354-6.
Ehrmann J, Kolˆr Z, Mokry J. Nestin as a diagnostic and prognostic
marker: immunohistochemical analysis of ist expression in different
tumours. J Clin Pathol. 2005;58:222-3.
Hanet MS, Yhomasen H, Westekemper H and coll. Expression von Nestin in malignem melanom der Bindehaut. DOG Congress Berlin. 2013; PS 05-02, sept 21.
Di Fonso A, Ghinassi B, Izzicupo P and coll. Novel evidence of ghrelin
and growth hormone segretagogue receptor expression by human
ocular tissues. Regul Pept. 2014 May 6;190-191C:1.
Goldstone AP, Prechtl CG, Scholtz S and Coll. Ghrelin mimics fasting
to enhance human hedonic,orbitofrontal cortex and hippocampal responses to food. Am J Clin Nutr. 2014; April 23 [Epub ahead of print].
Patologia molecolare - 1
Moderatori: F. Castiglione, A. Marchetti
The role of the epstein-barr virus in the
development of plasmablastic lymphoma:
new insights into cellular and viral micrornas
dysregulation
M.R. Ambrosio, G. De Falco, B.J. Rocca, V. Mourmouras, L.
Mundo, S. Gazaneo, S. Lazzi, L. Leoncini
Department of Medical Biotechnologies, University of Siena, Italy
Introduction. The pathogenesis of Plasmablastic lymphoma
(PBL) is not clearly understood, although a role for Epstein-Barr
virus (EBV) has been proposed as EBV DNA is detectable in 6075% of the cases. However, the exact mechanisms by which EBV
may contribute in promoting PBL is still an area of active debate.
The proposed mechanism for B-cell lymphomagenesis in the
setting of EBV infection includes EBV-induced B-lymphocyte
immortalization, leading to the emergence of dominant B-cell
clones, and transcriptional transactivation of viral and cellular
genes by EBV resulting in B-cell transformation. It is well known
that EBV encodes a series of genes products and microRNAs
interacting with or exhibiting homology to a wide variety of
anti-apoptotic molecule, cytokines, and signal transducers, hence
promoting EBV infection, immortalization and transformation.
Transformed lymphocytes are more susceptible to other genetic
mutations (i.e. MYC gene rearrangement) that may enhance EBV
lymphomagenesis. During latent infection, to maintain the viral
genome and to successfully evade the host cell immune surveillance, EBV expresses a small subset of genes. The differential expression pattern of these latent genes defines the distinct latency
programs (i.e. type I, II, and III latency). The latency I, which is
characterized by the expression of only EBNA1 and EBER, is
reported in most of PBL and Burkitt lymphoma (BL). Since BL
and PBL usually arise in the setting of immunodeficiency, one
should expect a latency III program; this may suggest that the
onset of these two diseases is not related to the imbalance of host
immunity but rather to other genetic mechanisms (i.e. MYC overexpression) or to EBV itself.
In this study we aimed at investigating the role of EBV in the
development of PBL in comparison to BL, in terms of latency
program expressed by the virus in the neoplastic cells, and of
microRNA (miRNA) profile to assess the contribution of viralencoded miRNAs in dysregulating host gene expression and
affecting key cellular pathways.
Material and methods. EBV positivity was detected by EBER in
situ hybridization. EBV latency was assessed by RT-qPCR, using
Taqman probes, and immunohistochemistry. MiRNA profiling
(including both cellular and viral miRNAs) was performed using
the MySeq Illumina platform.
Results. We found that, in addition to EBNA1, most of PBLs
and BLs expressed LMP-2 and lytic genes (i.e. ZEBRA and
EAD). LMP-1 was detected in one case of PBL and one case
of BL. These results suggest a non-canonical latency associated
gene expression program with a subset of viral episome genes,
corresponding to an abortive lytic cycle. Viral miRNA profile
indicated that several EBV-miRNAs, belonging to the BART
family, are expressed both in EBV positive PBLs and in BLs,
being expressed at a higher level in PBL. Cellular miRNAs
profile showed that only a few cellular miRNAs are differential
expressed between PBLs and BLs. In addition, when comparing
BL and PBL with the normal counterpart, principle component
analysis showed that PBLs and BLs clusterize together for cellular miRNAs and significantly differ from any normal counterpart.
If this our previous data will be confirmed, it would be difficult
to find for PBL and BL a normal cell to which they completely
correspond.
Conclusions. The reactivation of lytic cycle may play a role in
EBV-driven lymphomagenesis by increasing the total number
of latently infected cells. The detection of LMP-2 reflects the
molecular environment that MYC needs to unfold its oncogenic
potential. In addition, EBV-miRNAs may promote the transformation of primary B-lymphocytes by dysregulating physiologic
pathways.
References
1
Jones RJ, Seaman WT, Feng WH, Barlow E, Dickerson S, Delecluse
HJ, Kenney SC. Roles of lytic viral infection and IL-6 in early versus
late passage lymphoblastoid cell lines and EBV-associated lymphoproliferative disease. Int J Cancer. 2007 121(6):1274-81.
2
Vereide DT, Seto E, Chiu YF, Hayes M, Tagawa T, Grundhoff A,
Hammerschmidt W, Sugden B . Epstein-Barr virus maintains lymphomas via its miRNAs. Oncogene. 2014 33(10):1258-64.
3
Fish K, Chen J, Longnecker R. Epstein-Barr virus latent membrane
protein 2A enhances MYC-driven cell cycle progression in a mouse
model of B lymphoma. Blood. 2014 123(4):530-40.
4
Dittmer DP. Not like a wrecking ball: EBV fine-tunes MYC lymphomagenesis. Blood. 123(4):460-1.
224
Evaluation of GeneXpert HPV assay for HPV
detection in Formalin Fixed Paraffin Embedded
(FFPE) tissues
R. Cerutti, D. Furlan, C. Facco, A. Chiaravalli, F. Sessa, C. Riva
Anatomic Pathology, Department of Surgical and Morphological Sciences, University of Insubria and Ospedale di Circolo e Fondazione Macchi,
Varese
Background. The identification of Human Papilloma Virus
(HPV) in tissues is very useful to improve diagnosis and management of gynecological pre-malignant or malignant disorders
and to identify a subset of head and neck cancer, especially oropharyngeal, with peculiar biological characteristics and favorable
prognosis. In all these clinical settings it is important to have a
fast and reliable method to identify high-risk (HR) HPV types in
formalin fixed-paraffin embedded (FFPE) specimens.
Aim. We sought to evaluate the feasibility of HR-HPV detection
in a set of FFPE tissues of different sites using the cartridge-based
GeneXpert HPV assay on a Cepheid GeneXpert System, a qualitative real-time PCR assay validated to detect HR-HPV DNA in
cervical cytological specimens.
Materials and methods. We studied HPV in 69 different histological FFPE samples, mainly biopsies: 10 cervical adenocarcinomas, 3 anal squamous cell carcinomas, 35 oropharyngeal
squamous cell carcinomas, 19 cervical lesions (including ASCUS/CIN1, 10 cases, and CIN2-3, 9 cases) and two squamous
papillomas of the oral cavity. Oropharyngeal samples were
previously tested with HPV In Situ Hybridization (ISH) (Roche)
and p16 IHC; the 19 cervical samples were from HR-HPV-PCR
positive patients (PCR-hybridization array analysis, Genomica).
We also performed analysis on cytological cervical specimens of
10 patients already HPV-PCR tested. For the FFPE specimens,
deparaffinization with bioclear-ethanol and overnight lysis in
ATL Qiagen buffer with Proteinase K was done, and after 1 hour
incubation at 90¡C crude lysate was used to perform HPV assays.
Results. A total of 56 of 69 FFPE samples (81%) and 10/10 (100%)
fresh cytological specimens gave valid results on HR-HPV status.
In almost all FFPE cases (47/50) and in all cytologic samples with
a previous HPV analysis the results were completely concordant,
either for HPV presence and HPV genotype. In 13 cases where
no valid result were obtained we performed DNA extraction with
Qiagen FFPE tissue Kit, DNA quantification with Qubit Fluorimeter and evaluation of DNA fragmentation levels. DNA extraction
didn’t improve substantially the results, as amount of DNA didn’t
correlate with Ct of Sample Adeguacy Control (SAC) gene in Realtime PCR and only 3/13 samples gave better results with DNA vs
lysate. The DNA fragmentation analysis revealed that almost all
the samples that gave no results (or very high SAC Ct) had DNA of
poor quality (amplification of 200 bp fragment maximum).
Conclusions. GeneXpert HPV assay seems a fast, easy and reliable
method for HPV identification in FFPE samples, with good rates
of success even if working on lysate obtained from small biopsies.
The limitation of the technique resides in cases of FFPE tissue with
poor DNA quality, where the amplification of SAC is often inefficient giving invalid results especially in HPV negative samples.
Comparison of HER-2 status between primary
gastric cancer and metastatic sites:
a retrospective study on 31 patients
P. Cito1, G. Opinto2, A. Marzullo2, A. Napoli2, M. Liuzzi2, C.
Caporusso2, F. Silvestris1, E. Maiorano2
Department of Biomedical Sciences and Human Oncology – Section of
Clinical Oncology; 2 Department of Emergencies and Organ Transplantation – Section of Pathological Anatomy; Università degli Studi di Bari,
Aldo Moro - Bari
1
Background. HER-2 protein over-expression and gene amplification in gastric carcinoma are prerequisites for monoclonal
antibody-based therapies and, therefore, the assessment of HER-2
status in gastric carcinoma is mandatory for predictive purposes.
HER-2 status usually is evaluated in the primary tumor and
metastatic sites are rarely biopsed. Nevertheless, gastric cancer
shows intratumoral heterogeneity for HER-2 receptor, influenced
by histologic type and location of the tumor, age of patients and
sampling (biopsy vs. surgical sample). The aim of this study was
to verify HER-2 status variability among distinct biopsies and
the corresponding surgical samples and node metastases and to
evaluate the concordance rates.
Materials and methods. HER-2 status was assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization
(FISH), according to the TOGA Study recommendations, in 31
cases of gastric and gastro-esophageal junction cancers, in both
primary tumors (biopsies or surgical samples) and metastatic sites
(nodes or distant metastases).
Result. Matching HER-2 status between biopsies/surgical samples, biopsies/nodal samples, surgical/nodal samples and surgical
samples/metastatic sites showed high concordance rates, which
were higher with FISH analyses (range: 95-100%) in comparison with IHC procedures (range: 63-73%). Furthermore, HER-2
status, regardless of the method employed, remained stable in
the primary sites and in metastatic nodes. However, we found
significant HER-2 expression variability (5/31 cases) between
node/distant metastasis and primary tumors, thus confirming the
evidence of intratumoral heterogeneity.
Conclusions. The present study demonstrates that the status of
HER-2 at metastatic sites can be discordant
from that of the corresponding primary tumors. Our results highlight the heterogeneity and biological
complexity of gastric tumors and stress the importance of a reproducible and analytically standardized systems,
to clearly assess eligibility criteria for target therapies.
Kras mutation profiling of non-small cell lung
cancer: why testing it?
G. De Maglio1, L. Pagani1, M. Macerelli2, S. Cernic1, M.C. Barducci1, A. De Pellegrin1, G. Fasola2, S. Pizzolitto1
Department of Pathology, University Hospital Santa Maria della
Misericordia,Udine,Italy; 2 Department of Oncology, University Hospital
Santa Maria della Misericordia,Udine,Italy
1
Background. In the last decade, a deeper understanding of the
biological mechanism of non-small cell lung cancer (NSCLC)
led to targeted therapies development, changing the history of
patients with tumours harbouring EGFR mutations or ALK rearrangements [1].
Although KRAS mutations are the most common oncogenic alterations in NSCLC, with a reported frequency of approximately
25% of lung adenocarcinomas in Western countries, their prognostic and predictive roles remain still controversial [2,3].
Even though several retrospective studies demonstrated a poor
prognosis for patients with a KRAS mutant tumor, contrasting
results keep the debate alive [4,5]. Furthermore KRAS mutations
seems to have a negative predictive value for patients treated with
EGFR inhibitors [6] and predict an inferior response to platinumbased chemotherapy [7,8,9]. Probably, some KRAS point mutations may differently influence prognosis, metastatic progression
and responses to either chemotherapy or targeted-therapies [10].
KRAS mutations are usually mutually exclusive with EGFR or
BRAF mutations and ALK rearrangements [11,12].
Nowadays, several other driver mutations are being evaluated in
NSCLC, waiting for new intriguing targeted therapies [13,14,15].
We described EGFR/KRAS/BRAF mutation profile in NSCLC
tested by mean of a multitargeted assay.
Methods. A consecutive series of 334 advanced NSCLC (October 2012-May 2014) were tested for EGFR, KRAS and BRAF
mutational status upon Oncologist request, both in cytological
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COMUNICAZIONI ORALI
(93; 27.8%) and in histological samples (241; 72,2%). Cytohistological diagnosis were distributed as follows: 271 (81.1%)
adenocarcinomas, 18 (5,4%) squamous cell carcinomas, 39
(11.7%) NSCLC NOS and 6 (1,8%) adenosquamous carcinomas.
Molecular examination of EGFR/KRAS/BRAF were simoultaneously performed by mean of matrix-assisted laser desorption/
ionization-time of flight mass spectrometry (MALDI-TOF)
Sequenom¨ assay with CE-IVD Myriapod¨ Cancer status kit
(Diatech pharmacogenetics, Italy). Analitical sensitivity of the
test was up to 2,5% and 40 ng of DNA was enough for the whole
panel analysis.
Results. We found an EGFR/KRAS/BRAF mutation rate of
13,2%, 39,5% and 2,4%, respectively. All mutations in the three
genes were mutually exclusive.
Among 132 KRAS mutations detected, the most common point
mutations mapped on codon 12 (113; 85.6%) and codon 13 (13;
9.9%), followed by codon 61 (5; 3.9%) and codon 146 (1; 0.8%).
Frequencies of specific KRAS mutations are listed in Table 1.
KRAS mutations were almost equally distributed among sex
with a frequency of mutation of 40,3% and 37,8% in males and
females, respectively.
KRAS mutation frequency varied among age: 18.2% in patients
younger than 50 years-old, 50% in 50-65 years-old patients and
36,8% in elderly people (>65 years-old).
We mainly observed KRAS mutations in adenocarcinoma histotype (see Table 2). In squamous cell carcinomas any BRAF
mutations was observed, and only an EGFR p.L858R mutation
was detected.
Relative frequencies of EGFR mutations are described in Table 3.
All BRAF mutations found (8 cases) were p.Val600Glu.
Tab. I. Frequency of KRAS mutations.
p.G12C
p.G12V
p.G12D
p.G12A
p.G12S
p.G12R
p.G12F
p.G13C
p.G13D
p.G13R
p.Q61H
p.Q61R
p.Q61L
p.A146T
40,9%
27,3%
10,6%
3,0%
1,5%
1,5%
0,8%
5,3%
3,8%
0,8%
2,3%
0,8%
0,8%
0,8%
Tab. II. Frequency of KRAS mutations among different histotypes.
adenocarcinoma
squamous cell carcinoma
mixed adenosquamous carcinomas
NSCLC NOS
Total
271
18
6
39
132 (39.5%)
114
42,1%
3
16,7%
2
33,3%
13
33,3%
Tab. III. Relative frequencies of EGFR mutations
ex
ex
ex
ex
18
19
20
21
Total
2
29
3
10
44 (13.2%)
4,5%
65,9%
6,8%
22,7%
Conclusions. The frequencies of EGFR and BRAF mutations in
our case series are consistent with literature data [16]. However,
we found a higher rate of KRAS mutations than generally reported, while distribution among different mutations in KRAS exon
2 confirmed what previously described [2,3].
Early mutational profiling of all EGFR pathway components in
NSCLC patients could be useful for defining different molecular
subgroups of patients. Even if KRAS/BRAF mutational status
analysis does not still drive clinicians in treatment selection, it
remains an useful intermediate step between EGFR status assessment and ALK rearrangement analysis, thus for reasonably
excluding EGFR mutations and ALK rearrangements in KRAS
mutated tumours [18]. This was particularly effective to save a
further biopsy to EGFR-wild type patients eligible to ALK screening when small, low enriched or smear cytological samples were
the only available material.
References
1
Gridelli C, de Marinis F, Cappuzzo F, et al. Treatment of advanced
non-small-cell lung cancer with epidermal growth factor receptor
(EGFR) mutation or ALK gene rearrangement: results of an international expert panel meeting of the Italian Association of Thoracic
Oncology. Clin Lung Cancer 2014;15(3):173-81.
2
Riely GJ, Kris MG, Rosenbaum D, et al. Frequency and distinctive
spectrum of KRAS mutations in never smokers with lung adenocarcinoma. Clin Cancer Res. 2008;14(18):5731-4.
3
Rodenhuis S, Boerrigter L, Top B, et al. Mutational activation of the
K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study. J Clin Oncol. 1997;15(1):28591.
4
Slebos RJ, Kibbelaar RE, Dalesio O, et al. K-ras oncogene activation
as a prognostic marker in adenocarcinoma of the lung. N Engl J Med
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5
Mascaux C, Iannino N, Martin B, Paesmans M, Berghmans T, Dusart
M, et al. The role of RAS oncogene in survival of patients with lung
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D’Arcangelo M, Cappuzzo F. Erlotinib in the first-line treatment of non-small-cell lung cancer. Expert Rev Anticancer Ther.
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7
Loriot Y, Mordant P, Deutsch E, et al. Are RAS mutations predictive
markers of resistance to standard chemotherapy?. Nat Rev Clin Oncol
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8
Macerelli M, Caramella C, Faivre L et al., Does KRAS mutational
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9
Metro G, Chiari R, Bennati C, et al. Clinical outcome with platinumbased chemotherapy in patients with advanced nonsquamous EGFR
wild-type non-small-cell lung cancer segregated according to KRAS
mutation status. Clin Lung Cancer. 2014;15(1):86-92.
10
Conde E, Angulo B, Izquierdo E, et al. The ALK translocation in
advanced non-small-cell lung carcinomas: preapproval testing experience at a single cancer centre. Histopathology. 2013;62(4):609-16.
11
Suda K, Tomizawa K, Mitsudomi T. Biological and clinical significance of KRAS mutations in lung cancer: an oncogenic driver that
contrasts with EGFR mutation. Cancer Metastasis Rev. 2010;29(1):4960.
12
Thunnissen E, van der Oord K, den Bakker M. Prognostic and
predictive biomarkers in lung cancer. A review. Virchows Arch.
2014;464(3):347-58.
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Reungwetwattana T, Dy GK Targeted therapies in development for
non-small cell lung cancer. J Carcinog. 2013;12:22.
14
Paik PK, Arcila ME, Fara M, et al. Clinical characteristics of patients
with lung adenocarcinomas harboring BRAF mutations. J Clin Oncol.
2011; 29(15):2046-51.
15
Paolo M, Assunta S, Antonio R, et al. Selumetinib in advanced non
small cell lung cancer (NSCLC) harbouring KRAS mutation: endless
clinical challenge to KRAS-mutant NSCLC. Rev Recent Clin Trials.
2013;8(2):93-100.
16
Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the
epidermal growth factor receptor underlying responsiveness of nonsmall-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21):212939.
17
Rodenhuis S, Boerrigter L, Top B, et al. Mutational activation of the
226
18
K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study. J Clin Oncol. 1997;15(1):285-91.
Conde E1, Angulo B, Izquierdo E, et al. The ALK translocation in
advanced non-small-cell lung carcinomas: preapproval testing experience at a single cancer centre. Histopathology 2013 62(4):609-16.
Novel germline mutation of MLH1 gene in a case
of Lynch Syndrome with cancer of colon and
endometrium
D. Della Libera, A. D’Urso, F. Modesti, D. Florea, M. Gobbato
Unità Operativa Complessa Anatomia Patologica – Ospedale Santa Maria del Prato - Feltre / ULSS2 Veneto
Introduction. Lynch Syndrome (LS), formerly know as Hereditary Non Polyposis Colorectal Cancer (HNPCC), is a genetically
heterogeneous autosomal dominant disease, which determines the
predisposition to early development of colorectal cancer (CRC),
as well as tumors affecting endometrium, ovaries, stomach, small
intestine and genito-urinary tract. Endometrial carcinoma is the
most common non colorectal neoplasia occurring in women
with Lynch Syndrome (Watson and Lynch, 1993). In the female
population, the endometrial cancer incidence matches the colon
cancer one (Folkins et al., 2013). In more than 50 % of women
with HNPCC, endometrial carcinoma is the first or “sentinel”
cancer to develop (Lu et al., 2005). Lynch syndrome is the result
of germline mutation in one of the MisMatch Repair (MMR)
genes MLH1, MSH2, MSH6 and PMS2 (Boland et al. 2005).
Hundreds of unique mutations were reported in each of them
(http://www.insightgroup.org). Mutation leading to truncated
proteins, including nonsense, frameshift, splice-site, and large
deletion, are the most frequent cause of deficient MMR function (Peltomaki et al., 2004). According with Revised Bethesda
Guidelines (Umar et al. 2004) we investigated a case of 56 years
old woman presenting a synchronous colon and endometrial
cancer arising fourteen years ago and a metachronous colorectal
cancer arising last year. Matherial and Method Samples. The histology of the three tumors was reevaluated and blocks with a high
percentage of tumor cells were selected. DNA from tumor was
extracted with QIAamp DNA FFPE Tissue Kit (QIAGEN) by
microdissecting five μm tissue sections containing al least 80%
of tumor cell . All tumors were analyzed for Microsatellite Instability (MSI) and immunohistochemical staining for MMR genes,
methylation analysis of the MLH1 promoter and BRAF mutation
analysis. Germline mutation of MLH1 and PMS2 genes were
assessed on DNA extracted from blood with standard methods.
MSI Analysis. Twenty ng of DNA was subjected to MSI Analysis using a five mononucleotide markers panel represented by
NR21, NR22, NR24, BAT25 and BAT26. The amplified tumor
DNA and the matching normal DNA derived from blood were
analyzed by Capillary Electrophoresis equipment and GeneMapper software. MLH1 Methylation and BRAF mutation analysis.
MLH1 promoter methylation was conducted using Pyrosequencing technology on DNA extracted from five μm endometrial
cancer sections, Sodium Bisulfite treated, and amplified to assess
a region proximal to transcription initiation site located between
nucleotide -248 and -178. BRAF V600E mutation analysis was
conducted on DNA extracted from five μm colon cancer sections,
using Anti-EGFR MoAb response (BRAF Status) - Diatech Pharmaco Genetics. Immunohystochemistry. Immunohistochemical
staining was performed on formalin fixed, paraffin embedded
tissue. The primary antibodies were Anti-Human MutL Protein
Homolg 1 (clone ES05-Dako), Anti-Human Postmeiotic Segregation Increased 2 (Clone EP51-Dako), Anti-Human MutS Protein
Homolog 2 (Clone FE11Dako), Anti-Human MutS Protein Homolog 6 (clone EP49-Dako). Positive and negative controls were
considered. Sequencing analysis. Exon sequences and flanking
intronic regions of MLH1 and PMS2 genes were amplified using exons specific primers. PCR products were sequenced using
BigDyeTerminator v3.1 chemistry (Life Technologies). Capillary
Electrophoresis was conducted on AB3130 Genetic Analyzer
(Applied Biosystem-Life Technologies). Sequences obtained
were compared to the NCBI reference sequence NM_000249.3
and NM_000535.5.
Results. MSI analysis with five mononucleotide markers panel
(NR21, NR22, NR24, BAT25 and BAT26) presented a pattern of
high-grade instability (MSI-H) showing an allelic shift of al least
two markers. Immunohistochemical analysis for MMR proteins
(MLH1, PMS2, MSH2, MSH6) showed abnormal staining in all
three tumors with a loss of expression of MLH1 and PMS2 accompanied by a retention of MSH2 and MSH6 one. In order to exclude
a sporadic source of the pathology, endometrial tumor was subjected to MLH1 promoter methylation analysis by a Pyrosequencing assay to detect methylation events inside promoter region C as
described by Deng and coworkers as the real sequence responsible
of MLH1 protein expression silencing (Deng et al., 1999). At the
same time, colon tumor tissues were subjected to BRAF V600E
mutation analysis by Pyrosequencing assay. Both MLH1 promoter
methylation analysis and BRAF V600E mutation research were
negative, excluding the sporadic origin of the analyzed tumors. On
the basis of the obtained results, we proceeded to find the germline
mutation responsible for the pathology of the patient. Despite
the concurrent loss of MLH1 and PMS2 suggests a mutation in
MLH1 gene, due to the nature of heterodimers formed by the
MMR proteins, we decided to analyze the entire coding sequence
and the intronic flanking regions of both MLH1 and PMS2 genes.
Direct sequencing of MLH1 gene revealed the intronic variant
c.1558+1G>A not listed in the database of International Collaborative Group on HNPCC (http://www.insight-group.org). The presence of the mutation was confirmed both in forward and reverse
strand. This variant involves the donor site consensus sequence
in intron 13. The analysis of PMS2 showed two intronic variant.
The first, c.163+138A>G, is listed in LOVD database, while the
second, c.705+17A>G, is listed in LOVD-Insight database. Both
variants are described to have unknown pathogenicity.
Discussion. In the present study, a patient with HNPCC was
studied for a synchronous colon and endometrial cancer. The
genetic analyses executed on the tumor tissue of the colon, the endometrium and blood of the patient confirm the importance of the
study of MMR system on the diagnosis of Lynch Syndrome. The
presence of the same alterations on the MMR proteins expression
in the tumors of the colon and the uterus underlined the importance of considering the endometrial cancer as a sentinel tumor in
women potentially affected by LS. Infact in the Linch Sindrome
the risk to develop gynaecological malignancies is equal if grater
for devoloping a carcinoma of the colon. The described MSI
analysis confirmed the usefullness to use a panel composed by
five quasi-monomorphic mononucleotide markers (NR21, NR22,
NR24, BAT25 and BAT26) which appears to be more sensitive
and specific than the NCI panel in determining the MSI status
(Suraweera et al., 2002; Buhard et al., 2004). As it was already
observed in MSI-H gastrointestinal tumors, mononucleotide
repeats are much more unstable in MSI-H endometrial tumors
than dinucleotide repeats representing more accurate markers
in extra colonic malignancies (Suraweera et al., 2002). Despite
the quasi-monomorphic nature of the mononucleotide repeats in
Caucasian population, allowing the determination of MSI status
in gastrointestinal tumors without reference to matching-normal
DNA, it is already known that MSI-H endometrial tumors show a
lower intensity of instability compared with gastrointestinal ones.
According to this evidence, it is important to underline that the
MSI analysis conducted on the index patient involved both tumor
tissues DNA and the matching normal DNA obtained from the
blood sample. In the present work MSI analysis was accompanied by immunohistochemical characterization of MMR proteins
expression. These two approaches can be defined complementary
in the screening algorithm of LS suspicion, guiding the choice of
227
COMUNICAZIONI ORALI
the gene to investigate to highlight the molecular cause of the pathology. Finally the sequencing analysis presented here revealed
a novel germline intronic variant c.1558+1G>A of MLH1 gene
not previously described. It is interesting to note that the LOVDInsight database reports the variant c.1558+1G>T found by five
different authors (Viel et al., 1998; Benatti et al., 1998; Rossi
et al., 2002; Wagner et al., 2003; Pedroni et al., 2007) defined
as pathogenetic due to splicing defect demonstrated by MLH1
transcript analysis. The variant involves the same position in the
sequence of MLH1 gene, although with a different base substitution, related to the mutation c.1558+1G>A described in our
work. The new mutation involves the same donor site consensus
sequence in intron 13. In conclusion our observation of a novel
mutation in MLH1 gene suggests that it can be responsible for an
aberrant splicing event causing MLH1 protein loss, highlighted
by our immunohistochemical analysis, representing a new molecular variant capable of concurrent colon and extra colonic
Lynch Syndrome associated malignancies.
References
Benatti P, Roncucci L, Percespe A, et al. Small bowel carcinoma in
hereditary nonpolyposis colorectal cancer. Am J Gastroenterol. 1998
Nov;93(11):2219-22.
Berends MJW, Wu Y, Sijmons RH, et al: Toward new strategies to select
young endometrial cancer patients for mismatch repair gene mutation
analysis. J Clin Oncol 21: 4364-4370, 2003
Boland C.R. Evolution of the nomenclature for the hereditary colorectal
cancer syndromes. Familial Cancer, 2005. 4: p. 211-218.
Bonadona V., Bonaiti B., Olschwang S. et al. Cancer risks associated
with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch
syndrome. Journal of the American Medical Association, 2011. 305:
p. 2304-2310.
Buhard,O., Suraweera,N., Lectard,A., Duval,A. and Hamelin,R. (2004)
Quasimonomorphic mononucleotide repeats for high level microsatellite instability analysis. Dis Markers, 20, 251–257.
Deng G., Chen A., Hong J., Chae HS., Kim YS. Methylation of CpG in a
small region of the hMLH1 promoter invariability correlates with the
absence of gene expression. Cancer Research., 1999. 59: p. 2029-2033.
Folkins A.K., Longacre T. Hereditary gynaecological malignancies: advances in screening and treatment. Histopathology, 2013. 62: p. 2-30
Hampel H., Frankel W., Martin E. et al. Screening for Lynch syndrome
(hereditary nonpolyposis colorectal cancer). New England Journal of
Medicine, 2005. 352: p.1851-1860.
Hampel H., Frankel W., Panescu J. et al. Screening for Lynch syndrome
(hereditary nonpolyposis colorectal cancer) among endometrial carcinoma patients. Cancer research, 2006. 66: p. 7810-7817.
Lu K.H., Frankel W., Panescu J. et al. Gynecologic cancer as a “sentinel
cancer” for women with hereditary nonnpolyposis colorectal cancer
syndrome. Obstetrics Gynecology, 2005. 105: p. 569-574..
Lu K.H., Schorge J. O., Rodabaugh K.J. et al. Prospective determination
of prevalence of Lynch syndrome in young women with endometrial
cancer. Journal of Clinical Oncology, 2007. 25: p. 5158-5164.
Millar AL, Pal T, Madlensky L, et al: Mismatch repair gene defects contribute to the genetic basis of double primary cancers of the colorectum and endometrium. Hum Mol Genet 8: 823-829, 1999.
Pedroni M, Roncari B, Maffeio S, et al. A mononucleotide markers
panel to identify hMLH1/hMSH2 germline mutations. Dis Markers.
2007;23(3):179-87.
Peltomaki P, Vasen H: Mutations associated with HNPCC predisposition – Update of ICG-HNPCC/INSiGHT mutation database. Disease
markers 2004, 20(4-5):269-276.
Plazzer JP, Sijmons RH, Woods MO, Peltomaki P, Thompson B, DenDunnen JT, Macrae F: The InSiGHT database: utilizing 100 years of
insights into Lynch Syndrome. Fam Cancer 2013, 12:175–180.
Rossi BM, Lopes A, Oliveira Ferreire F, Nakagawa WT, Napoli Ferreira
CC, Casali Da Rochs JC, Simpson CC, Simpson AJ. hMLH1 and
hMSH2 gene mutation in Brazilian families with suspected hereditary
nonpolyposis colorectal cancer. Ann Surg Oncol. 2002 Jul;9(6):555-61.
Senter L., Clendenning M., Sotamaa K., Hampel H. et al The clinical
phenotype of Lynch syndrome due to germ-line PMS2 mutations. Gastroenterology, 2008. 135: p. 419-428.
Suraweera N., Duval,A., Reperant,M., Vaury,C., Furlan,D., Leroy,K.,
Seruca,R., Iacopetta,B. and Hamelin,R. (2002) Evaluation of tumor
microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology, 123, 1804–1811
Viel A., Genuardi M., Lucci-Cordisco E., et al. Hereditary nonpolyposis colorectal cancer: an approach to the selection of candidates to
genetic testing based on clinical and molecular characteristics. Community Genet. 1998;1(4):229-36.
Umar A., C. Richard Boland, Jonathan P., et al. Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome)
and Microsatellite Instability J Natl Cancer Inst 2004;96:261– 8
Wagner A, Barrows, Wijnen, et al. Molecular Analysis of Hereditary
Nonpolyposis Colorectal Cancer in the United States: High Mutation
Detection Rate among Clinically Selected Families and Characterization of an American Founder Genomic Deletion of the MSH2 Gene
Am. J. Hum. Genet. 72:1088–1100, 2003.
Sala Michelangelo – Piano inferiore –11,30-12,30
Patologia molecolare - 2
Moderatori: G. Caruso, A. Florena
Presurgical braf analysis in papillary thyroid
carcinoma: correlation with clinico-pathological
features and prognosis in a single monoinstitution
prospective experience
F. Galuppini4, C. Mian2, S. Barollo2, F. Vianello1, S. Sensi2, D.
Nacamulli2, M.R. Pelizzo3, M. Rugge4, G. Pennelli4
Radiotherapy Department; Istituto Oncologico Veneto – IRCCS, Padua;
Italy; 2 Endocrinology Unit; Department of Medicine-DIMED; University
of Padua; Italy; 3 II General Surgery; Department of Surgery Oncology
and Gastroenterology; University of Padua; Italy; 4 II Pathology Unit, Department of Medicine-DIMED; University of Padua; Italy
1
Introduction. Risk stratification in papillary thyroid carcinoma
(PTC) is based on postoperative parameters. Preoperative BRAFmutation testing could provide a novel tool to preoperatively
identify patients at higher risk for extensive disease. Aims of the
present prospective study, were 1) to correlate the BRAF status in
FNAB specimens of a large series of consecutive PTC followed
in a single institution with classical indicators of poor prognosis
and 2) to evaluate prospectively the BRAF status impact in patient’s prognosis.
Patients and methods. 186 consecutive patients (38 males and
148 females, mean age 48, median 49, range 22–81 years, mean
follow-up was 36± 8 months) with a histological diagnosis of
PTC and a BRAF analysis performed on the thyroid fine-needle
aspiration biopsy (FNAB) were included. We assessed the BRAF
mutational status in FNAB specimens before thyroidectomy for
PTC and BRAF mutational status association with clinicopathologic characteristics revealed postoperatively and with outcome
in a 36 ± 8 month-long follow-up.
Results. On a whole, 116 of 186 (62%) PTC carried a BRAF
mutation. The univariate analysis showed that BRAF status was
correlated with themore aggresive histological variants of PTC
(p=0.001), the cancer size (p=0.02), the extrathyroid extension
228
(P = 0.04) and advanced stages (p=0.03) at diagnosis. No associations were found with gender, age, multifocality, lymph node
involvement. On a whole 9% of BRAF mutated cases versus 4 %
of BRAF wild-type showed a persistent disease at the end of follow-up; anyway this difference was not statistically significant.
Conclusions. Our study confirmed, even in presurgical assessment, the association of BRAF mutation with clinical and pathological features of PTC classically associated with high risk PTC
patients.
Discordance rate of her2 status in primary gastric
carcinomas versus synchronous regional lymph
node metastases: a multicenter retrospective
investigastion
A. Ieni1, V. Barresi1, R. Caltabiano2, A. Caleo3, L. Reggiani
Bonetti4, S. Lanzafame2, R.A. Caruso1, P. Zeppa3, G. Tuccari1
1
Azienda Ospedaliera Universitaria “Gaetano Martino” and Department
of Human Pathology “Gaetano Barresi”, Section of Anatomic Pathology,
University of Messina, Messina, Italy; 2 Azienda Ospedaliera Universitaria “Gaspare Rodolico” and Department “G.F. Ingrassia”, Section of
Anatomic Pathology, University of Catania, Catania, Italy; 3 Azienda Ospedaliera Universitaria “S.Giovanni di Dio e Ruggi di Aragona” and Department of Medicine and Surgery, University of Salerno, Salerno, Italy;
4
Department of Diagnostic Medicine, Clinics and Public Health, Section
of Pathologic Anatomy, University of Modena, Italy
Introduction. Medullary thyroid carcinoma (MTC) is a rare
(sporadic or familial) neuroendocrine cancer originating from
parafollicular C-cells. MicroRNAs (miRNAs) are a class of small
non-coding RNAs involved in a large number of physiological
and pathological processes. Recently their role in thyroid tumor’s
carcinogenesis were investigated but only few studies evaluated miRNAs’ expression in MTC. Programmed cell death 4
(PDCD4) is a tumor suppressor gene involved in apoptosis, cell
transformation/invasion as well as in tumor progression.
Patients and methods. A consecutive series of 64 MTCs (sporadic = 56; familial = 8) was considered, with a median followup of 48 months (range=12-226). Four non-tumor thyroid tissue
samples were used as controls. RET exons 5, 8, 10, 11, 13, 14, 15
and 16 mutations, H-, N- and K-RAS exons 2 and 3 were assessed
by direct sequencing. qRT-PCR was used to detect/quantify mature hsa-miR-21, has-miR-127, has-miR-154, has-miR-224, hasmiR-323, has-miR-370, has-miR-375. PDCD4 immunostain was
automatically performed. The outcome of the neoplastic disease
was assessed by serum calcitonin (CT).
Results. MTCs were consistently associated to miR-21, miR-127,
miR-154, miR-323, miR-370, miR-375 up-regulation (p<0.001).
To further support these results, PDCD4 expression was significantly down-regulated in MTC samples, consistent with miR-21
over-expression. At the enrolment, higher miR-21 levels were
significantly associated to higher CT (p=0.0003), lymph node
metastases (p=0.001), and more advanced stages (p=0.0003).
Biochemically persistent disease at the end of the follow-up was
also associated to higher miR-21 levels (p=0.007). miR-224,
miR-323 and miR-375 up-regulation correlated with the absence
of nodal metastases (p=0.0005, p=0.0179, p=0.0259) and lower
stages at diagnosis (p=0.0439, p=0.0497, p=0.0159).
MiR-224 over-expression is associated with absent of distant
metastases (p=0.0005) and biochemical cure during follow up
(p=0.0246). Significantly lower miR-224 levels were observed in
sMTC carrying somatic RET mutations in comparison to sMTC
carrying a wild-type RET gene (p=0.0203). Differently miR-224
is upregulated in RAS mutated patients in comparison to sMTC
with wild-type RAS gene (p=0.0066).
Conclusion. In MTCs this study provides evidence that mirRNAs
expression correlate with both clinico-pathological variables and
prognosis. For this reason, evaluation of miRNA levels (especially up-regulation of miR-224) could represent a new prognostic
tool for stratification of MTC patients.
Introduction. Although the great majority of studies has shown
good overall concordance in HER2 expression between primary
and metastatic breast carcinomas, we documented a variable
discordance rate in HER2 status between breast carcinomas and
corresponding metastatic synchronous or asynchronous axillary lymph nodes, with positive or negative conversion. Taking
into account these data, as well as the frequent heterogeneity of
HER2 amplification in gastric carcinomas (GC), we have have
investigated a series of GC and their corresponding synchronous
regional metastatic lymph nodes to verify if the HER2 status may
change either with positive or negative conversion.
Materials and methods. One hundred-eight surgical specimens of
GC, obtained from an equal number of patients (M 67 - F 41; mean
age of the patients: 68.06 years; range 39-95 years) and corresponding synchronous lymph node metastases were retrospectively collected from files of different Italian units of Anatomic Pathology.
None of the patients had received neo-adjuvant chemotherapy or
other therapies before the surgery. Both primary tumors and the
lymph nodes had been collected during the same surgical procedure
and submitted to same fixation process to avoid any technical bias.
For each case, 4 µm-thick sections from the formalin-fixed paraffinembedded representative tissue blocks of the primary tumour from
two different sites and for each case, at least four metastatic lymph
nodes were tested for HER2 status by immunohistochemistry (IHC).
The analysis was carried out using the DAKO HercepTestTM kit
(Dako, Glostrup, Denmark) with an automated procedure (DAKO
Autostainer Link48), according to manufacturer’s instructions. An
antigen retrieval pre-treatment was performed by 3 changes in 0.01
M citrate buffer pH 6.0 in a microwave oven at 750 W. Each immunostained section was evaluated by the following score: 0 (absent
staining), 1+ (faint and discontinous membranous staining in < 10%
of neoplastic cells), 2+ (light to moderate lateral, basal-lateral or
complete membranous staining in > 10% of neoplastic cells), 3+
(strong, intense lateral, basal-lateral or complete staining in > 10%
of neoplastic cells). Fluorescence in situ hybridization (FISH) analysis was then performed in cases showing 2+ immunostaining using
HER2 FISH PharmDxTM kit (Dako, Glostrup, Denmark). Gene
amplification was recorded when ratio HER2/centromeric probe for
chromosome 17 (CEP17) signal was > 2.0.
Fleiss-Cohen weighted k statistics were used to assess the concordance rate between HER2 status of the primary BC and metastatic
synchronous lesions. K values between 0 and 0.2 were regarded as
no agreement, between 0.21 and 0.4 as fair agreement, between 0.41
and 0.6 as moderate agreement, between 0.61 and 0.8 as substantial
agreement, and between 0.81 and 1 as almost perfect agreement.
The statistical correlations between HER2 status and the other histopathological parameters were investigated by using Chi-squared test.
This test was also carried out to assess whether any difference in the
clinico-pathological parameters was present between discordant and
concordant GC. A P-value less than 0.05 was considered to be statistically significant. Data were analyzed by using the SPSS package
version 6.1.3 (SPSS, Chicago, IL, USA).
Microrna profiling in medullary thyroid carcinoma
F. Galuppini*1, G. Pennelli*1, S. Barollo2, M. Fassan3, V. Guzzardo1, M.R. Pelizzo4, C. Mian2, M. Rugge1
Department of Medicine (DIMED), Surgical Pathology & Cytopathology Unit, University of Padua; Padua-Italy; 2 Department of Medicine
(DIMED), Endocrinology Unit, University of Padua; Padua-Italy; 3
Department of Pathology and Diagnostics, ARC-NET Research Centre,
University and Hospital Trust of Verona, Verona, Italy; 4 Department of
Surgical Oncology and Gastroenterology Sciences (DiSCOG), Surgery
Unit, University of Padua; Padua-Italy; * The first two authors contributed
equally to this work
1
COMUNICAZIONI ORALI
Results. Tumour localization in the stomach was lower third in
52, middle third in 38 and upper-third 18 cases, seven of these
latter were located at gastro-oesophageal junction. Histopathological diagnoses, according to WHO’s criteria were: adenocarcinoma (tubular, papillary, tubulo-papillary and mucinous)
(61 cases), poorly cohesive carcinoma (34 cases) and mixed (13
cases). According to Lauren’s 61 cases were classified as intestinal type, 34 as diffuse and 13 cases as mixed. As for the grade, 55
GC were classified as low grade and 53 as high grade tumors. Regarding to the tumour stage, 1 case was stage I, 39 were stage II,
53 stage III and 15 stage IV. As far as the HER2 IHC assessment,
18 (17%) primary GC showed a 3+ score, 75 (69%) a 0 score (no
staining), while 7 (6.5%) were scored as 2+ and 8 (7.5%) as 1+.
In all of the cases scored as 2+, FISH analysis did not reveal any
amplification. Therefore among GC, HER2 was considered not
amplified in 90 cases (83.4%). Looking then to the synchronous
nodal metastases, HER2 was scored as 3+ in 16 cases (15%),
2+ in 4 cases (4%), 1+ in 5 cases (5%) cases and 0 in 82 cases
(76%) cases. FISH analysis performed in the 2+ tumours did not
show gene amplification. The K value for the concordance rate
in the HER2 status between GC and lymph node metastases was
0.651(substantial agreement).
The overall concordance rate was 90.74%; in detail, 86 cases
were concordantly HER2 not amplified in GC and in the corresponding nodal metastases, while 12 cases were HER2 positive
in both primitive and metastatic tumours. Discrepancy in HER2
status between primary GC and corresponding synchronous metastases was detected in 10 cases (9.26%). Four of the discordant
cases were HER2 not amplified in the GC and amplified in the
metastases, while 6 cases that were HER2 amplified in the GC
turned to be not amplified in the metastases. Thus, the concordance rate between primary GC and nodal metastases was 66.6%
for HER2 amplified tumours, and 95% for not amplified GC.
No significant differences in the various clinico-pathological parameters were evidenced between discordant and concordant GC.
As far as the histological classification and HER-2 expression
concerns, HER-2 amplification was significantly more frequent
in the intestinal-type GC than in the diffuse-type (P= 0.0496)
Discussion. The present multicenter retrospective study confirms
the high level of concordance between HER2 status in the GC and
in the corresponding lymph node metastases (90.74%), although a
discordance rate in HER2 has been documented (9.26%). The evidence that a defined percentage of synchronous metastases from
GC may have an HER2 status different from the primary tumour
may influence the therapeutic management and perhaps may
impact the prognostic evaluations of GC. This discrepant rate in
HER2 status might be referred to the tumour biological tendency
to develop new neoplastic cell clones. Therefore it is possible
that HER2 positive cells may appear as sub-clones in metastatic
lymph nodes as a product of the disease progression (positive
conversion). Conversely the absence of HER2 amplification
(negative conversion) in metastatic lymph nodes should not be
exclusively referred to the Trastuzumab therapy resistance, since
none of the patients had undergone to neo-adjuvant treatment.
Clinical-pathological features of 55 papillary
thyroid carcinomas bearing braf mutations other
than v600e
L. Torregrossa1, E. Sensi1, D. Viola2, M. Giordano1, G. Materazzi3, P. Miccoli3, R. Elisei2, F. Basolo1
Unit of Surgical Pathology, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy; 2 Unit
of Endocrinology, Department of Clinical and Experimental Medicine,
University of Pisa, Pisa, Italy; 3 Unit of Endocrine Surgery, Department
of Surgical, Medical, Molecular Pathology and Critical Area, University
of Pisa, Pisa, Italy
1
Background. Papillary thyroid carcinoma (PTC) represents the
229
most common type of thyroid cancer, accounting for more than
80% of all thyroid malignancies. BRAF (NM_004333) is a member of the RAF kinase family that promotes signaling through the
RAS-RAFMAPK signal-transduction cascade. An activating mutation located on exon 15 of the B isoform of the Raf kinase gene
results in a valine-to-glutamic acid substitution at amino acid 600
(BRAF V600E). Since its initial description in thyroid cancer,
BRAF V600E mutation has been widely found in PTCs, with a
prevalence of about 45% (1). In recent years, many researchers
have shown a significant association between BRAF V600E mutation and high-risk clinical-pathological features of patients with
PTCs, so this type of BRAF mutation has emerged as a promising
prognostic factor in the risk stratification of PTC (2-3). BRAF
mutations other than V600E are more rarely observed in PTC
(4-8). Although some authors have not found any association between tumor aggressiveness and these types of BRAF mutations
in PTC, their clinical significance remains still unclear (8).
Methods. 2,961 consecutive cases of PTCs were enrolled from
2006 to 2013. DNA samples were screened for BRAF mutations
in exon 15 using high-resolution melting analysis (HRMA) followed by Sanger sequencing.
Results. BRAF alterations were found in a total of 1,186 out of
2,961 PTC cases (40.0%). In particular, we found BRAF V600E
mutation in 1,131 cases and BRAF mutations other than V600E
in 55 cases. According to the histological variant of PTC, BRAF
V600E mutation was present in 41.4% of microPTCs (500 out
of 1,207), in 10.1% of follicular variants (83 out of 820), in
62.8% of classic variants (426 out of 678), in 80.6% of tall cell
variants (116 out of 144), and in 10.5% of PTCs with prevalent
trabecular and/or solid pattern of growth (6 out of 57). BRAF
mutations other than V600E were found in 18 microPTCs, 33
follicular variants, 3 classic variant, and 1 solid variant of PTC.
In the attempt to define the histological variant in the group of
microPTCs, we reclassified 16 out of 18 microPTCs as follicular
variants, 1 as classic variant and 1 as solid variant. In detail,
we found BRAF K601E mutation in 35 cases (63.6%), BRAF
V600_K601delinsE mutation in 6 cases (10.9%) and BRAF
T599I-V600_R603del in 2 cases (3.6%). Other 12 rare BRAF alterations (K601_S605delinsN, K601N+T599R, T599del, V600K,
A598_T599insV, V600delinsE, V600_K601delinsQ, T599delinsIYI, D594_T599dup, V600_K601insNTV, I582_A598dup, and
I592_A598dup) were found in single cases. The majority of these
last BRAF mutations have never been described in the literature.
Clinical-pathological features of the patients with PTC bearing rare BRAF alterations are shown in Table 1. At histological
evaluation, the large majority of these tumors resulted completely
encapsulated (53 out of 55, 96.4%) and only one case presented
lymph node metastasis (1.8%). Clinically, the majority of these
patients were classified as AJCC stage I (45 out 55, 81.8%).
Conclusion. BRAF alterations other than V600E are very rare in
PTC and in general seem to be more prevalent in PTCs with lowrisk clinical-pathological features (follicular variant, presence of
tumor capsule, absence of lymph node metastasis).
References
1
Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA,
Basolo F, Zhu Z, Giannini R, Salvatore G, Fusco A, Santoro M, Fagin
JA, NikiforovYE. BRAFmutations in thyroid tumors are restricted to
papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab
2003;88:5399–5404.
2
Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role,
molecular bases, and clinical implications. Endocr Rev 2007;28:742–762.
3
The BRAF(V600E) mutation is an independent, poor prognostic factor
for the outcome of patients with low-risk intrathyroid papillary thyroid
carcinoma: single-institution results from a large cohort study. Elisei
R, Viola D, Torregrossa L, Giannini R, Romei C, Ugolini C, Molinaro
E, Agate L, Biagini A, Lupi C, Valerio L, Materazzi G, Miccoli P,
Piaggi P, Pinchera A, Vitti P, Basolo F. J Clin Endocrinol Metab.
2012;97(12):4390-4398.
230
Tab. I. Clinical-pathological features of 55 patients with papillary thyroid carcinomas bearing BRAF mutations other than V600E.
Clinical-pathological features
Age at diagnosis (mean 46±13 yrs) Patients < 45 ys Patients ≥ 45 ys
Gender Male Female
Tumor size, mm (mean 17,6±12,9 mm) ≤ 10 11-20 21-40 >40
Histological variant * Follicular Classical Solid/trabecular
Multifocality Yes No
Bilaterality Yes No
Presence of tumor capsule Yes No
Lymph node metastasis Yes No
AJCC stages I II/III
n
27 28
17 38
18 24 9 4
49 4 2
22 33
13 42
53 2
1 54
45 10
%
49.0 51.0
30.1 69.9
32.7 43.6 16.4 7.3
89.1 7.3 3.6
40.0 60.0
23.6 76.4
96.4 3.6
1.8 98.2
81.8 18.2
* Histological variant has been defined even in PTCs sized ≤ 10 mm (defined as microPTCs according to World Health Organization Classification of
Thyroid Tumours, 2004).
4
5
6
7
8
Trovisco V, Vieira de Castro I, Soares P, Mximo V, Silva P,
Magalh‹es J, Abrosimov A, Guiu XM, Sobrinho-Sim›es M. BRAF
mutations are associated with some histological types of papillary
thyroid carcinoma. J Pathol 2004;202: 247–251.
Nikiforov YE, Biddinger PW, Thompson LDR Diagnostic pathology
and molecular genetics of the thyroid. In: Nikiforov YE, Ohori NP
(eds). Papillary carcinoma, 2nd ed. Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia, pp 183-246, 2012.
Trovisco V, Soares P, Soares R, Magalh‹es J, S-Couto P, SobrinhoSim›es M. A new BRAF gene mutation detected in a case of a solid
variant of papillary thyroid carcinoma. Hum Pathol. 2005;36(6):694697.
Lupi C, Giannini R, Ugolini C, Proietti A, Berti P, Minuto M, Materazzi G, Elisei R, Santoro M, Miccoli P, Basolo F. Association of
BRAF V600E mutation with poor clinicopathological outcomes in 500
consecutive cases of papillary thyroid carcinoma. J Clin Endocrinol
Metab. 2007;92(11):4085-4090.
Prevalence, tumorigenic role, and biochemical implications of rare
BRAF alterations. Barollo S, Pezzani R, Cristiani A, Redaelli M, Zambonin L, Rubin B, Bertazza L, Zane M, Mucignat-Caretta C, Bulfone
A, Pennelli G, Casal Ide E, Pelizzo MR, Mantero F, Moro S, Mian C.
Thyroid. 2014;24(5):809-819.
The molecular study of the pericentromeric
regions of chromosomes 9p21 locus 3,17,7 and by
determining UroVysion in urothelial carcinoma of
the bladder of high and low grade
A. Nocita, I. Putrino, A. Marasco, G. Pizzi, S. Mazza, M. Mauro,
N. Papaleo, F. Tallarigo
S.O.C. Anatomia Patologica e Citodiagnostica, P.O. San Giovanni di Dio,
ASP Crotone
Introduction. Diagnosis of primary and recurrent bladder cancer is one of the most difficult problems in urology and cytology. Bladder cancer is a chronic illness, and the patients need
continuous surveillance for early detection of recurrence and
progression. Patients with bladder cancer are typically divided by
urologists into 2 groups: those who have muscle invasive bladder cancer and those who do not. Patients with muscle invasive
cancer generally undergo cystectomy as there is a high risk of
developing metastatic disease if the bladder is not removed. “
Nonmuscle invasive “ tumors, often referred to as superficial
bladder cancer, are a pathologically and genetically heterogeneous group of tumors that includes patients with noninvasive
papillary tumors, carcinoma in situ (CIS), and tumors that invade
into the lamina propria but no deeper (T1 tumors). Noninvase
papillary urothelial tumors (pTa) recur in about 70% and progress
in abaut 5% to invasive cancer. The tumors with invasion limited
to the lamina propria (stage pT1) pose the greatest clinical problem. Local progression to potentially life-threatening muscleinvasive cancer (pT2-4) occurs in 20% to 30% of these tumors
after conservative surgical treatment. The aim of this study is to
evaluate and to detect cytogenetic abnormalites in urinary cells of
bladder cancer of low-grade papillary and high-grade tumors by
UroVysion fluorescence in situ hybridization (FISH).
Materials and methos. Fluorescence in situ hybrization (FISH)
is a technique that utilizes fluorescently labeled DNA probe to
detect chromosomal abnormalities in cells FISH is widely used
to aid in the diagnosis and management of patient with cancer.
UroVysion probe are fluorescently labeld nucleic acid probes
for use in situ hybridation assay on urine specimens fixed on
slides. The UroVysion kit consists of a 4-color,4-probe mixure
of dna probe sequence hhomologus to specific regions on chromosomes 3,7,9, and 17. The UroVysion probe mixture consist of
Chromosome Enumeration Probe (CEP) 3SpectrumRed, CEP 7
SprectrumGreen, CEP 17 SprectrumAcqua and Locus Specific
Identifier (LSI) 9p21 SprectrumGold. The probe are premixed
and predenatured in hybridization buffer for ease of use. Unlabeled blocking DNA is also included with the probes to suppress
sequence contained within the target loci that are cmmon to other
chromosomes. When hybridized and visualized, these probes
provide information on chromosomes copy number for chromosome ploidy enumeration. This UroVysion Kit is designed for
detection and quantification of chromosomes 3,7, and 17, and the
9p21 locus in human urine specimens by FISH. Cells recovered
from urine pellets are fixed on slides. The DNA is denatured to
its single stranded form and subsequently allowed to hybridize with the Urovysion probes. Following hybridization, the
unbound probe is removed by a series of washes, and the nuclei
are counterstained with DAPI (4,6 diamidino-2phenylindole),
a DNA-specific stain that fluoresces blue. Hybridization of the
UroVysion probes is viewed using a fluorescence microscope
equipped with appropriated excitation and emission filters allowing visualization of the intense red, green, acqua, and gold fluorescent signals. Enumeration of CEP 3,7, andd 17, and LSI 9p21
signals is conducted by microscopic examination of the nucleus.
Criteria for FISH Abnormality are: if, after 25 morphologically
abnormal cells have been analyzed, any of the following criteria
to select cells suspicious for malignancy include a) large nuclear
size; b) irregular nuclear shape; c) “patchy” DAPI staining; d) cell
clusters (do not count overlapping cells in clusters). A case can be
considered positive for 9p21 loss if 12 or more of 25 morphologically abnormal cells demonstrate homozygous 9p21 loss; or ≥ 4
of the 25 cells show gains for 2 or more chromosomes (3,7,or 17)
in the same cell.
Results. In this study were analyzed hundred urine samples, of
which fifty samples come from female patients and the remainder
of the male patients. Most of these patients undergo cytological
examination, because there is marked hematuria, or have recurrent
urinary tract infections, other patients are under control follow-up
for urothelial carcer The cytological diagnosis of one hundred and
twenty cases is characterized by the presence of inflammatory cellular elements such as neutrophils, istocyties, red blood cells and
urothelial cells reactive in nature with hyperchromatic nucleos. Of
COMUNICAZIONI ORALI
three hundred cases, sixty instead were negative for malignancy,
then while cases were characterized by the presence of a high
number of red blood cells and the presence of cylinders while ten
cases were considered inadequate for the diagnosis for the poor
cellularity. They were to be described in a hundred other cases, of
which fifty six were scored for the presence of different papillary
aggregates with reactive atypia or suspicious, while in the remaining forty-four cases reported year pavimentose cells that transitional urothelial cells with atypia very suspicious. The last hundred
percent of the patients are reported cases were under investigation
urologice, including the FISH assay with UroVysion. The FISH
detected in ten cases that were negative, twelve cases have trisomy
for the probe CEP7 (Green); six cases year the deletion of the locus
(LSI) 9p21, while fourtheen cases present contemporanety trisomy
for CEP17 (Water) and CEP3 (Red) and finally Eleven cases have
trisomy for CEP3 (Red) and CEP 7 (Green). While the forty-four
cases, only fifteen have polysomy for all probes examined, eighteen cases have tetrasomy in all four probes examined and finally
Eleven cases have trisomy / tetrasomy. (Figure 1). Pursued patients
were subjected to perform TUR Of the twelve cases with trisomy
for the probe CEP7, diagnosis, histology fragments bladder was
reactive hyperplasia in seven cases, while in five cases was of
urothelial dysplasia with aspects of chronic cystitis. Patients with
deletion of the locus ((LSI) 9p21, the TUR was characterized by
the presence of low-grade papillary urothelial carcinoma, whereas
cases with trisomy contemporary probe CEP17 and CEP 3, are
231
suffering from low-grade papillary urothelial carcinoma with focal
point and infiltration of the connective-sub-epithelial (pT1) while
the other trisomy CEP7 CEP3 and is characterized by the presence
of carcinoma Urothelial in situ (Ta). The cases with the mutation
of tetrasomy, the TUR is characterized both by diagnosis papillary
urothelial carcinoma of the upper (T2) (pT1), while cases with
trisomy / tetrasomy fortunately are low-grade papillary carcinoma
infiltrating (pT1) and without infilrazione. Finally the fifteen cases
characterized by polysomy for all four probes patients have a high
degree of urothelial carcinoma (G2-G3). I like because of ‘outcome
of’ histology and FISH are undergoing radical cystectomy.
Conclusions. Our data show that UroVysion multicolour FISH is
a highly sensitive and specific method for the detection f bbladder
cancer in urine specimens. FISH is easy to perform in routine cytology laboratories and has a high potential to improve the management of patients with symptoms of primary bladder cancer
or during surveillance for detection of recurrence. However this
study suggest that FISH may be a promising way to assess patient
for upper tract UC. However, additional larger studies are needed
to clearly define the criteria for positivity for upper tract UC.
References
1
Konety BR, Joyce GF, Wise M. Bladder and upper tract urothelial
cancer. J Urol. 2007;177:1636–1645.
2
Richter J, Jiang F, Gorog JP, et al. Marked genetic differences between
stage pTa and stage pT1 papillary bladder cancer detectedby comparative genomic hybridization. Cancer Res. 1997;57:2860–2864.
Fig. 1. Representative examples of normal and abnormal cells with UroVysion FISH probe set: (A) normal (‘‘disomic’’) cell demonstrating 2 signals for
all 4 probes, (B) polysomic cell with gains (3 or more copies) for 2 or more of the 4 probes. UroVysion probe set: CEP3 (red), CEP7 (green), CEP17
(aqua), and 9p21 (gold). (C) tetrasomic cell showing 4 copies of all 4 probes, and trisomy 7 cells showing 3 copies of CEP3 but 2 copies of the other
3 probes.
232
3
4
5
6
7
Sokolova IA, Halling KC, Jenkins RB, et al. The development of a
multitarget, multicolor fluorescence in situ hybridization assay for
the detection of urothelial carcinoma in urine. J Mol Diagn. 2000;
2:116–123.
Halling KC. Vysis UroVysion for the detection of urothelial carcinoma. Expert Rev Mol Diagn 2003;3:507–519 [Erratum appears in
Expert Rev Mol Diagn. 2004;4:266].
Yoder BJ, Skacel M, Hedgepeth R, et al. Reflex UroVysion testing of
bladder cancer surveillance patients with equivocal or negative urine
cytology: a prospective study with focus on the natural history of anticipatory positive findings. Am J Clin Pathol. 2007;127:295–301.
Kipp BR, Tyner HL, Campion MB, et al. Chromosomal alteration
detected by fluorescence in situ hybridization in urothelial carcinoma
and rarer histologic variants of bladder cancer. AJCP. [In press].
Marin-Aguilera M, Mengual L, Ribal MJ, et al. Utility of fluorescence
in situ hybridization as a non-invasive technique in the diagnosis of
upper urinary tract urothelial carcinoma. [see comment]. Eur Urol.
2007;51:409–415; discussion 415.
Intratumor heterogeneity of ALK-Rearrangements
and homogeneity of EGFR-mutations in the lung
mixed adenocarcinoma and adenosquamous
cancer
F. Zito Marino1, G. Liguori1, G. Aquino1, S. Ferrero2, S. Bosari2,
A. Morabito3, G. Rocco4, G. Botti1, R. Franco1
Pathology Unit, National Cancer Institute ‘G. Pascale’, Naples, Italy;
Division of Pathology, Fondazione IRCCS “Ca’ Granda” - Ospedale
Maggiore Policlinico, Milano Italy; 3 Medical Oncology Unit, Depart1
2
ment of Thoracic Surgical and Medical Oncology, National Cancer Institute ‘G. Pascale’, Naples, Italy; 4 Division of Thoracic Surgery, Department of Thoracic Surgical and Medical Oncology, National Cancer
Institute ‘G. Pascale’, Naples, Italy
Introduction. Adenocarcinoma (ADC) is the most frequently
diagnosed histological type of primary lung cancer accounting for
almost half of all lung cancers. ADC is a very heterogeneous tumor not only for the clinical and radiologic presentation but also
for histological and molecular features. Histologic intratumoral
heterogeneity is expressed both in term of a frequent minor heterogeneity due to the occurrence of several growth patterns, such
as lepidic, acinar, papillary, micropapillary and solid adenocarcinoma with mucin production, and an unusual major heterogenity
as in the adenosquamous lung carcinoma (AdSqLCs). The high
rate of morphological heterogeneity in ADCs could reflect an
heterogeneous molecular profiling. The most clinically relevant
aberrations in ADCs are EGFR-mutations, such as the in-frame
deletions E746-A750 in exon 19 and the L858R missense mutation in exon 21, and ALK-rearrangements (ALK-R). However,
few data are reported in the litterarture, about molecular features
of the mADCs and the AdSqLCs.
Aim and methods. The aim of our study was to assess EGFR-mutations and ALK-R in the different subtypes and/or growth patterns
in a series of mADCs/AdSqLCs. 590 NSCLCs were reviewed, 106
mADCs and 16 AdSqLCs out of 590 were analyzed. Comprehensive histologic subtyping was performed and the grade of mADCs
has been redefined according to the pattern-based scoring system
Fig. 1. Representative results of the different areas of a mADC with EGFR/ALK co-alterations. Each colour corresponds to a growth pattern represented in the sample. The first column: Hematoxylin and Eosin staining for each histological pattern. The second column: EGFR del exon 19 IHC results,
all patterns(B,F,L,P, T) were EGFR-positive. The third column: ALK IHC results, acinar (C), papillary(G) and micropapillary(M) were negative; solid(Q)
and solid-signet ring cell (U) pattern were positive. The fourth column: ALK FISH results, acinar (D), papillary(H) and micropapillary(N) were negative;
solid(Q) showed the classic break-apart pattern with one fusion signal and two separated orange and green signals; solid-signet ring cell (U) showed
the other pattern was a single red signal without a corresponding green signal in addition to fused signals.
COMUNICAZIONI ORALI
Tab. I. Clinical and histological features of ALK-rearranged and EGFR-mutated patients.
233
234
using the Student-Newman-Keuls Test.
Results. 10/122 were ALK-rearranged, 7 out of 10 showed
an intratumor heterogeneity of ALK-R. 12/122 were EGFRmutated, 10 carrying del exon 19 and 2 L858R mutation. All
12 EGFR-mutated were mADCs and expressed uniformly the
mutated receptor in all histologic patterns (Tab.1). Two patients
with mADC had concomitant ALK-R and del-ex19 EGFR. Both
cases showed an intratumor molecular heterogeneity of ALK-R,
on the contrary an homogeneous expression of del-ex19 EGFR in
all patterns (Fig. 1).
Conclusion. Our data suggest that EGFR mutations are generally
“driver mutations” that are acquired during early stages and provide a selective advantage to the neoplastic clone development.
On the contrary, ALK-R showed an intratumor heterogeneity
both in mADCs and in AdSqLCs. In addition ALK-R could be
likely associated with solid and solid signet-ring patterns. Moreover the presence of ALK-R is associated with high grade tumor
according Sica method. Finally Kaplan-Meier survival curve
based on univariate analysis combined II-III Sica grade and I-II
stage is significative (p value= 0.04).
proposed by Sica et al. Two TMAs were built selecting the different intratumor histological pattern. ALK-R were detected through
FISH (ALK probe Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe) and IHC (Ab anti-ALK D5F3). EGFR-mutations
were detected through IHC using EGFR mutation-specific antibodies and confirmed by RT-PCR (EntroGen’s EGFR mutation
analysis kit). All data were analyzed by SPSS software version 8.0
References
1
Sica G. et al. Am J Surg Pathol 2010.
2
Cadioli A. et al. Am J Surg Pathol 2014.
3
Travis W.D. et al Proc Am Thorac Soc 2011.
4
Chen Z.I.et al. The Oncologist 2012.
5
Gerashchenko T.S. et al. Biochemistry 2013.
6
Tomanga N. et al. Clinical Lung Cancer 2013.
7
Wang R. et al. Journal of Thoracic Oncology 2013.
PATHOLOGICA 2014;106:235-307
Apparato digerente
Anisakis appendicitis presenting as abdominal
pain: report of two paediatric cases
O. Bega, R. Scamarcio, M.A. Cascarano, S. Russo
Pathology Division, Medical School, University of Bari, (Italy)
Introduction. The infestation of anisakis was at first diagnosed
by Van Thiel in 1960.
It is caused by the third-stage larvae of Anisakis simplex, and
affects people eating raw or inadequately cooked fish. We report
two cases of paediatric appendiceal anisakiasis.
Case report. Case 1 A twelve-year-old boy presented to the paediatric surgery department with of right-sided abdominal pain.
He also complained of weight loss and decreased appetite.
He underwent laparoscopic surgical resection of the appendix without any significant postoperative complications. Histopathologic examination revealed the presence of intense inflammatory infiltrate
composed by neutrophils and leucocytes accompanied by abundant
eosinophils, surrounding a little larva of Anisakis simplex. Conclusive diagnosis was appendicitis secondary to Anisakis simplex.
Case 2 A eleven-year-old girl presented to the surgery department with brief history of abdominal pain.
Laparoscopic surgical resection of the appendicitis without postoperative complications. Histopathologic examination revealed
appendicitis secondary to multiple larvae of the genus Anisakis .
Discussion. The pathology of Anisakis is due mainly to two
mechanisms: allergic reactions and direct tissue damage.
Infestation by Anisakis should be investigated in patients with
abdominal pain after intake of raw fish, ileitis or cholitis of unclear origin, or eosinophilic gastroenteritis. The clue of gastric
anisakiasis is linked on the finding (and removal) of the worms
in the course of gastroscopy, or identifying them in the vomit
or stool of the patient. Sometimes the diagnosis is made in the
course of surgery, as well as a result of histological examination
of surgical specimens. It is also possible that this infestation is
underestimated by inadequate sampling of appendix. Infection
by larvae of genus Anisakis should be considered as a possible
cause of gastrointestinal disease, especially in countries where
raw or undercooked fish is a frequent food (such as Southern
Italy and Japan).
Conclusions. Public health authorities must be prepared to deal
with this unusual problem, due to lifestyle and alimentary habits:
anisakis is an index of this phenomenon, and the few diagnosed
cases may represent only the tip of the iceberg.
In this context, it seems worthwhile for specific training and diagnostic supports to be available for radiologists and pathologists.
References
Van Thiel et al., A nematode parasitic to herring, causing acute abdominal syndromes in man. Trop. Geogr. Med, 113, 1297,1960
del Pozo et al, Immunopathogenesis of human gastrointestinal infection
by Anisakis simplex.1999 J. Allergy Clin Immunology, 104:637-43.
Valle et al. Spontaneous splenic rupture and Anisakis appendicitis presenting as abdominal pain: a case report. Journal of Medical Case
Reports: 6: 114 2012.
Eosinophilic gastroenteritis muscular and serosal
form: a case report
G. Cipolloni1, M. Quaglieri2 , M. De Padova1, L. Sollima1, S.
Saltarelli3, P. Leocata1
1
Departement of life, healt & environmental sciences - Università degli Studi dell’Aquila; 2 Presidio ospedaliero Villa Letizia – Preturo,
L’Aquila; 3 UOC Anatomia e istologia patologica – ASL1 Abruzzo
Introduction. Eosinophilic Gastroenteritis (EG) was originally
described by Kaijser in 1937, as a disorder characterized by tis-
POSTER
sue eosinophilia that can affect different layers of the bowel wall,
anywhere from mouth to anus. Primary eosinophilic gastroenteritis is defined as disorders that primarily affect the gastrointestinal tract with eosinophil-rich inflammation in the absence
of known causes for eosinophilia such as parasitic and bacterial
infections (including Helicobacter pylori), inflammatory bowel
disease, hypereosinophilic syndrome, myeloproliferative disorders, chronic granulomatous disease, collagen vascular disease,
drug injury, and drug hypersensivity (1). The EG prevalence is 1
case per 100,000 people, the onset of disease in adulthood occurs
mainly in the third and fourth decade of life, with a higher prevalence in males. In 1970 Klein classified EG into three categories:
a mucosal, muscular and serosal form. The mucosal form of EG
(the most common variant) is characterized by vomiting, abdominal pain, diarrhea, blood loss in stools, iron-deficiency anemia,
malabsorption, protein-losing enteropathy, and failure to thrive
(2). The muscularis form is characterized by infiltration of eosinophils predominantly in the muscularis layer, leading to thickening of the bowel wall, which might result in gastrointestinal
obstructive symptoms mimicking pyloric stenosis or other causes
of gastric outlet obstruction (3). In these cases, the presence of
allergy or food intolerance is rare. The serosal form occurs in
a minority of patients with eosinophilic gastroenteritis, and it
is characterized by eosinophilc ascites (4). The GE diagnosis
necessarily requires histological (biopsy or surgical specimen)
or cytological (on ascites) confirmation of abnormal eosinophilic
infiltrate (5). There is no cut-off EG standardized diagnostic;
the majority of the authors considered that a minimum number
of eosinophils greater than 20 per high-power field. As in the
microscopic colitis, the diagnosis is difficult due to the presence
of patchy distribution, and to the absence of characteristic endoscopic pictures. In clinical suspicion is therefore recommended
multiple mucosa biopsies also seemingly unscathed (6).
Materials and methods. We report a case of a 34 year old man
with diffuse abdominal pain, not painful on palpation deep and
shallow, with a negative sign Blumberg and Murphy. There was
no remarkable feature in the physical examination. The abdomen
ultrasound examination showed an abundant Douglas effusion.
Red blood cells: 4.350.000/mm3, hematocrit: 41,70%, leukocyte:
6.900/mm3 (neutrophil: 78,90%, eosinophil: 2,80%, lymphocyte:
13,10%), platelet: 244.000/mm3 was counted in the blood examination. Liver and renal functions were in normal range. HIV
(human immuno-deficiency virus) ELISA-test (enzyme-linked
immunosorbent assay ) was negative. Parasitologic examination
and bacterial culture of stool were normal. The abdomen CT
scan without contrast confirmed the pelvic cavity effusion. The
omental adipose thickening plans was evident, mostly in the epigastric and left subfrenica sites, and in gastro-hepatic ligament.
The exam didn’t show bowel loops thickening. The chest X-ray
doesn’t evidence parenchymal inflammatory alterations and
pleural effusion. Esophagogastroduodenoscopy (EGD) revealed
distal esophagitis. In the body and in the antrum, the stomach
showed hypertrophic plicae and poorly distensible. The plicae
mucosa was normal, as well as duodenal mucosa. Antrum and
duodenum biopsies were completed. The laparoscopic investigation confirmed the presence of ascites and omentum edema. The
gastric wall appeared edematous and congested. At last, biopsy
of the gastric wall and omentum, ascites fluid withdrawal and
peritoneal washing were performed. The antrum, the
duodenum, the gastric wall and the omentum anatomic specimens were sampled and stained with haematoxylin-eosin. The
ascites fluid and the peritoneal washing were stained with
haematoxylin-eosin and papanicolau.
Results. Gastric mucosa biopsies showed a moderate chronic
gastritis, slightly active, not atrophic. Research of Helicobacter
Pylori was negative. Duodenal mucosa was regular. The cytological ascites and peritoneal washing examination showed
mesothelial cells, some lymphocytes and numerous eosinophils.
236
Fig. 1. Gastric mucosa H&E 10X.
Fig. 4. Peritoneal washing PAP 20X.
Fig. 2. Duodenal mucosa H&E 10X.
Fig. 5. Gastric wall H&E 10X.
Fig. 3. Ascites fluid H&E20X.
Fig. 6. Gastric wall H&E 20X.
Gastric wall biopsy showed an intense muscularis and serosa
infiltrate eosinophilic inflammatory. The fatty tissue fragment
corresponding to the omentum showed focal mesothelial cells
hyperplasia and nonspecific chronic inflammation with numerous eosinophils marginalized also in the vessels.
Conclusion. Eosinophilic Gastroenteritis is a rare disease, its
symptoms are related to the organ or to the organ affected area.
The EG diagnosis necessarily requires histological and cytological confirmation of abnormal eosinophilic infiltrate. The majority of the authors have considered as cut-off for the diagnosis a
minimum number of eosinophils exceeding 20 per high-power
field. Our case presents, in gastric muscle layer and in peritoneal
serous, intense eosinophilic infiltration in accordance with the
criteria described in the literature.
237
POSTER
Fig. 7. Omentum H&E 20X.
Fig. 8. Omentum H&E 40X.
References
1
Ahmad M, Soetikno RM, Ahmed A. The differential diagnosis of
eosinophilic esophagitis. J ClinGastroenterol 2000;30:242-4.
2
Kelly KJ. Eosinophilic gastroenteritis. J PediatrGastroenterolNutr
2000;30(suppl):S28-35.
3
Talley NJ, Shorter RG, Phillips SF, Zinsmeister AR. Eosinophilic gastroenteritis: a clinicopathological study of patients with disease of the
mucosa, muscle layer, and subserosal tissues. Gut 1990;31:54-8.
4
Antonini F, Saltarelli P, Frieri G, Latella G. Eosinophilic ascites. J
Gastroenterol Hepatol 2012;27(11):1759.
5
Rothenberg ME. Eosinophilic gastrointestinal disorders (EGID). J
Allergy ClinImmunol 2004;113:11-28.
6
Zhang L, Duan L, Ding S, Lu J, Jin Z, Cui R, McNutt M, Wang A.
Eosinophilic gastroenteritis: clinical manifestations and morphological characteristics, a retrospective study of 42 patients. Scand J
Gastroenterol 2011;46:1074-80.
Molecular features and methylation status in early
onset (≤40year) colorectal cancer: a population
based, case-control study
G. Magnani1, D. Furlan2, N. Sahnane2, L. Reggiani Bonetti3, F.
Domati1, M. Pedroni1
Dipartimento di Medicina Diagnostica, Clinica e Sanita` Pubblica, Universita` di Modena e Reggio Emilia, Italy; 2 Dipartimento di Chirurgia e
Scienze Morfologiche, Universitˆ di Insubria, Varese, Italy; 3 Divisione di
Anatomia Patologica, Policlinico, Modena, Italy
1
Background. Colorectal cancer is usually considered a disease
of the elderly in both sexes and increases with advancing age.
The mean age of affected individuals is estimated around 70
years. However, a small fraction of patients develops colorectal
cancer earlier (≤40 years). The reasons whereby some individuals
develop CRC at an unusual age are poorly understood.
Aim. We conducted a population based case-control study on
colorectal cancers diagnosed ≤40 years in order to characterize
genetic and epigenetic features of these tumors and to define the
frequency of those hereditary (Lynch Syndrome and Familial
Adenomatous Polyposis).
Methods. In the study we recruited 33 patients with disease ≤40
years of onset and a control group composed of 41 patients with
disease at ≥60 age, matched for the three characteristics of sex,
stage and tumor location. All tumors were investigated for MSI
test, Mismatch Repair proteins immunohistochemical analyses
and K-RAS and BRAF somatic mutations. Detection of germline
mutations was performed in cases with deficiency of Mismatch
Repair System and in patients with a clinical suspect of Familial
Polyposis, whereas MUTYH gene was investigated in all young
cases. Finally, we explored the role of aberrant DNA methylation (hypermethylation and hypomethylation) of all 74 colorectal
carcinomas. In particular hypermethylation was investigated by
MS-MLPA method and hypomethylation of long interspersed
element 1 (LINE-1) was evaluated by bisulfite DNA treatment
and pyrosequencing assay.
Results. Early onset colorectal cancer showed a high incidence
of hereditary forms (18%). K-RAS mutations were detected in
36% of early onset non hereditary cases. No BRAF mutations
were found in young patients. Hypermethylation analysis showed
a minor mean number of methylated genes in young cases. High
methylation frequency of ESR1, GATA5, WT1 genes were disclosed in both groups. Early onset colorectal cancer, excluding
Lynch Syndrome cases, showed an high hypomethylation frequency of LINE-1. Conclusion. We have found differences in the
genetic make-up of carcinomas from young and elderly patients.
Early onset tumors showed constitutional and somatic defective
of Mismatch Repair System, a similar frequency of somatic
mutations for KRAS to sporadic colorectal cancers (40%) and a
minor number of methylated genes. Hypermethylation of ESR1,
GATA5, WT1 genes suggests possible markers in the earlier
diagnosis of colorectal tumorigenesis.
CES2 and ABCG2 primary and synchronous
metastasis expression and clinical efficacy of
first-line FOLFIRI regimen in metastatic colorectal
carcinoma
G. Partipilo1, A. Mazzotta1, S. Dell’Endice1, N. Silvestris2, A.E.
Brunetti2, L. Palermo2, G. Simone3, A. Mangia1
Functional Biomorphology Laboratory; 2 Medical Oncology Unit, 3 Pathology Department; National Cancer Research Centre, Istituto Tumori
“Giovanni Paolo II”, Bari, Italy
1
Introduction. Irinotecan (CPT-11) belongs to the camptothecin
class of topoisomerase I (Topo-I) inhibitors. In combination with
leucovorin and 5-Fluorouracil (FOLFIRI), this drug was able to
improve the response rate and survival of metastatic colorectal
cancer (mCRC) patients. Enzymatic activation of CPT-11 is due
to carboxylesterase (CES), and its pharmacological behavior is
influenced by drug resistance-related proteins. Mechanisms of
resistance to CPT-11 have been identified as decreased Topo-I
levels, reduced drug activation via CES2, and increased efflux
of irinotecan and SN38 out of the cell by ATP-binding cassette
(ABC) transporters. We previously reported that the clinical response and prognosis of mCRC patients did not differ in tumors
with different thymidylate synthase (TS) or Topo-I expression.
Methods. Using immunohistochemistry we evaluated the biological role of CES2 and the expression of breast cancer resistance protein (BCRP/ABCG2) in 58 consecutive mCRC patients
238
Fig. 1. Immunohistochemical staining of CES2, ABCG2, TS, and Topo-I in metastatic colorectal cancer tissues. Representative images of positive (a)
cytoplasmic CES2 expression, (b) membranous and cytoplasmic ABCG2 expression, (c) cytoplasmic TS expression and (d) nuclear Topo-I expression
(original magnification x200).
who had undergone a first-line FOLFIRI regimen. The expression of these proteins was also examined in a group of synchronous lymph nodes and liver metastases. Furthermore, all samples
were revaluated for TS and Topo-I expression.
Results. High expression of CES2, ABCG2, TS, and Topo-I was
observed in 55%, 56%, 38% and 49% of patients, respectively
(Figure: a,b,c,d). There was a significant association between
high TS and high ABCG2 expression (P=0.049). Univariate
analysis showed that only TS expression significantly impacted
on time to progression (P=0.005). Moreover, Cox’multivariate
analysis revealed that TS expression was significantly associated
with overall survival, (P=0.01). There was no correlation with
response in any of the investigated markers. Topo-I expression
resulted significantly higher in liver metastases with respect to
the corresponding primary tumors (P=0.000), emphasizing the
role of Topo-I expression in metastatic cancer biology. CES2
expression tended to be higher in the primary tumor tissues than
in lymph nodes and liver metastases tissues (P=0.05).
Conclusion. We suggest CES2 overexpression may be a potential
marker of the malignant phenotype and that metastatic cancer
biology could predict the clinical outcome of the patients treated
with a first-line FOLFIRI regimen. A larger study would be necessary to evaluate the potential predictive and prognostic role of
CES2 and ABCG2.
References
1
Uchida K, Otake K, Tanaka K, et al. Clinical implications of
CES2 RNA expression in neuroblastoma. J Pediatr Surg. Mar
2013;48(3):502-509.
2
3
4
5
6
7
8
9
10
11
Marsh S, Hoskins JM. Irinotecan pharmacogenomics. Pharmacogenomics. Jul 2010;11(7):1003-1010.
Zhang W, Shannon WD, Duncan J, Scheffer GL, Scheper RJ,
McLeod HL. Expression of drug pathway proteins is independent of
tumour type. J Pathol. Jun 2006;209(2):213-219.
Xiao D, Yang D, Guo L, Lu W, Charpentier M, Yan B. Regulation of
carboxylesterase-2 expression by p53 family proteins and enhanced
anti-cancer activities among 5-fluorouracil, irinotecan and doxazolidine prodrug. Br J Pharmacol. Apr 2013;168(8):1989-1999.
Xie FW, Peng YH, Chen X, et al. Relationship between the Expression of CES2, UGT1A1, and GUSB in colorectal cancer tissues and
aberrant methylation. Neoplasma. 2014;61(1):99-109.
Zhang Q, Li K, Xu JH, et al. Role of ABCG2 expression driven by
cisplatin in platinum-containing chemotherapy for gastric cancer.
World J Gastroenterol. Oct 21 2013;19(39):6630-6636.
Shen B, Dong P, Li D, Gao S. Expression and function of ABCG2 in
head and neck squamous cell carcinoma and cell lines. Experimental
and therapeutic medicine. Nov 2011;2(6):1151-1157.
Wang X, Xia B, Liang Y, et al. Membranous ABCG2 expression
in colorectal cancer independently correlates with shortened patient survival. Cancer biomarkers: section A of Disease markers.
2013;13(2):81-88.
Langer R, Ott K, Feith M, et al. High pretherapeutic thymidylate synthetase and MRP-1 protein levels are associated with nonresponse
to neoadjuvant chemotherapy in oesophageal adenocarcinoma patients. Journal of surgical oncology. Oct 1 2010;102(5):503-508.
Popat S, Matakidou A, Houlston RS. Thymidylate synthase expression and prognosis in colorectal cancer: a systematic review and
meta-analysis. Journal of clinical oncology: official journal of the
American Society of Clinical Oncology. Feb 1 2004;22(3):529-536.
Azzoni C, Bottarelli L, Cecchini S, et al. Role of topoisomerase I and
239
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12
thymidylate synthase expression in sporadic colorectal cancer: Associations with clinicopathological and molecular features. Pathology,
research and practice. Feb 2014;210(2):111-117.
Paradiso A, Xu J, Mangia A, et al. Topoisomerase-I, thymidylate
synthase primary tumour expression and clinical efficacy of 5-FU/
CPT-11 chemotherapy in advanced colorectal cancer patients. Int J
Cancer. Aug 20 2004;111(2):252-258.
Evolution of intestinal polyps in familial
adenomatous disease in APC mice, related to
orlistat diet
A. Napoli, L. Polimeno, G. Arborea, G. Ingravallo, M. Mastromauro,
A. Tito, E. Maiorano, L. Resta
Dipartimento emergenze e trapianti d’organo. (DETO)
Introduction. The familial adenomatous polyposis (FAP) syndrome is characterized by a mutation of the APC (5q21-22) gene,
a suppressor gene, which inhibits cell proliferation and codifies
for beta-catenin, an important transcription factor. This mutation
is present in 70% of familial patients, while it arises de novo
in 30% of cases. Polyps may be present in the whole gastrointestinal tract, but are more frequent in the recto-sigmoid tract.
Adenomas may have different dimensions and a tubular, villous
or mixed architecture. Tumors usually arise in childhood and
growth up throughout adult life. Aim of this study was to prove
the eventual effects of a mutation of standard diet on intestinal
polyp growth in APC mice.
Methods. In order to record the effects of a diet additional
charged with orlistat, we utilized the following treatment experimental schedule (Table 1).
The standard diet consists of proteins (12.5%), soya oil (12%),
and fibers (3%); Orlistat was added to the standard diet in the experimental group. Orlistat (tetraidrolipostatin) is a drug utilized
in obese patient, which interferes with alimentary fat absorption,
reduces calories amount, thus leading to significant weight loss.
Moreover, it produces minimal decrease of blood pressure and
may prevent the occurrence of type-2 diabetes. Diarrhea is the
most frequently reported effect.
Samples of intestinal mucosa were fixed in formalin and embedded in paraffin. Four-micron thick sections were stained
with Hematoxylin-Eosin and separately observed by 2 different researchers, and the results obtained in the 2 groups were
compared. Epithelial dysplasia was classified as mild, moderate
o severe, on the basis of the glandular architecture, mucin depletion, nuclear atypia and mitotic activity.
Results. Cases 1-10 are referred to the intestinal mucosa of mice
fed with standard diet + Orlistat, while cases 11-14 to the mucosa
of mice fed with standard diet only (Table 2).
Conclusions. As already reported in the literature, intestinal
polyps from mice fed with standard diet showed intestinal
polyps with conventional histopathological features. Mice fed
with Orlistat-enriched diet manifested unequivocal and quicker
progression from intestinal polyps to adenocarcinoma. These
findings may suggest that statins should be used with caution in
consideration of several side effects, which potentially include
accelerated transition from adenomatous polyps to colonic adenocarcinoma, as highlighted in the current study.
Tab. II.
Cases Histological pattern
1
2 foci of moderate/severe dysplasia + 1 focus of
adenocarcinoma
2
Multiple foci of moderately differentiated
adenocarcinoma
3
1 focus of moderate dysplasia + 1 focus of severe
dysplasia/adenocarcinoma
4
Normal mucosa with isolated foci of carcinoma
5
1 focus of severe dysplasia with superficial
adenocarcinoma
6
Multiple foci of moderately differentiated
adenocarcinoma
7
Normal mucosa, foci of moderate dysplasia and
superficial adenocarcinoma
8
Small intestinal polyps
9
superficial adenocarcinoma
10
adenocarcinoma
11
High grade adenocarcinoma
12
Diffuse dysplasia of moderate degree
13
Intestinal mucosa with polypoid pattern
14
Intestinal mucosa with polypoid pattern
Immunohistochemical study about expression of
muc2 in colorectal cancer (crc)
O. Nappi1, M. Ammirabile1,2, C. Saporito1,2, A. D’Alterio1, C.
Della Ragione1, N. Mitilini1, M.R. Caruso1, A. Boscaino1
1
U.O.C. Anatomia Patologica, A.O.R.N. “A. Cardarelli”, Napoli; 2 U.O.C.
Genetica Medica, A.O.R.N. “A. Cardarelli”, Napoli
Background. Mucins are high-molecular-weight glycoproteins
that are widely expressed in epithelial tissues and are characterized by the presence of tandem repeat sequences rich in highly
O-glycosylated serine and threonine residues (1). Mucins types
include acid mucins and neutral mucins; at present, a total of 20
human mucins have been identified (2). These MUC proteins are
encoded by various MUC genes (3). It is well known that MUC
expression is down-regulated or up-regulated in most malignant
neoplasms of various organs (4). Interesting relations between
MUC2 expression and the pathogenesis of colorectal cancer
(CRC) have been disclosed.
MUC2 gene encode gel-forming mucins and is located in a
cluster on chromosome 11p15.5 (5). This gene codes for a typical secretory mucin, which is predominantly expressed in the
perinuclear cytoplasm of goblet cells (2), particularly in the deep
parts of the crypts, suggesting consistent presence of MUC2 in
the normal cryptal epithelium (4).
The primary function of the MUC2 gene product is to provide
a protective barrier between the epithelial surfaces and the gut
lumen (6).
Altered expression of MUC2 may be significantly correlated
Tab. I.
Groups
Dose:
mg/Kg food
N.
animals
Birth
date
Orlistat
standard
200mg/Kg
/
10
4
21/1/14
31/12/13
Age of
treatment
starting
6W
6W
Treatment
starting date
Treatment
time
Final
age
Sacrifice
date
11/3/14
18/2/14
10W
10W
16W
16W
20/5/14
22/4/14
240
Fig. 1. A: mucinous adenocarcinoma G2; B: mucinous adenocarcinoma G3; C: non-mucinous adenocarcinoma G2; D: non-mucinous
adenocarcinoma G3.
with the biological behavior and prognosis of CRC. (2). Particularly, MUC2 overexpression tended to be associated with the
T3 and T4 stage (7). Furthermore, MUC2 expression is present
in a normal tissue and generally decreased in tumor tissue of
non-mucinous adenocarcinomas, but is completely preserved in
mucinous adenocarcinomas (MUA), a distinct subtype of colon
cancer associated with microsatellite instability (8) and are often
located in the right-colon (9).
Has been found that right-sided CRCs were associated with
MUC2 expression, whereas MUC2 expression loss was more
frequently detected in left-sided carcinomas. This suggests
that there may be differences between the normal right and left
colonic segments that could favour malignant transformation
through different molecular pathways (10-11).
Has been also noted that the MUC2 expression’s level was decreased in the poorly differentiated cancers (G3) compared to
moderately differentiated cancers (G2) (12).
Furthermore, MUC2 expression showed correlation with gender
in that tumors of female patients expressed MUC2 more than
tumors of the male patients (13).
Methods. We analysed 40 cases of CRC: 18 were males and
22 were females. Of all the cases non-tumor tissues and tumor
tissues were analysed. Neoplastic samples were obtained from
right-colon (22 cases) and left-colon (18 cases). Among 22
samples arising from right-colon, 18 were MUA, while 4 were
non-mucinous adenocarcinomas. Among 18 samples arising
left-colon, 2 were MUA, while 16 were non-mucinous adenocarcinomas. The samples differ for the grading: 36 were moderately
differentiated (G2); 4 were poorly differentiated (G3).
Tumor specimens were fixed in 10% formalin for 24 h, processed
with processor LEICA for 1 night, embedded in paraffin and
cut into 3-5 μm thick sections. Some sections unstained were
mounted on superfrost glass slides for immunohistochemistry
(IHC), while others were stained with Hematoxyln-Eosin and
tumor’s area was identified by an experienced pathologist.
IHC analysis was done using the automatic immunostainer
(BenchMark¨ XT, Ventana¨). Monoclonal antibody prediluted
MUC2 (MQR-18) Cell Marque, was incubated for 32’ at 37¡ C.
Antigen-retrieval (HIR) was applied by heating sections with
buffer Ventana¨ CC1 (pH 8) at 99¡ C for 60’. Subsequently,
iViewDAB detection system was applied. All sections were
counterstained with Hematoxylin and mounted.
An IHC score was assigned to each case according to the following criteria: from 1+ to 3+.
Statistical analysis was performed using Med Calc software. The
Fisher’s exact test was used to compare the incidence of different
parameters between the two groups of patients. A p-value below
0.05 was considered as significant.
Results. The population analysed included 18 males (45%) and
22 females (55%) with a median age of 67 years (range 44-90).
Among 40 samples, was found a different expression of MUC2
at IHC. The samples were divided in two groups: low expression (+1, +2) and high expression (+3). 23 samples showed low
expression of MUC2, while 17 samples showed high expression
of MUC2.
Only in the mucinous subtype (n = 20) was found a same expression between normal and tumor tissue. In fact, in the nonmucinous adenocarcinomas (n = 20), the expression of MUC2
was different, resulting lower in tumor tissue (+2) than normal
tissue (+3).
We found that the MUC2 level in G3 was lower than G2 (Fig 1).
22 samples of right-colon showed a statistically different distribution in the expression of MUC2 (7 low and 15 high) to 18
samples of left-colon (16 low and 2 high) (P < 0.05).
Expression’s level of MUC2 did not show statistically significant
difference about gender (P >0.05).
Conclusions. In this study, we observed that MUC2 expression
was associated with tumor localization (right or left) (10-11) and
grading (G2 or G3) (12).
In disagreement with current literature, expression’s level of
MUC2 did not show a difference about gender.
The evaluation of MUC2 in IHC should be routinely included in
the diagnostic work-up of these patients, but further prospective
studies are needed to clarify the optimal management.
References
1
Gendler SJ, Spicer AP. Epithelial mucin genes. Annu Rev Physiol
1995; 57: 607-634
2
Yasuo Imai, Hidetsugu Yamagishi, Kazunori Fukuda, Yuko Ono,
Tohru Inoue, Yoshihiko Ueda. Differential mucin phenotypes and
their significance in a variation of colorectal carcinoma. World J
Gastroenterol 2013 July 7; 19(25): 3957-3968
3
Andrianifahanana M, Moniaux N, Batra SK. Regulation of mucin expression: mechanistic aspects and implications for cancer and inflammatory diseases. Biochim Biophys Acta. 2006 Apr;1765(2):189-222.
Epub 2006 Jan 27.
4
Tadashi Terada. An immunohistochemical study of primary signetring cell carcinoma of the stomach and colorectum: II.expression of
MUC1, MUC2, MUC5AC, and MUC6 in normal mucosa and in 42
cases. Int J Clin Exp Pathol 2013;6(4):613-621
5
Pigny P1, Guyonnet-Duperat V, Hill AS, Pratt WS, Galiegue-Zouitina
S, d’Hooge MC, Laine A, Van-Seuningen I, Degand P, Gum JR,
Kim YS, Swallow DM, Aubert JP, Porchet N. Human mucin genes
assigned to 11p15.5: identification and organization of a cluster of
genes. Genomics. 1996 Dec 15;38(3):340-52.
6
Allen A1, Hutton DA, Pearson JP. The MUC2 gene product: a human
intestinal mucin. Int J Biochem Cell Biol. 1998 Jul;30(7):797-801.
7
Li L1, Huang PL, Yu XJ, Bu XD. Clinicopathological Significance of
Mucin 2 Immuno-histochemical Expression in Colorectal Cancer: A
Meta-Analysis. Chin J Cancer Res. 2012 Sep;24(3):190-5
8
Byrd JC, Bresalier RS. Mucins and mucin binding proteins in colorectal cancer. Cancer Metastasis Rev 2004; 23: 77-99.
9
Verhulst J1, Ferdinande L, Demetter P, Ceelen W. Mucinous subtype
as prognostic factor in colorectal cancer: a systematic review and
meta-analysis. J Clin Pathol. 2012 May;65(5):381-8. doi: 10.1136/
jclinpath-2011-200340. Epub 2012 Jan 18.
10
Bendardaf R, Lamlum H, Ristamaki R, Algars A, Collan Y, Pyrhonen S. Response to chemotherapy (irinotecan plus 5-fluorouracil)
in colorectal carcinoma can be predicted by tumour DNA content.
Oncology. 2004;66(1):46–52.
241
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11
12
13
Bendardaf R, Lamlum H, Ristamaki R, Korkeila E, Syrjanen K, Pyrhonen S. Thymidylate synthase and microsatellite instability in colorectal cancer: implications for disease free survival, treatment response
and survival with metastases. Acta Oncol. 2008;47 (6):1046–53.
Sylvester PS, Myerscough N, Warren BF, Carlsted I, Corfield AP,
Durdey P, Thomas MG. Differential expression of the chromosome 11
mucin genes in colorectal cancer. J Pathol. 2001 Oct; 195(3):327-35.
Adam Elzagheid, Fatma Emaeting, Abdelbaset Buhmeida, Matti
Laato, Omran El-Faitori, Kari Syrjanem, Yrjo Collan, Seppo Pyrhonen. Loss of MUC2 expression predicts disease recurrence and poor
outcome in colorectal carcinoma. Tumor Biol. (2013) 34:621-628
“Intestinal Kaposi’s sarcoma. A case report”
R. Nenna1, D. Di Clemente1, T. Addati1, V. Losito1, G. Elicio1, P.
Miscioscia1, C. D. Inchingolo1
1
Anatomic Pathology Unit L. Bonomo Andria - ASL BT
Background. Kaposi sarcoma (KS) is a low-grade vascular tumor
associated with Human Herpes Virus 8 (HHV-8) infection. KS
lesions evolve from early (patch stage) macules to plaques (plaque
stage) that grow into larger nodules (tumor stage), these involve
skin, mucosa, lymph nodes and rarely visceral, as well as respira-
Fig. 1: a) Gross image radial section of large bowel, b) Mucosal and sub-mucosal localization of KS (EE: 2.5X), c) Mucosal ulceration (EE: 10X), d) KS (EE:
40X), e) CD34 (40X), f) CD31 (40X), g) HHV-8 (40X), h) KI-67 (40X), i) Perilinfonodal localization of KS (EE: 10X), l) CD34 (10X), m) HHV-8 (40X).
a)
b)
i)
c)
d)
e)
f)
g)
h)
l)
m)
242
tory and gastrointestinal tracts, developing in 1 of 4 different epidemiologic-clinical setting (classic, African, AIDS-associated and
iatrogenic). We described a case of 64 years old female submitted
to surgery (2013) to acute abdomen by suspected colon perforation.
Previously, patient had a diagnosis of chronic ulcerative colitis on a
rectal and colic biopsy and a nodular KS of the skin.
Materials and methods. Sixty-four years old woman was
presented in surgery to acute abdomen by suspected colon
perforation. Computed tomographic scan revealed free fluid in
the abdominal cavity and diffuse thickening of the walls colic,
compatible with injuries from rectum ulcerative colitis (RCU).
Patient was submitted to left colectomy surgery for perforated
abscess neoplasm suspected. We observed 55 cm left
colectomy. In left colectomy we appreciated numerous polypoid
nodules of the colic mucosa by necrotichemorrhagic appearance
(diameter ranging from 0.6 cm to 2.2 cm). In pericolic adipose
cellular there was a fistula between colon and abscess perforated
cavity. By pericolic cellular fat ten lymph nodes were isolated.
Microscopic analysis revealed a diffuse Kaposi sarcoma localizations in bowel and in two lymph nodes. Patient died two days from
after surgery for resistant thrombocytopenia to platelet transfusion.
Results. Histopathological final left colectomy diagnosis was:
“Visceral Kaposi Sarcoma with diffuse and ulcerated polypoid
nodular localization involving thickness of the colic wall complicated by fistula and perforated abscess and involving two lymph
nodes”. Microscopically, nodules examination showed several
fascicles of spindle-shaped tumor cells, with a chronic infiltrate
inflammatory composed of lymphocytes,
plasma cells and macrophages with hemosiderin in a bloody
background characterized by slit like spaces.
Spindle-shaped tumor cells showed pleomorphism and a significant number of mitotic figures. These features were confirmed
by immunohistochemistry. The neoplastic cells showed a positive reaction for Vimentina, CD31, CD34, HHV-8 and a negative
reaction for muscle actin HHF-35, CD117, DOG-1, S100 and
Desmina. Mitotic index was 10/10 HPF.
Conclusions. Kaposi sarcoma is a low-grade vascular tumor
HHV-8 associated. The biological behavior of KS depends to epidemiology-clinical settings and lost immunity. Histopathology
is the same in different settings and rare visceral involvement is
associated with poor prognosis. The colic localization of KS and
its complications are uncommon and critical, such as perforation
and obstruction. This report shows that visceral KS could mimic
RCU setting, mistaking diagnostic interpretation, if clinical history is not correctly reported. A clinic history and a previous
diagnosis of KS skin (HHV-8 correlated) and immunodeficiency,
as the RCU setting-like on endoscopic biopsy, could facilitate the
clinical identification of non-specific symptoms and
encourage early histological diagnosis in order to establish suitable therapies to prevent fatal complications.
References
Radu O., Pantanowitz L. “Kaposi sarcoma” Arch Pathol Lab Med-Vol
137, February 2013: 289-294;
Arora M., Goldeberg E.M. “Kaposi sarcoma involving the gastrointestinal tract” Gastroenterology & Hepatology-Vol 6, Issue 7, July 2010:
459-462;
Egwuonwu S. et al. “Gastrointestinal Kaposi sarcoma with appendiceal
involvement” South med J. 2011; 104: 278-281;
Weber J.N. et al. “Kaposi’s sarcoma of the bowel-presenting as apparent
ulcerative colitis” Gut, 2013August, 26: 295-300;
Majewski S. et al. “Nodular Kaposi’s sarcoma associated with colon
cancer” Acta Dermatoven Vol 20, 2011, 83-85;
Zoufaly A. et al. “Intestinal Kaposi’s sarcoma may mimic gastrointestinal stromal tumor in HIV infection” World Journal of gastroenterology 2007 September 7; 13: 4514-4516
Iron overload in gastric mucosa: a condition rarely
documented in pathology reports.
M. Onorati, P. Uboldi, M. Nicola, F. Di Nuovo
Pathology Unit, AO G. Salvini Garbagnate Milanese, MI
Introduction. Oral ferrous sulfate administration is commonly
used for prevention and treatment of iron-deficiency anemia.
Drug related gastrointestinal injury accounts for over 33 percent
of adverse drug reactions. Gastric injuries result from therapeutic
drug interactions or from exposure to heavy metals such as iron
salts, presumably from the oxidation of iron from the ferrous to
the ferric form. Especially gastrointestinal tract injury from iron
therapy is not widely recognized. Iron-related gastric mucosal
injury is related to oral iron pill ingestion and frequently occurs
in older patients. The purpose of this report is to document the
unusual morphological features of iron-related injury in gastric
biopsy specimens of an old woman and to argue about the role of
iron intake in iron-induced gastric inflammation and as potential
trigger in tumorigenesis.
Material and methods. A woman over 80 years of age presented
to our attention with anemia, fatigue and weight loss. She suffered from diabetes, chronic arthritis and cardiovascular diseases.
She had no past medical history of blood transfusions but she had
been treated with oral iron tablets at therapeutic dose. Clinical data related to blood analisys showed normal serum ferritin level so
the diagnosis of hemochromatosis and hemosiderosis were ruled
out. Due to the persistent anemia the patient underwent gastroscopy. Endoscopically, brownish and bluish discoloration mucosal
patches were present surrounded by mild hemorrhage in the antral
mucosa. In these areas of the stomach multiple biopsies were
taken. On microscopic examination, at a lower magnification,
histological staining for H&E showed the gastric mucosa with
superficial edema, chronic inflammation and a layer of brownish
granular pigment. It covered the epithelial surface and extended
into the superficial gastric pits and within the macrophages in the
lamina propria. Some of the pigment appeared crystalline in a
non-polarizing, refractile and coarsely fibrillar way, in contrast to
hemosiderin which is finely granular and intracellular. The bluish pigment was present in an extracellular location surrounded
by scattered multinucleated giant cells. Perls stain was performed
showing plurifocal positivity for iron deposition that gave a diffuse bluish tint to the macrophages cytoplasm and in the lamina
propria. Moreover a Giemsa stain showed numerous Helicobacter
Pylori curvet bacteria in the mucus overlying the mucosal surface. In consideration of the clinical data and of the morphological findings, the final histopathological diagnosis was: iron pill
chronic gastritis associated to Helicobacter Pylori infection.
Conclusion. Iron pill therapy induced gastric injury is probably fairly common. It is a lesion rarely documented in pathology diagnosis yet. These injuries often manifest as non-specific
histological changes so it is very difficult to make a definitive
diagnosis and a general diagnosis of medication-injury can be
challenging. Pathological changes due to iron injury have been
classified into four patterns by several previous series (luminal
pattern, lamina propria pattern, epithelial pattern and reticuloendothelial pattern according to the site of iron deposition). Clinical data, including medical drug history, predisposing condition
such as diets high in red meat, endoscopic findings and specific
histological patterns should alert the pathologists to make an accurate differential diagnosis list. Moreover, a correct diagnosis is
fundamental because several epidemiological studies underline
that iron pill therapy and high intake of red meat could be related
to increased gastrointestinal cancer risk. These studies were conducted on a mouse model of inflammation-associated colorectal
cancer. The hypothesis was that dietary iron and colonic mucosa
inflammation synergically activated colonic interleukins signalling promoting tumorigeneis. They showed that iron staining was
not detectable in the neoplastic cells suggesting that the increased
243
POSTER
Fig. 1. Gastric biopsy specimen showing iron deposition in lamina propria and in foreign body giant cells. Perls’ stain showing a bluish coloration
of iron deposits.
iron taken up by the tumors did not accumulate but was instead
utilized for cell proliferation. Finally they suggest that specifically oral iron therapy may be detrimental in inflammatory bowel
disease since it may be exacerbate colonic inflammation and increase colorectal cancer risk. In this case the gold standard would
be that the use of oral iron for the treatment of anemia should be
avoid as it is unlikely to improve anemia and may exacerbated
colonic inflammation increasing the risk of colorectal cancer in
patients affected by chronic inflammatory bowel diseases. Also in
gastric mucosa the iron has been detected in the interstitial space
or in the macrophages but not in the cytoplasm of the epithelial
gastric cells suggesting that it could be a factor inducing gastritis
with the same mechanism hypothesized for colonic mucosa and
could synergical activate gastric interleukins signalling promoting tumorigenesis. For these reasons, it is important that these
lesions would be documented in pathology reports to do a better
follow-up in order to reduce cancer risk. However, further studies
will be necessary to validate these theories
References
1
Morgan Jl et al. Increased dietary iron and radiation in rats promote
oxidative stress, induce localized and systemic immune system responses, an alter colon mucosal environment. Faseb J, 2014; 28 (3):
1486-98.
2
Chua ACG et al. Dietary Iron Enhances colonic inflammation and
IL-6/IL-11-Stat3 signaling promoting colonic tumor development in
Mice. PLOSE ONE, 2013; 8 (11): 1-12.
3
Kobayashi T et al. An autopsy case of secondary iron-overload cardiomyopathy. Intern Med, 2013; 52 (12): 1369-73.
4
IJssennagger et al. Dietary Heme Alters Microbiota and Mucosa of
5
6
mouse colon without fuctional changes in host-microbe cross-talk.
PLOSE ONE, 2012; 7 (12): 1-10.
Kaye P et al. Iron-induced mucosal pathology of the upper gastrointestinal tract: a common finding in patients on oral iron therapy.
Histopathology, 2008; 53 (3): 311-7.
Haig A et al. Iron-induced mucosal injury to the upper gastrointestinal
tract. Histopatholgy, 2006; 48 (7): 808-12.
Idiopathic cystoides intestinal pneumatosis:
an additional case.
M. Onorati1, G. Petracco1, P. Uboldi1, C. D’Urbano2, F. Di Nuovo1
Pathology Unit, AO G. Salvini Garbagnate Milanese, MI;
Unit, AO G. Salvini Garbagnate Milanese, MI
1
2
Surgical
Introduction. Cystoides intestinal pneumatosis (PCI) is an uncommon disease with an unknown etiology, characterized by the
presence of gas cysts within the submucosa or subserosa of the
intestine. Since it was first described by Du Vernoy in autopsy
specimens in 1730 and subsequently named by Mayer as PCI
in 1825, it has been reported in some reports. PCI is a physical
or radiographic finding, associated with a wide spectrum of diseases, ranging from life-threatening to innocuous conditions. For
this reason, the PCI management can vary from urgent surgical
procedure to clinical management. The presenting clinical picture
may be very heterogeneous and represent a challenge for the clinician. In some cases, PCI is not related to a specific disease and
its etiology remain unknown. This paper describes the case of a
idiopathic PCI in a patient with abdominal pain and stipsis in order to stress, more generally, the difficulties in the identification
of correct pathogenesis and consequent treatment. In fact many
244
patients undergo misdiagnosis and surgical mistreatment.
Material and methods. A 54-year-old woman suffering from recurrent intestinal subocclusive episodes, presented to our hospital
for severe abdominal pain. Nausea, vomiting, and constipation
were associated. Medical history and routine physical examination did not reveal any significant other disease. On physical
examination, abdomen was distended with no bowel sounds.
Serum calcium, kidney and liver function, and urinalysis results
were normal. Test for fecal occult blood was negative. During the
colonoscopy, several mucosal polypoid lesions, 3 mm to 20 mm
in diameter, were noted in the splenic flexure. Since the lesions
were easily indented with gentle pressure and had a bluish color,
PCI was suspected. Computed tomography confirmed multiple
air pockets in the intestinal wall at the splenic flexure. Since numerous occlusive episodes did occur, the patient underwent laparoscopic left colectomy. Grossly, the surgical specimen showed
numerous bubbles ranging in size from a a few millimetres to
2 centimetres. They were single and in clusters and involved
the mucosa, the submucosa and the subserosa. Microscopically,
several cysts were present into the mucosa, submucosa and subserosa. The cysts were partially lined by an endothelial layer. Inflammatory cells including leukocytes, eosinophils, plasmacells,
lymphocytes, foreign body cells and macrophages surrounded the
cysts. On the bases of the morphological findings the diagnosis of
a primary idiopathic PCI without any other underlying causes was
made. This patient required no other specific treatment and has
remained asymptomatic being healtly to the follow-up.
Conclusion. PCI has been associated with a wide variety of
underlining etiologies to explain the cyst formation and the abnormal accumulation of gas into them. Several theories has been
suggested. The most important of them are briefly described.
The mechanical theory based on intestinal obstruction, inflammatory bowel disease, ischemic bowel disease, gastroenteric
tumor, anorectal surgery, bowel preparation or colonoscopy that
result in intestinal wall injury or increased intraluminal pressure causing the intramural accumulation of gas. However, this
theory cannot explain how the cysts are maintained once they
have formed. The pulmonary theory (pulmonary diseases, such
as chronic obstructive pulmonary disease, asthma, and interstitial
pneumonia) may result in pulmonary alveolar rupture producing a
pneumomediastinum that dissects along the aorta and then along
the mesenteric vessels to the bowel wall. The bacterial theory: the
gas is produced by gas-forming bacteria that enter the mucosal
barrier through mucosal rents or increased mucosal permeability
and produce the gas within the bowel wall. Indirect support for
this theory was obtained by the successful treatment of PCI with
antibiotics. However, the presence of aerogenic bacteria in the
cysts has not yet been proven. Although Yale et al reported that
pneumatosis has been produced in germ-free rats by the injection of Clostridium species into the wall of the intestine, the
isolation and cultivation of these organisms is rarely possible.
Conversely, many of the patients who have pneumoperitoneum
resulting from the rupture of cysts show no signs of peritonitis, prompting the theory that in this population, the gas is not
caused by bacteria. The chemical theory or nutritional deficiency
theory: malnutrition can prevent the digestion of carbohydrates
and increased bacterial fermentation in the intestine, producing
large volumes of gas leading to distention and ischemia and
subsequently the submucosal dissection by the gas. Although
there are many symptoms of PCI, including abdominal pain,
abdominal distention, diarrhea, mucous stool and bloody stool,
none is disease specific. The cysts may cause obstruction by
internal or external compression of the bowel lumen when the
cysts become larger. Complications associated with PCI occur in
approximately 16.3% of cases and include intestinal obstruction
or intestinal perforation. In certain cases, pneumatosis intestinalis
may be considered a surrogate marker for intestinal ischemia and
impending perforation. However, the condition may also occur in
Fig.s 1. Gross features of cystoides intestinal pneumatosis. The air
bubbles are located along the entire colic mucosa. Haematoxilin
and eosin stains show variably sized cysts in the colic wall. Typical
macrophages and giant cells line some cysts filled of eosinophilic
amorphous material. Other cysts appear as optically empty spaces.
a benign context and is no longer considered a disease but rather
a sign of a variety of other abdominal pathologies conditions, and
its significance needs to be considered in accordance with each
patient’s clinical context. Clinicians believe that it does not have
a single etiology. However, rare cases of idiopathic PCI including
our case have been described. In general, the diagnosis of PCI is
based on endoscopy or plain radiography of the abdomen and
is usually not difficult because the typical radiolucency appears
as grape-like clusters or honeycomb-shaped shadows along the
wall of the intestine. After the identification of PCI, a prompt
further evaluation, including concomitant radiographic findings,
of the patient should be conducted. In conclusion, PCI is still a
poorly understood disease and the variety of treatments reflect the
poor knowledge regarding the complications of this disease. For
this reason management and treatment guidelines are required.
Conservative management is recommended in most patients
and surgery is usually indicated when acute and life-threatening
complications such as bowel necrosis, perforation or peritonitis
appear. A laparoscopically assisted approach is a good indication
for partial colectomy in cases of PCI that are non-responsive to
standard conservative treatment as our case.
References
Zorgdrager M, Pol R. Pneumatosis intestinalis associated with enteral
tube feeding. BMJ Case Rep. 2013 Dec 3;2013.
El Matiallah MA et al. Intestinal cystic pneumatosis revealed by stenosis
of a gastrointestinal anastomosis: report of a case. Pan Afr Med J.
2013 Aug 8;15:125.
Milone M et al. Computed tomography findings of pneumatosis and
portomesenteric venous gas in acute bowel ischemia. World J Gastroenterol. 2013 Oct 21;19(39):6579-84.
Wu LL et al. A systematic analysis of pneumatosis cystoids intestinalis.
World J Gastroenterol. 2013 Aug 14;19(30):4973-8.
Choi YA et al. A case of pneumatosis intestinalis associated with sunitinib treatment for renal cell carcinoma. Korean J Gastroenterol. 2013
Jun;61(6):347-50.
Adar T, Paz K. Images in clinical medicine. Pneumatosis intestinalis. N
Engl J Med. 2013 Apr 11;368(15):e19.
245
POSTER
Histological alterations induced by radiotherapy
in mesorectal tissue: evaluation of 90 consecutive
cases
L. Reggiani Bonetti1, F. Domati2, L. Maccio1, A. Farinetti3, A.
Manenti3
Anatomia Patologica – Az. Ospedaliero – Universitaria Policlinico di
Modena; 2 Dipartimento di Medicina Interna -Az. Ospedaliero – Universitaria Policlinico di Modena;3 Dipartimento di Chirurgia Generale -Az.
Ospedaliero – Universitaria Policlinico di Modena
1
Introduction. Radiotherapy has become an important tool in
the treatment of rectal cancers. Its relevance in rectal cancer
management is well known, as well as the post-treatment complications with their early and late clinical sequels.
Material and methods. Aim of our study was to evaluate the
histological alterations in a cohort of 90 patients who underwent a
50-Gy preoperative radiotherapy associated to FOLFOX scheme
chemotherapy (50 subjects), 50-Gy preoperative radiotherapy
alone (20 patients), 25-Gy preoperative radiotherapy alone (20
cases) and consequently submitted to a restorative low anterior resection of the rectum. Cases were extrapolated from a Specialized
Colorectal Cancer Registry instituted in 1984 in the Province of
Modena in which clinical and pathological informations about the
tumors and its behaviour were meticulous scheduled. In the study
were included only tumours preoperatively classified as Stage-II
at Computed Tomography (CT) or Magnetic Resonance (RM). On
the contrary, cases with a preoperative extended neoplastic infiltration of the mesorectum were excluded, in order to keep out the
possible histological alterations associated to neoplastic regression
and not induced by radiotherapy. The cases were compared with
10 controls represented by non-treated subjects underwent to rectal
surgical resection for neoplasia.
Results. We observed fibrosis of the mesorectal adipose tissue
(88%), vasculitis in mesorectal vessels (60%), fibrotized lymph
nodes the whole mesorectal adipose tissue (32%) and perineural
deposition of fibrosis (11%). These alterations were observed in
more than half of the cases but not in the controls (p<0.005) and,
although ubiquitously observed in the mesorectum of all subjects, were mainly observed in patients who underwent a 50-Gy
preoperative radiotherapy associated to chemotherapy. Conclusion. The detection of these histopathological alterations in the
mesorectum can give reason of the well-known post-operative
complications and long-term sequels.
Cavernous lymphangioma of the small-bowel
mesentery: a case report
N. Scibetta, N. Arena, E. Unti
U.O.C. di Anatomia ed Istologia Patologica, A.R.N.A.S. Ospedali Civico
e G. di Cristina, Palermo
Introduction. Lymphangioma (LA) is an uncommon malformation of lymphatic system, which shows benign proliferation of
lymph vessels in the mesenthery and all parts of the bowel wall,
the latter with the characteristics of submucosae tumors covered
with normal mucosa. The incidence of LA in the gastrointestinal
tract is low, and most cases are solitary. Herein, we present a
case of a LA of the small-bowel mesentery and discuss differential diagnosis.
Methods. A 16-year-old young-man with abdominal pain of the
lower abdomen and constipation was admitted to our hospital.
He had no significant past medical history. Physical examination revealed no remarkable abnormality in the abdomen. Laboratory rests (samples) showed hypochromic microcytic anemia.
Tumor marker levels (CEA, CA19-9) were within normal ranges.
X-ray findings on contrast study of the small bowel showed an
ileum loop dropped around a mesenteric mass. The patient underwent surgery.
Results. 49cm of the terminal ileum with the adjunct mesentery
was resected. The cross-sections of the resected segment showed
a mass of 5 cm in the mesenthery, involving all parts of the
bowel wall. There are several protruding ileal mucosal lesions
covered with normal mucosa, raging 3-5 mm in diameter. Microscopically the sections showed markedly dilatated lymphatic
channels, lined by a single very attenuated layer of flattened
endothelial cells with no atypia and contained small amounts
of proteinaceous fluid, with few erythrocytes. The wall of the
spaces was built up of fibroconnective tissue accompanied by aggregates of lymphoid tissue as well as normal arteries and veins.
Moreover, fascicles of smooth muscle as well as collagen bundles
could be seen. Endothelial cells were positive for all vascular
markers used (FVIII, CD31, CD34), negative for cytocheratin,
EMA, calretinin, HBME.
Spindle cells were found arranged in poorly delineated fascicles,
positive for vimentin, desmin, alpha smooth muscle actin, negative for PGR, ER, SI00, HMB45.
The final diagnosis was cavernous lymphangioma.
Conclusions. In this case there is not prominent synchronous
involvement of viscera, bones and internal soft tissues of the
trunk and pelvis.
The differential diagnosis include cavernous haemangioma,
there is no reliable endothelial markers that can distinguish
blood vessel from lymphatic endothelium, however there is an
important histologic difference as the former is characterized
by large thick-walled irregular spaces with extensive dissection
of collagen bundles. A conservative surgical treatment with segmental intestinal resection and termino-terminal anastomosis
was curative.
References
1
Ralf J Rieker, Armin Quentmeier, Carsten Weiss, Ulrich Kretzschamar, Kerstin Amann, Gunhild Mechtersheimer, Hendrik Blaker,
Herwart F. Otto. Cistic Lymphangioma of the Small-Bowel Mesentery.
Pathology Oncology Research,vol 6,N.2,2000.
2
Woo Chul Chung, Hye-Kang Kim, Jin Young Yoo, Jng Mo Yang.
Colonic lymphangiomatosis associated with anemia. World J Gastroenterol 2008 October 7;14(37):5760-5762
Incidental intestinal spirochetosis in colorectal
polyps
S. Serra, S. Hafezi, R. Vajpeyi, P. Gill, L.M. Wang, R. Chetty
Departments of Pathology, University Health Network, Toronto, Oxford
University Hospitals, Oxford, UK
Background. Intestinal Spirochetosis (IS) is a colonic infection
caused by two anaerobic intestinal spirochete species namely
Brachyspira aalborgi and B. pilosicoli. The infection is common in developing countries, while in western countries, occurs
mostly in HIV+ patients. We describe the clinicopathological
features of IS in a non-HIV cohort, in particular an association
with colorectal polyps.
Material and methods. An archival search for IS over a 9-year
period was performed in the Departments of Pathology, University Health Network, Toronto and John Radcliffe Hospital,
Oxford, UK. Clinical information recorded included: demographics, symptomatology, geographical location and association with
polyps. The slides were reviewed and the location of the microorganism was recorded.
Results. Ninety-one biopsies from 42 patients (44 biopsies were
from the right colon, 45 from the left colon and 2 site unknown)
were retrieved. The age range was 19 to 74 years (mean 52.67
years) and 25 (59.5%) were males. None of the patients were immunocompromised or HIV+. Thirty-four patients out of 42 were
residents in Northern Ontario. Screening for polyps or colorectal
cancer, altered bowel movement, diarrhoea and abdominal pain
were the reasons for the colonoscopy. There were 19 hyperplastic polyps (16 with IS), 4 sessile serrated polyps (SSPs) all with
246
IS,10 tubular adenomas (1/10 with IS), 4 patients had chronic
active colitis (0/4 IS), 3 haemorrhoids, 9 diverticular disease and
5 irritable bowel disease. Spirochetosis was diffusely identified
on the surface of the normal colonic epithelium in 65 biopsies,
focally in 13 and it was negative in 4.
Conclusion. The majority of the Canadian patients were from
Northern Ontario, slightly more in males and the infection was
asymptomatic. No patient was immunocompromised. This suggests the possibility of spread through contaminated water in
small communities in Northern Ontario. The presence of IS on
the surface of the normal colonic epithelium and on the surface
of hyperplastic and SSPs is most likely due to the glycoproteins
expressed on the surface of the epithelium, which function as
receptors for the microorganisms.
Fig. 1.
Lymphoepithelioma-like gastric carcinoma with
epithelioid granulomas: a case report
S. Squillaci1, R. Marchione1, M. Chiudinelli1, M. Piccolomini1,
E.Vielmi1, M. Ungari2
1
Unità Operativa di Anatomia Patologica, Ospedale di Vallecamonica,
Esine (Bs), Italia;2 Istituti Ospitalieri di Cremona, Anatomia Patologica,
Cremona
Introduction. Lymphoepithelioma-like carcinoma (LELC) of
the stomach is a rare histological type of gastric cancer with favourable prognosis that comprises less than 5% of all gastric carcinomas. More than 80% of gastric LELCs have been shown to
carry EBV genome with expression of several EBV-latent genes
(Latency I program) as opposed to 6% and 7% of diffuse and
intestinal-type ordinary adenocarcinomas respectively. Interestingly, geographic or racial influences do not seem important in
the relationship of EBV with gastric LELC. Another subset of
gastric LELCs shares a microsatellite-instability (MSI)-high
phenotype. From 7% to 39% of gastric LELCs have been found
to be associated with MSI-high with geographic variability in
this expression and a mutually exclusive connection with EBV
infection. The development of sarcoid-like associated reactions
is a rare event in LELCs.
Methods. A 45-year-old man presented to the Division of
Surgery of Hospital of Vallecamonica with epigastric pain and
anaemia. He underwent endoscopic biopsy that revealed a poorly
differentiated adenocarcinoma. A gastrectomy was performed
with lymph nodes dissection.
Results. A crateriform tumor of the gastric body measuring
7,6 cm in diameter with central ulcer was macroscopically observed. Microscopically, the neoplasm was composed of round
to polygonal cells with poorly defined cell borders prevalently
arranged into ribbons, nests and abortive glands within a dense
lymphohistiocytic stroma with numerous epithelioid and giant
multinucleated cells. Six of the dissected lymph nodes revealed
tumor metastasis and epithelioid granulomas with Langhanstype giant cells were noted both in the tumor stroma and in
three metastatic lymph nodes. Panoramic view of the H&E
sections showed a neoplastic expansive well-demarcated nodular growth, which extended into the adipose perigastric tissue
with focal visceral peritoneum invasion. The tumor cells were
positive for AE1/AE3 pan-cytokeratin whereas they were negative for chromogranin, synaptophysin, -HCG, CD45, CD3 and
CD20 . The stroma consisted of CD3 positive T lymphocytes
intimately intermixed with epithelial neoplastic cells and CD20
positive B lymphocytes in follicular aggregates at the borders
of the lesion.
The in situ hybridization for EBV-encoded small RNAs
(EBER-1 and 2) showed an intense nuclear hybridization signal
corresponding to the carcinoma cells whereas the neoplasm did
not stain for LMP1 (Fig. 1). The patient is well 7 months after
the operation, and there has been no recurrence until now.
Conclusions. Granuloma formation is quite common in the
regional lymph-nodes of the tumor tissue but rarely occurs in
the stroma of the neoplasms, particularly of LELC. Only two
reports about epithelioid granulomas occurring in LELC of the
stomach have been published to date (1,2). These studies suggest that the presence of sarcoid reactions in the stroma with
prominent lymphocyte infiltration could represent a host immune response to the carcinoma.
References
1
Tamura T, et al. Lymphoepithelioma-like carcinoma of the stomach
with epithelioid granulomas. Case Rep.Gastroenterol.2010;4:361-368.
2
Chaturika H, et al. Epstein-Barr virus-associated lymphoepitheliomalike gastric carcinoma. Arch.Pathol.Lab.Med.2008;132:706-709.
Citologia
Endoscopic ultrasound-guided fine needle aspiration
cytology in mediastinal masses: a personal
preliminary experience by cell block procedure
A. Ieni1, P. Todaro1, S. Crinò2, F. Monaco3, G. Speciale1, G.
Tuccari1
Azienda Ospedaliera Universitaria “Gaetano Martino” and Department
of Human Pathology “Gaetano Barresi”, Section of Anatomic Pathology,
University of Messina, Italy; 2 Azienda Ospedaliera Universitaria “Policlinico G. Martino” and Department of Internal Medicine – Digestive Endoscopy Unit, University of Messina, Italy; 3 Azienda Ospedaliera Universitaria “Policlinico G. Martino” and Thoracic Surgery Unit, University of
Messina, Messina, Italy.
1
Introduction. Endoscopic ultrasound-guided fine-needle aspiration cytology (EUS-FNAC) has been proven to be of significant
value in the diagnostic evaluation of benign and malignant
diseases as well as for the staging of malignant tumors of the
human body. It is noteworthy the diagnostic yield of EUS-FNAC
partially depends on the site, size and characteristics of the target tissues as well as certain technical and procedural factors;
moreover, it is mainly dependent on the expertise, training and
interaction between the endosonographer and cytopathologist.
We report herein a preliminary personal experience performed
in a small series of mediastinal masses retrospective archival
cytological pancreatic samples, a series of immunocytochemical
markers to assess. Moreover, the presumptive cytological diagnosis was strongly supported by the application of an appropriate
immunohistochemical algorithm on serial sections obtained by
the cell-block procedure.
Materials and methods. We analyzed a series of 9 mediastinal
neoplastic lesions, obtained from an equal number of patients
POSTER
(M:F=4:5; age range 34-73 years; mean age 59.3 yrs). EUSFNAC on mediastinal masses was performed by using a convex
array echoendoscope (EG 3870 UTK, Pentax – Tokio, Japan)
and by making three passes with a 22G needle. The specimens
were processed by an in-room cytopathologist and immediately
examined for adequate cellularity after staining by Haematoxylin and Eosin (H&E). A second slide was immediately fixed
in 98% ethanol and stained with Papanicolaou. Any excessive
material, including needle and syringe utilized in the procedure,
was rinsed in 10 ml of 50% ethanol in a specimen container. All
content was centrifuged in a 10 ml disposable centrifuge tube at
4,000 rpm for 6 minutes to create 1 or 2 pellets; the supernatant
fluid was decanted and the pelleted material was immediately
fixed in a freshly prepared solution of 4% neutral buffered formalin for 45 minutes. The, the cell pellets were placed in a
cassette and stored at 80% ethanol until ready for processing in
an automatic tissue processor (Leica TP 1020). The cell blocks
obtained were embedded in paraffin at 56¡ C and successively
3 µm thick sections were cut and routinely stained by H&E;
parallel serial sections of the same thickness were mounted on
poly-lysine-coated glasses and submitted to immunocytochemical procedures by standard method on a Ventana Benchmark
automatic immunostainer utilizing the following commercially
purchase antisera: broad spectrum cytokeratins AE1/AE3, cytokeratin 7, cytokeratin 20, EMA, ER, PgR, CD45, CD20, CD3,
CD5, CD30, TTF-1, CD56, NSE, Cromogranina A, Napsina-A,
CD99 and HER-2. The growth fraction was recorded as Ki67 LI.
Results. The smears of all cases generally exhibited a haemorrhagic background with isolated or aggregates of small cells,
with hyperchromic nuclei and differently represented cytoplasms, indicating thus a not uniform histogenesis. The presumptive cytological diagnosis was large cell diffuse B lymphoma
(three cases), thymoma (two cases), metastases (three cases); 1
case was attributed to disseminated sarcoidosis due to presence
of epithelioid cells. The immunohistochemical markers confirmed the nature of neoplastic lesions, clarifying that metastases
were attributable to ductal breast invasive carcinoma, small cell
lung carcinoma and cortico-surrenalic carcinoma.
Discussion. EUS-FNAC is widely considered an useful approach
in daily practice of major medical center around the world for diagnosing and staging mediastinal malignancies. By this method
the great majority of cases can be diagnosed with some difficulties on cytologic samples, either to differentiate benign from
malignant lesions, either to identify their histogenesis. However, additional diagnostic difficulties may be attributed to the
presence of extensive tumor necrosis, inflammatory associated
processes and limited sampling; in these cases, the cell block
technique can be of value to the pathologist in order to microscopically evaluate histological mimickers and to perform immunohistochemical stains in serial sections.
Primary effusion lymphoma: a cytological
diagnosis in an immunocompetent patient without
detectable tumor masses
M. Onorati1, G. Petracco1, M.M. Albertoni1, G. Pepe2, F. Di Nuovo1
Pathology Unit, AO G. Salvini Garbagnate Milanese, MI; 2 Lung Unit, AO G.
Salvini Garbagnate Milanese, MI
1
Introduction. Primary effusion lymphoma (PEL) is a complex,
rare and aggressive subtype of non-Hodgkin B-cell lymphoma
(NHL) that exhibits a non-B, non-T “null” phenotype. It is usually associated with human herpes virus 8 (HHV-8) in immunocompromised individuals, in the setting of human immunodeficiency virus infection, organ transplantation, or in rare cases of
advanced age. It was firstly described in 1989 as a body-based
cavity lymphoma. It is mainly found as a primary lymphomatous
effusion in the serous body cavities without clinically identifiable tumoral masses. The malignant effusion usually involves
247
only one body cavity: pleural, pericardial or peritoneal. PEL
is diagnosed when a cytological specimen shows characteristic
large pleomorphic, immunoblastic, plasmablastic, or anaplastic
cells with a peculiar “null-cell” phenotype. Demonstration of
human herpesvirus 8 latent antigens is required for diagnosis,
and treatment modalities are limited at this time. In rare cases,
patients are non-immunocompromised el¬derly individuals
from regions where human herpes virus 8 (HHV8) is endemic,
such as Mediterranean regions. Here, we report a cytological
diagnosis of a case of HHV-8-related PEL in a elderly, immunocompetent patient, without detectable solid lung and pleural
masses.
Matherial and methods. An immunocompetent 94-year-old
man was admitted to our hospital due to a one month history
of anorexia, fatigue, shortness of breath, and nonproductive
cough. He had no habit of regular alcohol consumption, he was
not a smoker and had not been previously exposed to asbestos.
His clinical family history was noncontributory. No history of
malignancy, HIV, hepatitis B or hepatitis C infection were noted
or other sources of immunosuppression. His blood pressure was
130/60 mm Hg, pulse was regular with a rate of 80 beats per
minute and body temperature was 37.5°C. Conjunctivae were
pale. Heart sounds were regular without murmur or friction
rubs. Chest auscultation revealed remarkably diminished breath
sounds at the left and the right thorax. There was no cyanosis,
clubbing, or edema at the extremities. Peripheral lymphadenopathy and organomegaly were not observed. Whole body Computed and Positron emission tomography-scan revealed bilateral
pleural effusion, without evidence of abnormal lymphoadenopaties, lung and pleural masses. Thoracentesis with continuous
pleural effusion drainage was immediately performed. The
culture of the effusion was negative. Smears were prepared from
the pleural fluid and were stained with hematoxylin and eosin
(H&E), Giemsa, and Papanicolaou (Pap) methods. Cell block
was fixed in 4% buffered formaldehyde and stained with H&E.
Pap and H&E revealed the presence of large atypical malignant
lymphoid cells with rounded nuclei, multiple, prominent huge
nucleoli and scant cytoplasm. Mitoses were frequent and atypical. Immunophenotypic profiles were determined according to
standard immunoperoxidase methods, using paraffin sections
from the cell block. The immunophenotyping of the neoplastic
cells evidenced a “null” phenotype with the expression of CD45
and CD30. The other immunohistochemical markers performed
(TTF-1, CK7, D2-40, calretinin, CD20, CD3, CD15, PAX5,
CD138) were negative. HHV-8 was detected in the neoplastic
cells confirming the suspicion of PEL.
Conclusion. The diagnosis of PEL is based on the combination
of clinical features, morphology, immunophenotype, virology
and molecular findings. The cytological preparations (smears,
cytospin and cell block) of the malignant effusion constitute the
main material for pathologic examination and diagnosis. The
cytologic features are usually large cells with moderate to abundant basophilic cytoplasm, round to irregular nuclei and prominent nucleoli with both morphologic features of immunoblastic
and anaplastic large cell lymphomas. Immunophenotypically,
and although PEL is B-cell clonal in origin, malignant cells
express leukocyte common antigen (CD45) and post-germinal
center B cell/plasma cell associated antigens (CD38, CD138)
suggesting a late stage of B-cell differentiation, but classic Bcell markers (CD19, CD20, CD79a) and T-cell markers (CD3,
CD4, CD8) are absent. In fact, it exhibits a non-B, non-T “null”
phenotype. PEL cells are commonly co-infected by EBV and
HHV8. However, EBV latent membrane protein 1 staining is
negative until reactivation. Molecular analysis usually demonstrates the post-germinal center B-cell origin of PEL by revealing a clonal immunoglobulin gene rearrangements and somatic
hypermutation. Therefore, cytomorphological differentiation of
PEL from other lymphomas involving the body cavities may
248
Fig. 1. Papanicolaou and H&E stains show atypical large lymphoid cells with irregular and lobulated nuclei. Immunocytochemistry of the pleural
effusion shows CD30 reactivity in cytoplasmic membrane and nuclear HHV8 reactivity of atypical lymphoid cells.
be difficult. The major differential diagnoses include anaplastic
large cell lymphoma (ALCL), diffuse large B-cell lymphoma
(DLBCL), Burkitt’s lymphoma, and pyothorax-associated lymphoma (PAL). Differential diagnoses include also benign
conditions like heart failure, volume overload, parapneumonic
effusion, tuberculotic pleuritis, uremic pleuritis and hemodialys.
Here we have documented a case of HHV-8-related PEL developed in a healthy, elderly patient. The association of clinical,
histologic, and radiologic findings was necessary to lead us to a
correct diagnosis. Moreover the immunophenotype and HHV-8
positivity were helpful in distinguishing this entity from other
lymphomas that can present as a body cavity effusion. The presented patient did not have any underlying immunodeficiency
conditions, except for advanced age. It is well known that in
the elderly, the T-cell response seems functionally decreased.
In fact, clonal CD8-positive T cells, although numerous and associated with mature, memory cell phenotype, are functionally
impaired due to a markedly restricted epitope-specific repertoire
and a 100-fold decrease of T-cell specificity. The importance to
distinguish PEL from other clinical benign condition affecting
pleural cavity in non-immunocompromised patients with an
unremarkable history is also underlined. This report may arouse
clinicians’ attention regarding the importance of evaluation for
pleural effusion in elderly patients, especially when the effusion
or symptoms associated with pleural effusion are refractory
to the therapy and thoracentesis volume control. In fact PEL
is generally resistant to chemotherapy and carries a very poor
prognosis with a median survival of less than 6 months. Due
to its rarity there is no clear standard of care established in
the treatment of PEL patients. Actually, studies in vitro and in
mouse models have been performed in preclinical trials to investigate the possibility of administration of targeted therapy. This
experimental studies should be explored in prospective human
clinical trials to improve patient’s survival. Lymphomas of body
cavity are rare and in presence of pleural or abdominal effusion
lymphomas without other detectable masses, we must keep in
mind the importance of HHV-8 immunostain. This one is the
best immunohistochemical tool to perform a correct cytological
and histological lymphoma diagnosis of the body cavity. Without HHV-8 positivity, PEL diagnosis could be underdiagnosed.
References
Kim Y. Current concepts in primary effusion lymphoma and other
effusion-based lymphomas. Korean J Pathol. 2014 Apr;48(2):81-90.
Sasaki Y. Primary Effusion Lymphoma in an Elderly HIV-Negative
Patient with Hemodialysis: Importance of Evaluation for Pleural Effusion in Patients Receiving Hemodialysis. Case Rep Nephrol Urol.
2014 May 21;4(2):95-102.
Antar A. Primary effusion lymphoma in an elderly patient effectively
treated by lenalidomide: case report and review of literature. Blood
Cancer J. 2014 Mar 7;4:e190.
Song JY. HHV-8-positive but EBV-negative primary effusion lymphoma.
Blood. 2013 Nov 28;122(23):3712.
Güven Karataş S et al. Human Immunodeficiency Virus (HIV)-Negative
249
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and Human Herpes Virus-8 (HHV-8)-Positive Primary Effusion
Lymphoma: A Case Report and Review of the Literature. Turk J
Haematol. 2013 Mar;30(1):67-71.
Comparison between thyroid fine-needle
aspiration biopsies classified with different italian
reporting systems: preliminary results of an
institutional series
P. Straccia, E. D. Rossi, T. Bizzarro, D. Damiani, M. Palumbo, C.
P. Lombardi1, A. Pontecorvi2, G. Fadda
Division of Anatomic Pathology and Histology, 1 Division of Endocrine
and Metabolic Surgery; 2 Division of Endocrinology Catholic University,
“Agostino Gemelli” School of Medicine – Rome (Italy)
Background. Nodular lesions represent a very common problem for the clinicians as well as a diagnostic challenge for the
pathologists. Up to 5% of the general population has a palpable
thyroid nodule though only approximately 5% of these clinically apparent thyroid nodules actually harbour malignancy. The
real challenge facing general practitioners, endocrinologists,
surgeons, and pathologists is to reach an accurate preoperative
diagnosis of malignancy and to ensure the patient receives a
timely and appropriate treatment. FNA is the only test that can
provide a definitive preoperative diagnosis of malignancy. The
sensitivity and specificity of FNA are reported to be 68–98%
and 56–100%, respectively. FNAB is also regarded as the most
accurate method for the selection of patients with thyroid nodules
for surgery or for the ‘wait and see’ management and it can be
considered a very cost-effective diagnostic test. The aim of the
present study is to compare the institutional series of the Catholic
University of Rome classified according to different reporting
systems. The first is the 5-tiered classification, proposed in 2007
by the Italian Society for Anatomic Pathology and Cytology,
joint with the Italian division of the International Academy of
Pathology (SIAPEC-IAP) and the two most important Italian
Societies of Endocrinology (SIE and AME). This year’s revision
in 6 tiers has been devised by the SIAPEC-IAP in agreement
with SIE and AME and the Italian Association of Thyroid (AIT).
The first reporting system included the following categories:
TIR 1-non diagnostic; TIR2 - negative for neoplasia; TIR3 - indeterminate/follicular neoplasia; TIR4- suspicious for malignant
neoplasm; TIR5- positive for malignancy. The latest reporting
system includes the additional subgroup of TIR1C (cystic) for the
non-diagnostic group and the subdivision of the indeterminate
category in TIR 3A (low-risk indeterminate lesion) and TIR 3B
(high-risk indeterminate lesion).
Materials and methods. From January 2014 to July 2014
in the Division of Anatomic Pathology and Histology of the
Catholic University “Agostino Gemelli” Hospital of Rome 957
patients underwent an ultrasound guided fine needle aspiration
cytology (FNAC) and subsequently the aspirated specimen were
processed using the ThinPrep liquid-based cytology (LBC)
method (Hologic Co., Marlborough, MA, USA). All FNAs were
performed with 25-gauge to 27-gauge needles without rapid adequacy assessment of the material. The aspirated material was
fixed with the hemolytic and preservative CytoLyt solution after
rinsing the needle in this solution. The cells were spun at 1500
revolutions per minute and the sediment was then transferred to
PreservCyt solution to be processed with the T5000 automated
processor according to the manufacturer’s suggestions. The
resulting slide was fixed in 95% ethanol and stained with the
Papanicolaou method. From January until March 2014, 609 cases
were diagnosed and classified according to the morphologic criteria adopted by SIAPEC-IAP classification of 2007, while from
April until July 2014, 348 cases were diagnosed according to the
new reporting system SIAPEC-AIT.
Results. The 609 FNAC samples of the first period were classified as follows: TIR1=53 (8,7%); TIR2=468 (76,84%); TIR3=48
(7,80%), TIR4=13 (2,1%), TIR5=27 (4,4%). Fifty-five patients
underwent surgery and 22 (38,2%) were histologically diagnosed
as carcinoma. Likewise the 348 cases samples of the second
period were classified as follows: TIR1=17 (4,8%); TIR1C=10
(2,8%); TIR2=249(71,5%); TIR3A=17 (4,8%); TIR3B=19 (5,4%);
TIR4=11(3,1%); TIR5=25 (7,1%). Thirty-four patients underwent
surgery and 12 (35,3%) of them resulted as malignant at the successive histological examination. The category TIR 3B (highrisk indeterminate lesion) is virtually represented by oxyphilic
neoplasms (ON, 16 cases: 84.2%) 2 out of them operated with a
diagnosis of adenoma. In the previous system ON accounted for
29.1% (14 cases) of the TIR 3 category.
Conclusions. This preliminary investigation emphasizes the
substantial reproducibility of the two reporting systems which
show similar rates of inadequacy (TIR 1) and suspicious for
carcinoma (TIR 4). As one may expect, the category of indeterminate low-risk TIR 3A in the recent reporting system
SIAPEC-AIT is composed either by cases which would have
been included in the benign category and by cases diagnosed as
follicular neoplasms. Although the histological follow-up is still
limited, the rate of malignancy in the surgical cases is similar.
This detail may find two possible explanations: 1) a possible difficulty in the application of the diagnostic criteria for the TIR 3B
category, 2) the high rate of oxyphilic neoplasms which result as
benign neoplasms at histology. In conclusion, there have been
limited changes in the inclusion criteria for cytological cases and
the new category of TIR 3A has likely decreased the number of
cases previously classified as benign.
References
Baloch ZW et al Diagnostic terminology and morphologic criteria for
cytologic diagnosis of thyroid lesions: a synopsis of the National
Cancer Institute Thyroid Fine-Needle Aspiration State of the Science
Conference. Diagn Cytopathol. 2008;36:425-37.
Fadda G et al. Cytological classification of thyroid nodules. Proposal
of the SIAPEC-IAP Italian Consensus Working Group. Pathologica
2010; 102:405-8.
Kocjan G et al. The interobserver reproducibility of thyroid fine-needle
aspiration using the UK Royal College of Pathologists’ classification
system. Am J Clin Pathol. 2011; 135: 852-9
Lobo C et al. The UK Royal College of Pathologists thyroid fine-needle aspiration diagnostic classification is a robust tool for the clinical management of abnormal thyroid nodules Acta Cytol 2011; 55:499-506
Fadda G, Rossi ED. Liquid based cytology in fine needle aspiration biopsies of the thyroid gland. Acta Cytol 2011; 55:389-400.
Rossi ED et al The cytological category of oncocytic (Hurthle ) cell
neoplasm mostly includes low-grade risk lesions at histology: an institutional experience. Eur J Endocrinol 2013: 169: 649-55
Nardi F, Basolo G, Crescenzi A, Fadda G, Frasoldati A, Orlandi F,
Palombini L, Papini E, Pontecorvi A, Vitti P: Italian consensus for the
classification and reporting of thyroid cytology. J Endocrinol Invest.
2014
Peritoneal cytology in gastric cancer
L. Vassallo1, C. Vindigni2, F. Roviello3, M. Di Martino3, G.
Flauti3, S. Pirisinu3, D. Marrelli3
Department of Medical Biotechnology, Pathological Anatomy Section,
University of Siena; 2 Unit of Pathology, Azienda Ospedaliera Universitaria Senese, Siena; 3 Department of Medical and Surgical Science and
Neuroscience
1
Introduction. Peritoneal dissemination is the most frequent pattern of metastases and recurrence in patients with gastric cancer.
In addition to macroscopic peritoneal disease, positive peritoneal
cytology has been included in the last edition of the America
Joint Committee on Cancer staging system as M1 disease. Infact, despite R0 resection, the majority of patients with positive
peritoneal lavage fluid develop peritoneal recurrence.
The aims of this study were to determine the incidence of positive peritoneal cytology (PLC) in patients with gastric cancer, to
250
Fig. 1.
Fig. 2.
identify risk factors for positive peritoneal cytology and to evaluate its prognostic significance.
Methods. From February 1998 to July 2011, the peritoneal
washing cytology of 220 patients with advanced gastric cancer
undergoing curative R0 resection at the Surgical Department
Fig. 3.
of Siena University was evaluated. The histotype was classified
according to Lauren and the TNM was reclassified according to
the VII edition TNM.
Differences between patients with positive or negative cytology
were assessed by Chi-square test. Survival was estimated using
Kaplan- Meier’s method (Figs 1, 2).
Results. Fifty-three patients (24%) had positive peritoneal cytology (Cy+). The positivity was correlated with the macroscopic
peritoneal metastases (17%), Borrmann type 3 (58%) and type 4
(23%), diffuse or mixed
histotype (68%), serosal surface infiltration or direct invasion of
adjacent organs (T4a and T4b) (77%), nodal metastases (98%),
prevalently N3 (77%) (p<0,001).
Cy+ was correlated with a poor prognosis. No significant difference in the survival was detected in patients with macroscopic
peritoneal carcinosis. In the patients without macroscopic peritoneal metastases, the 10-year survival in Cy+ patients was
statistically lower (Cy+ 20% vs Cy- 62%). The median survival
time was 12 months. The influence of Cy+ in the survival was
evident, although no statistically significant, in T4 patients
(16% vs 35%) (p=0,075). In Cy+ patients a worse prognosis was
observed in diffuse-mixed histotypes, although no statistically
significant (Fig. 3).
Conclusions. Positive peritoneal cytology (Cyt+) is an important
staging tool for patients with locally advanced gastric cancer. It
is a marker of poor prognosis and can predict relapse of disease
in gastric cancer patients.
Accurate staging of patients with locally advanced disease is
crucial for selecting the appropriate treatment strategy. In these
cases advanced multimodal treatments, such as intraoperative
hyperthermic chemotherapy can be appropriate.
References
De Andrade JP1, Mezhir JJ. “The critical role of peritoneal cytology
in the staging of gastric cancer: An evidence-based review”. J Surg
Oncol. 2014 May 22
Cabalag CS, Chan ST, Kaneko Y, Duong CP. “A systematic review and
meta-analysis of gastric cancer treatment in patients with positive
peritoneal cytology”. Gastric Cancer. 2014 Jun 3.
La Torre M, Ferri M, Giovagnoli MR, Sforza N, Cosenza G, Giarnieri E,
Ziparo V. “Peritoneal wash cytology in gastric carcinoma. Prognostic
significance and therapeutic consequences”. Eur J Surg Oncol. 2010
Oct;36(10):982-6
Wong J, Coit D. “Detection of gastric cancer peritoneal metastases by
peritoneal lavage: Current limitations and future perspectives”. Surgery. 2012 Jul;152(1):1-4.
Mezhir JJ, Shah MA, Jacks LM, Brennan MF, Coit DG, Strong VE.
251
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“Positive peritoneal cytology in patients with gastric cancer: natural
history and outcome of 291 patients”. Indian J Surg Oncol. 2011
Mar;2(1):16-23.
Fukagawa T, Katai H, Saka M, Morita S, Sasajima Y, Taniguchi H, Sano
T, Sasako M. “Significance of lavage cytology in advanced gastric
cancer patients”. World J Surg. 2010 Mar;34(3):563-8
Fig. 1.
Dermopatologia
A case of hidradenocarcinoma
A. Marinucci1, G. Cipolloni1, L. Melchiorri1, V. Ciuffetelli2, S.
Tambone3, P. Leocata1
Department of Life, Health & Environmental Sciences – Università
degli Studi dell’Aquila;, 2 Unità operativa di Anatomia e Istologia Patologica - ASL N°1 – Avezzano Sulmona L’Aquila; 3 Clinica Dermatologica
– Dipartimento Scienze e Tecnologie Biomediche, Università degli Studi
dell’Aquila
1
Introduction. Hidradenocarcinoma is a rare aggressive malignant tumor of the eccrine sweat glands, representing the malignant counterpart of hidradenoma [1].
Several synonyms for nodular hidradenocarcinoma have appeared in the literature: clear-cell hidradenocarcinoma, malignant clear-cell hidradenoma, solid-cystic adenocarcinoma,
malignant acrospiroma, malignant clear-cell myoepithelioma,
and clear-cell eccrine carcinoma.
It is mainly found within the head and neck, limbs and trunk
skin, rarely in inguinal and scrotal areas, but it can be found on
the skin in any localization [2].
The typical clinical presentation is a firm, erythematous , painless, subcutaneous nodule or plaque [3].
Microscopically, it shows a nodular growth pattern with presence
of solid structures and cystically dilated ductal and tubular structures. In its tissue texture there can be seen clear cells with pale
cytoplasm containing glycogen, eosinophilic cells with a centrally placed nucleus, squamoid cells and mucinous cells [4, 5].
Differential diagnosis between adenoma and carcinoma can
prove to be difficult.
The tumor has the potential for uncontrollable local recurrence
and tend to metastasize [6], namely there is little information
available on the natural history and appropriate management for
this disease [7].
We present a case of hidradenocarcinoma of a young man.
Methods. A 38-year-old man was seen for a nodular lesion.
Physical examination revealed a 1.5x1x0.8 cm erythematous,
firm, smoothly elevated nodule with normal overlying skin on
the right leg (Fig. 1).
No regional adenopathy was noted.
The tumor was resected and sampled.
Results. Histopathology showed that the tumor was a relatively
well-circumscribed, asymmetric, vascularized and lobulated
intradermal nodule, extending into the subcutaneous tissue with
capsule-like tissues. Margins were irregular and no connection
with the epidermis was seen (Fig. 2).
The tumor cells exhibited an expansile lobulated growth pattern
with focal tubular differentiation (Fig. 3), pleomorphism (Fig. 6)
and contained clear cells with distinct cell membranes (Fig. 4)
and squamoid cells with a central vesicular nucleus and eosinophilic cytoplasm (Fig. 5). Several focal areas of acrosyringeal
differentiation were seen (Fig. 5). The surrounding desmoplastic
stroma formed a pseudocapsule. Mitotic figures were 8 per 10
high-power fields (Fig. 6). Micro infiltration of a lymphatic vessel by neoplastic cells was also present (Fig. 7).
Immunohistochemistry revealed strong positivity for Epithelial
Membrane Antigen (EMA), which also decorated the luminal
border of ductal structures and for Ck-7. 50% of tumoral cells
express P53 (Fig. 8). CFDP-15 was negative.
Fig. 2.
Fig. 3.
252
Fig. 4.
Fig. 7.
Fig. 5.
Fig. 8.
Fig. 6.
A diagnosis of malignant hidradenocarcinoma was made.
Conclusion. Histologically there are currently no standardised
guidelines for diagnosis. It is often difficult to differentiate
hidradenocarcinoma from the benign equivalent hidradenoma.
The two types share the same composition of neoplastic cells.
However, clues to malignancy are asymmetry, deep extension, a
253
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conspicuous desmoplastic stromal reaction, infiltrative growth,
intravascular invasion, necrosis, cellular and nuclear pleomorphism and atypical mitotic figures,
There are no specific immunohistochemical features that establish the diagnosis.
References
1
Santa Cruz DJ. Sweat gland carcinomas: a comprehensive review.
Semin Diagn Pathol 1987;4:38–74.
2
Mehregan AH, Hashimoto K, Rahbari H. Eccrineadenocarcinoma: a
clinicopathologic study of 35 cases. Arch Dermatol 1983; 119: 104-14.
3
Hernandez-Perez E, Cestoni-Parducci R. Nodular hidradenoma and
hidradenocarcinoma. J Am Acad Dermatol 1985;12:15–20.
4
Ashley I, Smith-Reed M, Chernys A. Sweat gland carcinoma: case
report and review of the literature. Dermatol Surg 1997;23: 129–33.
5
Kersting DW. Clear cell hidradenoma and hidradenocarcinoma. Arch
Dermatol 1963;87:323–33.
6
Keasbey LE, Hadley GG. Clear cell hidradenoma: report of three
cases with widespread metastases. Cancer 1954;7:934–52.
7
Nazarian RM, Kapur P, Rakheja D, Piris A, Duncan LM, Mihm Jr MC,
Hoang MP: Atypical and malignant hidradenomas: a histological and
immunohistochemical study. Modern Pathology; 2009; 22: 600610.
PCR detection of Leishmania in granulomatous
dermatitis not otherwise specified
L. Moneghini1, C. Tomasini2, M. Falleni1, A.M. Barbui3, G. Bulfmante1
Division of Human Pathology, Department of Health Sciences, San Paolo
University Hospital, Milan, Italy; 2 Dermatopathology Section, Department of Medical Sciences, University of Turin, Turin , Italy; 3 Microbiology, Department of Medical Sciences, University of Turin, Turin , Italy
1
Leishmaniasis is a zoonosis caused by protozoan parasites of the
genus “Leishmania” (21-30 species) transmitted by the bite of
phlebotomine sandflies. Different types of clinical forms exist:
Visceral Leishmaniasis (VL), Cutaneous Leishmaniasis (CL)
and MucoCutaneous Leishmaniasis (MCL). CL in Italy is caused
mainly by dermatothrope L. Infantum (incubation time from 1-2
week to 7-8 months) (1).
The lesions, single or multiple, can present as papular eruption or
large ulcerative erosions. Chronic CL often presents as a tuberculoid granulomatous not distinguishable from other tuberculoid
diseases, such as lupus vulgaris, tubercoloid leprosy, syphilis,
atypical mycobacterioses, deep fungal infections, and Chron (3).
Polymerase chain reaction (PCR) for Leishmania DNA performed on paraffin-embedded tissue of a specimen should be
employed in cases where histopathology is not definitive (2).
The goal of this study was to evaluate the histologic granulomatous lesion not otherwise specified for the presence of Leishmania DNA.
Material and method. 48 histologic granulomatous skin lesion
(11 positive for Leishmania amastigotes and 37 without a specific
granulomatous diagnosis) collected in the Department of Dermatology of the University of Turin from 1993 and 2011, were
evaluated for Leishmania-DNA with real time-PCR on paraffin
embedded tissue sections.
Results. All the 11 skin biopsies histologically positive for
Leishmania amastigotes were positive for Leishmania-DNA.
Eleven out of 37 skin biopsies without a specific diagnosis were
positive for Leishmania-DNA (29.7%).
We identified 5 main histological patterns of chronic CL:
1) Nodular granulomatous pattern with discrete, round to oval
epithelioid aggregates surrounded by mantles of plasmacells
and lymphocytes
2) Diffuse granulomatous pattern (often V shaped), with epithelioid aggregates surrounded by confluent mantles of plasmacells
and lymphocytes.
3) Diffuse granulomatous pattern with interface lichenoid dermatitis
4) Pseudolymphomatous pattern with occasionally tubercoloid
granulomas
5) Pseudolymphomatous patter with mycosis-fungoid like aspects
While the histological diagnosis of the acute form of Leishmaniasis is based on the identification of the amastigotes, two main
patterns among those described in this study can be easily confused with other inflammatory lesions characterized by tubercoloid granulomata or pseudolymphoid aspect.
PCR Leishmania DNA detection in these of patterns of chronic
CL greatly useful to identify this parassitosis.
References
1
Biglino A1, Bolla C, Concialdi E, Trisciuoglio A, Romano A, Ferroglio E. Asymptomatic Leishmania infantum infection in an area
of northwestern Italy (Piedmont region) where such infections are
traditionally nonendemic. J Clin Microbiol. 2010 Jan; 48(1): 131-6
2
Bšer A1, Blšdorn-Schlicht N, Wiebels D, Steinkraus V, Falk
TM. Unusual histopathological features of cutaneous leishmaniasis identified by polymerase chain reaction specific for Leishmania on paraffin-embedded skin biopsies. Br J Dermatol. 2006
Oct;155(4):815-9.
3
Saab J, Fedda F, Khattab R, Yahya L, Loya A, Satti M, Kibbi AG,
Houreih MA, Raslan W, El-Sabban M, Khalifeh I. Cutaneous leishmaniasis mimicking inflammatory and neoplastic processes: a clinical, histopathological and molecular study of 57 cases- J Cutan Pathol
2012: 39: 251–262
Peculiar anatomic site of malignant melanoma in
black population
L. Viberti1, R. Tumino2-6, L. Daniele3, D. Fenocchio4-6, S. Guzzetti5-6
Casa di Cura Sedes Sapientiae, Turin, Italy;2 Anatomia Patologica e
Registro Tumori Azienda Sanitaria Provinciale di Ragusa (Italy); 3 Department of Medical Sciences, University of Turin, Turin, Italy; 4 Department of Pathology, Ospedale S. Maria della Misericordia, Perugia, Italy;
5
Department of Pathology, Ospedale Martini - ASL TO1, Turin, Italy; 6
Associazione Patologi Oltre Frontiera (APOF), NGO
1
Introduction. The incidence of cutaneous malignant melanoma
is considerably lower in pigmented skin populations than in
Caucasians, together with a particular prevalence in acral sites.
Aim of this study is to evaluate the incidence of cutaneous
melanoma in populations living in Sub-Saharan Africa, with
particular regard to the anatomic localization.
Materials and methods. We analyzed two retrospective series
collected in two different Sub-Saharan health facilities where
the NGO “Associazione Patologi Oltre Frontiera (APOF, or
“Pathologist Beyond Borders Association”) developed a cooperation project with local health staff for the implementation of
histological and cytological diagnoses. The cases of melanoma
have been extracted from the files of the Departments of Pathology of the Bugando Medical Center (BMC) in Mwanza,
Tanzania, and of the Mtendere Mission Hospital (MMH) in
Chirundu, Zambia.
Results. We have collected 83 diagnoses of melanoma out of
a total of 5,570 histological cases (1.5%) diagnosed from 2004
to 2007 at BMC and 14 cases out of 1,208 overall histological
cases (1,2%) diagnosed at MMH during the years 2007-2013. In
both hospitals the number of skin-located melanomas (excluding
lymph node metastasis and mucosal sites) was 89. For 13 cases
the anatomical localization was not specified. Of the remaining
76 cases with a proven primary skin location, 55 were located on
feet (72,4%) and 14 on legs (18,4%), with a total ratio of 90,8% of
cases localized on lower limbs.
Conclusions. Malignant melanomas of the skin in black populations show a particular anatomical distribution, with a particular
prevalence on feet. This finding could suggest a lack of correlation with a previous sun exposition.
Foot melanoma (together with hand melanoma) could represent a
specific subgroup of melanomas, with different genetic or environmental factors. Such a prevalence in acral sites in pigmented
skin populations needs to be studied further.
254
Interesse generale
Fig. 1. Lung: necrotizing bronchopneumonia of both bacterial and
fungal nature (HE, OM 50X).
Combined pathological and microbiological
analyses for the diagnosis of infection by
scedosporium prolificans: a case report
M.A.G.M. Butorano1, M. Caroli Costantini2, A. Galano3, D. Di
Palo3, M.G. Cusi3
Università degli Studi di Siena, Dipartimento di Biotecnologie Mediche,
Scuola di Specializzazione in Anatomia Patologica; 2 Azienda Ospedaliera Universitaria Senese, Dipartimento Oncologico, Sezione di Anatomia
Patologica; 3 Università degli studi di Siena, Dipartimento di Biotecnologie Mediche, Sezione di Microbiologia e Virologia
1
Introduction. We report a case of disseminated infection by Scedosporium prolificans in a heart transplant recipient. Although
antifungal prophylaxis is routinely practiced in such patients,
Scedosporium prolificans is an emerging, multidrug-resistant,
and often fatal pathogen, in immunocompromised patients. This
is, to our knowledge, the first case diagnosed in Italy in a heart
transplant recipient.
Materials and methods. A 51-year-old Caucasian man, with a
history of dilated cardiomyopathy, underwent orthotopic heart
transplantation. Subsequently, he developed an infection of the
sternal wound, pleural and pericardic effusions and progressive
hemodynamic dysfunction. Blood cultures were positive for Enterobacter aerogenes, whilst from the bronchoaspirate (BAS), E.
coli, and, successively, S. maltophylia and A. baumanni were isolated, as well as a filamentous fungus, whose identity, however,
was not defined. Despite continuous treatment and monitoring in
the intensive care unit, the patient died, four months after transplantation. A post-mortem examination was done and samples
from all the major organs were collected, fixed in formalin, and
routinely processed for pathological examination. Sections were
stained with Hematoxylin and Eosin, Grocott’s methenamine
silver stain (GSM) and PAS-D. For microbiological analysis,
DNA was extracted from paraffin-embedded sections of the
lung, amplified and then sequenced.
Results. Main findings at gross examination were a purulent collection between the pericardial folds and multiple foci of necrosis
in the lungs, suspicious for an infectious nature. Histological examination confirmed the presence of necrotizing bronchopneumonia (fig. 1), which proved to be of both bacterial and fungal
nature. The fungal microorganisms appeared under the form of
branching hyphae and yeasts. The same fungi were also present
in virtually all the organs examined, including heart (figure 2),
brain, kidneys, liver, colon, adrenal glands and peripheral nerves.
The cause of death was recorded as “necrotizing bronchopneumonia (bacterial and fungal) and disseminated fungal infection
leading to septic shock, in a heart transplant recipient”. The
results from the microbiological analyses identified the fungal
microorganism from the lung section, as well as from the BAS,
as Scedosporium prolificans.
Discussion. Scedosporium spp. are ubiquitously distributed
worldwide, commonly found in soil, sewage or polluted water1.
Currently, they represent emerging fungal pathogens, causing a variety of infections, primarily in immunocompromised
hosts2. In addition to solid organ transplatation, risk factors for
Scedosporium. prolificans infection include malignancy, cystic
fibrosis, and AIDS3, with morbidity and mortality rates of up to
75 - 100%1,4. Reports of Scedosporium infections in Australia5,
Spain6, and the United States7, have described the species’ distribution and clinical epidemiology. An early and accurate diagnosis of Scedosporium infection is difficult, because current mycological methods for detecting and identifying Scedosporium
spp. in clinical specimens are insensitive and time-consuming,
requiring up to 14 days in order to yield a fungal growth adequate
for morphological identification. Diagnostic molecular methods
Fig. 2. Myocardium: widespread colonisation by fungi, that present
mostly as septated hyphae (A: HE, OM 100X; B: GSM, OM 200X) x).
are not yet validated; they can be used only as an adjunct to conventional laboratory tests. The outcome of S. prolificans infection is very poor, because no drug appears to be effective, even if
some drugs have been reported to be of limited efficacy. In our
case, no specific treatment was given to the patient, also because
the precise identification of the fungus was delayed. Diagnosis
was confirmed by molecular methods, since mass spectrometry
was not able to identify it.
Conclusions. Multicentric studies are needed to develop appropriate techniques and protocols for the diagnosis and treatment
of this type of mycoses, with particular attention to immunosuppressed patients. Although still rare, infections by Scedosporium
prolificans are going to represent a problem, because of the aggressive clinical course and the lack of effective therapy.
References
1
Binder U, Lass-Flšrl C. Epidemiology of invasive fungal infections in the mediterranean area. Mediterr J Hematol Infect Dis.
2011;3(1):e20110016. http://www.ncbi.nlm.nih.gov/pubmed/21625305
2
Sayah DM, Schwartz BS, Kukreja J, Singer JP, Golden JA, Leard LE.
Scedosporium prolificans pericarditis and mycotic aortic aneurysm
in a lung transplant recipient receiving voriconazole prophylaxis.
Transpl Infect Dis. Epub 2013 Feb 6. http://www.ncbi.nlm.nih.gov/
pubmed/23387799
3
Rodriguez-Tudela JL1, Berenguer J, Guarro J, Kantarcioglu AS, Horre
R , de Hoog GS, Cuenca-Estrella M. Epidemiology and outcome of
Scedosporium prolificans infection, a review of 162 cases Med Mycol.
2009;47(4):359-70.
4
Husain S, Mu–oz P, Forrest G, Alexander BD, Somani J, Brennan K,
Wagener MM, Singh N Infections due to Scedosporium apiospermum
255
POSTER
5
6
7
and Scedosporium prolificans in transplant recipients: clinical characteristics and impact of antifungal agent therapy on outcome. Clin
Infect Dis. 2005; 40(1): 89–99.
Cooley L, Spelman D, Thursky K, Slavin M. Infection with Scedosporium apiospermum and S. prolificans, Australia. Emerg Infect Dis.
2007;13(8):1170-7.
Rojas R, Molina JR, Jarque I, Montes C, Serrano J, Sanz J, Besalduch
J, Carreras E, Tomas JF, Madero L, Rubio D, Conde E, Sanz MA, Torres A. Outcome of Antifungal Combination Therapy for Invasive Mold
Infections in Hematological Patients is Independent of the Chosen
Combination. Mediterr J Hematol Infect Dis. Epub 2012 . http://www.
ncbi.nlm.nih.gov/pubmed/22348193
Johnson LS, Shields RK, Clancy CJ. Epidemiology, clinical manifestations, and outcomes of Scedosporium infections among solid
organ transplant recipients. Transpl Infect Dis. 2014Jun 24. doi:
10.1111/tid.12244 [Epub ahead of print]. http://www.ncbi.nlm.nih.
gov/pubmed/24962102
Standardized health educational project : a
novel approach to improve formation in surgical
pathology and cytopathology
S. Erra1, S. Modena1, V. Magnelli2
1
SOC Anatomia Patologica P.O. Casale Monferrato; 2 Dipartimento di
Scienze e Innovazione Tecnologica, DiSIT, Università del Piemonte Orientale “Amedeo Avogadro”, Alessandria
Introduction. Students in life science and laboratory techniques
can choose to attend public hospital services in a pre-degree
period between six months and two years.
During last ten years, seventeen students have been welcomed at
our service of surgical pathology.
The mentoring experience gained in this period has led us to
adopt a method of teaching at the same time personalized but
standardized, without forcing steps of hands-on learning, but
strengthening the capacity of the individual student.
Methods. We evaluated the learning processes of seventeen
university students welcoming at our service between January
2004 and May 2014. Our students have been five men and twelve
women with between the ages of twenty and thirty-eight years.
Sixteen were attending the Faculty of Life Sciences, while one
was attending the faculty of biomedical laboratory techniques.
Four of them were working students. Fourteen students have
graduated, while the remaining are preparing their thesis at our
service.
In order to ensure equal learning opportunities to all students,
everyone has had time to attend actively in the various sectors
of the service pathology and cytopathology. In addition, each
student was involved in the drafting of case reports and in the
preparation of lectures on monothematic topics in predetermined
moments during company training.
The evaluation of the learning objectives was based on the
achievement of results considered more than good from the academics of degree courses attended by individual students.
Results. To objectify the training of students in our service
hospital of pathology, we designed an assisted route. After the
initial two months of frequency, our student is able to know and
understand the role of a service of pathological anatomy in a
medical multidisciplinary. This becomes possible through real
participation in the various activities of the service, in the wake
of the medical and technical staff, which provides useful and
practical explanations of the stages of labor. In the same time, the
student revises his/her knowledge of anatomy, comparing theory
and practical feedback facilitated by a daily relationship with the
staff of the team.
The initial period is useful not only to fit in a surgical pathology
work team, but to identify a personal interest in one specific sector of human pathology. Each student can choose the argument
for the thesis of degree, consistent with the ability of the service
and the expertise of the tutor teaching business, identified in the
context of team in our surgical pathology service.
Only after this student’s choice, he/she can start work for the
thesis, with a variable period for review of the scientific literature
regarding the selected argument.
The last educational training is represented by the acquisition of
the ability to convey certain concepts in a formal moment. For
this reason , each student is invited to prepare a lesson in the
context of a corporate training event, together with her/his tutor.
At the end of this standardized educational project, our student
writes his/her first case report under tutor supervision.
Our structured training has enhanced capacity and safety in the
students in our team work, helping to establish professional and
human correct relationship.
The success of the training method adopted is represented by the
excellent results that our students have achieved during university exams and interest raised in the discussion of the thesis from
the academic world.
Conclusion. Our surgical pathology service has a partnership
with the University for student attendance involving the preparation of the thesis.
Some of our team are recognized hospital tutors and we welcome
and prepare in the practice students to have a real approach to
their possible future in public health issues.
University demands practical formation to tutor entirely, in the
absence of agreed guidelines.
The results of this behavior is the confusion between tutors and
students on the ultimate goal of the practice frequency, due to the
enhanced customization of the preparation of trainees.
In the actual society “the disorder training” represents an unsustainable condition, above all in a public facility accredited.
For these reasons, we are trying to set up a software to be used
in the planning of a standardized training for the students that
choose to attend our service.
Our goal is to structure the present educational project in a
multidisciplinary software, through the integration of medical
sciences arguments with practical abilities in anatomic and laboratory pathology.
This educational software can be useful to objectify the training of university students during the internship period in public
hospitals.
References
1
Group-effort applied research: Expanding opportunities for undergraduate research through original, class-based research projects.
Moore SD, Teter K. Biochem Mol Biol Educ. 2014 Jun 4. doi:
10.1002/bmb.20802
2
Applying established guidelines to team-based learning programs
in medical schools: a systematic review. Burgess AW, McGregor
DM, Mellis CM.Acad Med. 2014 Apr;89(4):678-88. doi: 10.1097/
ACM.0000000000000162
3
Designing high-quality interactive multimedia learning modules.
Huang C. Comput Med Imaging Graph. 2005 Mar-Apr;29(2-3):223-33
Perinatal death and multidisciplinary management
S. Erra1, C.M.S. Tappero2, S. Beccio3, S. Barbero2
SOC Anatomia Patologica P.O. Casale Monferrato ASL AL; 2 SOC Diagnostica per immagini ed interventistica P.O. Casale Monferrato ASL AL; 3
SOC Pediatria P.O. Casale Monferrato ASL AL
1
Introduction. Perinatal deaths are unpleasant events involving
the obstetrics team and, sometimes, pathologists when autopsy
is required.
In regional referral hospitals, internal agreement has been established between medical specialities involved in the management
of perinatal mortality. In little provincial hospital, there aren’t
recognized guidelines. To improve the government of these
events, in our hospital a multidisciplinary team has been identified able to face the problems inherent cases of perinatal death.
Materials and methods. In the last five years, numerous motifs
256
Tab. I.
EVENT / PROTAGONIST
Perinatal death (from 24weeks+5days of
intauterine life to one month after birth)
Obstetrical team
Neonatologist
Pathologists
Radiologist
OBJECT/ ROLES
Medical record
Anamnestic history
Contact neonatologist
Microbiological samples
Request autopsy after parental consent
Request histological evaluation of placenta (intrauterine death)
Forms accepted and shared
Opening and compilation of medical record
Samples of blood (Guthrie test) and tissue (molecular tests)
External exam
Perinatal autopsy according to APEFA guidelines
Photos and evaluation indices of growth and maturation
Ancillary imaging studies (rx and virtopsy)
Placental histological evaluation in case of intrauterine death
Transmission of the histological reports to gynecology and pediatrics
Ancillary instrumental exams:
Rx for osseous morphologic alterations
Virtopsy with TC system
regarding incorrect management of perinatal mortality have led
to the need to build a multidisciplinary process. For this reason,
during last spring pathologists organized a training event on
perinatal death, finalized to establish roles and responsibilities
of each health profession involved in this event.
As result of our course, a document has been prepared in which
every task was established. This document has been approved by
responsible of the single medical specialities and finally shared
by the hospital management officially.
Results. Flow chart prepared to face perinatal death events correctly has been synthesized in the table 1.
Second our agreed division of roles, every health specialized
doctor knows what to do, when and how to do it in a correct way,
with saving of time and resources and with a real benefit for the
parents.
Conclusion. Management of perinatal death is difficult, above
all in absence of agreed guidelines. After the practical need for
a more effective management of issues, a medical multidisciplinary team decided to define local simple guidelines, finalized
to have a medical record for all cases of perinatal death.
The document identifies specific roles for each specialized doctor, in order to objectify and standardize procedures and methods and to give a real benefit to parents. Systematical study of
problems related to perinatal mortality is facilitated by orderly
and methodical data collection, guaranteed by a unique method
of procedure.
In this context, our medical team identified a diagnostic, therapeutic and welfare route (PDTA) regarding the management of
perinatal death in our hospital. In the reported guidelines pathologists have a central role, not only for the material organization of the route, but helping to introduce methods of diagnostic
imaging never applied to perinatal autopsy. In fact TC-virtopsy
is used in legal autopsy, without a concrete pathological findings,
in absence of collaboration through pathologists and radiologists.
In our work-team, the presence of radiologists experienced in
virtopsy and pathologists interested in perinatal pathology has
made possible a real collaboration between the two medical
specialty. Results of this team interaction are a more rapid and
reliable work, a greater attention during autopsy by pathologists,
guided from the virtopsy feedback. Finally parents longer feel
protected and reassured from the existence of a multidisciplinary
specialized team prepared to study and understand the motifs of
their baby death.
Collection and systematic analysis of a
comprehensive cohort of Triple-negative breast
cancers in southern Sardinia
S.A.M. Urru, R. Medda, T. Moi, E. Sollai, F. Sanges, E. Valle,
A. Bulfone, S. Orrù
Centro di Ricerca, Sviluppo e Studi Superiori in Sardegna (CRS4)
Background. Triple-negative breast cancers (TNBCs) are defined as tumors lacking the expression of the estrogen receptor
(ER), progesterone receptor (PR), and human epidermal growth
factor receptor 2 (HER-2). Approximately 15% of breast cancers
are triple negative breast cancer. Among breast cancers the TNBCs are the most aggressive malignancies and with the poorest
outcome since they cannot be treated with endocrine therapy or
targeted therapies. The aim of this study is to determine retrospectively the incidence, the clinicopathological features and
outcomes of this type of breast cancer in southern Sardinia. The
study is also aimed to retrospectively identify the TNBCs subtypes that showed to be most sensitive to therapy.
Methods. This is a retrospective study of confirmed triple negative breast cancer patients followed at the reference oncological
hospital of the Sardinia region (Cancer Hospital Regional Reference “Armando Businco” – Cagliari), between January 2000
and December 2013. Epidemiological, histological, clinical and
evolutive data were comprehensively collected. The statistical
analysis was performed using the Kaplan-Meier method.
Results. Collectively 540 patients were identified as having
triple-negative breast cancer in the period 2000-2013, representing the 10,52% of the total breast cancer patient population
diagnosed and cured at the Businco Hospital. The median age at
diagnosis was 56 years. 362 patients (67%) had invasive ductal
carcinoma, 58 patients (10,7%) had invasive lobular carcinoma
and 28 had medullar carcinoma (5,2%). Scarff-Bloom-Richardson (SBR) grade III was found in 422 cases (78%), while grade II
was found in 104 cases (19,25%). 510 patients received surgery,
297 (58,23%) had quadrantectomy surgery and 213 (41,76%) received radical mastectomy with axillary lymph nodes dissection
respectively in 220 and 188 patients. 456 of all patients (84,4%)
received chemotherapy: 384 patients (84,21%) received adjuvant chemotherapy, 18 patients (3,94%) received neoadjuvant,
while 54 (11,84%) patients received both. Women mean age at
menarche was 13 years, with almost two-thirds (62%, 317 of
257
POSTER
513) reporting menarche at ages 11, 12, or 13 years. Their mean
age at menopause was 49 years, with 9% (49 of 513) reporting
menopause before age 45 years, 41% (212) between the ages 45
and 54 years and 6% (28) at age 55 years or older. The complete
data analysis, including DFS/OS and molecular profiling will be
presented at the meeting.
Conclusions. These results suggest that most TNBC characteristics in Sardinian patients are in accordance with the existing
literature data. The immunohistochemical profiling will be performed in order to assess the distribution of the various TNBCs
subtypes. IHC and molecular data will be compared to clinical
data allowing a better understanding the clinical outcomes of the
different triple negative subtypes.
Osso e parti molli
Proteus Syndrome
O. Bega, R. Scamarcio, A. Cimmino
Dipartimento di Anatomia Patologica Policlinico di Bari
Background. Proteus syndrome was first described by Woedemann et al. in 1983. Although “the elephant man” Joseph Merrick, was originally believed to have neurofibromatosis, because
of the cerebriform or gyriform fibrous proliferation characteristically found on the volar surfaces. The body of J. Merrick was
entirely and grotesquely deformante by the disease exduding the
genital and left arm. Individuals with this syndrome have normal intelligence even if the effects of the disease can cause an
indirect damage to the intellect. The accumulation of tissue can
cause crushing of the blood vessels and a proper flow of blood
even into the brain. The Proteus syndrome is a rare congenital
disease. The etiology reported to the gene Alk 1 mosaic, which is
overexpressed, leading to an uncontrolled growth of tissues. The
type of mutation is spontaneous and it seems to occur in fetal
age and it causes several malformations due to an overgrowth of
tissues derived from germ layers and therefore a outsized growth
of tissues and bones.
Only 200 cases has been refered in literature.
Symptoms. Manifestations indude gigantism of the hands or
feet, asymmetry, skeletal abnormalities, swelling of skin, and tissues (including blood and lymph vessels) in various parts of the
body, often accompanied by tumor like masses over most of the
body surface (lipomatous, hemangiomatous tumors). Thickening
of the skin in the volar areas resulting in a coarse cerebriform or
guriform pattern.
Methods. A 51 years old man reached our attention with five
tumors. The lesions have grayish cerebriform surface and arises
in the soft tissues of the toes of the left foot.
Histopathological features. The lesions consisted of dense
fibrosis involving both the dermis and subacutis, with hyperkeratosis of the overlying skin defect in the production of collagenase, possibly related to a disturbed production of insulin-like
growth factors. Uitto et al. showed that these lesions consist
almost exclusively of type l collagen and suggested an underlying
defect in the production of collagenase.
Comment. The most famous case is undoubtedly that of Joseph
Merrick, known as the Elephant Man. Its skeleton, kept in England, might be helpful to understand the disease and to verify the
presence of the genetic mutation and to stimulate us to find useful therapies, at last to arrest the growth out of control of some
tumoral cells. The tests will not be a simple matter considering
that the responsible mutation of the syndrome is not evently
distributed in the body: the examination of a small sample could
not find cells carrying the gene and be fooled by the mystery of
the Elephant Man.
Solitary fibrous tumor in patient with concurrent
and recurrent glioblastoma multiforme
G. De Luca1, G. Maselli2, A. Di Lorito1, L. Ranieri1, C. Marinelli1,
R. Zappacosta3, G. Lattanzio3
Medical doctor, “SS Annunziata” Hospital, Unit of Pathology, Chieti,
Italy; 2 Medical doctor, “Spirito Santo” Hospital, Unit of Neurosurgery,
Pescara, Italy; 3 Pathologist, “SS Annunziata” Hospital, Chieti, Italy
1
Background. Age-adjusted incidence of glioblastoma (WHO
grade IV), the most common and most deadly glioma subtype in
adults, ranges from 0.59 to 3.69 per 100 000 persons, meanwhile
its 5-year relative survival varies by different countries from
0,05% to 4,7% [1]. This high grade malignant glioma has the
marked tendency to multifocality and intracranial recurrence,
but, probably also for its brief clinical history, evidences of
distant metastases appear extremely scarce. In literature there
are some case reports of neoplastic cells implantation on scalp
surgical scar and via ventriculoperitoneal shunts to the abdomen
[2] and of metastases to lymph nodes, cranial bones and other
organs [2,6]. To date only three described cases of distant cutaneous metastases can be enumerated to our knowledge, configuring
this eventuality as nearly anecdotal [3,4,5]. Our patient was a 63
year old man, undergone surgical excision of endocranial glioblastoma multiforme.
He was hospitalized after having warned headache irradiated to
right periorbital region and fever.
A cranial CT scan was performed and a frontal-temporal expansive lesion, with surrounding oedema, was observed. The
patient’s neurological examination was completely negative.
After some months since surgical excision of primitive malignancy, an endocranial recurrence was observed by follow-up CT
control, concurrently the patient underwent surgical removing of
subcutaneous nodule.
Methods. We sampled a biopsy of cutaneous and subcutaneous
tissue of the right thigh. In the subcutis macroscopically was observed a nodular formation of 0.9 cm in greatest dimension, with
apparently mucoid content. On hematoxylin and eosin stained
slides the nodule presented infiltrative margins and a wide central area of myxoid degeneration.
At higher magnification it was formed by short and ill-defined
fascicles of spindle cells intercalated with thick hyalinized collagen bands. Cytologically, the spindle cells showed abundant
eosinophilic cytoplasm with indistinct cellular borders and pale,
often vesicular, nuclei. The cellular atypia was mild, because for
only slight nuclear pleomorphism and inconspicuous nucleoli.
The neoplasm proliferative activity, assessed with Ki67 immunostaining, was inferior to 3% and there was no necrosis.
The overall histologic morphology was suggestive for a connective or nervous tissue neoplasm, of uncertain malignant potential.
Results. In order to exclude the rare eventuality of a cutaneous
metastasis from the concurrent glioblastoma, we perform a wide
immunohistochemistry panel, including S100, GFAP, chromogranin and synaptophysin. All these markers were negative,
driving us to investigate connective tissue tumors.
One of these, categorized into fibroblastic/myofibroblastic tumors by WHO, represents a definite pathological entity in
continuity to haemangiopericytoma. Like the lesion in exam,
solitary fibrous tumor could arise in thigh, is exclusive of adults
and could present cystic degeneration. Microscopically, myxoid
change is very common and its cellular population is spindle
shaped, characteristically often arranged in small parallel clusters next to dense collagen [7].
Immunohistochemical panel supported the morphologic observations, giving the data of negativity to smooth muscle actin and
desmin negativity, CD34 and Vimentin positivity.
Ultimately, the striking hyalinization and the absence of typical stag horn vessels led us to choose the diagnosis of solitary
fibrous tumor rather than of haemangiopericytoma.
258
Fig. 1. Myxoid areas, bundles of spindle cells.
Fig. 2. Hyaline collagen bands.
Fig. 4. Proliferation index, by Ki67.
Conclusion. Despite the possibility that a diffuse astrocytoma
metastasize to an extracranial site is extremely rare, we had
to consider it in differential diagnosis. The morphology was
suggestive for a mesenchymal tumor, but a biphasic variant of
glioblastoma, called gliosarcoma, has a metaplastic sarcomatous
component. Coherently, the sarcomatous component may show
bone, cartilage, fat or muscle differentiation, and is typically
negative for glial marker as GFAP or S-100. Notwithstanding
these speculations, the analyzed lesion lacked the pivotal features of astrocytoma.
References
1
Ostrom QT et al. The epidemiology of glioma in adults: a “state of the
science” review. Neuro Oncol. 2014 May 19
2
Allan RS Scalp metastasis from glioblastoma. J Neurol Neurosurg
Psychiatry 2004;75:559
3
Jain N et al. Cutaneous metastasis from glioblastoma. Br J Neurosurg.
2005 Feb;19(1):65-8.
4
Miliaras G et al. Multifocal glioblastoma with remote cutaneous
Fig. 3. Immunohistochemical panel, in clockwise: CD34, Vimentin, GFAP, AML.
POSTER
5
6
metastasis: a case report and review of the literature. Cent Eur Neurosurg. 2009 Feb;70(1):39-42
Figueroa P et al. Cutaneous metastasis from an intracranial glioblastoma multiforme. J Am Acad Dermatol. 2002 Feb; 46(2):297-300.
Kraft M et al. Parotid gland metastasis from glioblastoma multiforme:
a case report and review of the literature. Goldblum, Folpe, Weiss.
Enzinger and Weiss’s Soft Tissue Tumors, 6TH edition, ELSEVIER
Detection of chromosomal translocations in
soft tissue tumors: a comparison between
Fluorescence in situ hybridization (FISH),
qualitative RT-PCR and quantitative Real Time PCR
L. Marra1, A. Parolisi1, M. Cantile1, M. Cerrone2, M.P. Curcio1,
R. Franco1, G. Botti1, A. De Chiara1
S.C. Anatomia Patologica e Citopatologia, INT Fondazione «G. Pascale»; 2 Dipartimento Melanoma, Tessuti molli, Muscolo - Scheletrico e
Testa – Collo, INT Fondazione «G. Pascale»
1
Background. One third of soft tissue tumors are characterized
by chromosomal aberrations, in particular, translocations and
amplifications, which appear to be highly specific. The described chromosomal rearrangements in soft tissue tumors not
only aid in the diagnosis and classification of these tumours, but
are particularly useful in the differential diagnosis of patients
with an uncertain or dubious morphology. Routine techniques,
such as FISH and RT-PCR, can be within the reach of pathology laboratories, helping the pathologist in the diagnosis of such
neoplasms. Obviously, as for all biomolecular methods, an essential condition is the carefulness of all pre-analytical stages of
preparation of the biological samples.
Aim and methods. In this study we compared FISH, qualitative RT-PCR and Real Time PCR, for the detection of specific
genetic translocations in selected sarcomas synovial sarcoma [t
(X;18); SS18/SSX], myxoid liposarcoma [t(12;16);FUS/CHOP]
259
and Ewing sarcoma [t(11;22);EWSR1/FLI1- t(21;22); EWSR1/
ERG], of which we had both the FFPE and the fresh cryopreserved samples of our Institutional BioBank.
Break Apart Rearrangement Probe has been used in FISH analysis; in a normal cell a two fusion signal pattern will be observed
reflecting the two intact copies of gene, as one fusion, one green,
one orange signal pattern will be characteristic of abnormal
cells. In RT-PCR and Real Time PCR analysis we have used
appropriate primers to amplify regions containing the mutations
of interest. Annealing Temperature is specific for only primers.
Results. The FISH analysis was optimal in all analyzed sarcomas, while the poor quality of the purified RNA from FFPE
samples for RT-PCR analysis has allowed to obtain satisfactory
results only on fresh samples. In contrast, the analysis of RealTime-PCR has made to identify the presence of translocations in
all analyzed cases
Conclusions. FISH technique whilst remaining the technique of
choice for the detection of gene translocations in sarcomas, it is
a very expensive method. Molecular techniques instead, especially the Real-Time-PCR allows, at a lower cost but with high
sensitivity and specificity, to detect these molecular alterations.
Furthermore, the sampling of FFPE tissues, destined to diagnostic, should be accompanied by sampling of fresh cryopreserved
tissues to be used in all those cases in which the fixation may
affect the quality of nucleic acids.
References
1
Cerrone M, Cantile M, Collina F, Marra L, Liguori G, Franco R, De
Chiara A, Botti G. Molecular strategies for detecting chromosomal
translocations in soft tissue tumors (review). Int J Mol Med. 2014
Jun;33(6):1379-91.
2
Cantile M, Marra L, Franco R, Ascierto P, Liguori G, De Chiara A,
Botti G. Molecular detection and targeting of EWSR1 fusion transcripts in soft tissue tumors. Med Oncol. 2013 Mar;30(1):412.
Fig. 1. Different methods for EWSR1 gene translocation detection: (a) fluorescence in situ hybridization (FISH) (Vysis EWSR1 Break Apart FISH Probe
Kit in a Ewing Sarcoma sample); (b) RT-PCR (fusion transcript of 150 bp in Frozen and FFEP samples); (c-d) real-time PCR (melting curves associated
to fusion transcripts in samples).
Fig. 2. Different methods for SS18 gene translocation detection: (a) fluorescence in situ hybridization (FISH) (Vysis SS18 Break Apart FISH Probe Kit
in a Synovial Sarcoma sample); (b) RT-PCR (fusion transcript of 150 bp in Frozen and FFEP samples); (c) real-time PCR (melting curves associated to
fusion transcripts in samples).
260
Fig. 3. Different methods for CHOP/DDIT3 gene translocation detection: (a) fluorescence in situ hybridization (FISH) (Vysis DDIT3 Break Apart FISH
Probe Kit in a Myxoid Liposarcoma sample); (b) RT-PCR (fusion transcript of 150 bp in Frozen and FFEP samples); (c) real-time PCR (melting curves
associated to fusion transcripts in samples).
Sinonasal malignant haemangioendothelioma
in a young adult man previously treated with
radiotherapy
M. Onorati1, G. Petracco1, C. Facco2, I. Talamo1, F. Di Nuovo1
Pathology Unit, AO G. Salvini Garbagnate Milanese, MI; 2 Pathology
Unit, AO Ospedale di Circolo e Fondazione Macchi
1
Introduction. Malignant haemangioendothelioma of nose, paranasal sinuses and nasopharynx is relatively uncommon. It is more
common in men than women. Various epithelial and non-epithelial tumors have been reported in large series and individual case
studies however, malignant haemangioendothelioma was very rare. In fact frequently they result in inappropriate clinical management. The term haemangioendothelioma, first coined by Mallory
in 1908, has been used in the past for benign and fully malignant
tumors composed of endothelial cells. Nowdays, thanks to Weiss
and Enzinger, they have became a specific entity and the histological pattern of these vascular tumors occupy an intermediate
position between benign haemangioendothelioma and fully malignant angiosarcoma. Subsequently, haemangioendothelioma of
soft tissue has been stratified into two risk groups of malignancy,
classic and malignant haemangioendothelioma, based on mitotic
activity and size, in an effort to reflect different biologic subsets.
Molecular biology analysis has confirmed the statement of these
lesions. In fact, the t(1;3)(p36.23;q25.1) is a consistent genetic
abnormality in malignant haemangioendothelioma, of different
anatomic locations and grades of malignancy. The translocation
fuses CAMTA1 on 1p36.23 to WWTR1 on 3q25.1. This recurrent translocation has not been detected in any of the morphologic
mimics of malignant haemangioendothelioma, such as epithelioid
hemangioma, epithelioid angiosarcoma or epithelioid sarcomalike malignant haemangioendothelioma, and thus can serve as a
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261
Fig. 1. H&E: Highly cellular tumor composed of a complex network and lobules of small capillaries and vascular channels lined by relatively uniform
endothelial cells, having round nuclei and scanty cytoplasm. Some hystiocytes with pigment of hemosiderin are intermingled between the neoplastic
cells. Immunohistochemistry: Positivity for CD 31 and Ki67.
useful molecular diagnostic tool in challenging cases. Particularly
important is the lack of CAMTA1 and WWTR1 rearrangements
in epithelioid hemangioma, an under-recognized benign tumor,
that is often misdiagnosed as malignant haemangioendothelioma,
resulting in an overtreatment. Herein we report a case of nasal sinus malignant hemangioendothelioma in a 26 year-old-man with
a past history of bilateral intraocular retinoblastoma. We describe
this nasal sinus lesion for its rarity. It arose in the background of
previous radiation therapy for a bilateral intraocular retinoblastoma and this association has never been described.
Matherial and methods. A 26 year-old-man was admitted to
our hospital for massive nasal bleeding. He complained of a
gradually increasing monolateral nasal obstruction for one month
with a mass in the right nasal sinus. Attacks of severe headache,
nausea, vomiting and disorientation were frequent. He also had
a remarkable past medical history being affected by bilateral
intraocular retinoblastoma when he was child. Actually, he was
blind. On clinical examination no masses were found. Anterior
rhinoscopy revealed a firm, soft, pinkish-greysh mass (cm 4 in its
greater dimension) that bled on probing. At that moment, there
were no lymphadenopathy or any organomegaly. Computerized
Tomography scan of the nose and paranasal sinuses in axial and
coronal sections revealed a solid enhancing mass occupying the
right nasal sinus slightly protruding in the nasal cavity and extending to maxillary sinus, nasopharynx, ethmoid and the orbit.
There was also an intracranial extension of the mass into the right
front-parietal region. A punch biopsy of the mass was performed
and processed for routine histopathological examination. Haemotoxylin-eosin stained sections showed a highly cellular tumor
composed of a complex network and lobules of small capillaries
and vascular channels lined by relatively uniform endothelial
cells, having round nuclei and scanty cytoplasm. Mitoses were
frequent. Nuclear atypia and pleomorphism were seen. Cells
protruded into the neoplastic vessels in a hobnail fashion. Some
of the capillary lumina were filled with the neoplastic cells. Vascular channels were demonstrable by CD31 and CD34 stain. Cytogenetic analysis by FISH did not show mdm-2 gene and CDK4
gene amplification. On the bases of these findings a diagnosis of
malignant haemangioendothelioma of epitheliod type was given.
The patient underwent a wide local surgery. After treatment he
has gone to a specialized centre for rare tumors where he is doing
a follow-up.
Conclusion. Haemangioendothelioma belongs to the group of
vascular tumors of intermediate malignancy since it is histologically intermediate in appearance between a haemangioma and a
conventional angiosarcoma. An important feature of this tumor
is heterogeneity. In fact, it exhibits different histological features
sometimes unusual and ambiguous, still displaying all features
of a haemangioma but with a greater degree of cellularity and
mitoses. Not only their histology but also their biological behaviour separate them from haemangioma and angiosarcoma. Fu &
Perzin studied non epithelial tumors of nasal cavity, paranasal
sinuses and nasopharynx and found that the incidence of haemangioendothelioma was 3.8% out of 81 vascular tumors of these
262
sites. It can occur in the skin, subcutaneous tissue, liver, mammary glands, bones, striated muscles, pleura, uterus, orbit and
occasionally tonsils and central nervous system. Malignant nasal
sinus haemagioendothelioma is a very uncommon lesion. Differential diagnosis of malignant vascular tumors can be often quite
challenging, either at the low end of the spectrum, distinguishing
an epithelioid hemangioendothelioma from an epithelioid hemangioma, or at the high-grade end of the spectrum, between an angiosarcoma and a malignant epithelioid hemangioendothelioma.
Within this differential diagnosis both clinical and radiological
features (ie, size and multifocality) and immunohistochemical
markers (ie, expression of endothelial markers) are often similar
and cannot distinguish between benign and malignant vascular
lesions. These features cause problems in the establishment of the
patient’s treatment that is based on the possibility of a long survival noted for some rare facial localizations. This is in contrast
with the usually very rapid development of vascular sarcomas
in other localizations. In view of the low grade nature of this
tumor it has been suggested that complete and wide local excision even without adjuvant radiotherapy or chemotherapy should
be the preferred mode of treatment. For histological malignant
forms with regional lymph node metastasis a radical neck dissection should be considered. Radiotherapy should be reserved
for multifocal and recurrent disease and the choice of treatment
when bleeding is imminent. While the morphological patterns of
many vascular tumors have been well recognized, their genetic
hallmark is still under investigation. To date, only a few vascular
tumors have been analyzed cytogenetically, with different chromosomal translocations being reported. In 2001, Mendlick et al.
found an identical chromosomal translocation, t(1;3)(p36.3;q25)
in two cases of malignant haemangioendotheliomas, suggesting
that this may represent a recurrent abnormality in this subgroup
of tumors. These data could become a practical guide in the differential diagnosis of epithelioid vascular tumors using molecular
biology analysis and in the treatment of those cases as our own
that due to their rare site of origin and to their similar morphological appearances are very difficult to diagnose.
References
1
Wong BL et al. Kaposiform hemangioendothelioma of paranasal
sinus. Laryngoscope. 2014 Mar 12; doi: 10.1002/lary.24669. [Epub
ahead of print].
2
Errani C Genes Chromosomes Cancer. et al. A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioidhemangioendothelioma of different anatomic sites. 2011 Aug;50(8):644-53.
3
Lee CH et al. Kaposiform hemangioendothelioma arising from
the maxillary sinus: a case report. Kaohsiung J Med Sci. 2010
Mar;26(3):154-7.
4
Semino L et al. Endoscopic treatment of ethmoidal hemangioendothelioma: case report and review of the literature. Am J Otolaryngol.
2006 Jul-Aug;27(4):287-90. Review.
5
Birchler MT et al. Kaposiform hemangioendothelioma arising in the
ethmoid sinus of an 8-year-old girl with severe epistaxis. Head Neck.
2006 Aug;28(8):761-4.
6
Gupta SC et al. Haemangioendothelioma of paranasal sinuses with
intracranial extension. Indian J Otolaryngol Head Neck Surg. 2006
Apr;58(2):196-8. doi: 10.1007/BF03050789.
Coexisting sclerosing angiomatoid nodular
transformation of the spleen with lymphangioma
M. Onorati1, G. Petracco1, P. Uboldi1, C. D’Urbano2, F. Di Nuovo1
Pathology Unit, AO G. Salvini Garbagnate Milanese, MI; 2 Surgical Unit,
AO G. Salvini Garbagnate Milanese, MI
1
Introduction. The spleen has a complex and distinct vascular
structure compThe spleen has a complex and distinct vascular
structure compared with the vessel of other organs. So the vascular lesions, which are the majority of non-lymphoid primary
tumors of the spleen, can display a wide variety of histologic
features in this organ. Sclerosing angiomatoid nodular trans-
formation (SANT) is a recently recognized splenic vascular
lesion (first described by Martel in 2004) characterized by
multiple well-circumscribed individual and confluent vascularangiomatoid nodules with interstitial haemorrhage set mixed
with inflammatory cells. SANT appears to be a benign process
with a pathogenesis not fully understood. The lesion is often
discovered on imaging studies performed for other reasons and
splenectomy has been curative in all cases reported. Herein we
report a peculiar case of SANT of the spleen coexisting with a
lymphangioma. Coexistence of these two entities in a patient has
never been reported. Moreover we discuss differential diagnosis,
immunohistochemical profile and pathogenesis.
Material and methods. A 49- years- old man presented to our
hospital with acute abdominal pain. His past medical history
was unremarkable. He underwent a CT scan without contrast
enhancement and the radiological exam showed a diverticulitis
with perivisceritis and two splenic lesions of 44 mm and 13 mm
with evocative aspects of cavernous hemangioma. A repetead Ct
scan after 2 months with contrast enhancement confirmed the
diverticulosis and the two lesions of the spleen. The patient underwent surgical excision of the colic segment and splenectomy.
The macroscopic examination of the resected spleen revealed an
intraparenchimal, brownish, solid, lobular lesion, 58 mm in the
greater diameter and a subcapsular, grayish lesion, 15 mm in the
greater diameter. Microscopically the intraparenchimal lesion
displayed a vascular proliferation with very low mitotic rate (1
mitosis/10 HPF), angiocentric sclerosis and a inflammatory infiltrate formed of lymphocytes, plasma cells and histiocytes. The
immunohistochemistry showed positivity for CD34 and CD31
in cord capillaries, and positivity for CD8 in vascular sinusoids.
The subcapsular lesion displayed large and ectatic vascular space
filled with transparent pink material on Hematoxylin&Eosin sections and immunohistochemistry showed positivity for D2-40.
On the bases of these findings the final diagnosis was consistent
with sclerosing angiomatoid nodular transformation of the spleen
associated with lymphangioma.
Discussion. Splenic nodular lesions may pose diagnostic difficulties pre-operatively because splenic fine needle aspiration
biopsy is not usually performed due to the potential hemorrhagic
complications of the procedure, although it could be diagnostic.
Vascular tumors are the most common neoplasms of the spleen
and may mimic malignant tumors both clinically and radiologically. SANT usually affects middle-aged adults and shows a female predominance. It is a benign lesion and splenectomy has so
far proved curative. The relative high prevalence of synchronous
malignant tumor at other sites among the patients is likely to be
due to the fact that patients with malignancies undergo a variety
of imaging studies that lead to the detection of asymptomatic lesions like that. There is currently no pathognomonic finding for
the diagnosis of SANT on cross-sectional imaging, however the
literature suggests that the diagnosis can be made if a contrastenhanced MRI shows a “spoke-wheel pattern”, while the use of
contrast-enhanced ultrasonography needs to be further evaluated
as data is limited. The typical macroscopic feature of a SANT
lesion is of a well-circumscribed, non encapsulated, lobulated
mass with multiple dark brown nodules (hemorrhagic regions in
angiomatoid nodules) interspersed with stellate, whitish, fibrosclerotic scar. Microscopically SANT is composed of three basic
cellular components, spindle cells, inflammatory infiltrate and
endothelial vascular proliferation intermingled each other molding the angiomatoide nodules. In this lesion there are three distinct types of blood vessels, mirroring the normal composition of
splenic red pulp: a cord-capillary like type that co-express CD34
and CD31 but not CD8, a sinusoid-like type that express CD8
and CD31 but not CD34 and small veins that express only CD31.
These features allow the pathologist to differentiate it from
other splenic vascular lesions. In fact hamartoma of the spleen
is composed of disorganized overgrowth of sinus-like structures
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263
Fig. 1. Gross appearance of resected spleen showing an intraparenchimal red-brownish and white fibrosclerotic stellate scar and a subcapsular
graysh lesion. H&E of SANT showing multinodular vascular pattern. Immunoistochemistry showing positivity for CD8 and CD34 in different vascular
patterns of SANT.
of the red pulp, which is immunoreactive for CD8. Littoral cell
angioma usually presents as a nodular lesion with spongy aspect
and microscopically it displays anastomosing sinusoidal vascular
channels lined by tall endothelial cells with endothelial and histiocytic immunophenotype (CD34,CD31,CD68) but negative for
CD8. Inflammatory pseudotumor is a myofibroblastic prolifera-
tive lesion with an edematous myxoid background rich in blood
vessels and inflammatory cells. Hemangioendothelioma is a low
grade malignant vascular tumor that could have a sclerotic stroma

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