Journal of the Italian Society of Anatomic Pathology and Diagnostic

Transcript

ISSN 0031-2983
MYKEY
Cited in Index Medicus/MEDLINE, BIOSIS Previews, SCOPUS
MK
Key to Diagnosis
ESPERIENZA
conOSCENZA
EVOLUZIONE
Journal of the Italian Society of Anatomic Pathology
and Diagnostic Cytopathology,
Italian Division of the International Academy of Pathology
Società Italiana di Anatomia Patologica e Citopatologia diagnostica
Divisione Italiana della International Academy of Pathology
La DIAGNOSTICA che cambia
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. 105 October 2013
6° Congresso Triennale Siapec-Iap
ROMA - 26-30 Ottobre 2013
Mykey è un nuovo approccio metodologico e strumentale
La disciplina e il ruolo dell’anatomopatologo sono al centro
di una rapida e complessa evoluzione, che non può essere fronteggiata
con una visione tradizionale. MyKey è la soluzione
mykey
il sistema
MyKey è un sistema basato sull’esperienza che diventa conoscenza
per poi diventare nuovamente esperienza
È un sistema di navigazione, un GPS per la conoscenza
MyKey è uno strumento integrato di controllo dell’attività dei nostri servizi
Mykey consente una gestione più efficiente
ed un più attento utilizzo delle risorse sia umane che economiche
gestionale
esperto
per
l’anatomopatologo
26-30 Ottobre 2013
www.siapecroma2013.it
ROMA
Hotel Ergife Palace
Società Italiana di Anatomia Patologica e Citopatologia Diagnostica,
Simposio Roche
6° Congresso Triennale Siapec
Hotel Ergife
Palace
Hotel Ergife Palace
26-302013
Ottobre 2013
Roma,
26-30 Roma,
Ottobre
Hotel Ergife Palace
Roma, 26-30 Ottobre 2013
Simposio
Roche nella determinazione
La
qualità
La qualità
nella determinazione
dei
biomarcatori:
aspetti clinici e legali
dei biomarcatori: aspetti clinici e legali
• Il percorso Her2 positivo Vincenzo Arena
Il percorso Her2
positivo
Arena
•••L’importanza
di Braf
nel Vincenzo
melanoma
metastatico Daniela Massi
Il percorso Her2
positivo
Vincenzo
Arena
L’importanza
Brafalla
nelmedicina
melanomalegale
metastatico
Daniela
L’importanza
didiBraf
nel
melanoma
metastatico
Massi
•••Dal
rischio clinico
VaniaDaniela
Cirese Massi
• Dal rischio clinico alla medicina legale Vania Cirese
• Dal rischio clinico
allaall’errore:
medicina legale
Cirese
WORKGROUP
Caccia
causeVania
e conseguenze
WORKGROUP Caccia all’errore: cause e conseguenze
28 Ottobre 2013 • 13,00 - 14,00
28 Ottobre 2013 • 13,00 - 14,00
Sala Orange 2
28 Ottobre 2013
Sala Orange 2
Ore 13,00
- 14,00
La Sua opinione
sarà il punto
di partenza per la discussione
La Sua opinione sarà il punto di partenza per la discussione
Sala Orange 2
Hotel Ergife Palace
Simposio Roche
Collegarsi al sito www.editreespace.com/biomarcatori
La
qualità nella determinazione
Inserire password biomarcatori2013
Registrarsi
inserendo i propri dati
dei
biomarcatori:
aspetti
clinici
e
legali
Rispondere ai 4 quesiti scientifici
• Il percorso Her2 positivo Vincenzo Arena
la ricezione dell’e-mail di conferma
•Attendere
L’importanza
di Braf nel melanoma metastatico Daniela Massi
Le risposte saranno spunto di discussione durante il simposio
• Dal rischio clinico alla medicina legale Vania Cirese
WORKGROUP Caccia all’errore: cause e conseguenze
Hotel Ergife Palace
Cited in Index Medicus/MEDLINE, BIOSIS Previews, SCOPUS
Journal of the Italian Society of Anatomic Pathology
and Diagnostic Cytopathology,
Italian Division of the International Academy of Pathology
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,
Governing Board
SIAPEC-IAP
President:
C. Clemente, Milano
Vice President:
G. De Rosa, Napoli
General Secretary:
A. Sapino, Torino
Past President:
G.L. Taddei, Firenze
Members:
A. Bondi, Bologna
P. Dalla Palma, Trento
A. Fassina, Padova
R. Fiocca, Genova
D. Ientile, Palermo
L. Resta, Bari
L. Ruco, Roma
M. Santucci, Firenze
G. Zamboni, Verona
Associate Members
Representative:
T. Zanin, Genova
Copyright
Società Italiana di Anatomia
Patologica e Citopatologia
Diagnostica, Divisione
Italiana della International
Academy of Pathology
Publisher
Pacini Editore S.p.A.
Via Gherardesca, 1
56121 Pisa, Italy
Tel. +39 050 313011
Fax +39 050 3130300
[email protected]
www.pacinimedicina.it
Vol. 105 October 2013
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Società Italiana di Anatomia Patologica e Citopatologia diagnostica
26-30 Ottobre 2013
www.siapecroma2013.it
ROMA
Hotel Ergife Palace
Comitati
4
Sotto l’Alto Patronato
del Presidente della Repubblica
dell’Ordine Provinciale dei Medici-Chirurghi e degli Odontoiatri di Roma
PRESIDENTE
Claudio Clemente
PAST PRESIDENT
Gian Luigi Taddei
VICE PRESIDENTE
Gaetano De Rosa
SEGRETARIO
Anna Sapino
CONSIGLIERI
Arrigo Bondi
Paolo Dalla Palma
Ambrogio Fassina
Roberto Fiocca
Domenico Ientile
Leonardo Resta
Luigi Ruco
Marco Santucci
Giuseppe Zamboni
Rappresentante AITIC
Carmelo Lupo
PRESIDENTE DEL
CONGRESSO
Claudio Clemente
COMITATO
SCIENTIFICO
PRESIDENTE
Luigi Ruco
COMPONENTI
Piero Alò
Generoso Bevilacqua
Claudio Clemente
Gaetano De Rosa
Angelo Paolo Dei Tos
Carlo Della Rocca
Vittoria Donofrio
Alfredo Fabiano
Roberto Fiocca
Pietro Gallo
Felice Giangaspero
Antonio Marchetti
Oscar Nappi
Andrea Onetti Muda
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Guido Rindi
Anna Sapino
Gian Luigi Taddei
Fabio Maria Vecchio
Sabato 26 O
Sa
9.00-13.00
con il Patrocinio
CONSIGLIO DIRETTIVO
SIAPEC
Tavole s
13.00-16.00 Gr
St
Co
16.00-17-00
COMITATO
ORGANIZZATORE
17.00-18.00
PRESIDENTE
Giuseppe Santeusanio
19:30
COMPONENTI
Gerardo Botti
Franca Del Nonno
Maria Rosaria Giovagnoli
Vito Gomez
Mirella Marino
Ferdinando Quarto
Guido Pettinato
Antonio Rosario Ricci
Patrizia Rigato
Raffaele Rossiello
Steno Sentinelli
Stefania Uccini
Sa
Ce
Domenica
Sala
08.30-10.30 Sim
La p
dell
uter
10.30-11.00
11.00-12.00 Sim
La d
dell
endo
12.00-13.00 Lettu
13.00-14.30
SEDE
Hotel Ergife Palace
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00165 Roma RM
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SEGRETERIA ORGANIZZATIVA
Via Sassonia, 30 - 47922 Rimini
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14.30-16.30 Cors
La d
dei t
ovar
16.30-16.45
16.45-18.30 Slid
Gine
LEGENDA
= Simposi
= Gruppi d
omitati
a
li
hotel.com
NIZZATIVA
22 Rimini
ngrex.it
Tavole sinottiche
PROGRAMMA SCIENTIFICO
Sabato 26 Ottobre 2013
Sala Messalia Sala Orange 1 Sala Orange 2 Sala Terragona
Gruppo di
studio APCI
Gruppo di
Gruppo di
Gruppo di
13.00-16.00 Gruppo di
Studio Testa Studio GYM studio GIPAM Studio Pagine
Collo
Presentazione
16.00-17-00
FAD ai Gruppi
17.00-18.00
Sala Cesarea Sala Mileto
Sala Hama
9.00-13.00
19:30
Gruppo di
studio NAP
Gruppo di Studio
Gruppo di
Qualita’ Sicurezza e Ge- Studio GIC
stione del rischio in A.P.
Gruppo
di Studio
GIPAD
Sala Plenaria
Cerimonia Inaugurale e Saluto delle Autorita’
Domenica 27 Ottobre 2013
Sala Plenaria
08.30-10.30 Simposio
La patologia
della cervice
uterina
Sala Messalia Sala Orange 1
Simposio I
Simposio
Ematopatologia Neoplasie endocrine/
neuroendocrine
di grado intermedio ed
alto: stato dell’arte
Sala Tarragona
Simposio
Patologia
Pediatrica
Sala Cesarea
Comunicazioni
orali
Patologia
Mammaria 1
Simposio
Patologia
Pediatrica
Comunicazioni
orali
Patologia
Mammaria 2
Caffè
10.30-11.00
La diagnostica
della biopsia
endometriale
Sala Orange 2
L’Autopsia Oggi
Simposio II
Simposio
L’Autopsia Oggi
Ematopatologia Neoplasie endocrine/
neuroendocrine:
aspetti molecolari,
stato dell’arte ed
applicazioni pratiche
12.00-13.00 Lettura magistrale: Infiammazione e Cancro. A. Mantovani, Università di Milano
Lunch
13.00-14.30
14.30-16.30 Corso breve
La diagnostica
dei tumori
ovarici
Corso breve
Patologia
mammaria
Patologi Oltre Frontiera
Donne che aiutano le
donne: patologhe oltre
frontiera
Caffè
16.30-16.45
16.45-18.30 Slide Seminar Slide Seminar
Ginecopatologia Tumori degli
istiociti e delle
cellule dendritiche
e loro mimi
LEGENDA
= Simposio
= Gruppi di Studio
Corso breve
Diagnostica
Comunicazioni
Definizione di maligni- Ultrastrutturale orali
tà in neoplasie endoUropatologia
crine e neuroendocrine
Telepatologia con
Slide Seminar
Patologi Oltre Frontiera Casi difficili ed
Italia-Africa: prove
inusuali
generali di diagnostica
digitale
= Lettura Magistrale
= Corso Breve
= Slide Seminar
= Comunicazioni Orali
Incontro sui
controlli di
qualità FISH
Comunicazioni
orali
Patologia
dell’Osso e dei
Tessuti Molli
= Sessione Congiunta
= Assemblea Societaria
5
Tavole sinottiche
6
Lunedì 28 Ottobre 2013
Sala Plenaria
Martedì 29
Sala Messalia
08.30 -10.30 Simposio
Patologia digestiva
Sala Orange 1
Sala Orange 2
Simposio
Simposio
Management
Dermatopatologia Update in patologia in Anatomia
Novità sulle
toraco-polmonare Patologica:
lesioni
qualità, sicurezza
melanocitiche
e gestione del
rischio in Anatomia
Patologica
Sala Tarragona Sala Cesarea
Simposio
La diagnostica
pre-operatoria
nel distretto
testa-collo
Comunicazioni
orali
Ematopatologia
Simposio
11.00-12.00 Patologia digestiva
Simposio
Dermatopatologia
Non solo
melanoma
Tavola rotonda
La caratterizzazione
molecolare
del carcinoma
polmonare in
funzione della terapia
medica: i percorsi
diagnostici ottimali
Management
in Anatomia
Patologica:
qualità, sicurezza
e gestione del
rischio in Anatomia
Patologica
Simposio
Ruolo del
patologo nella
determinazione di
fattori predittivi
nelle neoplasie
del distretto
testa-collo
Comunicazioni
orali
Patologia
Troidea e
Neuropatologia
14.30-16.30 Corso breve
Corso breve
Il processo
La diagnosi dei
diagnostico nello
linfomi cutanei
screening del
carcinoma colorettale: dall’invio del
campione biologico
alla refertazione
Corso breve
Il contenzioso
Hot topics e
medico-paziente
controversie in
patologia toracica
12.00 -13.00 Lett
13.00-14.30
16.00-16.30 Nom
mia
Uno
sabi
13.15 - 14.00
Simposio Myriad
NON ECM
16.30-16.45
Comunicazioni
orali
Citologia
16.45-18-30 Slid
Pato
Mercoledì
Sala
Caffè
16.30-16.45
Slide Seminar
16.45-18-30 Patologia Digestiva
13.00 - 14.00
Simposio Roche
NON ECM
Simposio Dako
NON ECM
Lunch
11.00-12.00 Sim
Uni
mul
guid
14.30-16.00
12.00 -13.00 Lettura magistrale: Pre-Analytic, Quality Control and Predictive Molecular Pathology
M. Dietel (Berlin, Germany)
13.00-14.30
08.30 -10.30 Sim
Uni
mul
guid
10.30-11.00
Caffè
10.30-11.00
Sal
Slide Seminar
Slide Seminar
Dermatopatologia Patologia toracopolmonare
Attualità
legislative e
giurisprudenziali in
ambito sanitario
Comunicazioni
orali
Ginecopatologia e
Tecniche
08.30 -10.30 Sim
Urop
10.30-11.00
11.00-12.00 Sim
Urop
LEGENDA
= Simposio
= Gruppi di Studio
= Lettura Magistrale
= Corso Breve
= Slide Seminar
= Comunicazioni Orali
= Sessione Congiunta
= Assemblea Societaria
12.00 -13.00 Proc
nottiche
PROGRAMMA SCIENTIFICO
Martedì 29 Ottobre 2013
a Sala Cesarea
Comunicazioni
orali
Ematopatologia
Sala Plenaria
Sala Messalia Sala Orange 1 Sala Orange 2 Sala Tarragona
Simposio
08.30 -10.30 Simposio I parte
Unifocalità,
Citologia
multifocalità e linee vaginale
guida tumori mammari
Patologia
Simposio
Molecolare: Il patologo
le metodologie cardiovascolare oggi
“non in situ”
Comunicazioni
orali
Patologia
DIgestiva 1
Caffè
10.30-11.00
Comunicazioni
orali
Patologia
di Troidea e
Neuropatologia
Simposio
Patologia
dell’Osso
Sala Cesarea
Simposio
11.00-12.00 Simposio II parte
Unifocalità,
Nuove Tecniche
multifocalità e linee in citologia
guida tumori mammari
Simposio
sulla patologia
dei Tessuti
Molli
Patologia
Molecolare:
le metodologie
“in situ”
Simposio
Il consenso del Gruppo Italiano di Cradiopatologia per
la validazione di parametri
immunoistochimici nelle
cardiomiopatie ereditarie
Comunicazioni
orali
Patologia
Digestiva 2
12.00 -13.00 Lettura magistrale: Le cellule staminali tumorali. Ruggero De Maria, Istituto Tumori Regina Elena, Roma
13.00-14.30
Lunch
14.30-16.00
Assemblea Societaria
16.00-16.30 Nomenclatore di Anatomia Patologica - NAP.
Uno strumento indispensabile per il patologo
d
Caffè
16.30-16.45
Comunicazioni
orali
Citologia
Slide seminar
16.45-18-30 Slide Seminar
Patologia mammaria Citologia
Slide Seminar La formazione Comunicazioni
Osso e Tessuti in Anatomia orali
Molli
Patologica
Patologia Polmonare,
Molecolare e Paleopatologia
Comunicazioni
orali
Dermatopatologia e Patologia Testa-Collo
Mercoledì 30 Ottobre 2013
Sala Plenaria
Comunicazioni
orali
Ginecopatologia e
Tecniche
sione Congiunta
emblea Societaria
08.30 -10.30 Simposio
Uropatologia
Sala Messalia
Sala Orange 1
Sala Orange 2
Sala Tarragona
Simposio
Neuropatologia
SIAPEC-AIOM
Sessione AITICANTel-ASSIATEL
Simposio
Patologia dei
Trapianti
Sessione AITICANTel-ASSIATEL
Simposio
Patologia dei
Trapianti
Caffè
10.30-11.00
Uropatologia
Simposio
Neuropatologia
SIAPEC-AIOM
12.00 -13.00 Proclamazione del nuovo direttivo, consegna dei premi ai migliori Abstracts e chiusura
7
Sommario
Relazioni.................................................................................................................................... pag.147
Comunicazioni orali......................................................................................................................»224
Poster.............................................................................................................................................»259
Indice degli Autori........................................................................................................................»319
relazioni
Pathologica 2013;105:147-223
Domenica, 27 ottobre 2013
Sala Plenaria – ore 8.30-18.30
Ginecopatologia
Simposio: la patologia della cervice uterina
Moderatori: G.L. Taddei (Firenze), L. Resta (Bari)
Dalla displasia al carcinoma a cellule squamose
G.F. Zannoni
Anatomia Patologica, Policlinico A. Gemelli, Università Cattolica del
Sacro Cuore, Roma
All’inizio del secolo scorso il patologo inglese Sir William descrisse la presenza di anomalie cellulari accanto al carcinoma
invasivo, mentre fu Brodes che, intorno al 1930, introdusse il
termine di carcinoma in situ (CIS) per descrivere tali anomalie
che identificavano una precancerosi. Soltanto in un secondo
momento, tuttavia, Smith e Pemberton dimostrano la relazione temporale e spaziale del carcinoma in situ, descrivendo
casi di progressione verso il carcinoma invasivo.
Questi studi hanno consentito l’identificazione di un modello
di screening che permette di riconoscere una lesione preinvasiva da una lesione invasiva.
Intorno agli anni ’50 Reagan introdusse il termine di displasia per identificare quei quadri cito-istologici con anomalie
intermedie rispetto al normale e al carcinoma invasivo e
precisamente per descrivere un’alterazione della maturazione
cellulare caratterizzata dalla proliferazione di cellule squamose del tutto simili a quelle degli strati basali, ma con spiccate
alterazioni nucleari e con alterato rapporto nucleo-citoplasma.
In base al livello in cui tali alterazioni sono distribuite (strato
basale, parabasale e superficiale) la displasia assume il grado
rispettivamente di lieve, moderata ed invasiva. Nel frattempo
alcuni studi biologici avevano indicato come le alterazioni
cellulari della displasia e del carcinoma fossero qualitativamente simili: in entrambe le forme era infatti identificabile
una proliferazione monoclonale di cellule epiteliali squamose
anormali con contenuto di DNA aneuploide. Sulla base di tali
ricerche Richart, introducendo il concetto che tutti i precursori
del carcinoma cervicale rappresentino gradi differenti di un
unico processo che ha come risultato finale la invasione neoplastica dello stroma, propose la terminologia CIN (neoplasia
cervicale intraepiteliale). La CIN è suddivisa in tre categorie:
CIN1: fase iniziale della malattia con evidenzia delle atipie
cellulari nel primo terzo; CIN2: sovrapponibile alla displasia
moderata con evidenza di cellule atipiche fino al secondo
terzo compreso; CIN3: sovrapponibile alla displasia severa,
con cellule atipiche fino allo strato superficiale compreso.
Per cellule atipiche si intende allargamento nucleare e atipia
nucleare caratterizzata da pleomorfismo, cromatina dispersa a
zolle e contorni nucleari irregolari.
Molteplici studi condotti negli anni ’80 evidenziavano però
come la percentuale di CIN1 che evolveva a carcinoma fosse
relativamente bassa (circa 20%), poiché nella maggior parte
dei casi la malattia tende a regredire spontaneamente oppure
a persistere. Dunque, dal punto di vista biologico, non si può
considerare la CIN1 come una malattia evolutiva ma piuttosto
come una malattia biologicamente distinta, più blanda, che
nella maggior parte dei casi ha una prognosi eccellente. Tali
studi hanno cambiato la storia del trattamento della malattia:
attualmente, infatti, il clinico tende a curare soltanto le CIN2
e le CIN 3 mentre tende ad avere un atteggiamento d’attesa e
conservativo per le CIN1.
L’attuale sistema Betesdha, proposto all’inizio degli anni
2000, descrive molto bene lo iato fra le due patologie e il
diverso trattamento, dividendo la patologia preneoplastica
cervicale in due categorie: la SIL di basso grado designa le
coilocitosi e le CIN1, mentre le SIL di alto grado designano
le CIN2, le CIN3 e il carcinoma in situ. Tale sistema, ideato
per la gestione della citologia, trova applicazione anche nell’istologia, benché, in tale ultima disciplina risulti preferibile
l’uso della terminologia CIN. Il motivo risiede nel fatto che,
secondo alcuni studi, una quota di CIN2 regredisce, e pertanto
il clinico, conoscendo l’entità della displasia, può optare per
un trattamento più conservativo.
Nella nostra istituzione (Policlinico Gemelli) è d’uso riportare
nel referto la doppia classificazione (esempio: HIGH GRADE
SIL – CIN2 oppure LOW GRADE SIL – CIN1). Inoltre si
tende a separare, nell’ambito delle SIL di basso grado, le
coilocitosi e le CIN1. La coilocitosi rappresenta l’espressione
morfologica della infezione da virus HPV e le alterazioni
cellulari diagnostiche sono rappresentate dalla presenza di
coilociti che possono localizzatasi in tutti e tre gli strati. La
lesione CIN1 richiede la presenza di cellule basaloidi con
nucleo ipercromico e talora presenza di attività mitotica. In
base alla localizzazione di tali cellule (fino alla strato basale: CIN1, fino alla strato parabasale: CIN2 e fino allo strato
superficiale: CIN3) si gradua la malattia. In questi anni si è
tentato di identificare dei biomarcatori utili a selezionare le
CIN1 con comportamenti evolutivi. Poiché è stato dimostrato
che nelle lesioni infettive e in molti CIN 1 il virus dell’HPV
è in forma episomica, mentre nelle CIN2, CIN3 e carcinomi
il virus è in forma integrata, si è pensato di identificare le due
forme di infezione, prevedendo un differente intervento. In
tal senso ha avuto espansione lo studio di p16, una proteina che esprime l’integrazione del Virus, sebbene i risultati
clinici abbiano mostrato che la sua positività non è sempre
correlabile alla progressione. Numerosi studi concordano nel
considerare quale dato maggiormente attendibile la negatività
dell’espressione per la proteina in base alla constatazione che
i casi negativi per p16 tendono a non progredire.
Mentre la ricerca del DNA virus ha dato risultati scoraggianti,
in quanto il virus viene isolato in molte donne sotto i 30 anni
d’età, anche in assenza di lesione colposcopicamente rilevabile, più incoraggianti appaiono gli studi sulla identificazione
del RNA Virus, che è presente nei casi evolutivi in cui il virus
è integrato al DNA dell’ospite.
Lo studio morfologico della lesione ha un’importanza cruciale. A noi patologi, sulla base dell’istologia, ci viene richiesto
di differenziare le displasie che sono precancerosi da lesioni
148
simulanti, che hanno un impatto clinico completamente differente: metaplasia squamosa (presenza di glicogeno intracitoplasmatico, nucleo centrale e regolare senza atipie, assenza
di disordine di crescita, mantenimento della polarità nucleare,
alta espressione di Ki67 prevalentemente nello strato basale e
negatività per p16), metaplasia transizionale (donna anziana,
associata alla atrofia, assenza di atipie, assenza di mitosi). Il
carcinoma invasivo è dunque l’evoluzione finale della patologia preinvasiva. Si divide in due grandi categorie, per le quali
il clinico adotta comportamenti terapeutici completamente
differenti: carcinoma microinvasivo, neoplasia squamosa invasiva per meno di mm 5 ed estendentesi per una lunghezza
inferiore a mm 7 (suddisviso im IA1: meno di mm 3 e IA2 tra
mm 3 e mm 5, IB quando superiore per estensione a mm 7)
e carcinoma invasivo, neoplasia invasiva per 5 mm e più. La
diagnosi di carcinoma microinvasivo va evitata nelle biopsie
e può essere individuata soltanto nelle conizzazioni, avendo
avuto cura di includere ed esaminare l’intera cervice uterina. Il
carcinoma invasivo si gradua in base alle dimensioni nucleari,
ma, tra le pur numerose classificazioni proposte, nessuna ha
evidenziato correlazione con la prognosi. In genere i carcinomi non chreratinizzanti tendono a rispondere meglio alla
radio-chemioterapia rispetto ai carcinomi cheratinizzanti. Il
fattore di rischio più importante nella prognosi dei carcinomi
radiochemiotrattati rimane tuttavia lo stadio nel momento
della diagnosi.
Bibliografia
Richart RM. Cervical intraepithelial neoplasia: a review. In: Sommer
SC, editor. Pathology Annual. Appleton Century Crofts East Norwalk
1973, pp. 301-2328.
Cattani P, Siddu A, D’Onghia S, et al. RNA (E6 and E7) assays versus
DNA (E6 and E7) assays for risk evaluation for women infected with
human papillomavirus. J Clin Microbiol 2009;47:2136-41.
Negri G, Bellisano G, Zannoni GF, et al. p16 ink4a and HPV L1 immunohistochemistry is helpful for estimating the behavior of low-grade dysplastic lesions of the cervix uteri. Am J Surg Pathol 2008;32:1715-20.
Zannoni GF, Vellone VG, Carbone A. Morphological effects of radiochemotherapy on cervical carcinoma: a morphological study of 50 cases
of hysterectomy specimens after neoadjuvant treatment. Int J Gynecol
Pathol 2008;27:274-81.
Tumours of uterine cervix: small cells findings
D. Ientile, C. Mignogna, R. Genova
UO Anatomia Patologica, Ospedale Buccheri La Ferla FBF, Palermo
Current WHO (World Health Organization) classify epithelial
tumours of uterine cervix in squamous tumours, glandular tumours, other epithelial tumours and neuroendocrine tumours.
Finding a small cells tumour open a range of different diagnosis, ranging over the described classification.
Squamous cells tumours were first classified by Wenz and
Reagan in large cell keratinizing, large cell nonkeratinizing,
and small cell nonkeratinizing; they correlate these forms to
the prognostic outcome after radiation therapy. Because the
frequent confusion generating among the small cell nonkeratinizing squamous cell carcinoma and small cell undifferentiated carcinomas with neuroendocrine features, WHO classification include the last form in a separate class.
Neuroendocrine tumours of uterine cervix are rare, they are
6° Congresso Triennale Siapec-IAP • Roma, 26-30 OTTOBRE 2013
classified by WHO as typical (classic) carcinoid tumours,
atypical carcinoid tumours, large cell neuroendocrine carcinomas, and small (oat) cell neuroendocrine carcinomas.
Small cell neuroendocrine carcinomas account for 1-6%of
cervical carcinomas.. Morphological features are small anaplastic cells with scanty cytoplasm, finely granular chromatin, and inconspicous nucleoli. Mitotic rate is considerable
and necrosis is frequent. Vascular involvement is normally
present.
Small cell neuroendocrine carcinomas should be distinguished from poorly differentiated nonkeratinizing squamous
cell carcinomas composed of small cells. Usually nonkeratinizing squamous cell carcinomas have more cytoplasm
and cells are arranged in cohesive nests and have nucleoli.
Immunohistochemistry for neuroendocrine markers could be
useful to distinguish the neuroendocrine nature of the tumour,
but not in all cases, because 40% of nonkeratinizing squamous
cell carcinomas are positive to neuroendocrine markers and
40% of small cell neuroendocrine carcinomas are positive
for cytokeratins. The marker p63 could be helpful to identify
squamous differentiation.
So in this case it is important to observe the pattern of invasion of the tumour, because nonkeratinizing squamous cell
carcinomas colonize the stromal component in discrete nests,
whereas small (oat) cell neuroendocrine carcinoma cells
invade the stroma diffusely in trabecule and indefined nests.
An other area of differential diagnosis are lymphomas
involving uterine cervix. The most common type of lymphomas involving the cervix is the diffuse large B-cell
lymphoma, followed by the follicular lymphoma. In this
cases neoplastic cells infiltrates about, but does not replace
or destroy the endocervical glands. Cells are rounded, with
nuclear pleomorphism, vescicular chromatin, tiny nucleoli
and several mitoses. Immunohistochemical analysis with
a screening panel of antibodies against leukocyte common
antigen and neuroendocrine markers can easily support the
correct diagnosis.
Metastatic tumours to the uterine cervix can complicate the
differential diagnosis of small (oat) cell neuroendocrine carcinomas. Melanomas and metastatic tumour from the breast can
be excluded with immunohistochemistry. Although a careful
morphological evaluation can advance the diagnosis.
Inflammatory condition as follicular cervicitis could also
complicate the diagnosis; the dense infiltrate of lymphocytes present in the stromal component can mimic a small
cell tumour, but the presence of prominent follicles and the
specific morphology of inflammatory cells, quickly resolve
the question.
Recent literature highlight the presence of HPV 18 - DNA in
neuroendocrine tumours.
References
World Health Organization Classification of Tumours. Tumours of Breast
and Female Genital Organs. Internationale Agency for Research on
Cancer. Lyon 2003, p. 279.
Siriaunkgul S, Utaipat U, Suwiwat S, et al. Prognostic value of HPV18
DNA viral load in patients with early-stage neuroendocrine carcinoma of the uterine cervix. Asian Pac J Cancer Prev 2012;13:3281-5.
Abeler VM, Holm R, Nesland JM, et al. Small cell carcinoma of the cervix. A clinicopathologic study of 26 patients. Cancer 1994;73:672-7.
149
relazioni
Simposio: la diagnostica della biopsia endometriale
Moderatori: G.L. Taddei (Firenze), L. Resta (Bari)
La biopsia del carcinosarcoma (MMMT)
M.L. Carcangiu
Roma
I carcinosarcomi o tumori mulleriani misti maligni (MMMT)
costituiscono < 5% di tutti i tumori maligni dell’utero. È stata
riscontrata una associazione tra insorgenza del carcinosarcoma e trattamento con Tamoxifen o somministrazione di
estrogeni senza l’aggiunta di progestinici o trattamento radioterapico pelvico. In questo ultimo caso l’intervallo medio
tra il trattamento e la comparsa della neoplasia è tra i 10 e 20
anni. Inoltre le pazienti con carcinosarcoma uterino hanno gli
stessi fattori di rischio di quelle che sviluppano un carcinoma
dell’endometrio. C’è anche evidenza di una occasionale associazione con mutazioni del gene BRCA e con la sindrome
di Lynch.
I carcinosarcomi sono tumori tipici delle donne in postmenopausa e il sanguinamento vaginale è la presentazione
più frequente. Circa un terzo delle pazienti ha evidenza di
neoplasia extrauterina al momento della diagnosi. All’esame
clinico si apprezza spesso un ingrandimento dell’utero o una
massa pelvica e la neoplasia è riconoscibile a livello dell’ostio
cervicale in circa la metà dei casi.
Macroscopicamente, i carcinosarcomi appaiono come neoplasie polipoidi, generalmente di grandi dimensioni che
riempiono la cavità uterina talvolta prolassando nel canale
cervicale. Al taglio la neoplasia è tipicamente soffice e mostra
aree di necrosi, emorragia e degenerazione cistica. Inoltre, è
frequentemente evidente invasione del miometrio e talvolta
estensione alla cervice.
Istologicamente, i carcinosarcomi sono caratterizzati da una
intima commistione di epitelio e mesenchima, entrambe
maligni. Una delle due componenti può predominare. Le due
componenti sono di solito distinte e ben demarcate ma possono fondersi l’una con l’altra. La componente epiteliale è
più spesso di tipo endometrioide o sieroso ma possono essere
presenti altri tipi istologici. La componente mesenchimale è
classificata come omologa quando, come avviene nella maggior parte dei casi, è costituita da un sarcoma di alto grado
senza caratteristiche specifiche, ed eterologa quando sono
presenti elementi mesenchimali eterologhi come rabdomiosarcoma, condrosarcoma e, raramente, osteosarcoma. Una
differenziazione neuroectodermica è stata inoltre descritta.
Invasione miometriale e vascolare è un reperto istologico
comune.
Nelle biopsie o curettage endometriali la diagnosi differenziale tra un carcinosarcoma ed un carcinoma scarsamente
differenziato può essere difficile specialmente nei casi in cui
la proliferazione neoplastica del carcinoma è costituita da
elementi cellulari fusiformi che possono essere interpretati
come la componente mesenchimale di una neoplasia bifasica.
In genere le cellule fusate sia di tipo squamoso che ghiandolare hanno un grado di atipia citologica inferiore da quello
mostrato dalle cellule del carcinosarcoma ed in genere si può
osservare una transizione con elementi meglio differenziati. Il
riconoscimento di un pattern bifasico, con una ovvia componente sarcomatosa è il più importante criterio per la diagnosi
di carcinosarcoma. La presenza di una componente mesenchimale di tipo eterologo ovviamente facilita la diagnosi.
Fitogeneticamente, i carcinosarcomi sono ritenuti di deri-
vazione epiteliale ed esemplificano la transizione epiteliomesenchimale.
Numerosi studi genetici e molecolari hanno confermato
l’origine clonale dei carcinosarcomi dimostrando le stesse
mutazioni del gene TP53 nella componente epiteliale ed in
quella mesenchimale. Le mutazioni più comuni sono TP53
e PIK3CA. Altre alterazioni molecolari a carico di VEGFA,
HMGA2 e HPRT1 sono state recentemente descritte.
Le caratteristiche fenotipiche del carcinosarcoma sono state
attribuite a cambiamenti nella Akt/beta-catenina pathway ed
alla repressione transcrizionale dell’E-caderina.
I carcinosarcomi hanno una prognosi sfavorevole. Il tipo di
diffusione della neoplasia è simile a quello del carcinoma
endometriale di alto grado. Una alta proporzione di pazienti
con alla presentazione una neoplasia clinicamente stadio uno,
all’intervento hanno una malattia diffusa al di fuori dell’utero.
Le metastasi sono tipicamente ai linfonodi pelvici e para-aortici e quelle a distanza al polmone, cervello ed ossa. Tuttavia
la maggior parte delle pazienti muore come conseguenza di
una recidiva locale/addominale. Il rischio di malattia in uno
stadio avanzato è strettamente collegata alla profondità di
invasione del miometrio. La presenza di carcinoma di tipo
sieroso o a cellule chiare è più frequentemente associata ad
altri parametri sfavorevoli. La presenza di elementi eterologhi,
specialmente nel caso di una componente tipo rabdomiosarcoma, è statisticamente un fattore associato a cattiva prognosi
per le pazienti con un carcinosarcoma stadio I.
Bibliografia
Djordjevic B, Gien LT, Covens A, et al. Polypoid or non-polypoid? A
novel dichotomous approach to uterine carcinosarcoma. Gynecol
Oncol 2009;115:32-6.
Evans MJ, Langlois NE, Kitchener HC, et al. Is there an association
between long-term tamoxifen treatment and the development of carcinosarcoma (malignant mixed Müllerian tumor) of the uterus? Int J
Gynecol Cancer. 1995;5:310-3.
McCluggage WG, Abdulkader M, Price JH, et al. Uterine carcinosarcomas in patients receiving tamoxifen. A report of 19 cases. Int J Gynecol Cancer 2000;10:280-4.
Kloos I, Delaloge S, Pautier P, et al. Tamoxifen-related uterine carcinosarcomas occur under/after prolonged treatment: report of five cases
and review of the literature. Int J Gynecol Cancer 2002;12:496-500.
Hubalek M, Ramoni A, Mueller-Holzner E, et al. Malignant mixed mesodermal tumor after tamoxifen therapy for breast cancer. Gynecol
Oncol 2004;95:264-6.
Silverberg SG, Major FJ, Blessing JA, et al. Carcinosarcoma (malignant
mixed mesodermal tumor) of the uterus. A Gynecologic Oncology
Group pathologic study of 203 cases. Int J Gynecol Pathol. 1990;9:1-19.
de Brito PA, Silverberg SG, Orenstein JM. Carcinosarcoma (malignant
mixed müllerian (mesodermal) tumor) of the female genital tract:
immunohistochemical and ultrastructural analysis of 28 cases. Hum
Pathol 1993;24:132-42.
D’Angelo E, Prat J. Pathology of mixed Müllerian tumours. Best Pract
Res Clin Obstet Gynaecol 2011;25:705-18.
Euscher ED, Deavers MT, Lopez-Terrada D, et al. Uterine tumors with
neuroectodermal differentiation: a series of 17 cases and review of the
literature. Am J Surg Pathol 2008;32:219-28.
Sreenan JJ, Hart WR. Carcinosarcomas of the female genital tract. A
pathologic study of 29 metastatic tumors: further evidence for the
dominant role of the epithelial component and the conversion theory
of histogenesis. Am J Surg Pathol 1995;19:666-74.
Costa MJ, Guinee D Jr. CD34 immunohistochemistry in female genital
tract carcinosarcoma (malignant mixed müllerian tumors) supports a
dominant role of the carcinomatous component. Appl Immunohistochem Mol Morphol. 2000;8:293-9.
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Yoshida Y, Kurokawa T, Fukuno N, et al. Markers of apoptosis and angiogenesis indicate that carcinomatous components play an important
role in the malignant behavior of uterine carcinosarcoma. Hum Pathol
2000;31:1448-54.
Seidman JD, Chauhan S. Evaluation of the relationship between adenosarcoma and carcinosarcoma and a hypothesis of the histogenesis of
uterine sarcomas. Int J Gynecol Pathol 2003;22:75-82.
Szukala SA, Marks JR, Burchette JL, et al. Co-expression of p53 by epithelial
and stromal elements in carcinosarcoma of the female genital tract: an immunohistochemical study of 19 cases. Int J Gynecol Cancer. 1999;9:131-6.
Taylor NP, Zighelboim I, Huettner PC, et al. DNA mismatch repair and
TP53 defects are early events in uterine carcinosarcoma tumorigenesis. Mod Pathol 2006;19:1333-8.
Growdon WB, Roussel BN, Scialabba VL, et al. Tissue-specific signatures of activating PIK3CA and RAS mutations in carcinosarcomas of
gynecologic origin. Gynecol Oncol 2011;121:212-7.
Emoto M, Charnock-Jones DS, Licence DR, et al. Localization of the
VEGF and angiopoietin genes in uterine carcinosarcoma. Gynecol
Oncol 2004;95:474-82.
Romero-Pérez L, Castilla MÁ, López-García MÁ, et al. Molecular
events in endometrial carcinosarcomas and the role of high mobility group AT-hook 2 in endometrial carcinogenesis. Hum Pathol
2013;44:244-54.
Ferguson SE, Tornos C, Hummer A, et al. Prognostic features of surgical
stage I uterine carcinosarcoma. Am J Surg Pathol 2007;31:1653-61.
Sala Massalia – ore 8.30-18.30
Ematopatologia
Simposio I
Moderatori: S. Pileri (Bologna), L. Ruco (Roma)
Blastic plasmacytoid dendritic cell neoplasm
S.A. Pileri1, S.M. Rosaria1, F. Fuligni1, M.A. Laginestra1, C. Agostinelli1, T. Makeda2, L. Pagano3, A. Pileri Jr4,
N. Pimpinelli4, L. Cerroni5, C. Tripodo6, M. Paulli7, F. Facchetti8, C. Croce9, P.P. Piccaluga1
Unit of Haematopathology of Bologna University; 2 Division of Haematology of Harvard Medical School, Boston; 3 Institute of Haematology of Catholic University, Rome; 4 Department of Dermatological
Sciences of Florence University; 5 Department of Dermatology of
Graz University; 6 Tumour Immunology Unit of Palermo University;
7 Anatomic Pathology Section of Pavia University; 8 Department of
Pathology of Brescia University; 9 Department of Molecular Virology,
Immunology, and Medical Genetics of the Ohio State University
1 Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is
an extremely rare neoplasm that is currently included among
acute myeloid leukemias (AMLs) [Swerdlow et al., Fourth
Edition of the WHO Classification of Tumours of Hematopoietic and Lymphoid Tissues, 2008]. It actually represents an
orphan disease. This is due to its rarity on the one hand and the
clinical aggressiveness and dismal prognosis on the other. For
instance, PubMed quotes only 146 papers under the specific
item and most of them consist in case reports. About 90% of
patients survive no longer than 14 months and no consensus
does exist as to the most effective therapeutic approach. On
this respect, Pagano et al. (Haematologica 2013;98:239-46)
have recently reported that acute lymphoblastic leukaemia
(ALL)-adjusted schedules might be more effective than the
more commonly used AML-like ones. Only a few studies
have so far explored the genetics of the tumour by showing a complex karyotype and sporadic genetic alterations.
However, these contributions were usually based on small
cohorts, thus lacking functional relevance [Petrella et al.,
AJSP 1999;23:137-46; Leroux et al., Blood 2002;99:4154-59;
Reichard et al., AJSP 2005;29:1274-83; Dijkman et al., Blood
2007;109:1720-7; Jardin et al., Leukemia 2009;23:698-707;
Wiesner et al., JID 2009;130:1152-7; Lucioni et al., Blood
2011;118:4591-4; Alayed et al., Am J Hematol 2013; E-pub
ahead of print].
With this in mind, we decided to explore the pathobiology
of BPDCN by coordinating an international effort, aiming to
enrol: 1) a large number of tumours with optimally preserved
tissue, possible availability of normal DNA, and complete
clinical information, 2) samples of purified normal plasmacytoid dendritic cells, and 3) the CLA-1 cell line obtained from
a BPDCN. The planned approaches included gene expression
profiling (GEP), miRNA profiling, and next generation sequencing (NGS).
Materials and methods
Case collection. Stefano A. Pileri, Fabio Facchetti, Lorenzo
Cerroni and Marco Paulli critically reviewed the examples
of BPDCN collected in the tumour registries of the involved
Institutions. Thirty cases were selected that showed optimally
preserved formalin-fixed paraffin embedded (FFPE) tissue and almost exclusively consisted of neoplastic cells. In
four of these cases, both frozen samples and DNA extracted
from normal saliva of the patients were available. In further
seven cases, cryopreserved tissue but not saliva was obtained
besides FFPE blocks. In all instances, the local Ethical Committee had approved the collection of biological material.
Nine samples of normal PDCs were got from the buffy coats
of normal donors. Dr. Makeda provided the CAL-1 cell line.
Gene expression profiling studies. Frozen pathological tissue
samples and normal PDCs were profiled on the Affymetrix
platform according to previous experience (Piccaluga et al.,
JCI 2007, 117:823-834). For comparison, public databases
were used to obtain the profile of common myeloid precursors (MPs, N = 4), lymphoid precursors (LPs, N = 9), AMLs
(N = 132), ALLs (N = 155), resting (N = 2) and activated
(N = 2) pDCs.
The DASL Whole Genome Assay was used to profile both
FFPE and frozen tumour samples, along with normal PDCs
and the CAL-1 cell line on the Illumina HiScanSQ platform as
previously reported (Piccaluga et al., JCO 2013, E-pub ahead
of print). For the latter analysis, the samples were subdivided
into a training set and a test, respectively.
Micro-RNA expression profiling studies. These were performed by using nCounter® miRNA Expression Assay Kits
(NanoString Technologies, Seattle, WA, USA). Once again,
samples were subdivided into a training set and test set.
Immunohistochemistry. Validation studies with specific an-
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tibodies raised against products of deregulated genes were
carried out on automated platforms as previously detailed
(Piccaluga et al. JCO 2013; E-pub ahead of print).
CAL-1 cell line in vitro experiments. CAL-1 cells were separately incubated with Bortezomib, Cytarabine (ARA-C) and
BMS-345541. Cell viability, death and proliferation were
measured by Cell Titer-Glo Luminescent Cell Viability Assay, Annexin-V-FLUOS staining kit, and in situ cell proliferation kit FLUOS, respectively.
Transfection with mimic. The CAL-1 cell line was transfected
with mimic hsa-miR-720 and negative control.
Whole Exome Sequencing (WES). We used the Illumina NGS
platform HiScanSQ to carry out a WES study on the BPDCN
cases with frozen tissue and normal saliva available in order
to identify somatic mutations of diagnostic, prognostic and
possibly therapeutic value. The Sanger sequencing technology was used to validate in BPDCN cases the specific genetic
mutations found in WES experiments.
Bio-informatic analysis. For bio-informatic analyses and result interpretation, we used an “integrated approach” working
in close collaboration with the Center for Computational Biology at New York Columbia University and the Dana Farber
Cancer Institute / Harvard University in Boston.
Summary of the main results
Gene expression profiling studies. For the first time at the
molecular level, we definitively recognized the cellular derivation of BPDCN, which turned out to originate from resting
PDCs of myeloid origin. Intriguingly, despite the clear myeloid origin, BPDCN did not appear so closely associated to
AML, rather presenting an ambiguous molecular profile partially related to both AML and ALL. Based on that, it would
be definitely warranted to randomly compare AML-like vs.
ALL-treatments. Moreover, we identified in both the training set and test set a molecular signature consisting of 142
genes discriminating between tumors and controls. A careful
investigation of these genes provided several insights into
the functional alterations of BPDCN, revealing an extensive
deregulation of cell adhesion, matrix remodeling, vascular development, and proliferation. Finally, thanks to an integrated
bio-informatic approach we discovered aberrant activation of
the NF-kB pathway and suggested it as a novel therapeutic
target. We tested the efficacy of anti-NF-kB-therapy on the
BPDCN cell line, CAL-1, and successful evaluated by GEP
and IHC the molecular shut-off the NF-kB pathway.
In synthesis, we identified for the first time a molecular signature representative of transcriptional abnormalities of BPDCN
and developed a cellular model proposing the usage of a novel
therapeutic approach in the setting of this otherwise incurable
disease.
Micro-RNA profiling studies. We identified a signature of
114 discriminant microRNAs between BPDCNs and normal
PDCs and then, thanks to the integration with GEP data, we
recognized a network of miRNA - messenger RNA relationships possibly relevant for the tumor patho-biology. In particular, we found in both the training and test set the overexpression of hsa-miR-720, potentially responsible for the constant
down-regulation of DAPK2 and ZFHX3, involved in the
apoptotic pathway and cell cycle control. To better explore the
correlation between has-miR-720 and its putative target genes,
we transfected the CAL-1 cell line with mimic has-miR-720
and after 48 hours measured by Real Time quantitative PCR
the expression levels of hsa-miR-720, DAPK2 and ZFHX3.
As expected, after transfection, has-miR-720 levels increased
while DAPK2 and ZFHX3 expression diminished.
WES. WES revealed the presence of DNA mutations associated with myeloid neoplasms. These affected genes notoriously
involved in DNA epigenetic modifications and mechanisms of
alternative splicing. Interestingly, one patient reported a point
mutation of SUZ12 and another one a mutation on ASXL1.
Both genes belong to the Polycomb-group, responsible for
DNA chromatin modifications such as methylation and gene
silencing, and have already been found mutated in myeloproliferative and myelodysplastic disorders. Importantly, SUZ12
and ASXL1 alterations found in BPDCN patients occurred in
genomic locations never described before. Both are nonsense
mutations presumably resulting in premature stop codons and
truncated, usually non-functional protein products. Sanger
sequencing confirmed the presence of these mutations in the
same cases analyzed by WES. After molecular validation, we
decided to extend our investigation to the mutational status
of ASXL1 and SUZ12 as well as of additional 36 well-known
myeloid cancer-associated genes in a panel of 30 BPDCNs.
To analyze so many genes (each constituted by hundreds of
amplicons), we found that the Truseq Custom Amplicon of Illumina was the most appropriate approach. After the design of
a personalized assay, preparation of libraries and sequencing
run, we are currently analyzing the output data.
The relevance of ebv detection in the diagnosis
of malignant lymphoma
S. Uccini
Department of Clinical and Molecular Medicine, Sapienza University
of Rome, Ospedale Sant’Andrea, Roma, Italy
Epstein-Barr virus (EBV), a member of the human herpes
virus family, is a linear, double-stranded DNA virus that was
initially isolated from a cultured Burkitt lymphoma cell line
by Epstein et al. in 1964. Subsequent studies have proven that
it is causative agent in most cases of infectious mononucleosis. Currently, it is estimated that more than 90% of the adult
population worldwide are infected by the virus.
Primary infection is usually asymptomatic in childhood, whereas in adolescence or adulthood, it is associated with a selflimiting infectious mononucleosis syndrome in approximately
one third of the cases. The virus is secreted in the saliva, and
human infection occurs through oral transmission. Oropharyngeal infection results in a lytic (productive) infection followed
by infection of circulating B cells, leading to persistence of the
viral DNA as an episome in the nucleus, thus establishing latent
infection. In lytic infection, EBV-encoded genes selectively
replicate virion components including viral DNA genomes
and proteins. In latent infection, EBV-encoded genes, which
include 6 nuclear antigens (EBNA 1, 2, 3A, 3B, 3C and LP), 3
latent membrane proteins (LMP1, 2A and 2B), 2 small noncoding RNAs (EBER1 and EBER 2) and BamHI-A rightward transcripts, maintain the existence of the viral genome and enable
it to evade immune surveillance. Viral proteins and noncoding
RNAs can be easily detected in tissue samples by immunohistochemistry and in situ hybridization.
Despite its documented infection of T lymphocytes and
epithelial cells in certain circumstances, EBV has a major
predilection for B cells because they act as the reservoir for it
to persist. Consequently, the persistence of latent EBV genes
in B cells results in a carrier state and in certain occasions,
transformation into malignant B-cell lymphoma may occur.
Because of the preferential infection of B cells, B-cell lymphoma predominate among the EBV-related lymphoproliferative disorders.
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According to the World Hearth Organization (WHO) classification of lymphoid tumors, EBV-associated B-cell lymphoproliferative disorders (LPDs) include Hodgkin lymphoma,
Burkitt lymphoma, posttransplant lymphoproliferative disorders (PTLDs), lymphomatoid granulomatosis, pyothorax-associated lymphoma and senile EBV-associated B-cell LPDs.
Other LPDs, related to the EBV association with T-cell proliferation, are angioimmunoblastic T-cell lymphoma, extranodal
nasal type NK/T-cell lymphoma and EBV+ T-cell LPD of
childhood.
A spectrum of EBV-driven B-cell LPDs occurs in patients
with primary immunodeficiency, HIV infection, or with
iatrogenic immunosuppression for organ transplantation or
autoimmune diseases. Recently, EBV-associated LPDs of
the elderly have been described as a wide disease spectrum,
with different clinical behaviours, ranging from Japan
cases with aggressive behaviour and poor overall survival,
to a self-limited clinical condition known as EBV-positive
mucocutaneous ulcer. In these conditions, it has been postulated that the immunosenescence and the natural decay
of the immune system may represent the underlying pathogenetic events.
In conclusion, EBV-associated lymphoma represent a broad
spectrum of diseases, with a similar underlying condition
represented by an immune defect allowing aggressive viral
infection. It has been postulated that immaturity of the immune system in children, HIV-induced or drug-induced immunodeficiency in adults, a senile immunodeficiency in the
elderly, represent different but converging events leading to
the development of EBV-related LPDs. In this context, the
search for EBV-infected cells in tissue sections of LPDs is a
necessary requirement for recognition and proper classification of the disease.
Corso breve di patologia mammaria:
lesioni benigne che simulano la malignità e viceversa
Moderatori: V. Eusebi (Bologna), A. Sapino (Torino)
Benign locally infiltrating neoplasm
of the nipple with malignant clinical
presentation
E. Bonanno, M. Scimeca, M. Chimenti
Università di Roma Tor Vergata, Fondazione Policlinico Tor Vergata,
Dipartimento di Biomedicina e Prevenzione, Roma
Clinical Presentation. A 65-year-old woman with 2 months
history of inversion, retraction and congestion of nipple of the
right breast. Any significant history of breast diseases was referred. No risk factors were identified for breast cancer except
late age at first delivery. Physical examination revealed a firm
discrete mass, about 3 centimeters large in the right nipple.
Mammography confirmed the presence of a ill-defined and
high-density tumor with retraction of peripheral parenchimal
areas. Echography revealed a speculated nodule with indistinct margins and with a vascular pole in the center of the lesion and two additional more lesions, respectively 0,5 and 0,8
cm in diameter with similar characteristics in QSE of the same
breast. One axillary lymph node (2 cm in diameter) was clinically and radiologically evident and suspected for metastasis.
Any relevant finding in the controlateral breast.
Cytopathology and Histology. The cytological analysis of
the lesion, performed by thin layer cytology, showed few aggregates of epithelial duct cells sometimes with cytoplasmic
vacuolization and focal nuclear irregularity. A needle biopsy
of the lesion was requested. Due to the malignant clinical
presentation of the lesion the patient accepted a mastectomy
with nodes dissection.
Gross examination of the breast revealed the presence of multiple ill-defined firm gray nodules diffused to all breast lumps.
On microscopic examination the lesions were formed by ductules, tubules and strands of small uniform cells infiltrating
the surrounding tissue. Some ducts showed a characteristic
comma-shape. Cells showed nuclear pleomorphism, hyperchromasia. Mitotic activity was absent. Small keratinous cysts
containing well-developed lamellar keratin and bland appearance of cubic cell lining gland lumina were also reported. Immunohistochemical markers for myoepithelial layer such as
smooth muscle antigen, p63 and calponin were positive; inner
layer’s cells were positive for CK5 and CK14 whereas CK7
and oestrogen receptor were negative. Isolated foci of ductal
carcinoma in situ (cribriform and one only focus of comedonic type) 3 mm in diameter associated with ductal papillomatosis were found at the side of the main lesion. Axillary
lymph nodes were negative for metastasis. The morphological
findings of the lesions were consistent with syringomatous
adenoma of the nipple. The presence of double layer provided
additional evidence in favor of syringomatous tumour. No
recurrence had occurred in five years of follow-up.
Discussion and Conclusions. The term “syringomatous
adenoma of the nipple” was first coined by Rosen in 1983 1.
Jones et al. 2, in a clinical and pathological study of 11 cases
in 1989, introduced the term “infiltrating” to emphasize the
locally infiltrative nature of this benign but peculiar lesion.
The WHO classification 2012 repost the termo f “Syngomatous tumour” 3.
The histological derication of these lesions has been a matter
of debate; the epidermis covering the nipple, lactiferous duct,
and sweat gland ducts have all been postulated ad possible
sites of origin.
Patients with syngomatous adenoma of the nipple have been
reported to range in age from 11-76 years with an average age
of 40 years 4. The clinical presentation of infiltrating suringomatous adenoma of the nipple is variable. There is no specidic
radiological features characheristic to this unusual condition.
A clearer under standing of the spectrum of breast lesions with
syringomatous features will no doubt benefit both patients and
pathologists alike. It is important to differenziate syringomatous adenoma from tubular carcinoma and low-grade adenosquamous carcinoma 5. The glandular structures of tubular
carcinoma are mostly angolate with open lumina and are composed of single cell population as opposed to syringomatous
adenoma, which has variable amount of squamous metaplasia
and has characteristic “comma” or “tad pole” shapes. Ductal
carcinoma in situ is often associated with tubular carcinoma 6.
Surgical management varied from excisional biopsy to radical mastectomy although mastectomy is not indicated as
primary treatment. Local recurrence has been attributed to
be related to incomplete excision. Axillary dissection is not
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requie since any nodal metastasis has been found in patients
who had axillary dissection. Distant metastasis is unknown
with syringomatous adenoma on follow-up of as long as 24
years. Complete excision with free margins seems to be an
appropriate therapy and extensive surgical procedures should
be avoided. Due to the rarity of this disease, long-term followup is necessary.
Benign mesenchimal tumor mimicking a carcinoma. Clinical presentation. A 60 years-old woman undergoes her first
mammography for screening. An hyperdense spiculated
lesion with ill defined margins was found in the upper quadrants of right breast, near to chest muscle layer. Ultrasounds
examintion showed an hypoecogenic mass, with irregular
margins (1,47 cm of axial diameter). A magnetic resonance
examination was performed to evaluate the presence of multiple foci and to evaluate, in preoperative assessment, the
local extent to the pectoralis fascia. It depicts a nodular area,
hypointense, with speckled and irregular margins (1,5 cm of
axial diameter).
Cytopathology and Histology. Cytological smear was characterized by scant cellularity, with medium to large size and
polygonal elements, single or in small group with large, eosinophilic and granular cytoplasm and with uniform, round
to oval nuclei. Cell lacked of significant atypia or mitosis.
A needle biopsy of the lesion was requested. Patient refused
a core-biopsy, therefore an excissional surgical biopsy was
performed.
In the lumpectomy a 2 cm firm, homogeneous greyish yellow
mass, with ill-defined margins, fixed to the fascia was described. Frozen sections showed a neoplasia with large cells in
sheets and cords with a clear infiltrative pattern. Cells showed
numerous fine granules and scattered eosinophilic globules in
the cytoplasm; any nuclear atypia or mitotic activity was ob-
served. Exicision margins were free from neoplasia. Despite
the infiltrative pattern of the muscle due to the absence of
cellular atypia a conservative surgery was suggested and the
diagnosis was deferred to the paraffin embedded tissue.
The examination of the formalin fixed, paraffin embedded
tissue confirmed the presence of a neoplasia made by large
eosinophilic polygonal cells with well defined cell borders,
granular cytoplasm, bland nuclei and infiltrative pattern of
growth. Histochemical study demonstrated that the cytoplasmic
granules contained PAS positive and diastase resistant material. The immunohistochemistry revealed a positivity for S-100
protein and CD68 antigen a week and focal positivity for CEA
antin and Vimentin; cytokeratins and GFAP were negative. The
evaluation of Ki67 prolifetive antigen was low (< 10%).
A final diagnosis of granular cell tumours (GCT) was formulated. Any additional surgery was performed.
No recurrence had occurred in three years of follow-up.
Discussion and Conclusions. Granular cell tumour (GCT)
it’s a mesenchymal tumour that was first described in the
breast by Abrikossof in 1926 7. GCT occur throughout the
body with about 5% of them originating in the breast. With
very rare exceptions GCT is a benign neoplasm. Many authors 8-11 emphasize the importance of distinguish this benign
tumour from carcinoma.
Granular cell are rare lesions of probable Schwann cell origin,
which can occur in a variety of visceral and cutaneous sites 12.
The most common site is in the tongue, but they are present
in the breast around 6-8% of cases. GCT can occurs more
commonly in pre-menopausal woman. Clinically they present
as a mass, which may mimic malignancy if there is overlying skin tethering. In contrast to other breast tumours, which
occur predominantly in the upper outer quadrant, GCT occur
most commonly in the upper inner quadrant corresponding to
the cutaneous sensory territory of the supraclavicular nerve.
Synoptic table for Granular cells tumors.
Age
Clinical
Radiological
Cytological
Histology
Immunophenotype
Prognosis
17-74
Firm, hard, painless mass. Occur most commonly in the upper inner quadrant.
Stellate mass lacking calcifications (suggestive for carcinoma). Rarely well circumscribed
There could be a incongruity with clinical-radiological findings: tumour’s cells may be
mistaken for foamy macrophages or typical or atypical apocrine cells.
- Medium-large size cells with granular, eosinophilic, PAS-positive cytoplasm
- Uniform round to oval nuclei with sometimes prominent nucleoli
- Infiltrative growth pattern
- Collagenous stroma
- Mitosis or necrosis very rare
- No significant pleomorphism, with rare exceptions
Positive markers: S100, CD68, CEA (focal), VIM (focal)
Negative markers: panCK, ER, PR, CerbB2, GFAP
Excellent. Though non-metastasizing, it may recur locally if incompletely excised. Malignant
GCT is very rare (<1%) and prognosis in this case is very poor. Feature suggestive of
malignancy include large tumour size, pleomorphism, increased mitotic activity, necrosis.
DIFFERENTIAL DIAGNOSIS CLUES
Apocrine carcinoma
Intraductal carcinoma, nuclear pleomorphism; panCK+, S100-
Histiocytoid carcinoma (breast)
GCDFP+, panCK+, EMA+, S100-
Alveolar soft part sarcoma
Regular nests of cell, variable granularity, S100 variable.
Metastasis (kidney)
CD10+, VIM+.
WHEN SUSPECT A GRANULA
CELL TUMORS (GCT)
- Polygonal cell with granular and eosinophilic cytoplasm
- Infiltrative pattern lacking cellular atypia and pleomorphism
- Immunophenotypic definition
154
Multiplicity of lesions can be observed, particularly in black
patients. GCT can simulate carcinoma because of its firm
consistency, clinically in the breast adhesion to the pectoralis
fascia, and skin retraction. Some GCTs appear as an illdefined or stellate lesion on mammograms, which strengthens
the clinical impression of carcinoma 13.
GCTs must be distinguished from breast cancer, especially
from apocrine carcinomas and the histiocytic variant of invasive lobular carcinoma, histiocytic lesions, and metastic disease.
Immunoreactivity for S-100 protein, CEA, and vimentin,
with a negative reaction against cytokeratin are indicative
od GCT. Treatment of GCT is wide local resection 14. The
granular cell tumour is almost always benign. A malignant
type is, however, encountered in 2% of the cases. Even thug
malignant, this tumour rarely metastatizes. It responds poorly
to chemotherapy and radiation therapy. Because there is a
tendency for local recurrence and the remote possibilty of distant metastasis, follow-up is a crucial aspect of treatment and
management. The local recurrence rate of benign granular cell
breast tumour ranges between 2% and 8%, while the recurrence rate for malignant form is much higher, at around 35%.
Local recurrence is typically rapid, however, usually within 1
year of the original surgery 15. Distant metastasis of malignant
granular cell breast tumors i suite high, at just over 60%.
References
1
Rosen PP. Syringomatous adenoma of the nipple. Am J Surg Pathol
1983;7:739-45.
2
Jones MW, Norris HJ, Snyder RC. Infiltrating syringomatous adenoma of the nipple. A clinical and pathological study of 11 cases. Am
J Surg Pathol 1989;13:197-201.
3
Eusebi, Lester. Syringomatous tumor in WHO Classification of tumours of the breast 2012;151.
4
Carter E, Dyess DL. Infiltrating syringomatous adenoma of the nipple:
a case report and 20-year retrospective review. Breast J 2004;10:443-7.
5
Rosen PP, Ernsberger D. Low-grade adenosquamous carcinoma.
A variant of metaplastic mammary carcinoma. Am J Surg Pathol
1987;11:351-58.
6
Rosen PP, Caicco JA. Florid papollomatosis of the nipple. A study
of 51 patients, including nine with mammary carcinoma. Am J Surg
Pathol 1986;10:87-101.
7
Abrikossoff A. Uber Myome, ausgehend von der quergestreiften wilkuerlichen muskulatur. Virchows Arch Pathol Anat 1926;260:215-33.
8
Delaloye F, Seraj F, Guillou L, et al. Granular cell tumor of the breast:
a diagnostic pitfall. Breast 2002;11:316-9.
9
Haangensen CD, Purdy Stout A. Granular cell myoblastome of the
mammary gland. Ann Surg 1946;124:218-27.
10
Lelle RJ, Park H, Brow CA. Benign granular cell tumor mimicking
carcinoma of the breast. Report of the case. Eur J Gynaecol oncol
1992;13:390-3.
11
Adeniran A, Al-Ahmadie H, Mahoney MC, et al. Granular cell tumor
of the breast: a series of 17 cases and review of the literature. Breast
J 2004;10:528-31.
12
Adeniran A, Al-Ahmadie H, Mahoney MC, et al. Granular cell tumor
of the breast: a series of 17 cases and review of the literature. Breast
J 2002;10:528-31.
13
Delaloye F, Seraj F, Guillou L, et al. Granular cell rumor of the
breast: a diagnostic pitfall. Breast 2002;11:316-9.
14
Aneiros-Fernandex J, Arias-Santiago S, Husein-ElAhmed H, et al. Cutaneous granular cell tumor of the breast: a clinical diagnostic pitfall.
J Clin Med Res 2010;2:185-8.
15
Brown SC, Audisio RA, Regitnig P. Granular cell tumour of the
breast. Surg Oncol 2011;20:97-105.
Breast fibroadenoma containing multinucleated
stromal giant cells
L. Roncati, G. Ficarra, A. Maiorana
Department of Diagnostic, Clinical Medicine and Public Health, section
of Pathology, University of Modena and Reggio Emilia, Modena, Italy
Background. Multinucleated stromal giant cells (MSGC)
in breast tissue were reported for the first time by Rosen
in 1979 1. The author described the presence of these cells
both in normal and tumoral tissues, concluding that they had
no clinical value. Moreover, he suggested they might have
a reparative nature, being often found at the periphery of
mammary carcinomas. Among breast lesions, fibroadenoma
is labeled as a fibro-epithelial tumor, consisting of a stromal
component commingled with an epithelial component, hence
the term ‘biphasic lesion’. In spite of the high breast incidence
of fibro-epithelial tumors, MSGC are only rarely observed
inside these neoplasms 2 3. Therefore, the histopathological detection of these cells in a fibroadenoma can render difficult the
diagnostic framing, by placing the suspicion of malignancy.
Methods. A 40 years old woman presented with a new selfpalpable right breast nodule. After preliminary evaluation by
ultrasound and mammography, she underwent surgical nodulectomy. The patient’s post-surgical recovery was uneventful.
Thirty months after simple excision, the patient remained free
of disease recurrence. On gross examination, the grayish firm
nodule measured 2.7 cm in diameter and was characterized
by expansive borders. The specimen was routinely processed
for histopathological examination (4% formaldehyde fixation, paraffin embedding, sections staining with hematoxylineosin). Immunohistochemistry for CD34 (Ventana) and p53
(Ventana) was performed using a streptavidin-biotin peroxidase method.
Results. Microscopically, the nodule appeared to be biphasic
in nature. The epithelial component consisted of compressed
glands and haphazardly-arranged ducts, lined by benign
epithelial cells and surrounded by abundant stroma. In some
areas of the stromal component, an increased mitotic activity
(Fig. 1) with isolated atypical mitotic figures and scattered giant cells were noticeable (Fig. 2). Some giant cells exhibited
multiple nuclei (up to 15) with fine chromatin and sporadic
small nucleoli. Giant cells were found to be immunoreactive
for CD34 and p53. Based on morphologic features, a diagnosis of fibroadenoma with MSGC was rendered.
Discussion. The histopathological finding of MSGT in fibroadenoma is a rare occurrence 4. The large size (up to 100
um), coupled with the occasional bizarre appearance, of these
cells may alarm the pathologist, but so far there is consensus
about their benign biological behavior 5. The increased mitotic
activity and the isolated atypical mitotic figures in MGST do
Fig. 1. Increased mitotic activity in the stroma, HE, x240 (original
magnification).
Fig. 2. Multinucleated giant cells scattered in the stroma, HE,
x200 (original magnification).
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not correlate with malignancy and they should not influence
the tumor grading. In fact, also our follow-up data support
that the detection of MGST in a fibroadenoma has no adverse
clinical outcome. The differential diagnosis arises with a
benign, borderline or malignant phyllodes tumor containing
giant cells or with a primitive pleomorphic sarcoma of the
breast. The greatest attention should be reserved to a benign
phyllodes tumor with MGST 6: the presence of long epitheliallined clefts and of pseudoangiomatous stromal changes are
in favour of a phyllodes tumor. In the differential diagnostic
path, the absence of clear epithelial atypias allows to exclude a
carcinomatous component, while the limited number of pleomorphic stromal cells in a biphasic context permits to exclude
pleomorphic sarcoma. A single case of MSGC in adenoid
cystic carcinoma has been reported in the literature 7.
Although normal breast stroma, like most collagenous connective tissues, contains CD34+ resident dendritic interstitial
cells, the histogenesis of MSGCs in fibro-epithelial tumors
still remains obscure, even if they appear reactive in nature.
According to their immunohistochemical profile (vimentin+,
CD34+, p53+) and the results obtained by electron microscopy, a mesenchymal derivation (fibroblastic, myofibroblastic
or fibrohistiocytic) seems to be the most plausible origin 8.
References
1
Rosen PP. Multinucleated mammary stromal giant cells. Cancer
1979;44:1305-8.
2
Powell CM, Cranor ML, Rosen PP. Multinucleated stromal giant cells
in mammary fibroepithelial neoplasms. A study of 11 patients. Arch
Pathol Lab Med 1994;118:912-6.
3
Ryska A, Reynolds C, Keeney GL. Benign tumors of the breast
with multinucleated stromal giant cells. Immunohistochemical
analysis of six cases and review of the literature. Virchows Arch
2001;439:768-75.
4
Berean K, Tron VA, Churg A, et al. Mammary fibroadenoma with
multinucleated stromal giant cells. Am J Surg Pathol 1986;10:823-7.
5
Huo L, Gilcrease MZ. Fibroepithelial lesions of the breast with pleomorphic stromal giant cells: a clinicopathologic study of 4 cases and
review of the literature. Ann Diagn Pathol 2009;13:226-32.
6
Tse GM, Law BK, Chan KF, et al. Multinucleated stromal giant cells
in mammary phyllodes tumours. Pathology 2001;33:153-6.
7
Milentijević MJ, Basić M, Petrović A. Multinucleated stromal giant
cells in adenoid cystic carcinoma of the breast: a case report and
literature review. Vojnosanit Pregl 2011;68:178-80.
8
Silverman JS, Tamsen A. Mammary fibroadenoma and some phyllodes tumour stroma are composed of CD34+ fibroblasts and factor
XIIIa+ dendrophages. Histopathology 1996;29:411-19.
155
open lumens lined by a single layer of monotonous cells. Its
incidence is approximately 5% of all breast cancers 4. Patients
affected by tubular carcinoma have usually a good prognosis,
particularly when tumor size is less than 1 cm.
Methods. A 56 years old woman underwent a routine mammography that revealed the presence of a radial thickening of
approximately 1 cm localized at the supero-medial quadrant
of left breast. FNAC showed hyperplastic cells and an isolated
cellular cluster exhibiting mild nuclear atypia and lack of
myoepithelial cells (C1). Subsequently, vacuum-assisted core
biopsy was performed. Quadrantectomy and removal of the
sentinel lymph node were performed two months later. All
bioptic and surgical specimens were routinely processed for
histopathological examination (4% formaldehyde fixation,
paraffin embedding, sections staining with hematoxylineosin). Immunohistochemistry for p63 (Ventana) and miosin
(Ventana) was also performed using a streptavidin-biotin
peroxidase method.
Results. At microscopic examination, isolated tubules lined
by a single layer of monotonous epithelial cells with apical
snouts (Fig. 1), but devoid of myoepithelial cells, were detected adjacent to a sclero-elastotic parenchymal lesion (Fig. 2).
The absence of myoepithelial cells in tubules was confirmed
by immunohistochemistry (Fig. 3) and a diagnosis of tubular
carcinoma of the breast arising in a radial scar was rendered.
Pathological examination of the quadrantectomy specimen
confirmed the presence of remnants of a small tubular carcinoma. In the remaing tissue, isolated microcalcifications and
a minute in situ ductal carcinoma were also observed. The
sentinel lymph node was negative.
Discussion. In the breast, the collision of a carcinoma of
low-grade malignancy with a carcinoma-mimicker is a posFig. 1. Focus of tubular structures lined by a single layer of monotonous epithelial cells, HE, x150 (original magnification).
Fig. 2. Area with a scleroelastotic lesion, HE, x100 (original magnification).
Tubular carcinoma of the breast in collision
with radial scar
L. Roncati, G. Ficarra, A. Maiorana
Department of Diagnostic, Clinical Medicine and Public Health, section
of Pathology, University of Modena and Reggio Emilia, Modena, Italy
Background. Radial scars are stellate lesions characterized
by a sclero-elastotic center, that contains distorted tubular
structures lined by hyperplastic epithelium. A rosette-like arrangement (zoning phenomenon) is usually detected. Radial
scar was described for the first time by Hamperl in 1975 1
and it is characterized by several synonyms, which by themselves describe its nature, such as benign scleroelastotic lesion and radial sclerosing lesion. Its biological behaviour has
been extensively discussed by many authors with conflicting
points of view 2. It has also been argued that it could reflect
a pre-neoplastic condition towards the development of tubular carcinoma 3. Tubular carcinoma is a low-grade invasive
carcinoma consisting of a variable number of tubules with
Fig. 3. Neoplastic tubules lacking myoepithelial cell layer (a
non-neoplastic tubule with myoepithelial cells can be seen on
the right), immunohistochemistry for p63, x250 (original magnification).
156
sible occurrence: the challenge is to recognize both entities
by placing them in the differential diagnosis with other
similar diseases. In such cases, it becomes fundamental to
have a well-represented bioptic sample, as well as an immunohistochemical aid. The occurrence of a tubular carcinoma
in a radial scar offers a pathogenic cue: on the one side the
two lesions can be regarded as separate entities, while, on
the other side, a continuum from benignancy to malignancy
might be seen.
In tubular carcinoma, tubules are lined by a single layer of cuboidal to columnar cells with round nuclei and small nucleoli;
mitoses are uncommon. Tubules are devoid of myoepithelial
cells and the neoplastic cells stain for cytokeratins and Ecadherin 5. The differential diagnosis involves tubulolobular
carcinoma, low-grade adenosquamous (syringoid) carcinoma,
micro-glandular adenosis and the pure scleroelastotic lesion (radial scar). Tubulolobular carcinoma can be quickly
recognized because it combines the morphological features
and the immunohistochemical profile of both tubular carcinoma and invasive lobular carcinoma 6. In the low-grade
adenosquamous (syringoid) carcinoma, the comma-shaped
tubular structures present variable squamoid differentiation
and are lined at least by a double cell layer, the outer being
often actin rich 7. The presence of small quite uniform glands,
invested by a thin basal lamina and positive for S100 protein,
is instead typical for micro-glandular adenosis 8. In all benign
scleroelastotic lesions, ducts are encircled by myoepithelial
cells, well ascertainable by immunohistochemistry. At low
magnification, radial scar shows a central hyaline core with
compressed tubules from which hyperplastic ducts radiate
in a stellate configuration 9. This typical architecture and the
presence of myoepithelial cells in tubules, together with a
circumferential basement membrane, are absent in tubular
carcinoma. On the contrary, in duct ectasia, the duct are not
compressed but dilatated, with periductal inflammation and
progressive fibrotic changes 10.
References
1
Hamperl H. Strahlige narben und obliterierende mastopathie. Virchows Arch A Pathol Anat Histopathol 1975;369:55-68.
2
Jacobs TW, Byrne C, Colditz G, et al. Radial scars in benign breastbiopsy specimens and the risk of breast cancer. N Engl J Med
1999;340:430-6.
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Manfrin E, Remo A, Falsipollo F, et al. Risk of neoplastic transformation in asymptomatic radial scar. Analysis of 117 cases. Breast Cancer
Res Treat 2008;107:371-7.
4
Carstens PH, Greenberg RA, Francis D, et al. Tubular carcinoma of
the breast. A long term follow-up. Histopathology 1985;9:271-80.
5
Deos PH, Norris HJ. Well-differentiated (tubular) carcinoma of the
breast. A clinicopathologic study of 145 pure and mixed cases. Am J
Clin Pathol 1982;78:1-7.
6
Wheeler DT, Tai LH, Brathauer GL, et al. Tubulolobular carcinoma of
the breast: an analysis of 27 cases of a tumor with a hybrid morphology and immunoprofile. Am J Surg Pathol 2004;28:1587-93.
7
Soo K, Tan PH. Low-grade adenosquamous carcinoma of the breast.
J Clin Pathol 2013;66:506-11.
8
Koenig C, Dadmanesh F, Bratthauer GL, et al. Carcinoma arising in
microglandular adenosis: an immunohistochemical analysis of 20 intraepithelial and invasive neoplasms. Int J Surg Pathol 2000;8:303-15.
9
Alvarado-Cabrero I, Tavassoli PA. Neoplastic and malignant lesions
involving or arising in a radial scar: a clinicopathologic analysis of
17 cases. Breast J 2000;6:96-102.
10
Dixon JM, Ravisekar O, Chetty U, et al. Periductal mastitis and
duct ectasia: different conditions with different etiologies. Br J Surg
1996;83:820-2.
Sala Orange 1 – ore 8.30 – 18.30
Patologia endocrina
Intermediate and high grade endocrine/neuroendocrine neoplasms:
state of the art
Moderatori: M. Papotti (Torino), S. La Rosa (Varese)
Thyroid
F. Basolo
Pisa
Thyroid carcinomas account for less than 1% of all human
tumors with an annual incidence varies in different parts of the
world, from 0.5 to 10 cases per 100,000 population.
The papillary carcinoma (PTC) is the most frequent form of
thyroid cancer, with a range between 80 and 90% of all thyroid tumors.
The prognosis of PTC is usually good with a 10-year survival greater than 90%. However, 10% of patients with PTC
died of complications of the disease. Over the years able to
predict the worst prognosis parameters have been identified.
Among these we include the age, gender, presence of extrathyroidal tissue infiltration, nodal and distant metastasis,
histological variants, the size of the tumor. In addition to
these factors have recently been highlighted genetic factors
can affect the prognosis of PTC. First of all the gene mutation in BRAF can influence tumor progression in terms of
infiltration of extrathyroidal tissues and lymph node and
distance metastasis.
Rarer tumors but surely with worse prognosis are the medullary carcinomas, which account for approximately 10% of
malignant thyroid tumors. Originated by C-cells, have a typical clinical presentation, often familiarity and association with
other neuroendocrine disorders.
To date it is the only thyroid cancer that recognizes C cell
hyperplasia like a possible precursor. Most medullary carcinomas are sporadic, however there is a proportion, about 20%,
of familial cases that may be associated with multiple endocrine syndromes and which presents the RET proto-oncogene
mutations.
The anaplastic thyroid carcinoma, although rare (5-10% of
thyroid carcinomas), is one of the more aggressive tumors
in humans. It generally occurs in patients of advanced age
as massive fast-growing masses and, clinically, patients with
anaplastic carcinoma reach the exitus in a few months due to
massive local infiltration and distant metastases. Microscopically is a very heterogeneous tumor presenting epithelioid,
relazioni
squamous, or mixed patterns of growth. The cells always have
a marked anaplasia with bizarre shapes and numerous mitosis.
However, the diagnosis of anaplastic thyroid cancer is not a
problem because of its typical presentation, both clinical and
microscopic, and because of its immunohistochemical profile.
Anaplastic carcinoma in fact confirms its nature expressing
epithelial cytokeratins. Usually it is negative for thyroglobulin
or calcitonin.
The poor prognosis of anaplastic carcinoma pushed, very
recently, different authors to look for molecules able to act
on this type of tumor. The experimental results are sometimes
divergent but still leave a glimmer of hope in terms of overall
survival and quality of life of patients.
High grade neuroendocrine carcinomas –
rare locations
M. Papotti, N. Birocco*, E. Duregon, M. Volante
Department of Oncology, University of Turin at San Luigi Hospital,
Orbassano; * Medical Oncology, San Giovanni Hospital Torino
Introduction. High grade, poorly differentiated neuroendocrine carcinomas (NEC) occur in several extrapulmonary
(EPNEC) organs, as either small cell carcinomas (SCC) or
large cell NE carcinomas (LCNEC), apparently very similar
to their well-known respective pulmonary counterparts 1 2.
The impression is that EPNEC made of small or large cells
share many features with their pulmonary counterparts, but
also show peculiar pathological patterns in different organs,
including the possibility of combining NE differentiated components with conventional non-endocrine carcinomas of the
squamous, glandular or urothelial type.
Definition, incidence & terminology. EPNECs are malignant epithelial tumors having high-grade features and an
entire or predominant NE differentiated cell population. They
can develop in virtually any location. SCC were reported in all
the digestive tract, from oral cavity to anal canal, in the larynx,
thyroid, salivary gland, liver, pancreas, kidney, adrenal, breast
and in the urogenital tract of both male and female patients.
Extrapulmonary LCNEC has been occasionally reported in
similar anatomical sites to those of SCC (possible higher
incidence in the stomach) 3. High-grade NE carcinomas have
also been described in the skin since the original report of
Tang and Toker in 1972, under the generic term of trabecular
(Merkel cell) carcinoma. The morphology of classical Merkel
cell carcinoma is not different from that of small cell lung
cancer, but morphological and molecular features are heterogeneous, and clinical behavior and therapeutic implications
are variable and peculiar.
Overall, EPNECs account for no more than 3% of all
malignancies in individual anatomical sites (esophagous 4,
stomach 3 5 6, colon and rectum 7, pancreas 8, prostate 9 10,
bladder 11 and uterine cervix 12 13). A challenging diagnostic
(and also therapeutic) issue is the combination with a more
or less well-represented non-NE component. Such component
can be separated from or intermingled with the NE tumor cell
population.
Terminology – A relatively long list of different terms are
currently used to label these tumors (extrapulmonary small
or large cell neuroendocrine carcinoma, high grade NE
carcinoma, NEC G3, poorly differentiated neuroendocrine
carcinoma, poorly differentiated endocrine carcinoma). In addition, the occurrence of combined small and large cell NEC
has been reported in some locations (stomach, gallbladder,
bladder etc.), although this distinction is not considered of
157
clinical or prognostic relevance in these organs 14. For practical purposes, the nomenclature extrapulmonary NE carcinoma
(EPNEC) is advisable, with a sub-specification of either small
or large cell types.
Differential diagnosis of small and large cell nec. SCC
and LCNEC are defined by apparently clear-cut criteria, in all
similar to those proposed for their more common pulmonary
counterparts, although their differential diagnosis is not always easy, especially in cytology 15 or tiny biopsy specimens.
From a pathological point of view, the distinction between
small and large cell tumors is of relevance for the purpose of
a correct classification of the tumor itself and, above all, an
accurate differential diagnosis from other conditions made of
either small or large cells in individual organs. In practical
terms, it was claimed that the distinction between SCC and
LCNEC is an apparently useless exercise, not only due to
the similar clinical behavior reported above, but also because
many cases (indeed probably the majority of them) actually contain mixed cell populations, all sharing more or less
evident NE features, but displaying various cell sizes. As also
observed in the lung 16, it is therefore sometimes difficult to
address a single tumor to one or the other category.
The occurrence of EPNEC may also create differential diagnostic problems with poorly differentiated forms of their
respective non-endocrine carcinomas or other malignancies
such as hematological and mesenchymal neoplasms. As an
example, in the prostate, the most common EPNEC type is
small (oat) cell type, but a fraction of cases is classified as
“intermediate cell variant” of old classifications, and this latter feature may superficially address to a diagnosis of undifferentiated adenocarcinoma (Gleason score 5) 10. Because of
the possible superficial similarity with urothelial carcinoma,
this misdiagnosis is even more possible in the bladder, which
is the most common location of small cell EPNEC, also often
combined with a more or less represented urothelial component 17 18.
Concerning the differential diagnoses mentioned above, there
are no markers useful to discriminate small cell from large cell
cases. Both display an epithelial immunoprofile, with wide
spectrum cytokeratin expression, which may occasionally
have a paranuclear dot-like distribution, especially in the case
of small (oat) cell carcinomas. Conversely, high molecular
weight cytokeratins (types 1,5,10,14 of the Moll’s catalog)
are typically absent in the vast majority of NE differentiated
carcinomas of any location 19-22. Among NE markers, chromogranin A and synaptophysin are the most reliable molecules
supporting the morphological evaluation, while neuron specific enolase (NSE) and CD56 are sensitive, but much less
specific and therefore of limited usefulness, especially when
investigating a possible NE tumor in non-classical locations.
Differential diagnosis – An additional diagnostic issue is the
distinction of EPNEC from well-differentiated NE tumors
made of small cells. These are rare occurrences but may impact on the accuracy of diagnosis especially on small biopsies
or cytology samples. Among the various differential markers tested (including chromogranin A, TTF-1, cytokeratins),
Ki-67 index proved to be the most reliable for a differential
diagnosis of high grade carcinomas from carcinoid tumors in
pulmonary locations 23. No similar reports exist on extrapulmonary locations, but this approach may be useful in other
histo-cytological materials (e.g. liver biopsies), in the presence of marked artifactual cellular alterations or DNA crushing, which may hamper a correct interpretation.
Combined epnec and adeno-, squamous- or urothelial
carcinoma. At variance with the lung, pure forms of EPNEC
158
are extremely rare and more commonly a clear-cut small or
large cell NEC component is colliding or intermingled with
a non-NE carcinoma. In the case of such combined (mixed)
tumors, a first clue is to identify the NE cell population as
such, and then the relative proportions of the exocrine and
endocrine components are to be determined 24.
In the GEP area, this step is mandatory for the purpose of
identifying so called MANECs (mixed adeno-neuroendocrine
carcinoma), being at least 30% of each component required
for rendering such a diagnosis in any given mixed tumor.
In locations other than GEP tract, there are either different
diagnostic rules or none. NE breast carcinoma is one of the
several variants of this neoplasm, and is defined as a neoplasia having NE morphological features necessarily associated
to the immunohistochemical expression of NE markers in at
least 50% of the tumor as far as the WHO 2003 classification
is concerned 25. Among the variable morphological patterns,
small cell primary breast EPNECs contain a NE cell population largely exceeding the required 50% and reaching 90100% 26 27.
In the urogenital tract, combined carcinomas are as common
as pure forms of EPNEC. In the prostate, EPNECs occur as
either pure small cell carcinoma (57%) or combined with an
adenocarcinoma having a high (≥ 8) Gleason score, according
to the largest published series of 95 cases 10. In the bladder,
no criteria are mentioned for the definition of mixed neuroendocrine carcinoma: small cell carcinomas combined with urothelial, squamous or adenocarcinoma occur more frequently
(70%) than pure forms 28 29 and the trend is similar for the even
rarer LCNEC 30.
Similarly, in the ovary and in the uterine cervix there are no
specific rules. In the latter, the neuroendocrine component in
an otherwise squamous cell carcinoma has been described to
range from focal (17%) to half a tumor, but NE differentiation was associated to an adverse prognosis irrespective of the
extension 31.
Summary
–From a practical point of view, a common terminology is
advisable for pure extrapulmonary high-grade neuroendocrine carcinomas, using the acronym EPNEC and a subsequent distinction into small and large cell subtypes.
As in the lung, small and large cell EPNECs differ by cell
size, architecture and nuclear features, but morphologically intermediate cases exist and the general genetic background, and clinical behavior, are similar in the two forms.
– Immunohistochemistry is useful to distinguish EPNEC from
poorly differentiated non-NE carcinomas (using NE markers)
and from well-differentiated NE tumors (using Ki-67, especially in small tissue fragments/cytological samples), but not
to differentiate small and large cell EPNEC forms.
–The diagnosis of mixed/combined NE/non-NE forms in
extrapulmonary locations is based on morphology and
appropriate immunophenotype; due to its relatively high
frequency, it should be excluded by extensive sampling,
and the relative proportions on NE and non-NE components
should be reported in any case, to better understand the impact on clinical behavior and response to treatment(s).
References
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Travis WD. Update on small cell carcinoma and its differentiation
from squamous cell carcinoma and other non-small cell carcinomas.
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Jiang SX, Mikami T, Umezawa A, et al. Gastric large cell neuroendocrine carcinomas: a distinct clinicopathologic entity. Am J Surg
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Maru DM, Khurana H, Rashid A, et al. Retrospective study of clinicopathologic features and prognosis of high-grade neuroendocrine
carcinoma of the esophagus. Am J Surg Pathol 2008;32:1404-11.
5
Boo YJ, Park SS, Kim JH, et al. Gastric neuroendocrine carcinoma: clinicopathologic review and immunohistochemical study
of E-cadherin and Ki-67 as prognostic markers. J Surg Oncol
2007;95:110-17.
6
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.
7
La Rosa S, Marando A, Furlan D, et al. Colorectal poorly differentiated neuroendocrine carcinomas and mixed adenoneuroendocrine
carcinomas: insights into the diagnostic immunophenotype, assessment of methylation profile, and search for prognostic markers. Am J
Surg Pathol 2012;36:601-11.
8
Yachida S, Vakiani E, White CM, et al. Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and
distinct from well-differentiated pancreatic neuroendocrine tumors.
Am J Surg Pathol 2012;36:173-84.
9
Evans AJ, Humphrey PA, Belani J, et al. Large cell neuroendocrine
carcinoma of prostate: a clinicopathologic summary of 7 cases of a
rare manifestation of advanced prostate cancer. Am J Surg Pathol
2006;30:684-93.
10
Wang W, Epstein JI. Small cell carcinoma of the prostate. A morphologic and immunohistochemical study of 95 cases. Am J Surg Pathol
2008;32:65-71.
11
Alijo Serrano F, Sanchez-Mora N, Angel Arranz J, et al. Large cell
and small cell neuroendocrine bladder carcinoma: immunohistochemical and outcome study in a single institution. Am J Clin Pathol
2007;128:733-9.
12
Rekhi B, Patil B, Deodhar KK, et al. Spectrum of neuroendocrine
carcinomas of the uterine cervix, including histopathologic features,
terminology, immunohistochemical profile, and clinical outcomes in a
series of 50 cases from a single institution in India. Ann Diagn Pathol
2013;17:1-9.
13
Wang KL, Chang TC, Jung SM, et al. Primary treatment and prognostic factors of small cell neuroendocrine carcinoma of the uterine
cervix: a Taiwanese Gynecologic Oncology Group study. Eur J Cancer
2012;48:1484-94.
14
Quinn AM, Blackhall F, Wilson G, et al. Extrapulmonary small cell
carcinoma: a clinicopathological study with identification of potential
diagnostic mimics. Histopathol 2012;61:454-64.
15
Khalbuss WE, Yang H, Lian Q, et al. The cytomorphologic spectrum
of small-cell carcinoma and large-cell neuroendocrine carcinoma in
body cavity effusions: a study of 68 cases. Cytojournal 2011;8:18.
16
Travis WD, Brambilla E, Muller-Hermelink KM. WHO Classification
of Tumours of the Lung, Pleura, Thymus and Heart. Lyon 2004.
17
Thompson S, Cioffi-Lavina M, Chapman-Fredricks J, et al. Distinction of high-grade neuroendocrine carcinoma/small cell carcinoma
from conventional urothelial carcinoma of urinary bladder: an immunohistochemical approach. Appl Immunohistochem Mol Morphol
2011;19:395-9.
18
Zhao X, Flynn EA. Small cell carcinoma of the urinary bladder: a
rare, aggressive neuroendocrine malignancy. Arch Pathol Lab Med
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19
Sapino A, Righi L, Cassoni P, et al. Expression of the neuroendocrine phenotype in carcinomas of the breast. Semin Diagn Pathol
2000;17:127-37.
20
Sturm N, Rossi G, Lantuejoul S, et al. 34BetaE12 expression along
the whole spectrum of neuroendocrine proliferations of the lung, from
neuroendocrine cell hyperplasia to small cell carcinoma. Histopathology 2003;42:156-66.
21
Papotti M, Cassoni P, Sapino A, et al. Large cell neuroendocrine
carcinoma of the gallbladder: report of two cases. Am J Surg Pathol
2000;24:1424-8.
22
Viberti L, Bongiovanni M, Croce S, et al. 34betaE12 Cytokeratin Immunodetection in the Differential Diagnosis of Small Cell Tumors of
Lung. Int J Surg Pathol 2000;8:317-22.
23
Pelosi G, Rodriguez J, Viale G, et al. Typical and atypical pulmonary
carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy
specimens: a major pitfall in the management of lung cancer patients.
3
159
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Volante M, Rindi G, Papotti M. The grey zone between pure (neuro)
endocrine and non-(neuro)endocrine tumours: a comment on concepts
and classification of mixed exocrine-endocrine neoplasms. Virchows
Arch 2006;449:499-506.
25
Tavassoli FA, Devilee P. WHO Classification of Tumours of the
Breast and Female Genital Organs. Lyon 2003.
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Papotti M, Gherardi G, Eusebi V, et al. Primary oat cell (neuroendocrine) carcinoma of the breast. Report of four cases. Virchows Arch
A Pathol Anat Histopathol 1992;420:103-8.
27
Righi L, Sapino A, Marchio C, et al. Neuroendocrine differentiation
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Abrahams NA, Moran C, Reyes AO, Siefker-Radtke A, et al. Small
24
cell carcinoma of the bladder: a contemporary clinicopathological
study of 51 cases. Histopathology 2005;46:57-63.
29
Oesterling JE, Brendler CB, Burgers JK, et al. Advanced small
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radical cystoprostatectomy and adjuvant methotrexate, vinblastine,
doxorubicin, and cisplatin chemotherapy. Cancer 1990;65:1928-36.
30
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Urol 2011;64:105-13.
31
Horn LC, Hentschel B, Bilek K, et al. Mixed small cell carcinomas
of the uterine cervix: prognostic impact of focal neuroendocrine
differentiation but not of Ki-67 labeling index. Ann Diagn Pathol
2006;10:140-3.
Simposio: neoplasie endocrine/neuroendocrine: aspetti molecolari, stato
dell’arte e applicazioni pratiche
Moderatori: G. Rindi (Roma), M. Volante (Torino)
Molecular alterations in thyroid lesions:
a review of diagnostic applications
to fine needle aspiration (FNA) samples
D. de Biase, M. Visani, V. Cesari, A.L. Pession, G. Tallini
Scuola di Medicina e Chirurgia, Università di Bologna, Anatomia Patologica, Ospedale Bellaria, Bologna, Italy
Many studies published in the past several years have shown
that molecular analysis of fine needle aspiration (FNA) thyroid specimens is both feasible and useful 1-11. In fact, the type
of genetic alterations that occur in thyroid cancer, the clinical
context and the very nature of FNA samples, make the latter
ideally suited for molecular analysis. This is the case for several reasons. Thyroid tumors show a remarkable correlation
between phenotype (i.e. tumor type) and genotype (genetic
alterations), a correlation that is closer than that observed in
other epithelial neoplasms. Carcinomas with papillary architecture (classic papillary carcinoma or its tall cell variant) are
characterized by a high prevalence of two specific molecular
alterations: BRAF mutation (BRAFV600E), in 30-70% of
cases and RET/PTC rearrangement, in 20-40%. Encapsulated
tumors with follicular architecture (follicular adenomas, follicular carcinomas and follicular variant papillary carcinoma)
are characterized by Ras mutations (usually N-Ras codon
61) 12,13 in 20-50% of cases or by PAX8/PPARɣ rearrangement in 5-50% of cases. In spite of the variable prevalence
reported in different studies, these genetic alterations are (generally) mutually exclusive. One of them is present in ~70%
of carcinomas of follicular cell origin. The detection of these
molecular alteration is easily accomplished with assays based
on the analysis of DNA (BRAF and Ras) or RNA (RET/PTC
and PAX8/PPARɣ).
Thyroid nodules are extremely common in the general population. Although in the vast majority of cases they are benign,
a small subset of them is malignant. Conventional cytologic
examination with FNA is extremely cost-effective, but given
the high prevalence of nodular thyroid disease, any additional
tool that can add specific information is of significant value.
An additional consideration is that FNA samples are an ideal
source for molecular analysis. In fact, aspirated cells can be
immediately placed in special solutions to preserve nucleic acids at the time of the FNA procedure. An FNA passage dedicated to molecular analysis is preferred, but even the lavage
fluid obtained from rinsing the needles after the preparation
of the smears can give adequate results 10. Moreover, alcohol-
based fixation and staining (e.g. PAP) used for diagnosis
preserve nucleic acids well, so that molecular analysis can
be easily performed from the routinely processed slide after
removal of the coverslip 3. This procedure has the obvious
advantage of the selection of the slide (or the specific area on
the slide) where the cytologic alterations are located, allowing
a very precise correlation between morphology and genetic
changes. The archival slide is lost, but diagnostic areas can
be photographed for documentation and future review before
removing the coverslip and dissecting the cells for molecular
analysis.
The initial work on the application of molecular analysis to
the diagnosis of FNA samples was retrospective. In these
studies comparison of the results and interpretation of the
data presented were made difficult – if not impossible – by
the lack of a standardised nomenclature for cytologic diagnoses. More recent work has been careful in precisely defining
the diagnostic categories used. This is to be credited to the
application of the six-tiered Bethesda system for reporting
thyroid cytopathology 14 and to its incorporation into the
guidelines of cytopathology professional societies of several
European countries (e.g. Guidance on the reporting of thyroid
cytology specimens, November 2009, http://www.rcpath.org/
Resources/RCPath/Migrated%20Resources/Documents/G/
g089guidanceonthereportingofthyroidcytologyfinal.pdf, accessioned July 22, 2013) 15. In addition, the most recent
studies have been prospective rather than retrospective 1,11.
Some of them – particularly those of Nikiforov and collaborators 8 11 – have reported nearly 100% specificity in diagnosing
malignancy in thyroid nodules, a sensitivity that is higher than
that of cytology. However, what these studies are showing
is that molecular testing is not to replace conventional cytologic evaluation, but that molecular analysis plus cytologic
diagnosis can improve the identification of malignancy in
thyroid nodules. They have also shown that the improvement
is particularly significant for those very cases were cytologic
evaluation is equivocal or indeterminate. These are cases with
the FNA diagnosis of Atypia of Undetermined Significance
(Follicular Lesion of Undetermined Significance- AUS/FLUS
of the Bethesda classification, corresponding to the Neoplasm
possible – atypia/non-diagnostic-Thy3a category of the Royal
College of Pathology) 11.
Currently much of the work has focused on the four simple
genetic alterations commonly detected in thyroid carcinoma (BRAF, RET/PTC, N-Ras, PAX8/PPARɣ, see above).
160
Among these molecular markers, testing for the BRAFV600E
mutation is at the moment the most cost-effective. Virtually
all BRAF mutations in thyroid cancer are the result of a T
to A transversion (in exon 15, at nucleotide 1799, that results in substitution of a Valine with a Glutammate residue)
therefore the mutation can be very easily tested, at low cost,
even in laboratories with limited equipment. The test can be
performed with a variety of methods, of which allele-specific
PCR is probably the most rapid and least expensive 4. Our
group has recently developed ASLNAqPCR, a novel allelespecific quantitative test to accurately detect and quantify hot
spot mutations, including those of BRAF 16. It is important to
recognize that to date a handful of cases “false positive” for
the BRAFV600E mutation have been reported. Kim et al. 5
has shown five false positive cytology specimens in a large
prospective study that included 279 cases where the cytology
diagnosis was followed by the histologic examination of the
resected nodule. FNA material was analyzed using a highly
sensitive method. Direct sequencing of DNA extracted from
the five resected nodules, that were diagnosed as follicular
adenoma (one case) and hyperplastic nodules (four cases),
failed to confirm the presence of the mutation.
Given the high specificity and positive predictive value of
the BRAFV600E in cytology specimens (close to 100%) 17, a
thyroidectomy can be proposed to a patient whose FNA has
been diagnosed as AUS/FLUS with positive BRAF testing,
without the need to repeat the FNA 1 17. Another diagnostic
category that would benefit is that of lesions diagnosed as
Suspicious, since a thyroidectomy, without the need for
frozen sections or a diagnostic lobectomy, can be directly
recommended for a patient with a Suspicious FNA that is
BRAF positive 1 17. Many (although not all) studies have
shown that BRAFV600E is an indicator of poor prognosis:
papillary carcinoma with BRAFV600E mutations behave
more aggressively, with higher rates of extrathyroidal extension and lymph node metastases, higher rates of persistent
disease and higher rates of tumor-related death 18 19. Therefore the identification of BRAFV600E in cases diagnosed as
Malignant (or Suspicious) may modify the surgical approach
to include central compartment lymph node dissection,
although this should probably wait for more definite data
regarding the prognostic role of BRAF, particularly in the
case of small papillary carcinomas 20.
Several prospective studies have shown that the other molecular alterations common in thyroid carcinoma and mentioned
above (RET/PTC, N-Ras, PAX8/PPARɣ) can be tested as
a panel, together with BRAF. These studies, both from the
United States and Europe, have shown that FNA often provides enough material for all the tests to be performed and that
sensitivity can be significantly improved by adding markers
like RAS mutations and the PAX8/PPARɣ rearrangement
that are particularly relevant for follicular-patterned lesions
i.e. follicular adenoma, follicular carcinoma and the follicular
variant of papillary carcinoma 2 6 8 11 21. Potentially useful is
also the analysis of microRNA (miRNA) profiles 22, although
results are still preliminary.
One issue with the molecular analysis of BRAF, RET/PTC,
N-Ras, PAX8/PPARɣ and of other tumor-specific markers in
FNA should be noted. Although they may have a high positive predictive value (particularly BRAFV600E), the negative
predictive value is not as high: if the mutation is not there the
nodule can still be malignant.
Thus, a complementary approach that has been pursued by a
commercial company – Veracyte – is that of identifying markers not of malignancy, but of benignity in FNA samples. This
company has validated a proprietary test called the “Afirma
gene expression classifier” (AGEC) based on the expression
profile of 167 genes. Veracyte has made it available since
late 2010, and the test is recommended for use in cases with
indeterminate cytologic diagnoses (Bethesda categories III,
IV, V, corresponding to Thy3a, Thy3f and Thy4 of the Royal
College of Pathologists’s categories, respectively). The claim
is that if the cytologic FNA diagnosis is indeterminate and
the AGEC test negative, the likelihood of the thyroid nodule
being malignant is the same as that with a negative FNA
diagnosis 23-25.Indeed some studies are showing the use of the
AGEC test has reduced the number of diagnostic thyroidectomies performed on nodules with an indetermined cytologic
FNA diagnosis 26.
In conclusion, according to the current scientific literature
molecular analysis of FNA samples may be a useful complement to conventional FNA cytology in selected cases, with
the greatest in the AUS/FLUS (Thy3a) and the Suspicious
(Thy4) categories.
References
1
Adeniran AJ, Theoharis C, Hui P, et al. Reflex BRAF testing in thyroid
fine-needle aspiration biopsy with equivocal and positive interpretation: a prospective study. Thyroid 2011;21:717-23.
2
Cantara S, Capezzone M, Marchisotta S, et al. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules
improves the diagnostic accuracy of cytology. J Clin Endocrinol Metab
2010;95:1365-9.
3
Cohen Y, Rosenbaum E, Clark DP, et al. Mutational analysis of BRAF
in fine needle aspiration biopsies of the thyroid: a potential application for the preoperative assessment of thyroid nodules. Clin Cancer
Res 2004;10:2761-5.
4
Jin L, Sebo TJ, Nakamura N, et al. BRAF mutation analysis in fine
needle aspiration (FNA) cytology of the thyroid. Diagn Mol Pathol
2006;15:136-43.
5
Kim SW, Lee JI, Kim JW, et al. BRAFV600E mutation analysis in
fine-needle aspiration cytology specimens for evaluation of thyroid
nodule: a large series in a BRAFV600E-prevalent population. J Clin
Endocrinol Metab 2010;95:3693-700.
6
Moses W, Weng J, Sansano I, P et al. Molecular testing for somatic
mutations improves the accuracy of thyroid fine-needle aspiration
biopsy. World J Surg 2010;34:2589-94.
7
Nam SY, Han BK, Ko EY, et al. BRAF V600E mutation analysis of
thyroid nodules needle aspirates in relation to their ultrasongraphic
classification: a potential guide for selection of samples for molecular
analysis. Thyroid 2010;20:273-9.
8
Nikiforov YE, Steward DL, Robinson-Smith TM, et al. Molecular
testing for mutations in improving the fine-needle aspiration diagnosis
of thyroid nodules. J Clin Endocrinol Metab 2009;94:2092-8.
9
Salvatore G, Giannini R, Faviana P, et al. Analysis of BRAF point mutation and RET/PTC rearrangement refines the fine-needle aspiration
diagnosis of papillary thyroid carcinoma. J Clin Endocrinol Metab
2004;89:5175-80.
10
Troncone G, Cozzolino I, Fedele M, et al. Preparation of thyroid FNA
material for routine cytology and BRAF testing: a validation study.
Diagn Cytopathol 2010;38:172-6.
11
Ohori NP, Nikiforova MN, Schoedel KE, et al. Contribution of molecular testing to thyroid fine-needle aspiration cytology of “follicular
lesion of undetermined significance/atypia of undetermined significance”. Cancer Cytopathol 2010;118:17-23.
12
Basolo F, Pisaturo F, Pollina LE, et al. N-ras mutation in poorly differentiated thyroid carcinomas: correlation with bone metastases and
inverse correlation to thyroglobulin expression. Thyroid 2000;10:19-23.
13
Garcia-Rostan G, Zhao H, Camp RL, et al. ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid
cancer. J Clin Oncol 2003;21:3226-35.
14
Cibas ES, Ali SZ, Conference NCITFSotS. The Bethesda System For
Reporting Thyroid Cytopathology. Am J Clin Pathol 2009;132:658-65.
15
Tallini G, Gallo C. Fine-needle aspiration and intraoperative consultation in thyroid pathology: when and how? Int J Surg Pathol
2011;19:141-4.
16
Morandi L, de Biase D, Visani M, et al. Allele Specific Locked Nucleic
Acid Quantitative PCR (ASLNAqPCR): an Accurate and Cost-effective
161
relazioni
17
18
19
20
21
22
23
24
25
26
assay to Diagnose and Quantify KRAS and BRAF Mutation. PLoS
ONE 2012;7:e36084. doi:36010.31371/journal.pone.0036084.
Kim SK, Hwang TS, Yoo YB, et al. Surgical results of thyroid nodules
according to a management guideline based on the BRAF(V600E)
mutation status. J Clin Endocrinol Metab 2011;96:658-64.
Elisei R, Ugolini C, Viola D, et al. BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median
follow-up study. J Clin Endocrinol Metab 2008;93:3943-9.
Xing M, Westra WH, Tufano RP, et al. BRAF mutation predicts a
poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 2005;90:6373-9.
Soares P, Sobrinho-Simoes M. Cancer: Small papillary thyroid cancers--is BRAF of prognostic value? Nat Rev Endocrinol 2011;7:9-10.
Gupta N, Dasyam AK, Carty SE, et al. RAS mutations in thyroid FNA
specimens are highly predictive of predominantly low-risk follicularpattern cancers. J Clin Endocrinol Metab 2013;98:E914-22.
Nikiforova MN, Tseng GC, Steward D, et al. MicroRNA expression
profiling of thyroid tumors: biological significance and diagnostic
utility. J Clin Endocrinol Metab 2008;93:1600-8.
Alexander EK, Kennedy GC, Baloch ZW, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J
Med 2012;367:705-15.
Jameson JL. Minimizing unnecessary surgery for thyroid nodules. N
Engl J Med 2012;367:765-7.
Kloos RT, Reynolds JD, Walsh PS, et al. Does addition of BRAF
V600E mutation testing modify sensitivity or specificity of the Afirma
Gene Expression Classifier in cytologically indeterminate thyroid
nodules? J Clin Endocrinol Metab 2013;98:E761-8.
Duick DS, Klopper JP, Diggans JC, et al. The impact of benign gene
expression classifier test results on the endocrinologist-patient decision to operate on patients with thyroid nodules with indeterminate
fine-needle aspiration cytopathology. Thyroid 2012;22:996-1001.
Role of molecular testing in the diagnosis
of GEP-NETs
A. Scarpa
ARC-NET Research Centre and Department of Pathology and Diagnostic, University and Hospital Trust of Verona, Verona, Italy
The vast majority of GEP-NETs are clinically silent tumors
that are diagnosed when mass effects and liver metastasis
are present. Similar to other “rare” cancers, there is the lack
of both an appropriate prognostic algorithm and of effec-
tive systemic therapies for the treatment of advanced stage
diseases.
To explore the genetic events underlying these complex tumor
entities, we sequenced the exomes of 50 pancreatic neuroendocrine tumors (PanNET) and matched normal samples.
Somatic mutations in the chromatin remodeling pathway
(MEN1-DAXX-ATRX) were identified in 60% of tumors
(30/50). Genetic alterations of MEN1 accounted for about
40% of tumors analyzed and are associated with specific gene
expression profiles. The presence of ATRX/DAXX mutations
in 40% of samples is strongly associated with alternative
lengthening of telomeres (ALT) phenotype. Interestingly,
ALT tumors displayed specific transcriptional profiles. In
addition to mutations in chromatin remodeling pathways, somatic mutations in genes belonging to mTOR/PI3K pathways
(PTEN and TSC2) were identified in 14% of cases. Somatic
mutations of ATM were observed in 13% of samples. This
gene encodes for a master regulator of the DNA damage
response (DDR), thus representing an interesting target for
therapeutic approaches.
A recent massively parallel exome sequencing study on 48
small intestine neuroendocrine tumors (SI-NETs) pinpointed
a relatively low frequency of somatic single nucleotide variants (SNVs), which suggests that SI-NETs are stable cancers.
Integrative analysis of SNVs and somatic copy number
variations identified recurrently altered mechanisms of carcinogenesis: chromatin remodeling, DNA damage, apoptosis,
RAS signaling, and axon guidance. Candidate therapeutically
relevant alterations were found in 35 patients, including SRC,
SMAD family genes, AURKA, EGFR, HSP90, and PDGFR.
Mutually exclusive amplification of AKT1 or AKT2 was the
most common event in the 16 patients with alterations of
PI3K/Akt/mTOR signaling.
These preliminary findings are shedding new light on the
phenotypes associated with specific genetic events in GEPNETs, and provide several new targets that may be exploited
for both diagnostic purposes and for the development of novel
therapeutic strategies leading to a significant improvement in
the management of these diseases.
Associazione patologi oltre frontiera
Donne che aiutano le donne: patologhe oltre frontiera
Moderatori: M.R. Giovagnoli (Roma), P. Ceppa (Genova)
Sustainable interventions for breast cancer
diagnosis in low-resources countries:
the point of view of the pathologist
S. Guzzetti, A. Sapino*
S.C. Anatomia Patologica Ospedale Martini - Asl TO1, Torino; * Dipartimento di Scienze Mediche, Università di Torino, Anatomia Patologica III, AO “Città della salute e della scienza” Torino
In recent times, the word “sustainability” has entered into
our everyday vocabulary as a key parameter for evaluating
the effectiveness of an idea or a project: a “sustainable”
project takes into account not only the existing resources
and those available, but especially the preservation through
time of the benefits and of the objectives, once they have
been reached.
Overall, the concept of “sustainability” refers to “a quality and
system of life that allows people to meet their current needs
without compromising the resources available for future generations to meet their future needs”.
In health cooperation projects, the concept of sustainability
lies primarily in the ability of the project itself to be maintained and further developed by the local resources alone,
once the project objectives were achieved in the preset period
of time; this means that the implementation of the project
itself needs to take account of its own sustainability, precisely
to avoid to interrupt the produced benefits once over.
Based on these statements, we tried to build a path for the
management of patients with breast cancer in a specific context characterized by an extremely poor available resources;
only starting from the most essential path, from the point of
view both of effectiveness and ethics, we will be able indeed
to develop further improvements in time that will be specifically suitable for that particular situation.
Already a decade ago, in order to address this problem, an
162
international group of breast cancer experts and patient advocates met in Seattle on 2002, aiming to develop consensus recommendations for the diagnosis of breast cancer in countries
with limited resources ¹.
The panel stated, among others, that “a pathologic diagnosis
should not be bypassed, even when health care resources are
very limited, because a misdiagnosis of breast cancer can lead
to erroneous treatment of women without breast cancer, which
is harmful to the woman and wasteful of treatment resources”.
Consequently, the panel agrees “FNAB can play an important
role in the diagnosis of breast cancer in countries with limited
resources” and noted that “diagnostic capacity is critical to
the success of a comprehensive breast health care program in
countries with limited resources”.
Despite these recommendations, the proposed flow charts
for the management of breast cancer patients tended to be
very similar to those applied in high-resource countries, thus
requiring a certain level of health care which could not be
achieved in the majority of health facilities of developing
countries; the application of that protocols would have so been
necessarily limited to the populations living in urban areas
which, in these countries, are on the whole a minority ².
Our proposal for a “basic” path for the management of women
with breast cancer has started by the assertion that in countries
with very low human and economic resources a minimal effective pathological protocol must be strongly related to the
available treatments.
It implies that if the only available treatment is the radical
mastectomy (as still in many hospitals of the concerned countries), then diagnosis can be made by fine needle aspiration
cytology only, that means minimal technical requirement and
low costs, but also health staff with an optimal expertise.
Even the diagnostic categories should be adapted to this particular situation: the final goal of our protocol being to reduce
surgical overtreatment, only two categories should be given:
negative (encompassing the C3 category as defined by the
European Guidelines) and positive, which likewise ought to
encompass the C4 category.
The same rules could be also applied for the cytological diagnosis of suspect axillary lymph nodes detected by ultrasonography (a low cost diagnostic tool, therefore feasible even in
low-resource settings), because in our context the only useful
doubt to solve is if to perform a surgical dissection of nodes
or not.
Other “adapted rules” could be proposed also for the management of surgical specimens and for the post-operative diagnosis, always keeping in mind the minimal requirements for
an adequate specimen handling and for the assessment of the
histological type of the tumor respectively.
It is thus clear that all the above proposals are highly dependent on the operator’s expertise: the simplification of
diagnostic categories and of the procedures can lead to effective results even in low-resource settings only if the skill and
experience of health personnel will be able to compensate the
paucity of available technology.
In conclusion, in every health cooperation project dedicated to
poor countries, it will be possible to save money for technical
resources, but not for human resources!
References
¹ Vargas HI, et al. Diagnosis of Breast Cancer in Countries with Limited
Resources. The Breast Journal 2003;9(Suppl. 2):S60-S6.
² Anderson BO, et al. Breast Cancer in Limited-Resource Countries: An
Overview of the Breast Health Global Initiative 2005 Guidelines. The
Breast Journal 2006;12(Suppl. 1):S3-S15.
Cervical carcinoma: first test level in developing
countries: cytology or hpv test?
E. Ferrario1, L. Viberti2
1
Chirundu, Zambia; 2 Torino
In developing countries, where primary emergencies (malnutrition, conflicts) are overcome, health has to face, as well as
epidemic diseases, even cancer diseases.
The cervical cancer in African countries is the leading cause
of cancer death in women due to socio-economic factors and
the immuno-suppression caused by HIV infection.
Pap test cytology in the diagnosis of this cancer and its precursors in these contexts, with large technological and cultural
backwardness, has been the first choice in the projects implemented by “Patologi Oltre Frontiera ONLUS”, despite other
minimalist proposals from other organizations The use of viral
typing techniques, is currently questionable for both the high
cost of management that for the low specificity of the method
that increases much the number of patients to be sent to the
second level.
The search for alternative methods to the Pap test was determined essentially by the fact that, in the published experience
in developing countries, cervical cytology, which is a highly
specific screening test, showed a low sensitivity due to inadequate samples, laboratory errors or misinterpretation.
Our choice (relatively “upstream”) of using the Pap test, tries
to improve the sensitivity of the method through training
courses for the local technical staff in order to get better the
quality of preparation and reading cervical cytology. We have
organized residential courses on-site and in Italy, using also
e-learning systems with on-line teaching and lectures on the
web via a dedicated platform.
The aim, at present, is to manage from Italy only suspect or
positive cases while negative cases are directly reported from
African hospitals: the meaningful images selected by microscopic fields are sent to our web site and, with an organized
computer program we can ensure our advice and send the final
diagnosis.
In a project in Zambia, with Mtendere Mission Hospital we
have collaborated with the Italian Society of Colposcopy that
has provided to the hospital a new generation colposcope
and a radio frequency system for the treatment of lesions
histologically and cytologically diagnosed. The patients were
treated according to defined guidelines established taking into
account the particular context.
In conclusion, collaboration with colleagues gynecologists
in Africa also allowed to use the available resources for the
treatment and cure of cancer of the cervix, creating training
and professionalism, allowing to set a program of screening
and treatment of a level similar to that proposed in industrialized countries.
163
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Patologia endocrina
Corso breve: definizione di malignità in neoplasie endocrine e neuroendocrine
Moderatori: C. Capella (Varese), G. De Rosa (Napoli)
Definizione di malignità in neoplasie endocrine
e neuroendocrine: paratiroide
L. Torregrossa, F. Basolo
Anatomia Patologica Universitaria III, AOU Pisana, Pisa
Parathyroid tumors encompass a spectrum of proliferative
lesions including adenoma, atypical adenoma and carcinoma 1 2. Clinically, the most characteristic manifestation of
parathyroid tumors is primary hyperparathyroidism, which is
one of the most frequently encountered endocrine disorders,
with an incidence of about 22 cases per 100,000 persons per
year in Western countries, characterized by hypercalcemia
associated to elevated or unexpectedly “normal” values of
parathyroid hormone 3 4. Primary hyperparathyroidism is most
often caused by a single adenoma (80-85%) or four-glands
hyperplasia (10-15%), usually as a sporadic disease, rarely in
the context of hereditary syndrome (e.g., multiple endocrine
neoplasia types 1 and 2A) 3. Parathyroid carcinoma is a very
rare cause of primary hyperparathyroidism (< 1%). In some
Countries, including Italy, an incidence of parathyroid carcinoma more than 5% has been reported 5.
From the pathological point of view, often the differential diagnosis between benign and malignant endocrine lesions may
represent a diagnostic challenge: in this context, the parathyroid disease is no exception.
The definition and the applicability of morphological criteria
diagnostic of malignancy in parathyroid lesions are still controversial and object of debate. Many of the morphological
features that have been proposed as indicative of parathyroid
carcinoma (adherence to surrounding structures, local invasion, vascular and/or perineural invasion, fibrous bands, solid
growth or cellular organization of trabecular type, mitotic
activity) can also be observed in non-malignant lesions. Each
of these features has a diagnostic value in the definition of malignancy of a parathyroid lesion, but none is pathognomonic
of parathyroid carcinoma. Only the presence of metastases
(rarely observed as the initial clinical manifestation) is a clear
sign of malignancy 6. Bondeson and co-workers have noted
the presence of foci of coagulative necrosis, macronucleoli,
and mitotic activity > 5 mitoses per 50 HPF (High Power
Fields) is a triad indicative of more aggressive biological
behavior 7. The term “atypical adenoma” is used to describe
a group of parathyroid lesions showing some features of carcinoma (fibrosis, mitosis, cells within capsule) but lacking
unequivocal capsular and/or vascular invasion 8. The features
most frequently found in this group of lesions are: trapping of
tumor cells in the context of capsular thickness, intratumoral
fibrosis, hemosiderin deposits, cystic degeneration, mitosis
and peritumoral fibrosis. Pictures of capsular invasion without extension beyond the capsule and intratumoral vascular
invasion are present in individual cases, but lacking clear
signs of necrosis 8. It is clear, therefore, that the diagnosis of
malignancy in parathyroid disease can be very difficult. So,
new markers are necessary in order to support the diagnosis
of parathyroid carcinoma. Numerous studies have highlighted
the possible diagnostic role of parafibromin, a protein encoded by HRPT2 tumor suppressor gene 9 10. Germline mutations
of this gene able to inactivate parafibromin have been initially
identified in patients with the syndrome primary hyperparathyroidism-jaw tumors (HPT-JT). Then, somatic mutations
have been observed in the majority of sporadic parathyroid
carcinoma. Molecular analysis of the gene HRPT2 is certainly
a valuable aid in the diagnosis of parathyroid carcinoma, but
given the rarity of the disease this approach cannot be a real
help in many laboratories of Surgical Pathology. For this reason, immunohistochemical analysis of parafibromin has been
proposed as a rapid, cheap and efficient method of assessing
the state of the mutational gene HRPT2 9 10. In several studies,
the loss of parafibromin nuclear immunoreactivity shows very
high values of
sensitivity and specificity for the definitive
diagnosis of parathyroid carcinoma versus benign disease 9-11.
Additional markers that have shown some utility in this type
of differential diagnosis and which may be indicated in the
daily immunohistochemical practice are represented by p27,
MDM2, Bcl-2, cyclin D1 and Rb.
References
1
DeLellis RA, Mazzalia P, Mangray S. Primary hyperparathyroidism:
a current perspective. Arch Pathol Lab Med 2008;132:1251-62.
2
Bondeson L, Grimeluis L, DeLellis RA, et al. Parathyroid carcinoma.
In: DeLellis RA, Lloyd RV, Heitz PN, et al., eds. Pathology and genetics of tumours of endocrine organs (WHO classification). Lyon: IARC
2004, pp. 224-7.
3
Marcocci C, Cetani F. Primary hyperparathyroidism. N Engl J Med
2011;365:2389-97.
4
Wermers RA, Khosla S, Atkinson EJ, et al. Incidence of primary
hyperparathyroidism in Rochester, Minnesota, 1993-2001: an update on the changing epidemiology of the disease. J Bone Miner Res
2006;21:171-7.
5
Favia G, Lumachi F, Polistina F, et al. Parathyroid carcinoma: sixteen
new cases and suggestions for correct management. World J Surg.
1998;22:1225-30.
6
DeLellis RA. Parathyroid carcinoma. An overview. Adv Anat Pathol
2005;12:53-1.
7
Bondeson L, Sandelin K, Grimelius L. Histopathological variables
and DNA cytometry in parathyroid carcinoma. Am J Surg Pathol
1993;17:820-9.
8
Guiter GE, DeLellis RA. Risk of recurrence or metastasis in atypical
parathyroid adenoma. Mod Pathol 2002;15:115A.
9
Cetani F, Ambrogini E, Viacava P, et al. Should parafibromin staining
replace HRTP2 gene analysis as an additional tool for histologic diagnosis of parathyroid carcinoma? Eur J Endocrinol 2007;156:547-54.
10
Gill AJ, Clarkson A, Gimm O, et al. Loss of nuclear expression
of parafibromin distinguishes parathyroid carcinomas and hyperparathyroidism-jaw tumor (HPT-JT) syndrome-related adenomas from
sporadic parathyroid adenomas and hyperplasias. Am J Surg Pathol
2006;30:1140-9.
11
Wang O, Wang C, Nie M, et al. Novel HRPT2/CDC73 gene mutations
and loss of expression of parafibromin in Chinese patients with clinically sporadic parathyroid carcinomas. PLoS One 2012;7:e45567.
Malignancy definition in neuroendocrine
neoplasms of the gastroenteropancreatic tract
G. Rindi
Institute of Anatomic Pathology, U.C.S.C. Policlinico A. Gemelli,
Rome, Italy
First recognized as “carcinoids” in the gastroenteropancreatic
(GEP) tract, neuroendocrine tumors (NET) are a variety of
neoplastic lesions distributed in most organs and apparata.
The current World Health Organization (WHO 2010) define
164
low and high grade neuroendocrine cancer types with the
broad definition of neuroendocrine neoplasms and introduces
grading (G1-G3) and Tumour Node Metastasis (TNM) staging for class definition. The proposed WHO 2010 grading
system defines three classes (G1-G3) according to both mitotic count and Ki67 index. Three classes are defined as WHO
class 1, neuroendocrine tumor (NET), G1; WHO class 2, NET
G2 and WHO class 3, neuroendocrine carcinoma (NEC), by
definition G3. The definition of NET equalizes the previous definition of carcinoid (typical and atypical as variably
utilized in the pathology literature) and of well-differentiated
endocrine tumor/carcinoma as from the previous WHO classification. The WHO 2010 TNM substantially endorsed the
classification originally proposed by the European Neuroendocrine Tumour Society (ENETS) with two exceptions in
that i) it limits its application to NET G1-G2 (carcinoids) and
ii) it shows significant differences for pancreas and appendix
TNM definitions. In general a common staging system frame
was defined with stage I tumors with limited growth, stage
II larger or more invasive tumors, in absence of metastases,
stage III tumors invading the surrounding structures or with
loco-regional metastases and stage IV implying distant metastases. This classification system was first adopted by the
International Union Against Cancer (UICC) and subsequently
endorsed by both the American joint Cancer Committee
(AJCC) and the WHO. Current oncology guidelines for
Neuroendocrine neoplasms are largely based on proliferation fraction and stage to define specific therapeutic options.
Malignancy depends on grading and staging features and different survival has been demonstrated mostly in retrospective
series. Open questions entail the definition of specific cut-offs
between G1 and G2 for NETs at different anatomical places.
Indeed it appears that in some sites, i.e. pancreas, a higher cut
off of 5% may be more informative. On the same line within
G3 high grade NECs, limited data support different behavior
according to some NET-like aspects and Ki67 values between
20 and 99%. Data needs to be generated to support specific
indication. Finally, despite the value of the staging scheme
has been validated in large retrospective series, site-specific
adjustments have been proposed.
Mixed neuroendocrine/non-neuroendocrine
tumors
S. La Rosa, C. Capella, F. Sessa
UO Anatomia Patologica-Ospedale di Circolo e Dipartimento di
Scienze Chirurgiche e Morfologiche-Università dell’Insubria, Varese
Background and definition. Mixed neuroendocrine/nonneuroendocrine tumors are an emerging topic in surgical
pathology with several diagnostic, clinico-pathologic and
therapeutic implications 1. Although the systematic application of immunohistochemical techniques to the study of tumors has led to the recognition that neoplasms composed of
both neuroendocrine and non-neuroendocrine components are
not as rare as believed in the past, mixed neuroendocrine/nonneuroendocrine tumors are, by definition, neoplasms in which
each component represents at least 30% of the lesion 2 3. During the last 20 years, different terms have been used to classify
them creating some confusion among clinicians and pathologists 1. Although these tumors have been described in several
different organs, they have been better characterized in the
gastrointestinal tract, pancreas, and hepatobiliary sytem 4-7
where are now called “mixed adenoneuroendocrine carcinomas” (MANECs), following the criteria proposed in the
2010 WHO classification of tumors of the digestive system 3.
This last terminology, however, has not yet been officially
accepted for mixed tumors arising in other sites including
the pituitary gland 8, lung 9, prostate 10 11, urinary bladder 12 13,
kidney 14, breast 15, nasal cavity 16, and skin 17.
Pituitary gland. Mixed neuroendocrine/non-neuroendocrine
tumors arising in the sellar region are very rare. They are
formed of admixed craniopharyngioma and pituitary adenoma
components and account for only 6 well-documented reported
cases 8. Strangely, this specific tumor type was not included
as a separate entity in the WHO classification of endocrine
tumors 18, probably because of its rarity. However, this entity needs to be known for diagnostic reasons and should
be considered among the potential differential diagnoses
of neoplasms arising in this specific site. These tumors occurred more frequently in males with an average age of 48
years. While the non-neuroendocrine component was always
represented by an adamantinomatous craniopharyngioma,
the neuroendocrine component consisted of different types
of pituitary adenomas. Interestingly, the ultrastructural and
immunohistochemical observations of “hybrid” cells showing
morphologic characteristics of both craniopharyngioma and
pituitary adenoma, observed in one reported case, seem to
suggest that these tumors may originate from stem cells which
undergo divergent differentiation 8.
Nasal cavity. Mixed exocrine-neuroendocrine carcinomas of
the nasal cavity are extremely rare accounting for only eight
cases described in the English literature 16. This entity was not
included in the last WHO classification of tumors of the head
and neck, probably because of its rarity [19]. Since in most of
the reported cases the exocrine component was represented by
intestinal-type adenocarcinomas (ITACs) and considering the
close analogies between intestinal adenocarcinomas and ITACs, it would be reasonable to suggest to classify such mixed
tumors as MANECs, following the nomenclature proposed
for intestinal cancers. Sinonasal MANECs can show different types of components: with the exception of only one case
the neuroendocrine component was represented by poorly
differentiated neuroendocrine carcinomas (NECs) while the
exocrine component was most often represented by ITACs.
Based on the limited available information, it would seem
that sinonasal MANECs are not associated with occupational
risks in the same way as sinonasal ITACs and are aggressive
neoplasms associated with poor prognosis. Molecular analyses suggest a monoclonal origin of sinonasal MANECs from
a pluripotent cell undergoing a biphenotypic differentiation
and that the neuroendocrine differentiation may be from an
exocrine to an endocrine pathway 16.
Lung. Non-neuroendocrine differentiation in lung neuroendocrine tumors has been described. The morphological characteristics with the related clinical implications depend on the type
of neuroendocrine and non-neuroendocrine components 9. Mucin production has been described in lung carcinoids, but this
feature of divergent differentiation does not change the diagnosis of carcinoid and does not have prognostic significance 9.
Conversely, non-neuroendocrine components can be frequently
observed in both large and small cell high grade NECs. Up to
30% of large cell NECs contain a non-neuroendocrine component, usually represented by an adenocarcinoma and, less frequently, by squamous cell carcinoma, giant cell carcinoma and/
or spindle cell carcinoma. However, the clinical implication of
this morphological heterogeneity is not clear. About 10% of
small cell NECs contain a non-neuroendocrine component, that
may be squamous or glandular. These mixed lung carcinomas
appear to have a worse prognosis than the “pure” counterparts,
relazioni
possibly because of the relative chemotherapy resistance of
the non-neuroendocrine components. Interestingly, it has been
observed that the non-neuroendocrine components sometimes
emerged after therapy in either recurrent or metastatic locations
and this may be due to the effect of therapy 20.
Gastroenteropancreatic system. According to the 2010
WHO classification of tumors of the digestive tract 3 MANECs
are neoplasms with a phenotype that is morphologically recognizable as both gland forming epithelial and neuroendocrine, and are defined as carcinomas since both components
are malignant. A component of squamous cell carcinoma, although rare, can also be found. Both exocrine and neuroendocrine components can show different morphological features
mainly depending on the site of insurgence ranging from adenomas to carcinomas with different degrees of differentiation
in exocrine components and from well to poorly differentiated
neuroendocrine tumors in neuroendocrine components. In the
pancreas the most frequent combination is represented by acinar cell carcinoma and neuroendocrine tumor (NET) 6, while
mixed ductal adenocarcinoma-NET and mixed ductal/acinar
carcinoma-NET are very rare 5. In addition to MANECs, in
which both components are histologically malignant, rare tumors composed of adenoma and well differentiated neuroendocrine tumor (NET according to the nomenclature proposed
by the 2010 WHO classification) have been also described
in the colon and stomach. In the 2010 WHO classification,
these mixed adenoma-NET neoplasms were not specifically
categorized. Due to their low biological aggressiveness and
their morphological features, the term of mixed adenoneuroendocrine tumor (MANET) has been recently suggested
to diagnose these peculiar mixed neoplasms 4. This term,
although not included in the 2010 WHO classification, would
be more appropriate because it underlines the better prognosis
of this tumor category. MANECs and MANETs constitute
a diagnostic challenge, as often only one component of the
neoplasm is identified, which leads to an incomplete diagnosis
and suboptimal treatment. A provisional prognostic classification of gastrointestinal MANECs and MANETs is provided
in Table I.
The optimal strategy for the management of MANEC and
MANET is largely unknown, due to the rarity of these neoplasms. The more aggressive component should be taken into
account when considering their treatment. MANECs containing a NET and an adenocarcinoma component should be
treated as adenocarcinomas, whereas MANECs containing a
NEC component should be treated as NECs 21.
Few studies in the literature have addressed the histogenetic
issue of MANECs-MANETs and most of them reported variTab. I. Types of mixed exocrine-neuroendocrine neoplasms of
the gastrointestinal tract, grouped according to the grade of
malignancy 4.
Mixed adenoneuroendocrine carcinoma (MANEC)
High grade malignant
Mixed adenoma/adenocarcinoma-NEC
Intermediate grade malignant
Mixed adenocarcinoma-G1/G2 NET*
Amphicrine carcinoma
Mixed adenoneuroendocrine tumor (MANET)
– provisional category –
Low grade malignant
Adenoma-NET
NEC: poorly differentiated neuroendocrine carcinoma; * G1-G2
according to WHO 2010 classification 3; NET: neuroendocrine tumor.
165
ous and controversial data, thus making different histogenetic
hypotheses still open. These mixed tumors may represent either the simultaneous proliferation of multiple cell lineages or
the proliferation of stem cells capable of differentiating along
multiple cell lineages. The presence in MANECs and MANETs of amphicrine cells containing within their cytoplasm
both neuroendocrine granules and mucin droplets support the
hypothesis of a common precursor stem cell capable of divergent differentiation within an individual neoplastic cell. Molecular data on gastrointestinal MANECs indicating a close
genetic relationship between the two components suggest a
possible multistep progression from a common precursor lesion and that the neuroendocrine differentiation may be from
an exocrine to an endocrine pathway 22-24.
Urogenital system. Rare cases of mixed neuroendocrine/
non-neuroendocrine carcinomas have been described in organs of the urogenital system. Among the 12 renal cases
so far reported the neuroendocrine component was always
represented by NEC, while the non-neuroendocrine component was represented by urothelial, squamous, and papillary
carcinoma. Interestingly, none of the tumors originating in the
renal parenchyma showed a non-neuroendocrine component,
which was instead found in all tumors originating in the renal
pelvis 14. With the exception of only a few cases, the prognosis
was very poor with patients’death in a short time.
Rare cases of mixed cancers of the urinary bladder constituted
by adenocarcinoma and NEC have been reported 12 13. Due to
their rarity the clinical and prognostic characteristics still need
to be further clarified, even if these tumors seem to behave in
an aggressive fashion.
Although the immunohistochemically demonstrated divergent
differentiation in prostatic adenocarcinoma is not rare, true
mixed neuroendocrine/non-neuroendocrine carcinomas are
rare 11. Most tumors are characterized by an admixture of high
grade prostatic adenocarcinoma and NEC, more frequently of
the small cell type. Such mixed cancers have a ominous prognosis mainly related to the aggressiveness of NEC.
References
1
Volante M, Rindi G, Papotti M. The grey zone between pure (neuro)
endocrine and non-(neuro)endocrine tumours: a comment on concepts
and classification of mixed exocrine-endocrine neoplasms. Virchows
Arch 2006;449:499-506.
2
Solcia E, Klöppel G, Sobin LH. Histological typing of endocrine tumours. WHO international histological classification of tumours, 2nd
Edition. Berlin: Springer 2000.
3
Rindi G, Arnold R, Bosman FT, et al. Nomenclature and classification
of neuroendocrine neoplasms of the digestive system. In: Bosman FT,
Carneiro F, Hruban RH, et al. eds. WHO classification of tumours of
the digestive system, 4th Edition. IARC Press: Lyon 2010, pp. 13-4.
4
La Rosa S, Marando A, Sessa F, et al. Mixed adenoneuroendocrine
carcinomas (MANECs) of the gastrointestinal tract: an update. Cancers 2012;4:11-30.
5
Klöppel G. Mixed exocrine-endocrine tumors of the pancreas. Semin
Diagn. Pathol 2000;17:104-8.
6
La Rosa S, Adsay V, Albarello L, et al. Clinicopathologic study of 62
acinar cell carcinomas of the pancreas: insights into the morphology
and immunophenotype and search for prognostic markers. Am J Surg
Pathol 2012;36:1782-95.
7
Harada K, Sato Y, Ikeda H, et al. Clinicopathologic study of mixed
adenoneuroendocrine carcinomas of hepatobiliary organs. Virchows
Arch 2012;460:281-9.
8
Finzi G, Cerati M, Marando A, et al. Mixed pituitary adenoma/craniopharyngioma: clinical, morphological, immunohistochemical and ultrastructural study of a case, review of the literature, and pathogenetic
and nosological considerations. Pituitary 2013 Jan 24 [Epub ahead of
print].
9
Brambilla E, Lantuejoul S, Sturm N. Divergent differentiation in neuroendocrine lung tumors. Semin Diagn Pathol 2000;17:138-48.
10
Capella C, Usellini L, Buffa R, et al. The endocrine component of
166
prostatic carcinomas, mixed adenocarcinoma-carcinoid tumours and
non-tumour prostate. Histochemical and ultrastructural identification
of the endocrine cells. Histopathology 1981;5:175-92.
11
di Sant’Agnese A. Divergent neuroendocrine differentiation in prostatic carcinoma. Semin Diagn Pathol 2002;17:149-61.
12
Abenoza P, Manivel C, Sibley RK. Adenocarcinoma with neuroendocrine differentiation of the urinary bladder. Clinicopathologic, immunohistochemical, and ultrastructural study. Arch Pathol Lab Med
1986;110:1062-6.
13
Bertolini V, D’Ambrosio G, Consonni P, et al. Mixed adeno-neuroendocrine carcinoma (MANEC) of the urinary bladder: a case report.
Pathologica 2012;104:414-5.
14
La Rosa S, Bernasconi B, Micello D, et al. Primary small cell neuroendocrine carcinoma of the kidney: morphological, immunohistochemical, ultrastructural, and cytogenetic study of a case and review of the
literature. Endocr Pathol 2009;20:24-34.
15
Sapino A, Righi L, Cassoni P, et al. Expression of the neuroendocrine phenotype in carcinomas of the breast. Semin Diagn Pathol
2000;17:127-37.
16
La Rosa S, Furlan D, Franzi F, et al. Mixed exocrine-neuroendocrine
carcinoma of the nasal cavity: clinico-pathologic and molecular study
of a case and review of the literature. Head Neck Pathol 2013;7:76-84.
17
Zembowicz A, Garcia CF, Tannous ZS, et al. Endocrine mucinproducing sweat gland carcinoma: twelve new cases suggest that it is
a precursor of some invasive mucinous carcinomas. Am J Surg Pathol
2005;29:0-1339.
18
DeLellis RA, Lloyd RV, Heitz P, et al. Pathology & genetics of tumours of endocrine organs. WHO classification of tumours. Lyon:
IARC Press 2004.
19
Barnes L, Eveson JW, Reichart P, et al. WHO classification of tumours. Pathology & genetics of head and neck tumours. Lyon: IARC
Press 2005.
20
Gosney JR. Neuroendocrine tumors and other neuroendocrine proliferations of the lung. In: Hasleton P, Flieder DB, eds. Spencer’s
Pathology of the lung. Cambridge: Cambridge University Press 2012,
Chapter 31, pp 1151-85.
21
Hervieu V, Scoazec JY. Mixed endocrine tumors. Ann Pathol
2005;25:511-28.
22
Kim KM, Kim MJ, Cho BK, et al. Genetic evidence for the multi-step
progression of mixed glandular-neuroendocrine gastric carcinomas.
Virchows Arch 2002;440:85-93.
23
Vortmeyer AO, Lubensky IA, Merino JA, et al. Concordance of genetic alterations in poorly differentiated colorectal neuroendocrine
carcinomas and associated adenocarcinomas. J Natl Cancer Inst
1997;89:1448-53.
24
Furlan D, Cerutti R, Uccella S, et al. Different molecular profiles
characterize well-differentiated endocrine tumors and poorly differentiated endocrine carcinomas of the gastroenteropancreatic tract. Clin
Cancer Res 2004;10:947-57.
Short course: definition of malignancy
in endocrine and neuroendocrine neoplasms:
adrenal cortex
M. Volante, E. Duregon, M. Papotti
Department of Oncology, University of Turin at San Luigi Hospital,
Orbassano, Turin, Italy
Adrenocortical cancer (ACC) is a rare and aggressive disease
with an incidence of approximately one case per million.
The presence of distant metastases at the time of diagnosis
is relatively common and its differential diagnosis in this
clinical situation is mainly related to the exclusion of other
malignant neoplasms metastatic to the adrenal, especially
in inoperable cases 1. In the presence of adrenal-confined
disease, diagnostic difficulties in differentiating benign from
malignant adrenocortical neoplasms can be encountered, since
the pathological diagnosis of “carcinoma” does not rely on
clear cut morphological parameters but mostly on multiple
features, combined in scoring systems, suggestive but not
pathognomonic per se of malignancy 2-4.
The Weiss score. Among the different systems proposed, the
Weiss scoring system is the most widely employed 5 and is
based on the recognition at light microscopy of 9 morphological parameters, three related to tumor structure (presence
of eosinophilic cytoplasm in more than 75% of tumor cells, a
“patternless” diffuse architecture and necrosis), three related to
cytological features (presence of nuclear atypia, mitotic index
> 5/50 high power fields and atypical mitoses) and the remaining three related to invasive tumor properties (sinusoidal, venous and capsular invasion). In the general practice the diagnostic applicability of the Weiss system is not always easy, since
some of these features are subjective or biased by the adequacy
of tumor sampling 6. Moreover, it is scarcely reproducible in
the ACC morphological variants 7 8. In a large cohort of adrenocortical tumors, a recent French study on the reproducibility
of Weiss criteria showed that the inter-observer agreement was
“substantial” (κ = 0.70) in separating malignant (≥3 parameters)
from benign (≤ 2) tumors, and that - among all Weiss criteria necrosis was the most reproducible criterion 9.
The problem of borderline cases. In the majority of cases,
the differential diagnosis between adrenocortical adenoma
and carcinoma is easy to assess. However, in some cases
(approximately up to 10% of adrenal cancer series in the
Authors’personal experience) only one or two Weiss parameters are recognized, with a final score not sufficient for
a diagnosis of malignancy, but suggestive of a malignant
potential. These cases are worrisome from both pathological
and clinical standpoints, since, on the one hand, underline
the need of a more unequivocal pathological classification
and, on the other, pose questions on their appropriate clinical management and follow up. In most such “borderline”
cases, the commonly recognized Weiss parameters include
one or two of the following: nuclear atypia, eosinophilic
cytoplasm, diffuse growth or sinusoidal invasion. These are
parameters that generally have the lowest specificity when
correlated to malignant behavior; by contrast, conventional
malignancy-related parameters, such as high mitotic index,
atypical mitoses, necrosis, vascular and capsular invasion, are
usually not occurring alone, but are rather combined with the
other parameters, thus easily determining a high Weiss score.
A possible exception is represented by incidentally detected
“early” ACCs that may have small size and, although in the
presence of mitotic activity, might lack signs of invasion that
may be a late event during tumor progression. Based on the
above comments, a diagnosis of “borderline” or “uncertain
malignant potential” tumor should be rendered only after
extensive tumor sampling and the evaluation of an adequate
number of histological sections.
Beyond the Weiss score. An alternative view to the use of
diagnostic scoring systems is the definition of algorithmic approaches based on the recognition of the most relevant pathological features, such as mitotic index, and the exclusion of less
sensitive or poorly specific or equivocal features. Following
this alternative approach, a study suggested to select malignant
cases through a diagnostic algorithm based on mitotic count
(> 5/50HPF), followed by the recognition of other parameters
(including nuclear grade, diffuse growth pattern and tissue
reaction) 10, that proved to be more specific but less sensitive
as compared to the pathological scores proposed in Weiss and
van-Slooten systems. More recently, our group proposed a
diagnostic algorithm based on the observation that the disruption of the reticulin framework (highlighted by reticulin silverbased histochemical staining) is a consistent finding in ACC
and is only occasionally encountered in benign (Weiss score
≤ 2) adrenocortical tumors 11. This histochemical feature was
therefore considered as the primary step for the recognition of
167
relazioni
malignancy. The additional presence of at least one of the malignancy-related criteria conventionally considered in several
endocrine tumors (necrosis, mitotic rate >5/50HPF, and venous
invasion) raised up to 100% the sensitivity and specificity of the
proposed algorithm for a diagnosis of malignancy in a series of
92 cases. Very recently, this simplified scheme proved to be
highly reproducible also in a multicenter validation study 12.
The need of a grading system. Apart from diagnostic considerations, a critical reappraisal of the most relevant pathological
parameters in ACC diagnosis led to the recognition that some
of them have a prognostic impact being their expression also
correlated to survival. In this respect, a study by Assie and
coworkers on 124 metastatic cases 13 claimed the importance
of high mitotic index (and the presence of atypical mitotic
figures) as the most relevant factor significantly correlated
with clinical aggressiveness and poor survival. This same observation was also confirmed by other studies although with
heterogeneous cut off values 11 14 and supports the concept
that a grading system based on mitotic count might help in the
prognostic stratification of patients 15.
Molecular markers in the diagnosis and prognosis of ACC.
Apart from the prognostic impact of mitotic count already discussed above, none of the pathological parameters commonly
used for the definition of malignancy in adrenocortical tumors
is associated to a specific clinical behavior. Some immunohistochemical markers were shown to be independent predictors
of clinical aggressiveness, including Matrix Metalloproteinase
type 2 (MMP-2) 16, Glucose Transporter 1 (GLUT1) 17 and
Steroidogenic factor- 1 (SF-1) 18. However, none of them
has been validated enough to be considered a novel potential
prognostic biomarker in ACC. Ki-67 proliferation index can
provide additional help for establishing malignancy diagnosis,
although ACC with low proliferation index can exist and variability in Ki-67 staining procedure and interpretation might be
responsible for poor standardization and applicability of this
marker as a diagnostic tool, at least in the current practice.
References
1
Fassnacht M, Allolio B. Clinical management of adrenocortical carcinoma. Best Pract Res Clin Endocrinol Metab 2009;23:273-89.
2
van Slooten H, Schaberg A, Smeenk D, et al. Morphologic characteristics of benign and malignant adrenocortical tumors. Cancer
1985;55:766-73.
3
Weiss LM, Medeiros LJ, Vickery AL Jr. Pathologic features of prog-
nostic significance in adrenocortical carcinoma. Am J Surg Pathol
1989;13:202-6.
4
Aubert S, Wacrenier A, Leroy X, et al. Weiss system revisited: a clinicopathologic and immunohistochemical study of 49 adrenocortical
tumors. Am J Surg Pathol 2002;26:1612-9.
5
Lau SK, Weiss LM. The Weiss system for evaluating adrenocortical
neoplasms: 25 years later. Hum Pathol 2009;40:757-68.
6
VolanteM, Buttigliero C, Greco E, et al. Pathological and molecular features of adrenocortical carcinoma: an update. J Clin Pathol
2008;61:787-93.
7
Papotti M, Volante M, Duregon E, et al. Adrenocortical tumors with
myxoid features: a distinct morphologic and phenotypical variant
exhibiting malignant behavior. Am J Surg Pathol 2010;34:973-83.
8
Duregon E, Volante M, Cappia S, et al. Oncocytic adrenocortical
tumors: diagnostic algorithm and mitochondrial DNA profile in 27
cases. Am J Surg Pathol 2011;35:1882-93.
9
Tissier F, Aubert S, Leteurtre E, et al. Adrenocortical tumors: improving the practice of the Weiss system through virtual microscopy: a
National Program of the French Network INCa-COMETE. Am J Surg
Pathol 2012;36:1194-201.
10
Blanes A, Diaz-Cano SJ. Histologic criteria for adrenocortical proliferative lesions: value of mitotic figure variability. Am J Clin Pathol
2007;127:398-408.
11
Volante M, Bollito E, Sperone P, et al. Clinicopathological study of
a series of 92 adrenocortical carcinomas: from a proposal of simplified diagnostic algorithm to prognostic stratification. Histopathology
2009;55:535-43.
12
Duregon E, Fassina A, Volante M, et al. The Reticulin Algorithm for
Adrenocortical Tumor Diagnosis: A Multicentric Validation Study on
245 Unpublished Cases. Am J Surg Pathol 2013 Jun 14 [Epub ahead
of print].
13
Assie G, Antoni G, Tissier F, et al. Prognostic parameters of metastatic
adrenocortical carcinoma. J Clin Endocrinol Metab 2007;92:148-54.
14
Giordano TJ, Kuick R, Else T, et al. Molecular classification and
prognostication of adrenocortical tumors by transcriptome profiling.
Clin Cancer Res 2009;15:668-76.
15
Giordano TJ. The Argument for Mitotic Rate-based Grading for the
Prognostication of Adrenocortical Carcinoma. Am J Surg Pathol
2011;35:471-3.
16
Volante M, Sperone P, Bollito E, et al. Matrix metalloproteinase type
2 expression in malignant adrenocortical tumors: Diagnostic and
prognostic significance in a series of 50 adrenocortical carcinomas.
Mod Pathol 2006;19:1563-9.
17
Fenske W, Volker HU, Adam P, et al. Glucose transporter GLUT1
expression is an stage-independent predictor of clinical outcome in
adrenocortical carcinoma. Endocr Relat Cancer 2009;16:919-28.
18
Sbiera S, Schmull S, Assie G, et al. High diagnostic and prognostic
value of steroidogenic factor-1 expression in adrenal tumors. J Clin
Endocrinol Metab 2010;95:E161-71.
Sala Tarragona – ore 8.30-18.30
Patologia pediatrica
Moderatori F. Callea (Roma), F.M. Vecchio (Roma)
Pneumopatia da deficit di surfactante:
diagnostica ultrastrutturale e molecolare
R. Boldrini
Roma
Background. Il surfactante è costituito da fosfolipidi e proteine assemblati in corpi lamellari e secreti dagli pneumociti
alveolari di tipo II. Esistono 4 proteine associate al surfactant;
SP-A, SP-B, SP-C, SP-D ed un transporter di fosfolipidi: ABCA3. Mutazioni dei geni SP-B, SP-C e ABCA3 si associano a
patologie respiratorie anche gravi dell’età pediatrica.
Lo studio al microscopio elettronico di pazienti pediatrici con
patologie respiratorie rappresenta un buon mezzo per identi-
ficare questi disordini ereditari delle proteine del surfactante,
consentendo di effettuare una indagine genetica mirata.
In questi casi la microscopia ottica mostra una combinazione
variabile di pneumopatia interstiziale, caratterizzata da ispessimento dei setti interalveolari, diffusa iperplasia degli pneumociti di tipo II ed incremento dei macrofagi endo-alveolari.
Possono osservarsi in alternativa od in associazione al quadro
sopra-descritto, aspetti simil proteinosi alveolare con reperto
di materiale endo-alveolare PAS positivo.
La microscopia elettronica evidenzia una riduzione del numero dei corpi lamellari a livello del citoplasma apicale degli
pneumociti di tipo II ed anomalie morfologiche, talora molto
caratteristiche: mentre il reperto di corpi multivescicolari di
aspetto irregolare indirizza verso un deficit di SP-B, la pre-
168
senza di corpi lamellari piccoli, con “dense cores “eccentrici
suggerisce maggiormente un deficit di ABCA3.
Altre rare patologie polmonari dell’età pediatrica possono
essere identificate con l’ausilio della microscopia elettronica
quali la glicogenosi interstiziale polmonare, una pneumopatia
interstiziale recentemente decritta, caratterizzata dal reperto
di cellule mesenchimali contenenti glicogeno nell’interstizio
alveolare. L’eziologia è sconosciuta, ma l’ipotesi attualmente
più accreditata, anche in relazione ad un andamento clinico
generalmente favorevole ed ai benefici effetti della terapia
cortico-steroidea, è che si tratti di una patologia correlata
con un difetto di maturazione delle cellule interstiziali del
polmone.
Metodi. Vengono presentati i reperti clinici, istologici, ultrastrutturali e genetici di 9 pazienti dell’Ospedale Pediatrico
Bambino Gesù. La casistica studiata comprende 5 pazienti di
sesso maschile e 4 pazienti di sesso femminile che sono stati
ricoverati per distress respiratorio di grado severo associata
frequentemente ad ipertensione polmonare e quadro radiologico di interstiziopatia, sottoposti a biopsia polmonare a cielo
aperto per studio istologico ed ultrastrutturale.
Risultati. I quadri istologici mostravano aspetti variabili
caratterizzati da ispessimento dei setti alveolari in assenza di
infiltrazione infiammatoria, diffusa e marcata iperplasia epiteliale alveolare, presenza di aggregati di macrofagi alveolari
e talora presenza di materiale granulare PAS positivo endoalveolare. Un caso mostrava inoltre un quadro di anomalia
del letto vascolare polmonare riferibile a displasia alveolocapillare. In un caso l’ispessimento dei setti era dovuto ad
accumulo di glicogeno PAS positivo a livello delle cellule
interstiziali.
La microcopia elettronica ha evidenziato in 8 casi anomalie
dei corpi lamellari che variavano dal reperto di corpi multi
vescicolari irregolari a quello di corpi lamellari di piccole dimensioni con membrane addensate e “dense cores” eccentrici,
c. d. “fried egg”. In un caso l’aspetto ultrastrutturale dei corpi
lamellari risultava normale, mentre le cellule mesenchimali
fusate interstiziali evidenziavano un accumulo di glicogeno
monoparticolato che indirizzava verso la diagnosi di glicogenosi interstiziale polmonare.
Le indagini genetiche hanno evidenziato in 7 casi anomalie
correlabili con un deficit di ABCA3, in un caso anomalie
riferibili a deficit di SP-B, mentre nessuna anomalia è emersa
nel caso di glicogenosi interstiziale.
Conclusioni. I reperti ultrastrutturali associati con i difetti
di SP-B e ABCA3 sono altamente distintivi e consentono di
indirizzare verso indagini genetiche mirate ad identificare le
specifiche mutazioni.
La microscopia elettronica consente inoltre di identificare
anche altre rare patologie polmonari dell’età pediatrica quali
la glicogenosi interstiziale. Il riconoscimento di tale entità
e la diagnosi differenziale con le altre patologie respiratorie
pediatriche è importante, in relazione alla sua prognosi più
favorevole.
Patologia pediatrica
Moderatori: C. Gambini (Genova), V. Donofrio (Napoli)
Papillary carcinoma of the thyroid gland
in pediatric age: histopathologic and moleculargenetic characteristics
Tumori neuroblastici periferici: dall’istologia
alla genomica
P. Collini
UO Anatomia Patologica, Istituto G. Gaslini, Genova
Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Papillary carcinoma of the thyroid gland (PTC) is a rare tumor
in pediatric age, representing up to 3% of all pediatric cancers.
Among thyroid follicular-derived carcinomas, more than 90%
are PTCs, while follicular and poorly differentiated carcinomas
are exceptional, being anaplastic (undifferentiated) carcinoma
practically absent. In adult cases, some distinct morphologies,
such as the tall and columnar cell variants, mark a high-risk behaviour, with local and distant spread, and high mortality rates.
This fact seems not true in pediatric age, when survival is about
100% in spite of histology and stage. Very frequent is the solid/
trabecular variant of PTC, characterized by a high occurrence
of invasiveness into the neck soft tissue and nodal metastases
at onset. This variant has to be differentiated from the ominous
poorly differentiated carcinomas. A possible explanation for the
peculiar biologic behaviour of PTCs in pediatric ages has been
related to the higher rate of RET/PTC rearrangements, while
BRAF alterations are rare. The clinical and genetic characteristics of PTC arisen in pediatric age suggest the chance for a less
aggressive therapeutic approach.
Reference
Collini P Mattavelli F, Pellegrinelli A, et al, Papillary carcinoma of the
thyroid gland of childhood and adolescence: Morphologic subtypes,
biologic behavior and prognosis. Am J Surg Pathol 2006;30:1420-6.
A.R. Sementa, R. Defferrari, K. Mazzocco
I tumori neuroblastici periferici (PNTs, Peripheral Neuroblastic Tumours) sono un gruppo di neoplasie originate dal
sistema nervoso simpatico in via di sviluppo e rappresentano
i più comuni tumori solidi extracranici dell’infanzia. In Italia
si registrano circa 135 nuovi casi all’anno. Nell’80% dei casi
la malattia esordisce in età prescolare, con un’età media di
insorgenza intorno ai 17 mesi.
Secondo la International Neuroblastoma Pathology Classification (INPC), i PNTs sono classificati in quattro categorie:
Neuroblastoma (Schwannian stroma-poor), ganglioneuroblastoma, intermixed (Schwannian stroma-rich), ganglioneuroma
(Schwannian stroma-dominant) e ganglioneuroblastoma, nodular (composito, Schwannian stroma-dominant/stroma rich
e stroma poor). Nell’ambito della categoria “neuroblastoma”,
si riconoscono tre sottotipi: indifferenziato, scarsamente differenziato e differenziante.
I PNTs costituiscono un gruppo di tumori estremamente eterogeneo, dal punto di vista non solo istopatologico, ma anche
genetico-molecolare e clinico.
La grande variabilità determina un diverso comportamento
della malattia in base a caratteristiche quali età, istotipo, stadio. L’età del paziente alla diagnosi riveste un ruolo rilevante
per la prognosi: pazienti di età alla diagnosi inferiore ai 12-18
mesi mostrano una migliore sopravvivenza rispetto ai pazienti
più grandi (Fig. 1).
relazioni
Fig. 1.
I tumori neuroblastici sono suddivisi in differenti stadi in base
alla localizzazione, all’estensione del tumore primario e alla
presenza o meno di metastasi.
Nel 2009 l’International NB Risk Group Staging System
(INRSS) ha proposto una nuova metodologia di stadiazione
dei tumori neuroblastici periferici che tiene in considerazione,
oltre ai criteri sovraelencati, anche la presenza o meno di fattori di rischio chirurgico. I tumori neuroblastici sono risultati
raggruppati nelle seguenti categorie:
L1: tumore ad estensione locore-gionale che non presenta fattori di rischio chirurgico definiti radiologicamente (Image
Defined Risk Factors, IDRFs);
L2: tumore ad estensione locoregionale con presenza di uno o
più IDRFs;
M: metastasi a distanza (escluso stadio Ms);
Ms:metastasi a distanza a cute e/o cute e/o fegato e/o midollo
osseo (età < 12 mesi);
La sopravvivenza dei bambini in stadio localizzato risulta
superiore all’85%, mentre decresce drammaticamente nello
stadio M (ex stadio 4) attestandosi intorno al 40%.
Dal punto di vista genetico molecolare i PNTs sono caratterizzati dalla presenza di alterazioni cromosomiche numeriche e/o
strutturali. Mentre la presenza di alterazioni numeriche non
influenza negativamente la prognosi, diverso è il discorso per
quello che riguarda le alterazioni cromosomiche strutturali di
cui le principali sono: gain dei cromosomi 2p, 17q e 1q e loss
dei cromosomi 1p, 3p, 4p, 11q.
La prima alterazione genetica evidenziata nel neuroblastoma
nel 1984 è stata l’amplificazione dell’oncogene MYCN che
mappa sul cromosoma 2p24. Tale amplificazione, che si riscontra circa nel 20% dei neuroblastomi, con un incidenza che
aumenta fino a circa il 40% nei tumori a fenotipo più aggressivo, è considerata ancora oggi un potente fattore prognostico
sfavorevole.
Parallelamente all’amplificazione di MYCN, anche la delezione del cromosoma 11q, mutualmente esclusiva rispetto
all’amplificazione, correla con una cattiva prognosi, come si
può osservare nella Figura 2. Dalla figura si può chiaramente
vedere l’impatto negativo che hanno la amplificazione di
MYCN (MNA) e la delezione 11q (11q del) sulla sopravvivenza, rispetto alla presenza di alterazioni numeriche o altre
alterazioni strutturali. In una posizione intermedia si colloca il
gain del cromosoma 17q, spesso associato all’amplificazione
di MYCN.
L’incremento del numero di lavori scientifici in cui sono state
utilizzate tecniche di studio pangenomico, ha evidenziato
come le analisi dell’intero pattern genomico (piuttosto che
del singolo marcatore) siano essenziali per permettere l’iden-
169
Fig. 2. (da Caren H, Kryh H, Nethander M, et al. High-risk neuroblastoma tumors with 11q-deletion display a poor prognostic,
chromosome instability phenotype with later onset. Proc Natl
Acad Sci USA 2010;107:4323-8).
tificazione di diversi tipi di profilo di rischio in aggiunta alle
informazioni prognostiche dei markers convenzionali. Sulla
base di queste considerazioni, l’International Neuroblastoma
Risk Group (INRG) ha elaborato uno schema di trattamento
che tiene in considerazione, oltre a stadio, età alla diagnosi,
istologia e grado di differenziazione, anche lo stato di MYCN
e la presenza o meno di alterazioni strutturali ricorrenti nel
neuroblastoma (Tab. I).
Parallelamente alle indagini pangenomiche volte all’identificazione delle alterazioni strutturali nel NB, numerosi studi
sono incentrati sulla ricerca dei geni coinvolti e sul loro possibile ruolo nella tumorigenesi. Attualmente i geni MYCN e
ALK sono gli unici due oncogeni coinvolti nella genesi del
neuroblastoma.
Il gene MYCN appartiene alla famiglia MYC costituita da
fattori trascrizionali con struttura helix-loop-helix, che include
anche i geni c-MYC e MYCL. Questi fattori trascrizionali
regolano l’espressione di circa il 15% di tutti i geni. Il gene
MYCN forma un eterodimero con la proteina MAX e codifica
per un fattore trascrizionale prevalentemente espresso nello
sviluppo della cresta neurale. Questo fattore trascrizionale
controlla l’espressione di molti geni target, che a loro volta
regolano processi cellulari fondamentali, inclusi la proliferazione, la crescita cellulare, la sintesi proteica, il metabolismo,
l’apoptosi e la differenziazione. MYCN è vitale per proliferazione, migrazione e omeostasi delle cellule staminali mentre
livelli diminuiti sono associati a differenziazione neuronale.
Normalmente alti livelli di espressione di MYCN si associano
a un comportamento aggressivo del tumore e a ridotta sopravvivenza.
Il neuroblastoma è un tumore prevalentemente sporadico e
solo nell’1-2 % dei casi sono stati descritti casi familiari. Lo
studio di linkage nelle famiglie, seguito da sequenziamento
genico, ha portato ad evidenziare ALK come gene predisponente al neuroblastoma. ALK, localizzato sul cromosoma
2p23, è un recettore trans-membrana ad attività tirosinochinasica, le cui mutazioni causano un’attivazione costitutiva
del gene. Le mutazioni del gene ALK sono molto frequenti nel
neuroblastoma familiare (circa 80%), mentre sono piuttosto
rare (8%) nel neuroblastoma sporadico.
In seguito a studi recenti è risultato chiaro che ALK, coinvolto
in numerosi altri tipi di neoplasie, può rappresentare un target
per terapie innovative, in base all’inibizione selettiva della
170
Tab. I.
sua attività tirosino-chinasica. Attualmente presso il Children
Oncology Group è in corso un trial di fase 1 e 2 con l’utilizzo
di una piccola molecola inibitrice di ALK; si tratta del primo
agente diretto contro una specifica alterazione nel neuroblastoma, rappresentando il primo passo verso una terapia
personalizzata.
Negli ultimi anni molti gruppi si sono focalizzati sullo studio
dell’espressione genica, cercando di identificare delle signatures di espressione per la definizione dei gruppi di rischio. Una
delle prime pubblicazioni in questo campo ha riguardato una
signature di 59 geni per la stratificazione dei pazienti ad alto
rischio (Vermeulen et al, Lancet Oncology, 2009), seguita da
numerose altre, tra cui una signature riguardante i geni dell’ipossia, pubblicata da Varesio et al nel 2011.
Nel laboratorio di Anatomia Patologia dell’Istituto G. Gaslini
di Genova, centro italiano di riferimento per il neuroblastoma
vengono centralizzati campioni dei tumori della quasi totalità
dei pazienti italiani affetti da neuroblastoma e, con frequenza
in aumento, di pazienti provenienti da paesi esteri. Questo
materiale è analizzato dal punto di vista istologico al duplice
scopo di uniformare – quando necessario- la diagnosi agli
standards classificativi in uso internazionale fornendo i parametri istopatologici di significato prognostico e di certificare
la validità del campione utilizzato per le indagini biomolecolari. Si effettua inoltre la valutazione della ploidia tramite citofluorimetria, la determinazione tramite Ibridazione Fluorescente in Situ (FISH) dello stato dei cromosomi 1, 2, 3, 11, 17,
lo studio delle principali alterazioni cromosomiche strutturali
tramite Multiplex Ligation Probe Amplification (MLPA). Oltre alle indagini immunoistochimiche comunemente eseguite
a supporto diagnostico, si allestiscono colorazioni immunoistochimiche per MYCN e ALK.
Presso l’Istituto Gaslini è inoltre attiva una Biobanca Integrata
Tessuto- Genomica (BIT).
Per quello che riguarda la Biobanca dei Tessuti, l’esperienza
IGG nasce dalla presenza dell’anatomo-patologo al fianco
del chirurgo. I materiali bioptici e di exeresi sono esaminati
dall’anatomo-patologo che li campiona e avvia alla processazione e alla criopreservazione. Il patologo poi, nella sua
funzione diagnostica, certifica la conformità dei campioni ai
requisiti della Biobanca.
Il corretto campionamento del tumore è tappa fondamentale
per lo studio della neoplasia, in quanto garantisce la valutazione istologica e fornisce, al laboratorio che si occupa di studi
genetici e molecolari, materiale selezionato e correttamente
inquadrato.
La sezione Genomica della Biobanca Integrata si occupa della
gestione, lavorazione e conservazione del materiale tumorale
tramite estrazione di acidi nucleici e proteine. Parallelamente
alla conservazione, classificazione e distribuzione del materiale, vengono realizzati controlli di qualità e raccolta di dati
molecolari da inserire nella Biobanca. La condivisione dei
dati raccolti nella Biobanca Integrata consentirà di ottimizzare
e moltiplicare gli studi e le conoscenze su questo importante
gruppo di tumori anche a partire da un campione estremamente
limitato.
171
relazioni
Incontro sui controlli di qualità fish
Moderatori: A. Sapino (Torino), C. Clemente (Milano)
Presentazione dei risultati
L. Verdun di Cantogno
Dip. di Medicina di Laboratorio, Sez. di Anatomia Patologica, Città
della Salute e della Scienza, presidio Molinette, Torino
Il Controllo di Qualità (CQ) per la determinazione dello stato
di HER2, tramite metodiche di ibridazione in situ (FISH,
CISH, SISH) nasce nel marzo 2008 dal CQ Regionale per i
fattori prognostici predittivi della mammella, finanziato dalla
rete oncologica 2006-2008. L’esigenza, per i primi 9 centri
che vi partecipano, è quella di potersi confrontare su tre punti
fondamentali: allestimento del preparato, lettura ed interpretazione del risultato. La scelta ricade su di un sistema “a ring”
(Fig. 1) per due motivi: è l’unico che consenta di valutare
anche la fase pre-analitica (fissazione e taglio della sezione),
non grava su nessun centro in modo particolare (eccezion fatta
per il centro che si occupa della raccolta ed analisi dei dati).
L’iniziativa ha ottenuto da subito ampi consensi e si è passati
rapidamente dagli iniziali 9 centri aderenti, solo del nord/
centro Italia, ai 55 centri attuali che coprono tutto il territorio
nazionale.
Attualmente il CQ è così organizzato:
– i 55 centri partecipanti sono suddivisi in 5 gruppi: A, B, C,
D, E. Ogni gruppo può arrivare ad avere un massimo di 12
centri; oltre tale soglia si forma un nuovo gruppo, questo
perché altrimenti diventerebbe problematico trovare un
campione dello spessore sufficiente da poter essere inviato
a tutti i componenti del proprio gruppo;
– ogni mese un centro a turno (secondo un preciso calendario)
invia agli altri partecipanti del proprio gruppo 3 sezioni in
bianco su vetrino di un caso appositamente selezionato. Fatto il necessario per valutare lo stato di HER2 sul campione
in esame, (EE, ovviamente FISH ed eventualmente IHC), i
partecipanti devono compilare un datasheet elettronico, presente in un sito appositamente allestito per il CQ, al quale si
ha accesso tramite password. I dati richiesti sono: numerici
(numero nuclei letti, media HER2/nucleo, media CEP17/
nucleo e Ratio HER2/CEP17), ed interpretativi: valutazione
dello stato di HER2 (amplificato vs non amplificato) e di
CEP17. Per una interpretazione di HER2 (e CEP17) più
accurata lo stato di “non amplificato” comprende: gene (e/o
CEP17) nella norma (NN) per medie/nucleo > 1,8 e < 3,
Gain del gene (e/o CEP17) per medie/nucleo > 3 ma < 6 e
infine Loss del gene (e/o CEP17) per medie < 1,8. Èpossibile inoltre, nei campioni eterogenei, segnalare la % delle
cellule amplificate. In ultimo un’apposita casella invita ad
esprimere un giudizio sulla qualità della sezione esaminata
tra ottimo, buono, sufficiente ed insufficiente;
–l’invio del caso non viene effettuato solo nei mesi di Maggio e Agosto;
– nel mese di Maggio viene inviato a centri unificati un CMA
(Cell Macro Array) allestito con linee cellulari di carcinoma
della mammella che presentano stato di HER2 amplificato e
non amplificato;
–ogni due mesi i singoli centri ricevono via mail una tabella riassuntiva dove, tramite un codice identificativo
personale (ID), possono valutare il proprio operato confrontandolo con quello degli altri componenti del gruppo
cui appartengono;
Fig. 1. Schema a ring del CQFISH HER2 su carcinoma mammario.
Ogni mese, tranne che a Maggio e ad Agosto, un centro a turno
invia agli altri partecipanti un caso da analizzare. I dati ottenuti
vengono inseriti in un database condiviso.
–al termine di ogni anno di attività viene rilasciato un attestato di partecipazione che comprende una valutazione generale dell’operato in termini di concordanza sullo stato di
HER2 (amplificato/non amplificato), ed una valutazione più
dettagliata che tiene conto della sottostima/sovrastima delle
medie di HER2 e CEP17, della costanza di partecipazione
al CQ e della qualità delle sezioni inviate.
Oltre a tutto ciò, ogni 2 anni circa, si organizza un incontro
(accreditato ECM) tra tutti i partecipanti al CQ FISH HER2
in cui si presentano i risultati ottenuti: in questa sede si analizzano le diverse problematiche inerenti alla determinazione
di HER2: la metodica, la lettura, l’interpretazione, la stesura
del referto. Lo scopo è quello di individuare una linea di
azione condivisa, per rendere sempre più attendibili i risultati
ottenuti. In questo contesto ad un centro che risultasse avere
delle criticità viene fornito l’aiuto necessario per migliorare le
proprie performance di qualità.
Vengono qui presentati i risultati di concordanza del primo
CMA a centri unificati (CMA01-2013) e, a seguire, i risultati
dei casi CQ dal 47 al 59 (12 casi, un anno di attività) dei
gruppi A, B, C, D ed E (questo gruppo si è appena formato ed
è valutabile per soli due casi).
Il CMA01-2013 era costituito da 3 linee cellulari (linea I:
SKBR3, amplificata; linea II: T47D, non amplificata; linea
III: MCF7 non amplificata e con CEP17 > HER2). La partecipazione è stata alta, il 93% (51 su 55) dei centri che ha
ricevuto i campioni ha partecipato, mentre solo il 6% (3 su
51) ha ritenuto non valutabili i casi inviati (1, 2 o tutti e 3). La
concordanza sullo stato di HER2, amplificato vs. non amplificato, è stata del 100% (Fig. 2).
È stata poi effettuata una stima più dettagliata che tenesse conto, oltre che dello stato di HER2, anche dei singoli parametri
che hanno portato alla valutazione finale (Tab. I).
Con questi criteri di valutazione il punteggio massimo ottenibile dal singolo caso è 20 (10 per la concordanza amplificato/non
amplificato + 10 se anche tutti gli altri parametri sono corretti);
quindi il punteggio massimo per un CMA corretto è di 60 (20x3
casi valutabili). Si ottiene così una valutazione in 60esimi che,
espressa in decimi, genera un punteggio da 10 a 6 con cui valu-
172
Fig. 2. Valutazione della concordanza dello stato di HER2 amplificato (AMPL) vs non amplificato (GAIN, NN, LOSS) nelle 3 linee
cellulari del CMA01, analizzate dai 51 centri partecipanti. Linea
cellulare I amplificata, II non amplificata, III non amplificato. La
concordanza è risultata del 100%. NV: non valutabile.
Per quanto riguarda l`analisi dei dati relativi ai casi CQ dal
47 al 59, eseguita da 55 centri suddivisi in 5 gruppi, i risultati
sono i seguenti:
a)partecipazione: ponendo il cut off di partecipazione al 75%
dei casi da valutare, l`89% dei centri ha raggiunto e/o superato tale obiettivo; un solo centro si è iscritto senza mai
partecipare,
b)qualità del preparato: la stima di questo parametro ha reso
necessario individuare dei criteri di valutazione, esprimibili
con un punteggio. Si è optato per il calcolo della media
ponderata dando un valore numerico ai giudizi ricevuti
(ottimo = 3, buono = 2, sufficiente = 1, insufficiente = 0).
Individuando successivamente dei cut off per i valori delle
medie ponderate, si è potuta valutare la performance dei
singoli centri: il 25% ha un’ottima prestazione mentre più
del 50% buona (Fig. 4).
Fig. 4. Valutazione della qualità del preparato inviato, calcolata
per i soli 36 centri che hanno già inviato almeno un caso da
analizzare.
tare più sottilmente le performance dei 51 centri: tali punteggi,
per il 76% dei partecipanti, sono risultati compresi tra 10 e 8
evidenziando perciò ottimi risultati (Fig. 3).
Fig. 3. Valutazione dettagliata: percentuali dei punteggi ottenuti
dai 51 centri partecipanti.
a)Concordanza amplificato vs non amplificato: questo parametro è stato valutato per ogni centro, nell’ambito del gruppo di appartenenza, mediante percentuale dei casi risultati
conformi. Il cut off è pari al 90% (ASCO/CAP 2008). Il
dato è stato confermato mediante il calcolo della costante di
concordanza di Cohen-Fleiss:
Tab. I.
Tipologia di parametro
Parametri diagnostici
Codice
Cosa valuta
Punti
A/NA!?
stato di HER2 corretto/errato riferito ad Ampl./Non Ampl.
10/0
OK
tutti i dati del caso corretti
10
non valutabile
interpretazione numerica CEP17/HER2 non conforme
(riferito a Gain- Loss- Nella Norma)
caso ritenuto non valutabile
(10) -4
CEP17/HER2
sottostima rispetto alla media di CEP17/HER2
(10) -3
CEP17/HER2
sovrastima rispetto alla media di CEP17/HER2
(10) -3
no dati
dati di tutto il caso non pervenuti
(10) -5
compilaz.
errori o omissioni di compilazione/dati non conformi (per es. no
medie segnale/nucleo)
(10) -2
int. HER2/CEP17
Parametri tecnici/lettura
Parametri di partecipazione
Nota: se presenti più errori, il loro valore si somma. Il valore minimo dato ad un caso concorde è 3.
(10) -5
173
relazioni
Tab. II.
Gruppo
N. casi % concordanza
valutati ampl. vs. non ampl.
A
B
C
D
E
12
12
12
12
2
91%
91%
100%
100%
100%
K di CohenFleiss
(p < 0,05)
0,83
0,87
0,91
0,96
NV
Giudizio
sulla k
ottima
ottima
ottima
ottima
NV
NV: non valutabile
b)Valutazione dettagliata: come per il CMA anche per le
sezioni è stata effettuata una stima più dettagliata che
tenesse conto, oltre che dello stato di HER2, anche dei
singoli parametri che hanno portato alla valutazione finale
dello stesso:
Gruppo
Valutazione dettagliata (cut off 7,0)
A
B
C
D
E
91%
91%
82%
91%
NV
In questo contesto, utilizzando i sopra citati criteri di valutazione, il punteggio massimo attribuibile al singolo caso è 10;
ogni centro quindi può ottenere al massimo 120 punti (10x12
casi valutabili). Si ottiene così una valutazione in 120esimi
che, espressa in decimi, genera un punteggio da 10 a 6 con cui
stimare più sottilmente le performance dei 55 centri (Tab. II).
L’analisi delle prestazioni valutate sulle 12 sezioni (casi
47-59) conferma l’ottima performance complessiva dei vari
gruppi; tuttavia si individuano alcune situazioni di maggiore
difficoltà che andranno monitorate, allo scopo di raggiungere
un livello qualitativamente elevato in tutti i laboratori che si
occupano di questo tipo di indagini molecolari.
Lunedì, 28 ottobre 2013
Patologia digestiva
Moderatore: R. Fiocca (Genova)
Molecular predictors of response to targeted
therapy in the gastrointestinal tract
F.T. Bosman
University Institute of Pathology, Lausanne, Switzerland
It is not all that long ago that the work-up of a resection specimen of a malignant neoplasm in the gastrointestinal (GI) tract
required adequate macroscopical examination and sampling,
histomorphological examination, in case of need immunohistochemical staining – altogether culminating in a pathology
report with tumor type, grade and stage parameters as essential elements. That is rapidly changing. The basic workup of
these specimens does not change but the advent of targeted
therapies now requires essential additional information: which
cases run a high risk of recurrence (as they might require adjuvant therapy) and of those cases, which are the most likely to
respond to targeted therapy? Both issues require the development of reliable biomarkers, which are mostly molecular in
nature. We will briefly discuss which markers in the GI tract
neoplasms are under investigation and which have risen to the
status of standard of care.
Esophageal cancer. The treatment of esophageal carcinoma
remains a challenge. This is alike for squamous cell carcinoma
and adenocarcinoma, although the optimal modalities are different for these two main categories. For patients with locally
advanced squamous cell carcinoma neoadjuvant chemoradio-
174
therapy is the treatment of choice. A variety of studies has
addressed the question as to which parameters, such as p53,
proliferative cell nuclear antigen, EGFR, Ki-67, cyclin D1,
expression of thymidylate synthase, and microvessel density,
might predict response but none of these has resulted in useful
predictive markers. Complete pathological response, however, is a strong predictor of prolonged survival. For a subset
of patients chemoradiotherapy even appears to be definitive.
Neoadjuvant chemo(radio)therapy is also provided to patients
with locally advanced adenocarcinoma. Also for adenocarcinomas predictive biomarkers are lacking. Targeted therapy
has been tested for esophageal carcinoma only in the palliative setting. The use of the monoclonal antibody trastuzumab
in has proven effective in HER2 positive gastroesophageal
junction cancer. Addition of anti-VEGF-R and anti-EGFR
antibodies has shown some promise but this remains under
investigation, including eventual response predictive markers.
Gastric cancer. For gastric cancer, targeting of over-expressed HER2 with trastuzumab has been rather successful.
About 20% of gastric cancers overexpress HER2, using immunohistochemistry and fluorescence in situ hybridization
as detection methods in modalities similar to those in routine
use in breast cancer. Of HER2 overexpressing/amplified
gastric cancers, up to 50% will respond to a chemotherapy
regimen including trastuzumab. Additional targets are being
investigated. In addition various studies address the question
as to more accurate response predictive markers. As yet, other
targeted therapies in gastric cancer have fallen short or are
still in early clinical development. These include approaches
targeting FGFR2, MET and EGFR. Abnormalities in these
driver genes have a much lower frequency in gastric cancer
(although not finally established, all between 5 and 10%) and
therefore trials to test new therapeutic approaches are complicated, as are studies regarding response predictive markers.
In addition, optimal methods to define these abnormalities
in a clinical setting need to be defined. As an example, the
frequency of MET amplifications in gastric cancer varied
between 4 and 20%, depending on the method used.
Gastrointestinal stromal tumour (GIST). In the diagnosis
of GIST’s immunohistochemistry plays a major role as they
are mostly CD117 positive and almost all DOG1 positive.
CD117 represents c-KIT, the stem cell factor receptor. The
large majority of GIST’s has a mutation in c-KIT and the
remaining often a mutation in the PDGFR-A gene. However,
about 15% of the GIST’s is not mutated in either of these
genes and for these the search for other responsible genes is
still on. Low risk GIST’s need no further therapy after surgery. Advanced GIST’s are treated with the tyrosine receptor
kinase inhibitor imatinib. The use of imatinib as adjuvant
therapy for intermediate risk GIST’s is presently under investigation. The presence of KIT or PDGFRA mutations as well
as the type of mutation are predictive of response to imatinib.
Mutations in the juxta-membranous domain of c-KIT in exon
11 are most common. These have the best response rates
to imatinib, with a longer survival rates. Exon 9 mutations
in the c-KIT extracellular domain are specific for intestinal
GIST, are less responsive to imatinib and patients with such
mutations receive a higher dosage of the drug and yet have a
poorer progression free survival. PDGFRA mutations, usually
in exon 18 affecting the tyrosine kinase domain, are more
frequent in gastric GIST.
Small intestinal carcinoma. These tumours are relatively
rare, might behave like colorectal carcinomas but there is insufficient evidence for this notion. Large collaborative studies
are needed to elucidate optimal therapeutic modalities, including targeted therapy, for small intestinal carcinomas.
Colorectal cancer. The treatment of advanced colorectal
cancer has significantly changed with the introduction of
anti-EGFR antibody therapy (cetuximab, panitumumab). The
discovery that by and large KRAS mutated carcinomas do not
respond to this targeted approach has had a major impact on
the development of molecular pathology laboratories. KRAS
mutation testing has rapidly become one of the most frequently performed tests. Whether all KRAS mutations confer
resistance (some evidence suggests that the G13D mutation
does not and for KRAS mutations outside exon 2 insufficient
data exist) remains to be solved. KRAS mutations, however,
confer resistance but only about 40% of KRAS wild type carcinomas will respond. More effective predictors of response are
therefore needed. Some evidence exists that a combination of
molecular markers (in addition to KRAS, BRAF, NRAS, PIK3CA and PTEN, among others, have been tested) considerably
increases the predictive value regarding response. An additional issue is the identification of those patients that might
benefit from adjuvant therapy. As yet, stage III and high risk
stage II (notably T3/4 tumours) patients are offered adjuvant
treatment but a significant proportion of these patients would
not have relapsed without adjuvant treatment and of those that
do relapse only about 50% responds to the usual 5-FU/LV
treatment regimens. The use of markers such as microsatellite
instability and SMAD4 expression might be useful in defining
which patients might benefit most from adjuvant treatment.
Patologia digestiva
Moderatore: M. Rugge (Padova)
Corso breve: processo diagnostico
nello screening del carcinoma colo-rettale:
dall’invio del ampione biologico
alla refertazione
Unit of Pathology, IRCC, Candiolo-Torino; 2 Division of Pathology,
IEO, Milan; 3 Department of Biomedical Sciences and Human Oncology, University of Turin; 4 Department of Medical and Surgical Critical Care, Section of Pathological Anatomy, University of Florence;
5
AOU S. Giovanni Battista, CPO Piemonte, Turin, Italy
Coordinatore: M. Risio (Torino)
Screening is an important tool in cancer control in countries
with a significant burden of colorectal cancer (CRC), provided
the screening services are high quality. Pathology plays a key
role in CRC screening since the management of participants
in the programme depends on the quality and accuracy of the
diagnosis, which affects the decision to undergo further local
and/or a major resection as well as surveillance after screening.
Pathology in Colorectal Cancer Screening
Programmes: from Specimen Handling
to Histology Report
M. Risio1, A. Sonzogni2, O.Cassoni3, L. Messerini4, C. Senore5
1
relazioni
The range of pathology differs between the different programmes, with faecal occult blood-based programmes yielding more advanced disease than flexible sigmoidoscopy and
colonoscopy screening. Programme activities must focus on
the identification and appropriate management of invasive
colorectal cancer and its precursors. The management of preinvasive lesions involves surveillance to allow the prevention
of future disease, whereas management of adenocarcinoma
focuses on immediate treatment and decisions on local removal, or radical surgery with the potential for operative mortality.
Overuse of radical surgery must be avoided and recommendations for its use must be balanced with the risks to the patient.
Efforts are aimed at developing common diagnostic standards
to ensure quality, recognising areas where sufficient evidence
is still lacking, and initiating high quality studies to answer
these questions 1.
Management of endoscopic specimens: the interplay between
pathologist and endoscopist. The performance and handling of
tissue specimens obtained from endoscopic removal is a major
prerequisite to produce ultimate and accurate diagnoses in
colorectal cancer screening programs according to the axiom
“the best the handling of biopsies, the best the final report”.
Therefore, a close interplay is needed between pathologist and
endoscopist to optimize the management of endoscopic tissue
specimens 2. It is recommended to place complete specimens
in separate containers, one for each lesion, to avoid missing
the exact anatomical locations and offer pathologist the most
complete information about the samples being removed: orientation of small biopsies or polyps, identification of resection
margins for stalked or semi-stalked polyps and endoscopic
mucosectomy specimens, which should be pinned out by inserting pins at the periphery of them onto cork or thick paper.
The pathologist will have to accurately describe the gross
appearance of biopsies and sample them according to widely
agreed-upon procedures in order to allow the precise measurements of submucosal neoplastic extension, and distances from
deep and radial resection margins.
It is recommended to cut at least three hematoxylin&eosinstained sections for each paraffin block, with additional sections being prepared whenever deemed necessary 3.
Adenomatous and Non-adenomatous Colorectal Polyps: “Hot
Spots”. An objective, consistent, and reproducible histology featuring advanced adenoma is a prerequisite to fulfil
CRC screening aims. A first step towards achievement of a
substantial agreement in the histological identification of advanced adenomas was obtained by collapsing finer histologic
in broader diagnostic categories (i.e.: any villous component
> 20% vs. tubular; low versus high grade dysplasia). Still,
these “easier” binary systems account for an unsatisfactory
diagnostic inter-observer concordance, due to the presence of
gray areas (such as confounding histology conformation of
villi or extent of dysplasia) which cannot escape to subjective
interpretation. Recent data documented a persisting suboptimal agreement among colorectal cancer screening pathologists, with a potential impact upon patient management and
the efficient running of the screening service itself 4.
European guidelines for quality assurance provided indicators
(such as the expected maximum percentage of advanced adenomas among diminutive polyps) useful to “set” and address
the diagnostic criteria and limit misinterpretation with consequent under/overtreatment 1. However, additional complexity
in adenomas categorization based on controversial criteria
useful to achieve a satisfactory “serrated” morphologic algorithm effectively matching gland histology and estimated
biological evolution. Again, European guidelines attempted
175
to increase concordance by grouping serrated polyps in two
categories, distinguishable by the presence/absence of nuclear
dysplasia. Although apparently this should have improved
diagnostic agreement the efficacy of this simplified approach
probably does not correspond to an effective tool for a predictive stratification and needs to be optimized 5.
Cancerized adenomas and advanced colorectal adenocarcinoma: “Hot Spots”. Colorectal carcinoma invading the submucosa but not the muscular layer (pT1, early invasive cancer)
represents the earliest form of clinically relevant colorectal
cancer in most patients. Neoplastic invasion of the submucosa, in fact, opens the way to metastasis via the lymphatic
and blood vessels, and the choice between surveillance and
major surgery will turn on its metastatic potential. Adenomas
showing cancer cells confined to the mucosa are not considered malignant polyps and are classified as adenomas with
high grade dysplasia, high grade intraepithelial neoplasia or
mucosal high grade neoplasia 6.
The following histological features predict the risk of metastasis and the different clinical outcomes: grade of differentiation of carcinoma, lymphovascular invasion, and state of the
resection margin. Microstaging of invasive cancer, namely
the width and the depth of submucosal invasion together with
tumor budding at the advancing edge allow the metastatic risk
to be further stratified in minimal, low, and high. Despite their
proven prognostic value, there is a great heterogeneity in reporting, and there is no consensus on the assessment methods
and cut-off values 7, biassing or limiting their use in screening programmes. Diagnostic reproducibility is significantly
improved when using quantitative and selective methods of
evaluation, which are presently under investigation.
Screening should result in a reduction in the overall population incidence of late stage colorectal cancer to be used as an
early indicator of effectiveness: completeness, accuracy and
reproducibility of pathological staging are therefore required
to allow trends to be studied.
Translating Pathology Report into Screening Data Base. The
development of comprehensive systems for documentation
of the screening processes, monitoring of data acquisition
and quality, and accurate compilation and reporting of results are essential to the evaluation of population screening
programmes. Evaluation and interpretation of screening
outcomes, in turn, are essential to recognising whether a
colorectal cancer screening programme is achieving the
goals for which it has been established. Among the programme outcome variables, any pathological specimen
removed or biopsied at endoscopy or surgery should be
reported and tables presenting the following performance
indicators should be produced at regular intervals 8: 1) Lesion detection rate; 2) Adenoma detection rate; 3) Advanced
adenoma detection rate. In screening programmes the use
of the term “advanced adenoma” has developed, indicating
an adenoma that is either ≥ 10mm or contains high-grade
mucosal neoplasia or a villous component. Advanced adenomas detection rates of 4.9% to 8.6% have been reported in
population studies of colonoscopy; 4) Cancer detection rate;
5) Stage of screen-detected cancers. The staging of colon
cancer should use firstly the international TNM classification and secondly the Dukes classification.The proportion
of screen-detected cancers that were Dukes Stage A ranged
26-36%. A favourable stage distribution in screen-detected
cancers compared to clinically diagnosed cancers should be
observed: in the absence of this condition a screening programme could not be effective.
176
References
1
Quirke P, Risio M, Lambert R, et al. Quality assurance in pathology in
colorectal cancer screening and diagnosis—European recommendations. Virchows Arch 2011;458:1-19.
2
Cooper HS. Pathologic issues in the treatment of endoscopically
removed malignant colorectal polyps. J Natl Compr Canc Netw
2007;5:991-6.
3
Nash WJ, Niemann T, Marsh WL, et al. To Step or not to step. An
approach to clinically diagnosed polyps with no initial pathologic
finding. Am J Clin Pathol 2002;117:419-23.
4
Turner JK, Williams GT, Morgan M, et al. Interobserver agreement
in the reporting of colorectal polyp pathology among bowel cancer
screening pathologists in Wales. Histopathology 2013;62:916-24.
5
Bettington M, Walker N, Clouston A, et al. The serrated pathway to
colorectal carcinoma: current concepts and challenges. Histopathology 2013;62:367-86.
6
Lanza G, Messerini L, Gafà R, et al.; Gruppo Italiano Patologi Apparato Digerente (GIPAD); Società Italiana di Anatomia Patologica e
Citopatologia Diagnostica/International Academy of Pathology, Italian division (SIAPEC/IAP). Colorectal tumors: the histology report.
Dig Liver Dis 2011;43S:S344-55.
7
Risio M. The natural history of pT1 colorectal cancer. Front Oncol
2012;2:1-5.
8
Moss S, Ancelle-Park R, Brenner H. Evaluation and Interpretation of
screening outcomes. In: Segnan N, Patnick J, von Karsa L, eds. European Guidelines for quality assurance in colorectal cancer screening
and diagnosis. Lyon: IARC WHO 2010.
Sala Massalia – ore 8.30 - 18.30
Dermatopatologia
Dermatopatologia I sessione: novità sulle lesioni melanocitiche
Coordinatori: C. Clemente (Milano), G. Botti (Napoli)
Melanoma: interazione tumore-microambiente
G. Botti, P. Ascierto, M. Cantile, G. Scognamiglio, S. Staibano*
S.C. Anatomia Patologica e Citopatologia, Istituto Nazionale Tumori
IRCCS Fondazione G. Pascale, Napoli; * Dipartimento di Scienze Biomediche Avanzate, Università Federico II di Napoli
The formation and “transformation” of a tissue is guaranteed
by a dynamic interaction between their cells and the microenvironment, which is fundamentally made of extracellular
matrix and other cytotypes (immune cells, fibroblasts, and
vascular cells) (Fig. 1).
The out-of-control cell growth and the loss of cellular homeostasis play a key role in the genesis and progression of several
human cancers, and malignant melanoma certainly represents
the human disease more representative in terms of tumor progression process.
More than other tumors, melanoma also has an immuneFig. 1. Melanoma cell microenvironment.Interaction between
immune cells and ECM proteins (from Botti et al., 2013).
modulate behavior, which means that its entire pre-clinic
and clinic evolution is controlled by the patient’s immune
system. Even melanoma presentation (i.e., superficial diffusion or nodular type, characterized by an early and fast
vertical growth phase) reflects a high or low capacity of
the immune system to contain and control the spread of
tumor cells.
Considering the role of different lymphocyte subpopulations in antitumor surveillance, macrophages (TAM, “tumorassociated macrophages-”), recognized by the presence of
the antigenCD68 on their surface, are responsible directly
or indirectly, of the effect suppressor against the activated
T lymphocytes on tumor cells. The tumor-associated macrophages represent an essential component of tumor infiltrating
leukocytes and possess pro-tumor and anti-tumor properties,
with the first one that prevails in tumors. For a long time it
is known that macrophages and some of their products(IL-1,
TNF, IL-6) promote the metastatic process. The acquisition of
the pro-tumor functions is stimulated by cytokines produced
by the tumor microenvironment, inparticularIL-10, PGE2,
TGF-β andCSF-1.
The quantitative evaluation of macrophages in the context
of the tumors howed that a high representation is correlate with shorter survival of patients.This is due to the
establishment, by these cells, of a microenvironment that
favors the neo-vascularization and the development of a
fibrotic reaction, through the production of fibro-angiogenic
factors such as VEGF(VascularEndothelial GrowthFactor),
bFGF(basicFibroblastGrowth Factor)and TNF-α (Tumor Necrosis Factor-α). In vivo, macrophage infiltration correlates
positively with the depth of tumor infiltration, lymph node
involvement and clinical stage. Macrophages also secrete
immunosuppressive cytokines, that indirectly affect on tumor
infiltrating lymphocytes.
Regarding dendritic components(CD83 +), which are cells
that present antigens to CD4 + T lymphocytes(APC), their
number in the context of neoplasia is inversely correlated with
the depth of tumor invasion and the presence of lymph node
metastases. A reduced number of DCis, in fact, correlated
with a worse prognosis.
The regulatory T cells(CD4+/CD25+) have an inhibi-
relazioni
tory effect against the immune response mediated by
T cells CD4+/CD25-and CD8 + T cells, as they produce INF and secrete large quantities of IL-10, one of
the factors most responsible of immunosuppression.
The Natural Killer(CD57 +) are cells of the immune system
that actin a non specific manner against cancer. The infiltration of NK cells is, therefore, inversely related to the depth of
tumor invasion, clinical stage and the venous and lymphatic
invasion. The survival also appears to be significantly better
in patients with increased infiltration of NK cells.
Finally, the cytotoxic T lymphocytes(CD8 +) play a crucial
role in limiting the growth and spread of the tumor.
In light of these observations, the identification and localization of immune cells within the tumor tissue(intratumoral/
intraepithelial, peritumoral or stromal), commonly defined
as “immunoscore” is acquiring an important diagnostic and
prognostic value in melanoma disease. In fact, on the basis
of the location, the individual cell subpopulations can be exposed to the action of multiple substances, especially mediators of inflammation, which can alter their activity.
Moreover, in melanoma metastatic phenotype induction
process many changes are called into question in terms of
number and type of adhesion molecules and therefore in the
cells ability to bind one to the other and to the extracellular
matrix constituents.
Melanoma invasive capability takes place when their cells
are able to produce some molecules providing the genesis of
the vascular system. In this context, a very important role is
performed by the proteins of extracellular matrix.
The extracellular matrix (ECM) is comprised of fibrous proteins, glycos-aminoglycans, proteoglycans, and other structural glycoconjugates. Many of these, including osteopontin,
osteonectin, tenascin C, CCN3, and several MMP and integrins, are associatedmainly to melanoma progression.
Infact, more recently, a gene expression profiling of primary,
non-metastatic cutaneous tumors and metastatic melanoma
has resulted in the identification of several genes that may be
centrally involved in the progression and metastatic potential of
melanoma identifying several ECM molecules as essential in
this process. In particular, SPARC, involved in matrix remodeling during tumor progression and widely studied in melanoma
pathogenesis, shows an altered expression in cellular models of
melanoma characterized by an increased aggressiveness.
Recently, we demonstrated SPARC aberrant expression in
primary melanomas that preferentially metastasize to lungs. In
other sites, such as lymph node metastases or those associated
to the gastrointestinal tract, SPARC expression was very low
or even absent in both primary and corresponding metastasis
(Fig. 2).
Our data lead us to hypothesis that SPARC plays a role in predisposing some primitive tumors to preferentially metastasize
to the lungs compare to other anatomical sites.
In conclusion, it was profusely demonstrated that ECM
molecules through specific interactions with immune cells,
can regulate their migration and activity in tumor microenvironment.
177
Fig. 2. C) immunopositiveprimary melanoma (1) and immunopositive lung metastasis (2); D) immunonegativeprimary melanoma (1) and negative intestinal metastasis (2) (Botti et al., 2013).
This cascade of events, although common in all tumors,
plays a particular important role in melanoma progression.
References
Adair-Kirk TL, Senior RM. Fragments of extracellular matrix as mediators of inflammation. Int J Biochem Cell Biol 2008;40:1101-10.
Allavena P, Sica A, Solinas G, et al. The inflammatory micro-environment
in tumor progression: the role of tumor-associated macrophages. CritRevOncolHematol 2008;66:1-9.
Ascierto PA, Capone M, Urba WJ, et al. The additional facet of immunoscore: immunoprofiling as a possible predictive tool for cancer
treatment. J Transl Med 2013;11:54
Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow?
Lancet 2001;357:539-45.
Bancherau J, Steinman RM. Dendritic cells and the control of immunity.
Nature 1998;392:245-52.
Botti G, Cerrone M, Scognamiglio G, et al. Microenvironment and tumor
progression of melanoma: New therapeutic prospectives. J Immunotoxicol 2013;10:235-52.
Botti G, Scognamiglio G, Marra L, et al. Sparc/osteonectinleadsmetastaticprocessto thelungduringmelanoma progression. Submitted.
Braeuer RR, Zigler M, Villares GJ, et al. Transcriptional control of
melanoma metastasis: the importance of the tumor microenvironment.
Semin Cancer Biol 2001;21:83-8.
Chlenski A, Cohn SL.. Modulation of matrix remodeling by SPARC in
neoplastic progression. Semin Cell Dev Biol 2010;21:55-65.
Cipponi A, Wieers G, van Baren N, et al. Tumor-infiltrating lymphocytes:
apparently good for melanoma patients. But why? Cancer Immunol
Immunother 2011;60:1153-60.
Palmieri G, Capone M, Ascierto ML, et al. Main roads to melanoma. J
Transl Med 2009;14:86-102.
Prada F, Benedetti LG, Bravo AI, et al. SPARC endogenous level, rather
than fibroblast-produced SPARC or stroma reorganization induced by
SPARC, is responsible for melanoma cell growth. J Invest Dermatol
2007;127:2618-28.
Taylor RC, Patel A, Panageas KS, et al. Tumor-infiltrating lymphocytes
predict sentinel lymph node positivity in patients with cutaneous melanoma. J Clin Oncol 2007;25:869-75.
178
Corso breve di ematopatologia: linfomi cutanei e loro simulatori
Coordinatori: M. Santucci (Firenze), M. Paulli (Pavia)
Primary cutaneous B-cell lymphoma (PCBCL)
M. Paulli1, M. Lucioni1, A. Maffi1, M. Nicola1, E. Berti2
1
Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia and IRCCS Fondazione Policlinico “San Matteo”,
Pavia, Italy; 2 Dermatology Section, Università di Milano-Bicocca
and IRCCS “Cà Granda, Fondazione Ospedale Maggiore Policlinico”, Milan, Italy
Introduction. Primary cutaneous B-cell lymphomas (PCBCLs) are a heterogeneous group of lymphoproliferative disorders, which accounts for 25-30% of all primary cutaneous
lymphomas. PCBCLs exhibit a series of peculiar biological
and clinical-prognostic features with respect to their nodal/
systemic counterparts that may affect the skin secondarily.
Three major histotypes are now recognized: 1) primary cutaneous marginal zone B-cell lymphoma (PCMZL); 2) primary cutaneous follicular center cell lymphoma (PCFCL); 3)
primary cutaneous diffuse large B-cell lymphoma (DLBCL),
leg type (PCDLBCL-LT). PCMZL and PCFCL are indolent
lymphomas, with an excellent prognosis despite a high rate
of cutaneous recurrences; in contrast, PCDLBCL-LT is
clinically more aggressive often requiring multi-agent chemotherapy and anti-CD20 immunotherapy.
PCDLBCL-LT histologically consists of a uniform population of large round cells (centroblasts and immunoblasts),
resembling on both phenotypical and molecular grounds
the activated B-cell type of nodal DLBCL. Recently, the
term primary cutaneous DLBCL-other (PCDLBCL-O) has
been proposed to include diffuse lymphomas composed
of large transformed B-cells that lack the typical features
of PCDLBCL-LT and do not conform to the definition of
PCFCL. Some clinical studies suggested that such cases may
pursue an indolent clinical behaviour and may be treated in
a conservative manner; however, data regarding the actual
prognosis and behaviour of these peculiar cases are still
too limited. The spectrum of primary cutaneous DLBCL
also encompasses some rare morphological variants, such as
anaplastic or plasmablastic subtypes and T-cell rich B-cell
lymphoma.
Primary cutaneous marginal zone B-cell lymphoma. PCMZL usually affects adult over 40 years mostly involving
the upper extremities as red/violaceous plaques or nodules 1.
On histology, scattered residual reactive B follicles are
circumscribed by an expanded marginal zone, composed
by small to medium-sized cells varying in appearance from
centrocyte- to monocytoid cells. At the periphery lymphoplasmacytoid elements and monotypic plasma cells are frequently found 1. MZL cells express various B-cell markers
(CD19, CD20, CD22 and CD79a) as well as bcl-2 protein
being negative for CD10, bcl-6, CD23 and CD5.1 Molecular
biology assays usually highlight IGH genes clonal rearrangement and t(14;18)(q32;q21) IGH-MALT1 in one third
of cases 1. PCMZL is characterized by a favourable prognosis with 5-year-survival rates around 90-100% despite local
recurrences may occurr 1.
Primary cutaneous follicular center cell lymphoma.
PCFCL is the most frequent cutaneous lymphoma (25%
of skin lymphomas) affecting predominantly middle aged
adults. Its typical presentation is characterized by firm erythematous to violaceous plaques, nodules or tumours on the
trunk or head and neck region.1 PCFCL is morphologically
defined on the basis of cytological features including the
presence of centrocytes and centroblasts; its growth pattern
can be variable, from follicular to follicular and diffuse to
purely diffuse. PCFCL cells express B-cell markers and bcl6 protein. The positivity for CD10/CALLA is variable and
mostly associated with a nodular/follicular growth pattern.
PCFCLs usually lack IRF4/MUM1 and FOXP1 1 2. PCFCLs
are usually thought to be negative for bcl-2 protein as well
as for t(14;18) translocation and this criterion has been used
to differentiate PCFCL from cutaneous involvement during systemic follicular lymphoma 1 2. However recent data
seem question that statement showing that at least a quarter
of PCFCLs may express bcl-2 and/or bear the translocation
(unpublished data).
The outcome is favourable with a 5-year-survival rate of
95% despite local recurrences usually near the initial site of
presentation 1.
Primary cutaneous diffuse large B-cell lymphoma, leg
type. Within the group of primary PCDLBCL, this is the
best defined category, that represents about 4% of all
primary cutaneous lymphomas. It usually affects elderly
patients with a femal predominance (average age: 78 years,
M:F 1:3- 4) 2 3. The typical lesions usually involve the lower
legs but presentation at other sites (trunk, head-neck, and
upper extremities) has been reported in 10-15% of cases.
Patients usually present with solitary or multiple, rapidly
growing, violaceous nodular tumours 2 3. Histologically,
PCDLBCL-LT is characterized by a dense cellular infiltrate
that efface the adnexal structures and infiltrate the whole
dermis and often the subcutis. The lymphoma population
consists of large round cells usually with immunoblastic
cytologic features 2 3. Tumour cells express a B-cell activated
immunophenotype (CD19, CD20, CD22 and CD79a bcl-2,
MUM-1, bcl-6+/-) 4. Bcl-2 is overexpressed in about 90% of
PCDLBCL-LT but the molecular mechanisms responsible
for it are still partially unknown.2 Clonal rearrangement of
IGH genes is detected in most cases 2 3. The prognosis of
PCDLBCL-LT is poor, with frequent relapses and dissemination to extracutaneous sites 3 5. The 5-year disease survival
rates range from 40%-50% 6 7.
Primary cutaneous diffuse large B-cell lymphoma, other. The category of “primary cutaneous DLBCL, other”
(PCDLBCL-O) is heterogeneous encompassing rare, different entities. The term was introduced by EORTC/WHO
Consensus Classification, to identify those cases of DLBCL
composed of large transformed B-cells that lack the typical
features of PCDLBCL-LT and do not conform to the definition of PCFCL 8. Such cases usually present with solid nodules or tumours located on the leg in one-half of the patients,
on the head, trunk and arms in the remaining cases. Histologically, these tumours usually consist of a monomorphic
population of centroblast-like cells. In addition to B-cell
markers expression, lymphoma cells express bcl-6, MUM1
and FOXP1, while bcl-2 is usually negative 1 3. The precise
diagnosis and classification of these cases is still a matter of
debate, especially with respect to their differentiation from
PCFCL with diffuse growth pattern. Some clinical studies
suggested that such cases have an indolent clinical course
and may be treated in a conservative manner, in spite of the
179
relazioni
predominance of centroblasts 1 8 9. However, data regarding
the actual prognosis and clinical behaviour of these peculiar
cases are still too limited.
Conclusions. Primary cutaneous B-cell lymphomas are
a heterogeneous group of lymphoproliferative disorders,
with distinct clinico-pathologic features with respect to
their nodal counterparts. In less than a decade, significant
progress has been made in our understanding of clinical,
genetic and prognostical features. Their histological diagnosis usually poses little difficulties; however some cases
may present with unfamiliar clinical appearance or special
cytological features as among the group of DLBCL, other,
not otherwise specified type. In this contest, further studies
are requested to clarify whether the category of PCDLBCLO shows indeed peculiar clinico-pathologic features or represents only a morphologic or phenotypic variant of PCFCL
or PCDLBCL-LT.
References
1
LeBoit P, Burg G, Weedon D, et al., eds. Pathology and Genetics. Skin
Tumours. Lyon: IARC Press 2006.
2
Swerdlow SH, Campo E, Harris NL, et al., eds. WHO Classification of
3
4
5
6
7
8
9
Tumors of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press
2008.
Paulli M, Lucioni M, Maffi A, et al. Primary cutaneous diffuse large
B-cell lymphoma (PCDLBCL), leg-type and other: an update on morphology and treatment. G Ital Dermatol Venereol 2012;147:589-602.
Paulli M, Viglio A, Vivenza D, et al. Primary cutaneous large B-cell
lymphoma of the leg: histogenetic analysis of a controversial clinicopathologic entity. Hum Pathol 2002;33:937-43.
Grange F, Bekkenk MW, Wechsler J, et al. Prognostic factors in
primary cutaneous large B-cell lymphomas: a European multicenter
study. J Clin Oncol 2001;19:3602-10.
Senff NJ, Hoefnagel JJ, Jansen PM, et al. Reclassification of 300
primary cutaneous B-cell lymphomas according to the new WHOEORTC classification for cutaneous lymphomas: comparison with
previous classifications and identification of prognostic markers. J
Clin Oncol 2007;25:1581-7.
Grange F, Beylot-Barry M, Courville P, et al. Primary cutaneous diffuse large B-cell lymphoma, leg type: clinicopathologic features and
prognostic analysis in 60 cases. Arch Dermatol 2007;143:1144-50.
Willemze R, Jaffe ES, Burg G, et al. WHO-EORTC classification for
cutaneous lymphomas. Blood 2005;105:3768-85.
Senff NJI, NoordijkEM, Kim YH, et al. European Organization for
Research and Treatment of Cancer and International Society for Cutaneous Lymphoma Consensus recommendations for the management
of cutaneous B-cell lymphomas. Blood 2008;112:1600-9.
Sala Orange 1 – ore 8.30-18.30
Patologia toraco-polmonare
Simposio I parte: update in patologia toraco-polmonare
Moderatori: B. Murer (Mestre), O. Nappi (Napoli)
Update for Pathologists on Interstitial Lung
Disease
T.V. Colby
U.S.A.
I. Update: Idiopathic pulmonary fibrosis (IPF) and Idiopathic interstitial pneumonias (IIPs)
IPF: Guidelines for clinicians on the management and diagnosis of IPF that came out in 2000 were updated in 2011 with
evidence-based guidelines/key points:
–radiologic diagnosis of UIP possible when classic features
are identified;
–multidisciplinary discussion (MDD) seen as an integral part
and as the “gold standard” in the diagnosis of IPF and IIPs;
–emphasis on enrolling patients in clinical trials to identify
effective therapies,
– recognition of acute exacerbation of IPF.
In clinical trials, guidelines for patient accrual include radiologic confidence of diagnosis: Inconsistent with UIP, possible
UIP, definite UIP.
Somewhat parallel pathologic confidence levels are also suggested: Not UIP, possible UIP, probable UIP, definite UIP.
These “confidence levels” allow for a clinically workable
framework for entering patients into clinical trials. The criteria for the “pathology confidence levels” are not validated and
primarily of use for clinical trial accrual and not for a routine
diagnosis.
IIPs: The 2002 Consensus Statement on the IIPs is also being
updated and due to be published in late 2013 as evidencebased guideline with IIPs categorized as follows:
– major IIPs: IPF, Idiopathic NSIP, RBILD, DIP, COP, AIP;
– rare IIPs: Idiopathic LIP, Idiopathic pleuropulmonary fibroelastosis (PPFE);
– unclassified IIPs.
The lesions are also categorized as follows:
–chronic fibrosing IP’s (IPF/pattern UIP and idiopathic
NSIP/pattern NSIP);
– smoking related IPs (RBILD/pattern RB and DIP);
– acute/subacute IPs (COP/pattern OP and AIP/pattern DAD).
A clinical/managment classification based on disease behavior is included:
– reversible/self-limited, eg. some RB-ILD;
– reversible with risk of progression, eg. Cellular NSIP. COP;
– stable with residual disease, eg. some fibrotic NSIP;
–progressive/irreversible with potential for stabilization, eg.
some fibrotic NSIP;
– Progressive/irreversible despite therapy, eg. IPF.
Additional key points are as follows:
–transbronchial biopsy not useful in the diagnosis of most
IIPs (but see below);
– BAL not useful in the diagnosis of most IIPs;
– RBILD diagnosable without biopsy if CT and BAL fit;
–surgical lung biopsy and clinical/radiologic correlation (ie.
MDD) are most helpful and considered the gold standard;
–histologic patterns recognized: Brochiolocentric interstitial
pneumonias and acute fibrinous and organizing pneumonia
(AFOP); not considered IIPs yet.
The major new addition to IIPs is PPFE, probably the same
lesion described in the Japanese literature in the late 1990s as
idiopathic upper lobe fibrosis (see refs below). PPFE is typically characterized by upper lobe pleural and adjacent parenchymal elastotic fibrosis reminiscent of an apical cap. In the
180
largest series to date from London (12 pts): ~equal sex incidence, median age 57, and symptoms of dyspnea and cough. 7
reported recurring infections; 5 had non-specific autoantibody
positivity. 2 had a familial history of interstitial lung disease.
5 of the 12 died of the disease (over 4 mos to 2 yrs).
PPFE is also encountered in the setting of transplantation,
associated with the restrictive allograft syndrome post lung
transplantation and following bone marrow transplantation. In
the latter group, there was often a history of co-existing prior
pneumothoraces and obliterative bronchiolitis.
Pathologic diagnosis of IPF and IIPs. Current emphasis is on
multidisciplinary discussion and surgical lung biopsy in order
to identify the pathologic patterns associated with IPF and
most IIPs. Some recent evidence suggests that cryobiopsies
may be sufficiently large enough to identify such patterns and
surgical lung biopsies may become unnecessary, although
the results are preliminary and no definite conclusions can
be made.
References
American Thoracic Society; European Respiratory Society. American
Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial
Pneumonias. This joint statement of the American Thoracic Society
(ATS), and the European Respiratory Society (ERS) was adopted by
the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med 2002;165:277-304.
Erratum in: Am J Respir Crit Care Med2002;166:426.
American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and
treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am J Respir
Crit Care Med 2000;161:646-64.
Babiak A, Hetzel J, Krishna G, et al. Transbronchial cryobiopsy: a new
tool for lung biopsies. Respiration 2009;78:203-8.
Hetzel J, Eberhardt R, Herth FJ, et al. Cryobiopsy increases the diagnostic yield of endobronchial biopsy: a multicentre trial. Eur Respir
J 2012;39:685-90.
Hirota T, Fujita M, Matsumoto T, et al. Pleuroparenchymal fibroelastosis as a manifestation of chronic lung rejection? Eur Respir J
2013;41:243-5.
Kusagaya H, Nakamura Y, Kono M, et al. Idiopathic pleuroparenchymal
fibroelastosis: consideration of a clinicopathological entity in a series
of Japanese patients. BMC Pulm Med 2012;12:72
Larsen BT, Colby TV. Update for pathologists on idiopathic interstitial
pneumonias. Arch Pathol Lab Med 2012;136:1234-41.
Ofek E, Sato M, Saito T, et al. Restrictive allograft syndrome post lung
transplantation is characterized by pleuroparenchymal fibroelastosis.
Mod Pathol 2013;26:350-6.
Piciucchi S, Tomassetti S, Casoni G, et al. High resolution CT and
histological findings in idiopathic pleuroparenchymal fibroelastosis:
features and differential diagnosis. Respir Res 2011;12:111.
Raghu G, et al.; ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis. An official ATS/ERS/JRS/ALAT statement: idiopathic
pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med 2011;183:788-824.
Reddy TL, Tominaga M, Hansell DM, et al. Pleuroparenchymal fibroelastosis: a spectrum of histopathological and imaging phenotypes.
Eur Respir J 2012;40:377-85.
Shiota S, Shimizu K, Suzuki M, et al. [Seven cases of marked pulmonary
fibrosis in the upper lobe]. Nihon Kokyuki Gakkai Zasshi 1999;37:8796. Japanese.
Travis WD, et al. An official American Thoracic Society/European Respiratory Society Statement: Update of the international multidisciplinary
classification of the idiopathic interstitial pneumonias. In press 2013.
von der Thüsen JH, Hansell DM, Tominaga M, et al. Pleuroparenchymal
fibroelastosis in patients with pulmonary disease secondary to bone
marrow transplantation. Mod Pathol 2011;24:1633-9.
II. Interstitial lung disease in patients with connective tissue disease (CTD/ILD)
Compared to IPF, CTD/ILD has a more favorable prognosis.
CTD/ILD has a higher frequency of NSIP than IIPs; could
this might explain the more favorable prognosis of CTD/ILD?
Several studies comparing IIPs and CTD/ILDs pattern for pattern (i.e. UIP pattern vs. NSIP pattern) have still shown a more
favorable prognosis in CTD/ILD except for RA-associated
UIP which, in some studies, has a prognosis similar to IPF.
This is an area of controversy and further studies are needed.
From a histopathologic point of view, CTD/UIP differs from
IPF with increased overall inflammation, particularly germinal centers (p = .001). Anecdotally, one often notes multiple
patterns of injury in CTD/ILD in comparison to IIPs which
tend to show a relatively pure pattern from field to field and
slide to slide.
The concept of undifferentiated connective tissue disease
(UCTD) has been developed: presence of certain symptoms
and serologies associated with CTD without defined rheumatologic syndrome. Patients with IPF and features of UCTD
appear to have a favorable prognosis compared to IPF without
features of UCTD.
Some additional correlative findings regarding CTD/ILD and
IIPs include:
– among IPF patients, those with positive autoantibodies have
some pathologic similarity to CTD/UIP;
– the presence of autoantibodies in IIP is a significant predictor for the subsequent development of a CTD;
–patients with NSIP who also fulfill UCTD criteria have a
significantly better outcome than NSIP patients who lack
these features;
–patients with CTD/UIP have fewer fibroblast foci than patients with IPF;
– UCTD is typically associated with NSIP histology.
In some studies, greater than 50% of the patients diagnosed
with idiopathic NSIP develop some evidence of autoimmune
disease within two years.
References
Cipriani NA, Strek M, Noth I, et al. Pathologic quantification of connective tissue disease-associated versus idiopathic usual interstitial
pneumonia. Arch Pathol Lab Med 2012;136:1253-8.
Corte TJ, Copley SJ, Desai SR, et al. Significance of connective tissue
disease features in idiopathic interstitial pneumonia. Eur Respir J
2012;39:661-8.
de Lauretis A, Veeraraghavan S, Renzoni E. Review series: Aspects of interstitial lung disease: connective tissue disease-associated interstitial
lung disease: how does it differ from IPF? How should the clinical
approach differ? Chron Respir Dis 2011;8:53-82.
Kang BH, Park JK, Roh JH, et al. Clinical significance of serum autoantibodies in idiopathic interstitial pneumonia. J Korean Med Sci
2013;28:731-7.
Kim DS, Yoo B, Lee JS, et al. The major histopathologic pattern of pulmonary fibrosis in scleroderma is nonspecific interstitial pneumonia.
Sarcoidosis Vasc Diffuse Lung Dis 2002;19:121-7.
Kim EJ, Collard HR, King TE Jr. Rheumatoid arthritis-associated interstitial lung disease: the relevance of histopathologic and radiographic
pattern. Chest 2009;136:1397-405.
Kinder BW, Shariat C, Collard HR, et al. Undifferentiated connective
tissue disease-associated interstitial lung disease: changes in lung
function. Lung 2010;188:143-9.
Lee HK, Kim DS, Yoo B, et a. Histopathologic pattern and clinical features of rheumatoid arthritis-associated interstitial lung disease. Chest
2005;127:2019-27.
Nakamura Y, Chida K, Suda T, et al. Nonspecific interstitial pneumonia
in collagen vascular diseases: comparison of the clinical characteristics and prognostic significance with usual interstitial pneumonia.
Sarcoidosis Vasc Diffuse Lung Dis 2003;20:235-41.
Nakamura Y, Suda T, Kaida Y, et al. Rheumatoid lung disease: prognostic analysis of 54 biopsy-proven cases. Respir Med 2012;106:1164-9.
Park JH, Kim DS, Park IN, et al. Prognosis of fibrotic interstitial pneumonia: idiopathic versus collagen vascular disease-related subtypes.
Am J Respir Crit Care Med 2007;175:705-11.
Romagnoli M, Nannini C, Piciucchi S, et al. Idiopathic nonspecific inter-
relazioni
stitial pneumonia: an interstitial lung disease associated with autoimmune disorders? Eur Respir J 2011;38:384-91.
Song JW, Do KH, Kim MY, et al. Pathologic and radiologic differences
between idiopathic and collagen vascular disease-related usual interstitial pneumonia. Chest 2009;136:23-30.
Suda T, Kono M, Nakamura Y, et al. Distinct prognosis of idiopathic nonspecific interstitial pneumonia (NSIP) fulfilling criteria
for undifferentiated connective tissue disease (UCTD). Respir Med
2010;104:1527-34.
III. Hypersensitivity pneumonitis (HP)
The many causes of HP (primarily organic antigens) and its
histology are well known. A number of interesting aspects of
interest to pathologists have emerged:
– selman et al. postulate a two-hit hypothesis for the development of HP: 1. Genetic predisposition 2. Exposure to appropriate antigen;
–increased recognition of atypical mycobacteria in heated
water (e.g. hot tubs) as an antigen capable of inducing HP.
– usefulness of BAL in confirming the diagnosis of HP when
greater than 40% of the retrieved BAL cells are lymphocytes. This is one IP in which BAL retains utility;
–some antigens may cause more aggressive disease: Exposure to avian antigens (bird fancier’s lung) produces aggressive disease than exposure to other antigens;
– some cases of HP, notably those associated with aerosolized
mycobacteria (e.g. hot tub), are associated with larger and
more well-formed granulomas than in other HP cases;
–some cases of chronic HP are histologically indistinguishable from UIP and may indeed carry a similar prognosis. In
these cases, granulomas may be entirely lacking.
There have been some interesting studies from Japan comparing IPF to chronic HP. In an autopsy study, honeycombing
was found predominantly in the lower lobes in both IPF and
chronic HP, whereas cases that showed an asymmetric distribution or an upper lobe predominance of honeycombing were
more frequent in chronic HP. In all cases of chronic HP, central lobular fibrosis was a prominent feature and in some cases
“bridging fibrosis” (central to the periphery of the lobule)
clearly demonstrated pathologically and by three-dimensional
imaging. This finding was much more conspicuous in chronic
HP than IPF.
In a biopsy series, central lobular fibrosis, bridging fibrosis,
organizing pneumonia, bronchiolitis, granulomas, and giant
cells (including giant cells containing cholesterol clefts) were
characteristic features of chronic HP in comparison to IPF; a
UIP pattern was also seen in some cases of chronic HP.
References
Akashi T, Takemura T, Ando N, et al. Histopathologic analysis of sixteen
autopsy cases of chronic hypersensitivity pneumonitis and comparison
with idiopathic pulmonary fibrosis/usual interstitial pneumonia. Am J
Clin Pathol 2009;131:405-15.
Selman M, Pardo A, King TE Jr. Hypersensitivity pneumonitis: insights in
diagnosis and pathobiology. Am J Respir Crit Care Med.2012;186:31424.
Takemura T, Akashi T, Kamiya H, et al. Pathological differentiation of
chronic hypersensitivity pneumonitis from idiopathic pulmonary fibrosis/usual interstitial pneumonia. Histopathology 2012;61:1026-35.
IV. Aspiration
Gastroesophageal reflux disease (GERD) with “microaspiration” has been implicated as both a cause in IPF and as a
potential prognostic factor. Management of GERD has been
associated with a more favorable prognosis in IPF. If microaspiration is significant it is not something that pathologists
can appreciate.
Aspiration that is recognizable by the pathologist in the form
181
of foreign material (usually food) may produce diffuse interstitial lung disease. Foreign material may be easily missed
unless specifically sought. An airway granulomatous reaction
with neutrophils may be present.
Yousem and Faber described 10 patients with documented
aspiration in whom no food/foreign material was apparent
histologically. The authors found nonspecific pathologic
changes: 4/10 patients showed organizing pneumonia, 3 acute
diffuse alveolar damage, and 3 chronic bronchiolitis. 2 of the
patients had extensive scarring. Thus, the absence of identifiable foreign material in the lung biopsy does not exclude the
possibility of aspiration.
References
Lee JS, Ryu JH, Elicker BM, et al. Gastroesophageal reflux therapy is
associated with longer survival in patients with idiopathic pulmonary
fibrosis. Am J Respir Crit Care Med 2011;184:1390-4.
Mukhopadhyay S, Katzenstein AL. Pulmonary disease due to aspiration
of food and other particulate matter: a clinicopathologic study of 59
cases diagnosed on biopsy or resection specimens. Am J Surg Pathol
2007;31:752-9.
Raghu G, Freudenberger TD, Yang S, et al. High prevalence of abnormal
acid gastro-oesophageal reflux in idiopathic pulmonary fibrosis. Eur
Respir J 2006;27:136-42.
Raghu G, Yang ST, Spada C, et al. Sole treatment of acid gastroesophageal reflux in idiopathic pulmonary fibrosis: a case series. Chest
2006;129:794-800.
Yousem SA, Faber C. Histopathology of aspiration pneumonia not associated with food or other particulate matter: a clinicopathologic study
of 10 cases diagnosed on biopsy. Am J Surg Pathol 2011;35:426-31.
V. Lymphangioleiomyomatosis (LAM)
There has been a paradigm shift in the approach to LAM.
Lam is now on recognized as a low-grade destructive metastasizing neoplasm: LAM shows loss of heterozygosity for
tuberous sclerosis complex genes, similar clonality of lesions
at multiple sites, uncontrolled (albeit somewhat slow) growth,
the ability to recur in allografts, probably by a metastatic
mechanism, invasion, angiogenesis including exuberant lymphangiogenesis, and the ability to disseminate via the blood
stream and lymphatics. LAM cells have growth-promoting
DNA mutations and metabolic profiles that are consistent with
a neoplasm. This aligns with concept of PECOMA.
In the LAM uterus has been suggested as a primary site for
LAM cells.
As LAM cells proliferate, there is lymphangiogenesis and
LAM cell clusters may bud into effusions. There is an associated with increase in serum VEGF levels. Both VEGF
levels and the identification of LAM cell clusters in either fine
needle aspirations or in evaluation of effusion material have
diagnostic usefulness.
References
Hayashi T, Kumasaka T, Mitani K, et al. Prevalence of uterine and adnexal involvement in pulmonary lymphangioleiomyomatosis: a clinicopathologic study of 10 patients. Am J Surg Pathol. 2011;35:1776-85.
Kumasaka T, Seyama K, Mitani K, et al. Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis. Am J Surg Pathol 2004;28:1007-16.
Kumasaka T, Seyama K, Mitani K, et al. Lymphangiogenesis-mediated
shedding of LAM cell clusters as a mechanism for dissemination in
lymphangioleiomyomatosis. Am J Surg Pathol 2005;29:1356-66.
McCormack FX, Travis WD, Colby TV, et al. Lymphangioleiomyomatosis: calling it what it is: a low-grade, destructive, metastasizing
neoplasm. Am J Respir Crit Care Med 2012;186:1210-2.
Meraj R, Wikenheiser-Brokamp KA, Young LR, et al. Lymphangioleiomyomatosis: new concepts in pathogenesis, diagnosis, and treatment.
Semin Respir Crit Care Med 2012;33:486-97.
Mitani K, Kumasaka T, Takemura H, et al. Cytologic, immunocytochemical and ultrastructural characterization of lymphangioleiomyomatosis
182
cell clusters in chylous effusions of patients with lymphangioleiomyomatosis. Acta Cytol 2009;53:402-9.
Seyama K, Kumasaka T, Kurihara M, et al. Lymphangioleiomyomatosis: a disease involving the lymphatic system. Lymphat Res Biol
2010;8:21-31.
Seyama K, Kumasaka T, Souma S, et al. Vascular endothelial growth
factor-D is increased in serum of patients with lymphangioleiomyomatosis. Lymphat Res Biol 2006;4:143-52.
Xu KF, Zhang P, Tian X, et al. The role of vascular endothelial growth
factor-D in diagnosis of lymphangioleiomyomatosis (LAM). Respir
Med 2013;107:263-8.
VI. Smoking, fibrosis, and interstitial lung disease
Respiratory bronchiolitis, respiratory bronchiolitis-associated
interstitial lung disease, pulmonary lung and Langerhans cell
histiocytosis, eosinophilic pneumonia, and DIP are all well
recognized smoking-related ILDs. Several recent studies have
documented subclinical histologic fibrosis with or without associated emphysema and terms have included: smoking-related
interstitial fibrosis (SRIF), airspace enlargement with fibrosis
(AEF) and RB-ILD with fibrosis. Some recent large radiologic
studies of smokers who are asymptomatic have shown subtle
radiologic and functional abnormalities that may very well be
the correlate of this microscopic smoking realted fibrosis.
References
Doyle TJ, Washko GR, Fernandez IE, et al; COPD Gene Investigators.
Interstitial lung abnormalities and reduced exercise capacity. Am J
Respir Crit Care Med 2012;185:756-62.
Katzenstein AL. Smoking-related interstitial fibrosis (SRIF), pathogenesis and treatment of usual interstitial pneumonia (UIP), and transbronchial biopsy in UIP. Mod Pathol 2012;25(Suppl. 1):S68-78.
Kawabata Y, Hoshi E, Murai K, et al. Smoking-related changes in the
background lung of specimens resected for lung cancer: a semiquantitative study with correlation to postoperative course. Histopathology
2008;53:707-14.
Washko GR, Hunninghake GM, Fernandez IE, et al.; COPDGene Investigators. Lung volumes and emphysema in smokers with interstitial lung
abnormalities. N Engl J Med 2011;364:897-906.
Yousem SA. Respiratory bronchiolitis-associated interstitial lung disease with fibrosis is a lesion distinct from fibrotic onspecific interstitial pneumonia: a proposal. Mod Pathol 2006;19:1474-9.
An update on malignant pleural mesothelioma
pathology
P.G. Betta
Alessandria
Previously considered as a rare tumour, malignant pleural mesothelioma (MPM) has become an important public health issue because of increasing incidence (1422 persons diagnosed
with mesothelioma in Italy in 2008 vs 237 in 1993) 1 and poor
response to treatment, the median survival after symptom onset being less than 12 months. MPM is officially recognized as
an occupational cancer and as a signal disease for occupational asbestos exposure. There is sufficient evidence in humans
for the carcinogenicity of all forms of asbestos (chrysotile,
crocidolite, amosite, tremolite, actinolite and anthophyllite) 2.
The percentage of MPMs that are not associated with asbestos
is generally estimated at 10-20%. Current research suggests a
probable genetic predisposition (germline BAP1 mutations) to
mineral fibre carcinogenesis 3.
To obtain an earlier and reliable diagnosis of MPM is a crucial
issue. The clinical manifestations (chest pain, dyspnea…) of
MPM are usually non specific and insidious and cannot be used
alone as diagnostic criteria, even in case of previous asbestos
exposure. There are no specific diagnostic imaging criteria 4. In
fact, chest X-ray usually shows a unilateral pleural effusion or
thickening and it should not be used alone for the diagnosis of
MPM, whereas chest CT scan is unsuitable for definitive diagnosis of MPM, although diffuse or nodular pleural thickening
are suggestive of the disease. MRI is not relevant and PET scan
is now coming into wider use, its main advantage being greater
sensitivity for the detection of distant metastases.
When MPM is suspected on clinical or radiological data, thoracoscopy with multiple, deep and large biopsies is the best
diagnostic method (obtained in > 90% of cases). Therefore,
it is recommended, except in case of preoperative contraindication or pleural symphysis, to perform thoracoscopy for
the diagnosis of MPM. At times, MPM may be diagnosed by
cytological examination of the pleural effusion in combination
with characteristic radiological appearances and appropriate follow-up. However, the results are often equivocal and
therefore it is advised to forgo a diagnosis of MPM based
on cytology alone: cytological suspicion of MPM must be
followed by tissue confirmation. Conversely, disease recurrence and metastases can be ascertained on cytology alone.
Also fine needle biopsies are not primarily recommended for
the diagnosis of MPM because they are associated with low
sensitivity and risk of error 5.
The World Health Organization classification of tumors of the
lung and pleura recognizes three major histological subtypes
of MPM on the basis of the predominant histomorphological
growth pattern: epithelioid (~60%), sarcomatoid and mixed
(biphasic) 6. Accurate histological subtyping is important,
as histological subtype is a validated prognostic factor: survival of patients with epithelioid tumors is better than that
of patients with nonepithelioid tumors. Furthermore, some
clinicians believe that only patients with epithelial subtype
should be eligible for extrapleural pneumonectomy 7. Standard
staining procedures in diagnostic histopathology are insufficient in 7-15% of cases. A diagnosis of MPM of epithelioid
type should always be based on immunohistochemistry (IHC):
there is no single specific marker for mesothelioma and different combinations of markers are needed depending on the
differential-diagnostic questions to be answered. Two positive
mesothelial markers and 2 carcinoma markers positive for the
other tumor under consideration on the basis of morphology
(i.e. adenocarcinoma, squamous cell carcinoma), in addition
to a broad-spectrum anti-cytokeratin antibody, should be used
in the initial work up.8 Based on their sensitivity and specificity, calretinin, CK5 or CK5/6, WT-1, and D2-40 (podoplanin)
are the best positive mesothelioma markers. MOC-31, BerEP4, carcinoembryonic antigen (CEA), and Lewis(y) antigen
blood group 8 (BG8) are the best overall carcinoma markers.
TTF-1 and Napsin A are highly specific for lung adenocarcinoma and may assist in distinguishing MPM from lung adenocarcinoma, whereas p63, strongly and invariably positive in
squamous cell carcinomas, may help to differentiate between
epithelioid MPM and squamous cell carcinomas 8. The role of
IHC is more restricted in sarcomatoid MPM and in the initial
evaluation of a sarcomatoid tumor of the pleura cytokeratins,
calretinin, and D2-40 should be used. Multiple cytokeratin
antibodies including AE1/3, CAM 5.2 (or CK18), and CK7
are recommended, as cytokeratin
expression can be focal, weak, and/or variable. In the absence
of convincing cytokeratin staining, calretinin and/or D2-40
reactivity alone should not be construed as evidence of mesothelial histogenesis, because these markers show variable
positivity in many different types of sarcomas.
GLUT-1 (glucose transporter protein) and IMP3 (insulin-like
growth factor II mRNA binding protein 3) are two newly marketed tissue markers claimed to be valuable for identifying the
relazioni
benign or malignant nature of a mesothelial proliferation. As
for GLUT-1, preliminary results found 40 positive MPM vs
40 negative reactive mesothelial hyperplasia 9 but it recently
showed lower sensitivity than previously reported 10. GLUT-1
may be a helpful marker for MPM, both epithelial and sarcomatoid, but it is not informative when negative. IMP3 was
recently shown to be positive in 33 of 45 MMs (73%) and
negative in all 64 reactive mesothelial lesions tested 11. The
combination of both markers may be of greater diagnostic
value, but this hypothesis should be confirmed in further studies 12. Also additional investigations are needed to confirm the
initial claim concerning the ability of CD147 to distinguish
reactive mesothelial cells from MPM 13. One of the most
common genetic alterations in MPM is the homozygous deletion of the 9p21 locus within a cluster of genes that includes
cyclin-dependent kinase inhibitor 2A (CDKN2A), CDKN2B,
and methylthioadenosine phosphorylase (MTAP). The FISH
technique for detection of 9p21 deletions appears to be an effective technique for confirming the diagnosis of MPM vs reactive mesothelial hyperplasia in effusion and formalin-fixed,
paraffin-embedded tissue specimens 8 14.
MPM prognostic tissue markers (revised and updated from
Astoul P, et al.) 15
– p16/CDKN2A: deletion is associated with poor survival;
–Angiogenesis: high micro vessel density associated with
poor survival:
- A reduced level of BAX, a tumor-suppressor gene downregulated by tumor hypoxia, associated with a poor outcome;
-VEGF overexpression as monitored by IHC independently predicts short survival;
-High levels of VEGF and FGF2, or co-expression of
TGF-b, VEGF, FGF1, and FGF2 also associated with a
poor outcome.
–CD9: expression is an independent favorable prognostic
marker;
–c-MET plasma membrane localization predicts a longer
survival;
– Aquaporin 1: expression by ≥ 50% of tumor cells is associated with significantly enhanced survival, irrespective of
treatment or established prognostic factors;
–steroid receptor coactivators-2 (TIF-2): high TIF-2 expression correlates with improved patient prognosis;
–p27KIP1 and Rb loss are independent prognostic factors associated with reduced OS;
– hsa-miR-29c*: increased expression predicts a more favorable prognosis after surgical cytoreduction.
MPM predictive tissue markers (revised and updated from
Astoul P, et al.) 15
–ERCC1 (excision repair cross-complementation group 1):
low expression might predict increased sensitivity to platinum-based chemotherapy (CT). Combined negative ERCC1 and class III β-tubulin immunostaining is associated
with significantly prolonged PFS and OS in MPM patients
receiving cisplatin-vinorelbine therapy.
– TS (thymidylate synthase):
-in a series of carboplatin-/pemetrexed-treated MPM
patients, low TS protein and mRNA levels were significantly associated to disease control, improved PFS, and
OS;
- in a retrospective analysis, low TS protein levels were
predictive of improved time to progression and overall
survival;
- another retrospective analysis of TS and ERCC-1 protein
expression by IHC in 99 MPM patients treated with the
183
carboplatin/pemetrexed regimen found that TS expression was a predictor of clinical outcome;
- in a recent series of patients with MPM undergoing EPP,
TS expression was not associated with prolonged survival, but there was a trend for longer survival in patients
with high ERCC1 expression.
– FPGS (folylpoly-γ-glutamate synthetase):
- high FPGS protein expression was significantly associated with longer PFS, better objective tumor response,
and improved disease-control rate, but not with OS.
In addition, high TS protein expression was associated with progressive disease under pemetrexed-based
therapy, and shorter OS, but no association with FPFS
was observed;
- FPGS and reduced folate carrier (RFC) mRNA expression levels were strongly associated with clinical benefit
from pemetrexed treatment and overall survival. In addition, TS expression levels had no influence on patient
outcome;
- although previous retrospective data suggest that TS and
FPGS expression might be potential markers of pemetrexed efficacy in MPM, these markers lack sufficient
predictive value in individual patients and so for now
should not be used to guide therapeutic decisions in the
absence of prospective studies.
– SURVIVIN: significantly higher survivin expression in patients who responded to chemotherapy compared to patients
with progressive disease
References
1
INAIL. 4° Rapporto ReNaM (Registro Nazionale dei Mesoteliomi).
2012.
2
IARC monographs on the evaluation of carcinogenic risks to humans.
A review of human carcinogens. Part C: arsenic, metals, fibres, and
dusts. Lyon: IARC Press 2012, volume 100, C. 219-309.
3
Testa JR, Cheung M, Pei J, et al. Germline BAP1 mutations predispose
to malignant mesothelioma. Nat Genet 2011;43:1022-5.
4
Gill R. Imaging of mesothelioma. Recent Res Cancer Res
2011;189:27-55.
5
Scherpereel A, Astoul P, Baas P, et al. Guidelines of the European
Respiratory Society and the European Society of Thoracic Surgeons
for the management of malignant pleural mesothelioma. Eur Respir J
2010;35:479-95.
6
Travis WD, Brambilla E, Muller-Hermelink HK,et al. World Health
Organisation Classification of Tumours. Pathology and Genetics of
Tumours of the Lung, Pleura, Thymus and Heart. Lyon: IARC Press
2004.
7
Stahel RA, Weder W, Felip E. On behalf of the ESMO Guidelines
Working Group. Malignant pleural mesothelioma: ESMO clinical
recommendations for diagnosis, treatment and follow-up. Ann Oncol
2008;19(Suppl. 2):ii43-ii44.
8
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.
9
Kato Y, Tsuta K, Seki K,et al. Immunohistochemical detection of
GLUT-1 can discriminate between reactive mesothelium and malignant mesothelioma. Mod Pathol 2007;20:215-20.
10
Lagana SM, Taub RN, Borczuk AC. Utility of glucose transporter 1
in the distinction of benign and malignant thoracic and abdominal
mesothelial lesions. Arch Pathol Lab Med 2012;136:804-9.
11
Shi M, Fraire AE, Chu P, et al. Oncofetal protein IMP3, a new diagnostic biomarker to distinguish malignant mesothelioma from reactive
mesothelial proliferation. Am J Surg Pathol 2011;35:878-82.
12
Lee AF, Gown AM, Churg A. IMP3 and GLUT-1 immunohistochemistry for distinguishing benign from malignant mesothelial proliferations. Am J Surg Pathol 2013;37:421-6. 13
Pinheiro C, Longatto-Filho A, Soares TR, et al. CD147 immunohistochemistry discriminates between reactive mesothelial cells and
malignant mesothelioma. Diagn Cytopathol 2012;40:478-83.
14
Chung C T-S, Santos Gda C, Hwang DM, et al. FISH assay develop-
184
15
ment for the detection of p16/CDKN2A deletion in malignant pleural
mesothelioma. J Clin Pathol 2010;63:630-4.
Astoul P, Roca E, Galateau-Salle F, et al. Malignant pleural mesothelioma: from the bench to bedside. Respiration 2012;83:481-93.
Update in Thymic Epithelial Tumors diagnostics
M. Marino
Roma
In recent years, for every tissue and organ system, considerable progress has been reached in the diagnostic approach to
neoplasias, in surgical pathology procedures, in tumor epidemiology and classification, in their biological and molecular
genetic characterization. The increase in knowledge allowed
progresses in treatment and development of improved conventional/ targeted therapies. In rare tumors, all these steps
are hampered because of their rarity and for the inadequate
knowledge of the specific tumor characteristics, as well as for
limited interest among the scientific community. According to
the US National Cancer Institute (NCI) and the “International
Rare Cancers Initiative” (IRCI), a rare tumor is a cancer with
an incidence of ≤ 3 newly diagnosed persons out of a population of 100,000 persons per year (≤ 3/100,000 per year). Other
organizations and conference panels (e.g., ESMO Rare Cancers Conference 2012) have set the incidence rate for a rare
cancer slightly higher, at ≤6/100,000. In Europe, Rare cancers
are considered those with incidence < 6/100,000. Thymic
Epithelial Tumors (TET) account for < 1% of all adult cancers, with an incidence rate of 1-5 / million population / year.
Thymomas, the organotypic TET form, are the most frequent
primary mediastinal tumors in adults. Data from Surveillance,
Epidemiology, and End Results (SEER) cancer registries
in US showed an overall incidence of thymoma of 0.13 per
100,000 person/year. Among Thymic Epithelial Tumors
(TET), Thymomas represent a unique model among tumors
of the lympho hemopoietic organs as the neoplastic epithelial/
stromal component retain its capability to “educate” although
imperfectly the immature T-lymphocytes in their intratumorous development to mature peripheral T cells. Accordingly,
various and heterogeneous immunological derangements are
found associated to thymic epithelial pathology. Reactive,
neoplastic and age-related alterations of the thymic epithelial/
stromal compartment are known to be involved in the pathogenesis of early-onset Myasthenia Gravis (MG), in thymomaassociated MG and in late-onset MG. However, the complex
interplay of Thymus with immunity and autoimmunity is
still largely unknow. Therefore it is not surprising that the
incidence of extrathymic neoplasia is significantly higher in
patients with thymoma than in the general population. Paraneoplastic immune disturbance could account for the excess
risk for B non-Hodgkin’s lymphoma. Retrospective studies
showed that the overall incidence (number of new diagnoses
in a given year), of Thymoma is increasing, particularly for
MG-associated thymomas. In recent studies, Thymoma incidence in MG patients was 21%, the ethnic differences reported pointing to the possible role of genetic factors (e.g., human
leukocyte antigen, HLA) in thymoma pathogenesis. Due to
Thymoma rarity, risk factors are largely uncovered, although
it appears that smoking and radiation might constitute previously unrecognized risk factors. However, large multi-center
case-control studies would be required to evaluate adequately
environmental and genetic risk factors. In Thymomas, the
resection status and Masaoka stage represent the main prog-
nostic factors; the pathological classification according to the
World Health Organization (WHO) 2004, although debated,
has been recognized as a further relevant prognostic factor.
MG, WHO histology and Masaoka stage are inter-related.
MG is related to histotype and influences stage at presentation, myasthenic cases being generally recognized in lower
stages. Two clinical/histologic settings, irrespective of MG
status, have been recognized: an early Masaoka stage A/AB
WHO subtype and a high Masaoka stage/B WHO subtype.
Basing on a wide survey of literature data it appears that
Thymomas, originally considered mostly benign tumors,
mainly if diagnosed in early stages, have to be considered potentially invasive tumors with a long natural history and tendence to relapse even decades after primary tumor exeresis.
A multidisciplinary team is involved in thymoma diagnosis
and treatment: radiologists, thoracic surgeons, pathologists,
radiation oncologists, oncologists, statisticians. Our role as
Pathologists is to be responsible for accurate biobanking, so
precious in rare tumors, for clinically meaningful diagnoses
and for participating actively to thymoma case management.
In order to integrate professional specificities and knowledge,
the International Thymic Malignancy Interest Group (ITMIG,
www.itmig.org) was established in 2010 as a non-profit organization to involve and integrate the global thymic community
to discuss and develop a consensus regarding key problems in
neoplastic thymic pathology. Consistent standard definitions
and procedures have been proposed to the world thymic scientific community (J Thoracic Oncology, July 2011,Vol 6-Suppl
3). The main problems in thymoma diagnostics and treatment
are actually under discussion and several world-based projects
have been proposed. Among the ITMIG initiatives the creation of a global Retrospective web-based database has been
realized, as well as a TET Prospective database. The Retrospective database established at HUBzero platform (Purdue
University) (http://ccehub.org), with more than 8000 cases
collected all around the world (1996-2011), is contributing to
development of proposals to AJCC/UICC for the 8th edition of
the Stage classification system, in order to establish a TNMbased Thymic Epithelial Tumor Staging system. ITMIG has
also launched a web-based Prospective database, based on
definitions developed by an international consensus and on
accrual of extensive prospective clinicopathological data.
The Prospective database opened to the overall scientific and
health community in January 2013 to record and follow longitudinally new thymic epithelial tumor patients (2012-). The
Epithelial cells giving rise to thymoma have been referred to
an endodermal origin, although the occurrence of subsets with
medullary, cortical or other phenotypes cannot be excluded.
This possibility, formerly at the basis of the morphological
European “histogenetic thymoma classification” is actually the object of renewed interest. Recently, a proteasome
β subunit expressed exclusively in thymic cortical epithelial
cells has been described in mice and humans. This subunit,
designated β5t, is a component of the thymoproteasome, a
specialized type of proteasome implicated in thymic lymphocyte positive selection. A Japanese study 1 demonstrated
that β5t, when used in a panel of diagnostic markers, could
be a relevant marker in differentiating type B3 thymomas
from thymic carcinomas, being expressed in most type B and
in some type AB thymomas. A revision and update of the
2004 WHO classification of TET is in progress, basing on
the awareness of diagnostic difficulties and on new concepts
in thymoma differentiation and subtype heterogeneity. In
TET, over the past years, significant genetic and molecular
biological studies have been addressed to the definition of
185
relazioni
biological entities/molecular subsets with potential clinical
and therapeutic relevance. Among these studies, few gene expression profiles have been reported: by whole genome gene
expression analysis followed by unsupervised clustering of
data, four molecular tumor clusters correlating with histology
were reported 2. In the last years, the Department of Pathology at the Regina Elena National Cancer Institute, Rome, has
been participating to the international collaborative network
and Projects promoted by ITMIG 3, and has been promoting
own national/international studies in TET. Own findings contributing to the biological basis supporting targeted therapies
and new findings in MicroRNA as modulators of control gene
expression will be presented.
References
1
Yamada Y, Tomaru U, Ishizu A, et al. Expression of proteasome subunit beta5t in thymic epithelial tumors. Am J Surg Pathol
2011;35:1296-304.
2
Badve S, Goswami C, Gokmen-Polar Y, et al. Molecular analysis of
thymoma. PLoS One 2012;7:e42669.
3
Marchevsky A, Marx A, Strobel P, et al. Policies and reporting
guidelines for small biopsy specimens of mediastinal masses. J Thorac
Oncol 2011;6:S1724-9.
Simposio II parte - Tavola rotonda
La diagnosi molecolare nella pratica clinica
quotidiana del carcinoma polmonare non
a piccole cellule: opportunità e controversie
G. Fontanini
Dipartimento di Patologia Chirurgica, Medica, Molecolare e di Area
Critica, Università di Pisa
Il carcinoma polmonare è la principale causa di morte cancrocorrelata nel mondo con 1,4 milioni di decesso ogni anno. Nel
decennio passato sono state ottenute importanti acquisizioni
nella comprensione della patogenesi e della gestione di questo tumore con particolare riferimento all’adenocarcinoma.
Nello specifico, la scoperta dell’importanza biologica e terapeutica di alterazioni acquisite di alcuni geni (quali EGFR,
ALK, KRAS) che codificano per specifiche tirosino-chinasi,
coinvolte nella crescita tumorale e farmacologicamente aggredibili, ha aperto nuovi scenari nel percorso diagnostico e
terapeutico del carcinoma polmonare.
La pratica clinica quotidiana del laboratorio di anatomia
patologica si è modificata radicalmente con l’introduzione
di indagini molecolari per l’identificazione di quelle alterazioni geniche che, sulla base dei risultati di numerosi studi
clinici 1-4, rappresentano dei marcatori irrinunciabili nella
gestione del paziente con carcinoma polmonare non a piccole cellule. In particolare, la valutazione delle alterazioni dei
geni EGFR e ALK, visto il ruolo assunto nel trattamento di
prima linea dell’adenocarcinoma metastatico da alcuni inibitori tirosino-chinasici quali gefitinib, erlotinib e crizotinb,
inibitori dell’attività chinasica dei suddetti geni, è diventata
fondamentale nel percorso diagnostico-terapeutico dei carcinomi metastatici.
Il percorso di diagnostica molecolare all’interno del labora-
torio di Patologia Molecolare nella pratica clinica quotidiana,
dovrebbe seguire precise indicazioni e rispondere a specifiche
caratteristiche che siano in grado di definire l’appropriatezza
di tale percorso. I punti chiave nella definizione della appropriatezza della diagnostica di patologia molecolare sono:
a) integrazione nei Percorsi Assistenziali (PA); b) adesione
alle raccomandazioni delle società scientifiche di riferimento
(LG); c) adesione ai controlli di qualità nazional ed internazionali; d) adozione di procedure validate CE per IVD; e)
razionalizzazione e controllo dei costi.
Tali punti chiavi sono stati applicati in maniera specifica
all’interno del nostro laboratorio, nel percorso diagnostico
molecolare dei Carcinomi Polmonari non a Piccole Cellule,
tenendo conto delle necessità cliniche in relazione alla pianificazione terapeutica e alle opportunità metodologiche e
diagnostiche proprie del laboratorio stesso.
Bibliografia
1
Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-57.
2
Mitsudomi T, Morita S, Yatabe Y, et al.; West Japan Oncology Group.
Gefitinib versus cisplatin plus docetaxel in patients with non-smallcell lung cancer harbouring mutations of the epidermal growth factor
receptor (WJTOG3405): an open label, randomised phase 3 trial.
Lancet Oncol 2010;11:121-8.
3
Rosell R, Carcereny E, Gervais R, et al.; Spanish Lung Cancer Group
in collaboration with Groupe Français de Pneumo-Cancérologie and
Associazione Italiana Oncologia Toracica. Erlotinib versus standard chemotherapy as first-line treatment for European patients
with advanced EGFR mutation-positive non-small-cell lung cancer
(EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 2012;13:239-46.
4
Shaw AT, Kim DW, Nakagawa K, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med
2013;368:2385-94.
Corso breve: hot topics e controversie in patologia toracica
Coordinatori: B. Murer (Mestre), O. Nappi (Napoli)
L’attuale classificazione dell’adenocarcinoma
del polmone: criticità per il patologo
A. Cavazza, E. Tagliavini, M. Ragazzi, G. Rossi
UO Anatomia Patologica, Arcispedale S. Maria Nuova/IRCCS, Reggio Emilia
Gli importanti cambiamenti che negli ultimi anni sono intervenuti nel campo dell’adenocarcinoma polmonare hanno
portato alla pubblicazione nel 2011 di una nuova classificazione 1, che verrà incorporata nella prossima edizione della
classificazione WHO ma che già ora sostituisce l’attuale 2 per
la parte riguardante l’adenocarcinoma. Le principali novità
sono le seguenti:
–Viene eliminato il termine carcinoma bronchioloalveolare
(BAC).
Vengono introdotte le entità adenocarcinoma in situ e adenocarcinoma minimamente invasivo per quegli adenocarci-
186
nomi singoli, di dimensioni ≤ 3 cm, che hanno nel primo caso un esclusivo pattern di crescita lepidico, nel secondo caso
un prevalente pattern lepidico con aree infiltrative ≤ 5 mm.
Entrambe le neoplasie sono costituite di solito da cellule non
mucinose, raramente da cellule mucinose o da una commistione delle due. Nel caso di focolai infiltrativi multipli, deve
essere valutata la dimensione del focolaio maggiore e non la
somma dei diversi focolai. In alcuni casi, il riconoscimento
delle aree infiltrative può essere difficile 3.
–Così definiti, adenocarcinoma in situ e adenocarcinoma
minimamente invasivo sono poco frequenti ma sono associati a una sopravvivenza vicina al 100% se completamente
escissi. In generale, numerosi studi dimostrano che minore è
la componente infiltrativa di un adenocarcinoma polmonare
e migliore è la sua prognosi. Probabilmente questi studi
influenzeranno il prossimo TNM, dove il T potrebbe corrispondere alla dimensione della sola componente infiltrativa
valutata all’istologia e non alla dimensione complessiva
della neoplasia valutata macroscopicamente.
L’adenocarcinoma in situ è la lesione preinvasiva (probabilmente non l’unica) dell’adenocarcinoma periferico
del polmone, assieme all’iperplasia adenomatosa atipica.
Quest’ultima differisce dall’adenocarcinoma in situ per le
dimensioni più piccole (≤ 5 mm) e per il grado minore di
atipie citologiche, ma i limiti tra le due entità sono sfumati.
–Le diagnosi di iperplasia adenomatosa atipica, adenocarcinoma in situ e adenocarcinoma minimamente invasivo non
possono essere poste su biopsia o su citologico ma richiedono l’esame completo del pezzo chirurgico.
–L’adenocarcinoma invasivo viene classificato in base al
pattern prevalente (lepidico, acinare, papillare, micropapillare, solido-il pattern misto viene quindi eliminato), segnalando in diagnosi la percentuale di ogni pattern presente.
Si riconoscono inoltre quattro varianti di adenocarcinoma
invasivo: mucinoso (che corrisponde alla maggior parte
dei casi classificati in precedenza come BAC mucinoso),
colloide, fetale ed enterico. Pur non essendo inserite nella
classificazione, altre varianti (come ad esempio l’adenocarcinoma a cellule ad anello con castone) meritano a nostro
avviso di essere riconosciute. Questa più precisa sottoclassificazione dell’adenocarcinoma invasivo ha significato
prognostico.
–Al contrario delle precedenti, la nuova classificazione
prende in considerazione non soltanto i pezzi chirurgici,
ma anche le piccole biopsie e i campioni citologici 4 5: questo è particolarmente importante se si tiene conto che circa
2/3 dei carcinomi polmonari sono inoperabili e in questi
pazienti la biopsia e/o la citologia rappresentano l’unico
materiale disponibile. Sia su pezzo chirurgico che su
biopsia/citologico, il carcinoma del polmone deve essere
classificato in uno specifico istotipo ogni volta che è possibile farlo 6. In molti casi è per questo sufficiente l’ematossilina-eosina, ma quando non lo è (perché la neoplasia
è poco differenziata e/o le cellule neoplastiche sono poche
e/o presentano artefatti) è necessario ricorrere ad un limitato pannello immunoistochimico. Gli anticorpi più utili in
questo contesto sono TTF1 (positivo nella maggior parte
degli adenocarcinomi e dei carcinomi neuroendocrini e
negativo nel carcinoma squamoso), p40 (positivo nel carcinoma squamoso e negativo negli adenocarcinomi e nei
carcinomi neuroendocrini) e cromogranina, sinaptofisina
e CD56 (variamente positivi nei carcinomi neuroendocrini
ma occasionalmente positivi anche negli adenocarcinomi
e nei carcinomi squamosi-da utilizzare quindi soltanto
quando la neoplasia ha un “aspetto neuroendocrino” in
ematossilina-eosina). I termini generici “carcinoma non a
piccole cellule NOS” (per le biopsie/citologici) e “carcinoma a grandi cellule” (per i pezzi chirurgici) devono essere
limitati a quei pochi casi che rimangono inclassificabili,
che cioè non mostrano una chiara linea differenziativa
nemmeno quando indagati con appropriate indagini immunoistochimiche.
– Per la prima volta la classificazione è fatta non soltanto da/
per patologi, ma anche da/per clinici (oncologi e pneumologi), radiologi, chirurghi toracici, radioterapisti e biologi
molecolari, sottolineando in questo modo la necessità di
un approccio multidisciplinare al paziente con cancro del
polmone.
Bibliografia
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 Thor Oncol 2011;6:244-85.
2
Travis WD, Brambilla E, Muller-Hermelink HK, et al. Pathology and
genetic. Tumors of the lung, pleura, thymus and heart. Lyon, France:
IARC Press 2004.
3
Borczuk AC. Assessment of invasion in lung adenocarcinoma classification, including adenocarcinoma in situ and minimally invasive
adenocarcinoma. Mod Pathol 2012;25:S1-S10.
4
Travis WD, Brambilla E, Noguchi M, et al. Diagnosis of lung adenocarcinoma in resected specimens. Implication of the 2011 International Association for the Study of Lung Cancer/American Thoracic
Society/European Respiratory Society classification. Arch Pathol Lab
Med 2013;137:685-705.
5
Travis WD, Brambilla E, Noguchi M, et al. Diagnosis of lung cancer
in small biopsies and cytology. Implication of the 2011 International
Association for the Study of Lung Cancer/American Thoracic Society/
European Respiratory Society classification. Arch Pathol Lab Med
2013;137:668-84.
6
Rossi G, Pelosi G, Barbareschi M, et al. Subtyping non-small cell lung
cancer: relevant issues and operative recommendations for the best
practice. Int J Surg Pathol 2013;21:326-36.
187
relazioni
Gestione, qualità e sicurezza per l’anatomia patologica
Presentazione: G. Santeusanio (Roma) e R. Giardini (Milano)
Qualità e Sicurezza nei Sistemi Sanitari
A. Ghirardini, G. Leomporra, C. Seraschi, A. De Feo, R. Cardone
Ministero della Salute, Direzione Generale della Programmazione
Sanitaria, Ufficio Qualità delle Attività e dei Servizi
Il tema della qualità delle cure ha risvegliato, soprattutto in
questi ultimi anni, un notevole interesse e al miglioramento
della qualità e della sicurezza è stato dedicato un grande impegno. Nonostante il focus che i sistemi sanitari hanno concentrato su alcune aree quali la chirurgia, l’area materno-infantile
o l’uso dei farmaci, è evidente che il problema della qualità e
della sicurezza attraversa tutti gli ambiti sanitari.
Il miglioramento della qualità e della sicurezza quindi può
essere raggiunto mettendo in atto strategie che consentano
di realizzare cambiamenti positivi complessivi con ricadute
in ogni aspetto dell’assistenza e della cura. Esse devono
contemplare in una cornice unica, secondo il modello della
clinical governance, i molteplici aspetti che caratterizzano
la qualità quali l’efficacia, l’appropriatezza, la sicurezza,
il coinvolgimento e la partecipazione attiva dei pazienti,
l’equità di accesso ai servizi sanitari, l’efficienza e la sostenibilità. Ciascuna di queste dimensioni non può essere considerata come una entità separata dalle altre, sovrapponendosi
e potenziandosi l’un l’altra.
Il processo di sviluppo si avvale di alcuni elementi chiave che
attraversano tutte le dimensioni sopra citate quali la competenza, formazione e addestramento degli operatori, la continuità delle cure e la multidisciplinarietà, la comunicazione e la
gestione delle informazioni, la valutazione delle performance
e degli outcome, l’accreditamento dei servizi sanitari secondo
regole chiare e verificate.
È evidente che la qualità con le sue declinazioni e standard, va
considerato come un processo dinamico che muta in relazione
ai contesti, ai bisogni ed aspettative dei cittadini e alle evidenze fornite dalla ricerca scientifica.
Le politiche per la qualità e la sicurezza delle cure pertanto devono tener conto del contesto sociale, demografico, economico e storico del paese. Le principali variabili che influenzano
le scelte sanitarie sono rappresentate da: invecchiamento della
popolazione e aumento delle patologie cronico degenerative
che determinano maggiori bisogni per particolari fasce di
popolazione, sviluppo delle conoscenze scientifiche e nuove
tecnologie, complessità di modelli ed interventi basati su
evidenze, crescente impegno sul versante delle procedure e
degli esiti, pazienti più informati e consapevoli che richiedono al sistema sanitario trasparenza e accountability, nuove
esigenze di formazione tecnica e non tecnica, nuovi modelli
organizzativi per l’integrazione tra ospedale e territorio, la
nuova cornice istituzionale caratterizzata da devoluzione e
federalismo, situazione economica che impone maggiori contenimenti della spesa.
Soprattutto negli ultimi anni hanno ricevuto particolare attenzione gli aspetti finanziari e la valutazione dell’efficienza del
sistema, tuttavia, anche in momenti difficili sotto il profilo
economico, la qualità e la sicurezza delle cure devono essere
considerate la priorità per i professionisti, per i manager e
per i decisori. È in tali situazioni che più forte deve essere il
monitoraggio e la valutazione per riconoscere prontamente le
eventuali carenze e mettere in atto gli interventi necessari. Il
significato della valutazione e della misurazione delle attività e
dei servizi sanitari non è solo quello di consentire decisioni informate e consapevoli ai professionisti e decisori, di esaminare
e migliorare i processi organizzativi, ma attualmente ha anche
lo scopo di rendere conto ai pazienti e alla collettività delle
scelte e delle azioni secondo il principio dell’accountability.
Nonostante il tema del miglioramento della qualità sia comune a tutti i servizi sanitari moderni, nel nostro paese esso
presenta delle peculiarità a causa delle differenze legate alla
diversità dell’evoluzione dei diversi servizi regionali; pertanto
il superamento del divario strutturale e qualitativo dell’offerta
sanitaria tra le diverse realtà regionali costituisce ancora un
asse prioritario nella programmazione sanitaria nazionale.
Il Ministero della salute, alla luce del nuovo assetto costituzionale, è chiamato ad attuare la funzione di garanzia dell’effettiva erogazione dei livelli essenziali di assistenza, determinando la realizzazione di strumenti valutativi in grado di
rilevare le disomogeneità e le disuguaglianze della domanda
e dell’offerta, sia in termini quantitativi che qualitativi.
Tale funzione di garante nazionale dei livelli qualitativi del
sistema assume una forte valenza anche per poter rispondere
adeguatamente alle dinamiche di integrazione europea, e
soddisfare quanto richiesto dalla direttiva della Commissione
Europea sulla mobilità transfrontaliera EU/24/2011, il cui
recepimento è previsto per ottobre 2013.
In particolare, per consentire ai pazienti di esercitare i loro
diritti in materia di assistenza sanitaria transfrontaliera, ciascun Stato membro ove vengano prestate le cure, deve fornire
informazioni relative ai diritti dei pazienti, sulle procedure di
denuncia e sui meccanismi di tutela nonché informazioni sugli
standard e sulle indicazioni in merito a qualità e sicurezza definite dallo Stato membro di cura. La direttiva inoltre prevede
la costituzione di reti europee di centri di riferimento per malattie rare e alte specialità definendo una gamma di requisiti ai
quali corrispondere.
Tali scenari si riflettono sulle politiche per la qualità e la sicurezza delle cure promosse nel nostro paese.
In primo luogo si è reso necessario intervenire sul modello di
accreditamento. La normativa nazionale rappresenta l’accreditamento come uno strumento di selezione dei soggetti erogatori,
caratterizzato dalla necessaria corrispondenza ad una serie di
requisiti che sono direttamente correlati ai livelli di qualità
attesi, nonché dalla temporaneità del riconoscimento di adeguatezza agli stessi, che richiede una periodicità di controlli. La
gestione di tale sistema che richiede l’individuazione dei requisiti, la definizione delle procedure e delle modalità di verifica e
controllo, è demandato alle Regioni e alle Province Autonome,
ed ha comportato una differente evoluzione dei percorsi normativi e l’adozione di diverse modalità di implementazione.
È emersa quindi la necessità, anche in considerazione dei recenti indirizzi europei, di condividere degli elementi pregnanti
del sistema e definire un comune quadro di riferimento per
dare una nuova definizione all’oggetto dell’accreditamento,
svincolandolo da una dimensione prevalentemente strutturale
e focalizzando l’attenzione sulle diverse configurazioni che
188
concorrono all’espletamento di una funzione assistenziale.
Sono stati individuati otto criteri fondamentali declinati in
requisiti che devono far parte di tutti i sistemi regionali di
accreditamento, che divengono oggetto di verifiche da parte
di un organismo nazionale, che garantirà l’adeguato supporto
alle regioni e P.A. per lo sviluppo e l’applicazione del sistema
di accreditamento nazionale e delle bune pratiche. Questo
nuovo modello di accreditamento è stato condiviso è sottoscritto dal livello nazionale e regionale con l’Intesa del 20
dicembre 2012.
In questi ultimi anni grande spazio nelle politiche nazionali
per la qualità è stato attribuito alla sicurezza dei pazienti.
Seppure l’errore è insito in tutti i sistemi complessi e non può
essere eliminato ope-legis, la sicurezza dei pazienti deve essere continuamente presidiata e le criticità affrontate secondo
un approccio multidimensionale, considerando ed integrando
i vari aspetti: il monitoraggio e l’analisi degli eventi avversi,
la elaborazione e diffusione di raccomandazioni e buone pratiche per la sicurezza, il coinvolgimento di pazienti e cittadini,
la formazione degli operatori sanitari.
Seguendo questa logica, il Ministero della salute ha messe in
atto delle misure centrate sia sul versante della prevenzione
per adottare sistemi più sicuri e dall’altro sul miglioramento
delle capacità di analisi degli eventi avversi una volta verificatisi con l’obiettivo di identificare i “buchi” del sistema e
quindi i fattori che sono alla base degli eventi avversi.
Le azioni prioritarie hanno riguardato il monitoraggio e
l’analisi degli eventi sentinella attraverso l’istituzione di un
Sistema informativo ad hoc per il monitoraggio degli errori
in sanità (SIMES) ed è stato istituito l’Osservatorio nazionale
sul monitoraggio degli eventi sentinella, la elaborazione di.
Raccomandazioni per la prevenzione degli eventi sentinella
con l’obiettivo di diffondere informazioni relative alla sicurezza e offrire una strategia per aumentare la risposta a livello
aziendale, strategie per la formazione degli operatori e per il
coinvolgimento di pazienti, cittadini.
Qualità e gestione del rischio: una tautologia?
R. Giardini
Milano
L’ultima edizione di Eurobarometro segnala che la metà dei
cittadini europei intervistati ritiene molto probabile che le cure
mediche, ospedaliere e non, possano procurare un danno, quali infezioni ospedaliere (59%), diagnosi non corretta, mancata
o ritardata (58%), errori legati a farmaci e loro somministrazione (49%), chirurgici (46%), da strumentazione (39%). Secondo una commissione parlamentare nel 2012 in Italia sono
stati denunciati 34.000 medici ospedalieri e negli ultimi 15
anni le richieste di risarcimento sono aumentate del 250% ed
i contenziosi del 30%. È ormai chiaro che anche in medicina
errare è umano, ma anche in medicina, e particolarmente nei
sistemi complessi come l’attività diagnostica del patologo, il
problema non è dato dalla presenza di professionisti di scarso
livello che “errano troppo”, bensì da professionisti validi che
si trovano a lavorare in sistemi di scarsa qualità che debbono
essere resi più sicuri. Già Joseph Juran, l’evangelista della
qualità, aveva affermato che “per incamminarsi in maniera
rivoluzionaria sulla strada del miglioramento occorre che il
miglioramento diventi un dovere, una parte del lavoro quotidiano, scritto nel mansionario di ognuno”. In termini pratici,
un invito ad implementare un sistema che garantisca la qualità
delle nostre prestazioni. Ma con il sopravvenire della consapevolezza dell’errore, occorre riconoscere, con Atul Gawande,
che “un errore non deve necessariamente rimanere un errore”
e che la qualità non basta più, ma occorre “essere preparati
a riconoscere che qualcosa è andato storto a fare quello che
serve per rimetterlo a posto, perché la differenza tra la vittoria
e la sconfitta non sta nel non correre rischi, ma nell’essere in
grado di riparare gli errori”. Il risk management in laboratorio
rappresenta quindi l’insieme di varie azioni complesse, messe
in atto per migliorare la qualità, garantita dal sistema qualità,
delle prestazioni di laboratorio e garantire la sicurezza del
paziente, sicurezza basata sull’apprendere dall’errore. Le fasi
principali della gestione del rischio comprendono: il riconoscimento del rischio, la sua valutazione (gravità, probabilità),
gli accorgimenti per ridurlo ed il suo monitoraggio. Poiché
è dal 1926 che fu stabilito per la prima volta che in campo
anatomopatologico per un “minimum standard” “tutti i tessuti
rimossi durante intervento chirurgico dovessero essere esaminati in laboratorio e ne fosse di necessità stilato un referto, la
RCA (Root Cause Analysis) deve comprendere la disamina
e la valutazione di tutte le fonti di errore nell’attuare questo
standard minimo: accettazione, macroscopia, processazione,
trascrizione, analisi accessorie, interpretazione, refertazione.
Nella fase preanalitica, che manifesta caratteristiche per lo
più procedurali, vanno valutate preparazione e conservazione
del campione, identificazione del campione e del paziente, trasporto, accettazione e registrazione del campione; nella fase
analitica, a carattere, invece, tecnico-cognitivo-analitica, occorre prendere in considerazione esame macroscopico e prelievi, processazione, inclusione, taglio e colorazioni standard
o accessorie, l’esame microscopico ed il referto con la sua terminologia, accuratezza, completezza e tempestività analitica;
nell’ultima fase, quella postanalitica, la comunicazione è un
altro punto di rischio, con le modalità di consegna del referto e
di comunicazione delle diagnosi critiche. A titolo di esempio,
fra i tipi di errore che possono verificarsi nella fase analitica,
va considerata l’eccessiva fiducia (“overconfidence”) diagnostica, varia a seconda degli scenari professionali. È in effetti
un’errata calibrazione del proprio senso di accuratezza ed è
un fattore di rischio molto importante e difficile da valutare,
modificandosi a seconda della carriera dei professionisti: in
letteratura si è visto che gli studenti sono meno accurati e
scarsi “confidenti”, gli specializzandi sono accurati e altamente “confidenti”, mentre gli strutturati sono più “confidenti”.
ma meno accurati. I fattori che favoriscono gli errori possono
essere: 1. Input variabili (incompleta od inesatta storia clinica,
identificazione del campione o del paziente e conservazione
del campione); 2. Complessità della procedure (in un processo
il rischio di errore aumenta con l’aggiunta di passaggi: se un
passaggio ha un rischio di errore di 1%, con 25 il rischio di
errore aumenta a 22% e con 50 arriva a 39%); 3. Incongruenza
(a livello di informazione, di prestazioni individuali, di utilizzo delle procedure e del linguaggio diagnostico); 4. Processi
troppo ravvicinati o sovrapposti (non permettono di riconoscere e gestire minime discrepanze); 5. Intervento umano; 6.
Hand-off (trasferimento d’informazioni critiche: consegna del
campione biologico e del referto); 7. Vincoli di tempo (non
permettono di gestire improvvisi aumenti di carico di lavoro
od improvvisa carenza di personale). Sulla base di un solido
sistema di qualità, esistono numerose vie strategiche per
controllate e ridurre l’errore: ridurre il ricorso alla memoria,
migliorare l’accesso all’informazione, organizzare processi a
prova di errori, ridurre il ricorso all’attenzione ed alla vigilanza individuale, standardizzare procedure e terminologia,
semplificare i processi riducendo gli snodi critici, facilitare
il rilevamento degli errori nelle varie fasi dei processi. Per
esemplificare quest’ultima operazione, essa aiuta a risolvere i
189
relazioni
problemi relativi agli input variabili ed ad incongruenza se si
costruisce un sistema con molti controlli di qualità e con monitoraggio continuo per evidenziare le minime variazioni del
processo. Il sistema di qualità non è dunque una tautologia,
ma un indispensabile armamentario per gestire al meglio il
rischio anatomopatologico. L’obiettivo è avere sotto controllo
e migliorare in modo continuo tutti i processi; il vero risk
management è la prevenzione e l’eliminazione delle cause di
errore e di disturbo, nella convinzione che il miglioramento
continuo sia un obiettivo permanente dell’organizzazione. Il
risk management, sulla base della qualità garantita dal sistema
qualità, garantisce la sicurezza delle nostre azioni.
La specificità del referto anatomo-patologico
G. Raulli
Ravenna
Background. Post-analytical phase in surgical pathology is
mainly concerned with reporting and communication. Achieving quality communication means to underline the specificity
of surgical pathology report and the role pathologists play in
clinical pathways.
Methods. Literature review.
Results. The specificity of surgical pathology report is
outlined in the post-analytical phase of risk management in
surgical pathology. The post-analytical phase is essentially
the moment in which the surgical pathology report is communicated 1. In communication we realize the outstanding
features in surgical pathology: accuracy, completeness, timeliness and clinicians satisfaction 2. Besides, the complexity
of surgical pathology report for diagnostic, prognostic and
predictive information contained. Then, being at the end of a
diagnostic process, surgical pathology report is a fundamental
tool for quality control of all the steps that take place before
the surgical pathology contribute. Furthermore, it is used by
clinicians to get an informed consent aimed to therapeutic
treatment, becoming an educational tool for patient to be
aware of his disease 3. For this reason, being much of the
report highly technical and of interest only to physicians and
tumor registry, it requires a summary easy to be understood
by patient. Information addressed to the patient should focus
on easily understood concepts such as tumor type, size, extend
of spread. In other words a “patient-centered report” instead
of a “specimen-centered report” 4. Despite its inherent clinical
value, the ability of the surgical pathology report to convey
clinical information has been hindered by lack of uniformity
in format and content. Surgeons may misunderstand essential
features in up to 30% of their surgical pathology reports 5.
Errors in interpretations of these reports were less common
among experienced surgeons, but still present. On the other
side, in terms of communication between clinicians and pathologists, we can’t forget the necessity of clinical information in surgical pathology 6. Poor communication is frequently
at the root of medical errors that leads to patient injury 7. For
good communication surgical pathology report has to be well
formatted and complete. Four design principles have been
identified as useful in formatting to aid report comprehension: use headlines to emphasize key elements, maintain
layout continuity, optimize information density and reduce
clutter, nonessential information 4. As far as completeness,
use of synoptic checklists can assure adequacy of reports. Institutions in which checklists were routinely used reported all
required elements at a higher rate than those that did not use
checklist (88% versus 68%) 8. Finally, it is necessary to mea-
sure physician satisfaction with surgical pathology report in
terms of quality control and assurance 9. Satisfaction surveys
are simple tools that can provide the degree of perceptions
and expectations of clinicians. For achieving quality in surgical pathology, communication starts with surgical pathology
report, continues in clinical pathways with multidisciplinary
management 10 and requires a system of subspecialisatio 11 12
that convey trust and safety to the patient.
References
1
Zarbo RJ. The Oncologic Pathology Report. Arch Pathol Lab Med
2000;124:1004-10.
2
Nakheh RE. What is quality in surgical pathology? Journal of Clinical
Pathology 2006;59:669-72.
3
Strobel SL, Tatchell T. The Surgical Pathology Report as an Educational Tool for Cancer Patients. Annals of Clinical & Laboratory
Science2002;32:n. 4.
4
Valenstein PN. Formatting pathology reports: applying four design
principle to improve communication and patient safety. Arch Pathol
Lab Med 2008;132:84-94.
5
Powsner SM, Costa J, Homer RJ. Clinicians are from Mars and
pathologists are from Venus: clinicians interpretation of pathology
reports. Arch Pathol Lab Med 2002;124:1040-6.
6
Nakhleh RE, Gephardt G, Zarbo RJ. Necessity of clinical information
in surgical pathology: a College of American Pathologists Q-Probes
study of 771,475 surgical pathology cases from 341 institutions. Arch
Pathol Lab Med 1999;123:615-9.
7
Lingard L, Espin S, Whyte S, et al. Communication failures in the
operating room: an observational classification of recurrent types and
effects. Qual Saf Health Care 2004;13:330-4.
8
Idowu MO, Bekeris LG, Raab S, et al. Adequacy of surgical pathology
reporting of cancer: a College of American Pathologists Q-Probes
study of 86 institution. Arch Pathol Lab Med 2010;134:969-74.
9
Nakhleh RE, Souers R, Ruby SG. Physician satisfaction with surgical
pathology reports: a 2-year College of American Pathologists QTracks study. Arch Pathol Lab Med 2008;132:1719-22.
10
Nakhleh RE. Quality in Surgical Pathology Communication and Reporting. Arch Pathol Lab Med 2011;135:1394-97.
11
Black-Shaffer WS, Young RH, Harris NL. Subspecialisation of surgical pathology at Massachusetts-General Hospital. American Journal
of Clinical Pathology 1996;106(Suppl. 1):S33-42.
12
Sanders DS, Carr RA, Stores OP, et al. Subspecialisation in cellular
pathology in the DGH setting: the Warwick experience. Journal of
Clinical pathology 2006;59:884-6.
L’audit locale: uno strumento per la garanzia
della qualità del servizio di Anatomia Patologica
P. Dalla Palma
Anatomia Patologica, Ospedale S. Chiara, Trento
“We cannot change the human condition, but we can change
the conditions under which humans work” 1.
In queste parole è racchiuso tutto il senso di questa relazione.
È implicito nell’essere uomo la possibilità di sbagliare, però
possiamo modificare l’ambiente (correggere le procedure) per
evitare l’errore o almeno fare sì che sia meno probabile.
Quando si verifica un errore ci si deve innanzitutto porre due
domande:
aGraduazione dell’errore: evento sentinella o “near miss”
ovvero un “quasi errore” che siamo riusciti ad intercettare
e a correggere prima che divenisse importante per la salute
del paziente?
bRicostruzione dell’evento: verificarne le cause e le circostanze?
L’audit locale può essere (a mio parere è) la prima risposta
importante. A tal fine tutti gli attori coinvolti (medici, personale non medico e amministrativo) debbono essere convocati
per una fedele ricostruzione dell’avvento non tanto con la
finalità di trovare il/i colpevole/i (a questo semmai penserà
190
l’autorità giudiziaria o una commissione disciplinare) ma per
verificare se vi sono delle falle nell’organizzazione generale 3.
Troppo spesso i CTU si soffermano sulla correttezza o meno
della diagnosi cioè sull’errore umano mentre pongono poca
attenzione ai difetti insiti nell’organizzazione.
L’audit ha comunque oltre ad una fase istruttoria e una propositiva di eventuali modifiche organizzative anche una fase finale di verifica se le azioni correttive intraprese possono aver
effetto in futuro senza dover attendere un nuovo fallimento
alla ripetizione dell’avvento avverso.
Olson 6 in un bell’articolo dal titolo “Event management: the
heart of the quality program” ribadisce si deve far tesoro dei
propri errori anche dei più piccoli (i “near miss”) ma purtroppo sono ancora poche le organizzazioni che usano audit
strutturati per esaminarli. Gran parte di noi infatti tende a
minimizzare le non conformità ponendovi i più estemporanei
rimedi, fiduciosi che tale evento non si verificherà più e se del
caso ritenendo che è impossibile che si possano trovare rimedi
efficaci. Si allargano le braccia ritenendo l’errore insito nella
natura umana e giudicando una perdita di tempo fare audit per
piccoli incidenti proprio perché “si è sempre fatto in tal modo
e non vale la pena di cambiare”. Tutto bene fino a che non si
verifica l’evento sentinella cioè l’errore grave.
Con il Decreto Ministeriale dell’11 dicembre 2009 è stato istituito formalmente il Sistema Informativo per il Monitoraggio
degli Eventi Sentinella in Sanità. Tale sistema, denominato
SIMES, operando attraverso la piattaforma del Nuovo Sistema Informativo Sanitario (NSIS), consente a tutte le Aziende,
le Regioni e le Province Autonome di segnalare gli eventi occorsi, le cause e i fattori contribuenti individuati ma riportando anche le azioni predisposte al fine di ridurre la probabilità
di accadimento dell’evento stesso. In tal senso è parte fondante del sistema la riservatezza dei dati acquisiti che vengono
raccolti e resi anonimi ed utilizzati specificatamente ai fini
del miglioramento della sicurezza e della qualità nell’organizzazione dei servizi sanitari. Nel febbraio di quest’anno è stato
pubblicato il 4° rapporto degli eventi sentinella verificatisi
nelle strutture del Servizio Sanitario Nazionale (SSN) tra il
2005 e il 2011 5. Il numero riportato è di 1.723. Gli eventi
segnalati si sono verificati principalmente nei reparti di degenza (38,4%), seguiti dalla sala operatoria (19,6%), e l’area
di assistenza maggiormente interessata è stata la medicina
generale (13,0%), seguita da ostetricia e ginecologia (11,2%).
Purtroppo 1.723 eventi possono essere ritenuti un gran numero mentre si ritiene che in realtà il numero sia maggiore perché
la raccolta è basata sulla segnalazione volontaria dalle strutture del SSN e di tale fatto è conscio lo stesso Ministero quando
nel documento riporta: “Nonostante i buoni risultati raggiunti,
la sottosegnalazione rimane una problematica rilevante, evidenziando che le motivazioni culturali e organizzative alla
base del fenomeno sono ancora forti e diffuse”.
L’audit locale ha due livelli: uno più generale e strutturato
nei sistemi di qualità dell’intera struttura e uno limitato al
solo personale del servizio. Quest’ultimo esaminerà gli eventi
dovuti all’organizzazione interna del laboratorio e si rifletterà
nel manuale delle procedure che ogni servizio ha elaborato
magari prendendolo spunto dai numerosi lavori scientifici
apparsi nella letteratura e derivanti dalle linee guida delle
Società Scientifiche. Il referente della qualità del reparto
terrà un rapporto aggiornato dei singoli problemi che spesso
avvengono durante le normale routine e che sostanzialmente
attengono alla fase “analitica” dell’intero processo, notificandone la frequenza.
La responsabilità e i conseguenti eventuali cambiamenti
nell’organizzazione sono di esclusiva responsabilità del Ser-
vizio. La sempre maggiore diffusione dei sistemi informatici
che permettono di monitorare”chi fa che cosa” in altre parole
la tracciabilità del campione è uno step fondamentale nel processo di garanzia della qualità e della conseguente riduzione
del rischio clinico.
Tralasciando la correttezza e la completezza della fase analitica vera e propria, meno facilmente monitorabile è la fase
“post-analitica” e ancor più quella “pre-analitica”, la più uomo dipendente proprio perché coinvolge molti attori (medici,
personale sanitario non medico, amministrativi, operatori
tecnici,ecc) spesso a torto ritenuti meno importanti per l’esito
finale, cioè per l’impatto di una diagnosi patologica per la cura
del paziente 4.
I termini di cosa faccia parte della fase post-analitica sono
abbastanza chiari nella definizione non altrettanto semplici
da monitorare nel caso di avventi avversi. Nell’ottica generale della qualità vi è da chiedersi come essere certi che la
diagnosi, peraltro corretta e completa, pervenga celermente
all’indirizzo esatto (ospedale, reparto, medico curante, paziente nell’ordine) e che ne venga interpretato correttamente
il significato clinico. I mezzi informatici ci possono almeno
parzialmente aiutare: basti pensare alla posta certificata con
ricevuta di avvenuta consegna. Sull’effettivo recepimento
del significato clinico della diagnosi anatomopatologica possiamo intervenire formulando diagnosi semplici nella loro
estensione indipendentemente dalla complessità del quesito
diagnostico. A volte anche esprimere varie ipotesi diagnostiche può essere ugualmente utile perché possono indirizzare
il clinico sulle patologie più probabili. L’utilizzo dell’ultima
classificazione appena pubblicata sulle riviste specialistiche
non sempre manda messaggi univoci specialmente al medico
di medicina generale che non è e non può essere un tuttologo.
In tal caso un commento può risultare determinante per la cura
del paziente.
Tutti però concordano che la maggior fonte di errore con conseguenze almeno potenzialmente gravi per il paziente è nella
fase pre-analitica. Quest’ultima riguarda i dati anagrafici relativi alla richiesta, la motivazione della stessa, la modalità di
raccolta del materiale e l’adeguatezza dello stesso, il trasporto, il trattamento del materiale appena giunto in laboratorio,
l’etichettatura dello stesso con l’accettazione dello stesso nel
sistema di gestione del servizio. Chiaramente non tutte queste
fasi sono di responsabilità diretta del personale del servizio
ma coinvolgono piuttosto l’organizzazione generale della
struttura e spesso anche di strutture esterne a quella dove il
reparto opera.
Anche in questo campo un bel aiuto ci è stato fornito dai sistemi informatici. La pre-accettazione (order entry) direttamente
nei servizi nei quali è stato effettuato il prelievo del materiale
biologico e laddove il paziente è effettivamente presente può
ridurre molte delle difficoltà legate alla correttezza dei dati
anagrafici (così importanti per i raffronti nel data base dei
“precedenti” del servizio) e possibilmente nel corretto accoppiamento richiesta/materiale.
L’esperienza illustrata dalla collega Fabbretti su Pathologica 2 ha dato ottimi risultati (riduzione allo 0,27% di errori di
mismatching) per una situazione di una struttura che potremmo identificare come HUB a servizio di unità più piccole
identificate come SPOKE e dislocate in aree geografiche
diverse.
Dalla letteratura sembrerebbe che un gran numero di errori in
fase pre-analitica avvenga nel caso delle biopsie prostatiche
multiple. Nel corso del 2013 ho avuto esperienza diretta con
alcune situazioni di biopsie prostatiche che hanno condotto
in un caso a un evento sentinella, diligentemente segnalato
191
relazioni
al Ministero della Salute, dopo un audit locale e ad un “near
miss” risolto positivamente.
Il primo caso si riferisce a due pazienti (uno con neoplasia
maligna e uno con un quadro di atrofia) trattati lo stesso
giorno con dodici prelievi ciascuno con cognome simile ma
con scambio di attribuzione del caso. L’evento avverso si è
evidenziato tre mesi dopo allorchè una prostatectomia stata
eseguita in uomo relativamente giovane con diagnosi precedente di adenocarcinoma diffuso in realtà si è dimostrata
essere una atrofia con qualche focolaio di PIN sia pur di alto
grado. Vista l’incongruenza tra la diagnosi precedente e quella
sul susseguente pezzo operatorio è stata eseguita indagine molecolare per una compatibilità dei microsatelliti che ha dimostrato senza ulteriore dubbio trattarsi di due pazienti diversi.
Si è quindi risaliti a tutti i casi trattati nello stesso giorno e si
è individuato un paziente morfologicamente compatibile con
quello della prostatectomia. L’indagine molecolare ha dimostrato una perfetta compatibilità tra quest’ultimo paziente ed il
caso del paziente operato. L’errore formale è stato corretto ma
le conseguenze purtroppo sono state irreparabili. Per inciso è
stato avvertito e successivamente prontamente operato anche
il secondo paziente cui era stato attribuito erroneamente una
diagnosi di atrofia. L’audit interdisciplinare ha permesso di
identificare con certezza l’errore nella fase pre-analitica in
quanto il processo di tracciabilità in uso ha escluso che lo
scambio potesse essere avvenuto nella fase analitica o postanalitica. Non è stato però possibile avere la certezza se lo
scambio era avvenuto al momento del prelievo o al momento
dell’accettazione in laboratorio.
Per tale evento è stato istituito un audit in base al quale si
sono attuate delle modifiche al protocollo in uso. Si è fatta
una indagine sui “disposable” utilizzabili e si è trovato un
contenitore utilizzato negli Stati Uniti costituito da una piastra suddivisa in 12 piccoli compartimenti con già indicata
la sede del prelievo.Tale strumento è stato giudicato come
NON ottimale perché poco pratico e comunque NON scevro
da possibilità di errore nel trasferimento nelle biocassette da
inclusione. È stata perciò scelta un’altra politica quella della
pre-accettazione del caso già nelle biocassette da inclusione.
In pratica il giorno antecedente il prelievo sulla base della lista
di prenotazione del reparto di urologia, vengono prestampate
dal sistema del servizio delle biocassette riportanti il nominativo, il numero istologico generato dal sistema e il sottonumero indicante la sede standard del prelievo e consegnato
il tutto in una busta di plastica chiusa per ciascun paziente
all’ambulatorio dove vengono eseguite le biopsie. Avendo di
fronte il paziente, dopo aver eseguito il riconoscimento dello
stesso, le etichette prestampate in duplice copia in urologia
vengono apposte sia sulla richiesta standard che sulla busta
contenente le biocassette. In laboratorio non si farà altro che
trasferire il tutto nel processatore con riduzione completa della
possibilità di errore.
Questa modifica è stata adottata e verificata nei mesi successivi verificandone il miglioramento nella procedura e nello
stesso tempo intercettando alcuni piccoli problemi facilmente
rimediabili.
Una procedura sovrapponibile è già utilizzata nella nostra
struttura per la pre-accettazione dei numerosi esami di laboratorio nel servizio di biochimica clinica e di medicina
trasfusionale.
Sempre recentemente abbiamo però potuto constatare che
i controlli debbono comunque essere fatti e non ci si deve
fidare ciecamente dell’operato di altri operatori che magari
non fanno parte del Servizio. Una struttura vicina alla nostra
proprio per le biopsie prostatiche (sembrerebbe una male-
dizione!) usava una procedura simile a quella che stiamo
attualmente adottando. Venivano preparate in anticipo delle
biocassette numerate con la distinzione delle sottosedi ma
non vi veniva apposto anche il nominativo; in altre parole
veniva fatta una mezza accettazione lasciando al momento finale il match caso/paziente. Per un errore erano state
stampigliate due serie di biocassette aventi lo stesso numero
principale e quindi due pazienti successivi avevano avuto il
medesimo numero. Al momento dell’inclusione però ci si
era accorti dell’errore e conoscendo la precedente esperienza, il collega di un’altra Anatomia Patologica mi ha chiesto
di eseguire un’indagine molecolare che permettesse di attribuire correttamente il caso a ciascun paziente come è stato
effettivamente fatto.
Certo si tratta di indagini laboriose e costose e quindi la possibilità di ricorrere a tali indagini deve essere riservata a caso
specifici.
La conclusione di questa relazione è che non è sufficiente essere dei bravi patologi (nel senso di essere capaciti di formulare diagnosi corrette e complete) ma che bisogna saper governare tutto il processo. La segnalazione delle non conformità,
la graduazione delle stesse, il monitoraggio della frequenza
con cui avvengono, il controllo dell’efficacia delle soluzioni
attuate sono la base dell’audit locale che è veramente uno strumento per la garanzia della qualità totale nell’interesse finale
per la miglior cura possibile del paziente.
Bibliografia
1
Reason J. Human error: models and management. BMJ
2000;329:768-70.
2
Fabbretti G. Risk Management: correct patient and specimen identification in a surgical pathology laboratory. The experience of Infermi
Hospital, Rimini, Italy. Pathologica 2010;102:96-101.
3
Giard RWM. When is the practice of pathology malpractice? JClin
Pathol 2010;63:957-961.
4
Lippi G, Becan-McBride K, Behúlová D,et Al. Preanalytical quality
improvement: in quality we trust. Clin Chem Lab Med 2013;51:22941.
5
Ministero della Salute. Protocollo di Monitoraggio degli eventi sentinella. 4° Rapporto (Settembre 2005-Dicembre 2011) pubblicato febbraio 2013. http://www.salute.gov.it/imgs/C_17_pubblicazioni_1940_
allegato.pdf
6
Olson JD, Harrison CR. Event management: the heart of the quality.
Semin Thromb Hemost 2012;38:559-564.
Il contenzioso medico-paziente
Presidente: A. Didone (Roma); Moderatore: D. Ientile
(Palermo)
Schema di relazione introduttiva
1. Dopo la conversione in legge del decreto legge c.d. “sviluppo” n. 83/20121 – contenente l’introduzione nel codice di
procedura civile del nuovo art. 348 bis sull’inammissibilità
dell’appello – gli avvocati italiani potrebbero vedersi costretti
a spiegare al proprio cliente che il suo appello contro una sentenza ritenuta (a torto o a ragione) ingiusta è stato dichiarato
inammissibile perché “probabilmente” sarebbe stato rigettato.
In altri termini, dovranno spiegare al cliente – che ritiene di
avere ragione in una controversia – che ha avuto torto perché
“probabilmente” la controparte ha ragione e che non resta da
fare altro che proporre ricorso per cassazione.
Ma questo ricorso è ancor più problematico di prima.
Infatti, il testo del nuovo art. 348 bis c.p.c. così recita: «Fuori
dei casi in cui deve essere dichiarata con sentenza l’inammissibilità o l’improcedibilità dell’appello, l’impugnazione è dichiarata inammissibile dal giudice competente quando non ha una
ragionevole probabilità di essere accolta».
192
Sebbene la relazione di accompagnamento al D.D.L. di
conversione discorra di selezione delle impugnazioni meritevoli di essere trattate nel pieno merito, «tenendo conto che,
attualmente, nel 68 per cento dei casi il giudizio di appello
si conclude, nei processi civili, con la conferma di quello di
primo grado», non è neppure da prendere in considerazione
l’ipotesi che tra le varie concezioni probabilistiche sia stata
usata quella statistico-matematica.
Piuttosto la necessità che a mente dell’art. 348 ter c.p.c.
l’inammissibilità debba essere dichiarata «con ordinanza succintamente motivata, anche mediante il rinvio agli elementi
di fatto riportati in uno o più atti di causa e il riferimento a
precedenti conformi» fa sì che venga in considerazione il
concetto epistemico di probabilità, quale termine modale
dell’argomentazione, che fa riferimento pur sempre a dati,
ad una garanzia e ad un fondamento della garanzia. Anche
se, poi, la giustificazione dell’asserzione non è soggetta a
controllo alcuno.
Il nuovo art. 348 ter c.p.c., poi, prevede che quando è pronunciata l’inammissibilità dell’appello contro il provvedimento
di primo grado può essere proposto, a norma dell’art. 360,
ricorso per cassazione e, quando l’inammissibilità è fondata
sulle stesse ragioni, inerenti alle questioni di fatto, poste a
base della decisione impugnata, il ricorso per cassazione di
cui al comma precedente può essere proposto esclusivamente
per i motivi di cui ai numeri 1), 2), 3) e 4) dell’articolo 360.
In termini semplicissimi, non può essere denunciato il vizio
di motivazione.
Motivo, questo, che – in virtù delle nuove norme – è comunque escluso quando il ricorso è proposto contro una sentenza
di appello che conferma la decisione di primo grado ed è in
ogni caso limitato all’ipotesi di «omesso esame circa un fatto
decisivo per il giudizio che è stato oggetto di discussione tra
le parti» prevista dal nuovo art. 360 n. 5 c.p.c. per tutti i ricorsi
per cassazione proposti contro provvedimenti depositati dopo
l’11 settembre 2012.
2. Quale impatto può avere questa riforma del processo civile
nella materia del contenzioso “paziente-medico” (o meglio,
quando sarà approvato il nuovo codice deontologico, «persona assistita»-medico)?
A giudicare dai precedenti editi dopo la riforma dell’appello
civile non sembra che si tratti di materia nella quale le corti di
appello saranno propense a utilizzare il nuovo istituto dell’inammissibilità per manifesta infondatezza.
Solo i repertori di giurisprudenza dei prossimi anni consentiranno di verificare la bontà di questa previsione.
È, per converso, certa l’applicazione della riforma del giudizio
di cassazione nelle ipotesi in cui l’affermazione ovvero l’esclusione della responsabilità del medico da parte del giudice
di primo grado sia confermata da una sentenza di appello che
condivida la ricostruzione del fatto operata dal primo giudice.
In questo caso con il ricorso per cassazione non potrà essere
denunciato l’eventuale vizio di motivazione nella ricostruzione del fatto da cui siano affette le sentenze dei giudici di
merito.
Del pari certo è che, anche nell’ipotesi in cui il giudice di appello abbia riformato la sentenza di primo grado (es.: esclusa
la responsabilità in primo grado, su appello del paziente, essa
venga affermata in appello e viceversa), sarà applicabile il
nuovo testo dell’art. 360 n. 5 c.p.c., secondo il quale con il
ricorso potrà essere denunciato soltanto l’«omesso esame
circa un fatto decisivo per il giudizio che è stato oggetto di
discussione tra le parti».
Autorevolmente si è affermato che la responsabilità medica
è “il terreno di coltura elettivo e più efficace delle più au-
daci sperimentazione di nuove regole di responsabilità e ne
colpisce la evoluzione verso un vero e proprio sottosistema
di responsabilità autonomo” (Travaglino). Se fosse possibile
una quotazione, suscettibile di variazioni nel corso del tempo,
nei listini della “borsa del diritto” il titolo “responsabilità del
medico” si segnalerebbe in forte e costante rialzo (dal punto di
vista del paziente) (così Travaglino e De Nova).
Nel diagramma – sempre in salita – del listino vengono
indicate le pronunce: sul contatto sociale (Cass. 589/1999);
sull’onere della prova dell’inadempimento (Cass. ss. uu.
13533/2001; Cass. 11488/04: sussistendo un rapporto contrattuale – quand’anche fondato sul solo contatto sociale – in base
alla regola di cui all’art. 1218 cod. civ. il paziente ha l’onere
di allegare l’inesattezza dell’inadempimento, non la colpa né,
tanto meno, la gravità di essa, dovendo il difetto di colpa o la
non qualificabilità della stessa in termini di gravità (nel caso
di cui all’art. 2236 cod.civ.) essere allegate e provate dal medico); sul danno da perdita di chance (Cass. 4400/04 a Cass.
21619/07 e, più di recente, Cass. 23846/08); sull’inalterazione
della situazione pregressa (Cass. 8826/07: Il risultato “anomalo” o anormale – in ragione dello scostamento da una legge
di regolarità causale fondata sull’esperienza – dell’intervento
medico-chirurgico, fonte di responsabilità, è da ravvisarsi non
solo in presenza di aggravamento dello stato morboso, o in
caso di insorgenza di una nuova patologia, ma anche quando
l’esito non abbia prodotto il miglioramento costituente oggetto della prestazione cui il medico-specialista è tenuto, producendo invece, conseguenze di carattere fisico e psicologico);
sul contratto ad effetti protettivi verso i terzi (Cass. 14488/04
e Cass. 20320/05: la distinzione tra errore di diagnosi e somministrazione di farmaci+omessa informazione: l’ultima pronuncia: Cass. 10741/09); sulla nascita indesiderata da erronea
informazione post-intervento di interruzione di gravidanza
(Cass. 2793/1999, in passato, Cass. 6464/1994); sulla vicinanza della prova e la prova evidenziale (Cass.10297/04); sulla
responsabilità della struttura (Cass. ss.uu. 9556/2002; Cass.
13066/2004; Cass. 24791/0817, che, secondo Travaglino,
sovente trasformano la struttura sanitaria in una sorta di compagnia di assicurazione del paziente); sul consenso informato
(tra Cass. 5444/2006 e Cass. 2847/010, fino al caso Englaro)
e, in controtendenza, Cass. 975/09 (peraltro criticata in dottrina) sul frazionamento della responsabilità professionale
del medico tra inesatto adempimento e concomitante fattore
naturale costituito dalla pregressa situazione patologica del
paziente.
3. La salita del diagramma, peraltro, sembra essersi arrestata
con un contrasto che imporrà – a breve – l’intervento delle
Sezioni unite civili.
Infatti, con la sentenza n. 7269 del 22 marzo 2013, la terza
Sezione Civile della Cassazione ha affermato il principio per
il quale: «Nel giudizio avente ad oggetto il risarcimento del
danno cosiddetto da nascita indesiderata (ricorrente quando, a
causa del mancato rilievo da parte del sanitario dell’esistenza di malformazioni congenite del feto, la gestante perda la
possibilità di abortire) è onere della parte attrice allegare e
dimostrare che, se fosse stata informata delle malformazioni
del concepito, avrebbe interrotto la gravidanza; tale prova non
può essere desunta dal solo fatto che la gestante abbia chiesto
di sottoporsi ad esami volti ad accertare l’esistenza di eventuali anomalie del feto, poiché tale richiesta è solo un indizio
privo dei caratteri di gravità ed univocità».
In tal modo la Sezione ha creato un contrasto interno con la
precedente giurisprudenza secondo la quale «In tema di responsabilità del medico da nascita indesiderata, ai fini dell’accertamento del nesso di causalità tra l’omessa rilevazione e
relazioni
comunicazione della malformazione del feto e il mancato
esercizio, da parte della madre, della facoltà di ricorrere all’interruzione volontaria della gravidanza, è sufficiente che la
donna alleghi che si sarebbe avvalsa di quella facoltà se fosse
stata informata della grave malformazione del feto, essendo in
ciò implicita la ricorrenza delle condizioni di legge per farvi
ricorso, tra le quali (dopo il novantesimo giorno di gestazione) v’è il pericolo per la salute fisica o psichica derivante
dal trauma connesso all’acquisizione della notizia, a norma
dell’art. 6, lett. b), della legge n. 194 del 1978; l’esigenza
di prova al riguardo sorge solo quando il fatto sia contestato
dalla controparte, nel qual caso si deve stabilire – in base al
criterio (integrabile da dati di comune esperienza evincibili
dall’osservazione dei fenomeni sociali) del “più probabile che
non” e con valutazione correlata all’epoca della gravidanza –
se, a seguito dell’informazione che il medico omise di dare per
fatto ad esso imputabile, sarebbe insorto uno stato depressivo
suscettibile di essere qualificato come grave pericolo per
la salute fisica o psichica della donna» (Sez. 3, Sentenza n.
22837 del 10/11/2010 e altre conformi: n. 6735 del 2002, n.
11148 del 2004).
4. Riprendendo, ora, l’aspetto processualistico del tema
trattato e anticipato sub § 1, appare evidente l’influenza che
eserciterà la modifica normativa dei motivi denunciabili in
cassazione, posto che, qualunque soluzione sarà accolta dalle
Sezioni unite in ordine all’individuazione della parte gravata
dell’onere della prova, l’accertamento compiuto dai giudici
del merito in ordine all’esistenza della prova del nesso causale
tra l’omessa rilevazione e comunicazione della malformazione del feto e il mancato esercizio, da parte della madre, della
facoltà di ricorrere all’interruzione volontaria della gravidanza, in presenza di c.d. “doppia conforme”, non sarà censurabile in cassazione ai sensi dell’art. 360 n. 5 c.p.c.
In altri termini, la Cassazione non potrà mai censurare l’accertamento in fatto sorretto da una motivazione carente (contraddittoria o insufficiente).
Sembra, peraltro, da ammettere la censura – anche dopo la recente riforma – allorquando la motivazione sia solo apparente
ovvero sia a tal punto illogica da rivelarsi solo apparente.
Un esempio può essere – per restare al tema da ultimo evocato – l’affermazione (palesemente illogica) per la quale,
«secondo non precisabili regole d’esperienza, è noto che la
donna in stato di gravidanza normalmente richiede accertamenti diagnostici senza desiderare di essere informata delle
malformazioni del concepito».
Richiesta di risarcimento per danno
all’anatomopatologo: cause, frequenza
e impatto clinico degli errori in anatomia
patologica
G. Santeusanio
Università di Roma “Tor Vergata”, Roma, UOC Anatomia e Istologia
Patologica, Azienda USL Roma C, Roma
Il tasso di errore in anatomia patologica è relativamente
basso. Le stime disponibili, basate sull’incidenza di errori
evidenziati da studi retrospettivi e prospettivi 1-11, riportano
tassi complessivi di errore che variano dallo 0,5 1 al 43% 8,
mentre i tassi di errori clinicamente significativi variano
dallo 0,08 11 al 24% 7.
Le richieste di risarcimento agli anatomopatologi per errori
diagnostici sono in costante aumento 12, ma il numero assoluto
è relativamente basso quando viene paragonato alle altre specialità mediche 13. Questi dati possono sembrare rassicuranti,
193
ma in realtà non lo sono perché la media delle somme liquidate a titolo di risarcimento corrisponde approssimativamente
a 450,000 dollari per gli anatomopatologi che sono stati giudicati responsabili di errore diagnostico 14. Questa cifra, inoltre,
pone l’anatomia patologica al sesto posto, dopo la neurochirurgia, neurologia, medicina interna, la pneumologia e la
chirurgia generale, nella classifica di indennità di risarcimento
per specialità, prima della pediatria, ostetricia e ginecologia,
chirurgia toracica e cardiovascolare 13. Questa situazione può
essere spiegata dal fatto che la maggior parte dei trattamenti
terapeutici si basano sui risultati dei referti emessi dal servizio
di anatomia patologica e, di conseguenza, errori nel processo
tecnico e / o cognitivo diagnostico potrebbero tradursi in indicazioni fuorvianti la cura del paziente.
Inoltre, secondo gli studi pubblicati da David Troxel della
The Doctors Company, una compagnia assicuratrice della
responsabilità professionale con sede a Napa (California, US),
ogni anno, circa il 6% degli anatomopatologi californiani sono
citati in giudizio per richieste di risarcimento 15.
Gli errori in anatomia patologica hanno molte cause potenziali, spesso concomitanti. L’errore diagnostico può essere la
conseguenza di un “errore di sistema”, di un “errore cognitivo” e, infine, di un errore “senza colpa” che si riferisce all’incertezza circa lo stato dell’arte delle conoscenze scientifiche
anatomopatologiche al momento della diagnosi.
La richiesta di risarcimento per danni all’anatomopatologo
può essere avanzata per: (i) errori nella formulazione della
diagnosi da cui possono discendere in modo indiretto indicazioni, strategie e approcci terapeutici sbagliati, (ii) errori
nella formulazione di fattori prognostici cruciali nell’interpretazione dell’andamento clinico della malattia, (iii) errori
nell’indicazione di fattori predittivi con significato di esito
terapeutico, (iv) errore nell’attività di prevenzione di eventi
patologici e (v) ritardo nella comunicazione della diagnosi
anatomopatologica.
Da una ricerca riguardante sentenze e accordi giudiziari per
richieste di risarcimento per danno all’anatomopatologo per
errore diagnostico 16-21, troviamo al primo posto la mancata
diagnosi di melanoma; fanno seguito errori diagnostici riguardanti biopsie della mammella e casi di patologia ginecologica.
Tra i casi di diagnostica citopatologica, al primo posto troviamo la mancata diagnosi di carcinoma della cervice su PAP
test. Sono presenti anche contenziosi in cui gli errori sono
conseguenti a “errori di sistema “e non del patologo; questi
casi sono rappresentati da scambio di campioni biologici, errori di etichettatura dei contenitori dei campioni biologici, delle biocassette e dei vetrini portaoggetto, perdita del campione
biologico, contaminazione ed errori di trascrizione.
Nell’ottica di una “cultura organizzativa della sicurezza”
basata su un sistema di segnalazione non solo dei danni, ma
anche degli errori che non hanno causato danni, è auspicabile
la creazione di un database regionale degli errori e controversie giudiziarie in anatomia patologica. Ciò permetterebbe di
quantificare il fenomeno degli errori e dei relativi ricorsi da
parte dei pazienti all’istanza giudiziaria per ottenere il risarcimento per presunti danni.
Il database degli errori e controversie giudiziarie in anatomia
patologica, che può anche essere visto come strumento di
“formazione permanente” o di “ricerca clinica avanzata”, rappresenta quindi una scelta sia strategica che culturale.
Infatti i dati e le informazioni possono essere utilizzati per
formulare 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.
194
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Responsabilità medica in ambito penale
L’incremento della conflittualità medico/
paziente: analisi delle possibili cause
V. Cirese
Roma
In questi ultimi anni, si è assistito a un forte incremento delle
controversie giudiziarie (soprattutto penali) e dell’attività stragiudiziale delle compagnie assicurative, per casi di responsabilità civile avviati da pazienti, che si ritenevano insoddisfatti
o danneggiati dall’operato dei medici. Tanto in Italia come
negli altri Paesi dell’Europa, si osserva complessivamente un
aumento significativo della conflittualità nei confronti della
classe medica. Richiamando i dati contenuti nella relazione
del Comité Europeen des Assurances, dedicata al tema “La
responsabilité des professions medicales et son assurance
en Europe”, emerge evidente una richiesta di maggior tutela
dei diritti del paziente ovunque e in particolare in Germania,
Austria, Belgio e Francia. Inoltre, alcuni Tribunali (Svizzera e
Germania) applicano sempre più frequentemente l’inversione
dell’onere della prova o, comunque, rendono più facile l’onere
probatorio del danneggiato. Il numero dei casi e l’entità dei risarcimenti è ovunque in aumento, soprattutto in Belgio, Francia, Spagna e Lussemburgo. Nel Regno Unito si è verificata
una triplicazione delle richieste di risarcimento nel corso degli
ultimi 5 anni. In Portogallo, invece, il numero dei casi è rimasto relativamente esiguo, anche se di recente si è osservata
una tendenza all’aumento, sotto la spinta dell’organizzazione
dei consumatori. È interessante notare che in Svezia, a seguito
dell’introduzione del regime di “assicurazione paziente”, il
numero dei casi è minimo, circa 10 per anno.
Primarie fonti d’informazione riportano che in Italia le compagnie assicuratrici sosterrebbero la spesa globale di 2 miliardi al giorno per il risarcimento di danni ai pazienti, circa 800
miliardi di lire (più del doppio della raccolta). Le denunce
dei sinistri da responsabilità civile medica si agirebbero sulle
12.000 l’anno. Inoltre, le assicurazioni lamentano la sproporzione crescente tra i premi assicurativi incassati e le somme
destinate ai risarcimenti (rapporto 1/3, fonte ANIA) e per tale
motivo impongono tariffe sempre più alte, con maggiori oneri
per tutti.
Si assiste, inoltre, alla minaccia di uscita dal settore delle
assicurazioni più serie e qualificate (ad esempio, GENERALI, già Ina Assitalia, e RAS), con il contestuale ingresso di
compagnie straniere che, se da una parte impongono condizioni contrattuali di minor onere economico, dall’altra non
garantiscono una gestione corretta e qualificata del sinistro,
né si attivano per composizioni bonarie, stragiudiziali delle
controversie. Infine, queste “nuove” compagnie espongono
gli assicurati al rischio di una mancata o incompleta copertura, per dissesti, sofferenze temporanee, liquidazione della
società, etc..
Il fenomeno della citata conflittualità quale risultante di più
cause e fattori. La creazione, nell’immaginario collettivo, di
modelli e stili di vita incentrati sul benessere, sulla bellezza, sulla salute e sulla longevità costituisce sicuramente un
particolare fattore di incidenza sul fenomeno di incremento
della conflittualità nei confronti dei medici. Modelli creati ad
arte e supportati dai progressi della scienza e della medicina,
che hanno abituato ad attese di qualità della vita non sempre
possibili, si rilevano di indiscutibile condizionamento dei destinatari delle prestazioni.
L’attuale società, con la progressiva scomparsa di orientamenti tradizionali, appare molto concentrata sui problemi dei singoli, privilegiando beni materiali con un’ipersensibilizzazione
dei diritti individuali. Ciò ha mutato il sistema dei valori,
indirizzando verso un diverso stile di vita, verso l’autorealizzazione e la tutela del bene salute.
In tale contesto, l’operato del medico, che è considerato la
“chiave per accedere al benessere e a un futuro migliore”, è
messo in dubbio da una generazione di “consumatori”, cui è
stato insegnato a non accettare acriticamente nulla, ma a dubitare, controllare e far valere i propri diritti. Di conseguenza,
in ambito sanitario, la guarigione da una malattia viene considerata come un diritto indipendente dalle circostanze e dalle
reali possibilità di farlo valere come una prestazione, che per
essere stata in qualche modo pagata, deve essere esattamente
eseguita.
La mancata guarigione è quindi considerata “colpa” del sanitario, se non addirittura “inadempimento”.
Ne è scaturito il diffondersi di una “cultura del vittimismo”, in
relazioni
cui le “vittime” vengono rese popolari dalla televisione e dalla
stampa. Dal canto loro, i medici e le relative associazioni professionali, non hanno saputo costituire un “contraltare” con
una corretta informazione sui rischi e sui limiti della medicina, forse perché ancora non ne avvertono l’impellente necessità, sicuri di essere al riparo da ogni rischio per la semplice
operatività della copertura assicurativa (spesso insufficiente e
relativa al solo settore civile).
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
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 responsa-bile 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
195
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’istaurarsi 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
196
“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.
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’at-
tività 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).
Martedì, 29 ottobre 2013
Patologia mammaria
Simposio: unifocalità, multifocalità e linee guida tumori mammari
Moderatori: A. Sapino (Torino), V. Eusebi (Bologna)
Breast cancer multifocality: a prognostic
parameter
T. Tot
Falun, Sweden
The so-called first generation prognostic parameters (tumor
size, histology grade, and lymph node status – the components
of the TNM classification) represent the basis for staging and
prognostication in breast cancer 1. In addition, a few of the
numerous studied additional parameters have been introduced
into routine practice as prognostic and predictive markers
(estrogen and progesterone receptor status, HER2 status, Ki67
proliferation index) 2.
The prognostic information contained in subgross morphological parameters such as lesion distribution (unifocal, multifocal, or diffuse) and disease extent has remained largely
unexplored, although early publications pointed out its clinical importance decades ago 3-5.
These parameters have also been ignored by most international and national guidelines.
The latest developments in modern multimodality breast
imaging, especially introducing magnetic resonance imaging
into routine preoperative staging in some institutions, resulted
in the observation of multifocality in a considerable proportion of cases 6, thus influencing some clinicians to take this
information into account when planning surgical and oncological therapy even in the absence of adequate international
recommendations. On the other hand, lack of standardization
and generally-accepted definitions, the limited number of
related studies, and the practical difficulties in assessing these
parameters (conventional sampling method used in most
laboratories for histopathological work-up of breast cancer
specimens is insufficient for adequate assessing of lesion distribution and disease extent in many cases 7 8) hindered similar
progress of the histopathology approach.
For correct subgross characterization of a case, the following
parameters should be assessed: tumor size (defined as the largest diameter of the largest invasive focus), lesion distribution
(unifocal, multifocal, or diffuse distribution of the invasive
and in situ tumor components), disease extent (corresponding
to the tissue volume containing all the malignant structures
within the breast), intratumoral or intertumoral heterogeneity,
and the position of the tumor
within the breast 9. These parameters can be assessed with
radiological and histopathological methods, the most efficient
being a combination of these methods in the form of detailed
and systematic radiological-pathological correlation 10.
Diagnostic criteria. Multifocality is often understood to
represent foci of the same tumor in contrast to multicentricity
being the expression of multiple sites of origin of carcinoma
within the same breast consistent with the theory of the sick
lobe 11. Multifocality in breast carcinoma has been defined on
many ways in the literature focusing on the localization (same
or different quadrants), distance between the foci, or visibility on macroscopical examination. Most of these definitions
focused on invasive tumor foci not taking into account the in
situ component of the tumor. Diffuse distribution of the lesions in situ or invasive component was regularly disregarded.
In our approach 12 13, the distributions of the invasive and in
situ components of the same lesion are determined separately
but are also combined with each other to give an aggregate
pattern. Invasive lesions are considered to be ‘unifocal’if only
one invasive focus is observed in the large histological sections, which may or may not contain an in situ component.
‘Multifocal’ invasive lesions are characterized by the presence
of multiple, well-delineated invasive tumor foci separated
from each other by uninvolved breast tissue containing normal tissue, benign lesions or in situ carcinoma, regardless of
the distance between the foci. Tumors dispersed over a large
area in the section, much like a spider’s web, with no distinct
tumor mass are classified as ‘diffuse’. In situ carcinomas
are regarded as ‘unifocal’ if they appear to involve a single
terminal ductal lobular unit (TDLU) or several neighbouring
TDLUs. In situ carcinomas are designated ‘multifocal’ if they
involve several distant TDLUs with uninvolved breast tissue
in between and as ‘diffus if they involved mainly the larger
relazioni
ducts. The distribution of the invasive and in situ components
is then combined so that diffuse distribution of either the
in situ or the invasive component qualifies the lesion to be
categorized as ‘diffuse’. Multifocality of the invasive and/
or in situ component indicates a categorization ‘multifocal’.
The rest, comprising approximately a third of the cases, are
‘unifocal’tumors. The distribution of the lesions in a consecutive series of 1,000 breast cancer cases documented on largeformat histology sections is illustrated in Figure 1.
Multifocality and local control of the disease. The individual tumor foci in multifocal cases are often distant to each
other and a seemingly free surgical margin might be created
between these foci. This is a possible explanation of the observed increased local recurrence rates 14 and clearly increased
need for re-resection 15 in some published series.
Multifocality and lymph node status. Most of the related
references in the literature indicate a greater propensity of
multifocal cancers to metastasize to the lymph nodes 16 17.
Multifocality of the invasive component, as defined above, is
associated with doubled risk of axillary lymph node metastaFig. 1. Schematic illustration of the possible combined growth
patterns in breast carcinomas. (a) Unifocal in situ component, no
invasive component. (b) Multifocal in situ component, no invasive
component. (c) Diffuse in situ component, no invasive component. (d) Unifocal invasive component, no in situ component. (e)
Unifocal invasive component, unifocal in situ component within
the area of the invasive focus, and unifocal combined pattern. (f)
Unifocal invasive component, multifocal in situ component, and
multifocal combined pattern. (g) Unifocal invasive component,
diffuse in situ component, and diffuse combined pattern. (h)
Multifocal invasive component, no in situ component. (i) Multifocal invasive component, unifocal in situ component in one of
the invasive foci, and multifocal combined pattern. (j) Multifocal
invasive component, multifocal in situ component, and multifocal
combined pattern. (k) Multifocal invasive component, diffuse in
situ component, and diffuse combined pattern. (l) Diffuse invasive
component, no in situ component. (m) Diffuse invasive component, unifocal in situ component, and diffuse combined pattern.
(n) Diffuse invasive component, multifocal in situ component,
and diffuse combined pattern. (o) Diffuse invasive component,
diffuse in situ component, and diffuse combined pattern. (p)
Unifocal invasive component, unifocal in situ component outside
the invasive focus, and multifocal combined pattern. (q) Drawing
illustrating one of the possible mixed patterns with both diffusely
growing and well-delineated invasive foci, with a diffuse combined
pattern. The upper right image illustrates the sick lobe. Numbers
in the lower left corner of the drawings indicate the number of
cases in the series of 1,000 consecutive breast carcinomas belonging to that category. Reprinted from reference 13, open access
publication.
197
sis compared to unifocal cancers. The diffuse distribution of
the invasive component is associated with even higher rates of
axillary lymph node positivity. The differences are related to
large-volume macrometastatic disease. In one of our studies,
20.3% of unifocal, 48.2% of multifocal, and 61.9 % of diffuse
invasive carcinomas had lymph node macrometastasis. These
differences were independent of tumor size or tumor grade 18.
Multifocality and survival. Three independent recent publications evidenced the impact of lesion distribution on survival 19 21. The 10-year disease specific cumulative survival was
89.6% in carcinomas with unifocal, 76.0% in carcinomas with
multifocal and 63.6% in those with diffuse invasive component
in one study. The differences were statistically highly significant. Similar results were found with regard to aggregate (in
situ + invasive) growth patterns 21. The impact of multifocality
on survival seems to be independent of the applied therapy 20.
Conclusions. Multifocality is an important morphologic prognostic parameter in breast carcinoma with significant impact
on local recurrence rates, metastatic potential of the tumor and
patients’surviva
References
1
AJCC. Cancer Staging Handbook, 7 ed. New York- Londo:n SpringerDordrecht Heidelberg 2010.
2
WHO. Classification of Tumors of the Breast. Eds. Lakhani SR, Ellis
IO, Schnitt SJ, et al. International Agency for Research on Cancer,
Lyon 2012.
3
Gallager HS, Martin JE. The study of mammary carcinoma by mammography and whole organ sectioning. Early observations. Cancer
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4
Egan RL. Multicentric breast carcinoma: clinical-radiographic-pathologic whole organ studies and 10-year survival. Cancer 1982;49:1123-30.
5
Holland R, Veling SH, Mravunac M, et al. Histologic multifocality of
Tis, T1-2 breast carcinomas. Implications for clinical trials of breast
conserving surgery. Cancer 1985;56:979-90.
6
Tucker FL. Imaging-assisted large-format breast pathology: program
rationale and development in a nonprofit health system in the United
States. Int J Breast Cancer 2012;2012:171792.
7
Foschini MP, Tot T, Eusebi V. Large section (macrosection) histologic slides. In: Silverstein MJ, ed. Ductal Carcinoma in situ of the
Breast, 2nd ed. Philadelphia: Lippincott, Williams and Wilkins 2002,
pp. 249-54.
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Tot T. Cost-benefit analysis of using large-format histology sections in
routine diagnostic breast care. Breast 2010;19:284-8.
9
Tot T, Tabár L, Dean PB. Practical Breast Pathology. Stuttgart-New
York: Thieme 2002.
10
Tot T, Tabár L. The role of radiological–pathological correlation in
diagnosing early breast cancer: the pathologist’s perspective. Virchows Arch 2011;458:125-31.
11
Tot T. DCIS, cytokeratins, and the theory of the sick lobe. Virchows
Arch 2005;447:1-8.
12
Tot T. The clinical relevance of the distribution of the lesions in 500
consecutive breast cancer cases documented in large-format histological sections. Cancer 2007;110:2551-60.
13
Tot T. The role of large-format histopathology in assessing subgross morphological prognostic parameters: a single institution
report of 1000 consecutive breast cancer cases. Int J Breast Cancer
2012;2012:395415.
14
Joergensen LE, Gunnarsdottir KA, Lanng C, et al. Multifocality as a prognostic factor in breast cancer patients registeres in Danish Breast Cancer
Cooperative Group (DBCG) 1996-2001. Breast 2008;17:587-91.
15
Coopey S, Smith BL, Hanson S, et al. The safety of multiple reexcisions after lumpectomy for breast cancer. Ann Surg Oncol
2011;18:3797-801.
16
Andea A, Wallis T, Newman L, et al. Pathological analysis of tumor
size and lymph node status in multifocal/multicentric breast carcinoma. Cancer 2002;94:1383-9.
17
Coombs NJ, Boyages J. Multifocal and multicentric breast cancer:
Does each focus matter? J Clin Oncol 2005; 34:7497-502.
18
Tot T. Axillary lymph node status in unifocal, multifocal, and diffuse
breast carcinomas: Differences are related to macrometastatic disease. Ann Surg Oncol 2012;19:3395-401.
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. Boyages J, Jayashinghe UW, Coombs N. Multifocal breast cancer and
survival: each focus does matter particularly for larger tumors. Eur J
Cancer 2010; 46:1990-6.
20
Weissenbacher TM, Zschage M, Janni W, et al. Multicentric and mul19
21
tifocal versus unifocal breast cancer: is the tumor-node-metastasis
classification justified? Breast Cancer Res Treat 2010; 122:27-34.
Tot T, Gere M, Pekar Gy, et al. Breast cancer multifocality, disease
extent and survival. Hum Pathol 2011; 42:1761–9.
Simposio: unifocalità, multifocalità e linee guida tumori mammari
Moderatori: A. Sapino (Torino), V. Eusebi (Bologna)
Linee guida e indicatori di qualità
A. Rizzo
Castelfranco Veneto
Breast cancer is one of the most frequent cancer and accounts
for the largest number of cancer-related deaths in women in
Europe. Systematic screening of the female population based
on mammography offers the perspective of saving many lives
while reducing the negative side effects of treatment by detecting cancer at earlier stages.
These benefits can only be achieved, however, if the quality
of services offered to women is optimal – not only with regard
to the screening examination, but also the further diagnostic
procedures, and the treatment of women for whom the screening examination yields abnormal results. Quality assurance of
population-based breast screening programmes is therefore a
challenging and complex management endeavour encompassing the entire screening process.
By working together to develop and implement comprehensive guidelines, women will receive the same high level
services for breast screening 1.
It has also helped to reveal that implementation of high quality
standards in regional and national population-based screening
programmes naturally leads to further innovation and improvement in the quality of breast services provided outside
of screening programmes.
The scope of the guidelines so as to include quality assurance
of multidisciplinary diagnosis of breast cancer, standards for
specialist breast units and a certification protocol for diagnostic and screening services. We knew that modern medical
undertakings require specific training, accreditation, quality
assurance and evaluation, including audits by outside teams 2.
The European guidelines (LGE 2006) have greatly reduced
the differences among EU countries in the quality of care of
breast disease.
Training, multidisciplinary teamwork, monitoring and evaluation, cost-effectiveness, minimising adverse effects and timeliness of further investigations can be improved; accreditation,
rigorous quality assessment and evaluation by outside experts
can be implemented, reflecting their crucial place in any
breast unit. A major change will occur with more widespread
use of accreditation/ certification of clinics and hospitals providing breast services.
The pathologist is a key member of the multidisciplinary team
and must participate fully in preoperative and post-operative
case discussions. Accurate pathological diagnosis and the
provision of prognostically significant information are vital
to ensure appropriate patient management as well as accurate
programme monitoring and evaluation 3. A key objective of
the QA programme is to unify the terminology and diagnostic
criteria employed in reporting breast lesions. The pathologist
seek a second opinion in cases where doubt remains.
The GIPAM – Italian group of breast pathology study, born in
2012 on Italian Pathology Society (SIAPEC) – will provided
guidelines for italian pathology institutions that will may represent the base one of the requirements for the accreditation of
breast units at senonetwork. Non-operative diagnosis quality
assurance is very important to ensure appropriate patient management: Absolute and complete sensitivity, specificity, positive predictive value of a C5/B5 diagnosis, positive predictive
value of a C4/B4 diagnosis, false negative and false positive
rate, inadequate rate from cancers are key tools for the clinical evaluation of the effectiveness of FNAC or core biopsy.
Comparison of measures with advised targets then gives an indication of the performance of a unit. Achieving these targets
depends both on the pathologist or cyto-pathologist
interpreting the NCB or FNAC and on the operator taking the
specimen. It is strongly recommended to assess these parameters considering compared to standards proposed by the LGE
2006 and to perform periodic audit and re-check cases false
positive or negative diagnosis.
Multidisciplinary discussion of NCB and FNAC and audit
of preoperative diagnosis is an essential component of a QA
programme: all therapeutic resections should be discussed at
an MDM with correlation between pathologist, surgeon, radiation and medical oncologist prior to instituting appropriate
management.
Quality assurance of pathological diagnosis is well-developed
in some European member states but is absent or rudimentary
in others. There are two areas of pathological practice relating to breast cancer which are generally subject to quality
assurance in countries where a quality assurance program is
well-developed: 1. quality assurance of pathological diagnosis; 2. quality assurance of receptor testing and reporting on
immunohistochemistry and QA of in-situ hybridisation (ISH).
For example, regional QA for ER, PR, HER2 and Ki67 are
run in Piemonte region in Italy every four months and every
year in Emilia Romagna, Trentino Alto Adige and Veneto.
QA for HER2 gene analysis by FISH is performed in Italy
(www.siapecpiemonte.it/cqfish) in order to follow ASCO/
CAP HER2 guidelines.
A 1999 study at Johns Hopkins University’s School of Medicine 4 found that 86 of 6,171 pathology reports, or 1.4%, contained errors serious enough to change the diagnosis. These
errors included mistakes in staging, misidentification of the
type of cancer, and misdiagnosis of a benign tumour as malignant (and vice versa). External quality assurance schemes are
a method of ensuring that pathological diagnosis is as consistent as possible. The major schemes operating in the different
countries that run an EQA scheme mostly use slide circulation
or virtual EQA with virtual slides.
The largest slide circulation scheme is the UK National Health
Service Breast Screening Programme NHSBSP EQA scheme.
This has approximately 750 participants and uses blocks from
resection specimens sent in by the various participants to a
central laboratory where 70 slides are cut from each block
and checked for consistency of diagnostic criteria throughout
the cut sections. In Italy there isn’t national scheme, but some
regional scheme operate such as circulation of slide. The
199
relazioni
use of digital slides would represent a helpful alternative for
the EQA 5, e.g Emilia Romagna (http://anatomiapatologica.
ausl.bologna.it/pacs/index.php/telepatologia.html). In Veneto,
Italy, an Aperio ScanScope-XT slide scanner have been applied to breast EQA since 2009. 23/24 laboratories active in
Veneto joined the project. The images have been stored on a
virtual slide repository available online for a web consultation (https://servizi.azisanrovigo.it/). The discordant cases
have been widely discussed in a year plenary meeting. Cases
are selected to provide a range of diagnoses covering the
major diagnostic categories: the case material should consist
of good quality examples of common breast histopathology
conditions 6. The evaluation of the diagnostic concordance of
core biopsy (routine and consecutive set of 20 cases: 2011) of
each centre matched with gold standard was good-excellent
(k statistics weighted: 0.61-0.90; p < 0.001) for 16 institutions
(80%), moderate (k 0.54-0.57) for 4, low for 0.
In U.S.A., proficiency testing (PT) in cytopathology was
introduced nationally in 1988 as part of the Clinical Laboratory Improvement Amendments (CLIA ’88) to ensure ‘‘…
periodic confirmation and evaluation of the proficiency of
individuals involved in screening or interpreting cytological
preparations…’’ 7. However, implementation of this regulation did not occur until almost 20 years after the law because
there were many challenges in creating a test that adequately
addressed the subjective nature of cytologic interpretation and
that replicated normal working conditions, as the regulation
required 8. Unlike other areas of the clinical laboratory, where
test performance can change relatively quickly because of instrument calibration or reagent lot, cytological testing is based
on human interpretation. The current PT model has also been
criticized for not reflecting normal practice, which the CLIA
regulation requires: in fact the PT does not include established
Bethesda System categories for reporting uncertainty, such
as atypical squamous cells of undetermined significance. Pathology is also a very collaborative practice, which includes
regular rescreening of cases and frequent consultation with
colleagues on difficult cases; however, the current PT model
does not permit consultation and collaboration 9.
To conclude, the good laboratory pratices cannot be based
only to proficiency test but should include the set of actions
described above, this consideration globally considered and
conducted continuously over time.
References
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
Eusoma. The requirement of a specialist breast unit. Eur J Cancer
200;36:2288-93.
3
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.
4
Kronz JD, Westra WH, Epstein JI.: Mandatory second opinion surgical
pathology at a large referral hospital Cancer 1999;86:2426-2435
5
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.
6
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).
7
Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988 final rule. Fed Regist.
1992;57(40):7001–7186. Codified at 42 CFR §405 (42 U.S.C. 263A).
http://www.access.gpo.gov/nara/cfr/waisidx_04/ 42cfr493_04.html.
Accessed 5/10/12
8
Moriarty AT, Crothers BA, Bentz JS, et al. Automatic failure in
gynecologic cytology proficiency testing: results from the College
of American Pathologists’proficiency test program. Arch Pathol Lab
Med 2009;133:1757-60.
9
Howell LP, Nayar R, Savaloja L, et al. The Role of Proficiency Testing in Ensuring Quality: Findings From the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference
Working Group 3. Arch Pathol Lab Med 2013;137:183-9.
Androgen receptor and biological profile
in breast carcinoma
C. Riva, L. Cimetti, F. Sessa
Department of Surgical and Morphological Sciences, University of
Insubria-Varese, Italy
The role of estrogen (E) and estrogen receptors (ER) in the
pathogenesis and progression of breast carcinoma (BC) is
widely acknowledged. Over the past decades androgens (A)
and androgen receptors (AR) also have emerged as important factors involved in the pathogenesis of BC even if their
clinical significance and functional role have not been yet
completely elucidated. Studies attempting to establish a link
between A and BC started in earlies 1950.
Both E and A are important for normal breast development.
The balance between the stimulatory effect of E and the inhibitory effect of A functions as a critical factor that regulates
mammary cell proliferation, both in normal and in cancer tissues, even if in vitro studies reported that testosterone (T) and
DHT both stimulate and inhibit the growth of various breast
cancer cell lines 1-3.
Androgens, secreted by both ovaries and adrenal glands, are
necessary precursors for E synthesis in the ovary and they
constitute the dominant sex steroid hormones throughout a
woman’s entire life. After menopause, the imbalance between
A and E levels causes an adaptation in order to survive in a
low estrogenic milieu, leading the epithelial cells to a higher
capability of surviving, both increasing the local E synthesis
and the cells responsiveness to E (rising the levels of aromatase, ER and coactivators). These modifications could be at
the basis of steroid driven carcinogenesis 2.
Epidemiological evidences have shown the association between BC risk and A; in particular, it has been observed that
high serum T levels can precede BC occurrence in both postmenopausal and pre-menopausal women 3-6. Also, hormone
replacement with combined E and T had a significantly higher
risk for BC over E alone 7. Controversial data have been also
reported suggesting that A levels might have a protective role 3.
Androgens exert their effect in mammary epithelial cells via
two separate pathways, either directly via AR or indirectly
through aromatisation to E. Androgens binding to the intracellular AR is encoded by AR gene, a member of the nuclear
hormone receptor superfamily comprising ligand-responsive
transcription regulators. Finally AR functions as a transcription factor, which regulates the activity of other genes 1-3.
AR can also be activated by androgen-indipendent pathways
involving mutation, overexpression and postranslational modifications of AR and AR-associated coactivators. One of them
is associated with EGFR/HER-2 family through Akt or Ras/
Raf/MAPK pathway. In addition BRCA 1 has been identified
as a coactivator of AR 2.
Sparse epidemiological data suggested that a functional
polymorphism in the AR gene, in particular a long AR-CAG
repeat yielding a less active AR might be associated with increased breast cancer risk 1.
AR is expressed in various human tissues, including normal
breast of both pre- and postmenopausal women, where it is co-
200
localized with ER and PR in epithelial ductal and acinar cells,
but it is not found in mammary stroma or myoepithelium 8.
AR has been detected in some benign breast lesions, including
apocrine metaplasia and apocrine adenosis 8-10, papillomas, ductal
hyperplasia and epithelial component of fibroadenomas (personal observations). Male gynecomastia also shows AR positivity.
The presence of AR in BC has always been appreciated from
earlies 1990s 11 12. More recently, aided by more sensitive
methods of detection, a large body of evidences demonstrated
that AR, somewhat paradoxically, is the most commonly
expressed nuclear hormone receptor in BC. Infact several
immunohistochemical studies indicate AR positivity in a considerable proportion of BC, ranging approximately from 70%
to 90% of the cases 12-14 22 23, more frequently with a colocalization of ER and/or PR.
Some studies have evaluated the patterns of AR expression
in different histotypes of BC, even if the majority of the data
refer to ductal carcinomas 11-13. A strong and peculiar positivity has been described both in situ and invasive apocrine
carcinoma, and this intense AR+ was often accompanied
by a lack of ER and PR expression 10-13. AR expression is a
prominent feature of lobular neoplasias (87%) where ER and/
or PR are almost always coexpressed 14. AR expression has
been also found in BRCA-mutated tumors, in medullary 14, in
mucinous, in neuroendocrine BC (personal unpublished data),
in some sarcomatoid neoplasms and in mammary Paget’s disease along with ER-/HER-2+ 16. Approximately 80% of male
BC reveal AR positivity.
The vast majority of G1/G2 BC are AR positive, with a consistent coexpression of ER and PR 12 14; AR is much more
frequently expressed than ER and PR in G3 BC 13 17.
During last years AR has been diffusely investigated in
relation to molecular phenotype of BC, with the emerging
evidence of a variable frequence across various types: AR
is most common in luminal A (91%) and B (68%)BC 25. AR
is also frequently observed in HER2+/ER- (from 74% to 76
%) 12 15 18 20 26.
AR has been found in a significant subset of triple negative
basal-like cancers (ranging from 27 % to over 43% in different series) 15 25 26. The presence of AR in approximately one
third of basal like triple negative BC provides further evidence
that this is a heterogeneous group of tumors with peculiar
biological profiles.
The role of AR during the progression of breast cancer remains unclear, even if the expression of this hormone receptor has been documented from early to advanced stage of the
disease, moreover in about 25% of breast cancer metastases
AR are the sole sex steroid receptors observed. Early involvement of AR in DCIS is reported with a proportion of positivity ranging from 31 to 95% of the cases, coexpression of ER
and PR status in low- and intermediate grade tumors 9 12 22 25
and AR positivity along with ER negativity in over 60% of
high grade in situ lesions. Hanley found a strong correlation
between loss of AR and basal subtype of high grade invasive
BC compared to AR expression in high grade DCIS suggesting its possible role for progression to invasive neoplasia 17.
Some studies have focused on the AR and prognosis of
BC: in general, the AR expression is correlated with a better
outcome. Castellano et al. 23 demonstrated that AR is an independent prognostic factor of better outcome in ER+ patients;
in a subset of luminal B cancers (receiving chemo-endocrine
therapy) it was been observed a more favourable prognosis
for time to relapse and disease specific survival 23. Thus AR
expression could be used as a prognostic marker in luminal
B tumors 23. AR expression seems to be associated with a
more favourable prognosis in ER+ postmenopausal women 24.
Moreover it has been demonstrated in vivo that AR inhibit
ER-α by binding to estrogen-responsive element and preventing the activation of target genes that mediate the stimulatory
effects of 17-βestradiol on BC cells 28.
ER-negative/ AR positive tumors have a better biological behaviour than ER/AR negative cancers 18; survival after recurrence is longer in AR positive metastatic tumors 19. Rakha et
al. reported that AR positivity in lymph-node positive tumors,
along with tumor size and lymph node stage was the most useful prognostic marker in triple negative cancers 20.
Various studies investigated AR as a target for therapy strategies. AR positive BC patients have a better response to hormonal treatment than AR-negative patients. Both in vitro and
clinical studies have shown that synthetic progestins inhibit
the proliferation of ER/PR negative breast cancers via AR 21.
Inhibitory role of adrenal steroids and DHEAS in ER-/AR+
tumors emerged from preliminary data. The possible use of
A to treat ER-/AR+ BC should be combined with aromatase
inhibitors since they block in vivo conversion of A to E 27.
In HER2+/ER-/AR+ BC cell lines with molecular apocrine
features a functional cross-talk has been demonstrated between AR and HER-2 signaling pathways, promoting cell
proliferation; this finding indicates a possible advantage in the
blockage of both AR and HER-2 28.
Peculiar AR genotypes are been recently proposed to predict
response to endocrine treatment 30.
A clear and comprensive understanding of the mechanism of
AR signalling in the breast has yet to be revealed. Therefore
the potential role and the impact of AR in BC needs further
study; however AR are promising target for therapy strategies especially in ER negative and in triple negative tumors.
Finally, AR may be clinically useful predictors.
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Hanley K, Wang J, Bourne P, et al. Lack of expression of androgen
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Agoff SN, Swanson PE, Linden H, et al. Androgen receptor expression in estrogen receptor-negative breast cancer. Am J Clin Pathol
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Schippinger W, Regitnig P, Dandachi N, et al. Evaluation of the
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Rakha EA, El-Sayed ME, Green AR, et al. Prognostic markers in
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Park S, Koo J, Park HS, et al. Espression of androgen receptors in
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Qi J, Yang Y, Zhu H, et al. Expression of androgen receptor and its
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Nahleh Z. Androgen receptor as a target for the treatment of hormonereceptor negative breast cancer: an unchartered territory. Future
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Naderi A, Hughes-Davies L. A functionally significant cross-talk between androgen receptor and EerbB2 pathways in estrogen receptor
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Lundin K, Henningson M, Hietala M, et al. Androgen receptor
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patients. British Journal of Cancer 2011;105:1676-83.
Lettura Magistrale: le cellule staminali tumorali
R. De Maria Marchiano
Istituto Tumori Regina Elena, Roma
Tumours are composed of hetergenous cellular populations
with different morphological and functional features. This
heterogeneity is similar to that of normal tissues, in which different cells are steadily organized and inter-related. The differences existing between cell types from the same tissue derive
from the process of cell differentiation, a process sustained by
adult stem cells that, residing at the apex of the differentiation
process, give rise to a variety of cell types. Stem cell activity
ensures tissue homeostasis and maintains normal cell turnover.
Scientific evidence suggests that also tumors possess a pyramidal organization (hierarchical model), representing an
201
aberrant dysfunction of the normal architecture of normal
tissue due to the neoplastic transformation of adult stem cells.
According to this model, the development and progression of
cancer are caused by an exclusive contribution of a limited
number of cancer stem cells (CSTs), from which all other
cells derive. Since 1997 CSCs have been isolated from both
haematological malignancies and solid tumours.
From the very beginning the hierarchal model seemed to be
incompatible and alternative to the clonal evolution theory
previously formulated, claiming that tumours arise and develop
after casual genomic mutations that confer a selective advantage to some clones in comparison to others. This initial discord
between the two models seems to have subsided by an attempt
to match the two processes into a brief summary. The model
that combines both theories predicts that CSCs may evolve over
time according to clonal evolution mechanisms. In this way, diverse tumour stem cell clones may be generated so that they can
evolve and differentiate independently. Overall, novel evidence
allowed to postulate a combined clonal-hierarchical model.
For instance, microenvironmental conditions, a mainstay of
evolutionary principles, have been implicated in the generation
and maintenance of the CSC pool. Among the environmental
factors that regulate CST properties are hypoxia, low Ph and
paracrine-acting factors. Furthermore, the interaction of CSCs
with their microenvironment is bidirectional. Consistently, independent studies have demonstrated that CSCs generate new
vessels. Thus, the microenvironment influences the stem cell
phenotype and CST contribute towards creating a microenviroment favourable to cancer growth.
Biological Properties and Identifying Tumour Stem Cells.
Tumor stem cells share principal characteristics and biological
properties with their normal counterparts. They are endowed
with self-renewal ability (dividing into two daughter cells
where one of them contain the same original cell properties),
have great proliferating potential and can produce differentiated cells with diverse phenotypes.
One of the most commonly used parameters used for first
level characterization of CST is the expression of specific
cell-surface markers. Bonnet & Dick were the first to observe
leukemic stem cells express CD34. Thus, they consequently
defined a biomarker profile, that was CD34+CD38-, used
in studies with leukemic stem cells for a long period of
time. In regards to solid tumours, Al-Hajj et al. identified
ESA+C44+CD24−/low in breast CSC and a little later on Singh
et al. confirmed CD133 as a marker in CST derived from
glioblastoma multiforms. Subsequent independent studies
demonstrated that this glycoprotein identifies different types
of CSC in solid tumours including colorectal and lung cancer. Nevertheless, the identification of CSCs require more
sophisticated assays, given that markers do not necessarily
identify CSCs. An example derives from glioblastoma and
colon cancer studies, in which lack of CD133 identified CSC
associated with a different molecular subtype and with different metastatic ability, respectively.
As a second level of CSC characterization is the demonstration of the ability to grow indefinitely in vitro when cultured
under specific conditions, where they have a tendency to
aggregate in typical spheroid structures (tumourspheres).
Finally, the most important criterion for CSC identification is
the ability to generate a phenocopy of the parental tumor following their injection into immunocompromised mice.
Regarding CSC functional properties, they displayed higher
resistance against chemotherapy. The drug-resistant phenotype has been connected with various molecular mechanisms.
The most significant are a proficient activation of the DNA
202
damage repair machinery, alterations in mechanisms regulating apoptosis, high expression of ABC multidrug efflux
pumps and quiescence.
DNA integrity is constantly monitored by the cell; damage
associated with environmental factors or with the cell’s metabolic activity triggers the activation of a signal cascade that
recruit repair mechanisms or, when the damage exceeds the
cell’s repair capability, apoptotic pathways. The checkpoints
kinases 1 and 2 (Chk1 and Chk2) play a central role in regulating genomic integrity and in triggering repair processes. CSC
isolated form colorectal cancer, lung cancer and high-grade
primary brain tumors showed a more rapid activation of these
signals, which account for the increased chemoresistance of
CSCs compared to their differentiated counterpart.
Another molecular mechanism that enable CSCs to endure
stressful conditions such as exposure to chemotherapy is an
apoptotic balance favoring anti-apoptitic signalings. It has
been observed that in epithelial tumours interleukin-4 (IL-4)
plays an important role in the induction of antiapoptotic signals. This cytokine is produced in various tumours and seems
to be crucial in eliciting and maintaining a chemoresistant
phenotype in CST.
Next, it has been observed that similar to their normal cell
counterpart, CST express high levels of ATP-Binding Cassette Transporters, a system of transmembrane pumps that
extrude a wide variety of chemotherapeutic agents outside the
cells. Finally, cellular quiescence has been implicated in CSC
chemoresistance, as chemotherapy selectively kills rapidly
proliferating cells.
Targeted Therapies. Chemotherapy treatment contain many
limitations, the most obvious of these is not being directed
towards specific targets deregulated in tumours. CST isolation
has given a fresh boost in cancer research, allowing the investigations of novel pathway-targeted inhibitors targeting molecular signals preferentially activated in CSCs. The strategies
that have been used until now have aimed to block the signal
transduction pathways that promote self-renewal, inhibit
chemoresistant mechanisms, activate their differentiation or
interfere with the signals deriving from the microenvironment
to maintain the stem cell phenotypes.
In normal stem cells, the Hedgehoh (Hh), Wnt and Notch pathways play a central role in maintaining stem cell identity and
sustaining self-renewal process. These same pathways are often
deregulated in CST and have been the focus of clinical studies.
Hh antagonists are the self-renewal pathway inhibitors at the
most advanced stage of clinical development having revealed
promising activity against tumours carrying mutations in key
pathway components, such as basal cell carcinoma and medulloblastoma. Interestingly, Hh pathway inhibition significantly hampers clonogenicity of glioblastoma stem cells and
preferentially depletes pancreatic CSCs.
Abrogation of the Notch pathway via gamma-secretase inhibitors or anti-ligand antibodies have shown to be efficient
against CSCs isolated from glioblastoma multiforme, breast
cancer and colorectal cancers. Nevertheless, in preclinical
models antibody-mediated inhibition of Notch pathway has
been connected with the onset of vascular tumors and liver
histopathological alterations.
The therapeutic relevance of inhibiting the WNT pathway
is mostly indirect, and stemmed from the concept that interrupting the CSC-microenvironment crosstalk may have detrimental effects on CSC fitness. Consistently, myofibroblastsecreted factors instructed differentiated colon cancer cells to
activate the β-catenin-dependent transcription, leading to gain
the stem-like phenotype. 6° Congresso Triennale Siapec-IAP • Roma, 26-30 OTTOBRE 2013
A second class of potential anti-CSC agents are compounds
able to render them sensitive to standard chemotherapeutic protocols. The overexpression of ABC transporters represents one
of the mechanisms proposed to explain the multidrug resistant
feature of stem cells and CST. Therefore, various drugs have
been developed to interfere with these proteins. Although firstand second-generation inhibitors did not produce any significant clinical benefits, third-generation antagonists were synthesized and are under clinical investigation. To revert chemo- and
radioresistant, another potential therapeutic approach consists
in blocking DNA repair pathways. Preclinical studies demonstrated that the inhibition of Chk1 is capable of restoring CST
susceptibility to chemotherapy. Recently, various Chk1 and
Chk2 inhibitors entered clinical trials. Nevertheless, the clinical
development of some of these compounds was halted as safety
concerns, mostly cardiotoxicity, emerged from phase I studies
Next, the concept that CST are more resistant to cell death
compared to more differentiated cells prompted a series of
investigations aimed at interfering with the apoptotic machinery. For instance, blocking IL-4 by a specific antibody
significantly interferes with survival of colorectal CSCs and
their chemoresistance.
Finally, forcing differentiation has been proposed to restore
the sensitivity of CSCs to chemotherapy. Glioblastoma stem
cells exposed to the differentiation-inducing agent retinoic
acid underwent both growth arrest and expression of lineagespecific differentiation markers, in a process accompanying
by the down-regulation of Notch components. To a similar
extent, the pro-apoptotic/pro-differentiative bone morphogenetic protein 4 sensitizes colon CSCs to 5-fluorouracil
and oxaliplatin, whereas glioblastoma stem cells exposed to
BMP4 displayed reduced clonogenic ability coupled with an
increased expression of neural differentiation markers.
Conclusion. Given the central role of CST in tumors, these cells
have attracted much attention and efforts are underway to develop CSC-directed anticancer agents. The strategies that have
been used up until now to attack CSTs have produced promising results both in vitro and in animal models. Nevertheless,
the role of these molecules are yet to be defined in the clinical
setting. However, it is worth considering that these molecules
can inappropriately attack even adult stem cells of healthy tissues, with unpredictable side effects. Finally, the growing body
of knowledge on CSC biology allowed to test CSC-related
endpoints in clinical studies in the attempt of identifying novel
and more accurate prognostic and predictive indicators. Among
them are CSC-related gene expression profiles, specific CST
markers and stem cell-related polymorphisms.
References
Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer
stem cells. Nature 2001;414:105-11.
Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med
2006;355:1253-61.
Clevers H. The cancer stem cell: premises, promises and challenges. Nat
Med 2011;17:313-9.
Shackleton M, Quintana E, Fearon ER, et al. Heterogeneity in cancer:
cancer stem cells versus clonal evolution. Cell 2009;138:822-9.
Valent P, Bonnet D, De Maria R, et al. Cancer stem cell definitions and
terminology: the devil is in the details. Nat Rev Cancer 2012;12:767-75.
Schatton T, Frank NY, Frank MH. Identification and targeting of cancer
stem cells. Bioessays 2009;31:1038-49.
Zhou BB, Zhang H, Damelin M, et al. Tumour-initiating cells: challenges
and opportunities for anticancer drug discovery. Nat Rev Drug Discov
2009;8:806-23.
Maugeri-Saccà M, Vigneri P, De Maria R. Cancer stem cells and chemosensitivity. Clin Cancer Res 2011;17:4942-7.
Maugeri-Saccà M, Zeuner A, De Maria R. Therapeutic targeting of cancer stem cells. Front Oncol 2011;1:10.
203
relazioni
Sala Massalia – 8.30-18.30
Citologia
Simposio citologia vaginale
Moderatori: L. Di Bonito (Trieste), L. Resta (Bari)
L-II level cervico-vaginal Cytology: pilot projects,
results, and critical evaluations
Flow-Chart n. 1. Flow-chart describing pilot project starting with
enrolment until II level.
B. Ghiringhello*, M.R. Giovagnoli**
*
Torino, ** Roma
The pilot project, see flow chart n. 1 (below), which began in
2010, has screened a total of over 37000 women by subjecting
them to the test consisting on the research of viral HPV DNA;
a positive result was found in 7.8% of cases, amounting to
about 2900 women.
52% of women with a positive viral test showed no positive
cytology at the first level, while among positive cases (about
19%) the distribution of the lesions was expressed as follows: of all HPV tests performed, L-SIL were 0.95%, H-SIL
were 0.16%, ASCUS 2.24% and ASC-H 0.09%; of all HPV
positive tests, L-SIL were 12%, H-SIL were 2.15%, ASCUS
3.18% and ASC-H 1.19%.
When comparing data obtained in the pilot project with those
of traditional screening, there was a slight difference essentially dependent on the L-SIL category (1.2% vs 0.9%), the
category in which the viral test proves more specific.
The comparison between triage and II level cytology shows
good concordance for L-SIL (over 64%), for H-SIL (over
87%) and ASC-H (over 56%), while for ASCUS an equal
distribution between NEGATIVE and L-SIL diagnosis is
observed.
Similar results are observed in the comparison between triage
cytology and histology and between II level cytology and
histology, except for ASCUS diagnoses, which, in more than
64% of cases, prove negative at histology.
A noteworthy phenomenon is the increase in the “positive”
(ASCUS or more) diagnosis soon after the beginning of the
pilot test, almost certainly the result of a biased overdiagnosis
due to the knowledge of the HPV DNA positivity when reading triage cytology; the re-evaluation of these cases by the
supervisor, in fact, led to a reduction in the number of ASCUS
(from 9% of total positive test to 4%) with distribution of
cases between L-SIL (increased from 20% to 22%) and negative (from 61% to 66%).
The purpose of this experiment is to value the use of viral
test as a screening tool in alternative to traditional cytology,
allowing a increase in the interval of referral for women with
a negative viral test, and a cost reduction by eliminating
the I level cytology. In addition, viral test can be a tool for
monitoring the evolution of low-grade lesion and follow-up
of patients which have already received excisional treatment.
The most critical cases are the one characterized by absence of
abnormal colposcopic cervico-vaginal findings (colposcopic
grade = 0) and / or unsatisfactory colposcopy (colposcopic
grade = X), especially for categories negative or L-SIL first
level cytology.
In these cases, as suggested by international guidelines, women are subjected to endocervical curettage (see flow chart n. 2
below) in order to identify a lesion that cannot be indentified
at colposcopy. Anyway, this approach has some drawbacks:
Flow-Chart n. 2. Flow-chart describing II level in pilot project.
besides the invasiveness of the sampling, curettages determine
a substantial increase in workload in the services of pathology, with frequent diagnostic difficulties on one side and the
recognition of only a few cases of high-grade lesions on the
other.
Another criticism is represented by a lengthening of colposcopy referral time in women with a diagnosis of L-SIL because priority is given to women with a cytological diagnosis
of ASC-H and H-SIL.
204
Citologia di II livello: l’opinione del clinico
S. Costa
LILT, Bologna
Introduzione. Confrontato con la citologia convenzionale,
il test per la identificazione del DNA del papilloma virus
umano (HPV DNA test) mostra una sensibilità maggiore per
le lesioni intraepiteliali di alto grado (CIN2/3) e carcinoma.
Tale aumento non è dovuto ad una sovradiagnosi, ma ad
un’anticipazione diagnostica, dal momento che nei round di
screening successivi si osserva una riduzione di incidenza
della malattia 1. Inoltre lo screening eseguito con l’HPV
DNA test ogni 6 anni mantiene un Valore Predittivo Negativo
(VPN) doppio rispetto a quello del Pap test triennale. Infatti
l’incidenza cumulativa di CIN3+ dopo 6 anni tra le donne con
HPV DNA test negativo all’inizio dell’osservazione è risultata
dello 0,27% (IC al 95% 0,12-0,45%), mentre l’incidenza cumulativa di CIN3+ tra le donne con un Pap test negativo dopo
3 anni era dello 0,51% (IC al 95% 0,23-0,77%) 2.
Test per la ricerca di HPV DNA come test primario nello
screening. Seguendo queste considerazioni sono stati intrapresi diversi studi randomizzati che hanno mostrato come
l’utilizzo di HPV DNA come test primario, con la citologia
come esame di II livello per i casi HPV positivi e l’eventuale ripetizione del test dell’HPV a 12 mesi in caso di triage
negativo, incrementa il tasso di identificazione delle lesioni
intraepiteliali rispetto al Pap test di circa il 30-35% 3 4 con il
12% di aumento del numero dei test richiesti
Tematiche emergenti nel passaggio dalla citologia di screening alla citologia di triage. L’utilizzo di HPV DNA come
test primario nei programmi di screening comporta una serie
di tematiche nuove sia in termini di applicazione del test che
di gestione della positività del risultato. Infatti a) lo screening
basato sul test HPV non deve iniziare prima dei 30-35 anni;
al di sotto di tale fascia, dato l’elevato numero di HPV test
positivi, resta raccomandato lo screening citologico; b) la
positività prevista del test primario è di circa 8-12%, notevolmente superiore quindi al Pap test; c) la maggiore sensibilità
del test del DNA di HPV è coniugata ad una minor specificità
il che porta a un sostanziale aumento dei falsi positivi con
una non necessaria ripetizione del test che si aggira attorno
al 10-15%, d) per la minor specificità del test, le donne positive a HPV DNA non devono essere inviate direttamente a
colposcopia, ma è necessario utilizzare sistemi di triage, e) il
sistema di triage raccomandato è il Pap tes (2-5, f) il numero di
Pap test risulta quindi notevolmente ridotto rispetto agli attuali
protocolli. Ne consegue che, in seguito alla nuova strategia di
screening, il minor numero di citologie rende necessaria una
forte centralizzazione e la funzione di test filtro rende necessario un diverso approccio della lettura che, contrariamente
al TBS, valorizzi al massimo la specificità e tenga conto non
solo della maggiore frequenza di patologia ma anche della
garanzia di un successivo controllo a un anno 5 6. Èimportante
inoltre sottolineare che il Pap test di triage proviene da una
popolazione già selezionata e a maggior a rischio di patologia,
per cui aumenta notevolmente il tasso di anormalità, che si
stima essere attorno al 30-50%. Partendo quindi da un test di
base (HPV DNA) estremamente sensibile, con elevata quota
di falsi positivi, il triage citologico deve distinguere i veri dai
falsi positivi per riportare la specificità a livelli accettabili, distinguere cioè tra le donne già selezionate da un test estremamente sensibile, quelle che abbiano evidenti atipie citologiche
e quindi maggiore rischio di patologia.
Il Valore Predittivo Positivo (VPP) della citologia di triage
dovrà essere di gran lunga superiore alla citologia di screening in quanto i Pap test anormali contengono un numero
consistente di casi positivi per HPV a basso rischio(LR HPV)
e negativi per HPV ad alto rischio (HR HPV) a bassissima o
nulla possibilità di lesione CIN2+, il che attualmente comporta una riduzione del VPP.
Refertazione della citologia come triage dopo positività
del test HPV screening primario. La diagnostica cervicale
rappresenta un importante modello di interazione multidisciplinare, alla cui definizione concorrono, con strumenti
diversi, citopatologo, istopatologo e clinico, per cui il referto
cito-istologico rappresenta uno strumento fondamentale della comunicazione fra figure professionali diverse ma dedicate ad un medesimo obiettivo 7. Poiché il ruolo del patologo
è quello di trasformare le immagini in parole attraverso
categorie codificate, il nuovo scenario richiede una rimodulazione delle categorie diagnostiche considerate sempre
più come strumento fondamentale della comunicazione fra
patologo e clinico.
Considerato che le alterazioni citologiche dei campioni
HPV negativi non arriveranno alla lettura del citologo e
che quelle dovute a LR HPV saranno estremamente limitate
(solo le eventuali coinfezioni con HR HPV), si ritiene che
l’utilizzazione della categoria ASC-US debba essere limitata al massimo o addirittura abolita, classificando nel modo
più netto possibile i quadri morfologici, mentre la categoria
LSIL deve evitare una sovra-diagnosi ed inutili e costosi
controlli ad una paziente con modificazioni citologiche
aspecifiche 5 6.
Ruolo della colposcopia. È importante ricordare che la colposcopia fu introdotta agli inizi degli anni ’20 del secolo scorso
come primo mezzo di screening e diagnosi precoce del carcinoma del collo uterino in epoca anteriore al Pap test, con lo
scopo di identificare e di curare con successo le lesioni invasive
iniziali. Ovviamente nel corso degli anni le finalità dell’esame
sono notevolmente cambiate ma la colposcopia riveste ancora
un ruolo importante nei programmi di screening poiché rappresenta l’indagine diagnostica che, guidando il prelievo bioptico,
permette di conseguire una conclusiva definizione istologica.
L’utilizzo di HPV DNA come test primario comporta profonde modificazioni concettuali anche per il clinico in quanto la
colposcopia diventa un esame di III livello da eseguire su di
una popolazione con un maggior tasso di patologia, e poiché
sovente ci si trova di fronte a quadri colposcopici che pongono
quesiti di difficile soluzione, si raccomanda che l’esame avvenga presso presidi accreditati ove operi personale qualificato che
si sottoponga a periodici controllo di qualità, dovendo essere
assai limitata la possibilità di errore 8-11. Infatti un minor numero di colposcopie con maggior tasso di patologia richiede una
maggior appropriatezza per una minore possibilità di errore. A
questo proposito è di particolare interesse l’esperienza del controllo di qualità (CQ) web-based dello screening della Regione
Emilia-Romagna 12 per fornire una guida obiettiva, riproducibile e completa in grado di valutare la severità della lesione e
il suo eventuale potenziale di progressione neoplastica. Il CQ
via internet, basato sull’accesso a immagini colposcopiche sul
sito web della Regione, codificate secondo una classificazione
condivisa dagli operatori e derivata dalla terminologia della
Federazione Internazionale di Colposcopia, consente un aggiornamento permanente ed altamente qualificato che ha permesso
di accrescere notevolmente sia la capacità diagnostica che la
concordanza fra i vari operatori.
Conclusioni. I numerosi studi pubblicati dimostrano che l’uso
del test per la ricerca del DNA di HPV come test primario
seguito da triage con l’esame citologico standard e una ripeti-
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zione del test per il DNA dell’HPV ad 1 anno in caso di Pap
test negativo, è una strategia attuabile poiché incrementa la
diagnosi delle lesioni di alto grado di circa il 30% rispetto alla
sola citologia e mantiene un alto valore predittivo minimizzando la ripetizione di esami non necessari.
Bibliografia
1
Ronco G, Segnan N, Giorgi-Rossi P, et al.; New Technologies for Cervical
Cancer Working Group. Human papillomavirus testing and liquid-based
cytology: results at recruitment from the new technologies for cervical
cancer randomized controlled trial. J Natl Cancer Inst 2006;98:765-74.
2
Arbyn M, Sasieni P, Meijer C, et al. Chapter 9: Clinical applications
of HPV testing: a summary of metaanalyses. Vaccine 2006;24(Suppl. 3):78-89.
3
Kotaniemi-Talonen L, Nieminen P, Anttila A, et al. Routine cervical
screening with primary HPV testing and cytology triage protocol in a
randomised setting. Br J Cancer 2005;93:862-7.
4
Naucler P, Ryd W, Törnberg S, et al. Efficacy of HPV DNA testing
with cytology triage and/or repeat HPV DNA testing in primary cervical cancer screening. J Natl Cancer Inst 2009;101:88-99.
5
Health Tecnology Assessment (HTA), 2012.
La citologia di triage nei programmi di screening con HPV come test
primario. Documento GISCi, 2013
7
Zannoni GF. Il Sistema Bethesda: esempio di collaborazione fra patologo e clinico. In: Costa S, Syrjanen K, eds. Gestione delle pazienti
con Pap test anormale. Modena: Athena Ed. 2005.
8
Mariani L, Sideri M, Costa S, et al.; Italian HPV Study Group. Human papillomavirus DNA and Pap tests: the need for cotesting in
opportunistic setting during the transition time. J Low Genit Tract Dis
2013;17:362-5.
9
Origoni M, Cristoforoni P, Costa S, et al. HPV-DNA testing for
cervical cancer precursors: from evidence to clinical practice. Ecancermedicalscience 2012;6:258.
10
Costa S, Cristoforoni P, Mariani L, et al. Test HPV e prevenzione del
Carcinoma della Cervice uterina. Dall’evidenza alla clinica. Milano:
Elsevier 2010, pp. 1-55.
11
Costa S, Cristoforoni P, Mariani L, et al. Nuovi biomarcatori nella
diagnostica cervicale: aspetti clinici dell’utilizzo di p16 e k67. Torino:
Cosmopolis Ed. 2011, pp. 1-37.
12
Bucchi L, Cristiani P, Costa S, et al. Rationale and development of an
on-line quality assurance programme for colposcopy in a populationbased cervical screening setting in Italy. BMC Health Serv Res
2013;13:237.
6
Sala Orange 1 – 8.30-18.30
Patologia dell’osso e dei tessuti molli
Simposio sulla patologia dell’osso
Coordinatori: C. Della Rocca (Roma), F. Bertoni (Bologna)
Soft tissue tumors in the bone
A. De Chiara
INT G. Pascale Napoli
Primary bone tumours are mainly composed by: chondrogenic
tumours, osteogenic t., osteoclastic giant cell-rich t., notochordal t., adamantimomas, Ewing sarcomas, other tumors
specifically occurring in bone (p.is. NOF, ABC, fibrous dysplasia), and haematopoietic neoplasms.
Much less frequent in the bone, but for this reason sometimes
uncorrectly diagnosed, are those entities typically occurring
in soft tissue: fibrogenic tumours, myogenic t., lipogenic t.,
undifferentiated high-grade pleomorphic sarcomas, and the
rare neurogenic tumours, synovial sarcomas, alveolar sarcomas, and clear cell sarcomas (they are not even mentioned in
the 2013 WHO classification because of their rarity). In the
“blue book”, they are all classified as benign, intermediate and
malignant on the basis of their biological potential.
Fibrogenic tumours:
–Desmoplastic fibroma (intermediate, locally aggressive):
incidence is < 0,1% of all primary bone tumours. It is most
frequent in the mandible, followed by meta-epiphyseal
region of long tubular bones. Nuclear b-catenin is occasionally present but usually in < 10% of cells; MDM2 and CD4
are negative (in contrast to low-grade central osteosarcoma,
main differential diagnosis). Recurrences is 17% or 55-72%
of cases with or without resection, respectively.
– Fibrosarcoma (malignant): defined as intermediate- to highgrade spindle cell neoplasm, devoid of significant pleomorphism and any line of differentiation. This pattern of growth
is common to a wide variety of bone tumours, which have
to be carefully excluded. Therefore, it is mainly a diagnosis
of exclusion: myogenic, epithelial, and endothelial markers
should be negative, and the tumour should be devoid of
osteoid and cartilage. Using these criteria, fibrosarcoma is
extremely rare (if does exist on rare occasion).
Vascular tumours:
– Haemangioma (benign): common lesion (autopsy studies identified them in the vertebrae of approximately 10% of adults)
but rarely symptomatic (< 1% of primary bone tumours). Often
multifocal. Mainly vertebrae, craniofacial skeleton and methaphyses of long bones. Excellent prognosis.
–Glomus tumour (benign): extremely rare. Mainly distal
phalanx of a finger. Curettage is curative.
– Epithelioid haemangioma (intermediate, locally aggressive,
rarely metastasizing): uncommon, but the exact incidence
is unknown. Range in age from 1st to 9th decade of life
but a mean age of 35 years is reported. Mainly long tubular
bones, vertebrae, and small bones of hands. Multifocal in
18-25% of cases with a regional distribution. The treatment
usually consists in curettage and rarely in en-bloc resection.
Excellent prognosis, with local recurrence in 9% of cases.
–Epithelioid haemangioendothelioma (EHE) (malignant): rare, but the exact incidence is unknown. Range in age from 1st
to 8th decade of life but mainly in 2nd and 3th decade.The
skeleton can be the only organ involved or a component of
a multiorgan system. 50-64% are multifocal (within a single
bone or in separate bones). The translocation t(1;3)(p36;q25)
that results in fusion WWTR1 in 3q25 with CAMTA1 in
1p36 has been recognized. Breakpoint analysis of WWTR1CAMTA1 has established the monoclonal origin of “multifocal” EHE, indicating that they arise from local or metastatic
spread from a single primary as opposed to multiple independent primaries. Wide resection is the treatment of choise.
Mortality is 20%.
–Angiosarcoma (malignant): rare (<1% f malignant bone
tumours). Range in age from 1st to 8th decade. The majority are primary, but are sometimes associated with either
206
previous radiotherapy or bone infarction. Mainly long and
short tubular bones (74%). One third of cases are multifocal, and affect contiguous (64%) or distant bones (36%).
The combination of multifocality, epithelioid morphology
and epithelial markers can leads to a wrong diagnosis of
metastatic carcinoma. Usually, clinically extremely aggressive; the presence of macronucleoli, mitoses > 3/10HPF e
< 5 eosinophilic granulocytes seems to be associated with
an even worse survival, as well as the expression of D2-40
(indicating a lymphangiogenic differentiation).
Myogenic tumours:
– Leiomyoma (benign): about twenty cases reported in literature.
–Leiomyosarcoma (LMS) (malignant): wide age distribution (9-87 years) with a peak in 5th decade. Occasionally
associated to previous RT or EBV infection (in immunocompromised patients). Mostly lower extremity around the
knee. Metastatic disease from extra-osseous primary lesions
(visceral and soft tissue) should be excluded, as this is much
more common that primary LMS of bone. The histological
grade correlates with clinical outcome; high-grade tumors
often metastasize to lungs and 50% of patients succumb to
disease.
Lipogenic tumors:
–Lipoma (benign): rare. Intra-osseous lipomas account for
< 0,1% of primary bone tumours. From 2nd to 8th decade
but mostly in 5th decade. Mainly long bones. Parosteal
lipomas represent 15% of bone lipomas. The translocation t(3;12)(q28;q14) which results in the fusion transcript
HMGA2-LPP featured in soft tissue lipomas has also been
detected in parosteal lipomas. Recurrence is rare.
–Liposarcoma (malignant): extremely rare. Metastases to
the bone from a soft tissue primary is much more common. Long tubular bones, especially femur and tibia. The
histological variants in bone are similar to those seen in soft
tissue.
Undifferentiated high-grade pleomorphic sarcoma (UPS):
defined as high-grade malignant neoplasm with diffuse
pleomorphism, devoid of any specific line of differentiation.
They represent < 2% of all primary malignant bone tumours.
From 2nd to 8th decade of life with higher incidence in
adults > 40 years and 10-15% of patients < 20 years. The
majority arise as primary, but in about 28% of cases they
develop secondary to pre-existing conditions such as Paget
disease or bone infarct, or in irradiated bone. An autosomal
dominant predisposing condition has been identified: diaphyseal medullary stenosis (bone dysplasia characterized
by diffuse diaphyseal medullary stenosis with overlying endosteal cortical thickening, metaphyseal striations, scattered
infarctions and sclerotic areas throughout the long bones).
It is a diagnosis of exclusions, extensive sampling and immunophenotypic panel are mandatory to rule out other malignancies resembling UPS (SMA focally positive in about
50% of cases; keratin can be positive, which can confound
the differential diagnosis with metastatic sarcomatoid carcinoma). Highly aggressive with lung metastases in 45-50%
of cases. Generally, treatment is wide surgical resection
preceded by chemotherapy; the grade of tumor necrosis in
the resected specimen after CHT seems to be an important
prognostic factor. Better prognosis in patients < 40 years of
age and adequate surgical margins.
Neurogenic tumours:
–Schwannoma (neurilemmoma) (benign): very rare; in the
Mayo Clinic file < 1% of all benign bone tumors. Sporadic
or in association with NF2. Mostly in the 2nd-3rd decade
of life. Mostly sacrum and mandible, but also other bones.
Excellent prognosis.
– Neurofibroma and perineurioma (benign): sporadic cases in
the literature.
– Malignant peripheral nerve sheath tumours (MPNST) (malignant): sporadic cases in the literature. It is mainly a diagnosis of exclusion based mostly on morphological features.
Alveolar sarcoma: sporadic cases in the literature. Main differential diagnosis with a metastasis from a particular subtype
of renal cell carcinoma which involves young people: both
show immunohistochemical positivity for TFE3 e similar
genetic aberration (FISH for rearrangement of TFE3, or RTPCR for ASPSCR1-TFE3). Useful clinical history and immunohistochemical evaluation for cathepsin K (negative in renal
carcinomas) for the correct diagnosis.
Clear cell sarcoma (CCS): sporadic cases in the literature
(about ten cases). Main differential diagnosis with a metastasis from cutaneous or visceral melanoma: necessary clinical
history and molecular biology (FISH for rearrangement of
EWSR1, or RT-PCR for EWSR1-ATF1 or EWSR1-CREB1,
and BRAF).
Synovial sarcoma (SS): sporadic cases in the literature. Some
of the previously cases classified as hemangiopericytomas
are actually SS or solitary fibrous tumors. Necessary clinical
history (to exclude the possibility of a bone metastasis from a
soft tissue or visceral SS), immunohistochemical studies (differential diagnosis with other tumours) and molecular biology
(FISH for rearrangement of SYT, or RT-PCR for SS18-SSX1
or SS18-SSX2 or SS18-SSX4).
Simposio sulla patologia dei tessuti molli
Coordinatori: A. P. Dei Tos (Treviso), V. Ninfo (Padova)
Nuove entità in patologia dei tessuti molli
P. Collini
Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
The World Health Organization ‘Classification of soft tissue and
bone tumors’published in 2013 encompasses some new entities.
Among them, the pseudomyogenic hemangioendothelioma (PMHE) and the epithelioid inflammatory myofibroblastic sarcoma
(EIMS) represent two particularly interesting new clinico-pathologic entities. Their recognition has also therapeutic implications.
PSEUDOMYOGENIC HEMANGIOENDOTHELIOMA
(PMHE): cases of this rare entity had been reported before as
‘epithelioid sarcoma-like hemangioendothelioma’. This is a
rarely metastasizing vascular tumour typical of young males.
The 80% of cases are localized in the limbs, with a predilection for the lower ones. It is more frequently multinodular, involving skin, subcutis, and soft tissue. It can occur also in the
bone, with a lytic lesion. Histologically, PMHE is characterized by an infiltrative growth of spindle or polygonal, focally
epithelioid, eosinophilic cells, with vesicular nuclei with small
nucleoli. A neutrophilic infiltrate is also frequent. Rarely,
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marked atypia, high mitotic index, necrosis, and vascular
invasion occur. Immunophenotypically, PMHE shows coexpression of vascular, myogenic, and epithelial markers. The
recurrent t(7;19)(q22;q13) has been reported. The differential
diagnosis includes nodular fasciitis, myogenic neoplasms,
epithelioid sarcoma, epithelioid hemangioendothelioma, cellular fibrous histiocytoma, spindle cell squamous cell carcinoma. In some cases, a conservative surgical approach has
been proposed.
Epithelioid Inflammatory Myofibroblastic Sarcoma (EIMS):
this is a variant of inflammatory myofibroblastic tumour bearing a more aggressive clinical behaviour. It it more frequent
in males of any age. The site of origin is quite always intraabdominal. It shows a quite always fatal outcome. Histologically, it is characterized by epithelioid/histiocytoid round cells
in a myxoid matrix. A neutrophilic infiltrate is often present.
Immunophenotypically, neoplastic cells show ALK1 and
CD30 reactivity. A part of EIMS show the RANBP2-ALK
fusion gene. In these translocated cases response to crizotinib
was reported.
Reference
WHO. Classification of Tumours of Soft Tissue and Bone. Fletcher CDM,
Bridge JA, Hogendoorn PCW, Mertens F, eds. Lyon: IARCPress 2013.
Sala Orange 2 – 8.30-18.30
Patologia molecolare
Patologia molecolare: le metodologie “non-in situ”
Moderatori: G. Bevilacqua (Pisa), A. Marchetti (Chieti)
Next-generation sequencing diagnostics:
analysis of multigene panels
A. Scarpa, M. Fassan
ARC-NET Research Centre and Department of Pathology and Diagnostic, University and Hospital Trust of Verona, Verona, Italy
Plenty of targeted drugs have been developed following the
identification of driver mutations in malignant neoplasms.
Several are approved for clinical use and many are in clinical
trials. It is thus expected that the number of biomarkers that
will require diagnostic assessment will rapidly increase. This
calls for the implementation of time-effective methods that are:
(i) capable of probing simultaneously the mutational status of
multiple genes, (i) suitable to be applied to the limited cytological or bioptic material available for inoperable cancer patients.
Sanger sequencing is currently the most widely applied technique in the characterization of gene status in clinical practice.
Real-time PCR-based methods have been shown to efficiently
detect gene mutations in samples containing as low as 1%
mutated cancer cells. However, neither Sanger nor real-time
techniques permit the quantitation of mutant alleles in the
sample, i.e. the number of mutated cancer cells over the total
cancer cells in a sample. That is why there is no sufficient
information on the predictive ability of these techniques, and
no clear correlation could be established between the quantity
of mutated cancer cells in the neoplasm and the extent and
duration of response to therapy. More importantly, most methods have been developed and validated to assess single gene
alterations and have varying compatibility with the partially
degraded DNA from formalin fixed and paraffin embedded
(FFPE) tissues.
Massive parallel sequencing, also known as next-generation
sequencing (NGS) or deep sequencing, has been recently introduced and is the most sensitive approach to index multiple
genes starting from a limited amount of DNA from FFPE
samples. NGS can simultaneously investigate multiple potential
molecular targets, and represents a potent diagnostic complement to histopathological and immunophenotypic diagnosis.
To test NGS performance into clinical practice, we considered cytology specimens from 38 lung adenocarcinomas,
which were subjected to the simultaneous assessment of 504
mutational hotspots of 22 lung cancer-associated genes using
10ng of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In
24/36 cases (67%) at least one mutated gene was observed,
including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET,
SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS, respectively found in 6/36 (16%) and 10/36 (28%) cases, were
mutually exclusive. Nine samples (25%) showed concurrent
alterations in different genes.
Similarly, we considered gastric high-grade intraepithelial
neoplasia (HG-IEN) and early invasive gastric cancer (GC)
samples manually microdissected from a consecutive series of
15 surgically treated early GC. Forty ng of DNA were used for
multiplex PCR amplification using the Ion AmpliSeq Cancer
Panel that explores selected regions of 50 cancer-associated
genes. In GC samples, 12/15 presented at least one mutation
among the 50 investigated genes, and 6 of these cases showed
multiple driver gene alterations. TP53 mutations were observed
in 9 cases; 3 mutations were identified in APC, while ATM
and STK11 were mutated in 2 tumors. Seven HG-IEN lesions
showed an identical mutational profile to their matched GC and
13 mutations observed in the APC, ATM, FGFR3, PIK3CA,
RB1, STK11, and TP53 genes were shared by both non-invasive
and early invasive lesions. CDKN2A, IDH2, MET, and RET
mutations were observed only in the invasive counterpart.
NGS tests are superior to current standard methodologies, as
they also furnish quantitative data on the proportion of cancer cells bearing mutations over the total cancer cells in the
sample. Immunohistochemical (IHC) evaluation of p53 and
Smad4 in selected samples confirmed in fact that the proportion of cells showing abnormal IHC staining corresponded to
the proportion of mutated cancer cells in the samples indicated
by NGS testing.
In conclusion, NGS techniques are suitable for introduction
in a clinical setting as they: a) interrogate multiple genes in
a time-effective fashion and b) require limited amounts of
DNA. Their application to routine samples will allow a significant increase in the fraction of cancer patients eligible for
personalized therapy and permit tailoring clinical tials based
on the quantitation of the different mutations found in samples
from each single patient.
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Patologia molecolare: le metodologie “in situ”
Moderatori: A. Scarpa (Verona), G. Stanta (Trieste)
HER2 testing in breast cancer: advantages
and disadvantages of methods alternative
to fluorescence in situ hybridization
A. Di Benedetto, C. Ercolani, C.A. Amoreo, B. Antoniani,
V. Dimartino, V. D’Alicandro, E. Melucci, M. Mottolese
Pathology Department, Regina Elena National Cancer Institute,
Rome, Italy
Background. Normal glandular breast cells usually present
two/four copies of the HER2 gene depending on the cell
cycle phase whereas human breast cancer (BC) shows HER2
overexpression/amplification in about 15%-20% of cases 1. In
2000, the introduction of trastuzumab (Herceptin, Genentech),
the recombinant humanized monoclonal antibody (MoAb)
directed against HER2, into the metastatic setting and more
recently in the (neo)adjuvant setting, has completely changed
the natural history of HER2 positive BC patients both in terms
of time to recurrence and survival 2.
In addition to trastuzumab, other anti HER2 treatments such as
lapatinib, pertuzumab, and trastuzumab-emtansine (T-DM1),
have been approved by U.S. Food and Drug Administration
(FDA) for metastatic HER2 positive BC patients treatment in
combination with chemotherapy 3.
This scenario makes it increasingly important to correctly
identify HER2 positive BC patients who may benefit from
these targeted therapies.
HER2 testing at protein and DNA level. Immunohistochemistry: in the routine clinical practice, immunohistochemistry
(IHC), an easy and relatively cheap technique, is considered
the test of choice to detect HER2 protein overexpression.
HER2 positivity is usually determined following the ASCOCAP guidelines (4) and was scored as 0, 1+, 2+ or 3+. Only
score 3+ and score 2+ BC showing gene amplification are
eligible for anti HER2 based treatments.
FDA approved three anti HER2 antibodies directed to the
intracellular domain of the receptor: the polyclonal antibody
A0485 (Dako, Denmark A/S, Glostrup, Denmark), the mouse
monoclonal PATHWAY HER2 CB11 (PCB11) and the
monoclonal PATHWAY HER2 4B5 (Ventana Medical Systems, Tucson, AZ, USA). In addition, the monoclonal antibodies CB11 Oracle, CE-IVD approved (European Conformity-In Vitro Diagnostic; Leica Microsystems GmbH, Wetzlar,
Germany) and Sp3 (Spring bioscience, Pleasanton, CA) were
also currently used in many laboratories 5. It is not surprising
that this large availability of anti HER2 antibodies although
validated for diagnostic use, may create inter-laboratory analytical variability. In this context, standardization and external
quality control programs are mandatory in clinical practice in
order to minimize the potential bias mainly in reproducing the
two intermediate classes 1+ and 2+ scores 6.
Fluorescent in situ hybridization: BC with equivocal HER2
IHC results (score 2+) require further analysis using a gene
amplification test as fluorescence (FISH) or chromogenic in
situ hybridization (CISH/SISH). FISH is the most common
ISH technique among pathology laboratories. This testing
methodology is based on the use of complementary nucleic
acid sequences to the HER2 gene labelled with a fluorescent
reporter (Texas Red or Orange). A second probe, linked to
a different fluorochrome (FITC or Green) is used to detect
the centromeric region of chromosome 17 (CEP17). After
hybridization to the complementary DNA, the two probes
can be visualised using a fluorescence microscope. FISH is
a time-consuming technique that requires a microscope with
special multi-band fluorescence filters and trained personnel.
The fluorescent signals are prone to decay after a short period
and the slides cannot be archived. Therefore a high resolution
digital camera is necessary to record images.
Finally, FISH technique does not permit to observe gene
amplification in the context of the tumor morphology and the
heterogeneity can easily be missed owing the scoring is done
using the 100× oil immersion lens. Therefore, a correlation
with the histological sections, eventually through the use of
an automated FISH image analysis system, is strongly recommended to ensure that the invasive component is being assessed and also that heterogeneous HER2 gene amplification
areas are not missed 4.
Chromogenic in situ hybridization: Chromogenic in situ
hybridization (CISH) is a methodology introduced as an
alternative to FISH starting from 2000. In recent years, the
advantages of CISH, which allows a simultaneous evaluation of gene copy number (GCN) and detailed surrounding
tissue morphology on the same histological slide, have been
widely reported by several authors (7). CISH is a technique
that shares the benefits of a permanent record of the test
without signal decay, and the preservation of tissue architecture and morphology thereby facilitating the detection of
heterogeneous signals. CISH assay is usually monoprobe
but, recently, was introduced a new CISH (Dako DuoCISH
Kit) assay which involves a two-step chromogenic detection.
CISH allows observers to select only fields of invasive ductal
carcinoma, avoiding to analyze foci of intraductal carcinoma
more frequently amplified. Finally CISH, in contrast to FISH,
is a less costly assay.
Silver in situ hybridization: the FDA approved Silver in situ
Hybridization (SISH) test (Roche Diagnostics, Milan, Italy)
is a fully-automated assay for determining HER2 gene amplification. It allows a diagnosis within 8-10 hours and uses
an enzyme metallography methodology where the bound of
horseradish peroxidase (HRP) to silver ions catalyses the reduction of silver acetate to produce a black dot in cell nuclei
on the target site. The primary advantage of this technique
is the fully automation of the procedure which allows rapid
turnaround times. SISH can use single or dual probes. In the
latter, the HER2 signals are visualised as discrete black metallic silver dots and CEP17 as slightly larger red dots (Fast Red)
to achieve maximum contrast between the two signals.
Following the manufacturer’s guidelines, scoring of SISH
results are carried out assuming that a single signal is counted
as 1 gene copy, a small cluster as 8 gene copies, a large cluster
as 16 gene copies.
As CISH, this technology which may be successfully used
on cytological specimens, combines the brightfield benefits
of preservation of tumor morphology and an archivable slide
record.
ISH scoring criteria: according to the current ASCO-CAP
guidelines, a BC displaying a ratio between gene and CEP17
≥ 2.2 or with an average of more than six gene copies/nucleus
is considered as amplified. The equivocal range is defined as
HER2/CEP17 ratio from 1.8 to 2.2 or a GCN average between
4.0 and 6.0. All cases with a ratio under 1.8 or with less than
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4.0 GCN, are designated as negative. Polysomy 17, intended
as an increased CEP17 copy number, is considered to be present when a mean number of ≥ 3 signals is observed in at least
60 tumor cell nuclei. To overcome the problem of heterogeneous amplification, Hanna et al. 8 highlighted the possibility
that tumors with > 10% HER2-amplified cells (after counting
20–60 cells from multiple different fields) should be considered HER2+. The same authors recommend that the percentage of amplified cells is included in the pathology report.
IHC and ISH pitfalls: both IHC and ISH results can be influenced by pre-analytical conditions as detailed in the ASCOCAP guidelines. There are two key points: 1) the transfer time
of tissues from the surgical theatre to the pathology laboratory
(cold ischemia) and 2) the optimization of formalin fixation
time 9. Given the lack of alignment among specimen fixation
and processing protocols at different institutions, a central
question arises: could fixation and processing-related artifacts
skew IHC and ISH data and resultant conclusions? Published
data suggest that the answer is yes. Therefore, although difficult, pathologists should pay attention in controlling and
verifying all routine step procedures.
Fluorescent or chromogenic in situ hybridization: what is
the best test? At this time there is no consensus on which in
situ techniques is the better one, nor on the use of mono- or
duo-probe ISH systems, the usage of manual or fully automated staining and scoring-systems. A clinical diagnostic test
for HER2 should be reproducible and precise across different
laboratories and users. Nowadays there are new emerging
HER2 testing technologies at protein, RNA and DNA level.
Novel techniques for HER2 testing at Dna level: Multiplex Ligation-dependent Probe Amplification. Multiplex
Ligation-dependent Probe Amplification (MLPA) (MRCHolland, Amsterdam, The Netherlands), is a semi quantitative
technique which needs only minute quantities of DNA isolated from paraffin-embedded tissues. This technique is based
on amplification of pairs of hemiprobes that contain specific
target sequences (against specific genes) and universal PCR
primer sequences. The comparison with reference DNA samples derived from healthy individuals, is essential to obtain a
correct final result. The HER2 MLPA assay is made up by a
mix containing probes recognizing four different sequences
of the HER2 gene and other 27 probes specific for genes
located on chr17 including some close to the centromere. The
mean values of the 4 HER2 probe peaks that correlate with
the amount of target DNA are calculated using a dedicated
software (Coffalyser). Mean values < 0.7 is referred to gene
deletion, between 0.7-1.3 to normal gene status and values
between 1.3-1.5 and > 1.5 to gains and gene amplification
respectively. This high-throughput PCR-based technique is
easy to perform and might provide an alternative valuable tool
to evaluate HER2 gene status, although it presents two main
limitations potentially leading to some misleading results: a.
the inability to highlight the heterogeneity, b. the loss of tissue morphology which avoids to differentiate invasive from
in situ carcinoma 5.
209
Conclusions. The evaluation of HER2 status for every new
invasive BC is a settled concept but what is the best test both
in terms of accuracy and reproducibility? IHC, FISH, CISH/
SISH, all demonstrated good overall correlation with each
other in comparative studies and each technique has its own
advantages and disadvantages. Therefore, the testing choice
will likely be based on laboratory organization, on the basis
of practical and economic issues. Standardization of preanalytical, analytical and postanalytical (interpretation) factors
remains consistently difficult. Therefore, the implementation
of internal and/or external quality control programs become
even more essential. Among the emerging high-throughput
methodologies, MLPA, although with some limitations, appears to be a good complement to IHC and ISH, mainly in
equivocal cases. Genetic heterogeneity which can induce
misleading evaluation of HER2 status, underlines an urgent
need to homologate IHC and ISH positivity cut off. We
highlighted the clinical importance of testing HER2 status in
metastases, mainly when the PBC was HER2 negative. HER2
change during progression from negative to positive, open
up the possibility of significantly improving the prognosis
of these subsets of BC patients. An accurate definition of patients who can experience great benefit by trastuzumab or any
novel anti HER2 molecule, in adjuvant and metastatic setting,
represents a pivotal commitment in the clinical management
of BC patients.
References
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
Metro G, Mottolese M, Fabi A. HER-2-positive metastatic breast cancer:
trastuzumab and beyond. Expert Opin Pharmacother 2008;9:2583-601.
3
Chung A, Cui X, Audeh W, et al. Current status of anti-human epidermal growth factor receptor 2 therapies: predicting and overcoming
herceptin resistance. Clin Breast Cancer 2013;13:223-32.
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. Arch Pathol Lab Med 2007;131:18-43.
5
Moelans CB, de Weger RA, Van der Wall E, et al. Current technologies for HER2 testing in breast cancer. Crit Rev Oncol Hematol
2011;80:380-92.
6
Starczynski J, Atkey N, Connelly Y, et al. HER2 gene amplification
in breast cancer: a rogues’gallery of challenging diagnostic cases:
UKNEQAS interpretation guidelines and research recommendations.
Am J Clin Pathol 2012;137:595-605.
7
Vocaturo A, Novelli F, Benevolo M, et al. Chromogenic in situ
hybridization to detect HER-2/neu gene amplification in histological and ThinPrep-processed breast cancer fine-needle aspirates: a
sensitive and practical method in the trastuzumab era. Oncologist
2006;11:878-86.
8
Hanna WM, Rüschoff J, Bilous M, et al. HER2 in situ hybridization
in breast cancer: clinical implications of polysomy 17 and genetic
heterogeneity. Mod Pathol 2013 [Epub ahead of print].
9
Comanescu M, Annaratone L, D’Armento G, et al. Critical steps
in tissue processing in histopathology. Recent Pat DNA Gene Seq
2012;6:22-32.
210
La formazione in anatomia patologica
Moderatori: A. Sapino (Torino), L. Ruco (Roma)
La Scuola di Specializzazione in Anatomia
Patologica in Italia
L. Ruco
Sapienza Università di Roma
La Scuola di Specializzazione ha la durata di cinque anni durante i quali lo specialista in Anatomia Patologica deve aver
maturato conoscenze teoriche, scientifiche e professionali nel
campo della patologia sistematica anatomopatologica autoptica, macroscopica, microscopica, ultrastrutturale e molecolare,
finalizzate alla specifica attività assistenziale propria dell’anatomopatologo ed all’attività peritale.
Deve essere in grado di riconoscere e diagnosticare a livello
macroscopico e microscopico le alterazioni morfologiche
degli organi, dei tessuti e delle cellule nei preparati citologici,
nel materiale bioptico ed in corso di riscontro diagnostico. A
tal fine lo specialista in anatomia patologica deve acquisire
le necessarie competenze nell’applicazione e interpretazione
diagnostica in patologia umana delle metodiche anatomiche,
istologiche, citologiche, istochimiche, immunoistochimiche,
ultrastrutturali e di biologia molecolare, nonché capacità
nell’utilizzo di metodi relativi al controllo di qualità ed alle
valutazioni quantitative.
Sono specifici ambiti di competenza dello specialista in
anatomia patologica la diagnostica autoptica, macroscopica,
istopatologica, citopatologica, istocitopatologica intraoperatoria, ultrastrutturale e la caratterizzazione biomolecolare delle
lesioni di cellule, tessuti ed organi e dell’intero organismo,
finalizzate alla diagnosi di esse, utili anche per l’orientamento
prognostico e terapeutico, e per la valutazione epicritica della
diagnosi e dei correlati anatomo-clinici.
Obiettivi formativi integrati (ovvero tronco comune). Acquisizione delle conoscenze nell’ambito delle discipline che
contribuiscono alla definizione della Classe della Medicina
Diagnostica e di Laboratorio (Biochimica clinica, Patologia
clinica, Microbiologia e Virologia, Anatomia patologica), e
di quelle utili alla caratterizzazione del percorso formativo
comune alle diverse tipologie di Scuola (Patologia generale,
Medicina interna, Chirurgia generale) con particolare riguardo
alla patologia molecolare, fisiopatologia e patologia generale,
immunologia ed immunopatologia.
Obiettivi formativi di base:
– acquisizione di competenze nell’uso degli strumenti e delle
metodiche dei laboratori di istologia;
–acquisizione di conoscenze fondamentali di genetica, biochimica, biologia molecolare, statistica medica, epidemiologia, e di modalità di valutazioni morfometriche.
Obiettivi formativi della tipologia della Scuola:
– acquisizione di conoscenze approfondite di anatomia patologica sistematica, di tecnica e diagnostica delle autopsie, delle
basi teorico pratiche dell’istochimica e dell’immunoistochimica, della patologia ultrastrutturale, della citopatologia
diagnostica, compresa la citologia aspirativa con ago sottile,
e della biologia molecolare applicata alla diagnostica;
–la maturazione di completa capacità di diagnostica morfologica delle alterazioni cellulari, dei tessuti e degli organi, e
della valutazione patogenetica ed epicritica di esse anche in
rapporto al trattamento terapeutico;
– l’acquisizione degli elementi fondamentali utili alla valutazione, anche epicritica, delle correlazioni anatomo-cliniche
delle principali patologie subspecialistiche quali neuropatologia, patologia cardiovascolare, dermatopatologia, ematopatologia, nefrouropatologia, ginecopatologia, patologia
pediatrica, patologia ossea e dei tessuti molli, patologia
gastroenterologica, endocrinopatologia, patologia respiratoria, la patologia oncologica e tutto quanto concerne la
diagnostica morfologica relativa a cellule, tessuti e organi;
–l’acquisizione di esperienza di monitoraggio morfologico
dei trapianti d’organo. L’apprendimento di competenze
necessarie per l’organizzazione e la gestione del settorato
e dei laboratori di diagnostica istopatologica, di citopatologia diagnostica, di istochimica ed immunoistochimica, di
patologia ultrastrutturale e di biologia molecolare applicata
alla istocitopatologia, ivi compresi la sicurezza nel posto di
lavoro e lo smaltimento dei residui biologici e chimici.
Sono obiettivi affini o integrativi l’acquisizione di adeguate
conoscenze teoriche di genetica medica, di epidemiologia e
igiene generale, diagnostica per immagini, medicina del lavoro e
preventiva, tossicologia, medicina legale, deontologia e bioetica.
Sono attività professionalizzanti obbligatorie per il raggiungimento delle finalità didattiche della tipologia (revisione approvata dal CUN nel 2011, ma ancora non pubblicata sulla G.U.):
–partecipazione a corsi di aggiornamento, seminari, dimostrazioni, conferenze e congressi con tematiche direttamente pertinenti o comunque di completamento al percorso
formativo in anatomia patologica, accreditati dal ministero
della salute per l’educazione continua in medicina;
–maturazione critica nella ricerca bibliografica; scelta dei
manuali di tecniche istopatologiche, istochimiche, immunoistochimiche, di microscopia elettronica e di biologia
molecolare, e dei trattati e delle riviste scientifiche e professionali di anatomia patologica;
– l’informatica nella diagnostica istopatologica;
–raccolta e interpretazione di dati anamnestici e clinici utili
alla formulazione della diagnosi anatomopatologica macroscopica e microscopica, della prognosi e dei suggerimenti
terapeutici;
–organizzazione e gestione del settorato di anatomia patologica: acquisizione di dimestichezza con gli strumenti di uso
abituale; capacità di guida del personale tecnico addetto alla
manutenzione del settorato, alla ricomposizione delle salme
e allo smaltimento dei residui;
–apprendimento della tecnica e diagnostica delle autopsie,
dall’esame esterno della salma al prelievo e all’esame macroscopico dei visceri, compresi i dispositivi di protezione
per le salme infette;
–partecipazione ai lavori in sala settoria ed esecuzione delle
autopsie; partecipazione all’esecuzione di almeno 50 riscontri diagnostici, ivi compreso il relativo completamento
istopatologico;
–compilazione del verbale di autopsia e interpretazione dei
verbali di archivio;
–acquisizione della capacità di valutazione epicritica e correlativa anatomo-clinica, assieme ai clinici, delle lesioni
osservate nel riscontro diagnostico;
– maturazione della capacità di organizzare e condurre conferenze anatomo-cliniche;
–acquisizione di capacità diagnostica macroscopica e di descrizione dei prelievi operatori;
relazioni
– esecuzione dell’esame macroscopico di almeno 1.500 pezzi
operatori;
–organizzazione e gestione dei laboratori di istopatologia,
citopatologia, immunoistochimica e, ove esistenti, di patologia ultrastrutturale e di biologia molecolare;
–preparazione e manutenzione dei reagenti necessari per
l’allestimento dei preparati istologici, citologici, immunoistochimici e per l’estrazione e lo studio degli acidi nucleici:
fissativi e coloranti per il microscopio ottico e per quello
elettronico, reagenti e sieri per l’immunoistochimica, sonde
per l’ibridizzazione degli acidi nucleici, reattivi e metodiche
per la caratterizzazione dei profili di espressione genica
tissutale con tecniche di biologia molecolare;
– smaltimento dei reagenti chimici non più utilizzabili;
– esperienza nell’uso dei microtomi, degli ultramicrotomi e dei
criotomi; competenza nell’impiego del microscopio ottico a
luce, a contrasto di fase, a luce polarizzata ed a fluorescenza;
conoscenza dei principi della microscopia elettronica a trasmissione e a scansione e della microscopia multifocale;
– allestimento dei preparati istologici ed esecuzione di reazioni
istochimiche e immunoistochimiche su strisci e su sezioni;
–lettura al microscopio dei preparati istologici di almeno
5.000 biopsie e assunzione progressiva di capacità diagnostica autonoma in istopatologia;
– lettura al microscopio dei preparati citologici di almeno 2.000
pazienti e assunzione progressiva di capacità diagnostica autonoma in citopatologia ed in citologia aspirativa con ago sottile;
– esperienza nella valutazione dei preparati istochimici e immunoistochimici al fine della formulazione della diagnosi;
–esperienza nella valutazione dei risultati delle tecniche di
biologia molecolare finalizzate alla diagnosi, o alla caratterizzazione biomolecolare della lesione;
–interpretazione e refertazione di almeno 50 diagnosi molecolari effettuate su lesioni tissutali;
–partecipazione all’esecuzione e all’iter diagnostico di almeno 250 biopsie intraoperatorie su preparati istologici e
citologici per striscio ed apposizione;
–controllo preventivo del donatore e monitoraggio istocitopatologico dell’organo trapiantato;
– capacità di valutazioni morfometriche;
– aggiornamenti sul controllo di qualità in istopatologia.
Per il raggiungimento delle finalità della specializzazione è richiesta la partecipazione all’esecuzione e all’iter diagnostico,
con assunzione progressiva di responsabilità ed autonomia di
refertazione, dei seguenti atti specialistici:
– esame macroscopico di almeno 1.500 pezzi chirurgici;
–almeno 5.000 diagnosi istopatologiche, comprese quelle
con eventuali indagini istochimiche, immunoistochimiche,
ultrastrutturali e di biologia molecolare;
–almeno 2.000 diagnosi citopatologiche, incluse quelle di
citologia aspirativa con ago sottile;
– almeno 250 diagnosi intraoperatorie;
–almeno 50 riscontri diagnostici, ivi compreso il relativo
studio istopatologico;
– almeno 50 diagnosi molecolari su lesioni tissutali.
What do Italian pathology residents think about
their training programme?
M. Basciu1, L. Lorenzi2, S. Massa3, F. Pulcini4
Residency program in Anatomic Pathology. University of Milan Bicocca, University of Milan; 2 Residency program in Anatomic Pathology. University of Brescia, University of Milan; 3 Residency program
in Anatomic Pathology. University of Milan; 4 Residency program in
Anatomic Pathology. Sapienza University of Roma
1
211
During the last few years the Italian post-graduate and residency system has undergone several changes. The medical
residency contract has acquired the characteristics of typical
training-apprenticeship contracts as required for long time,
the salary has been adjusted to the European standards and,
in addition, similar disciplines have been added to the training program in order to enrich the background of the future
specialist (“Tronco Comune”).
Meanwhile, in order to reduce the expenses and to enrich the educational prospective, different Schools have been unified since
the 2008-2009 academic year. Schools of Anatomic Pathology
have also experienced these changes resulting in a decrease in
the number of School Headquarters and an enlargement of apprenticeship networks 1-3. Each network is now made up of one
Leading School and many other Affiliated Schools, generally,
but not always, geographically located close to the Leading one.
Concurrently, the “National observatory on medical specialization training” (“Osservatorio Nazionale”) has been activated in
order to define the standards for accreditation of University and
public Hospitals and to verify the suitability of the requirements
of each network and its institutions. Additionally, the National
Observatory was asked to monitor the training program outcomes and to define the procedure to assure the quality of training, in accordance with the European Union requirements 4 5.
As future pathologists we were curious to know: how is the
training planned around Italy? Is the educational program
similar in the whole country or is every school program different? Has the unification of different schools improved the
educational programs? What do the pathology residents think
about their training program?
In order to answer these questions a survey has been designed
by pathology residents with a particular focus on the last news
recently introduced in the Training system 6 7. In particular this
tool is intended to assess the stronger and better points of the
new system and to disclose its problems.
A questionnaire with 35 questions has been developed on the
SurveyMonkey® (http://it.surveymonkey.net) web program.
Questions are focused on the main fields of residency pathology training including those that should define the minimal
standards for school accreditation. The collection of these data
is, in our opinion, a starting point to develop useful proposal
to ameliorate the post-graduate training system in pathology.
From 5th June 2013 the questionnaire has been sent to residents
of all the 19 Leading and 17 Affiliated Pathology Schools in
Italy. Collection of questionnaires was completed on July 5.
The participation to the initiative was satisfactory with 31
over 36 schools joining the survey, with a total of 115 out
of the 236 pathology residents nowadays present in Italy.
Of notice, the participation rate was different depending on
the geographical distribution of the Schools. The event was
attended by 60% of the residents enrolled in Schools of the
Northern Italy and by respectively 38% and 36% of residents
in the Centre and Southern Italy/Islands groups.
References
1
D.M. 1-08-2005. Riassetto delle scuole di specializzazione di area sanitaria” e “Ordinamenti didattici scuole di specializzazione di area sanitaria.
2
D.M. 23-12-2010. Standard e i requisiti minimi delle scuole di specializzazione.
3
D.M. 24-04-2013. Assegnazione contratti di formazione specialistica
a.a. 2012/2013.
4
D.M. 6-11-2008. Osservatorio Nazionale della formazione medica
specialistica.
5
D.M. 29-03-2006. Accreditamento delle strutture facenti parte della
rete formativa delle scuole di specializzazione.
6
http://it.surveymonkey.net
7
Come si costruisce un questionario: alcuni spunti dalla ricerca operativa. http://www.dors.it/
212
Sala Tarragona – 8.30-12.00
Cardiopatologia
Simposio parte I: il patologo cardiovascolare oggi
Moderatori: A. Orlandi (Roma), A. Pucci (Pisa); Discussant: P. Gallo (Roma)
The role of the pathologist in diagnosis
and research…in the field of vascular disease:
Microvessels in Large Vessel Pathology
A.C. van der Wal
Academic Medical Center, Amsterdam, The Netherlands
Most pathologists are familiar with the microvascular pathology that is widespread and huge in variety in biopsies of many
organs, and especially in skin, GI tract, in kidney and in lung:
altogether a large burden in diagnostic pathology practice.
Several studies In the field of cardiovascular pathology, have
revealed an important contribution of microvessels also in
the pathology of large arteries and veins. In many cases they
are newly formed vessels (angiogenesis and/or vasculogenesis) in different stages of maturation, but also pre-existent
micovascular plexus can be involved. This notion is less well
known among general pathologists. Here, I will illustrate the
importance of a close consideration of the microvessels in large
vessel pathology (aorta and its major branching arteries) on the
basis of 3 different fields of interest: 1. the so called vulnerable (“high risk”) coronary atherosclerotic plaque; 2. immune
responses involved in the pathology of aortic aneurysms, and
3. episodic growth in congenital arteriovenous malformations.
Microvessels in vulnerable coronary artery plaques. Coronary atherosclerotic plaque disruption followed by thrombotic
occlusion is a widely accepted mechanism to explain the onset
of most acute ischemic coronary syndromes (unstable angina,
myocardial infarction and sudden coronary death). Many disrupted plaques show a large lipid core, a thin fibrous cap and
a tissue degrading inflammatory process. This initiated the
concept of vulnerable plaque: a subgroup of lesions with high
propensity to evoke ischemic cardiac disease. More recently,
interest emerged also for intraplaque microvessels in generating plaque vulnerability. Rupture of these microvessels cause
haematomas (IPH) inside the plaque, which in turn lead to rapid
volume expansion of plaque and consequent increase in lumen
stenosis. Tissue hypoxia, a major driving force for angiogenesis, is a crucial factor the formation of plaque micro vessels,
but also inflammatory processes are involved, which may induce the leakage of such vessels. Another important issue is that
IPH are significant contributors to lipid core expansion through
accumulation of free cholesterol from the lipid rich erythrocyte
membranes. This turn stimulates macrophage infiltration and
further increase of plaque vulnerability. Microvessels and related plaque hemorrhages are especially prevalent in the advanced
stages of atherosclerosis, as can be seen in the elderly population (50+) with severe tri-vascular coronary artery disease. In
this population of old patients with increased cardiovascular
risk, anticoagulants and antiplatelet drugs are widely used as
a prophylactic treatment against thromboembolic diseases.
The most frequently encountered and notorious complication
of these drugs is bleeding. Immune and histopathological
analysis of entire coronary arteries in relation to the use of the
anti-thrombotic drugs in our lab revealed that the total hemorrhagic burden (number and severity of IPH) was significantly
increased in arteries of patients using coumarin-type anticoagulants Moreover, anti-thromboembolic treatment appeared
associated with repeated and more severe types of IPH. Such
observations still need to be further evaluated clinically. By
the time that coronary plaque microvessels can be visualized
reliably with in vivo imaging techniques, which is now under
intense investigation and development, it can be anticipated
that they may serve as important surrogate markers for disease
severity and progression in coronary areteries.
Inflammatory aortic pathology: atherosclerosis and chronic aortitis. Atherosclerosis is by far also the most prevalent
and best studied inflammatory disease of aorta. In the earlier
pre-symptomatic stages of the disease, T-cell mediated immune responses and scavenger receptor mediated phagocytosis characterize the inflammatory infiltrate of intimal plaques,
but in the chronic and advanced stages of atherosclerosis there
is often an additional infiltrate present in the adventitia adjacent to intimal plaques. In a previous study we have observed
striking differences between intimal and adventitial inflammation. The adventitial response generates nodular structures
with follicular organization, the participation of B-cells and
plasma cells, and de novo expression of HECA-452 (which
is specific for high endothelial venules) in local microvessels.
Whether this response is specific for atherosclerosis or occurs
also in autoimmune chronic aortitis has been further investigated recently. The non-atherosclerotic chronic inflammatory
diseases are (idiopathic) giant cell aortitis, Takayashu aortitis,
luetic aortitis, and aortitis complicating several auto immune
disease such as reumatoid artritis, IBD and Behcet diseases.
Arteritis at these locations usually becomes symptomatic due to
formation of localized aneurysms or acute medial dissections,
which require surgical intervention followed by histopathological analysis. In 39 histologically proven cases of (autoimmune)
chronic aortitis and 11 atherosclerotic aneurysms we found
that 97% of cases of aortitis and 91% of atherosclerotic aortas
showed distinct adventitial lymphoplasmocellular (CD138,
CD20, CD45RA positive) inflammation. Inflammatory activity was severest in (non atherosclerotic) chronic aortitis, with
similar patterns of follicular organization and formation of high
endothelial venules (71% in aortitis and 40% in atherosclerosis). Such a structural organization of the adventitial lymphoid
tissue in atherosclerotic and non atherosclerotic inflammatory
aortic disease suggests that a local priming and maturation of
B-cells takes place, as is also a distinctive feature of the mucosa
associated lymphoid tissues of the body (MALT). In analogy,
the term VALT (vascular associated lymphoid tissue) has been
proposed, which introduces a novel insight in the inflammatory
response of large arteries. It will be of interest to see which antigens are involved in the generation of these antibody responses,
since at least in case of atherosclerosis there are indications that
circulating IgG antibodies may have an protective effect on the
course of disease. And, obviously it is also of importance to
recognize such inflammatory patterns in the pathological differential diagnosis of aortic aneurysms.
Angiogenesis in Vascular Malformations. Vascular malforma-
213
relazioni
tions (VM) are congenital anomalies that result from localised
errors of angiogenic development during embryonic life. They
may occur at any topographic site in the body, including the
heart (albeit rarely). According to current insights in the biology of benign vascular lesions, congenital vascular malformations represent slowly progressive growing vascular masses
composed of dysplastic but mature vessels. VM should be
distinguished from benign vascular tumors (i.e. distinct types
of angiomas), which show a different type of grow behaviour
due to episodes of angiogenesis followed by maturation of the
newly formed microvessels. And, this distinction also forms the
basis for the current classification set by the the International
Society for Study of Vascular Anomalies (ISSVA): either presence or absence of angiogenesis in hemangiomas versus vascular malformations. Despite this view we identified areas of
microvascular proliferation, interpreted as angiogenesis, in 30%
of cases of a large series of 107 surgically removed VM. The
areas of microvascular proliferation with high Ki67 labeling
index were multicentric (50%) or displayed a solid growth pattern, splitting up pre-existent adipose tissue and skeletal muscle
(38%). Pain, swelling, and rapid growth (in a period of weeks
to months) were the most frequently encountered symptoms in
this population, which suggests that the proliferative response
evokes symptoms due to a mass forming effect.
This observation suggests that high flow through arteriovenous
communications, which is a hallmark of AVM, plays a role in the
onset and/or outgrowth of microvascular proliferations. Nearly
all of them were AVM, of which is known that they have aberrant flow characteristics due to presence of direct arteriovenous
fistula. Hemodynamic forces can initiate or aggravate an angio-
genic response, at least in experimental studies. But also more
than 80% of the resections of AVM lesions with angiogenesis
appeared to clinically characterized as so called high/fast flow
lesions. This suggests suggests that aberrant flow relates to angiogenic responses in in vivo situations of vascular malformations.
Our finding of an angiogenic response in vascular malformations contrasts markedly with the original view of considering
vascular malformations as static lesions. This implicates the
the current classification should be revised, which is of importance for clinical diagnosis and related therapeutic strategies
for vascular anomalies.
References
Kolodgie F, Gold HK, Burke AP, et al. Intraplaque hemorrhage and
progression of coronary atheroma. N Engl J Med 2003;349:2316-25.
2
Li X, van der Meer J, van der Loos CM, et al. Microvascular endoglin
(CD105) expression correlates with tissue markers for atherosclerotic
plaque vulnerability in an ageing population with multivessel coronary artery disease. Histopathology 2012;61:88-97.
3
Burke A, Tavora F, Narule N, et al. Aortitis and ascending aortic
aneurysm: description of 52 cases and proposal of a histologic classification. Hum Pathol 2008;39:514-26.
4
Dingemans W, et al. Highly organized lymphoid infiltrates develop in
the aortic adventitia of aortic aneurysms due to either chronic aortitis
or longstanding atherosclerosis. Virchows Archive 2009; 455:239.
5
North P. Paediatric tumours and malformations. Surgical Pathology
210;3:455-94.
6
Meijer-Jorna LB, van der Loss CM, de Boer OJ, et al. Microvascular
proliferations in arteriovenous malformations relate to high-flow
characteristics, inflammation, and previous therapeutic embolization
of the lesion. J Am Acad Dermatol 2013;68:638-46.
1
Mercoledì, 30 ottobre 2013
Uropatologia
Simposio di uropatologia
Moderatori: M. Colecchia (Milano), R. Montironi (Ancona)
Neuroendocrine genitourinary tumors
A. Lopez Beltran
Cordoba, Spagna
Background. Two basic types of neuroendocrine (NE) tumours with diverse clinicopathological features and outcome
are identified in the urinary system and male genital organs;
carcinoid tumour and neuroendocrine carcinoma. Carcinoid, a
rare tumour, occurs in the kidney, bladder, prostate and testis.
It is morphologically, histochemically, immunohistochemically and ultrastructurally similar to its counterpart in other
organs, such as lung or gastrointestinal tract.
Metastases can be detected at the initial evaluation, although they have been reported up to several years after
removal, emphasizing the need for a long-term follow-up.
NE carcinoma occurs in the kidney, bladder and prostate,
and includes small cell carcinoma (SCC) and large cell NE
carcinoma (LCNEC), the latter being exceedingly rare. Both
show the morphology and immunophenotype of NE carcinoma originating in other organs. Although the occurrence
is rare, it is highly aggressive.
Neuroendocrine (NE) tumours represent a heterogeneous
group of neoplasms originating from NE cells. Such cells
are either present in endocrine organs or dispersed through
the body, and produce neurotransmitters, neuromodulators or
neuropeptide hormones. The NE cells are defined immunohistochemically by the presence in the cytoplasm of markers,
such as chromogranin A, synptophysin, CD56, or neuronespecific enolase (NSE), among others, and show dense-core
granules at the ultrastructural level.
Classification of Neuroendocrine Tumors. The classification of the NE tumours largely depends upon the anatomical
site and organ of origin. In the lung NE tumours include four
groups of neoplasms with diverse prognosis, i.e. typical and
atypical carcinoid, large-cell NE carcinoma (LCNEC) and
small cell carcinoma (SCC). The novel TNM classification
system for the gastroenteropancreatic NE tumours includes a
grading system based on the proliferative activity, with grades
1 and 2 referring to well-differentiated NE tumours and carcinomas, and grade 3 to poorly differentiated NECs. This new
classification system provides a powerful tool for prognostic
214
stratification of the gastroenteropancreatic NE tumours in
clinical practice.
In the urinary system and male genital organs the NE tumours
are subdivided into two basic types, i.e. carcinoid tumour and
SCC. Carcinoid tumour and SCC show diverse incidence,
clinicopathological features, origins and outcome.
Comments. Potential origins of the NE neoplasms of the
urinary system and male genital organs are: (i) Derivation
from NE cells of the diffuse NE system, e.g. those identified
in the normal urothelial tract and prostate and which might
increase in number in reactive or metaplastic settings; and (ii)
derivation from a multipotent stem cell, a concept crucial to
understanding the nature of NE tumours arising in conjunction with epithelial or germ cell malignancies, which might
express markers of both components. Only exceptional cases
of LCNEC have been published in genitourinary organs.
The role of the uropathologist is to establish the diagnosis
and identify features of prognostic importance. Based on
such an information, the urologist will select the follow-up
procedure and therapeutic options in each patient. A close
collaboration between the pathologist and urologist is also
of paramount importance to exclude a metastatic lesion from
more common sites.
The difficulty in distinguishing primary from secondary lesions is compounded by histochemical, immunohistochemical
and ultrastructural similarities, including cytoplasmic chromogranin A and synaptophysin positivity, and ‘dot-lik cytokeratin positivity. Although initially proposed as a specific
marker for pulmonary SCC, thyroid transcription factor-1
(TTF-1) positivity has been reported in 25-39% of bladder
SCC, limiting its utility in this differential diagnosis. Given
these similarities, high-grade pulmonary NEC secondarily
involving urothelium must be excluded on clinical and radiological grounds. The aim of this presentation is to discuss the
clinical features, origins, morphological and immunophenotypic profiles, and prognosis of the NE tumours of the urinary
system and male genital organs.
References
1
Murali R, Kneale K, Lalak N, et al. Carcinoid tumors of the urinary
tract and prostate. Arch Pathol Lab Med 2006;130:1693-706.
2
Hansel DE, Epstein JI, Berbescu E, et al. Renal carcinoid tumor: a
clinicopathologic study of 21 cases. Am J Surg Pathol 2007;31:153944.
3
Cheng L, Pan CX, Yang XJ, et al. Small cell carcinoma of the urinary bladder: a clinicopathologic analysis of 64 patients. Cancer
2004;101:957-62
4
Hailemariam S, Gaspert A, Komminoth P, et al. Primary, pure, largecell neuroendocrine carcinoma of the urinary bladder. Mod Pathol
1998;11:1016-20.
5
Alijo Serrano F, Sánchez-Mora N, Angel Arranz J, et al. Large cell
and small cell neuroendocrine bladder carcinoma: immunohistochemical and outcome study in a single institution. Am J Clin Pathol
2007;128:733-9.
6
Wang W, Epstein JI. Small cell carcinoma of the prostate. A morphologic and immunohistochemical study of 95 cases. Am J Surg Pathol
2008;32:65-71.
7
Tarle M, Ahel MZ, Kovacic K. Acquired neuroendocrine-positivity
during maximal androgen blockade in prostate cancer patients. Anticancer Res 2002;22:2525-9.
8
Tamas EF, Epstein JI. Prognostic significance of Paneth cell-like neuroendocrine differentiation in adenocarcinoma of the prostate. Am J
Surg Pathol 2006;30:980-5.
9
Segawa N, Mori I, Utsunomiya H, et al. Prognostic significance of
neuroendocrine differentiation, proliferation activity and androgen
receptor expression in prostate cancer. Pathol Int 2001;51:452-9.
10
Abbosh PH, Zhang S, Maclennan GT, et al. Germ cell origin of testicular carcinoid tumors. Clin Cancer Res 2008;14:1393-6.
miRNA nel carcinoma prostatico
A. Vecchione
Sapienza Università di Roma c/o Ospedale Sant’Andrea, Roma, Italia
Il carcinoma della prostata (PCa) è una delle cause principali
di mortalità e morbidità nei paesi sviluppati, con circa 237.800
uomini diagnosticati in Europa ogni anno. Il carcinoma prostatico è una malattia eterogena e la sua gestione potrebbe
essere migliorata in maniera considerevole dalla scoperta
di biomarcatori per la diagnosi, la prognosi e la risposta al
trattamento.
Oggi l’antigene prostatico specifico (PSA) rappresenta il
“gold standard” dei marcatori sierici per la diagnosi e la risposta al trattamento, ma presenta una serie di limitazioni. Infatti,
come marcatore diagnostico, elevati livelli sierici di PSA non
sono specifici per una malattia neoplastica maligna, con circa
il 50% di falsi positivi e il 15% di falsi negativi, determinando
un numero inaccettabile di procedure diagnostiche invasive
(biopsie). Inoltre il PSA ha uno scarso valore come marcatore
prognostico non consentendo di distinguere tumori indolenti
da quelli più aggressivi. Per questo motivo è necessaria l’identificazione di marcatori che possano accuratamente identificare neoplasie agli stadi iniziali e indicare quali tumori abbiano
bisogno di una terapia o no.
Recenti evidenze hanno dimostrato come una classe di geni,
chiamati microRNA (miR) controllino quasi tutte le funzioni
basilari della cellula. I miR rappresentano una classe di geni
non codificanti, conservati a livello evolutivo, della lunghezza
di circa 22 nucleotidi che vengono prodotti a livello endogeno
attraverso un processo di maturazione e trasporto. Queste molecole svolgono una funzione di controllo genico attraverso il
riconoscimento di geni bersaglio.
Numerosi studi hanno messo in evidenza un loro importante
coinvolgimento nello sviluppo, nella progressione e nella
prognosi del carcinoma prostatico. In particolare negli ultimi
anni, grazie a proprietà intrinseche di queste molecole (elevata
stabilità nel siero e molteplici funzioni biologiche), è stato
proposto un loro utilizzo come marcatori sierici di malattia.
Infatti è stato dimostrato che i miRs possono funzionare come
“RNA di comunicazione extracellulare” che potrebbe essere
particolarmente rilevante nel contesto delle malattie neoplastiche e rivelarsi di particolare ausilio nella diagnosi precoce
e nella predizione di risposta alla terapia.
Per testare l’ipotesi che modificazioni nei miR circolanti
possano essere utilizzati come marcatori non invasivi per la
diagnosi, stadiazione e risposta alla terapia nel carcinoma
prostatico, abbiamo collezionato prospetticamente nel corso
degli ultimi 18 mesi un totale di 300 campioni di plasma di
pazienti al momento della biopsia prostatica.
Di questi 300 campioni ne abbiamo selezionati 32 (16 adenocarcinoma e 16 malattie non neoplastiche) che sono stati
utilizzati come “training set”.
Abbiamo quindi analizzato il profilo di espressione dei miR
mediante “Next Generation Sequencing”. Recenti studi hanno
infatti messo in evidenza che l’utilizzo di questa metodica
rappresenta una soluzione all’avanguardia che oltre a verificare l’espressione di miR già noti, permette anche di scoprire
nuovi miR che potrebbero avere un ruolo importante al fine
di classificare i pazienti in base al sottotipo tumorale e alla
risposta al trattamento.
L’analisi dei dati effettuata sui campioni del training set ha
identificato una “firma” molecolare composta da 27 miR
significativamente deregolati nei campioni tumorali rispetto
ai controlli. In particolare, questa signature è correlata con il
relazioni
Gleason score dei campioni analizzati, mentre risulta essere
indipendente dai rispettivi valori sierici di PSA.
L’analisi dell’area sottesa dalla curva ROC ha inoltre evidenziato un sottogruppo ristretto composto da 6 miR con un
elevato potere predittivo in grado di discriminare i pazienti
con carcinoma della prostata (AUC = 0,805). I dati ottenuti
mediante sequenziamento sono attualmente in corso di validazione mediante Realtime PCR su una casistica indipendente
di 100 campioni (50 adenocarcinoma e 50 malattie non neoplastiche).
I risultati così ottenuti permetteranno di mettere a punto un
test diagnostico basato sull’analisi di specifici miR con un valore pratico nella classificazione dei pazienti che necessitano
o meno di una determinata terapia o di intervento chirurgico.
Inoltre permetteranno anche di ottenere nuove conoscenze
biologiche relative ai meccanismi della progressione tumorale, della metastatizzazione, della sensibilità ai farmaci e di
individuare nuovi target per disegnare farmaci più selettivi.
Usefulness of circulating tumor cell detection
in the diagnosis of adrenocortical carcinoma
C. Scatena1, P. Pinzani2, F. Salvianti2, M. Paglierani1, M. Luconi3, M. Mannelli3, D. Massi1, G. Nesi1
Division of Pathological Anatomy, Department of Surgery and
Translational Medicine, University of Florence, Florence, Italy;
2 Clinical Biochemistry and 3 Endocrinology Units, Department of
Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy
1
Adrenocortical carcinoma (ACC) is a rare and very aggressive
endocrine neoplasm with a poor prognosis, mainly dependent
on tumor stage at diagnosis. Early diagnosis followed by surgical tumor removal, possibly associated to adjuvant mitotane
therapy, has been proven the best option for ACC treatment 1.
The mean 5-year survival rate ranges between 16 and 38%,
although it drops to less than 10% in metastatic disease 2 3.
Considering that an early diagnosis is pivotal to the prognosis, the identification of sensitive, specific and noninvasive
biomarkers is mandatory to significantly improve the clinical
management along with the survival rate and life quality of
ACC patients. The best biomarkers should not only be able
to discriminate between benign and malignant adrenocortical
masses, but also to provide prognostic penetrance, enabling
noninvasive follow-up once the tumor has been surgically
removed.
ACC gains access to the systemic circulation very early during disease progression 4. Within this scenario, detection of
circulating tumor cells (CTCs), released into the peripheral
blood before any detectable metastasis is established, may
represent a promising diagnostic tool in ACC patients.
CTCs are defined as neoplastic cells originating from either
primary or metastatic tumors and circulating freely in the
peripheral blood of cancer patients 5 6. Isolation of CTCs from
the other circulating elements can be achieved with various
methods, either immunologic or physical 7. Immunologic
techniques are based on the separation of CTC through their
expression of epithelial cell-specific markers (epithelial adhesion molecules such as EpCAM) or tumor-specific markers.
Physical methods are based on cell separation according to
migration along a density gradient or to size (Isolation by Size
of Epithelial Tumor cells, ISET). The latter technique has the
advantage of isolating intact CTCs, but needs further morphologic analysis in order to identify them while immunocytochemistry is recommended for cell origin characterization.
215
Our group recently validated the use of ISET for CTC detection in cutaneous melanoma patients, demonstrating that
intact circulating melanoma cells are detectable by ISET in
the peripheral blood of patients with primary and metastatic
melanoma and that the presence of ISET-isolated CTCs correlates with peripheral blood tyrosinase mRNA levels detected
by real-time RT-PCR 8.
Among “isolation by size” techniques, ScreenCell® filtration
devices (ScreenCell®, Paris, France) represent new methods
of CTC isolation based on blood filtration which enable easy,
rapid (filtration of 3 mL of peripheral blood is usually completed within approximately 2 minutes) and open access to
CTCs avoiding the use of any dedicated instrument 9.
Currently, detection of CTCs in the peripheral blood is a reliable tool for prognosis and follow-up in breast cancer 5 and
represents a promising prognostic marker for patients with
other solid malignancies, including tumors of neuroendocrine
origin 10. So far, no attempt has been made to detect and characterize CTCs in blood samples of patients affected by ACC
or adrenocortical adenoma (ACA).
Our group recently evaluated CTC presence in blood samples from 14 patients with ACC and 10 patients with ACA
using a cytomorphologic technique based on filtration 11.
CTC analysis was performed through three sequential steps
consisting of:
–isolation from blood by filtration on ScreenCell® Cyto filtration devices;
– CTC identification through validated morphometric criteria,
i.e. cell size ≥ 16µm, nucleocytoplasmic ratio ≥ 50%, irregular nuclear shape, hyperchromatic nucleus and basophilic
cytoplasm 8 12;
–identification of cell origin by immunocytochemistry using antibodies against adrenocortical markers, such as
MART-1/MelanA, synaptophysin and steroidogenic factor
1 (SF-1).
CTCs were detected in all ACC but not in ACA patients. Tumor cells were observed mostly as single units and immunostaining confirmed their adrenocortical nature. These findings
provide the first evidence that CTCs may represent a valid and
sensitive marker to support the differential diagnosis between
malignant and benign adrenocortical tumors. In ACC patients,
the number of CTCs after surgery either remained stable or
significantly decreased compared to presurgical samples, confirming that mass removal may reduce the number of CTCs
entering the bloodstream.
As well as for liver 13 and gastric 14 tumors, the number of
postsurgical CTCs correlates with clinico-pathologic parameters of ACC such as stage (11.7 ± 14.5 vs 2.1 ± 2.1, for stage
= 4 and < 4, respectively) and tumor diameter (8.3 ± 11.2 vs.
1.8 ± 2.0, for diameter ≥ 8.8 cm and < 8,8 cm, respectively),
suggesting that this so-called “liquid biopsy” might be a useful mini-invasive tool for prognosis and for monitoring progression and response to treatments.
In the near future, evaluation of the molecular expression
profile of CTCs may help to develop tailored anti-metastatic
therapies in ACC. Further studies, performed on larger
cohorts of patients and on blood samples collected before
surgery and at different follow-up intervals, are required
to definitively validate the prognostic value of this novel
biomarker in ACC.
References
1
Terzolo M, Angeli A, Fassnacht M, et al. Adjuvant mitotane treatment
for adrenocortical carcinoma. N Engl J Med 2007;356:2372–80.
2
Fassnacht M, Allolio B. Clinical management of adrenocortical carcinoma. Best Pract Res Clin Endocrinol Metab 2009;23:273-89.
216
Icard P, Goudet P, Charpenay C, et al. Adrenocortical carcinomas:
surgical trends and results of a 253-patient series from the French
Association of Endocrine Surgeons study group. World J Surg
2001;25:891-7.
4
Turner HE, Harris AL, Melmed S, et al. Angiogenesis in endocrine
tumors. Endocr Rev 2003;24:600-32.
5
Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells,
disease progression, and survival in metastatic breast cancer. N Engl
J Med 2004;351:781-91.
6
Allard WJ, Matera J, Miller MC, et al. Tumor cells circulate in the
peripheral blood of all major carcinomas but not in healthy subjects
or patients with non malignant diseases. Clin Cancer Res 2004;
10:6897–904.
7
Sun YF, Yang XR, Zhou J, et al. Circulating tumor cells: advances in
detection methods, biological issues, and clinical relevance. J Cancer
Res Clin Oncol 2011;137:1151-73.
8
De Giorgi V, Pinzani P, Salvianti F, et al. Application of a filtration and isolation by size technique for the detection of circulating tumor cells in cutaneous melanoma. J Invest Dermatol
2010;130:2440-7.
9
Desitter I, Guerrouahen BS, Benali-Furet N, et al. A new device for
3
rapid isolation by size and characterization of rare circulating tumor
cells. Anticancer Res 2011;31:427-41.
10
Khan MS, Tsigani T, Rashid M, et al. Circulating tumor cells and
EpCAM expression in neuroendocrine tumors. Clin Cancer Res
2011;17:337-45.
11
Pinzani P, Scatena C, Salvianti F, et al. Detection of circulating tumor
cells in patients with adrenocortical carcinoma: a monocentric preliminary study. J Clin Endocrinol Metab 2013 as doi:10.1210/jc.20131396.
12
Vona G, Sabile A, LouhaM, et al. Isolation by size of epithelial tumor
cells: a new method for the immunomorphological and molecular
characterization of circulating tumor cells. Am J Pathol 2000;
156:57-63.
13
Xu W, Cao L, Chen L, et al. Isolation of circulating tumor cells in
patients with hepatocellular carcinoma using an asialoglycoprotein
receptor-based magnetic cell separation strategy. Clin Cancer Res
2011;17:3783-93.
14
Wu CH, Lin SR, Hsieh JS, et al. Molecular detection of disseminated
tumor cells in the peripheral blood of patients with gastric cancer:
evaluation of their prognostic significance. Dis Markers 2006;
22:103-9.
Sala Massalia – ore 8.30-12.00
Neuropatologia
Simposio di neuropatologia
Moderatori: F. Giangaspero (Roma), G. Cenacchi (Bologna)
Utility and limits of WHO Classification
of meningiomas
V. Barresi
Dipartimento di Patologia Umana “G. Barresi”, Università di Messina, Italy
Meningiomas are tumours derived from arachnoidal cap cells
in the meningeal coverings of the spinal cord and brain 1. They
represent the most common extra-axial primary tumors of the
central nervous system (CNS) 1, accounting for 34% of intracranial tumours. Though meningiomas are generally benign,
slow growing neoplasias, they display an inner tendency to
recur, the most relevant clinical factor in recurrence being the
extent of surgical resection, according to the scale proposed
by Simpson in 1957 2.
In clinical practice, meningiomas are diagnosed and classified
according to the criteria proposed by the World Health Organization (WHO) Classification of Tumours of the CNS 1. This
classification categorizes these tumors into several histotypes
and into three histological grades of malignancy: WHO I, or
benign; WHO II, or atypical and WHO III, or anaplastic 1.
In the current edition of WHO Classification (2007), grade I
meningiomas are recognized by their histologic subtype and
lack of anaplastic features. Grade II meningiomas are defined
by one or more of the following four criteria: 1) chordoid or
clear cell histologic subtype, 2) four to 19 mitoses per ten
high-power field (HPFs), 3) brain infiltration, and 4) three
or more of the following five histologic features: small cell
change, increased cellularity, prominent nucleoli, sheet-like
growth, or spontaneous necrosis. Grade III meningiomas
(anaplastic/malignant) are defined by rhabdoid or papillary subtypes, obvious malignant cytology, resembling that
of carcinomas, melanomas, or high grade sarcomas, or 20
or more mitosis per ten HPFs 1. In comparison to the 2000
WHO edition, the WHO 2007 introduced brain infiltration
as an independent criterion to classify an otherwise benign
meningioma as grade II 1. The effect of this change was an
increase in the percentage of tumours recognized as grade II.
At present, histological grade, established according to the
WHO guidelines, is the most useful morphological predictor
of recurrence of meningiomas. Indeed, grade I meningiomas
recur in 7-25% of cases, atypical meningiomas in 29-52% and
anaplastic in 50-94% 1. Hence WHO grade of meningiomas
is of help in the stratification of patients for recurrence risk
and in the identification of those who need closer radiological
follow up and possible radiation therapy. Nonetheless, a percentage of grade I meningiomas do recur despite benign histology and total resection, and, on the other hand, there is the
risk to overtreat totally resected grade II meningiomas which
may not recur. Lack of correspondence between grading and
prognosis may be related to the subjectivity and absence of
precise definitions which characterize the criteria given by the
classification. For instance, the assessment of mitotic count,
which represents the most important criterion for classifying
a meningioma as grade II 3, may be influenced by the heterogeneity of mitotic activity in different areas of the tumour, or
by sample size, which may vary the number of evaluable cells.
In addition, distinction between mitotic figures and pycnosis
or apoptosis may be challenging at times. Though still not recommended in routine practice, the use of additional staining
may be of help in the recognition of mitotic figures. In detail,
assessment of mitotic index by using immunohistochemistry
against proteins exclusively expressed during mitosis, such
as phosphohistone H3 (PHH3), seems to display higher interobserver reproducibility and increased prognostic value than
count on H&E stain 4.
Inter-observer variability also concerns the minor criteria
for the diagnosis of grade II meningioma. The presence of
sheeting, which means an interrupted patternless or sheet-like
growth 1, may be difficult to establish due to its similarity to
217
relazioni
the syncitial pattern of meningothelial histotype. Also, the
assessment of hypercellularity maybe highly subjective.
Concerning prominent nucleoli, the WHO classification does
not give a definition of this feature, nor it specifies whether
it should be considered regardless of its extent in the sample.
Inter-observer variability in the assessment of macronucleoli
may be reduced by using the description by Perry et al. 5,
who state that only nucleoli easily observed at 10x should be
considered. In addition, since focal macronucleoli, limited to
peri-necrotic areas, may be related to pre-operative (POE) embolization, only diffuse macronucleoli in the tumour sample
should be regarded as a sign of malignancy 5. With reference
to necrosis, only “spontaneous” foci or “geographic” necrosis
should be taken into account as a sign of malignancy in meningiomas according to WHO classification 1. Nonetheless,
a clear histological description of spontaneous necrosis is
not given 1, the distinction between from induced necrosis
remaining largely arbitrary. Thus, in a meningioma exhibiting
necrotic foci neurosurgical details should be requested, in order to exclude POE-induced necrosis. According to our recent
findings, spontaneous necrosis commonly shows a gradual
transition from the viable tumour tissue, in comparison to
induced necrosis which is characterized by an abrupt line of
demarcation from viable tumour tissue 3. Finally, also small
cell presence, corresponding to tumour cells with increased
nuclear/cytoplasmic ratio, may be difficult to assess due to its
similarity to lymphocytes.
Another issue has been raised as to the classification of chordoid histotype as grade II 6. Indeed, in a recent paper it was
shown that chordoid meningioma is characterized by a good
prognosis, unless it is submitted to incomplete resection or it
shows malignant morphological features 9.
Due to the inter-observer variability which characterizes
WHO grading and to its inability to identify all recurring
cases on the basis of the only H&E stain, many efforts have
been made in order to find a molecular signature of recurring
meningiomas or an immunohistochemical marker able to
precociously detect these cases. Recently, high expression of
the micro-RNA miR-190a and low expression of miR-29c-3p
and miR-219-5p has been shown to correlate significantly
with higher recurrence rates in meningioma patients 7. Also,
as gains at 1q seem to identify those atypical meningiomas
characterized by shorter-free survival, the assessment of 1q
copy number status can add clinically useful information for
the management of patients with atypical meningiomas 8.
In conclusion, grading of meningiomas according WHO Classification, provides significant prognostic information which
may be used in clinical practice for the management of these
tumours. Nonetheless, some of the histological parameters
in the Classification need a higher standardization to reduce
inter-observer variability and deeper understanding of the
molecular basis of this tumor is warranted in order to classify
meningiomas on the actual recurrence-risk and to provide
individualized therapies.
References
1
Perry A, Louis DN, Scheithauer BW, et al. Meningiomas. In: Louis
DN, Ohgaki H, Wiestler OD, et al., eds. WHO Classification of
Tumors of the Central Nervous System. Lyon: IARCC press, 2007,
pp. 164-72.
2
Simpson D. The recurrence of intracranial meningiomas after surgical treatment. J Neurol Neurosurg Psychiatry 1957;20:22-39.
3
Barresi V, Branca G, Granata F, et al. Embolized meningiomas: risk of
overgrading and neo-angiogenesis. J Neurooncol 2013;113:207-19.
4
Kim YJ, Ketter R, Steudel WI, et al. Prognostic significance of the
mitotic index using the mitosis marker anti-phosphohistone H3 in
meningiomas. Am J Clin Pathol 2007;128:118-25.
5
6
7
8
Perry A, et al. Meningiomas. In: Perry A, Brat DJ, eds. Practical
Surgical Neuropathology: A Diagnostic Approach. Philadelphia:
Churchill Livingstone Elsevier 2010.
Wang XQ, Mei GH, Zhao L, et al. Clinical features and treatment of
intracranial chordoid meningioma: a report of 30 cases. Histopathology 2013;62:1002-17.
Zhi F, Zhou G, Wang S, et al. A microRNA expression signature
predicts meningioma recurrence. Int J Cancer 2013;132:128-36.
Jansen M, Mohapatra G, Betensky RA, et al. Gain of chromosome arm
1q in atypical meningioma correlates with shorter progression-free
survival. Neuropathol Appl Neurobiol 2012;38:213-9.
The diagnostic role and the clinical relevance
of determination of BRAF status in brain tumors
M. Gessi
Inst. of Neuropathology, University of Bonn Medical Center, Bonn,
Germany
BRAF, a member of the serine/threonine kinase protein family, plays an important role in the RAS/RAF/MEK/ERK/
MAPK signalling cascade, a signal-transduction pathway that
modulates various processes, including cell proliferation and
survival. In several systemic cancers and leukemias, oncogenic activation of BRAF usually results from point mutations
rather than gene rearrangements. The most common BRAF
mutation is the T1796A point mutation, resulting in a valine
(V) -> glutamic acid (E) substitution at position 600.
BRAF mutations are also common in a wide spectrum of brain
tumors. BRAFV600E mutations have been found in about 1015% of pilocytic astrocytoma, in 5-10 % of pediatric diffusely
infiltrating gliomas, including low-grade (WHO grade II),
anaplastic (WHO grade III) astrocytomas and glioblastomas
(WHO grade IV) but in less than 2% of comparable adult
gliomas. Mutations occur frequently in gangliogliomas and in
pleomorphic xanthoastrocytomas (PXA). They have been also
described in a subgroup of dysembrioplastic neuroepithelial
tumors (DNT).
Besides mutations, BRAF activation can also result from gene
duplications. Duplications leading to a fusion between the
KIAA1549 and BRAF genes have been identified in 60% to
80% of pilocytic astrocytomas. Genomic sequencing has revealed a few breakpoint variants (KIAA1549 exon16–BRAF
exon 9, KIAA1549 exon 15–BRAF exon 9, KIAA1549 exon
16–BRAF exon 11, exon19–BRAF exon 9 and KIAA1549
exon18–BRAF exon fusions). These breakpoint variants
result in the formation of an oncogenic BRAF-KIAA1549
fusion resulting in a truncating BRAF without the N-terminal
auto-inhibitory domain. Other types of tandem duplication
with in-frame oncogenic fusions of BRAF or of other serine/
threonine kinase proteins (such as FAM131B-BRAF and
SRGAP3-RAF1) have been rarely described in pilocytic astrocytomas. Tandem duplications and BRAF point mutations
are mutually exclusive and do not occur in the same frequency
in pilocytic astrocytomas at all sites in the CNS. Cerebellar
pilocytic astrocytomas harbour frequent BRAF-KIAA1549
fusion (ranging from 60% to 90% of cases) in comparison
with BRAF mutations more frequently observed in hemispheric tumors. Moreover, BRAF fusions seem to be less frequent among tumors affecting older children and adults. The
prognostic significance of the presence of KIAA1549-BRAF
fusion in patients with pilocytic astrocytomas is still unclear,
even though some studies have found no differences in survival between tumors with and without BRAF duplication.
Although several studies have confirmed a strong association
between BRAF fusions and pilocytic astrocytoma histology,
218
cases of diffuse astrocytoma harbouring BRAF-KIAA1549
fusions have been reported indicating that such molecular
changes are very common in pilocytic astrocytomas but are
not absolutely specific for these tumors.
There is no standard method for testing BRAF status in diagnostic neuropathology. High sensitive standard tests for
detection of BRAF mutations in skin melanomas and thyroid
carcinomas are commercially available. Among sequencing
methods, pyrosequencing has a high sensitivity and represent
an ideal method for rapid detection of BRAF V600 mutations.
Multiplex ligation-dependent probe amplification (MLPA) is
a polymerase chain reaction (PCR)-based technique in which
probes hybridized to the sample DNA are amplified using
only one PCR primer pair. Mutation-specific MLPA probes
have been developed for the identification of the BRAFV600E
mutations. Besides molecular markers, a monoclonal antibody
recognizing the BRAFV600E mutated protein (clone VE-1) has
also been developed and is commercially available. Its reliability especially in comparison with other well-established
molecular based methods has been demonstrated in several
studies on different tumor subtypes, although false-negative
results may occur in tissues damaged by surgical coagulation
or freezing, and the staining intensity in mutated cases varies
greatly.
Several approaches to identify BRAF fusions are currently
used in molecular neuropathology laboratories. The presence
of duplication of the 7q34 region as a marker for BRAF fusion
can be assessed by FISH, which is a particularly attractive
method given their applicability to FFPE samples. Direct
identification of fusion transcripts using RT-PCR based methods is highly specific and further permits the identification of
specific fusion breakpoints. Although it can be also performed
on FFPE material, this method needs usually high quality cDNA often not available from FFPE.
Because target-based therapies are widely considered to be
the future of cancer treatment, attention has been focused on
developing inhibitors of the RAS-RAF-MEK-ERK/MAPK
signalling pathway and its upstream activators. Given the high
frequency of BRAF alterations among brain tumors, there is
great interest in targeted inhibition of the MAPK pathway, not
only in patients with glioblastomas but also in patients with
low grade gliomas whose tumors are in locations that prevent
full resection. Among the several MEK1/2 and RAF inhibitors
commercially available, some agents, such as sorafenib and
selumetinib (AZD6244), have been investigated or are currently under investigation in glioma patients.
In conclusion, BRAF alterations, leading to MAPK pathway
activation, are a common event in several types of gliomas
and the determination of its molecular status is become a diagnostic tool in neuropathology. The prognostic role of BRAF
alterations is still not clear, and analyses of large cohorts of
uniformly treated patients will probably be required to definitively evaluate the association with patient outcome. Because
investigations of the potential use of anti-BRAF agents in
management of gliomas are ongoing in clinical trials, it cannot be excluded that the molecular testing of BRAF status in
primary brain tumors will become more frequently requested
by neuro-oncologist in the future.
Sessione AITIC-ANTeL-ASSIATEL
Moderatori: C. Lupo (Palermo), B. Pini (Roma)
The molecular biology applied to tissue’s
pathology
E. Pilozzi
Roma
Until few years ago, the role of the pathologist was confined
to the morphological diagnosis of tissue abnomarlities integrated, when necessary, by the immunophenotypic characterization. Pathologist’s role in neoplastic disease consisted
mainly on morphological classification and staging of tumours, both crucial aspects for prognosis.
Relevant progress has been made in understanding the biology
of the molecular mechanisms underlying neoplastic transformation, tumour progression and metastasis. Malignancies
should be regarded as “multigenic” diseases since they accumulate numerous genetic or epigenetic abnormalities.
The genetic alterations that occur during the transformation
and neoplastic progression can be documented in the tissue
that is sent to Pathology Laboratory for histological examination. Tissues, that until recently were regarded merely as
source of morphological information, are currently regarded
as a huge content of molecular informations that can be
decoded.
The identification of gene alteration can be made by “in situ”
techniques (molecular morphology) or by molecular biology
techniques which require the extraction of nucleic acids (DNA
or RNA) from the tissue through its lysis. To the first group
belongs the FISH (fluorescent in situ hybridization), which
allows to assess increase in gene copy numbers, translocations
and deletions. To the second group belong the techniques
which provide amplification of the DNA sequence of interest
to highlight gene mutations. This goal can be achieved by
Sanger sequencing, pyrosequencing or through the use of Real
-Time PCR containing specific probes.
The molecular alterations that can be investigated in a tissue
can be roughly divided into: a) predictive information (fate of
the patient following the effect of a specific therapeutic intervention); b) prognostic information (fate of the patient regardless of the therapy); c) information of diagnostic value that
contribute to better classify morphologically complex lesions.
The molecular predictive markers.
As mentioned cancers are regarded as a “multigenic” disease
characterized by a number of genetic and epigenetic abnormalities. However it has been documented that alterations in
one or a few genes, so called drive-mutations, play a more
important role in the maintenance of the tumour. The identification of these genes has led to the development of highly
effective antitumour drugs, so-called “targeted therapy”. To
produce a therapeutic effect it is necessary that the molecular
alteration towards which it is directed is present in the neoplastic tissue of the patient being considered for therapy. In
fact, many target drugs developed on the biological evidence
219
relazioni
in preclinical studies proved to be unsuccessful when administered to a cohort of unselected patients. Patient selection is done through the identification, in neoplastic tissue of molecular
alteration of interest. So in the era of targeted therapy the role
of surgical pathology in the multidisciplinary management of
cancer patients has been enriched by a significant value even
in treatment choice. In fact the identification of predictive
biomarkers in neoplastic tissue has become mandatory for
therapeutic decision. The pathologist is the only specialist
able to identify the neoplastic tissue in which to look for the
molecular alterations and he is therefore able to integrate the
morphological report with a molecular report. Let’s now analyze what are the predictive molecular information essential
for the therapeutic treatment of cancer.
Molecular target drugs in use are monoclonal antibodies
directed towards receptors of growth factors (Growth Factor
Receptor) or proteins involved in the pathway of signal transmission of these receptors. The pathway of signal transmission through a receptor of growth can be “turned on” in many
ways: amplification of the gene coding for the growth factor
which leads to overexpression of the factor on cell membrane
(eg. HER2 amplification in breast cancer and in gastric cancer); mutation in the intracellular domain (typically the tyrosine kinase domain) which leads to activation of the receptor
even without the ligand (eg EGFR in lung carcinoma; C-KIT
and PDGFR-a in GIST); activation of kinase through gene
translocation (eg. ALK in lung cancer); activating mutation
in a gene encoding a protein involved in the cascade of signal
transmission (eg, K-RAS in adenocarcinoma of the colon, BRAF in melanoma).
The discovery of novel driver molecular alterations and of
drugs effective on them is leading to a almost daily increase
in the number of predictive biomarkers to be assessed in neoplastic tissues.
The molecular prognostic markers.
They represent informations that help to stratify the prognosis
of cancer patients regardless of the staging and in some cases
to direct the choice of treatment. We know from studies on
large cohorts of patients that BRAF mutation in lung and
colon cancer represents a negative prognostic factor. Another
example is the evaluation microsatellite instability (MSI) in
stage II colorectal carcinomas. Indeed, the presence of such
molecular phenotype reduces the possible benefit of a chemotherapy with fluorouracil.
Mutations in the IDH1 gene, chromosomes 1p and 19q codeletion, hypermethylation of MGMT gene represent a positive prognostic factor in glial neoplasms.
The molecular information of diagnostic value.
There are molecular alterations, in particular represented
by chromosomal translocations, which are almost tumourspecific. So their identification contributes to the histological
diagnosis. Examples are represented by lymphoproliferative
disorders (eg, mantle cell lymphoma t (11; 14), Burkitt’s lymphoma t(8; 14), follicular lymphoma t(14; 18)) and soft tissue
tumors (eg Ewing’s sarcoma t(11;22);round cell liposarcoma
t(12;16).
The study of clonality of lymphoid cells (B and T), through
gene amplification of IgH, TCRgamma, and TCRbeta, allows investigation of monoclonality or polyclonality of
lymphoid population. It helps in interpretation of complex
lesions in which morphology and immunophenotype are not
diriment.
The molecular documentation of infectious agents (eg mycobacterium, HPV) is another example of integration of a diagnosis that can be through of molecular biology techniques.
Conlusions. Increasing knowledge on molecular mechanisms
of disease, the development of target drugs and the technological progress conferred a new value to pathologic tissues as
source of large amount of molecular informations.
The new technologies will soon allow the simultaneous assessment of several genes at the same time providing prognostic and predictive informations.
Sessione AITIC-ANTeL-ASSIATEL
Moderatori: B. Pini (Roma), T. Zanin (Genova)
ALK clone D5F3 verso FISH ALK
L. Carmelo, E. Roz
Casa di Cura di Alta Specialità la Maddalena, Dipartimento Oncologico di III Livello, Palermo
La determinazione dello status del gene ALK si effettua su
sezioni di tessuto, fissate in formalina ed incluse in paraffina
di carcinoma polmonare non a piccole cellule (NSCLC), per
l’identificazione dei pazienti idonei al trattamento con Crizotinib. Di solito, nei pazienti affetti da NSCLC, la determinazione dello status di ALK, avviene dopo l’analisi mutazionale del
gene EGFR. I riarrangiamenti di ALK sono raramente coincidenti con le mutazioni EGFR, HER2 o KRAS. La patogenesi
molecolare dell’ALK inizia con ridistribuzioni cromosomiche
che si appaiano con le sequenze di codifica 3’per il dominio
di segnalazione intracellulare con elementi promotori 5’e
sequenze di codifica di altri geni. Un’inversione all’interno
del cromosoma 2p genera la formazione di un prodotto del
gene di fusione, comprendente porzioni del gene microtubuloassociato dell’echinoderma simil proteina 4 (EML4) e del gene ALK. Complessivamente, una serie di studi sui carcinomi
polmonari non a piccole cellule (NSCLC), ha indicato che
l’incidenza dei riarrangiamenti genici ALK varia dal 2 al 7%.
Per la determinazione dello status di ALK, presso il nostro
laboratorio è stato utilizzato il saggio FISH (ibridizzazione in
situ mediante fluorescenza) ALK Break-Apart su campioni
di NSCLC fissati in formalina ed inclusi in paraffina. Si è
proceduto ad allestire sezioni seriate di tessuto dagli stessi
campioni istologici testati in FISH e ad eseguire la colorazione immunoistochimica con l’anticorpo anti-ALK (D5F3), un
anticorpo monoclonale primario di coniglio. Questo anticorpo
si è dimostrato riproducibile nei suoi risultati di colorazione. I
casi positivi precedentemente testati in FISH, successivamente colorati con anti-ALK (DSF3) hanno mostrato un forte segnale granulare citoplasmatico. L’interpretazione dei risultati
dell’espressione della proteina ALK nei campioni di tessuto
è stata effettuata utilizzando l’algoritmo per il calcolo del
punteggio appropriato. Nel NSCLC è stato possibile osservare
anche una certa colorazione di fondo all’interno della mucosa
normale soprattutto in presenza di mucina e in aree tumorali
necrotiche. I casi negativi hanno mostrato l’assenza di forte
colorazione granulare citoplasmatica nelle cellule tumorali. I
220
risultati delle reazioni immunoistochimiche, positivi e negativi, sono concordanti con la determinazione di ALK mediante
tecnica FISH. Tuttavia, utilizzando l’anticorpo anti-ALK
(D5F3), si è evidenziata la necessità di operare su un preparato
istologico che abbia subito un accurato processo di fissazione
e processazione. L’antigene ALK risulta fortemente influenzato, ancor di più del gene ALK rilevabile mediante sonda
FISH, non solo dal tempo di fissazione, ma anche dal tipo di
fissativo utilizzato. A questo proposito, sarebbe opportuno
auspicare la creazione di un documento di accompagnamento
del preparato istologico da sottoporre alla determinazione
di ALK, qualora il campione da esaminare provenga da una
struttura diversa dal laboratorio che deve procedere all’esame.
Questo documento, redatto dall’ospedale di provenienza, attesterebbe le condizioni di preanalitica del campione da esaminare. Alla luce di queste informazioni si potrebbe procedere
con la tecnica più idonea: colorazione mediante l’impiego
dell’anticorpo anti-ALK (D5F3), nel caso in cui le condizioni preanalitiche rientrino nei requisiti procedurali richiesti
per questa tecnica; in caso contrario o nell’impossibilità del
reperimento di tali informazioni si dovrebbe procedere alla
determinazione dello status di ALK mediante tecnica FISH.
Bibliografia
1
Galetta D, Rossi A, Pisconti S, et al. The emerging role of ALK inhibitors in the treatment of advanced non-small cell lung cancer. Expert
Opin Ther Targets 2012;16(Suppl. 2):SS45-54.
2
Peled N, Palmer G, Hirsch FR, et al. Next-Generation Sequencing
ldentifies and lmmunohistochemistry Confirms a Novel CrizotinibSensitive ALK Rearrangement in a Patient with Metastatic Non-SmallCell Lung Cancer. J Thorac Oncol 2012:7:e14-16.
3
Yi ES, Boland JM, Maleszewski JJ, et al. Correlation of IHC and ÉtSn
ior ALK gene rearrangement in non-small cell lung carcinoma: IHC
score algorithm for FISH. J Thorac Oncol 2011;6;459-65.
4
Mcleer-Florin A, Moro-Sibilot D, Melis A, et al. Dual IHC and FISH
testing for ALK gene rearrangement in lung adenocarcinomas in a
routine practice: a French study. J of Thorac Oncol 2012;7:348-54.
5
Jokoji R, Yamasaki T, Minami S, et al. Combination of morphological feature analysis and immunohistochemistry is useful for
screening of EML4-AlK-positive lung adenocarcinoma. J Clin Pathol
2010;63:1066-77.
6
Mino-Kenudson M, Chirieac LR, Law K, et al. A novel, highly sensitive antibody allows for the routine detection of AlK-rearranged lung
adenocarcinomas by standard immunohistochemistry. Clin Cancer
Res 2010;16:1561-71.
Technical aspects of immunocytochemistry
and Fish analysis on cytological samples.
A point of view from SIAPEC-Campania
Molecular Pathology Group
C. Della Ragione, V. Cerbone, P. Galloro, N. Mitilini, G. Capasso
Division of Anatomic Pathology, Histology and Cytopathology,
“A. Cardarelli” Hospital, Naples, Italy
Background. Immunocytochemistry (ICC) and Fluorescent
in Situ Hybridization (FISH) need to be standardized on cytological samples. This is important not only to refine uncertain
microscopic diagnosis, but also considering the need to predict the response to treatment of targeted gene products. In
particular special care need to be taken to process lung cytological samples. These latter are usually obtained in advanced
lung cancer patients for which histological samples are not
available. ICC and FISH require specific pretreatment steps,
including heat induced epitope retrieval (HIER). These latter
have poorly been standardized. Differences include length of
time of pretreatment, temperature and type of enzymatic di-
gestion. This variability is enhanced by the fact that a number
of different samples and preparation types are used, including
direct smears, archivial stained slides, Thin-Prep and Cell
Blocks (CBs). These issues were discussed by the SIAPECCampania Molecular Pathology Group, taking into account
different experiences and opinions.
Materials and methods. Immunocytochemistry was carried
out testing a set of 31 antibodies, including both nuclear and
membrane/cytoplasmic targets on a wide selection (n = 100)
of different preparations and types of cytological specimens.
These included ethanol-fixed Papanicolaou stained direct
smears and Thin prep slides. HIER was performed with variable intensity by applying different lengths of time using
the Ventana Benchmark XT autostainer and/or enzymatic
digestion pretreatment. Positive and negative controls were
obtained by touch imprints of fresh surgical resections following the same fixation protocol of routine cytological samples.
FISH was carried out on 50 cytological samples relative to
pulmonary aspiration and pleural effusions tested by the
different set of probes (i.e, EGFR and ALK) able to detect
both gene-rearrangements and amplifications. Assays were
performed on alcohol or methanol-acetone fixed direct smears
and Thin-Prep slides and CBs; the slide were either unstained
or previously Papanicolaou stained. A slide area was marked
before slide decoverslipping with or without destaining 1.
Cell block from cytological specimens were also included
in the FISH analysis. Different pretreatment protocols were
employed; a pretreatment protocol similar to that used for
paraffin-embedded tissues was employed for CBs, while
on smears a range of different treatments ranged from direct probe incubation without any previous step to complex
protocols similar to the “Urovysion pretreatment Kit” or to
protocols that combined heat and enzymatic steps were tested.
Results and conclusions. Our results relative to ICC on cytological samples consistently shown: 1) HIER is mandatory
when nuclear antigen are detected; 2) HIER further enhances
the signal intensity of membrane/cytoplasmatic antigens; 3) in
any case HIER should be less intense than that requested on
paraffin-embedded histological samples; 4) Antigen Retrieval
by enzymatic digestion is less effective than HIER and may
lead to artifacts and should be therefore avoided; 5) Formalinfixed direct smears samples requires the same less-intense treatment of alcohol-fixed sample, as described by Fulciniti et al. 2.
Our results relative to FISH on cytological samples were
strongly dependent on sample preparation type: 1) On CBs
good results were achieved by the same conditions validated for
routine histological samples; 2) Additional unstained ethanolfixed direct smears, whose collection was dedicated prior to
FISH, did not require any pretreatment step; 3) FISH signal
obtained on stained routine archivial direct smears were not
always evaluable, mostly in preparation blood-cell rich or with
cells un-evenly smeared. Results on this kind of samples were
improved by destaining with 1% Hydrochloric acid in 70% ethanol for 1 hour; these destained samples benefited by pretreatment with 2X SSC (2 mins at 73°C) followed by a pepsin digestion at 37°C (time ranging from 5 to 30 mins. relying to pepsin
concentration utilized); 4) The same protocol did not succeed to
yield consistent results on Thin-Prep slides. Further studies are
ongoing to better address these issues on novel targets.
References
1
Betz BL, Dixon CA, Weigelin HC, et al. The Use of Stained Cytologic
Direct Smears for ALK Gene Rearrangement Analysis of Lung Adenocarcinoma. Cancer Cytopathol 2013 [Epub ahead of print].
2
Fulciniti F, Frangella C, Staiano M, et al. Air-dried smears for optimal
diagnostic immunocytochemistry. Acta Cytol 2008;52:178-86.
221
relazioni
Patologia dei trapianti
Moderatori: A. D’Errico (Bologna), A. Onetti Muda (Roma)
Role of pathologist in lung transplantation
L. Delsedime
Torino
The success of the transplant program requires a multidisciplinary approach; the role of the pathologist is carried out
mainly during follow-up with monitoring of post-transplant
complications obtained with transbronchial biopsy (TBB).
Although non-invasive methods have been tested, bronchoscopy with TBB remains the gold standard for detecting
and grading rejection episodes and for making differential
diagnosis between acute rejection and infection in Lung
Transplant patients.
The histopathological lesions evaluated at the biopsy should
be interpreted and integrated in the clinical context to allow
the management of the patient’s complications and clinical
decisions. Correlation with clinical and radiographic information is necessary for a conclusive diagnosis or in order
to refine interpretations when the biopsy shows more than
one process.
The main complications observed on TBB after lung transplantation in the early stages are:
Primary graft dysfunction (PGD), also known as ischemiareperfusion injury, is a peri-operative, multifactorial insult.
PGD is a syndrome encompassing a spectrum of mild to
severe lung injury that occurs within the first 72 hours after
lung transplantation (LTx). It is characterized by pulmonary
edema with diffuse alveolar damage that manifests clinically
as progressive hypoxemia with radiographic pulmonary infiltrates. The incidence of PGD ranges from 10% to 25% 1 2.
Patients with pulmonary hypertension and pulmonary fibrosis
have the highest incidence of ischemia reperfusion injury 3.
Severe PGD correlates with a significantly higher short term
mortality and in the long term it is associated with the risk of
chronic lung allograft dysfunction (CLAD) 4.
Acute Allograft Rejection (AR) remains a prevalent and
serious problem in LTx, with an incidence of 36% in the first
year post transplant according to the latest report from the
registry of the International Society for Heart and Lung Transplantation (ISHLT) 5. Specificity of clinical, functional and
radiographic abnormalities for acute rejection is low. Transbronchial biopsy is currently considered a “gold standard” for
distinguishing acute rejection from infection in lung allograft
recipients. The specificity and sensitivity of transbronchial
lung biopsy for detecting acute rejection are 93% and 77 %,
respectively 6.
For the definition, classification and grading of rejection on
TBB, the pathologist uses the recommendation provided by
the Working Formulation (WF) of the International Society
for Heart and Lung Transplantation (ISHLT) 7. This classification determines a standardized process between the
various centers, enables comparisons between case series
and allows for multi-center trials. Since the first WF in 1990,
two successive revisions (in 1995 and 2007) have followed,
leading to further simplifications of the original scheme.
These revisions have been enriched with some guidelines
on complex issues such as the significance of bronchiolar
inflammation and humoral rejection. The histologic lesions
associated with AR are well established: AR is defined by
the presence of perivascular mononuclear cell infiltrates,
which may be accompanied by sub-endothelial chronic inflammation (e.g., endotheliitis). The majority of the mononuclear cells in acute rejection are T cells but eosinophils
and occasionally neutrophils are common and their number
increases with the worsening of the grading. Four grades are
recognized, corresponding to minimal ((ISHLT grade A1),
mild ((ISHLT grade A2), moderate ((ISHLT grade A3) and
severe ((ISHLT grade A4) AR. The traditional threshold for
therapeutic intervention is Grade A2.
Minimal AR (A1) is characterized by a relatively thin chronic
mononuclear infiltrate surrounding scattered infrequent blood
vessels, particularly venules, in the alveolated lung parenchyma.
Mild AR (A2): although the perivascular infiltrate of lymphocytes is essentially confined to the perivascular adventitia
without infiltrating the interstitium, there are more layers of
lymphocytes surrounding the vessel and lymphocytes might
focally, minimally spill into the adjacent interstitium. The
infiltrates may include eosinophils and be associated with
endotheliitis, but there is no definitive extension of infiltrates
into the surrounding alveolar interstitium. Gradually the dense
perivascular infiltrates, associated with widespread endotheliitis extends into the alveolar septa and, subsequently, into the
alveoli (Moderate AR, A3).
Severe AR (A4): in severe rejection there are diffuse perivascular, interstitial, and air space infiltrates of mononuclear
cells with prominent alveolar pneumocyte damage and endotheliitis. This may be associated with intra-alveolar necrotic
epithelial cells, macrophages, eosinophils, hemorrhage, and
neutrophils and usually some evidence of acute lung injury
in the form of organizing pneumonia or hyaline membranes
disease. Grade A4 rejection is uncommon. Differentiating
grade A4 AR from other causes of Diffuse Alveolar Damage
(DAD)- pattern injury, such as reperfusion injury or infection,
may be challenging. The presence of widespread, well-defined perivascular infiltrates favours a diagnosis of rejection 8.
The interpretation of allograft biopsies may be difficult particularly because of frequent co-occurring of rejection and
infection. Viral infections in particular can cause mononuclear
inflammation, which represents a possible confounding cause
in the interpretation of histologic findings.
Antibody-mediated rejection (AMR) is a relatively infrequent
finding. The histopathology of AMR in lung allografts has
been poorly defined, in part because of the difficulty in identifying potential alloantibodies and in separating alloreactive
and nonspecific complement-mediated reactions 9.
The 2007 revised working formulation of the ISHLT concluded that histopathologic and immunophenotypic criteria of
AMR in LTx were not yet established or accepted by pathologists and the diagnosis and recognition of antibody-mediated
rejection of the lung is more controversial and less well developed than of other solid-organ grafts 7.
Recently there has been significant progress in understanding
the pathologic manifestations of pulmonary AMR since the
2007 ISHLT revised working formulation. At the workshop
of the 2012 ISHLT meeting in Prague consensus was reached
222
on a number of important items such as the necessity of a
multi-disciplinary approach for the diagnosis of pulmonary
AMR that includes the presence of clinical allograft dysfunction, the circulating donor-specific antibodies and pathologic
findings (the so-called “triple test”). A list of histopathologic
patterns of damage has been enumerated. Indications on the
interpretation of C4d capillary staining has been proposed 10.
Lymphocytic bronchiolitis (LB)
The ISHLT has promulgated a “B” scoring system for
rejection-related airway inflammation. In cases where small
airway inflammation is present but perivascular mononuclear
infiltrates diagnostic of AR are not, correlation with microbiology is necessary to exclude infections before ascribing acute
rejection as the cause. Rejection-related airway inflammation
should also be differentiated from bronchus-associated lymphoid tissue. Although the previous ISHLT working formulation assigned four grades of airway inflammation (B1–B4),
concerns over reproducibility resulted in simplification into
two grades (B1R and B2R) in the current classification 7.
The clinical meaning of LB in the absence of parenchymal
acute rejection is unclear and agreement is lacking concerning the clinical implications of this histological finding. Based
on experimental and clinical studies, LB is considered as a
possible manifestation of rejection and a likely risk factor for
bronchiolitis obliterans syndrome (BOS) when infection has
been excluded 11.
In conclusion, LB has a prognostic relevant significance, but
the arduous differential diagnosis with co-occurring infectious
forms, and the ineffectiveness of standard therapies, makes
this chapter still unresolved 12.
Late Complications
Infections. Lung transplant recipients have the highest
rates of infection among solid organ transplant patients 13.
Opportunistic infections increase in importance with ongoing immunosuppression. LTx recipients are at high risk for
cytomegalovirus (CMV) and herpesvirus (HHV) infections.
Among HHVs, some beta-Herpesviruses including Herpesvirus-6 (HHV-6) and Herpesvirus-7 (HHV-7) and gammaHerpesvirus including Epstein-Barr virus (EBV) have been
frequently isolated in the pulmonary alveolar microenvironment following lung transplantation 14. EBV infection has
also been associated with post-transplant lymphoproliferative disorders (PTLD). Many studies have demonstrated a
possible role of CMV on AR and post-transplant LB. In
particular CMV has been associated with allograft rejection
and transplantation-associated arteriosclerosis. CMV prophylaxis has been found to be beneficial in preventing CMV
infection after lung transplantation. Although relatively uncommon, adenovirus pneumonia results in significant morbidity and mortality in lung transplant recipients. Several
large, retrospective reviews have reported the incidence of
adenovirus pneumonia to be about 1% to 2% in this transplant population.
Chronic Rejection. Chronic rejection has remained a major
source of morbidity and mortality following lung transplantation. Survival data from the registry of ISHLT demonstrate a
significant improvement in the early (up to one year) survival
of transplant recipients over the past decade; however, the
subsequent decline in survival persists at virtually the same
rate. Chronic Airway Rejection affects > 50% of all lung
transplant survivors and is the leading cause of death. Mean
time to presentation is 1.5-2 yrs 5. The primary manifestation
of chronic rejection is obliterative bronchiolitis (OB), which
is thought to represent chronic airways rejection (grade C,
WF). OB is histologically characterised by submucosal and
intraluminal deposition of dense fibrous tissue in membranous and respiratory bronchioles with partial or complete
occlusion of the bronchiolar lumen. The lesions of OB may
be accompanied by chronic inflammation and nonspecific
post-obstructive changes such as mucostasis and foamy macrophages in the surrounding lung tissue. The current ISHLT
classification mandates grading based on the presence (C1)
or absence (C0) of evidence of obliterative bronchiolitis 7.
CR is usually a clinical diagnosis with lung function decline,
based on FEV1 decline (BOS), confirmed in a minority of
cases (15%) with TBB findings 5. Intensity and persistence of
AR, LB and decreased immunosuppression correlate strongly
with the development of OB. Recently there is a considerable
interest also in the role of gastric aspiration as a cause of graft
injury after lung transplantation. Micro-aspiration is common
in transplanted patients, it may cause pulmonary infiltrates
and may increase risk of BOS. The Leuven group reported the
roles of acid and nonacid gastroesophageal reflux and aspiration in lung transplant patients with or without BOS 15.
Chronic Vascular Rejection/accelerated graft vascular sclerosis (grade D, WF) refers to fibrointimal thickening of arteries.
Chronic vascular rejection, while less well characterized,
is also believed to occur in LT. It refers to the obliterative
process affecting arteries and veins, it occurs in most solid
organ transplants, and reflects accelerated atherosclerosis
with fibrointimal thickening of the subendothelial with loose
myxomatous connective tissue. A mononuclear cell and
foamy cell infiltrate is common. Chronic vascular rejection is
rarely identified on biopsies since they usually lack vessels of
sufficient size 8.
Restrictive allograftt syndrome (RAS) is a novel form of
chronic lung allograft dysfunction, described in 2011, characterized by a restrictive physiology together with radiologic
and pathologic characteristics of fibrosis in peripheral lung
tissue, including alveoli, pleura and interlobular septa 16.
Disease Recurrence: Recurrence of pre-transplantation lung
disease within the allograft lung is fairly common in lung
transplantation for sarcoidosis, lymphangioleiomyomatosis
and Langerhans cell histiocytosis and evidence of recurrence
may be identified in allograft biopsies.
Post-Transplant Lymphoproliferatve Disorders (PTLD) are a
long-term problem, with an incidence of 13% at 5 years and
28% at 10 years 5. PTLD is an uncommon complication after
LTx, and its incidence has declined remarkably in the era of
modern immunosuppression. Studies have reported incidence
rates between 2.5% and 8% in LTx recipients 17.
Although clinical signs and symptoms are helpful for alerting
to the possibility of a complication, they are not always present 18. Many centers choose to perform surveillance bronchoscopy with biopsy at specified time intervals after LTx (19-21).
In our Center for follow-up of transplanted patients surveillance bronchoscopy with TBBs is performed at 4 weeks, 12
weeks, 6,12,18 and 24 months post-transplant. Additional
TBBs are also performed during symptomatic episodes in
patients clinically compromised.
We will present the morphological aspects that characterize
the various types of damage observed on TBBs and some
results of the consecutive 911 TBBs obtained until December
2012 relative to the 203 lung transplants performed at the
Health and Science City Hospital of Turin, between September 1993 and December 2012.
Material for diagnosis was insufficient in 2% of the TBBs (according to the ISHLT general recommendations of specimen’s
adequacy). Among adequate TBBs, 33% were negative, 6%
showed relevant signs of ischemia-reperfusion injury; 29%
relazioni
Acute rejection (13% A1, 12% A2, 4% A3+A4). Significant
rejection was considered Grade 2 (mild) or higher; in contrast, patients with Grade A1 biopsies did not receive higher
level of immunosuppression. Lymphocytic bronchiolitis (B1R
sec. WF) was observed in 10% of TBBs, while another 10%
showed nonspecific features of interstitial inflammatory infiltrates accompanied in some cases by fibromyxoid plugs
within small airways and surrounding airspaces, as a pattern
of Organizing Pneumonia (OP); 4% showed histological
findings of established OB (C1 sec. WF); 5% had viral infections with pneumonia damage (CMV, HSV, Adenovirus), 2%
fungal infections and 1% PTLD. In our series the prevalence
of CMV infection (evaluated from bronchoalveolar lavage
(BAL) and TBB) was lower than that reported in the literature.
This confirms the importance of the universal prophylaxis
regime irrespective of serologic matching. Our preliminary
results seem to confirm the role of CMV in the pathogenesis
of acute rejection, while the role of other beta-herpesviruses
in this rejection needs to be further evaluated. CMV-IG in
addition to ganciclovir and valganciclovir in the first postoperative year after lung transplantation is effective in reducing episodes of AR and LB 22.
Therefore, the recognition at the surveillance TBB of acute
rejection of mild/ moderate grade or of viral infection, risk factors of chronic rejection has modified the therapeutic approach.
The TBB procedure exposes patients to a few procedurerelated complications; in our series pneumothorax occurred in
2%, and significant bleeding in 1% of the cases.
Interpretation of biopsies from LTx patients should take into
account the patient’s clinical situation and the results of other
laboratory tests and imaging.
In our Center the pathologist participates at the weekly multidisciplinary lung transplant staff meeting. During this meeting
the whole staff discusses the particular cases with open issues
and therapeutic decisions about treatment protocols (antibiotic
prophylaxis and immunosuppression) are taken by consensus.
Conclusions. The pathologist plays an important role in
the multidisciplinary management of transplant patients in
synergy with other professionals, monitoring the possible
complications post-LTx and contributing to modulate the appropriate therapies to clinical characteristics and history of
each individual patient.
The usefulness of morphological control through the TBBs is
in providing early evidences of complication, even before the
clinical manifestation allowing the modulation of therapies
(mainly anti-rejection and anti-infection mainly) at various
times. We therefore believe that the surveillance TBBs can
contribute to the increase in post-transplant survival and may
delay the onset of chronic rejection.
References
1
Lee JC, Christie JD, Keshavjee S. Primary graft dysfunction: definition, risk factors, short- and long-term outcomes. Semin Respir Crit
Care Med 2010;31:161-171.46.
2
Arcasoy SM, Fisher A, Hachem RR, et al; ISHLT Working Group
223
on Primary Lung Graft Dysfunction. Report of the ISHLT Working
Group on Primary Lung Graft Dysfunction. J Heart Lung Transplant
2005;24:1483-8.
3
Fiser SM, Kron IL, McLendon Long S, et al. Early intervention after
severe oxygenation index elevation improves survival following lungtransplantation. J Heart Lung Transplant 2001;20:631-6.
4
Whitson, BA, Prekker, ME, Herrington, CS, et al. Primary graft dysfunction and long-term pulmonary function after lung transplantation.
J Heart LungTransplant 2007;26:1004.
5
Christie JD, Edwards LB, Kucheryavaya AY, et al. International
Society of Heart and Lung Transplantation.The Registry of the International Society for Heart and Lung Transplantation: 29th adult
lung and heart-lung transplant report-2012. J Heart Lung Transplant
2012;31:1073-86.
6
Pomerance A, Madden B, Burke MM, et al. Transbronchial biopsy in
heart and lung transplantation: clinicopathologic correlations. J Heart
Lung Transplant 1995;14:761-73.
7
Stewart S, Fishbein MC, Snell GI, et al. Revision of the 1995 Working
Formulation for the Standardisation of Nomenclature in the Diagnosis
of Lung Rejection. J Heart Lung Transplant 2007;26:1229-42.
8
Hwang DM, Yousem SA. Approach to a lung transplant biopsy. J Clin
Pathol 2010;63:38-469
Yousem SA, Zeevi A. The histopathology of lung allograft dysfunction
associated with the development of donor-specific HLA alloantibodies.
10
Berry G, Burke M, Andersen C, et al. Pathology of pulmonary antibody-mediated rejection: 2012 update from the Pathology Council of
the ISHLT. J Heart Lung Transplant 2013;32:14-21.
11
Burton CM, Iversen M, Scheike T, et al. Is Lymphocytic Bronchiolitis a Marker of Acute Rejection? An Analysis of 2,697 Transbronchial Biopsies After Lung Transplantation. J Heart Lung Transplant
2008;27:1128-34.
12
Gordon IO, Bhorade S, Vigneswaran WT, et al. SaLUTaRy: survey of
lung transplant rejection. J Heart Lung Transplant 2012;31:972-9.
13
Stewart S. Pulmonary infections in transplantation pathology. Arch
Pathol Lab Med 2007;131:1219–31.
14
Costa C, Curtoni A, Bergallo M, et al. Quantitative detection of HHV-6
and HHV-7 in transbronchial biopsies from lung transplant recipients.
New Microbiol 201;34:275-80.
15
Hadjiliadis D, Duane DR, Steele MP, et al. Gastroesophageal reflux
disease in lung transplant recipients. Clin Transplant 2003;17:363-8.
16
Sato M, Waddell TK, Wagnetz U, et al. Restrictive allograft syndrome
(RAS): a novel form of chronic lung allograft dysfunction. J Heart
17
Kremer BE, Reshef R, MD, Misleh JG, et al. Post-transplant lymphoproliferative disorder after lung transplantation: A review of 35 cases.
J Heart Lung Transplant 2012;31:296-304.
18
De Vito Dabbs A, Hoffman LA, Iacono AT, et al. Are symptom reports
useful for differentiating between acute rejection and pulmonary infection after lung transplantation? Heart Lung 2004;33:372-80.
19
Chakinala MM, Ritter J, Gage BF, et al. Yield of surveillance bronchoscopy for acute rejection and lymphocytic bronchitis/bronchiolitis
after lung transplantation. J Heart Lung Transplant 2004;23:1396404.
20
McWilliams TJ, Williams TJ, Whitford HM, et al. Surveillance Bronchoscopy in Lung Transplant Recipients: Risk versus Benefit J Heart.
21
Glanville AR. Bronchoscopic monitoring after lung transplantation.
Semin Respir Crit Care Med 2010;31:208-21.
22
Solidoro P, Delsedime L, Costa C et al; Effect of CMV-immunoglobulins (cytotect biotest) prophylaxis on CMV pneumonia after lung
transplantation.New Microbiol 2011;34:33-6.
COMUNICAZIONI ORALI
Pathologica 2013;105:224-258
Domenica, 27 ottobre 2013
Sala Cesarea – ore 8.30-10.30
Patologia mammaria I
Moderatori: M.P. Foschini (Bologna), E. Vasquez (Catania)
Multiplex ligation-dependent probe amplification
(mlpa): a quantitative and sensitive method
to detect her2 status in breast carcinomas
with equivocal her2 gene amplification
C. Ercolani1, S. Di Benedetto1, E. Melucci1, C. Marchiò2, I. Terrenato3, C.A. Amoreo1, S. Buglioni1, V. Dimartino1, L. Perracchio1,
A. Sapino2, M. Mottolese1
Anatomia Patologica, Regina Elena-Ifo, Roma, Italia; 2 Scienze Biomediche e Oncologia Umana, Università di Torino, Torino, Italia; 3 Biostatistica-Direzione Scientifica, Regina Elena-IFO, Roma, Italia
1
Background. About 20% of current HER2 testing in breast
cancer (BC) may be inaccurate and the best method to assess
HER2 status remains controversial mainly in equivocal cases.
In this study we aimed to evaluate whether MLPA, a molecular
quantitative method able to detect gene gains/amplifications and
deletions, can identify misleading results in a series of 107 randomly selected BC with known HER2 status. We focused on the
42 cases defined as equivocal (ratio 1.8-2.2; gene copy number
4.0-6.0) and polysomic (≥ 3 signals/nucleus) by SISH.
Methods. We used MLPA to determine HER2 gene copy number gains and chromosome 17 (Chr17) centromere alterations
in parallel to immunohistochemistry (IHC) and Silver In Situ
Hybridization (SISH).
Results. We found a high concordance between IHC, SISH
and MLPA both in the 14 Non Amplified (NA) score 0/1+
BC and in the 19 Amplified (A) score 3+ BC (K = 0.94,
p < 0.0001). Among the 74 BC HER2 2+, 57 (78%) were
NA and 17 (23%) were A by SISH. Conversely, in the same
series, MLPA (cut off ≥ 1.5) detected HER2 amplification in
19 cases (26%).We found an increase in Chr17 centromere
copy number (≥ 3) by SISH, in 42 cases (56%). 18 out of these
42 (43%) BC showed gains/amplifications of the centromere
region genes (WSB1/NOS2A) by MLPA. All the 5 cases NA
by SISH and A by MLPA were denoted as polysomic by SISH
and displayed gain/amplification of the centromeric region by
MLPA.
In our series of 107 BC we found a high concordance rate (86,5%,
K = 0.63, p < 0.0001) between SISH and MLPA confirming that
this method is accurate to detect HER2 gene amplification.
Moreover, we evidenced a variable pattern of gains and losses
of Chr17 genes and none of our cases showed a true Chr17
polysomy. These findings are of particular clinical relevance in
equivocal cases in which an increased number of Chr17 centromere signals by SISH may provide misleading HER2 status assessment lowering the number of BC patients who can experience
great benefit by anti HER2 therapies.
Sentinel node tumor burden quantified based
on cytokeratin 19 mrna copy number predicts
non-sentinel node metastases in breast cancer
classified by molecular subtypes
S. Buglioni1, I. Terrenato2, B. Casini1, V. D’Alicandro1, E. Gallo1,
F. Di Filippo3, E. Pescarmona, M. Mottolese1
Anatomia Patologica, Istituto Nazionale Tumori Regina Elena, Roma,
Italia; 2 Direzione Scientifica, Biostatistica, Istituto Nazionale Tumori Regina Elena, Roma, Italia; 3 Chirurgia A, Istituto Nazionale Tumori Regina
Elena, Roma, Italia
1
Background. The OSNA assay is a molecular diagnostic technique to detect clinically relevant breast cancer (BC) metastases
in the sentinel lymph node (SLN) quantified based on cytokeratin
19 (CK19) mRNA copy number. Although the American College
of Surgeons Oncology Group Z0011 trial has defined a select
cohort of patients in whom a completion axillary lymph node
dissection (cALND) may be safely omitted, there are a still a
number of patients where prediction of non-SLN metastasis may
be helpful for cALND decision making. Multiple studies suggest
that specific pathologic characteristics of the primary tumor and
the SLN metastases are associated with an increased likelihood of
additional positive non-SLN. The aim of the present retrospective
study was to confirm (Buglioni et al, Plos One 2013) whether the
CK19 mRNA copy number detected by the OSNA assay in the
whole SLN, may predict the risk of a positive ALND across the
different BC molecular subtypes.
Methods. 242 BC patients with a positive SLN who underwent
complete axillary LN dissection were investigated. The SLN
tumor burden was classified as macrometastasis (CK19 mRNA
> 5000 copies/ml) or micrometastasis (250-5000 copies/ml). SLN
tumor burden was compared to axillary lymph node dissection
status and to the biological tumor profile.
Results. logistic regression evidenced that positive ALND was
significantly associated with a higher CK19 mRNA copy number
(> 5000; p < 0.0001), HER2 subtype (p = 0.007) and lymphovascular invasion (p < 0.0001). Conversely, BC patients with
CK19 mRNA copy number < 2000 mostly presented a luminal
subtype and a negative ALND. The OSNA assay, together with
a presurgical analysis of tumor subtype, could represent a valid
and objective tool to set up a novel model capable of more accurately predicting axillary involvement. Omission of ALND
could be proposed in patients with a micrometastatic SLN with
a low CK19 mRNA copy number (< 2000) and luminal tumor
phenotype.
A divergent prognostic role for p53 and BCL2
in 1099 early breast cancer classified according
to molecular subtypes: an observational
prospective study
A. Di Benedetto2, I. Terrenato1, C. Ercolani2, S. Vari3, P. Malaguti3, P. Vici4, L. Perracchio2, B. Antoniani2, C.A. Amoreo2, A. Fabi3, M. Mottolese2
Biostatistica-Direzione Scientifica, Regina Elena, Roma, Italia; 2 Anatomia Patologica, Regina Elena, Roma, Italia; 3 Oncologia Medica A, Regina Elena, Roma, Italia; 4 Oncologia Medica B, Regina Elena, Roma, Italia
1
Background. A limited number of biomarkers are currently
used to guide therapy and prognosis in breast cancer (BC).
Recently, p53 and BCL2 have been proposed as additional
prognostic markers, although their relationship with conventional parameters and patient outcome remains uncertain.
In particular, there are few data concerning p53 and BCL2
distribution within the molecular BC subtypes, Luminal A
(LA), Luminal B (LB/H- and LB/H+), HER2 subtype (H+) and
Triple Negative (TN).
Methods. We prospectively evaluated p53 and BCL2 expression
by immunohistochemistry in 1099 early BC patients operated
between 2000 and 2006 with at least 5 yrs follow-up data. None
of the H+ patients received trastuzumab in the adjuvant setting.
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Associations among p53 and BCL2, T, N, G and molecular subtypes were analyzed by multiple correspondence analysis (MCA),
while Kaplan-Meier method was applied to determine their impact on disease-free survival (DFS).
Results and Conclusions. p53 is highly positive in LB/H+
(38%), H+ (50%) and TN (33%), conversely, BCL2 is more
frequently expressed in LA (71%) and LB/H- (75%) BC (pvalue< 0.0001). MCA confirmed that p53+ and BCL2- are
located in the quadrant containing more aggressive tumor phenotypes and presence of relapse. Kaplan-Meier curves identified
BCL2 negativity as a significant discriminating factor for DFS
(p = 0.024). Of interest, in the subset of 595 N0 patients, p53
positivity and BCL2 negativity were significantly associated
to the lack of response to anthracycline (AC ± taxanes) based
chemotherapy (p< 0.0001). Visceral metastases are significantly
less frequent in LA (30%), LB/H- (37%) and TN (29%) as
compared to H+ (52%) and LB/H+ (58%) BC (p = 0.004). Our
data indicated that lack of BCL2, in contrast to p53 positivity,
appears to be a biomarker related to a more aggressive clinical
course across BC molecular subtypes although both biomarkers
may affect AC-based chemotherapy response in the subset of
N0 patients.
Adenoid cystic carcinoma of the breast with highgrade transformation
A. De Leo1, A. Rizzo2, L. Laurino3, M. Sironi4, M.P. Foschini1
Section of Anatomic Pathology “M. Malpighi”, of the Bellaria, Bologna, Italy; 2 Anatomic Pathology, S. James Hospital, Ulss 8, Castelfranco
Veneto, Italy; 3 Anatomic Pathology, General Hospital of Treviso, Treviso,
Italy; 4 Department of Pathology, General Hospital, Asl 4 Chiavarese, Sestri Levante, Genova, Italy
1
Background. Adenoid cystic carcinomas (AdCCs) constitute
0.1%-1% of all malignant breast tumours. These tumours, regardless of the anatomical site, are characterised by the expression of
CD117.Usually AdCC of the breast have good prognosis.
Salivary gland AdCC can transform into a high-grade adenocarcinoma (hgAdCC) leading to death of the patient within 5 years.
Similarly, it has been described a case of AdCC of the breast with
high-grade transformation.
The purpose of the present study is to describe features of high
grade transformation in AdCC of the breast.
Methods. The series consisted of four female patients aged from
47 to 65 years presenting a breast nodule measuring from 0.8 to 6
cm in greater axis. It has been performed immunohistochemistry
for cytokeratin7, cytokeratin14, CD117, oestrogen, progesterone,
Her-2, Ki67.
Results. On histology, the tumours were characterized by solid
growth pattern with focal necrosis; neoplastic cells showed cytological atypia and atypical mitoses. Focally, areas of conventional
AdCC were seen. All the cases were oestrogen, progesterone
receptor and Her 2 negative, while were strongly positive for
CD117. A variable positivity for CK7 and focal positivity for
CK14 were observed. CK 14 evidenced basal-myoepithelial cells
forming pseudolumina.
Lymph node metastasis were detected in 2/4 cases, whereas distant
metastasis were not observed. One of the patients relapsed with a
breast nodule of 9 cm, after 14 years, while the others are monitored
by follow-up and currently are alive with no evidence of disease.
In conclusion, hgAdCC differentiates from conventional
AdCC in morphological features and in clinical outcome. In
contrast to other “triple negative” breast carcinomas, these
tumours show a different morphology and molecular pattern
and clinically they have a less aggressive behaviour and a better prognosis.
Despite their rarity, a correct morphologic diagnosis of hgAdCC
is important for a proper management of the patients.
Identification and validation of a new set of five
genes for prediction of risk in early breast cancer
L. Morandi7, D. Bonifacio1, M.P. Sormani2, P. Bruzzi3, A. Gennari4, F. Zanconati5, A. Monzoni6, G. Mustacchi7
Clinico di Scienze Mediche, Chirurgiche e d(Units), Cattinara, Trieste,
Italy; 2 Department of Health Sciences (Unige), Cba, Genova, Italy; 3 National Cancer Research Centre, S. Martino, Genova, Italy; 4 Oncology
Unit, Galliera, Genova, Italy; 5 Clinico di Scienze Mediche, Chirurgiche
Salute, Cattinara, Trieste, Italy; 6 Alphagenics Biotech s.r.l., Area Science
Park Basovizza, Trieste, Italy; 7 Dibinem (Unibo), Bellaria, Bologna, Italia
1 Background. Molecular tests predicting the outcome of breast
cancer patients based on gene expression levels can be used to
assist in making treatment decisions after consideration of conventional markers.
Methods. In this study we identified a subset of 20 mRNA differentially regulated in breast cancer analyzing several publicly
available array gene expression data using R/Bioconductor package. Using RTqPCR we evaluate 261 consecutive invasive breast
cancer cases not selected for age, adjuvant treatment, nodal and
estrogen receptor status from paraffin embedded sections. The
biological samples dataset was split into a training (137 cases)
and a validation set (124 cases).
Results: The gene signature was developed on the training set
and a multivariate stepwise Cox analysis selected five genes independently associated with DFS: FGF18 (HR = 1.13, p = 0.05),
BCL2 (HR = 0.57, p = 0.001), PRC1 (HR = 1.51, p = 0.001),
MMP9 (HR = 1.11, p = 0.08), SERF1a (HR = 0.83, p = 0.007).
These five genes were combined into a linear score (signature)
weighted according to the coefficients of the Cox model, as:
0.125 FGF18 − 0.560 BCL2 + 0.409 PRC1 + 0.104 MMP9 −
0.188 SERF1A (HR = 2.7, 95%CI = 1.9–4.0, p < 0.001). The
signature was then evaluated on the validation set assessing the
discrimination ability by a Kaplan Meier analysis, using the same
cut offs classifying patients at low, intermediate or high risk of
disease relapse as defined on the training set (p < 0.001). Our
signature, after a further clinical validation, could be proposed
as prognostic signature for disease free survival in breast cancer
patients where the indication for adjuvant chemotherapy added to
endocrine treatment is uncertain.
Mammary microcalcifications: active phenomenon
or simple bystanders in breast cancer?
M. Scimeca, E. Giannini, C. Antonacci, E. Bonanno
Biomedicina e Prevenzione, Policlinico Universitario Tor Vergata, Roma, Italia
Background. Mammary microcalcifications are used as radiologic marker for breast cancer screening. We hypothesize that
microcalcifications are an active cellular process resembling the
mineralization occurring in the bone tissues by osteoblasts.
Methods. Paraffin-embedded sections of breast specimens with
and without microcalcifications, have been tested with antiVimentin, anti-BMP2 and anti-osteopontin (OPN).
Serial sections of the same samples were processed for electron
microscopy study.
Results. Breast tissue were classified as: benign lesions with
microcalcifications (BLm) (10 samples), in situ carcinoma with
microcalcifications (ISCm) (10 samples), infiltrating carcinoma
with microcalcifications (ICm) (10 samples), infiltrating carcinomas without microcalcifications (ICwm) (20 samples).
The amount of vimentin, a marker for mesenchimal cells, significantly increases in cells close to microcalcifications in ICm
samples.
BMP-2 detection discriminate ICm from ICwm allowing us to
associate the mineralization observed in the lesions of breast with
that occurring in the osteoblast cells.
OPN showed a focal distribution with an increase of the signal
close to microcalcifications made of HA.
226
OPN signal is very low in ICwm suggesting that during the formation of microcalcifications OPN plays a role quite similar to
that exerted during the mineralization in bone.
By the ultrastructural study we could identify numerous cells
with morphological characteristics typical of osteoblasts close to
microcalcifications.
They exhibit in their cytoplasm vescicles with electrondense granules referable to the vescicles of mineralization of the osteoblasts.
In conclusion our data let us to hypothesize that mammary epithelial cells, under specific stimuli, undergo towards the epithelial to
mesenchimal transition phenomen transforming themselves into
cells with an osteoblast-like phenotype.
Brown tumours of the breast (well differentiated
mammary invasive carcinoma with osteoclast like giant cells)
R. Panzacchi1, S. Foreid2, V. Agostini3, M. Del Vecchio3,
L. Costarelli4, S. Varga5, V. Eusebi1
Anatomia Patologica, Università di Bologna, Bologna, Italia; 2 Dept of
Pathology CHLC, Hospital de São José, Lisbon, Portugal; 3 Anatomia Patologica, Ospedale Mazzoni, Ascoli Piceno, Italia; 4 Anatomia Patologica,
S. Giovanni-Addolorata, Roma, Italia; 5 Dept of Pathology, University of
Zurich, Zurich, Switzerland
1 Background. Mammary carcinoma with osteoclast-like giant
cells (OGCs) are rare frequently pigmented tumours of the breast
first described by Holland et al. 1. OGCs, since the first report,
have been interpreted as reactive in nature.
Methods. We report four cases of mammary carcinoma with
OGCs, retrieved from the consultation files from one of us. The
age of patients ranged 40 to 48 yrs. H&E staining, immunohistochemistry (IHC) for ER and PR and data on the macroscopic
appearance of the tumours were available; we performed IHC
for mitochondria staining on all the cases and Mt DNA clonality
analysis of the neoplastic cells and the OCGs on two cases.
Results. Macroscopically, all tumours were well circumscribed
and three of them were reported to have dark brown colour.
Histologically, all cases were constituted by invasive duct carcinoma, G2 in one and G1 in three cases; sentinel lymph nodes
were obtained in 2 cases; in one a macro metastasis was present. OGCs were observed in the stroma in all cases, around the
epithelial neoplastic glands and occasionally inside the lumina.
The stroma was haemorrhagic in 2 cases and desmoplastic in
the other 2. Haemosiderin deposits were observed both within
the stroma and at the periphery of all tumours. All cases were
ER and PR positive. OGCs, as evidenced with IHC, were filled
with mitochondria, a finding consistent with oncocytic cells and
possibly accounting for the brown macroscopic colour of the
tumours together with haemorrhage and haemosiderin deposit
in the stroma. In the end, we were able to further emphasize
the reactive nature of OGCs in these tumours through Mt DNA
clonality analysis.
Reference
1
Holland R, Urbain JGM, Van Haelst JMG. Mammary carcinoma with
osteoclast-like giant cells. Additional observations on six cases. Cancer 1984;53:1963-73.
Nipple adenoma can simulate malignancy
on cytological secretion smears
F. Rosini1, S. Cinocca1, M. Del Vecchio2, M.C. Cucchi3, G. Saguatti4, M.P. Foschini1
Section of Anatomic Pathology “M. Malpighi”, of the Department of
Biomedical and Neuromuscular Disorders, University of Bologna, at Bellaria Hospital, Bologna (Italy); 2 Anatomic Pathology, General Hospital,
Ascoli Piceno (Italy); 3 Operative Unit of Surgical Oncology, ASL Bologna, Bellaria Hospital, Bologna (Italy); 4 Operative Unit of Breast Radiology, ASL Bologna, Bellaria Hospital, Bologna (Italy)
1
Background. Nipple adenoma is a rare benign tumour of the
breast that can be clinically mistaken for Paget’s disease because
of nipple ulceration and crusting. A non invasive diagnostic
method as cytology is suitable for the correct pre-operative
diagnosis and management of this lesion. Nonetheless nipple
adenoma occasionally can present atypical cytological features
leading to a misdiagnosis of malignancy. In this regard, we report
three cases of nipple adenoma with atypical features in nipple
secretion smears.
Methods. All the patients were female (aged 42, 67 and 81
respectively) and presented a palpable nodule below the nipple
associated with skin ulceration and nipple retraction. Cytological smears were obtained by gentle scraping of the nipple lesion.
The smears were fixed in ethanol 95% and stained by Papanicolaou method. All three lesions were subsequently surgically
removed and tissues were formalin fixed and paraffin embedded.
Immunohistochemistry was performed to confirm the diagnosis
of nipple adenoma.
Results. All cytological smears showed haematic background
with a moderate cellularity. Pluristratified epithelial clusters were
present, composed of plasmocytoid-like cells with enlarged nuclei. The clusters contained rare myoepithelial cells.
In all the three cases a diagnosis of atypia was performed.
Histological specimens showed epithelial proliferation involving
the nipple ducts, morphologically typical of nipple adenoma.
Immunohistochemical analysis confirmed the presence of myoepithelial cells (positive for p63, ck14).
In conclusion, nipple adenoma may present atypical cytological
features, thus leading to the false diagnosis of malignancy as
Paget’s disease and invasive carcinoma with skin ulceration. The
recognition of myoepithelial cells in the smear can be helpful for
the correct diagnosis and to prevent surgical overtreatment.
WT1 is a cytoplasmic marker of epithelioid cell
myofibroblastoma of the breast
F.R. Lonfo, L. Salvatorelli, G.M. Vecchio, E. Vasquez, G. Magro
G.F. Ingrassia, Policlinico Universitario “G. Rodolico”, Catania, Italia
Background. Immunohistochemical nuclear expression of
Wilms tumor transcription factor-1 (WT1) has been reported
in several tumors, especially in nephroblastoma and in ovarian
and mesothelial neoplasms. Recently, WT1 cytoplasmic immunoreactivity has been detected in some fetal (skeletal muscle,
endothelial cells, sympathetic neuroblasts, Schwann cells), adult
normal (endothelial cells) and neoplastic tissues (vascular tumors,
rhabdomyosarcoma), suggesting that this staining truly reflects
the intracytoplasmic presence of the protein. The aim of the
present study was to define whether WT1 may be exploitable as
diagnostic marker in mammary myofibroblastoma (MFB).
Methods. We investigated immunohistochemically the expression and distribution of WT1 in a series of 18 cases of mammary MFB, using antibody clone 6F-H2 directed to the WT1
N-terminus of the protein. Tissues samples included: 7 cases of
classic-type MFB, 1 case of collagenized/fibrotic-type MFB, 1
case of myxoid-type MFB, 2 cases of lipomatous-type MFB, 2
cases of Schwannian-like MFB, 5 cases of epithelioid cell MFB.
Results. All cases but epithelioid cell MFB were negative or
only focally (<5% of neoplastic cells) and weakly positive to
WT1. In contrast, all cases of epithelioid cell MFB exhibited
a strong and diffuse (> 90% of neoplastic cells) cytoplasmic
staining for WT1. No nuclear immunoreactivity was seen in any
of the tissues examined. As epithelioid cell MFB may mimic
invasive lobular or apocrine carcinoma, a total of 10 cases of
such tumors were also immunohistochemically tested for WT1,
with negative results. The present study first shows that WT1
is diffusely expressed in the cytoplasm of epithelioid cell MFB,
while it is absent or only focally detectable in the other vari-
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ants. These findings suggest that WT1 may assist in confirming
the diagnosis of the most diagnostically challenging variant of
MFB, namely the epithelioid cell variant, which can be confused with other malignant lesions.
Diagnostic pitfalls in mammary myofibroblastoma:
“old” and “new” unusual morphological variants
L. Salvatorelli, G.M. Vecchio, A. Bosco, L. Puzzo, V. Eusebi*,
G. Magro
G.F. Ingrassia, Policlinico Universitario “G. Rodolico”, Catania, Italia;
* M. Malpighi, Ospedale Bellaria, Bologna, Italia
Background. Mammary myofibroblastoma (MFB) is an unusual
benign tumor composed of fibroblasts and myofibroblasts. Since
the original description, its morphologic spectrum has been expanded by the recognition of several morphological variants. This
morphological heterogeneity may represent a potential diagnostic
pitfall, especially when evaluating fine-needle aspiration and/
or needle core biopsy. The aim of this study was to identify the
more unusual variants of mammary MFB, which may represent
potential diagnostic pitfalls.
Methods. Twenty-two cases of mammary MFB were revised
and classified accordingly to the current criteria. The following
unusual morphological variants were identified: i) 5 cases of epithelioid cell MFBs; ii) 3 cases of lipomatous MFBs; iii) 2 case
of atypical cell MFBs; iv) 2 cases of Schwannoma-like MFBs.
Results. Among the epithelioid cell MFBs, we identified: i) one
case composed of medium/large, atypical cells arranged in a micronodular pattern, closely reminiscent of an invasive lobular or
apocrine carcinoma; ii) in one case neoplastic cells were focally
arranged in nerve-like structures; iii) in one case neoplastic cells
were large in size and closely packed, resulting in a deciduoidlike appearance. Among the lipomatous MFBs, one case had morphological features closely reminiscent of desmoid-type fibromatosis. Among the atypical cell MFBs, in one case tumor stroma
was exclusively of myxoid-type, suggesting the possibility of a
myxoid sarcoma. Lastly, in 2 cases of MFB neoplastic cells were
predominantly arranged to form Verocay bodies-like structures,
closely resembling schwannoma. We emphasize that pathologists
should be aware of the wide morphologic spectrum exhibited by
MFB to avoid confusion with other benign or malignant lesions.
Although we think that the diagnosis of MFB is morphologically
based, we concede that immunohistochemistry plays a helpful
diagnostic role in ambiguous cases.
Patologia Mammaria II
Moderatori: L. Costarelli (Roma)
HER2 status between primary breast cancer
and its associated metastasis in 75 cases from
three institutions
E. Orvieto1, L. Alessandrini1, E. Roz2, M. Lo Mele1, P.L. Piovano3, R. Libener4, C. Lanza1, M. Rugge1
1
UOC Anatomia Patologica, AOU Padova, Padova, Italia; 2 Anatomia
Patologica e Patologia Molecolare Oncolog, Casa di cura la Maddalena
Palermo, Palermo, Italia; 3 Sc Oncologia, AO Nazionale “Ss. Antonio
E Biagi, Alessandria, Italia; 4 Sc Anatomia Patologica, AO Nazionale
“Ss. Antonio E Biagi, Alessadria, Italia
Background. Human epidermal growth factor receptor 2 (HER2)
has prognostic and predictive relevance in breast cancer. Differences in HER2 status between primary tumour and metachronous
metastasis could have a substantial impact on management of
patients. Retrospective studies suggest that discordance of HER2
status between primary and metastatic breast cancer exists, due
to clonal selection by therapy, tumor heterogeneity, technical
testing artifacts.
Methods. Matched primary and metachronous metastatic breast
cancer tissue samples relating to 75 cases referred to three Institutions, between 2002 and 2012 were collected. Histological assessment and HER2 immunohistochemical expression were reviewed
by pathologists from each Institution using standard scoring systems in both primary and metastasis. Cases with equivocal results
(score 2+) or with major discrepancies between primary tumor
and metastases were tested with FISH analysis.
Results. HER2 scores for primary breast cancer were: 0/1+ in 61
cases (81%), 2+ in 7 cases (9,5%) and 3+ in 7 cases (9,5%). In 4
cases (4/75;5,3%) the assessment of HER2 on metastases showed
a discordant result. Discordance from HER2-negative primary
cancer to HER2-positive paired metastasis (positive convertion)
was more likely (3/4; 75%) than the reverse in which the metastatic tumor lost HER2 expression (negative convertion). The
prevalent site of metastases in our series was liver.
Conclusions. Our lower rate of discrepant cases (5,3%) compared
with literature data (6.9-18.6%) could be explained by: accurate
review of HER2 status for each case, with additional FISH test
when necessary; low prevalence of bone metastases, in which the
decalcification could compromise HER2 result. Our data confirm
the utility to biopsy metastatic sites, if accessible, especially for
patients with HER2 primary negative tumor (positive convertion)
to allow appropriate selection of patients for whom additional
targeted therapy with Trastuzumab is indicated.
Concordance of her2 status between needle core
biopsies and paired surgical specimens of invasive
breast carcinoma in 248 cases
E. Orvieto1, L. Alessandrini1, M. Lo Mele1, P.L. Piovano2,
R. Libener3, G. Marchelle1, M. Rugge1
1 UOC Anatomia Patologica, AOU di Padova, Padova, Italia; 2 Sc Oncologia, AO Nazionale “Ss. Antonio e Biagi”, Alessandria, Italia; 3 Sc Anatomia Patologica, AO Nazionale “Ss. Antonio e Biagi”, Alessandria, Italia
Background. Needle core biopsy (NCB) has become the method
of choice for the diagnosis of breast cancer providing enough material for morphological diagnosis and for assessment of prognostic
and predictive factors. However, immunohistochemical (IHC)
evaluation on NCB may be less reliable than in surgical specimens
(SS) due to tumor heterogeneity. On the other hand formalin fixation protocol in NCB is more standardized than in SS.
Methods. A total of 248 NCB and paired surgically resected
specimens of invasive breast carcinoma were collected from two
Institutions, between 2009 and 2012. Patients who underwent
neoadjuvant chemotherapy were excluded. Both types of specimens were tested for HER2 status. Immunohistochemical expression was evaluated by the same pathologists using standard scoring systems and equivocal results (2+) were tested with FISH..
Results. HER2 scores determined by immunohistochemistry on
NCB were: 0/1+ in 188 cases (76%), 2+ in 41 cases (16%) and 3+
in 19 cases (8%). Concordant results were detected in 235 cases
(94.7%). One carcinoma, negative on NCB, was scored 2+ on
SS with FISH amplification. Nine cases scored 2+ on CNB were
positive on SS, either by immunohistochemistry (3+) or by FISH
228
analysis. Of three cases scored 3+ on CNB, two were scored 1+
on SS, the remaining being scored 2+ without FISH amplification.
Conclusions. Our concordance rate for HER2 status between
CNB and SS was similar to literature data and supports the routine use of NCB for HER2 assessment. Current recommendations
to retest HER2 in SS were confirmed: if NCB shows equivocal
results (immunohistochemistry and ISH) possibly representative
of tumor heterogeneity, not adequately sampled; if NCB shows
artifacts resulting in false positive immunohistochemistry (3+).
In addition, NCB should be performed on each invasive focus of
multiple tumors to avoid false negative results.
Murine mammary tumor virus (MMTV) in human
sporadic breast cancer
C. Scatena1, F. Lessi2, I. Armogida1, M. al-Hamad1, G. Fanelli1,
V. Ortenzi1, K. Zavaglia1, A.G. Naccarato1, C.M. Mazzanti2,
G. Bevilacqua3
1 Div of Surg, Molec and Ultrastruct Pathology, University of Pisa and
Pisa University Hospital, Pisa, Italy; 2 Pisa Science Foundation, Pisa,
Italy; 3 Pathology div, University, Pisa, Italy
Background. Even if breast carcinoma represents the most
frequent cancer in women and it has been largely studied
worldwide for many decades, its etiology is largely unknown.
On the other hand, in mice the causative role of MMTV in the
development of tumors of the mammary gland has been demonstrated for a long time. The strong similarities between the
human and the murine disease strongly suggest a possible viral
etiology of breast carcinoma in women. Previously, our group
demonstrated the presence of an exogenous MMTV envelope
gene-like sequence (MMTVels) in 33% of human infiltrating
breast carcinoma (IBC) 1. Moreover, we were able to show
MMTVels in all steps of breast cancer progression: normal
glandular epithelium close to IBC, atypical epithelial hyperplasia, ductal carcinoma in situ. MMTVels were also demonstrated
in the nuclei of tumor cells by chromogenic in situ hybridization 2. The present study investigated the presence of MMTVels
in IBC primary cell cultures.
Methods. Fragments were obtained fresh from 5 different cases
of IBC. The detection of MMTVels was performed by fluorescent
nested-PCR and confirmed by FISH analysis. Cells were examined for virus particles presence by electron microscopy (EM).
Results and conclusions. PCR and FISH analysis confirmed the
presence of MMTVels in 1 out of 5 cell lines. Moreover, MMTVlike particles were easily recognized at EM level. These data
represent a further element supporting the hypothesis of a viral
etiology for human breast cancer.
References
1
Zammarchi F, Pistello M, Piersigilli A, et al. MMTV-like sequences
in human breast cancer: a fluorescent PCR/laser microdissection approach. J Pathol 2006;209:436-44.
2
Mazzanti CM, Al Hamad M, Fanelli G, et al. A mouse mammary tumor
virus env-like exogenous sequence is strictly related to progression of
human sporadic breast carcinoma. Am J Pathol 2011;179:2083-90.
Stromal Caveolin-1 in human breast cancer
progression
G. Fanelli1, C. Scatena1, V. Ortenzi1, M. Menicagli1, M.P. Lisanti2, P. Aretini3, A.G. Naccarato1
1
Div of Surg, Molec and Ultrastruct Pathology, University of Pisa and
Pisa University Hospital, Pisa, Italy; 2 Breakthrough Breast Cancer Research Unit, Inst of Cancer Sciences, University of Manchester, Manchester, United Kingdom; 3 Pisa Science Foundation, Pisa, Italy
Background. Recent evidence suggests that a loss of expression
of Caveolin in the stromal compartment of human infiltrating
breast carcinomas (IBC) may be a predictor of disease recurrence,
metastasis and poor outcome. Tumor grade, stage, nodal status
and epithelial markers are considered important parameters for
predicting the prognosis of IBC cancer but, in many cases, this
classification “fails”. Incorporating stromal (sCav-1) as a possible
biomarker could be useful in distinguish high risk from low risk
tumors. On this basis, we studied sCav-1 expression in IBC and
in their respective axillary lymph nodes with the aim to find a
correlation with cancer progression.
Methods. 189 consecutive invasive IBC (53 with lymph nodal
metastatis and 136 without lymph nodal metastasis) were studied
by immunohistochemistry, using rabbit polyclonal anti-Cav-1
antibody, N20, Santa Cruz Biotechnology, 1:500. In IBC and in
metastatic lymph nodes sCav-1 was evaluated in the peritumoral
stroma, whereas in non metastatic lymph nodes beneath the capsule or along the trabeculae. The staining was scored semiquantitatively as 0 (negative, in stromal cells), 1 (diffuse weak or
strong; in less than 30% of stromal cells) or 2 (strong; 30% or
more of the stromal cells).
Results and conclusions. According to literature, sCav-1 is
less expressed in metastatic primary IBC than in non-metastatic
primary IBC. Moreover, we demonstrated for the first time that
sCav-1 expression is reduced in metastatic lymph nodes in comparison to primary IBC (p 11< 0,0001). On the other hand, sCav-1
is highly expressed in non metastatic lymph nodes. These data
strongly support a possible “key” role of stromal Cav-1 in tumor
metastatic progression.
The identification of a small but significant
subset of patients still targetable with anti-her2
inhibitors when affected by triple negative breast
carcinoma
E. Brunello1, G. Bogina2, M. Vergine3, G. Zamboni2, S. Beccari2,
B. Jasani4, E. Manfrin5, F. Bonetti5, G. Martignoni5, M. Brunelli5
Department of Pathology and Diagnostics, AOUI, Verona, Italy; 2 Pathology, Sacrocuore, Negrar, Italy; 3 Pathology, Sussex-Brighton, Brighton, Uk;
4 Pathology, Cardiff university, Cardiff, Uk; 5 Pathology, AOUI, Verona, Italy
1 Background. triple (ER-, PR-, HER2-) negative breast carcinoma
lack targeted therapies, making this group of tumors difficult to
treat. By definition, the lack of HER2 expression means a case
scoring 0 or 1+ after immunophenotypical analysis and make the
patients avoiding therapeutical chances with anti-HER2 inhibitors.
We sought to recruit from a group of triple-negative breast carcinoma, patients eligible for effective personalized targeted therapy
with anti-HER therapies on the basis of their HER2 gene status.
Methods. 135 patients diagnosed with IHC triple negative breast
carcinoma were studied. Whole tissue sections were used for in
situ hybridization analysis.
Results. 8/100 (8%) of ductal type triple negative breast carcinoma presented Her- 2/neu gene amplification versus 2/35 (5.7%)
non-ductal triple negative breast carcinoma. Three cases showed
a ratio 2.5. One case showed Her-2/neu heterogeneous gene amplification, ratio 2.3. The other six showed from 7 to 8 absolute
Her-2/neu gene copy number. Two cases staged pT1c and eight
cases staged pT2. Eight cases graded G3, two cases G2.
1) respectively 8% of ductal and 5.7% non-ductal type triple
negative breast carcinoma present Her-2/neu gene amplification; 2) the standard diagnostic flow-chart “do not FISH in 0-1+
(HER2-) breast carcinoma” should be replaced by “do FISH in
triple (ER-, PR-, HER2-) negative breast carcinoma”, to avoid
loss of therapeutical chances in a cohort of such a patients; 4) we
demonstrated the identification of a small but significant subset
of patients targetable with anti-HER2 inhibitors, giving patients
affected by (ex)triple negative breast carcinoma new personalized
therapeutical chances.
229
comunicazioni orali
Uropatologia
Moderatori: A. Vecchione (Roma), P. Alò (Roma)
Immunohystochemical expression of socs-3
seems to support the 2005 international society
of urological pathology modified gleason grading
system
F. Pierconti, T. Cenci, A. Calarco*, G. Petrone, F. Pinto,
L.M. Larocca, M. Martini
Anatomia Patologica, UCSC Policlinico A. Gemelli, Roma, Italia; * Urologia, UCSC Policlinico A. Gemelli, Roma, Italia
Background. In a recent report we demonstrated that epigenetic
silencing of gene of suppressor cytokine signaling 3 (SOCS3)
by real time PCR and immunohistochemistry method in radical
prostatectomy and prostatic biopsies, may be involved in the
pathogenesis of prostate cancer and identify a tumor subset with
an aggressive behavior.
Method. We analyzed immunohystochemical expression of
SOCS3 in 20 prostatic cancer biopsies Gleason score 6 (3+3); in
50 prostatic cancer biopsies Gleason score 7 (3+4) or (4+3) and
in 10 prostatic cancer biopsies 8 (3+5). The SOCS3 staining intensity were evaluated by two pathologists (FP and LML) in three
different ways: positive (+); negative (-); weak intensity staining
with negative neoplastic glands (+/-), on the basis of the intensity
of cytoplasmatic staining. Colonic mucosa were used as positive
control according to manufacturer’s instruction. In every case
analyzed within the focus of prostatic cancer no different SOCS3
immunohistochemical expression in Gleason grade pattern component was observed. In 18/20 of prostatic cancer biopsies Gleason grade 6 (3+3) positive SOCS3 staining were observed, while
we found 38/50 prostatic cancer biopsies Gleason grade 7 (3+4;
4+3) with negative staining (-) or with weak intensity staining
with negative neoplastic glands (+/-). All cases with Gleason
score 8 (3+5) showed neoplastic cells SOCS3 negative (-) or with
weak intensity staining (+/-).
Results. 1. At the same morphological Gleason grade pattern
correspond different molecular alteration of SOCS3 protein expression.
2. SOCS3 negative staining or weak intensity staining with negative neoplastic glands could identify in prostatic cancer biopsies
foci of Gleason pattern 3 in acinar adenocarcinoma Gleason grade
7 (3+4) or (4+3).
3. The study of SOCS3 immunohystochemical expression in little
foci of Gleason pattern 3 in prostatic biopsies could be useful to
reduce the Gleason upgrading in radical prostatectomy.
Immunohistochemical Study of βhemoglobin (HBB)
Expression in Prostate Carcinoma and Prostatic
Tissues
L. Ventura, M. Capulli*, C.A. Mercurio, A. Teti*, N. Rucci*
UO Anatomia Patologica, Ospedale San Salvatore, L’Aquila, Italia; * Dip.
Scienze Cliniche Applicate e Biotecnologiche, Università, L’Aquila, Italia
Background. Early studies on hemoglobin expression in breast
cancer revealed a pattern increasing with disease progression. We
also demonstrated a positive correlation between HBB expression
and tumor aggressiveness. Based on these observations and given
the similarities between breast and prostate cancer, the rationale
was shifted to investigate HBB in prostate carcinoma.
Methods. Slides from 22 patients undergone trans-urethral resection (3 cases), and radical prostatectomy (19 cases) were immunostained with a mouse monoclonal antibody against HBB. They
included normal tissues and prostatic diseases, such as adeno-
carcinoma, PIN, prostatitis, adenosis, hyperplasia and glandular
atrophy, as well as two nodal metastases.
Results. Weak and focal (1%) cytoplasmic positivity was noted
in one out of 22 adenocarcinomas. This particular case did not
show peculiar feaures. The neoplastic cells in the remaining 21
primary tumors and nodal metastases were constantly negative. A
weak positive signal was focally detected in normal (1 case) and
hyperplastic luminal cells (3 cases). Basal cells were negative.
Moderate and focal positivity was observed in atrophic glands
in a single case. Urothelial cells in normal urethra and urothelial
metaplasia were positive in 3 cases. Seminal vesicle epithelium
showed patchy positivity of expression in 1 case.
No significant expression of HBB was found in prostate carcinoma, in contrast with the results obtained in the breast. This
allows to speculate that prostate cancer may not be able to take
advantage of free radicals scavenging systems, giving a reason
for its indolent course in the majority of patients. Therefore,
the lack of HBB expression in prostate cancer may explain its
peculiar behaviour and the features of its progression and metastatic spread. Despite focal positivity in occasional cases, benign
lesions and normal tissues were negative. The positive staining
of urothelial and seminal cells may be related to the diffusion of
normal hemoglobin.
Role of p16 in prostate cancer and non malignant
lesions
A. Remo1, M. Pancione2, C. Zanella1, L. Astati1, A. Bellotti1,
I. Seghetto1, S. Pellegatti1, E. Manfrin3, R. Vendraminelli1
Pathology, “Mater Salutis”, Legnago (VR), Italia; 2 Science and Technology, University of Sannio, Benevento, Italia; 3 Pathology, University of
Verona, Verona, Italia
1
Background. Prostatic specimens occasionally may contain proliferative foci of small atypical acini that display some but not all
features of prostate carcinoma. p504s is the only prostate cancerspecific marker that, in combination with basal cell markers help
in the diagnosis of malignant lesions. Very little is known about
the diagnostic importance of p16 in primary prostate carcinoma
and non malignant elements.
Methods. We recruited 137 of routinely diagnostic prostatic
specimens (between 2009 and 2013), which consisted of 21 prostatectomy, 15 trans urethral prostatic resection (TURP) and 101
needle biopsy. We evaluated p16, in comparison with p504s, in
prostatic carcinoma (PC) and benign glands (BG). In this study,
both nuclear and cytosolic p16 expression were considered positive.
Results. We observed p16 expression in 86% of prostate cancer
specimens and 16% of benign elements (p = 0.001). Interestingly,
p16 alone retained a high diagnostic potential in prostatectomy
(95%) and in needle biopsy (84%) exhibiting a close association
with prostate cancer. p504s had a high sensitivity (97%) and PPN
(98%) but a low specificity (71%) and PPV (63%). In contrast,
P16 positive expression showed a higher specificity (84%) and
PPV (74%) than p504s. Two PC negative for p504s were positive
for p16, instead seven cases negative for p16 were positive for
p504s and notably none was negative for both markers. In prostatectomy, p16 showed a higher effectiveness completely loss in
TURP. In needle biopsies, both markers were complementary
indicating that their combined detection may help in performing
an accurate diagnosis.
In conclusion, our data suggest that p16 expression is sig-
230
nificantly enhanced in prostate carcinoma as compared to non
malignant elements. Our results provide evidence that p16 and
p504s together could improve the diagnosis of prostate cancer in
prostatectomy and needle biopsies. P16 detection might represent
a novel diagnostic biomarker in PC.
Dysregulation of mtor pathway in Upper Tract
Urothelial Carcinoma
E. Munari1, K. Fujita2, S. Faraj3, A. Chaux4, N. Gonzalez-Roibon3, J. Hicks3, A. Meeker3, N. Nonomura2, G. Netto3
1 Pathology, Johns Hopkins University, Verona, Italy; 2 Urology, Osaka
University, Osaka, Japan; 3 Pathology, Johns Hopkins Hospital, Baltimore,
Usa; 4 Office of Scientific Research, Norte University, Asunción, Paraguay
Background. Upper tract urothelial carcinoma (UTUC) accounts for 5-10% of all urothelial carcinomas. Despite many
shared features, key clinical and molecular genetic differences
between upper tract and bladder urothelial carcinomas are becoming apparent. We have previously demonstrated alterations
of mTOR pathway in bladder carcinoma with potential impact
on biological behavior. In the current study we evaluated the
expression status and prognostic significance of mTOR pathway
members in UTUC.
Methods. Archival formalin-fixed and paraffin-embedded tissues from 99 primary UTUC were retrieved from one of the
authors institution. Tissue microarrays (TMA) were constructed
with triplicate tumor samples and paired non-neoplastic urothelium. TMAs were analyzed using immunohistochemistry
for mTOR pathway members: PTEN, phos-AKT, phos-mTOR,
phos-S6, phos-4EBP1 and related markers p27 and c-MYC;
correlation with clincopathologic parameters and outcome was
performed. Results. We found significantly lower expression of PTEN,
phos-AKT, phos-mTOR, phos-S6, phos-4EBP1, p27, and c-MYC
in UTUC compared to paired benign urothelium (P < 0.0005).
We found a strong positive correlation between PTEN and phosAKT. Moderate correlation was observed between phos-mTOR
and phos-S6, PTEN and p27, phos-AKT and p27, phos-S6 and
p27, phos-mTOR and c-MYC, phos-S6 and c-MYC, and p27
and c-MYC. None of the evaluated biomarkers was associated
with increased hazard ratios for tumor recurrence or for cancerspecific mortality, when adjusting for relevant clinicopathologic
variables.
Conclusions. Dysregulation of the mTOR pathway was observed
in UTUC compared to normal urothelium, implicating a potential
pathogenic role in tumor development. In our cohort expression
of the evaluated biomarkers had no prognostic value.
Criteria for tumor tissue selection for research
biobanking of prostate adenocarcinoma
L. Cima1, E. Munari1, M. Brunelli1, G. Martignoni1, A.B. Porcaro,
W. Artibani2, V. Mori3, N. Sperandio1, R. Lawlor1, A. Scarpa1
1
Pathology and Diagnostics, AOUI, Verona, Italy; 2 Urology, AOUI, Verona, Italy; 3 Department of Pathology and Diagnotics, AOUI, Verona, Italy
1 Background. Promising new drug therapies are currently
available for patient affected with prostate adenocarcinoma.
Pilot or validation studies with molecular application need
cancer diagnosis on tissue worldwide. Tumor biobanking
for research represents a fundamental resource of neoplastic
tissue. Biobanking needs a pathology unit to identify altered
tissue but visual assessment and manual palpation does not often yield satisfactory results. We aimed to identify additional
criteria to improve the accuracy in obtaining cancerous tissue
suitable for biobanking.
Material and Methods. From July 2012 to July 2013, 211 patients, diagnosed with prostate adenocarcinoma undergoing radi-
cal prostatectomy, were recruited. Sample selection was based
on the pre-surgery ago-biopsy and criteria included presence of
prostate adenocarcinoma involving at least 50% bioptic cores
with a minimum of ten cores or prostate adenocarcinoma with
Gleason score 8 in at least 1 out of 14 biopsies. During grossing
samples were excluded due to low volum eor irregular margins.
The presence of tumor in the tissue harvested was evaluated using
frozen sections.
Results. Of the 190 patients identified, 76 (40%) were excluded as per criteria. Of the 114 remaining specimens 47 (41%)
were excluded for low volume or irregular margin. Prostate
adenocarcinoma was identified in 41 / 67 (61%) of harvested
specimens.
Conclusions. Our results demonstrate the usefulness of standard criteria such as number of involved bioptic cores in the
selection of specimens suitable for tumor harvesting reducing
the number of non neoplastic tissue and as such the effort required for biobanking cancer samples. The chosen criteria allow
appropriate and more effective harvesting of cancerous tissue
with cancer thus containing the economic and logistic burden
on hospital rountine.
Mir-183: a candidate biomarker for bladder cancer
diagnosis in voided urine?
V. Ortenzi1, A. Apollo1, K. Zavaglia1, C.M. Mazzanti2, S. Tomei3,
F. Lessi2, C.A. Sepich4, G. Bevilacqua1, A.G. Naccarato1
Div. of Surg, Molec and Ultrastruct Pathology, University of Pisa and
Pisa University Hospital, Pisa, Italy; 2 Pisa Science Foundation, Pisa,
Italy; 3 Department of Genetic Medicine, Weill Cornell Medical College,
Qatar Foundation, Doha, Qatar; 4 Div. of Urology, Ausl 12 “Versilia”
Hospital, Lido Di Camaiore, Italy
1
Background. Strategies for bladder cancer (BC) detection rely
on diagnostic combination of cystoscopy (92% sensitivity; 88%
specificity) and urinary cytology. However, cystoscopy is costly,
invasive and painful while urinary cytology has high specificity
(97%) but low sensitivity (17%-90%). New molecular urinebased tests like FISH and NMP22-test, compared with urinary
cytology, have higher sensitivity (95-100% for both tests) but
low specificity (48% and 66% respectively). miRNAs are small,
nonprotein-coding RNA regulators, that several studies proved to
exist in a stable form in various body fluids, such as blood, urine,
saliva. Deregulation of miRNA expression plays an important
role in many disorders, including cancer. Recently, miRNAs
have emerged as highly tissue-specific biomarkers with potential
diagnostic use.
Methods. We investigated whether miRNAs could be detected
in urine samples of BC patients and healthy controls, collecting
voided urine samples from patients with urothelial carcinoma
just prior to bladder tumor resection. Tumors were all urothelial
carcinomas of low and high grade. Total RNA was isolated and
quantitative reverse transcriptase-PCR was performed using specific primers to amplificate 3 miRNAs shown previously to be
deregulated in urothelial carcinomas: miR-183, miR-126 e miR96, with SNORD68 as endogenous control.
Results. Our findings suggest miR-183 as a possible diagnostic and prognostic marker for BC in voided urine, showing a
p-value of 83.33% to detect BC according to Artificial Neural
Network Analysis (ANN), with 100% sensitivity and 77.5%
specificity (p = 0.0001). Moreover, analysis of miR-183 ROC
curve for detection of LG lesions shows 100% sensitivity and
81% specificity while 100% sensitivity and 72.2% specificity
for detection of HG lesions. These preliminary results suggest
that miR-183 could be an alternative test to urinary cytology in
terms of sensitivity and can be a useful diagnostic tool in addition to cystoscopy.
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comunicazioni orali
SNAI2/slug is frequently down-regulated in human
prostate cancer and may function as a cellcycle regulator and marker of neuroendocrine
differentiation and tumor invasion
C. Sorrentino, M.G. Tupone, S. Esposito, S. Di Meo,
T. D’Antuono, E. Di Carlo
Department of Medicine and Sciences of Aging, ‘G. D’Annunzio’ University, Chieti, Italy
Backgound. SNAI2/Slug is a zinc-finger transcription factor that
acts as a master regulator of cell migration during both embryonic
development and tumor metastatization. Its role in prostate cancer
(PCa) onset and development is not fully understood. Here, we
investigated SNAI2 gene expression and regulation in human
PCa under normal and androgen deprivation therapy (ADT)
conditions.
Methods. We microdissected epithelium and stroma from cancerous and normal prostate specimens, from 165 prostatectomized patients, of whom 79 had received ADT, to detect SNAI2
gene expression levels by real-time RT-PCR. Genomic sequencing was performed to assess the involvement of epigenetic
mechanisms in the SNAI2 gene expression regulation. Immunostainings was used to localize the expression of SNAI2 and that of
its main down-stream target molecules and the Spearman’s rank
correlation coefficient (r) was used to examine the correlation
between them.
Results. SNAI2 expression was considerably down-regulated
in most of well and poorly differentiated malignant epithelia in
association with gene promoter methylation. By contrast, SNAI2
expression was confined to few cancer foci and to cell clusters
forming I. Synaptophysin/Chromogranin positive neuroendocrine differentiation areas, II. the expansion/invasion front of
high-grade PCa or, III. lymph-node metastasis. These cancer
cells had low proliferation and apoptotic activity and may express E-Cadherin. An inverse relationship was observed between
SNAI2 expression and the frequency of both Cyclin D1 or Ki-67
positivity and apoptosis. ADT up-regulated SNAI2 expression in
the malignant stroma of high-grade PCa foci and saved malignant
fibroblasts from apoptosis.
SNAI2 gene expression is differentially regulated in PCa epithelium and stroma and may function as a cell-cycle regulator and
marker of neuroendocrine differentiation and tumor invasion,
rather than as an E-Cadherin repressor.
The tumor suppressor function of il-27r
in prostate cancer patients
S. Di Meo, C. Sorrentino, M.G. Tupone, S. Esposito,
T. D’Antuono, A. Zorzoli*, I. Airoldi*, E. Di Carlo
Department of Medicine and Sciences of Aging, “G. D’Annunzio” University, Chieti, Italy; * Department of Experimental and Laboratory Medicine, IRCCS G. Gaslini Institute, Genoa, Italy
Background. Prostate cancer (PCa) is a typical age-related
malignancy and the second cause of cancer death in men. Immunotherapy may be of particular value for elderly PCa patients
and more readily acceptable than invasive management. Interleukin-27 (IL-27) has displayed anti-tumor activity in a variety
of preclinical models. Its effects, however, have never been
explored in human (h) PCa. We thus investigated the anti-tumor
potential of IL-27 in hPCa and analyzed the rationale for its clinical application.
Methods. Human prostate cancer cell line PC3 was tested for IL27 receptor (R) expression and then co-cultured with recombinant
(r) hIL-27. PC3 cells proliferation and apoptosis were assessed by
flow cytometry and angiogenesis-related gene regulation by PCR
array. In vivo effects of rhIL-27 were tested in PC3 tumors growing in athymic nude mice and investigated by histopathological
analyses. Finally, PCa samples from patients with different tumor
grades and stages were tested for IL-27R expression.
Results. rhIL-27 significantly inhibited proliferation of PC3
cells, up-regulated the anti-angiogenesis-related gene CXCL10/
IP-10, and down-regulated the pro-angiogenesis-related genes
FLT1, FGFR3, IGF1 and CCL11/eotaxin-1. PC3 tumors from
rhIL-27 treated animals displayed reduced proliferation index
and microvessel count in association with ischemic necrosis.
In patient’s prostate tissues, IL-27R was expressed by normal
epithelia and low grade PCa and lost by high tumor grades and
stages. Nevertheless, IL-27R was expressed by tumor and draining lymph node infiltrating leukocytes (T- and LN-IL).
In conclusion, IL-27 displays anti-proliferative and anti-angiogenic effects in IL-27R expressing PCa. The lack of IL-27R by
high grade and stage PCa may constitute a novel tumor evasion
mechanism, while its expression by TIL and LNIL suggest its
ability to condition tumor microenvironment.
Prostate carcinoma in renal transplants:
a single centre experience
R. Santi, D. Villari*, V. Saladino, G. Poggi, M. Sollai, G. Nicita*,
G. Nesi*
Division of Pathological Anatomy, University of Florence, Florence, Italy;
*
Division of Urology, University of Florence, Florence, Italy
Background. Renal transplant recipients are known to be at a greater
risk for de novo urological malignancies than the general population.
Several site-specific risk factors have been implicated in the development of urological malignancies in kidney recipients, prolonged
immunosuppressive therapy playing a major role. The aim of this
retrospective study was to determine the incidence, the clinicopathological features and outcomes of prostate carcinoma (PCa) in patients
subjected to kidney transplantation at our institution.
methods. Our series included 535 patients who underwent kidney
transplantation from 1991 to 2013. PCa was diagnosed in 15/535
(2.8%) recipients without a prior history of prostate malignancy.
Clinical characteristics of PCa patients, as well as therapeutic
strategies and outcomes were reviewed.
Results. At the time of PCa diagnosis, all patients had functioning grafts. A second neoplasia, most frequently affecting the skin,
was diagnosed in 6/15 patients (40%). Eleven patients underwent
radical prostatectomy, three patients were treated with a combination of androgen ablation and external radiotherapy, whereas
one patient was closely monitored using the watchful waiting approach. Mean follow-up was 51.6 months (range 4-170). Overall
survival rate was 35%: three patients died from causes unrelated
to PCa and one patient died after PCa progression. One patient
experienced chronic renal allograft rejection and started dialysis.
In conclusion, in renal transplant patients affected by PCa, radical
prostatectomy and radiotherapy were safe and effective treatments without compromise of graft functioning.
Non-organ-confined prostate cancer: predictors
of biochemical recurrence after radical
prostatectomy
R. Santi1, D. Villari2, B. Detti3, C. Saieva4, M. Sollai1, T. Chini2,
G. Poggi1, V. Saladino1, G. Nicita2, G. Nesi1
Division of Pathological Anatomy, University of Florence, Florence,
Italy; 2 Division of Urology, University of Florence, Florence, Italy; 3 Division of Radiotherapy, University of Florence, Florence, Italy; 4 Molecular
and Nutritional Epidemiology Unit, Cancer Research and Preventing Institute (ISPO), Florence, Italy
1 Background. Best therapeutic option for locally advanced
prostate cancer (PCa) is still a subject of debate. Clinicopathological features and risk factors such as positive surgical
margins and seminal vesicle involvement challenge individual
treatment options.
232
Methods. The aim of the present study was to retrospectively
evaluate the incidence of biochemical recurrence (BR) in a series
of 171 patients (pts) with locally advanced (pT3a/bN0M0) PCa,
treated with radical prostatectomy (RP) as monotherapy or in
combination with adjuvant radiotherapy (RTp).
Results. Mean age of pts was 67.9 years (range 45–80). Surgical
Gleason score was 7 in 85 (49.7%) pts, > 7 in 59 (34.5%) pts
and ≤ 6 in 27 (15.8%) pts. Pathological stage was pT3aN0M0 in
128 (74.9%) pts and pT3bN0M0 in 43 (25.1%). Surgical positive margins were detected in 72 (42.1%) specimens. Forty-four
(25.7%) pts underwent adjuvant RTp, whereas 127 (74.3%) had
RP as monotherapy. Mean follow-up was 3.0 years (range 0.36.0). During the follow-up period, 53 (31.0 %) pts experienced
BR. The mean time to relapse was 1.2 years (range 0.1-4.0). At
multivariate Cox regression analysis, surgical Gleason score > 7
(p = 0.004), seminal vesicle invasion (p = 0.013) and positive
margins (p = 0.002) proved to be independent predictors of BR.
Contrariwise, adjuvant RTp (p = 0.0001) was associated with
a lower risk of biochemical progression. In conclusion, these
results suggest the importance of optimizing pathological assessment in order to guide the decision making process, specifically
regarding the need for adjuvant therapy.
Clinical pathway organization for the treatment
of prostatic cancer
A. Colasante1, F. Ottaviani2, R. Iantorno3, G. Gaspari3, A. Vinciguerra4, A. Augurio4, R. Basilico5, J. Giampietro6, L. Mazzilli7,
A. Marchetti1
Anatomia Patologica, Clinicizzato Ss Annunziata, Chieti, Italy; 2 Case
Manager, Asl02, Chieti, Italy; 3 Clinica Urologica, Cinicizzato Ss Annunziata, Chieti, Italy; 4 Radioterapia e Medicina Nucleare, Clinicizzato Ss
Annunziata, Chieti, Italy; 5 Radiologia, Clinicizzato Ss Annunziata, Chieti, Italy; 6 Clinica Oncologica, Clinicizzato Ss Annunziata, Chieti, Italy;
7
Audit e Governo Clinico, Asl02, Chieti, Italy
1
Background. Every year in Italy about 9,000 people die due
to prostatic cancer (about 8% of total deaths from cancer). It
is the second leading cause of cancer death in males and is
the most frequently diagnosed cancer in men. Despite this, the
prostatic cancer mortality remains stable over time. Deaths for
cancer could be prevented not only through primary and secondary prevention, but also through timely interventions in terms of
diagnosis and treatment, in the context of appropriate diagnostic
and therapeutic care.
Methods. The PDTA (Percorso Diagnostico Terapeutico Assistenziale) is a plan of care, with multidisciplinary approach,
whose content is based on the EBM (evidence based medicine).
PDTAs explicitate both the goals of care with respect to a specific
clinical category of patients, and the actions, sorted in chronological sequence, which must be conducted in order to achieve the
above objectives, by the health care professionals involved in the
processing of the same type of patients. The path is an adaptation
to a local context of a guideline, with the precise identification
of the relevant organizational context: actors, responsibilities,
places, times, etc. Moreover, to us, it is absolutely necessary to
actively involve the patient in all phases of diagnostic-therapeutic
pathway and make him aware that you will provide the best
clinical and organizational approach. In this way his treatment is
as personalized as possible, with the best clinical practice today
known and with optimization of therapeutic tools available in our
national healt service.
Results. In our Institution, ASL-02 Lanciano-Vasto-Chieti, since
2011 the PDTA for patients with prostatic cancer has been activated. With this work we expose the personal experience from
the drafting of guidelines to the accomplishment of the GICO
(Interdisciplinary Group Cure for Oncology) and to the treatment
of patients with the determinant presence of the Case Manager,
of nursing provenance.
Sala Cesarea – Ore 16.45-18.30
Patologia dell’osso e dei tessuti molli
Moderatori: R. Alaggio (Padova), C. Di Cristofano (Roma)
Cyclin D1 as a diagnostic immunomarker for small
round blue cell tumors of childhood
L. Salvatorelli2, F. Brancato1, L. Puzzo2, R. Parenti3, E. Vasquez2,
R. Alaggio4, G. Magro2
1 Servizio di Anatomia Patologica, Ospedale Guzzardi, Catania, Italia; 2 G.F.
Ingrassia, Policlinico Universitario “G. Rodolico”, Catania, Italia; 3 Dipartimento di Scienze Biomediche-sez. Fisiologia, Università di Catania, Catania,
Italia; 4 Dipartimento di Patologia, Università di Padova, Padova, Italia
Background. Cyclin D1 amplification and/or overexpression
contribute to the loss of the regulatory circuits that govern G1-S
transition phase of the cell cycle, playing pivotal roles in different
human malignant tumors, including breast, colon, prostate cancer, lymphoma and melanoma. The aim of the present study was
to evaluate the immunohistochemical expression of cyclin D1 in
the most frequent small round blue cell tumors of childhood to
assess its potential diagnostic utility.
Methods. Immunohistochemical analyses were performed on
soft tissues Ewing’s sarcomas/PNET (30 cases), embryonal
rhabdomyosarcoma (13 cases), alveolar rhabdomyosarcoma (10
cases), untreated peripheral neuroblastic tumors (44 cases, including all types of neuroblastoma, ganglioneuroblastoma and
ganglioneuromas), lymphoblastic lymphomas (10 cases) and
Wilm’s tumors (10 cases).
Results. All cases of Ewing’s sarcoma/PNETs expressed diffuse-
ly (> 60% of cells) cyclin D1. In neuroblastic tumors a diffuse
(> 70% of these cells) staining was restricted to the neuroblastic
component, while it was lacking in differentiating, maturing and
mature ganglion cells. The mesenchymal/stromal component of
Wilm’s tumors exhibited only a focal immunoreactivity for cyclin
D1 (2-5% of neoplastic cells). In contrast all cases of rhabdomyosarcomas (embryonal and alveolar) and lymphoblastic lymphomas were not stained with cyclin D1. Our findings first show
that cyclin D1 is over-expressed in both Ewing’s sarcoma/PNET
and in the neuroblastic component of the peripheral neuroblastic
tumors, while it is absent or only focally detected in the other
small round blue cell tumors of childhood. Accordingly, cyclin
D1 may be exploitable as a helpful diagnostic immunomarker,
if evaluated in the appropriate morphological and immunohistochemical context.
Diagnosis of small round blue cell tumors using
an algorithmic approach
F.I. Ezejiofor1, B.J. Rocca2, A. Ginori2, A. Barone2, C. Cardone3,
S. Lazzi3, L. Leoncini2, M.R. Ambrosio2
1
Department of Pathology, ILE IFE Hospital, ILE IFE, Nigeria; 2 Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italia; 3 Sezione di Anatomia Patologica, AOU Senese, Siena, Italia
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comunicazioni orali
Background. Small round blue cell tumors category include
heterogeneous neoplasms composed of small, round to oval,
closely packed blue cells with high nuclear-cytoplasmic ratio, and round nuclei with small inconspicuous nucleoli. In
spite of similar morphological features, this group comprises
pathological entities from different lineage (epithelial, mesenchymal, hematopoietic, melanomatous) and represents a
great challenge to the pathologists for precise and conclusive
diagnosis. In this regard, the use of an algorithmic approach
may help to appropriately address the differential diagnostic
possibilities, reducing the costs and improving the diagnosis
and the treatment.
Methods and results. In this study we propose a clinical, morphological and FISH based algorithm that was employed in four
phases: phase 1 (clinical data and morphology), phase 2 (cytokeratins, TdT, desmin), phase 3 (CD20, CD99, Myogenin), phase
4 (FISH). We evaluated a total of 265 cases from Siena and Ile
Ige (Nigeria) Hospital. Using this algorithm, we determined a
specific diagnosis of the different types of SRBCT in 49%, 75%,
92%, in phase 1, 2 and 3 respectively. FISH analysis was necessary only in the 7% of cases. Only 2% of cases remained unresolved and additional immunostains (i.e. neuroendocrine markers
and HMB-45) were necessary.
Conclusions. We proposed a simple algorithmic approach
to diagnose the large majority of SRBCT to be used in both
resource-poor and resource-rich countries because it is inexpensive. The algorithm is of great help for a reliable diagnosis.
However, its importance should not be overemphasized and our
approach is neither meant to be comprehensive nor intended to
be an absolute method for immunohistochemical dissection of
that tumours.
Recurrent spindle cell lipoma: hitherto unreported
morphological features mimicking malignancy
G.M. Vecchio1, L. Salvatorelli1, F.R. Longo1, A. Bosco1, G. Angelico, D. Tricoli2, G. Magro1
Servizio Di Anatomia Patologica, AO Gravina-Caltagirone, Caltagirone,
Italia
1 2 Background. Spindle cell lipoma (SCL) is a relatively uncommon and histologically distinct variant of lipoma, typically arising in the subcutaneous tissue of the posterior neck, shoulder and
back of middle-aged men. Histologically, it is characterized by a
variable mixture of mature fat cells and CD34+ short bland-looking spindle cells set in a myxoid matrix with dispersed collagen
fibers. To the best of our knowledge, only four cases of recurrent
SCLs have been reported so far. Notably, one of such tumors
recurred as pleomorphic lipoma.
Methods. During a revision of 48 cases of soft tissue SCLs, we
selected two cases of recurrent SCLs exhibiting previously unreported/unrecognized morphological features which may represent
a diagnostic pitfall of malignancy.
Results. (Case 1) In addition to the areas (50% of the entire
tumor) with the classic features of SCL, there were alternating
fibrous areas in which were embedded numerous round to short
spindle cells closely packed. These areas also contained numerous small to medium-sized blood vessels with hyalinized walls.
The overall morphological picture was closely reminiscent of
fibrous-type recurrence as typically seen in (deep) aggressive angiomyxoma. Neoplastic cells were strongly and diffusely stained
with CD34 (desmin negative). (Case 2) Tumor was predominantly composed of highly cellular fibro-myxoid areas with only
a few interspersed thick collagen bands. These areas were closely
reminiscent of “well differentiated spindle cell liposarcoma”.
Notably numerous capillary-sized blood vessels were scattered
throughout the spindle cell component. After a careful search,
only focal (10% of the entire tumor) areas of otherwise typical
SCL were seen. Neoplastic cells were strongly and diffusely
stained with CD34. The present study emphasizes that recurrent
SCLs may exhibit areas with unusual morphology, which may be
diagnostically challenging if clinical information of a previously
excised SCL at the same site is missed.
Correlation of Rac1_RhoA pathway with ezrin
expression in osteosarcoma
F. Censi, C. Chiappetta, M. Leopizzi, C. Puggioni, V. Petrozza,
C. Di Cristofano, C. Della Rocca
Medical Surgical Sciences and Biotechnologies, Sapienza University of
Rome, Pathology Unit, ICOT, Latina, Italy
Introduction. Osteosarcoma is the most common malignant
tumor of bone. The major cause of death in osteosarcoma is the
increase of metastatic potential and the ezrin expression has been
correlated with the metastasis development. Ezrin links the actin
filaments with the cell membrane and interacts with RhoGDI by
dissociating it from RhoGTPases which allow GTPases to load
with GTP, activate RhoA and Rac1 to increase cell migration
and invasion. RhoGTPases have been found to contribute to
pathological process including cancer cell migration, invasion
and metastasis and overexpression of either the GTPases itself or
some elements of Rho signaling has been detected in many human tumors, including RhoA and Rac1.
Material and methods. We have analyzed Rac1 and RhoA
expression in osteosarcoma tissues to understanding the role of
ezrin-Rho family pathway in osteosarcoma metastatic progression. Moreover we have blocked the ezrin expression using
siRNA assay to investigate a possible correlation with RAC1 and
RHOA expression in ostesarcoma cell lines.
Result. Ours immunohistochemical data showed that many osteosarcomas presented cytoplasmatic positivity for both Rac-1 and
RhoA and cases, both ezrin positive than ezrin negative, showed
protein expression of Rac1 and RhoA. The results obtained by
ezrin siRNA transfection showed that ezrin expression in osteosarcoma cell lines might modulate mainly Rac1 expression.
Conclusions. It’s possible that the mechanism of cell motility
mediated by Rac1 and RhoA is maintained in osteosarcomas
and since ezrin, Rac1 and RhoA expression is not correlate with
metastasis progression in osteosarcoma, however osteosarcomas
without metastasis displayed a positivity for Rac1 and RhoA
expression compared to metastatic osteosarcomas, it’s could be
a protective factor.
Angioleiomyoma with unusual clinical
and morphological features: report of a rare case
occurring in the pre-sacral region
A. Bosco, G. Angelico, A. Giorlandino, F.R. Longo, G. Magro
G.F. Ingrassia, Policlinico Universitario “G. Rodolico”, Catania, Italia
Background. Angioleiomyoma (ALM) is a benign smooth muscle and vascular neoplasm, tipically found in the extremities and
more rarely in oral cavity. It occurs most frequently in women
ranging in age from 30 to 60 years. Pain is usually the prominent
symptom. Grossly, ALM appears as well-circumscribed, whitish nodule, which is histologically composed of mature smooth
muscle cells arranged in a fascicular pattern, which often seem
to arise from thick-walled vascular channels. Cytologic atypia
and mitoses are usually absent and rarely pleomorphism can be
present as a result of degenerative changes. We report the first
case of AML arising as a deep soft-tissue mass of the pre-sacral
region, showing as unusual morphological features the presence
of numerous keloidal-type collagen bands.
Methods. A 54-year old man underwent TC scan for lumbosacral
pain, which showed a deep-setaed soft tissue mass in the pre-sacral
region. The mass was completely surgically excised.
234
Results. At gross examination a nodular, well-circumscribed
mass, measuring 7 cm in its greatest diameter was observed. The
cut surface was closely reminiscent of an uterine leiomyoma.
Histologically tumor exhibited the typical morphological and
immunohistochemical (diffuse staining for desmin, a-smooth
muscle actin and h-caldesmon). Notably the most striking morphological feature was the presence of numerous keloidal-type
collagen bands scattered among neoplastic cells, a feature which
has not been previously reported/emphasized in this tumor. Due
to the abundant amount of these fibers, differential diagnostic
problems with solitary fibrous tumor and myofibroblastoma
may arise. Apart from the unusual site (pre-sacral region), the
other intriguing finding of the present AML was the presence
of numerous thick bands of hyalinized, frequently keloidal, collagen. Pathologist should be aware of the possibility that AML
may occur as deep-seated pre-sacral mass, exhibiting unusual
morphological features.
Lunedì, 28 ottobre 2013
Ematopatologia
Moderatori: LM. Larocca (Roma), S. Ascani (Terni)
Antibiotic therapy induced remission of bladder
malt lymphoma carrying t(11;18) (q21;q21) api2malt1: a case report
M. Nicola1, M. Lucioni1, R. Riboni1, G.A. Croci1, M. Arra1,
S. Cristina2, S. Valentini2, E. Dallera1, L. Arcaini3, M. Paulli1
Sez. di Anatomia Patologica, IRCCS Policlinico S. Matteo, Univ. di Pavia, Pavia, Italia; 2 Sez. di Anatomia Patologica, Ospedale Ss. Trinità, Borgomanero, Italia; 3 Divisione di Ematologia, IRCCS Policlinico S. Matteo,
Univ. di Pavia, Pavia, Italia
1 Introduction. Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) more
often involves the gastro-intestinal tract and is associated to
several translocations with t(11;18)(q21;q21) API2-MALT1
being most frequent one. Primary bladder MALT lymphoma is
rare and no translocation has ever been described. We report
a case of primary bladder MALT lymphoma carrying t(11;18)
(q21;q21).
Case report. In 2011, a 72-year-old woman, suffering from recurrent cystitis (10 years), was evaluated for persistent dysuria.
Urine cultures identified E. coli; cystoscopy revealed several
nodular lesions. Staging procedures did not detect systemic
disease.
On morphology an intense lymphoid infiltrate with frequent lymphoepithelial lesions was observed. Around reactive follicles, an
expanded marginal zone was composed by small cells and monocytoid elements. IGH genes were clonally rearranged. t(11;18)
(q21;q21) API2-MALT1 was identified by FISH. Since lymphoma was confined to the bladder and urine cultures detected E.
coli, the patient received ciprofloxacin alone. Cystoscopy showed
complete remission 6 months later.
Discussion. Our case developed in the setting of recurrent
cystitis and grossly mimicked urothelial carcinoma. Distinction
between MALT hyperplasia and MALT lymphoma is difficult:
marginal zone expansion with lymphoepithelial lesion pointed
to lymphoma. Clonal rearrangement of IGH and t(11;18)
(q21;q21) proved the suspect. As in the stomach, we suggest
that bladder MALT lymphoma could be related to chronic antigenic stimulation. Regression after antibiotic therapy endorses
this hypothesis.
Our case is the first bladder MALT lymphoma carrying t(11;18)
(q21;q21). In the stomach, t(11;18)(q21;q21) is associated to resistance to H. pylori eradication. Our patient achieved complete
remission after antibiotic therapy alone suggesting that in such
setting t(11;18)(q21;q21) does not adversely affect antibiotic
treatment and outcome.
Prognostic value of cd68 count in the treatment
of hodgkin lymphoma
M. Martini, S. Hohaus*, T. Cenci, M.T. Voso*, G. Leone*,
L.M. Larocca
Anatomia Patologica, A. Gemelli, Roma, Italia; * Ematologia, A. Gemelli,
Roma, Italia
Background. Despite recent advances in Hodgkin Lymphoma
treatment, about 20% of the patients still die from progressive
disease. Interim PET imaging has proved to be a useful prognostic tool when included in a response-adapted therapy setting (Gallamini, Blood 2012). Different studies showed that an increased
number of tumor associated macrophages strongly correlates
with a poorer prognosis in patients with cHL, thus providing a
new biomarker (Steidl, NEJM 2010). The aim of this study was
to evaluate the role of CD68 count as a prognostic factor in its
relation to early response evaluated by PET.
Methods. We studied 104 patients with HL (median age 38.5
years), diagnosed at our Institution between 2004 and 2013 and
treated with ABVD (70 patients), BEACOPP (25 patients) or other types of chemotherapy (9 patients). CD68+ cells were assessed
by staining with the PGM-1 antibody. In 50 patients, the CD68
count was < 5% and in 54 patients > 5%. PET was performed
after 2 cycles of chemotherapy in 91 patients: 70 patients were
PET-negative, 21 PET-positive.
Results. No association was found between CD68 count and patient
characteristics, as stage, bulky disease, monocyte and lymphocyte count, or IPS score. CD68 > 5% was associated to age ≥ 45
(p < 0.001). Patients with a higher CD68+ count had a higher risk
being interim PET positive (p < 0.05). In univariate analyses, that
was adjusted for the type of chemotherapy, high CD68 count and
interim PET positivity were independent predictors for reduced PFS
(p = 0.002 and p = 0.004, respectively). Moreover, in a multivariate
analysis adjusted for the type of chemotherapy, including, age, CD68
count, and interim PET, both CD68 count and interim PET retained
their prognostic significance (p = 0.003 and p = 0.004).
Conclusions. CD68 count was an independent prognostic marker, also when considering interim PET response. This may potentially allow for a risk stratification at diagnosis with further
treatment adaptation according to interim PET result.
Molecular and cellular alterations underlying
the impaired cross-talking between mds ecfc
and hematopoietic precursors
M. Martini, L. Teofili*, S. Capodimonti, A. Cocomazzi, E. Nuzzolo*, M.G. Iachininoto*, L.M. Larocca*
Anatomia Patologica, A. Gemelli, Roma, Italia; * Ematologia, A. Gemelli,
Roma, Italia
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comunicazioni orali
Background. Endothelial cells are relevant to the normal hematopoiesis. In facts, vascular niches within bone marrow support
hematopoietic stem cells proliferation and differentiation. Considering the common embryonic origin of hematopoietic and endothelial cells, we hypothesized that in MDS patients the vascular
niches might be damaged and unable to adequately sustain the
hematopoietic differentiation. To investigate this hypothesis, we
differentiated normal CD34+ cells over confluent layers of endothelial colony forming cells (ECFCs) as a surrogate of the vascular niche, obtained from patients with MDS or from healthy blood
donors. Our preliminary results suggest that in MDS does exist
an altered talking between endothelial and hematopoietic cells
(Teofili et al., Blood, ASH Annual Meeting, 2012;120:1718). The
aim of the present study is to analyze the molecular and cellular
alterations underlying the impaired talking between MDS ECFC
and hematopoietic precursors.
Materials. We compared through PCR Arrays the gene expression profiles of normal and MDS ECFCs, focusing our attention
on genes involved in Endothelial Cell Biology (PAHS-015Z,
QIAGEN). Then we analyzed the level of cytokines and growth
factors in the surnatants of co-coltures of normal and MDS ECFC
with normal CD34+ cells using the Bio-Plex pro-human cytokine
27-plex assay (Bio-Rad).
Results. We found that in MDS ECFCs several adhesive molecules such as ICAM-1, L-selectin and V-CAM were significantly
overexpressed. Moreover, we found that the cytokine milieu in
the surnatants of MDS ECFCs and normal ECFCs were significantly different. In particular, we observed that in MDS cultures
significant higher amounts of IL13, IL1b, PDGFb and VEGF
were detectable. Conversely, the levels of IL6, IL7, IL10, G-CSF
and CXCL-10 were significantly lower in MDS than in normal
ECFC cultures.
Conclusions. Our data suggest that a possible vascular niche
dysfunction may contribute to the altered hematopoiesis typical
of MDS.
The prognostic role of ebv in peripheral blood
of patients with diffuse large b cell lymphoma
M. Martini1, S. Hohaus2, M. Tisi2, M. Giachelia2, R. Santangelo,
T. Cenci1, M.T. Voso2, G. Leone2, L.M. Larocca1
Anatomia Patologica, A. Gemelli, Roma, Italia; 2 Ematologia, A. Gemelli,
Roma, Italia; 3 Microbiologia, A. Gemelli, Roma, Italia
1 Background. ‘EBV-positive diffuse large B-cell lymphoma of
the elderly’ was included as a provisional entity of DLBCL in the
revised 2008 WHO classification. We recently showed that the
plasma EBV-DNA load at HL diagnosis is an indicator of disease
activity and biological characteristics associated with negative
prognosis (Hohaus et al., Clin Cancer Res, 2011). We studied the
role of EBV-DNA copy number in different blood compartments
in patients with DLBCL at diagnosis, as a potential predictive
indicator for the presence of EBV in lymphoma cells and as a
prognostic marker in patients treated with immunochemotherapy
(R-CHOP).
Methods. We analyzed 136 patients with DLBCL (median age
62 years). EBV was detected using a commercial real-time kit
(Bio-Quant EBV, Italy) in peripheral blood (PB) compartments
(whole blood n = 133, plasma n = 55, and mononuclear cells
n = 52). Lymph node samples from 61 DLBCL patients were
analyzed for EBV infection through in situ hybridization for
EBV-encoded small RNAs (EBER).
Results. The presence and copy number of EBV in whole blood
and mononuclear cells were correlated (P < 0.05, P < 0.01, respectively), while there was no correlation to the detection of
EBV in plasma. We did not find any association between the
presence or viral load of EBV-DNA in any blood compartment
and the presence of EBV in the lymphoma cells. The presence of
EBV-DNA in peripheral blood was associated with a significant-
ly shorter event-free survival (EFS) at 2 years (P < 0.04). As well,
the EBV copy number was correlated with a worse outcome (HR
1.86, P<0.009). Correcting for IPI in a multivariate analysis, the
presence of EBV-DNA in peripheral blood retained its prognostic
significance (HR 2.02, P < 0.04).
Conclusion. Our findings suggest that EBV can be frequently
detected in peripheral blood at DLBCL diagnosis, which does
not reflect the EBV status of the lymphoma cells, but associates
with a worse outcome following standard immunochemotherapy.
Quantification of dapk1 promoter methylation
as biomarker in follicular lymphoma
M. Martini, M. Giachelia*, S. Hohaus*, F. D’Alo*, M.T. Voso*,
G. Leone*, L.M. Larocca
Anatomia Patologica, A. Gemellli, Roma, Italia; * Ematologia, A. Gemelli,
Roma, Italia
Background. Hypermethylation of the pro-apoptotic deathassociated kinase-1 (DAPK1) is a frequent epigenetic alteration
in Follicular Lymphomas (FL). We evaluated whether quantification of DAPK1 methylation in bone marrow (BM) and peripheral
blood (PB) at diagnosis and during follow-up provides important
prognostic information.
Materials. Aberrant methylation of the DAPK1 gene was quantified in 107 patients with FL, studying lymph node (n = 26), BM
(n = 107), and PB (n = 63) samples at diagnosis, at end of therapy
(n = 50) and during follow-up (n = 56), using a Methylight-PCR.
Information on BCL2-IGH and clinical characteristics for calculation of FLIPI were available for all patients.
Results. Aberrant DAPK1 methylation was found in 22/26 (85%)
lymph node biopsies, 62/107 (58%) BM samples and 25/63 (40%)
PB of FL patients at diagnosis. DAPK1 methylation levels were
higher in patients with histological signs of bone marrow infiltration and a higher FLIPI score, while there was no association
to the presence of the BCL2-IGH rearrangement. Patients with
aberrant DAPK1 methylation in BM had a significantly reduced
progression-free survival (PFS) following immunochemotherapy,
independent of the FLIPI score. DAPK1 methylation levels in BM
decreased following treatment. During follow-up, residual DAPK1
methylation and increases in methylation levels were associated
with an increased risk for relapse. In patients on watch-and-wait
strategy, higher levels of DAPK1 methylation at diagnosis were
associated with a shorter time to antilymphoma treatment.
Conclusion. Our study indicates that the quantitative analysis
of DAPK1 promoter methylation is a promising, prognostically
important biomarker for Follicular Lymphoma.
Cellular origin of lymphomatoid granulomatosis:
a look into the immunoglobulin v gene mutation
pattern
C. Bellan, T. Amato, A. Barone, L. Leoncini
Biotecnologie Mediche, Sez. Anatomia Patologica, Università di Siena,
Siena, Italia
Background. Lymphomatoid granulomatosis (LyG) is a rare
angiodestructive lymphoproliferative B-cell disorder often involving the lungs. LyG is an extranodal angiocentric and/or angiodestructive B-cell lymphoproliferative disorder composed of
polymorphic lymphoid infiltrate that consists of T lymphocytes,
plasma cells, and atypical larger neoplastic EBV(+) B-. The histologic grade of LyG is based on the respective proportions of the
EBV-infected B-cells, which may be monoclonal or oligoclonal,
and the reactive component. Molecular genetic techniques of the
tumor cells has revealed clonal Ig gene rearrangement in grade 2
and 3 cases of LyG. In situ hybridization techniques for EBER
have shown EBV infection in most cases. Although the immunophenotypic and genotypic characteristics of LyG confirm their B
236
cell lineage, the normal counterpart and stage of differentiation of
the tumor cells remain controversial.
Methods. Fifteen formalin-fixed and paraffin-embedded specimens were collected from the Department of Medical Biothecnology, University of Siena, Italy and from Pathology Department,
Hospital Universitario Marqués de Valdecilla, Santander, Spain.
IgH gene rearrangements analysis was performed to study of
clonality and sequence for comparison with known germ line VH
segments analysis. In addition estimation of antigen selection in
productive and mutated IgVH rearrangements and analysis of the
immunoglobulin heavy CDR 3 genes were performed.
Finally we carry out the comparison of the molecular results to
healthy controls, to address the possibility of a biased utilization
of immunoglobulin variable region.
Results. The results of the comparison with known germ line VH
segments revealed the presence of somatic mutations in all cases
analyzed, cloning of the IgH PCR products revealed identical VH
sequences in each cases, indicating that the mutation process is
not active.
The assignment of the VH segments of the lymphomas studied to
the VH families demonstrated that the VH usage was highly similar to that observed in peripheral blood from healthy individuals.
According to our molecular results, it appears that LyG cases may
originate from post-germinal center cells that have acquired somatic mutations in germinal center but in which the switched off.
Oncogene induced senescence distinguishes
indolent from aggressive forms of pulmonary
and non-pulmonary Langerhans’ cell histiocytosis
A. Caliò1, M. Chilosi2, F. Facchetti3, A. Zamò1, G. Martignoni1,
V. Poletti4, S. Tommasetti4, A. Dubini5, A. Tironi2, C. Doglioni6
1 Anatomia Patologica, Policlinico G.B. Rossi, Verona, Italia; 2 Anatomia
Patologica, G.B. Rossi, Verona, Italia; 3 Anatomia Patologica, Università
di Brescia, Brescia, Italia; 4 Pneumologia, Ospedale G.B. Morgagni, Forlì, Italia; 5 Anatomia Patologica, Ospedale G.B. Morgagni, Forlì, Italia;
6 Anatomia Patologica, Ospedale San Raffaele, Milano, Italia.
Background. The clonal/neoplastic nature of Langerhans’ cell
histiocytosis (LCH) has been recently confirmed by the demonstration of BRAF mutations in a large proportion of cases, including pulmonary forms (PLCH). Nevertheless, mutations are not
demonstrable in all cases, and the mechanisms leading to inflammatory cell recruitment and microenvironmental changes are not
completely understood. Mutations of BRAF, as other oncogenes,
are able to trigger cellular senescence, a p16INK4a dependent cell
cycle arrest involved in the suppression of tumorigenesis (the
best human example of oncogene-induced senescence is the melanocytic nevus). We hypothesized that this mechanism is also
involved in the pathogenesis of LCH.
Methods. We have investigated the occurrence of the BRAFV600E mutation and markers of cellular senescence in a series
of pulmonary (19 cases) and non-pulmonary (19 cases) LCH, including five aggressive cases. To overcome the low sensitivity of
sequencing analysis on archive samples we utilized a recently described and validated antibody (VE1), recognizing with elevated
sensitivity and specificity the V600E mutation. The expression of
p16INK4a and p21WAF1, two well established cell-senescence markers, were also investigated by immunohistochemistry.
Results. We demonstrated that a high proportion of cases
(47,4%), and the majority of PLCH (63%) were VE1 positive.
Langerhans cells in all cases, irrespective of BRAF mutation
status, expressed both p16INK4a and p21WAF1, with the exception
of the five aggressive cases (1/5 VE1+), that were all p16INK4a
negative. These data confirm that a large proportion of LCH,
including pulmonary cases, harbor BRAF mutations and should
be considered as clonal/neoplastic lesions. The possible involvement of oncogene-induced senescence and senescence associated
secretory phenotype may explain the peculiar pro-inflammatory
features that characterize this disease, and also their biological
and clinical heterogeneity.
Composite Langerhans cell sarcoma and marginal
zone lymphoma: evidence for clonal relationship
M.R. Ambrosio, G. De Falco, B.J. Rocca, T. Amato, A. Sacchini,
C. Bellan, S. Lazzi*, L. Leoncini
Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italia; * Sezione di Anatomia Patologica, AOU Senese, Siena, Italia
Background. Cells of the hematopoietic system are derived from
common precursors that differentiate into lineages with distinct
morphologic, immunophenotypic and functional characteristics.
As cells differentiate fully, they become “lineage committed”.
Tumors expressing the phenotype of one hematopoietic lineage
might turn into a genetically similar but phenotypically distinct
tumor of a different lineage. Recent studies have shown evidence
for phenotypic conversion of B-cell origin tumors to histiocytic
and dendritic-cell neoplasms, the most frequent association being
with follicular lymphoma and chronic lymphocytic leukemia. A
few possible pathways have been proposed to explain this phenomenon, including dedifferentiation, common progenitor, and
transdifferentiation mechanism. Herein we describe the development of a Langerhans cell sarcoma (LCS) in a marginal zone
lymphoma (MZL).
Methods. A 85-year-old male patient with a history of MZL
diagnosed 7 years before presented with a 2-month history of
progressive enlargement of a cervical lymph node. An excisional biopsy was performed. The surgical specimen underwent
routinely processing and examined under light microscopy.
Immunohistochemistry for CD20, PAX-5, C10. BCL-2, CD5,
CD23, Vimentin, CD68, S100, CD1a, Ki-67 was carried out as
well as immunoglobulin heavy chain gene (IGH) rearrangement
and comparative genomic hybridization (CGH) by laser capture
microdissection.
Results. The examination of the surgical specimen showed
pathologic features consistent with a diagnosis of LCS intermingled with a lymphoid proliferation sharing the morphological and immunohistochemical characteristics of MZL. IGH
gene rearrangement detected an identical FR1 and FR2 clonal
rearrangement in both LCS and MZL. CGH reveals a simple
karyotype with a few alterations mostly common to both tumors. Our findings provide evidence for transdifferentiation of
MZL to LCS suggesting that secondary genetic event may play
a role in this phenomenon.
A case of plasmablastic lymphoma
as transformation of a plasmacytoma:
a possible role of epstein-barr virus
M.R. Ambrosio1, G. De Falco1, B.J. Rocca1, A. Gozzetti2, V.
Mourmouras1, F. De Luca3, L. Leoncini1, S. Lazzi3
1 Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italia; 2 UO Ematologia, AOU Senese, Siena, Italia; 3 Sezione di Anatomia
Patologica, AOU Senese, Siena, Italia
Background. In the last WHO classification plasmablastic
lymphoma (PBL) is defined as a diffuse proliferation of large
neoplastic cells most of which resemble B immunoblasts, but
in which all tumour cells have the immunophenotype of plasma
cells. It occurs predominantly in immunodeficient patients, and is
characterized by the integration of EBV-RNA into tumour cells
in 60-100% of the cases.
Methods and results. A 70-year-old, non immunocompromised
man presented with a mass in the oral cavity. The examination
of the bioptic specimens showed pathologic features consistent
with a diagnosis of plasmacytoma. The disease relapsed one
year later and was treated surgically. Two years following the
237
comunicazioni orali
excision, the patient developed an ulcerated lesion of the oral
cavity in the same site of the first plasmacytoma. Histologic
examination revealed a diffuse proliferation of large, blastic
cells with prominent nucleoli intermingled with scattered cells
showing a plasmacytic differentiation. Mitotic rate was high.
The neoplastic cells expressed CD38, CD138, Mum-1. Monotypic restriction of lambda light chain was observed. CD45,
CD20, CD79a and PAX-5 were negative as well as HHV8. The
proliferative index was 95% and EBER was diffusely positive.
No translocation involving MYC, BCL-2 and BCL-6 genes was
identified by FISH analysis. The differential diagnosis included
PBL and plasmablastic plasmacytoma; however, considering
the site of involvement and the diffuse positivity for EBER, the
final diagnosis was PBL. Therefore, EBER detection was carried out also on plasmacytoma specimens; only scattered plasma
cells within the first lesion and less than 5% in the relapse were
positive for EBER.
Conclusions. This case shows the possibility that PBL may result
from the trasformation of a pre-existing plasmacytoma due to a
dedifferentiation induced by EBV. EBV may drive plasmacytoma cells to gain the plasmablastic cytomorphologic features
and a higher proliferation rate, probably interfering with B-cell
development.
The decreasing incidence of gastric malt
lymphoma over the last 20 years
A. Savio1, C. Zambelli2, P. Cesari2, G. Viviani3, F. Donato4,
F. Rolfi2, F. Zorzi1
Anatomia Patologica, Fondazione Poliambulanza, Brescia, Italia; 2 Endoscopia Digestiva, Fondazione Poliambulanza, Brescia, Italia; 3 Endoscopia Digestiva, Ospedale di Manerbio, Manerbio, Italia; 4 Medicina
Sperimentale ed Applicata, Università di Brescia, Brescia, Italia
1 Background. The discovery of Helicobacter pylori (Hp) in 1983
has changed the management and the clinical behaviour of many
gastrointestinal disorders. Hp plays a pivotal role in the development and progression of gastric MALT lymphoma (GML) and
that its eradication can cause complete and stable regressions
in about 80% of early stage cases. In our hospital treatment of
GML with eradication of Hp started in 1992. Hp eradication was
pursued in all the patients with dense lymphoid infiltration in a
gastritis setting.
After the first few years a dramatic reduction of newly diagnosed
cases was noticed.
Aims and methods. The aim of this study is to verify this drop
over the last 20 years and its hypothetical link to Hp eradication.
The rates of Hp infection and GML in patients undergoing EGD
in our hospital during three different 3-year periods have been
considered and compared with the data of the local Cancer Registry (CR).
The volume of the biopsy samples per patient was bigger during
the second and third triennium as a consequence of the adoption
of the 5-biopsies sampling protocol for gastritis evaluation in
place of 2-3 biopsies per patient during the first triennium.
5 of the patients diagnosed during the first period and 1 of the
third period underwent gastrectomy, two of them representing
collision tumours with adenocarcinoma. All the patients with
early stage disease were treated with eradication of Hp as first
line therapy.
A follow up longer than 2 years after antibacterial therapy was
available for comparison for 29 patients of the first triennium and
8 patients of the second triennium.
Results. The percentage of newly diagnosed cases of GML decreases from 0,23% (1992-1994) to 0,08% (2002- 2004) and to
0,05% (2010-2012) (Tab. I).
The rate of Hp+ve patients varies from 49% to 32% and to 20%.
Tab. I. Newly diagnosed cases of GML at Poliambulanza-HSO Hospital
in Brescia (Italy) compared with Hp+ve patients.
TRIENNIUM
EGD
GML
Hp+ve
1992-1994
15,983
38 (0.23%)
7,874 (49%)
GML/
Hp+ve
0.48%
2002-2004
10,628
9 (0.08%)
3,431 (32%)
0.26%
2010-2012
18,624
10 (0.05%)
3,766 (20%)
0.26%
The first interval drop of incidence of GML is much greater than
that expected on the basis of the Hp rate decline while it remains
aligned during the second interval.
The drop of GML is in contrast with the data of the non-Hodgkin
lymphomas from the CR (Tab. II).
Tab. II. Rate of non-Hodgkin lymphomas per 100,000 residents and per
year - Cancer Registry of Brescia (Italy).
M
F
1993-95
22.6
20.9
1999-2001
25.4
21.9
2004-2006
21.49
22.39
At follow-up the subset of GML either resistant to Hp eradication
or with delayed remission is stable during the first two periods, in
spite of the reduction of the total number of cases (Tab.3).
Tab. III. Follow-up of GML cases treated with eradication of Hp
(Legenda:NR non responders REL relapsing).
TRIENNIUM
GML
NR/REL
1992-1994
29
4
2002-2004
8
4
Moreover, the proportion of Hp-ve GML has increased from
initial 1/38 to final 4/10.
Discussion. Our data confirm the recent observation of a decreasing incidence of GML in two European studies 1 2 while being
discordant with the data from an epidemiologic study from East
Asia showing an increasing trend 3.
The effect of Hp eradication on the decreasing incidence of GML
is obvious. However GML drop is disproportionately higher during the first interval, even though possibly underestimated due to
the double amount of tissue examined by biopsy protocol during
the second and third periods.
In addition to antibiotics, the introduction of proton pump inhibitors, allowing a much stronger control of the gastric acid output
and causing a consequent downfall of the bacterial concentration,
may explain the excessive initial GML decline.
Eradication of Hp especially pursued in all the cases with dense
lymphoid infiltration in our hospital, could explain both the initial
disproportionate drop of GML and the tendency to select cases
either Hp-ve or resistant to antibacterial treatment.
References
1
Capelle LG, de Vries AC, Looman CW, et al. Gastric MALT lymphoma: epidemiology and high adenocarcinoma risk in a nation-wide
study. Eur J Cancer 2008;44:2470-6.
2
Luminari S, Cesaretti M, Marcheselli, et al. Decreasing incidence of
gastric MALT lymphomas in the era of anti-Helicobacter pylori interventions: results from a population-based study on extranodal marginal zone lymphomas. Ann Oncol 2010;21:855-9.
3
Huh J. Epidemiologic overview of malignant lymphoma. Korean J
Hemathol 2012,47:92-104.
238
Patologia tiroidea e neuropatologia
Moderatori: A. Maiorana (Modena), G. Magro (Catania)
Follicular variant of papillary thyroid carcinoma
with predominant spindle cell component:
report of two rare cases and discussion
on the differential diagnosis with other spindled
thyroid tumors
S. Corrado1, G. Papi2, S.M. Corsello3, L. Maccio1, A. Pontecorvi3,
A. Maiorana1
1 Istituto di Anatomia Patologica, Università di Modena e Reggio Emilia,
Modena, Italia; 2 Dipartimento di Medicina Interna, Azienda Usl, Modena,
Italia; 3 Cattedra di Endocrinologia, Università Cattolica, Roma, Italia
Background. Spindle cell metaplasia (SCM) represents a rare
entity in the context of both benign and malignant thyroid lesions.
We describe two rare cases of SCM in patients with papillary
thyroid carcinoma (PTC).
Patient 1. A 56-yr-old man underwent total thyroidectomy due to
euthyroid bilateral goiter. Cytological examination of fine needle
aspiration biopsy (FNAB) performed on the dominant 45 mm
nodule in the left lobe was consistent with follicular neoplasm.
Histological examination disclosed multifocal bilateral follicular
variant of PTC showing predominant (> 90%) spindle cell component in the dominant nodule only. The neoplasm stained positively for thyroglobulin and TTF-1, whereas was negative to p63,
chromogranin A, calcitonin, S-100 protein, NSE, actin, myosin,
desmin, EMA, CD1a, CD3, CD5, CD21, CD31, CD34, CD35,
CD68, CD117, bcl-2, melan A, HMB45. Staining with CK7,
CK19, Fil7+8, MNF116 was positive in the areas with follicular
differentiation only, was negative in the spindle cell areas of PTC.
Patient 2. A 53-yr-old woman underwent total thyroidectomy
for euthyroid bilateral goiter. On cytological examination, the
dominant 43 mm nodule in the right lobe was suggestive for
hyperplastic/adenomatous nodule, but histologically the picture
was diagnostic for follicular variant of PTC with SCM, in the
context of bilateral nodular hyperplasia. The tumor stained
positively for thyroglobulin, TTF-1 and CKMNF116, focally
positive for actin, whereas it was negative to CK Fil 7+8, EMA,
myosin, desmin, CD117, melan A, calcitonin. After thyroidectomy, both patients underwent radioiodine ablation therapy and
recombinant TSH (Thyrogen) test. Neither test detected residual
thyroid disease and both patients are still alive at 1 and 4 yrfollow-up, respectively.
Conclusions. We report two patients diagnosed with follicular
variant of PTC showing SCM, present their unusual pathological aspects, and discuss differential diagnosis with other thyroid
spindle cell lesions.
Dominant nodule and papillary thyroid carcinoma
in hashimoto thyroiditis: clinicopathologic
correlations in a series of 266 patients
L. Salvatorelli1, P. Amico1, M.G. Vecchio1, M. Castaing2, A. Torrisi2, G. Trovato3, G. Magro1
Registro Tumori Integrato CT-SR-ME, Policlinico Universitario “G. Rodolico”, Catania, Italia; 3 Medicina Interna, Policlinico Universitario
“G. Rodolico”, Catania, Italia
1 2 Background. Some patients affected by Hashimoto thyroiditis
(HT) may develop one or more nodules (> 1 cm), so-called
“dominant nodule” (DN). Due to the association between HT and
papillary thyroid carcinoma (PTC), the detection of DNs is alarming, with the indication to perform FNAC. Accordingly the aim
of the present study was to investigate: i) the clinicopathological
features of DN in HT; ii) the predictive value of DN for a concurrent PTC elsewhere in the thyroid gland.
Methods. We selected 266 cases of histologically proven HT.
Any nodule, grossly detected and histologically confirmed, with
a diameter ≥ 1.5 cm, merging from the surrounding thyroid parenchyma, was conventionally defined as a DN.
Results. DN was detected in 87 (32.7%) cases, while PTC was
found in 61 cases (22.9%) of HT. Histologically 72 (86.2%)
DNs resulted to be “hyperplastic follicular lesions (HFLs)”, 10
(11.5%) were PTCs and 2 (2.3%) were follicular adenomas.
Interestingly HFLs exhibited a variable cellular composition
(i.e. follicles with or without Hurthle cell change and with or
without inflammation). Among all cases of PTC, 10 (16.4%)
presented as DN, 20 (32.8%) were associated with a “histologically benign DN”, while most cases (31 cases; 49.2%) were
not related to DN. Interestingly among 87 cases of HT with
DN, PTC was detected in 30 (32.8%), in contrast to 31 cases
(17.3%) of PTC found in 179 cases of HT without any DN.
Our study first shows that: i) only a minority of DNs in HT
are actually PTCs, being most of them “hyperplastic follicular
lesions that could be clinically managed, avoiding unnecessary
surgery; ii) the predictive value of DN for a concurrent PTC
elsewhere in the thyroid gland (p = 0.02; RR = 1.34). Lastly,
this study emphasizes that a significant number of benign
DNs, being composed of follicles without associated inflammation, could be included in the category “Thy3” at FNAC
with indication to surgery, if pathologist is not aware of dealing with a patient affected by HT.
Prognostic and predictive role of vegf-a
and of the diffusible isoform vegf-121 in patients
with glioblastoma
M. Martini1, G.Q. D’Alessandris2, T. Cenci1, L. Ricci-Vitiani3,
S. Capodimonti1, R. Pallini2, L.M. Larocca1
1
Anatomia Patologica, A. Gemelli, Roma, Italia; 2 Neurochirurgia, A. Gemelli, Roma, Italia; 3 Istituto Superiore di Sanità, ISS, Roma, Italia
Background. VEGF-A is a key regulator of angiogenesis in
glioblastoma (GBM). It is released by bulk tumor cells and by
GBM stem-like cells (GSCs) in the “vascular niche”. The importance of VEGF-A in GBM is underscored by the effectiveness of
FDA-approved bevacizumab treatment. The VEGF-A gene can
be alternatively spliced to produce isoforms with different biological properties. VEGF-121 is freely diffusible, being devoid
of extracellular matrix (ECM)-binding domains. The prognostic
role of VEGF-A expression in GBM is controversial; moreover,
evidence is still lacking of the prognostic impact of VEGF-A
isoforms and of their predictive role for anti-angiogenic therapy.
Methods. In a cohort of 16 primary GBM patients, we determined the expression of VEGF-A and VEGF-121 both on deparaffinized tumor samples and on tumor-derived GSCs using
real-time RT-PCR. We also determined VEGF-A and VEGF-121
levels on an independent cohort of 17 patients treated with bevacizumab for recurrent GBM.
Results. A significant correlation was found for VEGF-121
levels (but not for VEGF-A) between tumors and GSCs.
VEGF-121 levels in tumor, normalized per the amount of
total VEGF-A, were significantly higher in patients surviving more than 1 year. A ratio VEGF-121/VEGF-A > 2.5%
was a significantly good prognosticator (p = 0.0025) for sur-
239
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vival. No correlations were found between tumor VEGF-A,
GSC VEGF-A, GCS VEGF-121, and survival. Surprisingly,
in the cohort of recurrent GBMs treated with bevacizumab,
VEGF-121 was inversely correlated with survival (p = 0.01),
and patients displaying progression-free survival>12 mos
had significantly lower amounts of VEGF-121 (p = 0.01) and
VEGF-A (p = 0.04).
Conclusions: This study suggests that ECM-bound isoforms of
VEGF are the most clinically relevant target of anti-angiogenetic
therapies. The role played by diffusible VEGF-121 is puzzling
because it seems to hold a favorable value for survival but to
predict poor response to bevacizumab therapy.
Tailored therapy in recurrent glioblastoma
M. Martini1, G.Q. D’Alessandris2, T. Cenci1, L. Ricci-Vitiani4,
A. Cocomazzi3, R. Pallini2, L.M. Larocca1
Anatomia Patologica, A. Gemelli, Roma, Italia; 2 Neurochirurgia, A. Gemelli, Roma, Italia; 3 Istituto Superiore Di Sanità, ISS, Roma, Italia
1
Background. The standard treatment of Glioblastoma (GBM)
is surgery followed by radio-chemotherapy with temozolomide
(TMZ). However, there is no consensus on the treatment of recurrent GBM. Recent data highlighted the key role of EGFR/PI3K/
Akt/mTOR pathway, inhibited by the phosphatase PTEN, and of
VEGF in the GBM pathogenesis. These molecular markers have
a prognostic value and are potential targets for therapies. The
present study aims to assess the efficacy of a targeted therapy
with bevacizumab, erlotinib, and sirolimus, selective inhibitors
of VEGF, EGFR and mTOR, in patients with recurrent GBM
tailored to their molecular profile.
Materials. In patients with recurrent GBM, we have choosen
a specific target therapy according to the tumor molecular profile. Bevacizumab was administered in cases of overexpression of VEGF; Erlotinib in cases of positivity for EGFRvIII;
Sirolimus, an mTOR inhibitor, in the cases of downregulation
PTEN.
Results. We have enrolled 16 patients (11 M, 5 F, mean age
55 years): 5 patients were treated with the combination of
bevacizumab and erlotinib, while 8 and 3 patients with Avastin
bevacizumab and sirolimus, respectively. In the 12 patients,
with follow-up> 6 months, the response rate (RR) and 6- month
progression-free survival (PFS-6) were 67% (8/12). The median
PFS was 8.5 months (range 3-39) and the median overall survival was 12 months (range, 5-39). RR and PFS-6 were 80%,
in patients treated with bevacizumab+erlotinib, 50% in patients
treated with bevacizumab and 50% with bevacizumab+sirolimus.
The serious adverse effects recorded were successfully managed
with medical therapy.
Discussion. In our study, RR and PFS-6 in recurrent GBM are
higher than those reported in the literature. We therefore conclude that the innovative approach used, ie the targeted therapy
tailored to the molecular profile of the tumor, is a promising
treatment in patients for whom there are no effective therapeutic
alternatives.
Overexpression of integrin 7 alpha identifies
a glioblastoma subgroup with a worse prognosis
M. Martini1, R. Pallini2, T.L. Haas3, T. Cenci1, G.Q. D’Alessandris2,
L.M. Larocca1
1 Anatomia Patologica, A. Gemelli, Roma, Italia; 2 Neurochirurgia, A. Gemelli, Roma, Italia; 3 Istituto Superiore di Sanità, ISS, Roma, Italia
Background. The integrins are involved in many biological
processes, including tissue development, immune responses, and
hemostasis. Integrin 7 alpha (INT7A) forms a heterodimer with
integrin b 1 in the plasma membrane and is responsible for communication between extracellular matrix and muscle cells. Altered expression and mutations of INT7A has been demonstrated
in some malignancies (human leiomyosarcoma and prostate cancer) where it appears to have a tumor suppressor role.
Methods. We analyzed the mRNA and protein expression of
INT7A, by real-time PCR and immunohistochemistry, in 62
glioblastoma (GB) and in 20 normal brain tissues. After surgical resection, all patients were treated with temozolomide and
radiotherapy. Results were correlated with clinical and molecular
features.
Results. The median expression of INT7A mRNA was significantly higher by 4 folds in GB respect to the normal brain tissue.
Moreover, INT7A mRNA expression analysis identified two
groups of GBs: a first group with a significant INT7A overexpression (30/62; 48%) and a second group with a relative lower
expression of INT7A (32/62; 52%). The different expression of
INT7A mRNA was confirmed by immunohistochemical analysis.
When we compared the INT7A mRNA level with overall survival, we found that GBs with INT7A overexpression were significantly associated with a worse clinical outcome (p = 0.048).
Conclusions. The higher level of INT7A in GB in comparison
to normal brain and the significant correlation between overexpression of this integrin with a worse clinical outcome seem to
suggest an oncogene role of INT7A in pathogenesis of this cancer
and an its involvement in the response to GB conventional radiochemotherapy.
Citologia
Moderatori: G. Fadda (Roma), P. Zeppa (Salerno)
Molecular typing of lung adenocarcinoma
on cytological samples using a multigene next
generation sequencing panel
K. Sikora1, M. Fassan1, E. Amato1, A.M. Rachiglio2, R. Cappellesso3, A. Mafficini1, M. Lambiase2, A. Fassina3, N. Normanno4,
A. Scarpa1
Dipartimento di Patologia e Diagnostica, Università di Verona, Verona, Italia; 2 Laboratorio di Farmacogenomica, INT-Fondazione PascaleCROM, Mercogliano, Italia; 3 Dipartimento di Medicina, Università di
Padova, Padova, Italia; 4 Farmacogenomica, INT-Fondazione PascaleCROM, Mercogliano, Italia
1
Background. EGFR mutated and ALK rearranged lung adenocar-
cinomas are currently treated with targeted drugs. Clinical trials
are ongoing for BRAF, PIK3CA or KRAS mutated cancers. The
number of additional targeted drugs entering into clinical practice
or trials is expected to rapidly increase. This calls for the mandatory implementation of methods probing the mutational status
of multiple genes on the limited cytological or bioptic material
available in most cases.
Methods. Cytology specimens from 38 lung adenocarcinomas
were subjected to the simultaneous assessment of 504 mutational
hotspots of 22 lung cancer-associated genes using 10ng of DNA
and Ion Torrent PGM™ next-generation sequencing.
Results. Thirthy-six cases were successfully sequenced (95%).
In 24/36 cases (67%) at least one mutated gene was observed,
240
including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET,
SMAD4, FGFR3, STK11, MAP2K1. The EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases,
were mutually exclusive. Nine samples (25%) showed concurrent
alterations in different genes.
Conclusions. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple
genes simultaneously and only requires as little as 10ng of DNA.
Its applicability to routine cytology represents a strong driver for
personalized therapy in lung cancer patients.
The intensity of braf v600e immunohistochemical
positivity compared with molecular analysis
in thyroid papillary cancer on histologic
and cytologic samples
E. Rossi1, M. Martini1, B. Angrisani1, P. Straccia1, T. Bizzarro1,
C. Ricci1, A. Pontecorvi2, C.P. Lombardi3, L.M. Larocca1, G. Fadda1
Anatomic Pathology and Histology, Catholic University, Rome, Italy;
Endocrinology, Catholic University, Rome, Italy; 3 Endocrine Surgery,
Catholic University, Rome, Italy
1
2
Background. The molecular detection of BRAF V600E mutation has been largely demonstrated as an effective diagnostic and
prognostic marker mainly for papillary thyroid cancer histotype
(PTC). Its detection has been typically performed using DNAbased techniques which include both direct DNA sequencing and
allele-specific PRC. The antibody VE-1 has been developed for the
immunoistochemical (IHC) detection in both histological and cytologic samples. The evaluation of the intensity of its positivity may
represent a problem mainly when a weak positivity is assessed. We
investigated the correlation between the molecular status of BRAF
V600E and the different intensity of its IHC counterpart
Methods. From January to June 2013, 55 cyto-histological samples were diagnosed as PTC including: 7 cases of tall cell variant
(TCV), 11 follicular variants (PCFV) and 37 classic variants
(PTC). All cases underwent mutational analysis (Pyrosequencing,
Diatech, Italy) for BRAF V600E and immunohistochemistry for
BRAF VE-1(UCS Diagnostics, Italy). The intensity was graded
from 0 to 3+ (strong positivity)
Results. Seventeen tumour showed no staining, 9 equivocal
staining (1+), 15 moderate staining (2+) and 14 with strong expression (3+). Thirty six were BRAF mutated and 19 wild type
(wt).Whereas all the 2+ and 3+ positive cases were BRAF mutated, 3 and 5 for the categories of negative and 1+ were BRAF
mutated, respectively. On the other hand, 4 wild type cases had a
2+ positivity and resulted histological PTC.
Conclusions. The use of VE1 BRAF antibody may represent
a feasible and valid first-line approach in the evaluation of
BRAF mutation. The present study highlights a good correlation
between molecular and immunohistochemical BRAF analysis
in thyroid cancers mainly when a moderate or strong positivity is present. Some discrepancies may be supported by another
mechanism of BRAF alteration, which involves copy number
gain. BRAF VE-1 may be used for selecting cases in which due
to the weak positivity or negativity the mutational analysis might
be performed.
References
Ghossein RA, Katabi N, Fagin JA. Immunohistochemical detection of mutated BRAFV600E supports the clonal origin of BRAF-induced thyroid
cancers along the spectrum of disease progression. J Clin Endocrinol
Metab 2013;98:E1414-21.
Pax8 immunocytochemistry is useful in fna from
anaplastic thyroid carcinoma
C. Bellevicine, E. Vigliar, U. Malapelle, A. Iaccarino, G. Troncone
Sanità Pubblica, Policlinico Universitario Federico II, Napoli, Italia
Objectives. Paired box gene 8 (PAX8) is a transcription factor
involved in the regulation of organogenesis of the thyroid gland,
kidney, and Müllerian system. PAX8 is frequentely expressed
in differentiated thyroid carcinomas and is retained in anaplastic
thyroid carcinoma. This candidate PAX8 as useful marker to recognize in FNA anaplastic carcinoma as of thyroid origin. The aim
of the present study is to investigate PAX8 immunohistochemical
expression and BRAF mutational status in cell-block from ATC,
comparing the results with the tests performed on the available
matching histological specimen.
Materials and methods. A computerized search was carried out
to identify cases of ATC diagnosed by FNA at our Department.
Three cases were found. In each case the original smears were
reviewed. CB and matched available histological sections were
tested both for PAX8 immunocytochemistry (ICC)and BRAF
mutations.
Results. On smears review, both Case 1 and 3 showed highly atypical dissociated cells with epitheliod morphology. Case 2 showed a
spindle-cell sarcoma-like cytomorphology with scattered pleomorphic large cells. In all cases, TTF1 and thyroglobulin expression
lacked. In the available histopathological samples the morphology
and immunophenotype profile was consistent with that reported on
CBs. In case 1 and 3 nuclei were strongly and diffusely positive
to PAX8. The Case 2 was also scored positive, with a moderate
signal. Both epitheliod ATCs from Case 1 and Case 2 harbored the
V600E BRAF mutation. Case 3 sequencing electropherogram was
wild-type. Cytological and matched histological samples yielded
concordant ICC and molecular results. Conclusion. In conclusion,
this study show that PAX8 is an useful ICC tool to aid the cytopathologist to diagnose ATC. BRAF V600E mutation was present
in two out of the three ATC cases. This patients would potentially
benefit from anti-BRAF(V600E) drugs, as observed in malignant
melanoma and in ATCs.
TIR-3 reporting in thyroid fine-needle cytology:
cytohistological correlations (2010-2013)
V. Ascoli, D. Bosco, B. Cerbelli, I. Cozzi, L. Marinelli,
D. De Mattia, L. Grillo
Laboratorio di Citodiagnostica, Dipartimento di Scienze Radiologiche,
Oncologiche ed Anatomopatologiche, Sapienza Università di Roma
Background. The SIAPEC working group for classification of
thyroid nodules has proposed in 2007 a 5-tier reporting system for
thyroid cytology, which includes the “indeterminate/inconclusive”
category (TIR-3). This category encompasses follicular-patterned
lesions. In such cases, only histology can provide a final diagnosis.
Methods. We assessed the distribution of 7624 thyroid aspirates in
the last 3 years (May-2010/May-2013) by the 5-tier system. Then,
we focused on TIR-3 cases by evaluating surgically treated cases
in our hospital and the histological diagnosis.
Results. TIR-1 were 2727 (35.7%), TIR-2 (4363; 57.2%), TIR-4
(153; 2%), TIR-5 (63; 0.8%). TIR-3 were 318 (4.2%), corresponding to 318 persons. Of these, 108 had surgical follow-up (44.4%).
Overall, the surgical yield of malignancy was 31.5% (26 papillary
carcinoma, 9 of which of the follicular variant; 6 follicular carcinoma; 2 medullary carcinoma). Adenomas represented the 33.3%
(19 follicular; 16 Hürthle cell lesion; 1 parathyroid adenoma). The
remaining 31.5 were benign lesions. There were also 10 occult
papillary carcinoma (9%).
Conclusions. Our results show that 70 persons (64.8%) with a
TIR-3 diagnosis had a neoplasm and correctly underwent surgery.
The malignancy rate is higher than that reported in the literature.
The benign rate (33%) indicates that, sometimes, patients could
rather benefit of a repeat aspirate instead of unnecessary surgery.
Our survey is limited because more than 60% of patients have no
follow-up. The SIAPEC has now proposed an update of the TIR-3
cytology thyroid reporting introducing a subclassification in TIR3-A and TIR-3-B with a lower and an higher risk of malignancy,
241
comunicazioni orali
respectively. Our study is in line with this update that will help
pathologists in better define indeterminate thyroid nodules. The
update system has the target to help clinicians in the therapeutic
approach to patients with thyroid nodules.
Digital cytology and tablet technologies:
an investigation based on a tool for the health
technology assessment
M.R. Giovagnoli1, E. Giarnieri1, M. Pochini1, A. Boschetto2,
D. Giansanti2
Dipartimento di Medicina Clinica e Molecolare, S. Andrea, Roma, Italia;
Dipartimento di Ingegneria Meccanica e Aereosp., Facoltà di Ingegneria, Sapienza Università di Roma, Italia; 3 Dipartimento di Tecnologie e
Salute, ISS, Roma, Italia
1 2 Background. E-slides are the product of a digitalization workflow by means of a heterogeneous process which starts from
scanning the slide by mean of a standard scope. Up to a few years
ago e-slides were mainly exchanged using a PC as client which
rendered it possible to successfully set-up effective networks for
tele-consulting and e-learning. In recent years greater technological advances have opened up new directions of research for
Digital Cytology: integration with DICOM, with 3D technologies, and with tablet technologies, the core field of this study. The
introduction of the tablet technology -ranging from cooperative
diagnosis up to the tele-consulting and tele-diagnosis- is critical
to the evolution of the Health Care System.
Methods. The specific aim of the study was to design a new,
wide-ranging HTA tool to assess, in terms of performance and
acceptance, the introduction of tablet technologies –portable
and non-portable– in the Health Care System. It has thus been
proposed a new HTA methodology in order to assess in terms of
performance and acceptance the integration of the tablet technologies with the digital cytology, useful both for cytology experts
and care-givers of an ICT technology.
It has thus been proposed a new HTA methodology based on a
word interactive document (HTA tool). Among all the functionalities this tool allows:
–investigate a wide range of tablet technologies ranging from
smart phones up to the very large cooperative virtual tables;
– investigate the parameters relevant to the acceptance/accuracy
and diagnostic power of the technology;
– collect data in a manner useful for a data-mining.
Results. The methodology, based on a word interactive document
(HTA-tool) has been successfully tested on 7 tablet systems in a
web-link of digital-cytology based on Aperio. The HTA tool has
shown to be very useful to investigate the tablet technologies and
thus to furnish to stake-holders valuable information for decisionmaking in the Health Care System.
Performance of egfr mutant specific antibodies
in different cytological preparation
and paucicellular specimen
C. Bellevicine, A. Bianco, U. Malapelle, E. Vigliar, A. Iaccarino,
G. Troncone
Background. Testing lung adenocarcinoma for EGFR mutations
is mandatory to select the most appropriate treatment. In addition
to DNA-based analysis, anti-EGFR mutant specific antibodies
(mt Abs) may be a cost-effective immunohistochemical (IHC)
tool. To date, mt Abs have mostly been applied to histological
specimens and only a few studies were carried out on cytology.
This may probably reflect the poor standardization and the high
variability in fixation and staining inherent to the cytological
sample. Since in most instances only the cytological specimen
is available, validating mt Abs on this sample is very relevant
to process cases lacking adequate amount/quality of DNA. Thus
this methodological study was undertaken by exploiting EGFR
mutant cultured human lung cancer cells subjected to different
preparation techniques used in routine cytology.
Materials and methods. Cell pellets from cultures of HCC827
and H3255 cell lines harboring respectively Exon 19 p. E746a750 deletion and Exon 21 p.L858R point mutation were obtained. The pellets were cytocentrifuged on charged slides and
then: 1) air dried; 2) air-dried and Diff-Quick stained; 3) alcohol
fixed; 4) alcohol fixed and Papanicolaou stained; 5) formalin
fixed and paraffin embedded cell block (CB); 6) suspended in
ThinPrep vials. To simulate paucicellular specimen with low
neoplastic content, the mutant cells were serially mixed with wild
type ones to reach a concentration of 10% mutant cell.
Results. The best IHC signal was observed in CBs and in alcoholfixed cells, even after Papanicolaou destaining. Air-dried slides
showed less intense membranous staining and a more granular
cytoplasmic signal. The mutated cells were well depicted by
EGFR mutant specific antibody, even when representing <10%
of the total population.
Conclusion. EGFR mutant specific Abs represents a reliable
screening tool for the most frequent EGFR sensitizing mutation
on cytological specimen, also enabling processing paucicellular
cytological specimens.
Cd10 is useful to differentiate gastrointestinal contaminats from pancreatic ductal
adenocarcinoma in cell block from eus-fna
E. Vigliar, A. Iaccarino, A. Bianco, G. Troncone, C. Bellevicine
Backgound. Endoscopic ultrasound (EUS) fine needle aspiration
(FNA) is an effective tool to preoperatively diagnose pancreatic
lesions, implemented in our Institution as early as 2006. Morphological criteria are usually sufficient to reliably diagnose most of
the pancreatic ductal adenocarcinoma (PDA). However, when
the a scant specimen has many gastric and duodenal epithelial
contaminating cells, the diagnosis is challenging. In most cases
CEA is useful, but addional marker are necessary to reliably diagnose well-differentiated PDA. The search for diagnostic markers
useful to discriminate on cell-block (CB) preparations between
contaminating and well differentiated PDA is worth pursuing.
Ancillary staining on CD 10 is a cell-surface metallopeptidase
normally expressed by numerous tissues, abundant in the gastrointestinal (GI) epithelial apical border. Its expression is decreased
or completely lost in malignancies; however, little investigation
focused on the CD10 expression in neoplasms arsing from exocrine pancreas. Here we assessed whether CD10 would be useful
to be included in a panel together with CEA to discriminate the
GI contaminant cells from neoplastic cells CBs.
Methods. Eight cases of EUS-FNA of pancreatic masses were
selected from our files, taking care to include cases whose diagnosis had been challenging due to the presence of contaminating
GI cells and whose CBs was available for immunostaining. Case
selected included both well differentiates (n = 6) and poorly differentiated (n = 2) PDAs. CD10 and CEA were stained on CBs
with standard methods.
Results. In all cases, the PDA cells stained for CEA. In malignant cells CD10 was negative in 7/8 (88%) cases; Only one case
showed faint cytoplasmatic signal, whereas the apical border
remained negative. Conversely, contaminant GI cells showed a
strong apical positivity for CD10. Thus, loss of CD10 is a consistent feature of PDA cells. This could aid the cytopathologist to
differentiate between well differentiated PDA and GI contaminant cells, in difficult cases.
242
Cervical vaginal screening using western blot:
interaction between e6-p53 and e7-prb
G. Di Benedetto1, V. Maccallini2, L. Coppola3
Anatomia Patologica Seconda Università Napoli, Moscati, Caserta, Italia; 2 Dipartimento Prevenzione, Asl 1, Pescina (AQ), Italia; 3 Anatomia
Patologica, Filippo Neri, Roma, Italia
1
Background. Two early HPV genes, E6 and E7, are known
to play crucial role in tumor formation. In summary, in the
low grade lesion squamous intraepithelial (LSIL), HPV DNA
penetrates into nuclei host and is present in episomal state
and e6 and e7 expression is regulated by viral gene and by
host cell. The mechanisms by which on the high grade lesion
(HSIL) integrates its DNA into the human genome are not
fully understood.The integration typically takes place in correspondence to the sequence of the E2 gene, with destruction
of the genes E2, E4, E5 and part of L2, while the sequences of
the E6 and E7 genes are joined to cellular sequences thereby
generating more stable transcripts (cfr.Video https://vimeo.
com/67874443). Western blot could be used to assess both the
presence of proteins E6 E7 and their possible interaction with
p53 and pRb respectively for the categories ASC-US – LSILHSIL), using liquid based cytology (LBC) and western blot
technique.
Material and methods. LBC was taken with a cytobrush.
All slides for LBC were stained and classified according
to the Bethesda classification 2001. On patients with paptest positive, the cell samples of Ascus-Lsil-Hsil, stored in
physiological solution were subjected to protein extraction.
When the electrophoresis identify an extra bands at molecular weight far superior to those of E6 and E7 induced us to
hypothesize that there was also the interaction between p53
and E6-E7-pRb and the samples reacted with p53-pRb antibodies (Fig. 1).
Results. Table I shows that on the ASC-US category, E6 E7 sensitivity was 1/32 (3,1%). On the LSIL category, sensitivity were
18/42 (42,8%), HSIL were 4 (100%). Fig 1 shows the presence
of extra bands at molecular weight far superior to those of E6 and
E7. With the use of monoclonal antibodies respectively primary
anti-p53 and anti- pRb was found in 1/42 (2,3 %) LSIL and in 2/4
(50%) HSIL (Tab. I).
The below figures relating to the results of the Western blot with
primary monoclonal antibody anti E6 and E7 antibody show, for
each sample, a dual-band response. The presence of extra bands
Monoclonal antibody E7
Monoclonal antibody p53
Fig. 1. Western blot obtained from some of the samples, where, in
addition to E6 and E7 was also found to their interaction with p53
and pRb respectively.
Monoclonal antibody pRb
at molecular weight far superior to those of E6 and E7 induced us
to hypothesize that there was also the interaction between E6-p53
and E7-pRb.
Tab. I.
Monoclonal antibody E6
Cytology
N = 2500
E6-E7
E6-p53 E7pRb
Asc-Us
32 (1,3%)
1(3,1%)
0 (0%)
Lsil
42 (1,7%)
18 (42,8%)
1 (2,3%)
Hsil
4 (0,2%)
4 (100%)
2 (50%)
78/2500 = (3,2%) 33/78 (42,3%) 4/78 (5,1%)
243
comunicazioni orali
Sala 6 – ore 16.45-18.30
Ginecopatologia e tecniche
Moderatori: G.F. Zannoni (Roma), R. Giardini (Cremona)
Uterine carcinoma as first malignancy in Lynch
syndrome: clinico-morphological and molecular
characterization
L. Cimetti, S. Freguia, N. Papanikolaou*, I. Carnevali1, N. Sahnane, A.M. Chiaravalli, M.G. Tibiletti, D. Furlan, F. Sessa,
C. Riva1
Dipartimento di Scienze Chirurgiche e Morfologiche, Università
dell’Insubria, Ospedale di Circolo, Varese, Italia; * Polo Scientifico e Tecnologico, Multimedica, Milano, Italia
Background. Early-onset gynaecological cancer frequently
represents the first malignancy in Lynch Syndrome (LS). Identification of these patients is important to propose surveillance
strategies as they show increased risk for synchronous and/
or metachronous colo-rectal cancer and other tumors. Since
the syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes, detection of LS cases is achieved
through immunohistochemistry (IHC) for MMR proteins,
microsatellite instability (MSI) assay and mutational analysis
of MMR genes.
Methods. Twenty uterine carcinomas (UC) from 17 LS families
classified according to Bethesda revised criteria (2007) were
studied for MMR proteins IHC, MSI and mutational status.
Clinico-morphological features including age at diagnosis, location in uterus, histological type, grade, tumor-infiltrating and
peritumoral lymphocytes (TIL, PL), mitotic index and stage were
assessed.
Results. Mean age at diagnosis was 46 y (range 30-68) and in 17
cases (85%) UC was the first manifestation of LS. Endometrial
cavity was the prevalent site (15 cases); other locations were
isthmus (2), cervix (1), endometrial cavity and cervix (1), isthmus and ovaries (1). Thirteen cases showed a pure endometriod
histotype. Abundant TIL and PL were observed in 11 and 13
cases respectively. IHC revealed MSH2 and/or MSH6 loss in 15
cases (75%) and MLH1/PMS2 loss in 5 cases. MSI was detected
in 18 cases (90%). MSH2, MLH1 and MSH6 gene pathogenetic
mutations were found in 9, 5 and 2 cases, respectively. Four cases
showed MSH6 mutations classified as “variants with unknown
significance”.
Conclusions. UC often represents the sentinel malignancy in
LS. Endometrial or isthmic endometrioid carcinomas with TIL
and PL in patients under 50 y 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.
Rnaset2 expression in ovarian epithelial tumors
and clinico-pathological correlations
L. Cimetti1, S. Freguia1, H. Reina1, F. De Stefano1, F. Acquati2,
R. Taramelli2, L. Monti2, F. Sessa3, C. Riva3
1 Dipartimento di Scienze Chirurgiche e Morfologiche, Università dell’Insubria, Ospedale di Circolo, Varese, Italia; 2 Dipartimento di Scienze Teoriche e Applicate, Università dell’Insubria, Varese, Italia; 3 Dipartimento
di Scienze Chirurgiche e Morfologiche, Università dell’Insubria, Ospedale di Circolo, Varese, Italia
Background. RNASET2 is a ribonuclease involved in cross-talk
between neoplastic cells and tumor microenvironment and it has
been found to possess a remarkable in vivo tumor suppressor
activity in transfected human ovarian cancer cell lines injected
in nude mouse. Recent experimental data suggest that control of
ovarian tumorigenesis mediated by RNASET2 occurs through
microenvironmental modifications.
Methods. Eighty-six paraffin-embedded surgical samples from
ovarian epithelial tumors have been immunohistochemically
tested using an anti-hRNASE2 rabbit polyclonal antibody. Histotype, grade, mitotic and proliferative index (Ki67/MIB1), p53 and
stage were also analysed.
Results. RNASET2 was expressed in 73/86 tumors (84%) and
in 2/3 of the positive cases immunoreactivity was detected in a
great proportion of cells (> 50%). Positivity was localized in the
cytoplasm with a prevalent granular (vs diffuse) pattern. RNASET2 was more frequently observed in serous (46/49, 88%), clear
cell (5/5, 100%) and endometrioid (9/9, 100%) than in mucinous
tumors (10/18, 55%). Among serous tumor, a more diffuse and
strong immunoreactivity was found in borderline and low-grade
tumors (73% of the neoplastic cells) vs high grade tumors (47%).
In malignant tumors with elevated mitotic index (> 20 x 10 HPF),
RNASET2 expression was mild and focal. No significant correlations with other clinico-pathological parameters were observed.
Conclusions. RNASET2 is widely expressed in ovarian epithelial
tumors including the most frequent serous histotype. Our results
confirm in vivo experimental data on RNASET2 involvement in
ovarian tumorigenesis. The decreasing trend of expression in malignant high grade and high mitotic index cancers suggests its role
in proliferation and aggressiveness control. Further studies on
immune cell response and patients outcome could better clarify
RNASET2 biological properties.
Endometrial carcinosarcomas: differentiation, p16expression and proliferation
J. Plank1, A. Kasal2, E. Egarter-Vigl1, F. Vittadello1, F. Rivasi2,
M. Riva2, G. Negri2
Claudiana Province College, College for Health-Care Professions, Bolzano, Italia; 2 Department of Pathology, Central Hospital, Bolzano, Italia; 3 Department of Pathology, University of Modena and Reggio Emilia,
Modena, Italia
1 Background. Carcinosarcomas (MMMT, malignant mesodermal
mixed tumors) are rare tumors that show both a carcinomatous
and sarcomatous differentiation. The mesenchymal component
may be of homologous (originated by tissues normally present
in the uterus) or heterologous (e.g. chondrosarcomatous, rhabdomyosarcomatous) type. Although most of the literature concerning carcinosarcomas describes that the biological behavior is
driven mainly by the carcinomatous tumor component 1, a recent
large study 2 describes a particularly poor outcome in carcinosarcomas with rhabdomyoblastic differentiation.
Methods. In this study we evaluated the correlation between differentiation and proliferation in 31 endometrial carcinosarcomas using
a dual stain immunohistochemistry technique including a panel of
differentiation antibodies (Cytokeratin AE1/3, Vimentin, Desmin,
CD10) and the proliferation marker Ki67. Moreover, we evaluated
the expression of the tumor suppressor protein p16 in 27 carcinosarcomas and 10 conventional adenocarcinomas of the endometrium.
Results. The mean proliferation rate of the cytokeratin, Vimentin, Desmin and CD10 positive tumor components was 54.8%,
41.5%, 20.9% and 28.6%, respectively. P16 was diffusely
expressed in the epithelial component of all carcinosarcomas
and adenocarcinomas in the stromal component of 96.4% of
carcinosarcomas (regardless of the differentiation) and never
in the non-neoplastic stroma of the adenocarcinomas. The re-
244
sults of this study are in agreement with the previous claims
that the behaviour of carcinosarcomas is mainly driven by the
carcinomatous component, whereas the kind of mesenchymal
differentiation, particularly when rhabdomyoblastic, does not
influence the tumor aggressivity. Moreover, we show that p16
may be a useful marker of mesenchymal tumor differentiation
in carcinosarcomas.
This study was funded by the Claudiana Province.
References
1
McCluggage WG, J Clin Pathol 2002.
2
Ferguson SE, Am J Surg Pathol 2007.
Early spontaneous abortion diagnostics:
considerations on two different series
and formulation of an unified protocol
P. Ceriolo, E. Fulcheri, F. Negri, Y. Musizzano*, C. Gambini
Anatomia Patologica, Istituto G. Gaslini, Genova, Italia; * Laboratoire
De Biopathologie, Centre Hospitalier Régional Universitaire, Montpellier, France
Background. Early spontaneous abortion (ESA) affect 14% of
pregnancies and up to 2% of women experience recurrent ESA.
Unfortunately, too many reports are non-diagnostic, leading to a
loss of confidence in histology and impairing the assessment of
real reproductive history. A reasoned approach to ESA should
aim at well-defined targets by keeping in mind the value of the
information provided.
Methods. Two series from S.Martino Hospital (1095 cases) and
G.Gaslini Institute (IGG, 266 cases) were reviewed, allowing
us to identify a shared diagnostic protocol. Specimens were received fresh and grossly examined looking for embryo, decidua,
chorion (sampled without cutting it). At IGG, frozen samples
were also stored in tissue bank. Specimens were fully examined
once the adequacy assessed on HE slides; further examination
of maternal tissues was performed by the help of PAS (stroma),
Masson’s trichrome, Weigert-fibrin, α-smooth muscle actin, desmin, vimentin (vessels), CD45 (inflammatory cells), CD146, and
CAM5.2 (trophoblast), while the fetal compartment was studied
by CD31, GLUT1 (vascularisation), CD45 (inflammatory cells),
p57 (paternal gene expression), MIB-1 (proliferation), CAM5.2,
human chorionic gonadotropin β, placental lactogen, placentallike alkaline phosphatise, p63 (trophoblast). FISH (probes for 9,
13, 15, 16, 18, 21, 22, X, and Y chromosomes) and flow cytometry were also applied.
Results. The majority of cases could be assigned to diagnostic categories, that is infection/inflammation, retroplacental
bleeding, defective implantation (including autoimmune and
endocrine diseases), karyotype abnormalities, and trophoblastic
diseases. In conclusion, modern ESA diagnostics should determine adequacy, identify trophoblastic disorders, and assign the
findings to diagnostic categories. This protocol could be seen as
expensive, but it can allow to reduce the overall cost by orientating further clinical investigations and by providing concrete
answers to the couples.
Endometritis and endometrial polyps:
role of growth factors
G. Arborea, G. Fiore, T. Montrone, R. Rossi, E. Cicinelli*, T. Lettini, L. Resta
DETO, Policlinico, Bari, Italia;
Bari, Italia
*
Ostetricia e Ginecologia, Policlinico,
Background. Endometrial polyps are a very common. They
are not always easily distinguished from more severe uterine
pathologies. Their etiology is still unknown, but the expression
of vascular endothelial growth factors (VEGF) and transforming growth factor-β (TGF-β) correlated with hormonal stimula-
tion, has been recently studied. Presence of micro-polips in endometritis (Resta et al. 2012) led us to study the correlation between
infiammation and genesis of macro-polips.
Methods. We studied immunohistochemical expression of VEGF
and TGFb 1-3 in 51 endometrial biopsies of women in different
ages, all of childbearing age. 17 concerned only endometrial polyps, 17 only endometritis and 12 cases with endometritis and endometrial polyps in the same sample. The positivity was recorded
with a semiquantitative method.
Results. The expression of VEGF and TGFbeta 1-3 for both
the glandular epithelium that for the stroma was significant
increased in endometritis and higher in cases associated with
the presence of macro-polyps. The cells positive to VEGF are
more numerous in the glands of the polyps correlated with
endometritis than those without infiammation. The TGFb3 is
expressed both in the glands in the stroma, in most polyps and
endometritis and also in the stromal cells with “spindle cell”
change, characteristics of inflammation. The demonstration of
the expression of TGFb3 in the spindle cells may assume a role
in the stimulation and growth of the stroma until the formation
of the polyp. We can therefore conclude that the expression of
angiogenic and fibrogenic factors both in glandular epithelium
and in the stroma of endometrial polyps and in the surrounding
endometrium seems to be important to determine the growth
and the formation of polyps.
Completely regressed metastases of cervical
carcinoma after neoadjuvant therapy:
a worrisome aspect with a relevant impact
on prognosis
V.G. Vellone1, G. Chiarello2, G. Amodio3, G. Scambia3, V. Masciullo3, G.F. Zannoni2
1 Anatomia Patologica, S. Martino, Genova, Italia; 2 Anatomia Patologica,
Gemelli, Roma, Italia; 3 Ginecologia, Gemelli, Roma, Italia
Introduction. Surgical pathology of cervical carcinoma following neoadjuvant treatment represents a challenging issue. The
impact of completely regressed metastases on overall (OS) and
disease free survival (DFS) is investigated.
Materials and methods. 238 consecutive patients which undervent to radio-chemoterapy followed by radical surgery for
cervical carcinoma were included in the study. Tumor regression
grade (pR), FIGO stage and evidence of completely regressed
metastases were evaluated according to OS and DFS.
Results. One hundred patients resulted pR0 (42%); 68 pR1
(28,6%); 70 pR2 (29,4%); 96 (40,3%) patients had No Residual
Disease (NRD: FIGO 0), 100 (44,6%) Local Residual Disease
(LRD: FIGO I-II) and 36 (15,1%) Metastatic Disease (MD: FIGO
III-IV); pR2 showed a worse OS and DFS when compared to pR0
e pR1 whereas MD appeared to have a poor OS and DFS when
compared to NRD and LRD.
19 patients (8%) showed completely regressed metastases with no
vital cells and histologic aspects similar to those of the primary
tumor with fibrous nodules, macrophages deposition, keratin
granulomas, necrotic areas and lakes of mucus; in 12 cases it resulted the only metastatic site: in 6 cases we reported lymphnodes
metastase, in 6 cases were perithoneal. These patients were considered as Regressed Metastatic Disease and compared to other
groups showed OS and DFS similar to MD.
Conclusions. Also in presence of complete regression following
radio-chemotherapy, metastases of the cervical carcinoma remain
an ominous sign significantly affecting both OS and DFS.
Risk management in anatomical pathology
M. Biancalani
Anatomia Patologica, S. Giuseppe, Empoli, Italia
comunicazioni orali
The topic of Risk Management in the Anatomical Pathology
Service is a subject that is encountering increasing interest
among the sector’s workers for a two-fold reason, contributing to improving the patient’s safety and that of the Service’s
workers.
A perfectly efficient Risk Management and Sample Tracking System cannot be confined to the single department but
must also provide for integrated procedures among the various
medical areas, thus registration of all processes/events that
from harvesting continue up to delivery of the report. In other
words from the formalisation of the computerised request by the
requesting party (operating theatre, ward, outpatients etc) that
has contact with the patient and that therefore can proceed first
of all with correct identification of personal details, to delivery
of the digitally signed report that will be sent to the company
repository in real time.
In addition to tracking all the steps foreseen in this procedure,
the Risk Management and Sample Tracking system must ensure
verification of the information that can allow, for example, recovery of Bio-cassettes and or microscope slides in the archive,
also after several years.
The sections below will contain an analysis of the various
phases that make up the operational workflow and that are the
focus of attention by the Risk Management and Sample Tracking system.
Production of computerised requests and check-in of
samples. While the request is being drawn up, and once the
patient has been “linked up” by accessing the company’s central database, the healthcare worker (operating theatre, ward,
outpatients etc) fills out the necessary information on the management software, such as – material sent, diagnostic query,
clinical information. The adhesive labels are then printed,
containing the patient’s ID data and that of the material in the
container, by using two-dimensional barcodes. When the material reaches Anatomical Pathology Department, the worker
checks in the requests by reading the two-dimensional barcode
on the label stuck to the container. This enables verification
of consistency between the request and the material sent,
therefore allowing the patient to be accepted and processed,
or to activate the procedures required for solving any critical
factors, if necessary.
Acceptance of managing the patient is carried out using the software, creating a unique alphanumerical code “type- year-number” that integrates with the two-dimensional barcode generated
by the requesting department. Should any non-compliances be
found during the check-in phase, or in any of the sample evaluation phases or diagnosis production (laboratory, reporting, etc),
the staff informs them about what has been detected and proceeds with the necessary corrective actions, which are explained
in the Service operational instructions.
Reduction, processing and inclusion. The bottles are taken
into the laboratory, where the pathologist and the technician
proceed with taking a reduction sample of the material. The
doctor extracts the samples from the bottles, carries out a macroscopic description, proceeds with reduction and gives the
technician instructions so that he/she can create the preparation
protocol set out for the case in question. Once the protocol has
been generated, the relative bio-cassettes can be printed out,
identified by the two-dimensional bar code containing the material coding and the alphanumerical code.
Once the macroscopic examination of the individual case or the
entire reduction session (it depends on organisation) has been
completed, a container is identified, once again with a data matrix code placed on it, which will store the bio-cassettes whilst
awaiting processing.
The bio-cassettes are then beeped and placed inside the specific racks or directly into the container as stated above by
the pathologist or the technician (depending on the laboratory
245
organisation). Once all the bio-cassettes have been inserted, the
container is beeped again to show tracking that the operation
has been completed.
Once the processing phase is completed, the technician then
identifies the container where the bio-cassettes came from, so
that the tracking system provides the list of the bio-cassettes that
were contained in it and which must be included. The operator
then proceeds with including the material, scanning the supports
by reading the two-dimensional barcodes. This step is carried
out a second time, after all the metal moulds have been removed
from the bio-cassettes, for two reasons: To allocate the inclusions to be cut to the technician(s) and to check that all the material included has been allocated for slicing. To carry out this
operation it is necessary to identify a container with a specific
data matrix where the bio-cassettes are housed. On completion
of the operation, as with processing, the second beeping is requested and the name of the technician is also requested, which
will take charge of the container.
Slicing and colouring. The technician can proceed with reading the two-dimensional barcode that identifies the container
in which the blocks to be cut are placed, or alternatively with
reading his/her own two-dimensional barcode shown on his/her
own badge. In this case, the tracking system directly provides
evidence of all the cases that have been allocated to the specific
technician beforehand.
The slides, printed according to the instructions set out in
the preparation protocol, are identified by using the twodimensional barcode. The slicing operation is carried out
as follows: The bio-cassette is beeped and subsequently the
case is identified with all the slides to be prepared according
to the protocol used. The section is performed and placed in
the thermostat-controlled tub. The corresponding slide must
be beeped before harvesting. If any non-correspondence
should be found, a warning will appear that will prevent any
further step being taken until the non-compliance has been
fully resolved.
The last phase of colouring is carried out by first of all identifying the basket into which the slides will be loaded, again
by reading the two-dimensional barcode. Once they have been
dried on the heat plate, the slides are beeped and placed in the
basked, ready for colouring, after a period in the oven to increase adhesiveness of the histological section to the glass. As
in the previous steps, the operation is completed in this case too
by the second beep of the basket.
Similarly to what is foreseen for the processing of laboratory
routine, should the pathologist require further investigations
during the report-writing stage, such as determination of the
histochemical or immune-histochemical type, the tracking system allows these requests to be identified quickly according to
the foreseen processing methods, via specific research filters.
Delivery for reading. Once colouring is completed and the
slides have been mounted, the technician then proceeds with
delivery of the cases for reading, according to what is indicated
by the software that in turn acknowledges the indications of the
pathologist in charge of allocating the cases to be read. Beeping of the slides has a dual purpose in this case too: It allows
verification that all colouring of the case has been completed
as in the protocol and also identifies the pathologist who will
deal with diagnosis, notwithstanding the possibility of stating
another name, also after allocation, for any other needs which
may have arisen.
Delivery of report. Once reporting has been completed, the
pathologist digitally signs the report using his/her own personal
certificate. In this way, the report produced assumes medicallegal value and is automatically sent to the department that
made the request and to the company clinical repository in order
to add to the patient’s digital medical records.
Filing/Storage. In order to correctly catalogue the items stored
246
in the Anatomical Pathology service, the tracking system allows
the operator in charge to indicate the storage position of the
bio-cassettes of a particular case, and any filing of the relative
slides (and therefore their presence in the histological file), by
reading the two-dimensional barcode printed directly onto the
support. It also allows specification of any delivery of the slide
or relative inclusions for consultancy, with an indication of
staff, date and time.
Monitoring of case progress. The tracking system allows
monitoring of the stage of processing and progress at any time,
and also checking of details of the relative bio-cassettes and
slides. In particular, the operator has a template on which the
various processing phases of the registered samples are specified, with indication of the date, time and staff that carried out
each operation. A summary of the supports generated and used
for each case is available, which specifies the entire procedure
completed for each bio-cassette and slide, also stating the containers that were used during the various processing phases.
able for in situ hybridization analysis; finally, the “penetration
speed” of ethanedial/alcohol fixative was comparable, although
kidney tissues demonstrated a faster fixation with ethanedial/
alcohol fixative formalin, if considering short time of analysis.
Conclusions. The search of an alternative fixative for formalin
must be considered a goal in Pathology Laboratories and, as
demonstrated in this study, alcohol based fixative can be considered as valid substitute, from morphological, immunohistochemical and biomolecular point of views.
The ethanedial/alcohol based alternative
fixative to formalin: impact on morphology,
immunohistochemistry and in situ hybridization
Background. In the context of Biobanking, histological QC
verifies that samples are representative of the whole lesion and
suitable for research purposes, according to their pathological
attributes. In this study we performed histological QC by evaluating the reproducibility of immunomorphological features on
frozen biobank samples and matching FFPE material.
Methods. 60 cases from our Institutional Biobank were enrolled, including both primary (29 breast, 9 colorectal, 5 gastric and 5 endometrial) and metastatic tumors (6 lymphnodes
and 6 liver metastases). At collection, each tissue sample
was cut into two “mirror-matching” halves: one was reduced
into aliquotes and frozen, while the other was prepared as a
“specular” FFPE block. For each case, tumor cellularity and
MIB-1 staining were evaluated on sections from both frozen
and FFPE tissue.
Results. Concordance of tumor cellularity varied from 55% to
71.7% of cases, depending on the tolerated discrepancy. Endometrial carcinoma and liver metastases showed the highest
concordance fractions.
Concordance of Ki-67 staining ranged from 52.1% to 81.2% of
cases, depending on the allowed deviation. Metastatic tumors
showed the highest concordance. 20% of cases resulted nonassessable because of technical artefacts.
Our data show that immunomorphological features are not as
reproducible between matching frozen and FFPE tissue as expected, due to the inherent heterogeneity of tumor tissue. Concordance seems to be influenced by anatomic site, histotype and
cytoarchitectural features. The occurrence of marked staining
artefacts in 20% of cases suggests a higher lability of molecular
components compared to morphological characteristics. Therefore, mirror-matching FFPE blocks can play a role in biobanking QC procedures; however, collection and storage procedures
need to be optimized to minimize sample heterogeneity and to
improve the preservation of molecular components.
Partially funded by PONa3-00134 ONEV.
M. Nebuloni3, L. Zawada1, P. Fociani1, E. Terron1, F. Pagano1,
L. Ermellino2, A. Grindati2, A. Ferri3, P. Zerbi3
1
Anatomia Patologica, Ospedale L. Sacco, Milano, Italia; 2 Anatomia Patologica, Ist. Auxologico Italiano, Milano, Italia; 3 DIBIC, Unimi, Anat.
Patol., Ospedale L. Sacco, Milano, Italia
Background. Formalin has been used as standard fixative for
histological samples in surgical pathology since the end of the
19th century. In recent years, the search for formalin-free fixatives has become a priority because of the risk of formaldehyde
as a chemical carcinogen. The aim of this study is to compare
formalin with an alcohol-based new fixative and to evaluate the
effects of this fixative on different tissues.
Methods. Gut, thyroid, kidney, breast and lymph node human
tissue were fixed in parallel with 10% bufferen formalin and
ethanedial/alcohol based fixative (GreenFix), for 24 hours at
room temperature. Tissue were stained with HaematoxylinEosin, Giemsa, Periodic Acid Schiff-Alcian, Wilder silver
staining. Immunohistochemistry and in situ hybridization with
24 different antibodies and c-erbB2 probe respectively were
performed and interpreted. Moreover, cytoinclusions from pleural and peritoneal effusions were performed with both fixatives
and evaluated.
Kidney, colon, placenta and liver tissues were used to analyze
fixation time and fixative “penetration speed”.
Results. No differences in morphology were seen on histological and histochemical slides. Cytoinclusions and immunohistochemical analysis were also comparable; just two nuclear
antibodies (Ki67 and TTF1) required modifications on antibody
dilution and detection system in comparison with standard
protocols.
Ethanedial/alcohol fixed tissues were also found to be suit-
Histological quality control (qc) of frozen samples
using matching formalin-fixed paraffin-embedded
(ffpe) tissue – preliminary results
of our institutional biobank
E. Mattioli, E. Foglia Manzillo, V. Rubini, F. Palma, A. Paradiso,
G. Simone
Anatomia Patologica e Citodiagnostica, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italia
247
comunicazioni orali
Patologia digestiva I
Moderatori: G. Tuccari (Messina), V. Villanacci (Brescia)
Intrahepatic cholangiocarcinoma: diagnosing
molecular heterogeneity
M. Fassan1, M. Simbolo1, A. Mafficini1, A. Ruzzenente2, D. Melisi3, A. Guglielmi2, A. Tomezzoli1, G. Tortora3, P. Capelli1,
A. Scarpa1
Dipartimento di Patologia e Diagnostica, Università di Verona, Verona,
Italia; 2 Dipartimento di Chirurgia, Università di Verona, Verona, Italia;
3
Dipartimento di Oncologia, Università di Verona, Verona, Italia
1
Background. Novel biomarkers for intrahepatic cholangiocarcinoma (ICC) are needed for impacting the diagnostic workout,
prognostic stratification, and design of novel tailored therapeutic
approaches. The use of conventional techniques for a wide ICC
molecular characterization is hampered by the high costs and time
needed to assess multiple molecular alterations, and the limited
amount of tissue consisting in formalin-fixed paraffin-embedded
(FFPE) liver biopsies.
Methods. Forty FFPE ICCs were evaluated using the multigene
Ion AmpliSeq Cancer Panel to investigate the mutational status
of 50 cancer-associated genes and to characterize ICC intratumor
heterogeneity.
Results. In 22/40 (55%) samples at least one mutation was observed, including BRAF, IDH1, JAK3, KRAS, NRAS, PIK3CA,
SMAD4, STK11, and TP53. IDH1 mutations were observed in
8 cases (20.0%). Four mutations (10.0%) were identified in the
KRAS, NRAS, and TP53 genes, while PIK3CA was mutated in
three tumors (7.5%). KRAS, TP53 and PIK3CA mutations were
confirmed at Sanger. Six cancers (15%) were found to have
multiple driver gene alterations. In these cases, significant differences were observed in the proportion of alleles affected for
distinct genes, supporting the presence of intratumor molecular
heterogeneity.
Conclusions. The implementation of multigene diagnostic tests
for prognostic stratification of ICC patients and identification
of predictive markers will be a strong driver for personalized
therapy.
Kras mutant allele-specific imbalance (masi)
in colorectal cancer patients
U. Malapelle, R. Sgariglia, C. Bellevicine, C. De Luca, M. Russo,
R. Moretto*, A. De Stefano*, C. Carlomagno*, G. Troncone
Sanità Pubblica, Università di Napoli Federico II, Napoli, Italia; * Oncologia, Università di Napoli Federico II, Napoli, Italia
Background. KRAS mutations in colorectal carcinoma (CRC)
are usually heterozygous. The mutant allele may become dominant when deletion of the wild-type allele and/or copy number
gain of the mutant allele leads to mutant allele-specific imbalance
(MASI).
Methods. Sequencing electropherograms of KRAS exon 2 from
451 advantage-stage CRC patients were reviewed; MASI was
calculated measuring the ratio between mutant (MUT) and wildtype (WT) allelic peaks. Tumors with MUT allele peak lower
than or equal to the WT peakwere considered negative for MASI
(MASI-), while those with MUT peak higher than WT, positive
for MASI (MASI+). Chi square test was used to measure the correlation between categorical variables; Kaplan-Meier with long
rank test was applied to compare survival.
Results. KRAS mutant rate was 35% (158/451). Codon 12 was
mutated in 128 cases, whereas codon 13 was involved in 30
cases. On the overall MASI occurred in 29/158 cases (18,4%).
Codon 12 MASI occurred in 20/128 (15,6%) instances, whereas
codon 13 MASI occurred in 9/30 (30%) p = 0.05 Complete
clinical data were available for 48 KRAS MUT patients (MASI
+ = n.10; MASI - = n. 38)treated with first line chemotherapy.
The difference in term of OS between two patient groups was
not statistically significant (p = 0.9). KRAS MASI was more
frequent in tumors with KRAS codon 13 than with codon 12
mutations. Our data did not show association between MASI
and overall survival. Further investigation is being carried out
to investigate the clinical significance of MASI in larger series
of codon 13 MT patients.
Kras mutation testing on endoscopic biopsy
of primary colorectal cancer
G. Giuffrè, V. Barresi, A. Ieni, A. Simone, R. Scarfì, G. De Luca,
G. Branca, G. Tuccari
Dipartimento di Patologia Umana “Gaetano Barresi”, Università di Messina, Messina, Italia
Background. In patients with metastatic colorectal cancer
(CRC) KRAS mutations are predictive of nonresponse to antiepidermal growth factor receptor therapy, therefore KRAS mutation testing is considered an essential prerequisite in therapeutic
choice. Mutational analysis is carried out on surgical as well
as biopsy specimens of primary tumour or metastases. In our
experience a lower KRAS mutational rate has been found in biopsy specimens (48 %) than surgical ones (52 %) of metastatic
CRC. In order to verify if this discordance might be due to endoscopic sampling of an heterogeneous tumour tissue we have
retrospectively analysed the first endoscopic biopsy of a series
of patients who were been previously tested for KRAS mutation
on surgical sample.
Methods. From 128 patients tested at our laboratory for KRAS
mutational status on surgically resected CRC we selected 42
patients (25 M, 17 F) for which the first diagnostic endoscopic
biopsy was available in archives of our department. All samples
were formalin-fixed and paraffin-embedded; in order to verify
the presence of CRC in endoscopic sample new histopathological
sections were prepared. Successively each sample was microdissected and DNA extraction was performed by QIAamp DNA
FFPE Tissue (Qiagen). Mutant KRAS was detected using a high
sensitive mutation kit (KRAS StripAssay, ViennaLab) that identifies ten more frequent somatic mutations located in codons 12
and 13 using mutant-enriched allele-specific PCR and reversehybridization.
Results. On surgically resected CRA a mutation of KRAS was
present in 18/42 patients (42.9 %); in particular mutations in
the codons 12 and 13 were found in 16 and 2 patients, respectively. On corresponding endoscopic biopsy the diagnosis of
CRC was confirmed in all cases while KRAS mutation was
detected in 17/42 patients (40.5 %). Therefore, an underestimation of KRAS mutational status has been found in 5,6 %
of biopsy samples which are less informative than surgically
resected samples.
248
Does biopsy reliably identify grade in gastroentero-pancreatic neuroendocrine tumors?
F. Grillo, M.P. Brisigotti, M. Albertelli*, T. Celiento, D. Ferone*,
R. Fiocca, L. Mastracci
UO Anatomia Patologica, Disc, Università di Genova, AUO IRCCS San
Martino IST, Genova, Italia; * Endocrinologia, DIMI, Università di Genova, AUO IRCCS San Martino IST, Genova, Italia
Background. A new proliferation-based grading system (ENETS/WHO) has proved important in establishing prognosis and
guiding therapy in gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs). Biopsies are often performed for grading but
little is known about their reliability in assigning grade. The aim
of this study was to determine the accuracy of grade identification
in virtual biopsies of GEP-NETs.
Methods. Twenty cases of resected grade 1 (11 cases) or grade
2 (9 cases) GEP-NETs (pancreas primaries or liver metastases)
were selected and grading assigned on the whole section by
counting Ki-67-positive cells out of 2000 cells in hot spots.
Virtual biopsies (VBs) were constructed by leaving different,
randomly selected, areas of the whole section unmasked. Progressively smaller areas (ranging from 30 mm2 to 0.25 mm2) were left
unmasked, using variable lengths (0.5-20 mm) and widths (0.51.5 mm) thereby simulating biopsy material. Four VBs were performed for each biopsy area: large – 15, 20 and 30 mm2, medium
– 4, 7.5 and 10 mm2 or small – 0.25, 0.5 and 1 mm2 in each case.
The number of neoplastic cells and Ki67 index were quantified in
each VB and compared with Ki67 assessed in the whole sample.
Results. Out of all (711) virtual biopsies performed, 50.7% of
them under-graded G2 lesions. In each G2 case at least one of
the 10 virtual biopsies performed under-graded the lesion. In all
11 G1 cases all VBs of medium or large size correctly identified
grade. In 1, G1 case, the ultra-small virtual biopsies (0.25 mm2)
over-graded the case as G2.
Conclusions. Clinicians must be aware that grade identified on
biopsy material can under-grade G2 cases (likely due to intratumor heterogeneity) or over-grade G1 cases (when little tissue is
available for evaluation). This may prove important in the clinical
management of patients as choice of therapy depends ever more
so on grade.
The crucial role of intestinal metaplasia
in the definition of barrett esophagus:
an essential factor to predict the risk of dysplasia
and cancer development
M. Salemme, C. Baronchelli, S. Manenti, R. Cestari , G.P. Cengia*, V. Villanacci
*
Anatomia Patologica, Spedali Civili, Brescia, Italia; * Endoscopia Digestiva, Spedali Civili, Brescia, Italia
Background. The histological definition of Barrett esophagus
(BE) is still controversial: in USA/Europe it is defined as the
presence of specialized intestinal metaplasia in the esophageal
epithelium 1, while in Japan/UK the definition of BE do not require the presence of intestinal metaplasia 2 3. Many authors have
tried to predict the risk of dysplasia/cancer development on the
basis of the presence/absence of intestinal metaplasia 4-6, stressing
the need of a clear definition of BE to plan an adequate surveillance program.
Materials and methods. We selected 647 cases with histological diagnosis of BE performed through an adequate biopsy sampling 7 at Endoscopy Unit of Spedali Civili in Brescia between
2000 and 2012. All cases were divided in two groups according
to the presence/absence of intestinal metaplasia on histology.
For each patient we analyzed all subsequent histological reports
performed during the follow-up period.
Results. Among 647 patients, we detected 537 cases with intesti-
nal metaplasia (IM+) and 110 cases without intestinal metaplasia
(IM-). During the follow-up period in IM- group none of the
patients developed dysplasia/cancer, while 72 patients belonging
to the IM+ group showed histological progression of the disease;
among these, 40 patients developed dysplasia, while 32 patients
developed dysplasia and subsequently cancer.
Conclusion. The majority (83%) of BE cases showed on histological examination the presence of intestinal metaplasia; in only
few cases (17%) this morphological element was absent. None
of IM- group patients developed dysplasia/cancer, while 13,4%
of IM+ patients showed histological progression towards dysplasia and/or cancer, emphasizing how the presence of intestinal
metaplasia gives an increased risk to malignant progression. The
correct definition of BE is therefore based on the histological
identification of intestinal metaplasia, an essential factor for the
progression towards dysplasia and cancer.
References
1
Sampliner RE; Practice Parameters Committee of the American
College of Gastroenterology. Updated guidelines for the diagnosis,
surveillance, and therapy of Barrett’s esophagus. Am J Gastroenterol
2002;97:1888-95.
2
Playford RJ. New British Society of Gastroenterology (BSG) guidelines for the diagnosis and management of Barrett’s oesophagus. Gut
2006;55:442.
3
Aoki T. Report of Research Committee on Definition of Barrett’s
Esophagus. Chiba: Japanese Society of Esophageal Diseases 2000.
4
Chandrasoma P, Wijetunge S, DeMeester S, et al. Columnar-lined
esophagus without intestinal metaplasia has no proven risk of adenocarcinoma. Am J Surg Pathol 2012;36:1-7.
5
Takubo K, Aida J, Naomoto Y, et al. Cardiac rather than intestinaltype background in endoscopic resection specimens of minute Barrett
adenocarcinoma. Hum Pathol 2009;40:65-74.
6
Riddell RH, Odze RD. Definition of Barrett’s esophagus: time
for a rethink--is intestinal metaplasia dead? Am J Gastroenterol
2009;104:2588-94.
7
Reid BJ, Blount PL, Feng Z, Levine DS. Optimizing endoscopic biopsy detection of early cancers in Barrett’s high-grade dysplasia. Am
J Gastroenterol 2000;95:3089-96.
The Gistogram: an at-a-glance graphic
presentation of the growing gist complexity
R. Ricci, A.P. Dei Tos*, G. Rindi
Anatomia Patologica, Università Cattolica del S. Cuore - Policlinico
“A. Gemelli”, Roma, Italia; * Anatomia Patologica, Ospedale regionale
di Treviso, Treviso, Italia
Background. GISTs are the most common mesenchymal tumors
of the GI tract. The groundbreaking discovery of GIST KIT mutations and KIT expression in 1998 rendered GISTs a paradigm of
molecular targeted therapies. The apparently linear pathogenesis
of GISTs opened the way to their successful treatment with the
TKI imatinib. Since then, an unexpected complex biology was
evidenced, eventually revealing the GIST rather heterogeneous
nature and the possible different diagnostic and therapeutic approaches. The identification of KIT-alternative molecular transforming pathways indicated new diagnostic clues and lead to
the development of drugs alternative to imatinib. To represent
the growing of genotype and phenotype evidence on GIST in
its increasing complexity we built up a progressive graph: the
“GISTogram”.
Methods. In the GISTogram a square represents the totality of
GISTs, and each feature is represented by a rectangular area proportional to the fraction of GISTs bearing it. The % values are set
by averaging quantitative data resulting from the recent literature.
The IHC features are represented by black and white textures, and
the genotype ones by colors.
Results. The descriptors are placed within the GIST square so
that overlaps generate mixed textures and/or colors, representing
features that may coexist in a single case. Separate areas indicate
249
comunicazioni orali
mutually exclusive descriptors. Although the term “WT GISTs”
is mostly used to classify GISTs without mutations in KIT and
PDGFRA, the so far “true” wild-type GISTs are represented by a
colorless area (accounting for about 10% of the total, coherently
with present knowledge) irrespective of textures reflecting immunohistochemical features, residual after all the progressive color
(i.e. mutation) adds. The GISTogram visually conveys most of
GIST characterizing features and their probability, either alone or
in combination, to be observed in a single case, giving at a glance
a view of the known GIST emerging complexity.
The pathway to the diagnosis of inflammatory
bowel disease (ibd): the results of a multicenter
observational study in piedmont
G. Canavese1, A. Sapino1, R. Suriani2, R. Rocca2, M. Daperno2,
I. Castellano1, R. Senetta1, P. Cassoni1, IBD group Piemonte3,
V. Villanacci4
Anatomia Patologica, AO Città della Salute della Città di Torino, Torino, Italia; 2 Gastroenterologia, Ospedale Mauriziano, Torino, Italia;
3 Piemonte, Italia; 4 Anatomia Patologica, Spedali Civili, Brescia, Italia
1
Background. The most widely followed guide lines highlight
that a reliable diagnosis of Inflammatory Bowel Disease (IBD) is
the result of a complex multidisciplinary collaboration between
gastroenterologist, endoscopist and pathologist: hence an accurate diagnostic approach is crucial for avoiding inappropriate
treatment and ineffective clinical management in patients that
have been investigated for IBD. Our aim was to evaluate the adherence to the current guide lines of a set of clinical, endoscopic,
and histopathological parameters in routine diagnosis of IBD in
Piedmont (Italy)
Methods. Histopathological reports and endoscopic reports if
available were collected from 311 patients of 11 centres. Data on
the clinical an endoscopic information, the sites of the endoscopic
sampling, the histopathological variables and the diagnostic conclusions present in each single report were analysed according to
the European Crohn’s and Colitis Organization requirements 1 2.
Results. The percentage of cases that satisfied the standards of
quality are summarised as follows: in 16% of the cases the timing
of the endoscopy (first diagnosis/follow up) was not reported in
the request form. The information about the symptoms, current
therapies and the onset of the disease was present in 24%, 15%
and 12% of the cases, respectively. Data on the laboratory examinations and other instrumental tests were accessible in a negligible number of cases (< 5%). In 10% of the records the standard
of sampling quality were fulfilled, and biopsies were performed
from ileum and each bowel district. In none of the cases the
biopsies were oriented on acetate strips. With regard to the histological examination, cryptitis/crypt abscess, crypt architectural
distortion, ulcers and basal plasmocytosis were described in 56%,
38%, 31% and 3% of the reports, while the final diagnosis was
conclusive for IBD in 47% of the cases.
This preliminary study shows that the standards of quality indicated by the international guide lines are rarely met in the clinical
practice in our cohort. Despite the poor adequacy of the information about clinic and endoscopy and the slight occurrence of suitable sampling, a conclusive diagnostic classification is achieved
in about a half of the records.
References
1
Stange EF, Travis SP, Vermeire S, et al.; European Crohn’s and Colitis
Organisation. European evidence based consensus on the diagnosis
and management of Crohn’s disease: definitions and diagnosis. Gut
2006;55(Suppl. 1):i1-15.
2
Stange EF, Travis SP, Vermeire S, et al.; European Crohn’s and Colitis
Organisation (ECCO). European evidence-based Consensus on the
diagnosis and management of ulcerative colitis: Definitions and diagnosis. J Crohns Colitis 2008;2:1-23.
Multiple inflammatory fibroid polyps, gists
and cd34-positive “plaque-like fibrous
hyperplasia” in a kindred bearing a novel germline
mutation in exon 14 of pdgfra
M. Martini1, T. Cenci1, P. Lanza1, F. Sicoli2, A. Larghi2, G. Rindi1, A. Cassano3, R. Persiani2, L.M. Larocca1, R. Ricci1
Anatomia Patologica, Università Cattolica S. Cuore-Policlinico “A. Gemelli”, Roma, Italia; 2 Scienze Chirurgiche, Università Cattolica S. CuorePoliclinico “A. Gemelli”, Roma, Italia; 3 Oncologia, Università Cattolica
S. Cuore-Policlinico “A. Gemelli”, Roma, Italia
1
Background. GISTs and inflammatory fibroid polyps (IFPs) are
among the most frequent mesenchymal tumors of the digestive
tract; both of them can be alone or multiple, can be part of various
syndromes, are often immunoreactive for CD34 and can harbor
PDGFRA mutations. Germline PDGFRA mutations have been
rarely reported, at the best of our knowledge only twice in familial form. So far, the possible relationships between GISTs and
IFPs remain an intriguing issue. Recently, Carney and Stratakis
reported CD34-positive fibrous tumors of uncertain classification
in association with GISTs and lipomas in a patient harboring a
germline mutation in PDGFRA exon 12.
Methods. CD117 (DAKO), DOG1 (Spring Bioscience) and
CD34 (Novocastra) were used for IHC. DNA was extracted using the QIAamp tissue kit (Qiagen) from ~100% tumor fragments
or normal gastric wall. KIT (ex. 9, 11 13, 17) and PDGFRA (12,
14, 18) were amplified and sequenced (ABI PRISM 3100-Avant
Genetic Analyzer, Applied Biosystems).
Results. We describe a kindred with familial IFPs bearing a
germline PDGFRA exon 14 mutation: P653L (so far unreported);
in one individual, two gastric GISTs and CD34-positive fibrous
tumours, which we named “plaque-like fibrous hyperplasia”
(PLFH), likely akin to those described by Carney and Stratakis,
were also present. Along with describing the clinical scenario
depending on this novel P653L PDGFRA mutation, we exploited
this this exceptional “baseline” genetic setting investigating the
additional molecular events involved in the arousal of IFP, GIST
or PLFH, looking into their possible relationships. Beside this, we
highlight a series of clinical, pathological and molecular features
overlapping with described cases of intestinal neurofibromatosis
(i.e. NF type 3b), postulating that this syndrome is actually a
disease with autosomal dominant transmission depending on
germline PDGFRA mutations, characterized by multiple gastrointestinal mesenchymal tumors (mostly IFPs, sometimes GISTs).
High-throughput mutation profiling identifies
novel molecular dysregulations in high-grade
intra-epithelial neoplasia and early gastric cancers
M. Fassan1, M. Simbolo1, E. Bria2, A. Mafficini1, S. Pilotto2,
P. Capelli1, S. Pecori1, C. Luchini1, G. De Manzoni3, A. Scarpa1
Dipartimento di Patologia e Diagnostica, Università di Verona, Verona,
Italia; 2 Dipartimento di Oncologia, Università di Verona, Verona, Italia;
3 Dipartimento di Chirurgia, Università di Verona, Verona, Italia
1
Background. Intestinal-type gastric adenocarcinoma (GC) is the
final step in a sequence of genotypic disruptions and phenotypic
alterations involving long-standing gastritis, gastric atrophy,
neoplastic intraepithelial transformation (intraepithelial neoplasia
[IEN]; formerly known as dysplasia), and invasive cancer. There
are still no molecular markers available to enable the distinction
between high-grade IEN (HG-IEN) and early invasive neoplasia.
Methods. HG-IEN and invasive GC areas were manually microdissected from a consecutive series of 15 surgically treated early
GC. Forty ng of DNA were used for multiplex PCR amplification
using the Ion AmpliSeq Cancer Panel (Life Technologies) that
explores selected regions of 50 cancer-associated genes.
Results. In GC samples, 12/15 presented at least one mutation
among the 50 investigated genes, and 6 of these showed multiple
250
driver gene alterations. TP53 mutations were observed in 9 cases;
3 mutations were identified in APC, while ATM and STK11 were
mutated in 2 tumors. Seven HG-IEN lesions showed an identical
mutational profile to their matched GC and 13 mutations observed in the APC, ATM, FGFR3, PIK3CA, RB1, STK11, and
TP53 genes were shared by both non-invasive and early invasive
lesions. CDKN2A, IDH1, MET, and RET mutations were observed only in the invasive counterpart.
Conclusions. The compatibility of next-generation sequencing
technologies with formalin-fixed and paraffin embedded tissues
represents a strong driver for the identification of novel molecular
biomarkers of cancer progression in gastric mucosa. This will lead
to the development of new molecular-based secondary prevention
approaches in the selection of patients at high risk for gastric cancer.
Histological grading based on the count of poorly
differentiated clusters in pre-operative biopsy
predicts nodal involvement and ptnm stage
in colorectal cancer patients
G. Branca1, L. Reggiani Bonetti2, A. Ieni3, L. Baron4, G. Tuccari5,
V. Barresi6
Patologia Umana, Policlinico G. Martino, Messina, Italia; 2 Attiv. Integr. di Lab., Anat. Pat. e Med. Leg., Policlinico Universitario Di Modena,
Modena, Italia; 3 Patologia Umana, Policlinico G. Martino, Messina, Italia; 4 UOC Anatomia Patologica, Ospedale S. Leonardo, Castellammare
di Stabia (NA), Italia; 5 Patologia Umana, Policlinico G. Martino, Messina, Italia; 6 Patologia Umana, Policlinico G. Martino, Messina, Italia
1 Background. Though histological grading is commonly assessed
in colorectal cancer (CRC) preoperative biopsy, its clinical impact
is limited by poor accuracy compared with the final resection
specimen and low inter-observer reproducibility. A grading based
on the number of poorly differentiated clusters (PDC) as the main
criterion was recently shown to be more reproducible and clinically
informative than conventional grading in CRC. Hence, the present
study aimed to investigate the reproducibility, accuracy and predictive value on lymph node status or pTNM stage, of this novel grading system in CRC tissue from preoperative endoscopic biopsy.
Methods. Grading based on the counting of PDC was assessed in
one hundred and sixty-three CRC endoscopic biopsies and corresponding surgical specimens.
Results. 93% of the biopsies could be graded with this system,
with a good interobserver agreement. In the comparison with
the surgical specimens, 75% of CRCs were correctly graded in
the biopsy, and 81% of poorly differentiated CRCs identified
at the time point of the initial biopsy. In addition, histological
grade based on PDC counting in the biopsy was predictive of the
lymph node involvement and pTNM stage of the CRC, and it was
significantly correlated with the presence of histological features
suggestive of malignancy (tumor budding, perineural invasion,
vascular invasion, infiltrating tumor border) in the surgical specimen. Our findings suggest that a grading system based on the
quantification of PDC is feasible in the majority of CRC endoscopic biopsies. In view of its good reproducibility, accuracy and
predictive value on the anatomical extent of the disease, it may be
taken into account value for decision making in CRC treatment.
Mgmt promoter methylation status in colorectal
cancer brain metastases
G. De Maglio1, S. Cernic1, E. Masiero1, M. Casagrande2,
M. Guardascione2, G. Aprile2, M. Skrap3, C.A. Beltrami4, G. Fasola2, S. Pizzolitto1
Soc Anatomia Patologica, AOU S.M. della Misericordia, Udine, Italia;
Dipartimento di Oncologia, AOU S.M. della Misericordia, Udine, Italia;
3
Soc Neurochirurgia, AOU S.M. della Misericordia, Udine, Italia; 4 Istituto di Anatomia e Istologia Patologica, AOU S.M. della Misericordia,
Udine, Italia
1
2
Background. Loss of MGMT protein expression due to promoter
hypermethylation is reported in 30-40% of colorectal carcinomas.
Ongoing phase II clinical trials are testing the activity of alkylating agents, suggesting that their usefulness is limited to colorectal
patients with MGMT methylation. Aim of this retrospective study
was to evaluate MGMT promoter methylation in brain metastasis
from colorectal cancer and their corresponding primary tumors.
Methods. We identified a cohort of 53 consecutive brain metastasis from colorectal cancer diagnosed in the period 1998-2013 in
our Surgical Pathology Department. We retrieved from archive
formalin-fixed, paraffin-embedded samples and matched primary
tumors, when available. Concordance analysis were performed
for 39 patients.
After genomic DNA extraction and bisulfite conversion, MGMT
promoter methylation status was assessed by pyrosequencing using a commercially available kit (MGMT plus®, Diatech
Pharmacogenetics) on a PyroMarkTM Q96 ID system (Qiagen)
with PyroMark CpG (Qiagen) software. The test is designed to
detect and quantify methylation level in ten CpG sites in exon 1
of MGMT gene.
Results. MGMT methylation was found in 34 (64.2%) brain
metastasis. Discordant results between metastatic and primary
lesions were found in 6 (15.4%) cases: 3 were methylated on the
primary site but not in the brain lesions while 3 cases acquired the
epigenetic modification during tumor progression.
Survival after brain surgery was calculated (median survival 163
vs 193 days) and revealed not to be statistically influenced by
MGMT promoter methylation.
Epigenetic MGMT promoter methylation was a common event
in our cohort of patients; this rate was higher than previously
reported for colorectal cancers.
The high concordance between primary and secondary lesions
suggests the hypothesis that colorectal cancer patients developing
brain metastasis could have a their own specific molecular and
epigenetic profile.
Interobserver reproducibility in the interpretation
of columnar lined esophagus
L. Mastracci1, N. Piol1, L. Molinaro2, F. Pitto2, C. Tinelli3,
A. De Silvestri3, F. Sarocchi1, F. Grillo1, ABRAM Study Group
1 Disc, Pathology Section, IRCCS S. Martino-IST, Genova, Italia; 2 Dep
of Biomedical Sciences and Human Oncology, AO Città della Salute e
della Scienza, Torino, Italia; 3 Clinical Epidemiology and Biometric Unit,
IRCCS S. Matteo, Pavia, Italia
Objective. Histology is mandatory for the confirmation of
endoscopically suspected esophageal metaplasia (ESEM) but
terminological confusion regarding the histological interpretation
of columnar lined esophagus (CLE) has kept pathologists wading
in murky waters for many years. Aim of this study is to evaluate
interobserver variability in the interpretation of CLE between
pathologists.
Methods. Thirty pathologists (10 gastrointestinal pathologists;
10 general pathologists; 10 pathology trainees) distributed nationwide and working in community and teaching hospitals
were invited to review three 10-case sets of CLE. A first set was
coupled with endoscopic description lacking a precise evaluation of ESEM extent. Endoscopy with detailed description and
extent of ESEM by using Prague CM criteria was provided in the
second and third steps. Moreover, participants were required to
use national guidelines (Fiocca et al. DigLivDis 2011;43 Suppl
4:S319-30) for evaluation of the third set. Agreement was statistically assessed using Randolph’s free-marginal multirater kappa.
Results. The overall concordance was low (K = 0.377, CI 0.339
-0.456), with better results among GI pathologists (K = 0.418)
compared to general pathologists (K = 0.378) and trainees
(K = 0.327). Concordance was lower for the first set (K = 0.263)
compared to the second (K = 0.399) and the third set (K = 0.463).
251
comunicazioni orali
The trainee group improved more (from K = 0.173 in the first step
to K = 0.425 in the third step) than GI and general pathologists.
Similar concordance rates were obtained by all three groups in
the third step. In general major problems in interpretation of CLE
were observed when only cardia or oxyntic-type epithelia were
present, mainly when a shorter extension of ESEM or an irregular
Z-line were endoscopically referred.
Conclusion. Precise endoscopic description and use of guidelines
increase consistency in interpretation and reporting of CLE in
esophageal biopsies; agreement is in general low; agreement is
worse when short ESEM or irregular Z-line are described in the
absence of intestinal metaplasia.
Appendix - ABRAM (Assessement of Barret and its Reproduc-
ibility with Aim on Management) Study Group participants: Alò
PL (Frosinone), Al Omoush TMM (Trieste), Asioli S (Forlì),
Buffelli F (Bari), Conforti F (Catanzaro), Cornaggia M (Paderno
Dugnano), Cossu S (Nuoro), D’Armiento FP (Napoli), De Marco
L (Reggio Emilia), Fiocca R (Genova), Foscolo AM (Verbania),
Fraternali Orcioni G (Genova), Ingravallo G (Bari), Locatelli F
(Bologna), Luinetti O (Pavia), Marchio C (Torino), Montinari
E (Ferrara), Melchiorri L (L’Aquila), Messerini L (Firenze),
Migliora P (Vercelli), Pizzi M (Padova), Rugge M (Padova),
Saragoni L (Forlì), Tamponi E (Cagliari), Tomezzoli A (Verona),
Trisolini MP (Lecce), Trombatore M (Palermo), Vanoli A (Pavia), Vellone VG (Campobasso), Villanacci V (Brescia).
Patologia Digestiva II
Moderatori: F.M. Vecchio (Roma), L. Saragoni (Forlì)
The chromatin remodeling component, ini1
influences aggressive behavior of colorectal
cancer through a gene signature mapping
to chromosome 22
High PER-2 Expression associated to low EGFR,
Erβ1, Erβ2, b-catenin, miR-206 expression represent
a biological profile predictive of chemotherapy
response in 59 metastic colorectal cancer
A. Remo1, M. Pancione2, C. Zanella1, L. Sabatino2, C. Laudanna2,
A. Di Blasi3, M. Ceccarelli2, R. Vendraminelli1, E. Manfrin5,
V. Colantuoni2
E. Melucci1, A. Torsello2, A. Ruzzo3, I. Sperduti4, M. Zeuli2,
A. Gelibter2, V. Ferraresi2, M. Diodoro1, C. Garufi2, M. Mottolese1
Pathology, “Mater Salutis”, Legnago (VR), Italia; 2 Sciences and Technologies, University of Sannio, Benevento, Italia; 3 Oncology and Pathology, “Rummo”, Benevento, Italia; 4 Pathology, University of Verona, Verona, Italia
1
Background. INI1 (Integrase interactor 1) is the most studied
subunit of chromatin remodeling complexes, very little is known
about its role in the colorectal cancer (CRC) progression. We hypothesized that its assessment might predict aggressive behavior
of tumors as a novel clinical biomarker.
Methods. We examined INI1 protein expression in 134 cases of
CRCs and 60 matched normal mucosa by using tissue microarrays, and categorized the results according to mismatch repair
(MMR) and CpG island methylator phenotype and biomarkers
of tumor differentiation, CDX2, CK20, Vimentin and p53. The
prognostic significance was confirmed in two CRC independent
series of microarrays.
Results. Herein, we show that negative INI1 expression (11 %
of CRCs) associates with, lack of CDX2, poor differentiation,
liver metastasis and shorter patients’ survival regardless of MMR
or tumor stage. Unexpectedly, even CRCs, displaying diffuse
nuclear INI1 staining (33%), show an adverse prognosis and
vimentin over-expression, in comparison to the group with low
expression (56%). Negative effects of INI1 on metastatic behavior are enhanced combining p53 status. By interrogating global
gene expression from two independent cohorts of 226 and 146
patients, we validated the prognostic results, and identified a gene
signature characterized by INI1 deregulation. Notably, the top
genes of the signature (BCR, COMT,MIF) mapping repeatedly
on the long arm of chromosome 22 closely associated to INI1.
Our findings suggest that, dysregulation of INI1 and genetic hotspots associated to chromosome 22, might play an important role
in the metastatic behavior. Its detection in CRC may be useful as
a novel biomarker.
1
Anatomia Patologica, Istituto Regina Elena, Roma, Italia; 2 Oncologia
Medica a, Regina Elena, Roma, Italia; 3 Biologia Molecolare, Università,
Urbino, Italia; 4 Statistica, Istituto Regina Elena, Roma, Italia
Background. Dysfunction and alterations of the circadian clock
genes are involved in tumor progression. The principal circadian
clock genes are the period genes PER-1, PER-2, PER-3. In particular, PER-2 loss induces β-catenin and cyclin-D1 activation in
colorectal cancer cell lines and in experimental in vivo models.
In metastatic colorectal cancer (mCRC), PER-2 loss is associated
to a worse prognosis. The aims of our study was twofold: 1. to
identify a PER-2 biological profile related to proliferative activity
and treatment response 2. to analyze the miRNA profile related
to clock genes.
Methods. In 59 mCRC patients treated with first line chronomodulated triplet combination (Irinotecan+Oxaliplatin+Folinic
Acid+5-Fluorouracil) ± Cetuximab, we evaluated, by immunohistochemistry (IHC), the expression of PER-2, EGFR, ERβ1,
ERβ2, Cyclin D1, β-catenin and, by direct DNA sequencing,
the mutations of K-Ras and B-Raf genes. The expression levels
of miRNAs -206, -132, -192, -194, and -219 were examined by
quantitative PCR. Results. A higher percentage of EGFR (73% p>0.0001), ERβ1
(77%, p = 0.07), ERβ2 (88.5%, p > 0.0001), Cyclin D1 (69.2%,
p = 0.06) and β-catenin (84.6%, p = 0.02) expression was observed in the 26 (44%) PER-2 negative mCRC with respect
to the positive ones (33 mCRC, 56%). Concomitantly, in the
same group of PER-2 low expressing tumors, a higher miR-206,
miR-192 and miR-219 expression was observed. MCA analysis
evidenced that response to chemotherapy is associated to high
expression of PER-2, low expression of EGFR, ERβ1, ERβ2,
β-catenin and miR-206.
Conclusions. Our data confirmed that PER-2 loss in mCRC, as
already shown in in vitro data, is associated to the expression
molecules related to proliferative activity whereas the biological profile made up by high PER-2, low EGFR, ERβ1, ERβ2,
β-catenin, miR-206 expression may identify a biological profile
predictive of chemotherapy response.
252
Serrated polyposis: high incidence of a rare
condition in northern italy
Histopathological parameters predictive of fistula
after pancreatic surgery
A. Savio, N. Olivari*, R. Rodella, L. Sparano*, C. Petruzzellis*,
F. Zorzi
G. Corradi, G. Riva, N. Sperandio, M. Fassan, G. Marchegiani*,
R. Salvia*, C. Bassi*, G. Martignoni, A. Scarpa, P. Capelli
Anatomia Patologica, Fondazione Poliambulanza, Brescia, Italia; * Endoscopia Digestiva, Fondazione Poliambulanza, Brescia, Italia
Anatomia Patologica, Policlinico G.B. Rossi, Verona, Italia; * Chirurgia
Generale b, Policlinico G.B. Rossi, Verona, Italia
Introduction. Despite the fact that serrated polyposis (SP) was
first described back in 1970, it was not until the late 1990s that it
was recognised as a condition carrying an increased personal and
familial risk for colorectal cancer (CRC).
Since then, for many years a limited number of cases have been
reported by referral centres from northern Europe, USA and New
Zealand. Moreover, research over the last 20 years seemed identify people of north-western European origin as the only ethnic
group affected by SP. It is only very recently that a high prevalence of serrated polyposis has been reported in Spain. So far no
cases have been reported from Italy or other southern european
countries.
Aims&methods. In our centre, endoscopist have been trained
in recognising the appearance of sessile lesions for many years.
Similarly, sessile serrated adenomas/polyps (SSA/P) have been
identified and distinguished from innocent hyperplastic polyps
by histopathologists. However, it is only recently that a real attention of the criteria for the identification of serrated polyposis, as
defined by WHO in 2010, has been reached and shared between
histopathologists and endoscopists. Our detection strategy has
been to make a simple check of the previous histology of all the
patients found with a SSA/P, a key lesion in SP.
Results. As a result, twenty cases have been identified during
the last 30 months (January 2011-June 2013), representing about
0.1% of the patients undergoing colonoscopy (Tab. I). All our
cases were fulfilling the 1st and/or 3rd criterium for the diagnosis
of serrated polyposis according to WHO 2010. Considering the
familial history of colorectal cancer and polyposis of patients
diagnosed with SSA/P, but not entirely fulfilling the criteria for
a diagnosis of SP, could even increase the detection rate in our
centre.
Background. Postoperative pancreatic fistula is still regarded
as a major complication after surgery and it has high morbidity,
ranging from 11% to 65%. In literature, the pancreas tissue consistency has been evaluated as a parameter to predict the onset
of this complication. Our aim was to identify histopathological
parameters in pancreatic surgical margin specimens related to the
onset of pancreatic fistula.
Methods. We retrospectively investigated pancreatic surgical
margin specimens of 200 patients who underwent major pancreatic resections between March 2010 and November 2011. For
each specimen we evaluated and scored the following histopathological parameters: fibrosis (score: 0-4), adipose tissue (0-4), size
and position (central or peripheral) of Wirsung duct. The adipose
tissue score was taken from literature, while the fibrosis score
was based on the observation of the fibrotic replacement of acinar
tissue. Finally, we analyzed the correlation between the onset of
pancreatic fistula and each parameter score.
Results. From observation and evaluation of these histopathological parameters, we classified each specimen into four diagnostic categories: fibrotic pancreas (50 cases), fatty pancreas (2
cases), acinar pancreas (61 cases) and pancreas with decreased
acinar component (87 cases). In the former two groups, none of
the patients developed pancreatic fistula, while in the latter two
groups incidence was respectively 30% and 25%.
Surgeon’s evaluation of hard pancreas associated with fibrosis
is a good indicator of no risk of pancreatic fistula development.
However, soft pancreatic consistency is not necessarily associated with this complication. Just a high acinar component is
related to the risk of pancreatic fistula, suggesting the important
role of the pathologist in identifying patients who may develop
fistula.
Tab. I. Details of twenty cases with serrated polyposis diagnosed
between January 2011 and June 2013.
AGE
N. CASES
First SSA/P
before
2011
CRC
Associated
dyspl
and / or
adenoma
50-54
5
3
55-59
4
1
60-64
5
1
65-69
2
1
70-74
3
2
75-79
1
1
1
5
0
3
0
4
2
2
1
2
0
1
Conclusion. The prevalence found in our single centre seems
even higher than that reported from north-western European
centres. However, this difference may not represent a real higher prevalence of this condition, but is more probably the consequence of its previous under-recognition in our geographic area.
In fact, the criteria for the diagnosis of an SP have been met with
a cumulative count of the polyps previously resected in 9 of our
13 cases. The belief that serrated polyposis was exceedingly
rare, or even absent in our country could have played a role
in lowering our attention to this condition. In our experience a
focused attention by histopathologists and endoscopists plays a
key role in increasing rates of SP detection.
Evidence for multipotent endodermal stem/
progenitor cell populations in human gallbladder
D. Bosco1, G. Carpino2, V. Cardinale3, R. Gentile3, R. Semeraro3,
P. Berloco4, M. Nuti1, L. Reid5, D. Alvaro3, E. Gaudio5
1 Medicina Sperimentale, Sapienza, Roma, Italia; 2 Scienze delle Salute,
Foro Italico, Roma, Italia; 3 Scienze e Biotecnologie Medico-Chirurgiche,
Sapienza, Roma, Italia; 4 Chirurgia Generale e Specialistic Paride Stefanini, Sapienza, Roma, Italia; 5 School of Medicine, University of North
Carolina, Chapell Hill, NC, Usa 5 Anatomia, Istologia, Med. Legale e
App. Locomotore, Sapienza, Roma, Italia
Background and aims. Multipotent stem/progenitor cells are
found in peribiliary glands throughout human biliary trees and
are able to generate mature cells of hepato-biliary and pancreatic
endocrine lineages. The presence of endodermal stem/progenitors
in human gallbladder was explored.
Methods. Gallbladders were obtained from organ donors and
laparoscopic surgery for symptomatic cholelithiasias. Tissues or
isolated cells were characterized by immunohistochemistry and
flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule)
cells were immunoselected by magnetic microbeads and plated
onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored
for differentiation to specific adult fates. In vivo studies were
conducted in an experimental model of liver cirrhosis.
Results. The gallbladder does not have peribiliary glands, but it
has stem/progenitors organized instead in mucosal crypts. These
can be isolated by immune-selection for EpCAM. Approximately
10% of EpCAM+ cells in situ and of immunoselected EpCAM+
253
comunicazioni orali
cells co-expressed multiple pluripotency genes and various stem
cell markers; other EpCAM+ cells qualified as progenitors.
Single EpCAM+ cells demonstrated clonogenic expansion ex
vivo with maintenance of stemness in self-replication conditions.
Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes,
or glucose-responsive, insulin/glucagon-secreting neoislets. Ep-
CAM+ cells transplanted in vivo in immune-compromised hosts
gave rise to human albumin-producing hepatocytes and to human
Cytokeratin7+ cholangiocytes occurring in higher numbers when
transplanted in cirrhotic mice.
Conclusions. Human gallbladders contain easily isolatable cells
with phenotypic and biological properties of multipotent, endododermal stem cells.
Patologia Polmonare, Molecolare e Paleopatologia
Moderatori: D. Vitolo (Roma), G. Fornaciari (Pisa)
Comparison of a new multiplexed multigene
approach with pyrosequencing in diagnostic
molecular pathology
G. De Maglio, E. Masiero, S. Cernic, S. Pizzolitto
Soc Anatomia Patologica, AOU S.M. della Misericordia, Udine, Italia
Background. As cancer research promote characterization of
individual diseases based on molecular profiles, it is becoming
essential to interrogate multiple genes for each tumour. This
study explored the use of a multiplexed multigene mutation detection panel using Sequenom MassARRAY® Platform compared
to pyrosequencing method for EGFR, KRAS and BRAF testing.
Methods. Formalin-fixed and paraffin-embedded sample of 142
cases, previously tested for mutational status of KRAS/BRAF
(46), BRAF (13) or EGFR/KRAS (83) by pyrosequencing, have
been blinded analyzed on Sequenom MassARRAY® System.
Pyrosequencing assays were performed using CE-IVD commercially available kits (Diatech Pharmacogenetics, Italy), according
to manufacturer’s instructions.
For mass array spectrometry, EGFR, KRAS and BRAF mutations
were simultaneously tested using CE-IVD Myriapod® Cancer
status kit (Diatech Pharmacogenetics, Italy) on Sequenom MassARRAY® System (Sequenom, Inc. California).
Results. Among all samples, 135 (95.1%) had concordant results with the two methods. In 4 samples, Sequenom® defined
mutations that were indeterminate by pyrosequencing. In 3 cases
pyrosequencing revealed rare mutations that Sequenom® kit is not
designed to detect.
Sequenom® needed 16uL of DNA (2.5-25ng/uL) for 3 gene assessment, compared to the requested amount of 15, 50 and 5 uL (5-20ng/
uL) for KRAS, EGFR and BRAF for pyrosequencing, respectively.
The main usefulness of Sequenom® System was the chance to
obtain much more results, with a sensitivity up to 2.5%, using a
great less amount of DNA. This is significantly helpfulness for
small biopsies and for cytological samples with very low amount
or with low enrichment of tumor cells.
The simultaneously detection of three genes in the same run, even
requiring sample batching, provided a high throughput flexibility.
This study demonstrated that the Sequenom MassARRAY® System is a robust target method for detection of known mutations.
Reliability of thinprep processed cytological
samples in detecting egfr mutations in lung
adenocarcinomas by direct sequencing and realtime pcr
V. D’Alicandro, S. Buglioni, E. Melucci, C. Ercolani, B. Casini,
C.A. Amoreo, P. Visca, M. Filippetti*, M. Mottolese
Anatomia Patologica, Istituto Nazionale Tumori Regina Elena, Roma,
Italia; * Endoscopia Toracica, Istituto Nazionale Tumori Regina Elena,
Roma, Italia
Background. Specific EGFR tyrosine kinase inhibitors (TKIs)
are recommended as first-line therapy in patients with non-small
cell lung cancer (NSCLC) that have active EGFR mutations. Although cytology is a very common approach to evaluate patient
eligibility to TKIs, the mutational test procedures on cytological
samples, due to the limited number of tumor cells, still needs to
be optimized, standardized and validated. In this context, the use
of sensitive methods to detect EGFR mutations may be of pivotal
clinical importance. The aims of this study were: 1) to verify
whether ThinPrep (TP) method may be helpful in standardizing
the molecular analysis in NSCLC cytological samples 2) to set
out the feasibility of real time PCR to assess EGFR mutations in
cytological TP specimens.
Methods. DNA was extracted from 132 TP samples including 30
computerized tomography-guided lung, 44 ultrasound-guided supraclavicular or mediastinal lymph nodes, 20 visceral metastases
fine needle aspirates, 21 pleural effusions and 17 bronchial washings. EGFR mutations were detected by direct sequencing (DS,
exons 19 and 21) and real time PCR (qPCR, exons 18, 19, 20, 21).
Results. The DNA, successfully extracted from all the samples
(mean 56,4 ng/ml, range 12-210 ng/ml), presented a mean purity
(A260/280) of 1.70 (range 1.56-1.90). The overall specimen insufficiency rate was higher by DS compared to qPCR (14.5% vs
12.7%). In the series of 59 valuable cases analyzed by DS, we
found 6 (10.2%) EGFR mutations whereas 13 EGFR mutations
(23.6%) were observed in the 55 NSCLC analyzed by qPCR. Our
data indicate that DNA extracted from TP samples is characterized by a good quality higher than that observed in 60 randomly
selected pleural or lung biopsy specimens. Furthermore, qPCR
enabled detection of EGFR mutations in samples for which
conventional Sanger sequencing was not informative due to low
tumor content.
Externalizing cytological samples for egfr testing
is fraught with a high rate of inadequate samples
C. Bellevicine, U. Malapelle, S. Russo, F. Pepe, C. De Luca,
M. Salatiello, A. Bianco, R. Sgariglia, G. Troncone
Background. Testing for epidermal growth factor receptor (EGFR) mutations is required in advanced non small cell lung cancer
(NSCLC). While in the United States there are many laboratories
performing mutational tests in-house, in Italy only a few pathology departments were equipped to run molecular diagnostics at
the time EGFR testing became mandatory for the prescription of
gefitinib in NSCCL patients, receiving various types of samples
from outside hospital. Here we reviewed our clinical practice ar-
254
chives and compared the sampling method to the rate of rejected.
cytological specimens.
Methods. From July 2010 to July 2012, we received 364 cytology and 318 histology samples. Exon 19 deletions and the
L858R point mutation in exon 21, detected by fragment assay
and TaqMan assay respectively, were confirmed by direct
sequencing.
Results. Overall 87.8% (599 of 682) of samples met the adequacy criteria and qualified for testing: 294 histological and 305
cytological samples. The mutation rate was similar in histology
(8.5%) and cytology samples (8.8%). Rejection rates were 7.5%
(n = 24/318) and 16.2% (59/364) for histology (all unsatisfactory
specimens were biopsies) and cytology, respectively. The sampling method of each of the 59 rejected cytological specimens is
shown in Table I.
Tab. I.
Specimen
Number
tested
Number
rejected
FNAB
235
TBNA
Pleuric
effusion
Bronchial
washing/
brushing
Sputum
Total
Results. Median age was 60 years (range 33-82); most patients
were male (63%) with advanced stage (III-IV) in 87% of cases.
Pattern of growth was assessable only in 24 cases: 11 solid, 5
acinar, 6 with signet ring morphology, 1 papillary and 1 lepidic.
Fourteen cases showed both ALK rearrangement and immunohistochemical expression of ALK and no false negative results were
observed by IHC (sensitivity: 100%). One case was positive by
IHC and not rearranged by FISH (specificity: 97%). In two cases
FISH was not available for likely pre-analytical DNA damage.
Signet-ring morphology was the most common subtype in ALK
rearranged cases (43%). There was no correlation between semiquantitative IHC score (1+, 2+, 3+) and ALK rearrangement by
FISH. In conclusion, IHC with antibody anti-ALK (clone D5F3)
can be used as a reliable screening tool for identification of
ALK rearrangements in lung ADC. An ALK testing algorithm is
recommended in ADC lung tumors to reduce costs of diagnostic
procedures.
39
Total
number
274
Per cent
rejections
14.20%
25
6
31
19.30%
24
6
30
20%
20
7
27
24.13%
1
Unit of Pathological Anatomy, AOU Pisana, Pisa, Italy; 2 Surgical, Medical, Molecular Pathology and Critical Area, AOU Pisana, Pisa, Italy;
3 Diatech Pharmacogenetics, Jesi, Italy
1
1
2
50%
305
59
364
Background. EML4-ALK translocation has been described in a
subset of patients with non-small cell lung cancer (NSCLC) and
has been shown to have oncogenic activity. Fluorescent in situ
hybridization (FISH) is used to detect ALK-positive NSCLC, but it
is expensive, time-consuming and difficult for routine application.
For these reasons, we evaluated the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for
FISH analysis and for ALK inhibitor therapy in NSCLC.
Methods. We performed FISH and IHC for ALK and mutational
analysis for EGFR and K-ras in 523 NSCLC specimens. We
conducted IHC analysis with the monoclonal antibody D5F3
and a highly sensitive detection system. We also performed a
MassARRAY-based analysis in a small subset of 11 samples to
detect EML4-ALK rearrangement.
Results. Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and Kras
mutations were identified in 70 (13.4%) and 124 (23.7%) out
of 523 tumor samples, respectively. ALK rearrangement, EGFR
and Kras mutation were mutually exclusive. Out of 523 analyzed
tumor samples, 18 (3.4%) were ALK positive by IHC. 18 samples
had concordant IHC and FISH results, and 2 ALK FISH-positive
cases failed to show ALK protein expression. In the two discrepant cases, we did not detect any mass peaks for EML4-ALK variants by MassARRAY.
In conclusion, our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.
Conclusions. Externalizing EGFR testing is fraught with a high
rate of inadequate samples. Since the primary cytopathologist is
often reluctant “to sacrifice” the morphology of malignant cells
for DNA extraction, the smear sent to centralized laboratories is
not always the “good one” and is often paucicellular. To improve
the rate of adequate samples, primary (cyto)pathologists should
be engaged as knowledgeable partners in the molecular diagnostic process.
Detection of alk rearrangement in lung
adenocarcinoma by immunohistochemistry
and fish: a comparative study
G. Bellezza, I. Ferri, M. Becce, A. Pireddu, R. Del Sordo,
R. Colella, V. Ludovini*, A. Sidoni
Department of Experimental Medicine, Section of Pathology, University of
Perugia, Italy; * Division of Medical Oncology, Perugia, Italia
Background. Approximately 3-5% of lung adenocarcinoma
harbor anaplastic lymphoma kinase (ALK) rearrangements.
Fluorescence in situ hybridization (FISH) is the standard procedure for detection of ALK rearrangement but is expensive
and time consuming. We tested a novel antibody to ALK and
compared with results by FISH to verify if immunohistochemistry (IHC) may represent a cost effective and efficient
means of screening for ALK detection in lung adenocarcinoma
(ADC).
Methods. We considered a series of 52 selected ADC previously
screened for EGFR and kRAS mutations. Histological material
was obtained by bronchial or fine-needle biopsy or surgical specimens. Twelve cases were send from neighboring hospitals. ALK
expression was assessed by using standard IHC with antibody
anti-ALK (clone D5F3, Cell Signaling) and scored as 0, 1+, 2+
and 3+, depending on the intensity of cytoplasmatic or membrane
stain. ALK rearrangement was evaluated by FISH using Vysis
Break Apart Probe Kit.
ALK rearrangement in a large series
of consecutive nsclc: comparison between a new
immunohistochemical approach and fluorescent
in situ hybridization for the screening of eligible
patients for crizotinib treatment
G. Alì1, A. Proietti1, S. Pelliccioni1, C. Niccoli2, C. Lupi1, E. Sensi1, R. Giannini2, N. Borrelli2, M. Menghi3, G. Fontanini2
Braf and alk assessment in a subset
of pulmonary enteric adenocarcinomas
A. Nottegar1, F. Tabbò2, M. Brunelli1, C. Doglioni3, C. Luchini1,
E. Gilioli1, A. Iannucci1, G. Martignoni1, G. Inghirami2, M. Chilosi1
1 Department of Pathology and Diagnostic, AO Integrata Universitaria di
Verona, Verona, Italy; 2 Department of Molecular Biotechnology, Cerms,
Torino, Italy; 3 Department of Pathology, San Raffaele Scientific Institute,
Milano, Italy
Introduction. Pulmonary adenocarcinoma with enteric differentiation is a new entity recently introduced by the International
Association for the Study of Lung Cancer (IASLC) in the clas-
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sification of lung adenocarcinomas. Emerging data demonstrate
that epidermal growth factor receptor (EGFR) and its downstream
factors KRAS and BRAF could be mutated in lung adenocarcinomas, affecting the clinical response to EGFR inhibitors. ALK
inhibitor has been approved by the Food and Drug Administration
for the treatment of ALK-rearranged non-small cell lung cancers.
Moreover, therapies targeting BRAF mutant tumors have recently
been identified.
We sought to identify targetable biomarkers in a subset of pulmonary adenocarcinomas with enteric differentiation, with focus to
BRAF mutation and ALK rearrangement.
Materials and methods. We collected a series of adenocarcinomas with enteric differentiation and CDX-2 expression. Using the DNA extracted from formalin-fixed paraffin-embedded
tumor samples, every case was tested for EGFR, K-RAS and
BRAF mutation. ALK locus was also assessed by a break-apart
FISH kit.
Results. We collected 22 adenocarcinoma with enteric differentiation. All the cases were immunoreactive for CDX-2. EGFR
mutation were observed in 9% of cases (2/22) and K-ras mutation
in 50% of cases (11/22). All the 22 tumors were wild type for
BRAF. 2/22 cases showed an ALK-translocation (9%).
We found that pulmonary enteric adenocarcinomas show an EGFR mutation rate similar to other subtypes (10-15% of NSCLC
in European population), while have a higher percentage of K-ras
mutation (50% of pulmonary enteric adenocarcinomas vs 20% of
other subtypes). In the era of personalized therapies, the majority of patients affected by pulmonary enteric adenocarcinoma
lacks the ad hoc predictive rationale for receiving the EGFR-TKI
therapy.
Our series demonstrated that patients affected by pulmonary
enteric adenocarcinoma could not benefit from therapies with
BRAF inhibitors whereas 9% of patients harbors ALK-rearrangements and may be eligible for therapies with crizotinib.
A multidisciplinary study of tissues from egyptian
canopic jars (New Kingdom, 1550-1069 a.C.)
L. Ventura1, L. Arrizza2, N. Rucci3, M. Capulli3, A. Maurizi3,
C. Mercurio1, M.C. Guidotti4, A. Teti3, G. Fornaciari5
UO Anatomia Patologica, Ospedale San Salvatore, L’Aquila, Italia;
Centro di Microscopie, Università, L’Aquila, Italia; 3 Dip. Scienze Cliniche Applicate e Biotecnologiche, Università, L’Aquila, Italia; 4 Museo
Egizio, Soprintendenza Archeologica della Toscana, Firenze, Italia; 5 Divisione di Paleopatologia, Università, Pisa, Italia
1
2 Background. Ancient Egyptians were well known for the
art of embalming. During the process, internal organs were
removed and some of them washed, dehydrated with natron,
perfumed, and stored in so-called canopic jars, buried with
the mummy. Each jar had established contents and its own
protective deity.
Methods. We studied the material from four canopic jars in
the Egyptian Museum of Florence. They were found in Thebes
and belonged to an anonymous individual of the New Kingdom (1550-1069 BC, XVIII-XX Dynasties). After binocular
stereomicroscopy (BSM), selected fragments were submitted to
rehydration and conventional histology. One sample from each
jar was imaged with microcomputed tomography (microCT)
prior to be cut and examined with scanning electron microscopy
(SEM), also with energy dispersive X-ray analysis (EDX). Additional histologic investigation was performed after methacrylate embedding.
Results. The first jar (FI2198, with a human-headed stopper,
expected to contain liver) was found to contain lung. The
second one (FI2199, baboon-headed stopper) also contained
lung as expected. Both lung specimens showed deposition of
carbon and small polarizable crystals, allowing the diagnosis
of pulmonary silico-anthracosis. The third jar (FI2200, jackal-
headed stopper, expected to contain stomach) held amorphous
material enclosing wide birefringent fibres. After methacrylate
processing the sample melted away leaving only entwined
fibers related to the linen fabrics used to wrap the organs. In
the last one (FI2201, hawk-headed stopper, expected to contain intestines) amorphous material enclosing starch particles
could be referred to intestinal content. Chemical constituents
of natron salts (sodium chloride, sulphate and carbonates)
were also identified. Our findings demonstrate that the evaluation of canopic jars contents by a multidisciplinary approach
allows identification of human organs and non-human materials, providing useful information about the diseases of ancient
Egyptians.
Application of different techniques to the
paleopathologic study of ancient renal stones
L. Ventura1, L. Arrizza2, M. Capulli3, N. Rucci3, S. Gemini Piperni3, V. Giuffra4, A. Teti5, G. Fornaciari4
Centro di Microscopie, Università, L’Aquila, Italia; 3 Dip. Scienze Cliniche Applicate e Biotecnologiche, Università, L’Aquila, Italia; 4 Divisione
di Paleopatologia, Università, Pisa, Italia; 5 Dip. Scienze Cliniche Applicate e Biotecnologiche, Università, L’aquila, Italia
1 2 Background. The renal stones found in the mummies of Pandolfo III Malatesta, Lord of Fano (1370-1427) and an anonymous
nobleman from Popoli (XVIII century) were investigated using
different techniques.
Methods. Both specimens were examined with binocular stereomicroscopy (BSM) and scanning electron microscopy (SEM),
also with energy dispersive X-ray analysis (EDX). Tiny fragments from surface and inner portions were submitted to X-ray
diffraction (XRD) analysis. Subsequently, the calculi were imaged with microcomputed tomography (microCT).
Results. The stone from Pandolfo had a mulberry-like surface
with honey brown colour and measured 12 mm in largest diameter. Along with the organic constituents (C, O, N), the following
chemical elements were detected: K, S, Si, Cl, Ca, P, Na and Ba.
The calculus was composed of ammonium acid urate (95%) and
calcium oxalate dihydrate (weddellite) (5%). Internal structure
consisted of large spheroidal crystals with different density values. In the Popoli case, the ovoidal mass with small superficial
spherical buds measured 22x16x15 mm. The cut surface showed
a central nucleus of sharp-edged crystals and concentric laminations. Detected chemical elements were: C, O, N,Ca, P, K, S, Cl,
Na. The stone composition was calcium oxalate monohydrate
(whewellite) (90%) and calcium phosphate (hydroxylapatite)
(10%). Internal structure detail revealed concentric laminations
and aggregates of similar density values. These observations
enabled us to propose an ideal protocol for the examination of
stones found in mummies and osteoarcheological material. After
BSM observation, the specimen should be imaged with microCT,
in order to trace a map of the surface and the whole calculus
and guide the following SEM-EDX measurements for elemental
analysis. Matching the results from these methods avoids destructive XRD analysis and may allow to obtain an affordable evaluation of chemical composition on the entire stone, following a
conservative approach.
When paleopathology meets cosmetics. A unique
example of nail care in a last century mummy
L. Ventura1, L. Arrizza2, G. Fornaciari3
Centro Di Microscopie, Università, L’Aquila, Italia; 3 Divisione di Paleopatologia, Università, Pisa, Italia
1
2 Background. Since the first exploration, a nice pair of hands
with clear, white nails was visible in a crypt of the church of San
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Michele Arcangelo in Sermoneta (Latina province, Lazio region).
They belonged to a partially mummified female of 32-40 years of
age at death and displayed all but one (right fifth) of the fingernails. At direct examination, these latter appeared well preserved
and diffusely coloured in white, with generally intact cuticles.
Methods. The fourth left fingernail was carefully extracted from its
bed and submitted to stereomicroscopy, scanning electron microscopy (SEM), also with energy dispersive X-ray (EDX) analysis, in
order to evaluate its features and establish the chemical composition of the white substance, following a conservative approach.
Results. Stereomicroscopy allowed to appreciate differences
between dorsal (pigmented) and ventral (unstained) surfaces, as
well as to closely inspect the nail root and the free edge contours.
SEM evidenced further details of such structures, enabling us
to select areas for EDX measurements. The following elements
were detected:
C, O, S, Mg, Cl, K, P and Ca in unstained areas of the dorsal
surface and in the ventral one, indicating the organic structures
of the nail;
O, S and Ca in the pigmented areas of the dorsal surface, suggesting the presence of calcium sulphate (CaSO4) used as a
nail polish;
Al, Fe and Si in the free edge of the nail, referred to remnants
from manicure devices.
The presence of calcium sulphate (chalk, plaster) allowed the
preservation of the fingernails, that often disappear following
death. According to the history of nail care, the chemical composition of the polish and the nail shape helped to date back the
death of the subject to the very first decades of XX century. At
that time women used to stain their nails with tinted powders,
buffing them shiny and naturally coloured, even though in this
case the procedure could be performed also after death.
Dermatopatologia e patologia della testa-collo
Moderatori: E. Maiorano (Bari)
Role of fk506 binding protein 51 in cutaneous
lymphomas
D. Russo, M. Mascolo
Department of Advanced Biomedical Sciences, University Bederico II of
Naples, Naples, Italy
Background. Over the past ten years, our knowledge of immunopathogenesis of cutaneous lymphoma (CL) has advanced
considerably, uncovering new diagnostic clues and therapeutic
targets. Although not common, skin represents the second most
frequent extra-nodal site involved by lymphoma, after the gastrointestinal tract. The CL, with Mycosis Fungoides being by far the
commonest single entity, are comprised of a wide spectrum of
tumours with a broad range of genetic, phenotypic and clinical
features with different prognosis. Heterogeneity constitutes the
major obstacle to an effective treatment for these tumours. Identifying different mechanisms that underlie the clinical heterogeneity of CL may contribute to more accurate risk stratification and
personalized treatment. During the last years, several studies have
demonstrated the link between the constitutive activation of Nuclear Factor-kβ (NF-kβ) signalling pathway and the survival of
CL cells. Recently, we have evaluated the expression of FK506binding protein 51 (FKBP51) in some human malignant tumours.
FKBP51, an immunophilin involved in gene expression, DNA
repair and replication of eukaryotic cells, controls drug-induced
NF-kβ activation in human leukaemia and influences Rx-induced
apoptosis in skin melanoma, correlating with its aggressiveness.
Methods. We evaluated the immunohistochemical expression of
FKBP51 in a proper selection of primary CL that differs in clinical behaviour, in order to determine its biological significance.
Results. Our preliminary results support the hypothesis of a
biological role of FKBP51 in primary CL. Further investigations
of a larger series of cases are in progress, to clarify whether this
protein may have an important role in CL and may be used as new
promising biomarker for this disease.
Il melanoma fish test tra delusioni e speranze
A.C. De Vanna, A. Di Blasi, G. Ferrara
Anatomia Patologica, Gaetano Rummo, Benevento, Italy
Introduzione. La diagnostica differenziale delle neoplasie melanocitiche è stata di recente approcciata mediante ibridizzazione
fluorescente in situ (FISH) con sonde per centromero del cromosoma 6 (Cep6), RREB1 (6p25), MYB (6q23) e CCND1 (11q13)
(Abbott Molecular, Des Plaines, Illinois, U.S.A.). È stato tuttavia
argomentato che, nelle lesioni melanocitarie morfologicamente
difficili, i valori di sensibilità e specificità del test sono troppo
bassi per un suo effettivo utilizzo routinario.
Materiali e metodi. Abbiamo rivalutato i risultati FISH di 19 neoplasie spitzoidi alla luce della morfologia convenzionale. Tutte le
lesioni testate, giudicate in partenza come morfologicamente inusuali o atipiche, sono state riclassificate come ‘più probabilmente
benigne’ (n = 15) o ‘più probabilmente maligne’ (n = 4).
Risultati. Con la morfologia convenzionale come gold standard,
la FISH è risultata positiva in 3/4 casi morfologicamente riclassificati come ‘più probabilmente maligni’ ed in 4/11 casi morfologicamente riclassificati come ‘più probabilmente benigni’
(sensibilità: 75%; specificità: 73.3%). Nella serie erano compresi
due melanomi spitzoidi pediatrici, uno dei quali risultato FISHpositivo.
Conclusioni. I risultati del melanoma FISH test non rappresentano un elemento che, da solo, deve indurre a cambiare una
diagnosi morfologica. Risultati migliori sono attesi dal test nella
sua nuova formulazione basata sulle sonde per 9p21, 6p25, 11q13
e 8q24.
Tumor heterogeneity in nodular melanoma
of the skin
N. Porta, C. Chiappetta, R. Zaralli, C. Puggioni, V. Petrozza,
C. Della Rocca, C. Di Ristofano
Dept. of Medical Surgical Sciences and Biotechnologies, Sapienza University of Rome, Pathology Unit, Polo Pontino, ICOT, Latina, Italy
Background. The progression of tumors develops from clonal
expansion of malignant cells secondary to genomic alterations.
Tumor genomic heterogeneity is potentially important because
it has the ability to influence the response to molecular targeted
therapies.
Mutations in the BRAF oncogene are commonly reported molecular alterations in cutaneous melanoma; the V600E mutation
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being currently the most important therapeutic target in metastatic melanoma.
Formalin-fixed paraffin-embedded (FFPE) tumoral tissue with
mutations of BRAF and NRAS in a population of primitive nodular melanoma was studied using laser capture microdissection
(LCM), isolating different areas of the primitive tumor.
Methods. Fifteen samples of FFPE nodular melanoma of the skin
were considered. BRAF (exons 11 and 15) and NRAS (exons 1
and 2) mutations were analyzed by direct sequencing of sections
of neoplastic tissue and on 3 areas of tumor derived from the
same section and isolated after LCM.
Results. Twelve/15 patients analyzed showed either BRAF and
NRAS mutated. Most samples had the BRAF mutation (11/12)
and the most frequent mutation was the V600E.
Over half of the samples (8/15, 53%) showed alterations on the
entire histological section which were present in most of the
LCM derived material from the same sample. However, in some
patients (5/15, 33%) related LCM samples evidenced mutations
which were not evident in the material derived from the entire
section. This fact has emerged analyzing both the alterations of
BRAF than - even if to a lesser extent- of NRAS.
Conclusions. In some cases, mutations in BRAF and NRAS
have emerged only in microdissected samples; LCM likely enriches the sample, thus allowing to reveal the alterations present
in neoplastic subclones that are not obvious analyzing the entire
section.
Studio preliminare sulla valutazione dello stato
mutazionale del gene BRAF sui melanomi primitivi
e metastatici e della cute mediante metodiche
immunoistochimiche e biomolecolari
G. Soda, G. Giannini
Medicina Molecolare, Azienda policlinico Umberto I, Roma, Italia
Background. In una sostanziale percentuale di pazienti con melanoma metastatico è stata individuata una mutazione puntiforme
(V600E) del gene BRAF. Allo stato attuale il gold standard per
la determinazione di tale mutazione prevede l’utilizzo di tecniche
biomoleocolari.
Recentemente è disponibile un nuovo anticorpo monoclonale
(VE1), diretto contro la specifica mutazione (V600E) della
proteina BRAF, mutazione che è diventata un importante target
terapeutico per i pazienti con malattia metastatica.
Metodi. In questo studio preliminare abbiamo analizzato 25 pazienti con melanomi metastatici e 25 con melanomi cutanei primitivi. Delle sezioni ottenute dai campioni selezionati (FFPE),
alcune sono state colorate con le normali tecniche di immunoistochimica utilizzando l’anticorpo monoclonale clone VE1”Spring-Bioscience” e con kit di rivelazione “Bond Polymer
Refine Red Detection”; altre sezioni sono state utilizzate per
l’estrazione del DNA, sottoposto a PCR e sequenziamento diretto per la determinazione molecolare dello stato mutazionale
del codone V600.
Risultati. Il confronto dei risultati sullo stato mutazionale del
gene BRAF ottenuti mediante le indagini molecolari con quelli
ottenuti con l’immunoistochimica ha mostrato una notevole
concordanza.
Correlation between p16ink4a protein expression
and radiotherapy treatment in Oropharyngeal
Squamous Cell Carcinoma
T. Addati1, M.A. Caponio1, A. Di Lauro2, G. Scognamillo3,
S. Petroni1, G. Giannone1, M. Centrone1, G. Simone1
1 Anatomic Pathology Unit, NCRC “Giovanni Paolo II”, Bari, Italy; 2 Otolaryngology Unit, NCRC “Giovanni Paolo II”, Bari, Italy; 3 Radiotherapy
Unit, NCRC “Giovanni Paolo II”, Bari, Italy
Background. Human papilloma viruses (HPV) were reported
in 25-60% of oropharynx squamous cell carcinoma (OSCC) and
HPV16-type is highly prevalent (≈ 90%) in OSCC, compared to
other HPV types. Several reports evidenced that HPV-positive
OSCC has a better clinical outcome than HPV-negative ones,
claiming a clinical predictive and prognostic role for HPV for
prevention and treatment of OSCC.
The aim of this study was: to analyze p16INK4A expression in oropharyngeal lesions and to value the follow up of patients receiving radiotherapy.
Methods. We analyzed oropharyngeal specimens of 32 patients: 17 out these were diagnosed as squamous cell carcinomas, 7 as dysplastic lesions and 8 benign lesions (negative
control), using, for immunohystochemical assay p16 INK4a
monoclonal antibody (clone E6H4, Histology V-Kit, ROCHE).
In 7 (F.U. mean 36 month) out of 17 patients follow up data
were available.
Results. P16INK4a expression, in our cohort were strongly expressed in 5 out of 17 OSCCs, in 2 of these p16INK4a expression
was positive, but its distribution pattern regarded dysplastic
areas rather then carcinomatous areas. Moreover, 3 out of
these 17 cases presented a sporadic p16INK4a immunoreaction
(IR) in superficial cells, whereas the last 9 out of 17 samples
were negative for p16INK4a expression. Regarding dysplastic
lesions, 1 out of 7 were positive for p16INK4a expression. Seven
out of 8 benign lesions were negative for p16INK4a expression.
Follow-up were data available only in 7 cases where strongly/
sporadic p16 INK4a IR was detected: longer d. f. s. was observed
3/5 positive cases and in 1/2 cases with sporadic p16INK4a IR,
whereas the other case with sporadic p16INK4a expression were
in progression. The last OSCC p16INK4a positive were still
in treatment.
According follow-up data p16INK4a expression in OSCC could be
a predictive marker of response to radiotherapy, however, it is
important to increase the number of the cases.
Reproducibility of p16 immunohistochemistry
in recurrent head and neck carcinomas
A. Punzo1, C. Mian2, E. Egarter-Vigl1, F. Vittadello1, A. Ambrosini-Spaltro2, E. Vattemi3, S. Mussari4, F. Gallmetzer5, A. Kasal2,
G. Negri2
1 Claudiana Province College, College for Health-Care Professions, Bolzano, Italia; 2 Department of Pathology, Central Hospital, Bolzano, Italia;
3 Divisione di Oncologia Medica, Central Hospital, Bolzano, Italia; 4 Divisione di Radioterapia, Ospedale S. Chiara, Trento, Italia; 5 Divisione di
Otorinolaringoiatria, Central Hospital, Bolzano, Italia
Background. Immunohistochemical evaluation of the tumor
suppressor protein p16 is a widely used technique in the first step
of the determination of HPV-associated cancers of the head and
neck. Little is known about the reproducibility of p16 in recurrent
disease, particularly after treatment.
Methods. This study included 83 cases of squamous cell cancers
with recurrences within 6 months-11 years from the first diagnosis. p16 was evaluated in all primary cancers and, if positive, also
in the biopsies of the recurrent disease.
Results. The primary tumor site was: oral cavity, oropharynx
and larynx in 40.96%, 36.14% and 22.89% of cases, respectively.
Overall, p16 was expressed in 18.07% of all tumors. After recurrence, p16 was still expressed in 86.66% (all oral cavity and
oropharynx cancers, 0% of larynx cancers) of these neoplasms. In
5 cases a radiotherapy had been performed before relapse. In 3 of
these cases p16 persisted at recurrence.
Our study shows that p16 immunohistochemistry is mostly reproducible in head and neck cancers, even in recurrent disease and
after radiotherapy.
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Micro-rna profile related to braf and kit
expression/mutational status in pigmented lesions
of the conjunctiva
S. Blandamura1, L. Alessandrini1, R. Cappellesso1, E. Bonandini2, E. Valentini1, F. Simonato1, M. Cassaro3, C. Giacometti3,
C. Camerin3, A. Fassina1
Dipartimento di Medicina (DIMED), AO Padova, Padova, Italia; 2 AO
Padova, Padova, Italia; 3 Ospedale di Camposampiero, Camposampiero
(PD), Italia
1
Background. Conjunctival pigmented lesions represent 53% of
all excised conjunctival tumors. They can be classified as nevus,
primary acquired melanosis (PAM) with or without atypia, and
melanoma. The activation of RAS-RAF-MEK-ERK pathway
constitutes an essential event in melanoma oncogenesis and
progression and BRAF and KIT mutations have been detected in
conjunctival melanomas. Recently, microRNAs (miRNA) have
been reported to play a role in the pathogenesis of malignant
melanoma.
Methods. We tested 47 cases out of 85 melanocytic conjunctival
tumors (previously investigated for KIT status) for BRAF mutation by pyrosequencing and immunohistochemistry (IHC) with
anti-human BRAFV600E antibody (mouse, clone VE1, diluition
1:50; Spring Bioscience, Pleasanton, USA). MiRNAs known to
be involved in the KIT-RAS-RAF-MEK-ERK pathway (let-7a,
miR-15a, miR-15b, miR-16, miR-214, miR-221, and miR-222)
were also analysed by qRT-PCR.
Results. Overall, 10/47 cases (21%), 7 melanomas, 2 nevi, and
1 PAM, showed positive IHC staining for BRAFV600E; among
these, BRAF mutation was confirmed by pyrosequencing only
in a melanoma. Discordant results were retested with Sanger sequencing, that confirmed former results. IHC showed high sensitivity (100%; 1/1) and good specificity (80%; 37/46) in detecting
BRAF mutated cases. Let-7a was significantly down regulated
in melanomas compared with nevi (p < 0.001). MiR-15a/b and
miR-16 expression was lower in melanomas and PAMs than in
nevi (p < 0.05). No statistically difference was found comparing
BRAF and KIT IHC expression in melanomas/PAMs and in nevi
with miRNAs’ expression.
Conclusions. BRAFV600E IHC is an accurate, rapid, and costeffective method that could be employed as first line test to
detect cases that should undergo sequencing. Let-7a, miR-15a/b,
and miR-16 expression profiles could be used (either alone or in
combination) to differentiate benign from malignant conjunctival
pigmented lesions.
Repetitive bilateral central giant cell granuloma
of the mandibular angles in women of the same
family
I. Cicchinelli1, T. Cutilli2, G. Cipolloni1, G. Calvisi3, P. Leocata1
Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita
e dell’ambiente San Salvatore, L’Aquila, Italia; 2 Department of Life,
Health & Environmental Science, San Salvatore, L’Aquila, Italia; 3 UOC
Anatomia Patologica, San Salvatore, L’Aquila, Italia
1
Background. Central giant cell granuloma (CGCG) is a very rare
localized benign but aggressive osteolytic lesion of the jaws, as
referred by WHO. The incidence of CGCG in the general population is estimated to be 0,0001%, women are more often affected
than men (1,5/2:1).
Methods. We describe a unusual observation of bilateral CGCG
of the mandibular angles in women of the same family. Lesions
showed a similar radiological aspect in all cases. Mater was treated in 1990 when she was 24 years old; a daughter in 2008 when
she was 9 years old, and another daughter in January 2013 when
she was 6 years old. Father, 47 years old, and son, 17 years old,
did not have lesions, as well as it seems in the ascendant. Surgical
conservative treatment was performed by the same surgeon. Istopathological examination of the specimen revealed the presence
of numerous multinucleated giant cells, spindle-shaped cells and
cells with vesicular nucleus also seen surrounding the giant cells.
Results. All three cases are now disease free (follow-up from
23 years to 6 months). A first singular aspect of our observation
is that lesions appeared only in the women of the family and
radiologic characteristics of lesions were overlapping. A genetic
analysis still in progress seems to indicate a gender expression
of CGCG. Another important aspect is that in the II generation,
CGCG appeared early. Based on histopathologically aspects,
usually we have to consider the differential diagnosis of CGCG
with so-called “brown tumor of hyperparathyroidism” (BTOH),
PGCG, and giant cell tumor of bone (GCTB) because their histologic appearance overlaps. The radiological appearance of CGCG
is not pathognomonic. The final diagnosis is histological. These
considerations are availed for the lesions of the mother and the
first daughter in which a biopsy was performed. For the last case,
the radiologic aspect of lesions oriented for a repetitive family
pathology.
The impact of large format sections in the study
of laryngeal carcinoma: report of 2 cases
A. Fornari1, G. Abbona2, D. Bellis2, L. Fulcheri1, E. Crosetti3,
G. Succo4, M. Papotti5
SCDU di Anatomia Patologica, Ospedale S. Luigi Gonzaga, Orbassano,
Italia; 2 SC di Anatomia Patologica, Ospedale Martini Asl TO1, Torino,
Italia; 3 SC di Otorinolaringoiatria, Ospedale Martini Asl TO1, Torino,
Italia; 4 Università di Torino, SCDU Otorinolaringoiatria, Ospedale
S. Luigi Gonzaga, Orbassano, Italia; 5 Università di Torino, SCDU Anatomia Patologica, Ospedale S. Luigi Gonzaga, Orbassano, Italia
1
Background. Large format sections (LS, macrosections) allow
histological study of a large part of the organ of interest, including
not only the lesion (usually a tumor) but also the surrounding tissues and the surgical margins. LS are routinely applied mainly to
improve staging of prostate and breast carcinomas. Slootweg and
Grot also used LS to stage tumors of the head and neck district,
including the larynx, where the evaluation of the extent of the tumoral growth is crucial to perform a correct staging, to assess the
involvement of specific anatomic regions and structures by the tumor and to correlate radiologic imaging to pathological findings.
Methods. In our laboratories, we routinely use LS in the study of
laringectomy specimens in order to evaluate the diagnostic accuracy of cartilage and soft tissues involvement obtained by CT and
MRI imaging and to correlate radiologic imaging to histologic
findings, as quality-control tests of the surgical strategies adopted. Here, we report two cases where this strategy was considered
particularly useful in confirming (case 1) or not confirming (case
2) radiologic findings about the extension of laryngeal carcinoma.
Results. Case 1. 63 yo man with an exophytic lesion of the left
vocal cord (VC) extended to the anterior commissure and to the
right VC, with suspected paraglottic space (PGS) involvement.
CT and MRI showed extension of the tumor to inferior left PGS
and anterior extralaryngeal spreading through the thyroid cartilage. Pathologic study of the laringectomy specimen using LS
well confirmed all these radiologic findings.
Case 2. 40 yo man with relapsing SCC of the left VC. CT and
MRI showed extension to inferior left PGS and involvement of
the left arytenoid cartilage. Pathologic study using LS did not
confirm these radiologic findings, leading to a diagnosis of rpT2
pN0 tumor. The surgical strategy adopted was considered a case
of overtreatment.
Pathologica 2013;105:259-318
Citologia
Cytological classification of pancreatic lesions
from eus-fna material. A proposal
P. Baccarini, A. Fornelli
Pathology, Bellaria, Bologna, Italy
Background. Pre-operative fine needle aspiration of pancreatic
lesions is of great help in planning medical decisions. It is thus
essential for the pathologist to provide a comprehensive report
with detailed microscopic description as well as a diagnostic
category, using a C1-C5 system, similar to that used in breast and
thyroid cytology.
We attempt to standardize cytological diagnostic categories
proposing a classification system which encompasses the issues
encountered when dealing with EUS-FNA material.
Results. The following categories are proposed:
PA1: inadequate/non diagnostic samples
– consisting entirely of blood
– composed of normal pancreatic cells (missed sampling)
–acellular
–technically unable to be evaluated (poorly spread, delayed
fixation).
PA1c: acellular material from proved cystic lesions
The samples could show
–“clean” background with rare macropahages or inflammatory
cells
– loose mucous of unknown provenience (contamination cannot
be excluded).
PA2: negative for malignancy
This category implies a specific benign process.
PA3: atypical/indeterminate samples
– atypia is mild and/or limited to few cells and cannot be distinguish from reactive cellular changes occurring in pancreatitis.
PA3m: abundant mucous with or without atypia.
This category include material from solid and cystic lesions
–presence of thick mucous that might contain epithelial cells
with mild nuclear atypia. Severe nuclear pleomorphism is not
admitted.
PA4: suspicious for malignancy
The diagnosis of malignancy cannot be made with certainty
because of:
– low cellularity
– very well differentiated neoplasms
– technical artefacts
– clinic-radiological significant discordance.
PA5: positive for malignant cells
A suffix could be added if a specific diagnosis can be achieved:
PA5a for ductal adenocarcinoma, PA5ne for neuroendocrine
tumor, PA5na for non ductal adenocarcinoma.
Inter- and intra-observers agreement studies will be necessary
to validated the present cytological classification of pancreatic
lesions.
Amphicrine carcinoma of the pancreas diagnosed
on eus-fna cytology: an extremely rare case
A. De Leo, P. Baccarini, C. Fabbri*, V. Eusebi
Section of Anatomic Pathology “M. Malpighi”, Bellaria Hospital, Bologna, Italy; * Gastroenteroly Unit, Bellaria Hospital, Bologna, Italy
Background. Amphicrine carcinomas are rare tumours defined
by the presence of tumour cells showing evidence of both exocrine and endocrine differentiation. We here report a case of
amphicrine carcinoma of the pancreas in a 59-year-old man. The
patient had a 8-cm lesion arising in the head of the pancreas.
Tumour markers were negative.
Poster
Methods. The patient underwent endoscopic ultrasound-guided
FNA. The EUS-FNA was perfomed using a 22-gauge Procore
needle with a linear echoendoscope. The FNA material was
processed for routine cytological analysis (including smears and
cell blocks) and stained with H&E, AB-PAS and MGG. On cell
block we performed immunohistochemistry for chromogranin,
synaptophysin, beta-catenin, cytokeratin 7, cytokeratin-MNF116,
cytokeratin 20, vimentin, Ki-67 and CD10.
Results. Microscopic examination of the cytological sample
revealed the presence of monomorphic epithelial cells with eosinophilic cytoplasm and rounded nuclei with dispersed chromatin. Futhermore, some tumour cells contained large cytoplasmic
vacuole positive with Alcian blue-PAS. Immunohistochemical
stains showed that the neoplastic cells were diffusely immunoreactive to CK-MNF116, chromogranin and synaptophysin, while
they were negative for CD10, CK20, beta-catenin and vimentin.
The proliferation index was about 8%. The amphicrine nature
of the lesion was ascertained by the combined demonstration of
mucus staining and synaptophysin expression in the same cells.
The identification of the amphicrine nature of an apparently endocrine tumor is of relevance, because of the poor prognosis of
amphicrine carcinomas as compared to endocrine carcinomas and
the requirement for aggressive therapy.
Comparison between braf v600e molecular
and immunohistochemical analysis in thyroid
papillary cancer on histologic and cytologic
samples
B. Angrisani1, P. Straccia1, T. Bizzarro1, C. Ricci1, A. Pontecorvi2,
C.P. Lombardi3, L.M. Larocca1, M. Martini1, E.D. Rossi1,
G. Fadda1
1 Division of Anatomic Pathology, Catholic University, Rome, Italy; 2 Division of Endocrinology, Catholic University, Rome, Italy; 3 Division of
Endocrine Surgery, Catholic University, Rome, Italy
Background: The BRAF V600E mutation is the most common
genetic alteration of papillary thyroid carcinoma (PTC). The
antibody VE-1 has been developed for detecting immunoistochemically (IHC) this mutation in both histologic and cytologic
samples. We investigated the correlation between the molecular
status of BRAF V600E and its IHC counterpart VE-1.
Methods. From January to June 2013, 44 histological and 18
LBC-processed cytologic cases were diagnosed as PTC. The
histologic diagnoses were classified as follows: 5 cases of tall
cell variant (TCV), 9 follicular variants (PCFV) and 30 classic
variants (PTC). All cases underwent mutational analysis (Pyrosequencing, Diatech, Italy) for BRAF V600E and immunohistochemistry for BRAF VE-1 (UCS Diagnostics, Italy)
Results. 15 of 16 PTC harbouring BRAF (94%) also yielded
positive for BRAF VE-1 whereas the remaining 14 samples
were wild type (WT) for both mutational and IHC analysis.
Four of 5 PCFV (80%) showed strong IHC staining (2-3+) and
harboured mutated BRAF, while the remaining 4 were wild type
for BRAF and showed a mild IHC positivity in half of cases. 4
of 5 TCV (80%) harboured BRAF and showed strong IHC staining whereas one sample, genotipically heterozygous for BRAF,
showed no detectable IHC stain. Of the 18 PTC diagnosed on
LBC samples 10 were mutated both at either molecular and IHC
analysis whereas 2 out of 8 (75%) resulted positive at IHC in
WT molecular cases.
Conclusions. The present study highlights a good correlation
between molecular and immunohistochemical analysis for BRAF
in thyroid cancers. BRAF VE-1 may be used for identifying cases
with BRAF mutation to be addressed to surgery in the indeterminate categories.
References
Ghossein RA, Katabi N, Fagin JA. Immunohistochemical detection of mu-
260
tated BRAFV600E supports the clonal origin of BRAF-induced thyroid
cancers along the spectrum of disease progression. J Clin Endocrinol
Metab 2013:98:E1414-21.
Koperek O, Kornauth C, Capper D, et al. Immunohistochemical detection
of the BRAF V600E mutated protein in papillary thyroid carcinoma.
Diagnosis of gastrointestinal tract lesions
endoscopic ultrasound-guided fine needle
aspiration cytology: a personal experience by cell
block procedure
P. Todaro1, S. Crinò2, A. Ieni1, A.M. Bonanno3, G. Tuccari3
1 Dipartimento di Patologia Umana, Policlinico Universitario G. Martino,
Messina, Italia; 2 Dipartimento di Medicina Interna, Policlinico Universitario G. Martino, Messina, Italia; 3 Dipartimento di Patologia, Policlinico
Universitario G. Martino, Messina, Italia
Background. Endoscopic ultrasound-guided fine-needle aspiration cytology (EUS-FNAC) has been considered particularly
helpful for both diagnosis and staging gastrointestinal and pancreatic masses (GPM). Moreover, this procedure increases its
accuracy when coupled with the cell block technique, making
also possible immunohistochemical algorithms for the differential diagnosis of lesions. We report herein our experience by
EUS-FNAC and cell block on a series of GPM occurred in our
institution between April 2012 and June 2013.
Methods. EUS-FNAC was performed using a convex array echoendoscope (EG 3870 UTK, Pentax-Tokio, Japan) and making two
passes with a 22 or 25G needle. The specimens were processed
by an in-room cytopathologist and immediately examined for
adequate cellularity; needle and syringe utilized was firstly rinsed
in 10 ml of 50% ethanol in a specimen container, centrifuged
and pelleted, with a subsequent fixation in 4% neutral buffered
formalin for 45 minutes. After embedding in paraffin at 56°C the
slides performed were stained by haematoxylin-eosin.
Results. Cytologic specimens were obtained from 39 patients, 20
males and 19 females, with a mean age of 63.7 years (range 3887). The lesions were localized in the stomach (6), pancreas (26),
esophagus (2), biliary tree (4) and colon (1); the diagnoses were
categorized as positive for a neoplasm (21), suspicious for a neoplasm (2), nonneoplastic/reactive process (16). Among neoplastic
cases there were 13 pancreatic ductal carcinomas, 5 gastrointestinal stromal tumours, 2 leiomyomas and 1 cholangiocarcinoma.
In some cases, immunohistochemistry was additionally applied
with the following antisera: broad spectrum citokeratins (CK),
CK7, EMA, CD117, SMA, S100, CEA, p53, MIB-1. On the light
of these observations, we suggest that EUS-FNAC joined to cell
block procedure should be considered useful in the diagnosis of
gastrointestinal tract lesions, achieving high rates of sensitivity,
specificity and diagnostic accuracy.
Results. Anal HPV was detected in 22 women (22.2%). Only 1
oral sample (1%) was positive for HR-HPV using HCII. A low
grade intraepithelial lesion (LSIL) was cytologically evident in
8 of the anal samples (8.08%), while no oral sample showed
dysplastic changes. No cytological high-grade lesion was found.
Although the detection of high-risk anal HPV with HCII is relatively common in women with persistent cervicovaginal lesions,
concomitant oral infections or cytological dysplasia are rarely
found.
Pancreatic foregut ciliated cyst: a benign lesion
with worrisome cea levels
C. Bellevicine, E. Vigliar, P. Pisapia, C. De Luca, G. Troncone
Background. Pancreatic cystic lesions represent a challenge for
the practicing cytopathologist. Ciliated foregut cysts, a congenital benign cyst, may rarely occur in sub-diaphragmatic region,
most frequently in the liver. Conversely, pancreatic foregut ciliated cysts are very uncommon. Noteworthy, pancreatic foregut
ciliated cyst strikingly features elevated intracystic CEA levels,
leading to a very worrisome clinical presentation. Thus, in this
setting the cytopathologist’s role is crucial. Therefore, a case of a
ciliated foregut cyst of the pancreas, recently encountered in our
endoscopic ultrasound (EUS) pancreatic fine needle aspiration
(FNA) practice, are here illustrated.
Methods. In a 50-year-old female, a cystic pancreatic lesion was
incidentally discovered by computerized tomography (CT) and
it was further investigated by EUS. The FNA, performed under
EUS guidance by yielded only a few drops of fluid. In our institution, all pancreatic cystic FNAs are routinely performed according to a reference protocol: a cytopathologist (CB) prepares on
site an ethanol-fixed smear for Papanicolaou staining, aliquoting
the residual material for CEA levels measuraments and KRAS
gene analysis.
Results. Microscopically, loosely cohesive clusters of columnar
cells were evident. At higher power, these columnar cells displayed
a ciliated apical pole and basal small round-to-oval normal nuclei.
Occasionally, a cytoplasmic mucin vacuole dislodged and compressed the nucleus. In spite of CEA levels being slight elevated
(190 ng/mL), the above reported cytology enabled the diagnosis
of pancreatic ciliated foregut cysts. Accordingly, the KRAS gene,
showed a native wild type sequence. Neoplastic mucinous cysts,
similar to solid pancreatic cancers, harbor KRAS mutations in up
to 80% of cases. However, KRAS mutation has also been detected
in benign lesions, questioning the specificity of KRAS as single
molecular diagnostic marker. In the present case KRAS was wildtype, a finding consistent with the benign nature of this lesion.
Anal and oral hpv status in women with persistent
cervicovaginal lesions
Occult hpv-related head and neck squamous cell
carcinoma diagnosed detecting hpv in a fnac
of metastatic laterocervical lymph nodes
M. Herz, C. Mian, G. Negri, F. Vittadello*, S. Messini*
A. Ginori, M.A.G.M. Butorano, A. Carducci, A. Disanto
Anatomia Patologica, Ospedale centrale di Bolzano, Bolzano, Italia, Centro Explora, Padova, Padova, Italia; * Divisione di Ginecologia, Ospedale
centrale di Bolzano, Bolzano, Italia
Biotecnologie Mediche-Anatomia Patologica, AOUS Santa Maria alle
Scotte, Siena, Italia
Background. HPV-induced carcinogenesis is shared by almost all
cervicovaginal and anal cancers and part of oral cancers. A higher
risk of HPV-associated anal lesions has been already described
in women with persistent cervicovaginal lesions. In this study we
evaluated the frequency of concomitant anal and oral HPV-infections in women with persistent cervicovaginal dysplasia.
Methods. Cytological liquid-based samples (ThinPrep) were taken
from the anal and oral (tongue) region in 99 women with persistent
low-grade cervical intraepithelial lesion. In all samples a high-risk
HPV-test with Hybrid Capture II (HCII) was carried out.
Background. Fine-needle aspiration cytology (FNAC) often is
the first diagnostic procedure performed in patients with head and
neck masses. Recent studies have indicated that human papillomavirus (HPV) infection is associated closely with oropharyngeal
basaloid squamous cell carcinoma (SCC) and have explored the
value of identifying HPV in neck metastases to determine the
origin of occult primary head and neck SCC. Herein, we report a
case of a patient with an occult HPV-related head and neck SCC,
diagnosed detecting HPV in a FNAC of metastatic laterocervical
lymph nodes.
Methods. A 54-year-old man presented to our hospital for a
261
Poster
right laterocervical lymphadenopathy, not associated with pain
or compressive symptoms and centrally hypoechoic at the ultrasonography. A FNAC of the lymph nodes was made; wet fixed
and air dried smears were made and stained with May-Grunwald
Giemsa (MGG).
Results. FNAC revealed few small lymphocytes and a monomorphic population of basaloid cells, forming clusters with peripheral palisading. The nuclear-cytoplasmic ratio was high, with
dense hyperchromatic nuclei. Immunohistochemically, the cells
showed a strong positivity for cytokeratins and p63, and were
negative for synaptophysin. Therefore, a diagnosis of metastatic
basaloid SCC, probably of the head and neck region, was made.
To confirm the diagnosis, Pyrosequencing was performed to detect and genotype HPV DNA on the MGG smears; HPV type 16
DNA was detected. Even if subsequent biopsies did not identify
the primary tumor, irradiation of the head and neck region was
made and currently the patient is in good clinical conditions. This
case supports previous studies on the utility of identifying HPV
in FNAC of metastatic cervical lymph nodes in the prediction of
an occult oropharyngeal primary SCC and it suggests that HPV
detection has to guide the clinicians to treat the head and neck
region, even in the absence of histological confirmation.
Value of pathologists’on site evaluation during
initial ultrasound-guided fine needle aspiration
cytology (fnac) in screen detected breast nodules
S. Cinocca, C. Baldovini, R. Panzacchi, P. Mainoldi*,
M.G. Cattani, R. Stamati*, G. Saguatti*, M.P. Foschini
Dipartimento di Scienze Biomediche e Neuromotorie, Sezione di Anatomia Patologica, Ospedale Bellaria, Bologna, Italia; * UOC Senologia,
Ospedale Bellaria, Bologna, Italia
Background. Fine needle aspiration cytology (FNAC) plays an
important role in the pre-operative diagnosis of breast carcinoma,
as it is a minimally invasive and cost-contained procedure. However, as screen detected lesions are often of small dimensions (<1
cm in greatest axis), FNA procedures usually result into a high
rate of inadequate samples (C1). To avoid this problem it is useful
to perform an “on site evaluation” (OSE) of cytological aspiration
material during the procedure. The purpose of the present study
is to assess if OSE of FNAC material can reduce the C1 rate also
in screen detected lesions.
Materials and methods. All the mammary ultrasound-guided
FNACs that have been performed between January 2010 and May
2013 (41 months) were reviewed. The C1 rate variation was considered. In particular, It has been taken into account that, starting
from February the 1st 2012, FNACs have been performed adding
the OSE executed by an experienced pathologist.
Results. Prior to the introduction of OSE, the registered inadequate
rate was 23.17% (n = 130/561) in 2010 and 31.09% (n = 268/862)
in 2011; afterwards, the rate decreased to 15.00% (n = 117/780) in
2012 and to 14,24% (n = 55/386) in the first five months of 2013.
As to 2012, the monthly C1 rate fell within a 9.20% to 25.36%
range (whereas this last value refers to the C1 rate in January,
which was the last month before OSE introduction).
Conclusions. Pathologists’OSE is useful to obtain a reduction in
the C1 rate, also in small, screen detected breast nodules.
Mammary analogue secretory carcinoma
is cytomorphologically indistinguishable
from acinic cell carcinoma, papillary-cystic variant
on salivary gland fna: a report of a case
with histological follow-up
C. Bellevicine, V. Natella, A. Somma*, E. Vigliar, G. De Rosa*,
G. Troncone*
Scienze Biomediche Avanzate, Policlinico Universitario Federico II, Napoli,
Italia; * Sanità Pubblica, Policlinico Universitario Federico II, Napoli, Italia
Background. Acinic cell carcinoma papillary-cystic variant (ACC-PCV) and mammary analogue secretory carcinoma
(MASC) are uncommon salivary gland neoplasms. Both on cytology and histology, their morphology is overlapping and only
immunohistochemistry (IHC) can make a reliable distinction.
Moreover, only MASC harbors the ETV6–NTRK3 translocation.
MASC and ACC share an indolent behavior and in most cases
conservative surgery is sufficient. Conversely it is crucial to rule
out aggressive neoplasms such as low-grade mucoepidermoid
carcinoma, whose treatment requires a more radical surgical
management.
Case. A 39 years old woman presented with a 1 cm right parotid
nodule, showing cystic features on ultrasound (US) scan. Fineneedle aspiration (FNA) was performed. The smears showed
the presence of microvacuolated clear cells aggregated into
small groups and into several papillary structures. These papillae showed a complex branching architecture. The cystic nature
of lesion, the presence of papillae and the clear microvacuolated cytoplasm appearance suggested the possibility of either
MASC or ACC-PCV. Since significant nuclear atypia was not
observed, more aggressive malignancies, such as low-grade mucoepidermoid carcinoma, were ruled out. Unfortunately, since
a CB was not available it was not possible to further refine the
diagnosis by IHC. However, in the cytopathology report the favourable course of both MASC and ACC-PCV was pointed out
and a conservative surgery was recommended. On histology,
IHC showed negativity for DOG1 and positivity for S-100 and,
focally, for GCDFP. A MASC diagnosis was rendered.
Results. ACC-PCV and MASC are morphologically indistinguishable both in cytology and histology. However, the correct recognition of the cytological features associated to these
morphologically overlapping neoplasms and the exclusion of
mucoepidermoid carcinoma, which may require a more aggressive surgical approach, can suffice for the correct patient
management.
Usefulness of on-site rapid evaluation (ROSE) in
pulmonary cytology in different clinical settings
E. Giarnieri3, R. Arduini1, C. Falasca2, P. Bruno3, N. De Rosa1,
M.R. Giovagnoli3, P. Micheli1, G. Antinolfi1
AO dei Colli, Monaldi, Naples, Italy; 2 Oncologia, AO Sant’Andrea,
Rome, Italy; 3 Medicina Clinica e Molecolare, AO Sant’Andrea, Rome,
Italy
1 Background. Lung cancer is the first cause of cancer death in
men and the second one in women in Italy. Lung cancer diagnosis is often performed in a late stage when has already spread to
local lymph-nodes. Transbronchial needle aspiration (TBNA) is
a bronchoscopic technique allowing the sampling of cytological/
histological material from mediastinal lymph nodes. It is mainly
used for the diagnosis and staging of lung cancer. Technique
sensitivity range between 60 and 85%. Among the factors influencing its sensitivity are operator skill and experience, type of
needle used, lymph node localization and diameter, number of
aspirations performed, nature of the lesion (lung cancer vs metastases vs non neoplastic diseases, etc.), use of guiding techniques
and rapid on site examination (ROSE).
Material and methods. We evaluated TBNA results in two different clinical setting: the Monaldi Hospital in Naples and the S.
Andrea Hospital in Rome, comparing cytological outcomes of
TBNA and trans-thoracic FNAB (fine needle aspiration biopsy)
when performed with or without ROSE. This techniques was use
to assess material adequacy both for diagnostic and immunocytochemical purposes. We also evaluated the economical gain
related to the application of this procedure.
Results. In the year 2011, at the Monaldi Hospital 40 out of 204
TBNA cases were carried out with ROSE. In ROSE cases diagnostic performance increased from 36% to 82%.
262
In the same period, at the S. Andrea Hospital all the 53 TBNA
cases were carried out with ROSE.
TBNA showed conclusive results in 85% of the cases (71%
positive).
ROSE can greatly improve cytological performance independently from the clinical setting where it is used. This technique,
by reducing the number of inconclusive exams or hospitalization
time length, may allows significant cost reduction.
Cytologic findings in pap-smears of women
with endometrial cancer
A. Palicelli1, G. Sartori2, G. Negri3, F. Vittadello4, F. Rivasi2
1
Laboratori S.C. Anatomia e Istologia Patologica, Maggiore della Carità
di Novara, Novara, Italia; 2 Medicina Diagnostica, Clinica e di Sanità
Pubblica, Università di Modena e Reggio Emilia, Modena, Italia; 3 Anatomia e Istologia Patologica, Ospedale di Bolzano, Bolzano, Italia; 4 Centro
Explora, Centro di Ricerca e Analisi Statistica, Padova, Italia
Background. Endometrial cancer is the most frequent gynaecological cancer. Although the utility of pap smear cytology for
the prevention of cervical cancer is undisputed, the sensitivity of
cervical smears for endometrial cancer is regarded as insufficient.
Our study retrospectively evaluates the cytologic findings in papsmears of women with endometrial cancer.
Materials and methods. The study included 308 women with
endometrial cancer. All women had had a pap smear in the previous 12 months. As a control population, 300 consecutive smears
of women in the menopause (50+ years) but without endometrial
cancer were evaluated. All the study smears were revised, with
particular emphasis on presence of endometrial cells, histiocytes,
naked nuclei, blood or degree of maturation of the squamous epithelia, and eventually compared with the control smears.
Results. The mean age of the women was 62.8 years. 295
(95.78%) cancers were of type I, 13 (4.22%) of type II. Overall,
atypical cells were found in 93 (30.19%) smears. The percentage
of positive smears was higher when the smears were taken short
before the histological diagnosis of cancer (39.89%, 14.00% and
15.71% at 0-3, 4-6 and 7-12 months, respectively). The cytological diagnosis had been ASCUS, HSIL and AGC in 9.68%, 1.07%
and 89.25%, respectively. Blood, histiocytes, naked nuclei and
normal endometrial cells were found in 40.26%, 40.58%, 24.68%
and 21.43%, respectively. A mature smear was evident in 54.55%
of cases.
Control smears showed blood and histiocytes in only 4.67% and
12.00% of smears, respectively; endometrial cells were never
reported. Mature squamous cells were reported in 9.33%.
Conclusions. Atypical glandular cells, blood, histiocytes and
an abnormal squamous maturation are significantly more often
found in smears of women with endometrial cancer than in
smears of women with negative follow-up.
Utility of immunocitochemistry in the cytological
diagnosis of papillary thyroid carcinoma
M. Lombardi, A. Savio, D. Bianchi, E. Padolecchia, R. Rodella,
C. Zambelli, F. Zorzi
Anatomia Patologica, Fondazione Poliambulanza, Brescia, Italia
Background. Papillary thyroid carcinoma (PTC) is the most
common malignant neoplasm at this site, accounting for approximately 80% of all thyroid cancers.
Fine needle aspiration cytology (FNAC) represents its only preoperative diagnostic method.
The cytological diagnosis of PTC is based on peculiar cytological
characteristics that, however, are not exclusive of PTC, occurring
in other benign and malignant thyroid lesions.
Thyroid nodules diagnosed with categories III and IV of the
Bethesda System for Reporting Thyroid Cytopathology have
demonstrated to be malignant in 1/3 of histological specimens 1-4.
Immunocytochemistry might play an important role in order to
reduce uncertain or suspicious FNAC results.
Several diagnostic and prognostic markers have been recently
proposed in order to improve the diagnostic accuracy of PTC:
CK19, HBME-1, Galectin-3, CD44, CD56, E-cadherin, c-erbB-2,
p21, p27 and p16 5-8.
The purpose of our study was the evaluation of the possible diagnostic contribution of immunocytochemistry to the morphological evaluation of PTC in FNAC (conventional method).
Materials and methods. 52 cases with FNAC of thyroid nodules
were evaluated including 25 cases with cytological diagnosis
Malignancy or Suspicious for Malignancy (Bethesda categories
V e VI), 10 cases with diagnosis of Follicular Neoplasm or Suspicious for a Follicular Neoplasm (Bethesda category IV) and
17 cases with diagnosis of Atypia of Undetermined Significance
or Follicular Lesion of Undetermined Significance (Bethesda
category III).
Further histology was available in 29 cases including 13 PTC,
10 Follicular adenomas, 3 oxyphilic adenomas, 2 oxyphilic carcinomas and 1 follicular carcinoma. Immunocytochemistry was
evaluated on all the cases with histological confirmation using the
following panel of antibodies: CK19, HBME-1, CD44 and CD56.
Results. CK19 was positive in many lesions (Tab.1). However,
its staining pattern was peculiarly strong and diffuse in PTC
cases, while being weak and sparse in benign and malignant follicular lesions.
Most PTC cases showed positive HBME-1 membranous pattern.
CD 44 showed a non specific diffuse expression in many different lesions.
CD 56 loss was demonstrated in the majority of PTC cases.
Tab. I. Immunocytochemistry results of 29 cases with FNAC categories III to VI and histological diagnosis (# strong and diffuse; * weak
and sparse).
FNAC
HISTOLOGY
CK19+ve
HBME-1+ve
CD44+ve
CD56-ve
13 cases
Bethesda
V and VI
13 Papillary
carcinoma
12#
12
13
9
6 cases
Bethesda
IV
3 Follicular
adenoma
3*
1
3
0
2 Oxyphilic
adenoma
2*
0
2
0
1 Follicular
carcinoma
1*
0
1
0
7 Follicular
adenoma
6*
3
7
1
1 Oxyphilic
adenoma
1*
0
1
0
2 Oxyphilic
carcinoma
2*
0
0
0
10 cases
Bethesda
III
Conclusion. Our data suggest that a combination of strong and
diffuse positivity for CK19, HBME-1 positivity and CD56 loss
can assist the cytological diagnosis of PTC.
References
1
Yang J, Schnadig V, Logrono R, et al. Fine needle aspiration of
thyroid nodules: a study of 4703 patients with histologic and clinical
correlations. Cancer 2007;111:306-15.
2
Deveci MS, Deveci G, Li Volsi VA, et al. Fine-needle aspirationof
follicular lesions of the thyroid. Diagnosis and follow-up. Cytojournal
2006;3:9.
3
Yassa L, Cibas ES, Benson CB, et al. Long-term assessment of a multidisciplinary approach to thyroid nodule diagnostic evaluation. Cancer
2007;111:508-16.
4
Schlinkert RT, van Heerden JA, Goellner JR et al. Factors that predict
malignant thyroid lesions when fine-needle aspiration is suspicious for
follicular neoplasm. Mayo Clin Proc 1997;72:913-6.
5
Nga ME, Lim GS, Son CH, et al. HBME-1 and CK19 are highly
263
Poster
discriminatory in the cytological diagnosis of papillary thyroid carcinoma. Diagnostic cytopathology 2008;36:550-6.
6
Prasad ML, Pellegata NS, Huang Y, et al. Galectin 3, fibronectin-1,
CITED-1, p63 and CK19 immunohistochemistry is useful for the differential diagnosis of thyroid tumors. Modern Pathology 2005;18:48-7.
7
El Demellawy D, Nasr AL, Babay S, et al. Diagnostic utility of CD56
immunistochemistry in papillary carcinoma of the thyroid. Pathology
Research and Practice 2009;205:303-9.
8
Pazaitou-Panayiotou K, Migdakos N, Boglou K, et al. The immunocytochemistry is a valuable tool in the diagnosis of papillary thyroid cancer in FNA’s using liquid-based cytology. J Oncol 2010;2010:963926.
Urovysion and urine cytology in the follow-up
of patients with history of urothelial carcinoma
E. Pegolo, F. Riosa, L. Peronio*, G. Raiti, I. Guiotto, C. Di Loreto
Istituto di Anatomia Patologica, AOU S. Maria della Misericordia, Udine,
Italia; * Chirurgia, San Antonio, San Daniele del Friuli, Italia
Background. UroVysion is a FISH assay that detects common
chromosome abnormalities in urothelial cancers. The aim of
this study was to assess the effectiveness of FISH and urine
cytology in the follow-up of patients with a history of urothelial
carcinoma.
Methods. An unselected cohort of 80 patients with a history
of urothelial carcinoma underwent surveillance by cystoscopy,
cytology, and UroVysion for a total of 170 follow-up events.
Urine samples both for cytology and UroVysion test were
placed and stored in Cytolyt solution and then processed by
the ThinPrep 2000 method. Final cytologic results were classified into 1 of 4 categories: inadequate, normal-benign, atypical-suspicious or malignant. The results of the UroVysion test
were signed out as inadequate, negative or positive. During
cystoscopy, a biopsy was performed depending on the findings. The results of FISH and cytology were correlated with
clinical outcomes derived from a combination of histologic
and cystoscopic information.
Results. The median age of the patients was 74 years with a
mean follow-up of 15.9 months (range: 4-29 months). The male
to female ratio was 2.7:1. UroVysion was positive in the 54.8% of
the clinically positive cases and cytology in the 42.1%; among the
clinically negative cases, UroVysion and cytology were negative
in the 74.3% and in the 53.4% respectively. The false positive and
false negative rate was 25.6% and 45.2% for UroVysion and 13.6
and 26.3 for cytology, respectively.
The sensitivity, specificity, PPV and NPV for UroVysion in
detecting urothelial carcinoma was 54.7%, 74.3%, 59.1% and
70.7% respectively. Cytology showed an overall sensitivity of
77.9% and specificity of 61.0%, PPV of 63.8%, and NPV of
75.8%. The difference in the diagnostic performances of the two
tests resulted to be statistically significant.
In summary, UroVysion is a valid ancillary assay in the follow-up
of urothelial cancer; it may be particularly useful as a reflex test
in suspicious cytology results.
Preoperative cytological evaluation of sentinel
lymph node status in breast cancer patients
“fnac-sln”
V. Angione, S. Pizzolitto, M. Zagami, A. De Pellegrin, A. Carbone*
Soc Anatomia Patologica, AOUD S. Maria della Misericordia, Udine,
Italy; * Istituto Clinica Radiologica, AOUD S. Maria della Misericordia,
Udine, Italy
Background. Axillary dissection (AD) is recommended in
patients with advanced breast cancer at presentation or a positive sentinel lymph node (SLN) biopsy. This study is aimed at
evaluating the role of fine needle aspiration cytology (FNAC) of
SLN in selection of patients potentially candidate to AD, with
consequent reduction of SLN biopsy.
Methods. 276 breast cancer patients who underwent ecografic
evaluation of axillary lymph node status were retrospectively
evaluated. 51 patients (18,48%) had clinical features suspicious
for nodal metastasis (E4-E5) and were investigated by pre-operative axillary FNAC. Aspirate smears were either routinely stained
with Papanicolaou, and collected in Lysis-Buffer as to obtain
paraffin-embedded material. Paraffin sections were stained with
hematoxylin and eosin, and antibodies against broad-spectrum
cytokeratins were applied in doubtful cases.
Results. Of 51 patients cytologically examined, 26 were positive
for axillary lymph node metastasis; 2 had suspicious features, and
18 had a negative smear. 5 cases were deemed inadequate.
SLN examination confirmed metastatic disease in all 26 cytological positive lymph node, in 1 of 2 suspected cases, in 4 of 5
inadequate cases. 1 cytologically suspicious case had a negative
SLN biopsy, yet this was documented after pre-operative chemotherapy. 1/5 inadequate cases was not examined histologically.
11/18 negative cases were histologically confirmed; 4 showed
micrometastasis and had a subsequent negative AD; 3 cases had
histologic evidence of macrometastasis.
In 7/51 cytologically investigated SLN with either an inadequate (4) or false negative (3 cases) result, subsequent biopsy
was positive. These results suggest that as much as 2/3 (35/51;
69%) of SLN biopsy may be avoided based on FNAC results.
This study also confirms the accuracy of cytological evaluation
of axillary lymph node status in terms of positive predictive
value and its efficacy in reducing the case-load of inappropriate
SLN biopsies.
Dermatopatologia
Subcutaneous dirofilariasis. First case observed
in the province of Rieti (Lazio region)
F. Liberati1, R. Brucchietti2, M. Capulli2, L. Ventura3
1 U. O. Anatomia Patologica, Ospedale San Camillo de’ Lellis, Rieti,
Italia; 2 Dip. Scienze Cliniche Applicate e Biotecnologiche, Università,
L’Aquila, Italia; 3 U. O. Anatomia Patologica, Ospedale San Salvatore,
L’Aquila, Italia
Background. Human subcutaneous dirofilariasis is a zoonosis
due to Dirofilaria repens, an abitual parasite of the dog, transmitted to man by Culicidae mosquitos. In the last two decades, an
increasing number of cases has been reported in Italy, mainly in
northern regions. We describe a case of subcutaneous dirofilariasis occurred in Rieti, a province to date free from reports of this
disease in humans.
Methods. In february, a 40-year-old man presented with a slightly itching subcutaneous nodule in the left flank region, noted a
few months earlier and not regressed after topic application of antibiotics and steroids. The only significant fact in his past medical
history was a travel in Milan some months before. An excisional
biopsy of the nodule was then performed.
Results. Surgical specimen consisted of a 4.3 x 2.9 x 1.7 cm
fibroadipous tissue, containing a hard, greyish nodule with illdefined borders, measuring 2 cm in largest diameter. Microscopically, the nodule was made of a central necrotic area containing
ten sections of the nematode, variously oriented and surrounded
by an inflammatory infiltrate rich in neutrophils and eosinophils.
External cuticular ridges and a single sexual tubule were visible,
compatible with a male of Dirofilaria repens.
To the best of our knowledge, this case represent the first one
reported in the province of Rieti and the fifth occurred in the
Lazio region. Recent studies in this region highlighted a higher
prevalence of canine filariosis along the seacost territory and at
the boundaries with Umbria region. Here, canine filariosis has
been increasing its geographic spread, with evident progression
264
along river valleys and a moderate prevalence in bordering Rieti
and Viterbo provinces. The presence of Dirofilaria repens in dog
population of these areas has been considered alarming for humans that could be infected by mosquitos. The description of this
particular case occured in Rieti represents a logical consequence
of this trend.
Extraosseous subcutaneous ewing sarcoma:
an indolent case
P. Viola, g. De Luca, A. Di Lorito, C. Marinelli, A. Colasante*
Medicina e Scienze dell’invecchiamento, UO ANA PAT, Clinicizzato
“Ss. Annunziata”, Chieti, Italia; * Oncologia, UO ANA PAT, Clinicizzato
“Ss. Annunziata”, Chieti, Italia
Background. Ewing Sarcoma (ES) is a biologically aggressive,
poorly differentiated tumour of bone and soft tissue, which affects more commonly Caucasian adolescents and young adults.
Because a common genetic locus is responsible for a large percentage of Ewing’s sarcoma and primitive neuroectodermal tumours (PNETs), these are grouped together in a category known
as the Ewing family of tumours, but PNETs are generally not associated with bones. The occurrence of extra osseous ES in deep
soft tissue has been well described but cases with subcutaneous
indolent presentation are unusual.
Methods. We report a case of a 51 years old woman who underwent local excision for a subcutaneous lesion on her right
shoulder. Clinically the lesion was small, palpable, mobile and
the skin above was unaffected. The patient reported no other
symptoms. Grossly we examined two skin ellipses with subcutaneous tissue measuring 15 x 8 mm and 14 x 1 mm with no
remarkable lesions on. Microscopically we found a dermal infiltrative tumour associated with sclerosis of collagen composed of
nest of cells. They had a pale cytoplasm and nuclei tended to vary
somewhat in size and a number of cells had a single nucleolus.
Mitotic figures were fairly frequent with no lymph vascular or
perineural invasion. Differential diagnosis based on morphology
included lymphomas, malignant myoepitheloima of soft tissues,
rhabdomyosarcoma and rhabdoid tumour so we performed immunohistochemistry.
Results. Immunohistochemical staining were positive for S100,
CD99, EMA, Chromogranin; focally positive for Desmin and
negative for keratins, GFAP, LCA, Melan-A, HMB45, SMA,
CEA, NSE, C-Kit and MIB-1 was 25%. These data oriented for
ES since negativity for LCA excluded lymphomas, negativity
for SMA and GFAP were against malignant myoepithelioma
while Keratins and S100 excluded rhabdoid tumour. FISH
analysis confirmed the diagnosis, the cytogenetic examination
showed reciprocal translocation t(11;22)(q24;q12) as in about
95% of cases ES/PNET. The patient underwent to a second
excision to clear up the margins and then received radiotherapy.
After almost a year of follow up she has not developed recurrence or metastasis.
Fournier’s gangrene: the paramount importance
of an early diagnosis
G. De Luca, A. Di Lorito, C. Marinelli, P. Viola, G. Lattanzio
Medicina e Scienze dell’invecchiamento, UO ANA PAT, Ss. Annunziata,
Chieti, Italia
Background. Fournier’s gangrene is clinical variant of necrotizing fasciitis which involves the penis, scrotum, perineum and
abdominal wall in men and is caused by a mixture of aerobic and
anaerobic enteric organisms. These bacteria are resident urethral
or gastrointestinal flora, rarely Streptococcus Pyogenes may be
the only responsible. Most cases occur in patients aged 30-60
years but the real incidence is unknown. Risk factors include
genitourinary trauma, periurethritis, diabetes, immunosuppres-
sion, malignancies, poor nutritional status, septic injection into
the penile vein and systemic varicella for children. The port of
bacterial entry is usually a trivial cut or skin abrasion and the
source is contact with carriers of the organism. The pathogenetic
mechanism consists of a necrotizing vasculitis that affects skin,
subcutis, fascia and muscle, probably triggered by an endarteritic
process involving the small branches of the internal pudendal
artery. A rapidly developing cellulitis, fasciitis, myositis, and
systemic toxicity may result. Progression to single-organ or multiorgan failure may occur, usually as a result of gram-negative
sepsis and is typically the cause of death. The mortality rate
ranges from 6% to 50%.
Methods. We analyzed scrotum cutis and subcutis of a 49 years
old male patient with massive painful erythematous swelling of
the scrotum.
Results. The histological appearances were those of a severe necrotizing process with edema, necrosis, and purulent
inflammation involving skin, muscular and adipose tissue.
Leukocytoclastic vasculitis and vascular thrombosis with
diffuse granulocyte infiltration involved all levels. The presence of large numbers of coccoid bacteria resulted in diffuse
basophilia of the tissue and was made very clear by GIEMSA
stain. However, morphology was indiscernible from a generic
case of necrotizing fasciitis, but only integration with anatomical site and the clinical data made possible the diagnosis of
Fournier’s disease.
Porocarcinoma in the Florence area: an 11-year
series
M. Sollai, C. Anichini*, A. Papucci*, D. Massi, C. Urso*
Division of Pathology, Careggi University Hospital, Florence, Italy; * Department of Anatomic Pathology, Dermatopathology, S. M. Annunziata
Hospital, Florence, Italy
Background. Although rare, porocarcinoma is the most frequent sweat gland carcinoma. It frequently occurs in elderly
patients, seems to equally affect both the sexes and is often
located in the head and neck region. Clinically, it appears as a
cutaneous ulcerated nodule or a verrucous plaque, sometimes
arising in a pre-existing benign poroma. In situ and invasive
porocarcinomas have been described. In situ porocarcinoma is
a lesion confined within the epidermis, estimated to account for
10% of all porocarcinomas. Invasive porocarcinoma is a dermal
neoplasm with solid-cystic architecture and infiltrative borders.
Porocarcinoma cells show eosinophilic or clear cytoplasm and
a variable grade of nuclear atypia; squamoid differentiation is
common.
Methods. Clinical and pathological data of porocarcinomas diagnosed between January 1, 1998 through December 31, 2008 at
the Division of Pathology, Careggi University Hospital and the
Department of Anatomic Pathology, S. M. Annunziata Hospital,
Florence, Italy, were retrospectively reviewed.
Results. The present series of 128 porocarcinomas represents
the largest series studied up to now. About 600 cases of porocarcinoma can be found in the world literature; a series of 69
cases being published by Robson et al. in 2001. In our study,
porocarcinomas showed a slight predilection for females (66
vs. 62) and were more frequently located in the head and neck
region (59%); other frequent sites were lower limbs (14%),
trunk (11%) and upper limbs (11%). The mean age of patients
was 79.9 years. Most tumors were invasive (92 cases, 72%),
and a relevant proportion, higher than that usually reported in
the literature (10%), were in situ porocarcinomas (36 cases,
28%). Histologically, porocarcinomas displayed the following
features: solid-cystic structure, duct formation, intracytoplasmic lumina, comedo-type necrosis, necrosis, clear cell change,
squamoid and poral differentiation, lymphovascular invasion,
intraepidermal pagetoid spread and intact basal layers.
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Poster
Trichoadenoma of Nikolowsky: a misleading lesion
D. Tacchini, M.A.G.M. Butorano, L. Vassallo, A. Ginori, R. Lio
Department of Medical Biotechnology, S. Maria alle Scotte, Siena, Italy
Background. Trichoadenoma is a rare benign, well differentiated
solitary and slowly growing tumour of the hair follicle. It is most
commonly present on the face and, to a lesser extent, on the buttocks of adults, occurring equally in men and women. The clinical
appearance is often misleading, suggesting a diagnosis of basal
cell carcinoma, epidermoid cyst or seborrheic keratosis.
Methods. A 24 year old woman presented with a nodular lesion on
her forehead for many years and that has grown slightly in recent
months. A complete surgical excision of the lesion was carried out.
Macroscopically, the lesion consisted in a skin coloured, slightly
raised and irregularly-shaped papule, measuring 4 mm in diameter. Sections of the lesion were stained with hematoxylin and
eosin. The histological evaluation revealed a well-circumscribed
dermis proliferation, composed of numerous mature horn cysts
with epidermal-type keratinization. Cysts show a well developed
granular layer and a flaky central amount of keratin. These cysts
were surrounded by eosinophilic epithelial cells, which in some
areas assumed a pseudo-glandular pattern, and were immersed
in a sclerotic stroma. Interspersed among cystic structures were
basaloid tubules and cords. All the lesion was well circumscribed
and had not infiltrative aspects. Moreover, small solid nests of eosinophilic cells were present. No hair follicle formation was noted. Focal areas showed neutrophilic abscesses and foreign-body
reaction to ruptured cysts. Areas of solar elastosis were not appreciated. At higher magnification, the cells were monomorphic,
not atypical, of medium-sized and with an inconspicuous nuclei.
Results. Based on the morphological aspects, the final diagnosis
was tricoadenoma of Nikolowski.
Ballon cell melanoma versus ballon cell nevus:
when the line is crossed?
L. Ranieri, P. Viola*, C. Marinelli*, G. De Luca*, A. Di Lorito*,
D. Angelucci*
Medicine and Science of Aging, Ss Annunziata Hospital, Chieti, Italy;
* Medicine and Science of Aging, Ss Annunziata, Chieti, Italy
Background. Balloon cell melanoma (BCM) is a rare form of
vertical growth phase melanoma characterized by a nodular proliferation of neoplastic balloon cells. Clinically, lesions are soft,
rubbery, or firm nodules with a polypoid contour. It is composed
by nests and sheets of large cells with abundant clear or vacuolated cytoplasm that displace the nucleus to the periphery. In
some areas, conventional epithelioid vertical growth phase melanoma can be identified. Balloon change is due to degeneration of
melanosomes and the phagocytosis within lysosomal structures:
it explains the lack of expression of Melan-A and HMB45. However the epithelioid zones show cytopasmic expression of MelanA. Balloon cells have been reported in intradermal, halo and
combined nevi. BCM shows mild pleomorphism and rare mitotic
figures but occasionally necrosis and high mitotic count can be
identified. Balloon cell nevi (BCN) have centrally located nuclei,
no nuclear pleomorphism, no mitotic activity and maturation.
Methods. A 25-year-old woman presented with a 0.5 x 0.2 cm
pigmented lesion located in the minor labia of the vulva. The lesion shows a melanocytic proliferation with some balloon cells
and a focally Brisk-type flogistic infiltrate. No mitotic figures
and significant nuclear atypia were present and the whole lesion appears quite symmetric. Melanocytic cell show maturation
gradient and absence of epidermal pagetoid invasion. The lesion
stains positively for HMB45 and p16.
Results. The main reason BCM can be mistaken for BCN is its
benign morphologic appearance. Though the lesion resembles a
balloon cell nevus, there are important distinguishing features.
According to this characteristic, our diagnosis was of compound
balloon cells nevus with immunological regression. Considering
that in some cases a sure differential diagnosis between BCM
and nevus with balloon cells aspects cannot be placed and that
the lesion was completely excised, we suggest second-look and
follow-up.
Diagnostica molecolare
That dunce of a naïve gist which, disregarding
canonical gist pathology, teaches that kit
and pdgfra mutations are not (always) mutually
exclusive
R. Ricci1, M. Martini1, T. Cenci1, A. Antinori2, A. Cassano3,
L.M. Larocca1
Anatomia Patologica, Università Cattolica del S. Cuore - Policlinico
“A. Gemelli”, Roma, Italia; 2 Chirurgia, Università Cattolica del S. Cuore
- Policlinico “A. Gemelli”, Roma, Italia; 3 Oncologia, Università Cattolica del S. Cuore - Policlinico “A. Gemelli”, Roma, Italia
1 Background. GISTs may harbor driving mutations in KIT, PDGFRA or, rarely, BRAF, RAS, SDH, NF1. This implies different
chemosensitivity, making genotyping a pivotal step for therapy in
advanced/M+ or HR resected cases. KIT ex.9,11,13,17 and PDGFRA ex.12,14,18 should be screened to classify a GIST as WT;
since KIT and PDGFRA mutations in naïve GISTs are believed
single and mutually exclusive, some labs act sequentially, ceasing
at the first mutation. But double primary mutations can occur, and
this procedure may miss relevant ones.
Methods. CD117 (DAKO) and DOG1 (Spring) were used for
IHC. KIT (ex.9,11,13,17) and PDGFRA (12,14,18) were sequenced in DNA from 4 single 100% tumor fragments or normal
rectal wall(ABIPRISM3100-AvantGeneticAnalyzer, Appl. Biosystems; QIAampTissueKit, Qiagen).
Results. The transanally removed, ruptured 6cm rectal tumor showed spindle cells with perinuclear vacuoles, CD117+,
DOG1+, necrosis, 80mit/50HPF, featuring a HR GIST. PCR
consistently found a del(1504-9), A502-Y503, Ins L, in KIT ex.9,
and a mut (A→T at 2526), D842V, in PDGFRA ex.18. WT KIT
and PDGFRA in rectal wall ruled out germline mutations.
Double primary mutations are rare in GISTs. Those involving diverse hotspot exons are exceptional and insidious: their members
can differ in TKI-sensitivity and may escape detection in sequential genotyping; to the best of our knowledge, only 5 such pairs
have been reported, 1 in both KIT and PDGFRA. The PDGFRA
(exceptional in rectal GISTs) and A502-Y503, Ins L KIT ex.9
(unreported) mutations we report belong to this type and, with the
latter pair, breaks the paradigm of mutual exclusivity of KIT and
PDGFRA primary mutations in GISTs.
Adjuvant imatinib was started trying to switch-off KIT exon9
mut., to lower the biological potential of our GIST to that of
PDGFRA-driven one (expectedly indolent). D842V, imatinibresistant, suggests Crenolanib in case of relapse.
Thus, exceptions to the commonly accepted GIST paradigms can
occur, with relevant consequences.
Immunohistochemistry for Alk gene detection
on histological samples and critical issues of Fish
analyses on cytological specimens
O. Nappi, C. Della Ragione, A. Postiglione, V. Cerbone,
N. Mitilini, P. Galloro, A. Boscaino, M. Palumbo, R. Monaco
Anatomia Patologica e Citoistopatologia, AORN “A. Cardarelli”, Napoli,
Italia
Background. A large discussion is ongoing on the validity of Immunohistochemistry (IHC) as first investigation tool for detection
266
of Alk gene rearrangement. Moreover there is a lack of information on specific protocols to be employed on cytological samples
when these latter are the only available samples for Alk analyses.
In this study we tested the validity of IHC as first investigation
tool and the robustness of Fish analyses on cytological samples.
Materials and methods. 50 cases of NSCLC from FFPE archivial tissues were tested for Alk in IHC using ALK01 clone
under “extended conditions” and enhancer method and 5A4
clone under “standard conditions”. Results were subsequently
compared to Fish analyses on the same specimens. Moreover
Forty-seven slides of cytological samples of pulmonary FNA and
pleural effusions under different preparing conditions (dedicated
slides, decoverslipped smears, destained slides, Thin Prep slides,
cell-blocks) were evaluated for Fish analyses testing different
pretreatment protocols: 1) Similar to the Urovysion Treatment;
2) 2XSSC followed by different time of pepsin incubation; 3)
Without Any Pretreatment.
Results. In between the 50 FFPE samples tested for ALK-IHC, 38
were negative and 12 showed a range of positivity going from 1+
to 3+. All IHC negative were also Fish negative (split signal = 0).
Among the IHC positive 1 was Fish positive (split signal > 15%),
7 were Fish negative (split signal > 5 but < 15%) and 4 were
partially Amplified. On 47 cytological samples tested for Fish,
Cell-Blocks achieved a successfull reading (Cells read > 50%)
using a protocol similar to the one used for histological samples;
Dedicated Smears except Thin Prep slide were read successfully
without any pretreatment; Decoverslipped Smears always failed
when None Pretreatment was applied but they were readable after
a mild pretreatment specially if destained previously.
Conclusions. Our findings show that, under appropriate conditions, IHC is able to predict Alk-Fish negative specimens. Fish
analyses could be than reasonably applied only on cases scored
from 1+ to 3+. Moreover, in cases were cytological samples are
the only available specimens, Fish analyses can be successfully
conducted on cell-blocks or on dedicated slides or on decoverslipped slides prior destained.
Clinicopathological features of colorectal cancer
harboring braf mutation
O. Nappi1, M. Ammirabile1, L. Pagano2, C. Saporito2,
A. D’Alterio1, C. Della Ragione1, N. Mitilini1, A. Boscaino1,
M. Biglietto3, M.L. Cavaliere2
UOC Anatomia Patologica, AORN “A. Cardarelli”, Napoli, Italia;
2
Uoc Genetica Medica, AORN “A. Cardarelli”, Napoli, Italia; 3 Uoc
Oncologia Medica, AORN “A. Cardarelli”Napoli, Italia
1
Background. The BRAF gene encodes a protein that plays a key
role downstream of KRAS in the cell signalling pathway from
the Epidermal Growth Factor Receptor (EGFR) that activates important cell functions. Information on the KRAS/BRAF genotype
is also highly useful when selecting systemic chemotherapy for
patients with advanced and recurrent colorectal cancer (CRC),
to identify patients with poor prognosis. In the BRAF kinase
domain, the most common mutation is the mutation c.1799T > A
(p.V600E). From 5% to 10% of patients with CRC harbor the
BRAF mutation; it is mutually exclusive with KRAS mutations
in CRC and is strongly associated to the microsatellite instability.
Especially, BRAF mutations occur more frequently in right-sided
tumors and is often (60%) associated with poorly differentiated
adenocarcinoma or mucinous carcinoma subtypes.
Methods. We analyzed 104 cases of CRC: fifty-seven were male
and forty-seven were female. Formalin-fixed paraffin-embedded
neoplastic samples were obtained from right-colon (39 cases),
left-colon (30 cases), rectum (18 cases), small intestine (10 cases), metastases (7 cases). Only 20 samples (20%) were mucinous
carcinoma subtypes.
An area of tumor on the Hematoxylin and Eosin-stained slide
was identified by an experienced pathologist and was marked
and macrodissected on a corresponding unstained slide for subsequent DNA isolation. DNA was extracted using a commercial kit
(Qiagen) and quantified by Qubit fluorometer. The KRAS-BRAF
stripAssay is based on reverse-hybridization (10 mutations in
codons 12 and 13 of the KRAS gene and the mutation V600E
in BRAF).
Results. Among 104 cases tested, 6.7% were V600E mutationpositive (n = 7). The clinicopathological features associated
with this genotype were right-sided tumors (6 right and 1 left
colon; p < 0.05), mucinous carcinomas (4 mucinous and 3 nonmucinous carcinomas; p < 0.05). In contrast with previous study,
no significant differences have been observed regarding age and
gender (P > 0.05).
Egfr mutation detection by microfluidic
technology: a validation study
U. Malapelle, S. Russo, F. Pepe, R. Sgariglia, A. Bianco,
C. De Luca, C. Bellevicine, P. Pallante*, G. Troncone
Sanità Pubblica, Università di Napoli Federico II, Napoli, Italia; * Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, Napoli, Italia
Background. Advanced non-small cell lung cancer samples are
tested for epidermal growth factor receptor (EGFR) gene mutations. Their detection by direct sequencing is time-consuming.
Conversely, the length analysis of fluorescently labelled PCR
products is easier.
Methods. To avoid labelled primers and the automated capillary
electrophoresis apparatus, we validated a fast and sensitive chipbased microfluidic technology. The limit of detection of fragment
length assay on microfluidic device was 5%, more sensitive than
direct sequencing (12.5%). The novel methodology showed high
accuracy in the analysis of samples whose mutational status was
known.
Results. The accuracy in quantifying mutated alleles (mA) was
evaluated by PCR products subcloning; the mA% provided by
direct sequencing of subcloned PCR products showed a close correlation with the mA% provided by the microfluidic technology
for both exon 19 (R2 = 0.9) and 21 (R2 = 0.9). Microfluidic-based
on-chip electrophoresis makes EGFR testing more rapid, sensitive and cost-effective.
Egfr mutations detected on cytology samples
by a centralized laboratory reliably predict
response to gefitinib in non-small cell lung
carcinoma patients
U. Malapelle, C. Bellevicine, C. De Luca, S. Russo, F. Pepe,
R. Sgariglia, A. Bianco, A. Iaccarino, G. Troncone
Sanità Pubblica, Università di Napoli Federico II, Napoli, Italia
Background. Epidermal growth factor receptor (EGFR) mutations are reliably detected by referral laboratories, even if most
lung cancer cytology specimens sent to such laboratories contain
very few cells. However, EGFR mutations may be distributed
heterogeneously within tumors, thereby raising concerns that
mutations detected on cytology are not representative of the
entire tumor and, thus, are less reliable in predicting response to
tyrosine kinase inhibitor (TKI) treatment than mutations detected
on histology.
Methods. To address this issue, the authors reviewed their clinical practice archives and compared the outcome of TKI treatment
among patients who were selected by cytology versus patients
who were selected by histology. From July 2010 to July 2012,
364 cytology samples and 318 histology samples were received.
Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were
confirmed by direct sequencing; discrepancies were resolved by
cloning polymerase chain reaction products. The response rate
267
Poster
(RR) and progression-free survival (PFS) at 12 months (range,
3-34 months) were evaluable in 13 EGFR-mutated patients who
were selected for treatment by cytology and 13 patients who were
selected by histology.
Results. The mutation rate was similar in histology samples
(8.5%) and cytology samples (8.8%). The RR (54%) and PFS
(9.2 months) were similar in histologically selected patients
and cytologically selected patients (RR, 62%; PFS, 8.6 months;
P = .88). The disease control rate (responsive plus stable disease) was 92% in histologically selected patients and 100%
in cytologically selected patients. EGFR mutations detected
on cytology specimens by a centralized laboratory can predict
TKI treatment response equally well as mutations identified on
histology samples.
ALK rearrangements detection in cytological
samples of lung cancer
A. Proietti, G. Alì, S. Pelliccioni, C. Niccoli, A. Servadio,
G. Fontanini*
Anatomia Patologica III, Santa Chiara, Pisa, Italia; * Patologia Chirugica, Molecolare e dell’Areacritica, Università di Pisa, Pisa, Italia
Background. Anaplastic lymphoma kinase (ALK) gene rearrangements are detected in approximately 5% of lung non-small
cell lung cancer (NSCLC). Patients positive for ALK rearrangements may be successfully treated with ALK inhibitor crizotinib.
Since advanced stage lung cancer are not suitable for surgical
resection, about 70% of patients are diagnosed on cytological
specimens. We evaluated the suitability of stained cytologic direct smears and cell blocks for fluorescence in situ hybridization
(FISH) to determine ALK status.
Methods. 124 consecutive patients with NSCLC were studied
by FISH test for ALK rearrangments. Sample analyzed were 79
(63.7%) cellular cell blocks and 45 (36.3%) Papanicolau stained
direct smear.
Results. FISH analysis was successful in 114 of 124 samples
(92%). Unsuccessful analysis were due to insufficiently cellular
and inadequate material and they were mainly CBs (68.8%).
ALK rearrangements was detected in 4 cases (3.2%), three direct
smears (75%) and one CB (25%). 71.8% of cases were negative
for ALK rearrangements, 35 smears (39.3%) and 54 cell blocks
(60.7%) respectively.
We found FISH to be comparably adequate for the detection
of ALK rearrangements on both cell blocks and direct smears
samples. Stained cytologic direct smears can be effectively used
for ALK rearrangements analysis by FISH. Moreover they can be
a useful source for ALK status study when insufficient cell block
is encountered.
Trop-2 expression and v600e braf mutation
in papillary thyroid carcinoma
T. Addati, M. Centrone, S. Petroni, R. Daprile, O. Popescu,
V. Rubini, G. Simone
Anatomic Pathology Unit, NCRC Istituto Tumori “Giovanni Paolo II”,
Bari, Italy
Background. BRAF mutational analysis is commonly used to
assess papillary thyroid carcinoma (PTC), but absence of mutation does not mean absence of carcinoma because only half of
PTCs expressed gene mutation. For this reason are usually used
immunohistochemical markers as indicators of alterations in normal process of cell growth. TROP-2 is a cell surface glycoprotein
product of the TACTD2 gene family, that evidenced a regulatory
role in tumor growth in other malignancies.
The aim of the study is to assess combination between BRAF
and TROP-2 in distinguish benign from malignant nodules; and
evaluate the overlap of expression of the two markers.
Methods. According the role of Thyroid Fine-Needle Cytology that is the gold standard for thyroid lesions evaluation Two
Hundred Thyroid Fine Needle Cytology, performed on LBC and
reclassified according to TBS, with determination of BRAF status
were collected from 2006 to 2007. Forty of these patients, who
underwent lobectomy or thyroidectomy, entered the study and
their histological materials was available for TROP-2 immunohistochemistry.
Results. V600E BRAF mutation and TROP-2 expression were
absent in all 26 cases classified as Benign. BRAF mutation was
found in 7 malignant lesions as follow: 6 PTCs cases (3 AFLUS
and 3 Suspicious in cytology) and one case of PTC-follicular
variant with extensive oncocytic features (FN/SFN in cytology).
TROP-2 was expressed in 8/14 PTCs. The agreement between
two markers was detected in 32/40 cases, a total overlap was
found in benign lesions, whereas it is shown an agreement of 50%
in malignancies.
The disagreement regarded 5 PTC TROP-2(+) and BRAF Wilde
Type, whereas 3 PTC-FV were TROP-2(-) and BRAF-mutated.
In conclusion, our data showed that TROP-2 and V600E mutation are not related in PTC and further investigations are needed
in this issue. TROP-2 could be an available diagnostic tool in
thyroid to detect PTC in BRAF Wild Type cases.
Partially funded by PONa3-00134 ONEV.
P16ink4a expression as differentiation marker
in benign and malignant melanocytic lesions,
and fish analysis
S. Petroni, T. Addati, O. Popescu, M. Centrone, V. Rubini,
G. Giannone, G. Simone
Anatomic Pathology Unit, NCRC Istituto Tumori “Giovanni Paolo II”,
Bari, Bari
Background. The incidence of malignant melanoma, compared to other cancers, is increasing by 4% every year in the
world. Different DNA aberrations were reported in 95% of
skin melanomas and they constitute a marker to distinguish
benign melanocytic nevi from melanoma. FISH, that is considered the gold standard in skin biopsy with uncertain diagnosis
of melanoma, has a sensitivity of 87% and a specificity of 95%
as reported in the literature. However, scientific data shows
that the loss of p16INK4a expression could be significantly correlated with the degree of malignancy, with the progression
from nevus to melanoma. The aim of this preliminary study
is to analyze the impact of FISH and IHC in a small series of
skin samples, in order to evaluate the behavior of dysplastic
melanocytic lesions.
Methods. Twenty skin samples (4 melanocytic nevi, 9 dysplastic
nevi and 7 melanomas in situ) were assessed with fluorescence
in situ hybridization (FISH) test, targeting 6p25 (RREB1), 6q23
(MYB), centromere 6 (Cep6), and 11q13 (CCND1), and with
p16INK4a immunohistochemistry reaction.
Results. Our data show that 4/4 melanocytic nevi, used as a negative control, were p16 INK4a (+) and FISH(-). Concerning dysplastic lesions 2/9 were p16INK4a(+) and FISH(+), 1 was p16INK4a(-)
and FISH(+).
Regarding melanomas in situ, used as positive control, 4/7 were
p16INK4a(+) and FISH(+), 2/7 were p16INK4a(-) and FISH(+) and
the last one was p16INK4a(+) but it was not valuable for FISH
analysis.
Our data revealed an immunophenotipic pattern p16INK4a(+)/
FISH(-) in melanocytic nevi, a prevalent pattern immunophenotipic p16INK4a(+)/FISH(-) (to note that 33% were FISH+)
in dysplastic lesions and a prevalent immunophenotipic pattern
p16INK4a(+)/FISH(+) in melanomas in situ. According to litterature, p16INK4a and FISH reactivity could indicate a progression to
malignancy.
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Mycn gene amplification detection by fish
on touch preparations in neuroblastoma
C. Caporusso, M. Liuzzi, G. Opinto, R. Scamarcio, L. Resta,
E. Maiorano
DETO, Policlinico, Bari, Italy
Background. Neuroblastoma is the most common extracranial
solid tumor of children. Prognostic variables include age, stage,
histopthological appearance and several prognostic markers, as
the amplification of MYCN gene. Here we reported a rapid and
direct method for the detection of MYCN amplification, using
FISH on tumor imprints, made directly on glass slides.
Methods. We studied 7 primary neuroblastomas, 5 males and 2
female, aged from 4 months to 6 year old. Primary neuroblastoma samples were obtained by surgery. Imprints were made
directly on clean slides as soon as possible. Imprints were dried
and examined at microscopy to ascertain the number of cells.
Then the slides were immersed in 3.7% of formaldehyde for 2’,
washed in diluted wash buffer for 5’and dehydrated with ethanol
crescent series. After completely air dried, the slide were submitted to application of probe mix with a coverslip. The slides were
added in hybridizer with two humidity control strips at 78°C for
5’ and then for hybridation at 37°C in overnight. The second
day, the coverslips were removed and put in several washes in
2XSSC/0.3%+NP40 and in purified water for 3’. Fluorescence
mounting medium was applied.
Results. Spectrum green labelled LSI-N-Myc and hybrized to
the band region 2p24, while spectrum orange labelled LSI CEP2
and hybridized the alpha satellite region of human chromosome
2. One out of 7 tumors showed MYCN amplification. In cases
without MYCN amplification, the probes generally appeared as
two distinct signals of each color, while amplification of MYCN
was considered where the number of signals exceeded of ten
copies per nucleus. FISH method can be used to detect MYCN
amplification in direct tumor imprints from neuroblastoma by a
simple rapid and reproductive test.
CD10, Bcl6, rare Bcl-2, EMA, and negative for CD3, PUI1, PD1,
CD30; Ki67index: 70%.The morphological and immunohistochemical data oriented for a diagnosis of large T-cell/histiocyte
rich large B-cell Lymphoma. Bone marrow biopsy are negative.
Clinical study: IIIB; International Prognostic Index: 3. Despite
chemotherapy with R-CHOP-14 protocol (cyclophosphamide,
doxorubicin, vincristine, prednisone and rituximab, every 14
days). The patient had an initial excellent response to chemotherapy (PETpost 4cycle was negative), with overall improvement in her physical condition, but unfortunately 2 weeks after
fourty cycle of chemotherapy he developed a Citomegalovirus
pneumoniae. Left inguinal lymph node showed a 35 days last
forty R-CHOP. The histological examination of the biopsy of
these lymph node revealed a diffuse infiltrate composed by uniform tumor cells with multiple small nucleoli and finely dispersed
chromatin in their nuclei. The tumour has an extremely high
proliferation fraction as well as a high fraction of apoptosis. A
“starry sky” pattern is usually present, which in imparted by numerous benign macrophages that have ingested apoptotic tumour
cells. Immunohistochemical analysis showed that the atypical
cells were positive for CD20, CD10, Bcl6, and negative for CD3,
Bcl-2, IRF4, EMA; Ki67index: 100%. Bone marrow biopsy are
negative. FISH procedures were carried out on paraffin sections
of inguinal lymph node according to the recomendation of the
manifacturers. FISH analysis was performed using theVysis LSIIGH/MYC and CEP8 Tricolor, Dual Fusion Translocation Probe
for the detection of the translocation (8;14). Fluorescence signals
were evaluated using a AX70 fluorescence microscope on 100
nuclei and confirmed the presence of the rearrangement. The new
diagnosis is of Burkitt Lymphoma. Conclusions: we have a questions for you: in these case, the Burkitt Lymphoma is a second
Lymphoma or a trasformation of the first Lymphoma?
About a case of sclerosing angiomatoid nodular
transformation of the spleen
A. Giorlandino, R. Caltabiano, S. Lanzafame
Ematopatologia
Dipartimento G.F. Ingrassia Anatomia Patologica, AOU Policlinico Ove,
Catania, Italia
One patient, two lymphomas: wath happened
here?
Sclerosing angiomatoid nodular transformation of the spleen is
a rare benign vascular lesion with extensive sclerosis and unknown etiology. Although the pathogenesis is not clear, it has
been postulated that it may represent a peculiar hamartomatous
transformation of red pulp in response to an exaggerated nonneoplastic stromal proliferation. However, it is unclear whether
SANT is the end stage of a variety of benign splenic conditions
including inflammatory pseudotumor, hamartoma, or hematoma.
Considering that the lesion is benign, splenectomy is curative.
We report a new case of SANT, discussing differential diagnosis,
immunohistochemical profile, and pathogenesis.
R. Chiacchio1, M. Cimminiello2, A. Benvenuto1, G. Del Vecchio3,
M. Pizzuti3, P. Tramutola3
1 UOC Anatomia Patologica e Citodiagnostica, AO Regionale San Carlo
di Potenza, Potenza, Italia; 2 UOC Ematologia con TMO, AO Regionale
San Carlo di Potenza, Potenza, Italia; 3 UOC Chirurgia d’Urgenza, AO
Regionale San Carlo di Potenza, Potenza, Italia
Background. Tcell/histiocyte-rich large B-cell and Burkitt Lymphoma are two distinct entities recognized by the current WHO
classification of lymphoid neoplasms. We report a case of a man
of 62 years old with Tcell/histiocyte-rich large B-cell Lymphoma
at diagnosis and Burkitt Lymphoma at relapse, four months to
onset.
Methods and results. Case report: a 62-year-old man presented
with an 20-day history of high-grade fever, decreased appetite
and a 7-lb weight loss during approximately the past 20 days. The
patient had no history of malignant disease; superficial and deep
lymphadenopathy. Biopsy of the mass in left inguinal region are
performed. Grossly the lymph node, 6 x 4 cm in size, had softe
cut surface. Microscopic examination revealed a lymph node
almost replaced by a diffuse proliferation of scattered, single,
large B-cells embedded in a background of small lymphocytes
that represent T-cells and variable numbers of histiocytes. The
tumour cells are always dispersed. Immunohistochemical analysis showed that the large atypical cells were positive for CD20,
Toid dendritic cell neoplasm: report of two cases
M. Arrondini1, G. Angeli1, P. Migliora2, A. Santagostino3
Anatomia Patologica, Policlinico di Monza, Presidio Clinica Santa Rita,
Vercelli, Italia; 2 Anatomia Patologica, Area Diagnostica e dei Servizi,
S. Andrea, Vercelli, Italia; 3 Ematologia, Dipartimento Oncologico Interaziendale, S. Andrea, Vercelli, Italia
1 Background. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of acute leukemia characterized by the
clonal proliferation of precursors of plasmacytoid dendritic cells
which in 2008 was recognized by WHO as a distinct entity. It is a
highly malignant disease, characterized by a striking skin tropism
and a quick extension, with or without leukemic phase, leading to
death within a few months.
Methods. Our first patient was a 78-year-old man who presented
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Poster
with multiple reddish, infiltrating skin lesions on the trunk. Two
years before a diagnosis of refractory cytopenia with multilineage
dysplasia was made with episodes of monocytosis in April 2011.
The second patient was a 77-year-old man who came to our attention for some skin nodules on the sternum and on the back.
In both cases biopsies of skin lesions were performed, showing
overlapping morphological features. There was a dermal infiltrate
of monomorphic medium-sized cells with scant cytoplasm, dispersed nuclear chromatin with indistinct nucleoli, which do not
show epidermotropism nor angiocentrism.
Immunohistochemical studies were positive for CD45, CD 56,
CD4, CD123, CD43, CD68 PGM1 (dot-like), BCL2 and negative for all lineage specific markers, including MPO. Proliferative
index was 30% in the first case and 70% in the second one.
On the basis of these findings we made diagnosis of BPDCN.
Both patients underwent bone marrow biopsies which were positive for BPDNC and then started a multidrug chemotherapy according to the ALL protocols. The first patient achieved complete
remission after 7 cycles but after 16 month of follow up he had
bone marrow relapse in July 2013. The second patient started
chemotherapy but died soon after for a pulmonary infection.
Results. Accurate diagnosis of BPDCN is essential in order to
carry on a prompt treatment, especially considering that the clinical presentation is often indolent at the beginning.
The triple positive CD4+CD56+CD123+ phenotype associated
with negativity for lineage-specific markers should represent a
minimum requirement for BPDCN definition.
It emerge that BPDCN is quite sensitive to chemotherapy initially, with complete response rates ranging from 53% to 89%.
However the prognosis is poor, even for patients reaching CR,
with a median survivals times between 12 to 27 months.
An unusual case of primary testicular
t-lymphoblastic lymphoma in an adult
M. Onorati, G. Petracco, F. Di Nuovo, F. Facchetti*
Anatomia Patologica, G. Salvini, Garbagnate Milanese, Italia; * Anatomia
Patologica, Spedali Civili, Brescia, Italia
Background. We report a case of primary testicular T-lymphoblastic lymphoma (LBL) in a adult male expressing both B-cell
and T-cell differentiation antigens and clonality of the T-cell
receptor gamma chain gene.
Methods. A 38-year-old male presented a painful solid left
testicular mass. The ultrasound exam showed a highly vascularized, hypoechogenic lesion, replacing the testicular parenchyma.
Orchiectomy was performed. The testis was replaced by a fleshy,
whitish mass, that was characterized by a proliferation of small
to medium-sized cells with round to oval sometimes convoluted
nuclei, showing dispersed chromatin, inconspicuous nucleoli and
scanty, faintly basophilic cytoplasm. Mitoses were frequent. These
cells coexpressed T-cell (CD2, CD3, CD7) and B-cell (CD79a,
Pax5) antigens, were strongly positive for TdT, CD34 and CD99
and negative for CD1a, CD4, CD5, CD8, CD10, CD20, CD23,
CD56, CD117, and EBV. Due to lineage ambiguity, JH and TCR
gene rearrangement analysis was performed using the Biomed-2
protocol, that showed a clonal rearrangement of the TCR gamma
chain. A bone marrow biopsy and CT scan performed for staging
were negative thus a diagnosis of primary testicular T-LBL was
carried out. Two years after diagnosis the patient is healthy.
Results. Primary T-LBL of the testis is extremely rare and only
two cases have been reported until now, both in young individuals. This represents the first case of primary T-LBL in an adult.
Aberrant coespression of cell lineage antigens, mostly lymphoid
and myeloid markers, is not infrequent in lymphoblastic lymphomas/leukemias. In this case lineage ambiguity was particular
significant, due to coespression of highly specific T (CD3) and
B-cell (CD79a, Pax5) antigens. The analysis of antigen-receptor
genes rearrangement provided support to recognize the nature of
the immature lymphoid neoplasm; this may be useful for more
appropriate choice of treatment and prognostic definition.
Splenic marginal zone lymphoma
with a simultaneous presence of deletion at 7q
and a gain of 3q in the same karyotype
M. Onorati1, M.R. Sansò2, A. Vismara3, G. Petracco1, F. Di Nuovo1
1 Anatomia Patologica, G. Salvini, Garbagnate Milanese, Italia; 2 Citogenetica, G. Salvini, Garbagnate Milanese, Italia; 3 Oncologia, G. Salvini,
Garbagnate Milanese, Italia
Background. Splenic marginal zone lymphoma (SMZL) is a
low-grade B-cell lymphoma with a micronodular pattern of
spleen involvement, occupying the marginal zone. SMZL accounts for fewer than 1% of the non-Hodgkin’s B-cell lymphomas. Cytogenetic abnormalities are commonly present in SMZL.
The most frequent is the deletion of long arm of chromosome
7. We present a peculiar case of SMZL showing both a deletion
of 7q and a gain of 3q in the same karyotype. To the best of our
knowledge this is the second case reported in the literature.
Methods. A 49 year-old female presented for a routine laboratory
analysis that disclosed a severe leucocytosis. According to this
finding, a bone marrow biopsy was performed showing tumor
cells with morphological and immunohistochemical features of a
SMZL. Moreover conventional and molecular cytogenetic analysis, performed on peripheral blood and bone marrow aspirate,
showed a derivative chromosome 7 resulted from a translocation
of the chromosome 3 segment distal to 3q22 to the long arm of a
chromosome 7 at band q32, with a deletion of 7q and a gain of 3q.
Results. It is worth mentioning that only two patients with a
deletion of 7q presented a gain of 3q. As widely explained in
the recently literature, patients with gain of 3q do not have deletion at 7q, so the rare coexistence of both abnormalities could be
explained as the presence of two different pathways, one related
to the amplification of 3q and the other related to a deletion at
7q. In the future it could be interesting to study the clinical and
histologic data of both groups of patients underlining the useful
help of cytogenetic analysis and correlate their outcome with a
correct therapy. Moreover, the deletion at 7q and a gain of 3q are
recurrent cytogenetic abnormalities suggesting that these regions
could contain genes involved in SMZL such as POT1 (MIR29A
and MIR29B) and BCL6.
Mediastinal myeloid sarcoma arising in a patient
treated for breast cancer: a possible case
of therapy-related myeloid neoplasm?
P. Viola, A. Di Lorito, C. Marinelli, G. De Luca, A. Mani*,
G. Lattanzio
Medicina e Scienze dell’invecchiamento, UO ANA PAT, Clinicizzato
“Ss Annunziata”, Chieti, Italia; * Thoracic Surgery Departement, Royal
Brompton Hospital, London, UK
Background. Myeloid sarcoma (MS) is a tumour mass consisting of myeloid blasts with or without maturation occurring at an
anatomical site other than bone marrow, however isolated mediastinal MS is extremely rare.
In 2007 Workshop of the Society for Haematopathology focused
on therapy-related myeloid neoplasms (t-MDS). Among solid
tumours treated with drugs associated with risk of development
of t-MDS, breast carcinoma is the first.
We present the case of a 57 years old woman, previously treated
for breast cancer, with a necrotic mediastinal mass to characterize.
Methods. Her radiologic exams showed a large mediastinal mass
(10x6,3cm) extending for 20 cm from the posterior part of the
trachea towards the oesophagus, to the right lung, compressing
the inferior lobe so she underwent surgical resection.
Specimen was composed by multiple pieces of necrobiotic tissue
270
yellow to green in colour. Microscopically there was necrosis
with numerous ghost cells at the periphery and very few preserved elements with scant cytoplasm and round to oval nuclei
with finely-disperse chromatin. We performed immunohistochemistry (IHC) to better characterize the lesion.
Results. Due to the type of material, it was very difficult to
carry out a diagnosis: IHC shows strong positivity for CD68 on
the rare atypical cells. The negativity for CD20, CD3, CD79a
was against lymphomas while CD1a excluded Langherans cells
histiocytosis. Reed-Stemberg cells were not identified. Also the
absence of epithelial cells CKAE1/3-positive, excluded any type
of carcinomas including breast recurrence. MPO was not available in our institution. According to the WHO, CD68 is the most
reliable marker for MS. This data along with the macroscopic
features, morphology and the age of the patient helped us. Since
she has been treated for breast cancer we speculated a connection
between her previous therapy and the recent disease according
to the literature, further investigations are required to assess the
correct diagnosis.
Lymphomatoid granulomatosis: a rare gingival
involvement
L. Sollima, E. Di Cola, P. Di Giosia, S. Saltarelli, L. Leocata
Medicina Clinica, Sanità Pubblica, Scienze della vita e dell’ambiente, San
Salvatore, L’Aquila, Italia
Lymphomatoid granulomatosis (LYG) is a rare angiocentricdestructive process presenting Epstein-Barr virus (EBV) positive
B-cells and reactive T-cells. This lesion prevails in adult male
and in patients with known immunodeficiency. LYG is graded
with 3 grades according to the number of EBV positive B-cells.
An unusual case of gingival involvement in an immune competent man is described. A 64-year-old man, without history of
smoke and alcohol abuse, developed a chronic painful ulcerative
lesion of vestibular gum. A CT-scan revealed a 15 mm nodule
in left inferior lobe of the left lung. Broncoscopy and cytological examination of bronchial washing were negative. 18F-fluoro-2-deoxyglucose positron emission tomography (FDG-PET)
showed a low standardized uptake value (SUV 0.95)in lung
nodule and hypermetabolic activity (SUV 5.8) in mandibulary
body. Biopsy of the gingival ulcer identified angiodestructive
polymorphous lymphoid infiltrate with extensive necrosis. Numerous CD3, CD4 and CD8 positive T cells and rare CD56 and
CD57 positive NK cells were observed. Numerous large atypical
mononuclear blastic B cells (CD20+), associated with the walls
of several arterioles in the sample, showed immunoreactivity for
LMP1 and EBV-induced protein gene 3. Bone marrow biopsy
showed nodular infiltration of 5% by blastic CD20, EBV induced
protein gene3 and LMP-1 positive B cells, with trilineage hematopoiesis. A diagnosis of lymphomatoid granulomatosis grade 3
was established. The patient was treated with rituximab, an antiCD20 monoclonal antibody, combined with a chemotherapeutic
regimen consisting of cyclophosphamide, doxorubicin, vincristin
and prednisone. LYG shares some histological similarities with
extranodal NK/T-cell lymphoma, with which it was sometimes
confused. The overwhelming majority of cases affect the lungs
and mediastinal lymph nodes. Oral mucosal involvement has
been documented in a very limited number of reports.
The role of ebv in the pathogenesis of Burkitt’s
lymphoma: a four italian hospital based survey
G. Pannone1, A. Santoro2, P. Mirella1, M.C. Pedicillo1, S. Cagiano1,
P. Somma3, M.E. Errico4, V. Donofrio4, R. Zamparese5, P. Bufo1
1 Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy; 2 Department of Laboratory, Fondazione di Ricerca
e Cura Giovanni Paolo II, Campobasso, Italy; 3 Section of Pathological
Anatomy, Ospedale Dei Colli – Monaldi, Napoli, Italy; 4 Section of Patho-
logical Anatomy, Paediatric Oncological Hospital Pausillipon, Napoli,
Italy; 5 Section of Pathological Anatomy, Ospedale di Ascoli, Ascoli Piceno, Italy
Background. The exact worldwide incidence of Burkitt lymphoma (BL) is not known. There are three distinct clinical
variants of BL, each manifesting differences in epidemiology,
clinical presentation, morphology, biology and genetic features:
the endemic (African), the sporadic (non-endemic), and the
immunodeficiency-associated form.
Methods. We have described clinicopathological data of 49
Burkitt’s Lymphomas occurred in Italy from 2003 to 2013, in 4
different hospitals, two of which located in east Italy: University
Hospital of Foggia and General Hospital of Ascoli Piceno, and
the other ones located in the west-coast of Italy: General Hospital
(AORN Ospedale dei Colli -‘Vincenzo Monaldi’, Napoli) and
Children University Hospital (Ospedale Santobono Pausillipon,
Napoli). Immunohistochemical Detection of LMP1 Expression and EBER In Situ Hybridization Procedures have been
performed. Lymphocyte B monoclonal spread has been demonstrated using a PCR based method to amplify FR1, FR2, and FR3
immunoglobulins heavy chains DNA fragments.
Results. 1 out 6 analyzed cases was positive for LMP-1, with
25% of stained neoplastic cells and only 1 out 2 analyzed cases
was positive for EBER with 50% of positive tumor cells. Overall,
we can conclude that, in our Italian study population, considering
both the methods of virus detection, the prevalence of EBV infection associated to Burkitt’s Lymphoma was 25% (2 out 8 cases).
EBV has been detected only in adult patients, one.
Primary soft-tissue extranodal b-lymphoblastic
lymphoma (b-lbl) with double hit rearrangement
of myc and bcl2, presenting as paravertebral
mass: a case report
A. Di Bernardo, E. Armiraglio, E. Lazzari*, E.S. D’Amore*,
A. Parafioriti
Department of Pathology, Orthopaedic Institute Gaetano Pini, Milan,
Italy; * Department of Pathology, San Bortolo Hospital, Vicenza, Italy
Aims. B-LBL in adults is a highly aggressive neoplasm characterized by TdT expression and immature morphology, although
rarely the tumor can simulate a Burkitt lymphoma (BL) or a
diffuse large B-cell lymphoma (DLBCL). Herein we describe
a case of B-LBL presenting as a soft tissue mass with peculiar
morphologic features and concurrent rearrangement of MYC and
BCL2 genes. This represents a very rare molecular event in this
type of tumor and is usually described, according to the WHO,
in B-cell lymphoma, unclassifiable with feature intermediate between DLBCL and BL, or in a subset of DLBCL.
Methods. A 69 year old patient presented with 3 month history
of back pain. Radiographic examination revealed a L5 paravertebral soft tissue mass of 15 cm suspicious for sarcoma, which
was biopsied.
Results. Histologically a diffuse lymphoid proliferation with
starry-sky areas was observed, consisting of relatively monomorphic medium/large cells with round nuclei, single eosinophilic
macronucleolus, moderate amount of cytoplasm and high mitotic
count. The morphology was more suggestive of a DLBCL with
plasmacytic/immunoblastic differentiation. At immunohistochemical examination neoplastic cells were positive for TdT and
also for CD20, CD79-a, MUM1, CD10, Myc, Bcl2, CD38, p53
(focal), with a proliferation rate (ki67/Mib1) of about 70%. FISH
analysis revealed both MYC and IGH/BCL2 rearrangements. In
conclusion, despite the unusual morphology, a diagnosis of extranodal double-hit MYC and BCL2 B lymphoblastic lymphoma
was made. Accordingly, the patient started chemotherapy and, at
the time we wrote this report, was still alive.
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Poster
Protein losing enteropathy due to intestinal tract
involvment by Langerhans cell histiocytosis:
a case report
A. Guadagno1, G. Fraternali Orcioni2, M. D’Armiento3,
M. Giordano3, R. Fiocca1, G. Pettinato3,
Dipartimento di Scienze Chirurgiche e Diagnostiche, IRCCS San Martino - IST, Genova, Italia; 2 UO Anatomia Patologica Ospedaliera, IRCCS
San Martino - IST, Genova, Italia; 3 Dipartimento di Scienze Biomediche
Avanzate, AOU Federico II, Napoli, Italia
1 Introduction. Langerhans cell histiocytosis (LCH) is a rare disorder characterized by abnormal proliferation and dissemination of
Langerhans cells. LCH with gastro-intestinal (GI) tract involvement
is rare and its prognosis is poor due to disseminated disease. The
GI tract is exceptionally the first site of presentation. We report a
case of protein-loosing enteropathy (PLE) caused by intestinal tract
involvement by LCH, clinically interpreted as allergic gastroenteropathy. The diagnosis of infants with refractory gastrointestinal
symptoms, especially PLE, is often challenging, and the possibility
of LCH should be considered in the differential diagnosis.
Patient. A 20 month-old female with edema of the extremities
and eyelids was admitted to the local pediatric hospital (A.O.
“Santobono Pausillipon”) and then to the Pediatric Department
of the University Hospital “Federico II” (Naples) for further diagnostic investigation. PLE was diagnosed.
Results. Laboratory investigation showed hypoalbuminemia,
hypoprotidemia, increased IgE for lattoalbumin while proteinuria
was within normal range. CT and MRI showed mesenteric lymph
node enlargement and moderate hepato-splenomegaly. Upper
and lower GI endoscopy with biopsies was performed. Duodenal
and ileal samples showed villous atrophy without intraepithelial
lymphocytosis. The sub-epithelial lamina propria was filled with
medium-sized cells, oval in shape, and non “dendritic” morphology. Nuclei showed a distinctive, complex, folded pattern. Such
cells were immunoreactive with S100, CD68 and showed mild
and multifocal positivity for CD1a, typical of Langerhans cells.
CD2, CD3, CD20, CD79a and CD138 were negative. Accompanying eosinophils and neutrophils were also found. Gastric and
colonic biopsies were normal. A diagnosis of small intestinal
localization of LCH was made.
Bone marrow histopathology of patients
with unexplained cytopenia reveals alterations
in myelopoiesis that warrant distincion from
myelodysplastic and myeloproliferative changes
A.G. Giannone1, L. Bongiovanni1, G. Franco2, A. Gulino1,
R. Porcasi1, R. Rizzi3, S. Ascani4, A.M. Florena1, V. Franco1,
V. Liso3
Department of Science for Promotion of Health and Mather and Child
Care, University of Palermo, Italy; 2 Department of Biopathology and
Medical and Forensic Biotechnologies, University of Palermo, Italy; 3 Hematology Section, Bari University Medical School, Bari, Italy; 4 Institute of
Pathologic Anatomy and Radiology, University of Perugia, St. Maria Hospital, Terni, Italy
1
Background. Patients with unexplained cytopenia (UC) often
undergo bone marrow (BM) biopsy to exclude underlying malignancies. The aim of the study was to evaluate BM abnormalities
in patients with cytopenia, some of which associated with autoimmune diathesis, and to compare these with normal controls
(CTRL), myelodysplastic syndromes (MDS) and early myeloproliferative neoplasms (MPN).
Methods. We studied 18 BM biopsies performed for unexplained
peripheral blood cytopenia. These included 12 cases of idiopathic
chronic neutropenia and 18 of primary immune thrombocytopenia. Nine cases arose in a background of autoimmune diathesis
(Sjögren Syndrome, Hashimoto thyroiditis and patients with high
ANA values). No therapy was administered prior to biopsy. For
each case, we assessed the whole blood counts, mean cellularity, myeloid:erythroid ratio, reticulin fibrosis, microvasculature
(CD34), density of myeloid cells (myeloperoxidase), eosinophils,
megakaryocytes (vWF), immature precursors (CD34,CD117),
mast cells (Tryptase), macrophages (CD68), myofibroblasts/
adventitial cells (SMA), B and T lymphocytes (CD20,CD3).
Histological findings were compared with 9 CTRL cases, 6 MDS
cases and 14 cases of early MPN.
Results. In our series, analysis of BM in UC group showed a significant hypocellularity for age (10 of 18), variable myeloid:erythroid
ratio, mild dysmegakaryopoiesis (9 of 18), dysmyelopoiesis (4 of
18), increased reticulin fibrosis (4 of 18) and a marked hypereosinophilia that proved to be significantly different (P < .05) from
all the control groups. Conversely, peripheral blood eosinophils
showed no significant differences. Since morphological and immunohistochemical features in UC showed some similarities with
MDS and early MPN, differential diagnosis could be a diagnostic
challenge. Our results suggest that an increased eosinophil density
could be a useful diagnostic clue to distinguish UC from myeloid
malignancies-related cytopenias.
Ginecopatologia
Endometriod carcinoma of the endometrium
hpv11 positive with signet ring cells: diagnostic
criteria
D. Morichetti, T. Pusiol, M.G. Zorzi
Istituto di Anatomia Patologica, Ospedale di Rovereto, Rovereto, Italia
Background. The presence of signet-ring cells in an endometrial
adenocarcinoma is extremely uncommon and it is always necessary to rule out a metastatic neoplasm.
Methods. Endometrial curettage was performed in a 53-yearold multiparous. An extensive search for an extrapelvic primary
cancer was undertaken but revealed no evidence of malignancy.
The patient underwent a radical hysterectomy. The patient was alive
and without evidence of recurrence 14 months after the surgery.
Results. FIGO grade 2 endometrioid adenocarcinoma of the usual
type with signet-ring cells component was diagnosed in endometrial curettage. The remaining component was composed by signetring cell morphology. The neoplastic proliferation was found in the
endometrium of a radical hysterectomy. The uterine neoplasm invaded less than one-half of the myometrium (FIGO stage I B). Special stains for periodic acid-Schiff diastase and mucicarmine were
positive in the SRCs. DNA extraction from paraffin-embedded
tissue revealed the presence of human papilloma virus (HPV) type
11. The results of imunostaining (CD56, Synaptophysin, Estrogen
Receptor, CEA, Ki-67, Progesteron Receptor, Chromogranin A,
Vimentin, E- Cadherine, HER-2, p16) were similar in the two
components. We reviewed 4 cases of endometrial adenocarcinoma
with signet-ring cells. We believed that an uterine neoplasm may
be defined as “signet-ring cell adenocarcinoma of the endometrium” when the lesion is composed exclusively by signet-ring
cells. To date any case of true “primary SRC carcinoma of the
endometrium” has been reported. In our case the DNA extraction
from paraffin-embedded tissue revealed the presence of type 11
HPV. This finding is very surprising because the demonstration of
the presence of HPV subtypes is used as support for establishing
the cervical primitive origin of the adenocarcinoma.
Primary serous carcinoma of peritoneum:
a case report
F. Pontieri2, G.F. Zannoni1
UO Anatomia Patologica, P.O. di Rossano (CS), Italia; 2 Anatomia Patologica, Gemelli, Roma, Italia
1
272
The papillary serous carcinoma of the peritoneum is a rare malignant epithelial tumor histologically indistinguishable from highgrade papillary serous carcinoma of the ovary. In this study, we
report the case of a woman 57 years old, previously operated by
bilateral hystero-annessiectomy because of uterine fibromatosis,
with a clinical history characterized by abdominal ache associated
with fever, weight loss and ascites. The laparoscopic exploration of the peritoneal cavity, revealed the presence of diffuse and
multiple nodular structures with papillary aspects. The histological
evaluation showed a high-grade papillary serous carcinoma. The
immuno-histochemical profile showed a diffuse and strong positivity to p53, WT-1, CK7 and CK18, a moderate nuclear positivity
to Estrogen receptors and a negativity to CK20, Calretinin and
Progesterone receptors. The first histological diagnosis was peritoneal carcinomatosis incidental to papillary serous carcinoma of
high-grade ovarian cancer. However, the revision of the uterine
annexes ruled out the presence of invasive or in situ carcinomatous
sores in bilateral ovarian site. These clinical and instrumental data
associated with the Gynecologic Oncology Group criteria relating
to the differential diagnosis, allowed to carry out the diagnosis of
primary peritoneal papillary serous carcinoma.
In conclusion, a careful clinical, anamnestic, histological and
immuno-histochemical evaluation is essential to express a correct
diagnosis and to plan a suitable treatment. The primary peritoneal
papillary serous carcinoma requires a close follow-up because
of poor prognosis that is comparable or worse than the papillary
serous ovarian carcinoma.
Gynecologic malignancies in geriatric patients.
The experience of regional referral center
V.G. Vellone*, M. Viviano, F. Grillo*, L. Mastracci*, C. Maganza,
E. Fulcheri*
Dinogmi, Università Di Genova, Genova, Italia; * Disc, Università di
Genova, Genova, Italia
Background. Life expectancy of the female population is
growing in Italy, with a parallel increase in the detection of the
gynecological cancers. The therapeutic options should take into
account the individual characteristics of these patients and the
histopathology of the tumor.
Methods. The archive of University of Genoa was retrospectively reviewed for the period 2008-2012. All patients older than
70 years undergoing major surgery for gynecologic malignancies
were considered. The study population included 164 patients
divided into three groups: 70-74ys (n = 63, 37.8%), 75-79ys
(n = 43, 26,21%) and > 80ys (n = 59, 35.97%).
Results. During the period, an increase of 70-74ys and 75-80ys
patients was observed while > 80ys patients remained stable.
These patients showed co-morbidities such as arterial hypertension (62.79%), diabetes mellitus (18.6%), obesity (11.63%), arrhythmias (11.63%)and thyroid diseases (9.3%). Endometrium
resulted the most common site (n = 94, 57.32%). In > 80ys
patients there has been an increased frequency of typeII (n = 9,
25%) more often presenting in advanced stages (stage III: n = 6;
22%). Ovary was the second site (n = 37, 22.57%), aggressive
histologies (serous G3) were more frequent in the 75-79ys
group (n = 6, 54.55%) witha greater frequency of advanced
stages (stage III: n = 5, 71.42%). Vulvar carcinomas, were frequent (n=24, 14.63%), in particular in > 80ys patients (n = 12,
50%). Cervix carcinomas were rare (n=8; 4.88%) and concentrated in the 70-74 group (n = 6, 75%). Gynecologic malignancies are constantly increasing in old age and pose many questions in clinical management, mainly for co-morbidities. The
increased incidence of aggressive histologies in the endometrial
carcinoma and an increased frequency of vulva carcinoma in the
> 80ys group rise the hypothesis of a fourth age in gynecology
oncology, when curative-intended therapies are still feasible and
more attention should be paid to prevention.
Cervical choriocarcinoma: a case report
D. Pretelli, F. Castiglione, V. Terrinazzi, M. Rotellini, I. Montagnani,
E. Projetto, D. Moncini, A.M. Buccoliero, G.L. Taddei
Dai Biomedicina, AOU Careggi, Firenze, Italia
Background. Choriocarcinoma is a malignant neoplasm composed of trophoblast arranged in a dimorphic pattern and lacking
chorionic villi. It has also been termed chorionepithelioma. The
reported prevalence of choriocarcinoma varies widely throughout
the world, being greatest in Asia, Africa and Latin America and
substantially lower in North America, Europa and Australia.
The frequency is higher in older women and in sexually active
women.
Choriocarcinoma that affects the female genital tract is categorized like gestational or non-gestational. Gestational choriocarcinoma is the result of a pregnancy and is exemplified by its location in the uterine corpus. Primary extra-uterine choriocarcinoma
is very rare, found mostly in the genital tract (cervix, tube, ovary)
in patients with coincident or antecedent pregnancy. Gestational
trophoblastic diseases have a varying potential for local invasion
and metastasis.
Non gestational choriocarcinoma can develop from germ cells but
also from metaplastic transformation of epithelium.
Most of the extrauterine reported cases are gestational choriocarcinomas located in uterine cervix but this tumour is however
a very rare entity. The diagnostic criteria for this tumor are: absence of disease in uterine cavity, pathologic confirmation of the
disease in cervix, exclusion of molar pregnancy and exclusion of
coexistence of normal intrauterine pregnancy.
Choriocarcinoma that appears as a primary process of the uterine
cervix can be easily misdiagnosed as cervical pregnancy, benign
or malignant uterine cervical neoplasm, and so on.
Methods. A 29 year old Chinese woman was admitted in our
hospital with abdominal pain and vaginal bleeding.
A gynecological examination showed a friable, hemorrhaging
mass occupying the uterine cervix.
Human chorionic gonadotropin levels has been done and the result was a high value, so the patient underwent an hysterectomy
for cervical pregnancy.
Results. Postoperative pathological examination of surgical
specimen has revealed a tumour composed of sheets of cytotrophoblast and syncytiotrofoblast without presence of villi, and the
diagnosis of cervical choriocarcinoma was made. According to
this criteria, we affirm that pathologic examination is the gold
standard to diagnose this neoplasm.
Hpv testing in conventional pap smear?
Yes, we can!
P. Pierotti, P. Crucitti, S. Lega, A. Bondi
Anatomia Patologica, Maggiore, Bologna, Italia
Objectives. The possibility to perform an additional test for
HPV markers without calling back the woman has been strongly
stressed as the key factor to switch from conventional Pap smear
to liquid phase cytology in cervical screening.
In lung cancer we are using dismounted cytological slides of
fine needle aspirates to detect biomarkers to address cancer
therapy using PCR technology, without further histological
sampling.
We have done a preliminary test to apply the same procedure on
screening conventional Pap smears to detect HPV markers.
Materials and methods. 48 women with borderline lesions (26
ASC-US, 3 ASC-H, 17 LSIL and 2 HSIL) detected with conventional Pap smears that have been called back to perform a
second sample with the STM Qiagen medium for HC2 test, were
randomly selected from the population screening files.
The original cytological slides, prepared at least six mounts ear-
273
Poster
lier, were dismounted in xylene and rehydrated. Half portion of
the cytologic material was gently scraped from the surface and
resuspended in methanol. The slide was mounted again with the
remaining smear sample and archived to maintain documentation
of the cytologic result.
The HPV nucleic acid was investigated with a PCR test using
a Cobas 480Z instrument (Roche), according to the ThinPrep
protocol.
Results. The cases studied are shown in the table, grouped according HC2 results vs PCR amplification.
The nine invalid cases observed are probably due to insufficient
scraping of the cellular slides, in order to maintain cytologically
evaluable samples. The two discrepancy cases observed can be
due to the occurrence of a documented cross reaction in HC2
method.
HC2
PCR
POS
NEG
INVALID
TOT
POS
25
2
5
32
NEG
0
12
4
16
TOT
25
14
9
48
Conclusion. The HPV test is referred to as triage of ASC-US
screening detected, before colposcopy.
This very preliminary test shows that it is not necessary to call
back the woman to perform an HPV test, even if the primary
screening was performed with the traditional Pap smear.
The liquid based cytology seems not essential as we are led to
believe in order to have information on HPV infection at the
biomolecular level.
References
Mayrand MH, Duarte-Franco E, Rodrigues I, et al. Human Papillomavirus DNA versus Papanicolaou Screening Tests for Cervical Cancer. N
Engl J Med 2007;357:1579-88.
Castle PE, Solomon D, Wheeler CM, et al. Human Papillomavirus Genotype Specificy of Hybrid Capture2. J Clin Microbiol 2008;46:2595-604.
Papillary proliferation of the endometrium:
a benign lesion simulating adenocarcinoma
D. Morichetti, T. Pusiol, G.M. Zorzi
Anatomia Patologica, S.M. del Carmine, Rovereto, Italia
Background. Papillary proliferation of the endometrium (PPE)
consisted of papillary fibrovascular core, that are devoid of cytologic atypia. The lesion was first described by Silverberg and
Kurman in 1992. Lehman and Hart in 2001 have described the
clinico-pathologic features in nine cases. These authors have
identified two sub-groups: simple PPE and complex PPE. In
recent a study of 59 cases even Ip et al (2013) have classified the
PPE in two groups. The group 1 consisted of lesions with localized simple papillae. The group 2 was composed by complex or
elongated papillae or diffuse intracystic proliferation. The PPE
doesn’t include the group of endometrial hyperplasias of WHO
classification. PPE is commonly seen in postmenopausal women;
it frequently involves endometrial polip and it is associated with
one or more types of epithelial metaplasia. In the endometrial
stromal localization the lesion may simulate endometrioid adenocarcinoma with papillary pattern especially in the small fragments
of the endometrium. PPE is very unusual and mimics well differentiated adenocarcinoma and consequently, the report of a single
case may be useful for the knowledge of this lesion published in
few papers.
Methods. The computerized files of the Institute of Anatomic
Pathology of the Rovereto Hospital were searched for examples of
PPE in the period January 1993-June 2013. Examples of syncytial
surface change were excluded. Only 1 case of PPE has been found.
Results. A 55-year-old woman presented with postmenopausal
bleeding. The operative hysteroscopy revealed stromal PPE
composed by simple papillae, with short stalk, covered by epithelial cells with bland nuclear features. The diagnosis of simple
hyperplastic PPE was performed. In the histological material
were present two endometrial polips with focal simple PPE and
mucinous, hobnail metaplasia. Hysterectomy was performed and
revealed diffuse simple cystic hyperplasia of the endometrium.
Endometrial stromal nodule with highly cellular
leiomyoma: the big trick
A. Di Lorito, C. Marinelli, G. De Luca, P. Viola, G. Lattanzio
Medicine and Science of Aging, Santissima Annunziata Hospital, Chieti,
Italy
Background. Endometrial stromal nodules are benign tumors
composed of endometrial stromal cells. They are reported in
women from 23 to 86 years old, but about 75% of patients are
premenopausal. For the diagnosis, it is really important to differentiate them from highly cellular leiomyomas, endometrial
sarcomas and, in young women, from uterine tumors with sex
cord-like elements (UTROSCT). However, it has been described,
in rare case, neoplasm containing combined stromal- smooth
muscle tumor.
Methods. We reported a case of a 24 years old woman who came
to clinical observation for abnormal bleedings, abdominal pain
and loss of weight. Clinical evaluation reported an intramural
uterine lesion with irregular pattern of vascularization, without
serous involving and without capsule. In order to preserve the
young woman fertility, the surgeon extracted the nodule without
removing the uterus.
Gross pathology revealed a yellow nodule, of 7,5 x 5 x 4,5 cm,
70 gr weighted. Microscopically, the tumor was made up of two
components: highly cellular leiomyoma and endometrial stromal
component. The stromal part had expansive, non infiltrative margins, with cells resembling the normal proliferative stromal cells.
Mitotic activity was less than 5% and there was highly vascularization, without tumor invasion. In order to well characterize this
component, a panel of immunohistochemistry was performed.
Results. The stromal part was strongly positive for CD10 and
Ki67 in this part was less than 10%. AML and Desmin were positive in the leiomyoma component. The inhibin and the calretinin
staining was negative, excluding the UTROSCT differentiation
of the nodule.
In view of the microscopic appearance and the immunohistochemical profile, we made the final diagnosis of combined
stromal and smooth muscle tumor and we suggested a prolonged
follow-up.
Cellular fibroma with normal appearing ovaries
and uterus: an unusual presentation in the pelvic
cavity
A. Di Lorito, P. Viola, C. Marinelli, G. De Luca, G. Lattanzio
Italy
Background. Ovarian fibroma is a benign tumor, accounting of
4-5% of all ovarian tumors. The cellular subtype shows hypercellularity, increased mitotic rate and sometimes nuclear atypia.
In particular, in young women, abdominal pain, associated with
a pelvic mass, is a common clinical emergency, mimicking a
neoplastic entity.
Methods. We report a case of a 40 years old woman, who came
to clinicians for abdominal pain and important loss of weigh;
clinical examination revealed a solid mass in the Douglas cavity,
suspected of ovarian cancer. Upon laparotomy, a mass of 23 cm
has been found in the pelvic cavity, without any connection to
274
the ovaries and the uterus. Grossly it was a tumor with yellow
surface, solid with some and focal cystic area, filled of a white
material. Microscopically, it was composed by spindle cells,
without atypia, mitosis and necrosis. Frozen sections carried out
the diagnosis of fibrothecoma and, according to the clinicians,
the ovaries and the uterus have not been removed. After formalin fixation, microscopic evaluation of multiple tumor sections
showed hypercellular area with spindle cells, slight nuclear atypia
and 4 mitosis in 10 high power field. No necrosis has been found.
A panel of different immunohistochemical markers have been
requested.
Results. The tumor cells were positive for vimentin, CD34,
CD56 and PR. They were negative for pankeratins, calretinin,
S100, desmin, smooth muscle actin, CD10, sinaptophisin and
chromogranin. Ki67 was positive in up to 5% of the tumor cells.
According to the immunohistochemical profile and the microscopic features, the diagnosis of cellular fibroma has been made.
Considering that some studies have been established that it has a
good prognosis, but local recurrences are possible, follow up has
been recommended. In conclusion, cellular fibromas can mimic
an ovarian tumor and only with the carefully examination of a lot
of sections and with a panel of markers it is possible to make a
right diagnosis.
A case of uterine pecoma and lam
A. Marinucci, L. Melchiorri, G. Coletti*, A. Chiominto*,
P. Leocata
Dipartimento di Medicina Clinica, Sanità Pubblica, scienze della vita e
dell’ambiente, Università, Ospedale Civile “San Salvatore”, l’Aquila,
Italia; * UOC Anatomia Patologica, Ospedale Civile “San Salvatore”,
l’Aquila, Italia
Background. WHO defines PEComas as mesenchymal tumors
composed of perivascular epithelioid cells (PECs) that coexpresse myoid and melanocytic markers. They include: Angiomyolipoma, Clear Cell Sugar Tumor, Lymphangioleiomyomatosis
(LAM) and a group of rare, morphologically and immunophenotypically similar tumors arising at a variety of visceral and soft
tissue sites, collectively termed PEComas-NOS.
Methods. We report a case of a 51-year-old woman with vaginal
bleeding. Gynecological examination found a reddish and protruding intravaginal polypoid lesion. This 5 x 3 x 2 cm uterine
mass was surgically removed. H-E staining revealed uterine
adenomyosis. A PEComa-NOS, composed of ill-defined spindle
cell fascicles, was found to mainly be distributed around the adenomyosis foci. In contrast to the deeply eosinophilic myometrium, the spindle cells had clear to lightly eosinophilic cytoplasm.
Their nuclei were oval to spindle-shaped and uniform, with no
mitotic activity. The endometrial stroma of adenomyosis was
focally replaced by the PEComa. LAM, characterized by poorly
defined nodular proliferation of spindle cell fascicles with many
tongue-like protrusions in dilated vascular spaces, was also noted.
All tumor cells showed diffuse immunoreactivity with Smooth
Muscle Actin, HMB45, MelanA, focally with Estrogen Receptor
and negative for CD10.
Results. Uterine PEComa lacks characteristic clinical and
imaging changes. The diagnosis mainly relies on pathological approach and should be differentiated from: Epithelioid
Smooth Muscle Tumor (HMB45 -), Endometrial Stromal Sarcoma (HMB45 -), Uterine Clear Cell Carcinoma (HMB45 - and
CK +), Metastatic Renal Clear Cell Carcinoma (HMB45 - and
CK, EMA +), Paraganglioma (when the cytoplasm is clear:
HMB45 – and Neuroendocrine markers +). There are some open
questions about PEComas: the histogenesis, the physiological
counterpart of PEC and the identification of the histological
criteria of malignancy. Although most cases are benign, a subset
behaves in a malignant fashion.
Metastatic lobular carcinoma to vaginal
fibroepithelial stromal polyp
P. Amico*, G.M. Vecchio, A. Bosco, F.F. Amore, A. Giorlandino,
G. Magro
* UOC di Anatomia ed Istologia Patologica, Umberto I, Enna, Italia
Background. Invasive lobular carcinoma of the breast has a tendency to metastasize to the abdominal cavity, including gastrointestinal tract, ovaries, bladder and serosal surfaces. Only rarely
uterine and lower female genital tract metastases have been reported in literature. We report the first case of a metastatic lobular
breast carcinoma to a vaginal fibroepithelial stromal polyp, which
closely mimicked an angiomyofibroblastoma.
Methods. A 48-year-old female underwent colposcopic examination for vaginal bleeding. A 1 cm polypoid lesion located at the
lateral wall of the lower one-third of the vagina was seen and
surgically excised.
Results. Histological examination showed a typical fibroepithelial stromal polyp. The underlying stroma contained spindle
to stellate cells embedded in a loose oedematous matrix with
numerous capillary-sized blood vessels. Notably the stroma
contained small foci of bland-looking round to epithelioid discohesive cells arranged in single-file pattern or in small nests.
Only rarely mitoses were found. The possibility of a vaginal
angiomyofibroblastoma was seriously considered. Interestingly
we become aware from the clinicians that the patient was affected by invasive lobular breast carcinoma (T2N1M0) diagnosed two years before and treated with surgery followed by
chemotherapy and radiotherapy. Immunohistochemically the
discohesive stromal cells were surprisingly positive to CKAE1/
AE3, CK7, CK19, mammaglobin, gross cystic disease fluid
protein-15, estrogen/progesterone receptors, HER2 (score 3+),
while they lack the expression of E-cadherin, vimentin, asmooth muscle actin, desmin, CD34, and HMB-45. Based on
morphological and immunohistochemical features, the diagnosis of “metastatic lobular carcinoma to vaginal fibroepithelial
stromal polyp” was rendered. Pathologists should be aware of
this possibility to avoid a misdiagnosis, assuring patient the correct treatment and prognostic information.
Adenocarcinoma of endometrium in abortion:
an incidental diagnosis
L. Sollima, E. Di Cola, G. Calvisi, G. Coletti, P. Leocata
Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell’Ambiente,
San Salvatore, L’Aquila, Italia
A case of endometrial and ovaric endometrioid adenocarcinoma
discovered incidentally during curettage of abortion is described.
A 43-year-old woman, gravida 3, para 2, was subjected to uterine
dilatation and curettage for a spontaneous abortion. Histologically, H-E staining showed dismorphyc and hypotrophic villous,
syncytial endometritis and foci of well differenrentiated tubularpapillary endometrioid adenocarcinoma. Neoplastic cells showed
immunoreactivity for Estrogen Receptor (95%), Progesteron
Receptor (60%), p53 (25%), p16 (5-10%), CEA (focally); they
were negative for Her-2 immunostaining. Two months later,
the woman underwent hysterectomy and bilateral adnexectomy.
The ovarian histological specimen showed multiple and bilateral foci of well differenrentiated endometrioid adenocarcinoma
associated with endometriosis. Ovarian neoplastic cells were
beta-catenina, cytokeratin, epithelial membrane antigen (EMA)
positive but p53 and Wilms Tumor-1 negative. Endometrium
displayed multifocal noninvasive tubular-papillary endometrioid
adenocarcinoma. Metastases of iliac lymph nodes were absent.
Six months later omentum was surgically removed and it showed
no alteration. Endometrial carcinoma is the most common neo-
275
Poster
plasia of the female reproductive system but is rarely associated
with pregnancy. In literature only a limited number of studies
describing few cases (10-15) of this condition are reported. The
present case demonstrate that routine histologic examination
after dilatation and curettage performed for spontaneous abortion
seems advisable.
Placental mesenchymal dysplasia: a case report
I. Montagnani, F. Castiglione, P. Pretelli, V. Terrinazzi,
E. Projetto, D. Moncini, A. Buccoliero, G.L. Taddei
Anatomia Patologica, AOUC Careggi, Firenze, Italia
Background. Placental mesenchymal dysplasia (PMD) is a rare
placental anomaly first described in 1991 by Moscoso and colleagues. PMD is characterized by placentomegaly with abnormal
stem villi showing marked cystic dilatation and grapelike vesicle
formation, fibroblastic stromal overgrowth and vascular abnormalities that effect vessel of all sizes.
Its reported incidence is 0.02% of all pregnancies.
The underlying cause of PMD is currently unknown. Some speculate that PMD is a congenital malformation of the mesoderm.
There is a definite association (estimated about 23%) of PMD
with Beckwith-Wiedemann syndrome (BWS), characterized by
macrosomia, exomphalos, macroglossia, omphalocele, internal
visceromegaly, placental enlargement and susceptibility to childhood tumors. At one end of the spectrum, PMD exhibits phenotypic changes limited to the placenta and BWS at the opposite
end with phenotypic changes affecting both placenta and fetus. In
recent data omphalocele and BWS are linked to abnormal expression of gene(s) on chromosome 11p15.5.
Pathologically in PMD the placentas are usually extremely large
in size for gestational age, often exceeding 1000g at term. The
stem villi show enlarge, edema, cystic degeneration or overgrowth of fibroblastic stroma with intact terminal villi and many
kinds of vascular anomalies, such as cirsoid or sinusoid chorionic
vessels, thrombosis, thickening of vessel wall, vascular stenosis,
villous chorangiosis, chorioangioma and fetal thrombotic vasculopathy. Absence of trophoblastic proliferation placentas is the
main histological difference from partial moles.
Methods. Has come to our attention a case of a 38 year-old
woman that was subjected to a cesarean section in emergency
care at 27 +2 weeks of gestation. The fetus showed anemia
and intrauterine growth restriction (IUGR). The differential
diagnosis included a complete molar pregnancy, a partial molar
pregnancy or PMD.
Results. We report a case of Placental Mesenchymal Dysplasia
Inguinal lymph node metastasis as the presenting
sign of an advanced tubo-ovarian serous
carcinoma
L. Abete, F. Pitto, E. Pacella, L. Mastracci, E. Fulcheri, V.G. Vellone
Disc, S. Martino, Genova, Italia
Background. Lymph nodes metastases usually represent a late
event in serous ovarian carcinoma, for they tend to spread mainly
by transcelomic exfoliation. Metastases to paraaortic lymph nodes
have been documented in 33% of the patients with all stages of
disease while the incidence of groin metastases is reported to be
less than 3%, almost always as part of a disseminated disease. We
report a case in which inguinal metastases was the presenting sign
and in which a methodic approach and a relatively limited IHC
panel allowed the detection of the tubo-ovarian primary.
Methods. A 63 y.o. woman was admitted for an inguinal mass
suspicious for malignancy. Histological examination showed a
lymph node replaced by a carcinoma constituted by solid-necrotic
areas and complex, branching papillae lined by atypical cells with
high mitotic count, with no nuclear pseudoinclusions or sign of
secretion consistent, on morphological and IHC basis (CK7+,
WT1+, ER+, PR+, CA125+, p53+, Vim-, CK20-, TTF1-, TG-,
GCDFP15-, CDX2-), with a serous carcinoma of tubo-ovarian
primary. Subsequent CT scans showed a widespread disease with
left ovary and pelvic lymph-nodes enlargement and solid pelvic
and omental nodules suggestive for carcinomatosis. In the following surgical restaging, after neoadjuvant chemotherapy with
apparent complete remission by imaging, histologic examination
confirmed the response to therapy with a single detectable 1 cm
residual focus of papillary serous carcinoma, immunomorphologically identical to the inguinal lymph-node counterpart located
on the left fimbriae.
Results. Our report demonstrated how a metastatic deposit of a
papillary carcinoma in an inguinal lymph node can be the unusual
presentation of an advanced tubo-ovarian serous carcinoma. In
this case the histological examination and few immunohistochemical stains proved to be an effective and inexpensive guide
for the clinician to a correct diagnosis with a consequent effective
therapy for the patient.
Aggressive angiomyxoma
R. Scamarcio, M.A. Cascarano, O. Bega, G. Musci, G. Renzulli,
L. Resta
DETO, Policlinico, Bari, Italia
Background Aggressive angiomyxoma is a rare vulvovaginal,
perineal or pelvic mesenchymal neoplasm with a marked tendency to local recurrence but does not metastasize. It has a tendency to recur after surgical excision so it is termed as aggressive.
Aggressive angiomyxoma is a rare neoplasm 150 cases has been
reported. The treatment is surgical resection.
Methods. a case of an aggressive angiomyxoma of the vulva
in a 40 years old women is presented. The tumour presented as
a slowly growing polypoid mass. The tumour was treated by
widely local excision. Histologically, the lesions were composed
of stellate and spindle shaped neoplastic cells embedded in a collagenous and hyaluronic acid- containing stroma nuclear atypia
and mitoses were absent. Typically, the lesions had an important
vascular component, often displaying medial hypertrophy and
vascular grouping. The tumour cells are characteristically positive for estrogen and progesterone receptors, suggesting a hormonal role in the development of the tumour. Surgical excision
is the treatment of choice, although treatment with gonadotropinrelasing hormone agonists is an emerging therapy.
Results. Aggressive angiomyxoma of the vulva must be distinguished from the more common benign and malignant myxoid
tumors including myxoma, myxoid lyposarcoma, myxoid variant
of malignant fibrous histiocytoma, sarcoma botryoies, and other
soft tissue tumours with secondary myxoid changes.
The treatment is surgical resection. Radiotherapy and hormonal
adyuvant is not fully stablished. Metastases are exceedingly rare,
and overall, the prognosis is good.
Sirenomelia report of a case
V. Terrinazzi, F. Castiglione, P. Pretelli, I. Montagnani,
E. Projetto, D. Moncini, A. Buccoliero, L. Messerini, C. Comin,
G. Taddei
Anatomia Patologica, AOU Careggi, Firenze, Italia
Background. Sirenomelia, also known as sirenomelia sequence,
is a very rare congenital abnormality of the lower body in which
the legs are fused togheter and a variable combination of visceral
abnormalities.
This sporadic defect occurs in about 1 in 100,000 live births and
is usually fatal. It is a severe developmental field defect of the
posteror axis caudal blastema, resulting in apparent fusion of the
lower limbs buds. It occurs in the primitive streak stage during
276
week 3 of pregnancy before development of the allantois, and the
allantoic vassels are usually absent.
It is commonly associated with renal agenesis or renal anomalies,
absent or malformed external and internal genitalia, a single umbelicar artery, imperforate anus and upper gastrointestinal tract
malformation. Other anomalies, like cardiovascular and respiratory tract malformations occur in 25% to 35% of cases.
More than half the cases of sirenomelia result in still-birth and
those born alive usually die within a day or two of birth becouse
of complications associated with abnormal kidney or bladder
development and function. This sequence is a defect most commonly seen in infants of diabetic mothers and in identical twins.
Prenatal sonographic findings of sirenomelia includes olygoidramnios, skeletal deformities, bilateral renal agenesis or
other renal anomalies and abdominal wall defects. In some cases
the oligoidroamnios severe can limit the display of other abnormalities.
Methods. Has come to our attention the case of a woman of 39
years, pregnant at 20th week subjected to therapeutic termination of pregnancy for severe oligohyidramnios and intra-uterine
growth restriction.
The clinical information in our possession included the finding
of bilateral renal abnormalities, bladder not visible, and normal
karyotype.
Results. We report here a case of sirenomelia associated with
absent external genitelia, bilateral cystic renal dysplasia, absent
bladder and onphalocele.
Mixed malignant mullerian tumor
with neuroendocrine features in an irradiated
uterus for cervical carcinoma. A unique
association? A case study
F. Sarocchi, R. Ponte, L. Abete, F. Grillo, E. Fulcheri, V.G. Vellone
UOC Anatomia Patologica, IRCCS San Martino IST, Genova, Italia
Background. Chemo-radiation represents an effective therapy
for advanced cervical carcinoma and patients often survive many
years. Endometrial cavity is very close to cervix and, during the
irradiation, receives a high dose of mutagenic stimuli so that,
when radical hysterectomy is not performed after the therapy, it
acquires an increased risk of developing a secondary malignancy.
Methods. We report a case of a 68yo women who 8ys before underwent chemo-radiotherapy for a cervical squamous cell carcinoma. Follow up showed no residual disease until TC/MRI detected
a lesion of the uterine body. Gross examination displayed a 5 cm,
solid, polypoid mass occupying most of the uterine cavity deeply
infiltrating the myometrium with involvement of the serosa. The
neoplasm owned the defining features of Mixed Malignant Müllerian Tumor (MMMT). The sarcomatous component consisted of
spindle cells with marked cytological atypia, high mitotic count,
positive for vimentin and negative for cytokeratins (AE1/AE3,
CAM 5.2, MNF-116), muscular markers (smooth muscle actine,
desmin, caldesmon), CD68 and CD10. The carcinomatous component was mixed: type I moderately differentiated endometrioid,
neuroendocrine and type II serous carcinoma. The neuroendocrine
component was characterized by typical insular pattern, wide areas
of necrosis, granular cytoplasms, irregular nuclei with vescicular
chromatin, incospicuous nucleoli and a high mitotic count. IHC
displayed strong and intense expression of neuroendocrine markers
chromogranin, synaptophysin, CD56 and cytokeratins (“dot-like”).
No residual cervical carcinoma was documented.
Results. Endometrial MMMT is a rare (3% of all uterine neoplasms), highly aggressive disease and neuroendocrine features
are even rarer; our case further demonstrates how the irradiation for cervical cancer carries an increased risk of developing
a second endometrial cancer, even many years after, often with
worrisome histologic features.
Placental chorangioma: report of a giant case
and of a seven years 16 cases collection
I. Cataldo3, S. Baggio1, G. Zanconato2, C. Luchini3, A. Parisi3,
E. Manfrin3
Scienze della Vita e della Riproduzione, AOUI G.B. Rossi, Verona, Italia;
Scienze della Vita e della Riproduzione Ostetricia, AOUI G.B. Rossi, Verona, Italia; 3 Patologia e Diagnostica, AOUI G.B. Rossi, Verona, Italia
1 2 Background. Chorioangiomas are the most frequent benign nontrophoblastic vascular tumors of the placenta. Small lesions tend
to remain asymptomatic; larger ones are often associated with
adverse fetal and maternal outcome.
Methods. Clinico-pathological data of 16 cases of placental
chorioangioma detected in our Unit from 2006 to 2013 were
analysed, focusing on the last largest case.
Results. Chorioangiomas were detected in 16 out of 2131 examined placentas. All cases were found in women over 30 years
of age, 7 (43.7%) being twin pregnancies. They were associated
with preeclampsia/eclampsia in 7 cases, IUGR or placental abruption in 4 cases, 3 cases were asymptomatic and 1 in a first
trimester abortion.
The largest lesion presented in a 32-years-old primipara with unremarkable medical history at 31+1 weeks of pregnancy, with a
non-reassuring cardiotocography, high blood pressure, headache,
diffuse oedema, uterine contractions and reduced fetal heart rate.
Ultrasound revealed a well circumscribed vascular placental
mass, giving a differential diagnosis between chorioangioma and
placental abruption. An urgent cesarean section was performed
due to the worsening of fetal and maternal symptoms. A 1,750
g female was delivered, with Apgar score 8 and 9 and normal
metabolic parameters. She stayed to the Neonatal Intensive Care
Unit for 20 days, then she was transferred for standard postnatal
care. Placental examination revealed a 9 cm exophytic placental
mass, grossly described as a well defined brownish-pale nodule,
with small haemorrhagic areas, microscopically composed of
capillaries, with or without thrombi, surrounded by perivascular
cells. No further lesions were detected.
Conclusions. The large placental tumor observed in this case
was associated with pre-term labor and eclampsia. The worsening
maternal and fetal condition imposed an urgent surgical delivery.
A causative role of placental tumors may be suspected in the
absence of other recognized contributing factors.
Vascular density in borderline ovarian tumour:
a new insight
P. Viola, T. D’Antuono, M. Liberatore, A. Di Lorito, G. Lattanzio
Medicina e Scienze dell’invecchiamento, Clinicizzato “Ss Annunziata”,
Chieti, Italia
Background. Epithelial ovarian cancer is the fourth most frequent cause of death in women and the leading cause of gynecologic cancer mortality. Traditionally, mucimous histology has
been associated with a worse overall survival compared with serous ones. Information on the prognostic significance of histology
is limited and issues arise when the tumour is borderline. We try
to compare morphological and immunohistochemical data in the
two main categories: serous and mucinous borderline tumours.
Methods. 19 borderline epithelial ovarian tumours (9 mucinuos
and 10 serous) were evaluated immunohistochemically with p53,
WT-1, MIB1 and Cd34. Vascular density (number of vessels/
mm2 within the subepithelial stroma) was assessed by Leica
DMD 108 microscope, and compared with histological features.
MIB1 positivity was expressed dividing tumours into three categories: < 5% (A), 5-10% (B) and > 10% (C).
Results. WT-1 confirmed the serous nature of the 10 cases. MIB1 showed prevalence of category C for mucinous cancers (4/9)
while serous filled A category (5/10) confirming a higher grade
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Poster
of proliferation in the former. P53 was negative in 6/9 mucinous
and in 4/10 serous tumours.
Vascular density showed a mean of 83.3 vessel/mm2 (range 70100) for mucinous tumours and a mean of 28,8 vessel/mm2 (range
15-40) for serous tumours. Althought further investigations are in
progress, we postulate that a possible cause for aggressiveness of
mucinous subtypes tumours could be explained with the higher
vascular density within the subepithelial connective tissue underneath the neoplasm.
Adenomatoid tumor of the uterus: a case report
R. Giannatiempo, L. Nugnes, M. Postiglione, M.P. Maione,
A. Russo, A. Nicastro, D. Oppressore
UOS Anatomia ed Istologia Patologica, Ospedale Venagelico Villa Betania, Napoli, Italia
Case Presentation. A 26 years old female was referred to our hospital because of recent onset of menorrhagia. The patient noticed
increased and irregular bleeding during menstruation with passage
of clots. There was no pain during menses and no history of pelvic inflammatory disease. A transvaginal ultrasound showed an
anterverted uterus that measured 10.8x5.9x7.1 cm with a discrete
intramural fibroid in the left lateral posterior aspect that measured
3 cm in largest dimension and a small hemorrhagic cyst in the left
ovary. An endometrial biopsy specimen showed proliferative endometrium and her Papanicolaou smear results were negative. She
underwent a vaginal miomectomy. A 3 cm in diameter nodule was
observed with ill defined borders, a picture rembling leiomyoma.
Hystological examination of this intramural lesion showed multiple small gland like spaces lined by flat to cuboidal epithelium.
The lining was attenuated at some places. Non mitosis or pleomorphism were seen. After provisional diagnosis of adenomatoid
tumor, the immunohistochemical stains were applied. Immunohistochemical study revealed weak positivity for cytokeratin and
strongly positivity for calretinin in glandular structures confirming the diagnosis of adenomatoid tumor of the uterus.
The immunohistochemical results support the theory of histogenesis of the adenomatoid tumor as a type of benign mesothelioma.
Sertoli leyding cell tumor of the ovary:
a case report
shaped nuclei, small nucleoli and indistinct cytoplasm intermingled
in places with clusters of pale cells, luteinized cells and aggregates
of Leydig cells. Acinar/tubular structures lined by cuboidal cells
with eosinophilic cytoplasm were focally noted and cord like arrangement of the hyperchromatic cells was also focally observed.
There was no evidence of heterologous components. Immunohistochemical staining showed Sertoli cells positive for inhibin and calretinin, but was negative for EMA, PLAP, CK7, CK-20, AFP, and
CA-125. Based on the histopathology a diagnosis Sertoli-Leydig
cell tumor without heterogeneous element was made.
Mitotically active leiomyoma: a case report
Case Presentation. A 40 years old woman was referred to our
hospital due to abnormal vaginal spotting for 4-5 months. Pelvic
ultrasound was performed and revealed a 4x3 cm uterine mass.
After simple total hysterectomy, we found an enlarged uterus
with a mss over the posterior wall. On gross examination the
tumor appeared to be unremarkable. Operative findings revealed
a smooth muscle tumor showing focal areas of ulceration and
granulation tissue. However, aside and apart from these areas the
tumor showed edema, hemorrhage and most importantly a high
mitotic count. Disregarding the endothelial cells and fibroblasts
and concentranting on the smoth muscle cells the mitotic count
was over 20/10 HPF. Additional IHC staining for KI67 was performed. The KI67 labelling index was higher than 20%. Despite
the high mitotic rate, this tumor appeared circumscribed and
displayed insignificant cytological atypia and without coagulative tumor cell necrosis. Furthermore no atypical mitotic figures
were identified. Final pathology demonstrated a mitotically active
leiomyoma of the uterus.
Smooth muscle tumor of unknown
or undetermined malignant potential (stump)
of the uterus: a case report
Case Presentation. A 39 years old (para 2 living 2) presented
to our hospital with history of pain in lower abdominal for the
past 2 months. Her menstrual cycle was normal. On transvaginal
ultrasonography discrete intramural fibroids in the posterior wall
of the uterus were noted. A diagnosis of symptomatic fibroid was
made. Vaginal hysterectomy with conservation of the adnexa was
performed. Grossly the uterus with cervix measured 9x7x4 cm, the
cervix was normal, the endometrium measured 3 mm in thickness
and myometrium measured 2 cm in thickness. The uterus showed
multiple intramural and subserosal solid masses, the largest of
which measured 4 cm. The outer surface was capsulated, smooth
with dilated prominent blood vessels. The cut surface was solid,
had whorled appearance with foci of hemorrhage at one end.
The largest mass revealed a cellular tumor composed of spindle
shaped smooth muscle cells without atypical features arranged in
fascicles and bundles. Nuclei of smooth muscle cells were elongated, had blunt ends, finely dispersed chromatin and small nucleoli.
The mitotic activity was < 2 mitosis event per 10 high-power fields
(HPFs) with mild to focally moderate nuclear atypia and there was
no evidence of tumor cell necrosis. A distinct and well demarcated
zone of coagulative necrosis was present adjacent to the cellular
zone of smooth muscles. Immunohistochemical staining for AE1/
AE3, CD34, actin, desmin, and S-100 was performed. IHC staining
Case Presentation. A 70 years old presented to our hospital with
menorrhagia. Vaginal examination revealed a firm and mobile
cystic mass in the left adnexa. An CT scan of the pelvis showed a
heterogeneous solid cystic mass replacing the left ovary. The right
ovary and the uterus were normal. The patient’s serum testosterone
was raised but markers of epithelial ovarian cancer (CA125) and
germ cell tumors (AFP) were normal. MRI revealed a normal
uterus and a heterogeneous solid mass with necrotic areas and intense contrast enhancement with high cellularity adjacent to the left
ovary. No evidence of continuity of the mass or infiltration into the
left ovary was seen. However, because there was a strong suspicion
of malignancy, the patient underwent a hysterectomy and bilateral
salpingo-oophoerectomy. Operative findings showed replacement
of the left ovary by a 4 cm by a 3 cm, grey-white, smooth-surface
mass with intact capsule without spill and with no remnant healthy
ovarian tissue. There was no spillage of tumor during surgery.
Microscopic examination of the tumor showed a circumscribed
neoplasm surrounded by loose fibroconnective tissue and variably
sized cellular nodules in an edematous/loose connective tissue
stroma containing thick walled blood vessels. The nodules were
composed of sheets of hyperchromatic cells with oval to spindle
278
results were positive for actin and desmin.. Additional IHC staining for Ki-67, p53, ER, and PR was performed. The Ki-67 labeling
index was less than 5%, and results were negative for p53. IHC
staining results for both ER and PR were positive.
Keeping in view the extensive zone of coagulative necrosis, cellular nature of the smooth muscle tumor and lack of atypia, a diagnosis of the smooth muscle tumor of unknown or undetermined
malignant potential (STUMP) of the uterus was given.
Mesonephric adenocarcinoma of the uterine
cervix: report of two cases
M.R. Ambrosio1, B.J. Rocca1, A. Barone1, A. Ginori1, G. Lobello1,
A. Ambrosio2, L. Barbagli3, R.C. Santopietro3
Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italia; 2 Università Magna Grecia, Catanzaro, Italia; 3 Sezione di Anatomia
1 Background. Adenocarcinomas derived from remnants of the
paired mesonephric ducts are rare variant of cervical carcinoma,
with relatively few, well-documented reported cases. Mesonephric remnants in the uterine cervix are vestiges of the embryonic
mesonephric system. They usually regress during early female
development but remnants of ducts and lobules may persist in
the lateral cervical walls and, occasionally, a mesonephric hyperplasia or, rarely, a mesonephric carcinoma may develop. We
describe the clinopathologic and immunohistochemical features
of two additional adenocarcinomas.
Methods. Two patients ageing respectively 64 and 39 years
underwent endometrial curettage for abnormal vaginal bleeding.
Since the diagnosis was severe atypical glandular hyperplasia a
total hysterectomy plus bilateral annessectomy were performed.
Results. Macroscopically, the tumors ranged from 6 to 8 cm in
maximum diameter and presented as an ill-defined mass that diffusely thickened the cervical wall. The tumors were firm, whitish
and focally hemorrhagic. In one case there is neoplastic infiltration
of uterine body. Histological features were consistent with the
diagnosis of mesonephric adenocarcinoma, tubulocystic variant
in one case and mixed (ductal and tubular) in the other one. Mesonephric hyperplasia was also detected. Immunohistochemically,
tumor cells expressed CK7, CAM 5.2, vimentin, EMA, p16.
The diagnosis of mesonephric lesions is difficult on routine section
as the tumors are often misdiagnosed. In this regard, since the differential diagnosis includes several endocervical lesions (exuberant
mesonephric hyperplasia, minimal deviation adenocarcinoma, endometrioid and clear cell adenocarcinoma) a panel of immunohistochemical stains is mandatory to perform the correct diagnosis and
help clinicians to employ the more appropriate treatment.
Primary precursor t-cell lymphoblatic lymphoma
of uterine corpus: case report and review
of the literature
M.R. Ambrosio1, B.J. Rocca1, A. Barone1, A. Ginori1, G. Lobello1,
A. Ambrosio2, S. Bartolommei3, R.C. Santopietro3
1 Dipartimento Di Biotecnologie Mediche, Università di Siena, Siena, Italia; 2 Università Magna Grecia, Catanzaro, Italia; 3 Sezione di Anatomia
Background. Primary lymphomas of the female genital tract are
rare. Most of them involve the cervix. The large majority of primary endometrial lymphoma are of B-cell origin. Two out of the three
cases of T-cell lymphomas described so far are peripheral T-cell
lymphoma, one is lymphoblastic lymphoma. We report the second
case of T-cell lymphoblastic lymphoma (T-ALL) of uterine corpus.
Methods. A 64 years-old female was admitted to Siena University hospital for persistent vaginal bleeding. The patient underwent
physical examination, abdominal ultrasonography, endometrial
biopsy and bone marrow biopsy.
Results. Physical examination revealed marked enlargement of
the uterus and abdominal ultrasonography showed a 6.0x4.0 cm
enhancing mass in uterine corpus. The microscopic examination
displayed a diffuse proliferation of lymphoid blasts characterized
by scant cytoplasm, irregular hypercromatic nuclei and occasionally prominent nucleoli. Numerous mitotic figures were present.
The malignant cells were positive for CD10, CD3, CD2 and
TdT. Molecular studies evidenced a clonal T-cell receptor gene
rearrangement. A diagnosis of T-ALL was made. The patient underwent bone marrow biopsy with negative results and complete
staging evaluation revealed that disease was limited to the endometrium. She was treated according to the R-CHOP protocol and
ten months after the initial diagnosis the woman is free of disease.
Primary endometrial lymphoma is a very rare entity. Fox and More
criteria establish that the diagnosis of primitivity is correct only if
the lymphoma is confined to the uterus and no signs of leukemia
are present. Our case is unusual in two respects: it manifested in a
single extra-nodal site and the patient was free of disease after 10
months. However, as two of the previously reported cases as apparent primary endometrial T-cell lymphoma developed a widespread
disease after several months, a longer follow-up is necessary.
Miscellanea
Significance of cd10 and p27 expression
in testicular germ cell tumours
R. Del Sordo, G. Bellezza, S. Ascani, I. Ferri, A. Sidoni
Department of Experimental Medicine, Section of Pathology, Perugia,
University of Perugia, Italy
Backgound. CD10, a cell surface zinc metalloendopeptidase,
and p27, a cyclin–dependent kinase inhibitor, are associated with
progression of several tumours. We recently observed a very
frequent expression of CD10 in seminomas 1. This observation
prompts us to investigate also the expression of p27 in adult
testicular germ cell tumours in order to assess their possible diagnostic, prognostic and biologic significance.
Methods. The immunohistochemical expression of CD10 and
p27 was semiquantitatively evaluated in 96 pure forms and 39
mixed forms of germ cell tumours. The clinicopathological features (age, histotype, stage and follow-up) were retrieved.
Results. Immunostaining for CD10 was positive in 68/74 (92%)
seminomas and 16/24 (67%) seminomatous component of mixed
germinal cell tumours. Spermatocytic seminomas and the nonseminomatous germ cells tumours were negative for CD10. p27
was overexpressed in all embryonal carcinomas, in 15/17 (88%)
embryonal component of mixed forms and only in 10/50 (20%)
seminomas. The intratubular germ cell neoplasia of the unclassified type (IGCNU) always expressed CD10 and p27. CD10
and p27 expression in seminoma was higher (p < 0,05) in pT1
tumours compared to pT3 tumours.
Our findings indicate that only seminomas and IGCNU, express
CD10 but when they differentiate along embryonal, somatic, trophoblastic, yolk sac lines they lose the CD10 expression. On the
contrary p27 is expressed by IGCNU and mainly by embryonal
carcinoma. This aspect suggests a possible early-phase differentiation of IGCNU to embryonal carcinoma.
In addition CD10 could be considered a useful marker to differentiate seminoma from other forms of testicular germ cell tumours
and for a better estimation of the seminomatous component in
mixed germ cell tumours.
Reference
1
Del Sordo R, Ascani S, Bellezza G, et al. CD10 is frequently expressed
in classical seminomas. Histol Histopathol 2013 Jul 16 [Epub ahead of
print].
279
Poster
Osseous metaplasia and hematopoietic bone
marrow, a rare condition in the aortic wall
M. Onorati, G. Petracco, P. Uboldi, F. Di Nuovo
Anatomia Patologica, AO G. Salvini, Garbagnate Milanese, Italia
Background. Osseous metaplasia with bone marrow formation
in the aortic wall is a rare condition and only few cases have
been described in the literature. Herein we describe an occasional
finding of osseous metaplasia with bone marrow formation in the
abdominal aortic wall, detected during an autopsy procedure. To
the best of our knowledge these findings were never previously
reported in literature.
Methods. A 84 years-old woman with a past history of gastric
cancer was admitted to our hospital with abdominal pain. Due to
her bad conditions she died after two days. The main necroscopy
finding was a transmural small bowel infarction. Moreover the
whole aorta was markedly abnormal showing a thickened wall
with severe patchy calcifications and evidence of fibroatheromas,
mainly in the abdominal segment, below the origin of the renal
arteries. Microscopically, the aorta showed the progressive atherosclerotic lesions features similar to advanced fibroatheromas
and mostly, unusual foci of mature bone containing normocellular haematopoietic marrow with all cellular lineages represented.
Results. Osseus metaplasia in the aortic wall with synchronous
presence of functioning marrow is rarely encountered. Recent
studies have demonstrated the expression of various bone-associated proteins in stenotic valves, such us bone sialo protein-2,
which indicates that valvular calcification may be an actively
regulated process. Moreover, it is well known that multilineage
tissue develops in the human artery wall, in vitro e in vivo, especially in association with atherosclerotic lesions, giving rise to
cartilage, bone, fat, muscle and vascular tissue. Our findings are
consistent with these theories hypothesizing, as some authors say,
that the artery wall is both a destination and a source of progenitor
cell that have regenerative potential and, due to unknown mechanisms, produce ectopic tissues.
Use of physician extenders in surgical pathology
practice: the pathology assistant
G. Raulli, T. Fabbri, B. Curcio Rubertini*
Patologia Clinica, S. Maria Delle Croci, Ravenna, Italia; * Agenzia Sanitaria e Sociale regione Emilia-Romagna, Assessorato alla Salute regione
Emilia-Romagna, Bologna, Italia
Background. Changes in health care economics and organization have resulted in increase use of non physician providers in
most health care setting. Use of a variety of technical and nursing professionals with advanced competence in NHS has been
debated in Italy since 1999 (l. 42/99) and in many specialties new
physician extenders have been developed. Regarding surgical
pathology, in USA and Canada, the use of physician extenders,
Pathologists’Assistants whose expertise lies in gross examination
of surgical specimens, is common.
Methods. Literature rewiew.
Results. Pathology Assistant is a laboratory professional who,
through appropriately documented delegation by a pathologist of specific responsibilities for which he has been properly
trained, performs a comprehensive gross examination of surgical
specimens 1-3. The use of Pathology Assistant has been measured
to examine the impact on laboratory efficiency and quality 4.
Specifically, the Pathology Assistant performance in lymph
node retrieval from a sample of axillary dissection and colorectal
specimens was equivalent or superior to pathology residents. In
hospital setting, is documented improvement of lymph node yield
in colorectal surgical specimen with a pathology assistant 5. Then,
is there a role and a need for appropriately trained and certified
non medical personnel (Pathology Assistant) to assist patholo-
gists in the preparation and examination of tissue in the setting
of surgical pathology in Italy? Naturally the practice of pathology is an integral part of medical care, it entails the diagnosis of
disease and often is the single most important factor upon which
decisions regarding therapy are made. These responsibilities must
remain those of a certified pathologist. The delegation of responsibilities to pathology assistant in gross specimens examination
should be similar to that given to histotechnologists who prepare
histological slides and perform other specialized techniques.
This particular delegation should always be performed under the
close supervision of a qualified pathologist. In this way, the use
of Pathology Assistant save pathologists time to perform other
necessary functions. For achieving quality in surgical pathology, pathologists need a system of subspecialization 6 7 to play
a role in clinical pathways with multidisciplinary management
of the patient 8. Despite increases in surgical pathology service
time for many pathologists during the last several years, there is
continuing pressure to improve pathology services (decreasing
turnaround times and lowering costs) from other providers and
health care organizations in managed care system 9. Workforce
modifications have been one response to managed care pressures
in many specialty practices.
References
1
Grzybicki DM, Reilly TL, Hart AR, et al. National practice characteristics and utilization of pathology assistants. Arch Pathol Lab Med
2001;125:905-12.
2
Grzybicki DM, Vrbin CM, Reilly TL, et al. Use of physician extenders
in surgical pathology practice. Arch Pathol Lab Med 2004;128:165-72.
3
Grzybicki DM, eVrbin CM. Pathology resident attitudes and opinion
about pathologist’assistants. Arch Pathol Lab Med 2003;127:666-72.
4
Galvis CO, Raab SS, D’Amico F, et al. Pathologist assistance practice
a mesaurement of performance. Am J Clin Pathol 2001;116:816-22.
5
Reese JA, Hall C, Bowles K, et al. Colorectal surgical specimen lymph
node harvest: improvement of lymph node yield with a pathology assistant. J Gastrointestinal Surg 2009;13:1459-63.
6
Black-Shaffer WS, Young RH, Harris NL. Subspecialisation of surgical pathology at Massachusetts - General Hospital. Am J Clin Pathol
1996;106(Suppl. 1):S33-42.
7
pathology in the - DGH setting: the Warwick experience. J Clin Pathol
2006;59:884-6.
8
Nakhleh RE. Quality in surgical pathology communication and reporting. Arch Pathol Lab Med 2011;135:1394-7.
9
Grzybicki DM, Galvis CO, Raab SS. The usefulness of pathologist’
assistants. Am J Clin Pathol 1999;112:619-26.
Subspecialisation as a system in anatomic
pathology unit in district general hospital setting
G. Raulli
Patologia Clinica, S. Maria Delle Croci, Ravenna, Italia
Background. Mayor changes have taken place in working
practice for surgical pathology from the mid to late nineties.
Particularly has come a trend towards subspecialisation in surgical pathology with perceived higher standards of diagnosis and
reporting. In Italy, besides university teaching hospital, there is a
need for subspecialisation as a system in surgical pathology also
in district general hospital setting.
Methods. Literature rewiew.
Results. Clinical specialties have been quick to respond to the
demand of increasing complexity of diagnosis, technological
advances and treatment with moves away from the generalist to
the specialist.
Following on from this, the pathology report ha an increase
demand for diagnostic accuracy and prognostic information on
tumors, including immunohistochemical results and molecular
biology 1 2. As well as in terms of clinicians satisfaction 3 and
patient safety and error reduction in surgical pathology 4. With
this has come a trend towards subspecialisation 5 6, conversely the
280
reasons for retaining general pathologist has been debated. First
of all, what define subspecialisation in surgical pathology? The
time a pathologist dedicates to a specific site of surgical pathology. This is underlined also by guidelines in screening program as
breast for example 7. Moreover, in all countries where the majority of anatomic pathology unit have subspecialisation as a system,
pathologists define their professional activity in terms of time
dedicated to specific subspecialties. This system can be extreme
if a pathologist is dedicated only to one site (superspecialisation)
but in general implies reporting of a limited range of grouped site
in teams, two or three sites. This is different from taking part to
rotations on the general signout service and to have one site of
subspecialty. We are speaking of subspecialisation as a system in
an organization. To have this in Italian district general hospitals
we need in general larger-size of anatomic pathology unit to have
a broad range of cytology and histopathology specimens. On the
other side we have to ensure equitable workload distribution between colleagues reporting different specimen types of different
complexity 8. At the same time, to promote the subspecialisation
system we need to communicate the advantages for individual
career progression and last but not least auditable measures of
improved pathologist performance 5 6. Subspecialisation as a system in surgical pathology requires also a workforce modification
introducing the use of physician extenders in surgical pathology
practice in gross examination as the pathology assistant 9 10. We
need to respond to increasing complexity of diagnosis, to time
for multidisciplinary meetings, to ever-increasing workloads, to
future problems in recruitment and retention of pathologists.
References
1
Nakhleh RE. What is quality in surgical pathology? J Clin Pathol
2006;59:669-72.
2
Horowitz RE. Expectations and essentials for the community practice
of pathology. Human Pathology 2006;37:969-73.
3
Zarbo RJ, et al. Customer satisfaction in anatomic pathology. A College of American Pathologistis Q-Probes study of 3065 surveys from
94 laboratories. Arch Pathol Lab Med 2003;127:23-9.
4
Nakhleh RE. Patient safety and error reduction in surgical pathology.
Arch Pathol Lab Med 2008;132:185-5.
5
Black-Shaffer WS, Young RH, Harris NL. Subspecialisation of surgical pathology at Massachusetts - General Hospital. Am J Clin Pathol
1996;106(Suppl. 1):S33-42.
6
pathology in the - DGH setting: the Warwick experience. J Clin Pathol
2006;59:884-6.
7
European Guidelines for Breast Cancer Screening and Diagnosis 2010.
8
The Royal College of Pathologists. Guidelines on staffing and workload for histopathology and cytopathology departments. 3rd ed. April
2012.
9
Grzybicki DM, Reilly TL, Hart AR, et al. National practice characteristics and utilization of pathology assistants. Arch Pathol Lab Med
2001;125:905-12.
10
Grzybicki DM, Galvis CO, Raab SS. The usefulness of
pathologist’assistants. Am J Clin Pathol 1999;112:619-26.
11
Grzybicki DM, Vrbin CM, Reilly TL, et al. Use of physician extenders
in surgical pathology practice. Arch Pathol Lab Med 2004;128:165-72.
Snapshots in digital cytology: a feasibility study
based on the software tool mathematica focused
on the image-enhancement and improving
D. Giansanti1, A. Boschetto2, M. Pochini3, L. Bottini2,
M.R. Giovagnoli3
1 Tecnologie e Salute, ISS, Roma, Italia; 2 Dipartimento di Ingegneria Meccanica e Aereospaz., Facoltà di Ingegneria, Sapienza Università di Roma,
Italia; 3 Dipartimento di Medicina Clinica e Molecolare, Facoltà di Medicina e Psicologia, Sapienza Università di Roma, Italia
Background. E-slides in digital-cytology shows serious problems of memory occupancy. For this reason, especially in the
countries with a lower development of technology (such as the
lacking of a high speed internet connection) it is preferred to use
the exchange of images with a low memory occupancy such as
the snapshots from the E-slides.
The improvement of the diagnostic power of the snapshots is a
key aspect in digital-cytology. The following issues to improve
the snapshots have been considered basic in previous studies:
–the contrast and resolution of the Chromatin detail should be
improved to better distinguish details in cancer diagnosis;
– it could be useful the emulation of the focus function, furthermore it could save memory occupancy;
–the application of methodologies for the feature-recognition
and segmentation methodologies could speed and improve the
cellular classification in cancer diagnosis.
We investigated the feasibility of the application of the tool
developed in Mathematica for the image-enhancement and improvement with reference to these issues.
Methods. 6 E-slides scanned by means of the scanner Aperio have been positioned in a server at the URL http://www.
digitalslide.it. We used Image-Scope which is compatible to the
Windows OS platforms for the connection and extraction of the
information. 15 significant snapshots have been extracted from
the WEB related to cervico-vaginal cytology.
Results. A panel operator has been developed in Mathematica
with the aim to allow the cytologist to interact with the effects
induced by the selection of level parameters. For the emulation
of the focus two sliders in the panel allow the user to navigate the
outcomes. The results of the study show the success of the methodology based on proposed procedures and a high acceptance
from the users. This methodology could be useful in telemedicine
as a tool to assist the cytologists and cytology technicians in the
remote and/or cooperative tele-diagnosis.
Localization and subcellular distribution
of the p53 activator homeodomain-interacting
protein kinase-2 (hipk2) in normal
and neoplastic thymus: preliminary data
of an immunohistochemical study
E. Gallo1, G. Deriu1, F. Facciolo2, V. Cerasoli2, M.E. Dabrowska1,
A. Papadantonakis1, E. Pescarmona1, S. Soddu3, C. Rinaldo4,
M. Marino1
1 Department of Pathology, Regina Elena National Cancer Institute, Rome,
Italy; 2 Thoracic Surgery, Regina Elena National Cancer Institute, Rome,
Italy; 3 Department of Experimental Oncology, Regina Elena National Cancer Institute, Rome, Italy; 4 CNR, Sapienza University of Rome, Italy
Background. Homeodomain-interacting protein kinase-2
(HIPK2) is a positive regulator of p53 oncosuppressor function,
acting as a p53 apoptotic function regulator either by phosphorylating its N-terminal serine 46 and facilitating Lys382 acetylation
at the C-terminus, either by affecting p53 function by different
mechanisms. Furthermore, HIPK2 is an apoptosis promoter by
acting on the p53 family members p73 and p63. HIPK2 is reported to be constitutively localized in Nuclear Bodies. HIPK2
can be deregulated in tumors by several still largely unknown
conditions. In Thymic Epithelial Tumors (TET), both organotypic (Thymoma) and non-organotypic (Thymic carcinomas)
HIPK2 role is unknown. We report here preliminary data on the
localization and subcellular distribution of HIPK2 in the epithelial and lymphoid component of TET and of normal peritumoral
thymic tissue.
Materials and methods. Thirty-two cases of TET and 8 peritumoral thymi have been investigated by immunohistochemistry
by a monoclonal antibody recently developed and set-up in our
Institute.
Results. In normal thymus, cortical and medullary epithelial cells
(EC) were found to express small nuclear granules HIPK2+. In
lymphocytes, medium-sized intranuclear granules were seen. In
TET, A and AB subtypes showed consistently HIPK2+ Nuclear
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Poster
granules, whereas in B subtypes HIPK2 granules were heterogeneously and inconstantly seen. However, in few cases of B2-B3
subtypes and in some Thymic carcinomas HIPK2 showed either
nuclear overexpression or partial cytoplasmic delocalization, thus
suggesting a possible inactivation.
Conclusion. Our preliminary data on HIPK2 first document the
presence and detectability of the p53 regulatory factor HIPK2 in
thymic normal/neoplastic EC and in their associated thymocytic
component. Immunohistochemical staining of HIPK2 could represent a new marker useful in the diagnostic work-up of TET.
Moreover, HIPK2 staining could give insight into the functional
state of apoptotic pathways in TET.
A rare systemic infection by thricosporon
capitatum without lung involvement
G. De Luca, C. Marinelli*, P. Viola*, A. Di Lorito*, G. Lattanzio*
Medicina e Scienze dell’invecchiamento, UO Anatomia Patologica,
Ss. Annunziata, Chieti, Italia; Medicina e Scienze dell’invecchiamento,
Ss. Annunziata, Chieti, Italia
Background. Trichosporon Capitatum is yeast commonly found
in the environment and may be recovered from the skin, gastrointestinal tract, and respiratory tract of healthy humans. Invasive
disease has been documented in immunocompromised patients,
especially those with blood malignancies and neutropenia. Disseminated infection can also occur in relatively immunocompetent
individuals, such as intravenous drug users, patients with prosthetic devices, and patients with severe burn injuries. Case reports and
case studies document widespread solid organ invasion including
lung, liver, spleen, kidney, bone, central nervous system, and
heart; accordingly focal nodular lesions may be seen by imaging
studies in patients with hepatic, renal or splenic involvement. In
systemic infections, hyphae and budding yeasts can also be observed in the walls of blood vessels and in the dermis. The 30-day
mortality associated with T. Capitatum ranges from 60-80%.
Methods. Autopsy report: 66 years old patient with a prosthetic
tricuspid valve dead after a surgical prosthetic replacement of
ascending aorta. The patients had necrotic gangrene of the right
foot, chronic cardiomyopathy, abdominal aortic aneurysm, multiple superficial scarring in kidneys and spleen, disseminated mycotic emboli, particularly in common iliac arteries, splenic, lienal
and superior mesenteric artery. Histologically, multiple arterial
microemboli consisting of true hyphae, pseudohyphae and anneloconidia were found in kidneys and spleen, where caused extensive areas of ischemic infarction. In lungs and heart there were
no pathologic findings of mycotic embolism. The identification
of the yeast T. Capitatum was performed through blood culture.
Results. We found a rare systemic infection in a patient with
weak risk factors consisting of valves replacement and moderate
debilitation due to cardiomyopathy. Despite the literature, there
was complete sparing of lungs thus suggesting another unusual
site of infection.
Certification according to uni en iso 9001 20002008 of the pathology unit of garbagnate
milanese during 10 years: past, present and future
M. Onorati, M. Galimberti, M.C. Scotti, I. Talamo, P. Uboldi,
F. Di Nuovo
Anatomia Patologica, G. Salvini, Garbagnate milanese, Italia
Background. On May 2002, the UNI EN ISO 9001: 2000-2008
certification was introduced on voluntary basis to standardize
operating procedures of the workflow process of our Pathology
Unit. This board certification was challenging and involved all
human resources. In this report we summarize the key points to
acquire it and focusing on benefits and negative aspects of this
long lasting process and its future implications.
Methods. Implementation of certification involved planning,
systems analysis and training of laboratory staff. The certification
programme was mainly based on pre-analytical phase (specimen
and patient identification, adequacy of clinical history, specimen
delivery) analytical phase (functions of instruments, specimen
handling, block and slide labelling, quality of histologic sections,
immunohistochemical analysis) and post-analytical phase (turnaround time, transcription error phases, cyto-histological correlations). Moreover, external and internal audits, preventive and
corrective actions and non conformities were also accomplished.
We employed extensive human resources to write a quality
manual defining and describing the quality management system
including standard operating procedures, instructions, technical
recording and job descriptions. Each document was indexed in a
unique way and all documents were rigorously used in the daily
routine and were also updated during the years.
Results. The certification should be a standard for each pathology
laboratory. The main and clear benefit of the board certification
is the possibility to obtain a global process governance with an
overall organization advantage correlated to correctness of histopathological diagnosis. We were able to detect and reduce technical errors, to ensure and control the quality of the overall process
and to improve patient’s safety. We suppose that the certification
board and the tracking technology could represent the strategic
solution in monitoring workflow process.
“Behind the glass slide”: meditations
of a pathologist
L. Baron
S.O.C. Anatomia e Istologia Patologica, S. Leonardo Asl NA3 Sud, Castellammare di Stabia, Italia
In this age of exaggerated technicality and high risk of dehumanization, health services are increasingly aiming at rationalization and efficiency. In this context, pathologists, often
already deprived of contacts with patients, risk, more than those
active in other branches of medicine, to incur in depersonalization of their role. We may consequently forget that behind each
glass slide, each diagnosis, there is an individual with his or her
anxieties, expectations and hopes of a lifetime. The risk is that
we are interested more in “our” diagnosis than in “his” or “her”
disease. We are becoming more and more molecular pathologists, devoted to research, and less and less doctors, devoted to
human welfare. This is what I had in mind when I thought of
fixing in writing a few basic principles, such as in a Decalogue,
to remember what thoughts should harbor in the mind of a pathologist when sitting at the microscope. Obviously this is not
meant to be a scientific contribution; rather, it is a modest contribution to the humanization of our profession and an invitation
to reflect on the fact that one should dismiss one’s position as
a pathologist the day one can no longer see a man or a woman
behind the glass slide.
That day
The day I will not remember that behind the glass slide
a person is wondering and a family is waiting,
the day I will not suffer for their suffering,
the day I will not decrypt in those colored glasses
the colors of life,
the day I will not realize that those tattered limbs
belong to a man or a woman
the day I will feel no emotion in diving into a world
so minute and mysterious,
The day I will have no desire to design ways to fight the enemy
into whose eyes I have looked so many times,
The day I will feel no heat from the microscope
penetrate my fingers;
that day I will, for the last time, extinguish the light of my microscope
282
and will pass to spend a new life, maybe a “better life”
in this or another world.
Expression of carbonic anhydrase ix in a selected
series of human solid tumors
G. Ilardi, S. Varricchio
Advanced Biomedical Sciences-Pathology section, AOU “Federico II”,
Napoli, Italy
Background. Drug resistance is responsible for the failure
of treatment regimens in advanced human cancers. It may be
linked to intrinsic tumor cells features, or may arise from microenvironmental influences. Tissue hypoxia has been associated
with the aggressiveness and metastasizing ability of human solid
cancers. Hypoxic cancer cells overexpress Carbonic Anhydrase
IX (CA IX), a protein that ensures a favorable tumor intracellular pH, contributing also to stromal acidosis, facilitating tumor
invasion and metastasis. Therefore, the overexpression of CA IX
is considered an epiphenomenon of the presence of hypoxic, aggressive tumor cells.
Methods. We evaluated the immunohistochemical expression of
CA IX in a series of human solid cancers, to explore its relationship with the biological behaviour of tumors. The selected tumors
were from stomach, colon, uter, ovary, urinary bladder, thyroid,
skin, oral mucosa and kidney.For all cases, tumor grade and stage
were recorded.
Results. According to most of literature data, we found CA IX
expressed in all the advanced tumors. A positive trend of correlation between CA IX overexpression, tumor stage/grade and poor
outcome emerged. The highest expression was found in the perinecrotic areas of tumors and correlated with a more aggressive
behavior of tumors. This finding supports the idea that CA IX
immunohistochemical expression may be considered an hypoxia
marker linked to a worse clinical behavior of tumors.
Lean six sigma metodology and anatomic
pathology workflow
E. Bonanno, F. Borri, M. Abbondanza, C. Tedoldi, R. Rossoni*,
A. Orlandi
Anatomia Patologica, Policlinico Tor Vergata, Roma, Italia; * RTD Business Project Manager, Roche Tissue Diagnostics, Monza, Italia
Background. The application of principles of Lean Six Sigma
and Kaizen economy to an Health Care structure optimized the
process of workflow through the improvement of work-place and
employee’s culture, wastes reduction and time saving.
Materials and methods. We analyzed every single aspect of
work processes at the Anatomic Pathology (PTV of Rome).
We made a graphic representation of the workflow designing
“spaghetti map” to overcame the workflow critical bottle-necks
and error risks nodes. The complex and innovative Workflow
implementation required the adoption of a specific IT Workflow
Solution for anatomic pathology (VANTAGE from Roche Diagnostics) fully integrated with the current LIS software. To evaluate the results we considered 1000 specimens in the same period
time of four different years (2010-2013) and the mean time for
the diagnosis (TD) was estimated.
Results. The mean value of TDs were 38,94 days in 2010; 10,88
days in 2011; 12,5 days in 2012 and 8,65 days in 2013. Furthermore, the daily quantity of paper utilized to attend the routine job
in our workflow was considerably decreased.
Discussion. The “spaghetti map” related to the years 2010- 2012
showed a complex workflow. The new workflow adopted in 2013
improved our work quality thanks to a reliable and safe contribution of the informatization process. Moreover the TDs of 2013
were appreciably lower than those observed in previous years.
These data, along with the paper saving, showed a significant
workflow improvement following the introduction of specific IT
Workflow Solution system and a significant switch to a different
job-culture, due to the application of Lean Six Sigma principles.
Autophagy and cancer. Evidence in culture in drug
trial
G. Musci, R. Rossi, A. Cavallini, B.I. Carr*, L. Resta
DETO, Policlinico, Bari, Italy; * Istituto IRCS, Castellana Grotte, Bari,
Italy
Background. Autophagy is a type of cellular reaction implemented by the organism in various physiological and pathological
conditions as the survival or suppression of a neoplastic clone and
resistance to drug therapies. Autophagy therefore can stimulate or
prevent cancer according to the context.
Methods. We research in vitro the hepatoma cell lines Hep3B
and PLC/PRF/5 by western blot, flow cytometry (FACS) and
electron microscopy, in order to verify the cellular response to
Regorafenib, fluorinated derivative of sorafenib, inhibitor of
the multi-kinase pathway and VEGFR mediated angiogenesis.
The study was performed with time-course and dose-response
methods and using a negative control sample. While considering the response to the drug three main aspects were evaluated:
(1) the MAPK pathway through the expression of ERK1 / 2 and
phospho-ERK1 / 2 (P-ERK), Jun and P-Jun, JNK and P-JNK,
genes involved in cell-division cycle regulation; (2) apoptosis
quantitatively by flow cytometry and qualitatively by study of the
expression of Bax, Bcl-2, caspase-3, -7, -8, -9 activated, Bcl-xL,
survivin genes and (3) autophagy through the study of the expression of Beclin-1 and LC3 I / II genes and electron microscopy.
Results. The study shows features of the drug on several fronts
like inhibition of cell proliferation, activation of pro-apoptotic
pathways, suppression of anti-apoptotic pathways and activation
of autophagy. Moreover, the study of electron microscopy documents various ultrastructural aspects of the autophagy process.
This study opens new perspectives for autophagy as a key element in chemotherapy treatment and it allows new considerations
on the involvement of autophagy in phenomena such as drug
resistance, chemotherapy damage limitation and post-treatment
tissue regeneration.
Malignant mesothelioma of the tunica vaginalis:
one case report studied by microarraycomparative hybridization (a-cgh)
C. Covelli1, A. Punzi1, A. Marzullo1, A. Scattone2, M. Gentile2,
A. Pennella3, T. Lettini1, F. Pezzuto1, V. Pagliarulo4, G. Serio1
1
DETO, Policlinico, Bari, Italia; 2 Medical Genetics and Deparment of
Pathology, di Venere, Bari, Italia; 3 Department of Surgery and Pathology,
di Venere, Bari, Italia; 4 DETO, Urology Division, Policlinico, Bari, Italia
Backgroung. Malignant mesothelioma (MM) of the tunica vaginalis represents only 0.3%-5% of all MM. Actually, since the first
cases described by Bayley et al (1955) and Barbera et al (1957),
approximately 224 cases are reported in the literature. Trauma,
previous herniorrhaphy and long-standing hydrocele have been
considered as predisposing factors whereas the asbestos association has been reported only in a few cases. In this site the tumor
is characterized by difficult and late diagnosis, local aggressiveness and poor prognosis. The median survival is 23 months.
Exceptionally patients survive longer. Patients present swelling
and rapid enlargement of the scrotum due to hydrocele with or
without a paratesticular or testicular mass. Only advanced MM
may invade the testis, the skin and spread into the peritoneum and
loco-regional lymph nodes. Retroperitoneal and supra-clavicular
lymph nodes and liver, lung and bone metastases are reported.
Radical orchiectomy is the mainstay of treatment. In MM growth
factors, glycoproteins, genetic mutations and enzymatic cata-
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Poster
lases have been investigated to find new insights into the genetic
background of MM and to offer some clues to develop a new
molecular target therapy.
Methods. Conventional comparative genomic hybridization (cCGH) and microarray-CGH (a-CGH) showed several genetic
defects, most commonly losses of chromosomal regions in 1p,
3p, 6q22, 8p, 9p21, 10pter→p13, 13q, 14q, 15q, 17pter→p12
and 22q. In literature none correlation between genetic changes
and survival has been documented. We describe a case of MM
of the tunica vaginalis asbestos related in a patient with long survival (45 months). The tumor sample was submitted to a-CGH.
The defects observed were 53 (26 gains and 27 losses). Largely
concordant cytogenetic aberrations were consistent with previous
genetic analysis performed in MM.
Results. a-CGH provides a high-resolution map of copy number
changes that may be critical to mesothelioma progression.
Cdkn2a deletion in peritoneal mesotheliomas
is correlated with loss of p16 protein expression
and poor survival
F. Pezzuto1, A. Marzullo1, C. Caporusso1, A. Scattone2, A. Punzi1,
A. Pennella3, F. Corsi3, C. Covelli1, T. Lettini1, G. Serio1
DETO, Policlinico, Bari, Italia; 2 Medical Genetics and Deparment of
Pathology, Di Venere, Bari, Italia; 3 Department of Surgery and Pathology,
Policlinico, Foggia, Italia
1
Background. Malignant mesothelioma (MM) is a tumor that
arises from serosal surface, in relation to the exposure to asbestos.
Without an aggressive therapy it is rapidly fatal. A preoperative
prognostic classification is necessary to predict survival and the
outcome of the clinical and surgical approaches to patients.
Methods. Histological samples of 45 malignant peritoneal mesotheliomas (MPM) were stained immunohistochemically and by
FISH assay for the p16 protein. The results were related to patient
survival. Statistical correlation was evaluated for tumor histotype,
nuclear grade, mitotic count, necrosis, lymphoid infiltration and
desmoplasia. Pure sarcomatoid tumour, high nuclear grade, mitotic count more than 5 per 10 high-power field and desmoplasia
were associated with a significantly worse prognosis. Patients
with MPM with low p16 expression (≤ 5% of positive cells)
survived less than those with high p16 expression (≥ 5% of positives cells) (p = 0.001). Patients with homozygous deletion of the
CDKN2A survived a shorter time compared to those with heterozygous deletion or without any deletion. The 50% survival time
of subjects with low p16 expression was 7.7 months (95% CI:
4.1-11.3) versus 23.2 months (95% CI: 14.5-31.9) for those with
high p16 expression. The same results were obtained comparing
the FISH assay and the survival. On multivariate analysis, tumor
histotype, nuclear grade, mitotic count and p16 expression were
found to be associated independently with survival (p = 0.0001;
p = 0.0001; p = 0.0001; p = 0.0002, respectively).
Results. Our study suggests that histotype, nuclear grade, mitotic
count and desmoplasia are negative prognostic factors in malignant peritoneal mesothelioma. P16 expression is an important
predictor of survival, related to the progression of tumor. CDKN2A deletion stained by FISH can be used in low and doubtful
immunohistochemically expression of p16 protein to determine
the risk of a poor outcome.
Immunophenotypes of primary and metastatic
nets tumors
T. Montrone, A. Maino, G. Arborea, M. Cives, F. Silvestris,
L. Resta
DETO, Policlinico, Bari, Italia
Background. Neuroendocrine tumors have a low proliferative index and an indolent behaviour. Metastatic dissemination of NETs
is a worsening factor for prognosis. Epithelial-to-mesenchymal
transition has been postulated as a pivotal mechanism driving
metastatic spread.
Major factors identified as players of EMT are trascription factors (SNAIL), growth factors (CTGF, TGFb1) and chemokine
(IL-11, PTHrP).
Moreover, several studies have revealed the predictive role of
membrane receptor (RANK, CXCR4) for bone metastasization,
but their role in NETs is currently unknown.
Methods. We collected 36 cases of NET, with or without metastases and we valuteted the immunohistochemical expression of
these markers using semiquantitative Allred score and Wilcoxon
test.
Results. 12 metastatic NET and 24 with local disease were
included. In the first group we found an higher expression of
RANK (8/12) with respect to the localized group (13/11); in
particular, 66% of cases with bone involvement showed higher
amounts of this protein. Despite no different expression of
CXCR4 was expressed at high levels in all bone-metastatic primary (6/6). Expression of TGFβ1 was variable in localized NET
(high: 12; low: 12), whereas in the metastatic group was low in
9/12. We found a high and homogeneous expression of SNAIL
in both the groups. IL-11, PTHrP and CTGF were minimally expressed without significant differences between the two groups.
Our study suggests that high expression of RANK and CXCR4 in
NET primaries is an early predictor of bone metastatization. The
role of TGFβ1 is still controversial. Since both anti-proliferative
and proliferative actions have been described in NETs, however,
we found a negative association with tumor metastatization.
Lack of DNA damage response as early anticancer
barrier in testicular tumorigenesis
F. Merolla, M.L. Vecchione
University of Naples “Federico II”, School of Medicine, Department of
Advanced Biomedical Sciences, Naples, Italy
As soon as a cell, otherwise normal, undergoes to neoplastic
transformation, it suffers from a dramatic stress that triggers several signaling pathways, in order to block cell cycle progression.
Within this response, crucial is the role exerted by the DNA damage response pathway (DDR), whose activation is an early event
in several human cancers.
DNA damage response acts, in fact, as an early anti-cancer barrier
in human tumorigenesis.
We evaluated the IHC expression of several DDR proteins in different human cancers. In particular, we focused on the expression
of CCDC6 protein in testicular germ cell tumors.
CCDC6 is a substrate of the kinase ataxia teleangectasia mutated
(ATM), which allows a proper DNA damage checkpoint in response to genotoxic stress. CCDC6 is found rearranged upon
fusion with different partners in several human cancer types.
To date, testicular germ cell tumors (TGCTs) have been reported
to lack the DDR pathway activation.
For this reason, we analyzed CCDC6 expression, along with other
crucial DDR players, on serial section of human testicular germ
cell tumors, by immunohistochemistry.
Our data show lack of DDR response in human testicular germ
cell tumors as well as lack of CCDC6 expression, confirming
CCDC6 as a DDR response protein.
Moreover, we analyzed both mouse testes tissues, by IHC and
western blot, and cultured murine testicular cells, by western blot.
We found that CCDC6 loss was the most consistent feature
among the primary tumors.
These results indicate that the loss of CCDC6 in germ cell tumors, together with lack of DDR response, may be considered as
a limiting event in testicular germ cell tumor formation.
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Left ventricula noncompaction: report of a case
E. Pegolo, N. Finato, C.A. Beltrami
Istituto di Anatomia Patologica, AOU S. Maria della Misericordia, Udine,
Italia
A 19-year-old man presented to our hospital for a self-limiting
loss of consciousness while playing a soccer match. After an
extensive clinical evaluation and radiological examination with
cardiac magnetic resonance, the diagnosis of isolated left ventricular non compaction (LVNC) was established. Two years
later, at the age of 21, the patient underwent successful heart
transplantation for a dilated cardiomyopathy with a severe left
ventricular disfunction.
The explanted heart weighted 450 g with a transverse diameter
of 11 cm and a vertical diameter of 13.5 cm; the thickness of
the left and right ventricle was 1,8 and 0,8 cm respectively.
Grossly, on cut sections, the left ventricular wall demonstrated
deep recesses and trabeculations involving two thirds of the
thickness of the wall.
Histological examination of formalin-fixed paraffin-embedded
tissue sections stained with hematoxylin and eosin showed
that the deep recesses and myocardial trabeculations extended
close to the epicardial surface and were lined by endothelium;
the trichrome special stain highlighted a marked endocardial
fibrosis and diffuse areas of interstitial and replacement fibrosis.
A panel of myocardial immunohistochemical markers was also
performed and the confirmatory pathologic diagnosis of LVNC
was established.
In summary, we have reported a case of LVNC, a rare form of
cardiomyopathy believed to be a result of an arrest in cardiac
development. Further studies are required to define the genes and
the proteins that are involved in the pathogenesis of this lesion.
Neuropatologia
Prognostic impact of mgmt promoter methylation
status in glioblastoma patients treated
with carmustine wafer implants after surgery
G. De Maglio1, T. Ius2, F. Marchesin1, F. Freguia3, L. Pagani1,
M. Skrap2, S. Pizzolitto1
Soc Anatomia Patologica, AOU S.M. Della Misericordia, Udine, Italia;
Soc Neurochirurgia, AOU S.M. Della Misericordia, Udine, Italia; 3 Università di Trieste, Trieste, Italia
1 2 Background. Glioblastoma is the most common primary malignant brain tumor. Standard treatment includes microsurgical
resection followed by concomitant chemotherapy and radiation
therapy. About 40% of patients affected by primary glioblastoma
has hypermethylation of the promoter of O6-methylguanine-DNA
methyl-transferase (MGMT) gene and benefit from alkylating
agents chemotherapy.
The prognostic impact of MGMT promoter methylation status in
patients with newly diagnosed glioblastoma who received Carmustine Wafers (Gliadel) implants after surgery has been poorly
investigated.
Methods. A cohort of consecutive 82 patients, resected for
glioblastoma in the years 2007-2013, who received the Stupp
protocol (Gliadel followed by radiotherapy plus concomitant
and adjuvant temozolomide) were analyzed for MGMT status. Genomic DNA was extracted from FFPE tissues and then
bisulfite conversion of unmethylated cytosines to uracils was
performed. MGMT promoter methylation status was assessed
by using a commercially available kit (MGMT plus®, Diatech
Pharmacogenetics) according to manufacturer’s instructions on
a PyroMarkTM Q96 ID system (Qiagen) with PyroMark CpG
(Qiagen) software. The pyrosequencing test detected and quan-
tified methylation level in ten CpG sites in exon 1 of MGMT
gene. Analysis of survival was done using Cox-proportional
hazard models. Clinical, radiographic and outcome parameters
were measured for each case.
Results. We observed MGMT methylation in 50 (60.24%) patients. Patients with MGMT hypermethylation had a longer overall survival compared to patients who had unmethylated MGMT
(p = 0.031). The extent of resection was the strongest clinical
predictor of outcome (p = 0.001). Proliferation index, IDH1-2
mutations and tumor location didn’t affect the overall survival.
We demonstrated the prognostic impact of MGMT promoter
methylation status in patients with newly diagnosed glioblastoma
who received Gliadel Wafers implant.
Histological evaluation of recurrences
of glioblastoma multiforme
V.P. Fabbri1, G. Marucci1, L. Morandi1, D. De Biase2,
E. Di Oto1, G. Tallini2, E. Franceschi3, C. Sturiale4, G.P. Frezza5,
M.P. Foschini1
Department of Biomedical and Neuromotor Sciences, Bellaria Hospital,
Bologna, Italy; 2 Dimes, Bellaria Hospital, Bologna, Italy; 3 Department of
Medical Oncology, Bellaria Hospital, Bologna, Italy; 4 Division of Neurosurgery, Bellaria Hospital, Bologna, Italy; 5 Department of Radiotherapy,
Bellaria Hospital, Bologna, Italy
1 Background. Glioblastoma (GBM) is the most frequent malignant primary tumor of the brain. Despite advances in neurosurgical treatments, radiotherapy, chemotherapy and “target therapy”
its prognosis remains poor and progression of disease is the rule.
Only a minority of patients are submitted to a second surgical
treatment.
Aim of the present study was to compare morphological, immunoistochemical and molecular findings of recurrent GBMs, at
first diagnosis and at the time of the reoperation.
Methods. Clinical data from 25 primary GBMs, diagnosed after
2005, and their recurrences, were collected. All patients were
submitted to an adjuvant treatment regimen (temozolomide and/
or local field radiotherapy). All cases were immunostained with
anti-GFAP, Olig2 and Nogo-A antisera. MGMT and IDH1 status
was assessed. Follow-up data were available in 17 cases.
Results. Cases have been morphologically subdivided in three
groups: GBMs NOS (13 cases), GBMs with oligodendrogliallike component (7 cases) and mixed group (including rare
subtypes of GBM) (5 cases). In the group of GBMs NOS the
recurrences showed morphological and immunohistochemical
findings similar to those observed in the first tumor. GBMs
with oligodendroglial-like component changed their morphology after adjuvant therapy, acquiring a gliosarcomatous aspect.
Cases included in the mixed group presented few neoplastic
cells, large amount of radionecrosis and a reduced expression
of GFAP. Correlations with follow-up data evidenced that
GBMs NOS group presented a mean time before recurrence of
11 months and a median overall survival of 24 months; on the
contrary in the mixed group these values were respectively 35
and 51 months. GBMs with oligodendroglial-like component
displayed intermediate values.
Conclusions. Evaluation of recurrent GBMs evidenced significant differences on outcome following to adjuvant therapies, also
depending on subtype of the first tumor.
A case of epidemic meningitis in drug abuser
A. Di Lorito, G. De Luca, P. Viola, C. Marinelli, G. Lattanzio
Italy
Background. The employ of heroin, a diacetyl derivative of
morphine, is common among drug users. Heroin may be taken in
different ways via intravenous or subcutaneous routes. It can be
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Poster
also smoked, sniffed or inhaled (less usually). Common complications of heroin addiction include encephalopathy, trasverse myelitis, cerebral infarctions, meningitis, cerebral abscess, mycotic
aneurysm and plexopathy.
Methods. A 53 years old man had a prolonged history of sniffed
heroin. He was admitted to our emergency department unconscious, with high fever. Computed tomographic scan (CT) of the
brain showed no significantly abnormal findings and the urine
drug screen was positive for morphine. Meningitis was suspected
and broad spectrum of antibiotics was prescribed. However, after
some hours, the patient died and the autopsy was required.
Results. Gross exam analysis showed acute myocardial infarction
and hepatic cirrhosis. In the exam of the brain, in the ventricular
spaces, choroid plexuses and arachnoid space, purulent material
has been detected. The same material was found in the trunk
and in the cerebellum. Apart of the microscopic analysis, in the
material there were diplococcus bacteria, but it was also sent for
microbiological evaluation. Usually, in the drug users, meningitis from Staphylococcus aureus and rhinopharyngeal bacterial
flora is the most common entity. In this patient, we also found
a perforation of the left eye orbit, caused by the chronic heroin
inhalation, for which we suspected a form of meningitis, caused
by Staphylococcus. However, microbiological exam showed
a positivity for Neisseria meningitidis, diplococci lanceolati,
colony- forming in the chocolate Agar. Although the heroin inhalation and the perforation of the eye orbit were strong risk factors
for meningitis caused by opportunistic pathogens, this patient was
affected by epidemic meningitis. The immunodeficiency, due to
the hepatic condition, could explain the type of infection.
A case of juvenile intracranial germinoma
E. Di Cola1, L. Sollima1, G. Coletti2, V. Ciuffetelli1, A.R. Vitale3,
P. Leocata1
1 Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita
e dell’Ambiente, Università dell’Aquila, Ospedale Civile S. Salvatore,
L’Aquila, Italia; 2 UOC Anatomia Patologica, Ospedale Civile S. Salvatore, L’Aquila, Italia; 3 UOC Anatomia Patologica, P.O. SS. Filippo e
Nicola, Avezzano (AQ), Italia
Background. Central Nervous System germ cell tumors accounted for 2-3% of primary intracranial neoplasm and for 8-15%
of specifically paediatric examples. Approximately, 80-90% of
these tumors afflict subjects younger than 25 years of age, incidence peaking in 10-14 year-olds and a clear excess of cases
involve males. Intracranial germinomas are thought to arise from
the midline totipotential cells of the rostral neural tube. Most common site is pineal region, also anterior or posterior third ventricle,
rarely fourth ventricle. Germinomas are the prevalent tumor type
in the suprasellar compartment and basal ganglionic/thalamic
regions, with non-germinomatous germ cell tumors dominating at
other sites. Rarely associated with dysgenetic syndromes.
Methods. A 21-years old man with a history of diabetes insipidus, polyuria, polydipsia presented to the emergency department
with generalized worsening weakness and visual difficulties. A
CT-scan showed a large parasellar mass, subsequently confirmed
by MRI. A campimetric examination detected a bitemporal hemianopsia. Patient subsequently underwent to neurosurgical enucleation of the mass. Histologically, H-E staining revealed 2 distinct
cell types: large round epitheliod cells with clear cytoplasm and
central nuclei, containing fine granular chromatin, prominent nucleoli and small lymphocytes, infiltrating the fibrous tissue. The
large round cells were positive for: PLAP, c-KIT, D2.40 and focally for B-HCG (some syncytiothrophoblastic giant cells). They
were negative for: a-FP, GFAP, CKAE1/CKAE3 and CD30.
Differential diagnosis includes: malignant gliomas (GFAP+),
embryonal carcinoma (CD30+) and metastatic carcinomas.
Results. We have reported this case to emphasize how less
common tumors, mimicking more aggressive lesions, should be
included in the differential diagnosis of cerebral tumors. Germinomas have a relatively good prognosis and are very sensitive to
radiotherapy and chemotherapy.
Pten haploinsufficiency correlates with other
prognostically relevant markers
R. La Starza, T. Pierini, V. Pierini, B. Crescenzi, C. Matteucci,
V. Nofrini, S. Romoli, D. Beacci, P. Giovenali*, C. Mecucci
Struttura Semplice di Genetic dei Tumori, S.M. della Misericordia, Perugia, Italia; * Citologia e Istologia Diagnostica, S.M. della Misericordia,
Perugia, Italia
Background. PTEN/10q23, a putative tumour suppressor oncogene, is mutated/deleted or downregulated in solid tumours and
T-cell acute lymphoblastic leukemias. Single-copy loss of PTEN
is more tumourigenic than complete loss because complete loss
triggers a fail-safe senescence mechanism that opposes tumourigenesis. As in glial tumours PTEN deletion (PTENdel) is more
frequent than PTEN mutations, we studied PTENdel in 52 glial
tumours prospectively collected and correlated it with immunohistochemical and genetic findings.
Methods. The study enrolled 21 females and 31 males (age
range: 30-86; median: 64) with glial or mixed neuronal-glial
tumours. Immunohistochemistry evaluated TP53 in 39 cases and
MGMT in 35. PTENdel, 1p36/19q13 deletions and EGFR/7p11
amplification (EGFRampl) were investigated by Fluorescence in
situ hybridization. Analysis of at least 100-200 well separated
cells was carried out with an Olympus fluorescence microscope.
Results. Overall EGFRampl was found in 19 cases and 1p36 and/
or 19q13 deletions in 12. PTENdel was detected in 28 cases: 2
biallelic, 1 monoallelic and 25 monosomy 10. PTENdel was associated with EGFRampl in 16 cases, 1p36/19q13 deletions in 5,
TP53 expression in 14, and MGMT methylation in 19.
Conclusions. In this Italian study PTENdel affected 53,8% of
glial tumours and was significantly associated with glioblastoma
grade IV (chi-square; P = 0,029) confirming previous reports. In
the majority of cases PTENdel underlay monosomy 10 whereas
biallelic deletion was rare. We found a significant association of
PTENdel with EGFRampl (chi-square; P = 0,002); TP53 expression
was inversely correlated with PTENdel without reaching statistical significance (chi-square; P = 0,059). In conclusion, PTENdel
might contribute to identify a highly aggressive subgroup of glial
tumours supporting a rationale for the development of therapies
targeting the PTEN-PI3K-AKT pathway
Acknowledgments. Fondazione Cassa di Risparmio di Perugia
(Grant no. 2011.0159.021).
Melanocitoma arising in the phylum terminale:
report of a case
D. Tacchini, L. Vassallo, M.A.G.M. Butorano, R. Lio, S. Cardillo*,
V.F. Muzii*
Department of Medical Biotechnology, S. Maria alle Scotte, Siena, Italy;
* Neurosurgical Clinic, S. Maria alle Scotte, Siena, Italy
Background. Meningeal melanocytomas are rare pigmented lesions originating from melanocytic cells in the leptomeninges and
they generally have a benign behaviour. Half of them arise in the
posterior cranial fossa or in the cervical or thoracic spinal tract;
phylum terminale is a very unusual location and only six cases
have been reported in literature. Differential diagnosis must be
made, in primis, with malignant melanoma.
Methods. We present a case of melanocytoma arising in the
phylum terminale in a 46 year-old man with bilateral lower limbs
weakeness and lumbar pain. RMN showed a mass at the level
of L1-L2, so the patient underwent to laminectomy. The mass
was fixed in 10% buffered formalin, embedded in paraffin and
representative sections (4 micron thick) were stained with H&E.
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Others sections were used for immunohistochemical techniques
(S100, HMB-45, Mel-A, Mib1).
Results. On light microscopy the lesion was formed by sheets
of spindle or epithelioid cells with vescicular or fusiform nuclei,
inconspicuous nucleoli and indistinct eosinophilic cytoplasms.
Intracytoplasmic melanotic pigment was evident. Necrosis and
mitoses were absent. Proliferative index was < 1%. Immunohistochemistry revealed positive reaction to HMB-45, S100 and
Mel-A. Final diagnosis of melanocitoma was made. No local
recurrences or metastases were found after 3 years follow up.
Osso e tessuti molli
Mesenteric desmoid-type fibromatosis with small
and large bowel involvement: a case report
R. Nenna, C.D. Inchingolo, V. Losito
UOC Anatomia Patologica, Bonomo, Andria (BT), Italia
Background. Mesenteric Fibromatosis (MF), also called intraabdominal desmoid tumour is a rare proliferative disease affecting the mesentery. It is part of the clinical-pathologic spectrum
of deep fibromatosis, that are clonal fibroblastic/myofibroblastic
tumor-like benign proliferations arised in the deep soft tissues,
locally aggressive with potential to infiltrate or recur, but not
metastasize. MF with intestinal involvement can be easily confused with other primary gastrointestinal tumours especially with
that of the mesenchymal origin such as GISTs to varied potential
malignant behavior. Misdiagnosis might result in inappropriate
therapeutic decisions.
Materials and methods. In June of current year (2013), a
29-year-old female was admitted to our hospital because of the
intense abdominal pain lasting from a few days. Her past medical
history was unremarkable. The physical examination revealed a
mass on palpation in the mid-abdomen that was easily movable.
The physical examination was otherwise normal. Laboratory
findings were unremarkable and the level of CEA was within
normal limits. An abdominal computer tomography (CT) showed
an 8x7x8 cm ovoid well-delineated hypodense mass located
within the leaves of the ileal mesentery attached to the ileal and
trasverse colonic bowel wall. The tumour enhanced poorly and
homogenously with an intravenous contrast. There was no evidence of loco regional lymph-nodal involvement. The patient was
underwent to laparotomy for surgical excision en-block of the
mesenteric mass together with a 58 cm segment of the ileal bowel
and a 6 cm segment of transverse colonic bowel.
Results. At gross examination, the specimen consisted of
8x7,6x8cm ovoid well-circumscribed, nonencapsulated mesenteric mass, lined externally by peritoneal serosa and attached
to the ileal and trasverse colonic bowel wall. It was firm with
an elastic consistency and cut with a gritty sensation; on cross
section, the cut surface revealed a glistening white, coarsely fasciculated surface resembling scar tissues. There no necrotic areas.
Histologically, the lesion was composed of enlongated, slender,
spindle or stellate cells of uniform appearance arranged in sweeping bundles, set in a stroma with extensive keloid-like collagen
deposition. The cells lack hypercromasia or nuclear atypia and
have small, pale-staining nuclei with minute nucleoli. No mitosis
were found in 50 HPF. These cells showed tentacular, melting
insinuation into the muscolaris propria of ileal and colonic bowel
wall without mucosal ulceration. At immunohistochemical study,
the cells stain positive for vimentin, focally for smooth muscle
actin and actin HHF35 but not for CD117, CD34, DOG1, desmin
and S-100. The first diagnostic hypothesis of a CD117-negative
GIST was finally abandoned by the finding of diffuse Betacatenin nuclear overexpression. So, our diagnostic conclusion
was “ Intra-abdominal Mesenteric Desmoid-type Fibromatosis “.
Discussion. MF is a intra-abdominal fibromatosis that develops in the mesentery or retroperitoneum. According to Stout,
fibromatosis has several distinguishing features: proliferation of
well-differentiated fibroblast, infiltrative growth pattern, presence of variable amount of collagen between the proliferating
cells, lack of cytological features of malignancy, scanty or absent
mitotic activity and aggressive clinical behavior characterized by
repeated local recurrences with no capacity of distant metastasis.
MF is relatively rare disease that may arise as an extracolonic
lesion in patient with familial adenomatous polyposis such as
FAP or especially Gardner’s syndrome, generally after previous
abdominal surgery, a trauma or a hormonal stimulation, but also
spontaneously. In these patients, MF is regarded as a proliferation of myofibroblasts that show APC gene mutations that lead
to the overexpression of beta-catenin. Nevertheless, MF has been
reported also de novo in otherwise normal patients.
Conclusions. MF with intestinal involvement can be easly
confused with GISTs. A panel of gross, microscopic and immunoistochimical features allow, in the majority of cases, a correct
identification. MF compared to GISTs is negative for CD34 and
S-100 protein while the expression of vimentin, CD117, actin
SMA, actin HHF35 and desmin do not seem to be remarkably different in the two neoplasm. In doubtful cases, it may be of diagnostic value to assess the nuclear beta-catenin that MF expresses
as distinctive characteristic from other mesenchimal tumours.
References
Fletcher C, Unni K, Mertens F. World Health Organization Classification of Tumours: Tumours of soft tissue and bone. Lyon: IARC Press
2002, pp. 83-4.
Giuliani A, Demoro M, Ciardi A, et al. Mesenteric fibromatosis. Case
report. J Exp Clin Cancer Res 2007;26:3.
Wronski M, Ziarkiewicz-Wroblewska B, Slodkowski M, et al. Mesenteric
fibromatosis with intestinal involvement mimicking a gastrointestinal
stromal tumor. Radiol Oncol 2011;45:59-63.
Levy AD, Rimola J, Mehrotra AK, et al. Benign fibrous tumors and
tumorlike lesion of the mesentery: radiologic-pathologic correlation.
AFIP Archives and RadioGraphics 2006;26:245-64.
Haddad J, Alam H, Keriakos K, et al. Reporting an unusual case of mesenteric desmoids tumor. J Med Liban 2012; 60:173-81.
Tamura K, Tani M, Kinoshita H, et al. Mesenteric desmoid tumor of the
interposed jejunal pouch after total gastrectomy. World J Surg Oncol:
2006;4:27.
Tubercolous abscess of the chest wall:
a case report
E. Unti*, A. Raimondi°, N. Scibetta*, R. Ruisi*, S. Giordano#
UO Anatomia Patologica, ° UOC Chirurgia Plastica,
Infettive, ARNAS Civico-Palermo
*
#
UOC Malattie
Background. Empyema necessitatis refers to a collection of exudative fluid that extends directly from the pleural cavity to the thoracic
wall to form a mass in the extrapleural soft tissue of the chest. It was
an uncommon complication of tuberculous pleural effusion even in
the pre-antibiotic era, and has also been associated with bacterial
lung abscess, actinomycosis, blastomycosis, and malignancies.
Methods. A 86 -year-old Italian immunocompetent man was
admitted to the hospital because of a tumor of the thoracic wall.
Computerized tomography (CT) of thorax and histopathology of the
biopsied unhealthy tissue from the cavity were consistent with the
diagnosis of TB. The patient was treated with excisional biopsy. The
specimen sent was formalin 4% fixed and paraplast plus embedded.
Sections have been prepared for H&E, PAS, Ziehl -Neelsen staining. A needle aspiration of the subcutaneous abscess was performed
and tested by polymerase chain reaction (PCR) for Mycobacterium
tuberculosis. We conducted the following surgical treatment: careful exploration of the abscess; complete resection; cavity washing
using rifamycin; coverage using muscle flap; continuous suction
and drainage; compression dressing, medical therapy.
Results. Tuberculous abscesses of the chest wall, are uncom-
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Poster
mon in European countries endemic to the disease. In the world
musculoskeletal TB occurs in 1%-3% of patients with TB, while
chest wall TB constitutes 1%-5% of all cases of musculoskeletal
TB. Grossly, the tissue resected, with maximum diameter of cm
8,2 x 3,8 x 2, consist of soft tissues and skin. Histopathological
examination showed caseous necrosis and epithelioid cell granuloma containing Langhans’giant cells and lymphocytes. Acid-fast
bacilli were demonstrated. A needle aspiration of the subcutaneous abscess was positive by polymerase chain reaction (PCR) for
Mycobacterium tuberculosis.
Expression of phosphoinositide-specific
phospholipase c enzymes in human osteosarcoma
cell lines
M. Leopizzi1, V.R. Lo Vasco2, C. Chiappetta1, C. Puggioni1,
C. Di Cristofano5, V. Petrozza3, C. Della Rocca3
Dep Medical and Surgical Sciences & Biotechnologies, Sapienza, University of Rome, Rome, Italy; 2 Department of Sensitive Organs, Policlinic
Umberto I, Rome, Italy; 3 Department of Medical and Surgical Sciences &
Biotechnologies, ICOT, Latina, Italy
1 Background. Osteosarcoma is the most common non-haematological primary malignant tumour of bone in childhood and
adolescence. The number and nature of signal transduction
pathways networking in the pathogenesis of osteosarcoma is still
uncompletely unknown. The delineation of this complex interaction among pathways raised great interest.
Intracellular calcium ions are important second messengers implicated in the control of cell death. The calcium concentration
is regulated by different signal transduction pathways, including the Phosphoinositides (PI) signaling. Phosphatydil inositol
(4,5) bisphosphate (PIP2) is critical for many cellular activities.
The levels of PIP2 are regulated by means of Phosphoinositidespecific Phospholipase C (PI-PLC) family of enzymes.
Methods. Using RT-PCR, we delineated the panel of expression
of PI-PLC enzymes in four human osteosarcoma cell lines: MG63, 143B, SaOS- 2 and Hs888.
Results. In MG-63 cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3
and ε resulted expressed. In 143B cell line, PI-PLC β1, β2, β3, β4,
γ1, γ2, δ1, δ3 and ε were expressed. In SaOS-2 cell line, PI-PLC
β1, β3, β4, γ1, γ2, δ1, δ3, ε and η1. In Hs888 cell line, PI-PLC β1,
β3, β4, γ1, δ1, δ3, δ4, ε and η1 the administration of U-73122 to
cultures briefly modifies the levels of PI-PLC transcripts.
The obtained complete expression panel of PI-PLC isoforms will
be a useful tool for further functional studies about the role of the
PI signal transduction pathway in osteosarcoma.
Congenital vascular malformations: our ten-years
experience
M. Onorati, A.A. Ianniello*, P. Uboldi, G. Petracco, F. Di Nuovo
Anatomia Patologica, AO G. Salvini, Garbagnate Milanese, Italia; * Radiologia, AO G. Salvini, Garbagnate Milanese, Italia
Background. Congenital vascular malformations (CVMs), according to the Hamburg system classification, are a genetic,
heterogeneous group of rare angiodysplasias subcategorized as
venous (VMs), lymphatic (LMs) and arteriovenous malformations (AVMs) and further subdivided into extratruncular (early in
embryonic life) or truncular forms (at a later stage in embryonic
life), based on the time of developmental arrest during embryonic
life. They are present at birth occurring only in 1.5% of the population. Radiological imaging and pathological findings play an
important role to confirm the clinical diagnosis of CVMs. We report our ten-years experience of CVMs diagnosis in our hospital.
Methods. We studied 250 cases of CVMs in our hospital during
ten years. Magnetic Resonance was considered the best imaging
examination to identify and characterize CVMs supported by his-
tological and immunohistochemical examination. VMs appeared
hypointense on T1 weighted sequences and typically hyperintense on T2. LMs were hyperintense on T2 while hypointense
or isointense on T1. AVMs appeared as an heterogeneous mass.
Sometimes the radiological differentiation with a vascular tumor
was difficult. Microscopically, these lesions were heterogenously
showing an anarchic development of the venous, arterial and
lymphatic vessels. They consisted of ectasic, thickened vessels
with a tortuous course and increased in number. There were also
arterialized veins with media layer thickening and fibrosis. The
presence of arteriovenous shunting and capillary or cavernous
hemangiomas were also observed.
Results. The accurate diagnosis of vascular malformations is
challenging for the pathologist. They can be confusion with
vascular tumours with significant implications for the patient’s
management and prognostic outcome. For this reason specific
vascular markers (GLUT-1 and VEGF) can allow a correct differential diagnosis as described recently in literature.
Ossificans myositis of platysma: an unusual site
for this pseudosarcomatous lesion
G.M. Vecchio*, A. Parafioriti, G. Angelico*, F.R. Longo*, G. Magro*
Dipartimento Di Patologia-Anatomia Patologica, Istituto Ortopedico
“G. Pini”, Milano, Italia; * G.F. Ingrassia, Policlinico Universitario
“G. Rodolico”, Catania, Italia
Background. Myositis ossificans (MO) is a pseudosarcomatous lesion characterized by heterotopic bone formation in soft tissues, especially in the musculature. Most cases arise in large muscle groups
of the upper and lower limbs. Only rarely MO may involve head and
neck region, with only one case reported in the platysma muscle.
Methods. We report a rare case of MO arising in platysma muscle of a 50-year old female with a history of recurrent temporomandibular joint dislocation. The patient presented with a painful
swelling in her left mandible. Orthopantomography showed a
soft tissue mass with peripheral calcifications. CT scan showed
a mass in the platysma muscle with peripheral ossification and a
low-density central area. Complete surgical excision of the lesion
was performed and confirmed that the underlying cortical mandible bone was not involved.
Results. Gross examination revealed a whitish, firm, nodular mass
measuring 2,9 x 2,2 x 1,6 cm. Histological examination revealed,
at low magnification, a spindle cell proliferation with bone formation, completely encircled by compressed normal-appearing
skeletal muscle. The “zoning phenomenon” typically seen in MO
was evident. The central area was composed of plump fibroblastic/myofibroblastic spindle-shaped cells displaying a mild/moderate degree of nuclear pleomorphism, high mitotic activity and a
fascicular arrangement. Numerous osteoclast-like giant cells were
also seen. Varying amounts of osteoid matrix appeared to arise
abruptly from the spindle-shaped cells or rimmed by osteoblasts.
At the periphery of the lesion, immature woven bone evolving
into mature lamellar bone was evident. Although the overall morphological appearance was typical of MO, it was the unusual site
that caused some diagnostic problems The differential diagnosis
mainly included extra-skeletal soft tissue osteosarcoma. Awareness of the possibility that MO may occur in the platysma muscle
is crucial to avoid a misdiagnosis of malignancy.
Presacral myelolipoma in a patient
with concurrent invasive ductal breast cancer
diagnosed by fnc and cnb
V. Varone*, I. Cozzolino, G. Ciancia*, G. Merola*, A. Vetrani,
G. Pettinato*
Sanità Pubblica, AOU “Federico II”, Napoli, Italia; * Scienze Biomediche
Avanzate, AOU “Federico II”, Napoli, Italia
288
Background. Myelolipomas are uncommon benign tumors
composed of mature adipose tissue mixed with hematopoietic
elements occuring both in adrenal glands and in extra-adrenal
locations; the presacral region is the most frequent extra-adrenal
location. We present the unique case of a presacral myelolipoma
diagnosed by fine needle cytology (FNC) and core needle biopsy
(CNB) in a women-with a concurrent breast cancer.
Methods. A 55 year-old women was admitted at our institution
with the cytological diagnosis of right breast cancer and a noncharacterized presacral lump. The images from a noncontrast
pelvic CT showed a 5 x 4 cm heterogeneous presacral mass
without invasion or erosion of the anterior sacrum and with mixed
fat and soft tissue attenuation. The patient uderwent a CT guided
FNC and CNB. The air-dried Diff Quick stained smears showed
trilineage hematopoietic elements in a background of mature
fat cells. The histological slides showed mature adipose tissue
admixed with cellular stroma in the absence of bone trabeculae.
The stroma consisted of myeloid, erythroid and megakaryocytic
forming cell lines.
Results. A diagnosis of presacral myelolipoma was made on the
basis of these morphological findings in conjunction with the radiological imaging. MR imaging, recommended for further characterization, showed a lobulated mass, with mixed fat/soft tissue
signal and without bony invasion. Pre-contrast fat-suppressed
images showed loss of signal intensity in the areas which were
previously iso-intense to fat, providing further confirmation of
a significant fat component of the mass. The patient was treated
only for the breast cancer with a right supero–external quadrantectomy and the ipsilateral axillary lymph node dissection; surgical biopsy of the presacral mass was not performed. Follow up
MR at 5 and 16 months of the presacral mass showed stability of
the lesion without significant interval change in size, appearance,
or signal characteristics.
Periprostatic perineurioma, description of a rare
case
M. Guerriero1, A. Di Lallo2, P. Santoro2, A.P. Dei Tos3, A.M. Pollio1
Anatomia Patologica, Ospedale Regionale A. Cardarelli ASREM, Campobasso, Italia; 2 Urologia, Ospedale Regionale A. Cardarelli ASREM,
Campobasso, Italia; 3 Anatomia, Istologia Patologica, Citodiagnostica,
Ospedale San Maria di Ca’ Foncello, Treviso, Italia
1 Background. Perineurioma represent approximately 1% of soft
tissue neoplasms, nearly always benign peripheral nerve sheat
tumours entirely composed of perineurial cells. Intraneural and
mucosal types also exist.
About 200 cases have been reported. These tumours are slightly
more common in females than males, children are rarely affected
but occur over a wide age range, with a peak in middle-aged
adults. These tumours are nearly always sporadic, most commonly arise in the lower limbs, followed by the upper limbs and
trunk; the head and neck region, visceral organs, and central body
sites are rarely affected.
Methods. We describe a rare clinical case of periprostatic perineurioma.
Results. We represent a case of a 62-years-old male patient,
came to our Institution with a two months history of pelvic and
scrotal pain.
The clinical examination showed a prostate gland with normal
morphology and size with negative digital rectal examination and
PSA within normal limits, on the contrary, trans rectal ultrasound
revealed a periprostatic oval lesion, of about 8 cm.
A first biopsy and a second biopsy were characterized by tiny,
filamentous and friable fragments; they consisted of small and
spindle cells embedded in a myxoid stroma, negative for S100,
EMA with doubtful positivity, atypia absent, proliferative activity absent. They were suggestive of a myxoid benign lesion with
spindle cells.
When the tumour was excised, it presented polylobate and pseudocapsulated of 8x5x4 cm and a weight of 55 g. When cut, the
tumour appeared sallow.
Microscopically we observed a spindle cell proliferation, consisting of elements with bipolar palely eosinofilic cytoplasm and
tapering nuclei, within a myxoid stroma, and presented immunoreactivity for EMA and CD34 (supporting perineural nature), but
lacked MSA, S100, CD68, CD117 and β-Catenin. There were no
histological signs of malignancy.
Overall, the morphological and immunohistochemical data were
most consistent with the diagnosis of perineurioma.
Retroperitoneal pecoma: a case report
F. Buffelli, C. Covelli, P. Potincasa, O. Bega, M.G. Fiore,
A. Cimmino, L. Resta
D.E.T.O., Policlinico Consorziale, Bari, Italia
Background. Perivascular epithelioid cell tumor (PEComa) is a
mesenchymal tumors of relatively recent acquisition in the WHO
classification of tumors of soft tissue and bone. PEComa class
includes a heterogeneous family of tumors, as the pulmonary
“sugar” tumor, a lymphangiomyomatosis and, in the abdominal
cavity, the renal angiomyolipoma and the clear cell myomelanocytic tumor of the falciform ligament/ligamentum teres. In the
English literature, about one hundred cases (in almost every body
site) have been reported: they often occur in middle-aged patients, with a female predominance (female-to-male ratio nearly
to 7:1). Moreover, this group of tumors has been associated to
genetic alterations of the tuberous sclerosis complex (TSC). Open
question about PEComa are histogenesis, the normal/physiological counterpart of PEC, the definition of epithelioid AML, and the
histological criteria of malignancy.
Methods. Case report. A 34 year old woman with swelling in
the right side and the right iliac fossa with a smooth surface and
in relation to the lower pole of the right kidney. Histological
evaluation showed a proliferation of epithelioid/spindled cells,
with clear to lightly granular eosinophilic cytoplasm or a diffused
uniform proliferation of polygonal cells, with a round to oval nuclei, arranged in a perivascular location, with radial spread around
the vascular lumen. High vascular density areas were observed.
Minimal necrosis and low mitotic activity were noticed in the
tumor. Immunohistochemically, the tumor cells were positive
for HMB45, vimentin, smooth muscle actin, MelanA/Mart1 and
calponin.
Result. The pathological findings in our case were consistent
with those of PEComa and immunohistochemical data further
confirmed the histological diagnosis.
Typical subcutaneous ossifying fibromyxoid tumor
of the left shoulder: a case report
S. Squillaci, A. Pitino*, C. Spairani*, M. Ferrari*, C. Cazzulino*,
M.F. Cosimi*
Anatomia Patologica, Ospedale di Vallecamonica, Esine, Italia; * Anatomia Patologica, Ospedale San Giacomo, Novi Ligure, Italia
Background. Ossifying fibromyxoid tumor (OFMT) is a rare
neoplasm of the soft tissues. The number of published cases is
relatively limited and no consensus has been reached regard its
exact histogenesis and cell differentiation lineage.
Methods. A 81-year-old white male was referred to Hospital
of Novi Ligure for a slowly growing, subcutaneous lump
from his left shoulder. The lesion was present for some years.
Grossly, a well-circumscribed mass, 6x5,5x4,5 cm in size,
with a gray-tan, solid cut surface containing a frame of calcified tissue, was seen.
Results. Microscopic examination revealed a well-demarcated,
encapsulated lesion, with a peripheral partial shell of mature
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lamellar bone. It consisted of monotonous round to spindle cells,
with rounded to ovoid vesicular nuclei, a small amount of clear
or pale eosinophilic cytoplasm, and poorly defined cellular outlines. These cells were arranged in sheets and ill-defined lobules
intermixed with irregular areas of collagenous stroma. No atypia,
mitosis or necrosis was identified. The tumor cells were positive
to vimentin, S-100 protein, desmin, calponin, CD56 and immunonegative for keratins, MART-1 and CD34. Scattered elements
showed immunoreactivity for actin.
Conclusions. The case presented herein showed histological and
immunohistochemical features consistent with typical OFMT,
that is a rare neoplasm of intermediate malignant potential
(rarely metastasizing). OFMT should be differentiated from
mixed tumor/myoepithelioma, plexiform schwannoma, sclerosing epithelioid fibrosarcoma, superficial acral fibromyxoma, and
extraskeletal osteosarcoma. Overlapping morphologic features of
the above entities sometimes make a correct diagnosis difficult.
Recently, in two cases of OFMT a novel fusion gene, EP400PHF1 was identified along with the compatible translocation t
(6;12)(p21;q24.3) in the karyotyping of one of them 1. The detection of this chimeric transcript could be a helpful diagnostic tool
in such difficult cases.
References
1
Endo M, Kohashi K, Yamamoto H, et al. Ossifying fibromyxoid tumor
presenting EP400-PHF1 fusion gene. Hum Pathol 2013 Jun 24 [Epub
ahead of print].
Epithelioid sarcoma: a case report
G. Arborea, A. Cimmino, A. Napoli, G. Ingravallo, A. Punzi,
L. Resta
D.E.T.O, Policlinico, Bari, Italia
Background. Epithelioid sarcoma (ES), is an uncommon malignant soft tissue tumor that constitute less than 1% of all soft tissue
sarcomas (which represent itself only about 8% of all malignant
tumors). Only few cases of ES has been described in literature.
Epithelioid sarcomas are in general very rare malignant tumors,
but they show preference for distal parts of the upper extremity,
particularly the hand. Since they have an indolent and sometimes
slow growth rate, they are often confounded with benign lesions.
A frequent property of these tumors is the loss or inactivation of
the tumor suppressor gene SMARCB1/INI1.
Methods. We report the case of a 5 year old child with a lesion of the palm. came under our observation a whitish-gray
fragment of 0.6 cm. Histologically it is a fragment comprising cutaneous nodular proliferation to seat deep dermal cells
in morphology fusiphorm and / or epithelioid, aggregates in
irregular fascicles, with aspects of sclerosis, discrete nuclear
pleomorphism and foci of degenerative collagen. The tumor
was immunoistochemically positive for Vimentina, EMA, CK
8.18 e CK 19 and negative for S-100, CD31, CD34, CD68 e
Melan A. According to the anatomic site, the patient’s age, the
morphological features and the immunoistochemical analysi,s
we made diagnosis of ES.
Results. The diagnosis is confirmed by the National Cancer
Institute of Milan, after a further deepening immunohistochemistry.
Orbital giant cell fibroblastoma: a rare entity.
Report of a case
D. Tacchini, L. Vassallo, M.A.G.M. Butorano, P. Toti
Department of Medical Biotechnology, Section of Anatomic Pathology,
S. Maria alle Scotte, Siena, Italy
Background. Giant cell fibroblastoma is a benign, locally recurrent soft tissue tumor commonly found in children. Primary
orbital involvement has been previously described only once.
Differential diagnosis includes dermatofibrosarcoma protuberans
(DFSP), rhabdomyosarcoma, myxoid malignant fibrous histiocytoma and myxoid liposarcoma. The case herein illustrated is of
interest not only for its rarity, but also because a correct diagnosis
enables appropriate, not aggressive treatment.
Methods. We present a case of a 13-year-old girl with proptosis
and right orbital mass. After surgical excision, the mass was fixed
in 10% buffered formalin, embedded in paraffin and stained with
H&E. Immunohistochemistry includes S100, vimentin, CD34,
Ki67, desmin, smooth muscle actin. Electron microscopy was
performed too.
Results. Histological evaluation revealed a proliferation with
poorly defined borders consisting of spindle cells with round
nuclei, arranged in discrete fascicles and embedded in a myxoid
stroma. Pseudovascular spaces lined by multinucleated giant
cells, in part in a floret-like distribution, were present. Single
giant cells were also visible into the spindle component. True storiform areas such as in DFSP were not appreciated. Necrosis was
absent. Proliferative index was about 20%. Electron microscopy
revealed cells with highly convoluted nuclei, marginal chromatin
and prominent nucleoli.
Final diagnosis of giant cell fibroblastoma avoided aggressive
treatment; the girl did not developed recurrences after 3 years.
Potential role of tenascin and periostin
as prognostic markers in giant cell tumor of bone
A. Di Bernardo, E. Armiraglio, P.A. Daolio*, R. Zorzi*,
A.C. Berardi, A. Parafioriti
Department of Pathology, Orthopaedic Institute Gaetano Pini, Milan,
Italy; * Oncologic Orthopaedic Surgery Center, Orthopaedic Institute
Gaetano Pini, Milan, Italy
Aims. Giant cell tumours (GCTs) of bone are lytic neoplasms with
variable biological aggressiveness and high recurrence rate that
occur mainly in the epiphysis of long bones of young adults. These
neoplasms can occasionally metastasize to the lung, but malignant
transformation into sarcoma is rare. According to the current state
of knowledge, histological features are not considered a valid predictor of recurrence and/or metastasis risk. The aim of this study
was to investigate the expression of different markers in classic primary GCT, including some components of the extracellular matrix,
P63 and P53 that may help to differentiate patients with increased
risk of local recurrence and/or distant metastasis.
Methods and results. 40 cases of GCT were selected and
immunohistochemical analysis of the expression of P63, P53,
Tenascin C (TNC) and Periostin (Pn) was performed. A correlation was found between different expression patterns and
clinical outcome identifying Tenascin C (TNC) and Pn as the
most promising prognostic biological markers. TNC and Pn immunoreactivity evaluation may complete and integrate the morphologic data that alone are insufficient to risk-stratify patients
and may lead to a more accurate classification and identification
of subgroups with different outcome. This evidence supports the
use of TNC and Pn as potential tools helpful in risk stratifying
GCT patients.
Conclusions. Our study provides encouraging results in the
search for potential biomarkers with relevant clinical impact
in GCT, suggesting the possible prognostic value of TNC and
Pn expression in the identification of those GCT patients with
a higher risk of relapse which, consequently, require a closer
follow-up and, possibly, an adjuvant medical therapy.
Pseudomalignant myositis ossificans mimicking
osteosarcoma: a case report
E. Unti, C. Miraglia, N. Scibetta
Dipartimento dei Servizi Diagnostici, Arnas-Civico, Palermo, Italia
290
Background. Myositis ossificans (MO) is a benign non neoplastic heterotopic bone formation that chiefly affects adolescents and
young adults, with a slight male predominance. Its presenting features can closely resemble those of a soft-tissue sarcoma, with the
resultant potential for inappropiate initial management.We report
such a disease in a young man and discuss pertinent features in
the differential diagnosis of this condition.
Methods. A 30 year-old man presented with a tender, firm,
and non-mobile mass in the muscles of the right plica axillaris
posterior. The patient had history of injury. Ultrasound imaging
showed a soft-tissue mass with calcification and increased vascularity. An excision biopsy was performed. The resected specimen
was fixed in 10% buffered neutral formalin.Tissue sections were
stained with H&E.
Results. The mass excised was firm to hard, measuring
3,8x2,8x2,7 cm. Gross examination showed a ovoid, greyishwhite lesion. Microscopically the mass showed proliferating,
immature fibroblasts in the center with hyperchromatic nuclei,
with no cytologic atypia and nuclear pleomorphism. The stroma
is richly vascular myxoid and contains osteoclast-like giant cells
and injured myocytes.The mass showed a partial “zoning phenomenon” a central layer of cells and peripheral layer of natural
osteoid, with new bone formation. The fibroblasts were positive
for SMA, vimentin, negative for S100 protein, desmin. The tumor was reported to be MO. In this case, considering the history
of traumatic injury,the absence of abnormal mitotic figures and
nuclear pleomorphism, the partial “zoning phenomenon”, the
tumor was reported to be MO, and was advised close togerther
follow-up. Although MO and soft-tissue sarcomas resemble
each other pathologically, the treatment approach for each lesion
differs greatly. The zoning phenomenon is the most important
feature for differentiating MO from osteosarcoma. Clinical, histopathological and radiological correlation should be performed
to avoid aggressive treatment of MO.
Inflammatory myofibroblastic tumor
of the omentum and mesentery: a case report
E. Unti, C. Miraglia, N. Scibetta
Dipartimento dei Servizi Diagnostici, Arnas-Civico, Palermo, Italia
Background. Inflammatory Myofibroblastic Tumor (IMF) is a
rare neoplasm composed of myofibroblastic spindle cells accompanied by inflammatory infiltrate of plasma cells, lymphocytes
and eosinophils. It is a tumor that affects children and young
adults and it can occur anywhere in the body. We report a case
of multifocal intraabdominal IMT involving the omentum and
mesentery, presenting as multiple peritoneal masses similar to
peritoneal carcinomatosis.
Methods. A 24-year-old girl presented with a 1-month history
of progressive pallor,anorexia,fevers and diffuse abdominal pain.
Ultrasound and contrast-enahanced CT showed multiple peritoneal -based lesions of varying sizes, with intralesional vascularity
on colour Doppler. The uterus and ovaries were visualized separately with minimal ascites. Radiologically the lesions mimicked
those of carcinomatosis. Exploratory laparotomy was performed
and intraoperatively excision biopsy was performed. The resected
specimen was fixed in 10% buffered neutral formalin. Tissue sections were stained with H&E.
Results. The tumor showed a predominantly spindle cell pattern
showing mild atypia, with a diffuse sprinkling of lymphocytes
and plasma cells and a few lymphoid aggregates. The mitotic
rate was 1to2 per 10 high-power fields; no atypical mitoses
has been seen. Immunohistochemical stains demonstrated diffuse cytoplasmic reactivity in tumor cells for ALK-1, vimentin, desmin, BCL2,WT1,RE.The tumor was nonreactive for
SMA,cytokeratin AE1/AE3, S100 protein, calretinin, EMA,
CD117, CD10, alpha-inibin. IMF in the current WHO classifi-
cation is classified as a neoplasm with a tendency for local recurrence in up to 25% of cases and a very low rate of metastasis.
The biologic behavior, are not predicted by histomorphology.
Genetic abnormalities described in IMT have in common rearrangements of the ALK gene on chromosome 2p. In this case,
this uncommon presentation as multifocal masses needs to be
distinguished from other causes of peritoneal carcinomatosis.
The top differentials include sarcoma, lymphoma, GIST, Castleman’s disease.
Is hemangiopericytoma a distinct entity?
C. Marinelli, P. Viola, P. Raimondi*, D. Angelucci
Medicina e Scienza dell’invecchiamento, Ss. Annunziata, Chieti, Italia;
* Scienze Sperimentali e Cliniche, Ss. Annunziata, Chieti, Italia
Background. Hemangiopericytoma (HPC) is a rare soft tissue
neoplasm first described in 1942. Over the years it was better
considered as a non specific growth pattern, rather a specific
entity. The current WHO classification, who included HPC as a
variant of the solitary fibrous tumor (SFT), has generate debate.
The two are closely related and part of a morphologic continuum.
The cellular end corresponds to classic HPC and the hyalinized
end to classic SFT. Most case have hybrid features, depending on
the relative proportion of cells and fibrous stroma. We report a
case of right inguinal pure form of classic HPC.
Methods. A large inguinal mass was incidentally discoverd in a 58
year old woman. Abdominal CT noted tortuously enhanced vessels around the tumor. Complete tumor resection was performed.
Grossly a well encapsulated mass of 10.7 cm x 5 cm x 2.7 cm,
without central necrosis or hemorrhage, was described.
Results. Microscopically it consisted of closely packed monomorphic cells arranged around an elaborate vascular network.
Some dilated sinusoidal vessels had a staghorn like appearance
and few largest channels were invested with a thin coat of collagen. By immunohistochemical analysis, cells were strongly
positive with CD34 and blc2; negative with Vimentyn, CD31,
FVIII, S100 and Desmin. Mast cells were highlighted by CD117.
Mitotic index was low (MIB1 < 1%). The differential diagnosis
included a severals neoplasms that may display HPC-like features
showing a pericytic vascular pattern, specially SFT. The type of
cells (spindle rather than round or fusiform) and their arrangement in SFT (“patternless pattern”) differed from our case. In
contrast to SFT, the neoplasm presented a striking number of
staghorn vessels, partially hyalinized; lacked hyalinized collagen
and contained mast cells. The pathologic diagnosis of HPC versus
SFT rests on a combination of architectural, cytomorphologic
and immunophenotypic features. Our case was a rare pure form
of classic HPC.
Desmoid-type fibromatosis of the Retzius space:
a case report
G. Palumbieri
U. O. C. Istologia Patologica e Citodiagnostica, Dimiccoli, Barletta, Italia
Background. Desmoid-type fibromatosis, also known as aggressive fibromatosis, is an infiltrative fibroblastic/myofibroblastic
tumor with a strong tendency for local recurrence, but without
metastatic potential; it is derived from mesenchymal progenitor
cells, usually in deep soft tissue, and is characterized by disregulation of Wnt signaling patway, with high level of nuclear
beta-catenin, where it may activate oncogenes. Primitive tumors
of the Retzius space are rare in woman; I describe the first case
of desmoid-type fibromatosis of the Retzius space, to my knowledge: a 32 year old female, during pregnancy, present a tumour
mimicking a subserosal uterine leiomyoma by ultrasound. Intraoperatively, a voluminous subperitoneal tumor, arising from the
Retzius space, was found near the anterior uterine surface;after
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tumorectomy,it was performed cesarean section. At gross examination, the tumor is a white-yellowish mass with pseudocystic
change, measuring 1 4x 13 x 10 cm.
Methods. The surgical specimen was fixed in 10% neutral buffered formalin and embedded in paraffin; section were stained
with E.E. stain, pas/pas-diastasi stain,Alcian blu stain and Gomori stain; for immunohistochemistry, the Envision System (based
on dextran polymer technology) was used: antibodies employed
are Ki67-MIB1,Vimentin,Actin-HHF35, Desmin,S100 protein,
CD34, CD117, Fattore VIII, CD99, Estrogeni, Progesterone,
EMA, CK7, CK-MNF116, p53, Beta-catenin.
Results. A poorly circumscribed, uniform neoplasm positive
for Vimentin (diffuse) Actin-HHF35, Desmin (focal) and betacatenin (diffuse, nuclear and cytoplasmic expression), composed
of spindled-to-stellate tumor cells with ill-defined eosinophilic
cytoplasm and vesicular nuclei, sometimes with small nucleoli,
arranged in long sweeping fascicles, set in collagenous stroma,
sometimes with keloidal hyalinization, or in mixoid stroma with
pseudocystic change and prominent blood vessels with perivascular oedema or hyalinization; KI67-MIB1 index is low and
rare mitotic figures are identifiable. In conclusion, preoperatively
ultrasound in the most case are not able to distinguish the rare
tumors of the Retzius space from the uterine leiomyoma. Histopatological differential diagnosis includes reactive myofibroblastic
proliferation, neurofibroma, low grade myofibroblastic sarcoma,
LGFMS, low grade MPNST.The recent literature suggest that
relantionship between recurrence rate and margin status of the
desmoid-type fibromatosis is inconsistent.
Patologia digestiva
Extremely delayed liver metastasis from duodenal
GIST
A. Ginori1, F. Scaramuzzino1, D. Tacchini1, F.S. Carbone2,
L. Alia3, S. Tripodi4
Department of Medical Biotechnologies, Pathology Unit, University of
Siena, Siena, Italy; 2 Department of Medical and Surgical Sciences and
Neurosciences, University of Siena, Siena, Italy; 3 Legatumori Senese, Siena, Italy; 4 Pathology Unit, “AOU Senese”, Siena, Italy
1
Background. Assessment of aggressive behavior of GISTs is difficult. A special attention should be paid to localization, serosal
invasion, mucous ulceration, size of the tumor and mitotic index.
It is extremely rare for liver metastases from GIST to appear more
than 10 years after surgical removal of the primary lesion. Herein
we report a case of a extremely delayed (29 years after resection
of the original tumor) giant liver metastasis from a duodenal
GIST initially diagnosed as schwannoma.
Methods. A 71-year-old man presented with abdominal pain,
fever, nausea and vomiting. In September 1984, the patient had
undergone resection of a duodenal tumor, diagnosed as schwannoma. A whole body CT scan revealed a 20x15 cm giant liver
mass in the right lobe and secondary lesions in lungs and bones.
A biopsy of the hepatic lesion was performed.
Results. The biopsy revealed a neoplasm composed of cells
with round or polygonal nuclei and an abundant acidophilic
cytoplasm, sometimes vacuolated, admixed to atypical spindle
cells. Immunohistochemically, the cells were positive to c-Kit
and Dog-1 antibodies. On these bases a diagnosis of GIST was
made. the rarity of primary hepatic GIST in the literature (only
one case have been reported to date), prompted us to reexamine
the duodenal tumor resected in 1984. It was finally reclassified as
low-grade GIST. Pyrosequencing revealed the same mutation in
the exon 11 of the c-kit gene in both duodenal and hepatic lesions.
To the best of our knowledge, this case represents the longest
interval between initial presentation of GIST and subsequent
isolated liver metastasis. Our molecular an morphological data
are not able to explain the impressive behaviour of this neoplasia.
Further investigations seems to be necessary to reveal the mechanisms responsible for these behavioral differences in apparently
similar tumors.
This case also underlines the need for a long term conservation of
formalin-fixed paraffin-embedded archives.
Gastric metastasis from breast cancer: case report
G. Dima, F. Potieri*
Oncologia, Po di Paola, Paola (CS), Italia; * Anatomia Patolgica, Po di
Rossano, Rossano (CS), Italia
Gastrointestinal metastases arising from breast cancer are rarely
found, with an estimated incidence rate of approximately 0.5%; in
60% of cases, they occur in stomach. They mainly originate from
lobular rather than ductal breast carcinoma. These metastases are
mostly detected within a linitis plastica image, during endoscopic
evaluations, while are extremely rare in the early gastric cancer
(EGC) setting. This case report deals with a gastric metastasis
from ductal breast cancer in a 62 year-old woman, who had previously undergone a left radical mastectomy for a moderately differentiated grade breast carcinoma. An endoscopic biopsy of gastric neoplasm was performed, as she reported epigastralgia associated with nausea. A histologic evaluation highlighted glandular
elements with a predominant tubular growth pattern infiltrating
the lamina propria stroma, the immunohistochemical evaluation
of which was ER-CK7 positive, and PR-CK20-CDX2 negative,
confirming the gastric metastasis from breast carcinoma.
Conclusion. Gastrointestinal metastases from breast carcinoma
are rare; in the presence of a carcinomatous gastric neoplasm in a
patient with a clinical history of invasive breast cancer, we should
suppose the probability of a secondary gastric localization; the
histomorphological and immunohistochemical evaluations are
essential to make a correct diagnosis and a consequent adequate
therapeutic protocol.
Cytomegalovirus gastritis in a elderly
immunocompromised patient affected by Horton
arteritis
M. Onorati, G. Petracco, P. Fociani*, F. Di Nuovo
Anatomia Patologica, AO G. Salvini, Garbagnate Milanese, Italia;
* Anatomia Patologica, AO L. Sacco, Milano, Italia
Cytomegalovirus (CMV) infection is a common cause of morbidity and mortality and it has been recognized as an important
pathogen among patients receiving immunosuppressive treatment, particularly corticosteroid treatment. Moreover, it is well
known that immunocompetence generally decreases with age
therefore old patients receiving immunosuppressive treatment are
at higher risk of developing CMV disease compared to younger
and immunocompetent patients. Herein we present a case of
CMV gastritis precipitated by immunosuppression, in an elderly
patient with Horton arteritis.
Background. A 77 years old man was admitted to our hospital
because of right optic neuropathy with temporary loss of vision. Horton arteritis was diagnosed and the patient underwent
a corticosteroid treatment. After treatment the patient returned
under clinical observation because of a very compromised health
condition. He underwent a chest x-ray and a chest and abdominal
CT scan that did not reveal any lesion. Moreover a gastroscopy
was performed because of a gastric tumor suspect in presence
of anemia and weight loss. The histological examination of the
specimen revealed a peculiar finding: large epithelial cells with
characteristic “owl’s eye”, eosinophilic intranuclear inclusion
body, surrounded by a clear halo. Morphology, supported by
immunohistochemical findings (CMV staining positive) and by
292
the results of Polymerase Chain Reaction, was consistent with
the diagnosis of CMV gastritis. A correct antiviral treatment was
performed and the patient is now healthy.
Results. CMV has been increasingly recognized as an important
common pathogen in an immunocompromised state especially
when caused by immunosuppressive therapies. For these reasons,
in an elderly immunocompromised patient with anemia and
weight loss, in absence of any other pathological findings, you
must remind a possible CMV gastrointestinal infection and set
up appropriate, prompt diagnostic and therapeutic interventions.
Predictive role of NHERF1 and P-gp expression
in a consecutive series of advanced gastric cancer
(GC) patients (pts)
A. Mangia1, C. Saponaro1, T.S. Dell’Endice1, A.E. Brunetti2,
V. Lorusso3, N. Silvestris3, G. Simone4
1 Functional Biomorphology Laboratory, NCRC Istituto Tumori “Giovanni Paolo II”, Bari, Italy; 2 Scientific Direction, NCRC Istituto Tumori
“Giovanni Paolo II”, Bari, Italy; 3 Medical Oncology Unit, NCRC Istituto
Tumori “Giovanni Paolo II”, Bari, Italy; 4 Pathology Pepartment, NCRC
Istituto Tumori “Giovanni Paolo II”, Bari, Italy
Background. No pre therapeutic parameters predicting response
in GC pts are available in clinical routine. Overexpression of
NHERF1 (Na+/H+ exchanger regulatory factor 1) has been reported in different tumors and plays a role in the malignant transformation of cells. MDR1/P-glycoprotein (P-gp) overexpression
in GC pts reduces intracellular anticancer drug concentrations
and is correlated with low remission rates. We investigated the
combination expression of these two proteins in a consecutive
prospective series of advanced GC pts treated with first line epirubicin, oxaliplatin and capecitabine.
Methods. Until now, 28 consecutive advanced GC pts have been
analyzed. The expressions of NHERF1 and P-gp were assessed
by immunohistochemistry on formalin-fixed paraffin embedded
tumor samples. The sections were stained for NHERF1 and P-gp
proteins and scored by two independent reviewers. Cytoplasmic
NHERF1 and membranous or cytoplasmic P-gp expression were
evaluated as percentage of stained cells with respect to the total
of tumour cells evaluated. Statistical analysis were performed
using the Prisma version 5.00 software package.
Results. All 28 patients showed a positive expression of NHERF1
and 26 (93%) showed an overexpression of Pg-p. Furthermore,
NHERF1 and Pg-p expressions were higher in 20 not-responsive
than 8 responsive patients (p = 0.0007, p = 0.0084; respectively). Interestingly, there was significant Spearman correlation between NHERF1 expression and Pg-p expression in all
tumors (p = 0.0007 r = 0.6016), and intriguingly, this association
was significant also in 20 not responsive patients (p = 0.0003,
r = 0.7270).
Conclusions. Based on the above findings, NHERF1 associated
with Pg-p expression seems to be interactively involved in the
tumor multidrug resistance of GC patients, thus representing a
promising predictive marker in this subset of patients.
Pancreatic intraductal papillary-mucinous neoplasm
(IPMN) associated with invasive carcinoma (ic)
in twin sisters: evaluation of programmed cell death
4 (pdcd4) and mir-21 expression and comparison
with five non familiar cases
S. Blandamura1, R. Cappellesso2, L. Alessandrini2, C. Sperti3,
S. Pizzi4, F. Simonato1, A. Fassina1
Dipartimento di Medicina-Dimed, Università di Padova, Padova, Italia;
Scuola di Specializzazione in Anatomia Patologica, Università di Padova, Padova, Italia; 3 Dipartimento di Chirurgia, AO Padova, Padova,
Italia; 4 UOC Anatomia Patologica, AO Padova, Padova, Italia
1
2
Background. IPMN encompasses a spectrum of pancreatic neo-
plasms showing distinctive architectural features and mucin expression. The etiology is not completely understood and they can
occur in the setting of inherited cancer syndromes and be associated with IC. PDCD4 is a novel tumor suppressor gene known to
inhibit malignant transformation and progression in many tumors,
including IPMN and to be a target of miR-21, which has recently
been reported as being upregulated in pancreatic neoplasms.
Methods. We describe two cases of familial non syndromic
pancreatic IPMN occurring in twin sisters associated with tubular
and colloid invasive carcinoma, without nodal metastases. Both
twin cases and, for comparison, five non familiar IPMNs with
associated IC were immunohistochemically screened for their
expression of CK7, CK19, CK20, MUC1, MUC2, MUC5AC and
PDCD4. The expression profile of miR-21 has been explored by
qRT-PCR analysis and by in situ hybridization (ISH).
Results. On the basis of immunohistochemistry, non invasive
IPMN was classified as of pancreatobiliary type in one twin
sister and in 4 non familial cases, whereas intestinal type was
found in the other twin sister and one non familial case. Regardless of histological subtype or familiarity, PDCD4 showed strong
nuclear and cytoplasmic staining in the intraductal component
and weak/absent staining in the invasive component. miR-21
showed opposite expression of PDCD4 with ISH-test, confirmed
by quantitative RT-PCR analysis. Survival was significantly different between familial and non familial cases.
Conclusions. PDCD-4 reduced expression in all IPMNs with
associated IC, confirmed its role as tumor suppressor gene in the
malignant transformation of IPMNs. Our data also confirm recent
studies indicating miR-21 overexpressed in pancreatic neoplasms
including IPMN. Its role as potential therapeutic target should be
studied in larger series.
Stem cell markers in pancreatic ductal
adenocarcinoma and in normal pancrea
S. Ronchi1, B.E. Leone1, F. Pagni2, C. Cattaneo3
Dipartimento di Chirurgia, Ospedale di Desio, Desio, Italia; 2 Dipartimento di Chirurgia, Ospedale San Gerardo di Monza, Monza, Italia;
3 Dipartimento di Chirurgia, Ospedale di Vimercate, Vimercate, Italia
1 Background. Stem cells differ from other cells because of their
capability of self-renewing and differentiation in a variety of
cellular types. Only few cells within the whole tumoral mass are
cancer stem cells. They have the ability of giving rise to a tumour
with the same phenotype, when stimulated to do it, and they are
characterized by intrinsic resistance to adverse condition, such as
changes of microenvironment. The “cancer stem cell theory” has
given rise to a new interpretation of tumour initiation, metastatization and recurrence after chemotherapy.
The aim of our study is to explore stemness phenotype in normal
pancreas and in pancreatic ductal adenocarcinoma.
Methods. The distribution of cells with the putative stem cell
markers CD133, ABCG2, SOX9, CD44, CXCR4 was assessed
immunohistochemically on tissue microarrays from 65 cases of
pancreatic ductal adenocarcinoma and normal surrounding pancreatic tissue.
Results. In normal pancreatic tissue, all stem cell markers were
positive in centroacinar cells, more selectively for CD133 and
ABCG2.
In tumoral tissues, CD133 was positive in 11, ABCG2 in 4,
SOX9 in 63, CD44 in 39 and CXCR4 in 34 out of 65 tumours. A
simultaneous expression of these markers was found in 2 cases.
The only statistically significant correlation was between CD133
and high grading.
Centroacinar cells could be considered the reserve of stemness,
because they express the five stem cell markers that we have
tested. Moreover, the number of these cells increases in regenerative areas.
In neoplastic tissue, CD133 and ABCG2 are likely to be consid-
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ered true stem cell markers, because positivity was found only in
a small percentage of tumours and, when they are expressed, in
small number and clustered. Moreover, ABCG2 is an interesting
marker since it is a membrane pump implied in chemoresistance
and so, a possible target for a therapy.
Synchronous occurrence of two primary
sporadic gastrointestinal stromal tumors
(gists) and periampullary adenocarcinoma (pa):
immunohistochemical and mutational analysis
and mirna-221/mirna-222 expression profile
S. Blandamura, L. Alessandrini, F. Simonato, R. Bertorelle*,
A. Fassina
Dipartimento di Medicina (DIMED), AO Padova, Padova, Italia; * Istituto
Oncologico Veneto, IRCCS, Padova, Italia
Background. GISTs originating multifocally in different GI
sites, in patients lacking familial syndromes, might be interpreted
as recurrent/metastatic disease. Multiple GISTs occurring simultaneously with other primary malignancies have been described.
We report the first case of synchronous GISTs in the stomach and
in the duodenum and concomitant PA.
Methods. A 72 year-old man, with negative family history, underwent duodenocephalopancreasectomy for PA. During gross
examination of the surgical specimen, two well demarcated
subserosal nodules, measuring 1,3 and 1,4 cm, were found in the
gastric and duodenal wall, respectively. Immunohistochemistry
for CD117 and mutational analysis for KIT and PDGFRA genes
of the two GISTs were performed. Mir-221 and miR-222 expression was analysed by qRT-PCR.
Results. PA histological assessment showed a G2 adenocarcinoma staged pT4 pN1. Both GISTs were of the spindle cell type,
without necrosis and with 0-1 mitoses/50 HPF. CD117 showed a
focal expression (dot-like pattern) in the gastric GIST, a strong
and diffuse staining (membranous pattern) in the duodenal
GIST. The former had a PDGFRA deletion in exon 12 (p. R558I562del), the latter carried a mutation of KIT in exon 9 (p. A502Y503dup), which is frequent in small bowel GISTs and associated with lower response to Imatinib. GISTs showed significantly
lower levels of miR-221/222 than normal tissue (p < 0.05) and
gastric GIST expressed two-fold increased levels of both miRs
compared with duodenal one.
Conclusions. Our data support an independent origin of the
two GISTs. Determining whether these tumors are multiple
primaries or recurrencies is helpful to predict the malignancy
of the tumors and to select the proper treatment. Although
miR-221/222 expression does not have a diagnostic impact, it
could be a new therapeutic tool especially for GISTs resistant
to conventional therapy.
A rare case of mesenteric lipoblastomatosis
in an adult with associated hydrocephalus
A. Ginori1, L. Vassallo1, C. Miracco2, M.A. Mazzei3, C. Vindigni3
Biotecnologie Mediche-Anatomia Patologica, AOUS Santa Maria alle
Scotte, Siena, Italia; 2 Scienze Mediche, Chirurgiche e Neuroscienze,
AOUS Santa Maria alle Scotte, Siena, Italia; 3 Anatomia Patologica,
AOUS Santa Maria alle Scotte, Siena, Italia
1 Background. Lipoblastoma and Lipoblastomatosis are the circumscribed and diffuse forms of a rare benign mesenchymal
neoplasm of the embryonal adipose tissue. Less than twenty cases
of these neoplasms have been reported in the literature. Predominantly they occur in infancy and early childhood.
Methods. A thirty-year-old man with congenital hydrocephalus,
limb spasticity and mental retardation was referred to our Surgery
for relapsing episodes of intestinal sub-occlusion. A radiological
diagnosis of venous occlusive ischemia from superior mesenteric
vein torsion was made and the ischemic ileal tract and related
mesentery were removed.
Results. Macroscopically the ileal segment showed a thickened,
translucid wall, with stenotic tracts and ulcerated mucosa. The
mesentery was thickened, whitish and myxoid. At microscopic
examination, the intestinal subsierosa and mesentery were diffusely infiltrated by lobules of adipose cells subdivides by fibrovascular septa. The cells, positive for S-100 protein and CD34,
ranged in differentiation from prelipoblasts to mature adipocytes
without atypia with a plexiform capillary network. A diagnosis of
mesenteric lipoblastomatosis was given.
Lipoblastomatosis, although rarely, has also been reported in a
small number of patients over 10 years of age. In our case the
patient age was older than reported by other Authors. Symptoms
are often secondary to the mass effect of the tumor, like subocclusion or intestinal infarction, requiring emergent surgery.
The association with central nervous system disorders, as in our
case, has been reported. A pathogenetic relationship between the
PLAG gene rearrangement or overexpression, detected in lipoblastomatosis and these nervous disorders has been hypothesized
because PLAG1 gene influences the development of the central
nervous system.
Solved and unsolved questions in the diagnosis of IBD
R. Suriani1, A. Sapino1, R. Rocca2, M. Daperno2, E. David1,
R. Senetta1, G. Canavese1, V. Villanacci3
Anatomia e Istologia, Città della Salute e della Scienza Molinette, Torino, Italia; 2 Gastroenterologia, Mauriaziano, Torino, Italia; 3 Anatomia
e Istologia, Ospedali Civili Brescia, Brescia, Italia
1 Background. The ECCO guidelines represent the most important
evidence-based statements on the IBD in Europe. However the
results of impact of ECCO guidelines on the clinical approach in
IBD hospitalized and ambulatory patients are lacking.
Aim. In order to improve the diagnosis and treatment of IBD in
Piedmont we proposed a permanent project training addressed to
Pathologists and Gastroenterologists. The project has as objectives: 1) increase the diagnostic skills specifications 2) identify
regional guidelines for the diagnosis of IBD 3) to promote a
continuous interdisciplinary discussion and information between
different specialists (Gastroenterologist, Pathologist, Surgeon,
Endoscopist).
We performed a series of meetings on the histopathology and
clinical management of the IBD among 12 gastroenterology and
pathology centers in Piedmont a northern region of Italy based
on the ECCO consensus statements. The aim of the project was
to reach a consensus on 27 items based on endoscopic features,
serological markers, diagnosis of IBD versus non IBD, inflammatory changes versus dysplasia, medical treatment.
Methods. For each center a minimum of two doctors specialized in the gastroenterology and pathology field were present. 17
experts and opinion leaders in the IBD proposed a lecture in six
monthly sessions form January to June 2013. Each session was
comprehensive of a overview of mucosal biopsy samples and
clinical records. Each item was subdivided in two categories:
solved and unsolved questions.
Results. We identified 18 (66%) of unsolved problems and we
proposed 3 prospective studies.
The main questions were identified in: A) the orientation of the
biopsy sample B) the histopathologic diagnosis on IBD versus
non IBD in children, C) the terminology and classification of dysplasia. The appropriateness and suitability of clinical recording
purchased to the pathologist was judged inadequate or deficient
in the major part of the cases. We identified this deficiency as the
main problem and we proposed a prospective multicenter study
to resolve it.
Conclusion. the clinical implementation of ECCO guidelines is
difficult in the routinary management of IBD patients.
294
Mesenteric fibromatosis mimicking
an intrabdominal carcinoid
P. Viola, C. Marinelli, G. De Luca, A. Di Lorito, A. Colasante
Medicina e Scienze dell’invecchiamento, UO Anatomia Patologica, Clinicizzato “Ss Annunziata”, Chieti, Italia; * Oncologia, UO Anatomia Patologica, Clinicizzato “Ss Annunziata”, Chieti, Italia
Background. The intra-abdominal fibromatoses (IAF) are a
group of related lesions that affects GI tract and includes pelvic
and mesenteric fibromatoses. Most commonly IAF arise in the
small bowel mesentery and they could be related to FAP, Gadner’s syndrome or following intra-abdominal surgical resection.
We present a case of a patient with history of colorectal cancer,
which developed an intra-abdominal nodule 4 years later, mimicking a carcinoid on PET/TC.
Methods. A 60 years old man, during his follow up, discovered
an intra-abdominal nodule (4x2,3 cm), with borderline SUV
uptake to PET/TC. It was located 2 cm far from the anastomosis
of the previous right colectomy, so a new intestinal resection
was performed. Ileum with mesenteric adipose tissue measuring
40 cm in length and up to 6.5 cm was examined. Within the fat,
there was a hard consistency nodule, grey to white in colour,
measuring 2.2 cm in maximum diameter. Histologically it was
composed of bland, spindle cells in collagenous or sometimes
myxoid stroma. Even if grossly it was well demarcated, its margins showed infiltration of the surrounding tissues. Immunohistochemistry were performed to characterize the lesion (Vimentin,
SMA, Desmin, S-100, CD34 and CD117).
Results. Intra-abdominal fibromatoses are lesions CD117 (c-Kit)
negative which differentiate them from gastrointestinal stromal
tumours; CD34 stains lesional cells weakly and in a patchy distribution and it is typically less intense than in solitary fibrous
tumours. Most fibromatoses are reactive with SMA, Desmin (specific markers for myogenic differentiation among soft tissue tumours) and Vimentin which is present in soft tissue tumours, but
is not considered to be cell type-specific. In this case, the PET/TC
result rised the possibility of a carcinoid because of a borderline
uptake value. The morphology, the IHC and the clinical history
excluded a neuroendocrine lesion favouring the fibromatoses.
Assessment of minimum biopsy set for HER2
status evalutaion in gastric and gastro-esophageal
adenocarcinoma
L. Mastracci1, I. Gullo1, N. Piol1, L. Molinaro2, M. Fassan3,
A. De Silvestri4, V.G. Vellone1, M. Rugge5, R. Fiocca1, F. Grillo1
Disc, Pathology Section, IRCCS S. Martino - IST, Genova, Italia; 2 Dep
of Biomedical Sciences and Human Oncology, Ospedale San Giovanni
Battista, Torino, Italia; 3 Dep of Pathology and Diagnostics, AOU Integrata Verona, Verona, Italia; 4 Clinical Epidemiology and Biometric Unit,
IRCCS S. Matteo, Pavia, Italia; 5 Dimed, Surgical Pathology & Cytopathology Unit, University of Padova, Padova, Italia
1
Background. Trastuzumab represents a therapeutic option in
advanced or metastatic HER2-positive adenocarcinoma of the
stomach (GC) or gastro-esophageal junction (GEC). The assessment of HER2 status in these patients is mainly based on
the availability of endoscopic biopsies. Several authors have
demonstrated that the immunohistochemical expression of HER2
in GCs and GECs is highly heterogeneous, thus introducing the
problem of reliability of endoscopic biopsies in assessing HER2
status in these tumors. The aim of the study was to identify the
minimum endoscopic biopsy set required to confidently evaluate
HER2-status in GC and GEC.
Methods. One hundred and three cases of resected GCs and
GECs were retrieved. For each case two different sections were
immunostained for HER2. Assessment of HER2 was performed
by three expert pathologists, reaching a consensus for each case.
HER2 stained slides were therefore digitally scanned (Aperio
XT). Ten random virtual biopsies (2.6 mm diameter) for each
case were drawn on the luminal part of the sample by using a digital image analysis software and HER2 expression was assessed
for each virtual biopsy separately. Finally the HER2-status, as
defined by biopsy sets composed of a progressively increasing
number of virtual biopsies, was compared with the overall value
of HER2 expression initially assessed for each case.
Results. A 5 biopsy set was shown to be the most accurate in
predicting overall HER2-status with 91.89% sensitivity and
96.97% specificity. Specificity did not significantly differ
(range from 96.43% to 97.56%) if fewer biopsies were considered while sensitivity increased from 61.54% for 1 biopsy to
89.19% for 4 biopsies. Above 5 biopsies no increase in accuracy
was seen, so suggesting that this is the minimum biopsy set
required in order to obtain maximal accuracy for HER2 assessment in GCs and GECs.
Mixed adenoneuroendocrine carcinoma (manec)
of the gallbladder: a case report
F. Pitto, F. Sarocchi, P. Calamaro, L. Mastracci, V.G. Vellone,
R. Fiocca, F. Grillo
Anatomia Patologica, IRRCS San Martino-IST, Genova, Italia
Background. Malignant tumors of the gallbladder are rare and
the vast majority are adenocarcinomas. Mixed adenoneuroendocrine carcinomas (MANEC) are found even more rarely in
the gallbladder and only few cases have been reported in the
literature.
Methods. A 76-year-old woman was admitted for gallstones. US
showed a voluminous gallstone and a 40 mm mass projecting in
the gallbladder lumen showing vascularization at color-Doppler.
Cholecystectomy with resection of IV/V liver segments, resection
of the round ligament and hepatic hilum and retro-portal lymphadenectomy was performed.
Results. On gross examination a polypoid, soft and friable mass
in the gallbladder fundus was observed. Microscopically this
mass corresponded to a polypoid adenocarcinoma, moderately
differentiated (G2) with a mixed structure, in part tubular and in
part papillary, which invaded the stromal axis of the lesion and
gallbladder wall up to the muscularis. In the invading part of the
neoplasm a poorly differentiated small cell neuroendocrine pattern (30% of the total lesion) was seen. This component showed
high mitotic activity (29/10 HPF) and proliferation (Ki67 index of
80% - G3) and expression of synaptophysin (100%) and chromogranin (2% to 50%). Furthermore, in the exocrine component disperse chromogranin-positive neuroendocrine cells were observed.
Lymphatic, perineural and vascular invasion were present. Peritoneal serous, round ligament and liver segments were free from
neoplastic infiltration. The patient died a few months later.
The histogenesis of these neoplasms has yet to be elucidated.
They may derive from a collision of two different neoplastic cell
types or the more likely neoplastic transformation of a common
precursor cell with differentiation/de-differentiation in two separate, exocrine and endocrine, cell lines.
Margin resection status in radical
pancreaticoduodenectomy: a case-control study
M. Amato, G. Perrone, G. Nappo*, D. Borzomati*, R. Coppola*,
A. Onetti Muda
Anatomia Patologica, Policlinico Universitario Campus Biomedico di
Roma, Roma, Italia; * Chirurgia Generale, Policlinico Universitario Campus Biomedico di Roma, Roma, Italia
Background. Resection Margin (RM) involvement is a key prognostic factor for patients with pancreatic duct adenocarcinoma 1;
recent studies suggest that standardizated and meticulous pathological examination increase the R1 rate 2. Aim of the study is
295
Poster
to evaluate RM status using a standardized protocol for surgical
specimens examination.
Materials and methods. 50 pancreaticoduodenectomy (PD) specimens were analyzed applying a standardized protocol (SP) for
pathological evaluation. Circumferential resection margin of the
specimen was inked with 6 different colors and completely examined. PD specimens were serially sliced (3 mm) in a single axial
plane perpendicularly to the longitudinal axis of the duodenum.
Data were compared with 50 PD resection specimens processed by
non standardized protocol (NSP). Also, two different cut-offs were
adopted to define a positive RM in both SP and NSP series: 0mm
and 1mm. The minimum tumour distance to the ink was established on the basis of microscopic evaluation of all slices.
Results. Using 1mm clearance, SP showed a significantly higher
number of R1 resections than NSP (p < 0,05) (SP: 66% vs NSP:
26%). Significant differences between SP and NSP were found
in terms of the number of lymph nodes (median 32, IQR 26,2540,25 vs median 9, IQR 5-11), number of metastatic lymph nodes
(median 5, IQR 2-7 vs median 0, IQR 0-2), pN+ cases (45 vs
23). No differences were found between the two groups in terms
of resection margins status (NSP:10% vs. SP: 14%) when 0mm
clearance was applied. NSP group also showed a greater R1 rate
when using as 1mm clearance.
Conclusion. Standardization of the histopathological examination of pancreaticoduodenectomy specimens coupled with 1 mm
clearance criteria, influences the reporting of RM status, thus allowing a better correlation with pathological staging.
Reference
1
Allema JH, Reinders ME, van Gulik TM, et al. Prognostic factors for
survival after pancreaticoduodenectomy for patients with carcinoma
of the pancreatic head region. Cancer 1995;75:2069-76.
2
Verbeke CS, et al. Br J Surg 2006;93:1232-37.
First case of amphicrine carcinoma
of the ampullary region
A. Ginori, L. Vassallo, G. Lo Bello, L. Alia*, M. Vestri,
M. Cintorino, S. Tripodi
Biotecnologie Mediche - Anatomia Patologica, AOUS Santa Maria alle
Scotte, Siena, Italia; * Lega Tumori Siena, Siena, Italia
Background. Amphicrine carcinoma represents a peculiar tumor
in which exocrine and neuroendocrine features are present in
the same neoplastic cell,which show a divergent differentiation
expressing mucus and neuroendocrine granules within the cytoplasm. In the 2010 WHO classification of tumors of the digestive
tract, they are included in the intermediate grade malignant category of Mixed Adenoneuroendocrine Carcinomas (MANECs).
They are extremely rare. At least 4 cases have been reported in
the stomach. Herein, we report the first case in the literature of
amphicrine carcinoma of the ampullary region.
Methods. A 69-year-old man presented with right hypochondrial
pain, nausea, anorexia and weight loss. A CT scan of the abdomen revealed a cefalopancreatic neoplastic lesion involving the
ampullary region, with some locoregional lymphnodes suspicious
for metastases. A duodenocefalopancreasectomy was performed.
Results. The gross specimen showed a solid, yellowish lesion
(2 cm in maximum diameter) of the ampullary region, infiltrating
the duodenal wall and the pancreas. The histologic examination
showed a neoplasm composed of medium-sized to large cells with
anaplastic nuclei, prominent macronucleoli, granular chromatin,
and finely granular cytoplasm. These cells were disposed mostly in
solid nests and trabeculae, admixed with areas showing a glandular
pattern with foci of signet ring cell carcinoma. The same neoplastic
cells had a bivalent differentiation, epithelial and neuroendocrine,
showing a strong positivity for synaptophysin, chromogranin, cytokeratin 7 and carcinoembryonic antigen. Furthermore the same
cells expressed neutral and acid mucin (Alcian-PAS). A final diag-
nosis of intermediate grade malignant MANEC of the amphicrine
type of the ampullary region was made. Amphicrine carcinomas
are extremely rare neoplasms but they must be correctly diagnosed
to allow the choice of a correct treatment.
Preliminary evaluation of ghrelin (gh) gene
and protein expression in the surgical specimen,
and ghrelin serum concentration after primary
or revisional laparoscopic sleeve gastrectomy (lsg)
N. Porta, F. De Angelis, J. Cacciotti, C. Chiappetta, O. Iorio,
G. Cavallaro, V. Petrozza, G. Silecchia, C. Della Rocca,
C. Di Cristofano
Medical Surgical Sciences and Biotechnologies, Sapienza University of
Rome, Pathology Unit, ICOT, Latina, Italy
Introduction. Gh plays a role in food intake and insulin secretion/
suppression and is involved in weight-loss after LSG. The aim of
this prospective study was to evaluate differences of the Gh-gene
expressions and Gh-cells density in the LSG resected specimens.
Methods. 62 patients (mean BMI 42.6) underwent LSG (primary
45, revision 17. DMT2 10). TaqMan real-time quantitative PCR
and IHC Gh expression were evaluated in fundus, body and prepyloric region of the specimens. RNA was extracted by using
SV Total RNA Isolation System. RNA was reverse-transcribed
in a final volume of 20 uL using High Capacity cDNA RT Kit.
Gh-gene expression in each areas of stomach was evaluated.
Gh-protein expression was detected by immunohistochemistry
in FFPE tissues using anti-ghrelin antibody. For each area of
resected stomach, Gh cells per high power field (x400) were
counted and mean values were calculated. Total plasma ghrelin
levels were obtained before and 1 month postoperatively in 31
Patients (Primary 25, revision 6,DMT2 4).
Results. The mean Gh-mRNA expression was 2,5 throughout
the stomach. IHC showed a mean Gh cells percentage of 24,7%
(fundus 27,3%, body 23,9%, antrum 22,8%) BMI was negatively
correlated with protein and Gh-gene in tissues specimens. Gh cell
density decrease from the proximal stomach to the distal stomach
(p 0,007). No significant differences in Gh-expression in primary
vs revisional LSG were found. A significant difference was found
in the gastric body Gh expression in diabetes vs no diabetes
27,5% vs 22,9 % respectevely (p. 0,008). One month after surgery, ghrelin serum levels decreased more in the revisional group
(6 patients) (from 76,3 pg/ml to 5,7 pg/ml) than in the primary
group (25 patients) (from 73,7 pg/ml to 15,1 pg/ml).
Conclusion. Diabetic patients showed an higher Gh cell density
in the gastric body than no diabetic. One month after surgery the
decrease of Gh serum concentration is similar to primary and
revisional group.
Immune response and intestinal adenomas:
role of T-lymphocytes and macrophages
A. Maglietta1, L. Resta1, A. Marzullo1, R. Maglietta2, G. Marra3
DET

Journal of the Italian Society of Anatomic Pathology and Diagnostic

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