SIOP Europe - Società Italiana di Chirurgia Pediatrica

Société Internationale d'Oncologie Pédiatrique
SIOP
Europe
International Society of Paediatric Oncology
Unresectable Neuroblastoma Protocol
Neuroblastoma Localizzato Non Operabile
MYCN non-amplificato
età > 1 anno
Protocollo Terapeutico
Versione finale: Aprile 2000
(Emendato: Ottobre 2000)
Neuroblastoma Localizzato Non Operabile
MYCN non Amplificato
età > 1 anno
Protocollo Terapeutico
Questo protocollo deriva dalla discussione di diversi gruppi cooperativi nazionali europei.
Il comitato di scrittura è stato costituito da:
Oncologia Pediatrica:
Jan Kohler
Victoria Castel
Alberto Garaventa
Herve Rubie
United Kingdom (Chairman)
Spain
Italy
France
Chirurgia Pediatrica:
Keith Holmes
Tom Monclair
Giovanni Cecchetto
United Kingdom
Norway
Italy
Anatomia Patologica:
Claudio Gambini
Italy
Biologia:
Gian Paolo Tonini
Italy
Statistica:
John Imeson
Paulo Bruzzi
United Kingdom
Italy
Comitato di monitoraggio:
Kate Matthay, Les Robison e Jean-Michel Zucker
Comitato biologico:
Peter Ambros
Inge Ambros
Jerome Couturier
Nick Bown
John Lunec
Comitato Anatomia Patologica: Michel Peuchmaur
Emanuele d’Amore
Klaus Beiske
Claudio Gambini
Inge Ambros
Traduzione italiana a cura di Alberto Garaventa, Massimo Conte e Paolo Magillo
COORDINATORI NAZIONALI
AUSTRIA
Dr Ruth Ladenstein
St Anna’s Children’s Hospital
Kinderspitalgasse t
1090 VIENNA Austria
Tel
+ 43 1 40470
Fax
+ 43 1 40170 759
E mail
[email protected]
BELGIO
Dr Genevieve Laureys
Universital Hospital Gent
De Pintelaan 185
9000 GENT Belgium
Tel
+ 32 9 2402111
Fax
+ 32 9 2403875
E mail
[email protected]
DANIMARCA
Dr Niels Carlson
Dept of Pediatrics
Odense University Hospital
DK-5000 Odense, Denmark
Tel
+ 45 66 11 33 33
Fax
+ 45 65 91 18 62
E mail
FRANCIA
Dr Herve Rubie
Hopital Des Enfants
330 avenue de Grande-Bretagne
B.P. 3119-31026 TOULOUSE, Cedex 3, France
Tel
+ 33 5 34 558609
Fax
+ 33 5 34 558612
E mail
[email protected]
GERMANIA
Prof Frank Berthold
University of Cologne
Dept. of Padiatrics
Joseph Stelzmann Straae 9
50924 KOLN Germany
Tel
+ 49 221 4784380
Fax
+ 49 221 4784689
E mail
[email protected]
ITALIA
Dr Alberto Garaventa
Dept Hematology/Oncology
G Gaslini Children’s Hospital
Largo Gerolamo Gaslini 5
16148 GENOVA Italy
Tel
+ 39 010 5636464
Fax
+ 39 010 3762322
E mail
[email protected]
NORVEGIA
Dr Ingebjorg Storm-Mathisen
University Hospital
Dept Paediatrics
00027 OSLO Norway
Tel
+ 47 22 869067
Fax
+ 47 22 422822
E mail
PORTOGALLO
Dr Ana Forjaz de Lacerda
Instituto de Oncologia
R Prof Lima Basco
1093 Lisboa Codex
PORTUGAL
Tel
+ 35 1 217 200 429
Fax
+ 35 1 217 200 417
E mail
SPAGNA
Dr Victoria Castel
Hospital Infanil “La-Fe”
Avenida Campanar 21
46009 VALENCIA Spain
Tel
+ 34 96 3862789
Fax
+ 34 96 3868700
E mail
[email protected]
SVEZIA
Dr Ildiko Marky
Dept Paediatrics
Ostra Hospital
Ostra-S-41685
GOTHENBURG Sweden
Tel
+ 46 31
Fax
+ 46 31 843010
E mail
[email protected]
SVIZZERA
Dr Maja Nenadov Beck
University Hospital (chuv)
Rue de Burgnon
1011 LAUSANNE Switzerland
Tel
+ 41 21 3143567
Fax
+ 41 21 3143332
E mail
[email protected]
GRAN BRETAGNA
Dr Jan Kohler
Dept Paediatric Oncology
Southampton General Hospital
Tremona Road
Southampton
SO16 6YD HAMPSHIRE UK
Tel
+ 44 23 80794101
Fax
+ 44 23 80794962
E mail
[email protected]
INDICE
Page
1.
Introduzione e Razionale
5
2.
Dati Europei
6
3.
Obiettivi
9
4.
Disegno dello Studio
10
5.
Eligibilità
11
6.
Inquadramento diagnostico
11
7.
Linee Guida Anatomo Patologiche
12
8.
Studi Biologici
12
9.
Linee Guida Chirurgiche
13
10.
Terapia
17
11.
Modifiche per tossicità
18
12.
Valutazione durante il trattamento
19
13.
Valutazione al termine del trattamento
20
14.
Elementi statistici
20
15.
Consenso informato
23
16.
Bibliografia
27
ASPETTI ORGANIZZATIVI
17.
Appendici: 1.
International Staging & Response Criteria
29
2.
Scintigraphy Protocol
33
3.
Drug Information
36
4.
Common Toxicity Criteria
39
5.
Treatment of Progressive Disease
42
6.
SIOP Pathology & Biology Guidelines
45
7.
Organisational Aspects
65
8.
National Reference Laboratory Guidelines
68
1.
1.1
1.
INTRODUZIONE E RAZIONALE
Età e stadio alla diagnosi sono considerati i fattori prognostici più importanti nei
bambini affetti da neuroblastoma. Recentemente vari studi hanno documentato il
ruolo prognostico di alcuni fattori biologici. In particolare, l’amplificazione dell’
oncogene MYCN appare fortemente associata a maggior rischio di recidiva. Su
questa base il protocollo europeo LNESG (94.01 TRIAL), da poco concluso, ha
trattato con la sola chirurgia i pazienti affetti da neuroblastoma stadio INSS 2 senza
amplificazione di MYCN.
Il ruolo della terapia adiuvante nei casi di malattia in stadio 3 (e dei pochi casi di
stadio 2) non resecabile alla diagnosi, ma con caratteristiche biologiche favorevoli
deve dunque essere valutato.
1.2
Solo il 15-20% di tutti i casi di neuroblastoma si presentano in stadio 3 e di questi il
10-15% presentano amplificazione di MYCN. Vi è inoltre un piccolo sottogruppo di
neoplasie in stadio 2 non resecabili alla diagnosi. Data la scarsa numerosità di
questa casistica solo uno studio cooperativo multinazionale può rispondere ad
interrogativi relativi all'approccio terapeutico. I dati derivati dal LNESG (che
comprende 11 gruppi nazionali) indicano che un simile studio può reclutare circa 40
casi di stadio 3 per anno, dei quali due terzi di età superiore all’anno alla diagnosi, il
10% dei quali avrà il gene MYCN amplificato.
1.3
Resecabilità del tumore primitivo
La discussione relativa alla terapia del neuroblastoma localizzato si è spesso
focalizzata sulla asportabilità della neoplasia sia alla diagnosi che dopo
chemioterapia. Matthay et al3 hanno riportato un'eccellente sopravvivenza per i
casi di neuroblastoma di Stadio 2, indipendentemente dal residuo chirurgico.
1.3.1 Strother et al4 hanno analizzato l’ event-free survival (EFS) a 2 anni di 49 pazienti di
età superiore all’ anno alla diagnosi con stadio 2B o 3 biologicamente favorevole
trattati con i protocolli POG 8742 and 9244. L’ EFS a due anni per 21 bambini con
neoplasia completamente asportata alla diagnosi (15 stadio 2B, 6 stadio 3) era
dell'85% e del 70% per i 28 pazienti con neoplasia asportata in modo non radicale
(5 stadio 2B, 23 stadio 3). Nonostante il trend le differenze in EFS non erano
statisticamente significative. Un numero inferiore di stadi 3 erano stati sottoposti ad
intervento radicale alla diagnosi, ma non vi era differenza nella EFS. Una
chemioterapia intensiva era somministrata a tutti pazienti (Vincristina, Cisplatino,
VP16, Ciclofosfamide + Doxorubicina o Carboplatino) e la radioterapia veniva
somministrata solo ai pazienti con malattia residua dopo intervento chirurgico
differito.
1.3.2 Powis et al5 hanno valutato gli effetti dell'escissione chirurgica radicale nei pazienti
con stadio 3 di Evans registrati nello studio dell’ ENSG tra il 1982 e il 1992. Nessun
dato biologico era valutabile. La sopravvivenza complessiva (OS) dei pazienti di età
> 1 anno con asportazione macroscopicamente radicale della neoplasia non
differiva significativamente da quella dei pazienti con asportazione non radicale (OS
67% vs 42%; p =0.06). Se tuttavia l'esame istologico confermava l'asportazione
chirurgica radicale (come fu documentato in 29 su 47 casi) vi era un vantaggio
significativo (OS 86% vs 44%; p < 0.007).
1.3.3 Questi dati concordano con quelli di Haase et al6 per il Children’s Cancer Study
Group (CCSG) che ha riportato una sopravvivenza: del 77% per i 26 pazienti nei
quali era stata ottenuta una asportazione chirurgica radicale contro il 28% per i 32
casi con chirurgia incompleta. Nella più recente analisi del CCSG 7 relativa a 136
pazienti, anche l'asportazione chirurgica radicale era associata ed una OS migliore
(86% vs 56%).
1.3.4 Kushner et al8 suggeriscono che per i casi di neuroblastoma localizzato o di stadio
4s con caratteristiche biologiche favorevoli la chemioterapia non sia necessaria.
Essi hanno descritto 31 pazienti di cui 17 di stadio 2 o 3. Sei dei 13 pazienti di
stadio 2 e due dei 4 di stadio 3, avevano un residuo macroscopico dopo l'intervento
chirurgico alla diagnosi. Curiosamente, l’unico paziente di stadio 2 che ricevette
chemioterapia fu il solo a presentare una recidiva. Il 53% dei pazienti di stadio 2 e
3 avevano < 1 anno alla diagnosi. Tuttavia 2 pazienti di stadio 3 con residuo
macroscopico avevano 14 e 72 mesi alla diagnosi. Kushner non ha documentato
nessun caso di neoplasia con caratteristiche biologiche favorevoli progredita da
stadio < 4 a stadio 4.
2. DATI EUROPEI
2.1
FRANCIA (SFOP)
La SFOP ha descritto 316 casi di neuroblastoma localizzato dei quali 164 di età > 1
anno alla diagnosi. L'outcome dei bambini sotto e sopra all'anno era simile ma
l’EFS per i pazienti con neoplasia resecabile era leggermente superiore a quella dei
pazienti con neoplasia non asportabile (89% vs 78%).
La presenza di
amplificazione di MYCN era il fattore prognostico sfavorevole più significativo.
Un ulteriore lavoro10 della SFOP si è concentrato sui 130 bambini della stessa
serie, nei quali la neoplasia era stata giudicata inoperabile. Di questi 130 casi 78
avevano > 1 anno alla diagnosi e 34 dei 41 valutabili per MYCN non avevano
amplificazione. La chemioterapia iniziale (2 cicli di Carboplatino e VP16 seguiti da 2
cicli di Ciclofosfamide + Vincristina + Doxorubicina [CADO]) era stata seguita dalla
chirurgia in 33 pazienti, risultata radicale in 21 (64%). I pazienti con residuo
postoperatorio, sia macroscopico che microscopico, ricevevano due ulteriori cicli di
chemioterapia. Alcuni di essi, persistendo un residuo macroscopico (n=16), sono
andati incontro ad un secondo intervento (n=4), o hanno ricevuto radioterapia sul
residuo (n=4). La tossicità della chemioterapia è stata moderata. Con questa
strategia l'OS e l'EFS dei 34 pazienti MYCN non amplificati di età superiore all'anno
alla diagnosi, sono risultate essere rispettivamente 94% + 8% e 91% + 14%,
confrontabili a quelli dei bambini con neoplasia primitiva resecabile.
Terapia attuale: Carboplatino/VP16 e CADO.
2.2
SPAGNA
Castel et al11 ha pubblicato il trattamento di 33 bambini di stadio 3 (secondo
Evans), di età > 1 anno alla diagnosi. La chemioterapia di induzione comprendeva
alte dosi di Cisplatino/VM26 alternate ad Endoxan /Doxorubicina. Dopo la chirurgia
differita il trattamento di mantenimento era protratto per 14 mesi.
La radioterapia è stata erogata solo ai pazienti con neoplasia residua. 15 pazienti
hanno ottenuto una remissione completa, 7 una buona remissione parziale, 6 una
remissione parziale e in 3 vi è stata progressione di malattia. La sopravvivenza
attuariale a 48 mesi è del 60%.
Un secondo studio esteso tra il 1992 e il 1998 includeva 36 casi di stadio 3 secondo
INSS, di età superiore ad 1 anno alla diagnosi. L'intervento chirurgico radicale
d'emblé fu eseguito in un solo paziente. L'LDH era elevato in 18/35 casi e MYCN
era amplificato in 5 dei 24 casi valutati.
La chemioterapia comprendeva due cicli di Endoxan alte dosi + Vincristina, due di
Carboplatino + VP16 e tre di Cisplatino + VM26 in un periodo di 96 giorni.
Dopo la chirurgia differita il trattamento di mantenimento durava 9 mesi, ma i
pazienti con MYCN amplificato ricevevano chemioterapia ad alte dosi seguita da
infusione di cellule staminali emopoietiche autologhe.
La OS a 5 anni è del 70%, ed è in particolare 73% per i casi con MYCN non
amplificati, 42% per i casi con MYCN amplificato (66% per quelli di MYCN non
noto). L'EFS è pari al 66%.
Terapia attuale: i pazienti di stadio 3 in età > 1 anno alla diagnosi senza
amplificazione di MYCN o MYCN non noto, ricevono 4 cicli del precedente
trattamento chemioterapico con aggiunta di Amifostina. Dopo la chirurgia ricevono 4
ulteriori cicli di chemioterapia.
2.3
ITALIA
Garaventa et al12 hanno pubblicato l'esperienza italiana riguardante 145 casi di
neuroblastoma localizzato inoperabile trattati dal 1979 al 1990. I quattro protocolli
utilizzati in quel periodo comprendevano una chemioterapia iniziale seguita da una
chirurgia differita e nei due protocolli più recenti i pazienti in remissione completa
dopo la chirurgia differita non ricevevano altro trattamento.
La progression-free survival (PFS) a 5 anni dell'intero gruppo è stata del 54% (43%
con i protocolli meno recenti che utilizzavano dosi standard vs 59% con i protocolli
più recenti che utilizzavano alte dosi).
La PFS era significativamente migliore nei 48 pazienti sotto l'anno rispetto ai 97
bambini di età superiore (72% vs 39%; p = <0.001). I risultati non sostenevano
l'ipotesi che la chemioterapia di consolidamento comportasse un vantaggio nei
bambini in cui il trattamento chirurgico differito avesse portato all'asportazione
completa della neoplasia ed inoltre anche i bambini con residuo post-chirurgico non
sembravano avere un vantaggio dalla radioterapia sulla sede del tumore primitivo.
Terapia attuale:
Pazienti a rischio standard (tutti i bambini di età < 1 anno alla diagnosi e quelli di
età superiore con neoplasia pelvica o toracica): ricevono 6 cicli di Vincristina +
Endoxan + Adriamicina.
Pazienti ad alto rischio (bambini di età > 1 anno con neoplasia addominale):
ricevono una chemioterapia simile a quella dei pazienti di stadio 4 senza terapia ad
alte dosi, se hanno ottenuto la remissione completa.
NB: i pazienti ad alto rischio con MYCN amplificato hanno un outcome
sfavorevole, mentre quelli senza amplificazione genica hanno una prognosi
favorevole anche se la remissione completa non è stata ottenuta. Questi
pazienti potrebbero trarre vantaggio dalla MIBG a dosi terapeutiche.
2.4
AUSTRIA
Terapia attuale: (A-NB 94)
Vi è una stratificazione dei pazienti in base allo stato di MYCN.
I pazienti MYCN non amplificati ricevono due cicli di VP16 - Carboplatino e
Endoxan -Adriamicina - Vincristina seguiti da chirurgia e ulteriore terapia secondo il
medesimo schema.
2.5
GERMANY
Nel trial NB90 124 pazienti con neuroblastoma localizzato sono stati registrati, 95
erano Stadio 3 INSS e 29 Stadio 2.
La OS e la EFS a 3 anni sono risultate rispettivamente al 94% e 82%. Il trattamento
è stato più breve che nei trials precedenti (NB 82 : 10 cicli, NB 85 : 4-7 cicli e NB 90
: 4cicli) ma non per questo associato ad un outcome inferiore. Le recidive sono
state osservate sino a 5 anni dalla diagnosi e l'outcome dei pazienti recidivati è
stato sfavorevole (per le recidive locali: sopravvivenza a 10 anni pari a 26%, per le
recidive metastatiche: sopravvivenza 10 anni pari al 10%). Questo dato è simile a
quanto già pubblicato in precedenza. Il rapporto tra recidive locali e metastatiche è
approssimativamente 1:113.
Terapia attuale: L'attuale trial NB 97 stratifica i pazienti in base all'età, sintomi e
amplificazione di MYCN. Il gruppo a rischio standard comprende pazienti di età
superiore all'anno con neoplasia non resecabile in Stadio 2 e 3 con MYCN non
amplificato insieme agli Infants con sintomi life- or organ-threatening. Il gruppo
standard raccoglie il 12% circa di tutti i neuroblastomi (gruppo solo osservazione:
49%, gruppo alto rischio: 38.6%). Il trattamento consiste in quattro cicli di Cisplatino
- VP16 - Vendesina alternati a Vincristina - DTIC - Adriamicina e chirurgia differita.
Se non si ottiene la remissione completa o VGPR i pazienti ricevono radioterapia
sulla sede del tumore primitivo.
2.6
GRAN BRETAGNA/ENSG
La sopravvivenza a 5 anni per pazienti Stadio 3 entrati nello studio ENSG è pari al
65%5. Ci sono alcune recidive dopo 5 anni che portano la sopravvivenza a 10 anni
al 63%. La sopravvivenza è correlata con l'età (OS 90% versus 50%; p < = 0.0001)
e alla sede (OS 85% per le forme non addominali versus 60% per i tumori a sede
primitiva addominale; p = 0.02). I dati su MYCN sono stati ottenuti in pochi casi.
L'ENSG 6 è stato aperto nel 1993 ed era uno studio pilota per ENSG 9 che è stato
poi riaperto nel 1995 ed è tutt'oggi in funzione. Entrambi i protocolli raccomandano
5 cicli di OPEC/OJEC seguiti dalla chirurgia e da due ulteriori cicli di OPEC/OJEC.
Nell'ENSG 6, la radioterapia veniva somministrata a chi non avesse acquisito la
remissione completa dopo la chirurgia, mentre non era raccomandata nell'ENSG 9.
24 pazienti sono entrati nell'ENSG 6, ma i dati su MYCN sono ancora una volta
insufficienti. Nell'ENSG 9, sono stati arruolati solo pazienti con MYCN non
amplificato. Dei 35 registrati MYCN è risultato amplificato in 9, normale in 16 e non
noto per 10. Tre pazienti con MYCN normale e 3 con MYCN amplificato sono
deceduti.
3.
OBIETTIVI DELLO STUDIO
3.1 Obiettivo principale
Determinare l'outcome, in particolare per quanto riguarda il controllo locale, l'eventfree survival e l'overall survival dei bambini con età superiore all'anno alla diagnosi
di neuroblastoma Stadio 2 non resecabile e Stadio 3 con MYCN non amplificato.
Non resecabile alla diagnosi significa non asportabile senza rischi (vedi 9.6 fattori di
rischio).
Il regime chemioterapico rappresenta una riduzione della terapia
somministrata nella maggior parte dei protocolli attualmente in corso in Europa.
L'obiettivo è quello di una event free survival a tre anni pari all'80% e una overall
survival a tre anni pari all'85%.
3.2 Obiettivi secondari
3.2.1 Trattare tutti i bambini arruolati nei diversi centri appartenenti al Gruppo
Cooperativo Europeo in modo uniforme e raccoglierne i dati centralmente.
3.2.2 Raccogliere dati biologici validati in tutti i casi così da stabilire il valore prognostico
dei singoli parametri studiati.
4. DISEGNO DELLO STUDIO
Neuroblastoma localizzato non operabile
↓
Biopsia
↓
Carboplatino/Etoposide (1)
↓
CADO (2)
→
↓
Rivalutazione
(se PM il paziente esce dallo studio)
Carboplatino/Etoposide (3)
↓
CADO (4)
↓
Rivalutazione
→
Chirurgia
↓
Carboplatino/Etoposide (5)
Rivalutazione
→
↓
CADO (6)
↓
Osservazione
All'esordio i pazienti verranno definiti in stadio 2 o 3 seguendo i criteri INSS
(Appendice 1). La chirurgia iniziale dovrà includere almeno una biopsia diagnostica
per tutti i casi e il materiale tumorale verrà rivisto centralmente per l'istologia e per
gli studi biologici. Pazienti affetti da tumore con MYCN amplificato verranno esclusi
dal presente studio (nota del Coordinatore Italiano: i pazienti verranno inseriti nel
protocollo per lo stadio 4 attualmente in uso [ossia AIEOP NB97.04 A] e
successivamente nel Protocollo Europeo High Risk quando questo verrà
avviato).
Se vi è progressione di malattia durante o dopo i primi due cicli di chemioterapia, il
paziente verrà considerato fuori studio. Tutti gli altri casi riceveranno due ulteriori
cicli di chemioterapia seguiti da un approccio chirurgico avente come obiettivo la
radicale asportazione della neoplasia primitiva
Due ulteriori cicli di chemioterapia verranno somministrati dopo la chirurgia e
successivamente il trattamento verrà interrotto qualsiasi sia l'entità del
residuo. I pazienti verranno monitorati attentamente e un ulteriore trattamento
verrà somministrato qualora vi sia progressione locale (vedi Appendice 5).
Gli end-points dello studio saranno il grado di resecabilità della neoplasia, l'eventfree survival e l'overall survival.
5.
CRITERI DI ELEGIBILITA'
5.1 Criteri d'inclusione:
Neuroblastoma Stadio 2 non resecabile o stadio 3
Età > 1 anno alla diagnosi
MYCN non amplificato (definito da un laboratorio di riferimento. Vedi Appendice 7)
Nessun trattamento precedente
Consenso informato
Registrazione entro le 4 settimane dalla diagnosi (Dati al Centro Nazionale)
Tutti i criteri di elegibilità rispettati
5.2 Criteri di esclusione:
Inquadramento diagnostico non completato secondo il protocollo
6. INQUADRAMENTO DIAGNOSTICO
6.1 Anamnesi.
6.2 Esame obiettivo completo con peso, altezza e PAO.
6.3 Emocromo completo e profilo coagulativo.
6.4 Funzione epatica e renale (Na, K, Ca, Mg, PO4, urea, creatinina, glicemia, proteine
totali, bilirubina, transaminasi). LDH, Ferritina, NSE sieriche.
6.5 VMA e HVA urinari .
6.6 Diagnostica per immagini:
Rx torace AP
Ecografia addominale
TC o RM del tumore primitivo (3D)
Scintigrafia con I123 (preferibile ) o I131 mIBG
(appendice 2)
Scintigrafia con Tc MDP se mIBG negativa
6.7 Aspirato midollare valutabile in 2 sedi e 2 biopsie osteomidollare valutabili.
6.8 Istologia del tumore primitivo (disponibile per revisione centralizzata)
6.9 Velocità di filtrazione glomerulare [(meglio se misurata con Cr 51 EDTA)nota del
coordinatore] ed esame audiometrico.
7. LINEE GUIDA ANATOMO-PATOLOGICHE
(Vedi Appendice 6)
Le linee guida debbono essere seguite strettamente. Le procedure di suddivisione del
materiale indicate al punto 1.1 dell'Appendice 6 debbono essere eseguite dal patologo
(Appendice 6, 7.1) e la metodica di suddivisione dipende dalla quantità di materiale
tumorale disponibile. Alcune opposizioni dovrebbero essere eseguite prima di fissare il
materiale tumorale, E' di cruciale importanza per la corretta interpretazione dei risultati
delle indagini di biologia molecolare che il contenuto di cellule tumorali dei campioni
bioptici sia chiaramente definito.
Biopsia chirurgica: 2 differenti zone della massa primitiva dovrebbero essere biopsiate. I
prelievi dovrebbero essere di almeno 1 cm3 (Appendice 6.1.2e 6.1.3)
Agobiopsia: preferibilmente 4 agobiopsie da zone diverse della massa, ma almeno 2 in
casi di lesioni piccole (Appendice 6, 1.3.2)
Agoaspirato: almeno 2 (possibilmente 10) (Appendice 6, 1.3.3)
Per informazioni su: - diagnosi istologica/citologica Appendice 6,1.4
- trattamento del materiale alla chirurgia differita Appendice 6, 1.5
- valutazione dei linfonodi regionali Appendice 6, 1.*
- studio immunoistologico e citologico 6.1.7
I Patologi di ogni gruppo nazionale costituiranno un panel di revisori che dovrà prendere in
esame tutti i campioni dei pazienti inseriti nello studio.
8.
LINEE GUIDA BIOLOGICHE
L'analisi di MYCN, della porzione cromosomica 1p36.3 e il contenuto cellulare di
DNA sono accertamenti obbligatori che dovranno essere eseguiti nel laboratorio di
riferimento nazionale o negli altri laboratori di riferimento ossia qualsiasi altro
laboratorio partecipante al European Neuroblastoma Quality Control Assessment
(ENQUA). I risultati provenienti da laboratori diversi da questi non verranno tenuti
in considerazione per l'assegnamento al gruppo di trattamento. Tutte le indagini
biologiche relative a MYCN, cromosoma 1p e contenuto di DNA dovranno essere
riviste centralmente.
L'esatta determinazione del contenuto cellulare è un prerequisito essenziale per eseguire
una interpretazione affidabile degli studi di Genetica Molecolare.
Il numero di copie di MYCN verrà analizzato con metodiche Southern blot (SB) e FISH. Lo
studio in PCR non è raccomandato se non accompagnato da una seconda metodica di
conferma. Per SB e PCR il contenuto cellulare tumorale deve essere superiore al 60%.
Per uteriori dettagli e per la terminologia usata per la descrizione del risultato dello studio
di MYCN vedere le Appendici 2.3.1.
Lo stato del braccio corto del cromosoma 1p36.3 deve essere determinato con esame
FISH e PCR o SB. Per SB e PCR, il contenuto cellulare tumorale deve essere superiore al
60%. Ulteriori dettagli e la terminologia usata per la descrizione del risultato dell'analisi del
cromosoma 1p36.3 vedere Appendice 6.2.3.2.
Il contenuto di DNA deve essere determinato con citometria a flusso o citometria per
immagine e i dettagli sono riassunti al punto 6.2.3.3.
Informazioni dettagliate sull'esecuzione e la valutazione degli aspirati midollari sono
raccolti nell'Appendice 6.3.
9.
LINEE GUIDA CHIRURGICHE
9.1 Obiettivi della chirurgia
L'obiettivo della chirurgia nel neuroblastoma localizzato è quello di ottenere
l'asportazione completa del tumore con morbidità minima.
La chemioterapia pre-operatoria viene somministrata nell'intento di facilitare
l'escissione completa della neoplasia.
9.2 Definizioni
9.2.1 Biopsia
a)
La biopsia deve essere la prima procedura chirurgica in tutte quelle neoplasie
che sono state valutate come non resecabili.
b)
La quantità di tessuto sufficiente deve essere ottenuta idealmente da 2 aree
diverse del tumore per consentire una corretta diagnosi istologica e studi
biologici affidabili. In particolare è essenziale che si ottenga materiale
sufficiente per la determinazione accurata dello stato di MYCN. La biopsia a
cielo aperto è la metodica più soddisfacente ma i rischi dell'intervento devono
essere attentamente considerati e confrontati con quelli delle procedure
percutanee.
c)
L'agobiopsia è accetabile se nel centro vi è adeguta esperienza e la possibilità
di eseguire una completa valutazione biologica sui campioni così ottenuti.
d)
La definizione del trattamento ottimale è dipendente in modo critico da un
corretto campionamento tumorale ed è qundi obbligatorio che il patologo sia
pronto a ricevere il campione. Il tumore a fresco dovrebbe essere consegnato
al patologo immediatamente in condizioni di sterilità. Formalina o altri
conservanti non debbono essere utilizzati.
9.2.2 Escissione completa
a)
L'escissione completa è definita come l'asportazione di tutto il tumore inclusa
l'asportazione dei linfonodi abnormi e il campionamento dei linfonodi normali. Il
giudizio di escissione completa è basato sulla refertazione chirurgica e sulla
diagnostica per immagini post-operatoria.
b)
E' importante definire il rischio di residui microscopici locali anche se si è
ottenuta l'asportazione macroscopicamente completa. Questo può essere
facilitato dallo studio anatomo patologico di biopsie prese dal letto tumorale
così come dallo studio dei margini del tumore.
9.2.3 Escissione con residui minimi (<5% del volume originale o volume residuo
<5ml volume)
Dopo l'intervento sono presenti residui macroscopici di malattia. La quantità
dovrebbe essere stimata dal chirurgo in ml o come percentuale del volume
originale e valutata dalla diagnostica per immagini post-operatorie.
9.2.4 Escissione incompleta
Più del 5% o più di 5ml di tumore rimangono dopo l'approccio chirurgico.
9.2.5 Biopsia
Il 50% o più del volume iniziale del tumore rimane dopo l'approccio chirurgico.
9.3
Criteri pre-operatori e Operabilità (asportabilità) prima del trattamento
chemioterapico
Poiché l'escissione completa è l'obiettivo principale dell'intervento, questo non deve
essere tentato prima della chemioterapia qualora sia presente uno qualsiasi dei
fattori di rischio (vedi 9.6).
Le tecniche di diagnostica per immagini attualmente disponibili dovrebbero
consentire una valutazione di resecabilità nella maggior parte dei casi. Quando
l'MR o la TC suggeriscono che la resezione completa è poco probabile o dubbia
l'intervento chirurgico di asportazione della neoplasia non deve essere preso in
considerazione. La TC spirale o la angiografia digitale possono fornire informazioni
accurate sulla anatomia vascolare.
9.4
Tempi della chirurgia
L'intervento immediato a scopo di asportazione non è considerato appropriato
quando il chirurgo, sulla base di dati clinici e radiologici in seguito alla discussione
con l'oncologo, non ritiene possibile una asportazione radicale del tumore (vedi 9.6)
[o che questa sia possibile solo al prezzo del sacrificio di strutture importanti].
Dopo 4 cicli di chemioterapia l'intervento chirurgico a scopo di asportazione della
neoplasia dovrebbe essere preso in considerazione a meno che non ci sia una
progressione tumorale o la diagnostica per immagini non suggerisca che
l'escissione completa è ad elevato rischio di essere associata a morte o mutilazione
importante (per quanto la nefrectomia può essere accettabile). In queste
circostanze l'opzione di ulteriore chemioterapia o di una terapia alternativa
dovrebbe essere discussa con il coordinatore nazionale [nota del coordinatore
italiano: per una motivata discussione del caso è comunque opportuno ottenere
plurimi campioni bioptici dal tumore primitivo].
A questo punto l'inglobamento dei vasi non è più una controindicazione alla
chirurgia.
L'asportazione chirurgica dovrebbe comunque essere eseguita anche se la
diagnostica per immagini suggerisce che il tumore primitivo è regredito
completamente.
9.5
Procedure chirurgiche
9.5.1 La chirurgia del neuroblastoma mira ad asportare completamente il tumore primitivo
e alla valutazione della sua estensione. La cavità addominale o toracica dovrebbero
perciò essere accuratamente esplorate e ogni lesione sospetta dovrebbe essere
biopsiata.
9.5.2 Valuazione linfonodale
Dipende dalla sede tumorale del tumore primitivo. I linfonodi delle seguenti regioni
dovrebbero essere esaminati e rimossi se appaiono anormali:
a)
Regione Latero-cervicale: catena giugulare e area sovraclavicolare;
b)
Torace: linfonodi mediastinici, sopra e sotto la neoplasia;
c)
Addome: linfonodi celiaci (infra-diaframmatici), medio-aortici (a livello
renale) e regione iliaca (bilateralmente).
9.5.3 Estensione Intraspinale
Se fattibile la massa extraspinale dovrebbe essere rimossa anche se la malattia
intraspinale rimane (vedi 9.6.4). I residui tumorali macroscopici possono essere
lasciati nei forami intravertebrali specialmente quando vi è un rischio di
diesseminazione attraverso il liquido spinale o se si rischia di danneggiare la
vascolarizzazione del midollo spinale.
9.5.4 Nefrectomia16
La nefrectomia non è associata ad un vantaggio di sopravvivenza e dovrebbe
essere evitata se è possibile. La nefrectomia unilaterale è accettabile solo dopo
chemioterapia se questo consente l'escissione chirurgica completa. In questo caso
il chirurgo dovrebbe assicurarsi che il rene controlaterale sia normale ed i suoi vasi
liberi da neoplasia.
9.5.5 Incisione tumorale
Dopo la chemioterapia la maggior parte delle neoplasie acquisiscono maggior
consistenza e appaiono più compatte cosicché la disseminazione neoplastica è
improbabile. L'incisione del tumore è dunque permessa se questo aiuta
l'escissione.
9.5.6 I rapporti del tumore con i grossi vasi
Allo scopo di avere utili
informazioni sulla accuratezza della diagnostica per immagini pre-operatoria, i
reperti intraoperatori dovrebbero essere descritti in dettaglio. Particolare attenzione
dovrebbe essere posta alle difficoltà tecniche incontrate quando il tumore è a
contatto con i vasi .
9.5.7 Clips
Qualora sia necessario l'uso di clips, queste dovrebbero essere in titanio o
assorbibili per evitare interferenze con la successiva diagnostica per immagini.
9.6
Fattori di rischio per l'asportazione prima della chemioterapia
La formulazione del giudizio di operabilità di una neoplasia dipende dall'attento
esame clinico unito ad uno studio radiologico accurato. La decisione se operare o
meno è la conclusione di una valutazione multidisciplinare che coinvolge chirurgo,
oncologo e radiologo. In linea di massima, la presenza di uno qualsiasi dei fattori di
rischio di seguito elencati dovrebbe sconsigliare un intervento immediato con lo
scopo di asportare la neoplasia. Questo vale anche se i fattori di rischio non sono
stati riconosciuti prima dell'intervento e vengono accertati in corso di chirurgia. In
questa situazione l'intervento chirurgico dovrebbe essere interotto dopo
l'esecuzione delle biopsie.
Fattori di rischio correlati alla sede della neoplasia
9.6.1 Collo
Inglobamento dell'arteria vertebrale
Inglobamento delle radici del plesso brachiale
Superamento della linea mediana
9.6.2 Torace
Inglobamento della trachea o di un bronco principale
Inglobamento dell'origine o di diramazioni delle succlavie
Tumore toraco-addominale, tumore fusiforme peri-aortico
Tumore del mediastino inferiore, infiltrante la giunzione costo-vertebrale fra
T9 e T12
9.6.3 Retroperitoneo
Tumore surrenalico infiltrante l'ilo epatico
Tumore surrenalico che ingloba i rami dell'arteria mesenterica superiore a
livello della radice del mesentere
Tumore surrenalico che circonda l'origine del tronco celiaco e dell'arteria
mesenterica superiore
Invasione di uno o di entrambi i peduncoli renali
Tumore fusiforme che circonda l'aorta infrarenale
Inglobamento dei vasi iliaci
Tumore pelvico che supera la incisura ischiatica
9.6.4 Neoplasia endorachidea con sintomi neurologici
La necessità urgente in questa situazione è la riduzione della compressione
sul midollo spinale più che l'asportazione del tumore primitivo.
Ciò è ottenibile preferibilmente con la chemioterapia.
10.
TRATTAMENTO TERAPEUTICO (Vedi Appendice 3)
Cicli di Etoposide + Carboplatino verranno alternati a cicli di Ciclofosfamide +
Adriamicina + Vincristina (CADO). I cicli di chemioterapia dovrebbero essere
somministrati ad intervalli di 21 giorni purché i PMN siano > 1 x 109/l e la conta
piastrinica > 100 x 109/l.
10.1 VP16/Carbo
Entrambi i farmaci dovrebbero essere somministrati nei giorni 1-3 dei cicli 1 , 3, 6
anche in regime ambulatoriale*, se non è richiesta un'idratazione addizionale.
VP16/CARBO
FARMACO
DOSE/DIE
mg/m2
MODALITA' DI
SOMMINISTRAZIONE
Carboplatino
200
In soluzione glucosata 5% in
100 ml/m2 in 1 ora, gg 1-3
Etoposide (VP16)
150
In soluzione fisiologica
250 ml/m2 in 2 ore, gg 1-3
*Idratazione 750 ml/m2 in 8 ore
10.2 CADO (Ciclofosfamide, Adriamicina, Vincristina)
I farmaci dovrebbero essere somministrati su 5 giorni ma in casi particolari è
ammessa la somministrazione di Ciclofosfamide e Adriamicina su 2 giorni nei giorni
1 e 2 mantenendo però la VCR al giorno 1 e 5.
CADO
FARMACO
DOSE/DIE
mg/m2
MODALITA' DI
SOMMINISTRAZIONE
Ciclofosfamide
300
In soluzione fisiologica
100 ml/m2 in 1 ora gg 1-5
Adriamicina
30
In soluzione fisiologica
100ml/m2 in 6 ore gg 4-5
Vincristina
1.5
(max 2 mg)
IV bolo
gg 1 e 5
Idratazione 1000 ml/m2 in 8 ore g 1 e 4 e 5. Nei giorni 2 e 3 abbondante idratazione per
os.
11. MODIFICHE DEL TRATTAMENTO PER TOSSICITA'
11.1
Iniziare il ciclo di chemioterapia con PMN > 1000/ml e Pia > 100.000. Se il recupero
ematologico al 21° giorno non è adeguato rinviare di 5-7 giorni la chemioterapia
previo nuovo controllo dell'esame emocromocitometrico.
11.2
In caso di infezioni severe (CTC grado 4 - Appendice 4) considerare la riduzione del
20% dei farmaci mielotossici. Ogni tossicità non ematologica di grado 4 costitutisce
un evento avverso severo e deve essere segnalato per fax al Coordinatore
nazionale entro 24 ore.
11.3 In caso di reazione allergica al VP16 prendere le opportune misure terapeutiche e
ripetere la somministrazione sotto copertura steroidea.
11.4 Nel caso di neuropatia periferica severa (ptosi o perdita di riflessi o altro), la
seconda VCR del CADO deve essere rinviata di 1 settimana.
11.5 Nel caso di tossicità renale grado 2, eseguire GFR e modulare la dose del
Carboplatino.
12. ESAMI DURANTE IL TRATTAMENTO
Ciclo N°
1
2
3
4
Prima
della
chirurgia
5
6
Stop
Terapia
Peso
Emocromo, funzionalità
epatica e renale
AVM e AOV urinari
(Dopamina)
Marcatori sierici:
LDH
Ferritina
NSE
Valutazione midollare
(2 aspirati e 2 biopsie)
Tumore Primitivo: Eco
TC o RM
Scintigrafia con MIBG
con se MIBG negativa
Tc99 MDP
Studi biologici
MYCN
delezione di 1p
DNA ploidia
citogenetica
GFR ( Clearance DTPA o
EDTA)
Audiogramma
()
Ecocardiografia
()
()
13. ESAMI DOPO IL TERMINE DEL TRATTAMENTO
13.1
Studio della neoplasia
In assenza di sintomi si raccomanda:
13.1.1 Diagnostica per immagini (Rx o Eco) della sede della neoplasia primitiva:
a)
ogni 2 mesi per i primi 6 mesi dallo stop
b)
ogni 4 mesi fino a 24 mesi dallo stop
c)
ogni 6 mesi fino a 4 anni dallo stop
d)
a 5 anni dallo stop
N.B. se allo stop terapia la scintigrafia è ancora positiva ripeterla a 12 mesi.
13.1.2 Progressione o recidiva di malattia:
Valutare adeguatamente la sede e l'estensione della recidiva o progressione e
notificarla tramite scheda al Centro dati nazionale entro 4 settimane dall'evento.
13.2 Valutazione della tossicità
La tossicità di questo protocollo dovrebbe essere modesta. In caso di alterazioni
eseguire gli opportuni accertamenti nel follow-up.
13.2.1 Effetti tardivi:
•
Studio della funzione renale
Al termine del trattamento e a 5 anni dalla diagnosi ripetere lo studio della GFR.
•
Studio audiologico
A 2 e 5 anni dalla diagnosi eseguire esame audiometrico
•
Valutazione cardiologica
A 2 e 5 anni dalla diagnosi eseguire ecocardiografia
14. STATISTICA
Reclutamento
Sulla base del reclutamento attuale nel LNESG il protocollo europeo SIOP per
neuroblastoma Stadio 3 o 2 non resecabile o 3, dovrebbe reclutare 30 casi per
anno di pazienti di età superiore ad un anno alla diagnosi. Circa il 15% di questi
casi nel LNESG hanno MYCN amplificato. Un reclutamento annuale di 25 pazienti e
l'ingresso di 100 pazienti in 4 anni appare dunque fattibile. Questo numero di
pazienti potrebbe essere acquisito in un periodo di tempo più breve se altri Centri o
Gruppi parteciperanno allo studio.
End-point primario
Attualmente nella maggior parte degli studi viene riportata una sopravvivenza a tre
anni per i pazienti di età superiore all'anno con MYCN non amplificato nell'ordine
del 70-80%.
L'end-point primario di questo trial è quello di una Event Free Survival a tre anni
dell'80% e di una Overall Survival dell'85%.
A causa dell'esiguità del numero di casi con MYCN noto riportati nei diversi studi
precedenti, è improbabile che un confronto storico diretto porti ad evidenziare una
differenza significativa (un livello convenzionato del 5%) a meno che il nuovo
protocollo porti a una sopravvivenza o inferiore o superiore.
Per esemplificazione la precisione delle stime di sopravvivenza consentite
dall'inclusione di 100 pazienti e da un follow-up ulteriore di 3 anni è presentata dalla
tabella seguente:
Numero di decessi
a 36 mesi
% di sopravvivenza
osservata a 36 mesi
Limite inferiore del
IC al 95%
0
100%
97.0%
5
95%
89.8%
9
91%
84.8%
10
90%
83.6%
13
87%
80.1%
15
85%
77.8%
Se la percentuale osservata di sopravvivenza a 3 anni è del 91% (9 decessi),
questa sarà coerente con una sopravvivenza reale a 3 anni > 84,4%.
Se la percentuale osservata di sopravvivenza a 3 anni è del 85% (15 decessi)
questa sarà coerente con una sopravvivenza reale a 3 anni uguale o superiore solo
al 77.8%.
Regole per l'interruzione dello studio
La riduzione della chemioterapia potrebbe portare a una più elevata incidenza di
recidive e quindi verrà utilizzata una regola di interruzione per prevenire un'eccesso
di recidive metastatiche o di progressione locale nei pazienti entrati nello studio. La
regola si basa sul monitoraggio del numero di eventi nel periodo dei 12 mesi sia
come recidive metastatiche o progressione locale o morte per qualsiasi causa. A
completamento di queste regole bisogna inoltre segnalare:
1. La decisione di continuare o interrompere lo studio è una decisione medica e le
regole per l'interruzione dovrebbero essere utilizzata solo come linee-guida per
consentire questa decisione.
2. Se durante un'analisi ad interim non tutti i pazienti hanno un follow-up minimo
perchè sono stati persi al follow-up, l'incidenza degli eventi sarà stimanta
secondo il metodo di Kaplan-Meier. La stima verrà utilizzata per confronti con
rejection boundary. (???)
3. L'uso di un'incidenza di eventi di 12 mesi implica per valutare la risposta che i
pazienti continueranno ad entrare nel trial per un anno prima di valutare la
risposta dei precedenti pazienti.
Quattro analisi ad interim verranno eseguite ad intervalli quando 25%, 50%,
75% e 100%
dei pazienti avranno raggiunto un potenziale follow-up di 1
anno. La regola si basa sulla procedura multi-stage di Fleming .
4. Ogni decesso, compresi quelli dovuti alla tossicità (chirurgica o chemioterapica)
o a progressione di malattia, verificatosi entro i primi 12 mesi dalla diagnosi,
sarà considerato un evento.
5. Il calcolo dei limiti è stato effettuato assumendo come accettabile una
percentuale di eventi a 12 mesi < 15%, e controllando la potenza allo scopo di
riconoscere nel 79% dei casi una percentuale di eventi a 12 mesi > 25%. Il
rischio di errore α di chiudere erroneamente il protocollo è limitato allo 0.04
(probabilità di interrompere il Trial e rifiutare il protocollo, con una percentuale di
eventi a 12 mesi reale= 15%).
6. I primi 25 pazienti inclusi nello studio saranno analizzati quando sarà stato
raggiunto un follow-up minimo di 12 mesi. Il limite d'interruzione sarà raggiunto
se fra questi 25 pazienti un numero > 11 eventi viene osservato durante i primi
12 mesi (percentuale di eventi osservati = 44%).
7. Se il numero di eventi osservati è < 10 il reclutamento potrà continuare fino alla
analisi successiva, eseguita non appena un altro gruppo di 25 pazienti sarà
stato seguito per almeno 12 mesi. Il limite d'interruzione verrà raggiunto qualora
sia stato osservato fra questi 50 pazienti un numero > 14 eventi durante i primi
12 mesi (percentuale di eventi osservati= 28%)
8. Una terza analisi sarà effettuata quanto altri 25 pazienti arruolati saranno stati
seguiti per almeni 12 mesi. Il limite d'interruzione sarà raggiunto se fra questi 75
pazienti un numero > 18 eventi sarà stato osservato durante i primi 12 mesi
(percentuale di eventi osservati = 24%).
9. Se sarà stato osservato un numero di eventi < 17, ultima analisi sarà eseguita
sui 100 pazienti, dopo un follow-up di almeno 12 mesi. Se, durante i primi 12
mesi, sarà stato osservato un numero di eventi > 22 (percentuale di eventi
osservati = 22%) il limite superiore sarà oltrepassato in favore di una
percentuale di eventi eccessivamente alta (percentuale a 12 mesi > 22%)
SU CARTA INTESTATA DEL CENTRO LOCALE
GRUPPO EUROPEO PER IL TRATTAMENTO DEL NEUROBLASTOMA
PROTOCOLLO TERAPEUTICO PER IL TRATTAMENTO DEL
NEUROBLASTOMA LOCALIZZATO NON OPERABILE MYCN NON AMPLIFICATO
IN BAMBINI D'ETA' > AD 1 ANNO.
CONSENSO INFORMATO
Cari Genitori,
il vostro bambino è stato diagnosticato affetto da neuroblastoma, una neoplasia maligna
che colpisce quasi esclusivamente i bambini.
Il neuroblastoma si sviluppa dalle cellule del sistema nervoso periferico che danno origine
alla catena dei gangli nervosi paravertebrali. La sede più frequente è l'addome e in
particolare le ghiandole surrenaliche. Le varie indagini eseguite hanno dimostrato che in
vostro figlio la neoplasia non ha sviluppato metastasi (localizzato) e gli studi istologici e di
biologia molecolare suggeriscono che la prognosi possa essere favorevole ma, al
momento, dalle indagini eseguite la neoplasia non sembra asportabile senza rischi. Vostro
figlio dovrebbe perciò essere inserito nel protocollo terapeutico europeo per il trattamento
di queste forme, protocollo che prevede di eseguire 4 cicli di chemioterapia alternando uno
schema che associa Carboplatino e Vepeside ad un ciclo di Vincristina - Endoxan e
Adriamicina.
Dopo 4 cicli di chemioterapia verrà eseguita un'accurata rivalutazione e se possibile verrà
effettuato un intervento chirurgico allo scopo di rimuovere la neoplasia.
Salvo casi particolari, verranno poi somministrati ancora 2 cicli di terapia ed il trattamento
verrà poi sospeso poiché anche se vi sono dei residui questi possono regredire
spontaneamente, lentamente o restare inattivi e non influiscono sulla prognosi.
Quali sono gli obiettivi dello studio? Fino a che non otterremo la guarigione di tutti i
bambini senza effetti tossici collaterali tardivi dovremo sviluppare degli studi clinici per
sviluppare trattamenti migliori e da un'ampia revisione dei risultati europei è emersa una
disomogeneità terapeutica con risultati diversi. Abbiamo dunque adottato il trattamento
che sembra dare i migliori risultati per trattare tutti i pazienti europei nello stesso modo.
Questo ci consentirà di raccogliere importanti informazioni sulle caratteristiche cliniche e
biologiche che influscono sulla prognosi e ci consentirà di modulare meglio il trattamento.
Le informazioni sulle condizioni cliniche di vostro figlio, la risposta al trattamento e i suoi
effetti collaterali verranno raccolti in un registro computerizzato per le successive analisi. I
dati sono anonimi,
La partecipazione allo studio è libera e in qualsiasi momento potrete chiedere spiegazioni
e decidere di ritirare vostro figlio dallo studio. In tal caso sulla base dell'esperienza del
centro presso il quale è in cura vostro figlio, potrete discutere quale strategia terapeutica
adottare.
Gli effetti tossici di questo trattamento sono quelli della maggior parte dei chemioterapici
ed in particolare la caduta dei capelli, nausea e vomito e la tossicità ematologica che può
richiedere un supporto trasfusionale o facilitare la comparsa di febbre e/o infezioni. In ogni
caso a vostro figlio verrà fornito il trattamento di supporto necessario. Altri effetti collaterali,
in particolare tardivi, non dovrebbero svilupparsi ma vostro figlio verrà comunque
sottoposto ad accurati controlli periodici.
In qualsiasi momento potrete chiedere ulteriori spiegazioni o chiarimenti sullo studio e sul
trattamento al personale medico del reparto.
Il Dott.
ci ha illustrato la malattia di nostro figlio e
ci ha proposto di trattarlo secondo il protocollo europeo "Unresectable localized NB".
Confermiamo che:
•
•
abbiamo letto e compreso le informazioni contenute in questo documento
•
consentiamo che tutte le informazioni cliniche vengano utilizzate a scopo di studio
mantenendone la confidenzialità
•
acconsentiamo a che nostro figlio venga trattato secondo questo protocollo
europeo.
abbiamo capito che la partecipazione a questo studio è volontaria e possiamo
ritirare il consenso senza che le cure mediche ne siano danneggiate
Cognome e nome _________________________________________________________
Data __________________
Firma __________________
Firma del Medico Responsabile ___________________
16.
REFERENCES
1.
Prognostic Factors in Neuroblastoma
Evans AE et al.
Cancer 59; 1853-1859 (1987)
2.
Clinical Relevance of Tumour Cell Ploidy and N-Myc Amplification in Childhood
Neuroblastoma : A Paediatric Oncology Group Study
Look AT et al.
J. Clin. Oncol. 13; 884-893 (1993)
3.
Excellent Outcome of Stage II Neuroblastoma is Independent of Residual Disease
and Radiation Therapy
Matthay KK et al.
J. Clin. Oncol. 7; 236-244 (1989)
4.
Event-free Survival of Children with Biologically Favourable Neuroblastoma Based
on the Degree of Initial Tumour Resection : Results from the Pediatric Oncology
Group
Strother D et al.
Eur. J. Cancer 33; 2121-2125 (1997)
5.
The Effect of Complete Excision on Stage III Neuroblastoma : A Report of the
European Neuroblastoma Study Group
Powis MR, Imeson JD, Homes SJK.
J. Pediat. Surg. 31; 516-519 (1996)
6.
Aggressive Surgery Combined with Intensive Chemotherapy Improves Survival in
Poor-risk Neuroblastoma
Haase GM et al.
J. Pediatr. Surg. 26; 1119-1124 (1991)
7.
Surgical Management and Outcome of Loco-regional Neuroblastoma : Comparison
of the Children’s Cancer Group and the International Staging Systems
Hasse GM et al.
J. Pediatr. Surg. 30; 289-295 (1995)
8.
Survival from Locally Invasive or Widespread Neuroblastoma without Cytotoxic
Therapy
Kushner BH et al.
J. Clin. Oncol. 14; 373-381 (1996)
9.
N-Myc Gene Amplification is a Major Prognostic Factor in Localised
Neuroblastoma : Results of the French NBL 90 Study
Rubie H et al.
J. Clin. Oncol. 15; 1171-1182 (1997)
10.
Unresectable Localised Neuroblastoma - Improved Survival after Primary
Chemotherapy Including Carboplatin - Etoposide
Rubie H et al.
Br. J. Cancer 77; 2310-2317 (1998)
11.
Treatment of Stage III Neuroblastoma with Emphasis on Intensive Induction
Chemotherapy : A report from the Neuroblastoma Group of the Spanish Society of
Pediatric Oncology
Castel V et al.
Med. Pediatr. Oncol. 24; 29-35 (1995)
12.
Localised but Unresectable Neuroblastoma : Treatment and Outcome of 145
Cases
Garaventa A et al.
J. Clin. Oncol. 11; 1770-1779 (1993)
13.
The Recurrence Patterns of Stages I, II, and III Neuroblastoma : Experience with
77 Relapsing Patients
Berthold F et al
Annals of Oncology 7;183-187 (1996)
14.
The International Neuroblastoma Pathology Classification (The Shimada System)
Shimada H et al
Cancer 86;364-372 (1999)
15.
The Treatment of Neuroblastoma with Intraspinal Extension with Chemotherapy
Followed by Surgical Removal of Residual Disease
Plantaz D, Rubie H, Michon J.
Cancer 78; 311-319 (1996)
16.
Renal Function Following Unilateral Nephrectomy for Neuroblastoma and Wilms’
Tumour
Schell M, Cochat P, Hadj-Aissa A, Bouffet E, Dubourg L, Brunat-Mentigny M.
Pediatr. Nephrol. 9; 579-582 (1995)
17.
Non-parametric Estimation from Incomplete Observations.
Kaplan EL, Meier P.
J. Am. Stat. Assoc. 53:457-481 (1958)
18.
One Sample Testing Procedure for Phase II Clinical Trials.
Fleming TR.
Biometrics 38: 143-151 (1982)
APPENDIX I
INTERNATIONAL CRITERIA FOR DIAGNOSIS, STAGING AND
RESPONSE TO TREATMENT
These definitions are taken from the Revised Guidelines published in 1993 (Brodeur et al
Journal of Clinical Oncology volume 11 pp 1466-1477)
Table 1
DIAGNOSIS OF NEUROBLASTOMA
A diagnosis of neuroblastoma is established if :
1.
An unequivocal pathological diagnosis is made from tumour tissue by light microscopy
(with or without immunohistology, electron microscopy, increased urine or serum
catecholamines or metabolites )Ψ
OR
2.
Bone marrow contains unequivocal tumour cells Ψ (eg syncytia or immunocytologically
positive clumps of cells) and increased urine or serum catecholamines or metabolites
*
.
Notes
Ψ
If histology is equivocal, karyotypic patterns or abnormalities in tumour cells
characteristic of other tumours [eg t(11;22)], then exclude a diagnosis of
neuroblastoma, whereas genetic features characteristic of neuroblastoma (1p
deletion, 0MYCN amplification) would support this diagnosis.
*
Catecholamines and metabolites include dopamine, HVA and/or VMA; levels must be
> 3 SD above the mean for age (measured in mmol per mmol creatinine)# to be
considered increased, and at least two of these metabolites must be measured.
#
Timed urine collections are difficult in young children so normalisation per mmol
creatinine does away with the necessity for a timed collection and also avoids the
problem of false negatives due to dilute urine.
Table 2
ASSESSMENT OF EXTENT OF DISEASE
Tumour Site
Recommended Tests
Primary Tumour
CT and/or MRI scan* with 3D measurements.
MIBG scan if available†.
Metastatic Sites
Bone marrow
Bilateral posterior iliac crest marrow aspirates and trephine
(core) bone marrow biopsies required to exclude marrow
involvement. A single positive site documents marrow
involvement. Core biopsies must contain at least 1 cm of
marrow (excluding cartilage) to be considered adequate
Bone
MIBG† scan; 99Tc scan is required if MIBG scan negative or
unavailable, and plain radiographs of positive lesions are
recommended.
Lymph nodes
Clinical examination (palpable nodes), confirmed
histologically. CT scan for non-palpable nodes (3D
measurements)
Abdomen/liver
CT and/or MRI scan* with 3D measurements
Chest
AP and lateral chest radiographs. CT/MRI necessary if chest
radiograph positive, or if abdominal mass/nodes extend into
the chest
Notes
*
†
Ultrasound examination considered suboptimal for accurate 3D measurements
The MIBG scan is applicable to all sites of disease.
Table 3
INSS STAGING
Stage 1
Localised tumour with complete gross excision, with or without microscopic
residual disease; representative ipsilateral lymph nodes negative for tumour
microscopically (nodes attached to and removed with the primary tumour may
be positive).
Stage 2A
Localised tumour with incomplete gross excision; representative ipsilateral nonadherent lymph nodes negative for tumour microscopically.
Stage 2B
Localised tumour with or without complete gross excision, with ipsilateral nonadherent lymph nodes positive for tumour. Enlarged contralateral lymph nodes
must be negative microscopically.
Stage 3
Unresectable unilateral tumour, infiltrating across the midline*, with or without
regional lymph node involvement; or localised unilateral tumour with
contralateral regional lymph node involvement; or midline tumour with bilateral
extension by infiltration or lymph node involvement.
Stage 4
Any primary tumour with dissemination to distant lymph nodes, bone, bone
marrow, liver and/or other organs.
(except as defined for Stage 4S).
Stage 4S
Localised primary tumour (as defined for Stage 1, 2A or 2B), with
dissemination limited to liver, skin, and/or bone marrow †.
(limited to infants < 1 year of age)
Notes
Multi-focal primary tumours (eg bilateral adrenal primary tumours) should be staged
according to the greatest extent of disease, as defined above, and be followed by a
subscript "M" (eg Stage 3M).
*
The midline is defined as the vertebral column. Tumours originating on one side and
crossing the midline must infiltrate to or beyond the opposite side of the vertebral
column.
†
Marrow involvement in Stage 4S should be minimal, ie less than 10% nucleated cells
on bone marrow biopsy or quantitative assessment of nucleated cells on marrow
aspirate. More extensive marrow involvement should be considered Stage 4. The
MIBG scan (if done) should be negative in the marrow for Stage 4S.
Table 4
INRC DEFINITION OF RESPONSE
Response
Primary tumour*
Metastatic Sites*†
CR
No tumour
No tumour; catecholamines normal
VGPR
Decreased by 90-99%
No tumour;
Residual 99Tc bone changes allowed
PR
Decreased by > 50%
All measurable sites decreased by > 50%
Bones and bone marrow : Number of
positive sites decreased by > 50%;
no more than 1 positive bone marrow site
allowed†.
MR
No new lesions; > 50% reduction of any measurable lesion (primary or
metastases) with < 50% reduction in any other; < 25% increase in any
existing lesion.
NR
No new lesions; < 50% reduction but < 25% increase in any existing
lesion.
PD
Any new lesion; increase of any measurable lesion by > 25% ; previous
negative marrow positive for tumour.
Notes
CR
VGPR
PR
MR
NR
PD
Complete Response
Very Good Partial Response
Partial response
Mixed Response
No Response
Progressive Disease
*
Evaluation of primary and metastatic disease as outlined in Table 2
†
One positive marrow aspirate or biopsy is allowed for PR if this represents a
decrease from the number of positive sites at diagnosis.
APPENDIX 2
SCINTIGRAPHY PROTOCOL FOR mIBG SCANNING IN PAEDIATRIC
NEUROBLASTOMA
Objectives
To obtain scans of best and constant quality, only nuclear medicine departments with
proper equipment and experienced nuclear medicine physicians should perform the
examinations. The departments should be able to perform SPET scanning, if possible
with a multiple head camera to reduce the acquisition time. The radioisotope of choice is
Iodine-123 (I123) and should be used wherever available, regardless of higher costs.
Information about preceding therapeutic procedures like surgery and chemotherapy and
the time when they were performed should be available to the nuclear medicine
physicians. There are many drugs and compounds which on a theoretical basis could
interfere with the concentration of mIBG by neuroblastoma, for example, by interfering with
the uptake mechanisms or acting as competitive inhibitors. Therefore the discontinuation
of these medicines two weeks before administration of the radiopharmaceutical should be considered if this is clinically possible. The classes of drugs
which have interfered with the uptake of mIBG are tricyclic antidepressants and related
drugs, some antihypertensives, phenothiazines, amphetamines and particularly some
nasal decongestants and cough preparations.
Patient Preparation
Thyroid blockade
There are quite different guidelines for thyroid blockade and different prescriptions, for
example, for Lugol’s solution within European countries. Thus it would not be easy to
achieve a uniform thyroid protocol. However, the different regimens yield comparable
results. In the case of I131 mIBG the thyroid has to be blocked at least 24 hours prior to
tracer injection. However, in I123 mIBG scintigraphy thyroid blocking should be done as
well.
We recommended the following protocol: Beginning on the day before tracer injections
until the day after injection, children from one month to three years should receive
32.5 mg potassium iodide daily, from three to thirteen years 65 mg, and over this age 130
mg daily. New-borns receive 16.25 mg potassium iodine only on the day before tracer
injection. Rapid blockade by intravenous injection of potassium and perchlorate
(Irenat®) respectively, is an alternative option.
Others
To obtain scans without motion artefacts sedation might be necessary in some patients.
In small children an intravenous line has to be inserted by the paediatrician prior to tracer
injection.
Central venous catheters should not be used for application.
Tracer Activity
Considering an activity of 370 Mbq I123 mIBG for adults the EANM paediatric task group
generally recommends calculation of the activity for children by the following formula:
370 Mbq*body weight (KG)
70
On the other hand in this patients group the radiation hazard is far less than the risk
resulting from false negative or positive scanning results. Longer scanning times provoke
motion artefacts. Even rescanning inadequate frames rarely improves the results in our
experience. To obtain scintigrams of good quality within a short scanning time a sufficient
count rate is necessary. Therefore, when using I123 mIBG, in children under
6 kg body weight we recommend an activity of 37 Mbq, in children from 6 to10 kg 50-75
Mbq, from 10 to15 kg 75-100 Mbq, and from 15 to 30 kg 150 Mbq. Above 30 kg the
formula can be applied.
Scanning Protocol
As a compromise between best image quality and limitation of scanning time the imaging
protocol should not exceed one hour. In the case of a double-head camera in children
with a body length under 100 cm the planar whole body scintigraphy can be done within
35 minutes. SPET of the primary tumour region is useful in evaluation after surgery and
chemotherapy but might also be important at the initial scintigraphic examination for later
comparison. Using a three-head SPET camera the tomography protocol described below
takes about 15 minutes for one region. The time for patient positioning and setting of the
camera is included in our estimations.
The 159 KeV photopeak of I123 has to be centred in a 20% energy window.
Planar Scanning
Scanning time:
Twenty-four hours after tracer injection; for all frames with equivocal
result a second time at 48 hours.
Collimator:
A double-headed gamma camera with low energy high resolution
parallel hole collimators (LEHR) is recommended. To limit scanning
time on all purpose parallel hole low energy collimator (LEAP) can be
used in single-headed gamma cameras.
Views:
Whole body: anterior and posterior frames of skull/thorax,
abdomen/pelvis, and upper extremities; lower extremities in anterior
view.
Acquisition:
Ten minutes for each frame. When time is restricted or in case of a
single-head gamma camera 6 minutes/view. The scans are stored in
a 2562 pixel matrix.
Single Photon Emission Tomography (SPET)
Time:
Twenty-four hour after tracer injections.
Views:
Region of the suspected primary tumour; other regions from skull to
pelvis with equivocal planar result.
Collimator:
Multiple-head camera system with LEHR collimators.
Acquisition:
360o in 6o steps, 30 seconds per step, 642 pixel matrix.
Processing:
Reconstruction using a ramp filter and 3D post-filter (Butterworth 6th
order) or alternatively pre-filtering with a Butterworth filter (6th order)
and back-projection with a ramp filter. Reorientation of the images in
trans-axial and coronal slices.
Scan Assessment
Each scan should be evaluated directly from the screen by two independent experienced
observers. Hard copies of the best possible quality should be archived. The raw data
have to be stored on appropriate media for further evaluation.
APPENDIX 3
DRUG INFORMATION
CARBOPLATIN
Formulation
Vials containing solution of 50 mg/5 mls, 150 mg/15 mls or
450 mg/45 mls.
Storage
At room temperature.
Reconstitution
Dilute in water for injection BP or 5% dextrose to a maximum strength
of 500 µgms per ml.
Stability
When reconstituted with water for injection or glucose 5%, 8 hours at
room temperature, 24 hours in refrigerator. After dilution in glucose
5% infusion stable for 24 hours in refrigerator.
Administrator
In this protocol as 1 hour intravenous infusion.
Toxicity
Myelosuppression, especially thrombocytopenia, nausea and
vomiting, low incidence of nephrotoxicity, ototoxicity, neurotoxicity
and abnormalities of liver function. May cause urinary loss of serum,
magnesium, potassium and calcium, so levels of these should be
monitored.
CYCLOPHOSPHAMIDE
Formulation
100 mg, 200 mg, 500 mg and 1 G vials for reconstitution.
Storage
At room temperature.
Stability
Unreconstituted vials stable for 5 years at room temperature. A
solution of Cyclophosphamide appears to be chemically stable for
at least 28 days when stored 4 oC. Reconstituted solution (20 mg/ml)
should be used within 3 hours when stored at room temperature,
unless prepared under strict aseptic conditions, when it may be used
within 8 hours.
Administration
Mesna is not required with the dose prescribed in this protocol. It is
required with the second line chemotherapy (see appendix 5).
Toxicity
Myelosuppression, nausea, vomiting, alopecia, haemorrhagic cystitis,
sterility, second malignancies including leukaemia or bladder cancer.
May exacerbate the cardiotoxic effects of anthracycline therapy.
DOXORUBICIN
Formulation
Vials containing 10 mg or 50 mg in solution (2 mg/ml).
Vials containing 10 mg, 50 mg as powder.
Storage
Solution at 2-8 oC in refrigerator.
Powder can be stored at room temperature.
Reconstitution
Each 10 mg should be reconstituted with 5 mls water for injection.
Solution should be further diluted in 0.9% saline or 5% dextrose.
Stability
Powder 4 years at room temperature. Solution 2 years (2-8 oC in
refrigerator) or 1 month at room temperature. Reconstituted solution
(100 µg/ml) in 5% dextrose or 0.9% saline is stable for 28 days when
stored in refrigerator. Solutions should be protected from light during
storage and administration unless concentration is > 500 µg/ml and
freshly prepared. Substantial photodegradation may occur at
concentrations below 100 µg/ml if exposed to light.
Administration
The drug should be mixed with 0.9% saline and given over 6 hours.
Toxicity
Local necrosis if extravasation occurs. Cardiotoxicity. Bone marrow
suppression, mucosal ulceration, nausea, vomiting, alopecia.
ETOPOSIDE (VP16)
Formulation
Vials containing 100 mg Etoposide in 5 ml.
Storage
At room temperature.
Reconstitution
Ideally dilute to a concentration of 0.25-0.4 mg/ml in 0.9% sodium
chloride.
Stability
Vials are stable for 5 years at room temperature. At concentrations of
0.4 mg/ml in 0.9% saline solutions are stable for 96 hours at room
temperature in normal fluorescent lighting, in PVA containers.
Solution in PVC infusion bags should be used immediately, to avoid
leaching out of potential carcinogenic plasticisers.
Administration
By intravenous infusion over 2-4 hours (protected from light).
Caution: Anaphylactic reaction usually manifested as severe
hypotension may occur if infusion given too rapidly. Avoid
extravasation.
Toxicity
Bone marrow suppression. Alopecia, headache, fever, hypotension,
nausea, vomiting, anaphylactic reactions, second malignancies
including leukaemia.
VINCRISTINE
Formulation
1 mg, 2 mg, 5 mg vials with 10 mls diluent. 1 ml, 2 ml, 5 ml vials of
solution 1 mg/ml.
Also available in pre-filled syringes containing 1 mg in 1 ml, 2 mg in
2 mls unpreserved.
Storage
At 2-8 oC in refrigerator.
Stability
Depends on formulation : Lyophilised powder (0.6 oC) 3 years.
Solution (0-6 oC) 2 years. Reconstituted injection is stable for 14
days (2-8 oC). Diluted infusion (in 0.9% saline, 5% dextrose or
Ringer’s lactate) is stable for 24 hours at 20 µg/ml.
Administration
By bolus intravenous injection. Ensure that needle is well into the
vein to avoid extravasation.
Toxicity
Local necrosis if extravasated. Jaw pain, paresis, constipation,
neurotoxicity and alopecia, alopecia, paralytic ileus and SIADH.
APPENDIX 4
RECOMMENDATIONS FOR GRADING OF TOXIC EFFECTS
Taken from CTC-NCIC criteria
Site
H
H1
H2
H3
H4
H5
D
D1
D2
Haematological
Haemoglobin (g/100 ml)
Leucocytes (1000/mm3)
Granulocytes (1000/mm3)
Platelets (100`0/mm3)
Haemorrhage
Grade 0
Grade 1
Grade 2
Grade 3
WNL
<4.0
< 2.0
WNL
None
> 10.0
3.0-3.9
1.5-1.9
> 75
Petechiae
8.0-9.9
2.0-2.9
1.0-1.4
50-75
Mild blood loss
6.5-7.9
1.0-1.9
0.5-0.9
25-50
Needing blood transfusion
or fundal haemorrhages
< 1.5 x N
2.6 -5 x N
1.5 - 3 x N
5.1 -20 x N
5.1 -20 x N
2.1 - 5 x N
Ulcerated lesions,
requiring liquid diet only
Moderate
Intolerable requiring
therapy
Severe, abdominal
distension
WNL
< 1.25 x N
1.26-2.5 x N
D3
D4
D5
Digestive
Bilirubin
Transaminases
(SGOT/SGPT)
Alkaline phosphatase
Amylase
Stomatitis
< 1.25 x N
< 1.25 x N
No change
1.26-2.5 x N
< 1.5 x N
Mild soreness, erythema
D6
Nausea/vomiting
None
Nausea
2.6 -5 x N
1.6-2 x N
Painful erythema,
oedema, ulcers but can
eat solids
Transient vomiting < 5
episodes in 24 hrs
D7
Diarrhoea
None
Transient < 2 days
Tolerable but > 2 days
D8
Constipation
None or no change
Mild
M
M
M
M3
M4
M5
M6
M7
Metabolic/Renal
Blood creatinine
Proteinuria
Haematuria
Na + mmol/L
K+ mmol/lL
Ca + mmol/L
Mg++ mmol/l
WNL
No change
No change
135-145
3.5-5.4
2.15-2.59
1.5-2.0
< 1.5 x N
1+ or < 3g/l
Microscopic
146-149/130-134
5.5-5.9/3.1-3.4
2.6-2.89/1.9-2.1
1.2-1.4
1.5 - 3 x N
2-3 + or 3-10 g/l
Gross no clots
150-155/125/129
6-6.4/2.6-3
2.9-3.09/1.7-1.89
0.9-1.1
3.1 - 6 x N
4+ or > 10 g/l
Gross + clots
156-164/116/124
6.5-6.9/2.1-2.5
3.1-3.3/1.5-1.69
0.6-0.8
P
P1
P2
P3
P4
Pulmonary
PA O2
DL CO
CV
Function
> 90
100-75%
100-75%
No change
80-89
74-65%
74-65%
Mild symptoms
65-79
64-55%
64-55%
Exertional dyspnoea
50-64
54-40%
54-40%
Dyspnoea at normal levels
of exertion
Transient vomiting > 5
episodes in 24 hrs
RECOMMENDATIONS FOR GRADING OF TOXIC EFFECTS
Site
Grade 0
Grade 1
Grade 2
Grade 3
A
Allergy
No change
Oedema, transient rash
Mild bronchospasm, urticaria, no
parenteral therapy needed
Bronchospasm, parenteral therapy requ
S
Skin
No change
Macular, papular eruption,
erythema, asymptomatic
Dry desquamation, vesiculation, pruritus
General symptomatic macular, papular
vesicular eruption or ulceration
I
Infection
C
CI
Cardiac
Rhythm
None
Asymptomatic, transient
,requiring no therapy
C2
Function
No change
Asymptomatic
C3
Ischaemia
None
Non specific T wave flattening
C4
> 30%
C5
Echocardiograph
y (FS)
Hypotension
C6
Hypertension
N
N1
Neurological
Seizures
N2
Minor infection:
1= culture negative fever without septic
2= central catheter related bacteraemia
epidermidis, staphylococcus
3= other
Recurrent/persistent requiring no therapy
Requires treatment
Mild congestive heart failure responsive
therapy
Angina without evidence of infection
> 25% and < 30%
Asymptomatic, decline of resting EF > 20%
of baseline
Asymptomatic St + T wave changes
suggesting ischaemia
> 20% and < 25%
None or no change
Changes requiring no therapy
Requiring therapy but no hospitalisation
Requiring therapy and hospitalisation. r
within 48 hours after stopping the agent
None or no change
Asymptomatic, increase< 20
mmHg or < 150/100 if previous
WNL. No treatment required
Recurrent/persistent increase > 20 mmHg
or > 150/100 if previous WNL. No
treatment required
Requires therapy
Cortical
None or no change
Mild somnolence and agitation
N3
Cerebellar
None
Slight incoordination
Severe somnolence, agitation, confusion
disorientation
Locomotor ataxia
N4
Sensory
None or no change
N5
Motor
None or no change
Mild paraesthesia and/or loss of
deep tendon reflexes
Subjective weakness, no
objective findings
Moderate somnolence (< 50% waking
hors) or agitation
Intention tremor, dysmetria, slurred
speech, nystagmus
Mild or moderate objective sensory loss;
moderate paraesthesia
Mild objective weakness, without significant
impairment
N6
N7
Vision
Neuro-hearing
None or no change
Asymptomatic; hearing loss on
audiometry only
Symptomatic moderate hearing loss
> 15 and < 20%
Severe objective sensory loss, or paraes
interfering with function
Objective weakness with significant imp
of function
Symptomatic subtotal loss of vision
Hearing loss interfering with function but
correctable with hearing aid
APPENDIX 5
Treatment of Progressive Disease
Patients should be fully restaged including biopsy of the tumour to study histology and biological
markers.
1.
Recommendation for local relapse
High dose Cisplatin and VP16 (Etoposide) x 2 courses
Evaluation
PR
NR
High dose Cyclophosphamide and VP16
Etoposide) x 2 courses
↵
Surgery
Local irradiation
or mIBG
2.
Recommendation for metastatic relapse
High dose Cyclophosphamide and VP16 (Etoposide)
↓
High dose Cisplatin and VP16 (Etoposide)
↓
High dose Cyclophosphamide and VP16 (Etoposide)
↓
High dose MIBG + chemotherapy with PBSCT
↓
Fenretinide x 6 months
3.
Administration of drugs
Give at 21 day intervals if WBC >1x109/l, neutrophils >0.5x109/l and platelets >100x109/l
Cisplatin – VP16
VP16 :
100mg/m2/day x 5 days as daily infusion over 1-4 hours
(total dose 500mg/m2) in 0.9%saline.
Cisplatin:
40 mg/m2/day x 5 days as continuous infusion (total
200mg/m2) Either give as a 3% saline solution, or with
mannitol (see national chemotherapy guidelines if available).
Hyperhydration is required (3l/m2/day).
Cyclophosphamide –VP16
4.
VP16:
50 mg/m2/day x 5 days (total dose 250mg/m2) as continuous
infusion in 0.9% NaCl
Cyclophosphamide:
2g/m2/day as 3 hour infusion on days 2,3 and 4 (total dose
6g/m2) with hyperhydration (2.5 l/m2/day) and Uromitexan (2.4
gm/m2/day) Maintain urine output above 125mls/m2/hour with
frusemide and empty bladder regularly.
References
Philip T, Ghalie R, Pinkerton R & al.
A phase II study of high-dose Cisplatin and VP16 in neuroblastoma : a report from the Société
Française d’Oncologie Pédiatrique.
J. Clin. Oncol. 1987, 5 : 941-50.
Meresse V, Vassal G, Michon J & al.
Combined continuous infusion Etoposide with high-dose Cyclophosphamide for refractory
neuroblastoma : A phase II study from the Société Française d’Oncologie Pédiatrique.
J. Clin. Oncol. 1993, 11 : 630-7.
APPENDIX 6
PATHOLOGY AND BIOLOGY GUIDELINES FOR RESECTABLE AND UNRESECTABLE
NEUROBLASTIC TUMOURS AND BONE MARROW GUIDELINES
FROM THE SIOP EUROPE NEUROBLASTOMA PATHOLOGY AND BIOLOGY
GROUP
Peter and Inge Ambros (Vienna, Austria): [email protected]
In co-operation with:
Gabriele Amann (Vienna, Austria)
Klaus Beiske (Oslo, Norway)
Emanuele d’Amore (Padova, Italy)
Laurence Faulkner (Florence, Italy)
Claudio Gambini, (Genoa, Italy)
Michel Peuchmaur (Paris, France)
Lisa Walaas (Oslo, Norway)
For specific questions contact:
Tru cut biopsies: Klaus Beiske: [email protected]
Fine needle aspiration cytology: Lisa Walaas: fax +47 23 07 14 10
Immunoflourescence detection of tumour cells in the bone marrow:
Peter Ambros: [email protected]
Immunocytochemical detection of tumour cells in the bone marrow:
Klaus Beiske: [email protected]
Laurence Faulkner: [email protected]
Biology Group:
Peter Ambros (Austria)
Inge Ambros (Austria)
Jean Benard (France)
Maria Boavide (Portugal)
Nick Bown (United Kingdom)
Huib Caron (The Netherlands)
Valerie Combaret (France)
Olivier Delattre (France)
Nicole Gross (France)
Per Kogner (Sweden)
John Lunec (United Kingdom)
Tommy Martinsson (Sweden)
Rosa Noguera (Spain)
Frank Speleman (Belgium)
GianPaolo Tonini (Italy)
Nadine Van Roy (Belgium)
Pathology Group:
Gabriele Amann (Austria)
Inge Ambros (Austria)
Klaus Beiske (Norway)
Catherine Cullinane (United Kingdom)
Emanuele d’Amore (Italy)
Claudio Gambini (Italy)
Samuel Navarro (Spain)
Michel Peuchmaur (France)
We are grateful to the members of the SIOP Neuroblastoma Biology and Pathology Group
who provided us with valuable comments and suggestions
1.1
GENERAL REMARKS AND RECOMMENDATIONS:
It is one of the major tasks of the local pathologist to save tumour material for
diagnostic purposes, assessment of prognosis based on histopathology and for
biological analyses.
To enable reliable interpretation of the molecular-genetic results, the exact tumour cell content
of the specimen used for these investigations has to be determined. This is possible only if the
pathologist evaluates the specimens adjacent to those used for molecular-genetic/biologic
analyses (for details see below).
The handling of the tumour tissue should always be performed by the pathologist according to
the following protocol which is essentially based on the LNESG protocol. Copies of these
guidelines ought to be available to all pathologists dealing with tumour material from
neuroblastoma patients.
The tumour material should be transferred immediately from the operation theatre to
the local pathology department under sterile conditions. The splitting of the tumour
material must be done by the pathologist as soon as possible after the operation. The
time of splitting up the tumour should be stated.
In all instances, concerning either tumour resection or biopsies, tumour material from
different tumour areas (nodules are of special interest!) ought to be taken for histologic
and molecular-genetic/biologic examination. The reason for this recommendation is
based on the observation of tumour heterogeneities at the genetic level (e.g. for the
MYCN and/or the chromosome 1p status) and/or at the histologic level
(Ganglioneuroblastoma, nodular subtype according to the International Neuroblastoma
Classification, INPC, (Cancer 86, 349-371; 1999) both of which have prognostic
implications. Close co-operation between pathologists and biologists is strongly
recommended. Pathologists should inform the biologists if morphologically unfavourable
looking areas are present in the paraffin embedded material but most likely not in the
specimens selected for molecular-genetic/biologic investigations. These areas should
also be specifically analysed using the paraffin material (see below).
The patient’s peripheral blood (5-10ml, with heparin or EDTA) is needed for molecularbiologic studies as reference and should be sent to the reference biology laboratory
together with the tumour specimens.
The material selected for molecular-genetic/biologic investigations should be sent
as fast as possible to the National Reference Biology Laboratory. Please refer to
Appendix 7 for details specific to your National Group, in case of queries please contact
the National group co-ordinator.
If the tumour pieces selected for molecular-genetic/biologic investigations were not
appropriate for getting reliable results, MYCN and chromosome 1p36.3 status and
ploidy can be determined on the paraffin embedded material. Laboratories which do not
perform this kind of investigation can send the paraffin blocks, ideally with H&E slides, to
Drs. Peter and Inge Ambros, Vienna, Austria.
1.2
SPLITTING AND SECURING OF TUMOUR MATERIAL IN
RESECTABLE TUMOURS:
The following procedure is recommended (it is necessary for the pathologist to have
help from a person, i.e. from a technician or the paediatric oncologist):
Cut the tumour at the largest diameter and take at least two samples from, if existing,
morphologically different appearing areas (1x1x1cm). Tissue from a suspected
nodule must always be sampled. Identify the samples specifically with capitals (A, B,
etc.) and cut each of them into four pieces which are marked with numbers (tumour
specimen A 1-4, specimen B 1-4). More material can be processed in the same way (C,
D, etc.), but material from two different areas is the minimum. Check carefully for the
presence of nodules!
A1 and B1: make 10 touch preparations (at least 5) from a freshly cut surface. The
slides are air-dried and unfixed and, if necessary, they can be stored at –20°C
(storage of the slides for one week at room temperature does not adversely affect the
following analyses); for fluorescence based in situ hybridisation (FISH) and image
cytometry (ICM).
After making the touch slides, these pieces have to be fixed in formalin for routine
histologic examination which also should include the determination and indication
of the tumour cell content versus content of normal cells, such as Schwann cells,
lymphocytes, fibrovascular stroma etc.; amount of necrosis should be indicated as
well. This information is crucial for the interpretation of the FISH, ICM and
cytogenetic results!
A2 and B2: put in sterile culture medium (RPMI 1640) for cytogenetics,
assessment of MYCN and chromosome 1p status (on cytospin preparations),
evaluation of ploidy, drug sensitivity, etc.
A3,4 and B3,4: snap freeze as soon as possible in separate vials in liquid nitrogen
or at –70°C carbon dioxide.
Tumour samples A, B, etc. (at least 1x1x1cm)
piece 1: Touch preparations
Formalin
piece 2: RPMI 1640
1
2
3
4
pieces 3 and 4: snap freeze
FREEZE AND SEND THE SAMPLES AS SOON AS POSSIBLE
After this procedure, the rest of the operation specimen can be fixed in formalin and
worked-up according to common guidelines. The whole central 4mm section of the
tumour at the plane of the largest diameter should be embedded. A minimum of one
tumour section per cm should be embedded from the whole specimen including central
and peripheral areas of the tumour. Surgical margins must be reliably and
reproducibly identifiable.
1.3
SPLITTING AND SECURING OF TUMOUR MATERIAL IN
UNRESECTABLE TUMOURS:
The general recommendations given in paragraph 1.1 should be followed. The splitting
procedure depends on the amount of tumour material available for diagnostic histology
and molecular-biologic investigations. Touch preparations can almost always be
performed before fixing and embedding the tumour material (for tru cut and fine needle
aspirations see below).
1.3.1 OPEN BIOPSY
In case of open biopsy, two different areas of the tumour should be biopsied by the
surgeon. Specimen size should equal at least 1 ccm.
In accordance with the recommendations given above, the tumour specimens are
labelled with capitals (A, B etc.), are cut into 4 pieces, and the procedure given in
paragraph 1.2 can be followed.
In case of smaller biopsy specimens, the material can be cut into only:
3 pieces:
piece 1: for touch preparations and histology
piece 2: for RPMI 1640
piece 3: for snap freezing
or into
2 pieces:
piece 1: for touch preparations and histology
piece 2: for snap freezing
1.3.2 TRU CUT BIOPSIES
General remarks
If only tru cut biopsies are available, preferably four biopsies (at least two biopsies in
case of small lesions) from different areas of the tumour should be obtained.
Recommended needle size: 18G.
Although tru cut biopsies are usually ultrasound-guided and performed by radiologists,
the pathologist should be present during the procedure and immediately assess
the quality of the specimens (macroscopically) in order to save trouble and frustration
due to delivery of mainly necrotic material (see below).
It is important that the tumour cell content is determined in the biopsy material used for
molecular-genetic/biologic analyses.
Handling of tru cut biopsies
The pathologist/radiologist should not try to scrape off the tissue from the biopsy needle
using a scalpel or other devices, but put the needle into a tube/glass with sterile RPMI
1640 or any sterile isotonic buffer and shake gently until the specimen is released into
the fluid. This procedure helps to avoid artefacts due to crushing and squeezing. In
addition, needle biopsies tend to stick to each other which may hamper the subsequent
splitting of the material. Therefore, each biopsy should be kept in a separate tube
containing RPMI 1640 or another sterile isotonic solution.
Assessment of quality
Specimens should be at least 1 cm long and 0.1 cm thick. Tiny and fragmented
material cannot be approved. If there is any doubt regarding the quality of the material,
shake the glass with the specimen thoroughly once or twice. If the specimen breaks to
pieces, it often indicates tissue necrosis. The pathologist should not hesitate to ask for a
new biopsy.
Since it is impossible to judge macroscopically, whether a needle biopsy contains
tumour cells or mainly stroma, tru cut biopsies should, if possible, be preceded by
fine needle aspiration which (a) informs about the tumour cell content of the elected
area after only a few minutes, and (b) produces cell suspensions for ploidy measurement
and large numbers of cytological specimens for FISH analyses.
Obtaining tru cut biopsies
Variant 1)
All pieces obtained are identified with capitals (A, B, etc.) and transferred from the tubes
into separate (!) Petri dishes, pouring the whole content of the tube into the dish, and are
divided carefully into two parts using a scalpel. This procedure should be done without
using tweezers to avoid crush artefacts.
One part of each piece is carefully transferred into a tube with formalin using a
Pasteur pipette (plastic, for single use) to avoid crush artefacts.
The other half of each piece is snap frozen. Put the specimen carefully on the wall of a
freezing tube in order to avoid a curved orientation of the piece and put the tube into
liquid nitrogen.
The pathologist and the biologist should discuss and agree on the size of the parts they
need for histology and for the biological analyses. Alternatively, one whole biopsy
specimen could be used for histology and the second (third, etc.) specimen can be split.
Touch preparations will be performed by the biologist using the snap frozen material.
Variant 2)
4 pieces:
2 pieces are transferred from the isotonic solution or the RPMI 1640 into 4%
buffered formalin in separate glasses and embedded in separate paraffin
blocks (marked A and B) for morphological and immunohistochemical
analysis.
2 pieces are gently transferred by tweezers into separate, flat-bottomed
(!) plastic tubes, carefully overlaid by a water-based embedding compound
for preparation of frozen tissue sections (try to keep the specimen localised
at the bottom level of the vial!) and must be snap frozen as soon as
possible in liquid nitrogen or at –70°C carbon dioxide for moleculargenetic/biologic analyses and immunocytochemistry. Make 10 touch
preparations as described below.
2 pieces:
1 piece is transferred into formalin and embedded in paraffin and the other
piece is snap frozen as described above followed by making 10 touch
preparations.
Procedure for making touch preparations
It is well known that preparations containing whole nuclei (touch or cytospin preparations)
are superior for FISH analyses. However, fresh unfixed needle biopsies only rarely yield
enough cells when used for touch preparations. This problem can be solved by using the
snap frozen material (see above) for making touch preparations. After careful
removing the plug with the forceps by warming the tube in the hands, cut one frozen
section starting from the bottom of the frozen plug, stain with H&E and assess for
morphologically intact tumour cells (representativity). The cold cut surface of the plug can
then be used for making touch preparations on warm (room temperature) glass slides,
but be careful to refreeze the plug in time, because complete thawing destroys the
morphology and renders the material inappropriate for any in situ-analyses at the single
cell level. However, such specimens can still serve as sources for DNA/RNA and protein
extraction (SB, PCR, comparative genome hybridisation, Northern blot, Western blot).
Further comment
If the frozen material does not contain sufficient numbers of tumour cells, FISH and
ploidy analyses can be assessed on nuclei extracted from the paraffin-embedded
bioptic material (see above).
1.3.3 FINE NEEDLE ASPIRATIONS CYTOLOGY (FNAC)
General remarks
In cases of unresectable tumours, the pathologists and biologists can also be confronted
with tumour material derived from fine needle aspiration (FNA). In general, at least two
separate punctures/aspirations should be performed from each tumour. Depending on
the size of the tumour, the needle should be moved backward and forward into different
areas under constant aspiration in order to sample from more than one region of the
lesion . FNA is also recommended to be done before tru cut biopsies are taken.
Recommended needle size: external diameter of 0.6-0.7 mm; 22-23 Gauge.
The FNA sampling is usually performed in close co-operation between radiologist and
cytopathologist. Due to the results of palpation and ultrasound examination of the tumour,
FNA is either performed directly by the cytopathologist (palpable tumours) or by means
of ultrasound guidance (non-palpable tumours). In this case, the radiologist guides the
needle into the lesion, while the cytopathologist aspirates.
Handling
Beware of the ultrasound gel! When aspirated, it might ruin the morphology of the cells.
The skin must be wiped carefully before puncture.
An adequate aspiration will often contain 104-105 cells.
The aspirate should always be utilised for preparation of smears and cell suspensions.
1.
2.
3.
4.
Depending on the amount of aspirate, one droplet is placed on each of at
least four slides and ordinary monolayer smears are produced and airdried.
Subsequently, the needle and the rest of aspirate are flushed through with
1 ml sterile phosphate buffered saline and the cell suspension is kept in
the Eppendorf vial.
One smear from each aspiration is stained by the DiffQuik method which
takes 3 min to render the smear light microscopically evaluable
(preferentially done by an assisting cytotechnician!).
A preliminary report on adequacy of the sample is given to the clinician
and the radiologist, and the cytopathologist decides whether the FNA has
to be repeated.
Recommendations for subsequent analyses
Smears are well suited for morphological evaluation (May GrÜnwald-Giemsa stain)
- immunocytology
- FISH (MYCN and 1p status etc.)
- image cytometry, e.g. for static ploidy analysis
Cell suspensions can be used for - FCM analysis of ploidy
- tissue culture for e.g. cytogenetics
- preparation of cytospins (about 103 cells per slide)
which are air-dried over night and used for the same
purposes as smears.
Quality assessment
A series of cytospin preparations often contains a more equal (i.e. predictable) number of
tumour cells as compared to a series of individually produced touch preparations and
smears. One smear/cytospin of each series/aspiration has to be analysed by
morphology/immunocytology in order to estimate the number of tumour cells. This
information must be sent together with the slide to the reference biology laboratory.
Cytospins, smears and cell suspensions may be stored frozen for future analysis.
1.3.4 BONE MARROW ASPIRATIONS (see Chapter 3)
In case of a high tumour cell number infiltrating the bone marrow in stage 4 patients,
these cells can also be used for determination of the genetic composition of the
circulating neuroblastic tumour cells. If the tumour cell content exceeds 60 per cent, the
MYCN copy number can also be determined by SB. Lower infiltrations should always be
analysed by FISH for evaluating the MYCN and 1p status. In unclear situations, a GD2
staining preceding the FISH is highly recommended.
1.4
HISTOLOGY/CYTOLOGY REPORT
1.4.1 Surgically resected tumours
Morphologic classification: the tumour should be classified according to two
classifications (Shimada 1984 and the International Neuroblastoma Classification, INPC,
Cancer 86, 349-371, 1999). The mitotic rate and calcifications should also be indicated.
Surgical margins of resection: it should be commented on if there are tumour cells
infiltrating the resection margins or not, without making any conclusion as to whether the
tumour residual is microscopic or macroscopic.
Histological report on the specimens A1, B1 etc: This report must clearly indicate the
estimated percentage of tumour cells, i.e. neuroblastic/ganglionic cells, versus Schwann
cells and other normal cells contained in the samples used for the biological studies. A
copy of this report should then be submitted to the molecular biologist.
1.4.2 Biopsies
In the case of limited biopsy material, the tumour material obtained is not necessarily
representative of the whole tumour. For example, the biopsy could be taken from either a
neuroblastic nodule or the ganglioneuromatous area of a nodular Ganglioneuroblastoma.
In such critical cases, the use of the following terms, according to the INPC, is
recommended:
Neuroblastic tumour, unclassifiable: this term relates to a tumour which belongs
unequivocally to the peripheral neuroblastic tumour entity, but which cannot be allocated
with certainty into one of the four basic categories which are:
Neuroblastoma (Schwann cell stroma-poor)
Ganglioneuroblastoma intermixed (Schwann cell stroma-rich)
Ganglioneuroma (Schwann cell stroma-dominant)
Ganglioneuroblastoma nodular (Schwann cell stroma-rich/-dominant and stroma-poor)
Other terms recommended by the INPC to be used for tumours giving rise to problems in
classification, are:
Neuroblastoma (Schwann cell stroma-poor), NOS: this term is used for tumours with
an unequivocal categorisation, but the subtype, i.e. undifferentiated, poorly differentiated,
differentiating, cannot be assessed due to poor quality of the sections, extensive
haemorrhage, necrosis, crush artefacts, etc. (see INPC).
Ganglioneuroblastoma, NOS: is used for a tumour with a stroma-rich/-dominant
appearance containing areas of extensive calcification which may obscure a stroma-poor
nodule.
1.4.3 Fine Needle Aspiration Cytology (FNAC)
Cytological preparations of neuroblastic tumours do not contain the information on tissue
architecture necessary for histological classification. If only FNAC material is available for
primary diagnosis, the report including number, morphology (differentiation status of
tumour cells, Schwann cells, necrotic debris) and immunophenotype of the cells
analysed must be submitted to the biologist.
1.5
TUMOUR MATERIAL OBTAINED AFTER CYTOTOXIC THERAPY
Splitting of the tumour material in resected tumours or biopsies after cytotoxic
therapy can be done following the same guidelines as for tumours resected or biopsied
at diagnosis before cytotoxic therapy (see 1.1, 1.2, 1.3). However, for sampling, it has to
be taken into consideration that necrotic areas and also calcifications can be massive.
Therefore, it is especially essential to exactly state the percentage of viable tumour
cells versus normal cells (see above). The amount of necrosis and calcification.
It is known that both chemo- and radiotherapy can induce marked morphological
changes and can also induce cytodifferentiation and maturation (with development of a
Schwann cell stroma), but probably do not change the original genetic characteristics of
the tumour. Therefore, tumours can be classified morphologically according to the
INPC. However, the statement on pre-operative therapy has to be included in the
diagnosis and no assignment to the prognostic subgroups must be made.
1.6
REGIONAL LYMPH NODE EXAMINATION
Biopsy of regional nodes is highly recommended whenever feasible despite their
appearance.
Histology report: the pathologist's report should include information on:
Site and number of positive nodes
Types of metastatic spread according to:
Presence of micrometastases (less than 2 mm)
Intranodal parcelled metastases
Intranodal massive metastases
Nodal metastasis with extracapsular extension in localisations not
adherent to the resected tumour specimen
Morphological description of the tumour infiltrate
1.7
IMMUNOHISTOLOGY/-CYTOLOGY
Differential diagnosis: In some cases, i.e. neuroblastomas, undifferentiated subtype
according to the INPC, the differential diagnosis can make difficulties. In these
instances, the use of the following antibodies is recommended:
MIC2 (CD99), actin, desmin, low molecular-weight cytokeratin, leukocyte common
antigen (CD45), and vimentin are usually negative in neuroblastic tumours.
CD56 (N-CAM), NB84a, (monoclonal neuron specific enolase (NSE), neurofilament
triplet protein (NF), synaptophysin, tyrosine hydroxylase, protein gene product 9.5).
However, it has to be kept in mind that these markers may also be negative in
undifferentiated neuroblastomas.
Comments: NB84a does also react with epithelial cell and endothelial cells (see also
below). Although GD2 is positive in virtually all cases of neuroblastic tumours and very
useful in e.g. detection of neuroblastic cells in the bone marrow, anti GD2 staining
cannot be recommended to be used for paraffin material (very high background
staining). Moreover, GD2 expression is not restricted to neuroblastic tumours!
Lymph node diagnostics: for differential diagnosis see above. In addition, the
morphological result can be controlled by immunohistology using anti CD56 and anti
NSE antibodies. If the NB84a antibody is applied, its reaction with endothelial cells
should be kept in mind.
Exact determination of the tumour cell content: especially in the samples used for
molecular-genetic/biologic investigations and DNA analyses, the use of S-100 for
unequivocal detection of Schwann cells and of leukocyte common antigen is
recommended.
Cytological material: for detection and quantification of tumour cells in bone marrow
and fine needle aspirates, anti GD2 for bone marrow diagnostics, and anti GD2, anti
CD56 and anti S-100 for material obtained by fine needle aspiration are
recommended. For differential diagnosis see above.
1.8
PATHOLOGY REVIEW
To be sent to the National reference pathologist:
H&E slides from all available paraffin blocks
It is most important to include the H&E slide from the paraffin block adjacent to the
tumour specimens used for molecular-genetic/biologic investigations (sample A)
10 unstained sections from the most representative paraffin blocks
Copy of the written report including immunohistological results
The tumours will be reviewed by a central review panel.
2
BIOLOGY GUIDELINES FOR RESECTABLE AND UNRESECTABLE NEUROBLASTIC TUMOURS
2.1
GENERAL REMARKS AND RECOMMENDATIONS
Analyses of MYCN, chromosome 1p36.3 status and tumour cell DNA content are
obligatory investigations which have to be carried out in the National Reference
Laboratories or other Reference Centres, i.e. laboratories participating in the
European Neuroblastoma Quality Control Assessment (ENQUA) study. Results from
other laboratories will not be used for treatment group assignment.
To enable reliable interpretation of the molecular-genetic results, the exact tumour
cell content of the specimen used for these investigations has to be determined. This is
possible only if the pathologist evaluates the specimens adjacent to those used for
molecular-genetic/biologic analyses (for details see below). An exact determination of
the tumour cell content and correct tumour sampling are crucial prerequisites and
absolutely necessary for obtaining reliable molecular-genetic/biologic results (see
Chapter 1).
To be eligible for the entry in the Trial it is essential that the results for the MYCN studies
are available within two weeks (latest four weeks) of the tumour resection or the
diagnostic biopsy.
Infants found to have tumours with MYCN amplification it is essential that this result is
confirmed by another technique.
A common terminology for reporting MYCN and chromosome 1p36.3 results was
developed by the ENQUA-group and is given below. For uniformity and comparability, it
is strongly recommended to use these terms.
A quality control study performed in 1998 made evident the pitfalls which are inherent to
each method. A leaflet giving recommendations how to avoid or to reduce the most
commonly observed problems and pitfalls will be circulated to all biology laboratories
involved.
2.2
PROCEDURES FOR THE DETERMINATION OF THE TUMOUR CELL CONTENT
Resected tumours and open biopsies (see also Chapter 1, Pathology Guidelines):
Piece 1: FISH/ICM is performed on touch preparations. In this case, the tumour cell
content is determined on the formalin-fixed paraffin-embedded material of piece 1
and is included in the pathology report. Don’t make any conclusion in case no
numeric and no structural chromosome aberrations are found by FISH and/or the ICM
indicates a diploid DNA content. First, the pathologist has to indicate whether the cells
analysed are tumour cells or not!
Piece 2: in RPMI 1640 is used for cytogenetic studies or for making cell suspension
for FCM and cytospin preparations. Therefore, determination of the tumour cell
content cannot be carried out using this specific piece. If there is any doubt about the
nature of the cells analysed, e.g. no numeric, structural chromosome aberrations
and/or presence of a diploid DNA content, a GD2, NB84a, S-100 and common
leukocyte staining is recommended (see also Chapter 1).
Pieces 3/4: are snap frozen by the pathologist and are used for immunohistological
investigations and DNA extraction. For interpretation of the SB and PCR results, the
knowledge of the exact tumour cell content is especially important. Therefore, the
following procedure is recommended:
The snap frozen piece is first used for making touch preparations (avoid complete
thawing of the piece by dipping the piece repeatedly in liquid nitrogen!) for FISH and
ICM. Then, frozen sections are made before extracting DNA for PCR, SB or other
investigations. The frozen sections are especially important for determination and
documentation of the tumour cell content.
Snap frozen pieces
1. Prepare touch preparations (avoid complete thawing), at least 5 if possible
2. Make frozen sections (tumour cell content should be determined by the
pathologist)
3. Extract DNA
Tru cut biopsies:
The snap frozen material received from the pathologist should be handled as
indicated above for pieces 3 and 4.
Fine needle aspirations:
Smears and cell suspensions will be provided by the cytopathologist who will also
indicate the tumour cell content.
2.3
GENETIC PARAMETERS TO BE ANALYSED
2.3.1 MYCN COPY NUMBER
Methods and general remarks
The MYCN copy number can be determined by Southern Blot (SB) or fluorescence
based in situ hybridisation (FISH). Polymerase chain reaction (PCR) is not
recommended to be used without a second method.
It has to be kept in mind that by using SB as the only method for MYCN evaluation, the
chance to miss possible heterogeneities in the MYCN status (see Chapter 1) or to
misinterpret low amplification is higher (due to the ‘dilution’ effect) as compared to
FISH analysis which is done on the single cell level.
For SB and PCR, the tumour cell content has to be over 60 per cent.
For FISH, at least 500 tumour cells from different areas on the slides have to be
analysed.
SB use either pNb1 (Dr Schwab, Heidelberg, FRG) or an equivalent probe and a probe
for a single copy gene which has to be located on chromosome 2 (e.g L2.3).
FISH: use either the Oncor (Vysis) MYCN probe or the pNb9 or pNb101 (Dr. Schwab,
Heidelberg, FRG) and a chromosome 2 specific probe either specific to the centromere
(D2Z, Oncor) or to chromosome 2pter (to avoid misinterpretation by centromeric
associations).
PCR: These studies are only accepted when the PCR data are compared with SB or
FISH results. A detailed protocol (written by Dr. Olivier Delattre) will be circulated
together with the pitfall protocol.
MYCN copy number
Irrespective of the method used, the copy number has to be indicated according to the
number of chromosomes 2.
MYCN definitions and report of results:
SB and PCR - MYCN
MYCN amplification =
over 4-fold increase of the
band intensity in relation to the band from the
internal reference (on chromosome 2!).
Imbalance =
2 to 4-fold increase of the band
intensity in relation to the band from the internal
standard (on chromosome 2).
Needs further clarification by FISH!
No MYCN amplification
No result =
Unclear or not interpretable result
Sample contains less than 60 % of tumour cells
No DNA/No tumour
FISH-MYCN
MYCN amplification =
over 4 fold increase of the MYCN signal number
in relation to the number of chromosomes 2.
MYCN gain =
2 to 4 fold excess of MYCN copies in relation to
the number of chromosomes 2. Needs further
clarification!
MYCN amplification focal
No MYCN amplification
No result =
Unclear or not interpretable result
Not enough tumour cells contained in the sample
No tumour
2.3.2 CHROMOSOME 1P36.3 STATUS
Methods and general remarks
The integrity of the short arm of chromosome 1 (1p36.3) can be determined by three
methods, i.e. SB, PCR and FISH.
It has to be borne in mind that investigations carried out with SB and PCR can be used
to answer the allelic status of the chromosomal region 1p36.3. In contrast, analyses
done by FISH give information on the chromosomal level, i.e. on the relation between
the numbers of centromeres and subtelomeric regions of chromosome 1. A 2 to 1 ratio
found by FISH (i.e. 2 centromeres to 1 subtelomeric region) most likely corresponds to a
loss of heterozygosity (LOH) found by SB or PCR. However, every other disproportion of
centromeres and subtelomeric regions but with more than 1 1p36.3 signals (i.e. a 3 to 2,
4 to 3, 5 to 3 ratio etc.) found by FISH, does not necessarily reflect the presence of an
LOH. Vice versa, lack of an LOH does not necessarily reflect lack of cytogenetic
aberrations on chromosome 1p36.3.
Therefore, it is strongly recommended to use both kinds of methods (SB/PCR and
FISH) for the detection of chromosome 1p36.3 aberrations in order to get the whole
information of possible changes in this important chromosomal region!
For SB and PCR, the tumour cell content has to be at least 60 per cent.
For FISH, at least 500 tumour cells from different areas on the slides have to be
analysed.
SB: use e.g. the probe CEB15 or another probe (e.g. D1S7, Dr. Jeffreys) within
the consensus region (i.e. 1p36.3)
PCR: use e.g. the primers specific for D1S76 and D1S80 or others within the
consensus region.
FISH: use e.g. the probe D1Z2 together with a centromeric probe (D1Z1) or a
probe located on the long arm of chromosome 1 (to avoid misinterpretation by
centromeric associations) in a double colour FISH approach.
Chromosome 1p36.3 definitions and report of results
PCR and SB – Chromosome 1p36.3
complete or almost complete disappearance of one
band
Allelic imbalance (=inconclusive) = one band weaker than the other
band. This can mean either allele disequilibrium
(e.g. two paternal and one maternal chromosomes 1)
or LOH. In this case, the experiment has to be
repeated, the tumour has to be checked by
FISH, and the tumour cell content has to be
re-evaluated!
No allelic loss, no allelic imbalance
No result = Unclear or not interpretable
Constitutional homozygosity
Sample contains less than 60% of tumour cells
No DNA or no tumour
Allelic loss (LOH) =
FISH – Chromosome 1p36.3
Deletion =
only one subtelomeric region of the short arm of
chromosome 1 present (ratio 2/1 possibly together
with 4/2 in the same tumour, 3/1, 4/1).
FISH imbalance = disproportion of the ratio of centromeres of
chromosome 1 to the subtelomeric regions of the
short arm with more than one subtelomeric
regions (ratio 3/2, 4/3, 4/2, 5/3 etc.).
Needs further clarification by PCR or SB.
No deletion, no FISH imbalance detected by FISH with the probes...
No result =
Unclear or not interpretable result
Not enough tumour cells contained in the sample
No tumour
Recommendations: if less than 50% of the tumour cells show deletion or imbalance by
FISH, more slides, samples or nuclei isolated from paraffin material shall be
analysed; paraffin material should also be used when FISH on touch slides was not
successful (e.g. ganglioneuroma).
Report of the molecular-genetic results
The molecular-genetic results should be reported in the following manner:
A detailed description of the results and the methods used including percentages of tumour
cells versus normal cells, hybridisation pattern etc. should be described in a report section.
In the conclusion section, the result should be given as briefly and precisely as possible
using the terminology given above.
2.3.3 DNA content
Methods and general remarks
Two methods, i.e. Flow Cytometry (FCM) and Image Cytometry (ICM) can be used for
the assessment of the tumour cell DNA content.
The number of chromosomes 1 obtained by FISH analysis should not be used for
estimation of the DNA content as triploid tumour can be disomic for chromosome 1 or
diploid tumours can be trisomic for chromosome 1 etc.!
FCM: use reference cells derived from the same patient.
In case, the tumour material is not appropriate for determining the DNA content by FCM
and the respective reference laboratory does not perform ICM analyses, touch
preparations can be sent to Dr. Per Kogner, Karolinska Institute, Stockholm, Sweden
for ICM investigation.
Report of the FCM/ICM results
The report should indicate the method used and specify the number of tumour cells
versus normal cells contained in the sample under investigation.
The results on the DNA content of the tumour cells should be given in absolute
numbers.
2.4
CENTRAL REVIEW
2.5
MYCN, chromosome 1p36.3 results and data on the DNA content will be centrally
reviewed.
ANALYSIS OF OTHER PARAMETERS TO BE CHECKED FOR THEIR CLINICAL RELEVANCE
Gene products to be investigated
High affinity nerve growth factor receptor (trkA or p140trk)
Low affinity nerve growth facto receptor (LNGFR)
CD44
Methods
Expression of the genes listed above can be investigated on the RNA level by Northern
blot or on the protein level by immunohistology.
For Northern blot analyses, snap frozen material is essential.
For analyses of these parameters, tumour samples can also be sent to Dr. Nicole Gross,
Lausanne, CH and Drs. Marie Favrot and Valerie Combaret, Lyon, France.
3
BONE MARROW ASPIRATIONS
3.1
Supply two to four syringes with plugs and 10 to 20 glass slides for the bone marrow
smears and one polished cover glass. In addition, prepare an adequate anticoagulant
[e.g. heparin, EDTA, ACD (1:5)]. Two aspirations are necessary: one for bone marrow
smears and the second for immunocytology, PCR or other techniques.
First aspiration and preparation of bone marrow smears:
Take bone marrow aspirates from at least two, but four different sites are strongly
recommended. Aspirate briefly to obtain 0.2 ml to 0,5 ml bone marrow and put the
whole amount on a glass slide. Put this slide in an upright position and allow the
excess blood to flow down. Dip into the drop of bone marrow a polished glass cover
slip and transfer this dipped cover slip to a new glass slide and immediately
afterwards pull this small droplet with a cover slip along the slide. Air dry the slides for
at least 10 minutes. Stain slides according to Pappenheim.
Second aspiration for immunocytology:
Anticoagulant marrow aspirates with heparin, EDTA, or ACD (1:5). Aspirate the
appropriate [e.g. 100µl Heparin (1000IE/ml) in 10 ml PB or 5 ml BM] amount of
anticoagulant into the syringe. To avoid excessive dilution with peripheral blood each
aspirate should be no larger than 4-5 ml, ideally 2-3 ml. Shake immediately to allow the
anticoagulant mix with the bone marrow. Repeat this procedure for each punctation site.
Close the syringe properly to avoid leakage. Transport the closed syringes to the
haematology or immunology laboratory at room temperature as fast as possible.
3.2
HANDLING OF THE BONE MARROW CELLS IN THE LABORATORY
Separation of MNC, preparation and storage of cytospins
Separate the mono-nuclear cells (MNC) by a Lymphoprep (Nycomed) or Ficoll density
separation (1.077; follow the instructions of the manufacturer). Ideally, separate all
different sites independently. When it is needed that the different sites are pooled, pool all
bone marrows from the left site and right site separately. Alternatively, all sites can be
pooled but this must be indicated. After careful taking out the MNC fraction, wash cells in
PBS or Tris pH 7.0, without FCS and spin them down gently (e.g. 10min at 252g). The
recovered mononuclear cell fraction (MNC) should be counted and tested for % of vitality
by trypan blue dye-exclusion test.
Make the cytospins either with a Hettich or Shandon centrifuge. A total number of at least
2x106 MNC should be analysed. Each investigator can choose the method he/she is
more comfortable with, as long the cell preparations are not overcrowded and MNCs lie
well separated from one another. In fact, cell clumps can give both false negative and
false positive results. This MNC cell preparation can be achieved in different ways: by
several (10-20) regular diameter (5 mm) (Shandon) or, preferably, 2-3 wide diameter (17
mm) (Hettich) cytospins, by MNC sedimentation on sticky slides (Poly-L-Lysine; CellLine, Cell-Tak).
Cell preparations are air-dried for 2 to 24 hours and fixed with either 1-4%
paraformaldehyde for 10 min (since GD2 is a lipid antigen alcohol fixatives should be
avoided). The samples can be immediately stained or stored at –20°C or at –80°C (for
prolonged storage) in air-tight plastic chambers or tightly wrapped in aluminium foil. After
thawing, let samples dry (in closed boxes) for at least 2 hours before processing.
GD2 Immunocytology
The following guidelines have been developed for the purpose of improving initial staging
accuracy, treatment response evaluation, and, ultimately, patient care. Presently, the use
of anti GD2 antibodies to detect NB cells gives the most consistent results in terms of
sensitivity and specificity. The GD2 antigen is a disialoganglioside highly and consistently
expressed on neuroblastoma cells and not on normal marrow cells. In addition, this
methodology is relatively simple, cheap and widely applicable. However, macrophages
with internalised GD2 positive tumour material as well as unspecific adherence of the
antibody to the nuclear or cellular membrane can give rise to difficulties. Therefore, the
use of further independent or complimentary techniques in order to confirm or to disprove
doubtful results is recommended (i.e. FISH for NB associated genetic aberrations, RTPCR for NB specific transcripts).
• As long as each positive event is directly examined and confirmed under microscopy
(this excludes flow cytometry), fluorescence-based immunocytology or immunocytochemistry techniques are equally acceptable, provided a permanent record is
possible and the adequate study concept is followed and quality controls are
performed (see .4).
• In BM with less than 0,1% of tumour cell contamination at least 2x106 MNC should be
analysed. In highly infiltrated BM samples a smaller number of MNC can be analysed
(e.g. 30 fields in infiltrates over 1%).
• At least three extra unstained cytospins should be stored frozen (ideally at - 70°C) for
quality control studies.
The primary monoclonal antibody employed is left to the preference of each investigator
within a list of antibodies:
14.G2a (Ralph Reisfeld, La Jolla)
3F8 (Nai-Kong Cheung, New York)
5A7 (Nicole Gross, Lausanne)
The primary antibody can be used in the purified form or as the hybridoma supernatant
as long as the lowest concentration which consistently stains the positive control without
background staining is employed.
The type of secondary antibodies and development systems employed are left to the
preference of each investigator as long as the detection system is validated with cell-line
scalar dilutions and the proper negative and positive controls are employed (see below).
Staining Protocols
Fluorescence staining (Peter F. Ambros, CCRI Vienna, Tel +43-1-40470-412)
Include always a positive and a negative slide (fixed as stated below) for control.
Use either freshly prepared slides - but fresh slides must be air dried over night to
obtain good results - or when frozen, thaw them for at least two hours at room
temperature in closed boxes or wrapped before being used.
Stain and fix slides with DAPI/paraformaldehyde for 10 min. Prepare a DAPI working
solution by diluting 10µl of the stock solution (2mg/ml in PBS) in 10 ml PBS (store in the
dark at 4°C). Use 50µl from the working solution, dilute in 850µl PBS and add 100µl 40%
paraformaldehyde. Do not stain/fix slides in a chamber as tumour cells may float off and
may adhere to other slides so obscuring the picture! Ideally, all staining and fixing
procedures should be carried out under a cover slip. After fixation wash with PBS.
Make sure that the cells do not dry out during the whole staining procedure!
•
•
•
•
Remove excess of PBS and incubate the cells for 20 min. at 37°C with the anti
GD2 antibody diluted in PBS including 3% BSA. Evaluate the correct staining
concentration of the antibody by serial dilutions.
•
Wash with PBS including 3% BSA.
Incubate with an anti mouse antibody (e.g. rabbit anti mouse, FITC labelled,
DAKO F0313) 1:60 in PBS including 3% BSA for 30 min.
•
Wash again as above.
Incubate with a swine anti rabbit FITC (DAKO F205) 1:60 in PBS including 3%
BSA.
Wash again with PBS, remove excess of liquid and mount (without drying the
cells) with Glycerol containing an antifade (e.g. Citifluor AF3; UKC, Chem. Lab.
Canterbury, CT2 7NH, UK in Glycerol+Citifluor+PBS, 8+1+1, or
Vectashield+DAPI))
For detecting the tumour cells scan the slide with a 20x or 25x lens with a
fluorescence microscope equipped with a FITC filter for detailed analysis change
to a 50 times or 100 times lens.
Pitfalls: To avoid misinterpretation of positive cells such as macrophages, plasma
cells, staining of the nuclear membrane after disruption of the cell membrane etc.,
control morphology by phase contrast and DAPI staining. When the tumour cell
infiltrate is less than 1 in 104 MNCs, check by FISH or another technique whether
the genetic features of these cells correspond to the tumour cells (e.g. MYCN
amplification, 1p deletion, or other chromosomal aberrations). This sequential
analysis can ideally be done using MRDetect (MetaSystems, Altlussheim, FRG).
Enzymatic staining
A. (Klaus Beiske, Norwegian National Hospital Oslo, fax 0047-22-868596)
For light microscopy microscopic demonstration of tumour cells, fix air dried cytospins in
4% paraformaldehyde for 10 min (without the DAPI solution) and incubate with the anti-
GD2 antibody followed by polyclonal rabbit anti-mouse Ig antibody (DAKO D 314,
Dakopatts, Copenhagen, Denmark) and swine anti-rabbit Ig antibody (DAKO D 306),
both conjugated with alkaline phosphatase. Each incubation step is abrogated by threefold washing with TRIS-HCL buffer 0.01M at pH 7.6.
Visualise specific binding by treatment with substrate solution (15 mg Naphtol AS-TR
phosphate [Sigma], 20mg Fast Red ITR salt [Sigma], and 12mg Levamisole [Sigma], in
50 ml Michaelis Veronal acetate buffer pH 7.6) for 45 minutes at 37°C providing a deeply
red staining product. Counterstain specimens with hematoxylin, mount from water with
glycerine jelly, and examine under a light microscope.
B. Labelled Avidin Binding (LAB) Method on Hettich Cytospins (106 MNCs each)
(Lawrence Faulkner, Children’s Hospital Florence, fax 0039-55-570380)
MNCs are resuspended at 1-2 x 106 cells/ml and cytospins containing up to 106 MNC
each on a single area of 17 mm diameter are prepared by centrifugation 0.4-0.8 ml of the
MNC suspension at 400g for 10’ (Hettich Centrifuge, Tuttlingen, Germany) on regular
glass slides. Cytospins are dried at room temperature for 4 to 24 hours. After fixation for
10’ in cold acetone slides are washed in PBS for 5’, dried for 5’, followed by incubation
for 30’ at room temperature in a humid chamber with the primary mouse monoclonal antiGD2 antibody. After rinsing, washing in PBS for 10’ and drying for 5’ the slides are
incubated for 30’ with a polyclonal biotinylated anti-mouse antibody followed by rinsing,
washing in PBS for 10’, drying for 5’ and by a third 10’ incubation with a streptavidinalkaline phosphatase complex (LSAB2, DAKO). After washing, incubation with new
fuchsin chromogen (DAKO) for 8’ is used for staining bound alkaline phosphatase
complexes. Counterstain by incubating 5’ with hematoxylin. For each staining procedure
process a negative control (substituting the primary antibody solution with PBS) and a NB
cell-line cytospin positive control.
Note: Enzymatically stained specimens are inappropriate targets for interphase
cytogenetics (e.g. FISH), since enzyme substrate complexes may cover the
nucleus and thus prevent hybridisation. Extra cytospins should be available for
staining with fluorescence labelled antibodies in order to increase the reliability of
micrometastatic disease detection.
3.3
TEST INTERPRETATION, RESULT REPORTING AND QUALITY CONTROL STUDIES
• For each test a negative control with a comparable number of MNC should be examined. A
total of at least 0.5 to 1 x 106 MNCs should be evaluated for both the negative control (in
which the primary antibody is substituted with a buffered saline solution) and the actual
sample. For each staining procedure a positive control (prepared with a NB cell line) should
be prepared. This number can be reduced in cases with a high tumour cell count (>5%).
• A positive cell should be called such if it has a well recognisable nucleus and a completely
stained cytoplasm. An interpretable test is a test in which the negative control has no
significant background staining such as to interfere with positive cell discrimination and/or
has no cells that would be scored as positive. The positive control has to be brightly stained.
In other circumstances the test should be reported as non-interpretable.
• In doubtful cases or in cases with a low tumour cell infiltrate, the genetic aberration of the
tumour cells should be visualised to ensure that the immunological stained cell displays the
same genetic aberration as the primary tumour.
• In some instances clumps of amorphous GD2-positive material can be detected (cell
membranes? micelles?), they should be reported as such and quantified as best as possible.
+
For highly positive samples (>1 GD2 cell per 200x field) the result can be reported as a
percentage after having counted at least 30 fields.
• The number of positive cells should be reported together with the total number of cells
examined. The investigator should know how many total cells he/she is actually examining
with an error no greater then 10-20% (the average cell loss during slide/cytospin preparation
and/or staining should be initially verified for each immunocytology method). Light
microscopically stained slides should be stored at room temperature. All positive and
negative stained slides should be stored. Fluorescence slides should be stored at 4°C.
Fluorescence signals due to bound antibody and/or hybridised probes should be registered
and archived either as optically or digitally processed microphotographs.
• Unstained slides from at least five different patients and NB cell line dilutions, will be
circulated to other laboratories performing GD2 immunocytology. The testing results will be
discussed at a meeting of the different groups.
APPENDIX 7
ORGANISATIONAL ASPECTS
The conduct of this international study will be according to the following agreed procedures:
1. Status of Study
This is a collaborative study between several equal participating national groups.
The core group includes Austria, Belgium, Denmark, France (SFOP), Italy, Norway, Portugal,
Spain, Sweden, Switzerland and UK and Eire (UKCCSG).
Other national groups may join the study after discussion with the study committee.
2. Steering committee
Paediatric oncologists representing each national group and at least two statisticians will
participate in the steering committee of the study.
The Steering Committee shall meet as appropriate to consider patient treatment, eligibility and
outcome to ensure the smooth running of the study.
The information given twice yearly to the steering committee is:
• Accrual rate, description of causes of ineligibility,
• Toxicity data
• Description of the events
• Survival curves (overall and event-free survivals)
Interim analyses will be given to the Steering Committee. All scientific decision concerning
stopping, continuation or any amendment of the study will be made by the Steering Committee,
after discussion with the D.M.C.
3. Data Monitoring Committee (DMC)
An independent Data Monitoring Committee composed of 3 international experts will monitor
the progress of the study on ethical and scientific grounds on an annual basis.
The role of the DMC will be:
a) To review accrual rate
b) To examine interim analysis
These interim analyses will remain confidential. On the basis of these analyses, the DMC may
recommend whether the study should continue or whether it should be changed or terminated
prematurely.
c) To monitor toxicity
Every 6 months the statistician for the study will circulate a report to members of the DMC about
toxicity. The DMC will review these interim toxicity data although this is primarily the responsibility
of the study committee.
This biannual procedure will demonstrate any major toxicity.
d) To examine other trials
The DMC will review reports of related studies performed by other groups or organisations to
determine whether such information materially affects the aims or preliminary findings of the study.
e) Other
The DMC will be asked to review any major alteration to the study proposed by the study committee
prior to its implementation.
4. The Protocol
a) One common protocol will be used for the international study by all national groups. The finalised
master protocol in the English language will be held by the British Data Centre (UKCCSG). A
literal translation of the English master protocol will be prepared, if necessary, by each national
group.
b) Each national group Data Centre will be responsible for distribution of protocols to centres within
that national group.
c) Addenda may be added independently by any of the national groups to address local needs,
provided they have no bearing on the essential aims of the international protocol.
d) Subsequent to finalisation, any amendments to the protocol must be agreed by all the national
groups. The Central Data Centre, as the co-ordinators of the protocol, will issue a revised version
of the protocol, as required.
5. Study forms and data collection
a) Data may be collected on one common set of forms and forwarded to the central data centre or
data may be collected in each national data centre and sent electronically using a common coding
system, format and identical cross-checks.
b) Each national group Data Centre will be responsible for distribution of forms to centre within that
national group.
c) Additional forms may be produced independently by any national group for the collection of data
additional to that required for the international study.
d) Subsequent to finalisation, amendments to the forms must be agreed by all national groups. The
central Data Centre will be responsible for the issue of amended forms.
e) Each national group shall collect forms for its own patients, and shall be responsible for data
quality according to local practice. The data will be sent every 6 months unless a special request is
made by the Central Data Centre.
f) Registration will be carried out by faxing the pre-registration and registration forms to the
National Data Centre.
g) The master database for the entire study will be held at the Italian Data Centre.
h) Address of the Italian Data Centre is:
Trial Center – Neuroblastoma Unit
National Institute for Cancer Research
Advanced Biotechnology Center
Largo R. Benzi n.10
16132 GENOVA
ITALY
Fax:
Phone:
E-mail:
00 39 010354103
00 39 0105737
[email protected]
6. Confidentiality of patient data
a) The use of names as patient identifiers on paper forms and on national databases will be according
to national practice.
b) An abbreviated patient identifier may be used for data transfer and for the master database.
7. Data Quality Control
a) On receipt of forms at the national data centre, common range and logical checks will be carried
out on data prior to transfer to the master database. Any amendment to the checking programs
shall require mutual agreement of all national groups.
b) Data verification shall be carried out according to current national practice. Cross checks of data
entry will be carried out occasionally, between national centres, on a sample of forms.
c) Errors noted on the master database, after receipt of the group data, shall be reported back to the
national centre.
8. Data analysis and monitoring
a) Results of interim analysis of outcome and toxicity shall be reported to an independent
international data monitoring committee (DMC) as scheduled by protocol. The DMC may
recommend early stopping, continuation or extension of the study to the international study
committee.
b) Reports prepared from the national databases on overall outcome may be circulated locally subject
to approval for release by the DMC. No other publication will be allowed.
9. Adverse Events
a) Any adverse event (death, relapse or Grade 4 non-haematological life threatening toxicity) shall
be reported immediately by the treating institution to the national centre and relayed to the other
national centres, for further reporting according to local practice.
b) The toxicity criteria will be the same for all participating groups and appear in Appendix 4.
10. Pathology Review
a) For all cases, diagnosis is based on local pathologist observation but slides must be reviewed by
one of the pathology panel.
b) National group pathology data shall be discussed at the meeting of the international study
committee. This committee will meet as appropriate.
11. Institutional/Local ethical approval and patient consent
a) Institution/local ethical approval shall follow accepted national practice.
b) Accepted national procedures for patient consent as documented shall be used.
12. Contacts and core committee members
Members of the core committee for each country are listed on page 3.