Relapse Cytogenetics Overview of ALLR3 genetics Introduction to the IntReALL trial. Anthony V Moorman Leukaemia Research Cytogenetics Group

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1 Relapse Cytogenetics Overview of ALLR3 genetics Introduction to the IntReALL trial Anthony V Moorman Leukaemia Research Cytogenetics Group

2 Overview of ALLR3 Parker et al, Lancet 2010; 376:

3 Mitoxantrone better than Idarubicin Parker et al, Lancet 2010; 376:

4 ALL97/99: Cytogenetic Risk Groups Moorman et al (2010) Lancet Oncology, 11:

5 ALLR3 Cytogenetics at diagnosis and relapse Diagnosis Haploid 2% Relapse Haploid 3% t(5;14) 2% TAL1 2% Other 28% RUNX1 17% HeH 25% iamp21 5% IGH 1% TCR 2% t(5;14) 1% TAL1 2% Other 29% TCR 1% ETV6- ETV6- RUNX1 16% HeH 24% iamp21 5% IGH 1% Normal 10% t(17;19) 1% MLL 4% t(1;19) 2% Normal 9% MLL 5% t(1;19) 2% t(17;19) 1% High degree of stability between diagnosis and relapse for primary chromosomal abnormalities. Among 139 patients appropriately tested only 1 (0.7%) did not show the presence of the same abnormality at relapse.

6 Distribution of ALLR3 patients by cytogenetic risk group Patients with good risk cyto significantly younger (10.4y v 8.9y) Poor Risk, 67, 19% Intermedi ate Risk, 115, 33% ETV6- RUNX1, 66, 19% HeH, 99, 29% No strong relationship between cytogenetic risk group: Site of relapse; but patients with high risk cyto slightly more likely to have marrow involvement. 30% testes relapses were ETV6-RUNX1 MRD at day 35 but patients with high risk cyto slightly more likely to be MRD positive Based on 347 patients classified using either diagnostic or relapse karyotype

7 Cytogenetic risk group linked to time to relapse Late Early Very Early 20 0 ETV6-RUNX1 HeH Intermediate Risk Poor Risk

8 Multiplex Ligation-dependent Probe Amplification (SALSA MLPA kit P335-A1 ALL IKZF1) Probes for genes most frequently deleted in BCP-ALL IKZF1 (8) Early B-cell differentiation gene (deleted in 29% high risk ALL) CDKN2A/B (3) Cell cycle G1 control -CDK inhibitor and p53 stabilizer (deleted in 20% BCP-ALL) PAX5 (6) B-cell differentiation gene (deleted in 32% of BCP-ALL) EBF1 (4) - Early B-cell differentiation gene activating PAX5 ETV6 (6) Transcription factor required for haematopoiesis (involved in translocations in ALL and deleted in 5%) BTG1 (4) Negative regulator of cell cycle RB1 (5) - Negative regulator of cell cycle (deleted in 5-11% B-ALL) CRLF2/CSF2RA/IL3RA (1 each) Deregulated in 6% BCP-ALL

9 ALLR3 cohort available for MLPA testing 416 B-lineage cases 227 tested by MLPA 184 (44%) tested at relapse Representative wrt age, sex, time to relapse Tested slightly more cases with high risk cytogenetics and marrow involvement. 35 had isolated extramedullary relapses only one positive for any deletion 149 (36%) had marrow involvement 145 (35%) tested at diagnosis Representative wrt age, sex, cytogenetics Tested marginally more cases who went on to have isolated CNS relapses and had early relapse. 102 (25%) tested at both time-points. 78 with marrow involvement

10 Incidence of micro-deletions in BCP-ALL patients Original diagnosis (n=145) Marrow Relapse (n=149) Data from ALLR3

11 Incidence of micro-deletions in BCP-ALL patients 78 matched pairs only Percentage of cases with micro-deletion IKZF1 CDKN2A/B PAX5 RB1 BTG1 ETV6 EBF1 P2RY8-CRLF2 Percentage of cases with micro-deletion IKZF1 CDKN2A/B PAX5 RB1 BTG1 ETV6 EBF1 P2RY8-CRLF2 Original diagnosis Marrow Relapse Data from ALLR3

12 Genetic changes between diagnosis and relapse Losses, 10, 13% Mix, 6, 8% No change, 34, 46% Gains, 25, 33%

13 Incidence of micro-deletions by cytogenetic risk group IKZF1 CDKN2A/B 0 ETV6-RUNX1 HeH Intermediate Risk Poor Risk Overall

14 Incidence of micro-deletions by time to relapse IKZF1 CDKN2A/B Very early Early Late Overall Excluding good risk cytogenetics 50% of late relapses have an IKZF1 deletion

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16 ALLR3 Cytogenetics - Summary Cytogenetic risk group predicts outcome post-relapse - among standard risk patients. Spectrum of micro-deletions similar at relapse compared to presentation. Gains and losses observed in matched pairs but gains more common. Incidence of IKZF1 and CDKN2A/B deletions increases at relapses. Preliminary analysis shows that there is no association between IKZF1 or CDKN2AB deletions and outcome post relapse among ALLR3 patients Future plans supplement cohort with ~30 UK cases and add 73 ANZCHOG cases

17 IntReALL 2010 consortium

18 Outline of treatment schedule of IntReALL 2010 Recruitment Target patients over 4 years BFM Relapse Arm ALLR3 Relapse Arm Under review

19 IntReALL Diagnostics & correlative science overview Relapse sample Diagnostics - Morphology - Immunophenotype - Genetics (cyto/mol) - MRD (PCR/Flow) National representative Central IntReALL Trial Database (Marvin) National Biobanking Xenograft bank Pop Gen Tech Targeted NGS Genomic annotation Clinical and Scientific Research Projects Central IntReALL Diagnostic, Biobanking & Research Database (Scopeland)

20 Cytogenetic Representatives Zuzana Zemanova Joelle Tchinda Oskar Haas Bertil Johansson Anthony Moorman Anna Poluha Valerie de Haas Marta Jeison / Batia Stark Helene Cave Hiroaki Goto Paula Gameiro Silivia Bungaro / Anna Leszl Rosemary Sutton / Tom Revesz

21 Guidelines for minimum standards & data Collect details of genetic testing performed at diagnosis/presentation and relapse Essential tests Full G/R-banded analysis (or SNP/CGH array) of all samples from marrow relapses. Targeted FISH / RT-PCR testing in all relapses for the principal genetic abnormality observed at diagnosis If no/failed diagnostic genetics then Screen relapse sample for all principal genetic abnormalities Recommended tests Screening for key secondary abnormalities Greatest variability across laboratories in terms of techniques and targets Should attempt to capture data that meets key standards

22 Guidelines for minimum standards & data Yes Was a PGA detected at initial diagnosis? No Perform cytogenetic analysis and test for that particular abnormality using an appropriate method. Yes Were all PGAs tested for appropriately at initial diagnosis? No Perform cytogenetic analysis. No more essential tests required. PGA Principal Genetic Abnormality Perform cytogenetic analysis. Test for any PGA that was not tested for at initial diagnosis.

23 Principal Genetic Abnormalities in BCP-ALL Abnormality t(12;21)(p13;q22) / ETV6-RUNX1 Detection Method FISH, RT-PCR t(1;19)(q23;p13) / TCF3-PBX1 MLL translocations* t(4;11)(q21;q23)/mll-af4; t(11;19)(q23;p13.3)/mll-enl etc t(9;22)(q34;q11.2) / BCR-ABL1 Cytogenetics FISH RT-PCR t(17;19)(q22;p13) / TCF3-HLF IGH@ translocations* IGH@-CRLF2, IGH@-CEBP, IGH@-ID4, etc Near haploidy (<30 chromosomes) Low hypodiploidy / near triploidy (30-39 / chromosomes) High hyperdiploidy (51-65 chromosomes) Intrachromosomal amplification of chromosome 21 (iamp21) FISH Cytogenetics, DNA index, Array CGH, SNP array, FISH (panel of centromeric probes) FISH with AML1/RUNX1 probe Array CGH, SNP array * In the case of MLL and IGH@ translocation it is not necessary to identify the partner gene.

24 Principal Genetic Abnormalities in T-ALL Abnormality TAL1 rearrangement* SIL-TAL1/del(1)(p32) TLX3 rearrangement* t(5;14)(q35;q32) / BCL11B-TLX3 t(5;14)(q35;q11) / TRA@/TRD@-TLX3 TLX1 rearrangement* t(10;14)(q24;q11) / TRA@/TRD@-TLX1 t(7;10)(q34~36;q24) / TRB@-TLX1 LMO2 rearrangement* t(11;14)(p13;q11) / TRA@/TRD@-LMO2 t(7;11)(q34~36;p13) / TRB@-LMO2 MLL translocations* t(11;19)(q23;p13.3)/mll-enl, etc HOXA translocations inv(7)(p15q34)/trb@-hoxa t(7;7)(p15;q34)/ TRB@-HOXA MYB translocations t(6;7)(q23;q34) CALM-AF10 / t(10;11)(p13;q14) Appropriate Test FISH RT-PCR Cytogenetics FISH RT-PCR * In the case of these rearrangements it is not necessary to identify the partner gene or promoter.

25 Guidelines for minimum standards & data Collect full details of genetic testing performed at diagnosis/presentation Essential tests Full G/R-banded analysis (or SNP/CGH array) of all samples from marrow relapses. Targeted FISH / RT-PCR testing in all relapses for the principal genetic abnormality observed at diagnosis If no/failed diagnostic genetics then Screen relapse sample for all principal genetic abnormalities Recommended tests Screening for key secondary abnormalities Greatest variability across laboratories in terms of techniques and targets. Should attempt to capture data that is comparable.

26 Recommended tests for secondary abnormalities BCP-ALL cases IKZF1 deletions MLPA P335 / P202 kit PCR assay / FISH CRLF2 abnormalities Genomic rearrangements only P2RY8-CRLF2 by MLPA (P335), FISH, RT-PCR BCP-ALL & T-ALL cases TP53 deletions/mutations by MLPA, FISH / PCR-Sequencing

27 Collection of Molecular Cytogenetic Data National representative Central IntReALL Trial Database (Marvin) Limited dataset of a few key variables from the relapse required for trial Research Data from PopGenTech, Genomics, etc. Central IntReALL Diagnostic, Biobanking & Research Database (Scopeland) Full genetic profile of both the diagnostic and relapse samples

28 Clinical and Scientific Research Questions Factors influencing the time to relapse Biology of extra medullary relapses (mouse model) Characterisation of rare genetic subtypes Genetics and biology of T-ALL Down Syndrome ALL Effect of primary therapy on type and curability of relapses Proteomics - RPPA Signalling pathways of different genetic subtypes

29 Acknowledgements UKCCG Laboratories DCOG Cytogenetic Laboratories ANZCHOG Cytogenetic Laboratories ALLR3 team especially Catriona Parker and Vaskar Saha IntReALL team especially Vaskar Saha, Julie Irving, Conny Eckert and Oskar Haas LRCG Amy Erhorn, Claire Schwab, Alannah Eliott, Stacey Richardson, Christine Harrison etc.