LA MALATTIA MINIMA RESIDUA NELLA LEUCEMIA ACUTA MIELOIDE

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1 LA MALATTIA MINIMA RESIDUA NELLA LEUCEMIA ACUTA MIELOIDE Francesco Lo-Coco Università Tor Vergata, Roma Progetto di Formazione Nazionale SIE Napoli, 25 Ottobre 2018

2 Disclosures Research Support/P.I. Employee Stockholder Consultant Advisory Board Speakers Bureau Other (Specify) No relevant conflicts of interest to declare No relevant conflicts of interest to declare No relevant conflicts of interest to declare Teva, Orsenix Novar5s, Teva, Orsenix Novar5s, Teva No relevant conflicts of interest to declare

3 Acute Myeloid Leukemia Advanced median age (70 yrs) Current therapy poorly tolerated in elderly Dismal prognosis (50-80% relapse rate) Targeted therapy strongly needed

4 AML Treatment Outcome Age CR % ED % Cure % <15 90 <5 >60 <60 75 <10 45 >60 50 ~20 <15

5 Gene=c heterogeneity of AML Dohner et al, European LeukemiaNet, 2017

6 Heterogeneity of NK AML Schlenk et al. N Engl J Med 2008

7 Muta=onal padern in AML by WGS The Cancer Genome Atlas Research Network. N Engl J Med 2013

8 Molecular Complexity of AML as defined by NGS: Implica=ons for Treatment Inhibitors against one target will not suppress all leukemogenic clones Clearing all mutauons increases overall survival Patel, et. al. NEJM 2012

9 Comprehensive prognosis assessment in AML Pre-treatment prognostic factors Karyotype Molecular genetics Clinical parameters Post-treatment prognostic factors MRD detection Flow-cytometry Molecular biology

10 Prognos=c factors in AML and type of failure Characteristic Unfavorable category Early death Death during remission Age Older Performance status >2 Comorbidities increasing Resistance/ Relapse Previous HD/t-AML Yes Cytogenetics Unfavorable Mutational status* FLT3-ITD; KMT2A WBC count Hyperleucocytosis Quality of response MRD+ *Other mutations have been recently added : RUNX1, ASXL1, TP53

11 Can MRD improve outcome determina=on in AML? No. of leukemic cells Relapse CR MRD Cure 10 0 Time This modality may not only capture differences in treatment response that reflect the underlying molecular heterogeneity, but also interpa=ent variability in drug availability and metabolism, which may also significantly influence outcome Grimwade, Best Pract Hematol 2012

12 Measurable Residual Disease dd-pcr QRT-PCR Differences according to! disease type! drug availability! individual metabolism NGS:! Clonal heterogeneity and evolution Hourigan, et al, Nature Reviews Clinical Oncology 2013

13 Clonal Heterogeneity and Clonal Evolu=on -> one subclone within the founding clone evolved to become the dominant clone at relapse by acquiring additional mutations Ding L, et al. Nature 2012

14 Technical Platforms for MRD detection in AML! PCR, RT-PCR! RQ-PCR! Digital PCR! NGS! Flow cytometry

15 MRD detec=on in AML: PCR vs MPFC PRO CONTRA SENSITIVITY APPLICABILITY Flow (MPFC) 1. Fast, less expensive 2. Single cell analysis 3. Cell viability 1. Less leukemia specific 2. Sub clones expansion 3. Phenotypic shi\ 4. Complex analysis Wide (>90%) PCR / RQ-PCR 1. High DNA stability 2. Specific 3. Very low background in normal cells 4. QuanUtaUon 1. Time consuming, expensive 2. False posiuve 3. RNA instability (sample quality) 4. Cell viability not determinable Specific subgroups: (~60%) CBF-AML, NPM1 mut WT1 Modified from Buccisano, CCO 2009

16 Standardiza=on of Q-PCR for fusion transcripts E2A/PBX1 RUNX1/ RUNX1T1 TEL/AML1 PML/RARα CBFb/MYH11 SIL/TAL BCR/ABL MLL/AF4 Gabert et al., Leukemia 2003

17 Molecular targets for MRD in AML Fusion genes Mutations Overexpression PML-RARA CBFB-MYH11 RUNX1-RUNX1T1 MLL-fusion partner DEK-NUP214 BCR-ABL Others NPM1 RUNX1 ASXL1 MLL-PTD CEBPA? FLT3? DNMT3a? WT1 BAALC ERG MN1

18 MRD determina=on in CBF-AML predicts relapse Upfront genetics and MRD determination in CBF acute myeloid leukemia. 198 patients RQ-PCR transcripts determination after first consolidation a less than 3-log MRD reduction or a level <0.1% was associated with a higher specific hazard of relapse Jourdan et al, Blood 2013

19 Recurrent Transloca=ons: t(8;21) (RUNX1-RUNX1T1) CBF 2006 trial, n=94 pts CIR OS BM PB! MRD >0.001% considered posiuve! BM persistently + at 2 yrs in 8% of pt! PB at the end of consolidauon predicuve, anucipaung relapse by 4 mos! During follow-up: 100% relapse rate in BM > 500 copies, PB > 100 copies! Rising MRD levels accurately predict relapse (AML-MRC15, Liu Yin, Blood 2012) Willekens et al, Haematologica 2016

20 RQ-PCR for NPM1mut: impact of MRD on survival Kronke et al, JCO 2009

21 Overall Survival NPM1 muta=ons MRD by RQ-PCR in PB a\er 2 Cycles of CHT Cumulative incidence of relapse The presence of MRD by quan=ta=on of NPM1- mutated transcripts provided powerful prognos=c informa=on independent of other risk factors. Ivey A et al., NEJM 2016

22 MRD as a Predictor of Outcome in Development and Valida=on Cohorts Ivey A et al, NEJM 2016

23 NPM1 mut is a stable marker, DNMT3A mut and FLT3 ITD are not Ottone et al, Am J Hematol 2018

24 UPN_243 Paralel MRD assessment of DNMT3A and NPM1 UPN_ Texto 1 NP M1 DNMT3A NP M1 DNMT 3A 10 1 Esordio Induz ione Post cons! Texto Esordio Induzione Post cons UPN_43 UPN_ Relapsed Relapsed NPM1 DNMT3A NPM1 DNMT3A ,1 Esordio Induzione Post cons Relapse

25 Clonal Hematopoiesis and Blood-Cancer Risk Inferred from Blood DNA Sequence Giulio Genovese, Ph.D., Anna K. Kähler, Ph.D., Robert E. Handsaker, B.S., Johan Lindberg, Ph.D., Samuel A. Rose, B.S., Samuel F. Bakhoum, M.D., Ph.D., Kimberly Chambert, M.S., Eran Mick, B.S., Benjamin M. Neale, Ph.D., Menachem Fromer, Ph.D., Shaun M. Purcell, Ph.D., Oscar Svantesson, M.S., Mikael Landén, Ph.D., Martin Höglund, M.D., Ph.D., Sören Lehmann, M.D., Ph.D., Stacey B. Gabriel, Ph.D., Jennifer L. Moran, Ph.D., Eric S. Lander, Ph.D., Patrick F. Sullivan, M.D., Pamela Sklar, M.D., Ph.D., Henrik Grönberg, M.D., Ph.D., Christina M. Hultman, Ph.D., and Steven A. McCarroll, Ph.D. N Engl J Med Volume 371(26): December 25, 2014 We analyzed data from whole-exome sequencing of DNA in peripheral-blood cells from 12,380 persons, unselected for cancer or hematologic phenotypes.

26 NGS and Clonal hematopoiesis (CHIP) Clonal hematopoiesis with soma=c muta=ons in: DNMT3A, ASXL1, and TET2, genes implicated in hematologic cancers. Risk of hematologic cancer increased in these individuals Genovese G, Jaiswal G, et al. N Engl J Med 2014

27 Clonal evolution of FLT3-ITD mutations in AML DIAGNOSIS RELAPSE UPN Karyotype FLT3-ITD FLT3-ITD/ABLx10 NPM1 4 Karyotype FLT3-ITD NPM1 routine RT-PCR routine RT-PCR RQ-PCR routine RT-PCR 104 negative 46,XY negative positive 46,XY positive positive 213 negative 46,XX negative positive 46,XX positive positive 241 negative 46,XX negative positive 46,XX positive positive Exon 14 Exon 15 ITD Probe Primer forward Patients specific Primer reverse FLT3-ITD patient-specific RQ-PCR

28 Iden=fica=on of an=gens associated with FLT3-ITD (MPFC analysis of total BM cells from FLT3-ITD mut pts) Antigen FLT3 mutated FLT3 wt p value* (% of pts/tot) (% of pts/tot) CD99 73% (27/37) 48% (44/91) 0.02 CD % (38/38) 83% (75/90) 0.02 CD11b 92% (35/38) 66% (59/90) CD7 68% (26/38) 46% (41/89) CD25 87% (33/38) 40% (36/90) *Pearson's chi-squared test Battistini, Ottone et al, Clin Cancer Res 2015

29 Receiver Opera=ng Characteris=c (ROC) curve analysis A threshold of 11.7% cells staining posi=ve for this an=genic profile predicts FLT3 mut with specificity and sensi=vity >90% Battistini, Ottone et al, Clin Cancer Res 2015

30 Suitability of alterations for MRD detection ITD-mutations Exons 14/15 Preleukemic Leukemic Post-Onset Drivers DNMT3A TET2 IDH1/2 SF3B1 NPM1 Translocations RUNX1 CEBPA FLT3-ITD FLT3-TKD RAS PTPN11 KIT Sensitive Potentially unspecific Sensitive specific Specific Lost in 10-90% by courtesy of C. Thiede

31 Are MRD studies still valuable and cost effective in APL?

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33 J Clin Oncol 2003

34 MRD to direct pre-emp=ve treatment in APL Molecular failure (n=16; 5 events) 0.7 Probability Hematological relapse (n=33; 24 events) 0.1 p= Years Lo-Coco et al. Blood, 1999 Esteve et al. Leukemia, 2006

35 MRC AML-15 Trial for Newly Diagnosed APL Pre-emptive ATO therapy Grimwade et al, JCO 2009

36 Kine=cs of PML/RARa clearance in APL 0406 PML/RARA +ve patients by RQ-PCR Diagnosis Post-induc=on Post III consolida=on Cicconi et al, Leukemia 2016

37 ORH-2014 in APL: Pivotal Registration Study A Randomized, Multi-center, Phase III Study to Compare ORH-2014 and i.v. ATO in Low-Intermediate Risk APL Agreement with FDA on study design, population, and endpoints Primary Endpoint: Rate of molecular complete remission Secondary Endpoints: 2 year EFS, Safety, QoL

38 Muta=onal padern in relapsed APL ATRA-CHT ATRA-ATO WT1 FLT3 -ITD DNMT3A ETV6 FLT3- TKD TET2 ASXL1 JAK2 RUNX1 SRSF2 TP53 U2AF1 PML RARA single mutation multiple mutations wild-type not analysed

39 PML and RARA muta=ons at relapse A216 T (1) PML RARA A216 V (2) B2 L224 I I222_D223in (1) se (1) L220 P (1) LBD W22 5C (1) C23 5F (1) A216 T (1) PML A216 V (2) B2 E224 G (1) R272 Q (1) T291 T285 S287 A I L L290 (1) (1) F286 (1) V del (1) (1) Madan et al, Leukemia 2016 Iaccarino et al, BJH 2016 Zhu et al, NEJM 2014 Lehmann-Che et al, NEJM 2014 Goto et al, Blood 2011

40 Early and sensi=ve detec=on of PML A216V by ddpcr posiuve by ddpcr posiuve by Sanger negauve by ddpcr negauve by Sanger

41 Op=mized RQ-PCR of WT1 (Cilloni et al, JCO 2009) 41 WT1 overexpressed in >90% AMLs Suitable universal MRD marker for AML Comparison of sensiuvity & specifiuy of 9 different RQ-PCR assays by 11 European labs (LeukemiaNet)

42 WT1 level in PB of AML pa=ents at the end of the induc=on therapy predicts for relapse Normal range (<20 copies/10 4 copies of ABL) Cilloni et al, Haematologica 2008

43 Global immunophenotypic approach to AML Multiple staining at diagnosis Identification of leukemia-associated phenotypes Definition of a patient s immunologic fingerprint Immunologic fingerprint used during follow-up Venditti, Blood 2000; Venditti, Leukemia 2003; Buccisano, Leukemia 2006; Maurillo, JCO 2008; Buccisano, Blood 2010

44 Leukemia-associated Immunophenotypes LAIP type Examples Asynchronous an=gen expression Average 3 LAIP per pa=ent 85% of pediatric AML Lack of an=gen expression 80-95% of adult AML Cross-lineage an=gen expression CD34 + CD14 + CD34 + CD15 + CD33 + CD34 + CD13 - CD33 + Hla-DR - CD34 + CD13 + CD34 + CD13 + CD19 + CD34 + CD13 + CD2 + An=gen overexpression HLA-DR ++ CD CD13 ++ CD64 ++ CD4 ++ CD45 ++ Pitfalls: immunophenotypic shifts, complex analysis

45 MPFC Laboratory diagnostics of hematological malignancies has three major applications: Diagnosis Prognostic (sub)classification Evaluation of treatment effectiveness via detection of MRD

46 MRD Assessment by Flow Cytometry in AML Terwijn M et al, JCO 2013

47 Enrollment, Randomization, Initial Risk Assignment AML02: A prospec=ve, mul=center study of risk/mrd-directed therapy H-ADE ADE MRD ADE ± GO MRD Final Risk Assignment LR SR (w/o donor) CI CII CIII HR SCT SR (with donor) Levels of MRD were used to allocate GO and to determine the Uming of inducuon 2 Both MRD and geneuc abnormaliues at diagnosis were used to determine the final risk classificauon Rubnitz et al, Lancet Oncology 2010

48 AML02: Main conclusions N=230 CR rate 94% MDR+ 37% (Ind1) MDR+ 20% (Ind2) 71% ± 4% OS 63% ± 4% EFS 19% ± 3% 9% ± 2% Years on Study Relapse Death St. Jude AML Trials Risk- and MRD-adapted therapy resulted in 71% OS Day 22 MRD >1% significantly associated with worse OS, EFS, CIR (Rubnitz et al, Lancet Oncology 2010

49 Integrated Risk-Score (Tor Vergata) Low-Risk Favorable K / MRD- Int K / MRD- High-Risk Adverse K FLT3+ Good K / MRD+ Int K / MRD+ Buccisano et. al. Blood 2010

50 GIMEMA AML1310: a study of risk-adapted and MRD-directed therapy for adult AML MRD marker LAIP Risk stratif CG, molecular MRD assess LAIP Diagnosis Low-risk Int-risk Induction (1 or 2 courses) CR Consolidation 1 MRD- MRD+ autosct allosct High-risk No CR FLA-Ida salvage CR Low-risk: CBF/Kit wt ; NPM1+/FLT3- Int-risk: all others High-risk: Adverse K; FLT3+ allosct: MRD, MUD, UCB, HRD

51 GIMEMA AML1310 AML1310: results 342 post consolidation 177 candidates to AutoSCT 165 candidates to AlloSCT 110 (62%) received AutoSCT 110 (67%) received AlloSCT 81 not in CR post induction 23 (CR post salvage) candidates to AlloSCT 16 (70%) received AlloSCT

52 GIMEMA AML1310 AML1310: intermediate-risk OS and DFS by MRD status 52 Venditti A et al, submitted

53 Molecular MRD: ELN 2017 recommenda=ons for AML Response to therapy Transcripts When Source Defini=on of failure CR MRD- as a new response criterion - NPM1 - RUNX1- RUNX1T1 - CBFB-MYH11 - PML-RARA - aoer 2 cycles of standard induc=on/ consolida=on - aoer the end of treatment - every 3 months for 24 months aoer end of treatment - in PB and BM or PB every 4-6 weeks - Failure to achieve MRD-nega=ve CR (molecular resistance) - Rising MRD levels (>1 Log) during or aoer therapy (molecular progression) - Molecular relapse! PB instead of BM? In par=cular at prolonged follow-up?! Absolute copy numbers should also be reported Modified from Schuurhuis GJ, et al ELN-MRD WP, Blood 2018

54 Clinical Impact of MRD in AML is Increasingly Recognized

55 Labnet AML A network of laboratories licenced for the molecular diagnosis of AML Aiming at defining and spreading diagnos=c standards for AML, including molecular gene=cs, cytogene=cs, and immunophenotype Research projects

56 Diagnos=ca LAM: Pannello Base Tempi Cario=po G-banding 15 gg QRT-PCR Q-LAMP PML/RARA (bcr1-2 e bcr3) BCR/ABL (p190 e p210) RUNX1/RUNX1T1 CBFb/MYH11 24 ore ore PCR-EC PCR-RLFP FLT3-ITD FLT3-D835 e I836 NPM ore

57 Prossimi Step: Pannello Avanzato!

58 Risulta= Standardizzazione MRD Undiluited BCR-ABL p190 BCR-ABL p210 b3a2 PML- RARA bcr1 PML- RARA bcr3 CBFb/ MYH11 type A RUNX1/ RUNX1T1 NPM1

59 Conclusive remarks (I) MRD assessment by MPFC / RQ-PCR allows better strategies for delivering treatment in AML RQ-PCR provides valuable prognostic information in CBF and NPM1 +ve AML NGS may provide in the future more comprehensive data on clonal heterogeneity / evolution and overall effects of treatments

60 Conclusive remarks (II) MRD ve remission as a new treatment objective in AML to accelerate drug approval Importance of reference laboratories

61 Tor Vergata Team Maria Teresa Voso Laura Cicconi Mariadomenica Divona Tiziana Opone Serena Lavorgna Emiliano Fabiani Giulia Falconi Nélida I. Noguera ValenUna Alfonso Serena Travaglini Licia Iaccarino Adriano Vendir Francesco Buccisano