ALL FINAL Measurable residual disease.pptx

ALL MRD analysis

1. MRD IN CLINICAL PRACTICE

2. BFM STUDY GROUPS

3. PILOT STUDY I-BFM-SG pioneered evaluation of MRD by PCR A prospective multicentric study To determine the predictive value or MRD at various TPs N=240 patients, MRD at nine time points van Dongen , Jacques JM, et al. "Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood."  The Lancet  352.9142 (1998): 1731-1738. MRD TERM No signal Negative <0.01 Low 0.01-0.1 Intermediate >0.1 High

4. BFM-90 RF>0.8,CNS,TestesMed mass MA L-asp 4x25000

5. Induction Consolidation Reinduction Maintenance Treatment Group Weeks Years

6. RESULTS MRD-independent prognostic factor TP1 and TP2-degree of MRD positivity Later TP any MRD+ significant TP1-40% SRG/MRG MRD neg , reduced by 50 % at each TP Maintenance –no added benefit Each log increase in MRD, two fold increase in relapse Later time point MRD-imminent relapse TP5--- 9 % vs 86% Relapse free rates 89% of relapsed pts had preceeding MRD+ MRD MRD Relapse Rates <0.01 LR 2% 0.01-0.1 IR 23% >0.1 HR 75% van Dongen , Jacques JM, et al. "Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood." The Lancet 352.9142 (1998): 1731-1738.

7. BFM 95

8. BFM 95

9. BFM 95 Reduction of anthracycline dose by 50% in SR MR-ID Ara -c Omission of pCRT in non T ALL Protocol II in HR Increase maintenance by 12 months in boys HR-PPR/No CR at D33/ Ph+,MLL reaaranged Risk stratification CNS per se-not HR Age/TC per se-not HR No MRD based risk stratification

10. AIEOP-BFM ALL 2000 Introduced MRD by qPCR –predictive value of relapse Post-Induction therapy modification based on MRD at TP2(D78) MRD-TP1 MRD-TP2 TERM <0.01 <0.01 MRD-SR Others MRD-IR Any >0.1 MRD-HR Conter , Valentino, et al. "Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study." Blood 115.16 (2010): 3206-3214

11. AIEOP-BFM ALL 2000 SR No HR criteria MRD neg at TP1,TP2 MR No HR/SR criteria HR MRD>0.1 at D78 PPR NR-D33 Ph+/MLL+ MRD

12. AIEOP-BFM ALL 2000 PCR-MRD discriminated prognosis even on top of WBC count, age,PR and genotype In B-ALL, MRD at TP1 -strongest predictor for 5-yr EFS TP2 PCR-MRD important in tailoring treatment in children with high MRD at TP1 MRD risk category % 5 YEAR EFS MRD-SR 42 92.3 MRD-IR 52 77.6 MRD-HR 6 50.1 Conter , Valentino, et al. "Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study." Blood 115.16 (2010): 3206-3214

13. AIEOP-BFM ALL 2000-T-ALL Slower clearance of leukemic cells in T-ALL compared with B-ALL  T-ALL ,n = 464 Schrappe , Martin, et al. "Late MRD response determines relapse risk overall and in subsets of childhood T-cell ALL: results of the AIEOP-BFM-ALL 2000 study." Blood 118.8 (2011): 2077-2084. MRD risk category % 5 YEAR EFS MRD-SR 16 91 MRD-IR 63 80 MRD-HR 21 49(40)

14. MRD in T ALL vs B ALL Clearance of leukemic blast cells is slower in T-ALL MRD detection at a late time point (d78) is more suited to define RR in T-ALL PPR retains significance in T-ALL High levels of MRD at day 78 in T-ALL are not only related to medullar relapses but also to relapses with extramedullary component Blast clearance pattern MRD risk category B ALL T ALL MRD-SR 42.3 16.2 MRD-IR 51.7 62.9 MRD-HR 5.9 20.9

15. FEATURES IN T ALL Cortical T ALL less likely to be MRD-HR PPR more common in ETP/Mature ALL ETP ALL has higher incidence of MRD-HR(14/19)

16. BFM 2002

17. BFM 2002

18. MINI-RISK PROJECT-BFM 2002 Flow based MRD at day 15,33 and before protocol M PCR-MRD at D33, D78 ( Additionaly at week 22,wk52) MRD was not a part of risk based treatment-Marrow response was considered Fronkova , E., et al. "Minimal residual disease (MRD) analysis in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: is it possible to avoid MRD testing?." Leukemia  22.5 (2008): 989.

19. MINI-RISK PROJECT Age: 6 years were more likely to be d33 MRD+ TC: More than 20 000 None of PPR achieved MRD- at d33 T vs B-ALL Multivariate analysis No significant impact of immunophenotype on d33 MRD status (P-0.26), which was caused by its correlation with WBC MRD correlated strongly with ALL IC risk ALL-IC Identifies most HRG patients. A large overlap between the IR and SR groups concerning MRD negativity at the end of induction. Factor Correlation with MRD-D33 At week 12 Age P-0.0007 Not sigfn TC P-0.0013 P-0.0028 Pred response P-0.0026 P-0.031 Fronkova , E., et al. "Minimal residual disease (MRD) analysis in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: is it possible to avoid MRD testing?." Leukemia  22.5 (2008): 989.

20. ALL-IC BFM 2009 Utilization of only FCM for MRD analysis Difficulty in establishing PCR-MRD Importance of Day 15 FCM-MRD

21. ALL-IC BFM 2009

22. ALL-IC BFM 2009

23. AIEOP-BFM ALL 2009 Time Site LR SR HR Day 15 Day 33 Week 7 Bone marrow No HR factors and day 33 and day 78 MRD negative by PCR, or Day15 <0.1% by FCM Others HR factors, or day 15 ≥10% by FCM or d ay 78 ≥0.05% by PCR or for B-ALL only, day 33 ≥0.05% and day 78 positive ,0.05% by PCR

24. AIEOP BFM 2009

25. ST. JUDE STUDY XV 2000 and 2007, N=498 MRD at D19,D46,week 7 Provisional risk stratification MRD based final risk stratification Additional MRD determinations on weeks 17, 48 and 120 Pui , Ching -Hon, et al. "Clinical utility of sequential minimal residual disease measurements in the context of risk-based therapy in childhood acute lymphoblastic leukaemia: a prospective study." The lancet oncology 16.4 (2015): 465-474

26. age 10 years or over wbc greater than 50,000 at diagnosis CNS /testicular leukemia hypodiploid T-cell t(1:19) MLL rearrangement ≥1% ≥1% HR-MRD >0.1% in week 7

27. ST. JUDE STUDY XV pLR -MRD of <1% on day 19 predicted a superior outcome, regardless of the MRD on day 46 (10-year EFS of 95.5% ) pSR -MRD - provided that MRD was <1%, levels did not predict inferior outcomes-on condition that D46 MRD was neg with MRD ≥1% on day 19 YrEFS = 69.2%( pLR ) 10 Yr EFS= 65.1%( pSR ) 10 Yr EFS= 95.5 % 10 Yr EFS= 82.9% pSR with MRD <1% on day 19, with peristent positivity on D 46 72.7% 84% Pui , Ching -Hon, et al. "Clinical utility of sequential minimal residual disease measurements in the context of risk-based therapy in childhood acute lymphoblastic leukaemia: a prospective study." The lancet oncology 16.4 (2015): 465-474

28. ST. JUDE STUDY XV POST REMISSION INDUCTION MRD re-emerged in 4 of 382 on week 7 1 of 448 on week 17 1 of 437 on week 48 Previous MRD positive,MRD neg at week 7-only 2 /11 relapsed Sequential MRD after remission induction is clinically useful only if MRD+ 5/6-died HSCT Chemo Pui , Ching -Hon, et al. "Clinical utility of sequential minimal residual disease measurements in the context of risk-based therapy in childhood acute lymphoblastic leukaemia: a prospective study." The lancet oncology 16.4 (2015): 465-474

29. ST. JUDE STUDY XVI SJCRH Total XVI Site LR SR HR D 15 D 42 WK 15 BM Favorable factors; day 15<1%; day 42≤0.01% Favorable factors; day 15 ≥1%; day 42<1%, Unfavorable factors; day 42<1% Day 42 ≥1% week 15 ≥0.1% High dose vs conventional PEG asp Intensifying induction(#cyclophosphamide,6TG), in patients with day 15 MRD > 5%

30. UK ALL STUDIES Previous studies-Prognostication UKALL 2003: First randomized study to demonstrate that post-remission intensification of treatment is of benefit to patients with high risk MRD at the end of induction High risk MRD = At least 0.01% on day 29 of induction Regimen C- 8 extra doses of asparaginase , 18 extra doses of vincristine, and escalated-dose intravenous methotrexate 5-year EFS was better with augmented therapy than with standard therapy (89.6% vs 82.8 % 5-year OS was also better with augmented therapy than with standard therapy, although not significantly (92.9% vs 88.9%;   P  = 0.16 ) Vora A, Goulden N, Mitchell C, et al. Augmented post-remission therapy for a minimal residual disease-defined high-risk subgroup of children and young people with clinical standard-risk and intermediate-risk acute lymphoblastic leukaemia (UKALL 2003): a randomised controlled trial. Lancet Oncol  2014; 15: 809-818.

31. UK ALL 2003

32. UK ALL STUDIES First study for therapy modfn based on MRD-ALL 2003 Redefine Induction failure as EOI MRD ≥5% and/or M3 marrow EBF1-PDGFRB in 10% of patients with IF Any patient B-other or T-ALL with EOI MRD >1% -screening for ABL-class fusions ETP ALL-less molecular targets,FCM better than PCR

33. UK ALL 2011

34. UK ALL 2011 UKALL 2011 Site LR IR HR D29 Wk14 BM LR Day 29 <0.005% Unfavorable factors, or Day 29 >0.005%; and week 14 <0.5% HR Week 14 ≥0.5%

35. COG/POG STUDIES

36. COG/POG STUDIES Earlier studies-MRD on prognostication A prospective study of MRD under P9900,(n=2143) MRD –FCM day 8 (PB) end-induction(D29)(BM) MRD + vs -,outcomes differed 59% vs 88% 5-year EFS Borowitz , Michael J., et al. "Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study." Blood 111.12 (2008): 5477-5485. P9904 P9905 P9906 NCI –SR with TEL-AML1 Double trisomy NCI-SR without fav factors NCI-HR with favourable genetics NCI-HR MLL  gene CNS3 status Testicular inv

37. COG/POG STUDIES Very favorable prognosis (5-year EFS of 97% ± 1%) pre B-cell phenotype NCI standard risk CNS1 status favorable cytogenetics (high hyperdiploidy with favorable trisomies / ETV6-RUNX1  fusion) ≤0.01% MRD levels day 8 (PB) end-induction (BM)

38. COG AALL0232 Borowitz , Michael J., et al. "Prognostic significance of minimal residual disease in high risk B-ALL: a report from Children’s Oncology Group study AALL0232." Blood 126.8 (2015): 964-971. SER BM MRD ≥0.1% at D29 BM blasts ≥ 5% at D15

39. COG AALL0232 Intensive therapy in MRD positive patients Altered timing of relapse did not overcome the poor prognostic significance of MRD.

40. COG AALL03B1 Time Site LR SR HR VHR D8 D15 D29 Cut off of 0.1 BM FCM-D29 NCI SR Favorable genetics; no unfavorable factors; D 8/D15-M1 D29-M1 D29-<0.1% NO CNS2/3 NO testes inv High NCI SR Favorable/ unfav genetics; D15-M2/M3 D29-0.1 to 1%% Or A verage Neutral genetics; no unfavorable factors; D 8/D15-M1 D29-M1 D29-<0.1% NCI SR Favorable genetics; no unfavorable factors; day 29 ≥1% NCI SR Neutral genetics; no unfavorable factors; day 29 ≥1% NCI HR SER/RER Day 29 <1% NCI SR or HR Unfavorable factors NCI HR Day 29 ≥1%, Ph+ Hypodiploidy MLL SER, Induction failure MLL rearrangement

41. COG AALL 08B110 CNS3 Hypodiploidy iAMP21, Induction failure MLL rearrangement Day 8 peripheral blood MRD. Day 29 bone marrow morphologic response and MRD. Morphology of early response in BM is no longer performed on days 8 and 15

42. COG AALL 08B110 Time Site LR AR HR VHR D8 D29 Blood BM NCI SR Favorable genetics; no unfavorable factors; day 8<0.01% day 29<0.01% NCI SR Favorable genetics; no unfavorable factors; Day 8 ≥0.01%; day 29 <0.01%, or Neutral genetics; no unfavorable factors; day 8 <1% day 29<0.01% NCI SR Favorable genetics; no unfavorable factors; day 29 ≥0.01% NCI SR Neutral genetics; no unfavorable factors; day 8 ≥1% day 29 <0.01% NCI HR Day 29 <0.01% NCI SR or HR Unfavorable factors NCI SR Neutral genetics; day 29 ≥0.01% NCI HR Day 29 ≥0.01%, Age ≥13 y

43. MRD IN HSCT SETTINGS

44. BFM –AIEOP HSCT Pre-HSCT MRD was Negative ( MRD- neg ) in 51/119 patients (43 %) Positive (MRD-low) in 46 (31 %) Positive (MRD-high) in 22 (18%)

45. HSCT in CR1 based on MRD Pulsipher MA, Peters C, Pui C-H. High Risk Pediatric Acute Lymphoblastic Leukemia: To Transplant or Not to Transplant? Biology of blood and marrow transplantation:Journal of the American Society for Blood and Marrow Transplantation. 2011

46. UK ALL 2011 MRD positivity at week 11 more than 0.5 indication to start on ALL R3

47. ALL R3 PROTOCOL Intermediated group form 70% of relapsed ALL Rapid MRD clearers have better outcomes Cut off is 0.01% by two loci at day 35 BFM protocols have a cut off of 0.1%

48. ALL R3 PROTOCOL

49. I-BFM- Bader et al MRD in marrow at day 30, 60, 100, 180 and 360 days post SCT in CR2 MRD>1/1,000 were universally associated with relapse. Immunotherapy failed in all 7 cases where MRD was >1/1000 at the initiation of immune modulation 8 of 18 children, in whom immunotherapy was initiated at a time when marrow MRD was less than 1/1000, are in remission.

50. I-BFM- Bader et al Regular marrow sampling presents major logistical difficulties in children after SCT. Blood can be substituted for MRD analysis? MRD at a level greater than 1/10,000 (30 of 95 patients) in the blood is associated with a 100% risk of relapse (Bader, I-BFM, 2005). ALL R3 proposes to monitor peripheral blood/marrow for MC and intervene

51. DLI DOSES

52. PRE AND POST HSCT MRD N=119 transplanted in CR MRD by PCR in BM collected pre-HSCT,post-HSCT1 and post-HSCT3 EFS was 50 In patients transplanted in CR1 only high MRD –increased risk of relapse In CR2, a low-level MRD positivity also increases risk Lovisa , Federica, et al. "Pre‐and post‐transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia ." British journal of haematology  180.5 (2018): 680-693. MRD 5 year EFS <0.1% 39% >0.1% 18% Neg 73%

53. PRE AND POST HSCT MRD N=82 5 year EFS=77% 1, 3, 6, 9 and 12 months MRDpos (0.01%)- 10 fold increased risk of relapse Risk decreased by 5 fold-if MRDneg by additional therapy MRDpos post HSCT-2.5x (earlier-higher chance of relapse) No effect of DLI/weaning immsuppression if post HSCT MRD>0.1% Balduzzi , Adriana, et al. "Minimal residual disease before and after transplantation for childhood acute lymphoblastic leukaemia : is there any room for intervention?." British journal of haematology 164.3 (2014): 396-408.

54. PRE AND POST HSCT MRD Most relapses occur after 3 months 54% of relapse occurred between the months 3 and 9 months 38% after 9 months Prompt immunological intervention Rapid tapering or discontinuation of the immunosuppressive treatment, Infusion of DLI Early-Before graft is tolerant of host Lovisa , Federica, et al. "Pre‐and post‐transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia ." British journal of haematology  180.5 (2018): 680-693.

55. GVHD prophylaxis Cylosporin aiming at trough levels of 150 ng /ml Short methotrexate 10 mg/m2/dose on days +1, +3, +6, +11 And ATG in MD or MMD methotrexate only in MSD If no acute GVHD - immunosuppression was tapered and discontinued in the third month

56. CAVEATS OF MRD(diagnosis and treatment) POST HSCT Standardization and sensitivity Rapid progression of the malignancy(mean 21 days from MRD positivity to overt disease) DLI might have limited role, esp when given late Role in ALL not well established MRD might not indicate extramedullary relapse

57. Patients with detectable MRD or MC 1% recipient signals in 2 consecutive samples or >1% of autologous signals in a single sample) with no or only mild signs of acute ( aGVHD ) Pre emptive IT discontinuation or tapering of IS DLI One-thirds-intervened, EFS similar to the entire cohort, no major events Source of Stem cells DLI dose(T cells) HLA matched related 1x 10 6 Matched unrelated o.5x 10 6 Haplo 0.1 x 10 6

58. QUESTIONS RAISED Does chimerism contribute to early prediction of relapse? Chimerism -less sensitive and specific What is the ideal MRD to start DLI(0.1%/any?) Ideal window-90 days(+55 to +200) No aGVHD as predictor of relapse risk? Balancing the art of alloreactivity -GVHD and GVL-careful titration of DLI doses, early weaning off IS DLI vs No DLI-RCTs are not possible to know the real effect.

59. MRD IN HSCT-INDIA

60. MRD IN HSCT-INDIA Trend towards decreased relapse in MRD pos comparedto MRD neg 5/19 vs 1/26 Overall Survival benefit was less as there was increased GVHD in the MRD neg group EFS 66% vs 63%

61. MRD IN HSCT-INDIA

62. NCI trials 1*10^8 MNC/kg MRD+ ,grade II/>II aGVHD by day +60 DLI once by day +60 and then based on MRD and GVHD status. MRD + ,no GVHD, DLI monthly until GVHD occurred or MRD became negative (4X) For patients with NR or PR pre-transplantation and MRD negative by day +60 DLI was given once by day +90 regardless of MRD

63. MRD in PH+ ALL

64. METHODS MRD-PCR RQ-PCR measurement of BCR-ABL1 transcripts, and MRD-flow cytometry CA180372 was a collaboration between the COG,-EsPhALL

65. HSCT MRD was analyzed 4 times -induction IA/baseline, end induction IA, start high risk block 1 (HR1), and end HR block3 (HR3 )) Pts were recommended for HSCT if: Start of HR1 ≥0.05% by PCR (or by Flow), less than 3-log reduction in MRD by RQ-PCR for BCR-ABL1 OR Start of HR1 0.005-0.05% by PCR (or by Flow) or any positivity by BCR-ABL1 AND MRD remains positive at any detectable level (providing the assay limit is at least 0.1%) at the end of HR3

66. CONCORDANCE HSCT recommendations could be made for most (98%) pts by Flow. Only 39% of pts could be recommended for HSCT based on BCR-ABL1 results . The main reason that BCR-ABL1 results were available for fewer pts was the requirement for at least 10,000 copies at baseline in order to assess if a 3-log reduction was achieved .

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