MRD in ALL: Correct interpretation in clinical practice. Deepak Bansal Prof., Pediatric Hematology-Oncology unit PGIMER, Chandigarh

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1 MRD in ALL: Correct interpretation in clinical practice Deepak Bansal Prof., Pediatric Hematology-Oncology unit PGIMER, Chandigarh

2 Minimal residual disease Subclinical level of residual leukemia Below the limits of detection by morphology

3 Complete remission: CR Eradication of visible leukemic cells < 5% blasts in bone marrow + blood Restoration of normal hematopoiesis > 25% cellularity + normal peripheral counts

4 Morphological CR: Concerns 1. Hematogones: Benign lymphoid precursors Recovery from chemo or ASCT ~ 10% of lymphoid cells 2. Operator error 3. Detection limit: ~ 1%

5 A patient in clinical remission can harbor upto leukemic cells

6 Technique Blasts per 100 nucleated cells Standard microscopy 5 Microscopy, expert 1 Flow cytometry 0.01 PCR 0.001

7 Reading a MRD report 0.01% 0.01 leukemic cells per 100 mononucleated cells 1 leukemic cell per 10,000 mononucleated cells 0.01% = 10-4

8 Level of MRD predictive of relapse? Likely to differ: Various malignant disorders Children & adults Technique

9 MRD: Sample? Bone marrow or peripheral blood BM preferred B-lineage ALL Blood MRD levels 1-3 logs lower than bone marrow T-lineage ALL Blood MRD levels comparable or upto 1 log lower than bone marrow

10 Volume of bone marrow? 2-5 ml volume diluted erroneously low MRD First aspirate EDTA preferred Heparin: Cannot do PCR

11 Methods for detecting MRD 1. Cytogenetics 2. Cell culture 3. FISH 4. Southern blotting 5. Multicolor flow cytometry 6. PCR: RQ & RT 7. Deep sequencing

12 Multicolor flow cytometry Laser specific immunophenotypic features Aberrant expression of antigens Most common: Pediatric ALL in USA USA: NOT standardized in most clinical labs Europe: Standardized/validated in many centers

13 Flow cytometry: sensitivity Classic: 4-6 color Limited sensitivity/specificity for MRD Current: 6-8 color Sensitivity: 0.5 to 1 log lower than PCR Investigational, next-generation: 8 colors

14 Flow cytometry Advantages Availability Speed: Same day reporting Immunophenotype of leukemic clone at diagnosis Recommended Not strictly required for all cases Limitations Immunophenotypic shift: False negative results Low cellularity during/after induction

15 Multicolor flow cytometry Applicability B-lineage ALL: 85% T-lineage ALL: 90%

16 Real-time quantitative PCR Amplify a DNA or cdna sequence unique to leukemic clone Detects 1 malignant cell among 10 4 to 10 5 normal cells Two targets 1. Immunoglobulin (Ig) or T cell receptor (TCR) gene rearrangements 2. Leukemia-specific chromosomal rearrangements (fusion gene transcripts)

17 Ig and TCR gene rearrangements Random deletions & insertions of nucleotides occur during Ig & TCR gene rearrangement Generation of unique junctional sequences Serve as leukemic clone-specific markers At diagnosis Precise nucleotide sequence of junctional region of Ig or TCR genes identified in leukemic cells Junctional region-specific oligonucleotides is then designed to use as patient-specific probes

18 RQ-PCR: Advantages sensitivity 0.5 to 1 log higher than flow cytometry applicability: ALL: 95% Minimal tissue requirement DNA samples stable during transport Standardized method

19 RQ-PCR: Limitations Product contamination: Strict quality control Diagnostic sample testing to determine unique clonal rearrangement Must Expensive Time consuming: up to 4-6 weeks False -ve & +ve Evolution of leukemic clone, subclone formation

20 RQ-PCR Restricted to experienced molecular hematology labs

21 Reverse transcriptase PCR RT-PCR Identify leukemia-specific fusion mrna created by DNA breakpoints BCR/ABL: Adults ~ 25%; Pediatric ~5% TEL/AML1, MLL/AF4 Qualitative Extraordinarily sensitive & rapid

22 Multiplex PCR with deep sequencing Investigational Amplify several (Ig & TCR) DNA sequences Sequences DNA with high depth (coverage) Faster, more sensitive than RQ-PCR Diagnostic sample not required sensitivity - clinical utility not yet proven

23 Flow cytometry To identify patients at high risk of relapse, with plans to escalate care Likelihood of relapse following a positive result is high RQ-PCR To identify patients with a low risk of relapse, for whom a reduction in treatment intensity may be an option UpToDate

24 MRD: Definitions Complete MRD response No MRD detected Complies with minimal technical requirements for method used MRD persistence Presence of a continuously quantifiable MRD positivity measurable at 2 time points, with at least one treatment block in between MRD reappearance Conversion from MRD negativity to quantifiable MRD positivity, ideally with confirmation from a 2 nd sample

25 MRD negative No residual disease Adequate sensitivity ( 10-4 ) Proper technique Adequate sample Typically definition < 0.01% leukemic cells in bone marrow after remission induction therapy

26 MRD & ALL MRD: One of the most powerful predictors of survival for children & adults with ALL Large, prospective studies in pediatric ALL have conclusively demonstrated: Probability of long-term relapse-free survival is directly related to the level of MRD, both early in course of treatment, & at later time points

27 However Not all patients with MRD positivity will relapse Blasts may persist for some time after treatment but lack the ability to recapitulate because: An inherited defect or the immune system Some patients will relapse despite negative MRD Immunophenotypic or genetic markers identified at diagnosis on bulk of malignant cells may not be identical to those expressed by other leukemia clones Recurrent disease may arise from occult malignant progenitor cells that lack markers assayed on their more populous progeny

28 How to avoid incorrect MRD results? Use > 1 assessment technique, either simultaneously or in tandem Assess MRD at multiple time points

29 455 children; B-lineage ALL, PCR for MRD; Day 46 St Jude Children's Research Hospital, Memphis MRD level % of patients 5-year cumulative incidence of relapse Undetectable 58% 4% < 0.001% 11% 8% to < 0.01% 14% 13% 0.01% 17% 23% 1% 45% Stow et al. Clinical significance of low levels of MRD at the end of remission induction therapy in childhood ALL. Blood 2010.

30 Widely accepted that MRD 0.01% at end of induction, mandates treatment intensification Should therapy also be intensified for patients in the MRD 0.001% to < 0.01% group? Stow et al. Clinical significance of low levels of MRD at the end of remission induction therapy in childhood ALL. Blood 2010.

31 AIEOP-BFM 2000 study 3184 children Ph neg, B-lineage ALL PCR for MRD 0.01% Day 33: End of induction Day 78: Start of consolidation Conter et al. Molecular response to treatment redefines all prognostic factors in children & adolescents with B-cell precursor ALL: results in 3184 patients of AIEOP-BFM ALL 2000 study. Blood 2010

32 AIEOP-BFM 2000 study Risk category Day 33 Day 78 EFS Standard (42%) 0.01% 92% Intermediate (52%) > 0.01% > < 0.1% 78% High (6%) 0.1% 50% Conter et al. Molecular response to treatment redefines all prognostic factors in children & adolescents with B-cell precursor ALL: results in 3184 patients of AIEOP-BFM ALL 2000 study. Blood 2010

33 COG: 7430 children, B-lineage ALL Flow cytometry Day 29 Threshold: 0.01 % MRD status at end of induction obviates the need for bone marrow analysis at day 14 Assessment of end induction MRD in childhood B precursor ALL to eliminate the need for day 14 marrow examination: A COG study (abstract 10001). J Clin Oncol. 2013

34 MRD & allogeneic HCT Positive MRD Prior to After BMT relapse

35 Pre-BMT MRD Children, adolescents with ALL Morphological remission n = 64 MRD prior to BMT 2-yr EFS Detected 17% Not detected 73% Knechtli et al. MRD status before allogeneic BMT is an important determinant of successful outcome for children and adolescents with ALL. Blood 1998

36 Higher MRD prior to HCT poorer survival (p 0.002) Very-high-risk ALL (n = 64) AML (n = 58) Risk of death with a similar increment of MRD was in ALL than AML A pretransplantation leukemia burden impact in ALL MRD prior to BMT ALL 5-yr OS AML 5-yr OS Detected 49% 67% Not detected 88% 80% Although MRD before HCT is a strong prognostic factor, its impact has & should NOT be regarded as a contraindication for HCT Leung et al. Detectable MRD before ASCT is prognostic but does not preclude cure for children with very-high-risk leukemia. Blood 2012.

37 MRD post BMT ALL-BFM-SCT 2003 trial Children with relapsed ALL n = 113 MRD, PCR Days +30, +60, +90, +180, & +365 post-hct MRD 10-4 : 3-yr incidence of relapse: 38%, 50%, 75%, 100%, 100%, and 100%, respectively ~ 1/4 th of consistently MRD-negative patients relapsed MRD positivity at day +60: Highly predictive of relapse Bader et al. Monitoring of MRD after ASCT in relapsed childhood ALL allows for the identification of impending relapse: results of the ALL-BFM-SCT 2003 trial. J Clin Oncol. 2015

38 MRD in relapsed ALL? Children with relapsed ALL who achieve a complete remission: MRD may predict risk of a 2 nd relapse n = 60; high-risk ALL Post-reinduction MRD: 3-5 weeks 3-yr EFS Nil 73% < 0.01% 45% 0.01% 19% Paganin et al. Leukemia. 2008

39 MRD Escalate therapy? UKALL 2003 trial; n = 533 Age: 1-24 yrs At diagnosis: Standard or intermediate-risk MRD 0.01%; end of induction (Day 29) Randomisation: Standard or augmented therapy Augmented therapy Superior 5-year EFS: 90% vs. 83% (p 0.04) Vora et al. Augmented post-remission therapy for a MRD-defined high-risk subgroup of children & young people with clinical standard-risk & intermediate-risk ALL (UKALL 2003): a randomised controlled trial. Lancet Oncol 2014

40 ALL-REZ BFM 2002 End of induction MRD: PCR MRD 10-3 (n = 99) HSCT EFS: 64% compared with 18% in earlier ALL-REZ BFM P95/96 study Conclusions Allogeneic HSCT prognosis of patients with intermediate risk of relapse & unsatisfactory MRD response Eckert et al. Use of ASCT based on MRD response improves outcomes for children with relapsed ALL in the intermediate-risk group. J Clin Oncol 2013.

41 MRD Minimize therapy? UKALL 2003 Std or intermediate risk Undetectable MRD at day 29, or Detectable day 29 undetectable at week 11 Randomly assigned: 1 or 2 DI courses 5-yr EFS (n = 521) 1 DI: 94 4% 2 DI: 95 5% Vora et al. Treatment reduction for children & young adults with low-risk ALL defined by MRD (UKALL 2003): a randomised controlled trial. Lancet Oncol 2013.

42 UKALL 2003 ALL: 1-24 yrs Rapid clearance of MRD by end of induction Treatment reduction is feasible Vora et al. Treatment reduction for children & young adults with low-risk ALL defined by MRD (UKALL 2003): a randomised controlled trial. Lancet Oncol 2013.

43 Long-term surveillance? Will treatment based on serial MRD after end of therapy affect clinical outcome? Time between detection of a +ve MRD & clinical detection of relapse? Theoretically Clinically evident relapses should be preceded by Appearance of MRD, or in MRD

44 ALL: Not all patients with MRD are destined to relapse MRD: detected as long as 9 yrs following end of therapy without clinical relapse Highly sensitive quantitative PCR assay Demonstrated MRD in 15 of 17 children with ALL Remission 2 to 35 months after end of treatment Br J Haematol N Engl J Med 1997, 98

45 Eradication of all leukemia cells may not be a prerequisite for cure N Engl J Med. 1997

46 Positive MRD after end of therapy..? It is unclear whether the identification of MRD should lead to subsequent therapy.. Persistently positive MRD Trigger preparation for potential disease relapse E.g., search for potential donors may be done

47 ALL: MRD in adults Less data Ph +ve ALL 30% Marker of poor prognosis Routinely treated with ASCT Unclear if MRD can be used to alter therapy

48 Ph negative ALL in adults MRD positivity at various time points after the initiation of treatment is a strong factor predictor for relapse

49 Adult ALL, MRD & transplant Patients who remain MRD +ve may benefit from an early ASCT Remains to be proven in an unbiased prospective study Role of MRD prior to ASCT is less certain MRD after HCT risk of relapse in patients: Ph -ve or +ve Less potential for MRD to allow for therapy

50 MRD: Everyday questions

51 Private labs? Accredited? National Accreditation Board for Testing & Calibration Laboratories (NABL) College of American Pathologists (CAP) Transport of sample 4-8 o C Upto hours

52 Can results of MRD of a lab be rechecked? Raw data List mode files FCS files as a link To generate fresh plots Plots alone not good enough Quality of raw data 6 vs. 8 vs. 10 color

53 Discrepancy: Results of MRD & morphology..? 7-year-old Standard risk ALL Day 35 MRD: < 0.01% Bone marrow morphology: 8% blasts

54 Pediatric T-ALL: Best timing for MRD? End of induction or end of consolidation? Persistent end of induction MRD alone is not an indication to alter therapy MRD at later time points more accurately predicts outcome End of induction minimal residual disease alone is not a useful determinant for risk stratified therapy in pediatric T-cell acute lymphoblastic leukemia. Pediatr Blood Cancer. 2015

55 Case scenario 5-year-F; intermediate risk B-lineage ALL WBC: 75,000/µL BCR/ABL1 negative t(12;21) +ve 4 drug induction Day 35 MRD: 0.15% Augmented BFM consolidation MRD: 0.1%

56 B-lineage ALL: End consolidation MRD +ve Sure of results? Confirm; recheck 2 nd robust method: PCR: Ig/TCR Cytogenetics Ph like? Hypodiploidy (< 44 chromosomes) Affordability?

57 B-lineage ALL with hypodiploidy (< 44 chromosomes): Management Intensive chemotherapy If MRD 1% at end of remission induction Allogeneic HSCT 5-year-EFS (n=20): 74% Campana et al. MRD guided therapy in childhood ALL. Blood 2017

58 Hyperdiploid (> 50 chromosomes) ALL If high levels of MRD at end of induction May continue treatment with intensive chemo without ASCT if MRD-negative after consolidation Campana et al. MRD guided therapy in childhood ALL. Blood 2017

59 Childhood B-lineage ALL BCR/ABL +ve High-risk protocol + imatinib/dasatinib How to monitor MRD? When to transplant based on MRD?

60 Ph +ve B-lineage ALL: children 1. IG/TR MRD monitoring is more reliable than MRD monitoring of BCR/ABL1 fusion transcript 2. Decision of treatment intensification may be made based on MRD performed after consolidation Cazzaniga et al. Predictive value of MRD in Ph-positive ALL treated with imatinib in the European intergroup study of post-induction treatment of Ph-positive ALL, based on immunoglobulin/t-cell receptor & BCR/ABL1 methodologies. Haematologica 2018

61 Ph +ve ALL in Children: Recommendations for HSCT 1. MRD at end of IB/start of HR % (PCR: Ig/TCR) If NA: < 3-log in MRD (RQ-PCR for BCR-ABL) 2. MRD at end of IB/start of HR: % (PCR: Ig/TCR) & MRD at end of consolidation block 3 (HR3)/start of reinduction block 1 remains positive at any detectable level UK interim recommendations for treatment of children with Ph+ ALL. 2014

62 Pediatric B-lineage ALL with Ph-like genetic subtypes If MRD < 0.01% at end of remission induction Intensive chemo, plus Appropriate TKI Ph + ALL or Ph-like ALL with ABL-class fusion transcript Campana et al. MRD guided therapy in childhood ALL. Blood 2017

63 MRD - ALL - transplant Children/adolescents with high levels ( 1%) MRD at end of induction & persistent MRD at subsequent time points: Very high risk of relapse with chemo alone ASCT MRD level before ASCT risk of relapse Additional treatment to MRD before transplant Campana et al. MRD guided therapy in childhood ALL. Blood 2017

64 Case scenario 10-years-M; T-lineage ALL WBC: 112,000/µL CSF: negative High-risk protocol: 4 drug induction Day 35 MRD: 0.12% Augmented BFM consolidation MRD: 0.12%

65 T-lineage ALL; end consolidation MRD +ve Sure of results? Confirm; recheck 2 nd method: PCR: Ig/TCR

66 Conclusions MRD: Independent prognostic factor for relapse in childhood ALL Treatment selected on the basis of MRD appears to improve outcome Sequential MRD monitoring after remission induction is warranted for patients with detectable MRD MRD evaluation in childhood ALL: A clinical evidence review. Ont Health Technol Assess Ser. 2016

67 References UpToDate Detection of MRD in ALL Clinical use of MRD in ALL Wendy Stock: University of Chicago Zeev Estrov: MD Anderson Cancer Center

68 Dr Man Updesh Singh Sachdeva Lab Hematology, PGI Dr Vaskar Saha: TMC, Kolkata Dr Anirban Das: TMC Kolkata Dr Ajay Vora: UK