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St James's Institute of Oncology

Flow- cytometric quantification of minimal residual disease (MRD) in myeloma: independent outcome prediction & sequential survival benefits per log tumour reduction. St James's Institute of Oncology. Andy C. Rawstron. MRD analysis for clinical trials in myeloma. Myeloma IX

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St James's Institute of Oncology

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  1. Flow-cytometric quantification of minimal residual disease (MRD) in myeloma: independent outcome prediction & sequential survival benefits per log tumour reduction • St James's Institute of Oncology • Andy C. Rawstron

  2. MRD analysis for clinical trials in myeloma • Myeloma IX • Using MRD as an endpoint: lessons from the FDA • Harmonisation • Quantitative MRD analysis • Measuring MRD for clinical trials

  3. MRC Myeloma IX: Trial design

  4. MRC Myeloma IX: MRD status post-ASCT is an independent predictor of PFS • MRDNEG improved PFS in CR patients (34.3 vs 14.1 months, P=0.0068) • MRDNEG but IFPOS similar to MRDPOS ? Sample quality ? Maintenance randomization • MRDNEGimproved OS in CR patients (NR vs 61.9 months, P=0.0928) • Best outcome if MRDNEG and IFNEG (P=0.0385)

  5. MRC Myeloma IX: MRD status after induction

  6. Immunofixation response depends on half-life Up to one year to see maximum M-protein response Davies et al (2001) Brit J Haem 112:814-9

  7. MRC Myeloma IX: Thalidomide maintenance improves PFS in patients with detectable MRD after HDM • Best outcome was demonstrable in MRD negative patients receiving thalidomide maintenance and worst in those MRD positive patients who did not receive maintenance therapy (P=0.0003)

  8. 96 Thalidomide maintenance 100 No maintenance 80 68.8 60 40 27.6 20 3.4 0 Become MRD negative Remain MRD negative No change in conventional response with thalidomide maintenance but clear differences in neoplastic plasma cell levels • “Using electrophoresis and immunofixation as a monitoring technique, there was no difference between the thalidomide maintenance and no maintenance arms in the percentage of patients that upgraded response status over time (P .19).” (1) (2) • Morgan et al, Blood 2012, 119(1): 7-15 • Rawstron et al, JCO 2013 in press

  9. Optimal laboratory technique for assessing disease levels varies according to the goal of the assessment Quantitative, direct and sensitive measure of bone marrow infiltration is optimal for response assessment

  10. MRD analysis for clinical trials in myeloma • Myeloma IX • MRD provides rapid and sensitive measure of response to induction, ASCT and maintenance. • Using MRD as an endpoint: lessons from the FDA • Harmonisation • Quantitative MRD analysis • Measuring MRD for clinical trials

  11. Is MRD suitable as an end-point for clinical trials in myeloma? • MRD analysis improves assessment of response compared to serum markers alone, particularly in multi-component strategies • Longer survival with increasing treatment options  need for biomarkers that predict clinical benefit and offer a rapid measure of treatment efficacy Flow cytometry detection of minimal residual disease in multiple myeloma: Lessons learned at FDA-NCI roundtable symposium Am J Hematol. 2014 Dec;89(12):1159-60

  12. Development of “MRD” as a regulatory end-point • Identify MRD Endpoint in Clinical Trials • 5-10 sub-group analyses, primarily ASO-IGH qPCR • Develop Assay • Disease-specific flow assay applicable to larger trials • Standardization of Assay (NIH Consensus Conference) • EMN consensus • Apply Standardized Assay Prospectively • Apply to Regulatory Action

  13. MRD is an independent prognostic factor for PFS/OS in studies using CD138 / CD38 / CD45 for gating CD19 / CD56 / CD27 / CD117 / (CD81) for identifying neoplastic PC *CR patients only

  14. MRD analysis for clinical trials in myeloma • Myeloma IX • MRD provides rapid and sensitive measure of response to induction, ASCT and maintenance. • Using MRD as an endpoint: lessons from the FDA • Would facilitate development of new treatments, needs harmonisation document and more independent OS data • Harmonisation • Quantitative MRD analysis • Measuring MRD for clinical trials

  15. Harmonised assay for MRD detection • Characteristics of assays that predict outcome • CD138/CD38/CD45 backbone for gating • CD19/CD56/CD27/CD117/CD81 for characterization • Reagent specification to permit rapid validation of LDT (lab-developed test) or IVD panels • Backwards compatible • Suitable for prospective studies • targeted acquisition of 3-5 million events

  16. MRD analysis for clinical trials in myeloma • Myeloma IX • MRD provides rapid and sensitive measure of response to induction, ASCT and maintenance. • Using MRD as an endpoint: lessons from the FDA • Would facilitate development of new treatments, needs harmonisation document and more independent OS data • Harmonisation • Nearly done… • Quantitative measure of outcome • Measuring MRD for clinical trials

  17. Direct quantitative measure of tumour burden allows better prediction of PFS

  18. Direct quantitative measure of tumour burden allows better prediction of overall survival Median OS: >1%  4.0yrs 0.1-1%  5.9yrs 0.01-0.1%  6.8yrs <0.01%  >7.5yrs ~1 year improvement in overall survival per log tumour depletion Myeloma IX 6year survival data

  19. Relationship between categorical response and MRD

  20. >1% residual disease = PR or worse

  21. Direct quantitative measure of tumour burden allows better prediction of outcome for patients in CR

  22. Relationship between categorical response and MRD

  23. Sample quality First aspirate  morphology Second aspirate  laboratory

  24. Differences in aspirate quality according to referring hospital (trial samples) Plasma cells (% of leucocytes) >10% 5-10% 1-5% <1% Proportion of cases Centre ranking (median % plasma cells from baseline samples)

  25. Differences in aspirate quality according to referring hospital (diagnostic with trephine) Plasma cells (% of leucocytes) >10% 5-10% 1-5% <1% Proportion of cases Centre ranking (median % plasma cells from baseline samples)

  26. Differences in practice according to the median plasma cell percentage in bone marrow aspirate samples

  27. Differences in practice according to the median plasma cell percentage in bone marrow aspirate samples

  28. Post-treatment aspirate quality acceptable in >95% of cases, no difference in quality according to baseline sample quality Neoplastic plasma cells (% of leucocytes) <0.01% 0.01-0.1% 0.1-1% 1-10% >10% After Induction 3 Months after end of treatment 100% 90% 80% 70% 60% Proportion of cases 50% 40% 30% 20% 10% 0% Baseline Median >8%PC (n=26) Baseline Median >8%PC (n=40) Baseline Median >8%PC (n=40) Baseline Median <2%PC (n=40) 3 months after ASCT / end of treatment, using % normal PC as a marker of sample quality (>0.01% adequate, >0.1% good): >8% PC baseline median: 96% adequate / 50% good <2% PC baseline median: 95% adequate / 48% good

  29. Is MRD relevant in PR?

  30. Quantitative MRD and cytogenetics are independent predictors of progression-free and overall survival

  31. Outcome depends on disease level not treatment

  32. Outcome depends on disease level not treatment Neoplastic plasma cells (% of leucocytes) <0.01% 0.01-0.1% 0.1-1% 1-10% >10% Proportion of patients Number of patients 140 100% 120 80% 100 60% 80 60 40% 40 20% 20 0 0% CVAD (n=91) CTD (n=123) CVAD (n=117) CTD (n=66) CVAD (n=91) CTD (n=123) CVAD (n=117) CTD (n=66) Not CR Not CR CR CR

  33. Achieving <0.01% MRD: impact of ASCT • Median (range) % neoplastic plasma cells at end of induction, data available in 253/397 cases, of which 47/253 had <0.01% MRD after induction and ASCT • ≥0.01% MRD after ASCT (n=96)  1.5% (0.02 – 25%) • <0.01% MRD after ASCT (n=110)  0.02% (0.02 – 14%) • ≥0.01% MRD after induction & ASCT  Median 0.67 log tumour depletion (range -1.4 – 2.6) • ≥0.01% MRD after induction & <0.01% MRD after ASCT  Median >1.7 log tumour depletion • Responsive patients achieve ~2log depletion to ASCT • >1% MRD after induction  unlikely to respond optimally

  34. MRD analysis for clinical trials in myeloma • Myeloma IX • MRD provides rapid and sensitive measure of response to induction, ASCT and maintenance. • Using MRD as an endpoint: lessons from the FDA • Would facilitate development of new treatments, needs harmonisation document and more independent OS data • Harmonisation • Nearly done… • Quantitative measure of outcome • independent predictors of progression-free and overall survival • Sample quality acceptable – first (or only) pull for lab studies • Measuring MRD for clinical trials

  35. High-throughput sequencing: >1 log errors ERIC harmonised approach Leukemia 2007, 21(5): & 2013, 27(1) Logan et al. Leukemia 12 March 2013; doi: 10.1038/leu.2013.52:

  36. MRD by high-throughput sequencing • Isolate DNA and combine with 3 IGHV reference standards • 1º PCR: 16 cycle amplification of IGHV with consensus V and J primers (optimised to minimally skew the repertoire frequency during amplification) and append annealing sites for 2º PCR primers • Second stage PCR: 22 cycles using 1/100 of the 1º PCR product append sample indices and cluster formation sequences • Pool samples and purify (QIAquick) • Amplify in situ on Illumina via bridging PCR and sequence • MAP sequences to IMGT database and correct for • differential amplification of IGHV rearrangements • replicate amplicons and minor clonal expansions • non-functional rearrangements • Calculate the number of neoplastic (and total B-cell) reads using the IGHV reference standard • Calculate total leukocytes (total DNA by picogreen and qPCR for β-actin)

  37. High throughput sequencing for MRD detection: negative result  substantial improvement in outcome http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4023416/

  38. MRD strategy for UK clinical trials • Median 30 million cells per BM aspirate • Flow 10-4 (LoD 0.002%)  2 million cells • Suitable LoD for substantial proportion of cases • CD138/CD38/CD45 + CD19/CD56/CD27/CD117/CD81 • DNA for HTS  10 million cells • Immunomagnetic CD138-selection and storage of CD138+ and CD138- fractions for HTS  10 million • If required and sufficient cells, flow 10-5  10 million

  39. MRD analysis for clinical trials in myeloma • Myeloma IX • MRD provides rapid and sensitive measure of response to induction, ASCT and maintenance. • Using MRD as an endpoint: lessons from the FDA • Would facilitate development of new treatments, needs harmonisation document and more independent OS data • Harmonisation • Nearly done… • Quantitative MRD analysis • independent prediction of progression-free and overall survival • Sample quality acceptable – first (or only) pull for lab studies • Measuring MRD for clinical trials • Combination of flow + HTS MRD optimal

  40. Acknowledgements MRC Leukaemia Trial Steering Committee MRC Leukaemia Data Monitoring and Ethics Committee NCRI Haematological Oncology Clinical Studies Group NIHR, through the National Cancer Research Network UK Myeloma Forum Clinical Trials Committee Myeloma UK Funding Medical Research Council Pharmion Novartis Chugai Pharma Bayer Schering Pharma OrthoBiotech Celgene Kay Kendall Leukaemia Fund Chief Investigators JA Child GJ Morgan GH Jackson CTRU, Leeds K Cocks W Gregory A Szubert S Bell N Navarro Coy University of Birmingham MT Drayson K Walker A Adkins N Newnham Wessex Regional Genetics Laboratory, Salisbury F Ross L Chieccio LTHT, Leeds G Cook S Feyler D Bowen HMDS, Leeds RG Owen AC Rawstron R de Tute M Dewar S Denman ICR, London FE Davies M Jenner B Walker D Johnson D Gonzalez N Dickens K Boyd P Leone L Brito A Avridromou

  41. Thanks!

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