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 Using MRD as an endpoint: lessons from the FDA Harmonisation Quantitative MRD analysis Measuring MRD for clinical trials 2

MRC Myeloma IX: Trial design

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

MRC Myeloma IX: MRD status after induction

Immunofixation response depends on half-life CVADCTD Post induction (n=252)13%25%P=0.004 Day 100 (n=397)54%71%P< LCOM (2-4 hours) IgA (6 days) IgG (23 days) CR post induction 33.3%20.3%10.3% Up to one year to see maximum M- protein response Davies et al (2001) Brit J Haem 112:814-9

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)

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) Become MRD negativeRemain MRD negative Thalidomide maintenance No maintenance (2) 1.Morgan et al, Blood 2012, 119(1): Rawstron et al, JCO 2013 in press

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

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 10

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 Dec;89(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

StudyPFSOSMultivariate PFSMultivariate OS Paiva et al (2008) GEM2000 Yes MRD Cytogenetics MRD Rawstron et al (2013) Myeloma IX Yes MRD Cytogenetics Cytogenetics Paiva et al (2012)* GEM2000 GEM05<65 Yes MRD Cytogenetics Paiva et al (2011) GEM05>65 YesNoMRD Myeloma XYes Insufficient events MRD Insufficient events *CR patients only 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

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 14

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

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 16

Direct quantitative measure of tumour burden allows better prediction of PFS

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

Relationship between categorical response and MRD 19

>1% residual disease = PR or worse 20

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

Relationship between categorical response and MRD 22

Sample quality First aspirate  morphologySecond aspirate  laboratory

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

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

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

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

Post-treatment aspirate quality acceptable in >95% of cases, no difference in quality according to baseline sample quality Proportion of cases <0.01% %0.1-1%1-10% Neoplastic plasma cells (% of leucocytes) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Baseline Median >8%PC (n=40) Baseline Median <2%PC (n=40) After Induction >10% 3 Months after end of treatment Baseline Median >8%PC (n=26) Baseline Median >8%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

Is MRD relevant in PR?

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

Outcome depends on disease level not treatment

32 CVAD (n=91) CVAD (n=117) CTD (n=123) CTD (n=66) Proportion of patients 0% 20% 40% 60% 80% 100% CRNot CR Number of patients CVAD (n=91) CVAD (n=117) CTD (n=123) CTD (n=66) CRNot CR <0.01% %0.1-1%1-10% Neoplastic plasma cells (% of leucocytes) >10%

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 & 1.7 log tumour depletion Responsive patients achieve ~2log depletion to ASCT >1% MRD after induction  unlikely to respond optimally 33

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 34

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

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)

High throughput sequencing for MRD detection: negative result  substantial improvement in outcome C /

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

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 39

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 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 Acknowledgements

41 Thanks!