Flow Cytometric Detection of Minimal Residual Disease (MRD) in Multiple Myeloma

Transcription

1 Flow Cytometric Detection of Minimal Residual Disease (MRD) in Multiple Myeloma Paul K. Wallace, Ph.D. Roswell Park Cancer Ins7tute Department of Flow & Image Cytometry Elm & Carlton Streets Buffalo, NY Phone: (716) FAX: (716)

2 Outline Diagnos(c assessment of myeloma Role of mul(parameter flow cytometry in the assessment of MRD Correla(on with outcomes (PFS/OS) in a couple of studies Development and Standardiza(on of MRD assays for mul(ple myeloma Selec(on and characteriza(on of mabs MRD analysis Number of events FDA Status

3 Progress in the Treatment of Multiple Myeloma Mr. McBean Melphalan Combination Chemo Vincristine Doxorubicin Dexamethasone Melphalan Glucocorticoids Thalidomide High-dose chemo Stem cell transplantation Bisphosphonates Bortezomib Lenalidomide Carfilzomib Pomalidomide 1990 s- high- dose therapy and autologous stem cell transplant berer than standard therapy First CR s 2010 Achieve universal responses and complete responses in a significant frac(on of pa(ents by combining Immunomodulatory drugs (thalidomide/lenalidomide/pomalidomide Proteasome inhibitors (bortezomib, carfilzomib) Using novel agents with consolida(on and maintenance therapy can further increase complete response rate and prolong dura(on of response Adapted from Paiva, Bruno. ICCS Web Presenta(on. 2012

4 Novel Therapeutics in Combination with HSCT has SigniDicantly Improved Overall Survival (OS) in Multiple Myeloma Relapsed before 1998 Relapsed before Relapsed before Relapsed before Relapsed before Exposed to new drug Not exposed to new drug OS before 2000 = 11.8 mo OS a\er 2000 = 23.9 mo OS with novel therapeu(cs = 30.9 mo OS no novel therapeu(cs = 14.8 mo Kumar et. al. Blood 111: (2008) - Mayo

5 Are There More Useful Ways to Monitor Response to Multiple Myeloma? The defini(on of CR is subop(mal and requires refinement With increased rates of CR with modern MM therapies, there is increasing need for more sensi(ve evalua(ons of disease responsiveness and disease burden over (me There are 3 methods for detec(ng MRD that focus on the malignant MM clone: Immunophenotyping by mul(parameter flow cytometry Allele- specific oligonucleo(de real (me quan(ta(ve polymerase chain reac(on (ASO- RT- qpcr) Next genera(on sequencing

6 Clinical Assessment of Multiple Myeloma Mul(parameter flow cytometry (MFC) Can detect 1 in 10 4 maybe 1 in 10 5 Rela(vely easy to perform Fast turn- around- (me Allele- specific PCR (ASO- PCR) Detect 1 in 10 5 Difficult to perform Requires sequencing of the myeloma specific IgH Requires pa(ents specific primers Sequencing failure in 30% of pa(ents Next Genera(on Sequencing Emerging technology Expensive Detects 1 in 10 6 No clinical data

7 Allele- specidic PCR vs. Multiparameter Flow Cytometry- GEM2000 MRD evaluated in pa(ents a\er high dose therapy and autologous stem cell transplant Flow Panel CD38/CD56/CD19/CD45 CD138/CD28/CD33/CD38 CD20/CD117/CD138/CD38 Applicable in 90% of pa(ents ASO- PCR applicable in 75% of pa(ents ASO- PCR Flow Cytometry Number of residual tumor cells detected was comparable in both techniques ASO- PCR detected MRD in 17 cases vs. FCM which detected 11 cases ASO- PCR is more sensi(ve, but (me consuming and applicable in a lower propor(on of pa(ents 1 Sarasquete ME, et al: Minimal residual disease monitoring in mul(ple myeloma: A comparison between allelic- specific oligonucleo(de real- (me quan(ta(ve polymerase chain reac(on and flow cytometry. Haematologica 90: , 2005

8 Next Generation Sequencing versus Flow Cytometry (GEM2000 or GEM05) PFS # of cases NGS < mo 25 NGS > mo 75 NGS >10 5, MFC- 50 mo 12 NGS <105, MFC+ 1 relapse 5 NGS applicability 91% Mar(nez- Lopez et al et. al. Blood 123: (2014) and Adapted from Pavia, Bruno FDA presenta(on 2014

9 Outline Diagnos(c assessment of myeloma Role of mul(parameter flow cytometry in the assessment of MRD Correla(on with outcomes (PFS/OS) in a couple of studies Development and Standardiza(on of MRD assays for mul(ple myeloma Selec(on and characteriza(on of mabs MRD analysis Number of events Repor(ng

10 FDA Requirements for New Drug Approval Regular Approval: Demonstra(on of clinical benefit or an effect on an established surrogate Accelerated Approval: Serious or life- threatening illness and meaningful benefit over available therapies Surrogate endpoint that is likely to predict clinical benefit or clinical endpoint that can be measured earlier than mortality or morbidity Requires post- approval trials

11 FDA Requirements for Surrogate Endpoint Biomarker Surrogate must be shown to predict clinical benefit: Accomplished for flow MRD in MM GEM/PETHEMA in Spain (4 color panel) MRC Trials in UK (6 color panel) PRIMeR Study in US underway at Roswell Park

12 Impact of MFC MRD Status on Progression Free Survival and Overall Survival Among Patients Achieving CR 1 Progression free survival (%) PFS at 5 yr. 62% PFS at 5 yr. 30% p< Overall survival (%) IFx nega(ve for M- protein, <5% PCs in BM by morphology Months from diagnosis 0 OS at 5 yr 87%. OS at 5 yr 59% p= MRD nega(ve (n=94) MRD posi(ve (n=53) Pa(ents who were MRD nega(ve at day 100 a\er transplanta(on had significantly longer PFS and OS Only MRD status by MFC at day 100 and FISH cytogene(cs were iden(fied as independent prognos(c factors for PFS, and only flow MRD status and age were iden(fied for OS Paiva, B. et al. Mul(parameter flow cytometric remission is the most relevant prognos(c factor for mul(ple myeloma pa(ents who undergo autologous stem cell transplanta(on. Blood 112: , 2008

13 Prognostic Value of MRD Assessment by Multiparameter Flow Cytometry in the MRC Myeloma IX trial MRC Myeloma IX trial design overview Intensive Pathway Randomiza(on n = 1,114 Nonintensive Pathway Randomiza(on n = 856 Na clodronate & CVAD n=278 Zoledronic acid & CVAD n=278 Na clodronate & CTD n=278 Zoledronic acid & CTD n=277 Na clodronate & MP n=211 Zoledronic acid & MP n=212 Na clodronate & CTDa n=212 Zoledronic acid & CTDa n=214 Hi Dose Melphalan HSCT n=747 Maximal Response Thalidomide n=408 No thalidomide n=410 BM obtained at presenta(on, a\er induc(on and at day 100 post HSCT in intensive pathway and a\er induc(on in the non- intensive pathway Flow Panel CD27/CD56/CD19/CD38/CD138/CD45 MRD detected if 50 abnormal cells in 500,000 event file (0.01%) <20 abnormal cells were MRD nega(ve Pa(ents with abnormal cells were further analyzed by acquiring more events or by analysis of: CD81, CD117, CD200 and/or CD52 1 Rawstron, A et al: Minimal Residual Disease Assessed by Mul(parameter Flow Cytometry in Mul(ple Myeloma: Impact on Outcome in the Medical Research Council Myeloma IX Study. J Clin Onc 31: , 2013

14 MRD Status, PFS and OS in the MRC Myeloma IX trial Impact of MRD a\er a\er induc(on, HSCT and high dose melphalan Those pa(ents who are MRD nega(ve at the end of induc(on do berer than those who become MRD nega(ve a\er HSCT and high dose melphalan Both groups do berer than those who do not become MRD nega(ve OS was not significantly different 1 Rawstron, A et al: Minimal Residual Disease Assessed by Mul(parameter Flow Cytometry in Mul(ple Myeloma: Impact on Outcome in the Medical Research Council Myeloma IX Study. J Clin Onc 31: , 2013

15 Impact of MRD Status 100 days after HSCT in Conjunction with Cytogenetic Risk Group in the MRC Myeloma IX trial The propor(on of pa(ents achieving MRD nega(vity was not affected by cytogene(c risk group Best outcome in those pa(ents with favorable cytogene(cs and MRD nega(vity Worst outcomes in those pa(ents with adverse cytogene(cs and persistent disease 1 Rawstron, A et al: Minimal Residual Disease Assessed by Mul(parameter Flow Cytometry in Mul(ple Myeloma: Impact on Outcome in the Medical Research Council Myeloma IX Study. J Clin Onc 31: , 2013

16 BMT CTN 0702 A Trial of Single Autologous Transplant with or without RVD Consolidation versus Tandem Transplant and Maintenance Therapy

17 PRIMeR Synopsis Mul(- center, Phase III, randomized 3- arm trial for upfront treatment of pa(ents with mul(ple myeloma Primary endpoint: 3 year progression- free survival. Pairwise comparisons will be used to compare the 3 arms 750 pa(ents will be enrolled, 250 per arm Accrual period is 3 years Pa(ents will be followed for 4 years post- randomiza(on

18 PRIMeR Schema

19 Abnormal Antigen Expression in Multiple Myeloma An7gen Normal Expression % normal in PC Abnormal Expression in MM CD19 Posi(ve >90% Nega(ve 92-95% CD56 Nega(ve <15% Posi(ve 60-75% CD117 Nega(ve 0% Posi(ve 30-32% CD27 Posi(ve 100% Neg to dim 40-50% CD81 Posi(ve 100% Posi(ve 52% CD28 Neg to dim <15% Posi(ve 15-45% CD20 Nega(ve 0% Posi(ve 17-20% CD200 Dim Posi(ve Posi(ve NA CD38 Bright Posi(ve Dimmer 80% % abnormal in MM Aberrant phenotypes are seen in more than 90% of MM pa(ents Rawstron, A et al. Report of the European Myeloma Network on mul(parametric flow cytometry in mul(ple myeloma and related disorders. Haematologica. 93: , Mateo, G. et al. Prognos(c value of immunophenotyping in mul(ple myeloma: a study by the PETHEMA/GEM coopera(ve study groups on pa(ents uniformly treated with high- dose therapy. J Clin Onc. 26: , 2008.

20 Flow- MRD in The Bone Marrow of Multiple Myeloma: Current Practice (Survey of 11/26 responders from 30 major ins(tu(ons) Variables Median Range Number of events: 3-5 x 10 5 (1x10 5-4x10 6 ) No abnormal PCs for MRD+: 25 (20-50)* Sensi(vity (Max): 0.005% (0.01% %) No. of markers studied: 8 (6-12) *Pathologist dependent Roschewski, M. et al. J Clin Oncol (2014) 22:1-2

21 Flow- MRD in The Bone Marrow of Multiple Myeloma: Current Practice (Survey of 11/26 responders from 30 major ins(tu(ons) Markers for MRD+ Number of Labs (%) sigk or sigl: 8/11 (73%) CD19- CD45- : 6/11 (55%) CD19- CD45- CD56+: 6/11 (55%) CD27- /low: 3/11 (27%) Other: 5/11 (45%) Some ins(tu(ons reported they solely rely on CD19 and CD45 nega(vity regardless of CD56 Up to 30% normal PCs CD19 are CD45- and 15% CD56+ so if you don t look at other an(gens will miss myeloma Only a few ins(tu(ons used CD81 and CD117 Roschewski, M. et al. J Clin Oncol (2014) 22:1-2 Haematologica 2008, 93:

22 Flow Cytometric Immunophenotyping of Multiple Myeloma Advantages Applicability 92-97% Single cell analysis Sensi(vity: 10-4 and lower Speed <2 hr Simplicity (with standardized methods) Assessment of non- PC BM cell compartments Disadvantages Only the Bone Marrow/Blood Compartment Heterogenous BM infiltra(on Quality of BM sample (PB dilu(on) No specific tumor an(gens Limited number of mabs studied Fresh (<48h) samples required More sensi(ve methods available Final product analysis: protein (antigen) Op(mal panel should include CD45, CD138, CD38, CD56, CD19, CD117 CD81 & CD27

23 Outline Diagnos(c assessment of myeloma Role of mul(parameter flow cytometry in the assessment of MRD Correla(on with outcomes (PFS/OS) in a couple of studies Development and Standardiza(on of MRD assays for mul(ple myeloma Selec(on and characteriza(on of mabs MRD analysis Number of events FDA Status

24 Flow Cytometric Minimal Residual Disease Testing in Multiple Myeloma Steps for development of flow MRD as FDA regulatory endpoint: Iden(fy MRD endpoint in clinical trials Develop assay for MRD (at least 2 validated assays available in Europe) Harmoniza(on/Standardiza(on of assay for MRD in the study Apply standardized assay prospec(vely Apply MRD assay to regulatory ac(on

25 A Consensus is Building: Recent History October 2013: (ICCS) MM MRD Consensus Group formed January 2014: First dra\ circulated for cmment March 2014: Joint FDA/NCI Conference April 2014: Final dra\ circulated for comments July 2014: IMF Conference October 2014: Manuscripts to Cytometry B due for special Issue Panel & Staining Analysis Valida7on & QC Stetler- Stevenson, MA Jorgensen, J Paiva, B NIH Arroz, M Lisbon Wallace, P RPCI MD Anderson Univ de Navarra Came, N Peter Mac BarneR, D UK NEQAS Chen, W Rawstron, A Leeds Constance, Y NIH Stoolman, L U Michigan de Tute, R Leeds Wang, S MD Anderson Lagoo, A Monreal, M UT Southwestern Duke InterpFlow Oldaker, T Genop(x Pei, L MD Anderson

26 Consensus Multiparameter Flow Cytometry Panels for Multiple Myeloma MRD Panel V450 # BV510 FITC PE PcPCy5.5 PECy7 APC APCC750 1 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 2 CD138 CD27 CD38 CD56 CD45 CD19 ckappa clambda

27 Fine Tuning Multiple Myeloma Flow MRD Panel Evalua(on of new an(body clones for iden(fica(on of normal and aberrant/clonal plasma cells (alterna(ves for CD38 and CD138): CD54, CD229, CD319 and VS38c Evalua(on of new CD138 fluorochrome conjugates: CD138- HV450 vs. CD138- BV421 Evalua(on of new an(body markers for detec(on of aberrant plasma cells: CD200 Produc(on of (commercially available) lyophilized an(body cocktails* *a task for industry

28 Multiple Myeloma MRD Gating Strategy

29 Setting Individual mab Regions Gated on R1&R2&R3!R4

30 Bone Marrow Suitability Assessment

31

32

33

34

35 Clonality by κ:λ : Limited Sensitivity Compared to Other Markers Courtesy of Bruno Pavia (University of Navarra, Spain)

36 Number of Events to Evaluate Myeloma IX 500,000 Sensi(vity of 0.01% 500,000 = 50 posi(ve events GEM ,000 Dr. Paiva has increased this to 1 x10 6 and would like to collect more PRIMeR: 500,000 increased to 1,000,000 Using standard stain/lyse wash procedures we have hard (me consistently achieving this number In dilu(on studies can reliable detect 0.01% Consensus Guidelines A minimum of 2 x 10 6 cells Ideal 5 x 10 6 cells

37 Collecting SufDicient Events At Roswell Park for the PRIMeR when we set our goal to collect 500,000 events ran into problems We use a wash/stain/lyse/wash/fix procedure. For MRD samples we were staining up 200 μl of blood or bone marrow Cell loses at each step 68% of tubes reached 500,000 events Moved to a wash/count/stain/lyse procedure (NIH/FDA consensus method) Add 2x10 6 cells/tube 46% of tubes reach 1x10 6 events Moving to a bulk lyse method (NIH/FDA consensus method) Goal is to collect a minimum of 2x10 6 to 5x10 6

38 Limit of Detection (LOD) and Lower Limit of QuantiDication (LLOQ) Total number of leucocytes analyzed LOD % LLOQ % 100, , , ,000, ,000, ,000, LOD = (30 / total number of leucocytes) x 100% LLOQ = (50 / total number of leucocytes) x 100%

39 Multiple Myeloma MRD Bulk Lysis Method (EuroFlow) To 50 ml tube containing 10x10 6 cells add AmmCl lysing buffer (no more than 2 ml BM per conical) Incubate 15 minutes on roller plate Spin (800 x g) and Wash 1X 50 ml FCM Buffer Resuspend at 1 x 10 8 cells/ml in FCM Buffer Combine 100 μl of cells with mab cocktail Incubate 30 RT in dark Add 2 ml FACSLyse Incubate 10 min Goal to obtain sufficient cells to acquire 5x10 6 cells by FCM For kappa and lambda A\er surface staining and 2 nd wash Add Caltag Perm B (0.1% saponin) mabs Incubate wash and run Wash 2X with FCM Buffer Acquire data on FCM FCM Buffer: PBS with 0.5% BSA, 0.1% Na azide and 0.004% EDTA

40 Lyse Before Stain vs. AmmCl Lyse Post Stain

41 Evaluation of CD138 Fluorochromes for Multiple Myeloma MRD CD138 HV500 FACSLyse CD138 V500 Bulk Lyse CD138 BV510 Bulk Lyse CD138 V500 CD138 V500 CD38 FITC CD38 FITC CD38 FITC CD138 V500 Stain Index 48.3 Stain Index 3.4 Stain Index 42.8 Courtesy of Bruno Pavia (University of Navarra, Spain)

42 Outline Diagnos(c assessment of myeloma Role of mul(parameter flow cytometry in the assessment of MRD Correla(on with outcomes (PFS/OS) in a couple of studies Development and Standardiza(on of MRD assays for mul(ple myeloma Selec(on and characteriza(on of mabs MRD analysis Number of events FDA Status

43 Where We Stand On March 24, 2014, an FDA- NCI Roundtable Symposium detec(on of mul(ple myeloma MRD held at the FDA in Silver Spring, Maryland The qualifica(on process for a surrogate endpoint biomarker is a formalized process based on eviden(ary standards For regulatory approval, a surrogate must be shown to be reasonably likely to predict clinical benefit It was proposed that MRD tes(ng by flow cytometry needs to be integrated into the response criteria for mul(ple myeloma Therefore MRD should be considered for regulatory purposes including drug approval in the field of mul(ple myeloma Consensus guidelines for flow- cytometry MRD tes(ng have been developed and will soon be available There is, however, urgent need for consensus within the mul(ple myeloma community for inclusion of MRD nega(vity as a surrogate endpoint in clinical trials American Journal of Hematology, 2014 in press

44 Should MRD Testing for MM MRD Become Standard of Care? Myeloma community is interested CR criteria is ready to be redefined The concept of Flow- CR is ready Consensus guidelines now exist Is flow cytometry ready?

45 Summary Over the past decade, the propor(on of mul(ple myeloma pa(ents achieving a complete response (CR) has risen to 50-75% Classifica(on of CR in mul(ple myeloma is sub- op(mal and requires improvement in the age of autologous HSCT and novel therapies Allele- specific oligonucleo(de real (me PCR is more sensi(ve than mul(parameter flow cytometry but difficult to perform and is uninforma(ve 30% of the (me All studies have demonstrated pa(ents who were MRD nega(ve by flow cytometry at day 100 a\er HSCT had significantly longer PFS and OS MDR detec(on of MM is not for the feint of heart and requires rigorous QC and aren(on to details FDA, MRD should be considered for regulatory purposes including drug approval in the field of mul(ple myeloma

46 Acknowledgments Roswell Park Cancer Institute Buffalo, NY MM MRD Consensus Committee Joseph D. Tario, Jr. Jan Hoffman Ed Podniesinski Aileen Cinquino Terry Donnahue Lynn Bender Sue Camacho Susan Macro Matt Smith Jennifer Piraino Francine Bellanti Terry Hahn George Chen Philip McCarthy Maryalice Stetler-Stevenson NCI Maria Arroz Lisboa Terry Oldaker Genopix David Barnett UK NEQAS and the entire group Medical College of Wisconsin Milwaukee, WI Marcelo Pasquini

47 Thank you! And see you at GLIIFCA 24 Sept 25-27, 2015