Jan Philippé. VAKB 3 februari 2014

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1 Jan Philippé VAKB 3 februari 2014

2 DISEASE (+ MRD) NR of Citations (pubmed) 1st cited Author / journal (1st) ALL Hoelzer / Blood AML Hagenbeek / Leukemia CLL Dighiero / Leuk Lymphoma Multiple myeloma Follicular lymphoma Yeh / BJH Miyamura / Rinsho Ketsueki HCL Filleul / Leukemia MCL Corradieri / Blood

3 MRD during and at the end of therapy is a powerful predictor of PFS and OS In CLL and MM, at least, this predictor appears to be independent of clinical response, type or line of therapy, and other known biological markers Clinical trials are running to assess whether MRD can be used for guiding therapies

4 Molecular analyses ASO RQ-PCR of the junctional region of the rearranged IgH genes Analytical sensitivity: Applicability: ~80% Expertise: ++ Cost: ++ Speed: weeks PCR of the chromosomal translocations t(11; 14) in MCL (RQ-PCR) Analytical sensitivity: Applicability: 50% t(14;18) in foll lymph (RQ-PCR or nested PCR) Analytical sensitivity: Applicability: ±70% Expertise: + Cost: + Speed: days week(s)

5 Flow Cytometric analyses Multiparameter FC Sensitivity: 10-4 Applicability: >90% Expertise: ++ Cost: + Speed: one or 2 days

6 Comparison ASO RQ PCR vs FC in CLL MRD in CLL1/9 FC & PCR are equally effective for MRD quantification with sensitivity 10-4 PCR is more sensitive for MRD <10-4 Böttcher et al. Leukemia 2009

7 MRD in CLL2/9 Leukemia (2007) (Rawstron A. et al) 5 tube 4 CLR setup ERIC (European Research Initiative in CLL) update : Leukemia 2013 (Rawstron A. et al) MRD screening with Full MRD analysis with 1 tube, 4 CLR 2 tubes, 6 CLR

8 MRD in CLL3/9 CD5+ B-cells:29% κ/λ: 0,33/1 sig neg 19+5+: 11%

9 MRD in CLL4/9

10 MRD in CLL5/9 Why are such large deviations from normal required? Regenerating B cells have high levels of CD5 Highly skewed κ:λ ratios in the early stages after treatment Lack of sig on the CD5+ B cells varies between pts How frequent is screening + in a series of analyses? if > 1% CLL cells (according to full MRD) 72% if < 1% CLL cells (according to full MRD) 37% on average 50% of screening is +, with 100% PPV!

11 MRD in CLL6/9

12 MRD in CLL7/9

13 When is this full panel needed? In about 50% of cases the criteria as defined for the MRD screening are not met What are the strenghts of 2x 6 CLR? cost, time, complexity, permits acquisition of events Detection limit: 10-4 à 10-5 Quantitative Weaknesses? Differences between labs? MRD in CLL8/9

14 MRD in CLL9/9 EUROFLOW will propose. Fully standardized instruments Fully standardized staining protocols Use of library of phenotypes Use of gatings in plots obtained by PCA A. Langerak & S. Böttcher developed a new panel: validation will follow soon in 3 centers among which UZ Gent EuroClonality (a division of ESHLO) NGS Consortium on Next-Generation Sequencing for IG / TR immunogenetic analysis Coordinator: A. Langerak Main objectives are to develop, standardize, and validate IG/TR NGS assays for clonality assessment, MRD analysis, and repertoire analysis

15 MRD in MM1/9 Molecular analyses ASO RQ-PCR of the junctional region of the rearranged IgH genes Sensitivity: Applicability: ~40% Expertise: ++ Cost: ++ Speed: weeks Multiparameter FC Sensitivity: 10-4 Applicability: >90% Expertise: ++ Cost: + Speed: days

16 MRD in MM2/9 Reasons for low applicability in ASO RQ- PCR Lack of clonality detection (20%) Unsuccessful sequencing (10%) Suboptimal ASO performance (30%)

17 MRD in MM3/9 Puig N. et al. Leukemia 2013

18 MRD in MM4/9

19 MRD in MM5/9

20 MRD in MM6/9 Robillard et al. Blood Canc J 2013

21 MRD in MM7/9 Conclusions of the Nancy study: Millions of cells are needed to start with 7 CLR analysis has a lower sensitivity (10-5 ) This approach does not depend upon the initial phenotype Aberrant phenotypes may be found in healthy donors Thus proof of clonality is mandatory! Open question Quantitative? (variability in MM cells in aspirate) Pre-analytical problem

22 MRD in MM8/9 EUROFLOW will propose. Fully standardized instruments/protocols Use of plots based upon PCA A disease-specific approach (not patient-specific) Usage of pre-defined data bases of normal reactive PC phenotypes Other markers? (CD27, CD81,..) EuroClonality (a division of ESHLO) NGS Consortium on Next-Generation Sequencing for IG / TCR immunogenetic analysis Coordinator: Anton W. Langerak Main objectives are to develop, standardize, and validate IG/TR NGS assays for clonality assessment, MRD analysis, and repertoire analysis

23 MRD in MM9/9 -A high correlation between NGS and PCR MRD, -MRD-negativity in PBSC autografts by NGS may be more closely associated with durable remission than that revealed by ASO-qPCR Takamatsu et al. Abstract ASH 2013

24 Dank voor uw aandacht Vragen??

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