Minimal residual disease of CLL in our everyday hematological practice

Transcription

1 Minimal residual disease of CLL in our everyday hematological practice M. Doubek, J. Chovancová, B. Tichý, O. Stehlíková, H. Francová Skuhrová, M. Klabusay, K. Malinová, M. Krejčí, Y. Brychtová, Š. Pospíšilová, J. Mayer

2 Is MRD negativity the goal of CLL treatment in everyday practice? Moreton et al. Eradication of Minimal Residual Disease in B-Cell Chronic Lymphocytic Leukemia After Alemtuzumab Therapy Is Associated With Prolonged Survival. J. Clin. Oncol. 2005

3 Is MRD negativity the goal of CLL treatment in everyday practice? Contra Some studies demonstrated significantly prolonged OS in MRD- patients (Moreton et al., 2005; Del Poeta et al. 2005; Tam et al. 2008; Bosh et al. 2008) Clinical trial data vs. everyday practice should not be comparble CLL is disease of elderly (CIRS score) CLL maintenace toxicity CLL is incurable by conventional therapy CLL relapses are not immediately treated

4 Which CLL patient is suitable for MRD evaluation in our practice? Clinical trial Allo Tx (First line intensive therapy in younger patients)?

5 Which method is suitable for MRD evaluation in everyday practice? Flow cytometry RQ-ASO IgH PCR

6 MRD analysis RQ-ASO IgH PCR probe probe qpcr MRD monitoring in CLL allele (patient) specific primers, V H family specific LNA probe at DNA level, sensitivity at least 10-4

7 MRD analysis flow cytometry Standardized flow cytometric analysis of MRD in CLL Rawstron Protocol Clinical trial (our clinical trials); all patients after allo Tx Three antigen combinations CD5/CD19 and CD20/CD38 CD5/CD19 and CD81/CD22 CD5/CD19 and CD79b/CD43 Correlation with RQ-ASO IgH-PCR in CLL patients underwenting allo Tx

8 Flow cytometer BD FACSCanto II

9 Flow cytometry gating strategy active CLL 1. step: gate of CD19 + SS low from Leu 2. step: CD19 + SS low FS low CLL after therapy

10 Detection of CLL cells using CD20/CD38 and CD5/CD19 protocol CLL cells: low expression of both CD20 and CD38

11 Detection of CLL Cells Using CD22/CD81 and CD5/CD19 Protocol CLL cells: low expression of both CD22 and CD81

12 Detection of CLL Cells Using CD79b/CD43 and CD5/CD19 Protocol CLL cells: low expression of CD79b, dim expression of CD43 (CD43 positive)

13 Active CLL MRD - MRD + ( 20/38/5/19 (Protocol

14 MRD our pilot analysis The aim of our work was to compare detection of MRD in CLL patients using multiparametric flow cytometry and molecular biology approaches with detection of IgVH specific clones (RQ-ASO IgH-PCR).

15 MRD our pilot analysis Patients and Methods 17 CLL patients underwenting allogeneic Tx for refractory/relapsed disease (FLAMSA-RIC conditioning, n=15; Cy+TBI conditioning, n=2) MRD evaluated bi-monthly after allo Tx 397 samples analyzed

16 MRD our pilot analysis Results MRD positivity detected repeatedly after allo Tx in 12 patients In 9 patients MRD positivity detected after previous negativity In 3 patients persisent MRD positivity

17 MRD our pilot analysis Results Samples positive by PCR: 66.7% Samples positive by flow: 58.7% PCR negative, flow positive 1.3% PCR positive, flow negative 9.3%

18 MRD our pilot analysis Results Median time from molecular relapse to hematologic relapse: 17 months (6-38); n=4

19 flow [%] PCR vs. flow R= y = 4E-05x - 0,2003 R 2 = 0, PCR [kopie/10**6 GE]

20 MRD case report I FT, male, transplantace PB MRD - flow % PB chimérismus % PB kopie/10x6 GE MRD - PCR Klouzavý průměr/2 (PB kopie/10x6 GE MRD - PCR) Klouzavý průměr/2 (PB MRD - flow %) Klouzavý průměr/2 (PB chimérismus %) stop imunosuprese CT negativní CT negativní CT Infiltrát 74 mm relaps po HSCT CT tumorózní masa v retroperitoneu cykly MP+R 14.7., 11.8., a

21 MRD case report II JP, female, 1946 PB MRD - flow % chimérismus % PB kopie/10x6 GE MRD - PCR Klouzavý průměr/2 (PB kopie/10x6 GE MRD - PCR) Klouzavý průměr/2 (PB MRD - flow %) Klouzavý průměr/2 (chimérismus %) transplantace stop MMF stop CsA podání DLI relaps po HSCT

22 Conclusions Disadvantage of flow cytometry consisted in limited sensitivity in contrast to ASO-PCR, where the sensitivity reaches to 1 cell in On the other hand, disadvantage of PCR remain in high costs and labor intensity of this technique. For this reason flow cytometry is technique of first choice in our practice. It ensures rapid sample processing and the sensitivity of detection reaches up to one tumor cell in leukocytes.

23 University Hospital Brno and University Campus