PARALLEL MOLECULAR AADULT ND IMMUNOLOGICAL MONITORING OF MINIMAL RESIDUAL DISEASE IN ALL PATIENTS. (ASH 2006)

Parovichnikova E.N., Davidyan Y.R., Galtzeva I.V., Surin V.L., Vorobjev I.A., Gretzov E.A., Isaev V.G., Savchenko V.G.

National Research Center for Hematology, Мoscow, Russia

INTRODUCTION

Though the initial response to induction therapy, indicating rapid tumor clearance, is currently considered to be one of the most important prognostic factors, detection of submicroscopic levels of minimal residual disease (MRD), by means of PCR or immunophenotyping after induction and during different stages of treatment, has also become important in predicting outcome in ALL patients. We initiated a study using parallel PCR and flow cytometry to monitor MRD in adult acute lymphocytic leukemia (ALL) patients treated by standard approach.

MATERIALS AND METHODS

Bone marrow samples of 34 ALL patients (27 B-ALL, 7 T-ALL) were analyzed for IgH and TCR-gamma gene rearrangement at diagnosis, after pre-phase, after 8-weeks induction, at consolidation, before maintenance (+7-8 mo), and during maintenance. Molecular markers (clonal rearranged V-D-J or V-J regions) were revealed by PCR with family-specific primers. The patient-specific primers were generated after the sequencing of amplified fragments. The second round of PCR was performed using patient-specific oligonucleotides, individual for each patient. Thus, minimal residual disease was evaluated using nested-PCR on DNA of bone marrows samples. The PCR products were analyzed by electrophoresis in 6% polyacrylamide gel. The sensitivity of the method was calculated as 10^-4 (1 tumor cell on 10 000 normal).

Flow cytometry monitoring is based on the detection of a leukemia-associated phenotype (LAP) defined as aberrant expression of the surface or intracellular antigens on leukemia cells at the time of diagnosis. BM samples of 15 pts were investigated at diagnosis using three-color flow cytometry analysis, a panel of 20 monoclonal antibodies was used for detection of the patient-specific LAPs. 10⁶ MNCs (resuspended in PBS/BSA/SA) per tube were incubated at 4⁰C for 30 minutes with monoclonal antibodies against CD. The cells were rinsed and suspended in CellFix medium. List mode date was acquired on a FACS Calibur flow cytometer (Becton Dickinson) using the CELL QUEST software. MRD was measured at 7-8 mo of CR basing on aberrant LAPs at diagnosis. A threshold level for the presence of MRD was defined as 0,1% positive cells, counted for 70000-100000 cells in a sample.

RESULTS

Specific molecular markers were identified in 88% of the patients (30/34). In 67% of patients (20/30) clonal IgH gene rearrangements were discovered. All these patients were with B-lineage ALL. At the same time  in 83% of patients (25/30) TCRG gene rearrangements were detected. 19 of them have B-lineage ALL and 6 - T-ALL. In most of the patients (22/30) two or more specific markers were revealed. Prolonged monitoring was carried out in 22 patients with a follow-up from 2 to 24 months. 15 of those patients were analyzed for patient-specific leukemia-associated phenotype by flow cytometry at diagnosis and before maintenance. Contrary to PCR analysis, LAPs were detected in all investigated patients. The most common LAPs were CD19/34/10, CD19/34/TdT and CD19/34/IgM.

Clinical CR was achieved in 85% of patients. Prednisone resistance after prephase was determined in 71% of patients.

MRD was detected by PCR in all patients after pre-phase, in 21/22 (95%) after 1^st induction phase, in 11/15 (73%) after 2^nd induction phase, in 10/15 (67%) after consolidation, and in 8/14 (57%) before maintenance.

All patients (n=3) whose PCR probes were positive more than twice, relapsed at different time points. No relapses were noted in once positive patients (n=3). And 1 of  8 PCR-negative patients relapsed.

Flow cytometry data coincides with molecular parameters: 6 of 10 patients examined before maintenance (+7-8 mo) had cells with LAP.

Comparing our parallel measurements we have noted discrepancies in 3 of 10 cases. Two immunologically positive patients were PCR-negative, and one PCR-positive patient was immunologically negative.

CONCLUSION

Our data indicate that both methods are effective and relevant in detection of MRD. They show very slow tumor clearance in ALL patients. MRD positivity reflects the higher relapse probability. More patients can be followed for MRD with flow-cytometry, due to higher detection rate of LAP.

National Research Center for Hematology, Moscow, Novozykovski pr., 4a, 125167      E-mail: elenap@blood.ru