Interest of Early Determination of Bone Marrow MRD by a Sensitive 6-Color Flow Cytometry in the Evaluation of Response: The Experience of the CLL2007FMP, An Intergroup Phase III Randomized Multicentric Trial Comparing Fludarabine Cyclophosphamide (FC) and Rituximab (FCR) Versus FC and MabCampath (FCCam) in Previously Untreated B-Chronic Lymphocytic Leukemia Patients

The FCGCLL/MW and GOELAMS intergroup have conducted a multicenter phase III trial, CLL2007FMP, to evaluate the efficacy of FCCam versus FCR in previously untreated medically fit CLL patients. This trial was discontinued after randomisation of 165 patients for unacceptable toxicity in the FCCam arm. A sensitive 6-color flow cytometry technique was used to evaluate the response at month 9 (M9) in blood and bone marrow and to subsequently monitor minimal residual disease (MRD).

Methods and patients: 178 medically fit patients (CIRS< 6 and creatinin clearance ≥ 60 ml/min), younger than 65 years old, were enrolled. 165 patients were subsequently randomized to receive six oral courses of FC (F: 40mg/m2 d1-3 and C: 250 mg/m2 d1-3; q 28 days) in combination with either R (n=83; 375 mg/m2 i.v. d 0 at first cycle and 500 mg/m2 d1 all subsequent cycles; q 28 days) or Cam (n=82; 30 mg s/c d1-3; q 28 days). Patients were stratified according to IGHV mutational status and presence of 11q deletion. Cases with 17p deletion were excluded. Clinical response was evaluated based on iwCLL classical criteria. We established a sensitive and specific approach for the evaluation of MRD, using a 6-color flow cytometry technique with 3 combinations, including characteristic markers and light chain expression. We determined the limit of detection (LOD) of the assay by studying normal blood samples, and LOD varied from 0.5 to 0.7×10⁻⁵, depending on the combination considered.

The overall response rate was 87.8% in the FCR arm compared to 85.5% in the FCCam arm (p: NS). CR rates were not significantly different between the two arms with 64.6% in FCR arm and 50.6% in FCCam arm (p=.08).

At M9, the rate of undetectable MRD was significantly lower in bone marrow as compared to blood but neither in blood nor bone marrow did reach a significant difference between FCR and FCCam arms. MRD was undetectable in blood in 60.2% of patients (53% of patients in the FCCAm arm and 66.7% in the FCR arm (NS)). In bone marrow samples, MRD was undetectable in 33.7% of patients (25% of patients in the FCCam arm and 41.7% in the FCR arm (NS)). The results were similar when considering exclusively the patients who reached CR. Moreover, MRD was never found detectable in blood when undetectable in bone marrow at the same time point. Therefore, irrespective of treatment arm, the bone marrow sample appeared more efficient than the blood sample for the detection of a persistent residual disease. We next combined blood and bone marrow results to define immunophenotypic remission, we considered MRD at M9 as undetectable when undetectable in bone marrow and MRD as positive when detectable either in blood or bone marrow. The rate of immunophenotypic remission was significantly lower in the FCCam arm as compared to the FCR arm (p=0.03) with our sensitive technique but, interestingly, was no longer significant when using the classical threshold of 10⁻⁴ to assess MRD status. The number of best responders, identified by MRD negativity and clinical complete response was significantly higher with FCR than with FCCam (p= 0.029).

MRD was again assessed in blood at month 12 and was undetectable in 36.8% of patients in the FCCAm and 41.7% in the FCR arm (NS). All cases with positive blood MRD at M9 remained positive at M12. Among the 21 cases with positive bone marrow MRD and undetectable blood MRD at M9, only 10 explorations in blood at M12 are available at the moment but 4 cases remained undetectable. Conversely, when MRD was undetectable at M9 in both blood and bone marrow, 16/18 remained undetectable at M12, corresponding to a positive switch rate of only 11%.

In the era of combined immuno-chemotherapy with the goal of achieving the best response, the most sensitive MRD technique is warranted. The sensitivity of 6-color flow cytometry has proved useful to uncover the differences between the two treatment arms. In this study, the evaluation of MRD in bone marrow by 6-color flow cytometry is the most sensitive technique for the early evaluation of persisting residual disease. It may not be supplanted by MRD determination in blood at M12 for the precise assessment of the immunophenotypic remission rate. Studies including sequential determinations and MRD kinetics are ongoing to determine which interpretation threshold and time-points are the best predictors of progression-free survival.

Leblond:Roche, Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cymbalista:Roche (d) Mundipharma (e) Genzyme (e): Honoraria, Research Funding.