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Rituximab maintenance therapy in CLL
first-line treatment for CLL, and to investigate the impact of rituximab maintenance therapy on the response rate and PFS following FCR. A key secondary objective was to analyze MRD status after chemoimmunotherapy and rit- uximab maintenance.
Methods
Physically fit patients between 18 and 70 years old with active CD20+ CLL according to the World Health Organization classifi- cation, with an Eastern Cooperative Oncology Group Performance Status ≤2, were recruited into the REM (rituximab in maintenance) trial and received treatment with fludarabine (25 mg/m2 iv on days 1-3), cyclophosphamide (250 mg/m2 iv on days 1-3) and rituximab (375 mg/m2 iv cycle 1 and 500 mg/m2 iv cycles 2-6) every 28 days, for up to six cycles. Major exclusion criteria were prior treatment for CLL, severe cardiac, pulmonary, neuro- logical, psychiatric, or metabolic disease, continuous systemic cor- ticosteroids, active autoimmune hemolytic anemia or thrombocy- topenia, active severe infection, creatinine clearance <50 mL/min, or transformation to an aggressive B-cell malignancy. All cases were CD20+ as analyzed by flow cytometry, with a mean fluores- cence intensity lower than the expression found in normal mature B lymphocytes in peripheral blood and bone marrow (BM).
At the 3-month post-induction clinical response evaluation, patients achieving a CR, partial response (PR) or nodular PR (nPR), based on International Workshop on CLL guidelines, were treated with rituximab 375 mg/m2 iv every 2 months for 3 years (18 cycles). Anti-microbial prophylaxis included trimethoprim-sul- famethoxazole and acyclovir during treatment and until the level of CD4+ lymphocytes reached 0.3x109/L. Patients achieving a CR and undetectable MRD in both peripheral blood and BM after four courses of FCR were allowed to stop fludarabine plus cyclophos- phamide and complete two courses of rituximab and continue with rituximab maintenance therapy.
The primary endpoint was the CR rate after FCR treatment. Secondary endpoints included PFS, OS, correlation of response with the level of MRD after FCR and rituximab maintenance ther- apy, adverse events, and the prognostic impact of the biological markers CD38 and ZAP70, IGHV mutational status, cytogenetic abnormalities and BM-MRD on the course of the disease. Fluorescence in situ hybridization and IGHV analysis were per- formed locally in accredited laboratories using standardized proce- dures.
The study protocol was approved by the institutional review board of each participating institution and complied with the Declaration of Helsinki, and existing Good Clinical Practice guide- lines, laws and regulations. All participants provided written informed consent before enrollment.
Flow cytometry and measurable residual disease analysis
Samples were stained and lysed using a direct immunofluores- cence technique as previously described.13 The following antibody combinations were used: (i) CD22/CD23/CD19/CD5; (ii) FMC7/CD43/CD19/CD5; (iii) CD103/CD25/CD19/CD5; (iv) CD10/CD11c/CD19/CD5; (v) CD20/CD38/CD19/CD5; (vi) CD81/CD22/CD19/CD5; (vii) CD20/CD49d/CD19/CD5; (viii) sIgk/sIgl/CD19/CD5, and (ix) ZAP70/CD3+CD56/CD19/CD5. All monoclonal antibodies except ZAP70 were provided by Becton Dickinson (San José, CA, USA). ZAP70 was purchased from Immunotech (Marseille, France). Samples were acquired in a FACSCalibur flow cytometer (Becton Dickinson) and analyzed using Paint-A-Gate PRO software (Becton Dickinson). At least 20,000 events were acquired. B-lymphocytes were identified
according to their SSC/CD19+ distribution and the total percent- age of pathological CD38 and CD49d B cells was reported. ZAP70 was quantified using a cut-off of ≥20% to define the ZAP70+ sub- set of B cells.14
MRD was analyzed in samples from peripheral blood and BM after induction and from BM during rituximab maintenance ther- apy, with a combination of monoclonal antibodies slightly modi- fied from that in the European Research Initiative on CLL (ERIC) protocol: (i) CD20/CD38/CD19/CD5; (ii) CD81/CD22/CD19/CD5; (iii) sIgL/sIgK/CD19/CD5; and (iv) CD22/CD79b/CD19/CD5. CD43 was not included in the analy- sis: we included the last combination instead of CD43/CD79b/CD19/CD5 based on our previous experience with that combination in the analysis of MRD in CLL.13 The minimum number of pathological B cells acquired was that in the ERIC rec- ommendations.15 To achieve a limit of detection of 0.01%, at least 200,000 events were acquired if the minimum population size was 20 and 500,000 events if the minimum population size was 50. We prepared the necessary number of tubes for each combination to acquire at least 200,000 events. The complete gating strategy is described in the Online Supplement.
Statistical analysis
This was a two-staged, Simon optimal phase II clinical trial. Based on a CR rate observed in previous trials of first-line therapy ranging around 30%, the inactivity cut-off was chosen to equal 30% and the activity cut-off at least 50%. Hence, the hypotheses of interest were H0: r≤0.3 against Ha: r≥0.5%, where r is the CR rate. Using a type I error rate (α, probability of accepting an insuf- ficiently active treatment, a false positive outcome) set at 0.05, and a type II error rate (β, probability of rejecting an active treatment, a false negative outcome) set at 0.20, we estimated that 90 patients should be enrolled into this trial, assuming a 10% loss.
A descriptive analysis of continuous and qualitative variables was performed. PFS, OS and duration of response were summa- rized descriptively and graphically using the Kaplan and Meier method in the overall population and separately by biological fac- tors, genetic profiles and MRD status. The log-rank test was used for comparisons of PFS and OS curves. The χ2 test was used to assess the frequencies and differences of biological and cytogenet- ic abnormalities. The relationship between these abnormalities and MRD level was analyzed using logistic regression models. Safety data were summarized for all treated patients during induc- tion, maintenance and combined. All hypothesis tests were two- sided and a P-value <0.05 was considered statistically significant. All statistical computations were carried out with SPSS version 14.0 or subsequent versions.
Results
We present the results of an end-of-study analysis at 3 years of follow-up after 36 months of rituximab mainte- nance therapy following FCR induction. Between October 2007 and December 2012, 90 patients were assessed for eligibility in 29 center across Spain, and 84 were assigned to FCR (6 patients did not meet the eligibility criteria, of whom 2 after 1 treatment infusion) and were evaluable for response in an intent-to-treat analysis (Figure 1). Overall, 79.8% (n=67) of the enrolled patients were aged 64 years or younger, 67.9% (n=57) were male and 83.3% (n=70) had Binet stage B or C disease. The median age of trial par- ticipants was 59.5 years (range, 37-70), and 70.5% (n=55) of participants were in a good state of health with an Eastern Cooperative Oncology Group Performance Status
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