Assessment of Minimal Residual Disease (MRD) In Relapsed CLL Patients Treated with Fludarabine and Cyclophosphamide with or without Rituximab (REACH)

Levels of minimal residual disease (MRD) have been shown to correlate with PFS in previously untreated patients with CLL (CLL8, Boettcher et al. Leukemia, 2009). Patients who remain MRD positive after treatment have a higher risk of relapse. Eradication of MRD is therefore a desirable clinical endpoint of treatment. We were interested to assess this correlation in REACH, a randomized international clinical study in previously treated CLL patients, randomized 1:1 for treatment with rituximab, fludarabine and cyclophosphamide© R-FC (276 patients) or FC alone (276 patients); (Robak et al. JCO 2010).

While MRD quantification by flow cytometry requires an identifiable stable phenotype and fresh blood samples, PCR based methods can be performed centrally on frozen samples. We have therefore developed a Realtime Quantitative (RQ) PCR method, using patient-specific IgVH (immunoglobulin variable heavy chain) gene rearrangements as targets. Briefly, genomic DNA was isolated from CD19 sorted B-cells. ASO (allele-specific oligonucleotide) primers were designed matching the hypervariable N-D-N region of the patient-specific leukemic clone and used with reverse consensus primers and hydrolysis probes annealing to the family-specific joining region of the IGH rearrangement (Brüggemann et al., Leukemia, 2004). Maximum sensitivity and quantitative range were defined for every RQ-PCR. Patients were categorized as molecular responders (MRD negative) if there was no detectable clonal IgH rearrangement, using a sensitivity cut-off of 1×10^-4. Molecular response was assessed at the time of CR confirmation and 6 months later (if CR was maintained).

Among the 103 patients who achieved CR during the study, 86 patients had at least one MRD assessment in peripheral blood, 92 patients in bone marrow. Since many patients had a CR confirmation at different time points during the follow-up period, we initially analyzed the MRD levels only in patients who had achieved confirmed complete response at end of treatment +/−3 month (“EOT - period”).

The rate of MRD negativity in blood (22 pts: 5(15) FC, 6(7)R-FC) at EOT was 33% for patients treated with FC, and 86% for patients treated with R-FC (p=0.06); In bone marrow at the EOT (61 patients: 5(27) FC, 20(34) R-FC) the rates were 19% and 59%, respectively (p= 0,02), indicating higher efficacy of the Rituximab containing regimen in eradication of residual disease; This is in line with the previously reported results using FACS analysis of MRD in the CLL8 trial; the differences in the detection rate in blood versus bone-marrow, suggest a higher sensitivity for detection of MRD in bone marrow.

We therefore compared the levels of MRD negativity in samples from blood and bone marrow in patients where both samples were taken at the same time point. Results were concordant in 8/9 patients, one patient had a positive result in bone marrow with no detectable signal in blood. This supports the notion that assessment of MRD in bone marrow of CLL patients may be more sensitive than assessment in blood only. However, for a definitive statement larger sample size would be needed.

We then correlated MRD status at EOT, regardless of treatment arm, with PFS: In line with previous reports, there was a clear trend to longer PFS in patients who had reached MRD negativity (median PFS not reached), while patients with residual disease had shorter PFS; however, due to small sample numbers, statistical significance could not be reached. We also analyzed the correlation of MRD negativity reached at any time during and after treatment with PFS, bearing in mind that this sample set is inherently biased, since patients with early progression will be lost from the analysis; the results are consistent with the EOT findings.

ASO IgVH RQ-PCR is a powerful method to detect residual levels of disease in CLL patients with clinical complete response and undetectable MRD correlates with longer PFS. Among patients in REACH achieving clinical CR on either study arm, a higher percentage achieved MRD negativity on the R-FC arm, consistent with the increased efficacy shown for the Rituximab treatment arm by the REACH clinical data.

Mundt:Roche: Employment. Smith:Roche: Employment. Lin:Genentech: Employment. Barrett:Roche: Employment. Hurst:Genentech: Employment. Geisler:Roche: Research Funding, Speakers Bureau. Hiddemann:Roche: Research Funding.