Multilayer profiling of MRD in patients with relapsed/refractory CLL treated with venetoclax-based regimens in a real-world setting

Abstract Background Undetectable minimal residual disease (uMRD) is a strong prognostic factor for progression-free survival (PFS) in venetoclax-treated relapsed/refractory chronic lymphocytic leukemia R/R CLL. However, the uMRD rate has not been systematically evaluated in real-world R/R CLL cohorts. Venetoclax exerts strong selective pressure on the MRD clone, but the extent and nature of clonal evolution under venetoclax remain poorly characterized. In particular, it is unclear whether BCL2 mutations emerge during MRD persistence in remission. Methods We analyzed peripheral blood (PB) from Swiss patients enrolled in the VeRVe study (NCT03342144), a prospective, non-interventional study evaluating effectiveness, safety, and quality of life in real-world R/R CLL patients treated with venetoclax monotherapy (Ven) or venetoclax plus rituximab (VenR) according to local label. The primary objectives of this study are to estimate the uMRD rate under ven-based therapy in a real-world setting, compare the performance of different assays for MRD assessment, and track clonal evolution and the development of drug resistance during ven-based treatment. MRD was assessed at baseline, 12 months, and 24 months using: (i) 8-color multiparametric flow cytometry (MFC) following ERIC guidelines (sensitivity 10⁻⁴); (ii) high-throughput sequencing of immunoglobulin genes (Ig-HTS, clonoSEQ®; sensitivity 10⁻⁶); and (iii) a CAPP-seq–based circulating tumor DNA (ctDNA) assay (LyV4.0; sensitivity 10⁻³). To overcome limitations due to low MRD abundance, we used ctDNA to profile clonal evolution and screen for BCL2 mutations. Tumor genomic DNA (gDNA) was obtained from pre-treatment sorted CLL cells, while germline DNA was derived from PB T-lymphocytes collected at the time of best MRD response. Results Twenty-eight patients were recruited; 19 (Ven=4, VenR=15) were evaluable at the 24-month landmark [19 by MFC, 18 by CAPP-seq (one sample ongoing), and 18 by Ig-HTS (one baseline not available)]. At 24 months, 12 patients (63.1%) were in complete remission (CR), five (26.3%) in partial response (PR), one (5.3%) had progressed, and one had (5.3%) died. uMRD rates at 24 months were 58% by MFC, 67% by CAPP-seq, and 28% by Ig-HTS. MRD concordance among the methods on the evaluable patients was: MFC vs. CAPP-seq: 83% (κ=0.64); MFC vs. Ig-HTS: 72% (κ=0.47). All patients with detectable MRD by MFC and/or CAPP-seq were also positive by Ig-HTS. Baseline mutations detected by CAPP-seq in >15% of patients included TP53, IGLL5, BRAF, ATM, SF3B1, IRF2BP2, NSD, NXF1, and ZMYM3. Mutation concordance between gDNA and ctDNA at baseline was 84%. No BCL2 resistance mutations were observed pre-treatment in either compartment. Longitudinal ctDNA profiling (up to six timepoints over two years) showed no emergent BCL2 mutations in the 18 patients with detectable ctDNA at 24 months. Conclusions In this real-world prospective cohort, venetoclax-based treatment achieved uMRD rates comparable to the MURANO trial. Ig-HTS demonstrated the highest sensitivity for MRD detection, outperforming both flow cytometry and CAPP-seq. Importantly, no BCL2 mutations emerged during two years of therapy, suggesting that time-limited venetoclax regimens are associated with a low risk of resistance development. Disclosures DR has received honoraria or research grants from AbbVie, AstraZeneca, Gilead, BeOne, BMS, Janssen, Lilly. IR has received honoraria or travel grants from BeOne, AC has received honoraria or research grants from AbbVie, AstraZeneca, BeOne, BMS, Janssen and Lilly. GS has received honoraria or research grants from AbbVie, Amgen, BeOne, Celgene/BMS, Gilead, Novartis, Roche. IS has received honoraria from AbbVie, Amgen, AstraZeneca, BeOne, Janssen, Roche, Servier. TN has received honoraria from AbbVie, AstraZeneca, BeOne, Gilead, Janssen, Lilly, Roche. AB, SB, KP, GF, MP, MG and HS have no conflicts of interest to declare. MO, XS and CL are employees of AbbVie and may own AbbVie stock. Honorarium paid by AbbVie was not related to authorship of this abstract. No honoraria or payments were made for authorship. AbbVie sponsored this study and contributed to the design, study conduct. AbbVie participated in the interpretation of data, review, and approval of the publication. All authors had access to all relevant data.