Validation and Implementation of Flow Cytometry Based Minimal Residual Disease (MRD) Assay for Chronic Lymphocytic Leukemia (CLL) Clinical Studies
Authors: Gonneau Christele , Brugarolas Marc , Van Haecht Lotje , Lenjou Marc , Fritsche Caitlyn , Linnik Yevgeniy , Rifkin Lucas , Yu Ting , Rule Simon , Munugalavadla Veerendra , Peretz Yoav 
Published: December 2024

Background: With the advances made in novel anti-cancer therapies, Chronic Lymphocytic Leukemia (CLL) patient remission rates have significantly improved over the years. Minimal or measurable residual disease (MRD) is emerging as an independent predictor of progression-free survival (PFS) and overall survival (OS) in several heme indications and has been proposed as a potential surrogate endpoint for long term CLL survival in clinical trials. While next generation sequencing (NGS) methods are emerging for establishing MRD measurements in CLL, flow cytometry remains as an option of choice for measuring MRD in CLL.
Methods: In order to assess MRD in CLL patients using flow cytometry, Labcorp Biopharma Services validated a CLL MRD assay that was initially developed by the European Research Initiative on CLL (ERIC) (Rawstron et al, 2013). Assay validation was performed in several Labcorp central laboratories across the globe using bone marrow and whole blood samples from both healthy donors and CLL patients, characterizing precision performance, sample stability and B CLL cells assay sensitivity. This validated assay has been utilized in several clinical studies including ELEVATE- TN (NCT02475681; Sharman JP, 2020) as part of exploratory endpoints with 10-4 cut-off.
Results: Validation of this assay demonstrated good performance for critical reportable measurement CLL B cells with a lower limit of quantitation (LLOQ) of 0.009%/of total nucleated cells (TNC) and 0.008% of TNC for whole blood and bone marrow respectively and when target total events is set at 2x106 cells in total. Using a similar setup, we also demonstrated an ambient specimen stability for CLL B cells of 72 hours for whole blood samples and 48 hours for bone marrow samples. Precision performance for repeatability and reproducibility for both matrices was below ≤10% CV, confirming high readout precision and suitability of the assay for implementation in multi-centric clinical trials. As part of the exploratory endpoints from the ELEVATE- TN study, peripheral blood samples were collected from patients in complete remission (CR) or complete remission with incomplete count recovery (CRi). Patients with CR or CRi in the acalabrutinib-obinutuzumab arm achieved higher rates of undetectable MRD (uMRD) in peripheral blood samples vs patients with CR or CRi receiving acalabrutinib monotherapy and chlorambucil-obinutuzumab (40.9% vs 8.8% and 8.3%, respectively).
Conclusions: Validated six- color flow cytometry CLL- MRD assay is a robust test that has been successfully implemented (exploratory) and proven useful for global clinical studies, as demonstrated by clinical results obtained in ELEVATE-TN study. This flow cytometry assay is being implemented in several global clinical studies as an endpoint (secondary) including in an ongoing phase 3 study AMPLIFY (ACE-CL-311; NCT03836261).