Minimal Residual Disease (MRD) Analysis Using Multiparametric Flow Cytometry (MFC) and Identification of Differences in Subpopulations Using Next Generation Sequencing (NGS) in the Bone Marrow (BM) of Multiple Myeloma (MM) Patients (pts)

Introduction: MM is characterized by the accumulation of aberrant BM plasma cells (aPCs). The use of novel agents for anti-MM treatment has enabled the achievement of deeper responses and prolonged progression free survival (PFS) and overall survival (OS). These advances have created the need for sensitive means to detect residual PCs. MFC allows aPC detection with high sensitivity and is used with NGS in clinical trials (CTs) (Paiva, 2015-2018). It allows the detection of residual cells after anti-MM therapies and is a risk denominator of pt subgroups and post-therapeutic outcome. The incorporation of cost-effective, readily available and standardized MRD tools into CTs and clinical practice seems a key requisite. It may allow the improvement of clinical decisions: 1) timing of stem cell transplantation (SCT), 2) need for consolidation, 3) duration of maintenance, 4) therapy modulations and 5) early relapse detection. Since MRD gains importance for novel agents' potency to achieve MRD negativity, including FDA/EMA-approvals, we assessed MFC for detection of aPCs, their change to treatment and over time.

Methods: We systematically validated a 6-, 8- and 10-color panel using CD38, CD138, CD19, CD45, CD27, CD56, CD28, CD81, CD117, CD200 and kappa/lambda (k/l) in 9 different MM cell lines (MMCLs) and 213 BM, PB, leukapheresis (LA), extramedullary (EM) samples of 122 different MM pts and 14 healthy individuals (HI). These were immediately analyzed after sampling, using a bulk-lysis protocol. To reach a high sensitivity, >3x10⁶ viable nucleated cells were analyzed. MFC validation included fluorescence-minus-one-(FMO), isotype- and spike-in-controls to evaluate the reliability and sensitivity of our panels. Spike-in controls were performed using a dilution assay to recover defined MMCL cells in the PB of HI. Data analysis was accomplished using the BeckmanCoulter software Kaluza®. We compared MFC using BM and PB samples of HI, MM pts at initial diagnosis (ID) or with progression (PD), after standard therapy (ST) and SCT. Additionally, BM samples at ID were sorted into each 4 aPC- and normal PC- (nPC) subpopulations for DNA and RNA sequencing and follow-up. The study was performed with written consent of all pts and HIs and approval of the ethics committee.

Results: The antigen expression levels in 9 MMCLs using our 6-, 8- and 10-color panel could be confirmed as reported. The expression levels of antigens included in all panels were similar in all MMCLs (RPMI 8226; U266; IM-9; MM1.S; MM1.R; L363; Karpas620; NCI-H929; OPM-2). For all panels, we established easy to adapt gating strategies, using commercially available software to identify aPCs vs. nPCs in all analyzed samples. Using the 6-color panel, differentiation into up to 4 phenotypic subpopulations of aPCs and nPCs in MM pts and up to 4 subpopulations of nPCs in HI was achieved, depending on the heterogeneous phenotype the individual was expressing. Moreover, both DNA and RNA of obtained subpopulations were collected and analyzed via sequencing. Since in 1% of our samples, aPCs remained undetected via 6-color panel using surface antigens exclusively, we added k/l and did detect aPCs in all pts. The 10-color panel identified aPC- and nPC-subpopulations in even more detail. We determined a MRD sensitivity of 10^-5, confirmed via independent dilution assays. In 48% of BM and PB samples, aPCs were determined at ID and PD; 52% were assessed for MRD after ST and SCT. Of the latter, 55% were BM samples, of which 5% showed MRD negativity of <0.001%. The median time from ST- or SCT-treatment to 1^rst MRD evaluation was 44 days; this being assessed at subsequent time points currently. These data will be shown at the meeting.

Conclusion: Our 6-, 8-, and 10-color panels allow highly sensitive detection of aPCs vs. nPCs in MM samples. These panels have been validated using various controls, dilution assays, 9 MMCLs, MM pt samples and HI and can be implemented into routine diagnostics. The analysis of DNA and RNA expression patterns in distinct subpopulations will generate relevant insight, which subclones contribute to response vs. resistance mechanisms. The EuroFlow Consortium have established a two 8-color tubes test for identification of aPCs, which - albeit used worldwide - may not be applicable for every center and every pt outside CTs. Therefore, meticulously validated alternatives as shown here are of interest. Our 8-color panel has been included into our CT portfolio.