A Novel 10-Fluorochrome Multiparameter Flow Cytometry (MFC) Panel for Precise Plasma Cell (PC) and MRD Assessment in Clinical Routine of Multiple Myeloma (MM) Patients and throughout the Disease Course

Introduction: Since the introduction of novel agents into the treatment of Multiple Myeloma (MM), much deeper remissions can be achieved. This leads to a demand for disease evaluation tools with higher sensitivity and clinical practicability. In various clinical trials the assessment of the MRD status has been proven to have strongest prognostic value for MM patients (pts). The Euroflow Consortium has pioneered a highly standardized next generation flow (NGF) approach with the establishment of two 8-color tubes tests (Flores-Montero 2017). However, due to cost-intensive and time-consuming preconditions, this MRD approach might not be feasible in every center. Thus, other readily available, validated MRD tools may also guarantee highly sensitive MRD evaluation which allows for early relapse detection and to steer treatment, such as decision upon need for consolidation, duration of maintenance, therapy pauses or even earlier FDA/EMA approval of novel antimyeloma agents. Methods: Our 10 color single tube MFC panel consisting of the antigens CD38, CD138, CD19, CD45, CD27, CD56, CD28, CD81, CD117 and CD200, was systematically validated using Fluorescence-minus-one controls, consecutive spike-in-controls, consistency analyses and measurement of 9 MM cell lines. Thereafter, we assessed 131 Bone Marrow (BM), peripheral blood (PB), and leukapheresis (LA) samples from 97 different MM, MGUS and SMM pts and 13 healthy individuals (HI) as controls. Samples were processed within 6-20 hours of aspiration and a bulk lysis was performed. Subsequently, >3x106 viable nucleated cells were acquired on a BD LRSFortessa Cytometer. Data analysis was performed using the BeckmanCoulter software Kaluza. BM was sampled at initial diagnosis (ID) or at progression (PD), after standard therapy (ST) or stem cell transplantation (SCT). The study was performed with written consent from all pts and approval from the ethics committee. Results: Our MFC approach confirmed the antigen expression in all MM cell lines (RPMI 8226; U266; IM-9; MM1.S; MM1.R; L363; Karpas620; NCI-H929; OPM-2) as previously reported. The spike-in-controls determined the limit of detection (LOD) at 10-5. This LOD was achieved in 89% of our MRD samples. An easy-to-adapt gating strategy to identify abnormal plasma cells (aPC) vs. normal plasma cells (nPC) was also established in our MM pt samples. In this cohort, we identified 10 distinct aPC and nPC subpopulations that differed in their expression pattern, suggesting clonal subtypes of MM. Moreover, in 19% of BM samples, two malignant subpopulations coexisting in the same pt, were identified. Since our BM cohort consisted of 46% samples at ID or PD vs. 54% posttreatment samples after ST and SCT, clonality variation in the former, differences of aPCs vs. nPCs ratio and to the latter group could be determined. Moreover, significant differences of much higher percentage of aPC in symptomatic MM vs SMM/MGUS pts were readily verified with our panel (p=0.0087). With a median time of 51 days from treatment to MRD evaluation, 24% of post treatment samples were MRD negative (MRD-) at the 10-5 level ( Since we also assessed pre- and post-treatment samples, antibody expression levels on PC from ID to post treatment demonstrated a significant normalization in Median Fluorescence Intensity (MFI) of 5 antibodies (CD56, CD81, CD45, CD200, CD27). These may thus serve as informative markers for the MRD assessment of these samples. Conversely, with progression from remission to PD, CD81 and CD45 antibodies showed significant decreases (p=0.0007 and p=0.0026, respectively) in expression, which suggest that both may serve as early relapse markers. Of the assessed LA samples at harvest before ASCT, 44% were MRD-, warranting future investigations into possible implications for pts' prognosis, treatment and PFS/OS data. Conclusion: Here we present a readily available, cost-effective, quick and highly validated MFC panel with an easy to adapt gating strategy that allows for precise aPC vs. nPC assessment in- and outside clinical trials. Our 10 color MFC panel is applicable in BM samples of MM pts and precursor diseases, as well as in LA samples. With the additional MFC information, ideally at ID and repeatedly after treatment, readily available individual treatment decisions seem possible to obtain for every MM patient. Disclosures Wäsch: Amgen: Other: travel, Research Funding; Sanofi: Other: Travel, Research Funding; Jazz: Other: travel, Research Funding; Celgene: Other: travel, Research Funding; Gilead: Other: travel, Research Funding; Takeda: Consultancy; Gilead: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy.