Next Generation Flow (NGF): A High Sensitive Technique to Detect Circulating Peripheral Blood (PB) Clonal Plasma Cells (cPC) in Patients with Newly Diagnosed of Plasma Cell Neoplasms (PCN)

Introduction: Previous studies have shown that cPC can be detected in PB by conventional flow cytometry (FC) in around 70% of multiple myeloma (MM) and 37% of monoclonal gammopathy of undetermined significance (MGUS) patients at diagnosis. Its presence in MGUS has been associated with a higher risk of malignant transformation. We here investigated the utility and sensitivity of the EuroFlow-IMF NGF-MM minimal residual disease (MRD) approach for detecting circulating cPC in PB of patients with PCN.

Methods: A total of 137 samples (including 71 PB and 66 bone marrow -BM- paired samples) from 71 newly-diagnosed PCN patients (37 MGUS; 21 MM; 5 SMM and 8 solitary plasmacytomas -SP-), plus 6 PB samples from healthy controls, were studied. Samples were processed following the EuroFlow Bulk Lysis Standard Operating Protocol (SOP) and stained with the EuroFlow-IMF MM MRD panel (Tube 1:CD138_BV421/CD27_BV510/CD38_FITC/CD56_PE/CD45_PerCP-Cy5.5/CD19_PE-Cy7/CD117_APC/CD81_APC-C750, and; Tube 2: identical to Tube 1 except for CyKappa_APC/CyLambda_APC-C750). A median of 10.6 x10⁶ events (range: 1.7 x10⁶- 15.7x10⁶) were measured for PB samples using a FACSCanto II (BD Biosciences, San Jose, USA) instrument. Data were analyzed using the Infinicyt software (version 1.8.0RC6; Cytognos SL, Salamanca, Spain). Risk stratification of MGUS patients was established by the Mayo Clinic index. ROC analysis was used to define a cut-off to distinguish between MM and MGUS cases according to the percentage and absolute number of circulating PB cPC.

Results: Overall, cPC were detected in the PB of all MM and SMM cases studied (100%) and more than half of MGUS patients (60%; p=0.005), while constantly absent in the eight patients with SP. Upon classifying MGUS patients according to the Mayo Clinic Index (n=32), positive PB samples were found in 25%, 62% and 73% of cases with scores of 0, 1 and 2, respectively. Median (range) percentage and absolute cPC numbers (per µL) were of 13 to 16 and 10 to 200 times lower (p<0.0001) in MGUS -0.0002% (<0.0001%-0.05450%) and 0.011 cPC/µL (range: <0.0001 cPC/µL -3.2 cPC/µL)- than in SMM -0.0026% (0.00020%-0.23%) and 0.14 cPC/µL (range: 0.022 cPC/µL - 14.58 cPC/µL) and MM -0.0033% (0.00064%-1.05%) and 2.01 cPC/µL (range: 0.043 cPC/µL -103.8 cPC/µL)-, respectively. Interestingly, a clear relationship was found between the presence of circulating cPC in PB of both MGUS, SMM and MM cases, and BM involvement by >60% of cPCs within the PC BM compartment (R²= 0.75; n=66). The cut-off obtained to distinguish between MM and MGUS cases according to the percentage and absolute number of cPCs circulating in PB was of 0.0009% and 0.055 cPC/µL with a sensitivity of 93% and 86%, and a specificity of 75% and 75% for relative and absolute numbers, respectively.

Conclusions: The EuroFlow-IMF NGF-MM MRD panel and approach are well-suited for high sensitive detection of circulating cPC in the PB of virtually every newly-diagnosed MM and SMM patient and the majority of MGUS cases, particularly among MGUS at higher risk of malignant progression; interestingly in both patients groups, the presence of PB involvement and its levels were closely associated with the degree of involvement of the BM PC compartment by cPC.

* Both authors have contributed similarly to this work and they should both be considered as first author.