Next Generation Flow Cytometry Provides a Standardized, Highly Sensitive and Informative Method for the Analysis of Circulating Plasma Cells in Newly Diagnosed Multiple Myeloma: A Single Center Study in 182 Patients
Authors: Terpos Evangelos , Kostopoulos Ioannis V. , Papanota Aristea-Maria , Papadimitriou Konstantinos , Malandrakis Panagiotis , Micheli Paraskevi , Ntanasis-Stathopoulos Ioannis , Fotiou Despina , Metousis Andreas , Kanellias Nikolaos , Migkou Magdalini , Eleutherakis-Papaiakovou Evangelos , Gavriatopoulou Maria , Kastritis Eftathios , Tsitsilonis Ourania E. , Dimopoulos Meletios A. 
Published: November 2019

Introduction: The apparent heterogeneity of multiple myeloma (MM) constitutes a key challenge in the clinical management and the design of effective therapeutic interventions, while it entails the identification of biomarkers with a strong prognostic value. In this context and taking into account patients' inconvenience to invasive bone marrow (BM) aspiration, the assessment of circulating plasma cells (CPCs) in liquid biopsies, at the time of diagnosis, has been proposed as a useful assay with prognostic value. Different methodologies have been applied for the detection of CPCs; the most common is the use of multicolor flow cytometry (MFC), mainly of 2-, 4-, or 6-color combination panels, which however yielded heterogeneous results due to variations in the detection efficacy of each approach. In the present study, we applied the standardized and highly sensitive Next Generation Flow Cytometry (NGF) approach, to detect CPCs in diagnostic MM peripheral blood (PB) samples, we compared their phenotypic characteristics with the aberrant clonal cells of BM matched samples and we correlated their presence with disease characteristics.
Patients and Methods: PB and BM matched samples from 182 consecutive MM patients, at diagnosis, were evaluated for the presence of aberrant plasma cells (APCs) following the standard operating procedures (SOP) of NGF, according to EuroFlow guidelines. All these patients were diagnosed and treated in a single center (Department of Clinical Therapeutics, N.K. University of Athens, Greece). Samples were collected in EDTA-anticoagulated tubes and treated with the bulk-lysis procedure. Recovered cells were stained with antibodies against surface CD19-PEC7, CD27-BV510, CD38-FITC, CD45-PERCP, CD56-PE and CD138-BV421 and the intracellular CyIgκ-APC and CyIgλ-APCC750 to verify clonality. Six to ten million cells were acquired per sample, thus reaching a median Limit of Detection (LOD) of 3.5x10-6. Optimal PMT voltages were set according to the EuroFlow SOP for instrument set-up and daily performance status of FACSCANTOII was monitored with both CS&T (BD) and Rainbow beads (Spherotech Inc, Lake Forest, IL).
Results: CPCs were detected in 158/182 (86.8%) MM diagnostic samples within a range of 0.0002% to 63.8% of total PB nucleated cells (PBNCs). The CPCs showed the same aberrant phenotype as the one detected in the BM for all cases, although with a significantly reduced intensity for the markers CD27, CD38, CD138 and CD56. When more than one phenotypically distinct subgroups were detectable in the BM, the same phenotypic subsets were present in the PB with the same relative frequency for >90% of bi/multi-phenotypic cases. The higher number of CPCs (>0.1% of all PBNCs) strongly correlated with an increased BM infiltration rate by myeloma cells (p<0.0001), with ISS-3 disease stage (p<0.0001) and with the presence of high-risk cytogenetics [t(4;14), t(14;16) and/or del(17p53); p<0.0001]. There was also weaker correlation between high number of CPCs and high serum creatinine levels (p=0.015). Inversely, the absence of CPCs or the presence of CPCs at numbers ≤0.001% correlated with lower serum β2-microglobulin (p<0.0001), with higher hemoglobin levels (p<0.0001) and with the presence of an elevated normal plasma cell compartment within the BM (i.e. ≥5% of all PCs; r2=0.84, p<0.0001). There was no association between the CPC number and the therapeutic response to induction treatment (IMWG criteria). Despite the short follow-up period (median of 16 months), there is a trend for inferior PFS in patients with high CPCs (p=0.16).
Conclusions: The NGF approach using the EuroFlow protocol enables the detection of even rare CPCs in diagnostic MM PB samples, due to the high number of cells acquired and the elegantly elaborated 8-color marker combinations which allows for the detection of CPCs with even a non-typical phenotype. Our matched PB and BM analysis revealed that BM APCs and CPCs share very similar characteristics suggesting that liquid biopsy offers a representative alternative for the phenotypic characterization of BM APCs. The correlation of high CPCs with adverse disease characteristics suggests that the quantification of CPCs by standardized NGF may emerge as a valuable surrogate prognostic biomarker which could replace other invasive methods or other less informative assays.

Disclosures
Terpos: Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria. Gavriatopoulou:Amgen: Honoraria; Janssen: Honoraria, Other: Travel expenses; Genesis: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses. Kastritis:Genesis: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding.