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)
Authors: Sanoja-Flores *Luzalba , Paiva *Bruno , Flores-Montero Juan A , Puig Noemi , Burgos Leire , García Omar , Prosper Felipe , Merino Juana , Vidriales Maria Belen , Mateos Maria-Victoria , Garcia Ramon , Palomera Luis , Rios Rafael , Cañizo María Consuelo , Durie Brian G M , van Dongen Jacques J.M. , San Miguel Jesus , Orfao Alberto 
Published: October 2019

Abstract
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:CD138BV421/CD27BV510/CD38FITC/CD56PE/CD45PerCP-Cy5.5/CD19PE-Cy7/CD117APC/CD81APC-C750, and; Tube 2: identical to Tube 1 except for CyKappaAPC/CyLambdaAPC-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.

Disclosures
Paiva: EngMab AG: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Binding Site: Consultancy; Sanofi: Consultancy; BD Bioscience: Consultancy; Onyx: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Mateos:Celgene: Consultancy, Honoraria; Takeda: Consultancy; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. Durie:Celgene: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Johnson and Johnson: Consultancy. van Dongen:BD Biosciences (cont'd): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics. ; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge.Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.. Royalty income for EuroClonality-BIOMED-2 Consortium ; Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium ; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences). , Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL.. San Miguel:Janssen-Cilag: Honoraria; Onyx: Honoraria; Millennium: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria.