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2030 Characterization of B-Cell and Plasma Cell Compartment By Eight-Color Multiparameter Flow Cytometry in Patients with Waldenstrom Macroglobulinemia Prospectively Enrolled in the Fondazione Italiana Linfomi (FIL) BIO-WM Trial

Program: Oral and Poster Abstracts
Session: 622. Lymphoma Biology—Non-Genetic Studies: Poster II
Hematology Disease Topics & Pathways:
Diseases, Non-Hodgkin Lymphoma, Technology and Procedures, Lymphoid Malignancies, flow cytometry
Sunday, December 6, 2020, 7:00 AM-3:30 PM

Marzia Varettoni1*, Simone Ferrero, MD2,3, Cristina Picone1*, Milena Gilestro4*, Noemí Puig, MD, PhD5*, Luigi Marcheselli6*, Irene Dogliotti, MD2,3*, Angela Ferrari7*, Michele Merli, MD8*, Giulia Zamprogna, MD9*, Luca Laurenti, MD10*, Simona Tomassetti, MD11*, Emanuele Cencini, MD12,13*, Giacomo Loseto, MD14*, Dario Marino, MD, PhD15, Monia Marchetti, MD16*, Francesca Re, MD17*, Antonello Sica, MD18*, Jacopo Olivieri19*, Cristina Jiménez, PhD5*, Silvia Zibellini, PhD1*, Daniela Drandi, PhD2*, Chiara Candido1*, Martina Ferrante, PhD20*, Nicole Fabbri1*, Federica Cavallo20,21*, Luca Arcaini1,22*, Mario Boccadoro2,3 and Ramon Garcia-Sanz5

1Division of Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
2Department of Molecular Biotechnologies and Health Sciences, University of Torino, Torino, Italy
3Division of Hematology, A.O.U. Città della Salute e della Scienza di Torino, Torino, Italy
4Division of Hematology, A.O.U. Città della Salute e della Scienza, Torino, Italy
5Hospital Universitario de Salamanca (HUSAL), IBSAL, IBMCC (USAL-CSIC), CIBERONC, Salamanca, Spain
6Fondazione Italiana Linfomi- c/o Centro Oncologico Modenese (COM), Università di Modena e Reggio Emilia, Modena, Italy
7Ematologia, Azienda USL-IRCCS Reggio Emilia, Reggio Emilia, Italy
8UOC Ematologia, Ospedale di Circolo e Fondazione Macchi – ASST Sette Laghi, Varese, Italy
9Department of Haematology, Niguarda Cancer Center, ASST Grande Ospedale Metropolitano Niguarda, Milano, Italy
10Sezione di Ematologia, Dipartimento Scienze Radiologiche Radioterapiche ed Ematologiche, Fondazione Policlinico Universitario A Gemelli, Roma, Italy
11Ematologia, Ospedale degli Infermi di Rimini, Rimini, Italy
12Unit of Hematology, Azienda Ospedaliera Universitaria Senese, Siena, Italy
13U.O.C. Ematologia, AOU Senese, Siena, Italy
14Hematology and Cell Therapy Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy
15Oncologia Medica 1, Istituto Oncologico Veneto IOV IRCCS, Padova, Italy
16Ematologia, Ospedale Civile SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy
17UO Ematologia e CTMO, Azienda Ospedaliero Universitaria di Parma, Parma, Italy
18Oncologia Medica ed Ematologia, AOU Università degli Studi della Campania Luigi Vanvitelli, Napoli, Italy
19Clinica Ematologica, Centro Trapianti e Terapie Cellulari "Carlo Melzi", Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy
20Department of Molecular Biotechnologies and Health Sciences, University of Torino, Turin, Italy
21A.O.U. Città della Salute e della Scienza di Torino, Torino, Italy
22Department of Molecular Medicine, University of Pavia, Pavia, Italy

Background. The World Health Organization defines Waldenström Macroglobulinemia (WM) as a lymphoplasmacytic lymphoma associated with a serum immunoglobulin M (IgM) monoclonal protein. Since bone marrow (BM) infiltration is always present, while extramedullary involvement is infrequent, trephine BM biopsy is mandatory based on currently used diagnostic criteria, while multiparameter flow cytometry (MFC) studies are recommended to support the diagnosis (Owen et al, 2003). Next generation flow cytometry using panels with 8 to 17 monoclonal antibodies (MoAb), high number of cell acquisitions and appropriate software have shown an increased sensitivity, being able to detect one tumor cell among 10.000 normal cells (10-4) or even more.

Aims of the study. Here we report an extensive phenotypic characterization by eight color MFC of a large prospective cohort of newly diagnosed WM patients, enrolled in the multicenter, observational trial BIO-WM (NCT03521596). In this study we evaluated the potential role of MFC on BM and peripheral blood (PB) samples as an alternative to BM biopsy in the diagnostic work-up of WM, with the final goal to assess the feasibility of a non-invasive or at least less invasive diagnostics.

Methods. The diagnosis of WM was made using the criteria of the Second International Workshop on WM. Patients without bone marrow biopsy were excluded from the analysis. Whole BM and PB samples were processed following the bulk lysis protocol and stained using 8-color combination of the following fluorochrome-conjugated monoclonal antibodies: cIgM, CD56, CD5, CD19, CyK, CyL, CD38, CD45 (screening panel). CD5- lymphoproliferative disorders with two populations in different maturation stage showing identical cytoplasmatic light chain restriction underwent further characterization with six 8 color-panels (characterization panel). A minimum of 1x106/L cells per tube was acquired. The full list of monoclonal antibodies is reported in Table 1.

Results. We analyzed 173 prospective patients with a histologic diagnosis of WM, either asymptomatic (n=46, 27%) or symptomatic (n=127, 73%). Their baseline characteristics are reported in Table 2. MFC results on BM samples collected at diagnosis were available in 165 patients. The median white blood cell (WBC) count was 13.5 x 109/L (IQR: 4.9-94) and the median number of cell acquisitions was 1.16 x 106 (IQR: 0.91-1.58). Clonal CD19+ B-cells were detected by MFC in 155/165 cases (94%) and showed k light chain restriction in 76% of cases. The median percentage of clonal CD19+ B-cells out of total BM WBC was 5.9% (IQR: 2.9-12.4). Twelve cases out of 165 (7%) were CD5+. The complete phenotype of clonal B CD19+ lymphocytes is shown in Figure 1. Clonal CD38+ plasma cells were detected in BM samples in 86% of cases and were CD20+ in 51% of cases. The median percentage of clonal CD38+ plasma cells out of total BM WBC was 0.18% (IQR: 0.05-0.47). The complete antigenic expression of clonal plasma cells is shown in Figure 2. The MYD88 (L265P) mutation was found with droplet digital polymerase chain reaction (dd-PCR) in 95% of patients with detectable clonal CD19+ B-cells in BM samples, without significant difference between CD5- and CD5+ cases (95% versus 93%, P=0.698). Paired BM and PB samples were available in 157 patients. Clonal CD19+ B-cells were detected in BM samples in 149/157 cases (95%) and in PB samples in 98/157 cases (58%). Overall, there was a slight agreement between BM and PB MFC results according to Landis and Koch scale (63%, k = 0.138).

Conclusions. The comparison of MFC results on BM samples with BM histology in this large prospective cohort of WM patients suggests that eight-color MFC is highly sensitive and is able to identify the majority of patients with WM. Conversely, the comparison of MFC results on paired BM and PB samples indicates that MFC on PB samples has a low sensitivity, missing approximately 40% of cases. The integration of MFC results on BM samples with MYD88 mutation status assessed with a highly sensitive method such as dd-PCR may be considered as an alternative, less invasive method for the diagnosis of WM. Further analyses within this trial will assess the role of MFC combined with molecular profile of patients for the differential diagnosis between WM and other mature B-cell lymphoproliferative disorders as well as for the discrimination between WM and IgM monoclonal gammopathy of undetermined significance.

Disclosures: Varettoni: Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Other: Travel/accommodations/expenses; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel/accommodations/expenses. Ferrero: Servier: Speakers Bureau; Gilead: Research Funding, Speakers Bureau; EUSA Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Puig: JANSSEN: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; TAKEDA: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; AMGEN: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; THE BINDING SITE: Consultancy, Honoraria; BRISTOL-MYERS SQUIBB: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding, Speakers Bureau; CELGENE: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding, Speakers Bureau. Ferrari: Abbvie: Honoraria. Laurenti: Janssen: Honoraria; Gilead: Honoraria; AbbVie: Honoraria; Roche: Honoraria. Marchetti: Takeda: Other: Sponsored meetings; Pfeizer: Other: Sponsored meetings; AbbVie: Other: Sponsored meetings; Gilead: Consultancy; Novartis: Speakers Bureau; Amgen: Speakers Bureau. Re: BerGenBio ASA: Research Funding. Boccadoro: GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Research Funding; Mundipharma: Research Funding; AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Garcia-Sanz: Novartis: Honoraria; Janssen: Honoraria, Research Funding; Incyte: Research Funding; Gilead: Honoraria, Research Funding; BMS: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria; Takeda: Consultancy, Research Funding.

*signifies non-member of ASH