Impact of Treatment on B-Cell Regeneration By Next Generation Flow Cytometry in Patients with Multiple Myeloma
Authors: Pontes Robéria , Flores-Montero Juan , Sanoja-Flores Luzalba , Puig Noemi , Magalhães Roberto José Pessoa , Mateos Alba Corral , Salgado Anna Beatriz , Maiolino Angelo , Mateos Maria-Victoria , Paiva Bruno , da Costa Elaine Sobral , van Dongen Jacques JM , Orfao Alberto 
Published: October 2019

Abstract
Introduction: Several studies have shown the role of the immune system in the development of MM, but there is no systematic description of normal B-cell regeneration during treatment points. Recently, EuroFlow consortium has developed NGF panel with high sensitivity to evaluated MRD and potentially, to assess of the normal B-cell compartment of patients with MM. Aims: Here we evaluated the B cell regeneration pattern of bone marrow (BM) from patients with MM by Next Generation Flow (NGF) cytometry at diagnosis and at minimal residual disease (MRD) time points of treatment. Material and Methods: Overall 190 samples of MM patients (45% female and 55% male, median age of 65 years - 37 to 87 years, of which: 16 at diagnosis, 30 post-induction, 76 D+100, 59 maintenance/consolidation and 9 out of treatment, and 8 samples of BM healthy donor - HD (≥ 50 years). At the moment treatment, D+100 were included samples from two research centers: HCS/USAL (n=64) and HUCFF/UFRJ (n=12). Induction regimens were composed of triple protocols (PI + IMIDs + Steroids); followed by high doses of melphalan with ASCT, while patients in maintenance/consolidation followed second line treatment regimens - IP+IMIDs and/or PIs+Steroids and/or monoclonal antibodies or INF or IPs. All samples of BM were subjected to bulk cell lysis with ammonium chloride solution for >107 cell acquisition and labeled with a combination of 10 antibodies - Panel EuroFlow MM MRD.Results: Of the 174 post treatment samples, 36% presented MRD- and 64% MRD+. At diagnosis, patients exhibit a significant reduction of precursors B cells and normal plasma cells (nPC) relative to HD, probably a reflection of the occupation of their binding sites by cPC. At post-induction, there was an increase in precursors B, compared to MM patients at diagnosis, associated with a reduction of mature B-cell (transitional/naïve and memory), regardless of MRD status. Concerning the nPC compartment, a reduction was observed, relative to HD. During treatment, reduction of the tumor burden leaves these sites free for precursors B, which rapidly recover while leads to a drastic decrease in mature B cells and regeneration of the precursors to mature B-cells is slower. On the other hand, in D+100, independent of the MRD status, there was a post treatment medullary recovery, with an increase in B precursors and transitional/naïve B-cells, in contrast, with a reduction of memory B-cells. Out of treatment, we observed a recovery of precursors B, mature B-cells and increase of nPC, but the immune recovery of these nPC is not sufficient to reach the levels of a healthy individual. Conclusion: NGF emerges as an optimal approach for simultaneous assessment of the BM regeneration profile of B-cells and the MRD status. After starting therapy, MM patients re-establish the compartments of B-cell precursors and transitional/naïve B-lymphocytes; however, memory B cells and nPC do not recovery until the end of treatment. This study is a starting point for exploring the importance of the B cells of the medullary microenvironment in the MM. Its potential impact on patient outcome deserving further investigations

Disclosures
Puig: Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.