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Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenström's Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (P<.001), with only 1% of IgM-MGUS patients showing >10% B cells or 100% light-chain-isotype-positive B-cells (P<.001). Complete light-chain restriction of the B-cell compartment was an independent prognostic factor for time-to progression in smoldering WM (median 26 months; HR: 19.8, P=0.001) and overall survival in symptomatic WM (median 44 months; HR: 2.6, P=0.004). The progressive accumulation of light-chain-isotype-positive B-cells accompanied the emergence of a characteristic Waldenstrom's phenotype (CD22(+dim) / CD25+ /CD27+ / IgM+) that differed from other B-NHL by negative expression of CD5, CD10, CD11c or CD103. In contrast to myeloma, light-chain-isotype-positive plasma cells in IgM monoclonal gammopathies show otherwise normal antigenic expression. Our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM.

Achieving complete remission (CR) in multiple myeloma (MM) translates into extended survival, but two subgroups of patients fall outside this paradigm: cases with unsustained CR, and patients that do not achieve CR but return into a monoclonal gammopathy of undetermined significance (MGUS)-like status with long-term survival. Here, we describe a novel automated flow cytometric classification focused on the analysis of the plasma-cell compartment to identify among newly diagnosed symptomatic MM patients (N=698) cases with a baseline MGUS-like profile, by comparing them to MGUS (N=497) patients and validating the classification model in 114 smoldering MM patients. Overall, 59 symptomatic MM patients (8%) showed an MGUS-like profile. Despite achieving similar CR rates after high-dose therapy/autologous stem cell transplantation vs other MM patients, MGUS-like cases had unprecedented longer time-to-progression (TTP) and overall survival (OS; ~60% at 10 years; P<0.001). Importantly, MGUS-like MM patients failing to achieve CR showed similar TTP (P=0.81) and OS (P=0.24) vs cases attaining CR. This automated classification also identified MGUS patients with shorter TTP (P=0.001, hazard ratio: 5.53) and ultra-high-risk smoldering MM (median TTP, 15 months). In summary, we have developed a biomarker that identifies a subset of symptomatic MM patients with an occult MGUS-like signature and an excellent outcome, independently of the depth of response.

Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy. [ABSTRACT FROM AUTHOR]