Search

Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered atwww.clinicaltrials.gov as #NCT01237249., (© 2016 by The American Society of Hematology.)

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.

The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM(+) CD27(-) naïve B-lymphocytes, plus surface-membrane IgG(+), IgA(+) and IgM(+) memory CD27(+) B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/μL (range: 0.0003-0.08 cells/μL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.

Circulating myeloma tumor cells (CTCs) as defined by the presence of peripheral blood (PB) clonal plasma cells (PCs) are a powerful prognostic marker in multiple myeloma (MM). However, the biological features of CTCs and their pathophysiological role in MM remains unexplored. Here, we investigate the phenotypic, cytogenetic, and functional characteristics as well as the circadian distribution of CTCs vs paired bone marrow (BM) clonal PCs from MM patients. Our results show that CTCs typically represent a unique subpopulation of all BM clonal PCs, characterized by downregulation (P < .05) of integrins (CD11a/CD11c/CD29/CD49d/CD49e), adhesion (CD33/CD56/CD117/CD138), and activation molecules (CD28/CD38/CD81). Fluorescence in situ hybridization analysis of fluorescence-activated cell sorter-sorted CTCs also unraveled different cytogenetic profiles vs paired BM clonal PCs. Moreover, CTCs were mostly quiescent and associated with higher clonogenic potential when cocultured with BM stromal cells. Most interestingly, CTCs showed a circadian distribution which fluctuates in a similar pattern to that of CD34(+) cells, and opposite to stromal cell-derived factor 1 plasma levels and corresponding surface expression of CXC chemokine receptor 4 on clonal PCs, suggesting that in MM, CTCs may egress to PB to colonize/metastasize other sites in the BM during the patients' resting period.

Cytogenetic studies in clonal plasma cell disorders have mainly been done in whole bone marrow or CD138(+) microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) -are primary oncogenetic events. The aim of this study was to determine cytogenetic patterns of highly purified aberrant plasma cells (median purity ≥ 98%) in different clonal plasma cell disorders. We analyzed aberrant plasma cells from 208 patients with multiple myeloma (n=148) and monoclonal gammopathy of undetermined significance (n=60) for the presence of del(13q14), del(17p13) and t(14q32) using multicolor interphase fluorescence in situ hybridization. Additionally, immunoglobulin heavy chain gene arrangements were analyzed and complementarity determining region 3 was sequenced in a subset of patients and combined multicolor interphase fluorescence in situ hybridization/immunofluorescent protein staining analyses were performed in selected cases to confirm clonality and cytogenetic findings. At diagnosis, 96% of cases with multiple myeloma versus 77% of monoclonal gammopathy of undetermined significance cases showed at least one cytogenetic alteration and/or hyperdiploidy. The cytogenetic heterogeneity of individual cases reflected coexistence of cytogenetically-defined aberrant plasma cell clones, and led to the assumption that karyotypic alterations were acquired stepwise. Cases of multiple myeloma and monoclonal gammopathy of undetermined significance frequently showed different but related cytogenetic profiles when other cytogenetic alterations such as deletions/gains of the immunoglobulin heavy chain or the fibroblast growth factor receptor 3 were additionally considered. Interestingly, in 24% of multiple myeloma versus 62% of monoclonal gammopathy of undetermined significance patients with an immunoglobulin heavy chain translocation, aberrant plasma cells with and without t(14q32) coexisted in the same patient. Our data suggest that recurrent immunoglobulin heavy chain translocations might be absent in the primordial plasma cell clone in a significant proportion of patients with clonal plasma cell disorders carrying these cytogenetic alterations.