FCGR3A F158V alleles frequency differs in multiple myeloma patients from healthy population.

FCGR3A presents a single nucleotide polymorphism at location 158 (V/F), which affects its binding to the fragment crystallizable (Fc) of antibodies (Abs). FcγRIIIa-158 V allotype has the highest affinity and is associated with a better clinical response to IgG1 monoclonal Abs (mAb) treatment. We compared the allele frequency of FCGR3A-F158V polymorphism in cohorts of patients with B-cell lymphoproliferative disorders, including multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), non-Hodgkin lymphoma (NHL), and B-cell chronic leukemia (B-CLL). FCGR3A-158F homozygous were enriched and tended to be in MM and MGUS patients, respectively; but neither in B-CLL nor in NHL patients. We identified a significantly lower concentration of CD8 T-cells and resting memory CD4 T-cells in MM patients bone marrow with the F/F genotype, associated with an increase in the macrophage percentage. In contrast, natural killer cells increased in V/V homozygous patients. This suggests a deregulation of the immune microenvironment in FCGR3A-F/F homozygous patients. However, we did not observe difference in response following treatment combining chemotherapy associated or not with daratumumab, an IgG1 mAb direct against CD38. Our findings suggest that FCGR3A F158V polymorphism can regulate the immune environment and affect the development of tumor plasma cells.

Extreme thrombocytosis with an aggressive evolution harboring a novel variant of calreticulin (CALR) in exon 3.

We describe the case of a patient with extreme thrombocytosis whose evolution was rapidly fatal. No cause of secondary thrombocytosis was found. There was no sign of myelofibrosis but the megakaryocytes were small and dysplastic. The patient presented a calreticulin (CALR) variant in exon 3 (C105S), as well as concomitant mutations of ASXL1, U2AF1, and EZH2. This variant of CALR has never been described before, and after sorting, all identified mutations were found in myeloid cells but not in lymphoid cells. Therefore, the diagnosis of a frontier case of myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) was made. A treatment with hydroxycarbamide was started because of a high risk of thrombosis. Upon worsening of the hematological status two new mutations appeared, SETBP1 and ETV6, and the CALR mutation was still detectable, as well as the three other mutations found in the chronic stage. Our results show that this variant could contribute to MDS/MPN pathogenesis in that patient.

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors' combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression.

The BLM helicase is a new therapeutic target in multiple myeloma involved in replication stress survival and drug resistance.

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom's syndrome (BS), is a central factor of DNA damage repair by homologous recombination (HR) and genomic stability maintenance. Using independent cohorts of MM patients, we identified that high expression of BLM is associated with a poor outcome with a significant enrichment in replication stress signature. We provide evidence that chemical inhibition of BLM by the small molecule ML216 in HMCLs (human myeloma cell lines) leads to cell cycle arrest and increases apoptosis, likely by accumulation of DNA damage. BLM inhibition synergizes with the alkylating agent melphalan to efficiently inhibit growth and promote cell death in HMCLs. Moreover, ML216 treatment re-sensitizes melphalan-resistant cell lines to this conventional therapeutic agent. Altogether, these data suggest that inhibition of BLM in combination with DNA damaging agents could be of therapeutic interest in the treatment of MM, especially in those patients with high BLM expression and/or resistance to melphalan.

Inhibition of the Protein Arginine Methyltransferase PRMT5 in High-Risk Multiple Myeloma as a Novel Treatment Approach.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Subsets of patients have high-risk features linked with dismal outcome. Therefore, the need for effective therapeutic options remains high. Here, we used bio-informatic tools to identify novel targets involved in DNA repair and epigenetics and which are associated with high-risk myeloma. The prognostic significance of the target genes was analyzed using publicly available gene expression data of MM patients (TT2/3 and HM cohorts). Hence, protein arginine methyltransferase 5 (PRMT5) was identified as a promising target. Druggability was assessed in OPM2, JJN3, AMO1 and XG7 human myeloma cell lines using the PRMT5-inhibitor EPZ015938. EPZ015938 strongly reduced the total symmetric-dimethyl arginine levels in all cell lines and lead to decreased cellular growth, supported by cell line dependent changes in cell cycle distribution. At later time points, apoptosis occurred, as evidenced by increased AnnexinV-positivity and cleavage of PARP and caspases. Transcriptome analysis revealed a role for PRMT5 in regulating alternative splicing, nonsense-mediated decay, DNA repair and PI3K/mTOR-signaling, irrespective of the cell line type. PRMT5 inhibition reduced the expression of upstream DNA repair kinases ATM and ATR, which may in part explain our observation that EPZ015938 and the DNA-alkylating agent, melphalan, have combinatory effects. Of interest, using a low-dose of mTOR-inhibitor, we observed that cell viability was partially rescued from the effects of EPZ015938, indicating a role for mTOR-related pathways in the anti-myeloma activity of EPZ015938. Moreover, PRMT5 was shown to be involved in splicing regulation of MMSET and SLAMF7, known genes of importance in MM disease. As such, we broaden the understanding of the exact role of PRMT5 in MM disease and further underline its use as a possible therapeutic target.

Comprehensive characterization of the epigenetic landscape in Multiple Myeloma.

Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand the molecular processes that drive MM biology. Epigenetic modifications are involved in MM development, progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. Methods: We performed chromatin immunoprecipitation sequencing to analyze histone mark changes (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3) on 16 HMCLs. Results: Differential analysis of histone modification profiles highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. Using histone modifications associated to enhancer regions, we identified super-enhancers (SE) associated with genes involved in MM biology. We also identified promoters of genes enriched in H3K9me3 and H3K27me3 repressive marks associated to potential tumor suppressor functions. The prognostic value of genes associated with repressive domains and SE was used to build two distinct scores identifying high-risk MM patients in two independent cohorts (CoMMpass cohort; n = 674 and Montpellier cohort; n = 69). Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on the H3K4me3 modification predicting MM cell response to lenalidomide and histone deacetylase inhibitors (HDACi). Conclusions: The epigenetic landscape of MM cells represents a unique resource for future biological studies. Furthermore, risk-scores based on SE and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision medicine in MM.

RNA-Sequencing-Based Transcriptomic Score with Prognostic and Theranostic Values in Multiple Myeloma.

Multiple myeloma (MM) is the second most frequent hematological cancer and is characterized by the clonal proliferation of malignant plasma cells. Genome-wide expression profiling (GEP) analysis with DNA microarrays has emerged as a powerful tool for biomedical research, generating a huge amount of data. Microarray analyses have improved our understanding of MM disease and have led to important clinical applications. In MM, GEP has been used to stratify patients, define risk, identify therapeutic targets, predict treatment response, and understand drug resistance. In this study, we built a gene risk score for 267 genes using RNA-seq data that demonstrated a prognostic value in two independent cohorts (n = 674 and n = 76) of newly diagnosed MM patients treated with high-dose Melphalan and autologous stem cell transplantation. High-risk patients were associated with the expression of genes involved in several major pathways implicated in MM pathophysiology, including interferon response, cell proliferation, hypoxia, IL-6 signaling pathway, stem cell genes, MYC, and epigenetic deregulation. The RNA-seq-based risk score was correlated with specific MM somatic mutation profiles and responses to targeted treatment including EZH2, MELK, TOPK/PBK, and Aurora kinase inhibitors, outlining potential utility for precision medicine strategies in MM.

Ecdysis-related neuropeptide expression and metamorphosis in a non-ecdysozoan bilaterian.

Ecdysis-related neuropeptides (ERNs), including eclosion hormone, crustacean cardioactive peptide, myoinhibitory peptide, bursicon alpha, and bursicon beta regulate molting in insects and crustaceans. Recent evidence further revealed that ERNs likely play an ancestral role in invertebrate life cycle transitions, but their tempo-spatial expression patterns have not been investigated outside Arthropoda. Using RNA-seq and in situ hybridization, we show that ERNs are broadly expressed in the developing nervous system of a mollusk, the polyplacophoran Acanthochitona fascicularis. While some ERN-expressing neurons persist from larval to juvenile stages, others are only present during settlement and metamorphosis. These transient neurons belong to the "ampullary system," a polyplacophoran-specific larval sensory structure. Surprisingly, however, ERN expression is absent from the apical organ, another larval sensory structure that degenerates before settlement is completed in A. fascicularis. Our findings thus support a role of ERNs in A. fascicularis metamorphosis but contradict the common notion that the apical organ-like structures shared by various aquatic invertebrates (i.e., cnidarians, annelids, mollusks, echinoderms) are of general importance for this process.

G9a/GLP targeting in MM promotes autophagy-associated apoptosis and boosts proteasome inhibitor-mediated cell death.

Multiple myeloma (MM) is an (epi)genetic highly heterogeneous plasma cell malignancy that remains mostly incurable. Deregulated expression and/or genetic defects in epigenetic-modifying enzymes contribute to high-risk disease and MM progression. Overexpression of the histone methyltransferase G9a was reported in several cancers, including MM, correlating with disease progression, metastasis, and poor prognosis. However, the exact role of G9a and its interaction partner G9a-like protein (GLP) in MM biology and the underlying mechanisms of action remain poorly understood. Here, we report that high G9a RNA levels are associated with a worse disease outcome in newly diagnosed and relapsed MM patients. G9a/GLP targeting using the specific G9a/GLP inhibitors BIX01294 and UNC0638 induces a G1-phase arrest and apoptosis in MM cell lines and reduces primary MM cell viability. Mechanistic studies revealed that G9a/GLP targeting promotes autophagy-associated apoptosis by inactivating the mTOR/4EBP1 pathway and reducing c-MYC levels. Moreover, genes deregulated by G9a/GLP targeting are associated with repressive histone marks. G9a/GLP targeting sensitizes MM cells to the proteasome inhibitors (PIs) bortezomib and carfilzomib, by (further) reducing mTOR signaling and c-MYC levels and activating p-38 and SAPK/JNK signaling. Therapeutic treatment of 5TGM1 mice with BIX01294 delayed in vivo MM tumor growth, and cotreatment with bortezomib resulted in a further reduction in tumor burden and a significantly prolonged survival. In conclusion, we provide evidence that the histone methyltransferases G9a/GLP support MM cell growth and survival by blocking basal autophagy and sustaining high c-MYC levels. G9a/GLP targeting represents a promising strategy to improve PI-based treatment in patients with high G9a/GLP levels.

Kinome expression profiling to target new therapeutic avenues in multiple myeloma.

Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.

The hydroxymethylome of multiple myeloma identifies FAM72D as a 1q21 marker linked to proliferation.

Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.

A wnt2 ortholog in the sea urchin Paracentrotus lividus.

Members of the wnt gene family encode secreted glycoproteins that mediate critical intercellular communications in metazoans. Large-scale genome and transcriptome analyses have shown that this family is composed of 13 distinct subfamilies. These analyses have further established that the number of wnt genes per subfamily varies significantly between metazoan phyla, highlighting that gene duplication and gene loss events have shaped the complements of wnt genes during evolution. In sea urchins, for example, previous work reported the absence of representatives of both the WNT2 and WNT11 subfamilies in two different species, Paracentrotus lividus and Strongylocentrotus purpuratus. Recently, however, we identified a gene encoding a WNT2 ortholog in P. lividus and, based on that finding, we also reanalyzed the genome of S. purpuratus. Yet, we found no evidence of a bona fide wnt2 gene in S. purpuratus. Furthermore, we established that the P. lividus wnt2 gene is selectively expressed in vegetal tissues during embryogenesis, in a pattern that is similar, although not identical, to that of other P. lividus wnt genes. Taken together, this study amends previous work on the P. lividus wnt complement and reveals an unexpected variation in the number of wnt genes between closely related sea urchin species.

Comprehensive characterization of the mutational landscape in multiple myeloma cell lines reveals potential drivers and pathways associated with tumor progression and drug resistance.

Human multiple myeloma tumor cell lines (HMCLs) have been a cornerstone of research in multiple myeloma (MM) and have helped to shape our understanding of molecular processes that drive tumor progression. A comprehensive characterization of genomic mutations in HMCLs will provide a basis for choosing relevant cell line models to study a particular aspect of myeloma biology, or to screen for an antagonist of certain cancer pathways. Methods: We performed whole exome sequencing on a large cohort of 30 HMCLs, representative of a large molecular heterogeneity of MM, and 8 control samples (epstein-barr virus (EBV)-immortalized B-cells obtained from 8 different patients). We evaluated the sensitivity of HMCLs to ten drugs. Results: We identified a high confidence list of 236 protein-coding genes with mutations affecting the structure of the encoded protein. Among the most frequently mutated genes, there were known MM drivers, such as TP53, KRAS, NRAS, ATM and FAM46C, as well as novel mutated genes, including CNOT3, KMT2D, MSH3 and PMS1. We next generated a comprehensive map of altered key pathways in HMCLs. These include cell growth pathways (MAPK, JAK-STAT, PI(3)K-AKT and TP53 / cell cycle pathway), DNA repair pathway and chromatin modifiers. Importantly, our analysis highlighted a significant association between the mutation of several genes and the response to conventional drugs used in MM as well as targeted inhibitors. Conclusion: Taken together, this first comprehensive exome-wide analysis of the mutational landscape in HMCLs provides unique resources for further studies and identifies novel genes potentially associated with MM pathophysiology, some of which may be targets for future therapeutic intervention.

BrdU incorporation in multiparameter flow cytometry: A new cell cycle assessment approach in multiple myeloma.

BACKGROUND: Mutiple myeloma (MM) is a neoplasia characterized by the accumulation of malignant plasma cells (PC) in the bone marrow. Although proliferation markers have been studied in MM, none of the current staging systems include them. Moreover, approaches used to analyze proliferation do not separate MM cells (MMCs) from normal PC. METHODS: In this study, we combined multiparameter flow cytometry and BrdU incorporation or Ki67 staining to analyze MM cell proliferation in 44 monoclonal gammopathy of undetermined significance (MGUS), 153 newly diagnosed MM patients and 69 MM patients at relapse. The prognostic value of proliferation assessment was analyzed in 60 newly diagnosed patients treated with high-dose chemotherapy supported by autologous hematopoietic stem cell transplantation. RESULTS: The median number of proliferating malignant PC significantly increases during MM disease progression. MM patients with a percentage of proliferating MMCs greater than 1.42% using BrdU/DAPI or greater than 1.1% using ki67/DAPI, are associated with a significantly shorter event free survival compared with patients with a lower percentage of proliferating MMCs. CONCLUSIONS: Combination of flow cytometry with BrdU or ki67/DAPI staining could become a standard for the determination of MM cell proliferation. Furthermore, in the context of new effective myeloma treatment options, assessment of MM cell proliferation may be valuable, in clinical trials, to identify novel agents that could significantly affect the small proliferative compartment of MM cells. © 2018 International Clinical Cytometry Society.

PRC2 targeting is a therapeutic strategy for EZ score defined high-risk multiple myeloma patients and overcome resistance to IMiDs.

BACKGROUND: Multiple myeloma (MM) is a malignant plasma cell disease with a poor survival, characterized by the accumulation of myeloma cells (MMCs) within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with drug resistance. METHODS: We identified a significant upregulation of the polycomb repressive complex 2 (PRC2) core genes in MM cells in association with proliferation. We used EPZ-6438, a specific small molecule inhibitor of EZH2 methyltransferase activity, to evaluate its effects on MM cells phenotype and gene expression prolile. RESULTS: PRC2 targeting results in growth inhibition due to cell cycle arrest and apoptosis together with polycomb, DNA methylation, TP53, and RB1 target genes induction. Resistance to EZH2 inhibitor is mediated by DNA methylation of PRC2 target genes. We also demonstrate a synergistic effect of EPZ-6438 and lenalidomide, a conventional drug used for MM treatment, activating B cell transcription factors and tumor suppressor gene expression in concert with MYC repression. We establish a gene expression-based EZ score allowing to identify poor prognosis patients that could benefit from EZH2 inhibitor treatment. CONCLUSIONS: These data suggest that PRC2 targeting in association with IMiDs could have a therapeutic interest in MM patients characterized by high EZ score values, reactivating B cell transcription factors, and tumor suppressor genes.

DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells.

BACKGROUND: Multiple myeloma (MM) is the second most common hematologic malignancy. Aberrant epigenetic modifications have been reported in MM and could be promising therapeutic targets. As response rates are overall limited but deep responses occur, it is important to identify those patients who could indeed benefit from epigenetic-targeted therapy. METHODS: Since HDACi and DNMTi combination have potential therapeutic value in MM, we aimed to build a GEP-based score that could be useful to design future epigenetic-targeted combination trials. In addition, we investigated the changes in GEP upon HDACi/DNMTi treatment. RESULTS: We report a new gene expression-based score to predict MM cell sensitivity to the combination of DNMTi/HDACi. A high Combo score in MM patients identified a group with a worse overall survival but a higher sensitivity of their MM cells to DNMTi/HDACi therapy compared to a low Combo score. In addition, treatment with DNMTi/HDACi downregulated IRF4 and MYC expression and appeared to induce a mature BMPC plasma cell gene expression profile in myeloma cell lines. CONCLUSION: In conclusion, we developed a score for the prediction of primary MM cell sensitivity to DNMTi/HDACi and found that this combination could be beneficial in high-risk patients by targeting proliferation and inducing maturation.

Loss of RASSF4 Expression in Multiple Myeloma Promotes RAS-Driven Malignant Progression.

RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression.Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155-68. ©2017 AACR.

CD24, CD27, CD36 and CD302 gene expression for outcome prediction in patients with multiple myeloma.

Multiple myeloma (MM) is a B cell neoplasia characterized by clonal plasma cell (PC) proliferation. Minimal residual disease monitoring by multi-parameter flow cytometry is a powerful tool for predicting treatment efficacy and MM outcome. In this study, we compared CD antigens expression between normal and malignant plasma cells to identify new potential markers to discriminate normal from malignant plasma cells, new potential therapeutic targets for monoclonal-based treatments and new prognostic factors. Nine genes were significantly overexpressed and 16 were significantly downregulated in MMC compared with BMPC (ratio ≥2; FDR CD24, CD27, CD36 and CD302) was associated with a prognostic value in two independent cohorts of patients with MM (HM cohort and TT2 cohort, n=345). The expression level of these four genes was then used to develop a CD gene risk score that classified patients in two groups with different survival (P = 2.06E-6) in the HM training cohort. The prognostic value of the CD gene risk score was validated in two independent cohorts of patients with MM (TT2 cohort and HOVON65/GMMGHD4 cohort, n=282 patients). The CD gene risk score remained a prognostic factor that separated patients in two groups with significantly different overall survival also when using publicly available data from a cohort of relapsing patients treated with bortezomib (n=188). In conclusion, the CD gene risk score allows identifying high risk patients with MM based on CD24, CD27, CD36 and CD302 expression and could represent a powerful tool for simple outcome prediction in MM.

Characterization of human FCRL4-positive B cells.

FCRL4 is an immunoregulatory receptor that belongs to the Fc receptor-like (FCRL) family. In healthy individuals, FCRL4 is specifically expressed by memory B cells (MBCs) localized in sub-epithelial regions of lymphoid tissues. Expansion of FCRL4+ B cells has been observed in blood and other tissues in various infectious and autoimmune disorders. Currently, the mechanisms involved in pathological FCRL4+ B cell generation are actively studied, but they remain elusive. As in vivo FCRL4+ cells are difficult to access and to isolate, here we developed a culture system to generate in vitro FCRL4+ B cells from purified MBCs upon stimulation with soluble CD40 ligand and/or CpG DNA to mimic T-cell dependent and/or T-cell independent activation, respectively. After 4 days of stimulation, FCRL4+ B cells represented 17% of all generated cells. Transcriptomic and phenotypic analyses of in vitro generated FCRL4+ cells demonstrated that they were closely related to FCRL4+ tonsillar MBCs. They strongly expressed inhibitory receptor genes, as observed in exhausted FCRL4+ MBCs from blood samples of HIV-infected individuals with high viremia. In agreement, cell cycle genes were significantly downregulated and the number of cell divisions was two-fold lower in in vitro generated FCRL4+ than FCRL4- cells. Finally, due to their reduced proliferation and differentiation potential, FCRL4+ cells were less prone to differentiate into plasma cells, differently from FCRL4- cells. Our in vitro model could be of major interest for studying the biology of normal and pathological FCRL4+ cells.