The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.

Interleukin-6 (IL-6), although often regarded as a B-cell differentiation factor, was recently described as the essential survival factor for human plasmablasts in vivo in reactive plasmacytosis. The present study reinvestigated the roles of IL-6 and IL-2 in the generation of plasma cells from human memory B cells in vitro. The cells involved in this differentiation process were identified as preplasmablasts (CD20^±CD38^±CD138⁻), plasmablasts (CD20⁻CD38⁺⁺CD138⁻), and early plasma cells (CD20⁻CD38⁺⁺⁺CD138⁺⁺⁺). IL-2 or IL-10 induced a strong generation of plasmablasts and early plasma cells (PCs). Compared to IL-2 or IL-10, IL-6 alone was inefficient at PC generation. However, when combined with IL-2 or IL-10, IL-6 enhanced generation of early PCs. Moreover, anti–IL-6 monoclonal antibody markedly reduced IL-2–induced generation of early plasma cells, but not of plasmablasts. These roles of IL-2 and IL-6 were consistent with the difference in the expression of their respective receptors (R). CD25 (IL-2Rα) was increased 72 ± 10-fold on activated B cells, but decreased and then disappeared on plasmablasts. Conversely, CD126 (IL-6Rα) was barely expressed on activated B cells, but increased 18 ± 2-fold on preplasmablasts. Finally, IL-6 enhanced the proliferation (2-fold increase) of IL-2–generated plasmablasts. In conclusion, the data indicate that IL-6 is a growth factor for nonmalignant human plasmablasts.

Background Multiple myeloma is a plasma-cell tumor with heterogeneity in molecular abnormalities and treatment response. Design and Methods We have assessed whether human myeloma cell lines have kept patients’ heterogeneity using Affymetrix gene expression profiling of 40 human myeloma cell lines obtained with or without IL6 addition and could provide a signature for stratification of patient risk. Results Human myeloma cell lines, especially those derived in the presence of IL6, displayed a heterogeneity that overlaps that of the patients with multiple myeloma. Human myeloma cell lines segregated into 6 groups marked by overexpression of MAF, MMSET, CCND1, FRZB with or without overexpression of cancer testis antigens (CTA). Cell lines of CTA/MAF and MAF groups have a translocation involving C-MAF or MAFB, cell lines of groups CCND1-1 and CCND1-2like have a t(11;14) and cell lines of group MMSET have a t(4;14). The CTA/FRZB group comprises cell lines that had no or no recurrent 14q32 translocation. Expression of 248 genes accounted for human myeloma cell line molecular heterogeneity. Human myeloma cell line heterogeneity genes comprise genes with prognostic value for survival of patients making it possible to build a powerful prognostic score involving a total of 13 genes. Conclusions Human myeloma cell lines derived in the presence of IL6 recapitulate the molecular diversity of multiple myeloma that made it possible to design, using human myeloma cell line heterogeneity genes, a high-risk signature for patients at diagnosis. We propose this classification to be used when addressing the physiopathology of multiple myeloma with human myeloma cell lines.

The aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1^Met) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD₅₀) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD₅₀ ranging from 4 μM to more than 200 μM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1^Met did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1^Met increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1^Met-induced cell death. PRIMA-1^Met depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1^Met LD₅₀ values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1^Met-induced cell death and overcame PRIMA-1^Met resistance. PRIMA-1^Met (10 μM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53^neg JJN3-xenograft model that PRIMA-1^Met inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo.