Catherine Pellat-Deceunynck

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

In multiple myeloma, the use of multiparametric flow cytometry in many laboratories is currently restricted to clinical research studies and the differential diagnosis of unusual cases. This article report the indications of the European Myeloma Network for flow cytometry in patients with monoclonal gammopathies, and the technical recommendations for the analysis of plasma cells. 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.

The absence of CD56 (NCAM) on malignant plasma cells is a hallmark of plasma cell leukemia and of a special subset of multiple myeloma

In this study, we show that malignant plasma cells from patients with either primary (n = 12) or secondary (n = 15) plasma cell leukemia (PCL) do not express CD56 at all, neither in the bone marrow nor the peripheral blood in 81% of cases. On the other hand, multiple myeloma (MM) at diagnosis overexpress it in 63 of 94 (67%) cases (P = 0.0001). In three secondary PCL evaluated serially, CD56 was also lacking at diagnosis showing that CD56 is not downregulated at the end stage of the disease but rather not upregulated in this subset of patients. This last concept is strengthened by the observation that 29% of MM patients lacking CD56 or weakly expressing it at diagnosis present a detectable leukemic phase vs 11% only in CD56+ MM (P = 0.06). Forty percent of all the CD56−/weak malignant plasma cell disorders present or develop a leukemic phase vs only 15% of CD56+ cases (P < 0.008). cd56−/weak MM subset is also associated with a significantly less aggressive osteolytic potential (P = 0.012). We conclude that the lack or weak expression of CD56 is a characteristic feature of PCL but also delineates a special subset of MM at diagnosis mainly characterized by a lower osteolytic potential and a trend for malignant plasma cells to circulate in the peripheral blood more overtly.

Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors

Multiple myeloma (MM) patients are strongly vulnerable to infections, which remain a major cause of death. During infection, human immune cells sense the presence of invading pathogens through the Toll-like receptor family (TLR), which recognizes pathogen-associated molecular patterns (PAMP). We hypothesized that MM cells also could sense the presence of microorganisms, thus promoting myeloma disease progression. Here, we report that human myeloma cell lines (HMCL) and primary myeloma cells express a broad range of TLR, and are sensitive to the corresponding PAMP. Toll-like receptor 1, 7 and 9 are most frequently expressed by HMCL. The expression pattern of TLR does not correlate with the one of B cells, as TLR2 and 10 are lost while TLR3, 4 and 8 are acquired by some HMCL. Culture with TLR7- and TLR9-ligands saves HMCL from serum-deprivation or dexamethasone-induced apoptosis. Similarly, both ligands increase myeloma cell growth. These effects are mediated by an autocrine secretion of interleukin-6 (IL-6) since the neutralization of IL-6 blocks the growth and survival of HMCL. Thus, TLR expression and function are not restricted to the cells of the immune system and could be of advantage for cancer cells. In MM, recurrent infections could promote tumor growth and favor escape from standard therapies.

The cancer germ-line genes MAGE-1, MAGE-3 and PRAME are commonly expressed by human myeloma cells.

In this study, we have investigated the mRNA expression of the cancer germ‐line genes MAGE, BAGE, GAGE, RAGE and the tumor‐overexpressed gene PRAME by human myeloma cell lines and malignant plasma cells from patients with multiple myeloma (MM). By reverse transcription‐PCR, we show that all myeloma cell lines (n = 16) express at least one of these genes, except RAGE‐1 that was never expressed. We show that malignant plasma cells from the majority of MM patients (n = 21) expressed MAGE‐1, MAGE‐3 and PRAME. On the contrary, polyclonal reactive plasma cells did not express any of these genes. By flow cytometry, we show that mage‐1 protein is expressed within myeloma cells and cell lines and that anti‐mage‐1.HLA‐A1 cytotoxic T lymphocytes efficiently killed MAGE‐1+HLA‐A1+ MDN myeloma cells. Taken together, our data show that mage‐1 and mage‐3 could constitute specific targets for tumor immunotherapy of MM patients.

A high-risk signature for patients with multiple myeloma established from the molecular classification of human myeloma cell lines

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 Bcl-2 specific BH3 mimetic ABT-199: a promising targeted therapy for t(11;14) multiple myeloma

The Bcl-2 specific BH3 mimetic ABT-199: a promising targeted therapy for t(11;14) multiple myeloma

Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells.

R115777, a nonpeptidomimetic farnesyl transferase inhibitor has recently demonstrated a significant antileukemic activity in vivo in acute myeloid leukemia. Multiple myeloma (MM) is a fatal hematological malignancy characterized by an accumulation of long-lived plasma cells within the bone marrow. In the present study, we have investigated the effect of the R115777 on growth and survival of myeloma cells. We have found that R115777 induced (1) a significant and dose-dependent growth inhibition of the three myeloma cell lines tested; and (2) a significant and time-dependent apoptosis. R115777 also induced apoptosis in the bone marrow mononuclear cell population of four MM patients, being almost restricted to the malignant plasma cells. Finally, we have investigated the effect of the R115777 in the Ras/MAPK and JAK/STAT pathways which are implicated in survival and/or proliferation in MM. The phosphorylation of both STAT3 and ERK1/2 induced by IL-6 was totally blocked at 15 μM of R115777 and partially blocked when R115777 was used at 10 and 5 μM. The induction of apoptosis by R115777 in myeloma cells and its implication in the regulation of JAK/STAT signalling suggest that R115777 might be an interesting therapeutical approach in MM.

ABT-737 is highly effective against molecular subgroups of multiple myeloma.

Multiple myeloma is a plasma cell malignancy that is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. The Bcl-2 protein family is critical for myeloma cell survival. ABT-737 is a cell-permeant compound that binds to Bcl-2 and Bcl-x(L) but not to Mcl-1. Using a myeloma cell line collection (n = 25) representative of different molecular translocations, we showed that ABT-737 effectively kills a subset of cell lines (n = 6), with a median lethal dose ranging from 7 ± 0.4 nM to 150 ± 7.5 nM. Of interest, all sensitive cell lines harbored a t(11;14). We demonstrated that ABT-737-sensitive and ABT-737-resistant cell lines could be differentiated by the BCL2/MCL1 expression ratio. A screen of a public expression database of myeloma patients indicates that the BCL2/MCL1 ratio of t(11;14) and hyperdiploid patients was significantly higher than in all other groups (P < .001). ABT-737 first induced the disruption of Bcl-2/Bax, Bcl-2/Bik, or Bcl-2/Puma complexes, followed by the disruption of Bcl-2 heterodimers with Bak and Bim. Altogether, the identification of a subset of cell lines and primary cells effectively killed by ABT-737 alone supported the evaluation of ABT-263, an orally active counterpart to ABT-737, for the treatment of t(11;14) and hyperdiploid groups of myeloma harboring a Bcl-2(high)/Mcl-1(low) profile.

Noxa controls Mule-dependent Mcl-1 ubiquitination through the regulation of the Mcl-1/USP9X interaction.

The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.

Differential expression of Bcl-2 in human plasma cell disorders according to proliferation status and malignancy.

Multiple myeloma (MM) is a malignancy characterized by a very slow proliferation of malignant plasma cells leading to their accumulation within the bone marrow. This suggests that resistance to apoptosis may play a critical role both in the pathogenesis and resistance to treatment of MM. Bcl-2 is a key protein for the regulation of apoptosis. However, it has been shown that this protein also regulates the state of proliferation. In the current study, we show that malignant plasma cells from both the bone marrow and peripheral blood express high levels of Bcl-2 and are slowly proliferating cells. In contrast, myeloma cells from extramedullary sites (ie pleural effusion, ascitis, mammary and gastric plasmacytoma) express Bcl-2 weakly while being highly proliferative. Normal non-dividing bone marrow plasma cells express high levels of Bcl-2 protein. In contrast, four highly proliferative reactive plasmacytosis express weak levels of Bcl-2. We conclude that there is an inverse correlation between Bcl-2 expression and the proliferation rate of both normal and malignant plasma cells. These data may be explained by the double function of Bcl-2, ie its well known function as an anti-apoptotic molecule and its intriguing function as an inhibitory molecule of cell proliferation.