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PDF file - 3577K, (A) Highest confidence networks of genes deregulated by decitabine treatment in HMCLs. (B) According to Ingenuity Pathway Analysis software, the network is relevant to "Interferon signaling". Genes that were deregulated by decitabine in HMCLs are shaded in grey. Each interaction is supported by at least one literature reference with solid lines representing direct interactions and dashed lines representing indirect interactions.

PDF file - 48K, Genes underexpressed in decitabine treated HMCLs in the studies conducted by Heller G et al. and the current study.

PDF file - 43K, Prognostic value of decitabine deregulated genes in primary MMC of newly-diagnosed patients.

PDF file - 40K, Genes overexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 μM decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly differentially expressed between control and decitabine treated cells were identified using SAM supervised paired analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set yielding to a maximum variance across control and decitabine treated cells.

PDF file - 54K, Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 μM decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly differentially expressed between control and decitabine treated cells were identified using SAM supervised paired analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set yielding to a maximum variance across control and decitabine treated cells.

Multiple myeloma is a plasma cell cancer with poor survival, characterized by the clonal expansion of multiple myeloma cells (MMC), primarily in the bone marrow. Novel compounds are currently tested in this disease, but partial or minor patients' responses are observed for most compounds used as a single agent. The design of predictors for drug efficacy could be most useful to better understand basic mechanisms targeted by these drugs and design clinical trials. In the current study, we report the building of a DNA methylation score (DM score) predicting the efficacy of decitabine, an inhibitor of DNA methyltransferase (DNMT), targeting methylation-regulated gene expression. DM score was built by identifying 47 genes regulated by decitabine in human myeloma cell lines and the expression of which in primary MMCs of previously untreated patients is predictive for overall survival. A high DM score predicts patients' poor survival, and, of major interest, high sensitivity of primary MMCs or human myeloma cell lines to decitabine in vitro. Thus, DM score could be useful to design novel treatments with DMNT inhibitor in multiple myeloma and has highlighted 47 genes, the gene products of which could be important for multiple myeloma disease development. Mol Cancer Ther; 11(12); 2685–92. ©2012 AACR.

PDF file - 48K, Genes overexpressed in decitabine treated HMCLs in the studies conducted by Heller G et al. and the current study.

PDF file - 61K, Clinical patient data for age, serum-β2-microglobulin, and plasma cell infiltration in the Heidelberg/Montpellier-group (HM) and the Arkansas cohort. Median value and range are given. NA, not available. ISS, International Staging System.

PDF file - 43K, Cell viability of HMCLs treated with either 0.5 μM decitabine for 7 days. Date are the mean percentages � SD of viable cells evaluated by trypan blue exclusion (3 experiments).

PDF file - 74K, Cox univariate and multivariate analysis of OS in HM and TT2 patients' cohorts.

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Purpose: Multiple myeloma is an incurable malignant plasma cell disease characterized by survival ranging from several months to more than 15 years. Assessment of risk and underlying molecular heterogeneity can be excellently done by gene expression profiling (GEP), but its way into clinical routine is hampered by the lack of an appropriate reporting tool and the integration with other prognostic factors into a single “meta” risk stratification.Experimental Design: The GEP-report (GEP-R) was built as an open-source software developed in R for gene expression reporting in clinical practice using Affymetrix microarrays. GEP-R processes new samples by applying a documentation-by-value strategy to the raw data to be able to assign thresholds and grouping algorithms defined on a reference cohort of 262 patients with multiple myeloma. Furthermore, we integrated expression-based and conventional prognostic factors within one risk stratification (HM-metascore).Results: The GEP-R comprises (i) quality control, (ii) sample identity control, (iii) biologic classification, (iv) risk stratification, and (v) assessment of target genes. The resulting HM-metascore is defined as the sum over the weighted factors gene expression–based risk-assessment (UAMS-, IFM-score), proliferation, International Staging System (ISS) stage, t(4;14), and expression of prognostic target genes (AURKA, IGF1R) for which clinical grade inhibitors exist. The HM-score delineates three significantly different groups of 13.1%, 72.1%, and 14.7% of patients with a 6-year survival rate of 89.3%, 60.6%, and 18.6%, respectively.Conclusion: GEP reporting allows prospective assessment of risk and target gene expression and integration of current prognostic factors in clinical routine, being customizable about novel parameters or other cancer entities. Clin Cancer Res; 17(23); 7240–7. ©2011 AACR.

Background: Multiple myeloma (MM) is an incurable disease characterized by clonal plasma cell (PC) proliferation within the bone marrow (BM). Next-generation flow cytometry has become the reference tool to follow minimal residual disease (MRD). We developed a new simpler and cheaper flow cytometry method to analyze bone marrow samples in patients with MM. Methods: To identify and characterize abnormal PC, we designed a simple panel composed of anti-CD38, anti-kappa and anti-lambda light chain antibodies, combined with two antibody pools with the same fluorophore (anti-CD19 and anti-CD27 for the negative pool, and anti-CD56, anti-CD117 and anti-CD200 antibodies for the positive pool). We also developed dedicated software for the automated identification of malignant PCs and MRD assessment. We then compared PC identification with our simple antibody panel and with the larger antibody panel routinely used at Montpellier University Hospital Center in 52 patients with MM (M-CHU cohort). Results: Results for total PC detection (r2=0.9965; P

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 appearedto 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.

Although functionally competent cytotoxic, T cells are frequently observed in malignant diseases, they possess little ability to react against tumor cells. This phenomenon is particularly apparent in multiple myeloma. We here demonstrate that cytotoxic T cells reacted against myeloma antigens when presented by autologous dendritic cells, but not by myeloma cells. We further show by gene expression profiling and flow cytometry that, similar to many other malignant tumors, freshly isolated myeloma cells expressed several carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) at varying proportions. Binding and crosslinking of CEACAM-6 by cytotoxic T cells inhibited their activation and resulted in T-cell unresponsiveness. Blocking of CEACAM-6 on the surface of myeloma cells by specific monoclonal antibodies or CEACAM-6 gene knock down by short interfering RNA restored T-cell reactivity against malignant plasma cells. These findings suggest that CEACAM-6 plays an important role in the regulation of CD8+ T-cell responses against multiple myeloma; therefore, therapeutic targeting of CEACAM-6 may be a promising strategy to improve myeloma immunotherapy.

Multiple myeloma patients? survival under treatment varies from a few months to more than 15 years. Clinical prognostic factors, especially beta2-microglobulin (B2M) and the international staging system (ISS), allow risk assessment to a certain extent, but do not identify patients at very high risk. As malignant plasma cells are characterized by a variety of chromosomal aberrations and changes in gene expression, a molecular characterization of CD138-purified myeloma cells by interphase fluorescence in situ hybridization (iFISH) and gene expression profiling (GEP) can be used for improved risk assessment. iFISH allows a risk stratification with presence of a translocation t(4;14) and/or deletion of 17p13 being the best documented adverse prognostic factors. A deletion of 13q14 is no longer considered to define adverse risk. Patients harbouring a t(4;14) seems to benefit from a bortezomib- or lenalidomide containing regimen, whereas patients with deletion 17p13 seem only to benefit from a high dose therapy approach using long term bortezomib (in induction and maintenance) and autologous tandem-transplantation as used in the GMMG-HD4 trial, or the total therapy 3 concept. Gene expression profiling allows the assessment of high risk scores (IFM, UAMS), remaining prognostic despite treatment with novel agents, and prognostic surrogates of biological factors (e.g. proliferation) and (prognostic) target gene expression (e.g. Aurora-kinase A). Thus, assessment of B2M and ISS-stage, iFISH, and GEP is considered extended routine diagnostics in therapy requiring multiple myeloma patients for risk assessment and, even now, to a certain extent selection of treatment.

Available transcriptome data accumulate in public repositories, individual web pages or as various supplemental data, but these published data cannot be routinely accessed. We have developed the web based tool Amazonia! to overcome this hurdle and provide the possibility to query and to visualize the expression of a given gene in representative and selected human transcriptome datasets. This expression atlas provides expression bar plots for single genes, across samples selected from a wide range of normal tissues and malignancies, including pluripotent stem cells. When produced by the same platform type, datasets were renormalized and combined in order to generate series of several hundreds samples. Samples types are colored and ordered, and grouped in thematic pages for ease of navigation. We also integrated gene lists provided by original publications describing these microarray data, allowing the scientific community to challenge the expression of genes in datasets other than those for which they were initially published. To illustrate the powerfulness of this simple tool, we show how Amazonia! reveals the specific expression of the tight junction protein Claudin 6 in human embryonic stem cells and human induced stem cells (iPS), or the tissue specific expression of some chemokines and their receptors such as CCL16 in liver and CX3CR1 in central nervous system samples. Thus, Amazonia! advantageously complements large public repositories by providing a simple way to query a compilation of selected human transcriptome data. The tool is freely available at http://www.amazonia.transcriptome.eu/

Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization. The aim of this study was to determine the gene expression profile of cumulus cells (CC) surrounding the oocyte as biomarkers for embryo potential and to identify genes to be used as prognostic indicators of successful pregnancy. CC from single oocytes were analysed using DNA microarrays. Gene expression profiles of CC surrounding the oocyte associated with good embryonic quality and pregnancy outcome were computed. We observed that CC issued from oocytes that developed into embryos with a good morphology had differing gene expression profile according to the pregnancy outcome of the embryo. We demonstrated that the expression of BCL2L11, PCK1 and NFIB in CC is significantly correlated with embryo potential and successful pregnancy. These results were confirmed by quantitative RT-PCR. The gene expression profiling of human CC correlates with embryo potential and pregnancy outcome. BCL2L11, PCK1 and NFIB genes are proposed as biomarkers for predicting pregnancy. Our findings suggest a non-invasive approach, offering a new potential strategy for competent embryo selection. This approach should be validated in single-embryo transfer programmes.

Background Identification of new markers assessing endometrial receptivity may help in improving the clinical outcome of IVF. This study aimed at identifying genes expressed in human endometrium during the implantation window that could be used as such markers. Methods A series of normoresponder patients (n = 31) underwent endometrial biopsies (n = 62, 2 per patient) during the early secretory phase, 2 days after the LH surge (LH + 2) and the mid-secretory phase (LH + 7) of the same natural cycle that preceded a new ICSI attempt for male infertility factor. Samples were analyzed using DNA microarrays and gene expression profiles at the time of the implantation window were computed. Systems biology analysis allowed the identification of biological pathways that were over-represented in this signature. A new approach for class prediction applied to microarray experiments was then used to identify biomarkers putatively involved in endometrial receptiveness. Results Five genes expressed during the implantation window were all up-regulated in the LH + 7 samples compared with LH + 2 [laminin beta3 (P = 0.002), microfibril-associated protein 5 (P = 0.009), angiopoietin-like 1 (P = 0.005), endocrine gland-derived vascular endothelial growth factor (P = 0.049) and nuclear localized factor 2 (P = 0.007)]. Increased expression was validated by quantitative RT-PCR. Conclusions Five genes have been identified for the first time as being up-regulated during the implantation window and are proposed as new biomarkers for exploration of endometrial receptiveness. As the endometrial biopsy procedure can be performed during a natural cycle, it would be worth testing this approach as a novel strategy in patients with poor implantation after IVF or ICSI.

T-cell-mediated immunotherapy is a promising therapeutic option for multiple myeloma (MM). Gamma-delta T cells (gammadelta T cells) recognize phosphoantigens and display strong anti-tumour cytotoxicity. The synthetic agonist Phosphostim (bromohydrin pyrophosphate, BrHPP) has been shown to selectively activate Vgamma9Vdelta2 T cells. This study aimed to evaluate the expansion capacity and anti-myeloma cell cytotoxicity of circulating gammadelta T cells from MM patients at different time points throughout the disease, using Phosphostim and interleukin 2 (IL-2). Circulating gammadelta T cell counts in patients with newly diagnosed MM or in relapse did not differ from those in healthy donors. A 14-d culture of peripheral blood mononuclear cells with Phosphostim and IL-2 triggered a 100-fold expansion of gammadelta T cells in 78% of newly diagnosed patients. Gammadelta T cells harvested at the time of haematopoietic progenitor collection or in relapsing patients expanded less efficiently. Expanded gammadelta T cells killed 13/14 myeloma cell lines as well as primary myeloma cells, but not normal CD34 cells. Their killing efficiency was not affected by 2-d IL-2 starvation. This study demonstrated the ability of Phosphostim and IL-2 to expand gammadelta T cells from MM patients, and the efficient and stable killing of human myeloma cells by gd T cells.

The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IkappaB kinase/nuclear factor-kappaB (IKK/NF-kappaB). The present study aimed at investigating the effect of a pharmaceutical IKK2 inhibitor, the anilinopyrimidine derivative AS602868, on the in vitro growth of 14 human MM cell lines (HMCL) and primary cells from 13 patients. AS602868 induced a clear dose-dependent inhibition of MM cell growth on both HMCL and primary MM cells, which was the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Combination of AS602868 with suboptimal doses of melphalan or Velcade showed an additive effect in growth inhibition of HMCL. AS602868 also induced apoptosis of primary myeloma cells. Importantly, AS602868 did not alter the survival of other bone marrow mononuclear cells (CD138(-)) co-cultured with primary MM (CD138(+)) cells, except for CD34(+) haematopoietic stem cells. The results demonstrate the important role of NF-kappaB in maintaining the survival of MM cells and suggest that a pharmacological inhibition of the NF-kappaB pathway by the IKK2 inhibitor AS602868 can efficiently kill HMCL and primary myeloma cells and therefore might represent an innovative approach for treating MM patients.

International audience; Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.

The DNA microarray technology enables the identification of the large number of genes involved in the complex deregulation of cell homeostasis taking place in cancer. Using Affymetrix microarrays, we have compared the gene expression profiles of highly purified malignant plasma cells from nine patients with multiple myeloma (MM) and eight myeloma cell lines to those of highly purified nonmalignant plasma cells (eight samples) obtained by in vitro differentiation of peripheral blood B cells. Two unsupervised clustering algorithms classified these 25 samples into two distinct clusters: a malignant plasma cell cluster and a normal plasma cell cluster. Two hundred and fifty genes were significantly up-regulated and 159 down-regulated in malignant plasma samples compared to normal plasma samples. For some of these genes, an overexpression or downregulation of the encoded protein was confirmed (cyclin D1, c-myc, BMI-1, cystatin c, SPARC, RB). Two genes overexpressed in myeloma cells (ABL and cystathionine beta synthase) code for enzymes that could be a therapeutic target with specific drugs. These data provide a new insight into the understanding of myeloma disease and prefigure that the development of DNA microarray could help to develop an 'à la carte' treatment in cancer disease.

A new way to identify tumor-specific genes is to compare gene expression profiles between malignant cells and their autologous normal counterparts. In patients with multiple myeloma, a major plasma cell disorder, normal plasma cells are not easily attainable in vivo. We report here that in vitro differentiation of peripheral blood B lymphocytes, purified from healthy donors and from patients with multiple myeloma, makes it possible to obtain a homogeneous population of normal plasmablastic cells. These cells were identified by their morphology, phenotype, production of polyclonal immunoglobulins, and expression of major transcription factors involved in B-cell differentiation. Oligonucleotide microarray analysis shows that these polyclonal plasmablastic cells have a gene expression pattern close to that of normal bone marrow–derived plasma cells. Detailed analysis of genes statistically differentially expressed between normal and tumor plasma cells allows the identification of myeloma-specific genes, including oncogenes and genes coding for tumor antigens. These data should help to disclose the molecular mechanisms of myeloma pathogenesis and to define new therapeutic targets in this still fatal malignancy. In addition, the comparison of gene expression between plasmablastic cells and B cells provides a new and powerful tool to identify genes specifically involved in normal plasma cell differentiation.

Objective Adult primary human hepatocytes (PHHs) support the complete infection cycle of natural HCV from patients’ sera. The molecular details underlying sera infectivity towards these cells remain largely unknown. Therefore, we sought to gain a deeper comprehension of these features in the most physiologically relevant culture system. Design Using kinetic experiments, we defined the optimal conditions to infect PHH and explored the link between cell organisation and permissivity. Based on their infectivity, about 120 sera were classified in three groups. Concentration of 52 analytes was measured in 79 selected sera using multiplexed immunobead-based analyte profiling. Results PHH permissivity towards HCV infection negatively correlated with cell polarisation and formation of functional bile canaliculi. PHH supported HCV replication for at least 2 weeks with de novo virus production. Depending on their reactivity, sera could be classified in three groups of high, intermediate or low infectivity toward PHH. Infectivity could not be predicted based on the donors’ clinical characteristics, viral load or genotype. Interestingly, highly infectious sera displayed a specific cytokine profile with low levels of most of the 52 tested analytes. Among them, 24 cytokines/growth factors could impact hepatocyte biology and infection efficiency. Conclusions We identified critical factors leading to efficient PHH infection by HCV sera in vitro. Overall, we showed that this cellular model provides a useful tool for studying the mechanism of HCV infection in its natural host cell, selecting highly infectious isolates, and determining the potency of drugs towards various HCV strains.

Human multiple myeloma (MM) purified tumour cells readily undergo apoptosis in vitro. Interleukin 6 (IL-6), a main growth factor of tumour cells, has enabled the development of IL-6-dependent MM cell lines. Recently, we developed anti-gp130 monoclonal antibodies (mAbs), two of which (B1 + I2) were able to dimerize gp130 and replace IL-6 in vitro. We show here that the injection of B1 + I2 IL-6 agonistic mAbs via the inguinal subcutaneous (SC) route efficiently produced tumours in severe combined immunodeficiency (SCID) mice grafted with IL-6-dependent myeloma cell lines compared with either the intraperitoneal (IP) or abdominal surgical bursa (SB) routes. The SC tumour graft, together with Matrigel and vascular endothelial growth factor (VEGF), leads to a strong vascularization and early detection of serum human immunoglobulins (huIgs). SCID mice treated with B1 + I2 mAbs were injected with fresh MM cells from five patients, four of whom had consistent levels of huIgs, and tumour growth was present in two. For one patient, tumour plasma cells that were passed several times subcutaneously in new SCID mice, still expressed their initial markers after several months. They remained unable to grow in vitro in the presence of B1 + I2 or IL-6. The nature of the SCID factors involved and the triggered genes are under investigation.

Non-Hodgkin's lymphomas (NHL) are B-cell malignancies which generally present molecular abnormalities, such as bcl-2 translocation t(14; 18) predominantly in the follicular subgroup. Other molecular events have been described in NHL, including p53 gene mutation and overexpression of one chemoresistance mechanism, the multidrug resistance system, P-glycoprotein (MDR 1/P-gp). In this study, we analysed samples from 44 NHL patients with the presence of the bcl-2 major breakpoint region (MBR) rearrangement in 29 and without in 15. Immunochemical analysis revealed that 39 samples were positive for bcl-2 protein expression in tumoral cells (88.6%). Seventeen (38.6%) patients expressed P-gp and 9 (20.5%) expressed p53 proteins. Eleven patients expressed both bcl-2 and P-gp proteins, four expressed bcl-2 and p53 proteins whereas four expressed bcl-2, p53 and P-gp proteins. Our results confirm the importance of p53 expression as a key prognostic factor, and no objective response (OR) was found in patients with p53 positivity. MBR rearrangement was not associated with poor response to chemotherapy (62.1% OR in MBR positive patients v. 60% OR in MBR negative patients). The clinical impact of P-gp cannot be identified because no relationship was observed between P-gp expression and prognosis (58.8% OR in P-gp positive patients v. 63% OR in P-gp negative patients).

SUMMARY Erosive rheumatoid arthritis (RA) is accompanied by synovial tissue hyperplasia associated with the proliferation of transformed-appearing synovial lining cells. In the present study we have analysed the expression of the p53 tumour suppressor gene in the synovial pannus tissue from patients at various stages of the disease. We used a combination of polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) on DNA and reverse transcription, PCR and sequencing on cDNAs from synovial tissues or purified synovial cell populations of 24 RA and three osteoarthritis (OA) patients. We also studied p53 expression by immunohistochemical analysis. Mutations suspected after SSCP were identified by systematic sequencing of the p53 exon 6, especially in the fibroblast-like, adherent synovial cell population, associated with an erosive disease. Some accumulation of the protein was detected in immunohistochemical analysis of the p53 tumour suppressor gene in the patients' synovial tissues. However, no sign of malignancy was seen in these patients after a 2-year survey. These results show some abnormalities in the p53 tumour suppressor gene in RA patients, but do not allow this to be related to characteristic proliferative features of the rheumatoid synovium.

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.

International audience; BACKGROUND: Multiple myeloma (MM) is still a fatal plasma cell cancer. Novel compounds are currently clinically tested as a single agent in relapsing patients, but in best cases with partial response of a fraction of patients, emphasising the need to design tools predicting drug efficacy. Histone deacetylase inhibitors (HDACi) are anticancer agents targeting epigenetic regulation of gene expression and are in clinical development in MM. METHODS: To create a score predicting HDACi efficacy, five MM cell lines were treated with trichostatin A (TSA) and gene expression profiles were determined. RESULTS: The expression of 95 genes was found to be upregulated by TSA, using paired supervised analysis with Significance Analysis of Microarrays software. Thirty-seven of these 95 genes had prognostic value for overall survival in a cohort of 206 newly diagnosed MM patients and their prognostic information was summed up in a histone acetylation score (HA Score); patients with the highest HA Score had the shorter overall survival. It is worth noting that MM cell lines or patients' primary MM cells with a high HA Score had a significant higher sensitivity to TSA, valproic acid, panobinostat or vorinostat. CONCLUSION: In conclusion, the HA Score allows identification of MM patients with poor survival, who could benefit from HDACi treatment.

International audience; High throughput DNA microarray has made it possible to outline genes whose expression in malignant plasma cells is associated with short overall survival of patients with Multiple Myeloma (MM). A further step is to elucidate the mechanisms encoded by these genes yielding to drug resistance and/or patients' short survival. We focus here on the biological role of the DEP (for Disheveled, EGL-10, Pleckstrin) domain contained protein 1A (DEPDC1A), a poorly known protein encoded by DEPDC1A gene, whose high expression in malignant plasma cells is associated with short survival of patients. Using conditional lentiviral vector delivery of DEPDC1A shRNA, we report that DEPDC1A knockdown delayed the growth of human myeloma cell lines (HMCLs), with a block in G2 phase of the cell cycle, p53 phosphorylation and stabilization, and p21(Cip1) accumulation. DEPDC1A knockdown also resulted in increased expression of mature plasma cell markers, including CXCR4, IL6-R and CD38. Thus DEPDC1A could contribute to the plasmablast features of MMCs found in some patients with adverse prognosis, blocking the differentiation of malignant plasma cells and promoting cell cycle.

Mathematical Assessment of Prognosis Diagnosis is the result of combined clinical and biological examinations. In most cases, it is refined by a classification of the patient in a disease group, which in turn contributes to prognostic assessment. Prognosis has long been the subjective result of experience. With the appearance of new technologies combining numerous samples with large-scale quantitative biological marker measurements, molecular assessment supplied new possibilities in cancer diagnosis and prognosis.

International audience; Diffuse gliomas are incurable brain tumors divided in 3 WHO grades (II; III; IV) based on histological criteria. Grade II/III gliomas are clinically very heterogeneous and their prognosis somewhat unpredictable, preventing definition of appropriate treatment. On a cohort of 65 grade II/III glioma patients, a QPCR-based approach allowed selection of a biologically relevant gene list from which a gene signature significantly correlated to overall survival was extracted. This signature clustered the training cohort into two classes of low and high risk of progression and death, and similarly clustered two external independent test cohorts of 104 and 73 grade II/III patients. A 22-gene class predictor of the training clusters optimally distinguished poor from good prognosis patients (median survival of 13-20 months versus over 6 years) in the validation cohorts. This classification was stronger at predicting outcome than the WHO grade II/III classification (P≤2.8E-10 versus 0.018). When compared to other prognosis factors (histological subtype and genetic abnormalities) in a multivariate analysis, the 22-gene predictor remained significantly associated with overall survival. Early prediction of high risk patients (3% of WHO grade II), and low risk patients (29% of WHO grade III) in clinical routine will allow the development of more appropriate follow-up and treatments.

BACKGROUND. Asymptomatic multiple myeloma (AMM) evolves from monoclonal gammopathy of unknown significance (MGUS) and progresses to symptomatic myeloma characterized by end-organ damage. Aim of our study was to address the determinants of evolution and progression of AMM, their molecular background, and whether they are present upfront or evolve de novoin a multistep process on the background of an ongoing genetic instability. METHODS . CD138-purified plasma cell samples of 2369 consecutive patients with MGUS, asymptomatic, and symptomatic myeloma were investigated by fluorescence-in-situ-hybridization (n=304/432/1633), 951 (n=62/259/630) by gene expression profiling. Sixty-five paired samples at AMM and disease progression were assessed by iFISH, 28 of these were further assessed by array-comparative-genomic-hybridization, as well as whole exome- (WES), and RNA-sequencing. Serum/urine samples (n=8398) allowed modelling of plasma cell accumulation in AMM and MGUS, respectively (n=322/196). RESULTS . Up-front tumor mass, plasma cell accumulation rate and molecular characteristics, including alterations in gene expression and presence of progression-associated chromosomal aberrations, i.e. t(4;14), deletions of 13q14, 17p13, 8p21, gains of 1q21, as well as hyperdiploidy, drive and predict evolution and progression of AMM. But for hyperdiploidy, the same factors drive progression from symptomatic to relapsed myeloma and also in AMM rather their number than the specific single aberration impact on time to progression. This means that the mechanisms driving progression to symptomatic myeloma are (at least in part) the same driving progression under treatment. Molecularly, all chromosomal aberrations, most transcriptomic changes, and most frequent mutations detected in symptomatic myeloma including NRAS, KRAS, DIS3, HIST1H1E are already present in MGUS or AMM. In paired AMM/MM samples, 22/27 (81%) show a stable clonal pattern, 5/27 (19%) the de novo appearance of expressed clones, including KRAS or FAM46C. No significant transcriptomic differences are found by RNA-sequencing. (Sub-)Clonal complexity with 4-5 discernable clusters of 103-363 single nucleotide variants with an allele frequency of ≥10% remains fairly constant during disease progression with most being detectable in both AMM and MM, incompatible with clonal outgrowth to any reason in these patients. In CONCLUSION, evolution and progression of AMM are driven and can be well predicted by factors being present upfront, i.e. tumor mass, plasma cell accumulation rate, and the set of molecular alterations. Progression is, contrary to current thinking, in the vast majority of patients not driven by de novo acquired expressed clonal alterations. This is proven in our set of paired samples on the level of chromosomal numeric or structural alterations (as per iFISH and aCGH), expressed clonal single nucleotide variants (as per whole exome- and RNA-sequencing), and remaining subclonal complexity. This in turn disproves other de novo alterations (e.g. methylation), as the subclone harboring these would then need to become clonal. Disclosures Hillengass: Sanofi: Research Funding; Amgen: Consultancy, Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hose:Takeda: Other: Travel grant; EngMab: Research Funding; Sanofi: Research Funding.

Multiple myeloma is a plasma cell cancer with poor survival, characterized by the clonal expansion of multiple myeloma cells (MMC), primarily in the bone marrow. Novel compounds are currently tested in this disease, but partial or minor patients' responses are observed for most compounds used as a single agent. The design of predictors for drug efficacy could be most useful to better understand basic mechanisms targeted by these drugs and design clinical trials. In the current study, we report the building of a DNA methylation score (DM score) predicting the efficacy of decitabine, an inhibitor of DNA methyltransferase (DNMT), targeting methylation-regulated gene expression. DM score was built by identifying 47 genes regulated by decitabine in human myeloma cell lines and the expression of which in primary MMCs of previously untreated patients is predictive for overall survival. A high DM score predicts patients' poor survival, and, of major interest, high sensitivity of primary MMCs or human myeloma cell lines to decitabine in vitro. Thus, DM score could be useful to design novel treatments with DMNT inhibitor in multiple myeloma and has highlighted 47 genes, the gene products of which could be important for multiple myeloma disease development. Mol Cancer Ther; 11(12); 2685–92. ©2012 AACR.

Annexin A2 (ANXA2) promotes myeloma cell growth, reduces apoptosis in myeloma cell lines, and increases osteoclast formation. ANXA2 has been described in small cohorts of samples as expressed by myeloma cells and cells of the BM microenvironment. To investigate its clinical role, we assessed 1148 samples including independent cohorts of 332 and 701 CD138-purified myeloma cell samples from previously untreated patients together with clinical prognostic factors, chromosomal aberrations, and gene expression–based high-risk scores, along with expression of ANXA2 in whole BM samples, stromal cells, osteoblasts, osteoclasts, and BM sera. ANXA2 is expressed in all normal and malignant plasma cell samples. Higher ANXA2 expression in myeloma cells is associated with significantly inferior event-free and overall survival independently of conventional prognostic factors and is associated with gene expression–determined high risk and high proliferation. Within the BM, all cell populations, including osteoblasts, osteoclasts, and stromal cells, express ANXA2. ANXA2 expression is increased significantly in myelomatous versus normal BM serum. ANXA2 exemplifies an interesting class of targetable bone-remodeling factors expressed by normal and malignant plasma cells and the BM microenvironment that have a significant impact on survival of myeloma patients.

International audience; BACKGROUND: Genetic abnormalities are common in patients with multiple myeloma, and may deregulate gene products involved in tumor survival, proliferation, metabolism and drug resistance. In particular, translocations may result in a high expression of targeted genes (termed spike expression) in tumor cells. We identified spike genes in multiple myeloma cells of patients with newly-diagnosed myeloma and investigated their prognostic value. DESIGN AND METHODS: Genes with a spike expression in multiple myeloma cells were picked up using box plot probe set signal distribution and two selection filters. RESULTS: In a cohort of 206 newly diagnosed patients with multiple myeloma, 2587 genes/expressed sequence tags with a spike expression were identified. Some spike genes were associated with some transcription factors such as MAF or MMSET and with known recurrent translocations as expected. Spike genes were not associated with increased DNA copy number and for a majority of them, involved unknown mechanisms. Of spiked genes, 36.7% clustered significantly in 149 out of 862 documented chromosome (sub)bands, of which 53 had prognostic value (35 bad, 18 good). Their prognostic value was summarized with a spike band score that delineated 23.8% of patients with a poor median overall survival (27.4 months versus not reached, P

International audience; Gene expression-based scores used to predict risk in cancer frequently include genes coding for DNA replication, repair or recombination. Using two independent cohorts of 206 and 345 previously-untreated patients with Multiple Myeloma (MM), we identified 50 cell cycle-unrelated genes overexpressed in multiple myeloma cells (MMCs) compared to normal human proliferating plasmablasts and non-proliferating bone marrow plasma cells and which have prognostic value for overall survival. Thirty-seven of these 50 myeloma genes (74%) were enriched in genes overexpressed in one of 3 normal human stem cell populations--pluripotent (18), hematopoietic (10) or mesenchymal stem cells (9)--and only three genes were enriched in one of 5 populations of differentiated cells (memory B lymphocytes, T lymphocytes, polymorphonuclear cells, monocytes, osteoclasts). These 37 genes shared by MMCs and adult or pluripotent stem cells were used to build a stem cell score ((SC)score), which proved to be strongly prognostic in the 2 independent cohorts of patients compared to other gene expression-based risk scores or usual clinical scores using multivariate Cox analysis. This finding highlights cell cycle-unrelated prognostic genes shared by myeloma cells and normal stem cells, whose products might be important for normal and malignant stem cell biology.

Purpose: Multiple myeloma is an incurable malignant plasma cell disease characterized by survival ranging from several months to more than 15 years. Assessment of risk and underlying molecular heterogeneity can be excellently done by gene expression profiling (GEP), but its way into clinical routine is hampered by the lack of an appropriate reporting tool and the integration with other prognostic factors into a single “meta” risk stratification. Experimental Design: The GEP-report (GEP-R) was built as an open-source software developed in R for gene expression reporting in clinical practice using Affymetrix microarrays. GEP-R processes new samples by applying a documentation-by-value strategy to the raw data to be able to assign thresholds and grouping algorithms defined on a reference cohort of 262 patients with multiple myeloma. Furthermore, we integrated expression-based and conventional prognostic factors within one risk stratification (HM-metascore). Results: The GEP-R comprises (i) quality control, (ii) sample identity control, (iii) biologic classification, (iv) risk stratification, and (v) assessment of target genes. The resulting HM-metascore is defined as the sum over the weighted factors gene expression–based risk-assessment (UAMS-, IFM-score), proliferation, International Staging System (ISS) stage, t(4;14), and expression of prognostic target genes (AURKA, IGF1R) for which clinical grade inhibitors exist. The HM-score delineates three significantly different groups of 13.1%, 72.1%, and 14.7% of patients with a 6-year survival rate of 89.3%, 60.6%, and 18.6%, respectively. Conclusion: GEP reporting allows prospective assessment of risk and target gene expression and integration of current prognostic factors in clinical routine, being customizable about novel parameters or other cancer entities. Clin Cancer Res; 17(23); 7240–7. ©2011 AACR.