Expressed fusion gene landscape and its impact in multiple myeloma.

Multiple myeloma is a plasma cell malignancy characterized by recurrent IgH translocations and well described genomic heterogeneity. Although transcriptome profiles in multiple myeloma has been described, landscape of expressed fusion genes and their clinical impact remains unknown. To provide a comprehensive and detailed fusion gene cartography and suggest new mechanisms of tumorigenesis in multiple myeloma, we performed RNA sequencing in a cohort of 255 newly diagnosed and homogeneously treated multiple myeloma patients with long follow-up. Here, we report that patients have on average 5.5 expressed fusion genes. Kappa and lambda light chains and IgH genes are main partners in a third of all fusion genes. We also identify recurrent fusion genes that significantly impact both progression-free and overall survival and may act as drivers of the disease. Lastly, we find a correlation between the number of fusions, the age of patients and the clinical outcome, strongly suggesting that genomic instability drives prognosis of the disease.

A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma.

Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma.

Gene expression profile alone is inadequate in predicting complete response in multiple myeloma.

With advent of several treatment options in multiple myeloma (MM), a selection of effective regimen has become an important issue. Use of gene expression profile (GEP) is considered an important tool in predicting outcome; however, it is unclear whether such genomic analysis alone can adequately predict therapeutic response. We evaluated the ability of GEP to predict complete response (CR) in MM. GEP from pretreatment MM cells from 136 uniformly treated MM patients with response data on an IFM, France led study were analyzed. To evaluate variability in predictive power due to microarray platform or treatment types, additional data sets from three different studies (n=511) were analyzed using same methods. We used several machine learning methods to derive a prediction model using training and test subsets of the original four data sets. Among all methods employed for GEP-based CR predictive capability, we got accuracy range of 56-78% in test data sets and no significant difference with regard to GEP platforms, treatment regimens or in newly diagnosed or relapsed patients. Importantly, permuted P-value showed no statistically significant CR predictive information in GEP data. This analysis suggests that GEP-based signature has limited power to predict CR in MM, highlighting the need to develop comprehensive predictive model using integrated genomics approach.

Minor clone provides a reservoir for relapse in multiple myeloma.

Recent studies have provided direct evidence for genetic variegation in subclones for various cancer types. However, little is known about subclonal evolutionary processes according to treatment and subsequent relapse in multiple myeloma (MM). This issue was addressed in a cohort of 24 MM patients treated either with conventional chemotherapy or with the proteasome inhibitor, bortezomib. As MM is a highly heterogeneous disease associated with a large number of chromosomal abnormalities, a subset of secondary genetic events that seem to reflect progression, 1q21 gain, NF-κB-activating mutations, RB1 and TP53 deletions, was examined. By using high-resolution single-nucleotide polymorphism arrays, subclones were identified with nonlinear complex evolutionary histories. Such reordering of the spectrum of genetic lesions, identified in a third of MM patients during therapy, is likely to reflect the selection of genetically distinct subclones, not initially competitive against the dominant population but which survived chemotherapy, thrived and acquired new anomalies. In addition, the emergence of minor subclones at relapse appeared to be significantly associated with bortezomib treatment. These data support the idea that new strategies for future clinical trials in MM should combine targeted therapy and subpopulations' control to eradicate all myeloma subclones in order to obtain long-term remission.

Impact of high-risk cytogenetics and prior therapy on outcomes in patients with advanced relapsed or refractory multiple myeloma treated with lenalidomide plus dexaméthasone.

This retrospective analysis investigated the prognostic value of del(13) and t(4;14) abnormalities and the impact of prior treatment on outcomes in 207 heavily pretreated patients with relapsed or refractory multiple myeloma (MM) treated with lenalidomide plus dexamethasone. Patients with relapsed or refractory MM who had either earlier received thalidomide or bortezomib, or for whom continuation of these agents was contraindicated, and who had fluorescence in situ hybridization data available were included in the analysis. Patients with relapsed or refractory MM who received treatment with lenalidomide plus dexamethasone in the presence of del(13) and t(4;14) chromosomal abnormalities had lower overall response rates (ORRs) and shorter median progression-free survival (PFS) and overall survival (OS) compared with those who did not have these abnormalities. The results also showed that prior treatment with bortezomib was associated with shorter median PFS and OS. Progression during thalidomide therapy was the only significant independent predictor for OS and that the presence of del(13) and hemoglobin levels <10 g per 100 ml were prognostic factors for ORR and PFS, but not OS, in these heavily pretreated relapsed or refractory MM patients treated with lenalidomide plus dexamethasone.

International Myeloma Working Group molecular classification of multiple myeloma: spotlight review.

Myeloma is a malignant proliferation of monoclonal plasma cells. Although morphologically similar, several subtypes of the disease have been identified at the genetic and molecular level. These genetic subtypes are associated with unique clinicopathological features and dissimilar outcome. At the top hierarchical level, myeloma can be divided into hyperdiploid and non-hyperdiploid subtypes. The latter is mainly composed of cases harboring IgH translocations, generally associated with more aggressive clinical features and shorter survival. The three main IgH translocations in myeloma are the t(11;14)(q13;q32), t(4;14)(p16;q32) and t(14;16)(q32;q23). Trisomies and a more indolent form of the disease characterize hyperdiploid myeloma. A number of genetic progression factors have been identified including deletions of chromosomes 13 and 17 and abnormalities of chromosome 1 (1p deletion and 1q amplification). Other key drivers of cell survival and proliferation have also been identified such as nuclear factor- B-activating mutations and other deregulation factors for the cyclin-dependent pathways regulators. Further understanding of the biological subtypes of the disease has come from the application of novel techniques such as gene expression profiling and array-based comparative genomic hybridization. The combination of data arising from these studies and that previously elucidated through other mechanisms allows for most myeloma cases to be classified under one of several genetic subtypes. This paper proposes a framework for the classification of myeloma subtypes and provides recommendations for genetic testing. This group proposes that genetic testing needs to be incorporated into daily clinical practice and also as an essential component of all ongoing and future clinical trials.

[DNA microarrays: technology and new insights in oncology]. / Puces à ADN: techniques et apports en cancérologie.

INTRODUCTION: Large-scale genomic studies based on DNA microarrays improve our understanding of the pathophysiology of cancers. The use of these research findings to improve diagnosis, prognosis and treatment is the current challenge of research in genomics. EXEGESIS: The article briefly describes the DNA microarrays technology and use in oncology and underlines the steps required to translate research findings of correlations between gene expression and type of cancer, outcome or response to chemotherapy into robust clinical tools. CONCLUSION: Genomic technologies available today are sufficient to develop useful molecular markers for individual patient management.

[Genetic abnormalities in multiple myeloma: role in oncogenesis and impact on survival]. / Anomalies génétiques dans le myélome: rôle dans l'oncogenèse et implications pronostiques.

PURPOSE: Recent development of interphase fluorescence in situ hybridization (FISH) allows analysis on non-proliferant plasma cells. We describe the most frequent genetic abnormalities in multiple myeloma and their prognostic value. CURRENT KNOWLEDGE AND KEY POINTS: Most frequent genetic abnormalities are illegitimate rearrangements involving the IGH gene at 14q32 (60% of patients), hyperdiploidy (50 to 60% of patients), chromosome 13 deletion (40 to -50% of patients), chromosome 1q gain (30 to -40% of patients) chromosome 17 deletion (10% of patients). Some of these genetics abnormalities are observed in monoclonal gammopathy of undetermined significance (MGUS), a pre-malignant state. t(4;14) and t(14;16) translocations and chromosome 17 deletion negatively impact the overall survival. Patients with these genomic aberrations should be treated with specific treatment. FUTURE PROSPECTS AND PROJECTS: Identification of genetic abnormalities is important for evaluation of prognosis and treatment protocol in multiple myeloma.

Ploidy, as detected by fluorescence in situ hybridization, defines different subgroups in multiple myeloma.

Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma. However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients) or a DNA index calculation by flow cytometry (not routinely performed in myeloma). We validated a novel method based on interphase fluorescence in situ hybridization that can be utilitized to analyze almost all the patients. The method was very specific and sensitive for the detection of hyperdiploidy. Extended studies showed that most recurrent 14q32 translocations occur in nonhyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48 vs 66%). Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma.

Genetic heterogeneity in multiple myeloma.

In the past decade, many progresses have been made in our knowledge of the genetics of multiple myeloma. The use of molecular cytogenetic techniques has led to the identification of several recurrent (cyto)genetic abnormalities, representing either prognostic markers, or novel therapeutic targets. More global analyses of this genetic heterogeneity using expression array technologies should further extend our understanding of the disease, hopefully enabling some improvements in the treatment of the patients. The goal of this minireview is to summarize these recent advances.

[Manufacture of DNA chips in a laboratory for internal use: legislation and quality assurance]. / Fabrication de puces à ADN par un laboratoire pour sa propre utilisation: réglementation et assurance qualité

Manufacturing and using DNA chips in a laboratory, while respecting legality and good practices, require a review of the regulatory framework and relevant documentation for implementing a quality assurance system. Using DNA chips, either as a research tool, or as an in vitro diagnostic medical device, does not come within the same regulations: none in the first case, and european directive 98/79/CE in the second one. It is the same for research practice, for which the law to be enforced has been primarily conditioned to ethics, while carrying out medical analyses has been framed in France by the GBEA. The regulatory approach laid down in the GBEA is a first step for implementing a quality assurance system, but this must be extended to the manufacturing process of DNA chips. International standards (ISO 9001: 2000, ISO/IEC 15189...) provide documentation to meet this last requirement, but also enable one to carry on the quality approach up to the certification of the laboratory or its accreditation.

Rearrangements of the c-myc oncogene are present in 15% of primary human multiple myeloma tumors.

Rearrangements of the c-myc oncogene have been found in most plasmacytomas induced in mice and human myeloma cell lines (HMCLs) analyzed so far. However, neither induced mouse plasmacytomas nor HMCLs represent relevant models for human multiple myeloma (MM). To evaluate the incidence of c-myc rearrangements in human plasma cell dyscrasias, sets of probes were generated to allow direct assessment of c-myc translocations on interphase plasma cells by using fluorescence in situ hybridization. After validation of these probes, a large cohort of patients with either newly diagnosed MM (n = 529), relapsed MM (n = 58), primary plasma cell leukemia (PCL; n = 23), monoclonal gammopathy of undetermined significance (n = 65), or smoldering MM (n = 24) were analyzed. C-myc rearrangements were identified in 15% of patients with MM or primary PCL, independently of the stage of the disease (ie, diagnosis or relapse and MM or primary PCL). Analysis of the 2 main translocations observed on karyotyping, ie, t(8;14) and t(8;22), revealed that these specific translocations represented only 25% (23 of 91) of c-myc rearrangements. c-myc rearrangements were then correlated with several other patients' characteristics: illegitimate IgH recombinations, chromosome 13 deletions, and serum beta2-microglobulin levels. The only significant correlation was with a high beta2-microglobulin level (P =.002), although a trend for association with t(4;14) was observed (P =.08). Thus, c-myc rearrangement analysis in patients with MM revealed a strikingly lower incidence than that in HMCLs and plasmacytomas induced in mice, indicating that data obtained with these models cannot be directly extrapolated to human MM.

Negative regulation of onconstatin M signaling by suppressor of cytokine signaling (SOCS-3).

Oncostatin M (OSM) is known to inhibit the growth of melanocytes and early-stage melanomas, but this ability is lost with melanoma progression. The biological effects of OSM involve the activation of Janus kinases (Jak) and signal transducer and activator of transcription (STAT) factors. Since SOCS (suppressor of cytokine signaling) a recently described family of regulatory proteins, has been shown to act through down-regulation of Jak-STAT signaling, we investigated their putative role in the inhibition of OSM signaling in the human melanoma cell line A375. We observed that, among the SOCS family members examined, only SOCS-3 mRNA was strongly and rapidly induced by OSM. SOCS-3 protein was present within 1h and rapidly declined thereafter. Constitutive expression of SOCS-3 protein completely abolished the activation of the Jak-STAT signaling pathway as well as the Ras-MAP kinase pathway. As a result, A375 cells acquired an OSM-resistant phenotype. Our findings demonstrate that SOCS-3 is a potent regulator of OSM response and suggest that dysregulation of SOCS-3 expression could provide a mechanism for OSM resistance acquisition during tumour progression.

Negative cross-talk between interleukin-3 and interleukin-11 is mediated by suppressor of cytokine signalling-3 (SOCS-3).

Previous studies have shown that addition of interleukin-3 (IL-3) abrogated the B-cell potential of primary colonies supported by IL-11, erythropoietin, IL-7 and steel factor. However, the mechanism by which IL-3 exerts its inhibitory role is not understood. Using a variant of the mouse pro-B cell line Ba/F3 which expresses both IL-3 and IL-11 receptors, we showed that pretreatment of these cells with IL-3 before stimulation by IL-11 suppressed the tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3). This inhibition occurred within 30 min and required the synthesis of a negative regulator. The onset of IL-3-dependent inhibition was correlated temporally with the appearance of SOCS-3 (suppressor of cytokine signalling-3) protein. In addition, overexpression of SOCS-3 in the pro-B cell line effectively blocked STAT3 activation induced by IL-11. These findings establish that a cytokine (IL-3) that has been shown to modulate its own signal of activation is also able to down-regulate signalling activated by a different cytokine (IL-11). This cross-talk involves activation of the JAK (Janus kinase)/STAT signalling pathway, but not mitogen-activated protein kinase pathways, and is mediated, at least in part, by SOCS-3.

[Klein-Levin syndrome: a neurological disease with psychiatric symptoms]. / Le syndrome de Kleine-Levin: une affection neurologique à symptomatologie psychiatrique.

Kleine-Levin syndrome belongs to the recurrent hypersomnias group. It is a rare and benignant disease, occurring in young men 10 to 25 years old. The diagnosis is first clinical and the hypersomniac episodes, joined to psychiatric symptoms, are irregularly recurrent during few years. Diagnosis is uneasy during the first episode and in the attenuated forms... 500 cases have been described all around the world but it's highly likely that many patients haven't been listed. This syndrome, just like Gélineau disease, stands in the group of primary pathological hypersomnias. In a clinical point of view, the cardinal and constant symptom is hypersomnia. Psychiatric symptoms can be irregularly joined: megaphagia, sexual behavioural disorders, thymic disorders, personality modifications. The clinical examination is poor and aspecific. During an hypersomniac episode, a polygraphic recording during 24 or 48 hours will give diagnosis informations (fragmented and unstable sleep, reduction in stages 3 and 4 of non-REM sleep, reduction in REM sleep latency) and a biological and radiological evaluation will be necessary to exclude organic etiology (tumoral progres, infectious disease...). In a therapeutic point of view, prescription of psychostimulant drugs is recommended during fits and some treatments are used in a preventive way (lithium and carbamazepine).

Monoclonal antibodies against the human interleukin-11 receptor alpha-chain (IL-11Ralpha) and their use in studies of human mononuclear cells.

A panel of 14 hybridoma cell lines secreting monoclonal antibodies against the human interleukin-11 receptor alpha chain (hIL-11Ralpha) was obtained using two different approaches. Two antibodies were raised against peptides of the N- and C-terminal sequences, respectively, of the extracellular part of the hIL-11Ralpha. Another group of 12 antibodies was generated against a hybrid protein consisting of the extracellular part of the hIL-11Ralpha fused to mature full-length human IL-2. All these antibodies recognized native hIL-11Ralpha and most also recognized the denatured receptor on immunoblots after SDS-PAGE. Four different epitopes were identified on the extracellular part of the hIL-11Ralpha. One epitope, defined by the E27 antibody, is located at the N-terminus and the other three epitopes are clustered in the membrane-proximal, C-terminal region. The antibodies defining epitopes I and II recognized membrane-bound hIL-11Ralpha expressed in gp130/hIL-11Ralpha-co-transfected Ba/F3 cells. The E27 antibody cross-reacted with murine IL-11Ralpha, in agreement with the fact that the N-terminal region is highly conserved between species. The other 13 antibodies all recognized a region between amino acids 319 and 363, which is the membrane-proximal part of the hIL-11Ralpha. This region, which is less conserved between mouse and human, is shown here to be an immunodominant region. Anti-IL-11Ralpha monoclonal antibodies, which have not been described previously enabled us to explore the expression and tissue distribution of IL-11Ralpha on human peripheral blood mononuclear cells and cell lines. The antibodies provide powerful tools for the study of the regulation and function of the receptor.

Cloning and expression of CIS6, chromosome assignment to 3p22 and 2p21 by in situ hybridization.

A family of negative regulators of JAK signaling pathway referred to as suppressor of cytokines signaling (SOCS) or cytokine-inducible SH2 protein (CIS) has been recently identified. In order to find additional members of this family, we have used a consensus amino acid sequence contained in the well-conserved central SH2 domain to search DNA databases. We isolated cDNA coding for the human homologue of SOCS-5, referred to as CIS6. Northern blot analysis revealed CIS6 mRNA expression in various tissues such as heart, muscle, spleen, and thymus and in all myeloma cell lines examined. The gene was assigned to human chromosome bands 2p21 and 3p22 by in situ hybridization. CIS6 is structurally related to other members of the CIS family and therefore could act as a negative regulator of signal transduction.