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Assessment of MEK-ERK pathway targeting by BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias [Patient samples]

ABSTRACT: Multiple myeloma (MM) is a malignant disorder characterized by the clonal proliferation of plasma cells (PCs) in the bone marrow (BM). The genetic background and clinical course of the disease are largely heterogeneous, and MM pathophysiology ranges from the premalignant condition of monoclonal gammopathy of undetermined significance (MGUS) to smoldering MM, symptomatic MM, and extramedullary MM/plasma cell leukemia (PCL). Recent genome-wide sequencing efforts have provided the rationale for molecularly aimed treatment approaches, identifying mutations that can be specifically targeted, such as those in the mitogen-activated protein kinase (MAPK) pathway, which represent the most prevalent mutations in MM. Among these, mutations affecting BRAF gene, detected in 4-15% of patients, are of potential immediate clinical relevance due to the availability of effective inhibitors of this serine-threonine kinase which are in fact being explored also in myeloma. In this study, we screened by next generation sequencing (NGS) a large and representative series of intramedullary and extramedullary MM patients, including primary and secondary plasma cell leukemia (pPCL and sPCL, respectively), for mutations in BRAF, NRAS and KRAS genes. We evaluated the relationship of identified variants with other clinical and biological features and determined the transcriptional signature associated with MAPK pathway activation in MM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE66291 | GEO | 2015/07/01

SECONDARY ACCESSION(S): PRJNA276449

REPOSITORIES: GEO

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Project description:Multiple myeloma (MM) is a malignant disorder characterized by the clonal proliferation of plasma cells (PCs) in the bone marrow (BM). The genetic background and clinical course of the disease are largely heterogeneous, and MM pathophysiology ranges from the premalignant condition of monoclonal gammopathy of undetermined significance (MGUS) to smoldering MM, symptomatic MM, and extramedullary MM/plasma cell leukemia (PCL). Recent genome-wide sequencing efforts have provided the rationale for molecularly aimed treatment approaches, identifying mutations that can be specifically targeted, such as those in the mitogen-activated protein kinase (MAPK) pathway, which represent the most prevalent mutations in MM. Among these, mutations affecting BRAF gene, detected in 4-15% of patients, are of potential immediate clinical relevance due to the availability of effective inhibitors of this serine-threonine kinase which are in fact being explored also in myeloma. In this study, we screened by next generation sequencing (NGS) a large and representative series of intramedullary and extramedullary MM patients, including primary and secondary plasma cell leukemia (pPCL and sPCL, respectively), for mutations in BRAF, NRAS and KRAS genes. We evaluated the relationship of identified variants with other clinical and biological features and determined the transcriptional signature associated with MAPK pathway activation in MM. To further elucidate the transcriptional programs modulated by BRAF activation in MM, we used the PLX4032 drug to inhibit BRAF activity in U266 human myeloma cell line (HMCL), carrying K601N mutation and showing constitutive activation of MEK/ERK signaling. After confirming its ability to suppress MAPK pathway and myeloma cell proliferation in culture in the U266 cell line, we investigated the specific modulation of gene expression induced by the drug. U266 cells were treated with PLX4032 (30 µM) or DMSO for 12 hours and subjected to gene expression profiling (GEP) analysis by using Affymetrix GeneChip Human Gene 1.0ST arrays.

Project description:Multiple myeloma (MM) is a malignant disorder characterized by the clonal proliferation of plasma cells (PCs) in the bone marrow (BM). The genetic background and clinical course of the disease are largely heterogeneous, and MM pathophysiology ranges from the premalignant condition of monoclonal gammopathy of undetermined significance (MGUS) to smoldering MM, symptomatic MM, and extramedullary MM/plasma cell leukemia (PCL). Recent genome-wide sequencing efforts have provided the rationale for molecularly aimed treatment approaches, identifying mutations that can be specifically targeted, such as those in the mitogen-activated protein kinase (MAPK) pathway, which represent the most prevalent mutations in MM. Among these, mutations affecting BRAF gene, detected in 4-15% of patients, are of potential immediate clinical relevance due to the availability of effective inhibitors of this serine-threonine kinase which are in fact being explored also in myeloma. In this study, we screened by next generation sequencing (NGS) a large and representative series of intramedullary and extramedullary MM patients, including primary and secondary plasma cell leukemia (pPCL and sPCL, respectively), for mutations in BRAF, NRAS and KRAS genes. We evaluated the relationship of identified variants with other clinical and biological features and determined the transcriptional signature associated with MAPK pathway activation in MM. To identify transcriptomic profiles possibly related to BRAF, NRAS and KRAS mutations found in our study, we investigated by Affymetrix microarray technology a large fraction (n=142) of the samples analyzed by NGS, including wild-type patients for all the three genes and cases carrying mutations in at least one of them. Assuming that alterations present in a very limited number of malignant PCs might not appreciably affect gene expression, we chose to arbitrarily establish 20% as the lower variant allele frequency cut-off to perform supervised analyses.

Project description:Plasma cell leukemia (PCL) is a rare form of plasma cell dyscrasia that presents either as a progression of previously diagnosed multiple myeloma (MM), namely secondary PCL (sPCL), or as the initial manifestation of disease, namely primary PCL (pPCL). Although presenting signs and symptoms include those seen in MM, pPCL is characterized by several aspects that clearly define more aggressive course. To provide insights into the biology of pPCL, we have investigated the transcriptional profiles of a cohort of 21 newly-diagnosed, homogeneously treated pPCL patients included in a multicenter prospective clinical trial. All but one pPCL had one of the main IGH translocations, whose associated transcriptional signatures resembled those observed in MM. A 503-gene signature was identified that distinguished pPCL from MM, from which emerged 28 genes whose trend in expression levels was found associated with the progressive stages of plasma cell dyscrasia in a large dataset of cases from multiple institutions, including samples from normal donors throughout PCL. The transcriptional pattern of the pPCL series was then evaluated in association with outcome. Three genes were identified having expression levels correlated with response to the first-line treatment with lenalidomide/dexamethasone, whereas a 27-gene signature was identified associated with overall survival independently of molecular alterations, hematological parameters and renal function. Overall, our data contribute to a fine dissection of pPCL and may provide novel insights into the molecular definition of a subgroup of high-risk pPCL. This series of microarray experiments contains the gene expression profiles of purified plasma cells (PCs) obtained from 21 pPCL and 55 MM at diagnosis. PCs were purified from bone marrow samples using CD138 immunomagnetic microbeads according to the manufacturer's instructions (MidiMACS system, Miltenyi Biotec); the purity of the positively selected PCs was assessed by morphology and flow cytometry and was > 90% in all cases. 5.5 micrograms of single-stranded DNA target obtained from 100 ng of purified total RNA was fragmented and then labeled using the WT Terminal Labeling Kit according to the standard Affymetrix protocol (GeneChipÂ¨ Whole Transcript (WT) Sense Target Labeling Assay Manual). The fragmented labeled single-stranded DNA target was hybridized for 16 hours and 30 minutes at 45Â¡C on GeneChipÂ¨ Gene 1.0 ST array according to the standard Affymetrix protocol. Washing and scanning were performed using GeneChip System of Affymetrix (GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450 and GeneChip Scanner 7G). Log2-transformed expression values were extracted from CEL files and normalized using NetAffx Transcript Cluster Annotations, Release 31 and robust multi-array average (RMA) procedure in Expression Console software (Affymetrix Inc.). Non-annotated transcript clusters were discarded. The expression values of transcript cluster ID specific for loci representing naturally occurring read-through transcriptions or mapped to more than one chromosomal location were summarized as median value for each sample. gene expression analysis of 21 primary Plasma Cell Leukemia and 55 multiple myeloma tumors

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Assessment of MEK-ERK pathway targeting by BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias [Patient samples]

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