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Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.
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Senescencia Celular , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Línea Celular Tumoral , Humanos , Melanocitos/citología , Melanocitos/metabolismo , Proteínas Proto-Oncogénicas B-rafRESUMEN
Overdiagnosis of early melanoma is a significant problem. Due to subtle unique and overlapping clinical and histological criteria between pigmented lesions and the risk of mortality from melanoma, some benign pigmented lesions are diagnosed as melanoma. Although histopathology is the gold standard to diagnose melanoma, there is a demand to find alternatives that are more accurate and cost-effective. In the current "omics" era, there is gaining interest in biomarkers to help diagnose melanoma early and to further understand the mechanisms driving tumor progression. Genomic investigations have attempted to differentiate malignant melanoma from benign pigmented lesions. However, genetic biomarkers of early melanoma diagnosis have not yet proven their value in the clinical setting. Protein biomarkers may be more promising since they directly influence tissue phenotype, a result of by-products of genomic mutations, posttranslational modifications and environmental factors. Uncovering relevant protein biomarkers could increase confidence in their use as diagnostic signatures. Currently, proteomic investigations of melanoma progression from pigmented lesions are limited. Studies have previously characterised the melanoma proteome from cultured cell lines and clinical samples such as serum and tissue. This has been useful in understanding how melanoma progresses into metastasis and development of resistance to adjuvant therapies. Currently, most studies focus on metastatic melanoma to find potential drug therapy targets, prognostic factors and markers of resistance. This paper reviews recent advancements in the genomics and proteomic fields and reports potential avenues, which could help identify and differentiate melanoma from benign pigmented lesions and prevent the progression of melanoma.
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Melanoma , Neoplasias Cutáneas , Humanos , Proteómica , Melanoma/metabolismo , Neoplasias Cutáneas/patología , Genómica , Biomarcadores , Diagnóstico PrecozRESUMEN
Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.
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Genoma Humano/genética , Melanoma/genética , Mutación/genética , ADN Helicasas/genética , GTP Fosfohidrolasas/genética , Genes p16 , Humanos , Melanoma/clasificación , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Neurofibromatosis 1/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Factores de Empalme de ARN/genética , Transducción de Señal/efectos de los fármacos , Telomerasa/genética , Telómero/genética , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta/efectos adversos , Proteína Nuclear Ligada al Cromosoma XRESUMEN
High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation "hotspot" at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.
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Genes Supresores de Tumor , Melanoma/genética , Melanoma/patología , Proteínas/genética , Línea Celular Tumoral , Amplificación de Genes , Eliminación de Gen , Homocigoto , Humanos , Mutación , Metástasis de la Neoplasia , Estadificación de NeoplasiasRESUMEN
BACKGROUND: HIV-associated dementia (HAD) is the most common dementia type in young adults less than 40 years of age. Although the neurotoxins, oxidative/metabolic stress and impaired activity of neurotrophic factors are believed to be underlying reasons for the development of HAD, the genomic basis, which ultimately defines the virus-host interaction and leads to neurologic manifestation of HIV disease is lacking. Therefore, identifying HIV fingerprints on the host gene machinery and its regulation by microRNA holds a great promise and potential for improving our understanding of HAD pathogenesis, its diagnosis and therapy. RESULTS: A parallel profiling of mRNA and miRNA of the frontal cortex autopsies from HIV positive patients with and without dementia was performed using Illumina Human-6 BeadChip and Affymetrix version 1.0 miRNA array, respectively. The gene ontology and pathway analysis of the two data sets showed high concordance between miRNA and mRNAs, revealing significant interference with the host axon guidance and its downstream signalling pathways in HAD brains. Moreover, the differentially expressed (DE) miRNAs identified in this study, in particular miR-137, 153 and 218, based on which most correlations were built cumulatively targeted neurodegeneration related pathways, implying their future potential in diagnosis, prognosis and possible therapies for HIV-mediated and possibly other neurodegenerative diseases. Furthermore, this relationship between DE miRNAs and DE mRNAs was also reflected in correlation analysis using Bayesian networks by splitting-averaging strategy (SA-BNs), which revealed 195 statistically significant correlated miRNA-mRNA pairs according to Pearson's correlation test (P<0.05). CONCLUSIONS: Our study provides the first evidence on unambiguous support for intrinsic functional relationship between mRNA and miRNA in the context of HIV-mediated neurodegeneration, which shows that neurologic manifestation in HIV patients possibly occurs through the interference with the host axon guidance and its downstream signalling pathways. These data provide an excellent avenue for the development of new generation of diagnostic/prognostic biomarkers and therapeutic intervention strategies for HIV-associated neurodegeneration.
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Complejo SIDA Demencia/genética , Axones/patología , Lóbulo Frontal/metabolismo , Infecciones por VIH/genética , VIH/fisiología , MicroARNs/metabolismo , Transducción de Señal/genética , Complejo SIDA Demencia/diagnóstico , Complejo SIDA Demencia/patología , Adulto , Diagnóstico Precoz , Lóbulo Frontal/patología , Lóbulo Frontal/virología , Perfilación de la Expresión Génica , Genoma Humano/genética , Genómica , Infecciones por VIH/complicaciones , Infecciones por VIH/patología , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In this modern era of precision medicine, molecular signatures identified from advanced omics technologies hold great promise to better guide clinical decisions. However, current approaches are often location-specific due to the inherent differences between platforms and across multiple centres, thus limiting the transferability of molecular signatures. We present Cross-Platform Omics Prediction (CPOP), a penalised regression model that can use omics data to predict patient outcomes in a platform-independent manner and across time and experiments. CPOP improves on the traditional prediction framework of using gene-based features by selecting ratio-based features with similar estimated effect sizes. These components gave CPOP the ability to have a stable performance across datasets of similar biology, minimising the effect of technical noise often generated by omics platforms. We present a comprehensive evaluation using melanoma transcriptomics data to demonstrate its potential to be used as a critical part of a clinical screening framework for precision medicine. Additional assessment of generalisation was demonstrated with ovarian cancer and inflammatory bowel disease studies.
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The heterogeneity of multiple case breast cancer families that do not carry mutations in BRCA1 or BRCA2 (non-BRCA1/2 families) poses a challenge to the identification of breast cancer susceptibility genes. The aim of this study was to determine whether intrafamilial concordance in breast cancer pathology could identify subgroups of non-BRCA1/2 families with consistent genotypic features. Invasive breast cancers were reviewed from 84 individuals belonging to 30 multiple-case families; BRCA1 (n = 9), BRCA2 (n = 10), and non-BRCA1/2 (n = 11). Hierarchical cluster analysis based on histopathology and age at first diagnosis was then used to specify three subgroups designated Clusters 1-3. The genomic features of non-BRCA1/2 families were examined by genome wide linkage and FGFR2 SNP genotyping, according to whether they showed cluster-concordant or cluster-mixed familial pathology. The majority of pathogenic BRCA1 mutation carriers (80%) fell into a single cluster. In contrast pathogenic BRCA2 mutation carriers were distributed across all three clusters and within families, cluster groups were also generally mixed. Most non-BRCA1/2 mutation carriers belonged to Cluster 3 (71%). Genome wide linkage data from five non-BRCA1/2 Cluster 3-concordant families were compared with four mixed cluster non-BRCA1/2 families. This revealed a number of distinct linkage peaks, including some regions previously associated with breast cancer susceptibility. The distribution of low risk alleles in FGFR2 was not different between these two subgroups (P = 0.237). The pattern of breast cancer pathology concordance amongst family members may assist the investigation of breast cancer susceptibility in multiple case families.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Análisis por Conglomerados , Estudios de Asociación Genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/epidemiología , Salud de la Familia , Femenino , Genes BRCA1 , Genes BRCA2 , Ligamiento Genético , Genotipo , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Riesgo , Factores de RiesgoRESUMEN
Mutations in the DNA mismatch repair gene MSH2 lead to increased replication error and microsatellite instability and account for a substantial proportion of hereditary non-polyposis colorectal cancer (Lynch syndrome). A recent international collaborative genome-wide linkage scan (GWS) for breast cancer susceptibility loci found some evidence for there being a breast cancer susceptibility gene in a genomic region on chromosome 2p close to MSH2. We sought to investigate the possibility that mutations in MSH2 might explain the multiple cases of breast cancer in some families that were included in the international GWS. DNA samples from the affected probands of 59 multiple-case breast cancer families, many of whom gave LOD scores >0.5 in the MSH2 region, were screened for large genomic alterations in MSH2 via the Multiplex Ligation-dependent Probe Amplification (MLPA) assay and for coding region mutations via exonic sequencing. Several of the families also contained cases of colorectal cancer in addition to breast cancer and had been included in the GWS that had identified a positive LOD score on chromosome 2p. Using MLPA, c.1236C > T was identified in one proband but this variant was not predicted to create an alternate acceptor/donor site within exon 7 MSH2 using in silico analyses. A c.1734T > C was identified in a second proband via exonic sequencing but testing of the variant in other family members did not support segregation of this variant with disease. Extensive screening of 59 multiple-case breast cancer families did not identify any coding region mutations or larger genomic alterations in MSH2 that might implicate MSH2 as a breast cancer susceptibility gene.
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Neoplasias de la Mama/genética , Proteína 2 Homóloga a MutS/genética , Australia/epidemiología , Neoplasias de la Mama/epidemiología , Estudios de Casos y Controles , Reparación de la Incompatibilidad de ADN , Femenino , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Mutación , Sistema de Registros , Medición de Riesgo , Factores de RiesgoRESUMEN
INTRODUCTION: BRCA1 is involved in numerous essential processes in the cell, and the effects of BRCA1 dysfunction in breast cancer carcinogenesis are well described. Many of the breast cancer susceptibility genes such as BRCA2, p53, ATM, CHEK2, and BRIP1 encode proteins that interact with BRCA1. BCL6 corepressor-like 1 (BCoR-L1) is a newly described BRCA1-interacting protein that displays high homology to several proteins known to be involved in the fundamental processes of DNA damage repair and transcription regulation. BCoR-L1 has been shown to play a role in transcription corepression, and expression of the X-linked BCoR-L1 gene has been reported to be dysregulated in breast cancer subjects. BCoR-L1 is located on the X chromosome and is subject to X inactivation. METHODS: We performed mutation analysis of 38 BRCA1/2 mutation-negative breast cancer families with male breast cancer, prostate cancer, and/or haplotype sharing around BCoR-L1 to determine whether there is a role for BCoR-L1 as a high-risk breast cancer predisposition gene. In addition, we conducted quantitative real-time PCR (qRT-PCR) on lymphoblastoid cell lines (LCLs) from the index cases from these families and a number of cancer cell lines to assess the role of BCoR-L1 dysregulation in cancer and cancer families. RESULTS: Very little variation was detected in the coding region, and qRT-PCR analysis revealed that BCoR-L1 expression is highly variable in cancer-free subjects, high-risk breast cancer patients, and cancer cell lines. We also report the investigation of a new expression control, DIDO1 (death inducer-obliterator 1), that is superior to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and UBC (ubiquitin C) for analysis of expression in LCLs. CONCLUSION: Our results suggest that BCoR-L1 expression does not play a large role in predisposition to familial breast cancer.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Cromosomas Humanos X/genética , Variación Genética/genética , Proteínas Represoras/genética , Inactivación del Cromosoma X , Análisis Mutacional de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Mutación/genética , Neoplasias Ováricas/genética , Neoplasias de la Próstata/genética , Células Tumorales CultivadasRESUMEN
Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4-8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibitors, alternate BRAF exon 4-8 splicing is not associated with this intronic branch point mutation. We also confirm that melanoma cells expressing BRAF splicing variants retain exquisite sensitivity to existing FDA-approved MEK inhibitors.
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Purpose: To examine the relationship between immune activity, PD-L1 expression, and tumor cell signaling, in metastatic melanomas prior to and during treatment with targeted MAPK inhibitors.Experimental Design: Thirty-eight tumors from 17 patients treated with BRAF inhibitor (n = 12) or combination BRAF/MEK inhibitors (n = 5) with known PD-L1 expression were analyzed. RNA expression arrays were performed on all pretreatment (PRE, n = 17), early during treatment (EDT, n = 8), and progression (PROG, n = 13) biopsies. HLA-A/HLA-DPB1 expression was assessed by IHC.Results: Gene set enrichment analysis (GSEA) of PRE, EDT, and PROG melanomas revealed that transcriptome signatures indicative of immune cell activation were strongly positively correlated with PD-L1 staining. In contrast, MAPK signaling and canonical Wnt/-ß-catenin activity was negatively associated with PD-L1 melanoma expression. The expression of PD-L1 and immune activation signatures did not simply reflect the degree or type of immune cell infiltration, and was not sufficient for tumor response to MAPK inhibition.Conclusions: PD-L1 expression correlates with immune cells and immune activity signatures in melanoma, but is not sufficient for tumor response to MAPK inhibition, as many PRE and PROG melanomas displayed both PD-L1 positivity and immune activation signatures. This confirms that immune escape is common in MAPK inhibitor-treated tumors. This has important implications for the selection of second-line immunotherapy because analysis of mechanisms of immune escape will likely be required to identify patients likely to respond to such therapies. Clin Cancer Res; 23(20); 6054-61. ©2017 AACR.
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Antígeno B7-H1/genética , Resistencia a Antineoplásicos/genética , Expresión Génica , Melanoma/genética , Melanoma/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Escape del Tumor/genética , Escape del Tumor/inmunología , Adulto , Anciano , Antígeno B7-H1/metabolismo , Biopsia , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Melanoma/tratamiento farmacológico , Melanoma/patología , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Mutación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Escape del Tumor/efectos de los fármacosRESUMEN
INTRODUCTION: Human RAD51 is a homologue of the Escherichia coli RecA protein and is known to function in recombinational repair of double-stranded DNA breaks. Mutations in the lower eukaryotic homologues of RAD51 result in a deficiency in the repair of double-stranded DNA breaks. Loss of RAD51 function would therefore be expected to result in an elevated mutation rate, leading to accumulation of DNA damage and, hence, to increased cancer risk. RAD51 interacts directly or indirectly with a number of proteins implicated in breast cancer, such as BRCA1 and BRCA2. Similar to BRCA1 mice, RAD51-/- mice are embryonic lethal. The RAD51 gene region has been shown to exhibit loss of heterozygosity in breast tumours, and deregulated RAD51 expression in breast cancer patients has also been reported. Few studies have investigated the role of coding region variation in the RAD51 gene in familial breast cancer, with only one coding region variant--exon 6 c.449G>A (p.R150Q)--reported to date. METHODS: All nine coding exons of the RAD51 gene were analysed for variation in 46 well-characterised, BRCA1/2-negative breast cancer families using denaturing high-performance liquid chromatography. Genotyping of the exon 6 p.R150Q variant was performed in an additional 66 families. Additionally, lymphoblastoid cell lines from breast cancer patients were subjected to single nucleotide primer extension analysis to assess RAD51 expression. RESULTS: No coding region variation was found, and all intronic variation detected was either found in unaffected controls or was unlikely to have functional consequences. Single nucleotide primer extension analysis did not reveal any allele-specific changes in RAD51 expression in all lymphoblastoid cell lines tested. CONCLUSION: Our study indicates that RAD51 is not a major familial breast cancer predisposition gene.
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Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Recombinasa Rad51/genética , Adulto , Edad de Inicio , Estudios de Casos y Controles , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Linaje , Células Tumorales CultivadasRESUMEN
INTRODUCTION: The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is a multidisciplinary, collaborative framework for the investigation of familial breast cancer. Based in Australia, the primary aim of kConFab is to facilitate high-quality research by amassing a large and comprehensive resource of epidemiological and clinical data with biospecimens from individuals at high risk of breast and/or ovarian cancer, and from their close relatives. METHODS: Epidemiological, family history and lifestyle data, as well as biospecimens, are collected from multiple-case breast cancer families ascertained through family cancer clinics in Australia and New Zealand. We used the Tyrer-Cuzick algorithms to assess the prospective risk of breast cancer in women in the kConFab cohort who were unaffected with breast cancer at the time of enrolment in the study. RESULTS: Of kConFab's first 822 families, 518 families had multiple cases of female breast cancer alone, 239 had cases of female breast and ovarian cancer, 37 had cases of female and male breast cancer, and 14 had both ovarian cancer as well as male and female breast cancer. Data are currently held for 11,422 people and germline DNAs for 7,389. Among the 812 families with at least one germline sample collected, the mean number of germline DNA samples collected per family is nine. Of the 747 families that have undergone some form of mutation screening, 229 (31%) carry a pathogenic or splice-site mutation in BRCA1 or BRCA2. Germline DNAs and data are stored from 773 proven carriers of BRCA1 or BRCA1 mutations. kConFab's fresh tissue bank includes 253 specimens of breast or ovarian tissue--both normal and malignant--including 126 from carriers of BRCA1 or BRCA2 mutations. CONCLUSION: These kConFab resources are available to researchers anywhere in the world, who may apply to kConFab for biospecimens and data for use in ethically approved, peer-reviewed projects. A high calculated risk from the Tyrer-Cuzick algorithms correlated closely with the subsequent occurrence of breast cancer in BRCA1 and BRCA2 mutation positive families, but this was less evident in families in which no pathogenic BRCA1 or BRCA2 mutation has been detected.
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Neoplasias de la Mama/genética , Genes BRCA1 , Genes BRCA2 , Neoplasias Ováricas/genética , Bancos de Tejidos , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Algoritmos , Australia , Estudios de Cohortes , Análisis Mutacional de ADN , Recolección de Datos , Estudios Epidemiológicos , Femenino , Mutación de Línea Germinal , Humanos , Estilo de Vida , Persona de Mediana Edad , Nueva Zelanda , Linaje , Investigación/organización & administración , Factores de Riesgo , Manejo de EspecímenesRESUMEN
BACKGROUND: The ATM gene encoding a putative protein kinase is mutated in ataxia-telangiectasia (A-T), an autosomal recessive disorder with a predisposition for cancer. Studies of A-T families suggest that female heterozygotes have an increased risk of breast cancer compared with noncarriers. However, neither linkage analyses nor mutation studies have provided supporting evidence for a role of ATM in breast cancer predisposition. Nevertheless, two recurrent ATM mutations, T7271G and IVS10-6T-->G, reportedly increase the risk of breast cancer. We examined these two ATM mutations in a population-based, case-control series of breast cancer families and multiple-case breast cancer families. METHODS: Five hundred twenty-five or 262 case patients with breast cancer and 381 or 68 control subjects, respectively, were genotyped for the T7271G and IVS10-6T-->G ATM mutations, as were index patients from 76 non-BRCA1/2 multiple-case breast cancer families. Linkage and penetrance were analyzed. ATM protein expression and kinase activity were analyzed in lymphoblastoid cell lines from mutation carriers. All statistical tests were two-sided. RESULTS: In case and control subjects unselected for family history of breast cancer, one case patient had the T7271G mutation, and none had the IVS10-6T-->G mutation. In three multiple-case families, one of these two mutations segregated with breast cancer. The estimated average penetrance of the mutations was 60% (95% confidence interval [CI] = 32% to 90%) to age 70 years, equivalent to a 15.7-fold (95% CI = 6.4-fold to 38.0-fold) increased relative risk compared with that of the general population. Expression and activity analyses of ATM in heterozygous cell lines indicated that both mutations are dominant negative. CONCLUSION: At least two ATM mutations are associated with a sufficiently high risk of breast cancer to be found in multiple-case breast cancer families. Full mutation analysis of the ATM gene in such families could help clarify the role of ATM in breast cancer susceptibility.
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Neoplasias de la Mama/genética , Genes Dominantes/genética , Pruebas Genéticas , Mutación/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Mapeo Cromosómico , Análisis Mutacional de ADN , Proteínas de Unión al ADN , Femenino , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Escala de Lod , Masculino , Repeticiones de Microsatélite/genética , Linaje , Proteínas Serina-Treonina Quinasas/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard. In this study of ten melanoma patients, we compared WES data produced from analysis of gDNA isolated from FFPE tumour tissue with that isolated from fresh-frozen tumour tissue from the same specimen. FFPE samples were sequenced using both Illumina's Nextera and NimbleGen SeqCap exome capture kits. To examine mutations between the two tissue sources and platforms, somatic mutations in the FFPE exomes were called using the matched fresh tissue sequence as a reference. Of the 10 FFPE DNA samples, seven Nextera and four SeqCap samples passed library preparation. On average, there were 5341 and 2246 variants lost in FFPE compared to matched fresh tissue utilising Nextera and SeqCap kits, respectively. In order to explore the feasibility of future clinical implementation of WES, FFPE variants in 27 genes of important clinical relevance in melanoma were assessed. The average concordance rate was 43.2% over a total of 1299 calls for the chosen genes in the FFPE DNA. For the current clinically most important melanoma mutations, 0/3 BRAF and 6/8 (75%) NRAS FFPE calls were concordant with the fresh tissue result, which was confirmed using a Sequenom OncoCarta Panel. The poor performance of FFPE WES indicates that specialised library construction to account for low quality DNA and further refinements will be necessary before this approach could be used for routine clinical decision making over currently preferred techniques.
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Toma de Decisiones Clínicas , ADN de Neoplasias/química , Exoma/genética , Melanoma/diagnóstico , Australia , ADN de Neoplasias/genética , Formaldehído , Secciones por Congelación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Melanoma/genética , Melanoma/patología , Mutación , Adhesión en Parafina , Análisis de Secuencia de ADNRESUMEN
INTRODUCTION: Mutations in known predisposition genes account for only about a third of all multiple-case breast cancer families. We hypothesized that germline mutations in FANCD2, BRIP1/BACH1, LMO4 and SFN may account for some of the unexplained multiple-case breast cancer families. METHODS: The families used in this study were ascertained through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab). Denaturing high performance liquid chromatography (DHPLC) analysis of the coding regions of these four genes was conducted in the youngest affected cases of 30 to 267 non-BRCA1/2 breast cancer families. In addition, a further 399 index cases were also screened for mutations in two functionally significant regions of the FANCD2 gene and 253 index cases were screened for two previously reported mutations in BACH1 (p. P47A and p. M299I). RESULTS: DHPLC analysis of FANCD2 identified six silent exonic variants, and a large number of intronic variants, which tagged two common haplotypes. One protein truncating variant was found in BRIP1/BACH1, as well as four missense variants, a silent change and a variant in the 3' untranslated region. No missense or splice site mutations were found in LMO4 or SFN. Analysis of the missense, silent and frameshift variants of FANCD2 and BACH1 in relatives of the index cases, and in a panel of controls, found no evidence suggestive of pathogenicity. CONCLUSION: There is no evidence that highly penetrant exonic or splice site mutations in FANCD2, BRIP1/BACH1, LMO4 or SFN contribute to familial breast cancer. Large scale association studies will be necessary to determine whether any of the polymorphisms or haplotypes identified in these genes contributes to breast cancer risk.
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Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Proteínas 14-3-3 , Proteínas Adaptadoras Transductoras de Señales , Adulto , Edad de Inicio , Anciano , Empalme Alternativo , Biomarcadores de Tumor/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Exonucleasas/genética , Exorribonucleasas , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Femenino , Mutación de Línea Germinal , Proteínas de Homeodominio/genética , Humanos , Proteínas con Dominio LIM , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Linaje , ARN Helicasas/genética , Factores de Riesgo , Factores de Transcripción/genéticaRESUMEN
The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.
Asunto(s)
Genes Supresores de Tumor , Melanoma/genética , Melanoma/metabolismo , Complejo Represivo Polycomb 2/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Factor de Transcripción Activador 3/genética , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Epigenómica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genotipo , Histonas/química , Humanos , Indoles/química , Concentración 50 Inhibidora , Mutación , Piridonas/química , Regulación hacia ArribaRESUMEN
BACKGROUND: Acquired resistance to BRAF inhibitors (BRAFi) is a near-universal phenomenon caused by numerous genetic and non-genetic alterations. In this study, we evaluated the spectrum, onset, pattern of progression, and subsequent clinical outcomes associated with specific mechanisms of resistance. METHODS: We compiled clinical and genetic data from 100 patients with 132 tissue samples obtained at progression on BRAFi therapy from 3 large, previously published studies of BRAFi resistance. These samples were subjected to whole-exome sequencing and/or polymerase chain reaction-based genetic testing. RESULTS: Among 132 samples, putative resistance mechanisms were identified in 58%, including NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAF(V600E/K) amplifications (13%), MEK1/2 mutations (7%), and non-mitogen-activated protein kinase pathway alterations (11%). Marked heterogeneity was observed within tumors and patients; 18 of 19 patients (95%) with more than one progression biopsy had distinct/unknown drivers of resistance between samples. NRAS mutations were associated with vemurafenib use (p = 0.045) and intracranial metastases (p = 0.036), and MEK1/2 mutations correlated with hepatic progression (p = 0.011). Progression-free survival and overall survival were similar across resistance mechanisms. The median survival after disease progression was 6.9 months, and responses to subsequent BRAF and MEK inhibition were uncommon (2 of 15; 13%). Post-progression outcomes did not correlate with specific acquired BRAFi-resistance mechanisms. CONCLUSIONS: This is the first study to systematically characterise the clinical implications of particular acquired BRAFi-resistance mechanisms in patients with BRAF-mutant melanoma largest study to compile the landscape of resistance. Despite marked heterogeneity of resistance mechanisms within patients, NRAS mutations correlated with vemurafenib use and intracranial disease involvement.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Resistencia a Antineoplásicos , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Australia , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Análisis Mutacional de ADN , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Europa (Continente) , Femenino , Predisposición Genética a la Enfermedad , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/enzimología , Melanoma/genética , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Mutación , Fenotipo , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Factores de Tiempo , Resultado del Tratamiento , Estados UnidosRESUMEN
Whole genome sequencing (WGS) of cancer patients' tumours offers the most comprehensive method of identifying both novel and known clinically-actionable genomic targets. However, the practicalities of performing WGS on clinical samples are poorly defined.This study was designed to test sample preparation, sequencing specifications and bioinformatic algorithms for their effect on accuracy and cost-efficiency in a large WGS analysis of human melanoma samples.WGS was performed on melanoma cell lines (nâ=â15) and melanoma fresh frozen tumours (nâ=â222). The appropriate level of coverage and the optimal mutation detection algorithm for the project pipeline were determined.An incremental increase in sequencing coverage from 36X to 132X in melanoma tissue samples and 30X to 103X for cell lines only resulted in a small increase (1-2%) in the number of mutations detected, and the quality scores of the additional mutations indicated a low probability that the mutations were real. The results suggest that 60X coverage for melanoma tissue and 40X for melanoma cell lines empower the detection of 98-99% of informative single nucleotide variants (SNVs), a sensitivity level at which clinical decision making or landscape research projects can be carried out with a high degree of confidence in the results. Likewise the bioinformatic mutation analysis methodology strongly influenced the number and quality of SNVs detected. Detecting mutations in the blood genomes separate to the tumour genomes generated 41% more SNVs than if the blood and melanoma tissue genomes were analysed simultaneously. Therefore, simultaneous analysis should be employed on matched melanoma tissue and blood genomes to reduce errors in mutation detection.This study provided valuable insights into the accuracy of SNV with WGS at various coverage levels in human clinical cancer specimens. Additionally, we investigated the accuracy of the publicly available mutation detection algorithms to detect cancer specific SNVs which will aid researchers and clinicians in study design and implementation of WGS for the identification of somatic mutations in other cancers.
Asunto(s)
Algoritmos , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/aislamiento & purificación , Melanoma/genética , Manejo de Especímenes/métodos , Análisis Mutacional de ADN/economía , ADN de Neoplasias/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , Análisis de Secuencia de ADN/economía , Análisis de Secuencia de ADN/métodosRESUMEN
Acquired resistance to BRAF inhibitors often involves MAPK re-activation, yet the MEK inhibitor trametinib showed minimal clinical activity in melanoma patients that had progressed on BRAF-inhibitor therapy. Selective ERK inhibitors have been proposed as alternative salvage therapies. We show that ERK inhibition is more potent than MEK inhibition at suppressing MAPK activity and inhibiting the proliferation of multiple BRAF inhibitor resistant melanoma cell models. Nevertheless, melanoma cells often failed to undergo apoptosis in response to ERK inhibition, because the relief of ERK-dependent negative feedback activated RAS and PI3K signalling. Consequently, the combination of ERK and PI3K/mTOR inhibition was effective at promoting cell death in all resistant melanoma cell models, and was substantially more potent than the MEK/PI3K/mTOR inhibitor combination. Our data indicate that a broader targeting strategy concurrently inhibiting ERK, rather than MEK, and PI3K/mTOR may circumvent BRAF inhibitor resistance, and should be considered during the clinical development of ERK inhibitors.