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1.
Nature ; 486(7403): 395-9, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22495314

RESUMO

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Evolução Molecular , Mutação/genética , Alelos , Neoplasias da Mama/diagnóstico , Células Clonais/metabolismo , Células Clonais/patologia , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Mutação Puntual/genética , Medicina de Precisão , Reprodutibilidade dos Testes , Análise de Sequência de RNA
2.
Genome Res ; 22(10): 1995-2007, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22637570

RESUMO

Loss of heterozygosity (LOH) and copy number alteration (CNA) feature prominently in the somatic genomic landscape of tumors. As such, karyotypic aberrations in cancer genomes have been studied extensively to discover novel oncogenes and tumor-suppressor genes. Advances in sequencing technology have enabled the cost-effective detection of tumor genome and transcriptome mutation events at single-base-pair resolution; however, computational methods for predicting segmental regions of LOH in this context are not yet fully explored. Consequently, whole transcriptome, nucleotide-level resolution analysis of monoallelic expression patterns associated with LOH has not yet been undertaken in cancer. We developed a novel approach for inference of LOH from paired tumor/normal sequence data and applied it to a cohort of 23 triple-negative breast cancer (TNBC) genomes. Following extensive benchmarking experiments, we describe the nucleotide-resolution landscape of LOH in TNBC and assess the consequent effect of LOH on the transcriptomes of these tumors using RNA-seq-derived measurements of allele-specific expression. We show that the majority of monoallelic expression in the transcriptomes of triple-negative breast cancer can be explained by genomic regions of LOH and establish an upper bound for monoallelic expression that may be explained by other tumor-specific modifications such as epigenetics or mutations. Monoallelically expressed genes associated with LOH reveal that cell cycle, homologous recombination and actin-cytoskeletal functions are putatively disrupted by LOH in TNBC. Finally, we show how inference of LOH can be used to interpret allele frequencies of somatic mutations and postulate on temporal ordering of mutations in the evolutionary history of these tumors.


Assuntos
Alelos , Neoplasias da Mama/genética , Perda de Heterozigosidade , Polimorfismo de Nucleotídeo Único , Desequilíbrio Alélico , Neoplasias da Mama/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Redes Reguladoras de Genes , Genoma Humano , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Estatísticos , Mutação
3.
N Engl J Med ; 363(16): 1532-43, 2010 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-20942669

RESUMO

BACKGROUND: Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS: We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI­SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS: ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS: These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital­University of British Columbia Hospital Foundation.).


Assuntos
Adenocarcinoma de Células Claras/genética , Carcinoma Endometrioide/genética , Endometriose/complicações , Mutação , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Fatores de Transcrição/genética , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Endometriose/patologia , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
4.
Bioinformatics ; 28(7): 907-13, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22285562

RESUMO

MOTIVATION: Identification of somatic single nucleotide variants (SNVs) in tumour genomes is a necessary step in defining the mutational landscapes of cancers. Experimental designs for genome-wide ascertainment of somatic mutations now routinely include next-generation sequencing (NGS) of tumour DNA and matched constitutional DNA from the same individual. This allows investigators to control for germline polymorphisms and distinguish somatic mutations that are unique to the tumour, thus reducing the burden of labour-intensive and expensive downstream experiments needed to verify initial predictions. In order to make full use of such paired datasets, computational tools for simultaneous analysis of tumour-normal paired sequence data are required, but are currently under-developed and under-represented in the bioinformatics literature. RESULTS: In this contribution, we introduce two novel probabilistic graphical models called JointSNVMix1 and JointSNVMix2 for jointly analysing paired tumour-normal digital allelic count data from NGS experiments. In contrast to independent analysis of the tumour and normal data, our method allows statistical strength to be borrowed across the samples and therefore amplifies the statistical power to identify and distinguish both germline and somatic events in a unified probabilistic framework. AVAILABILITY: The JointSNVMix models and four other models discussed in the article are part of the JointSNVMix software package available for download at http://compbio.bccrc.ca CONTACT: sshah@bccrc.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Modelos Estatísticos , Neoplasias/genética , Humanos , Mutação , Software
5.
N Engl J Med ; 360(26): 2719-29, 2009 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-19516027

RESUMO

BACKGROUND: Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS: We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS: All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS: Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.


Assuntos
Fatores de Transcrição Forkhead/genética , Tumor de Células da Granulosa/genética , Mutação de Sentido Incorreto , Neoplasias Ovarianas/genética , Sequência de Bases , Feminino , Proteína Forkhead Box L2 , Perfilação da Expressão Gênica , Marcadores Genéticos , Genótipo , Tumor de Células da Granulosa/diagnóstico , Tumor de Células da Granulosa/patologia , Humanos , Imuno-Histoquímica , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Mutação Puntual , Análise de Sequência de RNA , Taq Polimerase
6.
PLoS Comput Biol ; 7(5): e1001138, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21625565

RESUMO

Gene fusions created by somatic genomic rearrangements are known to play an important role in the onset and development of some cancers, such as lymphomas and sarcomas. RNA-Seq (whole transcriptome shotgun sequencing) is proving to be a useful tool for the discovery of novel gene fusions in cancer transcriptomes. However, algorithmic methods for the discovery of gene fusions using RNA-Seq data remain underdeveloped. We have developed deFuse, a novel computational method for fusion discovery in tumor RNA-Seq data. Unlike existing methods that use only unique best-hit alignments and consider only fusion boundaries at the ends of known exons, deFuse considers all alignments and all possible locations for fusion boundaries. As a result, deFuse is able to identify fusion sequences with demonstrably better sensitivity than previous approaches. To increase the specificity of our approach, we curated a list of 60 true positive and 61 true negative fusion sequences (as confirmed by RT-PCR), and have trained an adaboost classifier on 11 novel features of the sequence data. The resulting classifier has an estimated value of 0.91 for the area under the ROC curve. We have used deFuse to discover gene fusions in 40 ovarian tumor samples, one ovarian cancer cell line, and three sarcoma samples. We report herein the first gene fusions discovered in ovarian cancer. We conclude that gene fusions are not infrequent events in ovarian cancer and that these events have the potential to substantially alter the expression patterns of the genes involved; gene fusions should therefore be considered in efforts to comprehensively characterize the mutational profiles of ovarian cancer transcriptomes.


Assuntos
Algoritmos , Fusão Oncogênica , Neoplasias Ovarianas/genética , Análise de Sequência de RNA/métodos , Sequência de Bases , Carcinoma/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Melanoma/genética , Dados de Sequência Molecular , Mutação , Neoplasias da Próstata/genética , Sarcoma/genética , Neoplasias Cutâneas/genética
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