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1.
Nature ; 541(7637): 359-364, 2017 01 19.
Article in English | MEDLINE | ID: mdl-28068672

ABSTRACT

Prostate tumours are highly variable in their response to therapies, but clinically available prognostic factors can explain only a fraction of this heterogeneity. Here we analysed 200 whole-genome sequences and 277 additional whole-exome sequences from localized, non-indolent prostate tumours with similar clinical risk profiles, and carried out RNA and methylation analyses in a subset. These tumours had a paucity of clinically actionable single nucleotide variants, unlike those seen in metastatic disease. Rather, a significant proportion of tumours harboured recurrent non-coding aberrations, large-scale genomic rearrangements, and alterations in which an inversion repressed transcription within its boundaries. Local hypermutation events were frequent, and correlated with specific genomic profiles. Numerous molecular aberrations were prognostic for disease recurrence, including several DNA methylation events, and a signature comprised of these aberrations outperformed well-described prognostic biomarkers. We suggest that intensified treatment of genomically aggressive localized prostate cancer may improve cure rates.


Subject(s)
Genome, Human/genetics , Genomics , Mutation , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Chromothripsis , DNA Copy Number Variations , DNA Methylation , Exome/genetics , Humans , Male , Neoplasm Metastasis/genetics , Prognosis , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Recurrence
3.
Nature ; 538(7625): 378-382, 2016 Oct 20.
Article in English | MEDLINE | ID: mdl-27732578

ABSTRACT

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.


Subject(s)
Carcinogenesis/genetics , Carcinogenesis/pathology , Gene Rearrangement/genetics , Genome, Human/genetics , Models, Biological , Mutagenesis/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Carcinoma in Situ/genetics , Chromothripsis , DNA Copy Number Variations/genetics , Disease Progression , Evolution, Molecular , Female , Genes, Neoplasm/genetics , Humans , Male , Mitosis/genetics , Mutation/genetics , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Polyploidy , Precancerous Conditions/genetics
4.
Nat Genet ; 47(7): 736-45, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26005866

ABSTRACT

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.


Subject(s)
Prostatic Neoplasms/genetics , Cell Line, Tumor , DNA Copy Number Variations , Genetic Association Studies , Genetic Heterogeneity , Genome, Human , Humans , Male , Middle Aged , Neoplasm Grading , Point Mutation , Polymorphism, Single Nucleotide , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-myc/genetics
5.
Nat Genet ; 46(11): 1166-9, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25240283

ABSTRACT

Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.


Subject(s)
Adenocarcinoma/genetics , Models, Molecular , Protein Kinase C/genetics , Salivary Gland Neoplasms/genetics , Adenocarcinoma/pathology , Amino Acid Sequence , Animals , Humans , Immunohistochemistry , Immunoprecipitation , Mice , Microscopy, Confocal , Molecular Sequence Data , Mutagenesis , Mutation, Missense/genetics , NIH 3T3 Cells , Protein Kinase C/chemistry , Salivary Gland Neoplasms/pathology , Sequence Alignment , Sequence Analysis, DNA
6.
PLoS One ; 9(2): e88163, 2014.
Article in English | MEDLINE | ID: mdl-24551080

ABSTRACT

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76-139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.


Subject(s)
DNA, Neoplasm/isolation & purification , Genotyping Techniques , Polymorphism, Single Nucleotide , Prostatic Neoplasms/genetics , DNA, Neoplasm/genetics , Fixatives , Formaldehyde , Genetic Markers , Humans , Male , Microsatellite Repeats , Paraffin Embedding , Prostatic Neoplasms/diagnosis , Quality Control
7.
Chem Biol Interact ; 205(1): 63-71, 2013 Sep 05.
Article in English | MEDLINE | ID: mdl-23791969

ABSTRACT

BACKGROUND: Quantitative real-time PCR (qPCR) is the "gold-standard" technique for measuring mRNA abundances. To correctly compare samples and generate biologically valid results, qPCR data usually require comprehensive normalization to account for sample content variation between reactions. The most common normalization approaches use one or more endogenous controls (reference or house-keeping genes) to adjust the measured levels of experimental genes appropriately. Ideal reference genes are those that display minimal variation across experimental conditions, and thus can vary widely across different biological systems. In particular, toxicogenomic studies of transcriptionally-disruptive toxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. RESULTS: We examined seven candidate reference genes in 199 mice varying in genotype and time/dose of TCDD exposure. We assessed gene-stability in four ways: (1) the variance of the raw Cq values across biological replicates, (2) the fold-change from basal mRNA levels following treatment, (3) the inter- and intra-group stability evaluated using the NormFinder algorithm, (4) the comparative ΔCq method for each candidate gene. Univariate analyses showed Hprt and Eef1a1 are the two most stable individual reference genes. It has been suggested that using multiple genes would produce a more consistent normalization factor; multivariate analysis was performed using NormFinder. In general, stability increased with the number of genes used, but specific gene-combinations synergized. CONCLUSIONS: We have validated seven reference genes for use in analyzing mRNA abundances in mouse models of TCDD toxicity. The use of multiple reference genes increases stability, providing more consistent normalization and more reliable results. The number of reference genes used should be maximized, based on experimental capabilities (platform, sample availability, etc.). Our results show the benefit of validating reference genes using multiple methods prior to generating large biological datasets.


Subject(s)
Genes, Essential/drug effects , Polychlorinated Dibenzodioxins/toxicity , Algorithms , Animals , Female , Gene Expression/drug effects , Hypoxanthine Phosphoribosyltransferase/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Multivariate Analysis , Peptide Elongation Factor 1/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction
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