RESUMEN
Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Genoma Humano/genética , Neoplasias Ováricas/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Estudios de Cohortes , Ciclina E/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/genética , Metilación de ADN , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Femenino , Genes BRCA1 , Genes BRCA2 , Genes de Neurofibromatosis 1 , Mutación de Línea Germinal/genética , Humanos , Mutagénesis/genética , Proteínas Oncogénicas/genética , Neoplasias Ováricas/tratamiento farmacológico , Fosfohidrolasa PTEN/genética , Regiones Promotoras Genéticas/genética , Proteína de Retinoblastoma/genéticaRESUMEN
BACKGROUND: Using whole exome sequencing to predict aberrations in tumours is a cost effective alternative to whole genome sequencing, however is predominantly used for variant detection and infrequently utilised for detection of somatic copy number variation. RESULTS: We propose a new method to infer copy number and genotypes using whole exome data from paired tumour/normal samples. Our algorithm uses two Hidden Markov Models to predict copy number and genotypes and computationally resolves polyploidy/aneuploidy, normal cell contamination and signal baseline shift. Our method makes explicit detection on chromosome arm level events, which are commonly found in tumour samples. The methods are combined into a package named ADTEx (Aberration Detection in Tumour Exome). We applied our algorithm to a cohort of 17 in-house generated and 18 TCGA paired ovarian cancer/normal exomes and evaluated the performance by comparing against the copy number variations and genotypes predicted using Affymetrix SNP 6.0 data of the same samples. Further, we carried out a comparison study to show that ADTEx outperformed its competitors in terms of precision and F-measure. CONCLUSIONS: Our proposed method, ADTEx, uses both depth of coverage ratios and B allele frequencies calculated from whole exome sequencing data, to predict copy number variations along with their genotypes. ADTEx is implemented as a user friendly software package using Python and R statistical language. Source code and sample data are freely available under GNU license (GPLv3) at http://adtex.sourceforge.net/.
Asunto(s)
Variaciones en el Número de Copia de ADN , Exoma , Genotipo , Neoplasias/genética , Algoritmos , Aberraciones Cromosómicas , Biología Computacional/métodos , Femenino , Genómica/métodos , Técnicas de Genotipaje , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pérdida de Heterocigocidad , Neoplasias Ováricas/genética , Polimorfismo de Nucleótido Simple , Poliploidía , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The clinical management of patients with cancer of unknown primary (CUP) is hampered by the absence of a definitive site of origin. We explored the utility of massively-parallel (next-generation) sequencing for the diagnosis of a primary site of origin and for the identification of novel treatment options. DNA enrichment by hybridization capture of 701 genes of clinical and/or biological importance, followed by massively-parallel sequencing, was performed on 16 CUP patients who had defied attempts to identify a likely site of origin. We obtained high quality data from both fresh-frozen and formalin-fixed, paraffin-embedded samples, demonstrating accessibility to routine diagnostic material. DNA copy-number obtained by massively-parallel sequencing was comparable to that obtained using oligonucleotide microarrays or quantitatively hybridized fluorescently tagged oligonucleotides. Sequencing to an average depth of 458-fold enabled detection of somatically acquired single nucleotide mutations, insertions, deletions and copy-number changes, and measurement of allelic frequency. Common cancer-causing mutations were found in all cancers. Mutation profiling revealed therapeutic gene targets and pathways in 12/16 cases, providing novel treatment options. The presence of driver mutations that are enriched in certain known tumour types, together with mutational signatures indicative of exposure to sunlight or smoking, added to clinical, pathological, and molecular indicators of likely tissue of origin. Massively-parallel DNA sequencing can therefore provide comprehensive mutation, DNA copy-number, and mutational signature data that are of significant clinical value for a majority of CUP patients, providing both cumulative evidence for the diagnosis of primary site and options for future treatment.
Asunto(s)
Terapia Molecular Dirigida/métodos , Mutación , Neoplasias Primarias Desconocidas/genética , Adulto , Anciano , Variaciones en el Número de Copia de ADN/genética , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/genética , Medicina Basada en la Evidencia/métodos , Femenino , Perfilación de la Expresión Génica/métodos , Genes Relacionados con las Neoplasias/genética , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Primarias Desconocidas/diagnóstico , Neoplasias Primarias Desconocidas/terapia , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Cancer profiling studies have had a profound impact on our understanding of the biology of cancers in a number of ways, including providing insights into the biological heterogeneity of specific cancer types, identification of novel oncogenes and tumor suppressors, and defining pathways that interact to drive the growth of individual cancers. Several large-scale genomic studies are underway that aim to catalog all biologically significant mutational events in each cancer type, and these findings will allow researchers to understand how mutational networks function within individual tumors. The identification of molecular predictive and prognostic tools to facilitate treatment decisions is an important step for individualized patient therapy and, ultimately, in improving patient outcomes. Whereas there are still significant challenges to implementing genomic testing and targeted therapy into routine clinical practice, rapid technological advancements provide hope for overcoming these obstacles.
Asunto(s)
Genómica , Mutación , Neoplasias/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Genoma Humano , Humanos , Neoplasias/patología , OncogenesRESUMEN
BACKGROUND: Androgens are critical for specifying prostate development, with the fetal prostate sensitive to altered hormone levels and endocrine-disrupting chemicals (EDCs) that exhibit estrogenic or antiandrogenic properties. Prostatic inflammation (prostatitis) affects 9% of men of all ages, and > 90% of cases are of unknown etiology. OBJECTIVES: In this study we aimed to evaluate effects of in utero exposure to the antiandrogenic EDC vinclozolin, during the period of male reproductive tract development, on neonatal, prepubertal, and postpubertal prostate gland function of male offspring. METHODS: Fetal rats were exposed to vinclozolin (100 mg/kg body weight) or vehicle control (2.5 mL/kg body weight) in utero from gestational day 14 (GD14) to GD19 via oral administration to pregnant dams. Tissue analysis was carried out when male offspring were 0, 4, or 8 weeks of age. RESULTS: In utero exposure to vinclozolin was insufficient to perturb prostatic development and branching, although expression of androgen receptor and mesenchymal fibroblast growth factor-10 was down-regulated. Prostate histology remained normal until puberty, but 100% of animals displayed prostatitis postpubertally (56 days of age). Prostatic inflammation was associated with phosphorylation and nuclear translocation of nuclear factor-kappa B (NFkappaB) and postpubertal activation of proinflammatory NFkappaB-dependent genes, including the chemokine interleukin-8 and the cytokine transforming growth factor-beta1. Significantly, inflammation arising from vinclozolin exposure was not associated with the emergence of premalignant lesions, such as prostatic intra-epithelial neoplasia or proliferative inflammatory atrophy, and hence mimics nonbacterial early-onset prostatitis that commonly occurs in young men. CONCLUSIONS: These data are the first to unequivocally implicate EDCs as a causative factor and fill an important knowledge gap on the etiology of prostatitis.
Asunto(s)
Antagonistas de Andrógenos/toxicidad , Intercambio Materno-Fetal , Oxazoles/toxicidad , Efectos Tardíos de la Exposición Prenatal , Prostatitis/inducido químicamente , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Epitelio/crecimiento & desarrollo , Epitelio/patología , Femenino , Humanos , Interleucina-8/metabolismo , Masculino , Morfogénesis , FN-kappa B/metabolismo , Embarazo , Próstata/crecimiento & desarrollo , Próstata/patología , Prostatitis/metabolismo , Prostatitis/patología , Ratas , Ratas Sprague-Dawley , Maduración Sexual , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The potential therapeutic applications of stem cells are unlimited. However, the ongoing political and social debate surrounding the intellectual property and patenting considerations of stem cell research has led to the implementation of strict legislative regulations. In Australia the patent landscape surrounding stem cells has evolved considerably over the past 20 years. The Australian Patents Act 1990 includes a specific exclusion to the patentability of human beings and of biological processes for their generation. However, this exclusion has received no judicial consideration to date, and so its scope and potential impact on stem cell patents is unclear.
Asunto(s)
Patentes como Asunto/legislación & jurisprudencia , Investigación con Células Madre/legislación & jurisprudencia , Células Madre Adultas , Australia , Técnicas de Cultivo , Células Madre Embrionarias , Predicción , Humanos , Invenciones/legislación & jurisprudenciaRESUMEN
High-grade serous cancer (HGSC), the most common subtype of ovarian cancer, often becomes resistant to chemotherapy, leading to poor patient outcomes. Intratumoral heterogeneity occurs in nearly all solid cancers, including ovarian cancer, contributing to the development of resistance mechanisms. In this study, we examined the spatial and temporal genomic variation in HGSC using high-resolution single-nucleotide polymorphism arrays. Multiple metastatic lesions from individual patients were analyzed along with 22 paired pretreatment and posttreatment samples. We documented regions of differential DNA copy number between multiple tumor biopsies that correlated with altered expression of genes involved in cell polarity and adhesion. In the paired primary and relapse cohort, we observed a greater degree of genomic change in tumors from patients that were initially sensitive to chemotherapy and had longer progression-free interval compared with tumors from patients that were resistant to primary chemotherapy. Notably, deletion or downregulation of the lipid transporter LRP1B emerged as a significant correlate of acquired resistance in our analysis. Functional studies showed that reducing LRP1B expression was sufficient to reduce the sensitivity of HGSC cell lines to liposomal doxorubicin, but not to doxorubicin, whereas LRP1B overexpression was sufficient to increase sensitivity to liposomal doxorubicin. Together, our findings underscore the large degree of variation in DNA copy number in spatially and temporally separated tumors in HGSC patients, and they define LRP1B as a potential contributor to the emergence of chemotherapy resistance in these patients.
Asunto(s)
Cistadenocarcinoma Seroso/tratamiento farmacológico , Doxorrubicina/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Receptores de LDL/deficiencia , Receptores de LDL/genética , Anciano , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Aberraciones Cromosómicas , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 2 , Cromosomas Humanos X , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , ADN de Neoplasias , Regulación hacia Abajo , Resistencia a Antineoplásicos , Femenino , Eliminación de Gen , Dosificación de Gen , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Receptores de LDL/biosíntesisRESUMEN
PURPOSE: Ovarian clear cell adenocarcinoma (OCCA) is an uncommon histotype that is generally refractory to platinum-based chemotherapy. We analyze here the most comprehensive gene expression and copy number data sets, to date, to identify potential therapeutic targets of OCCA. EXPERIMENTAL DESIGN: Gene expression and DNA copy number were carried out using primary human OCCA tumor samples, and findings were confirmed by immunohistochemistry on tissue microarrays. Circulating interleukin (IL) 6 levels were measured in serum from patients with OCCA or high-grade serous cancers and related to progression-free and overall survival. Two patients were treated with sunitinib, and their therapeutic responses were measured clinically and by positron emission tomography. RESULTS: We find specific overexpression of the IL6-STAT3-HIF (interleukin 6-signal transducer and activator of transcription 3-hypoxia induced factor) pathway in OCCA tumors compared with high-grade serous cancers. Expression of PTHLH and high levels of circulating IL6 in OCCA patients may explain the frequent occurrence of hypercalcemia of malignancy and thromboembolic events in OCCA. We describe amplification of several receptor tyrosine kinases, most notably MET, suggesting other potential therapeutic targets. We report sustained clinical and functional imaging responses in two OCCA patients with chemotherapy-resistant disease who were treated with sunitinib, thus showing significant parallels with renal clear cell cancer. CONCLUSIONS: Our findings highlight important therapeutic targets in OCCA, suggest that more extensive clinical trials with sunitinib in OCCA are warranted, and provide significant impetus to the growing realization that OCCA is molecularly and clinically distinct to other forms of ovarian cancer.
Asunto(s)
Adenocarcinoma de Células Claras/tratamiento farmacológico , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Indoles/uso terapéutico , Interleucina-6/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Pirroles/uso terapéutico , Factor de Transcripción STAT3/metabolismo , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/metabolismo , Adulto , Anciano , Inhibidores de la Angiogénesis/uso terapéutico , Línea Celular Tumoral , Análisis por Conglomerados , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Interleucina-6/sangre , Interleucina-6/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Sunitinib , Análisis de Matrices Tisulares , Resultado del TratamientoRESUMEN
Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Neoplasias Ováricas/genética , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteína Proto-Oncogénica N-Myc , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vinclozolin is an endocrine-disrupting chemical (EDC) that binds with high affinity to the androgen receptor (AR) and blocks the action of gonadal hormones on male reproductive organs. An alternative mechanism of action of Vinclozolin involves transgenerational effects on the male reproductive tract. We previously reported in utero Vinclozolin exposure-induced prostatitis (prostate inflammation) in postpubertal rats concurrent with down-regulation of AR and increased nuclear factor-kappaB activation. We postulated the male reproductive abnormalities induced by in utero Vinclozolin exposure could be reversed by testosterone supplementation, in contrast to the permanent modifications involving DNA methyltransferases (Dnmts) described by others. To test this hypothesis, we administered high-dose testosterone at puberty to Vinclozolin-treated rats and determined the effect on anogenital distance (AGD); testicular germ cell apoptosis, concentration of elongated spermatids, and the onset of prostatitis. Concurrently we examined Dnmt1, -3A, -3B, and -3L mRNA expression. Consistent with previous reports, in utero exposure to Vinclozolin significantly reduced AGD, increased testicular germ cell apoptosis 3-fold, reduced elongated spermatid number by 40%, and induced postpubertal prostatitis in 100% of exposed males. Administration of high-dose testosterone (25 mg/kg) at puberty normalized AGD, reduced germ cell apoptosis, and restored elongated spermatid number. Testosterone restored AR and nuclear factor-kappaB expression in the prostate and abolished Vinclozolin-induced prostatitis. Altered Dnmt expression was evident with in utero Vinclozolin exposure and was not normalized after testosterone treatment. These data demonstrate in utero Vinclozolin-induced male reproductive tract abnormalities are AR mediated and reversible and involve a mechanism independent of Dnmt expression.
Asunto(s)
Oxazoles/farmacología , Prostatitis/inducido químicamente , Canal Anal/anatomía & histología , Antagonistas de Andrógenos/farmacología , Animales , Apoptosis/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Femenino , Masculino , Ratones , Ratones Noqueados , Embarazo , Próstata/efectos de los fármacos , Próstata/embriología , Próstata/patología , Prostatitis/prevención & control , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermátides/efectos de los fármacos , Espermátides/fisiología , Testículo/anatomía & histología , Testículo/efectos de los fármacos , Testosterona/uso terapéuticoRESUMEN
Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.
Asunto(s)
Aberraciones Cromosómicas , Cromosomas Humanos Par 20/genética , Cisplatino/farmacología , Ciclina E/genética , Proteínas Oncogénicas/genética , Antineoplásicos/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cromosomas Humanos Par 19/genética , Ciclina E/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Amplificación de Genes , Humanos , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions.
Asunto(s)
Cromosomas Humanos/genética , Dosificación de Gen , Neoplasias Ováricas/genética , Adulto , Anciano , Femenino , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/patologíaRESUMEN
Rodent models and immortalized or genetically modified cell lines are frequently used-but have limited utility-for studying human prostate development and maturation. Using rodent mesenchyme to establish reciprocal mesenchymal-epithelial cell interactions with human embryonic stem cells (hESCs), we generated human prostate tissue expressing prostate-specific antigen (PSA) within 8-12 weeks. This human prostate model shows species-conserved signalling mechanisms that could extend to integumental, gastrointestinal and genital tissues.