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1.
Oncologist ; 19(4): 336-43, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24664487

RESUMO

PURPOSE: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. METHODS: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. RESULTS: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. CONCLUSION: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , DNA de Neoplasias/análise , Genes Neoplásicos/genética , Sequência de Bases , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA/métodos , Formaldeído , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação/genética , Parafina , Fosfatidilinositol 3-Quinases/genética , Análise de Sequência de DNA , Inclusão do Tecido , Fixação de Tecidos
2.
Breast Cancer Res Treat ; 148(2): 315-25, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25338319

RESUMO

Breast cancers are categorized into three subtypes based on protein expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/ERBB2). Patients enroll onto experimental clinical trials based on ER, PR, and HER2 status and, as receptor status is prognostic and defines treatment regimens, central receptor confirmation is critical for interpreting results from these trials. Patients enrolling onto experimental clinical trials in the metastatic setting often have limited available archival tissue that might better be used for comprehensive molecular profiling rather than slide-intensive reconfirmation of receptor status. We developed a Random Forests-based algorithm using a training set of 158 samples with centrally confirmed IHC status, and subsequently validated this algorithm on multiple test sets with known, locally determined IHC status. We observed a strong correlation between target mRNA expression and IHC assays for HER2 and ER, achieving an overall accuracy of 97 and 96%, respectively. For determining PR status, which had the highest discordance between central and local IHC, incorporation of expression of co-regulated genes in a multivariate approach added predictive value, outperforming the single, target gene approach by a 10% margin in overall accuracy. Our results suggest that multiplexed qRT-PCR profiling of ESR1, PGR, and ERBB2 mRNA, along with several other subtype associated genes, can effectively confirm breast cancer subtype, thereby conserving tumor sections and enabling additional biomarker data to be obtained from patients enrolled onto experimental clinical trials.


Assuntos
Algoritmos , Biomarcadores Tumorais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , RNA Neoplásico/genética , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Ensaios Clínicos Fase III como Assunto , Feminino , Seguimentos , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Limite de Detecção , Estudos Multicêntricos como Assunto , Análise Multivariada , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Curva ROC , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida
3.
Trends Cell Biol ; 13(3): 151-6, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12628348

RESUMO

The advent of DNA microarray technology has ushered in an era of systems biology whereby researchers can study the transcriptional behavior of thousands of genes in parallel. Advances in manufacturing techniques and informatics, and the availability of several genome sequences have furthered these capabilities to the point where whole-transcriptome studies can be accomplished in yeast, flies and plants, and soon will be possible in mammals. Concomitant with the expanding ability of the technology has been the development of novel techniques and their application towards the study of cellular biology.


Assuntos
DNA/análise , Biologia Molecular/instrumentação , Biologia Molecular/tendências , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/tendências , Animais , Regulação da Expressão Gênica/genética , Biblioteca Genômica , Humanos , Modelos Biológicos , Neoplasias/genética , RNA Mensageiro/genética
4.
Clin Cancer Res ; 13(2 Pt 1): 458-66, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17255266

RESUMO

PURPOSE: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood. EXPERIMENTAL DESIGN: We developed an algorithm to identify secreted proteins encoded among approximately 22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression. RESULTS: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (<0.5- or >2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls. CONCLUSIONS: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.


Assuntos
Carcinoma/diagnóstico , Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/genética , Algoritmos , Carcinoma/sangue , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Feminino , Perfilação da Expressão Gênica , Humanos , Programas de Rastreamento/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/diagnóstico , Prognóstico
5.
Clin Cancer Res ; 12(1): 89-96, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16397029

RESUMO

PURPOSE: Current serum testing for the detection of prostate cancer (PCa) lacks specificity. On diagnosis, the optimal therapeutic pathway is not clear and tools for adequate risk assessment of localized PCa progression are not available. This leads to a significant number of men having unnecessary diagnostic biopsies and surgery. A search for novel tumor markers identified macrophage inhibitory cytokine 1 (MIC-1) as a potentially useful marker. Follow-up studies revealed MIC-1 overexpression in local and metastatic PCa whereas peritumoral interstitial staining for MIC-1 identified lower-grade tumors destined for recurrence. Consequently, we sought to assess serum MIC-1 measurement as a diagnostic tool. EXPERIMENTAL DESIGN: Using immunoassay determination of serum MIC-1 concentration in 1,000 men, 538 of whom had PCa, we defined the relationship of MIC-1 to disease variables. A diagnostic algorithm (MIC-PSA score) based on serum levels of MIC-1, total serum prostate-specific antigen, and percentage of free prostate-specific antigen was developed. RESULTS: Serum MIC-1 was found to be an independent predictor of the presence of PCa and tumors with a Gleason sum > or =7. We validated the MIC-PSA score in a separate population and showed an improved specificity for diagnostic blood testing for PCa over percentage of free prostate-specific antigen, potentially reducing unnecessary biopsies by 27%. CONCLUSIONS: Serum MIC-1 is an independent marker of the presence of PCa and tumors with a Gleason sum of > or =7. The use of serum MIC-1 significantly increases diagnostic specificity and may be a future tool in the management of PCa.


Assuntos
Biomarcadores Tumorais/sangue , Citocinas/sangue , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Fator 15 de Diferenciação de Crescimento , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade
6.
Oncogene ; 24(6): 1053-65, 2005 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-15558012

RESUMO

Profiles of gene transcription have begun to delineate the molecular basis of ovarian cancer, including distinctions between carcinomas of differing histology, tumor progression and patient outcome. However, the similarities and differences among the most commonly diagnosed noninvasive borderline (low malignant potential, LMP) lesions and invasive serous carcinomas of varying grade (G1, G2 and G3) have not yet been explored. Here, we used oligonucleotide arrays to profile the expression of 12,500 genes in a series of 57 predominantly stage III serous ovarian adenocarcinomas from 52 patients, eight with borderline tumors and 44 with adenocarcinomas of varying grade. Unsupervised and supervised analyses showed that LMP lesions were distinct from high-grade serous adenocarcinomas, as might be expected; however, well-differentiated (G1) invasive adenocarcinomas showed a strikingly similar profile to LMP tumors as compared to cancers with moderate (G2) or poor (G3) cellular differentiation, which were also highly similar. Comparative genomic hybridization of an independent cohort of five LMP and 63 invasive carcinomas of varying grade demonstrated LMP and G1 were again similar, exhibiting significantly less chromosomal aberration than G2/G3 carcinomas. A majority of LMP and G1 tumors were characterized by high levels of p21/WAF1, with concomitant expression of cell growth suppressors, gadd34 and BTG-2. In contrast, G2/G3 cancers were characterized by the expression of genes associated with the cell cycle and by STAT-1-, STAT-3/JAK-1/2-induced gene expression. The distinction between the LMP-G1 and G2-G3 groups of tumors was highly correlated to patient outcome (chi(2) for equivalence of death rates=7.681189; P=0.0056, log-rank test). Our results are consistent with the recent demonstration of a poor differentiation molecular 'meta-signature' in human cancer, and underscore a number of cell-cycle- and STAT-associated targets that may prove useful as points of therapeutic intervention for those patients with aggressive disease.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Perfilação da Expressão Gênica , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ciclo Celular , Diferenciação Celular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/biossíntese , Transativadores/genética , Transativadores/farmacologia
7.
Biochemistry ; 45(51): 15529-40, 2006 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-17176074

RESUMO

Overexpression of the ErbB2 receptor tyrosine kinase is common in human cancers and is associated with an increased level of metastasis. To better understand the cellular signaling networks activated by ErbB2, a phosphoproteomic analysis of tyrosine-phosphorylated proteins was carried out in ErbB2-overexpressing breast and ovarian cancer cell lines. A total of 153 phosphorylation sites were assigned on 78 proteins. Treatment of cells with Herceptin, a monoclonal antibody that inhibits ErbB2 activity, significantly reduced the number of detectable protein phosphorylation sites, suggesting that many of these proteins participate in ErbB2-driven cell signaling. Of the 71 proteins that were differentially phosphorylated, only 13 were previously reported to directly associate with ErbB2. The differentially phosphorylated proteins included kinases, adaptor/docking proteins, proteins involved in cell proliferation and migration, and several uncharacterized RNA binding proteins. Selective depletion of some of these proteins, including RNA binding proteins SRRM2, SFRS1, SFRS9, and SFRS10, by siRNAs reduced the rate of migration of ErbB2-overexpressing ovarian cancer cells.


Assuntos
Fosfoproteínas/análise , Proteômica , Receptor ErbB-2/química , Receptor ErbB-2/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Cromatografia Líquida , Humanos , Dados de Sequência Molecular , Fosfopeptídeos/análise , Fosforilação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
Cancer Res ; 62(22): 6418-23, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12438227

RESUMO

To discover novel metastasis suppressor genes that are clinically relevant in common human cancers, we used isogenic human bladder cancer cell lines and used DNA microarray technology to identify genes whose expression diminishes as a function of invasive and metastatic competence. We then evaluated the expression profile of such genes in 105 pathologically characterized tumors from seven common organ sites, and we identified one gene, RhoGDI2, whose expression was diminished as a function of primary tumor stage and grade. When RhoGDI2 was transferred back into cells with metastatic ability that lacked its expression, it suppressed experimental lung metastasis but did not affect in vitro growth, colony formation, or in vivo tumorigenicity. In addition, RhoGDI2 reconstitution in these cells blocked invasion in an organotypic assay and led to a reduction of in vitro motility. These results indicate that RhoGDI2 is a metastasis suppressor gene, a marker of aggressive human cancer, and a promising target for therapy.


Assuntos
Adenocarcinoma/genética , Genes Supressores de Tumor , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Perfilação da Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina/biossíntese , Humanos , Invasividade Neoplásica , Metástase Neoplásica , RNA Neoplásico/genética , Transfecção , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
9.
Cancer Res ; 64(21): 7813-21, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15520187

RESUMO

Pulmonary metastases frequently develop in patients with aggressive bladder cancer, yet investigation of this process at the molecular level suffers from the poor availability of human metastatic tumor tissue and the absence of suitable animal models. To address this, we developed progressively more metastatic human bladder cancer cell lines and an in vivo bladder-cancer lung-metastasis model, and we successfully used these to identify genes of which the expression levels change according to the degree of pulmonary metastatic potential. By initially intravenously injecting the poorly metastatic T24T human urothelial cancer cells into nude mice, and then serially reintroducing and reisolating the human tumor cells from the resultant mouse lung tumors, three derivative human lines with increasingly metastatic phenotypes, designated FL1, FL2, and FL3, were sequentially isolated. To identify the genes associated with the most lung-metastatic phenotype, the RNA complement from the parental and derivative cells was evaluated with oligonucleotide microarrays. In doing so, we found 121 genes to be progressively up-regulated during the transition from T24T to FL3, whereas 43 genes were progressively down-regulated. As expected, many of the genes identified in these groups could, according to the ascribed functions of their protein product, theoretically participate in tissue invasion and metastasis. In addition, the magnitude of gene expression changes observed during the metastatic transition correlated with the in vivo propensity for earlier lung colonization and decreased host survival. To additionally define which genes found in the experimental system were of relevance to human bladder cancer lung metastasis, we evaluated gene expression profiles of 23 primary human bladder tumors of various stages and grades, and then we compared these gene expression profiles to the altered profiles in our model cell lines. Here we found that the expression of epiregulin, urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)14, and tissue inhibitor of metalloproteinase (TIMP-2) were consistently and progressively up-regulated when viewed as a function of tumor stage in tissues of patients versus the metastatic potential seen in the mouse lung model. The strong correlation of these four markers between the experimental and clinical situations helps validate this system as a useful tool for the study of lung metastasis and defines targets of therapy that may reduce the incidence of this process in patients.


Assuntos
Perfilação da Expressão Gênica , Neoplasias Pulmonares/secundário , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Animais , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/genética , Epirregulina , Feminino , Humanos , Camundongos , Fenótipo , Inibidor Tecidual de Metaloproteinase-2/genética , Neoplasias da Bexiga Urinária/mortalidade
10.
Cancer Res ; 76(5): 1193-203, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26759240

RESUMO

Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110ß D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Mutação , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologia
11.
PLoS One ; 11(11): e0165856, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27846280

RESUMO

In the age of personalized medicine stratifying tumors into molecularly defined subtypes associated with distinctive clinical behaviors and predictable responses to therapies holds tremendous value. Towards this end, we developed a custom microfluidics-based bladder cancer gene expression panel for characterization of archival clinical samples. In silico analysis indicated that the content of our panel was capable of accurately segregating bladder cancers from several public datasets into the clinically relevant basal and luminal subtypes. On a technical level, our bladder cancer panel yielded robust and reproducible results when analyzing formalin-fixed, paraffin-embedded (FFPE) tissues. We applied our panel in the analysis of a novel set of 204 FFPE samples that included non-muscle invasive bladder cancers (NMIBCs), muscle invasive disease (MIBCs), and bladder cancer metastases (METs). We found NMIBCs to be mostly luminal-like, MIBCs to include both luminal- and basal-like types, and METs to be predominantly of a basal-like transcriptional profile. Mutational analysis confirmed the expected enrichment of FGFR3 mutations in luminal samples, and, consistently, FGFR3 IHC showed high protein expression levels of the receptor in these tumors. Our bladder cancer panel enables basal/luminal characterization of FFPE tissues and with further development could be used for stratification of bladder cancer samples in the clinic.


Assuntos
Bancos de Espécimes Biológicos , Regulação Neoplásica da Expressão Gênica , Microfluídica/métodos , Transcrição Gênica , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Simulação por Computador , Feminino , Formaldeído , Genes Neoplásicos , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Reprodutibilidade dos Testes , Fixação de Tecidos , Neoplasias da Bexiga Urinária/patologia
12.
Nat Commun ; 7: 11579, 2016 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-27174596

RESUMO

Mutations in ESR1 have been associated with resistance to aromatase inhibitor (AI) therapy in patients with ER+ metastatic breast cancer. Little is known of the impact of these mutations in patients receiving selective oestrogen receptor degrader (SERD) therapy. In this study, hotspot mutations in ESR1 and PIK3CA from ctDNA were assayed in clinical trial samples from ER+ metastatic breast cancer patients randomized either to the SERD fulvestrant or fulvestrant plus a pan-PI3K inhibitor. ESR1 mutations are present in 37% of baseline samples and are enriched in patients with luminal A and PIK3CA-mutated tumours. ESR1 mutations are often polyclonal and longitudinal analysis shows distinct clones exhibiting divergent behaviour over time. ESR1 mutation allele frequency does not show a consistent pattern of increases during fulvestrant treatment, and progression-free survival is not different in patients with ESR1 mutations compared with wild-type patients. ESR1 mutations are not associated with clinical resistance to fulvestrant in this study.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/análogos & derivados , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mama/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Intervalo Livre de Doença , Estradiol/farmacologia , Estradiol/uso terapêutico , Antagonistas do Receptor de Estrogênio/uso terapêutico , Receptor alfa de Estrogênio/antagonistas & inibidores , Estrogênios/metabolismo , Feminino , Fulvestranto , Humanos , Indazóis/farmacologia , Indazóis/uso terapêutico , Pessoa de Meia-Idade , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico
13.
NPJ Breast Cancer ; 2: 16022, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28721382

RESUMO

Breast cancer is a heterogeneous disease and patients are managed clinically based on ER, PR, HER2 expression, and key risk factors. We sought to characterize the molecular landscape of high-risk breast cancer patients enrolled onto an adjuvant chemotherapy study to understand how disease subsets and tumor immune status impact survival. DNA and RNA were extracted from 861 breast cancer samples from patients enrolled onto the United States Oncology trial 01062. Samples were characterized using multiplex gene expression, copy number, and qPCR mutation assays. HR+ patients with a PIK3CA mutant tumor had a favorable disease-free survival (DFS; HR 0.66, P=0.05), however, the prognostic effect was specific to luminal A patients (Luminal A: HR 0.67, P=0.1; Luminal B: HR 1.01, P=0.98). Molecular subtyping of triple-negative breast cancers (TNBCs) suggested that the mesenchymal subtype had the worst DFS, whereas the immunomodulatory subtype had the best DFS. Profiling of immunologic genes revealed that TNBC tumors (n=280) displaying an activated T-cell signature had a longer DFS following adjuvant chemotherapy (HR 0.59, P=0.04), while a distinct set of immune genes was associated with DFS in HR+ cancers. Utilizing a discovery approach, we identified genes associated with a high risk of recurrence in HR+ patients, which were validated in an independent data set. Molecular classification based on PAM50 and TNBC subtyping stratified clinical high-risk patients into distinct prognostic subsets. Patients with high expression of immune-related genes showed superior DFS in both HR+ and TNBC. These results may inform patient management and drug development in early breast cancer.

14.
Oncogene ; 23(51): 8392-400, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15517021

RESUMO

Cancer develops through the successive accumulation and selection of genetic and epigenetic alterations, enabling cells to survive, replicate and evade homeostatic control mechanisms such as apoptosis and antiproliferative signals. This transformation process, however, may create vulnerabilities since the accumulation of mutations can expose synthetic lethal gene interactions and oncogene-driven cellular reprogramming ('addiction'), giving rise to new therapeutic avenues. With the completion of the human genome project, it is anticipated that the identification and characterization of genetic networks that regulate cell growth, differentiation, apoptosis and transformation will be fundamental to decoding the complexity of these processes, and ultimately, cancer itself. Genomic methodologies, such as large-scale mRNA profiling using microarrays, have already begun to reveal the molecular basis of cancer heterogeneity and the clinical behavior of tumors. The combination of traditional cell culture techniques with high-throughput screening approaches has given rise to new cellular-genomics methodologies that enable the simultaneous interrogation of thousands of genes in live cells, facilitating true functional profiling of biological processes. Among these, RNA interference (RNAi) has the potential to enable rapid genome-wide loss-of-function (LOF) screens in mammalian systems, which until recently has been the sole domain of lower organisms. Here, we present a broad overview of this maturing technology and explore how, within current technical constraints, large-scale LOF use of RNAi can be exploited to uncover the molecular basis of cancer--from the genetics of synthetic lethality and oncogene-dependent cellular addiction to the acquisition of cancer-associated cellular phenotypes.


Assuntos
Neoplasias/genética , Oncogenes , Interferência de RNA/fisiologia , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Neoplasias/metabolismo
15.
Oncogene ; 23(39): 6621-9, 2004 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-15208666

RESUMO

Gene expression profiling of anatomically diverse carcinomas and their corresponding normal tissues was used to identify genes with cancer-associated expression. We show here that the ubiquitin conjugase, UbcH10, is significantly overexpressed in many different types of cancers and is associated with the degree of tumor differentiation in carcinomas of the breast, lung, ovary and bladder, as well as in glioblastomas. We also show that UbcH10 overexpression in gastro-esophageal, and probably other carcinomas may be a direct consequence of chromosomal amplification at the UbcH10 locus, 20q13.1, a region known to be amplified in diverse tumors. To evaluate whether inhibition of UbcH10 function may be therapeutically relevant in cancer, we used small interfering RNAs (siRNAs) to silence UbcH10 transcription selectively. Diminution of UbcH10 expression significantly inhibited both tumor and normal cell proliferation without inducing cell death. However, when combined with agonists of the DR5/TRAIL receptor, siRNAs directed against the UbcH10 transcript dramatically enhanced killing of cancer cells, but not of proliferating primary human epithelial cells or fibroblasts. Together, these data demonstrate that UbcH10 plays an important role in tumor development and that its inhibition in combination with agonists of the TRAIL receptor may provide an enhanced therapeutic index.


Assuntos
Amplificação de Genes , Neoplasias/genética , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Apoptose/fisiologia , Sequência de Bases , Western Blotting , Primers do DNA , Humanos , Imuno-Histoquímica , Neoplasias/classificação , Neoplasias/patologia , Neoplasias/terapia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/fisiologia
16.
Neoplasia ; 7(4): 407-16, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15967118

RESUMO

Identification of biomarkers to recognize individuals with Barrett's esophagus (BE) predisposed to develop malignancy is currently a pressing issue. We utilized gene expression profiling to compare molecular signatures of normal esophagus and stomach, BE, and adenocarcinoma (AC) to identify such potential biomarkers. Over 22,000 genes were analyzed by oligonucleotide microarrays on 38 unique RNA Unsupervised and supervised clusterings were performed on a subset of 2849 genes that varied most significantly across the specimens. Immunohistochemistry (IHC) for two of the significantly differentially expressed gene products was performed on tissue microarrays. Unsupervised clustering identified two discernable molecular BE profiles, one of which was similar to normal gastric tissue ("BE1"), and another that was shared by several of the AC specimens ("BE2"). The BE1 profile included expression of several genes that have been described as tumor-suppressor genes, most notably trefoil factor 1 (TFF-1). The BE2 profile included expression of genes previously found overexpressed in cancers, such as carboxylesterase-2 (CES-2). IHC demonstrated the loss of TFF-1 late in the progression of BE to AC. It also revealed CES-2 as being upregulated in AC documented to have arisen in the presence of BE. These potential biomarkers, as well as the relative expression of genes from BE1 versus those from BE2, may be validated in the future to aid in risk stratification and guide treatment protocols in patients with BE and associated AC.


Assuntos
Adenocarcinoma/enzimologia , Esôfago de Barrett/enzimologia , Hidrolases de Éster Carboxílico/biossíntese , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais , Antígenos CD13/biossíntese , Esôfago/metabolismo , Mucosa Gástrica/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Modelos Genéticos , Família Multigênica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Lesões Pré-Cancerosas , RNA/metabolismo , RNA Complementar/metabolismo , Transdução de Sinais , Fator Trefoil-1
17.
Trends Mol Med ; 9(1): 5-10, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12524204

RESUMO

With the development and application of DNA microarrays, the expression of almost all human genes can now be systematically examined in human malignancies. This can lead to the identification of candidate molecular targets for therapeutic intervention and biomarkers for early detection of these diseases. However, perhaps the most exciting result to come from this research has been the demonstration that patterns of gene expression can distinguish between tumors of different anatomical origin, and define new subgroups of cancer with similar histological appearance, but distinct molecular profiles. Some of these new molecular subclasses of tumor appear to correlate with clinical behavior. If substantiated in larger studies, this might form a basis for stratifying patients so that they receive optimal therapeutic treatment and follow-up.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/diagnóstico , Neoplasias/genética , Humanos , Neoplasias/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Software , Resultado do Tratamento
18.
Hum Pathol ; 34(6): 605-9, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12827615

RESUMO

The 40-kilodalton processed glycoprotein, mesothelin, is highly expressed in epithelial mesotheliomas and adenocarcinomas of the ovary (serous papillary) and pancreas, but its expression in a large series of other common carcinomas has not been completely explored. In the present study, we used oligonucleotide and tissue microarrays to profile the expression of the mesothelin gene (MSLN) and encoded protein, respectively. Among 150 carcinomas of multiple anatomic sites, we found the highest average expression of MSLN in serous carcinomas of the ovary and adenocarcinomas of the pancreas, consistent with previous reports, as well as measurable but less-striking expression in pulmonary, gastric/esophageal, and colorectal adenocarcinomas. On tissue microarrays containing 621 carcinomas derived from the same and additional sites as those profiled by gene expression, mesothelin immunoreactivity was highest in cancers of the ovary (serous papillary, endometrioid, and undifferentiated) and pancreas, with less frequent staining seen in adenocarcinomas of the endometrium, lung, and stomach/esophagus. Some immunopositivity was observed in 42% of pulmonary adenocarcinomas, including 18% that had >50% of tumor cells that were immunoreactive. Some 14% of breast and 30% of colorectal adenocarcinomas showed immunopositivity, but no case contained >50% tumor cells that were immunoreactive. Mesothelin was either entirely absent or present in <5% of carcinomas of the prostate, bladder/ureter, liver, kidney, and thyroid. Overall, we observed good concordance between the results obtained by oligonucleotide and tissue microarrays. This large study of the MSLN gene and protein expression in common carcinomas provides data for future investigations that evaluate the utility of mesothelin/megakaryocyte potentiating factor as a potential serum tumor marker or target of immunotoxin-based therapy in human cancers.


Assuntos
Carcinoma/genética , Perfilação da Expressão Gênica , Glicoproteínas de Membrana/genética , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Biomarcadores Tumorais/análise , Carcinoma/química , Carcinoma/patologia , DNA de Neoplasias/análise , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Ligadas por GPI , Humanos , Técnicas Imunoenzimáticas , Masculino , Glicoproteínas de Membrana/análise , Mesotelina , Neoplasias/química , Neoplasias/patologia
19.
Epigenomics ; 6(1): 59-72, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24579947

RESUMO

Lung cancer remains the primary cause of cancer-related deaths worldwide. Improved tools for early detection and therapeutic stratification would be expected to increase the survival rate for this disease. Alterations in the molecular pathways that drive lung cancer, which include epigenetic modifications, may provide biomarkers to help address this major unmet clinical need. Epigenetic changes, which are defined as heritable changes in gene expression that do not alter the primary DNA sequence, are one of the hallmarks of cancer, and prevalent in all types of cancer. These modifications represent a rich source of biomarkers that have the potential to be implemented in clinical practice. This perspective describes recent advances in the discovery of epigenetic biomarkers in lung cancer, specifically those that result in the methylation of DNA at CpG sites. We discuss one approach for methylation-based biomarker assay development that describes the discovery at a genome-scale level, which addresses some of the practical considerations for design of assays that can be implemented in the clinic. We emphasize that an integrated technological approach will enable the development of clinically useful DNA methylation-based biomarker assays. While this article focuses on current literature and primary research findings in lung cancer, the principles we describe here apply to the discovery and development of epigenetic biomarkers for other types of cancer.


Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Epigenômica/métodos , Histonas/fisiologia , Neoplasias Pulmonares/genética , RNA não Traduzido/genética , Ilhas de CpG , Epigênese Genética , Epigenômica/instrumentação , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Reprodutibilidade dos Testes
20.
Clin Cancer Res ; 20(8): 2080-91, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24573554

RESUMO

PURPOSE: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples. EXPERIMENTAL DESIGN: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer. RESULTS: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA. CONCLUSIONS: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Neoplasias/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase , Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Receptores ErbB/genética , Técnicas de Genotipagem , Humanos , Células MCF-7 , Neoplasias/metabolismo , Neoplasias/patologia , PTEN Fosfo-Hidrolase/genética , Inclusão em Parafina , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Reprodutibilidade dos Testes , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/genética
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