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1.
Inflamm Bowel Dis ; 24(12): 2555-2564, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30099509

RESUMO

Background: The risk of developing colorectal cancer (CRC) is increased in patients with inflammatory bowel disease (IBD) of the colon. The aim of the study was to evaluate the effectiveness of selected methylation gene panel for the early detection of CRC in high-risk IBD patients. Methods: In a discovery phase, 73 biopsies of 48 IBD patients (associated or not to CRC) were analyzed from genome-wide DNA methylation analysis using the Illumina Human Methylation 450K BeadChip. The panel of 5 genes selected (EYA4, SLIT2, FLI1, USP44, and SND1) was validated prospectively using methylation-specific melting curve analysis in biopsies of diseased and adjacent healthy tissue of 203 patients: 38 with IBD and associated neoplasia, 81 patients with IBD (25 of them with high risk), 48 with sporadic CRC, and 36 healthy controls. Results: The prevalence of methylation was higher in patients with IBD and associated neoplasia (both in diseased and adjacent healthy tissue, 71% and 52%, respectively) than in healthy controls (2/36, 6%; P = 6.72E-05). Methylation in IBD patients at high risk of dysplasia or cancer was more frequently detected than in patients at low risk (92% vs 57%; odds ratio, 8.63; P = 0.001). EYA4 and SLIT2 were the markers most frequently methylated. Differences in methylation levels were more evident in healthy mucosa (82% vs 15% high vs low risk, respectively; P = 1.25E-05). Conclusions: Analysis of this panel of methylation markers may help in the early identification of colorectal dysplasia or cancer in high-risk IBD patients.


Assuntos
Biomarcadores Tumorais/genética , Colo/patologia , Neoplasias Colorretais/diagnóstico , Metilação de DNA , Doenças Inflamatórias Intestinais/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Neoplasias Colorretais/etiologia , Detecção Precoce de Câncer , Endonucleases , Feminino , Humanos , Doenças Inflamatórias Intestinais/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteína Proto-Oncogênica c-fli-1/genética , Espanha , Transativadores/genética , Proteases Específicas de Ubiquitina/genética
2.
Biochim Biophys Acta Mol Basis Dis ; 1864(9 Pt B): 2992-3000, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29908233

RESUMO

Telomeres are repetitive sequences (TTAGGG) located at the end of chromosomes. Telomeres progressively shorten with each cell replication cycle, ultimately leading to chromosomal instability and loss of cell viability. Telomere length anomaly appears to be one of the earliest and most prevalent genetic alterations in malignant transformation. Here we aim to estimate telomere length from whole-exome sequencing data in colon tumors and normal colonic mucosa, and to analyze the potential association of telomere length with clinical factors and gene expression in colon cancer. Reads containing at least five repetitions of the telomere sequence (TTAGGG) were extracted from the raw sequences of 42 adjacent normal-tumor paired samples. The number of reads from the tumor sample was normalized to build the Tumor Telomere Length Ratio (TTLR), considered an estimation of telomere length change in the tumor compared to the paired normal tissue. We evaluated the associations between TTLR and clinical factors, gene expression and copy number (CN) aberrations measured in the same tumor samples. Colon tumors showed significantly shorter telomeres than their paired normal samples. No significant association was observed between TTLR and gender, age, tumor location, prognosis, stromal infiltration or molecular subtypes. The functional gene set enrichment analysis showed pathways related to immune response significantly associated with TLLR. By extracting a relative measure of telomere length from whole-exome sequencing data, we have assessed that colon tumor cells predominantly shorten telomeres, and this alteration is associated with expression changes in genes related to immune response and inflammation in tumor cells.


Assuntos
Neoplasias do Colo/genética , Encurtamento do Telômero/genética , Telômero/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Instabilidade Cromossômica/genética , Colo/imunologia , Colo/patologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Variações do Número de Cópias de DNA/imunologia , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Encurtamento do Telômero/imunologia , Sequenciamento Completo do Exoma
3.
Target Oncol ; 13(1): 81-87, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29177953

RESUMO

BACKGROUND: Gemcitabine plus sirolimus enhances apoptosis in vitro and increases anti-tumor efficacy in vivo in soft-tissue sarcoma (STS) models. OBJECTIVE: The objective of this study was to evaluate the activity and toxicity of the combination of gemcitabine plus sirolimus in patients with STS after failure of standard chemotherapy. PATIENTS AND METHODS: Advanced STS patients, previously treated with doxorubicin and/or ifosfamide, were included in this single-arm phase II study. Patients received gemcitabine 800 mg/m2 intravenously (iv) at 10 mg/m2/min on days 1 and 8 every 3 weeks plus sirolimus 5 mg daily orally (po). After enrolment of the first 12 patients, the study protocol was amended due to toxicity and the starting dose of sirolimus was reduced to 3 mg daily po. Archival tumor samples were analyzed for extracellular signal-regulated kinase 1 and 2 (ERK1/2) expression and correlated with outcome. The primary endpoint was progression-free rate (PFR) at 3 months. RESULTS: From May 2012 to May 2013, 28 patients were enrolled at eight centers. PFR at 3 and 6 months was 44% and 20%, respectively, with 12 patients being free of progression at 3 months. Median progression-free survival (PFS) was 1.85 months (95% confidence interval [CI] 0.73-2.97) and median overall survival (OS) was 9.2 months (95% CI 5.8-12.5). No responses were observed. The most common grade 3-4 hematologic toxicities were neutropenia (48%) and leukopenia (41%) and the most frequent grade 3 non-hematologic toxicities were infection (18.5%), transaminitis (15%), fatigue (11%), and pneumonitis (11%). ERK1/2 expression was significantly correlated with PFS (p = 0.026). CONCLUSIONS: The combination of gemcitabine and sirolimus is an active treatment in STS. Further investigation is warranted. ClinicalTrials.gov identifier: NCT01684449.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Sarcoma/tratamento farmacológico , Sirolimo/uso terapêutico , Adulto , Idoso , Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Feminino , Hispano-Americanos , Humanos , Masculino , Pessoa de Meia-Idade , Projetos de Pesquisa , Sarcoma/patologia , Sirolimo/farmacologia , Adulto Jovem
4.
Int J Cancer ; 141(7): 1365-1380, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28577310

RESUMO

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , DNA Glicosilases/genética , Metilação de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/deficiência , Molécula de Adesão da Célula Epitelial/genética , Exodesoxirribonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Perda de Heterozigosidade , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética
5.
Mol Cancer Ther ; 16(9): 1999-2007, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28626084

RESUMO

In metastatic colorectal cancer (mCRC), recent studies have shown the importance to accurately quantify low-abundance mutations of the RAS pathway because anti-EGFR therapy may depend on certain mutation thresholds. We aimed to evaluate the added predictive value of an extended RAS panel testing using two commercial assays and a highly sensitive and quantitative digital PCR (dPCR). Tumor samples from 583 mCRC patients treated with anti-EGFR- (n = 255) or bevacizumab- (n = 328) based therapies from several clinical trials and retrospective series from the TTD/RTICC Spanish network were analyzed by cobas, therascreen, and dPCR. We evaluated concordance between techniques using the Cohen kappa index. Response rate, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutant allele fraction (MAF). Concordance between techniques was high when analyzing RAS and BRAF (Cohen kappa index around 0.75). We observed an inverse correlation between MAF and response in the anti-EGFR cohort (P < 0.001). Likelihood ratio analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value. PFS and OS were significantly longer in RAS/BRAF wild-type patients, independently of the technique. However, the predictability of both PFS and OS were higher when we considered a threshold of 1% in the RAS scenario (HR = 1.53; CI 95%, 1.12-2.09 for PFS, and HR = 1.9; CI 95%, 1.33-2.72 for OS). Although the rate of mutations observed among techniques is different, RAS and BRAF mutational analysis improved prediction of response to anti-EGFR therapy. Additionally, dPCR with a threshold of 1% outperformed the other platforms. Mol Cancer Ther; 16(9); 1999-2007. ©2017 AACR.


Assuntos
Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
6.
Clin Cancer Res ; 23(19): 5936-5947, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28645942

RESUMO

Purpose: The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in APC have expanded a single founder clone. We have investigated if the initial source of ITH is consequent to multiple independent lineages derived from different crypts harboring distinct truncal APC and driver KRAS mutations, thus challenging the prevailing monoclonal monocryptal model.Experimental Design: High-depth next-generation sequencing and SNP arrays were performed in whole-lesion extracts of 37 familial adenomatous polyposis colorectal adenomas. Also, ultrasensitive genotyping of hotspot mutations of APC and KRAS was performed using nanofluidic PCRs in matched bulk biopsies (n = 59) and crypts (n = 591) from 18 adenomas and seven carcinomas and adjacent normal tissues.Results: Multiple co-occurring truncal APC and driver KRAS alterations were uncovered in whole-lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultrasensitive genotyping of bulk biopsies and crypts revealed novel undetected APC mutations that were prominent among carcinomas, whereas abundant wild-type APC crypts were detected in adenomas. KRAS mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Nonrandom heterogeneity among crypts was also observed.Conclusions: The striking degree of nonrandom intercrypt heterogeneity in truncal and driver gene mutations observed in adenomas and carcinomas is consistent with a polycryptal model derived from multiple independent initiation linages as the source of early ITH in colorectal carcinogenesis. Clin Cancer Res; 23(19); 5936-47. ©2017 AACR.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Carcinogênese/genética , Evolução Clonal/genética , Neoplasias Colorretais/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Biópsia , Carcinoma , Neoplasias Colorretais/patologia , Feminino , Heterogeneidade Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação
7.
Fam Cancer ; 16(4): 501-507, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28365877

RESUMO

The clinical spectrum of germline mismatch repair (MMR) gene variants continues increasing, encompassing Lynch syndrome, Constitutional MMR Deficiency (CMMRD), and the recently reported MSH3-associated polyposis. Genetic diagnosis of these hereditary cancer syndromes is often hampered by the presence of variants of unknown significance (VUS) and overlapping phenotypes. Two PMS2 VUS, c.2149G>A (p.V717M) and c.2444C>T (p.S815L), were identified in trans in one individual diagnosed with early-onset colorectal cancer (CRC) who belonged to a family fulfilling clinical criteria for hereditary cancer. Clinico-pathological data, multifactorial likelihood calculations and functional analyses were used to refine their clinical significance. Likelihood analysis based on cosegregation and tumor data classified the c.2444C>T variant as pathogenic, which was supported by impaired MMR activity associated with diminished protein expression in functional assays. Conversely, the c.2149G>A variant displayed MMR proficiency and protein stability. These results, in addition to the conserved PMS2 expression in normal tissues and the absence of germline microsatellite instability (gMSI) in the biallelic carrier ruled out a CMMRD diagnosis. The use of comprehensive strategies, including functional and clinico-pathological information, is mandatory to improve the clinical interpretation of naturally occurring MMR variants. This is critical for appropriate clinical management of cancer syndromes associated to MMR gene mutations.


Assuntos
Neoplasias Colorretais/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Mutação de Sentido Incorreto , Idade de Início , Estudos de Casos e Controles , Reparo de Erro de Pareamento de DNA , Feminino , Frequência do Gene , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Humanos , Masculino , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Linhagem
8.
Cancer Lett ; 386: 196-207, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27894957

RESUMO

Epigenetic modifications have been shown to be important in developmental tumors as Ewing sarcoma. We profiled the DNA methylation status of 15 primary tumors, 7 cell lines, 10 healthy tissues and 4 human mesenchymal stem cells lines samples using the Infinium Human Methylation 450K. Differential methylation analysis between Ewing sarcoma and reference samples revealed 1166 hypermethylated and 864 hypomethylated CpG sites (Bonferroni p < 0.05, δ-ß-value with absolute difference of >0.20) corresponding to 392 and 470 genes respectively. Gene Ontology analysis of genes differentially methylated in Ewing sarcoma samples showed a significant enrichment of developmental genes. Membrane and cell signal genes were also enriched, among those, 11 were related to caveola formation. We identified differential hypermethylation of CpGs located in the body and S-Shore of the PTRF gene in Ewing sarcoma that correlated with its repressed transcriptional state. Reintroduction of PTRF/Cavin-1 in Ewing sarcoma cells revealed a role of this protein as a tumor suppressor. Restoration of caveolae in the membrane of Ewing sarcoma cells, by exogenously reintroducing PTRF, disrupts the MDM2/p53 complex, which consequently results in the activation of p53 and the induction of apoptosis.


Assuntos
Neoplasias Ósseas/genética , Caveolina 1/genética , Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Genes Supressores de Tumor , Proteínas de Ligação a RNA/genética , Sarcoma de Ewing/genética , Animais , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Camundongos Nus , Fosforilação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais , Espanha , Transfecção , Carga Tumoral , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
10.
Clin Sarcoma Res ; 6: 10, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27358721

RESUMO

BACKGROUND: Most GISTs have mutations in KIT or PDGFRA. Patients with advanced GIST with KIT exon 9, PDGFRA mutation or WT for KIT and PDGFRA have a worse progression-free survival (PFS) compared to patients with KIT exon 11 mutated tumors. We evaluated the immunohistochemical (IHC) expression of p-IGF1R (Y1316) and MMP3 as predictors of PFS or overall survival (OS). METHODS: Ninety-two advanced GIST patients included in GEIS-16 study with KIT and PDGFRA mutational information were examined for p-IGF1R (Y1316) and MMP3 expression in a tissue micro-array. To study activation of the IGF1R system, we have used an antibody (anti-pY1316) that specifically recognizes the active phosphorylated form of the IGF1R. DNA was extracted from paraffin-embedded tissues and intronic PCR primers were used to amplify exons 9, 11, 13 and 17 of KIT, 12 and 18 of PDGFRA. Bidirectional sequencing with specific primers was performed on a ABI3100 sequencer using the Big Dye Terminator v3.1 kit. Multivariate model was built using a stepwise automated variable selection approach with criterion to enter the variable in the model of p < 0.10 and criterion to keep the variable in the model of p < 0.05. PFS was computed as the date of imatinib initiation to progression or death. Overall survival was defined as the time from imatinib initiation to death. RESULTS: Phospho-IGF1R was expressed only in 9 % (2/22) of cases without KIT mutation. MMP3 expression was detected in 2/5 patients (40 %) with PDGFRA mutation, 1/16 patients (6 %) with WT genotype and 7/71 patients (10 %) of KIT mutant patients. At univariate analysis KIT exon 11/13 mutation had better PFS than patients with exon 9 mutation, PDGFRA mutation or WT genotype (p = 0.021; HR: 0.46; 95 %CI (0.28-0.76). Less than 24 months disease free-interval (HR 24.2, 95 % CI 10.5-55.8), poor performance status (PS) (HR 6.3, 95 % CI 2.5-15.9), extension of disease; >1 organ (HR 1.89; 95 % CI 1.03-3.4) and genotype analysis (HR 0.57, 95 % CI 0.37-0.97) but not immunophenotype analysis (HR 1.53; 95 % CI 0.76-3.06) were the strongest prognostic factors for PFS in the multivariate analysis. CONCLUSIONS: Our results do not support p-IGF-1R and MMP3 evaluation in non-selected GIST patients but evaluation of this immunophenotype in WT and mutant PDGFR mutation in larger group of GIST patients, deserve merits.

11.
Mol Cancer Ther ; 15(5): 1106-12, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27037411

RESUMO

The clinical significance of low-frequent RAS pathway-mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106-12. ©2016 AACR.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Genes ras , Nanotecnologia , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Adulto , Idoso , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Análise Mutacional de DNA , Receptores ErbB/antagonistas & inibidores , Éxons , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Tipagem Molecular , Mutação , Metástase Neoplásica , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase/métodos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Retratamento , Estudos Retrospectivos , Sensibilidade e Especificidade , Análise de Sobrevida , Resultado do Tratamento
12.
Oncotarget ; 7(14): 17711-25, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-26871478

RESUMO

The aim of this study was to analyze the impact of the mutanome in the prognosis of microsatellite stable stage II CRC tumors. The exome of 42 stage II, microsatellite stable, colon tumors (21 of them relapse) and their paired mucosa were sequenced and analyzed. Although some pathways accumulated more mutations in patients exhibiting good or poor prognosis, no single somatic mutation was associated with prognosis. Exome sequencing data is also valuable to infer tumor neoantigens able to elicit a host immune response. Hence, putative neoantigens were identified by combining information about missense mutations in each tumor and HLAs genotypes of the patients. Under the hypothesis that neoantigens should be correctly presented in order to activate the immune response, expression levels of genes involved in the antigen presentation machinery were also assessed. In addition, CD8A level (as a marker of T-cell infiltration) was measured. We found that tumors with better prognosis showed a tendency to generate a higher number of immunogenic epitopes, and up-regulated genes involved in the antigen processing machinery. Moreover, tumors with higher T-cell infiltration also showed better prognosis. Stratifying by consensus molecular subtype, CMS4 tumors showed the highest association of expression levels of genes involved in the antigen presentation machinery with prognosis. Thus, we hypothesize that a subset of stage II microsatellite stable CRC tumors are able to generate an immune response in the host via MHC class I antigen presentation, directly related with a better prognosis.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Neoplasias do Colo/patologia , Humanos , Imunidade Ativa/genética , Instabilidade de Microssatélites , Mutação , Estadiamento de Neoplasias , Prognóstico
13.
Clin Cancer Res ; 21(20): 4709-18, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26071483

RESUMO

PURPOSE: Somatic mutations occur at early stages of adenoma and accumulate throughout colorectal cancer progression. The aim of this study was to characterize the mutational landscape of stage II tumors and to search for novel recurrent mutations likely implicated in colorectal cancer tumorigenesis. EXPERIMENTAL DESIGN: The exomic DNA of 42 stage II, microsatellite-stable colon tumors and their paired mucosae were sequenced. Other molecular data available in the discovery dataset [gene expression, methylation, and copy number variations (CNV)] were used to further characterize these tumors. Additional datasets comprising 553 colorectal cancer samples were used to validate the discovered mutations. RESULTS: As a result, 4,886 somatic single-nucleotide variants (SNV) were found. Almost all SNVs were private changes, with few mutations shared by more than one tumor, thus revealing tumor-specific mutational landscapes. Nevertheless, these diverse mutations converged into common cellular pathways, such as cell cycle or apoptosis. Among this mutational heterogeneity, variants resulting in early stop codons in the AMER1 (also known as FAM123B or WTX) gene emerged as recurrent mutations in colorectal cancer. Losses of AMER1 by other mechanisms apart from mutations such as methylation and copy number aberrations were also found. Tumors lacking this tumor suppressor gene exhibited a mesenchymal phenotype characterized by inhibition of the canonical Wnt pathway. CONCLUSIONS: In silico and experimental validation in independent datasets confirmed the existence of functional mutations in AMER1 in approximately 10% of analyzed colorectal cancer tumors. Moreover, these tumors exhibited a characteristic phenotype.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Exoma/genética , Mutação/genética , Proteínas Supressoras de Tumor/genética , Adenoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Transformação Celular Neoplásica/genética , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Fenótipo
14.
J Hepatol ; 62(4): 785-90, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25463546

RESUMO

BACKGROUND & AIMS: Decoding the myriad of interactions that hepatitis C virus (HCV) establishes with infected cells is mandatory to obtain a complete understanding of HCV biology and its associated pathogenesis. We and others have previously found that HCV infection disrupts the formation of P-bodies in cell culture. These are cytoplasmic RNA granules with key roles in post-transcriptional regulation of gene expression. Therefore, P-body disruption might have consequences beyond viral propagation. However, whether P-body disruption occurs also in vivo is unknown. Aim of this study was to address this important issue. METHODS: Formalin-fixed paraffin-embedded liver biopsies from four groups of patients (healthy donors, patients with non-virus related liver inflammation, HCV- and HBV-infected patients) were immunostained to detect DDX6 and Dcp1, two core P-body components. Changes in the localization of these proteins were assessed by confocal microscopy. RESULTS: HCV specifically inhibited P-body formation in hepatocytes from human livers regardless of viral genotype, inflammation grade or whether the infection was recent or long established. Importantly, this alteration was reversed once HCV was eliminated by therapy. Furthermore, we observed in vivo an unexpected heterogeneity in P-body composition, which might reflect functional specializations. CONCLUSIONS: This is the first comprehensive in vivo P-body analysis that links a pathogenic condition to P-body alterations. Because of their role in gene expression, the alteration of P-bodies should be further studied to understand fully complex HCV-associated pathologies.


Assuntos
Grânulos Citoplasmáticos/fisiologia , RNA Helicases DEAD-box , Endopeptidases , Hepacivirus , Hepatite C Crônica , Proteínas Proto-Oncogênicas , Adulto , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/imunologia , Endopeptidases/biossíntese , Endopeptidases/imunologia , Feminino , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/metabolismo , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/imunologia , Carga Viral
15.
PLoS One ; 9(9): e106748, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25215506

RESUMO

BACKGROUND: Accurate detection of characteristic proteins secreted by colon cancer tumor cells in biological fluids could serve as a biomarker for the disease. The aim of the present study was to identify and validate new serum biomarkers and demonstrate their potential usefulness for early diagnosis of colon cancer. METHODS: The study was organized in three sequential phases: 1) biomarker discovery, 2) technical and biological validation, and 3) proof of concept to test the potential clinical use of selected biomarkers. A prioritized subset of the differentially-expressed genes between tissue types (50 colon mucosa from cancer-free individuals and 100 normal-tumor pairs from colon cancer patients) was validated and further tested in a series of serum samples from 80 colon cancer cases, 23 patients with adenoma and 77 cancer-free controls. RESULTS: In the discovery phase, 505 unique candidate biomarkers were identified, with highly significant results and high capacity to discriminate between the different tissue types. After a subsequent prioritization, all tested genes (N = 23) were successfully validated in tissue, and one of them, COL10A1, showed relevant differences in serum protein levels between controls, patients with adenoma (p = 0.0083) and colon cancer cases (p = 3.2e-6). CONCLUSION: We present a sequential process for the identification and further validation of biomarkers for early detection of colon cancer that identifies COL10A1 protein levels in serum as a potential diagnostic candidate to detect both adenoma lesions and tumor. IMPACT: The use of a cheap serum test for colon cancer screening should improve its participation rates and contribute to decrease the burden of this disease.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias do Colo/sangue , Neoplasias do Colo/diagnóstico , Detecção Precoce de Câncer , Adenoma/sangue , Adenoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Colágeno Tipo X/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangue , Reprodutibilidade dos Testes
16.
Oncotarget ; 5(15): 6437-52, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25115384

RESUMO

Based on 108 differentially expressed genes between carcinoma-associated fibroblasts (CAFs) and paired normal colonic fibroblasts we recently reported, a 5-gene classifier for relapse prediction in Stage II/III colorectal cancer (CRC ) was developed. Its predictive value was validated in datasets GSE17538, GSE33113 and GSE14095. An additional validation was performed in a metacohort (n=317) and 142 CRC patients by means of RT-PCR. The 5-gene classifier was significantly associated with increased relapse risk and death from CRC across all validation series of Stage II/III patients used. Multivariate Cox regression analyses confirmed the independent prognostic value of the stromal classifier (HR=2.67; P=0.002). Post-test probabilities provided evidence of the suitability of the 5-gene classifier in clinical practice, identifying a subgroup of Stage-II patients who were at high risk of relapse. Moreover, the a priory worst prognosis mesenchymal subtype of tumours can be stratified according to the physiological status of their carcinoma-associated fibroblasts. In conclusion the CAFs-derived 5-gene classifier provides more accurate information about outcome than conventional clinicopathological criteria and it could be useful to take clinical decisions, especially in Stage II. Additionally, the classifier put into relevance the CAF's intratumoral heterogeneity and might contribute to find relevant targets for depleting adequate CAFS subtypes.


Assuntos
Neoplasias Colorretais/classificação , Neoplasias Colorretais/genética , Fibroblastos/metabolismo , Idoso , Estudos de Coortes , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Feminino , Fibroblastos/patologia , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
18.
World J Gastroenterol ; 20(30): 10591-8, 2014 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-25132780

RESUMO

AIM: To investigate the relationship between the methylation status in the SLIT2 and TGFB2 promoters and colonic inflammation in inflammatory bowel disease patients. METHODS: We evaluated the methylation status of 2 genes (SLIT2 and TGFB2) in 226 biopsies taken from 62 colonoscopies of 38 patients (29 ulcerative colitis and 9 Crohn's colitis) using methylation-specific melting curve analysis. The relationships between methylation status and clinical, biological, endoscopic and histological activities were evaluated. Twenty-three of the 38 patients had a second colonoscopy and were included in a longitudinal analysis. Numerical results were given as the means ± SD of the sample and range, except when specified. Student t analysis, U Mann Whitney and ANOVA factor were used to compare the means. Qualitative results were based on the χ(2) test. RESULTS: SLIT2 methylation was more frequent in samples with endoscopic activity than with endoscopic remission (55% vs 18%, P < 0.001). SLIT2 methylation was also higher in samples with acute inflammation (56.5%) than in samples with chronic (24%) or absent inflammation (15%) (P < 0.001). For TGFB2 methylation, the correlation was only significant with endoscopic activity. Methylation was higher in the distal colon for both genes (P < 0.001 for SLIT2 and P = 0.022 for TGFB2). In the multivariate analysis, only inflammation status (and not disease duration or extension) was independently associated with SLIT2 methylation [OR = 6.6 (95%CI: 1.65-27.36), P = 0.009]. In the longitudinal analysis, the maintenance of endoscopic remission was protective for methylation. CONCLUSION: Endoscopic and histological inflammation are predictive for SLIT2 methylation.


Assuntos
Colite Ulcerativa/genética , Doença de Crohn/genética , Metilação de DNA , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Fator de Crescimento Transformador beta2/genética , Adulto , Idoso , Biópsia , Distribuição de Qui-Quadrado , Colite Ulcerativa/patologia , Colite Ulcerativa/terapia , Colo/patologia , Colonoscopia , Doença de Crohn/patologia , Doença de Crohn/terapia , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Indução de Remissão , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento
19.
Clin Gastroenterol Hepatol ; 12(11): 1865-70, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24993368

RESUMO

BACKGROUND & AIMS: Histologic recovery of patients with ulcerative colitis (UC) often is incomplete, even among those in clinical and endoscopic remission. Persistent active microscopic inflammation is associated with an increased risk of relapse and colorectal neoplasia. A high level of fecal calprotectin (FC) is a reliable marker of endoscopic lesions in patients with UC. We evaluated the accuracy of FC in identifying patients with UC in clinical and endoscopic remission who still have histologic features of inflammation. METHODS: We performed a prospective observational study of 59 patients with UC in clinical and endoscopic remission undergoing colonoscopy. Several biopsy specimens were collected from each colonic segment. Endoscopic remission was defined as a Mayo endoscopic subscore with a grade of 0 or 1. Active histologic inflammation was defined by the presence of neutrophils infiltrating crypt epithelial cells. FC was determined by enzyme-linked immunosorbent assay analysis. RESULTS: Eighteen patients (30.5%) showed evidence of active histologic inflammation. Patients with active histologic inflammation had a significantly higher median level of FC (278 µg/g; interquartile range, 136-696 µg/g) than those without active histologic inflammation (68 µg/g; interquartile range, 30-172 µg/g) (P = .002). In multivariate analysis, the FC and Mayo endoscopic subscore (0 or 1) were each independent predictors of histologic inflammation. The level of FC identified active histologic inflammation in patients in clinical and endoscopic remission, with an area under the receiver operator characteristic curve value of 0.754. CONCLUSIONS: Histologic inflammation is common among patients with UC in clinical and endoscopic remission. Patients with histologic features of inflammation can be identified reliably based on their fecal level of calprotectin.


Assuntos
Biomarcadores/análise , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/patologia , Fezes/química , Complexo Antígeno L1 Leucocitário/análise , Adolescente , Adulto , Idoso , Biópsia , Colonoscopia , Feminino , Histocitoquímica , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
20.
Mol Cancer Res ; 12(9): 1254-66, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24829396

RESUMO

UNLABELLED: The differential gene expression patterns between normal colonic fibroblasts (NCF), carcinoma-associated fibroblasts from primary tumors (CAF-PT), and CAFs from hepatic metastasis (CAF-LM) are hypothesized to be useful for predicting relapse in primary tumors. A transcriptomic profile of NCF (n = 9), CAF-PT (n = 14), and CAF-LM (n = 11) was derived. Prediction Analysis of Microarrays (PAM) was used to obtain molecular details for each fibroblast class, and differentially expressed transcripts were used to classify patients according to recurrence status. A number of transcripts (n = 277) were common to all three types of fibroblasts and whose expression level was sequentially deregulated according to the transition: NCF→CAF-PT→CAF-LM. Importantly, the gene signature was able to accurately classify patients with primary tumors according to their prognosis. This capacity was exploited to obtain a refined 19-gene classifier that predicted recurrence with high accuracy in two independent datasets of patients with colorectal cancer and correlates with fibroblast migratory potential. The prognostic power of this genomic signature is strong evidence of the link between the tumor-stroma microenvironment and cancer progression. Furthermore, the 19-gene classifier was able to identify low-risk patients very accurately, which is of particular importance for stage II patients, who would benefit from the omission of chemotherapy, especially T4N0 patients, who are clinically classified as being at high risk. IMPLICATIONS: A defined stromal gene expression signature predicts relapse in patients with colorectal cancer.


Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , Recidiva Local de Neoplasia/genética , Transcriptoma , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Prognóstico
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