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1.
Clin Exp Gastroenterol ; 12: 219-229, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190949

RESUMO

Purpose: The incidence of esophageal adenocarcinoma (EAC) has increased by 700% in Western countries over the last 30 years. Although clinical guidelines call for endoscopic surveillance for EAC among high-risk populations, fewer than 5% of new EAC patients are under surveillance at the time of diagnosis. We studied the accuracy of combined cytopathology and MUC2 immunohistochemistry (IHC) for screening of Intestinal Metaplasia (IM), dysplasia and EAC, using specimens collected from the EsophaCap swallowable encapsulated cytology sponge from Canada and United States. Patients and methods: By comparing the EsophaCap cytological diagnosis with concurrent endoscopic biopsies performed on the same patients in 28 cases, we first built up the cytology diagnostic categories and criteria. Based on these criteria, 136 cases were evaluated by both cytology and MUC2 IHC with blinded to patient biopsy diagnosis. Results: We first set up categories and criteria for cytological diagnosis of EscophaCap samples. Based on these, we divided our evaluated cytological samples into two groups: non-IM group and IM or dysplasia or adenocarcinoma group. Using the biopsy as our gold standard to screen IM, dysplasia and EAC by combined cytology and MUC2 IHC, the sensitivity and specificity were 68% and 91%, respectively, which is in the range of clinically useful cytological screening tests such as the cervical Pap smear. Conclusions: Combined EsophaCap cytology and MUC2 IHC could be a good screening test for IM and Beyond.

3.
Surg Clin North Am ; 99(3): 403-418, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31047032

RESUMO

Esophageal cancer and gastric cancer are leading causes of cancer-related mortality worldwide. In this article, the authors discuss the molecular biology of esophageal and gastric cancer with a focus on esophageal adenocarcinoma. They review data from The Cancer Genome Atlas project and advances in the molecular stratification and classification of esophageal carcinoma and gastric cancer. They also summarize advances in microRNA, molecular staging, gene expression profiling, tumor microenvironment, and detection of circulating tumor DNA. Finally, the authors summarize some of the implications of understanding the molecular basis of esophageal cancer and future directions in the management of esophageal cancer.


Assuntos
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Neoplasias Gástricas/genética , Adenocarcinoma/epidemiologia , Esôfago de Barrett/epidemiologia , Esôfago de Barrett/genética , Variações do Número de Cópias de DNA/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Neoplasias Esofágicas/epidemiologia , Previsões , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Metástase Linfática , MicroRNAs , Mutação/genética , Estadiamento de Neoplasias , Fatores de Risco , Neoplasias Gástricas/epidemiologia , Microambiente Tumoral
4.
Biomol Detect Quantif ; 17: 100078, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30906693

RESUMO

Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.

5.
Sci Rep ; 9(1): 3503, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837525

RESUMO

Liquid biopsy and detection of tumor-associated mutations in cell-free circulating DNA often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage and polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. Theoretically, high fidelity enzymes will also reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. We find that barcoding itself displays largest impact on error reduction, even with low fidelity polymerases. Use of high fidelity polymerases in the barcoding step of library construction further suppresses error in barcoded NGS, and allows detection of variant alleles below 0.1% allele frequency. However, the improvement in error correction is modest and is not directly proportional to polymerase fidelity. Depending on the specific application, other polymerase characteristics such as multiplexing capacity, PCR efficiency, buffer requirements and ability to amplify targets with high GC content may outweigh the relatively small additional decrease in error afforded by ultra-high fidelity polymerases.

6.
Head Neck ; 41(5): 1351-1358, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30554450

RESUMO

BACKGROUND: Recommendations for perioperative therapy in head and neck cancer are not explicit and recurrence occurs frequently. Circulating tumor DNA is an emerging cancer biomarker, but has not been extensively explored for detection of recurrence in head and neck cancer. METHODS: Patients diagnosed with head and neck squamous cell carcinoma were recruited into the study protocol. Tumors were sequenced to identify patient-specific mutations. Mutations were then identified in plasma circulating tumor DNA from pre-treatment blood samples and longitudinally during standard follow-up. Circulating tumor DNA status during follow-up was correlated to disease recurrence. RESULTS: Samples were taken from eight patients. Tumor mutations were verified in seven patients. Baseline circulating tumor DNA was positive in six patients. Recurrence occurred in four patients, two of whom had detectable circulating tumor DNA prior to recurrence. CONCLUSION: Circulating tumor DNA is a potential tool for disease and recurrence monitoring following curative therapy in head and neck cancer, allowing for better prognostication, and/or modification of treatment strategies.

7.
J Thorac Cardiovasc Surg ; 154(2): 714-727, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28495058

RESUMO

OBJECTIVE: To determine whether microRNA (miRNA) profiling of primary lung and head and neck squamous cell carcinomas could be useful to identify a specific miRNA signature that can be used to further discriminate between primary lung squamous carcinomas and metastatic lesions in patients with a history of head and neck squamous cell cancer. METHODS: Specimens of resected primary head and neck and lung squamous cell carcinomas were obtained from formalin-fixed, paraffin-embedded blocks. Paraffin blocks were sectioned and deparaffinized, and total RNA was isolated and profiled. Quantitative polymerase chain reaction was performed to verify array results. RESULTS: Twelve head and neck and 16 lung squamous cell carcinoma samples met quality control metrics and were included for analysis. Forty-eight miRNAs were differentially expressed (P < .05) between the 2 groups. Of these, 30 were also significantly associated (q < .25) with tumor type in 2 independent sets of primary head and neck and lung squamous carcinomas profiled by The Cancer Genome Atlas consortium, including miR-34a and miR-10a. The ratio of miR-10a and miR-10b was especially predictive of primary cancer site in all 3 data sets, with area under the (receiver operating characteristics) curve values ranging from 0.922 to 0.982. Quantitative polymerase chain reaction confirmed the association of miR-34a expression and the miR-10:miR-10b ratio with tumor type. CONCLUSIONS: MicroRNA expression may be useful for discriminating between head and neck and lung squamous cell carcinomas, including miR-34a and the miR-10a:miR-10b ratio. This differentiation has clinical importance because it could help determine the appropriate therapeutic approach.


Assuntos
Carcinoma de Células Escamosas/diagnóstico , Perfilação da Expressão Gênica/métodos , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias Pulmonares/diagnóstico , MicroRNAs/genética , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Feminino , Marcadores Genéticos/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/diagnóstico , Metástase Neoplásica/genética , Reação em Cadeia da Polimerase em Tempo Real
8.
Nat Protoc ; 12(4): 664-682, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28253235

RESUMO

Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.


Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Primers do DNA/genética , Limite de Detecção
9.
Clin Exp Gastroenterol ; 10: 29-37, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28223834

RESUMO

Bile acid reflux in the esophagus plays an important role in the carcinogenesis of esophageal adenocarcinoma (EAC). The G-protein coupled bile acid receptor (TGR5) has been associated with the development of gastrointestinal cancer. However, little is known regarding the role of TGR5 in esophageal carcinoma and precancerous lesions. We analyzed genomic DNA from 116 EACs for copy number aberrations via Affymetrix SNP6.0 microarrays. The TGR5 gene locus was amplified in 12.7% (14/116) of the EACs. The TGR5 protein expression was also assessed using immunohistochemistry from tissue microarrays, including Barrett's esophagus (BE), low-(LGD) and high-grade dysplasia (HGD), columnar cell metaplasia (CM), squamous epithelium (SE), EAC and squamous cell carcinoma. The TGR5 protein was highly expressed in 71% of EAC (75/106), 100% of HGD (11/11), 72% of LGD (13/18), 66% of BE (23/35), 84% of CM (52/62), and 36% of SE (30/83). The patients with high expression of TGR5 exhibited significantly worse overall survival compared to the patients with nonhigh expression. TGR5 high expression was significantly increased in the males compared to the females in all cases with an odds ratio of 1.9 times. The vitamin D receptor (VDR) was significantly correlated with TGR5 expression. Our findings indicated that TGR5 may play an important role in the development and prognosis of EAC through a bile acid ligand. Gender differences in TGR5 and VDR expression may explain why males have a higher incidence of EAC compared to females.

10.
Nucleic Acids Res ; 44(11): e105, 2016 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-27060140

RESUMO

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.


Assuntos
Código de Barras de DNA Taxonômico , Análise Mutacional de DNA , Reação em Cadeia da Polimerase Multiplex , Mutação , Biópsia , Linhagem Celular , Primers do DNA/química , Primers do DNA/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA
11.
J Mol Diagn ; 18(2): 235-43, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26752305

RESUMO

Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications.


Assuntos
Análise Mutacional de DNA/métodos , DNA de Neoplasias/sangue , Neoplasias/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA de Neoplasias/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Frequência do Gene , Humanos , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Sensibilidade e Especificidade , Proteína Smad4/genética , Proteína Supressora de Tumor p53/genética
12.
J Gastrointest Surg ; 20(3): 500-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26715559

RESUMO

BACKGROUND: The presence of dilated intercellular spaces in the stratified squamous lining of the esophagus is the pathognomonic feature of reflux esophagitis secondary to gastroesophageal reflux disease (GERD). In addition to stomach acid, bile salts are major constituents of gastroesophageal refluxate. The aim of our study was to determine the effect of bile salts cocktail at different pHs on epithelial junctions in an in vitro transwell model of stratified esophageal squamous epithelium. DISCUSSION: Human telomerase reverse transcriptase (hTERT) immortalized primary esophageal EPC1 cells were grown on polyester transwell surfaces in calcium-enriched media. The cells exhibited gradual stratification into an 11-layered squamous epithelium over 7 days, together with epithelial barrier function as indicated by increased transepithelial electrical resistance (TEER). This stratified epithelium demonstrated well-formed tight junctions, adherens junctions, and desmosomes as visualized by immunofluorescence and electron microscopy. When exposed to short pulses of bile salts at pH 5, but not either condition alone, there was loss of stratification and decrease in TEER, concomitant with disruption of adherens junctions, tight junctions, and desmosomes, leading to the appearance of dilated intercellular spaces. At the cellular level, bile salts at pH 5 activated the Wnt pathway (indicated by increased ß-catenin Ser552 phosphorylation). CONCLUSION: In conclusion, in our in vitro transwell model bile salts at pH 5, but not bile salts or media at pH 5 alone, modulate Wnt signaling, disrupt different junctional complexes, and cause increased permeability of stratified squamous esophageal epithelium. These changes approximate the appearance of dilated intercellular space similar to that found in GERD patients.


Assuntos
Ácidos e Sais Biliares/farmacologia , Células Epiteliais/efeitos dos fármacos , Esôfago/efeitos dos fármacos , Esôfago/patologia , Espaço Extracelular/efeitos dos fármacos , Técnicas de Cultura de Células , Impedância Elétrica , Epitélio/efeitos dos fármacos , Epitélio/patologia , Humanos , Membrana Mucosa/efeitos dos fármacos , Membrana Mucosa/patologia , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismo
13.
Expert Rev Mol Diagn ; 15(8): 1085-100, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26132215

RESUMO

OBJECTIVE: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). METHODS: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. RESULT: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. CONCLUSION: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.


Assuntos
DNA Complementar/análise , DNA/análise , Linhagem Celular Tumoral , DNA/genética , DNA Complementar/genética , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase em Tempo Real
14.
Hum Pathol ; 45(8): 1744-51, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24951052

RESUMO

Bile acid reflux into the esophagus is important in the development of esophageal adenocarcinoma (EAC). Recently, vitamin D receptor (VDR) was recognized as a bile acid receptor as well as a vitamin receptor. Expression of VDR is reported to influence the development of various types of cancer, such as those of the breast, liver, and colon. However, little is known about the role of VDR in esophageal neoplasms. We investigated the clinicopathological role of VDR in esophageal tumors. We analyzed genomic DNA from 116 EACs for copy number aberrations. The VDR locus was amplified in 7% of EACs. Expression of the VDR protein was also detected by immunohistochemistry from tissue microarrays created from tissues of Barrett esophagus (BE), low-grade (LGD) and high-grade dysplasia (HGD), columnar cell metaplasia (CCM), squamous epithelium (SE), EAC, and esophageal squamous cell carcinoma (ESCC). The protein was highly expressed in 88% of CCM (58/66), 95% of BE (35/37), 100% of the 19 LGD, 94% of HGD (15/16), and 79% of EAC (86/109), but expression in SE and ESCC was rare. Female patients with EAC and CCM were significantly less likely to have high VDR expression than male patients. The overall survival rate was significantly different for patients with tumors exhibiting VDR amplification versus nonamplification. Our findings suggest that VDR plays a role in the early development of EAC through a bile acid ligand. The sex difference in VDR expression may help to explain why men have a high incidence of EAC.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Esôfago/metabolismo , Lesões Pré-Cancerosas/metabolismo , Receptores de Calcitriol/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Esôfago/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Prognóstico , Receptores de Calcitriol/genética , Caracteres Sexuais
15.
BMC Gastroenterol ; 14: 78, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24742107

RESUMO

BACKGROUND: Cyclin E is a cell cycle regulator which is critical for driving G1/S transition. Abnormal levels of cyclin E have been found in many cancers. However, the level changes of cyclin E in esophageal adenocarcinoma and its precancerous lesion have not been well studied. Here, we focus on the gene amplification and expression of cyclin E in these lesions, and aim to ascertain the relationship with clinicopathological characteristics. METHODS: Genomic DNA was analyzed from 116 esophageal adenocarcinoma and 26 precancerous lesion patients using Affymetrix SNP 6.0 arrays. The protein overexpression of cyclin E was also detected using immunohistochemistry from tissue microarrays containing esophageal adenocarcinoma and precancerous lesions. Patient survival and other clinical data were collected and analyzed. The intensity and percentage of the cyclin E expressing cells in tissue microarrays were scored by two pathologists. Fisher exact tests and Kaplan-Meier methods were used to analyze data. RESULTS: By genomic analysis, cyclin E was amplified in 19.0% of the EAC samples. By immunohistochemistry, high expression of cyclin E was observed in 2.3% of squamous mucosa tissues, 3.7% in columnar cell metaplasia, 5.8% in Barrett's esophagus, 19.0% in low grade dysplasia, 35.7% in high grade dysplasia, and 16.7% in esophageal adenocarcinoma. The differences in cyclin E high expression between neoplastic groups and non-dysplasia groups are statistically significant (p < 0.05). The prognosis for patients with high cyclin E expression appeared slightly better than for those with low cyclin E expression although this was not statistically significant (p = 0.13). CONCLUSIONS: The expression of cyclin E significantly increases from non-dysplasia esophageal lesion to low and high grade dysplasia, suggesting that cyclin E plays an important role in the early stage of carcinogenesis. Importantly, cyclin E is also amplified and highly expressed in a subset of esophageal adenocarcinoma patients, but this increase is not associated with worse prognosis.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Carcinogênese/genética , Ciclina E/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Oncogênicas/genética , Lesões Pré-Cancerosas/genética , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/metabolismo , Carcinogênese/metabolismo , Ciclina E/metabolismo , Neoplasias Esofágicas/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/metabolismo , Polimorfismo de Nucleotídeo Único , Lesões Pré-Cancerosas/metabolismo , Análise Serial de Tecidos
16.
Ann Surg ; 260(1): 72-80, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24509200

RESUMO

OBJECTIVE: To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. BACKGROUND: Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. METHODS: Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. RESULTS: Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. CONCLUSIONS: This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , DNA de Neoplasias/genética , Neoplasias Esofágicas/genética , Genes p16/fisiologia , Células Caliciformes/patologia , Mutação , Lesões Pré-Cancerosas , Adenocarcinoma/patologia , Idoso , Esôfago de Barrett/patologia , Biópsia , Análise Mutacional de DNA , Neoplasias Esofágicas/patologia , Esôfago/patologia , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Metaplasia , Reação em Cadeia da Polimerase , Estudos Retrospectivos
17.
J Gastrointest Surg ; 17(10): 1723-31, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23921815

RESUMO

BACKGROUND: Barrett's esophagus is a preneoplastic metaplasia in which the normal squamous epithelium of the esophagus changes to an intestinal, columnar phenotype due to long-term gastro-esophageal reflux. The major components of this reflux are bile and stomach acid. Previous in vitro studies on the effect of bile and acid on esophageal cells have predominantly relied on transformed esophageal squamous cells or cancer cells grown in monolayer culture. DISCUSSION: In this study, we expanded our previous work using an immortalized primary esophageal squamous cell line (EPC1). We demonstrate that EPC1 cells form a multi-layer, stratified epithelium when grown on polyester transwell filters in media supplemented with calcium. When exposed to short pulses of bile and pH 5, but not either condition alone, EPC1 cells demonstrate a reduction in stratification layers and reduced expression of squamous epithelium-specific genes. Bile at pH 5 also causes activation of epidermal growth factor receptor and down-stream pathways. Blocking epidermal growth factor receptor activation partially attenuates the effects of bile acid and pH 5. These results suggest that bile at low pH, but not bile or low pH alone, promotes loss of differentiation status of stratified squamous esophageal epithelium in vitro, possibly by initiating a mucosal repair response through epidermal growth factor activation.


Assuntos
Ácidos e Sais Biliares/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Receptores ErbB/fisiologia , Esôfago/patologia , Técnicas de Cultura de Células/métodos , Epitélio/patologia , Humanos , Concentração de Íons de Hidrogênio , Transdução de Sinais , Células Tumorais Cultivadas
18.
Otolaryngol Head Neck Surg ; 149(2): 261-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23625795

RESUMO

OBJECTIVES: To investigate the correlation between the percentage of metastatic tumor present in lymph nodes resected from patients with squamous cell carcinoma of the head and neck (HNSCC) and level of expression of 3 marker genes: pemphigus vulgaris antigen (PVA), parathyroid hormone-related peptide (PTHrP), and tumor-associated calcium signal transducer 1 (TACSTD1). In addition, we investigated whether the level of expression of these 3 markers was associated with clinical outcomes for patients with HNSCC. STUDY DESIGN: Retrospective analysis of previously harvested patient samples. SETTING: The University of Pittsburgh. SUBJECTS AND METHODS: A total of 448 lymph nodes from 92 patients with HNSCC were evaluated for expression of the gene markers PVA, PTHrP, and TACSTD1 using real-time polymerase chain reaction. Confirmation of metastasis was determined by histologic examination. The expression level of these markers versus tumor percentage was analyzed. RESULTS: All 3 markers were studied independently and were associated with tumor percentage in metastatic lymph nodes. PVA had the strongest correlation, followed by PTHrP and then TACSTD1. PVA levels had a trend toward association with clinical outcome, specifically time to death caused by cancer, but this was confounded by tumor stage. CONCLUSION: All 3 tumor gene markers were associated with percentage of tumor cells in metastatic lymph nodes. PVA had the strongest correlation. PVA may add prognostic utility beyond pathologic staging, but this requires analysis of a larger cohort. Prospective studies of tumor volume in metastatic nodes should determine a lower limit threshold of molecular marker detection.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , RNA Neoplásico/genética , Idoso , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Autoantígenos , Biomarcadores Tumorais/biossíntese , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundário , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Desmogleína 3/biossíntese , Desmogleína 3/genética , Molécula de Adesão da Célula Epitelial , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/secundário , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Proteína Relacionada ao Hormônio Paratireóideo/biossíntese , Proteína Relacionada ao Hormônio Paratireóideo/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Biópsia de Linfonodo Sentinela , Carcinoma de Células Escamosas de Cabeça e Pescoço
19.
Nat Genet ; 45(5): 478-86, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23525077

RESUMO

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ~15%, the identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome sequencing. We identify a mutational signature defined by a high prevalence of A>C transversions at AA dinucleotides. Statistical analysis of exome data identified 26 significantly mutated genes. Of these genes, five (TP53, CDKN2A, SMAD4, ARID1A and PIK3CA) have previously been implicated in EAC. The new significantly mutated genes include chromatin-modifying factors and candidate contributors SPG20, TLR4, ELMO1 and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 identifies increased cellular invasion. Therefore, we suggest the potential activation of the RAC1 pathway as a contributor to EAC tumorigenesis.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/genética , Exoma/genética , Genoma Humano/genética , Mutação/genética , Mapeamento Cromossômico , Rearranjo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Invasividade Neoplásica
20.
J Thorac Cardiovasc Surg ; 145(2): 505-12; discussion 512-3, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23321130

RESUMO

OBJECTIVE: The incidence of esophageal adenocarcinoma is rapidly increasing in the western population. Despite aggressive treatment, survival after esophagectomy is suboptimal. The main objective of the present study was to evaluate the gene expression profiles in esophageal adenocarcinoma and determine their association with survival after resection. METHODS: We conducted a prospective National Institutes of Health/National Cancer Institute funded study to evaluate the prognostic significance of gene expression in patients with esophageal adenocarcinoma undergoing esophagectomy. Gene expression in tumor tissue was analyzed using high-throughput oligonucleotide arrays. The association of gene expression and overall survival was analyzed using the tail-strength statistic and Cox regression analysis. Gene signatures were constructed with semisupervised methods using principal components. A cross-validated risk score was devised by conducting 10-fold cross-validation, 100 times. RESULTS: We evaluated the gene expression in 64 patients with esophageal adenocarcinoma who underwent esophagectomy. The median overall survival was 27 months (95% confidence interval 22 to not reached). After filtering, 10,214 probe sets were used for survival analysis. The tail-strength statistic for these probe sets (0.318) indicated a significant association with overall survival. Patients were classified into high- and low-risk groups, according to the gene signature. High-risk patients had a predicted median survival of 19 months, but the median was not reached for the low-risk group (P < .05). On multivariate analysis, the gene signature was independently associated with survival (hazard ratio, 2.22; P = .04). CONCLUSIONS: Global gene expression levels were significantly associated with overall survival after esophagectomy. Furthermore, individual genes could be successfully combined into a strongly predictive, internally cross-validated gene signature. If validated further, these results could help direct additional clinical trials of neoadjuvant and adjuvant therapies for esophageal adenocarcinoma.


Assuntos
Biomarcadores Tumorais/genética , Esofagectomia/mortalidade , Perfilação da Expressão Gênica , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise por Conglomerados , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/cirurgia , Esofagectomia/efeitos adversos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Análise de Componente Principal , Modelos de Riscos Proporcionais , Estudos Prospectivos , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
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