ABSTRACT
TP53 mutations are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor outcome. The prognostic and predictive relevance of EGFR/TP53 co-mutations in NSCLC is controversial. We analyzed lung tissue specimens from 70 patients with NSCLC using next-generation sequencing to determine EGFR and TP53 status and the association between these status with baseline patient and tumor characteristics, adjuvant treatments, relapse, and progression-free (PFS) and overall survival (OS) after surgical resection. We found the EGFR mutation in 32.9% of patients (20% classical mutations and 12.9% uncommon mutations). TP53 missense mutations occurred in 25.7% and TP53/EGFR co-mutations occurred in 43.5% of patients. Stage after surgical resection was significantly associated with OS (P=0.028). We identified an association between progression-free survival and poor outcome in patients with distant metastases (P=0.007). We found a marginally significant difference in OS between genders (P=0.057) and between mutant and wild type TP53 (P=0.079). In univariate analysis, distant metastases (P=0.027), pathological stage (IIIA-IIIB vs I-II; P=0.028), and TP53 status (borderline significance between wild type and mutant; P=0.079) influenced OS. In multivariable analysis, a significant model for high risk of death and poor OS (P=0.029) selected patients in stage IIIA-IIIB, with relapse and distant metastases, non-responsive to platin-based chemotherapy and erlotinib, with tumors harboring EGFR uncommon mutations, with TP53 mutant, and with EGFR/TP53 co-mutations. Our study suggested that TP53 mutation tends to confer poor survival and a potentially negative predictive effect associated with a non-response to platinum-based chemotherapy and erlotinib in early-stage resected EGFR-mutated NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Male , Female , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/surgery , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/surgery , Lung Neoplasms/drug therapy , Erlotinib Hydrochloride/therapeutic use , Brazil , Neoplasm Staging , Neoplasm Recurrence, Local/pathology , Mutation , ErbB Receptors/genetics , ErbB Receptors/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Tumor Suppressor Protein p53/geneticsABSTRACT
The common epidermal growth factor receptor (EGFR) mutations, such as the L858R point mutation in exon 21 and the in-frame deletional mutation in exon 19, have been definitively associated with response to EGFR-tyrosine kinase inhibitors (EGFR-TKI). However, the clinical outcome and response to treatment for many other rarer mutations are still unclear. In this study, we report the results of Brazilian patients in stage IB-IIIA non-small cell lung cancer (NSCLC) following complete resection with minimal residual disease and EGFR mutations treated with adjuvant chemotherapy and/or EGFR-TKIs. The frequency of EGFR mutations was investigated in 70 cases of early stage NSCLC. Mutations in exons 18 and 20, uncommon mutations in exons 19 and 21, as well as in exons 3, 7, 14, 16, 22, 27, and 28, and/or the presence of different mutations in a single tumor (complex mutations) are considered rare. EGFR mutations were detected in 23 tumors (32.9%). Fourteen cases carried rare mutations and were treated with platinum-based chemotherapy and two cases were treated with erlotinib. The clinical outcome is described case by case with references to the literature. Notably, we found two rare EGFR mutations and one of them with an unknown response to chemotherapy and/or EGFR-TKIs. We have provided complementary information concerning the clinical outcome and treatment of patients with early stage NSCLC for several rare EGFR mutations not previously or only rarely reported. Description of cases harboring rare mutations can support the decision-making process in this subset of patients.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Brazil , Protein Kinase Inhibitors/therapeutic use , Mutation/genetics , ErbB Receptors/genetics , ErbB Receptors/therapeutic use , Treatment Outcome , Retrospective StudiesABSTRACT
TP53 mutations are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor outcome. The prognostic and predictive relevance of EGFR/TP53 co-mutations in NSCLC is controversial. We analyzed lung tissue specimens from 70 patients with NSCLC using next-generation sequencing to determine EGFR and TP53 status and the association between these status with baseline patient and tumor characteristics, adjuvant treatments, relapse, and progression-free (PFS) and overall survival (OS) after surgical resection. We found the EGFR mutation in 32.9% of patients (20% classical mutations and 12.9% uncommon mutations). TP53 missense mutations occurred in 25.7% and TP53/EGFR co-mutations occurred in 43.5% of patients. Stage after surgical resection was significantly associated with OS (P=0.028). We identified an association between progression-free survival and poor outcome in patients with distant metastases (P=0.007). We found a marginally significant difference in OS between genders (P=0.057) and between mutant and wild type TP53 (P=0.079). In univariate analysis, distant metastases (P=0.027), pathological stage (IIIA-IIIB vs I-II; P=0.028), and TP53 status (borderline significance between wild type and mutant; P=0.079) influenced OS. In multivariable analysis, a significant model for high risk of death and poor OS (P=0.029) selected patients in stage IIIA-IIIB, with relapse and distant metastases, non-responsive to platin-based chemotherapy and erlotinib, with tumors harboring EGFR uncommon mutations, with TP53 mutant, and with EGFR/TP53 co-mutations. Our study suggested that TP53 mutation tends to confer poor survival and a potentially negative predictive effect associated with a non-response to platinum-based chemotherapy and erlotinib in early-stage resected EGFR-mutated NSCLC.
ABSTRACT
The common epidermal growth factor receptor (EGFR) mutations, such as the L858R point mutation in exon 21 and the in-frame deletional mutation in exon 19, have been definitively associated with response to EGFR-tyrosine kinase inhibitors (EGFR-TKI). However, the clinical outcome and response to treatment for many other rarer mutations are still unclear. In this study, we report the results of Brazilian patients in stage IB-IIIA non-small cell lung cancer (NSCLC) following complete resection with minimal residual disease and EGFR mutations treated with adjuvant chemotherapy and/or EGFR-TKIs. The frequency of EGFR mutations was investigated in 70 cases of early stage NSCLC. Mutations in exons 18 and 20, uncommon mutations in exons 19 and 21, as well as in exons 3, 7, 14, 16, 22, 27, and 28, and/or the presence of different mutations in a single tumor (complex mutations) are considered rare. EGFR mutations were detected in 23 tumors (32.9%). Fourteen cases carried rare mutations and were treated with platinum-based chemotherapy and two cases were treated with erlotinib. The clinical outcome is described case by case with references to the literature. Notably, we found two rare EGFR mutations and one of them with an unknown response to chemotherapy and/or EGFR-TKIs. We have provided complementary information concerning the clinical outcome and treatment of patients with early stage NSCLC for several rare EGFR mutations not previously or only rarely reported. Description of cases harboring rare mutations can support the decision-making process in this subset of patients.
ABSTRACT
Using cDNA microarray analysis, we previously identified a set of differentially expressed genes in primary breast tumors based on the status of estrogen and progesterone receptors. In the present study, we performed an integrated computer-assisted and manual search of potential estrogen response element (ERE) binding sites in the promoter region of these genes to characterize their potential to be regulated by estrogen receptors (ER). Publicly available databases were used to annotate the position of these genes in the genome and to extract a 5’flanking region 2 kb upstream to 2 kb downstream of the transcription start site for transcription binding site analysis. The search for EREs and other binding sites was performed using several publicly available programs. Overall, approximately 40 percent of the genes analyzed were potentially able to be regulated by estrogen via ER. In addition, 17 percent of these genes are located very close to other genes organized in a head-to-head orientation with less than 1.0 kb between their transcript units, sharing a bidirectional promoter, and could be classified as bidirectional gene pairs. Using quantitative real-time PCR, we further investigated the effects of 17β-estradiol and antiestrogens on the expression of the bidirectional gene pairs in MCF-7 breast cancer cells. Our results showed that some of these gene pairs, such as TXNDC9/EIF5B, GALNS/TRAPPC2L, and SERINC1/PKIB, are modulated by 17β-estradiol via ER in MCF-7 breast cancer cells. Here, we also characterize the promoter region of potential ER-regulated genes and provide new information on the transcriptional regulation of bidirectional gene pairs.
Subject(s)
Female , Humans , Breast Neoplasms/genetics , Estradiol/genetics , Gene Expression Regulation, Neoplastic/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Response Elements/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Estradiol/metabolism , Estradiol/pharmacology , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Response Elements/drug effects , Transcription Factors/genetics , Transcription, Genetic/geneticsABSTRACT
Using cDNA microarray analysis, we previously identified a set of differentially expressed genes in primary breast tumors based on the status of estrogen and progesterone receptors. In the present study, we performed an integrated computer-assisted and manual search of potential estrogen response element (ERE) binding sites in the promoter region of these genes to characterize their potential to be regulated by estrogen receptors (ER). Publicly available databases were used to annotate the position of these genes in the genome and to extract a 5'flanking region 2 kb upstream to 2 kb downstream of the transcription start site for transcription binding site analysis. The search for EREs and other binding sites was performed using several publicly available programs. Overall, approximately 40% of the genes analyzed were potentially able to be regulated by estrogen via ER. In addition, 17% of these genes are located very close to other genes organized in a head-to-head orientation with less than 1.0 kb between their transcript units, sharing a bidirectional promoter, and could be classified as bidirectional gene pairs. Using quantitative real-time PCR, we further investigated the effects of 17ß-estradiol and antiestrogens on the expression of the bidirectional gene pairs in MCF-7 breast cancer cells. Our results showed that some of these gene pairs, such as TXNDC9/EIF5B, GALNS/TRAPPC2L, and SERINC1/PKIB, are modulated by 17ß-estradiol via ER in MCF-7 breast cancer cells. Here, we also characterize the promoter region of potential ER-regulated genes and provide new information on the transcriptional regulation of bidirectional gene pairs.
Subject(s)
Breast Neoplasms/genetics , Estradiol/genetics , Gene Expression Regulation, Neoplastic/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Response Elements/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Estradiol/metabolism , Estradiol/pharmacology , Female , Humans , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Response Elements/drug effects , Transcription Factors/genetics , Transcription, Genetic/geneticsABSTRACT
BACKGROUND: Glutamine and proline are metabolized the liver and may collaborate on its regeneration. Parenteral nutrition (PN) containing either glutamine or proline was given to partially hepatectomized rats. The total RNA content and growth factor gene expression in hepatic remnants was measured, to determine the effects of these amino acid supplementation on the expression of growth factors during liver regeneration. METHODS: Wistar rats nourished (HN) and malnourished (HM) were hepatectomized and divided in two groups: 20 receiving PN enriched with Alanyl-Glutamine (HN-Gln and HM-Gln) and 20 PN enriched with proline+alanine (HN-Pro and HM-Pro). The control groups comprised 7 nourished (CN) and 7 malnourished (CM) rats that didn't undergo surgery. Growth factor and thymidine kinase mRNA levels were measured by RT-PCR. RESULTS: In nourished rats, total hepatic RNA levels were lower in the HN-Gln and HN-Pro groups (0.75 and 0.63 microg/mg tissue, respectively) than in control group (1.67 microg/mg tissue) (P<0.05). In malnourished rats, total hepatic RNA content was higher in the HM-Pro group than HN-Pro, HM-Gln, and CM (3.18 vs. 0.63, 0.93 and 1.10 microg/mg, respectively; P<0.05). Hepatocyte growth factor mRNA was more abundant in the HM-Gln group when compared to CM (0.31 vs. 0.23 arbitrary units) and also in HM-Pro in relation to HM-Gln, HN-Pro, and CM(0.46 vs. 0.33 and 0.23, respectively, P<0.05). CONCLUSIONS: Proline or glutamine supplementation in malnourished rats improves total RNA content in the remnant hepatic tissue. Amino acids administration increased HGF gene expression after partial hepatectomy in malnourished rats, with a greater effect of proline than glutamine.
Subject(s)
Amino Acids/pharmacology , Gene Expression Regulation , Intercellular Signaling Peptides and Proteins/genetics , Malnutrition/genetics , Amino Acids/physiology , Animals , Gene Expression Regulation/drug effects , Intercellular Signaling Peptides and Proteins/biosynthesis , Liver/metabolism , Male , Rats , Rats, WistarABSTRACT
The taxane docetaxel is currently the most effective chemotherapeutic drug for the treatment of advanced breast cancer. However, a considerable proportion of breast cancer patients do not respond positively to docetaxel. The mechanisms of docetaxel resistance are poorly understood. Overexpression of ERBB2 occurs in 15-30% of breast tumors and is associated with chemoresistance to a variety of anticancer drugs. In the present study, we sought to identify genes involved in ERBB2-mediated chemoresistance to docetaxel. We generated SAGE libraries from two human mammary cell lines expressing basal (HB4a) and high (C5.2) levels of ERBB2 before and after intensive exposure to docetaxel and identified potential ERBB2 target genes implicated in a variety of cellular processes including cell proliferation, cell adhesion, apoptosis and cytoskeleton organization. Comparison of the transcriptome of the cell lines before and after docetaxel exposure revealed substantially different expression patterns. Twenty-one differentially expressed genes between HB4a and C5.2 cell lines, before and after docetaxel treatment, were further analyzed by qPCR. The alterations in the expression patterns in HB4a and C5.2 cell lines in response to docetaxel treatment observed by SAGE analysis were confirmed by qPCR for the majority of the genes analyzed. Our study provides a comprehensive view of the expression changes induced in two human mammary cells expressing different levels of ERBB2 in response to docetaxel that could contribute to the elucidation of the mechanisms involved in ERBB2-mediated chemoresistance in breast cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Receptor, ErbB-2/genetics , Taxoids/pharmacology , Transcription, Genetic/drug effects , Blotting, Western , Breast Neoplasms/drug therapy , Cell Line, Tumor , Docetaxel , Drug Resistance, Neoplasm/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Neoplastic/drug effects , Humans , Polymerase Chain Reaction , Receptor, ErbB-2/metabolismABSTRACT
Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ERalpha and ERbeta, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17beta-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17beta-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 microM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17beta-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER.
Subject(s)
Breast Neoplasms/metabolism , Estradiol/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Receptors, Estrogen/metabolism , Transcription Factors/drug effects , Breast Neoplasms/genetics , Cell Line, Tumor/drug effects , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Transcription, Genetic/drug effects , Up-RegulationABSTRACT
Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ERá and ERâ, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17â-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17â-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 µM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17â-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER.
Subject(s)
Female , Humans , Breast Neoplasms/metabolism , Estradiol/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Receptors, Estrogen/metabolism , Transcription Factors/drug effects , Breast Neoplasms/genetics , Cell Line, Tumor/drug effects , Gene Expression Regulation, Neoplastic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Transcription, Genetic/drug effects , Up-RegulationABSTRACT
The estrogens play important role in the homeostatic maintenance of several target tissues including those in the mammary gland, uterus, bone, cardiovascular system, and brain. Most of estrogen's action is thought to be mediated through its nuclear estrogen receptors, ERalpha and ERbeta, which are members of the nuclear receptor superfamily that act as ligand-induced transcription factors. Acting via its receptors, estrogen also plays an essential role in the development and progression of human breast cancer. The ER and progesterone receptor (PR), which are regulated by estrogen via ER, have been used as prognostic markers in the clinical management of breast cancer patients. However, the prognosis of a patient with ER+/PR+ breast cancer can be highly variable and a significant proportion of hormone receptor positive breast cancers does not respond to endocrine therapy. The identification of estrogen receptor target genes may improve our understanding of the role played by estrogens in breast cancer making it possible to better tailor hormone treatments and improve a patient's response to hormonal therapy. In this review, we explore the literature for data regarding the identification of estrogen receptor-regulated genes in breast cancer cell lines and breast tumor biopsies using high throughput technologies such as serial analysis of gene expression (SAGE) and cDNA microarrays.
Subject(s)
Breast Neoplasms/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Receptors, Estrogen/genetics , Antineoplastic Agents, Hormonal/chemistry , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Estradiol/analogs & derivatives , Estradiol/chemistry , Estradiol/pharmacology , Fulvestrant , Gene Expression Regulation, Neoplastic/drug effects , Humans , Molecular Structure , Raloxifene Hydrochloride/chemistry , Raloxifene Hydrochloride/pharmacology , Receptors, Estrogen/chemistry , Receptors, Estrogen/drug effects , Tamoxifen/chemistry , Tamoxifen/pharmacologyABSTRACT
The CAG repeat within exon 1 of the androgen receptor (AR) has been associated with the development of prostate cancer. The shorter number of glutamine residues in the protein has been associated with a higher transcriptional activity of the AR and increased relative risk for prostate cancer. In an attempt to identify differentially expressed genes in prostate cancer in relation to AR CAG repeat length variation, in this study we used total mRNA from normal and tumor tissues from 2 prostate cancer patients with AR alleles containing 19 and 26 CAG repeats to perform differential-display RT-PCR analysis. We were able to identify 48 different transcripts that showed homology to several known genes associated with different biological pathways. Among the differentially expressed genes, ATRX and SFRP1 were further validated by quantitative RT-PCR. The transcripts of both ATRX and SFRP1 genes proved to be down-regulated in most of the prostate tumors analyzed by quantitative RT-PCR. Hypermethylation of the promoter region of the SFRP1 gene was found in 17.5% (7/40) of the cases analyzed and was associated with the loss of SFRP1 expression (p=0.014). The differentially expressed genes identified in this study are implicated in several cellular pathways that, when up- or down-regulated, might play a role in the tumorigenic process of the prostate.
Subject(s)
Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Prostate/metabolism , Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , Repetitive Sequences, Nucleic Acid , Aged , DNA Primers/chemistry , Gene Expression Profiling , Humans , Male , Middle Aged , Prostatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The oral cavity is the sixth most common anatomical localization of head and neck carcinoma in men. Detection of oral carcinomas in the early asymptomatic stages improves cure rates and the quality of life. Tobacco smoking and alcohol drinking are the most important known risk factors for the development of head and neck tumors, suggesting that the exposure to these risk factors may increase the predisposition for genetic and epigenetic alterations, such as DNA methylation. The presence of methylated CpG islands in the promoter region of human genes can suppress their expression due to the presence of 5-methylcytosine that interferes with the binding of transcription factors or other DNA-binding proteins repressing transcription activity. Hypermethylation leading to the inactivation of some tumor suppressor genes, such as p16, has been pointed out as an initial event in head and neck cancer. Our aim was to evaluate an early diagnostic method of oral pre-cancerous lesions through the analysis of methylation of the p16 gene. DNA samples from normal oral mucosa and posterior tongue border from 258 smokers, without oral cancer, were investigated for the occurrence of p16 promoter hypermethylation. The methylation status of the p16 gene was analyzed using MS-PCR (methylation-sensitive restriction enzymes and PCR amplification), MSP (Methylation-specific PCR) or direct DNA sequence of bisulfite modified DNA. Hyper-methylation was detected in 9.7% (25/258) of the cases analyzed. These findings provide further evidence that epigenetic alteration, leading to the inactivation of the p16 tumor suppressor gene is an early event that might confer cell growth advantages contributing to the tumorigenic process. Thus, the detection of abnormal p16 methylation pattern may be a valuable tool for early oral cancer detection.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA Methylation , Genes, p16 , Mouth Mucosa/physiopathology , Smoking/genetics , Adult , Aged , Aged, 80 and over , Alcohol Drinking , Female , Humans , Male , Middle AgedABSTRACT
Sex hormones may play an important role in the tumorigenic process of the head and neck. The aim of our work was to investigate whether the androgen receptor (AR) CAG repeat polymorphism is associated with an increased relative risk for head and neck cancer. Genomic DNA from 103 male patients with head and neck carcinomas and 100 male controls were analyzed for the AR CAG polymorphism by PCR amplification and direct sequencing or denaturing polyacrilamide gel electrophoresis. Logistic regression analysis showed a significant association between CAG repeat length and risk of head and neck cancer in individuals with more than 20 CAG repeats [OR=2.54 (95% CI, 1.3-4.8)]. For the group of individuals with oral and laryngeal cancer the estimated relative risk was increased to 2.79 (95% CI, 1.2-6.3) and 3.06 (95% CI, 1.0-9.6), respectively, in men with CAG repeat length >20. These results suggest, for the first time, that shorter AR CAG repeat alleles have a protective effect for head and neck cancer development.
Subject(s)
Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/genetics , Polymorphism, Genetic , Receptors, Androgen/genetics , Adult , Aged , Aged, 80 and over , Case-Control Studies , DNA, Neoplasm/genetics , Humans , Logistic Models , Middle Aged , Repetitive Sequences, Nucleic AcidABSTRACT
Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.
Subject(s)
Expressed Sequence Tags , Genome, Human , Open Reading Frames , Transcription, Genetic , HumansABSTRACT
The aim of this work is detecting the loss of heterozygosity (LOH) and its relationship with the development and progression of head and neck cancer. Matched normal and tumor DNA from 81 patients with head and neck cancer were examined for LOH using six microsatellite repeat markers mapped to chromosomal regions 3p13, 6q13, 9p21, 11p15, 17p13.1, and 17q22. LOH frequency at a locus ranged from 21% to 55%. The highest frequencies were at 3p (41%), 9p (48%), and 17p (54%). Thirty-two of 81 tumor samples showed allelic loss at more than one region. Significant associations were found between LOH at 3p and 9p (P = 0.001), 9p and 11p (P = 0.03), and 9p and 17p (P = 0.007). LOH at 11p was frequent in tumors from the oral cavity (5/17), oropharynx (2/7), and hypopharynx (5/10), but absent in tumors from the larynx (0/11) (P = 0.02), and LOH at 17q was observed in tumors from oral cavity (10/30) and hypopharynx (3/9), but not in tumors from the oropharynx (0/10) or larynx (0/13) (P = 0.003). In addition to that, the occurrence of allelic losses at 9p and 17p strongly correlates to tobacco smoking (P = 0.03 and P = 0.006, respectively) and alcohol intake (P = 0.01 and P = 0.005, respectively). These results suggest that tumors from different sites have different LOH patterns and corroborate with epidemiological data implicating tobacco and alcohol in the etiology of head and neck tumors.
Subject(s)
Carcinoma, Squamous Cell/genetics , Chromosome Deletion , Head and Neck Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/pathology , Female , Head and Neck Neoplasms/pathology , Humans , Loss of Heterozygosity/genetics , Male , Microsatellite Repeats/genetics , Middle AgedABSTRACT
Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).
Subject(s)
Chromosomes, Human, Pair 22 , Transcription, Genetic , Expressed Sequence Tags , Gene Expression Profiling , Humans , Open Reading FramesABSTRACT
Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.
Subject(s)
Transcription, Genetic , Animals , Breast Neoplasms/genetics , DNA, Complementary , Databases, Factual , Expressed Sequence Tags , Humans , Molecular Sequence Data , Open Reading Frames , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The p53 protein plays an important role in the control of the cell cycle and DNA repair. Mutations in the TP53 gene may be a prognostic factor for certain forms of human cancer, with specific mutation sites being associated with significantly worse prognosis, particularly for colorectal and breast cancer. Thus, standardization of accurate, rapid, and cost-effective techniques for the detection of TP53 mutations is a high priority. At present, the only widely available technology that reliably detects and defines all mutations is DNA sequencing. However, the routine sequencing of the entire TP53 gene in all breast and colorectal cancer cases in hospital laboratories is prohibitively costly, complex, and time consuming. In order for the analytical power of DNA to be accessed by the routine laboratory, initial screening using immunohistochemistry, which is widely used as a test for detection of accumulated, mutated protein, followed by heteroduplex analysis of exons 4 to 9 to detect frameshift mutations in immunohistochemistry-negative cases, is proposed. To illustrate the effectiveness of this approach, 28 cases of head and neck squamous-cell carcinomas that were known to contain TP53 mutations were retrospectively analyzed. All missense mutations stained positive on immunohistochemistry using the monoclonal antibody DO7, and all insertions and deletions, even those involving a single nucleotide, were positive using an extremely simple heteroduplex analysis. Only rare nonsense mutations were not detected by this strategy. Nevertheless, application of these results to published data suggests that the prescreening would detect 80% of mutations but would result in a 75% reduction in the sequencing load of the laboratory.
Subject(s)
Carcinoma, Squamous Cell/genetics , Genes, p53 , Head and Neck Neoplasms/genetics , Mutation, Missense , Point Mutation , Antibodies, Monoclonal/analysis , Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/pathology , DNA Mutational Analysis , DNA, Neoplasm/analysis , Head and Neck Neoplasms/chemistry , Head and Neck Neoplasms/pathology , Heteroduplex Analysis , Humans , Immunoenzyme Techniques/methods , Prognosis , Tumor Suppressor Protein p53/analysisABSTRACT
Inactivation of the p16 gene is believed to contribute to the tumorigenic process of several neoplasms, including head and neck tumours. In the present study, DNA samples from paired tumour and adjacent normal tissue from 47 patients with squamous cell carcinoma of the head and neck were investigated for the occurrence of p16 genetic alterations. Single-strand conformation polymorphism and direct DNA sequence analysis led to the identification of p16 mutations in six cases (13%). Southern blot analysis showed that homozygous deletion is a rare event in the group of tumours analysed. Loss of heterozygosity (LOH) analysis was performed by polymerase chain reaction (PCR) using two microsatellite markers (IFNA and D9S171) from the 9p21 region. Taking into account only the informative cases, 17 of 32 tumours (53%) showed LOH for at least one of the markers analysed. The methylation status of the CpG sites in the exon 1 of the p16 gene was analysed using methylation-sensitive restriction enzymes and PCR amplification. Hypermethylation was observed in 22 (47%) of the head and neck tumours analysed. In our series of head and neck tumours, evidence for inactivation of both p16 alleles was observed in 13 cases with hypermethylation and LOH, two cases with hypermethylation and mutation, four cases with mutation and LOH and one case with homozygous deletion. These findings provide further evidence that genetic alterations, especially hypermethylation and LOH, leading to the inactivation of the p16 tumour suppressor gene are common in primary head and neck tumours.