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Esophageal cancer is the eighth most common cancer and the sixth most frequent cause of cancer mortality worldwide. Exposure to polycyclic aromatic hydrocarbons formed by incomplete combustion of organic matter is an important risk factor. Genetic polymorphisms in genes encoding PAH-metabolizing enzymes like glutathione S-transferases (GSTM1, GSTP1, GSTT1) which conjugate glutathione to PAHs for reduction of oxidative stress may affect an individual's response to PAH exposure. Genomic DNA from 50 esophageal squamous cell carcinoma patients extracted from peripheral blood. PCR-RFLP technique was employed to determine GSTM1, GSTT1, and GSTP1 polymorphisms. Aberrant promoter methylation of CDKN2A was applied by methylation-specific PCR technique. Concentration of urinary 1-hydroxypyrene was determined using a HPLC system. About 38.7% showed the null GSTM1 genotype (54% cases and 13% controls), 23.7% showed GSTT1 null genotype (30% cases and 13% controls), and 62.5% were GSTP1 A/A genotype (66% cases and 56% controls). Polymorphic variants of GSTM1 and GSTT1 were significantly associated with aberrant methylation of CDKN2A gene. The null state of GSTT1 was significantly associated with high concentrations of 1-OHP in urea (p < 0.01). There was significant association between methylated states of CDKN2A and high concentrations of 1-OHP in urine (p < 0.01). We identified significant association between polymorphism of GSTs genes and epigenetic silencing of tumor suppressor gene CDKN2A in esophageal squamous cell carcinoma.
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Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Estudos de Casos e Controles , Epigênese Genética , Genes p16 , Predisposição Genética para Doença , Genótipo , Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Humanos , Polimorfismo Genético , Fatores de RiscoRESUMO
OBJECTIVE: To characterise the gut microbiome in subjects with and without polyps and evaluate the potential of the microbiome as a non-invasive biomarker to screen for risk of colorectal cancer (CRC). DESIGN: Presurgery rectal swab, home collected stool, and sigmoid biopsy samples were obtained from 231 subjects undergoing screening or surveillance colonoscopy. 16S rRNA analysis was performed on 552 samples (231 rectal swab, 183 stool, 138 biopsy) and operational taxonomic units (OTU) were identified using UPARSE. Non-parametric statistical methods were used to identify OTUs that were significantly different between subjects with and without polyps. These informative OTUs were then used to build classifiers to predict the presence of polyps using advanced machine learning models. RESULTS: We obtained clinical data on 218 subjects (87 females, 131 males) of which 193 were White, 21 African-American, and 4 Asian-American. Colonoscopy detected polyps in 56% of subjects. Modelling of the non-invasive home stool samples resulted in a classification accuracy >75% for Naïve Bayes and Neural Network models using informative OTUs. A naïve holdout analysis performed on home stool samples resulted in an average false negative rate of 11.5% for the Naïve Bayes and Neural Network models, which was reduced to 5% when the two models were combined. CONCLUSION: Gut microbiome analysis combined with advanced machine learning represents a promising approach to screen patients for the presence of polyps, with the potential to optimise the use of colonoscopy, reduce morbidity and mortality associated with CRC, and reduce associated healthcare costs.
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BACKGROUND: Epidermal growth factor receptor family members such as ErbB1 and ErbB3 are involved in tumor progression and metastasis. Although, there are various reports about the prognostic value of EGFR members separately in gastric cancer, there is not any report about the probable correlation between ErbB1 and ErbB3 co-expression and gastric cancer prognosis. In present study, we assessed the correlation between ErbB1 and ErbB3 co-overexpression (in the level of mRNA and protein expression) and gastric cancer prognosis for the first time. METHODS: ErbB1 and ErbB3 expressions were analyzed by immunohistochemistry and real-time PCR in 50 patients with gastric cancer. Parametric correlations were done between the ErbB1 and ErbB3 expression and clinicopathological features. Multivariate and logistic regression analyses were also done to assess the roles of ErbB1 and ErbB3 in tumor prognosis and survival. RESULTS: There were significant correlations between ErbB1/ErbB3 co-overexpression and tumor size (p = 0.026), macroscopic features (p < 0.05), tumor differentiation (p < 0.05), stage of tumor (p < 0.05), and recurrence (p < 0.05). Moreover, ErbB1/ErbB3 co-overexpression may predict the survival status of patients (p < 0.05). CONCLUSION: ErbB1 and ErbB3 co-overexpression is accompanied with the poor prognosis and can be used efficiently in targeted therapy of gastric cancer patients.
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Biomarcadores Tumorais/metabolismo , Genes erbB-1 , Receptor ErbB-3/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Regulação Neoplásica da Expressão Gênica , Genes erbB , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Receptor ErbB-3/genética , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Taxa de SobrevidaRESUMO
BACKGROUND: Epidermal growth factor receptor family members such as ErbB1 and ErbB3 are involved in tumor progression and metastasis. Although, there are various reports about the prognostic value of EGFR members separately in gastric cancer, there is not any report about the probable correlation between ErbB1 and ErbB3 co-expression and gastric cancer prognosis. In present study, we assessed the correlation between ErbB1 and ErbB3 co-overexpression (in the level of mRNA and protein expression) and gastric cancer prognosis for the first time. METHODS: ErbB1 and ErbB3 expressions were analyzed by immunohistochemistry and real-time PCR in 50 patients with gastric cancer. Parametric correlations were done between the ErbB1 and ErbB3 expression and clinicopathological features. Multivariate and logistic regression analyses were also done to assess the roles of ErbB1 and ErbB3 in tumor prognosis and survival. RESULTS: There were significant correlations between ErbB1/ErbB3 co-overexpression and tumor size (p = 0.026), macroscopic features (p < 0.05), tumor differentiation (p < 0.05), stage of tumor (p < 0.05), and recurrence (p < 0.05). Moreover, ErbB1/ErbB3 co-overexpression may predict the survival status of patients (p < 0.05). CONCLUSION: ErbB1 and ErbB3 co-overexpression is accompanied with the poor prognosis and can be used efficiently in targeted therapy of gastric cancer patients.
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Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Neoplasias Gástricas/metabolismo , Biomarcadores Tumorais/metabolismo , Genes erbB-1 , Receptor ErbB-3/metabolismo , Prognóstico , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Imuno-Histoquímica , Regulação Neoplásica da Expressão Gênica , Taxa de Sobrevida , Genes erbB , Receptor ErbB-3/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVES: Variation in microsatellite sequences that are dispersed in the genome has been linked to a deficiency in cellular mismatch repair system and defects in several genes of this system are involved in carcinogenesis. Our aim in this study was to illustrate microsatellite DNA alteration in esophageal cancer. MATERIALS AND METHODS: DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues from surgical and matched margin-normal samples. Microsatellite instability (MSI) and loss of heterozygosity (LOH) were studied in 50 cases of esophageal squamous cell carcinoma (ESCC) by amplifying six microsatellite markers: D13S260 (13q12.3), D13S267 (13q12.3), D9S171 (9p21), D2S123 (2p), D5S2501 (5q21) and TP53 (17p13.1) analyzed on 6% denaturing polyacrylamide gel electrophoresis. RESULTS: Statistical analysis indicated a near significant reverse correlation between grade and LOH (P= 0.068, correlation coefficient= -0.272). Specifically, increased LOH in tumor DNA has a significant correlation with increased differentiation from poorly differentiated to well differentiated tumors (P= 0.002 and P= 0.016 respectively). In addition, higher number of chromosomal loci with LOH showed a reverse correlation with lymph node metastasis (P= 0.026, correlation coefficient= -0.485). Furthermore, there was a positive correlation between addiction and MSI (P= 0.026, correlation coefficient= 0.465). CONCLUSION: Microsatellite DNA alterations may be a prognostic tool for detection and the evolution of prognosis in patients with SCC of esophagus. It can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci and addiction with any of opium, cigarette, water pipe or alcohol can be a susceptibility factor(s) for MSI.
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INTRODUCTION: Gastric cancer (GC) is one of the leading causes of cancer-related death in Iran. Genome stability is one of the main genetic issues in cancer biology which is governed via the different repair systems such as DNA mismatch repair (MMR). A clear correlation between MMR defects and tumor progression has been shown. Beside the genetic mutations, epigenetic changes also have a noticeable role in MMR defects. METHODS: Here, we assessed promoter methylation status and the level of hMLH1mRNA expression as the main component of MMR system in 51 GC patients using the methylation-specific PCR and real-time PCR, respectively. Moreover, we performed a promoter methylation study of the E-cadherin gene promoter. RESULTS: It was observed that, 12 out of 39 cases (23.5%) had hMLH1 overexpression. Hypermethylation of hMLH1 and E-cadherin promoter regions were observed in 25.5 and 36.4%, respectively. Although, there was no significant correlation between hMLH1 mRNA expression and clinicopathological features, there are significant correlations between E-cadherin promoter methylation and tumor stage (p = 0.028) and location (p = 0.025). The rate of hMLH1 promoter methylation in this study was lower than that in the other population, showing the importance of the other mechanisms, in gastric tumorigenesis. CONCLUSION: The results of this study indicate that DNA repair system is adversely affected by hypermethylation of hMLH1 in a fraction of gastric cancer patients. Additionally, E-cadherin hypermethylation seen in a subset of our gastric cancer patients is consistent with other reports showing correlation with aggressiveness and metastasis of gastric cancer.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Biomarcadores Tumorais/genética , Caderinas/genética , Metilação de DNA , Mucosa Gástrica/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Progressão da Doença , Epigênese Genética , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive disorder characterized by hyperkeratosis involving the palms, soles, elbows, and knees followed by periodontitis, destruction of alveolar bone, and loss of primary and permanent teeth. Mutations of the lysosomal protease cathepsin C gene (CTSC) have been shown to be the genetic cause of PLS. This study analyzed CTSC mutations in five Iranian families with PLS and modeled the protein for mutations found in two of them. METHODS: DNA analysis was performed by direct automated sequencing of genomic DNA amplified from exonic regions and associated splice intron site junctions of CTSC. RFLP analyses were performed to investigate the presence of previously unidentified mutation(s) in control groups. Protein homology modeling of the deduced novel mutations (P35 delL and R272P) was performed using the online Swiss-Prot server for automated modeling and analyzed and tested with special bioinformatics tools to better understand the structural effects caused by mutations in cathepsin C protein (CTSC). RESULTS: Six Iranian patients with PLS experienced premature tooth loss and palm plantar hyperkeratosis. Sequence analysis of CTSC revealed a novel mutation (P35delL) in exon 1 of Patient 1, and four previously reported mutations; R210X in Patient 2, R272P in Patient 3, Q312R in two siblings of family 4 (Patients 4 and 5), and CS043636 in Patient 6. RFLP analyses revealed different restriction fragment patterns between 50 healthy controls and patients for the P35delL mutation. Modeling of the mutations found in CTSC, P35delL in Patient 1 and R272P in Patient 3 revealed structural effects, which caused the functional abnormalities of the mutated proteins. CONCLUSIONS: The presence of this mutation in these patients provides evidence for founder CTSC mutations in PLS. This newly identified P35delL mutation leads to the loss of a leucine residue in the protein. The result of this study indicates that the phenotypes observed in these two patients are likely due to CTSC mutations. Also, structural analyses of the altered proteins identified changes in energy and stereochemistry that likely alter protein function.
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Catepsina C/genética , Modelos Moleculares , Mutação , Doença de Papillon-Lefevre/genética , Adolescente , Sequência de Aminoácidos , Estudos de Casos e Controles , Catepsina C/química , Criança , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Doença de Papillon-Lefevre/diagnóstico , Conformação Proteica , Adulto JovemRESUMO
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the second-most frequently diagnosed cancer in Northeast Iran, often diagnosed in advanced stages. No standard early diagnostic guideline has been proposed to date and current therapeutic modalities are not effective. Detection of tumor-specific biomarkers, which is the goal of this study, could prove useful in the diagnosis of ESCC. METHODS: To better understand the gene expression profile of ESCC, we analyzed tumor samples and corresponding adjacent normal tissues from ESCC patients by Chemiluminescent Human Cancer GEArrays. Candidate genes were verified by real-time PCR. RESULTS: Out of 440 cancer-related genes included in the array, 71 were overexpressed compared to normal tissue, with significant differences in 11 genes. There were 108 genes underexpressed, with significant differences in 5 genes. Until now, the AP2M1, FTL, UBE2L6, HLA-C, and HSPA8 overexpressed genes and XRCC5, TP53I3 and RAP1A underexpressed genes were not reported in ESCC. We chose the MMP2, HLA-G, and XRCC5 markers from 58 Iranian ESCC patients to verify the expression validity by real-time PCR. The microarray results were confirmed with two-tailed significance levels of P = 0.003 (MMP2), P = 0.000 (HLA-G) and P = 0.002(XRCC5). Analysis performed for the candidate genes using GNCpro online software highlighted two pathways, an immuno-modulatory response and DNA replication and repair. We successfully performed and validated Chemiluminescent GEArray gene expression profiling in ESCC. Several biomarkers that might be related to tumorigenesis in ESCC were identified. CONCLUSION: Immuno-modulatory and DNA repair pathways could be used as targets to locate specific diagnostic, prognostic, and therapeutic biomarkers for ESCC.
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Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Reparo do DNA/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Evasão Tumoral/genética , Análise de Variância , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/imunologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/imunologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Irã (Geográfico) , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Colorectal cancer (CRC) remains the third most common cancer in the world. Approximately in 50 percent of patients, metastatic disease is a major cause of death. Therefore, early diagnosis of CRC is crucial for a successful outcome. For the detection of circulating cancer cells, this study applied a sensitive method that employed specific tumor markers for early detection. METHODS: A total of 80 blood samples from 40 CRC patients and 40 age-matched healthy controls were collected for the study. The circulating mRNA levels of two CRC tumor markers, tumor endothelial marker 8 (TEM-8) and carcinoembryogenic antigen (CEA) were evaluated using an absolute quantitative real-time PCR assay in a Stratagene Mx-3000P real-time PCR system. GAPDH was used as the endogenous control. RESULTS: TEM-8 and CEA were primarily detected more in the CRC patients rather than in the controls: 22/40 vs 9/40, p=0.009 and 30/40 vs 11/40, p=0.00054, respectively. In the CRC patients, the mRNA level of these markers was significantly higher in comparison to the normal controls (p=0.018 and 0.01). The overall sensitivity of this panel was 65% with a specificity of 75%. Statistical analysis for demographic variants did not reach significant values. CONCLUSIONS: TEM-8 and CEA markers were detected more frequently and in significantly higher levels in the blood samples of patients compared with samples from age-matched healthy controls. The copy number of CEA and TEM-8 mRNA, as detected by a real-time quantitative PCR, appears to be a promising marker for evaluating the risk of tumor spread.
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Biomarcadores Tumorais/sangue , Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Proteínas de Neoplasias/sangue , Células Neoplásicas Circulantes/metabolismo , Receptores de Superfície Celular/sangue , Biomarcadores Tumorais/genética , Antígeno Carcinoembrionário/genética , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Demografia , Feminino , Dosagem de Genes/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Receptores de Superfície Celular/genéticaRESUMO
Cancer-testis antigens (CTAs) are often specifically expressed in cancer cells and under normal conditions are only considered to be expressed in the germ line cells and the placenta. CTAs are potential targets for cancer immunotherapy and therefore necessitates their expression profiling. The expression profile of LAGE1, MAGE-A4 and NY-ESO1, their possible correlations and interaction, and the clinicopathological associations of each marker were studied. RNA was extracted from fresh esophagectomy tissues of 41 esophageal squamous cell carcinoma (ESCC) patients prior to any other therapeutic intervention. The relative mRNA expression of LAGE1, MAGE-A4 and NY-ESO1 was assessed with the real-time reverse transcription-polymerase chain reaction (RT-PCR) 5' nuclease assay. The overexpression of LAGE1, MAGE-A4 and NY-ESO1 was found in 39, 90.2 and 41.4% of ESCC samples respectively. Of the patients, 97.5% showed an overexpression of at least one CTA. The relative expression of MAGE-A4 was directly associated with lymph node metastasis and the stage of the tumor (p < 0.05). A significant direct correlation was also detected between the MAGE-A4/LAGE1 and MAGE-A4/NY-ESO1 levels of gene expression. MAGE-A4 is identified as a specific biomarker of ESCC with a possible oncogenic role contributing to tumor progression. Interactions between MAGE-A4, LAGE1 and NY-ESO1 and their significant clinical consequences introduce these CTAs as appropriate targets for a polyvalent cancer vaccine.
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Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Testículo/fisiologiaRESUMO
BACKGROUND: A complex of genetic and environmental factors is involved in carcinogenesis of the esophageal squamous cell carcinoma (ESCC). Glutathione-S-Transferases (GSTs) are phase-II enzymes playing role in detoxification of carcinogen electrophiles. Genetic polymorphisms of GSTM1, GSTT1 and GSTP1 in association with some environmental factors and their impact on esophageal cancer susceptibility were assessed in the Iranian population. METHODS: Genomic DNA of peripheral blood leukocytes from 148 confirmed esophageal cancer cases and 137 healthy individuals as control group was assayed for restriction fragment length polymorphisms in the GSTP1 loci by PCR amplification followed by digestion with Alw26I. Deletion of the GSTM1 and GSTT1 genes was detected by multiplex PCR. A data-mining method based on decision trees was applied to produce a predictive model of interactions between genotypes. RESULTS: Smoking was independently associated with ESCC (p<0.05, OR: 2.286, 95% CI=1.311-3.983). Smoking along with GSTP1 Val/Val genotype was associated to ESCC (p<0.001, OR: 3.886, 95% CI=1.830-8.251), while non-smokers with GSTP1 Val/Val were significantly more frequent in non-cancerous group. (p=0.007, OR: 0.507, 95% CI=0.309-0.830). CONCLUSIONS: Data-mining methods are useful tools to map out a scheme for predicting complex relations and combinations of different genotypes. Genotyping analysis of GSTP1 together with assessment of smoking seems to be important in determining the risk of ESCC in the Iranian population.
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Carcinoma de Células Escamosas/epidemiologia , Neoplasias Esofágicas/epidemiologia , Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Idoso , Carcinoma de Células Escamosas/genética , Árvores de Decisões , Neoplasias Esofágicas/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fumar/efeitos adversosRESUMO
AIM: To determine p16 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS: Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction (MSP) was used to evaluate methylation status of p16 promoter. p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS: Methylation was detected in 44.2% (23/52) of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients (14/52). Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases (32/52), and was significantly associated with promoter hypermethylation (P < 0.001). Methylation was significantly more frequent in higher pathological grades (P < 0.05). Methylation was not associated with other clinicopathological features and environmental factors including H pylori infection and smoking. CONCLUSION: p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.
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Biomarcadores Tumorais , Metilação de DNA , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Neoplasias Gástricas/sangue , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Idoso , Inibidor p16 de Quinase Dependente de Ciclina , Feminino , Helicobacter pylori/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Reação em Cadeia da Polimerase , Sulfitos/químicaRESUMO
BACKGROUND: Colorectal cancer (CRC) is one of the most common forms of cancers in the world and is curable if diagnosed at the early stage. Analysis of DNA extracted from stool specimens is a recent advantage to cancer diagnostics. Many protocols have been recommended for DNA extraction from stool, and almost all of them are difficult and time consuming, dealing with high amount of toxic materials like phenol. Their results vary due to sample collection method and further purification treatment. In this study, an easy and rapid method was optimized for isolating the human DNA with reduced PCR inhibitors present in stool. METHODS: Fecal samples were collected from 10 colonoscopy-negative adult volunteers and 10 patients with CRC. Stool (1 g) was extracted using phenol/chloroform based protocol. The amplification of P53 exon 9 was examined to evaluate the extraction efficiency for human genomic targets and also compared its efficiency with Machiels et al. and Ito et al. protocols. RESULTS: The amplification of exon 9 of P53 from isolated fecal DNA was possible in most cases in 35 rounds of PCR using no additional purification procedure for elimination of the remaining inhibitors.inhibitors. CONCLUSION: A useful, rapid and easy protocol for routine extraction of DNA from stool was introduced and compared with two previous protocols.
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Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Adulto , Sequência de Bases , Estudos de Casos e Controles , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Primers do DNA/genética , Fezes/química , Genes p53 , Humanos , Biologia Molecular , Reação em Cadeia da PolimeraseRESUMO
AIM: To detect tumor-associated DNA changes in stool samples among Iranian patients with colorectal cancer (CRC) compared to healthy individuals using BAT-26, p16 hypermethylation and long DNA markers. METHODS: Stool DNA was isolated from 45 subjects including 25 CRC patients and 20 healthy individuals using a new, fast and easy extraction method. Long DNA associated with tumor was detected using polymerase chain reaction method. Microsatellite studies were performed utilizing denaturating polyacrylamide gel to determine the instability of BAT-26. Methylation status of p16 promoter was analyzed using methylation-specific PCR (MSP). RESULTS: The results showed a significant difference in existence of long DNA (16 in patients vs 1 in controls, P < 0.001) and p16 (5 in patients vs none in controls, P = 0.043) in the stool samples of two groups. Long DNA was detected in 64% of CRC patients; whereas just one of the healthy individuals was positive for Long DNA. p16 methylation was found in 20% of patients and in none of healthy individuals. Instability of BAT-26 was not detected in any of stool samples. CONCLUSION: We could detect colorectal cancer related genetic alterations by analyzing stool DNA with a sensitivity of 64% and 20% and a specificity of 95% and 100% for Long DNA and p16 respectively. A non-invasive molecular stool-based DNA testing can provide a screening strategy in high-risk individuals. However, additional testing on more samples is necessary from Iranian subjects to determine the exact specificity and sensitivity of these markers.