Rapid DNA Extraction Protocol from Stool, Suitable for Molecular Genetic Diagnosis of Colon Cancer

ABSTRACT 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. Conclusion: A useful, rapid and easy protocol for routine extraction of DNA from stool was introduced and compared with two previous protocols. Iran. Biomed. J. 1

Guanylyl Cyclase C as a tumor marker for detection of circulating tumor cells in the peripheral blood of colorectal cancer patients

Purpose: Guanylyl cyclase C (GCC) is one of the most frequent tumor markers to detect the circulating tumor cells (CTCs) in peripheral blood of colorectal cancer (CRC) patients. It has been proposed as a new marker for the molecular staging of CRC. The level of GCC mRNA expression in peripheral blood of CRC patients was evaluated to explore its probable correlations with the clinicopathological features.Methods: Relative quantitative expression analysis of GCC mRNA was performed on 80 blood samples (40 patients and 40 normal) using the Real-time RT-PCR.Results: GCC mRNA expression was detected in 70% of the CRC blood samples. The level of GCC mRNA expression in peripheral blood of patients was significantly higher than that in the normal cases (p = 0.031). Moreover, there was a significant correlation between the GCC copy number and advanced stages of tumor (p = 0.041). Furthermore, we have observed a significant correlation between tumor sizes and GCC copy numbers (p = 0.050).Conclusion: GCC can be a useful marker not only for detection of CTCs in CRC blood samples, but also for the molecular staging of colorectal cancer.

DNA damage in oral mucosa cells of patients with fixed orthodontic appliances.

The release of toxic metal ions from orthodontic alloys has induced concerns regarding the biocompatibility of fixed appliances. This study investigated the genotoxic effect of metal appliances in a sample of patients undergoing fixed orthodontic treatment.The study included twenty-five healthy individuals requiring orthodontic therapy in both jaws. The patients were treated by stainless steel orthodontic brackets and nickel-titanium or stainless steel arch wires. The oral mucosa cells were gathered just before the appliance placement and 9 months later. The cells were centrifuged, fixed and dropped onto slides. After staining, the micronucleus (MN) assay was used to determine genome alteration. The data were analyzed by paired sample t-test.The mean micronuclei frequency in the buccal mucosa was 10.6 ± 5.7 per 1000 cells before the appliance placement and 9.2 ± 6.37 per 1000 cells 9 months later. No significant difference was found in the MN count before and 9 months after therapy (p=0.336).Under the conditions used in this study, application of fixed orthodontic appliances did not expose healthy individuals to increased risk of DNA damage in oral mucosa cells

ErbB1 and ErbB3 co-over expression as a prognostic factor in gastric cancer

Abstract 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

Loss of heterozygosity and microsatellite instability as predictive markers among Iranian esophageal cancer patients

Objective(s): 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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p16 promoter hypermethylation: A useful serum marker for early detection of gastric cance