Detection of Microsatellite Instability by High-Resolution Melting Analysis in Colorectal Cancer

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. microsatellite instability (MSI) is a molecular marker of a deficient mismatch repair system and happens in almost 15% of CRCs. Because of a wide frequency of MSI+ CRC in Iran compared to other parts of the world, the importance of screening for this type of cancer is highlighted. Methods: : The most common MSI detection technique is a fluorescent PCR-based method in which fragments are analyzed by capillary electrophoresis (CE). This technique is very time-consuming, difficult, and expensive. We sought to develop and evaluate a proper method with high accuracy, specificity, and sensitivity to screen the MSI+ CRC. A high-resolution melting (HRM) analysis procedure is relying on the analysis of the melting curve attributes. Low cost, feasibility, high specificity, and sensitivity are outstanding attributes of HRM analysis. Results: Five mononucleotide microsatellite markers, including BAT-25, BAT-26, NR-21, NR-24, and NR-27, in 25 archival CRC tumor tissue samples were compared with normal tissue adjacent using HRM method. The specificity and sensitivity of BAT-25 with HRM method were 100% compared to CE, while other markers had lower sensitivity. However, when all the markers were considered together, the sensitivity and specificity became 100%. The number of MSI+ samples was 56%, which shows a higher ratio than previous Iranian studies. The highest MSI was related to BAT-26 (52%). Conclusion: The HRM method is much simpler and more cost-effective than current MSI techniques, and its sensitivity and accuracy are comparable. Therefore, it can serve as an alternative method in cases where CE is unavailable.

Characterization of pathways involved in colorectal cancer using real-time RT-PCR gene expression data.

AIM: Efforts to explore biomarkers and biological pathways involved in the disease are needed to improve colorectal cancer (CRC) diagnosis and alternative treatments. BACKGROUND: The fourth common malignancy in the world is colorectal cancer. The over-all burden is predicted to rise by 2030. METHODS: In the current study, nine genes were selected. Previously, a panel of genes by Agendia, a classifier of robust gene expression (ColoPrint), was determined to significantly improve the prognostic accuracy of pathologic factors in stage II and III colorectal cancer patients. Five genes, including Ppara, Mctp1, Pyroxd1, Il2r, and Cyfip2, from this panel and four other genes which were not in this panel but were cited abundantly in the literature were selected. Then, expression levels of the selected genes in CRC tissue were compared with levels in adjacent normal tissue. To identify the pathways involved in CRC, gene set enrichment analysis was subsequently performed. Furthermore, to illustrate the relationship between genes in this disease, the cross-shaped co-expression pattern and gene regulatory network were determined using computational methods. RESULTS: This research found that the pairs of genes: {IL2R, CYFIP2}, {FOXM1, PPARA}, {MCTP1, CTSC}, and {PYROXD1, CYF1P2} are functionally related. Furthermore, two differentially expressed gene pairs ({FOXM1, PPARA} and {IL2R, CYFIP2}) are involved in the vascular endothelial growth factor receptor signaling pathway and the purine ribonucleoside diphosphate metabolic process, respectively. CONCLUSION: This research found that the combination of computational analysis and laboratory data provided the opportunity to better characterize the relation between central colorectal cancer genes as well as possible pathways involved in the colorectal cancer.