A Comparison of the PIK3CA Gene Mutation Detection by COLD-HRM PCR and High-Resolution Melting Methods in Different Dilutions of DNA Mutations

Breast cancer is the most common female cancer and the second most common cause of cancer death in women. In 2016, it s estimated that just under 30% of newly diagnosed cancers in women will be breast cancers. About 85% of breast cancers occur in women who have no family history of breast cancer. These occur due to genetic mutations that happen as a result of the aging process and life in general, rather than inherited mutations. PIK3CA mutations with the highest frequency, appear to have a clinical significance in breast cancer, according to COSMIC Database. A third of breast cancers are caused by somatic mutations in the PIK3CA gene, the gene encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K). Somatic mutations in the PIK3CA gene are associated with the disruption of the normal regulation of cell growth, cell migration and maintenance of tissue morphology by PI3K. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. PIK3CA mutations were determined in 45% tumors in iran. Tumor heterogeneity is one of the major problems limiting the efficacy of targeted therapies and compromising treatment outcomes. Furthermore Up to 30% of nucleic acids may be lost during fixation. Kapp et al. reviewed the processing of FFPE samples and noted that the mutation detection failure was 11.9% with 80% of these attributable to pre-PCR error and other one of the major problems in solid tumors, purity varies widely with some tumor samples having low-level DNA mutations tumor. Direct sequencing is used as standard for detecting most mutations. However, sequencing is limited by high cost, time consuming and low sensitivity, especially it has high demandingness on sufficient amount of tumour tissue and high quality which is usually difficult to obtain from cancer patients. Therefore, a more cost effective, faster, easier to perform, and more sensitive method for mutations testing is required. Also traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer. Molecular profiling of somatic mutations in cancer often requires the identification of low-level DNA mutations within an excess of wild-type DNA. Advances in technology have resulted in the development of new techniques including novel sequencing technologies and enrichment techniques such as COLD PCR (CO-amplification at Lower Denaturation Temperature can also be used to improve detection limits. COLD-PCR) resolves several limitations in low-level mutation detection by using critical denaturation temperatures to enrich mutant-containing amplicons during PCR. In addition we need a method for monitoring solution reactions during COLD-PCR testing . On the other hand High-resolution melting analysis (HRM) is a highly sensitive, robust, rapid, cost-effective mutation analysis technique and powerful tool for screening and detecting genomic mutations efficiently Materials and Methods: DNA from bt-20 cell line harboring the heterozygous H1047R mutation and MCF-7 cell line for wild type DNA was used. Serial dilutions of PIK3CA mutated DNA from the BT-20cell lines in wild-type DNA of MCF-7 cell line were prepared in order to asses method sensitivity. We tested 100%, 50%, 25%, 12.5%, 6.2%, 3.1%, 1.5%, 0.8%, 0.4%, 0.2% and 0.1% (mutated/wild type) dilutions. Dilutions were amplified via HRM-PCR and full COLD-HRM PCR for H1047R mutation in PIK3CA gene and the 2 approaches (COLD-HRM PCR and HRM )were compared.Results: Dilution experiments indicated an approximate 4 fold improvement in selectivity with COLD-HRM PCR. PCR-HRM exhibited mutation-detection approximately 3.1% whereas COLD-HRM PCR exhibited approximately 0.8% mutant-to-wild-type ratio.Conclusion: In this study for the first time in the world, H1047R mutations were investigated by using COLD-HRM PCR.In this study illustrates that the correct identification of less-represented mutations in background of wild-type can be significantly improved with COLD-PCR combined with HRM, without requiring expensive and time consuming procedures and while maintaining a closed-tube approach.

کلیدواژه ها:

Breast cancer ، PIK3CA gene ، mutation ، COLD-HRM PCR ، HRM