The potential of PIK3CA, KRAS, BRAF, and APC hotspot mutations as a non-invasive detection method for colorectal cancer.

BACKGROUND: Early diagnosis of colorectal cancer (CRC) can lead to prompt treatment modalities. Circulating cell-free DNA (cfDNA) analysis provides an alternative non-invasive procedure for the study of the molecular profiles of the corresponding tumor tissue. In this study, we aimed to investigate PIK3CA, KRAS, BRAF, and APC hotspot mutations in CRC tumor tissue, besides evaluating the diagnostic performance of KRAS, BRAF, and PIK3CA mutations in the plasma cfDNA. METHOD: Primary CRC tissue samples and paired plasma samples were collected from 70 patients. After DNA extraction, PCR-direct sequencing was used to screen for mutations in PIK3CA exon 9 and APC exon 15 in tumor tissues. Amplification Refractory Mutation System (ARMS)-quantitative PCR (qPCR) was used to evaluate KRAS codon 12 and 13, BRAF V600E, and PIK3CA exon 9 hotspot mutations. RESULTS: PIK3CA exon 9 hotspot mutations were detected in 47.1% of tumor tissues and 20% of paired plasma cfDNA samples by ARMS-qPCR method, while Sanger sequencing did not identify any mutation in PIK3CA exon 9. The KRAS exon 2 mutations were detected in 71.4% and 34.3% of tumor tissue samples and paired plasma cfDNA respectively. BRAF V600E mutation was observed in 17.1% and 4.3% of tissue DNA and plasma cfDNA respectively. A panel of PIK3CA, KRAS, and BRAF showed a sensitivity of 61% and a specificity of 100% (AUC = 0.803). APC hotspot mutations were observed in 76.8% of CRC tissue samples. APC mutations were not analyzed in the plasma samples. The co-existence of KRAS/PIK3CA/APC gene mutations encompassed the highest frequency among all combinations of mutations. BRAF and PIK3CA mutations were significantly more frequent in older patients. CONCLUSION: We demonstrated that a panel consisting of PIK3CA, KRAS, and BRAF mutations showed good diagnostic performance for detecting CRC in the plasma cfDNA.

Methylation of FBN1, SPG20, ITF2, RUNX3, SNCA, MLH1, and SEPT9 genes in circulating cell-free DNA as biomarkers of colorectal cancer.

BACKGROUND: Investigating aberrant tumor-specific methylation in plasma cell-free DNA provides a promising and noninvasive biomarker for cancer detection. OBJECTIVE: We aimed to investigate methylation status of some promoter regions in the plasma and tumor tissues to find biomarkers for early detection of colorectal cancer. METHODS: This case-control study on seventy colorectal cancer patients and fifty matched healthy controls used Methylation-Specific High-Resolution Melting Curve analysis to evaluate the methylation of the selected promoter regions in converted genomic tissue DNA and plasma cfDNA. RESULTS: The methylation levels in selected regions of SPG20 (+24375 to +24680, +24209 to +24399, and +23625 to +23883), SNCA (+807 to +1013, +7 to +162, and -180 to +7), FBN1 (+223 to +429, +1 to +245, and -18 to -175), ITF2 (+296 to +436 and -180 to +55), SEPT9 (-914412 to -91590 and -99083 to -92264), and MLH1 (-13 to +22) were significantly higher in tumor tissues compared with normal adjacent tissues. The methylation levels of FBN1, ITF2, SNCA, and SPG20 promoters were significantly higher in the patient's plasma compared to patient's normal tissue and plasma of healthy control subjects. FBN1, SPG20, and SEPT9 promoter methylation had a good diagnostic performance for discriminating CRC tissues from normal adjacent tissues (AUC > 0.8). A panel of SPG20, FBN1, and SEPT9 methylation had a higher diagnostic value than that of any single biomarker and other panels in tissue-based assay (AUC > 0.9). The methylation of FBN1(a) and SPG20(a) regions, as the closest region to the first coding sequence (CDS), had a good diagnostic performance in plasma cfDNA (AUC > 0.8) while a panel consisted of FBN1(a) and SPG20(a) regions showed excellent diagnostic performance for CRC detection in plasma cfDNA (AUC > 0.9). CONCLUSION: Methylation of FBN1(a) and SPG20(a) promoter regions in the plasma cfDNA can be an excellent simple, non-invasive blood-based test for early detection of CRC.