Mutation profiling of KRAS and BRAF in primary tumours and circulating tumour cells of colorectal cancer patients using PNA-LNA molecular switch

Abstract

Identifying KRAS and BRAF mutation status is essential for guiding targeted therapies and enhancing treatment outcomes in colorectal cancer (CRC). This study employs the “PNA-LNA molecular switch” to detect mutations in KRAS codon 12 (c.35G>T/G12V) and BRAF codon 600 (c.1799T>A/V600E) from primary tumours and circulating tumour cells (CTCs) in CRC patients, correlating mutation status with clinicopathological parameters. DNA was isolated from 71 primary tumours and 37 CTC samples. Mutation profiles were generated using the PNA-LNA molecular switch. KRAS mutations were detected in 26 primary tumours (36.6 %) and 13 CTCs (26.8 %), while BRAF mutations were observed in 19 primary tumours (26.8 %) and 7 CTCs (19 %). No significant correlation was observed between mutation status and clinicopathological parameters in primary tumours. However, KRAS G12V mutations in CTCs significantly correlated with lymph node metastasis (p = 0.002), overall pathological stage (p = 0.005), and lymphovascular invasion (p = 0.034). BRAF V600E mutation status showed no significant clinicopathological associations. Validation of the PNA-LNA molecular switch against Next-Generation Sequencing (NGS) showed 89 % concordance with p-values <0.001 for both genes. This method is highly comparable to NGS for detecting KRAS and BRAF mutations and shows promise as a point-of-care diagnostic tool. Larger patient cohorts are required to confirm its clinical utility.