Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

Area of Science:

• Oncology
• Genetics
• Molecular Diagnostics

Background:

• Somatic mutations in KRAS, NRAS, and BRAF genes are critical biomarkers for predicting resistance to anti-EGFR antibody therapy in colorectal cancer (CRC).
• Accurate and sensitive detection of these mutations is essential for guiding treatment decisions and improving patient outcomes.

Purpose of the Study:

• To establish and validate an extended RAS and BRAF mutation assay utilizing next-generation sequencing (NGS) for comprehensive analysis of these key genes in CRC.
• To assess the clinical utility of the developed NGS assay in formalin-fixed, paraffin-embedded (FFPE) tumor tissues and plasma samples.

Main Methods:

• Multiplexed deep sequencing was employed to detect somatic mutations in KRAS, NRAS, and BRAF, including low-frequency variants.
• The assay's technical performance was validated using normal DNA and FFPE tumor samples.
• A variant calling approach based on Poisson distribution was implemented to define mutation-positive status with a stringent significance level (α = 2 x 10⁻⁵).

Main Results:

• The NGS assay successfully profiled 100 FFPE CRC tumor samples and 15 plasma samples.
• Mutations in KRAS, NRAS, or BRAF were detected in 59% of the FFPE tumor specimens.
• High concordance (92%) was observed between the NGS assay and Scorpion-ARMS for KRAS mutation detection.
• KRAS and BRAF mutations were identified in both plasma and tissue from 6 patients, demonstrating the assay's applicability to liquid biopsies.

Conclusions:

• The established extended RAS and BRAF mutation assay using NGS is a validated and robust method for detecting clinically relevant mutations in CRC.
• The assay demonstrates significant potential for clinical utility, enabling comprehensive genetic screening in both FFPE tissues and liquid samples for personalized cancer therapy.