Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells...

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• KRAS mutations occur in 30-51% of colorectal cancer (CRC) cases.
• CRC patients with KRAS mutations often show non-responsiveness to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatments.
• Accurate KRAS mutation detection is crucial for guiding anti-EGFR MoAb therapy in CRC.

Purpose of the Study:

• To investigate the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR) assay for KRAS mutation detection.
• To assess the assay's sensitivity and applicability in rare cell detection.
• To evaluate the assay's performance on peripheral blood and tissue samples from CRC patients.

Main Methods:

• Developed a real-time PCR assay using WT KRAS locked nucleic acid blockers.
• Optimized the assay protocol using cell lines.
• Applied the optimized assay to peripheral blood and tissue samples from CRC patients, comparing results with conventional PCR sequencing.

Main Results:

• The optimized assay demonstrated superior sensitivity, detecting as few as two mutated KRAS cells in 10^4 WT cells (0.02%).
• Analysis of 45 colorectal tissue samples revealed a mutational discordance in 4 of 18 WT cases compared to conventional PCR sequencing.
• The method proved feasible for assessing KRAS status in patient samples.

Conclusions:

• The developed method is simple, effective, and robust for KRAS mutation detection at the single-cell level.
• It enhances detection sensitivity for KRAS mutations in paraffin-embedded tissues and circulating tumor cells.
• The assay reduces the need for removing contaminating lymphocytes during analysis.