Development of a multiplex allele-specific qPCR approach for testing PIK3CA mutations in patients with colorectal...

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• Phosphatidylinositol 3-kinases (PI3Ks) are crucial lipid kinases regulating cell growth and division.
• Somatic mutations in the PIK3CA gene are prevalent in various cancers, including colorectal cancer (CRC).
• Current PIK3CA mutation screening methods often overlook less common mutations in exons 1-7, focusing primarily on exons 9 and 20.

Purpose of the Study:

• To develop and validate a multiplex allele-specific PCR (AS-PCR) assay for comprehensive PIK3CA mutation screening.
• To investigate the frequency and distribution of PIK3CA mutations in colorectal cancer (CRC) patients from Siberia and the Far East of Russia.
• To assess the assay's suitability for analyzing PIK3CA mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples.

Main Methods:

• Development of a multiplex allele-specific PCR (AS-PCR) assay targeting PIK3CA exons 1, 4, 7, 9, and 20.
• Validation of the assay using 515 colorectal cancer (CRC) patient samples from Siberia and the Far East of Russia.
• Determination of assay sensitivity at 0.05-0.5% mutant DNA.

Main Results:

• The multiplex AS-PCR assay demonstrated high sensitivity for detecting PIK3CA mutations.
• An overall PIK3CA mutation frequency of 13.01% was identified in the studied CRC cohort.
• Specific mutation frequencies included 9.32% in exon 9, 1.94% in exon 20, and 1.74% in exons 1-7.
• This study represents the first characterization of PIK3CA mutation frequency in CRC patients from eastern Russia.

Conclusions:

• The developed multiplex AS-PCR assay is effective for screening activating PIK3CA mutations, including those in less commonly analyzed exons.
• The assay is suitable for routine analysis of PIK3CA mutations in FFPE CRC tissues.
• This research provides valuable insights into the genetic landscape of PIK3CA in Russian CRC patients.