DEVELOPMENT OF A MULTIPLEX ALLELE-SPECIFIC REAL-TIME PCR METHOD FOR DETECTION OF PIK3CA GENE SOMATIC MUTATIONS AND ITS VALIDATION IN THE TUMORS OF BREAST CANCER PATIENTS

Abstract

Aim: To develop a highly sensitive real-time polymerase chain reaction (PCR) system for detection of somatic mutations in 542 and 545 codons of exon 9 and 1047 codon of exon 20 of PIK3CA gene comprising more than 80% of all somatic mutations in this gene for application on histological material without macroand microdissection, and to analyze associations between these mutations and clinical and pathological characteristics of breast tumors.Materials and methods: The Allele-specific real-time PCR method with signal detection by TaqMan probes was used. For determination of its analytical sensitivity, plasmids carrying the mutations studied were constructed by standard genetic engineering methods using mutagenesis. DNA samples carrying various mutated/wild type DNA ratios (5.0; 2.0; 1.0; 0.5, 0.25%) were prepared. Results: Multiplex allele-specific real-time PCR method for detection of most common mutations in PIK3CA gene: p.E542K c.1624G>A, p.E545K c.1633G>A, p.H1047R c.3140A>G, p.H1047L c.3140A>T – was developed and optimized.Analytical sensitivity of mutation detection comprised 0.5% for p.E542K and 0.25% for p.E545K, p.H1047R and H1047L enzyme.Conclusion: The method developed for detection of somatic mutations in PIK3CA gene is sufficiently sensitive, specific and efficient, that allows to propose it for a routine screening in clinical diagnostic laboratories for evaluation of disease prognosis and monitoring of response to therapy and its modification.

Keywords

• allele-specific real-time pcr (as-rtpcr)
• pik3ca gene
• mutations
• breast cancer