Detection of PIK3CA Mutations in Plasma DNA of Colorectal Cancer Patients by an Ultra-Sensitive PNA-Mediated PCR.

backgroundMutant Phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) has been shown to be associated with the occurrence, development and prognosis in colorectal cancer (CRC). However, its detection has been limited because of complicated procedures and the low sensitivity of the present approaches.

methodsWe established an ultra-sensitive peptide nucleic acid-mediated polymerase chain reaction (PNA-PCR) assay to detect PIK3CA gene mutation in exon 9 and exon 20 with cell-free DNA (cfDNA). Using this technology, we detected the mutation status of PIK3CA in 128 colorectal cancer patients. 6 CRC patients receiving targeted therapy were chosen at random to undergo continuous PIK3CA mutation detection.

resultsThe results showed that the sensitivity of PNA-PCR clamping method was 0.1% for the exon 9 and 0.2% for the exon 20 variant alleles. When the PIK3CA mutation status was determined by PNA-PCR plus sequencing, 38.3% (49/128) of CRC carried at least one mutation, either E545Kor H1047R. The clinic-pathological parameters of age (p = 0.358), gender (p = 0.622), disease stage (p = 0.353) and disease location (p = 0.307) were not associated with the PIK3CA mutation. In the continuous monitoring study, we found that the gene status was associated with the effect of treatment. Furthermore, when the PIK3CA variant was determined by only the PNA-PCR method, there was a good linear relationship between ΔCp values and the proportion of variant DNA. The accuracy of PNA-PCR was 93.75 and 92.27% respectively when the cut-off values of ΔCp at 9.0 and 8.0 were set for determining the E545K and H1047R mutations.

conclusionsA simple, noninvasive, ultra-sensitive PNA-PCR technology was developed and was especially suitable for the dynamic detection of PIK3CA variants using cfDNA.