Detection Of PIK3CA Mutations In Plasma DNA Of Colorectal Cancer Patients By An Ultra-sensitive PNA-mediated PCR

Background: Colorectal cancer(CRC) is one of the common gastrointestinal cancer in the world, which has become one of the primary tumor of a serious threat to the health of people in recent years. Mutant PIK3CA(phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) has been shown to associate with the occurrence, development and prognosis in CRC. Whereas, their detection is limited because of complicated procedures and the low sensitivity of the present approaches.Methods: We established a hypersensitive PCR assay to detect PIK3 CA gene mutation in exon 9 and exon 20 with cell free DNA(cfDNA). Using the technology to detect mutation status in 128 colorectal cancer patients. Furthermore, 6 CRC patients receiving targeted therapy were chosen randomly to detect PIK3 CA mutation continuously.Results: Our results showed that the sensitivity of PNA(peptide nucleic acid)-mediated PCR(polymerase chain reaction) clamping was 0.1%(exon 9) and o.2%(exon 20) mutant alleles, respectively. When PIK3 CA mutation determined by PNA-PCR followed sequencing, E545 K mutation was detected in 33, and H1047 R was detected in 29 of 128 cases. Thirteen of them were positive with both E545 K and H1047 R mutation, and 79 of them were both negative. Totally, 38.3%(49/128) of CRC carried at least one code mutation either E545 K or H1047 R. The clinicopathological 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 PIK3 CA mutation. In the continuous monitoring study, we found that the gene status was associated with the effect of treatment. However, when PIK3 CA mutation only determined by PNA-PCR method, there were good linear relationship between ?Cp values and the proportion of mutation DNA in the wild-type. The accuracy of PNA-PCR was 93.75% and 92.25% when set the cut-off values of ?Cp at 9.0 and 8.0 for determining E545 K and H1047 R mutation, respectively.Conclusion: A simple, noninvasive, ultra-sensitive real-time PCR technology was developed and especially suitable for detecting PIK3 CA mutation with cfDNA dynamically. Dynamic monitoring in patients with tumor gene mutation status is predictable tumor biomarkers of EGFR monoclonal antibody therapy effect.