Abstract C38: Comparative evaluation of three real-time PCR assays for the detection of PIK3CA somatic mutations in formalin-fixed paraffin embedded breast carcinomas.

Abstract

Abstract Background: Mutations of the PIK3CA gene encoding for the p110α catalytic subunit of Phosphatidylinositol 3-kinases (PI3K) are found in approximately 25% of breast carcinomas. Most of PIK3CA mutations are reported as activators of the PI3K/AKT/mTOR pathway and lead to resistance to anti-HER2 therapies, and have an interest in molecular diagnosis for anti-mTOR and/or anti-PI3K therapies. Highly sensitive methods are required to detect PIK3CA mutations in paraffin-embedded tumor tissues. Methods: Forty-seven formalin-fixed paraffin embedded breast carcinomas tissues samples were assessed using two allele specific assays (Cobas® PIK3CA Mutation Test and PCR ARMS Scorpions®) and one non-specific real-time high resolution melting PCR assay (HRM) after macrodissection and DNA extraction. Cobas® allow the detection of 17 mutations on exons 1, 4, 7, 9 and 20 of PIK3CA, ARMS focuses on the detection on the four common hotspots (E542K, E545K/D, H1047R and H1047L) representing 90% of PIK3CA mutations and HRM allows the detection of all mutations located on the whole exons 9 and 20 of PIK3CA. Kappa statistics were used to compare the three assays. Results: Among the 47 samples, 18 (38.3%) were found to bear a PIK3CA mutation with Cobas®, 13 (27.7%) with ARMS and 19 (40.4%) with HRM. One sample was found to bear 3 mutations of PIK3CA (E542K, H1047X and H1049R). Calculated kappa were 0.893 (p<1.10-9) between Cobas® and HRM and 0.659 (p<1.10-9) between Cobas® and ARMS. Because the ARMS assay only focused on the 4 most common PIK3CA mutations, more than 10% of the samples were identified as false negative wild-type tumors although they had a mutation of PIK3CA as identified with Cobas® and HRM assays. Conclusion: Cobas® and HRM assays allowed to detect more extensively PIK3CA mutations than ARMS assay. ARMS assay only focusing on the 4 hotspots PIK3CA mutations seems to be inadequate with routine use since generating false negative PIK3CA wild-type results. Cobas® and HRM assays are significantly comparable for the detection of PIK3CA mutations, but Cobas® enables the characterization of the mutation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C38. Citation Format: Alexandre Harlé, Maëva Lion, Marie Rouyer, Nicola Lozano, Jean-Louis Merlin. Comparative evaluation of three real-time PCR assays for the detection of PIK3CA somatic mutations in formalin-fixed paraffin embedded breast carcinomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C38.