PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method

Abstract

Somatic mutations in the PIK3CA gene have been discovered in many human cancers, and their presence correlates to therapy response. Three "hotspot" mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity, and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous, and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies, the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group), 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas, we report one PIK3CA mutation for the first time. In the selected group, 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive , reliable, cost-effective, and easy-to-perform, and therefore can be used as a screening test in a high-throughput pharmacodiagnostic setting. Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology.