A PIK3CA Pyrosequencing-Based Assay that Excludes Pseudogene Interference

Abstract

Phosphatidylinositol 3'-kinase gene (PIK3CA) encodes a lipid kinase that regulates signaling pathways downstream of epidermal growth factor receptor and is mutated in 10% to 30% of colorectal cancers. Activating mutations in this gene up-regulates the AKT signaling pathway, making it a potentially useful therapeutic target. Mutations in PIK3CA are not exclusive of mutations in KRAS, BRAF, or NRAS. We designed a pyrosequencing assay to detect mutations in all three positions of codons 542 and 545 in exon 9 and codon 1047 in exon 20 of this gene. The exon 9 reverse PCR primer was designed to avoid amplifying a pseudogene in chromosome 22 that has >95% homology with exons 9 through 13 in PIK3CA. Two hundred colorectal cancers from FFPE tissue previously characterized for KRAS mutation status were evaluated for PIK3CA mutations. In KRAS-mutated samples, 20% had an additional mutation in PIK3CA. The mutation rate in KRAS wild-type samples was 7.5%. When using our assay, pseudogene was not observed in any of these samples. In addition, pseudogene- and gene-specific amplification was performed on DNA from 40 additional colorectal cancers. Sequencing of these PCR products yielded the expected gene or pseudogene sequence in all cases. Thus, we have developed a PIK3CA pyrosequencing assay capable of detecting mutations in all three positions in the three hot spot codons with no pseudogene interference.