PI3KCA Mutations in Endometrial Cancer. A Comparative Study between Direct Sequencing and a Real-Time Based Technology
J Hernandez-Losa, R Somoza, C Teixido, E Lindo, A Solsona, J Castellvi, A Garcia, H Alazzouzi, S Ramon y Cajal. Vall d´ Hebron University Hospital, Barcelona, Spain; Fundación Institut Recerca Vall d´Hebron University Hospital, Barcelona, Spain
Background: Alterations in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway are common in endometrial carcinoma. Despite of PTEN status, the gene mutations or amplifications of the catalytic subunit of PI3K (PI3KCA) have been reported. The majority of the mutations map to three sites E542 and E545 in helical domain and H1047 in the kinase domain, and reflect a gain of enzymatic function of the PI3K conferring a proliferative advantage, which act as a dominant oncoprotein. The discordances in the percentage of PI3KCA mutation and the appearance of novel therapies against this pathway justify the incorporation of new methodologies to assess these mutations.
Design: We have compared two different methods for the detection of mutations in the helical and catalytic domains of PI3KCA gene. We used a commercial kit that include a combination of Scorpions and ARMS technologies (PI3K-Mutation Kit, DXS company) versus Bigdye terminator v3.1 Cycle Sequencing Kit in 71 human endometrial cancer samples. The PI3K Mutation Kit detects 4 somatic mutations including in exon 9 (E542K/D E545K) and exon 20 (H1047R). The DNAs were extracted from FFPE tissue samples. We compare the sensitivity of each technology with different dilutions from a positive control PI3KCA gene mutation cell line (HCT116).
Results: The PI3K Mutation Kit detected the H1047R mutation in less than 5% of DNA mutant whereas direct sequencing detected up to 25% of DNA mutant in a background of wild type genomic DNA in cell lines. This high sensitivity was confirmed in human endometrial cancer samples. The Real-time based technology detected a 14% (10/71) of mutants whereas direct sequencing detected only 11% (8/71) in the specific hot spots. On the other hand, the direct sequencing showed different mutations implicating other amino acids such as Q546 (4/71), and E545A (1/71) mutations no detectable by the PI3K Mutation Kit.
Conclusions: The higher sensitivity results obtained with the PI3K Mutation Kit support the use of this technology in different screenings to assess the status of PI3KCA mutations in human endometrial cancer samples. Nevertheless other amino acids substitutions such as Q546, must be evaluated and explore it incorporation in future kits
Monday, March 22, 2010 1:00 PM
Poster Session II # 255, Monday Afternoon