PIK3CA and AKT1 mutations are independent in invasive breast cancer.

Abstract #1046 Background: The PI3K-AKT pathway is the most commonly altered pathway in invasive breast cancer. Somatic mutation in PIK3CA occurs in 26-30%, while AKT1 mutation was recently noted in 4-8%, other mutations occur rarely in PIK3R1 and PTEN. The prognostic implication of PIK3CA somatic mutation is inconclusive in that moderately sized retrospective studies report differing conclusions. To explain these disparate results our hypothesis is that different PIK3CA mutations impart differences in tumor biology. In an attempt to definitively identify the prognostic importance and functional attributes that specific mutations impart to breast tumor biology we are performing a broad mutation analysis on 600 archival invasive breast tumors with greater than 10 years of follow-up. Methods: Archival formalin-fixed paraffin embedded (FFPE) invasive breast tumors were identified from an institutional database from 1992-1996 that had known vital status and primary breast tumor size greater than 1 cm. From the confirmed invasive tumor blocks, two 10µm sections are cut for DNA extraction, punch blocks are obtained for tissue microarray (TMA) construction for immunohistochemistry and clinical demographics are collected. DNA is extracted and quantitated. PIK3CA hotspot mutations and rare PIK3CA mutations less commonly observed but thought to impart an oncogenic phenotype along with the recently identified AKT1 (E17K) mutation are assessed using the Sequenom genome multiplex array. PIK3CA hotspot mutations and AKT1(E17K) are confirmed by additional analysis on an alternate multiplex array. The first 190 cases were also assessed by Sanger sequencing for all PIK3CA coding exons. Results: The multiplex arrays used for the Sequenom mutation analyses had been previously validated. Thus far 400 samples have been procured and 190 cases have undergone mutation analysis. Both native DNA and DNA that had undergone whole genome amplification (WGA) were assessed to identify the most informative method for FFPE specimens. Notably, PIK3CA amplification and Sanger sequencing or WGA and mutation analysis by Sequenom multiplex array was less sensitive for identifying mutations than using unamplified native DNA with Sequenom analysis. Native DNA was informative in greater than 98% samples (3/190 uninformative) for the majority of PIK3CA mutations. All samples were informative for the most common hotspot mutation at PIK3CA (H1047R). At this report, the incidence of PIK3CA hotspot mutations (E542K, E545K, H1047R, H1047L) is 26.3% (50/190 cases), with rare PIK3CA mutations (C420R, N345K) occurring in 2.6% of cases. The more recently identified AKT1 (E17K) mutation is identified in 4.2% of cases and occurs independently of those tumors which harbor a PIK3CA mutation. Discussion: Mutation analysis, demographic collection and statistical analysis will be completed and updated at the meeting. The work thus far demonstrates that assessment of small amounts of archival tissue can be easily procured and undergo mutation assessment for key mutations that may be targeted therapeutically. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1046.