Abstract P1-07-13: The mutation detection and a high throughput screening of driver mutations in PI3K/AKT pathway based on next generation sequencing

Background The deregulation of the PI3K/AKT signaling pathway is essential to malignant cellular processes of breast cancer, including proliferation, apoptosis, and drug response. Oncogenic activating somatic mutations in the PI3K/AKT pathway are pervasive. However, it remains difficult to discriminate between driver and passenger mutations. This study was conducted to identify the landscape of genetic mutations in the PI3K/AKT pathway using Amplicon Sequencing in a Chinese population. Notably, we developed a Gateway-based mutation barcoding (GaMB) library which enables a high-throughput mutation-phenotype screen for specific vulnerable mutations that contribute to the cancer development and drug resistance. Method We collected 149 breast cancer specimens in a Chinese population and performed Ion Torrent Amplicon Sequencing for the key genes in PI3K/AKT pathway: PIK3CA, PIK3R1, AKT1, AKT2, AKT3, PTEN, PDK1 , and the canonical tumor suppressor gene TP53 , at 1000× coverage. Next, we established a high-throughput GaMB library that contained all of the PIK3CA mutations, either newly identified in Chinese population or reported in TCGA and COSMIC database, and tagged each mutations with a specific barcode. We then applied this library to functional screening processes using proliferation and drug response selection (doxorubicin or BKM-120) assays through which we screened the functional mutations with specific characteristics. The genomic DNA of the pooled surviving cells from the library, as well as the original cells before the screenings, was extracted and used for PCR amplification of the barcode regions, and then detected using Illumina Miseq sequencing to analyze the functional mutations. We then validated the cellular 2D- & 3D- proliferation abilities and the status of PI3K/AKT pathway activation in presence of identified mutations, respectively. Result Mutations in the PIK3CA (44%), PIK3R1 (37%), AKT3 (15%) and PTEN (12%) genes were the most prevalent. Mutations in PIK3CA were present in 65 samples (43.6%) which is similar to that reported in TCGA database. PIK3R1 (37%) was found significantly mutated, with a novel recurrent mutation, N595S, being identified in 24 patients. Similarly, AKT1 (10.1%), AKT2 (10.1%), and AKT3 (14.8%) mutations were present at a higher frequency in our population than has been reported in the TCGA and COSMIC database. In the PIK3CA -GaMB library, our highest-ranking mutations included the previously validated deleterious mutations H1047R and E545K and several mutations of uncertain significance, including E39K, G1049R, N345I, N345K, M1043V, and H1047T. In the validation assays, we found a high phenotype-consistency of these identified mutations using these functional validation techniques. The breast cancer cells harboring identified mutations exhibit a relatively higher proliferation ability and tolerance to chemotherapy and pathway inhibitors. Conclusion This study identified the landscape of genetic mutations in the PI3K/AKT pathway using Amplicon Sequencing in a Chinese population. A novel developed GaMB screening platform may allow the rapid identification of significant mutations that dominate breast cancer development and drug responses during treatment. Citation Format: Chen L, Yang L, Yao L, Hu X, Shao Z. The mutation detection and a high throughput screening of driver mutations in PI3K/AKT pathway based on next generation sequencing [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-13.