Background

Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway is essential to malignant cellular processes of breast cancer, including proliferation and drug response. Oncogenic somatic mutations of the PI3K pathway are pervasive in breast cancer. However, identification of impactful mutations and determination of their relevance to major components of this pathway remains difficult. This study was conducted to identify the landscape of somatic mutations in the PI3K pathway in a Chinese population. Notably, we developed a recombination-based mutation barcoding (ReMB) library which enables a high-throughput mutation-phenotype screens for vulnerable mutations that contribute to the cancer development and drug resistance.

Methods

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 at 1000× coverage. To discriminate between 'driver' and 'passenger' mutations, we developed a recombination-based mutation barcoding (ReMB) library that contained the novel identified mutations and that reported in TCGA and COSMIC databases in PIK3CA and PIK3R1 genes, each mutation tagging with a specific barcode to identify their potential oncogenic and drug-resistant characteristics. The unique barcode representing each mutation was detected using Illumina Miseq sequencing following proliferation and drug response selection (doxorubicin and BKM-120) assays to screen the functional mutations.

Results

We identified that mutations in PIK3CA (44%), PIK3R1 (17%), AKT3 (15%) and PTEN (12%) were prevalent and diverse in Chinese patients with a high proportion of tumours harboring multiple mutations, especially PIK3CA plus PIK3R1 mutations (9.0%). With ReMB screening, we found 11 non-synonymous impactful mutations in PIK3CA; these included eight proliferation-driving mutations, nine doxorubicin-resistant mutations, and eight BKM120-resistant mutations. The highest-ranking PIK3CA mutations include the deleterious mutations E542K, E545K and H1047R/L as well as mutations of unknown significance, including E39K, N345I, E453K, and G1049R. We also identified six non-synonymous impactful mutations inPIK3R1, including five proliferation-driving mutations, six doxorubicin-resistant mutations, and five BKM120-resistant mutations. The PIK3R1 impactful mutations include E160D, Q329L, N564D and K674R. Most impactful mutations in PIK3CA and PIK3R1 occurred at residues lying at the interfaces between p110α and p85α, or between the functional domains within p110α. These PIK3CA and PIK3R1 impactful mutations exhibit a mutually exclusive pattern, leading to oncogenesis and hyperactivity of PI3K pathway. Additionally, impactful mutations in PIK3CA are tightly associated with hormone receptor positivity.

Conclusion

This study identified the landscape of somatic mutations in the PI3K pathway in Chinese breast cancer patients. A novel developed ReMB screening platform allows the rapid identification of impactful PIK3CA and PIK3R1 mutations in breast cancer and has important implications for PI3K-targeted therapy.

Citation Format: Chen L, Yang L, Hu X, Shao Z. High-throughput barcode screening elucidates the functional characteristics and mutual relevance of PIK3CA and PIK3R1 somatic mutations in Chinese patients with breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-04-07.