Background: Less than 20% of PI3KCA mutated late stage breast cancer patients achieved an objective response in a Phase III clinical trial with alpelisib, a recently approved PIK3CA inhibitor. This suggests biological factors other than PIK3CA status, such as aberrant GPCR-linked signaling, may be important to measure when identifying patients eligible for PI3K inhibitors. A new assay using an impedance biosensor was developed to measure GPCR-initiated signaling activity and the involvement of the PI3K node in transducing this activity in live tumor cells. The CELx PI3K Signaling Function (CELx PI3K) Test measures ex vivo live tumor cell response in real-time to a specific S1P agonist and PI3K antagonists to diagnose breast tumors with PI3K-involved hyperactive signaling. This study set out to: 1) characterize the involvement of the PI3K node in hyperactive S1P-initiated signaling in patient breast cancer cells and cell lines; and 2) assess whether PI3K-involved hyperactive S1P signaling is limited to breast cancer cells with PI3KCA mutations. Methods: A panel of 18 fresh HER2-/PIK3CA WT tumor cells from breast cancer patients and five PIK3CA mutated breast tumor cell lines were obtained. Real-time live cell response to an S1P agonist, an alpha-specific PI3K antagonist (alpelisib), a gamma-specific PI3K antagonist (IPI-549), and a pan-PI3K inhibitor (taselisib) were measured using an xCELLigence RTCA impedance biosensor. From these responses, SIP-initiated signaling was quantified and a previously determined cutpoint was used to identify abnormal levels of signaling activity. The relative and net amount of PI3K isoform participation in S1P-initiated signaling was also quantified. Results: Five of the 18 PIK3CA WT primary patient breast tumor cells and two of the five PIK3CA mutated breast tumor cell lines (BT20 and HCC1954) had abnormal levels of S1P-initiated signaling activity. The mean reduction of abnormal SIP signaling in the five primary breast tumor cells with IPI-549 was two times greater than with alpelisib (60% vs. 30%). In the two PIK3CA mutated cell lines with abnormal S1P-initiated signaling, the mean S1P signaling reduction with IPI-549 and alpelisib was not significant (0% and 28%), but the mean S1P signaling reduction with taselisib was 82%. Conclusions: The CELx PI3K test found hyperactive S1P-initiated signaling involves the PI3K node in PI3KCA mutated and WT breast tumor cells. The abnormal levels of PI3K-involved signaling in PIK3CA WT patient breast cancer tumors were comparable in magnitude to levels found in well-characterized PI3KCA mutated breast tumor cell lines. Unlike the PIK3CA mutated cells lines, the bulk of the PIK3-involved signaling in the PIK3CA WT patient tumor cells was associated with the PI3K-gamma isoform rather than the PI3K-alpha isoform. The superior reduction of S1P signaling by taselisib vs. alpelisib in the HCC1954 cell line (74% vs. 21%) as determined with the CELx PI3K test is consistent with previously reported xenograft studies that found taselisib induced reduction of HCC1954 tumors whereas alpelisib had no significant effect. This study thus suggests that measurement of PI3K-involvement in hyperactive S1P signaling in live patient breast cancer cells may provide a means to identify breast cancer patients who may benefit from treatment with PI3K inhibitors.

Citation Format: Lance Laing, Salmaan Khan, Ian MacNeil, Catherine Kuzmicki, Samantha Kharbush, Benjamin Rich, Abhay Shukla Shukla, Kelly Brass Brass, Brian Sullivan Sullivan. Sub-group of PIK3CA WT breast cancer patients have hyperactive S1P signaling tumor cells responsive to PIK3-inhibitors: Functional signaling profile test identifies new patient group who may benefit from PIK3-targeted therapies [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-09-07.