Abstract Background: Epidermal Growth Factor Receptor (EGFR) family signaling is commonly dysregulated in cancer by amplification or activating mutations. Although studies have investigated dual EGFR/PI3K inhibition in breast cancer, they have not determined biomarkers that predict success. Here, we present a subset of patients with EGFR amplification and PI3Kinase pathway mutations in breast cancer that can be synergistically targeted by dual EGFR/PI3K inhibition. Methods: Frequency of EGFR amplification and PI3K pathway mutations (PIK3CA, PIK3R1/2, PTEN, AKT1/2/3, TSC1/2/3) were studied through datasets from cBioPortal and Caris Life Sciences. Triple negative breast cancer (TNBC) cell lines used include BT20 (amplified EGFR, PIK3CA activating mutation), MDA-MB-468 (amplified EGFR, PTEN deletion), and MDA-MB-231 (no EGFR or PI3K alterations). EGFR amplification was determined by immunoblot and fluorescent in situ hybridization, and PI3K mutations by sequencing. Signaling was determined by immunoblot, drug synergy by cell viability, cell death by propidium iodide staining, cell cycle analysis by flow cytometry, and xenograft animal study. Caris Life Science CODEai was used to evaluate real-world overall survival (OS) obtained from insurance claims and calculated from date of tumor biopsy to last contact using Kaplan-Meier estimates. Statistical significances were determined by chi-square and Mann-Whitney U test, student’s t-test with 2 tailed comparisons assuming equal variance, one-way or two-way ANOVA were performed as indicated. P-values of ≤ 0.05 were considered significant. Results: EGFR amplification occurs in approximately 1-2.5% of breast cancer patients, more frequently in TNBC (2.45-6.7%) and ER-/HER2+ (1.3-6.5%) breast cancers, and in the molecular basal (2.33-8.1%) and HER2 enriched (1.87-5.4%) subtypes. EGFR amplified patients in cBioPortal and in the Caris datasets had worse median OS compared to those with non-amplified EGFR (mOS: 111.1 m vs 164.6 m, p=0.0269, and 21.6 m vs 32.9 m, HR 0.69, 95% CI 0.58 - 0.82, p0.0001 for the cBioPortal and Caris data, respectively). cBioPortal and Caris databases showed that 54-71% of EGFR amplified tumors have activating mutations in the PI3K pathway. Our in vitro studies showed combination EGFR and PI3K inhibitors more dramatically reduced MAPK, mTOR and AKT signaling in the BT20 and MDA-MB-468 cells, whereas the inhibition of downstream signaling was less significant in MDA-MB-231 cells. Combination of EGFR and PI3K inhibitors reduced cell viability in these three cell lines, but inhibition was greater, statistically significant, and synergistic in MDA-MB-468 and BT20 compared to MDA-MB-231. Only MDA-MB-468 and BT20 cells had an increased fraction of apoptotic cells with combined pathway inhibition. EGFR or PI3K inhibition alone in a BT20 xenograft model reduced tumor volume, however the combination induced the only statistically significant reduction in tumor volume when compared to vehicle control. Conclusions: EGFR/PI3K inhibitor combination causes apoptosis and reduction in tumor growth in cells with EGFR amplification and PI3K alteration. EGFR amplification with coincident PI3K pathway mutations are drivers in a subset of breast cancers and identify a subgroup of breast cancers that are more likely to respond to dual targeted therapy. Citation Format: D. J. Wiisniewski, D. J. Voeller, Y. A. Addissie, S. K. Deshmukh, S. K. Wu, M. B. Lustberg, D. Wangsa, D. Wangsa, K. Heselmeyer-Haddad, G. Sledge, S. Lipkowitz. EGFR amplification and PI3K pathway mutations identify a subset of breast cancers that synergistically respond to EGFR and PI3K inhibition abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-05-05.
Wiisniewski et al. (Tue,) studied this question.