Abstract Introduction: Alpelisib (ALP), an inhibitor and degrader of phosphatidylinositol-3-kinase α (PI3Kα), + fulvestrant (FUL) demonstrated efficacy in PIK3CA-mutated, HR+, HER2- ABC in the phase 3 SOLAR-1 trial, which included only 20 patients (pts) with prior CDK4/6 inhibitor (CDKi) in the PIK3CA-mutated cohort. BYLieve (NCT03056755), a phase 2, open-label, 3-cohort noncomparative study, evaluates ALP + endocrine therapy (ET; FUL or letrozole [LET]) in pts with PIK3CA-mutated, HR+, HER2- ABC, progressing on/after prior therapies, including CDKi + ET. Cohorts A and B were restricted to pts receiving CDKi + (aromatase inhibitor [AI] or FUL), respectively, as immediate prior therapy, but Cohort C included pts whose cancer progressed on/after AI (in adjuvant or metastatic setting), and who received chemotherapy (CT; any line), or ET (FUL or LET monotherapy or with targeted therapy, including CDKi + FUL, but not CDKi + AI) as immediate prior treatment. Cohorts A and B demonstrated efficacy and safety of ALP + ET after prior CDKi. Here, we report primary results and biomarker analyses from Cohort C.. Methods: Pts in Cohort C received ALP 300 mg orally QD + FUL 500 mg intramuscular Q28D + C1D15. The primary endpoint was assessed in each cohort separately and is the proportion of pts with centrally confirmed PIK3CA mutation alive and without disease progression at 6 mo per local assessment; 95% CIs are calculated using Clopper and Pearson (1934) exact method. The 95% CI lower bound of the primary endpoint >30% is clinically meaningful evidence of treatment effect. In an exploratory analysis of baseline biomarkers using ctDNA, progression-free survival (PFS) was estimated in pt subgroups per high (≥10%) or low (<10%) ctDNA fraction and ESR1 mutational status via Kaplan-Meier estimation.. Results: 127 pts were enrolled in Cohort C (1 pt discontinued prior to treatment start) with ≥6 mo follow-up by the cutoff date (14 Jun 2021); 115 had a centrally confirmed PIK3CA mutation. Median follow-up was 11.4 mo (range, 0-23 mo); 79 (62.7%) pts had ≥2 prior lines of therapy in the metastatic setting, 58 (46.0%) pts had prior CT exposure in the metastatic setting, and 41 (32.5%) pts had prior FUL exposure in the metastatic setting. The primary endpoint was met, with 48.7% (95% CI, 39.3%-58.2%) of pts alive and without disease progression at 6 mo. Median PFS (mPFS) was 5.6 mo (95% CI, 5.4-8.1 mo). The most common all-grade adverse events (AEs) by preferred term were hyperglycemia (n=82, 65.1%), diarrhea (n=66, 52.4%), nausea (n=51, 40.5%), and rash (n=49, 38.9%). Grade ≥3 AEs (≥10%) included hyperglycemia (n=30, 23.8%) and rash (n=17, 13.5%). AEs leading to discontinuation occurred in 15.1% (n=19) of pts; most frequent AEs leading to discontinuation were rash (n=5, 4.0%) and hyperglycemia (n=4, 3.2%). Exploratory biomarker analyses in 74 pts who had available baseline biomarker samples at data cutoff showed that pts with a low ctDNA fraction (n=23) had longer mPFS than pts with high ctDNA fraction (n=51; 16.7 [95% CI, 10.4-19.5] vs 5.4 [95% CI, 2.9-7.2] mo; p<0.001, HR=0.31 [95% CI, 0.2-0.6]). Efficacy was similar in pts with (n=20) and without (n=54) ESR1-mutated tumors (6.3 [95% CI, 2.8-8.3] vs 8.3 [95% CI, 5.5-16.7] mo; p=0.095, HR=0.59 [95% CI, 0.3-1.1]).. Conclusion: In Cohort A of BYLieve, efficacy of ALP + FUL suggests clinical benefit immediately after CDKi + AI; Cohort C confirms clinically relevant activity of ALP + FUL in a heterogeneous population, primarily treated in the third line or later, and potentially regardless of ESR1 status or ctDNA fraction, with no new safety signals detected. Together, these data confirm that ALP targets the effects of the PIK3CA-driver oncogene in HR+, HER2- ABC. Citation Format: Hope S Rugo, Patrick Neven, Isabel Saffie, Yeon Hee Park, Michelino De Laurentiis, Florence Lerebours, Eva Maria Ciruelos, Nicholas Turner, Dejan Juric, Ennan Gu, Christina H Arce, Mukta Joshi, Estelle Roux, Murat Akdere, Stephen Chia. Alpelisib + fulvestrant in patients with PIK3CA-mutated, HR+, HER2— advanced breast cancer (ABC) who received chemotherapy or endocrine therapy (ET) as immediate prior treatment: BYLieve Cohort C primary results and exploratory biomarker analyses [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD13-05.