Abstract Background: Aromatase inhibitor (AI) based hormone therapy combined with CDK4/6 inhibitors in hormonereceptor-positive (HR+), Her2-negative metastatic breast cancer (MBC) is considered the standard ofcare. Ligand-binding domain mutations in ESR1 gene is a well-established mechanism of resistancereported in these patients. The ESR1 gene mutation frequencies have been reported to be between15–50% in AI-treated cases. Limited data is available from South Asian population and to date, no studyhas reported the frequencies of ESR1 gene mutations in Pakistani cohort. Methods: We enrolled (n = 50) Pakistani women with endocrine-resistant HR+, Her2 – MBC to conduct aprospective study at Aga Khan University Hospital (AKUH), Karachi, Pakistan. Genomic DNA wasextracted from formalin-fixed paraffin-embedded (FFPE) biopsy blocks of these patients and allele-specific multiplex PCR assays were conducted to analyze the hotspot mutations (Y537S Y537C, Y537N,D538G, and E380Q) in ESR-1 gene. Clinicopathological data including age at diagnosis, metastaticpresentation (de novo vs recurrent), ER/PR via IHC, Her2 status, patient comorbidities, and priorchemotherapy were recorded. Data analysis and associations were evaluated using t, Fisher’s exact,Chi-square, Kolmogorov-Smirnov and Mann Whitney tests via SPSS v25 and GraphPad Prism 4software. A p-value of less than 5% was considered as significant. Results: Our data showed that median age at diagnosis was 48 years (23-75). Strong ER positivity was found tobe in 68%, intermediate 24% and only 8% patients had low ER positivity. PR-positivity was found in84% patients. Overall, 96% patients harbored the Y537S mutation, a frequency that is far exceedingthe global reports. This confers strong ligand-independent activation and profound endocrineresistance. Y537S prevalence was similar in recurrent vs de novo cases (26/28 vs 22/22; p=0.50). Thetwo Y537S-negative patients were significantly younger (mean age 30 vs 49 years; p0.01). All patientswith comorbidities (n=30) carried Y537S (vs 18/20 without comorbidity; p=0.16). Her2-low status (IHC1+/2+) was found in 52% and was associated with more recurrent disease (62% vs 33%), thoughsurvival was similar. Prior chemotherapy (in 38/50 patients) correlated with recurrent disease (58% vs17%; p0.01). Patients without prior history of receiving chemotherapy had a higher median numberof concurrent ESR1 gene mutations (3 vs 2), though not statistically significant. Conclusions: We report an exceptionally high ESR1 gene Y537S single nucleotide polymorphism in endocrine-resistant HR+, Her2– MBC Pakistani cohort. Thus, highlighting the importance of unique tumor biologyand clonal evolution. These mutations were enriched in patients with strong ER/PR expression andyounger age group. Our findings support routine ESR1 gene testing in our population, implying tailoredapproach and the need for region-specific therapeutic strategies. Citation Format: Y. A. Rashid, S. Khan, M. Anis, R. Kumari, R. Idress, S. I. Azam, I. Masroor, A. K. Sattar, A. Gauhar, M. Rafiq, A. A. Jabbar. Exceptionally High Frequency of ESR1 Mutations in Pakistani Women with Endocrine Resistant Hormone Receptor positive Her2 neu negative Metastatic Breast Cancer 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 PS1-12-22.
Rashid et al. (Tue,) studied this question.