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Introduction: Based on the SOLAR-1 study, the PI3Kα-specific inhibitor alpelisib has been approved in combination with fulvestrant for postmenopausal women, and men, with HR+/HER2-, PIK3CA-mutated, advanced or metastatic breast cancer (BC). Despite PIK3CA mutations occurring in ⁓40% of HR+/HER2- BC, PIK3CA molecular testing is a new task to be carried out in clinical practice. Adopting the most appropriate testing strategy (RT-PCR vs. NGS) on the most appropriate biological sample is of capital significance in oncologic pathology. Here, we sought to assess the concordance rate for PIK3CA molecular analysis between different technical platforms in both metastatic and matched primary tumors. Methods: From our Institutional registry, n=16 HR+/HER2- metastatic BC, which were found to harbor PIK3CA mutations via next-generation sequencing (NGS) assays [custom panel and Oncomine Comprehensive Assay (OCA) v3 (Thermo Fisher Scientific, Waltham, MA, USA)], were selected. In half of the cases (n=8), matched primary tumors were available and were subjected to PIK3CA testing with NGS. The analytical performance between NGS and a semi-closed RT-PCR (EasyPGX®, Diatech Pharmacogenetics, Italy) was assessed in 13/16 (81.3%) metastatic samples for which archival slides and blocks and/or residual extracted DNA were available, and all the primary tumors. Results: Overall, upfront testing of PIK3CA mutational status with NGS detected genetic alterations in exons 7, 9, and 20. A concordance rate of 100.0% was observed between primary and metastatic tumors. On the other hand, the analysis of primary tumors (n=8) and 13/16 (81.3%) metastatic samples with RT-PCR revealed a concordance of 42.9%. Analytical performance comparison showed that the two technologies were concordant in 7/13 metastatic cases (53.8%). In two of the discordant cases (15.4%), RT-PCR did not identify the PIK3CA mutations due to their absence from its reference range. Interestingly, visual inspection of the RT-PCR raw data increased the concordance to 76.9%. In primary tumors, consensus between the two testing methods was observed in 5 (62.5%) cases. Discussion: The concordance rate analysis shows that upfront PIK3CA molecular testing with NGS appears to be more efficacious compared with RT-PCR. Our data suggest that primary tissues reflect the PIK3CA mutational status when tested with NGS. RT-PCR is simpler with a shorter turnaround time, and less expensive than NGS approaches, however, trained personnel are required for accurate results interpretation. In addition, the limited reference range of this testing method should be taken into account for potential false negative results. Conclusion: In terms of PIK3CA molecular testing, NGS should be the preferred method in comparison with RT-PCR. Primary tumors represent a valid biospecimen to be used for this analysis in the absence of the metastatic sample. Citation Format: Konstantinos Venetis, Francesco Pepe, Elham Sajjadi, Giuseppina Bonizzi, Mariia Ivanova, Davide Vacirca, Alessandra Rappa, Massimo Barberis, Giuseppe Viale, Elena Guerini-Rocco, Elisabetta Munzone, Umberto Malapelle, Nicola Fusco. Comparison of next-generation sequencing and real-time PCR for PIK3CA testing in hormone receptor-positive/HER2-negative breast cancer on metastatic and matched primary tumor samples [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-14.