{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T01:16:05Z","timestamp":1793236565415,"version":"4.5.0"},"reference-count":40,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2022,11,9]],"date-time":"2022-11-09T00:00:00Z","timestamp":1667952000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Novartis"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Somatic mutations in PIK3CA are present in ~40% breast cancers (BC); their detection in hormone receptor (HR)+\/HER2\u2212 tumors allows for selecting patients with advanced disease eligible for PIK3CA targeting with alpelisib. The choice of what type of PIK3CA testing approach to adopt and which tissue sample to analyze is a new task in breast pathology. In this methodological study, we sought to assess the performance of next-generation sequencing (NGS) and RT-PCR for PIK3CA testing on archival formalin-fixed paraffin-embedded (FFPE) primary tumors and corresponding metastases. Sixteen HR+\/HER2\u2212 BC with known PIK3CA-mutated status (ex. 7, 9, and 20) on metastatic samples by means of amplicon-based targeted NGS were selected, and n = 13 of these samples were re-tested with a commercially available CE-IVD RT-PCR assay. All available primary tumors (n = 8) were tested with both methods. NGS detected mutations in all samples, while RT-PCR in n = 2 sample-pairs and overall, in n = 5\/8 (62.5%) primary tumors and 7\/13 (53.8%) metastases (\u03ba = 0.09; 95% CI, \u22120.69\u20130.87). Slight agreement (\u03ba = 0; 95% CI, \u22120.59\u20130.59) was observed between NGS and RT-PCR, with the former being generally more sensitive in cases with low DNA quality and quantity. Post hoc visual inspection of the RT-PCR data increased the concordance to 76.9%. Targeted NGS offers reliable and robust PIK3CA testing on both tumor and metastasis FFPE samples; the accuracy of RT-PCR depends on the DNA quantity and quality. In HR+\/HER2\u2212 BC, both the selection of the PIK3CA testing strategy of FFPE tissues and which sample to analyze should consider several technical parameters and should be tailored for each case.<\/jats:p>","DOI":"10.3390\/cells11223545","type":"journal-article","created":{"date-parts":[[2022,11,10]],"date-time":"2022-11-10T02:07:48Z","timestamp":1668046068000},"page":"3545","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Analytical Performance of Next-Generation Sequencing and RT-PCR on Formalin-Fixed Paraffin-Embedded Tumor Tissues for PIK3CA Testing in HR+\/HER2\u2212 Breast Cancer"],"prefix":"10.3390","volume":"11","author":[{"given":"Konstantinos","family":"Venetis","sequence":"first","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"},{"name":"Department of Oncology and Hemato-Oncology, University of Milan, Via Festa del Perdono 7, 20122 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2803-9586","authenticated-orcid":false,"given":"Francesco","family":"Pepe","sequence":"additional","affiliation":[{"name":"Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3371-3878","authenticated-orcid":false,"given":"Elisabetta","family":"Munzone","sequence":"additional","affiliation":[{"name":"Division of Medical Senology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7401-1843","authenticated-orcid":false,"given":"Elham","family":"Sajjadi","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"},{"name":"Department of Oncology and Hemato-Oncology, University of Milan, Via Festa del Perdono 7, 20122 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2698-9470","authenticated-orcid":false,"given":"Gianluca","family":"Russo","sequence":"additional","affiliation":[{"name":"Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6429-0620","authenticated-orcid":false,"given":"Pasquale","family":"Pisapia","sequence":"additional","affiliation":[{"name":"Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7636-1000","authenticated-orcid":false,"given":"Mariia","family":"Ivanova","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuseppina","family":"Bonizzi","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Davide","family":"Vacirca","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandra","family":"Rappa","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alberto","family":"Ranghiero","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergio Vincenzo","family":"Taormina","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuseppe","family":"Viale","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"},{"name":"Department of Oncology and Hemato-Oncology, University of Milan, Via Festa del Perdono 7, 20122 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1630-5805","authenticated-orcid":false,"given":"Giancarlo","family":"Troncone","sequence":"additional","affiliation":[{"name":"Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0943-4804","authenticated-orcid":false,"given":"Massimo","family":"Barberis","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Guerini-Rocco","sequence":"additional","affiliation":[{"name":"Division of Pathology, IEO, European Institute of Oncology IRCCS, Via Giuseppe Ripamonti 435, 20141 Milan, Italy"},{"name":"Department of Oncology and Hemato-Oncology, University of Milan, Via Festa del Perdono 7, 20122 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3211-9957","authenticated-orcid":false,"given":"Umberto","family":"Malapelle","sequence":"additional","affiliation":[{"name":"Department of Public Health, Federico II University of Naples, Via S. 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