{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:19:02Z","timestamp":1785417542390,"version":"3.56.0"},"reference-count":51,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T00:00:00Z","timestamp":1779494400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100022736","name":"Hospital Moinhos de Vento","doi-asserted-by":"crossref","award":["25000.170835\/2023-51"],"award-info":[{"award-number":["25000.170835\/2023-51"]}],"id":[{"id":"10.13039\/100022736","id-type":"DOI","asserted-by":"crossref"}]},{"award":["25000.170835\/2023-51"],"award-info":[{"award-number":["25000.170835\/2023-51"]}],"id":[{"id":"https:\/\/ror.org\/009gqrs30","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background\/Objectives: Formalin-fixed paraffin-embedded tissue is widely used in pathology and molecular diagnostics, yet its variable quality can critically influence the accuracy of sequencing-based analyses. This study investigated pre-analytical and analytical factors that can affect exome sequencing performance in a Brazilian multicenter cohort of patients with breast cancer and prostate adenocarcinoma. Methods: Tumor samples were reviewed for diagnostic confirmation, and those with a minimum cellularity of 20% underwent DNA extraction, fluorometric quantification, fragmentation analysis, and exome sequencing. Pre-analytical parameters, including tumor content, DNA yield, and fragmentation profile, were recorded and correlated with sequencing results. Results: Only 36.7% of all analyzed samples (163\/444) generated valid whole-exome sequencing data, corresponding to 55.6% of those that proceeded to sequencing (163\/293). Although 94.5% of specimens met the minimum \u226520% cellularity threshold and 66.0% advanced to sequencing, a substantial proportion failed to yield usable exome data. Successful sequencing was associated with shorter storage durations (p &lt; 0.001) and superior analytical parameters (higher autosomal coverage, longer read lengths, lower duplication rates, and higher target coverage at \u2265500\u00d7 and \u2265100\u00d7; p &lt; 0.001). Detailed fixation-related variables (e.g., formalin type, fixation time, ischemia time) were not consistently available across centers, representing a major limitation for causal interpretation of pre-analytical effects. Conclusions: Our study identified a high failure rate in sequencing archival tissue, highlighting the need to prioritize more recently collected specimens and refine standardized sample handling protocols to maintain DNA integrity. These improvements are essential for optimizing sequencing workflow performance and feasibility in real-world settings.<\/jats:p>","DOI":"10.3390\/diagnostics16111595","type":"journal-article","created":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T00:30:33Z","timestamp":1779669033000},"page":"1595","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Pre-Analytical\u00a0and Analytical Challenges in Whole-Exome Sequencing of Formalin-Fixed Paraffin-Embedded Breast and Prostate Cancer Tissue: A Real-World Multicenter Study"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8569-9751","authenticated-orcid":false,"given":"Mahira L.","family":"Rosa","sequence":"first","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cl\u00e1udia","family":"Bordignon","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2195-4407","authenticated-orcid":false,"given":"Jaqueline B.","family":"Schuch","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ang\u00e9lica C.","family":"Baumont","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Bessel","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giovana D.","family":"Curzel","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan A.","family":"Cadore","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Paula M.","family":"Varela","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giovana T.","family":"dos Santos","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7098-033X","authenticated-orcid":false,"given":"Tiago F.","family":"Andreis","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francine H.","family":"Oliveira","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vitor F.","family":"Vasconcellos","sequence":"additional","affiliation":[{"name":"Hospital Universit\u00e1rio Cassiano Ant\u00f4nio Moraes, Vit\u00f3ria 29040-090, ES, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lilian A. R.","family":"Barros","sequence":"additional","affiliation":[{"name":"Instituto Brasileiro de Controle do C\u00e2ncer, S\u00e3o Paulo 03164-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6412-8041","authenticated-orcid":false,"given":"Cristiano P.","family":"Souza","sequence":"additional","affiliation":[{"name":"Hospital de C\u00e2ncer de Barretos, Barretos 14784-400, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Williams F.","family":"Barra","sequence":"additional","affiliation":[{"name":"N\u00facleo de Pesquisas em Oncologia, Universidade Federal do Par\u00e1, Bel\u00e9m 66073-005, PA, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela L. C.","family":"Louzeiro","sequence":"additional","affiliation":[{"name":"Hospital de Oncologia Dr. Tarquinio Lopes Filho, S\u00e3o Lu\u00eds 65076-090, MA, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandra","family":"Notari","sequence":"additional","affiliation":[{"name":"Hospital Escola da Universidade Federal de Pelotas, Empresa Brasileira de Servi\u00e7os Hospitalares (EBSERH), Pelotas 96020-360, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juliana J.","family":"de Menezes","sequence":"additional","affiliation":[{"name":"Hospital Nossa Senhora da Concei\u00e7\u00e3o, Porto Alegre 91350-200, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8298-4765","authenticated-orcid":false,"given":"Pedro E. R.","family":"Liedke","sequence":"additional","affiliation":[{"name":"Hospital de Cl\u00ednicas de Porto Alegre, Porto Alegre 90035-903, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-4173-8613","authenticated-orcid":false,"given":"Gl\u00e1ucio A.","family":"Bertollo","sequence":"additional","affiliation":[{"name":"Associa\u00e7\u00e3o Feminina de Educa\u00e7\u00e3o e Combate ao C\u00e2ncer (AFECC), Hospital Santa Rita de C\u00e1ssia, Vit\u00f3ria 29052-010, ES, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aline B. 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C.","family":"Mancini","sequence":"additional","affiliation":[{"name":"Hospital Universit\u00e1rio Get\u00falio Vargas, Manaus 69020-170, AM, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela D.","family":"Rosa","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriel S.","family":"Macedo","sequence":"additional","affiliation":[{"name":"Hospital Moinhos de Vento, Porto Alegre 90540-000, RS, Brazil"},{"name":"Instituto D\u2019Or de Pesquisa e Ensino (IDOR), S\u00e3o Paulo 04538-132, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1200\/EDBK_174176","article-title":"Precision Oncology: Who, How, What, When, and When Not?","volume":"37","author":"Schwartzberg","year":"2017","journal-title":"Am. 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