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However, tissue fixation conditions needed to preserve DDR-specific phospho-biomarkers have not been previously investigated. Using xenograft tissues and tightly controlled formalin fixation conditions, we assessed how preanalytical factors affect phosphorylated DDR biomarkers pRAD50(Ser635), \u0263H2AX(Ser139), pKAP1(Ser824), and non-phosphorylated biomarkers cMYC and ATM. Cold ischemia times ranged from 15 min to 6 hr, and the fixation duration ranged from 24 hr to 4 weeks. Epitopes pRAD50 and pKAP1 appeared the most labile assessed with staining loss after just 15 min of cold ischemia time, while ATM was more robust showing consistent expression up to 1 hr of cold ischemia. Notably, \u0263H2AX expression was lost with formalin fixation over 48 hr. The use of core needle biopsies where possible and novel fixation methods such as the 2-step temperature-controlled formalin approach may improve phosphorylated biomarker preservation; however, practical challenges may affect wider clinical application. The most essential tissue-processing step when downstream analysis includes DDR phosphorylated biomarkers is immediate tissue submersion in formalin, without delay, upon excision from the patient, followed by room temperature fixation for 24 hr.<\/jats:p>","DOI":"10.1369\/00221554231174069","type":"journal-article","created":{"date-parts":[[2023,4,29]],"date-time":"2023-04-29T11:06:26Z","timestamp":1682766386000},"page":"237-257","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":3,"title":["Epitope Lability of Phosphorylated Biomarkers of the DNA Damage Response Pathway Results in Increased Vulnerability to Effects of Delayed or Incomplete Formalin Fixation"],"prefix":"10.1369","volume":"71","author":[{"given":"Elizabeth J.","family":"Wiseman","sequence":"first","affiliation":[{"name":"Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0923-229X","authenticated-orcid":false,"given":"Jennifer I.","family":"Moss","sequence":"additional","affiliation":[{"name":"Bioscience, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7979-4960","authenticated-orcid":false,"given":"James","family":"Atkinson","sequence":"additional","affiliation":[{"name":"CPSS, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hana","family":"Baakza","sequence":"additional","affiliation":[{"name":"Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"Hayes","sequence":"additional","affiliation":[{"name":"Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4341-4655","authenticated-orcid":false,"given":"Sophie E.","family":"Willis","sequence":"additional","affiliation":[{"name":"Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1231-7286","authenticated-orcid":false,"given":"Paul M.","family":"Waring","sequence":"additional","affiliation":[{"name":"Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaime","family":"Rodriguez Canales","sequence":"additional","affiliation":[{"name":"Translational Medicine, Oncology R&D, AstraZeneca, Gaithersburg, Maryland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gemma N.","family":"Jones","sequence":"additional","affiliation":[{"name":"Translational Medicine, Oncology R&D, AstraZeneca, Cambridge, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2023,4,29]]},"reference":[{"key":"bibr1-00221554231174069","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-14-0983."},{"key":"bibr2-00221554231174069","doi-asserted-by":"publisher","DOI":"10.3121\/cmr.2009.825"},{"key":"bibr3-00221554231174069","doi-asserted-by":"publisher","DOI":"10.1016\/j.semcancer.2017.12.008."},{"key":"bibr4-00221554231174069","doi-asserted-by":"publisher","DOI":"10.1186\/bcr2719."},{"key":"bibr5-00221554231174069","unstructured":"Varian BJ. 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