Epitope Lability of Phosphorylated Biomarkers of the DNA Damage Response Pathway Results in Increased Vulnerability to Effects of Delayed or Incomplete Formalin Fixation

Phosphorylated biomarkers are crucial for our understanding of drug mechanism of action and dose selection during clinical trials, particularly for drugs that target protein kinases, such as DNA-damage-response (DDR) inhibitors. 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), dH2AX(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, dH2AX 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.