Impact of warm ischemia on phosphorylated biomarkers in head and neck squamous cell carcinoma

Objectives: To quantitatively and visually characterize changes in phosphorylated biomarker expression in head and neck squamous cell carcinoma specimens from excision through 90 minutes of warm ischemia. Materials and Methods: Tissue biospecimens were procured prospectively. Head and neck squamous cell carcinoma specimens from 5 patients were subdivided into three parts upon excision, exposed to warm ischemia of 15, 30, or 90 minutes, and routinely biobanked. Relative change in biomarker expression of p-Akt, p-ERK, and p-Stat3 was measured by immunoblot densitometry. Immunofluorescent stains were performed to visually supplement the quantitative analysis. Results: From 15 to 30 minutes of ex vivo ischemia, there was a significant decrease in p-Akt (p = 0.045) as the mean intensity fell by 44.9%. This decrease in p-Akt remained significant at the 90 minute time point (p = 0.015). From 15 to 30 minutes of ischemia, there was a trend toward a decline in p-ERK, which became significant by 90 minutes of ex vivo warm ischemia (p = 0.008). These changes were supported by qualitative differences in p-ERK fluorescence at 0 and 90 minutes warm ischemia. Conclusion: Some phosphorylated biomarkers of HNSCC remain highly dynamic during the period of ex vivo warm ischemia after surgical excision but before biobanking. These findings have critical implications for studies that attempt to correlate protein phosphorylation with clinical outcome. We conclude that ex vivo warm ischemia time is a major determinant of tissue quality that may explain inconsistent results from biomarker research in head and neck squamous cell carcinoma.