Semiquantitative immunohistochemical analysis for hypoxia in human tumors

Abstract

Objective: The goal of this study was to develop a semiquantitative scoring system for measuring hypoxia in human tumors by an immunohistochemical marker approach. Methods and Materials: Eighteen patients diagnosed with squamous cell carcinoma of the uterine cervix or head and neck were infused intravenously with a solution of pimonidazole hydrochloride at a dose of 0.5 gm/m 2. Twenty-four hours later, four biopsies on average from each tumor were fixed in formalin, processed into paraffin blocks, and sectioned. Tissue sections were immunostained for the presence of pimonidazole adducts. Microscopic images (×200) of immunostaining were captured and quantitated by standard image analysis. Images with known amounts of hypoxia spanning ranges of > 0% to 5%, > 5% to 15%, > 15% to 30%, and >30% were assigned scores of +1, +2, +3, and +4, respectively. Three observers then used this calibrated scoring system to analyze hypoxia in tumor sections in a blinded fashion. Results: Excellent interobserver reproducibility was obtained with the calibrated, semiquantitative, immunohistochemical assay for hypoxia in squamous cell carcinomas. Conclusion: The calibrated, semiquantitative assay shows promise as an approach to simplifying the quantitation of human tumor hypoxia by immunohistochemical techniques.