HIF-1α, pimonidazole, and iododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors

Purpose: Tumor hypoxia measured by microelectrodes has been shown to indicate poor patient outcome. Here we investigated four potentially more widely applicable immunohistochemical parameters of tumor oxygenation and perfusion in human head-and-neck tumors. Methods: Twenty patients with squamous cell carcinomas of the head and neck treated with primary surgery were injected with pimonidazole and IdUrd the evening before operation. Consecutive paraffin-embedded sections were stained for blood vessels, pimonidazole, IdUrd, and HIF-1α. IdUrd labeling and Ki-67 labeling around individual blood vessels were scored. The spatial relationship between HIF-1α and pimonidazole was studied, as well as the distribution of both markers as a function of distance from the nearest blood vessel. Results: Measurement of all four parameters (diffusion-limited fraction, pimonidazole fraction, HIF-1α fraction, IdUrd-negative vessels) was feasible, and a significant difference between tumors was found for all parameters. IdUrd-labeled cells were absent around some vessels, indicating lack of perfusion, because these regions were positive for Ki-67. There was a positive correlation between diffusion-limited fraction and pimonidazole area for all images from all tumors, although no correlation for mean values per tumor. Colocalization of pimonidazole and HIF-1α was low (0.02%-25%). Most expression profiles showed a more homogenous distribution for HIF-1α than pimonidazole. There was no significant correlation between the pimonidazole and HIF-1α fractions in the 10 tumors studied. Conclusions: Simultaneous immunohistochemical measurements related to hypoxia and perfusion are feasible (and easily applicable) in resected human tumors. The different geographic distributions of HIF-1α and pimonidazole indicate that HIF-1α might not be suitable as a marker for chronic hypoxia. Each parameter will be correlated with outcome in a larger ongoing study on head-and-neck tumors treated with surgery with or without postoperative radiotherapy.