Quantitative analysis of hypoxia markers in cervical cancer biopsies

I N T R O D U C T I O N : Although tumour hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus on the method best suited for routine measurement. Recent studies have suggested that carbonic anhydrase 9 (CA9) and hypoxia-inducible factor-la (HIF-la) can be used as endogenous markers of hypoxia. Expression of these markers was compared to binding of the chemical hypoxia marker pimonidazole and oxygen microelectrode measurements in 78 tumor biopsies from patients with locally advanced invasive carcinoma of the cervix. M E T H O D S : Two or more biopsies were taken one day after i.v. infusion of pimonidazole. One biopsy was used for flow cytometry analysis of pimonidazole binding, while sequential sections from the other biopsies were used for immunohistochemical analyses of pimonidazole binding and expression of CA9 and HIFla! Images of stained sections were tiled and analyzed to determine the fraction of labeled tumor cells in tissue and marker colocalization. Hypoxic proportions, HP2.5 and HP5, were obtained from polarographic oxygen electrode measurements in tumours. R E S U L T S A N D DISCUSSION: Automated image analysis was found to be a reliable method for quantitative measurement of hypoxia marker expression or binding. The mean percentage of marker positive regions of the sections was similar for the 3 markers (between 5.5% and 7.4%). A significant but weak correlation was found between flow cytometry and image analysis methods for detecting pimonidazole (r = 0.45). Hypoxia marker analysis on multiple biopsies taken from the same tumour indicated that there was substantial intra-tumour marker heterogeneity. CA9 expression correlated with pimonidazole binding (r 0.60), while weaker but significant correlations were observed between both of these markers and HIFla (r = 0.34 for pimonidazole and HIF-la , and r = 0.42 for CA9 and HIF-la). Markers showed a high degree of colocalization, with 60%80% of the regions staining with all markers i f one marker were present. Nevertheless, regions of mismatch were identified and were of particular interest as they could be indicative of transient changes in tumor perfusion. No correlation was observed between oxygen electrode measurements and expression of the three hypoxia markers. Neither pimonidazole binding nor endogenous marker expression correlated with known clinical prognostic factors (Fib, nodal status, maximum tumour size, or FIGO stage). High HIFl a expression predicted shorter progression-free survival (p = 0.024), while CA9 (p 0.333) and pimonidazole (p = 0.488) had no apparent predictive value in this group of patients. Measurements of HIF-lev changes over, the course of radiotherapy for 15 patients evaluated did not correlate with disease progression. C O N C L U S I O N S : Quantitative comparisons between three hypoxia markers indicated significant but weak correlations between binding of pimonidazole and expression of two endogenous markers. Good colocalization of HIFl a and CA9 with pimonidazole stained regions was observed confirming the ability of these proteins to mark hypoxic cells. In addition, HIFl a emerged as a potential prognostic marker, and the predictive ability improved when two or more hypoxia markers were combined.