Microdensitometry of osteopontin as an immunohistochemical prognostic biomarker in colorectal carcinoma tissue microarrays: potential and limitations of the method in ‘biomarker pathology’

AIMS: To explore the potential and limitations of 'biomarker pathology' with quantitative immunohistochemistry on tissue microarrays, taking osteopontin and colorectal carcinoma as a model system. METHODS AND RESULTS: Microdensitometry for quantitative evaluation of osteopontin immunohistochemistry (clone OP3N) on digital microphotographs using the public domain software ImageJ was observed to be straightforward to perform and reliable. However, using colorectal carcinoma cell lines (n = 11) the correlation between densitometric evaluations of Western blots and microdensitometry of immunocytochemistry of slide cultures was only moderate. A virtual resampling method to simulate tissue microarrays showed that, due to heterogeneity of immunostaining, tumours were misclassified in nearly 20% of the arrays, even if four punches were used. With regard to prognosis, microdensitometric evaluation of a tissue microarray made of a clinicopathologically well-characterized series of colorectal carcinomas with long-term follow-up (222 cases evaluable in the tissue microarray, UICC Stages I-III/R0) showed a moderate survival advantage of patients with high osteopontin expression by microdensitometry. CONCLUSIONS: These results challenge the basic assumption that microdensitometry is a precise technique for the quantification of proteins detected by immunohistochemistry and delineate drawbacks encountered when working with tissue microarrays in clinicopathological studies.