Parametric mapping of immunohistochemically stained tissue sections; a method to quantify the colocalization of tumor markers

BACKGROUND: Automated analysis of immunohistochemically stained tissue sections is of great importance in cancer research to detect tumor-specific prognostic markers and make therapy decisions. Here, an automated quantitative analysis is presented to assess the colocalization of CAIX, a membrane-bound hypoxic marker and Ki-67, a nuclear proliferation marker. METHODS: Tissue sections of 104 biopsies from 89 patients were stained for CAIX and Ki-67 with diaminobenzidine and haematoxylin counterstain. Image scans of whole tumor sections were recorded and image maps were created with parametric mapping to quantify the markers and assess the colocalization. RESULTS: The fraction of CAIX showed a range of 0–93%. The interobserver correlation and the correlation between manual scores and automated analysis were both very strong (rs=0.96, p <0.0001, and rs=0.97, p <0.0001). The labelling index of Ki-67 exhibited a range of 0–42% with less strong interobserver and manual to automated analysis correlations (rs=0.90, p <0.0001, and rs=0.71, p <0.0008). The relative tumor area positive for both markers varied from 0 – 76%. CONCLUSION: Parametric mapping of immunohistochemically stained tumor sections is a reliable method to quantitatively analyze membrane-bound proteins and assess the colocalization of various tumor markers in different subcellular compartments.