Quantitative Immunohistochemical Evaluation

Immunohistochemical staining was quantitatively evaluated using computer-assisted image analysis software (Visiopharm, Hoersholm, Denmark) as described previously (14 (link)). In brief, the slides were scanned using a whole-slide scanner (NanoZoomer 2.0, Hamamatsu Photonics, Hamamatsu City, Japan) and imported into Visiopharm software (for Windows 7, version 4.5.1.324) using the TMA workflow. Staining intensity was categorized as 0, 1+, 2+, or 3+ according to the distribution pattern across cores. A brown staining intensity (0-negative, 1-weak, 2-moderate, or 3-strong) was obtained using the predefined algorithm and optimized settings. The overall immunohistochemical scores (histoscore) for AGR2, BRD7, and POM121 were expressed as the percentage of positive cells multiplied by their staining intensity (possible range, 0–300). For the survival analysis and hierarchical clustering, the expression values were dichotomized (High vs. Low, described in superscript) using cut-off values affording the most discriminative power (histoscore of 39 for AGR2, 46 for BRD7, and 204 for POM121). PAUF protein expression was evaluated as previously described (4 (link)) to compare with that of the three proteins identified.