The same locations used for primary cilia AbMole Ellipticine analysis were found by referencing the annotated H&E slide. These areas of interest were exported as TIFF files from the Dmetrix scan files, and as JPEG files from the BioImagene scan files, and uploaded into Definiens Tissue Studio 3.0 Software. Tissue Studio 3.0 Software was tested for absolute agreement with manual hand counts performed by two separate investigators. Images from six normal prostate and six prostate cancer locations were blindly scored for nuclear staining of Gli1, where each nucleus was scored as either positive or negative. For each image, Tissue Studio 3.0 was used in conjunction to quantify the number of positive and negative cells/nuclei. A statistical test that is used to measure the consistency and absolute agreement of measurements made by different observers was applied to the data obtained for the six normal and cancerous prostate tissues. The intraclass correlation coefficient was determined as 0.7, using SPSS 19, which is considered strong agreement. For Ki67 analysis, some 
patient tissue could not be used for analysis due to too many serial sections missing between the Ki67-stained slide and the H&E slide, making it impossible to locate the exact area used for primary cilia analysis. The number of patients used for cancer was 72, and the number of patients used for perineural invasion was 15. For the Ki67 analysis with Definiens Tissue Studio, a modified Nuclei solution was used. The epithelial/cancer and stromal compartments of cancer and perineural invasion areas were separately analyzed using the Manual ROI Selection segmentation tool, with a segmentation of 8. The hematoxylin and immunohistological threshold were set at 0.12 arbitrary units and 0.03 a.u., respectively. The IHC threshold was determined by identifying the lightest positively-stained nucleus in the sample set and using this value as the cutoff for positivity. From the exported results, positive indices were computed per tissue type per patient. Some patient tissue could not be used for ��-catenin analysis due to inadequacy of serial sections, or too many serial sections missing between the ��-catenin stained slide and the H&E slide, making it impossible to find the exact location. For normal, the number of locations utilized was 26, from 10 patients. For PIN, the number of locations was 19, from 13 patients. For cancer, 154 locations were used, from 64 patients. For perineural invasion lesions, the number of locations used was 23, from 11 patients. For the ��-catenin analysis with Definiens Tissue Studio, the percentage and intensity of nuclear staining in epithelial and cancerous tissue was acquired. Stroma was excluded from the analysis since ��catenin is mostly expressed in epithelial cells. For the analysis, a modified Nuclei, Membrane and Cells solution was used.