Six complete and non-overlapping tumor regions of interest (ROI) were selected from each case and saved as .tif files. The number of diaminobenzidine (DAB)-stained nuclei was determined in the ROI using color deconvolution and the particle analysis plug-in of the Fiji platform (ImageJ distribution, http://fiji.sc/Fiji). The mean number of CD8-positive lymphocytes in all six fields was used for statistical analysis. The number of diaminobenzidine (DAB)-stained blood vessels was counted in a ROI using the color deconvolution plug-in of the Fiji platform (ImageJ distribution, http://fiji.sc/Fiji). The mean number of CD31-positive blood vessels in all six fields was used for statistical analysis.
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