Immunohistochemical Profiling of Tumor-Infiltrating Lymphocytes

Immunohistochemical staining was performed on the four micrometer TMA sections. Immunostaining for CD3 (DAKO, Glostrup, Denmark), CD8 (Neomarkers, Fremont, CA, USA), and Foxp3 (Abcam, Cambridge, UK) were performed using a Leica BOND-MAX autostainer. For GRP94 (Abcam, Cambridge, UK) and CD4 (Ventana, Tucson, AZ, USA), immunohistochemical staining was performed using the BenchMark XT platform. For detection of EGFR mutations, we performed immunohistochemistry using the EGFR pharmDx kit (Dako, Glostrup, Denmark) following the manufacturer’s instructions. Moderate to strong immunostaining for GRP94 in more than 25% of tumor cells was defined as GRP94 positivity; EGFR-positivity was defined by the presence of any membranous immunostaining of tumor cells [34 (link)]. For quantitative TIL analysis, immunostained TMA slides were scanned using an Aperio ScanScope CS slide scanner (Aperio Technologies, Vista, CA, USA) at 40× magnification. After digitalization, the automated digital image analysis algorithm (ImageScope TM, Aperio Technologies, Vista, CA, USA) of Aperio was used to quantify CD3+, CD4+, CD8+, and Foxp3+ TILs, and the density of each TIL was recorded [35 (link)]. The density of each immunoreactive TIL was dichotomized into high and low groups based on the median values.