Immunohistochemical Evaluation of HLA-ABC

Immunohistochemical staining of HLA class I was performed on TMAs using HLA-ABC (Clone EMR8-5; 1:200 dilution; Abcam) with Dako EnVision detection kit (Dako, Carpinteria, CA, USA). Some of the IBC cases had been evaluated for HLA-ABC expression in the previous study^{36 (link)}, and those cases were re-evaluated during the study. HLA-ABC expression was evaluated in the tumor cell membrane for both intensity and proportion of positive cells. We adopted the interpretation method of HLA class I expression from the study by Na et al.^{19 (link)}. The staining intensity was classified into negative, weak positive, and strong positive. Lymphocytes and endothelial cells were used as internal control, and weak positivity was defined as staining weaker than internal control. Staining equivalent or stronger than internal control was considered strong positive. For statistical analysis, high expression was defined as 50% or more tumor cells showing strong positivity. Low expression was defined as < 50% of the tumor cells revealing strong positivity or ≥ 50% showing weak positivity.