In breast cancer, the frequency of PD-L1 expression by cancer cells has been reported to vary widely, ranging from 1.7% to over 50% [17,24,25,26], with more frequent expression reported in TNBC [17,24,25], as with our result, as well as in HER2 positive cancers . younger patients (components and microcalcification. High TIM-3 expression was significantly associated with a combinational immunophenotype group of high PD-L1 and high PD-1 (component; TILs=tumor infiltrating lymphocytes; MC=microcalcification; LN=lymph node. Tissue microarray We prepared a tissue microarray (TMA), possessing 109 TNBC cancer tissue punches from FFPE tumor samples according to a previously described format . Numerous cancer cell areas with dominant TILs on hematoxylin and eosin stained slides were identified and two 3-mm tissue cores from individual tumors were obtained. TMAs were constructed with a tissue arrayer (Unitma Co., Ltd., Seoul, Korea). Ten TMA blocks were constructed. Immunohistochemistry and interpretation Neohesperidin dihydrochalcone (Nhdc) IHC for TIM-3, PD-L1 and PD-1 was performed with 4-m thick TMA tissue sections by using a BenchMark XT automated immunostainer (Ventana Medical System Inc., Tuscon, USA). After deparaffinization, rehydration and antigen retrieval, diluted primary TIM-3 rabbit monoclonal antibody (1:100; D5D5R?; Cell Signaling Technology, Beverly, USA), PD-L1 rabbit monoclonal antibody (1:100; E1L3N; Cell Signaling Technology) and PD-1 mouse monoclonal antibody (1:50; NAT105; Abcam, Cambridge, UK) were incubated. The primary antibodies were detected with Ultraview Universal DAB Detection Kit (Ventana Medical System Inc.), according to the manufacturer’s instructions, followed by hematoxylin counterstaining. For the validation of these antibodies, Neohesperidin dihydrochalcone (Nhdc) we used tonsillar tissue for TIM-3 antibody and PD-1 antibody, and placental tissue for PD-L1 antibody as positive controls. Two independent pathologists (J.S.J. and M.H.J.) observed the slides in a blinded manner. The distribution of TIM-3 expression in TNBC was identified as the percentage of distinctly immune-stained TILs among total TILs, as represented in Figure 1 and divided into score 1 (5%), 2 (6%C25%), 3 (26%C50%) and 4 (51%), referring to a Neohesperidin dihydrochalcone (Nhdc) previous study . For the evaluation of PD-L1 expression of TNBC cancer cells, we assessed the staining intensity with a 4-tiered scoring consisting of negative (0), weak (score 1), moderate (score 2) and strong (score 3) as well as the distribution of stained cancer cells by percentage, finally multiplying intensity score by distribution percentage to obtain the expression score (range, 0C300). By using a modified Muenst’s scoring method , PD-L1 expression was categorized into two groups according to the final scores: low expression ( 100) and high expression (100). According to the distribution of PD-1 expression in TILs, scoring was divided into 0 (5%), 1 (6%C33%), 2 (34%C66%) and 3 ( 66%) and re-categorized into a low expression group (score 0 and 1) and high expression group (score 2 and 3). Open in a separate window Figure 1 Representative T-cell immunoglobulin and mucin domain-3 (TIM-3) expressions in triple-negative breast cancer by immunohistochemistry (400). (A) Occasionally, stromal tumor infiltrating lymphocytes (TILs) express TIM-3, analyzing into score 1 (5%). (B) A few TILs express TIM-3, analyzing into score 2 (6%C25%). (C) Some TILs and histiocytoid cells express TIM-3, analyzing into score 3 (26%C50%). (D) Many TILs and histiocytoid cells express TIM-3, analyzing into score 4 (51%). The IHC study for the expression of ER (1:50), PR (1:50), HER2 (1:200), Ki-67 Neohesperidin dihydrochalcone (Nhdc) (1:800), cytokeratin 5/6 (1:50), epidermal growth factor receptor (EGFR) (1:50) and p53 (1:1,200) was available in all tissues. The interpretation of staining intensity and distribution for ER, FLJ31945 PR, and HER2 expression was analyzed as described previously . Statistical analyses The MedCalc software program (version 18.2.1; MedCalc, Ostend, Belgium) was used for statistical analyses. The distributions of TIM-3 expression levels in TILs with clinicopathological characteristics and biomarkers were compared using the chi-square test. DFS and OS based on TIM-3 expression were assessed by the Kaplan-Meier method with the log-rank test. Univariate survival analysis was performed in individual covariate and multivariate survival analyses using the Cox proportional hazards regression model to assess whether the expression level of TIM-3 in TILs was an independent predictor of disease relapse or survival. In all of the tests, components and absence of microcalcification tended to be related with high TIM-3 expression, although not statistically significant ( em p /em =0.2101, em p /em =0.0706, em p /em =0.1174, respectively). There were no correlations with CK5/6, EGFR, Ki-67 and p53 expression, lymph node metastasis stage Neohesperidin dihydrochalcone (Nhdc) or TNM stage. High TIM-3 expression was closely correlated with increased PD-L1 expression in cancer cells ( em p /em =0.0019) and high PD-1 expression in TILs ( em p /em =0.0001). The results are summarized in Table 1. Furthermore, a correlation between TIM-3 expression levels and combinational immunophenotypes of PD-L1 and PD-1 expression was observed. High TIM-3 manifestation was significantly associated with the high PD-L1/high PD-1 group ( em p /em 0.0001) (Table 2). Table 2 Correlation of TIM-3 manifestation with combinational immunophenotypes of PD-L1/PD-1 manifestation in TNBC Open in a separate windowpane TIM-3=T-cell immunoglobulin and mucin website-3; PD-L1=programmed death receptor ligand.