5536 anonymized tumors, including germ cell, epithelial, mesenchymal, melanocytic/neuroectodermal, and lymphohematopoietic tumors, and normal tissues derived from surgical specimens were assembled to multitumor blocks containing 30 to 70 rectangular tissue samples as previously described. (26 (link)) The size of tumor tissue samples was estimated to exceed the size of a single 0.6mm2 core by a factor of 10–15. All tumors, selected for this study, were extensively characterized histologically and immunohistochemically. In addition, a 10-week-old fetus was immunohistochemically analyzed.
The rabbit monoclonal antibodies clone E1L3N (#13684) and 28-8 (ab205921) against PD-L1 were obtained from Cell Signaling Technology, Inc. (Danvers, MA) and Abcam Inc. (Cambridge, MA) , respectively. The antibodies were applied at a dilution of 1:200 (E1L3N) and 1:500 (28 (link)–8 (link)) to selected normal and tumor tissue arrays. Both of the antibodies showed almost similar staining patterns in placental trophoblast, choriocarcinoma, anaplastic large cell lymphoma (ALCL), and squamous cell carcinoma of lung. However, we selected clone E1L3N because of its lower background signal.
Immunostaining was performed with the Leica Bond-Max automation and Leica Refine detection kit. (Leica Biosystems, Bannockburn, IL) The protocol included in situ deparaffinization and high-pH epitope retrieval for 25 minutes, incubation with primary antibody for 30 minutes, polymer for 15 minutes, postpolymer for 15 minutes, and DAB as the chromogen for 10 minutes, followed by 5-minute hematoxylin counterstaining. MLH1, MSH2, MSH6, and PMS3 immunohistochemistry was performed to analyze mismatch repair (MMR) system status as previously reported. (27 (link)) For the detection of Epstein-Barr virus (EBV) infection, Bond™ Ready-to-Use ISH EBER Probe was used in Leica Bond-Max automation system according to the manufacturer instructions. (Leica Biosystems, Bannockburn, IL)
The stained sections were independently evaluated by two pathologists (SI and MM). PD-L1 immunoreactivity in placental trophoblasts and peripheral nerves were used as external and internal positive controls, respectively. PD-L1 has been reported to be expressed on not only tumor cells but also dendritic cells and TAIs, therefore, we evaluated PD-L1 expression in both neoplastic cells and TAIs with a detection cut-off of 5%.
Chi-square test or Fisher’s exact test were performed by SPSS software (IBM, Armonk, NY) to analyze the statistical correlation between PD-L1-expression and other tumor status such as MMR-deficiency, EBER-positivity, p16-, and ALK-expression.
The rabbit monoclonal antibodies clone E1L3N (#13684) and 28-8 (ab205921) against PD-L1 were obtained from Cell Signaling Technology, Inc. (Danvers, MA) and Abcam Inc. (Cambridge, MA) , respectively. The antibodies were applied at a dilution of 1:200 (E1L3N) and 1:500 (28 (link)–8 (link)) to selected normal and tumor tissue arrays. Both of the antibodies showed almost similar staining patterns in placental trophoblast, choriocarcinoma, anaplastic large cell lymphoma (ALCL), and squamous cell carcinoma of lung. However, we selected clone E1L3N because of its lower background signal.
Immunostaining was performed with the Leica Bond-Max automation and Leica Refine detection kit. (Leica Biosystems, Bannockburn, IL) The protocol included in situ deparaffinization and high-pH epitope retrieval for 25 minutes, incubation with primary antibody for 30 minutes, polymer for 15 minutes, postpolymer for 15 minutes, and DAB as the chromogen for 10 minutes, followed by 5-minute hematoxylin counterstaining. MLH1, MSH2, MSH6, and PMS3 immunohistochemistry was performed to analyze mismatch repair (MMR) system status as previously reported. (27 (link)) For the detection of Epstein-Barr virus (EBV) infection, Bond™ Ready-to-Use ISH EBER Probe was used in Leica Bond-Max automation system according to the manufacturer instructions. (Leica Biosystems, Bannockburn, IL)
The stained sections were independently evaluated by two pathologists (SI and MM). PD-L1 immunoreactivity in placental trophoblasts and peripheral nerves were used as external and internal positive controls, respectively. PD-L1 has been reported to be expressed on not only tumor cells but also dendritic cells and TAIs, therefore, we evaluated PD-L1 expression in both neoplastic cells and TAIs with a detection cut-off of 5%.
Chi-square test or Fisher’s exact test were performed by SPSS software (IBM, Armonk, NY) to analyze the statistical correlation between PD-L1-expression and other tumor status such as MMR-deficiency, EBER-positivity, p16-, and ALK-expression.
