Cytology samples of pleural effusions from adenocarcinomas and mesotheliomas were immersed in acetone (3 min) and xylene (10 min) to remove the coverslip and were then rehydrated with alcohol with decreasing concentration and immersed in distilled water. The slides were stained with PD‐L1 (clone SP263, Ventana Medical Systems, Tucson, AZ) on an automated staining platform (Benchmark ULTRA; Ventana) inclusive of antigen retrieval with CC1 solution (24 min) and incubation time with primary antibody (1 hr). An OptiView DAB IHC detection kit (Ventana) and an OptiView amplification kit (Ventana) were used according to the manufacturer's recommendations for the visualization of the primary anti PD‐L1 antibody. The analysis was performed on 40 samples (10 derived from mesothelioma and 30 from adenocarcinoma samples). All immunocytochemistry evaluations were blindly evaluated and by two independent experts.
Pd l1 clone sp263
Manufactured by Roche
Sourced in United States, Germany
The PD-L1 (clone SP263) is a laboratory equipment product manufactured by Roche. It is used for the detection and analysis of the PD-L1 protein, which plays a role in the regulation of the immune system. The product is designed for research purposes and its specific applications are not within the scope of this factual description.
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Lab products found in correlation
Benchmark ultra, by Roche (2 mentions)
Optiview dab ihc detection kit, by Roche (1 mentions)
Optiview amplification kit, by Roche (1 mentions)
Mcpyv clone cm2b4, by Santa Cruz Biotechnology (1 mentions)
Pd l1, by Roche (1 mentions)
Ab4059, by Abcam (1 mentions)
Cd20 clone l26, by Abcam (1 mentions)
Cd20 clone l26, by Roche (1 mentions)
Hematoxylin, by Roche (1 mentions)
Cd68 clone pg m1, by Agilent Technologies (1 mentions)
Cd8 clone sp57, by Roche (1 mentions)
Cd10 clone 56c6, by Leica (1 mentions)
9 protocols using pd l1 clone sp263
1
PD-L1 Immunocytochemistry of Pleural Effusions
2
Quantifying Tumor Infiltrating Lymphocytes
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Tumor infiltrating lymphocyte (TIL) quantification was performed using the International TIL Working Group Criteria [12 (link)]. Immunohistochemistry for CD3, CD4, CD8, and immune cell PD-L1 was performed. For PD-L1 testing, Ventana PD-L1 clone SP263 was used. A total of > 1% of PD-L1 expression on immune cell and > 1+ by IHC was considered positive in this study [13 , 14 ].
3
Immunohistochemical Evaluation of PD-L1 and CD8 in Tumor Biopsies
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4-μm histological sections were prepared from formalin-fixed, paraffin-embedded tumors and mounted on positively-charged glass slides. Baseline tumor biopsies from Study 1108/NCT01693562 were immunostained separately for PD-L1 (clone SP263, Ventana Medical Systems, Inc., Tucson, AZ, USA) and for CD8 (clone SP239, Spring Bioscience, Pleasanton, CA, USA), both performed on the Ventana BenchMark ULTRA staining platform (Ventana Medical Systems, Inc., Tucson, AZ, USA) [37 (link), 39 (link)]. For the non-ICT patient specimens, a CD8/PD-L1 dual immunostain using these antibodies was applied. All immunostained slides were digitally scanned and the image files were uploaded for digital processing as previously described [37 (link)].
4
Pathological Analysis of Merkel Cell Carcinoma
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A trained pathologist reviewed all specimens concerning the following morphological variables: mitotic index, fibroplasia, inflammatory infiltrate, tumor thickness, ulceration, necrosis, hemorrhage and subtype [15 ]. Primary tumor samples were preferentially used. If not available, metastases were analyzed.
MCPyV (clone CM2B4, Santa Cruz, 200 µg/mL dilution, 0.1 mL) expression was classified as positive when tumor cells exhibited a dark staining. PD-L1 (clone SP263, Ventana) was quantified based on the percentage of positive cells in 10 fields at 10 magnifications over the total cell number. Tumor cell staining was compared with positive and negative controls. Two cutoff points were employed for the PD-L1 evaluations, namely < 1% (negative) and ≥ 1% (positive) or < 5% (negative) and ≥ 5% (positive).
MCPyV (clone CM2B4, Santa Cruz, 200 µg/mL dilution, 0.1 mL) expression was classified as positive when tumor cells exhibited a dark staining. PD-L1 (clone SP263, Ventana) was quantified based on the percentage of positive cells in 10 fields at 10 magnifications over the total cell number. Tumor cell staining was compared with positive and negative controls. Two cutoff points were employed for the PD-L1 evaluations, namely < 1% (negative) and ≥ 1% (positive) or < 5% (negative) and ≥ 5% (positive).
5
PD-L1 Immunohistochemistry in Tumor and Immune Cells
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Mismatch-repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 immunohistochemistry, EBER in situ hybridisation (Bond ready-to-use probe Leica Biosystems) and PD-L1 (clone SP263, Ventana Medical Systems) immunohistochemistry were performed as part of previous studies (Dislich et al. 2020 (link), 2022 (link)).
For the purpose of the current study, the PD-L1 expression data from the previous study were used (Dislich 2022 (link)). Two CPS cut-offs were established in the current study and cases were regarded as PD-L1 positive with CPS ≥ 1 or CPS ≥ 5. Additionally, the PD-L1 expression of the tumour cells and the immune cells was scored separately. The proportion of tumour cells with PD-L1 expression was scored into five groups: 0% (no tumour cells with PD-L1 expression), less than 1% (i.e., > 0 to < 1), 1% to less than 5% (i.e., ≥ 1 to < 5), 5% to less than 50% (i.e., ≥ 5 to < 50), and 50% and more (i.e., ≥ 50). The same scoring system was used for scoring the immune cells.
For the purpose of the current study, the PD-L1 expression data from the previous study were used (Dislich 2022 (link)). Two CPS cut-offs were established in the current study and cases were regarded as PD-L1 positive with CPS ≥ 1 or CPS ≥ 5. Additionally, the PD-L1 expression of the tumour cells and the immune cells was scored separately. The proportion of tumour cells with PD-L1 expression was scored into five groups: 0% (no tumour cells with PD-L1 expression), less than 1% (i.e., > 0 to < 1), 1% to less than 5% (i.e., ≥ 1 to < 5), 5% to less than 50% (i.e., ≥ 5 to < 50), and 50% and more (i.e., ≥ 50). The same scoring system was used for scoring the immune cells.
6
Comprehensive Analysis of FFPE Tumor Samples
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Formalin fixed paraffin embedded (FFPE) pathology sections were selected per the determination of a board-certified pathologist (C.B.) determined to contain sufficient and representative cancer tissue within the resection specimen. Tumor H&E’s were scored, and then serial sections (5 μm) were cut for IHC staining and RNA extraction (see below). PDL1 (clone SP263, Ventana), CD3 (clone SP7, Roche), CD20 (clone L26, Abcam), CD8 (clone SP16, Roche) and GZMB (ab4059, Abcam) were stained as previously described22 (link) and developed using monochromatic DAB (Jackson Immunoresearch).
7
Multiplex IHC Analysis of Breast Tumor Microenvironment
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Tissue samples were treated as described above. Antigens were sequentially detected, and heat inactivation was used to prevent antibody cross-reactivity between the same species. Following each primary antibody incubation (CD8, clone SP57, Ventana, ready to use, cat#: 790-4460; PD-L1, clone SP263, Ventana, ready to use, cat#: 740-4907; CD20, clone L26, Ventana, ready to use, cat#: 760-2531; CD68, clone PG-M1, DAKO, dilution 1:100, cat#: M0876; pan-CK, clone AE1/AE3/PK26, Ventana, ready to use; cat#: 760-2595), DISCOVERY anti-Rabbit HQ, DISCOVERY anti-Mouse HQ, and DISCOVERY anti-HQ-HRP were incubated. Stains were visualized with DISCOVERY Yellow Kit, DISCOVERY Teal Kit, and DISCOVERY Purple Kit, respectively, counterstained with hematoxylin (Ventana), and coverslipped. The proportion of PD-L1-positive immune cells within the tumor stroma was assessed by a clinical, board-certified breast cancer pathologist.
8
Immunohistochemical Profiling of LUAD
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A total of 103 LUAD patients from LUAD-NCC were assessed by immunohistochemistry (IHC) staining with PD-L1 (clone SP263; Roche Ventana), CD4 (ZA-0519; Zsbio Tech), CD8 (ZA-0508; Zsbio Tech), CD19 (ZM-0038; Zsbio Tech), CD68 (ZM-0060; Zsbio Tech) and CD163 (ZM-0428; Zsbio Tech) antibodies. Formalin-fixed, paraffin-embedded LUAD specimens were cut into 4 mm slides for IHC staining. Slides were stained using an automated Leica Bond staining system according to the manufacturer’s protocol. PD-L1 expression scores were defined as the percentage of tumor cells with membranous staining. Staining status was stratified into three subgroups: negative (PD-L1 <1%), intermediate (PD-L1 1-49%), and high (PD-L1 ≧50%). For CD4 and CD8, the proportion of positive cells was assessed as low density (≦25%), intermediate density (25-50%), and high density (> 50%). For CD19, CD68 and CD163, the percentage of stained cells was classified as follows: low density (≦10%), intermediate density (10-20%), and high density (> 20%). All slides were evaluated by two pathologists and any disagreement was resolved by consensus using a multi-head microscope.
9
Immunohistochemical Analysis of INADA Tumor Markers
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Expression of the PD-L1 protein and cell differentiation markers in tumors from 54 patients with INADA was detected by immunohistochemistry (IHC). FFPE tissue sections with a thickness of 4 µm were prepared and subjected to IHC with an automated Bond-III slide stainer (Leica Biosystems, Wetzlar, Germany) in accordance with the manufacturer's protocol using mouse anti-human monoclonal antibodies against MUC6 (clone CLH5; 1:100; Novus, America), MUC5AC (clone CLH2; 1:100; DAKO, Denmark), MUC2 (clone CCP58; 1:100; DAKO, Denmark), CD10 (clone 56C6; 1:70; Leica, Germany), and PD-L1 (clone SP263; rabbit monoclonal primary anti-PD-L1 antibody, prediluted, Ventana Medical Systems, Tucson, AZ). For PD-L1, reactivity was evaluated separately for cancer cells and in ltrating immune cells. Cases with ≥ 1% of cells (membrane staining) being stained were considered to be PD-L1 positive (Pollack et al. 2021) (link). For mucin histochemical staining, cytoplasmic reactivity was determined to be a positive outcome for MUC6, MUC5AC and MUC2, and luminal membranous reactivity was determined to be a positive result for CD10. Gastric phenotypic markers (MUC6 and MUC5AC) and intestinal phenotypic markers (MUC2 and CD10) were evaluated as positive when immunoreactivity was observed in ≥ 10% of tumor cells (Yoshida et
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