Ventana bench mark xt autostainer

Sections of FFPE tissues were prepared and stained with hematoxylin and eosin. IHC was performed on 4-μm tissue sections using the Ventana Bench Mark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) and the SP142 antibody (dilution 1:100; Ventana). PD-L1 positivity was defined as a membranous staining intensity of ≥5%. IHC scoring was done on a 0–2 scale (0 = <5%, 1 = 5%–49%, and 2 = ≥50%) [6 (link)]. The semi-quantitative H score (maximum value of 300 corresponding to 100% of tumor PD-L1-positive cells with an overall staining intensity score of 3) was determined by multiplying the percentage of stained cells by the intensity score (0, absent; 1, weak; 2, moderate; and 3, strong). Two experienced pathologists (H.S.S and Y.J.C) blinded to the patients’ clinical information examined the PD-L1 expression. For specimens with discrepant results, two pathologists re-evaluated the PD-L1 positivity status to reach a consensus after consultation.

Formalin-fixed, paraffin-embedded (FFPE) pretreatment biopsies of all included patients were collected in a tissue microarray (TMA) as described before [23 (link)]. In short, sections of the FFPE blocks were stained with hematoxylin and eosin (H&E) and assessed by a dedicated head and neck pathologist (SMW) to mark representative tumor regions. For each patient, three 0.6-mm tissue cores were obtained randomly from the assigned area of the FFPE blocks and collected in a TMA. The TMA blocks were cut into 4-μm sections, which were immunohistochemically stained for CD57 (NK1; 1:20; Novocastra). Stainings were performed using a Ventana Bench Mark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA).

For the immunohistochemical detection of TOP2A and SIRT1, commercially available and validated monoclonal antibodies were used [23 (link),39 (link)] (Table 5). Antigen retrieval was carried out by heat treatment with Target Retrieval Solution Citrate (Agilent Technologies, Santa Clara, CA, USA). Staining was performed on a Ventana Benchmark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) with a DAB+ Kit (Agilent Technologies, Santa Clara, CA, USA). All slides were counterstained with hematoxylin (Vector Laboratories, Burlingame, CA, USA). An ImmPRESS Anti-Rabbit IgG Polymer Kit was used for detection (Vector Laboratories, Burlingame, CA, USA). To exclude unspecific staining, system controls were included. Tonsillar tissue served as a positive control for immunohistochemistry. Immunostaining of cells was evaluated and scored semi-quantitatively (0 = 0–9%, negative; 1 = 10–100%, positive) (Table 6). All immunohistochemical and pathologic evaluations were carried out independently and blinded together with an experienced pathologist with special expertise in sarcoma pathology (T.K.). In the case of discrepancy, the slides were reevaluated under a multiheaded microscope and consensus reached.

For the immunohistochemical detection of TIM-3, commercially available and validated monoclonal antibodies were used (TIM-3 D5D5R, Cell Signaling Tech., Danvers, MA, USA). Antigen retrieval was carried out by heat treatment with Target Retrieval Solution Citrate (Agilent Technologies, Santa Clara, CA, USA). Staining was performed on a Ventana Benchmark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) with a DAB+ Kit (Agilent Technologies, Santa Clara, CA, USA). All slides were counterstained with hematoxylin (Vector Laboratories, Burlingame, CA, USA). An ImmPRESS Anti-Rabbit IgG Polymer Kit was used for detection (Vector Laboratories, Burlingame, CA, USA). To exclude unspecific staining, system controls were included. Tonsillar tissue served as a positive control for immunohistochemistry. The immunostaining of cells was evaluated and scored semi-quantitatively (0 = negative; 1 = ≥5% positive and weakly stained, 2 = ≥25% positive and moderately stained, 3 = ≥50% positive and strongly stained). All immunohistochemical and pathologic evaluations were carried out independently and blinded with an experienced sarcoma pathologist (T.K.). In the case of discrepancy, the slides were reevaluated under a multiheaded microscope and a consensus was reached.

The expression of HLA-I complex and its subunits were confirmed by IHC using the OPTIVIEW universal DAB kit (Ventana), the Ventana Bench mark XT autostainer (Ventana) and antibodies against HLA-ABC (EMR8-5, 1:8000, Abcam, Cambridge, UK), HLA-A (EP1395Y, 1:5000, Abcam), HLA-B (1:700, Abcam), HLA-C (1:1000, Abcam) and B2M (B2M/961, 1:2000, Abcam). Immunostaining of TILs was performed with antibodies specific to CD3 (1:100; Dako, Glostrup, Denmark) and CD8 (1:100; Dako) using the Bond polymer kit (Leica Microsystems) and Leica BOND-MAX autostainer (Leica Microsystems). IHC of PD-L1 was performed on the Autostainer Link 48 with EnVision DAB Detection System (Agilent Technologies, Santa Clara, CA) and 22C3 pharmDx antibody (prediluted; Dako), according to the manufacturer’s instructions.