Msh6 clone 44

Immunohistochemical (IHC) labeling was performed at the IHC laboratory of the Department of Pathology. Briefly, 4-μm-thick tissue sections from TMAs and whole-section slides of cases showing MMR protein deficiency in TMAs were deparaffinized and hydrated in xylene and serially diluted alcohol solutions. Endogenous peroxidase was blocked by incubation in 3% H₂O₂ for 10 min; next, heat-induced antigen retrieval was performed. Primary antibodies with BenchMark auto-stainers (Ventana Medical Systems, Tucson, AZ, USA) were used following the manufacturer’s protocol. Primary antibodies for HER2 (4B5, mouse monoclonal, 1:8, Ventana), MLH1 (clone M1, mouse monoclonal, prediluted, Roche, Basel, Switzerland), MSH2 (clone G219-1129, mouse monoclonal, 1:1000, Cell Marque, Rocklin, CA, USA), MSH6 (clone 44, mouse monoclonal, 1:200, Cell Marque), and PMS2 (clone EP51, rabbit monoclonal, 1:100, Dako, Glostrup, Denmark) were used. After the evaluation of IHC-labeled TMA slides, additional immunolabeling was performed on sections on the whole-section slides if samples showed any loss of MMR proteins or an HER2 immunolabeling score of 2+ or 3+ on TMA slides.

PD‐L1 expression was evaluated in the tumor (TC) and immune cells (IC) using SP142 antibody (Ventana). Any PD‐L1 expression was considered positive if either TC or IC exhibited staining. AR (clone 441, Leica Biosystems, Buffalo Grove, IL), ER (SP1 clone, Ventana, Tucson, AZ) and PR (1E2, Ventana, Tucson, AZ) were analyzed using a ≥10% threshold for nuclear positivity. HER2 (4B5 clone, Ventana) was considered positive if >10% cancer cells showed complete, circumferential (3+) expression or exhibited HER2 gene amplification (see below). Nine cases (five vulvar and four scrotal) of EMPD and four MPD were explored for the expression of the splice variant of AR (ARv7) using immunohistochemistry (EPR15656, Abcam). Three EMPD cases were tested for mismatch repair proteins: MLH1 (Clone M1, Ventana), MSH2 (Clone G219‐1129, Ventana), MSH6 (Clone 44, Cell Marque) and PMS2 (Clone EPR3947, Cell Marque). Topoisomerase 2α (Clone 3F6, Leica) expression was considered positive if cancer cells exhibited nuclear positivity in ≥10%.7

Immunohistochemistry was performed on 2 μm paraffin sections by using monoclonal antibodies specific for MLH1 (clone G168-15, dilution 1:25, BD Pharmingen, Heidelberg, Germany), MSH2 (clone FE11, dilution 1:200, Calbiochem, Darmstadt, Germany) or MSH6 (clone44, dilution 1:50, Cell Marque, Rocklin, USA) for detecting loss of MMR protein as described previously [12 (link)]. An immunoperoxidase method was used to visualise the antibodies by labelling them with a chromogen (3-amino-9-ethylcarbazole, Dako, Glostrup, Denmark). The amount of mucosal length and surface analysed was calculated as described previously [12 (link)].
For immunohistochemical staining of immune cells in the vicinity of MMR-DCF the following antibodies were used: CD4 (clone RPA-T4, dilution 1:50, BD Pharmingen, Heidelberg, Germany), CD8 (clone 4B11, dilution 1:50, Novocastra, Wetzlar, Germany) and FoxP3 (clone 236A/E7, dilution 1:50, eBioscience, Frankfurt, Germany).
Immune cell infiltration was qualitatively assessed by two independent observers.