Pms2 clone a16 4

MMR screening status was determined by immunohistochemical analyses of TMA slides of the protein products of genes involved in the DNA mismatch repair system, as described previously [16 (link)]. Antibodies (all from Ventana Medical Systems Inc., Tucson, Arizona, US) against the four key mismatch repair proteins (MLH1 (clone M1), MSH2 (clone G219-1129), PMS2 (clone A16-4) and MSH6 (clone SP93)) were used on an automated Ventana BenchMark ULTRA instrument. Tumors with loss of nuclear expression of one or more of these proteins were described as dMMR, while samples with positive staining for all four antibodies were denoted pMMR. Tumors where internal staining was lacking, such as positive lymphocytes, were considered uninformative.

EUS-FNB specimens obtained from treatment-naïve patients were archived respecting formalin fixation and paraffin embedding protocols and were subjected to IHC staining. Briefly, Ventana BenchMark Ultra automated slide-staining system was used in order to stain 4 μm thick tissue sections utilizing the following antibodies: anti-PD-L1 (clone SP263 Ventana), MLH1 (clone ES05 Leica), MSH2 (clone G219-1129, Ventana), MSH6 (clone SP93 Novus Biological), and PMS2 (clone A16-4, Ventana). Diaminobenzidine was used as a chromogen for UltraView detection in order to visualize antigen–antibodies reactions. A specimen was considered adequate for evaluation only if a minimum of 100 tumor viable cells were found on the slides. Membranous staining was the hallmark for positive PD-L1 expression (Figure 1C). The tumor proportion score (TPS) has been calculated as the percentage of viable tumor cells with complete or partial membrane staining at any intensity. PD-L1 expression was considered in specimens with TPS > 1% and high PD-L1 expression was considered in patients with TPS > 50%. In the case of absent nuclear staining of DNA mismatch repair protein (PMS2, MSH2, MSH6, or MLH1), the tumor was targeted as dMMR (Figure 2).