A16 4

Immunohistochemistry (IHC) was performed on FFPE sections (4 μm in thickness) for CD3 (Dako, A0452, 1/200), CD4 (Dako 4B12 IR649, 1/100), CD8 (Dako C8/144B, IS623, undiluted), PD-L1 (Dako clone 22C3), MLH1 (Dako ES05 1/50), MSH2 (Dako FE11 1/50), MSH6 (Dako EP49 1/50), PMS2 (BD Phamingen A16-4 1/50). PD-L1 immunostaining with Dako clone 22C3 was performed by Merck Research Laboratory, Palo Alto, CA. IC (lymphocytic) infiltrates were scored according to the method of Rothermel et al.^{29 (link)}modified to include both (1) percentage of tumor area occupied by tumor-infiltrating lymphocytes (TIL) and (2) percentage of circumferential peri-tumoral (PT) area occupied by IC infiltrates. Scoring was as follows: “0”: absence of IC; “1”: rare, <5% of the tumor area or the PT area positive; “2”: 5 to 50% of the tumor area or PT area positive; and “3”: 50–100% of tumor area or PT areas positive. PD-L1 expression in a characteristic membranous pattern was recorded as (1) percentage of tumor area (tumor cells) positive and (2) percentage of IC area positive in the circumferential PT areas. The immunohistochemical staining was assessed and quantified independently by two anatomic pathologists (R.L.B. and S.G.). Any discordant results were reviewed microscopically and consensus reached.

Expression of MMR proteins was assessed in 16 WCM UTUC tumors and 14 matched archival WCM UCB. IHC was performed on 4-μm-thick formalin-fixed paraffin-embedded tissue sections using a Leica Bond III automated stainer. Mouse antibodies against MLH1 (G168-728, 1:25 dilution, BD Biosciences), PMS2 (A16-4, 1:100 dilution, BD Biosciences), MSH2 (FE11, 1:200, EMD Millipore), and MSH6 (44/MSH6, 1:200, BD Biosciences) were used. IHC slides were scanned at ×200 total magnification using a single z-plane via an Aperio AT2 whole slide scanner (Leica Biosystems, San Diego, CA, USA). The scanned images were loaded onto the HALO^TM imaging analysis platform (Indica Labs, Corrales, New Mexico, USA). Study pathologists manually selected tumor areas for automated image scoring, and the HALO^TM analysis software determined the staining intensity of each tumor cell (0, 1+, 2+, 3+) and percentage of tumor cells for each intensity level. H-scores were then calculated using the formula [1×(% of cells with intensity of 1+)+2×(% cells 2+)+3×(% cells 3+)] with possible scores thus ranging from 0 to 300.

Immunohistochemistry analysis was performed using a tissue microarray to evaluate the expression of tumor protein p53 (p53) and two MMR proteins (hMSH6 and PMS2). Staining for p53 was performed using a primary monoclonal antibody (pre-diluted DO-7; Dako, Santa Clara, CA, USA) as previously described [8 (link)]. Expression of p53 was considered aberrant if >75% of the cells were strongly positive for p53 (overexpression) or if 0% of the cells were positive (null phenotype). Staining for the MMR proteins was performed using primary monoclonal antibodies against MSH6 (GRBP.P1/2/D4, 1:200; Serotec Inc., Raleigh, NC, USA) and PMS2 (A16-4, 1:200; PharMingen, San Diego, CA, USA). Expression was defined as abnormal if the expression of at least one of the MMR proteins was completely absent from all tumor cell nuclei [16 (link)].