Automated staining platform

Immunohistochemistry for ATR was done on formalin-fixed, paraffin-embedded tissues using anti-ATR antibody from abcam (ab54793) at a dilution of 1:500 and tissues were stained using automated staining platform (Ventana). Envision + polymer (ready to use; Dako) was used as a secondary antibody. Color was developed with 3,3′-diaminobenzidine (DAB) and instant hematoxylin (Shandon) was used for counterstaining.

Immunohistochemistry for ATR was done on formalin-fixed, paraffin-embedded tissue using anti-ATR from Abcam company (ab54793) overnight at a dilution of 1:500, and they were stained using automated staining platform (Ventana). Envision + polymer (ready to use; Dako) was used as a secondary antibody. Color was developed with 3,3′-diaminobenzidine (DAB), and instant hematoxylin (Shandon) was used for counterstaining. The ATR level was evaluated and verified by two qualified pathologists, who scored both the proportion of positive cells as well as the intensity of ATR expression in both cancer cells and their stromal fibroblasts.

The programmed death-ligand 1 (PD-L1) protein was detected using IHC staining with two unique antibody clones (SP263, Ventana Medical Systems, Tucson, USA) on an automated staining platform (Ventana). For the IHC staining of CD8 and PD-1, we used the SP16 (Thermo Fisher Scientific, Runcorn, UK; 1:100) and the EPR4877 (Abcam, Cambridge, UK; 1:100) antibodies, respectively. These stains were applied via the Bond-Max automatic immunostaining tool (Leica Biosystems). For HER2 and Ki-67 IHC staining, we employed 4B5 (Roche Diagnostics, Tucson, USA; pre-dilution) and SP6 (Cell Marque, California, USA; 1:300) antibodies, respectively, using the same automated staining system. Placental tissue sections were positive controls for PD-L1, while tonsil tissue sections were used for PD-1, CD8, and Ki67. The primary antibodies were omitted to prepare negative controls, and a rabbit monoclonal immunoglobulin (Ventana) was used as a negative control for PD-L1 staining.

Immunohistochemistry on formalin-fixed paraffin-embedded tissues was performed using anti-Ki-67 and CD34 antibodies (Abcam) at dilutions of 1:100 and 1:150, respectively, and then slides were stained using automated staining platform (Ventana). Envision + polymer (ready to use; Dako) was used as a secondary antibody. Color was developed with 3,3′-diaminobenzidine (DAB) and instant hematoxylin (Shandon) was used for counterstaining.

Immunohistochemistry on formalin-fixed paraffin-embedded tissues was performed using anti-DNMT1 antibody from Abcam (Cambridge, MA) overnight at a dilution of 1:500 and were stained using automated staining platform (Ventana). Envision + polymer (ready to use; Dako) was used as a secondary antibody. Color was developed with 3,3′-diaminobenzidine (DAB) and instant hematoxylin (Shandon) was used for counterstaining.

Immunohistochemistry for vimentin and E-cadherin were done on formalin-fixed, paraffin-embedded tissue of previously described breast cancer samples [13 (link), 16 (link)]. Vimentin was detected using ready-to-use mouse anti-human vimentin antibody from Ventana (Cat# 790-2917) and were stained using automated staining platform (Ventana). Immunohistochemical staining of E-cadherin was done manually using rabbit anti E-cadherin antibody (clone EP00y) overnight at a dilution of 1:500. Envision + polymer (ready to use; Dako) was used as a secondary antibody. Color was developed with 3,3′-diaminobenzidine (DAB) and instant hematoxylin (Shandon) was used for counterstaining.

An automated staining platform (Ventana Medical Systems, Inc.) was used to detect PD-1/PD-L1 protein expression by IHC: the SP142 Ab (laboratory developed test) was used to stain for PD-L1 expression on TCs of prostate, colorectal, ovarian, and pancreatic cancers, with a positive threshold of ≥2+ intensity and ≥5% cell stained; the SP142 Ab (Ventana) was used to stain for PD-L1 on immune cells of breast cancer, with a positive threshold of ≥1% of cells stained; the 22c3 Ab was used in NSCLC and ovarian cancers, with a positive threshold of tumor proportion score (TPS) ≥1 and combined positive score (CPS) ≥1, respectively; the 28-8 Ab was used to stain for PD-L1 expression on TCs for NSCLC, with a positive threshold of ≥1+ intensity and ≥1% cell stained; and the PD-1 Ab was used for ovarian cancer, with a positive threshold of ≥1% cells stained. Benign tonsil samples served as a positive control. MMR protein expression was tested by IHC (Ventana Medical Systems) using Ab clones for the four mismatch repair proteins [MLH1, M1 Ab; MSH2, G2191129 Ab; MSH6, 44 Ab; and PMS2 (Abcam, catalog no. ab203457, RRID:AB 2889230), EPR3947 Ab (Ventana Medical Systems, Inc.)]. The complete absence of protein expression of any of the four proteins tested (0+ in 100% of cells) was considered deficient MMR as described previously (21 (link)).