Clone es05

PD-L1 status was estimated by IHC staining of FFPE tissue sections using anti-PD-L1 antibodies clone 22C3 (DAKO, cat#m3666) or 28–8 (Abcam, Cat#ab205921) [29 (link), 44 (link)]. The specimen was considered PD-L1 positive expression when the combined positive score (CPS) was either ≥ 1 [45 (link)–47 (link)]. IHC analysis was conducted to detect MMR-related proteins, including MLH1 (clone ES05, cat#IR079, DAKO), PMS2 (clone EP51, cat#IR087, DAKO), MSH2 (clone FE11, cat#IR08561-2, DAKO), and MSH6 (clone EP49, cat#IR086, DAKO). Tumors were classified as dMMR when the expression of at least one MMR protein was lost and as MMR-proficient when all four MMR proteins had positive nuclear staining in tumor cells [48 (link)].

Mismatch repair/microsatellite status of tumors was determined by immunohistochemistry (IHC) and/or polymerase chain reaction (PCR) as described previously (Hirsch et al. 2018 (link)). Briefly, IHC was performed using the following primary antibodies: MLH1 (1:25; clone ES05, cat # M3640, Dako, Agilent Pathology Solutions, Agilent, Santa Clara, CA, USA), MSH2 (ready-to-use; clone FE11, cat # IR085, Dako), MSH6 (ready-to-use; clone EP49, cat # IR086, Dako), and PMS2 (1:50; clone EP51, cat # M3647, Dako). Detection was done using the EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse (cat # K5007, Dako). IHC stainings were validated by internal and/or external positive controls as well as negative control specimens. IHC stainings were evaluated by two pathologists (DH, TG). Microsatellite PCR of tumor and corresponding normal DNA was done using a panel of five mononucleotide markers (BAT25, BAT26, NR-21, NR-24, and MONO-27; cf. MSI Analysis System, Promega), and a panel of two mononucleotide (BAT25 and BAT26) and three dinucleotide markers [D5S346, D2S123, and D17S250; so-called Bethesda panel; (Boland et al. 1998 (link))]. Tumors were classified as MSI-H when two or more markers of either the Bethesda panel or the Promega panel showed an allelic size variation (i.e., a band shift compared with corresponding normal DNA).

Archival formalin-fixed paraffin-embedded (FFPE) tumor specimens with the pathological report, which included HER2 status, were retrospectively collected for IHC analysis, if residual samples were available. IHC analysis, such as for PD-L1, MMR, and chromogenic in situ hybridization for EBV-encoded RNA (EBER-ISH) using fluorescein-labeled peptide nucleic acid probes (EBV PNA Probe/Fluorescein, Agilent), were performed on FFPE tumor samples and assessed by an established pathologist.
For PD-L1 evaluation, IHC staining was performed using PD-L1 IHC 28-8 pharmDx (Dako). The level of PD-L1 protein expression was determined using the combined positive score (CPS), which was calculated as the number of PD-L1-stained cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells and multiplied by 100. Tumor PD-L1 positivity was defined as CPS ≥ 1%.
MMR status was determined by IHC for the following proteins; anti-mutL homolog 1 (MLH1; Clone ES05, Agilent Technologies), anti-mutS homolog 2 (MSH2; Clone FE11, Agilent Technologies), anti-postmeiotic segregation increased 2 (PMS2; Clone EP51, Agilent Technologies), and anti-mutS homolog 6 (MSH6; Clone EP49, Agilent Technologies), in FFPE samples. MMR-deficient (dMMR) was defined as a tumor that lacked staining for at least one of the MMR proteins.

Immunohistochemistry (IHC) staining of non-tumor- and tumor-FFPE samples for MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed according to established laboratory protocol at the Department of Pathology at the Institute of Oncology Ljubljana, Slovenia. Briefly, IHC staining was performed on 2 to 4 µm FFPE tissue sections using fully automated IHC system Ventana Benchmark XT (Ventana Roche, Oro Valley, USA). For this purpose, commercially available monoclonal antibodies for MLH1 (clone ES05, #M3640) (DAKO Agilent, Santa Clara, USA), PMS2 (clone EP51, #M3647) (DAKO Agilent), MSH2 (clone G219-1129, #556349) (BD Pharmingen, Franklin Lakes, USA) and MSH6 (clone SP93, #287R) (CellMarque, Rocklin, USA) were used. Primary antibodies were visualized using a three–step multimer detection system OptiView DAB IHC Detection Kit (Ventana Roche) following the manufacturer's protocol. MLH1 and PMS2 detection was enhanced using OptiVew Amplification Kit (Ventana Roche). All protocols passed external quality assessment in NEQAS and NORDIQC. Normal appendix and colon carcinomas with confirmed protein expression or loss of protein expression were used as controls.