Biomarker analysis
Tumors, tumor
CPS, TUMOR, TUMORS
PD-L1 expression was assessed in formalin-fixed paraffin-embedded tumor samples before treatment by the PD-L1 immunohistochemistry (IHC) 22C3 pharmDx assay (Dako, Glostrup, Denmark). PD-L1 evaluation was performed using the CPS, determined as the number of PD-L1-positive cells—tumor cells, lymphocytes, and macrophages—divided by the total number of tumor cells × 100. PD-L1 positivity was defined as CPS ≥ 1 or 5. Mismatch repair (MMR) status was assessed locally by polymerase chain reaction (PCR) amplification and fragment analysis or IHC analysis of the DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. Monoclonal antibodies against MLH1 (Clone: ES05, dilution 1:100, Dako Denmark A/S, Denmark), PMS2 (Clone: EP51, dilution 1:100, Dako Denmark A/S, Denmark), MSH2 (Clone: FE11, dilution 1:100, Dako Denmark A/S, Denmark), MSH6 (Clone: EP49, dilution 1:150, Dako Denmark A/S, Denmark) were incubated with tumor sections in a humidified chamber at 4 °C overnight. Negative controls (without the primary antibody) and positive controls were included in each run to ensure the specificity and accuracy of the staining procedure. MSI analysis was performed using the Revised Bethesda Guidelines for two mononucleotides (BAT 25 and BAT 26) and 3 dinucleotides (D2S123, D5S346, and D17S250) microsatellite markers. Tumors were classified as MSI-H/dMMR with 2 or more unstable markersThe biomarker panel was decided by the trial committee through discussion when designing the trial based on guidelines