Tissue Age Affects Antigenicity and Scoring for the 22C3 Immunohistochemistry Companion Diagnostic Test.

Programmed death-ligand 1 (PD-L1) antibody 22C3 is the approved companion diagnostic immunohistochemistry test for treatment with pembrolizumab and cemiplimab in multiple cancer types. The 22C3 and 28-8 antibodies target the extracellular domain (ECD) of PD-L1, which is known to contain N-glycosylation sites. We hypothesize that antigenicity could be affected by the degradation of the glycan part of the epitope and thus change the scoring of the assay over time. Here, we test samples over time and assess the effects of time and deglycosylation on PD-L1 signal by comparing an antibody with an ECD antigen to an antibody with an intracellular domain (ICD) antigen. Ten whole-tissue sections of non-small-cell lung cancer (NSCLC) from 2018 were selected for testing. Fresh-cut serial sections for each case were stained on DAKO Link48 for 22C3 according to the label. In parallel, a previously described laboratory-developed test using E1L3N (an ICD antibody) was performed on the Leica BondRX. Tumor proportion scores for 22C3 and E1L3N were read by a pathologist and compared to the previous clinical diagnoses. To determine the effect using a quantitative approach, a tissue microarray (TMA) cohort with 90 NSCLC cases was similarly assessed. Finally, to determine whether the possible effect of epitope glycosylation, antibodies were tested before and after enzymatic deglycosylation of specimens. We found that 6 of 7 archival positive samples showed a significant reduction in positive staining with 22C3 compared to the original diagnostic sample assessed 3 years earlier. In an older archival TMA cohort, a quantitative significant difference in signal intensity was noted when staining with 22C3 was compared to E1L3N. This loss of signal was not noted in the fresh cell line TMA consistent with a time-dependent degradation of staining. Finally, quantitative assessment of the fresh TMA showed a significant loss of signal after a deglycosylation procedure when stained with 22C3, which was not seen when stained with E1L3N. We believe that these data show that the glycan part of the 22C3 epitope is not stable over time, and that this issue should be considered when assessing archival tissue for diagnostic or research purposes.