PDL1 immunohistochemistry in canine neoplasms: Validation of commercial antibodies, standardization of evaluation, and scoring systems

Abstract

Immuno-oncology research has brought to light the paradoxical role of immune cells in the induction and elimination of cancer. Programmed cell death protein 1 (PD1), expressed by tumor-infiltrating lymphocytes, and programmed cell death ligand 1 (PDL1), expressed by tumor cells, are immune checkpoint proteins that regulate the antitumor adaptive immune response. This study aimed to validate commercially available PDL1 antibodies in canine tissue and then, applying standardized methods and scoring systems used in human pathology, evaluate PDL1 immunopositivity in different types of canine tumors. To demonstrate cross-reactivity, a monoclonal antibody (22C3) and polyclonal antibody (cod. A1645) were tested by western blot. Cross-reactivity in canine tissue cell extracts was observed for both antibodies; however, the polyclonal antibody (cod. A1645) demonstrated higher signal specificity. Canine tumor histotypes were selected based on the human counterparts known to express PDL1. Immunohistochemistry was performed on 168 tumors with the polyclonal anti-PDL1 antibody. Only membranous labeling was considered positive. PDL1 labeling was detected both in neoplastic and infiltrating immune cells. The following tumors were immunopositive: melanomas (17 of 17; 100%), renal cell carcinomas (4 of 17; 24%), squamous cell carcinomas (3 of 17; 18%), lymphomas (2 of 14; 14%), urothelial carcinomas (2 of 18; 11%), pulmonary carcinomas (2 of 20; 10%), and mammary carcinomas (1 of 31; 3%). Gastric (0 of 10; 0%) and intestinal carcinomas (0 of 24; 0%) were negative. The findings of this study suggest that PDL1 is expressed in some canine tumors, with high prevalence in melanomas.

Programmed Cell Death-Ligand 1 (PD-L1) Immunohistochemical Expression in Equine Melanocytic Tumors

“…Furthermore, it is stated that cytoplasmatic PD-L1 cannot be reversed with commonly used anti-PD-L1 antibodies [ 73 ]. A recently published article on canine melanoma also follows the same score methodology, where only membranous staining was considered positive [ 74 ]. However, other recent studies regarding PD-L1 in canine melanoma considered both localizations as positive staining and reported that cytoplasmatic labeling was the most common [ 45 , 48 , 51 ].…”

Checkpoint Inhibitors in Dogs: Are We There Yet?

“…Validation of commercial antibodies designed for human samples but with proven cross-reactivity for canine tissues may increase their availability for routine testing. While there are no specifically established evaluation methods for canine tumours, the extrapolation of human criteria has been used by Maekawa et al (2021) and Muscatello et al (2023) in several canine neoplasms [4,59]. In these studies, the PD-1/PD-L1 tumour proportion score (TPS; percentage of positive neoplastic cells) and combined positive score (CPS; percentage of positive cells, including immune cells, among total neoplastic cells) have been investigated [4,59].…”

“…While there are no specifically established evaluation methods for canine tumours, the extrapolation of human criteria has been used by Maekawa et al (2021) and Muscatello et al (2023) in several canine neoplasms [4,59]. In these studies, the PD-1/PD-L1 tumour proportion score (TPS; percentage of positive neoplastic cells) and combined positive score (CPS; percentage of positive cells, including immune cells, among total neoplastic cells) have been investigated [4,59]. An alternative method, the immune cell density score (IDS), which considers the proportion of positive immune cells among all the cells in a specific area, has been reported [60].…”

“…Immunohistochemistry is currently considered the gold standard for selecting human cancer patients eligible for anti-PD-1 and anti-PD-L1 immunotherapy [ 58 ]. However, technical aspects such as the sample type, the antibody used, and the reference cutoffs may provide discordant results [ 59 ]. Validation of commercial antibodies designed for human samples but with proven cross-reactivity for canine tissues may increase their availability for routine testing.…”

“…An alternative method, the immune cell density score (IDS), which considers the proportion of positive immune cells among all the cells in a specific area, has been reported [ 60 ]. These scores may be used for PD-1 and PD-L1 analysis in dogs [ 4 , 59 , 60 ].…”