Development of Monoclonal Antibodies Targeting Canine PD-L1 and PD-1 and Their Clinical Relevance in Canine Apocrine Gland Anal Sac Adenocarcinoma

Abstract

Canine apocrine gland anal sac adenocarcinoma (AGASACA) is an aggressive canine tumor originating from the anal sac glands. Surgical resection, with or without adjuvant chemotherapy, represents the standard of care for this tumor, but the outcome is generally poor, particularly for tumors diagnosed at an advanced stage. For this reason, novel treatment options are warranted, and a few recent reports have suggested the activation of the immune checkpoint axis in canine AGASACA. In our study, we developed canine-specific monoclonal antibodies targeting PD-1 and PD-L1. A total of 41 AGASACAs with complete clinical and follow-up information were then analyzed by immunohistochemistry for the expression of the two checkpoint molecules (PD-L1 and PD-1) and the presence of tumor-infiltrating lymphocytes (CD3 and CD20), which were evaluated within the tumor bulk (intratumor) and in the surrounding stroma (peritumor). Seventeen AGASACAs (42%) expressed PD-L1 in a range between 5% and 95%. The intratumor lymphocytes were predominantly CD3+ T-cells and were positively correlated with the number of PD-1+ intratumor lymphocytes (ρ = 0.36; p = 0.02). The peritumor lymphocytes were a mixture of CD3+ and CD20+ cells with variable PD-1 expression (range 0–50%). PD-L1 expression negatively affected survival only in the subgroup of dogs treated with surgery alone (n = 14; 576 vs. 235 days). The presence of a heterogeneous lymphocytic infiltrate and the expression of PD-1 and PD-L1 molecules support the relevance of the immune microenvironment in canine AGASACAs and the potential value of immune checkpoints as promising therapeutic targets.

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

“…In published canine studies, custom antibodies from hybridomas are often used. 18,21,35 Although this may be an advantage for the specificity of the reaction, with a view to future use for diagnostic purposes, it does not allow for their widespread availability. A possible alternative is the validation of commercial antibodies for specific use in canine tissues.…”

“…1 In many of the studies previously investigating PDL1 expression in canine tumors by immunohistochemistry, cytoplasmic labeling was also considered positive, and a specific membranous subcellular localization was not considered mandatory. 17,18,21,35 Conversely, in our study, we only considered tumor cells with membranous labeling positive, aligning with the evaluations used in humans. 1 A possible limitation of this study was that the scoring was performed by a single board-certified pathologist; future concordance studies are needed to evaluate differences at this assessment stage.…”

“…A fundamental factor in defining the reproducibility of results is the use of nonarbitrary evaluation systems and guidelines, thereby reducing differences in assessment methods among studies. 18,21 In this work, we used the current human pathology evaluation systems. 1 TPS and CPS are scoring systems that provide quantitative data, which may prove helpful in future prognostic studies to establish specific cutoffs for therapeutic applications.…”

“…1 In many of the studies previously investigating PDL1 expression in canine tumors by immunohistochemistry, cytoplasmic labeling was also considered positive, and a specific membranous subcellular localization was not considered mandatory. 17,18,21,35 Conversely, in our study, we only considered tumor cells with membranous labeling positive, aligning with the evaluations used in humans. 1…”

“…There is variability in the types of antibodies used and/or in the criteria used when evaluating labeling for PDL1 in canine tumors through immunohistochemistry. 5,17,21,35 Achieving a uniform consensus in assessing cellular PDL1 immunolabeling, similar to what has been accomplished in human studies, is crucial. 1,19 In human medicine, only membrane expression is considered positive and tumor cells with cytoplasmic granular and nuclear expression are considered background labeling and fixation artifacts.…”