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.

Department of Veterinary Sciences University of Parma Strada del Taglio 10 43100 Parma Italy

Department of Veterinary Sciences University of Turin Largo Braccini 2 10095 Grugliasco Italy

Institute of Genetics and Cancer The University of Edinburgh Crewe Road Edinburgh EH4 2XU UK

International Centre for Cancer Vaccine Science University of Gdansk Kladki 24 80822 Gdansk Poland

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty kopec 7 65653 Brno Czech Republic

Royal School of Veterinary Studies and Roslin Institute The University of Edinburgh Easter Bush Campus Midlothian EH25 9RG UK

Zobrazit více v PubMed

Polton G.A., Brearley M.J. Clinical Stage, Therapy, and Prognosis in Canine Anal Sac Gland Carcinoma. J. Vet. Intern. Med. 2007;21:274–280. doi: 10.1111/j.1939-1676.2007.tb02960.x. PubMed DOI

Barnes D.C., Demetriou J.L. Surgical Management of Primary, Metastatic and Recurrent Anal Sac Adenocarcinoma in the Dog: 52 Cases. J. Small Anim. Pract. 2017;58:263–268. doi: 10.1111/jsap.12633. PubMed DOI

Wouda R.M., Borrego J., Keuler N.S., Stein T. Evaluation of Adjuvant Carboplatin Chemotherapy in the Management of Surgically Excised Anal Sac Apocrine Gland Adenocarcinoma in Dogs. Vet. Comp. Oncol. 2016;14:67–80. doi: 10.1111/vco.12068. PubMed DOI PMC

Potanas C.P., Padgett S., Gamblin R.M. Surgical Excision of Anal Sac Apocrine Gland Adenocarcinomas with and without Adjunctive Chemotherapy in Dogs: 42 Cases (2005–2011) J. Am. Vet. Med. Assoc. 2015;246:877–884. doi: 10.2460/javma.246.8.877. PubMed DOI

Valenti P., Menicagli F., Baldi A., Barella G., Catalucci C., Attorri V., Spugnini E.P. Evaluation of Electrochemotherapy in the Management of Apocrine Gland Anal Sac Adenocarcinomas in Dogs: A Retrospective Study. Open Vet. J. 2021;11:100–106. doi: 10.4314/ovj.v11i1.15. PubMed DOI PMC

Meier V., Besserer J., Roos M., Rohrer Bley C. A Complication Probability Study for a Definitive-Intent, Moderately Hypofractionated Image-Guided Intensity-Modulated Radiotherapy Protocol for Anal Sac Adenocarcinoma in Dogs. Vet. Comp. Oncol. 2019;17:21–31. doi: 10.1111/vco.12441. PubMed DOI

McQuown B., Keyerleber M.A., Rosen K., McEntee M.C., Burgess K.E. Treatment of Advanced Canine Anal Sac Adenocarcinoma with Hypofractionated Radiation Therapy: 77 Cases (1999–2013) Vet. Comp. Oncol. 2017;15:840–851. doi: 10.1111/vco.12226. PubMed DOI

Williams C., Parys M., Handel I., Serra J.C., Lawrence J. Minimal Late Radiation Toxicity and Transient Early Toxicity Following Postoperative Definitive Intent Conformal Radiation Therapy (20 × 2.5 Gy) for Canine Apocrine Gland Anal Sac Adenocarcinoma. Vet. Radiol. Ultrasound. 2022;63:224–233. doi: 10.1111/vru.13042. PubMed DOI

Swan M., Morrow D., Grace M., Adby N., Lurie D. Pilot Study Evaluating the Feasibility of Stereotactic Body Radiation Therapy for Canine Anal Sac Adenocarcinomas. Vet. Radiol. Ultrasound. 2021;62:621–629. doi: 10.1111/vru.12998. PubMed DOI

Wouda R.M., Hocker S.E., Higginbotham M.L. Safety Evaluation of Combination Carboplatin and Toceranib Phosphate (Palladia) in Tumour-Bearing Dogs: A Phase I Dose Finding Study. Vet. Comp. Oncol. 2018;16:E52–E60. doi: 10.1111/vco.12332. PubMed DOI

London C., Mathie T., Stingle N., Clifford C., Haney S., Klein M.K., Beaver L., Vickery K., Vail D.M., Hershey B., et al. Preliminary Evidence for Biologic Activity of Toceranib Phosphate (Palladia®®) in Solid Tumours. Vet. Comp. Oncol. 2012;10:194–205. doi: 10.1111/j.1476-5829.2011.00275.x. PubMed DOI PMC

Elliott J.W. Response and Outcome Following Toceranib Phosphate Treatment for Stage Four Anal Sac Apocrine Gland Adenocarcinoma in Dogs: 15 Cases (2013–2017) J. Am. Vet. Med. Assoc. 2019;254:960–966. doi: 10.2460/javma.254.8.960. PubMed DOI

Heaton C.M., Fernandes A.F.A., Jark P.C., Pan X. Evaluation of Toceranib for Treatment of Apocrine Gland Anal Sac Adenocarcinoma in Dogs. J. Vet. Intern. Med. 2020;34:873–881. doi: 10.1111/jvim.15706. PubMed DOI PMC

Pradel J., Berlato D., Dobromylskyj M., Rasotto R. Prognostic Significance of Histopathology in Canine Anal Sac Gland Adenocarcinomas: Preliminary Results in a Retrospective Study of 39 Cases. Vet. Comp. Oncol. 2018;16:518–528. doi: 10.1111/vco.12410. PubMed DOI

Simeonov R., Simeonova G. Quantitative Analysis in Spontaneous Canine Anal Sac Gland Adenomas and Carcinomas. Res. Vet. Sci. 2008;85:559–562. doi: 10.1016/j.rvsc.2008.03.009. PubMed DOI

Mosca A., Restif O., Dobson J., Hughes K. Expression of Phosphorylated Signal Transducer and Activator of Transcription 3 and Its Prognostic Significance in Canine Anal Sac Adenocarcinoma. J. Comp. Pathol. 2021;182:15–21. doi: 10.1016/j.jcpa.2020.11.002. PubMed DOI

Skorupski K.A., Alarcón C.N., de Lorimier L.-P., LaDouceur E.E.B., Rodriguez C.O., Rebhun R.B. Outcome and Clinical, Pathological, and Immunohistochemical Factors Associated with Prognosis for Dogs with Early-Stage Anal Sac Adenocarcinoma Treated with Surgery Alone: 34 Cases (2002–2013) J. Am. Vet. Med. Assoc. 2018;253:84–91. doi: 10.2460/javma.253.1.84. PubMed DOI

Wong H., Byrne S., Rasotto R., Drees R., Taylor A., Priestnall S.L., Leo C. A Retrospective Study of Clinical and Histopathological Features of 81 Cases of Canine Apocrine Gland Adenocarcinoma of the Anal Sac: Independent Clinical and Histopathological Risk Factors Associated with Outcome. Animals. 2021;11:3327. doi: 10.3390/ani11113327. PubMed DOI PMC

Morello E.M., Cino M., Giacobino D., Nicoletti A., Iussich S., Buracco P., Martano M. Prognostic Value of Ki67 and Other Clinical and Histopathological Factors in Canine Apocrine Gland Anal Sac Adenocarcinoma. Animals. 2021;11:1649. doi: 10.3390/ani11061649. PubMed DOI PMC

Yamazaki H., Tanaka T., Mie K., Nishida H., Miura N., Akiyoshi H. Assessment of Postoperative Adjuvant Treatment Using Toceranib Phosphate against Adenocarcinoma in Dogs. J. Vet. Intern. Med. 2020;34:1272–1281. doi: 10.1111/jvim.15768. PubMed DOI PMC

Ariyarathna H., Thomson N.A., Aberdein D., Perrott M.R., Munday J.S. Increased Programmed Death Ligand (PD-L1) and Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) Expression Is Associated with Metastasis and Poor Prognosis in Malignant Canine Mammary Gland Tumours. Vet. Immunol. Immunopathol. 2020;230:110142. doi: 10.1016/j.vetimm.2020.110142. PubMed DOI

Aresu L., Ferraresso S., Marconato L., Cascione L., Napoli S., Gaudio E., Kwee I., Tarantelli C., Testa A., Maniaci C., et al. New Molecular and Therapeutic Insights into Canine Diffuse Large B-Cell Lymphoma Elucidates the Role of the Dog as a Model for Human Disease. Haematologica. 2019;104:e256–e259. doi: 10.3324/haematol.2018.207027. PubMed DOI PMC

Aresu L., Marconato L., Martini V., Fanelli A., Licenziato L., Foiani G., Melchiotti E., Nicoletti A., Vascellari M. Prognostic Value of PD-L1, PD-1 and CD8A in Canine Diffuse Large B-Cell Lymphoma Detected by RNAscope. Vet. Sci. 2021;8:120. doi: 10.3390/vetsci8070120. PubMed DOI PMC

Igase M., Nemoto Y., Itamoto K., Tani K., Nakaichi M., Sakurai M., Sakai Y., Noguchi S., Kato M., Tsukui T., et al. A Pilot Clinical Study of the Therapeutic Antibody against Canine PD-1 for Advanced Spontaneous Cancers in Dogs. Sci. Rep. 2020;10:18311. doi: 10.1038/s41598-020-75533-4. PubMed DOI PMC

Maekawa N., Konnai S., Takagi S., Kagawa Y., Okagawa T., Nishimori A., Ikebuchi R., Izumi Y., Deguchi T., Nakajima C., et al. A Canine Chimeric Monoclonal Antibody Targeting PD-L1 and Its Clinical Efficacy in Canine Oral Malignant Melanoma or Undifferentiated Sarcoma. Sci. Rep. 2017;7:8951. doi: 10.1038/s41598-017-09444-2. PubMed DOI PMC

Maekawa N., Konnai S., Nishimura M., Kagawa Y., Takagi S., Hosoya K., Ohta H., Kim S., Okagawa T., Izumi Y., et al. PD-L1 Immunohistochemistry for Canine Cancers and Clinical Benefit of Anti-PD-L1 Antibody in Dogs with Pulmonary Metastatic Oral Malignant Melanoma. npj Precis. Oncol. 2021;5:10. doi: 10.1038/s41698-021-00147-6. PubMed DOI PMC

Choi J.W., Withers S.S., Chang H., Spanier J.A., Trinidad V.L.D.L., Panesar H., Fife B.T., Sciammas R., Sparger E.E., Moore P.F., et al. Development of Canine PD-1/PD-L1 Specific Monoclonal Antibodies and Amplification of Canine T Cell Function. PLoS ONE. 2020;15:e0235518. doi: 10.1371/journal.pone.0235518. PubMed DOI PMC

Jiang X., Wang J., Deng X., Xiong F., Ge J., Xiang B., Wu X., Ma J., Zhou M., Li X., et al. Role of the Tumor Microenvironment in PD-L1/PD-1-Mediated Tumor Immune Escape. Mol. Cancer. 2019;18:10. doi: 10.1186/s12943-018-0928-4. PubMed DOI PMC

Vojtĕsek B., Bártek J., Midgley C.A., Lane D.P. An Immunochemical Analysis of the Human Nuclear Phosphoprotein P53. New Monoclonal Antibodies and Epitope Mapping Using Recombinant P53. J. Immunol. Methods. 1992;151:237–244. doi: 10.1016/0022-1759(92)90122-A. PubMed DOI

Pinard C.J., Hocker S.E., Poon A.C., Inkol J.M., Matsuyama A., Wood R.D., Wood G.A., Woods J.P., Mutsaers A.J. Evaluation of PD-1 and PD-L1 Expression in Canine Urothelial Carcinoma Cell Lines. Vet. Immunol. Immunopathol. 2022;243:110367. doi: 10.1016/j.vetimm.2021.110367. PubMed DOI

Hartley G., Faulhaber E., Caldwell A., Coy J., Kurihara J., Guth A., Regan D., Dow S. Immune Regulation of Canine Tumour and Macrophage PD-L1 Expression. Vet. Comp. Oncol. 2017;15:534–549. doi: 10.1111/vco.12197. PubMed DOI

Nagaya T., Okuyama S., Ogata F., Maruoka Y., Knapp D.W., Karagiannis S.N., Fazekas-Singer J., Choyke P.L., LeBlanc A.K., Jensen-Jarolim E., et al. Near Infrared Photoimmunotherapy Targeting Bladder Cancer with a Canine Anti-Epidermal Growth Factor Receptor (EGFR) Antibody. Oncotarget. 2018;9:19026–19038. doi: 10.18632/oncotarget.24876. PubMed DOI PMC

Li C.-W., Lim S.-O., Xia W., Lee H.-H., Chan L.-C., Kuo C.-W., Khoo K.-H., Chang S.-S., Cha J.-H., Kim T., et al. Glycosylation and Stabilization of Programmed Death Ligand-1 Suppresses T-Cell Activity. Nat. Commun. 2016;7:12632. doi: 10.1038/ncomms12632. PubMed DOI PMC

Li S.-M., Zhou J., Wang Y., Nie R.-C., Chen J.-W., Xie D. Recent Findings in the Posttranslational Modifications of PD-L1. J. Oncol. 2020;2020:5497015. doi: 10.1155/2020/5497015. PubMed DOI PMC

Chen D.S., Mellman I. Oncology Meets Immunology: The Cancer-Immunity Cycle. Immunity. 2013;39:1–10. doi: 10.1016/j.immuni.2013.07.012. PubMed DOI

Chen D.S., Mellman I. Elements of Cancer Immunity and the Cancer–Immune Set Point. Nature. 2017;541:321–330. doi: 10.1038/nature21349. PubMed DOI

Von Rueden S.K., Fan T.M. Cancer-Immunity Cycle and Therapeutic Interventions—Opportunities for Including Pet Dogs With Cancer. Front. Oncol. 2021;11:4853. doi: 10.3389/fonc.2021.773420. PubMed DOI PMC

Haake A.F.H., Langenhagen A.K., Jovanovic V.M., Andreotti S., Gruber A.D. ‘Hot Versus Cold’—Can Transcriptome Analysis of Canine Perianal Tumours Help Illustrate Their Distinct Immunophenotypic Landscapes? J. Comp. Pathol. 2022;191:14. doi: 10.1016/j.jcpa.2021.11.032. DOI

Marconato L., Frayssinet P., Rouquet N., Comazzi S., Leone V.F., Laganga P., Rossi F., Vignoli M., Pezzoli L., Aresu L. Randomized, Placebo-Controlled, Double-Blinded Chemoimmunotherapy Clinical Trial in a Pet Dog Model of Diffuse Large B-Cell Lymphoma. Clin. Cancer Res. 2014;20:668–677. doi: 10.1158/1078-0432.CCR-13-2283. PubMed DOI

Riccardo F., Tarone L., Camerino M., Giacobino D., Iussich S., Barutello G., Arigoni M., Conti L., Bolli E., Quaglino E., et al. Antigen Mimicry as an Effective Strategy to Induce CSPG4-Targeted Immunity in Dogs with Oral Melanoma: A Veterinary Trial. J. Immunother. Cancer. 2022;10:e004007. doi: 10.1136/jitc-2021-004007. PubMed DOI PMC

Alonso-Miguel D., Valdivia G., Guerrera D., Perez-Alenza M.D., Pantelyushin S., Alonso-Diez A., Beiss V., Fiering S., Steinmetz N.F., Suarez-Redondo M., et al. Neoadjuvant in Situ Vaccination with Cowpea Mosaic Virus as a Novel Therapy against Canine Inflammatory Mammary Cancer. J. Immunother. Cancer. 2022;10:e004044. doi: 10.1136/jitc-2021-004044. PubMed DOI PMC

Cascio M.J., Whitley E.M., Sahay B., Cortes-Hinojosa G., Chang L.-J., Cowart J., Salute M., Sayour E., Dark M., Sandoval Z., et al. Canine Osteosarcoma Checkpoint Expression Correlates with Metastasis and T-Cell Infiltrate. Vet. Immunol. Immunopathol. 2021;232:110169. doi: 10.1016/j.vetimm.2020.110169. PubMed DOI

Shosu R. Programmed Cell Death Ligand 1 Expression in Canine Cancer. In Vivo. 2016;10:195–204. PubMed

Doroshow D.B., Bhalla S., Beasley M.B., Sholl L.M., Kerr K.M., Gnjatic S., Wistuba I.I., Rimm D.L., Tsao M.S., Hirsch F.R. PD-L1 as a Biomarker of Response to Immune-Checkpoint Inhibitors. Nat. Rev. Clin. Oncol. 2021;18:345–362. doi: 10.1038/s41571-021-00473-5. PubMed DOI

Li H., Kuang X., Liang L., Ye Y., Zhang Y., Li J., Ma F., Tao J., Lei G., Zhao S., et al. The Beneficial Role of Sunitinib in Tumor Immune Surveillance by Regulating Tumor PD-L1. Adv. Sci. (Weinh.) 2021;8:2001596. doi: 10.1002/advs.202001596. PubMed DOI PMC

Comparative characterization of two monoclonal antibodies targeting canine PD-1