Relationship between PD-L1 Expression and Tumor-Infiltrating Lymphocytes in Canine Mammary Tumor

Belarmino Eugênio Lopes-Neto, Diana Célia Sousa Nunes Pinheiro, Júlio Gil Vale Carvalheira, Fernando Schmitt, Maria de Fátima Gärtner


Background: Studies pointed out that the tumor-infiltrating lymphocytes (TILs) have considerable importance in canine mammary tumor (CMT). On the other hand, cancer cells sometimes find ways to use immune checkpoint proteins as a shield to avoid being identified and attacked by the immune system as programmed death 1 ligand 1 (PD-L1). In this study, it was investigated the relationship between PD-L1 expression, stromal tumor-infiltrating lymphocytes (TILs) in canine mammary tumor (CMT), and the association with clinical and pathological characteristics of the tumors.

Materials, Methods & Results: PD-L1 expression and TILs were assessed in 23 female dogs with CMT. The tumors were grouped into simple carcinoma (CA, n = 8) and complex carcinoma (CC, n = 15). Stromal TILs were assessed using two thresholds as TILs-Low representing < 50% of infiltrate within stromal area and TILs-High representing ≥ 50% of stromal area. Clinicopathological data of CMT was characterized according to key parameters, as well as survival rates. TILs evaluation within tumor stroma revealed that 65.2% (n = 15) of tumors had TILs-Low. PD-L1 expression and stromal TILs were significantly associated (P = 0.009). PD-L1 expression was observed in 39% (n = 9) of all tumors of which 17.4% (n = 4) were from CA group and 21.7% (n = 5) were from CC group. PD-L1 expression within TILs was observed in 39% (n = 9) of the tumors. PD-L1 in malignant epithelium was present in all lymph node metastasis (n = 5). PD-L1 was associated with involvement of regional lymph nodes (P = 0.034). Survival curves demonstrated TILs-Low had higher (P = 0.010) overall survival (OS) compared with TILs-High, and PD-L1+ and PD-L1 (P = 0.06) did not differed. The clinicopathological variables significantly correlated with OS by univariate analysis were the histological grade (P = 0.009), lymph node involvement (P = 0.004), stromal TILs (P = 0.016), and PD-L1+/TILs-High vs. PD-L1/TILs-Low (P = 0.010). Multivariate analysis revealed that group of tumors with grade II-III was independent and negative prognostic factors for OS.

Discussion: In this study, PD-L1 was differently expressed according to the histologic subtypes of TMC. Currently, has been showed the presence of PD-L1 in several canine cancer. Nevertheless, only a few studies have described PD-L1 protein expression in dog tumors and showed PD-L1 was constitutively expressed on canine tumor cell lines, although the levels of basal expression were very variable. This expression can be modulated by IFN-γ exposure. In the present study, it was found a strong PD-L1 expression on TILs. The increase in PD-L1 cell surface expression by tumor cells can lead to decreased T-cell proliferation and increased apoptosis. In human breast cancer (BC) the PD-L1 expression was expressed in TILs and tumor epithelium. It has been reported the association of stromal TILs and PD-L1 expression with aggressive types and stages of BC. In this study, it was detected PD-L1 expression in malignant epithelium in all lymph node metastasis. PD-L1 overexpression was significantly associated with a series of clinicopathological parameters. It was demonstrated that PD-L1+/TILs-High had higher risk of overall survival (OS) than another group of interaction. High PD-L1 expression may be a prognostic indicator for reduced OS, while tumor PD-L1+ was associated with poorer disease-free survival. The presence of TILs has shown to be potentially predictive and a prognostic factor in BC subtypes. In CMT, it has been reported that a high proportion of TILs was correlated to several malignancy characteristics. In relation to PD-L1, further research is necessary to clarify this immune checkpoint as a potential therapeutic target and its application in clinical practice in CMT.

Full Text:



Becht E., Giraldo N.A., Dieu-Nosjean M.C., Sautès-Fridman C. & Fridman W.H. 2016. Cancer immune contexture and immunotherapy. Current Opinion in Immunology. 39: 7-13.

Bense R.D., Sotiriou C., Piccart-Gebhart M.J., Haanen J.B., van Vugt M.A., de Vries E.G., Schröder C.P. & Fehrmann R.S.N. 2017. Relevance of tumor-infiltrating immune cell composition and functionality for disease outcome in breast cancer. Journal of the National Cancer Institute. 109: 1-9.

Carvalho M.I., Pires I., Prada J., Gregório H., Lobo L. & Queiroga F.L. 2016. Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors. Veterinary Immunology and Immunopathology. 178: 1-9.

Carvalho M.I., Pires I., Prada J. & Queiroga F.L. 2014. A role for T-lymphocytes in human breast cancer and in canine mammary tumors. BioMed Research International. 2014: 1-11.

Chen J., Jiang C.C., Jin L. & Zhang X.D. 2016. Regulation of PD-L1: A novel role of pro-survival signalling in cancer. Annals of Oncology. 27: 409-416.

Chiku V.M., Silva K.L.O., Almeida B.F.M., Venturin G.L., Leal A.A.C., Martini C.C., Rezende E.F., Santos P.S. & Lima V.M. 2016. PD-1 function in apoptosis of T lymphocytes in canine visceral leishmaniasis. Immunobiology. 221: 879-888.

Coy J., Caldwell A., Chow L., Guth A. & Dow S. 2017. PD-1 expression by canine T cells and functional effects of PD-1 blockade. Veterinary Comparative Oncology. 15(4): 1487-1502.

de Araújo M.R., Campos L.C., Ferreira E. & Cassali G.D. 2015. Quantitation of the regional lymph node metastatic burden and prognosis in malignant mammary tumors of dogs. Journal of Veterinary Internal Medicine. 29:1360-1367.

Elston C.W. & Ellis I.O. 1991. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 19: 403-410.

Emran A.A., Chatterjee A., Rodger E.J., Tiffen J.C., Gallagher S.J., Eccles M.R., & Hersey P. 2019. Targeting DNA Methylation and EZH2 activity to overcome melanoma resistance to immunotherapy. Trends in Immunology. 40(4): 328-344.

Gama A., Paredes J., Gärtner F., Alves A. & Schmitt F. 2008. Expression of E-cadherin, P-cadherin and beta-catenin in canine malignant mammary tumours in relation to clinicopathological parameters, proliferation and survival. The Veterinary Journal. 177: 45-53.

Garcia-Teijido P., Cabal M.L., Fernandez I.P. & Perez Y.F. 2016. Tumor-infiltrating lymphocytes in triple negative breast cancer: The future of immune targeting. Clinical Medicine Insights Oncology. 10: 31-39.

Goldschmidt M., Peña L., Rasotto R. & Zappulli V. 2011. Classification and grading of canine mammary tumors. Veterinary Pathology. 48: 117-131.

Hanahan D. & Weinberg R.A. 2011. Hallmarks of cancer: the next generation. Cell. 144: 646-74.

Hartley G., Faulhaber E., Caldwell A., Coy J., Kurihara J., Guth A., Regan D. & Dow S. 2017. Immune regulation of canine tumour and macrophage PD-L1 expression. Veterinary Comparative Oncology. 534-549.

Ikebuchi R., Konnai S., Okagawa T., Yokoyama K., Nakajima C., Suzuki Y., Murata S. & Ohashi K. 2014. Influence of PD-L1 cross-linking on cell death in PD-L1-expressing cell lines and bovine lymphocytes. Immunology. 142: 551-561.

Ishii K., Shimizu M., Kogo H., Negishi Y., Tamura H., Morita R. & Takahashi H. 2020. A combination of check-point blockade and α-galactosylceramide elicits long-lasting suppressive effects on murine hepatoma cell growth in vivo. Immunobiology. 225: 151860.

Jelinic P., Ricca J., Oudenhove E.V., Olvera N., Merghoub T., Levine D.A. & Zamarin D. 2018. Immune-active microenvironment in small cell carcinoma of the ovary, hypercalcemic type: rationale for immune checkpoint blockade. Journal of the National Cancer Institute. 110(7): 787-790.

Jiang D., Gao Z., Cai Z., Wang M. & He J. 2015. Clinicopathological and prognostic significance of FOXP3+ tumor infiltrating lymphocytes in patients with breast cancer: a meta-analysis. BMC Cancer. 5: 727.

Johnson D.B., Sullivan R.J. & Menzies A.M. 2017. Immune checkpoint inhibitors in challenging populations. Cancer. 123: 1904-11.

Karayannopoulou M., Anagnostou T., Margariti A., Kostakis C., Kritsepi-Konstantinou M., Psalla D. & Savvas I. 2017. Evaluation of blood T-lymphocyte subpopulations involved in host cellular immunity in dogs with mammary cancer. Veterinay Immunology and Immunopathology. 186: 45-50.

Kennedy L.B., & Salama A.K.S. 2020. A review of cancer immunotherapy toxicity. CA: A Cancer Journal for Clinicians. 70: 86-104.

Kim C, Kim EK, Jung H, Chon HJ, Han JW, Shin K-H, Shin K.H., Hu H., Kim K.S., Choi Y.D., Kim S., Lee Y.H., Suh J.S., Ahn J.B., Chung H.C., Noh S.H., Rha S.Y., Kim S.H. & Kim H.S. 2016. Prognostic implications of PD-L1 expression in patients with soft tissue sarcoma. BMC Cancer.16: 434.

Kim J.-H., Chon S.-K., Im K.-S., Kim N.-H. & Sur J.-H. 2013. Correlation of tumor-infiltrating lymphocytes to histopathological features and molecular phenotypes in canine mammary carcinoma : a morphologic and immunohistochemical morphometric study. Canadian Journal of Veterinary Research. 77: 142-9.

Kumaraguruparan R., Prathiba D. & Nagini S. 2006. Of humans and canines: Immunohistochemical analysis of PCNA, Bcl-2, p53, cytokeratin and ER in mammarytumours. Research in Veterinay Science. 81: 218-24.

Law A.M.K., Lim E., Ormandy C.J. & Gallego-Ortega D. 2017. The innate and adaptive infiltrating immune systems as targets for breast cancer immunotherapy. Endocrine-Related Cancer. 24: 123-44.

Liu D., Xiong H., Ellis A.E., Northrup N.C., Rodriguez C.O., O’Regan R.M., Dalton S. & Zhao S. 2014. Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer. Cancer Research. 74: 5045-5056.

Lopes Neto B.E., Souza S.C.B., Bouty L.M., Santos G.J.L, Oliveira E.S., Freitas J.C.C. & Nunes Pinheiro D.C.S. 2017. CD4+, CD8+, FoxP3+ and HSP60+ expressions in cellular infiltrate of canine mammary carcinoma in mixed tumor. Acta Scientiae Veterinariae. 45: 1-8.

Maekawa N., Konnai S., Ikebuchi R., Okagawa T., Adachi M., Takagi S., Kagawa Y., Nakajima C., Suzuki Y., Murata S. & Ohashi K. 2014. Expression of PD-L1 on canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade. PLoS One. 9: 1-14.

Maekawa N., Konnai S., Okagawa T., Nishimori A., Ikebuchi R., Izumi Y., Takagi S., Kagawa Y., Nakajima C., Suzuki Y., Kato Y., Murata S. & Ohashi K. 2016. Immunohistochemical analysis of PD-L1 expression in canine malignant cancers and PD-1 expression on lymphocytes in canine oral melanoma. PLoS One. 11: 1-13.

Maekawa N., Konnai S., Takagi S., Kagawa Y., Okagawa T., Nishimori A., Ikebuchi R., Izumi Y., Deguchi T., Nakajima C., Kato Y., Yamamoto K., Uemura H., Suzuki Y., Murata S. & Ohashi K. 2017. A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma. Science Reports. 7: 8951.

Maleki V. S, Garrigós C. & Duran I. 2017. Biomarkers of response to PD-1/PD-L1 inhibition. Critical Review Oncology Hematology. 116: 116-24.

Marisa L., Svrcek M., Collura A., Becht E., Cervera P., Wanherdrick K., Buhard O., Goloudina A., Jonchère V., Selves J., Milano G., Guenot D., Cohen R., Colas C., Laurent-Puig P., Olschwang S., Lefèvre J.H., Parc Y., Ghiringhelli F., de Reynies A. & Duval A. 2018. The balance between cytotoxic T-cell lymphocytes and immune checkpoint expression in the prognosis of colon tumors. Journal of the National Cancer Institute. 110(1): 68-77.

Meuten D.J., Moore F.M. & George J.W. 2016. Mitotic count and the field of view area. Veterinary Pathology. 53: 7-9.

Mittendorf E.A., Philips A.V., Meric-Bernstam F., Qiao N., Wu Y., Harrington S., Su X., Wang Y., Gonzalez-Angulo A.M.,. Akcakanat A., Chawla A., Curran M., Hwu P., Sharma P., Litton J.K., Molldrem J.J. & Alatrash G. 2014. PD-L1 Expression in triple-negative breast cancer. Cancer Immunology Research. 2: 361-70.

Mori H., Kubo M., Yamaguchi R., Nishimura R., Osako T., Arima N., Okumura Y., Okido M., Yamada M., Kai M., Kishimoto J., Oda Y. & Nakamura M. 2017. The combination of PD-L1 expression and decreased tumor-infiltrating lymphocytes is associated with a poor prognosis in triple-negative breast cancer. Oncotarget. 8:15584-92.

Muenst S., Schaerli A.R., Gao F., Däster S., Trella E., Droeser R.A., Muraro M.G., Zajac P., Zanetti R., Gillanders W.E., Weber W.P. & Soysal S.D. 2014. Expression of programmed death ligand 1 (PD-L1) is associated with poor prognosis in human breast cancer. Breast Cancer Research and Treatment. 146: 15-24.

Okazaki T. & Honjo T. 2006. The PD-1_PD-L pathway in immunological tolerance. Trends in Immunology. 27(4): 195-201.

Owen L. 1980. TNM Classification of tumours in domestic animals. World Health Organ. 1-52.

Peña L., Gama A., Goldschmidt M.H., Abadie J., Benazzi C., Castagnaro M., Díez L., Gärtner F., Hellmén E., Kiupel M., Millán Y., Miller M.A., Nguyen F., Poli A., Sarli G., Zappulli V. & de las Mulas J.M. 2014. Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry. Veterinay Pathology. 51: 127-45.

Polonia A., Pinto R., Cameselle-Teijeiro J.F., Schmitt F.C. & Paredes J. 2017. Prognostic value of stromal tumour infiltrating lymphocytes and programmed cell death-ligand 1 expression in breast cancer. Journal of Clinical Pathology. 70: 860-7.

Rasotto R., Berlato D., Goldschmidt M.H. & Zappulli V. 2017. Prognostic significance of canine mammary tumor histologic subtypes: an observational cohort study of 229 cases. Veterinary Pathology. 54: 571-8.

Salgado R., Denkert C., Demaria S., Sirtaine N., Pruneri G., Wienert S., Van den Eynden G., Baehner F.L., Penault-Llorca F., Perez E.A., Thompson E.A., Symmans W.F., Richardson A.L., Brock J., Criscitiello C., Bailey H., Ignatiadis M., Floris G., Sparano J., Kos Z., Nielsen T., Rimm D.L., Allison K.H., Reis-Filho J.S., Loibl S., Sotiriou C., Viale G., Badve S., Adams S., Willard-Gallo K. & Loi S. - International TILs Working Group 2014. 2015. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Annals of Oncology. 26: 259-71.

Siegel R.L., Miller K.D. & Jemal A. 2017. Cancer statistics, 2017. CA: A Cancer Journal of Clinicians. 67: 7-30.

Sorenmo K.U., Rasotto R., Zappulli V. & Goldschmidt M.H. 2011. Development, anatomy, histology, lymphatic drainage, clinical features, and cell differentiation markers of canine mammary gland neoplasms. Veterinary Pathology. 48: 85-97.

Timmermans-Sprang E.P.M., Gracanin A. & Mol J.A. 2017. Molecular signaling of progesterone, growth hormone, Wnt, and HER in mammary glands of dogs, rodents, and humans: new treatment target identification. Frontier in Veterinary Science. 4: 1-13.


Copyright (c) 2021 Belarmino Eugênio Lopes-Neto, Diana Célia Sousa Nunes-Pinheiro, Júlio Gil Vale Carvalheira, Fernando Schmitt, Maria de Fátima Gärtner

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.