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

Abstract

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.

PD-L1 mRNA and protein expression in canine mammary carcinomas: Correlation with histopathological grade and molecular markers

“…2,23 In our study, PD-L1 expression was positively correlated with CK5/6 expression and was not utilized from a PD-L1 study in human breast cancer, applying the criteria to dogs may require modification due to interspecies differences. To overcome these limitations, we modified the evaluation method to consider cases as PD-L1 positive when at least 1% of the tumor cells were immunolabeled, according to the study by Lopes-Neto et al 20 We selected a polyclonal antibody used by Ariyarathna et al, 3 which showed a correlation between PD-L1 IHC expression and the histopathological grade of CMCs. The lack of correlation between PD-L1 IHC expression and histopathological grade in this study, in contrast to a previous study that used the same antibody, may be attributed to the different evaluation methods employed.…”

“…Different studies have employed diverse evaluation methods, including varying scales for labeling intensity and cutoff values for determining PD-L1 positivity. 3,20 In previous studies on CMCs, Ariyarathna et al 3 quantified PD-L1 IHC expression by considering the labeling intensity using a scale ranging from 0 to 3, and Lopes-Neto et al 20 evaluated PD-L1 IHC positive or negative cells when 1% of the tumor cells showed labeling.…”

“…PD-L1 IHC expression was considered positive when more than 1% of the tumor cells exhibited labeling on the membrane and/or cytoplasm based on the study by Lopes-Neto et al 20 The IHC results for ER, ERBB2, and CK5/6 were assessed following the method described by Sassi et al 26 ER expression was considered positive when nuclear staining was observed in at least 5% of tumor cells. ERBB2 expression was considered positive when at least 10% of the tumor cells showed complete membranous labeling, and cytoplasmic CK5/6 labeling in at least 1% of the tumor cells was considered positive.…”

“…35 Recently, studies on PD-L1 expression in CMCs have been conducted. 3,20,21 Most CMCs show PD-L1 protein expression in tumor cells, 21 and high PD-L1 protein expression correlates with poor prognosis. 3,20 However, PD-L1 mRNA expression in CMCs has not been investigated.…”

Association of mast cell density, microvascular density and endothelial area with clinicopathological parameters and prognosis in canine mammary gland carcinomas

“…The results in CMTs are scarce and contradict those of BC. In one study, CMTs with low TILs a had significantly higher OS than neoplasm with high TILs count [ 48 ], while in another study tumours with intratumoural low TILs count also had significantly higher OS, while peripheral TILs count did not have an association with OS [ 49 ]. The cause for the difference in BC and CMTs results could be the much bigger sample sizes in studies done with BC with a better analyzation of the results by meta-analysis.…”