Abstract
Background/Aim: IL-35 has a prominent immunosuppressive role and its overexpression has been reported in human breast cancer. However, the impact of IL-35 in canine mammary carcinogenesis has not been addressed yet. The present study determined the clinicopathological significance of IL-35 immunoexpression and its correlation with overall survival (OS) in 72 malignant canine mammary tumor (CMT) patients. Materials and Methods: Formalin-fixed paraffin-embedded malignant CMT samples (n=72) were submitted to immunohistochemical staining to detect IL-35 expression. Survival curves were obtained by the Kaplan–Meier method and the log-rank test was used for the survival estimates. Cox proportional hazard model for multivariate analysis was also performed. Results: IL-35 overexpression was associated with: skin ulceration, tumor necrosis, mitotic index, nuclear pleomorphism, tumor differentiation, histological grade of malignancy (HGM), neoplastic intravascular emboli and lymph node metastasis. Additionally, IL-35 was also correlated with a worse overall survival in multivariate analysis, arising as an independent predictor of poor prognosis. Conclusion: IL-35 is associated with carcinogenesis and worse prognosis of CMT.
Interleukin (IL)-35 is a member of the IL-12 family of cytokines. This heterodimeric cytokine is composed of the IL-12p35 subunit and the Epstein- Barr virus-induced gene 3 (Ebi3) subunit and is preferentially secreted by mouse and human regulatory T cells (Treg cells) (1-3). IL-35 is well documented as an anti-inflammatory cytokine implicated in the regulation of autoimmune diseases (4-6). Previous studies revealed that IL-35, produced by Treg cells, suppresses the function of Th1, Th17 and Th2 cells (7). More recently, reports suggested the pivotal role of IL-35 in the pathogenesis of tumor development, malignant progression and prognosis, which has attracted even more the scientific community's attention (1, 2, 8-10).
Recent mouse experiments have shown that IL-35 produced by Treg cells and cancer cells promoted tumor growth via enhancing myeloid cell accumulation and angiogenesis, and reducing the infiltration of activated CD8+ T cells into the tumor microenvironment (11). In general, IL-35 expression is considered to be implicated in immunosuppression, and tumor progression and also associated with a poor prognosis (1, 2). In the tumor microenvironment, IL-35 induces the conversion of proliferative FoxP3− conventional T cells into a hyporesponsive, strongly suppressive IL-35-producing CD4+FoxP3− induced regulatory T cell population (iTr35 cells), which has been found to potently inhibit antitumor T cell responses (3, 7, 10). This iTr35 cell population, which mediates immune suppression via IL-35 was strongly suppressive and stable in vivo. Furthermore, it did not express FoxP3 and did not require the other key suppressive cytokines (IL-10 or transforming growth factor-β, TGF-β) for conversion, while it was distinct from the known induced regulatory populations of TGF-β–iTr cells and IL-10–iTr cells which require longer conversion protocols, multiple cell types and/or additional molecules for optimal generation (3, 10). In summary, IL-35 is a Treg cell–specific cytokine that is required for the maximum regulatory activity of human and mouse Treg cells, under both in vitro and in vivo conditions and iTr35 cells have a highly restricted genetic signature that results in a CD4+FoxP3−Ebi3+p35+IL-10−TGF-β− phenotype (7, 12).
Immunohistochemical analysis revealed that IL-35 is highly expressed in several human malignancies as: pancreas cancer, nasopharyngeal carcinoma, colorectal cancer, esophageal carcinoma, hepatocellular carcinoma, and cervical carcinoma (1, 2, 13, 14).
In human breast cancer patients, a significantly increased expression of IL-35 was observed (10, 15). Furthermore, Ki-67, p53 and epidermal growth factor receptor (EGFR) expression on breast cancer tissues increased when circulating IL-23:IL-35 ratio decreased. These findings suggest that IL-35 seems to be an important indicator of breast cancer progression and prognosis (9, 15).
While these findings in human tumors are important, the impact of IL-35 on canine mammary tumorigenesis, cancer development and clinical outcome, has not been addressed yet. In the present study, in order to clarify this question, we investigated the intratumoral IL-35 expression in malignant canine mammary tumor (CMT) by immunohistochemistry, and correlated the findings with several clinicopathological parameters and patient's overall survival.
Materials and Methods
Patient selection and tissue sample collection. A total of 72 female dogs were included in this study. The sample collection was composed of tumors excised by surgery from patients with malignant mammary cancer. None of the patients had received radiotherapy or chemotherapy.The clinical stages of animals were categorized into: local (without lymph node involvement), regional (metastasis at regional lymph nodes) and distant (presence of distant metastases) (16, 17) by the modified pathology tumor-node-metastasis (TNM) system (18). Tumor size (T1 <3 cm; T2 ≥3 and <5 cm; T3 ≥5 cm) and skin ulceration were evaluated for each tumor sample.
Histopathological examination. Tissue sections (4-μm) were prepared from paraffin wax tissue blocks and then subjected to haematoxylin and eosin (HE) following routine methods. Each slide was assessed by two pathologists to perform the pathological classification in accordance with the criteria proposed by the World Health Organization (WHO) for CMT (19). Tumors were also graded [mitotic index, nuclear grade, differentiation grade, histological grade of malignancy (HGM)] by the Goldschmidt and collaborators method (20). Additional clinicopathological characteristics evaluated included the presence of tumor necrosis, presence of neoplastic intravascular emboli and regional lymph node involvement.
Immunohistochemistry. Paraffin-embedded sections were deparaffinized and rehydrated in graded concentrations of alcohol. The IL-35 immunostaining was performed with the streptavidin-biotin-peroxidase complex method using the Ultra Vision Detection System kit (Lab Vision Corporation, Fremont, CA, USA), following the manufacturer's instructions. The sections were submerged in 0.01 M, pH=6.0, citrate antigenic retrieval buffer and subjected to a pressure cooker. Then, slides were incubated with the anti-IL-35 antibody (EBI3, sc-32868, Santa Cruz Biotechnology, Dallas, Texas, USA; diluted to 1:200) at 4°C overnight. The antibody reaction products were observed with the chromogen 3, 3’-diaminobenzidine tetrachloride (DAB) at 0.05% with 0.01% H2O2 (30%). Finally, sections were counterstained with Gill's haematoxylin and viewed under a light microscope. A negative control was obtained by replacing the primary antibody with an irrelevant isotype-matched antibody. Canine lymph node and thymus sections were used as positive control.
IL-35 staining evaluation. IL-35 immunoexpression was evaluated by two observers who were blinded to the clinicopathological characteristics of the patients. The staining evaluation was based on a semiquantitative method, adapted from previous published studies (1, 2). This method involved scoring according to the percentage of positive cancer cells (immunolabelling extension) and staining intensity. The percentage of positivity was scored as 0 (0-25% positive cells), 1 (26-50% positive cells), 2 (51-75% positive cells), 3 (>75% positive cells). The staining intensity was scored as 0 (no staining), 1 (weakly stained), 2 (moderately stained), and 3 (strongly stained). A final score was obtained by multiplying staining intensity and the percentage of positive cells. A final immunostaining score ≤2 indicated a low IL-35 class, whereas a final immunohistochemical score >2 was considered as a high IL-35 class.
Follow-up study. Patients whose cause of death was unknown were excluded from the study. Informed consent on the collection of samples and the clinical follow-up was obtained from each patient's owner. The clinical follow-up of the female dogs included in this study comprised an examination 15 days after surgery and every 90 days thereafter for a maximum period of 730 days. The clinical follow-up examination of the patients included a physical checkup, a radiological evaluation of the thorax and an abdominal ultrasound scan. For the animals that died within the 730 days period, the overall survival (OS) time was determined from the date of surgery to the date of animal death/euthanasia in consequence to the advanced stage of the disease. For the dogs that survived for more than 730 days the OS time was calculated from the date of surgery to the last clinical examination.
Statistical analyses. All statistical analyses were carried out using the SPSS software (Statistical Package for the Social Sciences, Chicago, IL, USA) version 19.0. The chi-square test was performed to analyze the association between the clinicopathological characteristics and IL-35 immunoexpression. Survival curves were obtained with the Kaplan–Meier method and the log-rank test was used for the survival estimates. Cox proportional hazard model for multivariate analysis was also performed. In all cases, p<0.05 denoted the presence of a statistically significant difference.
Results
Clinicopathological data. According to the WHO criteria, the 72 tumors included in this study were histologically classified as: tubulopapillary carcinomas (n=37; 51.4%), solid carcinomas (n=5; 6.9%), complex carcinomas (n=14; 19.4%), benign tumors (n=2; 2.8%), anaplastic carcinomas (n=2; 2.8%) and carcinosarcomas (n=12; 16.7%). Based on the modified pathology TNM staging system, 44 patients (61.1%) were in local stage, 28 patients (38.9%) were in regional stage and there were no patients in distant stage. All female dogs were free from distant metastasis at the time of diagnosis (confirmed throughout the realization of thorax X-ray and abdominal ultrasound). The HGM was classified as I (n=18; 25%), II (n=17; 23.6%), or III (n=37; 51.4%) and twenty-one cases (29.2%) demonstrated the presence of neoplastic intravascular emboli.
Immunoreactivity for IL-35 in (A) solid carcinoma, note the predominant diffuse staining in the epithelial cells, bar=200 μm; (B) carcinosarcoma, note the intense positivity in the mesenchymal component, bar=100 μm; (C) complex carcinoma, note that the myoepithelial area was negative or revealed a weak focal positivity, bar=500 μm; (D) neoplastic intravascular emboli, note that the labelling intensity of IL-35 in the primary tumor and neoplastic intravascular emboli was similar, bar=100 μm.
Expression of IL35 in malignant CMT. The IL-35 immunoexpression appeared as a brown color present in the cytoplasm of the neoplastic cells in a diffuse or granular pattern. The lymphocytes present in the tumor area revealed a weak staining, however, only tumor cells were considered in the immunoreactivity evaluation method. The staining intensity of the adnexal normal mammary gland was always weaker than that of the tumor. Prominent and diffuse IL-35 immunostaining was observed in the epithelial cells (Figure 1A). In tumors diagnosed as carcinosarcomas, in addition to the epithelial staining, intense positivity in the mesenchymal component was observed (Figure 1B). In complex carcinomas the myoepithelial area was negative or revealed a weak focal positivity (Figure 1C). The carcinomas in benign tumors were negative in the benign tumor section comprising bone or cartilage. The labelling intensity of IL-35 in the primary tumor and in the neoplastic intravascular emboli was similar (Figure 1D). Regarding IL-35 immunolabelling extension, briefly, 9 (12.5%) cases showed extension 0 (0-25% positive cells), 23 (31.9%) cases showed extension 1 (26-50% positive cells) and 20 cases (27.8%) were found both in extension 2 (51-75% positive cells) and 3 (>75%). For IL-35 labelling intensity, there was a relatively homogeneous distribution between the three classes: weak (n=19; 26.4%), moderate (n=25; 34.7%) and strong intensity (n=28; 38.9%).
Association of IL35 immunostaining with clinicopathological variables. To evaluate the clinical significance of IL-35 expression in malignant CMT, its association with several clinicopathological variables was studied. Our analysis identified an association between the presence of aggressive disease and high IL-35 immunoexpression. As summarized in Table I, tumors with higher levels of IL-35 were associated with skin ulceration (p=0.042), tumor histological type (p<0.001), tumor necrosis (p<0.001), high mitotic index (p<0.001), marked nuclear pleomorphism (p=0.001), poor tumor differentiation (p<0.001), high HGM (p<0.001), presence of neoplastic intravascular emboli (p<0.001) and presence of lymph node metastasis (p<0.001).
Relationship between IL-35 expression and clinicopathological parameters in malignant CMT.
Kaplan–Meier OS curves comparing IL-35 class in 72 dogs with malignant mammary tumors.
Follow-up study. The high intratumoral IL-35 expression was associated with poor clinical outcome (p<0.001 in Kaplan–Meier curves; Figure 2). Animals with high intratumoral IL-35 immunostaining demonstrated a lower OS time (n=42; 390,652±36,271 days) than animals who presented low intratumoral IL-35 expression (n=30; 686,019±27,572 days). The clinicopathological parameters related to shorter OS, included: tumor size (p=0.034); skin ulceration (p=0.033); nuclear grade (p<0.001); differentiation grade (p<0.001); mitotic index (p<0.001); HGM (p<0.001); tumor necrosis (p=0.001); lymph node metastasis (p<0.001) and neoplastic intravascular emboli (p<0.001). The multivariate analysis performed with the Cox proportional hazard model demonstrated that high mitotic index and high intratumoral IL-35 expression were independent predictors of OS (p<0.001 and p=0.029, respectively). More information is provided in Table II.
Discussion
IL-35 is well documented as an anti-inflammatory cytokine implicated in the regulation of autoimmune diseases (4-6). However, IL-35 production in the tumor microenvironment may contribute to tumor progression and tumor immune surveillance. Recently, IL-35 overexpression has been described to be involved in the development, progression and poor prognosis of several human tumors, including breast cancer (1, 2, 9, 10, 13, 15).
In human breast cancer patients a significantly increased expression of IL-35 (10, 15) was observed and findings suggested that IL-35 seems to be an important indicator of breast cancer progression (9, 15). In CMT, to the best of our knowledge, this is the first study to investigate the role of IL-35 in canine mammary tumorigenesis, cancer development and clinical outcome.
Relationship of clinicopathological variables and IL-35 expression with overall survival.
In the present study, we observed that IL-35 staining was localized in the cytoplasm of cancer cells. Moreover, IL-35 was highly expressed in cancer cells compared to cells in the adnexal non-tumoral mammary gland, indicating that this interleukin might be involved in the pathogenesis of mammary cancer. Immunohistochemistry demonstrated that higher expression of IL-35 was significantly associated with more aggressive tumor phenotypes: presence of skin ulceration, presence of tumor necrosis, high mitotic index, marked nuclear pleomorphism, poor tumor differentiation, high HGM, presence of neoplastic intravascular emboli and presence of lymph node metastasis.
Our results showed that, in CMT, IL-35 overexpression was significantly associated with advanced tumor stage and these findings were in agreement with several reports in human malignancies. In human lung cancer cells, siRNA silencing of Ebi3 inhibited cancer cell proliferation, whereas stable expression of Ebi3 in lung cancer cells confer a growth promoting activity in vitro (21). In human colorectal cancer and in nasopharyngeal carcinoma IL-35 levels were highly correlated to the severity of malignancy and the clinical stage of tumors (1, 2).
In human breast cancer an increased expression of IL-35 was also described (15). Interestingly Ki-67, p53 and EGFR expression increased when the circulating IL-23:IL-35 ratio decreased, suggesting that IL-35 seems to be an important indicator of cancer progression (9, 10).
To better elucidate the clinical significance of IL-35 in CMT, we further studied the correlation of IL-35 with clinicopathological parameters and the OS of patients with mammary tumors. Interestingly, as described in studies in human cancers (1, 2, 11), IL-35 overexpression was correlated with a shorter OS of animals. Moreover, in our study, multivariate analysis demonstrated that high mitotic index and high intratumoral IL-35 expression were independent predictors of survival.
IL-35 is a Treg cell-secreted cytokine that inhibits T cell proliferation and function (7). Treg cells suppress other immune effector cells by numerous mechanisms, one of which being the secretion of inhibitory cytokines (3, 22, 23). In the tumor microenvironment, IL-35 induces the conversion of conventional T cells into a suppressive IL-35-producing CD4+Foxp3− induced regulatory T cell population (iTr35 cells) which can contribute to the enhancement of tumor growth (3, 6, 7). Additionally, Treg cell-derived IL-35 promoted the expression of multiple inhibitory receptors, thereby facilitating intratumoral T cell exhaustion, limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment (12). The intratumoral expression of IL-35 leads to reduced T cells cytotoxic activities and decreased survival of the immunocompetent cells with anti-tumor properties (11, 12).
Although Treg cells were described as the primary source of IL-35, recent studies in human prostate cancer have also suggested that CD8+ Tregs may exploit IL-35 as a dominant regulatory mechanism (24). Furthermore, regulatory B cells have recently been shown to produce IL-35 (25, 26). Hence, the inflammatory cells in the tumor microenvironment facilitate the enrichment of an IL-35+ Treg population, which seems to be one of the mechanisms of tumor immune evasion (12).
Conclusion
The present study evaluated the immunohistochemical pattern of IL-35 in malignant CMT and its association with clinicopathological parameters and OS. Our data suggest that IL-35 is involved in malignant progression of mammary cancer in dogs. Moreover, high IL-35 expression was correlated with unfavorable prognosis by univariate and multivariate analysis. Therefore, IL-35 may be a new prognostic biomarker in CMT patients.
Acknowledgements
This work was supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the project FCT/MCTES - UID/Multi/00211/2019 and a PhD scholarship SFRH/BD/78771/2011.
Footnotes
Authors' Contributions
MI Carvalho carried out most of the practical work and the draft of the manuscript. FL Queiroga and I Pires participated equally in the study design. All the authors participated in the discussion of the data analysis and results and contributed to review the manuscript.
Conflicts of Interest
None of the Authors of this paper has a financial or personal conflict of interest to declare.
- Received February 1, 2019.
- Revision received February 27, 2019.
- Accepted February 28, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved