Clinical Relevance of the serum CTLA-4 in Cats with Mammary Carcinoma

Abstract

Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) serves an important role in breast cancer progression, which has led to the development of novel immunotherapies aimed at blocking tumor immune evasion. Although feline mammary carcinoma is increasingly recognized as a valuable cancer model, no studies on CTLA-4 function had been conducted in this species. The serum CTLA-4, tnf-α and IL-6 levels of 57 female cats with mammary carcinoma were determined by ELISA, and immunohistochemistry was performed to evaluate CTLA-4 and FoxP3 expression in tumor cells and interstitial lymphocytes. The results obtained show that serum CTLA-4 levels are increased in cats with mammary carcinoma (P = 0.022), showing an association with a number of clinicopathological features: smaller tumor size, P < 0.001; absence of tumor necrosis, P < 0.001; non-basal status, P < 0.02 and HER-2-positive status. Additionally, a strong positive correlation was found between serum CTLA-4 levels and serum tnf-α (R = 0.88, P < 0.001) and IL-6 levels (R = 0.72, P < 0.001). Concerning the CTLA-4 and FoxP3 expression, although detected in both interstitial lymphocytes and tumor cells, a positive association was found only between interstitial CTLA-4 and FoxP3 expressions (R = 0.387, P = 0.01), which is negatively associated with the serum CTLA-4 levels (P = 0.03). These findings provide a preliminary step in the characterization of immune profiles in feline mammary carcinoma, uncovering a molecular rationale for targeted therapy with CTLA-4 pathway inhibitors. Finally, by strengthening the hypothesis of an immunomodulatory role for this regulator, we further validate the utility of spontaneous feline mammary carcinoma as a model for human breast cancer. Although the dog has been the focus in comparative oncology, cats also have clear benefits over rodent laboratory models of cancer, namely, they are immunocompetent and share the same environment as humans, reflecting more accurately the complex interplay between genetics and environmental risk factors, as well as the role of the immune system and tumor microenvironment (TME). Furthermore, the higher similarity between the cat and human genomes, together with the increased frequency of several tumor types (e.g. injection-site sarcoma, oral squamous cell carcinoma, lymphoma and malignant mammary tumors) 1-3 , anticipate that the cat may be a superior model. Feline mammary carcinoma shares many epidemiological and histopathological characteristics with human breast cancer, in particular, the human epidermal receptor-2 (HER-2) positive and triple negative (TN) subtypes, having been proposed as a suitable model for their study 4-6 , and offers further opportunities for studying certain aspects of tumor biology, such as the crosstalk between the immune system and tumor development. Chronic inflammation is a well-established risk factor for several cancers, with lymphocytes playing a pivotal role in the development of chronic inflammatory conditions 7. The activation of T-lymphocytes requires two signals: af...

Serum PD-1/PD-L1 Levels, Tumor Expression and PD-L1 Somatic Mutations in HER2-Positive and Triple Negative Normal-Like Feline Mammary Carcinoma Subtypes

“…The concentration levels of the above molecules were calculated using appropriate standard curves, by making serial dilutions from a stock solution of recombinant PD-1, PD-L1, CTLA-4 and TNF-α (9, 18, 36, 78, 156, 312.5, 625, 1250, 2500, 5000 and 10,000 pg/mL). Serum PD-1 and PD-L1 levels (pg/mL) were calculated using a quadratic regression (y = ax 2 + bx + c, r 2 = 0.99 for PD-1 and r 2 = 0.96 for PD-L1) and serum CTLA-4 and TNF-α concentrations were determined using a curve-fitting equation (y = mx + c, r 2 > 0.99) [ 10 ].…”

“…In addition, to identify the TILs the pathologists observed their characteristic morphology, as a scarce cytoplasm, a round and small nucleus and the presence of a dense chromatin. The scores of percentages of positive cells were recorded as: 0 (<1%), 1 (1–5%), 2 (6–30%) or 3 (>30%) [ 10 ]. The intensity scores were as follows: 0 (negative), 1+ (weak), 2+ (moderate), 3+ (strong) and 4+ (very strong).…”

“…Furthermore, a surgical approach is often necessary for the treatment of FMC, as the adjuvant chemotherapy (cyclophosphamide, vincristine, doxorubicin) is not useful in some cases [ 7 ]. Thus, the identification of novel diagnostic biomarkers and therapeutic targets is needed, not only to improve the clinical outcome of cats with mammary carcinoma but also because FMC shares clinicopathological, histopathological and epidemiological features, as well as the molecular classification with human breast cancer [ 4 , 8 , 9 , 10 ]. Moreover, the use of animal models has been increasing, allowing researchers to understand the mechanisms underlying tumorigenesis; mouse species are the most used due to their small size and short gestation period.…”

“…Furthermore, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is another immune mediator that inhibits T-cell immune function, being also targeted in breast cancer. Accordingly, serum CTLA-4 levels are increased in cats with mammary carcinoma, as well as in human malignant tumors [ 10 ]. Some trials are evaluating the combination of anti-PD-1/PD-L1 antibodies with CTLA-4 inhibitors [ 17 , 26 ].…”

“…Additionally, it has been described that certain pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), can up-regulate PD-L1 expression in cancer cells, contributing to create an immunosuppressive tumor microenvironment [ 27 , 28 ]. Furthermore, TNF-α is described to be elevated in cats with mammary carcinoma, being positively correlated with serum CTLA-4 levels, as in humans [ 10 ].…”