Clinical relevance of serum cytotoxic t-lymphocyte associated protein 4 (CTLA-4) and correlation with the pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) in feline mammary carcinoma

Dissertação de Mestrado Integrado em Medicina VeterináriaThe association between CTLA-4 expression and cancer prognosis has been extensively investigated in recent years, pointing to the link with inflammation, and highlighting the role of the tumor microenvironment (TME), of which inflammatory mediators like cytokines are an important non-cellular component. To the best of our knowledge, no studies on immune checkpoint regulators had been conducted on cats with mammary carcinoma before, nor had cytokine profiles been previously assessed. Thus, we investigated the serum profiles of CTLA-4 and pro-inflammatory cytokines IL-6 and TNF-α in 57 female cats with mammary carcinoma and checked for associations between CTLA-4 and cytokine serum levels. Our results clearly demonstrate that serum CTLA-4 levels are increased in cats with mammary carcinoma when compared to healthy animals (P=0.022). Furthermore, we show a strong positive correlation with TNF-α (R=0.88, P<0.001) and IL-6 levels (R=0.72, P<0.001), advancing the concept of an immunomodulatory role for this regulator in breast cancer pathogenesis. We also show a statistically significant association between higher levels of serum CTLA-4 and less aggressive clinicopathological features: smaller tumors (P<0.001), lower stage (P=0.002), absence of necrosis (P<0.001), no lymph node involvement (P=0.007), no lymphatic vessel invasion (P=0.006), positive hormone receptor status (P=0.007), non-TN status (P=0.041), non-basal status (P<0.001) and low Ki67 index (P=0.001). Our findings further expand this concept by indicating an association with specific breast cancer subtypes, namely, HER-2 positive with CTLA-4 (P<0.001) and TNF-α (P=0.004) and luminal A-like with IL-6 (P=0.020). We could not confirm an association between serum CTLA-4 and cytokines levels and survival due to the small sample size. Nevertheless, our findings suggest a potentially concentration-dependent protective role for serum CTLA-4 and IL-6, as evidenced by higher median survival times in the CTLA-4high (28 vs 22 months for the CTLA-4low group) and IL-6high (28 vs 19 months for the IL-6low group) groups. Conversely, TNF-α seems to be a negative prognostic factor, as shown by the lower median survival in the TNF-αhigh group (16.5 vs 23.5 months for the TNF-αlow group). An intriguing question that remains is how serum CTLA-4 influences or is influenced by the pro-inflammatory cytokines. Assessment of CTLA-4 tumor expression, T-lymphocyte subtypes, and tumor associated macrophages and myeloid derived suppressor cell profiles in the microenvironment, are important features to evaluate in future studies.RESUMO - RELEVÂNCIA CLÍNICA DA PROTEÍNA 4 ASSOCIADA AO LINFÓCITO T CITOTÓXICO 4 (CTLA-4) E SUA CORRELAÇÃO COM AS CITOQUINAS PRÓ-INFLAMATÓRIAS INTERLEUCINA 6 (IL-6) E FACTOR DE NECROSE TUMORAL ALFA (TNF-α) NO CARCINOMA MAMÁRIO FELINO - A associação entre a expressão da CTLA-4 e o prognóstico no cancro tem sido amplamente investigada, valorizando o papel da inflamação e do microambiente tumoral (TME), do qual mediadores inflamatórios como as citoquinas são uma importante componente não celular. Até à data, não existem estudos sobre reguladores de checkpoint imunológico em gatos com carcinoma mamário, nem foram avaliados perfis de citoquinas. Assim, foram investigados pela primeira vez, os perfis séricos da CTLA-4 e das citoquinas pró-inflamatórias IL-6 e TNF-α em 57 gatas com carcinoma mamário e verificada a existência de associações entre os níveis séricos da CTLA-4 e das referidas citoquinas. Os resultados obtidos demonstram que os níveis de CTLA-4 estão aumentados no soro das gatas com carcinoma mamário, quando comparadas com animais saudáveis (P=0.022). Foi também demonstrada uma correlação forte com os níveis séricos do TNF-α (R=0.88, P<0.001) e da IL-6 (R=0.72, P<0.001), reforçando o papel imunomodulatório deste regulador. Adicionalmente foi encontrada uma associação significativa entre os níveis séricos elevados da CTLA-4, e várias características clinicopatológicas menos agressivas: tumores mais pequenos (P<0.001), estadiamento precoce, (P=0.002), ausência de necrose tumoral (P<0.001), sem envolvimento dos linfonodos (P=0.007), sem invasão linfática (P=0.006), com positividade para os receptores hormonais (P=0.007), subtipo não-TN (P=0.041), subtipo não-basal (P<0.001), e baixo índice Ki67 (P=0.001). Os resultados obtidos ainda revelaram uma associação com subtipos específicos de cancro da mama, nomeadamente o HER-2 positivo com sobre-expressão da CTLA-4 (P