Untitled

Funder

Organizational Unit

Publications

Serum levels and tumor expression of TIM-3 as a promising clinical biomarker in cats with mammary carcinoma

Publication . Valente, Sofia Seixas Morais; Correia, Jorge Manuel de Jesus; Ferreira, Fernando António da Costa

T-cell Immunoglobulin and Mucin-domain containing molecule-3 (TIM-3) is an inhibitory receptor with a key role in regulating inflammation, autoimmunity and cancer progression. Recently, in order to overcome resistance to current immune checkpoint blockade therapies, TIM-3 has emerged as a promising targeting molecule for cancer immunotherapy. Although feline mammary carcinoma (FMC) is a valuable cancer model, no studies on the TIM-3 receptor have been developed in this species. Thus, in this study, 48 cats with mammary carcinoma were evaluated. Firstly, immunohistochemistry was performed to assess the TIM-3 expression in total (t), stromal (s) and intratumoral (i) tumor-infiltrating lymphocytes (TILs) and in cancer cells. In parallel, serum TIM-3 levels were quantified and compared with healthy controls using enzyme-linked immunosorbent assay (ELISA). Finally, the TIM-3 gene was sequenced from 19 tumor tissue samples to identify putative mutations. In FMC, our results demonstrated that sTILs-TIM3+ are more frequent than iTILs-TIM-3+, which are more abundant in luminal A/B subtypes. A positive correlation between sTILs-TIM-3+ and cancer cells-TIM-3+ and higher tumor malignancy grade was found (p=0.044 and p=0.018, respectively), whereas higher iTILs-TIM-3+ densities were mostly associated with a benign tumoral behavior. Additionally, higher densities of cancer cells overexpressing TIM-3 were associated with lymph node metastasis (p=0.025) and triple negative basal-like (p=0.038) and luminal B carcinomas (p=0.010), in contrast with luminal A mammary carcinomas. Finally, higher tTILs-TIM-3+ densities were predominantly associated with more favourable clinicopathological features, namely the percentage of tTILs-TIM-3+ was negatively associated with tumor metastasis (p=0.010) and tumor recurrence (p=0.013). Moreover, the TIM-3 serum levels were lower in the cats with mammary carcinoma than in the healthy group (p<0.001), being positively associated with higher densities of tTILs-TIM-3+ (p=0.016) and sTILs-TIM-3+ (p=0.023). From sequence analysis, only one mutation was identified in one tumor sample (intron 2: NC_058368.1: g.190379339 G>A). Altogether, in line with previous findings in human reports, our results suggest that the expression of TIM-3 among TILs subpopulations and cancer cells may differently influence the clinical outcome of cats with mammary carcinoma, reinforcing the hypothesis that feline mammary carcinoma is a valuable cancer model

2023-01-04Master thesis

Open access

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

Publication . Nascimento, Catarina; Urbano, Ana Catarina; Gameiro, Andreia; Ferreira, João; Correia, Jorge Manuel de Jesus; Ferreira, Fernando

Tumor microenvironment has gained great relevance due to its ability to regulate distinct checkpoints mediators, orchestrating tumor progression. Serum programmed cell death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) levels were compared with healthy controls and with serum cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and tumor necrosis factor-alpha (TNF-α) levels in order to understand the role of PD-1/PD-L1 axis in cats with mammary carcinoma. PD-1 and PD-L1 expression was evaluated in tumor-infiltrating lymphocytes (TILs) and cancer cells, as the presence of somatic mutations. Results showed that serum PD-1 and PD-L1 levels were significantly higher in cats with HER2-positive (p = 0.017; p = 0.032) and triple negative (TN) normal-like mammary carcinomas (p = 0.004; p = 0.015), showing a strong positive correlation between serum CTLA-4 and TNF-α levels. In tumors, PD-L1 expression in cancer cells was significantly higher in HER2-positive samples than in TN normal-like tumors (p = 0.010), as the percentage of PD-L1-positive TILs (p = 0.037). PD-L1 gene sequencing identified two heterozygous mutations in exon 4 (A245T; V252M) and one in exon 5 (T267S). In summary, results support the use of spontaneous feline mammary carcinoma as a model for human breast cancer and suggest that the development of monoclonal antibodies may be a therapeutic strategy.

2020Journal article

Open access

Avaliação da expressão da topoisomerase II alfa em tumores mamários caninos

Publication . Valdevino, Helena Raquel Correia; Correia, Jorge Manuel de Jesus; Ferreira, Fernando António da Costa

As neoplasias mamárias representam a neoplasia mais frequente em cadelas não esterilizadas, assumindo grande importância em clínica veterinária. Neste estudo foram analisados carcinomas mamários de cadela por imunohistoquímica (IHQ) para quantificação e avaliação da expressão da Topoisomerase IIα (TOPIIA). Objetivo: Quantificar a expressão da TOPIIA em carcinomas mamários de cadela, e correlacionar a expressão desta com variáveis como o Ki-67, a expressão de HER-2, a dimensão, o grau de malignidade e o tipo histológico do carcinoma. Materiais e métodos: Cinquenta e seis carcinomas mamários e cinco amostras de tecido mamário normal, foram submetidos a IHQ para avaliar a expressão de TOPIIA, e em cada caso foram classificadas 1000 células de marcação negativa, fraca, moderada ou forte. Resultados: Dos 56 tumores mamários analizados, 53,57% foram considerados positivos para a expressão da TOPIIA. Comprovou-se a existência de correlação positiva entre a sobreexpressão da TOPIIA e do Ki-67 (p=0,0364), e entre a sobreexpressão da proteína e menor tempo de sobrevivência (p=0,0264). Contudo, não foi demonstrada correlação entre as variáveis grau de malignidade, dimensão, tipo histológico do tumor e a sobreexpressão do HER-2. Conclusões: Este estudo sugere que a expressão da TOPIIA é maior nas células de carcinomas mamários do que nas células de tecido mamário normal, sendo que as cadelas com tumores classificados como positivos apresentaram menor tempo de sobrevivência, podendo este marcador ser considerado como fator de prognóstico. Contudo é indispensável a realização de mais estudos sobre este assunto, que contribuam para um maior conhecimento da importância desta proteína em oncologia veterinária assim como em medicina humana.

2018-05-04Master thesis

Open access

Identification of circulating microRNAs as biomarkers of feline mammary carcinoma

Publication . Santos, Ana Rita Gonçalves Nascimento da Silva; Ferreira, Fernando António da Costa; Correia, Jorge Manuel de Jesus

Feline mammary carcinomas (FMC) are very aggressive and, even after radical mastectomy, are usually fatal due to metastasis. Therefore, diagnostic tools allowing earlier detection and more effective treatment options are urgent. MicroRNAs (miRNAs) regulate gene expression and have been found to be altered in several cancer types, including breast cancer (BC). There are no studies yet evaluating miRNAs in feline cancers, thus this study aimed to elucidate if there are differences in miRNA serum levels between FMC and healthy controls, if they followed the same patterns as in BC and, finally, if they were associated with clinicopathological features. Serum samples from 45 female cats with FMC and 5 female healthy controls were used for the relative quantification of 5 microRNAs (miR-21, let-7a, miR-10b, miR-200b and miR-200c), which are consistently reported to be dysregulated in BC. Real-time polymerase chain reaction was performed and results were normalized to 2 reference genes (miR-191 and miR-484) by application of the 2-ΔΔCT method. Circulating miR-200c and let-7a were significantly downregulated (p=0,045 and p=0,04, respectively) in cats with mammary carcinoma, comparatively to healthy cats. Moreover, let-7a levels were significantly associated with overall-survival (p=0,04), histological subtypes (p=0,04) and an inverse correlation was found with serum stromal derived factor-1 (SDF-1) levels (p=0,03; Spearman r=-0,34). Regarding miR-21, high serum levels were significantly associated (p=0,02) with shorter disease-free survival (DFS) and lymph node metastasis (p=0,01), whereas for miR-10b, higher levels were associated to a positive SDF-1 status (p=0,01). Furthermore, higher serum miR-200b levels were significantly associated with shorter DFS (p=0,02), necrosis (p=0,02), histological (p=0,0078) and molecular subtypes (p=0,04), and a positive correlation was found with the tumour size (p=0,04, Spearman r=0,31). In conclusion, results suggest that miR-200c and let-7a are candidate diagnostic biomarkers for FMC, and let-7a, miR-21 and, particularly miR-200b, appear to have value as prognostic biomarkers. Moreover, miRNA patterns in FMC were similar to what is observed in BC, supporting that the first is a proper model in comparative oncology.

2018-03-15Master thesis

Open access

Emerging biomarkers and targeted therapies in feline mammary carcinoma

Publication . Gameiro, Andreia; Urbano, Ana Catarina; Ferreira, Fernando

ABSTRACT - : Feline mammary carcinoma (FMC) is a common aggressive malignancy with a low survival rate that lacks viable therapeutic options beyond mastectomy. Recently, increasing efforts have been made to understand the molecular mechanisms underlying FMC development, using the knowledge gained from studies on human breast cancer to discover new diagnostic and prognostic biomarkers, thus reinforcing the utility of the cat as a cancer model. In this article, we review the current knowledge on FMC pathogenesis, biomarkers, and prognosis factors and offer new insights into novel therapeutic options for HER2-positive and triple-negative FMC subtypes.

2021-08-11Journal article

Open access