Improving feline mammary carcinoma treatment through HER2-related immunochemotherapy agents and biomarkers

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas, área científica - Morfologia e FunçãoABSTRACT - Improving feline mammary carcinoma treatment through HER2-related immunochemotherapy agents and biomarkers - The feline mammary carcinoma (FMC) is a common tumour, with the HER2-positive and triple-negative being the most aggressive subtypes, as in women. This tumour is usually diagnosed belatedly, with scarce information about its development, and limited therapeutic options beyond mastectomy, presenting the cat a low overall survival time. Thus, this project was developed in order to improve therapeutic options, as well as, to reveal new tumour diagnosis/prognosis biomarkers, related to HER2 protein and taking advantage for the extensive knowledge in protocols used in humans. In order to disclose different therapeutic options for diseased cats, several drugs (TKi – lapatinib and neratinib; mAbs – trastuzumab and pertuzumab; ADC – T-DM1; and mTOR inhibitor – rapamycin, as adjuvant) and combined protocols were tested using FMC cell lines (CAT-M, FMCp and FMCm). These assays revealed promising antiproliferative effects and conserved molecular responses, inducing lower phosphorylation levels of target proteins, when TKi were used, and apoptosis, when mAbs and ADC drugs were evaluated. In parallel, combined protocols presented excellent synergistic responses. Moreover, feline tumour clinical tissue samples were analysed in order to identify her2 mutations as prognosis markers or therapy resistance indicators. The obtained results correlates a her2 mutation, in exon 18 (c.19573 A>T), to larger tumour sizes, a poor prognosis feature. Furthermore, any of the mutations found are described, in woman, as inducing therapeutic resistance. Additionally, considering the close relationship between obesity with increased leptin levels and the development of HER2-positive breast cancer, in woman, and since in cat obesity is a frequent nutritional disorder, the leptin/leptin receptor (ObR) axis was evaluated as possible tumour biomarker. Interestingly, cats with mammary carcinoma presented a decreased free leptin index, being the higher leptin levels associated to poor prognostic features, and serum ObR levels were correlated to an immunosuppressive status. In conclusion, the results obtained support the use of therapeutic drugs targeting the HER2, in order to improve cats’ prognosis, which could contribute for an advance in the veterinary oncology practice. Furthermore, leptin and ObR were suggested as tumour biomarkers, being proposed its use as putative adjuvant therapeutic targetsRESUMO - Melhorar o tratamento do carcinoma mamário felino através de imunoquimioterapia dirigida para o HER2 e novos biomarcadores tumorais - O carcinoma mamário felino (CMF) é um tumor frequente, sendo os subtipos HER2- positivo e triplo-negativo os mais agressivos, tal como na mulher. No gato, este tumor é geralmente diagnosticado num estadio avançado, havendo pouca informação sobre o seu desenvolvimento e opções terapêuticas limitadas, para além da mastectomia, o que resulta num curto tempo de sobrevivência. Assim, este projeto foi desenvolvido de forma a melhorar as opções terapêuticas, na gata, bem como identificar novos biomarcadores tumorais, relacionados com a proteína HER2 e tirando vantagem dos protocolos validados em medicina humana. De forma a encontrar diferentes opções terapêuticas para o tumor de mama, diversos fármacos (TKi – lapatinib e neratinib; mAbs – trastuzumab e pertuzumab; ADC – T DM1; e inibidor da via mTOR – rapamicina, como adjuvante) e protocolos de conjugação, foram testados usando linhas celulares imortalizadas de CMF (CAT-M, FMCp e FMCm). Estes ensaios exibiram efeitos antiproliferativos promissores, apresentando mecanismos de ação conservados, induzindo baixos níveis de fosforilação das proteínas alvo, aquando do uso de TKi e, apoptose, quando os mAbs e ADC foram testados. Em paralelo, os protocolos de conjugação apresentaram excelentes respostas sinérgicas. Ainda, amostras de tecido de tumores de mama de gata foram analisadas, por forma a identificar mutações no her2, como indicadores de prognóstico, ou resistência à terapêutica. Os resultados obtidos revelaram uma mutação, no exão 18 (c.19573 A>T), associada a tumores de maiores dimensões, um fator de mau prognóstico. Além disso, nenhuma mutação caracterizada se encontra descrita na mulher, como responsável pela resistência terapêutica. Adicionalmente, considerando a correlação entre obesidade, com aumento dos níveis de leptina e o desenvolvimento do tumor de mama HER2-positivo, na mulher e, uma vez que na gata este é um problema nutricional comum, o eixo leptina/recetor (ObR) foi avaliado, como possível biomarcador tumoral. Os resultados obtidos mostraram que gatos com carcinoma mamário apresentam uma diminuição do índice de leptina livre, estando os níveis elevados de leptina associados a mau prognóstico e, os níveis séricos de ObR relacionados com um estado de imunossupressão geral. Em conclusão, os resultados obtidos suportam o uso de fármacos anti-HER2 e ainda, o uso da leptina e do seu recetor como biomarcadores tumorais e possíveis alvos adjuvantes à terapiaN/

Emerging biomarkers and targeted therapies in feline mammary carcinoma

Research Areas: Veterinary SciencesABSTRACT - : 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.info:eu-repo/semantics/publishedVersio

VISTA is a diagnostic biomarker and immunotherapy target of aggressive feline mammary carcinoma subtypes

Research Areas: OncologyABSTRACT - Feline mammary carcinoma (FMC) is a common neoplasia, showing aggressive clinicopathological features, without viable therapeutic options. The study of tumor microenvironment has gained importance, due to the ability to control tumor progression by regulating the immune response. Considering the lack of knowledge, feline serum VISTA levels from cats with mammary carcinoma were compared with healthy controls, and with serum levels of PD-1/PD-L1, CTLA-4, LAG-3, IL-6, and TNF-α. In parallel, VISTA tumor expression was evaluated in FMC samples. The obtained data revealed that serum VISTA levels were significantly higher in cats presenting HER2-positive (p = 0.0025) or triple-negative subtypes (p = 0.0019), with higher serum levels in luminal A (p = 0.0025) correlated to the presence of metastasis (p = 0.0471). Furthermore, in HER2- positive or triple-negative tumors, correlations were obtained between serum VISTA levels and the serum levels of the above-mentioned molecules. In tumors, VISTA expression revealed a stronger intensity in cancer cells, when compared to TILs (p < 0.0001). Stratifying the samples by subtypes, a higher number of VISTA-positive TILs was observed in the HER2-positive subtype, compared with triple-negative tumors (p = 0.0138). In conclusion, results support the development of therapeutic strategies for HER2-positive and triple-negative FMC subtypes, reinforcing the use of cats as a human oncology model.info:eu-repo/semantics/publishedVersio

Single and multiple antenna relay-assisted techniques for uplink and downlink OFDM systems

In this paper we propose and assess the performance of relay-assisted schemes designed for both the uplink and downlink OFDM based systems, using efficient distributed space-frequency block coding protocols. We consider the use of an antenna array at the base station and a single antenna at the user terminal. At the relay node we consider either single antenna or an antenna array. We assume that some of the user terminals deployed in a certain area could act as relaying-able terminals for the communication of other users. Two types of relay-assisted protocols are considered: equalize-and-forward and decode-and-forward. The optimal maximum ratio combining coefficients are derived for the proposed relay-assisted schemes. The performance of these cooperative schemes is evaluated under realistic scenarios, considering typical pedestrian scenarios based on WiMAX specifications and using channel convolutional turbo code. The proposed schemes are also compared against the non-cooperative OFDM based systems. +umerical results show that the availability of antenna arrays at the relays significantly improves the cooperative systems performance, which outperform the non-cooperative ones in most studied scenarios

HER2-Targeted immunotherapy and combined protocols showed promising antiproliferative effects in feline mammary carcinoma cell-based models

Research Areas: OncologyABSTRACT - Feline mammary carcinoma (FMC) is a highly prevalent tumor, showing aggressive clinicopathological features, with HER2-positive being the most frequent subtype. While, in human breast cancer, the use of anti-HER2 monoclonal antibodies (mAbs) is common, acting by blocking the extracellular domain (ECD) of the HER2 protein and by inducing cell apoptosis, scarce information is available on use these immunoagents in FMC. Thus, the antiproliferative effects of two mAbs (trastuzumab and pertuzumab), of an antibody–drug conjugate compound (T-DM1) and of combined treatments with a tyrosine kinase inhibitor (lapatinib) were evaluated on three FMC cell lines (CATMT, FMCm and FMCp). In parallel, the DNA sequence of the her2 ECD (subdomains II and IV) was analyzed in 40 clinical samples of FMC, in order to identify mutations, which can lead to antibody resistance or be used as prognostic biomarkers. Results obtained revealed a strong antiproliferative effect in all feline cell lines, and a synergistic response was observed when combined therapies were performed. Additionally, the mutations found were not described as inducing resistance to therapy in breast cancer patients. Altogether, our results suggested that anti-HER2 mAbs could become useful in the treatment of FMC, particularly, if combined with lapatinib, since drug-resistance seems to be rare.info:eu-repo/semantics/publishedVersio

Diagnostic value of VEGF-A, VEGFR-1 and VEGFR-2 in feline mammary carcinoma

Research Areas: OncologyVascular endothelial growth factor (VEGF-A) plays an essential role in tumor-associated angiogenesis, exerting its biological activity by binding and activating membrane receptors, as vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2). In this study, serum VEGF-A, VEGFR1, and VEGFR-2 levels were quantified in 50 cats with mammary carcinoma and 14 healthy controls. The expression of these molecules in tumor-infiltrating lymphocytes (TILs) and in cancer cells was evaluated and compared with its serum levels. Results obtained showed that serum VEGF-A levels were significantly higher in cats with HER2-positive and Triple Negative (TN) Normal-Like subtypes, when compared to control group (p = 0.001, p = 0.020). Additionally, serum VEGFR-1 levels were significantly elevated in cats presenting luminal A, HER2-positive and TN Normal-Like tumors (p = 0.011, p = 0.048, p = 0.006), as serum VEGFR-2 levels (p = 0.010, p = 0.046, p = 0.005). Moreover, a positive interaction was found between the expression of VEGF-A, VEGFR-1, and VEGFR-2 in TILs and their serum levels (p = 0.002, p = 0.003, p = 0.003). In summary, these findings point to the usefulness of VEGF-A and its serum receptors assessment in clinical evaluation of cats with HER2-positive and TN Normal-Like tumors, suggesting that targeted therapies against these molecules may be effective for the treatment of these animals, as described in human breast cancer.info:eu-repo/semantics/publishedVersio

Serum and tissue expression levels of leptin and leptin receptor are putative markers of specific feline mammary carcinoma subtypes

Research Areas: Veterinary SciencesABSTRACT - Obesity is an established risk factor for breast cancer in post-menopausal women, being associated with elevated serum levels of leptin. Although overweight is a common condition in cat, the role of leptin and its receptor in feline mammary carcinoma remains unsettled. In this study, serum leptin and leptin receptor (ObR) levels were investigated in 58 cats with mammary carcinoma and compared with those of healthy animals, as were the expression levels of leptin and ObR in tumor tissues. The results showed that the Free Leptin Index is significantly decreased in cats with mammary carcinoma (p = 0.0006), particularly in those with luminal B and HER2-positive tumors, and that these animals also present significantly lower serum leptin levels (p < 0.0001 and p < 0.005, respectively). Interestingly, ulcerating tumors (p = 0.0005) and shorter disease-free survival (p = 0.0217) were associated to serum leptin levels above 4.17 pg/mL. In contrast, elevated serum ObR levels were found in all cats with mammary carcinoma (p < 0.0001), with levels above 16.89 ng/mL being associated with smaller tumors (p = 0.0118), estrogen receptor negative status (p = 0.0291) and increased serum levels of CTLA-4 (p = 0.0056), TNF-alpha (p = 0.0025), PD-1 (p = 0.0023), and PD-L1 (p = 0.0002). In tumor samples, leptin is overexpressed in luminal B and triple-negative carcinomas (p = 0.0046), whereas ObR is found to be overexpressed in luminal B tumors (p = 0.0425). Altogether, our results support the hypothesis that serum levels of leptin and ObR can be used as biomarkers of specific feline mammary carcinoma subtypes, and suggests the use of leptin antagonists as a therapeutic tool, reinforcing the utility of the cat as a cancer model.info:eu-repo/semantics/publishedVersio

Uma visão crítica sobre a utilização de cortinas de ar em estabelecimentos comerciais climatizados

Tendo por base um conjunto de ensaios realizados numa instalação experimental especialmente concebida para o efeito, é avaliada a influência de alguns parâmetros geométricos e dinâmicos sobre a eficácia da vedação térmica alcançada com a utilização de aparelhos de cortina de ar. A informação recolhida permitiu estabelecer diversas recomendações sobre a selecção, instalação e operação deste tipo de aparelho, as quais, na prática, nem sempre são seguidas, conforme se pode antever dos dados preliminares recolhidos em vistorias técnicas realizadas a estabelecimentos comerciais que utilizam este tipo de solução

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

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).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.This research was funded by Fundação para a Ciência e a Tecnologia (Portugal) through the projects PTDC/CVT-EPI/3638/2014 and CIISA-UIDP/CVT/00276/2020. C.N. is receipt of a PhD fellowship from University of Lisbon (ref.C00191r) and A.G. is receipt of a PhD fellowship from Fundação para a Ciência e a Tecnologia (ref. SFRH/BD/132260/2017).info:eu-repo/semantics/publishedVersio