Feline Mammary Carcinomas Research Articles

HER2-Targeted Immunotherapy and Combined Protocols Showed Promising Antiproliferative Effects in Feline Mammary Carcinoma Cell-Based Models.

Simple SummaryMammary tumors are common in cats, presenting an aggressive behavior with high tumor recurrence. Therefore, new and efficient therapeutic protocols are urgent. Monoclonal antibodies (mAbs; ADC) are widely used in human breast cancer therapy, inhibiting the HER2 dimerization and leading to cell apoptosis. Furthermore, drug combinations, with tyrosine kinase inhibitors (TKi) are valuable in patients’ therapeutic protocols. In this study, two mAbs, and an ADC, as well as combined protocols between mAbs and mAbs plus lapatinib (TKi) were tested to address if the drugs could be used as new therapeutic options in feline mammary tumors. All the compounds and the combined treatments revealed valuable antiproliferative effects, and a conserved cell death mechanism, by apoptosis, in the feline cell lines, where the mutations found in the extracellular domain of the HER2 suggest no immunotherapy resistance.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 (CAT-MT, 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.

Quantification of the collagen fibers of healthy and neoplastic mammary tissue in cats by the Picrossirius Red histochemical method

Although feline mammary carcinoma is not the most prevalent among the species, its aggressive behavior represents a low life expectancy, compared with most undifferentiated types of breast cancer. Tissue stiffness induced by the accumulation of collagen fibers is related to a risk factor for carcinogenesis in healthy women and aggressiveness in those with breast cancer, which can also occur in cats. The objective of this work is to identify the relationship between stromal collagen density and aggressiveness of mammary carcinoma in cats, according to the peripheral and central tissue distribution by the Picrossirius Red histochemical method. Image.J® and MatLab® software were used for digital image processing. The mean values of kurtosis and entropy attributes were ​​grouped into a control group, and low and high-grade carcinoma groups, analyzed with one-way ANOVA and Bonferroni’s multiple comparison test (p <0.01). Interpretation of stromal dynamics is important to evaluate both central and peripheral locations. According to entropy, there was a significant increase in the peripheral density in the carcinoma groups in relation to the control group, which can be justified by blood support. The same can be said of the central region, with a significant gain in collagen fibers from the tumors, indicated by kurtosis. The results suggest the presence of increases in stromal density in mammary carcinomas of cats, regardless of their graduation, and occurring in both regions.

Genetic variants of BRCA1 and BRCA2 genes in cats with mammary gland carcinoma.

Mammary tumours are the first and third most incident neoplasm in women and cats, respectively. Approximately 85% of feline mammary gland tumours are malignant and aggressive, especially the triple-negative and HER-2+ molecular subtypes. Triple-negative basal-like feline mammary carcinomas (FMCs) are considered suitable models due to the clinical and morphological similarities with human basal-like triple-negative breast cancer (TNBC). In women, TNBC has a poor prognosis and is often associated with mutations in the tumour suppressor genes BRCA1 and BRCA2. In light of this, the aim of the present investigation was to screen somatic and germline variants of BRCA1 and BRCA2 in nine female cats bearing FMCs. Matched whole blood and FMC samples were obtained for genetic analysis. Additional tumour samples were obtained for histopathological and immunohistochemical evaluation. Genomic DNA was isolated and 27 exonic regions of BRCA1 and BRCA2 genes were amplified and screened by next-generation sequencing. A somatic variant with high functional impact was found in exon 11 of BRCA2 at a frequency of 4.34% in one FMC-bearing cat. Four germline variants with moderate impact were detected in three of the nine FMC-bearing cats and were restricted to exon 9 of BRCA1. It is concluded that the germline genetic variants found in one-third of FMC-bearing animals might be associated with a higher risk of hereditary mammary carcinogenesis.

Neutrophil-to-lymphocyte ratio is an independent prognostic marker for feline mammary carcinomas.

Blood leukocyte counts and respective derived ratios have been described as potential prognostic markers in several tumours in veterinary oncology. This study aimed to evaluate peripheral blood leukocyte subpopulations and neutrophil-to-lymphocyte ratio (NLR) as prognostic factors for feline mammary carcinomas (FMC). Medical records from cats diagnosed with FMC between 2017 to 2019 were reviewed. Cats were included if fully staged, classified as WHO stage I to III, and submitted to mastectomy. Cats were excluded if they had evidence of other diseases. Forty-nine cats were included. The study endpoints were disease-free interval (DFI) and tumour-specific survival (TSS). The median DFI and TSS were 389 days and 528 days respectively. In the univariate analysis, higher values of total white blood cell count (WBC), neutrophil count (NEU) and NLR were identified as significant prognostic factors for both endpoints (P < .05). On the multivariate analysis, NLR remained an independent prognostic factor for TSS (P=.024). In the receiver operating characteristic curve analysis, the estimated cut-off for WBC was 8.49 × 109 /L (DFI and TSS); for NEU was 4.62 × 109 /L (DFI) and 6.65 × 109 /L (TSS) and for NLR was 2.46. These cut-offs were significant prognostic factors for DFI and TSS (P < .05). NLR cut-off remained an independent prognostic factor for both DFI (P=.032) and TSS (P=.043) in the multivariable analysis. Our results suggest that NLR, NEU, and WBC can be important non-invasive presurgical prognostic markers, and that NLR is an independent prognostic marker for FMC. Prospective studies are warranted to validate its clinical use.

Evaluation of TFR-1 Expression in Feline Mammary Cancer and In Vitro Antitumor Efficacy Study of Doxorubicin-Loaded H-Ferritin Nanocages.

Simple SummaryTransferrin receptor one (TFR-1), recognized by ferritin, is overexpressed in many tumor cells. This feature has been exploited to produce a selective overload of drugs within tumor cells by creating an engineered ferritin nanocage loaded with doxorubicin (HFn(DOX)). This bionanotechnology has been tested in human cancer, but there are no studies in veterinary oncology. This work, after evaluating the expression of TFR-1 in feline tumors, demonstrated for the first time the effectiveness in vitro of this nanocage in animals. These results confirm that engineered bionanocages also offer unprecedented opportunities for animal cancer to be applied in veterinary medicine and in comparative studies including spontaneous animal models of cancer.The transferrin receptor 1 (TFR-1) has been found overexpressed in a broad range of solid tumors in humans and is, therefore, attracting great interest in clinical oncology for innovative targeted therapies, including nanomedicine. TFR-1 is recognized by H-Ferritin (HFn) and has been exploited to allow selective binding and drug internalization, applying an HFn nanocage loaded with doxorubicin (HFn(DOX)). In veterinary medicine, the role of TFR-1 in animal cancers remains poorly explored, and no attempts to use TFR-1 as a target for drug delivery have been conducted so far. In this study, we determined the TFR-1 expression both in feline mammary carcinomas during tumor progression, as compared to healthy tissue, and, in vitro, in a feline metastatic mammary cancer cell line. The efficacy of HFn(DOX) was compared to treatment with conventional doxorubicin in feline mammary cancer cells. Our results highlighted an increased TFR-1 expression associated with tumor metastatic progression, indicating a more aggressive behavior. Furthermore, it was demonstrated that the use of HFn(DOX) resulted in less proliferation of cells and increased apoptosis when compared to the drug alone. The results of this preliminary study suggest that the use of engineered bionanocages also offers unprecedented opportunities for selective targeted chemotherapy of solid tumors in veterinary medicine.

The role of COX expression in the prognostication of overall survival of canine and feline cancer: A systematic review.

Cyclooxygenase (COX) isoforms‐1 and ‐2 have been extensively investigated in cancer. Although COX‐2 is the isoform most studied and has been described in several malignancies associated with histologic criteria of malignancy and worse prognosis, COX‐1 has also been linked to some forms of cancer. With the present review our aim was to summarize the current state of knowledge and clarify if and in which type of tumours COX‐1 and/or COX‐2 expression have real prognostic implications. We searched PubMed database for prognostic studies using predefined inclusion criteria in order to ascertain the prognostic value of COX‐1 and COX‐2 in malignant neoplasia in dogs and cats. Eighteen studies were analysed. COX‐2 was shown to be a negative prognostic factor in canine and feline mammary tumours, canine mast cell tumour, canine melanoma, canine osteosarcoma and canine renal cell carcinoma. COX‐1 showed a negative prognostic value in feline oral squamous cell carcinoma (SCC). We found high heterogeneity among studies regarding COX immunohistochemical evaluation methodology even in the same type of neoplasia pointing out the need for its standardization at least by tumour type. The available data support the use of COX‐2 as a prognostic factor in canine (mammary carcinoma, mast cell tumour, melanoma, osteosarcoma and renal carcinoma) and feline (mammary carcinoma) cancers. For COX‐1, its use is advised in feline oral SCC.

Tyrosine Kinase Inhibitors Are Promising Therapeutic Tools for Cats with HER2-Positive Mammary Carcinoma

Feline mammary carcinoma (FMC) is a common neoplasia in cat, being HER2-positive the most prevalent subtype. In woman’s breast cancer, tyrosine kinase inhibitors (TKi) are used as a therapeutic option, by blocking the phosphorylation of the HER2 tyrosine kinase domain. Moreover, clinical trials demonstrated that TKi produce synergistic antiproliferative effects in combination with mTOR inhibitors, overcoming resistance to therapy. Thus, to uncover new chemotherapeutic strategies for cats, the antiproliferative effects of two TKi (lapatinib and neratinib), and their combination with a mTOR inhibitor (rapamycin), were evaluated in FMC cell lines (CAT-M, FMCp and FMCm) and compared with a human breast cancer cell line (SkBR-3). Results revealed that both TKi induced antiproliferative effects in all feline cell lines, by blocking the phosphorylation of EGFR members and its downstream effectors. Furthermore, combined treatments with rapamycin presented synergetic antiproliferative effects. Additionally, the DNA sequence of the her2 TK domain (exons 18 to 20) was determined in 40 FMC tissue samples, and despite several mutations were found none of them were described as inducing resistance to therapy. Altogether, our results demonstrated that TKi and combined protocols may be useful in the treatment of cats with mammary carcinomas, and that TKi-resistant FMC are rare.

Histone Deacetylase Inhibitors and Microtubule Inhibitors Induce Apoptosis in Feline Luminal Mammary Carcinoma Cells.

Simple SummaryFeline mammary tumors (FMT) are very common in cats, associated with very aggressive behavior and a short life expectancy. Surgery is the most used treatment but tumor recurrence is common. Currently, available therapies are insufficient, therefore, new molecular targets are needed to develop more efficient therapeutics. Histone deacetylases inhibitors (HDACis) have been developed to target tumor cells, by disrupting gene expression and leading to cell death. Microtubules inhibitors (MTIs) have also been a focus of research, to target polymerization of microtubules, and consequently disturbing the cytoskeleton and leading to cell cycle arrest and apoptosis. However, there are few studies on the use of HDACis and MTIs in cats. In this study, we addressed if these two drug classes could be used as new therapeutic options in FMTs. All HDACis and MTIs exhibited suitable and dose-dependent antitumor effects in FMT cell lines. Immunoblot analysis confirmed that the mode of action of HDACis is conserved in feline mammary tumor cell lines. Finally, flow cytometry showed that exposure with HDACis and MTIs lead to the induction of cellular apoptosis. In summary, HDACis and MTIs possess antitumor properties suggesting further studies on their use in the treatment of feline mammary tumors.Feline mammary carcinoma (FMC) is the third most common type of neoplasia in cats, sharing similar epidemiological features with human breast cancer. In humans, histone deacetylases (HDACs) play an important role in the regulation of gene expression, with HDAC inhibitors (HDACis) disrupting gene expression and leading to cell death. In parallel, microtubules inhibitors (MTIs) interfere with the polymerization of microtubules, leading to cell cycle arrest and apoptosis. Although HDACis and MTIs are used in human cancer patients, in cats, data is scarce. In this study, we evaluated the antitumor properties of six HDACis (CI-994, panobinostat, SAHA, SBHA, scriptaid, and trichostatin A) and four MTIs (colchicine, nocodazole, paclitaxel, and vinblastine) using three FMC cell lines (CAT-MT, FMCp, and FMCm), and compared with the human breast cancer cell line (SK-BR-3). HDACis and MTIs exhibited dose-dependent antitumor effects in FMC cell lines, and for all inhibitors, the IC50 values were determined, with one feline cell line showing reduced susceptibility (FMCm). Immunoblot analysis confirmed an increase in the acetylation status of core histone protein HDAC3 and flow cytometry showed that HDACis and MTIs lead to cellular apoptosis. Overall, our study uncovers HDACis and MTIs as promising anti-cancer agents to treat FMCs.

Serum and Tissue Expression Levels of Leptin and Leptin Receptor Are Putative Markers of Specific Feline Mammary Carcinoma Subtypes.

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-α (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.

Detection of Epithelial Cell Adhesion Molecule in Feline Normal and Tumor Cell Lines and Tissues With Selected Commercial Anti-human EpCAM Antibodies.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein expressed at intercellular junctions in epithelial cells. As an epithelial biomarker, it used for immunologic-based capture of epithelial-derived circulating tumor cells (CTCs) in human patients with different carcinomas. EpCAM expression has not been described in normal or neoplastic epithelial tissues in cats. Our goal was to find a commercial antibody that recognizes surface EpCAM expression for CTC detection. We tested two anti-human EpCAM antibodies, designated for use with flow cytometry, for detection of surface EpCAM expression on feline cell lines derived from normal mammary and renal epithelia and mammary and oropharyngeal squamous cell carcinomas in cats. Only one of the antibodies, a goat polyclonal antibody, labeled normal and neoplastic feline mammary epithelial cells and oropharyngeal squamous cell carcinoma cells; no labeling was observed for normal feline kidney epithelial cells. At low dilution, this antibody immunohistochemically stained the intercellular junctions of normal pancreatic, intestinal and mammary epithelium, as well as neoplastic mammary epithelium in feline tissues; however, oral mucosa, skin, and an oropharyngeal squamous cell carcinoma showed no positive immunostaining. The antibody only weakly bound feline squamous cell carcinoma cell lines under static adhesion. Our results indicate that EpCAM is expressed in specific epithelia in cats but is variably expressed in feline mammary tumors and oropharyngeal squamous cell carcinoma. A higher avidity cross-reactive or feline-specific antibody will be required to further investigate EpCAM expression in normal and neoplastic feline tissue or for detecting CTCs in the blood of tumor-bearing cats.

An Immunohistochemical Study of the PTEN/AKT Pathway Involvement in Canine and Feline Mammary Tumors.

Simple SummaryThe PTEN/AKT pathway is involved in several human and animal tumors’ pathogenesis. This study investigates the PTEN/AKT pathway’s biological and prognostic values in canine and feline mammary tumors. PTEN, phospho-AKT (p-AKT) and Rictor expression was determined by immunohistochemistry in canine mammary adenomas and carcinomas and feline mammary carcinomas. In mammary tumors of both species p-Akt was inversely correlated with PTEN expression and positively with Rictor expression; p-Akt and Rictor expression correlated with poorer prognosis. This data could provide a rationale for further studies of this pathway in veterinary oncology due to prognostic and therapeutic implications.Phosphatase and tensin homolog deleted on chromosome10 (PTEN), phospho-v-Akt murine thymoma viral oncogene homolog (AKT), and the Rapamycin-Insensitive Companion of mTOR (Rictor) expression was investigated by immunohistochemistry in 10 canine mammary adenomas (CMAs), 40 canine mammary carcinomas (CMCs), and 30 feline mammary carcinomas (FMCs). All the CMAs, 25 of 40 CMCs (63%) and 7 of 30 FMCs (23%), were PTEN-positive. In dogs, no CMAs and 15 of 25 CMCs (37%) expressed phospho-AKT (p-AKT), while 24 of 30 FMCs (82%) were p-AKT-positive. One of 10 CMAs (10%), 24 of 40 CMCs (60%) and 20 of 30 FMCs (67%) were Rictor-positive. In the dog, PTEN expression correlated with less aggressive tumors, absence of lymphatic invasion, and longer survival. P-AKT expression correlated with more aggressive subtype, lymphatic invasion, and poorer survival and Rictor expression with lymphatic invasion. In cats, PTEN correlated with less aggressive carcinomas, absence of lymphatic invasion, and better survival. P-AKT and Rictor expression correlated with poorer survival. PTEN expression was inversely correlated with p-AKT and Rictor in both species, while p-AKT positively correlated with Rictor expression. A strong PTEN/AKT pathway involvement in behavior worsening of CMT and FMTs is demonstrated, providing a rationale for further studies of this pathway in veterinary oncology.

Immunoexpression and Prognostic Significance of Multidrug Resistance Markers in Feline Mammary Carcinomas

Feline mammary carcinomas (FMCs) are commonly characterized by high clinical aggressiveness and poor prognosis. FMCs share many features with the corresponding human disease, allowing the comparative investigation of tumour biology and therapeutic strategies, including multidrug resistance (MDR) mechanisms. Although transporting/binding proteins, including permeability glycoprotein (P-gp), lung resistance protein (LRP) and metallothionein (MT), are frequently associated with tumour aggressiveness and unresponsiveness to chemotherapy in human breast cancer, they have not been analysed in FMCs. We investigated the immunoexpression of P-gp, LRP and MT in FMCs and their correlation with clinicopathological parameters and overall survival (OS) time in 46 FMCs, with a median follow-up period of 289 days. These markers were co-expressed in 85% of tumours. P-gp was expressed in 93.4% of FMCs and was positively associated with tumour grade (P=0.049). While unequivocally observed in all FMCs, LRP immunoexpression did not correlate with any clinicopathological parameters or OS. Expression of MT was significant in triple-negative basal- and normal-like molecular subtypes of FMCs (P=0.023). The concurrent expression of MDR proteins indicates the potential existence of chemotherapy resistance-related mechanisms in FMCs. The positive association between P-gp and MT immunoexpression and aggressive phenotypes could open new therapeutic and translational strategies for FMCs.

Diagnostic Value of VEGF-A, VEGFR-1 and VEGFR-2 in Feline Mammary Carcinoma.

Vascular 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, VEGFR-1, 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.

Adjuvant doxorubicin vs metronomic cyclophosphamide and meloxicam vs surgery alone for cats with mammary carcinomas: A retrospective study of 137 cases.

This study aims to evaluate the efficacy and side effects of low dose cyclophosphamide chemotherapy plus meloxicam as an adjuvant treatment, compared with high dose doxorubicin or surgery alone in cats with mammary carcinoma. Medical records of 228 female cats treated for mammary carcinoma between 2008 and 2018, were reviewed in eight veterinary institutions. Only cats with complete tumour staging and radical mastectomy were included in the study. One hundred and thirty-seven cats were divided into three treatment groups: group 1 (n=80) cats treated with surgery, group 2 (n=34) cats that had surgery and adjuvant treatment with doxorubicin, and group 3 (n=23) cats with surgery and adjuvant treatment with low dose metronomic cyclophosphamide and meloxicam. The study endpoints were disease free interval (DFI) and overall survival (OS). Toxicity was evaluated according to the VCOG-CTCAE criteria. The median DFI was 270, 226 and 372 days in groups 1, 2 and 3, respectively. The median OS was 338 (group 1), 421 (group 2) and 430 (group 3) days. The differences between groups were not significant (DFI P=.280 and OS P=.186). Toxicity was observed in 52.9% (n=18) of cats in group 2 and 39.1% (n=9) of cats in group 3, with mild to moderate intensity. Differences were not significant (P=.306). In conclusion, adjuvant chemotherapy treatment did not improve survival and the overall benefit remains unproven. Randomized prospective trials are necessary to clarify the effectiveness of adjuvant chemotherapy treatment for feline mammary carcinomas.

Serum Metabolomic Analysis of Feline Mammary Carcinomas based on LC-MS and MRM Techniques.

IntroductionTo date, there have been no panoramic studies of the serum metabolome in feline mammary carcinoma. As the first such study, metabolomics techniques were used to analyse the serum of cats with these tumours. Three important metabolic pathways of screened differential metabolites closely related to feline mammary carcinomas were analysed to lay a theoretical basis for further study of the pathogenesis of these carcinomas.Material and MethodsBlood in a 5–8 mL volume was sampled from twelve cats of the same breed and similar age (close to nine years on average). Six were feline mammary carcinoma patients and six were healthy. L glutamate, L alanine, succinate, adenine, hypoxanthine, and inosine were screened as were alanine, aspartate, and glutamate metabolism, the tricarboxylid acid (TCA) cycle, and purine metabolism. Data were acquired with LC-MS non-target metabolomics, multiple reaction monitoring target metabolomics, and multivariate statistical and bioinformatic analysis.ResultsExpression of five of the metabolites was upregulated and only inosine expression was downregulated. Up- and downregulation of metabolites related to glycometabolism, potentiation of the TCA cycle, greater content of lipid mobilisation metabolites, and abnormality of amino acid metabolism were closely related to the occurrence of the carcinomas.ConclusionThese findings provide a new direction for further study of the mechanisms associated with cat mammary neoplasms.

Metastatic feline mammary cancer: prognostic factors, outcome and comparison of different treatment modalities - a retrospective multicentre study.

Although feline mammary carcinomas (FMCs) are highly metastatic, the literature and treatment options pertaining to advanced tumours are scarce. This study aimed to investigate the clinical outcome of metastatic FMC with or without adjuvant treatment. The medical records of 73 cats with metastatic FMC (stage IV) were reviewed and included in this study. Metastatic disease was detected by distinct imaging techniques (radiography, ultrasound and CT) and confirmed by cytology and/or histopathology. Cats with adjuvant chemotherapy treatment (n = 34) were divided into three groups: group 1 (n = 9) cats receiving maximum tolerated dose chemotherapy; group 2 (n = 15) cats receiving metronomic chemotherapy; and group 3 (n = 10) cats treated with toceranib phosphate. The study endpoints were time to progression (TTP) and tumour-specific survival (TSS). Treatment-related toxicity was evaluated according to the Veterinary Co-operative Oncology Group's Common Terminology Criteria for Adverse Events version 1.1 (VCOG-CTCAE). Overall mean TTP and TSS were 23 and 44 days, respectively. Cats with clinical signs at the time of diagnosis had a lower TSS (14 days) than asymptomatic cats (128 days; P <0.001). Cats with pleural effusion had a lower TSS (16 days) than cats without (P <0.001). Median TSS was 58, 75 and 63 days in groups 1, 2 and 3, respectively (P = 0.197). Toxicity was observed in 66.7%, 20% and 30% of cats in groups 1, 2 and 3, respectively. To the best of our knowledge, this study includes the highest number of patients with metastatic FMC assessed. Despite the overall poor prognosis, some cats survived >6 months, indicating that adjuvant treatment may be an option to consider in metastatic disease. More studies are warranted for better understanding and management of stage IV patients.

One-year conditional survival of dogs and cats with invasive mammary carcinomas: A concept inspired from human breast cancer.

Numerous studies have described the prognostic factors of canine and feline mammary carcinomas (MCs), that is, variables that predict patient survival after diagnosis. But how does survival estimation evolve in patients that escaped early death from their cancer? In human oncology, conditional survival (CS), the probability of surviving X further years when cancer patients have already survived Y years, is used to analyse cancer outcomes in a long‐term perspective. In this cohort of 344 dogs and 342 cats with surgically removed stage I to III invasive MCs, with a minimal follow‐up of 2 years, we calculated the 1‐year CS, that is, the probability for patients that have survived 1 year, to survive or to die from cancer during the subsequent year. The 1‐year conditional specific survival probabilities were 59% and 48% at diagnosis of invasive MC respectively in dogs and cats, and 80% and 52% in 1‐year surviving dogs and cats respectively, suggesting that 1‐year surviving dogs were relatively protected from cancer‐related death, whereas feline MCs remained life‐threatening cancers for longer periods of time. Among the most significant parameters associated with CS in surviving dogs and cats were the nodal stage and lymphovascular invasion, as well as patient age, cancer stage and margin status in surviving dogs. By comparison, tumour size and the histological grade did not significantly alter CS probabilities in surviving dogs and cats. Conditional survival may be considered a very interesting tool for veterinary practitioners to estimate the likely outcome of cancer survivors.

Intratumoralcollagen signatures predict clinical outcomes in feline mammary carcinoma.

Breast cancer is the most common cause of cancer-related deaths in women worldwide. Identification of reliable prognostic indicators and therapeutic targets is critical for improving patient outcome. Cancer in companion animals often strongly resembles human cancers and a comparative approach to identify prognostic markers can improve clinical care across species. Feline mammary tumors (FMT) serve as models for extremely aggressive triple negative breast cancer (TNBC) in humans, with high rates of local and distant recurrence after resection. Despite the aggressive clinical behavior of most FMT, current prognostic indicators are insufficient for accurately predicting outcome, similar to human patients. Given significant heterogeneity of mammary tumors, there has been a recent focus on identification of universal tumor-permissive stromal features that can predict biologic behavior and provide therapeutic targets to improve outcome. As in human and canine patients, collagen signatures appear to play a key role in directing mammary tumor behavior in feline patients. We find that patients bearing FMTs with denser collagen, as well as longer, thicker and straighter fibers and less identifiable tumor-stromal boundaries had poorer outcomes, independent of the clinical variables grade and surgical margins. Most importantly, including the collagen parameters increased the predictive power of the clinical model. Thus, our data suggest that similarities with respect to the stromal microenvironment between species may allow this model to predict outcome and develop novel therapeutic targets within the tumor stroma that would benefit both veterinary and human patients with aggressive mammary tumors.

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.

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.

Expression of HIF-1\u03b1 and VEGF in feline mammary gland carcinomas: association with pathological characteristics and clinical outcomes

BackgroundThe microenvironment within solid malignant tumors, including feline mammary gland carcinomas (FMGCs), is commonly hypoxic, possibly due to the lack of functional blood vessels in rapidly proliferating neoplastic tissue. Malignant cells can undergo genetic and adaptive changes that prevent them from dying due to oxygen deprivation through expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF). Therefore, HIF-1α and VEGF are ideal biomarkers for cancer therapy and prognostic evaluation. The aims of this study were to evaluate the expression of HIF-1α and VEGF in feline mammary carcinomas and analyze their correlations with clinical and pathological factors, such as clinical stage, histologic grading, regional metastasis, and overall survival rate.ResultsParaffin-embedded tissue samples collected from 72 cats with FMGCs were retrospectively studied. Histologic pattern and histologic grading (Elston and Ellis grading system) of these FMGCs were determined. Our data indicated that grade II tubulopapillary carcinomas (43/72, 59.7%) prevailed in this study, and most FMCGs showed apparent necrosis, squamous metaplasia, and intratumoral stromal response. According to the results of immunohistochemical (IHC) stainings performed in tissue microarrays (TMAs), HIF-1α and VEGF overexpressions were respectively noted in 69.4% (50/72) and 77.8% (56/72) of FMGC cases. Chi-square test showed no correlation of HIF-1α overexpression with clinical and pathological factors. VEGF overexpression was significantly correlated with histologic pattern (p = 0.021), stromal response (p = 0.048), squamous metaplasia (p = 0.001), and lymphovascular invasion (p = 0.007). However, neither HIF-1α nor VEGF overexpression was correlated with histologic grading and metastasis. Of 38 cats with 1-year follow-up, IHC stainings of HIF-1α and VEGF were performed on whole tissue sections. The results showed that overexpression of HIF-1α was significantly correlated with the overall survival rate (p < 0.05) (log-rank test), whereas there was no significant correlation between VEGF overexpression and overall survival rate.ConclusionsThis study suggests that the overexpression of HIF-1α may indicate poor prognosis/overall survival rate in cats with FMGCs. Developing compounds that inhibit HIF-1α may be a potential approach to FMGC treatment.