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The use of tyrosine kinase inhibitors (TKI) has been growing in veterinary oncology and in the past few years several TKI have been tested in dogs. However, different from human medicine, we lack strategies to select patients to be treated with each TKI. Therefore, this study aimed to screen different tumor subtypes regarding TKI target immunoexpression as a predictor strategy to personalize the canine cancer treatment. It included 18 prostatic carcinomas, 36 soft tissue sarcomas, 20 mammary gland tumors, 6 urothelial bladder carcinomas, and 7 tumors from the endocrine system. A total of 87 patients with paraffin blocks were used to perform immunohistochemistry (IHC) of human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptors 1 (EGFR1), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet derived growth factor receptor beta (PDGFR-β), c-KIT, and extracellular signal-regulated kinase 1/2 (ERK1/ERK2). The immunohistochemical screening revealed a heterogeneous protein expression among histological types with mesenchymal tumors showing the lowest expression level and carcinomas the highest expression. We have demonstrated by IHC screening that HER2, EGFR1, VEGFR-2, PDGFR-β and ERK1/ERK2 are commonly overexpressed in dogs with different carcinomas, and KIT expression is considered relatively low in the analyzed samples.

An unusual variant of prostate adenocarcinoma (PC) expressing nuclear p63 in secretory cells instead of the typical basal expression has been reported in men. Nevertheless, the biological behavior and clinical significance of this phenomenon is unknown. In dogs, this unusual PC subtype has not been described. In this study, p63 immunoexpression was investigated in 90 canine PCs and 20 normal prostate tissues (NT). The p63 expression pattern in luminal or basal cells was confirmed in a selected group of 26 PCs and 20 NT by immunohistochemistry and/or Western blotting assays. Eleven canine PC samples aberrantly expressing p63 (p63+) in secretory cells were compared with 15 p63 negative (p63-) cases in the context of several molecular markers (high molecular weight cytokeratin-HMWC, CK8/18, CK5, AR, PSA, chromogranin, NKX3.1, PTEN, AKT and C-MYC). P63+ samples were positive for CK5, HMWC and CK8/18 and negative for PSA, NKX3.1, PTEN and chromogranin. Five p63+ PCs were negative for AR, and the remaining six samples had low AR expression. In contrast, p63- PC showed AR and PSA positive expression in all 15 samples. Only five p63- PCs were positive for CK5. Both p63+ and p63- PC samples showed higher cytoplasmic AKT expression and nuclear C-MYC staining in comparison with normal tissues. Metastatic (N = 12) and non-metastatic (N = 14) PCs showed similar immunoexpression for all markers tested. In contrast to human PC, canine PC aberrantly expressing p63 showed higher expression levels of HMWC and CK5 and lower levels of NKX3.1. Canine p63+ PC is a very rare PC group showing a distinct phenotype compared to typical canine PC, including AR and PSA negative expression. Although in a limited number of cases, p63 expression was not associated with metastasis in canine PC, and cytoplasmic p63 expression was observed in animals with shorter survival time, similar to human PC cases., Competing Interests: The authors have declared that no competing interests exist.

Background: c-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines. Results: Eleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive. Conclusion: The present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer. [ABSTRACT FROM AUTHOR]

The epithelial-mesenchymal transition (EMT) is a fundamental event responsible for the invasiveness and metastasis of epithelial tumours. The EMT has been described in many human cancers, but there are few reports of this phenomenon in veterinary oncology. Due to the importance of this process, the current study evaluated mesenchymal and epithelial marker protein expression in prostate lesions from dogs. Our results indicate both a loss of E-cadherin and translocation of β-catenin from the membrane to the cytoplasm and nucleus in the tumour group. Vimentin expression in the tumour group was higher than in normal tissue. All of the metastases were positive for prostate-specific antigen, pan-cytokeratin and E-cadherin, although fewer positive cells were present than in the primary tumours. The immunohistochemical results showed a loss of epithelial markers and a gain of a mesenchymal marker among metastatic cells, suggesting that the EMT occurs during the metastatic process of canine prostate carcinoma. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostatic diseases are important worldwide, being the prostate cancer (PC) the most common tumor in men. Among the factors associated with PC development, the preneoplastic lesions are well-recognized. Preneoplastic lesions are cellular morphological alterations, induced by different factors and present a potential to progression for PC. In this scenario, dogs are considered spontaneous models. Dogs naturally develops prostatic hyperplasia, preneoplastic lesions and PC. Among the preneoplastic lesions, the proliferative inflammatory atrophy (PIA) develops spontaneously in dogs. PIA is an epithelial lesion induced by prostatic chronic inflammation, leading to a proliferative atrophy of the prostate gland. Thus, this study aimed to perform a full PIA morphological, phenotypical and molecular characterization in dogs. After reviewing the archives of the veterinary pathology service, it was identified 171 dogs containing PIA in the prostate gland, and among the PC cases (N = 84), it was identified PIA lesions surrounding 60.7% of PC cases. Besides that, we identified loss of genes related to the maintenance of prostatic tissue and can predispose to malignant transformation. Moreover, mutations in androgen receptor gene were identified, demonstration alteration in DNA in PIA. Overall, these results support the hypothesis that PIA can be considered a preneoplastic lesion in canine prostate. Proliferative inflammatory atrophy (PIA) is an atrophic lesion of the prostate gland that occurs in men and dogs and is associated with a chronic inflammatory infiltrate. In this study, we retrospectively reviewed canine prostatic samples from intact dogs, identifying 50 normal prostates, 140 cases of prostatic hyperplasia, 171 cases of PIA, 84 with prostate cancer (PC), 14 with prostatic intraepithelial neoplasia (PIN) and 10 with bacterial prostatitis. PIA samples were then selected and classified according to the human classification. The presence of PIA lesions surrounding neoplastic areas was then evaluated to establish a morphological transition from normal to preneoplastic and neoplastic tissue. In addition, the expression of PTEN, P53, MDM2 and nuclear androgen receptor (AR) were analyzed in 20 normal samples and 20 PIA lesions by immunohistochemistry and qPCR. All PIA lesions showed variable degrees of mononuclear cell infiltration around the glands and simple atrophy was the most common histopathological feature. PIA was identified between normal glands and PC in 51 (61%) out of the 84 PC samples. PIA lesions were diffusely positive for molecular weight cytokeratin (HMWC). Decreased PTEN and AR gene and protein expression was found in PIA compared to normal samples. Overall, our results strongly suggest that PIA is a frequent lesion associated with PC. Additionally, this finding corroborates the hypothesis that in dogs, as is the case in humans, PIA is a pre neoplastic lesion that has the potential to progress into PC, indicating an alternative mechanism of prostate cancer development in dogs. [ABSTRACT FROM AUTHOR]

Basal cells have an important role in the carcinogenic process and few studies evaluate their role in animals cancer development. Due to this importance we evaluated by immunohistochemistry the basal cells in prostatic proliferative inflammatory atrophy (PIA) and prostatic carcinoma (PCa). We found higher number of cases with more than 75% of positive basal cells in CaP and PIA when compared to normal tissue. PCa and PIA showed 34βE12 protein expression when compared to normal prostatic tissue. Ki67 showed higher proliferative index in PCa and PIA compared to normal tissue. Higher p63, 34βE12 and Ki67 immunohistochemical staining in PIA and PCa indicate the involvement of basal cells in these lesions in dogs, different from humans. [ABSTRACT FROM AUTHOR]

In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines. • There are differences in follicular cryotolerance according to the cryopreservation technique used. • Primordial and immature follicles are less prone to degeneration when cryopreserved by slow freezing. • Mature follicles are more sensitive to cryodamage and better preserved by vitrification. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages (TAM) are related to poor prognosis in canine mammary tumors (CMT). An association between TAM and sustained angiogenesis has been implicated with malignancy; however, comparison of the expression of TAM and vascular endothelial growth factor (VEGF) in different histologic types of CMT has not been completely studied. The objective of this study was to evaluate the immunostaining of TAM and VEGF in four different types of malignant CMT. Ninety-nine mammary carcinomas [tubular (29), tubulopapillary (22), mixed-type (28) and solid carcinoma (20)] were evaluated by immunohistochemical staining using MAC387 and VG-1 to detect TAM and VEGF immunoexpression, respectively. The total number of positive macrophages within the tumor was used for TAM immunolabeling and a score of four categories was used for VEGF immunolabeling. TAM immunoexpression was found to be statistically higher in tubular carcinomas than in solid carcinomas (P=0.0015). Differences between other types of carcinoma were not statistically significant. VEGF score was higher in tubular, papillary and mixed-type carcinomas than in solid carcinomas (P<0.0001). A positive relationship between the highest mean value of TAM and VEGF immunoexpression was also found (P=0.0015). TAM and VEGF expressions can be lost in more aggressive CMT, as in solid carcinoma. Furthermore, regardless of histologic type, higher numbers of TAM are associated with higher scores of VEGF, thus favoring the relationship between TAM and angiogenesis. [ABSTRACT FROM AUTHOR]