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This study investigates the molecular and immune characteristics of equine ocular squamous cell carcinoma (eoSCC). Immunohistochemistry (IHC) and next-generation sequencing (NGS) were used to detect protein expression and TP53 mutations, respectively. T lymphocytes (CD3 + ), regulatory T cells (FoxP3 + ), B lymphocytes (CD20 + ), and macrophages (IBA-1 + ) were quantified. A total of 29 cases of eoSCC were evaluated, consisting of 3/29 carcinomas in situ (CISs) and 26/29 squamous cell carcinomas (SCCs). p53 positivity by IHC was detected in 19/29 cases while by NGS, 21 TP53 mutations were found in 13/29 cases (44.83%), of which 18/21 were C > T base substitutions, typical of ultraviolet (UV)-induced DNA damage. In tumors with TP53 mutations, IBA-1⁺ macrophages were significantly increased ( p  = 0.001) and CD3⁺ T lymphocytes were also more abundant ( p  = 0.028) than in wild type TP53 cases, whereas CD20⁺ B lymphocytes and FoxP3⁺ regulatory T lymphocytes showed no significant differences. Equus caballus papillomavirus type 2 positivity was detected in 6/29 cases (20.69%) via in situ hybridization (ISH), but viral presence did not impact immune cell infiltration. Ki67 scores were higher in SCCs/CISs with TP53 mutations, but the difference was not statistically significant. Overall, TP53 mutations appear to contribute to eoSCC development, potentially as a consequence of UV-light exposure, and to influence immune cell infiltration.

Necrosis seen in histopathology Whole Slide Images is a major criterion that contributes towards scoring tumour grade which then determines treatment options. However conventional manual assessment suffers from inter-operator reproducibility impacting grading precision. To address this, automatic necrosis detection using AI may be used to assess necrosis for final scoring that contributes towards the final clinical grade. Using deep learning AI, we describe a novel approach for automating necrosis detection in Whole Slide Images, tested on a canine Soft Tissue Sarcoma (cSTS) data set consisting of canine Perivascular Wall Tumours (cPWTs). A patch-based deep learning approach was developed where different variations of training a DenseNet-161 Convolutional Neural Network architecture were investigated as well as a stacking ensemble. An optimised DenseNet-161 with post-processing produced a hold-out test F1-score of 0.708 demonstrating state-of-the-art performance. This represents a novel first-time automated necrosis detection method in the cSTS domain as well specifically in detecting necrosis in cPWTs demonstrating a significant step forward in reproducible and reliable necrosis assessment for improving the precision of tumour grading.

Canine liposarcoma is an uncommon tumor that shares morphological similarities with its human counterpart. In dogs, the genetic features of this tumor are unknown and, based on immunohistochemical studies, amplification of the gene MDM2 and the mutation of TP53 are suspected. In this study 51 cases of primary liposarcomas were immunohistochemically stained for MDM2 and p53 and subjected to fluorescent in situ hybridization and next-generation sequencing to detect MDM2 amplification and TP53 mutations, respectively. MDM2 and p53 were expressed in 21 and 6 cases, respectively. MDM2 amplification and TP53 mutations were identified in 10 and 15 cases, respectively. Statistical analysis revealed an association of the myxoid subtype and the mitotic count with p53 expression and TP53 mutation. No association was found between  MDM2 amplification and MDM2 expression or tumor subtype. These results suggest that despite morphological similarities, canine liposarcoma differs from its human counterpart, for which MDM2 amplification is diagnostic for well differentiated and de-differentiated variants, and TP53 mutations are more common in pleomorphic liposarcoma rather than the myxoid one as occur in our cases. Furthermore, canine myxoid liposarcoma likely represents a distinct disease rather than a mere morphological variant.

Feline nasal carcinomas are rare but clinically aggressive neoplasms. This study characterizes their histopathological features and evaluates HER2, p53, Ki-67, and PCNA expression using immunohistochemistry and digital image analysis, aiming to provide a comprehensive biological characterization with potential prognostic and therapeutic implications. Tumors were classified into adenocarcinomas (AC) and non-adenocarcinomas (non-AC). Among the 23 cases examined, adenocarcinoma was the most common subtype (17 cases). HER2 was scored as 3+ in 7 cases, 2+ in 8 cases, 1+ in 5 cases, and 3 cases were scored 0. A statistically significant association was found between histological type and HER2 expression (Fisher’s exact test, p = 0.02), with a higher prevalence of HER2 positivity in adenocarcinomas. Evaluation of p53 expression according to histological grouping showed a trend toward significance (p = 0.0593), with p53 positivity observed exclusively in non-AC. The Ki-67 index had a median of 4.4 (min 0.5, max 21.06), and the PCNA index had a median of 82.26 (min 19.55, max 100). No significant associations were identified between the Ki-67 labeling index and HER2 expression, histotype, and the inflammatory infiltrate. Finally, Pearson correlation analysis revealed no significant correlation between Ki-67 and PCNA indices (p = 0.32). The overexpression of HER2 lays the groundwork for the possible use of anti-HER2 targeted drugs in this tumor type, particularly in adenocarcinomas. These findings provide baseline immunohistochemical data for feline nasal carcinomas and highlight HER2 as a relevant biomarker for future diagnostic and therapeutic research.

Canine anal sac gland adenocarcinomas (ASACs) are locally aggressive and highly metastatic to regional lymph nodes. Tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) can be effective prognostic and predictive markers in numerous human neoplasms and are increasingly investigated in dogs. The aim of this study was to characterize immune cells in canine ASACs and their relationship with tumor size, histologic metastatic status, and tumor clinical stage. Thirty ASACs with known tumor size, metastatic status, and clinical stage were immunolabeled for Iba1 (macrophages), CD20 (B cells), CD3 (T cells), and Foxp3 (regulatory T cells). With image analysis, two areas of 1 mm2 were analyzed for each case at the tumor core (TC) and invasive margin (IM) and immune cells were counted. Eighteen patients had metastasis at the time of diagnosis, of which fifteen were nodal only, and three were both distant and nodal. The median tumor size was 32.5 mm (range 11–70). The clinical stage was I in five cases, II in seven cases, III in fifteen cases, and IV in three cases. T cells and macrophages were the most abundant immune cells in all tumors. Tumor size did not influence the number or type of infiltrating immune cells. By contrast, significantly higher numbers of TC T lymphocytes were found in patients without metastasis, while significantly higher numbers of TC macrophages were found in dogs with metastasis. Immune cell infiltrate did not differ according to clinical stage. The results indicate that the tumor immune microenvironment, specifically TILs and TAMs, contribute to tumor behavior and may influence metastatic potential; in particular, high CD3 infiltration may prevent tumor progression, while increased macrophage infiltration could promote it.

HER2 overexpression has been reported in various human and canine tumours. The aim of this study is to investigate the expression of HER2 protein in different histotypes of canine carcinomas in order to identify potential tumours that could benefit from the HER2-targeted therapy. Eighty-two (82) canine carcinomas (squamous cell, gastro-intestinal, rectal, pulmonary, prostatic, urothelial, and ovarian) from paraffin-embedded samp les were immunohistochemically evaluated. The degree of HER2 expression was scored based on the ASCO/CAP 2018 guidelines. Intestinal carcinomas were those with greater HER2 overexpression (3+) with 81% of positive cases, followed by 42% of rectal carcinomas and 28% of squamous cell carcinomas. These observations suggest that HER2 overexpression could be a driver in the oncogenesis of several types of canine carcinomas and lay the foundations for the identification of different types of canine carcinomas that could benefit from HER2-targeted therapy.

In human medicine, p53 immunohistochemistry (IHC) is a common method that is used for the identification of tumors with TP53 mutations. In veterinary medicine, several studies have performed IHC for p53 in canine tumors, but it is not known how well it actually predicts the mutation. The aim of this study was to estimate the accuracy of the IHC method for p53 (clone PAb240) using a lab-developed NGS panel to analyze TP53 mutations in a subset of malignant tumors in dogs. A total of 176 tumors were analyzed with IHC and then 41 were subjected to NGS analysis; among them, 15 were IHC positive and 26 were negative, and 16 out of 41 (39%) were found to be inadequate for NGS analysis. Excluding the non-evaluable cases at NGS, of the remaining eight IHC-positive cases, six were mutants and two were wild-type. Among the 17 IHC-negative cases, 13 were wild type, and 4 were mutants. The sensitivity was 60%, specificity was 86.7%, and the accuracy was 76%. These results suggest that when using IHC for p53 with this specific antibody to predict mutation, up to 25% wrong predictions can be expected.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of increasing prevalence marked by poor prognosis and limited treatment options. Ca2+-activated KCa3.1 potassium channels have been shown to play a key role in the aberrant activation and responses to injury in both epithelial cells and fibroblasts, both considered key drivers in the fibrotic process of IPF. Pharmacological inhibition of IPF-derived fibroblasts is able to somewhat prevent TGF-β– and basic fibroblast growth factor-dependent profibrotic responses. In the current study, we investigated whether blockade of the KCa3.1 ion channel in vivo with a selective inhibitor, Senicapoc, was able to attenuate both histological and physiological outcomes of early fibrosis in our large animal (sheep) model for pulmonary fibrosis. We also determined whether treatment was targeting the profibrotic activity of sheep lung fibroblasts. Senicapoc was administered in established fibrosis, at 2 weeks after bleomycin instillation, and drug efficacy was assessed 4 weeks after treatment. Treatment with Senicapoc improved pre-established bleomycin-induced changes compared with vehicle control, leading to improved lung compliance, reduced extracellular matrix and collagen deposition, and a reduction in both α-smooth muscle actin expression and proliferating cells, both in vivo and in vitro. These studies show that inhibiting the KCa3.1 ion channel is able to attenuate the early fibrogenic phase of bleomycin-dependent fibrosis and inhibits profibrotic behavior of primary sheep lung fibroblasts. This supports the previous research conducted in human IPF-derived fibroblasts and suggests that inhibiting KCa3.1 signaling may provide a novel therapeutic approach for IPF.

Recently, human epidermal growth factor receptor 2 (HER2) has emerged as a therapeutic target of interest for non-small-cell lung cancer in humans. The role of HER2 in canine pulmonary adenocarcinomas is poorly documented. To address this gap, this study employed three methodologies: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS) to investigate the protein expression, gene amplification, and mutation of HER2 in 19 canine primary pulmonary adenocarcinomas. By IHC, 3 out of 19 cases were overexpressed 3+, 6 were 2+, and 10 were negative. With FISH, 2 cases were amplified (12.5%), 3 were inadequate for the analyses, and the others were non-amplified. With NGS, seven cases were inadequate. All other cases were wild-type, except for one IHC 3+ case, which was amplified with FISH and with a specific mutation already described in human pulmonary adenocarcinoma, V659E. This mutation is probably sensitive to tyrosine kinase inhibitory drugs. These results are similar to those in human medicine and to the few data in the literature on canine lung carcinomas; the presence of 12.5% of amplified cases in dogs lays the foundation for future targeted drugs against HER2 alterations.

Canine hepatozoonosis caused by Hepatozoon canis is an emerging disease in Europe. Clinical pictures vary from subclinical to life-threatening and non-specific clinical signs are predominantly reported. A 2-month-old female puppy originating from Southern Italy was adopted and moved to Northern Italy. Then, the dog was brought to a local veterinary practice for gastrointestinal signs, migrating lameness and pruritic dermatitis, and then tested positive for Hepatozoon spp. gamonts at the blood smear. After treatment with imidocarb dipropionate and doxycycline, the dog showed an initial clinical improvement. However, gastrointestinal signs recurred, and diffuse superficial pyoderma appeared on the thoracolumbar region, along with fever, lethargy, and weight loss. Eight months from the first onset of clinical signs, the dog was referred to a veterinary clinic and subjected to complete blood count, urine and fecal analysis, along with abdominal ultrasonography, whole-body CT and gastroduodenal endoscopy. Skin biopsies and blood samples were subjected to a PCR-coupled sequencing protocol, which scored both positive for H. canis. Alterations were consistent with a pre-existing cholangiohepatitis and multiple acquired extrahepatic shunts secondary to portal hypertension. The dog was euthanatized due to a clinical worsening two months later. The potential role of H. canis in the systemic disease observed, clinic-pathological findings and epizootiological implications are discussed.