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 withTP53 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.

Nasal hamartoma (NH) is a rare nonneoplastic excessive growth of native tissue of the nasal cavity. Four histological subtypes are described in cats and dogs: mesenchymal nasal hamartoma (MNH), epithelial hamartoma, mixed tissue (chondro-osseous and respiratory epithelial adenomatoid hamartoma/COREAH), and nasal vascular hamartoma. This descriptive, retrospective study aims to describe the computed tomographic (CT) features of feline NHs. Fourteen cats were included (six MNH, six COREAH, one epithelial, and one vascular). Twelve MNH and COREAH were soft-tissue and mineral-attenuating locally invasive nasal masses with expansile behavior (nine unilateral and three bilateral). The nasal turbinate architecture was replaced by exuberant/disorganized mineral densities arranged in a "cerebroid pattern" in all cases. Contrast enhancement was moderate in 75% of cats. Commonly, the mass extended into the rostral nasopharynx, contralateral choana/nasal cavity, and nostrils, more rarely into the orbital cavity and/or facial soft tissue. Focal osteolysis/atrophy of the paranasal bones was common (10/12 cases). The epithelial and vascular hamartomas were soft tissue-attenuating unilateral masses without mineral components. Contrast enhancement was marked in the vascular hamartoma. NHs are relatively rare, tumor-like growths in cats that mainly contain mesenchymal elements. Common CT features include expansile nasal lesions that are locally invasive, characterized by a mineral "cerebroid pattern." Despite the low incidence, it is crucial to be aware of their presence and include them in the differential diagnosis of feline nasal masses.

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.

Angiogenesis, a hallmark of cancer, supports tumour growth and metastasis by establishing an abnormal vascular network, and microRNAs (miRNAs) regulate this process post-transcriptionally. Because evidence in canine mammary tumours (CMTs) remains limited, we profiled 24 putative pro- and anti-angiogenic miRNAs by RT-qPCR in benign and malignant CMTs compared with normal mammary glands, and we predicted angiogenesis-related targets using multiMiR followed by Gene Ontology and KEGG pathway enrichment analyses. Intratumoral angiogenesis was quantified as microvascular density (MVD) and endothelial area (EA) on Factor VIII-immunolabeled sections using QuPath. MVD and EA were higher in malignant than in benign CMTs and peaked in grade III carcinomas. Malignant CMTs showed a progressive shift towards a pro-angiogenic miRNA profile, with significant upregulation of pro-angiogenic miR-9, miR-20a, miR-98, miR-210, and miR-21(p < 0.05). Conversely, anti-angiogenic miRNA displayed a heterogenous, context-dependent expression pattern: miR-152-3p and miR-542-3p were downregulated in benign CMTs relative to normal mammary tissue, whereas miR-205 and miR-34a were upregulated in malignant CMTs (p < 0.05). In malignant CMTs, MVD correlated with EA (r = 0.8, p = 0.0003), EA correlated with miR-98 (r = 0.67, p = 0.006), and tumour size correlated with miR-210 (r = 0.58, p = 0.03). In benign tumours, EA correlated with miR-497 (r = 0.81, p = 0.02). Target prediction identified 16,910 genes, with pro- and anti-angiogenic miRNAs sharing 86.5% of predicted targets, indicating extensive regulatory overlap. KEGG enrichment highlighted 100 significantly enriched pathways (FDR < 0.05), including MAPK, PI3K-Akt, HIF-1, VEGF, and breast cancer signalling, with MAPK1 and MAPK3 among the most frequently targeted genes. Finally, miR-34a showed the best diagnostic performance for distinguishing benign from malignant CMTs. Overall, findings support a substantial contribution of miRNAs to angiogenic regulation in CMTs, strengthen the utility of the canine model in comparative breast cancer research, and highlight the potential of miRNA-based biomarkers for tumour stratification and anti-angiogenic targeting.

The Sicilian Black pig is a well-adapted autochthonous breed, yet its susceptibility to respiratory disease remains poorly defined due to limited data on pathogen circulation and lesion patterns in extensive production systems. This study characterised the pathological features of Porcine Respiratory Disease Complex (PRDC) in this breed by examining lungs and tracheobronchial lymph nodes from 59 animals through pathological assessment, molecular detection of major respiratory pathogens, and evaluation of crystalline particulates using scanning electron microscopy coupled with energy-dispersive X-ray analysis to characterise infectious and environmental contributors. Gross examination revealed multifocal cranioventral bronchopneumonia in 35/59 pigs (59.32

Injections have been linked to feline sarcomas (feline injection-site sarcoma; FISS) and cutaneous lymphomas (cutaneous lymphoma at injection site; CLIS). Both tumors often exhibit lymphoplasmacytic inflammation ascribed to injected immunogenic material. CLIS is hypothesized to emerge from transformation and clonal expansion of lymphoid cells following persistent immune stimulation with feline leukemia virus (FeLV) reactivation and transformation. To further study whether the lymphocytic infiltrates associated with FISS can represent a suitable niche for the development of CLIS, 34 cases of FISS were examined. Lymphoid cell phenotypes were assessed using CD3 and CD79 immunohistochemistry. For cases with prominent inflammation, FeLV p27 and gp70 immunohistochemistry and PCR for antigen receptor rearrangements were performed. Male domestic shorthair cats predominated. The mean age was 12.2 years (range: 5-17 years). FISS developed in thoracic (8/34, 24%), flank (7/34, 21%), and interscapular (5/34, 15%) regions. Similar proportions of B and T lymphocytes were found in 11/34 (32%) cases; T-cells predominated in 12/34 (35%) cases, and B-cells predominated in 11/34 (32%). At least one FeLV antigen was expressed in lymphoid infiltrates in 10/18 cases (55%), and in neoplastic fibroblasts in 8/18 cases (44%), while both FeLV proteins were expressed in neoplastic cells in 3/18 cases (17%). One cat had clonal T-cell receptor-gamma and was diagnosed with concurrent FISS and CLIS. This case lacked FeLV expression. FeLV amplification from formalin-fixed paraffin-embedded material was unsuccessful. The expression of FeLV p27 and/or gp70 in neoplastic spindle cells and lymphoid infiltrates raises the possibility of FeLV involvement in the tumorigenesis of FISS and CLISs.

Equine penile tumors are common in horses and are often related to infection with equine papillomavirus type 2 (EcPV2). This study investigated the immune cell infiltrate (ICI) of these tumors in horses, focusing on the role of EcPV2. Using multiplex immunohistochemistry (mIHC) for CD3, CD20, and IBA-1 and immunohistochemistry (IHC) for FoxP3, 27 horses with papillomas (5/27), in situ carcinomas (CISs) (3/27), and squamous cell carcinomas (SCCs) (19/27) were evaluated. Eighteen cases tested positive for EcPV2 by either or both in situ hybridization (ISH) and polymerase chain reaction (PCR) (18/27 by PCR, of which 16 were ISH+). The ICIs were more abundant in EcPV2-positive tumors, although differences were not statistically significant. The number of FoxP3+ regulatory T-cells was significantly higher in EcPV2+ tumors, both in intraepithelial and stromal compartments. There were higher IBA-1+ macrophage densities in SCCs than in papillomas or CISs. p53 IHC was performed, and non-basal positivity was associated with malignancy. The TP53 mutational analysis with next-generation sequencing revealed that 13/21 cases had a wild-type TP53, while TP53 variants were detected in 4/21 cases. The ICIs did not vary according to TP53 status. Tumor proliferation was also assessed with Ki67, which indicated progressively higher proliferation from benign to malignant tumors. In conclusion, although the number and distribution of B-cells, T-cells, and macrophages did not vary according to EcPV2 status, FoxP3 regulatory T-cells were observed in significantly higher numbers in EcPV2+ neoplasms, indicating a different immune landscape compared to EcPV2-negative tumors.

Canine anal sac adenocarcinoma (ASAC) is an aggressive malignancy with high metastatic potential. Histologic and proliferation parameters such as mitotic count and Ki67 scores have limited prognostic value according to the published literature. Using pathologist-supervised digital image analysis methods with the image analysis software QuPath, we analyzed 58 cases of ASAC to evaluate mitotic count (MC) and Ki67 indices, explore relationships between different Ki67 indices [semi-automatic Ki67 digital hotspot score (Ki67-saHDS), Ki67 global digital score (Ki67-GDS), and fully automatic Ki67 digital hotspot score (Ki67-faHDS)] and MC, and to verify which method carries the most significant prognostic value. The MC did not impact median tumor-related survival (TRS) time. Although high correlation coefficients were observed between the 3 Ki67 scores, Ki67-GDS had more prognostic relevance than hotspot-based scores (Ki67-saHDS and Ki67-faHDS). Dogs with Ki67-GDS ≥ 26 had significantly shorter survival times (175, days 95%, confidence interval (95% CI) = 123-540) compared to dogs with Ki67-GDS< 26 (median survival time (MST) 650 days, 95% CI = 503->1579). No association was observed between TRS and Ki67-faHDS or Ki67-saHDS. On multivariate analysis, anisokaryosis and Ki67-GDS, but not tumor size, lymphovascular invasion, or MC, were independent prognostic markers for survival. These results demonstrate the advantage of Ki67 GDS over hotspot-based scores; however, these data need to be validated in a larger cohort of cases before clinical implementation.

Mitotic count (MC) is a well-established prognostic factor in many canine malignancies. While standardisation efforts have improved inter-pathologist agreement regarding the morphology of mitotic figures and the size of the counting area, the selection of the tumour region for MC assessment remains to be standardised. This study aimed to evaluate the spatial distribution of the most proliferative areas in selected canine tumour types, using Ki67 immunohistochemistry, to identify optimal candidate regions for MC assessment. Tumour types analysed included melanomas, cutaneous mast cell tumours (cMCT), canine mammary carcinomas (CMC) and soft tissue sarcomas (STS). Using image analysis, Ki67 density maps were generated from digital slides and classified according to their distribution pattern (focal/multifocal or diffuse) and location within the tumour (central, peripheral or scattered). A total of 202 cases were included: 43 melanomas, 30 cMCTs, 42 CMCs and 87 cSTSs. The vast majority of tumours (92.6%) exhibited a multifocal hotspot distribution. Peripheral hotspot localisation was predominant in 55% of cases, particularly in cMCTs (73.3%) and melanomas (76.7%). In contrast, cSTSs more frequently showed a scattered hotspot pattern (60.9%) (χ2 = 41.9; p < 0.001). CMCs had a higher proportion of centrally located hotspots (16.7%). These findings suggest that pathologists should focus on peripheral tumour regions when assessing MC in cMCTs and melanomas. Both central and peripheral regions should be considered in CMCs, while a more extensive, comprehensive evaluation may be required in STSs. The observed association between tumour histotype and proliferation pattern likely reflects inherent biological differences among the tumour types studied.

Oral mucosal melanomas (OMMs) are the most frequent oral malignancy in dogs, often characterized by aggressive local behavior and a high metastatic rate. The mechanisms that drive canine OMM metastasis are still largely unknown, providing for limited therapeutic approaches once the disease has spread to metastatic sites. The objective of this investigation was to evaluate the differences in gene expression between canine primary OMMs and their matched nodal metastases. Transcriptional profiling of formalin-fixed, paraffin-embedded biopsies of 4 canine OMMs and their respective lymph node biopsies was performed using exon microarrays. Confirmation of the differential expression of selected genes was subsequently sought by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) on 13 paired samples (primary tumor-metastatic lymph node). Results highlight the activation of pathways associated with actin cytoskeleton organization, and cellular motility and migration. In particular, transcriptional data indicated increased expression of genes associated with Rac1 signaling-regulated cell migration, including ELMO1, VAV3, and DOCK2, in nodal metastases. Overall, the results of this investigation point to a significant role for Rac1 signaling in the pathogenesis of OMM metastasis to regional lymph nodes. The Rac1 signaling-associated genes highlighted herein are indeed involved in the activation of cellular migration, and one, or more, may represent a future therapeutic target to prevent metastatic dissemination, or treat OMM with distant metastases.

Cyclooxygenase-2 (COX-2) is overexpressed in many human and animal cancers. Selective COX-2 inhibitors have shown antitumoral effects in tumors with a high expression of COX-2. This study evaluates (1) the expression of COX-2 in rabbit uterine adenocarcinomas, (2) the correlation between immunophenotypic expression and histopathological changes, and (3) the post-surgery response to therapy with COX-2 inhibitors. Forty rabbit uteri were divided into three groups: neoplastic, hyperplastic, and normal endometrium. A histological and immunohistochemical score was applied to investigate the tumor’s grade and the COX-2 expression. By histological evaluation, 30 cases of endometrial adenocarcinoma, 5 cases of endometrial hyperplasia and 5 normal endometria were found. Of the six cases of endometrial adenocarcinoma with follow-up available, four received a post-surgical treatment with meloxicam and two were treated by surgery alone. The survival time of the animals treated with meloxicam was longer than that observed in the untreated animals. A statistically significant difference in COX-2 IHS was observed between non-neoplastic endometrium and adenocarcinoma. The progressive increase in COX-2 expression from normal epithelium to carcinoma suggests that upregulation of COX-2 expression may play a role in tumor initiation and progression. Our findings suggest the possible use of COX-2 inhibitors in treating uterine adenocarcinoma in rabbits. Further study will be needed to confirm this hypothesis.

Rapid evaporative ionization mass spectrometry (REIMS) coupled with a monopolar handpiece used for surgical resection and combined with chemometrics has been previously explored by our research group (Mangraviti et al. in Int J Mol Sci 23(18):10562, 2022) to identify several mammary gland pathologies. Here, the increased sample size allowed the construction of three statistical models to distinguish between benign and malignant canine mammary tumours (CMTs), facilitating a more in-depth investigation of changes in cellular metabolic phenotype during neoplastic transformation and biological behaviour. The results demonstrate that REIMS is effective in identifying neoplastic tissues with an accuracy of 97%, with differences in MS spectra characterized by the relative abundance of phospholipids compared to triglycerides more commonly identified in normal mammary glands. The increased rate of phospholipid synthesis represents an informative feature for tumour recognition, with phosphatidylcholine and phosphatidylethanolamine, the two major phospholipid species identified here together with sphingolipids, playing a crucial role in carcinogenesis. REIMS technology allowed the classification of different histotypes of benign CMTs with an accuracy score of 95%, distinguishing them from normal glands based on the increase in sphingolipids, glycolipids, phospholipids, and arachidonic acid, demonstrating the close association between cancer and inflammation. Finally, dysregulation of fatty acid metabolism with increased signalling for saturated, mono- and polyunsaturated fatty acids characterized the metabolic phenotype of neoplastic cells and their malignant transformation, supporting the increased formation of new organelles for cell division. Further investigations on a more significant number of tumour histotypes will allow for the creation of a more extensive database and lay the basis for how understanding metabolic alterations in the tumour microenvironment can improve surgical precision.

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 splenic hemangiosarcoma has a high metastatic rate and short survival time. Currently, the main prognostic parameters are tumor stage and therapy, while data on histologic parameters, such as grade and Ki-67 expression, are scarce. The aims of this study were to compare two methods of assessment of Ki-67, verify their prognostic impact, and define a threshold value based on survival. Thirty-one cases of histologically diagnosed canine splenic hemangiosarcoma, which were treated with splenectomy and had full staging and follow-up information, were collected. Three were stage I, 17 stage II, and 11 stage III. The mean mitotic count (MC) was 23.9 (standard deviation [SD]: 22.1) and the median was 15 (range, 1-93). Immunohistochemistry for Ki-67 was performed, the Ki-67 labeling index (Ki-67LI) was assessed as a percentage of positive neoplastic nuclei per ≥500 cell, and the Ki-67 count (KI-67C) was defined as the average number of positive nuclei using a 1 cm2 optical grid performed in 5, 40× fields. The mean Ki-67LI and Ki-67C were 56.4% (SD: 38.7) and 27.2 (SD: 12.9) and medians were 51% (range, 8.2-55.2) and 26 (range, 5.5-148), respectively. Using a cut-off of 56% and 9, respectively, Kaplan-Meier survival curves showed an association of overall survival with Ki-67LI and MC. In addition to clinical stage, Ki-67LI maintained its prognostic value on multivariate analysis, supporting the role of Ki-67LI as an independent prognostic parameter. Based on these results, we propose a diagnostically applicable cut-off value of 56% for Ki-67LI as a prognostic parameter for canine splenic hemangiosarcoma.

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.

A cytological grading system for canine mast cell tumors (MCTs) has been developed, but its integration into clinical routine has been hindered due to its diagnostic limitations. The aim of this study was to assess the prognostic value of Ki-67 and argyrophilic nucleolar organizing region (AgNOR) markers in cytological MCT samples and to determine cut-off values for these markers in correlation with histopathological grading. Cytological samples were collected prior to surgical excision, and histopathological samples were obtained postsurgery from 45 dogs diagnosed with cutaneous mast cell tumors (MCTs). The cytological specimens were classified using a two-tier grading system, and their Ki-67 (average immunopositive nuclei per 100 cells) and AgNOR (average AgNOR counts per 100 nuclei) signaling was assessed. Through receiver operating characteristic (ROC) analysis, cut-off values for Ki-67 and Ki-67 × AgNOR were determined to better align with histopathological grading (classified as low or high grade according to Kiupel's scoring system). Without the inclusion of proliferative markers, there was a 73% agreement between cytological and histopathological grading. The prediction of histopathological grade was slightly more accurate when assessing Ki-67 and Ki-67 × AgNOR signaling in cytological specimens (75% and 80%, respectively) compared to the initial cytological grading. The cytological assessment of canine MCTs proves beneficial for the initial evaluation, and the incorporation of the evaluation of Ki-67 and AgNOR markers may assist in identifying diagnostically highly malignant MCTs.