Non-mosaic X monosomy (77,X) in a female dog with signs of virilization.

A 14-month-old female Miniature Poodle dog with an enlarged clitoris and asymmetry in the placement of the teats was subjected to clinical, histopathological, and genetic studies. Macroscopically, the uterus and fallopian tubes appeared normal, while both ovaries were diffusely altered. At histology, the ovarian parenchyma was almost completely effaced by a diffuse hyperplasia of theca cells with atretic primary follicles. Chromosome analysis showed pure (non-mosaic) X monosomy (77,X). This finding was confirmed by the highly sensitive droplet digital PCR (ddPCR) approach. Despite the observed virilization, molecular analysis did not show the presence of Y-linked genes (SRY, ZFY, and TSPY1) in the blood cells or ovary tissue. To the best of our knowledge, this is the first case of X monosomy in a dog associated with virilization.

Clinicopathologic features and biologic behavior of canine splenic nodules with stromal, histiocytic and lymphoid components.

The term fibrohistiocytic nodule has been discouraged in favor of specific pathologic entities, including complex nodular hyperplasia, splenic stromal sarcoma and histiocytic sarcoma. Nevertheless, the diagnosis of splenic lesions with mixed stromal, histiocytic and lymphoid components still remains a challenge due to lack of straightforward histologic criteria. Misestimation of the biologic behavior of these lesions may lead to detrimental consequences on the clinical management of patients. In this study, we retrospectively evaluated the clinicopathologic features and outcome of canine splenic nodular lesions with mixed components, to identify prognostic factors and histologic criteria of malignancy. Thirty-seven cases were included. Immunohistochemistry did not allow for further subclassification. Nine (24.3%) dogs died from disease-related causes after a median of 234 days (range, 48-1,247). One-, 2- and 3-year disease-specific survival rates were 80, 60, and 43%, respectively. When considering nodules with stromal cell atypia and at least one of mitotic count ≥9, presence of karyomegaly/multinucleated cells and lymphoid component <40%, half of these dogs died of disease-related causes with a median disease-specific survival time of 548 days (95% CI, 0-1216). In the remaining dogs, no disease-related death was reported (P < 0.001). Canine splenic nodular lesions with mixed stromal, histiocytic and lymphoid components and histologic criteria of malignancy may behave aggressively, leading to distant metastasis and death. In the absence of further criteria aiding their classification, and to better characterize their biologic behavior, we encourage the distinction of these complex splenic tumors from conventional sarcomas and histiocytic sarcomas.

Environmental risk factors for the development of oral squamous cell carcinoma in cats.

BACKGROUND: Risk factors for oral squamous cell carcinoma (OSCC) in cats are derived from a single study dated almost 20 years ago. The relationship between inflammation of oral tissues and OSCC is still unclear. OBJECTIVES: To investigate previously proposed and novel potential risk factors for OSCC development, including oral inflammatory diseases. ANIMALS: Hundred cats with OSCC, 70 cats with chronic gingivostomatitis (CGS), 63 cats with periodontal disease (PD), and 500 controls. METHODS: Prospective, observational case-control study. Cats with OSCC were compared with an age-matched control sample of client-owned cats and cats with CGS or PD. Owners of cats completed an anonymous questionnaire including demographic, environmental and lifestyle information. RESULTS: On multivariable logistic regression, covariates significantly associated with an increased risk of OSCC were rural environment (OR: 1.77; 95% CI: 1.03-3.04; P = .04), outdoor access (OR: 1.68; 95% CI: 1.07-2.63; P = .02), environmental tobacco smoke (OR: 1.77; 95% CI: 1.05-3; P = .03), and petfood containing chemical additives (OR: 1.98; 95% CI: 1.04-3.76; P = .04). Risk factors shared with CGS and PD were outdoor access and petfood containing chemical additives, respectively. A history of oral inflammation was reported in 35% of cats with OSCC but did not emerge as a risk factor. CONCLUSIONS AND CLINICAL IMPORTANCE: The study proposes novel potential risk factors for OSCC in cats. Although a history of inflammatory oral disease was not significantly more frequent compared with random age-matched controls, OSCC shared several risk factors with CGS and PD.

Longitudinal lymph node step-sectioning for the identification of metastatic disease in canine mast cell tumor.

Lymph node (LN) metastasis in canine mast cell tumor (MCT) can affect prognosis and postsurgical treatment recommendations; however, routine histological single-section examination may underestimate the incidence of metastases. This prospective study aimed at determining whether longitudinal step-sectioning of the entire LN allows for a more reliable detection of metastases. Dogs with MCT undergoing resection of the primary tumor and regional lymphadenectomy were enrolled. Formalin-fixed LNs were bisected longitudinally, both halves were embedded in paraffin and histological sections prepared at 200 µm steps. The nodal mast cells were classified according to the Weishaar classification. First-section evaluation (FSE; ie, examination of the first section obtained from the blocks) and whole LN step-section evaluation (SSE) were compared. Fifty-eight LNs were included. The median number of sections per LN was 6 (range, 3-28). FSE with toluidine blue (TB) revealed 27 (47%) nonmetastatic (HN0), 14 (24%) premetastatic (HN1), 9 (15%) early metastatic (HN2), and 8 (14%) overtly metastatic (HN3) LNs. SSE with TB resulted in upgrading the LN status in 2 cases (HN2 to HN3; HN0 to HN1). Evaluation of the first section plus an additional step-section resulted in 100% accuracy. Compared with SSE with TB, the accuracy of FSE with HE was 98% for HN3 LNs and 74% for HN2 LNs. FSE appears to reliably allow for the detection of LN metastasis in MCT, although examination of a further parallel section at a 200 µm step may increase the accuracy. A metachromatic stain is recommended for the identification of early metastases.

A retrospective study on prophylactic regional lymphadenectomy versus nodal observation only in the management of dogs with stage I, completely resected, low-grade cutaneous mast cell tumors.

BACKGROUND: While lymphadenectomy of metastatic lymph nodes (LNs) has been associated with improved outcome, the clinical utility of prophylactic lymphadenectomy in dogs with stage I cutaneous mast cell tumors (cMCTs) remains a controversial topic. To assess the therapeutic role of lymphadenectomy of uninvolved regional LNs, the long-term outcome of cMCT-bearing dogs with cytologically negative and surgically unresected regional LNs (observation only, OO) was compared with that of dogs with surgically resected and histologically negative regional LNs (prophylactic regional lymphadenectomy, PRL). RESULTS: A retrospective analysis of 64 dogs with a low-grade, completely resected stage I cMCT was performed: 35 (54.7%) dogs were subjected to OO and 29 (45.3%) underwent PRL. Dogs were monitored for a median of 813 and 763 days in the OO group and PRL group, respectively. The number of dogs undergoing MCT progression was significantly higher in the OO group (P = 0.028) and curve comparison revealed a tendency to a better time to progression in the PRL group (P = 0.058). No significant difference in survival time (P = 0.294) was observed between dogs in the OO and PRL groups. CONCLUSIONS: Our results showed that lack of immediate lymphadenectomy was associated with a higher risk for tumor progression. This preliminary judgement, reinforced by the findings that lymphadenectomy was well tolerated in all cases, and that histopathology provides the definitive assessment of the nodal pathological status, may suggest that prophylactic lymphadenectomy is indicated in the management of stage I MCTs. Larger prospective studies are warranted for generating clinical evidence of this latter hypothesis.

Periarticular histiocytic sarcoma with heart metastasis in a cat.

A 4-year-old intact female domestic short-haired cat was referred for recommendations about adjuvant medical treatment 1 month after left forelimb amputation due to periarticular histiocytic sarcoma (HS). At presentation, physical abnormalities were limited to enlarged ipsilateral superficial cervical and axillary lymph nodes. Routine blood analysis, abdominal ultrasound, and thoracic radiology were unremarkable. The cat initially received lomustine without any adverse events. Four weeks later, the cat developed severe acute respiratory distress. Results of thoracic radiographs and transthoracic echocardiographic analysis were suggestive of pulmonary and heart metastasis. Due to the cat's poor clinical condition and prognosis, the owner elected euthanasia, and a necropsy was performed. Based on gross pathology, histopathology, and immunohistochemistry, an HS with nodal, renal, pulmonary, and heart (right auricular and right ventricular) metastases was diagnosed. This case represents the first description of HS with a heart metastasis in a cat, providing further insight into the clinical course and metastatic behavior of this rare malignant neoplasm. Clinicians should be aware of this site of metastasis and consider HS in the list of differential diagnoses for secondary heart tumors in cats.

Flow Cytometric Assessment of Ki-67 Expression in Lymphocytes From Physiologic Lymph Nodes, Lymphoma Cell Populations and Remnant Normal Cell Populations From Lymphomatous Lymph Nodes.

Recent literature suggests conventional flow cytometric (FCM) immunophenotyping complemented by Ki-67 FCM assessment as a reliable tool to classify canine lymphomas. Ki-67 expression assessed by FCM is rarely reported in canine lymphoma cases and reference data for normal canine lymph nodes are missing. Moreover, nothing is known about the Ki-67 expression within the occasionally observed remnant cell population within the gates of normal lymphocytes in lymphoma cases. Aim of this study was to compare flow cytometric Ki-67 expression of lymphocyte populations from normal canine lymph nodes, lymphoma cells from World-Health-Organisation (WHO) classified lymphoma patient samples and their neighboring normal remnant cell population. Cryopreserved lymphocyte cell suspensions from normal lymph nodes from eight dogs free of lymphoma served as reference material. Fourteen cases diagnosed by cytology, FCM, clonality testing, histopathology including immunohistochemistry consisting of 10 DLBCL, 1 MZL, 1 PTCL and 2 TZL showed a residual small lymphocyte population and were investigated. The Ki-67 expression in normal canine lymphoid tissue was 3.19 ± 2.17%. Mean Ki-67 expression in the malignant cell populations was 41 ± 24.36%. Ki-67 positivity was 12.34 ± 10.68% in the residual physiologic lymphocyte population, which otherwise exhibited a physiologic immunophenotype pattern. This ratio was equivalent (n = 3) or lower (n = 11) than the Ki-67 expression of the malignant cell population within the sample. This is the first report of FCM derived Ki-67 expression combined with immunophenotype patterns in normal canine lymph nodes, compared with lymphoma cell populations and residual normal cell populations of lymphoma cases diagnosed by state of the art technology.

Unusual Presentation of Aortic Valve Infective Endocarditis in a Dog: Aorto-Cavitary Fistula, Tricuspid Valve Endocarditis, and Third-Degree Atrioventricular Block.

A 2-year-old Boxer with a history of subaortic stenosis and immunosuppressant therapy developed aortic valve infective endocarditis. On echocardiographic examination with simultaneous electrocardiographic tracing, multiple uncommon periannular complications of the aortic valve endocarditis were found, including aorto-cavitary fistula with diastolic left-to-right shunt, tricuspid valve endocarditis, and third-degree atrioventricular block. Necropsy confirmed the above echocardiographic findings. Although aortic valve endocarditis represents a well-known disease entity in dogs, the dynamic nature of this condition may allow development of complex and uncommon echocardiographic features.

Ki-67 assessment-agreeability between immunohistochemistry and flow cytometry in canine lymphoma.

Recent literature suggests a combination of flow cytometric determination of Ki-67 and immunophenotype as a reliable tool to classify canine lymphomas. Immunohistochemistry (IHC) on histological samples is the gold standard technique assessing Ki-67 index. Agreement between IHC and FCM derived Ki-67 indices has never been investigated. The aim of this study was to investigate the agreement between IHC and FCM in the assessment of Ki-67 expression/index, in order to evaluate whether FCM may serve as a non-invasive alternative method for the estimation of proliferative activity in canine lymphoma. Dogs with previously untreated canine lymphoma undergoing diagnostic lymphadenectomy were prospectively enrolled. Ki-67 expression/index was assessed by FCM and IHC and expressed as percentage of positive cells. 39 dogs classified by histopathology matched the inclusion criteria. With both methods, Ki-67 expression/index was higher in intermediate/high-grade lymphomas. Spearman's coefficient of correlation was ρ = 0.57; (95% CI0.33-0.75) suggesting a moderate correlation. A Bland-Altman plot revealed a negative constant bias of -3.55 (95% CI: -10.52 to 3.42) with limits of agreement from -45.71 to 38.61. The study confirmed agreement albeit with wide confidence intervals between the values of Ki-67 expression/index assessed with FCM and IHC. Discrepancies were observed in a subset of cases. Possible explanation could be that Ki-67 index in IHC is determined in the most proliferative areas of the slide, which could introduce kind of sampling bias, whereas FCM evaluates many more cells in cell suspension. Further studies are warranted to investigate this phenomenon.

Validation of oral brushing as a non-invasive technique for the identification of feline oral squamous cell carcinoma by DNA methylation and TP53 mutation analysis.

Feline oral squamous cell carcinoma (FOSCC) is a frequent and progressively invasive tumour. Early lesions are difficult to recognize based on the sole clinical examination and may be misinterpreted as non-neoplastic. Mutations of TP53 and epigenetic alterations of specific genes are present in FOSCC and may be early detected. Aim of this prospective study was to investigate the DNA methylation pattern of a 17-gene panel and TP53 mutational status of FOSCC cytological samples obtained by oral brushing. Results were compared with a control group, in order to validate this non-invasive procedure for the screening of FOSCC. In FOSCC, the same analyses were carried out on the corresponding histological sample, if available. Thirty-five FOSCC and 60 controls were included. Mutations of TP53 were detected in 17 FOSCC brushings (48%) and in none of the controls (P < .001). Six genes (ZAP70, FLI1, MiR124-1, KIF1A, MAGEC2 and MiR363) were differentially methylated in FOSCC and were included in a methylation score. An algorithm based on TP53 mutational status and methylation score allowed to differentiate FOSCC from controls with a 69% sensitivity and a 97% specificity (accuracy, 86%). In 19 FOSCC histological samples, TP53 mutational status was fully concordant with brushings and a positive methylation score was observed in all cases. These results are promising for the identification of FOSCC by oral brushing, although some factors may limit the accuracy of this technique and further studies are required to assess its reproducibility in clinical practice.

Expression of cannabinoid and cannabinoid-related receptors in the oral mucosa of healthy cats and cats with chronic gingivostomatitis.

OBJECTIVES: Feline chronic gingivostomatitis (FCGS) is an oral disease. Cats with FCGS experience intense oral pain. Some cats remain refractory to current therapies based on dental extraction and adjuvant medical treatment; it is therefore necessary to investigate alternative therapeutic targets involved in inflammatory mechanisms and pain, namely the endocannabinoid system (ECS). The present study investigated the expression of cannabinoid receptors type 1 (CB1R) and 2 (CB2R), and cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential ankyrin 1 (TRPA1) and serotonin 1a receptor (5-HT1aR), in the oral mucosa of healthy cats to determine whether there was altered expression and distribution in cats with FCGS. METHODS: Samples of caudal oral mucosa were collected from eight control cats (CTRL cats) and from eight cats with FCGS (FCGS cats). Tissue samples were processed using an immunofluorescence assay with cat-specific antibodies, and the immunolabelling of the receptors studied was semiquantitatively evaluated. RESULTS: The mucosal epithelium of the CTRL cats showed CB1R, TRPA1 and 5-HT1aR immunoreactivity (IR), while CB2R and GPR55 IR were generally not expressed. In the CTRL cats, the subepithelial inflammatory cells expressed CB2R, GPR55 and 5-HT1aR IR. In the FCGS cats, all the receptors studied were markedly upregulated in the epithelium and inflammatory infiltrate. CONCLUSIONS AND RELEVANCE: Cannabinoid and cannabinoid-related receptors are widely expressed in the oral mucosa of healthy cats and are upregulated during the course of FCGS. The presence of cannabinoid and cannabinoid-related receptors in healthy tissues suggests the possible role of the ECS in the homeostasis of the feline oral mucosa, while their overexpression in the inflamed tissues of FCGS cats suggests the involvement of the ECS in the pathogenesis of this disease, with a possible role in the related inflammation and pain. Based on the present findings, ECS could be considered a potential therapeutic target for patients with FCGS.

An Evolutionary Cancer Epigenetic Approach Revealed DNA Hypermethylation of Ultra-Conserved Non-Coding Elements in Squamous Cell Carcinoma of Different Mammalian Species.

BACKGROUND: Ultra-conserved non-coding elements (UCNEs) are genomic sequences that exhibit > 95% sequence identity between humans, mammals, birds, reptiles, and fish. Recent findings reported their functional role in cancer. The aim of this study was to evaluate the DNA methylation modifications of UNCEs in squamous cell carcinoma (SCC) from different mammal species. METHODS: Fifty SCCs from 26 humans, 17 cats, 3 dogs, 1 horse, 1 bovine, 1 badger, and 1 porcupine were investigated. Fourteen feline stomatitis and normal samples from 36 healthy human donors, 7 cats, 5 dogs, 5 horses, 2 bovines and 1 badger were collected as normal controls. Bisulfite next generation sequencing evaluated the DNA methylation level from seven UCNEs (uc.160, uc.283, uc.416, uc.339, uc.270, uc.299, and uc.328). RESULTS: 57/59 CpGs were significantly different according to the Kruskal-Wallis test (p < 0.05) comparing normal samples with SCC. A common DNA hypermethylation pattern was observed in SCCs from all the species evaluated in this study, with an increasing trend of hypermethylation starting from normal mucosa, through stomatitis to SCC. CONCLUSIONS: Our findings indicate that UCNEs are hypermethylated in human SCC, and this behavior is also conserved among different species of mammals.

Analysis of DNA methylation and TP53 mutational status for differentiating feline oral squamous cell carcinoma from non-neoplastic mucosa: A preliminary study.

Feline oral squamous cell carcinoma (FOSCC) is characterized by high local invasiveness and early bone lysis. The late diagnosis largely limits the efficacy of therapy and increases treatment-related morbidity. The aim of this exploratory study was to assess the methylation pattern of 10 candidate genes and TP53 mutational status in histologic samples of FOSCC. Results were compared with normal oral mucosa and oral inflammatory lesions, in order to establish a gene panel for FOSCC detection. For 10 cats, the above analyses were also performed on oral brushing samples, in order to explore the utility of these methods for screening purposes. Thirty-one FOSCC, 25 chronic inflammatory lesions and 12 controls were included. TP53 mutations were significantly more frequent in the FOSCC (68%) than in the non-neoplastic oral mucosa (3%; P <.001). Based on lasso regression analysis, a step-wise algorithm including TP53, FLI1, MiR124-1, KIF1A and MAGEC2 was proposed. The algorithm allowed to differentiate FOSCC with 94% sensitivity and 100% specificity (accuracy, 97%). When applying the proposed algorithm on 10 brushing samples, accuracy decreased to 80%. These results indicate that the altered DNA methylation of specific genes is present in FOSCC, together with a significant proportion of TP53 mutations. Such alterations are infrequent in normal oral mucosa and chronic stomatitis in cats, suggesting their involvement in feline oral carcinogenesis and their utility as diagnostic biomarkers. Further studies on a high number of brushing samples will be needed to assess the utility of a screening test for the early detection of FOSCC.

Preliminary Assessment of the Mucosal Toxicity of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis) Essential Oils on Novel Porcine Uterus Models.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2-0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.

Assessment of PDGFRß promoter methylation in canine osteosarcoma using methylation-sensitive high-resolution melting analysis.

Platelet-derived growth factor signalling pathways play a fundamental role in inducing and sustaining the proliferative and prosurvival stimuli in canine osteosarcomas (cOSAs). The increased expression of platelet-derived growth factor receptors (PDGFRs) α and ß, and their cognate ligands, were almost invariably observed in cOSAs and OSA-derived cell lines. In particular, overexpression of PDGFRß-mediated signalling pathways was found in both the tumour microenvironment, where it drives stromal cell recruitment, and in neoangiogenesis, such as in tumour cells where it triggers aberrant proliferation, migration and local invasion. The majority of the pathological consequences of PDGFRß signalling are because of aberrant expression. In fact, epigenetic dysregulation of oncogenes throughout demethylation of their promoter has emerged as a pivotal mechanism driving oncogenesis. The aim of this study was to assess the methylation status of the PDGFRß promoter and to clarify its role in modulating the expression of the tyrosine kinase receptor in canine osteosarcoma. The CpG island of the PDGFRß promoter was identified using a mixed in silico and experimental approach, and a method based upon the methylation-sensitive high-resolution melting assay for quantitatively and precisely assessing the methylation status of the promoter was then set up. The method herein described was then exploited to assess the methylation status of the promoter in a case series of cOSAa. COSAs consistently but variably expressed PDGFRß. However, the promoter was almost completely demethylated, and its methylation status did not correlate with the expression levels. This finding supported the hypothesis that post-transcriptional regulatory mechanisms may act in cOSAs.

Adjuvant medical therapy provides no therapeutic benefit in the treatment of dogs with low-grade mast cell tumours and early nodal metastasis undergoing surgery.

Lymph node (LN) metastasis is a negative prognostic factor in dogs with cutaneous mast cell tumours (cMCTs). While elective lymphadenectomy of metastatic LNs improves outcome, the benefit of adjuvant medical therapy in dogs with early metastatic (HN2) LNs is debated. The aim of this retrospective multicentre study was to evaluate the therapeutic benefit of adjuvant medical therapy following surgical removal of the primary low-grade cMCT (Patnaik grade 1-2 and Kiupel low-grade) and lymphadenectomy of HN2 LNs by analysing survival rates and patterns of recurrence. Seventy-three dogs were included: 42 received adjuvant medical treatment (chemotherapy and/or kinase inhibitors), and 31 did not. The median follow-up time for medically treated dogs was 619 days: two experienced local recurrence, three nodal relapse and four distant relapse. For dogs undergoing surgery only, the median follow-up time was 545 days. None of them experienced local recurrence, nodal, or distant relapse. Time to progression was significantly shorter in dogs receiving adjuvant medical treatment (P = .021). A similar tendency was observed for overall survival (P = .056). The current study shows that dogs with low-grade cMCTs, that undergo surgical excision of the primary tumour and elective lymphadenectomy of the HN2 regional LN harbour a good prognosis. The use of adjuvant medical treatment in these dogs does not seem to provide any benefit in terms of progression and survival.

Evidence of fish and human pathogens associated with doctor fish (Garra rufa, Heckel, 1843) used for cosmetic treatment.

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well-known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.

Prevalence of p53 dysregulations in feline oral squamous cell carcinoma and non-neoplastic oral mucosa.

Squamous cell carcinoma is the most common malignant oral tumor in cats. The late presentation is one of the factors contributing to the detrimental prognosis of this disease. The immunohistochemical expression of the p53 tumor suppressor protein has been reported in 24% to 65% of feline oral squamous cell carcinomas, but no study has systematically evaluated in this tumor the presence of p53 encoding gene (TP53) mutations. The aim of this retrospective study was to determine whether p53 immunohistochemistry accurately reflects the mutational status of the TP53 gene in feline oral squamous cell carcinoma. Additionally, the prevalence of p53 dysregulation in feline oral squamous cell carcinoma was compared with that of feline non-neoplastic oral mucosa, in order to investigate the relevance of these dysregulations in cancer development. The association between p53 dysregulations and exposure to environmental tobacco smoke and tumor characteristics was further assessed. Twenty-six incisional biopsies of oral squamous cell carcinomas and 10 cases each of lingual eosinophilic granuloma, chronic gingivostomatitis and normal oral mucosa were included in the study. Eighteen squamous cell carcinomas (69%) expressed p53 and 18 had mutations in exons 5-8 of TP53. The agreement between immunohistochemistry and mutation analysis was 77%. None of non-neoplastic oral mucosa samples had a positive immunohistochemical staining, while one case each of eosinophilic granuloma and chronic gingivostomatitis harbored TP53 mutations. Unlike previously hypothesized, p53 dysregulations were not associated with exposure to environmental tobacco smoke. These results suggest an important role of p53 in feline oral tumorigenesis. Additionally, the immunohistochemical detection of p53 expression appears to reflect the presence of TP53 mutations in the majority of cases. It remains to be determined if the screening for p53 dysregulations, alone or in association with other markers, can eventually contribute to the early detection of this devastating disease.

Evaluation of Ki-67 expression in feline non-ocular melanocytic tumours.

BACKGROUND: Melanomas are rare in cats. The eye is the most commonly involved site, whereas few data are available about feline non-ocular melanomas (NOMs). Ki-67 thresholds with prognostic relevance have been established for canine melanomas, but not in cats. This study was undertaken to investigate the relationship between Ki-67 index, tumour characteristics, and clinical outcome in feline NOMs. Histologic samples were retrospectively reviewed. Amelanotic tumours were admitted upon immunohistochemical positivity for Melan A or S100. Evaluated parameters included morphological diagnosis, histotype, junctional activity, degree of pigmentation, vascular invasion, lymphocytic infiltrate, necrosis, mitotic count (MC) and Ki-67 index. Pigmented tumours were bleached before evaluation. Clinical and follow-up information were retrieved via telephone interviews with the referring veterinarians. RESULTS: Fifty tumours located in skin (n = 33) and mucosae (n = 17) were included. Forty-eight percent and 95% of amelanotic tumours (n = 21) stained positive for Melan A and S100, respectively. Most achromic tumours were mucosal (P < 0.001, Fisher's exact test) and presented a spindle cell morphology (P = 0.002; Fisher's exact test). MC and Ki-67 index were significantly correlated (P < 0.001; R = 0.67; Spearman's rank correlation); median values were 15 (range, 0-153) and 28% (range, 1-78%), respectively. Both were significantly higher in spindle cell melanomas, in tumours lacking junctional activity and in poorly-pigmented tumours. Follow-up information was available for 33 cats (66%). Variables related with a poor clinical outcome included mucosal location, tumour size, spindle, balloon and signet ring cell histotypes, low pigmentation, MC > 5, Ki-67 > 20% and lack of treatment administration. On multivariable analysis, only tumour histotype and treatment retained prognostic significance. CONCLUSIONS: Although the majority of feline NOMs behave aggressively, Ki-67 index, together with other parameters, may contribute to prognostic assessment. Prospective studies on homogeneous populations are warranted to identify reliable threshold values for this marker.