Biomolecular techniques in support of histopathological diagnosis in veterinary oncology

Negli ultimi vent'anni, la medicina veterinaria ha focalizzato la propria attenzione verso il benessere animale e lo sviluppo di strumenti diagnostici e terapeutici più efficienti. In particolare, in campo oncologico sono stati adottati approcci multidisciplinari finalizzati a caratterizzare al meglio le neoplasie dei piccoli animali, per poterne migliorare la prognosi e validarli come modelli spontanei di patologia dell'uomo. Alle più aggiornate conoscenze sul comportamento biologico dei tumori e le loro caratteristiche istopatologiche si stanno infatti affiancando tecnologie d'avanguardia sia nel campo della ricerca che della diagnostica quotidiana. Lo scopo del presente studio è stato quello di indagare l'utilità diagnostica di tre tecniche biomolecolari in supporto all'esame istologico ed immunoistochimico per valutare se in grado di amplificare il potenziale diagnostico delle attuali tecniche gold-standard. In particolare, abbiamo indagato: (1) L'applicazione della PCR per il riarrangiamento del recettore antigienico (PARR) per determinare la clonalità delle lesioni linfoidi iperplastico/neoplastiche nelle milze di cane. L'idoneità di questa tecnica è stata confermata nei casi istologici dubbi attraverso una critica interpretazione dei risultati sulla base dei dati clinici, morfologici e di immunofenotipo. (2) La concordanza tra il gold-standard immunoistochimica e la meno invasiva citofluorimetria a flusso nella valutazione dell'attività proliferava (Ki-67 index) dei linfomi nodali del cane. E' stata dimostrata una buona concordanza complessiva tra le due tecniche, nonostante il ruolo prognostico del Ki-67 nel linfoma del cane sia ancora dubbio. (3) I pattern genetici ed epigenetici del carcinoma squamocellulare orale del gatto, per identificare criteri biomolecolari idonei alla diagnosi precoce di questo tumore, fatale anche a causa della tardiva diagnosi, sulla base di recenti scoperte in campo umano. E' stato proposto un test non invasivo basato su un algoritmo diagnostico preliminare per discernere lesioni orali neoplastiche da quelle non neoplastiche nel gatto.Within the last two decades, veterinary medicine has strongly increased its attention for animal best care practices and development of more efficient diagnostic and therapeutic strategies. Namely, in veterinary oncology multi-disciplinary approaches were adopted to better characterize small animals tumors, in order to improve prognosis and validate them as naturally occurring models for the human counterpart. In addition to refining the knowledge of biological behavior and histopatological features of neoplasms, cutting-edge biomolecular technologies are gaining ground in both research and everyday diagnostic workup. The aim of the present study was to investigate the diagnostic utility of three biomolecular technologies in support of histopathological and immunohistochemical examination, in order to evaluate whether they can enhance the potential of the current gold-standard diagnostic procedures. In particular, we investigated: (1) The application of PCR for antigen receptor rearrangement (PARR) for assessing the clonality of hyperplastic/neoplastic splenic lymphoid lesions in dogs. The suitability of this technique was confirmed in doubtful histological cases through a critical interpretation of the results on the basis of the clinical, morphological and immunophenotyping data. (2) The agreement between the gold standard immunohistochemistry and the less invasive flow cytometry in the evaluation of proliferative activity (Ki-67 index) of canine nodal lymphomas. We demonstrated an overall good concordance between the techniques, although the possible prognostic role of Ki-67 in canine lymphoma has still to be clarified. (3) The genetic and epigenetic modification patterns of feline oral squamous cell carcinoma, in order to provide biomolecular criteria for the early detection of this late-stage diagnosed and fatal tumor, on the basis of recent discoveries in the human counterpart. A preliminary diagnostic algorithm was proposed as a non-invasive test to discriminate between neoplastic and non-neoplastic feline oral lesions

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\u20138 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

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  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

Pathological and microbiological investigations in mortality outbreaks involving doctor fish (Garra rufa, Heckel 1843) used for cosmetic treatment

Introduction: Doctor fish are widely used in fish spa; their presence as living animals prohibits the use of conventional methods for water and equipment sterilization and disinfection. This has led to concerns about the potential transmission of infection to human beings and on fish welfare. The susceptibility to microbiological agents in this species is not well known, only few reports focused on episodes of Garra rufa mortality are available. The aim is to describe disease outbreaks occurred in a fish spa Materials and Methods: In the period 2016-2017 two outbreaks resulted in a chronic mortality. Collected fish were euthanized and sampled immediately for pathological, bacteriological and molecular (Infectious Pancreatic Necrosis Virus-IPNV) investigations. A classification based on timing of granulomatous lesions, in association with an acid-fast staining and immunohistochemistry against Mycobacterium bovis were performed. Results: Some animals showed rapidly growing erosive and ulcerative skin lesions. The main histopathological finding was otherwise visceral, intermediate or late stage, granulomatosis. Immunoreactivity to Mycobacterium bovis was registered. Phenotypic and genetic characterizations of isolated bacteria showed the presence of Aeromonas veronii and Vibrio cholerae in one batch (2016), Aeromonas veronii and Shewanella putrefaciens in another batch (2017). The IPNV- RNA was not found. Conclusions: The attention to granulomatogenous bacteria in aquatic species has increased because of their zoonotic potential. In the cases described, microbiological investigation associated to the pathological features led to the final diagnosis of mycobacteriosis. The presence of other bacteria has led some concerns about the use of these animals for cosmetic treatment

Comparison between May Grünwald-Giemsa and rapid stains in fine-needle aspirates of canine mast cell tumor: diagnostic and prognostic implications

Introduction Mast cell tumors (MCTs) are often diagnosed by cytology based on the identification of their characteristic purple intracytoplasmic granules with methanolic Romanowsky stains, including May Grünwald-Giemsa (MGG). In clinical practice, aqueous Romanowsky stains are commonly used because of their rapidity, but the risk exists that mast cell granules may not stain properly. The purpose of this study was to investigate the phenomenon of hypogranularity of MCT with rapid staining (RS) in terms of frequency and correlations with tumor characteristics, along with diagnostic and prognostic implications. Materials and Methods A prospective study was carried out on cytologic preparations of histologically-confirmed canine primary MCTs and metastatic lymph nodes. For each case, good-quality smears were stained with both MGG and RS and comparatively assessed. Results Fifteen out of 60 (25%) primary canine MCTs were hypogranular with RS (P=0.002). These were predominantly cutaneous high-grade tumors and in 7 of them (11.7%) a certain diagnosis could not be made with RS. Accuracy in grading assessment was not significantly different compared with MGG (79% vs 83%). Ten of 25 (40%) metastatic lymph nodes were hypogranular with RS (P=0.002); the negative predictive value in the detection of nodal metastases, assessed with three independent observers, was significantly lower (100% vs 73%; P=0.010). Discussion This study confirms that RS can be ineffective in staining MCT granules, especially in biologically aggressive tumors, and can occasionally prevent the achievement of a correct diagnosis. The use of RS is discouraged for the search of nodal metastases, as the identification of isolated mast cells can be particularly challenging

Liver expression in Cystic Fibrosis could be modulated by genetic factors different from the Cystic Fibrosis Transmembrane Regulator genotype.

During a multicentric study conducted in Southern Italy, we studied five sets of cystic fibrosis siblings bearing a strongly discordant liver phenotype, three with genotype ΔF508/R553X, one with genotype ΔF508/unknown, and one with genotype unknown/unknown. The siblings of each set were raised in the same family environment, and there were no interpair differences in nutritional state or in therapy compliance. All siblings had pancreatic insufficiency and moderate respiratory expression. One sibling of each of the five sets was free of liver involvement, and the other had severe liver expression. Other causes of liver disease (viral, metabolic, and genetic other than cystic fibrosis) were ruled out. Therefore, environmental factors, nutritional state, and therapy compliance are not involved in the liver expression of cystic fibrosis in the five unrelated sibships. This suggests that modifier genes, inherited independently of the cystic fibrosis transmembrane regulator gene, could modulate the liver expression in cystic fibrosis patient

Canine Splenic Nodular Lymphoid Lesions: Immunophenotyping, Proliferative Activity, and Clonality Assessment

Canine splenic lymphoid nodules are currently classified as indolent lymphomas (marginal zone lymphoma [MZL], mantle cell lymphoma [MCL]) or nodular hyperplasia (lymphoid [LNH] or complex [CNH] type). Their differentiation can be difficult on morphology, because of similar histologic appearance and poorly defined diagnostic criteria. Thirty-five surgical samples of splenic lymphoid nodules were reviewed in order to assess the diagnostic contribution of immunophenotyping, proliferative activity and clonality (PARR) in differentiating between hyperplastic and neoplastic lesions. Proliferative activity was evaluated by double immunolabeling for Ki-67 and CD79a, in order to separately assess the proliferative activity of B cells and non-B cells. Definitive diagnoses were MZL ( n = 11), MCL ( n = 4), LNH ( n = 10), and CNH ( n = 10). The overall concordance between histology and PARR was above 90%. Lymphomas had a significantly higher percentage of CD79a-positive areas (mean, 36.30%; P = .0004) and a higher B-cell proliferative activity (median Ki-67 index, 5.49%; P = .0012). The threshold value most accurately predicting a diagnosis of lymphoma was 6528% of B-cell areas, with a Ki-67 index above 3%. Dogs were monitored for a median follow-up time of 870 days (IQR, 569-1225), and no relapses were documented. Overall median survival time was 1282 days. The combination of histology, immunohistochemistry and PARR can improve the diagnostic accuracy for canine splenic lymphoid nodules, although the long-term behavior of these lesions appears similar

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

Abstract 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

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