Feline thymidine kinase 1: molecular characterization and evaluation of its serum form as a diagnostic biomarker.

BACKGROUND: Thymidine kinase 1 (TK1) catalyzes the initial phosphorylation of thymidine in the salvage pathway synthesis of dTTP, an essential building block of DNA. TK1 is a cytosolic enzyme with its highest level during the S-phase of the cell cycle. In cancer cells TK1 is upregulated and excess TK1 is leaked into the blood. Therefore, serum TK1 has been used as biomarker for cancer diagnosis and prognosis in human medicine. Feline TK1 shows high sequence similarity to TK1 from other species. The aim of this study was to characterize feline TK1 and evaluate if serum TK1 can be used as a diagnostic biomarker. RESULTS: Feline TK1 was cloned, expressed and affinity purified. The purified feline TK1 phosphorylated not only pyrimidine deoxyribonucleosides but also pyrimidine ribonucleosides and to some extent purine deoxynucleosides. A number of anticancer and antiviral nucleoside analogs also served as substrates with fairly high efficiency. ATP and dATP were the preferred phosphate donor. Serum TK1 activity in felines with malignant diseases was significantly higher than that in healthy individuals. ROC analysis revealed an area under the curve (AUC) of 0.98 with a sensitivity of 0.83 and a specificity of 0.95 for felines with lymphoma. Serum TK1 activity in felines with IBD or inflammatory disease was within the same range as healthy ones. Furthermore, in felines with lymphoma serum TK1 activity returned to normal levels in response to treatment. CONCLUSION: Feline TK1 has high specific activity and a broader substrate specificity in comparison with TK1 from other species. Serum TK1 activity in felines with malignant diseases is significantly higher than that in normal felines and in felines with inflammatory diseases. These results suggest that serum TK1 may be a promising biomarker for the diagnosis and monitoring of malignant diseases and for the differential diagnosis of certain inflammatory disease.

Histopathology and Feline Pancreatic Lipase Immunoreactivity in Inflammatory, Hyperplastic and Neoplastic Pancreatic Diseases in Cats.

The most common pancreatic diseases in cats are pancreatitis and exocrine pancreatic insufficiency (EPI). Non-invasive methods, such as serological quantification of feline pancreatic lipase immunoreactivity (fPLI), are often used in the diagnosis of pancreatitis. Previous studies have compared fPLI concentrations with histopathology, considered to be the gold standard for diagnosis of feline pancreatitis. However, fPLI concentrations in cats suffering from pancreatic tumours were rarely described. The aim of the present study was to determine the sensitivity and specificity of an in-house enzyme-linked immunosorbent assay (ELISA) for the quantification of fPLI in serum samples based on histopathological findings in cats diagnosed with various pancreatic diseases. Pancreatic biopsy samples from 80 cats were included. Five groups were defined on the basis of pancreatic histopathology: group 1, normal pancreas; group 2, nodular hyperplasia; group 3, mild pancreatitis; group 4, marked (moderate/severe) pancreatitis; and group 5, pancreatic neoplasia. Serum samples from all cats were tested by fPLI ELISA (<3.6 µg/l normal, 3.6-5.3 µg/l questionable, >5.3 µg/l pancreatitis). In group 1 (n = 19), serum fPLI values were within the reference interval in 74% of cases and in group 2 (n = 9) in 78%. Cats with mild pancreatitis (n = 23), marked pancreatitis (n = 11) and pancreatic neoplasms (n = 18) had significantly increased fPLI concentrations compared with group 1 (P = 0.004/0.001/≤0.0001). Cats with nodular hyperplasia had significantly lower fPLI values than cats with marked pancreatitis (P = 0.048) or tumours (P = 0.002). Serum fPLI concentrations in group 3 were <3.6 µg/l (n = 6), 3.6-5.3 µg/l (n = 4) and >5.3 µg/l (n = 13). Calculated test sensitivity for mild pancreatitis was fPLI >3.5 µg/l: 73.9% and fPLI >5.3 µg/l: 56.5%. In group 4 (n = 11), seven of nine cats (77.8%) with marked purulent pancreatitis had elevated fPLI. In group 4, a sensitivity of 81.8% was detected for fPLI >3.5 µg/l and 63.6% for fPLI >5.3 µg/l. Two cats with marked non-purulent pancreatitis had elevated fPLI, while two cats with marked purulent pancreatitis had normal fPLI values (<3.6 µg/l). In group 5, one cat with pancreatic adenoma and one with pancreatic acinar carcinoma had normal fPLI concentrations. The other cats with pancreatic adenoma (solid, n = 1; cystic, n = 4) or carcinoma (solid, n = 9; cystic, n = 2) had elevated or high fPLI values (4.1 to >40 µg/l, median 21.2 µg/l), probably caused by additional inflammation. The results of the present study confirm the importance of detailed histopathological characterization for the interpretation of clinical signs and fPLI values in feline pancreatitis. Primary pancreatic neoplasms may also lead to elevated fPLI concentrations as there is concurrent pancreatitis in most cases. However, severe pancreatic diseases, such as chronic non-purulent pancreatitis or tumours without inflammation, may result in normal fPLI values.

Alpha-enolase staining patterns in the renal tissues of cats with and without chronic kidney disease.

Renal α-enolase has variable expression in inflammatory and neoplastic diseases. Therefore, in order to define the distribution of α-enolase in renal tissues of cats, an immunohistochemistry assay was validated and described here. Tissues from 29 cats with IRIS Stage 2-4 CKD, 8 control cats < 2 years of age, and 4 control cats> 10 years of age were assessed. Interstitial nephritis was the predominant histopathological finding in the CKD group. The control cats < 2 years of age had moderate α-enolase immunoreactivity in tubular epithelium but staining was absent to mild in glomeruli. In contrast, α-enolase was moderate to high in tubular epithelium and glomeruli in control cats > 10 years of age. In cats with CKD, α-enolase was decreased in tubules that were degenerative or atrophic, similar to normal tubules in control groups, and moderate to high in glomeruli. When compared between the study groups, the results suggest that alpha-enolase decreases in damaged tubules and increases in the glomeruli of older cats prior to the development of detectable CKD. Further studies will be required to determine whether these findings relate to the pathogenesis or could be used in the diagnosis of feline CKD.

Significance of sphingosine kinase 1 expression in feline mammary tumors.

BACKGROUND: Sphingosine kinase 1 (SPHK1) is an enzyme that converts pro-apoptotic ceramide and sphingosine into anti-apoptotic sphingosine-1-phosphate. There is growing evidence that SPHK1 activation promotes oncogenic transformation, tumor growth, chemotherapy resistance, and metastatic spread. High SPHK1 expression has been associated with a poor prognosis in several human cancers. RESULTS: In the present study, the expression level of SPHK1 was examined in feline mammary tumor (FMT) specimens, and the IHC expression level of SPHK1 was associated with the histological grade of FMTs. IHC analysis of 88 FMT cases revealed that the expression level of SPHK1 was upregulated in 53 tumor tissues (60.2%) compared to adjacent mammary tissues. SPHK1 expression in FMTs was significantly associated with histological grade, presence of lymphovascular invasion, and estrogen receptor negativity. Treatment of primary FMT cells with SPHK1 inhibitors reduced cell viability, indicating that SPHK1 acts to promote FMT cell survival. These results indicate that SPHK1 may play an important role in FMTs and may be a therapeutic target in cats with FMT. CONCLUSIONS: SPHK1 over-expression in breast cancer tissues is associated with a poor prognosis in humans. SPHK1 over-expression in more aggressive FMTs provides support for a potential role of SPHK1 inhibitors for the treatment of FMTs. Targeting SPHK1 has potent cytotoxic effects in primary FMT cells. These findings suggest that further examination of the role SPHK1 plays in FMTs will pave the way for the investigation of SPHK1 inhibitors in future clinical applications.

Diagnosis of feline pancreatitis with SNAP fPL and Spec fPL.

OBJECTIVES: Pancreatitis is a frequent disease in cats for which the ante-mortem diagnosis remains challenging. Feline pancreatic lipase immunoreactivity (fPLI) has been reported to have a high sensitivity for the diagnosis of pancreatitis. The aim of this study was to compare the rapid in-house test SNAP fPL with the standard test Spec fPL and to evaluate the use of SNAP fPL to diagnose pancreatitis in an emergency setting. METHODS: fPLI of 111 cats with a clinical suspicion of pancreatitis was measured with both SNAP fPL and Spec fPL. Furthermore, clinical signs, haematological and biochemical changes, and abdominal ultrasound findings were recorded. RESULTS: Seventy-eight of 111 cats (70.3%) were tested below the cut-off level for pancreatitis with SNAP, as well as Spec fPL, whereas 21/111 (18.9%) were tested with values above the cut-off level with both tests. In 12/111 (10.8%) cats the results were discordant. The comparison of both tests revealed an agreement of 78/80 (97.5%) when Spec fPL was ⩽3.5 µg/l (negative) and 18/20 (90%) when Spec fPL was ⩾5.4 µg/l (positive). The most common clinical signs in cats with suspected pancreatitis (n = 21) were lethargy (95.2%), reduced appetite and vomiting (90.5% each), dehydration (81.0%), diarrhoea (57.1%), abdominal pain and weight loss (47.6% each). Hyperglycaemia and hyperbilirubinaemia (85.7% each), increased aspartate transaminase (76.2%) and alanine transaminase (47.6%), leucocytosis (61.9%), lymphopenia (57.1%), decreased sodium and chloride (57.1% each), and increased urea (52.4%) were the most common abnormalities in blood work. CONCLUSIONS AND RELEVANCE: Clinical signs, as well as routine blood-work changes, were non-specific and thus proved to be insufficient to diagnose pancreatitis. The combination of SNAP fPL and subsequent Spec fPL, if indicated, provided the opportunity to rule out or to diagnose pancreatitis with a higher certainty than previously known test methods. This study proved SNAP fPL to be a valuable tool to exclude or include pancreatitis in an emergency setting.

Cyclooxygenase-2 immunoexpression in intestinal epithelium and lamina propria of cats with inflammatory bowel disease and low grade alimentary lymphoma.

BACKGROUND: Cyclooxygenase 2 (COX-2) is an inducible isoform by cellular activation, proinflammatory cytokines and growth factors. The aims of the current study were to evaluate COX-2 immunoexpression in epithelial and lamina propria (LP) of cats with inflammatory bowel disease (IBD) and low grade alimentary lymphoma (LGAL), as well as to correlate them with clinical signs and histopathological scoring. Cats diagnosed with IBD and LGAL (2007-2013) were included in the current study. Feline chronic enteropathy activity index (FCEAI) was calculated for all cases. Control group was composed by 3 healthy indoor cats and 5 sick cats died or were euthanized (non-gastrointestinal illness). Diagnosis and classification of IBD and LGAL was established according to the WSAVA gastrointestinal standardization group template and the National Cancer Institute formulation, respectively. Furthermore, a modified WSAVA template was applied for LGAL evaluation. Immunolabelling for COX-2 (polyclonal rabbit anti-murine antibody) was performed on biopsy samples. Epithelial and LP (inflammatory or neoplastic cells) COX-2 immunolabelling was calculated according to the grade and intensity. The most representative segment scored by the WSAVA and the modified WSAVA were used for statistical analysis. RESULTS: Significant difference was found regarding COX-2 intensity overexpression in the epithelial cells of IBD and LGAL groups when compared to control cats, but not between the groups of sick cats, whereas no differences were found regarding the grade of immunoreactivity between groups. No difference was found for COX-2 immunoexpression at the LP between all groups. However, 3 cats from LGAL group showed COX-2 expression in neoplastic cells at the LP. There were no correlations between epithelial or LP COX-2 expression and FCEAI and histological alterations. CONCLUSIONS: Increased COX-2 intensity at the epithelial cells observed in cats with IBD and LGAL may be secondary to the inflammatory response or a protective function in the intestinal reparation. COX-2 expression at the LP was presented in 33% of LGAL. This result provides a reason for further investigation concerning the role of COX-2 expression in feline alimentary lymphoma.

Fatty acid synthase as a potential therapeutic target in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy in both feline and human patients. Recent work has demonstrated aberrant expression of fatty acid synthase (FASN) and an increased capacity for lipogenesis in human OSCC and other cancers. In human OSCC, inhibition of FASN decreased cell viability and growth in vitro, and diminished tumour growth and metastasis in murine preclinical models. This study aimed to characterize FASN as a therapeutic target in feline OSCC. Immunohistochemistry revealed high FASN expression in primary feline OSCC tumours, and FASN expression was detected in OSCC cell lines (3 feline and 3 human) by immunoblotting and quantitative real-time-polymerase chain reaction (qRT-PCR). Orlistat, a FASN inhibitor, substantially reduced cell viability in both feline and human OSCC lines, although feline cell lines consistently displayed higher sensitivity to the drug. FASN mRNA expression among cell lines mirrored sensitivity to orlistat, with feline cell lines expressing higher levels of FASN. Consistent with this observation, diminished sensitivity to orlistat treatment and decreased FASN mRNA expression were observed in feline OSCC cells following incubation under hypoxic conditions. Treatment with orlistat did not potentiate sensitivity to carboplatin in the cell lines investigated; instead, combinations of the 2 drugs resulted in additive to antagonistic effects. Our results suggest that FASN inhibition is a viable therapeutic target for feline OSCC. Furthermore, cats may serve as a spontaneous large animal model for human oral cancer, although differences in the regulation of lipogenesis between these 2 species require further investigation.

Feline platynosomiasis: analysis of the association of infection levels with pathological and biochemical findings / Platinossomiase felina: análise da associação dos níveis de infecção com achados patológicos e bioquímicos

Abstract Platynosomiasis is a common feline hepatic disease caused by Platynosomum fastosum (Trematoda - Dicrocoelidae), which is also known as ‘lizard poisoning’. Most reports of feline platynosomiasis show that this disease is sporadic and manifests with uncommon lesions; its pathogenicity is still not well understood. This study aimed to describe liver injuries and enzymatic changes associated with natural P. fastosum infection in 47 stray cats in an endemic area. Overall, 38.3% (18/47) of cats were parasitized, and 2,358 flukes (P. fastosum) were collected (131 – mean intensity of parasitism; 50.2 – mean abundance). The alanine transaminase (ALT) measure was significantly higher in parasitized animals, while alkaline phosphatase (ALP) showed no statistical difference between parasitized and non-parasitized animals. In infected animals, gross pathological lesions and microscopic liver injuries ranged from mild to severe, and were similar to those in previous descriptions of feline platynosomiasis. Nonetheless, the intensity of parasitism was not related to the severity of macroscopic or microscopic hepatic injuries. However, feline platynosomiasis should be considered in the differential diagnosis of feline liver disorders, as well as, in any program of helminth control, even if no clinical abnormalities are present.

Resumo Platinossomiase é uma doença hepática felina comum causada por Platynosomum fastosum (Trematoda - Dicrocoelidae), também é conhecida como “envenenamento por lagartixa”. A maioria dos relatos de platinossomiase felina mostra que esta doença é esporádica e se manifesta com lesões incomuns; sua patogenicidade ainda não é bem compreendida. Este estudo objetivou descrever as lesões no fígado e alterações enzimáticas associadas à infecção natural por P. fastosum em 47 gatos errantes em uma área endêmica. No total, 38,3% (18/47) dos gatos estavam parasitados, e 2.358 trematódeos (P. fastosum) foram coletados (131 – intensidade média de parasitismo; 50,2 – abundância média). A quantidade de alanina transaminase (ALT) foi significativamente maior nos animais parasitados, enquanto a fosfatase alcalina (ALP) não apresentou diferença estatística entre os animais parasitados e não parasitados. Nos animais infectados, lesões patológicas macroscópicas e microscópicas hepáticas variaram de leve a grave, e foram semelhantes a descrições anteriores de platinossomiase felina. No entanto, a intensidade do parasitismo não foi relacionada à gravidade das lesões hepáticas macroscópicas ou microscópicas. Contudo, a platinossomiase felina deve ser considerada no diagnóstico diferencial de distúrbios hepáticos em felinos, assim como, em qualquer programa de controle de helmintos, mesmo que nenhuma anormalidade clínica esteja presente.

Kit receptor tyrosine kinase dysregulations in feline splenic mast cell tumours.

This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c-KIT mutations) in cats affected with splenic mast cell tumours. Twenty-two cats were included. Median survival time was 780 days (range: 1-1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c-Kit mutations and tumour differentiation, mitotic activity or survival.

AKT capture by feline leukemia virus.

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

Is Serum Total LDH Evaluation Able to Differentiate between Alimentary Lymphoma and Inflammatory Bowel Disease in a Real World Clinical Setting?

CONTEXT: An increase in enzyme lactate dehydrogenase (LDH) in serum is a negative prognostic factor for survival in cats affected by lymphoma. Measuring LDH at the time of diagnosis has been studied for differentiating neoplastic disease from non-neoplastic disease in dogs. Inflammatory bowel disease (IBD) and alimentary lymphoma are common diseases in cats. OBJECTIVE: The aim of this study was to determine whether elevation of total LDH occurred in cats with alimentary lymphoma and non-neoplastic gastrointestinal disease, such as IBD, and to evaluate whether this enzyme is useful in supporting the differential diagnosis of these specific diseases. MATERIALS AND METHODS: A prospective non-randomized controlled study was carried-out in a real world setting of three Italian private veterinary clinics. Seventy-one client-owned cats with a history of chronic gastrointestinal symptoms were enrolled; 33 cats were histologically diagnosed as having alimentary lymphoma and 38 cats as having IBD. Serum samples of total LDH analysis were measured. RESULTS: Gender (P = 0.016) and age (P = 0.046) were found to be significant factors influencing the differentiation of serum total LDH between cats with alimentary lymphoma and those with IBD. Despite low diagnostic accuracy in the overall population (63%), a cut-off value of serum total LDH ranging from 0.85- to 1.04-times the upper reference limit showed good capability (accuracy >80%) of differentiating these two conditions in neutered males and cats younger than 8 years of age (AUC: 0.805, 0.833; sensitivities: 76.9%, 83.3%; specificities: 80.0%, 76.5%; PPV: 76.9%, 55.6%; NPV: 80.0%, 92.9%; respectively). CONCLUSIONS: Although our study showed that gender and age are significant factors in differentiating serum total LDH between cats with alimentary lymphoma and those with IBD, this test had poor diagnostic accuracy in differentiating between these two conditions in the overall population.

Serum HER2 levels are increased in cats with mammary carcinomas and predict tissue HER2 status.

HER2 is overexpressed in about 30% of feline mammary carcinomas (FMC) and in 15-30% of breast cancers. Women with HER2-positive breast tumors are associated with shorter survival. This study aimed to optimize the detection and quantification of serum HER2 (sHER2) in cats and to evaluate its potential in diagnosing cats with mammary carcinomas (MC) overexpressing HER2. A prospective study was conducted in 60 queens showing MC and 20 healthy animals. Pre-operative serum samples were collected for sHER2 quantification using two immunoassays: ELISA and Dot blot assay. sHER2 levels were compared with tissue HER2 status assessed by immunohistochemistry. Queens with FMC showed significantly higher mean levels of sHER2 by both ELISA and Dot blot assay. A significant difference in the sHER2 levels was also found between cats with HER2-positive MC and those with low-expressing HER2 MC. A significant correlation between sHER2 levels and tumor HER2 status was also found, particularly when ELISA was used (r = 0.58, p < 0.0001). The value of 10 ng/ml was proposed as the optimal cutoff for both immunoassays by ROC analysis. Like in humans, sHER2 levels are increased in cats with MC HER2-positive, strongly suggesting that evaluation of sHER2 levels can be very useful in feline oncology. The results show that ELISA and Dot blot assay can replace the immunohistochemistry technique, due to their efficacy and lower costs for diagnostic purposes and for monitoring the response to anti-HER2 therapies in cats.

Evaluation of prognostic factors and survival rates in malignant feline mammary gland neoplasms.

OBJECTIVES: The aim of the study was to investigate prognostic factors in feline mammary gland neoplasms, correlating them with overall survival (OS). METHODS: Fifty-six primary malignant mammary gland neoplasms and 16 metastatic lymph nodes from 37 female cats were analyzed. Clinical staging, histologic type and grade, and immunohistochemistry for Ki-67, progesterone and estrogen receptor, human epidermal growth factor receptor type 2 (HER-2), cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were evaluated. Follow-up was performed in order to correlate prognostic factors with OS. RESULTS: Lymph node metastasis was found in 35% of cases. Clinical stage III, tubulopapillary carcinomas and histologic grade II cases prevailed in the study. Most neoplasms were positive for hormonal receptors, negative for HER-2 overexpression and presented VEGF overexpression. Immunoreactivity for Ki-67 (P = 0.046) and COX-2 (P = 0.007) was higher in metastases than in primary tumors. COX-2 (P = 0.089), HER-2 (P = 0.012) and histologic grade (P = 0.080) were correlated with OS. CONCLUSIONS AND RELEVANCE: The data suggest that inhibition of ovarian hormones and COX-2 may represent a therapeutic option for malignant feline mammary gland neoplasms. When evaluating disease progression, COX-2 scores and Ki-67 index should be analyzed in primary tumors and metastases. Histologic grade, HER-2 status and COX-2 scores were found to have a direct influence on OS. Prognostic factors allow for a better understanding of disease outcome in a condition that is characterized by a poor prognosis. The present work highlights the need for further studies on endocrine therapy and COX-2 inhibitors, which could influence OS.

Feline intestinal mast cell tumours: clinicopathological characterisation and KIT mutation analysis.

OBJECTIVES: Feline intestinal mast cell tumours (FIMCTs) are rare and reportedly characterised by poor differentiation, aggressive biological behaviour and lack of reliable therapeutic aids. KIT proto-oncogene-activating mutations have never been investigated in these tumours. This study describes the main clinicopathological and microscopic features observed in 17 FIMCTs. METHODS: Tumour degree of differentiation, proliferative activity, Kit protein expression and KIT mutations were evaluated and correlated with survival to assess their prognostic relevance. RESULTS: Ten tumours were located in the small intestine, two in the ileocaecocolic junction, and five in the large intestine. Survival times ranged from 3-538 days. Fifteen tumours were evaluated histologically, and there were six well-differentiated, six moderately differentiated and three poorly differentiated FIMCTs. The last showed a medium-to-large deposition of collagen tissue (P <0.001), and significantly higher mitotic and Ki67 indexes compared with more differentiated tumours (P = 0.011). On survival analysis, tumour degree of differentiation (P <0.001) and a mitotic index >2 (P = 0.022) were significantly associated with decreased survival times. Twelve cases showed Kit protein immunoexpression. The Kit pattern was membranous in five cases (33.3%), focal paranuclear in five (33.3%) and diffuse cytoplasmic in two (13.3%). Cytoplasmic Kit patterns were associated with a lesser differentiation (P = 0.015). Mutation analysis was successfully performed on 12 primary tumours and four lymph node metastases; however, no encoding mutation was detected. CONCLUSIONS AND RELEVANCE: Contrary to reports in the literature, FIMCTs seem to have an extremely variable biological behaviour. We propose a classification based on tumour degree of differentiation and proliferative activity. These findings need to be confirmed in larger series, and exploration of further genomic regions of KIT is warranted to clarify its role in the development and progression of these neoplasms.

Protease expression in giant cell tumour of bone: a comparative study on feline and human samples.

Human giant cell tumour of bone (GCTB) is a rare low grade of malignancy tumour with tendency to recur. During tumourigenesis the bone remodeling balance is subverted by the tumour cellular components that interacting with bone matrix induce release of growth factors and cytokines, promoting cell proliferation and bone resorption. The master regulators of this positive feed-back are acid and neutral proteases that destroying extracellular matrix increase osteolysis. In contrast, in cats, very few data are reported on GCTB biological activity. In this study, histological features and metalloproteinase (MMPs) and urokinase plasminogen activator system (uPA) expression were compared in human and feline GCTB and differences in distribution and intensity related to histological pattern and clinical behaviour were determined. In both species, the overexpression of these molecules suggested a strong and complex cross-talk between tumour and microenvironment.

Therapeutic response in feline sandhoff disease despite immunity to intracranial gene therapy.

Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of ß-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and ß-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.

Prognostic significance of Kit receptor tyrosine kinase dysregulations in feline cutaneous mast cell tumors.

Feline cutaneous mast cell tumors (FeCMCTs) are characterized by variable biological behavior. Development of multiple nodules and potential visceral involvement, along with inconsistency of conventional prognostic aids, justify uncertainty in differentiating benign from malignant forms. c-Kit proto-oncogene activating mutations have been reported in feline mast cell tumors (MCTs), but their prognostic relevance was not investigated. This study was performed on FeCMCTs with variable clinical outcome to assess whether Kit cytoplasmic immunohistochemical labeling can be regarded as indicative of c-Kit mutations and to evaluate the relationship between Kit dysregulation and survival. Twenty-four cats diagnosed with a primary cutaneous MCT were enrolled. Kit immunohistochemical pattern and c-Kit (exons 8, 9, 11) mutational status were assessed in 34 tumor samples. Risk factors affecting survival were a number of mitoses greater than 5 per 10 HPFs (P = .017) and cytoplasmic Kit labeling (P = .045). Increased mitotic activity was associated with Kit cytoplasmic expression (P = .01). c-Kit encoding mutations were present in 19 (56%) tumors (exon 8, 19%; exon 9, 71%; exon 11, 10%), however, they were not significantly related to protein expression and they had no influence on prognosis. Additionally, in 6 of 9 (67%) cats, multiple nodules from the same cat had different mutational statuses. Mutations in the fifth immunoglobulin-like domain of Kit occur frequently in FeCMCT, but they are variably associated with aberrant protein expression and do not appear to be strictly correlated with biological behavior. These findings need to be confirmed in larger series, and exploration of further genomic regions of c-Kit is warranted.

Serum thymidine kinase activity in clinically healthy and diseased cats: a potential biomarker for lymphoma.

The thymidine kinases are enzymes that convert deoxythymidine to deoxythymidine monophosphate and have a function in DNA synthesis. Rapidly proliferating cells will have higher levels of thymidine kinase. Serum thymidine kinase activity (sTK) is a useful tumour marker in humans and dogs, with utility as a prognostic indicator in lymphoma. In the current study serum samples were collected from 49 clinically healthy cats, 33 with lymphoma, 55 with inflammatory disease and 34 with non-haematopoietic neoplasia (NHPN). sTK was measured using a radioenzyme assay and a reference interval (1.96 × SD) was established from the clinically healthy cats (<5.5 U/l). Mean sTK activity for healthy cats was 2.2 U/l (range 0.8-8.4, ± SD 1.7). Mean sTK activity for cats with lymphoma was 17.5 U/l (range 1.0-100.0 SD ± 27.4). Mean sTK activity for cats with NHPN was 4.2 U/l (range 1.0-45.0, SD ± 8.6). Mean sTK activity for the inflammatory group was 3.4 U/l (range 1.0-19.6, SD 3.9). Cats with lymphoma had significantly higher sTK activity than healthy cats or cats with inflammatory disease (P <0.0001) and cats with NHPN (P <0.0002). sTK activity is a potentially useful biomarker for feline lymphoma and further study is required to assess its utility as a prognostic indicator.

Exocrine pancreatic insufficiency in the cat.

Exocrine pancreatic insufficiency (EPI) is a syndrome caused by an insufficient amount of pancreatic digestive enzymes in the small intestine. Clinical signs most commonly reported in cats with EPI are weight loss, loose and voluminous stools, steatorrhea, polyphagia, and in some cases a greasy soiling of the hair coat in the perianal region. Serum feline trypsin-like immunoreactivity concentration is the diagnostic test of choice for the diagnosis of affected cats. Treatment of cats with EPI consists of enzyme supplementation with either a powdered pancreatic extract or raw pancreas. Most cats with EPI also have severely decreased serum cobalamin concentrations and may require lifelong parenteral cobalamin supplementation. Most cats respond well to therapy and can have a normal life expectancy and quality of life.

Neonatal gene therapy with a gamma retroviral vector in mucopolysaccharidosis VI cats.

Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.