Feline Oral Squamous Cell Carcinoma Research Articles

Computed Tomographic and Histopathological Characteristics of 13 Equine and 10 Feline Oral and Sinonasal Squamous Cell Carcinomas

Squamous cell carcinoma (SCC) is the most common equine sinonasal and feline oral tumour. This study aimed to describe the computed tomographic and histopathological characteristics of equine and feline SCC. Thirteen horses and 10 cats that had been histopathologically diagnosed with oral or sinonasal SCC and had undergone computed tomography (CT) of the head were retrospectively included in the study. CT characteristics of the mass and involved structures were noted. Histological examinations were evaluated according to a human malignancy grading system for oral SCC, which considered four grades of increasing aggressiveness. In horses, the masses were at the levels of the paranasal sinuses (n = 8), mandible (n = 3), tongue (n = 1), and nasal cavity (n = 1). In cats, the masses were at the levels of the maxilla (n = 4), mandible (n = 3), tongue (n = 1), and buccal region (n = 1) and were diffusely distributed (facial and cranial bones; n = 1). Masses in the equine paranasal sinuses showed only mild, solid/laminar, periosteal reactions with variable cortical destruction. However, maxillary lesions in cats showed severe cortical destruction and irregular, amorphous/pumice stone-like, periosteal reactions. CT revealed different SCC phenotypes that were unrelated to the histological grade. For morphologic parameters of the tumour cell population, a variability for the degree of keratinization and number of mitotic cells was noted in horses and cats. Concerning the tumour-host relationship a marked, extensive and deep invasion into the bone in the majority of horses and cats was seen. Most cases in both the horses and cats were categorized as histological grade III (n = 8); four horses and one cat were categorized as grade IV, and one horse and one cat were categorized as grade II. In this study, we examined the diagnostic images and corresponding applied human histopathological grading of SCC to further elucidate the correlations between pathology and oral and sinonasal SCC imaging in horses and cats.

Telomerase Reverse Transcriptase (TERT) Expression, Telomerase Activity, and Expression of Matrix Metalloproteinases (MMP)-1/-2/-9 in Feline Oral Squamous Cell Carcinoma Cell Lines Associated With Felis catus Papillomavirus Type-2 Infection.

Telomerase activity contributes to cell immortalization by avoiding telomere shortening at each cell division; indeed, its catalytic subunit telomerase reverse transcriptase (TERT) is overexpressed in many tumors, including human oral squamous cell carcinoma (hOSCC). In these tumors, matrix metalloproteinases (MMPs), a group of zinc-dependent endopeptidases involved in cell migration, contribute to invasive potential of cancer cells. A proportion of hOSCC is associated with infection by high-risk human papillomavirus (HR-HPVs), whose E6 oncogene enhances TERT and MMPs expression, thus promoting cancer progression. Feline oral squamous cell carcinoma (FOSCC) is a malignant tumor with highly invasive phenotype; however, studies on telomerase activity, TERT, and MMPs expression are scarce. In this study, we demonstrate telomerase activity, expression of TERT, and its transcriptional activator cMyc along with expression of MMP-1, -2, and -9 in FOSCC-derived cell lines SCCF2 and SCCF3, suggesting a contribution by these pathways in cell immortalization and invasion in these tumors. Recent studies suggest that a sub-group of FOSCC as well as SCCF2 and SCCF3 are associated with Felis catus PV type-2 (FcaPV-2) infection. However, in this work, FcaPV-2 E6 gene knock-down caused no shift in either TERT, cMyc, or MMPs levels, suggesting that, unlike its human counterpart, the viral oncogene plays no role in their regulation.

Detection of Felis catus papillomavirus type-2 DNA and viral gene expression suggest active infection in feline oral squamous cell carcinoma.

Papillomavirus (PV) infection is associated with development of epithelial cancer in different species, including domestic cat (Felis catus). Felis catus PV type-2 (FcaPV-2) is considered the causative agent of a proportion of feline cutaneous squamous cell carcinoma (SCC), through the transforming properties of its E6 and E7 oncogenes. However, the possible role of FcaPVs in the aetiology of feline oral SCC (FOSCC) is still unclear. The aim of this study was to assess the presence and gene expression of FcaPV-2 in FOSCC samples. We detected FcaPV-2 DNA in 10/32 (31%) of the analysed FOSCC by the use of PCR methods. Importantly, viral mRNA was detected by RT-PCR in 7/10 (70%) of DNA positive samples. In particular, FcaPV-2 L1, E2 and E6E7 genes were found to be expressed in 5/10 (50%), 3/10 (33%) and 5/10 (50%) samples, respectively. Viral DNA was also detected in non neoplastic oral ulcerative lesions (ULs) (4/11, 36%); qPCR suggested a difference in viral load between ULs and FOSCCs, particularly in those expressing E6E7, although it was not statistically significant. These data suggest, but do not definively prove, a possible role of FcaPV-2 in the development of a proportion of FOSCC. Moreover, L1 and E2 gene expression results indicate that FcaPV-2 infection associated with these tumours may possibly be productive.

A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumour growth of feline oral squamous cell carcinoma.

Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC.

A virome sequencing approach to feline oral squamous cell carcinoma to evaluate viral causative factors

Feline oral squamous cell carcinoma (FOSCC) may be the best naturally-occurring model of human head and neck squamous cell carcinoma (HNSCC). HNSCC can be broadly divided into human papillomavirus (HPV)-negative cancers and HPV-positive cancers where HPV is the causative agent. Previous studies in FOSCC have used both species-specific and species-nonspecific PCR primers that may be insensitive to the detection of PVs and other viruses that may be divergent from known sequences. ViroCap is a targeted capture and next generation sequencing tool that was designed to identify all known vertebrate DNA and RNA viruses. In this study we used a metagenomic approach using ViroCap for DNA viruses in 20 FOSCC, 9 normal feline oral mucosal, and 8 suspected PV positive control samples. We tested the hypothesis that viruses would be enriched in FOSCC compared to normal oral mucosa. The virome of the FOSCC and normal feline oral mucosa consisted of feline foamy virus in 7/20 and 2/9 (35% and 22%), feline torque teno virus in 2/20 and 0/9 (10% and 0%), alphaherpesvirus in 2/10 and 0/9 (10% and 0%), FIV (0% and 22%), Epstein-Barr virus in 1/20 and 0/9 (5% and 0%) and feline papillomavirus in 1/20 and 0/9 samples (5% and 0% respectively). Felis catus papillomavirus-3 was found in 1 of 20 FOSCC samples. A virus was not associated consistently with FOSCC. If PVs have a role in FOSCC it is at most a supplementary or uncommon role. FOSCC appears most closely related to HPV-negative HNSCC. Future research on FOSCC should focus on identifying genetic and environmental causes.

Immunohistochemical Features of Epithelial-Mesenchymal Transition in Feline Oral Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive malignancy with invasive and metastatic behavior. It is poorly responsive to chemotherapy and radiation. Neoplastic epithelial-mesenchymal transition (EMT) portends highly malignant behavior and enhances resistance to therapy. In transitioning to a more malignant phenotype, carcinoma stem cells undergo transformation mediated by expression of proteins, endowing them with mesenchymal properties advantageous to cell survival. The goal of the current study was to identify proteins associated with EMT in FOSCC. This study documents protein expression patterns in 10 FOSCC biopsies and 3 FOSCC cell lines (SCCF1, SCCF2, SCCF3), compatible with an EMT phenotype. As markers of EMT, P-cadherin, N-cadherin, vimentin, nuclear transcription factors Twist and Snail, hypoxia inducible factor 1α (HIF-1α), programmed death ligand 1, and vascular endothelial growth factor D, as well as E-cadherin, were examined using immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay. P-cadherin, Twist, HIF-1α, and programmed death ligand 1 were commonly expressed in biopsies and cell lines. N-cadherin, classically associated with EMT, was not highly expressed, and E-cadherin was coexpressed along with proteins characteristic of EMT in all specimens. Production of vascular endothelial growth factor A by cell lines, a process regulated by HIF-1α expression, was suppressed by the small-molecule inhibitor dasatinib. These data are consistent with EMT in FOSCC and shed light on cellular changes that could contribute to the aggressive behavior of FOSCC.

Increased p16CDKN2A, but not p53, immunostaining is predictive of longer survival time in cats with oral squamous cell carcinomas

Although oral squamous cell carcinomas (SCCs) are common in cats there are currently few prognostic markers for these cancers. This study used 52 feline oral SCCs to determine if prognosis can be predicted by the age or sex of the cat, the presence of bone within the diagnostic sample, or the anatomic location of the SCC. Additionally, as p16CDKN2A protein (p16) and p53 are prognostic for human oral SCCs, p16 and p53 immunostaining was evaluated.Only SCC location and p16 immunostaining were prognostic. Cats with oropharyngeal SCCs had an estimated median survival time (MST) of 151 days which was significantly longer than cats with maxillary (51 days P = 0.017), sublingual (33 days P = 0.011) and mandibular (34 days P = 0.029) SCCs. Overall, 19% of oral SCCs were p16-positive with intense nuclear and cytoplasmic immunostaining within most neoplastic cells, 69% had cytoplasmic immunostaining that was confined to the periphery of nests of neoplastic cells, and 12% had no p16 immunostaining. Cats with p16-positive SCCs had a MST of 87 days, which was significantly longer than cats with p16-peripheral SCCs (MST 37 days, P = 0.03), but not longer than cats with p16-negative SCCs (MST 51 days, P = 0.72). No papillomaviral DNA was amplified from the p16-positive SCCs. Twenty (39%) SCCs contained immunostaining for p53, but this was not prognostic (P = 0.31). These results suggest that feline oral SCCs develop by cellular mechanisms that result in one of three patterns of p16 immunostaining. Cancers which develop due to these mechanisms appear to have different clinical behaviors and p16 immunostaining predicts the behavior of these common feline cancers.

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.

Investigation of novel chemotherapeutics for feline oral squamous cell carcinoma.

Feline oral squamous cell carcinomas (FOSCC) are highly aggressive neoplasms with short survival times despite multimodal treatment. FOSCC are similar to squamous cell carcinomas of the head and neck (SCCHN) in humans, which also present therapeutic challenges. The current study was undertaken to identify novel chemotherapeutics using FOSCC cell lines. A high throughput drug screen using 1,952 drugs was performed to identify chemotherapeutics for further investigation. Two of the drugs identified in the drug screen, actinomycin D and methotrexate, and two drugs with similar molecular targets to drugs found to be efficacious in the screening, dinaciclib and flavopiridol, were selected for further investigation. Drug inhibition profiles were generated for each drug and cell line using an MTS assay. In addition, the effects of the drugs of interest on cell cycle progression were analyzed via a propidium iodide DNA labeling assay. Changes in caspase-3/7 activity after treatment with each drug were also determined. The findings demonstrated effectiveness of the drugs at nanomolar concentrations with sensitivity varying across cell lines. With all of the drugs except for actinomycin D, evidence for G1 arrest was found. Dinaciclib and flavopiridol were demonstrated to induce apoptosis. The results of the study suggest that the selected drugs are potential candidates for developing novel chemotherapeutic approaches to FOSCC. Through these studies, novel therapeutic strategies for the treatment of FOSCC can be developed to provide better care for affected cats which can also serve as proof of concept studies to inform translational studies in SCCHN in humans.

CD147 and Cyclooxygenase Expression in Feline Oral Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (OSCC) is a highly invasive form of cancer in cats. In human OSCC, cluster of differentiation 147 (CD147) contributes to inflammation and tumor invasiveness. CD147 is a potential therapeutic target, but the expression of CD147 in feline OSCC has not been examined. Immunohistochemistry was used to determine if cyclooxygenase 2 (COX-2) and CD147 expression in feline OSCC biopsies was coordinated. Tumor cells were more likely to express COX-2 (22/43 cases or 51%) compared to stroma (8/43 or 19%) and adjacent oral epithelium (9/31 cases or 29%) (p < 0.05). CD147 was also more likely to occur in tumor cells compared to stroma and adjacent mucosa, with 21/43 (49%) of cases having >50% tumor cells with mild or moderate CD147 expression, compared to 9/28 (32%) in adjacent epithelium and only 5/43 (12%) in adjacent stroma (p < 0.05). In feline OSCC cell lines (SCCF1, SCCF2, and SCCF3), CD147 gene expression was more consistently expressed compared to COX-2, which was 60-fold higher in SCCF2 cells compared to SCCF1 cells (p < 0.05). CD147 expression did not correlate with COX-2 expression and prostaglandin E2 (PGE2) secretion, indicating that they may be independently regulated. CD147 potentially represents a novel therapeutic target for the treatment of feline OSCC and further study of CD147 is warranted.

Investigation of immune cell markers in feline oral squamous cell carcinoma

Squamous cell carcinoma is the most common oral cancer in the cat and presents as a locally aggressive lesion for which an effective therapeutic protocol remains elusive. Feline oral squamous cell carcinoma (OSCC) shares many clinical characteristics with human head and neck squamous cell carcinoma (HNSCC). Accordingly, present studies were conducted to determine similarities for immune markers shared by feline OSCC and human HNSCC. Biopsies harvested from a feline patient cohort-1 (n = 12) were analyzed for lymphoid cell infiltrates by immunohistochemistry (IHC). Results revealed unique patterns of T cell infiltration involving both neoplastic epithelium and stroma that were detected in most patient tumor biopsies (92%) examined by IHC staining for CD3. Intratumoral B cell infiltrates were detected within tumor stroma only, based on IHC staining for CD79a and CD20 for all patients within the same cohort-1. Infiltration of tumors by a regulatory CD4 T cell subset (Tregs) defined by expression of the forkhead transcription factor FoxP3, was also detected in biopsies from 57% of patients and involved infiltration of neoplastic epithelium and stroma. Patient biopsies were also examined for expression of immunomodulator cyclooxygenase (COX)-2 and revealed positive but weak staining of neoplastic epithelium in a significant proportion of cases (75%). Interestingly, COX-2 expression was detected in both neoplastic epithelium and stroma. Blood collected from a second cohort of feline OSCC patients (n = 9) revealed an increased frequency of circulating CD4+FoxP3+ T cells when compared to healthy adult controls (n = 7) (P = 0.045), although frequencies of CD4+CD25+FoxP3+ T cells were comparable between patients and healthy pet cat controls. Lastly, biopsies from feline OSCC patients were characterized for histologic subtype using a classification scheme previously described for human HNSCC. This analysis revealed the conventional subtype as the predominant variant (75%) with conventional subtypes split evenly between well differentiated and moderately differentiated carcinomas. Two cases were classified as papillary and one case as basaloid subtypes. Correlations between subtype, immune marker scores or circulating Treg frequencies and clinical characteristics or outcome were not detected, most likely due to small patient numbers within patient cohorts. However, findings from these studies provide a preliminary step in the characterization of immune and histologic markers that will be critical to defining prognostic immune markers for feline OSCC and potential targets for testing of immunotherapeutics also relevant to human HNSCC in future studies.

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.

Inhibition of the PI3K/AKT Pathway Sensitizes Oral Squamous Cell Carcinoma Cells to Anthracycline-Based Chemotherapy In Vitro.

Anthracycline-based chemotherapy, such as doxorubicin (Dox), while effective against many solid tumors, is not widely used for head and neck cancers. In this study, we evaluated the efficacy of Dox, and its derivative AD198 in human, canine, and feline oral squamous cell carcinomas cells (OSCC) in vitro. Dox and AD198 had significant an anti-proliferative effect on human, canine, and feline OSCC cells in dose-dependent manner. AD198 inhibited cell proliferation more effectively than Dox in tested OSCC cells. In the human oral squamous cell carcinoma SCC25 cells, Dox and AD198 increased the production of reactive oxygen species and subsequently increased apoptosis through activation of caspase signaling pathway. Dox and AD198 increased activation of AKT, ERK1/2, and p38 MAPK signaling pathways in tested OSCC cells by dose-dependent manner. The efficacy of Dox and AD198 treatments in inhibition of cell proliferation was increased in tested OSCC when combined with PI3K/AKT inhibitor, LY294002 treatment. Inhibition of PI3K/AKT reduced Dox- and AD198-induced activation of ERK1/2 and further increased Dox- and AD198-induced phosphorylation of p38 MAPK in OSCC. Our results suggest that the anthracycline therapies, such as Dox or AD198, can be more effective for treatment of OSCC when combined with inhibitors of the PI3K/AKT pathway. J. Cell. Biochem. 118: 2615-2624, 2017. © 2016 Wiley Periodicals, Inc.

Therapeutic Targeting of Protein Kinase CK2 Gene Expression in Feline Oral Squamous Cell Carcinoma: A Naturally Occurring Large-Animal Model of Head and Neck Cancer.

Protein kinase CK2 (CK2) is a highly promising target for cancer therapy, and anti-CK2 gene expression therapy has shown effectiveness in rodent models of human head and neck cancer (HNC). To date, there has been no large-animal model of cancer in which to further explore anti-CK2 therapies. Feline oral squamous cell carcinoma (FOSCC) has been proposed as a large-animal model for human HNC, and we have previously shown that CK2 is a rational target in FOSCC. Here we have tested the hypothesis that a novel tenfibgen-coated tumor-specific nanocapsule carrying RNA interference (RNAi) oligonucleotides targeting feline CK2α and CK2α' (TBG-RNAi-fCK2αα') would be safe in cats with FOSCC; assessment of target inhibition and tumor response were secondary aims. Nine cats were enrolled and treated at two dose levels in a 3+3 escalation. Cats received a total of six treatments with TBG-RNAi-fCK2αα'. Pre- and posttreatment, tumor and normal oral mucosa biopsies were collected to assess CK2 expression, using immunohistochemistry (IHC) preparations evaluated by light microscopy. Toxicity and tumor response were assessed on the basis of standard criteria. The most common adverse events were grade 1 or 2 weight loss and anorexia. Grade 3 tissue necrosis was seen in association with tumor response in one cat, asymptomatic grade 4 elevations in aspartate transaminase and creatine phosphokinase in one cat, and asymptomatic grade 3 hypokalemia in one cat. Of six cats with evaluable biopsies, two had a reduction in CK2 IHC score in tumors after treatment. Four cats had progressive disease during the study period, three had stable disease, one had partial response, and response could not be evaluated in one cat. We conclude that the drug appeared safe and that there is some evidence of efficacy in FOSCC. Further investigation regarding dosing, schedule, target modulation, toxicity, and efficacy in a larger group of cats is warranted and may inform future clinical studies in human head and neck cancer.

Pharmacokinetics and derivation of an anticancer dosing regimen for the novel anti-cancer agent isobutyl-deoxynyboquinone (IB-DNQ), a NQO1 bioactivatable molecule, in the domestic felid species.

Isobutyl-deoxynyboquinone (IB-DNQ) is a selective substrate for NAD(P)H:quinone oxidoreductase (NQO1), an enzyme overexpressed in many solid tumors. Following activation by NQO1, IB-DNQ participates in a catalytic futile reduction/reoxidation cycle with consequent toxic reactive oxygen species generation within the tumor microenvironment. To elucidate the potential of IB-DNQ to serve as a novel anticancer agent, in vitro studies coupled with in vivo pharmacokinetic and toxicologic investigations in the domestic felid species were conducted to investigate the tractability of IB-DNQ as a translationally applicable anticancer agent. First, using feline oral squamous cell carcinoma (OSCC) as a comparative cancer model, expressions of NQO1 were characterized in not only human, but also feline OSCC tissue microarrays. Second, IB-DNQ mediated cytotoxicity in three immortalized feline OSCC cell lines were studied under dose-dependent and sequential exposure conditions. Third, the feasibility of administering IB-DNQ at doses predicted to achieve cytotoxic plasma concentrations and biologically relevant durations of exposure were investigated through pharmacokinetic and tolerability studies in healthy research felines. Intravenous administration of IB-DNQ at 1.0-2.0mg/kg achieved peak plasma concentrations and durations of exposure reaching or exceeding predicted in vitro cytotoxic concentrations. Clinical adverse side effects including ptyalism and tachypnea exhibited during and post-IV infusion of IB-DNQ were transient and tolerable. Additionally, IB-DNQ administration did not produce acute or delayed-onset unacceptable hematologic, non-hematologic, or off-target oxidative toxicities. Collectively, the findings reported here within provide important safety and pharmacokinetic data to support the continued development of IB-DNQ as a novel anticancer strategy for NQO1 expressing cancers.

Felis catus papillomavirus type 2 E6 oncogene enhances mitogen-activated protein kinases and Akt activation but not EGFR expression in an in vitro feline model of viral pathogenesis

A possible causative role of Felis catus papillomavirus type 2 (FcaPV2) in the development of feline oral and cutaneous squamous cell carcinomas (SCC) has been recently suggested by demonstrating viral gene expression in vivo and transforming properties by its putative oncogenes E6 and E7 in vitro. The activated molecules MEK (pMEK), ERK (pERK) and Akt (pAkt) are signaling transduction effectors regulating cell proliferation and inhibition of apoptosis, which are critical steps towards tumour formation. Here, we show by Western blotting (WB) that expression of FcaPV2 E6 in feline epithelial cells enhances pMEK, pERK and pAkt levels compared to control cells. Additionally, we demonstrated by real-time quantitative PCR on epidermal growth factor receptor (EGFR) transcripts and WB that activation of these signaling routes is independent from EGFR differential gene expression, total protein levels or phosphorylation, unlike in human papillomavirus associated tumours. This study contributes to define the molecular scenario underlying FcaPV2-triggered pathogenesis of feline SCC.

Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease.

P16, pRb, and p53 in Feline Oral Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is a highly aggressive head and neck cancer in cats, but the molecular pathogenesis of this cancer is still uncertain. In this study, p16, p53, and pRb proteins were detected and quantified by immunohistochemistry in forty-three FOSCC primary tumors and three FOSCC xenografts. p16 mRNA levels were also measured in three FOSCC cell lines (SCCF1, F2, and F3), which were consistent with their p16 immunoreactivity. Feline SCCF1 cells had very high levels of p16 protein and mRNA (55-fold greater) compared to SCCF2 and F3. A partial feline p16 cDNA sequence was amplified and sequenced. The average age of cats with FOSCC with high p16 immunoreactivity was significantly lower than the average age in the low p16 group. Eighteen of 43 (42%) FOSCCs had low p16 intensity, while 6/43 (14%) had high p16 immunoreactivity. Feline papillomavirus L1 (major capsid) DNA was not detected in the SCC cell lines or the FOSCCs with high p16 immunostaining. Five of 6 (83%) of the high p16 FOSCC had low p53, but only 1/6 (17%) had low pRb immunoreactivity. In summary, the staining pattern of p16, p53, and pRb in FOSCC was different from human head and neck squamous cell carcinoma and feline cutaneous squamous cell carcinoma. The majority of FOSCCs have low p16 immunostaining intensity, therefore, inactivation of CDKN2A is suspected to play a role in the pathogenesis of FOSCC. A subset of FOSCCs had increased p16 protein, which supports an alternate pathogenesis of cancer in these cats.

High Numbers of Stromal Cancer-Associated Fibroblasts Are Associated With a Shorter Survival Time in Cats With Oral Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAFs) are fibroblastic cells that express α-smooth muscle actin and have been identified in the stroma of numerous epithelial tumors. The presence of CAFs within the tumor stroma has been associated with a poorer prognosis in some human cancers, including oral squamous cell carcinomas (SCCs). Cats frequently develop oral SCCs, and although these are generally highly aggressive neoplasms, there is currently a lack of prognostic markers for these tumors. The authors investigated the prognostic value of the presence of CAFs within the stroma of oral SCC biopsy specimens from 47 cats. In addition, several epidemiologic, clinical, and histologic variables were also assessed for prognostic significance. A CAF-positive stroma was identified in 35 of 47 SCCs (74.5%), and the median survival time (ST) of cats with CAF-positive SCCs (35 days) was significantly shorter than that of cats with CAF-negative SCCs (48.5 days) (P = .031). ST was also associated with the location of the primary tumor (P = .0018): the median ST for oropharyngeal SCCs (179 days) was significantly longer than for maxillary (43.5 days; P = .047), mandibular (42 days; P = .022), and sublingual SCCs (22.5 days; P = .0005). The median ST of sublingual SCCs was also shorter compared with maxillary SCCs (P = .0017). Furthermore, a significant association was identified between site and the presence of stromal CAFs (P = .025). On the basis of this retrospective study, evaluating the tumor stroma for CAFs in feline oral SCC biopsy specimens may be of potential prognostic value.

Retrospective evaluation of toceranib phosphate (Palladia) in cats with oral squamous cell carcinoma.

Objectives The aim of the study was to determine the clinical benefit and adverse event profile of toceranib phosphate in the treatment of feline oral squamous cell carcinoma (FOSCC). Methods Data obtained from the medical records of cats with oral squamous cell carcinoma diagnosed between 2010 and 2014 treated with toceranib phosphate were compared with medical record data from cats that did not receive toceranib, cytotoxic chemotherapy or radiation, to determine the response to toceranib treatment and adverse event profile of toceranib in cats. Concurrent use of non-steroidal anti-inflammatory drugs (NSAIDs) was allowed. Results Forty-six cats with FOSCC were included; 23 received treatment with toceranib (group 1) and 23 did not (group 2). The overall biological response rate in group 1 was 56.5%. Median survival time of toceranib-treated cats was significantly longer at 123 days compared with 45 days in cats not treated with toceranib ( P = 0.01). Cats achieving stable disease or better on toceranib therapy had significantly longer progression-free survival ( P <0.0001) and median survival ( P = 0.0042) times than those with progressive disease on toceranib. Administration of NSAIDs was also associated with significantly improved survival time ( P = 0.0038) among all cats. Anorexia was common but may reflect the underlying disease in these patients. Toceranib was well tolerated in cats, with the most common side effect being mild gastrointestinal toxicity. Conclusions and relevance Toceranib was well tolerated in cats with oral squamous cell carcinoma and may lead to improved survival times, especially when combined with NSAIDs. NSAID administration was also associated with improved survival times, and the relative benefit of toceranib and NSAIDs is difficult to determine from this retrospective study. Despite improvement in survival times, long-term survival in this patient population remained poor. As toceranib was well tolerated and may improve survival time, prospective evaluation of toceranib alone is warranted to assess response as a single agent and as part of multimodal therapy in an effort to achieve a more durable response in FOSCC.