Evaluation of feline oral squamous cell carcinomas for p16 protein immunoreactivity and the presence of papillomaviral DNA

Oral squamous cell carcinomas (OSCCs) develop commonly in cats. While the cause of the feline neoplasms is unknown, a quarter of human OSCCs are caused by papillomavirus (PV) infection. As PV DNA has been previously detected in a feline OSCC, it was hypothesised that PV infection could be a significant cause of feline OSCCs. Human OSCCs that are caused by PVs contain increased p16^CDKN2A protein (p16), which can be detected using immunohistochemistry. In cats, increased p16 immunoreactivity has been reported within PV-associated skin lesions. This study evaluated p16 immunoreactivity within 30 feline OSCCs. Additionally, PCR was used to amplify PV DNA from the OSCCs. Increased p16 immunoreactivity was present within 2 OSCCs. However, as PV DNA was not amplified from any OSCC in this study, it cannot be confirmed that the increased p16 was caused by PV infection. Therefore, these results do not support the hypothesis that PVs are a significant cause of OSCCs in cats. Loss of p16 expression is considered an important process in the development of human non-PV-induced OSCCs. In contrast, loss of p16 immunoreactivity was only present in 2 feline OSCCs. This suggests that human and feline OSCCs develop due to different molecular mechanisms.     

Detection of two different papillomaviruses within a feline cutaneous squamous cell carcinoma: Case report and review of the literature

CASE HISTORY A 15-year-old, brown-and-white cat was presented to a veterinary clinic with an ulcerated, reddened 1-cm diameter lesion on the nasal planum.

CLINICAL FINDINGS AND DIAGNOSIS: Histology of a biopsy sample confirmed the lesion was a squamous cell carcinoma (SCC). PCR amplified DNA sequences from two different papillomaviruses. One sequence was from FdPV 2, which has previously been amplified from feline cutaneous SCC. However, the other sequence has not previously been reported, suggesting a novel feline papillomavirus.

CLINICAL RELEVANCE: There is evidence that papillomaviruses promote the development of SCC on sun-exposed skin in humans. This is the first report in a cat of a papillomavirus other than FdPV2 and the first time that multiple papillomaviruses have been detected within a single neoplasm in this species. Whether the papillomaviruses influenced the development and behaviour of this SCC is currently uncertain, but this case provides additional evidence of the association between papillomaviruses and feline cutaneous SCC. If papillomaviruses are found to influence the development of SCC this may allow novel strategies to prevent these common neoplasms in cats.     

Detection of papillomaviral sequences in feline Bowenoid in situ carcinoma using consensus primers

Feline Bowenoid in situ carcinoma (BISC) is a rare disease that presents as multiple discrete plaques of epidermal hyperplasia and dysplasia. Two studies using immunohistochemistry revealed papillomaviral antigens in 11% and 47% of BISCs. Additionally, a recent study detected papillomaviral DNA in 24% of BISC lesions. To further investigate the association between papillomaviruses and BISC, polymerase chain reaction using consensus primers was used to detect papillomaviral DNA in 18 formalin-fixed samples of BISC. Papillomaviral DNA was amplified from 11 of the samples but from none of the controls. Six amplicons were sequenced; one was homologous with a papillomavirus from a human patient with multiple cutaneous squamous cell carcinomas and the other five showed weak homology to human papillomavirus type 17. These five sequences were > 96% homologous over a 235 bp sequence, indicating the presence in all five BISCs of one papillomavirus type distinct from any previously sequenced and more closely related to human than animal papillomaviruses. The results confirm an association between BISC and papillomaviruses, and as all six papillomavirus sequences identified are closely related to human papillomaviruses, it is possible that the virus is transmitted from humans to cats or vice versa.     

Association between ovarihysterectomy and feline mammary carcinoma

The etiopathogenesis of feline mammary carcinoma is not well understood. Although putative, risk factors include breed, reproductive status, and regular exposure to progestins. An association between age at ovarihysterectomy (OHE) and mammary carcinoma development has not been established. Therefore, a case-control study was performed to determine the effects of OHE age, breed, progestin exposure, and parity on feline mammary carcinoma development. Cases were female cats diagnosed with mammary carcinoma by histological examination of mammary tissue. Controls were female cats not diagnosed with mammary tumors selected from the same biopsy service population. Controls were frequency matched to cases by age and year of diagnosis. Questionnaires were sent to veterinarians for 308 cases and 400 controls. The overall questionnaire response rate was 58%. Intact cats were significantly overrepresented (odds ratio [OR] 2.7, confidence interval [CI] = 1.4-5.3, P < .001) in the mammary carcinoma population. Cats spayed prior to 6 months of age had a 91% reduction in the risk of mammary carcinoma development compared with intact cats (OR 0.9, CI = 0.03-0.24). Those spayed prior to 1 year had an 86% reduction in risk (OR 0.14, CI = 0.06-0.34). Parity did not affect feline mammary carcinoma development, and too few cats had progestin exposure to determine association with mammary carcinoma. Results indicate that cats spayed before 1 year of age are at significantly decreased risk of feline mammary carcinoma development.     

A multi-institutional epidemiologic study evaluating environmental risk factors for feline oral squamous cell carcinoma

Feline oral squamous cell carcinoma (FOSCC) is an aggressive cancer in domestic cats that has no effective treatment option when advanced. Preventative or early diagnostic measures are thus crucial. FOSCC is also a model for human head and neck SCC (HNSCC); strong risk factors in HNSCC include exposure to alcohol, tobacco, areca nut, and high-risk human papillomavirus. Previous studies have identified flea collar and tobacco smoke exposure, feeding canned tuna, canned cat food and cat foods with chemical additives, living in a rural environment, and having outdoor access as risk factors for FOSCC but there was no overlap in the risk factors between studies. In our study, risks for FOSCC were evaluated in an online epidemiologic survey study in 67 cats with FOSCC and 129 control cats. Clumping clay cat litter and flea collar use were significant risk factors for FOSCC on multiple logistic regression with odds ratios of 1.66 (95% CI 1.20–2.30) and 4.48 (95% CI 1.46–13.75) respectively. Crystalline silica is a carcinogen that may be present in all clay cat litters and tetrachlorvinphos is a carcinogen that is present in the most commonly used flea collars in our study. We recommend further investigation into the association between FOSCC and clay-based litter and/or flea collars containing tetrachlorvinphos.     

Assessment of predictive molecular variables in feline oral squamous cell carcinoma treated with stereotactic radiation therapy

This study evaluated molecular characteristics that are potentially prognostic in cats with oral squamous cell carcinoma (SCC) that underwent stereotactic radiation therapy (SRT). Survival time (ST) and progression‐free interval (PFI) were correlated with mitotic index, histopathological grades, Ki67 and epidermal growth factor receptor expressions, tumour microvascular density (MVD), and tumour oxygen tension (pO₂). Median ST and PFI were 106 and 87 days, respectively (n = 20). Overall response rate was 38.5% with rapid improvement of clinical symptoms in many cases. Patients with higher MVD or more keratinized SCC had significantly shorter ST or PFI than patients with lower MVD or less keratinized SCC (P = 0.041 and 0.049, respectively). Females had significantly longer PFI and ST than males (P ≤ 0.016). Acute toxicities were minimal. However, treatment‐related complications such as fractured mandible impacted quality of life. In conclusion, SRT alone should be considered as a palliative treatment. MVD and degree of keratinization may be useful prognostic markers.     

Epidermal growth factor receptor expression in feline oral squamous cell carcinomas

Feline oral squamous cell carcinomas (SCC) have a poor prognosis despite aggressive treatment with surgery, radiation and anticancer drugs. Overexpression of the epidermal growth factor receptor (EGFR), a membrane‐bound tyrosine kinase receptor, has been found in many human epithelial neoplasms, including oral SCC. EGFR overexpression has been associated with advanced disease and a poor prognosis. The purpose of this study was to determine whether feline oral SCC express EGFR. Thirteen formalin‐fixed paraffin wax‐embedded biopsy samples from feline oral SCC were analysed for EGFR expression using immunohistochemistry. Nine of 13 tumours (69%) were positive for EGFR expression, suggesting that altered EGFR expression plays a role in feline oral SCC and provides a rationale for a potential clinical benefit using EGFR inhibitors in combination with conventional treatments.     

Detection of human papillomavirus DNA in feline premalignant and invasive squamous cell carcinoma

Squamous cell carcinoma (SCC) is the most common malignant cutaneous and oral neoplasm of cats. Papillomavirus (PV) DNA has been identified in a proportion of feline Bowenoid in situ carcinomas (BISCs), cutaneous SCCs and a single oral SCC, but its exact role in the pathogenesis remains unknown. In humans, it has been suggested that ultraviolet (UV) light and human PV (HPV) may act as cofactors in cutaneous SCC carcinogenesis. Little is known about the influence of UV light on PV prevalence in feline cutaneous lesions, including actinic keratosis (AK). Additionally, PV prevalence in noncutaneous feline lesions, including oral SCC, is largely not known. This study aimed to determine the presence of PV in 84 cats with premalignant and invasive SCC from cutaneous and noncutaneous sites using polymerase chain reaction and to investigate an association with UV light. Papillomaviral DNA was amplified from two of 12 cases of AK, seven of 22 BISCs, nine of 39 cutaneous SCCs and two of 35 non‐cutaneous SCCs. Of the PV DNA sequenced, 50% was most similar to HPV of the genus Betapapillomavirus, while the other 50% was most similar to Felis domesticus PV type 2. Exposure to UV was not associated with an increase in PV for cutaneous SCC. The results of this study suggest that in the cat, HPV DNA may be detectible within a higher percentage of squamous lesions than previously demonstrated, UV exposure may not be a confounder for PV presence, and noncutaneous lesions may have a low prevalence of PV.     

Amplification of papillomaviral DNA sequences from a high proportion of feline cutaneous in situ and invasive squamous cell carcinomas using a nested polymerase chain reaction

Squamous cell carcinomas (SCCs) are common skin tumours of cats. Previous studies have suggested that papillomaviral (PV) DNA is detectible within some feline SCCs. A PV DNA sequence has been previously amplified from five feline bowenoid in situ carcinomas (BISCs). Primers specific for this sequence were used in a nested polymerase chain reaction to compare PV detection rates in SCCs to rates within non-SCC skin lesions. Papillomaviral DNA was amplified from 20 of 20 BISC, 17 of 20 invasive SCC and 3 of 17 non-SCC controls. The rate of PV amplification from feline cutaneous SCCs was significantly higher than from non-SCC lesions. These results confirm that feline cutaneous SCCs are associated with PV infection. In humans, there is evidence that PVs promote SCC development within sun-exposed skin. The demonstrated association between PVs and feline cutaneous SCCs suggests, but does not prove, that PVs may also promote feline SCC development. If PVs are oncogenic in cats, prevention of PV infection may reduce feline cutaneous SCC development. To the authors' knowledge, this is the first time that PV DNA has been amplified from a non-SCC sample of feline skin.     

Gemcitabine as a radiosensitizer for nonresectable feline oral squamous cell carcinoma

Eight cats with locally advanced, oral squamous cell carcinoma (SCC) were treated with a combination of gemcitabine and palliative radiotherapy. Low-dose gemcitabine was administered twice weekly (25 mg/m2) in conjunction with megavoltage radiation in 6 Gray (Gy) fractions for a total dose of 36 Gy. Responses included two complete and four partial responses, and two cats had no response to therapy. Median duration of remission was 42.5 days (range, 11 to 85 days). Median survival time was 111.5 days (range, 11 to 234 days). This data suggests that a combination of low-dose gemcitabine and palliative radiation therapy may be tolerable for cats with oral SCC and may cause a therapeutic benefit.     

Predicting clinical outcome in feline oral squamous cell carcinoma: tumour initiating cells,telomeres and telomerase

Feline oral squamous cell carcinoma (SCC) has very poor prognosis. Here, a retrospective pilot study was conducted on 20 feline oral SCC patients who underwent stereotactic radiation therapy (SRT), to evaluate: (1) the value of putative tumour initiating cell (TIC) markers of human head and neck SCC (CD44, Bmi‐1); (2) telomere length (TL) specifically in putative TICs; and (3) tumour relative telomerase activity (TA). Significant inverse correlations were found between treatment outcomes and Bmi‐1 expression, supporting the predictive value of Bmi‐1 as a negative prognostic indicator. While TL exhibited a wide range of variability, particularly in very short fractions, many tumours possessed high levels of TA, which correlated with high levels of Bmi‐1, Ki67 and EGFR. Taken together, our results imply that Bmi‐1 and telomerase may represent novel therapeutic targets in feline oral SCC, as their inhibition – in combination with SRT – would be expected to have beneficial treatment outcome.     

Piroxicam and carboplatin as a combination treatment of canine oral non-tonsillar squamous cell carcinoma: a pilot study and a literature review of a canine model of human head and neck squamous cell carcinoma

Results of the treatment with a combination of carboplatin and piroxicam in seven dogs with advanced non‐tonsillar oral squamous cell carcinoma (SCC) were retrospectively analysed. This multi‐agent protocol was well tolerated by all dogs and resulted in a complete regression of the tumour without additional surgery in four of seven patients. Additional surgery was necessary to remove a metastatic lymph node in one dog and residual tumour in a second dog, which achieved a partial response following medical therapy. Median follow‐up for all the dogs was 534 days, while the time‐to‐recurrence, time‐to‐progression and overall survival for this group of patients have not yet been reached. Our study, although limited in number of animals, suggests that this multiagent approach is a useful treatment option for oral non‐tonsillarSCC in dogs and warrants wider application.     

Evaluation of 18F‐FDG PET/CT as a diagnostic imaging and staging tool for feline oral squamous cell carcinoma

18F‐fluorodeoxyglucose positron emission tomography combined with computed tomography (18FDG‐PET/CT) has been shown to be effective for staging human oral squamous cell carcinoma (SCC) but its application for cats with oral SCC is unknown. Twelve cats with biopsy‐proven oral SCC were imaged with whole body 18FDG‐PET/CT to determine its value as a diagnostic imaging and staging tool and fine needle aspirates were obtained of accessible regional lymph nodes. All tumors were FDG avid and conspicuous on 18FDG‐PET/CT images, with an average of the maximum standardized uptake value 9.88 ± 5.33 SD (range 2.9–24.9). Soft tissue infiltrative tumors that were subtle and ill defined on CT were highly visible and more extensive on FDG‐PET/CT. Tumors invading the osseous structures were more similar in extent on 18FDG‐PET/CT and CT although they were more conspicuous on PET images. Three cytologically confirmed metastases were hypermetabolic on PET, while two of those metastases were equivocal on CT.     

Corneal squamous cell carcinoma in a Border Collie

A 6-year-old, female, spayed Border Collie was presented to the Unit of Comparative Ophthalmology at the Animal Health Trust with a 6-month history of a progressive nonpainful opacity of the left cornea. A keratectomy was performed and the tissue submitted for histopathology. The diagnosis was squamous cell carcinoma. There has been no recurrence of the neoplasm to date (5 months). Canine corneal squamous cell carcinoma (SCC) has not been reported previously in the UK.     

Neoadjuvant chemoradiotherapy and surgery as treatment for oral maxillary squamous cell carcinoma in a dog

A gingival maxillary squamous cell carcinoma was diagnosed in a 12-year-old male Yorkshire Terrier. After a complete diagnostic work-up, including a computed tomography scan, the tumour was staged as T3bN1aM0 and considered non-resectable at presentation. The combination of neoadjuvant megavoltage radiotherapy and neoadjuvant and adjuvant chemotherapy with carboplatin and doxorubicin decreased the size of the tumour, allowing for surgery. The dog was free from local disease for 421 days after which it was euthanased at the owners' request.     

Detection of canine oral papillomavirus-DNA in canine oral squamous cell carcinomas and p53 overexpressing skin papillomas of the dog using the polymerase chain reaction and non-radioactive in situ hybridization

Nineteen cutaneous and mucocutaneous papillomas, as well as 29 oral and 25 non-oral squamous cell carcinomas of dogs were analyzed immunohistologically for the presence of papillomavirus (PV)-antigens. Canine oral papillomavirus (COPV)-DNA was detected in formalin-fixed, paraffin-embedded tissues by polymerase chain reaction (PCR) and non-radioactive in situ hybridization (ISH). Furthermore, the expression of the tumor suppressor protein p53 was investigated. PV-antigens were detectable in more than 50% of the oral and cutaneous papillomas, while no PV-antigens could be demonstrated in venereal papillomas. One squamous cell carcinoma was PV-antigen positive. Only two cutaneous papillomas of the head showed a strong p53-specific immunostaining, while overexpressed p53 was detectable in approximately 35% of all squamous cell carcinomas. It was possible to amplify fragments of the E6, E7 and L1 gene by polymerase chain reaction (PCR) from five of eight oral and from five of eight cutaneous papillomas as well as from three oral squamous cell carcinomas. Nine of 10 papillomas showed a strong nucleus-associated hybridization signal typical for COPV-DNA. In three squamous cell carcinomas COPV-DNA was located in nests of the epithelial tumor cells surrounding ‘horn pearls' or disseminated in the carcinoma tissue. These observations support the view that COPV may also induce non-oral papillomas in the dog and confirm the opinion that a progression of viral papillomas into carcinomas in dogs may occur.     

Radical Eye Exenteration and Second Intention Healing in Horses: A Case Series

Exenteration of an eye may be necessary in clinical practice for a variety of disease processes. Specifically, neoplastic processes may require wide excision margins that may not allow primary closure of the surgical site. This report describes a series of horses undergoing unilateral radical eye exenteration for which primary closure was not possible because of the extent of tissue excised. For each case, the underlying pathology, surgical technique, perioperative treatments, and outcome are described. Six cases were treated of which three survive at this time, two were euthanized because of recurrence, and one was euthanized for unrelated problem. Radical exenteration of the eye with wide margins and second intention healing is easy to perform and well tolerated in horses. It should be considered when extensive resection is required for instance in neoplastic processes. Aftercare is required as for all wounds that heal by second intention; time from surgery to epithelialization is approximately 4 months.