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Penile squamous cell carcinomas (SCCs) are common, potentially life-threatening neoplasms of horses. They are well-recognized to be caused by Equus caballus papillomavirus (EcPV) type 2, although EcPV2 cannot be detected in all cases. A 23-year-old standardbred gelding developed multiple penile in situ and invasive SCCs that contained histological evidence of PV infection. By using both consensus and specific PCR primers, these lesions were found to contain EcPV7 DNA, but not DNA from EcPV2 or any other PV type. To determine how frequently EcPV7 is present in equine penile SCCs, specific primers were used to detect EcPV2 and EcPV7 in a series of 20 archived samples. EcPV7 was the only PV detected in one, both EcPV2 and 7 were detected in five, and only EcPV2 was detected in 14 SCCs. EcPV7 DNA was also detected in three of 10 archived oropharyngeal SCCs, although only as a co- infection with EcPV2. This is the first report of EcPV7 causing disease in horses. These results suggest EcPV7 could cause a subset of equine penile SCCs, and this is the first evidence that PV types other than EcPV2 can cause these neoplasms. The detection of EcPV7 in the oropharyngeal SCCs suggests a potential role of this PV type in the development of these SCCs. There were no clinical or histological features that differentiated lesions containing EcPV7 DNA from those containing EcPV2 DNA. If EcPV7 causes a proportion of equine penile SCCs, vaccines to prevent EcPV2 infection may not prevent all equine penile SCCs. • EcPV7 is a detectible in penile and laryngeal SCCs in horses • EcPV7 was detected as a sole infection in two equine penile SCCs • EcPV7 probably causes a subset of equine penile SCCs • EcPV7 is a less common cause of penile SCCs than EcPV2 • Not all equine penile SCCs may be prevented by vaccines against EcPV2 [ABSTRACT FROM AUTHOR]

Equus caballus papillomavirus type 2 (EcPV-2) has been recognized as a potential cause of a subset of genital squamous cell carcinomas (SCCs) in horses. In the current study, we measured EcPV-2 seropositivity in 50 healthy horses from Western Canada, and these were compared to a herd of horses with known EcPV-2 exposure. Second, the presence of EcPV-2 DNA was measured using EcPV-2-specific PCR (polymerase chain reaction), performed on a variety of tissues collected at necropsy from 70 horses that lacked any history, gross, or histologic evidence of neoplasia or papillomavirus-associated disease. EcPV-2-specific RNA in situ hybridization (R-ISH) was performed on PCR-positive samples to identify the specific tissues infected. The prevalence of asymptomatic infection with EcPV-2 in Western Canadian horses was 20/70 (29%). Exposure to EcPV-2 as measured by seropositivity was 18/50 (36%). EcPV-2 positivity by anatomic location, as measured by R-ISH, was as follows: penis 10/29 (35%), vulva 5/34 (15%), eyelid 8/68 (12%), oral mucosa 7/65 (11%), skin from muzzle 7/68 (10%), and retropharyngeal lymph node 2/64 (3%). The youngest horses with EcPV-2 infection, based on PCR, were fetuses, suggesting for the first time that vertical transmission of EcPV-2 occurs in horses. The current study observed an increased prevalence of EcPV-2 as compared to previous studies. We suggest that this difference is due to our use of biopsies in place of superficial swabs. We propose that EcPV-2 infection in asymptomatic horses is more common than previously reported and that the virus' role in equine genital SCCs may be more complex than originally thought. [ABSTRACT FROM AUTHOR]

Equus caballus papillomavirus type-2 (EcPV-2) has been proposed as a causal factor in equine genital squamous cell carcinoma (SCC). This study had 2 objectives: first, calculate the frequency of papillomavirus (PV) and EcPV-2 infection in papillomas, carcinomas in situ (CIS), and SCCs in Western Canadian horses; and second, determine if EcPV-2 status of equine SCCs is associated with overall survival (OS). EcPV-2 status of 115 archived tissue samples, spanning 6 years, was determined using broad spectrum (MY09/11) and EcPV-2-specific polymerase chain reaction (PCR) assays, EcPV-2-E6/E7 chromogenic RNA in situ hybridization (R-ISH), and amplicon sequencing. A retrospective survey gathered data on history, outcome, breeding, treatment, and rationales of referring veterinarians when managing PV-associated diseases. Histologic grade and completeness of surgical margins of SCCs were also considered. EcPV-2 DNA was identified in 10/58 (17%) SCC, 8/27 (30%) papillomas, 0/5 CIS, and 0/11 lesions identified as "other." Overall, 18/101 (18%) of these lesions were positive for EcPV-2. EcPV-2 was identified in 10/35 (29%) SCCs arising from genital tissues but in 0/22 SCCs from other locations. There was no association between breeding history and EcPV-2 status of genital SCCs. EcPV-2 status of genital SCCs was not associated with OS (P =.76). The strongest negative predictors of OS were a lack of treatment (P <.01) and recurrence post-treatment (P <.01). Weaker predictors of OS included older age at time of diagnosis (P =.02). Completeness of margins at surgical excision, concurrent disease, treatment type, anatomic location of the SCC (anogenital vs other), and histologic grade of the SCC did not influence OS (P >.1). [ABSTRACT FROM AUTHOR]

Papillomaviruses (PVs) are well recognized to cause pre-neoplastic and neoplastic diseases in humans. Similarly, there is increasing evidence that PVs play a significant role in the development of pre-neoplastic and neoplastic diseases of the haired skin of dogs and cats, and the mucosa of horses. As the mechanisms by which PVs cause neoplasia are well studied in humans, it is valuable to compare the PV-induced neoplasms of humans with similar PV-associated neoplasms in the companion animal species. In the second part of this comparative review, the pre-neoplastic and neoplastic diseases thought to be caused by PVs in humans, dogs, cats, and horses are described. This includes PV-induced cutaneous plaques, cutaneous squamous cell carcinomas (SCCs) and mucosal SCCs within the four species. The review concludes with a discussion about the potential use of vaccines to prevent PV-induced diseases of dogs, cats, and horses. • Viral plaques develop when the body cannot inhibit papillomavirus replication. • Papillomaviruses are not a frequent cause of canine skin or oral cancer. • Papillomaviruses can be an important cause of feline skin cancer. • Papillomaviruses cause cancer of the equine external genitalia. • Vaccines can be able to protect against some papillomaviral diseases. [ABSTRACT FROM AUTHOR]

Abstract: Equus caballus papillomavirus type 2 (EcPV-2) infection has been associated with equine genital squamous cell carcinomas (SCCs). However, quantitative PCR (qPCR) has not been performed to determine viral copy numbers within these lesions. Additionally, the frequency with which EcPV-2 can be detected in other common sites of equine SCC development remains uncertain. The aim of this study was to develop a qPCR assay to estimate the viral load in a variety of equine tissue samples. These included 40 SCC lesions, 19 penile non-SCC or precursor disease lesions, and 222 tissues without observable lesions from SCC-prone sites on clinically normal horses. EcPV-2 DNA was present significantly more frequently, and in higher copy numbers, in equine penile SCC lesions than in either healthy penile mucosa or non-SCC penile lesions. This supports the hypothesis that EcPV-2 is involved in development of penile SCCs and suggests that penile EcPV-2 infection is rare in the absence of SCCs. Samples of normal vulval mucosa rarely contained EcPV-2 DNA and none of the nictitating membrane samples contained EcPV-2 DNA, indicating that asymptomatic EcPV-2 infection is uncommon at these sites. EcPV-2 DNA was detected in a proportion of both SCCs and normal samples from the oral cavity or pharynx, although there were no significant differences in the rate of infection or viral copy number between the SCCs and the normal mucosal samples. As such, the role of EcPV-2 in development of SCCs in this location remains to be established [Copyright &y& Elsevier]

Abstract: 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 p16CDKN2A 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. [Copyright &y& Elsevier]

There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is etiologically associated with the development of genital squamous cell carcinoma (SCC) and precursor lesions in equids. However, the precise mechanisms underlying neoplastic progression remain unknown. To allow the study of EcPV2-induced carcinogenesis, we aimed to establish a primary equine cell culture model of EcPV2 infection. Three-dimensional (3D) raft cultures were generated from equine penile perilesional skin, plaques and SCCs. Using histological, molecular biological and immunohistochemical methods, rafts versus corresponding natural tissue sections were compared with regard to morphology, presence of EcPV2 DNA, presence and location of EcPV2 gene transcripts and expression of epithelial, mesenchymal and tumor/proliferation markers. Raft cultures from perilesional skin harboring only a few EcPV2-positive (EcPV2+) cells accurately recapitulated the differentiation process of normal skin, whilst rafts from EcPV2+ penile plaques were structurally organized but showed early hyperplasia. Rafts from EcPV2+ SCCs exhibited pronounced hyperplasia and marked dysplasia. Raft levels of EcPV2 oncogene transcription (E6/E7) and expression of tumor/proliferation markers p53, Ki67 and MCM7 expression positively correlated with neoplastic progression, again reflecting the natural situation. Three-dimensional raft cultures accurately reflected major features of corresponding ex vivo material, thus constituting a valuable new research model to study EcPV2-induced carcinogenesis. [ABSTRACT FROM AUTHOR]

Background: There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is causally associated with the development of equine genital squamous cell carcinomas (SCCs). Early stages of disease present clinically as plaques or wart-like lesions which can gradually progress to tumoural lesions. Histologically these lesions are inconsistently described as benign hyperplasia, papilloma, penile intraepithelial neoplasia (PIN), carcinoma in situ (CIS) or SCC. Guidelines for histological classification of early SCC precursor lesions are not precisely defined, leading to potential misdiagnosis. The aim of this study was to identify histologic criteria and diagnostic markers allowing for a more accurate diagnosis of EcPV2-associated equine penile lesions. Results: A total of 61 archived equine penile lesions were histologically re-assessed and classified as benign hyperplasia, papilloma, CIS or SCC. From these, 19 representative lesions and adjacent normal skin were comparatively analysed for the presence of EcPV2 DNA and transcripts using PCR and RNA in situ hybridisation (RISH). All lesional samples were positive by EcPV2 PCR and RISH, while adjacent normal skin was negative. RISH analysis yielded signal distribution patterns that allowed distinction of early (hyperplasia, papilloma) from late stage lesions (CIS, SCC). Subsequently, the 19 lesions were further assessed for expression of p53, Ki67, MCM7 and MMP1 by immunohistochemistry (IHC). All four proteins were expressed in both normal and lesional tissue. However, p53 expression was up-regulated in basal keratinocyte layers of papillomas, CIS and SCCs, as well as in upper keratinocyte layers of CIS and SCCs. MCM7 expression was only up-regulated in upper proliferating keratinocyte layers of papillomas, CIS and SCCs. Conclusion: This study proposes combining a refined histological protocol for analysis of equine penile lesions with PCR- and/or RISH based EcPV2-screening and p53/MCM7 IHC to more accurately determine the type of lesion. This may help to guide the choice of optimum treatment strategy, especially at early stages of disease. [ABSTRACT FROM AUTHOR]

See also the original review article by Taylor and Haldorson. [ABSTRACT FROM AUTHOR]