Sabine Brandt

Background: Since its discovery in 2008, equine papillomavirus type 2 (EcPV2) has been detected in up to 100% of equine genital and up to 40% of equine oronasal squamous cell carcinomas (SCCs). Preliminary sequence data point to the existence of EcPV2 E6 and E7 oncogene variants compared to the British reference sequence from 2008. Objectives: To Identify E6 and E7 sequence variations in EcPV2‐positive tumours of horses from Continental Europe and Iceland. Study Design: Cross‐sectional study. Methods: EcPV2 E6 and E7 were amplified from DNA of 60 EcPV2‐positive SCCs/SCC precursor lesions. Amplicons were gel‐purified and submitted to bidirectional Sanger sequencing. Resulting 5′‐and 3′‐sequences were aligned with each other and then to EcPV2 sequences in the GenBank to determine sequence variations on DNA and predicted protein level. Results: Eight E6 gene variants were identified, predicting four E6 protein variants compared to the reference sequence. One E6 variant predominantly occurred in Icelandic horses imported from Iceland. Detected genetic variations in E6 were unlikely to affect known functional motifs. The E7 gene was highly conserved. None to three SNPs were detected per lesion, and only one E7 sequence contained a SNP predicted to be non‐synonymous. Main Limitations: Low number of oronasal SCCs ( n = 10) included in the study. Conclusions: The detected E6 and E7 variants point to the existence of different EcPV2 sublineages. Interestingly, one E6–E7 sequence was significantly associated with SCC‐bearing Icelandic horses imported from Iceland, suggesting that a specific EcPV2 sublineage may have evolved on the island. Provided that this theory can be confirmed, the long period between initial EcPV2 infection in Iceland and tumour development would indicate that EcPV2 can induce chronic infections that are reactivated in the older horse. Obtained sequence data are currently implemented in the generation of an immunotherapeutic vaccine targeting EcPV2 E6 and E7.

Bovine digital dermatitis (BDD) is an infectious claw disease chiefly induced by treponemes, while deep digital sepsis (DDS) is caused by pyogenic bacteria. It is currently unclear why persistent BDD-associated claw horn lesions (BDD-CHL) do not develop DDS. One possible explanation could be that the intralesional presence of treponemes makes it more difficult for pyogenic bacteria to colonize BDD-CHL. To lay the groundwork for exploring this possibility, we determined the presence and amounts of treponemes and selected pyogenic bacteria in BDD-CHL versus DDS at the DNA level. “Total Treponema” (TT-)PCR and type-specific qPCR were used to screen 18 BDD-CHLs from 15 cows and 18 DDS samples from 11 cows for bacterial DNA. TT-PCR identified Treponema DNA as most abundant DNA in 8/18 BDD-CHLs and 1/18 DDS samples. qPCR revealed Treponema pedis DNA in all BDD-CHLs, and one DDS lesion. Fusobacterium necrophorum DNA was significantly more abundant in DDS compared to BDD-CHL samples (p = 0.027). Both lesion types harbored similar mean levels of Trueperella pyogenes DNA. Interestingly, 7/18 DDS samples contained Parvimonas DNA. Our DNA-based data call for downstream studies involving living BDD-CHL-associated Treponema species and F. necrophorum, and, importantly, point to a possible pathogenic role of Parvimonas in DDS.

Sarcoids are benign, yet locally aggressive skin tumours that commonly affect horses and other equid species. The lesions are induced by bovine papillomavirus types 1, 2, and probably 13 in conjunction with other factors including trauma and a genetic predisposition. Although sarcoids have a substantial impact on the health and welfare of affected equids, information on the immune response to bovine papillomavirus infection and resulting sarcoids is limited. However, there is evidence that sarcoid disease is associated with an impaired immune response to bovine papillomavirus infection. This observation agrees with the common perception of sarcoids as persistent lesions. Contrary to this general view, there are reports on the spontaneous regression of skin tumours diagnosed as sarcoids, giving rise to the question whether sarcoids can self‐resolve, and if so, under which circumstances. This review addresses this unresolved issue.

Squamous cell carcinoma (SCC) is the most common malignant tumor disease in horses. It predominantly affects the ocular, oronasal, and anogenital region. Equine SCC is difficult to treat, also because important aspects of SCC development and metastasis are still unclear. We previously provided evidence that equine SCC cells can adopt a stem cell-like phenotype as a hallmark of malignant progression. Here, we investigated whether equine SCCs harbor endothelial-like tumor cells that form an alternative network of pseudo-vessels better known as vasculogenic mimicry (VM). Following histopathological diagnosis, 43 equine SCCs or precursor lesions (15 ocular, 14 genital, 14 oronasal tumors) were PCR-screened for equine papillomavirus (EcPV) infection. Subsequently, formalin-fixed paraffin-embedded (FFPE)-sections of all tumors were analyzed by Periodic Acid-Schiff (PAS) reaction and immunohistochemical (IHC) staining for endothelial cell marker CD31. Obtained micrographs were evaluated by a scientific board to unanimously identify sections of intact tumor tissue displaying PAS-positive, CD31-negative lumens harboring erythrocytes. Thirteen lesions exhibiting these features were subjected to triple immunofluorescence (IF) staining for CD31, pan-cytokeratin (KRT) and type 4 collagen (Col4) or alpha smooth muscle actin (αSMA) to confirm the presence of VM, and to determine whether pericytes have a role in this phenomenon. All genital and 50% of oronasal lesions scored positive for EcPV type 2, whilst ocular lesions tested negative. One mandibular SCC harbored EcPV type 5. Six genital, three oronasal, and four ocular tumors unambiguously exhibited VM as revealed by CD31-/PAS+ vessel-like structures containing erythrocytes, the detection of CD31-negative cells lining their lumens, and the presence of Col4 and αSMA in this lining. Detection of these two proteins in the context of VM suggests that VM-forming cancer cells recruit pericytes to enhance channel formation and stability. To our knowledge, this is the first report providing evidence of VM in equine cancer, and more generally, SCC in animals.

Summary: Aural plaques are a common condition but rarely need treatment. Diagnosis is usually straightforward by clinical examination. Differential diagnosis distinguishing aural plaques from sarcoid, if needed, is best achieved by PCR from surface samples or swabs. There is little evidence of any effect of autohaemotherapy. In contrast, Imiquimod is highly successful in the treatment of aural plaques. Purely cosmetic therapy of aural plaques is questionable.

Malignant melanoma (MM) affects not only humans but also animals, with gray horses being particularly predisposed to acquiring the disease. Multiomics have greatly advanced the understanding of human MM. In contrasty little is known regarding the pathogenesis of gray-horse melanoma and the unique phenomenon of melanoma “dormancy” in some animals. To help close this gap in knowledge, melanoma tissue and intact skin collected from gray horses were subjected to transcriptome analysis using RNAseq. In the next step, cultured primary tumor cells and normal skin fibroblasts were established from gray horses, and their protein expression profiles were determined. The obtained data unambiguously identified gray-horse melanoma (ghM) as a malignant tumor, as reflected by the overrepresentation of pathways typically activated in human melanoma and other human cancers. These included the RAS/RAF/MAPK, the IRS/IGF1R, and the PI3K/AKT signaling networks. In addition, the obtained data suggest that the key molecules RAC1, RAS, and BRAF, which are frequently mutated in human melanoma, may also contain activating mutations in ghM, whilst PTEN may harbor loss-of-function mutations. This issue will be subject to downstream analyses determining the mutational status in ghM to further advance the understanding of this frequent disease in gray horses.

Squamous cell carcinoma (SCC) is the most common malignant tumor disease in horses. It predominantly affects the ocular, oronasal, and anogenital region. Equine SCC is difficult to treat, also because important aspects of SCC development and metastasis are still unclear. We previously provided evidence that equine SCC cells can adopt a stem cell-like phenotype as a hallmark of malignant progression. Here, we investigated whether equine SCCs harbor endothelial-like tumor cells that form an alternative network of pseudo-vessels better known as vasculogenic mimicry (VM). Following histopathological diagnosis, 43 equine SCCs or precursor lesions (15 ocular, 14 genital, 14 oronasal tumors) were PCR-screened for equine papillomavirus (EcPV) infection. Subsequently, formalin-fixed paraffin-embedded-sections of all tumors were analyzed by Periodic Acid-Schiff (PAS) reaction and immunohistochemical (IHC) staining for endothelial cell marker CD31. Obtained micrographs were evaluated by a scientific board to unanimously identify sections of intact tumor tissue displaying PAS-positive, CD31-negative lumens harboring erythrocytes. Thirteen lesions exhibiting these features were subjected to triple immunofluorescence (IF) staining for CD31, pan-cytokeratin (KRT) and type 4 collagen (Col4) or alpha smooth muscle actin (αSMA) to confirm the presence of VM, and to determine whether pericytes have a role in this phenomenon. All genital and 50% of oronasal lesions scored positive for EcPV type 2, whilst ocular lesions tested negative. One mandibular SCC harbored EcPV type 5. Six genital, three oronasal, and four ocular tumors unambiguously exhibited VM as revealed by CD31-/PAS+ vessel-like structures containing erythrocytes, the detection of CD31-negative cells lining their lumens, and the presence of Col4 and αSMA in this lining. Detection of these two proteins in the context of VM suggests that VM-forming cancer cells recruit pericytes to enhance channel formation and stability. To our knowledge, this is the first report providing evidence of VM in equine cancer, and more generally, SCC in animals.

Background, aim: It is well accepted today that papillomaviruses can induce mucocutaneous tumours in vertebrates. Given the high metastatic potential of some of these tumours in humans, and their tendency to recur following treatment, there is a need of more effective therapeutics. Methods: Following the generation of live-attenuated influenza viruses co-expressing bovine papillomavirus type 1 (BPV1)-specific E6 and E7 peptides, we assessed the efficacy of this immunotherapeutic vaccine prototype in a natural tumour model, i.e., horses bearing BPV1 or 2-induced skin tumours termed sarcoids. So far, 21 horses with mild and moderate disease repeatedly received intralesional injections with the vaccine. Ten horses with severe disease (multiple/aggressive sarcoids; 64 in total) were treated by combined therapy, i.e., injections with the vaccine and electrosurgical tumour excision. Where possible, a follow-up was carried out by photo-documentation and BPV1/2 PCR-screening of hair roots/dandruff of the tumour sites. Results: Nine of 21 horses with mild or moderate disease showed complete tumour regression (CR) after six months, and all samples collected from seven individuals scored BPV1/2-negative. Further nine horses exhibited partial sarcoid regression (PR) that is still ongoing, and harboured BPV1 or 2. Three horses showed stable disease and BPV1/2 infection. In one of these horses, repeated surgery and additional booster injections led to freedom from sarcoids and BPV1/2. From the 10 severely affected horses, nine showed CR six months after combined therapy. Three recurrent lesions in two horses were eradicated by repeated immunotherapy or combined therapy. This led to 8/9 horses being tumour- and BPV1/2-free meanwhile. Conclusion: Sarcoid recurrence following standard therapy is a common event because current treatments do not target BPV1/2 infection. Flu-BPV1-E6E7-immunotherapy was effective in 86% of mild and moderate-type sarcoids as well as eradicating BPV infection underlying the disease. Similarly, combined therapy led to CR in 9/10 cases and significantly reduced the risk of post-treatment recurrence due to eradication of BPV infection: only 3/64 treated sarcoids recrudesced, corresponding to a unique recurrence rate of only 4.7%.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial–mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.