Longitudinal pilot study examining the effect of punch biopsy on equine sarcoid growth dynamics.

BACKGROUND: Nonexcisional tissue biopsies facilitate pre-operative confirmation of equine sarcoid yet fear of lesion deterioration currently limits its use in the diagnostic workup. OBJECTIVES: To evaluate the effect of a single punch biopsy on tumour growth dynamics [thickness, area, circumference, viral load (VL) and Visual Analog Scale (VAS)]. ANIMALS: Six client-owned horses with 11 sarcoids of various classification. MATERIALS AND METHODS: Growth dynamics were recorded on a weekly basis, 12 weeks pre- and 24 weeks post-biopsy. The effect of a single punch biopsy on growth dynamics was estimated by linear mixed-effect models. Spearman's rank correlation coefficient (SRCC) was calculated to investigate correlations with the number of weeks before and after the intervention. RESULTS: While statistically significant post-biopsy changes were recorded for all parameters except VL, no parameter was consistently increased after the biopsy intervention. In two of 11 sarcoids, the VL correlation pattern revealed significant strong correlations: sarcoid 6 (pre: r = -0.66, p < 0.05; post: r = 0.81, p < 0.001), increased VL; and sarcoid 8 (pre: r = 0.85, p < 0.001; post: r = 0.17, p > 0.05), no further increase after biopsy. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results indicate that post-biopsy lesion deterioration is not a general concept that applies to all sarcoids, and both deterioration or improvement are possible outcomes over a 24-week period. Further clinical studies with a larger sample size are needed before a definitive conclusion can be made.

Performance of fine-needle aspirate testing compared with superficial swab testing for quantification of BPV-1/-2 viral load in equine sarcoids.

Bovine papillomavirus (BPV) types 1 and 2 are causally associated with equine sarcoid, the most common mesenchymal neoplasm of horses, but the viral load (VL) differs between lesions. Sensitive and accurate BPV detection and quantification is essential for clinicians to confirm clinical suspicion, as well as in research settings for stratifying these skin lesions. Due to the limitations of histopathology in sarcoid diagnosis, PCR screening of superficial swabs constitutes the principal sampling method for BPV detection. This study aimed to investigate the ability of superficial swabs and fine-needle aspirates (FNA) to accurately detect the VL in equine sarcoids, considering the main clinical types: occult, nodular, verrucous and fibroblastic. Superficial swabs and FNAs from a series of sarcoid-affected horses were tested in parallel for BPV DNA quantification. Quantitative real-time PCR screening of postoperative tissue biopsies served as reference standard for the accuracy assessment of the viral titters. Our results indicate that VL is not a predictor of the clinical type. Student's t-test results gave evidence of a significant difference between both sample methods (P < 0.001) with FNA giving the best approximation of the actual VL (P < 0.01). In contrast to superficial swabs, the reference standard correlated moderately with FNA in general (P < 0.05; r = 0.39) and strongly with FNA results within the occult sarcoid group (P < 0.05; r = 0.59). In conclusion, the correlation of FNA with the reference standard was strong enough to suggest this is the preferred method for quantifying VL in sarcoids.

Immunogenicity analysis of BPV-1 positive equine sarcoid-derived cultured fibroblasts.

Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovine papilloma-virus (BPV)- 1 infected fibroblasts using a flow cytometric crossmatch assay. The presence of antibodies against exogenous bovine serum albumin (BSA) and fibroblast-like mesenchymal stromal cells (MSCs) was also evaluated by ELISA and flow cytometry, respectively. The viral load in the sarcoid explants, the corresponding cultured sarcoid fibroblasts, and matched peripheral blood mononuclear cells (PBMCs) from affected horses were determined by quantitative BPV-1/- 2 PCR analysis. Antibodies against autologous sarcoid cells were present in six out of twelve sarcoid-affected horses. Serum from all horses showed cross reactivity with allogeneic sarcoid cells, while only a part reacted with BSA or MSCs. Screening of host PBMCs demonstrated the absence of BPV E1 nucleic acids. Statistical analysis revealed a significantly higher mean viral load in the parental sarcoid tissue compared to the low passage fibroblasts (P < 0.001). These results support the hypothesis that sarcoid-affected horses may develop antibodies recognizing tumour-specific antigens. In contrast to sarcoid explants, equine PBMCs do not seem to contain complete BPV genomes. These results provide a basis for future investigations on the clinical relevance of these antibodies.

Cross-sectional comparison of superficial swab and fine-needle aspiration: Improving the diagnostic workup of horses with sarcoids.

Although bovine papillomavirus (BPV)-induced equine sarcoids are often identified solely by clinical examination, confirmation of the clinical diagnosis is essential for correct treatment selection. Only few approaches are presently available for this purpose. PCR-based screening for BPV in superficial swabs is widely-used to support clinical suspicion. While this method effectively detects sarcoid involvement in ulcerated lesions, sensitivity is lower in tumors with intact epithelium. This cross-sectional study compared the diagnostic characteristics of superficial swabs and fine-needle aspirates (FNA) with the aim to validate FNA as an alternative sampling method to detect BPV in suspect lesions. Among 63 lesions confirmed as sarcoids from 58 horses, compared to swabs FNA detected a greater proportion of BPV positives in general (swab, 70 %,;95 % confidence intervals [CI], 58.5 %-81.2 %; FNA, 98 %, 95 % CI, 95.3 %-100 %; P = 0.0001) and among a non-ulcerated subgroup (swab, 63 %, 95 % CI, 50.4 %-76.6 %; FNA, 98 %, 95 % CI, 91.4 %-100 %; P = 0.0001). Furthermore, the sensitivity, as well as the negative predictive value and accuracy of FNA for matched samples from 58 horses were superior to surface swabbing for the group of all horses and the subgroup that included only lesions with an intact epidermis (n = 48), with differences ranging from 23 % (95 % CI, 11.4 %-34.6 %) to 52 % (95 % CI, 25.9 %-78.1 %). Other advantages of FNA identified were a higher chance of adequate sampling and a reduced risk of unwanted detection of superficial contamination or latent BPV in keratinocytes. The ability to consistently detect BPV in all clinical lesion types indicates that FNA shows promise as a valid diagnostic tool to improve the consistency and quality of the diagnostic workup of equine sarcoids.

New approach for genomic characterisation of equine sarcoid-derived BPV-1/-2 using nanopore-based sequencing.

BACKGROUND: Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. METHODS: In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. RESULTS: We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. CONCLUSIONS: By generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.