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Lack of Correlation Between Papillomaviral DNA in Surgical Margins and Recurrence of Equine Sarcoids
Journal of Equine Veterinary Science xx (2014) 1–4
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Lack of Correlation Between Papillomaviral DNA in Surgical Margins and Recurrence of Equine Sarcoids Sandra D. Taylor DVM, PhD a, *, Balazs Toth DVM, MS a, Laura J. Baseler DVM, MS b, Virginia A. Charney DVM b, Margaret A. Miller DVM, PhD b a b
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2013 Received in revised form 18 December 2013 Accepted 24 December 2013 Available online xxxx
Bovine papillomavirus (BPV) types 1 (BPV-1) and 2 (BPV-2) are causally associated with the development of equine sarcoid tumors. Recurrence rates after surgical excision of sarcoids are estimated to be 30%–40%. We hypothesized that the presence of BPV DNA in histologically tumor-free surgical margins of sarcoids is associated with risk of recurrence, and increased quantity of BPV DNA is associated with increased risk of recurrence. Formalin-fixed sarcoids classified as “completely excised” histologically were obtained from two institutions. A total of 25 tumors were included, eight of which recurred within 1 year of excision. Qualitative and quantitative polymerase chain reaction (PCR) tests for detection of BPV-1 and BPV-2 were performed on neoplastic tissue and tumor-free surgical margins in formalin-fixed paraffin-embedded biopsy specimens following DNA extraction. Bovine papillomavirus-1 was found in all tumor samples and in histologically “clean” margins of 21 samples, whereas BPV-2 was found in only two tumor samples. Although quantitative PCR was more sensitive than qualitative PCR in detecting BPV DNA in surgical margins, there was no significant difference in the presence of BPV-1 or BPV-2 DNA in margins of tumors that recurred versus those that did not recur for either test. Although this study is limited by sample size, our results suggest that PCR analysis of surgical margins for BPV DNA is not a reliable method to predict equine sarcoid recurrence after resection. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Equine Papillomavirus Sarcoid Tumor
Sarcoids are locally invasive, fibroblastic skin tumors, and are the most common equine tumor worldwide [1–5]. Although sarcoids do not metastasize, they have a negative impact on equine function and aesthetics. Bovine papillomavirus (BPV) types 1 (BPV-1) and 2 (BPV-2) are causally associated with the development of equine sarcoids [4,6]. Bovine papillomavirus DNA has been detected in up to 100% of sarcoids [7–12], approximately 80% of which contain BPV-1 DNA and approximately 20% of which * Corresponding author at: Dr. Sandra D. Taylor, DVM, PhD, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 625 Harrison St, West Lafayette, IN 47907. E-mail address: [email protected] (S.D. Taylor). 0737-0806/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2013.12.012
contain BPV-2 DNA [7,10,12,13]. In addition, viral oncogenes and capsid gene transcripts have been identified in sarcoids, providing evidence for direct involvement of BPV in their development [9,14]. In contrast to BPV-1 and/or BPV-2 infection in cattle, which results in cutaneous fibropapillomas that produce progeny virus and regress after a cell-mediated immune response [15–17], equine sarcoids are locally aggressive tumors that are likely nonpermissive for virus production and occasionally regress [1,6,18–20]. Bovine papillomavirus types 1 and 2 are doublestranded DNA viruses with a 7900 base-pair genome [4]. As for other papillomaviruses, the genome can be divided into two major regions. The early (E) region encodes the replication and transcription regulatory proteins E1 and E2,
2
S.D. Taylor et al. / Journal of Equine Veterinary Science xx (2014) 1–4
as well as the transforming proteins (oncoproteins) E5, E6, and E7. The late (L) region encodes the structural capsid proteins, L1 and L2. The E and L regions are separated by a stretch of nontranscribed DNA, defined as the long control region, which contains the transcriptional promoters and enhancers, the origin of replication, and binding sites for transcription factors. Bovine papillomavirus-1 and BPV-2 demonstrate 87% DNA sequence homology (Basic Local Alignment Search Tool, National Center for Biotechnology Information, United States National Library of Medicine, Bethesda, MD), with disparate regions within both E and L genes. There is no uniformly effective therapy for sarcoids. Tumors that are easily accessible and in a location where skin closure is feasible are often treated with surgical excision. However, approximately 30% of equine sarcoids recur after surgical excision, on average within 6 months [1,21–24], and recurrent tumors are often more aggressive and rapidly growing than the original tumor [25,26]. This effect may be because of surgical disruption and activation of latent virus in adjacent skin of sarcoid-bearing horses, but this hypothesis has not been tested [8]. Management of recurring sarcoids is costly, and predicting which tumor will recur is currently not possible. One study determined that the presence of BPV DNA in apparently normal skin surrounding sarcoids was associated with local recurrence [10]. Importantly, histopathologic examination of surgical margins was not performed in that study, although sarcoid tumor cells typically extend beyond grossly visible tumors in the form of long rete pegs [24–28]. Furthermore, quantitative real-time polymerase chain reaction (PCR) was not used to quantify the BPV DNA in the surgical margins, results of which may have been useful to predict recurrence. The objective of the present study was to determine the association between the detection (qualitative and quantitative) and amount (quantitative; copies per cell) of BPV DNA in sarcoids and recurrence after complete excision. We hypothesized that the presence of BPV DNA in histologically tumor-free surgical margins of sarcoids correlates with risk of recurrence, and that increased quantity of BPV DNA is associated with increased risk of recurrence. A test that could predict the likelihood of recurrence would allow more aggressive and/or additional treatments in a timely fashion if recurrence was likely, thereby providing a more favorable prognosis because tumors that recur after surgical excision behave more aggressively and are not well circumscribed [29].
Formalin-fixed paraffin-embedded (FFPE) equine sarcoid samples with histologically “tumor-free” margins (uniformly >2 mm in thickness) were obtained from the University of California-Davis and Purdue University. Histologic sections stained with hematoxylin–eosin (HE) were evaluated from each tumor to confirm the diagnosis and to exclude any tumors with neoplastic tissue at the margins of the sections. Cases were included only if follow-up information was available for at least 1 year after surgery. A total of 25 tumors were included in the study, 32% (n ¼ 8) of which recurred within 1 year of excision and 68% (n ¼ 17) that did not recur during the study period. Samples for DNA extraction were obtained without knowledge of whether the tumor had recurred. Histologic sections stained with HE were used to map the tumor and tumor-free tissue in each unstained paraffin section or paraffin block. This was done by laying the HE-stained section directly under the unstained section or over the paraffin block to identify and mark neoplastic and nonneoplastic portions of the sample for DNA extraction. For the University of California-Davis cases (n ¼ 12), the samples of tumor and tumor-free tissue were scraped with a sterile scalpel blade from unstained 5-mm-thick paraffin sections. A new scalpel blade was used for each paraffin section, and tumor-free margins were sampled first to avoid contamination from tumoral tissue. For the Purdue University cases (n ¼ 13), four core samples of tumor-free margins and four core samples of each tumor were collected from the paraffin blocks with a sterile 16G needle. Needles were changed for each paraffin block, and tumor-free margins were sampled first. For PCR analysis, margin samples and neoplastic tissue samples were separately pooled from each specimen. DNA extraction was performed using QIAmp DNA FFPE Tissue Kit (Qiagen, Valencia, CA) according to manufacturer’s recommendations. Neoplastic tissue and tumor-free margins were tested for the presence of BPV-1 and BPV-2 DNA by traditional (qualitative) PCR amplification of the E2 gene region (234 bp amplicon) using primers that were complimentary to shared sequences between BPV-1 and BPV-2 (Table 1). The following reagents were used with a total volume of 20 mL per reaction: 10 Buffer B (1), MgCl2 (1.25 mM), dNTP mix (200 mM for each dNTP; ThermoFisher Scientific Fermentas, Waltham, MA), primers (10 pmol each), Taq DNA polymerase (1 unit; Fisher Scientific BioReagents, Waltham, MA), and DNA (2 mL). The reaction conditions were as follows using an Eppendorf Mastercycler pro thermocycler: 95 C for 2 minutes followed by 35
Table 1 Forward and reverse primer sequences for (a) qualitative and (b) quantitative PCR and TaqManÒ probe sequences for quantitative PCR amplification of BPV-1 and BPV-2 (a) Qualitative PCR 50 -ATAAGTTGCAAGATCATATAC-3’ 50 -GCTTGTGTCAAGCAAAGACC-3’
Forward primer Reverse primer (b) Quantitative PCR Forward primer Reverse primer TaqManÒ probe
BPV-1 50 -TCATCCACCTCTTCTGATTTTAGAGAT-3’ 50 -TCTCTTCTTTGCTCGGCTCC-3’ 50 -CTGGGTCGCATCCGAAGGACCT-3’
BPV, bovine papillomavirus; PCR, polymerase chain reaction. Note that primer sequences are identical between BPV types for each assay.
BPV-2 50 -TCATCCACCTCTTCTGATTTTAGAGAT-3’ 50 -TCTCTTCTTTGCTCGGCTCC-3’ 50 -AGACGGAGTTTCCGCATCAGAAGGACC-3’
S.D. Taylor et al. / Journal of Equine Veterinary Science xx (2014) 1–4
cycles of 95 C for 30 seconds, 55 C for 45 seconds, and 72 C for 30 seconds; following each cycle, reaction tubes were heated to 72 C for 10 minutes for final elongation. Similarly, real-time (quantitative) PCR was performed using primers that were complimentary to shared sequences between BPV-1 and BPV-2, and TaqManÒ probes that were unique to each BPV type (Table 1). The following reagents were used with a total volume of 15 mL per reaction: iQ Supermix (1; Bio-Rad Laboratories, Hercules, CA), primers (0.3 mM each), probe (0.2 mM), and DNA (100 ng). The reaction conditions were as follows using an Eppendorf Mastercycler pro thermocycler: 95 C for 3 minutes followed by 45 cycles of 95 C for 20 seconds, 60 C for 40 seconds, and 68 C for 20 seconds. Serial dilutions of a BPV plasmid were used to generate a standard curve. This 2,836 bp plasmid DNA was produced by GenScript (Piscataway, NJ) and contained a pUC57 insert. Serial dilutions of the plasmid DNA were performed using TE buffer (10 mM Tris-HCl; 1 mM Ethylenediaminetetraacetic acid; pH 7.7). An equine glyceraldehyde-3-phosphate dehydrogenase gene primer and/or probe set was used as an internal control for normalization of input DNA. Positive controls were derived from sarcoid tissues confirmed positive for BPV-1 (mean Ct [cycle threshold] ¼ 21). Negative controls included normal equine skin and nontemplate controls, all with negative Ct for BPV-1 and BPV-2. Data were reported as viral copies per cell. A Shapiro–Wilk test was used to assess normality of data, which was found to be nonparametric. Data were further analyzed using a chi-squared test and Wilcoxon rank-sum test. Logistic regression was used to determine whether the presence or quantity of BPV-1 or BPV-2 was associated with recurrence. A P value of <.05 was considered significant. Bovine papillomavirus-1 DNA was found in 100% (n ¼ 25) of FFPE tumor samples and in 84% (n ¼ 21) of samples from histologically “clean” surgical margins. Bovine papillomavirus-2 DNA was found in 8% (n ¼ 2) of tumors, and was not found in any margin sample. Bovine papillomavirus-1 DNA was more likely to be found in tumor and margins compared with BPV-2 (P < .001). There was no significant difference in the presence of BPV-1 (qualitative PCR) in tumors compared with surgical margins for tumors that recurred and those that did not recur (P ¼ .11). Although quantitative (real-time) PCR was more sensitive in detecting the presence of BPV-1 or BPV-2 DNA in surgical margins compared with qualitative PCR, there was no significant difference in the presence of BPV-1 or BPV-2 DNA in margins of tumors that recurred and those that did not recur for either test. There was no difference in the amount (copies/cell) of BPV-1 DNA in the surgical margins between the tumors that recurred (median, 0; range, 0–0.13) and those that did not recur (median, 0.02; range, 0–5.0)
Table 2 Median and range for BPV-1 copies per cell for surgical margins and bulk tumor samples in recurrent and nonrecurrent sarcoids
Surgical margins Bulk tumor
Recurrence
Nonrecurrence
P value
0 (0–0.13) 0.32 (0.04–13.61)
0.02 (0–5.0) 2.12 (0.13–29.62)
.52 .042
BPV, bovine papillomavirus.
3
(P ¼ .52) (Table 2). Surprisingly, the amount of BPV-1 DNA in tumors that recurred was significantly lower (median, 0.32; range, 0.04–13.61) than in those that did not recur (median, 2.12; range, 0.13–29.62) (P ¼ .042) (Table 2). In the present study, there was no evidence that a higher quantity of BPV DNA in either tumor or surgical margins of sarcoids considered “completely excised” by histopathology predicted recurrence; thus, the hypothesis was rejected. It is possible that the “clean” margins were, in fact, devoid of neoplastic cells despite the presence of BPV DNA. This is consistent with results of a study that found BPV DNA in normal skin from 73% of sarcoid-bearing horses and may reflect latent carrier status [30]. Another possibility is that histopathology is not sensitive enough for the detection of neoplastic cells in surgical margins of excised equine sarcoids and that some of the histologic sections with apparently “clean” margins contained neoplastic cells. It is also possible that clean margins were contaminated during sampling for DNA extraction. Pooling four marginal samples from each specimen could have increased the likelihood of BPV-DNA contamination from adjacent neoplastic tissue. Finally, a significant difference in marginal BPV-DNA quantity could exist between the two groups (recurrence vs nonrecurrence), but the sample size was too small to detect this difference. In the present study, a higher amount of BPV-1 DNA in the bulk tumor was associated with decreased recurrence. These results are inconsistent with a previous study that demonstrated a positive association between intratumoral viral load and disease severity, but the inclusion criteria for “severe” disease contained many variables, with recurrence or nonrecurrence not consistently reported [31]. Our results could reflect the small sample size and may not be significant, or they could indicate a more robust immune response triggered by a higher viral load. A study investigating feline cutaneous fibropapillomas with histologic features consistent with equine sarcoids found that tumor recurrence after surgical excision was not consistently associated with detection of papillomavirus DNA [32]. Furthermore, human papillomavirus viral loads have been shown to be nonassociated with recurrence of respiratory and anogenital papillomas [33,34]. The presence and quantity of BPV-1 DNA was much higher than that of BPV-2 DNA in equine sarcoids; BPV-2 was only found concurrently with BPV-1. These findings are consistent with previous reports [7,30,35]. The sample size for this study was small given the difficulty in achieving complete tumor resection and in following the patient for at least 1 year after surgery. Although increased BPV-1 DNA in bulk tumors was associated with nonrecurrence, the small sample size precludes us from making specific management recommendations based on this information. Excision with wide surgical margins is still likely important in decreasing the risk of recurrence, and close monitoring for regrowth is critical for early removal of recurrent sarcoids. Acknowledgments The authors would like to thank Dr. Alain Theon for providing tissue samples from the University of California-
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Davis, Anisa Dunham for outstanding technical assistance, and Dr. George Moore for statistical expertise. This project was supported by USDA-NIFA and the Purdue University College of Veterinary Medicine’s Competitive Equine Research Funds. References [1] Ragland WL, Keown GH, Spencer GR. Equine sarcoid. Equine Vet J 1970;2:2–11. [2] Cotchin E. A general survey of tumours in the horse. Equine Vet J 1977;9(1):16–21. [3] Marti E, Lazary S, Antczak DF, Gerber H. Report of the first international workshop on equine sarcoid. Equine Vet J 1993;25:397–407. [4] Nasir L, Campo MS. Bovine papillomaviruses: their role in the aetiology of cutaneous tumours of bovids and equids. Vet Dermatol 2008;19(5):243–54. [5] Schaffer PA, Wobeser B, Martin LE, Dennis MM, Duncan CG. Cutaneous neoplastic lesions of equids in the central United States and Canada: 3,351 biopsy specimens from 3,272 equids (2000-2010). J Am Vet Med Assoc 2013;242(1):99–104. [6] Gorman NT. Equine sarcoid–time for optimism. Equine Vet J 1985; 17(6):412–4. [7] Otten N, von Tscharner C, Lazary S, Antczak DF, Gerber H. DNA of bovine papillomavirus type 1 and 2 in equine sarcoids: PCR detection and direct sequencing. Arch Virol 1993;132(1-2):121–31. [8] Carr EA, Theon AP, Madewell BR, Griffey SM, Hitchcock ME. Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States. Am J Vet Res 2001;62(5):741–4. [9] Carr EA, Theon AP, Madewell BR, Hitchcock ME, Schlegel R, Schiller JT. Expression of a transforming gene (E5) of bovine papillomavirus in sarcoids obtained from horses. Am J Vet Res 2001;62(8):1212–7. [10] Martens A, De Moor A, Demeulemeester J, Peelman L. Polymerase chain reaction analysis of the surgical margins of equine sarcoids for bovine papilloma virus DNA. Vet Surg 2001;30(5):460–7. [11] Martens A, De Moor A, Ducatelle R. PCR detection of bovine papilloma virus DNA in superficial swabs and scrapings from equine sarcoids. Vet J 2001;161(3):280–6. [12] Bogaert L, Van Poucke M, De Baere C, Dewulf J, Peelman L, Ducatelle R, et al. Bovine papillomavirus load and mRNA expression, cell proliferation and p53 expression in four clinical types of equine sarcoid. J Gen Virol 2007;88(Pt 8):2155–61. [13] Angelos JA, Marti E, Lazary S, Carmichael LE. Characterization of BPV-like DNA in equine sarcoids. Arch Virol 1991;119(1-2):95–109. [14] Nasir L, Reid SW. Bovine papillomaviral gene expression in equine sarcoid tumours. Virus Res 1999;61(2):171–5. [15] Okabayashi M, Angell MG, Christensen ND, Kreider JW. Morphometric analysis and identification of infiltrating leucocytes in regressing and progressing Shope rabbit papillomas. Int J Cancer 1991;49(6):919–23. [16] Coleman N, Birley HD, Renton AM, Hanna NF, Ryait BK, Byrne M, et al. Immunological events in regressing genital warts. Am J Clin Pathol 1994;102(6):768–74. [17] Knowles G, O’Neil BW, Campo MS. Phenotypical characterization of lymphocytes infiltrating regressing papillomas. J Virol 1996;70(12): 8451–8.
[18] Amtmann E, Muller H, Sauer G. Equine connective tissue tumors contain unintegrated bovine papilloma virus DNA. J Virol 1980; 35(3):962–4. [19] Lancaster WD. Apparent lack of integration of bovine papillomavirus DNA in virus-induced equine and bovine tumor cells and virus-transformed mouse cells. Virology 1981;108(2):251–5. [20] Wilson AD, Armstrong EL, Gofton RG, Mason J, De Toit N, Day MJ. Characterisation of early and late bovine papillomavirus protein expression in equine sarcoids. Vet Microbiol 2013;162(2-4):369–80. [21] Brown MP. Surgical treatment of equine sarcoid. Philadelphia: WB Saunders; 1983. [22] Genetzky RMBRD, Myers RK. Equine sarcoids, causes, diagnosis, and treatment. Compend Contin Educ Pract Vet 1983;5:S416. [23] Carstanjen B, Jordan P, Lepage OM. Carbon dioxide laser as a surgical instrument for sarcoid therapy–a retrospective study on 60 cases. Can Vet J 1997;38(12):773–6. [24] Hallamaa RE, Saario E, Tallberg T. Macroscopical and histopathological changes in regressing primary and recurrent equine sarcoids during active specific bio-immunotherapy. In Vivo 2005; 19(4):761–7. [25] Tarwid JNFPB, Clark EG. Equine sarcoids: a study with emphasis on pathologic diagnosis. Compend Contin Educ Pract Vet 1985; 7:293. [26] McConaghy FF, Davis RE, Reppas GP, Rawlinson RJ, McClintock SA, Hutchins DR, et al. Management of equine sarcoids: 1975-93. N Z Vet J 1994;42(5):180–4. [27] Martens A, De Moor A, Demeulemeester J, Ducatelle R. Histopathological characteristics of five clinical types of equine sarcoid. Res Vet Sci 2000;69(3):295–300. [28] Baker JR, Leyland A. Histological survey of tumours of the horse, with particular reference to those of the skin. Vet Rec 1975;96(19): 419–22. [29] Knottenbelt DC, Kelly DF. The diagnosis and treatment of periorbital sarcoid in the horse: 445 cases from 1974 to 1999. Vet Ophthalmol 2000;3(2-3):169–91. [30] Bogaert L, Martens A, Van Poucke M, Ducatelle R, De Cock H, Dewulf J, et al. High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoid-affected and healthy horses. Vet Microbiol 2008;129(1-2):58–68. [31] Haralambus R, Burgstaller J, Klukowska-Rotzler J, Steinborn R, Buchinger S, Gerber V, et al. Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease. Equine Vet J 2010;42(4):327–31. [32] Schulman FY, Krafft AE, Janczewski T. Feline cutaneous fibropapillomas: clinicopathologic findings and association with papillomavirus infection. Vet Pathol 2001;38(3):291–6. [33] Sanclemente G, Herrera S, Tyring SK, Rady PL, Zuleta JJ, Correa LA, et al. Human papillomavirus (HPV) viral load and HPV type in the clinical outcome of HIV-positive patients treated with imiquimod for anogenital warts and anal intraepithelial neoplasia. J Eur Acad Dermatol Venereol 2007;21(8):1054–60. [34] Maloney EM, Unger ER, Tucker RA, Swan D, Karem K, Todd NW, et al. Longitudinal measures of human papillomavirus 6 and 11 viral loads and antibody response in children with recurrent respiratory papillomatosis. Arch Otolaryngol Head Neck Surg 2006; 132(7):711–5. [35] Bogaert L, Martens A, Kast WM, Van Marck E, De Cock H. Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors. Vet Microbiol 2010;146(3-4):269–75.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Lack of Correlation Between Papillomaviral DNA in Surgical Margins and Recurrence of Equine Sarcoids Sandra D. Taylor DVM, PhD a, *, Balazs Toth DVM, MS a, Laura J. Baseler DVM, MS b, Virginia A. Charney DVM b, Margaret A. Miller DVM, PhD b a b
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2013 Received in revised form 18 December 2013 Accepted 24 December 2013 Available online xxxx
Bovine papillomavirus (BPV) types 1 (BPV-1) and 2 (BPV-2) are causally associated with the development of equine sarcoid tumors. Recurrence rates after surgical excision of sarcoids are estimated to be 30%–40%. We hypothesized that the presence of BPV DNA in histologically tumor-free surgical margins of sarcoids is associated with risk of recurrence, and increased quantity of BPV DNA is associated with increased risk of recurrence. Formalin-fixed sarcoids classified as “completely excised” histologically were obtained from two institutions. A total of 25 tumors were included, eight of which recurred within 1 year of excision. Qualitative and quantitative polymerase chain reaction (PCR) tests for detection of BPV-1 and BPV-2 were performed on neoplastic tissue and tumor-free surgical margins in formalin-fixed paraffin-embedded biopsy specimens following DNA extraction. Bovine papillomavirus-1 was found in all tumor samples and in histologically “clean” margins of 21 samples, whereas BPV-2 was found in only two tumor samples. Although quantitative PCR was more sensitive than qualitative PCR in detecting BPV DNA in surgical margins, there was no significant difference in the presence of BPV-1 or BPV-2 DNA in margins of tumors that recurred versus those that did not recur for either test. Although this study is limited by sample size, our results suggest that PCR analysis of surgical margins for BPV DNA is not a reliable method to predict equine sarcoid recurrence after resection. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Equine Papillomavirus Sarcoid Tumor
Sarcoids are locally invasive, fibroblastic skin tumors, and are the most common equine tumor worldwide [1–5]. Although sarcoids do not metastasize, they have a negative impact on equine function and aesthetics. Bovine papillomavirus (BPV) types 1 (BPV-1) and 2 (BPV-2) are causally associated with the development of equine sarcoids [4,6]. Bovine papillomavirus DNA has been detected in up to 100% of sarcoids [7–12], approximately 80% of which contain BPV-1 DNA and approximately 20% of which * Corresponding author at: Dr. Sandra D. Taylor, DVM, PhD, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 625 Harrison St, West Lafayette, IN 47907. E-mail address: [email protected] (S.D. Taylor). 0737-0806/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2013.12.012
contain BPV-2 DNA [7,10,12,13]. In addition, viral oncogenes and capsid gene transcripts have been identified in sarcoids, providing evidence for direct involvement of BPV in their development [9,14]. In contrast to BPV-1 and/or BPV-2 infection in cattle, which results in cutaneous fibropapillomas that produce progeny virus and regress after a cell-mediated immune response [15–17], equine sarcoids are locally aggressive tumors that are likely nonpermissive for virus production and occasionally regress [1,6,18–20]. Bovine papillomavirus types 1 and 2 are doublestranded DNA viruses with a 7900 base-pair genome [4]. As for other papillomaviruses, the genome can be divided into two major regions. The early (E) region encodes the replication and transcription regulatory proteins E1 and E2,
2
S.D. Taylor et al. / Journal of Equine Veterinary Science xx (2014) 1–4
as well as the transforming proteins (oncoproteins) E5, E6, and E7. The late (L) region encodes the structural capsid proteins, L1 and L2. The E and L regions are separated by a stretch of nontranscribed DNA, defined as the long control region, which contains the transcriptional promoters and enhancers, the origin of replication, and binding sites for transcription factors. Bovine papillomavirus-1 and BPV-2 demonstrate 87% DNA sequence homology (Basic Local Alignment Search Tool, National Center for Biotechnology Information, United States National Library of Medicine, Bethesda, MD), with disparate regions within both E and L genes. There is no uniformly effective therapy for sarcoids. Tumors that are easily accessible and in a location where skin closure is feasible are often treated with surgical excision. However, approximately 30% of equine sarcoids recur after surgical excision, on average within 6 months [1,21–24], and recurrent tumors are often more aggressive and rapidly growing than the original tumor [25,26]. This effect may be because of surgical disruption and activation of latent virus in adjacent skin of sarcoid-bearing horses, but this hypothesis has not been tested [8]. Management of recurring sarcoids is costly, and predicting which tumor will recur is currently not possible. One study determined that the presence of BPV DNA in apparently normal skin surrounding sarcoids was associated with local recurrence [10]. Importantly, histopathologic examination of surgical margins was not performed in that study, although sarcoid tumor cells typically extend beyond grossly visible tumors in the form of long rete pegs [24–28]. Furthermore, quantitative real-time polymerase chain reaction (PCR) was not used to quantify the BPV DNA in the surgical margins, results of which may have been useful to predict recurrence. The objective of the present study was to determine the association between the detection (qualitative and quantitative) and amount (quantitative; copies per cell) of BPV DNA in sarcoids and recurrence after complete excision. We hypothesized that the presence of BPV DNA in histologically tumor-free surgical margins of sarcoids correlates with risk of recurrence, and that increased quantity of BPV DNA is associated with increased risk of recurrence. A test that could predict the likelihood of recurrence would allow more aggressive and/or additional treatments in a timely fashion if recurrence was likely, thereby providing a more favorable prognosis because tumors that recur after surgical excision behave more aggressively and are not well circumscribed [29].
Formalin-fixed paraffin-embedded (FFPE) equine sarcoid samples with histologically “tumor-free” margins (uniformly >2 mm in thickness) were obtained from the University of California-Davis and Purdue University. Histologic sections stained with hematoxylin–eosin (HE) were evaluated from each tumor to confirm the diagnosis and to exclude any tumors with neoplastic tissue at the margins of the sections. Cases were included only if follow-up information was available for at least 1 year after surgery. A total of 25 tumors were included in the study, 32% (n ¼ 8) of which recurred within 1 year of excision and 68% (n ¼ 17) that did not recur during the study period. Samples for DNA extraction were obtained without knowledge of whether the tumor had recurred. Histologic sections stained with HE were used to map the tumor and tumor-free tissue in each unstained paraffin section or paraffin block. This was done by laying the HE-stained section directly under the unstained section or over the paraffin block to identify and mark neoplastic and nonneoplastic portions of the sample for DNA extraction. For the University of California-Davis cases (n ¼ 12), the samples of tumor and tumor-free tissue were scraped with a sterile scalpel blade from unstained 5-mm-thick paraffin sections. A new scalpel blade was used for each paraffin section, and tumor-free margins were sampled first to avoid contamination from tumoral tissue. For the Purdue University cases (n ¼ 13), four core samples of tumor-free margins and four core samples of each tumor were collected from the paraffin blocks with a sterile 16G needle. Needles were changed for each paraffin block, and tumor-free margins were sampled first. For PCR analysis, margin samples and neoplastic tissue samples were separately pooled from each specimen. DNA extraction was performed using QIAmp DNA FFPE Tissue Kit (Qiagen, Valencia, CA) according to manufacturer’s recommendations. Neoplastic tissue and tumor-free margins were tested for the presence of BPV-1 and BPV-2 DNA by traditional (qualitative) PCR amplification of the E2 gene region (234 bp amplicon) using primers that were complimentary to shared sequences between BPV-1 and BPV-2 (Table 1). The following reagents were used with a total volume of 20 mL per reaction: 10 Buffer B (1), MgCl2 (1.25 mM), dNTP mix (200 mM for each dNTP; ThermoFisher Scientific Fermentas, Waltham, MA), primers (10 pmol each), Taq DNA polymerase (1 unit; Fisher Scientific BioReagents, Waltham, MA), and DNA (2 mL). The reaction conditions were as follows using an Eppendorf Mastercycler pro thermocycler: 95 C for 2 minutes followed by 35
Table 1 Forward and reverse primer sequences for (a) qualitative and (b) quantitative PCR and TaqManÒ probe sequences for quantitative PCR amplification of BPV-1 and BPV-2 (a) Qualitative PCR 50 -ATAAGTTGCAAGATCATATAC-3’ 50 -GCTTGTGTCAAGCAAAGACC-3’
Forward primer Reverse primer (b) Quantitative PCR Forward primer Reverse primer TaqManÒ probe
BPV-1 50 -TCATCCACCTCTTCTGATTTTAGAGAT-3’ 50 -TCTCTTCTTTGCTCGGCTCC-3’ 50 -CTGGGTCGCATCCGAAGGACCT-3’
BPV, bovine papillomavirus; PCR, polymerase chain reaction. Note that primer sequences are identical between BPV types for each assay.
BPV-2 50 -TCATCCACCTCTTCTGATTTTAGAGAT-3’ 50 -TCTCTTCTTTGCTCGGCTCC-3’ 50 -AGACGGAGTTTCCGCATCAGAAGGACC-3’
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cycles of 95 C for 30 seconds, 55 C for 45 seconds, and 72 C for 30 seconds; following each cycle, reaction tubes were heated to 72 C for 10 minutes for final elongation. Similarly, real-time (quantitative) PCR was performed using primers that were complimentary to shared sequences between BPV-1 and BPV-2, and TaqManÒ probes that were unique to each BPV type (Table 1). The following reagents were used with a total volume of 15 mL per reaction: iQ Supermix (1; Bio-Rad Laboratories, Hercules, CA), primers (0.3 mM each), probe (0.2 mM), and DNA (100 ng). The reaction conditions were as follows using an Eppendorf Mastercycler pro thermocycler: 95 C for 3 minutes followed by 45 cycles of 95 C for 20 seconds, 60 C for 40 seconds, and 68 C for 20 seconds. Serial dilutions of a BPV plasmid were used to generate a standard curve. This 2,836 bp plasmid DNA was produced by GenScript (Piscataway, NJ) and contained a pUC57 insert. Serial dilutions of the plasmid DNA were performed using TE buffer (10 mM Tris-HCl; 1 mM Ethylenediaminetetraacetic acid; pH 7.7). An equine glyceraldehyde-3-phosphate dehydrogenase gene primer and/or probe set was used as an internal control for normalization of input DNA. Positive controls were derived from sarcoid tissues confirmed positive for BPV-1 (mean Ct [cycle threshold] ¼ 21). Negative controls included normal equine skin and nontemplate controls, all with negative Ct for BPV-1 and BPV-2. Data were reported as viral copies per cell. A Shapiro–Wilk test was used to assess normality of data, which was found to be nonparametric. Data were further analyzed using a chi-squared test and Wilcoxon rank-sum test. Logistic regression was used to determine whether the presence or quantity of BPV-1 or BPV-2 was associated with recurrence. A P value of <.05 was considered significant. Bovine papillomavirus-1 DNA was found in 100% (n ¼ 25) of FFPE tumor samples and in 84% (n ¼ 21) of samples from histologically “clean” surgical margins. Bovine papillomavirus-2 DNA was found in 8% (n ¼ 2) of tumors, and was not found in any margin sample. Bovine papillomavirus-1 DNA was more likely to be found in tumor and margins compared with BPV-2 (P < .001). There was no significant difference in the presence of BPV-1 (qualitative PCR) in tumors compared with surgical margins for tumors that recurred and those that did not recur (P ¼ .11). Although quantitative (real-time) PCR was more sensitive in detecting the presence of BPV-1 or BPV-2 DNA in surgical margins compared with qualitative PCR, there was no significant difference in the presence of BPV-1 or BPV-2 DNA in margins of tumors that recurred and those that did not recur for either test. There was no difference in the amount (copies/cell) of BPV-1 DNA in the surgical margins between the tumors that recurred (median, 0; range, 0–0.13) and those that did not recur (median, 0.02; range, 0–5.0)
Table 2 Median and range for BPV-1 copies per cell for surgical margins and bulk tumor samples in recurrent and nonrecurrent sarcoids
Surgical margins Bulk tumor
Recurrence
Nonrecurrence
P value
0 (0–0.13) 0.32 (0.04–13.61)
0.02 (0–5.0) 2.12 (0.13–29.62)
.52 .042
BPV, bovine papillomavirus.
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(P ¼ .52) (Table 2). Surprisingly, the amount of BPV-1 DNA in tumors that recurred was significantly lower (median, 0.32; range, 0.04–13.61) than in those that did not recur (median, 2.12; range, 0.13–29.62) (P ¼ .042) (Table 2). In the present study, there was no evidence that a higher quantity of BPV DNA in either tumor or surgical margins of sarcoids considered “completely excised” by histopathology predicted recurrence; thus, the hypothesis was rejected. It is possible that the “clean” margins were, in fact, devoid of neoplastic cells despite the presence of BPV DNA. This is consistent with results of a study that found BPV DNA in normal skin from 73% of sarcoid-bearing horses and may reflect latent carrier status [30]. Another possibility is that histopathology is not sensitive enough for the detection of neoplastic cells in surgical margins of excised equine sarcoids and that some of the histologic sections with apparently “clean” margins contained neoplastic cells. It is also possible that clean margins were contaminated during sampling for DNA extraction. Pooling four marginal samples from each specimen could have increased the likelihood of BPV-DNA contamination from adjacent neoplastic tissue. Finally, a significant difference in marginal BPV-DNA quantity could exist between the two groups (recurrence vs nonrecurrence), but the sample size was too small to detect this difference. In the present study, a higher amount of BPV-1 DNA in the bulk tumor was associated with decreased recurrence. These results are inconsistent with a previous study that demonstrated a positive association between intratumoral viral load and disease severity, but the inclusion criteria for “severe” disease contained many variables, with recurrence or nonrecurrence not consistently reported [31]. Our results could reflect the small sample size and may not be significant, or they could indicate a more robust immune response triggered by a higher viral load. A study investigating feline cutaneous fibropapillomas with histologic features consistent with equine sarcoids found that tumor recurrence after surgical excision was not consistently associated with detection of papillomavirus DNA [32]. Furthermore, human papillomavirus viral loads have been shown to be nonassociated with recurrence of respiratory and anogenital papillomas [33,34]. The presence and quantity of BPV-1 DNA was much higher than that of BPV-2 DNA in equine sarcoids; BPV-2 was only found concurrently with BPV-1. These findings are consistent with previous reports [7,30,35]. The sample size for this study was small given the difficulty in achieving complete tumor resection and in following the patient for at least 1 year after surgery. Although increased BPV-1 DNA in bulk tumors was associated with nonrecurrence, the small sample size precludes us from making specific management recommendations based on this information. Excision with wide surgical margins is still likely important in decreasing the risk of recurrence, and close monitoring for regrowth is critical for early removal of recurrent sarcoids. Acknowledgments The authors would like to thank Dr. Alain Theon for providing tissue samples from the University of California-
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