Correlates of Human Papillomavirus Viral Load with Infection Site in Asymptomatic Men

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Short Communication

Roberto Flores,1 Lu Beibei,2 Carrie Nielson,3 Martha Abrahamsen,2 Kyle Wolf,2 Ji-Hyun Lee,2 Robin B. Harris,4 and Anna R. Giuliano2 1Cancer Prevention Fellowship Program, National Cancer Institute, NIH, Bethesda, Maryland; 2H.Lee Moffitt Cancer Center and Research Institute, Tampa, Florida; 3Oregon Health and Science University, Portland, Oregon; and 4Arizona Cancer Center, Mel and Enid Zuckerman College of Public Health, Tucson, Arizona

Abstract

Numerous studies have evaluated human papillomavi- HPV-16 viral DNA detection compared with the reverse rus (HPV) DNA load in women, especially HPV-16 line blot assay. Viral load varied significantly by viral load, and its role in cervical carcinogenicity. Few anatomic site (P = 0.019). Penile shaft specimens had studies have examined HPV viral load in men, none significantly higher viral load than any other anatomic among asymptomatic men. The aim of the current study site evaluated except for the anal canal. HPV-16 viral is to quantify HPV-16 viral load in male anogenital load was positively correlated between proximal ana- specimens and to explore its correlates with anatomic tomic sites: perianal and anal canal (P = 0.003), perianal sites. Two-hundred and ninety-four specimens from 42 and scrotum (P = 0.011), scrotum and glans/corona men who tested positive for HPV-16 at one or more (P = 0.045), and scrotum and penile shaft (P =0.037). anatomic sites were evaluated. HPV DNA was detected In conclusion, the penile shaft seemed to be the with PGMY 09/11 primer and genotyped with reverse preferred site for HPV-16 viral replication. Viral load line blot assay followed by HPV-16 viral quantification correlation between proximal sites suggested a possible using type-specific real-time PCR assay (TaqMan). The autoinoculation in male HPV transmission. (Cancer quantitative PCR assay showed a higher sensitivity in Epidemiol Biomarkers Prev 2008;17(12):3573–6)

Introduction

Our understanding of the natural history of human persistent infection with HPV-16 with persistently high papillomavirus (HPV) infection in males is still very viral load values are risk factors for the development limited due to the small number of publications on the of cervical cancer (13, 14).HPV-16 viral load has also topic.Male HPV infection is important in male to female been evaluated as a biomarker for the clearance of transmission of HPV and subsequent infection and HPV infection in women (15).Unfortunately, there are disease in women (1, 2).Case-control studies of women few publications that have reported HPV viral load with cervical cancer and their husbands have shown that in men (5-7), and none that have examined viral load men’s sexual behavior affects women’s risk of cervical by anatomic site.The purpose of the current study is neoplasia, even after controlling for female sexual to determine HPV-16 viral load in male anogenital activity (3, 4).HPV-16 viral load may be an important specimens and explore its possible association with factor in the natural history of HPV in men as it may anatomic site. determine risk of infection persistence and subsequent disease in men and transmission to women (5-7). In women, HPV viral load seems to be important in Subjects, Materials, and Methods disease progression.Recent studies suggest that HPV-16 viral load may be used as a molecular biomarker to Study design, clinical sampling, and HPV testing for the improve the prognostic value of HPV testing (8-10). current analysis have been previously described in detail Other studies found a positive correlation between high (16-18).Briefly, a cross-sectional study of HPV infection HPV-16 load and high-grade squamous intraepithelial in 463 men between 18 and 40 years old—359 in Tucson, lesions (11, 12).Retrospective studies observed that Arizona and 104 in Tampa, Florida—was conducted from 2003 to 2006.Participants completed a risk factor questionnaire that included questions on sociodemo- Received 5/21/08; revised 9/14/08; accepted 10/1/08. graphic, clinical, and sexual behavioral factors.Anogen- Grant support: Centers for Disease Control and Prevention, through the Association of American Medical Colleges (cooperative agreement; grant U36/CCU319276; ital specimens were obtained independently by rubbing AAMC identification no.MM-0579-03/03). separate saline-wetted Dacron swabs over the entire Requests for reprints: Roberto Flores, Cancer Prevention Fellowship Program, surface of the (a) glans penis/coronal sulcus, (b) penile National Cancer Institute, NIH, 6120 Executive Boulevard, EPS Suite T-41, Bethesda, c d MD 20892-7105.Phone: 813-745-6350; Fax: 410-955-7407.E-mail: [email protected] shaft (including the prepuce, if present), ( ) scrotum, ( ) Copyright D 2008 American Association for Cancer Research. perianal area, and (e) anal canal up to the anal verge.A doi:10.1158/1055-9965.EPI-08-0467 calcium alginate or Dacron urethral swab was used to

Cancer Epidemiol Biomarkers Prev 2008;17(12). December 2008

Table 1. HPV-16 viral load (per 103 CE) among specimens with a detectable viral load by reverse line blot genotyping result and anatomic site

Anatomic site Reverse line blot genotyping result no.(%)

HPV-16 positive Median viral load (range) HPV-16 negative Median viral load (range) P All sites 82 (67.2) 782 (3-373,782) 40 (32.8) 19 (0.8-990) <0.001* Anal canal 6 (62.5) 1,315 (665-43,797) 3 (37.5) 4 (0.8-9) 0.037* Perianal area 6 (50.0) 5,667 (26-82,742) 6 (50.0) 12 (2-178) 0.013* Glans/corona 17 (68.0) 138 (3-373,782) 8 (32.0) 78 (17-588) 0.336 Scrotum 18 (60.0) 251 (7-38,212) 12 (40.0) 28 (4-332) 0.005* Semen 2 (50.0) 114 (59-170) 2 (50.0) 40 (5-76) 0.699 Penile shaft 32 (84.2) 2,486 (12-75,701) 6 (15.8) 156 (3-990) 0.006* Urethra 2 (40.0) 590 (229-951) 3 (60.0) 2 (1-8) 0.149

*Denotes statistical significance (a = 0.05).

sample the first 2 cm of the urethral epithelium.Semen fragment of the L1 gene using the PGMY system (19). specimens were self-collected in a sterile cup. HPV genotyping was conducted using the reverse line blot method (20).This detection method uses the HPV L1 HPV DNA Detection and Genotyping. DNA consensus PCR products labeled with biotin to detect 37 extracted from swabbed cellular material and semen HPV types.The same DNA extracts were used for was used for HPV testing by PCR for amplification of a quantitation of viral load using HPV-16 type-specific real-time PCR. HPV-16 Viral Load. Specimens from seven anatomic sites of those men who were HPV-16–positive at one or more anatomic sites by the reverse line blot method were evaluated for HPV-16 viral load using a quantitative real-time PCR assay (TaqMan) as previously described (21).Briefly, the standard curve for absolute quantification of HPV-16 was generated using the E6-E7 region of HPV-16 that had been cloned into plasmid vectors (21).Real-time PCR reactions were done in a 20 AL volume using the ABI PRISM1 7900HT instrument (Applied Biosystems).A picogreen fluores- cence assay to determine DNA concentration was used to normalize viral load values with cell equivalents (CE) in exfoliated skin cells (Invitrogen).HPV-16 standard curves were generated by plotting the cycle threshold (Ct) values against known concentrations of input HPV-16 copy number.The calculated CE based on the picogreen assay was used to determine the viral copy number per CE for each unknown sample as described by van Duin et al.(15).Serial dilutions of CaSki DNA (HPV-16–positive cell line) ranging from 2.7 to 10.8 ng were used as positive controls 2to determine inter-run variability, as well as non- template controls as negative control. Statistical Analysis. Viral load (per 103 CE) was log10-transformed and summarized in box plots and scatter plots.The Kruskas-Wallis test and the Wilcoxon rank-sum test were applied to compare viral load by anatomic site and by reverse line blot genotyping results.The Spearman rank correlation was used to determine the correlation in viral load between anatomic sites. Figure 1. Distribution of HPV-16 viral load by anatomic site. The crude (A; 103 copies)and normalized ( B; no. of copies per 3 10 CE)HPV-16 viral load is log 10-transformed and summa- Results rized in the box plot. Columns, the interquartile range, with the upper boundary of the box representing the 75th and the lower Of 463 participants enrolled in the cross-sectional study, boundary representing the 25th percentile; horizontal line 42 participants who tested positive for HPV-16 at one or indicates the median value, bars, minimum and maximum. more anatomic sites by the reverse line blot method were

Cancer Epidemiol Biomarkers Prev 2008;17(12). December 2008

Figure 2. Correlation of HPV-16 viral load between anatomic sites in close proximity. Dots, the number of subjects with a detectable viral load (log10-transformed); slope, linear correlation approximated based on Spearman’s r. included in this analysis, contributing a total of 294 Discussion specimens.The mean age of the 42 participants was 27.5 years (SD, 6.3). The majority of men were nonsmokers To our knowledge, this is the first study to examine HPV- (57%) or former smokers (17%), and circumcised (86%). 16 viral load at seven different anogenital sites using Of the 294 specimens evaluated for HPV-16 DNA, type-specific real-time PCR in men.In this study, the 122 specimens were HPV-16–positive by quantitative real-time PCR assay detected HPV-16 viral copies in 122 PCR method, whereas only 82 of 122 specimens were specimens.Of 122 specimens, 40 (32.8%)specimens with HPV-16–positive by reverse line blot method.Of 122 significantly lower viral load had been classified as HPV- specimens with a detectable viral load, significantly 16–negative by reverse line blot genotyping assay.This higher viral load was found in reverse line blot HPV- may indicate that the threshold of detection in the real- 16–positive specimens than that in reverse line blot time PCR assay could be lower than the one observed for HPV-16–negative specimens (P < 0.001), and the the reverse line blot assay.Alternatively, our quantitative differences remained significant for individual sites: PCR assay could be less sensitive to PCR inhibitors anal canal (P = 0.037), perianal (P = 0.013), scrotum present in skin exfoliated cells of the male genital area. (P = 0.005), and penile shaft (P = 0.006; Table 1). Although PCR-based genotyping assays are the method The distribution of HPV-16 viral load by anatomic site of choice to detect HPV DNA, the use of more sensitive is shown in Fig.1.Among all specimens with a assays such as quantitative real-time PCR may be detectable viral load, the viral copy number ranged from advantageous in minimizing false negatives in HPV <1 to 4.65 Â 104, with the highest median viral copy of testing.HPV-16 viral load in male genital specimens in 133 copies detected in anal canal specimens and the the present study seems to be much lower compared lowest median viral copy of 1 copy observed in urethra with HPV-16 viral load found in female cervical speci- specimens.After cellular normalization, viral load (per mens, as shown in a previous study by our group (22). CE) showed a similar pattern in distribution.Normalized This finding is consistent with the results reported by viral load ranged from <1 to 373 copies per CE with the Bleeker and colleagues, in which 61.4% of male speci- highest median viral load of 1.6 copies per CE (range, mens had viral load values that were below the detection <1-75 copies per CE) found on the penile shaft followed threshold of the assay compared with 0% of female by the anal canal and perianal area, and the lowest cervical specimens (5).Based on these observations, we median viral load found in the urethra.Viral load varied hypothesize that the cervical epithelium may be a more significantly by anatomic site (P = 0.019). The penile shaft favorable environment for viral replication than the skin had a significantly higher viral load than any other epithelium, although it is likely that factors which anatomic sites except for the anal canal.Figure 2 presents restrain viral replication on skin epithelium remain the correlation of viral load between anatomic sites. unidentified. HPV-16 viral load was significantly correlated between In the present study, the penile shaft seems to be the specimens from anatomic sites in close proximity: preferred site for HPV-16 replication among all the sites perianal and anal canal (P = 0.003), perianal and scrotum evaluated.We observed significantly higher viral loads (P = 0.011), scrotum and glans/corona (P = 0.045), and in the penile shaft than in most anatomic sites.In scrotum and penile shaft (P = 0.037). addition, we have previously shown that infections with

Cancer Epidemiol Biomarkers Prev 2008;17(12). December 2008

multiple HPV types were most frequently detected on would be expected by chance and is associated with increased viral loads.Clin Infect Dis 2005;41:612 – 20. the penile shaft of men enrolled in this cross-sectional 6. De Marco F, Di Carlo A, Poggiali F, Muller A, Marcante ML. study (17).We observed significant positive correlations Detection of HPV in genital condylomata: correlation between in viral load between infection sites that were in close viral load and clinical outcome.J Exp Clin Cancer Res 2001;20: proximity such as perianal and anal canal, or perianal 377 – 83. 7. Sanclemente G, Herrera S, Tyring SK, et al.Human papillomavirus and scrotum, or the sites that are in regular contact with (HPV) viral load and HPV type in the clinical outcome of HIV- each other at a resting position such as the scrotum and positive patients treated with imiquimod for anogenital warts and glans/corona, or scrotum and penile shaft.This obser- anal intraepithelial neoplasia.J Eur Acad Dermatol Venereol 2007;21: vation suggests that previously HPV-uninfected genital 1054 – 60. 8. Cuzick J, Terry G, Ho L, Hollingworth T, Anderson M.Type-specific sites may be inoculated by existing infections from human papillomavirus DNA in abnormal smears as a predictor of proximal anatomic sites through direct contact between high-grade cervical intraepithelial neoplasia.Br J Cancer 1994;69: the sites and facilitated by environmental factors such as 167 – 71. humidity, temperature, and possibly poor hygiene. 9. Hart KW, Williams OM, Thelwell N, et al.Novel method for detection, typing, and quantification of human papillomaviruses in Autoinoculation has been observed in the transmission clinical samples.J Clin Microbiol 2001;39:3204 – 12. of other viruses such as the herpes simplex virus (23, 24). 10. Sun CA, Lai HC, Chang CC, Neih S, Yu CP, Chu TY.The significance In the case of herpes simplex virus, active virions can be of human papillomavirus viral load in prediction of histologic transported through scratching or body fluid such as severity and size of squamous intraepithelial lesions of uterine cervix.Gynecol Oncol 2001;83:95 – 9. saliva and lead to manual inoculation of proximal sites. 11. Beskow AH, Gyllensten UB.Host genetic control of HPV 16 titer in This hypothesis is supported by the lack of correlation of carcinoma in situ of the cervix uteri.Int J Cancer 2002;101:526 – 31. HPV-16 viral load among distant sites such as the 12. 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Castellsague X, Bosch FX, Munoz N, et al.Male circumcision, penile simplex virus on the face in the absence of ‘‘cold sores,’’ after trauma. human papillomavirus infection and cervical cancer in female J Oral Maxillofac Surg 2008;66:136 – 8. partners.N Engl J Med 2002;346:1105 – 12. 24. Wolf R, Davidovici B, Marcus B, Orion E, Lipozencic J.Autoinocula- 5. Bleeker MC, Hogewoning CJ, Berkhof J, et al.Concordance of specific tion and dissemination are two different forms of herpes virus human papillomavirus types in sex partners is more prevalent than spread.Acta Dermatovenerol Croat 2006;14:258 – 60.

Cancer Epidemiol Biomarkers Prev 2008;17(12). December 2008

Downloaded from cebp.aacrjournals.org on September 28, 2021. © 2008 American Association for Cancer Research. Cancer Epidemiology, Correction Biomarkers & Prevention Correction: Correlates of Human Papillomavirus Viral Load with Infection Site in Asymptomatic Men

In this article (1), which was published in the December 2008 issue of Cancer Epidemiology, Biomarkers & Prevention, an error resulted in an author's name being printed in the byline incorrectly as Lu Beibei. The author's name is Beibei Lu.

Reference 1. Flores R, Beibei L, Nielson C, Abrahamsen M, Wolf K, Lee J, et al. Correlates of human papillomavirus viral load with infection site in asymptomatic men. Cancer Epidemiol Biomarkers Prev 2008;17:3573–6.

Ó2011 American Association for Cancer Research. doi: 10.1158/1055-9965.EPI-10-1251

216 Cancer Epidemiol Biomarkers Prev; 20(1) January 2011 Correlates of Human Papillomavirus Viral Load with Infection Site in Asymptomatic Men

Roberto Flores, Lu Beibei, Carrie Nielson, et al.

Cancer Epidemiol Biomarkers Prev 2008;17:3573-3576.

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