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Journal of Clinical Oncology, Vol 25, No 29 (October 10), 2007: pp. 4550-4556 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.12.3182 Human Papillomavirus-16 Is the Predominant Type Etiologically Involved in Penile Squamous Cell Carcinoma
From the Department of Pathology, VU University Medical Center; and the Department of Urology and the Division of Diagnostic Oncology, the Netherlands Cancer Institute, Amsterdam, the Netherlands; the Division of Genome Modifications and Carcinogenesis, Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Heidelberg; and the Department of Dermatology, University Hospital Charité, Berlin, Germany Address reprint requests to Daniëlle A.M. Heideman, PhD, Department of Pathology, VU University Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, the Netherlands; e-mail: dam.heideman{at}vumc.nl
Purpose Human papillomavirus (HPV) infections are suggested to be involved in the development of penile squamous cell carcinoma (SCC), but comprehensive studies to define the association are limited. Therefore, we performed molecular and serologic analyses for a broad spectrum of HPV types on a large series of 83 penile SCCs, and we compared serological findings to those of age-matched male controls (N = 83). Methods Penile SCCs were subjected to detection and typing assays for mucosal and cutaneous HPVs and to subsequent, type-specific viral load and viral gene expression assays. Sera of patients and of controls were analyzed for type-specific mucosal and cutaneous HPV L1, E6, and/or E7 antibodies using bead-based, multiplex serology. Results HPV DNA of mucosal and/or cutaneous types was found in 46 of 83 (55%) penile SCCs. HPV16 was the predominant type, appearing in 24 (52%) of 46 of penile SCCs. The majority of HPV16 DNA–positive SCCs (18 of 24; 75%) demonstrated E6 transcriptional activity and a high viral load. Additionally, HPV16 molecular findings were strongly associated with HPV16 L1-, E6-, and E7-antibody seropositivity. Furthermore, serologic case-control analyses demonstrated that, in addition to the association of HPV16 with penile SCC, seropositivity against any HPV type was significantly more common in patients compared with in controls. HPV18 and HPV6 seropositivity were associated with HPV16-negative SCCs but were not correlated to molecular findings. Conclusion HPV16 is the main HPV type etiologically involved in the development of penile SCC. Although individuals who develop penile SCC show a greater prior exposure to a broad spectrum of HPV types, insufficient evidence was found to claim a role for HPV types other than HPV16 in penile carcinogenesis.
Squamous cell carcinoma (SCC) of the penis is a relatively rare disease in developed countries, where age-standardized incidence rates range from 0.3 to one per 100,000 men. However, in parts of South America (Brazil and Columbia) and Africa (Uganda), the incidence can reach four per 100,000 men. In Europe, about 4,000 cases are diagnosed each year, which comprises less than 0.5% of all cancers.1 The disease generally occurs late in life, with a mean age at diagnosis of 60 years and an age-related incidence that rises continuously to reach its highest level after 70 years of age. For future preventive and therapeutic strategies, it is of major importance to gain insight into the pathogenesis of penile SCC. The development is most likely a stepwise chain of events over a period of many years from preneoplastic lesions to SCC. The etiology of penile SCC appears multifactorial, with a history of smoking, phimosis, poor hygiene, and lack of circumcision during childhood commonly associated with this neoplasm.2,3 However, the exact etiologic mechanisms that lead to the development of penile SCC are largely unknown. Seroepidemiologic and DNA detection studies have suggested a functional role of high-risk human papillomavirus (hr-HPV) for a subset of penile SCCs.4-10 Penile carcinomas with basaloid features and those of the warty subtype displayed the highest hr-HPV prevalence, up to 100%.9,11,12 On the other hand, keratinizing or not-otherwise-specified penile SCCs, which represent the most common histotypes (> 95%) diagnosed in Europe and the United States, generally showed a lower HPV prevalence of approximately 30% to 40%.8,9 To prove causal involvement of HPV in carcinogenesis, specific criteria have been postulated,13,14 including the presence of intratumoral viral DNA, intratumoral oncogene transcripts, and serum antibodies to HPV proteins. In previous studies evaluating a role of HPV infections in penile carcinogenesis, only some of these criteria have been addressed. Comparative studies (ie, serologic v molecular analyses of cases) and the evaluation of types other than HPV16 gained limited attention. In addition, sample sizes were modest in most studies. As such, many aspects of the association between HPV infections and the development of penile SCC remain elusive. In view of current HPV vaccine developments, better definition of the precise contribution of HPV to the etiology of penile SCC regains interest. The family of HPVs now consists of approximately 100 completely characterized types.15 The mucosal hr-HPV types are acknowledged to have carcinogenic capacity, but recent data suggest oncogenic potential of some cutaneous HPV types as well (reviewed in Akgül et al16). Interestingly, DNA of cutaneous HPV8 has occasionally been detected in penile lesions.17 In the present study, we performed a comprehensive study on a unique, large series of 83 patients with penile SCC to detect the presence and biologic activity of a broad spectrum of mucosal and cutaneous HPV types by analyzing viral DNA, RNA, and antibodies to HPV L1, E6, and/or E7 proteins. A potential role of HPV types in penile carcinogenesis was further explored by a case-control study, using HPV-based serologic parameters.
Patients, Controls, Sera Samples, and Tissue Samples This study was approved by and followed the local ethical guidelines of the institutional review board of the Netherlands Cancer Institute. Investigations were carried out on 83 patients with primary, invasive, penile SCC treated in the Netherlands Cancer Institute between 1969 and 2000. The study represents the number of patients from whom formalin-fixed, paraffin-embedded (FFPE) material from the primary tumor was available, and sera were collected at the time penile SCC was diagnosed. The ages of the patients ranged from 27 to 92 years (median, 65 years). Survival and clinicopathologic data of the patients have been previously described.18 Corresponding frozen tissue specimens from 30 patients analyzed in this study were tested during the course of a previous study.8 Hematoxylin and eosin (H&E) –stained, FFPE sections were used for histologic subtyping according to standard histologic criteria.11 A control group of 104 sera of males not diagnosed with penile cancer was collected between 1985 and 1989.19 Twenty-one sera of controls (median age, 63 years; range, 37 to 85 years) comprised the training set to calculate cutoff values for serologic assays; 83 sera of males (median age, 64 years; range, 27 to 81 years) who were age-matched to the cases (< 40 years, 7 v 7; 41 to 65 years, 40 v 38; > 65 years, 36 v 38, respectively) comprised the control set. Informed consent was obtained from all participating men.
Tissue Preparation and Nucleic-Acid Isolation For each tumor specimen, we isolated DNA in duplicate by digestion of deparaffinized sections overnight at 37°C using proteinase K solution, followed by heat-inactivation of proteinase K and DNA extraction, using the high pure PCR template preparation (HPPTP) kit according to the manufacturer (Roche, Mannheim, Germany) recommendations. DNA isolates were frozen at –20°C until use. RNA was isolated from deparaffinized, FFPE sections by overnight lysis in a NucliSense Lysis Buffer (BioMerieux, Boxtel, the Netherlands) and by a subsequent NucliSense EasyMag isolation procedure (BioMerieux). RNA isolates were stored at –80°C until use.
β-globin and HPV DNA PCR Detection and Typing Mucosal HPV DNA was detected by L1-region–based, GP5+/6+-PCR-enzyme immunoassay (EIA) followed by typing by Reverse Line Blot (RLB) assay, as described previously.20,21 The GP5+/6+-PCR-EIA-RLB sequence was applied in duplicate with both DNA isolates of each tumor specimen. Typing data were highly concordant between duplicate measurements (type concordance, 100%) and between duplicate DNA samples (type concordance, 94%). For analysis, patients were scored as positive for mucosal HPV types when these were detected in at least three of four measurements. Beta-gamma–cutaneous (BGC) HPV DNA was detected and typed by L1-region–based BGC-HPV-PCR-RLB, as described previously.22 The assay was supplemented with probes for 36 (putative) types, subtypes, or variants submitted to GenBank (available at http://www.hpv-web.lanl.gov): HPV-DL231, -DL250, -DL253, -DL267, -DL284, -DL285, -DL287, -DL332, -DL297 (accession number [AC], Z95963 to Z95971), -DL428 (AC, AJ010823), -DL436 (AC, AJ010824), -RTRX2, -RTRX3, -RTRX4 (AC, L38919 to L38921), -X10b (AC, AF091446), -X11b (AC, AF091447), -X11c (AC, AF097701), -X13 (AC, AF054873), -X14 (AC, AF054874), -X20 (AC, AF054877), -X23 (AC, AF054879), -X24, -X25, -X26, -X27, -X29, -X32, -X33 (AC, AF054881 to AF054887), -x34 (AC, AF055710), -X35 (AC, AF055711), -5c (AC, AF091436), -15b (AC, AF091457), -22b (AC, AF091439), -24b (AC, AF091441), -38b (AC, AF091443), and -38c (AC, AF091444). Based on our previous study demonstrating a reduced reproducibility of the BGC-HPV-PCR-RLB assay in case of infections with multiple types or lower viral loads,22 the BGC-HPV-PCR-RLB assay was applied in triplicate with both DNA isolates of each tumor specimen, and patients were scored as positive for cutaneous HPV types, subtypes, or variants when these were detected in at least four of six measurements.
Viral Load Analysis
RT-PCR For detection of HPV16 oncogene transcripts, we performed RT-PCR, amplifying a small fragment (86 bp) using primers that were designed to detect the most abundant splice variant within the HPV16E6 open reading frame, namely E6*I. For that purpose, the forward primer was designed to cover the splice-site, favoring detection of spliced HPV transcripts and reducing possible interference by traces of HPV DNA that might still be present in the sample. The location of primers according to the HPV16 genome26 are nt211 to 225 and 409 to 413 (forward primer) and nt460 to 479 (reverse primer). PCR products were detected using an EIA with an HPV16E6*I-specific probe (nt425 to 451), essentially as described before.21 RNA from FFPE, HPV16-containing, SiHa cells was used as the positive control for RT-PCR. For detection of HPV8 oncogene transcripts, we performed two LightCycler-based RT-PCR assays designed to detect HPV8E6 or E7 mRNA, respectively, as described.27 RNA from HPV8-containing primary keratinocytes was used as positive control for RT-PCR. To assess the quality of all RNA specimens, RT-PCR was performed by LightCycler technology using primers specific for the gene encoding the U1 small nuclear ribonucleoprotein-specific A protein (snRNP U1A, 72bp, sequences on request) and by LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche, Mannheim, Germany). One of the specimens used in this study was negative in the snRNP U1A RT-PCR assay and is stated as such in the results section. None of the negative control reactions lacking RT were positive in any of the RT-PCRs, indicating the absence of interference by possible traces of residual DNA.
Fluorescent Bead Technology–Based Multiplex Serology for HPV
Statistical Analysis
Presence of HPV DNA and Oncogene Transcripts in Penile SCC HPV DNA of mucosal and/or cutaneous types, as a single or multiple infection, was detected in 46 of 83 (55%) penile SCCs: mucosal types in 24 of 83 (29%), cutaneous types in 14 of 83 (17%), and a combination thereof in eight of 83 (10%; Table 1). The majority of the HPV DNA–positive tumors (24 of 46; 52%) contained HPV16. HPV8 was the second most prevalent type (10 of 46; 22%); coinfection with HPV16 was noted in four of 10 (40%) cases of HPV8. The prevalence of all other HPV types among the HPV DNA–positive cases was less than 11% (  five of 46) each.
RT-PCR analysis of HPV16 DNA–positive cancers revealed HPV16E6*I mRNA expression in the majority of cases (18 of 24; 75%; Table 1). HPV16 viral load analysis demonstrated a median of 72 genome copies per cell (range, 8.5 to 2,667) in mRNA-positive cancers and 0.5 copies per cell (range, 0.04 to 2.0) in transcript-negative HPV16 DNA–positive SCCs. Viral load levels were below the detection limit of the quantitative real-time PCR assays (< 0.5 copies per cell) for cancers containing HPV types 5, 8, 15, 20, 21, and/or 23. Moreover, none of the HPV8-positive cancers demonstrated HPV8 E6 and/or E7 mRNA expression.
Serologic Findings in Penile SCC and Correlation to Molecular Data
For HPV16, seropositivity against L1, E6, and/or E7 was significantly associated with an (active) infection, as indicated by the presence of intratumoral HPV16 DNA and/or RNA (Table 2). Odds ratios (ORs) ranged from 4.5 (95% CI, 1.5 to 13) to 25 (95% CI, 2.7 to 227) for HPV16 DNA presence in the tumor and from 6.7 (95% CI, 2.1 to 21) to 44 (95% CI, 4.5 to 422) for intratumoral HPV16 RNA presence. Notably, the tumors of all seven patients that were seropositive for both HPV16 E6 and E7 displayed both HPV16 DNA and RNA. For all other HPV types, no significant association between seropositivity for any of the antigens and HPV-DNA presence in the tumor was observed.
Case-Control Evaluation of the Seroprevalence of HPV Antibodies
Analyses for individual antigen seropositivity revealed significantly elevated ORs for penile SCC among men who were seropositive for antibodies against HPV16 L1, HPV16 E7, HPV6 L1, and HPV3 L1 (Table 4). The association between HPV16 L1 and E7 antibodies and penile SCC could be attributed mainly to men with HPV16 DNA–positive SCCs (Table 4). In the group of patients who had HPV16 DNA–negative tumors, an additional association with seropositivity against HPV18 L1, next to HPV6 L1, was found, whereas that with HPV16 L1, HPV16 E7, and HPV3 L1 disappeared (Table 4). None of the other HPV antigens (n = 32) demonstrated a significant difference in antibody seroprevalence between cases and controls (data not shown). In addition, seroprevalence of antibodies against the poliomaviruses BKV, JCV, and LPV did not differ significantly between cases and controls (ie, 95% v 99%, 78% v 81%, and 39% v 41%, respectively).
Finally, seropositivity against any antigen for those HPV types of which more than one antigen was assessed (ie, HPV8, 9, 15, 16, 18, 33, 38, 52, and 58), was evaluated. This analysis revealed similar data as obtained following analysis of individual antigens of these types (ie, an association of HPV16 to mainly HPV16 DNA–positive tumors and of HPV18 to predominantly HPV16 DNA–negative tumors; Table 4).
The current study describes a comprehensive serologic and molecular examination of HPV infections in relation to penile SCC. This is the first study that included a large series of penile SCC patients, took into account a wide variety of mucosal and cutaneous HPV types from different genera, and integrated molecular and serologic findings. Both antibodies against early E6 and/or E7 proteins, which are for certain HPV types acknowledged to be associated with HPV-related cancer, and against capsid L1 proteins, which are generally recognized to represent current and/or past exposure to HPV infections,30 were measured. Our study provides strong evidence that HPV16 infections play a biologic role in the development of a subset of penile SCCs. Association of HPV16 to penile SCC has been suggested before by molecular3,8-10 and serologic studies.5-7 Herein, an active involvement of HPV16 infections in penile carcinogenesis is strengthened by detection of viral gene expression and of high viral load in the tumors as well as by a strong correlation between HPV16-based molecular and serologic parameters. Serology was found to underestimate HPV exposure, which agreed with results from a previous study.6 The failure to detect type-specific antibodies in patients whose tumors contained a current infection with the respective HPV type remains unclear, but differences in sensitivity between molecular and serologic tests and/or a deficiency in the HPV-specific immune response in cancer patients might be plausible explanations. Our serologic case-control analyses further revealed an association of antibodies against HPV18 and/or HPV6 to penile SCC in males without HPV16 infection. At the DNA level, tumors with single or multiple infections of these mucosal types also were found. Nevertheless, frequencies were low, and no correlation between molecular and serologic markers could be observed for these HPV types. As such, although our data and those of others3,8-10,18,31,32 indicate an association of HPV18 and HPV6 DNA or antibodies with penile SCC, the contribution of these types to penile pathogenesis is elusive. At present, it is also unknown whether cutaneous HPV types are causally associated with a subset of penile SCCs. High antibody frequencies against certain cutaneous types were found in the sera of patients, and DNA of cutaneous HPV types was detected in penile SCCs. However, antibody seropositivity in patients did not differ significantly from that in controls, and no direct correlation between serologic and molecular parameters in cases, as was found for HPV16, could be demonstrated. Furthermore, viral load levels were low, which agreed with the results from a previous study for HPV8 in penile Erythroplasia of Queyrat, a carcinoma in situ of the penile mucosa.17 Also viral gene expression was absent. Thus, our findings argue against an etiologic role of cutaneous HPV types in penile cancer. Nevertheless, our results are similar to previous studies23,27,29 that investigated serologic and molecular HPV markers in relation to nonmelanoma skin cancer, in which cutaneous HPV types are supposed to be pathogenetically involved. In addition to type-specific findings, any HPV seropositivity overall or per subgroup was found to be associated with penile SCC. Comparison of HPV with poliomavirus serology strengthened the conclusion that these HPV findings were penile SCC–or otherwise generally tumor-specific. The higher seroprevalence in cases, in addition to the association of HPV16 to penile SCC, suggests an increased prior exposure of penile cancer patients to HPV infections, most likely related to the sexual lifestyle.33 A potential history of increased exposure to a broad spectrum of HPV types is further underscored by the finding that cases demonstrated significantly more seropositivity to multiple HPV types compared with controls (data not shown). In conclusion, we have provided strong molecular and serologic evidence that mucosal hr-HPV16 acts as a carcinogen in a subset of penile malignancies. Consequently, prophylactic HPV vaccination with HPV16–virus-like particles might reduce the development of approximately one-third of penile SCCs. Furthermore, our case-control analysis supports a greater prior exposure to a broad spectrum of HPV types for males who develop penile cancer. At the individual level, however, insufficient evidence for a contribution of HPV types other than HPV16 in the pathogenesis of penile SCC was found.
The author(s) indicated no potential conflicts of interest.
Conception and design: Daniëlle A.M. Heideman, Simon Horenblas, Chris J.L.M. Meijer, Peter J.F. Snijders Financial support: Chris J.L.M. Meijer Provision of study materials or patients: Michael Pawlita, Joost A. Leijte, Johannes M.G. Bonfrer, Simon Horenblas Collection and assembly of data: Daniëlle A.M. Heideman, Tim Waterboer, Michael Pawlita, Pien Delis-van Diemen, Ingo Nindl Data analysis and interpretation: Daniëlle A.M. Heideman, Michael Pawlita, Ingo Nindl, Chris J.L.M. Meijer, Peter J.F. Snijders Manuscript writing: Daniëlle A.M. Heideman, Michael Pawlita, Peter J.F. Snijders Final approval of manuscript: Daniëlle A.M. Heideman, Tim Waterboer, Michael Pawlita, Pien Delis-van Diemen, Ingo Nindl, Joost A. Leijte, Johannes M.G. Bonfrer, Simon Horenblas, Chris J.L.M. Meijer, Peter J.F. Snijders
We thank D. Boon, A. Brink, D. Claassen, A. de Haan, A.T. Hesselink, B. de Jong, U. Koch, B. Kroon, A. Köhler, M. Oppenländer, and N. Yagçi for technical assistance; H. Berkhof and S. Wilting for statistical support; and E.M. deVilliers, M. Favre, L. Gissmann, G. Orth, and H. Pfister for the gift of HPV plasmids.
Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Carter JJ, Madeleine MM, Shera K, et al: Human papillomavirus 16 and 18 L1 serology compared across anogenital cancer sites. Cancer Res 61:1934-1940, 2001 7. Bjorge T, Dillner J, Anttila T, et al: Prospective seroepidemiological study of role of human papillomavirus in non-cervical anogenital cancers. BMJ 315:646-649, 1997 8. Ferreux E, Lont AP, Horenblas S, et al: Evidence for at least three alternative mechanisms targeting the p16INK4A/cyclin D/Rb pathway in penile carcinoma, one of which is mediated by high-risk human papillomavirus. J Pathol 201:109-118, 2003[CrossRef][Medline] 9. Rubin MA, Kleter B, Zhou M, et al: Detection and typing of human papillomavirus DNA in penile carcinoma: Evidence for multiple independent pathways of penile carcinogenesis. Am J Pathol 159:1211-1218, 2001 10. Levi JE, Rahal P, Sarkis AS, Villa L: Human papillomavirus DNA and p53 status in penile carcinomas. Int J Cancer 76:779-783, 1998[CrossRef][Medline] 11. 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J Clin Microbiol 35:791-795, 1997[Abstract] 21. van den Brule AJ, Pol R, Fransen-Daalmeijer N, et al: GP5+/6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes. J Clin Microbiol 40:779-787, 2002 22. Brink AA, Lloveras B, Nindl I, et al: Development of a general-primer-PCR-reverse-line-blotting system for detection of beta and gamma cutaneous human papillomaviruses. J Clin Microbiol 43:5581-5587, 2005 23. Nindl I, Koehler A, Meyer T, et al: Detection of human papillomavirus DNA in primary squamous cell carcinoma and metastases. Br J Dermatol 154:797-799, 2006[CrossRef][Medline] 24. Hesselink AT, van den Brule AJ, Groothuismink ZM, et al: Comparison of three different PCR methods for quantifying human papillomavirus type 16 DNA in cervical scrape specimens. J Clin Microbiol 43:4868-4871, 2005 25. 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Dillner J: Toward "serolomics": Papillomavirus serology is taking a technologic lead in high-throughput multiplexed antibody analysis. Clin Chem 51:1768-1769, 2005 31. Dorfman S, Cavazza M, Cardozo J: Penile cancer associated with so-called low-risk human papilloma virus. Report of five cases from rural Venezuela. Trop Doct 36:232-233, 2006 32. Senba M, Kumatori A, Fujita S, et al: The prevalence of human papillomavirus genotypes in penile cancers from northern Thailand. J Med Virol 78:1341-1346, 2006[CrossRef][Medline] 33. Dillner J, Kallings I, Brihmer C, et al: Seropositivities to human papillomavirus types 16, 18, or 33 capsids and to Chlamydia trachomatis are markers of sexual behavior. J Infect Dis 173:1394-1398, 1996[Medline] Submitted April 25, 2007; accepted July 17, 2007.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
