Human Papillomavirus Prevalence in Invasive Penile Cancer and Association with Clinical Outcome

Author's Accepted Manuscript Human papillomavirus prevalence in invasive penile cancer and association with clinical outcome Rosa S. Djajadiningrat, Ekaterina S. Jordanova, Bin K. Kroon, Erik van Werkhoven, Jeroen de Jong, Divera T.M. Pronk, Peter J.F. Snijders, Simon Horenblas, Daniëlle A.M. Heideman PII: DOI: Reference:

S0022-5347(14)04261-X 10.1016/j.juro.2014.08.087 JURO 11744

To appear in: The Journal of Urology Accepted Date: 8 August 2014 Please cite this article as: Djajadiningrat RS, Jordanova ES, Kroon BK, van Werkhoven E, de Jong J, Pronk DTM, Snijders PJF, Horenblas S, Heideman DAM, Human papillomavirus prevalence in invasive penile cancer and association with clinical outcome, The Journal of Urology® (2014), doi: 10.1016/ j.juro.2014.08.087. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain. All press releases and the articles they feature are under strict embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date.

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Original Article

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Human papillomavirus prevalence in invasive penile cancer and association

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with clinical outcome

4 Rosa S. Djajadiningrat (1), Ekaterina S. Jordanova (2), Bin K. Kroon (1), Erik van Werkhoven

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(3), Jeroen de Jong (4), Divera T.M. Pronk (5), Peter J.F. Snijders (5), Simon Horenblas (1),

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Daniëlle A.M. Heideman (5)

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From the Departments of Urology (1), Biometrics (3) and Pathology (4), The Netherlands

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Cancer Institute, Amsterdam, The Netherlands

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Centre for Gynaecologic Oncology Amsterdam, Department of Obstetrics and Gynaecology

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(2) and Department of Pathology (5) VU University Medical Center, Amsterdam, The

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Netherlands

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Address all correspondence to:

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Simon Horenblas, Department of Urology, The Netherlands Cancer Institute

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Plesmanlaan 121, NL-1066 CX Amsterdam, the Netherlands, Phone: +31 20 512 9083; Fax:

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+31 20 512 2554; e-mail: [email protected]

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Conflict of interest or funding: None

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Keywords: penile cancer, squamous cell carcinoma, human papillomavirus, survival

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Word count text (including abstract): 2186

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Word count text (excluding abstract): 1974

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Word count abstract: 212

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ACCEPTED MANUSCRIPT Abstract

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Purpose: Penile cancer incidence is rising and suggested to be explained by changes in sexual

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practice and increased exposure of males to sexually transmitted high-risk human

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papillomavirus (hrHPV) infection. Previously, we found in penile cancers from a Dutch

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population treated in the years 1963-2001, a hrHPV prevalence of about 30%. Here, we

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assessed the prevalence of hrHPV-DNA in a more recent, contemporary penile cancer cohort

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and association with patient survival.

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Materials and Methods: hrHPV-DNA presence was assessed by GP5+6+-PCR in 212 formalin-

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fixed, paraffin-embedded invasive penile tumor specimens of patients treated between

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2001 and 2009. Five-year disease-specific survival (DSS) was calculated using Kaplan-Meier

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method with log-rank test and Cox regression.

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Results: hrHPV-DNA was detected in a subset of penile cancer cases (25%; 95%-CI:19-31).

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HPV16 was the predominant type, representing 79% (42/53) of all hrHPV infections. Five-

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year DSS in the hrHPV-negative group and hrHPV-positive group was 82% and 96%,

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respectively (log rank test p=0.016). Adjusted for stage, grade, lymphovascular invasion (LVI)

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and age, HPV status was still prognostic for DSS (p=0.030) with a hazard ratio of 0.2 (95%-

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CI:0.1–0.9).

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Conclusions: hrHPV DNA was observed in one-fourth of penile cancer cases. No relevant

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increase in hrHPV prevalence over the past decades was observed. The presence of hrHPV-

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DNA in penile cancer confers a survival advantage.

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ACCEPTED MANUSCRIPT Introduction

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The etiology of penile cancer is multifactorial with smoking, phimosis and poor hygiene

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commonly associated with this tumor1. Other risk factors include the number of sexual

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partners, and a history of genital warts or other sexually transmitted diseases2. At least part

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of these risk factors is related to infection with human papillomavirus (HPV). In circumcised

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men, HPV prevalence is lower than in uncircumcised men3, and penile cancer is rare in

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populations that routinely practice circumcision4.

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The reported proportion of penile cancer associated with high-risk HPV (hrHPV) types ranges

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from 30% to 100%1,5–7; depending on the population studied, the methods used for HPV

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detection, and/or histological subtypes analyzed. In the Dutch population (years 1963-2001),

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we found approximately 30% of penile cancers to be hrHPV-associated8–10. It was also shown

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that patients with hrHPV-positive tumors carried a survival advantage over patients with

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hrHPV-negative tumors. However, other studies concerning the association between HPV

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status and patient survival report inconsistent results6,11–13. As such, the exact role of HPV as

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a prognostic factor in penile cancer remains unclear. Moreover, penile cancer incidence is

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rising and suggested to be explained by changes in sexual practice and exposure of males to

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sexually transmitted HPV infection14. Recent studies on head and neck squamous cell

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carcinomas (SCC) have shown a rising incidence of hrHPV-associated subtypes over the past

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decades in several Western countries, including the Netherlands15–17. It is conceivable that

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the percentage of hrHPV penile cancers may have increased over time as well.

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Hence, the current study was set out to assess the prevalence of hrHPV-DNA in invasive

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penile cancer in a contemporary Dutch cohort and its association with patient survival.

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Towards this goal, a large series of penile cancer cases of the years 2001-2009 was analyzed

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for hrHPV-DNA presence by GP5+6+-PCR, and the association between HPV status and

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patient survival was assessed.

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Materials and Methods

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Study population

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Our institutional cohort comprised 487 patients who were diagnosed with penile cancer

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between 2001 and 2009. To achieve hrHPV-DNA data of patients with primary invasive

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cancer treated between 2001 and 2009, formalin-fixed paraffin-embedded (FFPE) tissue

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blocks were retrieved from the archives of the Department of Pathology, the Netherlands 3

ACCEPTED MANUSCRIPT Cancer Institute, the Netherlands, for additional testing. Patients who were initially treated

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for their primary tumor elsewhere, neoadjuvantly treated or with carcinoma in situ were

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excluded. For 212 patients, sufficient material of the primary invasive tumor was left for

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HPV-DNA testing. The use of clinical material was in compliance with the respective

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institutional ethical regulations for surplus material18 and approved by the institutional

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translational research board.

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All patients were (re)staged according to the TNM 2009 classification for penile cancer, and

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had not received pre-operative radiotherapy or chemotherapy. The clinical and pathological

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characteristics were prospectively kept in our institutional penile cancer database. Follow-up

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until 2013 provided information concerning node-positivity, disease status and disease-

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specific mortality.

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Sample preparation, histopathology and hrHPV-DNA detection and typing

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Sample preparation and hrHPV-DNA detection and typing have been described before8–10.

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Briefly, of each FFPE specimen, a series of consecutive 5-µM sections were cut under safety

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measures to avoid cross-contamination. The first and last sections were haematoxylin–eosin

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stained for histopathology, including confirmation of tumor presence, subtyping, and

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grading according to Broders19. In-between sections were collected in a reaction vessel for

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DNA extraction and subsequent PCR analyses.

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Detection of hrHPV on the DNA extracts from FFPE sections was performed by GP5+/6+-PCR

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enzyme immunoassay (EIA), using a cocktail of 14 hrHPV types (i.e., HPV 16, 18, 31, 33, 35,

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39, 45, 51, 52, 56, 58, 59, 66 and 68) as described before8–10. Beta-globin PCR was performed

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on each DNA extract as a quality control. hrHPV-positive samples were subsequently

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genotyped by bead-based array on the Luminex platform20.

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Statistical analyses

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Differences in patient characteristics between hrHPV-positive and negative groups were

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tested for statistical significance. DSS was defined as the time since surgery until death of

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penile cancer, penile cancer metastasis or complications related to penile cancer treatment.

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Patients alive at the end of follow-up were censored. DSS rates were calculated using

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Kaplan-Meier method, with the log-rank test assessing equality of distributions.

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Multivariable analysis of survival was performed using the Cox proportional hazards model. 4

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All statistical analyses were performed with SPSS (v20.0, SPSS Inc., Chicago, Illinois) and R,

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version 3.0.2 (http://www.r-project.org/). Two-sided p-values of <0.05 were considered

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statistically significant.

4 Results

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HPV prevalence

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In total, 53/212 (25%, 95%-CI: 19%-31%) of the penile carcinoma cases were positive for

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hrHPV-DNA; 42 (79%) contained HPV16, 4 (8%) HPV33, 3 (6%) HPV18, 2 (4%) HPV45, 1 (2%)

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HPV31 and 1 (2%) HPV52.

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10 Clinicopathological characteristics

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Clinicopathological variables of patients stratified by tumor HPV status are presented in

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Table 1. HrHPV-positive patients tended to have smaller tumors than hrHPV-negative

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patients. In hrHPV-negative patients, 45% of tumors was well differentiated compared to

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17% of tumors in hrHPV-positive patients. Regarding tumor subtypes, no differences in the

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distribution of SCC vs. other subtypes were found between HPV-negative and -positive

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cases (Table 1). Though based on small numbers, warty and subtypes with basaloid

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features were observed more frequently amongst HPV-positive cancers, reaching

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significance for the latter group only (p=0.08 and p=0.010, respectively).

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No differences in age or other clinical and pathological characteristics were observed

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between the hrHPV-positive and -negative groups. Median follow-up was 5.2 years

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(interquartile range 4.7-6.1).

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Patient survival

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Five-year DSS rates according to clinicopathological variables were calculated using Kaplan-

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Meier and log rank test. The status of the lymph nodes (p<0.0001), pT-stage (p<0.0001),

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lymphovascular invasion (LVI, p=0.0014), grade of differentiation (p=0.0022), and hrHPV

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status (p=0.0160) were prognostic for DSS. As shown in figure 1, 5-year DSS of hrHPV-

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positive patients (n=53) vs. hrHPV-negative (n=159) patients was 96% vs. 82%, respectively.

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No differences in survival were found based on age (at cut off 60 years) or tumor diameter.

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In a Cox proportional hazard model, hrHPV (HR 0.2, 95%CI: 0.1-0.9, p=0.034) was still a

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significant prognostic factor for DSS, after adjustment for lymph nodes status, pT-stage

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group, grade of differentiation, LVI and age (table 2).

4 Discussion

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In the current study, we showed that the proportion of hrHPV-related invasive penile

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cancers in the Netherlands Cancer Institute was 25% in the last decade, with HPV16 being

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the predominant viral type accounting for 79% of all hrHPV infections. Furthermore, we

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demonstrated that hrHPV-positive tumors appear to provide a significant survival benefit

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over hrHPV-negative tumors in multivariable analysis. Our findings are consistent with

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previous observations in Dutch cohorts of penile cancer. Comparing current data to that of a

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former series of our institute (years 1963-2001) which was evaluated during the course of

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previous studies8 using the same method for hrHPV-DNA detection, we found no evidence of

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an increase in the prevalence of HPV in penile cancer over the past decades (i.e., 29%

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(50/171) in 1963-2001 vs. 25% in 2001-2009). Furthermore, both in the contemporary and

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former cohorts, HPV16 was the most predominant type and hrHPV-positive tumors showed

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a better prognosis.

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The proportion of HPV-related penile cancers has been reported to differ among several

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areas in the world. In two reviews concerning HPV prevalence and invasive penile cancer,

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Europe and South-America were found to have lower HPV-related penile cancer numbers

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than North-America and Asia6,21. This may explain the observed lower hrHPV prevalence of

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25-29% in the Dutch population than the reported prevalence in most other studies1,5,6. A

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recent, Swedish study reported an HPV prevalence of 82.9% in penile cancer (n=216), with

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the majority being hrHPV-positive and with multiple HPV infections in almost 30% of HPV-

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positive cases22. No associations with histopathological parameters were observed and

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association with patient survival was not studied. The hrHPV prevalence in that study is

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considerably higher than the prevalence described here. This large difference in hrHPV

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prevalence might depend on methodological differences for assessing HPV status, or

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differences among the investigated populations. In the Swedish study almost 30% of cases

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was carcinoma in situ, while we included only invasive tumors. It is known from vulvar

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cancer that carcinoma in situ lesions are more often HPV-positive than invasive lesions23.

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ACCEPTED MANUSCRIPT The high prevalence of HPV16 in our study, is consistent with all other penile cancer studies,

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except for two conducted in Argentina and Thailand21. Of interest, HPV16 is also the most

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common type detected in other HPV-associated anogenital cancers, as well as in HPV-

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associated oropharyngeal cancers24–26.

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Recent studies on head and neck squamous cell cancers have shown a rising incidence of

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hrHPV-associated subtypes over the past decades in Europe and North America15–17. Also in

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penile cancer, a recent study was performed, where HPV DNA was suggested to be more

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frequently detected in more recently diagnosed cases27; being 68% (46/68) in the period

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2004–2005 vs. 36% (4/11) in 1998–2003 (p = 0.05). Given the limited numbers in early time

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period, these findings however provide only very limited evidence of a more prominent and,

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possibly, increasing role of HPV infection in penile carcinogenesis over time. In the current

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study, we observed no signs of an increase in HPV prevalence in penile carcinoma over time,

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comprising an extensive period from 1963 till 2009. Due to low numbers in the early years

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(before 1980, n=18), it is hard to make an accurate estimation in those years, but also

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between 1980-2009 no clear increase in HPV prevalence was observed. So far, no other

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studies are available on time trends in HPV prevalence in other HPV-related anogenital

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tumors, such as anal, vaginal and vulvar cancer. In vulvar cancer however, incidence is rising

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among young women25, suggesting a possible causative role of HPV, similar to

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oropharyngeal cancer.

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In most other HPV related tumors, a difference in age distribution is observed with HPV-

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positive tumors more often in younger patients. In our penile cancer population – both the

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contemporary cohort as the population before 2001 – this was not observed. Our study

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cohort does not include the entire Dutch penile cancer population, but over 60% of all penile

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cancer patients are annually treated at our institute28. It is known that basaloid and warty

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subtypes are more likely to be HPV positive than other subtypes6. Though based on small

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numbers, we confirm a higher prevalence of basaloid subtypes in HPV-positive patients,

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but for warty subtypes a non-significant increase was observed. Furthermore, significantly

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less well differentiated tumors were observed among the HPV-positive tumors. This is in

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line with some previous studies where virus DNA was also more often associated with

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high-grade tumors29. The better prognosis of hrHPV-positive tumors in our cohort is in line

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with data on head and neck cancer, where HPV is known to be a good prognostic factor for

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survival and disease progression24. The survival benefit in the current study was seen over all

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ACCEPTED MANUSCRIPT time periods, and confirm our previous analysis8. Treatment differences are probably not

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the explanation for this survival benefit, as there was no significant difference in primary

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treatment between HPV-negative and HPV-positive patients. Remarkably, the survival

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benefit was demonstrated without any association between HPV and lymph node status,

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and significantly less well differentiated tumors among HPV positive patients. In other

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penile cancer studies concerning prognosis and HPV status, no differences in survival among

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HPV positive patients were observed12,13,27. Also in vulvar cancer studies concerning HPV

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infection and prognosis, data are contradictive25. However, in most of these studies limited

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numbers of patients were included, which may explain the non-discriminative role of HPV.

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This study is not devoid of limitations. Firstly, by using FFPE material, it may be that the

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proportion of HPV positivity is underestimated5. Furthermore, we only used tissue from

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patients not pre-treated with chemotherapy. Therefore it is possible that the proportion of

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patients with more advanced tumors is underrepresented in this study.

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Our results underscore previous observations in penile cancer. hrHPV infection is associated

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with a subset of invasive penile tumors (25%). The presence of hrHPV confers a survival

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advantage in multivariable analysis. No evidence of an increase in the prevalence of HPV in

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penile cancer over the past decades was observed.

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20 Acknowledgement

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The authors would like to thank the people from the Molecular Pathology & Biobank Core

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Facility from the Netherlands Cancer Institute for all their administrative and technical

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support.

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References

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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. 2003; 201: 109–18.

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Heideman DAM, Waterboer T, Pawlita M, et al: Human papillomavirus-16 is the predominant type etiologically involved in penile squamous cell carcinoma. J. Clin. Oncol. 2007; 25: 4550–6.

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Flaherty A, Kim T, Giuliano A, et al: Implications for human papillomavirus in penile cancer. Urol. Oncol. 2013: 1–8.

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Bezerra AL, Lopes A, Santiago GH, et al: Human papillomavirus as a prognostic factor in carcinoma of the penis: analysis of 82 patients treated with amputation and bilateral lymphadenectomy. Cancer 2001; 91: 2315–21.

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Lopes A, Bezerra ALR, Pinto CAL, et al: P53 As a New Prognostic Factor for Lymph Node Metastasis in Penile Carcinoma: Analysis of 82 Patients Treated With Amputation and Bilateral Lymphadenectomy. J. Urol. 2002; 168: 81–6.

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Miralles-Guri C, Bruni L, Cubilla AL, et al: Human papillomavirus prevalence and type distribution in penile carcinoma. J. Clin. Pathol. 2009; 62: 870–8.

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Kirrander P, Kolaric A, Helenius G, et al: Human papillomavirus prevalence, distribution and correlation to histopathological parameters in a large Swedish cohort of men with penile carcinoma. BJU Int. 2011; 108: 355–9.

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Smith JS, Backes DM, Hoots BE, et al: Human papillomavirus type-distribution in vulvar and vaginal cancers and their associated precursors. Obstet. Gynecol. 2009; 113: 917– 24.

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Hoots BE, Palefsky JM, Pimenta JM, et al: Human papillomavirus type distribution in anal cancer and anal intraepithelial lesions. Int. J. Cancer 2009; 124: 2375–83.

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Legends

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Table 1: Patient characteristics a

Wilcoxon rank sum test with continuity correction Linear-by-linear Association test c Fisher’s exact test b

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Table 2: Multivariable Cox proportional hazards model for disease-specific survival

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Figure 1: Disease-specific survival after primary tumor treatment; hrHPV-positive tumors (n=53) appear to have a significant survival benefit (96%) over hrHPV-negative tumors (n=159, 82%, log rank test p=0.016)

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ACCEPTED MANUSCRIPT Table 1: Patient characteristics hrHPV negative hrHPV positive

Total

n=159

n=53

N=212

67 (58-74)

63 (54-71)

63 (54-71)

pT1a (%)

38 (24)

14 (26)

52 (25)

pT1b (%)

5 (3)

5 (9)

10 (5)

pT2 (%)

99 (62)

29 (55)

128 (60)

pT3 (%)

11 (7)

5 (9)

16 (8)

pT4 (%)

6 (4)

0 (0)

p-value

Age Median (IQR)

Median (IQR)

3 (2-4.1)

Subtype

6 (3)

2.5 (1.5-3.5)

3 (2-4)

44 (83)

183 (86)

0 (0)

5 (2)

3 (6)

5 (2)

139 (87)

Verruceus (%)

5 (3)

Warty (%)

2 (1)

Basaloid (%)

1 (1)

4 (8)

5 (2)

Basaloid-SCC (%)

1 (1)

1 (2)

2 (1)

Papillar (%)

8 (5)

1 (2)

9 (4)

Cuniculatum (%)

1 (1)

0 (0)

1 (0)

Pseudohyperplastic (%)

1 (1)

0 (0)

2 (1)

Sarcomatoid (%)

1 (1)

0 (0)

1 (0)

71 (45)

9 (17)

80 (38)

Intermediate (%)

61 (38)

32 (60)

93 (44)

Poorly (%)

27 (17)

12 (23)

39 (18)

No (%)

135 (86)

44 (83)

179 (85)

Yes (%)

22 (14)

9 (17)

31 (15)

Missing (%)

2

0

2

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SCC (%)

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Tumor size (cm)

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Pathological T-stage

0.23a

0.025a

0.49c*

Grade of differentiation Well (%)

0.004b

LVI

0.66c

ACCEPTED MANUSCRIPT Lymph nodes Tumor negative (%)

107 (67)

39 (74)

146 (69)

Tumor positive (%)

52 (33)

14 (26)

66 (31)

0.49c

Penis preserving

72 (45)

26 (49)

98 (46)

0.64c

(partial) amputation

87 (55)

27 (51)

114 (54)

RI PT

Penile treatment

IQR = interquartile range SCC = squamous cell carcinoma

a

c

Fisher’s exact test *SCC vs. other

AC C

EP

TE D

M AN U

Wilcoxon rank sum test with continuity correction b Linear-by-linear Association test

SC

Crude percentages are presented

ACCEPTED MANUSCRIPT Table 2: Multivariable Cox proportional hazards model for disease-specific survival p value

1 0.2

0.1 – 0.9

0.034*

1 28.1

6.5 – 121.8

<0.001*

1 0.6 2.5

0.2 – 1.7 0.8 – 7.8

1 2.8

1.3 – 6.0

1 1.7

0.7 – 3.9

EP AC C

0.340 0.110

1.0-1.1

0.010*

SC

M AN U

1.0

RI PT

(95% CI)

TE D

hrHPV Negative Positive Lymph node status Negative Positive pT stage group pT1a-b pT2 pT3-4 Grade of differentiation Well / intermediate Poorly LVI No Yes Age in years *statistically significant

Hazard ratio (HR)

0.250 0.290

ACCEPTED MANUSCRIPT

1.0

RI PT SC M AN U

0.6

HR−HPV− HR−HPV+

EP

TE D

0.4

0.2

AC C

Disease specific survival probability

0.8

0.0 159 53

142 52

129 50

121 48

99 46

66 HR−HPV− 31 HR−HPV+

0

1

2

3

4

5

Time (years)

ACCEPTED MANUSCRIPT Key of abbreviations

DSS: disease-specific survival ENE: Extranodal extension

RI PT

FFPE: formalin-fixed paraffin-embedded IQR: Interquartile range HPV: Human Papillomavirus

SC

hrHPV: high-risk Human Papillomavirus LVI: Lymphovascular invasion

AC C

EP

TE D

M AN U

SCC: squamous cell carcinoma