Risk Factors for Squamous Cell Carcinoma of the Penis— Population-Based Case-Control Study in Denmark

Home , Penile cancer, Phimosis, Priapism

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Birgitte Schu¨tt Madsen,1 Adriaan J.C. van den Brule,2 Helle Lone Jensen,3 Jan Wohlfahrt,1 and Morten Frisch1 1Department of Epidemiology Research, Statens Serum Institut, Artillerivej 5, Copenhagen, Denmark; 2Department of Pathology, VU Medical Center, Amsterdam and Laboratory for Pathology and Medical Microbiology, PAMM Laboratories, Michelangelolaan 2, 5623 EJ Eindhoven, the Netherlands;and 3Department of Pathology, Gentofte University Hospital, Niels Andersens Vej 65, Hellerup, Denmark

Abstract

Few etiologic studies of squamous cell carcinoma female sex partners, number of female sex partners (SCC) of the penis have been carried out in populations before age 20, age at first intercourse, penile-oral sex, a where childhood circumcision is rare. A total of 71 history of anogenital warts, and never having used patients with invasive (n = 53) or in situ (n = 18) penile condoms. Histories of phimosis and priapism at least 5 SCC, 86 prostate cancer controls, and 103 population years before diagnosis were also significant risk controls were interviewed in a population-based case- factors, whereas alcohol abstinence was associated control study in Denmark. For 37 penile SCC patients, with reduced risk. Our study confirms sexually tissue samples were PCR examined for human papil- transmitted HPV16 infection and phimosis as major lomavirus (HPV) DNA. Overall, 65% of PCR-examined risk factors for penile SCC and suggests that penile- penile SCCs were high-risk HPV-positive, most of oral sex may be an important means of viral transmis- which (22 of 24; 92%) were due to HPV16. Penile SCC sion. The association with priapism was unexpected risk was positively associated with measures of early and needs replication. (Cancer Epidemiol Biomarkers and high sexual activity, including lifetime number of Prev 2008;17(10):2683–91)

Introduction

In the Western world, squamous cell carcinoma (SCC) of case-control study in Denmark, a country with a largely the penis is a rare malignancy occurring mainly among uncircumcised male population (19) to further address elderly men above age 60 years (1, 2) and with a the etiology of penile SCC. standardized annual incidence of <2 per 100,000 men (1- 4). In some developing countries, penile SCC is more common and affects a somewhat wider age range (5-9). Material and Methods The etiology of penile SCC remains incompletely understood (7, 10). Studies have consistently reported Participants. Three study groups were identified, neonatal or childhood circumcision to be associated with including case patients diagnosed with invasive or reduced risk (1, 4, 6, 10-12), which corresponds geo- in situ penile SCC between 1993 and 1998 and two graphically with reduced rates of penile SCC in frequency-matched control groups consisting of male populations practicing neonatal circumcision (13). The population controls and patients diagnosed with adeno- protective effect of childhood circumcision, but not of carcinoma of the prostate in the same time period as the circumcision in adulthood (4, 11), seems to be attribut- penile SCC patients. Information about newly diagnosed able to the elimination of inflammatory conditions cases of penile SCC and prostate cancer was obtained related to poor genital hygiene (2, 8-10), such as phimosis every third month from the files of the Danish Cancer (3, 4, 14-16) and balanitis (14). Accordingly, an intact Registry and the Danish Pathology Registry, as well as foreskin has been shown not to be associated with through manual searches in the files of pathology increased penile SCC risk in the absence of phimosis (4). departments across Denmark. Overall, we identified Other previously suggested risk factors include infection 259 men with penile SCC (215 invasive and 44 in situ with high-risk types of sexually transmitted human cases) diagnosed between 1993 and 1998. Before inviting papillomaviruses (hrHPV;refs. 4, 11, 17) and current these men to participate in the study, we contacted the tobacco smoking (4, 11, 18). We undertook a nationwide hospital department or the private practitioner responsible for their treatment to ensure that each eligible participant had been fully informed about his diagnosis, that he had no other health-related reason for nonpartic- Received 5/19/08;revised 7/14/08;accepted 7/18/08. ipation known to the doctor (e.g., psychosis, hearing Grant support: Danish Cancer Society (grant no. 96-100-17), Dagmar Marshall’s Foundation, Manufacturer Einar Willumsen’s Memorial Foundation and the Danish impairment), and that he was a Danish-speaking person. Medical Research Council. Of the original 259 invasive or in situ penile SCC patients, Requests for reprints: Birgitte Schu¨tt Madsen, MSc, Department of Epidemiology 88 patients (34%) had died, for 47 patients (18%), we Research, Statens Serum Institut, Artillerivej 5, Dk-2300 Copenhagen S, Denmark. Phone: 45-32683951;Fax: 45-32683165. E-mail: [email protected]. received no reply from the hospital department or Copyright D 2008 American Association for Cancer Research. private practitioner responsible for the patient’s treat- doi:10.1158/1055-9965.EPI-08-0456 ment, and 3 patients (1%) were inaccessible due to

Cancer Epidemiol Biomarkers Prev 2008;17(10). October 2008

emigration. Thus, we invited 121 (47%) penile SCC HPV-positive samples were subsequently analyzed for patients who had been informed about their cancer (or their HPV types using the reverse line blotting method. carcinoma in situ) and were alive at the time we aimed to The following hrHPV types were detected and identified approach them between 1997 and 2000. Prostate cancer as follows: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, controls were frequency matched to penile SCC patients 66, and 68;low-risk HPVs detected were HPV 6, 11, 26, on year of diagnosis and birth year (F5 y). From the Civil 34, 40, 42, 43, 44, 53, 54, 55, 57, 61, 70, 71 (CP8061), 72, 73, Registration System, a nationwide demographic database 81 (CP8304), 82/MM4, 82/IS39, 83 (MM7), 84 (MM8), (20), we obtained a list of all Danish men born on two and CP6108. specific dates for each year between 1911 and 1963. As Statistical Analysis. To ensure comparable exposure patients with penile cancer were invited to participate in ascertainment periods in all 3 groups of study partic- the study, this list was used to select possible population ipants, we assigned a pseudo-year of diagnosis between controls, by means of frequency matching to invited 1993 and 1998 to population controls according to the penile SCC patients on year of birth. We anticipated distribution of year of diagnosis among penile SCC somewhat lower participation rates among population patients. Specifically, the distribution of pseudo-year of controls, so to obtain the intended case-cancer control diagnosis among population controls was similar to the and case-population control ratios of 1:1, we invited distribution of year of diagnosis among penile SCC cases more population controls than patients with penile (i.e., similar proportions of population controls having cancer. Potential participants (121 penile SCC cases, 136 pseudo-year of diagnosis in 1993, 1994, 1995, 1996, 1997, prostate cancer controls, and 172 population controls) received a letter of invitation with information about the and 1998 as penile SCC patients with year of diagnosis in study and were asked to sign and return a consent form 1993, 1994, 1995, 1996, 1997, and 1998, respectively). We in a prepaid envelope to participate in the study. considered only those exposures that preceded the All consenting participants underwent a structured pseudo-year of diagnosis to be relevant in population telephone interview between March 1997 and April 2000. controls, similar to the situation in penile SCC cases and prostate cancer controls, for whom only exposures that The interviews, conducted according to written guide- preceded the year of cancer diagnosis counted as lines by six trained female medical students who were exposures of potential etiologic relevance. unaware of the study hypotheses and who were kept blinded to the case or control status of the participants, Univariate Analyses. Because most risk factors are covered a broad range of topics including basic school similar for both diagnostic entities (4), we considered as attendance and postschool education, weight and height, our case group all patients with invasive or in situ penile tobacco and alcohol consumption, issues related to SCC. Initially, we calculated univariate odds ratios (OR) general and genital health, fertility, and details about separately for penile SCC patients versus each of the two sexual experiences and hygiene as well as venereal control groups, adjusting only for age at diagnosis in 10-y history. Patients with prostate cancer were chosen as a age groups (< 40, 40-49,..., z70 y). ORs obtained with supplementary control group because most examined either of the two control groups were generally similar, risk factors for penile SCC are not suspected to be risk so we subsequently tested the appropriateness of factors for prostate cancer. Consequently, information combining the two control groups by doing a logistic provided by these control subjects was anticipated to be regression analysis restricted to prostate cancer controls comparable with the information obtained from popula- and population controls, with adjustment for age in 10-y tion controls. Moreover, we anticipated the reliability of age groups. This analysis revealed that none of the answers to be high among prostate cancer controls examined explanatory variables were associated with becausethesemenwereexpectedtobesimilarly statistically significantly increased risk of prostate cancer, motivated as case patients to provide honest answers so to reduce complexity and gain statistical power, we with respect to sexual and venereal history, genital combined the two control groups in all subsequent hygiene, and other sensitive matters. Finally, possible analyses. Logistic regression analyses were done to recall problems were expected to apply equally to penile calculate exposure ORs for penile SCC versus the SCC patients and prostate cancer controls because both combined group of control subjects, again with adjust- cancers originated in the male genitalia. ment only for age at diagnosis in 10-y age groups. The results of these age-adjusted analyses are called univar- HPV Status. For 37 (52%) of the 71 penile SCC cases iate ORs. Likelihood ratio tests for homogeneity deter- who participated in the study, we identified paraffin- mined possible differences in risk between exposure embedded archival tumor specimens in the relevant categories. For categorized continuous variables, we also pathology department. These tumor tissues were exam- did likelihood ratio tests to determine the appropriate- ined for HPV DNA by the PCR technique using general ness of reducing the observed associations to a linear GP5+/6+ primers (GP5+/6+ PCR-EIA assay) as de- specification. Trend tests were done only when the scribed in detail elsewhere (21, 22). In brief, paraffin- preceding likelihood ratio test for linearity yielded a embedded tissues were cut for PCR analyses;outer reasonable fit (P > 0.05). In tests for trend, categorized sandwich sections were H&E stained and analyzed for continuous variables were treated as continuous varia- the presence of tumor cells, whereas inner sections were bles, using the median for each category as the category used for DNA extraction and subsequent PCR analyses. value. All DNAs were tested for B-globin PCR positivity after agarose gel analysis to check for the quality of DNA for Multivariate Analyses. All explanatory variables with a PCR purposes (23). HPV was detected using the GP5+/ P value of <0.10 in the test for homogeneity in the 6+ PCR-EIA using cocktail probes for 14 hrHPV types univariate analysis were entered simultaneously in a and 23 low-risk HPV types as previously described (22). logistic regression model along with age in 10-y age

Cancer Epidemiol Biomarkers Prev 2008;17(10). October 2008

groups. Using backward elimination to remove the least 26.7% of invited population controls), illness or death significant variables one by one until all remaining (8.3%, 13.2%, and 7.0%, respectively), or lack of telephone variables in the model were statistically significant (10.7%, 8.1%, and 6.4%, respectively). (P value in test for homogeneity < 0.05), we arrived at Characteristics of the Study Population. Men who a multivariate model. Subsequently, excluded explana- agreed to participate in the study were slightly younger tory variables were given an extra chance to enter the than those who declined the invitation to participate. The final model by means of forward inclusion, but none of median age at cancer diagnosis was 62 years among the previously excluded variables reached statistical participating penile SCC patients (64 years among 53 significance. Tests for homogeneity and tests for trend patients with invasive SCC, 57 years among 18 patients were done as for the univariate analyses. Comparisons 2 with SCC in situ), and 68 years among prostate cancer of proportions were made using the m test or, when controls, whereas the corresponding median age at the numbers were small (<5 in any one cell), by means of pseudo-year of diagnosis was 62 years among population Fisher’s exact test. Throughout, two-sided P values of controls. Corresponding median ages among 50 penile <0.05 and 95% confidence intervals (CI) excluding unity SCC cases, 50 prostate cancer controls, and 69 population were considered indicators of statistical significance. All controls who declined the invitation to participate in the logistic regression analyses were carried out in Statis- study were, respectively, 4, 2, and 5 years older. Of the 37 tical Analysis System software Version 9.1 (SAS penile SCC patients whose tumor tissues were PCR Institute). examined for the presence of HPV DNA, 24 (65%) were Ethics. The study was approved by The Danish hrHPV positive, and 1 (3%) was positive to a low-risk National Committee on Biomedical Research Ethics, HPV type (HPV6;Table 1). By far, the predominant HPV approval no. C-1995-20, and The Danish Data Protection type was HPV16, which was detected in 22 (59.5%) of the Agency, approval no. 1995-1200-187. Delayed reporting 37 examined tumors, corresponding to 92% of the 24 was due to shortage of funding. hrHPV-positive tumors. All 9 examined lesions from patients with penile SCC in situ were hrHPV positive Results versus 15 (53.6%) of 28 lesions from patients with invasive penile SCC (Fisher’s exact test; P = 0.02, two Participation Rate. Of the 121 patients with invasive sided). Penile SCC lesions involving the preputium were or in situ penile SCC, 136 prostate cancer controls and 172 slightly more likely to be hrHPV positive (71% of 14) than male population controls who were invited to participate lesions of the penile glans (61% of 23) and lesions of in the study, 71 penile SCC cases (58.7%), 86 prostate unspecified penile location (60% of 5), but these differ- cancer controls (63.2%), and 103 population controls ences were not statistically significant (X2 = 0.47;degrees (59.9%) agreed to participate in the telephone interview. of freedom = 2; P = 0.79). A series of 12 tumor specimens The most common reasons for nonparticipation were from controls with prostate cancer subjected to the same unspecified unwillingness (22.3% of invited penile SCC PCR analysis as the penile SCC cases were consistently cases, 15.4% of invited prostate cancer controls, and negative for all examined HPV types (Table 1).

Table 1. Characteristics of 260 participants in case-control study of risk factors for invasive or in situ SCC of the penis, Denmark

Penile SCC (n = 71)* Prostate cancer controls (n = 86) Population controls (n = 103) Age at diagnosis (y) <40 y 4 (6) 0 (0) 3 (3) 40-49 9 (13) 1 (1) 11 (11) 50-59 18 (25) 21 (24) 31 (30) 60-69 18 (25) 22 (26) 28 (27) z 70 22 (31) 42 (49)c 30 (29) HPV status (n = 37) (n = 12) (n =0) HPV positiveb 25 (68) 0 (0) NA HrHPV positive 24 (65) 0 (0) NA x LrHPV positive 1 (3) 0 (0) NA HPV negative 12 (32) 12 (100) NA k Fisher’s exact test P < 0.001 { Anatomic localization Penile glans 34 (44) NA NA Penile preputium 21 (28) NA NA Unspecified** 21 (28) NA NA

NOTE: Percentages may not sum to 100 because of rounding. Abbreviations: lrHPV, low-risk human papillomavirus;NA, not applicable. *Penile SCC cases include 53 patients with invasive penile SCC and 18 patients with in situ penile SCC. cNone of 12 examined tumor tissues from controls with prostate cancer were positive for any of the examined HPV types. bThe 24 hrHPV-positive penile SCCs were positive for the following specific HPV types: HPV 16 (n = 22), HPV 31 (n = 1), and HPV 33 (n = 1). xThe lrHPV-positive penile SCC patient was positive for HPV 6. kFischer’s exact test for the comparison of tumor HPV positivity in penile SCC cases (25 positive of 37) vs prostate cancer controls (0 positive of 12). { One penile SCC had tumor involvement of glans, preputium, and corpus, and three had tumor involvement of glans and preputium. **Includes one patient with SCC of the penile corpus.

Cancer Epidemiol Biomarkers Prev 2008;17(10). October 2008

Table 2. Sexual and venereal risk factors for invasive or in situ SCC of the penis

No of subjects Univariate OR Multivariate OR (%) (95% CI)* (95% CI)*

Penile Prostate Population Penile Penile SCC cancer controls controls SCC vs controls SCC vs controls (n = 71) (n = 86) (n = 103) c Lifetime no. female sexual partners 0-1 4 (6) 16 (19) 16 (16) 0.20 (0.06-0.63) 0.32 (0.09-1.18) 2-4 18 (25) 29 (35) 28 (28) 0.50 (0.25-0.99) 0.41 (0.17-1.00) 5-9 8 (11) 12 (14) 19 (19) 0.43 (0.18-1.06) 0.32 (0.11-0.94) z10 41 (58) 26 (31) 37 (37) 1 (reference) 1 (reference) Test for homogeneity P = 0.009 P = 0.06 Test for trend P = 0.002 P = 0.01 No of female sexual partners before age 20 y 0 19 (28) 50 (60) 39 (40) 1 (reference) 1 (reference) 1 5 (7) 13 (16) 14 (14) 0.91 (0.31-2.68) 0.79 (0.23-2.73) 2-3 19 (28) 12 (14) 21 (22) 2.87 (1.31-6.29) 4.05 (1.44-11.4) z4 26 (38) 8 (10) 23 (24) 3.74 (1.69-8.28) 5.11 (1.89-13.8) Test for homogeneity P = 0.002 P < 0.001 Test for trend c P < 0.001 P < 0.001 Age at first sexual intercourse (y) V16 26 (37) 11 (13) 27 (28) 2.67 (1.26-5.67) 2.82 (1.09-7.32) 17-19 26 (37) 25 (30) 37 (38) 1.78 (0.89-3.54) 2.35 (1.01-5.47) z20 19 (27) 47 (57) 34 (35) 1 (reference) 1 (reference) Test for homogeneity P = 0.03 P = 0.05 Test for trend P = 0.009 P = 0.01 No of female partners for anal intercourse 0 58 (83) 75 (90) 89 (91) 1 (reference) 1 (reference) 1 4 (6) 6 (7) 6 (6) 0.87 (0.26-2.86) 1.09 (0.23-5.10) z2 8 (11) 2 (2) 3 (3) 3.72 (0.95-14.6) 2.05 (0.34-12.3) Test for homogeneity P = 0.15 P = 0.74 Test for trend P = 0.09 P = 0.45 No of female partners performing oral sex 0 21 (30) 51 (62) 51 (52) 1 (reference) 1 (reference) 1-2 23 (33) 24 (29) 28 (29) 2.41 (1.17-4.96) 1.72 (0.68-4.37) z3 25 (36) 7 (9) 19 (19) 5.47 (2.25-13.3) 3.65 (1.14-11.7) Test for homogeneity P < 0.001 P = 0.09 Test for trend P < 0.001 P = 0.04 No of visits to prostitutes 0 52 (75) 65 (80) 79 (82) 1 (reference) 1 (reference) 1 6 (9) 4 (5) 6 (6) 1.48 (0.50-4.42) 0.29 (0.05-1.63) z2 11 (16) 12 (15) 11 (11) 1.34 (0.60-2.96) 0.63 (0.20-2.00) Test for homogeneity P = 0.64 P = 0.29 Test for trend P = 0.45 P = 0.38 No of male sexual partners 0 67 (97) 80 (98) 99 (99) 1 (reference) 1 (reference) z1 2 (3) 2 (2) 1 (1) 1.66 (0.26-10.6) 0.12 (0.01-2.88) Genital washing before and after intercourse Never (0 times of 10) 4 (6) 7 (9) 9 (10) 0.55 (0.16-1.83) 0.83 (0.18-3.74) Sometimes (1-5 times of 10) 26 (38) 23 (29) 28 (31) 1.05 (0.53-2.06) 0.61 (0.24-1.54) Often (6-9 times of 10) 27 (39) 30 (38) 25 (27) 1 (reference) 1 (reference) Allways (10 times of 10) 12 (17) 19 (24) 29 (32) 0.48 (0.21-1.10) 0.41 (0.14-1.20) Test for homogeneity P = 0.18 P = 0.40 Test for trend b P = 0.52 P = 0.68 Venereal diseases Anogenital warts 23 (32) 5 (6) 7 (7) 6.34 (2.87-14.0) 7.01 (2.48-19.8) Chlamydia, syphilis or gonorrhea 13 (18) 13 (15) 13 (13) 1.37 (0.65-2.88) 0.76 (0.29-2.02) Genital herpes 3 (4) 1 (1) 2 (2) 2.79 (0.53-14.6) 3.76 (0.53-26.4) Crab lice 19 (27) 20 (24) 21 (21) 1.37 (0.72-2.61) 0.48 (0.18-1.26) Penile candidiasis 5 (7) 2 (2) 2 (2) 2.17 (0.51-9.29) 1.64 (0.22-12.3) Labial herpes 29 (41) 32 (37) 41 (40) 1.18 (0.67-2.10) 0.64 (0.30-1.37) HIV test (ever) 11 (15) 3 (4) 7 (7) 2.76 (1.06-7.23) 1.99 (0.55-7.21) Condom use (never) 22 (31) 22 (26) 17 (17) 1.81 (0.97-3.40) 2.45 (1.04-5.80) Marital status Unmarried without current female partner 1 (1) 4 (5) 9 (9) 0.19 (0.02-1.57) 0.07 (0.00-1.71) Unmarried with current female partner 3 (4) 1 (1) 1 (1) 2.85 (0.39-20.6) 5.62 (0.60-52.5) Married 47 (66) 58 (68) 76 (75) 1 (reference) 1 (reference) Widowed 7 (10) 12 (14) 8 (8) 1.13 (0.43-3.01) 0.56 (0.15-2.11) Separated/divorced 13 (18) 10 (12) 8 (8) 1.96 (0.87-4.39) 1.36 (0.45-4.15) Test for homogeneity P = 0.09 P = 0.09

(Continued on the following page)

Cancer Epidemiol Biomarkers Prev 2008;17(10). October 2008

Table 2. Sexual and venereal risk factors for invasive or in situ SCC of the penis

No of subjects Univariate OR Multivariate OR (%) (95% CI)* (95% CI)*

Penile Prostate Population Penile Penile SCC cancer controls controls SCC vs controls SCC vs controls (n = 71) (n = 86) (n = 103) Partner’s lifetime no. of other sexual partners 0 11 (17) 21 (28) 27 (30) 0.58 (0.25-1.37) 0.40 (0.13-1.27) 1-2 20 (31) 25 (33) 24 (26) 1 (reference) 1 (reference) z3 27 (42) 16 (21) 23 (25) 1.51 (0.71-3.20) 0.74 (0.28-1.91) No current female partner 7 (11) 13 (17) 17 (19) 0.57 (0.21-1.52) 0.38 (0.10-1.46) x Test for homogeneity P = 0.11 P = 0.28 Test for trend P = 0.05 P = 0.86 k History of venereal disease in female partner Anogenital warts 3 (5) 1 (1) 2 (2) 2.47 (0.46-13.2) 0.19 (0.01-2.52) Chlamydia, syphilis or gonorrhea 2 (3) 0 (0) 4 (4) 1.09 (0.18-6.46) 0.44 (0.04-4.63)

NOTE: Percentages are calculated based on respondents with nonmissing values. *Cases vs all controls. All univariate ORs are adjusted for age at the time of diagnosis. Multivariate ORs are adjusted for age at the time of diagnosis, number of female sexual partners before age 20 y, anogenital warts, condom use, phimosis, priapism, and cumulative alcohol consumption. cMultivariate ORs not adjusted for number of female partners before age 20 y. bNumbers and percentages of respondents who reported that they had had the disease in question (or who had ever been HIV tested or who had never used condom). In the calculation of ORs for these variables, respondents reporting no such disease (or those who had never been HIV tested or who had ever used condom) served as the reference group. xTrend and homogeneity tests are restricted to men reporting their current female partner’s lifetime number of other sexual partners. kNumbers and percentages of respondents who reported that their female partner had had the disease in question. In the calculation of ORs for these variables, respondents reporting no such disease in their female partner served as the reference group.

Univariate Risk Factor Analyses sexual intercourse (Ptrend = 0.009), number of female Sexual and Venereal Risk Factors. Measures of early and partners performing oral sex (Ptrend < 0.001), and female high sexual activity were positively associated with partner’s lifetime number of other sexual partners (Ptrend penile SCC risk (Table 2). Statistically significant trends = 0.05). A history of anogenital warts (OR, 6.34;95% CI, were seen for lifetime number of female sexual partners 2.87-14.0) and ever having been HIV tested (OR, 2.76; (Ptrend = 0.002), notably the number of female sexual 95% CI, 1.06-7.23) were also significantly linked with partners before age 20 years (Ptrend < 0.001), age at first increased penile SCC risk (Table 2).

Table 3. Penile conditions and risk of invasive or in situ SCC of the penis

No of subjects (%) Univariate OR Multivariate OR (95% CI)* (95% CI)*

Penile SCC Prostate Population Penile SCC Penile SCC (n = 71) cancer controls controls vs controls vs controls (n = 86) (n = 103) c Childhood circumcision No 71 (100) 85 (99) 99 (96) 1 (reference) 1 (reference) Yes 0 (0) 1 (1) 4 (4) — — Fisher’s exact testb P = 0.33 Penile pathology Phimosis 18 (27) 8 (10) 11 (11) 3.39 (1.62-7.11) 4.91 (1.85-13.0) x Paraphimosis 9 (13) 2 (2) 10 (10) 2.00 (0.79-5.05) 2.40 (0.77-7.53) Balanitis 14 (22) 7 (8) 7 (7) 3.07 (1.36-6.93) 2.37 (0.81-6.92) Priapism 7 (10) 3 (4) 1 (1) 4.90 (1.36-17.7) 9.23 (1.09-78.3) Penile trauma 7 (10) 8 (10) 8 (8) 1.04 (0.40-2.74) 0.80 (0.21-3.01) Glanular erythema >1 d 4 (7) 2 (2) 8 (8) 0.83 (0.24-2.88) 0.87 (0.17-4.42) Itching >1 wk 4 (6) 1 (1) 3 (3) 2.25 (0.52-9.73) 0.65 (0.07-5.63) k Ever noticed smegma under foreskin At age <20 y 25 (61) 46 (61) 51 (57) 1.25 (0.60-2.61) 1.38 (0.54-3.54) At age 20-39 y 12 (31) 25 (32) 41 (44) 0.83 (0.38-1.80) 0.86 (0.32-2.30) At age z40 y 10 (28) 24 (30) 31 (34) 0.80 (0.36-1.81) 0.64 (0.22-1.87)

NOTE: Percentages are calculated based on respondents with nonmissing values. *Cases vs all controls. Univariate ORs are adjusted for age at the time of the diagnosis. Multivariate ORs are adjusted for age at the time of the diagnosis, number of female sexual partners before age 20 y, anogenital warts, condom use, phimosis, priapism, and cumulative alcohol consumption. cChildhood circumcision includes all circumcisions before age 18 y. bPenile pathology at least 5 y before diagnosis. Respondents reporting not to have had the penile pathology in question served as the reference group. xParticipants were considered to have paraphimosis only if they did not also report having phimosis. kRespondents reporting no smegma in the specified age group served as the reference group.

Cancer Epidemiol Biomarkers Prev 2008;17(10). October 2008

Penile Conditions. There was no significant difference dose-response relationship between alcohol consumption in the proportion of participants who had undergone and risk of penile SCC, but individuals reporting no childhood circumcision between patients with penile alcohol consumption were at significantly reduced risk SCC (0 of 71) and controls (5 of 189;Fisher’s exact test, (OR, 0.27;95% CI, 0.07-0.98 for 0 versus >20 consump- P = 0.33, two sided). However, phimosis (OR, 3.39;95% tion-years). School attendance and postschool education CI, 1.62-7.11), balanitis (OR, 3.07;95% CI, 1.36-6.93) and were not associated with risk (Table 4). priapism (OR, 4.90;95% CI, 1.36-17.7) were each Multivariate Risk Factor Analyses significantly associated with risk of penile SCC (Table 3). For each of the 7 penile SCC patients who reported a Sexual and Venereal Risk Factors. Associations with the history of priapism, >10 years had passed from the lifetime number of female partners (Ptrend =0.01), priapic episode to the time of penile SCC diagnosis the number of female partners before age 20 (OR, 5.11; z (mean, interval 27 years;range, 10-62 years). All 4 95% CI, 1.89-13.8;for 4 versus 0 female partners, Ptrend controls reporting priapism were younger than 26 years < 0.001), age at first sexual intercourse (Ptrend = 0.01), (on average, 19 years;range, 16-25 years) at the time they and the number of female partners performing oral sex z experienced priapism, whereas the case patients were on (OR, 3.65;95% CI, 1.14-11.7;for 3 versus 0 oral sex average ages 32 years (range, 15-60 years) at their partners, Ptrend = 0.04) all remained statistically signifi- reported priapic episode. cant in multivariate analysis, as did a history of anogenital warts (OR, 7.01;95% CI, 2.48-19.8;Table 2). Social and Nonsexual Risk Factors. Although smoker Never having used condoms (OR, 2.45;95% CI, 1.04-5.80) status (lifelong nonsmoker, former smoker, or current became statistically significant after adjustment, whereas smoker) was not associated with risk, cumulative tobacco ever having been HIV tested and female partner’s history consumption was dose-dependently associated with of other sexual partners lost significance upon adjust- risk of penile SCC (Ptrend =0.009).Therewasno ment for confounders (Table 2).

Table 4. Social and nonsexual risk factors for invasive or in situ SCC of the penis

No of subjects (%) Univariate OR Multivariate OR (95% CI)* (95% CI)*

Penile SCC Prostate Population Penile SCC Penile SCC (n = 71) cancer controls controls vs controls vs controls (n = 86) (n = 103) Smoker status Lifelong nonsmoker 9 (13) 13 (15) 23 (22) 0.58 (0.24,1.40) 0.78 (0.25-2.43) Former smoker 29 (41) 38 (44) 39 (38) 1 (reference) 1 (reference) Current smoker 33 (46) 35 (41) 41 (40) 1.06 (0.57,1.94) 1.08 (0.50-2.34) Test for homogeneity c P = 0.36 P = 0.85 Cumulative tobacco consumption (pack-years) Lifelong nonsmoker 9 (13) 13 (15) 23 (22) 0.41 (0.17-1.00) 0.59 (0.19-1.84) <12.5 pack-years 5 (7) 9 (11) 17 (17) 0.22 (0.06-0.73) 0.39 (0.09-1.76) 12.5<25 pack-years 16 (23) 20 (24) 20 (19) 0.82 (0.41-1.67) 0.76 (0.29-2.00) z25 pack-years 41 (58) 43 (51) 43 (42) 1 (reference) 1 (reference) Test for homogeneity P = 0.02 P = 0.57 Test for trend b P = 0.009 P = 0.22 Cumulative alcohol consumption (consumption-years) 0 3 (4) 17 (22) 8 (9) 0.27 (0.07-0.98) 0.18 (0.04-0.84) <10 19 (28) 11 (14) 15 (16) 1.56 (0.73-3.34) 2.83 (1.09-7.32) 10-20 13 (19) 13 (17) 26 (28) 0.66 (0.30-1.44) 0.44 (0.16-1.24) >20 34 (49) 37 (47) 44 (47) 1 (reference) 1 (reference) Test for homogeneity P = 0.03 P < 0.001 Test for trend NA NA Years at school (y) <10 53 (75) 61 (72) 76 (74) 1 (reference) 1 (reference) z10 18 (25) 24 (28) 27 (26) 0.75 (0.39-1.46) 0.66 (0.28-1.56) Test for homogeneity P = 0.40 P = 0.33 Postschool education None 19 (27) 27 (32) 35 (34) 0.84 (0.44-1.61) 0.80 (0.35-1.86) Short (V3 y) 38 (54) 44 (52) 52 (50) 1 (reference) 1 (reference) Long (>3 y) 14 (20) 14 (16) 16 (16) 1.29 (0.60-2.74) 1.12 (0.41-3.04) Test for homogeneity P = 0.61 P = 0.82 No of children 0 12 (17) 9 (10) 13 (13) 1.36 (0.62-2.99) 0.97 (0.29-3.22) z1 59 (83) 77 (90) 90 (87) 1 (reference) 1 (reference)

NOTE: Percentages are calculated based on respondents with nonmissing values. Abbreviation: NA, not applicable. *Cases vs all controls. Univariate ORs are adjusted for age at the time of the diagnosis. Multivariate ORs are adjusted for age at the time of the diagnosis, number of female sexual partners before age 20 y, anogenital warts, condom use, phimosis, priapism, and cumulative alcohol consumption. cOne pack-year equals one pack of cigarettes (20 cigarettes) per day for a year. bOne consumption year is equivalent to 2 alcoholic drinks per day for 1 y.

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Penile Conditions. Associations with histories of phi- sexual details, the finding that men with same-sex mosis (OR, 4.91;95% CI, 1.85-13.0) and priapism (OR, experience are not at increased risk of developing penile 9.23;95% CI, 1.09-78.3) became stronger in multivariate SCC accords well with prior reports of no conspicuous analyses, whereas the association with balanitis lost penile SCC risk in gay men (4, 24). statistical significance (OR, 2.37;95% CI, 0.81-6.92). Most Circumcision in childhood is uncommon in Denmark participants had noticed the presence of smegma under with an estimated overall proportion of <2% of boys their foreskin at some point in their life. However, the having undergone the procedure before age 15 years in presence of smegma was not associated with penile SCC recent decades (19). No patient with penile SCC had been risk (Table 3). circumcised in childhood or adolescence versus 5 (2.6%) of 189 controls, a difference that was not statistically Social and Nonsexual Risk Factors. The significant dose- significant. Self-reported phimosis, however, was response relationship with cumulative tobacco consump- reported by 27% of penile SCC patients versus 10% to tion disappeared after adjustment for confounding 11% of controls, thus corroborating the current view that having an unretractable foreskin, notably in combination variables (Ptrend = 0.22;Table 4). Although alcohol abstinence remained inversely associated with the risk with penile hrHPV infection, may constitute the single of penile SCC (OR, 0.18;95% CI, 0.04-0.84;for 0 versus most important risk factor for penile SCC (1). Balano- >20 consumptions-years), there was no dose-response posthitis, inflammation of the penile glans and foreskin, relationship between alcohol consumption and penile has also been linked to penile SCC risk in previous SCC risk (Table 4). studies with ORs of 3.5 (95% CI, 1.5-8.5) and 9.5 (95% CI, 5.2-17.2), respectively (4, 14). With 22% of penile SCC patients versus only 7% to 8% of controls reporting a history of balanitis, our study confirms this association, Discussion although it was not statistically significant in the multivariate analysis. However, unlike in some other Our study adds to the accumulating international studies (4, 25) the occasional presence of smegma under evidence that sexually transmitted hrHPVs, notably the foreskin, which was noticed by a majority of all HPV16, are important etiologic factors in penile SCC participants, was not associated with penile SCC risk in (4). Of tumors tested in our study, more than half the present study. (59.5%) were DNA positive for HPV16, constituting 92% A history of penile trauma was not reported more of all hrHPV-positive tumors. These findings are in frequently by penile SCC patients than controls in our agreement with those of Daling et al. (4) who observed study. This is in contrast to findings in a case-control 69% of 94 examined penile SCCs to be DNA positive for study in Western Washington/British Columbia, where HPV 16 (87% of all HPV-positive tumors), and findings researchers reported statistically significant ORs of 3.2 in a recent study by Pascual et al. (17) who reported (95% CI, 1.5-6.8) and 5.2 (95% CI, 3.1-8.7) for penile injury that 65% of 49 penile SCCs were HPV 16 positive and penile tear, respectively (4). The reason for this (84% of all HPV-positive tumors). The central involve- difference between studies is unclear. ment of oncogenic HPV infection in the etiology of Tobacco smoking, particularly current smoking, has penile SCC was further emphasized by our finding that, been reported in a number of studies to be linked to among a number of sexually transmitted diseases, only increased risk of penile SCC (4, 10, 11, 14, 18, 26). For a history of anogenital warts obtained status as an instance, current smokers were at double risk of penile independent risk factor in our study. Specifically, a SCC compared with lifelong nonsmokers in the Seattle- history of anogenital warts was reported by 33% of based case-control study (4). However, not all studies are penile SCC patients as contrasted by only 6% to 7% of in support of tobacco smoking as an important etiologic controls, corresponding to an OR of 7.0, which is close factor. A case-control study in China failed to find to the OR of 7.6 reported for genital warts in a Seattle- support for a causal role of tobacco smoking (25), and based case-control study (4). researchers in Sweden reported only a weak, positive Our findings support the view that transmission of the association with current consumption of >10 cigarettes responsible etiologic agents, presumably HPV16 and per day (14). In the present study, current smoking was other hrHPVs, usually takes place through intimate not associated with penile SCC risk and cumulative sexual contact. We observed a statistically significant tobacco consumption was associated with risk only in trend of increasing risk with increasing numbers of univariate analysis, but upon adjustment for confound- female sexual partners. Notably the partner number ers in the multivariate model, there remained no before age 20 years seemed to differ between penile SCC significant role for tobacco consumption. Differences in patients and controls in our study. To the best of our reported ORs linking tobacco smoking to penile cancer knowledge, our study is the first to show a strong and risk is likely to be partly due to differences in smoker statistically significant association between the practice prevalence across countries. In the present Danish of heterosexual penile-oral sex and risk of penile SCC, study, 40% of population controls were current smokers, suggesting that oral sex, a common sexual practice which is almost twice the proportion (22%) of current reported by almost half of our population controls, may smokers among population controls in the Seattle-based be a major and previously underestimated means of viral study (4). Considering the much higher smoker preva- transmission from the female partner’s oral cavity/ lence in Denmark, smoking may be less strongly pharynx to the penis. Men who had practiced anal sex associated with other social and behavioral penile SCC with two or more female partners were also at increased risk factors among Danes than among citizens in areas risk, but numbers were unstable. Although some study with a lower smoker prevalence. Theoretically, some of participants may have been reluctant to report certain the previously reported effect of tobacco smoking might

Cancer Epidemiol Biomarkers Prev 2008;17(10). October 2008

reflect associations with behavioral risk factors that study prostate cancer controls, 6.4% of population controls), participants were reluctant to provide honest answers thus making telephone ownership an unlikely major about. biasing factor. However, penile SCC patients in our Our finding that priapism may be a risk factor for study were diagnosed during the study period 1993 to penile SCC is potentially interesting but requires cau- 1998 so, because interviews only started in 1997, tious interpretation. During interviews, a nontrivial 10% participating penile SCC patients might be somewhat of penile SCC patients affirmed a question of whether skewed toward patients with localized cancers. We they had ever experienced an unwanted penile erection cannot exclude that this might have introduced bias in of long duration that would not wear off. In contrast, some analyses. Specifically, to the extent that some of the only 4 (2.1%) of 189 controls reported ever having had studied risk factors have a negative effect on survival such an experience, thus resulting in a statistically after penile cancer, some of the ORs in our study may be significant multivariate OR of 9.2. We deliberately somewhat conservative. Another limitation is the limited excluded those priapic episodes that occurred <5 years number of penile SCC patients, which made findings in before the time of diagnosis to avoid inclusion of cancer- some of our statistical analyses unstable. related priapism (27-29). All seven penile SCC patients Our study also had some methodologic assets. It was with a positive history reported that the priapic episode population-based, which adds to the generalizability of antedated the cancer diagnosis by 10 or more years, thus our findings and, unlike other case-control studies, our reducing the possible distorting influence of priapism study benefited from having two independent control secondary to malignancy. The lack of prior supporting groups. Only after (a) having seen that risk factor evidence of a causal connection constitutes no strong associations were fairly similar in initial logistic regression argument against an association because to the best of analyses obtained with the two control groups separately, our knowledge, no prior penile cancer case-control study and (b) after having formally examined the appropriate- has examined the possible role of priapism. Obviously, ness of combining the two control groups in a logistic further data from other settings are needed before a regression analysis comparing the two control groups, did causal association of priapism with later penile SCC we combine them to gain statistical power, thus adding robustness and credibility to the reported risk factor development can be established. associations. Another advantage of our double control Approximately one third of tested penile SCCs were group study design, which has been used previously in HPV negative, suggesting that a proportion of penile case-control studies of risk factors for anal and vulvo- cancers may arise through causal pathways that do not vaginal cancers (21, 31), is that blinding of interviewers to involve infection with anogenital types of HPV. Theoret- the case or control status of participants is more easily ically, rare HPV types that were not included in the broad maintained. Expectations among interviewers may color panel of HPV types examined might have been present in the way answers are perceived and noted on the some of our negative cases. However, some subtypes of questionnaires, but interviewers in our study could not penile SCC are probably genuinely HPV negative, plausibly distinguish between interviews conducted with notably those with keratinizing SCC histology (30). Some penile SCC cases and those conducted with prostate cancers arising in the penile skin may resemble cutaneous cancer controls. Any influence of interviewer bias is SCCs elsewhere on the body, which are not etiologically therefore likely to have been small and nondifferential. related to HPV16 or other mucosotropic types of HPV. In conclusion, our study provides population-based Unfortunately, the limited number of HPV-tested penile evidence that sexually transmitted hrHPV infections, SCC patients in our study precluded meaningful risk notably infections with HPV16, constitute a central risk factor analyses stratified by HPV status. factor for penile SCC and that heterosexual penile-oral Our study has some limitations that need to be sex may be a hitherto unrecognized means of viral considered. Participation rates were modest (around transmission. Phimosis was confirmed as a strong risk 60%) among penile SCC patients and among prostate factor, but the novel association with priapism needs cancer and population controls. We share these difficul- replication in other studies. ties in recruiting penile SCC patients with other case- control studies whose participation rates among invited penile SCC patients ranged between 50% and 70% (4, 10, Disclosure of Potential Conflicts of Interest 11), although very high levels of participation (respec- Dr. Frisch has served as a member of a national advisory board tively, 90% and 91%) have been seen in case-control for Glaxo Smith Kline, the manufacturer of a vaccine against studies carried out in China (25) and Sweden (14). HPV 16 and HPV 18. The other authors disclosed no potential Theoretically, the f40% of invited penile SCC patients conflicts of interest. and controls who declined the invitation to participate in this and other studies could represent socioeconomic or Acknowledgments behavioral segments of the population that would make generalization to the entire population problematic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be Participants in our study were slightly younger than hereby marked advertisement in accordance with 18 U.S.C. those who declined the invitation to participate in the Section 1734 solely to indicate this fact. study, a general pattern that applied to all three groups of study participants. Also, by study design, participants were restricted to those invited subjects who were in References possession of a telephone, a criterion that precluded the 1. Wideroff L, Schottenfield D. Penile Cancer. In: Schottenfeld D, participation of only slightly different proportions of Fraumeni JF. Cancer Epidemiology and Prevention. New York: invited penile SCC patients (10.7%) and controls (8.1% of Oxford University Press;2006. p. 1166 – 72.

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