To the editor
Oral human papillomavirus type16 (HPV16), a sexually transmitted infection, is believed to be responsible for the rise in incidence of oropharyngeal squamous cell cancers in the United States.1 An association between self-reported number of cigarettes currently smoked per day and oral HPV prevalence has been observed.2,3 We investigated associations between objective biomarkers reflective of all current tobacco exposures (environmental, smoked and smokeless tobacco) and oral HPV16 prevalence.
Methods
The National Health and Nutrition Examination Survey (NHANES) is a cross-sectional, stratified multistage probability sample of the US population.4 The NHANES was approved by the National Center for Health Statistics Institutional Review Board and written informed consent was obtained from participants. In 2009–2012, the response rate was 73.4%. Mobile examination center participants 14–69 years old were eligible for oral HPV DNA testing. Exfoliated oral cells were collected using a 30-second oral rinse and gargle, and tested for HPV16 as previously reported.2
Computer-assisted self-interviews were used to ascertain self-reported tobacco use and sexual behaviors. Self-reported tobacco use for the past 5 days included any nicotine-containing product. Biomarkers of recent tobacco use included serum cotinine, a major nicotine metabolite, and urinary NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol), a tobacco-specific, carcinogenic metabolite.4
Of 20,293 participants interviewed, analysis was restricted to participants aged 18 to 59 years (n=8527) with data on number of oral sexual partners, oral HPV16 and recent tobacco use. The principal outcome was oral HPV16 infection. NHANES sample weights were used to estimate prevalence. Mean biomarker values were compared using Wald tests. For modeling, cotinine and NNAL were log-transformed and considered as continuous and categorical variables. The association of tobacco use with oral HPV16 was explored using logistic regression. All multivariable models included variables considered significant (2-sided P<0.05) in bivariable analysis and important factors based on the literature.2 Analyses were performed using SUDAAN software 11.0.1.
Results
Among 6887 participants, 2012 (28.6%, 95%CI=26.5%–30.9%) were current tobacco users and 63 (1.0%, 95% CI =0.8%–1.3%) had oral HPV16 detected. Current tobacco users were more likely than non-users to be male, younger, less educated, and to have a higher number of lifetime oral sexual partners (Table 1). In bivariable analysis, self-reported and biological measures of tobacco exposure as well as oral sexual behavior were significantly associated with prevalent oral HPV16 infection (Table 2). Oral HPV16 prevalence was greater in current tobacco users (2.0%, 95%CI=1.3–3.1) compared with never/former tobacco users (0.6%, 95%CI=0.4–0.9), p=0.004. Mean cotinine (157.7 vs. 57.2 ng/mL, p=0.002) and NNAL levels (0.36 vs 0.12 ng/mL, p=0.020) were higher in individuals with versus without oral HPV16 infection.
Table 1.
Description of NHANES study population included in analysis, by current self-reported tobacco use* (yes/no used any product containing nicotine in the past 5 days)
| Overall | Current tobacco user | No current tobacco use | PValue | |
|---|---|---|---|---|
|
| ||||
| N= 6887 (%) | N= 2012 (%) | N= 4875 (%) | ||
| Gender | <0.001 | |||
| Male | 3494 (51.2) | 1240 (61.4) | 2254 (47.2) | |
| Female | 3393 (48.8) | 772 (38.6) | 2621 (52.8) | |
| Age | <0.001 | |||
| 18–29 | 2219 (29.2) | 685 (32.5) | 1534 (27.9) | |
| 30–39 | 1551 (21.9) | 454 (20.9) | 1097 (22.3) | |
| 40–49 | 1596 (24.5) | 476 (25.4) | 1120 (24.1) | |
| 50–59 | 1521 (24.4) | 397 (21.2) | 1124 (25.6) | |
| Race/Ethnicityˆ | <0.001 | |||
| Non-Hispanic white | 2724 (64.2) | 996 (68.3) | 1728 (62.6) | |
| Non-Hispanic black | 1548 (12.2) | 498 (13.8) | 1050 (11.5) | |
| Hispanic/ Other/ Multi-race | 2615 (23.6) | 518 (17.8) | 2097 (25.9) | |
| Education | <0.001 | |||
| < High school graduate | 1599 (16.4) | 595 (23.9) | 1004 (13.4) | |
| High school graduate or equivalent | 1560 (21.6) | 604 (29.3) | 956 (18.5) | |
| Some college | 2142 (32.5) | 627 (33.7) | 1515 (32.0) | |
| ≥ College graduate | 1579 (29.5) | 185 (13.1) | 1394 (36.0) | |
| Annual household income | <0.001 | |||
| < $35,000 | 2723 (30.8) | 1069 (43.8) | 1654 (25.5) | |
| $35,000 to $64,999 | 1462 (24.5) | 391 (23.8) | 1071 (24.8) | |
| $65,000 to $99,999 | 954 (19.1) | 208 (16.0) | 746 (20.3) | |
| ≥ $100,000 | 1137 (25.7) | 186 (16.4) | 951 (29.4) | |
| Ever married | 4640 (75.5) | 1338 (72.4) | 3302 (76.7) | 0.009 |
| Smoked any cigarette past 5 days | 1725 (23.3) | 1725 (81.3) | 0 (0.0) | <0.001 |
| Smoked a pipe past 5 days | 16 (0.2) | 16 (0.6) | 0 (0.0) | 0.007 |
| Used snuff past 5 days | 34 (0.8) | 34 (2.8) | 0 (0.0) | <0.001 |
| Used chewing tobacco past 5 days | 112 (2.1) | 112 (7.3) | 0 (0.0) | <0.001 |
| Used marijuana or hashish in past month | 1009 (14.2) | 642 (30.4) | 367 (7.6) | <0.001 |
| Drank alcohol regularly# | 2923 (48.3) | 1141 (59.0) | 1782 (44.1) | <0.001 |
| Ever performed oral sex | 5393 (86.0) | 1727 (91.0) | 3666 (84.0) | <0.001 |
| Number of people performed oral sex on in lifetime | <0.001 | |||
| 0 | 1359 (14.1) | 254 (9.1) | 1105 (16.1) | |
| 1 to 2 | 2369 (35.4) | 531 (25.5) | 1838 (39.3) | |
| 3 to 5 | 1612 (26.0) | 547 (28.1) | 1065 (25.1) | |
| 6 to 10 | 696 (12.5) | 282 (16.2) | 414 (11.0) | |
| ≥ 11 | 684 (12.1) | 355 (21.1) | 329 (8.4) | |
This includes cigarettes, pipes, cigars, chewing tobacco, snuff and nicotine replacement.
Race and ethnicity were self-identified by participants using options defined by NHANES.
Regular alcohol use was defined as alcohol at least once per week, on average, past year for everyone twenty years and older. For those 18–19 years old, regular alcohol use was defined as at least one drink of alcohol on 40 or more days in lifetime.
Table 2.
Bivariable and multivariable associations of biomarkers and behavioral measures of current tobacco use with oral HPV16. Bolding represents results that are statistically significant (p<0.05).
| Oral HPV 16 | |||||
|---|---|---|---|---|---|
|
| |||||
| N | Weighted Prevalence | Bivariable OR (95%CI) | N | Multivariable£ OR (95%CI) | |
| Cotinine (log base 10; ng/mL) | 6598 | – | 1.41 (1.15, 1.72) | 5889 | 1.31 (1.07, 1.60) |
| Cotinine quartiles (ng/mL) | |||||
| Below detection limit | 1432 | 0.3% | Ref | 1281 | Ref |
| < 0.033 | 1207 | 0.9% | 2.98 (0.73, 12.28) | 1075 | 2.67 (0.63, 11.24) |
| 0.033 to 0.179 | 1298 | 0.7% | 2.11 (0.60, 7.41) | 1134 | 2.00 (0.57, 6.99) |
| 0.180 to 126 | 1426 | 1.2% | 3.88 (0.99, 15.23) | 1268 | 3.67 (0.88, 15.36) |
| >126 | 1235 | 2.0% | 6.49 (1.99, 21.18) | 1131 | 4.34 (1.14, 16.53) |
| p-trend | 0.003 | 0.02 | |||
| NNAL (log base 10; ng/mL) | 6681 | – | 1.80 (1.34, 2.44) | 5942 | 1.68 (1.23, 2.28) |
| NNAL quartiles (ng/mL) | |||||
| Below detection limit | 2174 | 0.4% | Ref | 1943 | Ref |
| < 0.002 | 1120 | 0.8% | 2.01 (0.60, 6.77) | 976 | 2.00 (0.53, 7.51) |
| 0.002 – 0.009 | 1095 | 0.4% | 0.95 (0.28, 3.19) | 964 | 0.96 (0.24, 3.81) |
| 0.010 – 0.190 | 1188 | 1.9% | 4.94 (1.57, 15.55) | 1037 | 4.52 (1.17, 17.38) |
| >0.190 | 1104 | 2.3% | 5.86 (1.68, 20.46) | 1022 | 4.54 (1.19, 17.30) |
| p-trend | 0.002 | 0.01 | |||
| Used nicotine in last 5 days | |||||
| No | 4875 | 0.6% | Ref | 4315 | Ref |
| Yes | 2012 | 2.0% | 3.54 (1.79, 6.98) | 1817 | 2.70 (1.35, 5.41) |
| Number of cigarettes per day | |||||
| Did not smoke cigarettes, pipes or cigars in last 5 days | 5007 | 0.7% | Ref | 4434 | Ref |
| <10 | 843 | 1.3% | 1.90 (0.53, 6.81) | 740 | 1.89 (0.56, 6.43) |
| 10 to <20 | 463 | 3.0% | 4.60 (1.94, 10.89) | 424 | 3.97 (1.56, 10.13) |
| 20 to <30 | 329 | 1.7% | 2.59 (0.73, 9.11) | 306 | 2.08 (0.57, 7.65) |
| ≥30 | 88 | 4.1% | 6.33 (1.25, 32.20) | 84 | 3.15 (0.70, 14.20) |
| p-trend | 0.05 | 0.22 | |||
| Pack-years of tobacco | |||||
| 0 | 4105 | 0.7% | Ref | 3637 | Ref |
| >0 to <5 | 1262 | 0.7% | 1.07 (0.26, 4.31) | 1115 | 0.84 (0.22, 3.23) |
| 5 to <10 | 368 | 2.6% | 4.04 (1.35, 12.11) | 337 | 2.88 (0.99, 8.41) |
| 10 to <20 | 442 | 1.7% | 2.65 (0.96, 7.29) | 395 | 1.77 (0.66, 4.76) |
| ≥20 | 545 | 2.6% | 4.09 (1.39, 12.01) | 505 | 2.79 (1.01, 7.72) |
| p-trend | 0.008 | 0.05 | |||
| Lifetime number of oral sex partners | |||||
| 0 | 1359 | 0.5% | Ref | 1177 | Ref |
| 1–2 | 2369 | 0.4% | 0.90 (0.22, 3.74) | 2160 | 0.76 (0.18, 3.18) |
| 3–5 | 1612 | 0.6% | 1.29 (0.32, 5.17) | 1503 | 1.06 (0.25, 4.61) |
| 6–10 | 696 | 1.4% | 3.03 (0.62, 14.76) | 655 | 2.24 (0.43, 11.56) |
| ≥11 | 684 | 4.0% | 9.07 (1.67, 49.24) | 637 | 5.89 (0.91, 38.10) |
| p-trend | 0.004 | 0.03 | |||
:Adjusted for age (18–29, 30–39, 40–49, 50–59), gender, race/ethnicity (non-Hispanic White, non-Hispanic Black, other), income (<$35000, 35000–64999, 65,000–99,999, ≥100,000) and number of lifetime partners performed oral sex on (0, 1–2, 3–5, 6–10, ≥11). Hosmer-Lemeshow goodness-of-fit tests had p-values >0.05 for all multivariable models using the Satterthwaite F tests, and for all models except lifetime number of oral sexual partners, NNAL quintiles and nicotine in the last 5 days using the Wald F tests.
All biomarkers of tobacco exposure remained associated with oral HPV16 infection after adjustment for other factors (Table 2). Significant dose-response relationships were observed between cotinine (p-trend=0.02) or NNAL (p-trend= 0.01) levels and odds of oral HPV16 infection (Table 2). Each log increase in cotinine, approximating 3 cigarettes per day, was independently associated with oral HPV16 prevalence, with an adjusted OR of 1.31 (95%CI=1.07–1.60). Each log increase in NNAL, approximating 4 cigarettes per day, was independently associated with oral HPV16 prevalence, with an adjusted OR of 1.68 (95%CI=1.23–2.28).
Discussion
In this large cross-sectional, population-based study, we demonstrated strong and consistent dose-response relationships between behavioral and objective biomarkers of current tobacco use and oral HPV16 infection. Tobacco use is an established co-factor for the development of cervical cancer, for which HPV infection is a necessary cause.5 Tobacco use has local and systemic immunosuppressive effects,6 however the specific biological mechanisms underlying our observed associations are unknown. Tobacco use may alter determinants of oral HPV16 prevalence, such as incidence, persistence or re-activation of infection.
These findings highlight the need to evaluate the role of tobacco in the natural history of oral HPV16 infection and progression to malignancy. Although adjusted for sexual behavior, we cannot entirely exclude the possibility that tobacco use is a marker for risky behavior. Additional limitations include some unstable estimates and a cross-sectional study design that precludes our ability to determine temporality or causation.
Acknowledgments
Grant Support: This study was supported by P50DE019032, R01DE021395, R01DE023175 (NIDCR), Milton J. Dance Head and Neck Center, and Merck. The funder had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; and preparation, review or approval of the manuscript; and decision to submit the manuscript for publication. Drs. D’Souza and Gillison had previous research support from Merck Inc. Dr. D’Souza had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Timothy S. McNeel, B.A. (Information Management Services, Inc.) assisted with statistical analyses and received compensation for this role.
References
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