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HPV Associated With Oropharyngeal Cancers
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Case-Control Study of Human Papillomavirus and Oropharyngeal Cancer
NEJM, May 10, 2007
Gypsyamber D'Souza, Ph.D., Aimee R. Kreimer, Ph.D., Raphael Viscidi, M.D.,
Michael Pawlita, M.D., Carole Fakhry, M.D., M.P.H., Wayne M. Koch, M.D., William H. Westra, M.D., and Maura L. Gillison, M.D., Ph.D.
Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health (G.D.); the Departments of Pediatrics (R.V.), Otolaryngology-Head and Neck Surgery (C.F., W.M.K.), and Pathology (W.H.W.), Johns Hopkins Hospital; and the Division of Viral Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University (M.L.G.) - all in Baltimore; the Division of Cancer
Prevention, National Cancer Institute, Bethesda, MD (A.R.K.); and the Infection and Cancer Control Program, German Cancer Research Center, Heidelberg, Germany (M.P.).
"...This epidemiologic study provides support for the association between HPV and a subgroup of oropharyngeal cancers. The strength of the evidence is underscored by the associations of high-risk sexual behaviors, oral HPV infection, and HPV-16 exposure (as determined from the results of serologic tests) with oropharyngeal cancer. ...Oral HPV infection is strongly associated with oropharyngeal cancer among subjects with or without the established risk factors of tobacco and alcohol use.... our data suggest two distinct pathways for the development of oropharyngeal cancer: one driven predominantly by the carcinogenic effects of tobacco or alcohol (or both) and another by HPV-induced genomic instability.....Oral-genital contact was strongly associated with oropharyngeal cancer, but we cannot rule out transmission through direct mouth-to-mouth contact or other means.... A history of squamous-cell carcinoma of the head and neck in a first-degree relative, a history of cancer in a sibling, a history of oral papillomas, and poor long-term oral hygiene (some or complete tooth loss or infrequent toothbrushing) were all associated with oropharyngeal cancer. A history of heavy tobacco use (20 pack-years or more), a history of heavy alcohol use (15 drinks or more per week for 15 years or more), and a history of regular marijuana use were also associated with oropharyngeal cancer.....Certain kinds of sexual behavior were significantly associated with oropharyngeal cancer after adjustment for confounding variables. The association with oropharyngeal cancer increased significantly with the number of vaginal-sex partners or oral-sex partners..."
ABSTRACT
Background Substantial molecular evidence suggests a role for human papillomavirus (HPV) in the pathogenesis of oropharyngeal squamous-cell carcinoma, but epidemiologic data have been inconsistent.
Methods We performed a hospital-based, case-control study of 100 patients with newly diagnosed oropharyngeal cancer and 200 control patients without cancer to evaluate associations between HPV infection and oropharyngeal cancer. Multivariate logistic-regression models were used for case-control comparisons.
Results
A high lifetime number of vaginal-sex partners (26 or more) was associated with oropharyngeal cancer (odds ratio, 3.1; 95% confidence interval [CI], 1.5 to 6.5), as was a high lifetime number of oral-sex partners (6 or more) (odds ratio, 3.4; 95% CI, 1.3 to 8.8). The degree of association increased with the number of vaginal-sex and oral-sex partners (P values for trend, 0.002 and 0.009, respectively). Oropharyngeal cancer was significantly associated with oral HPV type 16 (HPV-16) infection (odds ratio, 14.6; 95% CI, 6.3 to 36.6), oral infection with any of 37 types of HPV (odds ratio, 12.3; 95% CI, 5.4 to 26.4), and seropositivity for the HPV-16 L1 capsid protein (odds ratio, 32.2; 95% CI, 14.6 to 71.3). HPV-16 DNA was detected in 72% (95% CI, 62 to 81) of 100 paraffin-embedded tumor specimens, and 64% of patients with cancer were seropositive for the HPV-16 oncoprotein E6, E7, or both. HPV-16 L1 seropositivity was highly associated with oropharyngeal cancer among subjects with a history of heavy tobacco and alcohol use (odds ratio, 19.4; 95% CI, 3.3 to 113.9) and among those without such a history (odds ratio, 33.6; 95% CI, 13.3 to 84.8). The association was similarly increased among subjects with oral HPV-16 infection, regardless of their tobacco and alcohol use. By contrast, tobacco and alcohol use increased the association with oropharyngeal cancer primarily among subjects without exposure to HPV-16.
Conclusions
Oral HPV infection is strongly associated with oropharyngeal cancer among subjects with or without the established risk factors of tobacco and alcohol use.
Background
Infection with sexually transmitted human papillomavirus (HPV) is a cause of virtually all cervical cancers.1 Molecular evidence also provides support for a role for HPV, particularly HPV-16, in the pathogenesis of a subgroup of squamous-cell carcinomas of the head and neck.2 Genomic DNA of oncogenic HPV is detected in approximately 26% of all squamous-cell carcinomas of the head and neck worldwide,3 but the molecular evidence is most rigorous and consistent for oropharyngeal squamous-cell carcinoma, in which viral integration and the expression of viral oncogenes (E6 and E7) have been shown.4
The epidemiologic evidence of a causal role for HPV in a subgroup of squamous-cell carcinomas of the head and neck is less rigorous than the molecular evidence. The example of the relationship between HPV and cervical cancer5 indicates that high-risk sexual behavior and exposure to and infection with HPV will increase the risk of other cancers caused by HPV.6 Although each of these three factors has been found to increase the risk of squamous-cell carcinomas of the head and neck,7,8,9,10,11,12,13,14 no single study has shown an association of all three with the development of oropharyngeal cancer.
In this study, we focused exclusively on oropharyngeal cancer, for which the molecular evidence of a causal role for HPV is compelling. Strong epidemiologic data would provide additional support for a causal association between HPV and oropharyngeal cancers and might guide future cancer-prevention programs involving vaccination to prevent oral HPV infection or screening to detect it.
Patients
Our case-control study was nested within a longitudinal cohort study of patients with newly diagnosed squamous-cell carcinomas of the head and neck in the outpatient otolaryngology clinic of the Johns Hopkins Hospital in Baltimore from 2000 through 2005. Eligible case patients included those with a confirmed diagnosis of oropharyngeal squamous-cell carcinoma.
The control group consisted of patients without a history of cancer who were seen for benign conditions between 2000 and 2005 in the same clinic from which the case patients were enrolled (Table 1). Subsequent to enrollment of a case, eligible control patients within the same sex and 5-year age categories were approached until two control patients were individually matched to each case patient. The study protocol was approved by the institutional review board of the Johns Hopkins Hospital. Written, informed consent was obtained from all patients.
Results
We enrolled 130 consecutive patients with newly diagnosed oropharyngeal cancer in the longitudinal cohort study from 2000 through 2005, and 100 patients (77%) agreed to participate in our nested case-control study. Case patients who declined enrollment were similar to those who were enrolled with regard to age, race or ethnic group, and anatomical site of the tumor but were more likely to be female (P=0.001). Approximately 70% of eligible control patients (200) agreed to participate.
In the univariate analysis, case and control patients were similar with regard to age, sex, race or ethnic group, and education, but case patients were more likely than control patients to live outside of Maryland (Table 1). A history of squamous-cell carcinoma of the head and neck in a first-degree relative, a history of cancer in a sibling, a history of oral papillomas, and poor long-term oral hygiene (some or complete tooth loss or infrequent toothbrushing) were all associated with oropharyngeal cancer (Table 1). A history of heavy tobacco use (20 pack-years or more), a history of heavy alcohol use (15 drinks or more per week for 15 years or more), and a history of regular marijuana use were also associated with oropharyngeal cancer (Table 1). Similar percentages of case and control patients had no history of tobacco or alcohol use (13% and 14%, respectively; odds ratio, 1.0; 95% CI, 0.5 to 1.9).
Certain kinds of sexual behavior were significantly associated with oropharyngeal cancer after adjustment for confounding variables (Table 2). The association with oropharyngeal cancer increased significantly with the number of vaginal-sex partners or oral-sex partners (P for trend=0.002 and 0.009, respectively) and was markedly elevated among patients with a high lifetime number of such partners (Table 2).
Oropharyngeal cancer was also strongly associated with serologic measures of exposure to HPV-16 and with the presence of oral HPV infection (Table 3). Oropharyngeal cancer was significantly associated with seropositivity for the HPV-16 L1 capsid protein, a validated measure of lifetime HPV-16 exposure (odds ratio, 32.2; 95% CI, 14.6 to 71.3).25 The presence of an oral HPV-16 infection was strongly associated with oropharyngeal cancer (odds ratio, 14.6; 95% CI, 6.3 to 36.6), as was oral infection with any of 37 HPV types (odds ratio, 12.3; 95% CI, 5.4 to 26.4) (Table 3).
To explore whether the association between sexual behaviors and oropharyngeal cancer could be explained by HPV-16 exposure, we reevaluated the associations using multivariate models after adjusting for HPV-16 L1 serologic status. In this analysis, sexual behaviors were no longer significantly associated with oropharyngeal cancer (data not shown). However, associations of sexual behaviors with oropharyngeal cancer became stronger when the analysis was restricted to patients with an HPV-16-positive tumor (Table 2). A high lifetime number of oral-sex or vaginal-sex partners, engagement in casual sex, early age at first intercourse, and infrequent use of condoms each were associated with HPV-16-positive oropharyngeal cancer (Table 2).
The association between HPV-16 exposure and oropharyngeal cancer was investigated among patients with varied use of tobacco and alcohol. The association was greatly increased among patients without a history of smoking or drinking who were seropositive for HPV-16 L1 (odds ratio, 44.8; 95% CI, 5.9 to 338.5) or had an oral HPV-16 infection (odds ratio, 43.7; 95% CI, 4.2 to 452.7). HPV-16 L1 seropositivity and oral HPV-16 infection were also highly associated with oropharyngeal cancer among patients with a history of heavy tobacco and alcohol use and those without such a history (Table 4). Thus, measures of both lifetime and prevalent oral HPV-16 infection were associated with an increased risk of oropharyngeal cancer, whether or not there was a history of use of tobacco, alcohol, or both.
We evaluated whether combined exposure to HPV and tobacco or alcohol further increased the odds that oropharyngeal cancer would develop. No evidence of synergy was found (Table 4, top): combined exposure to HPV and heavy tobacco and alcohol use was not additive (synergy index <1). Moreover, when the analysis was restricted to patients who were seropositive for the HPV-16 L1 protein, the odds of oropharyngeal cancer were not increased among heavy users of tobacco or alcohol (Table 4, bottom). By contrast, among patients who were seronegative for the HPV-16 L1 protein, the odds of oropharyngeal cancer were increased among heavy users of tobacco or alcohol, and the odds of oropharyngeal cancer were further increased among heavy users of both tobacco and alcohol (synergy index >1) (Table 4, bottom). Similar relationships were observed in patients with and those without the presence of an oral HPV-16 infection (Table 4). Therefore, tobacco and alcohol were important risk factors for oropharyngeal cancer, but they may not have acted as cofactors in HPV-mediated carcinogenesis in the oropharynx.
In the multivariate analysis, oropharyngeal cancer was independently associated with HPV-16 L1 seropositivity (odds ratio, 32.2; 95% CI, 14.6 to 71.3), poor dentition (odds ratio, 4.1; 95% CI, 1.6 to 10.6), infrequent toothbrushing (odds ratio, 6.9; 95% CI, 1.6 to 30.3), history of squamous-cell carcinomas of the head and neck in a first-degree family member (odds ratio, 5.4; 95% CI, 1.0 to 30.8), and heavy tobacco use (odds ratio, 2.5; 95% CI, 1.1 to 6.0) after adjustment for age, sex, and alcohol use. These factors were collectively estimated to be responsible for 90% of cases of oropharyngeal cancers (the attributable risk; 95% CI, 72 to 96), with 55% of cases (95% CI, 45 to 63) attributable to HPV-16 exposure alone.
The percentage of oropharyngeal cancers in which HPV-16 genomic DNA was detected by in situ hybridization was 72% (95% CI, 62 to 81) (Table 3 and Figure 1). Of the 60 specimens of available fresh-frozen tumor, 35 (58%; 95% CI, 45 to 71) were positive for HPV-16, with a median of 1.2 viral copies per cell (interquartile range, 0.02 to 11) analyzed. Five fresh-frozen specimens were positive for a high-risk type of HPV other than HPV-16 (two for HPV-33, one for HPV-35, and two for both HPV-33 and HPV-16).
To corroborate the in situ data, we tested for serum antibodies against HPV-16 oncoprotein E6, E7, or both, which have high specificity but moderate sensitivity for the detection of invasive cancer associated with HPV-16.26 Such antibodies were found in 64% of the case patients and in 4% of the control patients (odds ratio, 58.4; 95% CI, 24.2 to 138.3; P<0.001) (Table 2).
Discussion
This epidemiologic study provides support for the association between HPV and a subgroup of oropharyngeal cancers. The strength of the evidence is underscored by the associations of high-risk sexual behaviors, oral HPV infection, and HPV-16 exposure (as determined from the results of serologic tests) with oropharyngeal cancer. Furthermore, we found that HPV-16 DNA was specifically localized to tumor-cell nuclei in 72% of 100 paraffin-embedded specimens of oropharyngeal cancers, a finding corroborated by the high prevalence of antibodies for HPV-16 oncoprotein E6, E7, or both (64%) in the patients with oropharyngeal cancer. Although a cause-and-effect relationship cannot be inferred from a single study, our findings confirm and extend those of other case-control studies.7,8,9,10,11,12,13,14 Our results are also consistent with a previous report of an increase in the subsequent risk of oropharyngeal cancer by a factor of 14 among HPV-16 L1 seropositive subjects,26 which provides strong evidence that exposure to HPV can precede the appearance of oropharyngeal cancer by 10 years or more.
The degree to which oral HPV infection may interact with tobacco use, alcohol use, or both to increase the risk of squamous-cell carcinomas of the head and neck has been unclear. A greater-than-additive risk has been reported, albeit inconsistently,8,9,10 for patients exposed to both HPV and tobacco9 and those exposed to both HPV and alcohol.10 We found that exposure to HPV increased the association with oropharyngeal cancer regardless of tobacco and alcohol use, but we uncovered no evidence of synergy between exposure to HPV and tobacco or alcohol use. For these reasons, our data suggest two distinct pathways for the development of oropharyngeal cancer: one driven predominantly by the carcinogenic effects of tobacco or alcohol (or both) and another by HPV-induced genomic instability.
Our data suggest that oral HPV infection is sexually acquired. Oral-genital contact was strongly associated with oropharyngeal cancer, but we cannot rule out transmission through direct mouth-to-mouth contact or other means. Certain sexual behaviors13,14 and a history of oral HPV infection7,10 were associated with an increased risk of squamous-cell carcinomas of the head and neck in previous studies in which 25% or more of the tumors from patients were positive for HPV DNA but not those in which less than 25% of the tumors from patients were positive for HPV DNA.8,9 Discrepant findings may be explained by the heterogeneity of the case populations, with variable percentages of cancer cases attributable primarily to tobacco and alcohol use, as compared with HPV infection. In our study, the heterogeneity of case patients was minimized by restricting enrollment to patients with oropharyngeal cancer, 90% of whom had tumors on the tonsil or base of the tongue.
Although HPV-16 alone accounts for more than 90% of cases of HPV-positive squamous-cell carcinomas of the head and neck,8 a more accurate and probably higher proportion might be found by testing for other types of HPV (e.g., types 18, 31, 33, and 35), which are infrequently detected in oropharyngeal cancers.
In our study, oropharyngeal cancer was independently associated with a family history of squamous-cell carcinoma of the head and neck and poor oral hygiene, findings that are consistent with other reports.27 The risk of cervical cancer is also increased in women with a family history of that cancer.28,29 Until specific genetic markers for the risk of an HPV-associated cancer are identified, familial aggregation due to shared environmental exposures cannot be ruled out as an explanation for these findings. Poor dentition,30,31 infrequent toothbrushing,31,32 and infrequent dental visits30,33 have been associated with an increased risk of squamous-cell carcinomas of the head and neck. Because tooth loss is commonly caused by chronic bacterial infections (e.g., periodontitis), it may serve as a surrogate for chronic infection and inflammation, which may be important in the pathogenesis of cancer. Particular coinfections in the cervix (e.g., infection with Chlamydia trachomatis) increase the risk of cancer,34 and our results suggest that bacterial coinfections could play a similar role in the oral region. The absence of data on diet, which is associated with the risk of squamous-cell carcinomas of the head and neck,35 is a limitation of our study but is unlikely to explain the observed associations with HPV infection.
The public health implications of our findings are underscored by the annual increases in the incidence of tonsillar and base-of-tongue cancers in the United States since 1973.36,37 The widespread oral sexual practices among adolescents may be a contributing factor in this increase.38 Our results and those of other studies provide a rationale for HPV vaccination in both boys and girls - since oropharyngeal cancers occur in men and women. If vaccination is as effective in preventing oral HPV-16 infection as it is in preventing cervical infection,39 a substantial reduction in the incidence of oropharyngeal cancer in vaccinated populations would provide the ultimate evidence of causality.
Supported in part by grants from the Damon Runyon Cancer Research Foundation (Clinical Investigator Award, to Dr. Gillison), the State of Maryland Cigarette Restitution Fund (to Dr. Gillison), the National Institute of Dental and Craniofacial Research (DE016631-01, to Dr. Gillison), and the National Institutes of Health (Training Grant T32AI50056, to Dr. D'Souza).
No potential conflict of interest relevant to this article was reported.
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