Human papillomavirus (HPV) associated oropharyngeal cancer incidence has been increasing in the United States.1–3 Despite the reduction in tobacco smoking and a decline by 50% of HPV-negative cancers from 1988 to 2004, the population-level incidence of HPV–oropharyngeal cancer in the United States has increased by 225% from 0.8/100,000 to 3.6/100,000 over the same period.1 The increase in cancer cases has been pronounced among men, whose cancer incidence rates are 2 to 4 times those among women.4 Infection with oncogenic HPV, in particular HPV16 and HPV18, has been recognized as a risk factor for head and neck squamous carcinoma arising in the oropharynx.5,6 Oral HPV infection in the United States is uncommon, and most infections clear within 24 months of viral acquisition.7 In the general population, the overall prevalence of any oral HPV infection is 11.5% among men and 3.2% in women aged 18–69 years.8 A pronounced sex imbalance is also evident among persons infected with oral HPV16 (1.8% among men vs. 0.3% among women).8
Despite the recent rise of HPV-associated oropharyngeal cancer incidence, routine screening for oral HPV infection is not recommended in the United States.9,10 However, there are several commercially available tests for qualitative and quantitative oral HPV detection. Oral rinse collection for HPV DNA detection through dental office is a feasible and an acceptable testing procedure, and persons are comfortable answering smoking or sexual history questions during dental visits.11 In contrast to screening strategies for HPV detection in the cervix, secondary prevention through clinical evaluation of oral HPV infections is challenging, as preneoplastic lesions for early intervention have not been identified and the role of routine oral HPV DNA testing in the general population is unknown.
Since November 2016, several dental care clinicians in Seattle area have been offering quantitative HPV16 and HPV18 (HPV16/18) testing for oral HPV infection as part of oropharyngeal cancer screening during preventative dental care visits. Using this extensive collection of oral samples, we aimed to describe the HPV16/18 prevalence in this population and assess quantitative HPV16/18 detection in oral rinses. We also evaluated HPV16/18 viral load (VL) dynamics in a subgroup of persons with oral rinses collected at 2 time points.
Study Population and Setting
Oral rinse for quantitative HPV16/18 detection was offered to all persons older than 18 years seeking routine dental health care from November 2016 to November 2018 as a fee for service (cost not covered by dental insurance) during the annual visual oral cancer screening. In this study, we included persons who provided demographic information (age and sex) and an oral rinse for HPV16/18 detection. Because testing was provided for clinical care, informed consent was not obtained. The analysis of the data was reviewed by the University of Washington Institutional Review Board.
Oral sample was collected by rinsing and gargling 10 mL of Scope for 30 seconds. Samples were stored at room temperature and transported to the FidaLab, a Clinical Laboratory Improvement Amendments–certified laboratory, for processing within 1 week of sample collection. The results were reported to the patient by e-mail if HPV VL was negative, or by telephone from the dental office and mail if HPV was detected. Persons with HPV VL of less than 10,000 copies/mL were recommended 6-month retesting, and persons with VL >10,000 copies/mL or those with oropharyngeal cancer-related symptoms were referred to an otorhinolaryngology provider.
Quantitative HPV16 and HPV18 Polymerase Chain Reaction
Oral samples were evaluated for the presence of HPV16 and HPV18 using quantitative polymerase chain reaction (PCR) as previously described.12,13 Briefly, oral rinses were centrifuged at 600g for 10 minutes to collect cell pellets. Cell pellets were resuspended in digesting buffer, and genomic DNA was isolated using QIAamp Blood DNA mini kit (Qiagen). Multiplex real-time TaqMan quantitative PCR assay was used to detect and quantify HPV16 and HPV18 in each sample on the QuantStudio 7 Flex using the following program: 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Dilution series of full-length HPV16 and HPV18 plasmids of known concentrations were used as standard curves. Exo internal control was spiked in each PCR reaction to monitor PCR inhibition. HPV16 and HPV18 quantities were reported as copy per milliliter oral rinse sample.
We used the χ2 test to compare demographic characteristics between persons with and without oral HPV16/18 and the Mann-Whitney U test to compare age and HPV16/18 VL between men and women with positive oral samples. We set the level of statistical significance at P value less than 0.05 (2-sided). To examine the association between HPV detection and sex and age, we used logistic regression and calculated odds ratios (ORs) and 95% confidence intervals (CIs). We included an interaction term to assess whether sex significantly modified the association between oral HPV16/18 detection and age.
To evaluate quantitative HPV infection patterns over time, we restricted our analysis to a group of persons who provided oral rinses at 2 time intervals. To examine the HPV16/18 VL and infection persistence (HPV16/18 detection in both oral rinses) or clearance (HPV16/18 detection in first oral rinse and HPV negative in second oral rinse), we plotted quantity HPV detected in log10 copies per milliliter at 2 periods. We used R program for data analysis.
Oral HPV Detection
A total of 15,344 oral rinses were collected from November 2016 to November 2018. Thirty-one persons were excluded from the analyses because of missing information on age or sex.
Overall, 6957 men and 8351 women were tested for oral HPV16/18; the mean age of the persons was 47 years (Table 1). We detected oral HPV16/18 in 152 (1%) unique persons; 127 had HPV16 and 25 had HPV18. The median age of persons with oral HPV16/18 was 53 years compared with 47 years (P = 0.47) in persons without oral HPV16/18. People with oral HPV16/18 were more likely to be men than women (1.6% were men vs. 0.6% were women, P < 0.001). Male sex was associated with higher risk of oral HPV16/18 infection in this population (OR, 3.2; 95% CI, 2.1–4.4). In contrast to women, the proportion of positive oral HPV16/18 rinses among men increased with higher age, suggesting that the relationship between oral HPV16/18 detection and age differed by sex (Fig. 1). The odds of having a positive oral HPV16/18 was 1.3 times as high for each additional decade of life among men (95% CI, 1.13–1.45). However, the odds of having a positive oral HPV16/18 among women did not change with each additional decade (OR, 0.9; 95% CI, 0.81–1.21).
HPV Quantification Among Persons With Oral HPV16/18
We detected oral HPV16 in 96 men and 31 women. In contrast, only 13 men and 12 women had oral HPV18. Compared with women with oral HPV16, men with oral HPV16 were older (median age, 55 vs. 48 years; P < 0.001) and had a higher median VL (39.7 vs. 1.1 copies/mL, P < 0.001; Table 2). Similarly, men with oral HPV18 tended to be older than women with oral HPV18 (median age, 51 vs. 47 years; P < 0.001), but the VL was similar for both sexes (9.0 vs. 1.4 copies/mL, P = 0.56).
HPV Detection in Persons With 2 Oral Samples
A total of 628 persons each had 2 oral rinses collected with a mean time between samples of 6 months. At baseline (rinse 1), 581 were HPV16/18 negative and only 1 person became HPV16 positive at second rinse. Thirty-nine persons had detectable oral HPV16 at baseline, and 13 remained HPV16 positive at the second rinse; 8 had HPV18 at baseline and 2 remained positive at the subsequent rinse. All persons with consecutive positive test results were men and had higher baseline median VL compared with those with first positive and second negative samples (Fig. 2). Persons whose first oral rinse was HPV16/18 positive were more likely to provide a follow-up oral rinse compared with those who had a negative HPV test result at first rinse (9% vs. 1%).
In this study, we evaluated a large number of oral samples collected for HPV16/18 detection in persons attending dental clinics in Seattle, Washington. Our findings largely support prior observations8,14–17 that the prevalence of oral HPV16/18 infection is low but higher among men than women, especially at higher VL levels, and higher at older age. Furthermore, HPV16/18 infection persistence was more common among men than women, and more likely among persons with higher VL at baseline. The HPV test used for this study has been extensively characterized and validated in several clinical and epidemiological studies.12,18,19 Although results generated by sampling with oral swabs versus oral rinse are generally comparable, oral rinses are more convenient and reach areas that are difficult to collect with oral swabs (e.g., back of the throat).18
A higher oral HPV prevalence has been reported among men than women in the general population.8,14,16 According to data from 2011–2014 National Health and Nutrition Examination Survey (NHANES), high-risk oral HPV infection was more common among men (7.3%) than women (1.4%).8 Similarly, oral HPV16 detection was significantly higher in men (1.8%) than in women (0.3%). Our analysis of a larger but geographically localized cohort also reflected a similar sex-specific prevalence pattern. The reasons for the observed sex imbalance in the prevalence of oral HPV infections are unknown, but it mimics the higher risk of HPV-driven oropharyngeal cancer in men. One hypothesis is that cervical HPV infection in women may lead to a more robust immune response, which could help to clear oral HPV infection.14,20 In a prior NHANES report from 2009 to 2010, a peak prevalence of oral HPV infection was noted in persons aged 30 to 34 and 60 to 64 years.14 However, most recent studies suggest no significant difference by age in the prevalence of high-risk-HPV infection in men8,21 and women.8 We found that persons with oral HPV16/18 infection were more likely to be older and the risk of detection increased with age among men but not women, suggesting that oral HPV detection across age varies by sex. The overall contribution of persistent oral infection or new viral acquisition among older persons is not well understood.
The observed findings of higher HPV16 load among men than women have not been consistently seen in other cohorts. Report from the NHANES 2009–2010 cycle showed that high-risk oral HPV load was marginally higher among men than women, although the difference did not reach statistical significance.14 Beachler et al.22 reported no difference in HPV16 VL among men and women in a cohort of HIV-infected and HIV-uninfected patients. Our findings of higher HPV16 VL mirror the observed sex and age distribution for HPV-associated oropharyngeal cancer.
The observation of persistent HPV detection preceding the development of cytological abnormalities that lead to neoplasia has been defined in both cervical and anal cancers. In contrast, the natural history of HPV-driven oropharyngeal cancer is not well understood. The duration of oral HPV detection in immunocompetent persons ranges from 3.5 to 20.7 months.15 The timing between oral HPV detection and cancer development is unknown, but oral HPV DNA can be detected on average of 4 years before the diagnosis of oropharyngeal cancer.23 In our study, men with higher HPV16/18 loads at baseline were more likely to have a second positive sample; however, most people with oral HPV infections cleared within 6 months. Given that HPV detection can be intermittent, with repeated detection after clearance, representing either reactivation of HPV or reacquisition, additional sampling over time might be informative. Additional longitudinal studies among older population are needed to define the association between detection of oral HPV16/18 and development of oropharyngeal cancer.
Primary prevention through immunization against HPV infection in HPV-naïve persons is a promising strategy to reduce both genital and oral HPV acquisition. HPV vaccines have showed efficacy of greater than 90% against HPV infection due to HPV vaccine types.24 At this time, the estimated population-level effect of HPV vaccination on oral HPV infection reduction is less than 20% in the overall population because of the low HPV vaccine uptake in the US population and only recent addition of male youth to target population.25 Importantly, new estimates suggest herd immunity protection against oral HPV infections in unvaccinated US men aged 18 to 59 years.26 Whether HPV vaccination can provide therapeutic effects in previously infected persons has not been formally assessed. However, reduction in oral HPV detection was reported in a clinical trial of quadrivalent HPV vaccine that aimed to reduce anal high-grade lesions in persons with HIV.27
Our study highlights that the general population is interested in diagnostic testing to detect oral HPV infection. Interestingly, a larger proportion of persons tested for oral HPV were women than men. This difference could be attributed to sex imbalance regarding regular oral health care, as women are more likely to receive regular dental evaluations than men.28 Although both oropharyngeal and cervical cancers are caused by HPV infection, several underlying differences prevent the direct adoption of cervical cancer screening strategies for oropharyngeal cancer. First, the prevalence of oral HPV and the incidence of oropharyngeal cancer are lower than cervical cancer in the absence of a screening program. In contrast to genital HPV prevalence, oral HPV16 infection is uncommon in younger persons. Second, the estimated lifetime risk of oropharyngeal cancer in persons with oral HPV16 infection is low (e.g., 0.7/100 in men and 0.2/100 in women).16 Lastly, precursor lesions for HPV-driven oropharyngeal cancer for early intervention that could parallel cervical cytologic screening have not been identified. As such, given the low prevalence of oral HPV infection and the lack of data showing that the natural course of the infection can be modified with an early intervention, molecular testing with PCR for oral HPV detection is not routinely recommended.
A potential use of oral HPV testing for disease surveillance has been described among patients with HPV-positive oropharyngeal cancer. For example, in a prospective cohort study of patients with incident HPV–oropharyngeal cancer in the United States, HPV16 DNA was found in more than half of oropharyngeal cancer cases at the time of diagnosis and in only 5% after treatment.29 A recent prospective study of 396 patients with oral cavity or oropharyngeal head and neck squamous cell carcinoma evaluated dynamics of oral HPV DNA detection using oral rinse samples at diagnosis and at completion of cancer treatment. Persistent detection of tumor-type HPV after cancer treatment was associated with increased risk of cancer recurrence and death.30 These data suggest that quantitative oral HPV detection may be useful for surveillance after tumor ablation. Although quantitative oral HPV detection is an attractive noninvasive and relatively inexpensive tool, the potential public health benefit of primary or secondary screening for oral HPV infection in the general population is unknown.
Although our study examined a large population in Seattle, this self-selected cohort is unlikely to be representative of all people residing in the state. Dental clinics offer oral HPV16/18 testing to all persons older than 18 years, but not all proceed with testing because the test is not covered by insurance, and we do not have estimates of the proportion of dental clinic patients who accepted the test. Despite these limitations, the prevalence of oral HPV16/18 in our study is similar to previously described population-based studies.8 Generalizability of these findings could be limited to persons residing in a metropolitan city with a large proportion of white population, although oropharyngeal cancer is most common among this racial group. Our study was also limited by lack of additional demographic, sexual behavior, and clinical information.
In conclusion, we reported a low prevalence of oral HPV16/18 infection among persons attending dental clinics in Seattle, Washington. Higher HPV16 load was found in men at an older age, and persistent infection was observed in those with higher HPV load at baseline. Defining the role of oral HPV testing as a strategy for prevention of oropharyngeal cancer, particularly in subgroups at highest risk of invasive disease, will require more detailed natural history data and effective strategies for early intervention.
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