HPV-Related Oropharynx Cancer in the United Kingdom: An Evolution in the Understanding of Disease Etiology

The developed world has experienced a dramatic rise in oropharyngeal squamous cell carcinoma (OPSCC) incidence (1, 2). In England, the age-standardized incidence rate (ASR) for OPSCC approximately tripled in men (from 2.0 to 5.8) and doubled in women (from 0.8 to 1.7) between 1995 and 2011 (3). Associations between tobacco and alcohol consumption and OPSCC are well established (4); however, sexual behavior is also a risk factor, with lifetime number of oral sex partners recognized as the behavioral measure most strongly associated with OPSCC development (5). Changes in sexual behavior appear to underlie the increasing proportion of OPSCC attributable to oncogenic human papillomavirus (HPV; refs. 1, 4, 6). Several North American and European studies have confirmed sharp rises in HPV-induced OPSCC incidence, although the exact proportion of HPV-positive tumors within the total disease burden varies considerably by geographical region (7–11).

In the United Kingdom, the proportion of OPSCC attributable to HPV has been assessed in several single-center studies; however, each was small, applied diverse methodology, and had restricted geographical coverage (12–14). The current pan-UK study aimed to assess the proportion of OPSCC attributable to HPV infection in a large contemporary sample (2002–2011 inclusive) using robust, standardized methods. There is a pressing need for these data to facilitate health economic analyses and to inform evidence-based policy making with regard to prophylactic male HPV vaccination, as has recently been implemented in Australia (15, 16).

The study received Research Ethics Committee approval (REC 11/NQ/0452). Northern Ireland samples were accessed under approvals from the Northern Ireland Biobank (NIB 11/001). OPSCCs were defined as cancers involving the base of tongue (C01), soft palate and uvula (C05.1 and C05.2), tonsil (C09), and oropharynx-not-otherwise-specified (C10.9). OPSCC cases diagnosed between 2002 and 2011 (inclusive) were collected from 11 recruiting centers distributed across the United Kingdom to ensure results were not distorted by effects in one area or center (Belfast, Bristol, Cardiff, Coventry, Edinburgh, Liverpool, London, Manchester, Newcastle, Poole, and Southampton). The overall target sample size (1,710) was sufficient to allow comparison of prevalence between years with 7.5% precision. The number of samples per center was determined pragmatically, based on the number of cases seen annually at each center. To avoid selection bias, the first 17 cases per year (11 cases for Coventry and Bristol) with available formalin-fixed paraffin-embedded (FFPE) tumor blocks were included (irrespective of the definitive treatment modality employed). A representative FFPE block, either from diagnostic or resection specimen, was selected. Gender, age at diagnosis, year of diagnosis, and histologic diagnosis, including anatomic subsite classification, were recorded.

Sections of each FFPE block were taken for DNA analysis. To prevent DNA contamination, the microtome was thoroughly cleaned between specimens, and a new blade was used for each block. Tissue microarrays (TMA) were constructed for p16 IHC and high-risk HPV DNA in situ hybridization (ISH) testing as described previously (17). Following construction, hematoxylin and eosin–stained sections of the TMAs were analyzed to confirm accuracy of sampling. Samples were considered adequate only if all three TMA cores included tumor.

For PCR, DNA was extracted from 2 × 10 μm FFPE whole sections by digestion for 16 hours in Tris 50 mmol/L/EDTA 1 mmol/L/Tween 0.5% with 1 mg/mL proteinase K at 56°C, followed by heat inactivation (100°C for 5 minutes) and centrifugation. Extracted DNA was tested for HPV DNA presence using the Optiplex HPV Genotyping Kit (Diamex GmbH) according to the manufacturer's instructions. This assay uses Luminex technology to detect 24 common HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). PCRs were performed using the Qiagen Multiplex PCR Kit (Qiagen GmbH). The assay includes primers for amplification of the human β-globin gene to confirm sample adequacy. Appropriate controls were included for sectioning (blank paraffin block), DNA extraction (reagent blanks), and HPV testing (HPV-positive Caski cell line DNA-positive control and water-negative control).

p16 IHC was undertaken as a surrogate marker of HPV oncogene expression (18) using a proprietary kit (CINtec p16 Histology, Ventana Medical Systems) on a Ventana Benchmark Autostainer. p16 IHC was scored as positive if there was strong and diffuse nuclear and cytoplasmic staining present in greater than 70% of malignant cells (18). All other patterns were scored as negative.

High-risk HPV DNA ISH was carried out using proprietary reagents (Inform HPV III Family 16 Probe (B), Ventana Medical Systems) on a Ventana Benchmark Autostainer. This test detects high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66, and tumors were scored as positive if any blue reaction product colocalized with the nuclei of malignant cells (19). Focal-specific staining of only part of the tumor section was regarded as positive. Diffuse staining of tumor and stromal tissues, representative of nonspecific chromogen precipitate, was scored as negative. Pale staining limited to the nucleoli of cells and staining of occasional leucocytes and stromal cells was also disregarded, in line with the manufacturer's instructions.

All p16 IHC and high-risk HPV DNA ISH testing was undertaken in UK hospital pathology laboratories holding Clinical Pathology Accreditation (CPA Ltd.). Both tests are registered in vitro diagnostic devices for clinical use and as such carry CE marking, with associated methodologic validation. Test results were scored by a panel of experienced head and neck pathologists, all of whom are accredited UK pathologists and members of the National Head and Neck Histopathology External Quality Assurance scheme administered by the Royal College of Pathologists. For quality assurance, the panel undertook a calibration exercise on a training set of OPSCC cases (whole sections and TMAs) prior to scoring the study material.

All cases were tested using the three HPV detection assays and were classified as HPV positive if they showed evidence of both HPV gene expression (indicated by p16 IHC) and HPV DNA (indicated by ISH and/or PCR). A diagnostic algorithm utilizing p16 IHC, HR HPV DNA ISH, and PCR in a stepwise fashion was applied (19, 20). The results of individual HPV diagnostic tests and clinically relevant combinations of tests are also reported.

Contemporaneous data on cancer incidence were obtained from the Office of National Statistics in England, NHS National Services Scotland, Northern Ireland Cancer Registry, and Welsh Cancer Intelligence & Surveillance Unit. ASRs were calculated for the following groups: oropharyngeal cancers (composed of C01, base of tongue; C05.1, soft palate; C05.2, uvula; C09, tonsil; C10.9, oropharynx not-otherwise-specified); laryngeal (C32); and mouth cancers [IARC definition, comprising C03, gum; C04, floor of mouth; C05, palate (excluding C05.1, soft palate and C05.2, uvula); and C06, other and unspecified parts of mouth], as representative head and neck subsites not associated with HPV infection (20). ASRs were calculated using the updated 2013 European Standard Population and mid-2012 UK population estimates (21).

The characteristics of included cases (gender, age at diagnosis, oropharyngeal subsite, year of diagnosis, and study center) were described using frequencies/percentages or means/SDs as appropriate. Characteristics were compared with excluded cases, and with Cancer Registry data using t tests or χ² tests as appropriate. The proportion of HPV-associated cases was calculated for the whole sample, then for each subset [with 95% confidence intervals (CI)]. Age was the only continuous variable and was categorized into five groups. The proportion of cases positive by p16 IHC, high-risk HPV DNA ISH, and HPV PCR was also calculated to allow comparison with studies reporting these endpoints, as was the prevalence of HPV types among cancers caused by a single HPV type. Trends in the proportion of HPV-associated cancers over time were assessed using Poisson regression with robust error variance (22). This approach was used because ORs (obtained from logistic regression) are poor approximations of risk ratios if the outcome prevalence is high. Models were fitted before and after adjusting for sample characteristics. Finally, HPV-positive proportions determined in the current study were applied to the UK incidence data to estimate the burden of oropharyngeal cancers caused by HPV over the period.

All analyses were undertaken in Stata 14.0 (StataCorp. 2015; Stata Statistical Software).

Figure 1 illustrates the application of study inclusion criteria and the HPV diagnostic testing algorithm. Valid results were obtained for 1,474 cases obtained from 11 centers. Sample characteristics (gender, age at diagnosis, oropharyngeal subsite, year of diagnosis, and study center) are shown in Table 1. The mean age of patients was 59.3 years, 75.0% of patients were male, and the majority of cases (57.9%) were tonsil cancers. Invalid results were obtained for 55 patients, and these were excluded from the analysis. The age, gender, and subsite distribution was similar for the included and excluded samples, and excluded samples were evenly distributed across the study period. The reasons for exclusion were either the absence of tumor in the TMA cores or loss of TMA cores during processing for staining.

The age and gender distributions were compared between OPSCC cases included in the current study, and those reported by UK Cancer Registries for the same period (Supplementary Table S1). The study sample included 8.3% of the 17,739 OPSCCs diagnosed in the United Kingdom from 2002 to 2011. The gender balance between the current study (75% male) and the UK OPSCC population (73.5% male) was similar. There was a higher proportion of younger patients (45–54.9 years) in the study sample (30.4%) than in the OPSCC population overall (25.7%; P = 0.001); however, the age distribution for cases in the two halves of the study period was similar (2002–2006 vs. 2007–2011, P = 0.5; Supplementary Table S2).

The prevalence of HPV infection in OPSCC was 51.8% (95% CI, 49.3–54.4). The prevalence of HPV infection within specific subgroups is shown in Table 2. The proportion of HPV-positive cases was higher in men than women (54.3% vs. 44.4%) and decreased with increasing age (69.2% in patients aged less than 44 years vs. 37.2% in patients aged over 75 years). The mean age of patients with HPV-positive disease was 57.4 years compared with 61.4 years for HPV-negative cases (P < 0.001). The prevalence of HPV infection varied depending on the tumor site, with the tonsil subsite showing the highest prevalence (61.8%) and soft palate/uvula showing the lowest (9.1%). HPV prevalence also varied between study centers, from 67.5% (95% CI, 59.7–74.4) in Liverpool to 35.4% (95% CI, 27.0–44.8) in Belfast. The variation in the overall proportion of samples defined as positive by each test (53.7% p16 positive, 45.0% ISH positive, 66.6% PCR positive) is consistent with published literature and reflects the established sensitivities and specificities of the assays (12–14).

There appeared to be little variation in prevalence across the 10-year study period (Fig. 2), either with HPV positives defined using the stepwise algorithm or for individual tests. Statistical models were used to assess change in the proportion of HPV-positive cases per year; the unadjusted risk ratio was 1.00 (95% CI, 0.99–1.02) for HPV infection for each year compared with the previous year (P_{linear trend} = 0.6), and the risk ratio adjusted for gender, age at diagnosis, anatomic subsite, and study center was 1.00 (95% CI, 0.98–1.03; P_{linear trend} = 0.7). This confirmed the absence of change in the proportion of HPV-positive samples over time.

Among the 764 HPV-positive OPSCCs, the Optiplex HPV Genotyping Kit identified a specific high-risk HPV type in 732 cases. In 710 (97%) of these cases, a single HPV type was present; the remaining 22 samples contained DNA of more than one HPV type. Among cases in which a single HPV type was detected, HPV16 was present in 684 of 710 cases (96.3%; 95% CI, 94.7–97.6) and HPV 18 in 11 of 710 cases (1.5%; 95% CI, 0.8–2.8; Supplementary Table S3). Among cases classified as HPV positive, HPV 16 and/or 18 were identified in 714 of 764 cases (93.5%; 95% CI, 91.5–95.1). HPV33 was detected in 20 cases (2.6%; 95% CI, 1.6–4.0); in 9 of these cases, HPV16 was also present.

Between 2002 and 2011, the UK incidence of OPSCC increased by 100.6%, whereas the incidence of laryngeal cancer increased by 9.3% (Fig. 3). The majority of OPSCCs occurred in men (2011 ASR: 6.3 male vs. 2.1 female). Laryngeal cancers were similarly more common in men than women (2011 ASR: 7.1 vs. 1.3). The incidence of mouth cancers increased by 45.4%, and there was a smaller difference in incidence between men and women than was observed with OPSCCs and laryngeal cancers (2011 ASR: 3.7 vs. 2.4).

To estimate the burden of oropharyngeal cancers caused by HPV over this period, the proportions determined in the current study were applied to the incidence data (Fig. 4). Figure 4 highlights increasing incidence of both HPV-positive and negative OPSCC, especially in men. It was notable that incidence curves for HPV-negative OPSCCs and mouth cancers show very similar trends. With regard to non-HPV–associated head and neck cancers in males, substantial absolute increases in ASR were observed for HPV-negative OPSCCs, and for mouth cancers, with a smaller increase in laryngeal cancers (1.72, 1.14, and 0.73/100,000 respectively).

This first nation-wide study investigating the prevalence of HPV in OPSCC within the United Kingdom showed that 51.8% (95% CI, 49.3–54.4) of cases diagnosed between 2002 and 2011 were HPV positive. Over the same period, the incidence of OPSCC in the broader population approximately doubled (ASR 2002: 2.1, ASR 2011: 4.1), while the relative proportions of HPV-positive and negative OPSCC remained stable over time. These data demonstrate a parallel rise in HPV-positive and negative disease incidence that has not previously been reported and show that, in the United Kingdom at least, the increasing incidence of OPSCC cannot be explained solely by an increase in HPV-associated disease.

The strengths of the study include large sample size, broad geographical representation, rigorous and systematic case selection, and use of well-validated commercial tests to identify HPV tumor status. The results are likely to reflect national trends, although there is potential for variation in OPSCC incidence, and in HPV prevalence, between different geographical areas in the United Kingdom. To assess potential bias in case selection, the records supplied by each center were formally reviewed. This showed that FFPE blocks from only 9 patients were unavailable due to use in other clinical studies. The study group included a higher proportion of younger patients (45–54.9 years) relative to the OPSCC population, but given the younger mean age for HPV-positive patients, this would be more likely to result in overestimation of the proportion of HPV-associated disease, rather than underestimation. The HPV testing regime included three independent, well-validated, commercial tests (IHC, ISH, and PCR), performed in independent laboratories, and the three tests showed highly similar trends in HPV prevalence (Fig. 2). The analysis of data pertaining to behavioral factors, such as smoking and sexual history, could potentially have allowed further interpretation of our results; however, due to the retrospective nature of sample and data collection, these data could not be reliably obtained.

It is important to stress that conclusions based on the data presented should not be generalized beyond the United Kingdom. Substantial variation has been reported in the proportion of OPSCCs attributable to HPV between countries and time periods (10, 11). This is likely to be a reflection of variations in multiple factors, including sexual behavior and rates of genital HPV infection, as well as tobacco and alcohol consumption. This highlights that trends in the etiology of OPSCC must be considered in a population-specific manner. Previous small, single-center studies from the United Kingdom reported HPV prevalence rates in OPSCC of 37.5% (95% CI, 28%–48%), 42.7% (95% CI, 36%–50%), and 55% (95% CI, 45%–66%; refs. 12–14). The current study is consistent with these but is based on a much larger sample with broader geographical representation, including centers in all four countries of the United Kingdom. We observed a consistent proportion of HPV-positive OPSCCs over time from 2002 to 2011, against a background of increasing incidence. This contrasts with previous data, detailing an increased proportion of HPV-positive OPSCCs associated with increasing incidence of OPSCCs overall (8). Recent data from North America (10) and Stockholm (23), however, suggest that plateaus in the proportion of HPV-positive OPSCC and HPV-positive tonsillar squamous cell carcinoma, respectively, have been observed from the year 2000 onwards.

The absence of change in the proportion of HPV-associated disease, despite a continued rise in incidence of OPSCC, implies that HPV-negative OPSCC, traditionally associated primarily with smoking (24), is also increasing in incidence. However, this contrasts with the more modest increase in incidence of other smoking-related head and neck malignancies, such as laryngeal cancer (Fig. 3; ref. 25), and suggests that another risk factor, in addition to HPV and smoking, may contribute to the increased overall incidence of OPSCC. Prior tonsillectomy appears to reduce risk of tonsillar carcinoma (26, 27), but although the current UK tonsillectomy rate is approximately 75% lower than in the 1950s (28), the absence of a disproportionate increase in OPSCCs specifically involving the tonsils, in our results and other published data (25), suggests this is not a major contributory factor to the increasing incidence of OPSCC. The observed increases in ASR among the different subsites (non-HPV OPSCC, mouth, and laryngeal) may reflect the degree of exposure of specific anatomic sites to individual carcinogens, including alcohol and tobacco smoke. In an analysis of Dutch HNSCC incidence, van Monsjou and colleagues (29) suggested that behavioral changes in the post-World War II generation included reduced smoking rates coupled with significant rises in alcohol consumption, and they suggested that excessive alcohol intake may be a more critical risk factor for OPSCC than smoking. In the United Kingdom, smoking rates have declined from 46% of adults in 1974 to 20% in 2010 (30, 31). However, since 1950, per capita alcohol consumption has increased from 3.9 L/year to a peak of 9.4 L/year in 2004 (32). This appears consistent with the increasing incidence of cancers at sites with greater exposure to alcohol (e.g., mouth) but smaller increases at sites more strongly associated with tobacco smoking (e.g., larynx).

It is probable that gender-neutral prophylactic HPV vaccination could prevent HPV-positive OPSCC (33). Indeed national bodies, such as the Joint Committee on Vaccination and Immunisation in the United Kingdom, are currently considering extending prophylactic HPV vaccination to include boys, as well as girls. Our data substantially expand the evidence base available to inform decisions such as this, particularly when viewed in the context of a projected substantial continued rise in OPSCC incidence of up to 239% in the next 20 years (34). The current bivalent and quadrivalent HPV vaccines protect against infection with oncogenic HPV types 16 and 18; these types were present in 714 of 764 cases (93.5%; 95% CI, 91.5–95.1) of HPV-positive OPSCCs in our study. Our data suggest that of the 1,781 cases of OPSCCs diagnosed in men in the United Kingdom in 2011, approximately 926 were HPV positive, and 866 were associated with HPV types included in current vaccines.

The parallel increase in both HPV-positive and HPV-negative tumors should be of concern to those involved in the clinical management of OPSCC, and to public health officials charged with developing strategies to reduce incidence. The data presented highlight that in the United Kingdom, increases in OPSCC incidence are not entirely due to HPV-associated disease. However, these findings should not be extrapolated to other developed world populations; rather, they emphasize the need to assess the etiology of head and neck cancers, and oropharyngeal cancers in particular, on a population-specific basis.

N.G. Powell reports receiving a commercial research grant from GlaxoSmithKline, has received speakers bureau honoraria from GlaxoSmithKline and Sanofi Pasteur MSD, and is a consultant/advisory board member for Sanofi Pasteur MSD. K.S. Cuschieri reports receiving commercial research grants from GeneFirst and Euroimmun. M. Robinson is a consultant/advisory board member for Leica Biosystems Ltd. H. Mehanna reports receiving commercial research grants from GSK Biologicals, GSL PLC, MSD, Sanofi Pasteur, and Silence Therapeutics and has received speakers bureau honoraria from MSD, Merck, and Sanofi Pasteur. H. Cubie has received speakers bureau honoraria from the GSK expert panel. E. Junor has provided expert testimony as a reviewer of Belgium Head and Neck Guideline. C.M.L. West has received speakers bureau honoraria from Merck. T.M. Jones reports receiving a commercial research grant from GSK and has received speakers bureau honoraria from Sanofi Pasteur. No potential conflicts of interest were disclosed by the other authors.

Study design, execution, interpretation, and manuscript preparation were undertaken by authorship as detailed without external influence.

Conception and design: N.G. Powell, M. Robinson, H. Mehanna, H. Cubie, E. Junor, K.J. Harrington, A.R. Ness, R.J. Shaw, M. Evans, T.M. Jones

Development of methodology: N.G. Powell, K.S. Cuschieri, M. Robinson, H. Mehanna, A. Long, C.M. Nutting, G.J. Thomas, D.J. McCance, J.A. James, R.J. Shaw, M. Evans, T.M. Jones

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.G. Schache, N.G. Powell, M. Robinson, H. Mehanna, D. Rapozo, E. Junor, H. Monaghan, K.J. Harrington, C.M. Nutting, U. Schick, A.S. Lau, N. Upile, J. Sheard, C.M.L. West, K. Oguejiofor, S. Thomas, M. Pring, G.J. Thomas, E.V. King, D.J. McCance, J.A. James, M. Moran, P. Sloan, R.J. Shaw, M. Evans, T.M. Jones

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.G. Schache, N.G. Powell, M. Robinson, S. Leary, K.J. Harrington, K. Brougham, S. Thomas, A.R. Ness, G.J. Thomas, J.A. James, M. Moran, R.J. Shaw, M. Evans, T.M. Jones

Writing, review, and/or revision of the manuscript: A.G. Schache, N.G. Powell, K.S. Cuschieri, M. Robinson, S. Leary, H. Mehanna, H. Cubie, H. Monaghan, K.J. Harrington, C.M. Nutting, A.S. Lau, C.M.L. West, S. Thomas, A.R. Ness, D.J. McCance, J.A. James, M. Moran, R.J. Shaw, M. Evans, T.M. Jones

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.G. Schache, N.G. Powell, K.S. Cuschieri, M. Robinson, H. Mehanna, A. Long, A.S. Lau, J. Sheard, K. Brougham, M. Pring, J.A. James, M. Moran, M. Evans, T.M. Jones

Study supervision: N.G. Powell, M. Robinson, E. Junor, C.M. Nutting, M. Evans, T.M. Jones

We would like to acknowledge the assistance of the staff of the respective National Cancer Registries within the United Kingdom, especially Jaroslaw Lang (Information Services Division, NHS National Services Scotland), Deirdre Fitzpatrick (Northern Ireland Cancer Registry - funded by the Public Health Agency), and Ceri White (Welsh Cancer Intelligence & Surveillance Unit).

T.M. Jones was the recipient of a research grant provided by GlaxoSmithKline Ltd. that funded the project research costs. In addition, the Northern Ireland Biobank, which provided access to tissue samples from that region, is funded by the Health and Social Research Development Division of the Public Health Agency. The University of Liverpool and Aintree University Hospitals NHS Foundation Trust jointly provided sponsorship for the research project.

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