Limited information is available about the involvement of human papillomavirus (HPV) in head and neck squamous cell carcinomas (HNSCCs) in Romanian patients.
Materials and methods
In total, 189 formalin-fixed paraffin-embedded tissue samples (99 oral cavity tumors, 28 oropharynx, 48 pharynx, and 14 larynx/hypopharynx) were analyzed for HPV DNA and RNA using Luminex-based assays, and for overexpression of p16INK4a (p16) by immunohistochemistry.
Of the 189 cases, 23 (12.2%) were HPV DNA-positive, comprising half of the oropharyngeal cases (14/28, 50.0%) and 9/161 (5.6%) of the non-oropharyngeal cases. HPV16 was the most prevalent HPV type (20/23, 86.9%), followed by HPV18 (5/23, 21.7%) and HPV39 (1/23, 4.3%). Only two (2/189, 1.1%) HNSCC cases were HPV-driven, i.e. positive for both HPV DNA and RNA.
Citation: Ursu RG, Danciu M, Spiridon IA, Ridder R, Rehm S, Maffini F, et al. (2018) Role of mucosal high-risk human papillomavirus types in head and neck cancers in Romania. PLoS ONE 13(6): e0199663. https://doi.org/10.1371/journal.pone.0199663
Editor: Maria Lina Tornesello, Istituto Nazionale Tumori IRCCS Fondazione Pascale, ITALY
Received: February 1, 2018; Accepted: June 12, 2018; Published: June 25, 2018
Copyright: © 2018 Ursu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This study was supported by the European Commission, grant HPV-AHEAD (FP7-HEALTH- 2011-282562) to MT. RGU is supported by the University of Medicine and Pharmacy “Grigore T. Popa”, Iasi, Romania (grant no. 30336 / 28.12.2017). RR and SR are employees of Roche Diagnostics, a company that commercializes in vitro diagnostic tests used in this study. The funder provided support in the form of salaries for authors RR and SR, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
Competing interests: RR and SR are employees of Roche Diagnostics, a company that commercializes in vitro diagnostic tests used in this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with an estimated annual burden of 355,000 deaths and 633,000 incident cases . Romania ranks second in mortality from HNSCCs in all-age males (32.4/100,000) among European countries .
HNSCCs are etiologically heterogeneous, being caused by tobacco use, alcohol consumption, poor oral hygiene, exposure to certain chemicals, and genetic features [2–4], as well as viral infections [5, 6]. High-risk (HR) human papillomavirus (HPV) infections have been associated with a subset of HNSCCs [7, 8]. HPV16 is the most common type, being present in more than 80% of HNSCCs [9, 10].
Chaturvedi et al. (2013) reported that the incidence of oropharyngeal cancer increased significantly in developed countries from 1983 to 2002 . The proportion of HPV-positive oropharyngeal cancers among HNSCCs has been increasing over the past decades in many parts of the world, whereas the overall incidence of HNSCC is decreasing, consistent with declines in tobacco use . Several studies reported a steady increase in the proportion of HPV-driven oropharyngeal cancer cases in the United States , in Sweden [14, 15], in Australia , and in New Zealand . HPV has also been associated, to a much lesser extent, with non-oropharyngeal cancers such as oral or laryngeal cancer. In central India, less than 2% of these cancers were HPV-driven .
The prevalence of HPV DNA in HNSCCs varies greatly by study, cancer site, and geographical area [19, 20], being high in oropharyngeal cancer cases from the United States (71.0%) , eastern Denmark (62%) , and the Czech Republic (57.0%) , whereas several studies reported the absence of HPV DNA in oropharyngeal cancer cases from Mozambique  and China , or a low or intermediate HPV prevalence in Germany (34.4%) and Brazil (15.5%) . All these studies are based on HPV DNA detection techniques. However, several independent studies have highlighted that the detection of HPV DNA alone is not sufficient to accurately define HPV-driven HNSCCs [18, 27–29]. The use of additional markers, such as viral RNA and p16INK4a (p16) expression as a surrogate for HPV-induced transformation, allows a more precise classification of HNSCC.
In a recent study, the HPV-attributable fraction based on positivity for HPV DNA and for either HPV E6*I mRNA or p16, was 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, oral cavity, and larynx, respectively . Similar rates have been obtained in Kazakhstan, where 25.7% of oropharyngeal cancer cases tested positive for HPV DNA and p16 , and in Northeastern Italy, where 20% of oropharyngeal cancer cases tested positive for HPV DNA and HPV RNA . In central India, HPV DNA/RNA double positivity was found in only 9.4% of oropharyngeal cancer cases . HNSCCs from the Philippines all tested negative for both HPV DNA and HPV RNA . In addition, in a recent study  based on 3680 HNSCCs from Europe, Africa, Asia, and the Americas, 22.4% of the oropharyngeal cancers tested positive for HPV DNA and for either HPV RNA or p16, and 18.5% were positive for all three markers. South America had the highest HPV-attributable fraction (53.6%) in oropharyngeal cancer, followed by Central and Eastern Europe (50.0%), Northern Europe (50.0%), Eastern Asia (22.4%), Central America (19.7%), Western Europe (19.4%), and Southern Europe (9.4%).
In Romania, limited information is available about the involvement of HPV in HNSCC. In this study, we aimed to determine the HPV-attributable fraction in HNSCC by analyzing HPV DNA and HPV RNA status, as well as by determining the p16 expression level, within a large retrospective cohort of HNSCC cases from Northeastern Romania.
Materials and methods
Patients and samples
Two hundred and three HNSCC patients were identified in the Departments of Oral and Maxillofacial Surgery, Otorhinolaryngology, and Plastic Surgery at the University of Medicine and Pharmacy “Grigore T. Popa” (Iași, Romania), from January 2010 to September 2014. All specimens were fixed for 18–24 hours in 10% neutral buffered formalin, at room temperature. The formalin-fixed, paraffin-embedded (FFPE) HNSCC blocks included squamous cell carcinoma of the oropharynx (International Classification of Diseases for Oncology [ICD-O] C01 –base of tongue, C02.4 –lingual tonsil, C09 –tonsil, C10 –oropharynx), pharynx (ICD-O C14 –other and ill-defined sites in the lip, oral cavity and pharynx, C14.8 –overlapping lesion of lip, oral cavity and pharynx), oral cavity (ICD-O: C00.0–C00.9, C01, C02.0–C02.9, C03.0–C03.9, C04.0–C04.9, C05.1–C05.9, C06.0–C06.9, C09.1–C09.9, C10), and hypopharynx and larynx (ICD-O: C13, C32). The FFPE tissue samples were retrieved from the hospital archives and comprised 34 HNSCC cases from the oropharynx and 169 HNSCC cases outside the oropharynx (16 larynx/hypopharynx, 51 pharynx, and 102 oral cavity cancer samples). All patients were diagnosed with keratinizing or non-keratinizing squamous cell carcinomas. Histological analyses on hematoxylin and eosin (H&E) stained slides were performed in order to confirm that all FFPE blocks contain cancer tissues. Clinical and epidemiological information was collected from the hospital databases using a form and questionnaire developed in the context of a European and Indian case study (HPV-AHEAD; http://hpv-ahead.iarc.fr). Ethical clearance for the investigations reported in this study was obtained from the Institutional Ethical Committee of the University of Medicine and Pharmacy “Grigore T. Popa”, Iași, Romania (reference number 7150). The study implied the use of archival material only, and it did not envisage any contact with the patients. Adequate measures to ensure data protection, confidentiality, patients’ privacy, and anonymization were taken into account. No informed consent was available, due to the retrospective design of the study and the large proportion of deceased and untraceable patients. All data were fully anonymized before access.
Preparation of paraffin sections and DNA extraction
Each FFPE block was sectioned according to the HPV-AHEAD protocol, which includes the preparation of 31 sections from each FFPE tissue block. Sections 1, 10, and 31 (S1, S10, and S31) were used for histology, S2 and S9 were used for p16 immunohistochemistry (IHC), and S11–S30 were stored for future IHC analyses in independent studies. In addition, S3–S5 and S6–S8 were collected in two different vials and subsequently used for DNA and RNA analysis . To minimize the risk of cross-contamination during sectioning, a new blade was used for each FFPE block and the microtome was extensively cleaned after each block with ethanol 70% and DNA Away (Dutscher, Brumath, France). In addition, to monitor possible cross-contamination during the sectioning, empty paraffin blocks were processed every 10th cancer specimen. DNA was extracted by an overnight incubation of the paraffin tissue sections in a digestion buffer (10 mM Tris/HCl pH 7.4, 0.5 mg/ml proteinase K, and 0.4% Tween 20) . The percentage of tumor cells (0%, <10%, 10–50%, 50–90%, >90%) was estimated by two pathologists (MD, IAS) on H&E-stained slides (S1 and S10) .
HPV DNA genotyping
HPV DNA positivity was determined by using a type-specific multiplex genotyping (TS-MPG) assay, which combines multiplex PCR and bead-based Luminex technology (Luminex Corporation, Austin, TX) as previously described [36, 37]. This assay detects 19 HR or probable high-risk (pHR) HPV types (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68a and b, 70, 73, and 82) and two low-risk (LR) HPV types (HPV6 and 11), as well as cellular beta-globin gene, which is used to control for DNA quality. After PCR amplification, 10 μl of each reaction mixture was analyzed by multiplex HPV genotyping (MPG) using Luminex technology (Luminex Corporation, Austin, TX) as described previously [37, 38]. All HPV DNA-positive FFPE specimens and a randomly selected subgroup of approximately 10% of HPV DNA-negative specimens were further analyzed for the presence of HPV E6*I mRNA and for overexpression of the cell-cycle inhibitor p16, which is considered a surrogate marker for HPV infection. The 10% of HPV DNA-negative cases were selected randomly and blindly, while the study was still anonymized.
HPV RNA analysis
Total RNA was purified from three pooled sections of the same tissue block using the Pure Link FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA) as described previously . RT-PCR was carried out using the QuantiTect Virus Kit (Qiagen, Hilden, Germany), in a total volume of 25 μl containing 5 μl of 5xQuantiTect Virus Mastermix, 0.25 μl of 100xQuantiTect Virus RT Mix, 0.4 μM of each oligonucleotide, and 1 μl RNA as described previously . The HPV type-specific E6*I mRNA assay developed for 20 HR- or pHR-HPV types  was applied for the detection of viral transcripts. The assay amplifies a 65–75 base pair amplicon of HPV and an 81 base pair amplicon of ubiquitin C (ubC) cDNA. Biotinylated amplification products are hybridized to ubC and HPV type-specific probes representing splice junction sequences on Luminex beads, followed by staining with streptavidin-phycoerythrin, and quantified in a Luminex analyzer. The use of a splice product sequence as detection probe makes this assay absolutely specific for RNA and avoids false positivity from residual viral DNA in the RNA preparation, which is a risk in RNA assays assessing unspliced RNA sequences.
The analytical sensitivity of the respective assays per reaction is 10,000 copies for HPV70, 1,000 copies for HPV67, and 10–100 copies for the remaining 19 HPV types and for ubC . The HPV RNA assay has been widely applied and validated as a marker for HPV transformation in carcinoma of the anogenital region, such as the cervix [39, 40], vulva , penis , lung , and scrotum , as well as carcinoma of the head and neck [30, 33], and specifically oropharynx [45, 46], unknown primary of the neck [47, 48], larynx , and esophagus .
All HPV DNA-positive specimens and the randomly selected 10% of HPV DNA-negative specimens were analyzed for the presence of (i) HPV16 E6*I mRNA and (ii) ubC mRNA as a cellular mRNA positive control. Tissues positive for DNA of a non-HPV16 type were, in addition, analyzed for E6*I mRNA of the respective type. Specimens that were HPV E6*I and/or ubC mRNA-positive (RNA+) in RNA analysis were considered HPV RNA valid.
Expression of p16 was evaluated manually by IHC in FFPE sections using the CINtec p16 Histology kit (Roche mtm laboratories AG, Mannheim, Germany) according to the instructions of the manufacturer. Briefly, slides were de-paraffinized in xylene and rehydrated in graded alcohol. The antigens were retrieved for 10 minutes using a pH 9.0 epitope retrieval solution (95–99 °C), followed by a 20 minute cool-down period at room temperature. Different from the instructions of the manufacturer, specimens were then microwaved in preheated Vector H-3300 unmasking solution (Vector Laboratories, Burlingame, CA) for 15 minutes. This step was followed by incubation of the p16 primary mouse anti-human antibody (clone E6H4) for 60 minutes. The samples were subsequently incubated with the goat anti-mouse IgG secondary antibody/peroxidase conjugate reagent, followed by signal generation using DAB. Finally, slides were counterstained with hematoxylin, dehydrated, mounted with permanent mounting medium, and cover-slipped. Immunoreactivity was visualized by light microscopy. Expression of p16 was evaluated by IHC in all HPV DNA-positive FFPE specimens and in a randomly selected subgroup of approximately 10% of HPV DNA-negative specimens. A continuous, diffuse staining for p16 within the cancer area of the tissue sections was considered as positive, and a focal staining or no staining was considered negative. Positive p16 expression was defined as diffuse nuclear and cytoplasmic staining in 70% or more of the tumor cells. The validity of the p16 IHC staining result was assessed by evaluating the presence of p16 internal control staining. IHC slides were evaluated by RR, FM, and DH blinded to any other clinical information or HPV DNA or RNA status, as specified in the HPV-AHEAD protocol . Discrepant cases were re-checked by a pathologist, and the final classification of the staining was based on the majority consensus of the working group.
Of the 203 HNSCC cases, 2 cases (1 oral cavity and 1 pharyngeal) were excluded due to insufficient DNA quality as evidenced by negative ß-globin results, and 11 cases were excluded due to invalid RNA and/or p16 data. One case was excluded as the tissue block did not contain cancer tissue. The final study therefore comprised 189 HNSCC patients, with a median age of 62.5 years (range, 35–89 years). The vast majority of the patients were male: n = 171 (90.5%) (Table 1). Only FFPE blocks where the first and last H&E sections reflected tumor tissue were included in the study. More than 36% of the samples showed >50% of invasive carcinoma in the section, while 47.6% and 15.9% of the samples showed respectively 10–50%, and <10% of tumor cells.
Table 2 shows the HPV DNA, RNA, and p16 detection in HNSCC cases. HPV DNA was detected in 23 of the 189 (12.2%) HNSCC cases. HPV16 was the most prevalent type, being present in 20 of the 23 HPV DNA-positive tumors (86.9%), followed by HPV18 (5/23, 21.7%) and HPV39 (1/23, 4.3%). The oropharynx cases showed higher HPV DNA prevalence (14/28, 50.0%), followed by cancers of the larynx (5/14, 35.7%) and of the oral cavity (4/99, 4.0%). Multiple HPV type infections were detected in 3 HNSCC cases; 2 cases were positive for both HPV16 and HPV18 (1 larynx case and oropharynx case), and 1 oral cancer was positive for both HPV18 and HPV39 (Tables 1 and 2). One larynx case was positive for a low-risk HPV type (HPV6).
HPV RNA and p16 expression was examined in all HPV DNA-positive cases (n = 23) and a randomly selected subset of HPV DNA-negative cases (n = 13). The percentage of HPV-related HNSCCs was 1.1% (2/189) for both HPV DNA and RNA positivity. The highest percentage of combined HPV DNA and RNA positivity was found in the oropharynx (1 of the 28 HPV DNA-positive cases, 3.6%). The corresponding tonsil case tested positive for HPV18. Only one non-oropharyngeal case (1/161, 0.6%) was positive for both HPV DNA and RNA. The corresponding posterior hypopharyngeal wall case tested positive for HPV16 (Table 2).
The p16 IHC data were stratified per HR-HPV DNA and RNA status (Table 3). Only one HPV DNA-positive case (1/23; 4.3%) was p16-positive, and 7.7% (1/13) of HPV DNA- and RNA-negative cases were p16-positive, regardless of the anatomical sub-localization. In addition, none of the HNSCC cases that were HPV DNA- and RNA-positive tested positive by p16 IHC. Moreover, 2 of the 34 HPV RNA-negative cases (5.9%) were p16-positive (Table 3).
Among the cases that tested positive for HPV DNA, the smoking status was available for only 10 patients, among whom 8 were current smokers and 2 were former smokers. Most importantly, the clinical information was available for the HPV-driven HNSCCs (n = 2). Both tumors (1 tonsil case and posterior hypopharyngeal wall case) were late-stage (III and IV) and were from current smokers: patients aged 54 years (female) and 55 years (male), respectively.
It is now well demonstrated that mucosal HR-HPV types, mainly HPV16, are causally involved in a significant proportion of oropharyngeal cancers and to a much lesser extent in a subset of other HNSCCs . However, the contribution of HR-HPV to the carcinogenesis of HNSCC appears to be subject to major geographical variability .
Compared with other European countries, cervical cancer rates are highest in Romania (28.6/100,000), highlighting the importance of HPV infections in this population. However, limited information is available about HPV-associated HNSCC in Romania . Here, we have evaluated the contribution of HPV to HNSCC development in a study in Northeastern Romania by analyzing HPV DNA and HPV RNA status within a large retrospective cohort of HNSCC cases, as well as by determining the p16 expression status. The most frequent HR-HPV type was HPV16, followed by HPV18 and HPV39. The high HPV16 DNA prevalence in this study was similar to other findings published in the literature [51, 52].
Approximately 12% of HNSCCs tested positive for HPV DNA, but only 1.1% of all cases (n = 2) were positive for both HPV DNA and RNA and thus considered as being HPV-driven [18, 53–56]. According to a recent review , the term HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) refers to carcinomas of the oropharynx presumed to be associated with HPV, on the basis of positivity to HPV DNA and p16 IHC. In this study, only one case tested positive for both markers: HPV DNA and p16. Thus, the fraction of HNSCCs attributable to transforming HPV infections in this Romanian region appeared to be considerably lower compared with various other geographical regions [10, 30, 57]. However, the low HPV prevalence in the FFPE samples is in line with the analysis of fresh tumor tissues from Romania, all being HPV-negative .
Both HPV DNA- and RNA-positive cases tested negative for p16. In addition, the great majority (95.6%) of HPV DNA-positive cases were p16-negative. The lack of expression of p16 in HPV DNA- and RNA-positive cases has been reported in other studies [18, 30]. These data contrast with the scenario observed in the Netherlands, where a double positivity for p16 and HPV DNA was shown to be valid to identify HPV RNA-positive cases [55, 57], and in Italy, where a fair agreement between HPV16 RNA-positivity and p16 overexpression in oropharyngeal cancer has been reported . The absence of p16 expression in HPV DNA-positive HNSCC could be due to the fact that HPV, despite its presence in the tumor, is biologically inactive and is present in the tumor as a passenger virus or viral contaminant. Loss of p16 expression is a frequent event in cancer, and it occurs by deletion, point mutation, or hypermethylation [60–63]. The inactivation of p16 by hypermethylation of its promotor is common in HNSCC [64–66]. Hypermethylation of p16 promotor has been reported to be an early event in the development of oral cancer [67, 68]. Exposure to certain carcinogens, such as tobacco, may lead to alterations of p16 expression . Indeed, hypermethylation of the p16 promoters was observed in several smoking-related human cancers, for example in non-small cell lung carcinoma [69–71] and cervical squamous cell carcinoma . In addition, increased methylation of p16 was observed in laryngeal squamous cell carcinoma , and in normal oral mucosa , in smokers. Therefore, loss of p16 expression by hypermethylation in HNSCC, due to smoking or to other exposure factors [75, 76], could precede HPV infection, which would not induce p16 accumulation in this specific circumstance. Alternatively, Halec et al.  suggested that increasing chromosomal instability induced by HPV oncoproteins may lead to the loss of p16 in these cancers.
Moreover, one HPV DNA- and RNA-negative case tested positive for p16. A similar result was reported in a recent worldwide HNSCC study, thus suggesting that p16-positivity is not a perfect surrogate for HPV .
A limitation of our study is that information on other HNSCC risk factors (e.g. alcohol consumption, smoking) was available for only a few patients. This limitation was mainly due to the fact that the study implied the retrieval of archived HNSCC specimens, which were often not associated with detailed clinical information. From the available data in clinical questionnaires, 75.6% of the patients declared that they were smokers and 81.3% that they were users of alcohol. According to the latest WHO Report on the Global Tobacco Epidemic, 2015, the smoking prevalence in Romanian male adults was 37.4% . This high percentage supports the idea that smoking can be an important risk factor for HNSCC in our study.
Tobacco smoking and alcohol consumption are important risk factors for HNSCC [12, 78]. More than 70% of HNSCCs are attributable to tobacco use and alcohol consumption . Cigarette smoking is a strong risk factor for HNSCC independent of alcohol consumption . The risk of developing laryngeal cancer was 10–20-fold higher in current smokers compared with non-smokers, and a 4–5-fold increased risk was observed for cancers of the oral cavity, oropharynx, and hypopharynx [79–81]. Alcohol consumption alone plays an independent role in approximately 4% of HNSCCs only . However, pooled data from 17 case–control studies in Europe and the USA highlighted a multiplicative joint effect, rather than an additive effect, of tobacco use and alcohol consumption on HNSCC risk .
Another limitation of the study was the limited number of oropharyngeal cancers analyzed. This was mainly due to the fact that the majority of archival HNSCCs were from the oral cavity.
The results of this study warrant additional analyses, to describe the risk factors, the natural history and the clinical role of oral HPV infections in Romania.
In conclusion, a very small subset of HNSCC cases within this cohort from Northeastern Romania appeared to be HPV-driven, as evidenced by a low concordance between HPV DNA status and HPV RNA or p16 status of the analyzed HNSCC cases. Our study provides novel insights into the contribution of mucosal HR-HPV types in the development of HNSCC from Northeastern Romania and highlights potential differences in the carcinogenesis of HNSCC in this region compared with other European and non-European countries.
We are grateful to Dr. Karen Müller and Jessica Cox for editing, and to Nicole Suty for her help with preparation of this manuscript.
- 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–86. Epub 2014/09/16. pmid:25220842.
- 2. Barul C, Fayosse A, Carton M, Pilorget C, Woronoff AS, Stucker I, et al. Occupational exposure to chlorinated solvents and risk of head and neck cancer in men: a population-based case-control study in France. Environmental health: a global access science source. 2017;16(1):77. Epub 2017/07/26. pmid:28738894; PubMed Central PMCID: PMCPMC5525363.
- 3. Stornetta A, Guidolin V, Balbo S. Alcohol-Derived Acetaldehyde Exposure in the Oral Cavity. Cancers. 2018;10(1). Epub 2018/01/19. pmid:29342885; PubMed Central PMCID: PMCPMC5789370.
- 4. Riaz N, Morris LG, Lee W, Chan TA. Unraveling the molecular genetics of head and neck cancer through genome-wide approaches. Genes & diseases. 2014;1(1):75–86. Epub 2015/02/03. pmid:25642447; PubMed Central PMCID: PMCPMC4310010.
- 5. Marur S, D'Souza G, Westra WH, Forastiere AA. HPV-associated head and neck cancer: a virus-related cancer epidemic. The Lancet Oncology. 2010;11(8):781–9. Epub 2010/05/11. pmid:20451455; PubMed Central PMCID: PMCPMC5242182.
- 6. Raab-Traub N. Epstein-Barr virus in the pathogenesis of NPC. Semin Cancer Biol. 2002;12(6):431–41. Epub 2002/11/27. pmid:12450729.
- 7. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst. 2000;92(9):709–20. Epub 2000/05/04. pmid:10793107.
- 8. Gillison ML, Castellsague X, Chaturvedi A, Goodman MT, Snijders P, Tommasino M, et al. Eurogin Roadmap: comparative epidemiology of HPV infection and associated cancers of the head and neck and cervix. Int J Cancer. 2014;134(3):497–507. Epub 2013/04/10. pmid:23568556.
- 9. Bratman SV, Bruce JP, O'Sullivan B, Pugh TJ, Xu W, Yip KW, et al. Human Papillomavirus Genotype Association With Survival in Head and Neck Squamous Cell Carcinoma. JAMA oncology. 2016;2(6):823–6. Epub 2016/03/25. pmid:27010835.
- 10. Ndiaye C, Mena M, Alemany L, Arbyn M, Castellsague X, Laporte L, et al. HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis. The Lancet Oncology. 2014;15(12):1319–31. Epub 2014/12/03. pmid:25439690.
- 11. Chaturvedi AK, Anderson WF, Lortet-Tieulent J, Curado MP, Ferlay J, Franceschi S, et al. Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2013;31(36):4550–9. Epub 2013/11/20. pmid:24248688; PubMed Central PMCID: PMCPMC3865341.
- 12. Sturgis EM, Cinciripini PM. Trends in head and neck cancer incidence in relation to smoking prevalence: an emerging epidemic of human papillomavirus-associated cancers? Cancer. 2007;110(7):1429–35. Epub 2007/08/29. pmid:17724670.
- 13. Chaturvedi AK, Engels EA, Anderson WF, Gillison ML. Incidence trends for human papillomavirus-related and -unrelated oral squamous cell carcinomas in the United States. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2008;26(4):612–9. Epub 2008/02/01. pmid:18235120.
- 14. Nasman A, Attner P, Hammarstedt L, Du J, Eriksson M, Giraud G, et al. Incidence of human papillomavirus (HPV) positive tonsillar carcinoma in Stockholm, Sweden: an epidemic of viral-induced carcinoma? Int J Cancer. 2009;125(2):362–6. Epub 2009/03/31. pmid:19330833.
- 15. Attner P, Du J, Nasman A, Hammarstedt L, Ramqvist T, Lindholm J, et al. The role of human papillomavirus in the increased incidence of base of tongue cancer. Int J Cancer. 2010;126(12):2879–84. Epub 2009/10/27. pmid:19856308.
- 16. Hong AM, Grulich AE, Jones D, Lee CS, Garland SM, Dobbins TA, et al. Squamous cell carcinoma of the oropharynx in Australian males induced by human papillomavirus vaccine targets. Vaccine. 2010;28(19):3269–72. Epub 2010/03/17. pmid:20226244.
- 17. Lucas-Roxburgh R, Benschop J, Lockett B, van den Heever U, Williams R, Howe L. The prevalence of human papillomavirus in oropharyngeal cancer in a New Zealand population. PLoS One. 2017;12(10):e0186424. Epub 2017/10/20. pmid:29049330; PubMed Central PMCID: PMCPMC5648183.
- 18. Gheit T, Anantharaman D, Holzinger D, Alemany L, Tous S, Lucas E, et al. Role of mucosal high-risk human papillomavirus types in head and neck cancers in central India. Int J Cancer. 2017;141(1):143–51. Epub 2017/04/04. pmid:28369859.
- 19. Chaturvedi AK. Epidemiology and clinical aspects of HPV in head and neck cancers. Head and neck pathology. 2012;6 Suppl 1:S16–24. Epub 2012/07/13. pmid:22782220; PubMed Central PMCID: PMCPMC3394159.
- 20. Gillison ML, Chaturvedi AK, Anderson WF, Fakhry C. Epidemiology of Human Papillomavirus-Positive Head and Neck Squamous Cell Carcinoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2015;33(29):3235–42. Epub 2015/09/10. pmid:26351338; PubMed Central PMCID: PMCPMC4979086.
- 21. Goodman MT, Saraiya M, Thompson TD, Steinau M, Hernandez BY, Lynch CF, et al. Human papillomavirus genotype and oropharynx cancer survival in the United States of America. European journal of cancer (Oxford, England: 1990). 2015;51(18):2759–67. Epub 2015/11/26. pmid:26602016; PubMed Central PMCID: PMCPMC4666760.
- 22. Carlander AF, Gronhoj Larsen C, Jensen DH, Garnaes E, Kiss K, Andersen L, et al. Continuing rise in oropharyngeal cancer in a high HPV prevalence area: A Danish population-based study from 2011 to 2014. European journal of cancer (Oxford, England: 1990). 2017;70:75–82. Epub 2016/11/27. pmid:27888679.
- 23. Tachezy R, Klozar J, Salakova M, Smith E, Turek L, Betka J, et al. HPV and other risk factors of oral cavity/oropharyngeal cancer in the Czech Republic. Oral diseases. 2005;11(3):181–5. Epub 2005/05/13. pmid:15888110.
- 24. Blumberg J, Monjane L, Prasad M, Carrilho C, Judson BL. Investigation of the presence of HPV related oropharyngeal and oral tongue squamous cell carcinoma in Mozambique. Cancer epidemiology. 2015;39(6):1000–5. Epub 2015/11/22. pmid:26590333.
- 25. Chen XJ, Sun K, Jiang WW. Absence of high-risk HPV 16 and 18 in Chinese patients with oral squamous cell carcinoma and oral potentially malignant disorders. Virol J. 2016;13:81. Epub 2016/05/22. pmid:27206495; PubMed Central PMCID: PMCPMC4875721.
- 26. Hauck F, Oliveira-Silva M, Dreyer JH, Perrusi VJ, Arcuri RA, Hassan R, et al. Prevalence of HPV infection in head and neck carcinomas shows geographical variability: a comparative study from Brazil and Germany. Virchows Archiv: an international journal of pathology. 2015;466(6):685–93. Epub 2015/03/31. pmid:25820374.
- 27. Halec G, Holzinger D, Schmitt M, Flechtenmacher C, Dyckhoff G, Lloveras B, et al. Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma. Br J Cancer. 2013;109(1):172–83. Epub 2013/06/20. pmid:23778529; PubMed Central PMCID: PMCPMC3708587.
- 28. Jung AC, Briolat J, Millon R, de Reynies A, Rickman D, Thomas E, et al. Biological and clinical relevance of transcriptionally active human papillomavirus (HPV) infection in oropharynx squamous cell carcinoma. Int J Cancer. 2010;126(8):1882–94. Epub 2009/10/02. pmid:19795456.
- 29. Chernock RD, Wang X, Gao G, Lewis JS Jr., Zhang Q, Thorstad WL, et al. Detection and significance of human papillomavirus, CDKN2A(p16) and CDKN1A(p21) expression in squamous cell carcinoma of the larynx. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc. 2013;26(2):223–31. Epub 2012/09/22. pmid:22996374; PubMed Central PMCID: PMCPMC3529982.
- 30. Castellsague X, Alemany L, Quer M, Halec G, Quiros B, Tous S, et al. HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients. J Natl Cancer Inst. 2016;108(6):djv403. Epub 2016/01/30. pmid:26823521.
- 31. Adilbay D, Adilbayev G, Kidirbayeva G, Shipilova V, Sadyk Z, Koyanbekova G, et al. HPV infection and P16 expression in oral and oropharyngeal cancer in Kazakhstan. Infect Agent Cancer. 2018;13:2. Epub 2018/01/19. pmid:29344081; PubMed Central PMCID: PMCPMC5767046.
- 32. Baboci L, Holzinger D, Boscolo-Rizzo P, Tirelli G, Spinato R, Lupato V, et al. Low prevalence of HPV-driven head and neck squamous cell carcinoma in North-East Italy. Papillomavirus research (Amsterdam, Netherlands). 2016;2:133–40. Epub 2017/10/28. pmid:29074172; PubMed Central PMCID: PMCPMC5886905.
- 33. Albano PM, Holzinger D, Salvador C, Orosa J 3rd, Racelis S, Leano M, et al. Low prevalence of human papillomavirus in head and neck squamous cell carcinoma in the northwest region of the Philippines. PLoS One. 2017;12(2):e0172240. Epub 2017/02/16. pmid:28199413; PubMed Central PMCID: PMCPMC5310881.
- 34. Mena M, Lloveras B, Tous S, Bogers J, Maffini F, Gangane N, et al. Development and validation of a protocol for optimizing the use of paraffin blocks in molecular epidemiological studies: The example from the HPV-AHEAD study. PLoS One. 2017;12(10):e0184520. Epub 2017/10/17. pmid:29036167; PubMed Central PMCID: PMCPMC5642890.
- 35. Gheit T, Vaccarella S, Schmitt M, Pawlita M, Franceschi S, Sankaranarayanan R, et al. Prevalence of human papillomavirus types in cervical and oral cancers in central India. Vaccine. 2009;27(5):636–9. Epub 2008/12/06. pmid:19056450.
- 36. Gheit T, Landi S, Gemignani F, Snijders PJ, Vaccarella S, Franceschi S, et al. Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types. J Clin Microbiol. 2006;44(6):2025–31. Epub 2006/06/08. pmid:16757593; PubMed Central PMCID: PMCPMC1489390.
- 37. Schmitt M, Dondog B, Waterboer T, Pawlita M, Tommasino M, Gheit T. Abundance of multiple high-risk human papillomavirus (HPV) infections found in cervical cells analyzed by use of an ultrasensitive HPV genotyping assay. J Clin Microbiol. 2010;48(1):143–9. Epub 2009/10/30. pmid:19864475; PubMed Central PMCID: PMCPMC2812266.
- 38. Gheit T, Abedi-Ardekani B, Carreira C, Missad CG, Tommasino M, Torrente MC. Comprehensive analysis of HPV expression in laryngeal squamous cell carcinoma. J Med Virol. 2014;86(4):642–6. Epub 2014/01/01. pmid:24374907.
- 39. Halec G, Schmitt M, Dondog B, Sharkhuu E, Wentzensen N, Gheit T, et al. Biological activity of probable/possible high-risk human papillomavirus types in cervical cancer. Int J Cancer. 2013;132(1):63–71. Epub 2012/04/20. pmid:22514107.
- 40. Halec G, Alemany L, Lloveras B, Schmitt M, Alejo M, Bosch FX, et al. Pathogenic role of the eight probably/possibly carcinogenic HPV types 26, 53, 66, 67, 68, 70, 73 and 82 in cervical cancer. J Pathol. 2014;234(4):441–51. pmid:25043390.
- 41. Halec G, Alemany L, Quiros B, Clavero O, Hofler D, Alejo M, et al. Biological relevance of human papillomaviruses in vulvar cancer. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc. 2017;30(4):549–62. Epub 2017/01/07. pmid:28059099.
- 42. Alemany L, Cubilla A, Halec G, Kasamatsu E, Quiros B, Masferrer E, et al. Role of Human Papillomavirus in Penile Carcinomas Worldwide. European urology. 2016;69(5):953–61. Epub 2016/01/15. pmid:26762611.
- 43. Anantharaman D, Gheit T, Waterboer T, Halec G, Carreira C, Abedi-Ardekani B, et al. No causal association identified for human papillomavirus infections in lung cancer. Cancer Res. 2014;74(13):3525–34. Epub 2014/04/25. pmid:24760422.
- 44. Guimera N, Alemany L, Halec G, Pawlita M, Wain GV, Vailen JSS, et al. Human papillomavirus 16 is an aetiological factor of scrotal cancer. Br J Cancer. 2017;116(9):1218–22. Epub 2017/04/05. pmid:28376081; PubMed Central PMCID: PMCPMC5418448.
- 45. Holzinger D, Wichmann G, Baboci L, Michel A, Hofler D, Wiesenfarth M, et al. Sensitivity and specificity of antibodies against HPV16 E6 and other early proteins for the detection of HPV16-driven oropharyngeal squamous cell carcinoma. Int J Cancer. 2017;140(12):2748–57. Epub 2017/03/21. pmid:28316084.
- 46. Baboci L, Boscolo-Rizzo P, Holzinger D, Bertorelle R, Biasini L, Michel A, et al. Evidence of the causal role of human papillomavirus type 58 in an oropharyngeal carcinoma. Virol J. 2013;10:334. Epub 2013/11/14. pmid:24220072; PubMed Central PMCID: PMCPMC3842782.
- 47. Schroeder L, Boscolo-Rizzo P, Dal Cin E, Romeo S, Baboci L, Dyckhoff G, et al. Human papillomavirus as prognostic marker with rising prevalence in neck squamous cell carcinoma of unknown primary: A retrospective multicentre study. European journal of cancer (Oxford, England: 1990). 2017;74:73–81. Epub 2017/03/25. pmid:28335889.
- 48. Schroeder L, Wichmann G, Willner M, Michel A, Wiesenfarth M, Flechtenmacher C, et al. Antibodies against human papillomaviruses as diagnostic and prognostic biomarker in patients with neck squamous cell carcinoma from unknown primary tumor. Int J Cancer. 2018;142(7):1361–8. Epub 2017/11/22. pmid:29159804.
- 49. Halec G, Schmitt M, Egger S, Abnet CC, Babb C, Dawsey SM, et al. Mucosal alpha-papillomaviruses are not associated with esophageal squamous cell carcinomas: Lack of mechanistic evidence from South Africa, China and Iran and from a world-wide meta-analysis. Int J Cancer. 2016;139(1):85–98. Epub 2015/11/04. pmid:26529033; PubMed Central PMCID: PMCPMC5772872.
- 50. Bleotu C, Popescu CR, Anton G, Plesa A, Grigore R, Welt L, et al. Tracking down of laryngo-pharyngeal metastasis. Roumanian archives of microbiology and immunology. 2010;69(3):153–63. Epub 2011/03/26. pmid:21434592.
- 51. Dayyani F, Etzel CJ, Liu M, Ho CH, Lippman SM, Tsao AS. Meta-analysis of the impact of human papillomavirus (HPV) on cancer risk and overall survival in head and neck squamous cell carcinomas (HNSCC). Head & neck oncology. 2010;2:15. Epub 2010/07/01. pmid:20587061; PubMed Central PMCID: PMCPMC2908081.
- 52. Michaud DS, Langevin SM, Eliot M, Nelson HH, Pawlita M, McClean MD, et al. High-risk HPV types and head and neck cancer. Int J Cancer. 2014;135(7):1653–61. Epub 2014/03/13. pmid:24615247; PubMed Central PMCID: PMCPMC4107082.
- 53. Boscolo-Rizzo P, Pawlita M, Holzinger D. From HPV-positive towards HPV-driven oropharyngeal squamous cell carcinomas. Cancer treatment reviews. 2016;42:24–9. Epub 2015/11/09. pmid:26547133.
- 54. Smeets SJ, Hesselink AT, Speel EJ, Haesevoets A, Snijders PJ, Pawlita M, et al. A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer. 2007;121(11):2465–72. Epub 2007/08/08. pmid:17680565.
- 55. Rietbergen MM, Leemans CR, Bloemena E, Heideman DA, Braakhuis BJ, Hesselink AT, et al. Increasing prevalence rates of HPV attributable oropharyngeal squamous cell carcinomas in the Netherlands as assessed by a validated test algorithm. Int J Cancer. 2013;132(7):1565–71. Epub 2012/09/06. pmid:22949073.
- 56. Boscolo-Rizzo P, Schroeder L, Romeo S, Pawlita M. The prevalence of human papillomavirus in squamous cell carcinoma of unknown primary site metastatic to neck lymph nodes: a systematic review. Clinical & experimental metastasis. 2015;32(8):835–45. Epub 2015/09/12. pmid:26358913.
- 57. Saraiya M, Unger ER, Thompson TD, Lynch CF, Hernandez BY, Lyu CW, et al. US assessment of HPV types in cancers: implications for current and 9-valent HPV vaccines. J Natl Cancer Inst. 2015;107(6):djv086. Epub 2015/05/01. pmid:25925419; PubMed Central PMCID: PMCPMC4838063.
- 58. Ribeiro KB, Levi JE, Pawlita M, Koifman S, Matos E, Eluf-Neto J, et al. Low human papillomavirus prevalence in head and neck cancer: results from two large case-control studies in high-incidence regions. Int J Epidemiol. 2011;40(2):489–502. Epub 2011/01/13. pmid:21224273.
- 59. Bussu F, Sali M, Gallus R, Vellone VG, Zannoni GF, Autorino R, et al. HPV infection in squamous cell carcinomas arising from different mucosal sites of the head and neck region. Is p16 immunohistochemistry a reliable surrogate marker? Br J Cancer. 2013;108(5):1157–62. Epub 2013/02/14. pmid:23403821; PubMed Central PMCID: PMCPMC3619072.
- 60. Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science. 1994;264(5157):436–40. Epub 1994/04/15. pmid:8153634.
- 61. Cairns P, Mao L, Merlo A, Lee DJ, Schwab D, Eby Y, et al. Rates of p16 (MTS1) mutations in primary tumors with 9p loss. Science. 1994;265(5170):415–7. Epub 1994/07/15. pmid:8023167.
- 62. Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 1995;55(20):4525–30. Epub 1995/10/15. pmid:7553621.
- 63. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, et al. 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nature medicine. 1995;1(7):686–92. Epub 1995/07/01. pmid:7585152.
- 64. Choudhury JH, Ghosh SK. Promoter Hypermethylation Profiling Identifies Subtypes of Head and Neck Cancer with Distinct Viral, Environmental, Genetic and Survival Characteristics. PLoS One. 2015;10(6):e0129808. Epub 2015/06/23. pmid:26098903; PubMed Central PMCID: PMCPMC4476679.
- 65. Rosas SL, Koch W, da Costa Carvalho MG, Wu L, Califano J, Westra W, et al. Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res. 2001;61(3):939–42. Epub 2001/02/28. pmid:11221887.
- 66. Riese U, Dahse R, Fiedler W, Theuer C, Koscielny S, Ernst G, et al. Tumor suppressor gene p16 (CDKN2A) mutation status and promoter inactivation in head and neck cancer. Int J Mol Med. 1999;4(1):61–5. Epub 1999/06/22. pmid:10373639.
- 67. Sinha P, Bahadur S, Thakar A, Matta A, Macha M, Ralhan R, et al. Significance of promoter hypermethylation of p16 gene for margin assessment in carcinoma tongue. Head Neck. 2009;31(11):1423–30. Epub 2009/05/12. pmid:19431196.
- 68. Ruesga MT, Acha-Sagredo A, Rodriguez MJ, Aguirregaviria JI, Videgain J, Rodriguez C, et al. p16(INK4a) promoter hypermethylation in oral scrapings of oral squamous cell carcinoma risk patients. Cancer Lett. 2007;250(1):140–5. Epub 2006/11/23. pmid:17113222.
- 69. Georgiou E, Valeri R, Tzimagiorgis G, Anzel J, Krikelis D, Tsilikas C, et al. Aberrant p16 promoter methylation among Greek lung cancer patients and smokers: correlation with smoking. European journal of cancer prevention: the official journal of the European Cancer Prevention Organisation (ECP). 2007;16(5):396–402. Epub 2007/10/10. pmid:17923809.
- 70. Zhang B, Zhu W, Yang P, Liu T, Jiang M, He ZN, et al. Cigarette smoking and p16INK4alpha gene promoter hypermethylation in non-small cell lung carcinoma patients: a meta-analysis. PLoS One. 2011;6(12):e28882. Epub 2011/12/17. pmid:22174919; PubMed Central PMCID: PMCPMC3236763.
- 71. Kim DH, Nelson HH, Wiencke JK, Zheng S, Christiani DC, Wain JC, et al. p16(INK4a) and histology-specific methylation of CpG islands by exposure to tobacco smoke in non-small cell lung cancer. Cancer Res. 2001;61(8):3419–24. Epub 2001/04/20. pmid:11309302.
- 72. Lea JS, Coleman R, Kurien A, Schorge JO, Miller DS, Minna JD, et al. Aberrant p16 methylation is a biomarker for tobacco exposure in cervical squamous cell carcinogenesis. American journal of obstetrics and gynecology. 2004;190(3):674–9. Epub 2004/03/26. pmid:15041998.
- 73. Pierini S, Jordanov SH, Mitkova AV, Chalakov IJ, Melnicharov MB, Kunev KV, et al. Promoter hypermethylation of CDKN2A, MGMT, MLH1, and DAPK genes in laryngeal squamous cell carcinoma and their associations with clinical profiles of the patients. Head Neck. 2014;36(8):1103–8. Epub 2013/06/28. pmid:23804521.
- 74. von Zeidler SV, Miracca EC, Nagai MA, Birman EG. Hypermethylation of the p16 gene in normal oral mucosa of smokers. Int J Mol Med. 2004;14(5):807–11. Epub 2004/10/20. pmid:15492849.
- 75. Takeshima M, Saitoh M, Kusano K, Nagayasu H, Kurashige Y, Malsantha M, et al. High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka. Journal of oral pathology & medicine: official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology. 2008;37(8):475–9. Epub 2008/02/21. pmid:18284544.
- 76. Tran TN, Liu Y, Takagi M, Yamaguchi A, Fujii H. Frequent promoter hypermethylation of RASSF1A and p16INK4a and infrequent allelic loss other than 9p21 in betel-associated oral carcinoma in a Vietnamese non-smoking/non-drinking female population. Journal of oral pathology & medicine: official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology. 2005;34(3):150–6. Epub 2005/02/04. pmid:15689228.
- 77. WHO report on the global tobacco epidemic—Romania 2017. Available from: http://www.who.int/tobacco/surveillance/policy/country_profile/rou.pdf?ua=1.
- 78. Hashibe M, Brennan P, Benhamou S, Castellsague X, Chen C, Curado MP, et al. Alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium. J Natl Cancer Inst. 2007;99(10):777–89. Epub 2007/05/17. pmid:17505073.
- 79. Tobacco smoke and involuntary smoking. IARC monographs on the evaluation of carcinogenic risks to humans / World Health Organization, International Agency for Research on Cancer. 2004;83:1–1438. Epub 2004/08/03. pmid:15285078; PubMed Central PMCID: PMCPMC4781536.
- 80. Vineis P, Alavanja M, Buffler P, Fontham E, Franceschi S, Gao YT, et al. Tobacco and cancer: recent epidemiological evidence. J Natl Cancer Inst. 2004;96(2):99–106. Epub 2004/01/22. pmid:14734699.
- 81. Tuyns AJ, Esteve J, Raymond L, Berrino F, Benhamou E, Blanchet F, et al. Cancer of the larynx/hypopharynx, tobacco and alcohol: IARC international case-control study in Turin and Varese (Italy), Zaragoza and Navarra (Spain), Geneva (Switzerland) and Calvados (France). Int J Cancer. 1988;41(4):483–91. Epub 1988/04/15. pmid:3356483.
- 82. Hashibe M, Brennan P, Chuang SC, Boccia S, Castellsague X, Chen C, et al. Interaction between tobacco and alcohol use and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium. Cancer Epidemiol Biomarkers Prev. 2009;18(2):541–50. Epub 2009/02/05. pmid:19190158; PubMed Central PMCID: PMCPMC3051410.