Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in India and Europe

Final Report Summary - HPV-AHEAD (Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in India and Europe)

Executive Summary:
The HPV-AHEAD consortium has generated a multidisciplinary team, including basic biologists, epidemiologists, virologists, and clinicians involved in different fields, e.g. head and neck surgery, screening programmes, and public health, to evaluate the role of human papillomavirus (HPV) infection and other cofactors in the development of head and neck cancer (HNC) in Europe and India. The achieved goals provided important insights that will have a beneficial impact on the development of novel strategies for screening, diagnosis, treatment, and prophylaxis of HPV-associated HNC in these geographical regions and elsewhere.
We have collected and analysed a large collection of plasma/sera (n=4000) in many European centres and HNC tissues (n=8000) from 42 centres in 16 European countries as well as from 6 Indian centres. Most of the HNC specimens were associated with epidemiological and clinical data.
The blood specimens were used in case–control studies. The obtained data showed that antibodies against mucosal high-risk HPV types are valid markers of viral infection. Most importantly, a prospective study showed that HPV antibodies can be detected many years before the development of the disease, highlighting their potential use as prognostic markers of oropharyngeal cancer development.
HNC tissues have been analysed by several laboratory assays, including detection of viral DNA and RNA, to precisely determine the fraction of HNCs attributable to mucosal high-risk HPV types in many geographical regions in Europe and India. Although the analyses of the results are still under way, preliminary data show that approximately 10–15% of HNCs in Europe are associated with mucosal high-risk HPV infections, while the proportion is lower, 5–10%, in India. In agreement with previous studies, HPV infection is mainly associated with oropharyngeal cancers in both geographical regions. Importantly, HPV-positive cases of HNC appeared to have a better prognosis than HPV-negative cases.
In Europe, HPV DNA/RNA positivity correlated well with high expression of the surrogate marker of HPV infection, p16INK4a. In contrast, in India, p16INK4a staining was found not to be a good marker for HPV infection. The HPV-AHEAD consortium has also investigated the identification of additional surrogate markers of HPV infection in HNC. Two potential candidates have been proposed, namely the SUMO-conjugating enzyme UBC9 and the transcription factor E2F1, which need to be validated in future studies.
In an independent subproject, the prevalence of HPV infection in the oral cavity of young populations was investigated in two European countries, Germany and Italy; the prevalence was found to be about 10–15%. Ongoing analyses are investigating how lifestyle and HPV vaccination status influence HPV infection in the oral cavity.
In addition to the activities described above, many efforts of the HPV-AHEAD consortium were focused on reviewing the scientific literature and generating meta-analyses on HNC-related topics.
Importantly, the consortium has organized many training activities and transferred technologies within and outside the HPV-AHEAD working group.
All achievements and findings of the HPV-AHEAD consortium have been disseminated through the generation of scientific articles and public webpages as well as the organization of international workshops and summer schools.

Project Context and Objectives:
Head and neck cancer (HNC), with an estimated global burden of approximately 700 000 incident cases per year, is the sixth most common malignancy reported worldwide and has a high case fatality rate of 380 000 deaths per year (GLOBOCAN 2012, IARC; http://globocan.iarc.fr/). Approximately 50% of HNCs occur in the oral cavity, followed by 30% in the larynx and 10% in the oropharynx. Alcohol consumption, smoking, poor oral hygiene, genetic features, and infection with mucosal high-risk human papillomavirus (HPV) types are key risk factors for HNC development (Marur et al., 2010). Among the various types of HNC, cancers of the lingual and palatine tonsils are most frequently associated with HPV infection (1, 2). Although the overall incidence of HNC is decreasing in developed countries due to increasing awareness of tobacco and alcohol as risk factors for human carcinogenesis, the proportion of oropharyngeal cancers among HNCs has been steadily increasing in the USA and Europe. A recent review highlighted that the percentage of HPV-positive oropharyngeal cancers varies by geographical region, ranging from 56% in North America to 17% in southern Europe (3), suggesting that environmental and/or lifestyle factors are implicated in the development of HPV-positive HNC.
The Indian subcontinent has the highest HNC incidence in the world, accounting for one third of the world burden. Many etiological factors are involved in HNC development in the Indian population, including alcohol consumption, smoking, and use of smokeless tobacco, e.g. pan masala, gutka, and zarda. A limited number of studies reported that the percentage of HPV DNA-positive cases can vary in different Indian geographical regions (4-12).
Establishing the HPV positivity of HNC is of high clinical relevance because it can influence prophylaxis, prognosis, and potentially the treatment modality. Thus, it is very important to establish diagnostic algorithms for the classification of HPV-positive HNC that can be adopted in routine clinical practice.
The main research activities of the HPV-AHEAD consortium were focused on the evaluation of the role of mucosal high-risk HPV types and other risk factors in the development of HNC in Europe and India. Specifically, the objectives of the HPV-AHEAD consortium are:
(i) To produce a formal systematic review on the available data and a comprehensive analysis of the descriptive epidemiology and time trends in HNC incidence and mortality in Europe and India, using the available data in the literature and data banks.
(ii) To conduct epidemiological studies in European and Indian populations in order to establish the overall proportion and type distribution of HPV-positive HNC at different anatomical sites in different geographical regions.
(iii) To determine whether HPV infections interact with additional HNC risk factors in European and Indian populations.
(iv) To perform retrospective studies using the follow-up information on HNC patients (including registry linkages) to establish whether HPV positivity confers a better prognosis and survival.
(v) To determine the prevalence of DNA and/or RNA HPV positivity in oral specimens (e.g. exfoliated cells and saliva) in young populations in order to describe risk factors and natural history of oral HPV infections, and to explore novel screening strategies that could be broadly used in the clinical routine.
(vi) To transfer technology to Indian research laboratories as well as to develop several strategies for the training of European and Indian researchers in topics related to infections and cancers.
The HPV-AHEAD study included 8 closely linked work packages (WP) that were associated with specific activities, as schematically illustrated in Figure 1.
Activities of WP1 were mainly focused on review of available data on HNC incidence and mortality in India and Europe. This subproject also aimed to evaluate the contribution of different risk factors, such as mucosal high-risk HPV infection, smoking, and alcohol, by performing a systematic review of the scientific literature and meta-analyses. Work in WP1 led to the generation of many scientific publications in high-ranking scientific journals and the creation of an interactive website (http://www.hpvcentre.net/) that will remain active and will be continuously updated after the end of the HPV-AHEAD study.
In WP2, the collection of specimens included more than 8000 formalin-fixed, paraffin-embedded (FFPE) HNC tissues from four anatomical sites (i.e. oral cavity, oropharynx, larynx, and hypopharynx) as well as blood samples from controls and cases. In Europe, to maximize the efforts and create new synergisms, the HPV-AHEAD consortium brought together three ongoing and well-established European studies on HNC: ARCAGE (Alcohol-Related Cancers and Genetic susceptibility in Europe), EPIC (European Prospective Investigation into Cancer and Nutrition), and ICO (Institut Català d’Oncologia). These three studies comprise 42 centres in 16 European countries. In particular, ARCAGE was associated with the collection of detailed information on exposure to risk factors (i.e. alcohol consumption and smoking habits) and outcome of the disease after therapy. In addition, two other European centres, namely the University of Antwerp (Belgium) and the European Institute of Oncology (Milan, Italy) have collected more than 2000 FFPE HNC tissues together with clinical information. Together, these 44 European centres provided approximately 4700 HNC tissue blocks.
In India, approximatively 3000 FFPE HNC tissues have been collected in six different urban and rural hospitals, namely Ambilikkai, Bangalore, Barshi, Guwahati, Sevagram, and Trivandrum, covering a large area of the country. Approximately 2000 specimens have been retrieved from existing archives together with limited epidemiological and clinical data. In addition, more than 1000 specimens have been prospectively collected together with information on exposure to risk factors and clinical outcome.
In addition to the FFPE HNC tissues, the HPV-AHEAD consortium has collected blood samples of HNC patients (n=2000) and controls (n=3000) to perform two case–control studies in the context of ARCAGE and EPIC. In particular, we aimed to determine the presence of antibodies against early and late HPV proteins in sera of cases and controls.
A database has been generated at IARC (Lyon) that includes all available information associated with the patients and specimens.
The work on WP3 aimed to analyse the specimens collected in WP2 by performing several laboratory assays/procedures, namely (i) detection of HPV DNA and RNA, (ii) immunohistochemical staining for the cell cycle inhibitor p16INK4a as surrogate marker of HPV infection, (iii) detection of antibodies against early and late proteins (n=38) of several mucosal high-risk HPV types, (iv) histological re-evaluation of HNC by six pathologists of the HPV-AHEAD consortium of the original histopathological diagnosis of the HNC cases. One of the strengths of the HPV-AHEAD consortium is that all collected human specimens have been analysed with a specific assay by a single laboratory. This centralization of the assays has avoided the risk of interlaboratory variability. All specimens collected in European centres were analysed in Europe, and those collected in India were processed at the Rajiv Gandhi Centre for Biotechnology (RGCB) in Trivandrum under the supervision of Partners 1 (Tommasino), 2 (Pawlita), and 10 (Pillai). Importantly, the same laboratory assays used in the European laboratories were transferred to RGCB. The level of agreement between the assays performed in Europe and India was constantly evaluated during the study by exchanging a small number of specimens (10%) between the two institutes.
Finally, a Pathology Review Panel was created, which includes six pathologists associated with the HPV-AHEAD consortium. The panel established the general criteria for a standardized histological review and generated an online Pathology Evaluation form. Activities in WP4 aimed to analyse the laboratory data and associate them with the clinical information. Due to the large amount of generated data, WP4-related work will continue for approximately 2–3 years after the official end of the HPV-AHEAD consortium.
The initial epidemiological data on FFPE HNC tissues show that approximately 10–15% of HNCs in Europe are positive for HPV DNA and HPV RNA, while in India the proportion of HPV-positive HNCs was 5–10%. In agreement with previous studies, HPV infection is mainly associated with oropharyngeal cancers in both geographical regions. In Europe, HPV DNA/RNA positivity correlated well with the p16INK4a staining. In contrast, in India, p16INK4a staining was found not to be a good marker for HPV infections. Many HPV-negative HNCs were positive for p16INK4a. In addition, a fraction of HPV DNA/RNA-positive cases were negative for p16INK4a. These findings led to the hypothesis that exposure of the oral cavity to additional carcinogens (e.g. betel quid mixed with tobacco products) may lead to genetic alteration in the INK4a locus that includes the p16INK4a gene.
The data also showed that a small proportion (1–2%) of oral and laryngeal cancers is associated with HPV infection in Europe and India.
Importantly, the detection of HPV DNA alone was found to be insufficient proof for viral causality and could lead to misclassification of the HNC. Thus, the detection of HPV DNA should always be combined with additional markers, e.g. detection of viral RNA.
The serological studies showed that antibodies against early proteins, E6 and E7, of mucosal high-risk HPV types are good markers for HPV-positive oropharyngeal cancers. In particular, HPV16 E6 antibody positivity has an odds radio of approximately 200. Interestingly, even if antibodies against the late HPV protein L1 are considered markers of previous exposure to the viral infection, they were positively associated with the development of oropharyngeal cancers. This scenario is quite different to the one observed in cervical cancer patients, where E6 and E7 antibodies are produced at late stages of the disease.
In WP5, many efforts were focused on the identification of procedures to determine the prevalence of mucosal high-risk HPV infection in the oral cavity.
We compared several methods to collect oral exfoliated cells and to perform HPV genotyping. Gargles were found to be a good method of specimen collection. Regarding the prevalence of viral DNA in the oral cavity, approximately 12% of oral specimens collected in the young populations in Germany and Italy were positive for mucosal high-risk HPV types. However, HPV positivity dropped significantly (to about 4%) when a less-sensitive HPV genotyping assay was used. When we restricted our analysis to a small region of the oral cavity by processing archival oral non-tumour tissue samples, the HPV DNA positivity decreased dramatically (to less than 1%). Together, these data show that studies aiming to determine the prevalence of HPV infection in the oral cavity should include a significant number of exfoliated cell samples and should use very sensitive HPV DNA detection assays.
The main goal of WP6 was to identify novel surrogate markers for HPV infection and to facilitate the development of laboratory assays that can be easily transferred to routine clinical settings. We tested several immunohistochemical protocols for the different cellular proteins of pathways known to be deregulated in cancer cells. The analysis of a small fraction of HNC specimens indicated that two cellular proteins are upregulated in HPV-positive HNC, i.e. the SUMO (small ubiquitin-like modifier) pathway-conjugating enzyme UBC9 and the transcription factor E2F1. We are currently planning a larger study to further validate our original data.
WP7- and WP8-related activities were focused on the generation of tools and strategies (i) to facilitate the interaction among the partners of the HPV-AHEAD consortium; (ii) to transfer technologies and know-how in India; and (iii) management of the consortium.
These goals were achieved by organizing annual meetings, telephone conferences, and a summer school on virus-associated cancers in India, and by organizing exchange visits of European and Indian researchers.
In conclusion, the HPV-AHEAD study has provided much important information and data on HPV-associated HNC in Europe and India. In particular, the findings have revealed the precise contribution of mucosal high-risk HPV infections to HNC development and support the use of novel markers for HPV-positive HNC in a vast geographical area.
This study will have an impact on screening, diagnosis, treatment, and prophylaxis of HPV-associated HNC in Europe, India, and elsewhere.

References
(1) Marur et al., Lancet Oncol. 2010 11(8):781-9.
(2) Gillison et al., Int J Cancer. 2014 134(3):497-507.
(3) de Martel et al., Lancet Oncol. 2012 13(6):607-15.
(4) Choudhury and Ghosh. 2015 10(6):e0129808.
(5) Patel et al., J Oral Pathol Med. 2014 43(4):293-7.
(6) Bahl et al., Head Neck. 2014 36(4):505-10.
(7) Jalouli et al., Anticancer Res. 2012 32(2):571-80.
(8) Jamaly et al., Tumour Biol. 2012 33(2):543-50.
(9) Barwad et al., Diagn Cytopathol. 2012 40(10):893-7.
(10) Jalouli et al., Acta Otolaryngol. 2010 130(11):1306-11.
(11) Gheit et al., Vaccine. 2009 27(5):636-9.
(12) Mitra et al., J Clin Pathol. 2007 60(9):1040-7.

Project Results:
The HPV-AHEAD study is a straightforward and objective-driven programme with a well-defined structure based on 6 scientific work packages (WP 1–6) and two WPs (WP7 and 8) focused on training, transfer of technology, and management.

WP1: Epidemiology and time trends analyses of HNC in Europe and India based on global analysis of the literature and existing databases
This WP aimed to evaluate (i) the rates and trends in the incidence and mortality of head and neck cancer (HNC) in European countries and Indian regions using the currently available data in the literature as well as (ii) the contribution of human papillomavirus (HPV) infection and other risk factors (e.g. alcohol and smoking).
In the framework of the Eurogin Roadmap, partners of the HPV-AHEAD consortium (Arbyn-UA, Castellsagué and de Sanjosé-ICO, Tommasino-IARC) collaborated with other experts in the field to estimate the burden of all HPV-related cancers for 2008 at the global level, with particular attention to oropharyngeal cancer. For the Eurogin 2012 Roadmap, a comparison was made between cervical and oropharyngeal cancer regarding the epidemiology, natural history, risk factors, trends, pathology, potential impact of screening and vaccination, and diagnostic and therapeutic issues. This collaborative effort resulted in the generation of two publications:
(i) Arbyn M, de Sanjosé S, Saraiya M, Sideri M, Palefsky J, Lacey C, Gillison M, Bruni L, Ronco G, Wentzensen N, Brotherton J, Qiao YL, Denny L, Bornstein J, Abramowitz L, Giuliano A, Tommasino M, Monsonego J. 2012. EUROGIN 2011 roadmap on prevention and treatment of HPV-related disease. Int J Cancer. 131:1969-82.
(ii) Gillison ML, Castellsagué X, Chaturvedi A, Goodman MT, Snijders P, Tommasino M, Arbyn M, Franceschi S. 2014. Eurogin Roadmap: comparative epidemiology of HPV infection and associated cancers of the head and neck and cervix. Int J Cancer. 134:497-507.
The findings presented in these two articles confirmed that approximately 75% of the HNC burden occurs in men. HNC is the 7th most common cancer in men and the 13th most common in women. Its incidence has a large worldwide geographical heterogeneity, highlighting the key role of environmental and lifestyle factors in the etiology of this malignancy. The proportion of oropharyngeal cancers that are attributable to HPV infection varies between studies and anatomical subsites. The percentage of HPV-positive oropharyngeal cancers was higher in North America (60%) compared with Europe (40%) and all other regions (33%). Collectively, data also showed that HPV prevalence in HNC increased significantly from 41% before 2000 to 72% after 2004. The mucosal high-risk (HR) HPV type 16 is responsible for the majority of these malignant lesions (80–100%). Tobacco smoking or chewing and alcohol consumption are well-established risk factors for HNC cancer. The fact that men are more exposed to these risk factors than women provides a possible explanation for the gender disparity in HNC incidence. For HPV-positive HNC, risk factors include ethnicity, sexual habits, and marijuana use. In some geographical regions (e.g. USA), an elevated risk for men has also been observed for HPV-positive HNC. This difference in risk may be explained by sexual behaviours, gender differences in the natural history of HPV infections in the oral cavity and/or genital tract, and sex hormones. Finally, analysis of the available data confirmed the involvement of HPV infection in a much smaller subset of oral and laryngeal cancers compared with oropharyngeal cancers.

In another collaborative effort, partners of the HPV-AHEAD consortium (Arbyn-UA, Alemany, Bosch, Castellsagué and de Sanjosé-ICO, Forman and Sankaranarayanan-IARC), together with other HPV experts, have synthesized in a Monograph (“Central and Eastern Europe and Central Asia regional report” of the ICO Monograph Series on HPV and Disease Prevention) the current burden and trends of HPV-related cancers (including HNC) and the current situation for screening and HPV vaccination for countries in central and eastern Europe. These activities resulted in the publication of four aticles in Vaccine (Bosch et al., 2013 Vaccine 31 Suppl 7:F1-31; Bosch et al., 2013 Vaccine 31 Suppl 6:G1-31; Bosch et al., 2013 Vaccine 31 Suppl 6:H1-31; I1-31).
An ongoing trend analysis of HNC incidence and mortality in several countries worldwide (including countries in Europe and India) performed by the IARC team was presented at the annual HPV-AHEAD meeting and summer school organized in Trivandrum in November 2012 by Partner 4 (Arbyn). This trend analysis included mainly information for the countries or areas where high-quality data separated by ICD-O code are available. Data used for this analysis are based on Cancer Incidence in Five Continents and available mortality datasets.
Partners 4 (Arbyn-UA) and 3 (Alemany, Bosch, Castellsagué, Mena, and de Sanjosé-ICO) in collaboration with researchers from McGill University in Montreal, Canada (Ndiaye and Trottier), have performed a systematic review and meta-analysis of the HPV DNA prevalence and HPV type distribution in HNC. Briefly, a literature search on PubMed was conducted to identify published studies on HNC that used PCR for HPV DNA detection and reported information on HPV genotype distribution. The search included papers published from 1990 up to March 2012. The search strategy was performed not only for Europe and India but was extended to English literature published in all countries worldwide and indexed in PubMed. In addition to the DNA information, data on causal linked markers, mRNA HPV E6/E7 and p16INK4a, was extracted and used to determine the HPV-attributable fraction of HNC. The study has been published in Lancet Oncology (please see Ndiaye et al. “HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis.” 2014, Lancet Oncology, 15:1319-31). The study showed that the highest HPV DNA positivity was observed for the oropharynx (45.8%; 95% CI, 38.9–52.9%), followed by the oral cavity (24.2%) and the larynx/hypopharynx (22.1%). The estimated HPV-attributable fraction in oropharyngeal cancer defined by expression of positive cases of E6/E7 mRNA was 39.8% and of p16INK4a was 39.7%. Of subsites, tonsils (53.9%; 95% CI, 46.4–61.3%) had the highest HPV DNA prevalence. HPV DNA prevalence varied significantly by anatomical site and geographical region, but not by sex habits, tobacco use, or alcohol consumption.
In conclusion, the research activities in WP1 resulted in the accomplishment of all planned deliverables and in the generation of many scientific articles published in high-ranking journals. Most importantly, all collected data on HNC and associated risk factors have been included in an interactive website (http://www.hpvcentre.net/) that will remain active and will be continuously updated after the end of the HPV-AHEAD study.

WP2: Collection of human specimens in European and Indian centres
The major goal of WP2 was to collect a large number of formalin-fixed, paraffin-embedded (FFPE) HNC tissues from four anatomical sites, namely the oral cavity, oropharynx, larynx, and hypopharynx, in Europe and India. In addition, we collected approximately 4000 blood specimens from controls and HNC patients to perform serological studies in Europe. The collection of specimens in Europe was significantly facilitated by the inclusion in the HPV-AHEAD study of three ongoing and well-established studies on HNC, namely ARCAGE (Alcohol-Related Cancers and Genetic susceptibility in Europe), EPIC (European Prospective Investigation into Cancer and Nutrition), and ICO (Institut Català d'Oncologia).
At an early stage of the study, several collecting centres agreed to deliver the FFPE blocks to IARC. However, some centres could not deliver the entire FFPE blocks since they need to be stored in the archive of the hospital for legal reasons. To overcome this problem, we developed a protocol for sectioning that was distributed to these hospitals. The protocol describes the generation of several sections for laboratory assays and histological analyses, as shown in Figure 2. In case the size of the tissue was the limiting factor, sectioning was stopped after S10 (indicated by the arrows), while if there was sufficient tissue the sectioning progressed until S31. The use of the different sections is explained in Figure 2.
The protocol also describes several procedures to minimize cross-contamination among the different FFPE blocks during the processing in the histology laboratory. Before starting to process the FFPE blocks, each histology laboratory received a test panel of HPV-positive and HPV-negative FFPE blocks. Sections were generated and shipped to IARC for HPV genotyping. Only after performing the quality test for the generation of sections did each collecting centre start processing FFPE HNC tissue blocks. The same protocol was followed by the IARC histology laboratory for sectioning the FFPE blocks sent to IARC. During the HPV-AHEAD study, six histology laboratories in Italy, Spain, Belgium, and India have successfully used this sectioning protocol. In addition, the protocol has been distributed to many other colleagues involved in similar collaborative studies. A technical manuscript is in preparation in order to disseminate the protocol and facilitate the organization of future multicentre studies, which imply the collection of human specimens from many hospitals.
Approximately 1200 HNC specimens from the oral cavity (n=327), oropharynx (n=248), larynx (n=511), and overlapping (n=76) were collected in the ARCAGE study from 14 centres across 8 participating countries in Europe, i.e. the Czech Republic, Germany, Greece, Hungary, Ireland, Italy, Norway, and Spain. These specimens have been associated with detailed clinical information and follow-up data.
A similar number of specimens (n=1261) was collected in the ICO study, from the Czech Republic, Greece, Hungary, Poland, Portugal, Spain, Slovenia, and the United Kingdom.
In the EPIC study, we identified 525 incident HNC cases (200 oral cavity, 100 oropharynx, and 325 larynx), but it was possible to retrieve only 220 specimens in five European countries (Germany, Italy, Spain, Sweden, and the United Kingdom).
The two other European centres, EIO-Italy (Partner 5) and UA-Belgium (Partner 4), collected 1000 and 800 HNC specimens, respectively.
In addition, during the HPV-AHEAD study a new collaborator (Dr Ramona Gabriella Ursu, Romania) joined the network and provided an additional 200 HNC specimens.
In conclusion, we were able to collect almost 4700 specimens in Europe. All HNC specimens were associated with clinical information. An online form was generated to collect the clinical information. All centres had the possibility of completing the form online for each collected specimen. A central database was generated at IARC that is accessible to all partners of the HPV-AHEAD consortium.
In India, HNC specimens were collected in six hospitals, as indicated in Figure 3.
Ambilikkai, Barshi, and Sevagram are in rural areas, while Bangalore, Guwahati, and Trivandrum are considered urban areas. This Indian network was created immediately after the initiation of the HPV-AHEAD study in September 2011. A kick-off meeting involving the six Indian centres and Partners 1 (Tommasino and Sankaranarayanan) and 10 (Pillai) was held in Trivandrum on 12 November 2011. This meeting was focused on the establishment of protocols and strategies for the collection and processing of specimens.
IARC signed a collaborative agreement with each of the six Indian collecting centres and provided economic support using the coordinator’s budget.
About 3000 FFPE HNC tissues were collected in India, representing four different anatomical regions, i.e. hypopharyngeal, oropharyngeal, oral cavity, and laryngeal cancers, with approximate percentages of 20%, 20%, 40%, and 20%, respectively.
A total of 2000 specimens were retrieved from existing archives together with limited epidemiological and clinical data. In addition, more than 1000 specimens were prospectively collected together with information on exposure to risk factors and clinical outcome. In particular, two questionnaires were generated to collect information on the disease and its evolution as well as information on lifestyle and habits, such as tobacco chewing and smoking, alcohol consumption, diet, and oral hygiene.
All samples were sent to RGCB in Trivandrum, where they were processed as described above using the HPV-AHEAD sectioning protocol, while clinical and lifestyle information was collected at IARC to generate a centralized database.
Finally, based on the initial call of the EC and the Indian Council of Medical Research (ICMR), Partner 10 (Pillai) with Partners 1 (Tommasino and Sankaranarayanan) and 2 (Pawlita) have collaborated on the preparation of a grant application that has recently been granted by ICMR. The application was fully based on the study design and protocols adopted by the HPV-AHEAD consortium. In the new study, five additional Indian groups have been involved, i.e. (i) Dr Jitendrakumar Singh, Mahavir Cancer Sansthan & Research Centre (MCSRC), Phulwarisharif, Patna; (ii) Dr M Nagarajan, V N Cancer Centre, GKNM Hospital, Pappanaickenpalayam, Coimbatore; (iii) Dr Shirly Sunder Singh, Cancer Institute (WIA), Chennai; (iv) Professor R K Grover, Delhi State Cancer Institute, Dilshad Garden, Delhi; and (v) Professor Ravi Mehrotra, Motilal Nehru Medical College 16/2, Lowther Road, Allahabad, in order to collect 5000 additional HNC specimens. It is expected that at least 2000 additional HNC specimens will be collected in the next years.
In addition to the FFPE HNC tissues, we collected blood samples of HNC patients (n=2000) and controls (n=3000) to perform two case–control studies in the context of ARCAGE and EPIC. In particular, we aimed to determine the presence of antibodies against early and late HPV proteins in sera of cases and controls.
In conclusion, all the goals of WP2 have been achieved. In addition to the collection of the human specimens, several protocols and procedures were developed that can be easily adapted to other novel multicentre studies. Most importantly, a large network has been created in Europe and India that is expected to continue to collaborate for the next years on topics related to HPV and HNC.

WP3: Laboratory analyses to determine HPV positivity
The research activities of WP3 were focused on the characterization of the specimens collected in WP2 by several laboratory assays/procedures, as shown in Figure 4.
In order to avoid the risk that the performance of assays could be influenced by inter-laboratory variabilities, we adopted the strategy that a single laboratory/group was responsible for a single assay/procedure. The only exception was for detection of HPV DNA, which was performed in two laboratories. IARC (Partner 1) and RGCB (Partner 10) processed all European and Indian specimens, respectively. This decision was based on the fact that only a limited number of Indian specimens (about 10%) could be shipped outside India. Importantly, the identical HPV DNA detection assay was used in both laboratories. Partners 1 (Tommasino and Sankaranarayanan) and 2 (Pawlita) coordinated the technology transfer from European laboratories to RGCB in India. In addition, to minimize the risk that the HPV DNA detection assay could have different performance at IARC and RGCB, the two laboratories shared throughout the entire study a single batch of reagents that was generated at IARC. Finally, 10% of the Indian specimens were sent to IARC and re-analysed for detection of HPV DNA. Perfect agreement was obtained between the two sets of data obtained at IARC and RGCB.

HPV DNA detection assay
DNA of the HPV types shown in Figure 4 was detected using an assay developed by Partner 1 (Tommasino) and extensively validated in many independent studies. Briefly, the assay includes a first step to amplify the viral DNA by a multiplex-PCR protocol and a second step for HPV genotyping using a Luminex-based assay. In addition, the assay includes assessment of β-globin gene to evaluate the quality of extracted DNA. The extremely high sensitivity of the assay in detecting viral DNA renders its use very suitable for analysis of FFPE tissue blocks that contained highly fragmented DNA.
All European FFPE HNC blocks have been processed, while in India the analysis is slightly delayed and 42.2% (1283/3089) of the specimens have been tested for HPV DNA positivity. The quality of DNA was excellent in most of the cases; only 4.5% and 6.2% of the European and Indian specimens, respectively, were found to be β-globin negative. The delay in the analysis of the Indian specimens is due to the laborious protocol for sectioning and the limited number of microtomes in the Indian histology laboratory. However, it is envisaged that the analysis will be completed in 2015. The quality of the sectioning facilities in collecting centres and of the HPV testing procedures has been also evaluated. The high quality of HPV testing has been maintained throughout the study (with no PCR contamination and with high reproducibility).
The HPV DNA prevalence in European countries was 21.3%. HPV16 accounted for 86% of the total infections, followed by HPV18 (5.9%) and HPV31, 33, 35, 45, 51, 52, and 56 (< 3% collectively). The HPV DNA prevalence in India was 16.0%. HPV16 was present in 83.7% of the high-risk HPV infections, followed by HPV31 (11.6%) and HPV18 (7.2%). HPV6, 11, 33, 35, and 56 collectively accounted for less than 2% of the high-risk infections.
Among the collecting centres in Europe based on only one hospital, i.e. UA-Belgium (Partner 4), EIO-Italy (Partner 5), and Iaşi-Romania (external collaborator), the percentage of HPV-positive cases was very similar, at 12.5%, 12.9%, and 14.2%, respectively, while in the ICO and ARCAGE collections the HPV prevalence was higher (22.7% and 30.5%, respectively). These differences may reflect the percentage of oropharyngeal cancers collected in the centres/studies.
All HPV DNA-positive cases and 10% of the HPV DNA-negative cases were sent to Deutsches Krebsforschungszentrum (DKFZ; Pawlita) for HPV RNA analyses, and the corresponding slides were sent to Roche mtm laboratories AG for p16INK4a/Ki-67 dual staining.

HPV RNA detection assay
Similarly to the HPV DNA method, the RNA detection assay developed by Partner 2 (Pawlita) is based on PCR and Luminex technology. To avoid the risk of false positives due to possible DNA contamination of the RNA sample, the assay detects the junction of a spliced form of HPV RNA, E6*I. In addition, the assay includes the detection of the ubiquitin C transcript, as mRNA quality control marker. About 1100 HPV-positive and 400 HPV-negative FFPE HNC specimens were processed for detection of HPV RNA. Less than 10% of the HNC specimens contained poor-quality RNA and gave invalid results. The results highlighted certain variability in the HPV E6*I RNA positivity of the HPV DNA-positive cases. For instance, about 60% of the HPV DNA-positive HNC specimens collected at EIO-Milan (Partner 5) or UA-Belgium (Partner 4) were found to be HPV E6*I RNA positive, while in one of the Indian centres (Sevagram) only 10% of the HPV DNA-positive cases were found to be positive for HPV RNA. Initial data for the other five Indian centres showed the same trend. This difference is in part explained by the proportion of oropharyngeal cancers collected at the single collecting centres and/or by the role of additional risk factors in the different geographical regions. The analysis of HPV RNA confirmed that HPV16 plays a major role in the development of HNC. However, a small percentage (<2–3%) of HPV DNA/RNA-positive HNCs were also found positive for HPV types HPV18, 31, 33, 39, 45, 56, 58, and 59.
Only 2 HPV DNA-negative cases out of 400 (0.5%) were very weakly positive for HPV RNA. Most likely the HPV RNA positivity for these 2 specimens may be simply due to cross-contamination among the specimens. Approximately 60% of HPV DNA-positive HNC specimens were found to be HPV RNA-negative, indicating that detection of HPV DNA alone is insufficient proof for viral causality.

p16INK4a assay
Studies led by Partner 9 (Ridder, Roche mtm Laboratories) on premalignant and malignant cervical lesions have shown that the cell cycle inhibitor p16INK4a is a good surrogate marker of HPV infection. Indeed, p16INK4a is strongly accumulated in HPV-infected cervical epithelial cells as an attempt to compensate for the cell cycle deregulation induced by the HPV oncoprotein E7.
In the HPV-AHEAD study, Partner 9 has determined the expression of p16INK4a and Ki-67 in all HPV DNA-positive specimens and 10% of HPV DNA-negative specimens by double immunostaining, using its p16INK4a research kit (Roche mtm Laboratories). This is a qualitative immunohistochemical assay for the evaluation of p16INK4a levels in FFPE tissue sections. Ki-67 is a marker of proliferation and enables a better identification of the highly proliferating cancer cells.
As described for the HPV RNA analysis, approximately 1500 HPV DNA-negative or DNA-positive HNC specimens were analysed by Ki-67/p16INKa double immunostaining.
Slides were independently read without knowing the status of HPV DNA and RNA. Initial analysis of the staining data of 650 HNC specimens showed a positivity of 35.6% for p16INK4a and Ki-67, ranging from 65% in Poland (ICO) to 21% in India (Bangalore). Overall, the European specimens showed higher p16INK4a positivity compared with the Indian specimens. The vast majority (97.3%) of the p16INK4a-positive sections showed a diffuse staining, while the remaining cases had isolated areas/patches of p16INK4a-positive cells.
Although this task was not originally included in the study design of HPV-AHEAD, a proportion (n=195) of specimens were processed with a single p16INK4a staining kit. The results showed an excellent agreement (98%) between the results obtained with single p16INK4a or double Ki-67/p16INK4a staining, indicating that for future studies the analysis can be simplified without compromising the quality of the data.

Histology review
Although all the HNC tissues were initially subjected to histological diagnosis, the study design of HPV-AHEAD implies the re-evaluation of the histology of all collected specimens. A Pathology Review Panel was created that includes six pathologists: Dr John-Paul Bogers (Belgium), Dr Nitin Gangane (Sevagram), Dr Rekha V. Kumar (Bangalore), Dr Maria Belén Lloveras Rubio (Barcelona), Dr Fausto Maffini (Milan), and Dr Thara Somanathan (Trivandrum). IARC hosted the first meeting of the Pathology Review Panel on 20–21 March 2012. During this meeting, the panel analysed several HNC sections, established the general criteria for a standardized histological review, and generated an online pathology evaluation form. It was agreed that each pathologist would review approximately 1000–2000 specimens. In addition, approximately 10% of the specimens reviewed by a single pathologist would be re-analysed by a second pathologist. In case of controversy, specific cases would be reviewed by the entire Pathology Review Panel.
Due to the purchase of a digital slide scanner at IARC after the start of the HPV-AHEAD study, the original procedure was slightly modified. In fact, it was decided to generate a database containing all digital images of the tissue sections. This modification of the protocol greatly facilitated the work of the Pathology Review Panel, enabling all members of the panel (i) to simultaneously access the digital images and (ii) to work online from any location in the world. In contrast, the previous plan required the rotation of glass slides among the members of the Pathology Review Panel.
Approximately 4000 specimens have been reviewed by at least one member of the Pathology Review Panel. In a recent exercise, the review panel of six pathologists completed the analysis of approximately 600 HNC cases from Sevagram. Each pathologist analysed 120 slides; of these, 60 (50%) were re-analysed by a second pathologist on the review panel. A very good concordance was reported among the pathologists for the classification of the main diagnosis (92.9%; PABAK=0.86). However, the concordance was not as high for the classification of cancer subtypes.

Serological assays
HPV antibody detection was performed by Partner 2 (Pawlita) by multiplex serology, which includes a glutathione S-transferase (GST) capture of bacterial recombinant fusion proteins of GST with 38 viral antigens, as shown in Figure 5, in combination with Luminex technology.
All blood specimens collected in the HPV-AHEAD study have been analysed, and the results are described in detail in WP4.
Overall, the work of WP3 achieved all planned goals. It generated several procedures/strategies to manage and distribute the collected specimens to several laboratories/groups in a very coordinated and well-controlled way.

WP4: Epidemiology of HPV-positive and HPV-negative HNC
This WP aimed:
(i) To evaluate HPV prevalence in different types of HNC in European and Indian regions
(ii) To analyse the time trend of HPV-positive HNC in European and Indian regions
(iii) To assess the attributable risk of HNC development based on HPV serology
(iv) To determine whether HPV positivity influences HNC prognosis.

In this subproject, we are following the strategy illustrated in Figure 6.
We are currently analysing the data obtained in the single cohort/population. Subsequently, we plan to compare the data within Europe and India, and finally between Europe and India. Additional time is required to complete all statistical analyses and preparation of the scientific manuscripts. It is envisaged that the HPV-AHEAD consortium will continue to work together for at least two additional years.
A few examples of the analysis of single populations/cohorts are given below.
In a study in the context of the ARCAGE cohort published in 2013 (Anantharaman et al., 2013 J Natl Cancer Inst. 105:536-45), we evaluated the association between antibodies against early and late proteins from several mucosal HR HPV types and risk of cancer development in the upper aero-digestive tract. Blood specimens from 1496 patients with cancers of the upper aero-digestive tract and 1425 control subjects were examined by the Luminex-based multiplex serological assay described in WP3. As shown in Figure 7, HPV16 L1 seropositivity was associated with increased risk of oral cavity cancer (OR, 1.94; 95% CI, 1.03–3.65) and oropharyngeal cancer (OR, 8.60; 95% CI, 5.21–14.20). HPV16 E6 antibodies were present in 30.2% of oropharyngeal case subjects and only 0.8% of control subjects (OR, 132.0; 95% CI, 65.29–266.86).
We also determined whether HPV antibody response correlated with markers of viral infection, i.e. HPV DNA and p16INK4a expression. An agreement of 67% was observed between HPV16 E6 serology and the corresponding presence of an HPV-related cancer: 4 of 6 HPV DNA-positive/p16INK4a-overexpressing tumours were HPV16 E6 antibody-positive.
In a second study on HPV antibody and HNC using the EPIC cohort, blood samples were identified in the EPIC biobank from (i) 638 patients with incident HNC (i.e. 180 oral cancers, 135 oropharyngeal cancers, and 247 hypopharyngeal/laryngeal cancers), (ii) 300 patients with esophageal cancers, and (iii) 1599 comparable controls. The presence of HPV antibody was evaluated with the assay described above. HPV16 E6 seropositivity was present in prediagnostic samples for 34.8% of patients with oropharyngeal cancer and 0.6% of controls (OR, 274; 95% CI, 110–681) but was not associated with other cancer sites. As shown in Figure 8, the increased risk of oropharyngeal cancer among HPV16 E6 seropositive participants was independent of time between blood collection and diagnosis and was observed more than 10 years before diagnosis (Kreimer et al. 2013 J Clin Oncol. 31:2708-15).
Together, these two serological studies highlight the value of HPV16 E6 antibody as a marker of HPV-induced HNC. Most importantly, the findings indicate that HPV E6 antibodies can be used as a diagnostic marker before the development of HNC.
Preliminary analysis of the association of tumour markers of HPV infection and HNC prognosis in the ARCAGE study included 502 HNC cases, of which 112 (22.3%) were oropharyngeal. All tumours were tested for the presence of HPV DNA and expression of surrogate protein p16INK4a, and the joint positivity to both markers was considered a HPV-positive tumour. HPV positivity was significantly associated with lower risk of death among oropharyngeal cancer patients (HR, 0.44; 95% CI, 0.23–0.86). The 5-year survival probability among HPV-positive oropharyngeal cancer patients was higher (0.60) than that among HPV-negative patients (0.41).
Initial data on the analyses of the samples collected in Italy (EIO; Partner 5) are shown in Figure 9. The majority of HNCs positive for HPV16 DNA/RNA are oropharyngeal cancers. Although these cases usually have a more advanced disease, these patients respond better to chemotherapy and/or radiotherapy and have a better prognosis. In addition, a high percentage of patients with HPV16 DNA/RNA-negative HNC were found to be current smokers (68%), while only 22% of the patients with HPV16 DNA/RNA-positive HNC were current smokers. Similar findings have been confirmed in the Belgium cohort (UA; Partner 4).
The validity of p16INK4a as a surrogate marker for HPV infection in HNC is under evaluation in several European cohorts. Initial findings indicate that p16INK4a overexpression correlates well with HPV DNA/mRNA positivity, although some HPV DNA/RNA-negative cases were positive for the expression of the cell cycle inhibitor.
Among the Indian studies, specimens from Sevagram have been fully characterized for HPV DNA and RNA as well as p16INK4a expression, and a manuscript is in preparation. The majority of the HNCs were from the oral cavity (n=342, 75.7%), larynx (n=71, 15.7%), oropharynx (n=33, 7.3%) and pharynx (n=6, 1.3%). HPV DNA was detected in 62 HNCs, with HPV16 being the most prevalent type (47/62). The other HR HPV types were HPV31 (7/62), HPV18 (4/62), HPV35 (2/62), and HPV56 (2/62).
No DNA from low-risk HPV types or from multiple HR HPV types were detected in all HNC specimens. After stratification by anatomical site, the highest HR HPV DNA positivity was found in cancer of the oropharynx (7/33; 21.2%) followed by the larynx (10/71; 14.1%) and oral cavity (45/341; 13.1%). Of the HR HPV DNA-positive HNC specimens, 15 were also positive for HPV RNA. Figure 10 summarizes the data. The highest percentage of double-positive cases for HPV DNA and RNA was in the oropharynx (9.1%). In agreement with other worldwide studies, we detected a low percentage of HPV DNA/RNA-positive cancers of the oral cavity and larynx. Interestingly, the initial findings in India suggest that HPV16 infection is involved in a lower percentage of HNC, in comparison to Europe. Five of the HNCs (33%) were positive for DNA and RNA of other mucosal HR HPV types, namely HPV18, 31, 35, and 56. These initial findings, if validated in the other Indian studies, may reflect important differences between Europe and India and influence HPV vaccination strategies that could be implemented in India.
As described above, all HPV DNA-positive HNCs (N=62) were checked for p16INK4a expression, together with 38 HPV DNA-negative cases. As shown in Figure 11, only 5 of the 15 HNCs positive for HPV DNA and RNA were also positive for p16INK4a, while the remaining HPV DNA/RNA-positive cases were p16INK4a-negative. Since HPV RNA is considered the gold standard for HPV infection, these data indicate that p16INK4a is not a good surrogate marker of HPV infection in this Indian population. These conclusions are also supported by the fact that (i) 14 cases of HPV DNA-positive and RNA-negative HNC and (ii) 5 cases of HPV DNA/RNA-negative HNC expressed p16INK4a. This scenario differs from the one observed in European cohorts, where p16INK4a expression appears to better correlate with positivity for HPV RNA. This difference could reflect the presence of different risk factors for HNC development in Europe and India. The analyses of the data obtained in the other five Indian regions in India are under way and will provide important insights into this specific issue.
In summary, the initial data provide important insights into (i) epidemiology and etiology of HNC in Europe and India, (ii) prognosis and survival of HNC associated with different risk factors, and (iii) novel biomarkers and assays for HNC screening and diagnosis.
In particular, the key messages are:
(i) Antibodies against E6 and L1 of several mucosal HPV types can be used as diagnostic markers for HPV-positive HNC.
(ii) Patients with HPV-positive HNC have E6 antibodies in their blood several years before the development of the disease, indicating that HPV E6 antibodies can be used as prognostic markers for HPV-positive HNC.
(iii) HNC linked to HPV infection has a better prognosis than HPV-negative HNC.
(iv) Detection of HPV DNA alone is not sufficient for a precise determination of the role of HPV infection in HNC.
(v) A higher proportion of HNCs in India appear to be associated with infection of mucosal HR HPV types other than HPV16.
(vi) p16INK4a appears to be a better surrogate marker for HPV-positive HNC in Europe than in India.
(vii) A small proportion (1–2%) of oral and laryngeal cancers are associated with HPV infection in Europe and India.
However, additional time is needed to fully analyse all the collected information and generated data on this large collection of HNC specimens in Europe and India, as well as to prepare all manuscripts associated with the HPV-AHEAD study. As already mentioned above, it has been agreed among members of the HPV-AHEAD consortium that we will continue to work together for at least two additional years.

WP5: Risk factors and natural history of HPV infections in the oral cavity
The main research activities of this WP aimed to investigate the natural history of HPV infection in the oral cavity and possible risk factors.
Our initial efforts were focused on the development of a robust, simple, and reproducible sampling protocol for oral exfoliated cells. In particular, we identified a procedure that preserves DNA and allows storage for several days at room temperature, greatly increasing the feasibility of the planned studies. Briefly, commercial mouthwash solutions (Listerine) were used for gargling. Gargles were immediately mixed with one volume of preservation buffer containing methanol and EDTA. The protocol was validated in a pilot study with approximately 20 volunteers.
DNA was extracted with an automated DNA extractor (MagNA pure, Roche). To identify the correct procedure for the storage of purified DNA at collecting centres, its stability has been evaluated at different temperatures. We included in the protocol two conditions: (i) short-term storage at +4°C and (ii) long-term storage at −20°C. No good-quality RNA was obtained from exfoliated cells collected by gargling. This is explained by the fact that gargling, being a non-invasive method, can mainly collect dead exfoliated cells of the superficial layers of the mucosa, in which most of the RNA is already degraded.
Gargles were collected from 999 individuals in Heidelberg (Germany) and Padua (Italy). The original plan was to collect approximately 500 oral samples in the student population of the medical school in Heidelberg. To increase the statistical power of the study, additional collecting centres were included in the HPV-AHEAD network. Partner 5 (Pawlita, DKFZ) initiated a collaboration with Professor Paolo Boscolo-Rizzo from the University of Padua, who coordinated the collection of an additional 500 oral specimens in three different hospitals in northern Italy (Treviso, Padua, Conegliano). The collection of samples in Italy and Germany was associated with a questionnaire, which provided several information, e.g. gender, age, education, and HPV vaccination status. Of the 999 recruited individuals, 174 had been vaccinated for HPV, with a higher proportion of vaccinated participants in Germany than in Italy (please see table below).
Gargles were analysed for the presence of viral DNA by two different Luminex-based multiplex HPV genotyping methods (MPG). The first includes the use of degenerate PCR primers in the late gene L1, while the second method uses specific primers of the early E7 gene from 21 mucosal HPV types (TS-E7). Results obtained with the L1 MGP showed a similar HPV prevalence in Germany and Italy, about 4%. However, a higher percentage of HPV16-positive oral specimens was detected in Germany (60%) than in Italy (37%). The prevalence of the different mucosal HPV types detected in all 999 oral specimens is shown in Figure 13.
In agreement with a key role in HNC, HPV16 was the most frequently detected HPV type. Only three oral specimens contained multiple HPV infections.
No significant association was observed between oral HPV status and gender, age, education, vaccination status, tobacco use, alcohol consumption, history of tonsillectomy, or number of sex and oral sex heterosexual and homosexual partners in the lifetime and in the past 12 months.
A total of 865 specimens were re-analysed with the TS-E7 assay. In accordance with its high sensitivity, the TS-E7 method detected a higher number of mucosal HPV types (113/865, 13%). The TS-E7 method detected a larger spectrum of mucosal HR HPV types as well as a higher number of multiple infections than the MPG assay (Figure 14). The increased prevalence of HPV types obtained by the TS-E7 assay allowed us to re-evaluate the impact of HPV vaccination on the presence of vaccine HPV types in the oral cavity of vaccinated and unvaccinated women. The data obtained with the TS-E7 assay showed that the percentage of vaccine HPV types in oral samples was 5.3% and 10.8% (p=0.01) in unvaccinated and vaccinated women, respectively. Ongoing analyses are focused on evaluating possible associations between HPV infection determined by the TS-E7 assay and other risk factors, e.g. education, tobacco use, alcohol consumption, and sexual habits.
In conclusion, we have been able to establish a robust and simple collection method of oral samples for the analysis of HPV infection. The comparison of two different HPV genotyping methods highlighted the concept that the sensitivity of the assay can significantly improve the detection of HPV infection in the oral cavity, where due to the continuous swallowing the concentration of free viral DNA can be very low.
We also performed analysis of HPV prevalence in archival oral non-tumour tissue samples. We observed in these specimens an extremely low prevalence of mucosal HPV types. These findings are confirmed in an independent study entitled “Study of papillomavirus & precancerous lesions in the tonsils (SPLIT)” and coordinated by Dr Silvia Franceschi (IARC). In this collaborative study, Partner 1 (Tommasino) has analysed approximately 700 tonsillar specimens, and HPV DNA was detected in only 2% of the samples. In contrast, other viruses (e.g. Epstein–Barr virus [EBV]) were highly frequent.
Overall, most of the goals of WP5 have been achieved.

WP6: Analyses of cellular gene and microRNA expression in HPV-positive and HPV-negative HNC
The tasks of this subproject were mainly focused on (i) determining the expression levels of several cellular proteins as well as microRNAs in a subgroup of HPV DNA-positive or HPV DNA-negative HNCs, and (ii) identifying surrogate markers for HPV infection. In the original proposal, we planned to determine the expression of cellular proteins involved in the regulation of key pathways in carcinogenesis, including SUMO pathway components and several signalling pathways, such as NF-κB, AKT, and mTOR.
HPV positivity was established considering the status of viral DNA and RNA and the expression of p16INK4a. Only HNCs positive for the three markers were classified as HPV-positive cases. In contrast, HNCs were classified as non-HPV-related cases when they were negative for the three markers.
At an early stage of the subproject, we observed that the immunohistochemical staining for the majority of these cellular proteins did not tightly correlate with the HPV status. Only a few cellular proteins appeared to be considerably deregulated in HPV-positive cancer cells and could potentially be used as surrogate markers of HPV infection. In particular, the expression of UBC9, a key enzyme of the SUMO pathway, appeared to be strongly upregulated in HPV-positive HNC. SUMO (small ubiquitin-like modifier) proteins are small proteins that are covalently and reversibly attached to other cellular proteins to modify their functions. SUMOylation is a post-translational modification controlled by an ubiquitin-like pathway and resulting in many different outcomes on protein stability, interaction, and localization, DNA repair and replication, transcriptional regulation, cell cycle control, apoptosis, cell signalling, and viral replication. Initial work of Partner 5 (Chiocca) showed that the expression of several enzymes of the SUMO pathway is strongly upregulated in low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively) of the cervix (Figure 15).
Importantly, the intensity of the staining for these enzymes increases according to the severity of the cervical lesion. Figure 16 illustrates the results obtained for the UBC9 staining of LSIL and HSIL. The intensity of UBC9 staining is significantly higher in HSIL than in healthy tissue or LSIL.
In agreement with these results obtained in cervical tissues, Partner 5 (Chiocca-EIO) also found a similar UBC9 expression pattern during head and neck carcinogenesis. As shown in Figure 17, UBC9 levels were low in normal epithelia, higher in low-grade dysplasia, and even higher in high-grade dysplasia.
In addition, analysis of FFPE HNC tissues showed that UBC9 positivity was higher in HPV-positive HNCs compared with their HPV-negative counterparts (Figure 18). In conclusion, these results support a possible use of UBC9 staining as an additional marker of HPV infection.
Partner 6 (Mosialos) assessed the activity of the canonical and non-canonical NF- B activation pathways in HNC samples by evaluating the nuclear localization of p65/RelA and p52/NF- B2, respectively. In addition, partner 6 evaluated the expression levels of CYLD, argininosuccinate synthase 1 (ASS1), cMyc and E2F1. The selection of these molecules was based on literature reports that indicated an HPV-dependent expression of these proteins in HNC. The slides were scored according to the procedure outlined by Partner 2 (Pawlita) in Kostareli et al J Clin Invest. 123(6):2488-501, 2013. Final expression scores ranged from 1 to 16. One of the antigens tested gave statistically significant differences between HPV-positive and HPV-negative HNCs. More specifically, the expression of E2F1 was higher in HPV-negative compared to HPV-positive samples (Figure 19). The median scores in the case of E2F1 were 6 for the HPV-positive samples and 8.5 for the HPV-negative samples. Representative images of samples stained for E2F1 are shown in Figure 19. No differences or statistically significant differences were noted in the median scores of HPV positive and HPV-negative samples that were stained for p65/RelA, p52/NF-kappB2, CYLD, cMyc and ASS1.
An additional task of this subproject was to determine the pattern of expression of microRNAs in HPV-positive and HPV-negative HNC. Unfortunately, due to the lack of support from the Indian Medical Research Council for the Indian studies, a considerable part of the coordinator’s budget was allocated to Partner 10 (Pillai) and six Indian hospitals in order to collect and process the Indian specimens. This change in the budget prevented the analysis of the microRNAs. The HPV-AHEAD consortium has agreed that additional funding will be sought to perform this work.

WP7: Training activities and transfer of technologies
An important activity of the HPV-AHEAD consortium was to facilitate the establishment of novel collaborative projects between European and Indian researchers. Immediately after the initiation of the HPV-AHEAD study, Partners 1 (Tommasino and Sankaranarayanan) and 10 (Pillai) established an Indian Consortium including six hospitals from different geographical areas, as shown in Figure 20. Three hospitals, Ambilikkai, Bangalore, and Barshi, are in rural areas, while the other three hospitals, Bangalore, Sevagram, and Trivandrum, are located in urban centres. A kick-off meeting involving the six Indian centres, Partner 10, and the HPV-AHEAD coordinator was held in Trivandrum on 12 November 2011. This meeting was focused on the establishment of protocols and strategies for the collection and processing of specimens. Collaborative Research Agreements were generated between IARC and the six centres in order to provide economic support for the collection of the HNC specimens. In addition, Partner 10 (Dr R. Pillai) together with Partners 1 (Tommasino and Sankaranarayanan) and 2 (Pawlita) have prepared a grant proposal that was submitted to the Indian Council of Medical Research (ICMR). This application has the same study design as HPV-AHEAD, including similar tasks and deliverables. Briefly, this novel study aims to collect an additional 4000–5000 HNC specimens from five additional Indian centres: (i) Mahavir Cancer Sansthan & Research Centre (MCSRC), Phulwarisharif, Patna (PI: Dr Jitendrakumar Singh); (ii) V N Cancer Centre, GKNM Hospital, Pappanaickenpalayam, Coimbatore (PI: Dr M Nagarajan); (iii) Cancer Institute (WIA), Chennai (PI: Dr Shirly Sunder Singh); (iv) Delhi State Cancer Institute, Dilshad Garden, Delhi (PI: Professor R K Grover); and (v) Motilal Nehru Medical College 16/2, Lowther Road, Allahabad (PI: Professor Ravi Mehrotra). Although the application has been granted by ICMR, funds have not yet been released and this new collaborative study has not yet been initiated. However, contacts have been established between the HPV-AHEAD consortium and these five additional Indian centres. The second HPV-AHEAD annual meeting was held in Trivandrum on 8–9 November 2012. In addition to partners of the HPV-AHEAD consortium, the invitation was extended to several researchers from the six collecting centres already participating in the HPV-AHEAD study, as well as from the five new centres included in the ICMR application. This event was an opportunity for the new collaborative Indian centres to become acquainted with the structure, aims, and the initial data of the HPV-AHEAD consortium. Approximately 30 Indian researchers attended the HPV-AHEAD annual meeting and discussed possible future collaborations in the context of HPV and HNC.
In addition, Partners 1 (Tommasino) participated in another independent grant proposal on HPV and HNC that was submitted to ICMR. The principal investigator of this application is Professor Rajesh Dikshit from Tata Memorial Hospital in Mumbai, and the project has similar tasks to the HPV-AHEAD study in evaluating the role of HPV infection and other risk factors in the development of HNC. This study has also been granted by ICMR and is awaiting the release of the funds. As an initial step, a researcher of the group from Tata Memorial Hospital has visited for a few weeks the HPV laboratory at RGCB in Trivandrum, to which the technology for HPV genotyping and HPV serology has been transferred from IARC (Partner 1) and DKFZ (Partner 2), respectively. The final aim of this additional collaborative effort is to establish a HPV diagnostic platform in Mumbai in order to perform additional HPV-related studies.
In summary, the HPV-AHEAD consortium has put in place several strategies that have generated several collaborations between European and Indian research groups. Furthermore, there is no doubt that the HPV-AHEAD consortium will continue to have a positive impact on future collaborative research projects within India and between Europe and India.
In addition to the activities described above, the HPV-AHEAD consortium organized a summer school on the role of infections in human cancer at RGCB in Trivandrum immediately before the HPV-AHEAD annual meeting (5–7 November 2012). The faculty included many partners of the HPV-AHEAD consortium and a few Indian researchers. The topics of the lectures were focused on epidemiology, biology, and preventive strategies of several viruses associated with human carcinogenesis, i.e. HPV, hepatitis B virus (HBV), and EBV. The HPV-AHEAD consortium provided support for travel and accommodation for approximately 20 young researchers from various Indian regions. Furthermore, at least 20 additional junior scientists from the local community attended the event.
A mini-symposium on topics closely related to the research activities of the students attending the summer school was organized on the last afternoon of the event (7 November 2012). Five Indian researchers presented their scientific projects closely related to the topics of the HPV-AHEAD programme. All presentations were followed by lively discussions between the faculty and participants.
In addition to the events in India, Tommasino and Brennan (Partner 1) organized an international scientific workshop in Italy on 2–3 June 2014 entitled “Emerging Issues in Head and Neck Cancer” (http://www.iarc.fr/headandneck2014/) in which many members of the HPV-AHEAD consortium were involved together with worldwide leaders in HNC and HPV. This event was followed by another congress entitled “Emerging Oncogenic Viruses” organized by Partners 1 (Tommasino) and 2 (Pawlita) and other colleagues, with 110 participants from Europe, the USA, South America, and Asia (http://www.iarc.fr/oncogenicviruses2014/). The HPV-AHEAD logo was displayed on the homepages of both event websites.
In regard to the transfer of technology, Partners 1 (Tommasino) and 2 (Pawlita) have worked together with Partner 10 (Pillai) to transfer the HPV genotyping and the serology assays based on the Luminex technology. Both assays have been used widely for detection of a broad spectrum of infectious agents and offer unique features: (i) have high throughput, specificity, and sensitivity; (ii) have versatility to use many types of specimens; and (iii) are relatively inexpensive, in order to be used in large-scale epidemiological studies.
As an initial step in the transfer of the technology, both European partners visited the RGCB and planned, together with local scientists, the organization of these new platforms. After RGCB purchased a Luminex apparatus, IARC and DKFZ hosted RGCB junior researchers for several months, who were trained on the use of the Luminex-based assays. Both multiplex Luminex-based platforms are now fully established at RGCB in Trivandrum. One Luminex-based platform is used to detect HPV DNA for 21 different mucosal HPV types. The other Luminex-based platform is used to detect antibodies in human sera against 38 proteins from many mucosal HPV types. Both platforms are currently used for HPV-AHEAD-related as well as independent studies. For instance, they are currently used in an independent study coordinated by Partner 1 (Sankaranarayanan) aiming to compare the efficacy of different prophylactic HPV protocols in the young Indian population. The study also involves Partners 1 (Tommasino), 2 (Pawlita), and 10 (Pillai). Initial data have been included in a manuscript by Sankaranarayanan et al. entitled “Immunogenicity and HPV infection following one, two and three doses of quadrivalent vaccine: Early results from a multi-center cohort study in India”, which is in press in Lancet Oncology and include in the list authors Tommasino (Partner 1) Pawlita (Partner 2) and Pillai (Partner 10).
In summary, the activities of the HPV-AHEAD consortium have had a profound impact on (i) the generation of novel collaborations between European and Indian groups, (ii) the transfer of technology to India, and (iii) the training of Indian researchers in the field of infections and human cancers.

WP8: Network management
As planned in the grant proposal, a number of strategies have been put in place for the coordination and management of the HPV-AHEAD consortium. The management structure is illustrated in Figure 21. A Steering Committee (SC) was created and included Partners 1 (Tommasino and Sankaranarayanan), 2 (Pawlita), 3 (Bosch), 5 (Chiocca), and 7 (Boeing). In addition, Dr Olaf Kelm, head of the IARC Grant Office, assisted the SC with all administrative issues. The SC was responsible for assisting the coordinator in the management of the HPV-AHEAD consortium together with work package leaders (WPL) and for communicating with the EC scientific officer.
The HPV-AHEAD consortium also appointed an Independent and Ethical Advisory Board (IEAB) that included four external scientists: (i) Professor Thomas Schulz (Chairman), Department of Virology, Hannover Medical School, Hannover, Germany; (ii) Professor Anna Giuliano, Director, Center for Infection Research in Cancer, Moffitt Cancer Center, Tampa, Florida, USA; (iii) Professor Moni Kuriakose, Division of Head and Neck Oncology, Narayana Hrudayalaya Health City, Bangalore, Karnataka, India; and (iv) Professor Matti Lehtinen, University of Tampere School of Public Health, Tampere, Finland. During the study, IEAB members have provided scientific and ethical advice and participated in two annual meetings of the HPV-AHEAD consortium (in Barcelona in 2014, and in Heidelberg in 2015) as well as meetings and telephone conferences of the SC.
The achieved goals in this WP include:
(i) Generation of the HPV-AHEAD consortium agreement.
(ii) Creation of a public and a consortium homepage of the HPV-AHEAD programme (http://hpv-ahead.iarc.fr/).
(iii) Organization of four consortium annual meetings: (a) 25–27 September 2011, Lyon, France; (b) 8–9 November 2012, Trivandrum, India; (c) 18–19 March 2014, Barcelona, Spain, with participation of IEAB; (d) 6–8 July 2015, Heidelberg, Germany, with participation of the chairman of the IEAB and the EC scientific officer Dr Jan-Willem Van De Loo.
(iv) Organization of a kick-off meeting for the Indian network,12 November 2011, Trivandrum, India.
(v) Organization of 14 SC events, namely nine telephone conferences and five meetings.
(vi) Preparation of all ethical applications and clearance by IARC and local ethical committees.
Finally, the IARC administrative office, the head of the IARC Grant Office (Dr O. Kelm), and the coordinator (Tommasino) have been responsible for administrative and financial management, which includes the following tasks:
(i) Periodic reporting and document production and archiving.
(ii) Establishing and maintaining financial records.
(iii) Coordination and consolidation of annual financial statements by all project partners, follow-up of EC payments, distribution of partner shares, and monitoring of payments according to the agreed procedures.
(iv) Executing and controlling global expenses, such as investments, subcontracting, etc.
(v) Obtaining audit certificates and bank guarantees as required.
(vi) Assistance to individual project partners on specific administrative issues. The financial management rules for the projects were agreed upon in the HPV-AHEAD Consortium Agreement.

Potential Impact:
HNC represents a serious health problem as the sixth most common malignancy worldwide, with an estimated global burden of approximately 700 000 incident cases and a fatality rate of 380 000 deaths per year (GLOBOCAN 2012, IARC; http://globocan.iarc.fr/). Due to the establishment of effective anti-tobacco and anti-alcohol campaigns, the overall incidence of HNC is decreasing in developed countries. However, it remains a severe problem in low- and middle-income countries where such strategies are not yet fully implemented. Infection with mucosal high-risk (HR) HPV types is an additional key risk factor for a subset of HNC. Importantly, HPV-positive HNCs have been steadily and significantly increasing in developed countries, e.g. the USA and Europe. For instance, in Sweden tonsillar cancers associated with HPV infection tripled in the past three decades. Together, these data indicate that changes in lifestyle and/or specific environmental factors may be responsible for the increase of HPV-positive HNC. The Indian subcontinent has the highest HNC incidence in the world, and it accounts for one third of the global burden. Although it is well known that in India alcohol consumption, smoking, and use of smokeless tobacco are key risk factors for HNC development, very little is known about the potential role of HPV infection in this pathological condition.
The main activities, achievements, and possible future impacts of the HPV-AHEAD programme are schematically represented in Figure 22.
The HPV-AHEAD consortium has generated a very large biobank, collecting more than 8000 FFPE HNC blocks in Europe and India to perform case studies and evaluate the role of mucosal HR HPV types and other risk factors in carcinogenesis at these anatomical sites. The strength of this study was also that all specimens and clinical information were processed in a centralized manner in order to have standardized performance of the laboratory assays and interpretation of the results.
Although the analyses of the obtained results is still under way and will continue for additional years, the initial findings provide important insights into: (i) the incidence of HPV-positive HNC in different geographical areas in Europe and India, (ii) the use novel viral markers in the diagnosis and prognosis of HPV-associated HNC, and (iii) the potential development of novel therapeutic and prophylactic strategies.
Establishing the HPV positivity of HNC is of high clinical relevance because it can influence prognosis and potentially the treatment modality, including the reduction of intensity of therapy compared with HPV-negative HNC. Survival rates for oropharyngeal cancer are strongly influenced by HPV status (Ang et al., 2010). In a North American study, the median survival of patients with HPV-positive oropharyngeal cancers was found to be significantly longer than that of patients with HPV-negative oropharyngeal cancers (Chaturvedi et al., 2011). In randomized clinical trials, patients with HPV-positive oropharyngeal cancer consistently have a 70% relative reduction in risk of death compared with HPV-negative patients (Ang et al., 2010). The identification of simple procedures for a precise classification of HPV-positive HNC is urgently needed in routine clinical practice and will have a strong beneficial impact on the management of patients. Our data confirm that detection of viral DNA alone is insufficient proof for viral causality and could lead to misclassification of the lesion. In Europe, and even more in India, the percentage of HPV-positive specimens significantly decreased when HPV DNA and RNA were considered compared with HPV DNA alone. Importantly, our findings provide evidence that HPV serological markers, i.e. mucosal HR HPV E6 antibodies, could be included in novel algorithms for a reliable classification of HPV-positive HNC. Although the serological findings need to be validated in independent and large-scale studies, due to the fact that antibody detection assays are relatively simple and are routinely used in clinical settings, it is highly likely that the detection of antibodies against mucosal HR HPV types can be transferred to routine clinical practice to improve the classification of HNC.
Another issue that has hindered the management of HNC is that, in contrast to those in the genital tract, HPV-associated premalignant lesions in the head and neck region are not yet clearly identified. Screening for premalignant cervical lesions has had a profound impact on the prevention of cervical cancers. Most premalignant cervical lesions arise in the transformation zone, which is relatively easily accessible for visual and colposcopic inspection, as well as for cell and tissue sampling. In contrast, most HPV-positive oropharyngeal cancers originate at anatomical sites that are not visible and not easy to access, e.g. the invaginating tonsillar crypt epithelium. Premalignant HPV-positive oropharyngeal lesions have been rarely identified using tonsillectomy specimens (Begum et al., 2005; Palmer et al., 2014). Small-scale studies in which exfoliated cells were directly collected from patients did not reveal any correlation between HPV16 and cytopathology when malignant lesions were not visible (Jordan et al., 2012). In HPV-AHEAD, we have provided evidence that antibodies against HR HPV16 E6, E7, E1, and E2 proteins were present in individuals’ blood more than 10 years before diagnosis of oropharyngeal cancers, suggesting a potential role for HPV serology as an early marker of oropharyngeal cancer (Kreimer et al., 2013). Thus, these data pave the way for new studies aimed at developing new screening strategies for individuals at higher risk for development of HPV-positive HNC. For instance, it would be important to evaluate whether the combination of serological assays with detection of viral DNA in the oral cavity could lead to the development of robust and highly specific screening procedures. These future studies can be considerably facilitated by our work in WP5, in which we have established a simple and sensitive protocol for the detection of mucosal HPV DNA in saliva. In addition, our study highlighted the importance of using a very sensitive assay for the detection HPV DNA in gargles.
Studies on cervical lesions demonstrated that the increased expression level of the cell cycle inhibitor p16INK4a in HPV-positive cells is a good surrogate marker for viral infection. Our data in the context of the HPV-AHEAD study indicate that p16INK4a positivity as a marker for HPV infection is less specific in HNC than in cervical lesions, in particular in India. Although the analyses of the Indian specimens are still under way, we have identified several HPV DNA and RNA-positive cases that were p16INK4a-negative. The completion of the ongoing analyses will further clarify this issue. In Europe, p16INK4a staining appears to better correlate with HPV DNA and RNA positivity, although initial data indicate a lower specificity than that observed in cervical lesions.
Our findings also provide information on the incidence of HPV-positive HNC in many geographical regions in Europe and India. Although HPV16 is involved in the majority of HNC, initial data in India indicate that approximately 30% of HNCs are associated with other mucosal HR HPV types, such as HPV31, 35, and 56, while in Europe the other mucosal HR HPV types appear to be only marginally involved in HNC. If these data are confirmed by the analyses of the findings obtained in other Indian regions, they will be extremely important for the implementation of HPV vaccination programmes covering multiple mucosal HR HPV types, such as the recently developed nonavalent HPV vaccine. However, our initial findings in India showed that HNC can be also associated with HPV types not included in the nonavalent vaccine, e.g. HPV35 and HPV56.
Another very important contribution of the HPV-AHEAD consortium is the continual review of the literature and the generation of meta-analyses on worldwide incidence and mortality of HNC and the role of the different risk factors. In addition to many scientific publications, these research activities have resulted in the creation of an interactive website (http://www.hpvcentre.net/) on HNC and associated risk factors that will remain active and will be continuously updated after the end of the HPV-AHEAD study.
In addition, the HPV-AHEAD consortium has been very successful in generating a multidisciplinary team, creating many synergisms and collaborations between the partners of the HPV-AHEAD study and additional researchers in Europe and India on closely related topics. These achievements will have a profound impact on novel collaborative studies. The consortium was also very productive in transferring technologies from Europe to India and training junior scientists in the fields of infections and cancer.
In summary, most of the achieved objectives are highly relevant for public health, providing important novel knowledge for diagnosis, treatment, and prophylaxis of HNC in Europe and India and other geographical regions.
Dissemination of the achievements outside the consortium has been carried out using several strategies:
(i) Presentation of the data at national and international meetings.
(ii) Organization of international workshops.
(iii) Publications in peer-reviewed journals.
(iv) Creation of a public website.
It is expected that the HPV-AHEAD consortium will continue working together for many years, and efforts are now focused on seeking additional funding to offer new developments and exploitation of the achievements obtained so far.

List of Websites:
http://hpv-ahead.iarc.fr/

Final Report Summary - HPV-AHEAD (Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in India and Europe)

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