Papillomaviruses replicate and cause disease in stratified squamous epithelia. Epithelial differentiation is essential for the progression of papillomavirus replication, but differentiation is also impaired by papillomavirus-encoded proteins. The papillomavirus E6 and E7 oncoproteins partially inhibit and/or delay epithelial differentiation and some of the mechanisms by which they do so are beginning to be defined. This review will outline the key features of the relationship between HPV infection and differentiation and will summarize the data indicating that papillomaviruses alter epithelial differentiation. It will describe what is known so far and will highlight open questions about the differentiation-inhibitory mechanisms employed by the papillomaviruses. Papillomaviruses and their host species have evolved together for tens of millions of years, and it is thought that the viruses have both adapted to specific host niches and co-speciated with their hosts . PapilloThe human papillomavirus (HPV) E6 and E7 proteins have been the most frequently characterized with respect to their effects on cellular proliferation and, to some degree, on differentiation. Some of the pro-proliferative mechanisms employed by HPV E6 and E7 are shared by other DNA tumor viruses that do not specifically infect stratified epithelia. For example, several DNA tumor viruses inactivate the retinoblastoma and p53 tumor suppressors to promote proliferation and evade apoptosis . The abiThis review will summarize the studies that have established the effects of HPV-encoded proteins, particularly HPV E6 and E7, on epithelial differentiation. It will highlight some of the mechanisms by which HPV E6 and E7 delay and impair differentiation. Impaired differentiation is a hallmark of some cancers and could contribute to HPV-mediated carcinogenesis. Open questions related to HPV control of epithelial differentiation and the possibility of differentiation therapy for HPV-positive cancers will be discussed.HPV have circular double-stranded DNA genomes of ~8 kbp that encode seven to nine open reading frames (ORF) depending on the specific virus. Genetic diversity among the papillomaviruses (PV) is high. Well over 200 HPV genotypes and hundreds more animal PV genotypes have been identified and sequenced and many more are pending characterization ,7. IndivSeveral features of the productive HPV life cycle depend on the biology of the stratified squamous epithelium. Upon initial infection, HPV particles bind to heparan sulfate proteoglycans present on the cell membrane or in the extracellular matrix ,9. A subFollowing deposition of the HPV DNA in the nucleus, a burst of viral genome amplification establishes HPV genomes in basal epithelial cells. Viral DNA replication requires the HPV E1 and E2 proteins as well as components of the host cell DNA replication machinery . In a prHPV transcription occurs from two major promoters: an early promoter present in the ~1 kbp viral non-coding region (variously referred to as the long coding region (LCR) or the upstream regulatory region (URR)) and a downstream late promoter. These have been extensively characterized in HPV31, where they are present at nucleotide 97 (p97) and nucleotide 742 (p742), respectively. The corresponding sites in HPV16 are p97 and p670 and in HPV18 are p55 and p811 ,13,14,15Epithelial differentiation is a constantly occurring regenerative process that forms the protective exterior barrier between an organism and its surrounding environment. Best studied in mammalian skin, the process of epidermal differentiation originates in proliferative basal keratinocytes that are separated from the dermis by a specialized form of extracellular matrix (ECM), the basement membrane . LamininThe first transition in the differentiation program is the delamination of a basal cell and its progression to a suprabasal (spinous) state ,23. Supr2), with or without the withdrawal of growth factors and/or the addition of serum. For many of the HPV-episome-containing cell lines, calcium treatment induces viral DNA amplification, transcription from the late promoter, and differentiation-dependent viral transcript splicing events. Culture in methylcellulose-based semisolid medium can promote many of the same changes. Three-dimensional cell culture systems (organotypic “raft” cultures) have been employed by HPV researchers for several decades. In such cultures, keratinocytes are grown to confluence on a matrix (often a collagen matrix containing mouse fibroblasts), then lifted to the air–liquid interface and allowed to stratify and differentiate for a period of 1–2 weeks. For cells that contain viral episomes, this allows for somewhat heterogenous differentiation and mimics some aspects of the viral life cycle. HPV virions are typically produced from organotypic cultures only rather inefficiently, especially in the case of immortalized cell lines. New PV model systems are sure to enable additional investigation. For example, a recently discovered murine papillomavirus has prompted additional studies on the relationship between cutaneous PV biology and epithelial differentiation [Several aspects of the differentiation-dependent HPV life cycle can be modeled in keratinocyte culture systems ,27,28. Hntiation ,32,33,34Experiments in these systems have demonstrated that differentiation is required for HPV replication. Some specific cellular pathways that regulate epithelial differentiation have been found to regulate HPV genome amplification and late transcription. Protein kinase C (PKC) inhibitors limit HPV late activities, and efficient HPV virion production from organotypic raft cultures requires PKC activation ,36. OtheJust over one dozen mucosal HPV, primarily found in genus alpha species 7 and 9, are the “high-risk” HPV genotypes that cause nearly all cervical cancer and many other anogenital cancers ,38. HighWAF1/CIP1 and p27KIP1 [The contributions of the HPV E6 and E7 oncogenes to carcinogenesis have been studied extensively. A few years after zur Hausen and colleagues published the initial reports that cervical cancer samples contain human papillomavirus DNA, zur Hausen’s and other groups found that the HPV E6 and E7 ORFs are specifically preserved in cervical cancer cells and that they have transforming activity in rodent cells ,48,49,50 p27KIP1 ,55,56. A p27KIP1 . To coun p27KIP1 ,59. In a p27KIP1 ,61,62,63 p27KIP1 ,65.INK4A/p14ARF epigenetically [INK4A induction.This existing model does not account for all of the available information concerning the activities of HPV E6 and E7 . Most HPetically ,80,81. RA recent sequencing study highlights the critical contribution of HPV16 E7 to cervical carcinogenesis . In thisUpdated models of oncogenic transformation by high-risk HPV have the potential to incorporate the new information provided by high-throughput studies and to account for activities of the viral oncoproteins that are not included in existing models. It is often stated that HPV oncoproteins act to uncouple proliferation from differentiation . HoweverThe recognition that a subset of HNSCC are caused by HPV has enabled the comparison of HPV-positive to HPV-negative tumors at a similar anatomical site. A finding from pathology reports is that HPV-positive HNSCC tend to be poorly differentiated compared to HPV-negative HNSCC ,86. A reSome previous studies in the pathology literature are consistent with this hypothesis even though they did not directly assess HPV status. A 1996 study observed a large range of involucrin expression in HNSCC and reported that poorly differentiated HNSCC are more likely to distantly metastasize . PropensSoon after observing that high-risk HPV oncogene DNA was preserved in cervical cancer cells, researchers began to conduct studies on the oncogenes and on the entire HPV genome in differentiation models in keratinocytes. They observed that HPV DNA-positive HFK or SCC-13 cells could stratify in raft cultures and that the cells expressed keratins but did not differentiate normally. Hematoxylin and eosin staining on HPV-positive raft cultures indicates that the abnormal morphology resulting from HPV DNA is comparable to the abnormal morphology in a cervical or penile intraepithelial neoplasia . InitialResearchers have examined high-risk HPV E6 or E7 alone and/or used mutant genomes that do not express E6 or E7 in order to assess the individual contributions of the oncoproteins. The strongest effects on differentiation occur when E6 and E7 are expressed together. Organotypic cultures that express high-risk HPV E7 consistently exhibit an increase in proliferation in suprabasal cells. Several of the initial reports concluded that high-risk HPV E7 has more differentiation-inhibiting activity than HPV E6 ,100,101.Transcriptional studies on normal and HPV-DNA-containing cells and on HPV-positive and negative cancer cells illustrate that HPV gene expression impairs epithelial differentiation. Consistent with the analysis of raft cultures, human cells harboring high-risk HPV genomes express lower levels of differentiation marker genes and both high-risk HPV E6 and E7 likely contribute to this repression. Most work has focused on the HPV16 oncogenes, with some analysis of HPV18 and minimal examination of low-risk HPV. Early cDNA array experiments examined a few thousand cellular genes and compared normal cells to cells expressing HPV16 E6 and E7 . The strAdditional transcriptional studies on HPV16 immortalized cell lines and on cervical cancer cell lines demonstrate that the repression of differentiation is maintained over time in HPV-positive cells. Wan and colleagues compared normal cells to early passage HPV16-immortalized and to late passage HPV16-immortalized cells and noted that downregulation of epithelial structural genes is reflected both in the early and late cell models . These aMore recent analyses have combined transcriptional data with pathway analysis. Overall, pathways related to cell cycle, cellular assembly and organization, DNA replication, and growth and proliferation are dysregulated by HPV16 E6 and E7. Basal keratins, such as keratin 5, are relatively unaffected by HPV16 E6 and E7, but HPV16 E7 tends to downregulate individual genes related to epithelial differentiation and a similar effect is observed with HPV16 E6 alone or with HPV16 E6 and E7 expressed together . RecentlGenus beta HPV infect the cutaneous epithelium. A subset of beta HPV cause hyperproliferative lesions in the skin of patients with the genetic disorder Epidermodysplasia Verruciformis, and links between beta HPV and skin cancer have also been proposed . SeveralElegant structural studies on papillomavirus E6 proteins demonstrate how HPV E6 achieve specific binding to their cellular targets ,124,125.CBF1/RBP-J , Su(H) (Drosophila), and Lag-1 (Caenorhabditis elegans)), displacing the repressors of CSL activity and allowing for the recruitment of transcriptional co-activators. RBP-J/CBF1 is the mammalian repressor and MAML1 is one such co-activator [Several signaling pathways are affected by MAML1, including those involving Notch, p53, MEF2C, and beta-catenin ,132. Howctivator ,135,136.ctivator ,116,137.Several groups used NOTCH-MAML1 reporter assays and analyzed endogenous NOTCH-responsive transcripts to find that beta HPV E6 inhibit signaling in response to a NOTCH stimulus ,117,137.These results have been interpreted to mean that beta HPV E6 proteins bind MAML1 to repress epithelial differentiation. Presumably, this would enable maintenance of a more proliferative state in the beta HPV infected epithelial cell and a cellular environment that promotes persistence and viral DNA replication. Meyers and colleagues demonstrated that HPV8 E6 significantly inhibits the calcium-responsive transcription of HES1 and the calcium-dependent inhibition of involucrin in keratinocytes . This inThe lack of robust life cycle models for the genus beta HPV means that these predictions regarding differentiation have not been tested in organotypic cultures. However, a recently discovered mouse papillomavirus, MmuPV1, shows promise as a model of cutaneous PV biology . MmuPV1 The effects of genus alpha HPV E6 on differentiation and the mechanisms by which they might act are less well established than for genus beta HPV E6. A differentiation-inhibitory effect of HPV16 E6 was identified in early studies that measured the ability of oncoproteins to induce resistance to calcium-mediated differentiation . Later vLike genus beta HPV E6, HPV16 E6 has been suggested to alter Notch signaling, although the underlying mechanism(s) are less clear than for the cutaneous HPV E6. MAML1 was initially identified in a yeast-two-hybrid screen for binding partners of HPV16 E6 , but theInactivation of the NOTCH signaling pathway by cutaneous HPV is consistent with the tumor-suppressive role of NOTCH in the skin ,155. NOTStudies in cervical cancer cell lines indicate that NOTCH signaling is dysregulated in cervical cancer, although there is not yet a consensus on whether manipulating NOTCH signaling in these cells promotes or inhibits cancer cell proliferation ,160,161.CIP1 (CDKN1A), p27KIP1 (CDKN1B), and p15INK4B (CDKN2B). The net effect of activation of these cell cycle inhibitors is cell cycle arrest in G1. TGFβ signaling controls epithelial differentiation and can have context-dependent tumor suppressive or oncogenic activities. The pathway is frequently mutated in epithelial tumors such that its activity is lost [In addition to NOTCH signaling, other pathways impact epithelial differentiation and are affected by HPV E6. TGFβ signaling begins with the TGFβ ligand binding to serine/threonine kinase receptor complexes. The intracellular binding partners of the TGFβ receptor are receptor-associated SMAD (R-SMAD) and co-activator SMAD (co-SMAD) proteins. Phosphorylation events allow for the assembly of an R-SMAD/co-SMAD complex on target gene promoters. The complex can transactivate the promoters of target genes, such as p21 is lost ,168,169, is lost .trans retinoic acid (ATRA).One of the early transcriptional studies on gene expression changes induced by HPV16 E6 and E7 noted that the viral oncoproteins alter the cellular response to TGFβ . The autThe mechanistic basis for effects on TGFβ signaling has been more clearly established for the genus beta HPV E6 proteins. Mendoza and colleagues found in a yeast-two-hybrid experiment that HPV5 E6, but not HPV9 E6, bound to SMAD3 . HPV5 E6INK4B (CDKN2B) promoter after TGFβ treatment.More recent experiments have further elucidated the interaction between genus beta HPV E6 and TGFβ signaling. The HPV E6 proteomics experiments identified several genus beta HPV E6 in complex with SMAD2 and SMAD3 ,115. MeyThe pro-proliferative effect of HPV E7 has been documented extensively . This seEpithelial differentiation in organotypic culture is impaired by episomal HPV16 DNA dependent on the HPV16 E7 ORF ,100. A rCIP1 links differentiation and proliferation in keratinocytes and the differentiation-inhibitory effect of HPV16 E7 has been ascribed to its ability to bind to p21CIP1 and block the inactivation of cyclin/cdk complexes [In monolayer culture, HPV16-E7-expressing cells resist differentiation induced by calcium treatment . p21CIP1omplexes ,56. In aomplexes . Low-risomplexes .Several experiments in mice indicate that HPV16 E7 inhibits differentiation and suggest that this is independent of RB1 binding ,176. Balmyc promoter [Some cellular pathways related to differentiation are known to be dysregulated by HPV E7. HPV16 E7 can abrogate some effects of TGFβ treatment, such as TGFβ-mediated inhibition of the promoter . Studiespromoter . Specifipromoter , but it High-risk HPV E7 modulate levels of p63, a master regulator of epidermal development in epithelial cells . An upstThe mutational analyses of HPV16 E7 suggest that its differentiation-inhibitory activity could be independent of RB1 binding, but the RB1-independent activities of high-risk HPV have remained poorly defined. The proteomic analyses published by several groups have suggested a new RB1-independent activity of high-risk HPV E7 ,115. DivOur recent work on PTPN14 and high-risk HPV E7 led us to discover that the loss of PTPN14 from human keratinocytes results in a striking downregulation of the epithelial differentiation program . An N-teMechanistically, we do not yet understand how PTPN14 degradation could limit epithelial differentiation. PTPN14 is a non-receptor protein tyrosine phosphatase that negatively regulates the transcriptional co-activator YAP1 ,189,190.Most effort to understand how HPV manipulates epithelial differentiation has focused on the E6 and E7 oncogenes and on signaling pathways, including Notch and TGFβ. Additional HPV-encoded proteins likely impact differentiation and additional differentiation-related signaling pathways are altered during HPV infection. For example, a recent study comparing HPV18 genomes to HPV18 genomes that do not express the E5 protein found that E5 contributes to a repression of involucrin expression upon calcium-mediated differentiation .Differentiation therapy, the induction of differentiation by a chemical agent, has been extremely successful in specific instances in the clinic. For example, acute promyelocytic leukemia can often be cured by treatment with ATRA and arsenic ,194. AltHPV-encoded proteins reprogram host cells to promote virus replication and enable persistent infection. In particular, HPV E6 and E7 drive the establishment of a pro-proliferative environment in the infected cell. They promote cell cycle re-entry in differentiating keratinocytes and restrict the epithelial differentiation program. Years of research on HPV E6 and E7 have enabled groundbreaking discoveries on the mechanisms that normally control the cell cycle and on how cell growth control pathways are dysregulated in cancer.HPV E6 and E7 lack enzymatic activity, meaning that their reprogramming activities are enabled by interactions with host cellular proteins. Unbiased proteomic analyses have begun to reveal the host cellular targets that are shared among and/or unique to a subset of the HPV oncoproteins. Some of these targets appear to be used by the HPV oncoproteins to manipulate the differentiation program. For example, the genus beta HPV E6 proteins engage MAML1 and SMAD to modulate signaling in the NOTCH and TGFβ pathways ,117,166.Studies on the mucosal HPV oncoproteins indicate that both high-risk HPV E6 and E7 have differentiation inhibitory activity. The accumulated evidence suggests that high-risk HPV E7 have a stronger inhibitory effect on differentiation relative to high-risk HPV E6, particularly in three-dimensional culture models. Furthermore, some mutational analyses are consistent with the idea that high-risk HPV E7 has a differentiation-inhibitory effect that is not dependent on its ability to bind RB1. PTPN14 is one host cell target of high-risk HPV E7 that is targeted independent of RB1 and shows early promise as a regulator of keratinocyte differentiation, but more work to understand how high-risk HPV repress differentiation is warranted. In addition, most of the work on how genus alpha HPV oncoproteins affect differentiation has been on the high-risk HPV. It remains unclear whether high-risk HPV oncoproteins, particularly high-risk HPV E7, have a greater capacity to decrease differentiation than their low-risk counterparts. An additional challenge to directly comparing high-risk HPV E6 and E7 comes from the fact that in cells that contain the HPV genome, E7-encoding transcripts are more abundant than E6-encoding transcripts ,17. A diTaken together, the results summarized in this review support the idea that cutaneous HPV E6 could have a greater differentiation-inhibitory capacity than cutaneous HPV E7, whereas mucosal HPV E7 is the more potent inhibitor of differentiation compared to mucosal E6. This hypothesis should be tested rigorously. Others have noted that when comparing HPV E6 and E7, E6 is the more potent oncoprotein in the skin whereas E7 is more potent in the mucosal epithelium . It willA final open question is whether inhibition of differentiation is required for HPV-mediated oncogenic transformation. It would seem logical that all HPV, not only high-risk HPV, must limit differentiation to enable viral genome replication in the stratified epithelium. However, differentiation is not only a normal process occurring in stratified epithelia, it is also a keratinocyte stress response. Oncogene-induced differentiation (OID) is the process by which keratinocytes differentiate in response to tumor suppressor (including p53) inactivation ,206 or o


In 2006, 4 years of planning was started by the Ministry of Health, Malaysia (MOH), to implement the HPV (human papillomavirus) vaccination programme. An inter-agency and multi-sectoral collaborations were developed for Malaysia’s HPV school-based immunisation programme. It was approved for nationwide school base implementation for 13-year-old girls or first year secondary students in 2010. This paper examines how the various strategies used in the implementation over the last 7 years (2010–2016) that unique to Malaysia were successful in achieving optimal coverage of the target population.Free vaccination was offered to school girls in secondary school (year seven) in Malaysia, which is usually at the age of 13 in the index year. All recipients of the HPV vaccine were identified through school enrolments obtained from education departments from each district in Malaysia. A total of 242,638 girls aged between 12 to 13 years studying in year seven were approached during the launch of the program in 2010. Approximately 230,000 girls in secondary schools were offered HPV vaccine per year by 646 school health teams throughout the country from 2010 to 2016.Parental consent for their daughters to receive HPV vaccination at school was very high at 96–98% per year of the programme. Of those who provided consent, over 99% received the first dose each year and 98–99% completed the course per year. Estimated population coverage for the full vaccine course, considering also those not in school, is estimated at 83 to 91% per year. Rates of adverse events reports following HPV vaccination were low at around 2 per 100,000 and the majority was injection site reactions.A multisectoral and integrated collaborative structure and process ensured that the Malaysia school-based HPV immunisation programme was successful and sustained through the programme design, planning, implementation and monitoring and evaluation. This is a critical factor contributing to the success and sustainability of the school-based HPV immunisation programme with very high coverage. HPV is the commonest sexually transmitted infection (STI) , 2. HPV Cervical cancer is the second most frequent cancer in the low-income countries and fourth in the world .It is thVarious strategies for prevention have been developed including vaccination, cervical cytology (Pap testing), and HPV DNA based screening, for early detection and effective treatment. HPV vaccines covering HPV16 and 18 can prevent up to 70% of cervical cancer cases without screening , 16. EarPrior to 2010, cytology-based screening was the main preventive measure for cervical cancer in Malaysia. The Papanicolaou smear (Pap smear) screening services initially aimed at post-partum mothers in family planning programs. In 1995, Pap smear screening was expanded to women aged between 20 to 65 years . DespiteThe HPV vaccines became available in 2006 . Since tThe Ministry of Health (MOH), through the school-based services, has introduced the HPV vaccination program for school girls at age 13. The school health service package provides school-based services from preschool until age 15 as shown in Table Intramuscular injection of 3 doses of quadrivalent HPV vaccine delivered on a standard dosing schedule .From 2010 to 2014, each recipient received 3 doses of HPV vaccine at the interval of 0, 1 and 6 months. However, in 2015 following the guideline from the World Health Organization (WHO), two doses of HPV vaccine were introduced under the new policy where by these doses of vaccine were given at 0- and 6-monthsinterval , 33. ThiHigh rates of school enrolment for 13-year olds (96.0%) and retention of female students in secondary schools have made it possible for the HPV vaccination to be integrated into the School Health Service Program and ensures equal access to the HPV vaccine between urban and rural area , 24.At the onset of the launch, 450 school’s health teams were readily available to deliver the services throughout the country for both public and private schools. To ensure the 3 doses of vaccination were able to be delivered within the set time frame, the nurses from the various MOH health clinics were mobilised to support the school health teams.The progress of implementation on the level of consent and coverage of immunization were reported to National Policy and Practices on Immunisation committee regularly. The year seven female students’ enrolment data were obtained from District Education departments at the end of the year and later verified with schools’ enrolment lists.The most crucial factor that determines supporting high vaccination coverage was program monitoring. The National, State and districts HPV Operation Rooms were set-up to closely monitor the implementation at all implementation levels. The operation room was spearheaded by the Director of the Family Health Development Division, with support from various technical officers responsible for technical documentation of the progress, adverse reactions, public enquiries, ICT requirements and daily operations. All public enquiries on the HPV immunization were responded to by a Public Health Physician and paramedics that were put on-call on a rotational basis to answer the hotlines from the public as well as any queries from implementers at school or health clinics.The National School based HPV Immunisation Program was implemented in 2958 public and private secondary schools registered under the Ministry of Education, Malaysia (MOE). A scheduled school visit by the school health team was carried out to ensure HPV vaccination spaced at month 0, 1 and 6 were completed within the same year before school end in November.Written parental consent was obtained prior to vaccination. The consent forms were distributed to parents through school teachers prior to scheduled vaccination visits. The parents returned the consent form to the school team. Whenever the children were refused immunization by their parents, the school health teams would call the parents to determine reasons for refusal and made are mark on the student registration form.Ethical issues surrounding HPV vaccination and parental rights to adequate information was not compromised in the process of obtaining parental written consent. Parents were provided with HPV vaccine information through print and electronic media to enable those making independent informed choices.Compliance to religious requirement was fundamental to HPV vaccine uptake amongst the Muslim population in Malaysia. MOH worked with Malaysian religious authority in having an Islamic ruling (fatwa) on HPV vaccination. The ruling stipulates “Immunising women with HPV vaccine that has been determined to have no element of doubt in its content and will not harm the recipient, is permissible for the prevention of cervical cancer” , 35.Introduction of HPV vaccine particularly involves supporting the acceptability of the vaccines to adolescent girls who may have little or no knowledge about cervical cancer and parents that may be suspicious of the vaccines, will give particularly if exposed to negative media attention and thus programs must proactively address communication requirements , 37. ThrElectronic, printed and social media were the main communication channels employed in creating public awareness towards accepting HPV vaccination –41. MajoOn the day of the vaccination, the school health nurses delivered a brief introduction on the HPV infection, cervical cancer, and HPV vaccine to the recipients. After the introduction, an assessment was carried out on health status to determine presence of illness or any contraindications for vaccination which require immunisation to be deferred to later dates. A guideline was developed by Family Health Development Division, MOH to carry out the mass vaccination . The HPVAfter the vaccination, recipients were observed for 20 min for any immediate adverse reactions. They were provided with an Adverse Event Following Immunisation (AEFI) monitoring form and vaccination card before returning to their classes. All recipients were advised to seek medical help if they experienced any suspected adverse reactions. The AEFI monitoring forms were collected from students at school either on the following visits or at any contact with the health providers in accordance to guidelines .Free vaccination was offered to eligible recipients (year seven) in Malaysia which was usually at the age of 13 in the index year. Only female year seven students are eligible to receive vaccination irrespective of their actual age. All recipients of the HPV vaccine were identified through school enrolment data obtained from education departments from each district in Malaysia. A total of 242,638recipients aged between 12 to 13 years studying in year seven were approached during the launch of the program in 2010. Approximately, 230,000 recipients were offered HPV vaccine annually by 646 school health teams throughout the country from 2010 to 2016.Parental consent, coverage by doses and vaccination completion were closely monitored as part of vaccination delivery indicators. Parental consent reflects the level of acceptance of the vaccine introduced. It was measured by the total number of parents consented for their child to be vaccinated over total number of year seven female student’s enrolment. A total of 232,645 from 242,638 (95.9%) parents consented to the immunisation in 2010. Subsequently, in 2011 a total of 229,021 from 234,668 (97.6%) and in 2012 a total 232,705 from 237, 017 (98.2%) were obtained from the parents. Throughout 2013, a total of 243,681 from 247,549 (98.4%) parents gave consent for immunisation. In 2014, 2015 and 2016, a total of 226,253 from 229,739 (98.5%), 220,789 from 224,761 (98.2%) and 215,090 from 218,590 (98.4%) parents consented to their child to be immunised respectively. Reasons for non-consent included unsure of vaccine safety, child has been immunised in private clinic and unable to retrieve consent forms from students. Figure Vaccination coverage provides information on vaccination uptake in the total population, including those girls not in school and therefore not offered vaccination. It was measured by total number of recipients who received a complete immunisation course over estimated total number of female students Fig. .Fig. 3PeCompleted vaccination refers to recipients completing 3 doses of HPV vaccination or 2 doses in 2015. It was observed that 93.35% students with parental consent completed their 3 doses of vaccination in 2010. It improved to 98.3% in 2011 and increased to 99.3, 99.4 and 99.6% in 2012, 2013 and 2014, 99.6% from 2015 and 99.1% from 2016 cohort who received 2 doses.First dose completion for 2010 was 99.5% for recipients with parental consent. The completion improved to 99.7, 99.8 and 99.9% from 2012, 2013 and 2015 respectively but declined to 83% in 2016. Main reason for non-immunised among the consented were fear, absenteeism, claimed to have been immunised before and due to vaccine being charged to the students attending private sector schools in 2016.The completion rate for the second dose was slightly lower than the first dose. The completion of second dose was 98.9% in 2010 and increased above 99% in 2011. The main reason stated by students for not continuing their vaccination course was the experience of side effects during first dose.Third dose completion rates showed a further drop of on average 0.5 to 1%. The highest drop for the third dose was in 2010 from 98.9 to 97.9% while the lowest drop was 0.3% in 2014. Third dose completion was not applicable in 2015 due to the two doses schedule. Reasons for not completing were similar to the reasons stated for the second dose.Active adverse event following immunisation (AEFI) monitoring was introduced to create students and parental awareness on vaccine and vaccination safety. Any adverse event would be reported for every dose of immunisation. Active reporting was encouraged by providing an AEFI form to all recipients. Reports of recipients experiencing AEFI were collected and monitored by the Malaysian Adverse Drug Reactions Advisory Committee (MADRAC).During the first year of implementation, the MOH managed to provide HPV vaccination services to almost 96% 231,869) of eligible recipients in all public, private and international secondary schools. The absence of mothers during vaccination in school did not reduce the HPV vaccination uptake among girls in Malaysia , 43. Thr31,869 ofThe World Health Organization recommends a school-based approach for effectively delivering HPV vaccine in countries that have a fairly high enrolment of girls in schools . The MalA sustainable national HPV vaccination programme is resource intensiveand requires a strong policyto ensure continuous financial support –47. AdvoPartnerships across stakeholders are necessary for the development of relevant and effective strategies in addressing issues for maximum efficiency and positive impact of national HPV vaccination implementation , 50. TheWhile anticipating potential media threats, MOH utilized multipronged strategies to communicate the messages across the public. MOH had successfully engaged various subgroups to talk and discuss regarding the introduction of HPV immunization through print, electronic and social media (face book and twitter) and telephone hotline. Other activities carried out were rumours surveillance, public forums, health talks, print and electronic media campaigns. The messages were tailored to local cultural context, religion and information needs of parents, target population and general public and to alleviate fear and misconception about vaccine being new, and reinforce vaccine safety , 53.School-based programmes require the stable commitment of and strong collaboration between health and education authorities. Two essential elements for such collaboration are policies and mechanisms. National policies and the corresponding managerial and operational mechanisms are the key elements for the school health programme to be successful. The approach used in Malaysian school-based immunisation program is recommended as it was able to deliver HPV vaccination with good coverage. The success of the implementation in Malaysia was due to careful program planning at national, states and district levels. Involvement of various stakeholders and gatekeepers facilitated the public acceptance of a new and potentially controversial vaccine. Highlighting the relationship of the vaccination and cervical cancer in the context of the Malaysian culture and religious belief further enhanced the uptake of vaccination in school. Periodic mass media campaigns created a positive supportive environment in changing beliefs and practices towards the new vaccine. Availability of health personnel to respond to public concerns further increased public confidence with the MOH new policy. Political will and a high-level commitment ensure the sustainability of the School-based HPV Vaccination Programme in Malaysia.


Human papillomavirus (HPV) epidemiological research has generally been individual based, typically focusing on women, with couple-based research mostly consisting of cross-sectional assessment of prevalent HPV infection in both partners.The HPV Infection and Transmission among Couples through Heterosexual activity (HITCH) study was set up to investigate the transmissibility of HPV among young, recently formed couples in Montreal, Canada. This paper provides an overview of the HITCH cohort study design and procedures as well as a narrative summary of the most important findings.HITCH is a longitudinal investigation of HPV transmission in recently formed heterosexual partnerships initiated within 6-month pre-enrollment, a time at which considerable transmission is believed to occur. A total of 549 newly formed dyads were recruited (2005-2011) from postsecondary institutions, including 502 young women and their male partners. An additional 46 males were enrolled at follow-up, as some women enrolled a subsequent partner at follow-up. Women aged 18-24 years were followed for 24 months for acquisition of HPV types not present at enrollment, whereas men returned for a single follow-up visit at month 4, for a sum total of 3361 clinic visits. The last follow-up visit occurred in January 2014. Extensive sociodemographic, sexual behavioral, and medical history data were collected every 2-4 months using computer-assisted, self-administered questionnaires. Furthermore, participants provided genital, blood, oral, and hand specimens for HPV assessment.Although in its early analysis stage, HITCH has produced important publications. Findings from HITCH have increased the available knowledge about the natural history of HPV transmission and its determinants, provided further evidence regarding oral-oral and oral-genital routes of HPV transmission, and supplied empirically valid epidemiological parameters of HPV transmission to assist mathematical modelers in health economic assessments. In addition, HITCH data were made available to several multistudy collaborations evaluating new HPV detection assays and evidence for-or-against HPV type replacement following the introduction of HPV vaccination.HITCH will continue to offer a unique resource for research on HPV transmission.RR1-10.2196/11284 Human papillomavirus (HPV) is the most common sexually transmitted infection and a necessary cause of all cervical cancer cases in the world ,2. Most Initiated in 2005, the overarching aim of HPV Infection and Transmission among Couples through Heterosexual activity (HITCH) cohort study was to further our understanding of the transmissibility of HPV among recently formed couples to better inform prevention strategies. When it was developed, no other study had specifically targeted newly forming couples, and some had excluded couples of <6-month duration ,11. ReseThe HITCH cohort study (referred to hereafter as HITCH) was conducted by a team of researchers at McGill University, led by two of the authors (ELF and ANB), in collaboration with the McGill Student Health Services Clinic, Concordia Health Services Clinic, and Centre Hospitalier de l’Université de Montréal, all located in the province of Québec, Canada. The study was conducted in accordance with the principles and articles stipulated by the Tri-Council Policy Statement Ethical Conduct For Research Involving Humans . It receThe inclusion criteria for women were as follows: those aged 18-24 years, enrolled at a university or junior college in Montreal, intending to remain in Montreal for the next 2 years, currently heterosexually active with a male partner with whom sexual activity was initiated within the previous 6 months, willing to comply with follow-up, having an intact uterus and no history of cervical lesions or cancer, and neither currently pregnant nor planning to become pregnant in the next 24 months. Men were eligible to participate if they were aged ≥18 years and willing to comply with follow-up.Recruitment was achieved through printed promotional materials and electronic advertisements on campuses and venues frequented by students. Interested couples were invited to visit the study website and contact study nurses.Couples were recruited for assessment of sexual histories and HPV testing . Women wMen and women self-completed separate Web-based questionnaires. Women attended a clinic visit at enrollment and 5 other follow-up visits, whereas men only attended a clinic visit at enrollment and at 4 months.Extensive efforts were made to prevent attrition, including prescheduled appointments, timely electronic reminders between administered questionnaires, in-person motivation by the research nurse at each visit, and monetary compensation (Can $50 per visit and Can $10 per questionnaire completed by a woman between visits). Reflecting in part the mobile nature of student populations, female attrition rates were 43.0% (216/502) by month 24; 48 women attended 1 visit (baseline) only, 38 attended 2, 38 attended three, 46 attended 4 and 44 women attended 5 visits. Among men who consented to return for a follow-up visit, 22.6% (124/548) were lost to follow-up . No statistically significant differences were noted between participants who dropped out and those who remained in terms of age, smoking status, lifetime number of sex partners, age at sexual debut, or HPV status.Information was collected using Web-based self-completed questionnaires at each visit, and is described herein in accordance with the Checklist for Reporting Results of Internet E-Surveys guidelines . A securWe collected biological specimens at each visit . ParticiGenital, oral, and hand specimens were tested using a polymerase chain reaction (PCR) protocol based on the amplification of a 450-bp segment in the L1 HPV gene using the Linear Array HPV genotyping assay (Roche Molecular Systems) . Using tHigh-resolution PCR typing of the DQB1 and DRB1 class II loci of the human leukocyte antigen (HLA) system was performed to identify specific alleles that may help explain variations in the HPV susceptibility. HLA testing was done in DNA samples extracted from oral specimens  of the enrollment visit or, in case these were unavailable or insufficient for analysis, using specimens from subsequent visits or genital samples. In addition, sequence-based typing with the AlleleSEQR DRB1 and AlleleSEQR DQB1 assays  was used to identify all possible alleles. Locus-specific exon 2 and 3 amplicons were produced by PCR amplification and then sequenced to determine allele-specific nucleotide sequence variants. To save costs, we tested only specimens from couples with an infection transmission opportunity .HITCH enrolled the first couple in May 2005 (McGill University site) and March 2006 (Concordia University site). It reached its enrollment target in January 2011 with a total of 1051 participants (502 women and 549 men) recruited. Of 502 women, 42 enrolled a second male partner and 4 enrolled a third male partner. In total, there were 3361 clinic visits and 4408 and 2495 person-months of follow-up among women and men, respectively. HITCH findings, to date, suggest that HPV is highly transmissible. Presented at local, national, and international scientific conferences and meetings, the spectrum of findings regarding HPV transmission have received broad acceptance in the scientific community and considerable press coverage in international media. Results are likely to influence prevention efforts for cervical cancer and other HPV-related diseases, including behavioral strategies to reduce risk.At enrollment, similar HPV prevalence 147/263, 55.8%) was detected among women and men, but couples were not necessarily concordant for the same HPV types [3, 55.8% Analysis of patterns of type-specific concordance and discordance at baseline revealed that the extent of concordance was far greater than expected and consistent with rapid transmission of HPV between partners . HPV wasUsing data from the first follow-up visit, HPV transmission rates were estimated among participants with documented sexual exposure to an infected partner in couples that were discordant on ≥1 HPV types . We obseCorrelates of genital HPV infection in partnerships were identified at baseline using the couple or “dyad” as the unit of analysis, rather than the individual . To the We estimated the prevalence of oral HPV and assessed risk factors among male partners at enrollment . The preIt has been hypothesized that following a reduction in HPV vaccine-targeted genotypes, an increase in the prevalence of other types might occur due to reduced competition during natural infection. Any apparent postvaccination increase must be distinguished from diagnostic artifacts consequent to consensus PCR assays failing to detect HPV types present in low copy numbers in coinfected specimens. The assumption is that with a decline in vaccine-preventable types, there might be increased detection of previously “masked” types. Using data from 6 epidemiological studies, including HITCH, a total of 1200 anogenital specimens (blinded to HPV status) were reanalyzed to evaluate unmasking of HPV52 that might have been caused by elimination of HPV16 . ResultsWe used data of 3200 females from 3 cohort studies, including HITCH [Pooling data from 5 prevaccination epidemiological studies, including HITCH, and applying a hierarchical Bayesian regression approach that uses shrinkage and adjustment for confounders  and other HPV types, we found HPV16 to be the most common type  . HPV typHPV DNA detection may not always represent true infections but maybe depositions from infected sexual partners. We examined whether sexual risk factors and a biomarker, Y chromosome DNA, were associated with genital HPV partner concordance using baseline genital specimens . HPV DNAWe observed that this biomarker may serve as a measure of recent condomless vaginal sex in HPV studies lacking data on sexual behavior, such as surveillance studies of HPV prevalence . Among fSexual mixing refers to whom individuals choose as sexual partners. It is described as assortative on a characteristic if individuals have the tendency to choose partners who are similar to them on that particular characteristic , and disassortative if individuals tend to choose partners who are different from them . Among 502 young adults in a new heterosexual dating partnership, we found a moderate to strong assortativity on partners’ demographic characteristics, as well as on their number of sexual partners, sexual partner acquisition rates, concurrency, and gap lengths between partnerships . These fThe utility of a generic HPV probe assay was compared with the commercial Linear Array  for the detection of HPV DNA in a multistudy collaboration . HITCH vThere are two unique features of HITCH that are novel for HPV research. The cohort is the first large-scale study of HPV acquisition involving sexual partners. Previous studies had typically sample sizes of ≤100 couples and some had as few as 25 couples ,38,39. MHITCH is one of the few studies that could provide empirical estimates of HPV transmission parameters to be used in models for assessing the population health impact and cost-effectiveness of vaccination strategies. There has been an increased awareness about HPV and its role in cervical cancer since the introduction of the first prophylactic HPV vaccine in 2006, 1 year after the initiation of HITCH. Gardasil  is a quadrivalent vaccine to prevent genital warts caused by genotypes HPV6 and HPV11, as well as anal, cervical, vulvar, and vaginal cancers caused by HPVs 16/18. Approved in 2009, Cervarix  is a bivalent vaccine against cervical precancerous lesions and cancer caused by HPVs 16/18, which represent 70% of all cervical cancer cases. In 2014, Gardasil 9 (Merck) vaccine was introduced to help prevent against diseases associated with HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 .In the postvaccine era, transmission studies such as HITCH are especially important to inform all possible prevention strategies. First, there are still some cancer caused by other high-risk HPV types, and the duration of immunity is unknown. Second, the benefits of HPV vaccination in young female adolescents will take years before being realized. Third, many teenage girls and women will still be susceptible to infection because vaccination outside of the government-funded program is only available privately at a high cost. Even for girls who may now receive the vaccine free-of-charge, implementation has not occurred without debate -43. If hA number of limitations of this study have to be underscored. These include loss to follow-up, volunteer nature of self-selected subjects limiting the generalizability, the unavailability of hand and oral specimens for all participants at all time points, and absence of anal specimens. In addition, the potential for self-report response bias needs to be acknowledged. However, the use of self-completed electronic questionnaires to collect repeated measurements of behavioral data was likely to reduce the potential for social desirability bias. If we were to re-establish HITCH, we would have opted to (1) actively follow men at all visits; (2) collect hand, oral, and anal specimens for both men and women throughout follow-up; (3) collect data on lifetime and recent number of oral sex partners; (4) not perform serology at each visit, as this might have influenced compliance rates; and (5) instruct participants to abstain from sexual activity for a minimum of 48 hours before specimen collection to prevent potential false-positive findings.Ongoing areas of active analysis and manuscript preparation, as well as planned investigations, from our extensive program of research with HITCH data include (1) influence of HLA types on HPV type-concordance and transmission; (2) hand-genital patterns of HPV type-specific auto-concordance and couple-concordance; (3) assessment of the association between circumcision and condom use and distribution, concordance, and transmission of HPV genotypes in genital specimens; (4) impact of HPV vaccination on the prevalence, concordance, and transmission of type-specific HPV infections; (5) concordance and transmission of specific variants within HPV genotypes; (6) transmissibility of HPV focusing on the viral load and type-specific concordance of HPV infection between young couples; (7) evaluation of HPV acquisition in women for whom HPV positivity was measured both before and after the first sexual activity with a new male sex partner; (8) development and validation of a rapid, easy-to-perform and inexpensive serologic noncompetitive, multiplexed Luminex- based platform to measure total immunoglobulin G antibody; and (9) association between type-specific serological antibodies and HPV prevalence, acquisition, and clearance.Information about HITCH findings and publications is available at the McGill University Division of Cancer Epidemiology website . A regul


The HPV DNA is more frequently found unintegrated in the cancers of the oropharynx compared to those that arise in the cervix, and may include novel HPV-human DNA hybrids episomes. Here, we review current understanding of HPV biology in the oropharynx and discuss the cell-based systems being used to model the HPV life cycle in tonsil keratinocytes and how they can be used to inform on HPV-driven neoplastic progression in the oropharynx.Most human papillomavirus (HPV) positive head and neck cancers arise in the tonsil crypts; deep invaginations at the tonsil surface that are lined with reticulated epithelium infiltrated by immune cells. As in cervical HPV infections, HPV16 is the most prevalent high-risk type in the oropharyngeal cancers, and a genital-oral route of infection is most likely. However, the natural history of HPV-driven oropharyngeal pathogenesis is an enigma, although there is evidence that it is different to that of cervical disease. It is not known if the virus establishes a productive or abortive infection in keratinocytes of the tonsil crypt, or if viral infections progress to cancer This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’. IntriThere is much interest in patients with HPV-OPSCC because they have overall increased survival compared to those with HPV-negative disease, and thus it has been proposed that this group of younger patients could benefit from deintensification of therapy to reduce the longer term toxicities in anticipation of long-term survival ,16. HoweThe high-risk (HR) HPV types found in cervical cancers are also present in OPSCC and HPV16 is the most prevalent HR type in both types of tumour . HoweverChromosomal integration of HPV genomes is a common feature of cervical cancers and at this body site is linked to a poorer clinical outcome. Integration of the viral DNA most often disrupts the E1 and E2 genes and deregulates virus gene transcription, leading to maintained and often high expression of the HPV oncoproteins E6 and E7 in the tumours. However, in HPV-OPSCC the primary form of HPV DNA is non-integrated episomes and viral DNA integrants are less frequent than some earlier studies indicated; most likely owing to a combination of the use of different methodologies to detect episomes and/or integrants, and ambiguities in data interpretation –29. As iPIK3CA, EP300, PTEN, FBXW7 and TGFBR2. However, several other genes found to be commonly mutated in HPV-OPSCC, including ZNF750, FGFR3, CASZ1, CYLD and RIPK4 have not been identified in cervical cancer [Recent analysis of the mutational landscape of HPV-positive (predominantly OPSCC) in comparison to HPV-negative  squamous cell carcinomas provides evidence that the mutation burden in HPV-positive tumours is indicative of apoliprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminase editing . Interesl cancer , indicat(a)The mucosa of the tongue base and tonsils  is invaginated and forms crypts lined with reticulated epithelium . It is wAnalysis of non-malignant tonsil tissue for indications of HPV infection suggest that HPV infection at this site is rare, even when the reticulated epithelia was sampled . This raMucosal squamous epithelia at different anatomical sites that are susceptible to HPV infection are at different risks of cancer development. The uterine cervix has a nearly 100% association with HR HPV whereas at other sites including anus, vagina/vulva and oropharynx, only a subset of the tumours are HPV-associated. This differential risk between mucosa sites could be explained by the nature of the target cell for HPV infection, which influences disease outcome. In the cervix, a small and discrete population of non-stratified cuboidal cells of embryonic origin in the metaplastic transformation zone at the squamocolumnar junction (SCJ) are highly susceptible to HPV infection and from which the majority of neoplasms arise . The SCJCervical cancer pathogenesis is characterized by a well-defined premalignant phase of low-grade to high-grade intraepithelial neoplasias (CIN1 to 3) that develop over many years, but dysplastic lesions have rarely been reported in the oropharynx. This, combined with lack of detection of HPV productive infections at this anatomical site, the dominance of HPV16-positivity and the difference in integration rates between the two sites suggests that the natural history of HPV-driven disease in the oropharynx is very different to the cervix, but as yet unexplained .2.One approach that has proved very successful in understanding the interaction between HR HPV types and cervical keratinocytes has been by the analysis of cells isolated from low-grade cervical neoplasias. The most highly researched are HPV16-positive W12 cells  and the (a)Transgenic mouse models engineered to express the HPV16 oncoproteins E6 and E7 in squamous epithelia and treated with chemical carcinogens have been very informative of the role of the viral oncoproteins to the development of skin, cervical, and head and neck (tongue and oesophagus) carcinogenesis –60. Thes(b)ex-vivo model maintains tissue cytoarchitecture, including the major lymphocyte subtypes and follicular dendritic network, and would enable HPV infection to be interrogated in the presence of a physiological immunological landscape.One approach that as yet has not been exploited for investigating HPV-tonsil interactions is the use of tonsil explants. Fresh human tonsils dissected into small blocks can be cultured on collagen rafts at the liquid–air interface . Tonsil (c)a).Another approach and one that has been used extensively in the papillomavirus field is the generation of ‘HPV infected’ epithelium equivalents from primary keratinocytes grown in three-dimensional (3D) organotypic raft culture. Keratinocytes harbouring the viral genomes are seeded onto a dermal equivalent formed from rat tail type 1 collagen embedded with fibroblasts (mouse 3T3-J2 fibroblasts are routinely used in the HPV replication model). After a short period to allow expansion of the keratinocytes into a confluent monolayer on the collagen plugs, they are lifted onto a solid support and cultured at the air-liquid interface for a period of about two weeks—the time it takes for a basal keratinocyte to fully differentiate. This involves feeding the rafts from underneath with serum-containing medium that is free of epidermal growth factor, providing nutrients and allowing cytokines and growth factors produced from the fibroblasts to be delivered to the keratinocytes. The ‘raft’ system enables analysis of the spatial and temporal organization of the virus life cycle—from early events to viral progeny assembly a.FigureEstablishment of HPV genome-containing keratinocytes has largely relied upon transfection of HR HPV genomes along with a drug selectable marker into primary keratinocytes, followed by a brief period of drug selection to remove non-transfected cells . The lifSeveral groups including our own, are using primary tonsil keratinocytes to investigate HPV biology at this site . Because(i)One important question is why the tonsil crypt is more susceptible to HPV transformation than the surface squamous epithelium. A very interesting study isolated tonsil progenitor cells; cells expressing high levels of cell surface antigens CD44 and nerve growth factor receptor (NGFR) from the crypt and the surface epithelium . In surf(ii)HPV16 does replicate in ‘unsorted’ primary tonsil keratinocytes following transfection of complete viral genomes or infection with HPV16 pseudovirions ,70,71. U(iii)The rarity of other HR HPV types in oropharyngeal tissues might be because these types are unable to infect these tissues or they differ in replication potential. However, HPV18 is very effective in extending the lifespan of tonsil keratinocytes and the cells support HPV18 productive events upon 3D culture ( figures).Figure(iv)b,c). However, tonsil surface and crypt epithelia undergo alternative differentiation programmes and in particular, there are marked differences in their keratin profiles [When grown in organotypic raft culture, keratinocytes from squamous epithelia of different body sites exhibit an intrinsic potential for tissue-specific differentiation. Thus, keratinocytes isolated from neonate foreskin tissue form a cornified structure with a strong keratinized corneum, whereas tonsil keratinocytes recapitulate the structure of non-keratinizing epithelium b,c. Howeprofiles . Cytokerprofiles . Anotherprofiles .a). However, intense CK8 expression occurred in superficial cells of isogenic HPV16 and HPV18 tonsil epithelium equivalents, with low-level staining in basal keratinocytes (a). This was specific to the site of origin of the keratinocytes because very low CK8 staining occurred in rafts of foreskin keratinocytes harbouring the HR HPV episomes (a). In the tonsil keratinocyte rafts, CK7 was restricted to the occasional superficial cell, but in HPV16 and HPV18 genome-containing isogenic lines, the levels of this cytokeratin were markedly increased in cells of the upper intermediate and superficial layers corresponding to the productive phase of the HPV life cycle (b). A similar effect on CK7 expression did not occur in HPV18-positive foreskin keratinocyte equivalents (b). These findings provide evidence that HR HPV alters the expression pattern of tonsil crypt-specific differentiation markers, a phenomenon that appears to be unique to this anatomical site. HPV is known to delay differentiation in the cervix to support virus replication, but in the tonsil perhaps the virus is able to reprogramme the keratinocyte to express the differentiation characteristics of the tonsil crypt. Since CK7 has been implicated in a translational mechanism of HPV mRNAs [So, do the tonsil epithelium equivalents show CK8 and CK7 expression patterns of the surface or the crypt, and does HPV replication affect the expression profiles of these cytokeratins? Our HR HPV tonsil keratinocytes models are beginning to answer these questions. Immunostained organotypic rafts of primary human tonsil keratinocytes using anti-CK8 antibodies showed patchy CK8 staining; it was largely absent from areas of full stratification, but in areas that were not full thickness, some basal/parabasal cells were positive for this cytokeratin a. Howeveinocytes a. This wepisomes a. In thefe cycle b. A simiivalents b. These PV mRNAs , this mi(v)E2 gene [Patients with HPV-positive oropharyngeal cancer often present with more advanced disease compared to those with HPV-negative OPSCC, but they have a better prognosis and overall survival, although recurrence is still a concern ,75. So wE2 gene ,30. IdenHPV DNA integration is far less frequent in HPV OPSCC than in the cervix; but in addition to integrated and unintegrated forms of the viral DNA, a third form of a viral DNA-human hybrid has been proposed . WhetherIntriguingly, in our models of HPV replication in primary tonsil keratinocytes, in the three donors investigated to date, HPV18 is maintained as an episome for between 40 and 70 population doublings after which there is a marked decrease in episomal forms of the viral genome as determined by Southern blotting. Whereas in the same three donors, HPV16 DNA either integrated very early upon establishment of the cell lines (in one donor), and in the two other donors remained episomal in extended cell culture (greater than 90 population doublings to date). While the donor sample size is too small to conclude that HPV16 is less likely to integrate than HPV18 in primary tonsil keratinocytes, these models are useful to investigate differences in HPV16 and HPV18 biology and virus–host interactions in isogenic tonsil keratinocyte backgrounds grown in short and long-term cell culture under non-stress conditions .3.It is a puzzle that despite the recognition of an association with HR HPV and OPSCC, infection at these sites in non-malignant tissue has not been conclusively found. Primary keratinocytes isolated from tonsil tissue are not refractory to HPV16 infection and are able to support the production of infectious progeny, but are the primary tonsil keratinocytes truly representative of the target cell for the virus? If stem cells in the crypt are the natural target and oral HPV infections may represent a source of virus to infect the tonsil, then these cells might be less permissive for HPV replication, leading to deregulated expression of E6 and E7. Since in the anogenital tract, the phenotype of the HR-HPV target cell is a determinant of carcinogenic risk, the molecular definition of the target cell of these viruses in the tonsil is needed .The reason for the prevalence of HPV16 in HPV-OPSCC is also unclear; but primary tonsil keratinocytes support the infectious cycle of HPV18, that is rarely found in tonsil cancers, so simply that this HPV type is less able to infect or replicate in the tonsil cells seems an unlikely explanation of why we see less HPV18-positive tonsil cancers. Perhaps the nature of the interaction between these viruses and the immune system in this lymphoepithelial tissue is different between the various HPV types, and this contributes to the different outcomes of infection. Further studies to understand the effects of HPV infection and replication in tonsil keratinocytes upon innate signalling and other arms of the immune system are needed. With regards to this topic, RNA-Seq of gingivial keratinocytes immortalised with the telomerase enzyme hTERT and HPV16 genomes showed downregulation of the interferon-regulated innate signalling complex, interferon-stimulated gene factor 3 (ISGF3) and a number of the gene targets of this complex . AlthougInvestigation of the biology of the HPV life cycle in primary tonsils is one level of understanding of the virus–host interaction. However, since the tonsil is a lymphoidal tissue with significant infiltration of lymphoidal cells into the tonsil crypt, future studies might advance these models by co-culturing keratinocytes with immune components in two-dimensional and 3D culture systems.in vivo [While cell lines have been established from cervical neoplasias and have been of huge importance in understanding the natural history of cervical HPV pathogenesis, equivalents from the oropharynx have not been established. This might be because at this anatomical site the natural history of HPV infection is very different to the cervix as either a lengthy premalignant stage does not occur , or the in vivo . Our isoPerforming global gene expression analysis on organotypic raft cultures of primary cervical keratinocytes harbouring HPV16 episomes revealed a number of host genes modulated by the virus  that had not been observed in HPV16-positive foreskin based models . The bioNeither should studies be limited only to HPV16, but include models harbouring HR HPV types found less frequently in HPV-OPSCC which might aid identification of the molecular reasons for the different pathogenicity in tonsil tissue.It is commonly assumed that papillomaviruses evolve slowly, but next-generation sequencing of HPV DNA isolated from lesions is challenging this assumption and in fact inter-host HPV genotype variability might be high, although conservation of E7 has been demonstrated in the cervix ,80,81. TIn summary, it is becoming recognized that virus-driven oropharyngeal disease is different to cervical pathogenesis: thus the refinement and enhancement of tonsil keratinocyte-based models will contribute to a greater understanding of HPV disease at this anatomical site.


In clinical oncology, only a few applications have been developed using HPV as a personalized tumor marker, a lack most probably related to the limited information obtained by the classical Polymerase Chain Reaction (PCR) approach. To overcome this limitation, we have recently developed the capture-based Next-Generation Sequencing (NGS) “CaptHPV” assay, designed to provide an extensive and comprehensive molecular characterization of HPV DNA sequences associated with neoplasias, ie the sequence of the viral genome (245 genotypes), its physical state, viral load, integration site and genomic alterations at integration locus. These data correspond to highly specific tumor markers that can be used to improve diagnosis and patient’s follow-up.We report here a case that is a straightforward and practical illustration of the power of the CaptHPV method. A patient developed successively a carcinoma of the anal canal and of the tongue. The two tumors were squamous cell carcinoma, found associated with HPV16 using PCR. In order to document a possible metastasis to the tongue from the anal cancer, we performed CaptHPV analysis on the two tumors. The analysis of the anal carcinoma found 55 viral/human hybrid reads allowing the identification of the HPV16 DNA integration in the 4q25 chromosomal band locus with a 178,808 bp deletion in the cell genome. Molecular analysis of the tongue tumor disclosed 6110 reads of HPV16, with a viral pattern strictly identical to that of the anal tumor. A total of 131 hybrid reads between HPV16 and the cell genome were found, corresponding exactly to the same locus of integration of viral DNA at the 4q25 site. The 178,808 bp genomic deletion was also found in the lingual tumor. The exact identity of HPV insertional signatures in the two tumors, demonstrates unambiguously that the tongue tumor derived from the anal cancer whereas neither histological immunophenotyping nor classical viral analysis using PCR could allow a definitive diagnosis.Our observation indicates that the establishment of a detailed cartography of HPV DNA sequences in a tumor specimen provides crucial information for the design of specific biomarkers that can be used for diagnostic, prognostic or predictive purposes. Specific genotypes of human papillomaviruses (HPV) are associated with a large number of carcinomas developed in the mucosa of the ano-genital and of the head and neck tracts. The prevalence of HPV is different according to tumor localization and has been described in 96% in cervical , 88% in This lack in developments of clinical applications based on HPV as a tumor marker in the follow-up of patients with HPV-associated invasive carcinoma is most probably related in part to the limited information obtained by the Polymerase Chain Reaction (PCR) approach, frequently used to determine the HPV genotype. This method is very sensitive but provides a qualitative result, meaning the presence or absence of HPV DNA, based on the detection of a short DNA sequence related to a specific or to a limited number of HPV genotypes. Information about the exact sequence of the HPV genome, its physical state - episomal or integrated-, the integration site and the presence of genomic change at the insertion site, such as amplification or deletion, are missing. Thus all features that constitute personalized biomarkers specific for each tumor are lacking. To overcome this limitation, we have recently developed the capture-based Next-Generation Sequencing (NGS) “CaptHPV” assay. The CaptHPV assay has been designed to provide an extensive and comprehensive molecular characterization of HPV DNA sequences associated with neoplasias. CaptHPV allows, in a single experiment, the identification of the physical state, viral load, insertion site and presence of genomic alterations in the specimen for 245 different HPV genotypes. This approach, validated on a retrospective series of 72 cases of invasive carcinoma of the uterine cervix , revealeWe report here a patient case who developed two HPV-associated tumors, first in the anal canal then in the tongue. Classical histological and virological analyses could not permit to conclude whether this corresponded to the development of independent tumors, or to the secondary spread to the tongue of the primary anal tumor. Using CaptHPV, we were able to show that the two tumors harbored identical HPV insertional signatures, demonstrating that the tongue tumor corresponded to the metastatic spread of the anal carcinoma. We think that, in the near future, this innovative approach will be of large use to determine a detailed cartography of the viral sequences that characterize any HPV-associated carcinoma, providing specific tumor markers useful for patients’ follow-up.In January, 2018, a 58 years old male patient was hospitalized for a tumor of the tongue  gene and CCDC34P1, RPS12P8, RPL36AP19 and TUBB8P3 pseudogenes . Whereas HPV53 DNA sequences corresponded to the full length viral genome, most probably related to viral episomes, HPV16 DNA showed a breakpoint on the E2/E4 ORFs with a deletion of 6 nucleotides. The LCR, E6 E7 L1 and L2 ORFs were conserved. The identified integration mechanism of HPV16 in the tumor cell genome was that of two junctions in a co-linear pattern (2J-Col). Analysis found 55 viral/human hybrid reads allowing the identification of the HPV16 DNA integration in the 4q25 chromosomal band locus with a 178,808 bp deletion in the cell genome corresponding to the deletion of On the whole, the perfect identity of the molecular features of the HPV16 DNA sequences and integration pattern in the two tumors demonstrates that the tongue tumor is a metastasis of the anal carcinoma.Using the innovative CaptHPV assay, we report here detailed cartographies of HPV DNA patterns, encompassing full viral sequence, integration signature, integration locus and genomic changes at the insertion site, established from anal and lingual SCCs sequentially developed in the same patient. We show that the identity of these cartographies in the two tumors unambiguously demonstrates that the lingual tumor derived from the anal carcinoma, whereas neither histological immunophenotyping nor classical viral analysis using PCR could allow a definitive diagnosis.The identity of viral insertion in primary and metastatic tumours has been reported in some cases of Merkel cell carcinoma associated with the Merkel cell polyomavirus (MCPyV) , but theAs illustrated in the present observation, the cartography of HPV insertion may be crucial to make the differential diagnosis between metastatic spread and occurrence of an independent tumor, with important consequences in the choice of therapeutic approaches. Diagnostic applications of the NGS-based characterization of HPV-associated tumors should multiply, in particular through the use of the method for the identification of circulating tumor DNA. Such an approach will highly facilitate the diagnosis of relapse when the suspected site of relapse is difficult to access for classical cytological or histological procedure. In such situations, the identification in the blood of the insertion signature, previously determined in the tumor, will constitute a highly specific surrogate for triggering treatment. In a pilot study, we have shown that the mutational insertion could be detected in the blood of cervical cancer patients with equivalent sensitivity as that observed for the detection of only viral sequences . More reIn certain situations, even the NGS-based approach can be of limited impact to make the differential diagnosis between metastasis and second primary tumors. It is particularly the case when only episomal HPV copies are detected, a situation encountered in 25–30% of cervical cancers . In thesFAM241A is a potential target gene at this site, deleted through integration in our case of anal carcinoma. No data are available on the potential implication of this gene in oncogenesis. The HPV integration loci were found largely dispersed in the genome but hotspots have been described, among which target genes such as MYC, KLF5/KLF12, TP63, VMP1, NFIX [The HPV insertion at the 4q25 locus detected in our observation had not been reported so far. P1, NFIX  have beeP1, NFIX , a minorOn the whole, our observation indicates that the establishment of a detailed cartography of HPV DNA sequences present in a tumor specimen provides crucial information for the design of specific biomarkers that can be used for diagnostic, prognostic or predictive purposes during the patient’s follow-up.


HPV infection renders oropharyngeal squamous cell carcinomas more radiosensitive, which results in a favorable prognosis for HPV-positive patients treated with radiation alone or with concurrent platinum-based chemotherapy. The degree of radiosensitivity in fractionated regimens has not yet been fully explored; therefore, in this study, the radiosensitivity of HPV-negative tumors (FaDu) was compared to that of HPV-positive tumors (2A3) subjected to concurrent cisplatin chemotherapy and fractionated versus isoeffective single-dose tumor irradiation in immunodeficient mice. HPV-positive tumors were approximately 5 times more radiosensitive than HPV-negative tumors, irrespective of the irradiation regimen. In both tumor models, concurrent cisplatin chemotherapy and the fractionated regimen induced significant tumor radiosensitization, with a 3- to 4-fold increase in the tumor growth delay compared to that of single-dose irradiation. Furthermore, the degree of radiosensitization induced by cisplatin chemotherapy concurrent with the fractionated irradiation regimen was much higher in HPV-positive tumors, where a synergistic antitumor effect was observed. Specifically, after combined therapy, a 26% higher survival rate was observed in mice with HPV-positive tumors than in mice with HPV-negative tumors. These data suggest that HPV-positive tumors are more radiosensitive to fractionated regimen than to single-dose irradiation with concurrent cisplatin chemotherapy acting synergistically to irradiation. In the clinic, it was observed that HPV-positive oropharyngeal SCC responds better to treatment with radiotherapy and concurrent platinum-based chemotherapy3.HPV-positive squamous cell carcinoma (SCC) of the oropharynx is an increasingly common disease and is in many ways distinct from its HPV-negative counterpart, which is caused by smoking and excessive alcohol consumption10. Furthermore, the underlying mechanisms were explored. These studies suggested that the reason for the better response of HPV-positive SCC to the treatment may be impaired DNA damage repair in HPV-infected tumor cells7. This impairment is probably caused by HPV-viral proteins, E6 and E7, which interfere with the host cell cycle to promote the reproduction of the virus13. Furthermore, the immune response to viral proteins may contribute to better treatment outcomes in HPV-positive tumors, as the presence of HPV antigens may render the tumors more immunogenic14.This observation was confirmed in a number of preclinical and clinical studies17.Due to the improved response of HPV-positive oropharyngeal SCC to nonsurgical treatments, several studies evaluating different protocols of treatment de-escalation were initiated in patients with these tumors. The results of these studies showed that an approximately 20–30% deintensification of radiotherapy in combination with concurrent chemo- or immunotherapy in HPV-positive tumors is feasible and safe and leads to fewer side effects, with preservation of tumor control and survival rates6. We proposed impaired DNA repair mechanisms and G2/M cell accumulation as possible underlying mechanisms6. However, the degree of radiosensitization in fractionated regimens with concurrent cisplatin treatment, a standard treatment for these tumors in the clinic, which may also add to radio-immunosensitization, has not yet been fully explored. Therefore, the aim of the present study was to compare HPV-positive tumors to HPV-negative tumors in response to concurrent cisplatin chemotherapy and fractionated irradiation in comparison to isoeffective single-dose tumor irradiation.In our previous study of experimental tumor xenografts, we demonstrated a 20% increase in radiosensitivity in HPV-positive 2A3 tumors compared to that in HPV-negative FaDu tumors by single-dose tumor irradiation2 humidified atmosphere. The pharyngeal SCC cell line FaDu  was cultured in RPMI 1460 medium . The HPV E6 and E7 viral protein-expressing cell line 2A3  was cultured in Dulbecco’s Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 1 mg mL−1 G418 disulfate salt solution . Both media were supplemented with 5% fetal bovine serum (Thermo Fisher Scientific), 10 mM L-glutamine (Thermo Fisher Scientific), 100 U/mL penicillin  and 50 mg/mL gentamicin . The methods used to determine the presence and expression of the HPV16 E7 gene in the 2A3 cell line and the corresponding results are described in Prevc et al. 2018, Supplementary Materials6.All cell lines were cultured at 37 °C in a 5% CO5 and incubated for 24 h. For the combined treatment, 0.2 µg/mL cisplatin was added to the cell medium 20 min prior to irradiation  with 0.55 mm copper and 1.8 mm aluminum filtering. Twenty-four hours after the treatment  and 0.5% Tween-20  for 20 min, centrifuged for 5 min at 470 g  and fixed in 3 ml of 70% alcohol at 4 °C. Twenty-four hours later, the cells were centrifuged , washed once with PBS, again centrifuged for 5 min at 470 g, resuspended in 50 µL of RNase A  and stained with the DNA binding dye propidium iodide . Then, cell cycle analysis of the samples was performed using a FACSCanto II flow cytometer (BD Biosciences). DNA histograms of the number of cells against the observed fluorescence intensity were obtained. The resulting cell cycles were analyzed with ModFit LT software (Verity Software House). For each sample, the distribution of cells in the G0/G1, G2/M and S phases was determined.The cells were plated on Petri dishes at a density of 4 × 10ion Fig. . The celent Fig. , the celad libitum. For induction of subcutaneous tumors, a 100 μL suspension of 2 × 106 FaDu or 5 × 106 2A3 cells, prepared from cell cultures in vitro, was injected subcutaneously into the shaved backs of the mice. At a 40 mm3 tumor volume, the mice were randomly divided into groups of at least 8.Female SCID mice  were maintained on a 12 h light–dark schedule under specific pathogen-free conditions at constant room temperature and humidity. Food and water were provided Treatment protocols were approved by the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (permission No. 34401-1/2015/16) based on the approval of the National Ethics Committee for Experiments on Laboratory Animals. Animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986, and in accordance with the guidelines for animal experiments of the EU Directive 2010/63/EU.−1) using a Gulmay 225 X-ray system  with 0.55 mm copper and 1.8 mm aluminum filtering. Mice were irradiated either by a single dose of 10 Gy or by 8 fractions of 2 Gy (one fraction per day in 8 consecutive days)  was injected intravenously, with the volume depending on the weight of the mice. Twenty minutes after the cisplatin injection, we performed either single-dose irradiation or the first fraction of the fractionated regimen Fig. . For locys) Fig. . The numTumor growth was monitored three times a week by measuring the tumor diameters using a Vernier caliper until the tumor volume reached 300 mm3 . Tumor volume was calculated using the Eq. ,2\documeet al.21. Survival estimates were obtained by the method of Kaplan-Meier and compared by the log-rank test23.Statistical analysis and graphical representation were performed using SigmaPlot Software . Data were tested for normality of distribution using Shapiro-Wilk test. The arithmetic mean (AM) and standard error of the mean (SE) were calculated. Statistically significant differences between experimental groups were determined using one-way analysis of variance (one-way ANOVA) followed by a Holm–Sidak test. The difference between experimental groups was considered significant if the p-value was <0.05. In addition, the mode of action  of treatments with independent mechanisms was evaluated by the method developed by Spector In this study, we used FaDu tumors as an HPV-negative tumor model and 2A3 tumors as an HPV-positive tumor model. Tumors were irradiated with two isoeffective regimens, i.e., 10 Gy single-dose irradiation or a fractionated regimen (8 × 2 Gy). Indeed, the tumor growth delays after 10 Gy single-dose irradiation and fractionated irradiation were similar in both tumor models. The tumor growth delay in FaDu tumors was 2.9 ± 1.0 days after single-dose irradiation and 3.4 ± 0.9 days after fractionated irradiation, p = 0.31; in 2A3 tumors, it was 15.6 ± 2.3 days after single-dose irradiation and 18.1 ± 6.6 days after fractionated irradiation, p < 0.05. However, 2A3 tumors were 5.3 times more radiosensitive to 10 Gy single-dose irradiation (p < 0.001) and 5.4 times more sensitive to fractionated irradiation (p < 0.05). In addition, 2A3 tumors were 2.6 times more sensitive to cisplatin treatment alone (p < 0.05) than FaDu tumors  in FaDu tumors, while in 2A3 tumors, it was 25.3 ± 2.6 days   Fig. . In factFurthermore, concurrent cisplatin treatment combined with the fractionated regimen also resulted in superior survival rates in both tumor models, while there were no cures after the combination of single-dose irradiation and cisplatin chemotherapy. A 26% difference in survival rates was observed between HPV-positive 2A3 tumors and HPV-negative FaDu tumors in favor of the former  . Skin reactions were observed only in the group of mice treated with cisplatin combined with single-dose irradiation. Thirty percent of animals presented skin reactions, which manifested as edema and mild erythema that completely resolved 25 days after the treatment.in vitro. In both schedules, cisplatin was administered only once, i.e., 20 min before the first 2 Gy irradiation. The cell cycle analysis in HPV-negative and HPV-positive cells demonstrated primarily S phase arrest 24 h after cisplatin treatment alone; compared to the irradiation and combined treatment a significantly higher proportion of cells was obtained in S phase (p < 0.05). The combined cisplatin treatment with single-dose as well fractionated irradiation resulted in G2/M phase arrest; compared to the control groups , a significantly higher proportion of cells was obtained in G2/M phase. This G2/M arrest was significantly more evident in HPV-positive 2A3 cells than in HPV-negative FaDu cells (p < 0.05) and was 72 h later even more pronounced. In addition, combined treatment with the fractionated irradiation regimen and cisplatin resulted in a higher proportion of 2A3 cells in G2/M phase than treatment with cisplatin and single-dose irradiation, which may contribute to increased sensitivity in vivo  or 24 h after the third fraction of irradiation in the fractionated regimen combined with cisplatin treatment vs. 30%, p < 0.05). These data indicate that HPV-positive tumors are more sensitive to fractionated irradiation than to single-dose irradiation with concurrent cisplatin treatment. A higher sensitivity of HPV-positive tumors to cisplatin treatment alone and cell cycle redistribution during fractionated irradiation even after the first fraction, i.e., higher accumulation of HPV-positive cells than HPV-negative cells in G2/M phase, which is most sensitive to irradiation, could be one of the underlying mechanisms for the increased efficacy of this combined treatment.Our previous study demonstrated that HPV-positive pharyngeal tumors were 30% more sensitive to single-dose irradiation and 20% more sensitive to concurrent cisplatin therapy than HPV-negative tumors. The current study confirms the previous findings and extends them to a clinically relevant fractionated regimen. These results suggest that HPV-positive tumors respond better to fractionated radiotherapy with or without concurrent cisplatin chemotherapy than HPV-negative tumors. A 3- to 4-fold increase in tumor growth delay was observed after fractionated irradiation compared to single-dose irradiation, together with superior survival when combined with concurrent cisplatin therapy. A difference of 26% was found between survival rates recorded in HPV-positive and HPV-negative tumors  protein, which both mediate proteasomal degradation of targeted proteins and consequently abrogate apoptosis and senescence in host cells or arrest cells in G2/M phase, which is the most vulnerable to irradiation33. Furthermore, irradiation of HPV-positive cells induces fast progression of cells through S phase with subsequent arrest in G2/M phase8. The cell accumulation in this phase of the cycle correlates with increased levels of apoptosis and DNA double-strand breaks and decreased levels of mitosis in vitro8. Consequently, this may lead, together with other mechanisms, to higher radiosensitivity (up to 30%) of HPV-positive tumors in vivo, as observed in our previous study on single-dose irradiation6. In the current study, we determined an increased proportion of HPV-positive cells in G2/M phase after a single-dose or fractionated irradiation regimen in vitro. Additionally, a better response of HPV-positive tumors in vivo to both single-dose (up to 5.4-times prolonged tumor growth delay) and fractionated irradiation regimens (up to 5.3-times prolonged tumor growth delay) was recorded compared to that of their HPV-negative counterparts, thus confirming our previous results and expending them to the fractionated regimen. These results are in line with recent findings in clinical studies where treatment of a reduced intensity in HPV-positive oropharyngeal tumors was found to be equally effective as standard treatment regimens17. The results of these studies showed that a 20–30% decrease in the total dose of fractionated irradiation (from standard 70 Gy to 56 Gy or 50 Gy) for HPV-positive tumors is safe and feasible, with local control and survival remaining as high as those observed after the standard irradiation dose17.Overall, patients with HPV-positive oropharyngeal SCC tumors respond better to radiotherapy than those with HPV-negative tumors and have a better prognosis27 but also to cisplatin compared to HPV-negative cells34, and the same was observed in the combined treatment setting. Thus, the degree of radiopotentiation can vary; therefore, one of limitations of our study is the use of only one tumor model. Further studies employing other tumor models are warranted to confirm the radiopotentiating effect obtained by cisplatin in combination with fractionated irradiation. In clinical practice, cisplatin adds a benefit to radiotherapy, with significant improvement of overall survival at five years in head and neck SCC patients36. It is known that cisplatin treatment increases the duration of S phase and arrests cells in the G2/M phase39, which could contribute to radiosensitization. In the present study we share similar observations. Furthermore, radiation-induced arrest in the G2/M phase occurred later and was more sustained in HPV-positive than HPV-negative cells4. Our data demonstrate a significant increase in G2/M arrest after concurrent cisplatin treatment combined with a single-dose or fractionated irradiation regimen, which is more pronounced (up to 2.9-fold) in the HPV-positive tumor model than in the HPV-negative model. The obtained results differ slightly from the previous ones6 and the reason might be the lower dose of irradiation (2 Gy compared to 5 Gy) and a slightly higher dose of cisplatin (0.2 μg/ml compared to 0.1 μg/ml). A higher dose of irradiation was found to induce higher G2/M arrest in our previous study6 and also shown in other studies8.In published studies, different HPV-positive cell lines have shown considerable variations in sensitivity not only to irradiationInterestingly, both tumor models responded much better when cisplatin treatment was combined with fractionated irradiation than with single-dose irradiation; in the former case, the effect was synergistic. In fact, the tumor growth delay after combined treatment with cisplatin and fractionated irradiation was improved up to 4 times in both tumor models compared to that of the single-dose radiation modality. The survival difference between the two tumor models when treated with fractionated irradiation was 26% in favor of HPV-positive tumors, which had a 1.5 times longer tumor growth delay.47. Radiation can induce an immunogenic death of tumor cells, which then serve as a source of tumor antigens. Additionally, cisplatin-induced major histocompatibility complex I expression, improved recruitment and proliferation of immune effector cells, upregulation of lytic activity of cytotoxic effector cells and downregulation of the immunosuppressive tumor microenvironment have been shown to add to the antitumor response42. Thus, an important shortcoming of our study remains the inability to evaluate the contribution of the immune response to the antitumor effects, as the tumors were growing on immunodeficient mice. Indeed, radiation therapy with concurrent cisplatin resulted in a partial response of tumors in the absence of an immune response (immunodeficient B6129S7-Rag1un1Mom/J mice), while 50% of the complete response was obtained in immunocompetent animals (C57Bl/6 mice)26. Furthermore, boosting the immune response with the modulated adenoviral vaccine that targets HPV oncogenes E6 and E7 in combined chemoradiotherapy treatment cured 90% of HPV-positive tumors in immunocompetent animals but none in immunodeficient animals49. Additional research revealed that irradiation of HPV-positive cancer cells induces the reduction of CD47 (cluster of differentiation 47) expression, which stimulates phagocytosis via dendritic cells and results in immune-mediated cell death of HPV-positive cancer50. Additionally, selectively decreased CD47 expression induced by radiation of HPV-positive tumor cells in combination with cisplatin treatment efficiently improved immune-mediated tumor clearance in vivo in immunocompetent but not immunodeficient Rag1 mice50. However, the chosen treatment regimen for HPV-positive and HPV-negative tumors in immunodeficient animals in these studies50, i.e., concurrent cisplatin treatment and 8 Gy irradiation given once a week for three weeks, was less effective than ours, resulting only in a tumor growth delay and no survivors. Similar results were obtained in our study with single-dose irradiation, with or without concurrent cisplatin therapy, which resulted in a prolonged tumor growth delay without surviving animals. However, cisplatin treatment before the first fraction of irradiation and fractionated irradiation  acted synergistically, indicating the role of irradiation as an inducer of immunogenic cell death. Previous studies have shown that low dose irradiation in itself has an effect on macrophages thereby supporting T cell responses51. In addition, oncoproteins E6 and E7 make HPV-positive tumors highly recognizable to the immune system, thus inducing HPV-specific T cell response, which favors tumor control52. However, in a proportion of patients with a HPV-positive tumor immune response to HPV is weak or even absent and subsequently has a worse outcome upon standard therapy than those a HPV-specific T cell response52. Therefore, we can only speculate that although immunodeficient SCID animals were used, the innate immune response was elicited, mediated mainly by natural killer cells, macrophages, and granulocytes that synergized with cisplatin therapy. Overall, local irradiation can act synergistically with other therapies, but the antitumor efficacy of combined treatment is determined by a fraction dose of the irradiation.One of the possible mechanisms responsible for the synergistic effects of cisplatin treatment concurrent with the fractionated regimen is the involvement of immunomodulation by the combined treatment. Immunomodulatory effects of radiation and cisplatin treatment have been previously confirmed in preclinical and clinical studies2/M phase, which could be one of the underlying mechanisms of the improved response of HPV-positive tumor cells to chemoradiation.In conclusion, we confirmed that the response of HPV-positive pharyngeal SCC is better than the response of HPV-negative tumors after fractionated irradiation. Furthermore, radiosensitization by concurrent cisplatin treatment at isoeffective irradiation doses was still present in both tumor models but was more pronounced after fractionation than after single-dose irradiation. Namely, the combined treatment acted synergistically, resulting in a 56% survival rate after the fractionated regimen in mice bearing HPV-positive tumors and only 30% in mice bearing HPV-negative tumors. The fractionated regimen increased the accumulation of cells in G