This cohort study assesses associations between sexual activity, the oral presence of human papillomavirus (HPV), and HPV vaccination among sexually active female adolescents vaccinated against 4 HPV types. What is the prevalence of oral human papillomavirus (HPV) in sexually active female adolescents receiving the quadrivalent vaccine?This longitudinal cohort study of female adolescents detected HPV DNA in 6.2% of oral rinse samples, with prevalence decreasing with time since initiation of sexual activity. Detection of HPV types targeted by the quadrivalent vaccine  was also significantly lower among those who received at least 1 dose of vaccine than among unvaccinated participants.This study’s findings suggest that detection of HPV in the oral cavity is not uncommon in sexually active female adolescents, but that HPV vaccination is associated with a significant decrease in detection of HPV types in the oral cavity. Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States, and oral HPV infection is associated with increased risk of oropharyngeal cancer.To describe the risk factors for oral HPV in sexually active female adolescents receiving the quadrivalent vaccine.Longitudinal cohort study involving repeated collection of oral rinse specimens from sexually active female adolescents conducted between October 19, 2007, and March 9, 2017, at a large adolescent health center in New York, New York, that provides free health care, including HPV vaccination.Human papillomavirus vaccination and self-reported history of sexual behavior.Prevalence of HPV in the oral cavity.Among the 1259 participants who were included in this study, median age at entry into the study was 18  years; 638 (50.7%) were of African American descent, 569 (45.2%) were of Hispanic descent, 43 (3.4%) reported another race/ethnicity, and race/ethnicity was unspecified for 9 (0.7%). The median (mode) age at first sexual activity was 14.8 (14) years, and 1161 (92.2%) reported having had oral sex. Human papillomavirus DNA was detected in baseline oral rinse samples of 78 of the 1259 participants . There was a significant decrease in oral HPV detection with time (in years) since first engaging in sexual activities, independent of age and concurrent detection of cervical HPV; comparing 4 or more years with 1 year or less, the odds ratio was 0.45 . Detection of vaccine types  was significantly lower among participants who had received at least 1 dose of the quadrivalent HPV vaccine at the time of enrollment compared with those who were unvaccinated .This study’s findings suggest that detection of HPV in the oral cavity is not uncommon in sexually active female adolescents. In addition, HPV vaccination is associated with a significant decrease in detection of HPV vaccine types in the oral cavity. Study participants included sexually active patients aged 13 to 21 years at time of enrollment who were planning to or had already received the quadrivalent HPV vaccine (4vHPV) targeting HPV types 6, 11, 16, and 18. Pregnant and menstruating women were invited to return at a later date after resolution of their pregnancy or their menstrual period. No selection was done by race/ethnicity. The study sample excluded 630 consented participants who had not completed a baseline visit as of March 9, 2017. The study was approved by the Institutional Review Board at Icahn School of Medicine at Mount Sinai, Manhattan, New York, and written informed consent was obtained from all study participants and from guardians accompanying minors before enrollment. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  at baseline (enrollment visit), with exact binomial 95% CIs. Cross-sectional associations with detection of HPV DNA in oral samples were assessed by logistic regression after adjustment for participant age in years.To study the association between oral HPV prevalence and risk associations with sexual behaviors, we fitted logistic regression models to assess the association of number of oral sexual partners, vaginal sexual partners, or both with oral HPV detection at enrollment. Additional models also assessed time since initiation of sexual activity. Models were adjusted for age in years, with further adjustment for statistically significant covariates, including concurrent detection of cervical HPV and time since initiation of sexual activity. Tests for trend for ordered categorical variables were conducted by including the variables in the model as ordinal values.Cumulative incidence was estimated by using the Kaplan-Meier method for incident infections detected in oral samples collected at follow-up visits. Time to event was estimated by taking the midpoint of the interval preceding incident events. Participants who were HPV negative at enrollment and who did not have an infection until the last visit were censored at the last follow-up date. Statistical calculations were conducted using Stata version 14 , and statistical significance was assumed at the 2-tailed α = .05 level.Of the 1259 participants, 638 (50.7%) were of African American descent, 569 (45.2%) were of Hispanic descent, 43 (3.4%) reported another race/ethnicity, and 9 (0.7%) were of unspecified race/ethnicity . The medOne oral rinse sample was obtained from each of the 1259 participants at enrollment. Human papillomavirus types were detected in the baseline oral rinse samples of 78 of the 1259 participants , with oncogenic HPV types detected in 21  and 4vHPV vaccine types  detected in 8 participants . The greatest prevalence of oral HPV was detected at age 16 years for all types (16 of 153 [10.5%]) and oncogenic types (6 of 153 [3.9%]) .P for trend = .03)  .Although marijuana use and cigarette smoking appeared to be inversely associated with detection of HPV in the oral cavity, these were not significant after adjustment for age and time since initiation of sexual activity . The most consistent risk factor associated with detection of HPV in the oral cavity was detection of HPV in the cervical sample collected at the same visit.P = .06).At enrollment, 192 of the 1259 study participants (15.3%) had not received the 4vHPV vaccine. eFigure 1 in the To further investigate the association of vaccination with detection of any 4vHPV types, we used a multivariable logistic regression approach to compare vaccinated with unvaccinated participants after adjustment for age, years since first sexual activity, and concurrent detection of 4vHPV types in the cervix . PrevaleWhen we assessed incident detection of oral HPV among vaccinated women in our cohort, rates of infection were low overall, ranging from 0.05  to 0.46  HPV infections per 100 person-years , ranging between 2% and 5%.13 We observed a decrease in detection of HPV in the oral cavity with years since the first sexual activity, potentially reflecting the highly transient nature of HPV infection.14 Concurrent detection of cervical HPV was itself significantly associated with detection of HPV in the oral cavity, suggesting that the anogenital region may be a source and that transmission could involve digital transfer or autoinoculation.16Our data do not provide strong support of a sexual transmission model for oral HPV acquisition, although detection was highest soon after initiation of sexual activity.17 there is little information about associations in sexually active adolescents. Consistent with previous studies,19 we report that any sexual activity, not just oral sex, may be associated with oral HPV detection. In comparison with previous studies, however, we observed no association between the likelihood of HPV detection in the oral cavity and number of oral sex partners, vaginal sex partners, or both.20 This may be owing in part to the lack of a reference group of individuals who had not engaged in sexual activity in our study cohort.21Although increased sexual activity has been identified as an important risk factor for oral HPV,22 The current cohort was also more likely to present with an oncogenic HPV type or vaccine type in the oral cavity and cervix compared with other groups.10 Despite these factors, we observed a significant vaccination benefit reflected by the lower prevalence of HPV vaccine types  in the oral cavity among vaccinated vs unvaccinated female adolescents. Results from the bivalent HPV vaccine trial conducted among women aged 18 to 25 years showed a vaccine efficacy of 93% against detection of oral HPV-16 and HPV-18 four years after vaccination.7 In comparison, Chaturvedi et al23 showed an 88% reduction in prevalence of oral HPV-16 and HPV-18 when assessing differences by self-reported vaccine status among participants in NHANES. Our estimates of vaccine benefit among sexually active female adolescents are in line with these population estimates. However, given the low prevalence of oral HPV and the inclusion of vaccinated participants, our study had insufficient statistical power to assess associations with individual HPV types.Our study population had a greater median number of sexual partners and an earlier initiation of sexual activity compared with previously reported cohorts, including the vaccine trial participants.24 Although we observed a decreased prevalence of HPV-16 and other 4vHPV vaccine types among vaccinated female adolescents, we also detected new HPV-16 infections in the oral samples at a rate of 0.2 per 100 person-years. In addition, repeated detection of 4vHPV vaccine types in oral rinse samples across a period of more than 1 year was observed in at least 2 participants, suggesting a subset of individuals with persistent infections that were likely acquired before vaccination. We also observed similar or higher incidence for other oncogenic nonvaccine HPV types 25; continued research is needed to determine whether the additional types targeted by the new 9-valent vaccine will also decrease.26A number of studies have shown HPV-16 to be the main HPV type associated with oropharyngeal cancers in the United States.Overall, we found that detection of HPV in the oral cavity is not uncommon in sexually active female adolescents, but it decreases with age and time since the initiation of sexual activity. Although our study population had a greater median number of sexual partners and a younger age at sexual initiation compared with previous studies, we observed a significant vaccination benefit reflected by the lower prevalence of 4vHPV vaccine types. Our findings suggest that concurrent detection of an HPV infection in the cervix may be a risk factor for detection of HPV in the oral cavity.


Metabolic changes in tumour cells are used in clinical imaging and may provide potential therapeutic targets. Human papillomavirus (HPV) status is important in classifying head and neck cancers (HNSCC), identifying a distinct clinical phenotype; metabolic differences between these HNSCC subtypes remain poorly understood.+ve and HPV−ve HNSCC, performed a meta-analysis on FDG-PET imaging characteristics and correlated results with in vitro extracellular flux analysis of HPV−ve and HPV+ve HNSCC cell lines. The monocarboxylic acid transporter-1 (MCT1) was identified as a potential metabolic target and tested in functional assays.We used RNA sequencing to classify the metabolic expression profiles of HPVSpecific metabolic profiles were associated with HPV status, not limited to carbohydrate metabolism. There was dominance of all energy pathways in HPV-negative disease, with elevated expression of genes associated with glycolysis and oxidative phosphorylation. In vitro analysis confirmed comparative increased rates of oxidative phosphorylation and glycolysis in HPV-negative cell lines. PET SUV(max) scores however were unable to reliably differentiate between HPV-positive and HPV-negative tumours. MCT1 expression was significantly increased in HPV-negative tumours, and inhibition suppressed tumour cell invasion, colony formation and promoted radiosensitivity.HPV-positive and negative HNSCC have different metabolic profiles which may have potential therapeutic applications. Aerobic glycolysis is an inefficient pathway for generating ATP, but is thought to confer a growth advantage for tumour cells, providing a biosynthetic advantage by generating carbon precursors required for the synthesis of macromolecules essential for cell division.3 While aerobic glycolysis has become a recognised ‘hallmark of malignancy’, its causal relationship to tumourigenesis remains unclear.The identification of common links between oncogenes and important regulators of metabolism has fuelled a resurgent interest in cancer cell metabolism, despite the Warburg effect being first described nearly a century ago.−ve) and -positive (HPV+ve) disease. HPV+ve tumours are most commonly found in the oropharynx (OPSCC), including the tonsil/base of tongue region, with HPV status usually determined by expression of a surrogate marker p16 (INK4A).5 HPV+ve and HPV−ve tumours have significantly different molecular profiles and clinical behaviour, particularly with respect to invasion, metastasis and response to chemoradiotherapy.8 PET imaging is routinely used for identification, staging and follow up of HNSCC, however no studies have examined how HPV status affects the metabolic phenotypes of these tumours, which may in turn affect the clinical ability to detect tumours. There is also increasing evidence of fundamental links between a tumour’s metabolic phenotype, its interactions with the tumour microenvironment and clinical outcome,12 suggesting that specific metabolic pathways may promote tumour progression and represent therapeutic targets. Understanding the metabolic profiles of HNSCC cancer subtypes may therefore impact on the clinical management of this disease.Head and neck squamous cell carcinoma (HNSCC) is currently classified into human papillomavirus-negative :Ham’s F12 , containing 10% (v/v) foetal bovine serum (FBS) and 2 mM L-glutamine. Two immortalised cell lines derived from the upper aerodigestive tract from suspected HPV+ve tumours, UD-SCC-2 and UPCI:SCC90,14 were kindly supplied by Susanne M. Gollin . These cell lines, along with Detroit 562, an additional HNSCC cell line used in functional analyses, were cultured in 10% FBS supplemented DMEM. Polymerase chain reaction (PCR) was performed to confirm the HPV status of cell lines used in functional assays by the presence of E6/E7 oncoprotein RNA  were plated in 80 µl of growth medium in a XF96 culture plate well, while the probes were immersed in XF Calibrant Solution  overnight at 37 °C in non-CO2 incubator 24 h prior to experimental run. Running medium for the assay was prepared using glucose-free DMEM supplemented with 10% serum, 2 mM L-glutamine (PAA) and 1 mM sodium pyruvate (PAA), and the pH was adjusted to 7.4 at 37 °C. Prior to performing the assay, drugs from mitochondrial and glycolysis stress kits (Seahorse Bioscience) were diluted in running medium and loaded into injection ports of the probe cartridge. Cells were washed twice with 200 µl of running medium, and the plate was placed in a non-CO2 37 °C incubator whilst sensor cartridges were calibrated. OCR and ECAR were measured over a 2-minute period as absolute rates (pmoles/min and mpH/min respectively), followed by 3-min mixing and re-oxygenation of the media. Experimental conditions in each assay were performed with a minimum of triplicate wells. Glycolytic capacity was calculated using the equation: maximum rate measurement after oligomycin injection—last rate measurement before glucose injection, expressed as mpH/min. A standard Bradford assay (Bio-Rad Laboratories) analysis was performed on end-point wells to normalise to total protein count per well (ug/well).OCR and ECAR measurements were performed using the XF96 Extracellular Flux analyzer  as described in Wu et al.Cells were lysed in NP40 buffer . Samples containing equal amounts of protein were electrophoresed under reducing conditions in 8–10% SDS-PAGE gels. Protein was electroblotted to PVDF membranes . Blots were probed with antibodies against MCT1 (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-rat or anti-mouse (Dako) was used as secondary antibodies. Bound antibodies were detected with the enhanced chemiluminescence western blotting detection kit system (Amersham). Blots were probed for Total FAK (Millipore) as a loading control.17 Briefly, a layer of Matrigel  diluted 1:2 with DMEM was placed on top of each insert prior to seeding 5 × 105 cells/200 µl in each chamber. A 72-h incubation at 37 °C was performed to allow invasion of cells to the lower chamber and counted using a CASY counter .Cell invasion was analysed using Transwell® assays  as previously described.17 A 1ml mixture comprising 3.5 volumes type I rat tail collagen (Merck Millipore), 3.5 volumes Matrigel (Becton-Dickinson), 1 volume 10x DMEM, 1 volume foetal calf serum and 1 volume of 10% DMEM together with 2.5 × 105/ml HFFF2 cells was allowed to polymerise. A mixture of HNSCC cells (5 × 105) and HFFF2 fibroblasts (25 × 104) in a combined volume of 1ml of DMEM was then added dropwise onto the top of the gel. After 24 h incubation, the gels were raised onto nylon sheet coated stainless steel grids. After 7 days incubation at 37 °C the gels were bisected, fixed in formal-saline and processed to paraffin. Sections (4 μm) were stained for H&E ± pan-cytokeratin.Organotypic cultures were prepared as previously described.SCC-25 cells were treated with 10 nM AZD3965 or a DMSO control for 48 h prior to the start of the survival assay, trypsinised, counted and seeded in 6-well plates at 2000 cells/well. AZD3965/DMSO containing media was replaced after 24 h and then left in situ for 10 days until conclusion of the experiment.To test radiosensitivity, the cell suspensions were exposed to a range of γ-ray doses in 2-Gy increments dose of γ-rays prior to plating at 2000 cells/well. Standard cell media was utilised for both control and AZD3965 pre-treated populations to homogenise post radiation exposure conditions.18 was utilised, allowing automated thresholding and analysis, to calculate both % area and intensity of colonies.At completion cells were fixed in a 3% crystal violet/10% formalin solution. Following collation of scanned images, the ‘ColonyArea’ plugin of Image J19 . At the time of the present study, 35 patients in this cohort initially had sequencing data available for analysis. The quality of raw SE read data was assessed and reads of low quality were removed using PRINSEQ and reads with low complexity were remove using DUST, complexity scores above 7 were removed. SE reads were then mapped to the human genome (hg19) using TopHat (version 2.0.9) allowing no mismatches. Only reads uniquely aligned were considered for further counting using HTSeq-count (version 0.5.4), yielding read count values for a total of 23,368 RefSeq annotated genes. The raw counts were further processed in Bioconductor package EdgeR (version 3.4.2). Nonspecific filtering of count data was performed such that genes with less than 2 read counts per million in 25% of the samples were removed. The remaining 14565 genes  were identified with ToppGene web tool22 and functional enrichment analysis was performed using ToppFun from the ToppGene suite. Statistical significance of different GO terms and pathways were estimated using hypergeometric testing with FDR adjustment for multiple testing using the Benjamini and Hochberg method. GO terms and functional pathways with FDR corrected p-value < 0.05 was considered significant. Significant GO terms were visualised in semantic similarity-based scatterplots generated by REVIGO.GO terms associated with biological processes and biological pathways that were significantly over-represented for DEGs (n = 260 (231 with confirmed HPV status)) from University Hospital Southampton (2000-10), Poole NHS Foundation (2000-6) and Bart’s and the London NHS Trust (2000-6) as described previously23 (REC references 09/H0501/90 and 07/Q0405/1). Tumours classified as HPV+ve were positive for both p16 immunohistochemistry and HPV ISH. Immunochemistry was performed using MCT1 antibody (Santa Cruz Biotechnology) and scored by two blinded investigators (KM/JF) as high (moderate/high strength staining) or low (absent/low strength staining).Immunohistochemical analysis was performed on a tissue microarray of consecutively-treated patients with oropharyngeal squamous cell carcinoma  was not reported, indicated mean values were estimated from reported median, range and sample size.24 A mean difference approach was used for meta-analysis and a Forrest plot to examine effect created using Review Manager version 5.0 (RevMan).25The following broad search strategy was utilised on Pubmed/Medline and Embase: ((FDG OR PET) AND (HPV OR papillomavirus)), with all fields included and including dates 1946-present. Titles and abstracts were scrutinised and full texts of relevant and related articles were retrieved to enable final selection. Only cases reporting SUV(max) scores were included. Case reports and commentaries were excluded. Only English language articles analysing the direct comparison between FDG-PET results in HPVT-test was used. For other datasets where normal distribution was not evident, non-parametric data analysis was performed . Error bars show standard deviation (SD) unless otherwise stated. For TMA/database analysis, SPSS Statistics  was used.For comparisons between experimental groups in functional assays, a Student’s The primary endpoint for survival analysis was death from OPSCC . An OPSCC-specific survival time was measured from the date of diagnosis until date of death from OPSCC or date last seen alive. Kaplan–Meier survival curves were produced using clinicopathological patient data. Death from other causes or loss to follow up was marked as censored for analysis.For all analyses, a p value of equal or less than 0.05 was considered to be significant.+ve, 24 HPV−ve) were analysed by RNA sequencing19 to produce a differentially expressed gene (DEG) list of 1587 genes based on viral status  analysis on the 111 differentially expressed, metabolism-related genes and summarised results in REVIGO26  associated with nucleotide biosynthesis and carbohydrate metabolism.To identify differences in specific metabolic pathways between HPVO26 Fig. . The top27 We found that there was upregulation of glycolysis genes  in HPV−ve HNSCC, which also expressed lower levels of the negative regulator, PDK1  and two HPV+ve  were analysed using a Seahorse XF96 Extracellular Flux Analyzer  . Four HNSCC cell lines, two HPVMA) Fig. , measuri+ve and HPV−ve HNSCC prompted us to investigate whether these differences were apparent on FDG-PET imaging. Prior research has been limited by HPV−ve sample size due to the emerging dominance of HPV+ve tumours in the oropharynx, and the previous relatively confined use of PET imaging to this anatomical site. We therefore performed a systematic review of the literature and produced a virtual data-series from several comparative studies, for analysis using SUV(max), which has been previously proposed as the most accurate and reproducible SUV measure.28The distinct in vivo and in vitro metabolic profiles between HPV−ve cohort  and 11.3 in the HPV+ve . A meta-analysis was performed on collated results, utilising a mean difference approach , with no overall difference between SUV(max) scores based on HPV status, although there was a strong trend for higher SUV(max) scores in the HPV−ve populations . Although the trend for higher SUV(max) scores in HPV−ve tumours is consistent with our in vitro findings, the result suggests that, at a single timepoint, SUV(max) does not accurately distinguish between HPV+ve and HPV-ve HNSCC.We identified a total of 305 studies; following de-duplication, 243 studies were screened by title ± abstract. 229 were excluded following initial screening. Full articles for the remaining 14 papers were reviewed and from this, 7 studies were excluded due to not fitting study aims (4) or insufficient data (3)  and MCT4  showed no differential expression between HPV−ve and HPV+ve HNSCC; MCT1 expression (SCL16A1) however, was significantly elevated in HPV−ve tumours (p = 0.012), suggesting a potential tumour subtype-specific, metabolic target 31 confirmed higher expression of MCT1 in HPV-ve tumour samples 23 also showed significantly stronger MCT1 expression in HPV-ve tumours , correlating weakly with Glut1 expression (Spearman’s Rho = 0.228 p = 0.001) and tumour cohesion , but not with tumour grade, perineural, lymphatic or vascular spread. radiotherapy , a selective inhibitor of MCT1,15 which is in early-phase clinical testing. We first confirmed MCT1 expression in a panel of HPV-ve  and HPV+ve (UD-SCC-2 and UPCI:SCC90) cell lines . Since highest expression of MCT1 was seen in SCC-25 cells, we analysed the effect of AZD3965 in more detail, using this cell line as representative of our target population of HPV−ve HNSCC. Initially we performed extracellular flux analysis to analyse real-time metabolic outputs, following MCT1 inhibition. AZD3965 produced a dose-dependent reduction of glycolytic rate as well as glycolytic capacity . Since the negative survival impact of MCT1 expression was most evident in patients treated with chemoradiotherapy, we hypothesised that AZD3965 may make SCC-25 cells more radiosensitive. SCC-25 cells were treated with AZD3965 for 48 h, and then exposed to incremental γ-rays doses up to a maximum of 6-Gy. AZD3965-treated cells were significantly more sensitive to irradiation .Targeting lactate balance has previously been suggested as a potential therapeutic option to target cancer cells.nes Fig. . Given tity Fig. . This wa32 and FDG uptake in PET imaging reflects this,33 however, it is not clear whether metabolic differences exist between HPV+ve and HPV-ve HNSCC, tumour subgroups that show very different clinical behaviours. We found that HPV−ve tumours have a more glycolytic phenotype compared to their HPV+ve counterparts, but also have a globally altered metabolic profile consistent with the greater requirements for macromolecule production, as well as alterations in other metabolic and biosynthetic pathways. In keeping with recent advances in diverse metabolic targeting , RNA sequencing data showed enrichment in HPV−ve tumours of additional metabolic pathways including those involved in fatty acid, lipid and nucleotide biosynthesis. Understanding the wider metabolic features of HPV−ve tumours, and how this may affect response to treatment may enable us to develop treatment combinations that exploit metabolic dependence.HNSCC has high rates of glucose uptake,−ve tumours  were supported by extracellular flux analysis confirming a higher rate of glycolysis and glycolytic capacity in HPV−ve cell lines, in keeping with the requirement for redox balance in the presence of rapid cell division. It may also explain why HPV−ve cell lines showed a greater glycolytic capacity in vitro, with their full glycolytic potential moderated by elevated PKM2 to allow for intermediate shuttling for nucleotide and NADPH production. Our finding of concurrent higher oxygen consumption rates in HPV-ve cell lines is consistent with other studies noting that mitochondrial function is not impaired in the majority of cancer cells.36 The reason for differences in glycolysis between HPV−ve and HPV+ve HNSCC is uncertain; the three rate limiting reactions in glycolysis are catalysed by hexokinase, phosphofructokinase and pyruvate kinase, and all are relatively upregulated in HPV−ve HNSCC. Recent studies show that TP53, a negative regulator of glycolysis, is mutated in around 75–85% of HPV-ve HNSCC.39Findings of increased expression of glycolytic genes in HPV40Hypoxia inducible factor 1 alpha (HIF1a) is also a potent promoter of glycolysis which is commonly expressed in HNSCC, being positively regulated by hypoxia and other factors including oncogenic signalling and reactive oxygen species.−ve tumours have a biosynthetic advantage over their HPV+ve counterparts.42 Altered tumour metabolism may also provide opportunities for therapeutic targeting of specific metabolism-related pathways. MCT1 expression is differentially upregulated in HPV−ve tumours and significantly associated with poor survival in OPSCC patients, notably in those with HPV−ve disease treated with chemoradiotherapy. Similar correlation of MCT1 expression with poor prognosis has been reported in tumours of the breast,43 ovary,44 stomach45 and colon.46 Functional flux analysis of response to MCT1 inhibition indicated a shift in the metabolism profile of HNSCC cell lines from glycolysis to oxidative phosphorylation, resulting in suppressed tumour invasion and colony formation. Although previously considered a lactate importer, MCT1 has demonstrated bi-directionality47 which may help to explain the variable effects on cell metabolism produced by MCT1 inhibition reported in the literature. For example, in various cancer cell lines Bola et al.48 demonstrated that AZD3965 resulted in an increase in glycolysis and glycolytic enzymes whereas, similar to the present study, Doherty et al.49 found that MCT1 inhibition rapidly abrogated glycolytic function. The presumed reduction in intracellular pH following reduction of lactate efflux measured helps explain the negative cell survival effects on HNSCC cell lines following MCT1 inhibition observed in clonogenic assays. Areas of low pH in the tumour microenvironment have been demonstrated to correspond with cancer cell migration and invasion,51 therefore the reduction in lactate efflux with the MCT1 inhibitor may also explain the functional effects on cell migration and invasion. However, while these data suggest that MCT1 targeted therapy could be of potential benefit to patients with HPV−ve tumours, further pre-clinical modelling is required to translate these finding.Ultimately the classification of metabolic phenotype in HNSCC may have important relevance in understanding the biology and clinical behaviour of the disease subgroups, indicating that glycolytic HPV52 but with these imaging metrics alone, there have been conflicting reports in the literature about the significance of HPV status to glycolytic phenotype.53 It is possible that dynamic PET imaging over time could pick up differences in the rate of glucose uptake between HPV-ve and HPV+ve tumours. However, imaging at a single, static and relatively late time point, as currently occurs in clinical imaging protocols, does not distinguish between tumour subgroups.FDG-PET is now a standard-of-care investigation in the diagnosis of primary and metastatic HNSCC, as well as being used for post-treatment surveillance. We found that SUV(max) alone cannot distinguish HPV status in primary tumours, especially given the range of reported metrics and variability of imaging parameters between centres. Standardised uptake value (SUV) measurements have been shown to be reproducible and have both diagnostic and prognostic value in HNSCC−ve and HPV+ve HNSCC; whilst this does not impact on the ability to clinically detect these subgroups using PET imaging, our data adds to the growing evidence that targeting glycolytic pathways may have therapeutic value in HPV−ve disease.In summary, we show that there are significant metabolic differences between HPVSigned letter approving additional authorSupplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Figure 5Supplemental Figure 6Supplemental Figure 7Supplemental Figure 8Supplemental Figure 9Supplementary Table 1Supplementary Table 2Signed author letter


These results show that the Tag/Catcher AP205 VLP system can be exploited to make a combinatorial vaccine capable of eliciting antibodies with dual specificity.In Africa, cervical cancer and placental malaria (PM) are a major public health concern. There is currently no available PM vaccine and the marketed Human Papillomavirus (HPV) vaccines are prohibitively expensive. The idea of a combinatorial HPV and PM vaccine is attractive because the target population for vaccination against both diseases, adolescent girls, would be overlapping in Sub-Saharan Africa. Here we demonstrate proof-of-concept for a combinatorial vaccine utilizing the AP205 capsid-based virus-like particle (VLP) designed to simultaneously display two clinically relevant antigens (the HPV RG1 epitope and the VAR2CSA PM antigen). Three distinct combinatorial VLPs were produced displaying one, two or five concatenated RG1 epitopes without obstructing the VLP’s capacity to form. Co-display of VAR2CSA was achieved through a split-protein Tag/Catcher interaction without hampering the vaccine stability. Vaccination with the combinatorial vaccine(s) was able to reduce HPV infection  This region also accounts for 86% of global malaria cases (219 million), including 435,000 malaria related deaths per year3. Women are particularly vulnerable to malaria during pregnancy when P. falciparum infections cause placental malaria (PM) (reviewed by4 and5). Consequently, both cervical cancer and PM constitute major public health challenges in Sub-Saharan Africa, where people often have limited access to health care , or the affected individuals may be incapable of paying for expensive vaccines or treatment6.There is a striking overlap in the global prevalence of cervical cancer and malaria, particularly in Sub-Saharan Africa. Cervical cancer is caused by a sexually acquired infection with certain types of Human Papillomavirus (HPV)7. The HPV vaccines based on the HPV L1 major capsid protein, which form Virus-Like Particles (VLPs), are extremely potent, possibly offering life-long protection after a single vaccine dose9. The high immunogenicity of the HPV vaccines is attributed to structural features of the L1 VLPs, which lead to the efficient generation of long-lived antigen-specific antibody-producing cells12. HPV L1 VLP vaccines strongly elicit neutralizing antibody responses against the included HPV genotypes represented in the vaccines, but offer little in the way of cross-protection against other HPV types (reviewed by13). The clinical data generated by the HPV L1 VLP vaccines has consequently stimulated a great interest in developing next-generation VLP-based vaccines with a similar capacity of inducing long-term antibody responses, but against a broader panel of HPV types.HPV vaccination programmes have been widely implemented in the developed world, which has caused a substantial decrease in the incidence of HPV infections14). In particular, the N-terminus of the HPV L2 protein contains a major cross-neutralizing epitope, RG115. Several studies have shown anti-RG1 antibodies confer complete type-specific protection and moderate cross-protection against heterologous HPV types17. Recently, it was shown that displaying multiple conjoined L2 RG1 epitopes, deriving from different HPV types on the surface of a VLP backbone, broadened the protective immunity against diverse HPV types18. The RG1 epitope, specifically concatenated constructs comprised of the RG1 epitope from several HPV types, represents a good target antigen for the development of a broadly protective HPV vaccine. However, the RG1 epitope is only transiently exposed to the immune system during the attachment of HPV to the basement membrane19 and therefore, an RG1-based vaccine will likely need to elicit long-lived, high levels of circulating antibodies to be effective.Current efforts to develop a broadly protective prophylactic HPV vaccine focus on targeting the HPV minor capsid protein, L2, which contains multiple highly-conserved and broadly neutralizing epitopes . VAR2CSA is responsible for the sequestration of P. falciparum parasites20 in the placenta of pregnant women by mediating binding to the placental chondroitin sulfate A (CSA)21. Accordingly, an effective VAR2CSA-based PM vaccine may need to elicit antibodies that inhibit the binding between placental CSA and VAR2CSA expressed on the surface of infected erythrocytes (iE)24.To date, there is no commercially available vaccine against PM, although two different vaccines, both including the minimal binding region of the 25. Both vaccines should ideally be administered to pre-pubescent girls and elicit durable (decades-long) immune responses. Combining the two disease targets on a common, highly immunogenic VLP platform could be a strategy to induce long-term protection against both diseases, and will be beneficial in terms of production costs and deployment.Vaccines against PM and HPV have overlapping target populations and need to be inexpensive in order to be broadly employed in the sub-Saharan region30 to generate VLP based vaccines  that display both VAR2CSA and HPV L2 RG1 epitopes at high densities. Using mouse models, this study examined whether the VLP-based combinatorial vaccine design would allow for induction of high titered, dual-specificity antibody responses against the co-displayed antigens.We have used the well-characterized AP205 capsid-based VLP platformP. falciparum protein, the SpyCatcher sequence and L2-HPV coding sequences were genetically fused to the 5′ and 3′ ends of the gene coding for AP205 capsid protein, respectively , allowed for covalent attachment of VAR2CSA to the VLPs resulting in the formation of VLPs displaying both the HPV L2 and VAR2CSA antigens at high density   Fig. . The lev51) Fig. . Finally51) Fig. .Figure 2in vivo, mice immunized with 2xL2-VLP or 5xL2-VLP were challenged with a panel of luciferase expressing HPV pseudovirions (PsV)  using a well-established PsV genital challenge mouse model18 . By contrast, the 2xL2-VLP vaccine were generally less effective than the 5xL2-VLP vaccine at preventing infection with HPV PsV. Vaccination with 2xL2-VLP only caused a significant 3- and 47-fold reduction in PsV35 (P = 0.04) and 45 (P = 0.04) infection levels, respectively while no significant reduction was seen for PsV31 (P = 0.5) and 52 (P = 0.12) infection Fig. .in vitro HPV L2-based PsV inhibition assay. The tested sera showed both homologous PsV16 as well as heterologous neutralizing capacity. As expected, the neutralizing titers against the homologous PsV were higher compared to the heterologous PsV45 neutralization titers.  . Inhibition was most pronounced towards parasites of the FCR3 genotype on which the VAR2CSA sequence was based. Some heterologous inhibition was seen towards parasites of the NF54 genotype, but not towards parasites of the 7G8 genotype  Fig. . IgG indype Fig. .Figure 4The aim of this study was to establish proof-of-concept for a VLP-based combinatorial vaccine eliciting functional antibody responses against both HPV and placental malaria. The unique attribute of the AP205 VLP, to accept genetic fusions at both the N- and C-terminus, was exploited to generate combinatorial VLP vaccines, which were designed to simultaneously display the placental malaria antigen (VAR2CSA) and between one and five concatenated HPV L2 sequences representing the cross-reactive RG1 epitope (aa 17–38). Specifically, L2 RG1 antigen sequences were based on the most prevalent oncogenic HPV types in the sub-Saharan African region.31 (NEF55) and co-display of antigenic polypeptides, accomplished by genetic fusion at both N- and C-terminus, has not previously been reported for any VLP. Remarkably, AP205 capsid proteins genetically fused to SpyCatcher (122 aa) at the N-terminus and to HPV L2 RG1 sequences (up to 118aa) at the C-terminus retained their ability to assemble into uniform, non-aggregated VLPs. These results confirm the exceptional capacity of the AP205 VLP to tolerate large genetic fusions at both the N-and C-terminus and underline the broad applicability of this VLP backbone for VLP-based vaccines.The first question we sought to answer was whether the large genetic fusions to the AP205 capsid protein would hamper VLP assembly. To our knowledge the largest polypeptide that has previously been fused successfully to the C-terminus of AP205 is 55aain vitro. Collectively, these results support the conceptual design of a VLP-based combinatorial HPV/PM vaccine.Next, we investigated the ability of the combinatorial VLP based vaccines to generate functional antibody responses against the co-displayed antigen targets. To this end, a theoretical concern of the vaccine design was that one of the co-displayed antigens could dominate the immune response over the other and prevent induction of protective levels of antibodies against the less dominant. In particular, we speculated that covalent attachment of the large PM antigen (VAR2CSA) may sterically hinder access to the RG1 epitopes and/or dominate the immune response due to the high number of B and T-cell epitopes present in the antigen. However, the combinatorial vaccines all elicited high serum levels of anti-RG1 antibodies and co-display of VAR2CSA did not have any measurable effect on the induction of these antibodies. Finally, mice challenged with HPV16 PsV experienced a similar reduction in infection level after immunization with 2xL2-VLP with and without co-display of VAR2CSA. Concerning the PM antigen target, all combinatorial vaccines elicited high levels of anti-VAR2CSA antibodies, which were able to inhibit binding of both homologous and heterologous malaria parasite strains to their human receptor in vivo challenge model it was demonstrated that immunization with 5xL2-VLPs  caused significant protection against a broader panel of HPV strains compared to immunization with 2xL2-VLPs . Whereas both vaccines caused a significant reduction in infection levels of PsV35 and PsV45 only the 5xL2-VLP vaccine induced significant protection against PsV31 and PsV52. Considering the RG1 sequence similarity across the five HPV genotypes included in the 5xL2-VLP vaccine design, HPV35 and HPV45 RG1 sequences show the highest similarity to the corresponding HPV16 and HPV18 sequences, respectively  or HPV16, 18, 35, 31 and 52 (QLYKTCKQAGTCPPDIIPKVEGgsDLYKTCKQSGTCPPDVVPKVEGgsQLYRTCKAAGTCPPDVIPKVEGgsQLYQTCKAAGTCPSDVIPKIEHgsQLYQTCKASGTCPPDVIPKVEG) was attached to the 3′ of the full-length SpyCatcher-AP205 gene27 by PCR. A small Glycine-Serine (GS) linker was inserted between L2 peptides followed by a Glycine-Glycine-Serine-Glycine (GGSG) linker between the SpyCatcher-VLPs and the L2 peptide(s). To create 1xL2-VLPs, the SpyCatcher-AP205 VLP served as template for the initial PCR, where GGSG-16L2 was added by including the nucleotide sequence for the L2 RG1 epitope of HPV16 in the reverse primer overhang.The RG1 epitope of HPV16 (QLYKTCKQAGTCPPDIIPKVEG), HPV16 and 18  containing the concatenated L2 RG1 epitope of HPV16, 18, 35, 31, 52 was used to create an amplicon with a 5′ overlap, capable of annealing to the 3′ end of the SpyCatcher-AP205 sequence and containing a 3′ NotI cloning site. For 5xL2-VLP, the full sequence was amplified, whereas only the 16 and 18L2 sequences were amplified for creating the 2xL2-VLPs. In a third PCR reaction, the amplicons of the first and second PCR were mixed in order to amplify the 5x and 2xL2-VLP constructs.The 5x and 2xL2-VLPs were created by PCR. The SpyCatcher-AP205 VLP sequenceE. coli One Shot® BL21 Star™ (DE3) cells (Thermo Scientific). The non-optimized small-scale upstream expression and downstream purification accomplished using Optiprep™ (Sigma) step gradients, as previously described28.The resulting 5xL2, 2xL2 and 1xL2 VLP genes were cloned into a pET-15b vector using NcoI and NotI restriction sites and the SpyCatcher-AP205-L2-pET-15b plasmid was transformed into 21 (herein termed VAR2CSA). The SpyTag-VAR2CSA-HIS gene sequence was subcloned into the pET-15b vector as described above, expressed in C3029H SHuffle T7 competent E.coli cells (New England Biolabs Inc.) and purified by IMAC and size exclusion chromatography .PCR added SpyTag (AHIVMVDAYKPTK) and a flexible GGS-linker 5′, as well as a V5 and hexa-histidine tag (HIS) 3′ to the gene coding for the DBL1-ID2A region (aa 9–1025) of VAR2CSA (FCR3 genotype)28. Briefly, for TEM, the 5x, 2x and 1xL2-VLPs were adsorbed to 200-mesh carbon-coated grids. Grids were stained with 2% uranyl acetate (pH7.0) and analyzed with an accelerating voltage of 80 kV, using a CM 100 BioTWIN electron microscope .The 5x, 2x and 1x-L2-VLPs were assessed using negative stain transmission electron microscopy (TEM) as well as dynamic light scattering (DLS) as previously describedFor DLS measurements, the distribution of particle sizes was acquired . The 5x and 2xL2-VLPs were analyzed with 20 runs and the 1xL2-VLP with 10 runs. The estimated diameter of the main particle population and the percent polydispersity (%Pd), was calculated for all three L2-VLPs.To generate the combinatorial HPV and PM VLP vaccines, purified SpyTag-VAR2CSA-His and 2x or 5xL2-VLPs were mixed in a 3:1 molar ratio (VAR2CSA:VLP) and incubated for 16 h at 4 °C in a phosphate-buffered saline (PBS), pH 8 and adjusted to 0.6 M NaCl.Excess, unbound antigen was removed by dialysing  against PBS pH8, 0.6 M NaCl.VLP vaccines  were formulated 1 hour prior to immunizations using 2%Alhydrogel (Brenntag) to a final concentration of 2 mg/ml aluminum hydroxide.in vitro HPV PsV challenge was performed on this serum. For the 5x and 2xL2-VLP vaccines (with and without VAR2CSA), 8 week old, female BALB/c mice  were immunized with 5 µg . Mice receiving the 5xL2 (n = 25) or 2xL2-VLP (n = 25) vaccines or the corresponding combinatorial vaccines (n = 6 in each group) were immunized in a prime-boost regimen on week 1 and 4  were immunized intramuscularly (thigh muscle) with 3.14 µg antigen dose 1xL2 VLPs  was used to detect the level of anti-VAR2CSA and anti-L2 immunoglobulin (IgG) levels. For VAR2CSA specific IgG titers, 96-well microtiter plates (Nunc MaxiSorp) were coated with 0.1 µg/well recombinant VAR2CSA protein (no SpyTag). The L2 peptide (aa 17–31) of the 16, 18, 31, 35, 45 or 51 HPV types was genetically fused to the N-terminus of an unrelated mycobacterium tuberculosis antigen . For 5xL2- and 2xL2-VLP studies, the recombinant fusion proteins were used as coat protein for all the ELISAs. For the 1xL2-VLP study, ELISA plates were coated with synthetic L2 peptides (aa 17–31) of the HPV16, 18 or 31, through conjugation of the biotinylated peptides to streptavidin as described elsewherePlates were developed with TMB and signal was measured at O.D.450. An O.D. cutoff 0.1 of was used to determine the endpoint titers of PM and HPV specific serum IgG.in vitro parasite binding-inhibition assay was employed to determine the capacity of antibodies induced by the 5x, and 2x-VLP-VAR2CSA vaccine to inhibit the binding between native VAR2CSA expressed on iE and CSA (decorin Sigma). For a detailed description of the assay see33. In short, panning of late-stage Plasmodium falciparum (homologous FCR3 or heterologous NF54 and 7G8 genotypes) parasites selected for a CSA-binding phenotype. CSA-binding parasites were labeled with Tritium and added to CSA-coated wells. Diluted, pooled VAR2CSA immune serum  was added in triplicates. Unbound iE were washed off by a pipetting robot (Beckman-Coulter) and remaining iE were collected onto a filter plate (Perkin-Elmer). Liquid scintillation counting  determined the number of iE not inhibited by the VAR2CSA immune sera. Each assay measured mean iE binding and the percentage of binding iE was calculated by dividing the test result with the mean value of iE in wells incubated without serum.An The assay was repeated three times for FCR3 and 7G8 and twice for NF54. The data points represent the mean percentage parasite binding across the technical repetitions of the assay. Error bars represent the standard deviation of this mean. BSA coated wells and naïve sera were used as a negative control.34.Type 16, 31, 35, 45 and 52 HPV PsV containing a reporter plasmid encoding for both GFP and luciferase were generated using HEK293TT cells as described elsewhereThe infectious unit (IU) count was determined by infecting HEK293TT cells for 48 h and measuring the percentage of infected (GFP-positive) cells by flow cytometry.35.The capacity of anti-L2 antibodies to inhibit infection by the homologous HPV16 PsV as well as heterologous HPV45 PsV was determined for sera obtained from mice with 1xL2-VLP vaccine using a previously described L2-based HPV PsV neutralization assayin vivo PsV genital challenge model, as described previously18. Briefly, Balb/C mice previously immunized with 5xL2-, 2xL2-VLP, or the corresponding combinatorial vaccines, were randomized into challenge groups  starting approximately 3 weeks after the final boost. Five days prior to challenge, mice received 3 mg progesterone. Mice were briefly anesthetized using isoflurane and vaginally challenged with 6.3*106 infectious units (IU) PsV16, 3.15*107 IU PsV31, 8.84*106 IU PsV 35, 1.19*107 IU PsV45 or 6.2*105 IU PsV52. PsV infection was assessed under inhalation anesthesia (isoflurane) 48 hours after challenge by instilling 0.4 mg luciferin vaginally and measuring radiance (five minute exposure) using a Caliper IVIS Lumina II . Background radiance was subtracted from this radiance value in order to determine average radiance (p/s/cm2/sr). Immunized mice were compared to groups of unvaccinated mice .Vaccine induced protection against HPV infection was evaluated in an All animal studies were performed in accordance with Federation of European Laboratory Animal Science Associations (FELASA) and American Association for Laboratory Animal Science (AALAS) guidelines and regulations and were approved by the Danish Animal Experiments Inspectorate  and the University of New Mexico Institutional Animal Care and Use Committee (IACUC), Animal Welfare Assurance # D16–00228 (A3350-01) USDA Registration # 85-R-0014, Protocol No. 16-200498-HSC.GraphPadPrism7.0 was used for calculating geometric mean titers, geometric standard deviation, preparing all graphs and statistical calculations. The non-parametric, two tailed, Mann-Whitney Rank Sum Test was applied in order to determine statistical significance (P < 0.05).Supplementary Dataset 1


The prevalence of HPV vaccine uptake was only 2.6% for bisexual/ homosexual males and 5.0% for heterosexual males. Only 45.8% of the overall subjects knew HPV vaccination is not for females only, with a significantly higher prevalence in females (49.7%) than in males (38.6%). The low prevalence of male HPV vaccine uptake and awareness called for the need to have more male-specific HPV campaigns to promote HPV vaccination awareness and uptake in males to reduce the overall prevalence of HPV infection.To date, empirical studies on HPV vaccine uptake are still limited in Chinese populations and mainly conducted in female cohorts. In order to inform health services planning and health promotion programmes for HPV vaccination, this cross-sectional study aimed to report the prevalence of self-reported HPV vaccination status and to examine gender and sexual orientation differences in the uptake of HPV vaccine in Chinese college students. The overall prevalence of HPV vaccine uptake was 27.6% ( Transmi in 2014 . There i in 2014 . The eco in 2014 .A systematic review conducted in 2010 on HPV vaccine awareness and acceptance among women in the Asia Pacific region found a large variation in HPV vaccine awareness among and within countries . For exaBesides, cultural beliefs and social sigma might also affect the HPV vaccine uptake in Asia. A recent qualitative study on Chinese college students identified several concerns about HPV vaccination . First, Currently, there are three registered HPV vaccines in Hong Kong. 4-valent Gardasil was licensed in Hong Kong in 2006; 2-valent Cervarix in 2008; and 9-valent Gardasil in 2015. Of these, 4-valent Gardasil and 9-valent Gardasil are approved for use in both males and females. Unlike some Western countries such as Australia , HPV vacTo date, empirical studies on HPV vaccine uptake are still limited in Chinese populations. Besides, most Chinese studies about HPV vaccine uptake, and knowledge and beliefs about HPV vaccination were exclusively conducted in females. Furthermore, study findings in mainland China might not be transferable to Hong Kong because HPV vaccines were just approved in mainland China in 2016 and HPV vaccines were gradually available community health centers across 17 provinces in mainland Chinese recently (after ten years of the HPV vaccine being available in developed counties including Hong Kong) . In ordeIt was the secondary analysis of a study about the health needs of emerging adults. The study details have been reported elsewhere .Study subjects were recruited in four major universities in Hong Kong by convenience sampling because we had no access to all students in the selected universities. In order to increase the representativeness of the study sample, we included both government-funded and private universities and recruited subjects in different academic terms (from July 2014 to November 2015).Potential participants were invited to complete a self-administered questionnaire. Questionnaires were available in English and Chinese. Subject were excluded if they refused to join the study or they did not speak or understand Chinese or English.Subjects were asked (i) if they had heard about HPV vaccination and (ii) if they had had HPV vaccination before. To assess their perception on HPV vaccination, they were asked if HPV vaccination is meant for females only. These questions were responded on the options of “true”, “false” and “unsure”.Written informed consent of study participants was obtained. The study protocol was approved by the institutional review board of all participating universities . The authors declare no conflicts of interest.According to the Centers for Disease Control and Prevention (CDC) Advisory Committee on Immunisation Practices, catch-up vaccination should be strongly encouraged for females aged 13 to 26 and male aged 13 to 21 who were not vaccinated previously or have not completed the 3-dose series. Therefore, only males under age 22 and females under age 27 were included in the data analysis. Chi-square test and simple logistic regression were conducted to examine gender and sexual orientation differences in the prevalence of HPV vaccination respectively.n = 582) were female. The mean age was 20.1 (standard deviation: 1.5). 4.5% of subjects (n = 40) were bisexual or homosexual males while 8.5% of subjects (n = 75) were bisexual or homosexual females. 74.9% of subjects (n = 665) were born in Hong Kong while 15.5% of subjects (n = 138) were born in mainland China.888 subjects were eligible to join this study. 65.5% of subjects  had heard of HPV vaccination, we observed a very low overall prevalence of HPV vaccine uptake among males (4.7%), which is much lower than the 22% reported in a US study on men aged 18 to 22 years . BesidesThe low HPV uptake rate in both males and females in Hong Kong can be attributed to the attitude and knowledge of parents because it is usually up to parents to decide whether their child is vaccinated  and thisThe prevalence of HPV vaccine uptake among bisexual/homosexual males was way lower than those reported in previous studies. A study in the US found that 21% of men who have sex with men had received ≥1 dose of HPV vaccine . This siDespite the limitation of the sampling method, the present study can serve the purpose of need assessment which can provide some pilot information for intervention development. Given the low prevalence of HPV vaccination among college students in Hong Kong, there is a need to increase vaccine uptake in this population. There are some possible strategies. First, all students should be screened for HPV vaccination status. For students who do not receive the first dose of HPV vaccine, HPV vaccination decision aid should be provided to them in order to facilitate their decision-making on HPV vaccination. Second, online education materials about HPV infections and HPV vaccination should be readily accessible to college student in order to increase the knowledge and awareness of HPV infections and the vaccines. Third, given the low prevalence of HPV vaccination among male students, especially for MSM, male-specific education materials about HPV infections and HPV vaccination should be developed in order to promote HPV vaccine update among males. A study found that MSM with a recommendation of HPV vaccination were over 40 times more likely to have been vaccinated . Fourth,This is one of the first studies to explore gender and sexual orientation differences in HPV vaccination and awareness among Chinese populations. We found a low prevalence of HPV vaccine uptake among Chinese young males, especially for MSM; and low awareness of male HPV vaccination. Our study findings urge the need to have more health promotions on male HPV vaccination in the public in order to increase the vaccine uptake rate. It is particularly important to MSM who are more susceptible to HPV infections and related adverse health outcome.


More than 25 years ago it was established that a HPV (Human Papilloma Virus) infection was the causal factor for cervical cancer. Based on this discovery HPV vaccines were developed and primary HPV screening proposed. The impact of 10 years prophylactic HPV vaccination with the bivalent and quadrivalent vaccines has been tremendous. There is a reduction of HPV infections 16/18, 31, 33 and 45 of respectively 89%, 94%, 79% and 83%. High grade lesions have been reduced by 85% and warts by 90%. Within 20 to 30 years a reduction in cervical cancer incidence, by 70-80%, is to be expected. The 9 valent HPV vaccine, which was introduced last year and is reimbursed for girls between 12 – 19 years, is expected to increase the figures by 14 to 18%.Recently, doubt has been created regarding primary HPV screening. Since 2017, the annual screening report in Belgium suggests that 15% of the cervical cancers were HPV negative. Previous published data in Belgium (period 2001 - 2008) showed that the number of HPV negative tumors is less than half of the suggested figure (7%). Frequent reasons for false negative HPV tumors are the used HPV testing methods and the misclassification of endometrial cancers or metastasis as cervical cancers. Other explanations are the loss of HPV expression and the existence of cervical cancers independent of HPV. The incidence of HPV negative tumors doesn’t give any information about the performance of primary HPV screening. Data from randomized controlled trials are very clear: if a woman has a normal cytology and no HPV infection or normal cytology and a HPV infection, then her chance of developing a CIN3 + lesion after 5 years is, respectively, 0,2% and 6%. In Belgium, primary HPV screening with dual-stain cytology triage would considerably reduce the incidence (36%) and mortality (40%) of cervical cancer. There is necessity to improve the screening as we are entering an era of vaccinated women who will get screened. Standardized high quality HPV testing is the key stone for improvement. HPV screening preferable with triage markers is superior to cytology, despite the fact that there are HPV negative cancers. The fact that there are HPV negative cancers should not undermine all idea’s regarding primary HPV screening. Cervical cancer remains a major health issue in Belgium with 4070 in situ cancers and 468 invasive cervical cancers in the 2013-2014 period . PrimaryThe primary prevention is the prophylactic HPV vaccination. At present, there are three prophylactic HPV vaccines: Gardasil, Cervarix and Gardasil 9. The school-based vaccination program in the Flemish part of Belgium has a coverage of 91% and in the French Community the coverage is around 36% . Recent We still have to continue cervical cancer screening because not all women are vaccinated and the vaccine does not protect against all types of cervical cancer. Cervical cancer screening should shift from primary cytology to primary HPV screening . IdeallyThe Belgian cancer register looked at all the performed HPV tests before or at the moment of cancer diagnosis . HPV resFrom a test point of view, there are multiple questions regarding this representation. For instance, it is unclear which test was used and if all these analyses were done on a smear or on a tissue sample. It is likely that a mix of different HPV tests was used. Also, it remains unknown if these tests were validated. The figure of 14,7% HPV negative cervical cancer mentioned in the Annual Report (period 2013-2014) should be compared with previous data form our country and worldwide . The firInvasive cervical cancer (ICC) can be divided in three groups. The first group are squamous cervical cancers (SCC) and they account for 75-90% of ICCs . The secThere are several reasons for HPV negative cervical cancers . First oSubtypes with a high prevalence of HPV are the usual type, intestinal, villoglandular, signet-ring cell and the endometrioid ADCs which originate from the cervical squamous columnar junction zone. Together they account for more than 90% of all ADCs. Subtypes with a low HPV prevalence are serous and clear cell ADCs. Due to the rareness of these subtypes only a low number of cases are tested. When comparing the different small studies, you notice that the HPV positivity figure vary, which is confusing. Subtypes which are typically HPV negative (unrelated to HPV) are the gastric type, the mesonephric type and the endometrioid ADC from the upper part of the endocervix and lower uterine segment. These subtypes are however rare. The minimal deviation adenocarcinoma of mucinous type is considered a well-differentiated form of gastric ADC . The incSupport for these HPV independent pathway(s) are the fact that these types have been linked to mutations. In clear cell ADC the PI3K-AKT pathway could be involved as in 50% of cases a positive p-AKT and p-mTOR immunostaining is observed . In oldeAnother group of tumors which can be regarded as false negative, are those tumors that lose HPV. More and more reports are describing the molecular features of cervical cancer . There iTo our knowledge, only one publication links cervical cancer and low risk HPV 6 . WhetherAdditionally, a group of misclassified endometrial cancers (direct extension) and metastasis from other primary tumors which test HPV negative should be considered. Central pathology reviews in major studies has reclassified multiple cancers , obscuring blood or lubricants, inflammation, fixation or cytolysis may result in false negatives. Thus, multiple reasons for questioning the representativeness of HPV testing in currently published studies exist. Some of these studies tested HPV on old (if not very old) stored HPV material, rendering impossible to determine if this material could be representative. A retrospective study showed that tumors from older patients and tumour samples stored for a longer time had a lower HPV prevalence . SamplesIt is very likely that false negative results arise due to way tissues were collected, stored and the used HPV test(s). In order to reduce the number of false negative results, it is necessary to standardize an operating procedure (SOP) for tissue collection and HPV testing. The used HPV tests should be validated and have a very high sensitivity. Special attention to the minimal viral load cutoff should be given, because low level persistent HPV infections exist . LaboratWhen a cervical tumor tests negative for HPV, the clinician should have two considerations. First, “could this be a secondary malignancy, either by direct grow or metastasis?”. In some clinical settings almost 68% of negative cervical cancers appeared to be misdiagnosed primary cervical cancers . Second,The sensitivity in cytology screening ranges between 50 to 70%. This means that 50 to 30% of abnormal lesions are missed. The sensitivity when testing for DNA on high risk HPV in RCTs  is more than 90% , meaningThere is a group of cancers which has a false diagnosis and group of cervical cancers which was missed  due to the test which was used, not representative sample, no endocervical cells, low viral load, or because of a small amount of DNA. Retrospective analyses without surgical staging overestimate the proportion of HPV negative cervical cancers . This nuThere is, however, a very small proportion of mainly rare adenocarcinomas which are HPV negative (true negative). A recent study which used next-generation sequencing to characterize primary cervical cancers, found that only 5% of cervical cancers were HPV-negative . HPV vacPrimary HPV screening with dual-stain cytology in Belgium would reduce the incidence of cervical cancer by 36% and the annual cervical mortality by 40% . We needFinally, the fact that there are HPV negative cancers should not undermine all idea’s regarding primary HPV screening.


Decision-making involves a tradeoff between pressures for caution and urgency. Previous research has investigated how well humans optimize this tradeoff, and mostly concluded that people adopt a sub-optimal strategy that over-emphasizes caution. This emphasis reduces how many decisions can be made in a fixed time, which reduces the “reward rate”. However, the strategy that is optimal depends critically on the timing properties of the experiment design: the slower the rate of decision opportunities, the more cautious the optimal strategy. Previous studies have almost uniformly adopted very fast designs, which favor very urgent decision-making. This raises the possibility that previous findings—that humans adopt strategies that are too cautious—could either be ascribed to human caution, or to the experiments’ design. To test this, we used a slowed-down decision-making task in which the optimal strategy was quite cautious. With this task, and in contrast to previous findings, the average strategy adopted across participants was very close to optimal, with about equally many participants adopting too-cautious as too-urgent strategies. Our findings suggest that task design can play a role in inferences about optimality, and that previous conclusions regarding human sub-optimality are conditional on the task settings. This limits claims about human optimality that can be supported by the available evidence. Substantial effort has been invested in understanding the optimality of perceptual decision-making  than most studies in the literature have used , FDT is the feedback display time common to all trials , and ET is the error timeout added to FDT following incorrect trials .We excluded trials with responses that were considered to be too fast to have come from decision-making processes < 100 ms), or so slow that people likely had lost attention during the trial (> 5000 ms). These exclusions eliminated approximately 0.3% of the data. Reward rate is the expected number of correct decisions per unit time, which can be calculated by Eq. 00 ms, orWe used the diffusion model , and the fourth version constrained the threshold to be lower than the optimal setting . We compared these four models using their marginal likelihoods , which were calculated with the TIDEz algorithm , followed by the too-cautious model , then the too-urgent model , and lastly the optimal model (9965). These findings indicate that (1) despite participants approaching optimality and occasionally being within the optimal region, there is decisive evidence in favor of participants being suboptimal, based on the non-optimal models all beating the optimal model, (2) participants seem to be more on the too-cautious side than the too-urgent side, as the too-cautious model was preferred over the too-urgent model, and (3) the preference for too-cautious responding may have been inconsistent over participants, as the completely free threshold was preferred over both the too-cautious and too-urgent models. This last conclusion is important, as it suggests that people were not, on average, too cautious.on average optimal, or very close to optimal, but the model comparisons suggest that free  threshold estimates are important. To investigate this more carefully, we performed the same analyses as above, but for each individual subject. The results for the first analysis—comparing estimated thresholds against the optimal threshold, across blocks of the experiment—are summarized in Fig. Taken together, the initial analyses and the model comparison analyses suggest an interesting pattern: Fig. n = 24), too-urgent (n = 24), and optimal (n = 21) models. The remaining 13% of participants (n = 10) were best fit by the freely-varying model.The second analysis compared the four model versions separately for each participant. Around 30% of the participants were best described by each of the too-cautious , and so generalizations about overly cautious strategies may not be warranted. Results reported by Simen et al., , who manurgent than optimal strategies  adopting more Lastly, with our task we attempted to make the optimal strategy very cautious. This resulted in participants’ strategies being centered around optimal. On the other hand, earlier experiments have used tasks that make the optimal strategy very urgent, and these have resulted in participants’ strategies centered around overly cautious responding. The combination of these findings may suggest that although it is easy to push participants to being overly cautious with task design, it is much harder to make them overly urgent. An alternative explanation is that participants have a preferred speed–accuracy tradeoff policy  and it is difficult to push them far from this strategy. It will require future research investigating other manipulations (or tougher criteria) to disentangle these accounts. In addition, future research should also explore other experimental factors that may influence participant strategy, such as the task instructions provided.


There are no HPV‐based measures for managing anal cancer (AC) in HIV‐infected (HIV+) men who have sex with men (MSM) because of the high positivity of high‐risk (HR)‐HPVs. As next‐generation sequencing (NGS) is able to describe the composition of HPVs as percent (%) reads rather than positive vs negative results, we used NGS approach to detect HPVs in anal samples of HIV+ MSM to test its ability to differentiate those who are diagnosed with atypical squamous cells of unknown significance or greater (ASCUS+) from those who are free of such lesions and to understand the burden of HPV infections in relation to HPV vaccines.Study included 81 HIV+ MSM characterized for demographics, patient‐reported outcome measures, HIV related laboratory measures and anal cytology. We summarized NGS HPV data using % read cut points (>0%‐>30%) and tested the relationship between % reads of HR‐HPVs and risk of ASCUS+ using logistic regression.P = .033).Forty‐six HPVs were detected at the >0% read cut point. The prevalence of any HR‐HPVs varied from 100% to 40% with >0% to >30% reads while ≥99% were infected with HR‐HPVs included or not included in the 9 valent HPV vaccine at the >0% read cut point. MSM with >30% HR‐HPV reads were 4.5 times more likely to be diagnosed with ASCUS+ compared to ≤30% reads (NGS‐based approach is more accurate than PCR‐based HPV testing for identifying HIV+ MSM at risk for developing AC. We raise the concern regarding the efficacy of current HPV vaccines for preventing AC in this high‐risk population. Next‐generation sequencing‐based approach is more accurate than PCR‐based HPV testing for identifying HIV+ MSM at risk for developing anal cancer. We raise the concern regarding the efficacy of current HPV vaccines for preventing anal cancer in this high‐risk population. In order to address these gaps in knowledge, we used a NGS approach to explore HPV genotypes present in anal samples of HIV+ MSM with any of the following cytologic findings: 1) negative for intraepithelial lesions or malignancy (NILM), 2) atypical squamous cells of unknown significance (ASCUS), 3) low‐grade squamous intraepithelial lesions (LSIL) and 4) high‐grade squamous intraepithelial lesions (HSIL).22.1Study included 81 HIV+ MSM with a history of anal receptive intercourse who were seen at the UAB's 1917 HIV Outpatient Clinic in Birmingham, AL. The clinic provides comprehensive core medical and social services to adult PLWH. All patients were characterized for demographics, patient‐reported outcome measures (PROs),® solution by agitating the swab in the solution 10 times and pushing it against the PreservCyt vial wall to further release cells. Vial caps were tightened so the torque line on the cap passes the torque line on the vial. These vials were labeled at the bedside and transported to the UAB cytopathology laboratory for cytological testing. Immediately after the cytology tests were completed, all samples were delivered to the laboratory of Dr Piyathilake for processing and storage for HPV NGS assay. The anal cytological diagnoses were obtained from the UAB patient database . The study protocol and procedures were approved by the UAB Institutional Review Board.A medical provider (physician or nurse practitioner) collected anal samples using clinic standard collection protocols. Briefly, cells were collected using a water‐moistened, synthetic‐fiber swab with a non‐scored stick. A swab was inserted into the anal canal past the dentate line until it abuts the distal rectal wall. Cells were harvested by using a circular motion as the swab was retracted while applying firm lateral pressure to sample the epithelium in the mucosal folds of the anal canal. Swabs were rinsed in Thin Prep Pap Test2.22.2.1DNA was isolated using the fecal DNA isolation kit from Zymo Research following the manufacturer's instructions. DNA concentrations/quality was determined based on 260/280 ratio using a nanodrop 2000 spectrophotometer. DNA with a 260/280 ratio of 1.7‐2.0 was considered satisfactory.2.2.2To ensure the quality of results generated from the proposed study, the HPV sequencing assay described below included two positive control DNA specimens (Hela Cell DNA/HPV 18 and Caski/HPV 16) in each PCR run. Sequencing results of >98% reads for each HPV genotype was required to accept the sequencing results for patient specimens in a given PCR run.https://pave.niaid.nih.gov (PaVE database). Megablast was used to search each read against the database and top hits with specified given identity cutoff of 90% match and coverage <70% were identified. The proportion of hits for a given sample described the composition of specific HPV genotypes as % reads for each genotype in a given sample that adds to 100% with a certain percentage of sequences that did not match with sequences in the PAVE database. Figure Analysis of various HPV sequences was accomplished with deep sequencing of 20 ng of DNA isolated from each anal sample. Methods followed those described previously in the human papillomavirus laboratory manual from the WHO2.3The differences in the characteristics of MSM diagnosed with NILM or ASCUS+ were tested using Pearson chi square test. The prevalence of HPV genotypes was summarized using the following % read cut points; >0%,>5%, >10%,>20% and >30%. Because of the universal presence of HR‐HPVs at ≤5% reads, to test the association between HR‐HPV genotypes and ASCUS+, we created the HR‐HPV variable by averaging the % reads of all HR‐HPVs with >5% reads in a sample. Afterward, the relationship between % reads of HR‐HPVs  and the risk of being diagnosed with ASCUS+ was tested using unconditional logistic regression models after adjusting for age, race, body mass index (BMI), smoking status, lifetime number of sexual partners and improvement in CD4  or the CD4 count and viral load at time of diagnosis/HPV testing.3P = .0269). Approaching significance (P = .0731), a higher percentage of MSM (82%) diagnosed with NILM had undetectable HIV viral loads (<20 copies/mL) compared to MSM diagnosed with ASCUS+ (61%). No other variables were significantly different between MSM diagnosed with NILM and those diagnosed with ASCUS+.Table As shown in the Table P = .033). MSM ≥ 32 years of age were 4.3 times more likely to be diagnosed with ASCUS+ compared to < 32 years (P = .041). MSM who did not achieve an improvement in CD4 levels on cART >500 were 8.3 times more likely to be diagnosed with ASCUS+ compared to those who improved >500 (P = .006). The association between HR‐HPV and risk of being diagnosed with ASCUS+ remained similar when we replaced the improvement in CD4 with either CD4 or viral load at the time of diagnosis/HPV testing , respectively. However, we did not observe a statistically significant association between CD4 or viral load at the time of diagnosis/HPV testing and risk of being diagnosed with ASCUS+ .There was a stepwise increase in the odds of being diagnosed with any abnormal anal cytology from >10% reads (OR = 1.70) to >30% reads of HR‐HPVs OR = 4.53)  and multiplex PCR reported that NGS is more sensitive and has the ability to detect multiple infections compared to traditional methods and, therefore, may have the potential for use as an alternative method for HPV genotyping and diagnosis of lesions.The most prevalent HR‐HPV genotype detected in our study was HPV 16 at all cut points, including the highest cut point, >30% cut point which was significantly associated with the risk of ASCUS+. Other HPV types with higher prevalence in general were HPV 18 followed by HPV 52, 35, 39 and 45. In contrast, a study that detected HPVs using 454 NGS and reported results based on the total number of HPV reads in anal specimens of HIV+ MSM in Mexico, documented that HPV 45 was the most prevalent followed by HPV 51, 58, 16, 52 and 59.Our observation that the prevalence of known HR‐HPVs in HIV+ MSM varies from 100%  to 40% with the lowest % read cut point to the highest cut point and there was a stepwise increase in the odds of being diagnosed with any abnormal anal cytology from low to high % reads indicated that NGS‐based HPV testing is likely to be more accurate than PCR‐based HPV testing to identify HIV+ MSM at risk for lesions that may progress to AC. This would allow for targeted referral of HIV+ MSM for high‐resolution anoscopy (HRA) in a cost effective way. A previous study conducted among HIV negative (HIV‐) MSM documented that having > 5 anal sex partners and PCR evidence of an anal HPV infection but not age or smoking status were significantly associated with any abnormal anal cytology diagnoses.With regard to the primary prevention of AC, HPV vaccination has been recommended in some countries for MSM under 45 years of ageOur study has several strengths including a population representative of the HIV+ MSM receiving care in the Deep South, which is disproportionately impacted by the HIV epidemic in the United States; a good assessment of anal cytology/risk factors and state of the art HPV testing. Even though NGS data may be relatively complex to analyze, it may serve as a cost effective and sensitive HPV genotyping method because of its highly sensitive detection capability of multiple HPV genotypes and the ability to associate HPV risk based on HPV sequence % reads, allowing for a accurate measure rather than a less accurate nominal “positive vs negative” measure for a given HPV genotype. However, discovery of simple biomarkers of HPV carcinogenesis that are strongly related to NGS HPV results are needed to make use of this approach effectively in clinical settings. Our results will form the foundation for discovery and validation of such biomarkers.Limitations of the study are the cross‐sectional study design which did not allow us to examine the temporal association between exposure to varying % reads of HPV sequences and risk of developing ASCUS+ and lack of histological diagnoses of lesions since only a few patients were referred for HRA directed biopsy in accordance with the current 1917 Clinic protocol. Since the ASCUS and LSIL cytology diagnoses are likely to be upgraded to higher grades on biopsy,In conclusion, we report that our NGS‐based approach is more accurate than PCR‐based HPV testing to identify HIV+ MSM at risk for developing AC as MSM with >30% HR‐HPV reads were 4.5 times more likely to be diagnosed with ASCUS+ compared to ≤30% reads. We also raise the concern regarding the efficacy of currently available HPV vaccines for preventing AC in this high risk population as HIV+ MSM are universally exposed to HR‐HPV genotypes that are included or not included in such vaccines. However, our results should not be interpreted in a way to discourage vaccination of HIV+ with currently available HPV vaccines. Replication of all our findings in other HIV populations is needed to increase the scientific credibility of these observations.Formulation of research goals and aims‐CJP. Generation of data and verification‐MRC, SB, CJP, GAB, JLR. Data analysis and interpretation‐RK, SB, CJP. Writing original draft and presentation of results‐CJP, SB. Review and editing of the final manuscript‐CJP, SB, RK, MRC, GAB, JLR. Click here for additional data file. Click here for additional data file.


Although head and neck squamous cell carcinoma (HNSCC) has in the past been largely associated with tobacco use, human papillomavirus (HPV+) oropharynx cancer has in recent years emerged as the fastest growing type of HNSCC. Patients with HPV+ HNSCC have a better prognosis; however, the 5-year survival for both HPV+ and HPV− subtypes with recurrent or metastatic disease is poor. To gain insights into the tumor microenvironments of both HNSCC subtypes and identify potential therapeutic targets, we performed epigenomic deconvolution on 580 HNSCC samples from the TCGA dataset. Deconvolution revealed distinct molecular and histoepigenetic profiles of the two tumor subtypes, including their cellular composition, epigenomic profiles and gene expression for constituent cell types, and potential cancer cell-specific targets. Our analyses show that high abundance of both CD8 T-cells and B-cells explains better prognosis in HPV+ HNSCC. Deconvolution of gene expression profiles revealed higher expression of the immunotherapy target PD-1 in HPV+ immune cells compared to HPV− cells, suggesting that HPV+ tumors may preferentially benefit from PD-1 targeted therapy. Further analyses identified HPV+ and HPV− cancer cell surface proteins that can also serve as potential targets for therapy. Specifically, Wnt pathway receptor ROR2 is preferentially overexpressed in HPV+ subtypes, suggesting opportunities for development of targeted therapy based on HPV status. In summary, the comprehensive molecular and histoepigenetic analysis of tumor microenvironments by epigenomic deconvolution reveals potential novel biomarkers and targets for precision therapy of HNSCC. Head and Neck Squamous Cell Carcinoma (HNSCC) arises from the squamous epithelial cells in the mucosal lining of the oral cavity . The annTargeted therapy has in the past few decades become an established approach for cancer treatment . MonocloComprehensive molecular profiling of HPV+ and HPV− HNSCC tumors revealed distinct molecular etiologies, with a high percentage of HPV− tumors carrying TP53 mutations, while a high percentage of HPV+ tumors showing overexpression of p16INK4a , 12. MosTo help develop precision therapies for HPV+ and HPV− HNSCC and to elucidate the factors that affect their prognosis, we set out to identify differences and similarities in HPV+ and HPV− HNSCC tumors at the molecular, cellular and microenvironment levels. We also identify potential new biomarkers or therapy targets. One of our target groups are cell surface proteins, which represent a group of genes widely used to develop targeted therapies –17 and iIdentification of therapy targets in tumors represents a challenge due to the presence of different cell types in the tumor microenvironment. Previous studies have attempted to look for targets in HPV+ and HPV− HNSCC without taking into consideration the complexity of the cell type composition of tumors . These sStage 1 of the EDec method Fig.  revealedr = 0.72 level  and stromal/normal epithelial proportions at r = 0.73 level   from Emory University. CDSA includes 255 HNSCC samples from the TCGA collection and provides proportions of stromal, epithelial, and cancer cells for each tumor biopsy . Since t27) Fig. . This leAnalysis of cell type proportions for 72 HPV+ HNSCC, 243 HPV− HNSCC and 50 adjacent normal samples shows that, as expected, the 50 normal samples have higher proportion of stromal/epithelial cells and negligible proportions of cancer cells compared to the tumor samples. Deconvolution models the cancer fraction by three distinct cancer cell types, one corresponding to HPV+ HNSCC, another to HPV− HNSCC and one present in both Fig. . The denTo further explore the epigenetic differences between the HPV+ and HPV− subtypes, we performed differential methylation analysis. We first identified differentially methylated genes using the RnBeads package  and thenThe set of genes with hypermethylated promoters showed enrichment for pathways involved in cancer, such as receptor signaling pathways and histone modifications. We also observed enrichment for genes targeted by the Polycomb proteins SUZ12 and EED, which are known to be repressed in histologically poorly differentiated tumors  Supplem. The setPrevious studies have shown that HPV+ has higher T-cell infiltration than HPV− HNSCC, however they have not compared tumor profiles with those of normal samples . Our anap-value = 0.014, 95% CI 0.249–0.857, and HR = 0.45, p-value = 0.010, 95% CI 0.248–0.828, respectively)  Fig.  and are p-value = 1.82E−01 and p-value = 0.01, respectively)  . Moreover, we observed the same survival pattern when comparing tumors with high vs low abundance of B-lineage cells   abundance of CD8− T-cells showed better survival than patients with low (bottom 25%) abundance of CD8 T-cells (55) Fig. .Since HPV+ HNSCC is more common in lymphoid-rich regions , we asked if the abundance of CD8 T-cells and B-cells in HPV+ HNSCC might be a result of confounding due to the tendency of HPV+ HNSCC to occur in those regions. Toward this goal, we compared the immune cell-type proportion differences between HPV+ and HPV− HNSCC tumors present only in these lymphoid-rich regions. HPV+ HNSCC tumors showed a significantly higher abundance of CD8 T-cells and B-cells compared to HPV− HNSCC  , consistp-value = 2.98E–07) and HPV− HNSCC . In contrast, PD-1 (PDCD1) is significantly overexpressed only in HPV+ tumors . This finding is interesting in light of the clinical trials targeting PD-1 in HNSCC (regardless of HPV status) that show that only a subset of patients show response [We next compared the gene expression profiles (deconvoluted by EDec) of the immune fraction in HPV+ and HPV− HNSCC. As expected, marker immune genes for T-cells and B-cells were more highly expressed in HPV+ HNSCC tumors compared to HPV− tumors Fig. . Interesresponse . Our resresponse . In contTo gain further insights into the immunological differences between HPV+ and HPV− tumors, we next compared the gene expression profiles in the immune compartment estimated by EDec between HPV+, HPV− and normal samples. We performed gene set enrichment analysis  on the iBecause of their accessibility, cancer cell surface proteins are potential targets for therapy using both antibodies and small molecules. We therefore analyzed the expression of cell surface proteins for HPV+ and HPV− subtypes of HNSCC by estimating the gene expression profiles of cancer cells (EDec stage 2) using the normalized TCGA HNSCC RNA-seq data. Toward this goal, we combined the three cancer epithelial estimated proportions from EDec stage 1. EDec stage 2 was independently applied to 72 HPV+ and 243 HPV− HNSCC tumors and 20 normal tissue controls. EDec predicted overexpression  of 439 genes in HPV+, 449 in HPV− and 163 genes in both HPV+ and HPV− cancer cells Fig. –c. To idWe next performed gene set enrichment analysis on the cell surface genes preferentially overexpressed in HPV+ or HPV− or both HNSCC subtypes. Cell surface genes in HPV− tumors were enriched for Nicotine pathway, consistent with the role of tobacco consumption in HPV− HNSCC. Another enriched pathway was cell–cell junction, implicated in the process of epithelial to mesenchymal transition (EMT), leading to invasion and metastasis . InteresIn HPV+ HNSCC we observed enrichment of cell adhesion and integrin pathways, which have previously been implicated in metastasis , 43, conIn order to select candidates for validation, we screened the cell surface proteins based on their subcellular localization, expression level in normal tissue according to the GTEx portal from the Broad Institute , literatTranscription of the cancer-testis antigen LY6K was validated in several HPV+ and HPV− HNSCC cell lines Fig. . LY6K-T3We next evaluated protein expression of LY6K by Western blot analysis. LY6K showed higher expression in HPV− when compared to HPV+ HNSCC Fig.  but showExpression of candidates CELSR3 and ROR2 was examined using qRT-PCR in HNSCC cell lines listed in Table We could not validate protein expression of ICAM-1, VCAM-1, and PTGIR . We observed that a five cell-type model showed the best reproducibility of methylation and proportion estimates.Gene set enrichment analysis was performed using GSEA . To perft-tests comparing the means of expression in HPV+ vs normal samples, and HPV− vs normal samples. The threshold to determine differential expression was FDR < 0.1, fold change of gene expression cancer vs normal samples >3. To identify subtype-specific overexpressed proteins we performed a t-test comparing the means of gene expression in HPV+ vs HPV−, using a fold change of gene expression >1.5 and FDR < 0.1.To estimate gene expression profiles of constituent cell types, we applied EDec stage 2 to the TCGA RNA-seq profiles and cell type proportions (estimated in stage 1). We applied the method independently to the 72 HPV+, 243 HPV− and 20 normal samples that have both DNA methylation data and RNA-seq profiles. To identify genes that are preferentially overexpressed in HPV+ and in HPV−, we performed To identify the cell surface proteins, we downloaded the Cell Surface Atlas . Gene sep-value < 0.05) and an immune gene is differentially expressed  between HPV+ and HPV−, t-test was performed between HPV+ vs normal samples, HPV− vs normal samples. To detect subtype specific overexpressed genes and subtype-specific immune cell-type abundance, a t-test was performed between HPV+ vs HPV−.To determine the abundance of constituent cell types of the immune compartment, we applied the previously described method MCP counter  to the TKaplan–Meier and Cox regression survival analyses were performed in R using the packages ‘survminer’ and ‘survival’. Gene set enrichment analysis was performed using GSEA  on the gAll our statistical analyses were performed using the R programming language and Bioconductor packages. The pairwise correlation analyses were performed with the two sided, Pearson method with a confidence level of 0.95. The Kaplan–Meier survival analyses were performed with the R package ‘survminer’, using the top and bottom 25% quartiles of CD8 T-cells and B-cells abundances. The multivariate Cox regression was performed with the R package ‘survival’, using the top and bottom 25% quartiles of abundance for CD8 T-cells and B-cells, we also included as factors the following values: HPV status, age of diagnosis, and cancer stage. The default method ‘Efron’ was used. The collinearity test was performed with the R package ‘car’ to calculate the variance inflation factors (VIF). The generalized linear model was performed using the family binomial (logit).2 in humidified incubator.The HPV− HNSCC cell lines DHEP3 , MDA1386TU, MDA1586, HN30, MDA686TU  were grown in DMEM (Sigma-Aldrich), 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptamycin, 1% non-essential amino acids, 1% sodium pyruvate and vitamin. The HPV+ HNSCC cell lines UM-SCC47, UPCI:SCC090, UPCI:SCC154, and UPCI:SCC152  are cultured in MEM with 10% FBS, non-essential amino acids and gentamicin. They were maintained at 37 °C with 5% COTotal RNA was extracted using TRIzol reagent (Invitrogen) and the concentration and the purity of RNA were measured by Cytation 3 (BioTek). cDNA was synthesized from 1 µg total RNA using qScript cDNA supermix (Quanta). The gene-specific PCR products were generated with PerfeCta SYBR Green FastMix (Quanta) and reactions carried out in a CFX96 real-time PCR machine (Bio-Rad). Primer sets for the candidate genes are listed in Supplementary Table Cell pellets were washed with ice-cold PBS twice then scraped from the T75 flask. Cell pellets were lysed in RIPA buffer (Biosciences), phenylmethanesulfonyl fluoride (PMSF), and cocktail inhibitor 2 (Sigma). Then the proteins (30 µg) were resolved by 4–12% SDS-PAGE pre-cast gels (Invitrogen) and subsequently electrophoretically transferred to PVDF membranes. Membranes were incubated with 3% BSA of blocking buffer for 1 h at room temperature then incubated with primary antibodies at 4 °C overnight. Membranes were incubated with corresponding species of secondary antibodies for 1 h at room temperature, followed by the detection with the enhanced chemilumiescence (ECL) system (Thermoscientific Fisher) and visualized by ChemiDoc (Bio-Rad). The relative densities of bands were quantified using ImageJ software (NIH). Sources of antibodies were as follows: anti-LY6K , anti-CELSR3 , anti-β-actin, anti-ICAM-1, anti-VCAM-1, anti-PTGIR , anti-ROR2 .All the authors of this manuscript state the material is original research, has not been previously published and has not been submitted for publication elsewhere while under consideration.Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Table 1aSupplementary Table 1bSupplementary Table 1cSupplementary Table 1dSupplementary Table 2Supplementary Table 3aSupplementary Table 3bSupplementary Table 3cSupplementary Table 4aSupplementary Table 4bSupplementary Table 4cSupplementary Table 5aSupplementary Table 5bSupplementary Table 5cSupplementary Table 6aSupplementary Table 6bSupplementary Table 6cSupplementary Table 7Supplementary Table 8