Detection of stage I HPV-driven oropharyngeal cancer in asymptomatic individuals in the Hamburg City Health Study using HPV16 E6 serology - A proof-of-concept study.

Background: The lack of detectable precancerous lesions poses challenges to the early detection of human papillomavirus-driven oropharyngeal cancer (HPV-OPC). Antibodies against HPV16 early proteins, especially E6, are uniquely sensitive and specific biomarkers detectable years prior to HPV-OPC diagnosis. Thus, HPV16 early protein serology warrants clinical investigation for HPV-OPC screening. Methods: Using multiplex serology, we analyzed HPV16 serum antibodies of the first 5000 participants (n=4,424 sera, recruited 2016-2017) of the Hamburg City Health Study, a population-based prospective cohort (45-74 years). Participants seropositive for HPV16 E6 and at least one additional early protein (E1, E2, E7) were considered at high risk for HPV-OPC development and invited to six-monthly non-invasive head and neck follow-up (FU) examinations (visual inspection, endoscopy, ultrasonography, performed 2019-2020). Participants with suspicious lesions were examined by magnetic resonance imaging and panendoscopy with biopsy. Histologically confirmed OPC cases were treated according to standard of care. Findings: In total, 35 out of 4,424 study participants (0·8%, 95% confidence interval (CI) 0·6-1·1%) were HPV16 E6 seropositive. Among these, eleven (0·3%, 95%CI 0·1-0·5%) were considered at high risk for HPV-OPC of which nine were successfully re-contacted and invited to regular clinical FU examinations. Two males and one female were diagnosed with stage I HPV-OPC within 1·3 years of clinical FU (3-4 years after initial blood draw), representing one diagnosis of prevalent advanced disease, one incident diagnosis of advanced disease, and one incident diagnosis of early disease. The remaining participants showed no detectable signs of cancer, and undergo regular examinations (median clinical FU: 1·0 years, median total FU from blood draw to last clinical FU visit: 4·7 years). Interpretation: HPV16 early antibodies allowed identifying three asymptomatic stage I HPV-OPC patients, out of eleven participants considered at high risk. However, two of the three cases already showed signs of advanced disease at diagnosis. Targeting multiple early proteins may considerably improve the positive predictive value of HPV16 serology and may have clinical utility for HPV-OPC screening. Funding: This work was funded by DKFZ and UKE intramural funding.

Accuracy of high-risk HPV DNA PCR, p16(INK4a) immunohistochemistry or the combination of both to diagnose HPV-driven oropharyngeal cancer.

BACKGROUND: The incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC. METHODS: The accuracy of three possible test strategies, i.e. (a) hrHPV DNA PCR (DNA), (b) p16(INK4a) immunohistochemistry (IHC) (p16), and (c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. RESULTS: Ninety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16 + DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16 + DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. CONCLUSIONS: Single hrHPV DNA PCR and p16(INK4a) IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Disease prognostication can be encouraged based on this combined test result.

Concordance of p16INK4a and E6*I mRNA among HPV-DNA-Positive Oropharyngeal, Laryngeal, and Oral Cavity Carcinomas from the ICO International Study.

Background: Tests or test algorithms for diagnosing HPV-driven oral cavity and laryngeal head and neck carcinomas (HNC) have not been yet validated, and the differences among oral cavity and laryngeal sites have not been comprehensively evaluated. We aimed to assess the utility of a diagnostic algorithm for the detection of HPV-driven oral cavity (OCC), oropharyngeal (OPC) and laryngeal (LC) carcinomas using HPV-DNA testing followed by p16INK4a immunohistochemistry, taking E6*I mRNA detection as the reference standard. Methods: Formalin-fixed paraffin-embedded OCC, OPC, and LC carcinomas were collected from pathology archives in 29 countries. All samples were subjected to histopathological evaluation, DNA quality control, and HPV-DNA detection. All HPV-DNA-positive samples (including 78 OCC, 257 OPC, and 51 LC out of 3680 HNC with valid HPV-DNA results) were also tested for p16INK4a immunohistochemistry and E6*I mRNA. Three different cutoffs of nuclear and cytoplasmic staining were evaluated for p16INK4a: (a) >25%, (b) >50%, and (c) ≥70%. The concordance of p16INK4a and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was assessed. Results: A total of 78 OCC, 257 OPC, and 51 LC were HPV-DNA-positive and further tested for p16INK4a and E6*I mRNA. The percentage of concordance between p16INK4a (cutoff ≥ 70%) and E6*I mRNA among HPV-DNA-positive OCC, OPC, and LC cases was 79.5% (95% CI 69.9−89.1%), 82.1% (95% CI 77.2−87.0%), and 56.9% (95% CI 42.3−71.4%), respectively. A p16INK4a cutoff of >50% improved the concordance although the improvement was not statistically significant. For most anatomical locations and p16INK4a cutoffs, the percentage of discordant cases was higher for HPV16- than HPV-non16-positive cases. Conclusions: The diagnostic algorithm of HPV-DNA testing followed by p16INK4a immunohistochemistry might be helpful in the diagnosis of HPV-driven OCC and OPC, but not LC. A different p16INK4a expression pattern was observed in those cases HPV-DNA-positive for types other than HPV16, as compared to HPV16-positive cases. Our study provides new insights into the use HPV-DNA, p16INK4a, and HPV-E6*I mRNA for diagnosing an HPV-driven HNC, including the optimal HPV test or p16INK4a cutoffs to be used. More studies are warranted to clarify the role of p16INK4a and HPV status in both OPC and non-OPC HNC.

Absolute Risk of Oropharyngeal Cancer After an HPV16-E6 Serology Test and Potential Implications for Screening: Results From the Human Papillomavirus Cancer Cohort Consortium.

PURPOSE: Seropositivity for the HPV16-E6 oncoprotein is a promising marker for early detection of oropharyngeal cancer (OPC), but the absolute risk of OPC after a positive or negative test is unknown. METHODS: We constructed an OPC risk prediction model that integrates (1) relative odds of OPC for HPV16-E6 serostatus and cigarette smoking from the human papillomavirus (HPV) Cancer Cohort Consortium (HPVC3), (2) US population risk factor data from the National Health Interview Survey, and (3) US sex-specific population rates of OPC and mortality. RESULTS: The nine HPVC3 cohorts included 365 participants with OPC with up to 10 years between blood draw and diagnosis and 5,794 controls. The estimated 10-year OPC risk for HPV16-E6 seropositive males at age 50 years was 17.4% (95% CI, 12.4 to 28.6) and at age 60 years was 27.1% (95% CI, 19.2 to 45.4). Corresponding 5-year risk estimates were 7.3% and 14.4%, respectively. For HPV16-E6 seropositive females, 10-year risk estimates were 3.6% (95% CI, 2.5 to 5.9) at age 50 years and 5.5% (95% CI, 3.8 to 9.2) at age 60 years and 5-year risk estimates were 1.5% and 2.7%, respectively. Over 30 years, after a seropositive result at age 50 years, an estimated 49.9% of males and 13.3% of females would develop OPC. By contrast, 10-year risks among HPV16-E6 seronegative people were very low, ranging from 0.01% to 0.25% depending on age, sex, and smoking status. CONCLUSION: We estimate that a substantial proportion of HPV16-E6 seropositive individuals will develop OPC, with 10-year risks of 17%-27% for males and 4%-6% for females age 50-60 years in the United States. This high level of risk may warrant periodic, minimally invasive surveillance after a positive HPV16-E6 serology test, particularly for males in high-incidence regions. However, an appropriate clinical protocol for surveillance remains to be established.

Prevalence of Transcriptionally Active HPV Infection in Tumor-Free Oropharyngeal Tissue of OPSCC-Patients.

Objectives: The natural history of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is still largely unknown. Since reports of second primary tumors (SPTs) in patients with HPV-related OPSCCs are increasing, a multifocal HPV infection, hinting a «virus-induced field effect¼, has been hypothesized. This study aimed to investigate the HPV-prevalence in normal appearing oropharyngeal tissue in patients with OPSCCs. Materials and Methods: 49 OPSCC patients undergoing panendoscopy were prospectively enrolled. Tumor specimens and biopsies of normal appearing oropharyngeal tissue adjacent to and distant from the index OPSCC underwent histopathological examination, p16INK4A immunohistochemical staining, HPV DNA and mRNA-detection. Patient characteristics and follow-up data on SPTs were obtained. Results: 26 of 49 (53%) OPSCC were positive for HPV DNA and p16INK4A. HPV mRNA was detected in 23 of 26 (88%) of these tumor samples. HPV DNA was detected in 36% adjacent mucosa and in 17% distant mucosa samples and only in patients with an HPV-related index OPSCC. HPV mRNA could not be detected in tumor-free distant and adjacent mucosa samples. No evidence of association between HPV detection in normal appearing mucosa and development of second primary tumors was found. Conclusions: HPV was detectable but not transcriptionally active in adjacent/distant tumor-free oropharyngeal tissue. This suggests that a multifocal HPV infection, hinting a «virus-induced fielcd cancerization¼, may not be pertaining to HPV-related OPSCC.

Human cytomegalovirus alters immune cell profile with potential implications for patient survival in head and neck cancer.

Cytomegalovirus (CMV) is a highly prevalent human herpes virus that exerts a strong influence on immune repertoire which may influence cancer risk. We have tested whether CMV immunoglobulin G (IgG) serostatus is associated with immune cell proportions (n = 132 population controls), human papillomavirus (HPV) co-infection and head and neck cancer risk (n = 184 cancer cases and 188 controls) and patient survival. CMV status was not associated with the proportion of Natural Killer cells, B cells or the neutrophil-to-lymphocyte ratio. However, CD8+ T cells increased with increasing categories of IgG titers (P =1.7 × 10-10), and titers were inversely associated with the CD4:CD8 ratio (P = 5.6 × 10-5). Despite these differences in T cell proportions, CMV was not associated with HPV16 co-infection. CMV seropositivity was similar in cases (52%) and controls (47%) and was not associated with patient survival (hazard ratio [HR] 1.14, 95% confidence interval [CI]: 0.70 to 1.86). However, those patients with the highest titers had the worst survival (HR 1.91, 95% CI: 1.13 to 3.23). Tumor-based data from The Cancer Genome Atlas demonstrated that the presence of CMV transcripts was associated with worse patient survival (HR 1.79, 95% CI: 0.96 to 2.78). These findings confirm that a history of CMV infection alters T cell proportions, but this does not translate to HPV16 co-infection or head and neck cancer risk. Our data suggest that high titers and active CMV virus in the tumor environment may confer worse survival.

Maternal HPV-antibodies and seroconversion to HPV in children during the first 3 years of life.

To assess the dynamics of human papillomavirus (HPV) serology, we analyzed HPV6-,11-,16-,18-, and 45 antibodies in infants during the first 36 months of their life. Serial serum samples of 276/327 mother-child pairs were collected at baseline (mothers) and at months 1, 2, 6, 12, 24 and 36 (offspring), and tested for HPVL1-antibodies using the GST-L1 assay. Concordance between maternal and infant HPV-antibody levels remained high until month-6 (p < = 0.001), indicating maternal antibody transfer. At 1 month, 40-62% of the infants tested seropositive to any of the 5 HPV-types. Between 1-3 years of age, 53% (58/109) of the children born to HPV-seronegative mothers tested HPV-seropositive. Times to positive seroconversion varied between13.4 and 18.7 months, and times to negative seroconversion (decay) between 8.5 and 9.9 months. Significant independent predictors of infants' seroconversion to LR-HPV were hand warts and mother's history of oral warts and seroconversion to LR-HPV. No predictors of seroconversion to HR-HPV were identified. Maternal HPV-IgG-antibodies are transferred to her offspring and remain detectable for 6 months, corroborating the IgG molecule's half-life. Seroconversion to HPV-genotypes 6, 11, 16 and 18 was confirmed among children born to HPV-seronegative mothers, implicating an immune response to these HPV-genotypes during early infancy.

DNA methylation-derived systemic inflammation indices and their association with oropharyngeal cancer risk and survival.

BACKGROUND: Head and neck squamous cell carcinomas are associated with systemic inflammation (SI). We evaluated whether DNA methylation-derived SI (mdSI) indices are associated with oropharyngeal cancer risk and survival. METHODS: Ninety-four oropharyngeal squamous cell carcinoma (OPSCC) cases and 57 controls with DNA methylation data were included. Logistic regression analysis and survival analysis were performed to test the association of mdSI indices with OPSCC risk and survival. RESULTS: Higher methylation-derived neutrophil-to-lymphocyte ratio (mdNLR) was associated with increased risk of OPSCC (OR = 1.21, 95%CI: 1.11-1.40) while no association was found with methylation-derived lymphocyte-to-monocyte ratio (mdLMR). For 5-year overall survival, higher mdLMR was significantly associated with decreased risk of death (HR = 0.25, 95%CI: 0.10-0.64) while the converse was observed for mdNLR (HR = 2.48, 95%CI: 1.04-5.92). CONCLUSION: We observed an association between mdSI indices and OPSCC risk and 5-year overall survival. It is possible to use mdLMR as an independent prognostic factor for OPSCC.

Seroprevalence of mucosal and cutaneous human papillomavirus (HPV) types among children and adolescents in the general population in Germany.

BACKGROUND: In Germany, HPV vaccination of adolescent girls was introduced in 2007. Nationally representative data on the distribution of vaccine-relevant HPV types in the pre-vaccination era are, however, only available for the adult population. To obtain data in children and adolescents, we assessed the prevalence and determinants of serological response to 16 different HPV types in a representative sample of 12,257 boys and girls aged 1-17 years living in Germany in 2003-2005. METHODS: Serum samples were tested for antibodies to nine mucosal and seven cutaneous HPV types. The samples had been collected during the nationally representative German Health Interview and Examination Survey for Children and Adolescents in 2003-2006. We calculated age- and gender-specific HPV seroprevalence. We used multivariable regression models to identify associations between demographic and behavioral characteristics and HPV seropositivity. RESULTS: We found low but non-zero seroprevalence for the majority of tested HPV types among children and adolescents in Germany. The overall seroprevalence of HPV-16 was 2.6%, with slightly higher values in adolescents. Seroprevalence of all mucosal types but HPV-6 ranged from 0.6% for HPV-33, to 6.4% for HPV-31 and did not differ by gender. We found high overall seroprevalence for HPV-6 with 24.8%. Cutaneous HPV type seroprevalence ranged from 4.0% for HPV-38 to 31.7% for HPV-1. In the majority of cutaneous types, seroprevalence did not differ between boys and girls, but increased sharply with age, (e.g., HPV-1 from 1.5% in 1-3-years-old to 45.1% in 10-11-years-old). Associations between behavioral factors and type-specific HPV prevalence were determined to be heterogeneous. CONCLUSIONS: We report the first nationally representative data of naturally acquired HPV antibody reactivity in the pre-HPV-vaccination era among children and adolescents living in Germany. These data can be used as baseline estimates for evaluating the impact of the current HPV vaccination strategy targeting 9-14-years-old boys and girls.

Epigenetic biomarkers of ageing are predictive of mortality risk in a longitudinal clinical cohort of individuals diagnosed with oropharyngeal cancer.

BACKGROUND: Epigenetic clocks are biomarkers of ageing derived from DNA methylation levels at a subset of CpG sites. The difference between age predicted by these clocks and chronological age, termed "epigenetic age acceleration", has been shown to predict age-related disease and mortality. We aimed to assess the prognostic value of epigenetic age acceleration and a DNA methylation-based mortality risk score with all-cause mortality in a prospective clinical cohort of individuals with head and neck cancer: Head and Neck 5000. We investigated two markers of intrinsic epigenetic age acceleration (IEAAHorvath and IEAAHannum), one marker of extrinsic epigenetic age acceleration (EEAA), one optimised to predict physiological dysregulation (AgeAccelPheno), one optimised to predict lifespan (AgeAccelGrim) and a DNA methylation-based predictor of mortality (ZhangScore). Cox regression models were first used to estimate adjusted hazard ratios (HR) and 95% confidence intervals (CI) for associations of epigenetic age acceleration with all-cause mortality in people with oropharyngeal cancer (n = 408; 105 deaths). The added prognostic value of epigenetic markers compared to a clinical model including age, sex, TNM stage and HPV status was then evaluated. RESULTS: IEAAHannum and AgeAccelGrim were associated with mortality risk after adjustment for clinical and lifestyle factors (HRs per standard deviation [SD] increase in age acceleration = 1.30 [95% CI 1.07, 1.57; p = 0.007] and 1.40 [95% CI 1.06, 1.83; p = 0.016], respectively). There was weak evidence that the addition of AgeAccelGrim to the clinical model improved 3-year mortality prediction (area under the receiver operating characteristic curve: 0.80 vs. 0.77; p value for difference = 0.069). CONCLUSION: In the setting of a large, clinical cohort of individuals with head and neck cancer, our study demonstrates the potential of epigenetic markers of ageing to enhance survival prediction in people with oropharyngeal cancer, beyond established prognostic factors. Our findings have potential uses in both clinical and non-clinical contexts: to aid treatment planning and improve patient stratification.

Cutaneous ß HPVs, Sun Exposure, and Risk of Squamous and Basal Cell Skin Cancers in Australia.

BACKGROUND: Sun exposure causes cutaneous squamous (SCC) and basal cell (BCC) carcinomas. Human papillomavirus (HPV) infection might cause SCC. METHODS: We examined associations of ß and γ HPV infection in skin-swab DNA and serum antibodies with skin cancer risk, and modification of the carcinogenic effects of sun exposure by them, in case-control studies of 385 SCC cases, 832 BCC cases, and 1,100 controls nested in an Australian prospective cohort study (enrolled 2006-2009). RESULTS: Presence of ß-1 and ß-3 HPV DNA appeared to increase risks for SCC and BCC by 30% to 40% (P adjusted <0.01). BCC was also associated with genus ß DNA, OR = 1.48; 95% confidence interval (CI), 1.10 to 2.00 (P adjusted <0.01). Associations were strengthened with each additional positive ß HPV DNA type: SCC (OR = 1.07; 95% CI, 1.02-1.12) and BCC (OR = 1.06; 95% CI, 1.03-1.10), Ptrend<0.01. Positivity to genus ß or γ in serology, and genus γ in DNA, was not associated with either cancer. There was little evidence that any ß HPV type was more strongly associated than others with either cancer. A weaker association of sun exposure with SCC and BCC in the presence of ß-3 HPVs than in their absence suggests that ß-3 HPVs modify sun exposure's effect. CONCLUSIONS: Our substantive findings are at the level of genus ß HPV. Like SCC, BCC risk may increase with increasing numbers of ß HPV types on skin. IMPACT: The consistency in our findings that HPV infection may moderate the effects of sun exposure, the main environmental cause of SCC and BCC, merits further investigation.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Cutaneous Human Papillomaviruses and the Risk of Keratinocyte Carcinomas.

Cutaneous human papillomavirus (cuHPV) infections may be novel targets for skin cancer prevention and treatment, but critical information regarding the development of virus-positive skin cancers following cuHPV infection has been lacking. In this study, baseline cuHPV infection was measured by serology and viral DNA detection in eyebrow hairs (EBH) and forearm skin swabs (SSW) among 1,008 individuals undergoing routine skin cancer screening exams and followed for incidence of basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cuSCC). Baseline ß-HPV detection, particularly in SSW, significantly predicted cuSCC (HR = 4.32; 95% confidence interval, 1.00-18.66), whereas serologic evidence of past ß-HPV infection was not associated with cuSCC. Less than 5% of baseline ß-HPV types detected in SSW were present in subsequent cuSCC tumors, and cuHPV detected in SSW with higher mean fluorescence intensity values were more likely to be present in cuSCC compared with those with lower levels (P < 0.001). ß-HPV-positive cuSCC occurred more often in areas of highly sun-damaged skin than did ß-HPV-negative cuSCC. Overall, no clear patterns were observed between baseline ß-HPV detection and subsequent development of BCC, or between baseline γ-HPV detection and either cuSCC or BCC. Collectively, these results demonstrate that ß-HPV detection in SSW is a significant predictor of cuSCC risk, although evidence suggests only a small subset of cuSCC is etiologically linked to ß-HPV infection. SIGNIFICANCE: ß-HPV positivity may be a useful biomarker for identifying individuals who could benefit from increased screening or novel cutaneous squamous cell carcinoma prevention strategies.

Seroprevalence of Chlamydia trachomatis, herpes simplex 2, Epstein-Barr virus, hepatitis C and associated factors among a cohort of men ages 18-70 years from three countries.

OBJECTIVES: To estimate the seroprevalence of Chlamydia trachomatis (CT), herpes simplex type-2 (HSV2), hepatitis C (HCV), Epstein-Barr virus (EBV) and nine human papilloma virus (HPV) types, and investigated factors associated with the seropositivity among men from three countries (Brazil, Mexico and U.S). METHODS: Archived serum specimens collected at enrollment for n = 600 men were tested for antibodies against CT, HSV2, HCV, EBV, and 9-valent HPV vaccine types (6/11/16/18/31/33/45/52/58) using multiplex serologic assays. Socio-demographic, lifestyle and sexual behavior data at enrollment were collected through a questionnaire. RESULTS: Overall, 39.3% of the men were seropositive for CT, 25.4% for HSV2, 1.3% for HCV, 97.3% for EBV, 14.0% for at least one of the seven oncogenic HPV (types: 16/18/31/33/45/52/58), and 17.4% for HPV 6/11. In the unadjusted models, age, race, smoking, sexual behavior variables, and seropositivity for high-risk HPV were significantly associated with the seropositivity for CT. In multivariable analyses, self-reported black race, higher numbers of lifetime female/male sexual partners, current smoking, and seropositivity to high-risk HPV were significantly associated with increased odds of CT seropositivity. Odds of HSV2 seroprevalence were elevated among older men and those seropositive for high risk HPV. CONCLUSION: Exposure to STIs is common among men. Prevention and screening programs should target high-risk groups to reduce the disease burden among men, and to interrupt the disease transmission to sexual partners.

Sustainability of neutralising antibodies induced by bivalent or quadrivalent HPV vaccines and correlation with efficacy: a combined follow-up analysis of data from two randomised, double-blind, multicentre, phase 3 trials.

BACKGROUND: Quadrivalent and bivalent vaccines against oncogenic human papillomavirus (HPV) are used worldwide with different reported overall efficacies against HPV infections. Although protective concentrations of vaccine-induced antibodies are still not formally defined, we evaluated the sustainability of neutralising antibodies in vaccine trial participants 2-12 years after vaccination and the correlation with reported vaccine efficacy. METHODS: We did a follow-up analysis of data from the Finnish cohorts of two international, randomised, double-blind, phase 3 trials of HPV vaccines, PATRICIA (bivalent, HPV16 and 18) and FUTURE II (quadrivalent, HPV6, 11, 16, and 18). In 2002 and 2004-05, respectively, Finnish girls aged 16-17 years participated in one of these two trials and consented to health registry follow-up with the Finnish Cancer Registry. The cohorts were also linked with the Finnish Maternity Cohort (FMC) that collects first-trimester serum samples from nearly all pregnant Finnish women, resulting in 2046 post-vaccination serum samples obtained during up to 12 years of follow-up. We obtained serum samples from the FMC-based follow-up of the FUTURE II trial (from the quadrivalent vaccine recipients) and the PATRICIA trial (from corresponding bivalent vaccine recipients who were aligned by follow-up time, and matched by the number of pregnancies). We assessed neutralising antibody concentrations (type-specific seroprevalence) to HPV6, 16, and 18, and cross-neutralising antibody responses to non-vaccine HPV types 31, 33, 45, 52, and 58 from 2 to 12 years after vaccination. FINDINGS: Up to Dec 31, 2016, we obtained and analysed 577 serum samples from the quadrivalent vaccine recipients and 568 from the bivalent vaccine recipients. In 681 first-pregnancy serum samples, neutralising antibodies to HPV6, 16, and 18 were generally found up to 12 years after vaccination. However, 51 (15%) of 339 quadrivalent vaccine recipients had no detectable HPV18 neutralising antibodies 2-12 years after vaccination, whereas all 342 corresponding bivalent vaccine recipients had HPV18 neutralising antibodies.. In seropositive quadrivalent vaccine recipients, HPV16 geometric mean titres (GMT) halved by years 5-7 (GMT 3679, 95% CI 2377 to 4708) compared with years 2-4 (6642, 2371 to 13 717). Between 5 and 12 years after vaccination, GMT of neutralising antibodies to HPV16 and 18 were 5·7 times and 12·4 times higher, respectively, in seropositive bivalent vaccine recipients than in the quadrivalent vaccine recipients. Cross-neutralising antibodies to HPV31, 33, 45, 52, and 58 were more prevalent in the bivalent vaccine recipients but, when measurable, sustainable up to 12 years after vaccination with similar GMTs in both vaccine cohorts. Seroprevalence for HPV16, 31, 33, 52, and 58 significantly correlated with vaccine efficacy against persistent HPV infections in the bivalent vaccine recipients only (rs=0·90, 95% CI 0·09 to 0·99, p=0·037, compared with rs=0·62, 95% CI -0·58 to 0·97, p=0·27 for the quadrivalent vaccine recipients). Correlation of protection with prevalence of neutralising or cross-neutralising HPV antibodies was not significant in the quadrivalent vaccine recipients. INTERPRETATION: The observed significant differences in the immunogenicity of the two vaccines are in line with the differences in their cross-protective efficacy. Protective HPV vaccine-induced antibody titres can be detected up to 12 years after vaccination. FUNDING: Academy of Finland and Finnish Cancer Foundation.

Association between Human Polyomaviruses and Keratinocyte Carcinomas: A Prospective Cohort Study.

BACKGROUND: A positive association between Merkel cell polyomavirus (MCPyV) infection and cutaneous squamous cell carcinoma (cuSCC) has been observed in at least one previous case-control study. To evaluate this association in a prospective context, we investigated infections with human polyomaviruses (HPyV), including MCPyV, as predictors of keratinocyte carcinomas, including cuSCC and basal cell carcinoma (BCC), among a cohort of immunocompetent individuals enrolled in the Viruses in Skin Cancer (VIRUSCAN) Study. METHODS: Associations between markers of baseline HPyV infection (serum antibodies and viral DNA in eyebrow hairs and skin swabs) and incident keratinocyte carcinomas were modeled using Cox proportional hazards regression. Proportions of baseline HPyV infections that were concordant with a subsequent tumor positive for the same HPyV type were assessed. RESULTS: No significant associations were observed between baseline markers of MCPyV or other HPyV infections and cuSCC or BCC. Less than 4.5% of baseline MCPyV infections were also detected in subsequently developed keratinocyte carcinoma tumors. CONCLUSIONS: HPyV infection was not a predictor of keratinocyte carcinoma risk in this prospective cohort. IMPACT: Cancer-associated infections represent attractive targets for cancer prevention; however, HPyV infections have limited potential as novel targets for cuSCC prevention.

HPV DNA genotyping, HPV E6*I mRNA detection, and p16INK4a/Ki-67 staining in Belgian head and neck cancer patient specimens, collected within the HPV-AHEAD study.

BACKGROUND: The main risk factors for head and neck cancer (HNC) are tobacco and alcohol use. However, an important fraction of oropharyngeal cancer (OPC) is caused by human papillomaviruses (HPV), a subgroup with increasing incidence in several western countries. METHODS: As part of the HPV-AHEAD study, we assessed the role of HPV infection in 772 archived tissue specimens of Belgian HNC patients: 455 laryngeal (LC), 106 oral cavity (OCC), 99 OPC, 76 hypopharyngeal (HC), and 36 unspecified parts of the head and neck. All specimens were tested for HPV DNA (21 genotypes); whereof all HPV DNA-positives, all HPV DNA-negative OPCs and a random subset of HPV DNA-negatives of the other HNC-sites were tested for the presence of type-specific HPV RNA and p16INK4a over-expression. RESULTS: The highest HPV DNA prevalence was observed in OPC (36.4 %), and was significantly lower (p < 0.001) in the other HNCs (OCC:7.5 %, LC:6.6 %). HPV16 was the most common HPV-genotype in all HNCs. Approximately 83.0 % of the HPV DNA-positive OPCs tested HPV RNA or p16-positive, compared to about 37.5 % and 44.0 % in OCC and LC, respectively. Estimation of the attributable fraction of an HPV infection in HNC was very similar for HPV RNA or p16 in addition to DNA-positivity; with 30 % for OPC, and 3 % for OCC and LC. CONCLUSION: Our study confirms the heterogeneity of HPV DNA prevalence across anatomical sites in HNC, with a predominance of HPV16 in all sites. The estimated proportion of HPV-driven HNC in Belgium, during the period 1980-2014, was 10 times higher in OPC compared to OCC and LC.

Survival advantage in patients with human papillomavirus-driven oropharyngeal cancer and variation by demographic characteristics and serologic response: Findings from Head and Neck 5000.

BACKGROUND: Patients with human papillomavirus (HPV)-driven oropharyngeal cancer (OPC) experience better survival than those with HPV-negative OPC. It is unclear whether this benefit varies by demographic characteristics and serologic response. METHODS: Records from 1411 patients with OPC who had HPV serology data were analyzed. HPV status was based on HPV type 16 (HPV16) E6 serology. Participants were followed for a median of 5.9 years, and Cox proportional hazards models were used to estimate hazard ratios (HRs). The association between HPV status and overall survival was analyzed by age group, sex, smoking status, tumor site, HPV antibody levels, and HPV antibody pattern. Models were adjusted for age, sex, smoking status, and comorbidity. RESULTS: For the overall association between HPV status and survival, the fully adjusted HR was 0.43 (95% CI, 0.33-0.56). The HR was 0.19 (95% CI, 0.10-0.35) for participants aged ≤54 years, 0.38 (95% CI, 0.25-0.56) for those aged 55 to 64 years, and 0.73 (95% CI, 0.47-1.13) for those aged ≥65 years (P for interaction = .023). There was no clear evidence for an interaction by sex, smoking status, or tumor site. Survival did not differ according to E6 antibody levels in those who were seropositive. All seropositivity patterns were associated with increased survival compared with a pattern of seronegativity for all antibodies. Patients who are positive for E1, E2, E6, and E7 may experience better survival. CONCLUSIONS: HPV status confers a survival advantage across all groups. This survival advantage is more marked for younger patients. The HPV antibody pattern, but not the antibody level, may also affect survival.

Elimination of HPV-associated oropharyngeal cancers in Nordic countries.

Incidence of human papillomavirus (HPV, most notably HPV type 16) associated oropharyngeal squamous cell carcinoma (OPSCC) among middle-aged (50-69 year-old) males has tripled in four high income Nordic countries (Denmark, Finland, Norway and Sweden) over the last 30 years. In Finland and Sweden, this increase was preceded by an HPV16 epidemic in fertile-aged populations in the 1980's. The recent implementation of school-based prophylactic HPV vaccination in early adolescent boys and girls will gradually decrease the incidence, and eventually eliminate the HPV-associated OPSCCs (especially tonsillar and base of tongue carcinomas) in the Nordic countries. However, beyond the adolescent and young adult birth cohorts vaccinated, there are approximately 50 birth cohorts (born in 1995 or before) that would benefit from screening for HPV-associated OPSCC. This article reviews the need, prerequisites, proof-of-concept trial and prospects of preventing HPV-associated OPSCC in the Nordic countries.

Inequality in survival of people with head and neck cancer: Head and Neck 5000 cohort study.

BACKGROUND: Explanations for socioeconomic inequalities in survival of head and neck cancer (HNC) patients have had limited attention and are not well understood. METHODS: The UK Head and Neck 5000 prospective clinical cohort study was analyzed. Survival relating to measures of socioeconomic status was explored including area-based and individual factors. Three-year overall survival was determined using the Kaplan-Meier method. All-cause mortality was investigated via adjusted Cox Proportional Hazard models. RESULTS: A total of 3440 people were included. Three-year overall survival was 76.3% (95% CI 74.9, 77.7). Inequality in survival by deprivation category, highest education level, and financial concerns was explained by age, sex, health, and behavioral factors. None of the potential explanatory factors fully explained the inequality associated with annual household income or the proportion of income of benefits. CONCLUSION: These results support the interventions to address the financial issues within the wider care and support provided to HNC patients.