We detected no significant difference in DCE-MRI parameters by HPV status. Based on a multivariate logistic regression model, the combination of clinical factors, such as tumor subsite and alcohol habits, with the perfusion-free diffusion coefficient Dt of LNs, may help to accurately discriminate OPSCC by HPV status.The aim of this paper is to define the pre-treatment radiological characteristics of oropharyngeal squamous cell carcinoma (OPSCC) using morphological and non-morphological magnetic resonance imaging (MRI), based on HPV status, in a single-institution cohort. In total, 100 patients affected by OPSCC were prospectively enrolled in the present study. All patients underwent 1.5T MR with standard sequences, including diffusion-weighted imaging with and intravoxel incoherent motion (IVIM-DWI) technique and a dynamic contrast-enhanced (DCE) MRI. For all patients, human papillomavirus (HPV) status was available. No statistically significant differences in the volume of primary tumors (PTs) and lymph nodes (LNs) were observed based on HPV status. When comparing the two patient groups, no significant differences were found for the PT radiologic characteristics  and LN morphology (solid/cystic/necrotic). Tumor subsite, smoking status, and alcohol intake significantly differed based on HPV status, as well as ADC and D Squamous cell carcinoma (SCC) is a common malignant tumor affecting the skin. A peculiar feature of this tumor is the abnormal and quick growth of keratinocytes in the epidermis, often secondary to ultraviolet or sunlight exposure . When thAt present, HPV-related OPSCC has been found to be a separate subtype of head and neck squamous cell carcinoma (HNSCC), characterized by peculiar biologic features, clinical course, and response to treatment . HPV-OPSIn this study, we analyze the pre-treatment MR characteristics of OPSCC in a single-institution patient cohort. In particular, we focus on morphological and non-morphological MRI features of both primary tumors and neck nodes, including diffusion-weighted and perfusion-weighted sequences, after separating patients by HPV status.The study protocol was authorized by the local Institutional Review Board , and specific, informed consent was obtained from each patient. The criteria for inclusion were: (i) age older than 18 years; (ii) OPSCC confirmed by biopsy; and (iii) availability of HPV status. Exclusion criteria included: (i) any contraindication to MR examination; (ii) prior surgery as excisional biopsy of the tumor or neck nodes; (iii) prior chemotherapy (CHT), including induction CHT or radiotherapy to the primary disease and the neck; (iv) unknown HPV status; and (v) distant metastases at presentation (M1). The disease was staged following the 8th edition of the AJCC (American Joint Committee on Cancer) Cancer Staging Manual for HNSCC .® Histology Kit .Two techniques (p16 immunohistochemistry and PCR-based detection) were used to identify HPV-positive patients, defined as those with lesions positive for both p16 and HPV-DNA . Dependi2). DCE-MRI was obtained through a 3D fast-spoiled gradient echo sequence . In total, 30 dynamic volumes were acquired after having optimized temporal resolution (5 s) and total scanning time (5 min and 15 s). After three dynamic volumes, a contrast agent (0.1 mmol/kg bodyweight of gadopentetate dimeglumine) was injected intravenously, with a rate of 3 mL/s. According to the standard acquisition protocol, the protocol also included post-contrast T1-w images with liver acquisition with volume acceleration sequences  on axial and coronal planes.MRI examinations were performed on a 1.5-T system  with 16-channel radiofrequency coils, including a head coil, a surface neck coil, and a spine coil. The MR examination included coronal fast spin-eco (FSE) T2-weighted (T2-w) images , followed by axial FSE T2-w images , and pre-contrast axial T1-w images , acquired from the level of the skull base to the thoracic inlet. DWI were obtained via single-shot spin-echo and echo-planar imaging , with multiple b values  in consensus. Anatomical T2-w images and/or post-contrast T1-w images were used for PT and LN segmentation and volume size quantification. Image visualizing and contouring were performed using 3D Slicer Software (Version 4.1.1). Based on their appearance on T2-w images and/or post-contrast T1-w images, PTs were described as having well-defined borders, as well as being exophytic, ulcerated, and necrotic, while LNs were classified as solid, cystic, or necrotic. Cystic change was defined as a homogeneous low signal intensity on T1-w images and high signal intensity on T2-w images relative to normal muscle, with the enhancement of the capsule after contrast medium with well-defined margins, homogeneous fluid content with no internal complexity, irregularity, or solid component by criteria of Goldenberg et al. , or with2 were used to estimate the tissue diffusion coefficient Dt (in mm2/s) based on a mono-exponential function; ADC was similarly calculated using b values of 0, 500, and 800 s/mm2 [f  and D * (perfusion-related diffusion coefficient), were not included in the analyses, considering that, in a previous investigation [The diffusion parameters were derived from homemade scripts created in the MATLAB programming language . To perform the data fitting, the median value of the signal within the entire lesion was calculated at each b value. Data points acquired at b values of 300, 500, and 800 s/mm00 s/mm2 . The pertigation , no assotrans, the transfer constant between plasma and the extravascular extracellular space (EES); Kep, the transfer constant between EES and plasma; and ve, the fractional volume of EES [Perfusion maps were derived from commercial software . A pharmacokinetic two-compartment model was applied to DCE-MRI images to extract: Ke of EES . The semt-test, as appropriate, while the relationships between categorical variables were evaluated using the chi-square or Fisher’s Exact tests, as appropriate. The Shapiro–Wilk test was applied to evaluate the normality distribution of the data.Median values and interquartile ranges were used to describe continuous variables, while frequencies and percentage values were used for categorical variables. Differences between continuous variables were assessed with the Mann–Whitney test or Student’s 2 test was used to evaluate potential differences in smoking status and alcohol intake by HPV status. Specifically, patients were defined as non-smokers if they smoked <6 packs/year, moderate smokers if they smoked 6 ≤ packs/year < 25, and strong smokers if they smoked ≥25 packs/year; patients were defined as a moderate alcoholic if they drank <1 L of wine/day and heavy alcoholic if they drank ≥1 L of wine/day.The chiAdditional tests were conducted considering three patient subgroups according to smoking status, alcohol intake, and HPV status: specifically, HPV-negative patients, HPV-positive non-smoker (<5 pack/year), non-alcoholic patients and HPV-positive smokers (≥6 pack/year), and moderate or heavy alcoholic patients. The Kruskal–Wallis test was applied to assess the differences between the groups of patients.A univariate logistic regression model was applied to identify variables that might play a role in the risk of HPV status in OPSCC. A multivariate logistic regression model was used with predictive variables that were significant in the univariate analyses following the forward selection method. The confidence interval for the area under the receiver operating characteristic curve (AUC) of each model was calculated with the bias-corrected and accelerated percentile bootstrap method, using 1000 replicates. The model classification ability was also evaluated in terms of accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).The statistical calculations were performed using SPSS statistical software version 21 .In total, 100 patients affected by OPSCC, 72 HPV-positive and 28 HPV-negative, were prospectively included in our investigation from January 2016 to September 2019. p = 0.025).Patients with HPV-related OPSCC tended to be younger and were more likely to have lower N-stage disease than patients with non-HPV-related OPSCC. The proportion of HPV-positive OPSCC was significantly affected by the tumor subsite (p < 0.001). Regarding alcohol intake, 45 HPV-positive and 9 HPV-negative patients were non-drinkers; 19 HPV-positive and 6 HPV-negative patients were moderate alcoholics; and 4 HPV-positive and 14 HPV-negative patients were heavily alcoholic (p < 0.001).Data on smoking status were missing in one HPV-negative patient, while data on alcohol intake were missing for two HPV-negative patients and one HPV-positive patient. Regarding smoking status, 31 HPV-positive and 4 HPV-negative patients were non-smokers; 13 HPV-positive and 4 HPV-negative patients were moderate smokers; and 22 HPV-positive and 25 HPV-negative patients were strong smokers  and LN volumes  were observed.No statistically significant differences in PT volumes  or the morphology of LNs .p = 0.107).The distribution of rECS was similar in HPV-positive and HPV-negative patients, with 38 cases (60%) of ECS and 14 (54%), respectively  and LNs , as shown in Conversely, ADC and Dtrans, Kep, ve, and IAUGC) between the two patient groups for both PTs and LNs  could significantly impact ADC and Dt  but not ADC and Dt parameters of LNs . Based on this, the differences in ADC and Dt of PTs by HPV status were further investigated in tonsil and base of the tongue groups, separately .Some interactions between imaging-based parameters, particularly ADC and DC and Dt . We founparately . Even thAnalogously, a comparison between diffusion parameters of HPV-negative, HPV-positive non-smoker, non-alcoholic patients, and HPV-negative versus HPV-positive smoker and alcoholic patients, for both PTs and LNs, was performed .t could be considered independent from tumor size and rECS . Due to the high collinearity between the ADC and Dt parameters, only Dt was included in the multivariate model.Univariate and multivariate logistic regression analyses for HPV positivity are reported in t of LN were the best predictor variables to determine HPV status. The resultant accuracy was 86.3% (95%CI: 76.7–92.9%), with sensitivity of 94.9% (95%CI: 85.6–98.9%), specificity of 61.9% (95%CI: 38.4–81.9%), PPV of 87.5% (95%CI: 80.1–92.4%), and NPV of 81.3% (95%CI: 57.8–93.2%)). The ROC curve of the model is illustrated in Tumor subsite, alcohol intake, and DTwo illustrative cases, respectively, of an HPV-positive OPSCC of the tonsil in a non-smoker and non-alcoholic man, and of an HPV-negative OPSCC of the base of the tongue in a moderate smoker and alcoholic man, are presented in Recently, several investigations have focused on the dissimilar characteristics of OPSCC in relation to HPV status, using multiple techniques, i.e., CT, MRI, and PET-CT, to obtain imaging biomarkers related to lesion volume size, margins, tumor microenvironment, cellular density, vascular perfusion, and metabolism ,25,26,27Consistent with previous studies, we reported that patients with HPV-related OPSCC had different smoking and drinking habits than HPV-negative patients .Regarding morphological features in our population, we did not observe significant differences in terms of PT characteristics between the two groups; although, in HPV-positive patients, lesions with well-defined and exophytic margins were slightly more prevalent, as reported by Chan et al.  using boRegarding the morphological features of OPSCC neck nodes, differences have been reported by HPV status, with the HPV-positive group showing a cystic appearance that was slightly predominant ,29,30,31Regarding rECS, in our series, we did not find a statistically significant difference between HPV-positive and negative patients. In terms of the significance of the ECS, in patients with HPV-positive OPSCC, there are contradictory opinions: some authors ,34 maintThe recent literature demonstrates a growing interest in using multimodality techniques to obtain a quantification of functional features of tumors that are potentially associated with tissue architecture, microvascularity, or metabolic activity ,36, intet to better investigate the role of pure cellularity [t values of PTs in HPV-positive patients than HPV-negative patients, which is consistent with the previous literature [t parameters of PTs, and only in the base of the tongue was the difference in Dt between HPV-negative and HPV-positive patients on the significance threshold.The potential of DWI to discriminate OPSCC by HPV status has been already reported in earlier papers ,24,27,36lularity . We fount and ADC for LNs in a larger population. Contrary to PTs, no differences in ADC and Dt parameters of LNs were found in relation to the tumor subsite, suggesting that ADC/Dt and tumor subsite may be considered independent predictors of HPV status; this is consistent with the results of the multivariate regression model, as discussed below.Compared to our previous work , a signit, in comparison with ADC, suggest that it may be more convenient to eliminate the perfusion contribution from the diffusion coefficient estimation to more accurately investigate cellular microstructure. These differences in Dt were also confirmed after dividing HPV-positive patients based on smoking and alcohol habits; in fact, it can be noticed that Dt values of HPV-positive non-smoker non-alcoholic patients and HPV-positive smoker and alcoholic patients substantially overlap for both PTs and LNs.The stronger statistical significance of DIt was hypothesized by Driessen et al.  that difThe possible impact of HPV status on DCE-MRI parameters was also investigated. Consistent with earlier literature, any difference in vascularity was reported based on HPV status for primary tumors and lymph nodes ,42,43.t of LN. In particular, tumors located in the tonsil, compared to those located in the base of the tongue, were associated with a higher risk of HPV positivity according to a recent investigation of Bos et al. [t values of lymphadenopathy were associated with a lower risk of HPV-positive OPSCC [The multivariate regression model had good predictive performance (AUC = 0.87) and indicated that the most significant factors associated with HPV status were tumor subsite, alcohol intake, and Ds et al. , while mve OPSCC ,46.t maps to explore the diagnostic performance of predictive multifactorial models of HPV positivity using an independent external cohort of patients to validate the models.Some potential limitations of our study need to be acknowledged. Even though the patient population was quite large, there was a lower proportion of HPV-negative patients, which may have introduced a potential statistical sample bias. Moreover, further quantitative analyses could have been proposed, particularly on DWI, which has been demonstrated to be more informative in relation to HPV status . Future t of lymphadenopathy may help to accurately discriminate OPSCC by HPV status.In conclusion, our study suggests that the combination of clinical factors, such as tumor subsite and alcohol habits, with the perfusion-free diffusion coefficient D


GHSR, SST, ZIC1, ASCL1, LHX8, ST6GALNAC5) in cervicovaginal secretions collected within the first part of the urine void (FVU) from a referral population. Cytology, histology, and HPV DNA genotyping results on paired FVU and cervical samples were available. Urinary median methylation levels from HR-HPV (n = 93) positive women were found to increase for all markers with severity of underlying disease. Significantly elevated levels were observed for GHSR and LHX8 in relation to high-grade cervical intraepithelial neoplasia , with area under de curve values of 0.80  0.59–0.92) and 0.76 (95% CI 0.58–0.89), respectively. These findings are the first to support the assertion that methylation analysis of host cell genes is feasible in FVU and holds promise as molecular, triage strategy to discern low- from high-grade cervical disease in HR-HPV positive women. Molecular testing on FVU may serve to increase cervical cancer screening attendance in hard-to-reach populations whilst reducing loss to follow-up and await further optimization and validation studies.Host cell DNA methylation analysis in urine provides promising triage markers for women diagnosed with a high-risk (HR) human papillomavirus (HPV) infection. In this study, we have investigated a panel of six host cell methylation markers ( These barriers can be diverse, including practical, emotional, and cognitive barriers2. Self-sampling could overcome part of these issues3 and HPV testing of self-collected cervicovaginal samples (SS) has shown similar accuracy compared to clinician-collected cervical samples (CS) using a validated PCR-based method4. Notwithstanding that SS are well-accepted by women, highest preference is given to urine self-sampling10. In recent trials, good HR-HPV DNA agreement and clinical sensitivity has been reported in first-void urine (FVU) compared to CS15. FVU allows for self-collection of cervicovaginal secretions that accumulate between the small labia and around the urethra opening, and are captured within the first part of the urine void. It is often mistaken for the first urine of the day (morning urine), which—in contrast to FVU—does not improve urinary HPV detection16.Cervical cancer screening based on cytology and/or (primary) detection of high-risk human papillomavirus (HR-HPV) DNA—the main etiological agent of cervical cancer—has successfully reduced cervical cancer incidence and mortality, but is hampered by suboptimal screening coverage. Though population based approaches such as sending personal invitation letters and reminders for a scheduled appointment have proven to be effective in increasing participation rates, barriers for attending cervical cancer screening have persevered19, FVU may also harbour other biomarkers. The use of urine to detect biomarkers for cervical screening has been receiving close appraisal in the last decade (previously reviewed20). Similar to the limitations of SS, FVU will likely not fulfil the high-quality cellularity standards required for morphological biomarkers such as cytology. Molecular biomarkers on the other hand have the potential to overcome this issue and are likely to yield high-throughput, objective, and reproducible results. Host cell methylation markers have shown promise as they are able to distinguish low-grade cervical intraepithelial neoplasia (CIN) with productive HR-HPV infections from high-grade CIN with transforming HR-HPV infections, especially those with a high short-term risk of progression to cancer, and detect all carcinomas22. Their clinical value to discern HR-HPV infected women with clinically relevant disease has been validated in CS26 and SS30. More recently, methylation of host cell genes in urine has shown promise as biomarker as well35. Combining primary HPV detection, and in case HR-HPV positive methylation marker triage on the same FVU sample could pose a non-invasive strategy to identify HR-HPV women with clinically relevant cervical disease in need of referral. Such strategy may be especially interesting in hard-to-reach populations where an approach based on clinician-collected samples is not effective, as well as in light of pandemics such as the current SARS-CoV-2 pandemic where access to healthcare is hampered and home-based self-sampling could aid prevention initiatives.Based on the same concept behind identifying more human and HPV DNA in FVU than in subsequent fractionsGHSR, SST, ZIC1, ASCL1, LHX8, and ST6GALNAC529) in FVU as biomarker for cervical cancer prevention. These markers were previously identified by unbiased genome-wide methylation analysis in HR-HPV transformed cell lines/tissue biopsies 26 and SS 29 and validated as triage markers for HR-HPV positive women. All markers revealed a very good diagnostic performance for the detection of CIN3 + in CS and/or SS29. This study more specifically aimed to study feasibility of testing for these six methylation markers in FVU to discern underlying high-grade cervical disease from normal tissue and low-grade lesions in women diagnosed with HR-HPV.Therefore, the primary objective of this study was to investigate the potential of methylation analysis of six host cell genes  cytology or (ii) histology outcomes as reference. The study protocol was registered on clinicialtrials.gov (NCT02714127) and approved by the central ethics committee of the University of Antwerp and UZA . All included participants signed informed consent prior to participating in study-related procedures which were in accordance with the Declaration of Helsinki (1964) and its later amendments as well as the ethical standards of the institutional review board.This cohort included 119 women (aged 25–64) referred to the Antwerp University Hospital  colposcopy clinic due to abnormal cytology and/or infection with one or multiple HPV genotypes (January-November 2016) as described previously18) in a 1:2 UCM:FVU ratio, whole UCM-buffered FVU samples were preserved on dry ice in individual aliquots within a median time span of 12 min (interquartile range (IQR): 11–16 min) after sample collection. FVU samples were subjected to batches in random order for DNA extraction, HPV DNA genotyping, and methylation of host cell genes.Women collected a FVU sample with a first-void urination device  at the hospital prior to their visit with the gynaecologist for a CS and colposcopy examination. Women were requested beforehand to not extensively wash their genitals before the visit at the clinic and to not urinate at least one hour prior to this visit. Upon addition of Urine Conservation Medium ) and colposcopy  were retrieved from the women’s medical records. CS  were transferred in 20 ml collection medium , analysed at UZA laboratory with the ThinPrep Pap Test (Hologic Europe), and graded according to the Bethesda classification. When indicated (according to the guidelines of the European Federation of Colposcopy), a biopsy for histological confirmation was taken during colposcopy by the clinician and graded at the UZA pathology lab using the CIN classification system. Women graded with different colposcopy and histological outcomes were classified according to the most severe stage.18 as previously described18. In brief, 4 ml of UCM-buffered whole FVU was transferred to an Amicon Ultra-4 50K filter , centrifuged , and incubated  after addition of 2 ml NucliSENS Lysis Buffer  to the retentate on the filter. After NucliSENS easyMag  DNA extraction, 35 of the 55 µl of DNA eluate was diluted with elution buffer  to a final volume of 75 µl used for HPV DNA genotyping. The remaining 20 µl DNA eluate was used for measuring methylation of host cell genes.DNA extraction was performed per in-house protocol developed by Vorsters A et al.11. Briefly, this assay quantifies 13 HR-HPV genotypes: IARC Group 1 (HPV16/18/31/33/35/39/45/51/52/56/58/59) and 2A (HPV68); three possibly HR-HPV genotypes: IARC Group 2B (HPV53/66/67); and two low-risk (LR) HPV genotypes: IARC Group 3 (HPV6/11)37. β-globin was amplified to assess the DNA quality and to determine the concentration of human (h)DNA present in the sample. The 75 µl FVU DNA extracts were directly pipetted into the 96-well plate, followed by qPCR. For HPV DNA genotyping in CS, 400–800 µl of the remaining LBC specimens were subjected to automatic nucleic acid preparation, followed by qPCR.HPV DNA genotyping data for FVU and CS were generated by Riatol quantitative PCR HPV genotyping assay (qPCR) as previously describedGHSR, SST, ZIC1, ASCL1, LHX8, and ST6GALNAC5) was performed as described before39. In brief, 250 ng of isolated FVU DNA (or 20 µl when concentration was < 12.5 ng/µl) was subjected to bisulphite treatment using the EZ DNA Methylation Kit . For multiplex qMSP, 2.5 µl of bisulphite converted DNA (≤ 50 ng) was added to 10 µl amplification mix39. The housekeeping gene ACTB was used to assess DNA quality and successful bisulphite conversion. Cycle threshold (CT)-values of methylation markers were normalised to the reference gene ACTB using the comparative method (2−ΔCT × 100)40, obtaining methylation marker ratios. As a threshold for sample validity, a CT-value below or equal to 32 for ACTB was required for each sample.Methylation analysis of a marker panel consisting of six host cell genes ) were visualized according to cytology and histology classification via scatter plots with overlying box plots, indicating median methylation levels and according interquartile ranges (25th and 75th percentile). For each methylated gene, differences in marker ratios according to disease outcome  were tested using the Mann Whitney U-test. The performance of each host cell methylation gene was visualized by a receiver operating characteristic (ROC)-curve, and evaluated by the area under the curve (AUC). The 95% CI for AUC were derived by using 5,000 bootstrap samples. Statistical analyses were performed at a significance level of 5% using the statistical software JMP Pro 13.The Cohen’s Kappa (κ) was calculated to assess the HPV genotype agreement between paired samples and was judged as follows: κ ≤ 0.20, poor; 0.21 ≤ κ ≤ 0.40, fair; 0.41 ≤ κ ≤ 0.60, moderate; 0.61 ≤ κ ≤ 0.80, good; and κ ≥ 0.81, very good agreement42. From the 114 women with HPV genotyping results available for both FVU and CS, a good Cohen’s Kappa (κ) agreement was observed for HR-HPV : 0.494–0.800), whereas this agreement was found to be excellent for HPV16/18 . Samples from 93 women with a positive test result in FVU and/or CS for HR-HPV  were selected for methylation marker analysis. Performance of each methylation marker was evaluated using either (i) cytology or (ii) histology outcomes as reference, available for 89 and 33 out of the 93 HR-HPV positive samples, respectively. Demographics and HPV genotype/viral load data are detailed in Supplementary Table 11.The median participant age in our referral population .Firstly, we analysed the methylation levels in relation to cytology using the complete series of HR-HPV positive samples. A set of 89 out of 93 HR-HPV positive samples with cytology endpoint available were selected Fig. , resultiSST Fig. . All tesGHSR, SST, ASCL1, and LHX8, but not for ZIC1 and ST6GALNAC5  was observed for ASCL1 and ST6GALNAC5 (0.05 < p ≤ 0.10) , and a similar trend for LHX8 (0.05 < p ≤ 0.10).Secondly, we assessed the presence of the methylation marker levels in FVU of HR-HPV positive women with known histological outcome. This resulted in a set of 33 samples; 6 normal/CIN0, 8 CIN1, 8 CIN2, and 11 CIN3  and LHX8 , followed by ST6GALNAC5, ASCL1, and SST/ZIC1, in the respective order  and LHX8 . Stratifying according to cytology (HSIL +) results in overall lower AUC’s, ranging between 0.66  and 0.57 , using HSIL + as endpoint  cytology and (ii) biopsy confirmed high-grade disease from normal/low-grade disease. To do so, an estimation of the clinical performance of each methylation marker in FVU was evaluated by the AUC, and visualized by ROC-curves , previously identified in HPV transformed cell lines, tissue biopsies, and cervicovaginal self-samples, in FVU. All six methylation markers showed an increase in median methylation levels in FVU for underlying disease (HSIL + and CIN2 +). Four out of six markers showed a gradual increase in median methylation levels with increasing lesion severity . The rise in methylation levels with increasing lesion severity was predominantly pronounced for GHSR, LHX8 and SST, where significant increases were observed using CIN2 + /CIN3, CIN2 +, and HSIL + as endpoint, respectively.To the best of our knowledge, this study is the first to report on the potential of methylation analysis of this panel of six host cell genes  cancer might allow us to identify methylated genes that predict high-grade cervical disease more accurately in FVU. Nevertheless, as we are using the first part of the urine void as liquid biopsy to capture cervicovaginal secretions, similarities in biomarker profile are expected. Secondly, CIN2 and CIN3 are a heterogeneous group of disease of which only a subset has a high risk of progression to cancer. Previous research has indicated that high methylation levels are associated with an advanced stage of disease and presumable high cancer progression risk. On the other hand, CIN2 and CIN3 with low methylation levels are suggested to have a low cancer progression risk26. We have no data on the duration and cancer risk of the CIN2 and CIN3 included in present study.The less pronounced difference between median methylation levels between normal/low-grade and high-grade cervical disease in FVU compared to CS/SSDAPK1, RARB, TWIST, CDH13) according to lesion severity 32. Significant different methylation levels between cancers and controls in (first-void) urine have also been described for ASCL1, FAM19A4, GHSR, LHX8, PHACTR3, PRDM14, SFRP4, SST, and ZIC135. An estimate of the diagnostic performance of the markers in our study showed similar AUC’s as those reported by van den Helder and colleagues (2020)35, between 0.62–0.80 and 0.56–0.77 to discriminate between CIN0/1 and CIN2 + and CIN0-2 and CIN3, respectively. Highest AUC’s were observed when comparing cancers versus controls. It is therefore of interest to investigate the discriminatory power of our methylation marker panel in FVU from cancer patients in future studies.Others have investigated difference in host cell methylation levels in urine between cancers and controls, for which larger differentiations between groups are anticipated. One study reported increasing number of hyper methylation-positive urine samples  (panels) are proposed as a triage strategy to discern high grade disease in women with a positive HR-HPV test, we believe that the controls used in this study align well with the intended clinical use, empowering the results.Furthermore, the controls used in this study to assess biomarker performance were all HR-HPV positive , which is different from other studies reporting on triage marker performance between (pre)cancers and controls with partial HR-HPV positivitySST) versus histology endpoints (i.e. GHSR and LHX8) is likely a reflection of the suboptimal performance of cytology to predict underlying disease and the relatively small sample size. For this reason and given the nature of the study being a feasibility study we did not investigate diagnostic performance of methylation marker combinations. Secondly, at the time of study initiation limited optimization experiments had been performed identifying the optimal FVU collection and pre-analytical processing method preceding methylation analysis by qMSP. Optimizations might potentially increase analytic sensitivity, and consequently distinctions between cases and controls and AUC’s. Yet, it is expected that this will not change the general conclusions from present study, showing that increases in methylation levels of host cell genes can be observed in FVU with increasing severity of underlying disease.Limitations of our study need to be acknowledged, and firstly involve the relative small sample size, potentially contributing to the insignificant differences observed, and heterogeneous character of our study population. This study was designed to identify promising triage markers for HR-HPV positive women. Hereto, a referral population of women was targeted attending colposcopy, either because of first abnormal screen result, or for follow-up of a persistent infection and/or associated lesion. Histology outcomes from biopsies (only taken when indicated) and cervical conization were only available for one third of the study population. To overcome this, performance of the markers was also assessed based on cytology, notwithstanding that histology is the golden standard reference test. Including both endpoints was reinforced by good κ-agreements observed between cytology (HSIL +) and histology CIN2 + (κ: 0.60 (95% CI 0.35–0.86)) and CIN3 (κ: 0.68 (95% CI 0.42–0.94)) endpoints. The difference between methylation markers showing significance in cytology (i.e. In summary, the data from this study propose that methylation analysis of host cell genes in FVU has the diagnostic potential to distinguish normal/low-grade from high-grade underlying cervical disease in HR-HPV infected women. Concomitantly, it is a promising liquid biopsy to offer a fully molecular screen and triage strategy based on primary HPV testing and methylation marker detection in the same sample. Its non-invasive nature, being well-accepted, and ability to be self-collected at home furthermore fortifies its use to potentially increase screening-attendance in hard-to-reach populations whilst reducing loss to follow-up. Further studies including a larger sample series with histological confirmation of underlying disease are ongoing to define the diagnostic accuracy of methylation marker testing in FVU and to extract the most discriminative methylation markers or marker combinations.Supplementary Information.


In Japan, government subsidies for human papillomavirus (HPV) vaccination of girls aged 13–16 commenced in 2010. By early 2013, vaccination had become a widely accepted national immunization program. However, in June of 2013, the Ministry of Health, Labor, and Welfare (MHLW), the government’s lead agency, suspended its recommendation for vaccination in response to reports of adverse vaccine events. The rate of HPV vaccination quickly dropped from 70% to almost zero, where it has lingered for eight years. In 2020, a new 9-valent HPV vaccine was licensed in Japan. The momentum seemed to be building for the resumption of HPV vaccinations, yet Japanese mothers remain widely hesitant about vaccinating their daughters, despite the well-proven safety and efficacy of the HPV vaccines. The Japanese government and our educational and medical institutions must work harder as a team to inform our parents and their children about the life-saving benefits of the HPV vaccine, and at the same time, we must respond to all their concerns and questions. The vaccine hesitancy of unvaccinated women born in 2000 and thereafter is a natural consequence of the suspension of the government‘s recommendation. We must also take every possible measure to reduce the significant risk for cervical cancer these women have. Cervical cancer is one of the most common cancers for women. In 2020, an estimated 600,000 women were diagnosed with cervical cancer worldwide, and about 341,000 deaths were attributed to it . Modern It is well established that the most critical risk factor for cervical cancer is having experienced a persistent infection with one of the high-cancer-risk versions of HPV . PersistMost cervical cancers are primarily preventable by an effective HPV vaccination. Secondary prevention approaches include early and consistent screening for and treatment of precancerous lesions. Almost inexplicably, although Japan has in hand all the technical, medical, policy, and fiscal tools with which to eliminate cervical cancer, they are almost totally failing to do so.Among its many problems is cervical cancer screening, which in Japan is recommended for all women over the age of 20. Incredibly, the screening rates for young women within three key age groups, of 20–24, 25–29, and 30–39, are only 10%, 10–20%, and 10–30%, respectively . Forty-tWe would like to share some relevant background information, some insights on the current states of HPV vaccinations and cervical screening in Japan, and give some consideration as to what directions Japan is now moving. We begin by noting that the quadra-valent HPV vaccine was approved in 2006 in the United States of America, the European Union, and Australia, and by 2020, it had been approved in more than 130 countries . The newThose were the ‘good old days’ when vaccination rates for girls of targeted age groups approached 70% compliance. However, soon thereafter, the news media began to sensationalize reports regarding the occurrence of diverse symptoms after HPV vaccination, including chronic pain, motor impairment, and other symptoms. As a result, on the 14th of June 2013, Japan’s Ministry of Health, Labor, and Welfare (MHLW) decided to temporarily suspend the national immunization program’s recommendation for routine HPV vaccination while an investigation into the safety of the vaccine was conducted.Such a safety study was carried out. In 2014, MHLW’s advisory group, the Vaccine Adverse Reactions Review Committee (VARRC), assessed the pathogenesis and the causality of the “diverse symptoms” reported to have occurred after HPV vaccination, and they defined them as “functional somatic symptoms”. Later that same year, the Japanese Pediatric Society submitted a request to the MHLW to resume its recommendation for HPV vaccinations, with no success. In 2015, positive results from an adverse vaccine events follow-up survey were released, but the suspension remained under “continued review”. Next, the WHO Global Advisory Committee for Vaccine Safety (GACVS) noted that the MHLW’s policy decisions were based on weak circumstantial evidence, causing the public to fail to use a safe and effective vaccine, potentially resulting in real harm . In ApriA year later, in April 2017, a nationwide epidemiological survey, led by a research team designated by the MHLW, concluded that “the diverse symptoms” occurred equally among unvaccinated girls of the same age groups and were therefore not specific to vaccinated girls . The detIn response to this last finding, in 2017 the VARRC discussed how to provide better information to the public to overcome their deeply entrenched HPV vaccine hesitancy. The following year, 2018, a HPV vaccine information leaflet developed by the MHLW was released; an evaluation of its results was reported in 2019 ,23.Most recently, in 2020, a 9-valent HPV vaccine was licensed in Japan. Combined with the pro-vaccine effort driving the COVID-19 vaccination program, momentum seems to be building for the resumption of HPV vaccinations as well. However, eight long years have now passed since the recommendation was suspended, and during those eight years, almost no one was vaccinated, leaving large numbers of Japanese girls and women at future risk of HPV-driven cancers and cancer deaths.In 2013, news of Japan’s suspension of its HPV vaccine recommendation due to its concerns about vaccine side-effects traveled globally through online news and social media networks . As a reIn Japan, two studies on the safety of HPV vaccines were conducted. The first was a nationwide epidemiological survey by a research team designated by the MHLW, the results which were reported to the VARRC in December 2016. Since the present Japanese vaccination system does not follow a systematic process for identifying adverse reaction causality, a questionnaire survey was conducted among participating medical institutions. That study had certain limitations: the temporal relationship between the vaccination and the symptom onset was unclear in many cases, and the age bias by vaccination history was too large to allow for age adjustment. In addition, because it is a questionnaire survey, it was suggested that the study held various biases, such as the content of the medical interviews. Although the study design did not allow for an in-depth analysis of symptom causality, it concluded that unvaccinated girls of the same age were getting the same “diverse symptoms” ,22.The second safety study was the Nagoya study, conducted in 2016 to analyze whether there was an association between HPV vaccination and the development of 24 reported post-vaccine symptoms included: irregular menstrual periods; an abnormal amount of menstrual bleeding; pain in the joints or other parts of the body; severe headache; fatigue; poor endurance; difficulty concentrating; visual field abnormalities; abnormal sensitivity to light; sudden vision loss; dizziness; cold feet; difficulty falling asleep; abnormally long sleep duration; skin problems; hyperventilation; memory decline; loss of ability to perform simple calculations; loss of ability to remember fundamental Kanji [Chinese characters indispensable for life in Japan]; involuntary uncontrollable body movements; loss of the ability to walk normally; becoming dependent on a walking stick or wheelchair; sudden loss of strength; and weakness in the hands and feet . The surThe next question is about the efficacy of the HPV vaccines, of which there have been many reports. Two studies, in particular, are noteworthy in that they explored the efficacy of the HPV vaccine in preventing the most invasive and deadly forms of cervical cancer. In 2018, Luostarinen et al. were the first to report on evidence obtained from an intention-to-treat trial showing that HPV vaccination strongly protects against HPV-associated invasive cervical cancer . They anIn 2020, Lei, et al., in another large-scale follow-up study, also investigated the efficacy of the HPV vaccine for the prevention of invasive cervical cancer . They usp = 0.016).In Japan, the preventive effect of the vaccine for stages of cervical cancer up to stage CIN3 has been reported. HPV vaccination led to a significant reduction in the incidence of abnormal cytology in vaccinated women born in 1994–1995 . In a cap = 0.0008) was afforded by the vaccine [An expanded survey showed similar results. Data was collected for women born from 1990 to 1997 who became eligible for their 20-year-old cervical cancer screening between 2010 to 2017. The incidence of CIN3+ that occurred in the generation before the HPV vaccine was introduced into Japan was 0.17% ; for the generation that was vaccinated, the CIN3+ rate was 0.02% (2/8155), indicating significant protection  vaccination triggered the suspension of DPT vaccination. It was resumed three months later, but the vaccination rate dramatically decreased. In 1979, 13,000 cases of DPT and more than 20 DPT-related deaths were reported . SimilarVaccine hesitancy was listed as the eighth of the “ten greatest threats to global health” that WHO announced in 2019 . In JapaBased on several assumptions, we have calculated the potential for future cervical cancer incidence and mortality numbers that will be increased as a result of the policy decision to continue not recommending HPV vaccination  Figure . The incIt is highly likely that these estimates of future disease will become a reality if sufficient measures are not taken to protect those women who missed their opportunity to be vaccinated due to the suspension of governmental recommendations. A significant rise in abnormal cervical cytology findings in the post-2013 cohort of unvaccinated women will demonstrate the effectiveness of the HV vaccine—in a very undesirable way. If this happens, there is deepening concern that the number of cervical cancer cases and deaths will increase in parallel in the future—all due to the decline in vaccination rates that has occurred under the suspension of the HPV vaccine recommendation.Eight years have passed since the government’s recommendation was discontinued in 2013. During this time, the population of mothers with daughters eligible for HPV vaccination has been almost completely replaced. What are now the attitudes toward HPV vaccination of this new generation of mothers?p < 0.05, p < 0. 05, p < 0.05, respectively). At any point in time, only about 20% of mothers would “inoculate” solely based on whether the MHLW would restart their recommendation.Following the suspension of governmental recommendation, we have been periodically conducting internet surveys of the mothers, which were done in March 2014, May 2015, March 2016, and December 2019, and compared the results of these surveys . The resp < 0.001). This was proof that an increase in HPV vaccination rate could be achieved (among the targeted girls born in 2003) by a leaflet sent to individuals by a local government entity.Most Japanese mothers are concerned about vaccinating their daughters against HPV, so there is still hope for a turnaround. We must plan now to quickly take effective measures to increase the vaccination rate, once the long-promised governmental recommendation is resumed. As part of that preparation, we have been investigating the effectiveness of providing different informational leaflets to girls of HPV vaccination age and/or to their parents . In Julyp = 0.02). Seven of the 21 mothers (33.3%) returned to the clinic to get their daughter vaccinated during the study period. Despite the small number of subjects observed, the study suggests that efforts to re-establish HPV vaccine acceptance should be focusing on mothers who are less likely to impose preconditions on their daughter’s vaccination.We have found that, even under the suspension of governmental recommendation, if their family doctor provided information using an information leaflet explaining the need for cervical cancer prevention, that act led to an increase in the number of vaccinations . Among tThe challenges Japan faces as a nation in reviving its national HPV vaccination program are immense. How can we possibly achieve the WHO’s lofty goal of 90% HPV vaccination by age 15 ? AccordiAccording to the “Chasm Theory”, there is a large chasm between the early market, consisting of Innovators and Early adopters, and the mainstream market, which consists of the Early and Late majorities, that cannot be easily crossed. The leaflet intervention by Isumi City resulted in a 10% vaccination rate, and the vaccination rate, if and when the recommendation is resumed, may be about the same. As the report by Ugumori et al. implies, to cross that chasm, first, medical workers will need a firmer understanding of the HPV vaccine so that they can confidently recommend vaccination to the targeted population of girls and their parents. That population will be expected to pass on that recommendation to their peers, resulting in the widening dissemination of the HPV vaccine among the majority.If the governmental recommendation is finally resumed in 2022, it will be the women born between 2000 and 2005 that have a very low vaccination rate because they have surpassed the targeted age (12–16) for the routine free vaccination program. When the future risk of cervical cancer incidence in women born in 1993 is considered to be 1 (these girls were never vaccinated because they were over 16), for receiving HPV vaccination when public subsidies started in 2010, the risk for the women born in 2000 is 0.916, 0.990 for women born in 2001, 0.998 for women born in 2002 to 2003, and 0.999 for women born in 2004 to 2005 . These wThe introduction of catch-up vaccinations has been somewhat effective, although it can never be as effective as the original preventive effect due to the increasing rate of sexual experience. In Sweden, the incidence rate ratio for the comparison of the vaccinated population with the maximally unvaccinated population was significantly reduced to 0.47  among women who had been vaccinated between the ages of 17 to 30 . In ordep < 0.001). The answer yes to the question “Did you ever talk with your family about cervical cancer?” was also significantly lower (25.0%) compared to 44.6% of the pro-vaccination generation (p < 0.001).Women who lost their opportunity to be vaccinated also lost the opportunity to know how to prevent cervical cancer. We analyzed two internet surveys, conducted in 2015 and 2020, to evaluate the differences in their understanding of the means of preventing cervical cancer in young Japanese women . We compFor those who missed their routine vaccination opportunity, we must promote providing them with catch-up vaccination opportunities, and we must encourage them to undergo routine cervical cancer screening. To avoid burdening them unfairly with the consequences of the suspension of the government recommendation, every measure must be taken to reduce their risk.Following the results of our risk predictions in 2016, we have strongly and persistently been appealing for a resumption of the governmental recommendation, but it has not . In FY 2Vaccine hesitancy is caused by a complex decision-making process that is affected by a variety of contexts, such as individual, group, and vaccine-specific factors that include media, history, religion, culture, and socioeconomics . In IrelIn Japan, hesitancy to use the COVID-19 vaccines was reported to be higher among young women, and the most common reason for their hesitancy was fear of adverse events . Other fIt will be difficult for Japan to catch up with countries like Australia that have successfully introduced the HPV vaccine. However, we must return to the fight for the elimination of cervical cancer as soon as possible. The government must resume its recommendation and strongly promote effective cervical cancer control measures.


Human papillomavirus (HPV) DNA integration is a crucial event in cervical carcinogenesis. However, scarce studies have focused on studying HPV integration (HPVint) in early-stage cervical lesions. Using HPV capture followed by sequencing, we investigated HPVint in pre-tumor cervical lesions. Employing a novel pipeline, we analyzed reads containing direct evidence of the integration breakpoint. We observed multiple HPV infections in most of the samples (92%) with a median integration rate of 0.06% relative to HPV mapped reads corresponding to two or more sequence breakages. Unlike cancer studies, most integrations events were unique (supported by one read), consistent with the lack of clonal selection. Congruent to other studies, we found that breakpoints could occur, practically, in any part of the viral genome. We noted that L1 had a higher frequency of rupture integration (25%). Based on host genome integration frequencies, we found previously reported integration sites in cancer for genes like FHIT, CSMD1, and LRP1B and putatively many new ones such as those exemplified in CSMD3, ROBO2, and SETD3. Similar host integrations regions and genes were observed in diverse HPV types within many genes and even equivalent integration positions in different samples and HPV types. Interestingly, we noted an enrichment of integrations in most centromeres, suggesting a possible mechanism where HPV exploits this structural machinery to facilitate integration. Supported by previous findings, overall, our analysis provides novel information and insights about HPVint. Papillomaviridae family, and more than 225 types have been characterized based on sequence information [Cervical cancer (CC) is the is the fourth most common malignancy and a leading cause of mortality in women worldwide . In Mexiormation . HPVs arormation ,7.HPVs are classified as low risk (LR) and HR, depending on the relative propensity to produce malignant progression . LR-HPVsWhile most HR-HPV infections are cleared spontaneously by the immune system, a few will develop persistent infections that could progress to invasive disease . TherefoSCAI and NR3C2), likely altering the original expression patterns and contributing to the complete loss of gene function [MYC, THEM49, and FANCC genes [HPV integrations (HPVint) are likely to occur through a microhomology-mediated DNA repair pathway, based on the evidence that the micro homologous sequence is significantly enriched around integrations sites . Some refunction . SeveralCC genes . HoweverCC genes . TherefoThe progression to CC by viral integration can also be affected by the HPV genotype. In invasive CC, the integration of HPV 31 and 33 was found less frequent than HPV 16, 18, and 45 . Thus, HBecause HPV is needed for tumorigenesis, understanding viral oncogenesis in pre-cancerous lesions is critical for clinical management and CC prevention. This study aimed to analyze HPVint in pre-tumor cervical lesions. HPVint status and locus have become important for discovering the mechanisms underlying virus infection, improving diagnosis, and cervical cancer treatment ,21,22,23Briefly, in this project, we selected liquid cervical samples from colposcopy consultation patients and carried out high-throughput viral integration detection (HIVID), next-generation sequencing (NGS), and bioinformatics methods to identify viral integration sites, viral and human genome breakdown sites, and HPV types based on capturing HPV sequences using a set of viral-specific probes. In the HIVID method, the fragments carrying an HPV sequence are enriched by a set of HPV probes and then processed to high-throughput sequencing ,27. We fA total of 24 cervical cell samples were taken from January 2014 to July 2016, DNA was extracted and stored at −20 °C until use. Samples were grouped according to Pap smear result: Normal, atypical squamous cells of undetermined significance (ASCUS), low-grade cervical lesions (LSIL), high-grade cervical lesions (HSIL), and Unknown (when Pap smear result was unavailable).The patients included in this study were referred to the colposcopy consultation of the Department of Gynecology and Obstetrics of the Hospital Universitario “Dr. Jose Eleuterio González” of the Universidad Autónoma de Nuevo León (HU-UANL) due to an altered Papanicolaou test, genital warts, or genital pathology detection. The median age was 36 years (SD = 13) and had a mean body mass index (BMI) of 26.3, where half of these patients had overweight. The patients selected for this study had a sexual debut at a median age of 20 years old with an average of 3 sexual partners. Fifteen patients had a previous sexually transmitted disease reported in their clinical history.HPV-positive samples were genotyped by multiplex qPCR. The method detects 14 HR-HPV types . To maximize detection, the reads were mapped to a de novo collection of HPV types (hpvDB) representing all know HPV sequences. The results are detailed in We observed 58% HPV-type agreement between both detection methods . The HPV2 = 0.00, p = 0.81), mapped reads , or % of mapping , suggesting that the number of types detected is not highly dependent on sequencing reads. Most samples showed multiple HPV infections (92%), similar to 93% found in liquid-based cytology specimens using HPV capture technology [We noted that the 24 samples obtained more than 140,000 total reads , 67% shochnology . We noteIn samples with a high number of reads, we observed complete coverage of reads all along the HPV genomes . Interesblastn) to human and HPV to determine specific read fragments at nucleotide level corresponding to each genome. Finally, reads were filtered as described in methods to diminish false positives per sample and only possible integrations involving HPV types reported in In our preliminary analyses, we tried common approaches that we have already used in cancer to estimate integrations . NeverthRAD51B [MACROD2 [RAB32. Overall, the putative integrated reads across samples are summarized in RAD51B  and MACR[MACROD2 . Figure We observed that the percentage of integrated reads per type highly corresponded to the overall mapping shown in To determine whether there are preferences for integration sites in HPV, we compared the overall mapping to the putative integrated reads. We noted that peaks of putative integrated reads highly correspond to peaks of mapped reads across samples, HPV types, and viral load, as shown in representative comparisons in RAB32 , followed by L2 (17%), E7 (16%), E1 (14%), E2 (13%), E5 (6%), and E6 (5%). The genome region that had the least rupture percentage was E4 (4%).CC remains a public health problem in Mexico since it is the second cause of death from cancer in women ,38. PersHPV genotyping by NGS and qPCR were in agreement in about 58% of patients. qPCR is limited by the primer sequences, whereas NGS is limited by the probe sets. Because the probe set is comprehensive among types and across the genome , NGS shoIn our study, 13 of 24 patients had LSIL, and 8 had multiple HPV infection (61%). In a study with LSIL patients, the most prevalent HPV genotypes were 66 (25%), 16 (21%), 53 (18%), 51 (17%) and 52 (14%) , while aAnother interesting finding in this study was that most all the patients had multiple HPV infections by the NGS method (92%). Our results are in good agreement with a previous study using NGS for HPV genotyping that also found that NGS had high sensitivity, in particular for multiple infections ,44. OtheHPV is frequently integrated into the human genome . The intWe noted an enrichment of integrations in most centromeres . There iOther authors noted that the integrations’ frequency increases with lesion progression ,53,54. TOther study in liquid-based cytology specimens found 10 genotypes  that were detected only in episomal form . They alConsensus reports reached that various portions of the HPV genome are deleted in the integrated HPV sequences . Common Viral genome integration into the host genome triggers various genetic alterations, such as oncogenes amplification, tumor suppressor gene inactivation, and inter- or intra- chromosomal rearrangements, as well as genetic instability ,33. The It has been suggested that HPV initially integrates into the human genome randomly, based on its accessibility to the genome ,13. HoweRAD51B, MACROD2, FHIT, CSMD1, LRP1B, and DLG2 .We selected 24 patients with gynecological alterations referred to the colposcopy consultation at the Gynecology and Obstetrics Department HU-UANL in Monterrey, Nuevo León, México. All of them agreed to participate in this study by signing an informed consent, previously approved on August 10, 2011 by the Institutional Review Board of the Hospital Universitario “Dr. Jose Eleuterio González” of the Universidad Autónoma de Nuevo León (Project identification code BI11-002). The PAP smear result was re-interpreted from transcripts in all but two samples. All the patients included were previously detected as HPV-positive .®), preserved in ThinPrep® PreservCyt solution, and stored at −70 °C until DNA extraction. Samples were collected from January 2014 to July 2016. DNA was extracted from cervical cells using the PureLink Genomic DNA kit from Invitrogen® , following the manufacturer’s instructions. DNA quantity and quality were measured by spectrophotometry. DNA was stored at −20 °C until use. Samples were grouped according to Pap smear as Normal, ASCUS, LSIL, HSIL, and Unknown.Cervical samples were taken using a cytobrush .HPV detection was performed using the consensus primers set PGMY 09/11, which amplifies a broad spectrum of HPV types. ® HPV HRC genotype-titer-FRT kit  according to the manufacturer’s instructions in a 7500 Fast Real-Time PCR System .qPCR was performed to genotype and quantify HPV types in samples, using AmpliSensβ-globin gene as an internal endogenous control, carried out in four separated reaction tubes. Each genotype is detected in a separate fluorescent channel  that allows its detection and quantification . Each genotype is detected in a separate fluorescent channel, making it possible to determine the genotype and viral load.This kit is based on simultaneous real-time amplification (multiplex PCR) of DNA fragments of 14 HR-HPV types  and a DNA fragment of the HIVID-NGS was performed by MyGenostics  Gene Technology Company, as reported previously . A targebwa-mem. Overall estimations of viral presence were estimated mapping raw reads to the hpvDB. The most abundant HPV type was always considered. To include other types, we integrated those whose counts were at least 1% of the most abundant type and higher than 50 reads or that the read count was at least 5000.A set of most dissimilar HPV types genomes was generated from Papillomavirus Episeme (PaVE) , a curathttps://github.com/samtools/hts-specs, accessed on 4 January 2021) taking reads with 30 nt or more not matched. Second, we generated pseudo-reads of 30 nt from 5′ and 3′ ends from NGS reads that did not fully map to hpvDB. Third, we identified candidate integration reads whose one end mapped to human (hg19) and the other end mapped to hpvDB. Fourth, original NGS candidate reads were aligned to human and hpvDB using Blastn [Viral integrations pipeline methods are focused on cancer, which are based on multiple reads supporting the same integration point ,29,30,31g Blastn . The proFHIT, CSMD1, and LRP1B and others such as CSMD3, ROBO2, and SETD3. We noted an enrichment of integrations in most centromeres, suggesting a possible mechanism where HPV utilizes centrosome or kinetochore machinery to facilitate integration. We observed that in precancerous cervical cells, there are already integrations in genes observed after clonal selection. The integration sites identified in the host genome could be used as possible biomarkers for early diagnosis in patients with cervical lesions.In this study, our results had revealed characteristics of HPVint in precancerous lesions. We observed that breakpoint in HPV can occur in any part of the viral genome, with a higher frequency in L1 gene. We rarely observed repeated reads for a specific integration site, contrary to what is observed in cancer samples. This observation is consistent with the idea that lesions still have not been through clonal selection. Based on host genome integration frequencies, we found previously reported integration sites like This study provides a theoretical basis to understanding the mechanism of tumorigenesis from the perspective of HPVint and its association with cervical lesions.


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 ) 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. Genetic susceptibility loci for oropharyngeal cancer have been reported but these studies have not always examined human papillomavirus (HPV) status. Here, the authors perform genome-wide analysis taking into account HPV16 serology status and report two independent loci in the HLA region, suggesting the protective role of HLA variants against HPV infection. HPV16-positive (HPV(+)) OPC are now considered a separate disease entity, with different treatment regimen, and different biological, epidemiological, clinical, and prognostic characteristics from HPV16-negative (HPV(−)) OPC6.Oropharyngeal cancers (OPC) are a heterogeneous group of tumors with some caused by smoking and excessive alcohol consumption and others by human papillomavirus (HPV) infections. The majority of HPV-positive OPC are caused by HPV type 16 (HPV16), with HPV surpassing tobacco exposure as the leading cause of OPC in many countries9. In our previous study, the effect of the human leukocyte antigen (HLA) region (6p21.3), specifically the HLA haplotype DRB1*13:01-DQA1*01:03-DQB1*06:03, was found to be protective for OPC9. The same haplotype was previously reported to be protective for cervical cancer10, and this further highlights the need for comprehensive genetic analyses of OPC by HPV status.Germline genetic variants are likely to play different roles in susceptibility to HPV(+) and HPV(−) OPC and identification of specific variants may help in the development of effective prevention, screening, and treatment strategies. Genome-wide association studies (GWAS) have identified several susceptibility loci for OPC, but have generally been limited by lack of information on HPV status13. Antibodies against HPV E6 and E7 oncoproteins occur in response to an underlying HPV-driven neoplastic process and are therefore expected to be at high levels among those with an HPV-driven tumor and low levels among cancer-free individuals. Large population-based cohort studies have demonstrated the potential of serology measures against HPV16 early viral proteins to detect individuals with HPV16‐driven OPC with a high sensitivity and specificity15. In particular, HPV16 E6 antibodies have been shown to occur in more than 90% of those with HPV16(+) OPC while seroprevalence among population controls is a rare condition (<1%)17. Consequently, antibodies against specific E proteins are useful in determining tumor HPV status16. In addition, they may also provide information on what humoral responses are activated in response to infection and malignant progression.To achieve this, correct identification of HPV-driven oropharyngeal tumors is critical. HPV serology is an appropriate solution in situations where tumor tissue for direct assessment is not available. It captures humoral immune response levels via measurement of antibodies against viral proteins. Antibodies against HPV L1 are considered to represent cumulative past and present HPV infection from multiple possible anatomic sites , and are relatively common in individuals without an HPV-related tumorIn this work, we conduct a GWAS in OPC stratified by HPV status based on HPV16 serology to examine genetic architecture, predisposition, and potential functional consequences of the associated loci to develop insights into OPC etiology. Our study identifies OPC genetic heterogeneity by HPV16 status and implicates the HPV(+)OPC protective HLA variants as major determinants of humoral response against viral proteins.9, 1643 OPC cases and 5256 cancer-free controls remained for analyses  were classified as HPV(+). Among the controls with serology data available (1543), 16 (1%) were classified as seropositive for HPV16; thus, the population specificity was 99.0% (95% CI: 98.3–99.4%). Median fluorescence intensity (MFI) values of HPV16 serology markers  were markedly higher in cases compared to controls  and 565 HPV(−) OPC cases, and 5256 controls) within each continent and meta-analyzed yielded 7,574,753 SNPS for risk association analyses. GWAs results of the two traits are presented in Fig. P = 4.5 × 10−11) mapped to 6p21.32 was identified for HPV(+)OPC cases  and their haplotype with decreased HPV(+)OPC risk  had R2 ≥ 0.9.The HLA region is highly polymorphic with extensive linkage disequilibrium (LD) and assessing the causality of identified associations is difficult. To fine-map the region, we therefore imputed sequence variation in classical HLA genes. The final set of imputed variants used in association analyses were of high quality; 92.5% of the variants had 9 in which HPV status data was available for only a limited number of cases. OPC extended into the class I and II regions Fig. . The mosP < 5 × 10−8). This mapped to an A allele of rs9269942, a multiple nucleotide variation that results in an amino acid change to glutamine in codon 71 (71-Glu) of HLA-DRB1 protein  for which a protective trend was also identified OPC (Table p > 5 × 10−8) and weaker when both are in the model  with lower E6 antibody levels and not with L1 MFI levels ; 71-Glu, which is located in the peptide-binding groove of HLA-DRB1*13:01, showed a robust association  with lower L1 MFI levels but no correlation with E6 MFI levels . This suggests that their association with HPV(+)OPC could potentially be mediated via influencing humoral immune response during viral tumorigenesis and corroborates the independence of these variants in their associations with lower risk of HPV(+)OPC.We conducted a protein quantitative trait loci (pQTL) analysis of 8291 MHC imputed variants using HPV16 E6 or L1 antibody levels in all OPC patients to explore associations between these variants and antibody levels. Rs4713462 and 71-Glu were the variants most strongly associated with E6 and L1 antibody levels, respectively . The association was less significant as expected given the smaller numbers and reduced range of data . This could again be explained by the more limited data range when one restricts to L1+ cases  expecting to find a similar association to the one using all OPC cases. Analyses restricting to HPV16 E6+ cases (>1000 MFI) captured again the association with rs4713462 . We therefore investigated the possible influence of E6 or L1 antibody levels on their association with the respective genetic variant by adjusting for the alternative serology marker. As shown in Supplementary Table Due to the functional connection and correlation of other E proteins  with HPV16 E6, we explored the relationship between the top associated HLA variants and antibodies against these proteins as a sensitivity analysis. We also explored association with other well-known but less common high-risk HPV types (E6 and E7 of HPV18/31/33/35/45/52/58) to assess the question of whether the identified HLA variants are E6 specific associated variants independent of HPV type. Our results suggest that the rs4713462 association might be specific to HPV16 E6 antibodies given that the strongest association was still the one detected with HPV16 E6 , a DNA-binding protein such as zinc-finger transcription factors. Interestingly, rs35189640 also maps into a repressed Polycomb-group protein (PcG) binding site specific to epithelial tissue based on chromatin states evaluated in more than 30 tissues OPC cases and controls, a risk variant at 12p23.3  cases with the 565 HPV(−)OPC cases and repeated the analyses. This was done under the assumption that OCC is a predominately HPV(−) disease and has risk factors in common with HPV(−)OPC, i.e., tobacco and alcohol usage.9 for both cancer sites, we identified two susceptibility loci at 6p21.32  and 15q21.2  common to both cancer entities. The rs3828805 is in LD with HLA-DRB1 71-Glu variant (r2 = 0.48) that we found to be associated with HPV16 L1 antibody levels in HPV(+) OPC. As expected, rs4713462, which was found exclusively associated with antibodies against the HPV16 E6 viral oncoprotein and is independent from rs3828805 (r2 = 0.0004), was not associated with risk of OC/OPC(−)  was identified in the meta-analysis of HPV(+) and HPV(−)OPC for rs4713462 at HLA region and for rs35189640 at BTBD11 locus suggesting an exclusive association with HPV(+) and HPV(−)OPC, respectively OPC and OCC analyses OPC OPC and OC share their top disease-related genetic associations while those for HPV(+) and HPV(−)OPC were different revealing a different genetic predisposition and probably genetic architecture OPC risk: rs4713462 and an amino acid polymorphism in 20. T-cell-mediated immune responses are essential for the clearance of both HPV infection and HPV-driven tumor cells and one may speculate that the associated HLA variants are promoting a targeted cell-mediated response to viral proteins which confers the protective effect against an HPV-driven cancer. Indeed, the relatively lower MFI levels of L1 or E6 in those individuals carrying the HLA-DRB1 71-Glu or the A allele of rs4713462 respectively may be explained by more active “consumption” of antibodies when the T cell sees its antigen reflecting an active immune reaction22. Consequently, the HLA-DRB1 71-Glu variant may implicate a more effective control of preventing an HPV16 infection that ultimately protects against HPV-driven cancers. This would be mimicking the effect of prophylactic vaccines which are exploiting the role of L1 capsid protein in initiating a tissue-specific infection and its antigenicity or maybe inducing a more effective T-cell response against L1 and thus preventing oncogenic transformation. The A allele of rs4713462 may be acting at a later stage through a targeted cell-mediated response to viral carcinogenesis provided by HPV16-infected cells (over)expressing E6. Therefore, our results provide evidence of natural immune response promoted by HLA germline variants against HPV-driven tumors at different stages of the infectious process that leads to cancer. This suggests, in the case of the E6-related variant, that a therapeutic vaccine targeting HPV16 E6 may attenuate viral impact after established HPV infections23. Several promising approaches have been developed, with some having achieved advanced clinical trial stages27. Oncoproteins E6 and E7 have been widely used in therapeutic HPV vaccine studies, given that the HPV16 E6 and E7 proteins are both drivers of unchecked cell proliferation in the most common oncogenic HPV types and are constitutively expressed. HPV vaccines using E7 alone have been more commonly used than E6. The fact that E7 is more abundantly expressed and highly conserved makes it a priori a better candidate, but it is a poorly immunogenic protein in reality. Our results put a particular emphasis on the potential of protein/peptide vaccines against E6. However, combination with other therapies targeting regulatory mechanisms and local immunosuppression in the tumor microenvironment, might be necessary for cancer therapeutic vaccination to become clinically successful.In our current understanding, HPV seropositivity is the result of a humoral immune response to a viral protein in those individuals trying to mount a T-cell response to eliminate affected cellsBTBD11 and showed shared genetic links with OCC identifying two risk loci in addition to those that were already known: 6p21.32 and 15q21.2. Functional evidence at 12p23.3 was limited, even though we found that the BTBD11 variant maps into a tissue-specific PcG protein binding site and nearby a transcription starting site. This suggests a potential impairment of transcription regulation increasing risk for epithelial malignancies30. At 15q21.2, rs10851478 showed a significant risk effect to develop HPV(−) oral cavity cancers and also tend to be a cis-eQTL for the FGF7 gene specific of fibroblasts. FGF7 is a potent epithelial cell-specific growth factor with an important role in morphogenesis of epithelium suggesting this variant as one of the malignancy-contributing factors from tumor stroma32.The HPV(−)OPC analysis revealed one association at 12p23.3 in A limitation of the current study is the relatively modest sample size for some of the analyses. However, the inclusion of a rich collection of data partially compensates this limitation. For HPV(+)OPC, convergent evidence of association from distinct approaches (GWAs and pQTL) identify the same variants within HLA as the best associated variants in relation with HPV infection. For HPV(-) cancer the statistical power conferred by using a second dataset with potential genetic links has allowed us to support the discovery of additional genome-wide significant SNPs.To conclude, the results generated in this study are important for understanding the biology of oral and OPC. Although future functional analyses are needed to confirm the impact of our findings, we provide extensive information on the likely consequences of relevant genetic variants putting forward a rich set of plausible gene targets and biological mechanisms for functional follow-up for both HPV(+)OPC and HPV(−)OPC and OCC cancers. In particular, our findings enhance the role of HLA variants in the immune pathogenesis of HPV(+)OPC, and may also have implications for cancer immunotherapies such as HPV-targeted vaccines that are currently undergoing clinical evaluation.9  and 5256 controls were included in this study OPC, namely tobacco and alcohol usage. We therefore hypothesized that HPV(−)OPC and OCC may share genetic risk factors and, to expand the study population for HPV(−) tumors, pooled together the series of 2,359 OCC cases and 565 HPV(−)OPC and performed a GWA analysis , C03.0–C03.9, C04.0–C04.9, C05.0–C06.9). In contrast to OPC, HPV is thought to play a very limited role in OCC, with an estimated prevalence of 5% or less in most populations9. In addition, the International Agency for Research on Cancer Institutional Review Board  evaluated and approved inclusion of each contributing study into the current studyEach included study was approved by their corresponding local ethics committee/Institutional Review Board, and informed consent was obtained from all study participants to undertake serological analyses which were not covered in our previous study9. Imputation was performed using the Michigan Imputation Server35 with the Haplotype Reference Consortium panel36. SNPs with minor allele frequency (MAF) < 0.01 or R2 < 0.3 in any of the batches were excluded before association analyses. Taking advantage of the high OncoArray SNP coverage in the HLA region, we used the genotyping data from 25 to 35 Kb at chromosome 6 (NCBI build 37) to impute 8,961 HLA variants  that were not directly genotyped. For this, we utilized the reference data collected by the Type 1 Diabetes Genetics Consortium (T1DGC)38 using the SNP2HLA v1.0.3 package in Beagle software v340. We applied post-imputation QC criteria of R2 < 0.3 to exclude variants from the association analysis. All the evaluated HLA variants were defined as binary markers in the analyses as follows: (a) For biallelic SNPs, classical HLA alleles and binary amino acid positions, the effect allele or variant was the minor allele, the presence of the HLA allele or the presence of the less frequent amino acid, respectively; (b) For multi-allelic amino acid positions, composite markers were defined where each possible individual allele and combination of alleles was tested for association.For all subjects, individual-level genome-wide SNP data were available41. Briefly, viral antigens were affinity-purified, bacterially expressed fusion proteins with N-terminal Glutathione S-transferase. Biotinylated secondary antibodies  were used in a 1:1000 dilution as reporters to detect bound plasma antibodies. Samples were evaluated at a final 1:100 dilution for antibodies against the HPV16 oncoproteins E6 and E7, other viral regulatory proteins , and the major capsid protein L1 as well as for antibodies against proteins from other high-risk HPV types (HPV18/31/33/35/45/52/58) (Supplementary Table 14. OPC cases were classified as HPV(+) or HPV(−) based on a previously validated HPV16 seropattern algorithm14 with HPV(+) defined as follows: HPV16 E6 > 1000 or 3 out of 4 E proteins greater than their cutoff value . Subjects who did not fulfill the criteria for HPV(+) were classified as HPV(−).Plasma samples were analyzed at the German Cancer Research Center  using multiplex serology by laboratory staff blinded to the case–control status and cancer site of the subjects42. The P value for heterogeneity was calculated using Cochran’s Q test. Conditional analyses within associated regions and meta-analysis of regional results were performed using glm funtion in R software43 (v.3.6.3).Regional meta-analysis (Europe and North America) was performed to evaluate the relationship between SNPs and OPC risk by HPV status and between SNPs and risk of OCC or HPV(−)OPC (pooled series of OCC and HPV(−)OPC). In brief, GWAs for each world region were performed for genotype dosages, using multivariable unconditional logistic regression assuming a log-additive genetic or dosage model with age, sex, and eigenvectors as covariates. Next, association statistics were included in a fixed-effect meta-analysis performed in PLINK v1.07Q) was used to evaluate the consistency of the effects of the top hits across GWAs by combining in a meta-analysis HPV(+) and HPV(−)OPC results or HPV(−)OPC and OC after dividing the control group into two equivalent random series.A test for heterogeneity (Cochran’s Phased genotypes obtained from the imputed data were used to generate population-based haplotypes with the Haplo.stats R package (v.1.7.7).45.Each haplotype, allele, variant, and associated amino acid was individually tested for an association with risk of OPC by HPV status within the statistical framework and covariates defined previously . Variables that were independently associated with risk  were assessed in multivariable risk models to search for the best overall HLA model. First, we used a forward selection stepwise regression of significant variants within the independently associated regions. For each gene or region we looked for the set of significant amino acid positions and we defined the classical alleles with consistent residues at those positions. We also included as covariates in these risk models the top independently associated markers identified within the HLA region from multivariable regression analyses. Model selection for the best overall HLA predictors associated with risk was conducted using the lowest BICThe aim was to find the best set of amino acids and/or HLA alleles that were independently associated with lung cancer as judged by the lowest AIC (Akaike Information Criterion) and BIC.Three-dimensional HLA ribbon models for the HLA-B and HLA-DR proteins were prepared using UCSF Chimera v1.11.P value < 10−6 was performed. Using the HaploR R package, both HaploReg46 and RegulomeDB47 were crosschecked to explore data reflecting transcription factor binding, open chromatin and the presence of putative enhancers. Further, the Genotype-Tissue Expression dataset (GTEx v8)48 was interrogated to identify potential genes influenced by disease-associated SNPs. For this purpose, GTEx v8 eQTL summary statistics, based on RNAseq analysis, were obtained for tissues best representing oropharyngeal and oral cavity tissue in this platform . Functional annotation for variants reaching P < 5 × 10−8 is summarized in Supplementary Table To identify potentially functional candidates a comprehensive bioinformatic assessment of the SNPs with GWAS 2) of genomic regions of interest were also examined in the reference European population from Phase 3 of the 1000 Genomes Project (1000G)49.Linkage Desequilibrium LD statistics (D′ and R50 were extracted on 2020-12-02 (containing 4795 publications and 222481 associations) and crosschecked against our results.To evaluate potential overlap with published GWAS findings, results reported in the NHGRI catalog53 while E6 antibody levels are accurate markers of malignant HPV-related disease54. Considering the connection between antibody-mediated immune responses and HLA-mediated antigen recognition, we explored the impact of MHC genetic variants on the humoral response to HPV proteins on the whole series of OPC cases. Linear multivariate models were implemented to test associations of genetic variants across the MHC region with HPV16 L1 or E6 log-transformed MFI levels, assuming a dosage model with age, sex, and eigenvectors as covariates in OPC cases.As mentioned, plasma antibody levels against HPV16 L1 are thought to represent cumulative past and present HPV infection from multiple possible anatomic sites9. In addition, the HPV(−)OPC-associated low-frequency variant at 12q23.3 (rs35189640) was successfully validated by TaqMan assays (Thermo Fisher Scientific) in 1810 individuals (concordance of 99.9%).Technical validation of imputed findings was performed in the recent GWAS studyFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Mississippi (MS) has among the highest rates of cervical cancer incidence and mortality in the United States, with disproportionately higher rates among Blacks compared to Whites. Here, we evaluate the prevalence of high‐risk human papillomavirus (HPV) and abnormal cytology in a representative baseline sample from a diverse statewide cohort of individuals attending cervical screening in MS from the STRIDES Study .We included individuals aged 21–65 years undergoing screening at the University of Mississippi Medical Center (UMMC) and the Mississippi State Department of Health (MSDH) from May to November 2018. We calculated age‐specific HPV prevalence, overall and by partial HPV16/18 genotyping, and abnormal cytology by race.A total of 6871 individuals (mean age 35.7 years) were included. HPV prevalence was 25.6% and higher in Blacks (28.0%) compared to Whites (22.4%). HPV prevalence was significantly higher in Blacks aged 21–24 years (50.2%) and 30–34 years (30.2%) compared to Whites in the same age groups . The prevalence of high‐grade cytologic abnormalities, a cytologic sign of cervical precancer, peaked earlier in Blacks (ages 25–29) compared to Whites (35–39). For comparison, we also analyzed HPV prevalence data from the National Health and Nutrition Examination Survey  and observed similar racial differences in HPV prevalence among women aged 21–24 years.Our findings suggest that Blacks undergoing cervical cancer screening in MS have higher prevalence of other high‐risk 12 HPV types at younger ages and experience an earlier peak of high‐grade cytologic abnormalities compared to Whites. We evaluated the age‐specific prevalence of human papillomavirus (HPV) and abnormal cytology in a baseline sample from a large, diverse statewide screening cohort in Mississippi. Overall, HPV prevalence was high (25.6%), and varied by race, with Blacks having significantly higher prevalence of other high‐risk 12 HPV types at younger ages compared to Whites, corresponding to an earlier peak of high‐grade cytologic abnormalities. To address this gap, we designed the STRIDES study  which includes over 30,000 individuals undergoing cervical cancer screening throughout the state of MS.22.1Data included in this study are from a representative baseline sample of a large statewide cohort study, STRIDES. Briefly, STRIDES includes individuals undergoing cervical cancer screening and management at the University of Mississippi Medical Center (UMMC) and the Mississippi State Department of Health (MSDH). UMMC is the sole academic medical center in the state, and all cervical pathology samples from MSDH clinics are sent to UMMC. Screening and management data are obtained from electronic health records (EHRs). Discard specimen collection began in May 2018, and follow‐up is ongoing. The current cross‐sectional study is a representative subset of the STRIDES cohort, including all individuals aged 21–65 years who underwent screening from 8 May 2018 to 26 November 2018. The Institutional Review Boards at UMMC and MSDH approved this protocol; a HIPAA waiver of consent was granted.2.2®). Specimens are processed and prescreened with automated image analysis, followed by full screening by the cytotechnologist prior to final cytologic interpretation by a cytopathology fellowship‐trained pathologist. Cytologic interpretation occurs without prior knowledge of the HPV result. Cytology results are interpreted using the Bethesda 2014 terminologyAll specimens are processed and interpreted in the UMMC Department of Pathology. Liquid‐based cytology (Pap) testing is conducted using the ThinPrep Pap 2000 System (Hologic2.3® HPV genotyping test (Roche Molecular Systems) . The assay provides type‐specific identification of types 16 and 18 and pools other high‐risk HPV genotypes: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. HPV66 is also assayed although its inclusion is of questionable value.Two HPV assays were included in this study: cobas 4800 for specimens with clinical HPV testing and the TypeSeq assay for specimens not sent for clinical HPV testing, described in detail below. At MSDH, screening transitioned from primary cytology with ASC‐US triage with reflex HPV testing to HPV and cytology co‐testing on 1 July 2018, for those aged 30–65 years. Individuals under 30 years of age undergo primary cytology screening with ASC‐US triage. UMMC clinics predominantly perform co‐testing among individuals aged 30 years and older and primary cytology with HPV ASC‐US triage for those under age 30; however, some UMMC providers continue to perform screening using primary cytology regardless of age. MSDH and UMMC specimens that were sent for clinical HPV testing were tested using the Roche Diagnostics cobas 4800Specimens that were not sent for clinical HPV testing were sent to the National Cancer Institute's Cancer Genomics Research Laboratory for testing with TypeSeq.2.4We collected demographic information including age and race from the EHR based on the intake information recorded from the patient at the screening visit. Race and ethnicity included the following categories from the EHR: White or Caucasian (“White”), Black or African American (“Black”), American Indian or Alaska Native, Asian, Native Hawaiian or Other Pacific Islander, Multiracial, Other Race, and includes Patient Refused or Unknown. Due to low sample size, American Indian or Alaska Native, Choctaw Indian, Asian, Native Hawaiian, or Other Pacific Islander, were collapsed into one category (“Other Race”). Age was categorized in 5‐year groups from 21 to 24 years to 60+ years. For some analyses where numbers were sparse, we combined older age groups as 40–49, 50–59, and 60+ years.2.5n = 36). We used descriptive statistics to summarize baseline characteristics and chi‐square and one‐way ANOVA testing to evaluate differences in baseline characteristics by race. Among Whites and Blacks where we had large enough sample size, we calculated the proportion and 95% confidence intervals (CIs) of individuals who tested positive for high‐risk HPV, overall and by HPV16/18 versus other HR12, by race, and stratified by age group. Among all individuals irrespective of HPV results, we evaluated the distribution (proportions and 95% CIs) of cytology by race, stratified by age group, grouping together ASC‐US and LSIL as low‐grade cytologic abnormalities and ASC‐H and HSIL as suggestions of underlying cervical precancer. We also computed proportions of overall HPV positivity by continuous age and race using a 5‐year moving average for individuals aged 21–65 years of age.We included data from the baseline visit, defined as the first observed screening record in the EHR as of May 2018. If HPV testing data were missing from the baseline visit but collected in a subsequent visit within 6 months, we used data from that second visit . Briefly, NHANES is an ongoing series of population‐based cross‐sectional surveys conducted by the National Center for Health Statistics at the Centers for Disease Control (CDC). The survey consists of a household interview and a physical examination in a mobile examination center. Cervicovaginal samples were self‐collected at a mobile examination center and sent to the CDC laboratory for HPV testing and genotyping with linear array.p values were two‐sided, and a p value <0.05 was considered significant. Statistical analyses were conducted using STATA/SE, version 16.0.All reported 33.1n = 4624; 67.3%) or UMMC  were included in this analysis. The mean age of all individuals was 35.7 years and varied by race, with Whites being slightly older on average compared to the other two groups . Differences in HPV positivity by race were not significant for other cytologic categories.As shown in Table 3.2p < 0.0001, respectively). The highest prevalence of HPV infection was observed around age 26 years (34.4%) among Whites, whereas the highest observed prevalence of HPV infection among Blacks occurred at age 21 years (51.3%)  Figure . In both3.3p < 0.0001) with the highest observed prevalence occurring between ages 25 and 29 years for Whites (30.6%) and ages 21–24 years for Blacks (48.4%). In contrast, both Whites and Blacks aged 21–24 years had very similar prevalence of HPV16/18 infections , with no significant differences observed by age group. Among Whites, the highest observed prevalence of HPV16/18 infection occurred between ages 25–29 years (6.5%) and among Blacks, between ages 30–34 years (7.1%). The distribution of HPV testing and partial genotyping results by race and age were similar across both assays (data not shown).Figures 3.4p < 0.0001,  Blacks aged 21–24 years had significantly higher prevalence of ASC‐US/LSIL compared to Whites in the same age group  Figure . However4Racially diverse populations from settings like MS have not been well‐represented in cervical cancer research. In our baseline sample from a diverse statewide cohort of individuals undergoing screening in MS, the prevalence of carcinogenic HPV infection was 26% and was higher in Blacks (28%) compared to Whites (22%). We observed a substantial difference in the prevalence of HPV infection in the youngest age group (21–24 years), with Blacks having significantly higher prevalence compared to Whites . While HPV16/18 prevalence was similar across racial groups, prevalence of other HR12 HPV types was significantly higher among Blacks compared to Whites. Age‐ and race‐specific prevalence of low‐grade cytologic abnormalities reflected similar patterns to those of HPV infection. With respect to high‐grade cytologic abnormalities, signaling cervical precancer, we observed an earlier peak in the prevalence in Blacks, occurring in ages 25–29, whereas this peak was observed in Whites 10 years later (ages 35–39). Collectively, our data suggest a younger age of onset of carcinogenic HPV infection and subsequent cervical precancer in Blacks compared to Whites in MS.Given that until recently, HPV‐based screening in the United States was only recommended for women aged 30 years and older, population‐based data on racial differences in the age‐specific prevalence of HPV infection and partial genotyping are rare in individuals under 30 years of age. In general, our results were in line with NHANES data, showing higher prevalence of HPV infection overall and for other HR12 HPV types in Blacks compared to Whites aged 21–24 years, although only our study was sufficiently powered to show statistically significant differences by race. In our study, the differences in the prevalence of other HR12 HPV infections between Blacks and Whites were much less pronounced in women aged 35 years and older. In contrast, in NHANES, Blacks had higher prevalence of other HR12 HPV infections across all age groups, albeit not statistically significant. The differences between our findings in MS and NHANES seem to be related both to a higher prevalence of other HR12‐HPV infections among older Blacks in NHANES as well as higher prevalence of other HR12‐HPV infections among older Whites in MS.It is possible that both behavioral and biological factors underlie the observed racial differences in the age‐specific HPV prevalence patterns in MS. Generally, the peak prevalence of HPV infection is tightly linked to age at sexual debut.The data reported here represent one of the largest studies focusing on HPV natural history in a diverse, US screening population, including over 4000 Blacks. Other large population‐based screening cohorts have a much lower proportion of Black individuals,Our study suggests that Blacks undergoing cervical screening in MS experience an earlier age of onset of other HR12 HPV infections and peak of high‐grade cytologic abnormalities compared to Whites. These findings may have implications for natural history modeling, risk estimation, and recommendations for HPV‐based screening. Studies of cervical carcinogenesis and screening are scarce in diverse populations like MS. STRIDES is addressing this critical gap and will continue to make important contributions to understanding HPV natural history and cervical precancer risk in a diverse high‐risk population to ensure that disease models and clinical guidelines are more inclusive and better reflect different target populations.The Institutional Review Boards at the University of Mississippi Medical Center and Mississippi State Department of Health approved this protocol. A HIPAA waiver of authorization and HIPAA waiver of informed consent were granted.The authors declare no relevant conflict of interest.The opinions or assertations contained herein are the private views of the authors and do not reflect the official policy of the Department of the Army or Department of Defense.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S3aClick here for additional data file.