Methylation markers have shown potential for triaging high-risk HPV-positive (hrHPV+) women to identify those at increased risk of invasive cervical cancer (ICC). Our aim was to assess the performance of the S5 DNA methylation classifier for predicting incident high-grade cervical intraepithelial neoplasia (CIN) and ICC among hrHPV+ women in the ARTISTIC screening trial cohort. The S5 classifier, comprising target regions of tumour suppressor gene EPB41L3 and L1 and L2 regions of HPV16, HPV18, HPV31, and HPV33, was assayed by pyrosequencing in archived hrHPV+ liquid-based samples from 343 women with high-grade disease (139 CIN2, 186 CIN3, and 18 ICC) compared to 800 hrHPV+ controls. S5 DNA methylation correlated directly with increasing severity of disease and inversely with lead time to diagnosis. S5 could discriminate between hrHPV+ women who developed CIN3 or ICC and hrHPV+ controls (p <.0001) using samples taken on average 5 years before diagnosis. This relationship was independent of cytology at baseline. The S5 test showed much higher sensitivity than HPV16/18 genotyping for identifying prevalent CIN3 (93% vs. 61%, p = .01) but lower specificity (50% vs. 66%, p <.0001). The S5 classifier identified most women at high risk of developing precancer and missed very few prevalent advanced lesions thus appearing to be an objective test for triage of hrHPV+ women. The combination of methylation of host and HPV genes enables S5 to combine the predictive power of methylation with HPV genotyping to identify hrHPV-positive women who are at highest risk of developing CIN3 and ICC in the future.

Human papillomavirus testing is increasingly central to screening programs for cervical cancer. Although HPV testing has excellent clinical sensitivity it suffers from modest clinical specificity. Cytology has taken an important role as a triage test, while more recently HPV16/18 genotyping has provided modest incremental benefits. Despite such progress issues remain to be addressed, some of which are how to: 1) improve access to the best methods, 2) decrease costs so more women can be screened, 3) simplify management algorithms, 4) Recognize the evolving diagnostic landscape created by widespread HPV vaccination. Part of the solution lies in improving the positive predictive value of the initial screen and reflex triage. An aspirational goal should be that most women attending colposcopy have true precancer; DNA methylation testing has a role to play here. Cervical cancers and CIN2/3 generally have high levels of methylation in target human and HPV genes. In contrast normal and healthy women with no intraepithelial lesions or malignancy usually have low methylation. Furthermore, there is an interesting and useful secular gradient of increasing cervical DNA methylation as women progress toward precancer and cancer. Recent results from clinical studies using commercially available routine qPCR-based DNA methylation tests have been encouraging, demonstrating the potential for higher sensitivity and specificity than current triage methods.

Purpose To our knowledge, there are very few studies evaluating if the levels of folate modify the risk of cervical intraepithelial neoplasia grade 2 and higher (CIN2+ and CIN3+) associated with the levels of HPV genome methylation, two cofactors related to single carbon metabolism and independently associated with cervical cancer in previous studies. We conducted a case–control study nested in a three-arm randomized clinical pragmatic trial (ASCUS-COL trial) to evaluate the risk of CIN3+ associated with methylation levels according to serum folate concentrations. Methods Cases ( n = 155) were women with histologically confirmed CIN2+ (113 CIN2, 38 CIN3, and 4 SCC) and controls were age and follow-up time at diagnosis-matched women with histologically confirmed ≤ CIN1 ( n = 155), selected from the 1122 hrHPV + women of this trial. The concentrations of serum folate were determined by the radioimmunoassay SimulTRAC-SNB-VitaminB12/Folate-RIAKit and the methylation levels by the S5 classifier. Stepwise logistic regression models were used to estimate the association between folate or methylation levels and CIN2+ or CIN3+. The joint effect of folate levels and methylation on the risk of CIN3+ was estimated using combinations of categorical stratifications. Results Folate levels were significantly lower in women with CIN3+ than in other diagnostic groups ( p = 0.019). The risk of CIN3+ was eight times higher (OR 8.9, 95% CI 3.4–24.9) in women with folate deficiency and high methylation levels than in women with normal folate and high methylation levels (OR 1.4, 95% CI 0.4–4.6). Conclusion High methylation and deficient folate independently increased the risk of CIN3+ while deficient folate combined with high methylation was associated with a substantially elevated risk of CIN3+.

PDF file - 219K. Please Note: Supplementary Table 1 was updated 06/20/12. Please see the published correction notice for details of modifications to the article made in order to correct author error.

Methylation of host and viral genes is promising for triage of women with high-risk human papillomavirus infections (hrHPV). Using a population-based sample of hrHPV positive women with cervical biopsies within 12 months after cervical screening, the clinical value of the S5 methylation classifier (S5), HPV genotyping and cytology were compared as potential triage tests, for outcomes of cervical intraepithelial neoplasia (CIN) grade 3 or greater (CIN3+), CIN2+ and CIN2, and the area under the curve (AUC) calculated. S5 scores increased with histopathology severity (P-trend < .001). For CIN3+, the AUC was 0.780 suggesting S5 provides good discrimination between <CIN3 and CIN3+. AUCs were significant for all pairwise comparisons of <CIN2, CIN2 and CIN3+ (P < .001). The positive predictive value (PPV) of HPV16/18 genotyping for women with any abnormal cytology was greater than S5 (25.36% vs 20.87%, P = .005) for CIN3+, while sensitivity was substantially greater for S5 (83.33% vs 59.28%, P < .001). Restricting to women with abnormal cytology, but excluding those with high-grade cytology, both S5 and HPV16/18 provided CIN3+ PPVs high enough to recommend colposcopy. Triage with S5 also appeared useful for hrHPV positive women negative for HPV16/18 (CIN3+ PPV: 7.33%, sensitivity: 57.52%). S5 provided increased sensitivity for CIN3+ compared to HPV16/18 genotyping for hrHPV positive women, overall and when restricted to women with abnormal cytology, suggesting S5 may improve colposcopy referral. S5 also has the ability to distinguish between <CIN2, CIN2 and CIN3+, a finding of importance for managing CIN2, given the complexity and uncertainty associated with this diagnosis.

High-risk human papillomavirus type 16/18 (HPV16/18) genotyping is unable to accurately discriminate nonprogressive infections from those that will progress to cervical cancer. Our study aimed to assesses if additional testing either with liquid-based cytology (LBC) or the putative progression markers p16/Ki-67 and HPV16/18 E6 oncoprotein (E6) can improve the efficiency of HPV16/18 genotyping for triaging high-risk HPV (hrHPV)-positive women through better cancer risk stratification. Women attending colposcopy after positive HPV16/18 genotyping results within the Forwarding Research for Improved Detection and Access for Cervical Cancer Screening and Triage (FRIDA) hrHPV-based screening study in Tlaxcala, Mexico, underwent further testing with LBC, p16/Ki-67 dual-stained (DS) cytology and E6. We calculated measures of test performance for detecting histologically confirmed cervical intraepithelial neoplasia grade 2 or higher (CIN2+) and grade 3 or higher (CIN3+). A number of 475 (64.3%) of 739 HPV16/18-positive women had complete results for all tests. Triage positivity rates were 14.1%, 18.5% and 24.4%, for LBC, E6 and DS, respectively. Compared with LBC, DS had higher sensitivity (24.4% vs 60.0%) although lower specificity (87.0% vs 79.3%) for CIN3+ (P < .001), whereas E6 had a sensitivity of 37.8% and a specificity of 83.5%. No invasive cancer was missed by DS or E6, but 75% were in normal cytology. DS test was associated with nearly 75% reduction of colposcopy referrals compared with the direct referral of all HPV16/18-positive women, giving the least number of colposcopies (n = 4.3) per CIN3+ detected. We show that adjunctive testing of HPV16/18-positive women with DS may greatly reduce unnecessary colposcopy referrals within HPV-based screening employing HPV16/18 genotyping while retaining acceptable sensitivity for CIN2+ and CIN3+.

Background Colorectal cancer (CRC) is associated with patient demographics, lifestyle exposures and molecular alterations. However, it is not possible to determine which adenomas will progress to CRC, as ethically it is unacceptable to leave and follow adenomas. We hypothesised that certain lifestyle exposures at high levels could precondition exposed bowel tissue by changing and aging it, increasing the risks of deleterious DNA methylation and genetic alterations. We used a novel study design comparing adenomas with concurrent CRC (thus more likely exposed to deleterious lifestyle effects) to single adenomas in bowels with no history of CRC; we called these high (HR) and low-risk (LR) adenomas respectively. Methods We carried out a discovery and replication epigenome-wide association study (EWAS) on 106 HR and 111 LR adenomas, profiled with MethylationEPIC BeadChips. In order, to identify differentially methylated positions (DMP), regions (DMR), and DNAm (DNAmethylation) lifestyle exposures and risks, with adjustment for confounders, and gene ontology (GO) and pathway enrichment. Then, two open-source gene expression omnibus (GEO) validation datasets (52, 57 and 49, 48 HR and LR normal bowel tissues respectively) were analysed for these DNAm lifestyle exposures and risks, with adjustment for confounders. Results Our EWAS found 5 Bonferroni significant DMPs with absolute delta betas ≥ 5%, and 14 significant DMRs with absolute mean DMR delta betas ≥ 5%, replicated in the GPX7, RGS3 and TMEM135 cancer-associated genes. DNAm high alcohol exposures were strongly associated with increased risk of HR adenomas (odds ratio (OR) per standard deviation (SD) = 2.16 (95% confidence interval (CI) 1.55 - 3.09, p-value = 9.7 × 10 -6 )). In the validation datasets, DNAm high alcohol (ORperSD = 2.12 (95% CI 1.35 - 3.55, p-value = 2.0 × 10 -3 ) and ORperSD = 1.79 (95% CI 1.14 - 2.96, p-value = 1.7 × 10 -2 )), and high body mass index (BMI) exposures (ORperSD = 1.72 (95% CI 1.13 - 2.73, p-value = 1.5 × 10 -2 )) were associated with increased risk of HR normal bowel tissues. Conclusions High alcohol and BMI exposures may precondition normal bowel tissues and adenomas for increased risk of DNA methylation alterations associated with CRC progression. The DNAm exposure signatures and our newly identified genes may be useful epigenetic biomarkers for CRC prevention.

Background: Human papillomavirus (HPV)-based screening is rapidly replacing cytology as the cervical screening modality of choice. In addition to being more sensitive than cytology, it can be done on self-collected vaginal or urine samples. This study will compare the high-risk HPV positivity rates and sensitivity of selfcollected vaginal samples using four different collection devices and a urine sample. Methods: A total of 620 women referred for colposcopy were invited to provide an initial stream urine sample collected with the Colli-Pee device and take two vaginal self-samples, using either a dry flocked swab (DF) and a wet dacron swab (WD), or a HerSwab (HS) and Qvintip (QT) device. HPV testing was performed by the BD Onclarity HPV Assay. Results: A total of 600 vaginal sample pairs were suitable for analysis, and 505 were accompanied by a urine sample. Similar positivity rates and sensitivities for CIN2+ and CIN3+ were seen for DF, WD, andurine, but lowervalueswere seenforQTandHS. Noclear user preferences were seen between devices, but women found urine easiest to collect, and were more confident they had taken the sample correctly. The lowest confidence in collection was reported for HS. Conclusions: Urine, a DF swab, and WD swab all performed well and were well received by the women, whereas the Qvintip and HerSwab devices were less satisfactory. Impact: This is the first study to compare five self-sampling methods in the same women taken at the same time. It supports wider use of urine or vaginal self-sampling for cervical screening.

IntroductionUrine self-sampling for human papillomavirus (HPV)-based cervical cancer screening is a non-invasive method that offers several logistical advantages and high acceptability, reducing barriers related to low screening coverage. This study developed and evaluated the performance of a low-cost urine self-sampling method for HPV-testing and explored the acceptability and feasibility of potential implementation of this alternative in routine screening.MethodsA series of sequential laboratory assays examined the impact of several pre-analytical conditions for obtaining DNA from urine and subsequent HPV detection. Initially, we assessed the effect of ethylaminediaminetetraacetic acid (EDTA) as a DNA preservative examining several variables including EDTA concentration, specimen storage temperature, time between urine collection and DNA extraction, and first-morning micturition versus convenience sample collection. We further evaluated the agreement of HPV-testing between urine and clinician-collected cervical samples among 95 women. Finally, we explored the costs of self-sampling supplies as well as the acceptability and feasibility of urine self-sampling among women and healthcare workers.ResultsOur results revealed higher DNA concentrations were obtained when using a 40mM EDTA solution, storing specimens at 25°C and extracting DNA within 72 hrs. of urine collection, regardless of using first-morning micturition or a convenience sampling. We observed good agreement (Kappa = 0.72) between urine and clinician-collected cervical samples for HPV detection. Furthermore, urine self-sampling was an affordable method (USD 1.10), well accepted among cervical cancer screening users, healthcare workers, and decision-makers.ConclusionThese results suggest urine self-sampling is feasible and appropriate alternative for HPV-testing in HPV-based screening programs in lower-resource contexts.

The shift towards primary human papillomavirus (HPV)-based screening has necessitated the search for a secondary triage test that provides sufficient sensitivity to detect high-grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings an improved specificity to avoid unnecessary clinical work and colposcopy referrals. We evaluated the performance of the previously described DNA-methylation test (S5) in detecting CIN3 and cancers from diverse geographic settings in high-, medium- and low-income countries, using the cut-off of 0.80 and exploratory cut-offs of 2.62 and 3.70. Assays were performed using exfoliated cervical specimens (n = 808) and formalin-fixed biopsies (n = 166) from women diagnosed with cytology-negative results (n = 220), CIN3 (n = 204) and cancer stages I (n = 245), II (n = 249), III (n = 28) and IV (n = 22). Methylation increased proportionally with disease severity (Cuzick test for trend, P < .0001). S5 accurately separated women with negative-histology from CIN3 or cancer (P < .0001). At the 0.80 cut-off, 543/544 cancers were correctly identified as S5 positive (99.81%). At cut-off 3.70, S5 showed a sensitivity of 95.77% with improved specificity. The S5 odds ratios of women negative for cervical disease vs CIN3+ were significantly higher than for HPV16/18 genotyping at all cut-offs (all P < .0001). At S5 cut-off 0.80, 96.15% of consistently high-risk human papillomavirus (hrHPV)-negative cancers (tested with multiple hrHPV-genotyping assay) were positive by S5. These cancers may have been missed in current primary hrHPV-screening programmes. The S5 test can accurately detect CIN3 and malignancy irrespective of geographic context and setting. The test can be used as a screening and triage tool. Adjustment of the S5 cut-off can be performed considering the relative importance given to sensitivity vs specificity.

Methylation biomarkers are promising tools for diagnosis and disease prevention. The S5 classifier is aimed at the prevention of cervical cancer by the early detection of cervical intraepithelial neoplasia (CIN). S5 is based on pyrosequencing a promoter region of EPB41L3 and five late regions of HPV types 16, 18, 31, and 33 following bisulfite conversion of DNA. Good biomarkers should perform well in a variety of sample types such as exfoliated cells, fresh frozen or formalin-fixed paraffin-embedded (FFPE) materials. Here, we tested the performance of S5 on 315 FFPE biopsies with paired exfoliated cervical samples using four different conversion kits (Epitect Bisulfite, Epitect Fast Bisulfite, EZ DNA Methylation, and EZ DNA Methylation-Lightning). The S5 values from FFPE biopsies for all kits were significantly correlated with those obtained from their paired exfoliated cells. For the EZ DNA Methylation kit, we observed an average increased methylation of 4.4% in FFPE. This was due to incomplete conversion of DNA (73% for FFPE vs. 95% for cells). The other kits had a DNA conversion rate in FFPE similar to the cells (95%-97%). S5 performed well at discriminating <CIN2 lesions from CIN2+ lesions on the FFPE with all kits given optimized adjustments to the cut-off. The area under the curve (AUC) for S5 on FFPE was not significantly different from the paired cells (0.74-0.79 vs. 0.81). The best sensitivity and specificity were obtained for EZ DNA Methylation after the adjustment of the cut-off to reflect its lower conversion rate. Consistent methylation results can be obtained from FFPE material regardless of the conversion kit used. The S5 classifier performed as well on FFPE material as on exfoliated cells with adjusted cut-off allowing easier clinical implementation.

The S5-methylation test, an alternative to cytology and HPV16/18 genotyping to triage high-risk HPV-positive (hrHPV+) women, has not been widely validated in low-middle-income countries (LMICs). We compared S5 to HPV16/18 and cytology to detect cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) and CIN3+ in hrHPV+ women selected from a randomized pragmatic trial of 2661 Colombian women with an earlier-borderline abnormal cytology. We included all hrHPV+ CIN2 and CIN3+ cases (n = 183) age matched to 183 <CIN2 hrHPV+. Baseline specimens were HPV-genotyped and tested by S5-methylation, blinded to cytology, histology and initial HPV results. We evaluated the test performance of predefined S5-classifier (cut-point 0.8) and a post hoc classifier at a different cut-point (3.1). S5 sensitivity for CIN2+ was 82% (95% confidence interval [CI] 76.4-87.5) and for CIN3+ 77.08% (95% CI 65.19-88.97). S5 sensitivity was higher than HPV16/18 sensitivity (48.1%, 95% CI 40.85-55.33) or cytology (31.21%, 95% CI 24.50-37.93) but with lower specificity (35%, 95% CI 28.1-42). At cut-point 3.1, S5 sensitivity for CIN2+ (55.2%, 95% CI 48-62.4) or CIN3+ (64.6%, 95% CI 51.0-78.1) was also superior to HPV16/18 (P < .05) or cytology (P < .0001). At this cut-point S5 specificity (76%, 95% CI 69.8-82.1 for <CIN2) was higher than HPV16/18 (67.21%, 95% CI 60.41-74.01, P = .0062) and similar to cytology (75.57%, 95% CI 69.34-81.79, P = 1). HPV16/18 plus cytology sensitivity was similar to S5 for CIN3+, however, false-positive rate was higher (50.27% vs. 24.04%). High sensitivity is crucial in LMICs, S5-methylation exceeded HPV16/18 or cytology sensitivity with comparable specificity for CIN2+ and CIN3+ in hrHPV-positive Colombian women. Furthermore, S5 triage had comparable sensitivity and significantly fewer false positives than cytology and HPV16/18 combination.

Summary Extremely large datasets are impossible or very difficult for humans to comprehend by standard mental approaches. Intuitive visualization of genetic variants in genomic sequencing data could help in the review and confirmation process of variants called by automated variant calling programs. To help facilitate interpretation of genetic variant next-generation sequencing (NGS) data we developed VisVariant, a customizable visualization tool that creates a figure showing the overlapping sequence information of thousands of individual reads including the variant and flanking regions. Availability and implementation Detailed information on how to download, install and run VisVariant together with an example is available on our github website [https://github.com/hugging-biorxiv/visvariant].

With a lifetime risk of high-risk human papillomavirus (hrHPV) infection around 80% and less than 1% of those infections leading to cervical cancer, the development of a cervical carcinoma is a rare complication of hrHPV infection. How this happens is discussed in detail in Section 1 of the book and in Chapter 19, Clinical trials of human papillomavirus vaccines. Most infections are cleared by the host immune system within 2 years and do not always produce a definite dysplastic lesion. Several determinants have been found to influence the progression of oncogenic HPV infection, such as smoking, number of sexual partners, contraceptive use, and an effective immune response. Cervical cancer develops through a series of stages and takes years. The large majority of HPV lesions, however, are productive lesions which produce and shed viral particles without signs of cellular transformation. The morphology of HPV-induced lesions is such that it is not possible to reliably distinguish which lesions are transforming to cancer from those with a high chance of spontaneous regression. The transforming process is driven by viral oncogenes E6 and E7 overexpression, but these genes are also relevant to productive infection, leading to formation and release of new virus particles. Which hrHPV infections lead to productive infections which will never progress and which ones lead to progressive transforming lesions and warrant treatment is not clear. The site of infection is the basis of one hypothesis that has been formulated. Squamo-columnar junction cells, which are localized ectocervical squamous epithelium and endocervical glandular epithelium, are thought to be highly susceptible to transforming HPV infection while productive infections are thought to arise from squamous epithelium from the ectocervix.

Low to moderate risk abnormal cytology of potential clinical importance has been given various names over the decades including borderline, mild and moderate dysplasia, class II/III Papanicolaou smear, condylomatous atypia, ASCUS, and many other variations. There was little disagreement that women with one or more of the moderate versions of cytological abnormality should be sent to colposcopy, however, there was constant angst regarding the much more numerous borderline and mild cytological results. High-risk (hr) HPV testing to triage ASCUS was proposed in the early 1990s but the idea faltered due to a few initially contradictory small publications. In the late 1990s some large well-designed studies were done, most notably the ASCUS LSIL Triage Study (ALTS). Data from ALTS and meta-analyses including numerous other studies finally established hrHPV DNA and RNA testing as an excellent method of triage for women with ASCUS / borderline cytology.

Establishment of routine testing for human papillomavirus had a very long and tortuous diagnostic development path, spanning more than 40 yearsfrom initial characterization of the first HPV types to widespread clinical use. The HPV success story was the work product of many differentinvestigators and required numerous seminal discoveries, one of which deserved the Nobel Prize in Medicine. Initial studies of the virus by electronmicroscopy and immunochemistry led to cloning of the HPV types and to early well-performing but complicated HPV DNA tests. Almost 70 differentHPV genomes had to be cloned before we arrived at the final set of HPV probes for cervical cancer screening. The newer HPV DNA tests employsignal and target amplification and their performance is so good that they are revolutionizing cervical cancer screening programs worldwide.

Background. There is no prognostic test to ascertain whether cervical intraepithelial neoplasias (CINs) regress or progress. The majority of CINs regress in young women, and treatments increase the risk of adverse pregnancy outcomes. We investigated the ability of a DNA methylation panel (the S5 classifier) to discriminate between outcomes among young women with untreated CIN grade 2 (CIN2). Methods. Baseline pyrosequencing methylation and human papillomavirus (HPV) genotyping assays were performed on cervical cells from 149 women with CIN2 in a 2-year cohort study of active surveillance. Results. Twenty-five lesions progressed to CIN grade 3 or worse, 88 regressed to less than CIN grade 1, and 36 persisted as CIN1/2. When cytology, HPV16/18 and HPV16/18/31/33 genotyping, and the S5 classifier were compared to outcomes, the S5 classifier was the strongest biomarker associated with regression vs progression. The S5 classifier alone or in combination with HPV16/18/31/33 genotyping also showed significantly increased sensitivity vs cytology when comparing regression vs persistence/progression. With both the S5 classifier and cytology set at a specificity of 38.6% (95% confidence interval [CI], 28.4-49.6), the sensitivity of the S5 classifier was significantly higher (83.6%; 95% CI, 71.9-91.8) than of cytology (62.3%; 95% CI, 49.0-74.4; P = 0.005). The highest area under the curve was 0.735 (95% CI, 0.621-0.849) in comparing regression vs progression with a combination of the S5 classifier and cytology, whereas HPV genotyping did not provide additional information. Conclusions. The S5 classifier shows high potential as a prognostic biomarker to identify progressive CIN2.

Oropharyngeal cancer (OPC) incidence is increasing significantly among men and often requires intensive therapy causing significant morbidity. Early detection of OPC is needed, when monotherapy can be safely delivered with less treatment‐associated morbidity, while maintaining high cure rates. We conducted a study of 101 pretreatment male OPC cases matched 1:1 to 101 disease‐free controls for age and smoking history. Oral gargles were collected from cases and controls with additional biopsies or aspirates from cases. The HPV SPF 10 ‐LiPA 25 PCR assay was utilized for HPV genotyping. Methylation of three CpG sites (438, 427 and 425) in the EPB41L3 gene and methylation status of the L1 (6,367, 6,389), L2 (4,257, 4,262, 4,266, 4,269, 4,275, 4,282) and E2 (3,412, 3,415, 3,417, 3,433, 3,436) CpG sites of HPV 16 positive specimens was assessed by pyrosequencing. Significant correlations were observed between tumor and oral specimens for all methylation biomarkers ( p < 0.01). EPB41L3 and HPV 16 L1, L2 and E2 methylation were significantly ( p < 0.0001) higher among cases than controls, regardless of early vs . late disease. When HPV 16 genes and EPB41L3 methylation status were combined in a logistic regression analysis, a sensitivity of 70.3% and a specificity of 90.9% were observed for the detection of OPC from an oral gargle. Our data suggest that methylation biomarkers measured in oral gargles may have utility in identifying OPC early. Future studies are needed to replicate these findings and to inform additional biomarkers that can maximize specificity and sensitivity for early OPC detection.

Objective: Assess oral gargle-tumor human papillomavirus (HPV) agreement among oropharyngeal squamous cell carcinoma (OPSCC) cases by several disease characteristics. Materials and methods: 171 treatment naive OPSCC were enrolled 2014-2017. Tumors were categorized as early or late disease with early disease defined as T1-2 with no nodal involvement or at most a single ipsilateral positive node < 3 cm. Oral gargle samples were obtained via a 30-second rinse and gargle. The RHA Kit HPV SP10-LiPA(25) was utilized for HPV genotyping of tumor (FFPE) and oral gargle specimens. Sensitivity, specificity, positive and negative predictive value, percent agreement, and 95% exact binomial confidence intervals were estimated. Multivariable logistic regression models were fit to predict agreement. Results: 83.0% and 93.0% of oral gargle and tumor specimens were HPV positive. Oral gargle-tumor agreement for any oncogenic HPV type and HPV 16 was 73.7%. High oncogenic HPV oral gargle-tumor agreement was observed for late disease presentation, p16 positive cases, and tumors at the tonsils (74.5-80.8%). Similar trends were observed for HPV 16. Agreement for any oncogenic HPV and HPV 16 was significantly higher for late vs. early disease (77.9% vs 57.1%, p = 0.01). Oral gargle-tumor oncogenic HPV and HPV 16 agreement was independently associated with age >= 50 years and late disease presentation. Conclusion: Overall, oral-tumor HPV agreement among OPSCC was relatively high. However, oral-tumor HPV agreement was significantly lower among younger cases and those diagnosed with earlier disease. Additional biomarkers are needed to improve oral HPV test characteristics to identify OPSCC early.