FAM19A4/miR124‐2 methylation constitute an alternative/supplement to cytology as triage method to be investigated in real‐life pilot implementation.In human papillomavirus (HPV) cervical cancer screening, cytology is used as triage to counter the low specificity of HPV testing. VALID‐SCREEN is a EU‐multicenter, retrospective study conducted to evaluate the clinical performance of the FAM19A4/miR124‐2 methylation‐based molecular triage test as a substitute or addition to cytology as reflex testing of HPV screen positive women. FAM19A4/miR124‐2 methylation test (QIAsure Methylation Test) was evaluated in 2384 HPV‐positive cervical screening samples, from women 29‐76 years of age, derived from four EU countries. Specimens were collected in ThinPrep or SurePath media, HPV‐status, concurrent cytology, and histology diagnosis were provided by the parent institutes. The control population consisted of women with no evidence of disease within 2 years of follow‐up. A total of 899 histologies were retrieved; 527 showed no disease, 124 CIN2 (5.2%), 228 CIN3 (9.6%) and 20 cervical cancers (0.8%); 19 of 20 screen‐detected cervical cancers were found methylation‐positive (sensitivity 95%). Overall specificity of FAM19A4/miR124‐2 methylation test was 78.3% . The negative predictive value of hrHPV positive, methylation‐negative outcomes were 99.9% for cervical cancer , 96.9% for ≥CIN3 (95%CI: 96‐98), and 93.0% for ≥CIN2 (95%CI: 92‐94). Overall sensitivity for CIN3 using FAM19A4/miR124‐2 methylation test was 77% . CIN3 sensitivity was uniform between centers independent of sample collection medias, DNA extraction methods and HPV screening tests. Being objectively reported compared to the subjectivity of cytology, equally performing across settings and screening methods, the  What's new?In HPV screening for cervical cancer, cytology has been used to triage those women who are hrHPV‐positive. However, cytology is subjective, and skilled cytologists are often in short supply. A molecular test using specific biomarkers might offer better results. In this large European study, the authors found that a new standardized test for the methylation biomarker combination FAM19A4/miR124‐2 did indeed yield results that were equal to or better than triage by cytology, in determining which hrHPV‐positive women should be referred for colposcopy. ASCUSatypical squamous cells of undetermined significanceCCcervical cancerCINcervical intraepithelial neoplasiaEUEuropean UnionFAM19A4TAFA4: TAFA chemokine like family member 4)family with sequence similarity 19 (chemokine [C‐C]‐motif)‐like), member A4 PPVpositive predictive valueqMSPquantitative methylation‐specific PCRTStumor suppressor1With superior sensitivity for ≥CIN2 detection and an improved protection against cervical cancer, high‐risk human papillomavirus (hrHPV) based cervical screening has replaced or is scheduled to replace cytology as primary screening method in several countries with several other expected to follow shortly.Current HPV screening strategies are challenged by overdetection of clinically irrelevant, transient HPV infections which can result in unnecessary follow‐up visits, colposcopy overreferral and overtreatment. The lower specificity of HPV testing is today countered by reflex triage testing with (repeat) cytology, either with or without HPV 16/18 genotyping. Yet, cytology triage holds challenges too; in the Netherlands triage by abnormal cytology (threshold ≥ASCUS) in screening led to a ~3‐fold colposcopy referral increase with a ~6‐fold increase in detection of benign or CIN1 lesions compared to cytology as screening method.HPV infection alone is insufficient for progression to cervical cancer and additional genetic and epigenetic alterations in the host genome are pivotal parts in the oncogenic process.FAM19A4/miR124‐2FAM19A4/miR124‐2 methylation analysis displays a clinical sensitivity and specificity similar to cytology.FAM19A4/miR124‐2 methylation assay detects 98.3% of all cervical cancers, independent of histotype, HPV genotype, geographical area and sample type.FAM19A4/miR124‐2 methylation‐negative women had a 14 year CIN3+ risk equal to that of concurrent negative cytology outcome yet an even lower risk for cervical cancer.FAM19A4/miR124‐2 negative result provides safety that cervical cancer is not present by its high sensitivity for cervical cancer and its low long‐term risk for cervical cancer and advanced CIN lesions.Recently, the FAM19A4/miR124‐2 methylation‐based triage in an HPV based screening setting, we conducted a large multicenter post hoc, retrospective, cross‐sectional study evaluating HPV‐positive women aged ≥29 years from different European screening settings covering the most common liquid‐based cytology (LBC) sample collection medias, DNA extraction methodologies and clinical validated HPV tests.To investigate the clinical performance of a 22.1FAM19A4/miR124‐2 methylation test for detection of histologically confirmed cervical cancer, CIN3, CIN2, and ≤CIN1 in hrHPV positive cervical specimens from women undergoing screening in Scotland, Denmark, Slovenia and the Netherlands. Data on hrHPV status and pathology diagnosis of the cervical cancer specimens were provided from the parent institutes. All participating institutes used clinically validated hrHPV DNA assaysA multicenter retrospective study was designed within the VALID‐SCREEN (EU‐HORIZON2020) framework to determine the clinical performance of the 2.2For each participating center, the cohort description, sample collection media, HPV screening assay and DNA extraction for methylation testing are summarized in Table Sample selection was performed locally by the parent hospital or institution. Additional, center‐specific exclusion criteria for the Slovenian cohort were (a) HPV vaccination recorded at screening visit, (b) early therapeutic intervention, or (c) no colposcopy data available. For the Netherlands, no follow‐up data was available until 2017. A group of 224 samples from the Danish cohort did not have concurrent cytology as per Regional guidelines at that time. These Danish women, age 60 to 64 years, were under the screening exit program and were referred directly to colposcopy upon the hrHPV positive screening sample.Scotland: Cervical samples were taken using the Cervex‐Brush  and collected in PreservCyt LBC . Cytology grading was according to British Society for Cytology and NHS Cervical screening program guidelines but translated into Bethesda nomenclature. Of 281 samples, a total of 161 samples were included in the final analysis. HPV testing was performed by Cobas4800 HPV test  as a consequence of the PaVDag evaluation).Denmark: All samples were collected using the Combi‐brush  in SurePath LBC media . A total of 424 samples were included in the final analysis. HPV testing was performed by BD Onclarity HPV test  or Genomica CLART HPV2 . From the SurePath vial, 10% volume equalling 1 mL of the total cervical screening sample volume was used for DNA extraction.Slovenia: Cervical screening samples were taken with the Cervex brush or Cytobush plus and collected in PreservCyt . A total of 928 samples were included in the final analysis. HPV testing was performed using the Hybrid Capture 2 assay  and the Abbott RealTime High Risk HPV Assay . From the PreservCyt samples, 5% volume equalling 1 mL of the total volume was used for DNA extraction.The Netherlands: Cervical samples were taken with a cytobrush and collected in 1 mL UCM (QIAGEN). Cytology grading was translated into Bethesda nomenclature. A total of 871 HPV‐positive samples were included in the final analysis. HPV testing was performed using HC2 (QIAGEN). From the UCM sample, 10% volume equalling 100 μL of the total volume was used for DNA extraction.2.3FAM19A4/miR124‐2 methylation analysis including sample DNA extraction was performed as previously described.FAM19A4 and miR124‐2 promoter methylation levels using the QIAsure Methylation Test (QIAGEN). For all centers, a sample input of 2.5 μL bisulfite‐converted DNA was used for PCR on the Rotor‐Gene Q MDx 5plex HRM instrument (QIAGEN) equipped with the AssayManager software (QIAGEN). This software runs the assay followed by automatic quality assurance and data analysis. The reported results were hypermethylation‐positive, negative or invalid. Additional quality assurance was employed using the housekeeping gene β‐actin (ACTB) as a reference for successful bisulfite‐conversion, sample quality and signal normalization. Methylation testing was performed by local technicians blinded for the clinical data.2.4Data were analyzed per parent institute as well as combined for a pooled analysis. To assess between‐country heterogeneity we compared the prevalence, obtained clinical sensitivity and specificity between centers using chi‐square test or Fisher's exact test (where expected cell counts were below 5). In case the overall test showed a significant difference between the four centers, post hoc tests for pairwise comparison of countries were performed using a Bonferroni correction. The 95% (exact) CI's were determined for the proportions of methylation‐positive samples. Bayesian analysis was used to estimate the posterior mean and 95% credible interval of absolute risk of disease for methylation/cytology strata. The model assumes a binomial distribution for the number of patients with disease in each of the subgroups and independent noninformative uniform priors for the absolute risks. Bayesian analysis was performed in R version 3.5.3. All other analyses were performed in Stata version 14. A two‐sided significance level of 5% was used for the heterogeneity analyses.3FAM19A4/miR124‐2 methylation comprised a total of 2656 hrHPV positive women (Table P < .001). Post hoc pairwise comparisons showed distribution for Slovenia to differ significantly from that for Scotland, Denmark and the Netherlands . No significant pairwise differences were found between Scotland, Denmark and the Netherlands.The cohorts evaluated for FAM19A4/miR124‐2 methylation for cervical cancer was 95%  varying between 88% and 100% (Table FAM19A4/miR124‐2 methylation test among hrHPV positive women was 78.3% (n = 2012); 95%CI: 76‐80) varying from 71.1% (Scotland) to 80.3% (the Netherlands). Overall, specificity and sensitivities for CC, CIN3 and CIN2 were not found to differ between the centers .The combined clinical sensitivity of The negative predictive value of hrHPV positive, methylation‐negative outcomes were 99.9% for cervical cancer , and 93.0% for ≥CIN2 (95%CI: 92‐94). The overall positive predictive value (PPV) of hrHPV positive, methylation‐positive outcomes for ≥CIN3 was 28.3%  and 36.7% for ≥CIN2 (95%CI: 33‐40). When methylation was stratified by HSIL cytology, the sensitivity for CIN3 was 85.2% (95%CI: 78%‐90%) and specificity was 46%  with good specificity  among HPV‐positive women. Our study represents the largest clinical evaluation of a defined methylation biomarker panel to date. The results confirm and expand earlier studies with these biomarkers in HPV‐positive women, as well as the earlier described high assay reproducibility.This large multicenter retrospective clinical performance study shows that triage of HPV‐positive women with the FAM19A4/miR124‐2 methylation assay detected >98% of cervical cancers, independent of histology type, geographical area, sample type and HPV genotype.FAM19A4/miR124‐2 appears to be a rather universal event in cervical carcinogenesis and that women testing methylation‐negative are unlikely to have a prevalent cancer. In addition, post hoc analyses of a large screening trial by De Strooper et alFAM19A4/miR124‐2 methylation‐negative women is similar or better for cervical cancer and ≥CIN3 endpoints compared to a negative cytology test. This implies that 3‐year to 5‐year screening intervals can be maintained with safety for HPV based screening with a negative FAM19A4/miR124‐2 methylation triage test.A key observation from our study is that 19 of 20 screen‐detected cervical cancers were found methylation‐positive , 3% (the Netherlands) and 24% (Denmark), invalid methylation samples could at first glance seen to be an issue for routine implementation. A driving factor for invalid test results were low‐cellularity/DNA, yet samples could in general not be repeated routinely with more sample input for example, 4× to 10× more as this was not available given the retrospective nature of our study using biobanked and residual material from completed routine screening. Looking into methylation testing reproducibility, we have previously shown a high degree of this as presented in Floore et al.A limitation of our large‐scale multicenter study is a verification bias for cytology because the majority of included samples were referred after abnormal cytology making a head‐to‐head comparison between cytology and methylation impossible. With only 18 ≥CIN3 cases after NILM cytology, the relative sensitivity of the P < .001, data not shown), and a trend toward a difference between Denmark and Slovenia . Yet, the PPV depends on the prevalence of CIN lesions, which was found to be lower in Slovenia compared to the other countries. Since detection of CIN lesions is based on the diagnosis of abnormal cytology which—especially for low grade cytology—shows limited reproducibility, these differences in cytology scoring may account for some of the differences observed between centers. Other factors affecting the PPV in our study are screening history. Here, Slovenia screens most often at 3 years, Denmark and Scotland in between at 3‐year and 5‐year intervals, with the Netherlands having 5‐year intervals. Finally, as PPV is also strongly associated to age, the mean age difference between the cohorts could also have influenced the PPV.Statistical comparisons revealed a significant lower PPV for ≥CIN3 in Slovenia compared to the Netherlands  cytology as triage test for colposcopy referral, with or without HPV16/18 genotyping. Yet, cytology is largely subjective in its nature and leads to overreferral especially for low‐grade cellular abnormalities and therefore remains a challenge from the clinical perspective. Substituting cytology in triage of hrHPV positive screening samples with an objective molecular biomarker test with a high PPV for ≥CIN2 or ≥CIN3 such as 5FAM19A4/miR124‐2 methylation test shows a high sensitivity and PPV for ≥CIN3 as a colposcopy triage test of HPV‐positive women exceeding the PPV thresholds for colposcopy referral in the US  and many other western countries (10%‐20%). A further advantage is the objective machine read results based upon a clinical cut off which will not be influenced that is, by HPV vaccination status. Since the long‐term safety of the FAM19A4/miR124‐2 methylation test is equal for ≥CIN3FAM19A4/miR124‐2 methylation test into cervical screening program's to provide further data and experiences to inform on how a fully molecular cervical screening program can be designed to the benefit of women and health care services both.The The project was funded by the SME Instrument of the European Commission in the HORIZON2020 .Jesper Bonde's institution has received research funding or consumables at reduced price or for free to support research from BD Diagnostics, Agena Bioscience, Genomica SAU, LifeRiver Biotech and QIAGEN. He has received honoraria for lectures from BD Diagnostics and Hologic Ltd. Jesper Bonde is an appointed member of the National Danish Cervical Screening Committee by the Danish Health Authority, and a member of the cervical screening steering committee of the Capital Region of Denmark.Kate Cuschieri institution has received research funding or gratis consumables to support research from the following commercial entities in the last 3 years: Cepheid, Genomica, LifeRiver, Euroimmun, GeneFirst, SelfScreen, Qiagen, Hiantis and Hologic.Grazyna Stanczuk received diagnostic tests from Roche and travel sponsorship and speaker's fee from Roche and Abbott. DAMH has been on the speaker's bureau of QIAGEN, serves occasionally on the scientific advisory board of Pfizer and Bristol‐Meyers Squibb, and has minority stake in Self‐screen B.V., a spin‐off company of VU University Medical Center . Self‐screen B.V. develops, manufactures and licenses the high‐risk HPV assay and methylation marker assays for cervical cancer screening and holds patents on these tests.Chris J. L. M. Meijer is minority shareholder and part‐time CEO of Self‐screen B.V., a spin‐off company of VUmc, which develops, manufactures and licenses the high‐risk HPV assay and methylation marker assays for cervical cancer screening and holds patents on these tests. CJLMM has a very small number of shares of QIAGEN and MDXHealth, has received speakers' fees from GSK, QIAGEN, and SPMSD/Merck, and served occasionally on the scientific advisory boards (expert meeting) of these companies.Anja Oštrbenk Valenčak has received reimbursement of travel expenses for attending conferences and honoraria for speaking from Abbott Molecular, Qiagen and Seegene.Arno Floore, Saskia Doorn and Albertus Hesselink are employed by Self‐screen B.V.All other authors report no conflict of interest.The work in our study with human derived material was conducted under national and international rules and legislation, as well as European standards of research ethics, as it is expressed in the applicable legislation/regulations  and guidelines for Good Clinical Practice. The study was approved by the local ethics committees where applicable. The Danish data contribution: Danish Data Protection Agency AHH 2015‐085, I‐suite: 04150 and AHH‐2017‐024, I‐Suite: 05356.Data S1 Supplementary Information.Click here for additional data file.


OGG1 prevents the accumulation of mutations and regulates the transcription of various oxidative stress–response genes. In addition to targeting DNA, oxidative stress can affect proteins like OGG1 itself, specifically at cysteine residues. Previous work has shown that the function of OGG1 is sensitive to oxidants, with the cysteine residues of OGG1 being the most likely site of oxidation. Due to the integral role of OGG1 in maintaining cellular homeostasis under oxidative stress, it is important to understand the effect of oxidants on OGG1 and the role of cysteines in its structure and function. In this study, we investigate the role of the cysteine residues in the function of OGG1 by mutating and characterizing each cysteine residue. Our results indicate that the cysteines in OGG1 fall into four functional categories: those that are necessary for (1) glycosylase activity (C146 and C255), (2) lyase activity , and (3) structural stability (C253) and (4) those with no known function (C28 and C75). These results suggest that under conditions of oxidative stress, cysteine can be targeted for modifications, thus altering the response of OGG1 and affecting its downstream cellular functions. More than 100 different oxidized lesions have been identified in DNA . The mos8-Oxoguanine glycosylase (OGG1), which has a canonical function in the base excision repair (BER) pathway, can efficiently recognize and excise 8-oxoG , 16, binCXCL1, CXCL2, CCL20, IL1B, TNFA), indicating that OGG1 glycosylase activity is not necessary for this function   . After OBecause OGG1 plays a central role in maintaining cellular homeostasis under oxidative stress, it is important to have a full understanding of the effect of oxidation on OGG1. In addition to DNA, proteins involved in the cellular response to stress such as OGG1 can also be oxidized, specifically at cysteine residues. Cysteine is a unique amino acid that is highly conserved but can be targeted for modification by oxidation due to the high reactivity of the sulfur atom . It has Previous studies have suggested that the glycosylase activity of OGG1 is sensitive to specific environmental toxins, like cadmium  and arseOGG1 contains eight cysteine residues that are highly conserved across species . C28 andA). NEM is specifically reactive with the thiol group of cysteine and blocks the sulfhydryl group. In the presence of 0.25 mM NEM (10× molar excess of OGG1), the glycosylase activity of OGG1 is completely abolished. This finding indicates that OGG1 contains reactive cysteine residues that are essential for glycosylase activity. Next, 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid (IAEDANS), a thiol-reactive fluorescent molecule, was combined with OGG1 to quantify the least number of reactive cysteines required to inhibit the activity. The absorbance and extinction coefficient of IAEDANS (ε336 = 6100 M−1 s−1) and OGG1  were used to identify the labeling ratio. In the presence of 0.25 mM IAEDANS (10× molar excess of OGG1), two cysteine residues were labeled, which was sufficient to inhibit the glycosylase activity (B). These results provide evidence that cysteines are important for the activity of OGG1.OGG1 contains eight cysteine residues. However, little is known about their role in the activity of OGG1. To identify whether OGG1 contains reactive cysteines and whether they are important for activity, we treated OGG1 with N-ethylmaleimide (NEM) and tested the effect of blocking the potentially reactive cysteines on glycosylase activity A. NEM isactivity B. These A and C). However, most of the cysteine mutants bind to the DNA with an altered affinity. Specifically, mutations at positions 28, 140, 146, 241, 253, and 255 have an ∼10-fold increase in the dissociation constant. The decreased binding affinity in C28S was unexpected due to its distal location from the DNA binding site and the active site. This finding indicates that C28 may influence DNA binding through long-range interactions. The increase in the dissociation constant of both C253S and C255S is expected due to their proximal location in the helix-hairpin-helix motif near the DNA binding site. Specifically, C253 has been shown to be part of the active site, which interacts with the lesion once it is extruded into the active site . The results indicate that OGG1 binds specifically to its substrate and that the cysteine mutations only influence specific binding but not nonspecific binding.In addition to exhibiting substrate specificity for the 8-oxoG lesion over other oxidized lesions, WT OGG1 also preferentially binds to 8-oxoG:C (specific binding) over 8-oxoG:A (nonspecific binding) substrates . This se−1  to ensure that the reaction is not limited by percent active enzyme and by the slow rate of DNA dissociation . The rea−1 .Figure 4−1). Thus, a modification at this site can decrease the glycosylase activity. C146 is located near the active site, but there are no direct interactions with the lesion and DNA. The shortest distance between C146 and the lesion is 5 Å, with no apparent intermediate molecules between them. Therefore, C146 might be important for stabilizing the extruded lesion or it might be involved in recognizing and flipping the lesion out of the duplex.The results indicate that the C146S mutant has a slow glycosylase rate . The mechanism is unclear because of the lack of direct interactions between C140 and the active site. This finding provides evidence that there could be several consequences to the modification of cysteines depending on the site and type of modification (bulkiness and charge).Interestingly, the C140S mutant led to an increase in the glycosylase rate . This finding indicates that the lyase activity is highly sensitive to the status of cysteines.The lyase activity of OGG1 results in the formation of a nicked DNA backbone at the 5’ end of the lesion, leaving a 3’ PUA and a 5’ phosphate. In the BER pathway, the PUA group is further processed by APE1 to form a 3’OH, which is the substrate for the subsequent nucleotide incorporation reaction by polymerase beta. The lyase activity was tested by mixing OGG1 with the 8-oxoG–containing DNA substrate at 37 °C and quenching the reaction at each time point with formamide dye followed by incubation at 85 °C for 5 min. The results in Figure 5253, 255 A. This fB). This finding indicates that the decrease in lyase activity is not due to a change in DNA binding.To ensure that the decrease in lyase activity is not resulting from a change in DNA binding to the abasic site, we measured the apparent binding affinity of the enzyme to a DNA substrate containing tetrahydrofuran (THF), which is a stable mimic of the abasic site. Our results indicate that WT OGG1 has a dissociation constant of 0.6 nM. Although a few cysteine mutants have higher dissociation constants, specifically C28S (2.2 nM), C146S (9.9 nM), and C241S (3.5 nM), most mutants have dissociation constants that are very similar to WT and they maintain their high binding capacity to the THF-containing substrate B. This fA). The WT has a melting point (Tm) of 43 °C, similar to previous reports . C140 is located within an ⍺-helix, and a mutation at this position may lead to instability. C253 is located at the active site and has been shown to be important for the catalytic activity  glycosylase activity (C146 and C255), (2) lyase activity ,  . MutatinIn addition to C255, we provide evidence of a novel role of C146 for the glycosylase activity. A mutation at C146 led to a decrease in the glycosylase activity. C146 is located near N149 and N150, which are important for filling the void in the duplex DNA upon extrusion of the lesion out of the duplex and into the active site. This proximity may explain why C146 is essential for the glycosylase activity .−1)  , giving Another factor by which the cysteines could influence the ability of OGG1 to participate in a specific oxidative stress pathway is by affecting protein–protein interactions. Specifically, several BER components such as APE1 and XRCC1 have been shown to interact with OGG1 and facilitate the repair of 8-oxoG. For example, the interaction of OGG1 with XRCC1 or APE1 has been shown to increase the turnover rate of OGG1 by a factor of 3× and 5×, respectively , 46. MorIn addition to maintaining genomic stability and preventing propagation of mutations through the BER pathway, OGG1 has several other functions in the cell. Most prominently, OGG1 has been shown to bind 8-oxoG in promoter regions of certain genes and alter their expression level , 28, 47.BCL2 because it induces topological changes in the promoter region necessary for its transcription (A) (B). Lastly, the third scenario suggests that only the binding of OGG1 at promoter regions but not its catalytic activity is necessary for the transcriptional activation of various proinflammatory genes, such as CXCL2 and TNFA (C) . T. TBCL2 btion (A) . This setion (A) B. Lastlyand TNFA C 47). D. DBCL2 bvia the first scenario (formation of break) can occur (A). Modifications at positions C140, C163, C241, and C253 maintain the glycosylase activity but inhibit lyase activity, potentially leading to the transcriptional activation described in the second scenario, where formation of abasic site is required but nicking of the DNA backbone is attenuated (B). Lastly, our results indicate that positions C146 and C255 are important for the glycosylase activity; thus, a modification at C146 or C255 can activate the pathway in the third scenario, where the nonproductive binding of OGG1 is required for transcription activation (C). Recent work by Hao et al. , which may further sensitize the protein to oxidation. Specifically, S326C is a frequently occurring mutation in OGG1 that has been associated with various cancers such as lung cancer , prostat280 of 37,000 M−1 cm−1 as the extinction coefficient for OGG1.OGG1 in pET30 vector with an N-terminal 6x-histidine tag was provided by Dr Sylvie Doublie. Site-directed mutagenesis was used to create the following eight mutants: C28S, C75S, C140S, C146S, C163S, C241S, C253S, and C255S. The protein was expressed in Rosetta (DE3). The Rosetta (DE3) cells were grown at 37 °C to OD600 of 0.6 to 0.8, induced with 1 mM IPTG, and left to shake at 18 °C overnight. Cells were harvested by centrifugation and resuspended in buffer A [50 mM Tris (pH 8.0) and 100 mM NaCl] and lysed by sonication. The clarified lysate was loaded onto a nickel-charged chelating column, washed with buffer A, and eluted with buffer B . The eluent was loaded onto an SP column, washed with buffer C , and eluted in buffer D . Protein concentration was estimated using ε32P]ATP and T4 polynucleotide kinase. Excess ATP was removed by a Microspin column. The radiolabeled strand and the complementary strand were annealed in 50 mM Tris-HCl (pH 7.6), 50 mM KCl, 1 mM EDTA, and 10% glycerol to generate a double-stranded DNA substrate with the lesion. Annealing was confirmed using a native gel.Oligonucleotides were purchased from the Keck Oligo Synthesis Resource at Yale University and gel purified. The following sequences were used in this study: 5’ CGTTCAACGTGCACTXACAGCACGTCCCAT and the complementary 5’ ATGGGACGTGCTGTYAGTGCACGTTGAACG (X is either 8-oxoG or THF and Y is either a templating cytosine or adenosine). The lesion-containing DNA strand was phosphorylated at the 5’ terminus using [γ-Kd) were extracted using Equation Bmax is the maximum bound fraction.DNA containing either 8-oxoG or THF 0.1 nM) was incubated with OGG1 at a concentration range of 0.5 to 4000 nM for 15 min at 4 °C in 25 mM Tris-HCl (pH 7.6), 175 nM NaCl, 1 mM EDTA, 1 mM DTT, 300 μg/ml bovine serum albumin, and 12% glycerol. Free DNA was separated from bound DNA by running the samples on a 10% native gel in Tris/Borate/EDTA buffer at 150 V for 2 h at 4 °C. Fraction-bound DNA (Y) was plotted as a function of OGG1 concentration (X). Dissociation constants ( nM was it). Points were fitted to a single-exponential equation (Equation kobs) for each of the mutants.Reactions were initiated by rapidly mixing 500 nM OGG1 with equal volumes of 50 nM DNA  at 37 °C in 50 mM Tris-HCl (pH 8.0), 20 mM NaCl, 2 mM DTT, and 10% glycerol. The reaction was quenched using a KinTek rapid quench-flow apparatus for short time points and by hand for time points longer that 120 s. The abasic site formed as a result of the glycosylase activity was quenched with 700 mM NaOH and then heated at 85 °C for 5 min. Reaction product was separated by gel electrophoresis using a 20% polyacrylamide gel containing 6 M urea. Gels were exposed to a phosphor screen, and the signal was detected by phosphorescence emission. The intensities of the product and substrate bands were quantified using ImageQuant , and the fraction product formed was plotted as a function of time  was estimated by fitting the points to a sigmoidal equation.The secondary structure of 6 μM OGG1 in 10 mM dibasic sodium phosphate was measured using the Applied Photophysics circular dichroism spectrometer at 195 to 250 nm. Thermal stability of the secondary structure was measured at 208 nm from 25 to 55 °C, and the melting temperature (All data are contained within the article. Raw gels are also available upon request.The authors declare that they have no conflicts of interest with the contents of this article.


As cervical cancer is one of the most common malignancies in women worldwide even with present screening methods, the incidence in most developed countries is not decreasing for the last 15-20 years. A shift has been observed in the age of diagnosis in favour of younger women, and treatment of already developed cervical cancer is a challenge for surgeons. It is imperative to find new diagnostic methods for accurately pointing out patients at high risk of developing malignant disease and developing personalized treatment. Since cervical cancer is almost exclusively associated with HPV infection, understanding changes happening in an infected cell may prove invaluable for search of such methods, but it may also prove helpful in the diagnosis and treatment of other anogenital and nasopharyngeal region cancers. This review follows HPV-related changes in infected cell biology to point what potential markers and targets for therapy are in option when dealing with HPV-related diseases. Papillomaviridae (PV) family, which includes over 200 genotypes specific to humans [L1. Taxonomy of PV is based on the nucleotide sequence of L1. Different genotypes of HPV present different affinities to either squamous epithelium or mucosal membranes, where most commonly they induce low-grade squamous intraepithelial lesions (LSIL), which regress within 2 years in 90% of cases [Cervical cancer is the 5th most common malignancy in women, with over 560 000 cases annually being diagnosed . The inco humans , 4. TheyIn some cases, progression to high-grade squamous intraepithelial lesions (HSIL), which are treated as a direct precancerous state, may occur. In this case, surgical intervention is necessary, as potential regression of these lesions is rare, as presented in Taxonomy of Papillomaviridae consists of 5 genera: Alpha, Beta, Gamma, Mu, and Nu. All of them have different tropisms . Most of the types from the Gamma genre have no oncogenic potential. This is associated with the ability of the virus to complete its life cycle within the maturing epithelial cells and release new virions to infect another host, without causing any symptoms like condylomas or intraepithelial neoplasia despite being present in mucosal or epithelial cells –8. This As HPV infection is the necessary but insufficient factor in cervical carcinogenesis, several other factors are at play in this oncogenic process : other sEarly genes found in the E regionE1 and E2 that modulate and initiate viral replicationRegulatory genes E5, E6, and E7 that play an important role in cancerous transformation of infected cellsOncogenic genes L1 and L2Late genes found in the L region, which code structural proteins forming viral capsid: Noncoding fragment that performs a control function (long control region (LCR)) –27In the HPV genome, the following genes are distinguished  viral DNA , 28, 29 ogenesis .The importance of immune system in cervical cancer may be proven on the example of patients with immune deficiencies. HIV infection compromises host immune response and accelerates cervical carcinogenesis. The risk of cancer in HIV-positive population is increased 10-fold (in comparison to seronegative women) . HPV inf2+ intracellular pathway, with MMP-9 being present in up to 100% of patients with HSIL or SCC [As progression to high-grade lesions occurs, CD8+ lymphocyte infiltration in the epithelium, as well in stroma, may be observed. The presence of CD8+ T cells is more frequent in regressing lesions supporting their positive role in self resolution of HPV infection . ChemoatL or SCC , 41. AnoL or SCC . This paL or SCC . This efα, β, and γ, with an ability to induce the JAK/STAT signalling pathway, essential for cell division [HPV-inflicted inflammatory response of the stroma may also induce cell proliferation through IL-2. Cervical cancer cells were proven positive for interleukin 2 receptor (IL-2R) subunits division . Howeverdivision . High ledivision . Along wdivision , thus podivision , 47.Retinoblastoma protein (pRB) is a suppressive protein, with an ability to arrest cell cycle . In a noIn HPV-infected cells where E6 and E7 proteins are expressed, this cell cycle control mechanism is lifted and cell replication is uncontrolled. The level of p53 rises after host cell DNA damage  but the E7 protein binds to unphosphorylated pRB and releases an active E2F transcription factor, initiating transcription and progression from the G1 phase to the S phase of the cell cycle  Figure .A suppressive pRB effect on p16INK4A is lifted, and overexpression of p16 protein may occur. Moreover, E7 inactivates p21 and p27 proteins and upregulates AKT activity . AKT hasAfter initial activation of JAK/STAT3 in high-grade intraepithelial lesions, development of cancer shifts those changes. Cancer cells lose receptor chain binding IL-6, and STAT3 is no longer expressed in cancer cell nests. This leads to induction of interferon regulatory factor 1 (IRF-1)—a proapoptotic transcription factor. With higher levels of IHC expression of IRF-1, response to chemotherapy and immunotherapy was observed to be better –60, thusα) (overexpressed in cancer cells) is degraded by proteasomes. However, this was observed only for normoxic HPV-positive cells [α which is also hypoxia dependent [α through regulated in development and DNA damage response 1 (REDD1) inhibits mTORC1 signalling and stops senescence in case E6/E7 is repressed [Phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB)-AKT/mammalian target of rapamycin (mTOR) signalling pathway has a key role in cell cycle regulation and is crucial for surviving cellular stress. Its disruption plays an important role in tumor development . With thve cells . Hypoxiaependent . HIF1α tepressed . This prepressed . Inhibitepressed . Even thepressed . It is pepressed . The pitepressed ; althougepressed , 69.As the cell cycle becomes deregulated in precancerous lesions and cancer, it is widely agreed that aneuploidy will occur in those cells with a question whether it is an effect or the cause of carcinogenesis , 71. ArgSince the discovery of relation between HPV presence and carcinogenesis, we have learned a great deal of biology and molecular interplay between host cells and HPV genome. Introduction of prophylactic vaccines brought another breakthrough in decreasing HPV-related cancers. However, it will not be possible to completely eliminate HPV infections. This is why further understanding of HPV-related cellular physiology adjustments is a key point in developing new diagnostic and therapeutic methods. It is crucial to evaluate diagnostic methods to prevent overtreatment of patients with a high chance of self-resolution of HPV infection on the one hand and accurately planning treatment in patients with already developed cancer for achieving highest possible disease-free survival (DFS) on the other. It may prove to be difficult when taking into account that most HPV infections are caused by more than one genotype, which may alter molecular changes observed in vitro in artificial cell lines.Detection of E6 and E7 mRNA presence is used in diagnosing HPV infection for some time now. Even though methods detecting HPV DNA are of the widest clinical usage, there is arising data pointing to detection of presence of p16 overexpression to be useful in increasing cytological primary screening accuracy.(1)Presence of CD8+ T cellsPositive prognosis markers for precancerous lesions—high chance of regression—useful in young patients where surgical treatment could be postponed or dropped altogether(2)αMMP-9, Th17 lymphocyte presence, IL-6, and HIF1Negative prognosis markers for precancerous lesions—high chance of progression(3)PD-L1Prognostic markers(4)IRF-1, IL-2, and PI3K/AKT/mTOR pathwayPredictive markers and targets for personalized therapyOur conclusion is that the following markers are eligible for further inquiry in clinical conditions:


In 2013, Jinan KingMed Diagnostics (JKD) first established a systematic cervical cytology training and quality control (QC) program in Shandong Province, China. We compared the efficacy of high-risk human papillomavirus (HR-HPV) detection, cytology, and their combination in routine clinical practice after the implementation of the training and QC program to identify the optimal first-line screening method in this region.The data of patients histologically diagnosed with cervical intraepithelial neoplasia (CIN) 1, CIN2/3, and invasive cervical cancer (ICC) between January 2014 and December 2017 were retrieved from the JKD database. Cytology and/or HR-HPV testing results within 3 months preceding the CIN1 diagnoses and 6 months preceding the CIN2/3 and ICC diagnoses were analyzed.P = 0.105) and ICC detection . Compared with either method alone, co-testing slightly increased the screening sensitivity for CIN2/3  and ICC  detection. In the CIN1 group, cervical cytology alone (92.9% [520/560]) was more sensitive than HR-HPV testing alone , and co-testing (95.3% [530/556]) did not significantly improve the screening sensitivity (P = 0.105).Prior screening data were available for 1829 CIN1 patients, 2309 CIN2/3 patients, and 680 ICC patients. Cytology alone and HR-HPV testing alone had similar rates of positive results for CIN2/3 (97.2% [854/879] vs. 95.4% [864/906], After the implementation of a systematic training and QC program, both cytology and HR-HPV testing may be adopted for primary cervical cancer screening in Shandong Province. Cervical cancer is the third most common malignancy in women, and approximately 85% of all cases of cervical cancer occur in low-resource countries, where there is a lack of trained personnel for cervical cancer screening . CervicaPersistent high-risk human papillomavirus (HR-HPV) infection, especially with the HPV-16/18 genotypes, is the leading cause of cervical cancer and its precancerous lesions , 11. IniCervical cancer is highly prevalent in China. In 2012, Chinese women accounted for 12% of all new cases and 11% of all deaths due to cervical cancer in the world . Both thKingMed Diagnostics (KD) is the largest independent operator of pathology laboratories in China, and has established China’s first ever training school for cytopathologists and formulated cytological QC measures at its headquarters in Guangzhou, in accordance with the College of American Pathologists (CAP) requirements . Jinan KJKD provides clinicopathological services to more than 900 hospitals, physical examination centers, and community clinics throughout Shandong Province. After a formal approval by KD’s ethics review board, patients with histological diagnoses of CIN1, CIN2/3, and ICC were identified from the pathology databases of JKD over a 4-year period from January 2014 to December 2017. In this study, we included only the cytology and/or HR-HPV testing results obtained within the 3 months preceding a CIN1 diagnosis or within the 6 months preceding a CIN2/3 or ICC diagnosis. The majority of the cases included in this study were collected from local hospitals, physical examination centers, and community clinics that serve mainly suburban and rural areas, where a large number of clinicians are not specially trained or qualified. Cytology alone, HR-HPV testing alone, and co-testing with cytology and HR-HPV testing were all used as screening modalities. The diagnoses of CIN1, CIN2/3, and ICC were rendered by histopathological examinations, including cervical biopsy, endocervical curettage, diagnostic excisional procedures, and hysterectomy. All LBC and histopathological examinations were performed at the Pathology Department of JKD, while HR-HPV detection was performed at the Molecular Department of JKD. Cytology and/or HR-HPV testing data from other hospitals or laboratories were not included in this study.LBC preparation was carried out strictly in accordance with the manufacturers’ instructions , 27, 28.JKD has established the largest standardized molecular laboratory in Shandong Province and has obtained the International Organization for Standardization certification for molecular diagnosis to ensure the diagnostic accuracy of molecular testing. Nearly 200,000 samples are tested for HPV every year in the JKD laboratory. In this study, all HR-HPV tests were carried out in the standardized molecular laboratory of JKD by using one of two methods: Hybrid Capture 2  and HPV genotyping  –29. The 2 test was used to compare differences in categorical data, and the one-way analysis of variance were used to compare differences in continuous data, with the Bonferroni test being carried out where appropriate. P < 0.05 was considered statistically significant.Statistical analysis was conducted using SPSS software . The Pearson χP < 0.001) and CIN2/3 patients (P < 0.001), but no significant difference was observed between the average ages of the CIN1 and CIN2/3 patients (P = 0.846). In each of these three groups, no significant differences in average age were found between the different screening modalities.During the 4-year study period, CIN1, CIN2/3, and ICC were histologically diagnosed in a total of 1829 patients, 2309 patients, and 680 patients, respectively, who had undergone prior cytology and/or HR-HPV testing (Table P < 0.001) and ICC groups . However, this rate did not differ between the CIN1 and ICC groups (P = 0.083). LSIL was the most common abnormal cytological result in the CIN1 group (reported in 65.4% of patients), followed by ASC-US (20.5%), HSIL (5.2%), and ASC-H (1.8%). In contrast, HSIL was the most common abnormal cytological result in the CIN2/3 (52.7%) and ICC (48.3%) groups. The other abnormal results in the CIN2/3 group were LSIL (24.1%), ASC-H (12.2%), ASC-US (8.0%), cancer cells (0.2%), and unsatisfactory cellularity (0.1%). In the ICC group, cancer cells (16.1%) was the second most common cytological interpretation, followed by ASC-H (16.1%), NILM (8.3%), AGC (7.0%), ASC-US (5.2%), unsatisfactory cellularity (2.6%), and LSIL (0.4%).The detailed results of cytology alone are summarized in Table P = 0.112) but significantly higher than that in the CIN1 group . The rate of positive HR-HPV results was similar for the two HPV testing methods in the CIN2/3  and ICC groups . In the CIN1 group, however, HC2 testing resulted in a significantly higher rate of positive results than did genotyping .Among patients who underwent only HR-HPV testing using the HC2 assay, the final histological diagnoses were as follows: CIN1, 309 (53.8%) patients; CIN2/3, 499 (55.1%) patients; and ICC, 69 (31.4%) patients (Table P = 0.008) and ICC groups . The overall HR-HPV prevalence in the CIN2/3 (92.7%) and ICC (94.8%) groups was similar to each other (P = 0.718) but significantly higher than that in the CIN1 group . HC2 testing and genotyping detection had similar rates of HR-HPV detection in the CIN1 , CIN2/3 , and ICC  groups. The rate of abnormal findings on co-testing was significantly higher in the CIN2/3  and ICC groups  than in the CIN1 group (95.3%), but the rates were similar in the former two groups (P = 0.549).Among the patients who underwent both cytology and HR-HPV co-testing, the histological diagnoses were as follows: CIN1, 556 patients; CIN2/3, 524 patients; and ICC, 230 patients. HC2 testing was performed in 194 (34.9%) of the 556 CIN1 patients, 213 (40.6%) of the 524 CIN2/3 patients, and 41 (17.8%) of the 230 ICC patients; the remaining patients underwent HPV genotyping (Table P < 0.001). In the same group, co-testing  was significantly more sensitive than HR-HPV testing alone (P < 0.001) but only slightly more sensitive than cytology alone (P = 0.105). In the CIN2/3 group, the sensitivity of cytology alone  was marginally higher than that of HR-HPV testing alone . However, the sensitivity of the combination  was significantly higher than that of cytology alone (P = 0.001) and HR-HPV testing alone (P < 0.001). In the ICC group, HR-HPV testing alone  was slightly, but not significantly more sensitive than cytology alone , while co-testing  was significantly more sensitive than cytology alone (P < 0.001) and HR-HPV testing alone (P < 0.001). Furthermore, when 6 cases of unsatisfactory cytological interpretation were excluded (these cases needed to be resampled according to the guidelines [P = 0.636).A comparison of the sensitivities of cytology alone, HR-HPV testing alone, and co-testing with both cytology and HR-HPV detection is shown in Table idelines , 15), cyThe present study demonstrated that cervical cytology alone and HR-HPV testing alone had similar efficiency for CIN2/3 and ICC detection, while cervical cytology alone was significantly more sensitive than HR-HPV testing alone for CIN1 detection. Additionally, the study showed that compared to either method alone, combined co-testing could further slightly increase the screening efficiency for CIN1, CIN2/3, and ICC. All of the above data were obtained after the implementation of a systematic training and QC program for cervical cytology screening in Shandong Province, China, and the population of women tested had not previously undergone intensive screening. These results suggest that a systematic cervical cytology training and QC program can improve the screening efficiency of cervical cytology for the detection of CIN1, CIN2/3, and ICC, so that it is equivalent to or even slightly higher than that of HR-HPV detection.A biopsy diagnosis of a CIN2/3 or worse lesion is the clinical threshold leading to ablative or excisional therapy. The treatment of CIN1 lesions, which have substantial rates of spontaneous regression, is discouraged, particularly in adolescents , 30. HowOur data revealed that the rate of abnormal results was marginally higher for cytology alone (97.2%) than for HR-HPV testing alone (95.4%) in the CIN2/3 group, which is comparable to the data from the Cytopathology Department of Guangzhou KD (GKD), which is the headquarters of KD with full CAP certification in China . GKD is The present screening data showed that HR-HPV testing alone (92.7%) was only slightly more sensitive than cytology alone (89.1%) and without statistical significance in ICC patients, especially when cases of unsatisfactory interpretation had been excluded (91.5%). One retrospective study of GKD data  examinedCo-testing has been shown to not only detect significantly more CIN2/3 or worse lesions but also results in significantly lower rates of ICC and its precursor lesions in subsequent rounds of screening , 14, 17.As a cervical cancer screening method, cytology has several advantages, such as simple preparation, low infrastructure requirements, and cost-effectiveness. In addition, the detection of different cytological abnormalities is helpful for the clinical triage of patients. Even now, cervical cytology (both LBC and CPS) is widely used for the large-scale screening and follow-up of high-risk groups, especially, in areas with poor economic conditions in China , 10, 25.In addition to accelerating the training of cytopathologists and establishing a cytological QC system to improve the efficiency of cervical cytology, it is necessary to seek alternative screening methods and gradually reduce the dependence on qualified cytopathologists. Compared with cytological screening, HR-HPV detection is easy to automate and establish QC processes for, which would ensure the accuracy of screening. Therefore, HR-HPV testing as a first-line screening method for cervical cancer has high application value in areas where there is a lack of qualified cytopathologists in China. However, we should also note that HR-HPV detection alone as a first-line screening method for cervical cancer has some limitations. Usually, most of the HPV load will automatically be eliminated by the host immune system within 8–10 months , 3. OnlyIn conclusion, the results of this study show that the screening effectiveness of cervical cytology after the implementation of a systematic training and QC program was similar or even slightly higher than that of HR-HPV testing for the detection of CIN1, CIN2/3, and ICC in a largely unscreened population from Shandong Province, China. The experience of JKD can provide a good example to create training programs for cytopathologists and QC standards for cervical cancer screening in this region. In addition, new screening methods, such as HR-HPV detection, can be adopted as a favored alternative to cervical cytology, which may gradually reduce the dependence on cytological screening and rapidly popularize cervical cancer screening among women in low-resource settings such as rural areas from Shandong Province, China.


The oropharynx has become the leading primary site for Human Papilloma Virus (HPV)-associated head and neck cancer. HPV positive oropharyngeal squamous cell carcinoma (HPV+ OSCC) has emerged as an epidemic not easily recognized by many physicians, resulting in delays in diagnosis and management. HPV+ OSCC traditionally refers to younger, healthier patients with high economic status and high-risk sexual behavior and is related to improved prognosis. De-intensification strategies are being evaluated in ongoing clinical trials and if validated, might help spare severe morbidity associated with current cisplatin-based chemoradiotherapy, which is the standard of care for all patients with locally advanced head and neck cancer. On the other hand, whether HPV status represents an important prognostic factor for non-oropharyngeal sites remains to be elucidated. High-risk Human Papillomaviruses (HPVs) are associated with the development of premalignant lesions and certain cancers due to their high oncogenic potential. Indeed, HPV is involved in head and neck cancer carcinogenesis and represents an important contributing factor particularly in cancer of the oropharynx. In developed countries, HPV currently accounts for the majority of patients with primary oropharyngeal squamous cell carcinoma (OSCC), taking precedence over traditional etiological factors, such as smoking and alcohol . Most imCDKN2A gene (cyclin dependent kinase inhibitor 2A) [p53, Rb up-regulation and p16 down-regulation [HPV life cycle is highly dependent on the cellular procedures of the host cell. Initially, HPV virus infects actively dividing basal keratinocytes and is subsequently subjected to viral genome amplification . Viral gitor 2A) . P16 noritor 2A) ,12. On tgulation . p53 (84%), mutation or loss of CDKN2A (58%), amplification of CCND1 (31%), which is an oncogene implicated in cell cycle regulation, amplification of MYC (14%) and overactivation of PI3K kinase pathway (30%) [TP53 mutations (3%) or CDKN2A alterations (0%). The aforementioned E6 and E7 viral proteins functionally inactivate p53 and pRb in HPV+ HNSCC [EGFR and CDKN2A genes. Moreover, mutations or alterations in the PI3KCA gene are commonly observed in HPV+ OSCC. On the other hand, HPV+ tumors are characterized by loss of TRAF3 (TNF Receptor-Associated Factor 3) (22%) and amplification of E2F1 (19%) [Interestingly, molecular data from recent genomic studies demonstrate that HPV+ and HPV negative (HPV-HNSCC) have distinct genomic profiles. The Cancer Genome Atlas (TCGA) reported the first results of sequencing data from HNSCC biopsies in 2015 . It is way (30%) . On the V+ HNSCC . In addiV+ HNSCC . On the F1 (19%) ,14.E6 oncogene, overactivation of PI3KCA and worse outcome, whereas the inflamed mesenchymal (HPV IMU) subtype is mainly correlated with profound immunogenicity and mesenchymal cell differentiation. However, this classification has not yet been incorporated in clinical practice [Of note, there has been a trend to categorize HPV+ OSCCs in clinical and prognostication subgroups based on their gene expression clustering. The classical or keratinocyte differentiation (HPV KRT) subtype includes cancers with primarily keratinocyte differentiation, higher proportion of spliced practice ,19. In apractice . HoweverImportant data also suggest a role of HPV integration. As described in detail earlier, initially in the HPV life cycle, HPV DNA is found in episomes. Carcinogenesis involves the integration of HPV viral DNA into the host cell genome, which leads to high expression of viral oncoproteins E6 and E7 . HoweverGenome-wide association analysis has demonstrated that genomic factors contribute to susceptibility to HPV+ OSCC. More specifically, the Class II haplotype DRB1*1301-DQA1*0103-DQB1*0603 has been shown to have a protective effect over HPV+ OSCC as compared to HPV− OSCC . These gMost importantly, HPV genomics that refers to sequencing of the HPV genome has emerged as an interesting field of research and has provided robust data regarding HPV16 genetic variation and cervical carcinogenesis, molecular characterization and characteristics of cervical cancer ,25. FutuDetermination of HPV status in OSCC is fundamental both as a contributor of diagnosis and a prognostication factor. HPV status has been established as an important and versatile biomarker and routine HPV testing is universally recommended by the College of American Pathologists (CAP), the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) and the National Comprehensive Cancer Network (NCCN) guidelines. CAP released formal guidelines in 2018 . Some crHPV status is currently an obligatory stratification factor in clinical trials of OSCC and it is used to determine eligibility for trials evaluating treatment de-escalation. Surprisingly, a substantial proportion of OSCC cases in the US remain untested, mainly due to lack of robust predictive significance and cost-effectiveness. In a recently published study that used National Cancer Database, it was revealed that 12% of OSCCs in the US were not tested between 2013 and 2015, mainly due to insurance issues (patients with private insurance were more likely to be tested) and diagnosis in low-volume hospitals, as large or academic centers were more likely to recommend testing. In addition, testing was performed less frequently in women and older people .E6/E7 HPV RNA-ISH, HPV DNA polymerase chain reaction (PCR) and E6/E7 HPV RT-PCR [Several methods are used to detect the presence of HPV including p16 immunohistochemistry (IHC), HPV DNA in situ hybridization (ISH), V RT-PCR . P16 IHCV RT-PCR . SeveralV RT-PCR .E6/E7 mRNA via PCR in tissue is deemed the gold standard, given the high sensitivity and feasibility of this method; nevertheless, its sensitivity is significantly decreased once the sample is of poor quality. RNA-ISH is currently being used more frequently, particularly in combination with p16 [Regarding the remaining HPV-specific testing modalities, each of them has several advantages and disadvantages. It is currently clear that DNA detection alone is not adequate for evaluation of HPV status, mainly due to low sensitivity . Most imwith p16 . Obviouswith p16 .Subsequently, HPV detection should be confirmed in p16+ cases using RNA PCR or HPV DNA detection strategies. In a study by Schache et al., the authors used several detection strategy combinations to evaluate HPV16 status in fixed and fresh frozen tissue derived from 108 OSCC cases. It was found that the combination of p16 IHC and DNA quantitative PCR (qPCR) yielded the highest sensitivity (97%) and specificity (94%) compared to the gold standard RNA qPCR . UnfortuDespite a steady decline in the incidence of non-oropharyngeal HNSCC during recent decades, we have been witnesses of an overall increase in the incidence of OSCC following the rise of OSCC cases attributed to HPV infection . From 19HPV16 accounts for almost 90% of OSCC cases . This suClinical presentation of HPV+ OSCC differs significantly from tobacco-related HNSCC, which traditionally affects older adults with tobacco or alcohol abuse . On the HPV+ OSCC display a significantly better prognosis compared to tobacco-related HNSCC and the risk of death has been reported to be approximately 60% of that of HPV− OSCC . ImproveThe latest edition of the AJCC/UICC tumor classification system has been reported in 2018 after assessment of research focused on molecular pathogenesis and prognostic factors of head and neck cancer . The devThis emerging HPV-specific classification, which represents the urgent need to appropriately portray the nature and prognosis of the new disease, is essential in many aspects. First, it is useful for stratification in de-escalation trials focused on HPV+ disease; second, it is clinically relevant for discussion with patients to properly understand the stage and prognosis of the disease. Last, it is a necessary tool for the future, as separate management recommendations may be applicable in HPV+ and HPV− tumors.The 8th edition of the clinical and pathological TNM staging is illustrated in In western countries, the gold standard for patients with localized or locally advanced OSCC is combined cisplatin-based chemotherapy and radiation, excluding patients with early-stage disease who may be qualified for surgical intervention. Chemoradiation has demonstrated impressive results, with a 5-year overall survival of 95% in HPV+ patients . In deveThe idea of replacing cisplatin, a toxic chemotherapy agent, with cetuximab, a monoclonal antibody against EGFR in locally advanced OSCC has been developed rather early, after the IMCL 9815 trial, that compared cetuximab in combination with radiotherapy with radiotherapy alone has shown good effectiveness in patients with OSCC . In thisHerein, we discuss several pitfalls of the De-Escalate HPV Trial. First, it included only patients with low-risk “favorable” HPV+ OSCC subgroup. This group selection was arbitrarily based on two parameters: a) the 7th edition of TNM AJCC/UICC staging system  and b) the stratification by Ang et al. in their analysis of the RTOG 0129 study cohort of HPV+ OSCC patients . Ang et p = 0·5056 for non-inferiority; one-sided log-rank p = 0·0163) and patients in the cisplatin arm showed superior OS (5-year OS 84.6% vs. 77.9% in the cetuximab arm) [On the other hand, Gillison et al. reported the results of the RTOG 1016 trial, a non-inferiority trial that randomized 849 “all comers” with HPV+ OSCC to receive either cisplatin or cetuximab in combination with IMRT . Althougmab arm) . In addimab arm) .2 and LN dissection was required after chemoradiation because pathologic complete response (pCR) was the primary endpoint, whereas in the second trial RT was administered in combination with either weekly cisplatin or weekly cetuximab and primary endpoint was 2-year Progression Free Survival (PFS) [These two randomized trials that yielded rather disappointing results have clearly shown that it is not the proper time, at least not yet, for de-intensification strategies for any HPV+ patient outside of clinical trials. However, this should not be the end of treatment de-escalation in locally advanced HPV+ OSCC. The lack of predictive biomarkers of response to cetuximab hampers proper design of clinical trials. However, other de-intensification strategies have shown good results in phase II trials. Chera et al. conducted two trials evaluating reduced radiation  in patients with low-risk HPV+ OSCC  ,53. In tal (PFS) ,53. Bothal (PFS) ,56, or ≥al (PFS)  to IC anE6/E7 oncogene expression by RT-PCR and p16 was assessed by IHC. It was shown that 3.7 % of patients were HPV16 positive and 2.2% were positive for other HR HPV subtypes. P16 IHC had good sensitivity (79.2%), excellent specificity (93%) and high negative predictive value (98%) but low positive predictive value (41.3%) for HPV detection [It is widely known that HPV and p16 are prognostic biomarkers for HPV+ OSCC. However, several studies have pointed out the prevalence of HPV infection in non-oropharyngeal areas, such as the oral cavity, larynx, and oropharynx. More specifically, 3%–5% of oral cavity and laryngeal cancers are HPV+ . The queetection .Studies suggest that there are differences regarding the role of p16 and HPV positivity in non-OSCC HNSCC. Chung et al. assessed HPV status by ISH and p16 expression by IHC in patients with non-OSCC HNSCC  included in three RTOG studies  . It was More recently, Fakhry et al. retrospectively tested HNSCC cases diagnosed between 1995 and 2012 for HPV status using DNA and RNA-ISH and p16 status by IHC. In non-OSCC patients, p16 and HPV status were not found to be prognostic . SimilarAltogether, studies focusing on prognostic impact of HPV status in non-OSCC HNSCC have shown conflicting results. In addition, p16 has been shown to be a poor surrogate biomarker for HPV infection in these cases. Thus, routine HPV testing is not recommended for non-OSCC cases. Prospective studies are required to elucidate the potential role of HPV status in non-OSCC HNSCC.HPV+ OSCC currently represents a global epidemic, a widely recognized increasingly prevalent entity. Similar to cervical cancer, the majority of HPV+ cases are attributed to high-risk HPV16 subtype. P16 positivity serves as a surrogate marker for HPV infection and is an independent prognostic factor for those patients. In addition to the differences in risk factors and clinical presentation, HPV+ OSCCs display a substantially improved survival. Thus, HPV status is now routinely used as a stratification factor in clinical trials and is a clinically relevant parameter for patient consultation. This has been depicted in the new edition of AJCC/TNM staging that includes a separate staging manual for HPV+ OSCC. However, randomized trials evaluating de-intensification strategies substituting cisplatin with cetuximab have failed to show non-inferiority in terms of outcome and current guidelines do not recommend a different treatment approach for HPV+ OSCC. HPV status has no confirmed prognostic role for non-OSCC HNSCC. Future studies are necessary to establish the role of de-escalation treatment approaches in HPV+ OSCC. Until then, the cornerstone of treatment is an interdisciplinary team approach with emphasis on quality of life during the treatment and survivorship periods.


Human papillomavirus (HPV) has been reported recently in surgical smoke generated by gynecological operations. The objective of this study was to investigate whether gynecologists who have performed electrosurgery including loop electrosurgical excision procedure (LEEP), are at risk of acquiring HPV DNA through surgical smoke.A related questionnaire was designed and 700 gynecologist nasal swab samples were collected in 67 hospitals. In addition, the flow fluorescence hybridization technique was used to detect HPV DNA, and the Chi-square test was applied to analyze whether related risk factors including electrical surgery, were correlated with HPV infection in surgeons’ nasal epithelial cells.P = 0.016). Additionally, the HPV detection rate was significantly lower in electrosurgery operators who used surgical mask  than in those who did not use protective masks . Furthermore, the N95 mask  significantly reduced the risk for HPV infection compared to that with the general mask . Furthermore, 46 participants infected with HPV were followed-up for 3–24 months, and approximately 43.48% (20/46) and 100% (41/41) became negative for HPV DNA, respectively.The HPV infection rate in the nasal epithelial cells of the participants who performed electrosurgery  or LEEP  was significantly higher than that in the remaining participants who did not perform electrosurgery  or LEEP , respectively. The most prevalent HPV genotype in the electrosurgery group was HPV16 . The HPV-positive rate was increased in the group that had a longer duration of electrosurgery  contains supplementary material, which is available to authorized users. Cervical cancer is the third most common gynecological cancer worldwide and one of the main reasons for cancer-related death in females in developing countries  who was on the scene when medical workers were canvassed through the self-administered questionnaire. Any uncertain items were queried by a quality control engineer (Tu Q.M.) by telephone.All the medical personnel voluntarily participated in the research and signed a written consent form. Participants were asked to provide nasal swab samples for DNA extraction and HPV detection. The nasal swab samples were collected by Zhou Q.F. and obtained from both sides of the operators’ nasal cavity with a depth of 2–3 cm using a sterile cotton swab which was then inserted into a 1.0 mL of phosphate buffered saline (PBS). The collected samples were subjected to HPV genotype detection by sequencing. All specimens were stored at − 20 °C immediately after collection.A high-purity viral nucleic acid kit  was applied to extract HPV DNA from human nasal epithelial cells. A total of 200 μL of digestion buffer was added to the sample solution. After incubation at 72 °C for 10 min, 100 μL of binding buffer was added and mixed immediately. After centrifugation, 500 μL of inhibitor removal buffer was added to the supernatant. The buffer was centrifuged at 8000 rpm for 1 min. The filter tube was inserted into a nuclease-free tube, and 50 μL of elution buffer was eluted in the purified DNA. Afterwards, the DNA was resuspended and stored at − 20 °C until needed  was applied to evaluate the quality of the DNA extracted by measuring the optical density (OD) of absorbance OD260/OD280 which followed the manufacturer’s recommendations. Additionally, the human β-globin gene from the collected specimen was amplified to monitor the quality of the DNA samples for subsequent DNA amplification.HPV DNA detection was performed using the Tellgenplex™ HPV DNA Test  according to the manufacturer’s instructions. Genotyping was carried out by a flow-through hybridization technique using HPV DNA amplified by polymerase chain reaction (PCR). This testing method can detect 27 HPV genotypes, including 17 high-risk HPV types , and 10 low-risk HPV types . Appropriate positive and negative controls were used in all PCR amplifications. The PCR system volume was 20 μL, including 5 μL of primer mixture, 9.2 μL of PCR premixed solution, 0.8 μL of Taq DNA polymerase and 5 µL of extracted DNA. In total, 40 cycles were performed  for each well. The negative control assay was performed at the same time. The product was detected by a Luminex 200 analyzer instrument . The HPV-genotype result was determined according to the position of the HPV-genotype probes on the microarray chip. Multiple dots indicate multiple infections.Surgeons whose nasal swab specimens tested positive for HPV DNA received additional HPV DNA tests for nasal swabs at 3, 12 and 24 months.P < 0.05.Statistical analysis was performed using SPSS 17.0 statistical software . Age is expressed as the mean and standard deviation (SD). A Chi-square test was conducted to identify the significance of differences between two groups. A logistic regression model was used to analyze the correlation between risk factors and HPV infection among doctors performing electrocardiosurgery of the cervix. There was statistical significance when A total of 700 gynecologists in 67 local hospitals throughout Zhejiang Province from January 2016 to December 2016 were recruited to the study for detection of HPV DNA by HybriMax; their mean age was 35.1 ± 8.8 years (ranging from 21 to 72). The included hospitals comprised 25 third-grade class-A hospitals and 42 township hospitals. In addition, the relevant hospitals included Women’s Hospital, School of Medicine, Zhejiang University; Zhejiang Provincial People’s Hospital; the First Affiliated Hospital of Wenzhou Medical University; the Second Affiliated Hospital of Wenzhou Medical University; Taizhou Hospital of Zhejiang Province; the Central Hospital of Lishui City; and others.As shown in Table P < 0.001).Among all the gynecologists, the prevalence of HPV was 6.57% (46/700), with 6.43% (45/700) of the participants having a single viral type and 0.14% (1/700) having multiple viral types. As shown in Table Additionally, the overall rate of HPV positivity among operators performing LEEP was further evaluated. As shown in Table P = 0.119) between the two groups.The difference in the HPV-positive rates between the LEEP group and the other electrosurgery group was further investigated. As shown in Table As shown in Fig. As shown in Fig. P = 0.083). In addition, whether an increasing duration of electrosurgery causes higher HPV infection rates was investigated. Participants who had performed electrosurgery for more than 15 years were more prone to HPV infection  than those who had performed electrosurgery for 0–5 years , 5–10 years  and 10–15 years . This result indicated that there was a significantly higher HPV risk in doctors who had performed electrosurgery for a longer period (P < 0.001).The possible risk factors for HPV positivity of operator nasal swabs were analyzed. As shown in Table P = 0.44). Most LEEP operators had over 15 years of LEEP experience. The HPV-positive rate was significantly higher in the group who had longer electrical surgery times with LEEP (P = 0.017).As shown in Table P < 0.001). As shown in Table P < 0.001). Moreover, the rate of HPV detection in LEEP surgeons who used surgical masks  was remarkably higher than that in surgeons who did not wear a mask during LEEP .Additionally, the most common protective measures during electrosurgery of the cervix included surgical masks and smoke suction devices. In our research, 432 doctors wore a protective mask during electrosurgery, and the remaining 37 participants  wore no surgical mask during the operation. The general mask was the most common protective mask , followed by the N95 mask . The HPV-positive rate was significantly lower in cervical electrosurgery operators who used a surgical mask  than in those who did not use a protective mask . Furthermore, use of the N95 mask in electrosurgery  significantly reduced the risk for HPV infection compared to that with the general mask . The other variable evaluated in this study was the risk consciousness pertaining to surgical smoke. Most of the participants  were aware of the hazards posed by surgical smoke to electrosurgery operators. However, the association between safety consciousness and rates of HPV positivity was not statistically significant (P = 0.642). As shown in Table P = 0.713, 0.642, respectively).Smoke absorbing devices were more commonly applied in electrosurgery . The use of smoke evacuation systems decreased the risk of HPV infection, but this difference had no statistical significance  who had been HPV positive in nasal swab samples turned negative. The other 26 doctors  still exhibited the same HPV genotypes in nasal swabs. Moreover, five participants who had a positive HPV test were withdrawn in the 24 months after the first survey. No HPV DNA could be detected in the remaining surgeons , which means that 100% (41/41) became negative for HPV DNA. This result indicated that the nasal HPV infection of surgeons was transient and not persistent. This may be because electrosurgery, including LEEP, caused inactivation of the HPV DNA in the surgical smoke. Therefore, although HPV DNA from surgical smoke was detected, it could not lead directly to HPV-associated diseases.Thirty-six gynecologists who performed LEEP and had a positive HPV test in nasal swabs were followed up. Approximately 47.22% (17/36) became negative for HPV DNA, and 52.78% (19/36) still exhibited the same HPV genotypes in nasal swabs in the 3 months after the first survey. One gynecologist was lost to follow-up at 24 months because of departure from the hospital, and 100% (35/35) of the other gynecologists became negative for HPV DNA.During the 24-month follow-up period, no HPV-related cancers or diseases, such as nasopharyngeal condyloma acuminatum, nasopharyngeal papilloma, and nasopharyngeal cancer, were found.In the current study, we found that (1) gynecologists who performed electrosurgery operations, including LEEP for cervical lesions, especially those who were exposed to surgical smoke for a long time, were at risk for HPV-DNA infection and that (2) surgical masks, especially the N95 mask, effectively decreased the hazard of acquiring HPV DNA from surgical smoke exposure during cervical electrosurgery, including LEEP.Electrosurgery  is one of the most common techniques in the diagnosis and treatment of gynecological diseases, such as various cervical lesions Below is the link to the electronic supplementary material.


Descriptive analysis, the chi-square test, and logistic regression analysis were used to answer different research questions. Health care providers’ overall knowledge scores are consistent with their perceptions about HPV and HPV vaccine. Occupation, specialty, the type of hospitals, and the frequency of participants’ search for information using professional informational sources are the most significant characteristics that are closely associated with their knowledge of HPV and its vaccine. Targeted educational interventions are needed to improve health care providers’ engagement in the promotion of the HPV vaccine. Such interventions, besides increasing knowledge, should also emphasize the importance of appropriate information sources to acquire information.The limited access to vaccination and vaccine hesitancy are prevalent even among health care providers in less developed countries. This study assessed the relationships between (i) health care providers’ knowledge on human papillomavirus (HPV) and vaccination for HPV and (ii) their perceptions, demographic characteristics, and the use of information sources. In this large-scale online survey, participants ( Human papillomavirus (HPV) is the most prevalent viral infection of the reproductive tract and is a major cause for cervical cancer . CervicaHowever, vaccine hesitancy, which is defined by the Strategic Advisory Group of Experts (SAGE) working group of the WHO, refers to delay in the acceptance or refusal of vaccination despite the availability of vaccination services . NowadayOne effective measure to overcome hesitancy among audiences and providers is to make providers thoroughly master knowledge of HPV and HPV vaccines. This way, they can have a good understanding of the benefits and risks of being vaccinated and thus have confidence to recommend vaccination to their patients. Therefore, it is necessary to examine the knowledge, perceptions, and information sources of HPV and its vaccine among health care providers.Being directly responsible for the recommendation and delivery of HPV vaccines to the public, the importance of health care providers has been emphasized by several studies ,11,12,13In terms of their subjective perception, some physicians tend to perceive that their knowledge is insufficient. When rating their knowledge about the risk of HPV infection and resultant cervical cancer, general practitioners (61%), obstetricians/gynecologists (28%), and pediatricians (56%) felt that their knowledge of this aspect was not sufficiently high . PhysiciRQ1: Are providers’ subjective perceptions consistent with their objective knowledge on HPV and the HPV vaccine? If not, is the variance significant?With respect to the acquisition of relevant knowledge, most surveys on this topic assessed whether providers’ knowledge is associated with their demographics. Particularly, specialty, hospitals’ attributes, and the experience with HPV and HPV vaccination are viewed as critical variables in physicians’ knowledge. For example, there was a greater possibility that obstetricians/gynecologists knew specific oncogenic strains of HPV than the general practitioners , and a sRQ2: Is the knowledge of providers associated with their demographic characters, especially specialty, hospital type, and practical experience? If so, which factors are the most significant?RQ3. Is the knowledge of providers associated with the source and frequency of information acquisition? If so, which information source predicts the most significant variance?The purpose of this study was to examine whether the knowledge of health care providers is consistent with their perceptions of HPV and HPV vaccine as well as whether the level of their knowledge is associated with their demographics, particularly their specialty, hospital attributes, practical experience, and the information acquisition frequency in three well-developed big cities of China.We conducted this survey in Shanghai, Guangzhou, and Shenzhen, China. Shanghai is a well-known international metropolis and China’s economic and trade center. Its rapid development makes people there more aware of the advantages of HPV vaccination and more receptive to new ideas and policies, especially those related to health. Guangzhou and Shenzhen are located at Guangdong Province in Southern China. Before the HPV vaccine had been officially approved in mainland China, the benefits of the HPV vaccine had been foreseen and introduced by commercial advertisements and word of mouth in Hong Kong and Macao, which are geographically close to Shenzhen and Guangzhou. In this way, many people living in these two cities had the convenience to go to Hong Kong or Macao to get vaccinated, so the acceptance of HPV vaccination in these two cities was relatively high. Moreover, Shanghai, Guangzhou, and Shenzhen are equipped with adequate and superior medical resources compared with most other cities in China. Thus, to investigate the health care providers in these three cities will lead to a better understanding of HPV and the HPV vaccine than in most other Chinese cities.It is very difficult to go into the Chinese hospitals to do an offline survey because of the administrative regulations and the busy work of the providers; therefore, some researchers have tried to conduct studies on Chinese medical personnel through online surveys. For instance, Zhang C and Liu Y conducted an online survey to study the salary of physicians in Chinese public tertiary hospitals, which implemented a self-administered questionnaire instrument using a smart phone platform (WeChat) . Niu ChiWe collected participants’ demographics by asking them about their personal information, including their city, gender, age, occupation, specialty, title, type of hospitals, and years of practical experience.We assessed participants’ perception and knowledge on HPV and its vaccine by a set of questions. The concept of perceptions sometimes related to their perceived knowledge about some facts, such as their perceived benefits or risks about HPV vaccines ; howeverFor the knowledge part, knowledge refers to participants’ actual knowledge level and is objective. Six questions about HPV and 4 questions about HPV vaccine were selected from the literature ,29,30,31During the design of the study, we wanted to assess the relationship between the knowledge level and the frequency of using different types of information sources, so we referred to available literature ,32 and tWe used SPSS  23.0 for analysis. Specifically, Pearson correlation analysis, chi-square, and logistic binary regression analysis were conducted.Pearson correlation analysis was conducted to answer RQ1. To answer RQ2, we examined the distribution of high and low knowledge scores for HPV (classified into the “higher” and “lower” groups) based on demographic characteristics of participants by the chi-square test. To realize the daily use of information sources by providers to acquire HPV and HPV vaccine-related information, descriptive statistics analysis was used. To answer RQ3, logistic binary regression analysis was conducted. The dependent variable is binary and categorized as “lower” and “higher” knowledge level of HPV or HPV vaccine; the independent variable is the frequency of different information sources use, scores of which range from 1 to 5.n = 1394), 66.3% were physicians and 33.3% were nurses ; of the respondents, 30.6%, 31.9%, and 37.3% were from Shanghai, Guangzhou, and Shenzhen, respectively . Regarding age, 18.3%, 47.0%, and 34.6% were 18–27, 28–37, and >38 years old, respectively ; majority were females . General practitioners, pediatricians, obstetricians, preventive care physicians, and physicians in other specialties represented 19.6%, 14.4%, 43.8%, 17.6%, and 4.5% of the study population . Primary and intermediate physicians both represented more than 40% , and senior physicians were a minority (11.5%) . Of the participants, 45.5% had worked for 0–9 years, 32.2% had worked for 10–19 years, and only 22.2% had worked for >20 years . Most participants worked in maternal and child health hospitals (39.0%) or community health centers (34.8%) , and about 30% of them vaccinated over 200 patients weekly .Of the participants (p < 0.01) and HPV vaccine knowledge scores were significantly correlated with the respondents’ perception of these aspects, and the overall knowledge score was similarly associated with the general perception (p < 0.01).The knowledge of respondents with regard to HPV and the HPV vaccine was surveyed by asking six and four questions, respectively. The mean score on HPV was 4.47, and 54.7% of the respondents achieved a score of 5 or 6 of 6. The mean score about the HPV vaccine was 3.12, and 75% of the respondents achieved a score of 3 or 4 out of 4. When asked “How do you judge your understanding or the level of your knowledge about HPV and HPV vaccine?”, around 52.2% and 46.5% of the respondents answered that they thought they know these two topics well (score 4) and excellently (score 5). The results were shown in As shown in As shown in The mean values (score range is from 1 to 5) of the frequencies of different kinds of information sources used are the following: 3.47 for professional sources , 3.21 for media , 3.04 for interpersonal discussion (including discussions with friends working in other fields and patients), and 2.70 for pharmaceutical companies. Clearly, professional and media sources are the most frequently used information sources for this topic.In this study, we analyzed the knowledge, perception of HPV and HPV vaccine, and the use of information sources for relevant resources from the health care providers’ perspective by conducting a large-scale survey with 1394 participants covering three big cities in China. First, we examined whether providers’ knowledge level were consistent with their perceptions about HPV and HPV vaccine; then, the variance of providers’ knowledge level based on their demographic characteristics such as city, specialty, and hospital type were analyzed according to 6 questions for HPV and 4 questions for HPV vaccine; finally, the relationship of providers’ knowledge level regarding HPV and its vaccine and their frequency of different information source use was verified.Nowadays, it has been emphasized repeatedly that receiving a recommendation from a health care provider is one of the most significant and consistent factors that influence the audience’s decision on HPV vaccination ,33,34. HGenerally, in this study, respondents had good knowledge of HPV and its vaccine, which fairly coincided with their own perceptions about their understanding of these topics.It is worth noting that far more providers from Shenzhen than from Shanghai and Guangzhou achieved higher HPV knowledge scores. However, it is evident that the medical resources and the level of medical treatment of the latter two cities are superior to those of Shenzhen according to China’s hospital rankings, which are professional and acknowledged widely in China . TherefoWith respect to different specialties where respondents worked, it is not surprising that most obstetricians and gynecologists and respondents who worked in preventive health care departments obtained better knowledge scores for HPV and its vaccine, respectively. However, most of the pediatricians obtained lower knowledge scores for both HPV and its vaccine, which would hinder their recommendation of vaccination to the parents of adolescents or teenagers who are the target group for vaccination promotion. Previous studies indicated that the pediatricians’ recommendation is a key factor that strongly influences the vaccination decisions of adolescents and parents for their children at the right age . HoweverIn terms of the type of hospital, it seems that participants who worked at general and specialist hospitals had the best understanding of HPV  respondents obtained higher scores on the knowledge of HPV vaccine. This difference may be caused by the fact that the participants who take charge of vaccination are normally younger. In this case, regular training about HPV should be offered to young and intermediate health care providers, which is a key step for reminding them of their deficiency on the knowledge of HPV pathology and inspiring them to take the initiative in this learning process. By this way, they can not only offer suggestions about vaccines to patients, but also can provide advice on the dangers of HPV. The resulting fears could probably be useful to decrease the potential vaccine hesitancy of the patients.Health care providers, as professionals, are the transmitters of medical information to the audience. Therefore, their knowledge and perceptions are important factors in influencing vaccine acceptability . Many stFurthermore, the use of more professional information sources was associated with more accurate HPV-related knowledge. Frequent discussions with peers on relevant issues could also be useful. Other information sources, such as media, may be crucial in information seeking, but could not facilitate a higher level of knowledge acquisition. Based on these results, we recommend that the physicians and nurses should search for knowledge from professional journals and other reliable professional sources.There were some limitations to this study. Due to the nature of the survey link distribution, we did not know the exact number of providers who has received the survey link or in fact browsed the link that we have provided. In addition, those who participated in the survey might be those who were interested in the topic and were more aware of the HPV situation in China and elsewhere. Thus, the results have limited generalizability and should be interpreted carefully. Another limitation of this study is that all the participants in our sample pool were health care providers from developed cities in China. More health care providers might be short of relevant knowledge in underdeveloped cities; we plan to investigate this population in our future studies. Additionally, all data collected in this study were self-reported, and that may generate bias.In this large-scale survey, we examined the health care providers’ knowledge and perceptions on HPV and the HPV vaccine, as well as whether their self-reported perceptions of their own knowledge were consistent with their knowledge in the context of vaccine hesitancy. We also analyzed whether their knowledge levels were associated with demographics, such as specialty, hospital attributes, practical experience, and the frequency of information acquisition from different sources. We found that participants’ perceptions of their knowledge of HPV and HPV vaccine were generally consistent with their knowledge scores. In addition, city, specialty, title, and type of hospital are significantly associated with the knowledge levels of health care providers either on HPV or HPV vaccine, or both, which reflects some phenomenon or shortage of current situations of HPV and HPV vaccination in China. The providers should use professional sources more often to search for knowledge on the two aspects.