This situation highlights the need to implement efficacious and inexpensive treatment methods. This study aims at evaluating the efficacy of a topical antiviral drug named AV2®or placebo. They will undergo control examinations after two months and after six months. In case of persistent lesions on VIA, treatment by cryotherapy will be done. The primary outcomes will be the change of lesions, the clearance of HPV DNA, and the correlation of the two 2 months after treatment with AV2®.Women will undergo cytology sampling, HPV testing and Visual inspection of the cervix after application of 5% acetic acid (VIA). VIA-positive women will be randomized to one of two groups to receive treatment by either AV2This study is the first large-scale study in Africa to evaluate systematically the efficacy and safety of a topical antiviral drug for the treatment of HPV— associated lesions of the cervix. Its findings will direct the planning of suitable algorithms for CC screening and treatment.ClinicalTrials.gov – Unique identifier: NCT02346227, registered on November 8, 2014. It is the most common cancer in women of Low and Middle Income Countries (LMICs) In the Democratic Republic of the Congo (DRC), 18.85 million women, aged 15 years and older are at risk of developing CC. Current estimates indicate that every year 6024 women are diagnosed with cervical cancer and 4719 die from the disease The link between human papillomavirus (HPV) infection and CC is well established Sauvaget et al. performed meta-analyses with different categories of studies to provide an updated estimation of the accuracy of visual inspection with acetic acid (VIA) in detecting true disease. The reference category consisted of 26 studies in which VIA was performed on asymptomatic women who all underwent confirmatory testing and in which the disease threshold was cervical intraepithelial neoplasia grade 2. They reported an 80% sensitivity  and a 92% specificity  for VIA. Study region, capacity of screener, or size of the study population did not modify VIA accuracy. In conclusion, it was shown that screening for precancerous and cancerous cervical lesions using VIA is a simple, low-cost, and efficient alternative to cytologic testing in low-resource areas. The more the severity of disease, the more sensitive and specific is VIA in detecting the disease In another meta-analysis by Sauvaget, Cryotherapy was shown to be an effective, safe, and acceptable treatment for CIN. Actually, on a equivalent of 28,827 cases of treated CIN, Cryotherapy achieved cure rates of 94.0% for CIN1, 92.0% for CIN2, and 85.0% for CIN3 Of all available and effective treatments of CIN, cryotherapy seems the most appropriate for LMICs, but is still hampered by major logistical problems such as unavailability of refrigerant gas. These feasibility issues highlight the need for a low-cost and minimally invasive method of treating CIN in LMICs ®, Cesa Alliance, Luxembourg) made of natural essential oils was developed and preliminary results suggest it to be effective in clearing or reducing the size of PLC related to HPV Recently, an antiviral drug called Antiviral 2  patients who received the active treatment, versus 0% for the comparable placebo pool. At the other end of the scale, only 2 (7%) participants in the AV2® group failed to respond positively compared to 80% of participants in the placebo group ® could offer minimally invasive treatment with minor morbidity and time constraints.In a recently published phase 2 clinical trial study conducted by Martinez et al. at Hospital de la Familia, in Juarez, Mexico, efficacy of AV222.1The present study is a double-blind randomized placebo-controlled clinical trial. It will include 400 patients in a health centre in Kinshasa, DRC: Centre Hospitalier du Mont-Amba. It is estimated to last 56 weeks considering final data collection for primary outcome measurements.2.2Inclusion criteria: The targeted population is all sexually-active women, aged at least 25 years old and who will give their informed consent to participate in the study.The reason for this target population is based on the natural history of HPV infection. HPV is acquired at the onset of sexual activity which occurs at around 15 years of age. Its prevalence is very high at a younger age and is often transient because most women will spontaneously eliminate the virus after some years. When it persists, HPV infection can take in average 10 years to lead to CC. Thus, testing is projected to start 10 years later after sexual debut in this case at 25 years old onward.Exclusion Criteria: Pregnant, breastfeeding and post-partum women will not be accepted in the study. In case of suspected or diagnosed cervical cancer, the women will be referred in a tertiary hospital for the proper management. Other exclusion criteria include: previous cervical surgery, medical history of any severe diseases, intake or application of other antivirals, known or suspected allergic or adverse response to the investigational product or its components and women who are unable to follow the study protocol.2.3Prior to the start of the study, a basic training of the doctors and nurses involved will be organized. This training will encompass Good Clinical Practice (GCP), collection of liquid-based cytology sample with cytobrushes, and VIA and cryotherapy procedures.® , immediately after LBC sampling, VIA will be performed. Its results will be reported according to the WHO criteria and will serve for randomization. The results for LBC and HPV testing will not be available at this moment.Women aged 25 and over will be invited to participate in a community cervical cancer screening programme. After giving their informed consent, eligible women will be examined by a trained doctor. The procedure will be performed while the woman lies in the lithotomy position on the examination table. After the cervix is exposed by a speculum inserted in the vagina, the doctor will collect a LBC specimen using the Thinprep PreservCyt solution® or placebo. All randomized women will be followed-up and recalled after two months for repeat LBC and VIA. Women remaining positive on VIA at this stage will be treated by cryotherapy.VIA-negative women will not be randomized and will receive no treatment. VIA-positive women will be randomized to one of two groups to receive either AV2At six months' follow-up, women will be assessed in the same manner regarding VIA and LBC. The study will end at this stage. At each stage, the examinators will be blind to the previous results concerning the patient. At the end of the study, All women who will test positive for cervical lesions or HPV will be properly treated according to standard protocols and resources.2.4VIA positive women will be randomized in a block wise manner, without stratification, to one of two treatment arms as shown in 2.5VIA involves the painting of the cervix with 5% acetic acid and the observation of the color change 1 min after. The most common form of reporting the VIA test outcome involves negative and positive categories, depending upon the absence or presence of acetowhite lesions and clinical signs of invasive cancer. A positive test is generally based on the detection of well-defined, densely opaque acetowhite lesions in the transformation zone closer to the squamocolumnar junction. The criteria for a negative test includes one or more of the following: no acetowhite lesions, faint ill-defined translucent acetowhite lesions, endocervical polyps, nabothian cysts, dot-like acetowhite lesions, acetowhite lesions far away from the transformation zone and a prominent squamo-columnar junction.2.6It is not possible to perform HPV testing locally as there are no laboratory facilities in DRC allowing this. Laboratory examinations will be done at the HPV and STIs Training Centre for Africa, Sefako Makgatho Health Sciences University, Pretoria, South Africa. To ensure quality, all the samples will be re-examined at Algemeen Medisch Laboratorium (AML) in Antwerp, Belgium.® solution, both cytology and HPV testing will be performed. HPV testing in this study will encompass both HPV detection and HPV genotyping as well as HPV viral particle load. Cytology results will be reported according to the 2001 Bethesda system terminology.From a single LBC specimen collected in the ThinPrep test vial containing the PreservCytSample collectionThe HPV and STIs Training Centre for Africa will receive Thinprep vials with 10 ml of the cervical sample. The samples will be stored at room temperature between 15 °C and 30 °C in a store room. The level 2 safety hood is set up and prepared by cleaning with alcohol followed by DNASe away for decontamination. The vials will be moved to dirty area (extraction room) for preparation. The pipettes will also be cleaned with alcohol and DNASe away for decontamination. The 2 ml tubes will be labeled according to the sample number on the ThinPrep vials. A 1000 μl pipette will be used to remove 2 ml of sample from the ThinPrep vials into the 2 ml test tubes for HPV testing avoiding any spillage or aerosol. The ThinPrep vials will be then filled to the ThinPrep vial mark (10 ml) with Preservcyt solution to prepare for cytology slide processing. The ThinPrep vials will then be capped and stored at room temperature until processing of the cytology slides on ThinPrep T5000 system.2.6.1® solutions using the ThinPrep T5000 cytology system. Labeled slides with corresponding number on the Vial together with a filter and the vial are placed on the carousel for processing. Once prepared, the slides will be stained and covered with cover using entelen. After staining, the slides will be screened by a qualified and trained cytotechnologist and reviewed by Pathologist. All the results will be reported on a form using the vial number only as identifier.Preparation of the cytology slides will be performed from the cervical samples in ThinPrep Preservcyt2.6.2We will use the Abbott system. The Abbott RealTime High Risk HPV is a qualitative, multiplex real time PCR for the detection of DNA from 14 High Risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 in clinical specimens, typing of HPV 16 and/or HPV 18, single and mixed infections . An endogenous human beta globin sequence is detected as sample validity control for cell adequacy, sample extraction and amplification efficiency. Two external controls (negative and positive controls) are included in every run. The viral load will be expressed as the number of HPV copies per million of normal human cells.2.7® is a synergistic combination of FDA-approved organic compounds, having a highly effective broad-spectrum anti-viral effect, both topically as orally. It is a mixture of natural essential oil components  in equal volumes diluted 50% in olive oil (Olea europea).AV2The components of AV2 have a long history of use in the Food and Fragrance industry. The components earned the GRAS  status in 1965. The present FDA status of the components is the following: Carvone: GRAS under 21 CFR 182.60; Eugenol: “Direct Food Substances Affirmed as Generally Recognized as Safe” status under 21 CRF 184.1257; Geraniol: GRAS under 21 CFR 182.60; Nerolidol: “Food Additives Permitted for Direct Addition to Food for Human Consumption”” status by FDA under 21 CFR 172.515 ® de-activates the HPV virus outside the cell by preventing endocytosis. Due to the broad-spectrum antiviral activity of AV2®, cervical lesions may regress due to deactivation of the virus. (8). It is administered as a topical spray to the cervix.It is postulated that AV2Side effects: Topically AV2® does provoke for about 30–60 s a warming or burning feeling similar to disinfecting alcohol on very sensitive areas such as Labia minora.The Placebo consists of 10% lemon (citrus limon) and 10% lime (citrus aurantifolia) essential oils in 80% olive oil. The oils are included to provide a fragrance to the placebo, similar to AV2®.2.8-The primary outcome will be the change of lesions 2 months after randomization.Change of lesions will be defined as regression or progression of the size of the lesion seen on VIA. Regression can be partial or complete when there is a total disappearance of the lesion also called remission. On cytology, regression will be defined as improvement from high-grade to low-grade CIN and remission will be defined as improvement from any grade of CIN to no CIN.-•Clearance of HPV at 2 months post-randomization,•Correlation between change of lesions change in HPV DNA at 2 months post-randomization,•Change in HPV viral particle load 6 months post-randomization.The secondary outcomes will be:The secondary outcomes will be-The tertiary outcome will be the number of patients with adverse effect.An adverse effect (AE) will be defined as any unfavourable and unintended sign, symptom, or disease temporally associated with the use of the product administered. A causality assessment of the AEs will be done in accordance with the guidelines of the WHO–Uppsala Monitoring Centre.2.9Assuming a lesion regression rate of 50% in the non-interventional group and 70% in the antiviral drug group at 2 months post-treatment, a minimum sample size of 163 patients is needed to show with 95% power a difference between both groups. To allow for up to 10% drop-outs or unevaluable patients, we will recruit at least 190 patients in each arm. We expect 20% of the women to be HPV positive, of whom 10% with and 10% without lesions, indicating that over 1900 women will have to be enrolled to obtain the required sample size.2.102 or Fisher's exact tests (when required). Student's t-test will be used for continuous variables. A Mc Nemar or paired ttest will be used for within-patient comparisons where approriate. Non-normally distributed variables will be transformed, or nonparametric tests  will be used. Stepwise multivariate analyses will be performed, and all possible interactions up to order 2 will be tested. All reported P values will be 2-sided. For all statistical analyses a P value of ≤0.05 will be considered as statistically significant.Data will be double-entered and cleaned in EpiInfo . Proportions will be compared using the xAll analyses will be performed using the STATA statistical analysis software packages .2.11The KINVAV study has been approved by the Institutional Ethical Committees /Institutional Review Board (IEC/IRB) of both the University of Kinshasa School of Public Health  and the Antwerp University Hospital , and registered in ClinicalTrials.gov (NCT02346227). It will be conducted in full agreement with the principles of the “Declaration of Helsinki” and subsequent relevant amendments. All participants will provide written informed consent before entering the trial.3In this trial, we will randomize women based on VIA which is known to be a subjective test. But according to the WHO studies, VIA does show better performance that allow its use as a cervical cancer screening test, especially in the frame of « see and treat approach » (SAT) ® test (Qiagen) is not available yet in DRC. Plus, we need to prioritize the SAT approach which minimizes lost to follow-ups and all the difficulties encountered when women have to be recalled for results. For instance, in Africa, mothers are the pivot in the life of the family, they go to the crops, they travel for small businesses and all these situations possibly tend to affect follow-up rates.For our study, the ideal process should be to start with HPV testing and secondarily randomize only HPV positive women. Unfortunately, real time HPV test such as Care HPVMoreover, in our institution we are not able to perform HPV testing needed in this study yet. The samples will be shipped to South Africa for both LBC and HPV testing and it will take some months to get the results back. So, we cannot rely on HPV testing for randomization. We will thus randomize based on VIA test whose results are immediate. According to the natural history of cervical cancer, it takes 7–20 years for cervical HPV infection or dysplasia to lead to cervical cancer and a high proportion of HPV infection and mild dysplasia may regress spontaneously. During the two-months follow-up period we will detect all women with cervical dysplasia, even in the placebo arm, and treat them accordingly by cryotherapy which is a standardized treatment method used for the treatment of precancerous lesions. For those reasons, placebo can be safely used.This study will be the first large-scaled study in Kinshasa to report on HPV prevalence and genotyping in DRC. One limitation for this study is the fact that instead of cytology or hpv testing results, randomization will be based on VIA which is a subjective test.Finally, when we will get all the results concerning LBC and HPV testing, we will recall women to inform them of their results and treat those in need with standard treatment until we clear all precancerous lesions.Sources of funding, institutional affiliations and conflicts of interest will be declared in the subsequent publications related to this study.The authors declare that they have no competing interests.ABM, YJ, JPVG have written and designed the study protocol. JB, CS, RT, RL have commented and delivered expert opinion on the protocol. ABM IS site principal investigator. YJ is coordinating the trial on behalf of the sponsor. All authors read and approved the final manuscript.Recruiting


Matrix metalloproteinases (MMPs) play a pivotal role in neuroinflammation that is associated with neurodegenerative diseases. Our group recently reported that MMP-8 mediates inflammatory reactions by modulating the processing of TNF-α. To improve the efficacy of the currently available MMP-8 inhibitor (M8I), we have synthesized structurally modified M8I derivatives  and compared their efficacy with original compound (comp 1). Among M8I derivatives, comp 2, 3, and 5 inhibited the production of NO, ROS, and IL-6 more efficiently than the original compound in lipopolysaccharide (LPS)-stimulated microglia. When we compared the anti-inflammatory mechanisms of the most effective derivative, comp 3, with comp 1, comp 3 suppressed the mRNA expression of iNOS and cytokines more efficiently than comp 1. Although comp 1 inhibits only TNF-α processing, comp 3 additionally inhibits the expression of TNF-α. Both compounds inhibited LPS-induced activity of MAP kinases, NF-κB, and AP-1, while they increased heme oxygenase-1 expression by upregulating AMPK-Nrf2 signaling. Overall, the effect of comp 3 on anti-inflammatory signaling was much stronger than comp 1. We verified the anti-inflammatory effects of comp 1 and 3 in the LPS-injected mouse brain and primary cultured astrocytes. Comp 1 and 3 suppressed microglial activation, astrogliosis, and proinflammatory gene expression in the brain. Moreover, the compounds inhibited proinflammatory gene expression in the cultured astrocytes. Collectively, our data suggest that the MMP-8 inhibitor may be a promising therapeutic agent for neuroinflammatory disorders. Microglia are resident innate immune cells in the central nervous system. They show high motility in the brain parenchyma and functionally interact with synapses. Microglia also phagocytose apoptotic cells and debris throughout the brain and release neurotrophic factors to support neuronal survival , 2. MicrMMPs belong to the zinc-containing endopeptidases, which are involved in the regulation of cell-matrix composition and processing of a number of bioactive molecules . Under pin vivo.When we examined the effects of the MMP-8 inhibitor (M8I) in LPS-stimulated microglia, it robustly inhibited TNF-α secretion. However, its inhibitory effect on other proinflammatory mediators such as NO, IL-6, and ROS was rather modest . TherefoTo find a more potent MMP-8 inhibitor compared to the original M8I comp 1), we tested four derivative compounds (comps 2-5) with the modified functional side chains attached to the phenylsulfone ring that is known to bind to the hydrophobic MMP S1' specificity pocket . Comp 1 , we testTo compare the effects of comps 2-5 with comp 1, BV2 cells were treated with each compound for 1 h prior to LPS stimulation. As shown in Figure For further study, we selected comp 3 and compared its effects with comp 1 because comp 3 had the strongest anti-inflammatory effects among the M8I derivatives and showed improved efficacy of NO, IL-6, and ROS inhibition. When we examined the effects of comp 3 on the expression of inflammatory molecule mRNA, we saw that it suppressed the expression of TNF-α, iNOS, IL-1β, and IL-6 that was induced by LPS. However, comp 1 did not significantly alter their expression, except for IL-6 Figure . The datWe previously reported that M8I prominently inhibits TNF-α processing in LPS-treated microglia . In the Given that the mitogen-activated protein kinases (MAPKs) regulate the inflammatory response in microglial cells, we examined the effects of comp 1 and 3 on MAPK activity. Western blot analysis revealed that comp 1 and 3 markedly inhibited the phosphorylation of MAPKs in LPS-stimulated BV2 cells Figure . NotablyThe AMP-activated protein kinase (AMPK) and nuclear factor-E2-related factor-2 (Nrf2) pathways are involved in the regulation of inflammation and redox signaling -19. Morein vivo neuroinflammatory conditions, mice were administrated with the compound before LPS injection. Because microglia are the main players in the neuroinflammatory response, the level of microglial activation was determined using Iba1 staining. As shown in Figure To verify the anti-inflammatory effects of comp 1 and 3 under There is accumulating evidence that astrogliosis is a characteristic feature of diverse neurodegenerative pathologies and that reactive astrocytes contribute to neuroinflammation-related disease progression , 24. In Next, we evaluated the effects of comp 1 and 3 on astrogliosis triggered by LPS in the brain of mice with systemic inflammation. Immunohistochemistry with GFAP staining showed astrogliosis in the cortex and corpus callosum of LPS-injected mice, as indicated by the increased number of GFAP-positive cells. Moreover, cell bodies of GFAP-positive cells were larger and more densely stained compared to the control. However, treatment with comp 1 or 3 reduced the number of GFAP-positive reactive astrocytes, along with causing a reduction in size and density of cell bodies Figure . CollectMMPs play an important role in cancer, arthritis, atherosclerosis, and various inflammatory and neurological disorders , 10, 25.At present, to improve the efficacy of M8I, we have synthesized several M8I derivative compounds by modifying the functional group attached to phenylsulfone ring (comps 2-5), or by changing the hydroxamate to a carboxylate structure (comp 4). The hydroxamate and carboxylate have a zinc chelating function, while the arylsulfone group binds to the hydrophobic MMP S1' specificity pocket . Our preIn subsequent studies, we selected comp 3, with the best anti-inflammatory effect among the derivatives, and compared its function and mechanism with comp 1. We observed that comp 3 regulated the expression of TNF-α, iNOS, IL-1β, and IL-6 at the mRNA level, whereas comp 1 inhibited only IL-6 mRNA expression Figure . As we pDespite the anti-inflammatory effects of M8I in activated microglia, antioxidant effects and impact on redox signaling by M8I have not yet been reported. In the present study, we found that comp 1 and 3 inhibited ROS production and upregulated antioxidant HO-1 expression Figure . ConsistIn the present study, we found that M8I inhibits the inflammatory response in activated astrocytes as well as in microglia. Although the involvement of astrocytes in neuroinflammatory diseases has been less extensively studied than that of microglial cells, astrocytes also participate in CNS immune responses and the progression of neural pathologies, which is considered critical in the modulation of neuroinflammation . Recent Through this study, we have developed multiple MMP-8 inhibitor derivatives and demonstrated their anti-inflammatory and antioxidant effects in microglia, astrocytes, and systemically inflamed mouse brains. Moreover, the structure-function relationships of the M8I derivatives have been explored. We found that chemical modification of the methoxyl group attached to the phenlysulfone ring to a phenyl group improved the efficacy of the original M8I. Therefore, the strong anti-inflammatory and antioxidant effects of M8I derivatives, such as comp 3, may provide improved therapies for various neuroinflammatory disorders.Escherichia coli serotype 055:B5) was obtained from Sigma-Aldrich . The reagents/enzymes for reverse transcription polymerase chain reaction (RT-PCR) and oligonucleotides for electrophoretic mobility shift assay (EMSA) were purchased from Promega . Antibodies against phospho-/total forms of MAPKs, AMPK, and β-actin were supplied by Cell Signaling Technology . Antibodies against TNF-α, HO-1, Nrf2, and lamin A were purchased from Santa Cruz Biotechnology  or Abcam . The antibodies against ionized calcium binding adaptor molecule 1 (Iba1) or glial fibrillary acidic protein (GFAP) were purchased from Abcam or Invitrogen .All reagents for cell culture were purchased from Gibco BRL . LPS   were syn2. Primary astrocyte cultures were prepared from mixed glial cultures by modifying a previously published method ATP. Nuclear proteins (5 μg) were incubated with a 32P-labeled probe on ice for 30 min, and DNA–protein complexes were resolved by electrophoresis on a 5% acrylamide gel and visualized using autoradiography. The DNA sequences of the probes were as follows: NF-κB (5′-AGTTGAGGGGACTT TCCCAGGC-3′), AP-1 (5′-CGCTTGATGAGTCAGCCGGAA-3′), ARE (5′-TGGGGAACCT GTGCTGAGTCACTGGAG-3′).BV2 cells were pretreated with comp 1 or 3 for 1 h and stimulated with LPS for 3 h. The nuclear extracts from cells were prepared as previously described . Double-BV2 cells were pretreated with comp 1 or 3 for 1 h prior to stimulation with LPS, and total cell lysates or nuclear proteins were prepared as described previously , 37. Thet-tests. Calculations were performed using GraphPad Prism software . A p-value less than 0.05 was considered to indicate statistical significance.Data were expressed as the mean ± S.E.M., and statistical analyses were performed using a one-way ANOVA followed by Newman-Keuls post-hoc tests or


Human papillomaviruses (HPV) are detected in 70–80% of oropharyngeal cancers in the developed world, the incidence of which has reached epidemic proportions. The current paradigm regarding the status of the viral genome in these cancers is that there are three situations: one where the viral genome remains episomal, one where the viral genome integrates into the host genome and a third where there is a mixture of both integrated and episomal HPV genomes. Our recent work suggests that this third category has been mischaracterized as having integrated HPV genomes; evidence indicates that this category consists of virus–human hybrid episomes. Most of these hybrid episomes are consistent with being maintained by replication from HPV origin. We discuss our evidence to support this new paradigm, how such genomes can arise, and more importantly the implications for the clinical management of HPV positive head and neck cancers following accurate determination of the viral genome status. Recently we published an analysis of head and neck cancer data from The Cancer Genome Atlas (TCGA) providing evidence that the three types of genomic status for human papillomavirus (HPV) in head and neck cancer (HNC) are episomal, integrated and virus–human hybrid episomes replicating autonomously; there was very little evidence for the presence of so-called mixed tumors that have both episomal and integrated viral genomes . In thisThe HPV life cycle is inextricably linked to the differentiation of the host epithelium ,3. InfecThere are two viral proteins that coordinate viral replication for all papillomaviruses in association with host proteins; E1 and E2. The E2 protein forms homodimers and binds to 12-bp palindromic DNA sequences surrounding the viral origin of replication in the LCR and recruits the viral helicase E1 to the origin of replication ,9. E1 inHigh-risk HPV activate a DNA damage response (DDR) that promotes the viral life cycle and several DDR proteins are involved directly with HPV replication ,25,26,27A large majority of cervical cancers are caused by HR-HPV . There aHead and neck cancer is the sixth most common worldwide, with around 600,000 cases diagnosed annually. Cancer incidence in the developed world is decreasing in general, due to smoking cessation; the one exception to this is the increasing cases of head and neck cancer. This increase is due to an increase in the incidence of HPV-positive head and neck cancer in the oropharyngeal region  over the past several decades; HPV16 is causative in 80–90% of these tumors ,79,80,81As described above, integration of the HPV genome into the host is a common feature of cervical cancer, and integration is an indicator of a poorer clinical outcome. The situation with HPV integration in head and neck cancer with relation to clinical outcome is much less clear. Early studies on HPV and integration in head and neck cancer employed fluorescence in situ hybridization (FISH) applied to interphase nuclei to visualize the viral genomes and concluded that the appearance of defined “dots” in the nuclei were indicative of viral genome integration ,86. ThisFull RNA and DNA genomic sequences for HPV-positive head and neck cancers became available following publication of data from The Cancer Genome Atlas Network . This orRecently we published a report detailing our genomic analysis of all of the head and neck cancer samples from The Cancer Genome Atlas . Here weAnalysis of the DNA-seq data supported our conclusion that Category 1 tumors were integrated tumors as hybrid viral–human reads that describe the integration point could be detected. Category 2 tumors were confirmed as predominantly episomal as there was no consistent detection of viral–human reads. The conclusion about our Category 1 and 2 tumors is no different from that arrived at by others. The difference in our interpretation is with the Category 3 tumors. Others would have called this a “mixed” tumor that contains both episomal and integrated viral DNA. This concept is difficult to reconcile with the observation that E1 can bind to and amplify DNA when it is integrated into that of the host ,31,95. TOur conclusion from analyzing TCGA Whole Genome Sequencing (WGS) data for Category 3 tumors is that the virus genome is not integrated into that of the host genome. Rather, the HPV genome replicates as an independent viral–human hybrid episome. This suggests that at some point the original episomal viral genome broke and ends joined to that of the host but subsequently the host DNA associated with the HPV DNA broke and a ligation event occurred resulting in excision of DNA that forms a viral–human hybrid circular episome. Potential mechanisms to explain this are shown in The conclusion from our analysis is that the viral genome is maintained episomally either as an intact HPV genome structure or an HPV–human DNA hybrid episome. If the HPV genome co-exists as both an integrated and episomal structure in tumors, it is not common. In The proposed initial driving force for integration is DNA breakage in which HPV is linearized in the E1 region and some DNA is removed from the ends to result in deletion. Breakage of the human DNA then leads to recombination of the free ends of the HPV DNA with the free ends of the human DNA, resulting in integration. For a monomeric HPV episome, the integrated HPV DNA will have lost the ability to express an intact E1 and any E2. The integrated HPV DNA will no longer initiate replication from its own origin within the LCR and will be replicated from human origins as part of the human genome. However, if the same breakage and integration events occurred for a dimeric HPV episomal genome, the result could be quite different. An integrated dimer of HPV DNA will retain the ability to express E1 and E2 from the unaltered copy of the HPV genome. This would allow the initiation of replication from the HPV origin while being within the human genome. Since HPV-origin firing can evade the one initiation per cell cycle control used for human origins, the HPV genome could go through multiple rounds of replication in a single cell cycle, which is predicted to form the proposed onion skin structure causing harm to the cell. This problem can be averted or solved by the excision of the HPV DNA to form a new episome. While we propose the excision encompasses the HPV DNA, there are possible options as to what DNA is excised. The HPV episome could be formed from excision of human DNA on both sides of the integrated HPV DNA encompassing all HPV DNA. The excised DNA could encompass human DNA on only one side of the integrated HPV DNA and most of the HPV DNA. The question that arises from our work  is why hWe are not claiming that the work of others is wrong in any way. What we are proposing is that, in light of data from The Cancer Genome Atlas, it may be worthwhile re-interpreting older results. Our results are not in conflict with those of others. HPV-positive head and neck cancer patients have a much improved overall survival when compared with HPV-negative patients . For thiOne indicator for worse outcome in HPV-positive cervical cancer is integration of the viral genome into that of the host. However, several reports have claimed that this is not the case in HPV-positive head and neck cancer ,94. We pBamH1 (a single cutter for HPV16) and bands are seen at 8 kbp and additional positions on the gel it is presumed that this represents a mixed tumor. The proposed viral–human hybrid genomes could give the same signal. In addition, Southern blotting of tumor DNA that does not cut the viral genome has been used to suggest integration but again, if the human DNA replicating with the virus in a hybrid episome is cut by the non-cutter enzyme confusing results can occur in addition to the size of the uncut virus–human hybrid also confusing the results. Two other assays that are used, APOT-PCR and DIPS-PCR, are used to monitor the presence of viral-human RNA and DNA products, respectively, and have been used to demonstrate integration. However, these techniques would give positive signals in Category 3 tumors where the viral genome is replicating as an episome as a viral–human hybrid. FISH has also been used to indicate the presence of integrated HPV DNA but this technique can also pick up episomal viral DNA and does not precisely define integration. Therefore, none of these techniques are appropriate for categorizing the genomic status of HPV in head and neck cancer.What is the best approach for characterizing tumors that have truly integrated viral DNA? We can easily eliminate some tests. Measuring an E2 to E6 DNA ratio as an indicator of integration is invalid for several reasons. Firstly, there are several head and neck tumors where the integration of the viral genome is in the E1 gene and the E2 gene is actually retained intact, therefore a 1:1 ratio would falsely suggest these tumors were episomal. Secondly, in our model we show that the viral genome exists as dimers/multimers in episomal forms and that in some occasions the E2 gene from one of the viral genomes can be deleted. This would give an E2 to E6 ratio of less than 1 suggesting integration but this would not be the case. Thirdly, in our Category 3 tumors where we propose the virus is replicating as a viral–human episome it is a viral dimer/multimer that fuses to the host genome and again E2 can be lost in one of the copies of the viral genome. Therefore, in these tumors the E2 to E6 ratio would be less than 1 but the viral genome is not permanently integrated into that of the host. Another technique that has been used to confirm the presence of integrated and/or mixed tumors is Southern blotting. But again, in our model the results from such analysis are open to misinterpretation. For example, if a viral–human hybrid episome was digested with From our preliminary analysis of outcome data from TCGA HPV-positive head and neck cancers the data demonstrates that our Category 1 patients (those that we predict only have integrated DNA) do worse clinically than those with tumors containing the virus as an episome, whether replicating with human DNA as a hybrid or not. This makes our challenge easier in many ways as, for clinical outcome concerns, a simple identification of HPV-positive tumors with truly integrated DNA is required. A simple assay for identifying these patients who are truly integrated is the absence of E2 through to E5 RNA. Indeed, a recent report looking at just this facet of HPV positive head and neck cancers demonstrated that, using the lack of E2 RNA as an indicator of integration, patients who lacked E2 expression had poorer clinical outcomes . TherefoTherefore, we propose that as part of de-escalation clinical trials, the status of HPV-positive head and neck tumors with regards to E2/E5 expression should be considered. Patients who lack E2/E5 expression should be followed very carefully in such trials.Looking at the head and neck cancer data from TCGA resulted in our model of the three different types of HPV genome status. Considering all possible explanations of what is present in the data, we do think it reasonable that “mixed” tumors are a category where the viral and human sequences replicate together as an episome. This matters as including this category as having integrated viral genomes has muddied the interpretation of outcome for HPV-positive patients. During de-escalation trials, it is important to identify patients who are at increased risk and we predict that these patients will be those who have truly integrated tumors. Much work remains to be done. We are currently working on establishing cell lines from head and neck cancers where we will look at the status of the viral genome at very early passage to ensure that the viral genome does not become integrated following medium-term cell culture. We are in the process of using E6/E2/E5 RNA in situ hybridization with tumor samples to determine if those that lack E2/E5 expression do have worse clinical outcomes. We conclude by asking the HPV and clinical communities to have an open mind with respect to our proposed model and consider it for testing.


In vitro, Ang2 aggravated high glucose-induced astrocyte apoptosis via GSK-3β activation. Ang2 directly bound to αvβ5 integrin, which was abundant in astrocyte, and the blockade of αvβ5 integrin, in vitro, effectively attenuated Ang2-induced astrocyte apoptosis. In vivo, intravitreal injection of anti-αvβ5-integrin antibody inhibited astrocyte loss in early diabetic retinopathy. Taken together, Ang2 induced astrocyte apoptosis under high glucose via αvβ5-integrin/GSK-3β/β-catenin pathway. Therefore, we suggest that Ang2/integrin signaling could be a potential therapeutic target to prevent the vascular leakage by astrocyte loss in early diabetic retinopathy.The vascular leakage in diabetic retinopathy leads to macular edema and vision loss. Although astrocyte play an important role in regulating blood-brain barrier integrity in the brain, the precise role of astrocyte in blood-retinal barrier was yet to be elucidated. This study aimed to investigate the role of angiopoietin 2 (Ang2) in astrocyte loss and vascular leakage in the early streptozotocin-induced diabetic retinopathy. We demonstrated that vascular leakage occurred with astrocyte loss in early diabetic mice retina as Ang2 increased. The astrocyte loss and vascular leakage were inhibited by intravitreal injection of Ang2-neutralizing antibody.  The perivascular endfeet of astrocyte contribute to the BBB features.7 Although astrocyte is limited to the ganglion cell layer, astrocyte plays an important role in vessel integrity both via direct contact and through humoral factors.9 In addition, astrocyte is closely related to the formation of tight junction in developing retina.12 Moreover, retinal astrocyte may decrease in number at sites where vessel is damaged with increased permeability of the BRB in pathologic conditions.15 Thus, in this study, we focused on the role of astrocyte regarding to the vascular leakage in diabetic retina.In the retina, both astrocyte and Müller cells are involved in vascular ensheathment. However, astrocyte and Müller cells are affected by diabetes in different ways.16 Hyperglycemia-induced Ang2 has a central role in pericyte apoptosis by Ang2/α3β1-integrin/p53 signaling pathway in streptozotocin (STZ)-induced diabetic mice. Local vascular leakage occurs early in the experimental diabetic mice even before the pericyte loss.5 In addition, Ang2 is upregulated by hyperglycemia in endothelial cells and diabetic retina.19 Moreover, Ang2 binds to integrin and regulates angiogenesis.20 In view of neurovascular unit, we postulated that Ang2 could affect astrocyte via integrin signaling before the pericyte loss in diabetic retina.Recently, we have demonstrated that angiopoietin 2 (Ang2) induces pericyte apoptosis via integrin signaling in DR.β/β-catenin pathway. Furthermore, we demonstrate both in vitro and in vivo that blocking αvβ5 integrin effectively attenuate Ang2-induced astrocyte apoptosis. Taken together, Ang2-induced astrocyte apoptosis via αvβ5-integrin signaling contributes to the vascular leakage in diabetic retina.Here, we provide evidence for the role of Ang2-induced astrocyte loss in vascular leakage in early diabetic retina. We demonstrate that Ang2 shows synergistic effects with high glucose on astrocyte apoptosis via GSK-3P<0.001; P<0.001; To determine whether retinal astrocyte coverage to retinal vessel is attenuated in diabetic mice retina, we performed immunohistochemistry in retinal flat mounts of diabetic mice. Retinal vascular coverage by glial fibrillary acidic protein (GFAP)-labeled astrocyte was evaluated in 3 weeks of STZ-induced diabetes and age-matched non-diabetic control mice. Intriguingly, focal loss of astrocyte coverage to retinal vessel was observed in diabetic retina . The relP=0.018 by one-way ANOVA; post hoc test, retinal Ang2 at 3 weeks was significantly increased compared to control  and Ang1/2. Retinal Ang2 level was increased at 1 and 3 weeks after diabetic induction -2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence-activated cell sorting (FACS) analysis. Ang2 leads to pericyte death under high-glucose conditions.16 Similarly, it aggravated astrocyte cell death under high glucose  and β-catenin phosphorylation . Inhibition of GSK-3β reduced Ang2-induced β-catenin phosphorylation at 30 min  was treated for the indicated time period. We found that Ang2 induced GSK-3rylation , which i glucose . Then, tt 30 min , and sub at 48 h . As expe21 astrocyte did not express Tie-2 or Tie-1 while HRMECs expressed Tie-2 and Tie-1. Compared to HRMECs with Tie-2 expression, astrocyte did not express Tie-2 in FACS analysis (data now shown). Next, to determine whether integrin may serve as receptors for Ang2 in astrocyte apoptosis under high glucose, we incubated astrocyte under high glucose for 30 min with Ang2 and H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) peptides. GRGDS (0.5 mg/ml) attenuated Ang2-induced β-catenin phosphorylation . Integrin α3- and αv-blocking antibodies attenuated Ang2-induced β-catenin phosphorylation  compared to the loss in PBS-injected diabetic mice  later than astrocyte change, whereas astrocyte progressively decreased in number and GFAP immunoreactivity.15 However, there was no study to investigate the role of astrocyte loss in diabetic retinal vascular leakage in terms of neurovascular unit. Importantly, the molecular mechanism of astrocyte loss in diabetes has not been elucidated yet. Thus, we approached with this concept and also suggested that Ang2-induced astrocyte apoptosis via integrin signaling as the precise molecular mechanism of astrocyte loss in DR.The early glial change in the diabetic retina has been described in diabetic animal models.21 Indeed, Ang2 could bind to integrin α3β1, αvβ5, and α5β1.20 Interestingly, integrin α3- or β1-blocking antibodies did not affect Ang2-induced astrocyte apoptosis, whereas they mediated Ang2-induced pericyte apoptosis.16 Because we showed integrin β1 was not responsible to Ang2-induced astrocyte apoptosis . Because survivin is a member of the inhibitor of apoptosis family of protein, decrease of β-catenin can lead to cellular apoptosis.30Regarding to the molecular mechanism of Ang2-induced astrocyte apoptosis, we demonstrated that Ang2 activated GSK-331 This approach replicates some of the early features of DR and has the advantage that the onset of diabetes can be defined as the time of STZ injection. However, toxin-induced diabetes in mice has been less successful because of strain-dependent resistance to STZ.32 In addition, DR features could be compounded by inflammatory effect of STZ. Despite the drawbacks of STZ-diabetic mice model, we showed high enough glucose levels during experimental periods  and human brain astrocytes (Applied Cell Biology Research Institute) were maintained in M199 medium and DMEM with 20% FBS containing growth factors , respectively. All cells were cultured at 37 °C in an incubator with a humidified atmosphere of 5% COαvβ5, phycoerythrin (PE)-conjugated anti-endothelial-specific receptor tyrosine kinase 2 (Tie-2) mouse IgG antibody, mouse Ang2 ELISA kit, mouse VEGF ELISA kit, human phospho-kinase array kit, anti-integrin αvβ3, and anti-integrin αvβ5  neutralizing antibodies were purchased from R&D systems . Mouse Ang1 ELISA kit was purchased from MyBioSource . Anti-Bax antibody was purchased from Epitomics . Anti-phospho GSK-3β (Ser9), anti-phospho β-catenin, anti-β-catenin, and anti-PARP antibody were purchased from Cell Signaling Technology . Anti-Tie-1 and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology . Anti-Tie-2 antibody, GRGDS peptide, and SB216763 were purchased from Millipore . MTT -2,5-diphenyltetrazolium bromide), STZ, and FITC-dextran (70 kD) were purchased from Sigma-Aldrich . FITC-conjugated annexin-V/PI assay kit were purchased from BD Biosciences . Anti-GFAP, anti-integrin αv, β1, β3, β5, and anti-Ang2 antibodies were purchased from Abcam . Anti-mouse Ang2-blocking antibody (Angy-2-1) was purchased from Adipogen . Anti-integrin α3 , anti-integrin β1 , and α-integrin-blocking and IHC kit  were purchased from Millipore and used for functional blocking.Recombinant human Ang2, human integrin 16 Age-matched controls received citrate buffer only. Mice with blood glucose levels >300 mg/dl 4 days after STZ injection were deemed diabetic. Diabetic and non-diabetic mice were killed 3 weeks after diabetes induction, and eyes were collected under deep anesthesia and immediately frozen at −80 °C for ELISA, or fixed in 4% paraformaldehyde for retinal flat mount. Glucose levels  and body weight were monitored consecutively.All animal experiments in this study were in strict agreement with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Seoul National University Animal Care and Use Committee. Six-week-old, pathogen-free male C57BL/6J mice were purchased from Central Lab. Animal Inc. . Diabetes was induced by a single intraperitoneal injection of freshly prepared STZ at a concentration of 180 mg/kg body weight in 10 mM citrate buffer (pH 4.5) as previously reported.N=6 per each group). Samples including retina, vitreous, and lens were homogenized and lysed with brief sonication in 500 μl RIPA buffer with a complete protease inhibitor cocktail (Roche). After centrifugation at 12 000 r.p.m. for 20 min, supernatants were collected. Ang1, Ang2, and VEGF levels were measured using ELISA kits according to the manufactures' instructions. The protein concentration was measured using a BCA protein assay kit . Ang1 (ng), Ang2 (pg), and VEGF levels (pg) were standardized to the retina protein (mg).Two retinas from mice were pooled  at a magnification of × 40 and × 100.μm x 0.5 μm) per each mice were selected (N=6–7). The mid-peripheral retina was designated as the mid-one-third of the retina from optic nerve head to ciliary body. The images were imported into ImageJ ; thereby, images were adjusted for color threshold based on the automatic isodata algorithm, and the interested area with FITC was marked with red. Then, the area of red was measured. The data were expressed as vascular leakage (%), which was normalized to those of control mice.To analyze vascular leakage quantitatively, the four representative sites of leakage at the mid-peripheral retina (0.5 N=6–8). The morphology of astrocyte was analyzed with maximal projection images of z-stack (magnification × 400). For the analysis of the vascular coverage by astrocyte, the colocalized area of astrocyte and retinal vessel was divided by the area of retinal vessel . The colocalized area was calculated by using the Leica Application Suite Advanced Fluorescence (LAS AF) software . The images were imported to Imaris software , and analyzed for the vascular area. Then, the vascular coverage by astrocyte was normalized to that of control . Briefly, the eyes were enucleated and fixed in 4% paraformaldehyde for 1 h. The retinas were dissected out in 2 × PBS and incubated in 100% methanol overnight at −20 °C. Then, the retinas were blocked in Perm/Block solution (0.3% Triton-X and 0.2% BSA in PBS) for 1 h at room temperature. Then, the retinas were incubated with anti-GFAP antibody (1 : 100) and Alexa Fluor 594-conjugated anti-IB4 antibody (1 : 100) in Perm/Block solution overnight at 4 °C. Then, the retinas were repeatedly washed with PBS and incubated with secondary antibody (1 : 200) for 1 h at room temperature. After washed with PBS, the retinas were mounted with Fluoromount aqueous mounting medium (Sigma-Aldrich). The representative site of astrocyte loss at the mid-peripheral retina per each mice was selected  was injected intravitreally under deep anesthesia at 2 weeks after diabetes induction with STZ. Sterile PBS was injected for control. After 7 days of injection, retinas were subjected to the retinal flat mount for the vascular leakage and/or astrocyte analyses.One microliter containing 1 4 cells were seeded into 96-well plates. After 24 h, cells were treated with Ang2 (300 ng/ml) under normal glucose (5 mM glucose), high glucose (25 mM glucose), and high mannitol (5 mM glucose plus 20 mM mannitol) as an osmotic control for 48 h. Cell viability was determined by MTT assay according to the manufacturer's instructions. Three independent experiments were performed for each experimental condition.In all experiments, 2.5 × 105 cells were treated with Ang2 (300 ng/ml) under normal glucose, high mannitol, and high glucose at 37 °C for 48 h. To determine the effect of integrin blocking, anti-integrin-blocking antibodies (10 μg/ml) were treated 1 h before the addition of Ang2. The cells were collected and washed two times in PBS. Cells were stained with FITC annexin-V and PI for 15 min, and analyzed by flow cytometry. Annexin V-positive/PI-negative cells were determined to apoptotic.In order to evaluate the apoptosis, 5 × 10μg) using 2.5 μM oligo-dT primers, 1 mM dNTPs, and MuLV reverse transcriptase. Quantitative real-time PCR (qPCR) assays were performed in qPCR master mix for SYBR Green PCR master mix  using 7900HT real-time PCR (Applied Biosystems). qPCR conditions were set as follows: (i) 95 °C for 10 min and (ii) 50 cycles of 5 s at 95 °C and 20 s at 60 °C. qPCR was performed using the primers in Total RNA was collected and isolated from cells using RNeasy Plus Mini kit . cDNAs were prepared from RNAs  according to the manufacturer's instructions. cDNA was prepared with High Capacity RNA-to-cDNA kit (Life Technologies). Real-time PCR was performed with StepOnePlus Real-Time PCR System (Life Technologies) with TaqMan Fast Advanced Master Mix (Life Technologies) and specific Gene Expression Assays . Product IDs of Gene Expression Assays for genes are as follows: for Angpt1, Mm00456503_m1; for Angpt2, Mm00545822_m1; for Vegfa, Mm00437306_m1; and for Rn18s, Mm03928990_g1. All procedures were performed in accordance with the MIQE guidelines.αvβ5 (500 ng) were precleared with G-Sepharose beads (Millipore) at 4 °C for 1 h. Then, the complex and anti-integrin αvβ5 antibody (2 μg) were incubated with G-Sepharose beads at 4 °C overnight. The immune complexes were collected by centrifugation and washed with buffer three times. For immunoblotting, cells were collected and lysed in RIPA buffer with a protease inhibitor cocktail. Protein lysates were resolved by SDS polyacrylamide gel and transferred onto nitrocellulose membrane. The membranes were incubated with primary antibodies (1 : 1000) overnight at 4 °C and secondary antibodies (1 : 10 000) for 1 h at room temperature. The membranes were incubated with enhanced chemiluminescent substrate (Pierce) and exposed to a film or LAS 4000 .For immunoprecipitation, Ang2 (500 ng) and integrin t-test for two groups, and one-way ANOVA and post hoc test with Bonferroni's multiple comparison for multiple groups, respectively . *P<0.05 was considered statistically significant. Quantitative data and figures are given as mean±S.E.M.Statistical analyses were performed using the standard two-tailed Student's


Cytokeratin (CK) intermediate filaments are demonstrated to have enormous potential in regulating cellular motility and cancer progression. There are more than 20 divergent CKs that have been identified, of which CK 8, 17, 18 and 19 are reported to be elevated in the tumour biopsies of head and neck cancer squamous cell carcinoma (HNSCC) patients. However, CK expression profiles in the saliva of HNSCC patients have not been investigated. We aim to investigate the mRNA expression profiles of CKs in saliva collected from healthy controls, HPV-negative and -positive HNSCC patients.Oral rinse samples were collected from 42 cancer-free healthy controls (age-matched) and patients who have been diagnosed with HPV-negative (n = 20) and -positive (n = 48) HNSCC.Here, we report that the mRNA expression profiles of CKs differed in saliva collected from healthy controls and HNSCC patients. The mRNA expression levels of CK 8 and 18 were significantly elevated in saliva collected from HPV-negative HNSCC patients; whilst, CK 17 and 19 were expressed at a higher mRNA level in saliva collected from HPV-positive HNSCC patients compared to healthy controls. Importantly, receiver operating characteristic (ROC) analysis showed salivary CK 8 and 18 to have superior sensitivity and specificity in discriminating the HPV-negative HNSCC patients from healthy controls (80% and 86%) as well as between HPV-negative and -positive HNSCC patients (75% and 81%).In summary, we have demonstrated that an aberrant expression of salivary CKs may serve as a potential non-invasive diagnostic biomarker in HNSCC. Head and neck cancer is a heterogeneous group of tumours arising from various anatomic structures including the nasal and oral cavity, oropharynx, larynx and hypopharynx . More thAlthough the major risk factors for HNSCC are identified and have been used as prognostic biomarkers in the recent decades, disappointingly, there is no significant improvement in the overall survival rates of HNSCC  compared to other cancer types like breast, colorectal and prostate –12. ThisCytokeratins (CKs) are a family of cytoskeletal intermediate filament proteins that are commonly found in epithelial tissues . CKs canThe aberrant expression of CKs has been reported to be associated with HNSCC tumour development and progression –30. IndeHealthy controls (n=42) and HNSCC patients (n=20 for HPV-negative HNSCC and n=48 for HPV-positive HNSCC) were recruited in this study (refer to Table According to the TNM classification system of AJCC for HPV-negative and -positive HNSCC patient staging was as follows: Stage I (5% and 4.2%), II (0% and 2.1%), III (30% and 10.4%), IVa (30% and 70.8%), IVb (20% and 10.4%) and IVc (0% and 2.1%) respectively. Most cases of HPV-negative and -positive HNSCC patients were diagnosed at an advanced stage IV . HPV-negative HNSCC cancer sites were mostly oropharyngeal and oral cavity (50% and 25%) whilst laryngeal, hypopharyngeal and nasopharyngeal were 5%, 15% and 5% respectively. Compared to HPV-negative HNSCC patients, the most common anatomical site found in HPV-positive HNSCC patients were oropharyngeal and oral cavity (91.7% and 8.3%). There were few cases of well differentiated squamous cell carcinoma found in both HPV-negative and -positive HNSCC patients. Most of the HPV-negative (75%) and -positive (50%) HNSCC patients were considered as moderate and poor squamous cell carcinoma.Although CK is one of the well-known tumour biomarkers for a number of tumour types including HNSCC, its expression level in saliva still remains to be elucidated. To address this question, we first investigated whether the saliva collected from healthy controls and HNSCC patients express the mRNA levels of CK 8, 13, 17, 18 and 19. As shown in Figure Current literature reports that the expression of CKs is able to distinguish the HPV status in HNSCC patients  and hencConversely, the mRNA expression levels of CK 17 and 19 were significantly elevated in saliva collected from HPV-positive patients compared to healthy controls as shown in Figure The clinical diagnosis in HNSCC patients is challenging compared to other cancers as they are difficult to radiologically resolve due to the tumour size and anatomical region . TherefoAmple evidence has suggested that the CKs expression profile is associated with a range of different cancers including breast, colorectal, prostate and head and neck , 35–37. in vitro and in vivo studies, high-risk type HPV-16 E1^E4 was found to interfere and collapse the structure of CK 8 and 18 networks in the cytoplasm [Recent studies reported that the majority of HPV-positive HNSCC tumours have distinct phenotypes. This was confirmed by our immunohistochemistry (IHC) results that most of them have a non-keratinizing morphology with basal cell features , 44  patients who have been diagnosed with HPV-negative and -positive HNSCC from the Princess Alexandra Hospital, Woolloongabba, Queensland, Australia. In addition, 42 cancer-free healthy controls (age-matched) were also recruited. Participants’ gave written informed consent, prior to obtaining samples. Clinical stages of HNSCC patients were classified according to the Tumour-Nodal-Metastasis (TNM) classification of malignant tumours of the American Joint Committee on Cancer (AJCC) and the HPV status was evaluated by a pathologist using standard routine diagnostic testing of p16INK4a immunohistochemistry (IHC) as described in our previous studies .Oral rinse samples were collected from participants as described previously . BrieflyTotal RNA was isolated from oral exfoliated cell pellets using combined method of Qiazol-Chloroform (Qiagen) and RNeasy Mini Kit (Qiagen) as per manufacturer protocol. Briefly, 130 μL of chloroform was added to Qiazol containing oral exfoliated cells and centrifuged at maximum speed for 15 mins at 4°C. The resulting aqueous phase was transferred to a clean eppendorf tube with an equal volume of 70% ethanol. Then, transferred to RNeasy Mini spin columns and following the manufacturer's instructions.RNA (200 ng) was used to synthesize cDNA using the iScript cDNA Synthesis Kit  following the manufacturer's instructions. qRT-PCR was carried out with the QuantStudio™ 7 Flex Real-Time PCR System . Sense and anti-sense primers targeted against the genes of interest are listed in The mRNA expression levels of individual CKs in saliva between two cohorts  were compared using non-parametric analysis using Mann-Whitney U-test; while among the three cohorts  were compared using the ordinary one way ANOVA. P-values less than 0.05 were considered significant. A receiver operating characteristic (ROC) curve was used to discriminate healthy controls from HPV-negative HNSCC patients as well as between HPV-negative and -positive HNSCC patients using the salivary CKs mRNA expression levels. All the statistical analysis was performed using GraphPad Prism 7 software version 7  and R Programming.https://software.broadinstitute.org/morpheus).The hierarchical clustering analysis was determined using Morpheus (


To evaluate the diagnostic accuracy of the 2011 International Federation for Cervical Pathology and Colposcopy (IFCPC) colposcopic terminology.The clinicopathological data of 2262 patients who underwent colposcopy from September 2012 to September 2016 were reviewed. The colposcopic findings, colposcopic impression, and cervical histopathology of the patients were analyzed. Correlations between variables were evaluated using cervical histopathology as the gold standard.Colposcopic diagnosis matched biopsy histopathology in 1482 patients (65.5%), and the weighted kappa strength of agreement was 0.480 (P<0.01). Colposcopic diagnoses more often underestimated (22.1%) than overestimated (12.3%) cervical pathology. There was no significant difference between the colposcopic diagnosis and cervical pathology agreement among the various grades of lesions (P=0.282). The sensitivity, specificity for detecting high-grade lesions/carcinoma was 71.6% and 98.0%, respectively. Multivariate analysis showed that major changes were independent factors in predicting high-grade lesion/carcinoma, whereas transformation zone, lesion size, and non-stained were not statistically related to high-grade lesion/carcinoma.The 2011 IFCPC terminology can improve the diagnostic accuracy for all lesion severities. The categorization of major changes and minor changes is appropriate. However, colposcopic diagnosis remains unsatisfactory. Poor reproducibility of type 2 transformation zone and the significance of leukoplakia require further study. Cervical cancer is the most common malignancy among women in China. Because of the widespread implementation of cervical cancer screening with cervical cytology and/or human papilloma virus (HPV) testing, colposcopy has become more frequently used globally. The use of colposcopy has led to a beneficial reduction in the number of unnecessary blinded biopsies and conization procedures as well as in the frequency of cauterization therapy for cervical erosions. Although colposcopic assessment continues to advance and evolve, universally accepted colposcopic diagnostic criteria and terminology are lacking. Most investigators believe that the traditional Reid colposcopic index (RCI)  is simpl2=3.813, p = 0.282). The colposcopic diagnosis was overestimated in 279 patients  and underestimated in 501 patients . Within two grades (referring to the same grade or to a difference of two grades) or more, 7 patients  were overestimated and 23 patients  were underestimated . The agreement of the colposcopic diagnosis and cervical biopsy pathology was perfectly matched in 1482 patients (65.5%), and the weighted kappa strength of agreement was 0.480 (p < 0.01). Moreover, agreement within one grade (referring to the same grade or to a difference of only one grade) was observed in 2232 patients (98.7%), and the weighted kappa strength of agreement was 0.980 (p < 0.01). In those patients who were histologically diagnosed as normal or benign, low grade squamous intraepithelial lesions (LSIL), HSIL, or cancer, the agreement between the colposcopy diagnosis and pathological diagnosis was 63.7% (440/691), 66.4% (624/940), 67.4% (366/543), and 59.1% (52/88), respectively. There was no significant difference among the four groups . Although there is no colposcopic overestimate, 115 patients  were underestimated.Of the 2262 patients, 340 patients underwent conization or hysterectomy. The agreement of the colposcopic diagnosis and final histopathologic diagnosis was perfectly matched in 225 patients (66.2%) .Overall, 1005 cases  were classified as type 2 transformation zone (TZ), 887  as type 1 TZ, and 370  as type 3 TZ. Among 370 cases classified as type 3 TZ, the age of 243 patients  was > 50 years, whereas the frequency of this TZ type was low in women aged 30-50 years  and rare in those aged < 30 years . The most frequently reported TZ type in women aged < 30 years was type 1 . Among 1005 cases classified as type 2 TZ, 665 patients  were aged 30-50 years. The linear trend test showed a correlation between TZ types and the three age groups .Of 2262 patients, 1955 patients demonstrated abnormal colposcopic findings on < 30% of the visible cervix , 148 patients had colposcopic abnormalities on 30%-67% of the visible cervix , and 159 patients had abnormalities on > 67% of the visible cervix . The linear trend test showed a correlation between the size of the lesion and pathological diagnosis: the larger the size of the lesion, the more serious the disease , followed by fine punctation  and fine mosaic . The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden index for the detection of LSIL are shown in Table Correlation between abnormal colposcopic findings and cervical histological diagnoses is shown in Table major changes are shown in Table Colposcopic findings graded as major changes had a high predictive value for HSIL/carcinoma. The sensitivity, specificity, PPV, NPV, and Youden index for the detection of HSIL+ for Of the non-specific signs, non-stained lesions were very common  and had a high sensitivity and NPV, but it was unsatisfactory for the specificity, PPV and Youden index. Although iodine-stained lesions had a high specificity, PPV and NPV, the sensitivity was very low (27.3%). Among 15 patients  with leukoplakia, LSIL was found in 4 patients, and HSIL in 8 patients. Leukoplakia had a considerable PPV for HSIL .suspicious for invasion, such as atypical vessels had a considerable PPV for HSIL+ .Invasive cancers were rare and found in only 88 patients . However, some colposcopic findings classified as miscellaneous findings had a low PPV  for HSIL+. Among 37 patients with condyloma, LSIL was found in 26 patients, and HSIL in 4 patients. Among 97 patients with a polyp, LSIL was found in 2 patients, and HSIL in only 2 patients.Several colposcopic findings classified as Some colposcopic findings in predicting LSIL/HSIL+ are summarized in Tables 2=19.935, p=0.000) between single HR-HPV infections and multiple HR-HPV infections were observed regarding the histology findings on colposcopically directed biopsy. HPV Type 16 was identified most frequently , followed by types 52, 58 , 56, 33, and 18 . The prevalence of HPV type 16 increased with the grade of cervical disease, as defined by biopsy-histological diagnosis: 18.2% (167/910) in LSIL, 51.6% (268/519) in HSIL, and 81.5% (66/81) in cervical carcinoma. On the basis of univariate analysis  and multivariate analysis , the presence of HPV type 16 was correlated with cervical HSIL+, and it was an independent predictor factor for cervical HSIL+ development. Although HPV types 18, 58, 52, and 33 were also identified frequently in HSIL+, presence of HPV types 18, 58, 52, and 33 was not associated with this risk.We also analyzed HPV genotyping data. Of the 2262 patients, 62 were not tested for HPV, 284 were negative for HPV, 23 were positive for Hybrid Capture II test, and 1893 were positive for HPV genotyping test. Single high-risk HPV (HR-HPV) infections were found in 993 of 1893 patients (52.4%), single low-risk HPV (LR-HPV) infections were found in 72 of 1893 patients (3.8%), and multiple infections  were present in 828 of 1893 positive samples (43.7%). Most of the multiple infections consisted of two HPV types , whereas the rest had three to eight HPV types. A high prevalence of single HR-HPV infection was found among HSIL  and micro invasive cervical cancer (MIC) / invasive cancer . Significant differences  . TatiyacIn the present study, the colposcopic diagnosis more frequently underestimated (22.1%) than overestimated (12.3%) the cervical biopsy pathology. For those patients who underwent conization or hysterectomy, although there is no colposcopic overestimate, 115 patients  were underestimated. An overestimated colposcopic diagnosis leads to an unnecessary cervical biopsy, whereas an underestimated diagnosis leads to missed diagnoses, possibly high-grade lesions/carcinomas. The primary task for colposcopists is to improve the accuracy of colposcopic diagnosing HSIL+ and prevent the growth of undiagnosed lesions.In the present study, we found that the colposcopic examination has a high sensitivity(80.3%) and is capable of differentiating a healthy cervix from any cervical lesion in 80.3% of the cases; however, the specificity was low (63.7%), probably leading to over-examination and thereby increased patients cost. Some studies have reported that such over-examination causes the patient to undergo an unnecessary cervical biopsy. We also found that colposcopy had a 71.6% sensitivity for the detection of HSIL+, similar to that reported in previous studies 30%-91.3%) 1.3% 9, –17. More. More17.Precise identification of the squamocolumnar junction and evaluation of the TZ are crucial steps during colposcopy. The 2011 IFCPC colposcopic terminology confirmed the classification of the TZ as an obligatory terminology. Our study showed a linear trend between the TZ type and three age groups, with type 3 TZ found more commonly in women > 50 years old and type 1 TZ found more frequently in women < 30 years old. Further, in the present study, univariate analysis revealed that TZ type was linked to the diagnostic accuracy of colposcopy. However, binary logistic regression analysis revealed that TZ type was not an independent predictor factor in diagnosing LSIL or HSIL/carcinoma. A prospective multicenter study by Luyten et al. compared the distribution of TZ types among women according to age . In theiLesion size has also been included in the new terminology system. Our study revealed a linear trend between lesion size and severity of the diagnosis, and a larger lesion size was more closely related to HSIL+. However, binary logistic regression analysis revealed that lesion size was not an independent predictor factor in diagnosing HSIL+. Several previous studies have shown that a large lesion size was more closely related to HSIL or more, and a small lesion size predicted the absence of a squamous epithelial lesion/CIN , 20, 21.minor changes and major changes, the incidence of a thin acetowhite epithelium was the highest (65.4%). However, a thin acetowhite epithelium was found in only 27.1% of cases with HSIL, and had little or no association with HSIL+. Some studies have reported that a thin acetowhite epithelium does not suggest the existence of a cervical vaginal lesion [The 2011 IFCPC terminology has revised the grading nomenclature by adding two new pathognomonic, objective colposcopic criteria: the “inner border sign” and “ridge sign”. We analyzed these specific signs in detail, and the specificity and PPV of both signs were high. Other studies have confirmed the clinical utility of these criteria and their strong association with HSIL –25. Withl lesion ; howeverl lesion . Massad l lesion . In the Another common sign, a non-stained lesion, was once classified as a major change in the RCI scoring system and previous IFCPC colposcopic terminology. However, non-stained lesions are currently classified as a non-specific sign in the 2011 IFCPC terminology. Our study further confirmed that the diagnostic value of non-stained lesions for HSIL+ is very low, and was not an independent predictor factor for HSIL+; although iodine-stained lesions had a high specificity, PPV, and NPV, the sensitivity was very low. Leukoplakia had a considerable PPV of 53.3% for HSIL+; however, our data revealed that leukoplakia was not an independent predictor factor for HSIL+. The present published studies are discordant on the significance of this terminology. In addition, it is worth noting that 30 of 37 cases suggestive of condyloma were related to LSIL/HSIL, suggesting that we should pay close attention to cervical condyloma during colposcopy, and that cervical biopsy should be performed if indicated.High risk HPV- related infections are a significant source of cervical morbidity and mortality. Our data revealed that HPV type 16 was identified most frequently, followed by HPV types 52, 58, 56, 33, and 18. The prevalence of HPV type 16 increased with the grade of the cervical disease as defined by biopsy-histological diagnosis, and the presence of HPV type 16 was an independent factor for predicting cervical HSIL+ development, whereas the presence of HPV types 18, 33, 52, 56, and 58 were not associated with this risk. This implies that HR-HPV infections, especially HPV type 16, increase the risk of developing severe lesions. As colposcopy is inadequate for patients or in the case of suspected cervical HSIL+, colposcopic examination should be combined with cervical cytology and HR-HPV genotyping, especially with HPV type 16, to further improve diagnostic accuracy.Our study had several limitations. First, the study design was retrospective in nature and enrollment bias may have occurred. Second, colposcopy diagnosis may have been biased by prior knowledge of the cervical cytology results. Third, although the conization/hysterectomy data had been used to strengthen the pathology diagnosis, the sample size is less. Finally, we did not compare the overall diagnostic accuracy of 2011 IFCPC criteria with that of other colposcopic diagnostic criteria (such as RCI) for the detection of high-grade CIN.The clinical application of the 2011 IFCPC terminology is in its preliminary stage. Our data revealed that the categorization of major changes and minor changes is appropriate; the accuracy of colposcopic diagnosis using the IFCPC terminology was stable and did not vary with lesion severity. However, there was only a moderate strength of agreement between colposcopic diagnosis and cervical pathology, and the diagnostic accuracy remains unsatisfactory. Therefore, we propose that colposcopic findings should be combined with cervical cytology and HR-HPV genotyping test, especially with HPV type 16, to further improve the diagnostic accuracy. Studies evaluating the 2011 IFCPC colposcopic terminology are limited and further prospective studies are needed. In future studies, we aim to investigate RCI and 2011 IFCPC criteria and obtain data with respect to accuracy of the two colposcopic diagnostic methods.We retrospectively reviewed the medical records of women who underwent colposcopic examinations and cervical biopsy at the colposcopy clinic of the General Hospital of Tianjin Medical University between September 2012 and September 2016. These women had no previous surgical procedures of the cervix, no history of pelvic radiation or hysterectomy, no sexual intercourse for 3 days before the examination, and no confirmed or clinically suspected immunosuppression  or other chronic disease that might have compromised their immune system. Colposcopy indications included atypical or abnormal cervical cytology results and/or positivity for HR-HPV and/or suspicious clinical findings. Positive cervical cytology included atypical squamous cells of uncertain significance; atypical squamous cell, cannot exclude HSIL; LSIL; HSIL; atypical glandular cells; and invasive cervical cancer. HPV genotyping was performed via the PCR-reverse dot hybridization method. Eighteen HR-HPV types  and five LR-HPV types  were identified. The data in this study were collected from the hospital’s archived database. This study did not influence the diagnosis or treatment of the patients. Informed consent was waived by the ethics review board of the General Hospital of Tianjin Medical University. The institutional review board approved this (No. IRB 2016-YX-073).At the colposcopy clinic, all colposcopies were performed by two expert colposcopists with more than five years of experience who had received the ASCCP professional colposcopy training. All colposcopic examinations were performed and recorded using the VIZ-YD system, which is a video exoscope-based system  that allows full HD video documentation of the colposcopic examination and is used for clinical records and research. Steps in colposcopic assessment of the cervix included the following: 1) clean the cervix with normal saline; 2) assess the cervix about 1 min after the application of 5% acetic acid, and observe margins of the lesion, epithelial color and vascular patterns; and 3) assess the cervix after the application of diluted Lugol’s iodine solution, and observe iodine staining. Colposcopic findings were described using the 2011 IFCPC terminology: (1) TZ types, lesion size in percentage of cervix, and normal colposcopic findings ; (2) abnormal colposcopic findings, grade 1( minor) , grade 2 (major) , non specific ; (3) suspicious for invasion, atypical vessels, additional signs; and (4) miscellaneous findings, such as condyloma, polyp, inflammation, and post treatment consequence. It is impossible that all abnormal findings (suggestive of the same grade disease) simultaneously exist in one patient. As a result, colposcopists made a preliminary colposcopic diagnosis for each colposcopic finding on the basis of the criteria of the IFCPC. It should be noted that the same colposcopic finding could be present in different grades of lesions. When doubtful diagnosis occurred, all the inconsistent diagnoses were analyzed by another senior colposcopist, who made a decision on the final colposcopic diagnosis.All colposcopically detected abnormal areas were biopsied, and large or multiple lesions were subjected to more than one biopsy. If the colposcopic examination showed no lesion in the cervix, a random biopsy specimen was obtained at the squamocolumnar junction in that quadrant at 3, 6, 9, or 12 o’clock. An endocervical curettage was performed after the cervical biopsy.All biopsy specimens were fixed in formaldehyde and embedded in paraffin according to the routine procedures of the pathology department. The biopsy specimens were examined by the pathologist. After examining the specimen, an experienced expert pathologist made a final diagnosis according to the 2012 Lower Anogenital Squamous Project’s two-tier squamous intraepithelial lesions (SIL) terminology (LSIL and HSIL). In uncertain cases, P16 immunohistochemistry should be used in the assessment of HSILs, particularly in dealing with a specimen that already has an H&E morphologic interpretation of HSIL(CIN 2) and in the distinction between HSIL(CIN2 or CIN3) and its mimics, such as immature squamous metaplasia, atrophy, reparative epithelial changes, and tangential cutting . LSIL re2 test) and weighted kappa values. The sensitivity, specificity, PPV, NPV, and Youden index were used to compare the colposcopic diagnosis and cervical pathology. P-values < 0.05 were considered statistically significant.SPSS 20.0 software  was used for statistical analysis. The significance of agreement between the colposcopic diagnosis and cervical pathology was assessed with the chi-square statistic (χ


Data regarding the anogenital distribution of and type-specific concordance for cutaneous β- and γ-HPV types in men who have sex with women is limited and geographically narrow. Knowledge of determinants of anogenital detection of cutaneous HPV types in different regions is needed for better understanding of the natural history and transmission dynamics of HPV, and its potential role in the development of anogenital diseases. Genital and anal canal samples obtained from 554 Russian men were screened for 43 β-HPVs and 29 γ-HPVs, using a multiplex PCR combined with Luminex technology. Both β- and γ-HPVs were more prevalent in the anal (22.8% and 14.1%) samples than in the genital (16.8% and 12.3%) samples. Low overall and type-specific concordance for β-HPVs (3.5% and 1.1%) and γ-HPVs (1.3% and 0.6%) were observed between genital and anal samples. HIV-positive men had higher anal β-  and γ-HPV  prevalence than HIV-negative men. Due to the lack of genital samples from the HIV-positive men, no comparison was possible for HIV status in genital samples. The lack of type-specific positive concordance between genital and anal sites for cutaneous β- and γ-HPV types in heterosexual men posits the needs for further studies on transmission routes to discriminate between contamination and true HPV infection. HIV-positive status may favor the anal acquisition or modify the natural history of cutaneous HPV types. Currently, the International HPV Reference Center lists 210 HPV types2. The majority of HPVs belongs to the Alphapapillomavirus (α), Betapapillomavirus (β), and Gammapapillomavirus (γ) genera3. HPV infections in humans are categorized as mucosal or cutaneous based on their epithelial tropism4. So far, 12 mucosal high-risk α-HPVs have been associated with the development of anogenital malignancies and classified by the WHO/International Agency for Research on Cancer as oncogenic to humans5.Human papillomavirus (HPV) is one of the most common sexually transmitted infections (STI) worldwide9 or γ-HPVs11, respectively. In fact, cutaneous β- and γ-HPVs can be ubiquitously found in swabs of normal skin13, eyebrow hair samples13, in the oral cavity15 and gargles16, nostril16 and oesophageal17 mucosal samples, and even in faeces, as a result of infecting or passing through the entire digestive system18. Additionally, recent studies confirmed the anogenital presence of β- or γ-HPVs on the genitalia21 and in the anal canal in men22. Both β- and γ-HPVs have been detected on the surfaces of male genital lesion samples23 and in vulvar squamous cell carcinoma24. Additionally, β-HPVs were also found in penile and anal intraepithelial neoplasia diagnosed in HIV-positive men who have sex with men (MSM), supporting the role of cutaneous HPVs in some cases of neoplasia development25. However, most studies on genital HPV infection have examined only α-HPV types20.The oncogenic potential of other genera has not been fully established but rather proposed or speculated in skin cancer for β-HPVs29 but the determinants of the anogenital presence of HPVs in males is largely based on geographically-concentrated populations of HIV-positive and HIV-negative MSM28. It is not clear how cutaneous HPVs are transmitted into the anal canal, i.e., if it can be due to sexual behavior or passing through the entire digestive system. A possible commensal colonization of the anogenital skin with cutaneous HPVs in HIV-positive MSM was posited25. Moreover, the risk of β-HPV infection in the anogenital region in men who have sex with women (MSW) may occur through direct skin contact30. Supportively, the first report on sequencing previously unclassified β- and γ-HPVs in the anal canal of men who have sex with women (MSW) from the HIM Study has suggested that other forms of transmission apart from penile-anal intercourse may exist29. However, these transmission hypotheses have yet to be fully established.The presence of β- and γ-HPVs in the anal canal of men has been established21. To further this etiological knowledge and better understand transmission dynamics of HPV and its role in the development of anogenital diseases, more evidence must be gathered to establish the spectrum of cutaneous HPVs in the anogenital area among diverse population groups worldwide. The aim of the current study is to analyse the prevalence, type-specific positive concordance and determinants for the presence of genital and anal β- and γ-HPVs in a large cohort of MSW from the Eastern hemisphere.Studies on genital-anal type-specific positive concordance of cutaneous HPVs involving MSW are nearly absent and represented with the recent report from the HIM Study on the diversity of β-HPV typesC. trachomatis infections was detected in 31 (5.7%) MSW. Up to one third of men (n = 153) reported having Chlamydia infections in the past. Over half of the men (56.3%) had their sexual debut when they were younger than 18 years old. Less than half of the men (47.2%) reported having more than 20 life-time sex partners.The characteristics of the remaining 554 (97.7%) men are presented in Table All samples included were successfully typed for β- and γ-HPVs. β-HPV prevalence was 33.0% n = 122) for either genital or anal samples, 16.8% (n = 76) for genital and 22.8% (n = 107) for anal sites , β-HPV-23 and β-HPV-38  and β-HPV-5 . The most commonly-detected β-HPV types in the anal canal were β-HPV-22 , and β-HPV-110 , and β-HPV-5 and β-HPV-12  , γ-HPV-108  and γ-HPV-4, γ-HPV-50 and HPV-95 , while γ-HPV-50 and γ-HPV-132 , γ-HPV-95  and γ-HPV-121  were the most common types found in anal specimens  and anal (ORs = 1.3 (95% CI: 0.8–2.2)) samples compared to those starting sexual activities below 18 years. The prevalence of anal β-HPVs increased with increasing number of lifetime sexual partners ; this association was strongest among the men with more than 20 lifetime sexual partners . For the γ-HPV types, no linear trend was observed in the genial or anal samples.C. trachomatis infection . Regarding γ-HPVs, only self-reported genital C. trachomatis infection in the past was associated with an increased anal γ-HPV-positivity .The detection of β-HPVs in genital samples was also slightly associated with present genital Due to the lack of genital samples from the HIV-positive men, no comparison was possible for HIV status in genital samples. Among the anal sites, both cutaneous HPV genera were more commonly detected in HIV-positive men than HIV-negative men  Table .Table 6CN. gonorrhea in one (0.2%), C. trachomatis in 36 (6.1%), herpes simplex virus in 3 (0.5%) and M. genitalium in 7 (1.2%) men, respectively (data not shown). The evaluation of patients with the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI)31 observed no association between the clinical symptoms and the prevalence of cutaneous HPVs in the anal and genital area (data not shown).Forty-seven (7.9%) recently diagnosed STIs were observed in the study, including HIV in one (0.2%), This is the first report of the determinants for anal and genital presence of a broad range of both β- and γ-HPV types in a cohort of 554 Russian heterosexual men investigated and compared. Importantly, despite the abundance of both HPV genera observed in the anogenital area in the current study, the type-concordant association between genital and anal sites was uncommon. Although some association was observed between the number of lifetime sex partners and genital and anal prevalence for either genera (in particular for β-HPVs), poor type-specific positive concordance between the two sites and higher but nonsignificant prevalence of both HPV genera in anal sites compared to genital samples provides more evidence to the existence of such transmission route as autoinoculation, which needs to be explored further.27. The current study supports the evidence that impairment of the host’s immune surveillance may impact β- and γ-HPV infections differently28. Further studies should be extended with genital and non-anogential sites, to better understand the association between HIV infection status and subsequent HPV acquisition and vice versa.Importantly, a substantially higher prevalence of β- and γ-HPVs was detected in HIV-positive than HIV-negative men. No significant association between the anal prevalence of the two cutaneous genera with HIV status or sexual behavioral factors were observed in a study among Italian MSM, applying the same diagnostic test29 from the USA, Brazil and Mexico. As the epithelium of male genitals is known to be rich with a broad range of HPVs20, the current study employed the proficient32 Luminex assay, which allowed for the detection of the broadest range of β- and γ-HPV types and compared genital and anal prevalence within the same individuals. Applying the Luminex assay, the study from the Western hemisphere assessed diversity of β-HPV types at oral gargles, anal canal and genital sites specimens obtained from 717 men21. The current study adds to this new evidence by including 554 MSW, the first such known sample in the Eastern hemisphere.To our knowledge, the only study that investigated the prevalence of two cutaneous genera in anal and genital specimens obtained among MSW was the HIM study. First, the study reported the high prevalence of β- and γ-HPVs in the anogenital skin by sequencing 25 β- and 3 γ-HPVs in the anal canal in 164 MSW33. The addition of urine samples to penile swabs in the detection of HPV in men has illustrated how useful the combination of genital samples could be for epidemiological or clearance studies34. The combination of the external genital and scrotum swabs was also employed in the detection of β-HPV types in the HIM study21.Another strength of the current study was the use of combined penile and urethral samples in the detection of cutaneous HPVs. Penile and urethral swabs have been found more likely to have the highest prevalence with α-HPV infections35 and the lack of retrospective genital samples obtained from the HIV-positive men, as only anal and serum samples were primary collected36 during the survey on Chlamydia LGV infection. Larger, preferably prospective, studies to further explore these issues would ideally include both penile and urethral specimens to elucidate the role of immunosuppression on the prevalence of genital β- and γ-HPVs among HIV-positive and HIV-negative MSW and MSM. To help in understanding the potential transmission routes of cutaneous HPVs, further behavioral studies should also include more detailed surveys on sexual behaviors.The obtained data also do not represent the general population. The study limitations also include a low number of MSM observations, as research on MSM populations in Russia remains delicate20. In this respect, self-inoculation or transmission from skin, mucosa or secretions from the partners may result in the detection of β- and γ-HPV DNAs in the anal canal of men. Identical cutaneous β-HPV types in both penile and anal intraepithelial neoplasia were found in a study on HIV-positive MSM, assuming the dissemination between different anogenital regions within a given patient25. In a study among 25 heterosexual couples, the man’s hand either β- or γ-HPV types were found in the anogenitals of the female partner in 25% of the visits, while the woman’s hand β- and γ-HPV types were found in the males’ anogenital area in approximately 50% and 25% of the visits, respectively30.The routes for the transmission of HPVs into the anal canal in MSW are not clear. Minimizing the risk of potential contamination from the anatomical sites close to the anal verge at the time of anal sampling, the procedure should be performed by a single trained physician in a standard manner, as in the present study. The presence of cutaneous HPV DNA in the anogenital area may reflect deposition of virions released from other body sites with productive infections21. In the current study, next-to-nothing type-specific concordance between genital and anal sites was observed for both HPV genera in HIV-negative MSW. It could also be envisioned that different transmission routes may result in the discrepancy on HPV prevalence between different sites, regardless their sexual practices. In the studies of heterosexual couples, transmission of α-HPVs between hands and genitals or apparent self-inoculation (primarily in men) events37 resulted in modest concordance rates between genital and non-genital sampled sites38. The further studies on extended number of anatomical sites may provide the answer.In the HIM Study, multiple β-HPV types were more likely to be detected at the genital than at the anal canal39, the current study could not distinguish new or persistent HPV infection. Detecting HPV transcripts could be seen as a useful options, since the detection of HPV DNA may be the result of transient deposition39. The presence of HPV DNA does not necessarily indicate presence of infectious virus42, although the relatively high rate of concordance of β- and γ-HPVs between sex partners found in the recent study of 25 couples has suggested that the anogenitals represents a more common area for infectivity than previous thought and that sexual transmission is possible30. β-HPV does seem to exhibit activities that can promote oncogenesis, although using distinct from α-HPV mechanisms such as cell transformation promotion and deregulation of pathways linked to the host immune response45. In addition, recently reported associations with the risk of incident head and neck cancer others than α-HPV types, which included γ11- and γ12-HPV species and β1-HPV-5 type46, advocates the needs for further, preferably prospective, studies on etiological role of cutaneous HPVs in carcinogenesis.Similar to othersIn summary, the current study extends our knowledge regarding β- and γ-HPV types and their distribution in diverse male populations, which is essential for developing a better understanding of the natural history, transmission dynamics, and the potential role of different HPV types as co-factors in the development of anogenital malignancies.22. Genital and anal samples originally obtained from 609 men in two clinic-based studies in St. Petersburg, Russia were available for testing. The first was a pilot case-control study investigating anal Chlamydia LGV infection performed in an infectious disease hospital providing treatment for HIV-infected, from December 2005 to January 200636. The second was conducted among men seeking routine STI testing at the urology units of two university outpatient clinics, from February 2006 to February 200947.The study settings and methods have been described in detail elsewhereFor both studies, men were eligible for enrolment if they were at least 18 years old and reported no anorectal disorders. A medical history and a standard physical examination were completed. Participants completed a questionnaire concerning sexual behaviour . Data on age and sexual preferences were only available from the infectious disease hospital participants.22.All men self-reported their sexual preferences. If a man reported having had sex  with at least one other man during his lifetime, he was categorized as MSM. If only sex with women was reported, a man was classified as MSW. The questionnaire was administered during a face-to-face interview, before anal sampling. Importantly for the local context, all participants were informed that this information would be unavailable for the third part. In addition, the responses were codedAfter excluding samples from 40 MSM (6.6%) and β-globin-negative samples from 13 (out of 569) individuals (2.3%), in total the genital and anal samples obtained from 554 self-reported as MSW were included in the analysis  under the Federal Agency of Public Health and Social Development of Roszdrav.DNA samples and questionnaire data were anonymized, and no study personnel besides the principal investigator had access to participants’ identifying information.47. The study clinician first sampled the distal urethra (inserted up to about 2 cm inside) followed by the penis  using two separate brushes. The swabs were rinsed in 1000 µl of phosphate buffer in two separate tubes. The anal canal was sampled with a third brush wetted with phosphate buffered saline, inserted about 2 cm into the anal canal and rotated 360 degree clockwise and anticlockwise directions. The swab was then rinsed in 1000 µl of phosphate buffered saline in a separate tube. The tubes were placed in a refrigerator at 4 °C. The samples were then transferred to a −20 °C freezer and stored until HPV testing.Samples from genital sites and anal canal were collected. To minimize the risk of potential contamination from the anatomical sites close to the anal verge at the time of anal sampling, the procedure was performed by a single trained physician in a standard manner. Before sampling, men were instructed to abstain from any form of sex for 3–5 days and from urination for 3–4 hours. Urethral sampling was performed as described elsewhere48. The DNA samples were shipped to the laboratory of the Group of Infections and Cancer Biology at IARC  for β- and γ-HPV-specific genotyping.Using a 100 µl aliquot of the original anal sample, DNA was extracted in the laboratory of the Department of Laboratory Medicine at Karolinska Institutet , using a freeze-thaw-boil procedure, as previously described53. The multiplex type-specific PCR method uses specific primers for the detection of 43 β-HPVs 21 and 29 γ-HPVs . HPV type species were classified according to de Villiers3. Two primers for the amplification of the β-globin gene were included to provide a positive control for the quality of the DNA in the sample55.Genital and anal samples were tested for the presence of HPVs using type-specific PCR bead-based multiplex genotyping (TS-MPG) assays that combine multiplex polymerase chain reaction (PCR) and bead-based Luminex technology , as described elsewhere52.In the current study, 10 μl of the anal sample and 10 μl of the genital samples were analysed. A 10 μl volume of genital sample was obtained by combining into one sample 5 μl of penile and 5 μl of urethral aliquots. Following multiplex PCR amplification, 10 μl of each reaction mixtures were analysed by multiplex genotyping using the Luminex technologyIn the current analyses we included only individuals, who had both genital and anal samples, whose samples were tested as beta-globin-positive. Paired genital and anal samples were collected in urology participants in parallel. For HIV-positive patients, only anal samples were obtained.56. Odds Ratios (OR) and their 95% confidence intervals (CI) were calculated using logistic regression to assess associations between β- and γ-HPV positivity and age, age at sexual debut, number of lifetime sexual partners, past and present C. trachomatis infection . Except HIV status , all variables were included in the adjusted regression model. All analyses were completed using Stata versions 14 .The prevalence  of β- and γ-HPVs by anatomical sites was presented as proportions. “Type-specific positive concordance” was defined as having the same HPV genotype in both genital and anal samples of a participant, with the Kappa-based extent of agreement between the two anatomical sites measured Ethical Committee of the Department of Clinical Investigations and Intellectual Property of St. Petersburg Medical Academy of Postgraduate Studies  under the Federal Agency of Public Health and Social Development of Roszdrav .