Cervical cancer prevention using cervical cytology is insufficiently sensitive, a significant proportion of HPV-infected women having normal cytology.The objective of the present study was to try to identify factors associated with abnormal cytology in HPV-infected women living in remote areas of French Guiana.A study was conducted in women aged 20–65 years having HPV infections confirmed by HPV DNA detection using the GREINER-BIO-ONE kit. In addition to HPV testing, cytology was performed and classified as normal or abnormal. Demographic and life history variables, and infecting genotypes were compared between the normal and abnormal cytology groups.P < 0.001.None of the demographic and life history variables were associated with cytology results. HPV genotype 53 was significantly associated with absence of cytological abnormalities whereas HPV 52, 58, 16 and perhaps 33 and 66 were independently associated with a greater risk of cytological abnormalities. When grouping HPV genotypes in different species, only species 9  was significantly associated with abnormal cytology AOR = 5.1 (95% CI = 2.3–11.2), It was not possible to predict which HPV-infected women will have cytological abnormalities or notfrom anamnesis. In this study HPV 53 seemed more benign than other HPV genotypes. On the contrary, species n°9, containing 5 of the genotypes contained in the nonavalent HPV vaccine, was significantly associated with more cytological abnormalities. HPV testing and vaccination with the nonavalent vaccine should be implemented in these remote parts of French Guiana. Cervical cancer follows Human Papilloma Virus (HPV) infection. In French Guiana, the standardized incidence rate of cervical cancer is 30.3 per 100,000 women, thus representingthe second most frequent cancer in females . These fThe objective of the present study wasto try to identify factors associated with abnormal cytology in HPV-infected women.The design was a cross sectional study which took place between Dec 2012 and Sept 2014 in the remote villages on the Maroni and Oyapock rivers, which can only be accessed by boat or air.The study included women aged 20–65 years living in the remote villages on the Maroni and Oyapock rivers. Hysterectomy, pregnancy >3 months, or never having had sex were exclusion criteria.All women presenting at the health center who wished to participate and fulfilled the inclusion criteria and did not have exclusion criteria. The source population was estimated to be 5 000women in our target age group and our aim was to include at least 300 women in order to be able to measure an HPV prevalence of 30% with a 5% margin of error and a 95% confidence interval.Communication in all the villages sensitized the local populations about this public health problem before beginning the inclusion of patients. Local authorities  and health center workers were also informed. Radio messages informed the population of the dates when the study would take place in the village. Women wishing to be screened came to the health center at the planned dates. Samples were stored in a cooler until the end of the mission. The samples were then sent to the Virology laboratory of Fort de France Hospital, in Martinique, where an automated method was used for extraction and genotyping. Sample DNA extraction was performed using a minimum of 2 ml in a liquid phase, followed by PCR, and genotyping using the GREINER-BIO-ONE PapilloCheck® kit discriminating between 24 genotypes among which, HR 16, 18, 31, 33, 45, 51, 52, 53, 58, 59, 66, 68… The kit has a high sensitivity and specificity , 8. The When HPV was positive and cytology was negative, a gynecological follow up was recommended to verify if HPV positivity disappeared or if cytological lesions appeared.Cytological examination of a cervical vaginal smear was performed in the Fort de France University laboratory (Bethesda 2001 criteria) . Liquid-The detection of HPV DNA was performed using the GREINER-BIO-ONE kit at the Virology laboratory in Fort de France University Hospital. A short questionnaire was used to collect socio economic and demographic data, gynecological and obstetrical history.The group of women with confirmed HPV infection was analyzed. They were then divided in 2 groups, one with normal cytology, and one with cytological abnormalities. These two groups were compared in terms of demographics, and sexual history, and in terms of infecting-HPV genotypes. Crude odds ratios were calculated and multivariate logistic regression was used to look for independent predictors of cytological abnormalities. Power calculations were performed to determine variable for which the study size may have been insufficient. Data was analyzed using Stata 13.0® .All patients received an HPV test and cervical cytology free of charge. All included subjects gave written informed consent. Approval was given by: the Comité d’Evaluation Ethique de l’Inserm (CEEI), approval n° 12–064; the Comité Consultatif sur le Traitement de l’Information en matière de Recherche dans le domaine de la Santé (CCTIRS), n° 12.310; and the Commission Nationale de l’Informatique et des Libertés (CNIL), n° 912,459.Data can be made available after requesting the authorization from the Commission Nationale de l’Informatique et des Libertés .Among 601 women with a cervical smear of satisfactory quality, 199 had a positive HPV test.Among the 61 women with abnormal cervical smears, 52 (85%) had a positive HPV test. Among the cytological anomalies there were 30 ASCUS, 7 ASCH, 18 LSIL, 7 HSIL, and 3 glandular anomalies. One woman had ASCH and LSIL, another had HSIL and glandular anomalies, another had LSIL and glandular anomalies, and one had ASCUS and glandular anomalies. Among the 540 women with normal cervical smears, 147 (27%) had a positive HPV test.Table P = 0.006.Fig. On the contrary, among HPV genotypes identified, HPV 52, 58, 16 and perhaps 33 and 66 were significantly associated with a greater odds of abnormal cytology. Genotypes found in less than 10 women were grouped in another category to run a multiple logistic regression model. Table HPV 18 was not significantly associated with cytological abnormalities but the number of positive samples may have led to insufficient statistical power.Among HPV-infected women, 27.2% had a normal cervical smear. This low sensitivity of smears as a screening method is a problem in a remote area where women may not have regular gynecological follow up. The normal demographic or life history variables were unable to predict whether cytology was normal or not. Differences in HPV genotypes, however, were significantly associated with having abnormal cytology. Thus, in our sample, HPV 53 was always associated with normal cytology whereas HPV 52, 58 and 16 were associated with abnormal cervical smears. This may have reflected differences in the number of positive samples for these viruses, with rare genotypes having insufficient numbers for meaningful statistical comparisons. The results may also reflect heterogeneous potential of different HPV genotypes to lead to cytological anomalies . Indeed HPV species 6, which includes HPV 53, is reported to be associated with high risk lesions , 15 but Given the relatively low sample size and the large number of different genotypes, the different cytological anomalies, further studies should compile additional data to give a more robust estimation of the differences between genotypes and HPV species.Overall, common demographic and life history variables could not predict which women have a higher risk of having positive or negative cervical cytology. On the contrary, different high risk HPV genotypes,notably those from HPV species 9, seemed to have different potentials to cause cytological abnormalities. Vaccination with the nonavalent vaccine thus seems preferable. Presently, given the large proportion of the population that does not have health insurance in these remote areas, the cost of the vaccine is prohibitive. These financial barriers should be removed to achieve sufficient HPV vaccination coverage. Finally, given the remoteness of the villages where the study was conducted and the difficulties of access to care and the low sensitivity of cervical smears alone, future screening strategies should rely on HPV tests alone or in combination with cytology, which have demonstrated their operational interest –22.


M = 8.32 visits, SD = 17.15) and were more likely to attend at least 8 psychotherapy visits, (15.7%) than veterans with MH diagnoses but no TBI history . PTSD and TBI history, but not depression or anxiety, were associated with a greater number of psychotherapy visits when controlling for demographic and clinical variables. PTSD, anxiety, depression, and TBI history were associated with number of psychotropic medication-management visits. TBI history was related to greater MH service utilization, independent of MH diagnoses. Future research should examine what MH services are being utilized and if these services are helping veterans recover from their disorders.Traumatic brain injury (TBI) and mental health (MH) disorders are prevalent in combat veterans returning from Afghanistan and/or Iraq (hereafter referred to as returning veterans). Accurate estimates of service utilization for veterans with and without TBI exposure (referred to as TBI history) are imperative in order to provide high quality healthcare to returning veterans. We examined associations between TBI history and MH service utilization in a subsample of returning veterans who were newly diagnosed with posttraumatic stress disorder (PTSD), depression, and/or anxiety in the 2010 fiscal year . Data were extracted from the Veterans Health Administration (VHA) National Patient Care Database. Veterans with MH diagnoses and TBI histories attended significantly more psychotherapy visits, ( Since 2001, over 2.6 million Operation Enduring Freedom (OEF), Operation Iraqi Freedom (OIF), and Operation New Dawn (OND) veterans (hereafter referred to as returning veterans) have left active duty and become eligible for health care through the Veterans Health Administration (VHA)  and are Traumatic brain injury (TBI) is another common condition among veterans, given that more than 300,000 service members have experienced a TBI since 2001 . TBIs ofModerate and severe TBI are less common but can result in significant activity limitations and participation restrictions . To addrPsychiatric symptoms such as those needed to diagnosis PTSD, depression, and anxiety are related to self-reported TBI symptoms . PTSD anAlthough less studied in veteran samples, depression and other anxiety disorders are also commonly comorbid among those with TBI histories. There is wide variability in the estimates of psychiatric diagnoses post-TBI because of the diverse samples studied, including epidemiological, inpatient, and outpatient. With this caveat in mind, between 23% and 38% of civilians with mTBI histories are diagnosed with an anxiety disorder ,18 withiVeterans with TBI histories may incorrectly attribute their symptoms to the head injury rather than a MH disorder or psychiatric distress. Recently, lay and medical attention has focused on the long-term effects of TBI, which may increase the concern veterans feel when they are diagnosed with a TBI history. However, accumulating evidence indicates that long-term mTBI symptoms (>12 months) are primarily related to psychological factors, such as premorbid or concurrent MH diagnoses, rather than solely to the original head injury , 26–28. If long term mTBI symptoms are primarily related to psychological factors, then MH service use may not be affected by TBI history because psychological symptoms may already be the focus of treatment for veterans identified as having a psychiatric disorder. Conversely, service utilization may be affected by TBI histories, as veterans with TBI histories can be more clinically complex with greater severity of psychological symptoms ,15. ConsIt is imperative to understand the service utilization patterns of this younger generation of veterans as their war experiences have differed from previous generations. Younger veterans are more likely to serve in multiple deployments of greater durations, have less time between deployments, and return to a bombardment of information about MH disorders and TBI sequelae . AlthougParticipants included a subset of OEF/OIF veterans who received at least one new mental health (MH) index diagnosis of depression, anxiety disorder, or PTSD in the VHA in the 2010 fiscal year . We restricted the sample to OEF/OIF veterans, including only those born in 1973 or later  codes to determine MH diagnoses of the most common mental health problems for returning veterans: depression, including major depressive disorder, dysthymia, depression not otherwise specified, and mood disorder due to a general medical condition ; anxiety disorders ; and PTSD and/or acute stress disorder . Veterans could have more than one MH diagnosis.We defined new “index” diagnosis as the presence of a new anxiety, depression, and/or PTSD diagnosis without any MH diagnoses in the six months (clean period) prior to the index date. The veterans could have had a MH diagnosis before the six month clean period or a diagnosis given by a non-VHA provider. The veteran’s symptoms likely began before the index date; however, the index date marks the first time veterans would have access to VHA MH services since each service encounter identifies the problem(s) treated during that visit. In order to capture the most comprehensive sample of veterans with psychiatric disorders, MH diagnoses were not restricted to MH clinics and could have been given by any VHA provider. We used the following International Classification of Diseases–9A retrospective database study was conducted to examine MH diagnoses and MH service use for returning veterans with and without TBI histories. The VHA maintains data extracted from veterans’ electronic medical records in the VHA National Patient Care Database (NPCD). It scrambles Social Security numbers to protect veterans’ identities while still allowing patient and service characteristics to be studied. The accuracy and validity of the NPCD data are monitored by the VHA VIReC . VeteranDemographics included age (continuous variable), sex (man or woman), ethnicity , marital status , service-connected disability percentage , distance to VHA in miles (continuous variable), and MH diagnoses (either present or absent).We used Current Procedural Terminology (CPT) codes to the veteran’s use of outpatient MH services, including individual psychotherapy , group therapy , family therapy , and medication-management visits .Diagnoses require a provider to give an actual diagnosis rather than rely on a screen that is non-specific, thus we selected TBI ICD-9 codes rather than TBI screen results. ICD-9 codes do not reliably distinguish between those with mTBI and those with moderate and severe TBI , so all To examine the potential effect of MH comorbidities (other than the index diagnosis), we examined substance-use disorders , psychotic disorders, , bipolar disorders , and personality disorders  in any VHA encounter in the year following the index diagnosis.t-tests examined whether veterans with and without TBI histories differed in number of therapy and medication visits, using adjusted degrees of freedom to account for unequal variances. A chi-square was used to detect differences in the proportion of veterans with and without TBI histories who completed eight or more psychotherapy visits, which was modeled after previous studies to identify if the veteran received the minimum number of sessions associated with an adequate dose of sessions for evidence-based psychotherapies (EBP) for PTSD, depression, and anxiety [Data analyses were conducted using SAS version 9.2 . Independent samples  anxiety ,41. Whil anxiety ,41,42. T anxiety . CategorOf the 583,733 veterans in the VHA system who received a new-onset MH diagnosis in 2010, 84,255 (14%) were born after 1973 (our OEF/OIF variable); and, of these, 55,458 (66%) had served in combat and comprised our study sample. Within this combat OEF/OIF subsample, 21,110 (38%) had a depressive disorder, 12,402 (22%) had an anxiety disorder, 31,709 (57%) had PTSD, and 11,351 (20%) had a TBI history (percentages sum to greater than 100% because of the comorbidity between the diagnoses). t-tests demonstrated that veterans with MH+TBI, on average, attended one more individual psychotherapy visit, two more group psychotherapy visits, and 0.28 more medication visits than veterans without TBI.Across the study sample, 49% of veterans attended at least one medication visit, and 46% attended at least one psychotherapy visit in the year after receiving their index diagnosis . There wX2  = 13.93, df = 2, p < .001, φ = .25. Average time from index diagnosis to the next MH psychotherapy or medication visit was 73.96 days . Veterans with MH  had a slightly longer but statistically significant time difference from the index diagnosis to next psychotherapy or medication visit than veterans with MH+TBI  = -3.13, p = .002, d = .04).F = 90.05, p < .0001; The regression model predicting number of psychotherapy visits with demographic variables, index diagnoses, psychiatric comorbidities, and TBI history as independent variables was significant, F = 158.72, p < .0001. Increasing age, female sex, being married, and not having a service-connected disability were significantly associated with greater number of medication visits. Having a PTSD (b = 0.47), anxiety (b = 0.34), or depression (b = 0.48) index diagnosis and all psychiatric comorbidities  were associated with having more medication visits. Finally, TBI history was significantly associated with greater number of medication visits (b = 0.34), while accounting for the influence of all other variables.We examined type and quantity of MH services used by a subset of OEF/OIF combat veterans with and without TBI histories who received a new MH index diagnosis in 2010. PTSD was the most common index diagnosis, followed by depressive and then anxiety disorders. This is expected in a sample of combat veterans for whom PTSD and TBI are the “signature injuries” . VeteranDemographic variables such as female gender, being married, and not having a service connected disability were all significantly related to greater number of medication and psychotherapy visits. Women are more likely to seek mental health treatment in general  and sociMH index diagnoses were also related to quantity of services. Of the three index diagnoses included , PTSD displayed the greatest association with the number of psychotherapy visits; whereas depression had the strongest association with medication visits. This difference may be explained, at least in part, by the fact that there are a greater number of evidence-based medications available for depression than for PTSD. Additionally, VHA has developed nationwide initiatives to increase access to EBP for PTSD, thus health care resources have focused on veterans with this disorder.All comorbid MH diagnoses were associated with more psychotherapy and medication visits. Substance use and personality disorders are generally chronic conditions that require long-term psychotherapy. If veterans presented with complex presentations that involved substances use and personality characteristics that prolong their recoveries, they used more services. MH comorbidity is common , and comEven after considering demographic characteristics and MH diagnoses, TBI history was associated with increases in MH service use. This is consistent with a previous study of OEF/OIF veterans who were screened for TBI history and MH diagnoses . Maguen The current data do not provide reasons why veterans with TBI histories used more MH services; however, there are possible explanations worth considering as this generation of veterans will continue to make up a larger proportion of the population served by VHA. Veterans with TBI histories may have used more services than veterans without TBI histories due to greater symptom complexity such as substance use disorders, easier access to services, an attempt to receive service connection disability, or a combination of these factors. The complexity of veterans with TBI histories  could beFinally, Veterans with TBI histories attended more psychotherapy visits and had shorter wait times until their first follow up visit than veterans without TBI histories. However, there remains opportunity to improve service utilization rates as only 46% of the entire sample attended at least one psychotherapy visit. This is consistent with civilian literature which suggests that 43% of diagnosed patients attend any psychotherapy visit , but alsThese results lead to clinical implications and hypotheses for future research. Clinically, it is important to be mindful of the index diagnoses in addition to MH comorbidities, such as personality and substance use disorders, as they are also related to greater service utilization. Veterans may also be more comfortable seeking TBI-related services than MH services as there is less stigma associated with medical than MH diagnoses. Regardless of TBI history, veterans with psychiatric diagnoses should be educated about the effective anxiety, depression, and PTSD treatments available at all VHA medical centers. Providers can correct any misunderstandings that a veteran may have about how a TBI history will impact his/her ability to benefit from a course of psychotherapy because, fortunately, veterans with and without TBI histories see symptom reduction with treatment ,34. In tA limitation of using national VHA databases for diagnostic information is that there is no independent verification of diagnostic validity through structured clinical interviews or assessments. Similarly, under recognition of TBI in the VHA may be a limitation. Although it is a national mandate that all veterans be screened for TBI, only 92% of veterans have a documented TBI screen , and theStrengths of this study include its use of a national subsample of OEF/OIF combat veterans, affording us abundant statistical power with a large representative sample and results that will likely generalize to other OEF/OIF veterans with PTSD, anxiety, and depression with and without TBI. Understanding the unique service utilization trends of this younger generation of veterans is imperative, as they will likely differ from those of previous veteran generations as the nature of their injuries and quantity and content of deployments are vastly different than other war eras . Having Among a national subsample of veterans with psychiatric diagnoses, those with TBI histories attended more psychotherapy and medication management visits than those without TBI histories. Veterans with TBI histories were more likely than veterans without TBI histories to receive eight sessions of psychotherapy, which provides the opportunity to participate in a full course of psychotherapy. Future research should examine if the increase in services used by veterans with TBI histories is resulting in reduction of MH symptoms and overall recovery for these veterans.


The current study aimed to investigate the incidence and survival patterns of HNSCCs arising from different anatomic sites, potentially related (the oropharynx) or unrelated  to HPV, to provide clues on possible growing impact of HPV in the epidemiology of HNSCC in Italy. Epidemiological data were retrieved from ten long-term Cancer Registries covering a population of 7.8 million inhabitants. Trends were described by means of the estimated annual percent change (APC) stratified by age and gender, and compared between HPV-related and HPV-unrelated anatomical sites. The data regarding 28,295 HNSCCs diagnosed in Italy between 1988 and 2012 were analyzed. In males, the incidence rate (IR) of cancers arising from sites unrelated to HPV infection significantly decreased in all age groups , whereas stable IR were observed for cancers arising from sites related to HPV infection. In females, IR for cancers from HPV-related sites increased significantly over the observed period; the largest increment was noted in those over 60 (APC:2.92%) who also showed a significantly lower number of HNSCCs from the larynx/hypopharynx (APC:- 0.84) and a significantly higher number of oral cavity tumors (APC = 2.15). The five-year relative survival remained largely unchanged in the patients with laryngeal/hypopharyngeal SCC and, conversely, significantly improved in the patients with SCC at HPV-related sites. The trends observed suggest a potential increasing impact of HPV infection on the epidemiology of HNSCC in Italy, but to a lesser extent and with a different pattern from that observed in other Western countries. In 2020, head and neck cancer (HNC) is expected to affect approximately 833,000 and 151,000 new patients worldwide and in Europe, respectively .According to recent estimates, worldwide 38,000–45,000 cases of HNC are yearly attributable to HPV ,8. The fInterestingly, a significantly increasing prevalence of HPV-driven OPSCC has been observed over recent decades in Europe and North America . MoreoveTo provide clues on a possible growing impact of HPV infection in the epidemiology of HNC in Italy, the current population-based study set out to investigate the patterns registered over the last quarter century in incidence and survival rates, stratified by age and gender, of HNSCCs arising from different anatomic sites potentially related or unrelated to HPV infection.Incident cases of HNC registered between 1988 and 2012 were retrieved from the Italian Network of Cancer Registries (AIRTUM) based on a historical pool of 10 population-based Cancer Registries covering a population of 7.8 million of inhabitants (13% of the whole country). All data were fully anonymized before access by the researchers. Only cancer registries that provided data over the entire 1988–2012 period were included in the present analysis .th edition (ICD-10). Information on the cancers’ morphology was codified in accordance with the International Classification of Diseases for Oncology (ICD-O) morphology codes (3rd edition).HNC cases were identified by extracting categories C00-C14 and C30-C32 of the International Classification of Diseases, 10Only malignant cases with morphology codes for squamous cell histology or morphologic variants of SCC were included in the analysis  and, depending on the anatomical site of the tumor origin, classified as HPV-related or unrelated.Anatomic sites that are HPV-related included: the tonsils (C09), the base of the tongue , other oropharynx sites (C10) and Waldeyer's ring (C14.2). Unrelated HPV sites included: areas of the oral cavity  and the larynx-hypopharynx . Cancers arising from the lip (C00), the nasopharynx (C11), the nasal cavity (C30), the sinuses (C31), and the salivary glands (C07-08) were not included in the analyses as they are linked to other etiological factors or to ill-defined sites .P value. The trend was considered to be significant when P was < 0.05.Incidence rates (IR) were reported as European age-standardized and expressed as the number of new cases per 100,000 person-year. Changes in the incidence rates of HPV-related and -unrelated anatomic sites were assessed in relation to the time period, age grouping  and gender using the annual percentage change (APC) with the corresponding 95% confidence interval (CI), indicating an increased or decreased trend with a 2-sided Relative survival was calculated for all the cases included in the cohort analysis (diagnoses formulated between 1988 and 2012) as the ratio of observed to expected survival. National life tables by registry, age, and gender were used for calculating expected survival according to the Ederer II method . Age -stStatistical analyses were performed using the SEER*Stat software, Version 8.3.4 and the Joinpoint Regression Program, Version 4.5.0.1 .Incidence of all HNSCCs. Between 1 January 1988 and 31 December 2012, a total of 28,295 new cases of HNSCCs were registered in the AIRTUM pool with a 99.9% proportion of microscopically verified cases. The number of incident cases classified according to the site and gender are reported in P<0.001; 1998–2012: APC -3.14, 95% CI -3.63 to -2.65; P<0.001), while it rose significantly in the females .P = 0.001) (P<0.001) and from 14.8% to 16.5% in the females (P = 0.35). Although the cancer registration process was never modified, we noted a decreasing tendency of registering unspecified sites over the observation period. To avoid a period effect in the trends, we omitted analyzing by subsites .During the observation period, 3984 cases of potentially HPV-related cancers, 81.6% in males, were diagnosed. The average IR of HNSCCs from HPV-related sites during that period was 3.1 in the males and 0.6 in the females. The incidence remained stable in the males over the observation period A. Conver= 0.001) B. The prP<0.001) (P<0.001). The average IR of HNSCCs from the laryngeal and hypopharyngeal sites was approximately 11.5 times higher in the males  with respect to the females  showing the greatest difference between genders among head and neck sites. The relative prevalence of laryngeal (C32) and hypopharyngeal (C12 and C13) SCCs remained stable across the observation period (P = 0.85). The IR of HNSCC from the larynx and hypopharynx dramatically declined in the males  falling from 18.3 per 100,000 in 1988–1991 to 8.7 per 100,000 in 2010–2012 (P = 0.28) . During the last three decades, cancer of the oral cavity significantly fell in the males  A. An oppP<0.001) B who sho010–2012 A. A not  = 0.28) .P = 0.009). The IR of SCC arising from HPV-related sites increased significantly in females in all the considered age categories (P = 0.003).In the males A, the IRtegories B. The laP<0.001), while and in the 60+ year olds it increased until 2006  and flattened thereafter . In the males (P<0.001). In the females (P = 0.048).In the males C, the IRhe males E, the IR females F, the IRP = 0.043). Conversely, relative survival in the patients with laryngeal and hypopharyngeal SCCs only increased by 3.5 percentage points (P = 0.20), although the relative prevalence of laryngeal and hypopharyngeal SCCs, with the latter carrying a relatively poor prognosis, remained stable across the entire observation period (P = 0.857).Age-standardized relative survival  significThis cancer registry-based study addressed trends in HNSCC incidence at different anatomic sites in Italy in relation to their potential association with HPV infection in Italy over a 25-year period. Overall, the analysis of APC basically identified three different patterns: 1. cancers whose incidence is increasing ; 2. cancers whose incidence is decreasing ; 3. cancers whose incidence was substantially constant over the observation period . Thus, the analysis confirmed that the incidence rates varied depending on the site and gender.As has been reported for other tobacco-related cancers, the marked decline in the incidence of laryngeal/hypopharyngeal SCCs across all age groups mirrors the significant reduction characterizing men's smoking habits over the past six decades . Cancer -1 daily alcohol intake was 2.79 and 6.10 for laryngeal and oral cavity cancer, respectively [The incidence of other non HPV-related cancers has decreased in all age groups in males. SCCs of the oral cavity have, in particular, declined steadily and significantly over the observation period, even if the decline was lower than that for laryngeal/hypopharyngeal SCCs. The trend in the females moved instead in the opposite direction, namely an overall increase in APC of 2.15% of SCC of the oral cavity, versus stable figures for laryngeal/hypopharyngeal SCC. Similarly to smoking habits, per capita alcohol consumption in Italy has declined in both genders during the last 50 years, a decline that parallels the fall in deaths linked to liver cirrhosis . While iectively . Furtherectively . Laryngeectively . It woulectively . Interesectively  and in vectively .Incidence trends for SCCs at HPV-related sites in Italians have remained stable in the males over the entire observation period, while a significant increase in the annual incidence was observed in the females . The stable incidence in OPSCCs in males despite the marked reduction registered in environmental-related HNSCCs suggests that increasing HPV infection rates may be counterbalancing the effects of reduced tobacco and alcohol consumption.Conversely, in females, the net increase in the incidence of SCCs at HPV-related sites may be due to the increasing role of HPV in these neoplasms in the absence of environmental-related OPSCC reduction. The geographic prevalence of HPV-related OPSCC is quite heterogeneous ,10. At tHPV-driven OPSCC is characterized by a highly significant better prognosis compared to the non HPV-driven counterpart. ConsistThe study’s strengths include the large sample size and the long study period (25 years) using population-based data in a defined region. Cancer-registry data, characterized by elevated standards of completeness ensure the generalizability of these results. In addition, to our knowledge, this is the first study conducted in Southern Europe aiming to analyze the incidence trends of HNSCC focusing on anatomic sites associated with transforming HPV infection.The study’s main weakness is the lack of individual patient information on HPV status in tumor tissues and tobacco and alcohol exposure. Indeed, using cancer site as a proxy of HPV-related cancer could leave the door open to misclassification. The results are nevertheless in line with those produced by a prospective study that rigorously defined HPV-driven cases . Other sIn addition, the tendency of registering unspecified sites declined over the observation period. This bias would mean that the IRs for the first study years were underestimated, thus producing an opposite distortion depending on the shape of the trend observed: underestimation of the slope for a decreasing incidence and overestimation for an increasing one. The proportion of cases with unspecified sites was nevertheless low and decreased by less than 1% during the observation period, from 2.7% in 1988–1992 to 2.0% in 2008–2012, meaning that, the effect of this bias was probably quite small.Finally, incident cases of SCC metastatic to the neck lymph nodes from an unknown primary were not included in the present analysis; as this entity is HPV-driven in a substantial proportion of cases , this maTopographically restricted to the oropharynx, HPV-driven HNSCCs, which exhibit a survival benefit compared to HPV-unrelated tumors, have been increasing rapidly in several Western countries. Although to a lesser extent and following different patterns with respect to those observed in other Western countries, the trends in HNSCC incidence and survival rates at the different sites examined here suggest a potential increasing impact of HPV infection on oropharyngeal oncogenesis in Italy. One thousand five hundred sixty-five SCCs are expected to arise from HPV-related sites in Italy in 2017. According to the recent analyses, the fraction of OPSCCs driven by HPV infection in Southern Europe is estimated at between 20 and 30% . BesidesS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


In this critical review, we trace the evolution of the intersection of science, politics, economics and gender norms during the original HPV vaccine approval, marketing era, and implementation. Given the focus on cervical cancer screening, women were identified as bearing the burden of HPV infection and its related illnesses, and the group responsible for prevention. We also describe the consequences of the feminization of HPV, which has resulted primarily in reduced protection from HPV-related illnesses for males. We propose a multilevel approach to normalizing HPV vaccines as an important aspect of overall health for both genders. This process must engage multiple stakeholders, including providers, parents, patients, professional organizations, public health agencies, policymakers, researchers, and community-based organizations.Human papillomavirus (HPV) can cause a number of anogenital cancers  and genital warts. A decade ago, the HPV vaccine was approved, and has been shown to be a public health achievement that can reduce the morbidity and mortality for HPV-associated diseases. Yet, the mistaken over-identification of HPV as a female-specific disease has resulted in the  Feminization occurs when an issue's social construction concentrates on females Vaccinations are one of the top public health achievements of the early twenty-first century, and the HPV vaccine has the potential to prevent morbidity and mortality from cervical and other HPV-related cancer and diseases feminization of HPV and fits within an existing cultural narrative of HPV being a woman's problem. We conclude by evaluating the impact of feminization of HPV by examining the HPV vaccine's current utilization and disparities in the United States, skewed economic discussions for HPV vaccine policy, and insufficient protection for males.Feminization has the potential to negatively impact public awareness and approaches to address social and health issues across multiple stakeholder levels  2As early as five centuries ago, the relationship between cervical cancer and sexual transmission was postulated By the year 2000, cervical cancer was the second most common cancer among women worldwide and approximately half of all women who had cervical cancer died annually After years of global vaccine research and development, within the United States the Food and Drug Administration (FDA) reviewed the primary evidence of a quadrivalent HPV vaccine  3may be vaccinated with 4vHPV to prevent genital warts – a “permissive” rather than “routine” approval for use in males routinely vaccinated, males 13-to-21 should be routinely vaccinated as a catch-up group, and males 22-to-26 who are in high-risk populations, such as men who have sex with men (MSM), should be vaccinated In addition to identifying HPV as a cause for cervical cancer, other studies investigated HPV's connection to anogenital cancers, including penile and anal cancers 4Evolving research during the decade since the vaccine was first approved has resulted in changes both in the vaccine itself, and in the dosing schedule. However, policy differences between the two federal agencies (the FDA and ACIP) responsible for the approval of the vaccine have resulted in confusing recommendations by age, sex, and vaccine type . In Dece5philosophical exemption) that makes enforcement impossible, and for both Virginia and the District of Columbia, only girls are included in the legislation, perpetuating the focus on protection of girls While a number of other countries utilize school-based approaches for nationwide vaccine programs Due to the separation of state and federal lawmaking in the United States, individual states can determine and implement state-specific legislation regarding HPV vaccination requirements and mandates. According to the National Conference of State Legislatures, 42 states and territories have introduced legislation related to HPV vaccine requirements, funding, or education. Yet some states are more successful than others in the passage of these initiatives 6From a scientific standpoint, the creation of the HPV vaccine was straightforward and an almost perfect case-study of the development of a highly efficacious and safe vaccine. A more critical investigation, however, reveals how the vaccine developed in tandem with pervasive gender biases. HPV vaccine research logically proceeded in women because of the known cytological site of cervical infection and an easily identified “surrogate” endpoint. There was not an identified cancer in males that had the prevalence – or the clinical endpoints – that rivaled cervical cancer, and although genital warts affect males and females, no males were involved in the initial vaccine trials and the primary impetus for vaccine development was cancer prevention. The initial vaccine was approved for adolescent females Although typically characterized as a woman's disease, HPV is sexually transmitted , implying contact with another person and transmitting agent of HPV Feminization of HPV reinforces the long-held belief that women are responsible for reproductive healthcare in heterosexual partnerships Heuristic beliefs that the HPV vaccine is associated with early sexual behavior among adolescents persist, yet scientific evidence repeatedly demonstrates that this is not the case Tell Someone and Make the Connection, targeted at adult women. These campaigns show women, including a mother and daughter dyad, expressing their shock at the connection between HPV infection and cervical cancer and their determination to ‘tell’ everyone in their lives they care about, including their sister and mother In addition to the scientific research trajectory, the initial marketing of the HPV vaccine to females in the U.S. further feminized HPV in the public mind. As noted above, the 4vHPV vaccine, marketed by Merck Pharmaceuticals, was approved by in 2006, a full 3 years before Glaxo Smith-Kline's 2vHPV vaccine, Cervarix, was approved. The GSK version of the vaccine has never had a strong presence in the U.S. In expectation of the 2006 licensure of the 4vHPV vaccine, Merck & Co., Inc. released two unbranded awareness campaigns in June of that year, 7The feminization of HPV has had numerous consequences, disadvantaging males from receiving a beneficial vaccine and continuing a sexist rhetoric of HPV primarily impacting females. From a public health standpoint, if vaccine uptake rates in females were adequate, a treatise on the feminization of HPV would be essentially unnecessary because of achieving herd immunity. Unfortunately, we are nowhere near that threshold. Data on 2014 4vHPV uptake showed that 60% of females and 42% of males 13-to-17 years had received at least one dose and 40% of females and 22% of males completed the vaccine series is it cost-effective to vaccinate both males and females compared to females only” rather than asking “is it cost-effective to vaccinate both genders compared to not vaccinating anyone?” Additionally, cost-benefit models tend to underestimate the benefit of male vaccination, as they include assumptions regarding high female vaccine coverage Because the initial approach in the United States focused on achieving high vaccination rates among females, cost-effectiveness models have been inadvertently skewed, basing the value of vaccination only upon one gender. This has mistakenly framed the question as, “There is an additional inherent limitation to the herd immunity thesis as it is based on heteronormative worldviews. The initial logic of the HPV vaccine and herd immunity ignores MSM, and therefore does not achieve sufficient protection for all males, especially among a sub-group at greater risk for HPV-related outcomes. Clinical trial data among MSM who have been vaccinated with 4vHPV demonstrate a reduced risk of anal cancer The connection of HPV to cervical cancers has overshadowed the importance of preventing other HPV-related cancers, including oropharyngeal cancers, which affect both males and females. Oropharyngeal cancer is the second most common HPV-related cancer in the U.S., and it is projected that by 2020, HPV-related oropharyngeal cancers will exceed HPV-related cervical cancers As a result of the delay in male HPV vaccine approval, studies have indicated that females have a greater awareness and knowledge of the HPV vaccine compared to males 8feminization of HPV involves a complicated story intersected by science, politics, economics and gender norms. Historical events leading up to the HPV vaccine were driven by both scientific and economic priorities, necessitating the initial licensure for females. Given these priorities, and the reliance upon scientific evidence to drive policy, retrospective decisions to approve the vaccine for both males and females from 9-to-26 years would have been untenable. Nonetheless, the failure to create consistent guidance between males and females at the earliest opportunity underscores the risk of fragmenting science and policy.The all young people. This is demonstrated by Healthy People 2020 objectives that call for increase in HPV vaccine 3-dose coverage among 13-to-15 year old females and males to 80% national movement is required to implement state-by-state legislation and policy for school-based entry for HPV vaccination that is gender neutral. This will require coordination from multiple stakeholders: healthcare providers, parents, young adults, state-level policymakers, public health agencies, and professional organizations. Normalization of HPV vaccination is needed for this prevention strategy, despite associated controversy. Moreover, if we fail to face the issue of feminization of HPV directly, we will continue to perpetuate these consequences.It is encouraging that science and public health policy have begun to bridge the gap between scholarly evidence and societal norms to recognize that HPV vaccination is important for Thus, in order to improve HPV vaccination, we suggest a multilevel approach in normalizing HPV vaccines as an important aspect of overall health for all individuals. This approach includes policy strategies, provider education and communication tools, targeted patient messages, and coordinated action among providers and scholars. Borrowing from the social ecological model of health, we assert that a gender-neutral strategy for HPV vaccination must involve key agents at every level of influence to normalize HPV as gender-neutral philosophical exemption that has largely contributed to the lack of improvement in vaccination rates At a policy level, we advocate normalizing HPV vaccination through school entry requirements with strict exemption guidelines, identical to those for other adolescent vaccines. In making the HPV vaccine a requirement for school entry, the vaccine can instead be viewed as one of the vaccines necessary for school-age children. While requiring HPV vaccination for school entry would potentially normalize the vaccine and increase uptake, initial resistance to normalizing HPV vaccination through schools may persist. Additionally, we must consider the implementation of previous HPV vaccine mandates that have been less than successful, and learn from these efforts. For example, Virginia enacted a school-entry mandate in 2008 for HPV vaccination among girls, but this mandate had a all people from HPV-related disease.In addition to normalizing HPV vaccination through policy, normalization must occur through primary care physicians . Existing evidence suggests these physicians may be hesitant to recommend HPV vaccines all vaccines for protecting against preventable causes of death and disease. Agencies should design these messages in consultation with academic researchers, providers, and community organization groups to ensure their relevance in advancing gender-neutral HPV ideals.Similarly, the emergence of 9vHPV provides an opportunity for public health agencies such as the CDC to implement messages targeting both unvaccinated individuals and parents with vaccine-eligible children. Rather than reproducing the gendered messages of 4vHPV, 9vHPV messages must include gender-neutral information that challenges existing views of HPV. Such messages can normalize HPV vaccination by emphasizing its importance for males and females in the same regard as other vaccines. These messages can simultaneously defeminize HPV and emphasize the importance of 9HPV has a long history of feminization that has been perpetuated by the intersection of science, politics, economics and gender norms. Not only has the feminization of HPV supported historic rhetoric of gender inequality, which adversely impacts females, but has also directly impacted males by resulting in lower-than-expected HPV vaccination rates. In our suggestions for normalizing HPV vaccination and making it gender neutral, we assert a need for multilevel approaches targeting providers, patients, parents, professional organizations, public health agencies, policymakers, researchers, and community-based organizations. This approach can combat the consequences of feminization and potentially increase the likelihood of changing existing assumptions about HPV.


Despite being more than ten years since its introduction, global acceptance to the human papillomavirus (HPV) vaccine is still low. The immunogenetic background of the host, and HPV antigen recognition, are important in natural HPV infection, and should be taken into account in the understanding of adverse autoimmune reactions by the HPV vaccine in certain groups. There is no doubt of the benefit of vaccines in the reduction of the incidence of infectious diseases, and in the case of HPV, the prevention of persistent infection that would lead to cervical cancer. Side-effects, however, should be closely monitored and reported without any bias, to ensure that the benefits of vaccines outweigh the risks of adverse reactions. In this article we bring the attention on certain adverse effects of the vaccine against HPV that have not been well studied as they are not well defined. We also compare the different approaches on HPV vaccine policies regarding its adverse reactions in countries like Japan and Colombia, vs. the recommendations issued by the WHO. The vaccine against the human papillomavirus (HPV) - HPV infection is the most common sexually transmitted viral infection. Several risk factors for HPV infection have been identified, and include genetic predisposition, immune status, co-infection with other sexually transmitted diseases, and smoking. Persistent infection with this virus is associated with squamous carcinoma of the cervix, oropharynx, anus, genitalia , head, and neck. Approximately 90% of HPV infections resolve spontaneously through the immune system. Persistent infection with high-risk types 16 and 18 contributes to 20% and 50% of cervical cancers, respectively . For Cervarix, local adverse reactions occurring in ≥ 20% of subjects are pain, redness, and swelling at the site of injection. The most common general adverse events in ≥ 20% of the subjects are fatigue, headache, myalgia, gastrointestinal symptoms, and arthralgia (FDA).The most frequent reported symptoms of HPV vaccination are chronic pain with paresthesia, headaches, fatigue, and orthostatic intolerance . Small sApparently, dysfunction of the sympathetic nervous system plays an important role in the pathogenesis of these syndromes . Ninety It should be mentioned that chronic arthropathy has also been observed with other vaccines, such as the rubella vaccine.HPV vaccine safety - The recommendation for the use of HPV vaccine by the WHO is based on efficacy and effectiveness data Fig. 1).Despite these pronouncements on the safety of HPV vaccine, regions in Colombia have reported a disproportionate number of neuropathic pain cases  .It is important to note that the occurrence of demyelinating disease after vaccination, despite being low, is not negligible. This type of complication has been reported for multiple vaccines such as influenza, HPV, hepatitis A or B, rabies, measles, rubella, yellow fever, anthrax, meningococcus, and tetanus.Post-vaccination syndromes: beyond adjuvants - The presence of viral nucleic acids can cause autoimmune phenomena (henomena . HPV vachenomena . The Cerhenomena .The term “ASIA” (Autoimmune/inflammatory Syndrome Induced by Adjuvants) describes a spectrum of clinical conditions that share similar signs and symptoms, including somatoform and dysautonomic post-vaccination phenomena . With reIt would not be the first time that adjuvants in a vaccine cause an adverse reaction. The use of AS03 as an adjuvant in the Pandemix vaccine against the influenza virus was linked to the development of autoimmune narcolepsy.A recent study, however, shows that aluminum present in the adjuvant does not play a role in cellular hypersensitivity . On the Equally important, the absence of symptoms does not exclude the presence of an inflammatory phenomenon, with epipharingitis being found in all women examined after HPV vaccination in Japan . This phThis evidence justifies the call made in countries such as Colombia for the identification of predictive markers to develop autoimmune diseases in the population to be vaccinated against HPV.In conclusion - Apart from cultural and religious barriers, the adverse effects to HPV vaccines must be re-evaluated, since the initial clinical trials of the first vaccines were tested in different populations than women in Latin America or Japan. The role of the host immunogenetic background in HPV infection and the recognition of HPV antigens (antigens  are impo


This study aimed to provide more information for cancer prevention strategies by determining the distribution of human papilloma virus (HPV) genotype prevalence in invasive cervical carcinoma (ICC) and precancerous lesion patients in the Yangtze River Delta area in China.This multi-centre descriptive cross-sectional study involves four university hospitals in the Jiangzhehu area. Women with histologically confirmed cervical intraepithelial neoplasia (CIN) 1, CIN2, CIN3 or ICC who were diagnosed and treated in the four selected hospitals between February 2012 and April 2014 were eligible for recruitment. The average age of the patients was 40.93 ± 11.87 years old, among whom the youngest was 17 years old and the oldest was 76 years old.Those with immunodeficiency diseases or a previous history of cancer or CIN were excluded. HPV genotyping was performed by a central laboratory. The distribution and age and disease specificity of the HPV genotype prevalence were analysed.Of the 2181 collected samples, 251 were ICC and 1930 were CIN. The mean age of cervical cancer and CIN patients was 40.93 ± 11.8 years . The five most commonly identified HPV types in each lesion class were as follows: CIN1: 52, 58, 16, 33, and CP; CIN2: 16, 58, 52, 33, and 31; CIN3: 16, 58, 33, 52, and 31; and ICC: 16, 58, 18, 52, and 33. CIN1 had an earlier age of onset (30–40 years) than CIN2, CIN3, and cervical cancer. The age of onset of cervical cancer exhibited two peaks at 40–44 and 50–54 years of age. In all infected patients, the frequency of HPV infection with a single type was 62.9%, and with multiple types, it was 38.1%. There was no difference in the frequencies of multiple types amongst the different cervical lesions.The most prevalent genotypes in the investigated area  justify the necessity of anti-HPV vaccination in teenagers and young girls under 24 years old in the Yangtze River Delta area in China. Infection with multiple high-risk HPV types versus single infection does not increase the risk for ≥ CIN2 in ICC development. Cervical cancer is the third most common cancer amongst women worldwide, with an estimated 500,000 new cases annually and a 50% mortality rate. Human pAlthough infection with HPV is common while ICC is rare, persistent infection with high-risk or oncogenic types increases the risk of developing cancer. International prevalence studies estimate that approximately 70% of ICCs are attributed to either HPV-16 or − 18 –8. A metThe Yangtze River Delta region (Jiangzhehu area) includes Jiangsu Province, Zhejiang Province, and Shanghai; it is the most economically developed region in China. The total GDP in the Yangtze River Delta area was approximately $1 trillion USD in 2010 , and theIn this cross-sectional study, cervical specimens from ICC and CIN patients residing in the Delta region were collected. HPV genotypes were determined, and the type distribution was analysed amongst the population. This study aimed to understand the distribution of HPV types and to provide comprehensive scientific evidence to aid the development of control strategies and prevent cervical cancer in the region.Suitable patients who visited one of four university-affiliated hospitals within the region were enrolled in this study. These hospitals included the Women’s Hospital, Zhejiang University School of Medicine, the Affiliated Drum Tower Hospital of Nanjing University School of Medicine, the Obstetrics and Gynaecology Hospital of Shanghai Fudan University, and the First Affiliated Hospital of Nanjing Medical University. The cases came from 4 centres of the Yangtze River Delta area in China. These four central hospitals represent the top hospitals of Zhejiang, Jiangsu and Shanghai. According to the number of patients with local illnesses, more than half of the local women in this area undergo abnormal and opportunistic screening for cervical cancer in the clinics of these four hospitals. Approximately 15% of the local population participated in cervical cancer screening. All hospitals have sufficient patient resources to reflect the HPV classification of cervical cancer and precancerous lesions and suitable clinical and laboratory facilities to undertake this study.The study population comprised patients diagnosed with CIN1, CIN2, CIN3, or ICC based on pathology who were treated at one of the four aforementioned hospitals between February 2012 and April 2014. To be included in the study, patients were required to meet the following inclusion criteria: untreated histopathological diagnosis of CIN1, CIN2, CIN3, or ICC; no previous ICC or CIN history; negative for immunodeficiency diseases, including HIV; informed consent; and permanent residency in the Yangtze River Delta region. The average age of the patients was 40.93 ± 11.87 years old, among whom the youngest was 17 years old and the oldest was 76 years old. There were no patients younger than 16 years old. There were 10 excluded patients whose postoperative diagnoseis didn’t accord with preoperative diagnoses .Pathological diagnosis of cervical lesions was used as the gold standard, and the diagnosis was conducted and reviewed in each hospital independently. Uniform diagnostic review was not conducted. Diagnosis of cervical cytology was classified by the 2001 revised Bethesda system. Pathology experts interpreted the results as follows: absence of intraepithelial lesion or malignancy (WLM); atypical squamous cells of undetermined significance (ASCUS or AGUS); atypical squamous cells, high-grade intraepithelial lesions cannot be excluded (ASC-H); low-grade squamous intraepithelial lesion (LSIL); high-grade squamous intraepithelial lesion (HSIL), including those caused by HPV infection; and SCC, ACa, and AdCa. Histological grading was based on Broder’s classification.A cross-sectional study design was employed to determine the distribution of HPV genotypes amongst ICC and CIN patients in the Delta region. According to the histopathological results, patients were divided into 4 groups: the CIN1, CIN2, CIN3 and ICC groups. The patient information was collected and then tested for HPV typing.All participating hospitals followed the same patient registration protocol and obtained the following information: patient name, assigned patient number, completed HPV patient information collection form (CRF), and signed informed consent. The survey was approved by the Medical Ethics Committee of Affiliated Obstetrics and Gynecology Hospital of Zhejiang University Medical School. Cervical cytology was conducted for all patients following a gynaecological examination. The study was conducted over a period of 26 months. For consistency of results and to ensure proficiency, all registrations were completed by a physician and a trained investigator at each hospital.All participating hospitals sequentially registered patients, and physicians recorded all information in the HPV-CRF. According to the histopathological results, patients were divided into 4 groups: the CIN1, CIN2, CIN3 and ICC groups. The cervical exfoliated cells collected before operation were genotyped for HPV, 10 cases were excluded because of the inconformity of the pre- and post-operative diagnoses. A Cytobrush was inserted into the endocervical canal until the brush bent against the ectocervix. The brush was then rotated clockwise five times. After specimen collection, the brush head was placed into a collection vial that was labelled with the collection date, patient name, and patient number before the HPV types was checked. The storage vial with the brush head was stored immediately at − 20°C. All specimens were sent to a central laboratory within weeks of collection for determination of the HPV genotype.HPV genotyping was conducted as reported previously .An HPV GenoArray test was performed for the HR-HPV samples. The HPV genotype was determined by DNA amplification, flow-through hybridization, and a gene chip using the 21 HPV GenoArray Diagnostic Kit  according to the manufacturer’s instructions. Detailed protocols for this assay have been described previously . The genAll results were verified using the 37 HPV GenoArray Diagnostic Kit from the same company. The chip could identify 15 HR-HPVs  and 24 LR-HPVs .t-test was used for variable data. A p value of < 0.05 was considered significant.A database was established using Excel 2003, and the results were analysed using SPSS 17.0 software . A Chi-square test was used for the counting analysis, and a A total of 2181 qualified specimens were collected. The specimens included 251 ICC cases and 1930 CIN cases. HPV infection was not detected in 200 cases. Infection with one, two, three, four, five, six, and eight types of HPV was detected in 1372 cases, 454 cases, 119 cases, 29 cases, 5 cases, 1 case, and 1 case, respectively. One 50-year-old patient with a CIN2 pathological diagnosis tested positive for HPV types 52, 39, 45, 51, 35, 53, 66, and CP8304.The distribution of HPV infection with a single type and multiple types in the study population from the Yangtze River Delta region is shown in Table The five most prevalent HPV types in ICC and its precursor patients from the Yangtze River Delta region were 16, 58, 52, 33, and 31. Of these, the most prevalent types were 52, 58, 16, 33, and CP8304 in CIN 1; 16, 58, 52, 33, and 31 in CIN 2; 16, 58, 33, 52, and 31 in CIN 3; and 16, 18, 58, 52, and 33 in ICC.The distribution of infection with a single HPV type in the study population from the Yangtze River Delta region is shown in Fig. The five most prevalent HPV types involving a single infection were 16, 58, 52, 33, and 31 in ICC and CIN patients from the Yangtze River Delta region. Of these, the five most prevalent HPV types of single infection were 52, 58, 16, 33, and CP8304 in CIN 1; 16, 58, 52, 33, and 31 in CIN 2; 16, 58, 33, 52, and 31 in CIN 3; and 16, 18, 52, 33, and 31 in ICC.The most prevalent HPV types were similar between single and multiple infections, suggesting that these types possess the strongest oncogenicity.In all infected patients, the frequency of HPV infection with a single type was 62.9%, and with multiple types it was 38.1%. The most common number of multiple infections involved two different types, and the maximum number of types was eight. There were no differences in the frequencies of multiple types amongst different cervical lesions, suggesting that increased numbers of HPV types do not elevate the risk for high-grade CIN in ICC development, as shown in Fig. HPV types with the frequency less than 1% are not shown.Age-stratified HPV distribution in the study population from the Yangtze River Delta region Fig HPV types with a frequency less than 1% are not shown.HPV types with the frequency less than 1% are not shown.There was no significant difference in HPV typing in Jiangsu, Zhejiang and Shanghai Fig. . HPV typIn the Yangtze River Delta region, over 10% of cervical cancer patients are under 35 years of age, and over 25% of patients with CIN and cervical cancer are under 35 years of age. The prevalence of CIN1 in the Delta region reached a peak in patients between 30 and 40 years of age, which is approximately 10 years earlier than the peak for other high-grade lesions. The prevalence of CIN3 and ICC peaked simultaneously and later than the peak for CIN2. ICC presented with two peaks at 40–44 and 50–54 years of age, as shown in Fig. The Yangtze River Delta region = YR,Zhejiang Province = ZJ,Jiangsu Province = JS,Shanghai = SH,Of = −.For example (e.g.): Total of the Yangtze River Delta region(%) = Total-YR,CIN1 of Zhejiang Province = CIN1-ZJ.In Zhejiang Province, the incidence of cervical cancer in patients under 35 years old was 9.09%. The incidence of ICC and CIN in patients under 35 years old was 26.05%.The age distributions of patients with CIN1, CIN2, and ICC in Zhejiang Province all showed two peaks. The first peaks for CIN1 and CIN2 were 10 years earlier than that for ICC, and the first peak for CIN3 was 5 years earlier than that for ICC.In Jiangsu Province, the incidence of cervical cancer in patients less than 35 years old was 18.05%. The incidence of ICC and CIN in patients less than 35 years old was 30.1%.The age distribution of patients with ICC showed two peaks in Jiangsu Province. The first peak of Jiangsu Province for ICC was 5 years earlier than those of Zhejiang Province and Shanghai.In Shanghai, the incidence of cervical cancer in patients less than 35 years old was 4%. The incidence of ICC and CIN in patients less than 35 years old was 21.93%.2 = 9.75, p < 0.01) than that in Shanghai. The age distribution of patients with ICC also showed two peaks in Shanghai, whereas those of CIN1 and CIN2 were 5 years earlier than that of ICC.The age distribution of patients with ICC showed two peaks in Shanghai. The prevalence of cervical lesions in patients less than 35 years old in Jiangsu Province was significantly higher . In a study from Nanjing, Jiangsu Province, Hong et al. reported overall prevalence of HPV DNA amongst pregnant women of 13.4% (422/3139). The most frequently detected HPV genotypes were HPV-16 , − 18 , and − 58  [Our present study identified that the most common HR-HPV types in CIN and ICC patients from the Delta region are HPV-16, 58, 52, 33, and 31. The distribution of infected HPV types shows substantial differences amongst healthy women, CIN and ICC patients. In a study in Zhejiang Province, Ye et al. reported an HPV prevalence of 13.3% using a cervical pap test. In the same population, the prevalence of HR-HPV was 10.2% . The preThe distribution of infected HPV types shows substantial regional differences amongst CIN and ICC patients. Yang et al. previously investigated 1715 cervical neoplasia outpatients and identified HPV infection in 57.1% of those patients. They also reported that 76.9% of cancer patients were HPV-positive whereas 98.1% of CIN3 patients were HPV-positive. The most common types of HPV infection in these patients were HPV-16, 58, 52, 33, and 31 .A meta-analysis of the HPV type distribution identified a total of 14,595 and 7094 cases of ICC and HSIL, respectively [In the Yangtze River Delta region, the HPV type distribution of women aged over 35 years was similar to that of the HSIL type in women with CIN2 and CIN3 . FurtherWe hope that this cohort study provides information to support vaccination before the peak age of HPV infection. The best time to vaccinate women against HPV is before engaging in sexual activities, or at minimum prior to 13–24 years of age.The HPV distribution frequency is correlated to some extent with pathogenicity. For example, women with persistent infection were more likely to have a higher pathogenicity. Our current findings suggested that in groups aged under 30 and over 40 years old, the highest frequency distribution was consistent with high pathogenicity and was observed in HPV types 16, 18, 58, 52, 33, and 31.According to the most prevalent genotypes in our area, vaccination is a strong tool in the prevention of cervical cancer , and new vaccines are necessary to cover some genotypes that are missing from current formula.In CIN patients, the most prevalent HPV type was HPV-52, and infections with multiple types were identified in 10% of individuals in the Yangtze River Delta Region. Jiangsu Province was found to have the most multiple-type infections. Our findings differ from those of the Beijing University First Hospital, Sichuan University, and Munoz, which all suggest a regional difference in HPV distribution between CIN and ICC. Our study indicates that HPV types 58 and 52, in addition to types 16 and 18, play important roles in the development of cervical cancer in the Yangtze River Delta Region.There was no significant difference in the number of sexual partners or sexual behaviour of women in different areas of the Yangtze River Delta region. With respect to the economy, the GDP of Jiangsu Province was greater than that of Shanghai, which was greater than that of Zhejiang Province, whereas the national per capita income was the highest in Shanghai, followed by Zhejiang Province and Jiangsu Province. The per capita income is relatively low in Jiangsu Province; to increase this, the region has observed a large influx of personnel, resulting in an increased likelihood of HPV infection. Moreover, investigations into cervical cancer screening suggest an overall lack of health care knowledge and indicate that women do not pay attention to screening and early diagnosis and treatment of disease. This appears to be most serious in Jiangsu Province, followed by Zhejiang Province and Shanghai Province. Comparing the differences in the most common HPV types together with aspects of infection in the three different regions, we can conclude that the Jiangsu area has a higher proportion of composite infection. This likely reflects the rapid economic development of Jiangsu Province, the relatively low per capita income, the increased flow of personnel, the lack of health care knowledge, and the low importance placed on cervical cancer screening and early diagnosis and treatment of disease.HPV58 and HPV52, in addition to HPV16 and HPV18, may be involved in the occurrence of cervical cancer in the Yangtze River Delta area in China. Infection with multiple high-risk HPV types does not increase the risk for high-grade CIN in ICC development.In the Yangtze River Delta Region, the peak age of the onset of cervical cancer was between 40 and 54 years, whereas that of CIN was between 30 and 39 years of age. On average, the peak age of onset of CIN was 5–10 years earlier than that of cervical cancer. The most common HPV types identified were 52, 58, 16, 33, and CP8304 in CIN1 patients; 16, 58, 52, 33, and 31 in CIN2 patients; 16, 58, 33, 52, and 31 in CIN3 patients; and 16, 18, 58, 52, and 33 in ICC patients. The age at which infection with HR-HPV most commonly occurs is the same as that in CIN and is earlier than the peak onset of cervical cancer. This is consistent with current epidemiological models of viral carcinogenesis.According with the most prevalent genotypes in our area, vaccination is a strong tool in the prevention of cervical cancer, and new vaccines are necessary to cover some genotypes that are missing from the current formula.


According to international guidelines, HPV DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more severe lesions. The aim of this study was to assess whether REALQUALITY RQ-HPV Screen, a new assay based on real time PCR that targets the E6-E7 region of 14 high-risk human papillomaviruses, meets the criteria for primary cervical cancer screening.As required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with that of a clinically validated reference test . The reproducibility of the device was assessed as well. The clinical samples used to test the hypothesis of non-inferiority and to asses reproducibility comprised 910 and 536 cervical specimens respectively. All specimens were originating from a population-based screening cohort.The study demonstrates that both the clinical sensitivity and specificity of REALQUALITY RQ-HPV Screen are non-inferior to those of HC2. In addition, an adequate intra- and inter-laboratory reproducibility has been reached by the test.REALQUALITY RQ-HPV Screen fulfils all the requirements of the international guidelines and can be considered clinically validated for primary cervical cancer screening purposes. Cervical cancer screening based on Pap Test represents one of the most successful public health interventions in the last 60 years, having led to the decrease of both cervical cancer incidence and mortality . RecentlIn order to perform the non-inferiority test, a total of 910 women, who attended for the Cervical Cancer Screening Program in the district of Bari in the period from September 2015 to October 2016, were selected retrospectively from the files in the Cytopathology Department of the Hospital Di Venere, that represents a reference laboratory for cervical cytology. Of the selected women, 73 had histologically confirmed ≥CIN2, 600 had a negative cytology result in this as well as the previous screening round, 192 had a negative cytology result in this as well as the two previous screening rounds and 45 had an abnormal cytology result with <CIN2 histology. Cytologic cervical samples belonging to the first group of women  were tested to assess the clinical sensitivity of REALQUALITY RQ-HPV Screen for ≥CIN2, while cytologic cervical samples belonging to the second, third and fourth group of women  were tested to assess its clinical specificity. Samples were selected consecutively within each group of women.The reproducibility of REALQUALITY RQ-HPV Screen was assessed by testing 536 samples, 168 (31.3%) of which were HC2 positive. These samples, selected to provide an adequate positivity rate according to guidelines [After collection, samples had been stored in ThinPrep PreservCyt Solution  at room temperature throughout the whole duration of this study.HC2 was performed right after cytology on all samples with abnormal cytological results, according to the screening algorithm implemented at Hospital Di Venere. Samples with normal cytology were tested with HC2 between May and October 2016, in parallel with the REALQUALITY RQ-HPV Screen analysis. HC2 allows the detection of 13 hrHPV genotypes  by hybridization with RNA probes and signal amplification. Briefly, 6 mL of each PreservCyt sample were centrifuged with a conversion buffer, the pellet was resuspended in a denaturing reagent and incubated at 65 °C for 45 min. The hybridization step with the cocktail of RNA probes was performed in a microtiter plate at 60 °C for 60 min. The samples were then transferred to another microtiter plate coated with anti-hybrid antibodies and incubated for 60 min. The immobilized hybrids were detected by adding peroxidase-labelled anti-hybrid antibodies and a chemioluminescent substrate. Light emission was measured semiquantitatively as Relative Light Unit (RLU) in a luminometer. Results were expressed in terms of RLU of the sample divided by the mean RLU of three positive controls, each containing 1.0 pg/mL of HPV DNA. Samples were reported as positive when producing concentration values equal to or greater than 1.0 pg/mL. Positive and negative controls, provided by the manufacturer, were included in each run.DNA extraction and first round DNA testing with REALQUALITY RQ-HPV Screen were performed between May and October 2016. DNA was extracted from 400 μL of PreservCyt samples using the GENEQUALITY X120 Pathogen kit  with the fully automated platform GENEQUALITY X120. When required, the platform also performs the setup of the PCR plates and handles the loading of the DNA from the elution plates automatically. Real time PCR with REALQUALITY RQ-HPV Screen was performed, according to manufacturer instructions, on an Applied Biosystems 7500 Fast Dx Real-Time PCR System . This in vitro diagnostic device allows the detection of 12 hrHPVs  as a pool and the individual genotyping of HPV 16 and 18 by real time PCR targeting E6 and E7 regions in HPV genome; human beta-globin gene is detected as well to allow monitoring of both extraction and amplification processes. A ready to use mastermix is provided, containing a cocktail of hydrolysis probes marked with four different fluorochromes: FAM for the hrHPV group, JOE for HPV 16, Cy5 for HPV 18 and ROX for the control gene. A positive control, which allows monitoring fluorescence detection from all channels, is also provided by the manufacturer and was included in each run; a negative control (molecular grade water) was included as well. All samples were amplified once and fluorescence signals from each of the four channels were read independently. Sample positivity was assigned when at least one HPV amplification curve intersected the threshold line, set according to manufacturer instruction, and the corresponding Ct value was < 40. Sample negativity was assigned when no HPV amplification curve intersected the threshold  and the control gene Ct was ≤ 34.All samples with discordant results were additionally analyzed using AMPLIQUALITY HPV-TYPE EXPRESS v.3.0 . This genotyping assay, based on the amplification of L1 viral region followed by Reverse Line Blot, is capable of recognizing 40 different HPV types separately –10.P value < 0.05 was considered statistically significant. The Cohen’s kappa statistic was used to test intra- and inter-laboratory reproducibilities.To compare the clinical sensitivity and specificity for ≥CIN2 of REALQUALITY RQ-HPV Screen to that of HC2, a non-inferiority score test with a power of at least 80% was performed , 11. TheNo samples resulted unsuitable for analysis with REALQUALITY RQ-HPV Screen .REALQUALITY RQ-HPV Screen as well as HC2 were positive for the same 71/73 ≥ CIN2 samples, showing an absolute sensitivity of 97.3%  , while HC2 was negative for 757/837 samples with an absolute specificity of 90.4% (CI 88.3% to 92.4%) .The non-inferiority score test demonstrated that both the clinical sensitivity and specificity for ≥CIN2 of REALQUALITY RQ-HPV Screen were non-inferior to those of HC2  with a kappa value of 0.987 Table .Table 2IThe inter-laboratory reproducibility for the specimens tested in the laboratories of Bari and Messina was 99.6%  with a kappa value of 0.991 , kappa values associated to the intra- and inter-laboratory agreement ranged from 0.931 to 1.000 Table .Table 4IREALQUALITY RQ-HPV Screen, a novel real time PCR assay targeting E6 and E7 regions in HPV genome, has been assessed in the present study according to Meijer et al. international guidelines  and was P = 0.002) and specificity (P = 0.021). Only two ≥CIN2 samples resulted negative for both assays. The genotyping of these two cases confirmed the absence of HPV DNA in the case histologically classified as adenocarcinoma. This finding is in agreement with recent works reporting cases of HPV absence in this type of cancer [Our findings show that REALQUALITY RQ-HPV Screen performs at least equally well as HC2, as the null hypothesis of inferiority was rejected for both sensitivity  and cobas® 4800 HPV Test  have already been validated according to Meijer et al. international guidelines [Devices with assay designs more similar to REALQUALITY RQ-HPV Screen than HC2, such as Abbott RealTiidelines , 19 corrREALQUALITY RQ-HPV Screen fulfils all the requirements of the international guidelines and can be considered clinically validated for primary cervical cancer screening purposes.


The major transformation activity of the high-risk human papillomaviruses (HPV) is associated with the E7 oncoprotein. The interaction of HPV E7 with retinoblastoma family proteins is important for several E7 activities; however, this interaction does not fully account for the high-risk E7-specific cellular immortalization and transformation activities. We have determined that the cellular non-receptor protein tyrosine phosphatase PTPN14 interacts with HPV E7 from many genus alpha and beta HPV types. We find that high-risk genus alpha HPV E7, but not low-risk genus alpha or beta HPV E7, is necessary and sufficient to reduce the steady-state level of PTPN14 in cells. High-risk E7 proteins target PTPN14 for proteasome-mediated degradation, which requires the ubiquitin ligase UBR4, and PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cell lines. Residues in the C terminus of E7 interact with the C-terminal phosphatase domain of PTPN14, and interference with the E7-PTPN14 interaction restores PTPN14 levels in cells. Finally, PTPN14 degradation correlates with the retinoblastoma-independent transforming activity of high-risk HPV E7. High-risk human papillomaviruses (HPV) are the cause of cervical cancer, some other anogenital cancers, and a growing fraction of oropharyngeal carcinomas. The high-risk HPV E6 and E7 oncoproteins enable these viruses to cause cancer, and the mechanistic basis of their carcinogenic activity has been the subject of intense study. The high-risk E7 oncoprotein is especially important in the immortalization and transformation of human cells, which makes it a central component of HPV-associated cancer development. E7 oncoproteins interact with retinoblastoma family proteins, but for several decades, it has been recognized that high-risk HPV E7 oncoproteins have additional cancer-associated activities. We have determined that high-risk E7 proteins target the proteolysis of the cellular protein tyrosine phosphatase PTPN14 and find that this activity is correlated with the retinoblastoma-independent transforming activity of E7. Human papillomaviruses (HPV) are the etiologic agents for cervical cancer, many other anogenital cancers, and an increasing proportion of oropharyngeal cancers . The 8-k1–The high-risk HPV oncoproteins are important for their role in the development of HPV-associated cancers and also because they represent simple and tractable research tools that can be used to study tumor suppressor pathways in human cells. The cellular pathways revealed through the studies of oncoproteins encoded by DNA tumor viruses, including the HPVs, have proven to be critical in many non-virus-associated cancers . HPV E7 1/S checkpoint  and score in several transformation assays –19, but –20–25–E6 and E7 do not have enzymatic activities and thus act through physical interaction with specific host cellular factors. To better understand E6 and E7 activities, we conducted systematic proteomic analyses of interactions between HPV proteins and their host cellular targets using a panel of E6 and E7 proteins from diverse HPV types –32. Othe–PTPN14  is a classical non-transmembrane protein tyrosine phosphatase (PTP) . It has 36–The association of PTPN14 with several cancers led to our particular interest in PTPN14 as a target of HPV E7 proteins from diverse virus types. In this study, we confirmed the interaction of PTPN14 with several HPV E7 proteins and further discovered that high-risk HPV E7 proteins promote the proteasome-mediated degradation of PTPN14. We map the interaction sites on E7 and on PTPN14 and show that overexpression of the E7-binding fragment of PTPN14 can block the PTPN14-E7 interaction and restore PTPN14 levels. Finally, we find that BPV1 E7 but not HPV1a E7 triggers PTPN14 degradation. Our results are consistent with the model that the targeted degradation of PTPN14 by high-risk E7 could account for some of the retinoblastoma-independent transformation activity of E7 that has been described in the literature for many years.Our previous proteomic analysis of the cellular interacting proteins of diverse HPV E7 proteins identified the interaction of the cellular protein PTPN14 with 15 different HPV E7 proteins. These PTPN14-binding E7 proteins included all 10 genus alpha E7 proteins and 5 of the 7 genus beta E7 proteins that we examined . Twelve To validate this interaction, we performed immunoprecipitation (IP)-Western blot (WB) analysis using a subset of the N/Tert cell lines from our initial E7 study. N/Tert-1 cells are human foreskin keratinocytes immortalized with hTert  that we To confirm that the reduced PTPN14 levels observed in the N/Tert cells expressing the high-risk E7 proteins was a consequence of E7 expression rather than an artifact that might have resulted from the long-term culture of the N/Tert-1 cell lines used in the proteomics studies, we transduced N/Tert-1 keratinocytes with the retrovirus encoding HPV18 E7 and assessed PTPN14 levels 24 to 72 h posttransduction . The redTo determine whether E7-mediated PTPN14 protein reduction also occurs in HPV-positive cervical cancer cell lines, we examined PTPN14 protein in HPV18-positive HeLa cell and HPV16-positive Caski cells . PTPN14 To determine the regions of E7 and of PTPN14 that contribute to their interaction and to the reduction of PTPN14 protein levels, we performed a series of binding experiments using several well-defined mutants of HPV16 E7 and deletion mutants of PTPN14 . We chosWe assessed the ability of a panel of HPV16 E7 mutants expressed in stable pools of N/Tert-1 keratinocytes to bind and degrade PTPN14 in IP-WB experiments and selected several of them for further study. Having found that several C-terminal mutations eliminated the ability of HPV16 E7 to bind to PTPN14, we chose to focus on the N- and C-terminal mutations that we predicted would be least disruptive to HPV16 E7, namely, the H2A N-terminal mutant and the L82R L83R C-terminal mutant. Wild-type HPV16 E7 and HPV16 E7 Δ21–24 were also included as controls. This experiment assessed the coimmunoprecipitation of E7 and PTPN14 and the effect of the E7 mutations on the reduction of PTPN14 levels. Both wild-type HPV16 E7 and the RB1-binding mutant of E7 (HPV16 E7 Δ21–24) reduced the steady-state levels of PTPN14 protein, but cells producing HPV16 E7 H2A or HPV16 E7 L82R L83R contained levels of PTPN14 protein equivalent to those in the empty-vector-containing cells , 42, andFinally, we used a panel of PTPN14 deletion mutants very similar to a previously described collection of PTPN14 constructs . These mTo further confirm the binding site of E7 on PTPN14 and to determine the effect of interfering with this interaction, we overexpressed either the PTP domain fragment that includes the E7 binding site, full-length PTPN14, or a control linker 2 domain fragment (L2) of PTPN14 that does not bind to E7 . In N/TeTo confirm that the PTP domain acts to interfere with the binding of E7 to endogenous PTPN14 and that the increase in PTPN14 levels is observed with other HPV E7s, we transduced N/Tert-HPV18 E7 cells with lentiviruses encoding wild-type PTPN14, the PTPN14 L2 domain, or the PTPN14 PTP domain. Again, full-length PTPN14 was detected in these cells only when it was overexpressed or when the PTP domain was present  might be required for this reduction in PTPN14 protein levels, we assessed PTPN14 stability in cells with siRNA-mediated depletion of UBR4 or of CUL3. CUL3 is the scaffolding component of many E3 ligases, and it interacts with several HPV E7 proteins , but CULTo confirm that UBR4 targets PTPN14 for degradation only in the presence of E7, we examined UBR4’s effect on PTPN14 stability in a cycloheximide chase experiment in cells without E7 . When E7RB1 binding and degradation have long been recognized to contribute to the cellular transformation activity of the high-risk HPV E7 proteins. However, it is equally well appreciated that high-affinity RB1 binding by E7 does not correlate with the ability of E7 proteins to act in cellular transformation assays. For example, HPV1a E7 binds RB1 with high affinity but does not transform , 46. In To determine whether PTPN14 degradation might better correlate with E7 transformation activity than does RB1 binding, we assessed the ability of several HPV E7 proteins from diverse genera as well as BPV1 E7 to target PTPN14 for degradation. In IP-WB experiments using E7 proteins stably expressed in N/Tert-1 keratinocytes, we observed that BPV1 E7, but not HPV1a E7 nor the genus gamma HPV4 E7, was able to reduce the steady-state level of PTPN14 . ConsistPTPN14 was difficult to detect on a Western blot after immunoprecipitation of BPV1 E7 likely because of its efficient degradation in the presence of this E7. Consistent with the ability of BPV1 to reduce PTPN14 levels in N/Tert-1 cells, we observed that PTPN14 was readily detectable by Western blotting in mouse C127 cells but not in ID13 cells . ID13 ceDecades of study have helped to explain how high-risk E6 and E7 act in immortalization and transformation assays, yet there are still open questions regarding the mechanisms by which these proteins dysregulate host cellular functions. For more than 25 years, there has been evidence in the literature that high-risk HPV E7 proteins have retinoblastoma-independent immortalization and transformation activities , 46. ManOur previous report on the E7 protein interaction data focused on aspects of E7 function related to RB degradation. We described the cullin 2/Zer1 E3 ubiquitin ligase complex that is required for the HPV16 E7-mediated degradation of RB1, and this together with an earlier study established the mechanism of RB1 degradation by HPV16 E7 , 50. NexPTPN14 degradation by E7 is an excellent candidate to account for the data supporting the retinoblastoma-independent transformation activity of E7. High-risk E7 is necessary and sufficient for PTPN14 degradation, and PTPN14 degradation is not an activity of low-risk E7  and 2. PWe also see a close correlation between PTPN14 degradation and RB-independent transformation in two assays. In a previously published study, we found that HPV16 E7 and some mutants of HPV16 E7 have activity in an assay of retinoblastoma-independent transformation . The HEK47–INK4A, a negative regulator of the cell cycle that is upregulated in high-risk HPV-associated cancers. The induction of p16INK4A and subsequently p53 would normally lead to cell cycle arrest and oncogene-induced senescence mediated by RB1. To avoid this, E7 proteins inactivate RB1 and bypass the senescence that would otherwise result from the initial oncogenic hit. This model could account for the RB1-independent transformation functions of high-risk HPVs and could explain why high-risk but not low-risk E7 proteins are oncogenic, but the initial oncogenic event has not been identified. Our future studies will examine the possibility that PTPN14 degradation provides this initial oncogenic event.It is interesting to consider how PTPN14 degradation might address the limitations of the current HPV-associated transformation model. A revised model of transformation has proposed that an initial oncogenic event occurs when high-risk E7 proteins are expressed in cells . This leFinally, there are other binding partners of E7, and we do not discount the possibility that they may also act in RB-independent transformation by E7. p190RhoGAP binds to the C terminus of several HPV E7 proteins, although this interaction is not specific for high-risk HPVs . We also56–PTPN14 is an appealing candidate to contribute to transformation by E7 because of its involvement in cell growth controls. PTPN14 can act as a negative regulator of YAP1 in the Hippo signaling pathway, which regulates proliferation and apoptosis , 60, 61.Our results demonstrate that high-risk HPV E7 bind and target PTPN14 for proteasome-mediated degradation. This degradation requires UBR4 and can be inhibited by blocking the interaction between E7 and PTPN14. The ability of E7 to target PTPN14 for degradation maps to the N and C termini of E7 and is independent of RB1 binding by E7, making it consistent with previous suggestions in the literature that there is a retinoblastoma-independent transformation activity of E7. PTPN14 is mutated in several cancers, and our subsequent studies will investigate the effect of restoration of PTPN14 levels in HPV-positive cancer cells. We also plan to investigate the effect of the E7-PTPN14 interaction on host cellular pathways, including Hippo signaling and other pathways impacted by YAP.The murine stem cell virus (MSCV)-empty vector and MSCV-HPV18E7-Flag-HA plasmids used in transient transduction experiments have been previously described , 65. MSC4Cl treatments, cells were treated with 30 µM MG132, 10 mM NH4Cl, or dimethyl sulfoxide (DMSO) control in culture medium for 8 h prior to harvest. For cycloheximide treatment, cells were treated with 40 µg/ml cycloheximide in culture medium and harvested at the indicated time points.N/Tert-E7 cell lines and their propagation have been previously described, and new N/Tert-1 cell lines were established and selected in the same way . HeLa, CN/Tert-1, HeLa, Caski, and C33A cells were transfected with siRNAs as previously described , 66 usinWestern blots were performed as previously described  using NuGel filtration chromatography was performed essentially as previously described . BrieflyFigure S1 Quantification of PTPN14 levels in cycloheximide chase experiments. ImageJ software was used to quantify the intensity of the bands in the PTPN14 Western blots shown in Figure S1, EPS file, 0.4 MBTable S1 Plasmids used in this study.Table S1, XLS file, 0.02 MB