To characterize the cervicovaginal microbiota of HPV-positive and HPV-negative asymptomatic Peruvian women, by identifying the presence of 13 representative bacteria genus.A total of 100 HPV-positive and 100 HPV-negative women were matched by age for comparison of microbiota. The following bacteria were more frequently identified in HPV-positive patients compared to HPV-negative: Eubacterium (68 vs 32%), Actinobacteria (46 vs 33%), Fusobacterium (11 vs 6%) and Bacteroides (20 vs 13%). A comparison between high-risk and low-risk genotypes was performed and differences were found in the detection of Actinobacteria (50 vs 33.33%), Bifidobacterium (50 vs 20.83%) and Enterococcus (50 vs 29.17%). Human papillomavirus (HPV) infection is the most common sexually transmitted infection worldwide . It is rAccording to the World Health Organization (WHO) and the Global Cancer Observatory (GLOBOCAN), cervical cancer represents the fourth most common cancer reported in women, accounting for approximately 6.6% of all female cancers , 5. It iSeveral studies have shown that the prevalence of HPV is the accumulated number cases of incident and persistent infections combined due to the lack of clearance . More thIt is still unclear why some HPV infections resolve clinically, and others progress to cervical dysplasia and ultimately, neoplasia. Recently, it has been proposed that women with a specific composition of cervico-vaginal microbiota may be more susceptible to acquire HPV or display a rapid progression to cancerous lesions , 9.Lactobacillus, accompanied by other anaerobic bacteria including Actinobacteria, Fusobacterium, Gardnerella, Atopobium, Leptotrichia, Sneathia, Prevotella spp. and other bacterial species [The cervico-vaginal microbiota is composed by a variety of microrganisms, usually dominated by different species of  species . Moreove species .Lactobacillus strains present in the cervico-vaginal microbiota; however, the role of other anaerobic bacteria requires further investigation. Therefore, the objective of this study was to characterize the cervico-vaginal microbiota of HPV-positive and HPV-negative asymptomatic Peruvian women, by identifying the presence of 13 representative bacteria genus.Most of the studies have assessed the impact of the relative abundance of Hospital Regional Docente de Cajamarca, in northern Peru [A case–control study was carried out, using samples and data collected from a previous study performed in ern Peru . Women wA total of 833 women were studied in the context of a screening performed in asymptomatic women from Cajamarca, Peru. Among the 833 samples, 200 samples were positive for HPV and 633 were negative at baseline. For methodological convenience, we selected 100 positive cases and 100 negatives cases matched by age for the comparison of cervico-vaginal microbiota in this study.Samples were collected from the ectocervix and endocervix using a disposable cytobrush, they were stored in a tube containing phosphate buffered saline (pH: 8.6) for preservation. The tubes were maintained at − 4 °C until later processed. All tubes were vortexed and centrifuged to pellet the cells, which were resuspended in 1 mL of phosphate buffered saline. Three aliquots of each sample were stored at − 20 °C until testing.Samples were kept at − 20 °C until analyzed. DNA extraction from 200 µL of cervico-vaginal samples was performed using the High Pure PCR Viral Nucleic Acid Kit (Roche), according to manufacturer’s instructions. The DNA extracted was stored at − 20 °C until used.HPV amplification was carried out using primers and conditions previously described . AmplifiActinobacteria, Bacteroides, Bacteroidetes, Bifidobacterium, Clostridium, Enterococcus, Eubacterium, Firmicutes, Fusobacterium, Lactobacillus, Prevotella, Proteobacteria and Veilonella. Conditions of the PCR were an initial incubation of 95 °C for 5 min, followed by 45 cycles at 95 °C for 1 min; 52 °C for 45 s and 72 °C for 1 min; with a final extension at 72 °C for 10 min. The amplified products were later analyzed by gel electrophoresis on 2% agarose .The amplification of 13 representative bacteria genus was performed using primers previously described . The genHospital Regional de Cajamarca, Peru. The samples were obtained in the Specialized Oncological Preventive Service of the Hospital Regional de Cajamarca, as part of the cervical cancer screening program. The collection of the samples was done with a prior informed consent, which allowed the use of this samples for further studies.This study was approved by the Research Ethics Board of the Demographic characteristics and gyneco-obstetric records of the two groups were compared. HPV-positive patients were aged 35.06 ± 8.86 and HPV-negative were aged 35.72 ± 8.72. The marital status of the patient was also assessed, most of the patients were cohabiting (40 and 46% respectively) in both groups. Also, gynecological and obstetric background information was obtained, as shown in Table Eubacterium (68 vs 32%), Actinobacteria (46 vs 33%), Fusobacterium (11 vs 6%) and Bacteroides (20 vs 13%).Different bacteria genus were compared between the two groups as shown in Table Actinobacteria (50 vs 33.33%), Bifidobacterium (50 vs 20.83%) and Enterococcus (50 vs 29.17%), respectively. Moreover, it can be observed that some bacteria were more frequently identified in high-risk genotypes compared to HPV-negative samples: Bacteroides (21 vs 13%), Actinobacteria (50 vs 33%), Fusobacterium (67.1 vs 32%).The variations in microbiota between high-risk (including possibly oncogenic) and low-risk was also evaluated as reported in Table HPV prevalence is a function of both the incidence and the overt persistence of infection –3. ThreeIt has been proposed that women with a certain composition of cervicovaginal microbiota may be more susceptible to acquire HPV infection, lack clearance of the infection or show a more rapid progression to cervical neoplasia . Most stEubacterium, Actinobacteria, Fusobacterium and Bacteroides were more likely to be identified in HPV-positive patients. The main difference was found in the genus Eubacterium, being identified in 68% HPV-positive samples and in 32% HPV-negative samples. Similarly, high-risk genotypes showed a higher percentage of Bacteroides, Actinobacteria and Fusobacterium compared to HPV-negative samples. When comparing high-risk and low-risk genotypes, Actinobacteria, Bifidobacterium and Enterococcus, where more likely to be identified in women infected with high risk genotypes. It has been established that these bacteria are usually found in the gut microbiota, however, they are normal transient colonizers of the vaginal microbiota [We found important differences in the frequency of bacteria identified between HPV-positive and HPV-negative Peruvian women, as well as in high-risk and low-risk HPV genotypes. In the present study some important anaerobic bacteria such as, crobiota .Gardnerella vaginalis and Lactobacillus gasseri were detected more frequently in women with HPV infection. Similar to our study, Chao et al. [Bacteroides plebeius, Acinetobacter lwoffii, and Prevotella buccae were detected significantly more frequently in HPV-positive women.A variety of studies have found an association between cervico-vaginal microbiota and HPV infection, however most of the results reported in literature differ from ours. Gao et al.  performeo et al. , reporteEubacterium eligens was associated with HPV clearance. Di Paola et al. [Lactobacillus depletion and bacterial vaginosis, was a risk factor for HPV persistence. Particularly, the identification of Atopobium spp. and G. vaginalis could be useful as microbial markers of HPV persistence. In the current study we could not determine whether the presence of the bacteria identified were associated with persistence or clearance of the infection, however, we our findings could lead to future studies to determine the role of these bacteria.Also, several studies have examined the dynamics in HPV infection and the role of the cervical microbiota. In contrast with our study, Arokiyaraj et al.  reporteda et al.  found thThe composition of the cervico-vaginal microbiota is dynamic and responds to different endogenous and exogenous factors , such asThe human microbiota plays a pivotal role in the development of mucosal immune system and metabolism, and also participates in the wellbeing of the human host , 24. TheIn conclusion, we found significant differences in the frequency of identification of different bacteria genus among HPV-positive and HPV-negative Peruvian women, also between high-risk and low-risk genotypes. A better characterization of the microbiota composition and its impact on HPV infection is still required. The identification of different bacterial species found in the cervico-vaginal microbiota may act as biomarkers for the identification of susceptible women for persistent infections and progression to neoplasia. Also, the microbiota could have a future role as a therapeutic target for the treatment of this disease.One of the main limitations is that we did not have information regarding antibiotic or probiotic before sampling. Moreover, we did not have the histological evaluation of neoplasia or pre-cancerous lesions in the women studied. Also, because of the nature of our study, we were unable to determine whether the changes in cervicovaginal are induced by HPV infection or the other way around. Moreover, it would be crucial to examine whether the cervical microbiota may have an impact on HPV infection dynamics, such as acquisition, regression, clearance or progression of the infection to neoplasia. Finally, we did not measure the relative abundance of each bacterium, only the frequency of identification.


Rapid, high-volume screening programs are needed as part of cervical cancer prevention in China.In a 5-day screening project in Inner Mongolia, 3345 women volunteered following a community awareness campaign, and self-swabbed to permit rapid HPV testing. Two AmpFire™ HPV detection systems (Atila Biosystems) were sufficient to provide pooled 15-HPV type data within an hour. HPV+ patients had same-day digital colposcopy (DC) performed by 1 of 6 physicians, using the EVA™ system (MobileODT). Digital images were obtained and, after biopsy of suspected lesions for later confirmatory diagnosis, women were treated immediately based on colposcopic impression. Suspected low- grade lesions were offered treatment with thermal ablation (Wisap), and suspected high-grade lesions were treated with LLETZ.Of 3345 women screened, 624 (18.7%) were HPV+. Of these, 88.5% HPV+ women underwent same-day colposcopy and 78 were treated. Later consensus histology results obtained on 197 women indicated 20 CIN2+, of whom 15 were detected and treated/referred at screening .Global control of cervical cancer will require both vaccination and screening of a huge number of women. This study illustrates a cervical screening strategy that can be used to screen-and-treat large numbers of women. HPV self-sampling facilitates high-volume screening. Specimens can be tested rapidly, promoting minimal loss-to-follow-up. Specifically, the AmpFire™ system used in this study is highly portable, simple, rapid (92 specimens per 65 min per unit), and economical. Visual triage can be performed on HPV+ women with a portable digital colposcope that provides magnification, lighting, and a recorded image. Diagnosis and appropriate treatment remain the most subjective elements. The digital image is under study for deep-learning based automated evaluation that could assist the management decision, either by itself or combined with HPV typing. Infection with human papillomavirus (HPV) is a leading cause of cancer among women worldwide with approximately 500,000 new cervical cancer cases and 250,000 deaths each year . CervicaCervical cancer screening strategies have evolved from cytology-based to HPV-based . FollowiWithout compromising yield of disease, self-sampling can increase participation and reach of cervical cancer screening programs. Self-sampling is comparable to clinician-obtained sampling for HPV-based screening, is well accepted in many populations, and has been incorporated into screening programs to improve coverage , 5.Currently, there are no national cervical cancer screening programs in China and only 10–30% of Chinese women report having ever had cervical cancer screening . AlthougThe Inner Mongolia Minority Autonomous Region (Inner Mongolia), is a vast territory that stretches in a great crescent for 1500 miles across northern China. Inner Mongolia was part of the ancient Silk Road region and is bordered to the north by Mongolia and Russia. Inner Mongolia is a geographically diverse and relatively underdeveloped province, with a population of over 24 million in the 2010 census. Forty-nine ethnic groups live in Inner Mongolia though the majority are Mongolian or Han. Additional ethnicities include Manchu, Hui, Daur, Ewenki, Oroqen, and Korean. More than 58% of the population lives in rural areas.Due to the level of development, complex geography, and dispersed population across the rural parts of the majority of Inner Mongolia, the conventional, multi-step screening process for cervical cancer is not feasible. The traditional process of screening with cytology, colposcopy, biopsy and subsequent treatment of women diagnosed with cervical precancer is too resource and expertise-intensive for low-income, vast regions such as Inner Mongolia. Accordingly, techniques such as visualization with acetic acid (VIA) and HPV testing have been studied as alternative methods.The prevalence of HPV varies amongst different ethnic and geographic regions , 12. In This study demonstrates the feasibility of a rapid, high-volume screening approach that combines self-sampled HPV testing and digital colposcopy triage to reach unscreened populations, as exemplified by Inner Mongolia, China.This is a cross-sectional analysis using data from women attending a same-day, high-volume screening demonstration in Inner Mongolia, China. Women were contacted and, following an awareness campaign, 3345 agreed to participate and provided consent Fig. . ParticiDuring a 6-day period in May 2019 that consisted of 4 clinic days and 2 travel days, 3345 Chinese women aged 30 to 65 were screened in three different medical clinics in the Inner Mongolia Minority Autonomous Region. IRB approval was obtained from United Family Hospitals Investigational Review Board, Beijing, China. Local health officials notified and registered potential participants in the preceding weeks. The vast majority of women had never been screened for cervical cancer. After obtaining informed consent, participants received a brief explanation of HPV and cervical cancer via a pre-recorded video, as well as instructions on how to obtain a self-sampled vaginal specimen.We screened 460–990 women per day Fig. . The firSelf-collected vaginal specimens were collected using dry brushes and tested for 15 high-risk HPV  on site using two AmpFire™  machines running concurrently. While waiting for test results, women participated in a joint breast cancer screening effort. All women positive for high-risk HPV (hrHPV+) results were contacted via text message and returned for colposcopic examination the same day or the following day.Digital Colposcopy (DC) was performed with the EVA system 90 s after the application of acetic acid. All DC was performed by one of six physicians and the EVA system was used to obtain 1–3 DC images per patient. Thin acetowhite lesions were considered suspicious for CIN1. Thick acetowhite lesions, rapidly appearing lesions, lesions with course mosaicism or punctuation, and lesions with sharp borders were considered suspicious for CIN2 + .[27] The limitations of visual assessment for grading are acknowledged and discussed below.If DC was positive for cervical abnormalities (suspected CIN1+), patients underwent cervical biopsy of the exocervix using a SoftBiopsy™  brush for subsequent confirmatory diagnosis, but were treated presumptively with thermocoagulation or loop electrosurgical excision procedure (LLETZ) the same day. The SoftBiopsy™  brush obtains tissue from the entire exocervix to be processed in a single slide. Compared with multiple biopsies from biopsy forceps, it is faster to obtain tissue from multiple areas of the exocervix and it causes less trauma and bleeding, making it easier to perform thermocoagulation (if needed) immediately after biopsy.Thermocoagulation was performed when DC findings were suspicious for CIN1 lesions using one of two different thermocoagulation systems: C3 thermo-coagulator  or TC thermocoagulator™ . A LLETZ was performed when findings were highly suspicious for CIN2+ or when thermal ablation was not technically possible.If no acetowhite changes were seen and the entire transformation zone was visible, the patients were informed of the findings and not immediately treated. Repeat HPV testing in 1 year was advised; although the availability of such follow-up was recognized not to be assured, immediate treatment in the absence of a visible lesion was judged not to be warranted. If the transformation zone was not fully visible on DC or if the lesion extended in the endocervical canal, endocervical curettage (ECC) was performed.Overall, at colposcopy, clinician colposcopic impression led to 216 women biopsied and, of those, 84 immediately treated. Immediate treatment included thermal ablation, LLETZ, and none . All biopsy specimens were subsequently processed, and read twice, by two pathologists . The original histopathologic diagnoses included CIN1, CIN1–2, CIN2, CIN2–3, CIN3, and inadequate. For this research analysis, precancer case status among the HPV-positive women was defined as follows: CIN3 by either pathologist or CIN2 by both pathologists. The remaining adequate samples were considered <CIN2.The few women with unanticipated high-grade lesions (CIN2+) were later contacted with their histology results and were counseled to follow up at the regional hospital for appropriate management. As mentioned, while women were waiting for the results of their HPV tests, they were screened for breast cancer with a portable ultrasound unit (results reported separately). Throughout the screening and treatment process, for educational purposes, local doctors and nurses were taught colposcopy techniques by the visiting physicians and received training to evaluate DC images.Using self-sampled HPV testing, 3345 women were screened. Most women screened were ethnically Han (64.4%) and Meng 20.1%)  were invited for colposcopy and 552 attended the medical examination. Colposcopic impression was inadequate for 20.8%. Of the rest, 78.5% impressions were normal and not requiring biopsy.The characteristics of the 197 HPV-positive women that underwent colposcopy and had a biopsy are listed in Table From the 78 women who received treatment, 71 (89.7%) were treated with thermal ablation and 7 (9.0%) with LLETZ.This rapid, high-volume program yielded 0.60% of precancer (20 out of 3345) in the screening population. From the 20 cases identified by histology, 10 were infected with HPV16, 1 with HPV18, and 8 with other types (HPV status was unknown for 1 case)  .As the success of cervical cancer screening programs has dramatically decreased the rates of cervical cancer in developed nations, the global burden of this disease falls mainly in areas of limited resources. It is estimated that the number of 35–64-year-old women in rural China exceeds 150 million. As the vast majority of these women have never been screened, strategies must be developed that allow for the screening and treating of large numbers of women. We demonstrate a screen-and-treat model that is low-cost, rapid, and capable of being implemented on a large scale, as evidenced by the ability to screen 3345 women in less than one week. The authors have previously described a similar strategy for cervical cancer screening in the Yunnan Province .The first component of this model consists of self-obtained HPV specimens for rapid testing. It is practical for women in China to obtain self-collected HPV specimens, which then undergo rapid testing. Self-swabbing for HPV appears to be acceptable to Chinese women, regardless of their ethnicity.Rapid testing is a critical aspect of this model as it allows for same-day treatment. The highly portable isothermal PCR based HPV AmpFire™ testing system takes 1 h to run 94 specimens and provides partial genotyping (another test kit is available for complete typing). It is minimally labor intensive and does not require a high degree of technical expertise to run. The self-swab specimens can be stored dry (without collection media) and the reagents can be stored at room temperature for several weeks. Furthermore, the price per specimen is approximately $US 7 and the same system can be used to test for other sexually transmitted infections.The second component of this proposed model is to perform DC on all HPV+ women with a highly portable DC system. Our program included 6 physicians, a level of expert involvement that is often not available. Given the huge number of women in China who have never been screened as compared to the number of physicians available, DC is a modality that could be performed (if authorized) by mid-level providers such as nurses or midwives. Digital images obtained have excellent resolution and areas of question can be magnified for better interpretation. In addition, captured images can be used for continued education of mid-level providers, and the images can be sent electronically, through a secure, cloud-based portal, to an expert colposcopist for consultation of difficult cases.Our study supports assistive technology to aid clinical decision making based on human visual impression. Even when performed by experienced specialists, colposcopic impression shows subjectivity and limited reliability or inter-observer agreement . In addiOngoing efforts using Automated Evaluation (AVE) of the cervix show promising results using archived images from a consortium of sites , 20. AVEThe third component of this screen-and-treat model is to use the clinical impression obtained from the DC images, perhaps assisted by AVE, to determine immediate and appropriate treatment. Thermocoagulation is inexpensive, highly portable, and a highly effective treatment modality for cervical intraepithelial lesions caused by HPV.Cervical cancer screening programs could be combined with same-day vaccination efforts for HPV and other medical interventions. For example, we combined cervical and breast screening as part of the same program.The screening model proven to be feasible by this effort is promising, but might not be widely applicable at the present time. The health camp approach Fig.  is, by i


Persistent infection with certain types of human papillomaviruses (HPVs), termed high risk, presents a public health burden due to their association with multiple human cancers, including cervical cancer and an increasing number of head and neck cancers. Despite the development of prophylactic vaccines, the incidence of HPV-associated cancers remains high. In addition, no vaccine has yet been licensed for therapeutic use against pre-existing HPV infections and HPV-associated diseases. Although persistent HPV infection is the major risk factor for cancer development, additional genetic and epigenetic alterations are required for progression to the malignant phenotype. Unlike genetic mutations, the reversibility of epigenetic modifications makes epigenetic regulators ideal therapeutic targets for cancer therapy. This review article will highlight the recent advances in the understanding of epigenetic modifications associated with HPV infections, with a particular focus on the role of these epigenetic changes during different stages of the HPV life cycle that are closely associated with activation of DNA damage response pathways. Human papillomaviruses (HPVs) are small, non-enveloped, double-stranded DNA viruses that exhibit a strict tropism for cutaneous and mucosal  epithelium . More thIn infected cells, the HPV genome exists as an extrachromosomal element (episome) of approximately 8 kilobase pairs that encodes for six to eight open reading frames (ORFs) A. Due toFollowing initial infection of exposed basal cells, the early promoter becomes active, resulting in expression of the E1 viral helicase, which along with E2, facilitates establishment replication, whereby viral episomes are quickly amplified to 50–100 copies per cell ,15. In tEpigenetics is defined as a post-translational modification process that affects gene expression but does not alter the underlying DNA sequence. In the virion and infected cells, HPV genomes are organized in the form of nucleosomes packaged into chromatin ,30. HPV The viral promoters are bound by nucleosomes in an ordered arrangement and are subjected to chromatin remodeling at different stages of the viral life cycle ,35. DNasHistone acetylation is a dynamic and reversible process that modulates gene expression by altering the spatial density of chromatin and is regulated by histone-modifying enzymes, including histone acetyltransferases (HATs) and histone deacetylases (HDACs) . The actSeveral HPVs express an E8^E2C protein, which functions as a transcriptional repressor from both promoter-proximal and distal E2-binding sites (E2BS) in the LCR and also negatively affects viral replication ,56,57,584CGGT-3′) (DNA methylation is a post-replicative DNA modification that involves methylation of the 5’-position of cytosine residues located in CpG dinucleotides and is mediated by DNA methyltransferases (DNMTs) . HPV DNACGGT-3′) A 70]. H. H4CGGT-HPV infection can also induce modifications of host cellular chromatin, including DNA and histone methylation, resulting in aberrant expression of cellular genes. In high-risk HPV-associated lesions, epigenetic silencing of tumor suppressor genes such as p53 by DNA methylation in CpG island regions of gene promoters is often observed . DisruptThe histone methylation status of cellular chromatin can also be modified by HPV proteins, leading to aberrant cellular and viral gene expression. Studies by Smith et al. demonstrated that E2 recruits the H3K4 demethylase SMCX (JARID1C/KDM5C) to the HPV18 URR to transcriptionally repress early promoter activity, resulting in decreased E6/E7 expression ,62. In aIn addition, human foreskin keratinocytes (HFKs) expressing HPV16 E7 exhibit increased levels of activating marks such as H3K9 acetylation and H3K4 methylation, with both histone marks requiring E7’s Rb- and HDAC-binding domains . Using CStudies over the past decade have established a critical role for the DNA damage response (DDR) in productive replication of high-risk HPV types  15,87].,87.15,87Following induction of cellular DSBs, ATM is recruited to the break site and activated by the MRN complex (MRE11/Rad50/Nbs1) as well as acetylation by TIP60 . The MRNDuring the HPV life cycle, DDR activation is accompanied by significant alterations of viral chromatin that allow access of DNA repair proteins required for viral replication  109]. γ. γ109]. Although TIP60 acetyltransferase has been implicated in negatively regulating HPV gene expression ,62, TIP6The Structural Maintenance of Chromosomes family protein SMC1 is phosphorylated by ATM in response to DNA damage and plays a central role in recruitment of HR repair factors to DSBs ,125. SMCRecent studies have shown that the HPV life cycle is also epigenetically regulated by SETD2 methyltransferase . SETD2 iEven though high-risk HPVs utilize DDR pathways to facilitate viral replication, genomic instability is a hallmark of HPV-associated cancers, indicating that disruption of DNA damage repair mechanisms contribute to the initiation and progression of cancer . HPV-indRecent studies in HPV pathogenesis have revealed critical roles for epigenetic modifications in HPV infection and cervical carcinogenesis. Epigenetic modifications of HPV DNA are important for establishment replication, genome maintenance, and productive replication during the viral life cycle. It is clear that chromatin remodeling surrounding areas containing damaged DNA is required to allow access to DNA repair proteins that are necessary for HPV replication. Future investigation may focus on studying the specific histone marks that correlate with distinct DNA damage response pathways. As the incidence of HPV-associated oropharyngeal carcinoma continues to rise, expanding our knowledge of epigenetic drivers that regulate HPV pathogenesis in the oropharyngeal epithelium is an important area of future research. Even though epigenetic modifications in HPV-associated oropharyngeal cancers, including DNA methylation and histone modifications, have been reported , the rol


During 2013 and 2016 the region of Skåne, Sweden started to analyse human papillomavirus (HPV) and cytology in postmenopausal women 60–65 years of age. Our aim was to evaluate high-risk (HR) HPV mRNA testing for the triage of HPV DNA-positive postmenopausal women with normal cytology.A total of 271 women, 60–65 years of age, underwent liquid-based cytology (LBC) and HPV testing by using the HR-HPV DNA MGP-PCR-Luminex assay. HR-HPV DNA-positive women with normal cytology underwent complimentary HPV mRNA testing . Over a period of 49 months (SD 11.0) the women received regular follow-ups at intervals of 12–18 months. Women with abnormal cytology and/or a positive HR-HPV DNA and/or mRNA result at two subsequent visits were scheduled for colposcopy and clinical examination.p = 0.002).Over the surveillance period, 3.6% (10/271) of the HR-HPV DNA-positive women developed histologically confirmed high-grade squamous intraepithelial lesions (HSILs) or worse. The cumulative incidence rates (CIR) were 29.7% (CI 24.8–30.1) for HSIL or worse among HPV mRNA-positive women at enrolment (39.5% 107/271) and 0% among HPV mRNA-negative women , , in contrast to women with a negative HR-HPV mRNA outcome. The HR-HPV mRNA APTIMA assay detecting 14 HR-HPV types may be a useful triage method among HPV DNA-positive postmenopausal women with normal cytology. International consensus on when to stop cervical cancer screening among postmenopausal women does not exist . In SwedIn a recent audit in the region of Skåne (southern Sweden) it was observed that 24% (31/177) of squamous cervical cancer (SCC) or cervical adenocarcinoma cases between 2016 and 2017 were over 65 years of age .Most of the affected older women have symptoms at the time of diagnosis due to an advanced cancer stage, and the mortality is as high as 70% . This daAmong postmenopausal women it has been shown that combined screening of high-risk (HR) human papillomavirus (HPV) DNA testing and cytology offers a higher sensitivity than screening with cytology alone , 4.Therefore, between 2013 and 2016 the organised cervical cancer screening programme in the Skåne region performed a double test, consisting of both HR-HPV testing and cytology in postmenopausal women aged 60–65 years. It was considered as a last control before leaving the screening program. The double test consisted of a liquid-based cytology (LBC) specimen which was co-tested for HR-HPV DNA. In HR-HPV DNA-positive women with normal cytology the LBC specimen was further investigated for the presence of HR-HPV mRNA. Among such women with normal cytology we recently reported, in a prospective, one year follow-up study, that the specificity of the HPV mRNA-based APTIMA assay (60.2%) was superior to that of the Luminex HPV DNA assay (42.3%) regarding the detection of cervical pre-cancer lesions, while the sensitivities were similar [However, the aim of the present study was to prospectively evaluate if the presence of HR-HPV mRNA at enrolment could predict the future development of cervical abnormalities among HPV- DNA-positive, postmenopausal women with normal cytology over a four-year follow-up period. Another aim was to evaluate to what extent HR-HPV mRNA testing can be used as a triage method in postmenopausal women.n = 5925) were tested for the presence of HR-HPV.Between 2013 and 2016, women 60–65 years of age, with normal cytology, and living in the southern region of Sweden (Skåne)  individuals with a mean age of 61.9 years (SD +/− 1.7).Criteria indicating exclusion from further follow-up were history of cervical neoplasia and/or treatment of cervical disease such as the loop electrical excision procedure (LEEP), hysterectomy or trachelectomy and ongoing oncological treatment at the time the double test was performed. A total of 271 HR-HPV DNA-positive women with normal cervical cytology were eligible for inclusion  that was analysed for HR-HPV DNA by using the MGP-PCR-Luminex assay , 7. In wAt all further follow-up controls which were performed at intervals of 12 to 18 months, the same selection criteria were applied to determine which women were in need of a further clinical investigation. During our surveillance period at least three consecutive follow-ups could be documented.Women presenting with normal cytology and negative HR-HPV DNA results left the routine screening service.LBC results were defined as normal, atypical squamous cells of undetermined significance (ASCUS), atypical glandular cells (AGCs), low grade squamous intraepithelial lesions (LSILs), and high-grade intraepithelial lesions (HSILs) according to the Bethesda classification . HistopaIn women with HSIL lesions on LBC and corresponding colposcopic findings, an LEEP was performed for therapeutic management. Also, patients with cytological ASCUS or LSIL but a colposcopic picture suggestive of an underlying pre-cancerous lesion were scheduled for a LEEP procedure.In the case of an inaccessible transformation zone located within the cervical channel, a cervical biopsy or conisation specimen was obtained for diagnostic reasons.The MGP-PCR Luminex HPV DNA assay detects several HPV types simultaneously , 7.x g for five minutes and then liquid was removed so that 500 uL remained. From each sample, DNA was purified by total NA-kit (200 uL input and 100 uL output) using Magna Pure LC (Roche) and then HPV DNA was amplified by PCR with modified GP5+/6+ (MGP) primers [From each LBC vial (Thinprep) 2 ml was centrifuged at 3500  primers .After amplification, the Luminex-based HPV genotyping allows the identification of the following HR-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and the probable high-risk type 68 (A and B) as well as the possibly high-risk types 26, 53, 66, 67, 69, 73 and 82 as described by IARC classification from year 2012 . In the The HPV E6/E7 mRNA (APTIMA) assay detects qualitatively E6/E7 mRNA from 14 HR-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.From each LBC vial (Thinprep) 1 ml was automatically transferred to 2.9 ml APTIMA transfer solution  by a Tomcat instrument . Thereafter an aliquot of 400 ul was further processed for the HPV E6/E7 mRNA (APTIMA) assay by the Panther system We calculated the proportion of HPV E6/E7 mRNA positivity for each of these 14 HR-HPV types as well as for HPV67 , as determined by the MGP-PCR Luminex HPV DNA assay.The presence of the same HR-HPV genotype at inclusion and at follow-up was defined as a persistent infection. At follow-up, for women with benign cytology who tested negative for HR-HPV DNA but were positive for low-risk (LR) HPV DNA according to the Luminex assay, the HR-HPV status was considered as negative or as cleared infection.The endpoint was the development of histologically confirmed HSIL or worse over a follow-up period of four years.Statistical comparisons were based on two-sided chi-square tests. All comparisons were two-sided, and a 5% level of significance was applied. The absolute risk among among HPV mRNA positive women for the development of cervical abnormalities was presented with the corresponding 95% confidence intervals (CI).Cumulative incident rates (CIR) during the follow- up period were calculated according to Kaplan-Meier survival analysis and presented as percentages with the corresponding 95% CI.The statistical analyses were performed using SPSS version 19.0 or higher  and Omnistat .Over a total surveillance period of 49 months (SD 11.0), the detection rate of histologically confirmed HSIL or worse was 3.6% (10/271).Low grade lesions were detected in 13.3% (36/271) of the women. In 33 cases the diagnosis of ASCUS or LSIL lesions was based on cytological results and in the remaining three cases it was histologically confirmed.p = 0.002)   in the subgroup of HR-HPV mRNA-positive individuals and 0% in those women with a negative HR-HPV mRNA result (02) Fig. . The corThe absolute risk for the development of HSIL or worse was 9.3%  among HR-HPV mRNA positive women and 0%  among those individual with a negative HR-HPV mRNA result at baseline. The corresponding figures for ASCUS or worse were 31.8%  and 7.31%  respectively.At baseline, the overall HPV mRNA positivity rate was 39.5% (107/271) and HR-HPV mRNA was present in all cases of histologically confirmed HSIL and in 67% (24/36) of the women diagnosed with ASCUS or LSIL.p = 0.68). The CIRs for ASCUS or worse were 64.9% (95% CI 47.05–72.9) for women infected with HR-HPV DNA types 16/18 and 51.1%  in the other subgroup (Table Among women testing positive for HR- HPV DNA types 16/18 the CIR for HSIL or worse was 14.6% (95% CI 13.3–22.7) and among those with other HR-HPV DNA types the corresponding figure was 18.5%  of the HR-HPV DNA-positive women had cleared their infection, whereas 59% (159/271) were still persistently infected with the same HR-HPV DNA type(s). Over the entire follow-up period, persistent HR-HPV type(s) were observed among 16.9% (46/271) of the women whereas clearance of the HR-HPV infection was found in 73.8% (200/271) of the cases. Twenty-five women were lost during follow-up after two years   had no longer detectable HPV DNA, whereas the corresponding clearance rate among the HPV mRNA-positive women was 7%   who remained HR-HPV mRNA-negative but HPV DNA-positive after the first control, were found to have negative co-testing results. The corresponding figure among the HR-HPV mRNA-positive women was 32%  or histological (n = 3) results.In our cohort of HR-HPV DNA-positive postmenopausal women with normal cytology we were able to detect 10 cases of histologically confirmed HSIL lesions. Another 36 women were diagnosed with ASCUS or LSILS lesions based on either cytological (Women who tested positive for HR-HPV mRNA at enrolment were found to have a significantly increased risk for HSIL or worse (CIR 29.7%) and also for ASCUS or worse (CIR 59.9%) over a total surveillance period of 49 months.In contrast, none of the HPV DNA-positive women with a negative HR-HPV mRNA result at baseline developed HSIL or worse (CIR 0%) during follow-up, although a risk regarding the development of low grade lesions such as ASCUS or LSIL was still evident (CIR 26.1%). Concerning the persistency of HR-HPV types we observed that after four years of follow-up, 16.9% of the women were still diagnosed with a persistent HR-HPV infection while 73.8% had cleared their infection spontaneously.It is already shown in the literature that HR-HPV testing is a safe screening option in postmenopausal women  as it inAlso according to the current Swedish screening guidelines, women 30 years or older are screened with HR-HPV testing until the age of 64 while cytology is the screening method of choice in women 23 to 29 years old. A double test including cytology and HR-HPV testing is only indicated in women at 41 years of age. Only women with at least one negative HR-HPV DNA test at the age of 64 are allowed to exit the screening programme.Even though most of the HR-HPV assays approved for primary cancer screening are based on HR-HPV DNA detection, like the Hybrid Capture 2 (HC2) method or the GP5+/6 + EIA assay or the COBAS® there is now growing evidence that HR-HPV detection methods targeting the mRNA of the oncoproteins E6/E7 are also effective and reliable alternative screening tools –18.A further indicator underlining the limited sensitivity of cytology in the postmenopausal age group is the observation that we were able to detect a discrepancy of about 20% between LBC results and the corresponding histological findings in our series. In three women with ASCUS or LSIL, histopathological analysis of the matching LEEP specimen revealed the presence of underlying HSIL lesions, while in three other women with high-grade cytology no pre-cancerous findings could be confirmed on conisation material. According to the literature, the discrepancy level between cytology findings and the corresponding histological outcomes varies between 5 to 55%. Factors that may cause those elevated rates of false negative results are the subjective interpretation of the specimen and/or the absence of diagnostic cells , 20.A clinical circumstance that may further contribute to the limited sensitivity of cervical cytology is the higher probability of sampling errors as the transformation zone tends to be located higher up in the cervical channel. Furthermore, ageing effects such as a decline in oestrogen can lead to the presence of atrophic cells which in their turn can be mistakenly interpreted as ASCUS or LSIL lesions , 21. TheOur data show that HR-HPV mRNA-positive women aged 60 years or older are at risk of developing cervical abnormalities and are therefore in need of regular follow-up controls including HPV mRNA analyses. Additional gynaecological examination is indicated if a persistent HR-HPV mRNA infection is found at two subsequent annual controls and/or cytology shows abnormal results. Also, Johannson et al. demonstrated that among HPV DNA-positive women aged 35 years or older with either cytological ASCUS or LSIL at baseline, a positive HR-HPV mRNA result could predict the development of CIN 3 or worse with a sensitivity of 100% within the following four years . As in oPostmenopausal women represent a special patient group, as they tend to have a higher risk for persistent HR-HPV infections than younger individuals, who have a higher acquisition frequency but also a faster clearance rate , 24. FurIt is of clinical importance to distinguish between those individuals with active viral replication who are at risk of developing cervical pre-cancer lesions and those with latent HR-HPV DNA infections lacking any clinical significance.There is evidence in the literature that the level of the HPV mRNA copies increases proportionally to the severity of the cervical lesion . On the Those data are comparable to the results of other studies reporting clearance rates of about 40% in older women within an average time span of four months , 26. TheA cohort study with long-term follow-up was able to demonstrate that the five-year cumulative risk of developing CIN 3 or worse was comparable between the cohort of HPV DNA-negative and HPV mRNA-negative women . Those cRegarding the risk stratification of cytological ASCUS and LSIL lesions, it is stated in the literature that the APTIMA assay offers a higher specificity when compared to other DNA based HR-HPV detection methods , 16, 31.Furthermore, the results of a long-term follow-up study demonstrated that a negative APTIMA co-testing result among HPV DNA-positive women with minor cytological abnormalities at baseline was associated with a high negative predictive value of 100% for CIN 3 or worse within the following four years . Even thRegarding the distribution of the HR-HPV genotypes, we observed that HR-HPV types 16, 68A and 31 were the most frequent ones in our cohort. We observed a relatively high frequency of HPV68A infections (9.5%), which is rather unusual in younger women and not covered by the current vaccination programmes. Those data are in line with other studies showing that the HR-HPV profile in postmenopausal women to some extent differs from those encountered in younger individuals , 33. FurOur data favour the use of HPV mRNA detection as triage method in HPV DNA-positive postmenopausal women with normal cytology Fig. .Since postmenopausal women testing positive for HR-HPV mRNA and normal cytology have a substantial risk (9.3%) of developing severe cervical abnormalities we suggest that these women should be scheduled for regular, annual follow-up examinations. In the case of a persisting HR-HPV mRNA infection at two subsequent controls and/or the presence of cytological abnormalities, a cervical tissue biopsy/curettage or diagnostic LEEP specimen should be obtained, especially if the transformation zone is inaccessible for colposcopic evaluation.On the other hand our data indicate that postmenopausal women with a negative outcome for HR-HPV mRNA are no longer in need of further follow-up for at least four years.


Cervical cancer is the second-most common gynecological cancer, early screening plays a key role in the diagnosis and treatment of cervical intraepithelial neoplasia (CIN). Sustained E7 protein expression is the pathological basis for CIN and cervical cancer.We collected the cervical cell samples of women who visited the gynecological clinic of Peking Union Medical College Hospital between September 2018 and September 2019 and submitted them to the high-risk human papillomavirus (Hr-HPV) test. We performed a magnetic particle–based chemiluminescence enzyme immunoassay to analyze the HPV16/18 E7 protein level in CIN of different severities and compared the results with those of cervical pathology (gold standard) and the HPV test.The positive rate of HPV16/18 E7 protein increased with the severity of CIN: 26.6% in normal tissue, 58.3% in CIN1, and 70.6% in CIN2 or higher (CIN2+). For CIN2+, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the E7 protein were 70.6, 67.9, 52.2, and 82.3%, respectively. These values of the HPV test were 86.8, 44.5, 43.7, and 87.1%, respectively. With the combination of the E7 protein assay and HPV test, the specificity for diagnosing CIN2+ was 78.1%, which was significantly higher than that of the HPV test alone.HPV16/18 E7 protein level is correlated with the severity of CIN and has a high concordance rate with the pathological result. For cervical cancer screening, the combination of HPV16/18 E7 protein assay and HPV test improves the CIN diagnostic specificity, detection rate, and detection accuracy. Cervical cancer is the second-most common gynecological cancer . Early sThe study was approved by the Ethics Committee of Peking Union Medical College Hospital  and was conducted after obtaining the patient’s informed consent. Cervical cell samples of 23,772 women aged 18 or above who visited the gynecological clinic of Peking Union Medical College Hospital between September 2018 and September 2019 were collected and submitted to the HPV test. A total of 963 samples with HPV16/18 positive and 325 HPV negative patients with remarkable cytological abnormalities  were included in the study. Moreover, a total of 159 patients underwent colposcopy and biopsy for pathological examination, among which, 135 patients were positive for HPV16/18 and 24 samples were ASC-US+ with HPV negative. Besides, 46 randomly selected samples who did not undergo colposcopy or biopsy due to negative HPV test and cytologic test were included in the negative control group for statistical analysis  and the HPV test.Cytologic specimens were processed with the ThinPrep liquid-based cytology systems . Cytology results were classified with the Bethesda System for Reporting Cervical Cytology as revised in 2014 .The Cobas 4800 HPV test , which is a qualitative test for detection of HPV DNA, was used to analyze the samples. This test amplifies target DNA in cervical epithelial cells by PCR and nucleic acid hybridization  to detect 14 Hr-HPV types, of which HPV 16 and HPV 18 are of the greatest importance. This analysis also enables the detection of Hr-HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 at clinically significant levels of infectivity.The HPV16/18 E7 magnetic particle–based chemiluminescence enzyme immunoassay kit from AMID Biotech (Tianjin) Co., Ltd. was used to analyze the E7 protein level according to the instructions. The patients were instructed to refrain from intercourse and vaginal medication. The supernatant was collected for analysis within 2 h. The assay used the double-antibody sandwich method, where magnetic particles were used as the solid phase of the immune response, and highly specific antibodies were used to detect high-risk E7 protein in cervical cell samples.The pathological result was used as the gold standard. CIN was pathologically classified as mild (CIN1), moderate (CIN2), or severe (CIN3).P < 0.05 was considered statistically significant.SPSS v25.0 was used for statistical analysis. Count data are expressed as a percentage or n (%). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were evaluated with the receiver operating characteristic (ROC) curve and area under the curve (AUC). In this study, 46 patients were negative on both the cytologic test and HPV test. Pathologically, 67 patients were normal, 24 patients were CIN1, and 68 patients were CIN2/CIN3. E7 protein level was analyzed in 205 cell samples.When the ROC curve of E7 protein concentration was drawn, the AUC was 0.718 (95% CI 0.628–0.808) for the HPV16/18 E7 protein assay, and 8.270 was the optimal cut-off value .  in pathologically normal/inflammation cases, including 18 cases of positive HPV16/18 test results and 7 cases with both negative cytologic test and HPV test results. The positive rate was 58.3% (14/24) in CIN1 cases, 70.6% (48/68) in CIN2+ cases and 67.4% (31/46) in CIN3+ cases. The results indicated that the positive rate of E7 protein expression increased with the severity of CIN: it was 32.1% in normal and CIN1 patients (the negative control group) and 70.6% in CIN2+ patients  (13 in patP = 0.021) but was significantly more specific (P < 0.001). The combination of E7 protein assay and HPV test had a specificity and PPV of 78.1 and 58.3%, respectively, which were significantly higher than those of the HPV test alone  (Table P < 0.001).For CIN2+ detection, the E7 protein assay had a sensitivity, specificity, PPV, and NPV of 70.6, 67.9, 52.2, and 82.3%, respectively. For the HPV test, the values were 86.8, 44.5, 43.7, and 87.1%, respectively. The E7 protein assay was significantly less sensitive ; the PPV was 58.3%, which was also higher than that of the HPV test alone. Besides, similar with CIN2+ cases, when combined the E7 protein assay with the HPV test in CIN3+ cases detection, the specificity was 72.3%, which was significantly higher than that of the HPV test alone (40.9%) (P < 0.001). If the HPV test is used alone for CIN screening, many patients with transient HPV infection (rather than precancerous lesions) will undergo colposcopy and biopsy, which causes anxiety and panic and increases healthcare costs. The combination of E7 protein assay and HPV test improves the specificity of CIN detection and reduces the colposcopy referral rate and invasive examination rate.This study showed that E7 protein level was correlated with the severity of CIN. With the cut-off expression value of 8.27, the positive rate was 26.6% in pathologically normal/inflamed cases, 58.3% in CIN1 cases, and 70.6% in CIN2/CIN3 cases, indicating that the positive rate increased with the severity of CIN. In CIN2 and CIN3 cases, the sensitivity was 70.6%, which was lower than that of the HPV test (86.8%), but the specificity was 67.9%, which was significantly higher than that of the HPV test (44.5%). With the combination of the E7 protein assay and the HPV test, the specificity was 78.1%, which was significantly higher than that of the HPV test alone , including 12 cases with negative HPV tests. This may be due to technical limitations. Moreover, 18 pathologically negative cases were positive on the HPV16/18 test. E7 protein level may increase before positive pathological findings or may be more sensitive than a pathological examination. In a previous study, E7 protein expression was positive in 14.3% of pathologically normal cases . The 18 In this study, E7 protein expression was negative in 20 CIN2/CIN3 cases . The false-negative results may be related to sampling, laboratory procedures, or technical limitations. In our preliminary experiment, we analyzed the false-negative results of E7 protein in seven CIN2+ samples. The samples were tested using the BD Accuri C6 flow cytometer, and the results showed that each of the seven samples contained 16,000 to 760,000 cells. Moreover, streptavidin-peroxidase immunocytochemical staining detected E7 protein expression in two samples, however, the expression was detected in only a few cells. Specifically, one of the seven samples (CIN3) showed substantially intact cells, with low E7 protein expression in a few cells (brown particles in the nucleus and cytoplasm), as well as some cell fragmentation and protein release (Fig. E6 and E7 are the most important oncogenes of HPV . StudiesIn summary, this study shows that the combination of E7 protein assay and HPV test improves the diagnostic specificity and PPV for diagnosing CIN, which reduces the colposcopy referral rate and maybe one of promising strategies for minimizing regional variation in screening for CIN, and reduces healthcare costs. Large multicenter studies are needed to validate the results and continue the search for more effective screening tools for CIN.


TP53 was the most frequently mutated gene. The presence of mutation in NOTCH1 and PTEN, significantly decreased patient’s recurrence-free survival, but only NOTCH1 mutation remained significant after stepwise selection, with a risk of recurrence of 4.5 . These results show that Brazilian OpSCC patients exhibit a similar clinical and genetic profile in comparison to other populations. Molecular characterization is a promising tool for the definition of clinical subgroups, aiding in a more precise tailoring of treatment and prognostication.Tobacco- or human papillomavirus- driven oropharyngeal squamous cell carcinomas (OpSCC) represent distinct clinical, biological and epidemiological entities. The aim of this study was to identify genetic variants based on somatic alterations in OpSCC samples from an admixed population, and to test for association with clinical features. The entire coding region of 15 OpSCC driver genes was sequenced by next-generation sequencing in 51 OpSCC FFPE samples. Thirty-five percent of the patients (18/51) were HPV-positive and current or past tobacco consumption was reported in 86.3% (44/51). The mutation profile identified an average of 2.67 variants per sample. Sixty-three percent of patients  were mutated for at least one of the genes tested and  Excessive tobacco and alcohol consumption and high-risk human papillomavirus (HPV) infection (mainly HPV-16 and HPV-18) in the oropharynx are the main risk factors6. Besides etiology, HPV-positive and HPV-negative oropharyngeal squamous cell carcinoma (OpSCC) cases configure diseases with remarkably different clinical presentation, epidemiological and molecular profiles6. Regarding prognosis, HPV-positive OpSCC have a far more favorable outcome when compared with HPV-negative cases6. For this reason, the tumor-node-metastasis (TNM) staging for HNC was adapted in the eighth edition to include p16INK4A immunostaining as a surrogate for HPV status in OpSCC7.The most prevalent site is the oral cavity, followed by the oropharynx and larynx, and more than 95% are squamous cell carcinomas (SCC)8. This treatment frequently has long-term side effects, impacting patients’ quality of life9. Several treatment de-escalation trials for HPV-positive OpSCC have been initiated, which may lead to personalized treatment based on HPV status10.Treatment for OpSCC has evolved in the past decades and often includes approaches for organ preservation based on concomitant chemotherapy and radiotherapy followed by salvage surgery in non-responders13 and at least 50% of them will relapse locally, regionally or at distant sites, within the first two years after treatment15. While for HPV-positive OpSCC cases, 5-years overall survival rates (OS) are close to 80%, regardless of stage18, TNM stage has a significant impact in OS for HPV-negative OpSCC: 70%, 58%, 50%, and 30% for stage I, II, III, and IV18. Therefore, there is an increasing need for predictors of treatment response and disease progression/relapse.Around 2/3 of HNC patients are diagnosed with advanced disease in different parts of the world22. A high level of intertumoral heterogeneity has been observed, confirming the biological complexity of these tumors24. A common finding is that HPV-negative tumors have a higher mutation burden while HPV-positive tumors have far fewer genes mutated and variants per tumor, regardless of smoking status23. Results from The Cancer Genome Atlas (TCGA) consortium on the molecular profile of 279 HNSCC tumors confirmed the differences at the molecular level between HPV-positive and negative cases, with frequent activating helical domain mutations in PIK3CA in HPV-positive cases and TP53 mutations and CDKN2A loss-of-function mutations in HPV negative cases24. These findings suggest that the absence of the oncogenic effect from HPV oncoproteins in HPV-unrelated tumors requires the accumulation of multiple genetic aberrations to allow malignant transformation23.Studies characterizing the spectrum of genetic alterations in cancers have been enabling a better understanding of the molecular alterations playing a role in head and neck carcinogenesisDespite the different strategies for diagnosis and treatment of OpSCC patients, and the advent of HPV as an important prognostic marker, there is no molecular biomarker to guide selection among treatment and follow-up options that directly have an impact on patient survival. Therefore, the lack of risk categories based on clinical features and/or biomarkers that can be used for personalized treatment approaches for OpSCC has propelled research into the molecular landscape of these tumors. Moreover, there is an underrepresentation of genomic data on the mutation profile of HNC patients from admixed populations. Therefore, this study aims at identifying molecular alterations in a set of HNC patients from Brazil and to better characterize, together with clinical variables, OpSCC cases according to their outcome.25. Finally, after sequencing, only samples with acceptable scores of DNA sequencing quality were included (as described below).This study included pre-treatment formalin-fixed paraffin embedded (FFPE) tissue biopsies from 51 patients with primary oropharyngeal squamous cell carcinoma (OpSCC) diagnosed at the Department of Head and Neck Surgery of Barretos Cancer Hospital in Brazil between 2009 and 2017. These cases were selected based on very stringent criteria to ensure reliability of the clinical and molecular data as follows: pre-treatment FFPE tumor biopsies available from patients treated with curative intent by chemo-radiation protocols and with available follow-up data; samples with at least 60% of tumor cells at the histopathological examination and, with enough DNA for library preparation at an acceptable level of integrity as evaluated by multiplex PCRHematoxylin & eosin stained sections of paraffin blocks containing the tumor tissue from the patients included were reviewed by two expert pathologists (CSN and GCA) for diagnosis confirmation and characterization of cellular components. Scrapings from regions containing at least 60% of tumor cells were processed using the QIAamp DNA FFPE Tissue Kit . Isolated DNA samples were eluted in 35 µL of water and quantified in the Qubit fluorometer  prior to storage at −20 °C until use.28. Moreover, HPV-DNA detection of types HPV-16 and HPV-18 was performed in a subset of cases (n = 12) using droplet digital PCR as previously described and detailed in the Supplementary Methods29.Information regarding tobacco and alcohol consumption, clinical and pathological features were retrieved from patient’s charts. p16 immunohistochemistry was conducted as a surrogate marker for high-risk HPV . Samples with strong and diffuse nuclear and cytoplasmic staining in more than 75% of the tumor cells were considered positive for p16The present study was approved by the Barretos Cancer Hospital Institutional Review Board  and all methods were performed in accordance with the relevant guidelines and regulations.TP53, NOTCH1, CDKN2A, PTEN, PIK3CA, FBXW7, HRAS, TP63, CASP8, FAT1, KMT2D, RB1, IRF6, EZH2 and NSD1, based on a previous study30. Bearing in mind the consensus regarding DNA fragmentation when recovered from FFPE, primers were designed to amplify fragments with sizes ranging from 125–175 base pairs, to minimize the effect of DNA fragmentation in the efficiency of library preparation. Therefore, three pools of primers were used for the simultaneous amplification of 923 amplicons from the 15 genes.To identify somatic mutations, we performed NGS of a panel of 15 genes, including some of the most frequently mutated genes in HNC, Ten nanograms of genomic DNA from each sample were subjected to library preparation using the Ion AmpliSeq Library Kit 2.0  and an AmpliSeq Custom Panel  designed to specifically amplify the entire coding region of the 15 genes, with a coverage of 99.26%. Samples were barcoded using IonXpress Barcode Adapters . Automated template preparation and enrichment using an input of 30pM of purified library were performed in an Ion Chef System  and sequencing was conducted in an Ion Torrent PGM  using the Ion PGM Hi-Q View Sequencing Kit .35. A more detailed description of the methodology used is in the Supplementary Methods.The genetic ancestry of a subset of patients (n = 11) was determined using AIMs (Ancestry Informative Markers) as previously reportedhttp://wannovar.wglab.org/) and Cancer Genome Interpreter36. After quality filters were applied per sample 37, variants with the following characteristics were selected: non-synonymous and frameshift variants occurring within exons; variants with a depth of coverage of at least 200x; variants with an allele frequency of at least 10%; frequency observed in population databases of germline variants  lesser than 1%; classified as drivers or predicted drivers using the CGI tool (https://www.cancergenomeinterpreter.org/home); and previously reported at the COSMIC (https://cancer.sanger.ac.uk/cosmic) and/or TCGA databases .Sequencing results obtained in the Ion Torrent PGM run were processed in the Torrent Server. Reads generated were aligned to hg19 human reference genome using the Torrent Mapping Alignment Program (TMAP) and the Torrent Variant Caller (TVC) plugin version v5.8.0.19 (464) was used to call for variants. Variants were annotated using wANNOVAR (TP53 status by using the Evolutionary Action score of TP53-coding variants (EAp53) to stratify patients with tumors harboring TP53 mutations as high or low risk for unfavorable outcome, based on a model of the genotype-phenotype relationship described in39.All remaining variants were manually confirmed on the Integrative Genomics Viewer (IGV) version 2.7. We further explored https://xenabrowser.net). Mutation frequencies of the 15 genes tested in this study were compared between both cohorts.Tobacco, HPV status and mutational data from 39 OpSCC patients evaluated in The Cancer Genome Atlas (TCGA) were accessed with the online Xena Browser  under study/dataset identifiers EGAS00001004430/EGAD00001006151.Statistical analysis was performed using the software IBM SPSS Statistics 23 for Windows. Categorical variables were compared using Fisher’s exact or Chi-square test. Survival curves were calculated by Kaplan-Meier method and differences between groups were compared using the log-rank test. Recurrence-free survival was defined as the interval between the date of initial treatment and the diagnosis of recurrence. For all analysis, we considered statistical significance when p value <0.05.th edition40) at diagnosis (84.3%), T3/T4 tumors  and with clinically positive lymph nodes at diagnosis . All patients were treated with a platin-based chemotherapy concomitant to radiotherapy between 2009 and 2017.Clinical and histopathological data of the 51 oropharyngeal cancer patients enrolled in this study are presented in Table The median follow-up was 26 months (range: 1.00 to 86.00 months) and 44.3% of the cases were alive in 5-years. Recurrence occurred in 14 cases (27.5%). Tobacco smoking was significantly associated with reduced recurrence-free survival , while HPV-status association with increased recurrence-free survival was marginally significant  . For a small subset of cases with available DNA (n = 12), droplet digital PCR (ddPCR) was used for the detection of HPV-DNA for types HPV-16 and HPV-18 as part of another study (unpublished data). The agreement between p16-IHC and HPV-DNA results was of 91.7% : 6 were p16+ /HPV-DNA+, 5 were p16-/HPV-DNA-, but 1 had discordant a result, being p16 + /HPV-DNA- (Supplementary Table in-situ hybridization) from the TCGA study recovered from the Xena database . The majority of variants were classified as missense , followed by nonsense variants  and frameshift . We found an average of 1.65 (ranging from 1–12) genes mutated per sample and the majority of the patients  were mutated for at least one of the genes tested: 17/32 (53.1%) had only 1 gene mutated, 9/32 (28.1%) had between 2 and 5 genes mutated, and 6/32 (18.8%) had more than 5 genes mutated.TP53, 21.6% (11/51) for NOTCH1, 17.6% (9/51) for FAT1, 13.7% (7/51) for NSD1 and PIK3CA, 11.8% (6/51) for CDKN2A, 9.9% (5/51) for RB1, 7.8% (4/51) for KMT2D and PTEN, 5.9% (3/51) for HRAS, 3.9% (2/51) for IRF6 and FBXW7, and 2.0% (1/51) for EZH2  for 3/51 for TP53, the most frequently mutated gene in our cohort, most of the mutations were missense (28/51), 5 were truncating (3 frameshift and 2 nonsense) and the majority occurred within the p53 DNA-binding domain (between codons 95 and 288)  and all variants observed were missense with a high fraction happening in the N-terminal epidermal growth factor (EGF)-like ligand binding domains and another fraction in the C-terminal end, mainly around and within the Ankyrin repeat domain , followed by PIK3CA , RB1 , KMT2D , NOTCH1 and NSD1 , FAT1, FBXW7 and PTEN , and CDKN2A  , a very similar rate to the one we observed in our cohort . 5/39, a vBesides somatic variants characterization, for a subset of patients (n = 9) the genetic ancestry was determined  for FAT1, 1/18  for NOTCH1, and 3/18  for TP53   for TP53  mutated for CDKN2A and 7/9  for FAT1   were exposed to at least one of the risk factors evaluated (tobacco smoking and/or positivity for HPV infection): 3 (15.8%) cases were never-smokers, but HPV-positive; 8 were smokers, but HPV-negative; and 7 were smokers and HPV-positive Fig. .TP53 , NOTCH1 , FAT1 , and CDKN2A ; while PIK3CA was mutated in 20.5% of the samples (8/39). This agrees with the lower prevalence of ever smokers (69.2% versus 86.3%) and higher prevalence of HPV-positive cases in the TCGA cohort  had a higher T-stage (T3/T4).We tested whether the presence of mutation in the genes evaluated could be associated with the following demographical and clinical features: age at diagnosis, gender, tobacco consumption, drinking status, HPV status, T stage, N status and clinical stage. The only statistically significant association found was between TP53  , the same was observed for the presence of NOTCH1, significantly decreased the recurrence-free survival (RFS) of mutated cases    Table . In a muPTEN mutation was also significantly associated with decreased recurrence-free survival  (Table The presence of 5) Table . However5) Table .TP53 mutations. Although not statistically significant, we observed that the presence of TP53 variants reduced recurrence-free survival .As mentioned earlier, 45.1% (23/51) of the samples tested harbored TP53 missense mutations by using the Evolutionary Action score of TP53-coding variants (EAp53)39 to predict the impact of TP53 mutations on outcome: 34.8% (8/23) of the mutated cases were scored as having low-risk variants, while 65.2% had high-risk variants. High-risk variants carriers had an increased risk of 2.540-fold of having a recurrence  and a marginally significant decreased recurrence-free survival   and only 35.3% were HPV-positive (by p16-IHC); even within the HPV-positive group, the majority of cases were smokers (66.7%). This information shows that tobacco smoking still seems to have a bigger impact in the onset of OpSCC treated at our institution. Despite the small number of cases evaluated in our study, tobacco smoking was significantly associated with reduced recurrence-free survival (RFS), while HPV-status association with increased RFS was marginally significant. It is noteworthy that the majority of HPV-positive cases in our cohort were current or former smokers . HPV-positive OpSCC has a clearly improved outcome; however, smoking has a reportedly adverse effect on prognosis in both HPV-positive and HPV-negative cases6. This may have impacted the survival rates we observed, since although higher than in the HPV-negative group, the difference was not statistically significant.OpSCC were classically associated with heavy tobacco and alcohol consumption; however, a significant decrease in cases related to tobacco/alcohol and an increase in cases related to infection by high-risk human papillomavirus (HPV) is being observed, especially in high and middle-income countries44, colorectal31 and lung45), as well as in the healthy population34. All these studies found similar results, with a confirmed mixture of ancestry markers for the populations examined and a higher proportion of European ancestry markers, followed by African and then Asian and/or Native American in most of the patients. We were unable to test all our patients for genetic ancestry, but the small subset tested confirmed these results. Previous studies have shown differences in prevalence of mutation in specific genes according to genetic ancestry, such as an enrichment of TP53 mutations in AFR and of PIK3CA in EUR46. However, statistically significant confirmation of the genetic ancestry profile of the patients evaluated in this study and testing for any associations with the prevalence of gene mutations would require the evaluation of a substantially larger number of cases.Brazilian population has been frequently characterized by a considerable admixture of different ancestries in the genetic composition within individuals. Several studies have explored this admixture in large numbers of patients with the most prevalent tumor types  HPV-related; (ii) tobacco-related; and (iii) HPV-negative/tobacco-negative47. p16 detection by immunohistochemistry (p16-IHC) was included in the clinical routine as the gold-standard test for HPV-positive oropharyngeal SCC with proved clinical impact48 due to its easy-of-use, low cost and high sensitivity rates (near 100%)49. However, it frequently yields not ideal specificity rates (79% and 95%), detecting a positive p16 signal in tumors not associated with HPV50. For this reason, several studies have suggested additional HPV-specific tests to ensure accurate classification of OpSCC as HPV-related52. In our study, we performed HPV-DNA detection in a subset of the cases and observed a high agreement with p16-IHC results. Moreover, molecular characterization closely agreed with other studies that also used HPV-status to describe results. HPV-positive tumors usually have a lower mutational burden, are usually WT for TP53 mutations and frequently harbor activating PIK3CA mutations and amplifications (mainly the HPV-KRT)53. In accordance to this, in our cohort, HPV-negative cases had 2.5-times more variants than HPV-positive cases . Moreover, even though most of our HPV-positive cases were smokers , the frequency of TP53 mutation in this subgroup was significantly lower . Conversely, we found 2 HPV-positive case with PIK3CA mutation ; a classic C > T mutation in the helical domain, frequent in viral-associated tumors (p.E545K) and commonly associated with the apoliprotein B mRNA-editing enzyme catalytic subunit (APOBEC)-induced mutational signature54. Hayes and colleagues reported in a review article a frequency between 22–56% of PIK3CA activation in HPV-positive HNC; however, we only evaluated somatic mutations in the present study and did not explore copy-number alterations (CNA). This might explain the lower frequency in comparison to other studies that relate this activation both to mutation and amplification in HPV-positive cases53. As expected, besides TP53, genes frequently mutated in tobacco-related HNC had a low frequency of mutation in the HPV-positive group, namely NOTCH1 , CDKN2A  and FAT1 55.For HPV-positive cases, transcriptionally-active human papillomavirus leads to cell cycle deregulation through the abrogation of p53 and pRb pathways by the viral oncoproteins E6 and E7 (HPV E6 and E7). Further oncogenic events may lead to differentiation in HPV-KRT (HPV-keratinocyte differentiation and oxidative reduction process) or HPV-IMU  tumorsTP53 and CDKN2A. CDKN2A encodes the p16INK4A protein and is lost through deletion, inactivating mutations and hypermethylation in 15–22% of HNC53. Together with the frequent amplification of cyclin D1 (CCND1), also common in tobacco-associated HNC, CDKN2A LoF contributes to unscheduled DNA replication and leads to DNA damage and p53 activation56; however, TP53 inactivation is also found in 60–80% of HNC cases24, thus abrogating cell cycle arrest and apoptosis57. Consequently, these tumors have a higher mutational burden and frequent copy number alterations42. In our cohort, we found an overall rate of CDKN2A and TP53 mutation of 9.8% and 45.1%, respectively. Dogan and colleagues found similar rates, when using a targeted exome sequencing approach to characterize 83 HPV-negative and 74 HPV-positive OpSCC58: 49% of the cases were mutated for TP53, while 22% harbored CDKN2A mutations and deletions. Moreover, we did not test for CDKN2A inactivation through deletion or hypermethylation. However, among mutated cases, these genes were more frequently mutated in current/former smokers with rates of 83.3% for CDKN2A and 95.7% for TP53. The breakdown rates according to HPV status were: 78%/16% for TP53 mutations in HPV-negative/positive cases and 39% versus 4% for CDKN2A mutations in HPV-negative/positive cases.It is already known that the presence of TP53 mutations has an important impact in disease progression, treatment response, specially to platinum-based therapy, and survival62. Although not statistically significant, we observed that the presence of TP53 variants reduced recurrence-free survival . Next, we explored whether the algorithm Evolutionary Action (EAp53), that stratifies patients according to TP53 variants associated with especially poor outcomes39, could be used as a classificatory in our cohort. The association of TP53 variants classified with EAp53 still did not reach significance, but it is clear that, for our patients, high-risk variants carriers had an increased risk of 2.540-fold of having a recurrence  and a marginally significant decreased recurrence-free survival . Previous studies have tested this algorithm in HPV-negative HNC and validated the association of high-risk TP53 variants, as classified by EAp53, with decreased sensitivity to cisplatin, decreased survival and increased distant metastases in HNC39. Here we focused our analysis in OpSCC patients with a significant representation of HPV-positive cases and treated by chemoradiation. These differences should be taken into consideration in the interpretation of the algorithm results.The second and most classic subgroup of HNC is associated with tobacco-smoking and also involves deregulation of the cell cycle mainly through loss-of-function (LoF) of two tumor suppressor genes: NOTCH1, impacting squamous differentiation and cell polarity, while 5–23% of cases harbor mutations in FAT1, an important component of the WNT signaling53. This route seems to impact beta-catenin signaling and keratinocyte transformation, and tobacco-smoking is a known risk factor42. We found similar rates of mutated cases for these genes in our cohort, and the majority of cases mutated for NOTCH1 and FAT1 were current or former smokers. Moreover, we found and association between NOTCH1 mutation and a higher T stage, consistent with previous studies showing that functional NOTCH1 inhibits proliferation in SCC cells and that loss of canonical NOTCH increases tumorigenesis in both HPV-positive and p53-mutant mice63. In addition, the presence of NOTCH1 mutations significantly impacted recurrence-free survival. Emerging evidence indicates that Notch effects are dependent on the cellular context in which it is activated, with aberrant Notch signaling being associated with cancer recurrence, metastasis and treatment resistance in different tumor sites64. In a recent study, Lim and colleagues tested the feasibility of using targeted NGS to guide treatment of HNC patients, among the genes with association with a poorer overall survival they found NOTCH1 mutation as a predictor of worse outcome65. Similarly, Dong and colleagues found that in HPV-positive OpSCC cases, the presence of NOTCH1 mutations contributed to a worse overall-survival58. Vettore and colleagues also found an association between the presence of mutations in genes of the Notch pathway and a lower disease-free survival in patients with oral tongue carcinoma66. In a cohort of esophageal carcinomas from China, Song and colleagues found that patients with mutations in NOTCH1 had shorter survival and failed to respond to chemotherapy67. These results agree with our findings that the status of NOTCH1 mutations is a promising predictive biomarker for patient outcome and treatment response in OpSCC.After cell-cycle abrogation, HPV-negative/tobacco-positive cases usually acquire more alterations that drive different routes towards the progression of these tumors. These pathways usually involve squamous differentiation, oxidative stress and WNT signaling. Between 10–21% of HNC demonstrate LoF mutations in PTEN is a tumor suppressor gene mutated in 9–23% of HPV-negative HNC, leading to oncogenic activity through the activation of the PI3K/AKT/mTOR signaling42. We found PTEN mutations in 7.8% of the cases tested; once again, this rate does not include CNAs. Interestingly, we observed a significant decrease in recurrence-free survival in the presence of PTEN mutation . A recent study found that patients with high-expressing PTEN had an improved progression-free survival in response to cetuximab, in comparison to patients with low expression of this gene68. A similar scenario was observed in a Brazilian study on HNC patients treated with cetuximab, with a worse progression-free and overall-survival in patients with loss of PTEN69.Finally, NOTCH1 and PTEN mutations and survival in OpSCC patients treated with platin-based chemotherapy plus radiation.To our knowledge, this is the first study showing the association between HRAS mutations and LoF mutation in CASP870. This subgroup is usually described as diploid, as having fewer CNAs, DNA mismatch repair proficient and to occur more frequently in women with oral cavity tumors without a history of alcohol and smoking consumption70. The prevalence rates of this subgroup and the clinical impact is yet to be studied42. We did not detect any case with CASP8 variants in our cohort; moreover, all 3 cases identified with HRAS mutation were current or former smokers and one of them was also HPV-positive.The third subgroup of HNC comes mainly from previous studies that identified a subset of HNC with a strong correlation between gain of function 24. Besides including data from all subsites within head and neck cancer, the mutation data reported in the article compiled results from whole exome sequencing, while we only tested a panel of a limited number of genes. This can explain the similarities between mutation rates in the two cohorts of OpSCC samples explored in this article in comparison to the frequency reported in the TCGA publication.Although differences in the mutation rates of specific genes were observed, mostly due to different prevalence of risk factors (HPV and tobacco smoking), when comparing the results on our cohort and the TCGA OpSCC samples explored, the frequency of cases mutated for at least one of the genes was similar (62.7% versus 64.1%). In the TCGA publication with genomic data on head and neck cancer, only 14% of the samples tested did not present mutation in any gene30. The study reports that 25% of the cases were from the oropharynx (45/180), and only 8% of the entire cohort was HPV-positive (15/180)30. Therefore, their cohort was enriched for HPV-negative cases, and, although the number of OpSCC tested was similar to ours, only around 8% were HPV-positive, in contrast to our 35.3% frequency of HPV-positivity. This might explain the slightly lower rates of TP53 mutations and other genes associated with tobacco-smoking in our cohort in comparison to their findings. In addition, they also explored somatic CNA, that are also included in their higher rates.Recently, Perdomo and collaborators, evaluated a similar panel of genes in a cohort comprised of 180 HNC from 3 multicentric studies from South America and EuropeOur study has some limitations being the small number of cases evaluated and the short follow-up (median of 26 months) possibly the most important ones, directly impacting the statistical significance of the molecular findings in patient outcome. Besides that, we also did not evaluate copy-number alterations of the genes included, hindering comparisons to other genomic studies that include amplification and deletion in the mutational frequencies. It is important to point out that there is a lack of genomic studies including Brazilian cases; therefore, our study focused in the profile of somatic variants of a set of genes in a cohort of Brazilian OpSCC patients submitted to a homogeneous treatment and with sufficient and well-annotated clinical data. Despite of differences in geographic, economic and social habits when compared to other populations, the results were comparable to those reported before and new insights into possible molecular mechanisms associated with outcome were suggested.In conclusion, we report here the results on the genetic characterization of a set of HNC-related genes in a cohort of OpSCC patients. Some of the findings suggest the clinical relevance of genomic approaches to better classify subgroups of patients with different outcomes. Validation of these data could lead to a more refined prognostic stratification and contribute to a personalized treatment approach of OpSCC patients.Informed consent was obtained from each individual prior to sample collection and the study protocol was approved by the Barretos Cancer Hospital Institutional Review Board.Supplementary information.