Most endocervical adenocarcinomas (EAC) are associated with high-risk HPV (hrHPV) infection, with HPV genotypes 16, 18 and 45 accounting for >90% of the cases. Among endocervical glandular lesions, screening with hrHPV test has previously shown to predict the outcome better than cytology, although around one-fifth of the EAC remain negative both in hrHPV testing and cytology. The study consists of two consecutive HPV-primary screening rounds, conducted in 2012–2015 and 2017–2020. Of the 87 women aged 35 to 60 years of age diagnosed with Atypical endocervical cells, NOS or Atypical endocervical cells, favor neoplastic cytology during the first screening round, 63 (72.4%) were hrHPV positive and 24 (27.6%) were hrHPV negative. Among hrHPV positive patients, three EAC, two adenocarcinomas in situ (AIS), one AIS + high-grade intraepithelial lesion (HSIL) and 13 HSIL were found. Of the histologically verified lesions, 68.4% (13/19) were purely of squamous origin. All the EAC and AIS were HPV16 or HPV 18 positive. No high-grade histological lesions were found among the hrHPV negative patients with cytological glandular atypia. A later database search revealed one HPV-negative, gastric-type mucinous EAC that was missed by the HPV primary screening. In Finland, national cervical cancer screening has been organized since the 1960s. Women from the age of 30 to the age of 60 are invited to participate every fifth year, and in some municipalities 25- and 65-year-olds are also included. Since the beginning of the organized screening, the number of cervical cancer deaths in Finland has decreased to one-fifth of its original number [Traditionally, the conventional Pap smear has been the primary test. In 2012, the city of Tampere, and later all municipalities in Pirkanmaa region, started HPV primary screening with high-risk HPV (hrHPV) test and cytological smear as a triage in women aged ≥35 years ,5,6.The important role of hrHPV infection leading to the development of invasive cervical squamous cell carcinoma (SCC) and behind the most of the endocervical adenocarcinomas (EAC) has been well documented ,8,9,10. In the previous studies, the clear cell adenocarcinomas (AC) accounted for 3.9% to 4%, the serous AC for 3% to 3.2%, the endometrioid AC for 1.4% to 3%, the minimal deviation AC 1.6% to 6% and the AC, not otherwise specified, for 4.7% to 8% of the EAC . These lIn previous studies, HPV testing identified earlier more women with cancer or precancerous lesions than cytology alone ,14,15,16The aims of the present study are to evaluate the detection and outcome of endocervical atypia in an HPV primary screening programme.The study cohort represents women participating in an HPV primary screening programme organized by Fimlab Laboratories during 2012–2015 in the Pirkanmaa region, Finland. During these years, altogether 93.439 women aged 35 to 60 years were invited to cervical cancer screening, which included both the hrHPV test and the conventional pap test. All the pap tests of the hrHPV positive women were analyzed and, as a quality assurance, 10% of the HPV negative patients were assessed cytologically in the 2012–2016 period.In total, 66.147 (70.8%) of the invited women participated in the screening. Out of the participants, 87 (0.13%) were diagnosed with endocervical glandular atypia  with or without a squamous atypia .At the beginning of the study, according to the screening protocol and the national guidelines, women with a positive hrHPV test result and/or a cytological diagnosis of Atypical Squamous Cells, Undetermined Significance (ASC-US) and/or Atypical Endocervical Cells, NOS  were referred to repeat sampling after 12 months . The repIn 2016, there was a change in the national guidelines after which patients with cytological diagnosis of AEC, NOS with or without a positive hrHPV test result were referred for a colposcopy immediately.Women with a cytological diagnosis of Atypical Endocervical Cells, Favor Neoplastic  and/or a squamous cytological diagnosis of Low-Grade Squamous Intraepithelial Lesion (LSIL) or worse were always immediately referred for a colposcopy despite the hrHPV status of the patient. After the colposcopy, the patient was treated according to the national guidelines . DespiteAt the first screening round in 2012–2015, the patients were between 35 to 60 years old, and at the second screening round in 2017–2020, they were between the ages of 40 to 65 years.All diagnoses on cervical cytological samples were provided according to the Bethesda Classification for Reporting Cervical Cytology 2014 ,19,20. TThe HPV status, genotype, cytological and histological data were retrieved from the Laboratory Information System (LIS) of the Fimlab Laboratories Oy. Later, an additional LIS- search was conducted to find possible EACs missed by the primary HPV screening during the study period 2012 to 2020. For the statistical analysis, SPSS version 25 was used .Because the samples of the study include those produced by the national cervical cancer screening protocol and its follow-ups, the individual consent of each participant was not requested. The study was approved by the Ethical committee of Pirkanmaa Health Care District (R13094 and R16022). The study was conducted according to the Declaration of Helsinki.After the first screening rounds in 2012–2015, 61 patients were diagnosed as AEC, NOS on cytology . Of thosIn 2012–2015 a total of 17 patients were diagnosed with AEC, NOS in combination with a squamous cytological diagnosis . Out of A cytological diagnosis AEC, FN was provided seven times in the first screening rounds in 2012–2015 and AEC, FN + LSIL was diagnosed two times . All patAltogether, behind the 87 cytological endocervical glandular diagnoses with or without combined squamous cytological atypia, one EAC, two AIS, two EAC + LSIL, one AIS + HSIL and 13 HSIL were found reflecting that 68.4% (13/19) of the high-grade lesions verified histologically were purely of squamous origin . There ap-value > 0.05) to endocervical malignancies or to squamous neoplasias.Of the endocervical malignancies, 33.3% (2/6) were HPV18 positive and 66.7% (4/6) were HPV 16 positive . Among HAside for the two above-described AIS cases with the wide time lag before the histological diagnosis, all the high-grade histological lesions were diagnosed during the first screening round. No high-grade histological lesions were found among patients with cytological endocervical glandular atypia and a negative hrHPV test result , Table 2The diagnosis of this gastric-type mucinous EAC was made two years after the negative HPV primary screening result. The diagnosis was immunohistochemically confirmed, and the patient was treated with hysterectomy, combined with salpingo-oophorectomy and lymphadenectomy. There were no metastases. The tumor itself was not tested for hrHPV, but the vaginal hrHPV tests have remained negative since. A 5-year follow-up has been conducted, and no additional tumors were observed.During the first screening round, 17.5% (11/63) of the hrHPV positive patients and 29.2% (7/24) of the hrHPV negative patients left follow-ups unattended . Of the The results of the present study are in agreement with the previously published series in the sense that no endocervical glandular malignancies were found among patients with endocervical cell atypia and a negative hrHPV result, reflecting that the majority of the endocervical adenocarcinomas are associated with hrHPV infection ,9,10. InIn addition to the lack of glandular malignancies, in the present study, there were no histological HSIL or SCC among the cases with endocervical glandular atypia and a negative hrHPV result. This is supporting the previous findings, according to which over 60% of the mild cytological glandular atypias with or without mild squamous atypia represent benign changes, or low-grade squamous histological lesions ,27,28,29In our study, behind the cytological endocervical glandular diagnoses, with or without combined squamous cytological atypia, 68.4% of the clinically significant lesions  were purely of squamous origin. This might be caused by the extension of HSIL to endocervical glands, which is known to be a common cause of false cytological glandular diagnoses, or simply the result of the everyday struggle pathologists have in separating the cytological glandular features from squamous features ,26,30. NIn previous studies, the hrHPV test has been more often positive for cancer and precancerous lesions than cytology, and the difference in performance has been more pronounced among cases subsequently diagnosed with AIS or EAC than CIN3 or SCC ,12,15,16Similarly, in the present study, only two malignancies were found during the second screening rounds. Both lesions represented AIS, and their histological diagnoses were not reached until 5 years and 9 months and 6 years and 11 months after the initial hrHPV positivity and cytological glandular atypia at the study baseline. Although the participation rate for screening (70.8%) in the present study was similar to that previously reported in Finland , the higOur database search revealed one hrHPV-negative mucinous, gastric type adenocarcinoma case missed by the primary HPV screening during the study years 2014–2021. Since the hrHPV test at the screening was negative, no follow-ups were scheduled for the patient. The cancer was later diagnosed on a gynecological check-up for other reasons.Since, in previous studies, 22% of the EAC and around 15% of the AIS were negative both in cytology and in hrHPV test, it can be only speculated if including the cytological sample to the screening protocol would have led to an earlier diagnosis in this case ,12. It hIn our series, all AIS and EAC were positive with either HPV16 or HPV18. Out of the HSIL, 42.9% were positive with an hrHPV type other than HPV16 or HPV18. In the statistical analyses, the HPV types  or combinations of them did not provide a prediction specifically relating to endocervical malignancies or to squamous lesions. This may have been due to the small number of positive cases, which may also have been the reason why the positive cases were not analysed further.In conclusion, in this study of two consecutive HPV-primary screening rounds, among 87 women diagnosed with AEC, NOS or AEC, FN in cytology, three EAC, two AIS, one AIS + HSIL and 13 HSIL in were found in histology. All the EAC and AIS were either HPV16 or HPV 18 positive. No high-grade histological lesions were found among the hrHPV-negative patients diagnosed with cytological endocervical cell atypia. A later database search revealed one HPV-negative gastric-type mucinous adenocarcinoma that was missed by the primary HPV screening.


Many rural American Indian and Alaska Native (AIAN) veterans receive care from the Indian Health Service (IHS). United States Department of Veterans Affairs (VA) has reimbursement agreements with some IHS facilities and tribal programs and seeks to expand community partnerships in tribal areas, but details of how AIAN veterans use IHS are unknown. We aimed to assess the health status, service utilization patterns, and cost of care of veterans who use IHS.We used comprehensive and integrated IHS data to compare health status, health service utilization and treatment cost of veterans  to a matched sample of non-veterans . We employed logistic, linear, or negative binomial regressions as appropriate, by sex and overall.Compared to non-veterans, veterans had lower odds of having hypertension, renal disease, all-cause dementia, and alcohol or drug use disorders, but had similar burden of other conditions. In service utilization, veterans had lower hospital inpatient days; patterns were mixed across outpatient services. Unadjusted treatment costs for veterans and non-veterans were $3,923 and $4,145, respectively; veteran adjusted treatment costs were statistically lower. Differences in significance by sex were found for health conditions and service use.AIAN veterans, compared to AIAN non-veterans, were not less healthy, nor did they require more intensive or more costly care under IHS. Our results indicate the viability and importance of expanding IHS-VA partnerships in community care. American Indian and Alaska Natives (AIAN) have served in the United States (U.S.) military since the American Revolution , and theMany rural AIAN veterans can access care through both the U.S. Department of Veterans Affairs (VA) and IHS. Kramer and colleagues found that 25% of AIAN veterans used both VA and IHS care, while about 46% used IHS services only . In 2010Despite the progress made in increasing collaboration between the two systems, research regarding optimization of care has been slim. Since health records are not shared between the agencies, utilization patterns of dual users have not been explored, with two notable exceptions. Using data from 2002–2003, Kramer and associates linked IHS and VA records and found that the likelihood of dual use increased with the number of diagnoses, and compared to those using IHS only, dual users were more likely to use intensive specialty health care services, suggesting medical need contributes to dual use , 20. In While efforts to link data comprehensively across the two systems have not materialized, the need for coordinated care has likely increased. Research using data from 2008 indicated that rural AIAN veterans accessing VA care were more likely to have experienced combat, have higher ratings of service-connected disability, and higher mean number of visits compared to their rural non-Native counterparts . Other wDifferences between IHS and VA in operational systems and in mandate shape veteran health care experiences and associated costs. IHS is charged with providing health care to 2.6 million AIANs . As a reThe IHS is not an insurance program, but instead receives appropriations from Congress. It is chronically under-funded ; IHS expThe VA, in contrast, has a budget of $68 billion for health care to serve 9 million veterans, with a per capita expenditure of approximately 3 times that of IHS , 44. ThiHere, we analyze data from IHS to assess the health status, service utilization patterns, and cost of care of AIAN veterans in the IHS system. Absent system-wide VA-IHS linked records, our goal is to compare the health status, utilization, and cost of care of AIAN veterans to their AIAN non-veteran counterparts in order to inform the process of expanding VA support for local IHS services in the care of rural AIAN veterans. Because health conditions and care utilization patterns differ substantially by sex among veterans, our analyses disaggregate results by sex also –49. Our Data were extracted from IHS Improving Health Care Delivery Data Project infrastructure. . The study sample represents the broad geographic distribution of the AIAN population. Using regional classifications employed elsewhere, one project site was located in the East, four in the Northern Plains, two in the Southern Plains, five in the Southwest, two in the Pacific Coast, and one in Alaska. The IHS Data Project population was identified by project site, rather than by random sampling, to create important site treatment cost measures not available elsewhere . The IHS service delivery system includes IHS operated hospitals and clinics, tribally operated hospital and clinics, and urban Indian clinics. The 15 project sites include 9 sites where IHS directly provides health services and 6 sites that are tribally operated. The IHS Data Project includes little data for urban Indian clinics; we refer to IHS and tribal (I/T) services and other (non-I/T) services below. The project population was comparable to the national IHS service population in age and sex .IHS electronic data from three sources are included in the data infrastructure. The sources, described below, include the: 1) National Data Warehouse for patient registration and IHS and Tribal (I/T) service utilization data; 2) Purchased/Referred Care data for patient service utilization data at non-I/T providers, paid for by IHS and Tribal Health Programs; and 3) Centers for Medicare & Medicaid Services Cost Reports (Cost Reports) for project site cost data. Patient data from National Data Warehouse and Purchased/Referred Care sources are linked though use of computer-generated numbers that are unique to each patient. Hereafter, we use the general term “patient data” to refer to these two sources of data.Project personnel collaborate with IHS and the Tribal organizations that participate in IHS Data Project. This collaboration takes place through the project’s Collaborative Network, which includes three advisory committees  and a process to obtain written approvals from IHS National Institutional Review Board (IRB), Tribal IRBs, Tribal Councils, or Tribal Authorities, in addition to the collaborating university’s IRB, the Colorado Multiple Institutional Review Board. Specific approving Tribal entities are not named to protect community confidentiality, as requested by participating sites.We identified 12,428 AIAN adults who obtained services at least once during FY 2011–2013 and who also self-identified as veterans. We created a matched sample of non-veterans who were matched with veterans by birth year, sex, and project site. Since age and sex influence health status, service utilization, and treatment costs, and since the age and sex distribution of AIAN veterans did not match that of AIAN non-veterans in the FY2013 Data Project population, matching samples was important to ensure meaningful comparisons. We identified a non-veteran match for 98.5%  of the veterans. Over 60% of the veterans who were not matched with a non-veteran in their project site were aged 85 years and older. Analyses conducted for this report include FY 2013 data for the 12,242 veterans and the 12,242 matched non-veterans. Using the 2013 National Center for Health Statistics urban-rural classification scheme, approximately 60% of the sample resided in small, micropolitan, or non-core counties .TM DxCG Risk Solutions software , renal disease , end-stage renal disease , all-cause dementia , and alcohol or drug use disorders . They had similar odds of having of diabetes, CVD, chronic obstructive pulmonary disease, asthma, malignant cancer, liver disease, depression, and other mental health disorders.Differences in health status between veterans and non-veterans varied by sex. Among females, veterans had lower odds of having diabetes , renal disease , malignant cancer , and tobacco use disorders  than non-veterans. Among males, veterans had lower odds of hypertension , renal disease , end stage renal disease , all cause dementia , alcohol or drug use disorders , and tobacco use disorder .Utilization patterns, shown in Patterns of use of particular services were also similar by veteran status, including average number of visits to emergency departments, primary care, and behavioral health . CompareWe found few differences in cost of care between veterans and non-veterans using IHS. During FY 2013, unadjusted treatment costs for veterans were $3,923 and for non-veterans were $4,145 , a diffeVeteran adjusted treatment costs for all ages were significantly lower (p<0.05) than non-veteran costs. Among both females and males of all ages, adjusted treatment costs for veterans were similar to those for non-veterans. By age group, the only statistically significant difference found overall was among those aged 18–34 years, with veterans having had lower adjusted treatment costs compared to non-veterans (p<0.01). However, among those aged 18–34 years, adjusted costs for veterans were significantly lower than costs for non-veterans for both females (p<0.05) and males (p<0.05). About 40% of AIAN veterans did not have Medicare, Medicaid, or private insurance (no information was available about VA coverage). The likelihood of having Medicare, Medicaid, or private insurance was similar for veterans and non-veterans , more IHS facilities may serve as useful partners to provide such services to veterans residing in remote rural areas where distance or transportation are imposing barriers to VA care. Our findings can inform the scope and types of care VA could support for optimal impact.Finally, our findings contribute to the literature examining the tensions of race, historical trauma, and veteran benefits for Indigenous veterans cross-nationally , 65. TheOur findings should be viewed with some caution. These IHS data did not indicate which veterans were also eligible for or used VA care or other care outside of IHS. They also did not include those who did not use IHS at all during the study period. Thus, we cannot ascertain relative health status across users and non-users, so our analysis does not present a full picture of care for some. While sites were accounted for in analysis, they vary in number of veterans served. The health status, service utilization, and treatment cost results are based on cross-sectional data limited to one year; the interpretation of findings must remain descriptive, not causal. Our results also do not account for current reimbursement agreements. Indeed, these data are from 2013; however, they are unique in their detail and comprehensiveness, and until the recent pandemic, our findings likely represent patterns sustained since FY 2013. Our data are complex linked administrative data associated with care and do not contain the details that may be obtained from a medical record review. The lack of complete medical information on the study sample may have biased the prevalence of conditions downward . Additionally, these data do not include information on health care services and related costs that were obtained at non-I/T facilities and not paid for through the Purchased/Referred Care program . Sites also varied by types of services provided, proportion of veterans served, distance to non-I/T facilities, and completeness of data.Despite these limitations, these analyses provide important insights to a population largely ignored in the literature. The unique and comprehensive dataset detailed health status, utilization, and cost for a large and geographically diverse sample of AIAN veterans using IHS, finding more similarities than differences between AIAN veterans and non-veterans. The analysis here also serves as a helpful baseline relative to changes in the policy environment, for example, increased numbers of reimbursement agreements via the VA-IHS memorandum of understanding, or changes attendant to Veterans Community Care Program.As VA considers the viability of community partners, assessing increased partnership with IHS facilities, often the only accessible health care option for rural AIAN veterans, will be critical. Our results indicate the viability of such partnerships, with the added benefit of likely improving care for all patients that IHS serves. These analyses also reveal the challenge of coordinating care efficiently and effectively across systems; efforts to link veterans’ health records so health care providers can access medical histories and current treatment plans will most certainly improve care for veterans.S1 File(DOCX)Click here for additional data file.


Oral infection by Human Papillomavirus (HPV) has recently gained great attention because of its involvement in the development of a subset of head and neck squamous cell carcinoma. The role of specific Alpha-HPVs in this regard has been well established, whereas the contribution of other genera is under investigation. Despite their traditional classification as “cutaneous” types, Beta and Gamma HPVs are frequently detected in oral samples. Due to the lack of a standardized protocol, a large variety of methodologies have been used for oral sample collection, DNA extraction, HPV detection and genotyping. Laboratory procedures influence the evaluation of oral HPV prevalence, which largely varies also according to the population characteristics, e.g., age, gender, sexual behavior, Human Immunodeficiency Virus (HIV) status. Nevertheless, oral infection by Beta and Gamma HPVs seems to be even more common than Alpha-HPVs. The latter is 5–7% in the general population, and increases up to 30% approximately in HIV-infected men who have sex with men. Despite major advances in the evaluation of oral HPV prevalence, its natural history is still little understood, especially for Beta and Gamma HPVs. The latest technologies, such as Next Generation Sequencing (NGS), can be exploited to gain new insights into oral HPV, and to improve the identification of novel HPV types. Papillomaviridae family, are small, non-enveloped viruses with a circular double-stranded DNA encapsulated within an icosahedral capsid. To date, around 220 HPV types have been completely sequenced, and there are numerous potential new types that remain unclassified. Based on variations in the sequence of the L1 open reading frame, which encodes for the major capsid protein, HPVs are classified into five genera  [Human Papillomaviruses (HPVs) cause the most widespread sexually transmitted infection . HPVs, wHistorically, HPVs have also been divided into two different groups based on their tropism for either cutaneous or mucosal epithelia. Thus, in the literature, the Alpha-HPVs are still referred to as “mucosal” and the Beta-HPVs and Gamma-HPVs are defined as “cutaneous”. However, the growing evidence that demonstrates “cutaneous” HPV broad distribution at mucosal sites ,4,5,6,7 Alpha-HPVs include Low-risk and High-risk types, as classified by the International Agency for Research on Cancer (IARC) based on their carcinogenic potential . The HigThe contribution of HPV to the above-mentioned neoplasias ranges between 25% (vulvar carcinoma) and virtually 100% of cases  ,15,16,17Because of the causal association of this virus with HNSCC, current studies in the HPV field largely focus on oral infection. In this review, we will firstly discuss the most commonly used methods for oral sample collection, detection and genotyping of Alpha, Beta and Gamma HPVs. We will then outline the epidemiology of the infection caused by the three genera. Finally, a brief overview of the lesions caused by these HPV genera at the level of the head and neck region will be provided.The laboratory workflow for the assessment of oral HPV infection is depicted in 3 human diploid cells are generally used. A higher amount of human genomic DNA could negatively affect the sensitivity of HPV-DNA detection by inhibiting the amplification of the HPV target region [So far, no gold standard for oral sample collection has been identified. Many kinds of samples have been used for oral HPV detection, the main being: saliva, oral rinse-and-gargle, tonsillar washing, mucosal scraping or brushings and tissue biopsies. It has been estimated that approximately 100,000 cells should be processed to obtain reliable results with Southern blot hybridization . More set region ,24.Saliva samples are usually shaken and incubated at 50 °C–56 °C for 1–2 h before nucleic acid extraction ,26,27,28De Souza et al.  comparedComparison of oral gargle and tonsillar washing (performed by a clinician using a specific device) was carried out by Choo et al.  to invesBesides the wide spectrum of oral specimens that can be collected, and the variability of the pre-analytic phase, a large variety of methods are available for extraction and purification of nucleic acids. Notably, the extraction method can influence the sensitivity of HPV detection. D’Souza et al.  comparedThe assays used to assess the presence of Alpha-HPVs in oral samples are primarily those validated/employed for the diagnostics of this infection in cervical and anal samples. Currently, more than 250 commercial tests are available for the molecular detection of Alpha-HPVs . Some ofMoreover, broad range SPF10 primers ,40 have The methods available for the detection of Beta and Gamma-HPVs in oral samples can often identify only a restricted number of types. This may make it difficult to assess their actual prevalence in asymptomatic subjects and HNSCC.Currently, there are different PCR-based methods that can be used for Beta and Gamma HPV-DNA detection in skin and oral samples . These iAnother approach to improve the sensitivity of systems in Beta and Gamma HPV detection is Rolling Circle Amplification (RCA). This is a mechanism employed by some viruses to multiply their circular genomes, and it has been applied in the amplification of circular plasmid vectors used in cloning. The RCA protocol uses random hexamer primers to amplify the complete genomes of PVs without knowing their DNA sequences beforehand. The multiple-primed RCA method can be used to discover previously unknown PVs, and also other circular DNA viruses. The polymerization process is primed by exonuclease-resistant random hexamers that bind onto the circular DNA template at multiple sites, and, in doing so, they generate multiple replication forks . This meOther investigators developed a novel method combining a multiplex PCR based on E7-specific primers for Beta and Gamma HPVs with an array primer extension (APEX) for typing ,57. ThroThe majority of the HPV testing methods used in current research and/or clinical settings are designed to identify a restricted group of HPVs. Recently, new technologies have become available that can be used to discover novel HPV types. In this context, Next Generation sequencing (NGS) can also be used as a high-throughput assay for HPV genotyping . NGS invTo overcome the problem of sensitivity and specificity linked to consensus primer homology, a metagenomic approach could be chosen. For example, Pastrana et al. combined the RCA method with NGS technology to improve PV detection . Viral DNotably, the aspecific sequences/reads represent a problem for NGS determination: one milliliter of saliva might contain 10–100 million microbes and their presence is thus relevant when gargle or saliva samples are used for HPV testing. Consequently, the extracted DNA might be mostly bacterial DNA, masking the few copies of HPV-DNA contained in the samples. The considerable amount of human DNA and RNA can also reduce the reads that actually represent viral genome. Proficiency testing is required in order to assess the reliability of different NGS methods used for HPV detection.From a clinical point of view, the most relevant site of HPV infection at the level of the head and neck is represented by the oropharynx. However, HPV may also infect the oral cavity .Certain types of samples have been proven to be the most suitable for the purposes of epidemiological studies. However, these samples are often not site-specific, as in the case of oral rinse-and-gargles. This means that the precise site of HPV infection within the head and neck region cannot be established. Even using site-specific sampling techniques, e.g., directly brushing or swabbing the specific head and neck site, contamination by HPV-positive cells possibly deriving from the contiguous areas cannot be excluded. Therefore, it is not entirely correct to define the HPV infection as “oral”, especially when using non site-specific samples, since the term “oral” solely, and specifically, refers to the oral cavity. Notwithstanding this clarification, the expression “oral” HPV infection will be used hereafter for simplicity.The majority of the epidemiological studies on oral HPV infection have analysed Alpha-HPVs, rather than Beta or Gamma genera. Therefore, limited data on the prevalence and natural history of the latter, as well as their determinants and potential transmission routes, have been collected. Regardless of the genus investigated, it must be highlighted that oral HPV prevalence, incidence and clearance are largely influenced by the characteristics of the study population  and also by the methods used for oral sample collection, processing, and HPV detection .In the following paragraphs, we will focus on oral infection by Alpha, Beta and Gamma HPVs in “healthy”, i.e., cancer-free, subjects. Other reviews can be consulted for comprehensive data regarding oral HPV in HNSCC patients ,66.Firstly, the epidemiology of oral HPV in the general population, at average risk of infection, will be addressed. Subsequently, this will be described for men who have sex with men (MSM) and HIV-infected individuals, which are populations at increased risk for oral HPV infection. Data about the prevalence and corresponding predictors will be reported in more depth, and a brief overview of the natural history and the risk factors associated with incidence and clearance will be also provided. Taylor et al. more comprehensively reviewed the incidence, clearance and persistence of oral HPV infections .Oral infection by Alpha-HPVs is uncommon in the general population, so a thorough analysis of its epidemiology and natural history is challenging due to the large number of individuals that need to be enrolled. A systemic review that included 18 studies conducted between 1997 and 2009 reported a prevalence of any of the Alpha-HPVs among 4581 healthy individuals below 5% . PrevalePredictors, or determinants, of oral HPV infection have been more extensively investigated in the context of Alpha-HPVs, whereas much less is known regarding Beta and Gamma types. The most relevant predictors for the oral infection by the three different genera will be reviewed.(i) Age. Conflicting data concerning the prevalence of Alpha-HPVs according to age have been reported. In their study on the U.S. general population, Gillison et al. highlighted a bimodal pattern, with a first peak in 30–34 year-old individuals, and a second peak in 60–64 year-old individuals . SimilarSome evidence revealed older age as significantly associated with an increased detection of Beta-HPV types in the oral cavity of heterosexual males ,71,76.(ii) Gender. Gillison’s study revealed that any Alpha-HPV prevalence among men was almost three times higher than in women (10.1% vs. 3.6%) . The subCutaneous HPVs also seem to be more abundant in oral samples from men than in those from women. Indeed, a prevalence of 29.3% for Beta-HPVs was found in men of HIM study , whereas(iii) Sexual behavior. Sexual behavior represents a major contributor to the transmission of Alpha-HPVs at oral level, although other transmission modalities are possible. Briefly, vertical transmission seems to be possible, given that the same genotypes found in the genital tract of mothers have been detected in the oral cavity of their children ,87. SelfThe type of sexual intercourse , partnership, as well as the number of lifetime and recent partners are among the variables most frequently investigated. Gillison et al. showed that Alpha-HPV prevalence tends to increase significantly with the number of sexual partners . InteresNotwithstanding the central role of oral sex, open-mouthed kissing may also contribute to oral HPV infection. Significant associations of Alpha-HPV prevalence with lifetime ,91 and rSimilarly to Alpha-HPVs, Beta and Gamma types could also be sexually transmitted, although data in this regard are inconsistent. Analysing 21 stable heterosexual couples, Moscicki and colleagues demonstrated a concordance of 27% and 20% for Beta and Gamma HPVs, respectively, more than that observed in the non-couples  . Others (iv) Smoking. Among the behavioral factors associated with oral HPV infection, the role of smoking has been debated. In the study by Wong et al., tobacco use emerged as a predictor of Alpha-HPV infection . High-ri(v) Oral health. A large cross-sectional study, comprising more than 3000 participants that analysed four different measures of oral health has defined self-rated, poor-to-fair oral hygiene as an independent risk factor for oral HPV prevalence, irrespective of smoking and receptive oral sex intercourses . ImportaData on the natural history of Alpha-HPVs in the general population mainly derive from the HIM cohort. Over a 12-month period, Kreimer et al. observed incident oral infections in less than 5% and less than 1% of men for any HPV and HPV16, respectively, . Median Smoking appeared to be significantly associated with new oral High-risk HPV infections . ImportaA prospective population-based study performed in Hong Kong examined the persistence and clearance of Alpha, Beta and Gamma HPV oral infection among 458 individuals that were followed up for 24 months . A higheFurther longitudinal studies, especially on Beta and Gamma HPVs, are needed in order to elucidate the natural history of oral HPV infection, which is still marginally understood. Findings from prospective studies may also be key to the underestimation of the role of certain HPV types in the etiology of HNSCC.Quite a large amount of data is available regarding oral HPV infection among MSM. They typically harbor a 3 to 5-fold higher prevalence of Alpha-HPV types compared to the general population, mainly because of their sexual behavior . Alpha-HPVs are even more commonly detected in MSM with a concomitant HIV infection. It has been consolidated in the evidence that HIV-infected individuals have a higher prevalence, incidence and persistence of Alpha-HPV types both in oral and ano-genital sites ,104,105.In agreement with the findings already outlined above for the general population, Beta and Gamma HPV types are more abundant than Alpha types also in the oral cavity of HIV-infected and HIV-uninfected MSM . More spIn the following paragraphs, the most relevant predictors of oral infection among MSM and HIV-infected individuals will be reported. Regarding Alpha-HPVs, it must be noted that the majority of the studies investigated the associations for any HPV, while a few focused on High-risk types only.(i) Age. Age has been found to be associated with Alpha-HPV infection in MSM , especiaPrevalence of oral infection by Beta and Gamma HPVs in MSM also tend to increase with age . Explana(ii) Sexual behavior. Numbers of lifetime and/or recent any sex, oral sex, rimming or tongue-kissing partners all emerged as predictors of Alpha-HPV oral infection in MSM ,112,113.(iii) Smoking. A few studies evidenced a significant association between Alpha-HPV infection and smoking in MSM ,107,112 No significant association between smoking and prevalence of Beta/Gamma HPVs has been found either in HIV-infected or HIV-uninfected MSM in the OHMAR cohort .(iv) Oral health. The OHMAR study investigated the role of both self-reported and clinician-rated oral hygiene and health as possible determinants of oral HPV infection in MSM. This investigation failed to observe a significant association between these factors and prevalence of Alpha-HPVs . Althoug3 [(v) HIV status. The interplay between HIV and HPV is very complex and far from clear. A recent meta-analysis defined the interaction between HPV and HIV as synergistic. Indeed, it has been demonstrated that acquisition of ano-genital HPV infection significantly increases in HIV-infected subjects . In addi3 . However3  or Gamma3 .Notably, it has been reported that the low number of recent CD4+ T-cells is a risk factor for the acquisition of Alpha-HPVs  and signThe impact of anti-retroviral therapy (cART) on the epidemiological measures of oral HPV infection still needs to be dissected. Although it is clear that cART, especially currently available drug combinations, is effective in restoring the immunological status of HIV-infected individuals, its protective effect against oral HPV infection has not been demonstrated. A U.S. study found a similar prevalence of Alpha-HPVs in ever vs. never cART users , which wSeveral longitudinal studies investigated the natural history of Alpha-HPVs in MSM ,122,123,In terms of oral HPV clearance, some authors reported no differences between HIV-infected and HIV-uninfected MSM ,124, whiSeveral factors may affect the acquisition of oral infections by Alpha-HPVs, such as the HIV status, as already mentioned. In HIV-infected MSM, it has been observed that those with more lifetime oral sex partners show an increased incidence of any Alpha-HPVs, and the risk of acquiring High-risk HPVs is independently associated with receptive oral sex practices without a condom . InteresThe natural history of oral infections of Beta and Gamma HPVs among MSM and HIV-infected subjects has not been explored.HNSCCs encompass malignancies located in the oral cavity, the pharynx and the larynx. These cancers are mostly attributable to the use of tobacco and alcohol. However, as already highlighted, HPV plays a significant role in the development of OPSCCs, especially those arising at the tonsils and base of the tongue. HPV role in OPSCC developing at non-lymphoepithelial subsites of the oropharynx, e.g., soft palate and the oropharyngeal walls, is less certain . CompareBesides ano-genital warts, low-risk HPVs may also cause lesions at the level of the oral mucosa. For instance, HPVs 6 and 11 are the primary causative agents of the Recurrent Respiratory Papillomatosis (RRP), a chronic disease (most often with juvenile onset) characterized by recurrent exophytic papillomas in the respiratory tract . DespiteBeta and Gamma HPVs have also been detected in benign lesions of the head and neck area, such as squamous cell papillomas, with a higher prevalence in those arising in the oral cavity compared to the oropharynx . NotablyA large variety of methodological approaches have been used to investigate oral HPV infection by Alpha, Beta and Gamma types. New insights may come from the novelest technologies. The NGS approach, which has often been applied to cervical and anal samples, is very promising for HPV-DNA detection in oral-oropharyngeal samples, allowing the detection of a much larger HPV panel than traditional PCR methods. However, concordance between PCR and NGS is not absolute  and falsHPV types of Alpha, Beta and Gamma genera are frequently detected in oral samples of cancer-free individuals, but prevalence of infection largely varies according to the population characteristics, as well as the methodological procedures used for sample collection, processing and testing. Oral infection by Beta and Gamma HPVs seems to be even more common than Alpha-HPVs, although the clinical significance of the infection caused by the former genera needs to be clarified in more depth.Among the different head and neck sites that can be infected by HPV, the most relevant one from a clinical point of view is the oropharynx. Interestingly, HPV seems to have a specific tropism for the reticulated epithelium of the tonsillar crypts, where HPV-driven OPSCC mostly arises. Recent data suggest that this type of epithelium is not permissive for HPV infection, i.e., it cannot support the viral life cycle, making it difficult for the establishment of a productive infection. Compared to the squamous stratified epithelium of other head and neck sites, the reticulated epithelium of the tonsils may be more prone to transforming than productive HPV infections . A recenSeveral factors have been described as determinants of oral HPV prevalence, incidence or clearance. Relevant predictors include demographic , lifestyle (smoking), behavioral  and clinical variables (HIV status). While many of these have been extensively investigated, with either consistent or conflicting results, it is important to collect new and more solid data regarding the role of other factors, such as oral hygiene.Although major advances have been made in the understanding of oral HPV infection, it is clearly important to better elucidate its natural history. Given HPV involvement in the development of HNSCC, this knowledge is pivotal to clarify the precise steps from infection to cancer, and to face the challenges ahead in the fight against HPV-driven tumors.


Human papillomavirus (HPV) is a well‐established mucosotropic carcinogen, but its impact on urothelial neoplasm is unclear. We aimed to clarify the clinical and pathological features of HPV‐related urothelial carcinoma (UC).Tissue samples of 228 cases of UC were obtained from the bladder, upper and lower urinary tract, and metastatic sites to construct a tissue microarray. The samples were analyzed for the presence of HPV by a highly sensitive and specific mRNA in situ hybridization (RISH) technique (RNAscope) with a probe that can detect 18 varieties of high‐risk HPV. We also conducted immunohistochemistry (IHC) for a major HPV capsid antibody and DNA‐PCR.p = 0.002). In addition, HPV infection showed a significant association with tumor grade  and stage  of UC. Moreover, the metastatic rate was higher in HPV‐positive than in negative UC (OR =3.4).The HPV detection rates varied among the methods; probably due to low HPV copy numbers in UC tissues and the insufficient specificity and sensitivity of the IHC and PCR assays. The RISH method had the highest accuracy and identified HPV infection in 12 (5.2%) of the cases. The histopathological analysis of the HPV‐positive UC showed six cases of usual type UC, five cases of UC with squamous differentiation (UC_SqD), and one case of micropapillary UC. The HPV detection rate was six‐fold higher in the cases of UC_SqD than in the other variants of UC (odds ratio [OR] =8.9, These data indicate that although the incidence of HPV infection in UC is low, it is significantly associated with squamous differentiation and poor prognosis. Furthermore, our observations show that RNAscope is an ideal method for HPV detection in UC compared with the other standard approaches such as IHC and PCR assays. The etiological role of HPV infection in urothelial carcinoma is controversial. Moreover, its clinicopathological presentation and association with squamous differentiation are still undetermined. With highly specific and sensitive mRNA in situ hybridization assay, we detected HVP in a small subset of cases associated with poor prognosis and squamous differentiation. We also showed that methodologies variance was behind the discrepancy reported in previous studies. Compared with both immunohistochemical (IHC) and polymerase chain reaction (PCR) analyses, RISH is a robust method with the highest sensitivity (95%–98%) and specificity (93%–100%) in the detection of HPV infection.22.1We used our institutional pathology database to obtain the data of 228 FFPE tissue blocks from 217 patients with UC who consulted our institution between 2013 and 2019. Cases of pure squamous cell carcinoma, adenocarcinoma, or neuroendocrine tumors of the bladder were excluded. Tissue samples were obtained by means of transurethral resection of the bladder, cystectomy, or biopsy. Information on patient demographic, clinical, and pathological characteristics were collected. The selected cases were reassessed for their histological variant, grade, and stage classification in accordance with the 2016 WHO Classification.2.2First, we selected the most morphologically representative region of the tumor by examining hematoxylin–eosin‐stained slides. A tissue microarray (TMA) was constructed from the 228 FFPE tissue blocks according to the method described in our previous study.2.3Homo sapiens peptidylprolyl cis‐trans isomerase B and bacterial 4‐hydroxytetrahydrodipicolinate reductase (dapB) probes were used as positive and negative controls, respectively. Cervical squamous cell carcinoma tissue, which contained HR‐HPV, was also used as a positive control. In addition to the TMA sections, whole‐tissue sections of 40 selected cases, including the 12 positive TMA cases, were prepared and stained for RNAscope analysis using the same procedures. Slides of available preinvasive lesions from RISH‐positive cases were also evaluated for the presence of HPV infection with RNAscope®. Malignant cells with a higher intensity of punctate staining in the cytoplasm and/or nucleus compared with that of the negative control slide were regarded as positive. The RNAscope results were interpreted and classified using a 0 to 3+ scale by three of the authors  of this manuscript following the manufacturer's instructions.The HR‐HPV E6/E7 mRNA was detected using the RNAscope® technology with HPV‐HR18 probe cocktail  that was used to recognize 18 HR (high‐risk) HVP genotypes, which included the following: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. In the RNAscope® procedures, the RNAscope 2.5 HD‐BROWN manual assay kit (Advanced Cell Diagnostics) were performed according to the manufacturer's instructions. Briefly, 4‐mm thick TMA sections were air‐dried overnight, baked at 60℃ for 1 h, deparaffinized, and air‐dried again before pretreatment. The TMA sections were then boiled for 15 min in the target retrieval reagent and incubated for 30 min in RNAscope Protease Plus at 40℃ before hybridization. After pretreatments, the sections were hybridized with each probe in a HybEZ oven ) for 2 h at 40℃. The signal was visualized using the RNAscope 2.5 HD‐BROWN reagent kit. The 2.4HPV IHC detection was carried out on 5‐mm thick TMA sections using a Bond‐III automated IHC/ISH stainer . A primary polyclonal rabbit antibody against a major HPV capsid protein  was used to detect the following 11 HPV genotypes: 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58. HPV‐positive cervical carcinoma sections were used as positive controls. The presence of a cytoplasmic staining was regarded as positive for HPV. The detection intensity was interpreted and classified as 0 to 3+ by three of the authors  of this manuscript.2.5HPV genotyping was conducted by means of PCR analysis, which was performed on 55 samples, including 32 HPV‐positive and 23 randomly selected HPV‐negative samples that were confirmed by the results of either the RNAscope® technique or IHC. In one case (case 5), two FFPE samples of the same tumor taken at different stages and times were simultaneously analyzed.DNA was extracted from FFPE tissue sections of the corresponding cases and analyzed with uniplex E6/E7 PCR method following a previously described procedure.2.6t‐test was used for the comparison of means. Results were considered statistically significant if the p value was <0.05. The JMP Start Statistics version 13  was used for statistical analyses.The association between HPV infection and clinicopathological parameters was statistically assessed using the Fisher's exact test. The sample 33.1The cohort study N = 228 cases) was composed of 182 (79.8%) male and 46 (20.2%) female patients with a mean age of 74.8 years (Table 28 cases 3.2RISH with HPV‐HR18 probe cocktail detected 12/228 (5.2%) HPV‐positive cases. In the positive tissue samples, RISH examination revealed various numbers of brownish dots in the cytoplasm and nucleus of the majority of tumor cells . IHC with polyclonal antibody for HPV capsid antigen showed positive staining in 22/228 (9.6%) cases. IHC analysis revealed weak to strong cytoplasmic reaction in positive tissue samples  was also IHC‐positive. In one patient , PCR assay showed a variation of results of the same tumor (PCR‐negative in the early pT2 tumor sample and PCR‐positive in the late pT3 sample) while RISH showed a constantly positive tumor. The PCR assay did not detect HPV DNA in any of the RISH‐negative cases. RISH assay detected HPV in 9/52 (17%) of PCR‐negative/invalid cases.3.3The clinical characteristics of the 12 HPV RISH‐positive patients are summarized in Table 3.4The 12 HPV‐positive cases, which were confirmed by RISH, were high‐grade tumors. Among these, 75% (9/12) were muscle‐invasive or metastatic Table . HistopaSquamous differentiation was found in almost half (5/12) of the positive cases. The areas of squamous differentiation were predominant (>50%) in one case and focal (<50%) in the other four.Of the 12 HPV RISH‐positive cases, associated preinvasive urothelial lesions were histologically available in seven cases. In RISH, HPV was detected in 6/7 (85%) of associated preinvasive lesions. The six HPV ‐positive preinvasive lesions included five (83%) flat high‐grade lesions consistent with urothelial carcinoma in situ (UCIS) and one high‐grade noninvasive papillary urothelial carcinoma with squamous differentiation. The only negative associated preinvasive lesion was morphologically consistent with a flat lesion with atypia of unknown significance.3.5p = 0.49) and male to female ratio  . The HPV detection rate was sixfold higher in cases of UC_SqD than in usual and other histological subtypes of UC (23.8% vs. 3.5%).The proportion of UC with squamous features (squamous differentiation and divergent squamous and glandular differentiation) was significantly higher in HPV‐related UC than in HPV‐negative cases . Furthermore, the proportion of muscle‐invasive tumors (pT2 to pT4) was significantly higher in HPV‐positive than in HPV‐negative cases .Compared with HPV‐negative cases (71.8%), all HPV‐positive cases (100%) showed a high histological grade  and systemic  host factors that promote the persistence, integration, and replication of the viral material. To assess the presence of potentially associated risk factors of HPV infection in UC, patients were screened for the presence of underlying bladder condition, smoking history, or diabetes mellitus.Sobti et al.Interestingly, one patient had multiple bladder diverticula and UC with divergent squamous and glandular differentiation. In the literature, bladder neoplasm has been identified in 0.8% to 10% of patients with bladder diverticula.Analysis of HPV RISH‐positive patients showed the same demographic characteristics as HPV‐negative patients in terms of age and sex in our study. A previous study reported differences in HPV detection with age.Almost half of the positive cases in our study had squamous differentiation. The HPV detection rate was six‐fold higher in UC_SqD cases compared with the usual and other histological variant of UC. To our knowledge, this study is the first to show a significant association between HPV infection and squamous differentiation in UC. Among the known special variants of UC, UC_SqD is the most common type reported in up to 21% of invasive urothelial carcinoma.The most significant prognostic factors of UC include tumor grade, stage, and the presence of CIS.Further, in the current study, 5/6 (83%) of HPV‐positive associated preinvasive lesions consisted of UCIS which are known to be associated with an increased risk of tumor recurrence and progression.In conclusion, there were contradictory data in past reports regarding the incidence of HPV infection in UC and the association between HPV infection and clinicopathological factors. This may be due to differences in sensitivity and specificity of the detection method used. To detect low HPV copy numbers in non‐anogenital or oropharyngeal malignancies such as UC, highly specific and sensitive methods must be used, such as RNAscope®, which we used in the present study. Based on our data, HPV infection is detected in only a subset UC in association with squamous differentiation and higher histological grade and tumor stage. Our study provided new insights into the clinical and pathological presentation of HPV‐related UC and their potentially associated risk factor. However, larger case‐control and multicenter studies are needed to clarify the etiological role and carcinogenesis of HPV infection in UC.Institutional review broad approval was obtained at Wakayama Medical University Hospital, and informed consent was obtained in the form of opt‐out on the website.The authors declare no conflict of interests.FYM, IM, and SM conceived and designed the study. IM, MO, and AS collected, organized, and reported data and performed the laboratory work. MO did PCR analysis. FYM, IM, and SM analyzed and interpreted the data. FYM, IM, MO, YM, RI, YT, FK, and SM wrote or reviewed the manuscript. SM supervised the study. All authors read and approved the final manuscript.


Objective: This study aimed to determine human papillomavirus (HPV)-related awareness and willingness to receive HPV vaccination among college students, in Wenzhou, and its associated factors. Methods: A cross-sectional epidemiological study was conducted among college students in Wenzhou to investigate their knowledge, attitude, and factors affecting their willingness to receive HPV vaccination. Results: A total of 1035 questionnaires were collected, of which 1002 were valid . In total, 904 (90.2%) college students had heard of HPV, with a lower rate among males than females  and 693 (69.2%) had heard of the HPV vaccine, with a significantly lower rate among males than females . Overall awareness of HPV and HPV vaccine among males and females was moderate, with lower awareness among males. A total of 55.9% of males and 80.4% of females indicated that they would be willing to receive the HPV vaccine, a significant difference (p < 0.001). The price, safety of HPV vaccine, and lack of knowledge about HPV and HPV vaccine were the major barriers to HPV vaccination for college students. Compared to females, inadequate knowledge of HPV was the main barrier factor for HPV vaccination among male college students. Conclusions: The overall knowledge level of males is lower than that of females. For male college students, providing more knowledge about HPV infection is helpful to promote their willingness to vaccinate. It is necessary to promote HPV-related knowledge for male and female college students, respectively. Human papillomavirus (HPV) is a highly infectious virus that is transmitted primarily through sexual intercourse and is one of the most common sexually transmitted infections (STI) in the world ,2. HPV iVaccines have been one of the most effective interventions for infectious diseases . The intThere is now growing evidence that sexually transmitted infections among college students are on the rise and that the college student population is bearing a high burden of sexually transmitted infections. It is clear that they are in the high-risk age group for HPV infection ,14,15. IThe main objective of this study was therefore to understand the current state of knowledge about HPV and HPV vaccine among male and female college students and the factors that influence vaccination intentions, and to compare the differences between the genders. We hope to provide a scientific basis for policy formulation to improve HPV vaccination rates among college students.d is the permissible error and takes the value of d = 0.04 × p. p is the estimated HPV acceptance rate and based on previous studies [p was 73%, taking into account issues such as invalid questionnaires and increasing the sample size by 10%, the final sample size was at least 977. Due to the COVID-19 epidemic, face-to-face investigations were restricted. We generated a link to the online questionnaire via the survey platform , then posted the survey link and respondents completed the questionnaire online. We first randomly selected three universities within the university town and then recruited students by convenience sampling. Participants were required to fill in the questionnaire and submit it within a limited period of time. Participants in this study included both males and females. The inclusion criteria were: (1) college students; (2) Chinese citizens, and (3) Chinese reading ability. Participants were informed of the purpose and content of the survey and informed consent was obtained prior to completion. To prevent response bias, participants were guaranteed anonymity and their personal information was kept strictly confidential. In addition, participants received no compensation for their cooperation.We conducted this cross-sectional study in August 2020 in Wenzhou university town, which has the highest concentration of college students in Wenzhou, with five universities in the region. We calculated the sample size based on the following formula: studies , we deteThrough a review of relevant literature and expert discussion, we designed a questionnaire consisting of four sections, including demographic information, knowledge of HPV infection, knowledge of HPV vaccine and attitudes towards HPV vaccination and reasons for it. In order to quantify the level of knowledge of HPV-related knowledge and HPV vaccine knowledge among college students, we assigned scores to the questions on HPV infection knowledge and vaccine knowledge on the questionnaire, where each question was scored out of 3 or 5 points, with correct answers scored and incorrect answers not scored, out of a total of 55 points. In total, 16 questions were scored out of 44 points for HPV-related knowledge and 4 questions out of 11 points for HPV vaccine knowledge. Details of the questionnaire are attached.t-test and chi-square test, stratified by gender. Attitude towards HPV vaccination was used as the dependent variable, which was categorized as “yes” and “no”, and a multivariable logistic regression analysis was conducted to identify the influencing factors related to the attitude towards HPV vaccination among male and female college students. Statistical significance was assessed by two-tailed tests with α level of 0.05.Participants’ demographic characteristics, knowledge of HPV and HPV vaccine, access to knowledge, attitudes to vaccination and related factors were expressed as frequencies and percentages. HPV infection knowledge scores and HPV vaccine knowledge scores were expressed as mean ± standard deviation. Differences between groups were compared by A total of 1035 questionnaires were returned in the end, after excluding 33 invalid questionnaires due to incomplete responses, 1002 questionnaires were included in the final analysis. The demographic characteristics of the study participants are shown in p < 0.001). Among college students who had heard of HPV, both males and females mostly learned about HPV through the internet, with 69.4% and 81.6% of college students choosing this option, respectively. Among all college students, the two most desired ways for males and females to learn about HPV were the same: the internet (61.0% vs. 75.6%) and school lectures (60.7% vs. 69.3%), as shown in In the study, 90.2% and 69.2% of college students had heard of HPV and HPV vaccine respectively. The percentage of males who had heard of HPV and HPV vaccine was 85.3% and 53.7%, respectively, compared to 93.2% and 78.3% of females. The difference was statistically significant (p < 0.001). Specifically, females were more likely than males to know the transmission route of HPV, with a statistically significant difference (p = 0.001). As for other questions regarding knowledge of high-risk HPV and HPV-related disease treatment, the scores of males were lower than those of females, with statistically significant differences. In terms of HPV vaccine knowledge, only when asked: “Are you aware of the current status of the HPV vaccine for men today?”, males scored higher than females, with a statistically significant difference (p = 0.01). The rest of the HPV vaccine questions were answered better by women than men, with a statistically significant difference.p < 0.001). A multivariate logistic regression analysis of the factors associated with college students’ willingness to receive HPV vaccination are shown in In this study, we found that the knowledge of HPV and HPV vaccine among college students in Wenzhou was at a medium to low level, even though awareness of HPV and HPV vaccine was high. The overall knowledge level of males is lower than that of females. In addition, for male college students, providing more knowledge about HPV infection is helpful to promote their willingness to vaccinate. The main findings of this study may help to provide effective educational guidance and interventions for improving the vaccination rate of the target population in the future.The HPV vaccine does not prevent people who are already infected with HPV from developing cervical cancer, so the highest efficacy of the HPV vaccine is when it is administered before sexual debut. In fact, many Western countries have school-based vaccination programs for 12–13 years old boys and girls . In the Since the development of the HPV vaccine, numerous research articles have appeared in China on the knowledge, attitudes, and behavioral tendencies of college students . AccordiFrom the results of this study, the scores of college students’ knowledge of HPV and HPV vaccine were 22.73 ± 9.01 (44 points) and 5.06 ± 3.03 (11 points), respectively, which are at a low to moderate level, indicating that although the awareness rate has increased, college students’ knowledge of HPV and HPV vaccine is still low. In our study population, a large proportion of college students, both males and females, were unaware that HPV can infect men, and that more education intervention targeted at men is needed. In answering the question on HPV and diseases associated with high-risk HPV infection, males performed worse than females, although both scores were low, indicating to some extent the lack of knowledge of HPV infection among college students, especially among male students. The fact that the HPV vaccine is not approved for use in males in mainland China and that HPV infection is mainly associated with cervical cancer in females has contributed to this phenomenon. In a survey conducted in 2015, there was a need to improve the education of young males about HPV infection, its associated diseases, and the benefits of the vaccination . FurtherProportion of college students with the intention to vaccinate in several studies in China ranged from 33 to 70% ,23,24,33In exploring factors influencing attitudes towards HPV vaccination, our findings were similar to those of some other studies ,33. The Studies in several countries have shown that extending the current HPV vaccination program to the male population is a highly cost-effective preventive intervention that can better prevent diseases such as anogenital cancer, a proportion of head and neck cancer, cancer precursors, and genital warts in both men and women ,40. HighIt is necessary for government and universities to take targeted measures such as establish a national financial subsidy, increasing health education related to HPV infection in the school curriculum, and promoting knowledge about HPV and HPV vaccine through the internet and social media. Vaccine health education and the development of healthy behaviors among college students depend not only on the support of the government, medical institutions, and schools, but also on the increased awareness of college students to prevent HPV infection. Only by recognizing the seriousness and consequences of HPV infection and the effectiveness of the HPV vaccine can the overall HPV vaccination rate among college students be increased.There are several limitations to this study. Firstly, this study used self-reported data collected using a questionnaire and may be subject to self-report bias and recall bias. Secondly, due to the cross-sectional nature of the study, strong causal relationships cannot be provided and caution should be exercised in interpreting the influences. Finally, as the majority of college students in this study had medical backgrounds, the study population was limited in its representativeness.This study shows that college students of both sexes in Wenzhou have a medium level of willingness to accept HPV vaccine, a medium to low level of knowledge about HPV and HPV vaccine, and a lower overall level of knowledge among males than females. The low level of knowledge about HPV and HPV vaccine and the high price of the vaccine are the main barriers to HPV vaccination among college students. For male college students, providing more knowledge about HPV infection is helpful to promote their willingness to vaccinate. Strengthening health education on HPV-related knowledge and lowering the price of the vaccine would help promote the HPV vaccine among the college student population.


LRP1B mutation and HPV status via rewiring tumor samples of HNSCC (n=1478) and CC (n=178) from the TCGA dataset. Here, we found that LRP1B mutation was associated with HPV status in CC (P=0.040) and HNSCC (P=0.044), especially in HPV 16 integrated CC (P=0.036). Cancer survival analysis demonstrated that samples with LRP1B mutation showed poor disease outcomes in CC (P=0.013) and HNSCC (P=0.0124). In addition, the expression status of LPR1B was more favorable for prediction than TP53 or RB1 in CC and HNSCC. Mutation clustering analysis showed that samples with LRP1B mutation showed higher mutation count in CC (P=1.76e-67) and HNSCC (P<10e-10). Further analysis identified 289 co-occurrence genes in these two cancer types, which were enriched in PI3K signaling, cell division process, and chromosome segregation process, et al. The 289-co-occurrence gene signature identified a cluster of patients with a higher portion of copy number variation (CNV) lost in the genome, different tumor HPV status (P<10e-10), higher mutation count (P<10e-10), higher fraction genome altered value (P=2.078e-4), higher aneuploidy score (P=3.362e-4), and earlier started the smoking year (P=2.572e-4), which were associated with shorter overall survival (P=0.0103) in CC and HNSCC samples. Overall, LRP1B mutation was associated with tumor HPV status and was an unfavorable prognostic biomarker for CC and HNSCC.Human papillomavirus (HPV) infection and gene mutations were reputed as key factors in cervical carcinoma (CC) and head and neck squamous cell carcinoma (HNSCC). However, the associations of HPV status and gene mutations remain to be determined. This study aims to identify molecular patterns of  Human Papillomavirus (HPV) infection was reported to be responsible for almost all cervical cancer (CC), more than 90% of anal cancer, almost 70% of vaginal and vulvar cancers, about 60% of penile cancer, 5% to 30% of head and neck squamous cell carcinoma (HNSCC), and 60% to 70% of oropharynx cancer In general, almost all sexually active people will be infected with at least one type of HPV, and around half of those infections are high-risk HPV types LRP1B) is reputed as a tumor suppressor gene on chromosome 2. Gene mutations in LRP1B are frequently detected in many cancers, with 12% in non-small cell lung cancer, 11% in head and neck cancer, 9% in cervical cancer, 8% in bladder cancer, and 8% in prostate cancer LRP1B mutation was enriched in responders (34%) to immune checkpoint inhibitors (ICIs) compared with non-responders (3%)  LRP1B mutation may be an independent predictor for the response to ICI. Previous studies have led to the notion that the diverse clinical outcomes of CC and HNSCC are likely a reflection of the genetic heterogeneity of the tumor subclones from the gene expression profiles and the intratumor metabolic features LRP1B mutation was identified as a biomarker for ICIs in melanoma, non-small cell lung cancer Low-density lipoprotein receptor-related protein 1B  curve between normal and tumor, which was synchronously compared to TP53 or RB1. Furthermore, the co-occurrence mutation gene of LRP1B was performed to identify a co-occurrence mutation signature with different clinical characteristics in CC and HNSCC. These results suggested the associations of HPV status and LRP1B mutation, and the prognostic value of LRP1B in HPV-related CC and HNSCC.Here, we correlated HPV status, somatic mutations, disease outcomes, HPV expression, mutation gene expression, and clinical information of CC and HNSCC samples from TCGA to systematically unravel the HPV-associated mutation gene, that is 2.Cell resource and cell culture of SiHa and HeLa  were described previously LRP1B wild type, Hela, Caski, and Me180 were LRP1B mutation cell lines (4) were seeded in glass coverslips in 24-well plates after 24 h and were fixed in 4% paraformaldehyde (30 min) at room temperature, then penetrated in 0.3% Triton X-100 (30 min) before the blocking step  for 30 min in 37 °C) LRP1B  and Alexa Fluor 594 . Phalloidin  and DAPI  were used to stain microfilaments and the nucleus. Images were taken using a microscope  equipped with FITC and Cy3 filters. The mean intensity of staining was calculated by ImageJ (1.48v).According to the Catalogue of Somatic Mutations in Cancer (COSMIC) dataset, SiHa and MS751 were ll lines -3. Cellshttps://www.cbioportal.org/) with default settings in situ hybridization (ISH) status. Different research used different detection methods. 12.9% of samples with high-risk HPV were detected in RNA-Seq analysis LRP1B and corresponding clinical information were downloaded from the Gene Expression Omnibus (GEO), including GSE107591, GSE9750, GSE7803, and GSE63514.Integrated genomic and molecular data of 178 cervical carcinoma samples  from HNSCC cohorts LRP1B mutation status of samples was download from the dataset.According to the previous study HPV-E6 splices from RNA-seq were conducted by the previous piplines LRP1B was detected as an HPV integration hotspot in CC LRP1B integrated samples in LRP1B gene with transcription regulation elements  chromatin state discovery and characterization (ChromHMM)). Samples were downloaded from 135 samples with over 3,667 integration sites. HPV-LRP1B integrated samples were indicated by arrows. LRP1B gene and regulation elements were downloaded from the UCSC browser (http://genome.ucsc.edu/).LRP1B (n = 6), samples with HPV integration in other genes (n = 91). All integration sites and scores were annotated with default settings.IHC score of LRP1B in cervical carcinoma (n = 97) and normal cervix (n = 7) were downloaded from the published dataset  LRP1B mutation and the other group with LRP1B wild type. Overall survival analysis of HNSCC was performed by cBioPortal  via Log Rank test. Samples were clustered into one group with LRP1B mutation and the other group with LRP1B wild type. The Log Rank test gives greater weight to distant differences in outcome events, i.e., it is sensitive to distant differences, while the Wilcoxon test gives greater weight to near-term differences in outcome events.The Kaplan-Meier method was used to assess the difference in the disease survival of patients. Overall survival analysis of cervical carcinoma samples was performed by Gehan-Breslow-Wilcoxon test according to the follow-up information from TCGA cohort 1 LRP1B, TP53, and RB1 were performed by GraphPad Prism (version 6.02) from GSE107591, GSE9750, GSE7803, and GSE63514. GSE107591 was expression profiling of 23 normal and 24 HNSCC tissues. GSE9750 was 24 normal cervical epithelium and 33 primary cervical tumors. GSE7803 was 21 invasive cervical squamous cell carcinomas, 7 high grade squamous intraepithelial lesions (HSIL) and 10 normal squamous cervical epithelial samples. GSE7803 included 24 normal, 62 HSIL lesions, 40 CIN3 lesions, and 28 cancers.ROC curve analysis was employed to demonstrate the sensitivity and specificity of genes by risk score. ROC curves of LRP1B were performed by cBioPortal , the bioinformatic tool of TCGA, with default settings. Briefly, mutation genes of samples were performed in CC and HNSCC, respectively. Co-occurrence mutation genes and mutual exclusive genes were analyzed by Fisher's Exact Test LRP1B mutation or not. Description of 289 genes was showed in Co-occurrence mutation genes of https://www.cbioportal.org/) with default settings, including clinical information, tumor type, tumor HPV status, patient smoking history, mutation count, CNV status, aneuploidy score, et al. P-value <0.05 was considered statistically significant. All significant results were showed in Molecular characteristics of 289 co-occurrence mutation genes were performed in CC and HNSCC via cBioPortal  and Kyoto Encyclopedia of Genes and Genome (KEGG) pathways of 289 co-occurrence mutation genes of LRP1B mutation (n = 12) and the other group with LRP1B wild type (n = 166). Then, the top 255 mutation genes of all samples were displayed to analyze the mutation count of those two groups. Mutation count of CC , HNSCC (n = 1477) and pan-cancer studies (MSKCC) (n = 1676) were analyzed by clustering samples into LRP1B altered group and LRP1B unaltered group in cBioPortal tool .The cervical carcinoma samples of a TCGA cohort (n = 178) were downloaded P-value <0.05 was considered statistically significant.Data were analyzed by GraphPad Prism  and SPSS 25.0 . Data were expressed as the mean ± SD (standard deviation). Data were analyzed by Student's t-test (E6 counts) one-way analysis of variance (ANOVA) test (IHC score), Gehan-Breslow-Wilcoxon test  for CC), Log-rank test (OS for HNSCC), Chi-Square test  ≥5, N ≥40, Chi-square test; 1 ≤ T (minimum) ≤5, N ≥40, adjusted Chi-square test; T (minimum)<1 or N<40, Fisher's exact test), and Kruskal Wallis Test , followed by least significant difference (LSD) testing, detail information was showed in LRP1B mutation was associated with HPV-A9 integration status (P = 0.040), and PTEN mutation (P = 0.040), TSC2 mutation (P = 0.040), SMARCB1 mutation (P = 0.049), and MSH2 mutation (P = 0.049) were preferred in HPV-A9 non-integration samples.To explore the associations between gene mutation and tumor HPV status, we integrated 169 CC samples from the TCGA cohort to investigate the associations between tumor HPV status and gene mutation in CC, we then performed correlation analysis in HPV-A9 positive Figure  samples.LRP1B was found to be associated with HPV 16 integration status (P = 0.036) and TSC2 was preferred in HPV 16 non-integration group (P = 0.045). To further validate the correlation of LRP1B and TSC2 in HNSCC, we analyzed them in 1478 HNSCC samples and found that LRP1B was associated with tumor HPV status in HNSCC  infections are the most prevalent type in cervical cancer patients LRP1B mutation and E6 expression in CC  was lower than that of SiHa cells  =14, N (wild type) = 164) and identified that LRP1B mutation was associated with poor disease outcome  was associated with poor disease outcome in 1478 samples (N (wild type) = 913, P = 0.0124, Figure In CC, we performed survival analysis in 178 samples withLRP1B expression between tumor and normal in CC and HNSCC showed no statistical differences  and stable microsatellite instability (MSI) LRP1B mutation, we performed co-occurrence mutation analysis in CC and HNSCC, respectively. Several co-occurrence mutation genes were identified, such as TTN, MUC4, KMT2C, SYNE1, DMD, USH2A, ADGRV1, and SYNE2 in CC, TP53, TTN, CSMD3, MUC16, STNE1, FLG, and PCLO in HNSCC , positive regulation of phosphatidylinositol 3-kinase (PI3K) signaling (P = 9.77e-05), microtubule-based movement (P = 4.79e-05), and chromosome segregation (P = 0.0002), et al.  were co-occurred in LRP1B mutation samples of CC and HNSCC , and different CNV status in genome. CNV lost as preferred in LRP1B co-occurrence mutation signature group, especially in 3q (P = 1.07e-5), 18q (P = 1.69e-3), 13q (P = 0.0112), 9p (P = 0.0181), 5q (P = 0.0181), 8q (P = 0.0325), 21q (P = 0.0325), and 4q (P = 0.0446) , higher altered fraction genome (P = 2.572e-4), and longer smoking year (P = 2.572e-4), and higher aneuploidy score (P = 3.362e-4) were found in LRP1B co-occurrence mutation signature group  were found in LRP1B co-occurrence mutation signature group  receptor, which plays multi-roles as a tumor suppressor gene in normal cell function and development LRP1B was reported to promote cell growth and migration in colon cancer cells LRP1B mutation tended to down-regulated LRP1B expression and showed poor disease outcome in CC and HNSCC. In addition, LRP1B mutation serves as a good predictor for ICIs in multiple cancer types, largely due to the correlation with higher tumor mutation burden LRP1B mutation, as well as LRP1B co-occurrence mutation signature, were associated with higher mutation count. Higher tumor mutational burden shows distinct clinicopathologic features and is a biomarker for good efficacy and clinical response to ICIs in diverse cancers LRP1B was also detected as an HPV integration hotspot in CC LRP1B integration was associated with the downregulation of LRP1B. Additionally, the LRP1B IHC staining score could serve as a sensitive biomarker for cervical lesion detection, with 0.801 area under curve (AUC), 90% sensitivity, and 56.76% specificity  LRP1B was identified as a biomarker for normal and neoplasm samples of CC and HNSCC, with AUC 0.8007 in HNSCC and 0.9024 in CC, which suggested that LRP1B could serve as a biomarker in CC and HNSCC. Furthermore, LRP1B co-occurrence mutation signature tended to identify a cluster sample with multiple molecular bases of CC and HNSCC. The identification of molecular patterns or biomarkers of CC and HNSCC that could not only predict the tumor biology but help to further classify HPV-related cancers.LRP1B mutation characteristics. Firstly, the correlation between LRP1B mutation and tumor HPV status was lack of molecular evidence and underlying mechanisms in the interaction process. Secondly, the biomarker role of LRP1B in ICIs was showed in other cancer types, there was no ICIs treatment cohort of CC or HNSCC included for clinical analysis, making the biomarker role of LRP1B only focus on cancer survival, mutation count, AUC analysis, and molecular bases of CC and HNSCC. The only way to demonstrate the molecular function of LRP1B was cell biology tests, which need to be further explored in HPV-related CC and HNSCC.Still, with multi-molecular analyses in CC and HNSCC, our studies had several limitations in LRP1B mutation, as well as co-occurrence mutation signature of LRP1B, was associated with tumor HPV status, higher mutation count, and poor cancer survival in CC and HNSCC. Also, LRP1B was showed as a good biomarker between normal and neoplasm samples than TP53, and RB1 in CC and HNSCC. These results provided molecular insight into the correlation between HPV status and LRP1B mutation, and the prognostic value of LRP1B in HPV-related CC and HNSCC.Overall, by integrating molecular data of CC and HNSCC, including gene mutation, tumor HPV status, gene expression, and clinical information, we identified Supplementary figures.Click here for additional data file.Supplementary tables.Click here for additional data file.


E6/E7 polymerase-chain-reaction method to detect all high-risk and potentially high-risk HPV types. The HPV typing results were confirmed in 52 of 73 cancer cases; among the 21 remaining cases, 15 were discordant and 6 were partially concordant. In total, 8 of 16 (50%) HPV-negative samples became positive; 6 were positive for HPV16 and 2 were positive for HPV67. Moreover, two samples previously positive for HPV6 and HPV53 were negative for HPV. All nine cancers with multiple HPV types were found to harbor only a single HPV type. In total, 63 cancer tissues exhibited a single HPV type. HPV16 and HPV18 were detected in squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Alpha-5 (HPV82), -6 (HPV56), and -9 (HPV31/52/67) HPV types were detected in SCC, whereas Alpha-7 (HPV59/68) types were detected in ADC and adenosquamous carcinoma (ADSCC). These findings suggested that the different HPV types induced different histological cancers. Furthermore, all SCCs and 10 of 11 usual-type ADCs were positive for high-risk HPV types, supporting the use of HPV screening for the detection of these cancers and associated premalignant lesions. HPV16 is likely to remain undetected in some cervical cancer tissues because of low viral-copy-numbers. Putative high-risk HPV types  might be high risk in Japan.In our previous study, an L1-based human papillomavirus (HPV) test using liquid-based cytology revealed that some invasive cervical cancers (ICC) exhibited multiple HPV types or harbored no HPV DNA. Here, molecular mapping of formalin-fixed paraffin-embedded cancer tissue specimens from the same patients were conducted to confirm these observations. Among 377 ICC cases, 73 eligible specimens  were reexamined by manual microdissection of cancer lesions, then subjected to HPV genotyping using the uniplex  Cervical cancer is the fourth most common malignancy in women, with 570,000 new cases reported in 2018  . We estiAlthough the Pap test contributed to decreases in the incidence and mortality of cervical cancer up to 1990, its low sensitivity for premalignant and malignant lesions in young women is widely recognized . This pret al. , and incubated in a thermomixer . Equal amounts of neutralization reagent [0.04 mM Tris-HCL (pH 5.0)] were then added, and the samples were vortexed for 30 s. The samples were then centrifuged at 13,523E6/E7 PCR, which can detect 100 copies of DNA from each HPV type [E6 or E7 genes of the 13 high-risk types and 11 possible high-risk types, with β-globin used as an internal control gene. The PCR method was described in a previous study [E6 or E7 gene of 24 HPV types, including 11 possible high-risk HPV types (HPV26/30/34/53/66/67/69/70/73/82/85) and 13 high-risk HPV types (HPV16/18/31/33/35/39/45/51/52/56/58/59/68). HPV16 E6 gene primers were confirmed to have no mismatched sequences with HPV16 E6 variants, except for some African variants .HPV genotyping was performed using uniplex HPV type . HPV DNAus study . BrieflyE6/E7 PCR in some cancer cases showing discrepant results from previous typing and determined using L1-based PCR. Amplified E6 DNA was purified with the MinElute PCR purification kit  and examined for the sequence using a 3500/3500xL system  in both directions using forward and the reverse primers.We verified HPV-typing results obtained via uniplex E6 DNA was undetected in many discordant cases, we applied the short-fragment E6 PCR method to target HPV16 and HPV18 using the following primers:Because TGACTTTGCTTTTCGGGATTs16E6-F-primer, CAACGGTTTGTTGTATTGCTGs16E6R-primer, GGTGCCAGAAACCGTTGAATCCs18E6-F-primer, TACTTGTGTTTCTCTGCGTCGTTGs18E6-R-primer, E1 and E2 genes of HPV16, HPV18, and HPV67 using HPV-type-specific PCR. Fragmented E1 and E2 genes across the HPV16 and HPV18 genomes were amplified as previously reported [E1 and E2 genes for HPV67 were developed as follows.To assess the integration status of HPV genes in some ICC samples, we determined the presence of the reported . The priHPV E1 and E2 primers for HPV67:AGGACCCGGAAGGTACAGAC67E1a-F, CTCTACCTGCACCATTGTCTCTTG67E1a-R, TCAAGAGACAATGGTGCAGGTAGA67E1b-F, GTGTGTACAACGTTTGTGGCTTGA67E1b-R, AATAACACCATCAGTAGCAG67E1c-F, CAAAACTATGTTGCAGTACC67E1c-R, GGATAGAAAGACTAACGGTACTG67E1d-F, GTGGTCCGCATATTACCATAC67E1d-R, GGACCACCAAACACAGGGAA67E1e1-F, CTAATTGGCACCACGTCCTTGA67E1e1-R, GGACCACCAAACACAGGGAA67E1e2-F, AAAGCACATTCAATGCGTCTCA67E1e2-R, GAGGACAAGGAAAACCATGGAG67E2a-F, TCCCATATAGTCCACCTTTCCC67E2a-R, GGGAAAGGTGGACTATATGGGA67E2b-F, GCACTATAGGTGCAGTGTTAGG67E2b-R, CCAGACTTCTGCAACAACTC67E2c-F, TTAGCAGCACATATTGGCTT67E2c-R, β-globin was used as an internal control for DNA quality assessment but was also not detected in ~50% of cases, suggesting that DNA samples extracted from FFPE samples were likely fragmented. Therefore, we examined shorter fragments of L1 and E2 genes for HPV16, HPV18, and HPV67 using the following primers.Although we did this experiment, most of these genes were undetected in ICC cases showing discrepant results. Short HPV-L1 fragment primers for HPV16, HPV18, and HPV67:GATACTACACGCAGTACAAATATGTCs16L1-F, CCATGTCGTAGGTACTCCTTAAAGs16L1-R, CGCAGTACCAATTTAACAATATGTGCs18L1-F, CAAATCATATTCCTCAACATGTCTGCs18L1-R, ACCCTAGCCAGCCTGGTACTs67L1-F, AAAATCCATGCAGCCAAAACs67L1-R, Short HPV-E2 (sE2) primers for HPV16, HPV18, and HPV67:ATGCGGGTGGTCAGGTAATAs16E2-F, TCGCTGGATAGTCGTCTGTGs16E2-R, GAAGGAAACCCTTTCGGAACs18E2-F, GTATGCCATGTTCCCTTGCTs18E2 -R, GCACAAACAAAGGACGGAGTs67E2-F, GGGGGTATGCGTACAGAGTTs67E2-R, L1, E2, and E6 genes for HPV16, HPV18, and HPV67 was performed similarly to uniplex E6/E7 PCR using the following program: 10 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C to 60°C, and 30 s at 72°C, with a final 5-min extension step.PCR for the short fragments of the L1 gene (Genosearch-31) were used in the previous study. In the present study, areas of carcinoma in FFPE tissue specimens from the same patients were excised using a manual microdissection procedure; HPV genotype was then determined using the E6/E7-PCR method.Among the 377 ICC cases, 73 underwent reevaluation of HPV genotype by molecular mapping of FFPE tissue specimens . Among tComparison of the HPV genotyping results showed that 52 cases (71.2%) had concordant results for HPV type, whereas the other 21 cases (28.8%) were not in agreement. Among the discordant cases, 6 (28.6%) exhibited partial concordance because one of the previously reported multiple HPV types was detected in the present analysis . AdditioL1 gene in cervical LBC samples. Notably, all HPV-positive cancer cases harbored a single HPV type; all were high-risk or possible high-risk HPV types.Among the 15 cases that exhibited discordance, 9 (60.0%) were positive for HPV16 in this study, while 3 (20.0%) were positive for HPV67, 1 (6.7%) was positive for HPV18, and 2 had no HPV. HPV16 and HPV67 were the two most common HPV types missed by the previously employed HPV test targeting the HPV Among six cases that exhibited partial discordance (multiple to single) , one casE6 genes for HPV16, HPV18, and HPV67 in these samples; however, the E6 genes for these HPV types generally were not amplified in the second round of experiments using uniplex E6/E7 PCR .We identified a single HPV type in 19 cases that exhibited discordant and partially concordant results for HPV type . To veri6/E7 PCR . AdditioE1 and E2 genes in 13 discordant cases and in 5 control cases with concordant results (3 HPV16-positive and 2 HPV18-positive cases)  for HPV16, HPV18, and HPV67 using type-specific PCR. The L1 genes of the HPV types were detected in all 13 cases, whereas E2 was detected in 12 of the 13 cases; there was no band in 1 HPV18-positive case (#31).To determine the status of HPV gene integration into the host genome, we investigated the preservation statuses of the e cases) . All or controls . The onlet al. [et al. [E6/E7 PCR were effective and accurate for determining HPV in cervical and vaginal tissue specimens [The aim of this study was to investigate oncogenic HPV types in Japanese cervical cancer patients. Previous studies reported that 93–99.7% of ICC cases were positive for HPV , 26. Furet al. , 30 dete [et al.  reportedpecimens , as wellpecimens .et al. [In the present study, all 45 SCC cases, including 9 previously shown to harbor multiple HPV types , were poet al.  indicateet al. [Henceforth, the primary HPV test will be incorporated into cervical cancer screening programs worldwide; it is more sensitive than the Pap test and shows similar specificity. However, there are some limitations to the HPV test because HPV-negative cancer exists in some geographical areas. Furthermore, possible high-risk HPV types such as HPV67 and HPV82, identified as a single type in some ICC tissues, cannot be detected using current commercial HPV tests . The Intet al.  reportedet al. . In Japaet al. , 40. Excet al. , which met al. , 42. Howet al. [Arryo et al.  reportedet al. . In the et al. .L1-based PCR test, whereas the present study used FFPE specimens and an E6/E7-based PCR test. To elucidate the reason for the discrepant results, we determined the status of HPV gene integration into the host genome according to the presence or absence of the E1 and E2 genes, considering that these genes are frequently deleted in some cervical cancers (a marker of HPV gene integration) [E1 and E2 were undetected in 12 of 13 discordant cases, as well as 3 of 5 controls. Moreover, these genes were preserved in two of three controls with HPV16, but not in either of the two controls with HPV18. Further analysis revealed the shorter L1, E2, and E6 genes in all cases, except for E2 in an HPV18 ADSCC case and E6 in two NEC cases. HPV gene integration is unlikely to be associated with the discrepant results observed in the present study, considering that the L1 gene  was identified in all discordant cases. HPV gene integration might be responsible for the maintenance of low copy numbers of the HPV genome in cancer tissue [E6 gene during the second round of testing.In the present study, a single HPV type was identified in 13 of 21 cases with results that differed from findings in a previous study. For HPV genotyping, the previous study used LBC samples and an gration) . The prer tissue . AnotherE6 genes for each HPV type in the FFPE tissue specimens. Unexpectedly, the E6 gene, which was positive in the first round test, could not be amplified in most cases with discrepant HPV results from the findings according to the L1-based HPV test. The reason for this phenomenon is unclear. However, shorter E2 and E6 genes (100–200 bp) were amplified—and the sequences were confirmed—in almost all of these samples; the detection limit of target genes in FFPE was shorter than we had expected. In a previous study, it was stated that, “while FFPE blocks stored for years have been used successfully for DNA analysis, it is important to note that the length of amplifiable gene fragments may decrease over time” [E6 gene may occur because of DNA fragmentation during the interval between the first- and second-round tests.We verified the sequences of er time” . TherefoThe present study had some limitations. First, we used a small number of samples, which included a high proportion of HPV-negative samples and a high proportion of samples with multiple types of HPV. A larger sample size more representative of the general population will be needed to confirm the present findings. Second, the inclusion of poor-quality DNA in some FFPE samples may have skewed the results; it also precluded the derivation of clear conclusions regarding the HPV gene integration status in some cases.In some studies, HPV16/18- and HPV31/45-positive women screened for cervical cancer appeared to be at higher risk of high-grade cervical lesions . TherefoE6/E7-based HPV PCR represents an improved approach for detection of oncogenic HPV types in cancer. The behaviors of specific HPV types have been clarified in this study: HPV16 and 18 are likely to induce both SCC and ADC, while HPV types belonging to the Alpha-5 (HPV82), -6 (HPV56), and -9 (HPV31/52/67) groups predominantly induce SCC. Finally, Alpha-7 HPV types (HPV59/68) induce ADC or ADSCC. Additionally, we found that all SCCs and usual-type ADCs were positive for high-risk HPV types, suggesting that HPV screening is a useful approach for the detection of these cancers and associated premalignant lesions. Moreover, some ICCs were induced by possible high-risk types, such as HPV67 and HPV82, suggesting that the choice of HPV test is crucial for accurate cervical cancer screening; current commercial HPV tests might be limited in this regard. Accurate HPV typing based on tissue samples may gain greater clinical importance in the future.The combination of cancer microdissection and application of