As part of the human papillomavirus (HPV) vaccination strategy in South Africa, it is essential to have information on HPV prevalence, and HPV types distribution among the unvaccinated population. Information on the prevalence of HPV and the distribution of HPV types in adolescents and young women in South Africa’s Eastern Cape Province is minimal. Therefore, this study investigates the prevalence, distribution of HPV types, and factors associated with HPV infection amongst unvaccinated female learners. A sample composed of 213 sexually active female learners attending high schools in the Eastern Cape Province of South Africa; median age 18 years, who provided self-collected vaginal specimens. Roche Linear Array HPV genotyping assay that detects 37 HPV genotypes was used to detect HPV infection. HPV infection was detected in 76.06% (162/213) of participants. Of these 14.55% (31/213) were positive for HPV types targeted by the Cervarix® HPV vaccine (HPV-16 and/or 18), 20.66% (44/213) by Gardasil®4  and 37.09% (79/213) by Gardasil®9 . HPV-35, commonly detected in cervical cancer cases among women of African ancestry, was frequently detected (9.40%). Participants who reported to have ever consumed alcohol had a significantly higher risk of HPV infection . High HPV prevalence was observed among participants. The high prevalence of HPV types targeted by the Gardasil®9 vaccine encourages the introduction of the Gardasil®9 vaccine. Data from this study will inform both vaccination campaigns and monitor the impact on HPV types after vaccination. Sexually transmitted infection (STI) prevalence is very high in developing countries where routine STI screening and treatment have inadequate coverage. Self-sampling for STIs, including human papillomavirus (HPV) testing, is a promising method to increase the STI screening coverage ,2. Self-HPV is the most common sexually transmitted virus –11, withCervical cancer is the second most common cancer in South African women. The National Cancer Registry (NCR) of South Africa reported an age-standardized rate (ASR) of 22.56 per 100,000 for all South African women in 2014 and ASR of 27.01 per 100,000 for black African women . UnfortuCurrently, there are three HPV vaccines approved by the U.S Food and Drug Administration. They are Cervarix® (GlaxoSmithKline), Gardasil®4 (Merck Inc), and Gardasil®9 (Merck Inc). They target a different combination of HPV types; Cervarix® targets HPV-16/18, the most carcinogenic HPV types associated with approximately 70% cervical cancer cases; Gardasil®4 targets HPV-16/18 as well as two low-risk (LR) types, HPV-6/11, associated with genital warts and recurrent respiratory papillomatosis; and Gardasil®9 (Merck Inc) targets same types as Gardasil®4 and the other five high-risk (HR) HPV types HPV-31/33/45/52/58 ,30–32. AMany countries have implemented HPV vaccination programs since the first licensure of Gardasil®4 in 2006 ,32. In SThis study was approved by the University of Cape Town Human Research Ethics Committee (HREC: 369/2015). Permission to conduct research in the Eastern Cape was granted by the Eastern Cape Provincial Health Research Committee (EC_2016RP29_562). Both the Provincial Department of Health and Education in the Eastern Cape granted permission to investigators to conduct this study. Participation in the study was voluntary, with written informed consent and parental assent obtained for participants younger than 18 years.Participants of this study were recruited from grade 8 to 12 learners who participated in the HPV education intervention study conducted between April and May 2019 in two high schools situated in Chris Hani District Municipality of the Eastern Cape Province, South Africa. The participating schools belong to quintile one (no-fee paying schools) South African Department of education quintile ranking. The high schools were randomly selected. After HPV education intervention, females  were invited to the nearest primary care facilities where the study was conducted. Participants received information on study procedures, objectives, and other important information. After which, they responded to closed-ended questionnaires enquiring about their demographics, sexual practices, contraceptive use, smoking habits, and alcohol consumption. Questionnaires were self-administered while the researcher was reading out questions and explaining where necessary. Pre and post-HIV counseling, rapid HIV tests were conducted by a qualified clinic staff member or HIV lay counselor. All participants with a positive HIV test had necessary follow-up according to the Health Department’s protocol guidelines.The study reports on results only for those participants who were sexually experienced. A total of 257 female learners responded to the invitation, of whom 221 were sexually experienced, while 36 were not . Self-coThe white brush part was detached from the Evalyn® Brush device and placed in sterile 2ml cryo-tubes. One milliliter of Digene specimen transport medium  was added and vortexed three times, 10 minutes apart, to detach the cells from the white brush. A total of 400μl were used for nucleic acid extraction that was conducted using an automated procedure of MagNA Pure Compact  and MagNA Pure Compact Nucleic Acid Isolation Kit .Roche Linear Array HPV Genotyping Test  was used to detect HPV genotypes in extracted nucleic acid from vaginal specimens and manufacturer instructions were followed. The Linear Array HPV Genotyping Test amplifies the target HPV DNA for 37 anogenital HPV genotypes and include 24 low risk (LR) HPV types  and 13 high risk (HR)-HPV types . The Roche Linear Array HPV Genotyping Test also amplifies β-globin gene to monitor sample adequacy, extraction, amplification and hybridization.All variables were captured and coded in Microsoft excel 2013 and exported to Stata 14.1 for analysis. Participants were counted more than once when determining the prevalence of LR-HPV, HR-HPV, and probable HR-HPV if they have types that belong to more than one category. Multiple HPV infections were defined as the detection of two or more HPV types in the same sample. Numerical variables were explored using the Shapiro Wilk test, histogram, and/or box-and-whisker plot. The median and interquartile range (IQR) are used to summarise age in years since they were not normally distributed. Age was later categorised into three categories. The Wilcoxon rank-sum test was used to compare the median ages of males and females. Categorical variables are presented using frequency tables, percentages, and graphs. The two-sample test of proportions was performed to compare demographic characteristics of males and females, HPV status by the study site, and the age gap in years of current and/or previous sexual partner(s). Logistics regression was used to determine the bivariate association of HPV and the other variables to determine the Odds Ratio (OR). The unadjusted model (bivariate associations) is presented. The 95% Confidence Interval (CI) was used to estimate the precision of estimates. The level of significance was set at 5%  for statistical significance.A total of 220 sexually experienced females provided self-collected vaginal specimens; however, 3.18% (7/220) were β-globin negative and excluded from the analysis. Participants with valid (positive β-globin) vaginal specimens had a median age of 18 years (IQR: 17–19 years). A proportion of 32.39% study participants reported a sexual debut of age 16 years, 53.99% had 2–3 current sexual partners, 35.68% used condoms during last sexual intercourse, 79.44% had vaginal sex in the past month, 20.66% had anal sex in the past month, 16.43% had oral sex in the past month, 62.91% were on contraceptives, 22.07% had been pregnant, 65.73% had experienced vaginal discharge, and 21.13% had experienced genital warts/blisters/ulcers .HPV infection was detected in 76.06% (162/213) high school female learners . HPV preIn Female learners who reported having ever consumed alcohol had a significantly higher risk of HPV infection . Participants who reported ≥2 number of lifetime sexual partners  sexual partners past three months  had a higher risk of HPV infection but not statistically significant. Age, sexual debut age, and school grade were not found to be associated with HPV infection among female learners .According to our knowledge, this is the first peer-reviewed report on HPV prevalence and genotype distribution among female learners attending high schools in the rural areas of Eastern Cape Province of South Africa. High overall HPV prevalence (76.06%) and infection with 3–10 different HPV types (42.72%) are of public concern in this population. Among those that tested for HIV infection, the HPV prevalence was still found to be high among the HIV-negative group (74.10%). Giuliano et al. report similar HPV prevalence (71.4%) among HIV-negative women between the age of 16 and 24 years residing in the Western Cape Province of South Africa. The 94% of the Giuliano et al. study participants were of black ethnicity . The obsGirls who were vaccinated in 2014 when South Africa initially introduced the HPV school-based vaccination program were 14 years of age in 2019 when this current study was conducted. Therefore, the probability that some of the study participants would have received the HPV vaccination a few years before is low as the current study participants were between the age of 15 and 22 years. None of the study participants reported having received HPV vaccination at primary school. The high prevalence of HPV types targeted by Cervarix® HPV vaccine  and Gardasil®9  was similar to the one previously reported among HIV-negative Western Cape (Cape Town) and Gauteng (Soweto) adolescents and young women (18.6% and 38.5% respectively) , was comIt is not clear why the overall HPV prevalence was found to differ between school-clinic-1 and school-clinic-2 as the participants’ demographic data was not found to differ; both the school-clinic sites are located in rural areas far from town. Participants who reported having ever consumed alcohol had a higher risk of HPV infection, which could indicate high-risk sexual behaviour . HoweverIt is acknowledged that the study population was from two communities and does not represent the population of Eastern Cape Province, and cannot be generalised. Despite these limitations, the information reported remains essential for this province and South Africa as there is currently limited HPV information on this population. There are few chances that the participants could have received the HPV vaccine during the national school HPV vaccination program or elsewhere. However, the possibility of receiving the HPV vaccine is not completely ruled out because the vaccines were available in the country.High HPV prevalence was observed among high school female learners. The high prevalence of HPV types targeted by the Gardasil®9 vaccine encourages the introduction of the Gardasil®9 vaccine. It is envisaged that these results will contribute to HPV baseline data among adolescents and young women of Eastern Cape Province and will be utilized to evaluate the impact of HPV vaccination. Research or surveillance projects to monitor HPV prevalence and distribution among HPV vaccinated and unvaccinated adolescents and young women are necessary to monitor the impact of HPV vaccination in South Africa.S1 Data(XLSX)Click here for additional data file.


Conclusion as published.In the original article, there was an error in TTN c.3092C greater than G (p.Leu6494Arg)” should be changed to “TTN c.19481T greater than G (p.Leu6494Arg)”. The corrected section appears below.“TTN c.19481T greater than G (p.Leu6494Arg) and TRAPPC11 c.3092C greater than G (p.Pro1031Arg) were observed in a LGMD family and co-segregated with the disease phenotype, which may be responsible for the LGMD phenotype. However, our study cannot exclude the missed inspection such as complex rearrangement, gross deletion and gross duplication, as well as deep pathogenic point variants in introns involved in monogenic LGMD, presenting autosomal dominant or pseudo-dominant phenomenon. Our study provides a possibility of a digenic mechanism in unsolved families with muscular dystrophies.In summary, digenic variants TTN c.3092C greater than G (p.Leu6494Arg)” should be changed to “TTN c.19481T greater than G (p.Leu6494Arg)”. The corrected caption appears below.In addition, there was a mistake in the caption for Figure 1 as published. Within the description for Figure 1B, “(A) Pedigree of a Han Chinese three-generation family with LGMD. Arrow symbolizes proband; N, normal allele; V1, TTN c.19481T greater than G variant; V2, TRAPPC11 c.3092C greater than G variant. (B) The sequencing diagram of heterozygous TTN c.19481T greater than G (p.Leu6494Arg) variant. (C) The sequencing diagram of heterozygous TRAPPC11 c.3092C greater than G (p.Pro1031Arg) variant. (D) Sequence of normal control in the TTN gene. (E) Sequence of normal control in the TRAPPC11 gene. LGMD, limb-girdle muscular dystrophies; TTN, the titin gene; TRAPPC11, the trafficking protein particle complex 11 gene.. Pedigree and genetic data of the individuals in this study. The authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated.


Due to its higher sensitivity than cytology to identify precursor lesions of cervical cancer, detection of high-risk human papillomavirus (HR-HPV) DNA has been implemented as the primary screening approach. However, a high referral rate for colposcopy after HR-HPV DNA detection due to its low specificity in HR-HPV screening often leads to overtreatment and thus increases the healthcare burden. Emerging evidence has demonstrated that detection of host cell gene and/or HPV DNA methylation represents a promising approach for the early triage of cervical cancer in HR-HPV-positive women owing to its convenience and comparable performance to cytology, particularly in LMICs with limited healthcare resources. While numerous potential markers involving DNA methylation of host cell genes and the HPV genome have been identified thus far, it is crucial to define which genes or panels involving host and/or HPV are feasible and appropriate for large-scale screening and triage. An ideal approach for screening and triage of CIN/ICC requires high sensitivity and adequate specificity and is suitable for self-sampling and inexpensive to allow population-based screening, particularly in LMICs. In this review, we summarize the markers of host cell gene/HR-HPV DNA methylation and discuss their triage performance and feasibility for high-grade precancerous cervical intraepithelial neoplasia or worse (CIN2+ and CIN3+) in HR-HPV-positive women.Cervical cancer is the most prevalent gynecologic malignancy, especially in women of low- and middle-income countries (LMICs). With a better understanding of the etiology and pathogenesis of cervical cancer, it has been well accepted that this type of cancer can be prevented and treated  Invasive cervical cancer (ICC) represents the fourth most prevalent gynecologic cancer with an estimated 604,000 new cases, which causes approximately 342,000 deaths worldwide in 2020, especially in low- and middle-income countries (LMICs) . To achiMany studies have revealed that DNA screening of high-risk human papillomavirus (HR-HPV) is superior to cervical cytology in the detection of CIN2 or worse (CIN2+) lesions –11, whicDuring persistent HR-HPV infection and cervical oncogenesis, aberrant DNA methylation of human host cell genes or HPV genomic DNA is tightly associated with dysregulated functions of various tumor-suppressor genes –18. MoreE1, E2, and E4–7), which facilitate the expression and replication of the viral genome, and two late genes (L1 and L2) for capsid formation  that could spontaneously regress in 1–2 years, likely due to viral clearance. In contrast, transforming infections are associated with some CIN2 and most CIN3 lesions that are highly heterogeneous in regard of lesion regression versus oncogenesis, with a varied expression of E6 and E7 as well as patient survival , the Tumor Cervical Squamous Cell Carcinoma - TCGA dataset (TCGA ID: CESC) on the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl), and the Cervical Cancer dataset on the Human Protein Atlas platform (www.proteinatlas.org), respectively. Of note, mutations of these DNA methylation-modifying genes are relatively rare (DNMT1 (5%), DNMT3A (2.7%), DNMT3B (4%), TET1 (4%), TET2 (2%), and TET3 (2.4%); however, DNMT3A and TET2 are relatively high at the protein level but vary with different ages, while the levels of DNMT1, DNMT3B, and TET3 are moderate or relatively low  or negatively , with the overall survival of ICC patients  before progression to ICC. Therefore, the consequences of HR-HPV infection vary upon how cervical epithelial cells are exposed to HR-HPV, including transient, productive, and transforming infections . Transiesurvival . During 6 and E7 –27. Moreinvolved –30. For involved , 17. How lesions . E6 and ing TP53 , while Ees DNMT1 . Overexp and pRb . It also and pRb , 34–36.  and pRb , 38. In  and pRb . MechaniIn addition to host cell DNA methylation, HPV DNA can also be methylated during malignant transformation. Viral genome methylation has either negative or positive effects on viral gene transcription. The HPV genome has highly condensed and conserved CpG sites . AlterinAn ideal biomarker involving DNA methylation is able to distinguish the majority (>90%) of cancer cases from non-malignant ones . A greatCADM1  is a tumor-suppressor gene that encodes an intracellular adhesion protein of the immunoglobulin superfamily  and adenocarcinoma (AdCA), as well as their precancerous lesions  and carcinomas (143.5- to 454.9-fold) than those in CIN1 or normal cervical tissues; their methylation levels further increase (11.5- and 13.6-fold) in CIN2/3 with long-term HR-HPV infection (>5 years)  . On the ly CIN3+ . TherefoHsa-miR-124, a well-recognized epigenetic silencer, has been observed in many types of cancer, including lung, breast, hepatocellular, gastric, and colon cancer, as well as leukemia  and higher specificity (62.1% versus 47.6%) when compared with cytology. However, in women with age <30 years, its sensitivity is lower (50% versus 86.7%) than cytology, while the specificity is significantly higher (81.7% versus 52.4%). One explanation for such inconsistency between women with different ages is that younger women are more likely to have early CIN lesions and relatively short-term HR-HPV infection  and moderate specificity (49.4% and 46.3%) in women with either CIN3+ colposcopy results or age >30 years, better than either methylation test or HPV genotyping alone  displays significantly different methylation patterns between CIN2/3 lesions and normal cervical samples. The combination panels of C13ORF18/JAM3/ANKRD18CP exhibited comparable sensitivity and specificity to cytology for CIN2+ in HR-HPV-positive scrapings  for diagnosing CIN3+ have been evaluated in women who underwent colposcopy. While all three methylation panels  exhibited high NPVs with sensitivities in a range of 68% to 95%, the SOX1/ZSCAN1 panel has higher specificity (84%) than the other two  have excellent triage performance for women with cervical abnormal cytology (m) alone has also shown a triage performance comparable to cytology and better accuracy, with higher specificity than HPV16/18 as the triage tool for CIN3+ in HR-HPV-positive women  in a community-based study involving 2,609 women, which has thus been considered to have the screening performance superior to HR-HPV detection or abnormal cytology. The triage capacity of gene methylation  for CIN3+ is comparable between African and non-African populations, suggesting potential use of these gene methylations in different populations , a methylation-specific real-time PCR assay targeting ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671, could identify all cancer cases with an overall sensitivity of 67.7% for detecting CIN3+, particularly in women with age ≥30 years probably due to their long-term HR-HPV infection  have been observed in a subset of ICC samples but not in control samples  is significantly increased in CIN3+ but not lesions with grade <CIN2. Among them, methylation of seven genes  has high accuracy for detecting CIN3+, despite the differences in methodology  and HPV16 L1/L2 correlates to CIN lesion, with good performance in the triage of advanced CIN  is associated with 3q gain as well as the severity of CIN  could also be methylated in SCC and adenocarcinoma , while other types include adenosquamous carcinoma and several rare ones . In thishan AdCA , while mfrom SCC . Methyla lesions , 76. Otharcinoma . Howeverstotypes .Together, methylation of numerous host cell genes, either alone or in combination, have been considered powerful biomarkers to predict the progression of cervical cancer via the triage of HR-HPV positive women.L1, L2, E2-E4, E5, and URR has been well established. Particularly, a positive correlation between the level of L1 methylation and CIN2+ lesions appears to be consistent among multiple studies  compared to hypomethylation. Moreover, an increase in their methylation levels is associated with high-grade squamous intraepithelial lesion (HSIL) and the risk of advancing to CIN2+ in HPV16-positive women (L1 and L2 is particularly higher in pre-diagnostic CIN2+ samples (median time to diagnosis = 3 years) than control samples (L1 CpG 6457 methylation to detect CIN2+ (sensitivity of 91.1% and specificity of 60.2%)   is compaf 60.2%) . Howeverlication , 122. The option . Combinive women . MoreoveL1, L2, and E2 genes between CIN and ICC or CIN3 and SCC, while the methylation levels of URR, E1, E6, and E7 are considerably low. Methylation of HPV16/18/51 CpG sites is significantly higher in L1 than in URR for all three HPV forms, while the degree of methylation positively correlates with the severity of cervical neoplasia  and for CIN3+ . As consequence, the S5 classifier could reduce colposcopy referrals by 30%–50% without affecting sensitivity for CIN2+ and CIN3+, therefore significantly improving cost-effectiveness to allow identification of women with a true risk of ICC (A test (named S4 classifier) combining the host gene cytology . Then, tcytology . Due to cytology . Furthercytology . These ok of ICC .Together, the assays for host cell gene and HR-HPV DNA methylation alone and particularly in combination represent a promising triage approach with high sensitivity, specificity, and PPV for screening premalignant cervical diseases in HR-HPV-positive women. Although most evidence has been obtained from cross-sectional or retrospective studies with a limited sample size thus far, such an approach warrants further investigation in prospective screening and intervention studies, especially to optimize host cell gene and HR-HPV DNA methylation markers.CIN2 is an equivocal diagnosis and thus considered as the least reproducible histopathologic type , 127, whANKRD18CP, C13ORF18, EPB41L3, JAM3, SOX1, ZSCAN1, GHSR, SST, ZIC1, FAM19A4, PHACTR3, and PRDM14 genes). A study has revealed that three-quarters of CIN3 samples and half of CIN2 samples display a cancer-like hypermethylation pattern, suggesting a high risk of progression to ICC  has shown that the percentage of methylation reference is significantly higher in CIN3+ than those for the normal cervix and CIN1 or CIN2  were used to detect CIN3+  to cancer precursors (transforming infections) as well as some early-transforming infections with insignificant risk of short-term progression to ICC , 136. Whn to ICC . Another or CIN2 . Notablyct CIN3+ . These oCADM1/MAL methylation are often older than those with methylation-negative CIN2+, in association with the fact that CIN2+ lesions in young women could spontaneously regress and thus belong to early CIN lesion , there are approximately 30% of women who have not participated in annual cervical screening programs, while these women may however face a high risk of development to CIN and ICC –142. Mor history . This pr history –147. A s history , 148.C13ORF18, JAM3, EPB41L3, and TERT using a self-sampling lavage device has the diagnostic performance non-inferior to cytomorphology and the HR-HPV tests , Natural Science Foundation of the Jilin Province (grant numbers 20190201042JC and 20190201163JC), Science and Technology Development Program of the Jilin Province (No. 20210509010RQ), and Interdisciplinary Integration and Innovation Project of JLU.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objectives: Women with normal pathology screened from abnormal cervical cytology are a special population with higher progression risk than women with normal cytology. However, the associations between genotype distribution and changes of high-risk human papillomavirus (HR-HPV) infection and cervical progression risk in this special population remain unclear.Methods: A total of 1232 women with normal pathology screened from abnormal cervical cytology were enrolled into this cohort with 2-year follow-up. HPV genotyping detection was performed through flow-through hybridization. Hazard ratios (HRs) and Odds ratios (ORs) were calculated using Cox proportional hazard regression and logistic regression models, respectively.Results: Overall HR-HPV prevalence at baseline was 29.0%, with HPV16, 52, 58, 53 and 51 the top five genotypes. The 2-year persistence rate of HR-HPV infection was 31.9%. Compared with HR-HPV negative, the adjusted HRs of overall HR-HPV, HPV16, 31/33, 58, 51, and 53 infections for the progression risk of normal cervix were 5.31, 7.10, 6.95, 5.74, 5.04, and 4.88, respectively. Multiple HR-HPV infection cannot lead to an additional risk of progression relative to single HR-HPV infection. In comparison with HR-HPV persistently negative, same-type HR-HPV persistence was positively associated with progression risk of normal cervix (adjusted OR: 22.26), but different-type HR-HPV persistence was not linked to cervical progression.Conclusion: Genotypes and persistence of HR-HPV infection could stratify the cervical progression risk in women with normal cervical pathology and abnormal cytology and provide evidence for development of next generation of vaccines. HPV51 and 53 deserved attention apart from HPV16, 31, 33, and 58. Cancers caused by Human papillomavirus (HPV) infection, including cancers of the cervix, anus, vulva, vagina, penis and oropharynx and so on, account for approximately 4.5% of all cancers globally The causal role of HPV infection, especially high-risk HPV (HR-HPV) infection has been established in cervical carcinogenesis by previous studies In this CIN screening cohort study, we aimed to describe the genotype distribution and changes of HR-HPV infection and evaluate the relationship between them and the progression risk of pathologically normal cervix screened from abnormal cytology, in order to provide evidence for risk stratification of women with normal pathology and abnormal cytology in Chinese and for the development of next generation of vaccines with broader coverage of significant genotypes.Figure 1). All of the enrolled participants were married, Han ethnicity, Shanxi native, non-pregnant, and had no history of HPV vaccination, cervical and vaginal lesion treatments, hysterectomy, and other malignant tumors. Written informed consents were signed by all women and the study was approved by Shanxi Medical University Science Research Ethics Committee. (No: 2013-003).A CIN community cohort study including 39,988 women aged 18-65 years was established in two counties (Jiexiu and Yangqu) in Central Shanxi Province between June 2014 and December 2014. Baseline screening procedures have been described in detail previously Information of age, smoking, drinking, education level, yearly income, medical history, age at first intercourse, contraception history, parity, menopause history, hygiene habits, and family history of cancer was collected using a uniform structured questionnaire from an in-person interview. Information of HPV typing and pathological diagnoses was collected from laboratory tests.After removing the cervical secretions with a cotton swab, exfoliated cervical cells were collected using a HPV special cervical brush. Cervical biopsy tissues were collected under colposcopy  according to the Preventive Oncology International microbiopsy protocol.Pathological diagnoses were completed together by two gynecologic pathologists using cervical biopsy tissues. If the two pathologists reached different diagnoses, a third senior pathologist was invited to discuss the difficult or equivocal cases to arrive at a consensus diagnosis. In addition, immunohistochemical detection of p16 and Ki-67 was performed to assist the pathological diagnosis. Pathological results were classified as normal cervix, low-grade CIN (CIN1), high-grade CIN (CIN2/3), and cervical squamous cell carcinoma.HPV genotyping testing was performed using the 21 HPV GenoArray Diagnostic Kit  using exfoliated cervical cells. Compared with standard Roche Linear Array and hybrid capture II, most HPV genotypes detected with this kit shows good interassay agreement From June 2016 and December 2016, 2 years after enrollment, all women with normal pathology screened from abnormal cervical cytology were followed up except for only LR-HPV positive women. The follow-up contents included an in-person interview, HPV typing detection, colposcopy and histological examination. The outcomes of follow-up included the prognosis of normal cervix and the changes of HR-HPV infection. Persistent HR-HPV infection (persistence), including persistent same-type HR-HPV infection and persistent different-type HR-HPV infection, referred to HR-HPV positive at baseline and follow-up. Persistent same-type HR-HPV infection meant that at least one HR-HPV genotype was identical at baseline and follow-up; persistent different-type HR-HPV infection meant that none of the HR-HPV genotypes detected at follow-up was the same as those detected at baseline. The change from HR-HPV positive to negative was considered as the clearance of HR-HPV. Conversely, the change from HR-HPV negative to positive was regarded as the acquisition of HR-HPV. The prognosis of normal cervix included progression (transiting to CIN1 and above (CIN1+)) and persistence .The SPSS 22.0 software was used for data statistical analysis. Categorical variables were presented as frequency or proportion and continuous variables as mean and standard deviation. We used chi-square test to examine the differences among different groups. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox proportional hazard regression models to present the effect of baseline HR-HPV genotype-specific infection in progression of normal cervix. Odds ratios (ORs) and 95% CIs were estimated using binary unconditional logistic regression models to present the associations between changes of HR-HPV infection and progression of normal cervix. Statistical significance was set at α = 0.05 on the basis of 2-sided tests.Table 1). The mean ages of the 1232 women enrolled into this cohort were 48.73 ± 8.56 years (range 20-65 years). Of the 1232 participants, 1025 (83.2%) women had completed middle school or above, 20 (1.6%) women smoked, 52 (4.2%) women drank alcohol, 882 (71.6%) women had an income of more than 10,000 ¥RMB annually, 409 (33.2%) women had a history of female sterilization, 112 (9.1%) women had a family history of cancer, 463 (37.6%) women's age at first intercourse was under 23 years old, 82 (6.7%) women had a history of oral contraceptive use, 303 (24.6%) women's parity was 3 or more and 608 (49.4%) women had a history of menopause.We examined the distribution of demographic characteristics between the 1232 participants and the 271 women who were excluded at follow-up and found no significant difference  than at baseline (29.0%). Single HR-HPV prevalence, either at baseline or follow-up, was much higher than that of multiple HR-HPV. The most common HR-HPV genotype was HPV16 at baseline and HPV58 at follow-up. However, HPV16, 52, 58 were the three most frequently observed genotypes in this screening sample at baseline and follow-up. HPV16 and 58 occurred mainly as single infection. HPV52 occurred mainly as multiple infection at baseline while single infection at follow-up. HPV53, 51, 39, and 33 were also common at baseline and follow-up, with HPV39 and 53 mainly in the form of single infection and HPV33 and 51 mainly in the form of single infection at baseline and multiple infection at follow-up.The distribution of various HR-HPV genotypes differed, as shown in Compared with baseline, overall clearance and persistence rates of HR-HPV infection at 2-year follow-up were 68.1% (243/357) and 31.9% (114/357), respectively. Of the 875 women with HR-HPV negative at baseline, 113 (12.9%) turned positive for HR-HPV and 762 (87.1%) remained negative for HR-HPV at follow-up. Of the 114 women with HR-HPV persistence, 67 (58.8%) were persistent same-type infection and 47 (41.2%) were persistent different-type infection.2 = 5.87, p = 0.015) and age , while there was no significant difference in the distribution of yearly income, education level, smoking, drinking, menopause history, parity, age at first intercourse, frequency of vulva cleaning, frequency of underwear cleaning, history of female sterilization, and family history of cancer (p > 0.05). Women aged 20-35 years old and those who had used oral contraceptives for more than 1 year were more likely to progress.Of the 1232 participants, 22 (1.8%) progressed to CIN1, 10 (0.8%) progressed to CIN2/3, and 1200 (97.4%) persisted still as normal cervix at follow-up. Therefore, a total of 32 (2.6%) women progressed. Between the progression and persistence groups of normal cervix, there were statistically significant differences in oral contraceptive use , either single HR-HPV infection  or multiple HR-HPV infection , significantly increased the risk for the progression of normal cervix in relative to HR-HPV negative group. Compared with women with HR-HPV negative, the aHRs for the progression risk of normal cervix were 7.10 (95% CI: 3.05-16.53) for HPV16 infection, 6.95 (95% CI: 2.01-26.26) for HPV31/33 infection, 5.74 (CI: 1.94-16.96) for HPV58 infection, 5.04 (95% CI: 1.06-23.94) for HPV51 infection, and 4.88 (95% CI: 1.06-22.43) for HPV53 infection. We did not found significant associations between HPV18, 35, 39, 52, 56, 59, 66, or 68 infections and the progression of normal cervix.Table 3 compares the role of single and multiple HR-HPV infections in the progression of normal cervix by using Cox proportional hazard analyses. Compared with single HR-HPV infection, multiple HR-HPV infection  did not lead to a significant increased progression risk for normal cervix. Meanwhile, no significant differences associated with the risk for the progression of normal cervix were observed between single infection and multiple infection of HPV16, 31/33, 51, 53, or 58.Table 4 displays the associations between the changes of HR-HPV infection and the risk of cervical progression among the 1232 women with normal histology but abnormal cervical cytology through the use of binary unconditional logistic regression analyses. After fully adjusting for oral contraceptive use and age, we found that persistent HR-HPV infection , especially persistent same-type HR-HPV infection , was a risk predictor for the progression of normal cervix. No significant difference was observed in the cervical progression risk between the HR-HPV persistently negative group and the HR-HPV clearance group or the HR-HPV acquisition group. In addition, we also compared the cervical progression risk between the HR-HPV persistence group, especially the persistent same-type HR-HPV infection group, and other groups. Compared with the HR-HPV clearance group and the new acquisition group, the aORs for the progression risk of normal cervix in the HR-HPV persistence group were 6.64 (95% CI: 2.52-17.52) and 5.00 (95% CI: 1.46-17.19), respectively, and the aORs for the cervical progression risk in the persistent same-type HR-HPV infection group were 8.21 (95% CI: 3.06-23.01) and 7.19 (95% CI: 2.05-25.21), respectively. In relative to the persistent different-type HR-HPV infection group, the aOR for the progression risk of normal cervix in the persistent same-type HR-HPV infection group was 4.61 (95% CI: 1.10-20.31).HPV prevalence is dramatically different by geographical region and increase notably with the severity of cervical lesions. In the present study, overall HR-HPV prevalence was 29.0% at baseline, higher than 18.2% HPV prevalence in Xinjiang, China In our cohort, HPV16 was the most prevalent HR-HPV genotype at baseline, followed by HPV52, 58, 53, 51, 39, 33, 56, 66, 18, 68, 35, 59, and 31. HPV16 was the most common HPV type, which was consistent with those reported in previous studies worldwide The occurrence of cervical cancer has a “classic” process, from normal cervix, through CIN1, to CIN2/3, and finally to cervical cancer. In this study, the 2-year progression rate of normal cervix to CIN1+ was 2.6%, posing a risk for subsequent invasive cervical cancer. Our analysis results showed that women aged 20-35 and those who had used oral contraceptives for more than 1 year were more likely to develop CIN1+. Oral contraceptive use has been reported to increase the risk of developing cervical cancer in women with normal cytology in a retrospective population-based cohort study HR-HPV infection is widely known as the major cause of cervical lesion progression Compared with single HR-HPV infection, the role of multiple HR-HPV infection in cervical progression is controversial. Most cohort studies have shown that multiple HPV infection offers no additional risk than single HPV infection Although persistent HR-HPV infection has been considered as the main cause of cervical progression Several strengths need to be mentioned in this study. First, this is a prospective cohort that screened subjects from a population including 39,988 women. Second, the subjects were women with normal pathology screened from abnormal cervical cytology, and the effect of type-specific HR-HPV infection in their progression has been rarely studied. Last, we compared the cervical progression risk between the persistent same-type HR-HPV infection group and the persistent different-type HR-HPV infection group, which was rarely studied previously. There are also several limitations in our study. First, the follow-up period was 2 years, not long enough to observe the occurrence of cervical cancer. Second, we did not assess the association between persistent type-specific HR-HPV infection and the progression risk of normal cervix, as the number of women with persistent infection of a certain HR-HPV genotype was small. Third, it should be noted that some persistent HR-HPV infection might be the reinfection with these same genotypes, which would underestimated the relationship between HR-HPV persistence and cervical progression.In conclusion, HPV16, 52, 58, 53 and 51 were the top five HR-HPV genotypes in this screening population; Women infected with HPV16, 31/33, 58, 51, and 53 were at high risk for progression of normal cervix; There were no significant difference between multiple HR-HPV infection and single HR-HPV infection in the progression of normal cervix; Same-type HR-HPV persistence was positively associated with the progression risk of normal cervix, but different-type HR-HPV persistence was not linked to cervical progression. The combination of genotype distribution and changes of HR-HPV infection and their potential role in the progression of women with normal cervical pathology and abnormal cytology could be of great significance to the implementation of more precise regional type-specific cervical lesion screening programs and the development of next generation of vaccines in the future. In the future, we plan to follow up this population at 5-year intervals to explore the long-term impact of type-specific HR-HPV infection on cervical progression in women with normal pathology and abnormal cytology.


To investigate the course of health-related quality of life (HRQOL) from diagnosis to 2 years follow-up among patients with oropharyngeal cancer (OPSCC), in relation to human papilloma virus (HPV) status.This study included 270 OPSCC patients. Age, sex, tumor sublocation, tumor stage, HPV status, treatment modality, comorbidity, smoking, and alcohol use were retrieved from medical records. HPV status was positive when p16 and HPV DNA tests were both positive. HRQOL was assessed using the EORTC QLQ-C30/QLQ-H&N35 pretreatment and at 6 weeks, 6, 12, 18, and 24 months after treatment. To compare the course of HRQOL between patients with an HPV-positive versus HPV-negative tumor, linear and logistic mixed models were used.Patients with an HPV-positive tumor (29%) were more often male, diagnosed with a tumor of the tonsil or base of the tongue, treated with single treatment, had fewer comorbidities, were less often current smokers and had lower alcohol consumption. Adjusted for confounders, the course of global quality of life, physical, role, and social functioning, fatigue, pain, insomnia, and appetite loss was significantly different: patients with an HPV-positive tumor scored better before treatment, worsened during treatment, and recovered better and faster at follow-up, compared to patients with an HPV-negative tumor. The course of emotional functioning and oral pain was also significantly different between the two groups, but with other trajectories.The course of HRQOL is different in patients with an HPV-positive tumor versus an HPV-negative tumor, adjusted for sociodemographic, clinical, and lifestyle confounders. Head and neck cancer (HNSCC) is the sixth most common cancer worldwide . The maiThe study population consisted of patients diagnosed with primary squamous cell carcinoma of the mucosal surfaces of the oropharynx (OPSCC) and treated with curative intent between January 1999 and January 2011 at the Amsterdam UMC, location VUmc in Amsterdam. Exclusion criteria were distant metastases, second primary tumors, previous surgery or radiotherapy for HNSCC, brachytherapy, serious cognitive impairment or lack of basic knowledge of the Dutch language. We also excluded patients who did not complete pretreatment HRQOL questionnaires and of whom HPV status was not assessed.INK4A immunostaining followed by high risk HPV DNA-detection on formalin-fixed paraffin-embedded (FFPE) tumor specimen [Sociodemographic , clinical , tumor sublocation: tonsil, base of tongue, soft palate/uvula, other oropharyngeal locations), TNM tumor stage (stage I – IV according to the American Joint Committee on Cancer 7th edition) , HPV staspecimen . HPV staHRQOL data was collected as part of standard clinical care. Since 1999 (department of Radiotherapy) and 2016 (department of Otolaryngology-Head and Neck Surgery), HNC patients are asked to complete patient-reported outcomes measures (PROMs) on HRQOL using a paper-and-pencil version of the PROMs and a touch screen computer-assisted data collection system called OncoQuest respectively. Patients are asked to complete these PROMs in the clinic before start of treatment and at every follow-up visit at one of the two departments. Information on durable usage of these PROMs in our clinic can be found elsewhere . All patHRQOL was assessed using the EORTC QLQ-C30 and EORTC QLQ-H&N35. The 30-item EORTC QLQ-C30 is a generic HRQOL questionnaire that consists of nine domains and six single items. These nine domains include global quality of life, physical functioning, role functioning, emotional functioning, cognitive functioning, social functioning, fatigue, nausea and vomiting, and pain. Single items encompass: dyspnea, insomnia, appetite loss, constipation, diarrhea, and financial difficulties. The scores of the QLQ-C30 are linearly transformed to a scale of 0–100, with a higher score suggesting a higher  level of functioning or global HRQOL, or a higher  level of symptoms or problems , 29. Thet test. To compare the longitudinal course of HRQOL between patients with an HPV-positive versus HPV-negative tumor, linear, and logistic mixed models were used with fixed effects for group (HPV-positive or HPV-negative) and measurement  and their two-way interaction, and a random effect for subject (patients with an OPSCC tumor). To investigate potential confounding , clinical , lifestyle ) variables, these variables were included one by one in the analyses. Variables associated with HPV status and HRQOL that changed the regression coefficient of HPV status with more than 10% were included in the final model (starting with the strongest confounder) [p value of 0.05 or less was considered statistically significant and indicates a significant different trajectory of quality of life over time for HPV-positive versus HPV-negative patients.Descriptive statistics were generated for the range of patient characteristics and outcome variables. Chi-square tests were used to examine differences between patients with an HPV-positive versus HPV-negative tumor, with respect to sex, comorbidity, tumor sublocation, tumor stage, treatment modality, smoking, and alcohol use. Age was compared with the independent samples founder) . StatistIn total, 270 patients met the in- and exclusion criteria, of whom 29% had an HPV-positive tumor (78/270) Fig. . PatientThe course of the HRQOL scores (mean and standard deviation) from pre-treatment to 24 months follow-up on the various subscales of the EORTC QLQ-C30 and QLQ-H&N35 is presented in Table Multivariate mixed model analyses revealed that, after correcting for confounders , the course of HRQOL was significantly different between patients with an HPV-positive tumor versus patients with an HPV-negative tumor, regarding global quality of life physical functioning, role functioning, emotional functioning, social functioning, fatigue, pain, insomnia, appetite loss . With respect to global quality of life, physical functioning, role functioning, social functioning, fatigue, pain, insomnia, and appetite loss, patients with an HPV-positive tumor scored equal or better before treatment, worsened more during treatment, and recovered better and faster at follow-up compared to patients with an HPV-negative tumor Fig. . A seconIn this study, we found significant differences in the course of HRQOL from diagnosis to 2 years follow-up between OPSCC patients with an HPV-positive tumor compared to those with an HPV-negative tumor, when adjusted for confounders as sex, comorbidity, tumor sublocation, tumor stage, and treatment modality. These differences concerned the course of global quality of life, physical, emotional, social, and role functioning, and the symptoms fatigue, pain, insomnia, and appetite loss  and oral pain . No differences between both groups were found for the course of cognitive functioning, nausea/vomiting, dyspnea, constipation, diarrhea, financial difficulties , and, except for oral pain, all other HNC-specific symptoms .Of the ten studies that investigated HPV status in relation to HRQOL among HNSCC patients –23, six All ten studies investigated the association between HPV status and HRQOL during or after treatment or at follow-up. One study reported worse HRQOL in the two last weeks of treatment in patients with an HPV-positive tumor compared to those treated for an HPV-negative tumor . SeveralIn the “Introduction” section, based on these previous studies –23, we sDifferences in the course of HRQOL between patients with an HPV-positive versus HPV-negative OPSCC, especially the deterioration shortly after treatment in HPV-positive patients, may be explained by acute toxicity. Ringash et al.  examinedStrength of this study was the study design with general cancer as well as HNC-specific HRQOL assessments from baseline up to 2 years after treatment among a large sample of patients with OPSCC only. Also, HPV status was tested based on p16 as well as HPV-DNA, in contrast to previous studies that used p16 as surrogate marker of HPV status only. In addition, several sociodemographic and clinical confounders were taken into account. We also investigated the role of possible lifestyle confounders , but this did not change the outcome and was not relevant to the significance of the results. Further research is, however, warranted on the potential confounding role of continued smoking and alcohol use during follow-up, as well as other potential sociodemographic confounders such as education and living situation. These factors were not included in our study, which should be seen as a limitation. Another limitation of this study is that we excluded patients of whom HPV status was not known and those patients without pretreatment HRQOL data, which may have induced selection bias. Furthermore, our data represent a small proportion (estimated on 10–15%) of the total number of OPSCC patients in the Netherlands. Due to the study design and privacy regulations, it was not possible to compare responders and nonresponders of this study. Another limitation is that the QOL data was acquired as part of routine clinical care and therefore depended on timing of the follow-up visits and was therefore not exactly at 6 weeks, 3, 6, 12, and 24 months follow-up. As a comparable time, window was used for both HPV+ and HPV− OPSCC patients, we, however, expect that this does not have influenced our findings. Another limitation is the existence of missing surveys due to death and, in minority, to loss to follow-up. Finally, a potential limitation of this study is that HRQQOL measured at the end of treatment was not taken into account while acute toxicities such as fatigue and appetite loss may be highest at this time point.The findings in this study may contribute to the current discussion about treatment deescalation. Efforts to improve treatment outcome in this patient group should also focus on preventing HRQOL deterioration during and shortly after treatment when possible –34. FurtAmong OPSCC patients, the trajectory of the course of HRQOL is significantly different in patients with an HPV-positive versus an HPV-negative tumor, when adjusted for sociodemographic and clinical confounders. Regarding global quality of life, physical functioning, role functioning, social functioning, fatigue, pain, insomnia, and appetite loss, patients with HPV-positive OPSCC score better before treatment, deteriorate more during and shortly after treatment, and recover better and faster at follow-up, compared to patients with HPV-negative OPSCC. Furthermore the course of emotional functioning and oral pain is significantly different between the two groups, but with other trajectories.


PIK3CA and TP53 were commonly altered genes in cervical cancer, while SMAD4, RET, EGFR, and APC were NECC-specific altered genes. Of note, 11 NECC cases showed at least one actionable mutation linked to molecular targeted therapies, and 14 cases showed more than one combined positive score for PD-L1 expression. These results may boost the generation of effective treatment strategies for NECC in the future.Patients with neuroendocrine carcinoma of the cervix (NECC) have limited treatment options due to its rarity and aggressiveness. In this study, we performed a comparative genetic analysis between 25 NECC and other cervical cancer types . Furthermore, the expression of programmed cell death-ligand 1 (PD-L1) was assessed by immunohistochemistry. PIK3CA, 32%; TP53, 24%) and distinct  alterations compared with other histological types. The GENIE cohort had similar profiles and RB1 mutations in 27.6% of NECC cases. Eleven (44%) cases had at least one actionable mutation linked to molecular targeted therapies and 14 (56%) cases showed more than one combined positive score for PD-L1 expression. HPV-positivity was observed in all NECC cases with a predominance of HPV-18. We report specific gene mutation profiles for NECC, which can provide a basis for the development of novel therapeutic strategies.Neuroendocrine carcinoma of the cervix (NECC) is a rare and highly aggressive tumor with no efficient treatment. We examined genetic features of NECC and identified potential therapeutic targets. A total of 272 patients with cervical cancer  were enrolled. Somatic hotspot mutations in 50 cancer-related genes were detected using the Ion AmpliSeq Cancer Hotspot Panel v2. Human papillomavirus (HPV)-positivity was examined by polymerase chain reaction (PCR)-based testing and in situ hybridization assays. Programmed cell death-ligand 1 (PD-L1) expression was examined using immunohistochemistry. Somatic mutation data for 320 cases of cervical cancer from the Project GENIE database were also analyzed. NECC showed similar ( Neuroendocrine carcinoma of the cervix (NECC) is an uncommon and highly aggressive tumor. Based on Surveillance, Epidemiology, and End Results (SEER) data from the National Cancer Institute, the annual incidence of this cancer is 0.05/100,000 . ApproxiGenomic data of NECC are expected to provide basis for novel and more efficient treatment options for this aggressive tumor. In fact, molecular targeted therapies have contributed to the improvement of patient outcomes in various cancers . HoweverThe relationship between genotypes of human papillomavirus (HPV) and mutational profiles of NECC remains to be elucidated. High-risk HPV is reportedly associated with most cervical cancers, including SCC, ADC, and adenosquamous carcinoma (ASC) of the cervix . RecentlIn this study, we aimed to decipher the genetic characteristics of NECC compared with cervical cancer of other histological types using targeted sequencing and analysis of data available in a public database. Furthermore, we explored the links between the identified mutations and targeted therapies. We also elucidated the HPV genotypes in NECC and their association with genetic alterations.The study protocol was approved by the Institutional Review Board of the National Cancer Center Research Institute (2017–136) and the study was conducted in accordance with the ethical guidelines of the Helsinki Declaration. Written informed consent was obtained from all patients through an opt-out form. Patients who refused to provide consent were excluded from the study.Two-hundred and eighty-seven patients with pathologically confirmed cervical cancers, including NECC (n = 26), SCC (n = 191), ADC (n = 55), and ASC (n = 15), who received treatment between 2002 and 2018 at the National Cancer Center Hospital, Japan were retrospectively enrolled. Of the 287 cases, sequencing data from 272 patients met the quality control criteria and were included in this study. All the cases were reviewed by at least two gynecological pathologists, and the pathological diagnoses were confirmed according to the World Health Organization (WHO) tumor classification .TM Cancer Hotspot Panel v2 , which targets approximately 2800 Catalog of Somatic Mutations in Cancer (COSMIC) mutational hotspot regions of 50 cancer-related genes. An Ion AmpliSeqTM Custom Panel that was designed for the TP53 gene  using Ion AmpliSeqTM Designer  was also used. Sequencing was performed using the Ion Proton platform (Thermo Fisher Scientific). For quality control, samples with a mean read depth of coverage over 1000 and a base quality score of 20 (≤1% probability of being incorrect) were selected, which accounted for 90% of the total reads.Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissues using the QIAamp DNA FFPE Tissue Kit , according to the manufacturer’s instructions. Library construction was performed using purified genomic DNA (50 ng) obtained from the tumor tissues and the Ion AmpliSeqhttp://evs.gs.washington.edu/EVS/ (accessed on 1 July 2019)) or the integrative Japanese Genome Variation Database ), and (3) the mutations were registered as “pathogenic/likely pathogenic variants” in the ClinVar or as “oncogenic/likely oncogenic variants” in OncoKB (http://oncokb.org) databases using oncokb-annotator, commit 8910b65 (accessed on 29 June 2019). All the selected variants were then evaluated manually using the Integrative Genomics Viewer ).The sequencing reads were mapped to the University of California, Santa Cruz (UCSC) human reference genome GRCh37, and data analysis was carried out using the Torrent Suite Software v5.0.4 (Thermo Fisher Scientific). Somatic mutations were initially selected using the following criteria: (1) variant allele frequency of somatic mutations was >4% in tumor tissues, (2) single nucleotide polymorphisms were removed if they showed a threshold allele frequency ≥0.01 in either the National Heart, Lung, And Blood Institute (NHLBI) Grand Opportunity Exome Sequencing Project  were selected from among the 50 targeted genes in the Ion AmpliSeq™ Cancer Hotspot Panel v2, and the frequency of copy number alterations in these four genes was detected to be >5% in the TCGA dataset of cervical cancer patients within cBioPortal. All TaqMan probes, including PIK3CA (ID Hs02202946_cn), ERBB2 (ID Hs02803918_cn), PTEN (ID Hs05128032_cn), STK11 (ID Hs04013006_cn), and RNase P (cat. no. 4403328), which was used as a reference, were purchased from Thermo Fisher Scientific. Genome data were analyzed using the ABI PRISM 7900HT Sequence Detection Software CopyCaller v2.1 (Thermo Fisher Scientific) for copy number analysis. Copy number amplification was defined as the process resulting in the presence of >8 copies, whereas copy number loss was defined as the process resulting in the presence of <1.2 copy.Copy number alterations were detected by real-time genomic polymerase chain reaction (PCR) using the TaqMan copy number assay and the ABI 7900HT real-time PCR system (Applied Biosystems). Four genes (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 17 June 2019)).HPV genotypes were identified as follows: Genomic DNA (10 ng) was amplified using PCR for two distinct HPV genomic regions. The E6/E7 region of HPV was amplified using the primer set pU-1M/pU2R (HPVpU-1M: 5′-TGTCAAAAACCGTTGTGTCC-3′ and HPVpU-2R: 5′-GAGCTGTCGCTTAATTGCTC-3′); the region containing the HPV L1 gene was amplified using the primer set GP5+/GP6+ . PCR reactions were performed using the TaKaRa PCR Human Papillomavirus Typing Set . PCR products were purified using the NucleoSpin Gel (TaKaRa Bio Inc.) or a PCR Clean-up Kit (TaKaRa Bio Inc.). Sanger sequencing was performed using an ABI 3130xl DNA Sequencer , according to the manufacturer’s instructions. Similarity between the obtained sequences and various HPV genotypes in the GenBank database was determined by Basic Local Alignment Search Tool (BLAST) analysis  using HPV-III High Risk probes , according to the manufacturer’s instructions. The HPV-ISH assay is able to detect high-risk HPV genotypes in cervical cancer specimens, including HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-45, HPV-52, HPV-56, HPV-58, and HPV-66.Immunohistochemistry for retinoblastoma protein, RB1, was performed on 4 μm thick paraffin-embedded tissue sections, which had the same tumor areas provided for genetic analysis, using an RB1-specific mouse monoclonal antibody . Tumors were scored as negative for RB1 protein if more than 95% of cells showed no RB1 staining, with positive nuclear staining in the surrounding endothelial cells serving as an internal control. The expression of programmed cell death-ligand 1 (PD-L1) was examined in tumor cells and infiltrating histiocytes by immunohistochemistry using a rabbit polyclonal antibody (SP142). The expression of PD-L1 was scored by both tumor proportion score (TPS) and combined positive score (CPS), as previously described .http://www.synapse.org/genie (accessed on 2 July 2020)).Somatic mutations called from targeted sequencing data in the American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia information Exchange (GENIE) database (GENIE version 8.0-public) were downloaded as MAF files via the Synapse Platform  was defined as the time from the start of treatment to death from any cause. Survival curves were computed using Kaplan–Meier estimates with log-rank tests. Cox regression analysis was applied to test the predictors of OS and to calculate hazard ratios with 95% confidence intervals (95% CI).Statistical analysis was performed using the SPSS  and R  software. Categorical variables were compared using the chi-square test or Fisher’s exact test for small sample sizes. Continuous variables were compared using the unpaired Student’s The clinicopathological characteristics of 272 patients are summarized in PIK3CA and TP53 variants were detected in 6/25 (24%) of the patients each, followed by SMAD4 variants in 5/25 (20%), PTEN variants in 4/25 (16%), and RET variants in 4/25 (16%) patients. Copy number losses were detected in 4/25 (16%) patients for STK11 and in 3/25 (12%) patients for PTEN. Copy number amplifications were detected in 3/25 (12%) patients for PIK3CA. The PI3K pathway is frequently activated in cervical cancer, and the PI3K pathway-related gene PIK3CA was frequently altered in all histological types  of the cases presented more than 1 CPS .RET mutation is a therapeutic target not found in other histological types.Actionable mutations registered as evidence levels of 1–3B in OncoKB were detected in 11 (44%) patients with NECC . AccordiApproximately 80% of the patients with NECC had detectable HPV-18, and HPV-18-positivity was statistically more frequent in patients with NECC than in those with other histological subtypes . Next, wp = 0.002; KRAS mutations had significantly shorter OS than those without these mutations . The OS was significantly worse among patients with advanced FIGO stages  in the helical domain of PIK3CA. In Project GENIE, PIK3CA mutations in patients with NECC were less frequent (9.1%), whereas those in patients with other histological types were comparable to our results. Activation of the PI3K pathway through PIK3CA regulates various transformed phenotypes as well as the growth and differentiation of HPV-16 and HPV-18-immortalized cells; thus, it may play a pivotal role in HPV-induced carcinogenesis [TP53 mutations, more frequently observed in patients with NECC than in those with SCC and being similar to that in patients with ADC. Moreover, mutations in the MAPK pathway genes, including KRAS, BRAF, and NRAS, were observed at similar frequencies among the histological types. This observation was comparable to that reported previously [Our targeted sequencing results, comparing NECC with other subtypes of cervical cancers, reveal some similarities. Alterations in eviously . Of noteEGFR, RET, SMAD4, APC, CTNNB1, and PTPN11, are limited in NECC. These gene mutations are uncommon in cervical cancer [EGFR regulates multiple intracellular target pathways and affects a wide range of biological processes [EGFR mutations are significantly correlated with a highly differentiated grade in patients with cervical cancer [SMAD4, which regulates the canonical transforming growth factor (TGF)-β signaling pathway in porcine granulosa cells, was mutated in 5 (20%) patients with NECC. SMAD4 plays a critical role in tumor progression and induces proinflammatory cytokines, such as interleukin (IL)-5, IL-6, and IL-13, which might induce a tumor-promoting microenvironment [RET mutations were first identified in 4 (16%) patients with NECC in this study. To date, only two cervical SCC cases harboring RET mutations have been reported in Project GENIE. Somatic RET mutations has been reported in about 25–45% cases of sporadic medullary thyroid carcinoma (MTC), a type of carcinoma with neuroendocrine differentiation. Similarly, RET mutations may be associated with the neuroendocrine differentiation in some patients with NECC. In this study, APC, which is a negative regulator of the Wnt/β-catenin pathway, was mutated in 3 (12%) patients with NECC. The Wnt/β-catenin pathway plays a key role in the sequential development  of tumors into cancer [Notably, we identified that several recurrent gene mutations, such as those in l cancer . EGFR rerocesses . EGFR mul cancer . SMAD4, ironment . Oncogeno cancer .KRAS mutation appears to be a poor prognostic factor. Interestingly, Lyons et al. reported that a mitogen-activated protein kinase 1 inhibitor (trametinib) achieved complete radiologic response in a patient with recurrent NECC with KRAS mutation [These genetic differences and similarities between NECC and other types of cervical cancer seem to be consistent with the current understanding of NECC; NECC derives from major histological subtypes, such as SCC or ADC, showing trans-differentiation of the neuroendocrine phenotype ,25. We hmutation .PIK3CA mutations are the most frequent actionable mutations in cervical cancers and have been approved by the US Food and Drug Administration (FDA) as a predictive biomarker for the use of the PI3K inhibitor, alpelisib [RET mutations, R886Q, S891L, A883T, and C611Y, were first identified in four patients with NECC. Patients with oncogenic RET mutations reportedly benefit from treatment with RET inhibitors, BLU-667 and LOXO-292 [RB1 were observed in three patients with NECC in Project GENIE. Although loss of RB1 is not registered in OncoKB, two recent studies described that the deregulation of cell cycle transitions upon loss of RB1 can represent a high dependency on aurora kinases, which can be targeted therapeutically [RET mutations, were first identified in our study. Therefore, the data seem to be insufficient to elucidate the true nature of NECC and, thus, the published data must be integrated and utilized to identify a novel therapeutic target in future studies.We also evaluated NECC for actionable mutations with OncoKB evidence levels of 1 to 3B. Actionable mutations were detected in all histological types of cervical cancer, including 11 patients with NECC (44%). lpelisib . Variouslpelisib . One stulpelisib . Four onLOXO-292 . Loss-ofutically ,32. ReceWe first provided both TPS and CPS for PD-L1 expression in patients with NECC. Notably, 56% of patients with NECC showed more than one CPS, whereas no cases presented more than 1% TPS. The expression of PD-L1 on tumor and/or immune cells is predictive of the benefit of anti–PD-1 and anti–PD-L1 therapy in several tumor types . Althougp < 0.001) [Our results show that all patients with NECC were positive for high-risk HPV and 76% of them were positive for HPV-18; however, no significant correlation between HPV genotypes and specific gene mutations was observed. Although HPV-positivity was observed in more than 90% of all cervical cancer patients, NECC was significantly more associated with HPV-18 than other histological types. A large proportion of NECC cases are reportedly caused by high-risk HPV, primarily HPV-16 and HPV-18 . A previ< 0.001) . Further< 0.001) . Althoug< 0.001) ,40,41. IFrequent HPV-positivity in NECC also has diagnostic utility for detection of a primary site of metastatic NEC. A significant subset of patients with advanced neuroendocrine carcinomas (NECs) are diagnosed after the detection of distant metastases. However, 11–22% of NECs are reportedly of unknown origin . ConsideRET, even for limited genes and their regions. Plans are currently underway to test therapies targeting the candidate identified in this study.This study has several limitations that need to be addressed. First, the number of patients with NECC included in this study was limited. The survival analysis was not robust due to the small number of cases, and further external validation should be performed in a future study. However, considering its rarity, the mutational analysis of 25 cases of NECC has been a relatively large cohort in Asia. Second, targeted sequencing for mutational analysis used in this study contained only hotspot mutations in 50 cancer-related genes. Nevertheless, we could identify new therapeutic targets for NECC, such as alterations in PIK3CA (32.0%), TP53 (24.0%), SMAD4 (20%), PTEN (20.0%), STK11 (16.0%), and RET (16.0%) were frequently altered in patients with NECC. Nineteen patients (76%) with NECC could potentially benefit from molecular targeted therapies and immune checkpoint inhibitors. Furthermore, high-risk HPV infections, particularly HPV-18, may play a critical role in the carcinogenesis and aggressive behavior of NECC. Therefore, the current HPV vaccines could prevent a large proportion of NECC cases. Collectively, this study reveals specific gene mutation profiles and HPV status in NECC, which can provide a basis for the development of novel therapeutic strategies for this highly aggressive malignancy.We demonstrate genetic similarities and differences between NECC and other histological types of cervical cancers.


In 2015, Botswana introduced quadrivalent human papillomavirus (HPV) vaccine for girls aged 9–13 years. To establish a baseline HPV prevalence for future HPV vaccine impact monitoring, we evaluated HPV prevalences among the youngest unvaccinated women in Botswana and compared HPV prevalences among women living with HIV (WLHIV) and without HIV.Women aged 18–22 years were recruited from the University of Botswana and HIV clinics in Gaborone from October 2019–January 2021. Demographic and behavioral characteristics were self-reported during structured interviews; HIV clinical characteristics were abstracted from medical charts. Self-collected vaginal swabs were tested for 28 HPV types using Seegene Anyplex II HPV28. We compared prevalence of any HPV, high risk (HR)-HPV, and quadrivalent HPV vaccine types (HPV6/11/16/18) among WLHIV and women without HIV and evaluated risk factors for prevalence of HR-HPV.A total of 306 WLHIV and 500 women without HIV were recruited. Compared to women without HIV, WLHIV were more likely to be sexually experienced (86.6% versus 74.4%) and have ≥ 3 lifetime sex partners (55.3% versus 27.8%). All HPV type prevalences were significantly higher among WLHIV compared to women without HIV, including prevalence of any HPV (82.7% versus 63.0%), HR-HPV (72.9% versus 53.8%), and quadrivalent vaccine HPV types (34.3% versus 21.0%). Among WLHIV, there were no differences between those perinatally and non-perinatally infected for HPV prevalences, number of HPV types detected, CD4 count, or viral load.Over one-third of WLHIV and nearly a quarter of those without HIV had vaccine-type HPV detected. This study supports need for the national HPV vaccination program in Botswana and provides important baseline data for future evaluation of impact of the program. Cervical cancer is the most common cancer among women in Botswana and elsewhere in Sub-Saharan Africa . Human pIn 2015, Botswana introduced HPV vaccine into their national immunization program for the prevention of cervical and other HPV-associated cancers . QuadrivImpact of HPV vaccination programs in many high-income countries has been observed through the reduction in prevalence of HPV vaccine targeted types and other HPV attributable outcomes . Little We analyzed data from female participants in the Baseline HPV Prevalence for Vaccine Impact Monitoring in Botswana Study, a cross-sectional study of HPV prevalence among young adults aged 18–22 years. Although HPV vaccine was introduced in the national immunization program in Botswana in 2015, a baseline HPV prevalence could still be established among young adult women because girls vaccinated in the first year of the national program were 13–17 years-old in 2019, when the study recruitment began. Participants were consecutively recruited from the University of Botswana and HIV clinics in Gaborone, Botswana, from October 2019–January 2021, with the majority of participants recruited by July 2020. The inclusion criteria were documented HIV status in the prior 12 months  and age 18–22 years. Exclusion criteria included unknown HIV status, refusal of access to medical records to obtain information on HIV treatment and care, or known current pregnancy. Informed consent was provided by all participants prior to any data collection. Participants self-reported demographic and behavioral characteristics during structured interviews and self-collected a vaginal swab. Clinical data were abstracted from medical records for WLHIV.DNA was isolated from specimens with proteinase K lysis followed by automated extraction in the NucliSENS easyMAG  following manufacturer’s instructions. Swabs in the specimen transport medium (STM) were agitated on an orbital shaker for 45 min at ambient room temperature. For specimen lysis, 200 µL of vaginal swab STM was incubated with 20 µL proteinase K  and enough NucliSENS easyMAG Lysis Buffer  to bring the total processing volume to 800 µL. After incubation at 65 °C for 1 h, extraction was completed on NucliSENS EasyMAG (bioMérieux). DNA was eluted in 100 µL of buffer 3 at a pH > 8.0 and temperature of 70 °C. Water aliquots were processed in parallel with specimens as negative controls for DNA contamination.Extracts were tested according to manufacturer’s End Point-CMTA protocol with Anyplex II HPV28 assay . This assay detects 28 HPV types  and human internal control DNA. The assay was conducted on CFX96 Dx System . Automated analysis by Seegene Viewer software interpreted results as HPV positive, HPV negative or invalid.3), most recent viral load , and age of antiretroviral therapy (ART) initiation  were abstracted from medical charts. Perinatal infection was ascertained via self-report . A small number of participants (n = 18) reported perinatal infection status that appeared implausible in light of other self-reported or chart-verified information and were reclassified accordingly: participants who started ART at age < 15 years but self-reported non-perinatal or unknown infection were re-classified as perinatally infected (n = 6). Participants who started ART at age ≥ 15 years and within 1 year of first sex, and self-reported perinatal infection, were re-classified as not perinatally infected (n = 12). Although study participants were not expected to have received HPV vaccination based on age eligibility, self-reported HPV vaccination status was obtained to identify any bias in baseline HPV prevalence results. Only 4% of study participants reported plausible HPV vaccination based on age and geographic location; due to the small number, these participants were included in the analysis.This analysis was restricted to participants with valid HPV typing results. Demographic variables included age, sex, marital status (single versus married/living together), and education . Behavioral characteristics included ever having sex , number of sex partners in past 12 months , lifetime number of sex partners , age of first sex , age of first sex partner (< 20 versus ≥ 20 years), age of current sex partner , sexual orientation , condom use in past 3 months , tobacco use , and ever pregnant . HIV status  was based on documentation obtained at enrollment. Among WLHIV, the most recent CD4 count , any low-risk (LR)-HPV , quadrivalent vaccine types , HR-HPV vaccine types , and LR-HPV vaccine types . Among participants with any HPV detected, number of HPV types detected were categorized as 1, 2–4, and ≥ 5.Demographic and behavioral characteristics were described by HIV status. HPV prevalences were also described by HIV status for both individual HPV types and HPV type categories, and among sexually experienced participants for HPV type categories. HPV prevalences and HIV clinical characteristics were also described by perinatal HIV infection status among sexually experienced WLHIV.3, and age of ART initiation as < 15 versus ≥ 15 years. A sensitivity analysis excluding perinatally-infected WLHIV was conducted, but no meaningful differences in model findings were observed (data not shown). All analyses were conducted using SAS 9.4 .Because HR-HPV infections can result in more rapid neoplastic changes in WLHIV, we were interested in identifying characteristics associated with HR-HPV prevalence by HIV status. Among sexually experienced participants, unadjusted prevalence ratios (PR) and 95% confidence intervals (CI) were calculated using log-binomial regression to evaluate associations between selected participant characteristics and HR-HPV. Multivariable models were developed using backward selection  to identify characteristics associated with having a prevalent HR-HPV infection in this population. For the models, lifetime number of sex partners were categorized as 1–2 versus ≥ 3, CD4 count as ≤ 350 versus > 350 cells/mm3, and 33.7% were perinatally infected; 99.7% of WLHIV were on ART at the time of study enrollment.Among 806 female participants, 306 were HIV-infected and 500 were HIV-uninfected; all participants had valid HPV typing results. Compared to women without HIV, WLHIV were more likely to be sexually experienced (86.6% versus 74.4%), have ≥ 3 sex partners in the past 12 months (20.0% versus 9.7%) and in their lifetime (55.3% versus 27.8%), and have a current sex partner ≥ 24 years-old (71.7% versus 33.2%)  and quadrivalent vaccine-type HPV (34.3% versus 21.0%)  and ever being pregnant ; having ≥ 3 compared to ≤ 2 lifetime partners remained in the final model but was not statistically significant . Among women living without HIV, ≥ 3 compared to ≤ 2 lifetime partners  and always compared to sometimes or never using condoms  remained significantly associated with HR-HPV in adjusted analyses.Characteristics significantly associated with prevalence of any HR-HPV in unadjusted analyses included lifetime number of sex partners, ever being pregnant, and CD4 count ≤ 350 cells/mmIn this study, vaccine-type and non-vaccine-type HPV prevalences among women aged 18–22 years living with and without HIV in Botswana were characterized for the first time and established as a baseline for future evaluation of HPV vaccine impact in Botswana. HPV prevalences among WLHIV were significantly higher than women without HIV, including prevalences of HR-HPV (72.9% versus 53.8%) and quadrivalent vaccine-type HPV (34.3% versus 21.0%). There were some differences in factors associated with HR-HPV by HIV infection status. No difference in HR-HPV prevalence was observed between perinatally and non-perinatally WLHIV.HIV infection and HIV-associated immunosuppression increase both susceptibility to and persistence of HPV infection . The higBecause some WLHIV in this study were perinatally infected, we had the ability to compare HPV prevalences in perinatally and non-perinatally infected WLHIV. Among sexually experienced WLHIV, no differences were found in HPV prevalences in perinatally infected compared to non-perinatally infected; both groups had a high prevalence of any HPV . In two prior reports that directly compared perinatally and non-perinatally infected WLHIV, there were similarly no differences between the two groups in prevalence of any HPV , 21. AllWe found that there were some differences in factors associated with HR-HPV by HIV infection status. Among women living with and without HIV, having ≥ 3 lifetime sex partners was associated with HR-HPV detection and this was statistically significant among those without HIV. Increasing number of lifetime sex partners is a well-documented risk factor for HPV infection, including in other studies of university students in sub-Saharan Africa . Among wThis study had some limitations. A convenience sample of university students and women attending HIV clinics in Gaborone, Botswana was obtained. Although these were recruited sequentially, the findings may not be fully generalizable to all females aged 18–22 years in Botswana. However, using similar recruitment methods in a subsequent study should produce a comparable population that can be used to assess vaccine impact. Next, the survey was administered by trained study staff, but social desirability bias could have impacted the reliability of self-reported sexual behavior risk factors. Further, because this study was not powered to evaluate risk factors for HPV detection, adjusted analyses should be interpreted with caution; we described some associations based on a higher than conventional p < 0.05 threshold and variables not selected in these models could possibly be associated with HR-HPV. Lastly, perinatal HIV infection status was self-reported and although status was verified in medical records if available, some participants may have been misclassified.In conclusion, the findings from this study indicate a high prevalence of HPV, including HPV types targeted by the quadrivalent vaccine, in women living with and without HIV in Botswana. The Botswana HPV vaccination program is expected to have a substantial impact on reducing HPV infection and HPV-associated cancers. The data from this study will enable the first evaluation of HPV vaccine impact in Botswana in 3–4 years, when vaccinated girls reach the ages of 18–22 years. Importantly, the data will also allow future monitoring of HPV vaccine impact among WLHIV, including both perinatally and non-perinatally infected women, for which there is a critical gap of evidence. This report is a first step in understanding HPV vaccine impact in Botswana, which will inform and strengthen HPV vaccination programs in low- and middle-income countries worldwide.


N = 153) from a large university in the south participated. Descriptive statistics, chi-squared tests, and independent t-tests (p ≤ 0.05) were used to characterize the students’ vaccination status. Logistic regression was used to identify factors associated with vaccination status. HPV vaccination rates were low. Students who were older and married or living with a partner were less likely to have completed the HPV vaccine series. The most commonly cited reason for non-initiation and non-completion was the lack of provider endorsement. Vaccination status did not differ significantly according to race/ethnicity, religion, skills, or intention to counsel parents. While intentions to counsel parents on HPV vaccination are high among student nurses, interventions to improve vaccination rates among student nurses are needed. We identified factors associated with student nurses’ Human Papillomavirus Vaccine (HPV) vaccination status and their intention to counsel parents on HPV vaccination. Undergraduate student nurses ( Human papillomavirus (HPV) is the most prevalent sexually transmitted infection (STI) in the United States . Before Sexually active adolescents and young adults have the highest prevalence of HPV infection, with 50–80% of cases occurring within 2–3 years of sexual debut . It is eThe Advisory Committee on Immunization Practices (ACIP) recommends the HPV vaccine to 11- to 12-year-old adolescents, with catch-up vaccination for those aged 13–26 years .Although HPV affects all individuals, college-aged persons face substantially greater risks of HPV-related diseases than older adults; yet, many remain under-vaccinated . In 2016College-aged youth are a priority group for HPV catch-up vaccinations . ExistinStudent nurses, as future professionals in the largest segment of the health care system, are uniquely positioned to provide vaccine education, address parental concerns, and offer strong vaccine endorsements. Research has shown that nurse-led vaccine promotion improves uptake ,17. StudThe primary aim of this study was to identify factors associated with student nurses’ HPV vaccination status, their preferred counseling strategies, perceptions of parental vaccination hesitancy, and to assess whether their skills and intentions to counsel parents differed by their vaccination status. We assessed student nurses’ HPV vaccination status in a diverse sample of student nurses recruited from a large urban nursing school in the southwest. The data in this study were derived from the baseline survey of a large randomized controlled intervention trial. Undergraduate student nurses were recruited between 2015 and 2018. Data collection began after approval from the university’s Institutional Review Board. Survey items were drawn from measures used in previous published studies of HPV vaccination uptake ,21, skilDemographics pertaining to age, race/ethnicity, marital status, and religion were collected. The students’ vaccination status, including vaccination non-initiation, initiation, and completion, were assessed. Non-initiation was defined as not receiving any HPV dose. Initiation was defined as receiving at least one dose of HPV vaccine. Completion was defined as having completed all three doses of the HPV vaccination series (the recommended dosing at the time of this study). Factors associated with vaccination status were assessed using questions adapted from Kessel et al. . ParticiThe HPV-endorsement outcome expectation scale was used to assess students’ beliefs about HPV vaccine endorsement; using an eight-item scale developed by McRee, Gilkey, and Dempsey . StudentIntention to counsel parents on HPV vaccination was assessed via two items developed by investigators. Perceived parental barriers to HPV vaccine uptake and preferred counseling strategies were assessed using items from the Minnesota Health Care Provider Survey . p values of less than 0.05 were considered statistically significant for all analyses. Bivariate frequencies and chi-squared tests were then calculated to compare HPV vaccination status by demographic variables. Statistically significant variables were included in the final multivariable analyses of HPV vaccination initiation and completion. Bivariate associations of age and marital status with HPV vaccination completion were examined using logistic regression and were expressed as unadjusted odds ratios (OR). For the logistic regression analyses, HPV vaccination completion was dichotomized. The relations between age, marital status, and HPV vaccination completion were then examined using a multivariable logistic regression model. Data were analyzed using IBM Statistical Package for Social Sciences . Descriptive statistics were used to analyze demographic distribution and to stratify the participants by vaccination status. t-tests were used to determine the mean differences between non-initiators and initiators and between initiators and completers. The rest is on your table. Finally, we examined differences in intentions to counsel parents on HPV vaccination status between non-initiators and initiators and between initiators and completers. The Mann–Whitney U test was used due to a non-normal distribution of the data.Univariate descriptive statistics were computed to describe the distribution of each outcome variable. Next, independent-samples N = 153) was 24.5 years (SD = 5.95), with 73% (n = 112) aged between 18 and 26 years. The majority of participants were non-Hispanic  and self-identified their race as either Caucasian , Asian , African American  or Other . Nearly three-fourths of the participants  were single, with 30% (n = 46) stating that they were married or living with a partner. The vast majority of the participants identified as religious ; 73% (n = 111) self-identified as Christian (The mean age of participants (hristian . n = 64) of the students reported that they had not initiated HPV vaccination (n = 65) reported that they had received all three doses of the HPV vaccine. Caucasian  and Hispanic  participants were more likely to initiate the vaccine than Asian  or African American  students. Caucasian and Hispanic students had higher rates of HPV vaccination completion  than Asian  and African American  students.Forty-two percent  among those who had received at least one dose of HPV vaccine (initiators). Specifically, married students were less likely to have received the HPV vaccine than were single students. In addition, the relationship between vaccination completion and age was significant  among completers and non-completing initiators; older students were less likely to have completed the HPV vaccine series than younger students. The percentage of students who were non-completing initiators and completers did not differ by race , ethnicity , or religion  (data not shown). Therefore, only age and marital status were included in the logistic regression. Chi-squared tests indicated a significant relationship between HPV vaccination completion and marital status (Xp = 0.031) were almost three times more likely to have completed HPV vaccination than married students. Age  was significantly associated with a decreased likelihood of vaccination completion; an increase of one year in age was associated with a 14% reduction in the odds of vaccination completion. The Hosmer–Lemeshow fit statistics indicated an acceptable model fit   .n = 89) reported that the most important factors influencing vaccination initiation were a provider’s recommendation , confidence in the HPV vaccine , having a parent who recommended the vaccine , and having adequate knowledge of HPV disease and the vaccine . The most commonly cited factors that influenced vaccination completion were age (<26) , a provider’s recommendation , and the convenience of receiving the vaccine . The most common reasons for not completing the HPV series among non-initiators and non-completing initiators were age eligibility (>26 years) , lack of provider recommendation , having an intention to complete the HPV series but not having done it yet , and not being sexually active . Students who had received at least one HPV vaccine dose  or between vaccination initiators and completers   . n = 73, 57%) and their discomfort in talking with their children about sex . Perceived factors that contributed “a little” or “not at all” to parental decisions to delay or refuse HPV vaccination for their child included the parents’ belief that the HPV vaccine is not very effective , that parents let their children decide and the children refuse the vaccine , and that the child was sick at the time of the visit . In addition, student’s perception that parental concern that their children would suffer immediate short-term effects  and/or long-term complications from the HPV vaccine  contributed “a little” or “not at all” to parental delay or refusal of the HPV vaccine. Most students perceived that the factors contributing to parental decisions included parents’ beliefs that their children are not sexually active  and specific parental concerns  and providing information separately to parents and adolescents . Participants perceived that providing information to parents about the HPV vaccine prior to the clinical visit  and giving interactive decision aids to prioritize their health values  would also be beneficial when counseling parents. Students perceived that the following tools and strategies would be most helpful in counseling parents who hesitate to vaccinate their children for HPV: information tailored to the patient’s cultural background  . Of thosWhile the number of young adults receiving the HPV vaccine has increased over the last several years, there are still disparities by race/ethnicity . In thisIn our study, we found that higher vaccination completion was associated with being unmarried. This may be related to a greater perceived risk of HPV infection among unmarried student nurses. Those reported being married or living with a partner were less likely to have completed vaccination. This may be due to a belief that they are not at risk of contracting STIs. The literature suggests that single women were significantly more likely than married women to be interested in HPV vaccination . TherefoAdditionally, provider’s recommendation was the most influential factor associated with vaccine initiation and completion among student nurses. One study found that young adults who received a provider’s recommendation were over 35 times more likely to receive at least one dose of HPV vaccine than those who received no recommendation . Differences in vaccination status associated with the students’ intentions to counsel, their outcome expectations, and their skills satisfaction as both a sexual-health parent-communication counselor and as an adolescent sexual-health educator were also assessed. Surprisingly, there was no significant difference between the two groups (non-initiators vs. initiators and initiators vs. completers). These findings suggest that vaccination status did not have a negative or positive impact the students’ beliefs about counseling, skills satisfaction, outcome expectations, or intentions to counsel parents about the HPV vaccination. This may be due the low variability in the overall sample scores on skills satisfaction, outcome expectations, and intentions to counsel parents about the HPV vaccination which were high among the total student nurses ,37.While there is some evidence that a nurse’s personal beliefs and self-confidence in addressing vaccine concerns drive their vaccine-endorsement practices ,39, we dOur findings indicate that the student nurses perceived parental barriers—such as the belief that their child is not sexually active, discomfort in talking with their child about sex, and the belief that their child was unlikely to get an HPV-related disease—as factors that impact HPV vaccination rates. This is consistent with the previous literature, which suggests that factors such as reluctance to discuss sexual health, a child’s age, and a perception of low risk are common barriers to HPV vaccine uptake and completion among patients ,42. The limitations of this study include the use of previously collected data and self-reported measures without confirmatory HPV titers or confirmation of vaccination records. Self-reported measures  may reflect what student nurses felt to be the most desirable answer and may not indicate their actual future practices. Additionally, participants were recruited using a purposive sample of student nurses from one Bachelor of Science nursing school in an urban area, which may limit the generalizability of the findings. While data on gender were not collected, it is important to note that most student nurses were female. Gender differences in vaccination rates are well documented, with females reporting higher HPV vaccination rates . Despite these limitations, the focus on student nurses is an innovative approach to deepening our understanding of the HPV vaccination status of student nurses and of the barriers to and facilitators of vaccine endorsement. This research provides insight on student nurses, who are transitioning to a nursing role in which they will have the opportunity to promote HPV vaccination and to reduce missed opportunities for HPV prevention. This study’s findings provide direction for future programs and interventions aimed at young adults and emerging health care providers that address vaccination concerns and may increase awareness, HPV vaccine uptake, and vaccination completion. Given the growing epidemic of HPV infection, more research is needed, particularly among high-risk populations and future health care providers that may increase vaccine-promoting behaviors and impact clinical practice. Although the current acceptance of HPV vaccination is promising, uptake and completion rates remain low among college students, specifically student nurses. Therefore, it is important to develop targeted interventions to increase vaccination rates among student nurses to protect them from HPV-related diseases. Student nurses are uniquely positioned to promote HPV vaccination acceptance and completion despite their own personal vaccine status.