LATE-PCR) and melting curve analysis. The 23 most prevalent HPV strains  are assayed in two sealed reaction tubes within 2 h. Good sensitivity and specificity was evaluated by testing cloned HPV DNA and clinical samples. The detection limit was 5–500 copies/reaction depending on the genotype. No cross-reactivity was observed with the other HPV types that are not covered by our method or pathogens tested which were commonly found in female genital tract. When compared with the HPV GenoArray Diagnostic kit, the results from 1104 clinical samples suggest good overall agreement between the two methods, and the kappa value was 0.954. Overall, this new HPV genotyping assay system presents a simple, rapid, universally applicable, sensitive, and highly specific detection methodology that should be useful for HPV detection and genotyping, therefore, is potentially of great value in clinical application.We describe a new assaying system for the detection and genotyping of human papillomavirus (HPV) based on linear-after-the-exponential-PCR( Cervical cancer is one of the most prevalent cancers in women worldwide, with estimated 527,624 new cases and 265,672 deaths in 2012. Over 85% cervical cases were diagnosised in developing countries . The majPCR and Melting curve analysis (LPM) for the DNA detection and genotyping of the 23 most common HPV strains  [In this study, we developed a new assay that uses LATE- and 83) . We evalOne liquid-based cytology (LBC) sample was collected from each of 1108 patients aged 19–89 years  who visited the DiAn Medical Laboratory for routine HPV screening or genotyping from August to December 2017. Additional 920 HPV carrying samples , which diagnosised positive using the HPV GenoArray test kit or by type-specific PCR sequencing , were selected and stored at −80°C until use. The chosen samples were collected from 920 patients aged 23–92 years  from January 2016 to May 2017. All samples were collected using a cervical cytobrush and stored in PreservCyt solution (U.S. Cyt Company)tubes along with the cytobrush. The cervical sample collection and followup studies were approved by the ethics committee of DiAn Medical Laboratory (Reference number DA-YXLC-20170619-1).The LBC samples were used for genomic DNA extraction using a QIAamp DNA Mini kit  as per manufacturer’s instruction. DNA was eluted from the columns with 100 μL AE buffer and stored at −20°C.http://unafold.rna.albany.edu/?q=DINAMelt/Quickfold) and the probe–target hybrid folding program DINAMelt  was used to predict the possible hybrid structures and Tm values. Sample quality is monitored by amplifying a 100-base pair (bp) fragment of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene using the primers G100f 5′-CTGCTCACATATTCTGGAGGAG-3′ (forward) and G100r 5′- AAAAGCAGCCCTGGTGACC-3′ (reverse). All molecular beacon probe sequences and corresponding targets are listed in Various consensus primers have been reported for the amplification of a specific fragment of L1 the region of HPV DNA, including GP5+/6+, MGP, MY09/11, and PGMY09/11 . After c2 , 12.5 nM dU plus deoxynucleotide triphosphates , 37.5 nM limiting primer MY11 (5′-GCMCAGGGWCATAAYAATGG-3′), 500 nM excess primer GP6+ (5′-GAAAAATAAACTGTAAATCATATTC-3′), two units Hot-Start Taq polymerase (Takara), and one unit Uracil DNA Glycosylase . Probes in reaction A were P6(25 nM), P58 (15 nM ), P68(15 nM), and P59(20 nM), and for reaction B there were P51(25 nM), P66(25 nM), P73(15 nM), P83(15 nM), P45(20 nM) and P53(20 nM). Reaction B also contained internal control primers G100f /R (50 nM) and probe GAPDHP(20nM). All primers and probes were ordered and purchased from Invitrogen Life Technologies . A LightCycler480  real time quantative thermo cycler was used for LATE-PCR. The PCR was programmed as below: 10 min at 50°C and 5min at 95°C, followed by 55 cycles , preceded by a melting program starts at 37°C with 1°C increment per 30 sec interval until 85°C. Fluorescence signals in four different channels were collected and analyzed: FAM (465–510), HEX (533–580), TXR (533–610), and CY5 (618–660). To generate the Tm values, melt-curve analysis using the first derivative of fluorescence at temperatures between 37°C and 85°C was used to process the fluorescence data of the probe–target hybridizations.LATE-PCR was performed in a volume of 40 μl with 5 μl of extracted DNA as template. Each sample was tested in two reactions: A and B. Both reaction mixture contains 1× PCR Buffer , 2 mM MgClm values were collected. The nonparametric rank percentile method determinations of the 2.5th and 97.5th individual percentiles, as well as 95% confidence intervals for the upper and lower limits of each reference interval was used to calculate the reference interval. Results were evaluated using SPSS 16.0 statistical software. The normal distribution was examined by Kolmogorov–Smirnov Z-test.The 920 HPV-positive samples were further tested using the LPM genotyping assay described above and TRecombinant plasmids containing the cloned full-length L1 gene were used to assess the analytical sensitivity of our method.The inserted DNA fragments of each HPV type were PCR amplified from corresponding HPV positive samples that were diagnosised by the HPV GenoArray test. The pMD19-T vector(Takara) was used as the carrier and the construction of pMD19-T-L1 was performed following the manufacturer’s instruction. The accuracy of cloned L1 fragments were examined by DNA sequencing follow by BLAST, the results were 100% identical to the reference sequences. An ABI3130 genetic analyzer sequencing instrument was used for sequencing in our experiment.6 copies/ml. Equal amount (40 ng/ml) of human genomic DNA was added into each dilution. Five μl of each plasmid dilution were used as template in LATE-PCR and each HPV type run in 20 replicate reactions per dilution.The concentration of purified plasmids were quantified using a NanoDrop1000 Spectrophotometer . Copy numbers of the cloned HPV L1 genes in the stock were calculated using the molecular weights of the recombinant plasmids; Quantified plasmids were diluted with TE buffer (pH 8.0) to prepare serial of dilutions with concentration from 1 to 10Neisseria gonorrhoeae (ATCC49226)/ Candida albicans (ATCC90028)/ Escherichia Coli (ATTCC25922)/ Staphylococcus aureus (ATTCC25923)/ Herpes simplex virus type II/ Treponema pallidum/ Ureaplasma urealyticum,/Mycoplasma hominis positive using the QIAamp DNA Mini kit. Five microliter of HPV26, 40, 43, 44, 54, 61, 67, 69, 71, and 72 plasmids at a concentration of 108 copies/ml purchased from National Institutes for Food and Drug Control were used as templates in LATE-PCR .To evaluate the specificity of our method, DNA was purified from LBC samples that were diagnosised P value≥0.05 was considered not statistically significant.We next compared our method with the GenoArray test, 1108 LBC samples were tested by both method in parallel. The GenoArray test is an L1 consensus primer-based PCR assay that covers 21 HPV genotypes, including type16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 53, 66, 6, 11, 42, 43, 44 and 81. The low-risk HPV genotypes 43 and 44 were out of the detection range of the LPM assay and genotypes 70, 73, 82, and 83 could be detected, but not quantified by LPM assay. All GenoArray detection procedures were performed according to the manufacturer’s instructions, and LPM genotyping was conducted as described above. All data were analyzed with SPSS statistical software(version 16.0). The coincidence rate between the two detection methods was assessed using absolute agreement and Cohen’s kappa. The nonparametric McNemar test was used to analyze the complementarity of the two methods and if there was significant difference between the results obtained by the two methods. Difference with et al. [http://www.ncbi.nlm.nih.gov/BLAST).Sequencing was performed when there was discrepancy in the results between the two assays as described by Peng et al. . The PGMm values were recorded. Each Tm value were normal distribution tested; The reference Tm values were generated and evaluated using SPSS 16.0 software. Reference Tm value intervals for each HPV type are shown in m value overlapping was observed.For each HPV type, the results from forty HPV-positive clinical sample were used to establish reference intervals that are with statistically significance. All samples were tested with the LPM assay as described above and the T6 copies/ml) and added human genomic DNA were used as mimic of HPV DNA extracted from clinical sample to assess the sensitivity of LPM assay.Constructed L1 gene carrying plasmids with gradient concentration  that ≥ 95% of test runs produced positive results. The detection limit for HPV6, 11, 16, 18, 33, 35, 45, 52, 56, 58, 66, 68, 73, and 81 was 50 copies/reaction; 500 copies/reaction for HPV31, 42, 51, 53, and 59; and 5 copies/reaction for HPV39, 70, 82, and 83.Both assay detects largely overlapping sets of HPV genotypes, and HPV genotyping results from 1108 LBC samples were compared; the results were mostly concordant or compatible. Out of the 1108 samples tested, four failed to generate melting peak in the control channel of the LPM assay, suggesting that there was either insufficient cells in the collected sample or presence of PCR inhibitor in the extracted DNA. Therefore, results from 1104 samples with used to compare the performance of the two methods. Overall, the absolute agreement between the tests was 98.37% (95% CI: 97.44%–98.97%) and the kappa value was 0.954, indicating good agreement. The positive concordance rate was 98.94% (95% CI: 98.00%–99.44%) and the negative concordance rate was 96.43% (95% CI: 93.35–98.11%) . SubsequAmong the 1104 cases studied, 73 had discordant GenoArray and LPM assay results, six exhibited discordant sequencing and LPM assay for HPV70, 73, 82, and 83. The 73 discordant cases were further analyzed by type-specific PCR followed by sequencing . AccordiSome patients suffered from infection from more than one type of HPV, in the aspect of detecting multiple HPV infection, minor variations were observed between the GenoArray/Sequencing and LPM assay (P≥0.05) . Both asSince the discovery of HPV as the major cause of cervical cancer, various methods have been developed for HPV detection and genotyping. The majority of which are based on molecular biology techniques. The Hybrid Capture 2 system  detects 13 HR-HPV types  and five LR-HPV types  by signal amplification technique. It distinguishes high-risk from low-risk types, but incapable of HPV genotyping . Typing Currently, HPV GenoArray test is one of the most used methods for HPV genotyping in China. This method covers 21 HPV genotypes including 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 81. Numerous studies have proven its reproducibility and clinical sensitivity. Therefore, we used it as reference for the LPM assay in this study . Howeveret al. performed rapid universal identification of bacterial pathogens from a list of 111 species in 64 different genera using a molecular beacon melting temperature signature technique [et al. described a molecular beacon based system that successively distinguished 27 different Mycobacterium species [Real-time PCR has long been used in Vitro pathogen detection and identification, its limitation is only limited number of targets can be detected in one reaction, due to the limited fluorescence channel provided by the real-time PCR instrument –26. Howeechnique . Additio species . These a species . The senIn conclusion, in our study, the LPM assay showed similar clinical performance for genotyping as the HPV GenoArray test and seemed to be a promising alternative approach for HPV testing. It is sensitive, economical and high throughput that could be used for rapid and specific detection of 23 high- and low-risk HPV genotypes.


We evaluated the prognostic and diagnostic ability of p16/Ki-67 immunocytochemistry, HPV E6/E7 mRNA testing and HPV DNA assay in triaging ASCUS to find a way to manage cervical lesions more effectively.We conducted a prospective study through follow-up. The detection methods of the three factors: p16/Ki-67 immunocytochemistry conducted by using the CINtec® Plus Kit, E6/E7 mRNA testing by QuantiVirus®HPV E6/E7 mRNA assay and DNA by Hybrid Capture 2 assay.χ2 = 5.718, P = 0.023) and HPV DNA assay vs. p16/Ki-67 immunocytochemistry . The specificity of E6/E7 mRNA testing, p16/Ki-67 and DNA assay in triaging ASCUS was 44.33, 75.26 and 11.34% respectively and is all statistical difference , χ2 = 19.297, P < 0.001(HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry), χ2 = 80.707, P < 0.001(HPV DNA assay vs. p16/Ki-67 immunocytochemistry). The expression level of 2097.09 copies/ml was the optimal cut-off value for HPV E6/E7 mRNA testing to diagnose CIN2+, the sensitivity and specificity was 61.1 and 68.2%.One hundred three women with ASCUS satisfied requirements and completed the entire follow-up process. All CIN2+ occurred in women who were mRNA positive at baseline, none in mRNA negative. 100% (6/6) patients with CIN2+ were HPV DNA assay positive, 100% (6/6) were HPV E6/E7 mRNA testing positive and 50.0% (3/6) were p16/Ki-67 immunocytochemistry positive. The risk ratio of E6/E7 mRNA test was 57.306 . For endpoint of CIN2+, the sensitivity between HPV DNA assay and HPV E6/E7 mRNA testing is no statistical difference, but statistical difference exists between HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry (High expression of HPV E6/E7 mRNA could be a good candidate as a diagnostic biomarker to triage ASCUS superseding HPV DNA. p16/Ki-67 immunocytochemistry is suggested to be a good tool to triage ASCUS, but it reduced the sensitivity of diagnosis when improves the diagnostic specificity. The aim in primary screening for cervical cancer is to find out the high-grade cervical intraepithelial lesions and lesions which may develop into high-grade cervical intraepithelial lesions in the future. Liquid-based cervical cytology acted as the most important strategy for cervical cancer screening have reduced cervical cancer incidence and mortality during the last decades . AccordiBiomarkers of cervical cancer are needed to identify those HPV infections which may develop into a high-grade lesion. The most important process in cervical carcinogenesis progression is that HPV insert its sequence, especially the abnormal expression of E6 and E7 oncogenes, into the host genome. p16INK4a (p16) -overexpression is thought as a sign for deregulating E7 expression , 11. Ki-HPV E6/E7 mRNA test and p16/Ki-67 immunocytochemistry has been proposed as promising biomarkers for HPV oncogene expression. Previous studies had showed that HPV E6/E7 mRNA test and p16/Ki-67 immunocytochemistry have a large clinical value in cervical cancer screening , howeverThe study population consisted of female who visited outpatient clinic of gynecology department of the third affiliated hospital of Zhengzhou University from September 2015 to September 2017. Follow up was performed according to local colposcopy protocols. 300 women with ASCUS entered our study and all of them underwent Pap test, colposcopy biopsy and histopathological examination. These patients with ASCUS and histological diagnosis of cervical inflammation or mild cervical intraepithelial neoplasia  and no cervical destructive treatment or local and/or systemic medical treatment were recruited in this follow-up study. During the follow-up, 54 cases were excluded because the histological result is CIN2, CIN3 or cervical cancer. 28 cases were excluded due to cervical destructive treatment, 18 cases were excluded because positive cytology or HPV was found but not followed by colposcopy, 81 cases were excluded due to local and/or systemic medical treatment, 16 cases were excluded due to loss of follow-up. At last, 103 cases satisfied the requirements and completed the entire follow-up process. All subjects were followed up until 1 September 2018 and the duration of follow-up is at least 12 months (12 months–24 months). one or more cervical cancer screening was carried out and the last follow up episode determined the final follow-up result. When the last follow up episode is a negative cytology and HPV DNA, the final follow-up result defined as negative. When the last follow up episode is a positive cytology or HPV DNA, the final follow-up result was defined by colposcopy and histopathology. For women with cervical inflammation or CIN1, the final follow-up result of CIN2 or more serious defined as progression of cervical lesion. This study was reviewed and approved by the Ethics Committee of The Third Affiliated Hospital of Zhengzhou University. All women signed the consent forms and were informed regarding the purpose of the proposed study.The technology of LBC was used for the cytology test. Thinlayer slides were prepared using the Thin Prep 2000 Processor  according to the manufacturer’s instructions. The cytological specimens were reported using the 2001 Bethesda Reporting System Criteria [A second cytology slide was prepared from the residual LBC specimen using a T2000 slide processor . Immunostaining of cervical cytology slides for p16/Ki-67 was performed using the CINtec® Plus Kit  according to the manufacturer’s instructions. A case was considered positive if one or more cervical epithelial cell(s) stained both with a brown cytoplasmic stain (p16) and a red nuclear (Ki-67) irrespective of the interpretation of morphologic abnormalities. Slides without any double-stained cells were called negative for p16/Ki-67 dual-stain cytology .Residual LBC samples were also used for the detection of E6/E7 mRNA of 14 HR-HPV types by QuantiVirus®HPV E6/E7 mRNA assay  according to the manufacturer’s instructions. If the copy number was greater than or equal to 1.0, the QuantiVirus® HPV assay result for the patient was positive. Otherwise, the result was negative .HPV DNA was detected by Hybrid Capture 2 assay , according to the manufacturer’s instructions. RLU/CO ≥1 was identified as positive.Women with ASCUS Pap smears underwent colposcopy biopsy within 4 weeks after cytology test, according to colposcopy operating norms. All histological slides were diagnosed according to current World Health Organization classification. In this study, the diagnoses of CIN2, CIN3 and carcinoma are referred as ≥ CIN2 (CIN2 +). Histology was regarded as the “gold standard”, but for those whose cytology and HPV DNA were negative, the final results of follow-up were considered to be negative. The diagnoses of cytology and HPV DNA negative (no biopsy), cervical inflammation and CIN1 are referred as < CIN1- (CIN1-).p value < 0.05 was considered the cut-off level for statistical significance for all analysis.Statistical analysis was performed using SPSS 21.0 software. Chi-square tests were used to compare differences between groups when type of date was qualitative variable. Youden’s index was calculated as sensitivity% + specificity% - 100. Receiver operating characteristic (ROC) curve was used to assess the optimal diagnosis of E6/E7 mRNA expression testing. The risk of CIN2+ in ASCUS was evaluated by Cox’s proportional hazards regression model. All tests were two-sided and One hundred three women with ASCUS satisfied requirements and completed the entire follow-up process. Table We calculated the positive rate of different methods in different levels of cervical tissue. The overall test positivity was 89.3% 92/103) in HPV DNA assay, 58.3% (60/103) in HPV E6/E7 mRNA testing and 26.2% (27/103) in p16/Ki-67 immunocytochemistry. HPV E6/E7 mRNA testing and p16/Ki-67 immunocytochemistry was less frequently positive in ASCUS than HPV DNA test. 82.3% (51/62) patients with no biopsy were HPV DNA assay positive, 46.8% (29/62) were HPV E6/E7mRNA testing positive and 16.1% (10/62) were p16/Ki-67 immunocytochemistry positive. 100% (17/17) patients with inflammation were HPV DNA assay positive, 70.6% (12/17) were HPV E6/E7 mRNA testing positive and 5.9% (1/17) were p16/Ki-67 immunocytochemistry positive. 100% (18/18) patients with CIN1 were HPV DNA assay positive, 72.2% (13/18) were HPV E6/E7 mRNA testing positive and 72.2% (13/18) were p16/Ki-67 immunocytochemistry positive. 100% (6/6) patients with CIN2+ were HPV DNA assay positive, 100% (6/6) were HPV E6/E7 mRNA testing positive and 50.0% (3/6) were p16/Ki-67 immunocytochemistry positive. The positive rates of HPV DNA assay, HPV E6/E7mRNA testing and p16/Ki-67 immunocytochemistry in CIN1- and CIN2+ were statistically compared in Table /103 in HP < 0.05).The expression level of HPV E6/E7 mRNA testing in different cervical lesions is shown in Fig. χ2 = 5.718, P = 0.023) and HPV DNA assay vs. p16/Ki-67 immunocytochemistry . The specificity is all statistical difference , χ2 = 19.297, P < 0.001(HPV E6/E7 mRNA testing vs. p16/Ki-67 immunocytochemistry), χ2 = 80.707, P < 0.001(HPV DNA assay vs. p16/Ki-67 immunocytochemistry). ROC curve was used to determine an optimal cut-off value and further demonstrate the diagnostic performance of HPV E6/E7 mRNA testing for detecting CIN2+ . Johansson et al. . found tThe positive rates of different tests were statistically different in different cervical lesions and the more serious the lesion, the higher the positive rates, which can be confirmed by many studies , 25. HPVχ2 = 26.277, P < 0.001, HPV DNA assay vs. HPV E6/E7 mRNA testing; χ2 = 80.707, P < 0.001, HPV DNA assay vs. p16/Ki-67 immunocytochemistry). The sensitivity between HPV DNA assay and HPV E6/E7 mRNA testing is no statistical difference , but statistical difference exists between HPV DNA assay vs. p16/Ki-67 immunocytochemistry . This support that compared to HPV DNA assay, HPV E6/E7 mRNA testing improves the diagnostic specificity without reducing the sensitivity, while p16/Ki-67 immunocytochemistry improves the diagnostic specificity in a situation of reducing the sensitivity of diagnosis. However, Miriam Reuschenbach et al. [P = 0.32), but a higher specificity(P < 0.0001). This difference may happen because the different of study method .Our study found that the specificity of p16/Ki-67 immunocytochemistry and E6/E7 mRNA testing for detection of CIN2+ was higher than HPV DNA test . Thus, a ROC curve was used to further demonstrate the diagnostic performance of HPV E6/E7 mRNA testing, and the optimal cut-off value of mRNA testing is selected at 2097.09 copies/ml. Tong-Yu Liu’s and Ye-li Yao’s finding also supported that when an optimal cut-off value of mRNA testing was found out, the diagnosis accuracy will be improved [The expression level of HPV E6/E7 mRNA was calculated in this study. The E6/E7 mRNA showed a higher expression levels in CIN2+ than in CIN1−, and the differences between the lesions were statistically significant (Z = − 3.407, improved . As the The follow up after negative and CIN1 histology of ASCUS should be intensive for women with positive HPV E6/E7 mRNA, positive p16/Ki-67 immunocytochemistry and positive HPV DNA, and women with negative result of these test may be retested after longer intervals than currently performed. High expression of HPV E6/E7 mRNA could be a good candidate as a diagnostic biomarker to triage ASCUS superseding HPV DNA. p16/Ki-67 immunocytochemistry is suggested to be a good tool to triage ASCUS, but more research is needed to support this view.


Among cervical cancer patients in the U.S., a disproportionate number are Hispanics/Latinos. Also, about a third of patients diagnosed with cervical cancer annually in Mexico die of the disease. Vaccines are available to protect against HPV, the cause of cervical cancer.A cross-sectional study was conducted with 200 mothers of Mexican origin in the U.S. Midwest and Xalapa, Veracruz, Mexico. Based on a validated bilingual questionnaire, this study elicited information about knowledge and attitudes regarding HPV vaccination and cervical cancer.p < .0001. Logistic regression revealed that receiving information about the HPV vaccine from medical providers was a significant predictor of mothers’ willingness to vaccinate their children.Mothers living in Mexico showed better knowledge about HPV and HPV vaccine (77.8%) than participants living in the U.S. (48%) A need for increasing public health education of Mexican mothers in the Midwest on HPV/HPV vaccination, may lead to improving utilization of the vaccination and eventually a reduction of cervical cancer. HPV vaccination for boys is critical for reducing the risk of transmission to sexual partners and decreasing the risk of HPV- related diseases in the population. Therefore, we recommend increasing efforts to vaccine boys and increasing knowledge that boys must also be vaccinated, especially in Mexico. Cancer is a major global health problem. As one of the leading causes of mortality, cancer was responsible for an estimated 8.2 million deaths in 2012. Additionally, an estimated 14.1 million new cases were identified worldwide [The Human Papilloma Virus (HPV) is a causative agent of many cancers , 3. The According to the Center for Disease Control and Prevention (CDC), HPV has currently infected about 79 million individuals across the U.S. and annually infects 14 million individuals . In MexiOne of the most effective primary prevention efforts in decreasing HPV infection and consequently cervical cancer is HPV vaccination. The HPV vaccine was first introduced in the U.S. in 2006 and in Mexico in 2008. The cancers prevented with the vaccine are cervical (66.2%), anal (79.4%), oropharyngeal (60.2%), vaginal (55.1%), and vulvar (48.6%). HPV vaccines are therefore recommended for all preteens, including boys, and preferably before engaging in sexual activities to protect against infection . In 2012First-dose rates and completion rates for the vaccine vary across the U.S. by racial and ethnic groups , 15. In Previous studies have documented low levels of awareness and knowledge about HPV, the vaccine, and HPV’s role in the etiology of cervical cancer among Hispanics/Latinos living in the U.S. , 19. StuConsidering these differences in vaccination rates and the identified knowledge gaps in Hispanic/Latino groups, we developed a two-site parallel study in Xalapa, Veracruz, Mexico and Omaha, Nebraska, U.S. These cities were selected due to their similarities in population size and their relationship as sister cities. We conducted this study to examine the knowledge and beliefs about the HPV vaccine and the potential reasons for the HPV vaccination uptake rates among Mexican nationals. We initiated this study with the intention of generating new insights that may lead to interventions to improve vaccination rates of individuals in the U.S. and Mexico.This cross-sectional study was conducted between May and July of 2017 to assess how knowledge about HPV and the HPV vaccine plays a role in a mother’s decision to vaccinate her child. In the U.S., recruitment was made possible through a partnership between the University of Nebraska Medical Center (UNMC), OneWorld Community Health Centers Inc., and the Mexican Consulate at Omaha. In Mexico, recruitment was performed at the Dr. Gaston Melo Health Center through a partnership between UNMC and the University of Anáhuac Xalapa, School of Medicine. In both settings, the interviews were conducted by research members of our team who are native Spanish speakers.In the U.S., our participants were recruited from the waiting room of the Mexican Consulate in Omaha, Nebraska. The Mexican Consulate has a health office called Ventanilla de Salud (VDS) which, in coordination with OneWorld Community Health Centers, Inc. offers health care education and promotion for disease prevention to people who visit the consulate. The VDS facilitated the private space used to interview the participants of the study.In Mexico, participants were recruited from the Dr. Gaston Melo Health Center, a community health center in Xalapa, Veracruz. Clinicians provided assistance in identifying potential participants by introducing the study in a private medical office at the end of a patient’s appointment which, also served as the private space for the interview.In the U.S., to be eligible for the study, respondents needed to be female, mothers of Mexican origin living in Nebraska or a neighboring state, be at least 19 years of age, have a daughter and/or son between the ages of 9 and 18 living with her. In Mexico, the inclusion criteria were the same, with the exception that the participants had to be of Mexican origin living in Xalapa, Veracruz. In both settings, verbal consent was obtained before beginning each questionnaire.In the U.S., 132 potential participants were approached and invited to participate in the study. A total of 100 participants agreed and completed the survey.In Mexico, participants were invited from the family medicine service area of the clinic. A total of 118 individuals were approached to complete the questionnaire. 12 of those individuals refused to participate and six did not complete the questionnaire. The data analysis was conducted on the remaining 100 completed questionnaires. Anonymity was guaranteed at all times.A questionnaire was developed in both, English and Spanish, based on validated bilingual instruments used in the U.S., , 23–28. Three of the research members of our study who are of Hispanic/Latino origin and native Spanish speakers separately reviewed the Spanish and English versions to assure correct translation and cultural adaptation. After consensus, the questions were modified and adapted.The study questionnaire used in the U.S. consisted of 76 questions and only differed from the questionnaire used in Mexico in its demographic characteristics section. In the U.S., participants were given the option to complete the English or Spanish version of the questionnaire. Most of the questionnaires were administered in Spanish. In Mexico, the questionnaire consisted of 72 questions due to the adaptation of the demographic questions. The rest of the sections remained the same as the instrument used in the U.S.N = 197, Cronbach’s alpha = 0.818). One point was assigned for correct answers to the first 15 questions and three points were assigned to the last question for which more than one answer was allowed. We scored the “I do not know” option as zero. The maximum total possible score was 19 points. The level of knowledge was assessed by adding the points. The total score was divided into three categories according to the 75th, 50th and 25th percentile: high (15 or more points), medium (9 to 14 points) and low (8 or fewer points). Due to low frequency in the results for the high level of knowledge, we used two separate categories for level of knowledge: poor (score of 7 points or less) and good (score of 8 points or more).HPV knowledge was assessed by affirmation or negation of 16 statements. These statements related to the perception of risk, detection, transmission, prevention, treatment and consequences of HPV infection. The answers to 14 statements were pre-coded as: yes, no, and I do not know, and two questions had multiple choice options . Each statement asked about the importance, need, and benefits of vaccinating children in general. In addition, participants were asked about the benefits, safety, and appropriateness of HPV vaccination for children in the age group of 9–12 years. We assessed the beliefs about adverse behavioral consequences of HPV vaccination by using two questions on the Likert scale. We asked if vaccinating their children would send either of these messages: 1, it is okay to have sex and 2, she/he does not have to use safe sex practices.HPV vaccination beliefs were assessed from 12 statements and answers were yes/no/I do not know options  was conducted to identify potential predictors of mothers’ willingness to give at least one dose of the HPV vaccine to at least one of their children in the age range 9–18. A stepwise selection model was used. We controlled for country of residence, health insurance status of mothers and if mothers’ received information about the HPV vaccine from their medical provider or not as well as their HPV knowledge. Because of the statistical significance level of the LRM, we conducted statistical tests for the items that assessed the knowledge and beliefs to see if independent variables were associated or different between these two groups.p-value less than 0.05 was considered statistically significant. For the statistical analysis, SPSS version 23.0 was used.Two-sided tests were used for testing all the hypotheses. A This research was approved by the UNMC Institutional Review Board (IRB) and by the Academic Research Review Committee of the University of Anáhuac Xalapa.A total of 200 participants were recruited (100 from Mexico and 100 from the U.S.). The mean age of the mothers in the U.S. was 40.76 years (SD 6.2). The mean age of mothers in Mexico was 38.95 years (SD 6.6). Most participants had basic education levels of up to 9 years (64% in the U.S and 72% in Mexico). The majority had a stable life partner and were either married (57% in the U.S and 26% in Mexico) or cohabiting (45% in Mexico and 23% in the U.S) and almost half were dedicated to housework (47.5%). Mothers with at least one eligible child vaccinated against HPV were higher in Mexico (49%) than in the U.S (35%). Characteristics of the study population by country of residence are shown in Table N = 124) of the participants. However, when analyzing data separately by country, mothers living in Mexico showed better knowledge about HPV and the vaccine  than participants living in the U.S.  . However, the knowledge level between the two countries among mothers who did not vaccinate their children showed statistical significance . Table N = 33) of the participants selected the three correct answers: 1. By practicing abstinence, 2. By using condoms and 3. By being vaccinated. Most participants  selected two right answers, 28.5% (N = 57) only one right answer and 15.5% (N = 31) of the participants indicated that they didn’t know the answer or selected a wrong option.Overall, the knowledge of HPV and HPV vaccination was good with scores above eight points in 62.9% N = 24 of theMost of the women surveyed in Mexico (99%) and the U.S. (94%) believe that vaccines are beneficial. Similarly, 95% of the respondents in Mexico and 94% in the U.S. indicated that vaccinating their children against diseases that can be spread person-to-person is important.Furthermore, 88% of Mexican and 78% of U.S mothers indicated that if there were a vaccine that prevented cancer, they would want their children to get vaccinated. Mothers living in Mexico were more likely to respond that HPV vaccines are beneficial, safe and appropriate for preteens between 9 and 12 years old than mothers living in the U.S. (84 and 41% respectively). Additionally, they also were more likely to indicate that HPV vaccines are appropriate only for girls 40% vs. 13%) (Table % (Table p < 0.01). Additionally, the variable ‘Level of HPV knowledge’ is shown in the model with a borderline statistical significance (Table p = 0.05).The LRM indicated that receiving information regarding the HPV vaccine from medical providers was a significant predictor of mothers’ willingness to vaccinate their adolescents against HPV Table . Accordice Table . The levp = 0.95 (>.05).However, the country of residency of mothers was not a significant predictor of mothers’ willingness to vaccinate their children against the HPV. The Hosmer-Lemeshow goodness of fit test indicated that there is not a significant lack of fit as Health is a core element for the well-being of all individuals. While vaccinations have been instrumental in successfully protecting the health of communities globally, differences in knowledge, beliefs, and practices still influence the acceptability of vaccines.Results from our study report important contrasts. While there was an overall good level of knowledge about HPV and the vaccine among participants, mothers in Mexico showed better knowledge than mothers in the U.S. This is consistent with a previous finding in a study among Hispanic/Latino parents .We found small proportions of participants from both countries recognizing that practicing abstinence, using condoms and being vaccinated are methods for preventing HPV infection. This observation is consistent with results from a mixed-methods study that showed misinformation about HPV and cervical cancer prevention among adolescent of Hispanic/Latino origin .Participants in both countries chose health care professional recommendations as one of the most important factors in their decision to vaccinate their children. This finding is also consistent with previous studies in the U.S. where Hispanics/Latinos consistently cited health care professional recommendations as one of the most important factors in their decision . AdditioThis study has its own strengths and limitations. In the U.S., the Mexican Consulate facilitated recruitment due to its high number of visitors. The study minimized bias in the U.S. by using a recruitment site not related to a health care institution which may have been more likely to have access to participants already vaccinated. This allowed the group of obtained participants to be very diverse.Participants from Mexico were patients from the Dr. Gaston Melo Health Center. However, in our intent to have a representative sample of the community and minimize bias, we selected a community health center which is free of cost and available to all patients regardless of health insurance status or appointment.Our study was limited by small sample size and the restriction of sampling to the Midwest U.S. and Xalapa, Veracruz. Thus, we cannot generalize the findings from this study to the overall Mexican population in both the U.S. and Mexico. We recognize that there is tremendous variability within the Mexican population across the U.S as there is across Mexico. This means that, as done here, caution must be taken when comparing both groups while recognizing their distinct environment and access to resources.Results from the present study suggest a need for enhancing public health education among Mexican mothers in the Midwest on HPV/HPV vaccination and its association with cancer in males and females. Separately, given that mothers in Mexico were more likely to consider the vaccine as only appropriate for girls, it is necessary to use a gender-neutral approach when developing prevention programs in Mexico. HPV vaccination for boys is critical for reducing the risk of transmission to sexual partners and decreasing the risk of HPV-related diseases in the population. Therefore, we recommend increasing efforts to vaccine boys and increasing knowledge that boys must also be vaccinated, especially in Mexico.Given the importance of prevention efforts, there is a need for more reliable measures to estimate levels of knowledge, awareness and beliefs about HPV and HPV vaccination. These estimates are essential tools for developing educational programs.U.S. policies should also support vaccination in school settings. In fact, schools in both the U.S. and Mexico should include more health-based education delivered in a culturally and linguistically appropriate manner to parents of Hispanic/Latino origin.


After sample and SNP quality control, 166,505 SNPs in 161 samples  were examined. None of the SNPs reached genome-wide significance, and several candidate SNPs for future study were selected, including rs10999435 on chromosome 10q22, rs1322054 on chromosome 9q32-33, and rs10902171 on chromosome 11p15. Luciferase assay identified rs1322054 as the primary functional variant to regulate gene expression in immune cell. Further studies are needed to determine the genetic background of different integration patterns of HPV in cervical cancer patients.The present study investigated the association between single nucleotide polymorphisms (SNPs) in immune- or DNA repair-related genes and the integration pattern of human papillomavirus (HPV), a promising prognostic marker in cervical cancer. The HPV integration patterns of cervical cancer patients were determined by polymerase chain reaction and  However, the response to radiotherapy varies widely among patients, underscoring the need to identify biomarkers to predict patients’ treatment outcome. Recently, the integration pattern of human papillomavirus (HPV) has been investigated as a promising biomarker for cervical cancer patients2. The survival rate is higher in patients with tumors containing abundant episomal HPV DNA than in those with completely integrated HPV DNA or no HPV DNA2. We hypothesized that impairment of the DNA damage response and immune response, which affect the response to radiotherapy, may be associated with the HPV integration pattern. The bases of this hypothesis are as follows. First, the presence of high-risk HPV for a long period of time is dependent on its resistance to immune clearance mechanisms in the body, and episomal HPV DNA may be differently targeted by the immune surveillance system. High-risk HPV develops several mechanisms to avoid host immune response and persists as episomes in the HPV-infected cells in transformation zone of the cervix before progression to cervical cancer. One of the strategies to avoid detection is to prevent pro-inflammatory signal by blocking recruitment of antigen-presenting cells and the release of cytokines that mediate the immune response. In W12 keratinocyte model where HPV16 is naturally infected into normal keratinocyte and sustain as episomal status, it was shown that HPV16- integrated cells emerged at the same time episomes are cleared, and complete deregulation of viral oncogenes did not occur without complete reduction of E2, which is expressed by viral episomes. Gene expression comparison between the W12 cells with episomal HPV and integrated HPV by microarray analysis showed that disappearance of episome was associated with rapid increase in type I interferon pathway with rapid change in genes related to antiviral response3. In the same W12 model, it was also shown that episomal clearance caused by interferon-beta resulted in de-repression of viral integrants and hastened changes of HPV physical status from episomes to integrant4. The persistence of episomal HPV DNA in transformed cells provides high amounts of viral antigen through active replication, particularly E2. These cells are preferentially targeted by the immune system5 compared with cells with no or lower rates of viral replication. The viral antigen E2 is a major target of T cells during HPV infection6. Second, some individuals may be more vulnerable to HPV integration, which requires homologous recombination (HR) between HPV and the host genome7. These individuals may be similarly vulnerable to radiation-induced cell killing, because both HPV integration and repair of radiation-induced DNA breakage involve homologous recombination repair. HPV genome integration is frequently observed in important DNA repair genes such as Rad51B8, supporting the relationship between DNA damage repair and HPV integration. In a previous study, we showed that genetic variants of excision base repair proficiency genes are strongly associated with cervical cancer compared with the healthy control population. This finding also suggests that an individual host’s base excision repair or nucleotide excision repair ability may facilitate HPV viral persistence and cervical carcinogenesis9. Based on these, we hypothesized that genomic susceptibility to DNA damage may explain the association between differential HPV integration and radiotherapy outcome in cervical cancer patients.Integration of HPV DNA into the host genome is associated with cervical carcinogenesis. However, in our previous study, HPV DNA is present in episomal form in approximately 20% of patients with locally advanced cervical cancerHere, we investigated the potential relationship between genomic variants of DNA damage repair- and immune-related genes and different HPV integration patterns using a customized single nucleotide polymorphism (SNP) array system.1, we observed that the combination of in situ hybridization and PCR is effective for discriminating the different integration patterns of HPV DNA.A comprehensive association study of immune- and DNA repair-related genes was performed in 192 advanced cervical cancer patients treated by radiotherapy ± chemotherapy between July 2003 and August 2010 at the National Cancer Center, Korea. In a recent studyA PubMed search identified 1,284 candidate immune-related or DNA repair-related genes, which were used to generate a custom immune chip. Then, 981,980 single nucleotide variants located within 50 kpb upstream and downstream of the targeted genes were identified, and 561,049 SNPs validated by Affymetrix were loaded on the custom-made array.DNA was extracted from whole peripheral blood using a QIAamp DNA Blood Mini Kit  according to the manufacturer’s instructions. For the genetic association study, 192 samples were genotyped using the custom immuno-array described above according to the manufacturer’s instructions for the Affymetrix Axiom Array platform. All samples passed a sample quality control (QC) metric with a Dish QC > 0.82, and genotype calling was performed using Genotyping Console v4.2 software. All samples passed an overall call rate threshold >97%, and the average overall call rate was 99.71% (99.19–99.87%). No duplicates or first-degree relatives were detected in identity-by-descent testing and principal component analysis using HapMap data as controls showed that all Korean cervical cancer samples formed a single cluster together with the HapMap Asian population samples (Japanese in Tokyo [JPT] and Han Chinese in Beijing [CHB]) Fig. .p-values < 5E-04 from the association test were checked by visual inspection, and SNPs with ambiguous genotype calls were excluded. Candidate regions for further analysis were selected using a p-value threshold of 5E-04  and loss of heterozygosity (n = 270) were first assessed, and SNPs with a call rate ≤ 95%  and a minor allele frequency ≤ 5%  were excluded. Finally, SNPs that deviated from Hardy–Weinberg Equilibrium (p ≤ 0.001)  were further filtered out, resulting in the inclusion of 166,505 SNPs on autosomal chromosomes or X chromosome in the association test. All cluster plots for SNPs with 04 Table .1. According to this criterion, 38 (23.6%), 111 (68.9%), and 12 (7.5%) of 161 patients were classified into groups A, B, and C, respectively.The integration pattern of 30 patients could not be determined because PCR results were not available for these patients. Moreover, duplicated result was found for one patient. After excluding these patients, 161 patients were included for further analysis. The integration pattern of patients’ HPV was classified as episomal (group A), single-copy or multi-copy tandem repetition integrated (group B), and undetectable HPV (group C)10. Tumor size, which was the only significant variable at p < 0.05 in all logistic regression models, was adjusted in the logistic regression models before evaluating the association of SNPs with outcome.The distribution of patient characteristics was summarized as proportions for categorical variables and median (range) for continuous variables. The differences between groups were tested using Pearson’s Chi-squared or Fisher’s exact test for categorical variables and Wilcoxon rank-sum or Kruskal–Wallis test for continuous variables as appropriate. Differences in the genotype distribution of SNPs between HPV integration patterns were tested using additive and dominant genetic models, and the smallest p-value in the two tests was used to order the significance of the association of SNPshttp://www.regulomedb.org/index)11. The expression of candidate genes was searched on the Human Protein Atlas database (http://www.proteinatlas.org)12. The correlation between the SNP genotype and the expression of surrounding genes was examined using available data from the GTEx portal 13, DICE (https://dice-database.org/)14, blood eQTL browser (https://molgenis58.target.rug.nl/bloodeqtlbrowser/)15, and iMETHYL (http://imethyl.iwate-megabank.org/)16. The possible impact of nonsynonymous variants on the structure and function of proteins was evaluated using the PolyPhen-2 tool (http://genetics.bwh.harvard.edu/pph2/)17.Potential functional variants for transcription regulation were evaluated using the RegulomeDB database . All experiments were performed in accordance with the relevant guidelines and regulations. All subjects provided written informed consent.Table 18.To identify SNPs with different genotype distributions between HPV integration patterns, comparisons were made based on the results of logistic regression as follows: (1) A vs. B, (2) A vs. C, (3) B vs. C, and (4) A vs. B + C. In addition, an ordinal regression model was applied using the HPV integration patterns A, B, and C as the ordinal outcome variables. Fig. TNFSF8 gene. In total, 39 SNPs (effective number: 33.6) in 13 loci were significant with a p-value < 5E-04.Comparison of integration patterns A and B identified rs3181372, rs3181365, and rs1322054 on chromosome 9q32-33 as the SNPs with the smallest p-value at 1.34E-04 Fig. . These tHRAS gene showed an association, with a minimum p-value of 9.89E-04  and asp523-to-his (D523H), respectively. The D523H substitution was predicted to be damaging for the LRRC56 protein with a score of 0.983 according to PolyPhen-2.Comparison between groups A and C did not identify any significant SNPs with a p-value < 5E-04. This could be attributed to the small sample size of these two groups. Two SNPs, rs10902170 and rs10902171, that were in complete LD on chromosome 11p15 and located near the -04 Fig. . These SHRAS and LRRC56 genes, with a p-value of 1.19E-04  in two loci with a p-value < 5E-04. The minimum p-value was observed for four SNPs in complete LD near the -04 Fig. . These SComparison of integration pattern groups A and B + C identified 54 SNPs (effective number: 48.2) in 14 loci with a p-value < 5E-04. The minimum p-value of 1.16E-04 was observed for SNPs rs3181372, rs3181365, and rs1322054, which showed a minimum p-value in the comparison between groups A and B Fig. .PRF1) gene on chromosome 10q22  on 28 loci. The minimum p-value of 5.53E-05 was observed for rs10999435 located at 13 kbp 5′ of the perforin 1 . This tendency was concordant with the e-QTL data of TNFSF8 expression in NK cells  of cervical cancer development or HPV seropositivity have been reportedPRF1 gene, and is predicted to generate a transcription regulatory element characterized by a DNaseI hypersensitivity cluster and transcription factor binding with a score of 2a on the RegulomeDB database. The endogenous expression level of PRF1 in whole blood was decreased by a minor allele homozygous genotype  Data Portal  and the GTEx web portal (Genotype-Tissue Expression project4). Cervical cancer was shown the highest immune cytolytic index along with kidney and lung. Cytolytic activity has been previously shown to correlate with oncogenic viruses such as with HPV in cervical cancer, with EBV infection in stomach cancer, and with HBV and HCV infection in liver cancer24, and our results of different PRF1 genotypes in the order of group A, B, and C may suggest the possibility of different amount of tumor infiltrating lymphocytes due to different availability of HPV viral antigen.The most significant association observed in the present study was for rs10999435 based on the ordinal comparison of the three integration patterns. This SNP is located upstream of the ype Fig. , althougTNFSF8, TNFSF15, and Tenascin C) are located in this locus, and the cis-eQTL association of rs1322054 with the TNFSF8 gene is registered in DICE database and blood eQTL browser15. Recently published article also reported rs1322054 as one of the TNFSF8 regulatory variants with the function of TNFSF8 as general controller of inflammatory responses25. TNFSF8  is a cytokine that promotes cell proliferation in some lymphoma cell lines, whereas it induces cell death and suppresses cell proliferation in other lymphoma cell lines. The engagement of TNFSF8 expressed on the B cell surface plays an inhibitory role in modulating immunoglobulin class switch recombination (CSR), and CSR triggers humoral immunity against papillomavirus26. Interestingly, TNFSF8 was previously shown to be highly upregulated by HPV 16 E2 expression in cells27. The interaction between TNFSF8-mediated immunity and HPV might affect the physical status of HPV DNA in patients with cervical cancer.The minimum p-value in the comparison of integration patterns A vs. B, and A vs. B + C was observed on chromosome 9q32-33, and rs1322054 was predicted to generate a transcription regulatory element with a score of 2b on the RegulomeDB database and its C allele increased enhancer/suppressor activity compared to T allele in our luciferase assay. Three known immune-related genes  stably transfected with the HRAS gene with its activating mutation progress through the cell cycle faster than control cells by reducing the G1 phase, indicating that HRAS could play an important role in the development of cervical cancer together with the presence of HPV28. Although the identified intergenic variant was independent from the reported activating mutation of the HRAS gene, different expression levels of HRAS may affect the stability of HPV DNA in the cell. HRAS is located in short arm of chromosome 11 and loss of heterozygosity (LOH) at 11p is frequently observed in cervical cancer29. In one report where 38 cervical cancer samples were analyzed for LOH, 11p and 11q was 16% and 22%, respectively, in its and 3 out of 5 samples which have LOH at 11p was HPV-negative30. It had been reported that loci on the short arm of chromosome 11 between 11p11 and 11p15 where HRAS gene is located are likely to be involved in regulation of the HPV-16 enhancer-promoter strength and this may suppress HPV-16-induced transformation by down-regulating the activity of the viral early promoter31. If tumors that have functional segment of chromosome 11, the activity of the viral early promoter should be down regulated, thereby suppressing HPV-induced transformation. If this segment of chromosome is not functional in cervical carcinoma, this suppression does not occur, and may show more unfavorable outcome because viral early promoter activity would not be suppressed. Our results on different distribution of HRAS genotype between HPV-negative (group C) and HPV-positive (group A and B) group may be associated with aggressive clinical outcome of patients with HPV-negative tumors. Further in vitro studies are required to determine the primary gene controlling the integration status of HPV.The most significant result of the comparison between groups A vs. C, and B vs. C was observed on chromosome 11p15. Although the targeted gene based on known immune- or DNA repair-related functions was HRAS, the most significant SNP, rs10902171, induced an amino acid substitution of the LRRC56 protein with potentially damaging consequences. Although the eQTL data for cervix tissues is limited, strong eQTL associations of rs10902171 and its surrounding SNP rs112360405, which was predicted to affect a transcription regulatory element, were observed in testis against both C56 Fig. . No signPossibly due to the small sample size and small number of disease progression, none of the candidate SNPs showed significant association with the patients’ survival outcome (data not shown). The HPV integration pattern is a promising biomarker which was shown to be associated with patients’ survival outcome, and the goal of the current study was to examine genetic difference between patients with differential HPV integration patterns. Further larger cohort study is required to investigate SNPs directly associated with patients’ survival outcome.In conclusion, the present comprehensive genetic association study of Korean patients with cervical cancer identified potential markers associated with the HPV integration pattern in patients with cervical cancer. Further confirmation studies are necessary.Table S1Table S2


Does the cross-reactivity of T cells against the human papillomavirus (HPV) oncoproteins in HPV-positive cancers support current immunotherapeutic vaccine clinical trial designs in which the HPV type of the tumor is not matched with the HPV type of the vaccine?In an analysis of 16 samples, HPV-reactive tumor-infiltrating lymphocytes from patients with HPV-positive cancers displayed HPV-16/HPV-18 oncoprotein cross-reactivity in 1 of 16 samples. None of the 10 HPV-reactive T-cell receptors from the researchers’ library exhibited HPV-16/HPV-18 oncoprotein cross-reactivity.The low frequency of HPV-16/HPV-18 T-cell cross-reactivity supports clinical trial designs that match the HPV type of therapeutic vaccines to that of patient tumors. This study characterizes the prevalence of human papillomavirus 16 (HPV-16) and HPV-18 metastatic cervical cancers among National Cancer Institute (NCI) trial participants and the cross-reactivity of archived T cells between the 2 HPV types to determine whether treating patients with an HPV-16–targeted vaccine would be effective for women with HPV-18 cancers. Clinical trials are testing vaccines that target human papillomavirus 16 (HPV-16) oncoproteins for the treatment of cervical cancer regardless of the HPV type of the tumor. For patients with HPV-18–positive cancers, this strategy relies on cross-reactivity of HPV-16–reactive T cells against the HPV-18 oncoproteins.To determine the prevalence of HPV-16 and HPV-18 metastatic cervical cancers in women enrolling in clinical trials at a US medical center and to assess whether HPV oncoprotein–targeting tumor-infiltrating lymphocytes (TILs) and T-cell receptors (TCRs) possess HPV-16/HPV-18 oncoprotein cross-reactivity.This study was conducted at the National Institutes of Health Clinical Center, a tertiary care research hospital in the United States. The HPV type of the tumors from 65 consecutive patients with cervical cancer who were evaluated for participation in clinical trials was determined by retrospective medical record review. Immunological assays testing HPV cross-reactivity were conducted on all available archived samples of oncoprotein-reactive TILs from HPV-positive tumors (n = 16) and on a library of previously identified TCRs (n = 10).The HPV genotype of each patient’s tumor was determined. The cross-reactivity of archived TILs and a library of TCRs was assessed.The main outcomes were the prevalence of each HPV genotype and the frequency of TILs or TCRs with HPV oncoprotein–T-cell cross-reactivity. Cross-reactivity was assessed by enzyme-linked immunospot assays and interferon-γ production assays.The median (range) age of 65 referred patients was 44 (24-64) years. Ethnicity was recorded for 39 of 65 patients; 35 (89.7%) were white, 3 (7.7%) were Asian, and 1 (2.6%) was American Indian/Alaskan Native. Histologic tumor subtype was recorded for 41 of 65 patients; 25 (61.0%) were squamous cell carcinomas, 12 (29.3%) were adenocarcinomas, 2 (4.9%) were adenosquamous cell carcinomas, and 2 (4.9%) were neuroendocrine tumors. Thirty-nine of 65 patients (60.0%) had HPV-16–positive tumors and 21 patients (32.3%) had HPV-18–positive tumors. In the analysis of cross-reactivity, 1 of 16 oncoprotein-reactive archived TILs  displayed HPV-16/HPV-18 cross-reactivity. None of the 10 oncoprotein-reactive TCRs displayed HPV-16/HPV-18 cross-reactivity.Cervical cancers that tested positive for HPV-18 were common in this study and may be common in other US clinical trial populations. Results showed that HPV-16/HPV-18 intergenotype T-cell cross-reactivity of T cells from HPV-16–positive and HPV-18–positive cancers was uncommon. These findings support clinical trial designs in which the HPV type targeted by a therapeutic vaccine is matched with the HPV type of a cancer and suggest a change is necessary in the design of active clinical trials. In some clinical trials, including active multicenter studies,7 vaccines that target HPV-16 E6 and/or E7 are undergoing testing in patients with cervical cancer regardless of the HPV type harbored by the patient’s tumor.6Cervical cancer is a difficult-to-treat malignant neoplasm that causes the deaths of more than 4000 women in the United States each year.11 In the United States, HPV-16 is the most common (56%) and HPV-18 is the second most common (13%) type of cervical cancer.12 In other common HPV-associated cancers, such as head and neck and anal cancers, HPV-16 is overwhelmingly (>80%) the dominant oncogenic type.14 The viruses are members of different species (HPV-16 from species 9 and HPV-18 from species 7), and their oncoprotein sequences are nonidentical.11 For patients with HPV-18–positive cancers, a clinical trial strategy of vaccinating all patients with an HPV-16 oncoprotein–targeted vaccine therefore relies on cross-reactivity against nonidentical T-cell epitopes. A certain level of cross-reactivity is inherent to the T-cell arm of adaptive immunity because T-cell receptors (TCRs) can exhibit promiscuity in peptide recognition.15 However, whether robust intergenotype HPV-16/HPV-18 oncoprotein T-cell cross-reactivity exists in patients with metastatic cervical cancer is not well studied.At least 13 HPV types cause cancer, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.We sought to examine the immunological foundation for the strategy of treating all comers with metastatic cervical cancer with an HPV-16–targeted vaccine. Because research on the prevalence of high-risk HPV genotypes in HPV-associated cancers has mainly been based on the study of primary rather than metastatic tumors, we first determined the frequency of HPV-16–positive and HPV-18–positive tumors among patients with metastatic cervical cancer referred for immunotherapy clinical trials at our cancer center. Next, we evaluated whether a panel of archived HPV oncoprotein–specific tumor-infiltrating lymphocytes (TILs) expanded from HPV-16–positive or HPV-18–positive tumors possessed HPV-16/HPV-18 oncoprotein cross-reactivity and could therefore potentially be stimulated to attack tumors caused by both HPV types. Finally, to assess the potential cross-reactivity of individual TCRs, rather than mixed T-cell populations, we tested the HPV-16/HPV-18 cross-reactivity of an archived panel of HPV-16 and HPV-18 oncoprotein–specific TCRs.16 The 16 samples tested for cross-reactivity represented our complete bank of TILs with known HPV oncoprotein reactivity and sufficient material for assays. Of the 16 TILs tested, 10 TILs were generated from HPV-16–positive tumors  and 6 TILs were generated from HPV-18–positive tumors .The 65 patients included in the HPV type–prevalence analysis were screened for clinical trials according to National Cancer Institute (NCI) protocol 99-C-0128 at the NCI in Bethesda, Maryland, between April 24, 2012, and March 18, 2015. The 16 samples used to generate archived TILs were separately procured according to NCI protocol 03-C-0277 between May 9, 2012, and September 2, 2014, with approval of the Office of Human Subjects Research Determination 11503. All protocols were approved by the NCI institutional review board, and written consent was obtained from all participants. Race and/or ethnicity was self-reported from predetermined options. Patient leukapheresis and tumor samples were collected and processed by the National Institutes of Health Surgery Branch TIL laboratory. Archived TILs used in this study were grown from fresh tumor samples and selected as previously described.The HPV genotype of the 65 patients screened was determined by a review of medical records and/or by testing of formalin-fixed, paraffin-embedded tumor samples at the Massachusetts General Hospital Department of Pathology using the cobas 4800 System (Roche).18 From our library of TCRs, only those with strong HPV oncoprotein reactivity were included in this analysis  and 300 IU/mL interleukin 2. Stimulated cells were transduced using plates coated with RetroNectin (Clontech). Transduction was verified by flow cytometry using an anti–mouse constant region beta antibody (eBioscience). Transduced T cells were used in immunological assays 6 to 10 days after transduction.We isolated TCR sequences from archived TILs or cervix-infiltrating lymphocytes by methods previously described.+ cells isolated from PBMCs with CD14 MicroBeads (Miltenyi Biotec) and subsequent culturing with 1000 IU/mL recombinant human granulocyte-macrophage colony-stimulating factor (Peprotech) and 500 IU/mL human recombinant interleukin 4 (Peprotech), as described previously.18 Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs) were generated from patient PBMCs using the viral supernatant from the B95-8 producer cell line (ATCC) using standard procedures and cultured in Iscove’s Modified Dulbecco’s Medium (Life Technologies) supplemented with 10% fetal calf serum (HyClone) and standard additives.Dendritic cells (DCs) were generated from CD14To test for reactivity of TILs and TCR-transduced T cells against the HPV-16 and HPV-18 E6 and E7 antigens, DCs or EBV-LCLs were pulsed overnight with pools of 15-mer peptides overlapping by 11 amino acids spanning the entire proteins (Miltenyi Biotec), or electroporated with in vitro transcribed RNA (Invitrogen) generated per the manufacturer’s protocol from the full-length HPV oncoprotein genes. For TIL analysis, autologous DCs were used exclusively as targets. For TCR analysis, EBV-LCLs generated from the patient from whom the TCR was isolated were used when possible; otherwise, human leukocyte antigen–matched EBV-LCLs for that particular TCR were used  age of 65 referred patients with cervical cancer was 44 (24-64) years. Ethnicity was self-reported for 39 of 65 patients; 35 (89.7%) were white, 3 (7.7%) were Asian, and 1 (2.6%) was American Indian/Alaskan Native. Histologic tumor subtype was recorded for 41 of 65 patients; 25 (61.0%) were squamous cell carcinomas, 12 (29.3%) were adenocarcinomas, 2 (4.9%) were adenosquamous cell carcinomas, and 2 (4.9%) were neuroendocrine tumors. Assessment of HPV genotype revealed that 39 of 65 patients (60.0%) had metastatic cervical cancers that harbored HPV-16 and 21 patients (32.3%) had cancers that harbored HPV-18. Thus, a substantial proportion of patients with cervical cancer had HPV-18–positive cancers, and for these patients it would be important to know whether an HPV-16–targeted vaccine might stimulate HPV-18–reactive T cells.16 In initial assessment, TILs from 0 of 10 HPV-16–positive tumors  demonstrated specific HPV-18 E6 and/or E7 reactivity, and TILs from 0 of 6 HPV-18–positive tumors  demonstrated specific HPV-16 E6 and/or E7 reactivity (+ T-cell clonotype that targeted both HPV-18 and HPV-16 E6 (eFigure in the To investigate the theoretical potential for oncoprotein vaccines to induce HPV-16/HPV-18 cross-reactive T-cell responses, we studied archived HPV oncoprotein-reactive TILs from the tumors of 16 patients with HPV-16–positive or HPV-18–positive metastatic cancers.activity A. Furtheactivity B. ExaminBecause TILs are composed of a polyclonal population of T cells and thus TCRs with potentially diverse HPV reactivity, we next evaluated the potential HPV-16/HPV-18 oncoprotein cross-reactivity of single-epitope targeted HPV-specific T cells using a panel of HPV-oncoprotein specific TCRs (eTable in the 19 Another strategy is to genotype each patient’s tumor and give an HPV-16–directed vaccine only if the tumor is HPV-16 positive.20 A third approach is to administer an HPV-16–directed vaccine regardless of the HPV type of the cancer.7 For this approach to successfully treat patients with HPV-18–positive cancers, robust HPV-16/HPV-18 T-cell cross-reactivity must be present. Our findings that HPV-16/HPV-18 oncoprotein cross-reactivity occurred in only 1 of 16 TILs from HPV-positive cancers and in 0 of 10 HPV oncoprotein–specific TCRs suggest that vaccination with HPV-16 oncoproteins is unlikely to elicit the strong T-cell responses against the HPV-18 oncoproteins that would be necessary for successful treatment. These data therefore support a strategy either of vaccinating with both HPV-16 and HPV-18 oncoproteins or of HPV genotype matching the patient’s tumor and administered oncoprotein vaccine.Various clinical trial designs have been used in the study of HPV oncoprotein vaccines. One strategy is to vaccinate with both the HPV-16 and HPV-18 oncoproteins and thereby target the 2 most common types of high-risk HPV.23 Only study of T-cell reactivity in patients with HPV-18–positive tumors treated with HPV-16 oncoprotein vaccines could definitively assess the potential for HPV-16 oncoprotein vaccines to induce T-cell responses against HPV-18 oncoproteins. Nonetheless, the available evidence, including the findings presented here, suggests that vaccination of patients with HPV-18–positive tumors with an HPV-16 vaccine may confer risk with small chance of benefit.24 Genotype matching of HPV oncoprotein vaccines with patient tumors may optimize the clinical benefit of the therapeutic vaccines and their chance for successful development.The results of this study are consistent with studies of healthy participants and of vaccinated patients, which have shown low T-cell cross-reactivity between different HPV types.The immunological assays in the study are limited by sample size, with 16 TILs and 10 TCRs evaluated. It is also noteworthy that our data do not rule out T-cell cross-reactivity against every possible complex of an HPV-16/HPV-18 oncoprotein peptide with a human leukocyte antigen molecule. The TCR studies are particularly limited as they are intended to assess cross-reactivity in the context of a single human leukocyte antigen molecule. Nonetheless, the marked paucity of HPV-16/HPV-18 cross-reactive T cells strongly suggests that cross-reactivity is uncommon and that vaccine HPV type should be matched to tumor HPV type.Human papillomavirus 18 appears to be common among patients with metastatic cervical cancer seeking enrollment in immunotherapy clinical trials. Cross-reactivity against HPV-16/HPV-18 oncoproteins is uncommon in T cells that infiltrate metastatic HPV-16–positive and HPV-18–positive cancers and in T cells that target the HPV oncoproteins. These data support a change in immunotherapy clinical trial practice to match the HPV type of the oncoprotein vaccine with that of the tumor. These findings have implications for the design of active, multicenter, cooperative group clinical trials.


Although human papillomavirus (HPV) positive oral and oropharyngeal cancers have distinct epidemiologic and molecular characteristics compared to HPV-negative cancers, all patients with oral and oropharyngeal cancers received same standard regimen regardless of HPV status. For these reasons, specific regimens for patients with HPV-positive oral and oropharyngeal cancer are needed. Differentially expressed genes (DEG) between HPV-positive and HPV-negative oropharyngeal cancers were re-analyzed and categorized from public database. Then, druggable targets to HPV-positive oral and oropharyngeal cancer were identified and were validated with E6/E7, which is oncogene of HPV, transfected oral and oropharyngeal cancer cell lines and HPV infected cell lines. In DEG analysis, HPV-positive oral and oropharyngeal cancer showed distinct disease entity from HPV-negative cancers. Unlike HPV-negative oral and oropharyngeal cancer, thymidylate synthase (TS) and topoisomerase II (Topo II) were overexpressed in HPV-positive cancers. Transfection of Lenti-virus containing E6/ E7 to HPV-negative oral and oropharyngeal cancer cells induced upregulation of TS and Topo II in those cells. Although cisplatin, which is standard regimen in head and neck cancers, showed more effectiveness in HPV-negative cells, 5-FU and pemetrexed, which are TS inhibitors, or etoposide, which is Topo II inhibitors, worked more effectively in HPV-positive cells. In addition, cisplatin/etoposide and cisplatin/pemetrexed combination regimens showed synergic effects in HPV-positive cells. Pemetrexed or etoposide alone, or in combination with other chemotherapeutic agents such as cisplatin, can be used as novel substitutes in a regimen of concurrent chemoradiotherapy or a palliative regimen for HPV-positive oral and oropharyngeal cancer patients. However, a well-designed clinical trial is needed. Worldwide, more than 550,000 cases of head and neck cancer are newly diagnosed each year and approximately 380,000 deaths are attributed to the disease. In the UIn comparison with HPV-negative oropharyngeal cancer, 59 differently expressed genes have been already identified in HPV-positive oropharyngeal cancer, so it is predicted that HPV-positive oropharyngeal cancers have distinct epidemiologic, pathologic, and molecular characteristics., 12, 13 In this article, to meet the needs for separate treatments based on HPV status, we identified the differently expressed genes between HPV-positive and HPV-negative oral and oropharyngeal cancers, determined drugs that specifically targeted the overexpressed genes, and selected candidate drugs for a novel regimen of HPV-positive oral and oropharyngeal cancers.YD10B cells and Ho-1-N-1 were provided by Korean Cell Line Bank and Japan Cell Line Bank, respectively, and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) and 2% penicillin/streptomycin. HNSCC (human Head and Neck Squamous Cell Carcinoma) cell lines  were a gift from Dr. Jong-Lyel Roh . UM-SCC-1 and 93-VU-147T cells were maintained in DMEM containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Hela, SiHa and SKOV3 are provided from Korean Cell Line Bank and cultured in Roswell Park Memorial Institute medium (RPMI) with 10% FBS and 2% penicillin/streptomycin. Cisplatin, paclitaxel, pemetrexed, 5-FU, and etoposide were purchased from Sigma Aldrich (U.S.) and dissolved in DMSO for treatment to cells. The DNA vectors for E6 and E7 were obtained from Addgene (U.S.).5'-ATGCACCAAAAGAGAACTGC-3', Reverse (R) 5'-TTACAGCTGGGTTTCTCTAC-3'; HPV-16 E7, (F) 5'-GTAACCTTTTGTTGCAAGTGTGACT-3', (R) 5'-GATTATGGTTTCTGAGAACAGATGG-3'; thymidylate synthase (F) 5'-TTACCTGAATCACATCGAGC-3', (R) 5'-ATATCCTTCGAGCTCCTTTG-3'; topoisomerase II: (F) 5'-TGCCTGTTTAGTCGCTTTC-3', (R) 5'-TGAGGTGGTCTTAAGAAT-3; and GAPDH: (F) 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', (R) 5'- ATGTGGGCCATGAGGTCCACCAC-3'. cDNA was amplified by using the Accu Power Hot Start PCR Pre Mix  with the following conditions: 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 40 s at 72 °C. The amplified products were then separated on a 1.0% agarose gel, stained with 0.1 μg/mL ethidium bromide, and photographed under UV light Total cellular RNA was extracted from each cell by using a RNeasy plus mini kit (Qiagen) and the complementary DNA product was synthesized by using Transcriptor First Strand cDNA Synthesis Kit  in accordance with the manufacturer’s instructions. The following primers for PCR were used: HPV-16 E6, Forward (F) Original gene expression data was obtained from Martinez et al.  Based onThe DEG dataset was inserted as an input list for Cytoscape 2.0 and KEGG and STRING were called as template data.3 cells/well were seeded in 96-well plates. After treatment with the test chemicals in the presence or absence of hydroxyurea pre-treatment (10 μmol/mL), the cells were incubated with 5 mg/mL MTT for 4 h. The medium was subsequently removed; 150 μL solubilization solution and 150 μL stop solution were added and incubated at 37 °C for 4 h. The absorbance of the reaction solution was measured at 570 nm. The cell growth inhibition was calculated from the following equation:  × 100%.The cell viability was measured by using an MTT assay (Promega Ltd) in accordance with the manufacturer’s protocol. Briefly, 5 × 104 cells/well and treated with each of the test chemicals for 2 days. The media were prepared separately. The LDH Cytotoxicity Assay kit  was used as described by the manufacturer. Briefly, the cells were grown at 37 °C and 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin and seeded at 2 × 104 cells/well in 96-well plates. After 48 h, 100 μL of the supernatant of the cultured cells was transferred from each well to the corresponding wells of a new plate and 100 μL of reaction solution was added to each well. The plates were incubated with gentle shaking in an orbital shaker for 30 min at 18–21 °C (room temperature) and the absorbance of each well at 490 nm was measured by using a plate reader.The cells were seeded into 24-well plates at 5 × 10In Situ Cell Death Detection Kit, TMR red, Roche) and analyzed by a confocal microscopy.UM-SCC-1 and 93-VU-147T cells were seeded on cover slip and treated with drug or vehicle for 24 h. Then, the cells were fixed with 4% paraformaldehyde for 1 h at room temperature and permeablized with 0.1% Triton X-100 in PBS for 10 min at 4 °C. Cell death was stained by TUNEL assay kit  were treated with cisplatin, which is used as the mainstay of head and neck cancer chemotherapy and concurrent chemoradiotherapy (CCRTx), and examined for any differences in response by HPV status. In everyHo-1-N-1 and YD10B were treated with 5-FU, which is a TS inhibitor and used with cisplatin at CCRTx or in a palliative setting, depending on the concentrations. The use of 5-FU decreased oral cancer cell viability, regardless of the cell line or HPV status. AlthoughHo-1-N-1 and YD10B were treated with pemetrexed, which is another TS inhibitor, in the range from 0–10 μM. The MTT HPV-positive and HPV-negative oral cancer cells were treated with etoposide, a Topo II inhibitor, at different concentrations. EtoposidLow dose (1 μM) cisplatin was administered with various concentration of etoposide or pemetrexed in HPV-positive or -negative oral cancer cell lines. Although each combination regimen exerted gradual cytotoxicity in a dose-dependent manner, the effects were more prominent in HPV-positive oral cancer cell lines . The cytIn this article, we revealed the differences in the gene expression patterns between HPV-negative head and neck cancer and HPV-positive head and neck cancer and identified druggable targets, such as TS and Topo II, in HPV-positive head and neck cancer from big data derived patients with head and neck cancer patients. To mimic HPV infection in HPV naïve oral and oropharyngeal cancer cells, the transfection of E6 and E7, which are oncogenes of HPV, was performed. As shown in the bio-informatics data, the insertion of E6 and E7 increased the expression of TS and Topo II, which contributed to the development and progression of cancer. Although the HPV-negative oral cancer cell lines are more susceptible to cisplatin, which is the mainstay of previously established chemotherapy regimens of head and neck cancer, the inhibition of TS by 5-FU or pemetrexed and the inhibition of Topo II by etoposide exerted greater cytotoxicity in HPV-positive oral and oropharyngeal cancer cell lines in a dose-dependent manner. Furthermore, the addition of pemetrexed and etoposide to cisplatin showed synergistic effects on HPV-positive oral and oropharyngeal cancer cell lines.As previously mentioned, because there are insufficient data to justify a change the treatment modalities based on upon HPV status, patients with head and neck cancer currently receive the same treatment modalities and chemotherapeutic regimens, regardless of their HPV status. AlthoughIn previous study, Ijuin et al studied correlation between prognosis of advanced oropharyngeal cancer patients and TS or thymidylate phosphorylase. However,Etoposide was also previously used for the treatment of patients with head and neck cancers. Although oral etoposide showed dramatic efficacy : 9%) with radiation therapy in a study with a large percentage of patients with oropharyngeal cancer (50%), oral etoposide showed modest efficacy  in a study with a smaller percentage of patients with oropharyngeal cancer (11%)., 38 AlthWe identified why different approaches should be used, depending on HPV status, and identified TS and Topo II as druggable targets for HPV-positive oral and oropharyngeal cancer using “big data” derived from patients with head and neck cancer and bio-informatics techniques. In addition, we demonstrated differences in the efficacy depending on HPV status in model HPV oral and oropharyngeal cancer cell lines. We suggested that pemetrexed or etoposide in combination with cisplatin, can be used in a regimen of definitive concurrent chemoradiotherapy instead of cisplatin alone regimen for HPV-positive oral and oropharyngeal cancer patients. In addition, in case the cancer progresses despite the use of immunotherapy, pemetrexed or etoposide can be used a palliative regimen for recurrent or metastatic HPV-positive oral and oropharyngeal cancer patients. Although HPV prevalence in other head and neck cancers is lower than that in oropharyngeal cancer, if presence of HPV is confirmed, pemetrexed or topotecan need to be considered as another treatment option. However, a well-designed clinical trial is needed to test this in patients with HPV-positive oral and oropharyngeal cancer.S1 Fig(A) up-regulated gene list (B) Down-regulated gene list. Thymidylate synthase (TS) and topoisomerase II (Topo II) are highlighted.(TIF)Click here for additional data file.


In the English pilot of primary cervical screening with high-risk human papillomavirus (HR-HPV), we exploited natural viral clearance over 24 months to minimise unnecessary referral of HR-HPV+ women with negative cytology. Three laboratories were permitted to use 16/18 genotyping to select women for referral at 12-month recall. We estimated the clinical impact of this early genotyping referral.The observed numbers of women referred to colposcopy and with detected high-grade cervical intraepithelial neoplasia (CIN2+), and of women who did not attend early recall in the three laboratories were compared with those estimated to represent a situation without an early genotyping referral. The 95% confidence intervals (CI) for the differences between the protocols were calculated by using a parametric bootstrap.Amongst 127,238 screened women, 16,097 (13%) had HR-HPV infections. The genotyping protocol required 5.9% (95% CI: 4.4–7.7) additional colposcopies and led to a detection of 1.2% additional CIN2+ (95% CI: 0.6–2.0), while 2.3% (95% CI: 2.1–2.5) fewer HR-HPV+/cytology− women did not attend the early recall compared with the non-genotyping protocol.In a screening programme with high quality of triage cytology and high adherence to early recall,16/18 genotyping of persistent HPV infections does not substantially increase CIN2+ detection. Women become eligible for screening at age 25 years. Thereafter, they are recalled for cytological screening every 3 years until age 50 years, and then five yearly until the age of 64 years. Nationwide roll-out of primary high-risk human papillomavirus (HR-HPV) screening triaged with cytology is planned to be implemented by the end of 2019. In 2013, a pilot of primary cervical screening with HR-HPV testing was set up in six large CSP laboratories, accounting for about 13% of the nationally screened population.3 In these cases, the decision to refer HPV 16/18 positive women is made on a single screening sample.The aim of substituting cytology with HR-HPV testing is to achieve greater sensitivity and increase screening intervals. Because of relatively poor specificity, however, reflex cytology is required to identify those HR-HPV+ women who require colposcopy referral. In several countries including the USA and Australia, HPV 16/18 genotyping is being used at baseline to identify women with negative cytology at increased risk of underlying cervical intraepithelial neoplasia (CIN), for immediate referral.The English pilot also recognised a potential value of HR-HPV genotyping in triage, but it was considered that implementing it in the same way as those other countries, i.e. based on a single sample, would lead to an unsustainable increase in the demand for colposcopy. Hence, women with HR-HPV infections have been managed as shown in Table 6 The non-genotyping protocol with two early recalls within 24 months after screening, aimed to maximise the reduction in the need for colposcopy but was potentially vulnerable to the risk of non-adherence with an additional early recall. The genotyping protocol, expediting a referral of women with the most high-risk infections and reserving the second early recall for those whose infections are less likely to progress to cancer, aimed to reduce loss to follow-up at the second early recall and to maximise the detection of CIN2+ lesions requiring treatment. Hence, we evaluated the differences between the two protocols in the overall frequency of referral for colposcopy, detection of CIN2+ and CIN3+, and the loss to follow-up at early recall.Both of these triage protocols were aimed at reducing the need for colposcopy by exploiting the substantial natural clearance rates of all HR-HPV infections, including HPV 16/18.7 Briefly, six English CSP laboratories converted around a third of their screening population from primary liquid-based cytology (LBC) to primary HR-HPV screening. Conversion was population-based. The selection of administrative areas for conversion was not determined in a random process. Rather, the laboratories considered practical issues such as maintaining a single clinical management protocol in colposcopy practices serving each administrative area. During the pilot, the population age range and recommended screening intervals remained unchanged.The pilot started in May 2013 and the main outcomes have been described previously in detail.Screening samples were taken within primary care and were collected in either SurePath  or ThinPrep  LBC media. SurePath was used in three laboratories, while ThinPrep was used in the other three. In 2013–2014, two laboratories used Cobas 4800 ; two used RealTime  and the remaining two used APTIMA . Cobas and RealTime are HR-HPV DNA genotyping assays that report HPV 16 and HPV 18 separately from the 12 other HR-HPV genotypes, which are reported in combination. APTIMA is an HR-HPV mRNA assay detecting the 14 HR-HPV genotypes in combination.All HR-HPV assays had previously been approved for primary screening within the CSP. Triage cytology was read under routine conditions with knowledge of a HR-HPV infection, and was quality controlled to CSP standards. Colposcopy was conducted according to national clinical practice guidelines. All diagnoses reflect routine cytopathology and histopathology in the CSP.The present study was designed to compare the outcomes of screening in the pilot with and without HPV 16/18 triage at the 12-month early recall. As the first screening invitation is sent at age 24.5 years, we included women aged 24–64 years at the time of the screening test. Additionally, women were included if they had been screened during the first  round of primary screening with HR-HPV testing from the beginning of the pilot in May 2013 until December 2014 in the three Cobas or RealTime laboratories that used the HR-HPV genotyping information for the management of HR-HPV positive women  the latter type of infections. It has indeed been observed that this assay typically detects fewer HR-HPV infections than DNA assays, which ultimately leads to lower colposcopy rates in a routine screening programme.The prevalence screening episode for each woman was defined as starting with the first test recorded during the pilot period, i.e. the primary (baseline) test, and closed with any early recall tests or colposcopies. If the first recorded pilot test was preceded by another test within the two prior years, or if the test’s management code identified it as a follow-up to a recent cervical abnormality, the episode was excluded from further analysis. This is because those tests were unlikely to have been taken for the purpose of primary screening. Tests were linked using each women’s unique English NHS numbers.In this analysis, the infecting HR-HPV genotype was determined at the primary test and remained fixed even if the genotype changed by the 12-month early recall. The effect of a genotype change on the studied outcomes was addressed in a sensitivity analysis (see below). Women were included in the 16/18 category regardless of any co-infecting genotypes.10Our primary endpoints were (1) the total number of colposcopies performed, (2) the number of HR-HPV-positive/cytology-negative women not adhering to early recall and (3) the number of detected CIN2+ lesions for each triage protocol. CIN2+ was chosen as one of the primary endpoints as this is the threshold for treatment, but the results are also presented for the more reproducible endpoint of CIN3 + .11These outcomes were estimated based on aggregated observed data from the three genotyping laboratories Table , and theN = 98). We varied the proportion of women with persistent infections as:  the lower 5% confidence limit; or  the upper 95% confidence limit.Two sensitivity analyses were undertaken to assess the robustness of the findings. In the first of these, we addressed a subgroup of women with HPV 16/18 infections and persistently negative cytology at 12 months. Among these women, a relatively large proportion did not have a record of referral for colposcopy  and the total number in the non-genotyping protocol (denominator). For the number of women not adhering to early recall, the total number with HR-HPV-positive cytology - negative samples at baseline was used as the denominator. The positive predictive value (PPV) of colposcopy for CIN2+ and CIN3+ was calculated using the number of women attending colposcopy as the denominator. Detailed formulae are reported in Supplementary information Tables –3.12We obtained 95% confidence intervals (CI) for detection of CIN2+, number of colposcopies and loss of adherence to follow-up at the 12- and 24-month early recall using a parametric bootstrap. More precisely, following the flows in Fig. In total, 127,238 women were screened in the three genotyping laboratories in 2013–2014. Of these, 16,097 (13%) had a positive HR-HPV test result, 5287 (4%) with positive and 10,810 (8%) with negative cytology .Half of all CIN2+50%, 1423/2859) and 55% (968/1763) of CIN3+ were diagnosed in women infected with HPV 16, whereas 9% (247/2859 and 153/1763) of CIN2+ and CIN3+ were detected in women with HPV 18 without HPV 16  had a positive HR-HPV test result, 1274 (8%) with negative and 440 (3%) with positive cytology. This was similar to the screening results in the three substantially larger laboratories included in the main analysis. Among the 98 women with HPV 16/18 infections and negative cytology persisting at 12 months, the infection persisted until 24 months in 73 (74%). This proportion was virtually constant across age groups (data not tabulated). Among women with HPV 16/18 infections who attended colposcopy after the 24-month early recall, the observed PPV for CIN2+ was 27% (19/71), with 15% (8/54) if they had negative cytology, and 65% (11/17) if they had positive cytology.The genotyping protocol generated detection of 2869 CIN2+ and 1769 CIN3+ resulting from 8750 colposcopies among the 127,238 screened women Table . More thAn estimated 1741 cytology-negative women with a positive baseline HR-HPV test result did not attend the 12-month early recall. Additionally, 637 women who attended the 12-month early recall did not attend a recommended 24-month early recall. In total, we estimate that 22%  of HR-HPV-positive cytology - negative women did not attend or complete early recall.With this protocol, a total of 2835 CIN2+ and 1751 CIN3+ would be detected as a result of 8260 colposcopies among the 127,238 screened women Table . Again, Referring all persistently HR-HPV-positive women with negative cytology at 12 months to an additional 24-month early recall would result in 8%  of women not attending, in addition to the 16%  not attending the 12-month early recall. In total, we estimate that 24%  of HR-HPV-positive cytology-negative women would not have completed the recall under the non-genotyping triage protocol.The PPVs for CIN2+ were high when a colposcopy was undertaken following a positive cytology triage test result: 41% (2135/5163) at baseline and 35% (479/1369) after the 12-month early recall  at the 12-month early recall. At 24 months, the PPV for persistent HPV 16/18 infections, regardless of cytology, is estimated at 18% ((221–123)/(1728-1198), Table In all cases, the PPVs for CIN3+ were approximately half those for CIN2+.We estimate that the genotyping protocol would detect an additional 34 (95% CI: 26–43) CIN2+ and 18 (95% CI: 13–24) CIN3+cases among the 127,238 screened women, representing 1.2% (95% CI: 0.9–1.5) of CIN2+ and 1.0% (95% CI: 0.8–1.4) of CIN3 + cases detectable by the non-genotyping protocol , the latter would increase the need for colposcopy by 6.1% (95% CI: 5.2–7.0) and CIN2+ detection by 1.6% (95% CI: 1.3–1.9). Under the unfavourable scenario , the estimates would be lower at 4.7% (95% CI: 3.8–5.6) and 0.3% (95% CI: 0.1–0.6), respectively. Varying the proportion of women infected with HPV 16/18 who remain HR-HPV-positive by 24 months produced a range in the extra demand for colposcopy between 6.6%  and 5.3% .Using data from the English HPV pilot we estimated there would be a small increase in CIN2+ detection for HPV 16/18 genotyping compared with non-genotyping triage protocols for women with persistent HR-HPV infections and negative cytology. However, more rapid referral of persistently HPV 16/18 positive women with negative cytology would increase the number of colposcopies by 6%, which appears to be disproportionate with respect to an estimated increase in detected CIN2+ of 1%. This is a consequence of both reasonably high compliance with repeated testing in early recall observed in the pilot (close to 80%), and highly sensitive stratification of risk by cytology triage. The latter identified 75% of all CIN2+ at baseline and an additional 17% at 12-month early recall, with a high PPV on both occasions of over 30%. A very small pool of CIN2+ remained to be identified solely by HR-HPV genotyping but the PPV was substantially lower at around 10%.15 our finding of a 1% higher detection of CIN2+ and CIN3+with a faster referral of HPV 16/18 positive women warrants consideration. This relatively small additional increase in the number of detected CIN2+ achieved by genotyping persistent HR-HPV infections would be observed on top of the ~50% increase achieved in the pilot by substituting cytology with HR-HPV testing,7 and most of these cases would be detected in women below 30 years of age, when the likelihood of regression of CIN2+ is highest.16 Persistently negative cytology is often associated with early infections and lesions detectable only through HR-HPV testing have been hypothesised to be small.17 Given the long duration of progression of CIN lesions to cervical cancer,19 a delay of 12 months in diagnosing these cytologically negative lesions is unlikely to be associated with a significant risk of interval cancer, provided women adhere to early recall.As HPV 16/18 lesions are more likely to progress to cancer,20 and Australia.3 In Europe, the attitude towards using genotyping in this manner has so far been more conservative,23 and baseline referral was not tested in the English pilot out of concern that it would lead to an unsustainable demand for colposcopy. When the switch was made from cytology to HR-HPV screening in the pilot, the demand for colposcopy increased by about 80% in the prevalence round.7 Had direct referral of all HPV 16/18 positive women been recommended, we estimate that referral would increase by an additional 15–20% (Supplementary information). As expected, viral clearance, however, was substantial . The immediate colposcopies in women destined to clear their infections are likely to have contributed to the very high average number of colposcopies needed to detect each CIN2 + case in the ATHENA study, which evaluated a setting with immediate colposcopy of all women aged ≥25 years with HPV 16/18 infections; this number was eight.11 In the English pilot, where cytologically negative women were only referred in the presence of a persistent infection, the number of colposcopies to detect a case of CIN2+ was three (8750/2869, Table HPV 16/18 genotyping has been recommended for an immediate referral of HR-HPV positive/cytologically negative women in countries such as the USA24 As a result, the overall number of screened women who will require triage and colposcopy will decrease. The value of using genotyping for HPV 16/18 in the remaining persistent infections will probably decrease in line with the expected decrease in CIN2+ lesions associated with HPV 16/18.25Birth cohorts vaccinated against HPV 16/18 in the catch-up programme did not start entering the CSP until 2015, which means that our analysis is representative of an unvaccinated population. Through cross-protection, vaccination has the potential to decrease not only the prevalence of HPV 16/18 but also of certain other HR-HPV genotypes.11 assumptions on infection dynamics and the prevalence of CIN in women when managed following the non-genotyping protocol. Nonetheless, the sensitivity analyses showed that our conclusions were robust against a variety of assumptions. Additionally, using the data from the same three laboratories for both triage protocols meant that the background characteristics of the women, the catchment areas’ screening coverage and the cytology reading practices were constant. Finally, while our study compared two defined triage protocols, it cannot provide a conclusive answer as to what the optimal triage strategy would be for English HR-HPV positive women. A full optimisation study would require a substantially different approach comparing a number of alternative strategies, varying e.g. the eligibility criteria for triage, the number of early recalls, their timing, the tests and their positivity thresholds, and any age stratification.26 This is beyond the scope of our analysis.The large size and prospective protocol are key strengths of our study, as well as a population-based, routine HR-HPV-based screening setting using national standards and clinical guidelines, with quality assured HR-HPV testing, cytology, colposcopy and histology. The patterns of detection of CIN2+ by genotype Table  were conIn population-based screening programmes with good quality of triage cytology and where most women adhere to early recall, HPV 16/18 triage of persistently HR-HPV-positive and cytologically negative women 12 months after primary screening can add very little in terms of a clinical benefit such as additional detection of CIN2+.Supplementary material