TaME-seq2: tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling.

BACKGROUND: Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA. RESULTS: Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. CONCLUSION: TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.

HPV self-sampling among long-term non-attenders to cervical cancer screening in Norway: a pragmatic randomised controlled trial.

BACKGROUND: Cervical cancer screening participation is suboptimal in most settings. We assessed whether human papillomavirus (HPV) self-sampling may increase screening participation among long-term non-attenders in Norway. METHODS: A pragmatic randomised controlled trial with participation as the primary outcome was initiated in the national cervical screening programme in March 2019. A random sample of 6000 women aged 35-69 years who had not attended screening for at least 10 years were randomised 1:1:1 to receive either (i) a reminder to attend regular screening (control), (ii) an offer to order a self-sampling kit (opt-in) for HPV testing or (iii) a self-sampling kit unsolicited (send-to-all) for HPV testing. RESULTS: Total participation was 4.8%, 17.0% and 27.7% among control, opt-in and send-to-all (P < 0.0001; participation difference (%) send-to-all vs. control: 22.9 (95%CI: 20.7, 25.2); opt-in vs. control: 12.3 (95%CI: 10.3, 14.2); send-to-all vs. opt-in: 10.7 (95% CI: 8.0, 13.3)). High-risk HPV was detected in 11.5% of self-samples and 9.2% of clinician-collected samples (P = 0.40). Most women (92.5%) who returned a positive self-sample attended the clinic for triage testing. Of the 933 women screened, 33 (3.5%) had CIN2 + (1.1%, 3.7%, 3.8% among control, opt-in, and send-to-all, respectively), and 11 (1.2%) had cervical cancer (0%, 1.2%, 1.3% among control, opt-in, send-to-all, respectively). CONCLUSION: Opt-in and send-to-all self-sampling increased screening participation among long-term, higher-risk non-attenders. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03873376.

Substantial Decline in Prevalence of Vaccine-Type and Nonvaccine-Type Human Papillomavirus (HPV) in Vaccinated and Unvaccinated Girls 5 Years After Implementing HPV Vaccine in Norway.

Background: In 2009, quadrivalent human papillomavirus (HPV) vaccine was introduced in a school-based single-cohort program targeting 12-year-old girls in Norway. We estimated the impact of the Norwegian HPV immunization program. Methods: Three birth cohorts of 17-year-old girls, 2 nonvaccine-eligible cohorts (born 1994 or 1996) and 1 vaccine-eligible cohort (born 1997) were invited to deliver urine samples. The samples were analyzed for 37 HPV genotypes. HPV prevalence was compared between birth cohorts and between vaccinated and unvaccinated girls within and across birth cohorts after linkage to the Norwegian Immunisation Registry. Results: In total, 17749 urine samples were analyzed. A 42% (95% confidence interval [CI], 37%-47%) reduction in any HPV type and 81% (95% CI, 76%-85%) reduction in vaccine types (HPV-6/11/16/18) were observed in the vaccine-eligible cohort compared to the 1994 cohort. Vaccine types were reduced by 54% (95% CI, 39%-66%) and 90% (95% CI, 86%-92%) in unvaccinated and vaccinated girls, respectively, from the 1997 cohort, compared with unvaccinated girls born in 1994. A significant reduction was also observed for several nonvaccine types. Vaccine-type prevalence was reduced by 77% (95% CI, 65%-85%) in vaccinated compared with unvaccinated girls from the 1997 cohort. Conclusions: In this largely HPV-naive population, we observed a substantial reduction in vaccine and nonvaccine types in vaccinated and unvaccinated girls following introduction of HPV vaccination.

Quality assurance of human papillomavirus (HPV) testing in the implementation of HPV primary screening in Norway: an inter-laboratory reproducibility study.

BACKGROUND: Human papillomavirus (HPV) testing as primary screening for cervical cancer is currently being implemented in Norway in a randomized controlled fashion, involving three laboratories. As part of the quality assurance programme of the implementation, an evaluation of the inter-laboratory reproducibility of the HPV test was initiated, to ensure satisfactory HPV test reliability in all three laboratories. METHODS: The HPV test used is the cobas 4800 HPV Test, detecting 14 high-risk types with individual HPV genotype results for HPV16 and HPV18. In addition to the three laboratories involved in the implementation, the Norwegian HPV reference laboratory was included as a fourth comparative laboratory. A stratified sample of 500 cervical liquid based cytology (LBC) samples was used in the evaluation, with an aim towards a high-risk HPV positivity of ~25%. Samples were collected at one laboratory, anonymized, aliquoted, and distributed to the other laboratories. RESULTS: Comparison of the test results of all four laboratories revealed a 95.6% agreement, an 86.3% positive agreement and a kappa value of 0.94 (95% CI 0.92-0.97). For negative cytology specimens, there was a 95.8% overall agreement, a 67.4% positive agreement, and a kappa value of 0.88 (95% CI 0.80-0.93). For abnormal cytology specimens, there was a 95.8% overall agreement, a 95.5% positive agreement, and a kappa value of 0.86 (95% CI 0.71-0.97). CONCLUSIONS: The study showed a high inter-laboratory reproducibility of HPV testing, implying satisfactory user performance and reliability in the laboratories involved in the implementation project. This is important knowledge and we recommend similar studies always to be performed prior to the introduction of new screening routines.

Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance.

BACKGROUND: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03 % and 15 % between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL). METHODS: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process. RESULT: Standard PCR testing detected HPV in 55.2 % (64/116) of initially "HPV-negative" samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2 % HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented. CONCLUSION: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will help support high performance and quality improvement of HPV testing services. We encourage implementation of QA strategies based on re-analysis of "HPV negative" HSIL+ samples.

Correction: Self-collected and clinician-collected anal swabs show modest agreement for HPV genotyping.

[This corrects the article DOI: 10.1371/journal.pone.0250426.].

Differences in integration frequencies and APOBEC3 profiles of five high-risk HPV types adheres to phylogeny.

Persistent infection with Human Papillomavirus (HPV) is responsible for almost all cases of cervical cancers, and HPV16 and HPV18 associated with the majority of these. These types differ in the proportion of viral minor nucleotide variants (MNVs) caused by APOBEC3 mutagenesis as well as integration frequencies. Whether these traits extend to other types remains uncertain. This study aimed to investigate and compare genomic variability and chromosomal integration in the two phylogenetically distinct Alpha-7 and Alpha-9 clades of carcinogenic HPV types. The TaME-seq protocol was employed to sequence cervical cell samples positive for HPV31, HPV33 or HPV45 and combine these with data from a previous study on HPV16 and HPV18. APOBEC3 mutation signatures were found in Alpha-9 (HPV16/31/33) but not in Alpha-7 (HPV18/45). HPV45 had significantly more MNVs compared to the other types. Alpha-7 had higher integration frequency compared to Alpha-9. An increase in integration frequency with increased diagnostic severity was found for Alpha-7. The results highlight important differences and broaden our understanding of the molecular mechanisms behind cervical cancer induced by high-risk HPV types from the Alpha-7 and Alpha-9 clades.

Randomized Implementation of a Primary Human Papillomavirus Testing-based Cervical Cancer Screening Protocol for Women 34 to 69 Years in Norway.

BACKGROUND: Cervical cancer screening programs are facing a programmatic shift where screening protocol based on human papillomavirus testing (HPV-Screening protocol) is replacing the liquid-based cytology (LBC-Screening protocol). For safe technology transfer within the nationwide screening programme in Norway, HPV-Screening protocol was implemented randomized to compare the real-world effectiveness of HPV-Screening protocol and LBC-Screening protocol at the first screening round. METHODS: Among 302,295 women ages 34 to 69 years scheduled to attend screening from February 2015 to June 2017, 157,447 attended. A total of 77,207 were randomly allocated to the HPV-Screening protocol and 80,240 were allocated to the LBC-Screening protocol. All women were followed up for 18 months. RESULTS: The HPV-Screening protocol resulted in a relative increase of 60% in the detection of cervical intraepithelial neoplasia (CIN) grade 2 or worse [risk ratio (RR) = 1.6, 95% confidence interval (CI) = 1.5-1.7], 40% in CIN grade 3 or worse (RR = 1.4, 95% CI = 1.3-1.6), 40% in cancer (RR = 1.4, 95% CI = 1.0-2.1), and 60% in colposcopy referrals (RR = 1.6, 95% CI = 1.5-1.6) compared with LBC-Screening. The performance of both protocols was age dependent, being more effective in women ages under 50 years. CONCLUSIONS: The HPV-Screening protocol was well accepted by women in Norway and detected more CIN2, CIN3, and cancers compared with the LBC-Screening protocol. IMPACT: A randomized implementation of the HPV-Screening protocol with real-world assessment enabled a gradual, quality assured, and safe technology transition. HPV-based screening protocol may further be improved by using HPV genotyping and age-specific referral algorithms.

HPV16 and HPV18 type-specific APOBEC3 and integration profiles in different diagnostic categories of cervical samples.

Human papillomavirus (HPV) 16 and 18 are the most predominant types in cervical cancer. Only a small fraction of HPV infections progress to cancer, indicating that additional factors and genomic events contribute to the carcinogenesis, such as minor nucleotide variation caused by APOBEC3 and chromosomal integration. We analysed intra-host minor nucleotide variants (MNVs) and integration in HPV16 and HPV18 positive cervical samples with different morphology. Samples were sequenced using an HPV whole genome sequencing protocol TaME-seq. A total of 80 HPV16 and 51 HPV18 positive samples passed the sequencing depth criteria of 300× reads, showing the following distribution: non-progressive disease (HPV16 n = 21, HPV18 n = 12); cervical intraepithelial neoplasia (CIN) grade 2 (HPV16 n = 27, HPV18 n = 9); CIN3/adenocarcinoma in situ (AIS) (HPV16 n = 27, HPV18 n = 30); cervical cancer (HPV16 n = 5). Similar numbers of MNVs in HPV16 and HPV18 samples were observed for most viral genes, with the exception of HPV18 E4 with higher numbers across clinical categories. APOBEC3 signatures were observed in HPV16 lesions, while similar mutation patterns were not detected for HPV18. The proportion of samples with integration was 13% for HPV16 and 59% for HPV18 positive samples, with a noticeable portion located within or close to cancer-related genes.

Evaluation of HarmoniaHPV test for detection of clinically significant Human Papillomavirus infection using the VALGENT framework.

AIM: The VALidation of HPV GENotyping Tests (VALGENT) is a framework for comparison and validation of HPV tests with genotyping capabilities. In this study, the clinical performance of a single tube HPV test -HarmoniaHPV- was assessed in SurePath™ samples and compared to a clinically validated reference test, the GP5+/6+ Enzyme ImmunoAssay (GP5+/6 + EIA). METHODS: HarmoniaHPV test is a real-time, PCR based, limited genotyping HPV test which detects 14 high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 with HPV16, and HPV 18 reported individually. Clinical performance was assessed using 998 unselected, cervical screening samples enriched with 297 cytologically abnormal specimens (100 atypical squamous cells of unspecified significance, 100 low-grade squamous intraepithelial lesions, 97 high-grade squamous intraepithelial lesions). Cases were defined as women diagnosed with histologically confirmed cervical intraepithelial neoplasia 2 or more (≥CIN2, N = 122). RESULTS: Using the manufacturer recommended (un-adjusted) cut-offs, HarmoniaHPV had non-inferior sensitivity for detection of ≥ CIN2 but showed inferior specificity. A cut-off optimisation exercise was therefore carried out and optimised cut-offs for each individual channel rendered a sensitivity and specificity of HarmoniaHPV that was non-inferior to GP5+/6 + EIA. Analytically, the test showed excellent intra- and inter-laboratory reproducibility, which improved further with the use of the optimised cut-offs. CONCLUSION: HarmoniaHPV when operated with optimised cut-offs fulfils the international clinical criteria for use in cervical cancer screening on SurePath samples. The optimised cut-offs warrant additional testing and independent validation.

Self-collected and clinician-collected anal swabs show modest agreement for HPV genotyping.

BACKGROUND & AIM: Women with HIV/HPV coinfection and cervical lesions are at increased risk of developing HPV related anal cancer. Self-collection of anal swabs may facilitate HPV molecular testing in anal cancer screening, especially in high-risk groups, and yet it is not adequately studied. We evaluated level of agreement between self-collected anal swabs (SCAS) and clinician-collected anal swabs (CCAS) when used for HPV genotyping. We also described the anal HPV genotype distribution and HIV/HPV coinfection. METHODS: We performed a cross sectional study with participants from a visual-inspection-with-acetic-acid and cervicography (VIAC) clinic, in Harare, Zimbabwe. In a clinic setting, the women aged ≥18 years provided anal swabs in duplicate; first CCAS and then SCAS immediately after. HPV detection and genotyping were performed using next generation amplicon sequencing of a 450bp region of the HPV L1 gene. Level of agreement of HPV genotypes between CCAS and SCAS was calculated using the kappa statistic. McNemar tests were used to evaluate agreement in the proportion of genotypes detected by either method. RESULTS: Three-hundred women provided 600 samples for HPV genotyping. HPV genotypes were detected in 25% of SCAS and in 22% of CCAS. The most common genotypes with CCAS were HPV52, HPV62 and HPV70 and with SCAS were HPV62, HPV44, HPV52, HPV53 and HPV68. Total HPV genotypes detected in CCAS were more than those detected in SCAS, 32 versus 27. The agreement of HPV genotypes between the two methods was 0.55 in kappa value (k). The test of proportions using McNemar gave a Chi-square value of 0.75 (p = 0.39). Multiple HPV infections were detected in 28/75 and 29/67 women for CCAS and SCAS respectively. CONCLUSIONS: SCAS and CCAS anal swabs showed moderate agreement, with no statistically significant difference in the proportion of genotypes detected by either methods. Although the differences between the two methods were not statistically significant, CCAS detected more HPV genotypes than SCAS and more HPV infections were detected in SCAS than in CCAS. Our data suggest that self-collected anal swabs can be used as an alternative to clinician-collected anal swabs for HPV genotyping.

Clinical validation of the Cobas 4800 HPV assay using cervical samples in SurePath medium under the VALGENT4 framework.

BACKGROUND: The VALidation of HPV Genotyping Tests (VALGENT) framework is an international cooperation designed for comparison and clinical validation of HPV assays with genotyping capabilities. OBJECTIVES: Here we addressed the accuracy of the Roche cobas 4800 HPV test using SurePath samples from the Danish cervical cancer screening program under the VALGENT framework. MATERIAL AND METHODS: The VALGENT4 panel comprises 998 consecutive SurePath cervical samples from routine screening and 297 SurePath samples enriched for disease (100 ASC-US, 100 LSIL, 97 HSIL). The cobas HPV test is a real-time PCR assay which detects HPV16 and 18 individually and 12 other high-risk (hr) HPV genotypes in one bulk. RESULTS: The clinical performance of the cobas test was assessed relative to that of the comparator assay GP5+/6 + PCR Enzyme ImmunoAssay (GP-EIA) by a non-inferiority test. The relative sensitivity for ≥ CIN2 was 1.00 (95% CI: 0.97-1.04) and relative specificity for the control group was 1.02 (95% CI: 1.01-1.04). The cobas test was found non-inferior to that of GP-EIA for both sensitivity and specificity (p-0.0006 and p < 0.0001, respectively). The type specific performance of the cobas test was evaluated using the GP5+/6 + PCR with Luminex genotyping (GP-LMNX) as comparator. The cobas test showed excellent to good concordance (Kappa: 0.70 to 0.90) with GP-LMNX for all three genotype groups in the overall VALGENT population but good to moderate concordance in the Screening population (kappa from 0.56 to 0.80). CONCLUSIONS: The cobas HPV test demonstrated non-inferiority to the comparator assay on cervical SurePath screening samples using the VALGENT4 panel.

HPV16 whole genome minority variants in persistent infections from young Dutch women.

BACKGROUND: Chronic infections by one of the oncogenic human papillomaviruses (HPVs) are responsible for near 5% of the global cancer burden and HPV16 is the type most often found in cancers. HPV genomes display unexpected levels of variation when deep-sequenced. Minor nucleotide variations (MNVs) may reveal HPV genomic instability and HPV-related carcinogenic transformation of host cells. OBJECTIVES: The objective of this study was to investigate HPV16 genome variation at the minor variant level on persisting HPV16 cervical infections from a population of young Dutch women. STUDY DESIGN: 15 HPV16 infections were sequenced using a whole-HPV genome deep sequencing protocol (TaME-seq). One infection was followed over a three-year period, eight were followed over a two-year period, three were followed over a one-year period and three infections had a single sampling point. RESULTS AND CONCLUSIONS: Using a 1% variant frequency cutoff, we find on average 48 MNVs per HPV16 genome and 1717 MNVs in total when sequencing coverage was >100 × . We find the transition mutation T > C to be the most common, in contrast to other studies detecting APOBEC-related C > T mutation profiles in pre-cancerous and cancer samples. Our results suggest that the relative mutagenic footprint of HPV16 genomes may differ between the infections in this study and transforming lesions. In addition, we identify a number of MNVs that have previously been associated with higher incidence of high-grade lesions (CIN3+) in a population study. These findings may provide a starting point for future studies exploring causality between emerging HPV minor genomic variants and cancer development.

TaME-seq: An efficient sequencing approach for characterisation of HPV genomic variability and chromosomal integration.

HPV genomic variability and chromosomal integration are important in the HPV-induced carcinogenic process. To uncover these genomic events in an HPV infection, we have developed an innovative and cost-effective sequencing approach named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). TaME-seq combines tagmentation and multiplex PCR enrichment for simultaneous analysis of HPV variation and chromosomal integration, and it can also be adapted to other viruses. For method validation, cell lines (n = 4), plasmids (n = 3), and HPV16, 18, 31, 33 and 45 positive clinical samples (n = 21) were analysed. Our results showed deep HPV genome-wide sequencing coverage. Chromosomal integration breakpoints and large deletions were identified in HPV positive cell lines and in one clinical sample. HPV genomic variability was observed in all samples allowing identification of low frequency variants. In contrast to other approaches, TaME-seq proved to be highly efficient in HPV target enrichment, leading to reduced sequencing costs. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. Efficient identification of both HPV variability and integration sites will be important for the study of HPV evolution and adaptability and may be an important tool for use in cervical cancer diagnostics.

An observational study comparing HPV prevalence and type distribution between HPV-vaccinated and -unvaccinated girls after introduction of school-based HPV vaccination in Norway.

BACKGROUND: Many countries have initiated school-based human papillomavirus (HPV) vaccination programs. The real-life effectiveness of HPV vaccines has become increasingly evident, especially among girls vaccinated before HPV exposure in countries with high vaccine uptake. In 2009, Norway initiated a school-based HPV vaccination program for 12-year-old girls using the quadrivalent HPV vaccine (Gardasil®), which targets HPV6, 11, 16, and 18. Here, we aim to assess type-specific vaginal and oral HPV prevalence in vaccinated compared with unvaccinated girls in the first birth cohort eligible for school-based vaccination (born in 1997). METHODS: This observational, cross-sectional study measured the HPV prevalence ratio (PR) between vaccinated and unvaccinated girls in Norway. Facebook advertisement was used to recruit participants and disseminate information about the study. Participants self-sampled vaginal and oral specimens using an Evalyn® Brush and a FLOQSwab™, respectively. Sexual behavior was ascertained through a short questionnaire. RESULTS: Among the 312 participants, 239 (76.6%) had received at least one dose of HPV vaccine prior to sexual debut. 39.1% of vaginal samples were positive for any HPV type, with similar prevalence among vaccinated and unvaccinated girls (38.5% vs 41.1%, PR: 0.93, 95% confidence interval [CI]: 0.62-1.41). For vaccine-targeted types there was some evidence of lower prevalence in the vaccinated (0.4%) compared to the unvaccinated (6.8%) group (PR: 0.06, 95%CI: 0.01-0.52). This difference remained after adjusting for sexual behavior (PR: 0.04, 95%CI: 0.00-0.42). Only four oral samples were positive for any HPV type, and all of these participants had received at least one dose of HPV vaccine at least 1 year before oral sexual debut. CONCLUSION: There is evidence of a lower prevalence of vaccine-targeted HPV types in the vagina of vaccinated girls from the first birth cohort eligible for school-based HPV vaccination in Norway; this was not the case when considering all HPV types or types not included in the quadrivalent HPV vaccine.

The Valgent4 protocol: Robust analytical and clinical validation of 11 HPV assays with genotyping on cervical samples collected in SurePath medium.

BACKGROUND: The VALidation of HPV GENoyping Tests (VALGENT) is an international initiative designed to validate HPV assays with genotyping capability. The VALGENT4 protocol differs from previous VALGENT installments as the sample collection medium is SurePath, and exclusively includes samples from women ≥30 years of age which is concordant with the majority of HPV primary screening guidelines. Here we present the protocol for the fourth installment of the VALGENT framework. OBJECTIVES: In VALGENT4 11 HPV assays will be evaluated using two comparator assays based on PCR with the GP5+/6+ primers. STUDY DESIGN: Overall, the VALGENT4 panel consists of 1,297 routine samples comprised of 998 unselected, consecutive samples, of which 51 samples had abnormal cytology with 13 women diagnosed with ≥CIN2, and 299 consecutive samples enriched for ≥ASCUS cytology (100 ASCUS, 100 LSIL, 99 HSIL) with 106 ≥CIN2 upon follow up. Manipulated and DNA extracted panel samples were characterized with respect to human beta globin (HBB) and overall DNA content and composition to quality assess the panel prior to distribution to the collaborating sites. RESULT: The relative cellularity (mean CT value of HBB from the Onclarity assay) on the 1,297 LBC samples (CT=24.8) was compared with 293 un-manipulated routine cytology screening samples (CT=23.8). Furthermore, the DNA extracted panel samples was characterized using the Exome iPLEX pro assay, which reports amplifiable copies on individual samples as well as copies of five different base pair lengths. Here the data showed a slightly lower number of amplifiable DNA copies (ratio: 0.7, p=<0.01)) in the VALGENT4 panel samples compared to routine extracted cervical DNA samples CONCLUSION: The present manuscript details the manipulation, processing and quality assessment of samples used in VALGENT-4. This methodological document may be of value for future international projects of HPV test validation.

Intra-host sequence variability in human papillomavirus.

Human papillomaviruses (HPVs) co-evolve slowly with the human host and each HPV genotype displays epithelial tropisms. We assessed the evolution of intra HPV genotype variants within samples, and their association to anogenital site, cervical cytology and HIV status. Variability in the L1 gene of 35 HPV genotypes was characterized phylogenetically using maximum likelihood, and portrayed by phenotype. Up to a thousand unique variants were identified within individual samples. In-depth analyses of the most prevalent genotypes, HPV16, HPV18 and HPV52, revealed that the high diversity was dominated by a few abundant variants. This suggests high intra-host mutation rates. Clades of HPV16, HPV18 and HPV52 were associated to anatomical site and HIV co-infection. Particularly, we observed that one HPV16 clade was specific to vaginal cells and one HPV52 clade was specific to anal cells. One major HPV52 clade, present in several samples, was strongly associated with cervical neoplasia. Overall, our data suggest that tissue tropism and HIV immunosuppression are strong shapers of HPV evolution.

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

Transcriptionally active regions are the preferred targets for chromosomal HPV integration in cervical carcinogenesis.

Integration of human papillomavirus (HPV) into the host genome is regarded as a determining event in cervical carcinogenesis. However, the exact mechanism for integration, and the role of integration in stimulating cancer progression, is not fully characterized. Although integration sites are reported to appear randomly distributed over all chromosomes, fragile sites, translocation break points and transcriptionally active regions have all been suggested as being preferred sites for integration. In addition, more recent studies have reported integration events occurring within or surrounding essential cancer-related genes, raising the question whether these may reflect key events in the molecular genesis of HPV induced carcinomas. In a search for possible common denominators of the integration sites, we utilized the chromosomal coordinates of 121 viral-cellular fusion transcripts, and examined for statistical overrepresentation of integration sites with various features of ENCODE chromatin information data, using the Genomic HyperBrowser. We find that integration sites coincide with DNA that is transcriptionally active in mucosal epithelium, as judged by the relationship of integration sites to DNase hypersensitivity and H3K4me3 methylation data. Finding an association between integration and transcription is highly informative with regard to the spatio-temporal characteristics of the integration process. These results suggest that integration is an early event in carcinogenesis, more than a late product of chromosomal instability. If the viral integrations were more likely to occur in destabilized regions of the DNA, a completely random distribution of the integration sites would be expected. As a by-product of integration in actively transcribing DNA, a tendency of integration in or close to genes is likely to be observed. This increases the possibility of viral signals to modulate the expression of these genes, potentially contributing to the progression towards cancer.