A genome-wide association study (GWAS) was conducted to unravel these loci [marker–trait associations (MTAs)] using 35,648 genotyping-by-sequencing-derived single nucleotide polymorphisms in 123 SHWs. We identified 90 novel MTAs  and haplotype blocks associated with grain yield and yield-related traits including root traits under drought stress. The phenotypic variance explained by the MTAs ranged from 1.1% to 32.3%. Most of the MTAs (120 out of 194) identified were found in genes, and of these 45 MTAs were in genes annotated as having a potential role in drought stress. This result provides further evidence for the reliability of MTAs identified. The large number of MTAs (53) identified especially on the D-genome demonstrate the potential of SHWs for elucidating the genetic architecture of complex traits and provide an opportunity for further improvement of wheat under rapidly changing climatic conditions.Synthetic hexaploid wheat (SHW; 2 Zea mays L.), sorghum (Sorghum bicolor L.), and soybean (Glycine max L.), and the agriculture loss due to drought was estimated to be $30 billion  using the Bayesian clustering algorithm implemented in Structure software and the results showed that these lines were divided into three subgroups . The detThe GWAS identified novel genomic regions for GY and yield-related traits and the MTA explained the high phenotypic variance. The Fixed and random model Circulating Probability Unification algorithm (FarmCPU), with kinship, population structure (Q) or PC, best linear unbiased predictors (BLUPs) for each trait, and 35,648 GBS-derived SNPs was used to identify MTAs. The GBS-derived SNPs were well distributed across each of the chromosome . We idenThe 29 MTAs for GY were observed in 29 different genomic regions on seven chromosomes including 1B, 2B, 3A, 3D, 5B, 7A, and 7B with PVE ranging from 7.6% to 17.9% . EarlierThe present study identified four major haplotype blocks (from 19 bp to 433 kb) on chromosome 7A with two to six SNPs associated with GY in 2016 . First hUlp1) gene family -specific protease that affects several important biological processes in plants including response to abiotic stress [Arabidopsis thaliana) [Oryza sativa) [One MTA  present in-between the second 537 kb away) and third (837 kb) haplotype blocks was within the gene, TraesCS7A01G158200.1, and PVE on GY was 12.8% . This gee family . The Ulpc stress . It has haliana)  and rice sativa) ,37. This kb away Another major haplotype block 18 kb) of three MTAs was observed on chromosome 3A in 2017  and the  kb of thThe GY haplotype blocks and other MTAs identified in the present study have not been mapped to date and four MTAs were in the genes, of which functional annotations suggested that they are likely involved in drought tolerance. This result implied that haplotype blocks observed on chromosome 3A (3 MTAs) and 7A (16 MTAs), and one MTA on chromosome 3D (1) for GY are novel and may potentially be used in a marker-assisted breeding program, focusing on improving drought tolerance in wheat after validating them in different populations and environments.A total of 10 SNPs significantly associated with HI were identified on chromosomes 1D, 2A, 2D, 3A, 3D, 5B, 6B, 6D, and 7B  with PVESix MTAs for HI detected on chromosomes 2A, 3A, 6B, 6D, and 7B were found in genes and two of these genes have annotations suggesting their involvement in drought stress . The twoThe 15 MTAs responsible for BMWT were identified on chromosomes 1D, 2B, 3A, 4A, 6D, and 7B  with PVEA novel haplotype block (38 kb) of three SNPs on chromosome 3A associated with BMWT was identified in 2017  with PVEAll three MTAs present in this haplotype block were within genes  and one A total of 20 MTAs responsible for TKW were detected in 19 different genomic regions on chromosomes 1A, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5B, 6D, 7B, and 7D  with PVETwelve MTAs responsible for TKW detected on chromosomes 2A, 2B, 3A, 3B, 4A, 4B, 5B, and 6D were found in genes  and fiveThirteen MTAs responsible for GVWT were identified on chromosomes 1A, 2A, 2B, 2D, 3A, 4A, 5A, 6A, and 7A  with PVEEight MTAs responsible for GVWT detected on chromosomes 1A, 2A, 2B, 4A, 6A, and 7A were found in genes  and two Twenty MTAs responsible for AWNLN were observed on chromosomes 1D, 2A, 2B, 3B, 4A, 4B, 4D, 5A, 5B, 5D, 6B, and 7A  with PVEEleven MTAs responsible for AWNLN detected on chromosomes 1D, 2A, 4D, 5A, 5B, 6B, and 7A were found in genes  and fourThirteen MTAs responsible for FLLN were detected on chromosomes 1B, 1D, 2A, 2B, 2D, 4A, 6D, and 7B  with PVEEleven MTAs responsible for FLLN detected on chromosomes 1B, 1D, 2B, 2D, 4A, 6D, and 7B were found in genes  and fourSixteen MTAs responsible for FLW were detected on chromosomes 1A, 1B, 1D, 2B, 2D, 4B, 6B, and 6D  with PVEThirteen MTAs responsible for FLW detected on chromosomes 1A, 1B, 1D, 2D, 4B, 6B, and 6D were found in genes  and threTwenty MTAs responsible for FLW were detected on chromosomes 1A, 1B, 1D, 2A, 2D, 4D, 5A, 6B, and 7D  with PVEThree novel haplotype blocks were observed for FLA on chromosomes 1A (two MTAs), 6B (two MTAs) and 7D (three MTAs) with PVE by these haplotype block ranging from 5.5% to 8.6% . FourteeThe multi-trait marker associated with FLW and FLA was located on chromosome 1A (S1A_516732460) with PVE ranging from 8.0% to 9.5% . AnotherIn the present study, 23 MTAs responsible for STMDIA were identified on chromosomes 1A, 1D, 2B, 2D, 3A, 3B, 3D, 4D, 5A, 5B, 6A, 6B, 6D, 7A, and 7B  with PVEFour MTAs were detected on chromosome 3B and two of them (1 bp apart) were observed in one haplotype block in 2016 with PVE was 9.2% . Fifteen® . This trait is very unique compared to previous studies where they focused on the roots of seedlings [RTLN is one of the most important traits under drought stress. We have measured RTLN 3–4 days after anthesis (Zadoks 60 growth stage) under the drought-stressed field condition using WinRhizoeedlings ,68,69,70eedlings ,72.In the present study, 15 MTAs responsible for RTLN were identified on chromosomes 2B, 2D, 3B, 5B, 6A, 6D, and 7A  with PVEMatE family genes such as HvAACT1 in barley [TaMate in wheat [Seven out of eight MTAs responsible for RTLN were present on chromosome 6D. Two haplotype blocks (the haplotype block1 with a size of 64 kb and the haplotype block2 with a size of 5kb) were identified from five out of seven MTAs for RTLN on chromosome 6D with PVE ranging from 5.0% to 11.8% . One SNPn barley  and TaMain wheat , encode in wheat  and requin wheat . In the Excluding MTAs on haplotype block, eight MTAs for RTLN detected on chromosomes 2D, 3B, 5B, 6A, and 7A were found in genes  and fourThis study identified ~194 MTAs present on different chromosomes and associated with multiple traits. These 62 MTAs were associated with either the same trait in multiple years (MTA stability in different environments) or multiple traits within the same year or across years (suggesting epistasis) . AdditioA field experiment was conducted during two growing seasons (2016 and 2017) under drought stressed conditions (rainfed) at the research farm located at the Bahri Dagdas International Agricultural Research Institute in Konya, Turkey . This site was characterized by a low precipitation (below 300 mm), low humidity, and slightly alkaline clay loam soil .Ae. tauschii accessions resulting in 14 different lines  with replicated checks (‘Gerek’ and ‘Karahan’) in the 2016 growing season and modified alpha-lattice design (plot size: 1.2 m × 5 m) including 123 SHWs and replicated checks (‘Gerek’ and ‘Karahan’) with two replications in the 2017 growing season. The SHWs were planted on 20 September in 2015 and harvested on 18 July 2016 for the 2016 growing season, whereas the SHWs were planted on 15 September in 2016 and harvested on 21 July 2017 for the 2017 growing season.2  and reported in g⋅m−2. The HI, BMWT, TKW, GVWT, AWNLN, FLLN, FLW, and FLA (0.8 × FLLN × FLW) were measured using previously reported protocols [The GY was obtained by harvesting four middle rows of 1.008 mrotocols ,32,59,61yijklm is the GY and yield-related trait; μ is overall mean; Yri is the effect of ith year; R(Yr)ji is the effect of jth replication within the ith year; B(R(Yr))kji is the effect of kth incomplete block within jth replication of ith environment; Cl is the lth checks; Gmkji is the effect of mth genotypes  within the kth incomplete block of jth replication in the ith year; GXYrmi is the interaction effect of mth genotype and ith year; and eijklmn is the residual. In the combined ANOVA, year and check were assumed as fixed effects, whereas genotype, genotype × year interaction, replication nested within a year, and incomplete block nested within replications were assumed as random effects.A combined ANOVA was performed using the following model:yijkl is the trait, μ is the overall mean; Bi is the effect of ith incomplete block; Cj is the jth check; Gk(i)  is the effect of kth genotypes within the ith block; eijkl is the residual. In ANOVA calculated for the 2016 datasets, the check was assumed as a fixed effect, whereas genotype and incomplete block were assumed as random effects.Individual analyses of variance were performed because most of the traits had highly significant genotype × year interaction and therefore will be discussed hereafter. An augmented design was analyzed using the following model for the estimation of BLUPs in the year 2016:yijk is the trait, μ is the overall mean; Ri is the effect of ith replication; B(R)ji is the effect of jth block within the ith replication; Ck is the kth checks; Glji  is the effect of kth genotypes within jth incomplete block of ith replication; eijkl is the residual. In ANOVA calculated for the 2017 datasets, the check was assumed as a fixed effect, whereas genotype, replication, and incomplete block nested within replication were assumed as random effects.An alpha (α) lattice design with two replications was analyzed using the following model for the estimation of BLUPs in the year 2017:All phenotypic data were analyzed using PROC MIXED in SAS 9.4   using th2g, σ2yr, σ2gxyr, and σ2e are the variance components for genotype, year, genotype × year, and error, respectively, and n and r are the numbers of years and replications, respectively.Broad-sense heritability for each trait in each year was calculated based on entry mean basis using Equations (4–6) for 2016, 2017, and combine0d experiments, respectively:Pearson’s correlation of GY and yield-related traits was calculated based on BLUPs for each trait in each year using PROC CORR in SAS. The PC biplot analysis (PCA-biplot) was performed based on the correlation matrix to avoid any variation due to the different scales of the measured variables using ‘factoextra’ package in R software .https://bitbucket.org/tasseladmin/tassel-5-source/wiki/Tassel5GBSv2Pipeline) [Genomic DNA was extracted from two to three fresh young leaves of 14-day-old seedlings using BioSprint 96 Plant Kits , as described in Bhatta et al. . The GBSipeline)  with phyipeline)  using dehttps://web.stanford.edu/group/pritchardlab/structure_software/release_versions/v2.3.4/html/structure.html) [http://www.maizegenetics.net/tassel) [Population structure of 123 genotypes was assessed using the Bayesian clustering algorithm in the program STRUCTURE v 2.3.4 (available online: re.html)  and prin/tassel) , as desc/tassel) .1 and Q2) or first three principal components  as covariates by looking at the model fit using Quantile-Quantile (Q-Q) plots and FarmCPU-calculated kinship [p-value = 9.99 × 10-5 (−log10p = 4.00) was selected for MTAs considering the deviation of the observed test statistics values from the expected test statistics values in the Q-Q plots [Many GWASs were previously performed using the mixed linear model (MLM), where the population structure (Q) or PC was set as a fixed effect and kinship (K) as a random effect to control false positives ,92. Howe kinship  implemen-Q plots ,80 from 2 > 0.2) in adjacent regions (<500 kb) of significant MTAs were visualized and plotted using default parameters  of Haploview software (available online: https://www.broadinstitute.org/haploview/haploview) [Haplotype blocks with linkage disequilibrium (LD) values  . PVE by http://snpeff.sourceforge.net/) was used for SNP annotation and predicting the effects of SNPs on the protein function. The MTAs present within genes or flanked (5 kb) by genes were investigated [The genes underlying the MTAs and subsequently their annotations were retrieved using a Perl script and the IWGSC RefSeq v1.0 annotations  providedstigated .The present study showed SHWs have large amounts of genetic variation for GY and yield-related traits. The GWAS in 123 SHWs using 35,648 SNPs identified several novel  genomic regions or haplotype blocks associated with GY and yield-related traits in drought-stressed conditions. Most of the MTAs (120 MTAs) were present in genes ad several of them (45 MTAs) were annotated with functions related to drought stress. This provided further evidence for the reliability of the MTAs identified. We also identified MTAs on different chromosomes associated with multiple traits but within genes having the same annotation. This resulted in the identification of candidate genes belonging to the same gene family that likely have a major role in affecting GY and yield-related traits under drought stress in SHWs. The large number of MTAs, especially on the D-genome (53 MTAs with 34 MTAs being novel) identified in the present study, demonstrate the potential of SHWs for elucidating the genetic architecture of complex traits and provide an opportunity for further improvement of wheat under rapidly growing drought-stressed environment worldwide.


To ensure meaningful results in a clinical audit, as many hospitals as possible should participate. To optimise participation, the data collection process should either be performed by additional staff or be automated. Active participation may be promoted by offering relevant external parties insight into the actual quality of care.The aim of the study was to identify which facilitators and barriers experienced by hospital staff are associated with participation in the ongoing nationwide multidisciplinary Dutch Hip Fracture Audit (DHFA).A survey including questions about the respondents’ characteristics, hospital level of participation and factors of influence on DHFA participation was sent to hip fracture surgeons. The factors were based on results of semi-structured interviews held with hospital staff involved in hip fracture care. Univariable and multivariable logistic regression analyses were used to establish which respondent characteristics and factors were associated with participation and active participation (≥ 80% of patients registered) in the DHFA. Factors significantly increasing the (active) participation in the DHFA were classified as facilitators, and factors significantly decreasing the (active) participation in the DHFA as barriers.One hundred nine surgeons filled out the questionnaire. The factors most agreed on were availability of staffing capacity for data collection and automated data import. A lower intention to participate was associated with being an academic surgeon  and an orthopaedic surgeon . Data sharing with relevant external parties was associated with active participation .To improve participation in a nationwide clinical audit, it seems that the data collection should either be performed by additional staff or be automated. Active participation is facilitated if audit data is made available to other parties, such as insurers, healthcare authorities or policymakers. In a clinical audit, prospective data of the treatment process and of patient outcomes is collected in a systematic way. Based on these data, feedback is given to provide hospitals insight into their own performances. Also, in a clinical audit, hospitals are benchmarked on pre-established quality indicators to define best practices that can serve as examples for underperforming hospitals. The aim of this continuous process is that hospitals implement changes in the treatment process based on the feedback, with the ultimate goal to improve the quality of care .In the last decade, audits for different diseases have been initiated –8. The pThe aim of this study was to identify which factors (facilitators and barriers) experienced by hospital staff are associated with hospital participation in a nationwide multidisciplinary clinical audit on hip fracture care that was recently started in the Netherlands, the Dutch Hip Fracture Audit (DHFA).The DHFA is a continuous nationwide multidisciplinary hip fracture audit, which was started in April 2016 at the Dutch Institute for Clinical Auditing . In the To identify factors that may influence participation by medical professionals in the DHFA, the first author conducted semi-structured interviews with 8 trauma surgeons, 3 orthopaedic surgeons and 1 geriatrician/internist between 1 June 2017 and 8 November 2017. All the interviewees were members of at least one of the medical associations involved in the multidisciplinary DHFA.The structure of the interviews was developed specifically for the DHFA according to the Theoretical Domains Framework , 13 , years of experience, annual number of hip fracture operations, type of hospital , familiarity with the DHFA (yes or no) and hospital level of participation in the DHFA. The level of participation was divided into four categories: not participating in the DHFA, and having no intention to do so; not participating in the DHFA, but intending to do so; partially participating in the DHFA with < 80% of the patients are registered; and actively participating with ≥ 80% of the patients are registered. If the respondent indicated in the first part not to be familiar with the audit, the second part of the questionnaire was not shown.The second part of the questionnaire included the factors identified in the interviews that may influence participation in the DHFA. To limit the length of the questionnaire, the 76 factors were grouped into 21 overarching factors  and ‘agree’ , because for many factors, some categories were chosen by less than five respondents. For similar reasons, the four categories of participation were combined into two different dichotomous variables: the participation variable with ‘not participating’ versus ‘participating’ and the active participation variable with ‘partially participating with < 80% of patients registered’ versus ‘actively participating with ≥ 80% of patients registered’.p < 0.10) were entered in a forward stepwise multivariable logistic regression analysis, with the factors/respondent characteristics as independent variables, and participation/active participation in the DHFA as dependent variables. In the multivariable model, predictors for participation or active participation were considered statistically significant if the p value was less than 0.05. Factors significantly increasing the participation in the DHFA were classified as facilitators, and factors significantly decreasing the participation in the DHFA as barriers.For both participation and active participation in the DHFA, the associations with respondent characteristics and the 21 overarching factors obtained from the interviews were first studied in a univariable logistic regression analysis. Next, respondent characteristics and factors found in the univariable analysis to be associated with participation or active participation  109 surgeons completed the questionnaire, when the questionnaires received at the congress and by mail were combined. Seven surgeons (four orthopaedic surgeons and three trauma surgeons) who completed the questionnaire did not know about the existence of the DHFA. The characteristics of the respondents across the different categories of participation are shown in Table The three factors obtained from the interviews that were most agreed on in the questionnaire by both participants and non-participants concerned organisational aspects: ‘staffing capacity should be made available for the data collection in the DHFA (95.1%)’, ‘data import from the EHR into the DHFA should be automated (95.1%)’ and ‘participation in the DHFA should be supported financially by the hospital board (94.1%)’ Table .Table 3Rp > 0.10). Of the respondent characteristics, type of hospital , type of surgeon (orthopaedic versus trauma) and annual number of hip operations had a univariable association with participation in the DHFA (p < 0.10) and were entered in the multivariable analysis (Table p < 0.01) and being an orthopaedic surgeon  were independently associated with lower participation in the DHFA.In the univariable analyses, none of the 21 overarching factors as identified in the interviews was significantly associated with actual participation in the DHFA (is Table . Of thosp < 0.10) with active participation (i.e. registration of more than 80% of the hip fracture patients) in the univariable regression analyses and therefore entered in the multivariable analysis (Table p = 0.03) in the multivariable regression analysis.Within the subgroup of surgeons who participated in the DHFA, three factors  were associated . The most often agreed on factors influencing participation were related to the organisational context of the audit, i.e. how the data collection of the DHFA should be organised. However, only physician characteristics , and no factors were independently associated with participation in the DHFA. For active participation (≥ 80% of patients registered), the opinion of surgeons that the DHFA data provides relevant external parties with insight into the actual quality of hip fracture care was significantly associated with the level of participation.The factors identified through the semi-structured interviews are in line with the possible facilitators and barriers of a clinical audit previously described in literature , 10. TheThe primary goal of a clinical audit is to improve the quality of care. Similar to our results, most respondents in a survey among US surgeons also felt that an audit  would contribute to a better quality of care . InteresIn literature, a previously reported facilitator for a successful audit is easy data collection, through the use of modern medical record systems . A studyAs the factors most agreed on in the questionnaire were not specific to hip fracture management, our findings are applicable to upcoming clinical audits in general, regardless of their topic. Therefore, it is advisable that data collection is automated as much as possible and that time and resources are made available before the start of any audit. Hospitals in which the organisation of ongoing audits is well arranged can serve as examples for other hospitals where clinical auditing has yet to be implemented. Hospitals lacking the financial means to hire adequate staff for the DHFA data collection solved this by training of a research nurse in their employ, how to collect the data. They are now responsible for the data collection, but under the direct supervision of a surgeon in case of uncertainties.Orthopaedic surgeons and trauma surgeons working in an academic hospital might be less likely to participate in the DHFA than orthopaedic surgeons and trauma surgeons working in a general hospital, respectively. This finding may be explained by the fact that in the Netherlands, orthopaedic and academic surgeons operate considerably lower numbers of hip fracture patients. For this reason, they might consider the effort of implementing an audit into the routine working procedures for relatively few patients per year not be worthwhile. This consideration is also true from a statistical point of view. Low-volume hospitals/wards are less interesting for benchmarking, as analyses for hospitals/wards with low patient volumes are underpowered to detect variation in outcomes of care . One lesA facilitator for active (≥ 80% of patients registered) participation in an audit might be to make data available to relevant external parties  and give them insight into the actual quality of hip fracture care by benchmarking hospitals. A possible explanation for this finding is that making hospital-specific data available soon after the start of an audit works as a trigger for hospitals to start registering, as they do not want to lag behind other hospitals. In the survey of Cornish et al., the national benchmarking facility also appeared to be the main reason to submit data to the bowel cancer audit . Also, tThe optimal timing to make data available for relevant external parties is hard to define. From the second year of the DHFA on, the results of process indicators have been made available to the National Health Care Institute and the healthcare insurance companies. From the third year on, hospitals can choose to also make hospital-specific outcome indicators available, but from the fourth year onwards, this will be mandatory. The National Hip Fracture Database (NHFD) in the UK also made outcome indicators publicly transparent at hospital level in the third year after its start in 2007 .In our opinion, it is important for all kinds of upcoming clinical audits to realise that data being available to external parties can be a determining factor for hospitals to participate actively in an audit. Therefore, it is advisable to agree at an early stage on how and when data will be disclosed to other relevant external parties.Interventions in other audits that proved to be helpful in their implementation included financial rewards and obligatory participation. The need of financial support by the hospital board was also the third most agreed-on factor in our study. The NHFD has also shown that a financial reward, the Best Practice Tariff, can serve as a facilitator for hospitals to participate in a nationwide audit . The BesGovernmentally imposed participation has proven to facilitate participation. After the participation in the Dutch Surgical Colorectal Audit had become a quality indicator for the Health and Youth Care Inspectorate in the Netherlands, the participation rate rose to almost 100% .The grouping of the 76 factors identified in the semi-structured interviews into 21 overarching factors for the questionnaire has induced loss of information detail; it may have caused loss of the value of individual factors. The reason to group the factors was that, although we identified so many factors in the interviews, the questionnaire still should be clear and feasible and preferably be filled out within 10 min. We felt that sending out a questionnaire with all 76 factors would not render us the responses we needed. Another reason to group factors was from a statistical point of view: 76 factors and 5 respondent characteristics would induce the risk of multiple testing. Even with the 21 overarching factors this cannot be explained.Another limitation is that the questionnaire was only sent to members of the Dutch Trauma Society, and therefore included only trauma and orthopaedic surgeons. Consequently, physicians from other medical disciplines such as geriatricians and internists were not invited to fill out the questionnaire. As a result, facilitators and barriers that are specific to non-surgeons were not included. This was a deliberate choice, since the setup of the DHFA data registry in a hospital is mainly the responsibility of the orthopaedic and trauma surgeons.Regarding the representativeness of the respondents to the survey, the ratio between trauma surgeons and orthopaedic surgeons in our study (80/20) was comparable with that in the DHFA data for 2017 (71/29). However, the surgeons working in an academic hospital were overrepresented (14%) in the survey compared with the DHFA data for 2017 in which only 1% of the hip fracture patients were treated in an academic hospital. In addition, as responses were anonymous, possible clustering of respondents working at the same hospital may have occurred ,leaving unknown whether the respondents geographically represented the whole country. Both issues imply a possible selection bias.Yet another limitation is that two of the three orthopaedic surgeons who were interviewed were not familiar with the DHFA. We may therefore have missed possible factors that are especially important for orthopaedic surgeons.We learned from this study that the data collection process of a nationwide clinical audit should either be performed by additional staff or be automated in order to avoid an increase in registration load for physicians. Physicians treating low numbers of audited patients are less likely to participate in an audit. Active participation in an audit may be promoted by offering relevant external parties, such as insurers, healthcare authorities or policymakers, insight into the actual quality of care. The use of these results will accommodate the successful implementation of new audits and the provided tools may offer opportunities to optimise participation in running audits.


HPV test appears to be more effective in cervical cancer (CC) screening. However, the decision of its adoption as a primary screening method by substituting the established cytology lies in the evaluation of multiple criteria. Aim of this study is to evaluate the economic and clinical impact of HPV test as primary screening method for CC.A decision tree and a Markov model were developed to simulate the screening algorithm and the natural history of CC. Fourteen different screening strategies were evaluated, for women 25–65 years old. Clinical inputs were drawn from the HERMES study and cost inputs from the official price lists. In the absence of CC treatment cost data, the respective Spanish costs were used after being converted to 2017 Greek values. One-way and probabilistic sensitivity analyses were conducted.All screening strategies, that offer as primary screening method triennial HPV genotyping (simultaneous or reflex) alone or as co-testing with cytology appear to be more effective than all other strategies, with regards to both annual CC mortality, due to missed disease (-10.1), and CC incidence(-7.5) versus annual cytology (current practice). Of those, the strategy with HPV test with simultaneous 16/18 genotyping is the strategy that provides savings of 1.050 million euros annually. However, when the above strategy is offered quinquennially despite the fact that outcomes are decreased it remains more effective than current practice (-7.7 deaths and -1.3 incidence) and more savings per death averted (1.323 million) or incidence reduced (7.837 million) are realized.HPV 16/18 genotyping as a primary screening method for CC appears to be one of the most effective strategies and dominates current practice in respect to both cost and outcomes. Even when compared with all other strategies, the outcomes that it generates justify the cost that it requires, representing a good value for money alternative. Cervical cancer represents the fourth most frequent cancer in women worldwide and the eighth in Europe, with annual new incidence of 569,847 and 61,072, respectively .Since its introduction, in 1954 .In the late 1970s, the human papillomavirus (HPV) was associated with the development of squamous cell carcinomas . Twelve The association of HPV with cervical cancer led to the development of diagnostic methods for the detection of the virus’s DNA (HPV DNA test), which can ultimately be used as a primary screening method for cervical cancer screening and substitute or complement the Papanicolaou test. The performance of the HPV DNA test has been proven superior to the Pap test in the detection of CIN2 or greater, as the latter has demonstrated sensitivity that varies from 44% to 74%, with an average of 53%, while the sensitivity of the HPV DNA test reaches 100% . The speHowever, according to Cochrane and Holland , 18 beyoThe aim of the study is the investigation of the latter by exploring the economic efficiency of the HPV test as a primary screening method for cervical cancer in Greece. The cobas HPV test was used for the current analysis which detects 14 high-risk types  and simultaneously the 16 and the 18 genotypes. The study is conducted from the social insurance’s perspective and compares the budget impact, the cost per death averted and the cost per incidence reduced of screening with a) cytology annually (current practice) and triennially, b) the HPV test with simultaneous HPV 16 and 18 genotyping every three and every five years, c) the HPV test with reflex 16 and 18 genotyping every three and every five years, d) the HPV test with no genotyping every three and every five years, e) Co-testing with cytology and the HPV test with simultaneous 16 and 18 genotyping every three and every five years, f) Co-testing with cytology and the HPV test with reflex 16 and 18 genotyping every three and every five years, g) Co-testing with cytology and the HPV test with no genotyping every three and every five years. The aim of the study was achieved by providing a comparative evaluation of both the clinical and economic impact of all the above screening strategies, under two reimbursement scenarios.A decision tree was developed to model the screening and diagnosis of cervical cancer. The baseline assumption of individuals’ compliance to cervical cancer screening was 30.3% . The modThe compared screening algorithms and strategies are:a) Cytology: Cytology is offered and reimbursed annually by the social insurance fund in Greece. The model’s algorithm determines that a woman with negative cytology results returns to routine screening in one or three years. Women with intermediate results—referred to as atypical squamous cells of undetermined significance (ASCUS)—are re-tested with cytology in 6 months, and then, in case of cytology results of ASCUS or worse they are referred to immediate colposcopy, otherwise they return to routine screening. In case of positive results at the primary test  women are referred to colposcopy .b) Primary HPV test with 16/18 genotyping: HPV screening is assumed to be offered every 3 or 5 years. Women with negative results return for routine screening in 3 or 5 years. Women who are HPV 16/18 positive are referred for immediate colposcopy. Women who are HPV 16/18 negative, but positive to the rest of the 12 high risk HPV types are referred to cytology. In case of abnormal results, women are referred to colposcopy. Normal results are retested in 12 months and half of them that test hrHPV positive receive cytology and the rest colposcopy .c) Primary HPV test with no genotyping: HPV screening is provided every 3 or 5 years. As above women with no HPV findings return to routine screening. HPV positive women are referred for cytology and those with negative results are retested with HPV test in 12 months. Women with intermediate (ASCUS) and or positive results  are referred for colposcopy .d) Co-testing with cytology and HPV with genotyping. Both cytology and HPV test are offered every 3 or 5 years. Women with normal cytology results who are HPV negative return to routine screening. Those with positive HPV results and/or ASCUS cytology results and those positive to 16/18 genotypes are referred for colposcopy. Women with borderline cytology results (<ASCUS) and positive to HPV but negative to 16/18 are retested in 12 months .e) Co-testing with cytology and HPV with no genotyping. Both tests are offered every 3 or 5 years. As above, women with negative cytology and HPV results return to routine screening. Those with cytology results worse than ASCUS or ASCUS and HPV positive are referred for colposcopy. In the case of borderline results (<ASCUS) and HPV positive are retested in 12 months with co-testing .As mentioned above a Markov 7-state transition model with 1-month cycle  was developed to simulate women’s transition from one state of the disease to another . Women mThe vast majority of clinical, epidemiological and diagnostic tests’ performance inputs were drawn from the HERMES study  , which wAll costs were discounted at 3.5% and included all interventions reimbursed by the social insurance fund (EOPYY) for the diagnosis and the treatment of CIN and invasive cervical cancer. The HPV test is currently partially reimbursed by the social insurance fund, as users’ coinsurance rate has been set at 15% (12€). However, the analysis is conducted by estimating the economic impact in both the scenarios of partial and full reimbursement. On the contrary, cytology-based screening is fully reimbursed and is typically offered by the employment of two different examinations, i.e. 1) cytological examination of cervicovaginal smear, and 2) particular examination of endocervical smear, which are charged separately at 6.66 euros each . Unit costs for the resources used were obtained from the official price lists  Table 3Table 3.For strategies with the HPV test with simultaneous HPV 16 and 18 genotyping—no additional cost for reflex genotyping was included. In the absence of relevant data, the cost of treating cervical intraepithelial neoplasia (CIN2+) and cervical cancer was based on the respective Spanish costs , which wThe present study is an economic modelling study, which employs published data from the literature. In this context, it does not involve human participants and there were no direct or indirect interactions with patients or human specimens or tissue. The clinical data that were used  were draThe comparison of the under investigation strategies reveals that current practice  is inferior with regards to the incident of cancer and mortality due to missed disease from the majority of the comparators. With regards to cervical cancer deaths, it appears that all but three strategies have a significant positive impact on mortality from missed cases . The mosFrom a strict economic perspective, this analysis proves that, in most of the cases, current practice consumes more resources for inferior outcomes in both the partial and the full reimbursement scenarios, leading to inefficient resource allocation. Irrespective of the outcomes generated, under the partial reimbursement scenario all strategies appear to provide savings to social insurance, apart from those that are offered triennially and incorporate reflex genotyping. When full reimbursement is applied, the co-testing strategies with 16/18 genotyping and without genotyping provided every three years are added to the strategies that demand more resources than annual cytology.By evaluating cost and outcomes it becomes evident that annual cytology is dominated by the majority of screening strategies. All strategies that include genotyping show better efficacy in reducing cancer incidence and among them, the optimum outcomes are generated by the screening strategies that are offered triennially and are accompanied by HPV genotyping. However, even in the scenario of full HPV test reimbursement, the strategy of HPV test with simultaneous 16/18 genotyping every 3 years appears to be a cost saving strategy  that provides maximum cancer protection by considering both annual cancer incidence and mortality Tables  and 8. AThe difference in the total annual cost between the strategies is attributed to the compared screening algorithms’ clinical effectiveness and cost. All HPV strategies detect more CIN2+  and thusBoth OWSA and PSA were conducted for all screening strategies and proved that the results of the model are robust. The outcomes of both the sensitivity analyses for the screening strategies of 3-year and 5-year HPV testing with simultaneous 16/18 genotyping versus annual cytology are presented in Figs The analysis supports that from a public health perspective, adopting the HPV test with genotyping (simultaneous or reflex) with or without cytology in first line screening for cervical cancer is more clinically effective than cytology alone. However, from the most effective strategies, the screening algorithm of triennial HPV testing with simultaneous 16/18 genotyping appear to provide to the social insurance fund the opportunity to save resources  by achieving the optimum outcomes, at the same time. These savings are realized in both the partial and full reimbursement of the HPV test scenarios, though higher in the former .Nevertheless, it appears that the aforementioned strategy when offered quinquennially instead of triennially still provide better outcomes than current practice, but it generates significant higher savings (up to 10 million euros) at the expense of lower outcomes than when offered more frequently. The dilemma the decision makers face in such cases is whether the increased savings can justify or compensate for the lower health outcomes they come with. From an economist’s point of view, this decision could be justified in the case that these savings can be invested in another intervention that will provide more health outcomes than those lost from this choice. An example of this could probably be investing in interventions to increase participation in the HPV vaccination program. However, this still remains a political decision and is outside of the scope of this study. Despite that, the adoption of 5 year screening interval is supported by relevant studies that present efficiency gains and outcomes improvement , 43.In contrast to cost-utility analyses, budget impact and cost per death averted or per incidence reduced analyses are not usually accompanied by an established national threshold regarding the additional cost for the outcomes provided and thus, efficiency in such cases is mostly subject to current political views and the economic environment. Though, from an economic perspective, the current analysis provides strong arguments towards the adoption of HPV test with simultaneous 16/18 genotyping every 3 or 5 years and is in parallel supported by increasing evidence that adopting the HPV test as a primary screening method is a cost-effective or even cost-saving option compared to cytology –51. MoreBy considering Cochrane’s and Holland’s screening program criteria , 18, theRegarding its acceptability by the individuals, the HPV test can be safely assumed to be as acceptable as the cytology, due to the fact that women are going through the same procedure in order to perform the test.In terms of test’s accuracy, even though the HPV test and cytology examine different clinical outcomes, the HPV test initiated screening algorithms proved to be more accurate than cytology alone. This is attributed to HPV test’s superior performance and to the fact that in contrast to cytology, it provides the opportunity to better identify individuals’ risk of developing CIN3 –16, 53 iRepeatability is an additional criterion under which the tests should be evaluated and as described above the HPV test is more capable of providing consistent results in repeated trials compared to cytology for which interpretation involves a significant level of subjectivity as shown in relevant studies , 54.In terms of the under comparison tests’ performance, it has been demonstrated in numerous clinical studies that HPV test dominates cytology when sensitivity is examined, but shows slightly lower specificity –16. HoweBased on the discussion above, it becomes evident that HPV 16/18 genotyping according to Cochrane’s and Holland’s criteria is superior to cytology. On the contrary, any discussion regarding the adoption of an HPV test with reflex genotyping or co-testing with simultaneous or reflex genotyping appears to be redundant, since these strategies demand more resources compared to current practice for the same outcomes that the HPV 16/18 genotype provides.Adjusting the discussion to the Greek public health setting, the adoption of HPV 16/18 genotyping provides the opportunity to improve cervical cancer prevention even with low and inadequate levels of screening compliance rates, as those currently observed. Individuals’ adherence to all screening guidelines in Greece is quite low over time and hence, from a health policy perspective several interventions should be adopted in order to optimize secondary prevention. In line with the above, any intervention that improves access, awareness or screening effectiveness even for the few screened, should be placed as a priority in the health policy agenda. This is also enforced by the fact that in times of economic turmoil and fiscal restrictions health care resources should be shifted to primary care and public health in order to increase the efficiency of the resources invested in health care and to optimize population health status –57. In aHPV vaccination can contribute to the reduction of HPV infection and consequently to the burden of CIN2+ and CC. Therefore, as vaccination coverage increases, the marginal benefit of screening decreases due to the fact that fewer women will be tested positive to the virus. In this sense, this could be perceived as limitation of the present study as the vaccine’s impact has not been taken into account. Though, the vaccination coverage in Greece is quite low and thus the value of screening remains high. Nonetheless, regardless of the vaccination coverage, HPV screening still appears to be of great importance and of good value for money versus cytology . HoweverIn the absence of relevant data, the screening compliance rate and the attendance rate at re-testing have been assumed equal. Given that in light of positive or equivocal results, the attendance rate at retesting can be assumed increased, this could be seen as a limitation and a parameter that impacts the results of the model, as well, as shown by the OWSA. However, the PSA conducted included both high (80%) and low (20%) attendance rates and revealed that the model’s results remain robust.The prevalence of CC was assumed equal to that observed in the ATHENA trial  as in thIn addition, the present study estimates a specific incidence outcome which is (the difference in) incidence and mortality due to missed disease and resulting progression from the different screening strategies under investigation. Currently, there are no relevant local real world data of such an estimate  that could be used to compare our results and to validate the disease natural history model in this way.The transition probabilities used for CIN are not stratified by HPV type due to our currently limited relevant knowledge and as Wright et al. argue inThe present study was conducted for both HPV partial reimbursement (current reimbursement status) and full reimbursement scenarios. In this context, the adherence rate in HPV screening under the scenario of partial reimbursement may be overestimated as it has been proved that co-payment negatively impacts health services utilization –62. For Finally, despite the fact that HPV screening is being discussed for age groups >25 and >30 years old, the present study examined the first scenario as there is evidence to suggest that HPV screening is more effective when it begins earlier, at the age of 25 , which iScreening with the HPV test is an issue that has received a lot of international attention from academic societies, physicians and health care systems. In this direction, the international discussion of whether the HPV test should replace cytology as a primary screening method for cervical cancer concludes in favor of the former –68. In lOur analysis appears to support the outcome of the international dialogue and sets ground to the point that HPV screening is the most effective intervention to adopt in a National Screening Program. Primary screening with HPV test 16/18 genotyping alone every 3 or 5 years is the strategy that provides the opportunity of optimal resource allocation and health outcomes improvement. However, the adoption of the most cost-effective strategy is a health policy decision, which will be based on the current economic environment and the cost and benefits trade off. In this context, the present study could serve as an input to the decision making process and contribute to the discussion regarding cervical cancer prevention in Greece.S1 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


HPV DNA is found in almost 80% of VIN/VaIN. Current management is inadequate, with high recurrence rates. Our objective was to review the literature regarding the role of HPV vaccine in secondary prevention and treatment of VIN/VaIN.Clinicaltrials.gov. Search terms included HPV vaccine AND therapeutic vaccine* AND VIN OR VAIN, published in English with no defined date limit. Searches were carried out with a UCL librarian in March 2018. We included any type of study design using any form of HPV vaccine in the treatment of women with a histologically confirmed diagnosis of VIN/VaIN. We excluded studies of other lower genital tract disease, vulval/vaginal carcinoma and prophylactic use of vaccines. The outcome measures were lesion response to vaccination, symptom improvement, immune response and HPV clearance.Database searches included Ovid Medline, Embase, Web of Science, The Cochrane Library and We identified 93 articles, 7 studies met our inclusion criteria; these were uncontrolled case series. There were no RCTs or systematic reviews identified. Reduction in lesion size was reported by all 7 studies, symptom relief by 5, HPV clearance by 6, histological regression by 5, and immune response by 6.This review finds the evidence relating to the use of HPV vaccine in the treatment of women with VIN/VaIN is of very low quality and insufficient to guide practice. Further longitudinal studies are needed to assess its use in prevention of progression to cancer.The online version of this article (10.1186/s12905-018-0707-9) contains supplementary material, which is available to authorized users. Vulval/Vaginal Intraepithelial Neoplasia (VIN/VaIN) can precede the development of invasive cancer by a variable period. Usual-type VIN (uVIN) is commonly associated with carcinogenic subtypes of human papillomavirus (most commonly HPV 16) , 3.The incidence of VIN is thought to be around 1–2/100,000 . AlthougVaginal Intraepithelial neoplasia is an uncommon  usually asymptomatic disease , often a2 ablation) or medical  [The current treatments for VaIN are either surgical (excision or COtrogens) . Most trtrogens)  and is ctrogens) . A revieWomen with natural antibodies to HPV have less frequent recurrence of VIN than those who are antibody negative (22.9% vs 52%) . Three vProphylactic vaccines act by inducing neutralising antibodies to the viral L1 capsid proteins. It is thought that treatment of already established HPV infection requires activation of T-cells capable of killing virus infected cells, rather than producing antibodies against the virus itself, so therapeutic vaccines (in development), target viral genome E6 and E7 oncoproteins (necessary for initiation and maintenance of transformation to cancer) .Protein-based vaccines, which can be long or short peptide chains, and comprise TA-CIN  a fusion protein of HPV 16 L2, E6 and E7 , 19;DNA based vaccines that induce cytotoxic lymphocytes, T helper cells, and increase B cell immunity;A vaccine containing a mixture of two plasmids encoding HPV 16/18 E6 and E7 antigens;TA-HPV (therapeutic antigen HPV) a vaccinia vector-based vaccine expressing modified forms of HPV 16 and 18 E6 and E7 proteins andCell-based vaccines such as dendritic cells and tumour infiltrating T-cells .Different types of therapeutic vaccines have been tested in other HPV driven diseases. These include;The currently licensed HPV vaccines have an established role in the primary prevention of HPV infection generally in younger populations. The role of the HPV vaccine in secondary prevention and treatment has not yet been fully established. Women with VIN may have symptoms which are recurrent and difficult to treat. Clinicians who treat these patients  look for ways to improve the effectiveness of current treatment.We performed a review of the literature regarding the role of any HPV vaccination for secondary prevention, in the treatment of women with HPV-related VIN and VaIN.We conducted a systematic search of the literature with the strategy to review best available evidence. We searched for systematic reviews, and randomized controlled trials. If these were not available, we would include controlled non-random studies, and uncontrolled studies. We looked into the use of any form of HPV vaccine in the treatment of women with a histologically confirmed diagnosis of HPV related VIN and/or VaIN, versus control/standard treatment. These vaccines included the commercially available licensed vaccines prepared from the VLPs of the L1 protein of common HPV types, or the experimental vaccines against the HPV oncoproteins E6/E7. Our search also included vaccines as adjuvant to usual care. We excluded studies of other lower genital tract disease (pre-invasive or invasive), vulval/vaginal carcinoma and those studies concerning prophylactic use of vaccines.We excluded studies whose participants were pregnant, immunocompromised, or had a history of allergy to vaccine products. In the included studies, patients with VIN or VaIN were given any form of HPV vaccination.The outcome measures we wished to evaluate were lesion response to vaccination, symptom improvement, immune response, and HPV clearance. A Core Outcome Set protocol was not used as this is not currently available but is in development for vulva.Clinicaltrials.gov. Search terms included HPV vaccine OR Human Papilloma Virus vaccine OR Papilloma virus vaccines OR HPV Vaccin* AND therapeutic vaccine* AND VIN OR VAIN OR Vulval intraepithelial neoplasia OR Vaginal intraepithelial neoplasia, published in English with no defined date limit. Database searches were carried out with the aid of a UCL librarian in March 2018. Additional papers were identified via the reference lists of included studies  a vaccinia-based vector vaccine, or TA-CIN , a fusion protein vaccine comprising HPV16 viral proteins L2, E6 and E7 – the latter 2 responsible for inactivation of the tumour suppressor proteins p53 and pRb respectively, leading to hyper-proliferation of host cells and overexpression of p16 and p14 [Change in lesion size pre and post HPV vaccination was an outcome reported by all 7 studies. Symptom relief was reported by 5 studies, HPV clearance by 6 studies, histological regression by 5 studies, and immune response by 6 of the 7 studies. Follow-up was short - most commonly from 1 month to 1 year.Regression of the lesion was assessed by measuring lesion size before and after vaccination, commonly in two dimensions and taking the average of both sizes. Most of the studies categorized these into partial response (if more than a 50% reduction in size), no response (if less than 50% reduction) or complete response if no lesion was visible following vaccination. In these studies, lesion regression varied from no response up to an overall partial and complete response of 83%.Clinical improvement in symptoms was measured by either direct questioning at each visit or by asking participants to keep a symptom diary. Although no formal validated tool was used, commonly, symptoms were subjectively graded into none/mild/moderate/severe, depending on how it affected patient’s daily lives. The effects on symptom relief varied from no overall change in symptoms to 68.4% of women becoming symptom free after the follow up period.Immune response to vaccination ranged from 30 to 94%. Various different markers were used to determine immune responses (T-cell responses vs lymphoproliferative vs cytokine). The types of experimental vaccines, schedules and in some cases delivery method, also differed and this may contribute to the heterogeneity seen between study findings.HPV clearance is defined as testing HPV negative post vaccination having previously tested positive. The rate of HPV clearance varied between 8 and 74%. Histological regression is defined as the index VIN/VaIN lesion being downgraded from a high grade to a low-grade lesion. The rate of this occurring varied from 0 up to 63%. HPV status and histological regression did not seem to correlate with regression of the lesion nor the patient’s symptoms.There was a wide variation in results in relation to the above outcomes, which may reflect the differences in patients and the types of vaccines and schedules used.This study is the first systematic review of the use of any HPV vaccination in the treatment of HPV related vulval and vaginal disorders. This review has highlighted that no high-quality studies using commercially available vaccines have addressed this research question - available vaccines are designed for prevention. Studies that have been carried out are small, case series without control groups, rather than randomized controlled trials. They have used experimental therapeutic vaccines rather than the available vaccines. The study designs are of low methodological quality and a high risk of bias. Systematic reviews of randomized control trials indicate that available HPV vaccines are safe and effective for prevention. Effectiveness seems to decrease with age and exposure to HPV. The role of these vaccines as adjuvant to treatment has not been studied. The efficacy and safety of the experimental vaccines is yet to be established and it is likely to be a number of years before therapeutic vaccines become available for clinical use. Overall the evidence to answer the clinical questions of the role of HPV vaccination in the treatment of VIN/VAIN is of very low-quality.When comparing other systematic reviews and studies evaluating treatment outcomes for vulval disease, reporting centers primarily on response to treatment, with response being described as resolution, persistence or progression. This could be assessed clinically as a reduction in size of the lesion, symptoms response as reported by the patients, histological resolution (this assessment is invasive because it requires repeat biopsies), HPV clearance or persistence.A number of patients experienced relief of their symptoms post vaccination. A large number also experienced a reduction in the size of their lesions - it is unclear whether this correlates with symptom relief. Current treatments for VIN may be effective in the short term but with significant side effects and a high risk of recurrence.Of the studies which we identified, one reported no response in any of the initial outcome measures after 3 months . In thisOur review examines the role of HPV vaccination as an adjuvant to treatment for women who have HPV related VIN/VaIN. Whilst Miltz et al have examined the role of vaccination in anogenital pre-cancer , our revWomen with VIN/VaIN are living with chronic conditions, requiring prolonged and often repeated treatments which can have intolerable side effects, and yet high relapse rates. More effective treatments are needed that will reduce symptoms, reduce progression and reduce the rate of relapse. Having been introduced almost a decade ago as part of the childhood vaccination program, we should begin to see a reduction in the incidence of VIN/VAIN and other anogenital pre-cancers. In the meantime, for patients who have not been vaccinated, newer treatments with fewer side effects and morbidity are warranted. The evidence for the use of HPV vaccination in VIN/VaIN is of low quality and insufficient to guide practice. All of the vaccines used in these non-randomized studies were experimental and we did not find any research evaluating the use of commercially available vaccine as an adjuvant to usual treatment of VAIN/VIN. We aim to deliver such a study following on from this review. More studies are needed to investigate clinically important patient outcomes  with the use of HPV vaccine in a therapeutic setting. It would be prudent to also investigate its use in secondary prevention of cancer, which would need to be assessed through further longitudinal studies.Additional file 1:Appendix S1 – searches. (PDF 516 kb)Additional file 2:Figure S1 – Prisma Flow Diagram. (DOCX 108 kb)


FHIT, KLF5, and LINC00392 were the hotspot genes integrated by HPV in the DNA samples. RAD51B, CASC8, CASC21, ERBB2, TP63, TEX41, RAP2B, and MYC were the hotspot genes integrated by HPV in RNA samples. Breakpoints of DNA samples were significantly prone to the region of INTRON , whereas in the RNA samples, the breakpoints were prone to EXON. Pathway analysis had revealed that the breakpoints of RNA samples were enriched in the pathways of transcriptional misregulation in cancer, cancer pathway, and pathway of adherens junction. Breakpoints of DNA samples were enriched in the pathway of cholinergic synapse. In summary, our data helped to gain insights into the HPV integration sites in DNA and RNA samples of cervical cancer. It had provided theoretical basis for understanding the mechanism of tumorigenesis from the perspective of HPV integration in the HPV-associated cervical cancers.High-risk HPV is clearly associated with cervical cancer. HPV integration has been confirmed to promote carcinogenesis in the previous studies. In our study, a total of 285 DNA breakpoints and 287 RNA breakpoints were collected. We analyzed the characteristic of HPV integration in the DNA and RNA samples. The results revealed that the patterns of HPV integration in RNA and DNA samples differ significantly. HPV is a DNA virus that has been widely detected in humans and animals. High-risk HPV is clearly associated with cervical intraepithelial lesions and cervical cancer. Generally, about half of HPV infections could be eliminated within one year. However, infection by high-risk HPV usually could persist for several years and these types of HPV are also associated with reduced removal efficiency . MoreoveThe microscopic HPV particle is 50-60nm in diameter, and its surface consists of 72 capsomere . WrappedIn recent years, high-throughput sequencing technology had provided robust means to investigate the characteristic and biological significance of HPV integration. Previous study had revealed that HPV integration could trigger genome instability; for instance, it results in genome structure rearrangement and copy number variation . A recenIn this study, a total of 285 DNA breakpoints and 287 RNA breakpoints were collected from previous studies , 8–12. OHPV integration sites were collected from 6 recent studies . FunctioBecause HPV integration was considered a strong cis-activator of flanking genes and cis-acting enhancers can influence their target genes over long distances , 18 (up  FHIT(8), KLF5(6), and LINC00392(4) were the most integrated genes in the DNA samples. In contrast, RAD51B(9), CASC8(5), CASC21(5), ERBB2(5), TP63(5), TEX41(5), RAP2B(4), and MYC(4) were the most integrated genes in RNA samples  integrated by HPV in the DNA and RNA samples, respectively  in our RNA and DNA samples showed similar distributions in fragile, CpG, TFBS sites. However, the HPV integration sites in the RNA samples were more prone to fragile, CpG, and TFBS than that of the DNA samples  from second-generation sequencing in the existing databases. Therefore, most of the breakpoints that we used to carry out the analysis were from unpaired RNA/DNA samples.http://cancer.sanger.ac.uk/cancergenome/projects/cosmic). Further, mutations in ERBB2 had been known as therapeutic targets in lung and breast cancer in vitro [ RAD51B gene belongs to the RAD51 family, which is known to play important roles in DNA repair. Frequent HPV integrated into RAD51B might disrupt the DNA repair mechanism, which could partially explain the HPV-rendered genomic dysfunction and chromosome instability in cancers [As suggested by our results, the hotspots of HPV integration in the genome and transcriptome appeared to locate in different genes. Intriguingly, certain high frequency genes  in RNA samples appeared to have higher mutation frequencies  in the COSMIC database . This leads to a speculation that HPV integration into such region might trigger great chromosomal instability, probably via chromosomal translocation [location . Moreovelocation –25. TherAs observed in the DNA samples, the HPV integration sites were inclined to INTRON region. In contrast, the HPV integration sites found in the RNA samples were enriched in EXON, CpG, and transcription factor binding sites (TFBS). Interestingly, a large portion of HPV integration sites in RNA samples was located on the no-coding region . It might suggest that HPV integration could directly trigger the abnormal transcription and these functions of novel transcript kept unclear. Further, we found that the ratios of HPV integration sites within ALU and LINE were significantly higher in the DNA samples than those of RNA samples. Most importantly, the overall HPV breakpoints in the RNA samples suggested specific enrichments on the pathways of transcription regulation, cancer, and adherent junction. Furthermore, we noted that Xu et al. had compared the DNA junctions with the paired RNA junctions and they found that 12 of the 20 carcinomas (60%) contained a single transcriptionally active HPV16 integrate. The other 8 tumors (40%) are featured by a transcriptionally active HPV16 integrate together with one or two probably silent HPV16 integrates [Due to the significant difference observed while comparing the breakpoint profiles of the DNA and RNA samples, it raises the speculations that the genomic and transcriptomic breakpoints might play the different role in tumorigenesis.In this study, our results had revealed characteristics of HPV integration sites in the DNA and RNA samples. Additionally, the breakpoints in the RNA samples suggested tumorigenesis might arise from disrupting transcription and interrupting DNA repair mechanism. Altogether, this study had provided theoretical basis for understanding the mechanism of tumorigenesis from the perspective of HPV integration in the HPV-associated cervical cancers.


We investigated the role of tobacco and alcohol consumption on the occurrence of head and neck squamous cell carcinomas (HNSCC), and the joint effects of these factors with oral human papillomavirus (HPV) infection in the French West Indies, in the Caribbean. We conducted a population‐based case‐control study (145 cases and 405 controls). We used logistic regression models to estimate adjusted odds ratios (OR) and their 95% confidence intervals (CI). Two‐way interactions were assessed on both multiplicative and additive scales. Current smoking , drinking more than five glasses of alcohol per day , and oral infection with High‐risk HPV  were significantly associated with HNSCC. The combined exposure to tobacco and alcohol produced a significant synergistic effect on the incidence of HNSCC. Oral infection with High‐risk HPV increased the risk of HNSCC in never smokers and nondrinkers. The effects of tobacco, alcohol, and of the combined exposure of tobacco and alcohol were substantially lower in HPV‐positive than in HPV‐negative HNSCC. This is the first case‐control study to investigate the role of tobacco smoking, alcohol drinking and oral HPV infection in an Afro‐Caribbean population. Although each of these risk factors has a significant effect, our findings indicate that tobacco and alcohol play a less important role in Hr‐HPV‐positive HNSCC. Further investigations are warranted notably on the interaction of these three risk factors by cancer site. This is the first case‐control study to investigate the role of tobacco smoking, alcohol drinking and oral human papillomavirus (HPV) infection in an Afro‐Caribbean population. We showed that these risk factors have significant independent effects on the occurrence of head and neck squamous cell carcinomas (HNSCC). A significant less than multiplicative interaction was found between alcohol drinking and high‐risk oral HPV infections. The concurrent exposure to tobacco and alcohol had a synergistic effect on HNSCC risk but only for high‐risk HPV‐negative cases.  The population is predominantly Afro‐Caribbean. Their incidence rates of head and neck cancer, especially in men, are among the highest in Latin America and the Caribbean,In order to elucidate the etiology of HNSCC in the FWI, we conducted a population‐based case‐control study. It was previously shown that oral infection with high‐risk HPV was associated with an increase in risk of HNSCC. Although oral infection with HPV16 was associated with oropharyngeal cancer, HPV16 was not the predominant genotype and there was a higher prevalence of other high‐risk HPV genotypes in cases than in controls.In this study, we aimed to investigate the role of tobacco and alcohol consumption on the occurrence of HNSCC, and the joint effects of these factors with oral HPV infection. To our knowledge, this is the first study on this topic in an Afro‐Caribbean population.22.1We extended a large population‐based case‐control study previously conducted in mainland France to the overseas French departments Guadeloupe and Martinique. The details of this study, the ICARE study, have been described elsewhere.® OG‐500 kits (DNA Genotek) were used to collect a saliva sample from participants who gave their consent.Face‐to‐face interviews were conducted with cases and controls using a standardized questionnaire to ascertain sociodemographic characteristics and risk factors, in particular lifetime tobacco and alcohol consumption. During these interviews, OrageneWe initially identified 257 cases as potentially eligible, and 192 (74.7%) gave their consent to participate and were interviewed. After further review of clinical and pathological reports, 22 did not satisfy the inclusion criteria and were excluded from the study. Among the remaining 170 cases, 114 (72.3%) agreed to provide a saliva sample. Among the 497 eligible controls, 405 (81.5%) responded to the questionnaire and among them, 311 (76.2%) provided a saliva sample.2.2® kit, according to the manufacturer's instructions . The INNO‐LiPA HPV genotyping assay allows the detection of 32 HPV genotypes, which were classified as high‐risk HPV types , probable high‐risk , low‐risk , and other . The method for HPV detection has been described in detail elsewhere.Human papillomavirus‐integrated DNA from saliva samples was detected with the INNO‐LiPA2.3Detailed information on lifetime cigarette smoking history was recorded, for each period of identical smoking habits. The questionnaire included information on age at start and end of the period, number of cigarettes per day or per week, type of tobacco (blond vs black), filtered or not, inhalation pattern, and whether or not the product was manufactured or hand‐rolled. Ever cigarette smokers were defined as persons who smoked at least 100 cigarettes in their lifetime. Ex‐smokers were defined as persons who stopped smoking for at least 2 years. Smoking quantity was defined as the average number of cigarettes per day over the lifetime, and categorized into three groups . Smoking duration was expressed in years and was divided into 4 categories . Never smoker was the reference category used for all smoking‐related variables in our analyses. Dichotomous variables (ever/never) were created for smoking pipes, cigars, chewing, and snuffing tobacco.Lifetime alcohol drinking information was recorded as well, with for each period of regular consumption, the age at beginning and end, and the number of standard glasses per day, week or month for each type of alcoholic beverage . For each type of beverage, ever daily alcohol drinking was defined as at least one glass per day during at least 1 year. The average number of glasses per day was calculated over the lifetime, regardless of the type of beverage, and categorized into three groups . The reference category comprised subjects who never drank alcohol or who had drunk less than one glass per week. Alcohol duration was expressed in years and was divided into three categories . Exposure to HPV was assessed in several manners. Subjects with at least one HPV infection of any type were classified as HPV‐positive, others were referred as HPV‐negative. The group of HPV‐positive was further divided in two categories: high‐risk‐HPV‐positive (at least one HPV type in the high‐risk group) and non‐high‐risk‐HPV‐positive. A final binary variable was used for the exposure to high‐risk‐HPV: high‐risk‐HPV‐positive vs high‐risk‐HPV‐negative, the latter category grouping HPV‐negative and non‐high‐risk‐HPV‐positive.2.411/(OR01*OR10). The 95% CI for Ψ was determined using the CI for the interaction term in the multivariate model. Two‐way interaction on an additive scale was assessed using the relative excess risk due to interaction (RERI), RERI = OR11 − OR10 − OR01 + 1. Asymptotic 95% CI were calculated for the RERI as described elsewhere.The current analysis was restricted to squamous cell carcinomas of the oral cavity , the oropharynx , the hypopharynx  and the larynx . Our analysis included 145 cases and 405 controls. The association between smoking, alcohol and oral HPV infection and the occurrence of HNSCC was assessed by estimating odds ratios (ORs) adjusted for age, sex and recruitment site, and their 95% confidence intervals (CIs), using logistic regression models. The models for tobacco smoking were further adjusted for alcohol consumption. Models estimating the effect of alcohol were adjusted for smoking quantity and duration. Odds ratios associated with oral HPV were adjusted for smoking quantity, duration, and alcohol consumption. Two‐way interaction on a multiplicative scale was assessed by estimating Ψ, the multiplicative interaction parameter as follows, Ψ = ORHuman papillomavirus status was missing for 147 (27%) subjects (53 cases and 94 controls) who refused to provide a saliva sample, and for three controls for whom the quality of the specimen was considered inadequate for HPV detection. In addition, missing data were observed for smoking status (one case) smoking quantity , smoking duration , alcohol quantity (4 controls), and alcohol duration (27 cases 56 controls). We used multiple imputations by chained equations to deal with missing data.33.1Table 3.2Table Compared to never smokers, the risk of HNSCC was slightly greater for the persons who smoked only black tobacco than blond tobacco alone  (data not shown). The ORs were higher for those who inhaled deeply cigarette fumes  than for those who never inhaled  or inhaled a little  (data not shown). Cigarettes without filters (2.5%), hand‐rolled cigarettes (2.8%), pipe (5.0%), and cigars (3.6%) were uncommon in our study population and were not associated with the risk of HNSCC (data not shown). It should be noted that all cigar smokers and all pipe smokers but one case had also smoked cigarettes. No subject had chewed tobacco and only one case had snuffed.We observed a significant inverse association for those persons who drank less than one glass per day in relation to HNSCC, when compared to nondrinkers. We found that drinking more than five glasses of alcohol per day increases the risk of HNSCC by two‐fold. We observed as well an increase, although not significant, in HNSCC risk for persons who drank between 1 and 5 glasses per day. Compared to persons who drank alcohol for 30 years or less, the risk of HNSCC increased significantly among persons who drank for more than 45 years.Rum was the most frequently consumed alcoholic beverage in our study population regardless of case‐control status. The daily consumption of rum and beer increased the risk significantly by two fold compared to the persons who never drank rum or beer daily. In contrast, daily consumption of wine and other strong spirits did not increase the risk significantly compared to nondaily drinkers.In terms of oral HPV infections, no significant association with HNSCC was found for persons tested positive for HPV when compared to HPV‐negative subjects. Non‐high‐risk HPV types as well did not show any significant difference in risk to HPV‐negative subjects. On the other hand, subjects positive for Hr‐HPV types were twice as likely to develop HNSCC compared to Hr‐HPV‐negative subjects. Among Hr‐HPV types, we detected HPV16 in 4 cases and 2 controls. HPV18 was found only in 2 cases and 1 control. The other Hr‐HPV types which were detected in greater proportion among cases than controls were HPV33 (3 cases 1 control), HPV51 (3 cases 0 controls), HPV52 (8 cases 12 controls), and HPV56 (5 cases 6 controls).We analyzed tobacco, alcohol, and oral HPV risk among oropharyngeal and non‐oropharyngeal subsites separately; these results did not change in terms of direction of the association observed in the analyses with all HNSCC cases (data not shown).3.3Table Table 4Our findings provide new insight into the role of tobacco, alcohol, and oral HPV infection and their combined effects on the occurrence of HNSCC in the FWI.Similarly to other studies, we found that the risk of HNSCC increased with the duration and intensity of smoking, and the duration had a greater effect than the average number of cigarettes per day.Oral Hr‐HPV infections were significantly associated with HNSCC, regardless of Hr‐HPV genotype. A study conducted in Canada did not find any significant association with HNSCC and Hr‐HPV types excluding HPV16.Concerning the joint effect of tobacco and HPV, and alcohol and HPV, we found some evidence of a negative interaction on both the additive and multiplicative scale. In particular, the combined effect of alcohol and HPV was significantly less than multiplicative contrary to previous studies which revealed more synergistic relationships.We studied the prevalence of oral HPV infection and we are not able to distinguish between recent and long‐term infections, which complicates the interpretation of the results on the interactions. Smoking has been shown to increase the likelihood of acquisitionAnalyses by subsite did not reveal important differences with regards to the effects of tobacco and alcohol; although point estimates were higher in oropharyngeal cancer than in non‐oropharyngeal cancer, the confidence intervals were wide and the effect of traditional risk factors was similar in both subsites, as previously shown.Our data on the joint effect of tobacco, alcohol and Hr‐HPV on the occurrence of oropharyngeal cancer were supported by previous reports.Our study presents some limitations. The statistical power was low to detect interactions. In addition, the small sample size limited the detail in our analyses. In particular, we were not able to assess three‐way interactions by subsite as we would have liked with tobacco, alcohol and Hr‐HPV. We had 27% missing data for HPV in our sample. To handle missing data, we used a multiple imputation procedure that has been shown to result in less biased and more precise estimates than the exclusion of individuals with missing data.Considering the participation of the local cancer registries in the selection of cases and the methods employed to select the controls, we believe that no major selection bias has occurred in this study. Cases included in our study had a distribution by age, sex and cancer site similar to that of the cases recorded in the cancer registries; therefore, our sample can be considered representative of the HNSCC cases. The selection method for the controls was previously shown to produce unbiased samples, thus our controls could be regarded as being representative of persons of similar age and sex from the general population.5This is the first case‐control study to investigate the role of tobacco smoking, alcohol drinking and oral HPV infection in an Afro‐Caribbean population. We showed that these risk factors have significant independent effects on the occurrence of HNSCC. Overall, our findings suggest a less important role of tobacco and alcohol in Hr‐HPV‐positive HNSCC. The precise mechanisms driving these interactions on HNSCC risk are yet to be elucidated and further investigations are warranted notably on the interaction of these three risk factors simultaneously.The authors have no conflict of interest.JD, CJ, SG, SD, and DL participated in the study concept and design, and collected the data. AA, LM, and DL conducted the quality control of data. AA, SG, CH, and DL participated in the interpretation of data. AA and DL performed cleaning of final dataset, statistical analysis, and prepared the manuscript draft. All authors participated in manuscript editing, review, and approved the final version.French Data Protection Authority  no. DR‐2015‐2027; IRB INSERM no. 01‐036.Tables S1‐S3Click here for additional data file.