Differences in prognosis in HPV-positive and HPV-negative oral  squamous cell carcinomas (OSCCs) and increasing incidence of HPV-related cancers have spurred interest in demographic and temporal trends in OSCC incidence. We leverage multistage clonal expansion (MSCE) models coupled with age—period—cohort (APC) epidemiological models to analyze OSCC data in the SEER cancer registry (1973–2012). MSCE models are based on the initiation—promotion—malignant conversion paradigm in carcinogenesis and allow for interpretation of trends in terms of biological mechanisms. APC models seek to differentiate between the temporal effects of age, period, and birth cohort on cancer risk. Previous studies have looked at the effect of period and cohort on tumor initiation, and we extend this to compare model fits of period and cohort effects on each of tumor initiation, promotion, and malignant conversion rates. HPV-related, HPV-unrelated except oral tongue, and HPV-unrelated oral tongue sites are best described by placing period and cohort effects on the initiation rate. HPV-related and non-oral-tongue HPV-unrelated cancers have similar promotion rates, suggesting similar tumorigenesis dynamics once initiated. Estimates of promotion rates at oral tongue sites are lower, corresponding to a longer sojourn time; this finding is consistent with the hypothesis of an etiology distinct from HPV or alcohol and tobacco use. Finally, for the three subsite groups, men have higher initiation rates than women of the same race, and black people have higher promotion than white people of the same sex. These differences explain part of the racial and sex differences in OSCC incidence. In 2013, the National Cancer Institute published its Annual Report to the Nation on the Status of Cancer, 1975–2009, highlighting the trends in the burden of human papillomavirus (HPV) associated cancers in the United States. Although total cancer incidence has recently declined, incidence of HPV-positive oropharyngeal (OP) cancers have increased proportionally , and oneAnalysis of HPV-related oral  squamous cell carcinomas (OSCC) incidence in the Surveillance, Epidemiology, and End Results (SEER) cancer registries, have identified gender disparities but diminishing racial differences in the United States , 8. OverWe consider a subset of head and neck cancers reported to the Surveillance, Epidemiology, and End Results (SEER) cancer registries. We use the International Classification of Diseases (ICD) codes, as in Chaturvedi et al.  and BrowWe use SEER 9 data 1973–1991, SEER 13 data 1992–1999, and SEER 18 data 2000–2012, in order to leverage the increased sample size in later years. We refer to this data set as SEER Max. Concerns that can arise when combining these data sets—relating to changing racial composition, urban/rural divides, and the geographical distribution of new SEER registries— are minimized in this study by performing separate analyses for white men, black men, white women, and black women. Only white and black races are considered in this analysis, and we do not stratify by Hispanic/non-Hispanic ethnicity; SEER reports incidence rates by ethnic origin only for all races combined, white, and non-white. We consider only ages 0–84, as all cases for ages 85 and over are aggregated in the database. X(0), initial mutation rate μ0(t), growth rate α(t) and death rate β(t) of initiated cells, and malignant mutation rate μ1(t). In The two-stage clonal expansion (TSCE) model was developed by Moolgavkar, Venzon, and Knudson , 10 to cα, β, μ0, and μ1 are not age-dependent, the closed form solutions for the survival and hazard areIf the parameters r, p, and q constitute identifiable combinations. Note that −(p + q) is the net clonal cell proliferation α − β − μ1, and pq = −αμ1. Further, p ≈ − (α − β) [r with effects on initiation, effects on p with effects on promotion, and effects on q with effects on malignant conversion.The model parameters themselves are not all identifiable given age-specific incidence data, but  (α − β) . Hence, Ts of a tumor, the time between the time of tumor onset  and the time of clinical detection can be approximated byμ1 ≪ 1 and μ1 < p2/α) [Under the TSCE model, the sojourn time  < p2/α) , 15, 18.α, β, μ0, and μ1 are arbitrary functions of t, numerical solutions can be found [In the general case of age-dependent parameters, that is be found , 20, altThis model formulation may be extended to three stages and other more complex models and has been applied successfully to a variety of cancer types , 21–27. A), period (P), and birth cohort (C) [β0 is a constant and βA, βP, and βC are some functions to be determined, often constrained to be discrete functions or splines.Age—period—cohort (APC) models are a class of epidemiological models used to disentangle effects of age, period , and birth cohort  given prevalence  or incidence . The traditional model posits that incidence rates λ are described by a multiplicative model with age (hort (C) –31. ThisP = A + C. To resolve the unidentifiability, one may consider only two effect models, typically age—period or age—cohort, or constrain the age effects to have a given shape, such as the hazard function of a MSCE model, as we do in this analysis [One drawback of full APC models is their inherent unidentifiability: analysis , 15, 29.xi} with corresponding population-at-risk sizes {ni}, we derive a likelihood for the APC model in the following way. We assume that observed incident cases xi for a given population all of age Ai at time Pi from birth cohort Ci = Pi − Ai are Poisson distributed with mean μi = ni ⋅ λ, where λ is the incident rate function dependent on parameter β0 and functions βA, βP, and βC. Observations are assumed to be independent, and thus the likelihood for the whole data set of observations {xi} is given byGiven a set of observed cases {In this context of cancer diagnosis, interpretation of age and cohort effects is straightforward. In contrast, the period effect on its own represents a time-of-diagnosis effect, which may not accurately describe the historical effect of period on cancer. However, in concert with cohort effects, it becomes a surrogate measure of a person’s cumulative period effects. We follow this approach rather than explicitly modeling a historic period trend—i.e. period effects covering all calendar years since birth—as this would introduce an additional identifiability problem.In a general APC model, the age effects are not constrained, but if we are working within the TSCE framework, we can restrict the age effects to have the shape of the TSCE hazard:μ0 since r = μ0X(0)/α and X(0) and α are considered fixed:This added constraint theoretically resolves the non-identifiability problem in the full APC model , 29. In However, depending on the mechanism of carcinogenesis for a given cancer and the nature of the risk factors captured by the temporal trends, it is possible that the effects on promotion or malignant conversion rates rather than initiation rates are more realistic. Thus, by considering slightly different models with period or cohort effects acting on the promotion or malignant conversion parameters, one can investigate the impact of period and cohort on different stages of carcinogenesis. In this analysis we consider models of the formr = r · θP(P) · θC(C) where θP and θC are natural splines, r is the value of r at the reference period and cohort, and p and q are defined similarly.Here, xi} with corresponding population-at-risk sizes {ni} under these models is given byμi = ni ⋅ λ and {θP, θC} are the parameters of the natural spline functions. The negative log-likelihood, under the assumptions of each model, was minimized using a Davidon-Fletcher-Powell optimization algorithm in R (v. 3.1) [k) where k is the number of parameters. Hence, a more negative AIC represents a better fit, and one point gain in the negative log-likelihood is equivalent to reducing model complexity by one parameter.The negative log-likelihood (NLL) for observed cases {(v. 3.1) , and 95%Uncertainty quantification was investigated using Markov chain Monte Carlo (MCMC) methods; in particular, a covariance matrix for the parameters was estimated and used to create confidence intervals for the hazards and period and cohort effects. Results and additional details are found in the supplement .r, promotion p, and malignant conversion q. Further, to force the cohort and period effects into a more realistic form, we used natural splines with eight degrees of freedom for cohort effects and five for period, corresponding to approximately one degree of freedom for twelve and eight years respectively. We chose one model for all demographics in each of the three cancer subsite groups. We determined that period and cohort effects on initiation (r) gave the best model fits for all three subsite groups as this model gave the lowest AIC for most demographics in each category. A table of AIC for the considered models is shown in the supplement , since the asymptotic value of the hazard h(t) is r ⋅ (−p) and the deviance in p is relatively small. Further, a larger promotion p corresponds to an earlier increase in the hazard. We estimate mean sojourn time Ts from the biological parameters: black men and women have shorter sojourn times, and, generally, the sojourn time is shorter for women than for men. White men and women for HPV-related OSCCs are the one exception.The estimates of the biological parameters for initiation  hazards . As mentTrends in incidence of carcinoma at the three groups of subsites of the oropharynx and oral cavity, namely HPV-related sites, HPV-unrelated sites , and oral tongue sites, appear to be primarily driven by period and birth cohort effects on the cancer initiation rate rather than the cancer promotion rate or malignant conversion rate. For all three subsite groups, too, men had higher estimated initiation rates than women of the same race, and black men and women had higher estimated promotion rates than white people of the same sex. Cancer at the HPV-related and HPV-unrelated sites had very similar estimated promotion rates, which were different from those of cancers of the oral tongue.The three subsite groups have largely distinct patterns of period and birth cohort effects on their estimated initiation rates. HPV-related carcinomas, for instance, are found to be strongly cohort driven in this analysis, a result that is consistent with other findings that prevalence of sexually transmitted infections are usually related to cohort factors . In partThe separation of cohort and period effects in this analysis reveals some trends that were not apparent from the age-adjusted incidence rates alone. In particular, the age-adjusted rates for HPV-related OSCCs in white women remain nearly constant, but this analysis suggests that this seeming lack of trend belies a combination of increasing cohort and decreasing period trends. A similar effect is seen for the age-adjusted rates of oral tongue cancer for white men. Trends in the age-adjusted incidence may be a result of factors that affect everyone in a given time period or, more subtly, be caused by changes between one birth cohort to the next. One can begin to see the effects of these factors when plotting age-specific incidence stratified by different time periods or birth cohorts, as we do for white men for the HPV-related and HPV-unrelated subsite groups in As we saw in p in particular, suggests that HPV-related and HPV-unrelated cancers are distinct from the cancer of the oral tongue. Interestingly, the estimated rates of promotion p and the sojourn times are very similar between the HPV-related and HPV-unrelated OPSCCs and are quite different from those of oral tongue cancer, which seems to progress more slowly; the mean sojourn time for the oral tongue sites is about 2–5 years longer than the other two. The similarity between the HPV-related and HPV-unrelated promotion parameters and estimated sojourn times suggest that the tumor dynamics are very similar for these sites once the tumor has been initiated, whether by HPV, alcohol and tobacco use, or other cause. This similarity may appear to be in contrast with the known differences in cancer survival between HPV-related and HPV-unrelated cancers [Analysis of the estimated biological parameters for the three groups, the promotion parameter  cancers , though p for esophageal adenocarcinoma (SEER) in white men and women range from −0.16 to −0.20 [To better understand if the differences between the promotion parameters at the different subsite groups of the oropharynx and oral cavity are significant , we compare with findings from colorectal and esophogeal adenocarcinoma. Estimates of the promotion parameters for colorectal adenocarcinomas in United States (SEER) were −0.14, −0.19, and −0.20 for men for the proximal colon, distal colon, and rectum, and −0.14, −0.18, and −0.18 for women . Estimatto −0.20 . Hence, That the promotion parameter for cancer of the oral tongue is significantly different from that of the other two is consistent with the findings of other recent studies , 38, 39 As with any mathematical model, the modeling framework underlying this analysis is a simplification of the complex biological underpinnings of tumorigenesis and thus neglects a number of relevant factors. Additionally, as with other SEER-based studies, our work is limited by the uncertainty in classification of sites as presumed HPV-related or HPV-unrelated. Similarly, this analysis is limited by the lack of alcohol and tobacco consumption data in SEER, which precludes the possibility of controlling for these important risk factors.The use of a multistage model rooted in the biology of the system, a model which has been previously developed and validated, offers several advantages in this context. In particular, in addition to assessing trends in the data, we are able to pose hypotheses on the biological implications ; previous studies have not included biologically motivated carcinogenesis models in their analyses. Additionally, the large sample size afforded by the SEER database allows analysis stratified by both sex and race.This analysis suggests that cancer at HPV-related and HPV-unrelated sites have similar tumor growth dynamics once initiated. More work is needed to investigate these dynamics, as survival rates for HPV-positive and HPV-negative tumors are drastically different. Testing for HPV in oropharyngeal carcinomas should become routine and the results recorded in cancer registries.Although we cannot explicitly test the relationship between alcohol and tobacco use and oral cancer—SEER does not contain these covariates—the differences in cohort and period trends support the hypothesis that oral tongue cancer may have a different etiology from either HPV or alcohol and tobacco use. Although, there is little evidence as to what this etiology is, our analysis offers some additional information that may be useful to future studies. In particular, the birth cohort trends for oral tongue cancer appear similar to that of cancer of the HPV-related subsites, suggesting trends in sexual behavior may be relevant. That white women have distinctly different period effect trends for HPV-related (decreasing) and oral tongue (increasing) cancer while the three other demographics have similar decreasing trends for both groups may offer additional clues, though it is not clear at this time what those might be.Further, work is needed to understand why men have higher rates of initiation than women, for both white and black Americans, at all three subsite groups, a phenomenon that may be biologically rooted, and why black men and women have higher promotion rates than their white counterparts, a result of risk factors more likely influenced by socioeconomics and behavior than biology.Future studies may be able to include a joint analysis of HPV prevalence and incidence of HPV-related oropharyngeal squamous cell carcinomas using extensions of the two-stage carcinogenesis model. Indeed, such models may be able to shed light on the similarities and differences in initiation and growth of HPV-related and HPV-unrelated tumor as well as help quantify the additional risk for oral cancer associated with HPV infection.S1 FileProvides additional details on subsite classification and model selection, fit, and uncertainty quantification.(PDF)Click here for additional data file.S2 File(CSV)Click here for additional data file.S3 File(CSV)Click here for additional data file.S4 File(CSV)Click here for additional data file.S5 File(CSV)Click here for additional data file.S6 File(CSV)Click here for additional data file.S7 File(CSV)Click here for additional data file.S8 File(CSV)Click here for additional data file.S9 File(CSV)Click here for additional data file.S10 File(CSV)Click here for additional data file.S11 File(CSV)Click here for additional data file.S12 File(CSV)Click here for additional data file.S13 File(CSV)Click here for additional data file.S14 File(CSV)Click here for additional data file.S15 File(CSV)Click here for additional data file.S16 File(CSV)Click here for additional data file.S17 File(CSV)Click here for additional data file.


Identification of human papillomavirus (HPV) DNA in cervical tissue is important for understanding cervical carcinogenesis and for evaluating cervical cancer prevention approaches. However, HPV genotyping using formalin-fixed, paraffin-embedded (FFPE) tissues is technically challenging. We evaluated the performance of four commonly used genotyping methods on FFPE cervical specimens conducted in different laboratories and compared to genotyping results from cytological samples.10-PCR DEIA LiPA25 (version 1), Inno-LiPA, Linear Array and the Onclarity assay. Percentage agreement, kappa statistics and McNemar’s chi-square were calculated for each comparison of different methods and specimen types.We included 60 pairs of exfoliated-cell and FFPE specimens from women with histologically confirmed cervical intraepithelial lesions grade 2 or 3. Cytology specimens were genotyped using the Linear Array assay. Four expert laboratories processed tissue specimens using different preparation methods and then genotyped the resultant sample preparations using four different HPV genotyping methods: SPF25, respectively; 81.7 and 85.0 % for Linear Array versus Inno-LiPA and SPF-LiPA25, respectively; and 86.7 % for SPF-LiPA25 versus Inno-LiPA. Type-specific agreement was >88.3 % for all pair-wise comparisons. Comparisons with cytology specimens resulted in overall agreements from 80 to 95 % depending on the method and type-specific agreement was >90 % for most comparisons.Overall agreement with respect to carcinogenic HPV status for FFPE samples between different methods was: 81.7, 86.7 and 91.7 % for Onclarity versus Inno-LiPA, Linear Array and SPF-LiPAOur data demonstrate that the four genotyping methods run by expert laboratories reliably detect HPV DNA in FFPE specimens with some variation in genotype-specific detection.The online version of this article (doi:10.1186/s12879-015-1281-5) contains supplementary material, which is available to authorized users. The causal role of HPV in cervical carcinoma has been well established. Persistent infection with one of 13–15 oncogenic types of human papillomavirus (HPV) is a necessary cause of cervical cancer . DiagnosDetection and classification of HPV infection through polymerase chain reaction (PCR)-based methods has been successfully implemented in cervical cytology specimens . GenotypGiven the importance of accurately identifying HPV present in FFPE materials and the lack of studies comparing HPV assays in tissue specimens, we evaluated HPV genotyping methods in FFPE specimens using four HPV genotyping methods, representing frequently-used assays with well-documented performance in cervical specimens. The laboratories participating in this comparison have long-standing experience in HPV genotyping and they have participated in many inter-laboratory comparisons, including WHO HPV LabNet proficiency testing.The materials for this study were obtained from the Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED), a cross-sectional study including women referred to the University of Oklahoma from November 2003 to September 2009 for abnormal cervical screening results. SUCCEED design and methodology, including the details on enrollment, questionnaire data, HPV DNA genotyping, histology, and cytology procedures, have been described in depth elsewhere , 20. ParFor the present study, we selected 60 women diagnosed with CIN2+ (29 CIN2 and 31 CIN3) for whom tissue blocks were available and who had cytology-based HPV DNA genotyping results. The median duration between cytology sampling and LEEP was 27.5 days. In 26 women, cytology and LEEP were done on the same day.Institutional Review Board approval for this study was provided by University of Oklahoma and the U.S. National Cancer Institute.L1 primer PCR-based assay for 37 HPV types, 13 high- and 24 low-risk HPV types . HPV 52 is not determined directly by a type-specific probe but rather by a probe that cross hybridizes with HPV 33, 35, 52, and 58. The presence of HPV 52 was inferred only if the cross-reactive probe was hybridized but there was no hybridization detected for the HPV 33, 35 and 58 type-specific probes. Notably, concurrent infections of type 52 with the three other types cannot be detected. The procedure followed recommendations of the manufacturer with the variations that DNA was isolated from 1 mL of exfoliated cells in PreservCyt using Qiagen DNeasy Blood and Tissue Kit, 10 μL of template DNA was amplified, and the amplified products were hybridized and detected using an automated Auto-line probe assay (LiPA) staining system using 2.5 mL of each reagent per strip. The Linear Array results on the strips were evaluated by two independent observers. An unambiguous, continuous band was judged to indicate a positive result.HPV detection and genotyping in cytology specimens was done using the Linear Array HPV Genotyping Test (Roche Molecular Diagnostics), as previously described , 20. BriA series of 4-μm-thick tissue sections was cut from each paraffin block. The first and last sections were stained with hematoxylin and eosin (H&E) to confirm the diagnosis of CIN2+. Each participating laboratory received two unstained 4um sections for HPV genotyping. Standard measures to avoid cross-contamination were taken during tissue sectioning and processing.10-DEIA and LiPA25 (denoted as “SPF-LiPA25”). The main features of each HPV method are listed in Table One of four HPV genotyping methods was used to genotype tissue specimens in each specific laboratory: 1) the The BD Onclarity™ HPV Assay (denoted as “Onclarity”); 2) the Inno-LiPA system (denoted as “Inno-LiPA”); 3) the PGMY09/11 Linear Array (denoted as “Linear Array”); and 4) SPFE6 and E7 genomic DNA. It simultaneously detects all 14 high-risk HPV types, and can provide genotyping information on six individual genotypes , reporting the remaining HPV types in three distinct groups . Each FFPE patient sample was extracted using the automated workflow on the Viper™ LT system. The section was combined with 0.5 mL of distilled water and added directly to a tube with pierceable cap containing a proprietary diluent. The sample was then lysed directly using the Viper™ LT Pre-warm station before being transferred onto the deck of the instrument where it underwent automated sample processing and PCR detection. Briefly, the DNA was extracted using BD FOX™ magnetic particles and the eluate-containing DNA was used to set up three PCR genotyping reactions: G1 detects HPV 16, HPV 18 and HPV 45 plus the internal beta globin control; G2 detects HPV 31, HPV 33_58 and HPV 56_59_66 plus the internal beta globin control; G3 detects HPV 51, HPV 52 and HPV 35_39_68 plus the internal beta globin control. After 40 PCR cycles, any Ct score for a specific genotype and/or the internal beta globin control was considered positive for that channel.The BD Onclarity™ HPV Assay  is a real-time PCR assay that detects type-specific 10 consensus primer system to amplify a 65 bp fragment of the L1 region of HPV, followed by reverse line blot hybridization to HPV type-specific immobilized probes for 18 high-risk/ possibly high-risk  and 7 low-risk  HPV types. In addition to the internal assay control, testing for human DNA was conducted from all specimens (ERV3). The line probe assays are evaluated by two independent observers and were adjudicated by a third observer when different results are reported, which did not occur in this study.DNA was purified from paraffin-embedded material as previously described . PurifieWithout removal of paraffin wax, the tissue sections obtained for HPV genotyping were resuspended (50–125 μl) in 10 mM Tris pH 8.0 containing 1 mm EDTA, 0.1 % Laureth-12 and 1 mg/ml proteinase K (PK) and digested with shaking at 65 °C for 4 h followed by overnight at 37 °C. Prior to polymerase chain reaction (PCR)-based HPV genotyping, PK was inactivated at 95 °C for 15 min. Microfuge tubes were immediately centrifuged briefly at 13,000 X g while the paraffin-wax was liquefied and an aqueous-wax interface formed upon cooling. Two and five microliters of the aqueous digest from each tissue specimen were used for genotyping with the LINEAR ARRAY HPV Genotyping Test . The LA HPV Genotyping Test is a qualitative test for 37 HPV genotypes incorporating selective PCR amplification with biotinylated PGMY 09/11 L1 region consensus primers and colorimetric detection of amplified products bound to immobilized HPV genotype –related oligonucleotide probes on a LINEAR ARRAY HPV genotyping strip. PGMY-based HPV genotyping with the HPV LA have been previously reported in detail , 23. UsiTotal DNA was isolated from FFPE tissue material by proteinase K treatment, 250 μl proteinase K lysis buffer was added and incubated at 56 °C for 16–24 h. Proteinase K was heat-inactivated by incubation at 96 °C for 10 min. Each DNA isolation run and PCR run contained HPV positive and negative controls.10 PCR DEIA and LiPA25 version 1 assay  based on licensed Innogenetics technology [10 PCR primer set detects a broad spectrum of HPV genotypes by amplification of a small fragment of 65 bp from the L1 region of HPV. Reverse primers contain a biotin label at the 5’ end, enabling capture of the reverse strand onto streptavidin-coated microtiter plates. Captured amplimers are then denatured by alkaline treatment, and detected by a defined cocktail of digoxigenin-labeled probes, allowing detection of a broad spectrum of HPV genotypes. This method is designated an HPV DNA enzyme immunoassay (DEIA), and the results are an optical density value. When a sample is HPV positive by DEIA, the same SPF10 amplimer is also used to identify the HPV genotype by reverse hybridization to the LiPA25 genotyping strip (version 1). This line probe assay contains probes for 25 different HPV genotypes . The test results, purple lines on a LiPA strip are visually scored by two independent readers. In case of a discrepant line score a third reader determines the final outcome. In this study there were no different scores.Specimens were tested for HPV DNA by PCR amplification/typing using the HPV SPFchnology . The SPFIn SPF10 DEIA positive samples the presence of HPV 16 and 18 was also tested by type-specific (TS) PCR primer sets. TS16 and TS18 amplimers were detected by DEIA, similar to the method for SPF10 amplimer detection. The final genotyping result is the outcome of the testing algorithm . As a reAnalyses were limited to 14 HPV types classified as carcinogenic  [25) for types 33, 35, 39, 56 and 58, and 66. HPV type 68 genotyping was only compared between Linear Array and Inno-LiPA. HPV genotyping concordance was tested by calculating overall and type-specific percentages of agreement and kappa values. McNemar’s chi-square was calculated to test for significance (p < 0.05) in the pattern of disagreement.To evaluate the performance of each method in FFPE tissue samples, we compared FFPE HPV genotyping results between different HPV methods. However, pair-wise comparisons were restricted to those carcinogenic types for which a specific probe was available across all of the different methods. Thus, comparison across all four assays was possible for types 16, 18, 31, 45, 51 and 52, and across 3 assays  Identical: when the same number and type of HPV was identified in both specimens; (ii) Compatible: when at least one HPV type was found in common; and (iii) Discrepant: when no type was identified in common or the FFPE sample was HPV negative. This classification was performed, first by including all single and combined HPV type probes available in each of the assays and second, by including only single probes.All 60 exfoliated cell specimens were positive for at least one of the 14 carcinogenic HPV genotypes tested by Linear Array Table . The mos25 method). The SPF-LiPA25 assay identified more types at both the overall and the type-specific level. Detection of multiple types was rare among FFPE specimens by all methods (from 5.0 % in the Inno-LiPA to 18.3 % in the SPF-LiPA25). There was a notable difference in the detection rate of multiple types between tests with the Onclarity and SPF-LiPA25 method detecting 2–3 times more multiple types than those detected by the Inno-LiPA and Linear Array methods . Percentage of agreement for the two most common HPV types, 16 and 18, ranged from 88.3 % (SPF-LiPA25 vs. Onclarity) to 95 % (Onclarity vs. Linear Array) for HPV 16 and from 95 % (SPF-LiPA25 vs. Linear Array and Onclarity vs. Linear Array) to 98.3 % (Onclarity vs. Inno-LiPA and SPF-LiPA25 vs. Inno-LiPA) for HPV 18, but none of the differences were statistically significant. Summarized across all types, the percent identical, compatible and discrepant results for each assay combination is shown in Additional file Overall concordance between tissue-based HPV results across laboratory methods was good Table ; the ove25) of the cases , 56.7 % (Inno-LiPA), 53.3 % (Linear Array), and 61.7 %  to 100 % (HPV33 and 59) for the Linear Array method. Detection for HPV16 and 45 tested for the Inno-LiPA method and for HPV16 for the Linear Array were significantly lower in FFPE samples compared to detection in exfoliated specimens .The overall agreement of HPV status between exfoliated-cell and FFPE specimens across the four tested methods was 90 % for Onclarity, 85.0 % for Inno-LiPA, 80 % for Linear Array, and 95 % for SPF-LiPA25 Table . When coVery few studies have evaluated the performance of two or more methods in parallel in FFPE specimens –30. In t25, Inno-LiPA and the Linear Array method. Previous studies evaluating the SPF-LiPA25 assay in FFPE and cytology specimens found an overall percentage agreements greater than 90 %[25 has been also compared with other methods, such as the Genomica assay (primers MY09/1) [10-LiPA25 and the other tests. The SPF-LiPA25 method uses the general primer set, designed as SPF10, which amplifies a fragment of only 65 bp of L1 region of the HPV genome [With the exception of the Onclarity assay, the genotyping performance of these HPV methods has been previously evaluated in FFPE specimens. Our findings are very much in agreement with other reports indicating a good to excellent performance of SPF-LiPAthan 90 %, 15, 31. MY09/1)  and the  MY09/1) , with siV genome . This prGood performance for the Inno-LiPA method in FFPE versus cytology specimens has been also reported in previous studies. One Italian study compared the Inno-LiPA genotyping results from FFPE samples with the Linear Array results from the paired cytology specimens and reported an overall kappa value of 0.85 . Similar25 and with Inno-LiPA in FFPE cervical [To our knowledge, no previous study has compared FFPE samples tested by Linear Array with cytological results by Linear Array or any other method. We saw similar HPV genotyping results for FFPE and cytology specimens tested by Linear Array (agreement 80 %). As mentioned above, Linear Array has been directly compared with SPF-LiPAcervical  and vulvcervical , respectcervical , 35 and cervical . A limitOur study is the first one to test the Onclarity genotyping method using FFPE specimens. Previous studies have found that this method performs well in cytology specimens in comparison with other methods such as Hybrid Capture 2, Linear Array and Line Blot Assay . Likewis25 tests detecting more multiple types than either the Inno-LiPA or Linear Array tests. This may be related to smaller target sequences of these two assays, or to the previously described reduced performance of select L1 primer designs in detecting multiple infections [We observed that independent of the genotyping method used, overall HPV prevalence (any type versus none) in the FFPE specimens (between 80 to 95 % across methods) was lower than in cytology specimens (100 % with Linear Array), as has been found in some previous studies , 15, 31,fections . While aDNA fragmentation and DNA-protein cross-linking by formaldehyde exposure, as well as the presence of paraffin, can impact both DNA yield and PCR amplification efficiency. Our study evaluated combinations of DNA extraction methods and HPV genotyping assays performed in different laboratories. Therefore, the differences observed in this study cannot be directly attributed to assay performance versus the DNA extraction approach used. However, generally, the differences between results from different laboratories were minor and seemed to be related mostly to amplicon size.Strengths of the current analysis are the complete histological characterization of the selected specimens, and the short interval between cytological and histological specimen collection. A limitation of our study was the lack of parallel genotyping data from cytology specimens for all the tested genotyping methods.HPV genotyping from FFPE tissues showed high intra- and inter-laboratory reproducibility in our study; the use of any of the tested HPV genotyping methods in expert laboratories can provide reliable identification of the most important high risk HPV types in FFPE specimens. However, there remains concern that reliability of tissue-based genotyping may be worse in less experienced and less rigorous laboratories, as indicated by a WHO HPV laboratory comparison study including 29 centers across the world, which reported lower proficiency measures for HPV detection despite the use of standardized DNA samples , rather


The high-risk Human Papillomavirus (HPV) E6 oncoproteins are characterised by the presence of a class I PDZ-binding motif (PBM) on their extreme carboxy termini. The PBM is present on the E6 proteins derived from all cancer-causing HPV types, but can also be found on some related non-cancer-causing E6 proteins. We have therefore been interested in investigating the potential functional differences between these different E6 PBMs. Using an unbiased proteomic approach in keratinocytes, we have directly compared the interaction profiles of these different PBMs. This has allowed us to identify the potential PDZ target fingerprints of the E6 PBMs from 7 different cancer-causing HPV types, from 3 HPV types with weak cancer association, and from one benign HPV type that possesses an ancestral PBM. We demonstrate a striking increase in the number of potential PDZ targets bound by each E6 PBM as cancer-causing potential increases, and show that the HPV-16 and HPV-18 PBMs have the most flexibility in their PDZ target selection. Furthermore, the specific interaction with hScrib correlates directly with increased oncogenic potential. In contrast, hDlg is bound equally well by all the HPV E6 PBMs analysed, indicating that this is an evolutionarily conserved interaction, and was most likely one of the original E6 PBM target proteins that was important for the occupation of a potential new niche. Finally, we present evidence that the cell junction components ZO-2 and β-2 syntrophin are novel PDZ domain–containing targets of a subset of high-risk HPV types. The cancer-causing Human Papillomavirus (HPV) E6 oncoproteins have a unique carboxy terminal PDZ binding motif (PBM), which interacts with a number of different cellular PDZ domain-containing substrates. The PBM has important functions in both the viral life cycle and in HPV-induced malignancy. In this study we have used a proteomic approach to compare the ability of multiple HPV E6 oncoproteins to interact with different cellular PDZ proteins. We show a striking increase in the number of PDZ proteins recognised as the oncogenic potential of the individual E6 increases. Furthermore, we define combinations of PDZ proteins that are predictors of oncogenic potential, whilst others represent evolutionarily conserved targets bound across the evolutionary spectrum of low and high-risk PBM-containing E6 proteins. Taken together, these studies shed light on the functional conservation in the E6 PBM across multiple HPV types and support the hypothesis that ancestral PBM evolution originally conferred association with a restricted number of PDZ targets, which has, over time, evolved, to provide increased levels of flexibility and hence increase the number of cellular PDZ partners that can be bound by the cancer-causing E6 oncoproteins. High-risk human alpha-papillomaviruses are the causative agents of cervical cancer, other anogenital cancers, and an increasing proportion of head-and-neck cancers ,2. They http://pave.niaid.nih.gov/). The HPV E6 PBM plays a role in multiple E6 functions. It contributes directly towards increasing proliferation in the infected epithelium, and its loss results in a decrease of viral genome amplification and ultimately in loss of viral episomes ). The cells were removed from the plate by scraping and the debris removed by centrifugation at 14000rpm in a benchtop centrifuge for 2min at 4°C.Soluble proteins were extracted from 80% confluent HaCaT or NIKS cells by incubation for 10min on ice in lysis buffer  by incubation at 4°C on a rotating wheel for 1h, then washed three times with lysis buffer.The cell extract was pre-cleared by incubation with empty streptavidin conjugated magnetic beads at 4°C on a rotating wheel for 1h.After removal of the pre-clearing beads, the extract was incubated at 4°C on a rotating wheel for a further 2h with each of the biotinylated peptides bound to streptavidin-conjugated magnetic sepharose beads.The beads were washed three times with lysis buffer without protease inhibitors, transferred to fresh eppendorf tubes and washed twice more with lysis buffer without either protease inhibitors or Triton-x-100.5% of the beads were taken for western blot analysis and the remainder were subjected to trypsin-digest and the products analysed by mass spectroscopy, as described previously .The cloning and use of the GST Dlg1 fusion proteins have been described previously . The GSTThe following siRNAs were purchased from Dharmacon: si-Luciferase, si-HPV18 E6/E7, si-ZO-2, si-SNTB2, and were transfected using the RNAimax reagent (Invitrogen) according to the manufacturer's instructions.HeLa cells were transfected with siRNA, as indicated, and after 48h the confluent cells were scratched with a sterile Artline p2 pipette tip (Thermo Scientific). The cells were washed twice with PBS to remove any loose cells and immediately photographed using a Nikon COOLPIX995 camera. After 24h incubation the scratches were photographed again and the cell-free area quantified using the Image J and Prism programmes. The cells were then harvested in lysis buffer and the ZO-2 and SNTB2 levels analysed by western blot. Alpha actinin was used as a control for total cellular protein levels.The following antibodies were used: Mouse anti-DLG1 (Santa Cruz); goat anti-hScrib (Santa Cruz); rabbit anti-ZO-2 ; mouse anti-SNTB2 (Pierce); mouse anti-HA ; goat anti-CASK (Santa Cruz).Appropriate HRP-coupled secondary antibodies were purchased from DAKO.S1 FigNo material differences in the profiles were seen compared with those shown in (TIF)Click here for additional data file.


Inconsistent trends in HPV prevalence by age have been described in Africa. We examined the age prevalence pattern and distribution of 37 HPV-DNA types among urban Nigerian women.The study population was a sample of 278 women who presented to cervical cancer screening programs in Abuja, Nigeria, between April and August 2012. Using a nurse administered questionnaire, information on demographic characteristics and risk factors of cervical cancer was collected and samples of cervical exfoliated cells were obtained from all participants. Roche Linear Array HPV Genotyping Test® was used to characterize prevalent HPV and log-binomial regression models were used to examine the association between potential correlates and the prevalence of HPV infection.The mean age (SD) of the women enrolled was 38 (8) years. The overall prevalence of HPV was 37%. HPV 35 was the most prevalent HPV type in the study population. Among women age ≤ 30 years, 52% had HPV infection compared to 23% of those women who were older than 45 years (p = 0.006). We observed a significant linear association between age and the prevalence of HPV infections. The prevalence ratio (PR) and 95% confidence interval (CI) was 2.26  for any HPV infection, 3.83  for Group 1 HPV (definite carcinogens), and 2.19  for Group 2a or 2b HPV (probable or possible carcinogens) types, among women aged 18–30 years, compared to women who were older than 45 years.The prevalence of HPV infection was highest among younger women and decreased steadily with age among this population of urban Nigerian women. Human papilloma viruses (HPV) are small DNA viruses, of which there are several types associated with benign and malignant conditions of the cervix, penis, vulva, vagina, anus and oropharynx,2. ThesePrevious studies have shown that the prevalence of HPV infection rises soon after the onset of sexual activity,5 and atIn Africa, the age-specific prevalence of HPV differs across countries. HPV prevalence was highest in younger women and decreased steadily with age in Kenya, Uganda and Zimbabwe-10 but gIn this study, we examined the age prevalence pattern, distribution and socio-demographic correlates of HPV types among women in an urban, ethnically diverse population in Central Nigeria.Between April and August 2012, we enrolled women from cervical cancer screening clinics at National Hospital, Abuja and University of Abuja Teaching Hospital, Nigeria. Abuja is the Federal Capital of Nigeria and it is located in the central part of the country. Its population is comprised of individuals from almost all ethnic groups in Nigeria, thus it is the most ethnically diverse city in the country. The stuHPV DNA was extracted using Gentra Puregene Cell Kit® according to the manufacturer’s instruction. HPV genotyping was done using Linear Array HPV genotyping test® (Roche Diagnostics), a qualitative in vitro test which utilizes amplification of target DNA by the Polymerase Chain Reaction and nucleic acid hybridization, and detects 37 HPV genotypes - , 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39, and CP6108. It has been validated and offers a reliable and sensitive approach for HPV DNA detection and genotyping in cervical specimens, using standardized quality-controlled reagents-23.We classified HPV types according to the World Health Organization International Agency for Research on Cancer (IARC) Monographs Working Group assessment of the carcinogenicity of different HPV types. HPV typt-tests were used to assess differences in the distribution of continuous variables between groups while χ2 and Fishers exact tests were used for categorical variables. We created age categories as follows: ≤ 30, 31 – 35, 36 – 40, 41 – 45 and > 45 years and calculated the Prevalence Ratio (PR) and 95% confidence intervals (CI) by age categories and by HPV group classification using univariate and multivariable log-binomial regression models. In separate models, we examined the association between HPV positivity and the following covariates: age, marital status, age at sexual initiation, total number of sexual partners, HIV status, contraceptive use, douching and body-mass index (BMI). To test for a linear trend, we assigned each age category the median value and modeled this variable as a continuous variable. All P-values were 2-sided and the analyses were performed using SAS 9.3 for UNIX statistical software.Descriptive analyses were performed to characterize the sampled population. Student The study was conducted according to the Nigerian National Code for Health Research Ethics and the Declaration of Helsinki. Ethical approval to conduct this study was obtained from the IHVN Health Research Ethics Committee and the University of Maryland, Baltimore, Institutional Review Board. Informed consent was obtained from all participants before enrollment in the study.A total of 278 participants were enrolled in this study. We excluded 3 persons who had missing data on HPV genotype or several demographic variables, data from 275 participants were available for analysis. The mean age (SD) of the participants was 38 (8) and the median age (inter-quartile range) was 37 31–42) years. Table –42 yearsThe prevalence of any of the 37 HPV types in the study population was 37% (101/275). Group 1 HPV types (definite carcinogens) were found in 22% (61/275) of the study participants. HPV 35, a group 1 type, was the most prevalent type of HPV found in this study, it was found in 5% (14/275) of the women studied. Other group 1 HPV types found among the women studied were HPV 56, 58, 45 and 59 in decreasing order of prevalence. The prevalence of HPV16 and 18 in this population was low; HPV 16 was found in 2% (6/275) and HPV 18 was found in 2.5% (7/275) of the women studied. The prevalence of group 2a (Probable carcinogens) HPV type in the study participants (HPV68) was 3% (8/275). Group 2b HPV types (Possible carcinogens) were found in 24% (67/275) of the study participants. HPV81 and HPV42 were the most prevalent group 2b types, each was found in 4.7% (13/275) of the women studied.Among women who had HPV infection, the age group distribution was 32% 33/101) for ≤30 years; 27% (27/101) for 31 – 35 years, 20% (20/101) for age group 36 – 40 years, 13% (13/101) for 41 – 45 years and 8% (8/101) for women older than 45 years (Table 01 for ≤3We found multiple HPV infections in 12.7% (35/275) of the women. Multiple HPV infections were more prevalent among younger women. Compared to women in the highest age group (>45 years), the PR (95% CI) of multiple HPV infection was 4.38 , 5.00 , 2.15  and 0.94 , p for linear trend = 0.0004, for age group ≤ 30, 31 – 35, 36 – 40 and 41 – 45, respectively (Table The PR (95% CI) of any HPV infection was 2.54 1.69, 3.81) p = < 0.001, comparing women who were HIV positive with those who were HIV negative. In multivariable analysis, we found a significant association between HIV and prevalent HPV infection, the PR (95% CI) of HIV for any HPV infection was 2.27 (1.48 to 3.48). HIV status was also associated with the prevalence of group 1 and group 2 HPV types in multivariable analysis, adjusting for age, marital status, age at sexual initiation, number of sexual partners, use of contraceptives, douching and body mass index. The multivariable adjusted PR (95% CI) was 4.01  for HIV and group 1 HPV, and 1.87 (1.11 to 3.17) for HIV and group 2 HPV (Table .81 p = <2 to those with BMI 18.5 - < 25 kg/m2. There was no significant association between BMI and group 1 or group 2 HPV infections. The multivariable PR (95% CI) was 0.97  for BMI and group 1; and 0.97  for BMI and group 2 HPV infections, comparing women with BMI ≥ 25 kg/m2 to those with BMI 18.5 - < 25 kg/m2.The multivariable PR (95% CI) of any HPV infection was 0.96 (0.93 to 1.00), comparing women with BMI ≥ 25 kg/mIn this cross-sectional study of urban Nigerian women, the prevalence of HPV infection was 37%. We observed a significant inverse linear association between age and the prevalence of HPV infections. This prevalence was approximately 4 times higher in women aged 18 – 30 years compared to women who were older than 45 years and younger women were more likely to have carcinogenic HPV types.The prevalence of HPV infection among women in our study population was higher than had been reported in previous studies from Nigeria. Among 932 women surveyed between 1999 and 2000 in southwestern Nigeria, the prevalence of HPV was 26.3%. Our finDifferent trends have been reported on the age prevalence of HPV in Africa. The results of some studies showed there was a high peak of HPV prevalence among younger women which decreased with age, but among older women the prevalence either decreased,9, reachThe results of some studies suggests that HIV infection is a known risk factor for HPV infection. In our We found a marginal inverse association between any HPV infection and BMI in univariate analysis but this association was null in multivariable analysis. Prior studies of the association between body weight and HPV infections have yielded conflicting results. Among female sexual workers in Spain, there was an inverse association between high-risk HPV prevalence and BMI. In contrast, a study based on the US National Health and Nutrition Examination Survey (NHANES) showed that BMI was not associated with prevalence of HPV. The relOur study has several important limitations. First, the sample size was small hence we were unable to examine the prevalence of HPV infection among women age 60 years and above. We are therefore unable to determine whether the prevalence of HPV in this study population would have continued to fall, remained stable or risen. Second, because of the cross sectional design of our study we could not examine temporal trends of potential correlates of HPV and the risk factors for persistent HPV infections. With continued follow up of this population and repeated sampling, we will be able to report the pattern and spectrum of persistent HPV infection in Nigeria in future.The age prevalence pattern of HPV infection among women in cervical cancer screening programs in Nigeria is similar to that in many developed countries. Replication of this study in other urban African populations is required. Further studies to investigate epidemiological correlates of persistent high risk HPV infection are also needed. These studies will be useful in providing evidence for policies relating to the use of HPV DNA tests for screening, utility and coverage of current HPV vaccines and allocation of resources for HPV infections and cervical cancer prevention and treatment.Cross-sectional survey.BMI: Body mass index; CI: Confidence interval; CIN: Cervical intraepithelial neoplasm; HIV: Human immunodeficiency virus; HPV: Human papillomavirus; IARC: International Agency for Cancer Research; IHVN: Institute of Human Virology Nigeria; NHANES: National Health and Nutrition Examination Survey; PCR: Polymerase Chain Reaction REDCap Research Electronic Data Capture; PR: Prevalence ratio; SD: Standard deviation.CMW has received through the University of New Mexico, funds from grants and cooperative agreements from the US National institutes of Health related to cervical screening, funds from GSK for HPV vaccine studies and reimbursements for travel related to publication activities and equipment and reagents from Roche Molecular Systems for HPV genotyping.Other authors report no competing interest.SNA analyzed the data and drafted the manuscript. AOF, EOD, MA and IL coordinated the study and managed data accrual. OO and RU, site investigators, contributed to the study design, implementation and provided revisions of the manuscript. CMW performed HPV genotyping, contributed to data interpretation and provided revisions of the manuscript. CAA conceived the study, obtained funds, contributed to the study design and provided critical revisions of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/656/prepub


In Norway, repeat cytology and HPV testing comprise delayed triage of women with minor cytological lesions. The objective of this study was to evaluate HPV DNA and HPV mRNA testing in triage of women with an ASC-US/LSIL diagnosis.We used repeat cytology, HPV DNA testing (Cobas 4800) and HPV mRNA testing (PreTect HPV-Proofer) to follow up 311 women aged 25–69 years with ASC-US/LSIL index cytology.Of 311 women scheduled for secondary screening, 30 women (9.6%) had ASC-H/HSIL cytology at triage and 281 women (90.4%) had ASC-US/LSIL or normal cytology. The HPV DNA test was positive in 92 (32.7%) of 281 instances, and 37 (13.2%) were mRNA positive. Of the 132 women with repeated ASC-US/LSIL, we received biopsies from 97.0% (65/67) of the DNA-positive and 92.9% (26/28) of the mRNA-positive cases. The positive predictive values for CIN2+ were 21.5% (14/65) for DNA positive and 34.6% (9/26) for mRNA positive (ns). The odds ratio for being referred to colposcopy in DNA-positive cases were 2.8 times (95% CI: 1.8–4.6) higher that of mRNA-positive cases. Compared to the mRNA test, the DNA test detected four more cases of CIN2 and one case of CIN3.The higher positivity rate of the DNA test in triage leads to higher referral rate for colposcopy and biopsy, and subsequent additional follow-up of negative biopsies. By following mRNA-negative women who had ASC-US/LSIL at triage with cytology, the additional cases of CIN2+ gained in DNA screening can be discovered. Our study indicates that in triage of repeated ASC-US/LSIL, HPV mRNA testing is more specific and is more relevant in clinical use than an HPV DNA test. The goal in primary screening for cervical cancer is to detect and treat high-grade cervical intraepithelial lesions before invasive cancer develops In Norway, cytological high-grade lesions are detected in 1.0–1.2% in each screening round of the national cervical cancer screening program In the ASC-US/LSIL triage study (ALTS) women were randomized to repeat cytology, direct referral to colposcopy or HPV triage As only a small proportion (8–12%) of women with ASC-US or LSIL harbor high-grade histologically confirmed disease (CIN2+) In this study we performed a direct comparison of an HPV mRNA and an HPV DNA test in secondary screening of ASC-US/LSIL related to referral rates for colposcopy, biopsy rates, histological outcomes and sojourn time back to the screening program.For most women (81%) the index cytology represented the first ASC-US/LSIL diagnosis, whereas 10% and 9% of the women had a history of one or two to five ASC-US/LSIL diagnoses, respectively. The positivity rate of the HPV DNA test was 36.7% (114/311) relative 18.3% (57/311) for the HPV mRNA test .Thirty of the 311 women had ASC-H/HSIL cytology at triage, and 22, 20 and 8 of the ASC-H/HSIL cases were HPV DNA positive, HPV mRNA positive and HPV DNA/mRNA double negative, respectively. All 20 HPV mRNA positive cases were DNA positive. Among ASC-US/LSIL cases at triage, 68 were HPV positive for one or both tests: 27 were double positive (DNA+/mRNA+), 40 were DNA+/mRNA- and one woman was DNA-/mRNA+.A total of 281 women (90.4%) had ASC-US, LSIL, normal or inconclusive cytology at triage. Of these 281 women, 92 (32.7%) were HPV DNA positive and 37 women (13.2%) were HPV mRNA positive . Among tThe status at triage by diagnostic test are displayed in The positive predictive values for CIN2+ were 21.5% (14/65) for DNA positive and 34.6% (9/26) for mRNA positive. The positive predictive values for CIN3+ were 6.2% (4/65) for DNA positive and 11.5% (3/26) for mRNA positive. The odds ratio (OR) for being referred to colposcopy were 2.8 (95% CI: 1.8–4.6) in the HPV DNA group compared with the HPV mRNA group. The increased referral rate resulted in an additional diagnosis of four more cases of CIN2 and one more case of CIN3.Since we used the HPV DNA as a reference test, the sensitivity of the HPV DNA test is 100% (14/14) relative 64.3%  for the HPV mRNA test. The corresponding estimates of specificity are 70.8%  and 89.5% .Our study shows that HPV mRNA is more specific than HPV DNA in triage of women with repeated ASC-US/LSIL. A low positivity rate translates into a low referral rate for colposcopy, which is very appealing for triage situations. The referral rate for colposcopy was significantly higher for HPV DNA positive relative to HPV mRNA positive, winning only four more cases of CIN2 and one more case of CIN3. Thus, compared with the mRNA test, the use of DNA tests in triage more than doubled the workload for gynecologists and laboratories. As long as women with repeated ASC-US/LSIL and negative HPV mRNA tests are followed up with a new cytology after 12 months, very few cases of CIN2+ captured by DNA at triage will be lost The higher specificity of the HPV mRNA test translates into 39 more direct referrals to colposcopy/biopsy after ASC-US/LSIL diagnosis and positive HPV DNA and negative HPV mRNA. Among these women, four cases of CIN2 and one case of CIN3 were diagnosed, in addition to 20 extra cases of CIN1 and 14 extra cases with normal histology . All womThe HPV DNA test has higher sensitivity for CIN2+ than the HPV mRNA test. Women with repeated ASC-US/LSIL and a negative HPV mRNA test cannot be considered free of CIN2+ and need follow-up with another smear within 12 months. In two recently published studies from Norway, this is also the case for women with a negative HPV DNA test in delayed triage of ASC-US/LSIL Several studies have compared performance of different HPV diagnostic tests in direct referral to colposcopy after an abnormal cytology of repeated borderline/mild dyskaryosis or worse www.kreftregisteret.no).At the time of recruitment of Predictors 2 study, there were no guidelines for HPV testing. In 2010 the British recommendations implemented HPV testing in triage of women with borderline and mild dyskaryosis (reflex testing). Women with a negative HPV test are returned to screening. Women with a positive HPV test, but a normal colposcopy without having any biopsies collected, are also returned to screening. This is not the case in Norway. The Norwegian guidelines recommend follow-up of women with a negative cervical biopsy  and insufficient follow-up time in the cytology arm as limitations of our study. According to the Norwegian guidelines, women with normal cytology and a positive HPV result at triage should be followed up with a new cytology and HPV test within 12 months after triage. According to the Norwegian guidelines, women with repeated ASC-US/LSIL and a negative HPV result are returned to screening even though the risk of CIN2+ is more than 2.0% The Norwegian follow-up strategy for ASC-US/LSIL reflects the natural history of duration of primary HPV 16/18 infections/lesion formation, as there is a 6–12 month window before triage is undertaken There is a trade-off in any screening program how to find the most efficient way of diagnosing the long standing CIN2+ which have the potential to progress to cervical cancer. HPV type 16 in particular, HPV 18 and HPV 45 have demonstrated the highest progression rates to CIN3 and cervical cancer over a 10–20 year perspective Participation in a screening program is voluntarily. If there are too many “false” alerts, the program will lose legitimacy among women, which again may lead to lower attendance rates. In this respect we consider high specificity to be more important than high sensitivity in overall CIN2/3 detection. Within the referral algorithm for the Norwegian cervical cancer program, our study shows that the referral rate to colposcopy was more than doubled for the DNA versus the mRNA tested women, and the sojourn rate back to regular screening was significantly higher. The importance of a screening program is to treat the women in such a way that they remain confident in the program and continue attendance. So far these issues have not been discussed from the point of view of medicine, societal costs, ethics or women. There is a need for additional studies on head-to-head comparison of HPV tests in both primary and secondary screening that target different molecular sites.The Regional Committee for Medical and Health Research Ethics, Northern Norway, approved the study as a quality assurance study in laboratory work fulfilling the requirements for data protection procedures within the department (REK Nord 2012/276). Written consent from the patients for their information to be stored in the hospital database and used for research was not needed because the data were analyzed anonymously. The ethics committee specifically waived the need for consent.Our study contains two different HPV tests to triage women with minor cervical lesions: the HPV mRNA test PreTect HPV-Proofer (NorChip AS), which detects E6/E7 mRNA  of 5 HPV types, and the HPV DNA test Cobas 4800 (Roche), which detects 14 HPV types.http://www.roche.com).The HPV DNA test Cobas 4800 is designed as a qualitative single tube multiplex assay based on the real time PCR technology that simultaneously detects 14 high-risk genotypes. The assay identifies HPV type 16 and 18 with concurrent detection of twelve other HPV types  using L1 primers, and ß-globin is used as an internal control. DNA is isolated from a scrape of cells from a woman's cervix and is subsequently mixed in reaction wells with primers and probes that specifically recognize and amplify HPV DNA. This reaction produces fluorescence, which is then measured to determine the presence of HPV in the cervical sample. Specialized pipetting technology combined with AmpErase enzymes reduces cross contamination risk (http://www.norchip.com/).The HPV mRNA test PreTect HPV-Proofer is an E6/E7 mRNA-based real-time nucleic acid sequence-based amplification assay (NASBA), focusing on the reportedly most common oncogenic HPV types 16, 18, 31, 33 and 45 using specific E6/E7 primers for each HPV-type. To avoid false negatives due to degradation of mRNA, primers and probes against human U1A mRNA are included in the PreTect HPV-Proofer kit as a performance and integrity control. Artificial oligonucleotides corresponding to the viral mRNA were used as positive controls. Negative controls consisted of Rnase-free water and were included in each run  for cytological examination. DNA/RNA was isolated from 5 ml of the leftover material and analyzed with PreTect HPV-Proofer. The mRNA testing was performed according to the manufacturer's instructions (NorChip AS). The HPV DNA testing (Roche Cobas 4800) was performed and in accordance with national guidelines for HPV testing st, 1991, and March 31, 2013, 98 579 female residents were reported at the county level, with 494 400 valid cervical smears in the clinical database SymPathy. Within this database we identified 2394 women with at least one ASC-US/LSIL diagnosis from January 1st, 2010, through September 30th, 2012. After excluding women who had at least one HSIL diagnosis (n = 326) or cervical biopsy referral (n = 294) prior to index ASC-US/LSIL diagnosis, women who were 15–24 (n = 271) or 70–91 (n = 20) years of age at index ASC-US/LSIL, 1523 women were eligible for analysis. Furthermore, we excluded women with no follow-up (n = 199), who had direct biopsy (n = 79), who had triage less than 3 months (n = 65) or more than 18 months (n = 20) after index ASC-US/LSIL. After these exclusions 1169 women within the age span of the Norwegian cervical screening program and who had no prior HSIL or cervical biopsy constituted the preliminary study population.Our department analyses annually 23 000 cervical cytology samples. Between January 1st, 2012, at which time our department switched from HPV mRNA testing to HPV DNA testing in secondary screening of ASC-US/LSIL. Included were women who had their index ASC-US/LSIL cytology back in 2010 because of varying intervals in scheduled follow-ups. Our protocol expanded the time window and included all women who had a diagnosis ASC-US/LSIL over the last 3 to 18 months, different from the national screening program's recommended 6–12 month interval Our study began on January 1All statistical analyses used SPSS version 21 to perform Chi-square tests, Mann-Whitney tests and survival analysis. A p-value <0.05 was considered statistically significant.Our study indicates that in triage of repeated ASC-US/LSIL, HPV mRNA testing is more specific and is more relevant in clinical use than an HPV DNA test.Data S1Data file (SPSS): Data_PlosOne_ms-D-14-28018.(SAV)Click here for additional data file.


Oncogenic human papillomavirus (HPV) infection, particularly multiple HPV types, is recognized as a necessary cause of anal cancer. However, a limited number of studies have reported the prevalence of anal HPV infection in Asia. We determined the prevalence, genotypes, and risk factors for anal HPV infection in Japanese HIV-positive men who have sex with men (MSM), heterosexual men, and women.Chlamydia trachomatis, Entamoeba histolytica). Anal swabs were collected for oncogenic HPV genotyping.This cross-sectional study included 421 HIV-positive patients. At enrollment, we collected data on smoking, alcohol, co-morbidities, drugs, CD4 cell counts, HIV RNA levels, highly active anti-retroviral therapy (HAART) duration, sexually transmitted infections (STIs), and serological screening  rate was 54.6% in MSM, 8.8% in heterosexual men, and 0% in women. In univariate analysis, younger age, male sex, MSM, CD4 <100, HIV viral load >50,000, no administration of HAART, and having ≥2 sexually transmitted infections (STIs) were significantly associated with oncogenic HPV infection, whereas higher smoking index and corticosteroid use were marginally associated with oncogenic HPV infection. In multivariate analysis, younger age , MSM , CD4 <100 , and having ≥2 STIs  were independently associated with oncogenic HPV infection. These 4 variables were also significant risk factors for multiple oncogenic HPV infection.age, MSM, ≥2 STIs, and immunosuppression confer a higher risk of infection with oncogenic HPV and multiple oncogenic types.Among Japanese HIV-infected patients, approximately two-thirds of MSM, one-fifth of heterosexual men, and one-fifth of women have anal oncogenic HPV infection. Younger  Human papillomavirus (HPV) infections are the most common sexually transmitted infections (STIs) worldwide. HPV typMost data on anal HPV infection have been obtained in Western countries. A limited number of studies have reported the prevalence of anal HPV infection in Asia including Taiwan,11, IndiCompared with HIV-positive MSM, few studies have reported the prevalence of anal HPV infections with different genotypes in HIV-positive heterosexual men or HIV-positive women,12. In aTo address these issues, we prospectively collected information on oncogenic HPV infection as well as STIs and HIV-related factors, and we then determined the prevalence, genotypes, and risk factors for anal HPV infection in Japanese HIV-positive MSM, heterosexual men, and women.This prospective, cross-sectional study was carried out between September 2009 and March 2015 in the Department of Gastroenterology and Hepatology at the National Center for Global Health and Medicine (NCGM), Tokyo, Japan. Eligible patients were HIV-positive patients, at least 18 years old, who were willing to receive gastrointestinal (GI) tract cancer screening, provide anal swabs and blood for tests, and physically able and willing to provide written informed consent. NCGM has one of the largest HIV clinics in Japan with >3,500 registered patients as of May 2013. These patients were the first patients enrolled in the HIV-GI cohort (V-GI protocol) and were systematically screened for anal HPV infection and STIs during the study period. Written informed consent was obtained from all participants. This study was approved by the ethics committee of the National Center for Global Health and Medicine (Nos.1440) and was implemented in accordance with the provisions of the Declaration of Helsinki.Chlamydia trachomatis (C. trachomatis), and Entamoeba histolytica (E. histolytica). Positive infection was defined as current or prior infection. Syphilis infection was defined as positive Treponema pallidum latex agglutination (TPHA) test and rapid plasma reagin (RPR) titer ≥8[C. trachomatis infection was determined by anti-C. trachomatis IgG and IgA antibodies using an Enzyme-Linked ImmunoSorbent Assay . C. trachomatis infection was defined as positive IgA or IgG results; negative infection was defined as both negative IgA and IgG results. Amebic infection was assessed by anti-E. histolytica antibody , as described previously[Blood samples were collected for syphilis, hepatitis B virus (HBV),  titer ≥8. HBV inf titer ≥8, most ad titer ≥8. C. tracreviously. Serum areviously. Patientreviously. BecauseA dedicated brush  was used for sampling of the anorectal area. The anal brush was inserted 3–5 cm into the anal verge and the dentate line and it was then used to scrape the anal walls by repeatedly rotating it clockwise and counterclockwise. Nucleic acids were extracted from 500-μL analsamples in the Sure Path solution with a commercial kit . HPV DNA2 test (or Fisher’s exact test) for continuous or categorical variables, respectively. We used logistic regression analysis to compute the odds ratios (ORs) and 95% confidence intervals (CIs) as an estimate of anal oncogenic HPV infection associated with clinical factors. For multivariate analysis, we used a multiple logistic regression model that included all factors with p <0.05 on univariate analysis .Baseline characteristics were compared between patients with and without anal oncogenic HPV infection using the Mann–Whitney U test or χWe also elucidated the association between infection with multiple oncogenic HPV types and clinical factors in uni- and multivariate logistic regression analyses. Sex was not included in the logistic regression model because all patients infected with multiple oncogenic HPV types were males.The Cochran–Armitage test was used to determine trends in the proportion of oncogenic HPV infection according to CD4 count , HIV VL , and duration of HAART .p values <0.05. All statistical analyses were performed using Stata version 13 software .Statistical significance was defined as two-sided C. trachomatis, and E. histolytica infection were 41.3%, 54.4%, 50.1%, and 26.1%, respectively. The proportion of subjects with ≥2 STIs was 58.2%.During the study period, 421 HIV-infected patients were recruited. Patient characteristics are shown in The prevalence of anal HPV infection and the distributions of genotypes are shown in Risk factors for oncogenic HPV infection are shown in Risk factors for infection with multiple oncogenic HPV types are shown in p<0.001 and p<0.001, respectively; p<0.001 and p<0.001, respectively; p<0.001 and p<0.001, respectively; Oncogenic and multiple oncogenic HPV infection rates tended to increase with the decrease in CD4  in HIV-infected MSM. Supindham et al. recently reported that HPV-58 infection rate was 29% in HIV-infected MSM in Northern Thailand , which iCo-infection with multiple oncogenic HPV types has been associated with persistent infection and infection with a longer duration, and it thus represents an important risk factor for anal cancer,4,5. We C. trachomatis serology[Previous studies have shown specific risk factors for anal HPV infection including younger age,37, HBV  serology. These f serology,11,37–40 serology,11,38, s serology. These fC. trachomatis, and E. histolytica infection, information on the number of sexual partners, receptive anal intercourse, condom use, and Neisseria gonorrheae infection was not collected. In addition, the number of subjects in this study was relatively large (n = 421), but the study population was a convenience sample of patients attending the Department of Gastroenterology; therefore, it remains unknown if this sample population is representative of the general HIV-positive community.There are several limitations to this study. Although we collected information on syphilis, HBV, In conclusion, this prospective, cross-sectional study of Japanese HIV-infected patients demonstrated that approximately two-thirds of MSM and one-fifth of heterosexual men and women had anal oncogenic HPV infection. In the risk analysis, younger age, MSM, CD4 <100, and ≥2 STIs were independently associated with oncogenic HPV infection and multiple oncogenic HPV types. Our findings support the need for establishing an anal cancer screening system and regular follow-up strategies, especially in HPV high-risk HIV-positive patients, for preventing HPV transmission and detection of early anal neoplasia in Asia as well as Western countries.


Multiple-type human papillomaviruses (HPV) infection presents a greater risk for persistence in asymptomatic individuals and may accelerate cancer development. To extend the scope of HPV types defined by probe-based assays, multiplexing deep sequencing of HPV L1, coupled with an HPV-QUEST genotyping server and a bioinformatic pipeline, was established and applied to survey the diversity of HPV genotypes among a subset of healthy men from the HPV in Men (HIM) Multinational Study. Twenty-one HPV genotypes (12 high-risk and 9 low-risk) were detected in the genital area from 18 asymptomatic individuals. A single HPV type, either HPV16, HPV6b or HPV83, was detected in 7 individuals, while coinfection by 2 to 5 high-risk and/or low-risk genotypes was identified in the other 11 participants. In two individuals studied for over one year, HPV16 persisted, while fluctuations of coinfecting genotypes occurred. HPV L1 regions were generally identical between query and reference sequences, although nonsynonymous and synonymous nucleotide polymorphisms of HPV16, 18, 31, 35h, 59, 70, 73, cand85, 6b, 62, 81, 83, cand89 or JEB2 L1 genotypes, mostly unidentified by linear array, were evident. Deep sequencing coupled with HPV-QUEST provides efficient and unambiguous classification of HPV genotypes in multiple-type HPV infection in host ecosystems. Human papillomaviruses (HPV) are globally ubiquitous DNA viruses found in mucosal and cutaneous anatomical sites and comprised by more than a hundred genotypes. Low-risk HPV genotypes are associated with genital warts, while high-risk HPV genotypes, in particular HPV16, cause a number of cancers in men and women ,4,5,6,7.Studies of multiple-type HPV infection by conventional probe-based assays are hampered by the limited scope of type-specific probes and/or cross-hybridization by probes for related HPV genomes ,28,29,30The study included 22 samples from 18 men from the United States (n = 15), Brazil (n = 1), or Mexico (n = 2), representing a subset of healthy males from the HPV in Men (HIM) Multinational Study recruited between 2005 and 2008 to examine the HPV prevalence in asymptomatic males ,35,36,37Nested PCR with PGMY  followedStandard operating procedures were followed at every step of PCR amplifications to prevent cross-contamination, and no template controls were always negative.Gel-purified amplicons were sequenced from adaptor A using a Genome Sequencer FLX  with Titanium chemistry (Roche) according to the manufacturer’s protocol in the University of Florida Interdisciplinary Center for Biotechnology Research . Multiplex deep sequencing was performed on HPV L1 libraries pooled at equal molar ratio at the bead recovery step, a critical modification as pooling samples at the DNA level produced skewed sequencing output (data not shown). The first 14 samples were pooled in one region of an 8-region PicoTiter plate to obtain a median (QR) of 1,562  raw reads per sample. To increase the sequence depth and accelerate the turnaround time in the ICBR Sequencing Core Facility, the second set of 11 samples, including 8 samples from 4 participants, 2 control cell lines, and 1 HPV16 molecular clone were sequenced in two regions of a 4-region PicoTiter plate to generate a median (QR) of 26,193  raw reads per sample . All rawThe bioinformatic pipeline for analysis of L1 deep sequences is outlined in To assess deep sequencing errors, 25,928 quality L1 deep sequences from a molecular clone of HPV16 were aligned to an HPV16 reference sequence (GI│333031) using MUSCLE in Geneious package, followed by BioEdit . Errors were evaluated using an in-house code. Overall sequencing error rate was 0.46%  with 0.09%, 0.03%, 0.08% and 0.26% for transitions, transversions, and insertions or deletions (indels), respectively. After correction of indels, rate of misincorporation was 0.12% , which is 18 times lower than 2%, the maximum dissimilarity within an HPV genotype ,53,54.Quality sequences were clustered at 3% pairwise distance  PV Specific Blastn [Consensus sequences were queried in HPV-QUEST, a custom HPV-genotyping server which collected 150 cutaneous and mucosal HPV reference sequences containing the L1 region obtained from National Center for Biotechnology Information (NCBI) Genebank , Los Alac Blastn  was usedc Blastn  was applHPV genotype infection and diversity were investigated in cross-section DNA samples from 18 asymptomatic participants. A total of 21 (12 high-risk and 9 low-risk) HPV genotypes, representing 9 species  were detected . Seven iA majority  of the cohort had exclusively high- or low-risk genotypes, including seven individuals  and S17) with high-risk HPVs16, 51, 52, 59, 70 and 73 genotypes, and five  with low-risk HPVs6b, 81, 83, 87 or cand89 genotypes . In contResults of the NGS/HPV-QUEST typing were concordant with LA genotyping for 10 high- or low-risk genotypes  in 8 individuals  . While tGenotypes defined by PaVE PV Specific Blastn were virtually identical to those classified by HPV-QUEST with exactly the same local identities to the same reference sequences used in both databases. Minor differences between the two genotyping tools, including HPVJEB2 typed by HPV-QUEST and classified as HPV72 by PaVE PV Specific Blastn, were due to different reference sequences used by the two programs . HPVJEB2Across the study group, L1 regions from a variety of genotypes were identical to reference sequences at the nucleotide level . In contrast, a number of L1 variants displayed nucleotide differences from the corresponding reference sequences in more than 77% (14 of 18) of individuals . Both syLongitudinal changes of HPV genotypes were evaluated in S15 and S16 based on the ability to generate amplicon libraries from a series of three samples collected at six-month intervals  . Similar+ HNSCC are emerging as a separate biological entity among all HNSCC. HPV-positive HNSCC are less likely to harbor p53 mutations, are more sensitive to radio/chemotherapy, and have a lower risk of recurrence and a better chance of survival than HPV-negative HNSCC [Much recent attention has highlighted the necessity of HPV genotyping in clinical pathology. An increasing line of evidence implicates HPV viral diversity and genotypes as substantial factors in cancer grade, response to therapy, recurrence, and a patient’s survival in HPV-related cancers, e.g., cervical cancers (CC) and head and neck squamous cell carcinomas (HNSCC) ,18,30,64ve HNSCC ,16,17. Pve HNSCC . 10-LiPA25 [10 for SPF10-LiPA25, and GP+ for HPV DNA chip and GP+ reverse line blot, resulting in large differences in sensitivity of detecting HPV infection and individual genotypes among the tests [HPV-genotyping methods vary considerably across laboratories, resulting in substantial disagreement among studies ,66,67. H0-LiPA25 ,69, HPV 0-LiPA25 , and GP+0-LiPA25  detect a0-LiPA25 ,64,72,73he tests ,27,28. With the rapid revolution in technology, NGS applied to HPV genotyping provides high sensitivity in capturing even low frequency types, reflecting unprecedented coverage of viral copies and almost no limit in the number of types detected in a sample by querying reference sequence databases ,74,75,76In this study, we applied nested PCR using PGMY, followed by GP+ primers, to generate HPV L1 amplicon libraries since PGMY/GP+ provided greater sensitivity and broader coverage of HPV genotypes compared to MY alone, PGMY alone, GP+ alone, or MY/GP+ ,47. OtheHPV genotyping of deep sequences relied mainly on querying to NCBI Blastn ,75. NCBISequencing allowed the identification of signature polymorphisms among L1 genotypes in more than 75% of individuals in this exploratory study with relatively small sample size, providing a framework to apply to future studies of HPV clearance, persistence, and super-infection by variants of the same HPV genotypes in a larger cohort. In addition, other regions of the HPV genome that may provide more robust molecular information can be incorporated into a deep sequencing platform to track genotypes over time or evaluate HPV gene expression . A high In the current study, deep sequencing was applied to define the range of HPV genotypes in asymptomatic individuals. Our data clearly demonstrate that healthy men can carry a range of high-risk and/or low-risk types HPV genotypes, including the vaccine types. Infection by multiple-type high-risk HPV, alone or in combination with low-risk genotypes, is a significant clinical issue for prognosis or treatment for a variety of HPV cancers ,16,17,18


Type-specific high-risk HPV (hrHPV) infection is related to cervical carcinogenesis. The prevalence of hrHPV infection varies geographically, which might reflect the epidemiological characteristics of cervical cancer among different populations. To establish a foundation for HPV-based screening and vaccination programs in China, we investigated the most recent HPV prevalence and genotypic distributions in different female age groups and geographical regions in China.In 2012, a total of 120,772 liquid-based cytological samples from women enrolled for population- or employee-based cervical screening in 37 Chinese cities were obtained by the Laboratory of Molecular Infectious Diseases of Guangzhou KingMed. A total of 111,131 samples were tested by Hybrid Capture II and the other 9,641 were genotyped using the Tellgenplex™ HPV DNA Assay.The total positive rate for hrHPV was 21.07 %, which ranged from 18.42 % (Nanchang) to 31.94 % (Haikou) and varied by region. The regions of Nanchang, Changsha, Hangzhou, Chengdu, Fuzhou, Guangdong, and Guiyang could be considered the low prevalence regions. Age-specific prevalence showed a “two-peak” pattern, with the youngest age group (15–19 years) presenting the highest hrHPV infection rate (30.55 %), followed by a second peak for the 50–60-year-old group. Overall, the most prevalent genotypes were HPV16 (4.82 %) and HPV52 (4.52 %), followed by HPV58 (2.74 %). Two genotypes HPV6 (4.01 %) and HPV11 (2.29 %) were predominant in the low-risk HPV (lrHPV) type, while the mixed genotypes HPV16 + 52 and HPV52 + 58 were most common in women with multiple infections.This study shows that HPV infection in China has increased to the level of an “HPV-heavy-burden” zone in certain regions, with prevalence varying significantly among different ages and regions. Data from this study represent the most current survey of the nationwide prevalence of HPV infection in China, and can serve as valuable reference to guide nationwide cervical cancer screening and HPV vaccination programs.The online version of this article (doi:10.1186/s12879-015-0998-5) contains supplementary material, which is available to authorized users. HPV infection can cause a variety of genital diseases, and type-specific persistent infection of high-risk HPV (hrHPV) cases is strongly associated with cervical carcinogenesis . CervicaTo date, more than 200 HPV genotypes have been identified, and ~40 HPV genotypes have been detected in the female genital tract. HPV16 and HPV18 are well known as oncogenic genotypes; additionally, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV69, and HPV 82 are also closely associated with cervical cancer. Therefore, all of these genotypes are classified as “high-risk” HPV. Meanwhile, “low-risk” genotypes, including HPV6, HPV11, HPV42, HPV43, and HPV44 are the causative agents for benign or low-grade changes in cervical cells, such as genital warts , 8.The current hrHPV detection is supposed to serve as an additional approach for the early diagnosis of cervical cancer, and was implemented to complement less sensitive and non-objective cytology-based methods . The higThe prevalence of HPV infection and type-specific distribution vary greatly both between nations , and betObtaining large-scale information about the epidemiological features of HPV in China is critical for global HPV prevention strategies, because of the need to understand the geographic diversity and age distribution of HPV infections. Although a pilot project aimed to measure the hrHPV infection rate and cervical cancer screening was conducted in a few Chinese areas in 1999 , and sevThis study was approved by the Ethics Committee of Tianjin Medical University in accordance with the Ethical Principles for Biomedical Research Involving Human Subjects  and Declaration of Helsinki for Human Research of 1974 (last modified in 2000). Samples were originally obtained from clinical settings for laboratory diagnoses. After diagnostic testing, excess samples were anonymized and kept for this study. Informed consent was obtained from each female participant. For those individuals younger than 18 years old, the consent form was signed by the parents of each participant.KingMed Diagnostics is the largest reference laboratory in China and provides diagnostic testing services for over 13,000 hospitals in 18 provinces and 4 municipalities across the nation. From January to December of 2012, a total of 120,772 samples were obtained for population- or employee-based screening from 37 cities, belonging to 18 regions (Guangdong province was regarded as one region as 20 cities in this investigation were located in that province). Fig. The women enrolled from population- or employee-based cervical cancer screening programs ranged in age from 15 to 60 years old, were sexually active, and had no history of cervical treatment before the screening. Exclusion criteria included current pregnancy, <3 months post-partum, HIV-seropositivity, and a history of either hysterectomy or treatment for cervical cancer., then were resuspended in a standard transport medium (STM) and stored at 4 °C . All speHPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68) was examined using the HCII HPV DNA test . Data were calculated as a ratio of mean relative light unit (RLU) for the sample to the mean RLU values for the assay of a positive calibrator (PC). A RLU-to-PC ratio >1 (~1.08 pg DNA/ml) was defined as a positive result [Liquid-based samples were processed by following the instructions included in the Digene sample conversion kit. The hrHPV DNA panel of 13 pooled types  and 7 lrHPV genotypes . The Tellgenplex HPV™ kit was used to simultaneously examine the presence or absence of the most common 26 HPV genotypes in a single test by multiplex PCR combined with Luminex technology [HPV genotyping was performed using the Tellgenplex™ HPV DNA Test . The assay can be used to detect and genotype 26 HPV genotypes including 19 hrHPV genotypes  tests were used to compare differences among all regions and every two regions.The HPV infection rate in each region was calculated by dividing the number of HPV-positive samples by the total number of samples that were successfully tested for HPV. A binomial 95 % confidence interval (95 % CI) was estimated for each calculation of the prevalence of HPV. Chi-squared (χ2) test. Multiple comparisons were performed using the Bonferoni step-down procedure to minimize an inflated risk of type 1 error [P < 0.05 was considered statistically significant.The HPV infection rate was estimated within 5 age groups . A binomial 95 % confidence interval (95 % CI) was estimated, and P-values for age trends of HPV infection were analyzed using the linear-by-linear association test. Differences between each pairing of two age groups were compared by the chi-squared .P < 0.001). The regions with the highest hrHPV prevalence were Haikou (31.94 %) and Chongqing (27.29 %). By contrast, Nanchang, Changsha, Hangzhou, Chengdu, Fuzhou, Guangdong, and Guiyang could be grouped into the low prevalence regions . The prevalence of hrHPV infection differed significant among the various regions , moreover, HPV6 (45.80 %), HPV11 (26.15 %), and HPV61 (14.20 %) were common in lrHPV.Among the well-recognized 26 HPV genotypes, all genotypes except HPV73 were examined using the Tellgenplex technique. The total infection rate of each genotype among all of the samples and the distributions of each genotype in the HPV-positive individuals were respectively evaluated. In descending order, the infection rate and distribution are shown in Table Regarding the region-specific distribution of hrHPV, the top three genotypes were analyzed in each region. HPV16, HPV58, and HPV52 were dominant in six regions—Guiyang, Xi’an, Guangdong, Nanning, Changsha, and Shenyang—although the orders of the three genotypes showed variation in different regions , among which 434 16.82 %) and 52 (6.16 %) were infected with hrHPVs and lrHPVs, respectively. In the multiple hrHPV infection individuals, the frequencies of 6, 5, 4, 3, and 2 genotypes were 0.23 %, 1.84 %, 4.61 %, 17.74 %, and 75.58 %, respectively, and three genotypes showed higher positive rates, i.e., HPV16 (35.02 %), HPV52 (32.26 %), and HPV58 (21.20 %)  and Nanning (3.13 %), respectively. The distribution of dual, triple, quad and up multiple hrHPV infections in each city showed in Fig. 9.51 % anThis is one of the few nationwide investigations of high- and low-risk HPV in a large-scale screen of the Chinese population. Because of regional differences, the population composition, and the sampling periods, the reported prevalence varies from study to study, but the most the heavily burdened HPV regions are Sub-Saharan Africa (24.0 %), Eastern Europe (21.4 %), Latin America (16.1 %), and Southeastern Asia (14 %) . In thisIt is possible that the variable HPV prevalence in China occurs because of the large Chinese population and its territories. Meanwhile, economic conditions, cultural habits, and population migrations have affected Chinese lifestyles and health , 15, 26.Compared with region-based data, the rates obtained in this present study were higher than those previously reported for Shanghai , 29, SheRegarding the hrHPV prevalence in different age groups, a meta-analysis conducted by Bruni et al.  showed aData concerning the distribution of HPV genotypes is important for both vaccine development and HPV-based screening design, particularly for selecting the testing spectrum of HPV genotypes  and deteBeijing, Shanghai, Xinjiang, Henan, and Shangxi) in China, the infection rates of HPV16 (4.82 %), HPV52 (4.52 %), and HPV58 (2.74 %) were all higher than in the HPV16 (2.9 %), HPV52 (1.7 %), and HPV58 (1.5 %) strains obtained in this present study. Regarding the proportion of these three genotypes, except for HPV52 , which is higher than that reported in the present study, there were no significant differences detected in the proportion of HPV16  or HPV58 . The proportion of HPV16 in this survey was even lower than that among the normal cytology samples reported by Guan et al. and Bruni et al.  [P = 0.871) [Consistent with the data generated by some Chinese population-specific investigations, HPV16, HPV52, and HPV58 were found to be the dominant hrHPV types , 38. Comctively) , 39. Notctively)  reportedctively) , 41, 42.ctively) . However= 0.871) . These fIn addition to carcinogenesis, many benign cutaneous warts, mucosal lesions, and low-grade cervical intraepithelial lesions generate a considerable health burden that is associated with lrHPV infection . SpecifiCharacterization of the prevalence of multiple HPV infections might be important for understanding its effects on cervical carcinogenesis. Herrero et al.  reportedThis present study confirmed the high overall incidence of HPV in China and strongly argues for the necessity of developing national population-based screening programs. However, the appropriate management of this HPV screening program for a large number of women with HPV-positive specimens and the absence of cytological evidence of cervical pre-cancer or cancer remains a major concern . HPV genIn conclusion, China has large population and a variety of territories; meanwhile, economic conditions, cultural habits, and population migrations have dramatically affected Chinese lifestyle and health, for instance, cancers related to sexual transmitted diseases. Therefore, attention should be paid to the prevalence of HPV infection in a timely and regional basis because it has been commonly recognized that HPV infection plays a critical role in the occurrence of cervical cancer and its increase incidence. These surveillance findings indicated that a national plan for a cervical screening program is urgently needed, not only because of the increase in hrHPV infection rate in some previously reported regions, but also because of the high infection rate in most newly investigated regions. In brief, the most significant findings of this study are as follows: (1) the prevalence of hrHPV infection has reached a level that cannot be ignored, and the rate of increase is growing with time; (2) the prevalence of hrHPV infection showed population variations in age and region, and also reflected economic, cultural, and lifestyle relevance; (3) the HPV16, HPV52, and HPV58 strains consistently constituted the three dominant genotypes in different Chinese populations, a characteristic pattern that was significantly different from the epidemiological features in most of industrial countries. These findings defined principles for future proposals for cervical screening and vaccination in China.


Under the optimistic assumption of high-prophylactic HPV vaccine coverage, a significant reduction of cancer incidence can only be expected after decades. Thus, immune therapeutic strategies are needed for persistently infected individuals who do not benefit from the prophylactic vaccines. However, the therapeutic strategies inducing immunity to the E6 and/or E7 oncoprotein of HPV16 are more effective for curing HPV-expressing tumours in animal models than for treating human cancers. New strategies/technologies have been developed to improve these therapeutic vaccines. Our studies focussed on preparing therapeutic vaccines with low-cost technologies by DNA preparation fused to either plant-virus or plant-toxin genes, such as saporin, and by plant-produced antigens. In particular, plant-derived antigens possess an intrinsic adjuvant activity that makes these preparations especially attractive for future development. Additionally, discrepancy in vaccine effectiveness between animals and humans may be due to non-orthotopic localization of animal models. Orthotopic transplantation leads to tumours giving a more accurate representation of the parent tumour. Since HPV can cause cancer in two main localizations, anogenital and oropharynx area, we developed two orthotopic tumour mouse models in these two sites. Both models are bioluminescent in order to follow up the tumour growth by imaging and are induced by cell injection without the need to intervene surgically. These models were utilized for immunotherapies with genetic or plant-derived therapeutic vaccines. In particular, the head/neck orthotopic model appears to be very promising for studies combining chemo-radio-immune therapy that seems to be very effective in patients. All cervical cancers and precancerous lesions (CIN III) are caused by HPV infection. Since the early findings in the 1970s of HPV in cervical cancer by zur Hausen’s group, HPV has been linked to other anogenital cancers including vulvar, penile, and anal neoplasia. Recently, high-risk HPV (HR-HPV) infection has been detected in head and neck cancers (HNCs), with high frequency in oropharynx and tonsils, unveiling another aetiological factor in addition to tobacco and alcohol 55]. ThisAlthough this preliminary work regarding AT-84 E7 model for immunotherapy was not designed to assess immunological responses, the results provided information on the efficacy of heterologous prime-boost regimens compared with homologous prime-boost regimens, as reported in previous studies , 56. MicHowever, several aspects need to be improved in order to have a completely reliable model. For instance, information on the biological significance of the E7 expression in these tumours is lacking, but preliminary data indicate that the presence of E7 can improve cell growth rate. Nevertheless, our model holds a valid place in monitoring responses to immunotherapy, as it is orthotopic and derives from naturally occurring OSCC.In conclusion, we strongly believe that our oral HPV-associated mouse tumour represents an innovative, realistic, and new pre-clinical model for HPV-related OSCC. Moreover, the possibility of applying chemo-radiotherapy to this model together with easy surgical removal of tumour masses offers new perspectives to test current available patient therapy in a pre-clinical model. It is well known that the better prognosis of HPV-positive OSCC could allow less aggressive chemo-radiotherapy, and in this regard, our model will be very useful, and we are performing several protocols of combined therapy (chemo/radio and immunotherapy). Furthermore, the localization of this tumour permits easy access to intratumoural therapy. Preliminary interesting results of E7GGG/SAP DNA intratumoural injection are shown in Fig. Our studies are aimed at the development of low-cost technologies, such as DNA vaccines as well as plant-produced vaccines, and have proven that these immunotherapies are ready for clinical use. The oral pre-clinical model of HPV-associated cancer will have a pivotal role in designing new combined intervention in HPV-positive OSCC. Ongoing works is aimed at the development of new DNA/plant-derived vaccines against other HPV targets like the E5 oncogene of HR-HPV  in assoc