Epidemiological and laboratory-based studies have identified infection with one of 15 high-risk human papillomavirus (HPV) types as a necessary but not sufficient cause of cervical cancer. The prevalence of genital HPV infections is high in young women, but most of the infections regress without interventions. Host genetic variations in genes involved in immune response pathways may be related to HPV clearance, and HPV E6/E7 oncoproteins interacting or downstream genes, both coding and non-coding, may contribute to the outcome of high risk HPV infection and cervical cancer. Of specific interest for this review has been the selection of genetic variants in genes involved in the above-referred pathways with a summary of their applications in association studies. Because the supportive and opposing data have been reported in different populations, well-designed international collaborative studies need to be conducted to define the consistency of the associations, paving the way to better define the patients at high risk of developing cervical cancer. In spite of substantial declines in both incidence and mortality rates in the past 50 years, cervical cancer remains the second most common cancer among women worldwide, with estimated 493,000 new cases and 274,000 deaths in 2002in situ is up to 39%Persistent cervical infection with one of 15 carcinogenic human papillomavirus (HPV) types causes invasive cervical cancer and also precursor abnormalitiesHPV infection is necessary but not sufficient for the development of cervical cancer, because most individuals (70% to 90%) eliminate the virus 12 to 24 months after initial diagnosis without interventionRemarkable progress has been made in this field, but overwhelming criticism is also received because of an initially overoptimistic view on the magnitude of the effects of inherited genetic variants. Given mounting evidence that long-term HPV infection of carcinogenic types is a prerequisite for cervical carcinogenesis, host genetic differences in immune-responsive genes and HPV E6/E7 oncoprotein-interacting and downstream genes may be mainly modifiers for cervical cancer susceptibility. Of specific interest in this review has been the selection of several well-characterized genetic variants involved in immune-responsive genes and HPV E6/E7 oncoprotein-interacting or downstream genes to give a summary of their applications in association studies.in situ carcinomas evading immunological control seem to be influenced by the cellular antigen presentation system and the HLA receptorsAvailable data indicate that cervical cancer develops through infection to preinvasive lesions to invasive cancer, a process that on average takes decadesIL-1B gene, is a potent pro-inflammatory cytokine and an important part of the innate immune system. Studies have shown that IL-1β plays an important role in cervical carcinogenesisP = 0.0002), and plasma IL-1β levels above the 75% quartiles in controls were associated with a 1.74-fold significant increase in the risk of cervical cancerIL-1BIL-1B promoter single nucleotide polymorphisms (SNPs) in the development of cervical cancer, especially for C-511T (rs16944)IL-1B-511T and -31C alleles have been found to confer an increased risk for the development of cervical cancer, especially among subjects having higher levels of IL-1βIL-1β, encoded by the TNFA gene, is another potent pro-inflammatory cytokine that has been implicated in the control of HPV infection. HPV-harboring cervical keratinocytes constitutively produce active TNF-αTNFA polymorphisms, the majority of published studies have been focused on G-308A (rs1800629) in the promoter region, because the -308A allele is thought to increase the transcriptional activity of the TNFA geneTNFA G-308A and risk of cervical cancer are shown in TNF-α, encoded by the HLA have been hypothesized to be involved in the pathogenesis of cervical neoplasia via their role in the immunological control of HPV infection. Case-control studies have shown associations between specific HLA alleles and cervical cancer risket al. reported that seven alleles  were positively associated with, while four other alleles  were negatively associated with cervical squamous cell carcinomaHLA DRB1*1501-DQB1*0602 haplotype was reported to be protective against the high-grade CIN, especially in individuals infected with oncogenic HPVHLA DQB1*0302 and cervical disease has also been demonstrated in several populations, including the cohort in Costa RicaHLA is essential for the presentation of viral antigens. The activity of HLA molecules seems to be one of the determining factors in the induction of an adaptive immune responseIL12A) and a 40,000 Dalton heavy chain (known as p40 encoded by IL12B), which induces the production of interferon-γ (IFN-γ), favors the differentiation of Th1 cells and forms a link between innate and adaptive immunityIL-12A rs568408 [3′ untranslated region (UTR) G>A] and rs2243115 (5′UTR T>G) and IL-12B rs3212227 (3′UTR A>C) in a hospital-based study of 404 cervical cancer cases and 404 cancer-free controls. The IL-12A rs568408 GA/AA and IL-12B rs3212227 AC/CC variant genotypes were associated with increased risks of cervical cancer , compared with their corresponding wild-type homozygotesIL12B rs3212227 is also supported by other published studiesIL-12 is a heterodimeric pro-inflammatory cytokine formed by a 35,000 Dalton light chain , located at 2q33, encodes a receptor expressed by activated T lymphocytes. SNPs in CTLA4 have been reported to be associated with susceptibility to both autoimmune disease and cancerCTLA4 G49A (rs231775) variant causes 17Thr-to-Ala substitution in the leading peptide of the CTLA4 receptorCTLA4 G49A was found to be significantly associated with risk of multiple types of cancerCTLA4 G49A variation and cervical cancer risk but have not found any significant associationsCTLA4 G49A was associated with a 1.66-fold (95% CI=1.13-2.44) increased cervical cancer riskCytotoxic T-lymphocyte antigen-4  for arginine homozygotes, compared with heterozygotes, and 1.13 (95%CI = 0.94-1.35) for arginine homozygotes versus proline homozygotes. However, further subgroup analyses showed that significant excess risks were only observed in studies where controls were not in Hardy-Weinberg equilibrium, in non-epidemiological studies, and in studies where the p53 genotype was determined from tumor tissuesBRCA1 Pro871Leu (rs799917), BARD1 Pro24Ser (rs1048108) and Arg378Ser (rs2229571) and found that the BRCA1 variant rs799917 TT genotype was associated with a significantly decreased risk of cervical cancer in a recessive genetic model , but this association was not observed for the two BARD1 SNPsIt was reported that both the E6 and E7 oncoproteins interacted directly with and functionally antagonized BRCA1miR-218 by E6 and the consequent over expression of LAMB3 may promote viral infection of the surrounding tissue and eventually contribute to cervical carcinogenesis. In a recent case-control study, we identified one SNP rs11134527 located in the pri-miR-218 sequence and one SNP rs2566 in the 3′ UTR of LAMB3 and found that the pri-miR-218 rs11134527 variant homozygote GG was associated with a decreased risk of cervical cancer , while the LAMB3 rs2566 variant CT/TT genotypes were associated with a significantly increased risk of cervical cancer , although the functional relevance of these two SNPs have not well characterizedExcept for affecting protein coding genes, the expression of the E6 oncoprotein of the high-risk HPV16 could also reduce microRNA-218 (miR-218) expressionP = 0.01) and for CIN3 or worse (P = 0.07). Excluding HPV16, the P values would be 0.04 and 0.37, respectively. For HPV16, non-European viral variants were significantly more likely than European variants to cause persistence  and CIN3 or worse Globally, overall HPV infection has accounted for the persistence , EXO1 Thr439Met (rs4149963), CYBA 3′UTR (rs7195830), and FANCA Gly501Ser (rs2239359), were demonstrated to increase risk for CIN3 or cervical cancer, whereas immune-responsive gene TLR2 Ser450Ser (rs3804100) and DNA repair gene XRCC1 Gln399Arg (rs25487) polymorphisms were demonstrated to decrease risk. Furthermore, IRF3 S427T and XRCC1 Gln399Arg polymorphisms were significantly associated with HPV persistence, whereas TLR2 Ser450Ser, EXO1 Thr439Met, CYBA 3′UTR, and FANCA polymorphisms were associated with the risk of progression to CIN3 or worseA population-based cohort study of 10,049 women in Guanacaste, Costa Rica, performed analyses on several candidate SNPs in selected immune response and DNA repair genes to evaluate variants involved in HPV persistence and progressionOAS3, SULF1, and IFNG; the DNA repair genes DU, DMC, and GTF2H4; the EVER1 and EVER2 genes. OAS3, SULF1, DUT, and GTF2H4 SNPs were associated with HPV persistence, whereas IFNG and EVER1/EVER2 SNPs were associated with progression to cervical cancerIn a subsequent study by the same group, genes/regions statistically significantly associated with CIN3 or cervical cancer included viral infection and cell entry genes Although previous studies of potentially functional polymorphisms in candidate genes and cervical cancer susceptibility showed lack of consistence, they have advanced our knowledge of the genetic basis of the etiology of this cancer. For the association of low-penetrance genetic variants and cancer risk, heterogeneity is a great challenge, possibly coming from the variations in study design, ethnic diversity of target populations, lack of adjustment for known risk factors, small or modest sample sizes, and issues related to multiple significance testing. Therefore, future studies should call for a more careful study design, study execution, and data analysis. Specific for cervical cancer, a better understanding of the natural history of oncologic HPV infection and factors related to HPV persistence and progression will facilitate the identification of susceptible subjects.With high-throughput genotyping methods available, genome-wide association (GWA) approaches are popular in recent years, which may provide us the opportunity to give a comprehensive genetic view of the disease. However, up to now, GWA study was reported for neither cervical cancer nor HPV persistence. Most published studies have not presented sufficient information on environmental exposure other than HPV infection, which may also contribute to the heterogeneity between studies and populations. An important aspect of epidemiological studies is the ability to form improved multicenter research consortia, which may be beneficial for not only increasing study sample sizes, but also for providing finance, ensuring data quality and generalizing the findings and conclusions.The past years have seen that the GWA approach is well-suited for the identification of common SNPs with modest or small effects on the well-defined phenotypes


There are no data available on human papillomavirus (HPV) infections in women living in the Mississippi Delta, where cervical cancer incidence and mortality among African American women is among the highest in the United States. The aim of this analysis was to report the age-specific prevalence of HPV in this population.We recruited 443 women, 26–65 years of age, from the general population of women living in the Mississippi Delta to participate; 252 women had been screened for cervical cancer within the last 3 years while 191 had not. Women underwent a pelvic exam and had clinician-collected Pap sample taken for the routine cervical cancer screening by cytology. Women were asked to collect a self-collected specimen at home and return it to the clinic. Both specimens were tested for HPV genotypes.trend< 0.01), the prevalence of non-carcinogenic HPV did not, especially the prevalence of HPV genotypes in the alpha 3/4/15 phylogenetic group.Four hundred and six women (91.6%) had HPV genotyping results for the clinician-collected and self-collected specimens. The prevalence of carcinogenic HPV was 18.0% (95% CI: 14.4%-22.1%) for clinician-collected specimens and 26.8% (95% CI: 22.6%-31.4%) for self-collected specimens. The concordance for the detection of carcinogenic HPV between clinician-collected and self-collected specimens was only fair (kappa = 0.54). While the prevalence of carcinogenic HPV in either sample decreased sharply with increasing age (pThe prevalence of carcinogenic HPV in our sample of women living in the Mississippi Delta was greater than the prevalence reported in several other U.S. studies. The high carriage of HPV infection, along with lack of participation in cervical cancer screening by some women, may contribute to the high cervical cancer burden in the region. Persistent cervical HPV infection by cancer-associated or carcinogenic HPV is the necessary cause of cervical cancer ,2. DNA tThe Mississippi Delta is a little studied population that has historically had several-fold higher annual rates of cervical cancer incidence and mortality than those for the general U.S. ,17. The Recruitment and clinical methods were detailed previously . Brieflyhttp://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=6,475,165.PN.&OS=PN/6,475,165&RS=PN/6,475,165) and a vial of Scope™  mouthwash for specimen transport [For all participants, cervical specimens were collected into PreservCyt  for routine cervical cytology and the residual was retained for HPV testing. Women were given a kit for self-collection at home, including a self-collection device ,21 ; 2) HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51–56, 58, 59, 64, 66, 67, 68, 69, 70, 73, 82, and 82v, which are classified in the alpha 5, 6, 7, 9, and 11 group . All the carcinogenic and borderline carcinogenic HPV genotypes are found in these species; and 3) HPV genotypes 61, 62, 71, 72, 81, 83, 84, and 89, which are classified in the alpha 3, 4, and 15 group . There is some evidence that these HPV genotypes in these species have a predilection for vaginal tissue [Specimens were tested for 37 HPV genotypes  using Linear Array  ,24. HPV1l tissue -31.Logistic regression was used to calculate odds ratios (OR), 95% confidence intervals, and p values to test for differences between study groups (screened vs. under-screened) and linear trends with age groups  for testing HPV positive for categories of HPV. For categories of HPV prevalences, both crude and adjusted for study group and age group, were calculated. Binomial exact 95% confidence intervals were used where noted. An exact version of McNemar chi-square test or a symmetry chi-square test was used to test for differences in prevalence for individual or specific group of HPV genotypes from the same specimen or for same individual or specific group of HPV genotypes from different specimens.post-hoc power calculation based on a McNemar chi-square test indicated that 1) a sample size of 406 pairs for the whole analysis population achieves 80% power  to detect a difference in prevalence of 4.5% between specimens collections if the discordance was 10%, of 6.4% if the discordance was 20%, and of 7.8% if the discordance was 30%, and 2) a sample size of 312 pairs for women 30 years and older achieves 80% power  to detect a difference in prevalence of 5.2% between specimens collections if the discordance was 10%, of 7.3% if the discordance was 20%, and 8.9% if the discordance was 30%.A A p value of < 0.05 was considered statistically significant. STATA Version 11.1 was used for these analyses .There were HPV testing results available for 440 (99.7%) clinician-collected specimens and 409 (92.3%) self-collected specimens. The analyses were restricted to the 406 (91.6%) women for whom both results were available, which included 231 screened women and 175 under-screened women. Of the 406 subjects included in this analysis, 370 (91.1%) identified themselves as Black/African American, 33 (8.1%) as White/Caucasian, and 3 (0.7%) as other. The mean and median ages were 37.5 years and 36 years, respectively.We used logistic regression to show the impact of the two study groups and age on detection of HPV. As shown in Table Increasing age was statistically associated with a lower likelihood of testing positive of any HPV, carcinogenic HPV, and alpha 5/6/7/9/11 genotypes, but not for alpha 3/4/15 genotypes, for both specimen types  for clinician-collected specimens and 26.8% (95% CI: 22.6%-31.4%) for self-collected specimens. Self-collected specimens were more likely to test positive for any HPV, carcinogenic HPV, non-carcinogenic HPV, alpha 5/6/7/9/11, and alpha 3/4/15 genotypes than clinician-collected specimens (p < 0.001). As a consequence of the differences in HPV detection by specimen type, the concordance between the two specimens for HPV detection in any HPV category was only fair, with kappa values around 0.50.We calculated the prevalence of HPV categories by collection method, and the concordance between specimens, as shown in Table post-hoc power was 98.7%.The prevalence of carcinogenic HPV for women 30 and older (n = 312), the ages at which HPV and cytology cotesting is acceptable by national screening guidelines -34, was The concordance for HPV detection, categorizing hierarchically according to cancer risk, for the two specimens is shown in Table The agreement statistics for detection of all 37 HPV genotypes individually in self-collected vs. clinician-collected specimens were a kappa value of 0.56 (95% CI: 0.52-0.61), the total agreement of 97.8%, and the positive agreement of 40.0%, with self-collected specimens more likely to test positive for any of the HPV genotypes than clinician-collected specimens (p < 0.0001). The prevalence for individual HPV genotypes is shown in the Additional file trend = 0.0002) and self-collected (ptrend = 0.009) specimens decreased with increasing age, the prevalence of non-carcinogenic HPV did not . Grouping of HPV genotypes based on branches in the phylogenetic tree highlighted these differences in age trends. While the prevalence of alpha 5/6/7/9/11 detected in clinician-collected (ptrend = 0.001) and self-collected (ptrend = 0.007) decreased with increasing age, the prevalence of alpha 3/4/15 did not .Shown in Figure We found the prevalence of carcinogenic HPV to be relatively high in this population compared to other U.S. populations. The prevalence of carcinogenic HPV in clinician-collected specimens from women 30 and older in this population was approximately 2-fold higher than what has been observed at Kaiser Permanente Northern California (6.5% in women 30 and older) , and 40%We note that this study was only cross-sectional and therefore could not assess the total lifetime exposure to HPV, nor could we measure HPV persistence, which precedes and predicts the development of cervical precancerous lesions . Yet HPVThe prevalence of HPV and the HPV genotypes detected was strongly influenced by both the age of the patient and the type of sampling (clinician vs. self). The higher prevalence of carcinogenic HPV in women under 30 (vs. 30 and older) suggests may preclude the use of self-collection and HPV testing in these younger women although the decision to use it has to be weighed against the possibility that these higher-risk women may not get the screening they need in the future.While we did not observe an increase in alpha 3/4/15 genotypes in cervical specimens with age as observed in other studies , the prevalence of these HPV genotypes remained relatively unchanged with increasing age. As a consequence, the proportion of alpha 3/4/15 genotypes in the cervical specimens among any HPV infections increased significantly with age so that the prevalence of alpha 5/6/7/9/11 and alpha 3/4/15 genotypes were approximately the same in women 50 and older. To our surprise, the age pattern in the vagina as measured by self-collection was similar to that of the cervix, with higher prevalence of alpha 5/6/7/9/11 genotypes than alpha 3/4/15 genotypes in young women rather than the preponderance of alpha 3/4/15 genotypes at all ages. The differences in this study versus other studies could be due to the relative small sample size in this study or unmeasured differences in sexual behaviors. Another possibility is that self-collection device used in this study did a better job of sampling the cervix, making the self-collected specimens more representative of the cervical milieu of HPV genotypes than observed in other studies.We note that the one of our limitations for this study is that we used non-FDA approved HPV test, self-collection device, and transport medium. Linear Array, the HPV test used in this study, is one of the standard methods for HPV genotyping  and has Our data illustrated how self-collected specimens can result in significantly higher point prevalence of HPV (lower specificity), including carcinogenic HPV, than a cervical specimen. The point prevalence of HPV using self-collected specimens in this study was comparable to what was recently reported  in a natDr. Castle serves on a data and safety monitoring board for Merck. He has received HPV tests and testing for research at a reduced or no cost from Qiagen Roche Norchip, and MTM. Dr. Castle has served as a paid consultant to BD, Cepheid, and GE Healthcare and has received an honorarium for speaking from Roche.PEC participated in the design and oversaw the execution of the study, conducted the primary analysis, and drafted the manuscript. JCG assisted in the analysis and the drafted the manuscript. EEP participated in the design of the study and the conduct of the study. AR participated in the design and was responsible for the clinical care of the patients. PEG did the HPV testing and helped to draft the manuscript. ICS participated in the design and oversaw the execution of the study, and helped to draft the manuscript. All authors read and approved the final manuscript.The intramural research program of the NIH/NCI and the NCI’s Center for Reducing Cancer Health Disparities supported this work.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/5/prepubThe prevalence of 37 HPV genotypes in clinician-collected and self-collected specimens. Bold type indicates statistically greater prevalence of that type as determined using an exact version of the McNemar’s tests.Click here for file


Worldwide, cervical cancer is the second most common cancer in women. High-risk human papillomavirus (HPV) play a crucial role in the etiology of cervical cancer and the most prevalent genotype is HPV16. HPV 16 intratypic variants have been reported to differ in their prevalence, biological and biochemical properties. The present study was designed to analyze and identify HPV type 16 E6 variants among patients with cervical cancer in Morocco.A total of 103 HPV16 positive samples were isolated from 129 cervical cancer cases, and variant status was subsequently determined by DNA sequencing of the E6 gene.Isolates from patients were grouped into the European (E), African (Af) and North-American (NA1) phylogenetic clusters with a high prevalence of E lineage (58.3%). The Af and NA1 variants were detected in 31.1% and 11.6% of the HPV16 positive specimens, respectively, whereas, only 3% of cases were prototype E350T. No European-Asian (EA), Asian (As) or Asian-American (AA) variants were observed in our HPV16-positive specimens. At the amino acid level, the most prevalent non-synonymous variants were L83V (T350G), H78Y (C335T), E113D (A442C), Q14D (C143G/G145T) and R10I (G132T), and were observed respectively in 65%, 41.8%, 38.8%, 30.1% and 23.3% of total samples.Moreover, HPV16 European variants were mostly identified in younger women at early clinical diagnosis stages. Whereas, HPV16 Af variants were most likely associated with cervical cancer development in older women with pronounced aggressiveness.This study suggests a predominance of E lineage strains among Moroccan HPV 16 isolates and raises the possibility that HPV16 variants have a preferential role in progression to malignancy and could be associated with the more aggressive nature of cervical cancer. Cervical cancer is the second most predominant cancer worldwide, and more than 85% of the global burden occurs in developing countries, where it accounts for 13% of all female cancer . PersistGiven that the prevalence of cervical cancer varies in different regions and countries, a number of studies have addressed the possible association of HPV16 variant status with the risk for progression to malignancy. HPV variants are defined as isolates with primary DNA sequence differences that total no more than 2% of the L1 open reading frame (ORF) of the prototype sequence . The seqMany authors confirm that distribution of HPV variants is related to geographic or race distribution . HoweverFurthermore, HPV sequence variations studies can be used in epidemiological studies as a marker to track the spread of the virus in contact networks through populations.One hundred twenty nine fresh frozen uterine cervix biopsies were recruited for the variant analysis in the present study. The tumor samples were from biopsies collected for the routine diagnosis of cervical cancer from Gynaecology department of the National Institute of Oncology in Rabat and Oncology Centre Mohammed VI pour le Traitement des Cancers, CHU Ibn Rochd in Casablanca, that receive patients from all regions of Morocco. The mean age of patients was 52 ± 12 years, with extreme ages at 28 and 80. Each sample was divided into two portions: one portion was put in neutral buffered formalin and processed for routine histopathological examination; the other portion was stored at −80°C immediately after surgical removal, for HPV genotyping. Protocols were approved by Ethical Review Committees of the Faculty of Medicine of Fez and the Hassan-II University Hospital, and written informed consent was obtained from each study subject.CaSki and SiHa cervical cancer cell lines were obtained from the American Type Culture Collection (ATCC) and used as positive controls. These cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS).-eosin stain on 5 μm paraffin sections. Histopathological results of cervical cancer lesions were classified according to the International Federation of Gynaecology and Obstetrics (FIGO).Samples were fixed in 10% buffered-formaldehyde. Pathology diagnosis was made using the routine hematoxylinGenomic DNA was extracted from fresh frozen tissue and cervical cancer cell lines using a standard technique of digestion with proteinase K in the presence of sodium dodecyl sulfate (SDS) at 37°C overnight, followed by phenol/chloroform purification . The tisHPV detection and genotyping of cervical cancer cases were performed using a nested PCR, with the consensus MY09/11 and GP5+/6+ primers (Table http://www.ncbi.nih.gov/blast). In accordance with established guidelines, a nucleotide sequence was assigned to an HPV type if it corresponded with a known HPV genotype by >90% [For DNA sequencing, the PCR products were purified by the ExoSaP-IT clean up system  and sequenced directly on an ABI 3130XL DNA analyzer , using GP6+ primer as the sequencing primer and BigDye® Terminator v3.1 Cycle Sequencing Kit , according to manufacturer’s protocol. For each sample, PCR amplification and DNA sequencing were performed twice. Nucleotides sequences were aligned and compared with those of known HPV types available through GenBank by using the online BLAST 2.0 software server  (Table http://www.ncbi.nih.gov/BLAST/), MEGA v4.0 and SeqScape v2.6  programs.PCR amplifications were performed according to Lizano et al. . For evehttp://www.medcalc.org). The statistical relationship was considered as significant if the derived p value was < 0.05.The HPV16 variants according to other clinical features were compared using a Fisher’s exact test using the MedCalc statistical software  that represents 96.89% of cases (125/129). The remaining 4 cases (3.1%) were diagnosed as adenocarcinoma. According to stage, to the most important percentages were found in stage II and III representing 48.8% (61 cases) and 38.4% (48 cases), respectively. Moreover, 14 cases were classified as stage I (11.2%), whereas only 2 cases (1.6%) were classified as stage IV.+/6+primers, revealed the presence of viral DNA in 91.47% (115 of 129 SCC and 3 of 4 adenocarcinoma).The presence of amplifiable DNA was confirmed for all 129 cases by PCR based-technique using primers for a fragment of β-globin gene and therefore all DNA samples were adequate for further analysis. Molecular detection of HPV DNA, using nested PCR amplification of a conserved region of the HPV L1 gene DNA with the consensus MY09/11 and GP5In this study, we have identified 5 carcinogenic HPV genotypes: 16, 18, 31, 33 and 35. The most frequently found was HPV16, detected in 87.29% of all positive samples (103 cases), followed by HPV 18, present in 5.08% of samples (6 cases), HPV 31 in 2.54% of samples (3 cases), HPV 33 in 1.69% of samples (2 cases) and HPV 35 in 1 specimen (0.85% of cases). Undetermined HPV genotypes were identified in 3SCC cases.The 103 HPV16 single infected specimens were selected for E6 intratypic variation analysis. Overall, DNA sequences were compared to the HPV16 reference sequence . Accordingly, HPV16-positive specimens were classified into E, Af1, Af2 and NA branches  revealed the prototype sequence, while 97.1% of samples (100 out 103) showed at least one specific nucleotide variation in the HPV16 E6 region , African (Af) and the North-American (NA1) variant classes. The European variants HPV16 were the most frequently detected isolates in our population and represent 58.3% of total HPV16 isolates (n=60), followed by Af characterized in 30.1% of cases (n=31). Whereas the NA variants were found only in 11.6% of cases (n=12) (Table p > 0.05).The distribution of the different E6 variations in the HPV16 positive samples with respect to FIGO staging of cervical neoplasia is reported in Table p < 0.0001).The distribution of HPV16 E6 variants according to patients’ age was also assessed and is reported in Table Correlation between HPV16 E6 variants and malignant phenotype showed that E variants are mostly associated with high degree of differentiation, moderately and well differentiated carcinoma, whereas Af variants are more frequently found in poorly differentiated carcinoma Table .The present study, entirely realized in Morocco, highlighted the distribution patterns of intratypic variants of HPV16 among women with cervical cancer. A total of 129 samples of cervical cancer cases were collected. Molecular detection of HPV, using nested PCR amplification of a conserved region of the HPV L1 gene with the consensus MY09/11 and GP5+/6+ primers, revealed the presence of viral DNA in 91.47% of cases 118/129). We identified 5 carcinogenic HPV genotypes . Globally, HPV prevalence and distribution is in concordance with previously reported data in Morocco ,37. More/129. We et al. [et al.[Lalaoui et al.  have fou. [et al., HPV16 w. [et al.,33.To examine the prevalence of naturally occurring sequence variations in the HPV16 E6 gene in cervical cancer from Moroccan subjects, all HPV16 positive samples (n=103) were subjected to intratypic variant analysis by nucleotide sequencing of the E6 gene and comparison with the prototype sequence of HPV16 E6.Of all HPV16 positive samples, only 3 cases exhibited a prototype E6 gene sequence and 97.1% of cases (100/103) showed at least one specific nucleotide variation in the HPV16 E6 region. This finding is in agreement with published results reporting that approximately 90% of the E6 genes in invasive cervical cancer contained variations ,40.et al.[It is widely accepted that the distribution of HPV variants is related to geographic or race distribution ,27 and tet al. have repHPV16 European variant is a highly spread variant around the world, a meta-analysis conducted on HPV16 positive tumors samples collected globally from different ethnical group reported that European lineage was the most predominant lineage and was well distributed among the different geographical regions. This widespread presence suggests biological relevance of this variant .et al.[A large number of HPV16 variants circulating in our population belong to African branches (30.1% of cases). The association of Af variants with an increased risk of cervical cancer development is controversial. Some authors have reported a greater association between infection with HPV16 Af variants and an increased risk of cervical carcinogenesis ,43,44. Het al. have repet al.,42.Interestingly, we have detected NA1 lineage in 11.6% of our patients’ population. The occurrence of NA1 classes, which is a derivative of the AA and Af2 variants , is diffAccording to clinico-pathological data, HPV16 European variants  were mostly identified in younger women at early clinical stages (I and II) at diagnosis. However, most of the cancer patients, harboring the HPV16 Af variants, were older (over 45 years) and exhibited a pronounced aggressiveness of the disease, as the majority of cases were at advanced stage (III) with poorly differentiated carcinoma. These results raise the possibility that HPV16 variants have a preferential role in progression to malignancy and could be associated with the more aggressive nature of cervical cancer. Similar results were obtained in Tunisia reporting a higher proportion of non-European HPV16 variants in advanced stage (III). In contrast, the non-E variants were more frequently detected at earlier average age at diagnosis than European variants . This diMost nucleotide changes reported in our study have been previously described and some of them are of particular interest and may have considerable biological impact ,46. The Additionally, E6 variants R10G/L83V and Q14H/H78Y/L83V which are frequently detected in our study, were more prone to undergo cell-detachment-induced apoptosis than E6 prototype in a model of human normal immortalized keratinocytes (NIKS)  and has Interestingly, the E113D (A442C) was detected in 37.9% of samples and was exclusively found in the European variant (E350G/442C). This point mutation is largely reported in Asian populations and is implied in invasive cervical carcinoma -55.The majorities of the E6 amino acid switches in our samples were located at amino acid residues10, 14, 78 and 83 and were shown to be under positive selective pressure ,57. ThisTherefore, the observed amino acid variations in HPV16 E6 detected in our study may impact on the viral life cycle, which is tightly linked to the differentiation program of the cell .The introduction of molecular sub-typing of HPV allowed better genotyping and therefore a better characterization of strains circulating in Morocco. Thereby, the identification of HPV16 E, Af and NA1 variants suggests a different sexual mixing behavior. Their circulation in our population may involve a change in the biological properties and a change in the transforming potential, increasing the risk of cervical cancer development. Currently, Morocco is a destination of many sub-Saharan migrants looking for better life, and is also a gateway to Europe, that warrants the need for monitoring a wider range of HPV variants involved in cervical cancer in Morocco. Thus, in order to better understand the dynamics of the disease and to optimize current control programs and policies for a better survey of cervical cancer, it is useful to develop routine epidemiological surveillance, including molecular sub-typing.The authors declare that they have no competing interests.ZQ: Carried out the molecular studies, participated in project design and drafted the manuscript; MK: participated in the conception and design of the study; MME: Participated in the conception and design of the study; MB: Specimen collection and data analysis; CN: Specimen collection and data analysis; LB: participated in the molecular analysis; NB: Sample collection, clinical data acquisition and anatomy pathology analyses; AB: Sample collection, clinical data acquisition and anatomy pathology analyses; MA: Conceived the study and participated in the design and coordination of the project and drafted the manuscript; MEM: Participated in the design and coordination of the project and review of the final manuscript. All authors read and approved the final manuscript.Senior authors: Mohammed Attaleb and Mohammed El Mzibri.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/378/prepub


Cervical cancer is the second most common cancer of women worldwide and is the first cancer shown to be entirely induced by a virus, the human papillomavirus . Two recently developed prophylactic cervical cancer vaccines, using virus-like particles (VLP) technology, have the potential to prevent a large proportion of cervical cancer associated with HPV infection and to ensure long-term protection. However, prophylactic HPV vaccines do not have therapeutic effects against pre-existing HPV infections and do not prevent their progression to HPV-associated malignancy. In animal models, therapeutic vaccines for persisting HPV infection can eliminate transplantable tumors expressing HPV antigens, but are of limited efficacy in inducing rejection of skin grafts expressing the same antigens. In humans, clinical trials have reported successful immunotherapy of HPV lesions, providing hope and further interest. This review discusses possible new approaches to immunotherapy for HPV associated cancer, based on recent advances in our knowledge of the immunobiology of HPV infection, of epithelial immunology and of immunoregulation, with a brief overview on previous and current HPV vaccine clinical trials. Other cancers that are attributable to HPV infection include cancers of the vulva, vagina, anus and penis. The HPV family also includes the causative agents of genital and skin warts.Papillomaviruses are non-enveloped, double-stranded DNA viruses that infect the epithelium. Their genome encodes six non-structural proteins  and two structural proteins [Late 1 (L1) and L2]. Early proteins control the DNA-replication machinery of the infected cells by altering cell cycling and promoting viral genome replication, while late proteins, which form the capsid, are involved in the packaging of the virion [The epithelium is a multilayered stratified tissue with a vertical organization correlated with keratinocyte differentiation and maturation: at the basement are dividing stem cells in contact with the dermis, followed by a layer of non-dividing keratinocytes, a squamous layer and finally, the mature squamous layer that is in contact with the external environment .i.e., types -6 and -11, associated with anogenital warts and mild dysplasia), intermediate- and high-risk categories , depending on their association with cancer. The two most studied HPV are HPV-16 and -18, which contribute to almost 70% of all cervical cancers. Three novel papillomaviruses, HPV-109 from an SCC, HPV-112 from a condyloma and HPV-114 from a cervical lesion, have been identified recently, with more HPV types still to be characterized in HPV-associated lesions [More than 100 HPV types have been identified, almost 40 of which infect the anogenital region. Genital HPV are divided into low-  episomal form and replicate using the cellular DNA-replication machinery. At an early stage, the proteins E1 and E2 delay cell differentiation and promote cell replication and thus viral genome amplification. When epithelial cells mature and stop dividing, virion particles are formed and mature virions can be released from the epithelial cells. HPV-induced genital lesions often regress spontaneously within a year, but may persist in about 2% of cases for five years or more, with the histological hallmarks of CIN1 . As the infection persists, these lesions can progress to CIN2 and CIN3. In one-third of cases, CIN3 lesions can develop into cancer. In cervical cancer lesions, the HPV DNA is mostly found integrated in the cellular genome [2.2.1.The incidence of some cancers is higher in immunocompromised patients. Immune suppression after organ transplantation is connected with increased risk of virus-associated cancers  includinImmunosuppression is often reflected in low CD4 T-cell numbers in individuals with HIV. In a recent study, data from individuals diagnosed with AIDS over twenty five years were collected and analysed for HPV–associated cancers. A low CD4 T-cell count measured at AIDS onset was associated with significantly increased risks of HPV-associated invasive cancers, including anal cancers among men and vaginal or vulvar cancers among women. In the same study, in situ cervical cancer risk was lower and invasive cervical cancer risk was higher in patients with AIDS, although differences were not statistically significant due to the lack of sufficient data for cervical cancer patients. In contrast to the high risk of anal and other HPV-associated cancers, the risk of invasive oropharyngeal cancer was only moderately elevated among persons with HIV when compared with the general population. This modest increase in risk may reflect the fact that only a few oropharyngeal cancers  are caused by HPV  or the l2.2.Most HPV infections are transient, becoming undetectable within the first two years in immunocompetent individuals. While the spontaneous regression of latent HPV infection is difficult to assess due to the sensitivity of assays and challenge in discriminating between positive and negative samples (discussed in 3.2), lesions have been shown to often regress spontaneously in young women ,10. It h2.3.2.3.1.Adaptive immune responses to HPV infection include humoral  responses specific for viral structural (L protein) or non-structural (E protein) antigens. The L1 major structural protein of HPV assembles into particles or capsids indistinguishable from native virions. Although papillomavirus capsids have been shown to be quite immunogenic when injected into animals ,16, the There is considerable variability in the timing of the HPV-specific antibody response in relation to HPV infection. Nevertheless, serum IgG antibodies, which have a neutralizing potential, develop in response to infection in more than 50% of women. The kinetics of antibody development are slow (6 to 12 months), and the peak titres are low, but antibodies can persist for decades unless HPV-associated lesions resolve. Secretory IgA antibodies can also be detected in the cervical secretions of HPV-positive women with a similar time course of appearance to IgG antibodies, but with a shorter persistence time . HoweverHPV antibody assays are problematic because the levels of antibody are low, the threshold for positivity is difficult to define, and correct antigens (with the exception of L1 VLP) are not well characterized. Spontaneous clearance of HPV infection occurs in the majority of patients, and has been largely attributed to the cellular immune response rather than the humoral immune response, as further described below and in 2.3.2.High-risk HPV infections can progress to high-grade CIN due to impaired T-cell immunity . HPV earThere are several studies providing information about CD4+ and CD8+ cytotoxic T-cell responses to HPV-16 early proteins . Using fNatural killer T (NKT) cells are a heterogeneous group of T cells that recognize self or microbial lipid antigens presented by CD1d molecules. CD1d is a major histocompatibility complex (MHC) I-like glycoprotein present on the surface of not only antigen presenting cells but also intestinal epithelial cells, keratinocytes and reproductive tract epithelial cells .The most widely studied ligand of NKT cells is α-galactosylceramide  . A recenIFN-γ +874 polymorphism with an increased risk of cervical cancer in patients at stages III + IV. However, this study did not include all clustered polymorphism sites of IFN-γ and was not free from selection bias [MHC cell surface downregulation is one of the mechanisms employed by a variety of tumours to evade immune detection. Cell surface CD1d, a MHC-Ib molecule, is down-regulated in HPV-related lesions and in HPV-negative cervical cancer cell lines stably transfected with HPV-6 E5 and HPV-16 E5, thus linking decreased CD1d expression in the presence of HPV infection with evasion of NKT cells . On the ion bias .Based on the literature, NKT cells may play two different roles, depending on the type of HPV lesions. In established HPV-associated cancerous lesions, activation of NKT cells may provide therapeutic value, while inhibition of NKT cell activity or recruitment in precancerous lesions may allow a protective immune response.Regulatory T cells (Treg) are CD25hi Foxp3+ CD4+ T cells that have been involved in the failure of the immune system to control the development of numerous cancers both in humans and in mice ,45. TherIndeed, human, high-grade CIN lesions (CIN3) and cervical carcinomas have been shown to contain higher numbers of infiltrating lymphocytes and FoxP3+ Treg compared to colon carcinomas, skin melanomas, and bronchial carcinomas -48. MoreNotably, Treg may also be induced by therapeutic vaccines. In a study comparing patient groups with small or large HPV-16-positive vulvar lesions, larger lesions were found to contain higher frequencies of vaccine-induced HPV-16-specific Foxp3+ Treg cells, correlating with early treatment failure . A similin vivo depletion of Treg has already proved to be efficient in allowing the rejection of established tumours in mice and humans [The d humans -58. In ad humans . Moreoved humans . In patid humans . As a co2.3.3.Macrophages are one of the three types of phagocytes in the immune system and are distributed widely in the body tissues, where they play a critical part in innate immunity. Macrophages are found in many areas of lymph nodes, particularly in the marginal sinus and in the medullary cords. Here they can actively phagocytose pathogens and antigens, and so prevent them from entering the blood. Activated macrophages undergo changes that greatly increase their antigen-presenting function and anti-pathogen effectiveness, and amplify the immune response.Regarding their role in HPV infection, studies in women infected with HPV have shown a positive correlation between lesion grade, the number of infiltrating macrophages and IL-10 expression by these cells -63. Howein situ through the macrophage colony-stimulating factor (CSF-1), as its over-expression correlates with poor prognosis in breast cancer [The tumor microenvironment has been shown to influence tumor immune privilege, and this has become a new field of research in tumor immunology. Macrophages are enriched in several types of human cancer, including breast, ovarian, non-small cell lung cancer, and Hodgkin's lymphoma, and their presence correlates with a poor clinical outcome. Tumor-associated macrophages (TAM) have been therefore identified as regulators of tumor development. As an example, TAM have been shown to play a major role in colorectal cancer by recruiting Treg to the tumor mass, thus favouring an immunosuppressive microenvironment that leads to tumor growth . TAM cant cancer . TherefoMyeloid derived suppressor cells (MDSC) are a heterogeneous population of cells that have been associated with cancer, inflammation and infection, and been shown to suppress T-cell responses. A regulatory role for MDSC, as well as regulatory T cells, has been highlighted in animal models using transplantable tumour cells expressing the E7 protein, TC-1. One recent study found reduced MDSC and Treg numbers in both the spleen and transplantable tumour itself through the use of a tri-therapy combination HPV vaccine, leading to the restoration of a potent E7-specific CD8 T-cell response and the control of tumour growth . AnotherDespite no demonstrated association with HPV-associated cancers in humans, MDSC have been observed with increased prevalence in the peripheral blood and tumor microenvironment of patients with head and neck squamous cell carcinomas  and panc, allergy, parasitic and virus (HIV) infections have each been shown to recruit mast cells into the skin.Mast cells (MC) are localized at body sites that interface with the environment, such as the skin and mucous membranes, which are also the site of HPV infection. Mast cells are highly specialized innate immune effector cells that contain secretory granules in which large amounts of proteases are stored in complexes with serglycin proteoglycans . The preA major point linking mast cells to cancer is that mast cells accumulate in the stroma surrounding certain tumors, especially mammary adenocarcinoma, where they have been shown to synthesize and secrete potent angiogenic cytokines, such as vascular endothelial growth factor (VEGF). These molecules facilitate tumor vascularisation by a direct angiogenic effect and by stimulating the stroma and inflammatory cells of the tumor microenvironment ,80. In rDendritic cells (DC) are potent antigen-presenting cells (APC) that play a fundamental role in the induction and regulation of innate and adaptive immune responses against microbial pathogens. DC in human and mice can be broadly categorised into two major populations: plasmacytoid DC (pDC) and conventional DC (cDC), which can be further divided into migratory DC (Langerhans cells (LC) and interstitial DC) residing in peripheral tissues and lymphoid tissue-resident DC.In humans, pDC are present in cervical cancer lesions, primarily in the stroma underlying the tumor rather than the dysplastic epithelium, and produce the anti-viral cytokine, IFN-α, in response to HPV VLP ,82. ThesEpithelial LC are the model migratory DC, initiating immune responses to infecting pathogens by capturing Ag and delivering it to the T cell areas of the draining lymph nodes. Several studies have documented reduced numbers of LC in human HPV-associated cervical lesions -86, suggA unique population of interstitial DC has been recently defined in the cervical stroma, with elevated numbers of these cells in human cervical cancer relative to normal cervix. This stromal DC subset is distinct from LC and expresses immuno-suppressive factors IL-10, TGF-β and indoleamine 2,3-dioxygenase (IDO) ,75,89. HDC are regarded as the master regulators (chef d'orchestre) of the immune response. They have been utilized in numerous vaccination protocols to boost antigen presentation and T-cell costimulation and thus, augment antigen-specific T-cell responses. However, their multiplicity of phenotypes and functions make it difficult to know which population(s) of APC should be targeted.3.3.1.Virus-like particles (VLP) are a useful tool for development of vaccines and are often used in studies to identify viral assembly proteins. They resemble the native virus immunologically but are non infectious as they don't contain viral genetic material. Two prophylactic HPV vaccines (Cervarix and Gardasil) that are currently in use are based on the L1 major capsid protein. The HPV L1 open reading frame translates a 55-kD protein that efficiently self assembles into viral capsomeres and empty capsids, when expressed in eukaryotic cells . In papiCervarix™ is a bivalent vaccine manufactured by Glaxo-SmithKline that incorporates two HPV types  responsible for 70% of cervical cancers. Gardasil™, manufactured by Merck, is a quadrivalent vaccine that additionally incorporates HPV-6 and HPV-11. HPV-6 and -11 cause an estimated 75-90% of genital warts cases.HPV-16 and -18 VLP are produced in Trichoplusia ni Rix4446 cell substrate using a baculovirus expression vector system and formulated with the adjuvant system ASO4 that is comprised of 3-O-desacyl-4′ monophosphoryl lipid A (MPL) and aluminum hydroxide salt. The vaccine is administrated intramuscularly in three dose schedule - months 0, 1 and 6.Saccharomyces cerevisiae and formulated with adjuvant aluminum hydroxyphospate sulfate. The vaccine is administrated intramuscularly in three dose schedule - months 0, 2 and 6 [HPV -6, -11, -16 and -18 VLP are produced in yeast  2 and 6 .These prophylactic vaccines are licensed for use to prevent HPV infection and anogenital cancers in females and males aged 9–45 in many countries, and are administrated to teenage girls as a part of the routine immunization schedule in some. In addition, the quadrivalent vaccine is licensed for use to prevent genital warts in some developed countries. The use of these vaccines in developing countries is limited by cost-related issues. Thus, provision of prophylactic HPV vaccines to a wider population through development of biosimilar vaccines is under consideration in developing countries.As long as women are sexually active they remain at risk of cervical HPV infection. Therefore, it is essential that HPV vaccines provide long-lasting protection. Clinical efficacy has been observed up to 6.4 years for the bivalent vaccine  and up tBased on phase III clinical trial results , the Adv3.2.To allow meaningful approaches to vaccine development, both prophylactic and therapeutic, it is important to identify the age at which the population is infected, the extent of exposure to human papillomavirus within the general population, how long the infection persists and if the immune response to HPV is consistent throughout life. One of the key questions also remains to understand the first transmission of the virus and whether sexual transmission is the only route for high-risk HPV.The detection of human papillomaviruses including HPV-16 and -18 in neonates have been described in about 50% of cases where the mother is infected at the time of delivery. In two early studies, HPV-16 and -18 DNA were detected in buccal and genital swabs in more than 70% of infants at 24 hours post delivery and persisted in more than 80% of contaminated infants at 6 weeks through to 6 months of age ,101. Othversus caesarean, may play a role, as a significantly higher rate of HPV-16/-18 infection was found at birth when infants were delivered vaginally [The mode of delivery at birth, vaginal aginally . Ten yeaaginally ,107. Oneaginally . These daginally .If the only possible method of genital HPV infection is sexual transmission, non-infected individuals could be found within virgin girls and young women. On the other hand, if nonsexual transmission can occur, some virgins might have genital HPV-specific antibodies, and this would increase the cut-off and reduce test sensitivity if virgin females were used to provide a reference standard for antibody testing. Therefore, it would be informative to determine the antibody status in pre-pubertal children. Here, the results from different studies are controversial, as it is crucial to define the antibody detection methodology, and what would be a “positive” and a “negative” control for an assay.One large study in Edinburgh examined more than 1,000 serum samples from a cohort of 11–13 year-old virgin school girls for the presence of antibodies to HPV-1, -2, and -16 VLP. The study reported that 7% of study subjects had antibodies to HPV-16 VLP, 52% to HPV-1 and 38% to HPV-2. In contrast, a Swedish study showed that virgin teenage girls were not seropositive in an HPV-16 VLP-based ELISA, while 14% of early sexually experienced girls were detected HPV seropositive and positive for HPV-16 DNA, leading to a conclusion that non-sexually transmitted infections are rare or nonexistent among adolescent girls . AnotherAltogether these data show the difficulty in defining an HPV antibody positive from a negative serum. This makes interpretation of data on early HPV infection problematic. However, the balance of evidence suggests that there are only very few, if any, HPV infections caught before the onset of sexual activity.The prevalence of high risk HPV in the general population is well known. Women acquire HPV infection soon after onset of sexual activity  and morevs. 17.9%, respectively), with lower titres of antibodies [Cancers of the penis, anus, and oropharynx in males can be due to high risk HPV infection, although less than 25% of HPV-related cancers occur in males. However, HPV infections and related non-malignant diseases are common in males , 115. Thtibodies ,117.A recent study enrolled more than 4,000 healthy boys and men between 16 to 26 years of age from 18 countries in a randomized, placebo-controlled, double-blind trial, and demonstrated that the quadrivalent HPV vaccine used as a prophylactic vaccination can reduce the incidence of some HPV-related infections . There aDespite the success of preventive HPV vaccines, such vaccines are unlikely to reduce the global burden of HPV-associated cancers in the next few years due to their high cost and limited availability in developing countries, where there is a high incidence of cervical cancer. Moreover, existing preventive HPV vaccines do not generate therapeutic effects. Therefore, it is worthwhile to consider alternative strategies to treat HPV-associated premalignancy and malignancy.3.3.With age, the immune system undergoes dramatic changes. From fetal to neonatal periods, naïve T-cells are extensively produced and migrate from the thymus to secondary lymphoid organs in the periphery, where they accumulate and display a broad and polyclonal repertoire. From youth to adulthood, thymic export is balanced by cell death at the periphery, leading to an equilibrium in the apparent quantity of cells. With age, the thymus involutes and numbers of newly generated naïve T-cells gradually fall. Together with this thymic involution, an immune decline, termed immunosenescence, progressively alters B- and T-cell functions. In counterpart, an accumulation of antigen-experienced peripheral T-cells is observed, both in the CD4+ and CD8+ compartments, likely due to an accumulation of memory responses following antigen activation and from homeostatic proliferation to maintain T-cell levels . It is rAnother fact arguing in favour of an early therapeutic vaccination comes from the analysis of vaccination strategies against transplantable tumours in mice. Indeed, it has been shown in mice that a vaccine is more potent and will generate a strong anti-cancer effector response when it is administered within a certain time frame . In humaWe have previously discussed in Section 2 the deleterious role of Treg in cancer. Moreover, it has been described in mice that, at the very time of tumour emergence, self-specific Treg were activated early and briskly by self-antigens expressed by tumours, driving a secondary-type immune response in essence more rapid and efficient than the primary-type response of naive effector T-cells specific for tumour neoantigens . This meTherefore, here again, it seems that early vaccination after the detection of cancer might be the preferred option to promote a maximum anti-tumour response. In that case, an improved effort in the screening of cervical cancers or skin cancers induced by HPV infection is also a key in the success of immunotherapy for HPV-associated cancers.4.Current treatments for HPV-associated lesions rely primarily on excision or ablation of the infected lesion. Ablative therapies are effective when the disease is localized as in the case of CIN but the possibility of recurrence remains, as treatment does not always eradicate the underlying HPV infection. A successful immunotherapy might therefore be a preferred mode of treatment because it can target all HPV-associated lesions irrespective of their location. Ideally, it would also induce long-lasting immunity, thus preventing recurrence.Therapeutic cancer vaccines are anticipated to treat an existing cancer by enhancing the naturally occurring immune response to the cancer. Unlike prophylactic vaccines, which protect against HPV infection by generating neutralizing antibodies, therapeutic vaccines would likely require induction of antigen-specific T-cells for clearance of HPV-associated lesions (reviewed in ). Most o4.1.Live-vector based vaccines are highly efficient in delivering relevant antigens or DNA encoding antigens of interest. These vaccines have advantages including the possibility of choosing a desirable vector from a wide range of vectors to deliver antigens. Moreover, replication and spreading of live vectors in the host results in potent immune responses. However, using live vectors poses safety concerns in clinical applications. Additionally, neutralizing antibodies to the live vector, generated upon vaccination, may limit the efficiency of repeated immunizations with the same vector. The most common types of live vectors used for vaccinations are bacterial and viral vectors.4.1.1.Listeria and Salmonella. Listeria is a gram-positive intracellular bacterium that occasionally causes disease in humans. Listeria monocytogenes (LM) has the ability to replicate in the cytosol of APCs after escaping phagosomal lysis by secreting a factor called listeriolysin O (LLO). This unique feature allows peptide antigens derived from LM to be processed and presented via both MHC class I and class II pathways, resulting in potent CD4+ and CD8+ T-cell-mediated immune responses.The two most promising bacterial vectors for therapeutic HPV vaccines are Listeria-based vaccines targeting E7 have been shown to cause regression of solid implanted tumors in HPV-16 E6/E7 transgenic mice [Salmonella enterica serovar Typhimurium expressing HPV-16 antigens induced transient inflammatory responses in the genital mucosa and conferred protection against subcutaneously implanted HPV-16 tumors [nic mice . These vnic mice . Intrava6 tumors .Listeria-based vaccines, using HPV-16 E7 antigen fused to a fragment of LLO, was found to be well tolerated and did not have any side effects in end-stage cervical cancer patients [Listeria-based vaccines as an efficient vaccine for HPV infection. Although a promising candidate for many cancer vaccines, Salmonella has yet to enter clinical trials for therapeutic HPV vaccine.The first clinical patients . However4.1.2.Many recombinant viral vectors  have been used for therapeutic HPV vaccine development due to their high infection efficiency and expression of antigens in the infected cells (reviewed in ).A replication-deficient adenovirus encoding fusion proteins comprised of calreticulin, known to enhance MHC-I expression on cell surface, and E7 antigen (CRT/E7) has been shown to generate a potent and protective cellular response to E7, in an established tumour model in mice . This stVaccinia virus is another viral vector that has been tested in both preclinical and clinical trials . Phase I4.2.Protein and peptide-based vaccines are the most popular form of HPV therapeutic vaccines. Vaccination with HPV antigenic peptides involves the uptake and presentation of the peptide antigen in association with MHC molecules by DC. The polymorphic nature of MHC molecules in genetically diverse populations makes it difficult to identify one immunogenic epitope which would cover all individuals. However, the use of overlapping, long peptide vaccines based on HPV E6/E7 antigens has been effective in generating antigen-specific T-cell responses. This limited use of peptides is reduced when the entire protein is used. Since protein antigens can be processed by the patient's DC, which contain the relevant human leukocyte antigens (HLA), vaccines based on protein antigens can evade the limitation of MHC specificity associated with peptide vaccines. Adjuvants and fusion with immunostimulatory molecules, such as IL-2, are often used to overcome poor immunogenicity associated with protein and peptide-based vaccines. Several protein and peptide vaccines against HPV E6 and/or E7 have been successfully tested in preclinical and clinical models.In a E7-expressing TC-1 tumor model, mice were vaccinated subcutaneously with HPV-16 E7 peptide, together with a pan HLA-DR epitope (PADRE) peptide and the TLR3 ligand poly(I:C) adjuvant. PADRE peptide and poly (I:C) were used to enhance the activation of CD4+ T helper cells and dendritic cells, respectively, which altogether generated anti-tumor effects against TC-1. This therapeutic effect was further enhanced when the vaccine was administered in the tumour mass itself, generating higher frequency of E7-specific CD8+ T-cells and leading to a better survival .Bordetella pertusis adenylyl cyclase (CyaA), which targets proteins to APC [Pseudomonas aeruginosa exotoxin A, which facilitates their translocation to enhance MHC class I presentation [Like peptides, proteins have been tested as candidates for therapeutic HPV vaccines. To enhance the efficacy of HPV protein-based vaccines, adjuvants such as liposome-polycationic–DNA (LPD)  and sapos to APC , or withentation , have leMycobacteria bovis derived heat shock protein (HSP), known to enhance CTL responses, was used in patients with high-grade anal intraepithelial neoplasia (AIN) [Several trials have demonstrated the successful use of peptide/protein based vaccines against HPV. A phase I trial involving overlapping HPV-16 E6 and E7 peptides with Montanide ISA51 adjuvant in end-stage cervical cancer patients and VIN grade III patients elicited broad IFN-γ associated T-cell responses . In a siia (AIN)  and showia (AIN) . Finallyia (AIN) . The use4.3.4.3.1.DNA-based vaccines are another candidate for HPV-vaccine. They have been extensively studied in preclinical models, in order to optimize the cell targeting, DNA uptake and processing, presentation to MHC molecules...Strategies to deliver DNA directly into DC may vary. The strategy using Gene gun can efficiently deliver DNA to Langerhans cells in the epidermis, which then express the antigens, become mature and migrate to the draining lymph nodes, where they prime naïve T cells. This method has been shown to elicit HPV antigen-specific T cell immunity and antibody responses .As with protein-based vaccines, DNA encoding HPV antigens are fused with molecules such as FMS-like tyrosine kinase 3 (flt3) ligands  and HSP In a recent study, a DNA vaccine encoding herpes simplex virus type 1 (HSV-1) glycoprotein D genetically fused to human HPV-16 oncoproteins E5, E6, and E7, induced antigen-specific CD8+ T-cell responses and conferred preventive resistance to transplantable murine TC-1 tumor cells . Co-admiIn another study, a DNA vaccine encoding calreticulin (CRT) linked to human HPV-16 E7 (CRT/E7) showed increased intercellular uptake and processing of the DNA. With the TLR7 agonist Imiquimod, this vaccine generated E7-specific antitumor effects and prolonged the survival in treated mice, probably by also decreasing the number of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment of tumor-bearing mice . This CRM. tuberculosis HSP70 was tested in CIN2/3 patients. Fifteen CIN2/3 patients were given three different doses of vaccine . The vaccine led to complete regression of the lesions in three of nine patients treated with highest dose of the vaccine. E7-specific CD8+ T cell responses were detected in patients treated with vaccine at 1 mg and 3 mg [Several DNA vaccines have been tested in clinical trials for CIN2/3 patients (reviewed in ). A receand 3 mg ,157.in vivo. Their use has been shown to be well-tolerated by patients, quite safe, and good immunogenes. Nevertheless, more studies are needed to further improve the efficiency, safety, and cost of potential DNA vaccines.DNA-based vaccines are stable and easy to produce. They can lead to sustained cellular antigen expression compared to protein-based vaccines, which makes them a strong candidate for therapeutic HPV vaccines. However, they have limited potency to invoke innate defence mechanism because of lack of intrinsic ability to amplify and spread 4.3.2.RNA replicons are vaccines based on RNA viruses. They have the ability to self replicate in the infected host cell. Therefore, they can sustain the cellular antigen expression and as a result, can produce more antigenic protein than conventional DNA vaccines. However unlike DNA vaccines, RNA replicons cannot reproduce by themselves and therefore, can circumvent the possibility of integration into the host genome and cellular transformation. RNA replicons are customized to lack the viral structural genes and thus, the vaccines may be repeatedly administrated in patients without the generation of neutralizing antibodies against viral capsid proteins.A DNA-launched RNA replicons vaccine, called “suicidal vaccine” has been made to increase the stability of RNA replicons. In this vaccine, suicidal DNA is transcribed into RNA replicons and cells uptaking the suicidal DNA vector eventually die due to apoptosis, thereby minimizing the concerns associated with potential integration of vaccine DNA into the host genome and cell transformation. However, the expression of inserted genes in these vectors is transient and it can shorten the functional lifespan of DC if DNA is targeted to DC, thereby reducing their effectiveness in stimulating the immune system. In one of the preclinical models, a suicidal DNA vector, pSCA1, encoding HPV-16 E7 was fused with BCL-xL, an antiapoptotic protein, to enhance the survival of APC . This ve4.4.4.4.1.ex vivo to enhance their immunogenicity . HoweveIn a recent preclinical study in mice, forced expression in tumors of LIGHT, a ligand for the lymphotoxin-beta receptor, resulted in an increased expression of IFN-γ and chemoattractant cytokines such as IL-1α, MIG, and MIP-2. This correlated with an increased frequency of tumor-infiltrating CD8+ T cells and eradication of large well-established tumors .These vaccines are mainly used when tumor antigens are not identified; however they may not be relevant in case of HPV as tumor antigens associated with HPV-infection are largely known and this could be the reason that these vaccines have not yet tested in clinical trials for HPV-associated cancers.4.4.2.DC vaccines are another way to enhance T-cell mediated immunity against HPV-associated lesions.DC-based vaccines are the preferred choice for vaccine development and a number of methods have been used including the usage of different vectors, pulsing of DC with proteins and peptides or transfecting DC with DNA or RNA (see above and reviewed in ). In a TIn humans, autologous DCs were pulsed with HPV16 or HPV18 E7 recombinant proteins and E7-specific CD8+ T cell responses were observed in four out of 11 late stage cervical cancer patients . In anot®, a DC vaccine incorporating prostatic acid phosphatase, in patients with advanced prostate cancer has generated strong interest for DC-based vaccines [DC vaccines are patient-specific, where clinicians harvest DC from the patient, load them with tumor antigens such as E6 or E7, and then inject these DC back into the patient, where they elicit antigen-specific potent antitumor immune response. The success of Provengevaccines ,166. How4.5.In Section 4 we discussed the different formulations that can potentially been used to create a therapeutic vaccine. In the examples we cited, some of the studies already used combinational approaches, by adding an adjuvant or a co-stimulatory signal to the vaccine itself. In this paragraph, we just want to highlight it a bit more.Prime-boost vaccination strategies use several of the available therapeutic vaccines in combination to induce higher levels of tumor specific immune response. Some of these have been evaluated in clinical trials for therapeutic HPV vaccines. In one trial, nine patients developed HPV16 specific T cell responses and three out of 10 patients showed a significant reduction in the size of the lesion, when high grade VIN were primed with TA-HPV and boosted with TA-CIN .Therapeutic HPV vaccines may potentially be combined with other therapeutic methods such as radiotherapy and chemotherapy to enhance the clinical outcome. In an experimental model, treatment with low-dose radiotherapy made the TC-1 tumor cells more susceptible to lysis by E7-specific CD8+ T cells, and enhanced antitumor effects in tumor bearing mice . In a siTherapeutic HPV vaccines may also be combined to co-stimulatory signal delivery or cytokine adjuvantizing. The use of CTLA-4 antibodies has been shown to be effective in murine TC-1 tumor models . More reAnother strategy to improve HPV-vaccine is to combine them with the depletion of regulatory cells such as Treg, MDSC, or NKT cells, as discussed in Section 2. However in a recent study, depletion of Treg did not enhance the immune response induced by SFV-based HPVE6/E7 vaccine against murine tumors, suggesting that the SFVeE6/7 vaccine may not require additional immune interventions .Thus, as it has already been described in many other cancer types, combinational approaches might be the gold strategies to generate a successful anti-HPV therapeutic vaccine.5.Although preventive HPV vaccines are now available to use, the high prevalence of HPV-associated malignancies worldwide suggests a potential benefit from developing therapeutic HPV vaccines. New leads, news targets and new research axis have to be investigated to maximize the chances to find a way towards therapeutic vaccines.5.1.i.e., plant cell suspension, hairy root cultures, microalgae, etc) provides a powerful alternative for the production of recombinant therapeutic molecules, such as IgG, IL-12 or IFNs. Plant cell suspension can be derived from tobacco, rice, soybean, tomato, alfalfa and carrot plants; hairy root cultures are generated from the interaction between Agrobacterium rhizogenes, a Gram-negative soil bacterium, and a host plant; and microalgae are photosynthetic heterotrophic microorganisms found in wet environments [The HPV-derived VLP obtained by the self assembly of the viral L1 capsid protein have been generated in yeast and insect cell lines. However, the development of contained plant systems  compared to mammalian or eukaryotic systems , and also some post-translational modifications need to be adapted .www.biolex.com). The results of this phase II clinical trial for the treatment of patients with chronic hepatitis C with Locteron, an alpha-IFN produced in duckweed, were published in March 2011 [www.dowagro.com/), obtaining US Department of Agriculture (USDA) approval in 2006. Although not yet available commercially, this novel product represents the first approval for a plant-derived vaccine.There is recent evidence of the successful usage of plant-produced pharmaceuticals by Biolex Therapeutics  associated with conventional therapeutic approaches . With the increasing discovery of new drugs, development of new adjuvants, and the better understanding of tumor biology, we will have more opportunities to develop improved therapeutics against HPV-associated cancers. Successful clinical trials for therapeutic HPV vaccines based on positive preclinical data have now been published ,123, andWe thank Graham Leggatt, Fiona McMillan and James Wells for critical review of the manuscript. This work was supported by funding from the National Health and Medical Research Council (Program Grant No. 569938) and the NIH grant No. 5U01CA141583, Cancer Council Australia, Cancer Research Institute (New York), Lions Medical Research Foundation, Australian Cancer Research Foundation and a Queensland Government Premiers Fellowship. IHF derived royalty income from prophylactic vaccines against cervical cancer based on HPV VLP technology.


PEG3 methylation status by bisulfite pyrosequencing. Multinomial logistic regression was used to estimate odds ratios (OR) and confidence intervals (CI 95%) for associations between PEG3 methylation status and CIN or ICC. After adjusting for age, gravidity, hormonal contraceptive use and HPV infection, a 5% increase in PEG3 DNA methylation was associated with increased risk for ICC . HPV infection was associated with a higher risk of CIN1-3  and ICC . Infection with high risk HPV was correlated with mean PEG3 differentially methylated regions (DMRs) methylation (r = 0.34 p<0.0001), while the correlation with low risk HPV infection was weaker (r = 0.16 p = 0.047). Although small sample size limits inference, these data support that PEG3 methylation status has potential as a molecular target for inclusion in CIN screening to improve prediction of progression.Cytology-based screening for invasive cervical cancer (ICC) lacks sensitivity and specificity to discriminate between cervical intraepithelial neoplasia (CIN) likely to persist or progress from cases likely to resolve. Genome-wide approaches have been used to identify DNA methylation marks associated with CIN persistence or progression. However, associations between DNA methylation marks and CIN or ICC remain weak and inconsistent. Between 2008–2009, we conducted a hospital-based, case-control study among 213 Tanzania women with CIN 1/2/3 or ICC. We collected questionnaire data, biopsies, peripheral blood, cervical scrapes, Human papillomavirus (HPV) and HIV-1 infection status. We assessed PEG3 DMR is an important co-factor in the development of Invasive cervical carcinoma (ICC), especially among women infected with high risk HPV. Our results show that a five percent increase in DNA methylation of PEG3 is associated with a 1.6-fold increase ICC risk. Suggesting PEG3 methylation status may be useful as a molecular marker for CIN screening to improve prediction of cases likely to progress.We present the first evidence that aberrant methylation of the  Approximately half a million women throughout the world are diagnosed with cervical cancer annually and slightly over half of these women die from the disease; 80% are diagnosed in resource-poor settings PEG3 is a paternally expressed imprinted gene on chromosome 19q13.43 that encodes a protein with tumor suppressive function that plays a role in facilitating p53/c-myc-mediated apoptosis. PEG3 is regulated by allele-specific DNA methylation whereby only the maternally derived allele is normally methylated. Hypermethylation at the PEG3 regulatory differentially methylated region (DMR) leads to a decrease in PEG3 transcription, which in turn is presumed to inhibit the pro-apoptotic function of this gene PEG3 expression of this tumor suppressor as has been observed in other gynecologic cancers, including such as ovarian and endometrial cancers PEG3 DMR hypermethylation and transcriptional silencing has also been shown to occur in gliomas PEG3 DMR are associated with HPV infection, CIN and ICC.Epigenetic mechanisms of gene regulation, including DNA methylation, have an important role in coordinating gene expression changes in response to viral infection Between November 2008 and March 2009, eligible study participants were recruited from the Reproductive Health Clinic (RHC) at KCMC, a Cervical Cancer prevention clinic funded by the World Health Organization. Written informed consent was obtained from each study participant prior to enrollment. Research Ethics Boards at Kilimanjaro Christian Medical Centre (KCMC), the University North Carolina at Chapel Hill and Duke University approved this study.A trained nurse-interviewer obtained informed consent and administered a standardized in-person 40-minute questionnaire. Socio-demographic characteristics collected included age, martial status, type of marriage (polygamy vs. monogamy), tribe, educational attainment, cigarette smoking, alcohol consumption, reproductive history , sexual history , and medication and supplement use.Two cervical scrapes were obtained from each participant. One was prepared on a glass slide for cytological evaluation for CIN and ICC diagnosis, data also used as outcome for this study. The second specimen was collected using a cytobrush and rinsed into Preserv-Cyt ™ media . One-third of the specimen was reserved for HPV analysis and stored at 4°C, and the remaining two-thirds were centrifuged to pellet the cells, which were stored as aliquots at −80°C. DNA extracted from these cells was later used for DNA methylation analysis. Biopsy specimens were collected only when clinically indicated. Routine cervical screening by visual inspection with acetic acid (VIA) was performed. If there were positive findings by VIA or through direct examination, the patient was triaged and treated accordingly. Patients with negative findings were given follow up appointments within two weeks to provide results.http://www.asccp.org/). A gynecologist (BV) also reviewed medical charts monthly for HIV-1 test and cyto-pathological results to classify the cases using the Bethesda classification system. The results were then coded based on pathology and medical record findings. They were coded as 1) no evidence of cytological abnormality, 2) mild dysplasia including LSIL and CIN1, 3) moderate dysplasia including HSIL and CIN2-3, or 4) cancer that was primarily squamous cell carcinoma with the exception of three adeno-squamous carcinomas of the uterine cervix. None of the specimens were classified as atypical cells of uncertain significance (ASCUS). The results were available via the patient's clinic records, and the pathologist entered them into the database. The records were then compiled and securely shared with Duke University.The pathologist at KCMC (BS) processed and read the Papanicolaou smears and biopsy specimens using standard conventions according to ASCCP guidelines as appropriate . The standard clinical practice of Western blot was used for specimens that were reactive .Genomic DNA was prepared from cells isolated from the cervical scrapes or from biopsy specimens using PureGene protocol reagents  and treated with sodium bisulfite using the Zymo Easy-96 DNA methylation kit . Bisulfite treatment modifies the DNA by converting unmethylated cytosines to uracils, and leaves methylated cytosines unchanged. Pyrosequencing was performed using a Qiagen Pyromark Q96 MD Pyrosequencer.PEG3 promoter region at chromosome 19q13.43. The pyrosequencing assay for PEG3 was used as previously described The DMR analyzed is located within the PEG3 DMR differed significantly by infection with high risk (HR) versus low risk (LR) HPV and other HPV genotypes. Classification of high risk and low risk genotypes was based on FDA-approved HPV molecular tests (CDC). HR HPV genotypes included HPV 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,59 and LR HPV included HPV 6, 11, 26, 66, 68, 70, 73 and, 82 as described previously The mean DNA methylation fractions at the individual CGs were analyzed and compared among controls, women without CIN1/2/3 or ICC (n = 147) and the three case groups  using F-tests. Principal components analyses (PCA) were applied to determine if a single mean represents the methylation fraction at the CG dinucleotides within the DMR region. Methylation of the CGs was sufficiently correlated thus allowing a single mean to be used. F-tests were used to determine if DNA methylation at individual CGs within th or >75th quartiles, since both hypermethylation and hypomethylation of this locus can lead to deregulation of PEG3 expression and lead to loss of imprinting To estimate odds ratios (OR) and corresponding 95% confidence intervals (CIs) for the association between CIN/ICC and changes in DMR methylation, we used multi-polynomial logistic regression models. All models included methylation using 5% increments with adjustments made for HPV-infection, pregnancy history, HIV-1 status, and hormonal contraceptive use. Aberrant methylation was defined as <25Case groups and controls differed significantly by age, HPV infection and gravidity . The meaWe first validated the performance of the pyrosequencing assay in quintuplicate using fully methylated and unmethylated DNAs in defined proportions. At each increase in the amount of methylated DNA input, there was also an increase in the amount of methylation measured . The average standard deviation between these replicate measures was 1.59% . These results indicate that this assay has the ability to reproducibly detect differences in methylation values .PEG3 DMR in relation to HPV genotype. As shown in PEG3 DMR and HPV infection . LR-HPV genotypes had a sizably weaker correlation with PEG3 methylation . Infection with other HPV genotypes was not correlated with methylation fractions within the PEG3 DMR (mean correlation coefficient = 0.0163 p = 0.876).We examined methylation fractions of 10 CpG sites within the PEG3 DMR methylation and CIN and ICC status, adjusted for age, hormonal contraceptive use and any HIV-1 infection. We found little or no association between mean PEG3 methylation status and CIN1/2/3, ; p-value = 0.80). However, a 5% increase in methylation levels at the PEG3 DMR was associated with a nearly two-fold increase in the risk of ICC . As expected, infection with any HPV genotype was also associated with a higher risk of CIN1-3 (OR = 15.7 95% CI(5.1–48.6)) and ICC ).PEG3 DMR was associated with a 1.6 fold increase in ICC risk. We also found that PEG3 DMR hypermethylation was correlated with HPV infection; a correlation that was stronger for high risk as compared to low risk HPV infection. As would be expected, HPV infection was associated with increased risk of CIN1/2/3 and ICC. We present the first evidence in support of the hypothesis that aberrant methylation of the PEG3 DMR is an important co-factor in the development of ICC, especially among women infected with HR HPV.Our key findings in this case-control study of Tanzanian women are that after adjusting for HPV infection, age, OC use, and HIV-1 status, a 5% increase in DNA methylation at the PEG3 DMR hypermethylation and is consistent with DNA methylation-mediated repression of PEG3 as found in previous studies. Hypermethylation of various genes  in ICC case control sudies has been reported PEG3. To our knowledge this is the first study done with a human population that investigated the relationship between PEG3 DMR status and HPV infection and how this plays a role in CIN and ICC. Although cause and effect cannot be established in this case-control study, our findings suggest that PEG3 DMR methylation is a potential mechanism by which susceptibility to progression to ICC may occur, and thus may be a useful marker to identify CIN cases likely to progress.Our data suggests that increasing grade of lesion from CIN to ICC correlates with HPV infection and PEG3 DNA methylation increases risk of ICC are unclear. However, there is evidence suggesting that PEG3 plays an important biological role in p53/c-myc mediated apoptosis, implicating PEG3 as a gene whose function may be in part to prevent carcinogenesis PEG3 functions as an important tumor suppressor in carcinogenesis.The mechanisms by which in vitro and in vivo studies showing that the PEG3 promoter is hypermethylated with consequent transcriptional repression in ovarian and endometrial cancers PEG3 is silenced suggesting that during carcinogenesis, hypermethylation may be selected for in order to inhibit the pro-apoptotic function of PEG3. Our case control study shows an association between hypermethylation of PEG3 and ICC but not CIN, suggesting that these methylation alterations take place during transformation rather than in pre-cancerous lesions. Alternatively, the attenuation in risk may be due to combining low grade CIN largely comprised of lesions likely to regress, with higher-grade CIN cases, the majority of which have potential to progress and become ICC. Intriguingly, the correlation of HPV infection, an etiologic agent of ICC, and PEG3 hypermethylation is consistent with a multi-step process that starts with epigenetic mechanisms and HPV infection.The association found here is consistent with findings from PEG3 DMR methylation in relation to grade specific CIN, after accounting for the effect of HPV infection. It is possible that our inability to find associations between PEG3 methylation and CIN was due to combining CIN (in whom the majority or women are likely to regress) and CIN2 and CIN3  PEG3 and ICC risk. Another limitation is the case-control design, limiting our ability to infer PEG3 methylation as an important factor in progression. However, identifying methylation marks associated with case-control differences is a necessary step allowing for examination of this marker in longitudinal studies currently under way by several groups The main limitation of this study is the small sample size to examine PEG3 DMR increased the risk for ICC after adjusting for known confounders. We also found a strong correlation between HPV genotype and DNA methylation at the PEG3 DMR. Cytosine methylation is a stable modification in human tissue samples, and therefore PEG3 DMR methylation status could potentially be used as a marker to identify CIN likely to progress to ICC. Larger studies in a more diverse study population are required to replicate these findings.Despite these limitations, we found hypermethylation of the