The Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. However, the assay is limited to a restricted number of HPV, and sequence variations in the detection region of the HPV probes could give false negatives results. Recently, 454 Next-Generation sequencing (NGS) technology has been efficiently used also for HPV genotyping; this methodology is based on massive sequencing of HPV fragments and is expected to be highly specific and sensitive. In this work, we studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS.n = 48), with Cervical intraepithelial neoplasia grade 1 , or with Cervical cancer  were recruited, DNA was extracted, and HPV positivity was determined by PCR amplification using PGMY09/11 primers. All HPV- positive samples were genotyped individually by LA. Additionally, pools of amplicons from the PGMY-PCR products were sequenced using 454 NGS technology. Results obtained by NGS were compared with those of LA for each group of samples.Two hundred thirty three cervical samples from women Without cervical lesions  contains supplementary material, which is available to authorized users. Cervical cancer (CC) is the most frequent female cancer worldwide, occupying fourth place among cancers in women. In Mexico, the incidence of this pathology is high , reaching second place for cancers in women and causing 4769 deaths each year according to Globocan 2012 . GloballAll of these data suggest the need to develop detection and prevention strategies consistent with the characteristics of the geographic population.There are few commercial methods available for HPV genotyping , the majn = 87) of the samples were infected with HPV, 67.8 % with SI and 32.2 % with MI.From the 48 WCL samples, 12 (25 %) were positive for HPV. Considering only HPV-positive samples, 58.3 % were reported as SI and 41.7 % as MI  or CIN (−). As depicted in Fig. Comparing the number of reads obtained by group, it was observed that the greatest number of reads was obtained in the WCL (−) group, followed by the CIN I (−), CIN I SI, CC SI, CC MI, WCL (+), and CIN I MI groups Fig. . HoweverAs depicted in Table For a better overview of all NGS results, a summary of HPV genotypes found according to diagnosis group are depicted in Table Finally, consensus sequences (contigs) of each HPV genotype found exclusively among the Mexican samples analyzed were aligned and a phylogenetic tree was constructed Fig. . As expeCervical cancer remains a public health problem in Mexico, despite the prevention and vaccination campaigns implemented by the government. Vaccination is expected to decrease the incidence of this malignancy in the near future, and this probably would impact differentially, depending on the geographic region, because it has been shown that HPV genotype frequency varies among the different countries –22. ThusUsing 454 NGS, we could detect genotypes not included in LA: 32, 44, 74, 102, and 114 . We found HPV-32 only in the WCL group; this genotype has been associated with oral focal epithelial hyperplasia , 24 and With respect to HPV-44, we detected this genotype in WCL or CIN I samples, but not in CC. This HPV was isolated for the first time from a vulvar condyloma, and was found in five cases of 439 normal squamous epithelium samples and in three cases of 112 CIN I samples, but was not found in any of the 56 CC analyzed . HoweverHPV-74 has been found in low-grade vaginal intraepithelial neoplasia in immunosuppressed women and has been related phylogenetically with HPV-6, 11, 44, and 55 , 36. We alpha species 3  type, its complete genome must be cloned and its  variant . By analRegarding the classification of carcinogenicity for humans used by the International Agency for Research on Cancer (IARC) , there aOne limitation of this study was that groups of samples (according to the diagnosis), but not individually samples, were sequenced. Additionally, the HPV-genotypes detected were limited to the amplicons obtained by using the PGMY09/11 set of primers.We continue to recruit and analyze additional cervical samples in order to corroborate and report these observations. Genotyping of HPV by NGS will permit the detection of more possible HPV variants, whose sequences could increase or diminish HPV recognition by the immune system or modify the oncogenic potential of HPV proteins .We continue to need more studies with methodologies such as NGS to determine the HPV genotypes and variants circulating in Mexican population and at the different stages during CC progression.To our knowledge, this is the first time that data has been reported on HPV genotypes circulating in Mexico by using Massive parallel sequencing (MPS). Our data provide helpful information because we were able to find not previously detected HPV genotypes in Mexican population. The methodological strategy employing the PGMY09/11 primers appears to be a very good system.Approximately 250 cervical samples were obtained from the Centro Médico Nacional de Occidente  located in Guadalajara, Jalisco, Western Mexico. The women whose samples were taken attended a medical examination due to a cervical ailment. Diagnosis was first performed only by colposcopy observation ; when cervical lesions/cancer were (was) observed, a biopsy was taken for diagnosis confirmation by histopathology. From the 250 samples obtained, 17 samples were excluded , therefore, 233 samples were included in this study: 48 WCL; 98 CIN I, and 87 CC. Ages of the participating women ranged from 25–65 years. Samples were obtained by inserting a cytobrush into the endocervical canal, rotating it for 3–5 full turns, and placing it into ThinPrep PreservCyt® transport medium solution .Total DNA was extracted and purified from each cervical sample using the AmpliLute Liquid Media Extraction Amplicor Kit . A first screening for HPV positivity was performed by conventional single-round PCR utilizing PGMY09/11 primers, which amplified a 450-bp fragment of L1 . HPV-posForward 5′-[Universal Multiplicom tail A: AAGACTCGGCAGCATCTCCA] – [individually PGMY11 primer]-3′, and Reverse 5′-[Universal Multiplicom tail B: GCGATCGTCACTGTTCTCCA] – [individually PGMY09 primer]-3′. The PGMY sequences used were those reported in Table All HPV-positive samples identified by conventional PCR utilizing PGMY09/11 primers were included for MS. Amplicons for the NGS Library were generated by taking approximately 500–1000 ng of DNA from each sample and amplification by conventional PCR employing the following specifically designed primers: Purified amplicons generated from each sample were quantified with the Qubit® dsDNA HS fluorometry assay Kit  to carry out 5-ng amplicon pools per sample. Amplicon pools were generated for the NGS Library, taking into account diagnosis, HPV negativity or positivity observed by LA, and the SI- or MI-HPV infection detected in a same sample. The following groups were generated: WCL (−), samples WCL-negative for HPV by LA; WCL (+), samples WCL-positive for HPV by LA; CIN I (−), CIN I samples negative for HPV by LA; CIN I SI, CIN I samples with single HPV infection; CIN I MI, CIN I samples infected with 2 or more HPVs, and CC SI and CC MI.A second PCR reaction was performed to add the Multiplex identifiers (MID) barcodes, the key sequence (TCAG), and A or B sequencing adapters by using Multiplicom 454 MID Kits  according to the manufacturer’s instructions. Amplicons where then purified with Agentcourt® AMPure® XP beads  and evaluated with the 2100 Bioanalyzer system .The emulsion Polymerase chain reaction (emPCR) was performed with the GS Junior Titanium emPCR kit Lib A , and amplicons were sequenced in a GS Junior Sequencer utilizing the GS Junior Titanium Sequencing Kit (Cat. no. 05996554001) and the GS Junior Titanium PicoTiterPlate Kit (Cat. no. 0599661900), following the manufacturer’s instruction. All kits were supplied by Roche Diagnostics, Basel, Switzerland.alpha and beta papillomavirus reference sequences, downloaded from Papillomavirus Episteme (PaVE) [Quality control for sequencing data was performed, sequences with a Phred quality score of <20 were eliminated, and primer sequences were trimmed. The Roche GS Reference Mapper (ver. 2.9) was used to identify HPV genotypes by comparing fragment sequences with the L1 region of e (PaVE) . SpecifiConstruction of a phylogenetic tree with consensus sequences of each HPV was performed. First, all consensus sequences were aligned utilizing MEGA6 software , followed by the construction of the phylogenetic tree with the maximum likelihood method. Traditional tree-branch style was chosen and the Topology display was selected.This study was approved by the Ethics and Research Committees of the Instituto Mexicano del Seguro Social (IMSS), with registration number R-2014-785-036. All women provided written informed consent before the sample collection.


Cervical cancer ranks third in prevalence and fourth as cause of death in women worldwide. In Brazil, 17,540 women were diagnosed in 2012 with the disease. Persistent infection with high-risk HPV types is a necessary condition for the development of pre-invasive and invasive cervical neoplasia. Currently, over 100 HPV types have been identified, but HPV16 and 18 are recognized as the mayor culprits in cervical carcinogenesis. Our objective was to assess the relationships between single- (ST) and multiple-type (MT) HPV infections with patients’ age and lesion pathological status.328 patients with either squamous or glandular intraepithelial or invasive cervical lesion were selected. All subjects were tested for HPV genotypes with reverse hybridization for 21 high- (hr-HPV) and 16 low-risk (lr-HPV) probes. Prevalence of ST and MT HPV infections was compared across histological types and age strata.287 (87%) women had at least one HPV type detected and 149 (52%) had MT infections. The most prevalent HPV type was HPV16, present in 142 cases , followed by HPV58, 52, 31, 35 and 33. HPV18, in single or multiple infections, occurred in 23 cases (8% of hr-HPV cases). Almost all glandular lesions were associated with HPV16 and 18 alone. Multiple infections were significantly more prevalent in squamous than in glandular lesion for HPV16 and 18 (P = 0.04 and 0.03 respectively). The prevalence of MT infections followed a bimodal distribution; peaking in women younger 29 years and in those aged 50 to 59.Our data indicate that prevention strategies for pre-invasive and invasive squamous lesions should be focused on HPV16 and a few alpha-9 HPV types. It is clear to us that in young women, prophylaxis must cover a large amalgam of HPV types beyond classic HPV16 and 18. Cervical cancer is the third most prevalent cancer worldwide, being recognized as the fourth cause of death due to cancer in women. It is the second more prevalent type of cancer in women 44 years old or younger. BraziliHigh-risk human Papillomavirus (hr-HPV) persistent infection is considered the causal factor for pre invasive and invasive cervical carcinoma. More thin situ adenocarcinoma (AIS)) and invasive squamous/glandular cervical cancer (CC). MT-HPV has been observed in women with persistent infections, but its relationship with cervical carcinogenesis has not been established cet al. (2006)[Simultaneous presence of multiple HPV genotypes may be associated with a increased risk of high-grade squamous lesions or invasive cancer than the presence of single-type HPV infection. The classic study from Trottier . (2006) showed t. (2006). However. (2006), the difet al.[th. In the metanalysis[In our sample, glandular lesions were associated essentially with HPV16 or 18 ST infections. Quint et al. also detet al.. In our analysis, HPV18 wet al. (2010) who examined the HPV prevalence in archival samples obtained from patients with cervical pre-malignant and malignant lesions from Northeast Brazil. In that study, among the HPV-positive samples, 86.7% had a single infection, while 13.3% had a double infection. These findings contrast with ours, since we detected a higher prevalence of MT infections in all lesion groups[Another recent study examined the prevalence of HPV type distribution in Brazilian women with and without cervical lesions. From a total of 132 women with amplified HPV-DNA the frequency of HPV HR-types was 75%, with top appearing types being HPV16 (28%), followed by HPV18 (14.4%), HPV45 (7.6%), HPV58 (6.8%), HPV66 (6.8%), HPV31 (3.8%) and HPV33 (3.0%). The mean age of the 132 women were 39.5 years, ranging from 25 to 59 years of age. A significantly lower proportion of LSIL and HSIL was found among women infected by HPV16 and/or HPV18 when compared to the ones infected by other HR-types. Their data also showed that infection by alpha-7 and alpha-9 species presented a significant distinct distribution by age at diagnosis respective to women positive for other HPV types. However, separate comparisons among alpha-7 infections, alpha-9 infection, and infections by other HPV types, did not show a significant different distribution by age[n groups.Our results may bear significance in the era of prophylactic HPV vaccination. Both bivalent and quadrivalent prophylactic HPV vaccines target hr-HPV16 and 18 and had demonstrated different cross protection against HPV31, 33, 45, 52 and 58, potentially diverting 90% of cervical cancer risk. In addition to a new nonavalent vaccine against HPV16, 18, 31, 33, 45, 52 and 58 - now in phase 3 testing - protection against the most prevalent HPV types is in sight.The main strength of this study is that all women had a complete histological evaluation, giving to our sample a strong reliability related to the gravity of the cervical lesion. We also took care to assess all known HPV types using reverse line blot hybridization assay, which is an accurate and sensitive method. On the other hand, the sample is modest in size, especially women with glandular lesions, which unfortunately is a common situation in most recent papers on the subject.Collectively, our data indicate that prevention strategies for pre-invasive and invasive squamous lesions should be focused on HPV16 and a few alpha-9 HPV types. It is clear to us that in young women, prophylaxis must cover a large amalgam of HPV types beyond classic HPV16 and 18. For the prevention of glandular lesions, ST HPV16 or 18 infections should be addressed, with other alpha-9 or alpha-7 HPV types being of marginal importance.The authors declare that they have no competing interests.LSAR, SHRS and SD designed the study and wrote the manuscript. SD coordinated the study group. LOS analysed the data and wrote the manuscript. RRFA and AAR assisted with the experiments. LOS, LCZ and SD revised the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/214/prepub


The HPV viral lifecycle is tightly linked to the host cell differentiation, causing difficulty in growing virions in culture. A system that bypasses the need for differentiating epithelium has allowed for generation of recombinant particles, such as virus-like particles (VLPs), pseudovirions (PsV), and quasivirions (QV). Much of the research looking at the HPV life cycle, infectivity, and structure has been generated utilizing recombinant particles. While recombinant particles have proven to be invaluable, allowing for a rapid progression of the HPV field, there are some significant differences between recombinant particles and native virions and very few comparative studies using native virions to confirm results are done. This review serves to address the conflicting data in the HPV field regarding native virions and recombinant particles. Human papillomaviruses are the etiologic agent of cervical cancer and other anogenital and oral cancers ,2,3,4. THPV virions consist of an icosahedral capsid containing histone-associated dsDNA. The viral genome is circular and approximately 8000 bp in length. There is an average of 8 open reading frames (ORFs), which are divided into early and late gene expression classes. HPV replication is tightly linked to the differentiation program of host epithelial cells. Infection occurs in basal epithelial cells via microabrasions. Once infected, early non-structural proteins involved in activities such as genome maintenance and transcription activation are expressed. As the daughter cells divide and become increasingly differentiated in the suprabasal layers, viral genomes are amplified, the late structural proteins are expressed, and new virions assembled ,11,12,13Because the HPV life cycle is tightly linked to the host cell differentiation, the virus has been hard to grow in culture. Therefore, systems to create viral particles bypassing epithelial differentiation were developed. Recent research studying the HPV life cycle including infectivity, transmission, and viral structure predominantly relies on the use of recombinant papillomavirus particles. These recombinant particles such as virus-like particles (VLPs), pseudovirions (PsVs), and quasivirions (QVs) all bypass the need for stratifying and differentiating human epithelium. While data collected using recombinant particles has allowed for a rapid progression of the field of HPV research, there are few comparative studies utilizing native virions produced in organotypic raft culture, xenografts, or native tissue to confirm results in the context of stratifying and differentiating human epithelial tissue.The main focus of this review will be to compare native virions produced in stratifying epithelium to recombinant particles . All aspin vitro system preserves the molecular events that occur during differentiation with the convenience of not having to use animals such as in the xenograft system. In the organotypic system, human keratinocytes that stably maintain HPV viral genomes are grown above a dermal equivalent made of fibroblast cells embedded in a collagen matrix. Keratinocytes grow at the air liquid interface and are fed via diffusion from cell culture media below the dermal equivalent [Organotypic raft culture is the only system outside of the xenograft system that allows for the production of infectious HPV in its natural environment, a differentiating epithelium ,16,17,18uivalent . After ain vivo or in vitro system created a need for the development of the recombinant system  of many papillomaviruses. However, some HPV types, such as HPV16 and HPV18 actually have upstream, in frame, methionines that allow for the production of more than one L1 species. Specifically, HPV16 has one upstream methionine that allows for production of an L1 26 amino acids longer then L1 started at the consensus methionine and HPV18 has two upstream methionines enabling production of L1 proteins that are 61 and 26 amino acids longer then the L1 produced from the consensus methionine. These methionines were removed from the expression plasmids utilized in the recombinant system because only production of L1 from the consensus methionine allowed for efficient expression of L1 and production of particles . A HPV18During the process of developing into fully infectious virions, many viruses undergo structural changes, called maturation ,43. In tAnalysis of both 10-day and 20-day tissue sections show a difference in virion localization with 10-day virions predominantly localized in the nuclei of suprabasal cells and 20-day virions being found mainly in the cornified layers of the epithelium. Human epithelium has a natural redox gradient with the lower part of the tissue being a reducing environment and the upper layers being an oxidizing environment . This naWhile not absolutely essential for particle formation, a series of disulfide bonds in L1 and L2 molecules form both intra- and interpentameric interactions, stabilizing particles and making them more resistant to environmental influences from nucleases and proteases ,47. Withvs. differentiating epithelium.In addition to disulfide bonds in L1, L2 plays an important role in capsid formation and stabilization. L2 forms heterotypic interactions with L1 on the inside of the capsid as well as homotypic interactions with other L2 molecules. When L1 disulfide bonds were unable to form, L1 only capsomeres did not assemble into capsids. However when L2 was present, VLPs were formed . Within One of the remaining unknowns of HPV assembly and structure is the concentration of L2 in the viral capsid. L1 itself is able to self-assemble into capsids, however, including L2 into recombinant particles has been shown to increase yield, capsid stability, DNA encapsidation, and infectivity of particles ,56,57,58Organotypic raft culture allows for the generation of not only single base mutant virions but also for the generation of chimeric HPVs composed of different components of different HPV types in the natural environment of differentiating tissue. This whole gene replacement method has aimed at highlighting areas of conservation among different HPV types via complementation experiments. A chimeric virus, named HPV18/16, was created with the L1 and L2 ORF of HPV16 swapped into HPV18 in place of HPV18 L1 and L2 . SubstitIn addition to L1 and L2, a variety of components comprise the viral capsid including viral DNA and histones. There are other possible viral and cellular components that either directly or indirectly affect the final structure and/or yield of capsids. These components could include HPV early proteins such as E2, E4, E5, and E7 or cellular proteins such as chaperones or karyopherins. There are undoubtedly unknown viral and/or cellular factors necessary for the assembly of native virions. These proteins could act by direction of proper subcellular localization, initial interaction of capsid proteins, mediation of the correct temporal formation of disulfide bonds, and regulation of capsid protein expression.Our current understanding of the PV infection strategy proposes that, via microabrasions, virus particles gain access to the basal cells as well as the basement membrane. Utilizing recombinant particles, most papillomaviruses have been observed to infect cells by first binding to a glycosaminoglycan (GAG), heparan sulfate (HS), via L1 on either the basement membrane or the cell surface ,65,66,67Due to minor structural differences between different virus types, it is possible that initial attachment to and interaction with the ECM and cell surface might be HPV type specific. Though the L1 protein displays overall sequence homology and structural conformation, both structural diversity and conformational differences in the loop structures of the pentamer surface have been demonstrated for HPV types 11, 16, 18 and 35 . It is fThe entry process for HPV is slow, with cell surface events such as interaction with several receptors and conformational changes thought to be responsible ,80. HPV1To date, a multitude of receptors have been identified as potential primary and secondary HPV internalization receptors. One of the first identified was α6-integrin, an epithelial adhesion protein . An α6-iAfter endocytosis, acidification of the endosomal lumen allows for disassembly of the viral capsid and cellular sorting allows for transport to the nucleus where viral replication occurs ,99,100. in vitro rarely utilize primary keratinocytes. This is in part due to the extremely low infection efficiency of recombinant particles in primary cells [In a natural infection, HPV infects keratinocytes. However, experiments ry cells . Conversry cells . HPV18 iOf equal importance when looking at infection is the ability to look at virus neutralization. This is especially important due to the use of VLPs in the HPV vaccine that is now being administered to prevent infection. Almost all of the antibodies generated for HPV have been made using recombinant particles. Many of these antibodies generated, such as H16.V5 and H16.E70, have been shown to neutralize both recombinant particles and native virions ,114,115.There are two approved vaccines against the high-risk HPV types HPV16 and HPV18—Gardasil and Cervarix. Gardasil also provides protection against low-risk HPV types HPV6 and HPV11. These vaccines, however, are not efficiently cross protective against other high-risk HPV types. Therefore, another HPV vaccine, Gardasil 9 was recently approved that, in addition to the four types of HPV previously mentioned also provides protection against high-risk types HPV31, HPV33, HPV45, HP52, and HPV58. While these vaccines have been shown to be highly effective at preventing infection and the development of lesions, their high cost limits their use in developing countries. Additionally, the vaccine rate is low in some developed countries. Thus, the development of less expensive microbicides that would offer protection against a multitude of HPV types would offer women an additional protection against contracting HPV infection.in vitro screen of compounds that may effectively block infection by high-risk HPV types [50 for carrageenan inhibition of various HPV PsV types was in the ng/mL range [Due to the importance of GAGs, specifically HS, in HPV attachment to and infection of host cells, agents targeting GAGs are of great interest. Both high molecular weight sulfated or sulfonated polysaccharides and polymers such as cellulose sulfate, dextran sulfate, and polystyrene sulfonate showed microbicidal activity against BPV1 in mouse cells and HPV11 and HPV40 in human A431 cells without showing cellular toxicity . More rePV types . In thismL range . In contmL range . These rmL range . Taken tPreventing the incidence of HPV infection through the use of vaccinations and microbicides is invaluable. Of equal importance is the use of chemical disinfectants to keep surfaces and medical equipment free of virions that could be spread between patients. Determining the efficacy of chemical disinfectants against HPV is important because it includes real world scenarios of disinfection protocols on equipment in hospital settings that are possibly contaminated with HPV. Currently, what little information that is available on HPV disinfection has been based mainly on studies from surrogate viruses, such as hepatitis B virus (HBV), which were deemed, without experimental evidence, to have similar resistance to HPV. Additionally, the United States Centers for Disease Control and Prevention (CDC) currently acknowledges the lack of research regarding effective HPV disinfectants . DisinfeTaken together, all of the data has provided great insight into the structure of HPV as well as the method of binding to, entering, and infecting cells. The use of recombinant particle technology is invaluable, as it allows for rapid data generation in studies looking at multiple parts of the HPV life cycle. However, as this review points out, there are some significant differences between native virions and recombinant particles that necessitate conformational studies utilizing native virions.


Yunnan has one of the oldest and the most severe human immunodeficiency virus (HIV) epidemics in China. We conducted an observational study to evaluate the human papillomavirus (HPV) genotype distribution in relation to cervical neoplastic disease risk among HIV-infected women in Yunnan.We screened 301 HIV-infected non-pregnant women in Mangshi prefecture in Yunnan province. All consenting participants underwent simultaneous and independent assessment by cervical cytology, colposcopy-histopathology, and HPV genotyping. Unadjusted and multivariable-adjusted multinomial logistic regression analysis was conducted to evaluate factors associated with single or multiple carcinogenic HPV genotypes.p = 0.012) but not with the presence of multiple carcinogenic types in the multivariable-adjusted models.HPV genotypes were present in 43.5% (131/301) overall, and carcinogenic HPV genotypes were present in 37.5% (113/301) women. Among women with carcinogenic HPV genotypes, 80 (70.8% of 113) had a single carcinogenic HPV type, while 33 (29.2%) women had multiple (2 or more) carcinogenic HPV types. Overall, the most common carcinogenic HPV types were HPV52 (7.3%), HPV58 (6.6%), HPV18 (6.3%), HPV16 (6.0%), and HPV33 (5.3%). In women with cervical precancerous lesions , the most commonly detected genotypes were HPV16 (28.6%), HPV52 (25.0%), HPV58 (17.9%), HPV18 (10.7%) and HPV31 (10.7%). Increasing age was an independent risk factor associated with presence of single carcinogenic HPV types (adjusted odds ratio: 1.04, 95%CI: 1.01-1.07, As HIV-infected women continue to live longer on antiretroviral therapy in China, it will be increasingly important to screen for, and prevent, HPV-associated cervical cancer in this population, especially given the wide diversity and multiplicity of HPV genotypes. Invasive cervical cancer (ICC) is a leading cause of cancer related morbidity and mortality afflicting women globally, with over 527,000 annual incident cases and over 265,000 annual deaths . An estiWomen with immunosuppressive conditions, such as human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS), have been shown to have a higher prevalence of cervical HPV infection and higher frequency of multiple HPV genotypes than women from the general population . The advBy the end of 2011, it was estimated that there were 7.8 million persons living with HIV in China . The oveAt enrollment in the study, the mean age of the 301 HIV-infected women was 34 years (standard deviation ±8.9). A large majority (78.1%) were married or cohabiting with their husband. About half (50.2%) had fewer than 4 years of education (primary school or below), and under half of them (44.5%) reported a family income ≤80.5 US Dollars per month. Less than a quarter (24.3%) self-reported ever having a sexually transmitted infection (STI). A majority (62.5%) of participants reported their partners being HIV-infected. One-sixth of the participants (15.9%) reported exposure to smoking , about two-fifths (58.8%) reported condom use with regular partners, more than two-thirds (70.8%) had 2 or fewer lifetime sexual partners, and the mean age of first sexual intercourse was 20 years (standard deviation ±3.2). The mean CD4+ cell count was 571 cells/μL, more than half  were taking ART, and the mean duration of being on ART was 29 months.Cytology results revealed 252/301 (83.7%) women with ‘negative for intraepithelial lesion or malignancy’ (NILM), 33 (11.0%) with atypical squamous cells of undetermined significance (ASC-US), 11 (3.7%) with low-grade squamous intraepithelial cells (LSIL), and 4 (1.3%) with high-grade squamous intraepithelial cells (HSIL). One sample was unsatisfactory for evaluation. Colposcopic-histopathologic diagnosis revealed 251/301 (83.4%) women with no cervical intraepithelial neoplasia (CIN), 22 (7.3%) with CIN1, 16 (5.3%) with CIN2, and 12 (4%) with CIN3 lesions. The composite cytology-colposcopic-histopathological diagnosis (n = 301) showed no CIN  in 251/301 (83.4%) women, CIN1  in 22/301 (7.3%), CIN2  in 16/301 (5.3%), and CIN3 (on colposcopy-histology) in 12/301 (4.0%) women. Thus, CIN grade 2 or worse  lesions were present in 28/301 (9.3%) women.Over two-fifths  of the HIV-infected women in the study had presence of at least one HPV genotype. A total of 21 out of the 23 HPV genotypes identifiable by the HPV genotyping kit were detected among the 131 participants in the study with HPV, including all 13 carcinogenic HPV genotypes   had at least one carcinogenic HPV type. Among these, 80 women (70.8%) had a single carcinogenic HPV type infection, while 33 (29.2%) women had more than 2 carcinogenic HPV infections. Only 15/301 (5.0%) women were reported to be infected with ‘possibly-carcinogenic’ HPV types, and 34 (11.3%) women were detected with non-carcinogenic types or types of unknown carcinogenicity.p-for trend <0.001). The most common carcinogenic HPV genotypes in individual cervical disease categories were HPV18 (5.6%) in women with no CIN, HPV35 (13.6%) in CIN1, and HPV16 (28.6%) in CIN2+. The trend for increasing prevalence of HPV genotypes with increasing severity of cervical disease grades was significant for carcinogenic HPV genotype (p < 0.001), any single or multiple carcinogenic HPV (both for p < 0.001), ‘possibly carcinogenic’ HPV (p = .035), HPV16 (p < 0.001), as well as non-HPV16 carcinogenic HPV (p < 0.001) .The age distribution of HPV infection prevalence in the study population is depicted in Figure p < 0.05) only for the detections of carcinogenic HPV and multiple carcinogenic HPV genotypes.The relative proportion of HPV infection by CD4+ cell count categories, after adjusting for ART status, is graphically displayed in Figure p < 0.05) lower HPV detections associated with categories of longer duration on ART were observed for the groupings of carcinogenic HPV and multiple carcinogenic HPV genotypes.Figure p = 0.012) and having a cervical lesion (≥CIN1)  while presence of multiple carcinogenic HPV infections were independently associated with having a cervical lesion (≥CIN1) .Table Persistent infection with carcinogenic HPV is a necessary, though not sufficient, cause for invasive cervical cancer. HIV-infected women are more susceptible to HPV than HIV-uninfected women. Within China, the HIV/AIDS epidemic is diverse, with some of the highest rates in southwest China, especially in Yunnan province .The current study is the first study from China to provide a comprehensive assessment of the distribution of HPV genotypes among HIV-infected women in Yunnan using rigorously-ascertained cervical disease diagnoses. To avoid unnecessary invasive diagnosis procedures and increase the accuracy of CIN diagnosis than relying on any one approach (cytology or colposcopy-histology) alone, our study combined both cytology results and colposcopy-histologic diagnosis to compute composite disease endpoints. Thus we maximized the detection of cervical lesions, limited any selection bias, and increased the representativeness of our findings. The results from our cohort are therefore representative of HIV-infected women who seek cervical cancer screening services in Yunnan and our findings could add to the national and global data of HPV distribution among HIV-infected populations.Overall, at least one HPV genotype was detected in over two-fifths of women and at least one carcinogenic HPV genotype was present in over one-third of HIV-infected women. The prevalence of any HPV infection (43.5%) was almost three times higher than the corresponding rate among the general (HIV-uninfected) population in China , and theThe prevalence of HPV infection has ranged widely in studies among HIV-infected women in Asia ,8,13-18.p-for trend = 0.029) and shorter duration of ART (p-for trend = 0.004), consistent with the hypothesized loss of immune control over non-HPV16 types. However, HPV16 was still the most commonly detected carcinogenic type among women with cervical disease. Also, apart from non-HPV16 carcinogenic HPV types, we found significant differences regarding ART duration and CD4+ counts in any HPV and any carcinogenic HPV . Our results point to the need for further exploration of differences in immune control between HPV16 and non-HPV16 carcinogenic genotypes, and evaluating these associations with cervical cancer risk. In addition, it may be important to study the effect of early ART initiation on reduction in risk of carcinogenic HPV-induced neoplastic lesions, and if it differs by HPV genotypes involved. It will also be important to study the etiologic significance of concurrently present non/unknown-risk genotypes, some of which such as HPV43 had higher prevalence than even other carcinogenic HPV genotypes.Consistent with meta-analysis of HPV types distribution among HIV-infected women , HPV16 wWe noted that 29.2% of carcinogenic HPV positive women were infected with multiple carcinogenic HPV genotypes. This is similar to the rate of multiple HPV infections (32.2%) among carcinogenic HPV positive women in the general population when using GP5+/GP6+ polymerase chain reaction (PCR) assay as the HPV detection method . The proIt was interesting to note that age was a significant factor only with the presence of single carcinogenic infection but not multiple carcinogenic infections in the multivariable-adjusted analysis. While this lack of statistical association of age and multiple carcinogenic infections is intriguing, it likely reflects the underpowered nature of the study for evaluating this outcome, since we only had 33 cases of multiple carcinogenic infections overall and only 8 among women with CIN2+ disease. Indeed, we did not find any significant differences in multiple carcinogenic HPV infections relating to lower CD4+ cell counts, sexual behaviors, or ART therapy. .We noted that there was no clear peak of carcinogenic HPV prevalence in young (<30 years) women, unlike age-specific HPV prevalence surveys in China and globally ,24. TherThe currently available prophylactic HPV vaccines offer protection against HPV16 and HPV18 and some cross-protection to a few associated genotypes, and the future nonavalent vaccine could extend the protection against HPV 31/33/45/52/58. Although the vaccines have been shown to be safe and immunogenic, there are no studies yet on the efficacy of these vaccines among HIV-infected women . Hence, The strengths of our study included the choice of combined cytological and colposcopy-histological diagnosis, to compute the final disease categorization for women. This allowed for better disease ascertainment than relying on the results of cytology or colposcopy-histology alone. Our study also had several limitations. Firstly, subtle immunosuppression could not be measured by the enumeration of circulating CD4+ cells. Secondly, this study was cross-sectional, so results only could show an association but no evidence of causality. Thirdly, some behavioral factors such as drug use and commercial sex work were not captured in the questionnaire of our study, both of which are known to be significant risk factors for HIV-infection for many women in China, particularly in Yunnan. Finally, we note the absence of data on plasma HIV RNA data, nadir CD4 count, and duration of HIV infection while analyzing risk factors as a limitation of the study.In summary, in this cross-sectional study, we evaluated the diverse distribution of HPV genotypes and factors associated with HPV infection among HIV-infected women in Yunnan province. These results are similar to those reported among HIV-infected women in countries with similar levels of the HIV epidemic. The diversity of HPV genotypes in this population emphasizes the need for broad spectrum  HPV vaccines for eventual primary prevention, as well as the immediate necessity of regular and vigilant screening for cervical disease in this population.Four Frees and One Care” program that provided ART for HIV-infected women, was responsible for recruitment and retention of participants. For our study, we selected a cohort of HIV-infected women based on the following eligibility criteria: (i) 18 years of age or older (for informed consent); (ii) not currently pregnant; (iii) no history of prior treatment for cancer of the cervix; (iv) no history of hysterectomy; (v) no prior history of cervical cancer screening; and (vi) mentally and physically fit for undergo a pelvic examination. Participants were recruited regardless of CD4+ cell counts or current status of receiving ART. The study protocol was approved by the Institutional Review Boards at Cancer Institute and hospital, Chinese Academy of Medical Sciences (CICAMS) and Vanderbilt University. All participants gave written, informed consent.The study was conducted in a HIV-clinic in the Mangshi prefecture of Yunnan province, where HIV-infected women have been provided with free cervical cancer screening services since March 2009. Study subjects were recruited based on linkages with HIV voluntary counseling and testing (VCT) centers and referrals of known HIV-infected women from the provincial and prefecture-level Center for Disease Control (CDC)-affiliated ART clinics. The program staff, in close linkages with the CDC staff and the federally-funded “After detailed explanation of study procedures, written informed consent was obtained. A structured questionnaire was used to collect socio-demographic information and key bio-behavioral risk factors relevant to HIV/AIDS or cervical cancer. All enrolled participants underwent a complete external physical examination and venous blood collection for CD4+ T-lymphocyte count. A pelvic examination was performed with sample collection by Ayre’s spatula and endocervical cytobrush . All participants underwent visual screening and diagnostic colposcopy by trained gynecologist. Colposcopically-directed cervical biopsy, endocervical curettage (ECC), and loop electrosurgical excision procedures (LEEP) were advised and performed on consenting participants with clinical evidence of cervical abnormalities.Biopsy specimens were processed and interpreted by consulting pathologists affiliated with the Yunnan Cancer Hospital. Colposcopy and histology results were reported as per the Richart cervical intraepithelial neoplasia [CIN] grading system  and cerv3 viral copies/μL. According to Bouvard classification [We performed HPV genotyping on cervical specimens using polymerase chain reaction (PCR)-based amplification and commercial DNA-chip technology kit (Yaneng Bioscience (Shenzhen) Co. Ltd, P20120401), an SFDA-approved assay that has been extensively used in the past . The kitThe statistical analysis was conducted using IBM SPSS 17.0. The distribution of HPV infection and genotypes were presented as proportion. Chi-square tests were used for assessing the relationship between the distribution of HPV genotypes and different grades of cervical disease. Unadjusted and multivariable-adjusted multinomial logistic regression were used to assess the effect of socio-demographic, sexual, reproductive and HIV-infection related characteristics on the presence of single and multiple types of carcinogenic HPV, and adjusted odds ratios (AOR) and 95% confidence intervals (95%CI) of factors independently associated with single and multiple carcinogenic HPV infection among HIV-infected women were calculated. A two-tailed p-value of <0.05 was considered significant.The study protocol was approved by the institutional ethics committees of the collaborating institutions and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All study participants gave written, informed consent.


The human papillomavirus (HPV) vaccines have been widely introduced in immunization programs worldwide, however, it is not accepted in mainland China. We aimed to investigate the awareness and knowledge about HPV vaccines and explore the acceptability of vaccination among the Chinese population.A meta-analysis was conducted across two English  and three Chinese  electronic databases in order to identify HPV vaccination studies conducted in mainland China. We conducted and reported the analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.I2 = 98.9 %) and 17.55 % , respectively. The female population  and mixed population  exhibited higher HPV vaccine awareness than the male population . Populations of mixed ethnicity had lower HPV vaccine awareness  than the Han population . Among different regions, the HPV vaccine awareness was higher in EDA  and CLDA  than in WUDA . Furthermore, 67.25 %  of participants were willing to be vaccinated, while this number was lower for their daughters . The general adult population  was more willing to vaccinate their daughters than the parent population . Safety  was the main concern about vaccination among the adult population whereas the safety and efficacy  were the main concerns for unwillingness to vaccinate their daughters.Fifty-eight unique studies representing 19 provinces and municipalities in mainland China were assessed. The pooled awareness and knowledge rates about HPV vaccination were 15.95 %  contains supplementary material, which is available to authorized users. Cervical cancer, one of the most common cancers observed in females , affectsVaccines have always been among the most effective interventions for infectious diseases . ProphylThe HPV vaccine has been widely introduced in the vaccination programs of Hong Kong, however, is not popularly accepted in Mainland China at present. In addition, despite the numerous published studies focusing on the topic of HPV and vaccination in recent years, there is no comprehensive information concerning the acceptance and obstacles associated with vaccination among the population of Mainland China. In order to develop a practical vaccination program in the future, it is imperative to assess the level of awareness and knowledge about HPV, and the general attitude towards HPV vaccination among the Chinese population, as they are important behavioral determinants that will ultimately affect the acceptance of vaccination among the Chinese population. Therefore, we conducted a meta-analysis in order to gain a better understanding of this issue that may help generate new ideas to make future generalization of HPV vaccination possible in China.The meta-analysis was conducted in compliance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The ChiThe inclusion criteria for the epidemiological studies were the following: (1)study involved at least one of the key terms “HPV vaccine awareness”, “knowledge”, and “acceptability”for any region of Mainland China , (2) original data was available regardless of whether it was obtained directly from the article or traced from secondary data in the article. Studies that examining the effects of health educational interventions were excluded.A data abstraction form was constructed after scanning the selected articles. For each included study, we extracted the following information: author, publication year, region, study instrument, study subject , sampling method, sample size (N), the number of participants for the assessment of HPV vaccine awareness, knowledge, and acceptance, or the rate percentage proportions for these studied factors. We also extracted the reasons for unwillingness to be vaccinated if this information was available. The number of studied cases(n) and sample size(N) were the two necessary parameters for the calculation of the pooled rates of HPV vaccine awareness, knowledge, and acceptance of vaccination in the meta-analysis. In particular, the number of studied cases (n) was obtained directly from the original studies or by multiplying the sample sizes (N) with the proportions (%) associated with the investigated factors reported in the original studies.We employed a flexible appraisal scale suggested by Iain Crombie  for the We used “rate” to evaluate the studied items. The rate for HPV vaccine awareness was calculated by dividing the number of cases who were aware of HPV vaccine (n1) by the sample size (N); the rate for HPV vaccine knowledge was calculated by dividing the number of cases who knew the relationship between HPV (vaccine) and cervical cancer (n2) by the sample size (N); the rate for acceptance to be vaccinated was calculated by dividing the number of cases who were willing to get vaccinated (n3) by the sample size (N); the rate for acceptance of parents to vaccinate their daughters was calculated by dividing the number of cases who were willing to vaccinate their daughters (n4) by the sample size(N); the rate for reasons of unwillingness to be vaccinated was calculated by dividing the number of cases who gave a reason (n5) by the number of cases who were unwilling to be vaccinated (N-n3).P < 0.10, and using the I-square (I2) statistic to quantify the heterogeneity of the results. Publication bias was detected by Egger’s test  [Meta-analysis was conducted using a random effects model. Given the requirement for normalization of single rate in meta-analysis, an arcsine transformation for the original rate was performed to meet the requirement . StatistAs this study was a meta-analysis, we did not include any humans and animals. This study was approved by the Ethics Committee of Huazhong University of Science and Technology.Our search returned 1683 articles. A flow diagram of the selection process is shown in Fig. We included 58 individual studies –68 repreI2 = 98.9 %), and 17.55 % , respectively  while their willingness to get their daughters vaccinated was 60.32 %   respondents believed that they had a low risk of developing HPV infection, genital warts, or even cervical cancer. Other respondents were worried about the limited use of HPV vaccine in China . Respondents who were concerned with the safety and the efficacy of HPV vaccination accounted for 50.46 %  and 30.18 % , respectively. Participants who questioned the source of the vaccine and communicated a concern regarding the high price of the vaccine were 32.17 %  and 23.72 % , respectively  respondents were concerned regarding the limited use of HPV vaccine in China to date. Some respondents  doubted the safety and efficacy of the HPV vaccine. Only 17.24 %  of the respondents doubted the vaccine source. In addition, 28.37 %  of the respondents considered their children to be too young for vaccination , ethnicities (P = 0.017), and regions (P = 0.031). We observed a higher HPV vaccine awareness among the female population  and mixed population  relative to the male population . We also found that populations of mixed ethnicity have lower HPV vaccine awareness  compared to population of Han . Among different regions, the HPV vaccine awareness was higher in EDA  and CLDA  compared to WUDA . Subgroup analysis revealed that acceptability to be vaccinated varied among studies conducted using different sampling methods (P = 0.022). The acceptability of vaccination among cluster-sampled population  was higher compared to the convenience-sampled population . The subgroup analysis showed that acceptability for parents to vaccinate their daughters differed across ages (P = 0.014) and sampling methods (P = 0.038). General adult population  was more willing to vaccinate their daughters than parent population . Randomized sampling method showed a higher acceptability for vaccination of daughters  compared to cluster sampling method    Table .This is the first meta-analysis study conducted for the assessment of HPV vaccine related awareness, knowledge and acceptability among the Chinese population. Our meta-analysis identified low awareness (15.95 %) and low knowledge (17.55 %) of HPV vaccine among the Chinese population. The rates were lower compared to many other countries. Studies conducted in Turkey showed that HPV vaccine awareness among undergraduate students in Turkey was 44.5 % , while 2In addition, we found a relative high acceptability of HPV vaccination (67.25 % for themselves and 60.32 % for daughters). However, this rate declines in the high-end of the level across the world (59 % to 100 %) –79. In sThe primary obstacles concerning vaccination acceptability for responders were the safety and efficacy of the HPV vaccine. HPV vaccines have been proved safe and efficient against HPV infection , 83. WHOSufficient scientific evidence has clarified many of the misunderstandings related to vaccine safety, however, the concerns related to vaccination are still increasing . Public Mainland China has not introduced HPV vaccination into the routine immune vaccination program, experiences from other countries that implemented HPV vaccination program can be taken as an example. In many counties, an organized vaccination program is recommended to increase the vaccination coverage. It is now widely believed that the most urgent public-health issue is to increase HPV vaccination coverage and improve completion of the vaccination schedule, especially among sexually active females . Thus, mFurther research to perfect the existing HPV vaccines is needed. Moreover, as a measure of primary prevention, HPV vaccination should be performed alongside cervical screening (secondary prevention) as a clear strategy for the prevention of cervical cancer.The strength of our analysis is that the evaluation of the recently published papers about HPV vaccination among the Chinese population allowed us to offer evidence-based advice for the implementation of HPV vaccination in Mainland China in future. However, there were some limitations in this study. Obvious heterogeneity existed in the meta-analysis. We tried to perform meta-regression analysis to explain the source of heterogeneity, however, significant heterogeneity remained unexplained after an exploration of the relative factors, such as sampling method and population characteristics. In fact, for observational studies that involve proportions, substantial heterogeneity is a common dilemma . AlthougIn conclusion, this meta-analysis proved low HPV vaccine awareness and knowledge among the Chinese population. HPV vaccine awareness differed across sexes, ethnicities, and regions. However, given the limited quality and number of included studies, future studies with improved design are necessary for the verification of our findings.


Tongue squamous cell carcinoma (TSCC) is most aggressive head and neck cancer often associated with HR-HPV infection. The role of AP-1 which is an essential regulator of HPV oncogene expression and tumorigenesis is not reported in tongue cancer. One hundred tongue tissue biopsies comprising precancer, cancer and adjacent controls including two tongue cancer cell lines were employed to study the role of HPV infection and AP-1 family proteins. An exclusive prevalence (28%) of HR-HPV type 16 was observed mainly in well differentiated tongue carcinomas (78.5%). A higher expression and DNA binding activity of AP-1 was observed in tongue tumors and cancer cell lines with c-Fos and Fra-2 as the major binding partners forming the functional AP-1 complex but c-Jun participated only in HPV negative and poorly differentiated carcinoma. Knocking down of Fra-2 responsible for aggressive tongue tumorigenesis led to significant reduction in c-Fos, c-Jun, MMP-9 and HPVE6/E7 expression but Fra-1 and p53 were upregulated. The binding and expression of c-Fos/Fra-2 increased as a function of severity of tongue lesions, yet selective participation of c-Jun appears to promote poor differentiation and aggressive tumorigenesis only in HPV negative cases while HPV infection leads to well differentiation and better prognosis preferably in nonsmokers. Head and neck squamous cell carcinoma (HNSCC) is an extremely heterogeneous group of cancers arising from different subsites such as tongue, lips, larynx and other intra-oral locations13Among HNSCCs, tongue squamous cell carcinoma (TSCC) is one of the most common but highly aggressive cancer particularly in younger patients and is associated with a higher rate of metastasis with poor prognosis566767910111213It is well established that transcription factor activator protein-1 (AP-1) formed by homo or hetero-dimerization between Jun  and Fos  family proteins161418192021141920Several studies have demonstrated differential expression and high DNA binding activity of specific members of AP-1, particularly c-Fos, c-Jun and JunB during development of variety of carcinomas including oral carcinoma18192020221924181920A total of one hundred prospectively collected fresh tongue tissue biopsy specimens comprising precancer (n = 20), cancer (n = 50) and adjacent normal controls (n = 30) and two tongue cancer cell lines (UPCI:SCC090 and AW13516) were employed for the detection of HPV infection, HPV genotypes and analysis of expression and activation of AP-1 family proteins. The Clinico-epidemiological and demographical details along with status of HPV infection are presented in +ve cases out of 50 TSCCs studied and all of them were positive exclusively for HR-HPV type 16 and no other known HPV type could be detected (+ve TSCCs (n = 14), majority  of them were females. The majority  of HPV infection was found in well differentiated tumors (WDSCCs) in comparison to MDSCCs and PDSCCs  assay demonstrated only 14 (28%) HPVdetected . All predetected . Of twenSCCs see  and belo using γ32P-labeled probe harboring AP-1 consensus sequence. As presented in The DNA binding activity of AP-1 was analyzed by band shift assay in nuclear extracts derived from fresh tongue tissue biopsies of precancer (n = 20), cancer (n = 50) and normal adjacent control tissues (n = 30) as well as in two tongue cancer cell lines   . In contnd Fra-2 . In almond Fra-2 . HPV+ve of Fra-2  and no ive cells .−ve tongue cancer cases as compared to precancer and normal controls  in all spectrum of tongue tissue biopsies from normal, precancer to cancer including TSCC cell lines. AP-1 proteins, c-Jun , c-Fos  and Fra-2  showed significantly a higher level of expression specifically in majority of HPVcontrols , Table 3controls . Also, Jcontrols . InteresV status . OverallV status .−ve cells (figures not provided).To compare AP-1 family protein expression, the gene transcripts were evaluated in total RNA isolated from tongue tissue biopsies from cancer, precancer and normal controls and tongue cancer cell lines by RT-PCR. As shown in +ve and HPV−ve tumors. As depicted in the −ve tongue tumors. These target genes were also found to be upregulated in both the tumor cell lines but they are more pronounced in HPV−ve cells   was obseve cells .−ve aggressive tongue cancer tissues and cell lines, we examined the extent of functional contribution of Fra-2 in aggressiveness of the disease by silencing Fra-2 using Fra-2 specific siRNA in both HPV +ve and HPV−ve tongue cancer cell lines. As presented in the Since we observed significantly a higher expression and transactivation of Fra-2 particularly in HPV+ve cells  invasion after treatment with Fra-2 siRNA when compared to untreated  or scrambled siRNA treated cells   invasion after siRNA treatment as compared to untreated (±95.6 cells) or scrambled treated groups  (−ve) cells for 18 hours showed an ~63% migration rate while untreated or scrambled treated control cells showed ~100% migration  . In HPV1ls; 91%) . The untigration . Interesectively . These rHuman oral tongue cancer is a most aggressive disease and generally associated with a very high invasive and metastatic potential. Transcription factor, AP-1 plays a critical role in regulating wide variety of cellular processes including cell differentiation, apoptosis, transformation and signal transduction pathways, specifically during progression and metastasis of several cancers including oral cancer151920Since high-risk HPV type 16 is a major oncogenic virus often associated with tongue cancer and its oncogenic expression is again controlled by AP-1 and its family proteins, we have molecularly dissected the role of AP-1 and its family proteins and HPV during tongue tumerigenesis. We have also examined the role of AP-1 downstream target genes Bcl-2, MMP-9 and Cyclin D1 in tongue cancer.+ve cases belonged to well differentiated tumors (78.6%) which are expected to show better prognosis when treated. This was demonstrated first in oral cancer9293031The prevalence of HPV infection in TSCCs has been reported to vary between 0 to as high as 82%, depending on the geographic regions, tumor sites and techniques applied for detection of the virus6726277+ve and 75% (6/8) of them were nonsmokers. Similarly, out of 40 male TSCC patients, all were smokers but only 15% (6/40) of them were HPV+ve. It suggests that female patients who are generally non-smokers or mild smokers in India and are more likely to contract HPV infection leading to well differentiated tumors which would show better prognosis while males, in contrast, are often heavy smokers and are likely to have HPV−ve poorly differentiated tumors with worst prognosis. It gains support from recent studies34When we correlated HPV positivity with the tumor location, majority (85.7%) of HPV infection was found to be located in the base of the tongue and are most aggressive than those located in the mobile tongue or other sites of the tongue (14.3%). Our data are in good agreement with earlier studies which showed HPV16 as the most predominant type specifically found at the base of the tongue628+ve and HPV−ve tongue carcinoma. In sharp contrast to almost all other carcinomas, preferential heterodimerization between c-Fos and Fra-2, both of which are known to have highly oncogenic potential24A strong DNA binding activity of AP-1 observed in tongue cancer cases as compared to precancer or normal adjacent controls corroborate several earlier studies in variety of other carcinomas including oral and esophageal carcinomas15192022362238−ve tongue cancer cases which are shown to be highly aggressive and have worst prognosis, there is a selective participation of c-Jun along with c-Fos/Fra-2 heterodimer mainly in poorly differentiated tumors (PDSCCs/MDSCCs). Furthermore, Fra-2 silencing also downregulates c-Jun. It is indicative of a crosstalk between c-Jun and Fra-2/c-Fos that makes this subtype of tongue cancer highly aggressive. It gains support from recent report of Mariani et al. (2007) who demonstrated amplification and overexpression of c-Jun can block adipocytic differentiation leading to undifferentiated highly aggressive tumorset al. (2013) also showed that changes in BRCA1 gene expression is regulated by c-Jun and Fra-2 that bind to the CRE element in early stages of ovarian carcinoma that suppress BRCA1 expression in sporadic tumors and promote tumor progressionInterestingly, in majority of HPV+ve cases appears to induce tumor differentiation as majority of the HPV+ve cases were found to have well differentiated tumors (WDSCCs) that show better prognosis. It gains support from the study by Natio et al. (2005) who showed that JunD protein plays an important role in bone formation by stimulating differentiation in osteoblastsOn the other hand, participation of JunD observed specifically in HPV16o et al. 05 who sho et al. 05 who sh+ve and HPV−ve tongue cancer cell lines also provided similar results except that it showed preferential participation of JunB instead of c-Jun observed in HPV negative tongue cancer tissues. Ohyama et al. (2008) reported that overexpression of JunB along with c-Fos can enhance 4Nitroquinoline 1-oxide-induced tongue carcinogenesis in rat indicating an important role of JunB/c-Fos crosstalk in tongue cancer+ve cells indicates that JunD facilitates tumor differentiation leading to better prognosis.Both HPVet al. unpublished observation). This gains strong support from our present findings that knocking down of Fra-2 (responsible for aggressive tumorigenesis and drug resistance) led to upregulation of p53 , cancer (n = 50) and adjacent normal controls (n = 30) were collected from the Department of ENT Surgery of Dr. Ram Manohar Lohia Hospital, New Delhi. None of these patients received any pre-operative radiation and chemotherapy. The clinico-pathological and epidemiological details of the patients were collected using standard proforma. Biopsy samples obtained immediately after surgery in sterile vials containing cold phosphate buffer saline (PBS), transported to lab on ice and stored in −80 °C deep freezer till further analysis.The study was approved by the Institutional Ethics Committee of Dr. B. R. Ambedker Center for Biomedical Research (ACBR), University of Delhi, Delhi (India) and Dr. Ram Manohar Lohia Hospital, New Delhi. Written informed consent was obtained from all the subjects prior to their inclusion in the study. The study was carried out in accordance with the guidelines and principles of the Helsinki Declaration.2.The two tongue cancer cell lines; one HPV16 positive, UPCI:SCC090  was derived from the base of tongue squamous cell carcinoma2, 125 μM of each dNTP , 5 pmol of each oligonucleotide primer and 0.5 U Taq DNA polymerase . β-globin gene was used as an internal control. HPV positive samples were subjected to comprehensive HPV genotyping assay using type-specific PCR and PGMY-reverse line blot assay (RLB) as per recommended protocol provided under the WHO HPV LabNet program+ve cell lines have previously been characterized, we reconfirmed the HPV status in these cell lines by PCR.High molecular-weight genomic DNA was isolated from precancer, cancer and adjacent normal tissues as controls and two tongue cancer cell lines by standard procedure of proteinase K digestion, phenol-chloroform extraction and ethanol precipitation as followed in the lab. The initial HPV diagnosis was performed by using a pair of consensus degenerate primers (MY09 and MY11) derived from the highly conserved L1 region of HPV genome . PCR was32P] ATP  by T4 polynucleotide kinase and gel purified in a 15% polyacrylamide gel32P] ATP 5′-end labelled double-stranded AP-1 oligonucleotide probe was added and the incubation was continued for additional 25 min at room temperature and the DNA-protein complex was resolved in a 4.5% non-denaturing polyacrylamide gel. The gel was then dried and visualized by Phosphorimager (Fujifilm FLA-5100) using Multi Gauge-ver 3.X anlaysis software. For monitoring composition of AP-1 and Oct-1, following antibodies from Santa Cruz Biotechnology  were used: c-Jun (sc-45), JunB (sc-73), JunD (sc-74), c-Fos (sc-253), FosB (sc-48), Fra-1 (sc-605), Fra-2 (sc-171). The quantitative densitometric analysis was performed using Alpha Ease FC version 4.1 .Nuclear extracts from all spectrum of tongue tissue biopsies and cell lines (UPCI:SCC90 and AW13516) were prepared by the method of DignamProtein extracted from tumor tissues and cell lines (40 μg/lane) were separated on 8–12% SDS-PAGE, electrotransferred to PVDF membrane and immunodetected using respective antibodies as described earlierTotal RNA was isolated from fresh tongue biopsies comprising pre-cancer, cancer and normal adjacent control tissues and two tongue cancer cell lines using TRI reagent as per manufacturer’s protocol . The quality and integrity of RNA was checked spectrophotometrically in a Nanodrop  and on ethidium bromide-stained 1% agarose gel. For reverse transcription-PCR (RT-PCR) analysis, the isolated RNA (2 μg) was employed to prepare cDNA using first strand cDNA synthesis kit . RT-PCR reactions were carried out in duplicates using AP-1 specific primers. The primers used for RT-PCR are listed in 5 cells/well (AW13516 & UPCI:SCC090) and 2 ml antibiotics-free normal growth medium supplemented with 10% FBS was added and incubated at 37 °C in a CO2 incubator for 24–48 hours until the cells were 40–60% confluent followed by microscopic examination and protein expression analysis by immunoblotting.For Fra-2-siRNA interference assay, transient transfection of commercially available Fra-2 siRNA oligonucleotides  was performed according to the manufacturer’s protocol with some minor modifications5 cells were plated in upper chamber of the transwell with a matrigel-coated membrane . Medium with serum was added to the lower chamber as a chemoattractant. After incubation for 24 hours, cells on the lower surface of the membrane were fixed with formaldehyde and stained with Giemsa. Non-migratated cells were mechanically removed by a cotton swab. The images of invaded cells were acquired by an inverted microscope with a magnification of 20X. The number of invaded cells was quantified by counting cells in five randomly selected fields under microscope. Comparisons were made between the untreated or scramble siRNA treated and Fra-2 knockdown wells. All experiments were performed in triplicate.The effect of Fra-2 silencing on invasive characteristics of tongue cancer cells was evaluated using Matrigel invasion assay. After 24 hours of Fra-2 siRNA or scramble siRNA transfection, TSCC cells were trypsinized and resuspended in FBS-free medium. A total of 1 × 10+ve and HPV−ve TSCC cells were transfected with 80 nM Fra-2 siRNA and scramble siRNA as control as described above and incubated under standard conditions to achieve knockdown of Fra-2. After 24 hours, a scratch was carefully made by scraping through each well using a sterile pipette tip. The cells were then washed with PBS to remove detached cells. Scratches were monitored with an inverted microscope immediately after wounding and after incubation in an incubator  at different time points. Images were taken exactly at the same position before and after the incubation and migration rate was calculated by using image J software.To investigate the migratory capacity of TSCC cells, cell scratch assay was performed. Both HPVThe data analysis was performed using the statistical software Graph Pad Prism (version 6.0) and image J software. The association between HPV infection and expression profile of AP-1 proteins among different histopathological grades and clinico-pathological parameters in TSCC cases was determined using Fischer’s exact test and students t-test (two tailed). The p value ≤ 0.05 was considered as statistically significant.How to cite this article: Gupta, S. et al. Selective participation of c-Jun with Fra-2/c-Fos promotes aggressive tumor phenotypes and poor prognosis in tongue cancer. Sci. Rep.5, 16811; doi: 10.1038/srep16811 (2015).


Behavioral risks such as age at first sexual intercourse, number of sexual partners and partner’s sexual behavior are associated with an increased risk of HPV infection, persistence of the infection and the development of neoplastic precursor lesions. The objective of this study was to evaluate the risk factors associated with HPV positivity and with a diagnosis of cervical neoplasia in women referred with an abnormal cervical smear.This study evaluated a series of 198 women referred with an abnormal cervical smear. Risk factors for HPV infection were investigated using a questionnaire. All cervical specimens were tested for 27 HPV genotypes using the Roche polymerase chain reaction reverse line blot assay.p = 0.01). A significant association was also found between this risk factor and CIN 1 lesions or worse .The overall prevalence of HPV was 87 %. First sexual intercourse before 16 years of age was significantly associated with a positive HPV test (OR 4.41; 95 %CI: 1.20 − 19.33; The age at which a woman begins to be sexually active is associated with HPV infection and with a diagnosis of cervical neoplasia. Human papillomavirus (HPV) infection of the genital tract is thought to be the most common sexually transmitted virus. The prevalence of this infection is age-dependent and is higher in women between 15 and 25 years of age. The decrease in the rate of HPV infection with increasing age likely results from some combination of decreased HPV exposure, the self-limiting nature of most infections and a resistance to re-infection [Persistent infection with human papillomavirus (HPV) is a prerequisite for cervical cancer and its precursor lesions , 4. The Behavioral risks such as age at first sexual intercourse, the number of sexual partners and the partner’s sexual behavior are associated with an increased risk of HPV infection , 9. StudCo-factors that would act by affecting the way in which HPV is acquired, its persistence and development, as well as its progression to neoplastic lesions, are common . In addiTherefore, the objective of this study was to evaluate the risk factors associated with HPV infection and with neoplastic diagnoses in women referred with an abnormal cervical smear.This study was conducted at a colposcopy referral clinic in Goiânia, central Brazil. Between January 2006 and March 2009, a series of 198 women with an abnormal cervical smear were included. The study protocol was approved by the Internal Review Board of the Pontifical Catholic University of Goiás with the registration CEP 003/05.All the participants answered a complete standardized questionnaire concerning age, smoking, age at first sexual intercourse, number of lifetime sexual partners, oral contraceptive use and number of pregnancies. Exclusion criteria consisted of pregnancy and clinical signs of immunosuppression. The study protocol was approved by the institute’s internal review board and all patients signed an informed consent form.At colposcopy, a cervical sample was taken for a second conventional smear using a cervical brush, and the residual material was rinsed with and then stored in 1.0 mL Universal Collection Medium  for HPV DNA testing. Biopsies were taken from any colposcopically abnormal area. Women with a suspicious image penetrating the cervical canal and those in whom colposcopy was unsatisfactory and a second cervical smear was abnormal were submitted to cervical conization. Women with invasive carcinomas were treated in compliance with the appropriate clinical guidelines. When a woman was submitted to more than one histological examination, the most severe diagnosis was the one taken into consideration.During the study period, 193 biopsies were performed and analyzed according to the criteria defined by the World Health Organization, being classified as normal/cervicitis, CIN 1, CIN 2, CIN 3, invasive squamous cell carcinoma (SCC) or invasive adenocarcinoma. The remaining 5 women in the study tested negative at colposcopy and negative in their repeat cervical smear; therefore, the final diagnosis in these cases was negative for neoplasia. For the purposes of analysis, they were classified together with the women with normal results at histology or those with a finding of cervicitis. These 5 women were scheduled to return for follow-up visits every 3 months, and at the cut-off date for this analysis the duration of follow up in these cases ranged from 3 months to 2 years.The samples were processed as previously described . HPV ampAccording to their epidemiological association with cervical cancer and consolidated by biological studies, 12 types of HPV  have now been consistently classified as hrHPV . HPV-68 has been classified as probable high-risk , and another seven types have been classified as possible high-risk  , 15.The specimens were reviewed in accordance with the World Health Organization criteria   and werep) was set at 0.05.A database including demographic and behavioral factors was analyzed using the SPSS statistical software package . Crude and adjusted odds ratios (OR) for trends with 95 % confidence intervals (95 % CI) were calculated and the chi-square test was used whenever appropriate. The significance level for the tests  had their first sexual intercourse prior to 16 years of age. Forty of the women (20.2 %) had had a single sexual partner during their lifetime, while 158 (79.8 %) reported having had two or more partners over their lifetime. Most women  reported having been pregnant at least once. Oral contraceptive use was reported by 49 women (24.7 %). Forty-two women (21.2 %) reported smoking. Of the women referred with abnormal cervical smears, 87 % (171/198) had an HPV infection, including 42.4 % of women infected with HPV types 16 and 18 and 43.9 % of women infected with other HPV types.The distribution of HPV genotypes and histological diagnosis is shown in Table A significant association was found among women who had their first sexual intercourse prior to 16 years of age and HPV infection . In addition, a positive association was found between a woman having initiated her sexual life prior to 16 years of age and infection by HPV types 16 and 18 . The same analysis was performed with respect to these risk factors and other types of HPV; however, no such association was found. Furthermore, no association was found between any of the other risk factors  and HPV infection  1 or worse and women who had had their first sexual intercourse prior to 16 years of age . CIN 1 or worse was also significantly associated with HPV infection  and with HPV 16 and 18 infections . However, no association was found with any of the other HPV types  (Table Age at first sexual intercourse prior to 16 years of age was found to be significantly associated with HPV infection, particularly with high-risk oncogenic HPV 16 and 18 types, and with CIN 1 or worse in women referred because of an abnormal cervical smear. The diagnosis of CIN 1 or worse was also associated with HPV infection, particularly HPV types 16 and 18.Several studies have shown that cervical infections occur shortly after sexual debut, emphasizing the importance of sexual intercourse in transmission . The risWomen who begin to have sexual intercourse before the age of 16 are more vulnerable to HPV infection because during puberty the cervix undergoes cellular changes at the transformation zone that are known as ectopy . During Early age at first sexual intercourse, which may be a marker for the total number of sexual partners, is also associated with the risk of progression of the disease among HPV-positive individuals . An actiIn the present study, no association was found between the number of sexual partners and HPV infection or lesions of CIN 1 or worse. A study conducted in Latin America and the Caribbean showed that women tend to remain monogamous once married or cohabiting, while men do not ; therefoStudies have consistently shown an increased risk of male genital HPV positivity with sexual behavior, which could lead to an increased risk of cervical cancer in their female partners. More studies are needed in order to increase understanding of the natural history of HPV infection in males and its relationship with the risk of acquiring HPV and the development of cervical cancer in women .Cervical neoplasia associated with infection by individual HPV types has been evaluated up to a certain point and there are indications that the specific types lead to different risks with respect to persistence and progression . HPV typSignificant associations were found between women who had their first sexual intercourse prior to 16 years of age and HPV 16 and HPV 18 infections and lesions of CIN 1 or worse. HPV 16 and 18 are the types most commonly associated with cervical cancer . A recenKnowledge on the incidence of the different HPV genotypes is needed in order to focus appropriately on preventive, diagnostic and therapeutic measures. Mazarico et al.  showed tThis study showed that women who began to have sexual intercourse before the age of 16 had a 2.2-fold higher risk of developing CIN 1 lesions or worse. These data are in agreement with the findings of Flores et al.  who repoIn relation to risk factors such as high parity, the use of oral contraceptives, number of pregnancies and smoking, no statistically significant association was found with HPV infection or lesions of CIN 1 or worse. Other investigators have also reported a negative association between HPV infection and these risk factors . On the In fact, the use of oral contraceptives and smoking were the two principal factors found to accelerate maturation and are also known to be important risk factors for cervical neoplasia , 31. OneAs cervical cancer precursor lesions are the target of cervical cancer screening programs, it is important to improve understanding of the association between age and precancerous lesions of the cervix according to HPV type .Early events that can modify the carcinogenic potential of HPV infection include early age at first intercourse, which is a possible proxy of longer duration of persistent HPV infection. Understanding the epidemiology of genital HPV infection is important for the development of preventive actions against this infection and against HPV-related neoplasia.