Cervical cancer and other human papillomavirus- (HPV-) related cancers are preventable, but preventive measures implemented in developing countries and especially in low-income rural regions have not been effective. Cervical cancer burden derived from sexually transmitted HPV infections is the heaviest in developing countries, and a dramatic increase in the number of cervical cancer cases is predicted, if no intervention is implemented in the near future. HPV vaccines offer an efficient way to prevent related cancers. Recently implemented school-based HPV vaccination demonstration programmes can help tackle the challenges linked with vaccine coverage, and access to vaccination and health services, but prevention strategies need to be modified according to regional characteristics. In urban regions WHO-recommended vaccination strategies might be enough to significantly reduce HPV-related disease burden, but in the rural regions additional vaccination strategies, vaccinating both sexes rather than only females when school attendance is the highest and applying a two-dose regime, need to be considered. From the point of view of both public health and ethics identification of the most effective prevention strategies is pivotal, especially when access to health services is limited. Considering cost-effectiveness versus justice further research on optional vaccination strategies is warranted. Human papillomavirus (HPV) is the main cause of cervical cancer , the thiOf the estimated 530000 annual cases of cervical cancer, 86% occur in developing countries . Its staIt has been established that the HPV attributable proportion in cancers of the anus, vagina, penis, vulva, oropharynx, and oral cavity is 25% or higher , 19–21. Most of the genital infections with hrHPV type(s) are asymptomatic and heal without treatment. The risk of cervical cancer increases as the hrHPV infection persists. Over 70% of cervical cancers are attributed to HPV types 16 and 18 and approximately 20% to HPV types 31, 33, 35, 45, 52, and 58 . PrecancPrevention of hrHPV infections could decrease the incidence of numerous cancers in both sexes , 31. In Chlamydia trachomatis and HIV increase the risk of acquisition of hrHPV infection [Various studies indicate that a high number of lifetime sexual partners, tobacco smoking, parity, oral contraceptive use, and coinfections with nfection –42. The nfection , 44.STIs are a serious health problem in developing countries, and several studies indicate that conventional STIs increase the likelihood of HIV transmission . HPV infCervical cancer is a disease which can be prevented. Similar to liver cancer that is secondary to hepatitis B infection, cervical cancer has a recognized, single necessary cause, HPV. Application of cervical cytology in population-based screening programmes has significantly lowered the incidence of cervical cancer in developed countries . CervicaMortality rates of cervical cancer are lower than incidence rates with a ratio of mortality to incidence of 55% . SurvivaCurrently there are two licensed prophylactic HPV vaccines, a bivalent vaccine (Cervarix) against HPV types 16 and 18 and a quadrivalent (Gardasil) vaccine against HPV types 6, 11, 16, and 18 available . In order to be prophylactic, both vaccines need to be administered before the individual is exposed to HPV types covered by the vaccine. According to reports from the major phase III trials, the vaccines prevent from 97 to 98% of infections caused by HPV types 16 and 18 , 60. BotIt is known that the existing vaccines are most efficient for antibody production when administered to early adolescents. Both males and females had higher antibody responses at the age of 9–15 compared to the age of 16–26 , 63. TheAccording to WHO, 22 countries in low-resource regions have included an HPV vaccine in their vaccine programmes . AdoptedThe assumptions behind these recommendations and the superior cost-effectiveness of female vaccination strategy are that the health service system covers all the regions equitably and that adolescent girls have both access to the health services and are willing to use it. However, the probability of preventing other HPV-related cancers both in females and males has not been taken into account. Emerging information on HPV-related cancers in men may change the conclusions of cost-effectiveness modelling. In Australia, it is estimated that one quarter of the preventable cancers are in men , but thiThere is not enough information on how vaccinating males would change the transmission of HPV, but it is possible that vaccinating only females could result in an increase in HPV transmission like in the case of Rubella vaccination and Rubella acquisition by young adult females in the UK . BritishThe questions concerning HPV vaccine efficacy on males and the possible effect of vaccination on HPV transmission are valid as it is known that the vaccine against herpes simplex virus type 2 is not effective on males  but it cHigh vaccine coverage is needed to produce herd immunity, and the key question is whether sufficient coverage can be achieved by vaccinating only females. Sufficient coverage has been achieved only in the UK (75%) and Australia (70%). In the Netherlands (50%), Germany (40%), and USA (25%), the coverage is neither enough to protect significant proportions of females nor to produce herd immunity. Protection is effective on an individual level, but in the absence of herd immunity the unvaccinated remain unprotected. At present, the questions concerning coverage in both sexes and the magnitude of herd immunity remain open , 87, 90.The need for cervical cancer prevention is the greatest in developing countries where the burden of cervical cancer and other HPV-associated cancers is the heaviest, and preventive measures have not been/cannot be implemented consistently. From the point of view of global justice, the prevention of cervical cancer should be a priority in countries where its burden is the heaviest. The Global Alliance for Vaccines and Immunisation (GAVI) considers HPV vaccines among the vaccines that would have the biggest impact on the disease burden in developing countries. Within the developing countries (and in some developed or middle-income countries), the situation in rural regions with major problems of access to health services poses various questions concerning equity and justice . In the Due to the assortative nature of common sexually transmitted infections like HPV, the sufficient coverage to produce herd immunity effect is relatively low . In the In addition to access to health services (in this case vaccination or screening), it should be noted that the success of a vaccination or a screening programme is partly dependent on decision making by the objects of the intervention or other parties like parents, spouses, other family members, influential persons in the community, and politicians . In the HPV is such a common infection that a risk-group intervention alone is not likely to produce good results and have an impact. A high-risk group strategy based on behavioural characteristics would also be problematic to implement for a variety of reasons, most notably the inability to identify people at risk. Moreover, HPV does not have a tight core group as many classical STIs . A usefuCervical cancer is associated with poverty on both the global  and regiThis regional aspect has its implications in terms of judging different methods and optional preventive strategies that can guarantee access to and coverage of primary prevention. In the urban areas the recommended prevention strategies might be efficient to significantly reduce HPV disease burden because there are fewer problems connected to access and coverage. The challenges of implementing HPV vaccination programmes in practice are similar to some elements of implementing screening programmes, that is, financial constraints, competing health needs, and limited human resources. The infrastructure and logistical capacity needed are much more limited in rural regions and will require investments.The question is whether we can opt for a single strategy for a country or a group of countries or we should look at regional characteristics linked to access in deciding which strategy to adopt case by case. Malmqvist et al.  have staIn case mass vaccination would be offered, several strategies exist. A few relevant regional or even community-level strategies from the point of view of developing countries and/or regions are described as follows:vaccinating females at the age of 12–15: unrealistic targeting a coverage of 70%,questionable herd immunity effect,marginalized females excluded;(2)vaccinating females when school attendance is at the highest (10–12 years of age):unrealistic targeting a coverage of >80%, prioritizing vaccinating younger girls before school attendance drops—need for boosters? questionable herd immunity effect,marginalized females excluded;(3)vaccinating females and males at the age of 12–15:targeting a coverage of >40% in all regions,cost effectiveness,acceptance of the vaccine for boys?marginalization tackled by the herd immunity effect?(4)vaccinating females and males when school attendance is at the highest (10–12 years of age):targeting a coverage of >50% in all regions,acceptance of the vaccine for boys?marginalization tackled by the herd-immunity effect. All strategies would be school based with community outreach activities in regions where school attendance is low and equally there would be a need for information campaigns to adolescents, service providers, decision makers, schools, and parents . InformaIn terms of HPV vaccine coverage in developed countries, the highest rates have been achieved by school-based vaccination programmes . School-The challenges of a school-based programme are many. School-based programmes may not be feasible if sufficient resources are not allocated to providers . School The project funded by PATH, an American NGO, in Peru, Uganda, India, and Vietnam reached coverage of 80–95% in 9–14-year-old girls in selected schools demonstrating a high acceptability of the HPV vaccine . The PATOptional preventive strategies, regional strategies, or mixed strategies, offering vaccination only to girls in some wealthier regions, and to both sexes in certain low-income regions could tackle the problems linked with coverage and access. In some areas, it might be more feasible to have boys vaccinated because of higher school attendance, and this could lessen problems of coverage even though fewer girls would be reached. Other preventive acts such as male circumcision  and use The questions concerning dosage are closely linked with logistics, vaccine storage, vaccine acceptability, and cost effectiveness. The present vaccines are administered in three doses, but it is possible that a two-dose regimen could be enough to provide protection. This would have a significant effect mainly on costs but equally to vaccine acceptability and accessibility. In a study on Kenyan women, the acceptability of a three-dose vaccine regimen was only 31% compared to 86% for a one-dose regimen . It is kFurther cost-effectiveness studies are needed taking into account other HPV-related cancers besides cervical cancer. Regional characteristics and problems linked with access and coverage should be addressed as well. Vaccination strategies including catch-up vaccination in older females or addressing early adolescents after the sexual debut should be linked with the most accessible screening and treatment methods such as the single visit approach (VIA and cryotherapy) to ensure that the means of prevention would protect those who may be already HPV infected and for whom the prophylactic vaccine cannot be effective.Information about HPV vaccination and HPV-related cancers continues to emerge, but more research is needed especially on the long-term impact of vaccination, duration of protection, male vaccination, and reduction of HPV transmission. The GAVI Alliance subsidises the provision of vaccines to the poorest countries and is currently reviewing HPV vaccine as a candidate for sustainable financing. Even with secured financing, there is no simple answer concerning which strategy should be adopted in the developing countries.HPV vaccination is the most promising way to prevent cervical and other HPV-related cancers in developing countries. The vaccine is effective, safe, and widely accepted. Vaccination strategies have an important effect on the success of any vaccination programme. In the case of HPV, it is crucial to reach at least 70% of females or 40–50% of both sexes before sexual debut. Currently, only the option of vaccinating females is recommended by WHO.Optional preventive strategies, regional strategies, or mixed strategies in rural low-income regions could solve the problems linked with HPV vaccination coverage, access, and acceptability. Regional characteristics affect fundamentally the feasibility of HPV vaccination strategies as it has already been proven with screening. Differences between regions in terms of access to health services increase the need to adopt region-specific HPV vaccination strategies that are not currently deemed as cost effective. Emerging information on HPV-related cancers in both women and men and the feasibility of achieving high vaccine coverage in rural regions might produce different results in terms of cost effectiveness, and this might result as a change in strategic aims and recommendations. Targeting rural low-resource regions with specific vaccination strategies should be a priority from the point of view of ethics and public health. The number of cervical cancer cases is estimated to increase dramatically in developing countries if no intervention is implemented, and the trend is probably even stronger in regions where access to health services is limited. Whatever option is chosen, it is vital to merge any vaccination strategy with appropriate screening methods and sexual education.


Human papillomavirus (HPV) infections have been linked to a subset of oral and oropharyngeal cancers. However, little is known on the natural history of oral HPV infections. We designed the prospective Finnish HPV Family Study to assess the dynamics of HPV infections in parents and their infants. This study reports HPV genotype distribution and virus persistence in oral mucosa of the mothers.rd trimester of pregnancy and followed-up for 6 years. Oral scrapings taken with a brush were collected and HPV-genotyping was performed with nested PCR and Multimetrix® test . The predictors of persistent oral HPV species 7/9 infections were analyzed using generalized estimating equation models.Totally, 324 pregnant women were enrolled at the 3The point prevalence of oral HPV varied from 15% to 24% during the 6-year follow-up. Altogether, 18 HPV genotypes were identified either as single or multiple-type oral infections. HPV16 was the most prevalent type at 9.7%–18.4%, followed by HPV18, HPV6, and multiple infections. Altogether, 74 women had persistent oral HPV infection determined as at least two consecutive samples positive with the same HPV genotype. HPV16 and HPV6 were the two most frequent types to persist (76% and 9%) for a mean of 18.6 and 20.2 months, respectively, followed by multiple infections (8%) for 18.3 months. An increased risk for persistent oral HPV infection with species 7/9 was associated with being seropositive for low-risk (LR)-HPV-types at baseline, whereas the use of oral contraceptives and a second pregnancy during follow-up were protective. Clinical oral lesions were detected in 17% of these women, one-third of whom had persistent oral HPV-infections.HPV16 and HPV6 were the most common genotypes in oral HPV-infections and were also most likely to persist. Use of oral contraceptives and a second pregnancy protected against oral HPV persistence. Emerging evidence points to a causal role for human papillomavirus (HPV) in oral carcinogenesis Cross-sectional studies on asymptomatic oral HPV-infection report conflicting results on HPV-DNA prevalence, ranging from 0% to 81% with the mean of approximately 11% Only a few prospective studies on oral HPV-infections are available. Kurose et al. (2004) found HPV-DNA in 0.6% of oral scrapings from 662 subjects and two of them had a persistent infection over two years The Finnish Family HPV Study was designed to elucidate the dynamics of oral and genital HPV- infections within families The Finnish Family HPV Study is a prospective cohort study conducted at the Turku University Hospital and the University of Turku. The study protocol and its amendment (#2/1998 and #2/2006) have been approved by the Research Ethics Committee of Turku University Hospital. Altogether, members of 329 families were enrolled, comprising 329 mothers, 131 fathers and 331 newborns as described in detail previously Oral scrapings for HPV-testing were taken at baseline as well as at 2, 6, 12, 24 and 36-month and 6-year FU-visits. Oral scrapings were taken with a brush  from the buccal mucosa of both cheeks as well as the upper and lower vestibular areas. The brush was immersed in 80% ethanol and then immediately frozen and stored at −80°C until use. Cervical samples were collected as described earlier HPV-DNA was extracted from the oral scrapings with the high salt method as described previously HPV genotyping was performed with a Multimetrix kit®  which detects 24 LR- and HR-HPV-genotypes as follows: LR-HPV6, 11, 42, 43, 44, and 70; and HR-HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82. The earlier nested PCR products to detect any high risk HPVs was biotinylated by re-amplified with GP05+/bioGP06+-primers. The assay was performed in half of the volume given in the protocol in all steps except the final one. The labeled hybrids were analyzed with a Luminex LX-100 analyzer . A median fluorescence intensity (MFI) of at least 100 beads was computed for each bead set in the sample. The cut-off value for each run and HPV-type was 1.5× background MFI (negative control)+5MFI.If any sample was positive for HPV16, then the protocol was repeated from the original sample using nested PCR and a bead-based HPV16 genotyping assay All statistical analyses were run using the SPSS®  and STATA  software packages  by KS. Frequency tables were analyzed using the χ2-test with the likelihood ratio or Fisher's exact test for categorical variables. Differences in the means of continuous variables were analyzed using non-parametric  tests for two and more independent samples, respectively.At the first level, the genotype-specific outcomes of HPV infection in each woman were assessed by comparing the viral events at each follow-up visit to the baseline HPV status. Genotype-specific persistence was denoted when any case with two or more consecutive follow-up samples were positive for the same individual HPV genotype as a single infection or as a part of multiple-type infection.To analyze the predictors of genotype-specific HPV persistence, we determined the predictors of persistence only for the most prevalent HR-HPV-types, i.e., those of species 7  and species 9 .A generalized estimating equation (GEE) analysis was used with panel data, clustered by women-ID and run using population-averaged (PA) model At the 6-year visit, a careful examination of the oral mucosa was performed for 179 women by two dentists (JW and LW). Mucosal changes were recorded and photographed using a digital intra-oral camera . All lesions in the skin of the face and hands  were also carefully inspected and photographed.The point prevalence of oral HPV infection varied from 15% to 24% during the 6-year follow-up. Totally, 18 different HPV genotypes were identified in oral mucosa. Of these genotypes 12 HPV types were present as single infections Table 2.nd visit (median: 2.5 months), the HPV-prevalence increased to 21.7% (n = 65); it increased even further by the 3rd visit (6.7 months) to 24.1% (n = 70). At the 4th (12.9 months) and 5th (25.1 months) visits, the overall prevalence slightly decreased, but multiple infections increased from 3.1% (n = 9) to 5.6%. At the time point of 37.3 months, the overall prevalence decreased to the level of 15.6% (n = 41) with HPV16 comprising 12.2% (n = 32). After 6 years (77.9 months), HPV- prevalence was unchanged at 15.1% (n = 27), with HPV16 comprising 13.4% (n = 24) of these infections. Multiple infections decreased to the baseline level, 1.7% (n = 3). Species 9 was the most frequent HPV-species through the 6-year FU, followed by species 10 and 7.At the 2Totally, genotype specific oral HPV-persistence was found in 20 women (6.1%) when the HPV-positivity only of the baseline and last sample was regarded as selection criteria. However, during the follow-up 74 women were identified who had two or more consecutive follow-up samples positive for the same individual HPV genotype as a single or as a part of multiple infection Table 3.In the univariate GEE-model, being seropositive for LR-HPV at baseline increased the risk of persistence , whereas the second pregnancy during FU  decreased the persistence Table 4.At the 6-year FU-visit (n = 181), clinical oral lesions were found from 56 (31.3%) women. The most common clinical findings were lumps , hyperplasia , hyperkeratosis , and papilloma . Oral scrapings tested HPV-positive in 28.6%, 28.6%, 100% and 40%, of these women, respectively. The results of oral HPV-testing in women with hyperplastic lesions are summarized in Persistent HR-HPV-infections are the single most important risk factor in cervical carcinogenesis and have been recently implicated in oral and oropharyngeal carcinogenesis as well Before delivery, 17% of the mothers-to-be had HPV-DNA in their oral samples. This prevalence is much higher than that (2.5%) previously reported among pregnant women In the present cohort, HPV16 was the single most frequent genotype in oral mucosa at all-time points, followed by multiple-type infections. This finding is in agreement with previous reports Altogether, six HPV-genotypes were found to persist in 21% of these women. HPV16, followed by multiple-type infection as well as HPV6, were the most frequent genotypes to persist. According to our preliminary 2-year FU-data of this cohort, 9% of the mothers and fathers had persistent oral infection by HR-HPVs Oral sex has often been implicated among the risk factors of oral HPV-infections To elucidate the clinical relevance of these persistent oral HPV-infections, all women were subjected to clinical oral inspection at their 6-year FU. Although never reported previously, such an approach gives new insights into the potential causal role of persistent HPV-infections in the development of different oral pathologies. Interestingly, all women with hyperkeratotic changes in their oral mucosa also tested HPV-positive during the FU. During this longitudinal setting, many other clinical changes characterized by mucosal proliferation were also detected among women who tested HPV-positive in their oral scrapings. Importantly, almost all of these positive cases included the HPV16 genotype as well. This leads us to hypothesize that persistent HPV16 infection in the oral mucosa is a risk factor for different types of clinical lesions on gross inspection. However, we analyzed HPV only in the oral scrapings and not from clinical lesions. Analysis of the latter would lend additional support to the causal association of HPV with these lesions.Taken together, the Finnish Family HPV study was designed to create foundations for a better understanding of the natural history and different outcomes of oral HPV-infections at the genotype level. The present results showed that totally 18 different HPV genotypes were found in oral mucosa and HPV could also persist in oral mucosa. Because of the wide HPV-genotype spectrum which includes many rare genotypes, a significantly larger cohort will be needed to give the study enough power to conduct the risk analysis for individual genotypes.Table S1Studies reporting HPV prevalence in normal oral mucosa of healthy individuals since the reviews of Syrjänen and Syrjänen 2000, Kreimer and co-workers (2010) and Syrjänen et al. 2011.(DOCX)Click here for additional data file.


Complexes between cartilage oligomeric matrix protein (COMP) and the complement activation product C3b have been found in the circulation of patients with rheumatoid arthritis and systemic lupus erythematosus. In systemic sclerosis (SSc) COMP expression in the skin is upregulated both in lesional and non-lesional skin, which is also reflected in an increased amount of circulating COMP. We investigated the presence of COMP-C3b complexes in serum and skin biopsies of patients with SSc.The presence of COMP and COMP-C3b complexes in the serum of 80 patients with limited cutaneous SSc  and diffuse cutaneous SSc  and 97 healthy controls was measured by ELISA and correlated to different clinical parameters. Samples were collected both at baseline and after three to five years to assess longitudinal changes in COMP-C3b complex levels. Furthermore, skin biopsies from seven patients with dcSSc and three healthy controls were analyzed for expression of COMP and deposition of C3b and IgG.Serum levels of COMP-C3b were found to be elevated in both dcSSc and lcSSc compared to healthy controls and decreased at the second measurement in patients on immunosuppressive therapy. No co-localization of COMP and C3b was found in the skin biopsies, indicating that the COMP-C3b complexes are formed upon release of COMP into the circulation.COMP-C3b complexes are found in the serum of patients with SSc. The lack of co-localization between COMP and C3b in the skin suggests that COMP does not drive complement activation in the skin in SSc. Systemic sclerosis (SSc) is a multi-organ disease characterized by fibrosis of the skin and internal organs as well as vasculopathy . SSc is Activation of the immune system is a critical feature of SSc. Infiltrates of activated T-cells and macrophages are found in skin lesions early in disease ,18. Inflin vivo complement activation by COMP, complexes between COMP and the complement activation product C3b can be found in the serum of patients with SSc [We showed previously that COMP can activate the complement system, which occurs exclusively through the alternative pathway . Furtherwith SSc . Whetherwith SSc . Intereswith SSc  and it awith SSc . TherefoIn this study we set out to verify the presence of COMP-C3b complexes in the serum of SSc patients using a larger patient cohort and to study the relation of COMP-C3b to different clinical parameters as well as to examine presence of complexes longitudinally. We have further investigated the presence and co-localization of COMP and deposited complement components in the skin of SSc patients.Serum was collected from 80 patients with SSc who all fulfilled the American College of Rheumatology (ACR) criteria for SSc . The disSerum was furthermore collected from 97 healthy volunteers with no history of rheumatologic disease, from Lund and Malmö. All serum samples were retrieved at a standardized fashion (non-fasting) and were stored at −80°C after centrifugation. Further characteristics of the patients and controls are described in Table Informed written consent was obtained from all participants involved in the study and permission was obtained from the regional ethical review board for Lund University. In the case of the patient under 18 years of age, oral consent was obtained from the parents for taking the first blood sample as part of clinical diagnostic routine as well as for storage of the sample for use in the research project, and informed written consent from the patient herself was obtained at the time point of the second sample when she was an adult. These procedures were fully in line with the ethics regulations in Sweden at the time of sampling.Punch-biopsies, 3 or 4 mm in size were collected from the dorsal part of the right or left forearm, 2 to 4 cm proximal of the wrist. Skin samples were fixed in 4% formaldehyde, dehydrated with ethanol, embedded in paraffin and sliced to 4.5-μm sections. After mounting onto Superfrost Plus glass slides, samples were de-paraffinized and rehydrated as described . Antigen2 overnight at +4°C. The plates were washed with 50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20, pH 8.0 between each step in the assay. Plates were blocked using 1% BSA  diluted in 50 mM Hepes, pH 7.4 with 2 mM CaCl2 (blocking buffer) to prevent unspecific interactions. Serum samples were diluted 1:70 in 50 mM Hepes pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 with 50 μg/ml BSA, added to the wells and incubated for 2 h at RT. A biotinylated polyclonal anti-C3 antibody  was diluted in blocking buffer and incubated in the wells for 1 h at RT followed by a streptavidin-HRP conjugate . The plates were developed with o-phenylenediamine (OPD) substrate  and H2O2 and the absorbance at 490 nm was measured using a Cary 50 MPR microplate reader . Each sample was analyzed in duplicate and values obtained from the uncoated wells were subtracted from values obtained from antibody-coated wells. Obtained readings were then normalized against an internal control sample rendering data presented as arbitrary units (AU).Serum COMP-levels were measured using a commercially available COMP-kit . Serum COMP-C3b was measured on maxisorp plates  coated with a monoclonal antibody against COMP (home made) at a concentration of 5 μg/ml in 50 mM Hepes pH 7.4 with 2 mM CaClThe statistical significance of differences between groups was measured using the Kruskal-Wallis test with Dunn’s post hoc test or the Wilcoxon matched-pairs signed rank test, where appropriate. Two-parameter correlations were performed using Spearman’s correlation test. The significance of co-localization of staining in tissue was evaluated using Manders’ overlap coefficient.P <0.0001) corroborating our earlier results  . Prednisolone treatment did not affect the serum COMP-C3b levels in either patient group. Baseline COMP was high in the dcSSc-group and decreased significantly during follow up   in the dcSSc group in the first time-point sample , but not to the change in mRSS . This indicates that COMP-C3b is more related to the general inflammatory response in the patients than to the actual skin fibrosis. The change in COMP, however, correlated to the change in mRSS  in agreement with earlier results [COMP-C3b correlated with mRSS in the dcSSc group at baseline .rs = 0.5808, P <0.0001). As we detect no correlation between COMP-C3b and COMP, we can hypothesize that only certain fragments of released COMP are able to activate complement or that a certain environment is required for the complexes to form. This is the case also in other diseases, such as rheumatoid arthritis, where no correlation was found between serum COMP and COMP-C3b [We have confirmed in a large patient cohort that COMP-C3b levels are elevated in patients with SSc compared to healthy controls. The fact that there is no difference in serum COMP-C3b between patients with dcSSc and lcSSc is interesting, as these groups have quite different disease phenotypes and the systemic inflammatory component is more pronounced in dcSSc. The higher COMP levels seen in dcSSc are most likely a reflection of more extensive fibrotic skin involvement, as the COMP found in the circulation in SSc patients is thought to be released from the skin. Supporting this, we found a positive correlation between serum COMP and mRSS (COMP-C3b . We founNo correlation between serum COMP-C3b and C3 was found, most likely due to the fact that no apparent complement consumption was observed in the patients. In the dcSSc subset we saw a weak positive correlation between COMP-C3b and C4. Such positive correlation has previously been seen in patients with systemic lupus erythematosus . ReasonsMost skin biopsies of dcSSc patients studied showed COMP-staining, corroborating results published by others. Since we found very weak C3b staining in the skin of these patients, and the deposited C3b did not seem to co-localize with COMP, we can conclude that COMP in the skin does not drive measurable complement activation. Therefore, we can assume that the COMP-C3b complexes found in SSc patients do not originate from the skin but are formed in the blood after COMP is released into the circulation. It is likely that COMP needs to be cleaved or processed in a specific way or have a specific conformation for it to be able to trigger C3b-deposition, and that this does not occur until COMP is detached from the skin. These alterations might occur due to a specific inflammatory environment with local production of proteases and therefore may be down-regulated when the inflammatory reaction is diminished. This could explain the reduction of serum COMP-C3b in SSc patients at follow up. As the C-terminal globular domain of COMP, which is the domain responsible for complement activation , is engaComplexes between COMP and C3b are found in the circulation of both patients with dcSSc and lcSSc, although their levels relate more to individual inflammatory parameters in dcSSc. These complexes seem to form when COMP is released from the skin into the circulation and therefore it seems that COMP itself does not drive complement activation and deposition in the skin in SSc.AU: Arbitrary units; BSA: Bovine serum albumin; COMP: Cartilage oligomeric matrix protein; CRP: C-reactive protein; dcSSc: Diffuse cutaneous systemic sclerosis; ELISA: Enzyme-linked immunosorbent assay; lcSSc: Limited cutaneous systemic sclerosis; mRSS: Modified Rodnan skin score; PBS: Phosphate-buffered saline; RT: Room temperature; SSc: Systemic sclerosis.The authors KEO, AMB, TS, and DH have filed a patent application on a method to detect tissue degradation leading to inflammation. Authors TS and DH are co-founders and own stocks in AnaMar.KEO carried out the COMP-C3b ELISAs, stained and analyzed skin biopsies, did the statistical analysis, and drafted the manuscript. EH stained and analyzed skin biopsies, TS participated in the study design, and helped draft the manuscript. DH participated in the study design. RH participated in study design, provided patient material and revised the manuscript. AMB participated in the study design, helped draft the manuscript, and supervised the study. All authors read and approved the manuscript.Kaisa E Otteby has previously published under the name Kaisa E Happonen.


Nonmelanoma skin carcinoma (NMSC) is the most frequent cancer in the USA with over 1.3 million new diagnoses a year; however due to an underappreciation of its associated mortality and growing incidence and its ability to be highly aggressive, the molecular mechanism is not well delineated. Whereas the molecular profiles of melanoma have been well characterized, those for cutaneous squamous cell carcinoma (cSCC) have trailed behind. This importance of the new staging paradigm is linked to the ability currently to better clinically cluster similar biologic behavior in order to risk-stratify lesions and patients. In this paper we discuss the trends in NMSC and the etiologies for the subset of NMSC with the most mortality, cutaneous SCC, as well as where the field stands in the discovery of a molecular profile. The molecular markers are highlighted to demonstrate the recent advances in cSCC. Nonmelanoma skin carcinoma (NMSC) is the most frequent cancer in the USA and worldwide ; it has Multiple etiologies exist for cSCC, including environmental, genetic, viral and altered host immunity and virally mediated. The high incidence of cSCC and BCC is caused by the mutagenic effects of ultraviolet (UV) light which is intensified by geographic latitude , 7. cSCCWith a cure rate of greater than 90% for the routine lesion, and the large number of low risk lesions, the significance of the diverse spectrum and numerous subtypes of cSCC has been underappreciated given the often quoted 5-year recurrence and metastatic rates of 8% and 5%, respectively , 18, 19.Prognostic features have not been universally accepted in cSCC and the histologic features delineated above are advised for uniformity in standardizing clinical staging guidelines. Since cSCC is typically deemed to have a good prognosis, the variants with the worst biology, by not being grouped together, are underpowered numerically in an outcome analysis. Thus the variety of subtypes of cSCC certainly necessitates a comprehensive clinicopathologic classification system to group variants of SCC based on their biologic aggressiveness or indolence. The 7th Edition introduces the histologic features that need to be tracked and classified; yet a clinicopathologic classification system would allow lesions to be stratified based on biologic behavior and thus patients accordingly to optimize therapy. Clinicopathologic classification of cSCC has been characterized by Cassarino et al.  to stratβ-papillomavirus species 2, is more often associated with cSCC as opposed to the total amount of HPV DNA present [The increased incidence of cSCC in immunocompromised patients compared to BCC suggests a mechanism of viral pathogenesis. HPV is important in immunosuppressed patients, which are known to have higher levels of HPV DNA in cSCC lesions. Evidence of a higher viral load has been reported in cSCC in organ transplant patients with up to 80% of lesions containing HPV DNA . However present . Three t present , 26, (2) present , 25. Vir present .β papillomaviruses (β-HPVs) also play a role in the tumorigenesis of cSCC as shown by both European and US studies [β species 1 in SCC. Other viruses, such as polyomavirus (MCPyV) have been shown to be causative agent in Merkel cell carcinoma [Association of viral pathogens such as human papillomavirus (HPV) with head and neck squamous cell cancer (HNSCC), especially oropharyngeal cancer has been recognized over the past two decades. HPV16 is the most common genotype in these tumours, whereas HPV6 and HPV11 can also be found in a minority of these cancers, implying that these low-risk HPV types are not entirely benign in HNSCC. HPV DNA is closely associated with poorly differentiated cancers, positive lymph nodes, and late-stage disease, which portend a worse diagnosis. HPV status is also associated with p16 expression and HPV+ tumours are less likely to harbour p53 mutations . A subse studies . Howeverarcinoma . The genetic progression model for head and neck squamous cell carcinoma (HNSCC) demonstrates that loss of heterozygosity (LOH) is common during the progression from premalignant lesion to malignant tumors . Tumor sSeveral regions of chromosomal loss are identified in HNSCC. One of the most common regions, 9p21, has been reported in both HNSCC  and cSCCEpigenetics refers to the molecular mechanisms that regulate gene expression without changes in the DNA sequence. Epigenetic alterations include DNA methylation and histone modifications, which consist of methylation, acetylation, phosphorylation, ubiquitination, and sumoylation, chromatin remodeling and microRNAs –38. ChanEpigenetic dysregulation is thought to be involved in tumor biology and progression and thus may be relevant biomarkers for clinical prognosis . PromoteMessenger RNA (mRNA) and microRNA (MiRNA) profiles have been described in both HNSCC and cSCC . SeveralMitochondrial mutation in HNSCC has been well reported; however, only a few studies show the association of mitochondrial DNA mutation and cSCC . SeveralThe molecular mechanisms that underlie the development of cutaneous skin cancers are poorly understood. Even the spectrum of biologic behavior has been slow to be characterized given the previously very generic clinical criteria used to distinguish low risk lesions from a more aggressive lesions. Recent changes in the classification of the staging paradigm has better captured the histologic features of this more aggressive subset to allow for a more precision in identifying the worst subset. Incorporating a clear clinicopathologic classification system into stratifying lesions is necessary to better cluster them by their biology besides just histologic features.Thus molecular analysis can potentially profile that subset with biomarkers chosen to best correlate with the biologic phenotype.


A large proportion of vaginal and vulvar squamous cell carcinomas (SCCs) and intraepithelial neoplasias (VAIN and VIN) are associated with HPV infection, mainly type 16. The purpose of this study was to identify HPV genotypes, as well as E6/E7 mRNA expression of high-risk HPVs  in 56 histology samples of VAIN, VIN, vaginal, and vulvar SCCs. HPV was identified in 56% of VAIN and 50% of vaginal SCCs, 71.4% of VIN and 50% of vulvar SCCs. E6/E7 mRNA expression was found in one-third of VAIN and in all vaginal SCCs, 42.9% of VIN and 83.3% of vulvar SCCs. Our data indicated that HPV 16 was the commonest genotype identified in VAIN and VIN and the only genotype found in SCCs of the vagina and vulva. These findings may suggest, in accordance with other studies, that mRNA assay might be useful in triaging lesions with increased risk of progression to cancer. Human papillomavirus (HPV) infection of the female genital tract, is particularly frequent worldwide and its majority is transient, while at the same time, the persistent infections caused by the oncogenic types of HPV are responsible for cancer development. This oncogenic action of HPV is a result of the transformation ability of the high-risk (hr) HPV types' oncoproteins E6 and E7. The oncogenic properties of high-risk HPV reside in the E6 and E7 genes, which if inappropriately expressed in dividing cells deregulate cell division and differentiation .The causal relation of HPV infection with development of cervical cancer has been firmly established. The same does not necessarily apply to vulvar and vaginal squamous cell carcinomas (SCCs), where HPV is responsible for only a smaller percentage, namely, 60–70% of vaginal SCCs , 3 and 3The purpose of the current study was the HPV genotyping, as well as the expression of E6/E7 mRNA from the hrHPV types  in Greek women with VAIN, VIN, and SCCs of the vagina and vulva.PapilloCheck HPV-Screening, Greiner Bio-One GmbH, Germany, cat no. 465 060) as well as the expression of HPV 16, 18, 31, 33, and 45 E6/E7 mRNA, using the commercial real-time NucliSENS EasyQ assay .The sample of this study consisted of 56 paraffin-embedded tissue sections of VAIN, VIN, or vaginal and vulvar SCCs obtained from the Pathology Laboratory of the Regional Anticancer Oncology Hospital of Athens “St. Savvas”. The tissues were deparaffinized, and total nucleic acid was extracted using automated extraction . Then, genotyping of 24 HPV types was performed with microarray-based assay  and 5 μL 50% Tween20 solution ] and 5 μL proteinase K solution  at 65°C overnight. Proteinase K was deactivated in heat block at 80°C for 15 minutes.Fifteen micrometer sections of paraffin-embedded tissues were deparaffinized by incubation with 100% xylene  at 50°C and washings twice with 100% ethanol . Then, the pellets were dried at room temperature for 45 minutes. Finally, deparaffinized samples were digested with 100 μL of elution buffer. DNA quality test was carried out using Human Globin, Beta, Primer set kit  according to manufacturer's instructions. To assess RNA integrity, 5 μg of RNA per sample was separated on 1% formaldehyde-agarose gel.Tissue samples were transferred in lysis buffer  for 30 minutes, then total nucleic acid was extracted by the off-board protocol with the NucliSENS easyMAG platform (bioMérieux Hellas S.A), according to the manufacturer's instructions. The nucleic acids were eluted in 55 PapilloCheck HPV-Screening was used. This technology is based on a DNA chip for the type-specific identification of 24 types of HPV (18 high-risk and 6 low-risk types). E1-based PCR was performed according to the manufacturer's guidelines. For each sample, we mixed 19,8 μL PapilloCheck MasterMix, 0,2 μL HotStarTaq plus DNA polymerase , and 5 μL DNA from the tissue sample. Hybridization is followed by mixing 30 μL of the PapilloCheck hybridization buffer in a fresh reaction tube with 5 μL of the PCR product at room temperature and transferring 25 μL of the hybridization mix into each compartment of the chip. We incubated the chip for 15 minutes at room temperature in a humid atmosphere. The chip was washed in 3 washing solutions, centrifuged for 3 minutes at 5000 rpm, and scanned on the CheckScannerTM.The EasyQ assay  was performed for the qualitative detection of HPV E6/E7 mRNA of five hrHPV types  according to the manufacturer's instructions. Firstly, three premixes were made by adding reagent sphere diluent  into reagent spheres . In each premix, we added U1A/HPV 16, HPV 33/45, or HPV 18/31 primer and molecular beacon mixes, KCl stock solution, and NASBA water. Secondly, 10 μL of this premix was distributed to each well in a reaction plate, and the addition of 5 μL RNA followed. The plates were incubated for 4 minutes at 65°C to destabilize secondary structures of RNA, followed by cooling down to 41°C. The reaction was started by addition of enzymes  and measured in real time using the Lambda FL 600 fluorescence reader  at 41°C for 2 hours and 30 minutes.A commercial real-time NucliSENS t-test was used, along with relevant descriptive statistics  to compare the average age among patients depending on the prevalence of HPV and histology. In all the statistical tests, 5% level of significance was used. Concordance between DNA and mRNA tests was evaluated using the Cohen's kappa statistic.Our data were analyzed using SAS v9.0. Absolute and relative frequencies were used to present the HPV prevalence according to age and histology. Chi-squared tests were performed to assess the statistical significance of any differences in prevalence. P = 0.006).This group consisted of 18 patients with VAIN (mean age 46.2 years) and 4 patients with SCCs (mean age of 61.3 years). 8 VAIN cases were classified as VAIN I and 10 as VAIN II/III. HPV infection was detected in 56% of VAIN (10/18) and in 50% of vaginal SCCs (2/4). Detectable HPV DNA from at least one of the 24 genotypes was found in 75% (6/8) of cases of VAIN I and 40% (4/10) of VAIN II/III. In VAIN cases, the presence of HPV infection was strongly associated with younger patient age  and lrHPV types in 30% (3/10) of VAIN cases. Only hrHPVs were detected in vaginal SCCs (2/2). Multiple HPV types were present only in one case (one VAIN II/III sample contained HPV 33 and 53).The commonest HPV genotype was HPV 42 for VAIN I samples , followed by HPV 16  and HPV 39 , while HPV16 and HPV33 were the most common types for VAIN II/III . HPV 16 was the only type associated with HPV-infected vaginal SCCs .Regarding the hrHPV types 16, 18, 31, 33, and 45, 33.3% (6/18) of the VAIN and 50% of vaginal SCCs were HPV DNA positive for at least one of the above types. The detection rates of the five hrHPV types in samples from women with different grades of VAIN and SCCs are provided in P = 0.01 and P = 0.002), respectively  and 60% (6/10) of VAIN II/III were positive for HPV E6/E7 mRNA expression. This was statistically significantly higher than VAIN I samples, where no HPV E6/E7 mRNA expression was detected  and HPV E6/E7 mRNA test (NucliSENS EasyQ HPV assay) results was poor for patients with VAIN I  and vaginal SCCs , whereas it was good for patients with VAIN II/III  .P = 0.001).We included 28 patients with VIN, half of which had a low-grade lesion (mean age 35 years) and 6 patients with vulvar SCCs (mean age 62.5 years). VIN cases were not associated with lichen sclerosus, differentiated vulvar intraepithelial neoplasia, or squamous cell hyperplasia. PapilloCheck DNA Microarray detected HPV infection in 71.4% of VIN samples (20/28) and in 50% of vulvar SCCs (3/6). HPV DNA from at least one of the 24 genotypes was detected in 64.3% (9/14) of women with VIN I, and 78.6% of those with VIN II/III (11/14). In VIN cases, the presence of HPV infection was associated with younger patient age .HPV16 and HPV59 were the most commonly observed for infected VIN I samples , while HPV16 was the commonest genotype for VIN II/III  followed by types 18, 51, 52, 53, and 66 . In HPV-positive vulvar SCCs, only HPV 16 was present .Regarding the hrHPV types 16, 18, 31, 33, and 45, 53.6% (15/18) of VIN and 50% of vulvar SCCs were HPV DNA positive for at least one of the above HPV types by PapilloCheck DNA Microarray. The detection rates of the five hrHPV types in samples from women with different grades of VIN and SCCs are provided in P = 0.04).42.9% of VIN samples (12/28) and 83.3% of vulvar SCCs (5/6) were positive for HPV E6/E7 mRNA expression. There was a higher prevalence of E6 and E7 mRNA expression in patients with higher-grade lesions as shown in HPV16 was the commonest type revealed by NucliSENS EasyQ HPV assay for VIN I, VIN II/III, and vulvar SCCs  .The concordance between the HPV DNA test (PapilloCheck DNA Microarray) and HPV E6/E7 mRNA test (NucliSENS EasyQ HPV assay) results was very good for samples classified as having VIN I  but was poor for patients with VIN II/III . In the case of samples classified as having vulvar SCCs, the concordance was fair  .The goal of this study was to investigate HPV typing and HPV E6/E7 mRNA expression with intraepithelial neoplasia and squamous cell carcinomas of the vagina and vulva. This is the first study to report the association between HPV infection with oncogenic expression and vulvovaginal disease in Greek women. The role of HPV infection in vulvar intraepithelial neoplasia and squamous cell carcinoma has been confirmed through multiple studies worldwide. Although a similar link exists in vaginal precancerous and cancerous lesions, this has not been firmly established. This in part is due to the fact that VAIN and vaginal carcinomas are less common than their vulvar and cervical counterparts, due to the absence of a susceptible transformation zone and the protective effect of the keratinized vaginal mucosa . FurtherAs mentioned above, several studies have previously described the HPV prevalence and genotype distribution in VIN and SCCs of the vulva , 10–16. According to our results, hrHPV infection was the most frequently observed in VIN I cases. This is contrary to data obtained from an older study , where l35.7% of VIN I samples harbored hrHPV  and this rose to 71.4% in VIN II/III samples. This is expected as women with hrHPV infection are more likely to progress to high-grade lesions. Interestingly, the percentage of hrHPV dropped to 50% in vulvar SSCs. This suggests that the virus was present only at very low copy numbers and/or that only a specific region of viral DNA was integrated into the host's genome .In our study, HPV prevalence in VAIN samples was 56%. This was significantly lower than those reported by De Vuyst et al.  and Smith et al. (95.6%). In vaginal SCCs, HPV prevalence was 50%. This was lower but more comparable to those found in the studies mentioned above   Table 3Table 3.In a worldwide meta-analysis, the most frequent genotypes were HPV 16, 56, and 51 in patients with VAIN I lesions, HPV 16, 18, and 58 in those with VAIN II/III, and HPV 16, 18, and 31 in those with vaginal SCCs .Contrary to what was observed in vulvar samples, the proportion of patients with detectable hrHPV  increased progressively as the grade of the vaginal lesion progressed.Using the NucliSens EasyQ HPV assay, we investigated HPV oncoprotein expression in different grades of dysplasia and carcinoma. The results from E6/E7 mRNA test related well with the grade of lesion. The lowest rates of hrHPV types  E6/E7 mRNA expression were for patients with low-grade vulvar lesions (VIN I), whereas the higher rates were seen in high-grade lesions (VIN II/III), which is in accordance with what is seen in cervical lesions . It is pIn our series, there was a significantly higher detection of HPV 16 by NucliSens EasyQ HPV assay when compared to other hrHPV types for both vaginal and vulvar cases. This indicates that HPV 16 may be related to a different nature of persistent infection and oncoprotein expression in the vagina and vulva in comparison to HPV types 18, 31, 33, and 45.On the basis of DNA and mRNA assays, DNA from HPV was detected more frequently in vulvar low-grade lesions than E6/E7 mRNA expression. This data possibly reflects an episomal state or low number of copies of the virus. However, E6/E7 mRNA expression in a number of VIN I indicates that hrHPV may be oncogenically active even before it produces detectable changes in the cell .For vaginal high-grade lesions and carcinomas, as well as vulvar carcinomas, a higher rate of E6/E7 mRNA expression was observed, compared to HPV DNA. This suggests that the presence of E6/E7 oncoproteins is a specific marker for high-grade lesions. Interestingly, 2 cases of VAIN II/III, 1 case of VIN II/III, 2 cases of vaginal SCCs, and 2 cases of vulvar SCCs were positive only for mRNA expression. This may be explained by the fact that total viral DNA has been integrated to the host genome, and therefore, it cannot be detected by the DNA test. It is important to notice that in vulvar high-grade lesions a higher detection rate for HPV DNA was observed compared to E6/E7 mRNA expression. It is possible that these results were due to a very low level of viral transcriptional activity.The sample in our study was relatively small, primarily due to the low incidence of vulvar and vaginal intraepithelial neoplasias and carcinomas. Thus, we can only provide a rough estimate of the relative importance of each HPV type with regards to vaginal and vulvar cancer and precancer in our population.Another limitation of our study was that histology types of vulvar SCCs were not available, so a correlation between histology types and HPV infection was not possible. Nevertheless, it has been suggested that differentiated keratinizing SCCS, which occurs more frequently in elderly women, is not associated with HPV infection, whereas nonkeratinizing SCCS, which primarily affects younger women, is caused by hrHPV infection . This waThis study described the detection rates and attribution of genital HPV types, as well as the E6/E7 mRNA expression of intraepithelial neoplasias and squamous cell carcinomas of the vagina and vulva in Greek women. In summary, our results showed that a very crucial percentage of HPV was associated with VIN, VAIN, and vaginal/vulvar SCCs, and HPV 16 accounted for most HPV-positive cases. The fact that some cases of vulvar low grade lesions were positive for E6/E7 mRNA expression is also of interest, as it may identify these lesions as more clearly precancerous. A striking increase especially in the incidence of VIN in young women has been reported in the last decades in some high-resources countries , 23, 24.Further research is required to better assess the role of mRNA testing as a molecular marker for vaginal and vulvar carcinogenesis.


Nasopharyngeal carcinoma (NPC) is a malignant tumor which arises in surface epithelium of the posterior wall of the nasopharynx. There's is evidence that Epstein Barr virus (EBV) is associated to NPC development. However, many epidemiologic studies point to a connection between viral infections by the human papillomavirus (HPV) and NPC.Seventy Moroccan patients with NPC were screened for EBV and HPV. EBV detection was performed by PCR amplification of BZLF1 gene, encoding the ZEBRA (Z Epstein-Barr Virus Replication Activator) protein, and HPV infection was screened by PCR amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 35, 45 and 59.The age distribution of our patients revealed a bimodal pattern. Sixty two cases (88.9%) were classified as type 3 (undifferentiated carcinoma), 6 (8.6%) as type 2 (non keratinizing NPC) and only 2 (2.9%) cases were classified as type 1 (keratinizing NPC). EBV was detected in all NPC tumors, whereas HPV DNA was revealed in 34% of cases (24/70). Molecular analysis showed that 20.8% (5/24) were infected with HPV31, and the remaining were infected with other oncogenic types . In addition, statistical analysis showed that there's no association between sex or age and HPV infection (P > 0.1).Our data indicated that EBV is commonly associated with NPC in Moroccan patients and show for the first time that NPC tumours from Moroccan patients harbour high risk HPV genotypes. Nasopharyngeal carcinoma (NPC) is a tumour that arises in the epithelium surface of the posterior nasopharynx, and shows a peculiar geographic and ethnic distribution. The highest incidence rates of NPC are found among the southern Chinese population and in isolated northern populations such as Eskimos and Greenlanders , an ubiquitous human herpesvirus, and the development of different malignancies, such as Burkitt's lymphoma, 40%-50% of Hodgkin's disease, B-cell lymphoma in immunocompromised individuals, and NPC .The papillomaviruses are small double-stranded DNA viruses which infect squamous epithelia and display a very high selectivity for the specific epithelium infected ,8. More According to their ability to transform epithelial cells, HPV genotypes are divided into low-risk and high-risk types. Low-risk types are associated with benign lesions such as warts, while infections with high-risk types progress to malignant lesions -10. It hThe demographic characteristics of the 70 patients showed that the mean age of patients was 39.65 with extreme ages at 10 and 87 years old. The age distribution of our patients is represented in Figure The pathological analysis was performed according to the World Health Organization (WHO) classification and revealed that among the 70 cases, 62 (88.9%) were classified as type 3 (undifferentiated carcinoma), 6 (8.6%) as type 2 (non-keratinizing NPC) and only 2 (2.9%) cases were classified as type 1 (keratinizing NPC)   Table .The distribution of HPV DNA in the 24 HPV positive NPC cases is reported in Table . MoleculDistribution of HPV genotypes according to the anatomy pathology status showed that among the 62 NPC type III, 22 were HPV positive with a predominance of HPV31 which was present in 22.7% of cases (5/22), whereas, the only HPV-positive NPC type II biopsy was HPV-18 positive and the only HPV positive NPC type I was HPV-33 positive.No statistically difference was observed in the HPV prevalence between males 34.7%) and females (33%) Table , and no 4.7% and et al. have shown that HPV DNA was detected in 38.8% of 36 southern Indian NPC cases [et al. in Eighty-eight fresh tissue samples of NPC showed that HPV DNA was detected in 51% of the specimens [It is widely accepted that EBV is etiologically associated with NPC; but it is proven that other co-factors might be involved in the carcinogenesis process. HPVs are considered to be one of those factors since they possess the ability to transform epithelial cells and a significant number of NPC biopsies harbour HPV DNA ,13. We rPC cases . Tung etpecimens .et al. showed that among 88 fresh NPC specimens from Chinese population, coexistence of EBV and HPV DNA was observed in 42% of samples [Coinfection by HPV and EBV has not been well documented and the significance of the presence of both viruses in nasopharyngeal cells has not been determined. In our study, coinfection with both viruses was observed in 34% of patients. Tung  samples .Of interest, EBV was detected also in CC specimens from Indonesian patients, and 68% of analysed cases are co-infected with HPV and EBV . In our Our results show no correlation between HPV status with either age or gender in NPC patients. These findings are supported by other studies that reported no significant differences ,18. HoweWith regard to HPV genotypes, HPV31 was the most frequent genotype in Moroccan NPC patients 20.8%). The same genotype was also frequently found in tonsils and nasopharyngeal cells in western Mexico NPC cases .8%. The ,22, wereGeographic and racial distinctions have been identified between NPC of the Far East versus those diagnosed in Caucasian American patients with regard to the interrelationship of histologic subtype and HPV infection. In fact, HPV are detected more often (50%) in type I NPC from American Caucasian patients than type III . In our Taken together, these data suggest that i) HPV genotypes associated with NPC are different from those consistently found in CC and ii) the HPV genotypes associated with NPC are not geo-specific.HPV and EBV co-infections have not been well documented and the significance of the presence of both viruses in nasopharyngeal cells has not been determined. It has been shown that ZEBRA, an EBV immediate early protein expressed during lytic replication that activates early EBV genes, binds to p53 . The phyOn the other hand, further studies to evaluate the impact of EBV and HPV coinfection in cervical and nasopharyngeal carcinogenesis and molecular mechanisms implicated in tumor development are warranted.The analyses were performed on biopsies collected from 70 patients with histopathologically confirmed NPC at the Oncology Centre in Casablanca, Morocco, after informed written consent, as approved by the local Ethical Committee. The biopsies were stored at -80°C until used.-1 of proteinase K. The samples were digested for 3 h at 55°C. Proteinase K was heat-inactivated at 95°C for 15 min. The samples were spin down and the supernatants were collected and stored at -20°C until used. In order to evaluate the efficiency of DNA extraction, all samples were polymerase chain reaction (PCR)-amplified using PC04 and GH20 primers specific for human β-globin gene  containing 200 μg mlβ-globin and HPV assay. Positive controls containing the SiHa and Caski cell lines always amplified β-globin and HPV DNA, respectively.To avoid contamination leading to false positive results, all PCR-related work was carried out in specialized zones within a PCR laboratory that undergoes UV purification at least once every 24 h. To detect crossover contamination negative controls (PCR reagents containing no DNA) were included in each PCR amplification. All negative controls were negative for Z-Epstein-Barr Virus Replication Activator (ZEBRA) gene was amplified by PCR using ZES (forward) and ZEAS (reverse) primers [ primers . Primer DNA samples were amplified by PCR using consensus primers MY09 and MY11 for a 450 bp target sequence in the L1 region that is highly conserved on a broad spectrum of HPV genotypes . Each reThe results were analyzed statistically by the chi2 test. The level of significance was set at 95% (α = 0.05) for all tests.NPC: Nasopharyngeal carcinoma; EBV: Epstein-Barr virus; HPV: Human papillomavirus; CC: Cervical cancer; ZEBRA: Z-Epstein-Barr Virus Replication Activator; WHO: World Health OrganizationThe authors declare that they have no competing interests.NL: designed experiments, contributed and analyzed data, Carried out the molecular studies and drafted the manuscript. MA: designed the primers, the approach to validation, including the selection of appropriate materials and revised the manuscript. MK: participated in study design and performed the statistical analysis. FN: participated in PCR protocol design and performed the typing test. TM: participated in PCR protocol design and performed the typing test. RD: participated in PCR protocol design and performed the typing test. KB: Participated in the conception of the study and in its coordination. NB: Collected NPC specimens and carried out the anatomy pathology analyses. MEM: designed the laboratory procedures, coordinated the laboratory quality control and revised the manuscript. MK: Conceived the study, supervised this work, participated in its design and coordination, and revised the manuscript. All authors read and approved the final manuscript.


E6/E7 type-specific oncogenes are required for cervical carcinogenesis. Current PCR protocols for genotyping high-risk HPV in cervical screening are not standardized and usually use consensus primers targeting HPV capsid genes, which are often deleted in neoplasia. PCR fragments are detected using specialized equipment and extra steps, including probe hybridization or primer extension. In published papers, analytical sensitivity is typically compared with a different protocol on the same sample set.Human papillomavirus (HPV) E6/E7 genes of two low-risk and 19 high-risk genotypes  and the resulting short fragments were directly genotyped by high-resolution fluorescence capillary electrophoresis.A single-tube multiplex PCR containing type-specific primers was developed to target the ® imprinted with cut tumor surfaces, swabbed cervical cancers or ejected aspirates from nodal metastases of head and neck carcinomas. Between one and five long oligonucleotide targets per sample were detected without false calls. Each of the 21 genotypes was detected in the clinical sample set with up to five types simultaneously detected in individual specimens. All 101 significant cervical neoplasias (CIN 2 and above), except one adenocarcinoma, contained E6/E7 genes. The resulting genotype distribution accorded with the national pattern with HPV16 and 18 accounting for 69% of tumors. Rare HPV types 70 and 73 were present as the sole genotype in one carcinoma each. One cervical SCC contained DNA from HPV6 and 11 only. Six of twelve oropharyngeal cancer metastases and three neck metastases of unknown origin bore E6/E7 DNA; all but one were HPV16. One neck aspirate contained atypical squames with HPV26.The method was validated using long oligonucleotide templates, plasmid clones and 207 clinical samples of DNA from liquid-based cytology, fresh and formalin-fixed specimens and FTA MicrocardsAnalytical sensitivity in dilute plasmid mixes was variable.E6/E7 oncogenes of 21 HPV types in cellular and fixed tissue specimens. The method is straightforward, robust and reproducible and avoids post-PCR enzymatic and hybridization steps while detecting HPV with high clinical sensitivity in significant HPV-related neoplasia regardless of specimen type.A primer-rich PCR readily detects the  Alpha-human papillomaviruses (HPV) are sexually transmitted and may infect the anogenital and oral mucosae. They either resolve without sequelae or cause condyloma (HPV types 6 and 11 and their relatives) or rarely carcinoma, particularly cervical cancer and its precursors . HPV16 and 18 account for 70% to 80% of cervical cancers with the remainder due to other alpha- HPV genotypes. HPV are labeled high-risk (HR-HPV) if found in cancers in disproportion to their prevalence; uncertainty exists as to whether certain genotypes  should be classified as high-risk.E6/E7 in cell nuclei as dysplasia progresses. Although no "gold standard" for type-specific HPV genotyping has been agreed upon, current methods generally employ the polymerase chain reaction (PCR) with consensus primers aimed at the L1 capsid gene [E6/E7 genes which are most reliably conserved in cervical cancer [E6/E7 junction is also less prone to sequence variation than other genes, which like the capsid genes may be lost during integration of viral DNA into the host cell genome [Cervical carcinogenesis depends on the deregulation of HPV l cancer . The typl genome -5.E6/E7 primers in a multiplex PCR (MPCR) to yield products which are size and color-specific for each HPV genotype on capillary electrophoresis, the need for nested PCR, amplicon sequencing, probe hybridization or enzymatic detection steps [E6/E7 MPCR capable of detecting 19 HR-HPV in a test set of highly significant lesions (CIN 2 to carcinoma) submitted as cytology specimens, including aspirates from metastases, as well as fresh and formalin-fixed tumor embedded in paraffin (FFPE).By combining labeled E6/E7 targets  for determination of electrophoretic product size, (ii) for analytical sensitivity a test panel of 14 plasmid-cloned HPV genotypes [® used for ThinPrep cytology , which specimens were obtained with a cytobrush , (iv) FFPE specimens  and (v) Indicating FTA Microcards®  with imprinted cut tumor surface or applied swabs/aspirates . FTA cards significantly expedite sample collection, storage and preparation and have been described previously in lymphoma MPCR [We targeted in a single microfuge tube 19 definite or probable HR-HPV genotypes  [enotypes , courtesE6/E7 genes from types 6 and 11 but not from any HR-HPV. This was the only specimen to contain HPV11.Results in clinical material are presented in Table About one-fourth of 37 consecutive samples with normal cytology were infected with HR-HPV and three of these (8%) had multiple genotypes. In 15 consecutive exfoliative samples of high-grade CIN (grades 2 and 3), one or more HR-HPVs occurred in all in the following order of frequency : types 16, 18/31, 33/58/66 and 45/53/59/73/82.E6/E7 genes were detected in 75 of 76 primary or metastatic cervical carcinomas , as single infections in 91%. The one negative sample was a β-globin+ ADCA . HPV16 and 18 accounted for 69% of the 75 HPV-associated cervical cancers, and other genotypes were in order of frequency: 31, 45, 56/59, 33/52, 58/66 and 35/53/70, which accords with the national statistics [HPV atistics .Coinfection with more than one HPV type occurred less often in carcinomas in contrast to dysplastic cytology specimens, consistent with decreased rates of multiple infections among the generally older patients with carcinoma.+ tumors was oropharyngeal in six instances, consistent with previous studies [Nine of 31 nodal aspirates of SCC metastases from head and neck tumors .The MPCR was robust in clinical samples, producing readily interpretable peaks and results were reproducible on repeat runs. An occasional small spurious peak due to saturation of the laser detector disappeared after diluting the product.Secondary peaks attributable to primer cross-annealing appeared occasionally, mainly in cervical cytology specimens (presumably the result of higher virus loads in productive infections), but did not correspond in size and color to true peaks and had amplitudes usually below our cutoff. In theory, mispriming of a forward primer could result in an additional peak for a given HPV type, while a mispriming reverse primer could present a new peak of the same or different size and color  as the target peak.Forward primers in types 39, 59, 68 and 70, which are all 20-mers and share 3 to 7 nt in their 3' end sequence, might offer potential mispriming among their intended targets, which would not necessarily produce a peak shift related to anomalous product sequence and none was seen in electropherograms with these genotypes. Moreover, use of the "touchdown" technique in initial PCR cycles probably abrogated any tendency of this limited sequence similarity to competitively inhibit visualization of mixed infection. For example, two mixed HPV68/70 infections were easily detected.In silico analysis of individual E6/E7 target sequences queried for significant complementarity  with any of the primers in the master mix yielded 29 potential misprimed products of varying size and color derived from 16 of the 21 genotypes when testing clinical material, none of which were of the same size but different color from that expected for the target. Complete complementarity was noted only for HPV31, where its forward primer amplified type 31 E6/E7 in conjunction with the predicted homologous type 16 reverse primer. Thus, HPV31 gave the most significant and consistent secondary peak  [L1 primers (GP5+/6+) in a PCR on the same patient sample followed by bead-based hybridization typing identified HPV6 but failed to identify HPV11, whereas a singleplex PCR with our type 11 E6/E7 primers repeatedly confirmed the original result.Cervical cancer cell nuclei contain viral DNA either integrated in the host cell genome, as free episomes or both. If viral episomes disappear, which occurs in 20% to 50% or more of tumor specimens, most consistently in HPV18-induced tumors [ncogenes . A recenLuminex) . In the When compared with cytology, HPV genotyping by PCR offers higher sensitivity but lower specificity in detecting intraepithelial neoplasia  and PCR Our detection of rare HR-HPV in high-grade CIN and several suspected HR-HPV types in carcinoma points out that a relatively broad-spectrum multiplex PCR capable of typing all cervical carcinomas offers a desirable level of coverage but could still miss the rare tumor related to a low-risk HPV not covered in the analysis. In this regard, primers for additional genotypes could probably be added to the 46 already present in our MPCR without adversely affecting efficiency.Although multiplex PCR with capillary electrophoresis gives lower analytical sensitivity, which would militate against its use in detecting infection per se or minimal residual malignant disease , in detection scenarios in the present study clinical sensitivity is more relevant and this was retained. If appropriately automated, scaled and tested on a larger population sample, our MPCR might be less likely to overburden triaging schemes with excessive detection of infections destined to resolve .The cost of materials for the MPCR (unoptimized for use in screening) including primers and reagents, was about USD 7 per sample as of the year 2008.E6/E7 oncogenes of 21 HPV types in fresh and fixed cytology and tissue specimens. The method is straightforward, robust, avoids post-PCR sequencing and hybridization steps by using fragment size analysis and detects HPV with high sensitivity in significant HPV-related neoplasia regardless of specimen type.A primer-rich PCR readily detects the http://www.ncbi.nlm.nih.gov/tools/primer-blast/ to confirm the sequence's uniqueness for each HPV genotype and to adjust primers for minor polymorphisms in primer binding sites reported for subtype variants. All reverse primers were synthesized with 5' labels , which were alternated with each increase in corresponding amplicon size. Primers for the human β-globin control gene were designed to yield a green 268 nt product. The result was 46 unique primers designed to yield short products suitable for both high-molecular weight DNA and formalin-fixed material; expected HPV product sizes ranged from 94 to 258 nt with all but two being≤190 nt. Primer sets were then synthesized and purified by HPLC .Primer parameters were adjusted to yield as many amplicons separated in size by ~3 or more nucleotides as possible while maintaining high PCR efficiency. Primer pairs were chosen to avoid potential duplex formation within and between pairs. Each primer pair with amplicon was used in a BLAST query of human and viral databases DNA was extracted from sections and from cytology fluid using a kit  according to the manufacturer's instructions. DNA was precipitated in isopropanol, the pellet resuspended in 10 mM Tris-buffer, pH 8.5 and 50-100 ng was used as template.®  in a 50 μL reaction, which included 5 μL of a solution containing 0.3 μM of each forward and reverse primer for β-globin and HPV types 16, 39, 66 and 68 with 0.2 μM for all other primers. After 15 min at 95°C, 8 "touchdown" cycles  were run starting with annealing at 65°C for 90 s in the first cycle, then 95°C, 30 s; 65°C -1°C, 90 s for seven cycles; 72°C, 45 s, and 22 cycles with constant parameters  with a final extension at 68°C for 15 min. All E6/E7 oligonucleotide targets were detected singly and in mixed combinations of 4 or 5 genotypes at estimated concentrations of 1.5 × 103 to 1.5 × 105 virus equivalents per genotype per μL.After extensive testing in >400 PCR using single and combined primer pairs, an optimal MPCR, capable of detecting at least 5 genotypes simultaneously in a single tube, was determined. It used 25 μL of (obligatory) Multiplex PCR Kit® 3130 Genetics Analyzer whose capillaries were filled with POP7 polymer (minimum runtime 18 min). Data files were analyzed in GeneMarker®, vers. 1.75. Peaks were called if they were ~1 nt or less in width, corresponded to the predetermined size (± 0.5 nt) and color of a given genotype and were ≥500 fluorescence units to allow ready distinction from baseline.One μl of PCR product was added to a mixture of 12 μl formamide and 0.5 μl ROX 500 size standard prior to heat-denaturation and loading on an ABI PrismThe authors declare that they have no competing interests.MD designed the primers, the approach to validation, including the selection of appropriate materials and drafted the manuscript. JW designed the MPCR protocol and performed the analyses. Both authors collaborated in the interpretation of data for each sample. Both authors read and approved the final manuscript.