Current prophylactic vaccines against human papillomavirus (HPV) target two oncogenic types (16 and 18) that contribute to 70% of cervical cancer cases worldwide. Our objective was to quantify the range of additional benefits conferred by second-generation HPV prophylactic vaccines that are expected to expand protection to five additional oncogenic types .A microsimulation model of HPV and cervical cancer calibrated to epidemiological data from two countries (Kenya and Uganda) was used to estimate reductions in lifetime risk of cervical cancer from the second-generation HPV vaccines. We explored the independent and joint impact of uncertain factors  and vaccine properties , compared against currently-available vaccines.Assuming complete uptake of the second-generation vaccine, reductions in lifetime cancer risk were 86.3% in Kenya and 91.8% in Uganda, representing an absolute increase in cervical cancer reduction of 26.1% in Kenya and 17.9% in Uganda, compared with complete uptake of current vaccines. The range of added benefits was 19.6% to 29.1% in Kenya and 14.0% to 19.5% in Uganda, depending on assumptions of cancers attributable to multiple HPV infections and unidentifiable HPV types. These effects were blunted in both countries when assuming vaccine cross-protection with both the current and second-generation vaccines.Second-generation HPV vaccines that protect against additional oncogenic HPV types have the potential to improve cervical cancer prevention. Co-infection with multiple HPV infections and unidentifiable HPV types can influence vaccine effectiveness, but the magnitude of effect may be moderated by vaccine cross-protective effects. These benefits must be weighed against the cost of the vaccines in future analyses. Prophylactic vaccines against human papillomavirus (HPV), responsible for all cervical cancers and a smaller proportion of other anogenital and oral cancers, represent one of the major breakthroughs in cancer prevention. The two currently available HPV vaccines, which target two of the most oncogenic types, HPV-16 and -18, have the potential to prevent about 70% of cervical cancer cases worldwide; the 4-valent vaccine also targets two non-oncogenic types, HPV-6 and -11, responsible for genital warts. In settings that have not successfully implemented organized cervical cancer screening programs, the introduction of HPV vaccination may provide a critical opportunity for primary prevention of cervical cancer; in settings with organized screening, vaccination could also provide the opportunity to reduce the intensity of screening programs. Indeed, HPV vaccination of young adolescent girls has been shown to be cost-effective in both developed and developing countries Despite the great promise for prevention of cervical cancer and other HPV-related cancers, there are several characteristics of current HPV vaccines that may limit their success. Although the 4-valent HPV vaccine has been offered at an unprecedented price ($5 per dose) to the GAVI Alliance, the vaccines remain unaffordable in many parts of the world. Furthermore, the requirement of cold chain in storage and distribution adds another logistical challenge in vaccine accessibility and feasibility. Given regional variation in the proportion of cervical cancers attributable to HPV-16 and -18, the maximum impact of vaccination may be lower than 70%. For example, in Kenya where about 60% of cervical cancers are attributable to HPV-16,-18, reduction in cancer cases could be 10–20% lower than in Uganda, where up to 75% of cervical cancers are attributable to HPV-16,-18.Second-generation prophylactic HPV vaccines, currently in clinical trials, may hold several advantages over the current vaccines, including protection against additional oncogenic HPV types, less dependence on cold chain storage and distribution, and more efficient delivery mechanisms , facilitating integration with other childhood vaccines in routine immunization programs The objective of this study is to provide preliminary estimates of the potential health benefits from the anticipated 9-valent HPV vaccine, exploring the independent and combined effects of these factors. Since reductions in cervical cancer cases are not expected to be observed for several years post-vaccination, disease simulation models that are carefully developed to incorporate information on HPV epidemiology and cervical carcinogenesis can be used to project a range of estimated benefits in the absence of long-term empiric data and under conditions of uncertainty. Used in a decision-analytic framework, such disease simulation models can inform policy makers on how to design new or restructure existing cervical cancer prevention programs in the rapid wake of new technologies We used an established cervical cancer disease simulation model that had been previously calibrated to two separate countries, Kenya and Uganda, to estimate health benefits in terms of reductions in lifetime risk of cervical cancer from the 9-valent prophylactic HPV vaccine. We selected these two representative countries because of their high cervical cancer incidence rates, the lack of organized cervical cancer screening or HPV vaccination programs, and the different proportions of cervical cancer cases attributable to HPV types 16 and 18. These models had previously been calibrated to fit to epidemiological and clinical data from the respective countries and used to explore the cost-effectiveness of HPV vaccination and cervical cancer screening in five Eastern African countries We conducted a series of exploratory analyses to estimate both the absolute and incremental benefits of the 9-valent HPV prophylactic vaccine, compared with the benefits from currently-available HPV vaccines . The influence of several important factors was explored, separately and concurrently:The higher the prevalence the vaccine-targeted HPV types are in cervical cancer, the greater the expected reductions in cases from the 9-valent vaccine. Globally, HPV types 16, 18, 31, 33, 45, 52 and 58 are detected in approximately 91.6% (95% CI: 88.5–95.1%) of squamous cell cervical cancers total benefit in cervical cancer prevention from the 9-valent vaccine may not vary much across settings, the incremental benefit compared with the benefit of the current vaccines that target only HPV-16 and -18 could vary greatly across populations, depending on the prevalence of these types and the newly-targeted HPV types  in cervical cancer. For example, in Kenya, where HPV-16 and -18 contribute to roughly 62.2% of cervical cancer cases, HPV types 31, 33, 45, 52 and 58 are detected in 30.1% of all cases. In contrast, in Uganda, HPV-16 and -18 contribute to 73.9% – and the five other oncogenic types to 17.4% – of cervical cancer cases. Consequently, the added benefits of the 9-valent vaccine over the current vaccines in Kenya could be nearly double the added benefits in Uganda.While the To estimate the potential benefit of the 9-valent vaccine in reducing cervical cancer risk, we obtained and used the prevalence of each of the seven oncogenic HPV types targeted by the 9-valent vaccine as the basis to estimate their joint contributions to cancer cases. In Kenya, 62.2% of cervical cancers are caused by HPV-16 and/or -18; For Uganda, 73.9% of cervical cancer cases are attributable to HPV-16 and/or -18; When there is more than one HPV type present as the possible cause of cervical cancer, the estimation of vaccine efficacy solely on HPV type distribution could be inaccurate. It is plausible that when more than one HPV type is detected, only one of them is the actual cause of cervical cancer while the other HPV type(s) is(are) just an incidental finding. However, it is also plausible in some cases that there is a required synergistic effect between more than one HPV type in causing cervical cancer. With these diverse biological possibilities, the added benefit of a 9-valent vaccine could be overestimated or underestimated in the cases where multiple HPV types are identified  there is no overlap between unidentifiable HPV types and multiple infections with HPV types that are targeted by the 9-valent vaccine, and therefore, the 9-valent vaccine does not offer any benefit in these cases  none (0% cross-protection), (2) low (7.4% cross-protection), (3) moderate (37.4% cross-protection), and (4) high (58.2% cross-protection) Several assumptions were required for the analysis: (a) girls receive the vaccine  by age 12 prior to sexual debut; and (b) all vaccine recipients complete the full three-dose series (100% vaccine compliance rate) that is required for current vaccines and was assumed for the 9-valent vaccine. The primary outcomes were absolute and relative reductions in lifetime risk of cervical cancer, compared to no intervention.For the current vaccines, the absolute reductions in lifetime risk of cervical cancer were 60.2% in Kenya and 73.9% in Uganda. Assuming vaccine cross-protection against non-vaccine types, these reductions increased to 81.9% in Kenya and 88.2% in Uganda.In Scenario A, the 9-valent vaccine yielded absolute risk reductions of approximately 79.8% in Kenya and 87.9% in Uganda, when there was no cross-protection against non-vaccine types. The reductions were as high as 91.0% in Kenya and 94.5% in Uganda, under assumptions of high cross-protection against non-vaccine types.In Scenario B, the 9-valent vaccine increased absolute risk reductions to approximately 86.3% in Kenya and 91.8% in Uganda, without cross-protection against non-vaccine types, and were as high as 93.9% in Kenya and 96.2% in Uganda, with high levels of cross-protection against non-vaccine types.In Scenario C, the reductions in lifetime risk of cervical cancer associated with the 9-valent vaccine were greatest, ranging from 89.3% to 95.2% in Kenya and 93.4% to 96.9% in Uganda, depending on assumptions of cross-protection.Across varied scenarios of uncertainty, our simulation model projects that the 9-valent vaccine could reduce lifetime risk of cervical cancer by 79.8% to 95.2% in Kenya and 87.9% to 96.9% in Uganda. In comparison, currently-available vaccines could offer protection against 60.2% to 81.9% of cervical cancers in Kenya and 73.9% to 88.2% of cervical cancers in Uganda. The added benefits of the 9-valent vaccine over current vaccines therefore ranged from 9.2% to 29.1% in Kenya and from 6.3% to 19.5% in Uganda.Our study provides evidence that uncertainties surrounding HPV type distribution in cancer, unidentifiable HPV types, co-infection with multiple HPV infections, and cross-protection against non-vaccine types can lead to variable estimates of both the absolute and incremental benefits of the 9-valent vaccine. The added benefits were most favorable when the 9-valent vaccine could fully protect against unidentifiable HPV types and multiple infections (Scenario C) with an assumption that none of the vaccines provide cross-protection against non-vaccine HPV types. The added benefits were least favorable when cases with unidentifiable types and multiple infections were not attributable to types targeted by the 9-valent vaccine (Scenario A) with an assumption that all the vaccines provide high cross-protection against non-vaccine types.As expected, HPV type distribution in cancer had a greater impact on the added benefits of the 9-valent vaccine (compared to current vaccines) than the absolute benefits. Results showed that the added benefit of the 9-valent vaccine in Kenya, where cervical cancer is less attributable to HPV-16 and -18, was approximately 1.5 times greater than in Uganda, where cervical cancer is more commonly caused by HPV-16 and/or -18. The greater magnitude in added benefits in Kenya persisted in all scenarios from no cross-protection to a high level of cross-protection and from no benefit (Scenario A) to full benefit (Scenario C) against unidentifiable types and multiple infections.http://www.who.int/hpvcentre/en/), roughly 68.0%, 68.5%, 70.3%, 73.0% and 75.1% of cervical cancer cases were associated with HPV-16 and -18 in Asia, Africa, the Americas, Europe and Oceania continents, respectively This finding suggests that in a geographic area where cervical cancer is less attributable to HPV-16 and -18, the 9-valent vaccine could offer greater added benefits, compared to the current vaccines. Based on epidemiological data of HPV type distribution in cancer from the WHO/ICO Information Centre on Human Papilloma Virus (HPV) and Cervical Cancer  with the varying degrees of cross-protection against non-vaccine types. The impact of the uncertainty from unidentifiable types and co-infection with multiple types decreased as the level of cross-protection against non-vaccine types increased.Cross-protection against non-vaccine types was expected to reduce the added benefits of the 9-valent vaccine over the benefits from the current vaccines. Our results demonstrated that, with moderate cross-protection against non-vaccine types, the added benefits from the 9-valent vaccine could decrease by 6.4–9.8% in Kenya and by 4.8–6.7% in Uganda, compared to no cross-protection against non-vaccine types. With high cross-protection against non-vaccine types, the added benefits from the 9-valent vaccine were reduced by 10.5–15.8% in Kenya and by 7.7–10.8% in Uganda, in comparison to a scenario with no cross-protection.The findings from our exploratory analysis underscore that estimates of the incremental benefits from the 9-valent HPV vaccine compared to the current HPV-16/18 vaccines may vary widely subject to several uncertainties, ranging from cancer reductions of 9.2% to 29.1% in Kenya and 6.3% to 19.5% in Uganda. Despite the utility of our disease simulation model in projecting preliminary estimates of population-level vaccine benefits under complex interactions of various uncertainties, our analysis has several limitations. First of all, we did not model the infection from each of the HPV types individually. We classified all non-16, non-18 HPV types into either the group of five additional types targeted by the 9-valent vaccine or the group of the remainder types. We then made a simplifying assumption that the contributions to cervical cancers from all of these non-16, non-18 types were directly proportionate to their prevalence in non-16, non-18 cancer cases. This estimation may be imprecise if some oncogenic HPV types are more likely than others to be incidental infections, but in the absence of such information, we only modeled the contributions to cervical cancer based on prevalence.Secondly, we did not vary the levels of vaccine uptake and adherence to the full three-dose vaccine series. Perfect vaccine uptake is unlikely even in developed countries. Indeed, a recent national survey in the United States showed that fewer than 1 out of 3 teenage girls between 11 and 17 years old had received at least one dose of currently available HPV vaccines To date, there is no evidence suggesting that the 9-valent HPV vaccine will result in differentially higher uptake than currently available vaccines. While protection against more HPV types could be a strong incentive for individuals to obtain the vaccine, there is not yet a clear advantage in how the vaccine will be administered  or in the number of doses required. Vaccine uptake will continue to be a concern for the 9-valent vaccine as well as for currently available vaccines.In this study, we did not examine the impact of screening programs on the absolute and added benefits from the 9-valent vaccine; therefore, generalizability to settings that have screening programs may be limited. In settings where effective screening programs exist, we expect that the absolute and added health benefits from the 9-valent vaccine would be reduced. Generally, with more effective screening programs, the benefits of vaccination are expected to be lower. This trend applies to both currently available vaccines and the 9-valent vaccine. Countries without an effective cervical cancer screening program are likely to achieve greater added benefits from the 9-valent vaccine, compared to countries where effective screening programs are in place.We did not explore the benefits of vaccinating both boys and girls with the 9-valent vaccine relative to vaccinating both sexes with currently available vaccines. We expect that vaccination of both sexes with the 9-valent vaccine would further reduce lifetime risk of cervical cancer. Male HPV vaccination would also result in direct health benefits to males , as well as contribute to reduced HPV transmission in the population, thus providing “indirect” benefits to sexual partners. However, the burden of HPV-related diseases in males is relatively low, and we have learned from previous analyses that the added benefits from including boys in the vaccination program would likely be less than the added benefits from increasing uptake in girls There are other noteworthy limitations. The estimated benefits from HPV vaccination were restricted to squamous cell carcinomas of the cervix, as there is limited clinical and epidemiological information on non-squamous cell types of cervical cancer. As a result, the estimates may not be applicable to prevention of non-squamous cell cancers. In addition, we did not make any assumptions regarding the correlations between the four sources of uncertainty in estimating vaccine benefits. For example, we did not assume that cancer cases with unidentifiable types and multiple infections occurred with a positive correlation. Information from clinical trials may reveal negative or positive correlations among these factors, and modeling the interactions between different uncertainties may be feasible with such data.Other factors should be considered in policy decisions regarding adoption of the 9-valent vaccine. In this analysis, we assumed that neither the current vaccines nor the 9-valent vaccine have serious adverse side effects and compared only vaccine benefits in terms of cancer reduction. Costs of the vaccines and implementation are important factors that should be incorporated in future studies to assess the value of the added benefits of the 9-valent vaccine.The 9-valent HPV prophylactic vaccine is expected to offer greater protection against cervical cancer than current vaccines. The magnitude of this added benefit, however, is uncertain due to the complex interactions of several factors; important among them are HPV type distribution in cancer cases, cases that are attributable to co-infections with multiple HPV types or unidentifiable type(s), and vaccine cross-protection against non-vaccine types. In this study, we leveraged a disease simulation model of HPV-induced cervical cancer to estimate health gains in terms of cervical cancer reductions associated with the 9-valent vaccine under different scenarios. Type distribution of HPV in cancer cases, and cancers attributable to multiple HPV infections and unidentifiable HPV types can influence vaccine effectiveness individually and jointly, but the magnitude of influence may be moderated by vaccine cross-protective effects. These benefits must be weighed against the cost of the vaccines in future analyses.Ethics committee approval was not required for this study.Appendix S1Multiple HPV infections and potential vaccine benefits. This supplemental appendix gives examples of scenarios to show how co-infection with multiple HPV types can influence the estimation of vaccine benefits of the 9-valent HPV vaccine.(DOC)Click here for additional data file.


Rosalind Howes and colleagues present a map of glucose-6-phosphate dehydrogenase deficiency prevalence and severity. Individuals with the deficiency are at risk of mild to severe hemolysis when taking the antimalarial primaquine. Plasmodium vivax, primaquine is the sole effective treatment of infectious P. falciparum gametocytes, and may interrupt transmission and help contain the spread of artemisinin resistance. However, primaquine can trigger haemolysis in patients with a deficiency in glucose-6-phosphate dehydrogenase (G6PDd). Poor information is available about the distribution of individuals at risk of primaquine-induced haemolysis. We present a continuous evidence-based prevalence map of G6PDd and estimates of affected populations, together with a national index of relative haemolytic risk.Primaquine is a key drug for malaria elimination. In addition to being the only drug active against the dormant relapsing forms of Representative community surveys of phenotypic G6PDd prevalence were identified for 1,734 spatially unique sites. These surveys formed the evidence-base for a Bayesian geostatistical model adapted to the gene's X-linked inheritance, which predicted a G6PDd allele frequency map across malaria endemic countries (MECs) and generated population-weighted estimates of affected populations. Highest median prevalence (peaking at 32.5%) was predicted across sub-Saharan Africa and the Arabian Peninsula. Although G6PDd prevalence was generally lower across central and southeast Asia, rarely exceeding 20%, the majority of G6PDd individuals (67.5% median estimate) were from Asian countries. We estimated a G6PDd allele frequency of 8.0% (interquartile range: 7.4–8.8) across MECs, and 5.3% (4.4–6.7) within malaria-eliminating countries. The reliability of the map is contingent on the underlying data informing the model; population heterogeneity can only be represented by the available surveys, and important weaknesses exist in the map across data-sparse regions. Uncertainty metrics are used to quantify some aspects of these limitations in the map. Finally, we assembled a database of G6PDd variant occurrences to inform a national-level index of relative G6PDd haemolytic risk. Asian countries, where variants were most severe, had the highest relative risks from G6PDd.G6PDd is widespread and spatially heterogeneous across most MECs where primaquine would be valuable for malaria control and elimination. The maps and population estimates presented here reflect potential risk of primaquine-associated harm. In the absence of non-toxic alternatives to primaquine, these results represent additional evidence to help inform safe use of this valuable, yet dangerous, component of the malaria-elimination toolkit.Please see later in the article for the Editors' Summary Plasmodium falciparum is the most deadly and P. vivax is the commonest and most widely distributed. Malaria parasites have a complex life cycle. Infected mosquitoes inject “sporozoites” into people, a form of the parasite that replicates inside human liver cells. After a few days, the liver cells release “merozoites,” which invade red blood cells where they replicate rapidly before bursting out and infecting other red blood cells. This increase in the parasitic burden causes malaria's characteristic fever and can cause organ damage and death. Infected red blood cells also release “gametocytes,” which infect mosquitoes when they take a blood meal. In the mosquito, gametocytes multiply and develop into sporozoites, thus completing the parasite's life cycle. Malaria can be prevented by controlling the mosquitoes that spread the parasite and by avoiding mosquito bites by sleeping under insecticide-treated bed nets. Treatment with effective antimalarial drugs also decreases malaria transmission.Malaria is a parasitic infection that is transmitted to people through the bites of infected mosquitoes. Of the four parasites that cause malaria, P. vivax “hypnozoites,” which hibernate in the liver and cause malaria relapses. Unfortunately, primaquine induces mild to severe destruction of red blood cells (hemolysis) in people who have a deficiency in the enzyme glucose-6-phosphate dehydrogenase (G6PD). G6PD deficiency (G6PDd) is common in some ethnic groups but the global distribution of individuals at risk of primaquine-induced hemolysis is unknown and there is no practical field test for G6PDd. Consequently, it is hard to design and implement primaquine treatment practices that balance the benefits of malaria transmission reduction and relapse prevention against the risk of hemolysis. Here, the researchers use a geostatistical model to map the prevalence (frequency in a population) of G6PDd in malaria-endemic countries and to estimate how many people are affected in these countries. They also develop a national index of relative hemolytic risk.The Global Malaria Action Plan aims to reduce malaria deaths to zero by 2015 and to eradicate malaria in the long-term through its progressive elimination in malaria-endemic countries . Primaquine is a key drug for malaria elimination. It is the only treatment effective against the gametocytes that transmit malaria between people and mosquitoes and against The researchers fed data from community surveys of the prevalence of phenotypic G6PDd (reduced enzyme activity) for 1,734 sites  into a geostatistical model originally developed to map global malaria endemicity. The model predicted that G6PDd is widespread across malaria-endemic regions, with the lowest prevalences in the Americas and the highest in tropical Africa and the Arabian Peninsula, but that most G6PDd individuals live in Asian countries. The predicted prevalence of G6PDd varied considerably over relatively short distances in many areas but, averaged across malaria-endemic countries it was 8%, which corresponds to about 350 million affected individuals; averaged across countries that are currently planning for malaria elimination, the prevalence was 5.3% . Finally, the researchers used data on the geographical occurrence of G6PD variants classified according to their enzyme activity levels as mild or severe to derive an index of hemolytic risk from G6PDd for each malaria-endemic country. The greatest risk was in the Arabian Peninsula and west Asia where the predicted prevalence of G6PDd and the occurrence of severe G6PD variants were both high.These findings suggest that G6PDd is widespread and spatially heterogeneous across most of the malaria-endemic countries where primaquine would be valuable for malaria control and elimination. The accuracy of these findings is limited, however, by the assumptions made in the geostatistical model, by the accuracy of the data fed into the model, and by the lack of data for some malaria-endemic countries. Moreover, there is considerable uncertainty associated with the proposed index of hemolysis risk because it is based on phenotypic G6PDd enzyme activity classifications, which is presumed, but not widely demonstrated, to be a surrogate marker for hemolysis. Nevertheless, these findings pave the way for further data collection and for the refinement of G6PDd maps that, in the absence of non-toxic alternatives to primaquine, will guide the design of safe primaquine regimens for the elimination of malaria.http://dx.doi.org/10.1371/journal.pmed.1001339.Please access these Web sites via the online version of this summary at malaria; its 2011 World Malaria Report provides details of the current global malaria situation Information is available from the World Health Organization on malaria (in English and Spanish), including a selection of personal stories about malariaThe US Centers for Disease Control and Prevention provide information on global control of malaria and on the Global Malaria Action PlanInformation is available from the Roll Back Malaria Partnership on the Malaria Atlas Project website where G6PD deficiency prevalence maps, population estimates and the data used in this study can also be accessedInformation on the global mapping of malaria is available at the G6PD deficiency for affected families can be found on KidsHealth from the Nemous Children's Health System and the G6PD Deficiency Association websiteInformation about malaria; the MedlinePlus Encyclopedia provides information about G6PD deficiency (in English and Spanish)MedlinePlus provides links to additional information on  A third of malaria endemic countries  now plan for malaria elimination P. falciparum malaria treatment recommend a single dose of primaquine alongside artemisinin-based combination therapy (ACT) to prevent parasite transmission, particularly as a component of pre-elimination or elimination programmes The 2010 World Health Organization (WHO) guidelines for uncomplicated P. vivax and P. ovaleKey to sustaining progress towards malaria elimination is the prevention of parasite reintroduction from the relapsing malarias Primaquine can cause mild to severe haemolysis in G6PDd patients. The mechanism of primaquine-induced haemolysis is not fully understood. Reduced G6PD enzyme activity levels are likely to create a redox equilibrium within red blood cells that favours oxidised species of highly reactive primaquine metabolites. In one hypothesis, the 5-hydroxyprimaquine metabolite would be dominated by its oxidised quinoneimine species in G6PDd red blood cells, which may then react with the haem moiety of haemoglobin and cause its displacement to the lipid bilayer of red blood cells Understanding the distribution and prevalence of this genetic risk factor in any given area may substantially inform risk and thus better equip policy makers and practitioners alike in designing and implementing primaquine treatment practices. We respond here to demands from the malaria community for a prevalence map of this genetic condition In addition to the public health importance of G6PDd in the context of malaria elimination, the clinical burden of this genetic condition includes a range of haematological conditions, including neonatal jaundice and acute haemolytic anaemia in adults triggered by a range of foods, infections, and other drugs In this study, we compile data from available sources of G6PDd prevalence surveys, and use these as the evidence-base to inform a Bayesian geostatistical model specifically adapted to the gene's X-chromosome inheritance mechanism. This model generates spatially continuous G6PDd prevalence predictions, and allows quantification of prediction uncertainty. The model predictions are then matched with high-resolution population data to estimate numbers of deficient individuals within MECs, accounting for the predicted sub-national heterogeneity in deficiency rates. Finally, we assemble a database of G6PDd variant occurrences and propose here an index for how the prevalence map could be used to stratify haemolytic risk at the national level.This study's methodological objectives involved the assembly of representative community G6PDd prevalence surveys and the development of a Bayesian geostatistical model used to derive (i) maps of G6PDd prevalence within MECs, (ii) sex-specific estimates of the populations affected by this deficiency, and (iii) associated uncertainty metrics. These results were then combined with information on the distribution of the underlying G6PDd variants to generate an index for stratifying haemolytic risk from G6PDd. Each of these aspects is discussed briefly here, and in more detail in A literature search of online bibliographic databases was conducted to identify published community surveys of G6PDd. Existing databases published by Singh et al. in 1973 2) were included to ensure that sub-national variation could be represented Inclusion criteria were applied to ensure: (i) community representativeness: all potentially biased samples were excluded ; (ii) gender representativeness: only surveys reporting sex-specific raw data were included; (iii) spatial representativeness: only surveys that could be mapped with relatively confined extents  gives an estimate of the expected prevalence of homozygous females (q2). Phenotypic expression of female heterozygous (2q(1−q)) deficiency ranges across a spectrum of enzyme activity levels. Expression is variable due to irregular Lyonization rates h) varied between surveys; h was modelled as a spatial variable, with values learned from the data, but not modelled as a spatially structured variable. The deviance value represents both the proportion of heterozygotes diagnosed as phenotypically normal, as well as actual deviance from expected Hardy-Weinberg equilibrium due to factors such as selection, consanguinity, migration, or small population sizes.A Bayesian geostatistical framework p(d) = q+q2+2q(1−q)h; where p(d) is the probability of an individual being phenotypically deficient, and q is the allele frequency for deficiency. From this equation, frequencies of hemizygotes , homozygotes , and all deficient females h could be estimated. The model was fitted to the data and 1 million Markov chain Monte Carlo (MCMC) iterations The model framework is thus To validate the model predictions, an independent model iteration was implemented with a 95% subset of the dataset, allowing comparison of the predicted frequencies with observed frequencies from the 5% hold-out data. The hold-out data sample was selected to preferentially include spatially isolated data points, so as to ensure that the full prediction surface was included in the validation. Moreover, isolated areas are harder to make predictions for, and are therefore a conservative assessment of model reliability. Further details about validation methodology and derived statistics are given in beta version, adjusted to United Nations (UN) population estimates for the year 2010 To quantify the prevalence of G6PDd across national and regional populations, areal estimates  In order to stratify the potential haemolytic risk associated with G6PDd, a simple index was developed that incorporated both the national prevalence of the trait and the severity of the local genetic variants.Predicted national prevalence was stratified into three categories . Stratifying the severity of the local forms of G6PDd was more involved. A second online literature review was conducted to assemble all reports of genetic and biochemical variants, using the same search methods as for assembling the prevalence data. All occurrences of named G6PDd variants were abstracted into a database and mapped to the country where they had been observed. Variants were then grouped according to their severity: the only severity classification widely applied to all variants is that proposed by Yoshida et al. The prevalence and variant severity scores were then multiplied to give a stratified measure of the relative haemolytic risk of G6PDd in each country. A similar uncertainty index was determined on the basis of the uncertainty in the prevalence estimates, and the availability and heterogeneity of variant data in each country. The variant data, risk scoring tables, and uncertainty estimates are presented in more detail in Literature searches were conducted to collate all available reports of representative community G6PDd prevalence. A total of 17,272 G6PD abstracts were identified from online bibliographic databases, together with 472 potential data sources found in existing G6PDd databases n = 1,289)  were from 23 Asian countries; 10% of surveys (n = 132) represented 23 African countries; data from only nine countries in the Americas were identified, corresponding to 4% of surveys (n = 56). Male data were reported from 99% of the surveys, while 62% presented female data. Overall numbers of individuals sampled were 2.4 million males and 2.0 million females.The total number of surveys identified that met the inclusion criteria was 1,734 globally, with 74% from MECs  . SurveysThe database is described in more detail in The survey database formed the evidence-base for the geostatistical model, which predicted both the spatially continuous map of G6PDd allele frequency  and the Large swathes of the American MECs were predicted to have median G6PDd frequencies ≤1% (40.8% land area), with G6PDd being virtually absent from northern Mexico, Costa Rica, Peru, Bolivia, and much of Argentina . PrevaleAt the continental level, G6PDd was most prevalent across sub-Saharan Africa: 65.9% of the land area was predicted to have median G6PDd prevalence ≥5%, and 37.5% a median prevalence ≥10%. Predictions ranged from <1% at the continental extremities  to >20% in isolated pockets of Sudan, coastal west Africa, and around the mouth of the river Congo. These broad patterns were interspersed with some striking sub-national variation within countries with deficiency hotspots, including Nigeria . More broadly, rates across this disparately populated peninsula as a whole were heterogeneous, for example, dropping to prevalence of 3% (IQR: 2–4) in the central Al-Kharj and Riyadh area of Saudi Arabia. Further east, predicted prevalence remained high into southern Pakistan.This region had the highest uncertainty of the entire map (IQR exceeding 30%). No surveys were available from the south of Pakistan, and the closest neighbouring surveys in southern Iran, Oman, and western India reported prevalence of >20%, contrasting data from northern Pakistan. Prediction uncertainty dropped across central and southeast Asia, and predicted prevalence remained largely <10%, with three notable G6PDd prevalence hotspots in the central and southeast Asia regions peaking to >20%: (i) among the tribal, endogamous groups of Orissa province in east India, (ii) a patch along the northern Lao/Thai border, and (iii) much of the Solomon Islands archipelago. Underlying the broadly smooth continental-level variation, some areas were predicted to have highly heterogeneous sub-national G6PDd prevalence. Across Lao People's Democratic Republic (PDR), for instance, frequencies were predicted to range from 1% (IQR: 0–2) to 23% (16–32); predictions in Indonesia were from 0% (0–1) to 15% (10–21) in Nusa Tenggara; in Papua New Guinea, frequencies ranged from 1% (0–2) along the southern coast to 15% (10–22) along the East Sepik northern coast .The predicted allele frequency surface was evaluated against a hold-out subset of the data selected with spatially declustered randomization that preferentially selected data sparse sites where model predictions would be inherently most difficult The second modelling process related the allele frequency predictions to population distribution, generating sex-specific aggregated estimates of G6PDd populations, weighted by population distribution across the spatial regions of interest: national, malaria endemic, and the subset of 35 MECs targeting malaria elimination. These population-weighted estimates were modelled separately from the mapping process, and used the full model predictions, not just the summary median allele frequency map . As withWe estimated overall G6PDd allele frequency across MECs to be 8.0% (IQR: 7.4–8.8); using 2010 population data , this coNational frequency estimates ranged from 0.1% in Cape Verde (IQR: 0.0–0.5) and the Democratic People's Republic of Korea (0.0–0.4) to 22.3% in the Solomon Islands (15.7–30.9), 22.5% in the Congo (17.3–29.6) and 23.0% in Benin (17.0–30.1). Reflecting the prevalence map, national allele frequency estimates were generally lowest in the Americas and highest in Africa . ConvertData searches for reports of G6PDd variants identified 527 occurrences of class II variants and 405 class III variants from a total of 54 countries out of 99 MECs . OccurreThis index of risk is predicated on the current state of knowledge of G6PDd variant occurrence and the relationship between variants and haemolysis, as outlined in the Combining these variant severity scores with the scores of G6PDd prevalence gave an index of overall risk from G6PDd for each MEC. Greatest haemolytic risk from G6PDd was found in the Arabian Peninsula and across west Asia, where both prevalence and variant severity (dominated by the class II Mediterranean variant) were high. Across the Asian continent, risk remained high . In contrast, despite high prevalence, the low severity of the variants reported from sub-Saharan Africa resulted in the lowest risk categorizations from G6PDd globally, which was a moderate risk .The uncertainty inherent in this synthesis is considerable; however, the index indicated that according to the metrics employed in this study, uncertainty ranked highest in many sub-Saharan countries and most countries in the Americas (where 19 of 21 countries had uncertainty ranked 5–6 out of 6). Further data from these regions would substantially improve reliability both of the modelled prevalence predictions, as well as of the variant severity categorisations, many of which had to be inferred from neighbouring countries.The framework proposed here can be updated and refined as new data about variant occurrence and haemolytic risk become available.G6PDd is widespread across malarious regions, where we estimated the deficiency to have an overall allele frequency of 8.0%. We have developed here an evidence-based, geostatistically modelled, and spatially continuous prevalence map of G6PDd, together with uncertainty metrics and population estimates of affected individuals. Although highest levels of G6PDd frequency are predicted in sub-Saharan Africa, high population density makes Asia the centre of weight of G6PD deficiency-burdened populations. We discuss our results first in relation to existing G6PDd maps, and then in their public health context in relation to the coincident severity of local variants. Important limitations to the maps and population estimates stem from weaknesses in the underlying database of surveys. These are also discussed, in relation to the difficulties of predicting deficiency in females, in assessing the robustness of the model predictions, and in overcoming the barriers to predicting the severity of primaquine-induced haemolysis.Previous G6PDd maps have been published by the WHO G6PD Working Group in 1989 The evidence-based nature of the analysis leaves the model predictions vulnerable to weaknesses in the underlying database. While some of these limitations can be quantified, such as prediction uncertainty in areas with very scarce data, others cannot. The current study presents a methodological advance over previously published maps for being the first to quantify any aspect of prediction uncertainty. In brief, our mapping procedure involved 500 repeated predictions being made from the optimised Markov chain Monte Carlo (MCMC) algorithm . The med2), 20% were less specific and mapped as polygons up to 35 km in radius of which centroid coordinates were used in the model  high prevalence of carriers of the clinically silent and diagnostically invisible P. vivax hypnozoite; and (iii) the predominance of asymptomatic carriers of sexual and asexual blood stages despite low transmission intensity. The problem of P. vivax resistance to chloroquine is discussed elsewhere P. vivax parasitaemia in the 63 d following therapy for acute P. falciparum malaria . Those rates seem to support rational and pragmatic use of anti-hypnozoiticidal primaquine treatment for all malaria patients where these parasites occur together Malaria eliminating countries  face stePrimaquine is the only chemotherapeutic tool currently available for attacking hypnozoites and mature gametocytes. As explained elsewhere There is no immediate prospect of relief from the serious constraints to chemotherapeutics for malaria elimination. A new drug in phase IIb/III trials in 2012, Tafenoquine, is strategized as a successor to primaquine, but it is also likely to come with haemolytic toxicity in G6PDd patients, and thus the same constraints would apply The prominence of G6PDd represents a barrier to current options for malaria elimination therapy. Nevertheless, the unique properties of primaquine are increasingly in demand as communities target depletion of their parasite reservoirs. It is evident that no measures are currently in place to ensure safe delivery of primaquine within the context of G6PDd risk. The complexity and diversity of both malaria epidemiology and G6PDd mean that no single solution will be applicable for ensuring safe and effective primaquine treatment. The maps and population estimates presented here represent one component of this treatment decision-making framework, and pave the way for further data collection and refinement of mapping studies of G6PDd severity. The relative urgency of this important component to determining appropriate elimination therapy may be determined by the relative prevalence of G6PDd and malaria endemicity in any given area http://www.map.ox.ac.uk/).All maps at national and regional scales and in GIS and image formats, population estimates, as well as the input surveys database are freely available on the Malaria Atlas Project website Click here for additional data file.Protocol S1Assembling a global database of G6PD deficiency (G6PDd) prevalence surveys. (S1.1) Overview of database requirements. (S1.2) Library assembly. (S1.3) Dataset inclusion criteria. (S1.4) Survey diagnostic methods. (S1.5) The final G6PDd survey dataset. (S1.6) Defining MECs' limits.(DOCX)Click here for additional data file.Protocol S2Model based geostatistical framework for predicting G6PDd prevalence maps. (S2.1) Model requirements in relation to G6PD genetics. (S2.2) The model. (S2.3) Model implementation. (S2.4) Overview of mapping procedure. (S2.5) Uncertainty.(DOCX)Click here for additional data file.Protocol S3Model validation procedures and results. (S3.1) Creation of the validation datasets. (S3.2) Model validation methodology. (S3.3) Validation results.(DOCX)Click here for additional data file.Protocol S4Demographic database and population estimate procedures. (S4.1) GRUMP-beta human population surface. (S4.2) Areal prediction procedures.(DOCX)Click here for additional data file.Protocol S5Mapping the prevalence of G6PDd in females. (S5.1) Overview of G6PDd in females. (S5.2) Heterozygous G6PDd expression and diagnosis. (S5.3) Overview of female data in the G6PD database. (S5.4) Modelling phenotypic G6PDd prevalence in females. (S5.5) Maps of G6PDd in females and population estimates. (S5.6) Improving the map of G6PDd in females.(DOCX)Click here for additional data file.Protocol S6Developing an index of overall national-level risk from G6PD deficiency. (S6.1) G6PDd variants database. (S6.2) Generating an index of national-level risk from G6PDd. (S6.3) Generating an uncertainty index of the national-level risk index categories.(DOCX)Click here for additional data file.Table S1National-level demographic metrics and G6PDd allele frequency and population estimates.(PDF)Click here for additional data file.Table S2National areal prediction summary statistics and Monte Carlo standard error (SE) for each model output.(PDF)Click here for additional data file.Table S3Reported observations of class II and III G6PD variants from malaria endemic countries.(PDF)Click here for additional data file.


HPV encoded proteins can elicit ectopic protein–protein interactions that re-wire signaling pathways, in a mode that promotes malignancy. Moreover, accumulating data related to HPV is now providing compelling substantiation of a central role played by HPV in escaping immunosurveillance and impairment of apoptotic response. What emerges is an intricate network of Wnt, TGF, Notch signaling cascades that forms higher-order ligand–receptor complexes routing downstream signaling in HPV infected cells. These HPV infected cells are regulated both extracellularly by ligand receptor axis and intracellularly by HPV encoded proteins and impair TRAIL mediated apoptosis. We divide this review into different sections addressing how linear signaling pathways integrate to facilitate carcinogenesis and compounds that directly or indirectly reverse these aberrant interactions offer new possibilities for therapy in cancer. Although HPV encoded proteins mediated misrepresentation of pathways is difficult to target, improved drug-discovery platforms and new technologies have facilitated the discovery of agents that can target dysregulated pathways in HPV infected cervical cancer cells, thus setting the stage for preclinical models and clinical trials. There is an overwhelming list of research work that underlines the fact that HPV encoded proteins control cell cycle progression, apoptosis and cell differentiation, and have emerged as fundamental regulators of cervical cancer. Recent studies have revealed a complex network of protein interactions in HPV infected cells, and have connected HPV encoded proteins with other key signaling pathways. Such crosstalk has uncovered novel roles for signalings, including regulation of TGFβ/SMAD, WNT/β-catenin and Notch signaling cascades by HPV encoded proteins during carcinogenesis. This review highlights recent findings and trends in the HPV infected cervical cancer with an emphasis on how the HPV encoded proteins integrate with other pathways to promote cervical cancer. Moreover, several clues related to role of TRAIL mediated signaling in HPV infected cervical cancer cells are discussed. It also provides a better understanding of role of miRNAs in HPV infected cervical cancer cells. We also review recent patterns and approaches which have been used to induce apoptosis in HPV infected cervical cancer cells. Oncogenic proteins, such as those encoded by HPV, frequently form ectopic signaling complexes to re-constitute cellular behavior and exemplify how improved understanding of the HPV associated mechanisms might be translated into clinical benefit.Histological studies provide classification of cervical cancer. Therefore it can be characterized into different sub-categories, including squamous cell carcinomas (SCC) and adenocarcinomas (AdCAs). SCCs develop via well-defined precursor stages, called cervical intraepithelial neoplasia , however precursor stages for AdCAs are less well characterized. The precursor lesions to cervical cancers are known as cervical intraepithelial neoplasia (CIN) and noatably CIN1 lesions are referred to as low-grade CIN whereas CIN2 and 3 lesions together are considered high-grade CIN . Co. Co83]. Targeted inhibition of HPV16-E6 resulted in restoration of sensitivity to TRAIL . There iResearchers are targeting the anti-apoptotic machinery and associated signaling cascades that impair TRAIL mediated apoptosis. Wogonin, a flavonoid isolated from the root of the medicinal herb Scutellaria baicalensis Georgi was reported to be useful in sensitizing cervical cancer cells to TRAIL . It has Phenylethyl isothiocyanate (PEITC) increased the expression of the DR4 and DR5 in cervical cancer cells . LikewisDR5 promoter contains multiple Sp1 binding sites, which may contribute to the increased DR5 expression ,110. Sp1It is becoming successively more understandable that nanoparticles (NPs) have become an important tool in many industries including healthcare. Substantial fraction of information has revealed that compared with free antitumor drugs, drug-loaded long-circulating nanovectors show prolonged circulation time in plasma, enhanced accumulation in tumor tissues, and better-quality therapeutic activity. Functionalizing nanovectors with targeting moieties can promote specific receptor-mediated endocytosis, limiting non-specific uptake into the normal tissues. TRAIL has also been conjugated to different nanocarriers to improve the specificity of the delivery system and it has been shown that a nanocomplex system between the positively charged TRAIL and the negatively charged chondroitin sulfate (CS) (CS/TRAIL) was designed and applied in poly (lactide-co-glycolide) (PLGA) microspheres (MSs). The results indicated that TC-loaded PLGA MSs significantly inhibited tumour growth . FurtherTRAIL resistance has been frequently observed in cancer cells and different approaches are being tested to overcome the TRAIL resistant phenotype. There are different subsets of anti-apoptotic proteins which are over-expressed thus inducing resistance against TRAIL. Results have shown that natural flavonoid chrysin inhibited STAT3 phosphorylation thus repressing transcriptional regulation of Mcl-1. Proof of the concept was provided by treating cervical cancer cells with STAT3-specific inhibitor, cucurbitacin-I, which decreased Mcl-1 levels and enhanced TRAIL-induced cell death . LikewisIt is essential to investigate role of HPV encoded proteins in suppressing TRAIL mediated apoptosis. How HPV encoded proteins mediate expression of TRAIL, DR4/DR5 and DcRs is insufficiently studied. It is astonishing to note that HPV16 E2 and E6 are RNA-binding proteins and contain a protein-RNA interaction domain in their C-terminal regions. In addition, E2 and E6 interact with multiple cellular splicing factors like serine/arginine (SR) proteins . This reIntegrative genomics and genetics approaches have proven to be a functional tool in elucidating the complex relationships often found in gene regulatory networks and reconstitution of tumor-suppressive miRNA, or sequence-specific knockdown of oncogenic miRNAs by ‘antagomirs,’ has produced favorable antitumor outcomes in experimental models. We discuss existing knowledge gaps that need to be bridged prior to the consideration of miRNA-based experimental cancer gene therapy. These include our incomplete understanding of rate-limiting cellular components that impact the efficiency of this posttranscriptional gene-silencing phenomenon in HPV expressing cervical cancer cells. We partition this section into regulation of miRNAs by p53 and miRNA subsets which are documented to suppress and promote cervical cancer. We partition this section into regulation of miRNAs by p53 and miRNA subsets which are documented to suppress and promote cervical cancer.It is now clear that HPV encoded proteins target p53 to inhibit apoptosis of host cells. In the next section we discuss subsets of miRNA which are known targets of p53 and are inhibited by degrading p53. Detailed studies suggested that cortisol induced HPV-E6 expression and suppressed p53 and miR-145 in cervical cancer cells. MiR-145 expression in cervical cancer cells was wild-type p53-dependent, and cortisol down-regulated miR-145 expression . miR-23bHPV encoded proteins use methylation machinery to suppress tumor suppressor miRNAs and there is a direct piece of evidence that reveals hypermethylation of miR-124a and miR-203 in the precursor lesions . There iPhosphoinositide 3-kinase catalytic subunit delta (PIK3CD) is a miR-125b target and cells reconstituted with miR-125b represented inhibition of PI3K/Akt/mTOR pathway , while Bid was up-regulated in miR-125b-overexpressing cells . MiR-384miR-17-5p and miR-143 act as tumor suppressors in cancer cells by targeting TP53INP1 and Bcl-2 respectively –142]. F. F142]. It is noteworthy that transiently transfecting pre-miR-34c-3p, in HPV positive cervical cancer cells caused S-phase arrest and apoptosis . It is wmiR-10a, miR-205 and miR-133b are upregulated in cervical cancer and promote migration and invasion –156]. C. C156]. There is a complicated network by which miRNA subsets are transcriptionally triggered by downstream effectors of various signaling cascades and in turn miRNA subsets regulate modulators of signaling cascades. How HPV encoded proteins reconstitute signaling, transcriptional and epigenetic machinery to regulate tumor suppressor miRNAs and oncomirs still is a mystery.On a similar note, Arsenic trioxide induced cervical cancer apoptosis by downregulating HPV-E6 and upregulating p53 . There iIt was shown that targeted inhibition of E6 and E7 resulted in rescue of p53 Lee et al. , Maher eGRIM-19 has been acclaimed as tumor suppressor as cells reconstituted with GRIM-19 displayed ubiquitination and degradation of E6AP, and disrupted the E6/E6AP complex. The abrogation of E6/E6AP complex protected p53 from degradation and promoted cell apoptosis . It is iIn line with this approach, it has been proved that HPV E2 is negative transcriptional modulator of HPV E6 and E7 oncogenes, and also an apoptosis-inducing agent. There is an increasing trend of transiently transfecting tumor suppressor genes into cancer cells to enhance the efficacy of chemotherapy and radiations. A recent report indicated that oncolytic adenovirus armed with human papillomavirus E2 gene in combination with radiation demonstrated considerably augmented antitumor efficacy –83]. Si. Si83]. Signaling networks in cells are composed of upstream and downstream subnetworks. The upstream subnetwork contains the intertwined network of signaling pathways, while the downstream regulatory part controls expression of tumor promoting, tumor suppressing, pro-apoptotic, anti-apoptotic and microRNA subsets in a context dependent manner. HPV encoded proteins have emerged as centrally positioned hubs in regulation of signaling cascades in cervical cancer. Recent studies have revealed an extraordinarily complex network of proteins that is regulated by HPV encoded proteins. This highly interconnected network contrasts our conventional view of the cervical cancer as a linear sequence of events. It has lately been shown that Hh signaling is not induced directly by HPV-encoded proteins instead Hh-activating mutations are selected in cells initially immortalized by HPV . DarinapUnquestionably, the growing interest in this class of regulatory RNAs will lead to continued classification of miRNA expression particularly in cervical cancer and recognition of novel miRNA subsets that may act as oncogenes and tumour suppressors. Moreover, it is essential to develop a multi-level cross-talk network of the Notch, Wnt, TGF-β and SHH pathways, identify mutual and pathway-specific components/regulators and predominantly how HPV encoded proteins mastermind cross-talk between these pathways and other pathways.All the authors declare that they do not have any conflict of interest.TAH and FZ conceived the idea and did literature search on specific points. AAF and TAH did the literature search, integrated different points, and drafted the manuscript. TAH and FZ were involved in discussion and editing the manuscript. AAF designed the diagrams. All authors read and approved the final manuscript.


KIT were upregulated in specimens from patients with poor prognosis. Our pilot study identified several gene expression differences which could be used in the future as prognostic biomarkers in MCC patients.Due to the rarity of Merkel cell carcinoma (MCC), prospective clinical trials have not been practical. This study aimed to identify biomarkers with prognostic significance. While sixty-two patients were identified who were treated for MCC at our institution, only seventeen patients had adequate formalin-fixed paraffin-embedded archival tissue and followup to be included in the study. Patients were stratified into good, moderate, or poor prognosis. Laser capture microdissection was used to isolate tumor cells for subsequent RNA isolation and gene expression analysis with Affymetrix GeneChip Human Exon 1.0 ST arrays. Among the 191 genes demonstrating significant differential expression between prognostic groups, keratin 20 and neurofilament protein have previously been identified in studies of MCC and were significantly upregulated in tumors from patients with a poor prognosis. Immunohistochemistry further established that keratin 20 was overexpressed in the poor prognosis tumors. In addition, novel genes of interest such as phospholipase A2 group X, kinesin family member 3A, tumor protein D52, mucin 1, and  Merkel cell carcinoma (MCC) was first described in 1972 by Toker, which was initially thought to be derived from sweat glands . In factPrognosis of MCC is poor. Patients presenting with distant disease have two-year survival of only 32% . Only 50Due to rarity of MCC, prospective clinical trials have proven unfeasible . More reWe interrogated the Beaumont Hospital tumor registry for all patients treated for MCC within the last 10 years; 62 patients were identified. Patients were excluded who did not have tumor resection or tissue diagnosis at our institution due to lack of access to specimen. Patients were also excluded with inadequate followup or if their specimens failed to pass quality control measures for gene expression studies. All tissue paraffin block specimens were retrieved from anatomic pathology for validation and identification of disease by a single dermatopathologist (RM). After verification of tissue, MCC patients were stratified into one of three groups based upon status at 24 months following diagnosis. Poor prognosis patients either presented with or progressed to distant metastasis. Patients were considered to have a moderate prognosis if they presented with recurrent local disease or nodal disease at presentation but had no progression during followup of fewer than 24 months. Patients with favorable prognosis had local disease with no nodal disease at presentation and no progression during followup of at least 24 months . Seventeμm sections were cut from formalin-fixed paraffin-embedded (FFPE) tissue and mounted onto polyethylene naphthalate membrane glass slides (two sections per slide). Prior to deparaffinization, slides were placed into an oven at 60°C for 25 minutes then stained and dehydrated through a series of graded ethanol and xylene steps. Within 3 hours of sectioning, the pathologist-identified tumor areas were microdissected with both UV and IR lasers, where appropriate, using the ArcturusXT Microdissection System (Molecular Devices) onto CapSure HS LCM Caps (Molecular Devices). Four caps were used per graded region from the dissection of 1–4 sections representing approximately 5,000 tumor cells (1-2 mm2 area) captured per cap.For LCM, 5 Total RNA was isolated from the LCM tissues using the PureLink FFPE RNA Isolation Kit (Invitrogen). The transfer film with the attached dissected material was separated from each CapSure HS LCM Cap and placed in deparaffinization melting buffer at 72°C for 10 min and then treated with Proteinase K at 60°C for 45 min. Following the manufacturer's protocol, total RNA was purified using spin cartridge technology, treated with DNase, quantified , and stored at −80°C. RNA integrity was determined just prior to processing for expression microarray analysis. RNA integrity numbers ranged from 2.0 to 2.4, as determined by Bioanalyzer analysis (Agilent).μg amplified cDNA and the Encore Biotin Module following the manufacturer's recommendations. Hybridization, washing and staining , and scanning (with GeneChip Scanner 3000) were performed following manufacturer's protocols. Array scans from this process yielded signal intensities comparable to arrays prepared from high quality RNA from cell lines (data not shown). This method has also been independently reported recently to provide optimal RNA hybridization [Total RNA was amplified and labeled using the WT-Ovation FFPE System V2, WT-Ovation Exon Module, and the Encore Biotin Module , which enables amplification and target preparation of the low quality RNA from FFPE, LCM samples. Amplifications were performed with 100 ng total RNA input following procedures described in the WT-Ovation FFPE System user guide. Target preparation for Affymetrix Human Exon 1.0 ST Arrays  was performed using 5 dization .The CEL files containing the raw intensity data from the Affymetrix GeneChip arrays were imported into Partek Genomics Suite  and normalized using the robust multichip average with a guanine-cytosine content background correction, quantile normalization, log2-transformation, and median polish probeset summarization. Exons were then summarized to genes using the average of the probesets. Differentially expressed genes were identified using ANOVA with two factors: prognosis and scan date (random variable). Unsupervised hierarchical clustering was carried out using Partek software . Hierarchical clustering analysis was performed using Euclidean distance as similarity measure and average linkage for the agglomerative method. Gene set enrichment analysis (GSEA) and pathway analysis were performed using Pathway Studio 9.0 . The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are Tissue sections from the corresponding FFPE tissue blocks were placed on slides, underwent high pH antigen retrieval, and were subsequently incubated with one of the following primary antibodies: mouse monoclonal anti-human keratin 20 , mouse monoclonal anti-human mucin 1 , and rabbit polyclonal anti-human KIT , rabbit polyclonal anti-human KIF3A . The slides were then linked and visualized using the Dako Envision Flex system on a Dako Autostainer. Immunohistochemistry was scored based on intensity of staining with a 4-tier system . To address interobserver variability, scoring was performed by 2 independent investigators, and scores were averaged. Fisher's exact probability test was conducted comparing good and poor prognosis cohorts .The patient demographics and lesion characteristics are outlined in Laser capture microdissection was used to isolate and remove as pure a population of tumor cells as possible based on morphological identification by a dermatopathologist. n = 7) versus those with good prognosis (n = 5), 191 genes were found to be differentially expressed . This includes 127 genes that are overexpressed in tumors from poor prognosis patients and 64 genes that are underexpressed. NEFM , PLA2G10 , and KRT20 (keratin 20) are upregulated more than 3-fold in tumors with poor prognosis. CYP2A6 , MCART1 , and KRTAP19-5 (keratin associated protein 19-5) were downregulated at least 2.8-fold in poor prognosis samples.The 17 samples of MCC were analyzed using the Human Exon 1.0 ST array from Affymetrix. Two arrays (one good prognosis and one poor prognosis) failed to pass array quality control metrics. In a comparison of the patients with poor prognosis . This agrees with the overexpression seen in the gene level analysis. KIF3A protein was detectable in 1 of 6 poor prognosis patient samples (P ≤ 0.54).Several differentially expressed genes were further explored at the protein level. Immunohistochemistry was utilized to examine the protein products of MUC1, KIT, KIF3A, and KRT20 . While t samples , but no  samples . MUC1 prDiagnosis of Merkel cell carcinoma can be difficult. Current treatment consists of surgical removal with very wide excision borders. Virtually, all patients receive radiation following wide surgical excision, while patients with positive lymph nodes or metastatic disease at time of diagnosis may be candidates for chemotherapy. No standard chemotherapy protocol has yet been established for the treatment of MCC. Because of the morphological and immunohistochemical similarity of MCC to small cell lung carcinoma (SCLC), chemotherapy has been performed with protocols based largely on agents active in SCLC. A wide variety of chemotherapeutic agents have been discussed, including cytostatic drugs such as cyclophosphamide, doxorubicin, epirubicin, vincristine, etoposide, cisplatin, carboplatin, 5-fluorouracil, dacarbazine, mitoxantrone, bleomycin, and iphosphamide. Unfortunately, reports to date consist of only small studies and anecdotal evidence –24. TherThe ultimate goal of the study was to find markers that could identify poor prognosis patients at the time of diagnosis; these patients could benefit from increased or altered postsurgical therapy. In order to discover these novel biomarkers, expression microarray technology was utilized to examine global gene expression differences between patients that demonstrated a known prognosis (good prognosis or poor prognosis) based upon status 24 months following resection. This analysis resulted in a set of 191 genes being identified as differentially expressed. Hierarchical clustering of the patient samples based upon the expression of a subset of these genes  demonstrKRT20, tumor protein D52 (TPD52), MUC1, and KIT are upregulated in the poor prognosis tumors while homeobox B1 (HOXB1) is downregulated. TPD52, upregulated nearly 2-fold in tumors of poor prognosis patients, has been shown to be overexpressed in colorectal cancer [TPD52 has been seen in prostate cancer [KIT is also upregulated in the poor prognosis specimens. KIT is a transmembrane receptor for mast cell growth factor, also known as stem cell factor. Studies with MCC cell lines show that KIT is activated by paracrine or autocrine tumor cell-derived SCF which stimulates growth of MCC in vitro [HOXB1, which is downregulated in poor prognosis MCC, is a highly conserved transcription factor that plays an important role in morphogenesis and has been shown to be involved the metastatic potential of pancreatic cancer [Further characterization of the expression microarray data using subnetwork enrichment analysis revealed that gene expression associated with neoplasm metastasis and neurite outgrowth was highly regulated between samples of patients with a good and poor prognoses. The neoplasm metastasis associated genes l cancer  and ovarl cancer , and gene cancer  and breae cancer . KIT is in vitro . In one in vitro . HOXB1, c cancer .NEFM and PLA2G10. Both of these genes are upregulated more than 3.5-fold in tumors of poor prognosis patients. NEFM encodes a type IV intermediate filament medium chain and is commonly used as a biomarker of neuronal damage. In one study, neuroendocrine cell lines expressed NEFM while nonneuroendocrine cell lines did not [Genes associated with neurite outgrowth include the genes for  did not . Along w did not .DDAH1), kinesin family member 3A (KIF3A), and MUC1 which are all upregulated in the poor prognosis samples. The enzyme encoded by DDAH1 is involved in nitric oxide generation through regulation of methylarginines. Nitric oxide metabolism has been implicated in carcinogenesis, tumor progression, angiogenesis, and response to therapy [DDAH1 resulted in increased tumor growth and vascularization in a model of glioma tumorigenesis [In addition to these genes, there were other genes that were of interest and associated with cancer development or progression including dimethylarginine dimethylaminohydrolase 1 , KRT20, and KIT were upregulated in MCC compared to SCC just as these 3 genes were upregulated in poor prognosis patient samples compared to good prognosis patient samples. In addition, functional classes found to be altered between MCC and SCC included categories such as nervous system development, neuron development, and neuron projection morphogenesis which is similar to those found in The results of this study further develop a recently published gene expression analysis by Harms et al. . While oKRT20, KIT, MUC1, and NEFM has been identified in earlier MCC studies. In addition, several new potential biomarkers were identified. Genes such as TPD52, HOXB1, and KIF3A have been implicated in other cancer types and are connected with MCC-associated signaling. Furthermore, KRT20 demonstrated elevated protein levels in poor prognosis patient samples. This complements results from Harms et al. showing elevated KRT20 protein in MCC compared with SCC. From these results, we conclude that genes such as KIT, HOXB1, and KIF3A may provide valuable insights into the biology of MCC and the possibility remains that novel therapeutic modalities may be discovered with continued understanding of these biomarkers. Additionally, protein expression levels of KRT20 will be of importance in evaluating the prognosis of MCC. Given the limited sample numbers in this pilot study, we aim to expand this study through collaborations with other institutions. Ongoing studies, including further analysis of data with longer patient followup and an assessment of the influence of polyomavirus in these patients, will be necessary in order to elucidate the ultimate potential of these new biomarkers.In recent years, research has made rapid progress in identifying biological features of Merkel cell carcinoma; nevertheless, MCC represents a considerable challenge due to its relative rarity. While multiple studies have identified several biomarkers, prognostic value of these biomarkers has yet to be determined. This study is among the first to use LCM-captured MCC cells to investigate specific gene expression changes associated with prognosis of the tumor. Several of the gene expression changes presented here further implicate their role in MCC. Gene expression of Table S1: lists the 191 genes that are differentially expressed between patients with good prognosis and those with poor prognosis (p ≤ 0.05 and 1.5-fold cutoff). Genes selected for follow-up protein analysis by immunohistochemistry are noted.Click here for additional data file.


Extensive experimental work has conclusively demonstrated that infection with certain types of human papillomaviruses, the so-called high-risk human papillomavirus (HR-HPV), represent a most powerful human carcinogen. However, neoplastic growth is a rare and inappropriate outcome in the natural history of HPV, and a number of other events have to concur in order to induce the viral infection into the (very rare) neoplastic transformation. From this perspective, a number of putative viral, host, and environmental co-factors have been proposed as potential candidates. Among them oxidative stress (OS) is an interesting candidate, yet comparatively underexplored. OS is a constant threat to aerobic organisms being generated during mitochondrial oxidative phosphorylation, as well as during inflammation, infections, ionizing irradiation, UV exposure, mechanical and chemical stresses. Epithelial tissues, the elective target for HPV infection, are heavily exposed to all named sources of OS. Two different types of cooperative mechanisms are presumed to occur between OS and HPV: I) The OS genotoxic activity and the HPV-induced genomic instability concur independently to the generation of the molecular damage necessary for the emergence of neoplastic clones. This first mode is merely a particular form of co-carcinogenesis; and II) OS specifically interacts with one or more molecular stages of neoplastic initiation and/or progression induced by the HPV infection. This manuscript was designed to summarize available data on this latter hypothesis. Experimental data and indirect evidences on promoting the activity of OS in viral infection and viral integration will be reviewed. The anti-apoptotic and pro-angiogenetic role of NO (nitric oxide) and iNOS (inducible nitric oxide synthase) will be discussed together with the OS/HPV cooperation in inducing cancer metabolism adaptation. Unexplored/underexplored aspects of the OS interplay with the HPV-driven carcinogenesis will be highlighted. The aim of this paper is to stimulate new areas of study and innovative approaches. These additional events could be either the consequence of long-term viral protein expression, or the results of non-viral factors. In the search for such co-factor(s), many viral, host, and environmental factors have been investigated [2 to H2O during mitochondrial oxidative phosphorylation, as well as during microsomal and peroxisomal oxidations. In addition, RONS are also generated during a number of processes such as inflammation, infections, and immune reactions [Cervical cancer is one of the leading causes of cancer death among women. The identification of HR-HPV infection as the etiologic agents of cervical cancer, as well as other non-genital carcinomas, is undoubtedly among the greatest medical advances that have been made in the last twenty years . Accordistigated . The roleactions , mechanieactions ,5, chemieactions , exposureactions ,8, and ieactions .Epithelial tissues, the elective target for HPV infection, are heavily exposed to all named sources of OS. RONS, through oxidative damage, cause functional alteration of cell membrane lipids, proteins, and nucleic acids . In addi2 to H2O. Although highly efficient, the mitochondrial electron transport chain (ETC) is imperfect [Mitochondrial respiration is a fascinating biochemical process operating a coordinated four-electron reduction of Omperfect  and leakGeneration of the superoxide ion. The single electron (e-) reduction of molecular oxygen generates the radical superoxide ion (O2•- ) [ (O2•- ) , accordi2•- has the potential to react with almost all biological molecules, its direct contribution to cell damage is scarce, owing to its limited rate of diffusion and its comparatively low reactivity. Nonetheless, O2•- is able to sustain other cellular reactions generating extremely powerful RONS accountable for most of its toxicity. With the reaction (2)2O2). H2O2 and O2•- can further react according to the reaction (3), the “Haber–Weiss reaction,”1O2), is also generated as a by-product by the superoxide ion through the reactions (2) and (3) and by superoxide interaction with OH• [1O2 attacks the double bond in lipids with formation of hydroperoxides and endoperoxides. It also attacks and oxidizes methionine, tryptophan, histidine, and cysteine.This radical species is also produced within the cell by the action of some oxidases such as aldehyde oxidase, NADH oxidase, and xanthine oxidase, as well as under UV irradiation. Although Owith OH• . SingletGeneration of hydrogen peroxide. In addition to being generated by the divalent reduction of O2 and protonation during electron transports (reaction 4)2O2 is the primary product of many microsomal and peroxisomal oxidases. This non-radical ROS, with an intermediate reactivity between those of O2•- and OH•, is involved in thiol groups oxidation/inactivation and in redox potential imbalance [2O2 reduction to OH- plus OH• and oxidized metal ion. The transition metal ions Me(n+1)+ can then be reduced back to Men+ by the superoxide anion with the reaction (6)mbalance . Moreove2•- equivalents, thus demonstrating a potentially beneficial effect, but, on the other hand, it reconstitutes the pool of reduced metal ions, thus allowing a further cycle to the Fenton reaction and further generating hydroxyl radicals.This reaction, on one hand, scavenges OThe hydroxyl radical (OH•) is also formed under γ and x irradiation by direct lysis of water according to the reaction (7).OH• is the strongest oxidizing species; it reacts with almost all biological molecules with rates close to the theoretical diffusion limit. It gives place to reactions of hydrogen extraction, hydroxyl addition, and electron transfer. OH• is the major cause of oxidative adducts on purine and pyrimidine bases, as well as on the DNA sugar moiety, resulting ultimately in nucleic acid point mutation, cross-linking and strand interruption. The hydroxyl radical further concurs to DNA modification by the activity of lipid peroxidation products (LPO) . The mosThese are generated by OH• hydrogen extraction from unsaturated lipids in membranes and, following their direct purine attack, are able to produce exocyclic DNA adducts with high miscoding potentials .The nitric oxide (•NO), a radical RNS, is produced endogenously from L-arginine, oxygen and NADPH by several nitric oxide synthase (NOS) enzymes in response to a number of physiological stimuli. •NO is a very highly reactive species. With its short lifetime (a few seconds) and its ability to diffuse freely across membranes, •NO represents the ideal molecular messenger. As a paracrine or autocrine signaling molecule , it and itration .2O2 as a by-product. [2O2 leakage appears to be a physiological occurrence contributing significantly to OS as indicated by a direct correlation between the number of peroxisomes and their proliferation rate with the level of total cell OS and damage [2O2 leakage may be enhanced during rapid cell proliferation [Another major source of RONS is the peroxisome fatty acid beta oxidation which generates Hproduct. . Despited damage . Moreoved damage  suggestsferation .2 to O2•- [2 with generation of O2•- and organic intermediate restoration [Microsomal cytochrome P-450 enzymes metabolize xenobiotic compounds, usually drugs or alkaloids of plant origin, by catalyzing their univalent oxidation or reduction. Depending on which P450 isoenzyme is involved, these reactions consist either in the direct reduction of O to O2•-  or in thtoration .2•-; H2O2; •NO and hypochloric acid (HClO) [Inflammation increases the risk of cancer development and, at the same time, is a constitutive trait of any cancer tissues, thus cancer itself represents a cancer-promoting agent . During d (HClO) ,33, at lIn addition to its central role in ischemic conditions such as myocardial infarction and brain stroke, ischemia-reperfusion stress is, less obviously, a relevant mechanism in peripheral areas of proliferating lesions. OS represents a major pathogenetic momentum in ischemia-reperfusion and, surprisingly enough, its generation requires both hypoxia/anoxia and reperfusion phases . During etc. [RONS are also generated during the activation of a large number of signaling pathways induced by growth factors, cytokines and tumor promoters such as: TGF alpha; TNF alpha; TGF Beta-1, etc. . In turnetc. .More than one hundred oxidatively induced DNA lesions have been reported so far, and the current number may reasonably be even larger . Howevertrans-4-hydroxy-2-nonenal (HNE) is another one. These aldehydes, by direct DNA interaction, generate a number of DNA etheno-adducts such as 1N6 etheno-adenine (ε-A) and 3N4 etheno-cytosine (ε-C) [All of these have high miscoding potentials ,44. Modine (ε-C) ,22. Oncene (ε-C) , while tne (ε-C) . In addine (ε-C) ,51.2O2 accounts for a very minor part of it. Oxidized proteins are not repaired and must be removed through proteolytic mechanisms. The accumulation of oxidatively modified proteins in the cell lead to altered/disrupted functions, which can obviously induce, concur, or potentiate the homeostatic derangement which is the ultimate cause and the hallmark of cancer [It is clear that DNA is not the only molecular target of OS. Proteins are obviously prone to oxidative modification as well, and a number of oxidative modifications have been described . The mosf cancer ,56.2•− act as direct signaling factors; H2O2 represents the major oxidant signal [etc. [In addition to the structural alterations on DNA and proteins, RONS can interact with the carcinogenic process by modulating the gene transcription and the signal transduction pathways. The NF-kB; Nrf2 are two major cancer-relevant systems modulated by RONS ,58 and, t signal , and sigal [etc. ,60,61. Tal [etc. ,30,39,62In conclusion, aerobic life implies the generation of RONS. Due to their important physiological functions and sharp toxic activities, RONS represent major carcinogenic agents. It is widely accepted that HPV infection, through the expression of the E6 and E7 proteins, promote the degradation of the cellular proteins p53 and pRB, thus creating the conditions for the accumulation of cancer permitting genetic alteration. Thus, two different contributions of OS to HPV-driven carcinogenesis can be hypothesized: i) the mutagenic potentials of OS cooperate nonspecifically with the transforming activity of an HR-HPV infection. Such an additive mode can have important practical consequences, such as a shorter latency period or a higher efficiency in cancer induction; however, it does not imply any specific mechanism, rather, it represents a particular form of co-carcinogenesis; ii) OS can specifically modulate, or activate, or suppress, directly or indirectly, one or more HPV-specific molecular mechanisms orientating the viral/cell unit toward the transforming–carcinogenic process. The evidence about the latter hypothesis will now be reviewed and discussed.versus persistent infection, and neoplastic transformation. The first data suggesting a possible involvement of OS in these early phases initially came from indirect clinical–epidemiological observations: in a study aiming to identify additional conditions for cervical cancer, the concentration of diamines and polyamines was evaluated in 187 seminal plasmas and the concentration of diamine oxidases and polyamine oxidases was evaluated in 126 cervical mucus samples. The seminal amines are oxidized by amine oxidases in the cervical mucus and the hydroxyl radical, hydrogen peroxide and LPOs are generated. The results indicated that highly variable concentrations of amines and enzymes were found in seminal plasmas and mucus, respectively [et al., in a very large population-based study [PRDX3  is associated with higher persistence and higher progression risk. Molecular data consistent with the above indirect evidences were recently reported. In a very interesting study on viral replication in organotypic “raft” viral cultures, it was shown that, following HPV16 infection, complete viral progeny assembly is dependent on a tissue spanning redox gradient. Exposition to both reducing and oxidant condition is sequentially needed for infectivity maturation and redox balance modulation in the cell microenvironment and sharply affects the infectious titer of viral progeny [It is commonly assumed—although not proven—that cellular conditions at the early steps of viral infection  represent critical events for the outcome of HPV infection: spontaneously healing, productive infection ectively . It was ectively . The auted study , reporte progeny . These rAfter the viral infection, the core points of carcinogenesis are efficiency and accuracy of DNA replication. OS can strongly affect both, eventually creating the conditions for efficient viral integration. The first observation connecting OS with DNA papillomavirus replication came at the early beginning of the HPV and cancer story. In a BPV/mouse cell system, it was reported  that OS,in vitro and in vivo, while SV40 replication is [The connection between DNA damage sensing and HPV replication was further strengthened by the report of Moody and Laimins , showingation is . This laTaken together, the data above outline an OS-driven putative mechanism consistent with the viral DNA amplification and integration observed in cell lines derived from invasive cancers . Namely,i.e. above 10−6 M, a concentration easily attained during the macrophage/neutrophils activation, •NO induces apoptosis by nitrosating thiol groups of cellular proteins and enzymes [−8 M are essentially mutagenic [Apoptosis suppression is a fundamental mechanism for cancer development, permitting the survival of transformed clones and their selection on the basis of their fitness to the specific microenvironment. HPV-transformed cells achieve this goal expressing the E6 viral protein that promote the ubiquitination and the proteolytic degradation of the p53 cellular protein. A major contribution to the modulation of apoptosis is provided by the activation of the inducible form of nitric oxide synthase (iNOS), directing the production of •NO. As mentioned above, •NO is a powerful chemical messenger and cell regulator with a double-edged pro-survival and pro-apoptotic effect. This seemingly contradictory profile, however, can be resolved by considering the level of •NO concentration involved in each specific condition and the temporal–spatial mode of its release. At high concentration,  enzymes . Conversutagenic  and induutagenic ,78. Thusutagenic . In the utagenic ,80. Thisutagenic ,82. Accoutagenic . Meanwhiutagenic , thus coutagenic . Although the suppression of p53 functions is a pivotal event in HPV carcinogenesis, it has to be remarked that the HPV E6 expression in the context of its natural promoter does not suppress p53 activity completely, which remains measurable both in cell lines  and in c2O2-induced cell death through the up-regulation of catalase activity, the activation of NF-kB, and the orchestrated modulation of the cell factors: Bcl-xL; IL-18; Fas; Bad and Cytochrome-C [Other specific cooperative connections between OS and HPV carcinogenesis are sustained by the E7 protein. This oncogene increases the resistance to Hchrome-C . In addichrome-C . Thus, tA further OS-mediated regulation of viral oncogenes functions takes place at the transcription activation level. Early genes transcription is controlled by the E2 viral protein and by a wide array of cellular transcription factors . Among t+/H+ exchanger-dependent alkalinization of the intracellular compartments that, presumably by modifying the activity of the many redox sensitive cytoplasmic proteins, directly modulate the cell proliferation rate, the growth rate in low serum, and the anchorage-independent growth induced by E7 [An elevated rate of aerobic glycolysis, the so-called “Warburg effect” , is a veed by E7 .Data on OS in advanced cervical cancers has been steadily increasing since HPV infection was first proposed as a possible etiologic factor . In a faIndirect evidence also support the hypothesis that OS, in addition to being a hallmark of neoplastic growth, also has an active part in lesion progression. A large perspective cohort study showed that among women with a HPV-16 cervical infection, cigarette smoking, a powerful carcinogen with a wide range of different toxic activities, was associated with an increased risk of progression to CIN-III/invasive cancer . CigaretComplex data regarding advanced cancer and OS appear largely dispersed, somewhat recalling a patchwork of poorly related observations rather than an organic and consistent picture. Nonetheless, a direct correlation between an increasingly marked OS signature and the progression from mild dysplasia to severe dysplasia to invasive cancer is generally suggested by the majority of reports. Recent data, however, suggest a more articulated scenario indicating that, although an increasingly oxidant micro-environment is associated with tumor progression, tumor cells are characterized by a rather efficient control on RONS generation and oxidative damage attained by the appropriate up-regulation of antioxidant enzymes and detoxifying/pro-survival proteins ,122. ThiTheoretical consideration suggests OS and HR-HPV, two powerful initiating and promoting carcinogens, can act synergistically. Indirect clinical epidemiological evidence and initial biochemical data support the conclusion that viral infection, establishment of persistent-chronicle infection, and viral integration are indeed potentiated by OS. Both pro-survival and pro-apoptotic mechanisms have been described for OS; however, in viral dysplastic lesions, OS actually behaves as a pro-survival factor promoting the AP-1 mediated expression of E6 and E7, specifically reinforcing their anti-apoptotic mechanisms. A number of antioxidant enzymes and detoxifying pathways are consistently associated with HPV transformed cells that seem to be well equipped to fit highly oxidant environments. A number of questions, however, remain unanswered: No experimental evidence is available about the effect OS has on the regulation and functions of the E4, E5, L1 and L2 genes, as well as their proteins. Conversely, no data are available on the effect that E2, E4, E5 L1 and L2 viral proteins have on the OS generation, scavenging and repair. Apart from data on VEGF-mediated neo-angiogenesis, no conclusive data are available on the mechanisms involved in tissue invasion and remodeling, immune escape, cell shedding and metastasis homing. Only preliminary, non-systematic results are available on the role of OS in chemotherapy response/resistance and only initial attempts have been made to exploit OS signature as a potential marker for diagnosis and for clinical outcome of lesions. A large amount of work remains to be done.


Cancer is among the leading contributors to morbidity and mortality in the Pacific, but the magnitude of the problem and the potential for prevention have not been comprehensively studied. Over the past decade, cervical cancer has been the most common cancer among women in Fiji with an age standardised cervical cancer incidence rate of 51 per 100,000. This rate is among the highest in the South Pacific region and in the world. This high cervical cancer incidence rate is likely linked to the low cervical screening rate, but it points also to the possibility of a high burden of human papillomavirus (HPV) infection.We conducted a population-based survey in Fiji to provide information on human papillomavirus (HPV) prevalence, and the distribution of individual HPV types in a Fijian health-sub-district. We included 1,261 women aged between 16 and 64 years. A general primer GP5+/6+ mediatedpolymerase chain reaction (PCR) assay was used for HPV testing of 44 HPV types.The crude HPV prevalence in 1,244 women with an adequate HPV sample was 24.0% , 21.7-26.4%) and the corresponding age standardised prevalence was 25.5% . The prevalence of high-risk HPV types was 13.6% . Among 1,192 women with adequate cytological results, 13 (1.1%) showed cervical abnormalities, the majority of which were high-grade intraepithelial lesions or worse. HPV prevalence declined from 35.8% in women aged <25 years to 18.6% in those aged 55–64 years of age. After adjustment, the only variables significantly associated with HPV-positivity were age (ranging from odds ratio (OR) 0.57  for 25–34 year-old-women to OR 0.43  for 55–64 year-old-women) and ‘husband’s extramarital sexual relationships’ .These findings on HPV provide key information for future policy decisions on the most appropriate methods of cervical cancer prevention in Fiji and in the Pacific region. There has been considerable interest and research on non-communicable diseases throughout the South Pacific over the last 40 years . This haThe Fiji Islands consist of 322 islands and 840,201 inhabitants . The BriOver the past decade, cervical cancer has been the most common cancer among women in Fiji with an age standardised cervical cancer incidence rate of 51 per 100,000 . This raHPV genotyping of cervical cancer and cervical intraepithelial neoplasia specimens taken in 2003 to 2007 from 296 women at Fiji’s Colonial War Memorial Hospital, showed that HPV16 and 18 were the most common genotypes (77%), followed by HPV 31 (9%) and HPV 52 (7%) [We therefore conducted a study to determine the prevalence of HPV infection, cervical abnormalities, and other cervical cancer risk factors among a sample of women in the Valelevu health-sub-district of Suva, the Fijian capital. The study was a collaboration between Massey University and the International Agency for Research on Cancer (IARC), in partnership with the Fiji Ministry of Health’s Cervical Cancer Screening Programme.A total of 1,261 women aged 16–63 years (mean 36.9 years) participated in the study, and 944 (75%) lived in the study area. All 1,261 women provided a sample for HPV analysis, 17 were beta-globin negative, leaving 1,244 available for HPV analysis. A total of 1,192 cervical samples were adequate for, and assessed for, cytology.Table Figure Among the 1,244 women who had a valid HPV result, 39 (3%) had inadequate Pap smear results. Among women with adequate cytological results, cervical abnormalities were diagnosed in 13 women, of whom 84.6% were HPV-positive. The proportion of cytological abnormalities varied from 1.7% in women aged 25–34 years, down to 0.34% in women aged 35–44 years.Cytological abnormalities included four ASCUS , one low-grade abnormality (HPV45), one atypical squamous cells - cannot exclude high-grade squamous intraepithelial lesion (ASC-H) (HPV 42), and six high-grade abnormalities, , and one clinically detectable invasive cervical cancer (HPV16). All of the women with cytological abnormalities were recalled for follow-up.versus currently married women  and report of husband’s extramarital sexual relationships . A non-significant positive association was found between HPV positivity and nulliparity (compared with one full-term pregnancy) but no significant difference by number of full-term pregnancies. The association with being separated/divorced/widowed was mainly driven by findings in women aged 45 or older  in the general female population, similar to that found in the only other HPV prevalence survey conducted (to date) in the Pacific, in Vanuatu  . However, we did not have detailed information on the women’s sexual behaviour as it was considered inappropriate to ask questions about number of sexual partners to Fijian women in the current study. A positive association with being separated/divorced/widowed was present in the age-adjusted analysis and it was consistent with findings in previous IARC surveys . NotablyThe strengths of this study include the relatively large number of women surveyed and the comparability of study methods and our findings with those of other IARC surveys ,18-21. TOur study found that only 1% of Pap smears were abnormal, which is much lower than would be expected given the high HPV prevalence and from previous reports in Fiji where one in 10 Pap smears were abnormal , and in Cervical cancer is the most common cancer for women in Fiji. Our findings provide essential information for future policy decisions on the most appropriate methods of cervical cancer prevention in Fiji and in the Pacific region as a whole, particularly in light of the recent development of vaccines against HPV16/18 , were included in the study.A standardised questionnaire, similar to that used in other IARC-coordinated HPV surveys , was admDuring community meetings, and individually prior to sample collection, clients were given an information sheet about the study, which was also explained in English, Fijian or Hindi by one of the public health nurses. The nurses also did the Pap smears and took the specimens for HPV testing.The cervical cell sample collection methodology has been described elsewhere . BrieflyThe Pap smears were read and classified at the main tertiary hospital  laboratory of Fiji. During a one month period between September and October 2010 (due to acute laboratory staff shortage) 70 smears were sent for reporting at Medlab Pathology, Auburn, Australia which collaborates with the Fiji laboratory. Women with visible abnormalities or abnormal cytology were managed according to local protocols.HPV DNA analysis was performed at the VU University Medical Centre, Amsterdam, The Netherlands according to a protocol similar to that for previous IARC HPV prevalence surveys ,18. BetaAge-standardised HPV prevalence was estimated to allow comparisons with other IARC surveys , using fASC-H: Atypical squamous cells – cannot exclude high-grade squamous intraepithelial lesion; ASCUS: Atypical cells of undetermined significance; EIA: Enzyme immunoassay; IARC: International agency for research on cancer; OR: Odds ratio; HPV: Human papillomavirus; HR: High-risk; LR: Low-risk; PCR: Polymerase chain reaction.The authors declare that they have no competing interests.SF led the daily field work, data collection and data entry, and contributed to the literature review and writing of manuscript. NB undertook the data analysis, produced the tables and figures, and contributed to the writing of the manuscript. NP provided a major contribution to the study design, data analysis and writing of the manuscript. PJFS & CJLMM undertook the HPV typing and DNA analysis of the cervical cell specimens. LW contributed to the survey field work and specimen collection at the health clinics. GC contributed to the development of the proposal, the literature review, data analysis and writing of the manuscript. SF contributed to the development of the proposal, the literature review and writing of the manuscript. All authors read and approved the final manuscript.