Background and objectives: Human papillomavirus (HPV) is the most prevalent etiological agent of viral sexually-transmitted infection. This study retrospectively evaluated the impact of a switch to a real-time PCR assay in the HPV prevalence and genotypes distribution by a quasi-experimental before-and-after approach. Materials and Methods: In total, 1742 samples collected from 1433 patients were analyzed at the UOC Microbiology and Virology of Policlinico of Bari, Italy. HPV DNA detection was performed using initially nested PCR and subsequently multiplex real-time PCR assay. Results: Statistically significant difference in HPV overall prevalence after the introduction of the real-time assay was not detected (48.97% vs. 50.62%). According to different extraction-DNA amplification methods, differences were observed in the prevalence rates of HPV-45, 68, 40, 42, and 43. The lowest prevalence for HPV-45 was observed in the Magna Pure-Real Time PCR group, while HPV-68, 40, 42, and 43 were less observed in the Qiagen-Real Time PCR group. After, a multivariate logistic regression, an increase in the prevalence of HPV-42  was associated with the multiplex real-time PCR assay. Conclusions: Although this study is a not a direct comparison between two diagnostic methods because it has a sequential structure, it serves to verify the impact of a new molecular assay on HPV distribution. Moreover, the stability of HPV prevalence over time suggests that the population composition and the behavioral variables did not likely change during the observation period. Our study proposes that the introduction of a molecular test for HPV detection may be related to changes of HPV genotypes distribution. Human papillomavirus (HPV) is still the most prevalent viral sexually-transmitted infection either in men or women. Clinically, it is characterized by a wide spectrum of manifestations, including premalignant lesions that regress spontaneously and malignant lesions evolving to cervical cancer (CC) .Worldwide, although cervical screening programs have contributed to a decrease in the incidence, CC continues to be the second most common cancer among women, with an estimated 266,000 deaths for year [Nevertheless, the use of combined tests to detect the presence of HPV DNA together with conventional cytology examination has been shown to greatly improve the ability to detect the pre-cancerous states ,4. NowadUntil a few years ago, PCR followed by the nucleic acid hybridization techniques were used to detect HPV genotypes. When compared to conventional cytology, these techniques provided more detailed information regarding HPV genotypes .Afterwards, for HPV diagnosis real-time PCR techniques were introduced. Their performance has significantly improved both the hands-on time and decreased contamination rates.This study covers three years of routine diagnostic data on HPV DNA and aims to retrospectively evaluate the impact on HPV prevalence and HPV genotypes distribution of a switch from a nested-based PCR to a real-time based PCR on genital samples collected from patients in the Apulia region. To address this issue, a quasi-experimental approach for evaluating the effect of the real-time based PCR on the HPV prevalence and the prevalence of the single viral genotypes was based on the application of multiple logistic regression.From January 2012 to December 2014, 1742 consecutive samples, including 1605 cervico-vaginal swabs from 1328 females and 137 urethral swabs from 105 males were collected. Multi samples for some patients were due to retesting in different times.Specimens were transferred to the laboratory of Molecular Biology, U.O.C. Microbiology and Virology, Azienda Ospedaliera-Universitaria, Policlinico of Bari, where they were analyzed.All procedures performed in studies involving human participants were in accordance with the ethical standards.Sample information  together with the data of patients for whom molecular testing was performed  were recorded in an anonymous database by changing sensitive data into alphanumeric codes. No clinical data associated with these specimens were available.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For this type of study, formal consent is not required. This study was approved by Ethics Committee  Azienda Ospedaliero-Universitaria “Consorziale Policlinico,” Bari.g for 15 min at 7 °C. The majority of supernatant was discarded but 200 µL of supernatant was retained to resuspend the pellet.A total of 2 mL of phosphate-buffered saline (pH 7.4)  was added to cervical-vaginal swabs, collected by a rigid cotton-tipped swab applicator , and vortexed. Then, 1 mL of phosphate buffered saline  was added to urethral swabs and vortexed. Finally, all samples were transferred to microcentrifuge tubes and they were stored at −20 °C until processing. To extract viral nucleic acids, microcentrifuge tubes were centrifuged at rcf = 15,700× From January 2012 to December 2013, DNA extraction was performed by automated QIAcube System , following the manufacturer’s protocols.From January 2014 to December 2014, the vilrral nucleic acids were extracted from the resuspended pellet using the automated MagNa Pure 96 system  according to the manufacturer’s instructions.From January 2012 to June 2013, the extracted DNA samples were subject to a nested polymerase chain reaction (PCR) amplification, using Ampliquality HPV-HS Bio Kit  following the manufacturer’s instructions.The method provides for a first amplification of the viral genome L1 region, followed by a nested PCR with biotinylated primers. PCR products were analyzed using 3% agarose gel electrophoresis with ethidium bromide to display DNA under ultraviolet light. Subsequently, PCR products were typed by using Reverse Line Blot Hybridization Ampliquality HPV-Type Kit .To assess the suitability of extracted DNA, the thiosulfate sulfurtransferase (TST) gene region (202 bp) was amplified at the first amplification.TM II HPV 28 Detection System , which targets the viral L1 region and provides simultaneous detection and genotyping of 28 HPV-types. Briefly, the detection consists of two PCR reactions (panel A and B). The panel A includes 14 high-risk HPV (HR/HPV)-types, while the panel B includes 5 HR and 9 low-risk (LR)-types. PCR was performed on the CFX96 Real-Time PCR system .From July 2013 to December 2014, the extracted DNA samples were subject to multiplex real-time PCR (mRT-PCR) by Anyplexp-values were corrected by Benjamini and Hochberg’s (BH) procedure with False Discovery Rate (FDR) <1% [Differences in HPV prevalence were evaluated by Chi-Squared test and Fisher’s test as appropriate. FDR) <1% . Pairwisp-values collected from the logistic regression models were corrected for multiple comparisons by BH procedure with FDR < 1%.To assess the association of the real-time assay on the prevalence of the HPV overall infection and the prevalence of each HPV genotype (dependent variables) on the analyzed samples (samples dataset), logistic regression analysis was performed. Due to the lack of birth date for 102 patients, missing ages were imputed by multiple imputation by fully conditional specification implemented in the Multivariate Imputation by Chained Equations (MICE) package implemented in the environment R. A predictive mean matching imputation model was specified on the assumption of missing at random ages and the number of iterations was set to 20. In particular, 50 imputed data sets were generated. For each dataset, a logistic regression model was generated, and the 50 models were pooled together by the function pool of the package mice. Globally, 27 logistic regression models were evaluated. All Logistic regression analysis is based on the assumption of independence of the variables. To verify this assumption, a reduced dataset only containing the first sample for each patient (patient dataset) was generated and all analyses were repeated on it. Odds ratio estimations of the logistic regression models based on the samples and the patients’ datasets were compared.Calculations of all statistical tests were performed by the open source environment R .From 1 January 2012 to 31 December 2014, 1742 samples  from 1433 patients  were analyzed.During the observation period, the number of analyzed samples increased with time from a minimum value of 306 in 2012 to an intermediate value of 666 and to a maximum value of 770 in 2014. Moreover, after the introduction of the real-time assay an increase of analyzed cervico-vaginal swabs (87.66% vs. 94.12%) and a percentage decrease of urethral swabs (12.34% vs. 5.88%) was observed.p-value = 0.719).From January 2012 to the end of June 2013, 535 samples were extracted by QIAcube System  followed by a nested-PCR technique. From July 2013 to the end of December 2013, 437 specimens were extracted by QIAcube followed by mRT-PCR. From January to December 2014, 770 samples were extracted by MagNa Pure 96 system (Roche) and amplified by mRT-PCR. The HPV prevalence in the three groups was 48.97% (262), 49.65% (217), and 51.16% (394), respectively  while HPV-68, 40, 42, and 43 were less observed in the Qiagen-Real Time PCR group  .In particular, after pairwise comparison, QIAcube-real time combination resulted in higher HPV-45 prevalence compared to Magna Pure-Real Time PCR (2.99% vs. 0.00%), in lower HPV-68, HPV-40, HPV-42 prevalence rates compared to both Magna Pure-Real Time PCR and QIAcube-Nested PCR  and in lower HPV-40 prevalence compared to QIAcube-Nested PCR (0.00% vs. 1.82%).On logistic regression analysis, the introduction of real-time assay was associated with an increase of HPV-42  . MoreoveThis study evaluated the HPV positivity rates and the HPV genotypes distribution before and after the switch from a nested-based PCR to a real-time PCR on a three-year observation window. Multivariate analysis showed that the HPV prevalence was not affected by the introduction of the new real-time assay. The sensitivity of a nested PCR compared to a real-time amplification method was analyzed by several authors, but the conclusions were not unique ,13,14.On the other hand, some differences in the distribution of HPV genotypes were detected. In particular, the pairwise comparison showed some statistically significant differences in the distribution of HPV-45, 68, 40, 42, and 43. The analysis also highlighted that these differences were due to the QIAcube-Real Time PCR group. Logistic regression analysis only confirmed that the Real Time introduction was associated with an increase of the prevalence of HPV-42 .Currently, more than 100 distinct molecular tests are available on the global market for the detection of HPV DNA . HoweverAn important source of variations is that the detection of specific HPV genotypes may be affected by the different sensitivities of the currently available genotyping methods.TM II HPV 28 Detection System, Linear Array HPV genotyping test, Gp5+/6+ PCR-EIA-RH, CLART HPV2 Assay) were compared with Hybrid Capture 2 and a concordance about 80% or higher was reported. In particular, the comparison of the genotype distribution between AnyplexTM and Linear Array, Gp5+/6+ and CLART2, respectively, showed completely different genotypes in five (4.0%), two (2.3%), and three (2.9%) samples.In a study of Del Pino et al.  several TM was compared with MolecuTech REBA HPV-ID and HPV DNAChip and the percentage of HPV genotype agreement ranged from 93.7% to 100.0%. However, relatively high rates of discordance between the three assays were reported for HPV 31, 42, and 44. The authors also examined the performance of the assays in the detection of five common HPV genotypes  and reported that the sensitivity rates varied according to the detection method. In particular, MolecuTech showed a low sensitivity for HPV 52 (42.9%) while AnyplexTM and HPV DNAChip had low sensitivities for HPV 45 (25.0%).In a study of Lim et al. , AnyplexTM with Euroarray on 150 samples by Latsuzbaia et al. [TM assay.Comparison of Anyplexa et al.  showed aTM, compared with HPV Sign Genotyping Test, detected more high risk and low risk genotypes. In particular, better agreements were reported for HPV 16, 18, 35, and 70. Statistically significant differences were reported for HPV 31, 51, 52, 53, 56, 58, 59, 66, 73, 6, 42, 44, 54, and 61. These genotypes, except HPV 73, were more frequently detected by AnyplexTM assay.Marcuccilli et al.  reportedTM with PGMY-CHUV assay. In particular, HPV 40, 42, 54, and 68 were significantly more frequently detected by AnyplexTM. On the contrary, HPV 51 was more frequently detected by the PGMY-CHUV assay.Differences in HPV genotype detection were also reported by Estrade et al.  after coThe sequential design does not permit to infer the different sensitivities or specificities of the two diagnostic tests. However, the quasi-experimental approach of the study may be advantageous to acquire some preliminary information to better design subsequent studies or to speculate regarding the clinical usefulness of a new diagnostic assay in a relatively cheap manner. In particular, the latter option may be of some interest in a lack of funds context. To our knowledge, this is the first time that a quasi-experimental before-and-after approach has been applied to the evaluation of a new assay for the HPV evaluation. In particular, the data suggest that the introduction of Real-Time PCR did not affect the HPV prevalence, but it was associated with modification of the distribution of HPV-42. Other studies will be needed to clarify the reasons for these variations. However, other limits of this study must be considered. In particular, the temporal window is quite short and the influence of pluriannual trends cannot be excluded. Moreover, the composition of the population analyzed is not known and the differences in the distribution of the HPV genotypes may reflect differences in the behaviors or risk factors of the analyzed patients. However, the presence of background trends was also excluded by the logistic regression analysis and this makes it possible to assume that the behavioral and social parameters of the analyzed population have not changed over time.Despite these limits, such study suggests to carefully evaluate the introduction of a new type test for HPV also regarding the impact on the distribution of HPV genotypes. In particular, it may be considered as a potential confounding factor for evaluation of public health programs to control HPV spreading in the populations. At the same time, our study highlights the importance to carefully evaluate temporal dynamics with multivariate methods. Related to this, the accumulation of more rich surveillance data should be encouraged to ensure that demographic shifts in infection patterns could be accurately monitored.Having in mind that any technical innovation is usually related to new perspectives for both clinical and technical development over time, the results obtained suggest that the temporal distribution of HPV genotypes might be influenced by the diagnostic methods that are performed.This study is not a direct comparison of two diagnostic methods because it has a sequential design. In summary, with univariate analysis significant differences were observed in the prevalence rates of HPV-45, 68, 40, 42, and 43. The lowest prevalence for HPV-45 was observed in the Magna Pure-Real Time PCR group, while HPV-68, 40, 42, and 43 were less observed in the Qiagen-Real Time PCR group.With logistic regression analysis, the introduction of the real-time assay was associated with an increase in HPV-42. Furthermore, the logistic regression analysis did not reveal the presence of underlying trends. It is possible that the difference in the HPV-42 distribution detected after the switch to the real-time test could be related to the different sensitivities of the two diagnostic assays or to the presence of cross-reactions.An important limitation of this kind of study is the potential confounding effect of some variables . Unfortunately, these data were not available for the analysis. However, despite the increase in number of subjects tested, the stability of HPV prevalence over time suggests that the population composition and the behavioral variables did not likely change during the observation period.Therefore, this parameter should be taken into the consideration when a multivariate statistical model is represented, and further clinical studies could increase knowledge on the prevalence of HPV based on the use of different methods.


Because both pollution emissions and production policies often are international in scope, it is necessary to find optimal coordination strategies for international production planning and pollution abating. Differential game models are developed for multiple neighboring countries to reach optimal decisions on their production planning and pollution abating under cap-and-trade regulations. Non-cooperative and cooperative differential games are presented to depict the optimal tradeoffs between production planning and pollution abating. Hamilton-Jacobi-Bellman (HJB) equations are then employed to analyze the asymmetric and symmetric feedback solutions. Numerical simulations are used to illustrate the results. Five different dividends are also discussed. With the proposed strategies, more improvement will be directed toward production supplies and environmental issues than ever before. Since the middle of the 20th century, the rapid population growth and economic development have led to serious environmental pollution that has critical impacts on crop yields, climate change, biodiversity, and ecosystems, amongst others. As a result, these impacts have significant social and economic consequences affecting values, customs, human health, economic development as well as social welfare . In receThe Paris climate agreement, a new global agreement to combat climate change within the United Nations Framework Convention on Climate Change (UNFCCC), was signed in 2016. The agreement aims at holding global warming to well below 2 °C and to “pursue efforts” to limit it to 1.5 °C . As of MPollution emissions often show the feature of being international, that is, there exists transboundary pollution which is a typical example of a negative externality and is also an essential instance of global environmental agreement failure . When a Given the concerns mentioned above, the key contribution of this study is on developing differential game models for multiple neighboring countries to reach optimal decisions by optimizing their production planning and pollution abating under cap-and-trade regulations. By using Hamilton-Jacobi-Bellman (HJB) equations, the impacts of the production efficiency and the initial allocations of emission permits on the optimal production plans and the pollution abating decisions can be determined by analyzing the feedback equilibria of the proposed models.The remainder of this paper is organized as follows. The relevant literature is reviewed in As a powerful analytical tool, the differential game has been widely used in many scientific fields. You, Jiang and Li  employed2 emissions through a capture and storage mechanism. This study employs differential games to balance optimally business production planning and pollution abating to optimize income.Many researchers have made significant efforts in searching for optimal strategies of pollution control, as shown in 2 capture and storage and transboundary pollution and provided its explicit short-run dynamics with symmetric open-loop and a specific Markovian Nash strategy. Li [2 pollution and fossil fuel exhaustibility. Li [Transboundary/international pollution emission and abatement are now becoming one of the hottest topics in scientific research. A differential game is one of the powerful tools for studying transboundary pollution emission and abatement. Long  employedtegy. Li  extendedtegy. Li  by takintegy. Li  considertegy. Li  studied tegy. Li  studied tegy. Li  studied lity. Li  studied lity. Li  involve Although at first glance there are many similarities in the literature mentioned above, there are always some subtle differences from an economic point of view, mainly reflected in their objective functions and stock dynamics equations of carbon or pollution. In this study, to depict the complex scenarios of coordination strategies for international production planning and pollution abating under cap-and-trade regulations, we try to integrate the pollutant stock level, the abatement effort, the production quantity, the initial allocation of pollution emission permit and the emission permit trading price into the objective function and stock dynamics equation. All notations used through the paper are summarized in Although usually only two countries are involved in transboundary pollution emissions, a more general case is considered in this study. Assume The production quantity The production activity of each country generates not only net revenue but also a level of pollution with negative effects on its welfare. The environmental damage cost As we know, the higher the pollutant concentration is, the more costly the damage is to the country. The welfare of one country depends not only on its own policies but also on those of other countries . HoweverAnalogous to human beings, a country’s abatement action also has egoistic and altruistic sides, i.e., the former acts for the country’s self-interest and directly benefits the country’s revenue, and the latter is helpful for the others. The total pollution abatement amount of country The trading revenue Apparently, the following three cases holds: From (2)–(6), the instantaneous revenue of country The more goods the In a non-cooperative differential game, each country independently chooses its production plan and pollution abatement to maximize its own discounted infinite-horizon utility as stated in (9) in the followingIn a cooperative differential game, In the following, From (8) and (9), the HJB equation for country The following optimal feedback production and abatement strategies of country Although the coefficients of the value functions in the following propositions can be explicitly characterized, for simplicity and regularity, they are characterized implicitly for all, including the symmetric, asymmetric, cooperative and non-cooperative, scenarios.Proposition 1.In the asymmetric non-cooperative differential game, the Nash equilibrium solutions of the value functions and instantaneous levels of production and abatement, represented by Proof of Proposition 1.Substituting the optimal solutions from (12) and (13) into the HJB equation in (11) and simplifying yieldDifferentiating (14) w.r.t. Substituting (14) and (21) into (20), then equating the coefficients of The ordinary differential equation in (22) in the following is obtained by directly substituting (15) and (16) into (8)The optimal trajectory of the pollutant stock in (23) in the following is obtained by solving (22)Remark 1.For the symmetric case, let From (8) and (10), the HJB equation for any country can be written as The following optimal feedback production and abatement strategies of the country are obtained by taking the first-order conditions of the HJB equation in (24) w.r.t. Proposition 2.In the symmetric non-cooperative differential game, the Nash equilibrium solutions of the value functions and instantaneous levels of production and abatement, represented by Proof of Proposition 2.Substituting the optimal solutions from (25) and (26) into the HJB equation in (24) and simplifying yieldDifferentiating (27) w.r.t. Substituting (27) and (34) into (33) and equating the coefficients of The ordinary differential equation in (35) in the following is obtained by substituting (28) and (29) into (8)The optimal trajectory of the pollutant stock in (36) in the following can be obtained by solving (35)The following HJB equation for country The following optimal feedback production and abatement strategies for country Proposition 3.In the asymmetric cooperative differential game, the optimal solutions of the value functions and instantaneous levels of production and abatement, represented by Proof of Proposition 3.Substituting the optimal solutions in (41) and (42) into the HJB equation in (37) and simplifying yieldDifferentiating (40) w.r.t. Substituting (40) and (47) into (46) and equating the coefficients of The ordinary differential equation in (48) in the following is obtained by substituting (41) and (42) into (8)The optimal trajectory of the pollutant stock in (49) in the following is obtained by solving (48)The HJB equation for the countries in (50) in the following is from (8) and (9) together with Remark 1The following optimal feedback strategies of production and abatement of the countries are obtained by taking the first-order conditions of the HJB equation in (50) w.r.t. Proposition 4.In the symmetric cooperative differential game, the optimal solutions of the value functions and instantaneous levels of production and abatement, represented by Proof of Proposition 4.Substituting the optimal solutions of (51) and (52) into the HJB equation in (50) and simplifying yieldDifferentiating (53) w.r.t. Substituting (53) and (60) into (59) and equating the coefficients of The ordinary differential equation in (61) in the following is obtained by substituting (54) and (55) into (8)The optimal trajectory of the pollutant stock in (62) in the following is obtained by solving (61)In the following, numerical results of the case of a duopoly game, i.e., https://www.theice.com/market-data/end-of-day-reports), NCS and CS scenarios, The selection of important parameter values in the models is similar to that in List and Mason . The folvidences ,35,36,37The optimal solutions of the value functions, instantaneous levels of production and abatement, and the optimal trajectories of the pollutant stock levels are obtained by substituting the above parameter values into (14)–(16) and (23) for NCA, (27)–(29) and (36) for NCS, (40)–(42) and (49) for CA and (53)–(55) and (62) for CS, respectively. After simplification, these optimal solutions are presented in the following.CaseNCACaseNCSCaseCACaseCSNCA, NCS, CA and CS as time Obviously, NCA, NCS, CA and CS as time Apparently, NCA, NCS, CA and CS as time Apparently, NCA, NCS, CA and CS as time Apparently, Definition 1.The cooperative dividend is the difference between optimal payoffs of the cooperative game and the non-cooperative game.Definition 2.A cooperative agreement is feasible if and only if the total optimal cooperative payoffs are greater than the sum of the individual non-cooperative payoffs.The so-called agreement feasibility refers to the collective rational choice of many parties. Definition 2 shows that a cooperative agreement is feasible if sum of individual payoffs of the cooperation strategy is more than that of the current non-cooperative strategy.CPsc denotes the cooperative dividend of a symmetric game, CPca denotes the cooperative dividend of an asymmetric game, CPcs denotes the symmetric dividend of a cooperative game, CPncs denotes the symmetric dividend of a non-cooperative game, and SCPcs denotes the cooperative and symmetric dividend of a game.Definition 3.The cooperative dividend of an asymmetric game is the difference between optimal payoffs of its asymmetric cooperative scenario and its asymmetric non-cooperative scenario.According to Definition 3, the cooperative dividend of an asymmetric game can be written as Definition 4.The cooperative dividend of a symmetric game is the difference between optimal payoffs of its symmetric cooperative scenario and its symmetric non-cooperative scenario.According to Definition 4, the cooperative dividend of a symmetric game can be written as Definition 5.The symmetric dividend is the difference between optimal payoffs of the symmetric game and the asymmetric game.Definition 6.A symmetric agreement is stable if and only if the total optimal symmetric payoffs are greater than the total optimal asymmetric payoffs.Definition 7.The symmetric dividend of a non-cooperative game is the difference between optimal payoffs of its symmetric non-cooperative scenario and its asymmetric non-cooperative scenario.According to Definition 7, the symmetric dividend of a non-cooperative game can be written asDefinition 8.The symmetric dividend of a cooperative game is the difference between optimal payoffs of its symmetric cooperative scenario and its asymmetric cooperative scenario.According to Definition 8, the symmetric dividend of a cooperative game can be written as Definition 9.The cooperative symmetric dividend of a game is the difference between optimal payoffs of its symmetric non-cooperative scenario and its asymmetric cooperative scenario.Definition 10.A cooperative symmetric agreement is stable if and only if the sum of the optimal cooperative symmetric payoffs is greater than the sum of the optimal asymmetric non-cooperative payoffs.According to Definition 9, the cooperative symmetric dividend of a game can be written asObviously, Results mentioned above show that symmetry and cooperation are not only helpful in reducing both pollutant stock levels and pollutant emission levels but also helpful to payoffs of all countries. These results also prove the Paris agreement’s idea which is that as each country implements its pledge, others can learn what is feasible, and that collaborative global climate protection will emerge. So, we can preliminarily reach the following five necessary judgments.First, the competition between countries is integral and an essential strategic factor. A country should consciously put competition factors into consideration in its global strategy.Second, in global competition, a country needs to build its comparative advantage, which is the basis of cooperation. Only by taking the initiative in the competition can it better carry out international cooperation.Third, from the perspective of global governance, the compromise between countries in international competition does not necessarily mean clandestine collusion against the value of other countries. On the contrary, it is likely to lead to a more effective allocation of global resources.Fourth, the formation of regional alliances similar to the European Union will help in improving the symmetry of participants, and also be more conducive to production efficiency and pollution reduction.At last, the future global climate agreements following the Kyoto Protocol and the Paris Climate Agreement should strive to enhance cooperation, symmetry, and transparency in international production and pollution reduction. In this study, infinite-horizon cooperative and non-cooperative differential games are proposed to describe strategies of production planning and pollution abating of These countries in the game simultaneously make two decisions—how much product to produce and how much pollution to abate under cap-and-trade regulations. In other words, each country makes decisions about how much pollution to abate or how much pollution emission right to buy or sell when deciding how much product to produce. Furthermore, the equilibrium solutions of the value functions, instantaneous levels of production and abatement, and the optimal trajectories of the pollutant stock levels vary at the beginning and finally reach their steady levels.Both symmetry and cooperation are helpful in reducing pollutant stock levels, abating pollution emission levels, and improving value functions.Dividends increase in the following order: The cooperative dividend of a symmetric game, the cooperative dividend of an asymmetric game, the symmetric dividend of a cooperative game, the symmetric dividend of a non-cooperative game, and the cooperative and symmetric dividend of a game.Both symmetry and cooperation are shown to have significant impacts on the production planning and pollution abating decisions. These impacts are:As concluded above, the cooperation of production plan and pollution emission significantly affect the decisions of the countries. However, how to share their information and how to improve their production cooperation among the The deterministic differential games are based on assumptions of specific environmental damages which make the problem tractable. Further development is to consider uncertain environmental damages for which a stochastic differential game will be appropriate.The ordinary differential games are based on the assumption that the environmental damage cost depends on the total pollutant stock level without considering its spatial diffusion. Partial differential games may be employed to consider the spatial diffusion.The differential games are only constrained by the dynamical pollutant stock levels. If the pollution emission permits can be traded with dynamical prices, two or more dynamical constraints may be considered as shown in Xin and Sun .The following extensions are of interest for future research:


Primary care practitioners (PCP) play key roles in cervical cancer prevention. Human papillomavirus (HPV) knowledge is an important influence on PCPs’ cervical cancer prevention-related behaviours. We investigated HPV knowledge, and associated factors, among general practitioners (GPs) and practice nurses.A survey, including factual questions about HPV infection and vaccination, was mailed to GPs and practice nurses in Ireland. Multivariable logistic regression was used to determine which PCPs had low knowledge . Questions least often answered correctly were identified.697 PCPs participated. For HPV infection, GPs and practice nurses answered a median of nine and seven questions correctly, respectively (p<0.001). Significantly associated with low HPV infection knowledge were: being a practice nurse/male GP; working fewer hours/week; not having public patients; and having never taken a cervical smear. For HPV vaccination, both GPs and practice nurses answered a median of six questions correctly (p = 0.248). Significantly associated with low HPV vaccination knowledge were: being a practice nurse/male GP; working more years in general practice, fewer hours/week, in a smaller practice or in a practice not specialising in women’s health; and having never taken a smear. Six HPV infection questions, and seven HPV vaccination questions, were not answered correctly by >⅓ of PCPs.There are important limitations in HPV infection and vaccination knowledge among PCPs. By identifying factors associated with poor knowledge, and areas of particular uncertainty, these results can inform development of professional education initiatives thereby ensuring women have access to uniformly high-quality HPV-related information and advice. The causal relationship between cervical cancer and “high-risk” human papillomavirus (HPV) infection is reshaping cervical cancer prevention strategies internationally . PrimaryPCPs can significantly impact women’s cervical cancer prevention behaviours. They may positively or negatively influence women’s screening participation decisions and decision-making around HPV vaccination –6. For eThe success of the new prevention strategies also requires that women (and the wider population) are adequately informed about HPV, understand new protocols/programmes, and find them acceptable. However, public awareness of HPV is poor, understanding is limited, and misconceptions are common –18. WomePrevious research suggests some PCPs have significant gaps in their HPV-related knowledge –28. HoweThe study setting was Ireland, which has a mixed public-private healthcare system. Citizens pay for primary care services unless they possess a medical card, eligibility for which is based on means and age. General practitioners (GPs) may choose whether to include medical card holders (“public patients”) on their patient list. The cervical screening programme, CervicalCheck, began national roll-out in 2008 and provides women aged 25–60 with free cytology tests every 3–5 years . Women mDuring May-August 2011, we conducted a cross-sectional postal survey of GPs and practice nurses.In total 1760 practitioners (880 GPs and 880 practice nurses) were sampled to be invited to take part in the survey. The GP sampling frame was the Irish Medical Directory, which is Ireland’s most comprehensive listing of healthcare professionals. Simple random sampling was used to select the sample. Since there is no national practice nurse register, half of the nurse sample was selected, using simple random sampling, from a list of members of the Irish Practice Nurses Association and half from practice nurses registered with CervicalCheck. All of the individuals on both nurse sampling frames were female.Sample size was based on statistical power to detect, as significant, associations between a personal or practice-related characteristic or cervical screening-related behaviour and risk of low HPV knowledge. “Low” knowledge was defined as the lowest quintile of respondents; we aimed for ≥100 practitioners in this group. Assuming the characteristic had a prevalence of 25%, 100 respondents with a “low” score and 400 with a “higher” score would have 82% power to detect, as statistically significant, an odds ratio of 2.0 . Based on assumption that the response rate would be 30% (consistent with other PCP surveys in Ireland), and allowing for some practitioners not being contactable, we decided to sample 1760 practitioners, evenly split between GPs and practice nurses.The questionnaire was 12 pages long, including the front and back cover and the embedded consent form; a copy is available from the corresponding author on request. Content was theoretically informed based on findings from our qualitative work . Ten facWe used several strategies that have been shown to increase response rates to postal surveys . SubjectEthical approval was provided by the Irish College of General Practitioners (ICGP). The ICGP Ethics Committee has responsibility for reviewing and approving research in primary care in Ireland.Practitioners were included in the analysis if they had answered at least half of the HPV infection or HPV vaccination factual questions. Two knowledge scores, one for infection and one for vaccination, were generated for each participant comprising the number of correctly answered questions. Correct/incorrect answers were defined based on what was known about HPV infection and vaccination at the time of the fieldwork. Hence, if an individual answered 8 of the 11 HPV infection questions, and their responses to 5 of these were correct, their infection knowledge score was 5 out of a possible 11. Similarly if they answered all 11 HPV infection questions and responses to 5 were correct, their knowledge score was 5/11.Respondents’ personal and practice characteristics were summarised in terms of numbers and percentages or means (with standard deviations) overall and for GPs and practice nurses separately. The knowledge score distributions were skewed so we summarised average scores for GPs and practice nurses in terms of medians and compared GPs’ and practice nurses’ knowledge score distributions using the non-parametric Wilcoxson rank-sum test. Practitioners with scores in the lowest quintile of the overall distribution were designated as having “low” knowledge. Multivariable logistic regression was used to identify factors associated with low knowledge, separately for infection and vaccination. Practitioners’ personal and practice characteristics and cervical screening behaviours  were con697 PCPs participated ; practice nurses: 459 (53%)). Of the 690 practitioners who answered at least half the questions, there were four (0.6%) who either answered zero or one question correctly, and 28 (4%) who answered all 11 questions correctly.The median number of correctly answered questions was eight. GPs answered more questions correctly than practice nurses  were not answered correctly by almost half of practitioners . More prFor HPV vaccination, of the 687 practitioners who answered at least half the questions, 10 (1.5%) answered zero or one question correctly and six (0.9%) answered all 10 correctly. The median number of questions answered correctly was six. There was no difference between GPs and practice nurses  is consistent with those typically reported . We do nThere are important limitations in PCPs’ HPV infection and vaccination knowledge particularly among male GPs and practice nurses. Characteristics of practitioners most likely to need support, and specific aspects of HPV-related knowledge about which practitioners are most often uncertain or incorrect, have been identified. The results can inform development of professional education initiatives to ensure that women have access to uniformly high-quality HPV information and advice.S1 Table(DOCX)Click here for additional data file.


And cervical exfoliated cells of high-grade squamous intraepithelial lesion (HSIL), the most severe precancerous lesion of cancer, were used in the study. However, it takes a long time to collect enough specimens to conduct statistical analysis. Therefore, in the present study, microscope slides, cervical exfoliated cells on glass slides, were attempted. The results of the analysis demonstrated that hTERT and Ki67 expression levels were useful in distinguishing between cancerous and normal specimens, exhibiting a higher sensitivity and specificity than conventional HPV E6/E7 testing. And the study suggests clinical slide cell samples could be effectively used in the context of retrospective studies to identify novel biomarkers.After breast and colon cancer, cervical cancer is the third most common cancer of women worldwide. Since human papillomavirus (HPV) infection is known to be the predominant cause of cervical cancer, molecular HPV screening is currently used along with cytological and histological examination methods for precancer diagnosis. Nevertheless, the sensitivity of the current HPV test is less than 80%; thus, many cervical cancer cases are not able to be diagnosed by HPV screening alone, and likewise, patients with cervical cancer are often determined to be HPV-negative by the current screening methods. Therefore, human telomerase reverse transcriptase ( Cervical cancer is the third most common cancer of women worldwide, after breast and colon cancer . The WorKnown risk factors for cervical cancer include human papillomavirus (HPV) infection, promiscuous intercourse, sexually transmitted disease infection, long-term hormonal contraceptive use, and smoking . Of thesCurrent “gold-standard” methods for precancer diagnosis are cytological and histological examinations. For cytological examination, exfoliated cervical cells are collected by swabbing the cervix (as part of a “Pap-smear” test), before being placed onto a slide to be inspected for abnormalities. In case of histological examination, it is diagnosed via a microscopic examination of a stained tissue biopsy . Both ofRecently, a molecular method via the identification of HPV nucleic-acid sequences was developed for use in conjunction with standard cytological and histological examination techniques, commonly .L1, E6, and E7, along with the additional cancer markers human telomerase reverse transcriptase (hTERT) and Ki67. hTERT represents the catalytic subunit of telomerase. Telomeres are highly specialized structures that are located at chromosome ends and are known to be essential for genome stability [hTERT is known to be the rate-limiting factor for human telomerase activity, and as such, likely a more sensitive indicator of telomerase function and activity than the expression levels of other telomerase subunits that are constitutively expressed in both normal and cancer cells [Currently, commonly used diagnostic markers include the HPV-related proteins L1, E6, and E7. Of these, L1 is a major viral capsid protein that is produced in the cytoplasm, before being translocated into the nucleus of intermediate and superficial squamous epithelial cells, as previously visualized using immunochemical staining. E6 and E7 are primary HPV oncoproteins with numerous cellular targets including p53, and the retinoblastoma tumor suppression protein (pRB). E6 inhibits p53 to prevent apoptosis, whereas E7 is the primary transforming protein, and inhibits pRB to regulate cell-cycle arrest , 10. In tability . In facttability . The exper cells . On the er cells .Screening of these diagnostic markers may also be of use in assessing the progression of cervical cancer past the midstage, as demonstrated by a previously conducted prospective study of their expression in formalin-fixed paraffin-embedded (FFPE) clinical histological samples . CytologIn the present study, HPV and cancer markers mentioned above were analyzed with 110 HSIL and 50 normal microscope slides.A total of 110 and 50 slides with exfoliated cervical-cell samples mounted with Canada balsam  were retrospectively obtained from patients diagnosed to HSIL and normal, respectively, between 2000 and 2004, from the Department of Pathology, Yonsei University Wonju Severance Christian Hospital, Wonju, Republic of Korea. Strictly speaking, normal means negative for intraepithelial lesions or malignancy (NILM) in this context. To reduce interpretive diagnostic error, we only utilized HSIL specimen confirmed with cervical intraepithelial neoplasia grade 2 or worse (CIN2+).All subjects provided written informed consent for their participation in the study, which was approved by the Institutional Ethics Committee at Yonsei University Wonju College of Medicine .Clinical diagnosis was predominantly determined cytologically using the 2001 Bethesda System terminology; however, cases with available tissue biopsies were also histologically reviewed.μL of chloroform, the tube was shaken vigorously, incubated , and then centrifuged . The resultant aqueous layer was transferred to a new tube and mixed with an equal volume of isopropanol by inverting the tube. The mixture was incubated  and then centrifuged  before the resulting supernatant was removed, and 1 mL of 75% ethanol was added to the remaining pellet. After mixing via tube inversion, the mixture was centrifuged , and the supernatant subsequently removed. The remaining RNA pellet was dried and eluted in 25 μL of diethylpyrocarbonate- (DEPC-) treated water . The purity and concentration of the extracted total RNA was determined by measuring its absorbance at 260 and 280 nm using an Infinite 200 plate reader . The isolated total RNA was finally stored at −70°C until use. Note that all preparation and handling of total RNA were performed in a laminar flow hood, under RNase-free conditions.Slides with exfoliated cervical cells (microscope slides) were used for total RNA extraction. Slides were initially placed in coplin jars with xylene  for 4 days to remove their cover clips . They were then dried (5 min) and placed into a six-well culture plate . The Isol-RNA Lysis Reagent  was added onto each slide, and a 1 mL exfoliated cervical-cell sample was then collected from each slide via scraping (twice) with an autoclaved slide glass. Each collected exfoliated cervical-cell sample was transferred to an RNase-free 1.7 mL tube, lysed via vortexing/repeated pipetting, and allowed to incubate in the reagent . After the addition of 200 μL of total RNA was added to a master mix containing 1 μL of 10 mM dNTP mix , 0.25 μg of random hexamers, and 1 μL of DEPC-treated water in PCR tubes. The reaction mixture was incubated  and then quickly chilled on ice. A mixture of 4 μL of 5× First-Strand Buffer, 2 μL of 0.1 M dithiothreitol (DTT), and 1 μL of M-MLV reverse transcriptase (RT) was added to the reaction mixture, and the cDNA synthesis reaction then performed via cycling at 25°C for 10 min, 37°C for 50 min, and 70°C for 15 min.Complementary DNA (cDNA) was synthesized using an M-MLV Reverse Transcriptase kit  and random hexamers (Invitrogen), according to the manufacturer's recommendations. Briefly, 10 μL reaction mixture  consisting of 2× master mix, 1× primer mixture, 3 μL of sample DNA, and sterile deionized water (DW). This mixture was subjected to PCR cycling conditions comprising 94°C for 5 min (predenaturation), followed by 15 cycles of 94°C for 30 s (denaturation) and 55°C for 30 s , 45 cycles of 94°C for 30 s (denaturation) and 52°C for 30 s , and a final cycle of 72°C for 7 min (strand synthesis). The amplified biotin-labeled PCR products were then denatured  in denaturation solution, diluted in 970 μL of hybridization solution, applied to the REBA membrane strip in the blotting tray, and hybridized  to the desired probes. The membrane strips were then washed twice with 1 mL of washing solution , before being incubated  with a streptavidin-alkaline phosphatase (AP) conjugate  diluted (1 : 2 000) in a conjugate diluent solution (CDS). After two final washes with 1 mL CDS , colorimetric hybridization signals were visualized via incubation with an NBT/BCIP solution  for sufficient time to detect the enzymatic conversion of the NBT/BCIP substrate to its colored form. The resulting band patterns were then read and interpreted.A REBA HPV-ID® PCR-REBA test , in which a “nested PCR” method was used to amplify target regions between MY11-MY9 and GP5-GP6 using two primer pairs, was used for HPV genotyping. PCR was performed using a 20 E6/E7, hTERT, and Ki67 mRNA expression in cervical specimens was assessed via a multiplex RT-qPCR TaqMan assay that was performed using the CervicGen HPV E6/E7 and hTERT-Ki67 mRNA RT-qDx assay kits  and the CFX-96 real-time PCR system  for thermal cycling and fluorescence detection. Real-time PCR amplification of HPV E6/E7 mRNA was performed in a reaction mix containing 10 μL of 2× Thunderbird probe qPCR mix , 5 μL each of primer and TaqMan probe mixture, and 5 μL of template cDNA. Real-time PCR amplification of hTERT and Ki67 mRNA was performed in a reaction mix containing 10 μL of 2× Thunderbird probe qPCR mix , 3 μL of primer and TaqMan probe mixture, 2 μL of template cDNA, and sufficient DW to produce a final volume of 20 μL. Positive and negative controls were included throughout the procedure, and likewise, no-template controls (containing sterile DW instead of template DNA) were amplified with each PCR. The utilized PCR cycling conditions comprised 95°C for 3 min, followed by 41 cycles of 95°C for 3 s, and 55°C for 30 s. Each mRNA expression level was quantified by determining the “cycle threshold” (CT), which is the number of PCR cycles required for the fluorescence to exceed a value significantly higher than the background fluorescence. To avoid the generation of false negative results due to mRNA degradation, the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The six samples of HSIL group and four samples of normal group did not show GAPDH value; therefore, they were excluded from the following experiments. In other words, researcher performed experiments with 104 HSIL and 46 normal samples. Target gene mRNA expression levels relative to GAPDH were automatically calculated according to the comparative Ct method, using CFX Manager v1.6 (Bio-Rad) or Genex (Bio-Rad) Software. Gene expression was assessed using the comparative Ct (ΔΔCt) method, in which mRNA expression levels are represented relative to the expression level of the reference gene. hTERT and Ki67 expression levels in analyzed slide samples from patients without HSIL were considered to indicate the “baseline” expression level for each gene.HPV t-test, 95% confidence interval (CI), and ROC curve were used to assess the statistical significance of generated data. Cohen's kappa coefficient which measures agreement between two raters for qualitative items is also used.Statistical analyses were performed using GraphPad Prism software version 5.02 . Student's All analyzed slide samples were confirmed to exhibit the precancerous condition, cervical intraepithelial neoplasia (CIN) III. Furthermore, histological and cytological methods were used to confirm a diagnosis of HSIL among the 110 relevant slides. Patients with HSIL (range 10-79 years-of-age) were found to be predominantly aged between 30 and 39  or 40 and 49  years. A lesser number of HSIL patients were aged between 20 and 29  or 50 and 59  years, and very few were aged less than 20 or greater than 59 years .Of the 104 slides with exfoliated HSIL cervical-cell samples, 83 (79.81%) were found to be infected with at least a single HPV genotype, including 26 (25%) that were infected with multiple  HPV genotypes. Among these 83 cases, 56 (53.85%) were determined to be infected with a high-risk (HR) HPV genotype, while a single case (0.96%) was shown to be infected with a low-risk (LR) HPV genotype . Among t16 (cumulative proportion 40.22%), followed by HPV 52 (55.43%), 58 (66.30%), 31 (77.17%), 18 (84.78%), 33 (90.22%), 35 (94.57%), 66 (97.83%), 84 (98.91%), and 45 (100%).As shown in the constructed cumulative graph , the moshTERT and Ki67 expression levels in the 104 HSIL-diagnosed exfoliated cervical-cell samples. After excluding samples with a no GAPDH expression level, we determined that 98 (94.23%) and 49 (47.12%) of the 104 remaining HSIL samples were positive for hTERT and Ki67 mRNA expression, respectively  were positive for HPV E6/E7 only, 18 (17.31%) were positive for hTERT only, and three (2.88%) were positive for HPV E6/E7, but negative for hTERT  were positive for HPV E6/E7 expression only, 11 (10.58%) were positive for Ki67 expression only, and ten (9.62%) were positive for HPV E6/E7, but negative for hTERT expression  HSIL samples, 80 (76.92%) were positive for both HPV or hTERT . Converspression .HPV E6/E7 and Ki67 mRNA expressions, 96.15% (100/104) for hTERT and Ki67 mRNA expression, 97.12 (101/104) for HPV E6/E7 and hTERT mRNA expressions, and 98.08% (102/104) for the HPV E6/E7, hTERT, and Ki67 mRNA expressions (The positivity rates were 90.38% (94/104) for a combination of ressions .hTERT and Ki67 expression as diagnostic markers of cervical cancer. Exfoliated cervical cells before being placed onto a slide are currently used for screening test specimen because they accompanied with less invasive and less labor-intensive procedure than other tests [The previous study was conducted with FFPE clinical tissue samples . It was er tests , 18. Theer tests , 19. In er tests –23. For 16, and notably, detection rate of HPV 18 of slides is lower than that of histological samples.The results showed that HR-HPV infection is more closely associated with cervical cancer progression than LR-HPV both in the context of a single or of multiple HPV infections . In addiE6/E7 mRNA transcripts has been shown to be associated with a significantly increased risk of both precancerous group (CIN) and of cervical cancer [E6/E7 expression levels may be specific and effective predictors of cervical cancer risk was supported by the results of the present study, which showed the sensitivity of the utilized E6/E7 mRNA RT-qPCR assay to 79.81% in the 104 analyzed exfoliated cervical-cell samples. It indicate cervical cancer occurrence could be affected other factors not only HPV. Therefore, hTERT and Ki67 confirmed by tissue samples [hTERT mRNA RT-qPCR screening of the 104 clinical samples was 94.23%, whereas that of Ki67 screening was only 47.12%. Interestingly, a previous study demonstrated hTERT mRNA expression to be higher in cytological than in tissue samples from high-grade cervical lesions, while conversely, Ki67 mRNA expression was found to be higher in tissue than cytological samples from the high-grade cervical lesions [Cervical cancer oncogenesis is initiated and mediated via the upregulation of the HPV oncoproteins E6 and E7, such that the overexpression of l cancer . The hyp samples  were als lesions . The mechTERT and Ki67 mRNA expression was only detected in 94.23% and 47.12% of the analyzed cytology samples, respectively, combined screening for HPV E6/E7 and Ki67, HPV hTERT and Ki67, and HPV E6/E7 and hTERT mRNA expression identified 90.38%, 96.15%, and 97.12% of samples, respectively  and/or as indicators of the progression of cervical lesions.


INK4a overexpression to confirm involvement of HPV in tumorigenesis. Out of 287 screened studies, 14 studies with data from a total of 1819 OPSCC patients treated between 1988 and 2015 were included in the data synthesis. The reported average HPV‐AF varied considerably between the studies, ranging from 11.5% (1988‐2008) to 55.0% (2004‐2009). Two of the included studies did not only provide the HPV‐AF for the entire observed calendar period but also for separate years, allowing to more accurately assess changes over time. These studies reported increases in the HPV‐AF from 21% in 2000 to 53% in 2015 and from 38% in 2004 to 71% in 2013, respectively.The prevalence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing globally while the prevalence of other head and neck cancers is decreasing. The most likely reasons for this are a decreasing influence of smoking and the growing relevance of infections with the human papilloma virus (HPV) as a risk factor. A rise in the HPV‐attributable fraction (HPV‐AF) of OPSCC has been observed in many countries, yet a comprehensive review of prevalence rates and trends in Germany is lacking. To determine the current HPV‐AF of OPSCC in Germany and to assess whether it has changed during the last decades, we performed a systematic literature review. We screened Medline and EMBASE for studies that reported the tumor HPV status of newly diagnosed OPSCC patients treated at medical centers in Germany by testing for both HPV DNA and p16 While a recent publication assumed an HPV‐AF for OPSCC of only 16% for estimations of disease burden in Germany,1.3INK4a immunohistochemistry on pretreatment tumor biopsies. The combined detection of HPV and p16INK4a has demonstrated good sensitivity and specificity in a recent meta‐analysis on the detection of HPV‐driven OPSCC.The aim of this systematic review was to determine the most recent HPV‐AF of OPSCC in Germany and to assess whether this fraction has changed during the last decades. For this purpose, the available literature was systematically screened for prospective and retrospective studies including patients with newly diagnosed OPSCC that were treated at a German medical center. The HPV status of the tumor had to be determined by HPV DNA PCR combined with p162We followed the PRISMA guidelines in our literature search and in the preparation of the manuscript.2.1The PICOTS criteria  that were used for the selection of studies eligible for inclusion into the review are shown in Table 2.2A literature search was conducted in Medline and EMBASE using the search string ([HPV AND oropharynx] AND Germany) OR ([HPV AND head and neck] AND Germany). No restrictions regarding language or publication dates were applied. The last search was run on January 10, 2018. Authors with more than one included study were contacted to confirm that study populations of multiple publications from the same medical center did not overlap, and authors were asked to provide additional data for individual years if these were not reported in the published study results.2.3INK4a overexpression, and the following variables: study site, anatomic tumor location, case selection, calendar period of inclusion, and the type of HPV DNA PCR and p16INK4a test used. Tumor stage, age, gender, tobacco use, and alcohol consumption were additionally extracted if available.The process of study selection including reasons for exclusion is illustrated in the PRISMA flow diagram Figure . A data 2.4INK4a overexpression to indicate an actual involvement of HPV in tumorigenesis were included. Testing only for the presence of HPV DNA or overexpression of p16INK4a was considered insufficient. Additionally, we only included studies that reported results for the OPSCC cases separately from other HNC cases, that included patients at the time of diagnosis or treatment without evident selection for stage or histology, which provided separate data for a German medical center if an international cohort was analyzed and that tested tumors from at least 10 individual patients. To reduce bias resulting from studies with overlapping cohorts, we only included the most recent or most comprehensive publication from centers that repeatedly reported on the same patient population.Only studies that tested tumors for both HPV DNA and for p1633.1Out of 287 screened studies, 14 fulfilled the inclusion criteria and were included in the data synthesis. These 14 studies covered a total of 1819 individual OPSCC patients with no overlap between cohorts. A PRISMA flow diagram of the process of study selection is presented in Figure INK4a immunohistochemistry to determine the HPV status of the tumor tissue. The studies cover varying time periods from 1988 to 2015 as shown in Table All included studies report the HPV‐AF of OPSCC in newly diagnosed patients that were treated at medical centers in Germany, using both HPV DNA PCR and p163.2The reported overall HPV‐AF varied considerably between the individual studies, ranging from 11.5%Two of the included studies did not only provide the HPV‐AF for the entire cohort of the observed period but also for separate years, allowing to more accurately assess the change in the HPV‐AF over time Figure . One stu3.3. To assess the risk of bias across the included studies, a funnel plot was used  or one‐way analysis of variance with Tukey's test (three or more parameters). Out of the 14 studies included into the data synthesis, 13 reported the DNA‐ and p16INK4a‐based detection method they used . Still we were not able to separate the results for oropharyngeal sublocations, which might be important to account for the higher HPV‐AF known for tumors located at the palatine tonsils compared to base of tongue and other sites.INK4a immunohistochemistry to minimize this bias. Still, HPV PCR techniques are poorly harmonized and interpretation of p16INK4a immunohistochemistry can be heterogeneous. While testing for viral E6/E7 mRNA expression is a reliable alternative to testing for HPV association in OPSCC, it is less commonly used and still lacks standardization. It has been demonstrated that the combination of HPV DNA plus p16INK4a yields results comparable to E6/E7 mRNA testing.The second limitation of data on HPV‐AF is related to the method used to determine the HPV status, which is likely to contribute to heterogeneity between the individual studies. We only included studies that determined the HPV status by combining HPV PCR with p16The third limitation is related to the varying calendar periods addressed in the studies. Since it is assumed that the HPV‐AF increased over the last decades and thus is influenced by the time period of diagnosis, data synthesis for several calendar periods would be desirable. However, the observation periods varied strongly between the individual studies, and only two studies report changes of prevalence over time. It was thus decided to restrict data synthesis and to report the prevalence of HPV in the individual studies without performing a meta‐analysis.4.3INK4a and HPV mRNA, and 19.1% if determined by HPV DNA and p16INK4a as in the present review.In a large international study from 2016, the HPV‐AF of OPSCC in Europe was reported to be 16% if determined by HPV DNA, p16The same study that calculated the average European HPV‐AF of OPSCC to be 19.1% also reported four distinct HPV‐AF values for different geographic regions of Europe as defined by the GLOBOCAN classification.4.4INK4a expression is necessary to assume an actual involvement of HPV in tumorigenesis of OPSCC.INK4a expression testing in addition to HPV DNA among included studies. The combined analysis of p16INK4a plus HPV DNA has been demonstrated to accurately identify HPV E6/E7‐expressing OPSCC.Concerning a possible change in the HPV‐AF of OPSCC over time, our results indicate a strong increase during the last decades, particularly based on the results of two studies by Tinhofer et al and Würdemann et al.While the reported increase in the HPV‐AF of OPSCC in Germany fits into the general observation of an increase in developed countries worldwide, the reasons for this increase remain unclear. Part of a possible explanation is the fact that the prevalence of tobacco use, which is a major risk factor for HPV‐negative OPSCC, has decreased markedly in developed countries since the 1980s (from 33.1% in 1980 to 23.5% in 2012), that is in parallel to the increase in HPV‐positive OPSCC.5The majority of studies included in the present review report an HPV‐AF of OPSCC in Germany that is considerably higher than the previously assumed 16.0%. This is especially true for studies covering recent time periods, which report HPV‐AF values of up to 55%.Miriam Reuschenbach is an employee of MSD Sharp & Dohme GmbH and MSD Sharp & Dohme GmbH Germany provided funding for this work. Jens Peter Klussmann received a research grant from MSD Sharp & Dohme GmbH Germany, and Claus Wittekindt and Steffen Wagner are participants of this granted research project. Ingeborg Tinhofer has nothing to disclose. Click here for additional data file.


Human Papilloma Virus (HPV) DNA tests are highly sensitive and can triage women with mild lesions, improving the prognosis and diagnosis of cervical lesions. However, additional efficient strategies should be developed to improve the specificity of these tests.This study aimed to evaluate the clinical value of HPV DNA load in improving the diagnosis and prognosis of cervical lesions by p16/Ki-67 testing. Histological samples were collected from 350 women with HR-HPV genotyping and analyzed by qRT-PCR. Immunohistochemical staining was used to assess p16 and Ki-67 expression and clinical performance characteristics were calculated.P = 0.016) and HPV-58 (P = 0.004) viral loads significantly correlated with lesion severity. The P16/Ki-67/HPV DNA load co-test indicated an increased sensitivity for the detection of cervical intraepithelial neoplasia (CIN) lesions compared to p16/Ki-67 staining in HPV-16 and/or 58 positive cases. Viral load did not improve the sensitivity of p16/Ki-67 co-test in non-HPV-16 or 58 positive cases. The clinical performance of the p16/Ki-67/HPV DNA load co-test was limited for the prediction of the outcome of CIN1 lesions. However, amongst the 12 HPV-16 and/or 58 positive CIN2 cases in which return visit results were obtained, the behavior of the lesions could be predicted, with a sensitivity, specificity, positive prediction rate (PPV), and negative prediction rate (NPV) of 0.667, 1, 1 and 0.5, respectively.Of the cases, 271 had detectable HR-HPV infection, in which HPV-16 was most prevalent (52.0%), followed by HPV-58 (22.5%). P16/Ki-67-positivity increased with histological severity but not for HR-HPV infection. Amongst the 13 HR-HPV genotypes, only HPV-16 (Combination of the assessment of HPV DNA load with the intensity of p16 and Ki-67 staining could increase the sensitivity of CIN lesion diagnosis and predict the outcome of CIN2 in patients with a HPV-16 and/or 58 infection. Human papillomavirus (HPV) is a major cause of cervical cancer (CCa) and its screening can improve the diagnosis of both CCa and precancerous lesions , 2. HPV P16 is a tumor suppressor that inhibits cyclin-dependent kinases (CDK) 4 and 6, which regulate the checkpoint of the G1 to the S phase. Overexpression of p16 is associated with functional inactivation of the retinoblastoma (Rb) protein that is induced by the deregulation of HPV E6 and E7 proteins . ImmunohRecently, p16/Ki-67 staining has been proposed as a primary screening strategy to triage women with atypical cells of undetermined significance (ASCUS) and Low-grade squamous intraepithelial lesion (LSIL). The use of p16/Ki-67 as CCa biomarkers yields high sensitivity for CIN2+ and CIN3+ and decreases the inter-observer variability of cytology scoring –12. The Specimens were collected from women who underwent biopsy from January 2016 to June 2018 in the First Affiliated Hospital of Southwest Medical University. Over 600 specimens were collected during the two-year period, of which 50% of samples were excluded. Exclusion criteria encompassed previous operation for cervical disease , cold-knife conization, hysterectomy, laser therapy, chemotherapy/radiation treatment, pregnancy, and malignant diseases outside the cervix. A total of 350 eligible specimens were collected from women ranging from 20 to 72 years with an average age of 43.49 ± 9.56. Prior to biopsy, cervical cells and fragments were obtained from the internal genitalia with a cytobrush. Smears were collected for DNA extraction. Biopsy specimens were fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin. Sections were used for subsequent p16 and Ki-67 immunohistochemical analysis.Liquid-based cytological samples (50 μL) were pelleted and 200 μL of denaturing reagent was added to the pellet. Tubes were incubated at 100 °C for 10 min, followed by centrifugation. Supernatants were collected and the nucleic acid concentration measured using a NanoDrop 2000 spectrophotometer. HPV DNA was amplified via qRT-PCR and screened using the Slan-96P Real time PCR System . The following HR-HPV genotypes were assayed: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. For HPV DNA quantification assays, the levels of the dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1a) was employed as reference. Viral loads were calculated according to the formula: viral load = 2 ^ (reference CT – objective CT). CT: cycle threshold.In this study, cytological samples and biopsies were taken simultaneously. Histological results were collected from the records of pathologists. Results were interpreted by two experts and if an agreement was not obtained, a third expert was used to finalize the diagnosis. When multiple results were recorded in single patients, the most severe histological diagnosis was used for analysis. A consensus diagnosis was established on most available cervical specimens. Histological diagnoses were performed according to the WHO 2014 classification criteria and were classified as cervicitis , CIN1 (LSIL), CIN2, and CIN3 , and invasive CCa. To investigate the differences between CIN2 and CIN3, we did not abandon the CIN terminology and classified intraepithelial lesions into CIN1, CIN2, and CIN3.Serial sections (4 μm) from formalin-fixed paraffin embedded blocks were sliced and mounted onto glass slides. After sequential deparaffinization and rehydration, antigen retrieval was performed in a water bath following heating in a microwave. Slides were treated with 0.3% H2O2 for 15 mins to quench endogenous peroxidase. After blocking in 3% goat serum, slides were labeled with primary antibodies overnight at 4 °C. A secondary antibody reagent was employed for visualization after washing. Slides were incubated with DAB . Substrate chromogen solution and counterstaining was performed with Mayer’s hematoxylin and slides were mounted. Negative control slides lacking primary antibodies were included for each staining protocol. The recommendations of WHO considered a positive p16 staining as a case with a strong diffuse staining in at least one third of the thickness of the epithelium, starting from the bottom. For quantitative analysis of both p16 and Ki-67 immunostaining, we scored immunohistochemical results according to the description by Koo et al. . The perDescriptive statistics were employed for data characterization. A Pearson’s χ2 test was used to evaluate significant differences between the designated groups. A Pearson’s correlation analysis was performed to analyze the relationship between HPV DNA load, the severity of intraepithelial neoplasms, and p16/Ki-67 staining scores. During the analysis, CIN1, CIN2, and CIN3 cases were scored as 1, 2, and 3, respectively, and CCa was scored as 4. Those lacking intraepithelial lesions were scored as 0. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) with 95%-confidence intervals were calculated for two different endpoints. Sensitivity and specificity of the confidence intervals were plotted on a ROC characteristics graph using MedCalc software. For p16 and Ki-67 tests, the ROC curve was plotted by using the p16 score or Ki-67 score. However, for Ki-67/p16, HPV load/Ki-67, HPV load/p16, and HPV load/Ki-67/p16 test, the logistic curve was calculated first, then the predicted values on the curve were adopted to generate ROC curve.A total of 350 women were included in the analysis. The median age was 43.5 (± 9.56) years (complete range: 20–72). Of the 271 women with available HR-HPV results, 141 (52.0%) were positive for HPV-16, 61 (22.5%) were positive for HPV-58, and 56 (20.7%) were positive for HPV-52 (Table We stratified women by HPV status into three age categories: (1) < 30; 2) ≥30 and < 45; and (3) ≥45. In total, an increasing proportion of p16/Ki-67 positive subjects with increasing histological severity were observed. These ranged from 9.5% in women with cervicitis, 22.1% in women with CIN1, 73.5% in women with CIN2, 88.8% in women with CIN3, to 96.9% in women with CCa (Table  ≥30 and P = 0.015) immunohistochemistry staining, but did not significantly correlate with lesion severity or Ki-67 staining scores (Table P = 0.016) and HPV-58 (P = 0.004) loads significantly correlated with lesion severity. The HPV-16 load also significantly correlated with the p16 staining score (P = 0.047), but did not correlate with Ki-67 staining. The HPV DNA load of any subtype did not significantly correlate with Ki-67 staining score, though a strong correlation between the p16 and Ki-67 scores were noted (P < 0.001).The total HPV DNA load significantly correlated with p16 (es Table . RegardiWe analyzed the clinical performance characteristics of the HPV load, p16 and Ki-67, to detect CIN amongst the study population, stratified by HPV-16 and/or 58 infection (Fig. We estimated the performance of the HPV DNA load in combination with p16/Ki-67 staining to stratify women with CIN lesions. For HPV-16 and/or 58 positive women, the combined HPV DNA load/p16/Ki-67 co-test most efficiently detected the lesions. The sensitivity and specificity were 0.908 and 0.87 for CIN1+, 0.889 and 0.922 for CIN2+ and 0.866 and 0.775 for CIN3+, respectively Table . HPV/p16The behaviors of CIN1 and CIN2 lesions were predicted by p16, p16/Ki-67 or p16/Ki-67/HPV co-tests Table . DespiteHPV screening is recognized as a necessary but insufficient factor during CCa development. Whilst HPV DNA tests are highly sensitive for the detection of precancerous lesions, they lack the ability to differentiate precancerous cases from HPV infected individuals . The coeFollowing stratification by age and HPV status, p16/Ki-67 positivity increased with the severity of CIN and worse lesions, in accordance with previous reports , 19. ComThough persistent infections are required for the conversion of low-grade to high-grade lesions or cancer, the clearance of HR-HPV infections rarely occurs in patients with high viral loads . As suchThough CIN1 lesions with p16 staining had a higher tendency to progress to high grade lesions , it was In this study, we determined that the p16/Ki-67/HPV load co-test may have higher clinical value than the p16/Ki-67 co-test for detecting lesions in HPV-16 and/or 58 positive cases. However, the effect of the combination was not very strong, especially it was hard to improve the specificity of detection in those cases. We estimated the main cause may be that the genome variation and gene expression deregulation in HR-HPV infected tissues are very complex. Even for the same HR-HPV, the DNA load of the HR-HPV is not simply a linear function with genomic and expression abnormalities. Therefore, additional factors and assays should be considered to improve the combination if the combination could be employed as an alternative method to triage patients with HR-HPV infection.In a population with clear disease ascertainment due to biopsy and histological diagnosis, we demonstrate that the combined p16/Ki-67/HPV load co-test has a higher sensitivity than p16/Ki-67 co-test for the detection of cervical precancers in HPV-16 and/or 58 positive cases. We further show that the P16/Ki-67/HPV load co-test is an effective method to predict the progression of CIN2 lesions in these cases. Further studies are now required to evaluate the potential role of the P16/Ki-67/HPV load co-test as a triage marker, and larger numbers of CIN cases are required to identify optimal cut off points to effectively triage CCa patients.Additional file 1: Table S1. Dignoses, qRT-PCR amplifications and immunostaining analyses of the 350 cases.Additional file 2: Table S2. The data of the p16/Ki-67/HPV co-test to predict the progression of CIN1 and CIN2 for the 48 cases.


Papillomaviridae is a family of small non-enveloped icosahedral viruses with double-stranded circular DNA. More than 200 different human papillomaviruses (HPVs) have been listed so far. Based on epidemiological data, a subgroup of alphapapillomaviruses  was referred to as high-risk (HR) HPV types. HR HPVs are the etiological agents of anogenital cancer and a subset of head and neck cancers. The cutaneous HPV types, mainly from beta and gamma genera, are widely present on the surface of the skin in the general population. However, there is growing evidence of an etiological role of betapapillomaviruses (beta HPVs) in non-melanoma skin cancer (NMSC), together with ultraviolet (UV) radiation. Studies performed on mucosal HR HPV types, such as 16 and 18, showed that both oncoproteins E6 and E7 play a key role in cervical cancer by altering pathways involved in the host immune response to establish a persistent infection and by promoting cellular transformation. Continuous expression of E6 and E7 of mucosal HR HPV types is essential to initiate and to maintain the cellular transformation process, whereas expression of E6 and E7 of cutaneous HPV types is not required for the maintenance of the skin cancer phenotype. Beta HPV types appear to play a role in the initiation of skin carcinogenesis, by exacerbating the accumulation of UV radiation-induced DNA breaks and somatic mutations (the hit-and-run mechanism), and they would therefore act as facilitators rather than direct actors in NMSC. In this review, the natural history of HPV infection and the transforming properties of various HPV genera will be described, with a particular focus on describing the state of knowledge about the role of cutaneous HPV types in NMSC. Sparus aurata papillomavirus 1 (SaPV1) to 8,607 bp for canine papillomavirus type 1 (CPV1). Papillomaviruses (PVs) infect basal keratinocytes of the mucosal and cutaneous epithelia of both animals  and humans  has classified the PVs into 53 genera, among which only 5 genera include PVs that infect humans (HPVs). To be classified as a novel PV type, the nucleotide sequence of L1 must share < 90% similarity with other PVs (www.hpvcenter.se)  , and thinter.se) .n = 65) and beta (n = 54). The genera mu and nu include only 3 and 1 types, respectively . A recent study reported the identification of the complete genome of 83 previously unknown HPV types, among which 69 were classified as gamma , i.e., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, referred to as high-risk (HR) HPV types, has been classified as carcinogenic (IARC Group 1), and eight other HPV types, i.e., HPV26, 53, 66, 67, 68, 70, 73, and 82, have been classified as probably or possibly carcinogenic (IARC Groups 2A and 2B)  . The HR  and 2B) . In 2012 and 2B) . Epidemi and 2B) . These H and 2B) , in chil and 2B) –18.The genera beta, gamma, mu, and nu contain HPVs that infect cutaneous epithelia. The genus alpha also contains some HPV types, such as HPV2, 3, and 10, that colonize the human skin. The mu and nu genera include only a few HPV types, whereas the genus beta includes more than 54 HPV types, which are subdivided into five species (β1–5), and the genus gamma includes 98 types subdivided into 27 species . In the In this review, we describe the natural history of mucosal and cutaneous HPV infections, and discuss the transforming properties of a subset of them that have been shown or are suspected to play a causative role in various human cancers. In particular, we focus on describing the state of knowledge about the role of cutaneous HPV types in NMSC, because many existing reviews already cover all the aspects of mucosal HPV type infection and its role in transformation.The HPV genome is organized into three major regions: (i) a long control region (LCR), also called the upstream regulatory region (URR), located between the L1 and E6 open reading frames (ORFs), which contains the early promoter and regulatory element involved in viral DNA replication and transcription; (ii) the early region, which encodes the E1, E2, E4, E5, E6, and E7 proteins involved in viral gene expression, replication, and survival; and (iii) the late region, which encodes the structural proteins (L1 and L2). This organization is shared among all alpha HPVs . However∧E2C transcript , encoding for a protein that includes the E8 domain fused to the hinge and DNA-binding domains of E2  , and HPVThe DNA integration event could contribute to the HPV-driven malignant transformation of cervical cells, by affecting the expression of key host genes near the integration site . In addiAlthough it was commonly accepted that integration occurred randomly into the host genome, several studies have shown that non-random HPV integration occurs. Integration of HPV16 sequences appears to take place preferentially in common fragile sites in human chromosomes , 62, in Carcinogenesis can also be enhanced by epigenetic events. In cervical cancer, HPV16 E2 binding sites (E2 BS1 and E2 BS2), which play a role in transcriptional repression of E6 and E7 oncoproteins, are heavily methylated. The binding of E2 protein on these sites is then prevented, which leads to the upregulation of E6 and E7 oncoproteins . Host orData on HPV integration in HNC are still sparse and contradictory. Parfenov et al.  reportedCutaneous HPV types are ubiquitous and are widespread in the general population. Up to 90% of healthy individuals tested positive for beta HPV types –74. The Epidemiological studies in humans showed that the cutaneous HPV population present in hair follicles mirrors the HPV prevalence in the skin of the same individual, which makes eyebrow hair an excellent sampling method to characterize the individual cutaneous HPV population , 82. StuExposure to cutaneous HPV is common. Serological studies measuring antibodies against the type-specific L1 major capsid protein of HPV showed that 52% of the Dutch population and 67% of the Italian population were exposed to beta HPV infections . A recenCutaneous HPVs are highly prevalent in the rare hereditary disease epidermodysplasia verruciformis (EV), which tends to progress to cutaneous SCC (cSCC), frequently located at sun-exposed anatomical sites . HPV5 anIn organ transplant recipients, a 65–250-fold increased risk of developing SCC compared with the general population was reported , 95. In This observation is corroborated by growing evidence showing an association between cutaneous infection and the risk of developing NMSC in immunocompetent individuals under certain conditions  , inEpidemiological and biological data suggested that beta HPV types, and species β1 and β2 in particular, may be linked to cSCC development in immunocompetent individuals , 100 . This plication –162. Brdlication . ChIR1, lication –166. Thelication . In addilication , 169.Upon cell differentiation in stratified epithelium, vegetative or productive viral replication is initiated, with the subsequent production of progeny virions , 171, . For exin vivo  also have the ability to interact with p300 with high efficiency . HPV5 anE6 of HPV8 may also play a role in skin carcinogenesis by down-regulating CCAAT/enhancer-binding protein α (C/EBPα), a transcription factor with a role in cell differentiation. MicroRNA-203 (miR-203), previously shown to be an important regulator of epidermal proliferation and differentiation, is induced by C/EBPα. In HPV8 E6 expressing keratinocytes, downregulation of C/EBPα via E6-mediated p300 degradation led to loss of miR-203 expression and induction of the ΔNP63α protein, altering the proliferation abilities of the infected epithelial cells .Similar to E6s of HPV species β1, E6 of HPV38 (species β2) was able to prevent p300-mediated acetylation of p53, an event that resulted in its inhibition and thus led to the immortalization of primary keratinocytes , indicatE6 has additional cellular targets that play an important role in apoptosis  –197.The anti-apoptotic protein Bak, a member of the Bcl-2 family of proteins, plays an important role in regulating the apoptotic process by forming pores with Bax to permeabilize the outer mitochondrial membrane. Bak has been shown to be targeted for degradation by different HPVs from the alpha and beta genera, an event that contributes to preventing UV radiation-induced apoptosis. The ability of the E6 proteins of beta HPVs  to target Bak for degradation, is important for promoting the survival of DNA-damaged cells and therefore for the progression of NMSC. Bak interacts with the ubiquitin ligase E6AP; however, E6 proteins of HPV5 or 8 that do not interact with E6AP  can stilThose experiments highlight the ability of beta HPVs to enhance UV radiation-induced DNA damage, supporting a role of beta HPVs at the early stage of skin carcinogenesis.Beta HPVs display additional features for increasing the oncogenic potential of UV radiation exposure by attenuating DNA damage repair. For instance, the binding and destabilizing of p300 by beta HPV5 and 8 E6s also lead to lower levels of BRCA1 and BRCA2, critical to the homology-dependent repair of double-strand breaks . ViarisiAll HR HPV E6 proteins contain C-terminal PDZ domain-binding motifs (PBMs) that enable interaction with several cellular PDZ domain-containing proteins involved in the regulation of different processes, such as cell polarity, migration, and attachment . The intA phosphor-acceptor site for protein kinase A (PKA) is located within the PBM. Phosphorylation at this site negatively regulates the PDZ domain-binding activity. The PKA recognition sequence is highly conserved among E6 of HR HPV types and is not present in LR HPV E6 oncoproteins, further suggesting that the PDZ domain-binding activity is crucial in HR HPV-mediated transformation , 207. InDrosophila discs large 1, and hScrib, a homolog of the Drosophila scribble protein  and one nuclear export sequence (NES), which enable E7 to be located in both the nuclear and cytoplasmic compartments, where E7 has different functions .The CR1 domain has an UBR4/p600 binding site; the 600-kDa pRb-associated factor p600 is required for membrane morphogenesis, anchorage-independent growth, and cellular transformation , 239. MoThe CR2 domain comprises serine residues at positions 31 and 32 that are susceptible to phosphorylation by casein kinase II (CKII). Phosphorylation at these sites contributes to modulating some of the E7 functions. For example, CKII phosphorylation of E7 increases the binding affinity of HPV16 E7 for the TATA box-binding protein (TBP) , and it CR2 also comprises a conserved LXCXE motif located at residues 22–26 in HPV16 E7. This motif is essential for the interaction with pRb and related pocket proteins (p107 and p130) . An intaThe functionally and structurally pRb-related proteins, p107 and p130, are also targeted by the HPV E7 via the LXCXE motif, and both have the ability to bind to and inactivate E2Fs. These proteins are important negative regulators of the cell cycle, and they are believed to also act as tumor suppressors. However, unlike pRb, their role as tumor suppressors is still controversial, because related genetic alterations are not frequent in human cancers. Both HR and LR HPV E7s target p130 for proteasomal degradation, most likely by different mechanisms, thus highlighting an important role of this pocket protein in the HPV life cycle .Analysis of HPV E7-interacting proteins showed that HPV16 E7 interacts with E2F6, which has the particularity of lacking the binding domain for pRb and acts as a transcriptional repressor. The association of HPV16 E7 with E2F6 abrogates its repressive activity on target genes, which results in increased DNA replication of HPV16-infected cells .Using an organotypic culture of skin, Akgül et al.  showed tin vitro colony formation and tumor sphere assays, Hufbauer et al.  are able to trigger pRb hyperphosphorylation, an event that leads to release of E2F and to the transcriptional activation of E2F target genes [reviewed in ]. In addAlthough the transforming activities of E6 and E7 oncoproteins are well characterized, the role of the E5 oncoprotein is still poorly understood . E5 is aThe expression of E5 increased the immortalization effects of HPV16 E6 and E7 . E5 is iKip1 levels, which promotes cell-cycle progression  lack the E5 ORF, suggesting that the corresponding functions are performed by other viral proteins.E5 forms a complex with EVER1 and EVER2, proteins encoded by the TMC6 and TMC8 genes, respectively, . Most ofIn the past decades, epidemiological and biological studies have clearly demonstrated the role of mucosal HR HPV types in human carcinogenesis. While co-evolving with the human host, HPVs have developed strategies to evade the immune system and create the optimal conditions to persist in the host for many years. During HPV chronic infection, the E6 and E7 oncoproteins interact with different cellular proteins to prevent apoptosis, counteract the cellular senescence program, and promote unscheduled cell growth. These activities of HR HPV E6 and E7 are part of a viral strategy to complete the viral life cycle and generate progeny. The expression of the oncoproteins is kept under the tight control of E2. Loss of E2 repressive functions, by viral integration or by other mechanisms, which are discussed in this review, leads to constitutive high expression of E6 and E7, a key event in HPV-mediated transformation . AlthougOf note, recent studies identified HPV16 E6 antibody as a good diagnostic and prognostic marker of oropharyngeal cancer. HPV16 E6 seropositivity was detected more than 10 years before the diagnosis , 292.Moreover, although the available prophylactic vaccines have demonstrated their effectiveness in the context of prevention of anogenital cancers , very liProphylactic vaccines show a good efficiency in covering targeted HPVs, but they are inefficient in existing infections. Therefore, therapeutic vaccines are needed to fill this gap. Several studies have reported the development of promising therapeutic vaccines. Their use in the near future could be expected; however, their cost could be high. This limitation could be overcome by the use of inexpensive technologies  –299.One of the most recurrent questions regarding the prophylactic vaccine is whether HPV vaccination will result in the occupation of a newly vacant ecological niche by non-targeted HPV types. This hypothesis has been referred as “type replacement. ” To date, the available data do not support this hypothesis. However, as reported in recent studies, a few types  may need to be monitored after vaccination , 301.The bacterial microbiome composition may also play a role in the outcome of mucosal HPV infection. A chronic mycoplasma infection was shown to promote cervical dysplasia induced by HPV . Therefohttps://pave.niaid.nih.gov/). There are also important gaps in the understanding of the life cycle of cutaneous HPV types, which also deserve further research.Our knowledge of the biology of beta and gamma HPV types is still uncomplete. For example, the expression patterns of cutaneous HPV types and associated splicing patterns are largely unknown, except for HPV5 and 8 , whereas the other cutaneous HPV types were each determined to be “not classifiable as to its carcinogenicity to humans” (IARC Group 3), on the basis of insufficient epidemiological and biological data available at that time . In factMastomys coucha) have provided strong in vivo evidence for a synergism between UV radiation and cutaneous HPV types in the development of cSCC (Since that 2009 meeting, strong epidemiological and experimental data in favor of a role of cutaneous HPV types as cofactors in skin carcinogenesis have been published, as described in this review. Indeed, recent findings from our group and others have shown that unlike the expression of oncoproteins of HR HPV types, cutaneous HPV E6 and E7 expression appears to be required only at the initial step of skin carcinogenesis by exacerbating the deleterious effects of UV radiation . Over th of cSCC , 107. MoIn addition to these experimental data, a recent prospective study of two organ transplant recipient cohorts showed that having five or more different beta HPV types in eyebrow hair, and a high beta HPV viral load, was associated with cSCC carcinogenesis, providing strong evidence for a role of beta HPVs in this cancer, whereas no evidence was found for basal cell carcinoma . The devin vitro and in vivo models (Another open question regards the tropism of certain HPVs. As previously specified in this review, a dual tropism of HPV species β3 has been proposed. Moreover, this species displays biological similarities with mucosal HR HPV types , 120. Ouo models . In addio models , 306. Moo models . Those fo models , 119.A large number of gamma HPV types have been isolated; however, their biological activities are poorly studied and deserve more investigation. Finally, some of the known but not yet biologically characterized beta or gamma HPV types may display transforming activity and also deserve attention. In addition to these not-yet-studied types, one may also consider the large number of novel HPVs found in recent years, as well as the rapid evolution of viral detection and screening techniques and large data set analysis pipelines.For all these reasons, we believe that in the coming years the field of HPV research will continue to be a source of surprises and excitement for the scientific community.The author confirms being the sole contributor of this work and has approved it for publication. TG wrote the manuscript and designed the figures.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.