Research on perennial staple crops has increased in the past ten years due to their potential to improve ecosystem services in agricultural systems. However, multiple past breeding efforts as well as research on traditional ratoon systems mean there is already a broad body of literature on perennial crops. In this review, we compare the development of research on perennial staple crops, including wheat, rice, rye, sorghum, and pigeon pea. We utilized the advanced search capabilities of Web of Science, Scopus, ScienceDirect, and Agricola to gather a library of 914 articles published from 1930 to the present. We analyzed the metadata in the entire library and in collections of literature on each crop to understand trends in research and publishing. In addition, we applied topic modeling to the article abstracts, a type of text analysis that identifies frequently co-occurring terms and latent topics. We found: 1.) Research on perennials is increasing overall, but individual crops have each seen periods of heightened interest and research activity; 2.) Specialist journals play an important role in supporting early research efforts. Research often begins within communities of specialists or breeders for the individual crop before transitioning to a more general scientific audience; 3.) Existing perennial agricultural systems and their domesticated crop material, such as ratoon rice systems, can provide a useful foundation for breeding efforts, accelerating the development of truly perennial crops and farming systems; 4.) Primary research is lacking for crops that are produced on a smaller scale globally, such as pigeon pea and sorghum, and on the ecosystem service benefits of perennial agricultural systems. The breeding of perennial staple crops, such as perennial wheat, has emerged as a novel approach to sustainable agriculture and food systems. Perennial crops could reduce fuel dependency and better protect soil quality compared to conventional production of annual crops ,2. In adCajanus cajan) and cereals such as rice (Oryza sativa) and sorghum (Sorghum bicolor), which are more commonly used as annuals [Sorghum bicolor and S. halpense created several sterile offspring, but a handful of lines were picked from this initial effort for further development and use in the field [This notion has stimulated widespread interest amongst researchers and farmers alike . Yet, th annuals –9. Hill  annuals  even sug annuals  summariz annuals ,12. Intehe field –13.Oryza spp. had been done by the International Rice Research Institute (IRRI) but was abandoned due to changing research priorities. More recently, the Yunnan Academy of Agricultural Sciences has successfully revived these efforts [DeHaan, et al.  and Pime efforts ,15.While these reviews provide some information on the history of perennial crop research, their focus is primarily on the potential of perennial crops and theoretical approaches to breeding rather than providing a detailed analysis of existing literature. A review that evaluates the publication histories of different perennial crops and ratooning systems is missing from the literature. Such a systematic review will help identify gaps in the literature by comparing the successes and failures associated with developing perennial staple crops as well as how research trajectories affect the current status of each crop analyzed.Phaseolus coccineus, as we were more interested in how previous literature might explain the current status of perennial lines that are actively being developed. After an initial analysis of bibliographic metadata, we applied a topic modeling approach to identify topics in the perennial crops literature. We looked for changes in the topics across the perennial crops library, as well as within the collections of literature for each crop of interest. Topic modeling is a novel approach to objectively evaluating the development of a research area by identifying emergent themes and topics in a body of literature via statistical algorithms.Our objective was to assemble a comprehensive library of literature on principal staple crops for which perennial forms are under development in order to study the progression of perennial crops research over time. We excluded crops that have perennial forms of potential interest, such as We analyzed the scientific literature on a total of five crops: pigeon pea, rice, rye, sorghum, and wheat. These crops have viable perennial forms that are under active development and could have the potential to be used in both commercial and smallholder cropping systems. In addition, we analyzed the literature containing the keywords “perennial grain” to capture more general reviews on the concept of perennial cropping systems. The term “perennial grains” is frequently used to refer to the concept after seminal work from researchers associated with the Land Institute.We began by assembling a comprehensive library on the perennial forms of the target crops using the advanced search capabilities of four databases to ensure adequate coverage of the scientific literature: Web of Science , Scopus Searches were composed of two inclusion terms linked by a proximity operator For the most part, the proximity operator was set to “1,” which meant that the two inclusion terms were within one word of each other. This approach was less restrictive than searching for exact phrases . But it did better at reducing false hits than the "AND" Boolean operator, such as instances where inclusion terms might appear in unrelated parts of the text. The proximity operator was increased to “5” for the inclusion term “ratoon.” As a cultural practice, the term ratooning retains relevance even if it is separated by a greater number of words from the crop of interest. The term “ratoon” was paired with the terms “rice” and “sorghum,” as ratooning is a common practice with these crops. The searches for rye were further refined to exclude results that referenced perennial forage and weed perennial ryegrass. This conservative approach may have inadvertently excluded some relevant literature on perennial rye.Zizania spp.) and kernza (Thinopyrum intermedium). However, as we were primarily interested in examining how historical literature potentially influenced current status, we chose to exclude emerging crops that did not have a sufficient development history, as well as extraneous terms that may have yielded interesting, relevant literature but were not directly related to the crops of interest.In choosing our search criteria, we also made certain inadvertent and intentional omissions. Searches were centered on American English to the neglect of foreign languages and other English dialects. Potentially useful terms like “perennial cereals,” “rhizome,” and “stolon” would have likely contributed additional material of relevance, as would have searches for emerging perennial crops like wild rice  were retained. Articles on less relevant aspects of their genetics (i.e. production of particular enzymes or compounds) were excluded.Finally, the search term “perennial grain” returned a handful of review papers that addressed the concept of perennial grain systems or utilized it in service of another argument. Reviews that primarily explored perennial grains and their possible utility were retained, while reviews that only mentioned perennial grains in passing were excluded.We exported the library from Zotero as a CSV file for analysis in R . SeparatIn addition to analyzing bibliographic data, we analyzed publication abstracts using a topic modeling approach. First proposed in 1998 , topic mThe workflow that follows was applied to the library for all journal articles, to collections of journal articles for each crop, and to collections of journal articles published in three time periods . We patterned a workflow after Chavalarias and Cointet  and Grüna priori since we expected research to generally segregate into the three fields of genetics and breeding, agronomy, and environmental sciences. These three fields represent what we believed might be a hypothetical progression of perennial crop research: breeding to develop a viable crop, agronomic testing to understand performance, and environmental science to test for potential impacts on agroecosystems. We then examined topics by extracting the terms with a likelihood greater than 1% of being associated with a given topic.Topic models with three topics each were then generated using four different methods similar to Grün and Hornik : VEM, VEFor the purposes of this article, we discuss the results of the models using the LDA-Gibbs approach (Latent Dirichlet Allocation and Gibbs sampling). LDA is a generalization of probabilistic latent semantic indexing that allows journal articles to contain a mixture of multiple topics . Gibbs sThe final library of 914 records was retrieved primarily from WoS, followed by Scopus, ScienceDirect, and Agricola Agriculture, Ecosystems, and Environment, a publication that focuses on the interface between agroecosystems and their environment.Journal articles on perennial crops appeared as early as 1930, and the publication rate since has steadily increased Selektsiya i Semenvodstvo, which translates to Breeding and Seed ProductionCrop Science, Field Crops Research, Crop and Pasture Science and Agriculture, Ecosystems and Environment.Individual crops displayed unique patterns of research activity over time. The early period from 1930 to 1959 were largely represented by perennial wheat research from the former Soviet Union in such publications as Rice Research Newsletter, a publication of IRRI. Indexed, international journals in the agronomy and crop science fields such as Field Crops Research began to feature articles on perennial crops research in the 1990s.The period from 1960 to 1989 highlighted publications from India, the Philippines, and the International Rice Research Institute (IRRI), with a focus on perennial rice since the 1980s Agroforestry Systems. Journal articles on perennial sorghum are mostly published in agronomy journals since the 1960s, as well as a few Indian journals such as Madras Agricultural Journal and Journal of Maharashtra Universities.Perennial pigeon pea and sorghum have received comparatively less attention than the previously mentioned crops, however their rates of publication increased in the 1990s as well (data not shown). Research on perennial pigeon pea was predominantly from India where it is a native crop important to culture and cuisine. Additionally, perennial pigeon pea research has appeared in international publications with foci on agroecology and agroforestry such as The efficacy of topic models for the library and collections varied considerably. The analysis of the entire library resulted in the highest posterior probabilities for topic terms and journal articles . The effSplitting the library by time period had a similar effect to splitting the library into collections by crop. Earlier time periods had many fewer publications compared to the last 30 years, reducing the overall effectiveness of topic modeling. Furthermore, since research within each time period was often dominated by one crop , splitting the library by time did not necessarily provide further insights beyond the other approaches. Here we present the results of the topic models of the entire library across time and the topic models of the rice and wheat collections as these were most successful in generating multiple coherent topics that matched the papers with high posterior probabilities ratoon, main crop, and ratoon crop. Articles strongly associated with topic 2 are almost exclusively about the physiology and agronomic management of ratoon rice systems. Topic 3 is a more general topic that includes several agronomic terms, such as plant, product, crop, and soil. Topic 3 articles generally discuss interactions between perennial crops and their environment. Topic 1 is comprised of terms that refer to the breeding of perennial crops through hybridization of distantly related species, such as perennial, annual, hybrid, and resist. Strongly associated articles with topic 1 are primarily from the early Soviet literature in the 1930s.Topic 2 for the entire library includes terms that are primarily about ratoon rice production, such as The distribution over time of topics 3 and 1 matched the publication patterns for wheat and rice outlined above perennial, type, number and fertility, as well as the genus name agropyron (Agropyron glaucum) and the species name elongatum (Thinopyrum elongatum). These two species were used by breeders in the Soviet Union to introduce perennial characteristics into their breeding lines. Articles associated with topic 2 reflect these terms in that they cover the selection, phenology, and physiology of those lines. Terms for topic 3 were more generally about distant hybridization, including terms such as hybrid, variety, and cross. Topic 1 included a broad range of general agronomic terms such as perennial, annual, crop, yield, and soil. Despite how broad these terms were, the articles created a cohesive group focused on various aspects of the physiology of perennial plants and their potential implications for agronomic performance.Topic terms for the wheat collection largely reflected the fields of breeding and agronomy on wheat. Topic 2 included general terms, such as The distribution of topics over time for the wheat collection reflected the bimodal pattern of research on perennial wheat ratoon high on the list of probable terms. The other terms for either topic emphasize different aspects of ratooning systems. Topic 2 includes general terms such as yield, main crop, product, and field, while topic 3 includes such terms as height, stubble, panicle, and nitrogen. Journal articles associated with topic 2 were focused on the effects of nutrient management on ratoon rice, and its integration into cropping systems, while journal articles strongly associated with topic 3 were primarily about the physiological response of ratoon rice to harvest height, timing, and nitrogen applications. Topic 1, on the other hand, was concerned with characterizing perennial cultivars and wild relatives, with terms including perennial, population, and genetic.Both topics 2 and 3 for the collection on perennial rice were concerned with ratoon systems with the term All topics saw a steady increase over the past century with a large jump in the number of publications in the 1980s Overlaps in research between some crops are likely due to their agronomic compatibility or genetic relatedness. For example, literature on wheat and rye often overlapped since they are closely genetically related and were both used as source material in various perennial crop breeding efforts.While research on perennial crops is increasing overall, there appear to have been distinct periods of active research for each crop. We observed that research on perennial crops has generally transitioned from exploratory phases to eventually gain prominence through publications with a broader readership. At the exploratory, initial phase, dialog was primarily confined within small groups and specific communities of researchers, with articles primarily appearing in special interest or crop-specific publications. Rice provides a clear example, where the publication of research during the 1980s and 1990s was led by IRRI, and primarily consisted of newsletter and technical articles, followed later by the appearance of articles in more broadly circulated crop physiology journals. Likewise, journal articles on perennial wheat were primarily from the Soviet Union in the early 20th century and published in Russian language journals, while the recent increase in publication over the past 10 years has been primarily in prominent agronomy and agroecology journals.Field Crops Research and Agriculture, Ecosystems, and Environment emerge as primary publishers for the crops evaluated and for the more general topic of perennial grains. Both publications have impact factors around 3, and are top journals in their research areas. Further, they are often target publications for the fields of agronomy and breeding. The consistency with which such journals have articles on perennial crops in recent decades may indicate a favorable editorial environment that may help advance progress in perennial crops research.Certain publications have played an important role in fostering particular research areas. Contrary to our hypothesis, topic models for the entire library did not sort out into the three fields of research we expected—breeding, agronomy, and agroecology. Instead the higher numbers of rice and wheat publications guided topic models of the full library, generating topic terms that were highly specific to either crop and the approach researchers took to developing their perenniality or ratooning characteristics for agronomic purposes. Perennial crops research in the early 20th century was dominated by USSR breeding efforts and their technique of distant hybridization (i.e. topic 1). While research on rice was primarily about the physiology and management of ratoon rice systems (i.e. topic 2). Topic models of the rice and wheat collections also did not produce topics that matched our hypothesized categories.Topic 3 from the topic model of the entire library is the only topic that covered a field similar to our hypothesized category on agroecology. Papers associated with this topic included research on pest dynamics, soil moisture dynamics, and greenhouse gas budgets. Overall, however, the almost complete lack of topics dealing explicitly with soil biology and soil carbon is notable since some of the most prominent arguments for perennial cropping systems posit dramatic improvements in soil quality and ecological functions in agricultural landscapes . EnvironThis bias in the literature toward developing crops that achieve both high yield and high annual survival may be coming at the expense of research that better reflects growers’ motivations for choosing such crops. Adebiyi et al.  found inTopic modeling for the rice and wheat collections reveal differences in the patterns of research on these two crops that highlight interesting differences in strategies to develop and exploit perenniality. The comparatively large number of publications on rice is largely due to the inclusion of literature on ratoon rice systems, and topic models were dominated by different aspects of ratoon system research. The importance of rice ratooning should not be understated, as it is a system often practiced in different rice growing regions of the world in both commercial and smallholder contexts. Institutional efforts to support the development of ratoon systems are apparent in the large number of publications on rice that appear in the International Rice Research Newsletter, a publication of IRRI. Although ratoon rice systems are not perennial in the sense that they last for several years, the existence of such a broadly practiced, traditional system has arguably provided support and a starting point for institutional breeding efforts. Recent research at the Yunnan Agricultural University in China has produced a variety of rice that successfully ratoons from year to year ,15.Agropyron glaucum. Decades of effort managed to produce some promising lines, but only after considerable difficulty with issues of sterility, low annual survival, and unfavorable agronomic characteristics [Literature on wheat, on the other hand, was dominated by breeding efforts until much more recently when research topics on the physiology of perennial wheat cultivars and the agroecological potential of such systems appear to emerge. A practice analogous to ratooning rice arguably does not exist for wheat. Lacking a similar system to utilize as a starting point, efforts to breed perennial wheat may have been more handicapped. Breeding efforts in the Soviet Union required hybridizing wheat with more distantly related species, such as eristics ,35. Simieristics . Despiteeristics . Or, as eristics  argue, pThe comparison of rice and wheat also serves to highlight potential gaps in perennial crop breeding efforts with other crops. If a foundation of research on ratooning rice systems has provided a comparative advantage for developing successful perennial cultivars of rice, other crops, such as pigeon pea and sorghum, that have been similarly utilized as ratooning crops in traditional systems, may be primed for development, Although we included them in our searches, fewer publications for pigeon pea and sorghum were retrieved despite their historic and continued use as perennials in smallholder production systems. Pigeon pea originated as a perennial species and is still used as such in agroforestry systems, and is a widespread garden species in tropical gardens; however breeding for short duration varieties is the dominant trend in pigeon pea crop development. Sorghum is managed as a ratoon crop in some regions of Africa, however its documentation and development in the scientific literature is limited. This lack of publications is likely due various causes, but is perhaps best explained by it being primarily produced under semi-arid and marginal soil conditions. The same bias towards developing high yielding, commercially relevant perennial crops discussed above might explain the lack of interest in sorghum and pigeonpea among researchers. If greater effort were focused on developing these crops, existing knowledge systems and varieties could be effectively leveraged to generate truly perennial lines as appears to be the case with rice.International Rice Research Newsletter. Topic modeling of the entire library was strongly influenced by the high numbers of rice and wheat publications but also produced a topic about the agroecology of perennial and ratooning systems. No topics explicitly concerning soil biology or carbon dynamics were produced, reflecting a dearth of publications in this area and potential bias amongst researchers. A comparison of topic models for rice and wheat revealed differences between their histories of development. The existence and widespread use of ratoon rice systems has provided source material for developing perennial rice cultivars, while the lack of an analogous system for wheat has meant breeding efforts had limited material and research on which to base their research. Comparatively little has been done to develop some other perennial crops, such as pigeon pea and sorghum. But their use as perennials in some contexts may make them prime candidates for such development. However whether they receive such attention may depend on the priorities of researchers and the development sector.Analysis of perennial crops publications has highlighted interesting research development trajectories and publishing patterns over the past century. Each crop has seen episodic waves of interest with higher publication rates, but some crops are considerably less researched. The dominance of certain journals in the libraries indicates the importance of individual journals to supporting a field of research, especially journals that are crop-specific, such as the S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 PRISMA ChecklistCompleted PRISMA checklist for meta-analyses.(DOC)Click here for additional data file.


To assess how girls' preferences have changed almost 3 years after the much debated start of the human papillomavirus (HPV) vaccination program.A discrete choice experiment (DCE) was conducted among girls aged 11–15 years who were invited, or were not yet invited, to get vaccinated. A panel latent class model was used to determine girls' preferences for vaccination based on five characteristics: degree of protection against cervical cancer; duration of protection; risk of mild side-effects; age of vaccination; and the number of required doses of the vaccine.The response rate was 85% (500/592). Most girls preferred vaccination at age 14 years (instead of at age 9 years) and a 2-dose scheme (instead of the current 3-dose scheme). Girls were willing to trade-off 7% (CI: 3.2% to 10.8%) of the degree of protection to have 10% less risk of mild side-effects, and 4% (CI: 1.2% to 5.9%) to receive 2 doses instead of 3 doses. Latent class analyses showed that there was preference heterogeneity among girls, i.e., higher educated girls and HPV vaccinated girls had a higher probability to opt for HPV vaccination at a higher age than lower educated girls or non-vaccinated girls.not to lower the current target age of 12 years. A 2-dose scheme may result in a higher uptake and we recommend that if this scheme is introduced, it needs to receive adequate publicity.Three years after the start of HPV vaccination program the risk of mild side-effects and age at vaccination seem to have become less important. For the Dutch national immunization program, we recommend  Human Papillomavirus (HPV) infection is a necessary factor in the development of cervical cancer Many Western countries have included HPV vaccination in their immunization program. For example, the United Kingdom, Canada, Australia and the Netherlands offer the HPV vaccine to girls at an age between 11 and 14 years; in these countries, the uptake rates range from 50–80%. The willingness to accept HPV vaccination can largely be influenced by general preferences for healthcare interventions In the Netherlands, the bivalent HPV vaccine is offered free of costs to 12-year-old girls by sending a personal invitation. These girls do not need their parents' permission when deciding about uptake. Since the introduction of the vaccine in the Netherlands in 2009, uptake rates increased from 52% in 2009 after the media debates have ended and in the absence of reports of serious side-effects. To our knowledge, this is the first study to compare preferences for HPV vaccination as expressed in DCEs. We will look at the differences in preferences as measured in 2009 versus 2011. This comparison may provide insight into girls' motivation to be vaccinated or not, how this motivation can change over time, and how to improve dissemination of information about the vaccine.Therefore, the present study assesses which attributes of HPV vaccination have influenced preferences for HPV vaccination uptake The selection of HPV vaccination attributes and their levels was based on our previous study The final set consisted of the following attributes: 1) the degree of protection against cervical cancer; 2) the duration of protection; 3) the risk of mild side-effects; 4) the age of vaccination; and 5) the number of doses of the vaccine . The lev4×21) hypothetical HPV vaccination alternatives. We generated a subsample of these alternatives using priors available from De Bekker-Grob et al. http://www.choice-metrics.com/) The combination of four attributes with three levels each, and one attribute with two levels, resulted in 162 (3http://www.choice-metrics.com/).Calculation of the optimal sample size for estimating discrete choice models from DCE data is complicated, as it depends on the true values of the unknown parameters estimated in the choice models The first page of the questionnaire provided basic information about HPV vaccination. Next, respondents were asked to indicate per choice set which option appealed to them most. Pictographs were used to illustrate the percentages of the degree of protection and the risk of mild side-effects.To assess respondents' understanding of the DCE task we included a dominant choice set as a rationality test. In this choice set age of vaccine administration was similar in both alternatives, while one alternative was characterized by logically preferable levels on all other attributes. Also we included four items on a 5-point Likert scale to evaluate whether respondents considered the DCE questions ‘clear-unclear, ‘difficult-easy’, ‘annoying-pleasant’, and the number of questions as ‘too many-not too many’. Convergent validity was checked by asking the respondents to rank the five attributes of HPV vaccination from most important to least important. This ranking is compared with the trade-offs respondents were willing to make between the degree of protection and the other attributes.The questionnaire used in our 2009 study was pilot tested to check for face validity and for problems in interpretation (n = 16). Because the number of attributes are the same as in the present study and only the attribute ‘risk of serious side-effects’ is replaced with ‘number of doses of the vaccine’, we did not expect problems in interpretation and therefore did not pilot test the questionnaire of the present study.Respondents completed the questionnaire in the classroom or auditorium during school time. First, general information was given about HPV and vaccination and about the way DCE questions should be completed. Completion of the written questionnaire lasted about 20–30 min. Questionnaires were completed in November and December 2011.Beforehand, girls' parents had received an information letter covering the purpose, the voluntary nature and anonymity of the study, and an opt-out form. Parents that did not want their daughter to participate could sign the opt-out form. Girls' parents who approved participation did not have to sign an informed consent form. The Medical Ethics Committee of Erasmus MC, University Medical Center Rotterdam declared that this research (number MEC 2011-059) did not fell under the Medical Research Involving Human Subjects Act, because Participants were not subject to procedures or are required to follow rules of behavior.http://www.limdep.com/), the observations were analysed by a panel latent class model Panel latent class model means that the model accounts for the pseudo panel nature of the DCE data since each respondent completed 16 choice tasks. To determine the number of classes to impose on the model structure, we selected the model with the best fit based on the Akaike information criterion (AIC) The DCE was analyzed by taking each choice among the three options  as an observation. The utility for “no vaccination” was normalized to zero: V(no vaccination) = 0. Using NLogit software (We tested a number of different specifications for the utility (preference) function. After testing for linear continuous effects of the attributes, the following final specification of the utility model was estimated:|cV represents the observable utility (preference score) that respondents belonging to class segment c have for an HPV vaccination. β7|c1|c− are class specific coefficients of the attributes indicating the relative weight individuals place on a certain attribute (level). The unobserved component, The statistical significance of a coefficient  indicates that conditional to belonging to a class, respondents differentiated between one attribute (or attribute level) and another in making stated choices about HPV vaccination programs. A priori, we expected all attributes to be significant. The sign of a coefficient reflects whether the attribute has a positive or negative effect on the preference score (utility). We expected that the attributes ‘risk of mild-effects’ and ‘the number of doses of the vaccine’ would have a negative effect. The value of each coefficient represents the relative importance respondents assign to an attribute level. Sensitivity analyses were conducted to explore the impact of excluding respondents who failed the rationality test by excluding their data from the sample and re-running the analysis In terms of the class assignment parameters, statistically significant parameter estimates indicate that the associate covariate (i.e. ‘respondent's history of HPV vaccination’ and ‘education’) can be used to help in understanding the different segments. For example, if the education parameter associated with a particular class in the assignment model is positive and significant, then this is indicative that people who have a higher educational level are more likely to belong to that particular class and, hence, have preferences associated with the utility function belonging to that class as given in The trade-offs respondents were willing to make between the attributes were calculated by the ratios of the coefficients of the different attributes with the degree of protection as the denominator. These trade-offs were weighted by the probability that a respondent belongs to a given class. Confidence intervals were calculated in Excel using the Krinsky and Robb method Since our 2009 study is a point of reference for this study, we compared the similarity of the present sample to the 2009 sample. Mann-Whitney U tests were used for continuous variables and Chi-square tests were used for categorical variables.p-value<0.01); more respondents had a lower or intermediate educational level and less girls had a higher educational level ; and less respondents were vaccinated against HPV .The response rate was 85% (500/592). The mean age of the respondents was 12.9 years; most had a higher level of secondary education (38%) and no religious affiliation (68%). Of the respondents, 63% had already been invited to get vaccinated against HPV of whom 70% had opted for vaccination . CompareBased on the AIC criterion, three classes were identified . The aveOverall, respondents were willing to trade-off 7% (CI: 3.2% to 10.8%) of the degree of protection to have a 10% less risk of mild side-effects. To obtain protection against HPV for 25 years instead of 8 years, they were willing to trade-off 18% (CI: 8.6% to 29.6%), and to obtain lifetime protection instead of 8 years of protection, they were willing to trade-off 21% (CI: −0.1% to 37.2%). Respondents were willing to trade-off 4% (CI: 1.2% to 5.9%) to receive 2 doses instead of 3 doses. To get a vaccination at age 12 or 14 years, instead of at 9 years, respondents were willing to trade-off 4% (CI: −2.4% to 8.6%) and 8% (CI: −0.6% to 16.7%), respectively .The dominant choice set was answered correctly by 490/500 (98%) of the respondents; 83 respondents completed the ranking test incorrectly (e.g. giving the same rank to multiple attributes) and were excluded from this ranking analyses. The most important attributes according to the ranking test were: the degree of protection (70%); the duration of protection (17%); the risk of mild side-effects (8%); the number of doses (4%); and the age of vaccination (2%) (n = 407). The trade-offs respondents were willing to make between the degree of protection and the other attributes indicated the following order of importance of attributes: duration of protection, followed by the risk of mild side-effects, the number of doses of the vaccine, and age at vaccination . Thus, tThe mean evaluations of the DCE questions were (range 1–5): ‘unclear-clear’ , ‘difficult-easy’ , ‘annoying-pleasant’ , and ‘too many questions-not too many questions’ .We used a DCE to determine girls' preferences for HPV vaccination almost 3 years after the much debated start of the HPV vaccination program. Overall, girls were willing to trade-off 18% of the degree of protection to obtain a vaccination with 25 years protection instead of 8 years protection, and trade-off 7% to have a 10% less risk of mild side-effects. To receive 2 doses of the vaccine instead of 3 doses, they were willing to trade-off 4% of the degree of protection. Furthermore, it appeared that higher educated girls and HPV vaccinated girls have a higher probability to opt for HPV vaccination if it is offered at age 12 years instead of at age 9 years, than girls with lower education levels or girls who were not vaccinated.When comparing these reported trade-offs with those of our previous study in 2009 In summary, almost 3 years after initiation of the HPV vaccination campaign on the Netherlands, the risk of mild side-effects and age at vaccination seem to have become less important. Potentially, the girls had a better idea about which mild side-effects to expect and were less concerned about them. Also, the importance of the degree of protection may have gained value for the girls. The age of vaccination might be less of an issue in 2011 given the longer duration of protection, i.e. 8 years in 2011 compared with 6 years in 2009.There was preference heterogeneity among the girls, i.e. higher educated girls and HPV vaccinated girls have a higher probability to opt for HPV vaccination if it is offered at age 12 years instead of at age 9 years, than girls with lower education levels or girls who were not vaccinated. Furthermore, the majority of girls (including higher educated girls and HPV vaccinated girls) also preferred vaccination at age 14 years to vaccination at age 9 years. In other words, most girls did not prefer vaccination at the age of 9 years. Overall, girls were willing to trade-off 3.5% of the degree of protection to receive 2 doses instead of 3 doses, and most girls also preferred a 2-dose scheme to the current 3-dose scheme. Recently, the Netherlands National Institute for Public Health and the Environment decided that a 2-dose scheme will be introduced, because 2 doses are found to provide as much protection as 3 doses as long as the vaccination is given before girls turn 15 years of age A strength of the present study is the large number of respondents (n = 500) and the high response rate (85%). A limitation might be that we did not include protection against genital warts as an attribute.In conclusion, this study shows that, almost 3 years after the much debated start of the HPV vaccination program in the Netherlands, trade-offs that girls are willing to make have not changed substantially. The risk of mild side-effects and age at vaccination still influenced the girls' preferences, but seem to have become less important. This study shows that there was preference heterogeneity among the girls, with higher educated girls and HPV vaccinated girls having a higher probability to opt for HPV vaccination at a higher age, than girls with lower education levels or girls who were not vaccinated. Also, since most of the girls preferred vaccination at age 14 years to vaccination at age 9 years, we recommend not to lower the current target age of 12 years in national immunization program in countries such as the Netherlands, Denmark, Sweden, Norway and United Kingdom. We also recommend to introduce a 2-dose scheme (instead of the current 3-dose scheme), because the girls are far from indifferent to the choice between 2 and 3-dose scheme.


New Zealand initiated HPV vaccination in 2008, and has attained 3-dose coverage of ~50 % in 12–13 year old girls. Due to the success of program initiatives in Māori girls, higher coverage rates of ~60 % have been achieved in this group. We have previously reported a benchmark overall pre-vaccination prevalence of oncogenic HPV infection in high grade cervical lesions in New Zealand. The current extended analysis provides separate pre-vaccination benchmark prevalence for Māori and non-Māori women.The National Cervical Screening Programme Register (NCSP-R) was used to identify any woman aged 20–69 years of age with an index high grade cytology report from 2009–2011. Extended recruitment was performed until 2012 in clinics with a high proportion of Māori women. Ethnicity status was based on self-reported information by participating women through phone contact supplemented by recordings on the study questionnaire (the NCSP-R was not used to extract ethnicity status). A total of 730 women consented to participate and had a valid HPV test result; 418 of these had histologically-confirmed cervical intraepithelial neoplasia (CIN) 2/3 lesions . The prevalence of any cervical oncogenic HPV infection, HPV16, and HPV18 was calculated in women with CIN2/3.interaction = 0.02), with a lower prevalence in younger vs. older Māori women (57 % in 20–29 years vs 75 % in 40–69 years) but a higher prevalence in younger vs. older non-Māori women (70 % in 20–29 years vs 49 % in 40–69 years); the difference in the age-specific patterns of infection for HPV16/18 was not significant either when considering confirmed CIN2 alone (p = 0.09) or CIN3 alone (p = 0.22).In confirmed CIN2/3, the prevalence of any oncogenic HPV, HPV16 and HPV18 was 96 % (95 % CI:91–99 %), 54 % (95 % CI:46–63 %), 11 % (95 % CI:7–18 %) in Māori and 96 % (95 % CI:93–98 %), 54 % (95 % CI:48–60 %), 11 % (95 % CI:7–15 %) in non-Māori women, respectively. Age-specific patterns of infection for HPV16/18 in confirmed CIN2/3 differed between the two groups  contains supplementary material, which is available to authorized users. It is also possible that we did not detect an interaction between age and ethnicity for specific lesion grades because of a lack of power to detect such an interaction. Note also that we did not control for deprivation or other factors potentially underlying our findings for Māori vs. non-Māori women. The primary aim of the study was to describe potential differences in type-specific HPV prevalence by ethnicity, rather than explore mechanisms that underlie ethnic differences in the prevalence. The importance of doing so is that in New Zealand targeted programs are implemented for specific ethnic groups (irrespective of deprivation). We have therefore configured the analysis to be directly relevant to this approach.Our study has some limitations. It is possible that women who consented to participate in the study might differ from those who did not in terms of demographic characteristics or cervical screening history, which may have impacted the type-specific rates of HPV in high grade lesions, and that this difference might vary between ethnic groups. However, in our previous analysis we showed that the sample of women consenting to the study was representative of those with high grade lesions in the New Zealand NCSP Register . It is pvia laboratory reports of CIN2/3 for which biopsy may have been originally performed for a range of indications) [In line with our previous analysis , the vercations) . Despitecations) . The moscations) .It is known that type-specific prevalence of HPV varies substantially by geographical regions , 17. HowAlthough the results of general population HPV prevalence studies are not directly comparable with our findings, we identified two such studies assessing type-specific prevalence of HPV by ethnicity or Indigenous status. In Canada, HPV prevalence was assessed among women with aboriginal, Caucasian and other ethnic backgrounds  . The stuOur study, therefore, represents one of the first detailed investigations of the relative prevalence of vaccine-included HPV types which can directly compare findings across ethnic groups. Our finding that the overall prevalence of vaccine-included HPV types in CIN2/3 is similar in Māori and non-Māori women is reassuring, and implies that the longer term effects of vaccination will be similar in the two groups, or even (given the higher coverage rates achieved), that better post-vaccination outcomes might be achieved in Māori groups. We conclude that ongoing high coverage of both Māori and non-Māori women in the National HPV Immunisation Programme in New Zealand is required to achieve high overall program effectiveness, and our results also underpin the ongoing importance of initiatives to further increase participation in cervical screening in Māori women. In the future, repeat cross-sectional surveys of the type reported here will provide an ongoing mechanism of monitoring the impact of HPV vaccination in New Zealand. To facilitate ongoing monitoring of the impact of HPV vaccination on the National Cervical Screening Program, individual record linkage between the NCSP-R and immunisation registries is under active discussion.


The pathogenesis of sinonasal polyposis remains unclear, in spite of several investigative approaches. Antrochoanal polyps, a subgroup of sinonasal polyposis along with allergic- and chronic-inflammatory nasal polyps, mostly originate from the maxillary sinus and develop as a unilateral, pedunculated mass towards the nasopharynx. The human papillomavirus (HPV) is discussed as a possible causative and influencing factor in development and progression of sinonasal polyposis. This study aims to elucidate HPV frequency in nasal polyps and antrochoanal polyps.Genomic DNA from 257 tissue specimens  was subjected to three different established HPV- polymerase chain reaction assays, testing for 37 low- and high-risk HPV. In addition, immunohistochemical analyses for HPV16 were carried out, as well as immunohistochemistry and western blots of p16, a biomarker for HPV induced cancer.HPV-DNA was detected in 53.8% of antrochoanal polyps, 15.1% of nasal polyps, and 5.8% of nasal turbinates. HPV16 was the predominant type with a detection rate of 76% in nasal polyps and 62% in antrochoanal polyps. Immunohistochemically, HPV positive tissues stained positive for HPV16 antigens and p16 in epithelial cell layers. No significant p16 overexpression was traceable in antrochoanal polyps, nasal polyps and nasal turbinates by western blot. There was no correlation of HPV-status with sex, age, smoking, alcohol consumption or allergic background.The present study shows a significant frequency of high-risk type HPV16 in antrochoanal polyps. Absence of oncogenic transformation or correlation of the HPV-status with clinical data suggests a latent superinfection, possibly because of anatomical proximity to the oropharynx. Polyposis nasi et sinuum is a common disease affecting 1–4% of the general population . It is dNasal polyps are characterised as multifocal polypoid mucosal swellings, typical bilateral and arising mainly from the middle meatus and ethmoid sinus region. They occur more frequently in patients with persistent asthma, aspirin-exacerbated respiratory disease or cystic fibrosis and often show an allergic background , 4. FurtIn contrast, characteristic antrochoanal polyps usually constitute a larger solitary unilateral mass. They originate from the maxillary sinus, pass through the nasal cavity, extend into the choanae and further into the nasopharynx. Antrochoanal polyps consist of two components, a cystic antral and a solid polypoid part, linked by a long stalk. Patients suffering from antrochoanal polyps are more commonly children and young adults, with an average age of 27 in our cohort and other studies , 10. TheAlthough sinonasal polyposis is a well-investigated entity, the aetiology still remains unclear. To date, quite a few studies were carried out screening nasal polyps for causative factors, including human papillomavirus (HPV) –23. HPV Between 2000 and 2014 only six studies investigating the association of HPV with nasal polyps and sinonasal mucosa have been published. The results are highly inconsistent and showed prevalence of HPV-DNA in nasal polyps ranging from 0% to 40%, while no viral DNA was found in healthy controls , 28–31.Obviously data concerning the prevalence of HPV in sinonasal polyposis is lacking. Previous studies have not investigated subgroups separately, so no references exist about a possible association of HPV in particular with antrochoanal polyps. Therefore, the current study investigated the HPV frequency in a large series of inflammatory nasal polyps and antrochoanal polyps via well-established HPV-polymerase chain reaction (PCR) arrays. To provide further information about the localisation and activity of viral infection HPV 16 and p16, a biomarker for HPV induced cancer, immunohistochemistry, as well as p16 western blot was performed.166 archived fresh frozen specimens of nasal polyps from 2008 to 2012 were analysed. Overall, antrochoanal polyps were obtained from 39 patients, between 2000 and 2012. 31 of these were paraffin-embedded archive material from the Institute of Pathology, Friedrich-Alexander University of Erlangen-Nürnberg. Sixteen fresh frozen antrochoanal polyps were collected from patients who underwent surgical treatment at ENT-Hospital, Head and Neck Surgery, University of Erlangen-Nürnberg. There was an overlap of eight antrochoanal polyps which were available as fresh frozen as well as paraffin-embedded tissue  according to the manufacturer’s instructions. DNA concentration was evaluated using standard photometer and afterwards adjusted to 50–200 ng/μl.Genomic DNA (gDNA) from paraffin-embedded tissue was extracted by using peqGOLD Tissue DNA Mini Kit (C-Line) provided by peqlab  following the manufacturer’s protocol. DNA isolation and purification of cryopreserved tissue was conducted with AquaGenomic® Green I No ROX  were used. PCRs were implemented with the iCycler . Distilled water and DNA from reliable HPV negative tissue served as negative controls. Three HPV- positive oropharyngeal carcinomas were included as positive controls. The amplicons were detected by gel electrophoresis on 2.5% agarose gel and analysed with the Lumi-Imager . HPV type identification was pursued by direct sequencing analysis conducted by LGC Genomics GmbH  with primer GP6+ . Afterwards they were incubated at 4°C for 2 hours and centrifuged at 16000g for 1 hour. The total protein concentration was determined with bicinchoninic acid assays . After denaturation through 5 minutes at 90°C with SDS loading buffer including mercaptoethanol, 50 μg of lysate was applied on 12% Criterion™ XT Bis-Tris Precast Gel  and transferred to nitrocellulose membranes . The blotting control was Ponceau-S stained and immunostaining of β-Actin reference protein  served as control for the loading amount of protein. The primary antibody for detection was the monoclonal mouse antibody against p16 , followed by the secondary antibody, a peroxidase-labeled anti-mouse IgG, F(ab’)2 antibody . The blot was incubated with SuperSignal® West Dura Extended Duration Substrate  and the signals were detected with the Lumi-Imager .The homogenisation  of 30–50 mg of each tissue sample was accomplished in 1.0 mL lysis buffer [50 mmol/L Tris, pH 7.4; 300 mmol/L NaCl; 10 mmol/L EDTA; 1% NP40; 0.1% Triton-X100; 0.1% SDS; protease inhibitor cocktail Complete® GmbH . P-values less than 0.05 were considered statistically significant.To reveal differences between subject attributes or clinical data, as sex, smoking, alcohol consumption, allergic background and HPV- status, these variables were analysed by non-parametric Kruskal-Wallis analysis with post hoc multiple comparison rank analysis, non-parametric Mann-Whitney U test and chi-square test, as required. The statistical analysis was implemented using STATISTICA provided by StatSoftIn all 257 obtained tissue specimens  gDNA was extracted and studied by broad spectrum PCR assays GP5+/6+, SPF and NMPCR, relative to tissue fixation method Click here for additional data file.


Accurate identification of the microscopic risk factors of oral and oropharyngeal (OP) squamous cell carcinomas (SCC) and their morphologic variants is of at most importance, as these generally determine treatment modalities, prognosis and overall patient outcome. The great majority of oral and oropharyngeal squamous cell carcinomas are microscopically described as kerartinizing squamous cell carcinoma (KSCC). They bear certain resemblance to keratinizing stratified squamous epithelium. Tobacco habits and excessive consumption of alcoholic beverages have been considered to be the main etiologic agents in these carcinomas. The tumors occurred in older patients more commonly affected the oral tongue and floor of the mouth with well established morphologic risk factors including tumor grade, pattern of invasion and perineural involvement. Within the last 30 years however, the advent and expanding prevalence of high risk human papillomavirus (HPV) as an important etiologic agent for head and neck squamous cell carcinoma, particularly in the OP, has resulted in a significant change in the established morphologic criteria for risk assessment. The majority of HPV relate carcinomas of the OP are nonkeratinizing squamous cell carcinoma (NKSCC). These tumors are found to be more responsive to treatment with a favorable patient outcome and good prognosis. Consequently, alterations in treatment protocols aimed at de-escalation are currently being evaluated. More recently, other morphologic variants that are HPV positive are reported with increasing frequency in the OP and other head and neck sites. As a result, several clinical and pathologic questions have emerged. Importantly, whether the virus is biologically active in these tumors and involved in their pathogenesis, and second, what are the clinical implications with regard to patient management and outcome in the HPV-related variants. Examples of HPV-related squamous cell carcinoma variants that will be addressed here are: basaloid squamous cell carcinoma (BSCC), undifferentiated carcinoma (UCa), papillary squamous carcinoma (PSCC) and small cell carcinoma. Some studies have suggested favorable prognosis in some variants, analogous to that of the (NKSCC), while others showed poorer outcome. So far the number of studies on this subject is limited and the number of cases evaluated in each investigation is few. Because of that, it is prudent at this stage, not to alter management protocols as a result of identification of HPV in these variants and to await additional information Key words:Histopathologic risk-factors, oral cavity, oropharynx, squamous cell carcinoma variants, keratinizing squamous cell carcinoma, nonkeratinizing squamous cell carcinoma, HPV, basaloid squamous cell carcinoma, undifferentiated carcinoma, papillary squamous cell carcinoma, small cell carcinoma. Traditionally KSCCs have been graded according to their state of differentiation and resemblance to normal squamous epithelium into well, moderate and poorly differentiated variants. The grading system which was originally proposed by Broder  was baseA newer risk model was developed by Brandwein-Gensler and associates -6 as an During the last 30 years accumulating epidemiologic and clinical evidence has shown that high risk human papillomavirus (HPV) is a major etiologic factor in a subset of head and neck squamous cell carcinomas. These tumors have distinct clinical, microscopic and molecular features. The greatest majority of the carcinomas occur in the OP, particularly the palatine and lingual tonsils areas. They are characterized by younger age at onset , weak oret al,  for HPV DNA. While highly specific it is not very sensitive. On the other hand, PCR analysis for HPV DNA is highly sensitive but not necessarily specific. Detecting viral DNA by PCR does not indicate whether the virus is trancriptionally active (driver) or a bystander (passenger).E6/E7 mRNA expression is considered the “gold standard” for identification of clinically significant HPV infection in tumor specimens. Reverse transcriptase polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (RT-qPCR) are the main methods used for detection and quantitation of E6/E7 mRNA ,23. MoreA small minority of HPV+ OP SCC show atypical clinical behavior following treatment including poor OS, DSS and Disease free survival (DFS) ,26,27. SThe exact causal mechanisms involved in poor clinical outcome and patient survival in a minority of HPV positive SCC of the oropharynx are not clear. However, certain relevant molecular profiles have been proposed. These include over-expression of Bcl2, EGFR, and BclxL, ,30,31. EUnlike the OP the oral cavity proper is a rare site for HPV related SCC. El-Mofty and LU , using PThe great majority of HPV relate SCC of the OP is nonkeratinizing as described above. However, more recently increasing numbers of variants of squamous cell carcinoma, that are HPV-positive, are reported in the oropharynx as well as in other head and neck sites. As a result, several clinical and pathologic questions have emerged. Importantly, whether the virus is biologically active and involved in the pathogenesis of these tumors, and whether there are clinical implications with regard to patient outcome and treatment modality changes that may be needed in HPV related variants. Examples of HPV-related squamous cell carcinoma variants that will be addressed here include: basaloid squamous cell carcinoma, undifferentiated carcinoma, papillary squamous carcinoma, small cell carcinoma and keratinizing squamous cell carcinoma. Some investigations have suggested favorable prognosis in some HPV positive variants, analogous to that of the nonkeratinizing carcinoma, while others showed poorer outcome. So far the number of studies on this subject is limited and the number of cases evaluated in each investigation is few. Because of this, it is prudent that at this stage, not to alter management protocols as a result of identification of HPV in these variants and to await additional studies.In the upper aerodigestive tract, BSCC is rare variant of conventional SCC. It occurs more commonly in the hypopharynx and larynx and less frequently in the oropharynx. Like SCC the tumor is typically associated with traditional risk factors like tobacco smoking and alcohol abuse. It is generally considered a high grade malignant neoplasm with poor prognosis. Microscopically, it is characterized by a biphasic pattern. A basaloid component intimately associated with elements of keratinizing SCC. The basaloid cells are small, crowded with hyperchromatic round nuclei and scant cytoplasm. They form sheets and lobules that produce a “jigsaw puzzle” growth pattern with cystic spaces containing PAS –positive myxoid material. Stromal hyalization may be present Fig. . The squet al,  is best recognized in the nasopharynx where it is referred to by a variety of names including; lymphoepithelioma, nasopharyngeal type undifferentiated carcinoma and nonkeratinizing undifferentiated carcinoma (WHO type III). Microscopically identical carcinomas are also identified in the oropharynx. While undifferentiated carcinoma of the nasopharynx has a strong etiologic relationship to Epstein-Barr virus (EBV) , undiffeThe microscopic features of oropharyngeal undifferentiated carcinoma are indistinguishable from those of the nasopharyngeal type as defined by the WHO. The tumors are composed of solid sheets, trabeculae, nests and single neoplastic epithelial cells intimately intermingled with lymphocytes and plasma cells. The epithelial tumor cells are large with indistinct cell borders forming a syncytium Fig. . The nucPapillary squamous cell carcinoma (PSCC) is a poorly understood variant of SCC of the upper aerodigestive tract that is often confused with other exophytic mucosal malignancies such as verrucous carcinoma and squamous cell carcinoma with verrucous features. As defined by the WHO, PSCC is characterized by a predominant papillary growth pattern with thin fibrovascular cores covered by immature basaloid cells or dysplastic cells with minimal or no keratinization. PSSC is generally believed to have a better prognosis than conventional squamous cell carcinoma.et al.  respectively  KSCC of the OP overexpressed p16. HPV E6/E7 RNA ISH was positive in 5 tested cases. The OS and DSS was significantly better in the p16 positive than the p16 negative KSCC cases (ectively .The histopathologic features of keratinizing SCC of the upper aerodigestive tract have been used by Pathologists as tools for prediction of tumor behavior and patient prognosis. Consequently, Clinicians have relied on this information for determining treatment modalities to be used in each particular case. The greatest majority of these keratinizing neoplasms shared morphologic risk features that are used to classify them in a range of grades, from low to high. Less frequently, variant forms of KSCC such as BSCC, PSCC, adenosquamous carcinoma, spindle cell carcinoma and UCa are encountered. These variants have distinct morphology and clinical behavior and are not graded.Within the last few decades an HPV related nonkeratinizing variant of SCC was identified. The majority of which occurred in the oropharynx. The neoplastic cells in these tumors show an immature basaloid appearance and are mitotically active. Such phenotype is not limited to oropharyngeal sites but it is also observed in other HPV related tumors in variety of sites including the sinonasal tract, larynx the and anogenital sites ,49.The eAs the numbers of reported HPV positive head and neck squamous cell carcinoma variants are increasing, particularly in the oropharynx, it is of importance to establish the status of the virus in the tumors cells and to distinguish between a causal agent “driver” and a bystander “passenger”. Currently, the number of cases of SCC variants with established HPV causal relationship is still limited. Based on these studies, there is some evidence to suggest that variants such as BSCC, undifferentiated carcinoma and PSCC may have favorable prognosis similar to that of conventional NKSCC and better than their HPV negative counterparts. On the other hand, HPV related small cell carcinoma has been shown to be associated with early distant metastasis and poor overall survival. More over a minority of NKSCC of the oropharynx have been recently found to be associated with atypical clinical behavior following treatment, including poor OS, DSS and DFS ,26,27.It


Head and neck carcinomas have long been linked to alcohol and tobacco abuse; however, within the last two decades, the human papillomavirus (HPV) has emerged as a third etiology and is specifically associated with head and neck squamous cell carcinomas (HNSCC). In this anatomical region, the oncogenic HPV-16 mediates transformation and immortalization of epithelium, most commonly in the oropharynx. Nevertheless, the recent identification of novel HPV mechanisms thought to be specific to oropharyngeal carcinogenesis has coincided with observations that HPV-associated HNSCC has differing clinical behavior—in terms of natural history, therapeutic response, and prognosis—than HPV-negative head and neck tumors. Taken together with the growing incidence of HPV transmission in younger populations, these discoveries have sparked a rapid expansion in both laboratory and clinical studies on the infection and disease. Herein, we review the clinical characteristics of HPV-associated HNSCC, with particular emphasis on recent advancements in our understanding of the management of this infectious malignancy. With an annual incidence of nearly 600,000 cases, head and neck carcinomas constitute the sixth most common cancer group worldwide and account for more than 200,000 deaths each year . Over 90A 2013 global statistical analysis demonstrated a 36% overall prevalence of HPV in HNSCC, which is nearly double the figure reported one decade ago, while other sources have reported prevalence rates as high as 90% . It is eThe high-risk oncogenic HPV-16 is the most common type detected in oropharyngeal HNSCC; an analysis of a Canadian pathology database showed 57% of tonsillar cancer biopsies tested positive for HPV-16 ,9. From Patients with HPV-positive OPC tend to be younger at time of presentation than that of patients with HPV-negative oropharyngeal tumors . In one Furthermore, oropharyngeal HPV infection is transmitted through bodily fluids, such as during kissing and oral sex. Individuals with six or more lifetime sexual partners are at an increased risk of HPV-related OPC . Other cHPV-driven OPC predominantly arises from the reticulated epithelium of the oropharynx, which lines the crypts of the palatine tonsils and base of tongue . The reaOn initial presentation, oropharyngeal tumors may present with dysphagia, referred otalgia, tonsillar asymmetry, or impaired ability to protrude the tongue. However, patients often will have no pharyngeal complaints, since HPV-driven OPC is more likely to present with sudden identification of locally advanced disease in the neck in the presence of early stage occult disease in the oropharynx. It has been postulated that there may be a differential rate of neoplastic growth between the primary oropharyngeal mass and nodal disease, as the metastatic lesion may have more access to nutrients due to its proximity to vasculature .This presentation of abruptly appearing cervical nodal disease may also be explained by the association between HPV-driven OPC and the presence of large cystic nodal metastases . One stuAlthough HPV testing is performed for prognostic purposes, there is not currently sufficient data to support alteration of therapy based on HPV status. Strategies include definitive radiation therapy (RT) for early stage disease or, for more advanced disease, surgical resection and concurrent chemoradiation therapy with agents such as cisplatin. Given the younger associated age of diagnosis and favorable survival outcomes in HPV-positive patients relative to those who are HPV-negative, it is important to develop treatments to reduce late treatment morbidities while maintaining efficacy. The “de-escalation” strategies have been developed to decrease treatment intensity, with the goals of either replacing chemotherapy with more specifically targeted agents, reducing the chemoradiation dose, or replacing chemoradiation with radiation alone . MoreoveThe phase 3 EXTREME trial showed that the addition of cetuximab, a monoclonal antibody that inhibits ligand binding to the epidermal growth factor receptor (EGFR), to the first-line agents of platinum/5-fluorouracil significantly improved overall and progression-free survival (PFS) in patients with recurrent or metastatic HNSCC . A retrovs. Standard Chemotherapy early And Late Toxicity Events) trial involves 304 patients with Stage III or IV p16-positive oropharyngeal carcinoma who are randomized to conventionally fractionated IMRT to 70 Gy with either cisplatin or cetuximab [Radiation Therapy Oncology Group (RTOG) 1016 is a multi-center phase III trial involving 706 patients with Stage III or IV p16-positive OPC with the goal of evaluating the replacement of cisplatin with cetuximab . Patientetuximab . The stuetuximab .in situ hybridization) [Another set of trials examines the effect of induction chemotherapy followed by decreased chemoradiation dose in good responders. The Eastern Cooperative Oncology Group (ECOG) 1308 trial is a multi-center, phase II study in 90 patients with Stage III or IV HPV-positive OPC (with p16 immunostaining and HPV ization) . Patientization) . SimilarThe third major group of trials aims to investigate the role of deintensification of chemoradiation after surgical management in Stage III or IV p16-positive OPC patients. In the ADEPT  phase III study, 496 patients with prior trans-oral resection but current metastatic lymph nodes with extracapsular involvement will be randomized to receive either 60 Gy of radiation alone or radiation with concurrent cisplatin and evaluated for 2-year disease-free survival and locoregional control . In the In younger individuals, vaccination plays an increasing role in prevention strategies for overall HPV-mediated disease, including reduction in the oncogenic risk for OPC. It is believed that HPV-related HNSCC is preventable with vaccination, using either the Gardasil or Cervarix vaccines; however, further studies are needed ,35. CervListeria monocytogenes-based HPV vaccine ADX11-001 in patients with Stage II through IV OPC who have undergone transoral robotic surgery [Several vaccination strategies are currently under development in phase I trials, including one peptide and one vector vaccine. A pilot study with five patients diagnosed with advanced HPV-positive HNSCC examined a Trojan peptide vaccine consisting of HLA-I and HLA-II T cell epitopes of HPV16 and Melanoma Antigen E (MAGE-A3) tumor suppressor antigen found in epithelial cell malignancies ,37. Four surgery .vice versa, the development of a low-cost, non-invasive diagnostic approach for HPV-positive saliva would have a beneficial effect in reducing incidence of both HNSCC and cervical cancer. One issue that remains unaddressed, however, is whether the detection of HPV in an otherwise asymptomatic individual should be treated. Studies must be done to investigate how other risk factors, such as cigarette and alcohol abuse, along with the presence of HPV in the saliva correlate with development of OPC. Certainly, such a study stratifying various risk factors would be useful in developing an algorithm for clinicians to treat asymptomatic HPV-positive patients who meet criteria for intervention.In addition to the growth in our understanding of therapy for HPV-driven OPC, several ongoing studies are investigating novel methods of diagnosis and prognostic determination. One such study identified aberrant gene methylation of characteristic regions as being a potential non-invasive marker for detection of HPV infection . One of versus non-metastatic HNSCC, and thus, SLPI may be a potential biomarker for identifying individuals at risk of developing metastases [Additionally, several markers are under investigation as potential prognostic indicators. Preliminary results from a recent retrospective study suggest that cyclin D1 overexpression is a potential prognostic marker of primary OPC . Similartastases . Detectitastases ,44.PI3KCA, a downstream protein that is part of the EFGR signaling cascade [TRAF1 and amplification of the cell cycle regulator gene E2F1. These markers may serve as targets for future therapeutic agents against HPV-driven tumorigenesis.A recent study by the Cancer Genome Atlas profiled the genomes of 279 HNSCC samples, and found that HPV-associated tumors contain mutations in the oncogene  cascade . The autHPV is currently the most well-known infectious etiology of malignancy, garnering most of its attention from its role in cervical cancer. However, epidemiological trends appear to point towards a growth of incidence in HNSCC that will usurp cervical cancer by the year 2020 as the most common malignancy associated with HPV. Clinically, the characterization of HPV in individual tumors appears to be critical, since HPV-driven tumors are associated with a distinct presentation and better prognosis relative to HPV-negative OPC. While the identification of specific genes associated with both therapeutic sensitivity and prognosis are still in process, methods are already available to characterize the genetic expression of tumors to design a personalized plan of management. Additionally, it will be important to identify potential patient candidates using this personalized management approach who may benefit from the proposed de-escalation treatment regimens in the future. It should be emphasized that possible candidates are likely to include those with the lowest risk of likely recurrence or resistance to this de-intensified treatment. Lastly, the future likely also holds the development or identification of vaccinations that are capable of immunizing against HPV-mediated carcinogenesis of the oropharynx. Overall, our understanding has progressed tremendously over the last two decades and, in the next two decades, will hopefully continue to expand rapidly in a quest to find a consistently effective cure for this infectious malignancy.


In Italy, free-of-charge HPV vaccination is offered to 11-year-old girls since 2007. The National Immunization Plan established the target coverage at a minimum of 70%; it should increase to 95% within 3-year time frame. In 2012, four year after the introduction of HPV vaccination, coverage was stable at 69%. We conducted a national cross-sectional study to explore barriers to vaccination in Italy.Vaccination services selected, through the immunization registries, a sample of unvaccinated girls born in 1997 or 1998 and posted to their families a 23-items questionnaire inquiring barriers to vaccination, HPV knowledge, source of information on HPV, perception of risk of contracting HPV, advice from consulted health professionals on HPV vaccination.We analysed 1,738 questionnaires. Main barriers were fear of adverse events (reported by 80% of families), lack of trust in a new vaccine (76%), discordant information received by health professionals (65%) and scarce information on HPV vaccination (54%). Overall, 54% of families replied correctly to more than half of 10 questions exploring knowledge on HPV vaccination. Families with a high knowledge score were more likely to live in Northern and Central Italy, be Italian, have a high educational level, include a mother who attended cervical screening regularly and consult more information sources. Although paediatricians/general practitioners and gynaecologists were considered the most trusted source of information by 79% and 61% of respondents, they were consulted only by 49% and 31%. Among parents who discussed vaccination with a physician, 28% received discordant advices and 31% received the recommendation of accepting vaccination.Fear of adverse events, discordance of information and advices from physicians, and scarce information were the more commonly reported barriers to HPV vaccination. Health professionals played a key role as information providers, thus they must be better trained to provide clear notions. Training needs to include the development of communication skills; transparent discussion about the pros and cons of vaccination may reduce fear of adverse events and increase trust in vaccination. The creation of a public health network around vaccination would allow sharing information and attitudes on vaccinations, so that homogeneous messages could reach the target population.The online version of this article (doi:10.1186/s12879-014-0545-9) contains supplementary material, which is available to authorized users. Human Papillomavirus (HPV) vaccination represents an important opportunity for primary prevention of cervical cancer (CC). HPV vaccines have a high efficacy against cervical pre-cancer lesions if given to females before they are exposed to the virus , therefoAs of December 2012, 22 of 31 EU/EEA countries had implemented HPV vaccination. Target age, financing and vaccine delivery differ among countries  ind ind10] iIn Italy no research on barriers to HPV vaccination targeting parents of unvaccinated girls was available at national level. We conducted a survey among a sample of families of unvaccinated girls to explore reasons for non-vaccination. Understanding parental reasons for not vaccinating their daughters can help public health authorities to implement interventions increasing HPV vaccine acceptance.We conducted a cross-sectional study in the period November 2011-July 2012 as part of a national project (VALORE), coordinated by the National Institute of Public Health  and funded by the Ministry of Health.The survey addressed families of unvaccinated girls born in 1997 or 1998 who were offered vaccination in 2008-2010. We invited all 143 LHUs to participate (distributed in 21 regions/autonomous provinces). We excluded LHUs that were implementing an HPV vaccine catch-up programme targeting birth cohorts 1997 or 1998 at the time of the study.LHUs identified unvaccinated girls through the immunization registries. For calculating the sample size we assumed a precision of 10%, an alpha error of 5% and, given that current reference literature data was not available, a prevalence of 50%. According to these parameters, we needed a sample of at least 96 units for each LHU/region to provide sufficient power to generate estimates at local/regional level. Assuming a response of 30%, we randomly selected 320 girls from each list of unvaccinated girls. If unvaccinated girls were fewer than 320, we invited all of them.We developed a 23-item questionnaire  inquiring about: 1) demographic information, 2) vaccination status, 3) barriers/reasons for non-vaccination, 4) HPV knowledge, 5) source of information on HPV, 6) perception of risk that their daughter could contract HPV, 7) intention to have their daughter vaccinated in the future, 8) advice from consulted health care workers (HCWs) on HPV vaccination, 9) parents' socio-demographic and behavioural characteristics. Copies of the questionnaire  are available from the authors.To investigate barriers to vaccination, we proposed a list of 23 possible reasons for non-vaccination and asked participants to indicate how much each factor had influenced their decision of non-vaccination on a 4-point scale . To explore HPV knowledge, we proposed ten statements on HPV infection/vaccination, with three options available . Regarding sources of information, we proposed a list and asked participants who provided them information (they could indicate more than one source) and which sources they considered more trusted (they could indicate a maximum of three sources). For the other questions, answers with multiple options were proposed and parents could indicate one.We administered the questionnaire to a convenience sample of parents to ensure clarity and ease of administration, collected their comments and modified accordingly.In the period January-March 2012, vaccination services posted to the selected families a letter explaining the purpose of the study, the self-administered questionnaire, a stamp-addressed reply envelope to return the questionnaire to ISS and the invitation to the immunization service to get free-of-charge HPV vaccination. Since the survey was voluntary and anonymous, completing the questionnaire was considered as consent to participate. The national ethics committee of the ISS approved the study protocol.p-value of <0.05. For the purpose of the uni-variate and multivariable analysis, we dichotomized the following variables: educational level (at least one parent with high school or college degree/other), age (at least one parent <45 years/other), occupation (at least one employed parent/other), mother's attitude toward Pap test (regularly undergone/done once or never) and other accepted paediatric vaccinations . We computed a dichotomized knowledge score based on the number of correct answers on HPV infection and vaccination (≤5/>5) (outcome). We calculated odds ratios and their 95% confidence intervals (95% CI) to assess the association between selected variables and the outcome (knowledge score). We included into a logistic regression model the variables showing potentially interesting associations with the outcome and we used a forward stepwise procedure with the method of the likelihood ratio test for goodness-of-fit in order to control for possible confounding factors. At each step, a p-value of 0.10 was used as entry criterion and a p value of 0.15 as removal criterion. We used the statistical package STATA 11.2 to analyse data .We summarized categorical variables using frequencies and proportions and used Chi-square test, Fisher's exact test and Chi-square for trend to compare proportions. We defined statistical significance as a 2-tailed Fifty-six LHUs in ten regions participated in the survey: 32 in the North, 16 in the Centre and 8 in the South. Vaccination services posted 14,099 letters and 2,110 questionnaires returned to ISS, with a response of 15% . We excluded 372 questionnaires: 25 were filled incorrectly, 57 were sent to wrong cohorts and 290 to girls already vaccinated against HPV. Overall, we analysed 1,738 questionnaires.The number of letters returned to the sender because of unknown recipient was available for 39/56 LHUs; 7.3% of letters  returned to the vaccination centre, with a wide range among LHUs (0-22%).Ninety-nine percent of girls were Italian; 80% lived in the North of Italy; 99% had received other paediatric vaccinations. In most cases (74%) the mother filled the questionnaire; 81% of girls had at least one parent with secondary school or university degree; 88% of mothers underwent Pap-test regularly , lack of trust in a new vaccine , discordance of information received by HCWs on HPV vaccination  and scarce information on HPV vaccination   and 28% (95% CI 25.8-28.5) of parents; combining these two questions, 39% (95% CI 36.9-41.7) of families reported that a negative advice from a physician influenced the decision of non-vaccination. The belief that the age for vaccination was too young was reported by 45% (95% CI 43.1-48.1). Only 16% (95% CI 14.4-18.1) feared that vaccination would encourage more risky sexual activity. Issues linked to the access to vaccination services influenced marginally the acceptance  confirmed the intention to refuse HPV vaccination for their daughter in the future, 504 (29%) had not taken a decision yet and 377 (22%) intended to accept vaccination.Most respondents were aware of the causal relationship between HPV and CC 93%), the high frequency of HPV infections (70%), the sexual transmission of HPV (77%) and its asymptomatic nature 72%). Few respondents were aware of the transient nature of most HPV infections (19%), the possibility for men to be infected (37%) and the connection between HPV and genital warts (29%)  considered their daughter at risk of contracting HPV, 226 (14%) did not perceive any risk and 877 (53%) were not able to reply. The proportion of families perceiving the risk increased from 9.1% among those families that gave one correct answer to 59.2% among those who replied to all questions correctly .Ninety-three parents (5%) had never heard about HPV. Paediatrician/general practitioner represented the most commonly consulted source of information on HPV vaccination; the most trusted sources of information included paediatrician/general practitioner and gynaecologist. Although 79% and 61% of families trusted more their paediatrician/general practitioner and their gynaecologist, only 49% and 31% of the respondents had consulted these specialists  families consulted health professionals and other sources of information, while 358 (23%) had not received information by any physician; 906 (53%) indicated learning on HPV vaccination from 2-3 sources Table .Of 1,710 families, 600 (35%) felt sufficiently informed regarding HPV infection and vaccination; 787 (46%) did not feel enough informed and 323 (19%) were not able to give an opinion. Of 1,562 families, the information received by health professionals were considered appropriate to decide on HPV vaccination by 622 (40%); scarce and generic by 314 (20%); unclear regarding the vaccine mechanism by 278 (18%) and unclear on safety issues by 623 (40%).Out of 1,703, 345 20%) parents did not discuss about HPV vaccination with any health professional. Among the 1,358 parents who addressed the issue of HPV vaccination with a physician, 377 (28%) received discordant advices; only 421 (31%) parents received the recommendation of accepting HPV vaccination  feared the occurrence of adverse events of HPV vaccination, 2) were not well informed and aware of HPV infection and vaccination, 3) had received discordant advices and information on HPV vaccination from health professionals.Regarding the first point, concern about side effects was the main barrier to acceptance, in accordance with previous surveys -12]. Th. Th12]. Concerning the second point, the level of knowledge on HPV infection/vaccination was low. Since responders had received an invitation for their daughter's vaccination, they could have been more likely to seek HPV-related information. However, only 54% of families replied correctly to more than half questions. Our data suggest that many respondents do not understand the link between HPV and genital warts, the possibility for men to be infected and the transient nature of most HPV infections; other surveys conducted in Italy on the same topic -16] con con16] cOnly one third of families felt enough informed and the same proportion perceived the risk that their daughter could acquire the infection. Although knowledge does not predict behaviour's modification, it is a prerequisite for an aware decisional process on vaccination. A higher vaccine-related knowledge was positively associated with HPV vaccination uptake ,9] or i or i9] oRegarding the third point, discordant information and advice received by different health professionals hindered vaccination for 65% of families. Only 31% received positive advice from a physician. Studies reported that a proportion of clinicians had substantial concerns about promoting vaccination, particularly for younger age groups . NationaIn our study, family doctors and gynaecologists were considered the most trusted sources of information. However, there was a discrepancy between consulted and preferred sources of information on HPV vaccination: 30% of families that considered them as the most trusted source of information did not receive information on HPV from these professionals. A similar discrepancy emerged from a knowledge-attitude-practice survey among young adult Italian women . Since pOnly 25% of families got information on HPV vaccination from immunization services providers and 34% mentioned them among the most trusted sources of information. Another Italian survey reported that only 12% of young adult women would have liked to receive information on HPV vaccination by physicians working in vaccination service . Even thHCWs represented the most used source to get information on HPV, but 56% of parents actively searched other sources and 23% of families did not consult any health professional and obtained information from alternative sources only. Overall, 34% of families used the Internet to obtain information on HPV. The vaccine criticism movement took advantage of the Internet's ability to reach parents . A 2012 Censis[Caution should be exercised when comparing our results to other studies exploring knowledge on HPV vaccination because of different methodologies and target population. Contrarily to ours, a survey on this topic conducted by Censis, an ItalCensis. A possiCensis; the autFinally, we realized that invitation for HPV vaccination from LHUs did not reach some adolescents. In fact, 7% of families had not received the active call for HPV vaccination and 7% of our letters returned to the sender. This suggests that data from the immunization archives could be outdated. The improvement of computerized immunization registries linked to resident/health lists, as addressed in the National immunization Plan , represeThe limitations of our study include: scarce geographic representativeness of the sample , lack of a comparative group of parents of vaccinated girls and low response (15%). The low response might have introduced selection bias; respondents may not represent all unvaccinated girls' families (socio-demographic information of non-respondents are unknown). Because of the low response, the minimum sample size was achieved only in few LHUs; as a result, data could only be analysed at the national level. Previous experiences suggested that posting a self-administered questionnaire could lead to a lower response than face-to-face interviews. However, self-administration represented the easiest procedure to propose participation at the national level without overloading vaccination services.Fear of adverse events, discordant information received by HCWs, and scarce information were the more commonly reported barriers to HPV vaccination. It suggests that, in our study, the decision of non-vaccination might come from lack or discordant information on HPV vaccination rather than from a conscious intention to decline vaccination. As HCWs played such a key role as information providers, they must be better trained to provide clear and homogeneous information to adolescents and their parents. Such training should include the development of communication skills; openness and transparent discussion about the pros and cons of HPV vaccination as well the use of appropriate communication strategies may help reducing fear of adverse events and increasing trust in vaccination among parents. Efforts are also needed to stimulate collaboration among health professionals; the creation of a public health network around vaccination would allow sharing information and attitudes on vaccinations, so that homogeneous messages could reach the target population. Such network would enforce the role of public vaccination clinics as reference point for HCWs and general population and encourage an active role of general practitioners, paediatricians and gynaecologists for vaccination promotion. Finally, offering to adolescents a “package of vaccinations”  and using all access points in the health system to verify their coverage and offer lacking vaccines could represent an integrated method to increase acceptance among adolescents.http://www.epicentro.iss.it/problemi/hpv/pdf/Allegato3_Gruppo%20Lavoro%20VALORE.pdf.Multiple affiliations of local representatives for VALORE listed at: