As the natural boundary between the Yunnan-Guizhou Plateau and the Hengduan Mountains in China, the large-scale prominent terrain of Ailaoshan Mountain leads to obvious regional differences in climate and vegetation. In this article, a comparative analysis is made on both species composition and floristic components of spore pollen (SP) in two sample soil plots near the main peak of Ailaoshan Mountain. The results show that there is a remarkable difference between the western and eastern sample plots. The climate or meteorology characteristics are likely the main reasons accounting for the differences in habitat characteristics and plant sample investigation results. The tropical genera elements of sediment SPs are higher in the western sample plot (6.8%) than in the eastern sample plot (4.7%) because the western plot is controlled by the Indian summer monsoon at all times. The north temperate elements of sediment SP are lower in the western plot (13.4%) than in the eastern plot (26.4%) because the eastern plot is evidently sometimes influenced by the East Asian winter monsoon. In general, Ailaoshan Mountain may be one of important physical geographical boundaries between the interacting regions influenced by both the Indian summer monsoon and the East Asian winter monsoon. However, due mainly to the increasing disturbance by human activities, the habitat, composition, structure and function of primary vegetation have changed, which has resulted in the distortion of their corresponding relationships. Therefore, primary habitat and environmental information can no longer be correctly obtained according to modern vegetation conditions2. How to acquire the species compositions of the original vegetation is one of the important issues in modern vegetation restoration. SP is preserved in the stratum for a long period of time with features such as high yield, small individual size, resistance to acids or alkalis, and resistance to high temperature. Thus, SP has already become an important source of information for restoring species composition, structural composition, systematic function, and habitat status of primary vegetation5.Vegetation is the result of the comprehensive effects of environmental factors such as water, climate, soil, topography and other factors on different scales. Since the early 206. The air SPs have low research value due to the extreme instability of their spatial positions, while the surface SPs also have low research value due to their close relationships with modern vegetation, who can be seen or obtained for everyone. As an indicator of past habitat conditions, sediment SPs and their combinations can have important implications for the determination of sedimentary age, regional environmental change, inference of human ecological processes, and the correlation between plant communities and their habitat factors9.SPs include flowering plant pollen and cryptophyte spores. The former are mainly male germ cells of angiosperms and gymnosperms, while the latter are germ cells of cryptic plants that develop directly or indirectly into new individuals after detachment from their parents. While SPs are not exactly the same in function, they can effectively reflect the compositions of vegetation species and infer the primary habitat. According to the spatial location, SPs can be divided into three categories, i.e., air SPs, surface SPs and sediment SPs13. With the improvement of segregation, identification, statistical techniques, and improvement in the integration of numerical analysis methods, SP combinations have been used to study the correlations between vegetation components and ecological factors15. That is, research on SP-vegetation-environment correlations is already a hot topic. To date, a large number of scientific studies have been conducted on the correlations between SP and vegetation, while studies on soil sediment SPs in forests are rare16. This present study did not follow the routine application of SP analysis. The relationship between plant communities and habitats was also not explored, nor were the age of deposition or the history of environmental change determined. Human ecological processes were also not explored; however, the species composition and floristic components of SPs in soil sediments in an alpine forest are compared between two sample plots beside a high mountain known as Ailaoshan Mountain. The conclusions of this study could be used to restore or reflect the ecological effects of topography, climate and other environmental factors on primary vegetation.Recently, researchers have conducted extensive and in-depth scientific research on SP as a medium, involving the representativeness of SP, the correlations between SP and modern vegetation, and the restoration of primary habitat by using the correlations among SP, vegetation and environmentBased on the data in Table In the eastern plot, a total of 1155 woody pollens, 48 herb pollens and 107 fern spores were identified. Among the woody pollens, Castanopsis, Pinus, Alnus and Lithocarpus were ranked the top four, with percentages of 29.4%, 24.9%, 16.0% and 14.6%, respectively, which accounted for 84.9% of the total pollens, and their respective proportions are all higher than 10%, but the fifth proportion is simply 2.6%. Herbaceous pollens were dominated by Artemisia and Araliaceae, which accounted for 58.3% of the total. The fern spores were fewer, and three dominant genera included Drynaria, Polypodium and Selaginellacea, which accounted for 87.4% of the total.In terms of the quantity of SPs, the woody pollens and herbaceous pollens in these two plots were roughly the same, but the fern spores differed considerably, and the western plot was more than five times that of the eastern plot. There were 228 SPs from the western plot and 49 from the eastern plot that were not identified but were included, the total amount of SPs in the western plot was 37.6% higher than that in the eastern plot, which indicates that the original habitat of the western plot is more resistant to external disturbances and richer in biomass to a certain extent. In terms of the species classification of the identified SPs, woody pollens, herbaceous pollens, and fern spores in the western plot, there were 17 families, 8 families, and 5 families, respectively, and in the eastern plot, there were from 20 families, 7 families, and 6 families, respectively. This species composition of SPs indicates that the western and eastern plots are very similar in terms of species diversities of primary vegetation. In addition, for the woody pollens, the predominant families of the two sample plots are Fagaceae, including Castanopsis, Lithocarpus and Quercus, and both Pinaceae families, including the Pinus and Betulaceae families, which includes Alnus, accounted for a large proportion. The herbaceous pollens were dominated by Araliaceae and Artemisia families. However, there were some obvious differences. For instance, as is known that the Quercus woody can be classified as deciduous and evergreen, and the ratio of evergreen to deciduous pollens in the western plot was 25:27, while that in the eastern plot was 20:30. The ratio of evergreen components in the western plot was significantly higher than that in the eastern plot, which reflected the adequacy of precipitation and solar energy in the western plot and the superiority of the hydrothermal combination. The Ailaoshan Mountain interception of the warm and humid air currents from the Bay of Bengal may be the main reason, which has resulted in abundant water and heat and their good combination in the western plot, but the opposite case in the eastern plot.18. The research results of Zhu showed that with increasing latitude, the world’s widespread species and temperate flora components in the adjacent area of Ailaoshan Mountain are increasing, while the components of tropical flora show a decreasing trend21. However, the vegetation changes in floristic composition caused by nonlatitude factors and genetic analyses need to be strengthened22; this research addresses this issue.In general, flora is a general term for all types of vegetation in a certain region and is a natural part of plant species. Research on the plants of contemporary flora in China has mainly focused on the analysis of the geographical components of all plants in a natural geographical area and ultimately determined the sources of these species, but there have been few reports on the causes of vegetation pattern formation. Many studies have shown that the flora in the Yunnan Province belongs to the East Asia flora originating from tropical or subtropical regions, while the flora in this study area belongs to the old-world tropical flora in tropical Asia, which is divided into western and eastern subregions by Ailaoshan Mountain as the boundaryet al.23, SPs in the eastern and western plots of Ailaoshan Mountain belong to 9 types of flora  and accounted for 13.4% and 26.4% of the total SPs, respectively, which indicated that the eastern plot was significantly affected by the East Asia winter monsoon, while the western plot was less affected. Third, the tropical components of the SPs in the western plot, including the Discontinuous Zone of East Asia and tropical South America, the Pantropical Zone, and the Tropical Asia to Tropical Africa Zones, are larger than those in the eastern plot, which were 6.8% to 4.7%, respectively. Therefore, the western plot was more affected by the Indian summer monsoon than the eastern plot. Based on the above analysis, the tropical components in the western plot are relatively high, probably due to the control of the hot-humid airflow of the Indian summer monsoon from the southwest. However, the SP flora in the eastern plot may be not controlled by this hot and humid vapour system. The main reason for the differences in SP flora compositions of two sampling plots should be the barrier effect of the great Ailaoshan Mountain on atmospheric movement, i.e., Ailaoshan Mountain should be the boundary between the Indian summer monsoon and the East Asia winter monsoon in the study area.25, so there have been few related studies except for the following studies. Based on the SP data of different soil profile sediments at both Gaishan Mountain and Pingshan Mountain in Fuzhou of the Fujian Province, China, Hao et al. concluded that there were different characteristics of SP assemblages in different mountain locations and different soil depths26. That is, the location of the mountain and the soil depth can be indicated to a certain extent by the SP assemblage means of the soil sediments. Additionally, Chang et al. and Li et al. showed that the SP in the soil sediments in the subtropical evergreen broad-leaved forests was dominated by local SPs as a supplement to foreign SPs because the latter can only be transported by passing through fluid materials, such as the atmosphere and rivers28. The results of these studies are the theoretical basis of this study. The results of these studies indicate that the effect of some systemic habitat factors on the vegetation components can be re-inferred through the assemblage characteristics of mountainous soil SP. To compare the influence degree of the relevant research areas, the humid region exerted more influence than the arid region, while the high-cover area was stronger than the low-cover area30. In short, this comparative research on the species compositions and floral elements of the SPs in the subtropical evergreen broad-leaved forest soil can reflect the differences in the environmental characteristics of native vegetation to a certain extent.This comparative research on the species compositions and floral elements of the SPs in mountainous soil sediment are not conventional contents of modern SP analysis31. Second, in relation to modern vegetation and ecological factors, compared with those in the eastern region, the average annual precipitation and annual average solar radiation on the western side of Ailaoshan Mountain are relatively high, and both water and heat are sufficient, and the combination is good. Therefore, the coniferous forests are broad-leaved evergreen plants, which are high in composition32. Third, in the comparison of the beginning of the rainy season, the stations on the eastern side of Ailaoshan Mountain in 2001 entered into the rainy season mostly before the fifth five-day of April, while the stations on the west side were mostly in the first or second five-day of May. In other words, the trapping effect of the large mountain body of Ailaoshan Mountain on the strong, hot and humid marine monsoon airflow is one of the main reasons accounting for the disparity in the beginning of the rainy season between the western and eastern stations33, basically bounded by the main peak of Ailaoshan Mountain. Finally, based on basic data such as topography, climate, and vegetation, the mathematical model of self-organizing feature mapping, known as SOFM, was applied to study the ecogeographic demarcation of the area adjacent to Ailaoshan Mountain. The results showed that the great Ailaoshan Mountain not only blocked the southwest summer monsoon from the Indian Ocean, but the mountain is also a barrier for the southwest mountainous region in China from the northern Asia cold dry air34. These four conclusions agree with the differentiation in the species composition and floristic components of SP between the eastern and western sample plots in this study.The research team’s previous study on the effects of large-scale barriers at Ailaoshan Mountain has been fruitful, and some of the conclusions are similar to the results of this study, or which can partly explain the results of this study. First, in the comparison of regional climate complexity measures, we used a complex parameter, known as permutation entropy, for time series based on a comparison of neighbouring values and explained the dual effects of the great terrain and atmospheric circulation on the regional climate. Both the temperature permutation entropy and the precipitation permutation entropy displayed a decreasing trend from west to eastIn the present study, the SPs in soil sediments of the subtropical evergreen broad-leaved forests were used as media. The composition of plant species and floristic components between the western and eastern plots of the location near the main peak of Ailaoshan Mountain were compared and analysed. The following four main conclusions were obtained. First, on the west side of Ailaoshan Mountain, the main habitat features of the western plot were humid, fog, and massive bryophytes attached to the surface or trunks. The numbers or types of arbours, shrubs, grasses, and ferns were greater than those in the eastern plot. Differences in climate or meteorological characteristics may be one of the main reasons accounting for the differences in habitat features and plant sample investigation results. Second, the differences in the species composition between the eastern and western plots indicated hydrothermal heterogeneity. Compared with that in the eastern plot, the total amount of SPs in the western plot was 37.6% more than that of the ferns and mosses, which comprised the main differences. The evergreen and deciduous components of the Quercus plant in the SPs were approximately the same as those in the western plot, but the evergreen composition was lower in the eastern plot. Furthermore, there were significant differences in the floral component of the SPs in these two sampling plots. Relatively speaking, the higher component of the Tropical Zone in the western plot indicated that the southwest Indian summer monsoon had a stronger affect, while the SPs in the eastern pot had a higher composition of the North Temperate Zone, which indicated that the East Asian winter winds from the north had a significant impact. Finally, the barrier function of Ailaoshan Mountain was the main cause of the large differences in SP characteristics between the two plots. This barrier function mainly manifested itself in the interception of water vapor and air barrier. In this sense, Ailaoshan Mountain should be one of the regional boundaries under the joint action of both the Indian summer monsoon and the East Asian winter monsoon.The elevation of Ailaoshan Mountain is more than 2 000 metres above sea level. The mountain belongs to the north vein of the south branch of Yunling Mountain and is oriented in the northwest-southeast direction. The mountain comprises a combination of three natural geographical units of the Qinghai-Tibet Plateau, the Hengduanshan Mountains, and the Yunnan-Guizhou Plateau in China. The main peak is considered to be the boundary. The western and eastern sides are not only different in terms of climatic characteristics but also distinct in vegetation differentiation. In terms of climate, the study area has an obvious dry season with the replacement characteristics of the rainy season in one year. The November to April dry season is dominated by the plateau winter monsoon and the south branch westerly winds, and the weather is relatively dry. The May to October rainy season is significantly affected by two water vapour sources: one is from the Bengal Bay and belongs to the Indian Ocean while the other source is from the South China Sea and belongs to the Pacific Ocean, and the weather is relatively hot and humid. In terms of vegetation, the study area is the largest evergreen broad-leaved forest in the tropical-subtropical regions of China. The study area is not only located at the demarcation zone of the Pan-Arctic and paleoplants but is also at the intersection of various geographical flora components.2, where the dry season radiation is 195.8 MJ/m2 higher than that during the rainy season. However, in terms of the extreme temperatures, temperature extremes are greater in the east plot than in the west plot. If the altitude factor influence on temperature is considered, the extreme temperatures of the two sites are basically the same because the altitude of the western plot is 291 m higher than that of the east plot. To a great degree, this difference in climatic factors may be one of the main reasons accounting for the differences in habitat characteristics and vegetation sample survey results.In this study, we mainly compared the species compositions and faunal compositions of SP in soil sediments on the west and east sides of the main peak of Ailaoshan Mountain  at the junction of Xinping County and Zhenyuan County. For comparison, two 10 m × 10 m sampling plots are selected within the zonal vegetation type, i.e., subtropical evergreen broad-leaved forest. Ecogeographical features are similar: sunny slopes, large forest openings, and vegetation coverage of approximately 60%. The soil type is dark brown soil without exposed bedrock and with weak erosion. The sample survey results of the eastern and western plots of Ailaoshan Mountain are as follows. The western plot is located at 24°03′35.0″N, 101°23′57.3″E, and 2 525 m above sea level within Zhenyuan County. The local microclimate characteristics are cold, humid and foggy, and the prominent ecological feature is a large number of mosses. First, the arbour trees in this sample plot include 7 Anneslea fragrans from family Theaceae, 5 Osmanthus fragrans from family Oleaceae, 5 Machilus yunnanensis and 4 Lindera communis from family Lauraceae, and one Castanopsis orthacantha from family Fagaceae. In general, there are a total of 22 plants. Second, there are more shrub plants, mainly including Litsea pungens, Arrow bamboo, Camellia tsaii, Illicium majus, and so on. Third, the herbs are widely grown and mainly include Tupistra fimbriata from the family Liliaceae, Tetrastigma napaulense from the family Vitaceae, and so on. Lastly, the most prominent feature of this sample plot is the characteristics of numerous fern plants, including Asplenium, Sword Ferns, Sweet Fern and others. The eastern plot is located at 24°19′30.3″N, 101°19′01.1″E, and 2 234 m above sea level, within Xinping County. The local microclimate is characterized by low humidity without fog. The prominent ecological feature is the litter layer under the forest in this sample plot. First, there are 15 Schima superba in the family Theaceae, 4 Alnus nepalensis trees in the family Betulaceae, one Castanopsis fargesii in family Fagaceae, which is a total of 20 tree types. Second, the shrub layer is dominated by Radix Berberidis with an average plant height of 1.5 m. Third, the herb layer is dominated by Chromolaena Odorata in the family Asteraceae and a large number of Phyllagathis cavaleriei and Hispid Arthraxon. However, there are few fern plants. In contrast, relative to those of the eastern plot, the habitat of the western plot is mainly characterized by more moisture or fog, so a large number of mosses are attached to the surface or trunks. Although the number and types of trees in the west plot are 22 and 5, and those in the east plot are 20 and 3, the difference is rather small. However, there are many shrubs, herbs and ferns that flourish in the western plot. Table The field sampling timings in this study were as follows: field sampling on the eastern plot and western plot were conducted on November 22 and November 24, 2014, respectively. Only one soil profile was collected for each plot. Each soil cross-section profile was approximately 40–50 cm in depth, and the soil sample was collected every 10 cm, i.e., four soil samples were collected for each cross-section profile. Each soil sample was evenly mixed, and 10 g was taken for subsequent laboratory measurements.10–15% hydrochloric acid was added to disperse the soil cement to remove calcium carbonate, and the grass roots, fine leaves and other debris were removed by sieving through a 40-mesh screen. Fresh water was added, and samples were washed after 12 h, then the water was changed at least six times. Next, 40% hydrofluoric acid was added so as to remove minerals, and the samples were rinsed with water. Finally, the sample solution was sieved through a 7 μm diameter sieve cloth for approximately 3 minutes in an ultrasonic cleaning dish.All SPs were identified by Mr. Luo Yunli, a major professional operator in this study and a research associate from the State Key Laboratory of Systematic and Evolutionary Botany in Institute of Botany, Chinese Academy of Sciences.All SP assemblages identified from the 4 samples in each plot were used as data of each plot for laboratory analysis.


Data on human papillomavirus (HPV) prevalence around pregnancy were inconsistent. We assessed HPV prevalence before and after pregnancy, HPV incidence after pregnancy, and risk factors for HPV infection.Data from 15 754 women in control arms of 5 AS04-HPV-16/18 vaccine efficacy trials were analyzed, including 3001 women with at least 1 pregnancy. Results of HPV deoxyribonucleic acid testing on cervical samples were available. We analyzed risk factors, including age, region, pregnancy and its outcomes, duration from pregnancy resolution to collection of first postresolution cervical sample, previous HPV infection, cigarette smoking, and number of sexual partners with Cox regression.Prevalence of high-risk oncogenic (hr)-HPV types was similar before and after pregnancy (20.8% vs 19.8%). Incidence of hr-HPV was 40.1 per 1000 person-years  at 0–3 months, 266.7  at 3–6 months, and 95.7  at >6 months after pregnancy. Risk factors associated with HPV infection after pregnancy are previous HPV infection, elective abortion, and younger age at pregnancy resolution.Pregnancy could not be confirmed as a risk factor for HPV infection in this population despite an increased incidence detected 3–6 months after pregnancy resolution. Most women remained HPV negative after pregnancy.NCT001226810 , NCT00294047 , NCT00316693 and NCT00929526 , and NCT00779766 . Persistent human papillomavirus (HPV) infection is a prerequisite for cervical cancer , a diseaThe efficacy of vaccination against persistent HPV infection and the prevention of cervical intraepithelial neoplasia have been demonstrated widely , 16. In The current study, which is an analysis of pooled data from 5 vaccine efficacy trials, provided the opportunity to assess HPV prevalence and incidence before pregnancy and after pregnancy resolution. The primary objective was to estimate the HPV prevalence in the same group of women before and after pregnancy. The secondary objective was to evaluate the incidence of HPV infection after pregnancy in women who were HPV-negative at their last visit before pregnancy and who continued with the study postpartum. Lastly, we assessed the effect of potential risk factors such as pregnancy, history of HPV infection, cigarette smoking, number of sexual partners, age, region, and outcome of pregnancy on the incidence of HPV infection.https://clinicaltrials.gov under numbers NCT001226810 , NCT00294047 , NCT00316693 and NCT00929526 , and NCT00779766 . Results of the studies have been published previously [This study is a pooled analysis using data from women enrolled in the control arms of 5 AS04-HPV-16/18 vaccine efficacy trials . The effeviously . Women weviously , in Japaeviously , 20, andeviously .We defined the following cohorts for the analyses : Cohort Ethical approval from independent institutional review boards was obtained for all the clinical trials included in this work, and each woman provided written informed consent before the performance of any study-specific procedure. All studies followed the Declaration of Helsinki. Only deidentified data were used in the pooled analysis.3) placebo (HPV-015 and HPV-039).Included women were enrolled previously in 1 of the clinical trials , regardless of their cytological, serological, and HPV DNA status, where they were randomized to the control arm of the study and received a hepatitis A vaccine (HPV-008 and HPV-032/HPV-063) or an aluminum hydroxide  using broad-spectrum polymerase chain reaction (PCR), and type-specific PCR for detecting HPV-16 and HPV-18, were performed as described previously . No cervThe 5 trials were sponsored by GSK, allowing authors to extract data from the database owned by the company.We analyzed demographic and baseline characteristics with descriptive statistics. The primary objective of our analysis was to estimate HPV prevalence in cohorts 2 and 3, that is, before and after pregnancy. We determined HPV prevalence at the women’s last visit before pregnancy and at the first visit after pregnancy for 5 groups of HPV types—any HPV, any hr-HPV, HPV-16/18, HPV-16, and HPV-18. We calculated the incidence of HPV infection after pregnancy in cohort 4 (pregnant HPV-negative women) overall and stratified by the duration between pregnancy resolution and the first postresolution visit , age at pregnancy resolution, region, and outcome of pregnancy.We used the Cox proportional hazards model to analyze the potential risk factors for HPV infection in cohorts 4 and 5. Potential risk factors included the following: age at start of follow-up, history of previous HPV infection, pregnancy and its outcome, duration of follow-up, and region. Analysis was performed separately on the pooled HPV-008 and HPV-015 studies and the pooled HPV-032/063 and HPV-039 studies because these 2 groups of studies differed by the availability of the study entry questionnaire data, targeted age groups, and the assays used for HPV detection. A behavioral questionnaire was self-completed only for 2 studies (HPV-008 and HPV-015), providing information about smoking status and number of sexual partners. These variables were collected and assessed as potential risk factors in the pooled HPV-008 and HPV-015 studies. We used SAS 9.2  for all statistical analyses.A total of 15 754 women were enrolled in the control arm of the 5 efficacy studies . Of thosIn the HPV prevalence analyses, 2862 pregnant women were included in cohort 2; of them, 2448 (in cohort 3) also could be analyzed after pregnancy . The oveMost subjects , 357 cases of hr-HPV infection (incidence of 108.4 per 1000 person-years), including 99 cases of infection with HPV-16 or HPV-18 (27.3 per 1000 person-years), were detected . The incPotential risk factors for HPV infection were analyzed with the Cox proportional hazards model in the pregnant HPV-negative women (cohort 4) and in the nonpregnant HPV-negative women (cohort 5).In pregnant HPV-negative women from the HPV-008 and HPV-015 trials , incidenIn the HPV-032/063 and HPV-039 trials where information about sexual behavior was not captured, history of previous hr-HPV infection was associated with higher risk of acquiring infection with hr-HPV  in pregnant HPV-negative women. Risk of infection by HPV was lower in China than in Japan , history of previous HPV infection, elective abortion, and the number of sexual partners were risk factors for postresolution HPV infection. Smoking increased the risk of acquiring HPV infection in the nonpregnant women, though this was not observed in the pregnant cohort. The risk also varied among regions, being the highest in the Americas and lowest in Asia. Analysis using the Cox proportional hazards model did not confirm pregnancy as a risk factor in our study population.The most significant risk factor for any hr-HPV infection was a previous infection with HPV . This risk factor may be explained by the other mentioned lifestyle factors , genetic susceptibility to HPV infection, or dormant HPV infection that was not cleared and remained in basal layer of the cervix. Such dormant HPV infection could be reactivated by pregnancy or delivery and be detected as new infection.There are several limitations to the current study. First, there were no cervical samples collected during pregnancy. The value of taking such samples would be difficult to justify due to the potential risk of the procedure during pregnancy. Therefore, we were unable to assess the prevalence, incidence, and clearance of HPV infection during pregnancy. It would be valuable to determine these parameters during the pregnancy versus before and after pregnancy. For instance, a lower HPV prevalence during pregnancy could be due to a lower infection rate if fewer sexual partners are encountered. Second, initially HPV-negative women with their first postresolution HPV-positive cervical sample could have been infected any time between the last test before pregnancy and the first postpartum test. They were all considered as incident cases with the infection occurring after pregnancy, even though sexual activity usually continues, but at a lower frequency, during the first 2 trimesters of pregnancy , 33. In The results of this pooled analysis add to the current understanding about HPV prevalence and incidence around pregnancy. Prevalence of HPV was similar before and after pregnancy, suggesting that despite an altered immune system, the risk of infection by HPV may not change significantly during pregnancy . Another finding was that HPV incidence in previously HPV-negative women was the highest in the second 3-month period after pregnancy. This may reflect the timing of resumption and frequency of sexual activities.Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ofz486_suppl_Supplementary_MaterialClick here for additional data file.


VSCC) and precancerous vulvar intraepithelial neoplasia (VIN) can develop through human papillomavirus (HPV)‐dependent and ‐independent pathways, indicating a heterogeneous disease. Only a minority of VIN progress, but current clinicopathological classifications are insufficient to predict the cancer risk. Here we analyzed copy number alterations (CNA) to assess the molecular heterogeneity of vulvar lesions in relation to HPV and cancer risk. HPV‐status and CNA by means of whole‐genome next‐generation shallow‐sequencing were assessed in VSCC and VIN. The latter included VIN of women with associated VSCC (VSCCVIN) and women who did not develop VSCC during follow‐up (VINnoVSCC). HPV‐testing resulted in 41 HPV‐positive  and 24 HPV‐negative (11 VSCCVIN and 13 VSCC) lesions. HPV‐positive and ‐negative VSCC showed a partially overlapping pattern of recurrent CNA, including frequent gains of 3q and 8q. In contrast, amplification of 11q13/cyclinD1 was exclusively found in HPV‐negative lesions. HPV‐negative VSCCVIN had less CNA than HPV‐negative VSCC (P = .009), but shared chromosome 8 alterations. HPV‐positive VINnoVSCC had less CNA than VSCCVIN (P = .022). Interestingly, 1pq gain was detected in 81% of HPV‐positive VSCCVIN and only in 21% of VINnoVSCC (P = .001). In conclusion, HPV‐dependent and ‐independent vulvar carcinogenesis is characterized by distinct CNA patterns at the VIN stage, while more comparable patterns are present at the cancer stage. Cancer risk in VIN seems to be reflected by the extent of CNA, in particular chromosome 1 gain in HPV‐positive cases.Vulvar squamous cell carcinoma ( Given that vulvar (pre)cancerous lesions present as a heterogeneous disease with only a minority of vulvar intraepithelial neoplasia's (VIN) progressing to cancer, there is a clinical need to identify women with VIN at risk for cancer. Using whole‐genome shallow‐sequencing on HPV‐positive and HPV‐negative VIN and vulvar squamous cell carcinoma (VSCC), we were able to identify chromosome 1 gain as a strong indicator for cancer risk in HPV‐positive VIN.1The development of vulvar squamous cell carcinomas (VSCC) is recognized to be heterogeneous.TP53 mutations are frequently (~30%) detected in HPV‐negative VSCC as well as HPV‐negative VIN (~21%), suggesting that TP53 mutations might be involved at an early stage.Given that only a minority of VIN will progress to cancer and most affected women are treated similarly with often mutilating interventions,ve VIN ~2%, suggesive VIN ~%, suggesTo obtain better insight in the molecular events underlying vulvar carcinogenesis, this study was set out to comprehensively analyze HPV‐status and DNA copy number alterations (CNA) in a well‐characterized series of VIN and VSCC. To allow identification of specific CNA that may reflect the cancer risk for women with VIN, CNA in VIN of women with VSCC were compared with VIN of women without VSCC.22.1noVSCC) and 27 VIN were of women with associated VSCC (hereafter referred to as VINVSCC). In the VINnoVSCC group, the follow‐up period in which no VSCC developed was at least 8.3 years (range 8.3‐14.8 years). In the VINVSCC group, 13/16 cases presented with both VIN and VSCC and 3 cases developed VSCC during follow‐up (range 1.5‐4.2 years). Collection, storage and use of archival tissue were performed in compliance with the “Code for Proper Secondary Use of Human Tissue in the Netherlands” (https://www.federa.org).Cases of VIN and VSCC were retrieved from the pathology archives of the Departments of Pathology of VU University Medical Center (n = 26), the Academic Medical Center (n = 25) and the Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital n = 4), Amsterdam, The Netherlands. VIN adjacent to VSCC was considered to be a surrogate of the most advanced precancerous stage of VIN. Selected cases were enriched with p16 positive cases to achieve comparable group numbers as in regular care only a minority of VSCC cases is p16 (and HPV) positive. Selected cases were reviewed and tissue blocks were selected for sufficient VSCC and adjacent VIN tissue. For the selected VIN cases, long‐term pathology follow up data were available from PALGA, the nationwide pathology registry, to evaluate for progression to VSCC., Amsterd2.2DNA from VIN lesions was isolated following dissection of dysplastic regions (>70% dysplastic cells). For VSCC, whole tissue sections were used (>70% dysplastic cells). Laser capture micro‐dissection was performed using the Leica LMD6500  as described before,2.3INK4a followed by HPV DNA‐testing for p16INK4a‐positive samples, a test algorithm previously defined for the GP5+/6+ PCR test, and validated for HPV detection in FFPE tumor specimens.INK4a was performed on 3‐4 μm thick sections of FFPE specimens. Slides were considered positive when the lesion showed diffuse staining for p16INK4a.Determination of HPV‐status was performed using immunohistochemical staining of p16Human papillomavirus DNA‐testing including detection of all HR‐HPV types and typing for HPV 16 and HPV 18 was performed using HPV risk assay2.4Two hundred and fifty nanogram DNA was fragmented by sonication . Samples were prepared using the TruSeq nano‐kit  according to the manufacturer's instructions and sequenced on the Illumina HiSeq 2000 (Illumina San Diego) using the 50 bp single read (SR50) modus. Up to 24 samples were multiplexed on a single lane to yield ~8 million reads per sample.2.5Sequencing results were analyzed as described before.2.6Segmentation  and subsequent calling of gained, amplified and lost regions was done using CGHcall. Segments with a probability score of ≥0.5 were considered gained, amplified or lost.Unsupervised hierarchical clustering of different subsets of vulvar neoplasms was performed using a modified version of Weighted Clustering of Called Array CGH data (WECCA).2.7R (version 3.2.5) were used. The average total number of altered regions was compared between subgroups using the nonparametric Mann‐Whitney U test. To compare alterations between subgroups, a chi‐square‐test was performed using CGHtest,P‐value <.05 with an FDR <0.2 was considered statistically significant.P‐values below .05 were considered statistically significant.For statistical analysis IBM SPSS Statistics  and 33.1VSCC and VINnoVSCC, respectively) were analyzed for HPV‐status using p16INK4a‐immunohistochemistry followed by detection of HR‐HPV DNA by PCR. This resulted in a series of 13 HPV‐negative VSCC, 11 HPV‐positive VSCC, 11 HPV‐negative VIN and 30 HPV‐positive VIN. All VINnoVSCC (n = 14) were HPV‐positive. A total of 35 patients had HPV‐related lesions, 24 of these were HPV16‐positive, none were HPV18‐positive, and 11 were positive for other high‐risk HPV‐types.A series of 65 vulvar lesions, including 24 VSCC and 41 VIN with and without VSCC during follow‐up  than HPV‐negative VSCC . Interestingly, HPV‐positive VINnoVSCC displayed significantly less chromosomal alterations than VINVSCC  being detected in 100% of cases. In particular chromosome 3 and 8 isochromosome formation (3p/8p loss simultaneously with 3q/8q gain) was very common in HPV‐negative VSCC , but also detected in a subset of HPV‐positive VSCC . Comparison of HPV‐negative and ‐positive VSCC revealed 4 regions on chromosome 8p, of which 3 consecutive, that were significantly more frequent in HPV‐negative VSCC  resulted in 3 clusters  and VINnoVSCC  and is particularly characterized by gains at chromosomes 1, 3, and 20. Cluster II showing the least alterations contained more VINnoVSCC  than VINVSCC . Cluster III is characterized by gains at chromosome 1, 3 and 20, similar to cluster I, as well as losses at chromosome 4 and 11. Cluster III included mostly VINVSCC  and only one VINnoVSCC . The major differences in chromosomal alterations occurring in VIN lesions of the 3 clusters were determined by the maximum pairwise symmetrized Kullback‐Leibler divergence. Figure VSCC (81%), compared with VINnoVSCC . BCL2 (P = .007) and TP63 (P = .039) gains were more frequent in HPV‐positive lesions. Focal deletions were found at RBFOX1/3 and PTPRD, the latter mainly occurring in HPV‐negative lesions , located on 8q11.21, which was gained in 100% of HPV‐negative VSCC. Slug is an epithelial‐mesenchymal transition promoting factor reported to be expressed in ~50% of VSCC, and primarily associated with HPV‐negative tumors.Both HPV‐negative and HPV‐positive VSCC displayed frequent CNA at chromosome 3 and 8, with a highest frequency in HPV‐negative VSCC. These alterations are most likely of the isochromosome type since 3p/8p loss almost always occurred concurrently with 3q/8q gain in the same cases. Isochromosome formation of chromosome 8 has been reported previously for VSCC.VSCC showing similar CNA patterns as HPV‐negative VSCC , but HPV‐positive VINVSCC being very distinct. First, HPV‐positive VINVSCC displayed more alterations than HPV‐negative VINVSCC. Second, the affected regions differed between HPV‐positive and ‐negative VINVSCC. HPV‐positive VINVSCC frequently showed chromosome 1 gains (discussed below), chromosome 3q gain, chromosome 4 loss and chromosome 20 gain. In agreement with this, 2 previous studies on HPV‐positive VIN lesions demonstrated frequent gains at chromosome 1pq, 3q, 19, and/or 20q.In contrast to the relatively similar CNA patterns in HPV‐positive and ‐negative VSCC, there were striking differences between their precursor lesions, with HPV‐negative VINTP63, CD44, cyclin D1, and BCL2. Altered CD44 protein and TP63 mRNA expression in vulvar lesions has been reported by others.BCL2 and TP63 were more frequently affected in HPV‐positive cases. Gains of TP63 and cyclin D1 have also been described in HNSCC.INK4a‐negative VSCC being more frequently cyclin D1 positive than p16INK4a‐positive cases.Both VIN and VSCC displayed several focal alterations of well‐known cancer‐related genes, including VSCC and VINnoVSCC have been compared to identify potential copy number aberrations associated with cancer risk. Unsupervised hierarchical clustering analysis showed that these HPV‐positive VIN lesions displayed distinct copy number profiles. Particularly, gain of the complete chromosome 1 was significantly associated with the development of VSCC. Similar to our observation, Bryndorf et alVSCC, but infrequent , in VINnoVSCC. Interestingly, of the 3 VINnoVSCC cases with 1pq gain, 2 were radically resected indicating that the aggressive treatment might have prevented progression of these lesions into cancer. Of the 11 VINnoVSCC cases without 1pq gain, only 2 were radically resected indicating a low cancer risk in the remaining 9 lesions. Given the relatively low proportion of women with VIN that develop VSCC (ie 3%‐16%), many women with HPV‐positive VIN presumably have a low cancer‐risk profile and will therefore benefit from more conservative, for example, noninvasive management, whereas women with a high cancer‐risk profile should be approached more aggressively. Testing for chromosome 1 gain in HPV‐positive VIN, for example by fluorescent in situ hybridization, might help to identify VIN at risk for cancer progression and affected women may benefit from a more extensive treatment.Most importantly, this is the first study in which HPV‐positive VINVSCC showed frequent 1pq gain, none of the HPV‐negative VIN showed gain of 1p and only 2 cases had gains of 1q. This may implicate a causative role for HPV‐infection in the gain of this specific region. Indeed, gains of chromosome 1 are also frequent in HPV‐positive anal and cervical intraepithelial neoplasia, and cervical carcinomas.VSCC. Loss of 11p has been previously reported for both vulvar and cervical cancer,While HPV‐positive VINIn conclusion, we demonstrate a considerable heterogeneity of CNA in HPV‐positive and ‐negative VIN lesions, whereas HPV‐positive and ‐negative VSCC show more similar chromosomal profiles. Consistent with their more aggressive clinical course, HPV‐negative VIN show similar alterations as VSCC, affecting amongst others chromosome 3, 8, and 11q13.VSCC and VINnoVSCC, with both an increased number of CNA and, most importantly, gain of chromosome 1 as a possible indicator of progression risk towards VSCC. These molecular distinctions may provide biomarkers that predict VSCC risk and would fulfill an urgent clinical need to improve current treatment modalities of women affected by VIN. To explore the clinical significance of our findings, further prospective studies are needed.We found clear differences between HPV‐positive VINDAMH, PJFS, RDMS and CJLMM are minority shareholders of Self‐screen B.V., a spin‐off company of VUmc; (2) Self‐screen B.V. holds patents related to the work ; (3) DAMH has been on the speaker’s bureau of Qiagen and serves occasionally on the scientific advisory boards of Pfizer and Bristol‐Meyers Squibb; (4) PJFS has been on the speakers bureau of Roche diagnostics, Gen‐Probe, Abbott, Qiagen and Seegene and has been a consultant for Crucell B.V.; (5) CJLMM has received speakers fee from GSK, Qiagen, SPMSD/Merck, Roche diagnostics, Menarini and Seegene, served occasionally on the scientific advisory board (expert meeting) of GSK, Qiagen, SPMSD/Merck, Roche and Genticel and has been by occasion consultant for Qiagen and Genticel; (6) CJLMM has small number of shares of Qiagen, was minority shareholder of Diassay B.V. until April 2016, and until 2014 he held a small number of certificates of shares in Delphi Biosciences, which went into receivership in 2014; (7) CJLMM is part‐time director of Self‐screen B.V. since September 2017; (8) The other authors declare no conflicts of interest. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


We examined the association between anogenital human papillomavirus (HPV) infection and sexual networks in men who have sex with men (MSM).A total of 253 MSM, 20 years of age and older, were recruited from the community in Southern Taiwan in 2015–2016. At baseline and at each follow-up visit, MSM were screened for HPV to identify 37 HPV genotypes. At the six-month follow-up, MSM were asked to fill out an egocentric network assessment and to report the last five persons with whom they had sex regarding the characteristics of sexual behavior with each network member.A total of 182 participants (71.9%) returned for the follow-up and one third had at least one HPV type detected. A higher level of bridging network position calculated by the level of constraints in the network was significantly less likely to have HPV detection at the anal site. A high level of concurrency was associated with penile HPV detection .Identifying network-related characteristics can advance our understanding of high-risk populations and for prioritizing HPV vaccine recommendations. Human Papillomavirus (HPV) is significantly associated with cancers and diseases in both men and women. HPV is responsible not only for cervical cancer but also for 90% of anal cancers, 40% of penile cancers, and 12% of oral and pharyngeal cancers [The prevalence data for HPV infection in men varies by continent, country, ethnicity, recruitment method, age, HIV history, sexual orientation, and sample size . SeveralA high prevalence of HPV infection was also reported in European countries, but it was not as high in Australia. Two studies in European countries recruited MSM from STD clinics and found anal HPV infection prevalence around 69%–77% –7. In AuA cost-effectiveness study found that providing HPV vaccine to 12-year-old girls can be cost-effective in preventing many cases of cervical cancer and death ; howeverMSM is a highly neglected population in the current recommendation of girls-only vaccination programs in most countries, including Taiwan. Although the disease burden of HPV is higher in women than in men, by continuing to center HPV attention on women, a social environment was created that potentially reinforced the stigma of HPV infection, which may hinder HPV prevention efforts .Under the circumstances in several countries in which resources are limited and the cost for HPV vaccines are still high, prioritizing high-risk male populations for raising awareness and updating of vaccinations may help reduce the burden associated with HPV. Therefore, it is important to identify target populations that are at higher risk for HPV infection for interventions or policy enactment.In the current literature, risk factors of HPV infection often include individual-level factors, such as having a higher number of lifetime receptive anal sex occurrences between male partners , 8. UsinThe concept of social networks, which describes the existence of social ties, has been used to understand the spread of sexually transmitted infections (STIs). Social networks among MSM function as an immediate social environment that shapes network members’ shared behavioral norms and high-risk behavioral characteristics . Social HPV infection provides an interesting platform to study the complexity of social and sexual networks. Unlike HIV, people who are detected with HPV subtypes at one point does not mean that they will still be detected in six months because of effective immunity , 20. In In this study, we used HPV infection as a vehicle to study the association between sexual networks and STI transmission in a community cohort of MSM. The aims include: (a) to collect baseline sexual network characteristics via egocentric network assessment, and (b) to identify network characteristics associated with anal and penile HPV detection in a community cohort of adult MSM. Studies that examined prevalence and risk factors for HPV infection often report anal and penile sites separately because potential risk factors may predict one over the other , 23. An th and 12th month) and then annually in the 2nd and 3rd year. For each visit, MSM received HPV screening, including anal and penile swabs, and a self-administered paper-and-pencil survey assessing factors related to HPV infection. HPV DNA testing and genotyping was performed by polymerase chain reaction. In the follow-up surveys, we included a complete sexual network assessment to generate names and interpret the network relations. For this paper, we used data from the six-month follow-up collected during March to December 2016. A total of 182 participants (71.9%) returned for the six-month follow-up. The study was approved by the Ethics Committee of the National Cheng Kung University Hospital (reference number: A-BR-103-075). All participants received clear information about the study, fully understood the study purpose, and signed a written informed consent.A total of 253 MSM, 20 years of age and older, were recruited from the community. To be eligible, participants needed to have had sex with another male—mutual masturbation, oral or anal sex—and be willing to give consent to participate in the study. The major recruitment channel was through LGBTQ community health centers in Southern Taiwan. Study participants received three HPV screening tests in the 1-year frame  first via ROCHE cobas 4800 HPV Amplification Detection Kit, a qualitative multiplex assay detecting HPV in patient specimens. (b) If results showed positive, samples were further processed by ROCHE LINEAR ARRAY HPV genotyping test, a qualitative test that detects 21 high-risk  and 16 low-risk HPV genotypes .Name generator was used to elicit names of sexual network members. We asked participants to list the last five persons with whom they had had sex. The number of sexual partners was capped at five considering time constraints and respondent burden, which is also the number suggested for egocentric network data collection . Under eWe asked participants regarding time at first and last sex with each sexual network member , the frequency of sex and using a condom when they had sex, and substance use during sex. We also asked participants whether each sexual network member was HIV positive on a scale of definitely yes, very likely yes, not sure, very likely not, and definitely not. We dichotomized the answers into definitely yes/very likely yes vs. not sure/very likely not/definitely not.Concurrency was determined via the date of the first and last time they had sex with each alter. It was categorized as having no overlap partners, had two or more alters overlapped in any time period.We asked participants to report whether they thought any two of their sex partners may have had sex previously on a scale of definitely yes, very likely, not sure, very likely not, or definitely not. We created a tie between two alters indicating potential sex partners if they answered definitely yes or very likely. Structural constraint was calculated with igraph package in R software . The levWe described the characteristics and sociodemographics of egos and their alters. All relevant data are within the paper and its Supporting Information files  Tables. p < .05). For the anal site, 25.3% had at least one high-risk type detected and 22.0% had one low-risk type. For the penile site, 10.4% had at least one high-risk type and 7.7% had one low-risk type.At the six-month follow-up, among 182 MSM from the community (71.9%), 178 participants had valid anal HPV results and 171 participants had valid penile HPV results. Almost half of men (44.4%) had at least one HPV type detected . HPV detnd wave, 77.5% were within one year, 10.1% were between 1 to 2 years, and 9.8% were between 2 to 5 years.Out of 182 egos, 631 alters were reported. Almost half of the participants reported five alters (49%), 14% reported four or three alters, and 36% reported 1 or 2 alters. In molluscum cotagiosum, and 5% had been diagnosed with HIV. The majority of alters were also below age 35 (81.6%) and had a college degree (84.7%). When egos were asked to report their alter’s HIV status, 2% reported a confirmed positive status, about 60% were not sure about their status, and 38% thought that their partner was a confirmed negative.In For concurrency, 54% of the egos had no overlap partners, 15% had two alters overlapped, and 30% had more than two. On average, in 40% of the ego network, the ego was the receptive partner during anal sex. When the ego and alter first had sex, 42% had either never met before or had exchanged sex for money, drugs, housing, or other benefit. Twenty percent used alcohol when they had sex. In 3% of the ego network, alters were definitely or very likely to be HIV positive.In In To the best of our knowledge, this is the first study to examine the association between network-related factors in association with HPV infection. The study demonstrated sexual network-related risk factors in association with anogenital HPV detection in a community sample of MSM. Using egocentric network analysis to quantify sexual behaviors and characteristics between study participants and their most recent five sex partners, a few network-related factors were in line with previous understanding of STI transmission, such as whether high levels of concurrency were significantly associated with penile HPV detection. Based on whether two previous sex partners might potentially have a sexual relationship, a higher level of bridging was significantly less likely to have HPV detection at the anal site. Other commonly reported risk factors in HIV and STI research were found to be associated with either high- or low-risk types of anal HPV detection; for instance, a study participant being the receptive partner during anal sex, having HIV-positive sex partners, or alters with whom the first sex between them was more likely when they had just met or had exchanged sex for drugs or other benefits. Our study provides information to identify MSM populations at high risk for HPV infection.The often asymptomatic and undiagnosed nature of HPV infection offers an opportunity to examine the association of social links on sexual behaviors and sexual health. Instead of asking participants to report a proportion of risky sexual behaviors, the egocentric network approach requires participants to recall details for behaviors and characteristics for each partner. A large number of sexual network studies related to STI and MSM were conducted in the context of Black MSM in the U.S. . A revieBridging is a concept that has been addressed in several ways in the literature of STI research. Bridging populations contribute to the spread of STI to the general population; without the bridging population, the infection would have been contained in the high-risk core population . The posIn a network formed by communities as network nodes and a network connection formed by two individuals from different neighborhoods forming a sexual relationship, the community sexual bridging can be defined in various domains, such as walk-betweeness or flow-betweeness . CertainMoreover, given the high prevalence of online dating in MSM in Taiwan , it may Study findings should be interpreted in the context of the following limitations. First, the study utilized only cross-sectional data; hence, we can only identify HPV detection, and not account for chronical, recurrent, or definite infection. Temporal order cannot be fully established, especially regarding when participants were first exposed to HPV. Second, the majority of the study sample were recruited from a community-based NGO. Study findings may not be generalized to other populations, such as a sample with a higher number of sex partners or a higher prevalence of HIV and other STIs. Third, the egocentric network approach provides the benefit of having detailed information for each partner, yet, due to the burden in recalling too many partners, participants can only report sex partners up to five. It provides a snapshot of sex life, but may not be fully representative of a complete history. Fourth, because HPV screening is not part of a routine STI checkup, it is rare for MSM to know whether their male sex partners have HPV infection. Thus, we did not ask participants to self-report whether their partner had HPV infection. Our study also did not provide HPV screenings for sex partners. Considering that anogenital HPV infection is spread through sexual practices between partners, a lack of information on partners’ HPV status is a major overlook for network factors related to HPV detection.Our study demonstrated that an individual’s sexual network can be associated with his HPV status, although not all of the network factors were consistent with those associated with HIV or other STIs. An assessment of sexual network characteristics in association with HPV infection can advance our understanding of HPV transmission patterns. Our study results may help related authorities evaluate the current policies for HPV vaccination and reconsider the needs of this vaccination-targeted population. Our findings can help inform the design of targeted prevention and intervention programs for future studies.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


HPV test implementation as a primary screening tool has the potential to decrease cervical cancer incidence as shown by several studies around the world. However, in many low-resource settings, the HPV test introduction has been backed down mainly due to its price. In this study, we present a novel low-cost strategy involving simple devices and techniques for high-risk human papillomavirus (HR-HPV) detection. The analytical performance to detect HR-HPV infections of this novel strategy was assessed by comparing it with the Hybrid Capture 2 system (HC2), which is used as gold standard.Paired-cervical samples were collected from 541 women assisting to gynecological services in an outpatient clinic. One sample was transported in the Hybrid Capture Standard Transport Medium for HR-HPV detection by the HC2. The second sample was transported on glass slide for detection by PCR-based techniques .The level of agreement between the PCR-based techniques and HC2 system was determined with the Cohen’s kappa value. The kappa values between HC2 and GP-EIA, BSGP-EIA and pU 1 M-L/2R were 0.71 (CI 95% 0.63–0.78), 0.78 (CI 95% 0.71–0.84) and 0.63 (CI 95% 0.55–0.72), respectively. However, when the results from both BSGP-EIA and pU 1 M-L/2R were combined, the level of agreement with HC2 was increased to 0.82 (CI 95% 0.76–0.88), reflecting a very good agreement between the two HR-HPV detection strategies. Furthermore, the sensitivity of both techniques combined was also increased compared to the BSGP-EIA (88.7% vs 77.4%) and the pU (88.7 vs 60.9%) without penalizing the specificity obtained with the BSGP-EIA (95.1% vs 96.9%) and the pU (95.1% vs 96.5%).This novel strategy, combining two PCR-based techniques for HR-HPV detection, could be useful for cervical cancer screening in self-collected samples in low-income countries. Cervical cancer (CC) has the fourth highest rate for cancer incidence and mortality around the world. However, in many low-resource countries, CC becomes the first cause of female cancer and death . AlthougThe discovery that an infection by the human papillomavirus (HPV) is a necessary cause for CC development has represented a milestone in the prevention of this pathology . Twelve Although most of the commercially available HPV tests have excellent clinical sensitivity and specificity values , they arIn this study, we present a novel low-cost strategy that includes the use of a glass slide to transport cervical cells, and the use of two PCR to detect HR-HPV efficiently. The choice of these two PCR was based on the results obtained with PCR GP5+/6+ coupled with an EIA (GP-EIA), PCR BSGP5 + 6+ coupled with an EIA (BSGP-EIA) and PCR pU 1 M-L/2R (pU) compared to the results obtained by the HC2 system, used as gold standard for HR-HPV detection.The Bio-ethical Committee of the “Universidad Mayor de San Simón” approved the study protocol  and each participant signed an inform consent form before enrollment. A group of 541 women attending gynecological services were recruited in the outpatient clinic “CIES Salud Sexual y Reproductiva” in the city of Cochabamba, Bolivia. Paired-cervical samples were collected from each woman. One sample was taken for HR-HPV detection with the HC2 system  using the Digene® HC2 DNA Collection Device . A second sample was collected using a cyto-brush  and was smeared over a glass slide. Each slide was put inside a small cardboard box specifically designed for it. The box was then transported in a zippered storage bag to the laboratory. The patients received only the results obtained with the HC2 system.The HC2 system was used as gold standard in this study. This technique does not require DNA extraction and the HR-HPV DNA detection was performed exactly as indicated in the manufacturer’s protocol (Qiagen). The HC2 system allows the identification of 13 HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.Cells were detached from the glass slide using 400 μl of a solution containing 2,5% Chelex100  and 50 μg/ml of proteinase K , placed in an Eppendorf tube and then vortexed for 15 s. Cell lysis was performed at 56 °C for 1 h and then the proteinase K was inactivated by heating at 95 °C for 10 min. Tubes were centrifuged at maximal speed and supernatants containing crude DNA were recovered. A quality control for DNA extraction was performed by a PCR using the primers PC04 (5′-CAACTTCATCCACGTTCACC-3′) and GH20 (5′-GAAGAGCCAAGGACAGGTAC-3′), amplifying a 260 bp fragment from the human β-globin gene . Amplifi2, 0.2 mM dNTP mix, 1 μM of each primer, 0.625 U of GoTaq® G2 Hot Start Polymerase . The total reaction volume was 25 μl with 5 μl of crude DNA extract. PCR was performed in a T100™ Thermal Cycler . The amplification protocol used for this PCR was described by Fontaine et al. [General HPV DNA was detected using the GP couple of primers: GP5+ (5′-TTTGTTACTGTGGTAGATACTAC-3′) and GP6+ (5′-GAAAAATAAACTGTAAATCATATTC-3′). The GP6+ primer was biotynilated in order to perform an enzyme-linked assay afterwards. These primers amplify a 150 bp fragment from the L1 region of the HPV genome . The PCRe et al. . Amplifi2, 0.2 mM dNTP mix, 0.2 μM of each forward primer, 0.4 μM of each reverse primer, 0.625 U of GoTaq® G2 Hot Start Polymerase  and 5 μl of crude DNA extract. The PCR conditions were the same described by Schmitt et al. [HPV DNA was amplified using the modified set of primers BSGP5+/GP6+ (BS primers) which include 9 forward and 3 reverse primers . The 3 rDetection of HR-HPV DNA was performed using an EIA as described previously . This as2, 0.2 mM dNTP mix, 0.5 μM of each primer, 0.625 U of GoTaq® G2 Hot Start Polymerase  and 5 μl of crude DNA extract. The PCR conditions were slightly modified from the original protocol. Briefly, 40 cycles of amplification were used including a denaturation step at 94 °C for 1 min, an annealing step at 55 °C for 45 s and an elongation step at 72 °C for 1 min.HPV DNA was also amplified using the pU-1 M-L (5′-TGTCAAAAACCGTTGTGTCCAGAA GAAAA-3′) and pU-2R (5′-GAGCTGTCGCTTAATTGCTC-3′) pair of primers . The PCRTo determine the HPV genotypes in samples with discordant results for HR-HPV detection, two techniques were used: INNO-LiPA HPV Genotyping Extra  PCR products sequencing. The INNO-LiPA test allows the detection of 28 HPV genotypes  by reverse hybridization of biotinylated amplicons with specific oligonucleotides probes which are immobilized on membrane strips. The test was performed exactly as indicated in the manufacturer’s manual. The HPV DNA amplification was done using the INNO-LiPA HPV Genotyping Extra Amp kit  according to the manufacturer’s instructions, using the T100™ Thermal Cycler  and thermocycler program as previously described [Statistical analyses were performed using SPSS software. Cohen’s kappa value was calculated to determine the level of agreement for HR-HPV detection results between the different techniques used in this study. Kappa values of < 0.20, 0.21 to 0.40, 0.41 to 0.60, 0.61 to 0.80 and 0.81 to 1.00 were considered poor, fair, moderate, good and very good agreement, respectively .The sensitivity and specificity of the evaluated PCR tests were calculated considering the results obtained with the HC2 system as gold standard. A true positive (TP) correspond to a positive result yielded by the PCR test and the HC2 system. A true negative (TN) correspond to a negative result yielded by the PCR test and the HC2 system. A false positive (FP) correspond to a positive result yielded by the PCR test but negative for the HC2 system; and a false negative (FN) correspond to a negative result yielded by the PCR test but positive for the HC2 system. The sensitivity is calculated as follows: TP/(TP + FN). The specificity is calculated as follow: TN/(TN + FP) .The presence of HR-HPV DNA was evaluated on 541 samples with four techniques: HC2 (gold standard), GP-EIA, BSGP-EIA and PCR pU 1 M-L/2R. The results from the PCR-based techniques were compared with the gold standard to determine the kappa value (degree of agreement) between these techniques. The HR-HPV detection results from all comparisons are summarized in Table We first performed HC2 and GP-EIA and compared the result agreement by calculating the kappa value between them. The kappa value obtained was 0.71 (CI 95% 0.63–0.78) which can be interpreted as a good level of agreement. The sensitivity and specificity obtained were 70.4% (CI95% 62.1–78.7) and 96.2% (CI95% 94.3–98.1) respectively , representing a good level of concordance between the two methods even though it was clearly lower than the kappa value observed between HC2 and the PCR-based-EIA. However, when we merged the results for HR-HPV detection obtained by the BSGP-EIA and the PCR pU (i.e. a sample is considered positive if BSGP-EIA or PCR pU test is positive and a sample is considered negative if both BSGP-EIA and PCR pU tests are negative) we observed an increased kappa value of 0.82 (CI 95% 0.76–0.88), representing a very good level of agreement between this new HR-HPV detection strategy and the commercial gold standard HC2 system. The sensitivity of both techniques combined (BSGP-EIA + pU) was also increased compared to the BSGP-EIA (88.7% vs 77.4%) and the pU (88.7 vs 60.9%) independently  than the HC2 system.In this study, we present a low-cost strategy to detect HR-HPV for cervical cancer screening in low-resource settings. We searched for simple robust and cheap method to extract cell DNA and detect specifically HR-HPV DNA. The efficiency of this strategy to detect infections by HR-HPV types was evaluated by comparing it with the HC2 system.Our laboratory has recently presented evidence supporting the use of a simple cervical brush and a glass slide to efficiently collect and transport cervical cells sample for HPV DNA detection by PCR . FurtherConcerning the HR-HPV detection, we chose to perform PCR, as it is a simple, sensitive, effective and low-cost technique. The GP-EIA method has been one of the most used PCR-based technique to detect HR-HPV DNA and compared with the HC2 system it gave good results to detect CIN2+ lesions . HoweverAnother concern in the GP system is that it amplifies the L1 region of the viral genome. This region is thought to be disrupted when the viral DNA integrates into the host genome. The presence of only a viral integrated DNA form in cervical cells, in the absence of episomal forms, could occur in cervical cells, especially in high degree lesions. However, it is believed to be a rare event , 41. As Several studies have performed a comparison between the GP-EIA technique and the HC2 system for HR-HPV detection. Kappa values in these studies range from 0.59 to 0.74 , 42–44, Furthermore, we genotyped and/or sequenced the samples with discordant results between the HC2 system and our PCR-based techniques in order to analyze which kind of HPV infections were missed or over-detected by our strategy compared to the reference technique. It is noteworthy that among the 13 HPV-infected samples that were missed by our PCR strategy, there were only 2 infections by HPV16 or 18 (15%) which are the most oncogenic genotypes. On the other hand, 43% (9/21) of the samples that tested positive by our PCR strategy but negative for HC2 system contained infections by either HPV16 or 18. These results altogether show that our PCR-based strategy is missing fewer infections by HPV16 and 18, which is an important key feature for a HPV test in a primary screening program.We present analytical evidence supporting the use of a low-cost strategy involving simple devices and techniques for cervical sample collection and transport, and for HR-HPV DNA detection with two PCR-based techniques used together (BSGP-EIA and pU).This novel strategy could represent an interesting option to screen rural population for cervical cancer specially by self-collection of samples. Indeed, self-sampling with our simple devices (cotton swab and glass slide) seems well accepted among the Bolivian population, especially in rural areas, and could therefore increase the screening coverage rate of this population, as suggested by our preliminary study . Further


Therefore, TKTL1/CIP-2a/B-MYB protein levels could objectively predict oncoprogression risk in twice (HR-HPV- and Pap smear)-positive women. Further studies will assess the translatability of these findings into clinical settings.High oncogenic risk human papillomaviruses (HR-HPVs) promote cervical carcinoma development, the fourth most common feminine cancer. A slow oncodevelopmental phase—defined histopathologically as Cervical Intraepithelial Neoplasia (CIN) grades 1–3, or cytologically as Low- or High-grade Squamous Intraepithelial Lesions (LSIL or HSIL)—precedes the malignancy. Cervical carcinoma screenings through HR-HPV genotyping and Pap smears are regularly performed in Western countries. Faulty cytology screening or genotyping or patients' non-compliance with follow-ups can let slip an oncoprogression diagnosis. Novel biomarker tests flanking HR-HPV genotyping and cytology could objectively predict the risk of disease progression thus helping triage LSIL/ASCUS patients. Here, anonymized leftovers of fresh cervical epithelium scrapings from twice (LSIL/ASCUS and HR-HPV DNA)-positive and twice (Pap smear- and HR-HPV DNA)-negative (control) patients in a proteome-preserving solution served to assess the biomarker worth of three cervical carcinoma-related proteins, i.e., B-MYB (or MYBL2), Cancerous Inhibitor of PP2A (CIP-2a), and transketolase-like1 (TKTL1). Leftovers anonymity was strictly kept and storage at −80°C, protein extraction, immunoblotting, and band densitometry were blindly performed. Only after tests completion, the anonymous yet code-corresponding HR-HPV-genotyping and cytology data allowed to assign each sample to the twice-positive or twice-negative group. Descriptive statistics showed that the three proteins levels significantly increased in the twice-positive vs. twice-negative scrapings. Diagnostic ROC curve analysis identified each protein's Optimal Decision Threshold (OTD) showing that TKTL1 and CIP-2a are stronger risk predictive biomarkers  than B-MYB. Logistic Regression coupled with Likelihood-Ratio Tests confirmed that a highly significant relation links increasing TKTL1/CIP-2a/B-MYB protein levels in twice-positive cervical scrapings to the risk of HR-HPV-driven oncoprogression. Finally, a 3 year clinical follow-up showed that 13 patients  of the twice-positive group with biomarker values over OTDs compliantly underwent scheduled colposcopy and biopsy. Of these, 11  received a positive histological diagnosis, i.e., CIN1 ( So far,LR-HPVs) . Additio cancers –10. An aof cases –14. Notaof cases , 15. The (ASCUS) . Typical (ASCUS) , 18. The (ASCUS) , 20. Mor (ASCUS) , 22. Hen (ASCUS) . Convers (ASCUS) . Then ag (ASCUS) . Finallyobjectively predicting the actual risk of progression for any cytological precancerous lesion in HR-HPV DNA-positive and Pap smear-positive patients  binding guidelines emphasize the need for using first the HR-HPV genotyping followed, if positive, by the cytology test and again, if positive, by colposcopy; negative women should be retested after 1–3 years , 27. Howpatients , 28.twice-positive) patients and, as controls (Ctr), scraped leftovers from HPV DNA-negative and Pap smear-negative  patients. Our herein reported biochemical results integrated by the results of a subsequent 3-year follow-up of the compliant fraction (50%) of the same twice-positive patients indicate that increases in TKTL1, CIP-2a, and B-MYB proteins above their respective Optimal Decision Threshold (ODT) values may robustly predict the risk of an active HR-HPV-driven oncogenesis.Therefore, the present work aimed at assessing the potential worth of three proteins—i.e., B-MYB (or MYBL2), Cancerous Inhibitor of PP2A (CIP-2a), and transketolase-like1 (TKTL1)—as risk predictive biomarkers of disease progression. We chose these proteins because they are over expressed in advanced dysplastic lesions and cervical carcinomas  samples, and 26 LSIL/ASCUS and HR-HPV-DNA positive (“twice-positive”) samples constituted the main populations studied. Their mean age was for both groups was 34.5 years with a range from 16 to 56 years. Cytological diagnoses proportions were: LSIL, 2/3 and ASCUS, 1/3 of the total cases. LSIL and ASCUS data were pooled together because of the limited numbers of the latter. Moreover, ASCUS HPV-DNA-positive patients were categorized together with LSIL ones because GISCI's guideline indicates that the ASCUS diagnostic category, being borderline, must be avoided or limited to a minimum, and the relative samples classified as clearly as possible as LSIL or negative  –38. Evernegative . No sampg for 10 min at 4°C. The resulting pellets were resuspended in 1.0 ml of phosphate buffer saline (PBS) added with complete EDTA–free protease inhibitor cocktail . The cells' morphological integrity was checked under an inverted phase-contrast light microscope . Next, after three washings with PBS + anti-proteolytic cocktail, cell pellets were homogenized in T–PER™ tissue protein extraction reagent  also added with the complete EDTA–free protease inhibitor cocktail. Finally, to achieve total cell lysis samples were subjected to one freeze/thaw cycle at −20°C and ice-homogenized with an UltraTurrax™. The protein contents of the extracts were assayed according to Chiarini et al. . The membranes were probed with the following rabbit polyclonal primary antibodies: (a) anti-TKTL1 , diluted 1:1000 in TBST/1% BSA; (b) anti-B-MYB , at the concentration of 0.5 μg ml−1 in TBST/1% BSA; (c) anti-CIP-2a  diluted 1:1000 in TBST/1% BSA; (d) anti-β actin mouse monoclonal antibody (Santa Cruz Biotechnology) which served as loading control (LC) and was used at 1.0 μg ml−1 in TBST/1% BSA. After overnight incubation at 4°C, the membranes were probed with the secondary, alkaline phosphatase conjugated, donkey anti-rabbit IgG (Abcam), diluted 1:3000 in TBST or goat anti-mouse IgG (Thermo) diluted 1:1000 in TBST. BCIP/NBT  was used as chromogenic blue substrate to detect alkaline phosphatase activity. The membranes were incubated in the substrate solution for 10-min until color development; thereafter, the reaction was stopped by washing the membranes in several changes of bidistilled water. The membranes were scanned in an Odissey™  using the 700 channel. A digital image of each western blot was saved in TIFF format. The densitometric integrated intensity values of the specific bands for the B-MYB, CIP-2a, and TKTL1 antigens and of the loading controls (LC) were evaluated by means of the Image Studio™ Software, version 5.2, developed by LI-COR Inc. for WB analysis. The densitometric analysis of the specific protein bands pertaining to each sample was performed on three independent immunoblots and the results for each tested protein were presented as the mean of signal intensities ± SEM. To ease calculations all densitometric values were multiplied by E4. In detail, the “Add Rectangle” tool in Image Studio Software served to draw a rectangular-shaped box around the bands of interest to quantify the target proteins. The “Median background method” allowed to compute the median pixels intensity at the border contouring the band's shape and to subtract the background from the shape intensity. Finally, the “Signal value” for each shape was calculated by determining its “Total signal” and by subtracting from the latter the product of the “Background” multiplied for the “Area.”WB was performed as detailed in Chiarini et al. , 40. Equ2 plastic flasks , and incubated at 37°C in 95%v/v air/5%v/v CO2 in a complete medium consisting of 95%v/v Dulbecco's modified Eagle's Minimum Essential Medium , 5%v/v heat-inactivated (56°C for 30 min) FBS , and gentamycin . Before reaching confluence, cultures were split at a ratio of 1:6 after briefly incubating them at 18 ± 2°C with 0.025% (w/v) trypsin (Sigma-Aldrich). Protein extracts from the cells of six culture flasks were processed as previously detailed  to analyze the genotypes of the infecting HPVs using the INFINITY HPV QUADTM assay developed by AutoGenomics Inc, , which detects HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 . Shapiro-Wilk's test revealed that a normal distribution could not be assumed for most of the data groups. Welch's t-test or Wilcoxon rank Z test  specimens served as controls. To fully preserve the protein complement of the leftovers an ad hoc freezing medium (90%v/v FBS + 10%v/v DMSO) was devised and thoroughly tested prior to starting the study. The results proved its clear superiority over any other standard commercial solutions containing organic agents or fixatives, e.g., Thin PrepTM , CytofastTM , etc., which were also tested in parallel (not shown). All the leftovers were processed blindly, and their belonging to the twice-positive or twice-negative group became known only after immunoblotting procedures and specific band protein immunoblot densitometry evaluations had been carried out for all the specimens involved.The present work aimed at assessing the worth of three proteins, i.e., B-MYB, CIP-2a, and TKTL1, as risk predictive biomarkers of HR-HPV-driven precancerous disease progression. Anonymized fresh cervical epithelium scrapings leftovers which were “ypes see , multiplypes see . “Twice-P = 0.0023); CIP-2a, 7.7-fold (P < 0.0001); and TKTL1's 14.6-fold (P < 0.0001)—in the twice (LSIL/ASCUS and HR-HPV)-positive samples than in their twice-negative (control) counterparts  differed from the value (= 0.5) of the no discrimination or chance area (P < 0.001) differed from each other, and both TKTL1's and CIP-2a's AUC values were significantly (P < 0.001) higher than B-MYB's. This was the first indication that TKTL1 and CIP-2a likely are more robust biomarkers of HR-HPV-driven risk of disease progression than B-MYB in the twice-positive cervical epithelium scrapings.Receiver operating characteristics (ROC) curves plot the diagnostic accuracy of a putative biomarker across the whole range of potential decision thresholds , 46. Fignce area ; Table 3Precision statistical analysis showed how the values of ROC curve Sensitivity and Specificity and Youden's J index (or Youden's statistic) concomitantly changed for each of the proteins tested along with increasing densitometric values of specific protein bands . Notablyi.e., 0.472  of 2.02, a Predictive Value (+) of 0.48, and a Cost of 0.189) moved to the third rank B-MYB's candidacy as a standalone clinically useful biomarker of an active HR-HPV-driven oncogenesis to be looked for in cervical epithelium scrapings of twice (LSIL/ASCUS and HR-HPV)-positive patients.Remarkably, B-MYB's Youden's J index peak value, Conversely, CIP-2a's peak value of Youden's J index was 0.678  of 3.94, Predictive Value (+) of 0.64, and Cost of 1.40), which put CIP-2a's biomarker candidacy in the second rank.CIP-2a, 2380 with a false positive (FP) proportion of 0.231 and a false negative (FN) proportion of 0.091; and TKTL1, 1720 with a FP proportion of 0.167 and a FN proportion of 0.067  of 5.60, Predictive Value (+) of 0.97, and Cost of 0.103) . Moreoveof 0.067 . Thus, t0 and β1x Logistic Regression parameters estimates, with their corresponding 95% CIs, and SEs.An assessment of how the probability of HR-HPV-driven active oncogenesis changed with increasing densitometric values of B-MYB, CIP-2a, and TKTL1 in the cervical epithelium scrapings and its statistical level of significance was carried out via Logistic Regression  combined0+β1x predictor data was significantly smaller than the Log Likelihood of the Null Model with only the β0 intercept and no β0+β1x predictor data  of the Full Model with all the βtor data . TherefoFinally, a pilot study involving six HR-HPV DNA-negative cervical epithelial scrapings with a generic cytological diagnosis of inflammation but with a negative result of the complete TVAG  test showed that no significant difference could be detected in the expression of TKTL1, CIP-2a, and B-MYB proteins and in ROC curve and Logistic Regression/LRT diagnostic parameters vs. 16 double-negative control samples (data not shown). Though preliminary, these findings further strengthen the specificity of TKTL1 and CIP-2a as robust biomarkers of HR-HPV-driven active oncogenesis detectable in cervical LSIL/ASCUS scrapings.whose TKTL, CIP-2A, and B-MYB markers values were above corresponding OTDs , followed by CIN2+  and by CIN2 . These follow-up histology results strengthen the view that the biomarkers we analyzed in the cervical scrapings are endowed with a risk predictive value of disease progression.A total of 13 patients (50%) of twice-positive patients, ing OTDs , compliaThe sexually transmitted oncogenic HPVs are a real bane of human life. It is estimated that in the USA from 28 to 46% of the women under 25 years have been or are infected with HPVs; novel infections are ~6.2 million per year; chronic HPV carriers are ~20 million; histopathology reveals that ~330.000 women present a high-grade (CIN 2/3) cervical dysplasia, and ~1.4 million a low-grade (CIN 1) one; and ~3,700 women die of cervical carcinoma each year; of course, worldwide data have a much greater impact . PresentMost important, HR-HPVs can drive the development of cancers in both sexes at the level of vulva, anus, penis, prostate, oropharynx, skin, and neck, particularly in HIV-infected patients , 60–62. IGF2AS and PEG10 gene regions associates with a chance of CIN 1 → CIN 2 progression. Also, Bhatia et al.  of the mitotic cycle. Immunohistochemically, being weakly expressed B-MYB is hard to detect in most normal cervical epithelial cells. Conversely, B-MYB is over expressed and localizes in the cell nuclei of CIN 2+, cervical glandular intraepithelial neoplasia, and invasive cervical carcinoma specimens , 29, 33.In turn, CIP-2a promotes tumorigenesis and, in general, tumor progression by inhibiting the activity of protein phosphatase 2A (PP2A)—a prerequisite for the oncogenic transformation of human cells—and by stabilizing c-MYC in different tumors. CIP-2A is overexpressed in cervical carcinomas as it positively correlates with HPV-16 E6 and E7 proteins expression in CIN3 and cervical cancer tissues and cells . CIP-2a Finally, TKTL1 is a key enzyme in the pentose phosphate pathway, which plays as a crucial role in the progression of cervical neoplasia . TKTL1 ead hoc equipment and software (this is a current undertaking of our lab). Arguably, such automated procedures could be used in mobile labs crisscrossing underdeveloped countries to screen for HR-HPV-infected women at impending risk of cervical carcinoma.The present findings prove that our method of preserving cervical epithelium scraping leftovers to obtain from them integral protein lysates brings about results agreeing with histopathological data reported in the Literature –35. The Previously, the majority cervical carcinoma-related biomarkers were discovered in histological sections of cervical tissue cone biopsies taken at colposcopy. Although such biomarkers may increase the accuracy of the histological diagnosis of cancer, histopathology cannot be the primary methodological approach in cervical carcinoma screenings. This would be impractical as it could hardly deal with large numbers of LSIL/ASCUS bioptic samples particularly in underdeveloped countries. Conversely, using the very same cervical epithelium scrapings to perform standard cytology, HR-HPV DNA genotyping, and biomarker(s), such as TKTL1 and/or CIP-2a expression levels analysis via standard protein lysates Western immunoblotting and specific band densitometry is a feasible undertaking.In keeping with the just mentioned Literature, our present findings reveal for the first time that it is feasible to detect an increased expression of TKTL1, CIP-2A, and B-MYB in the cervical epithelium scrapings of HR-HPV DNA-positive women having a LSIL/ASCUS cytological diagnosis. Notwithstanding B-MYB's weaker expression, its statistically significant diagnostic indexes make it a still potentially translatable risk predictive biomarker of HR-HPV-driven disease progression. B-MYB's “weakness” may be due to its role as a transcription factor , which dStrengths of this works are: the novel diagnostic approach using cervical epithelium scraping leftovers; feasible and practical methodology used for protein expression analysis; use of biomarkers of the cervical epithelium as objective indicators of HR-HPV-driven disease; the precise assessment of the ODT values for each biomarker which reduces to a minimum the size of false-positive and false-negative findings; and the matching of the biomarker data with follow up data in 85% of the compliant patients.Limitations of this work are: grouping of ASCUS and LSIL patients in one category because, according to Italian GISCI directive, LSIL or negative diagnoses have mostly supplanted the ASCUS one; the relatively small numbers of patients examined which warrants further studies and follow-ups with larger cohorts.In conclusion, our biochemical, statistical and three-year follow up findings indicate TKTL1, CIP-2A, and B-MYB as potential robust risk predictive biomarkers of an active onco-developmental process driven by HR-HPV subtypes.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.We considered our study exempt from the above ethics requirements because (1) the anonymized cervical epithelium scrapings leftovers we used for our study would have been otherwise totally destroyed and scientifically wasted; (2) we could strictly maintain the complete anonymity of the same scrapings leftovers along all the procedural steps of our research: no subject can be identified from our data; (3) upon our preliminary verbal query Vicenza's Hospital Ethics Committee members answered that there was no need to submit a research protocol for approval since we would have been using anonymized leftovers and keeping them anonymous; and (4) FDA's regulations which exempt the use of leftovers from ethics requirements are taken as reference indications by Hospital Ethics Committees of Veneto and Trentino .AC, DL, and ID equally contributed to design the study and its methodology, to carry out the laboratory work, to collect and interpret the data, and to draft the manuscript. UA and MR supervised the technological aspects of the project and contributed to the interpretation of the data. MR and CE assisted in the clinical supervision and sample collection. UA and CE performed the statistical descriptive and diagnostic analyses of the data and wrote substantial parts of the manuscript. UA revised the manuscript draft. All authors read and approved the manuscript in its present final form.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salivary gland malignancies are a very heterogeneous group of cancers, with histologically > 20 different subtypes, and prognosis varies greatly. Their etiology is unknown, however, a few small studies show presence of human papillomavirus (HPV) in some subtypes, although the evidence for HPV having a causal role is weak. The aim of this study was to investigate if HPV plays a causal role in the development of different parotid salivary gland tumor subtypes.INK4a overexpression by immunohistochemistry.DNA was extracted from 107 parotid salivary gland formalin fixed paraffin embedded tumors and 10 corresponding metastases, and tested for 27 different HPV types using a multiplex bead based assay. HPV DNA positive tumors were stained for p16INK4a. The HPV positive primary tumor was a squamous cell carcinoma; neither mucoepidermoid nor adenoid cystic tumors were found HPV positive.One of the 107 malignant parotid salivary gland tumors (0.93%) and its corresponding metastasis on the neck were positive for HPV16 DNA, and both also overexpressed p16In conclusion, HPV DNA analysis in a large number of malignant parotid salivary gland tumors, including 12 different subtypes, did not show any strong indications that tested HPV types have a causal role in the studied salivary gland tumor types.The online version of this article (10.1186/s13000-018-0721-0) contains supplementary material, which is available to authorized users. Salivary gland malignancies are a very heterogeneous group of cancers, where histologically more than 20 different subtypes have been identified and prognosis varies greatly . These tINK4a) in, to our knowledge, the largest cohort of parotid salivary gland tumors, including 12 different subtypes, in patients diagnosed between 2000-2009 in the counties of Stockholm and Gotland.It has been well established by us and others that human papillomavirus (HPV) plays a causative role in a large proportion of oropharyngeal squamous cell carcinomas (OPSCC) –6 and thBetween 2000-2009, 145 patients in the counties of Stockholm and Gotland were identified and diagnosed with the following types of malignant parotid salivary gland tumors: acinic cell carcinoma, adenocarcinoma not otherwise specified (NOS), adenoid cystic carcinoma, basal cell adenocarcinoma, epithelial- myoepithelial carcinoma, mucoepidermoid carcinoma, myoepithelial carcinoma, oncocytic carcinoma, poorly differentiated carcinoma, salivary duct carcinoma, secretory carcinoma and squamous cell carcinoma (ICD-10: C07.9). From these, haematoxylin and eosin stained tumor sections were histologically evaluated to confirm presence of malignant cells and diagnosis. In total 117 biopsies (including 10 corresponding metastases) from 107 unique patients had sufficient tumor material for inclusion in this study and were collected as formalin fixed paraffin embedded (FFPE) tumors from the Clinical Pathology Department at Karolinska University Hospital. Sections from the FFPE tumors with, when possible, > 70% tumor cell area were taken for DNA extraction and immunohistochemistry (IHC) staining.DNA was extracted from 10 μm FFPE tumor tissue sections using the QIAamp DNA FFPE Tissue Kit  following the manufacturer’s suggested protocol. In short, the FFPE sections were dissolved in Xylene followed by rehydration in absolute ethanol. After protein degradation the samples were purified in several steps using spin columns, thereafter the purified DNA was stored at − 20 °C. For each tumor sample a blank paraffin section was included as a negative control in order to detect possible cross-contamination between samples. Presence of HPV DNA was detected, following a 40 cycle PCR amplification using the QIAGEN multiplex PCR kit  together with broad-spectrum GP5+/6+ primers  . PCR proINK4a tumor suppressor protein (p16) overexpression. p16 overexpression was determined by IHC using the mouse monoclonal antibody CINtec® p16 Histology , as previously described [The tumor that tested positive for HPV was also evaluated for p16escribed . The secFisher’s exact test was used to compare HPV numbers per sub-group of salivary gland tumors.p = 0.06, Table In total 117 tumor samples, including 10 corresponding metastases, from 12 different salivary gland tumor types, were analyzed for presence of HPV DNA  using a multiplex bead based assay. One of the 107 malignant salivary gland tumors and its corresponding metastasis on the neck were positive for HPV16 DNA, and both also overexpressed p16 and therefore could be defined as being HPV positive. All other subtypes tested were HPV DNA negative. In total, only one of seven squamous cell carcinoma salivary gland tumors was HPV positive and due to the small sample size of squamous cell salivary gland tumors it was not possible to draw any reliable conclusion to whether HPV was involved in the development of this tumor type. Furthermore, all other subgroups lacked high risk HPV DNA and did not express presence of any of the other tested HPV types, suggesting that HPV does not play a major role as a causative agent in these subgroups. The latter was shown in a more limited study by Skalova et al. .Nonetheless, HPV positive head and neck cancers often present with an HPV positive neck metastasis, and it has been shown that HPV positive cancer of unknown primary of the head and neck region often originate from an HPV positive OPSCC , 23. HenThere are some limitations to this study. Firstly, salivary gland tumors are a rare and heterogeneous group of tumors including more than 20 different subtypes, making it difficult to gather representable amounts of material for this type of analysis. Furthermore, although, to our knowledge, the presence of HPV was analyzed in the largest cohort published so far, the number of samples per subgroup was still limited, which makes it difficult to draw any clear conclusions. Secondly, the presence of p16 overexpression in samples was only tested on the one sample, and its corresponding metastasis, that both had tested positive for HPV DNA. Nevertheless, previous studies have shown p16 overexpression in malignant salivary gland tumor cohorts, still lacking or having a low correlation to presence of HPV , 13. TheIn conclusion, HPV DNA analysis in a large number of malignant salivary gland tumors, including 12 different subtypes, did not show any strong indications that tested HPV types have a causal role in the studied salivary gland tumor types.Additional file 1:Table S1. Summary of studies on salivary gland tumors in relation to HPV infection as referred to in the background section. (DOCX 33 kb)