HPV was identified in 42% (n=22) and high-risk (HR) HPV in 17% (n=9), with 18 different HPV types identified. The most common types were the HR HPV 33 (14%) and low-risk HPV 67 (14%), followed by the HR HPV 58. More than one HPV type was identified in 27.3% of cases. HPV 33 was frequently associated with other HPV types. No statistically significant differences in gender (P=0.58) and age (P=0.12) were observed between HPV-positive and HPV-negative cases. HPV was identified in almost half of the tongue cancer samples, with subtypes 33 and 67 being the most common. This suggested that HPV played an important role in this disease in the population studied. Given these results, current HPV vaccines may not be as effective in reducing tongue cancer rates in this population.The relationship between human papillomavirus (HPV) and oropharyngeal squamous cell carcinoma has been established. However, data from Ecuador is limited. The objective of this study was to characterize HPV infection in Ecuadorian patients with tongue cancer. Fifty-three patients with tongue cancer treated at the tertiary referral center Sociedad de Lucha Contra el Cancer (SOLCA), Guayaquil, between 2006 and 2011 were identified. Linear Array More than 680,000 cases of head and neck cancer (HNC) are diagnosed worldwide each year, and approximately 375,000 patients die annually from this disease . An estiTobacco and alcohol use are risk factors strongly associated with the development of OPC. In combination, these risk factors are thought to have a multiplicative effect, rather than just an additive effect . RecentlHuman papillomavirus (HPV) infection has been proposed as a cause for certain HNC, particularly OPC ,7. A conThere is no data showing the impact of HPV-related OPC and the specific HPV types associated with this pathology in Ecuador. The objective of this study was to identify the presence and prevalence of HPV types in tongue cancer cells from a cohort of Ecuadorian patients, and to compare these results with HPV type prevalence in other countries.All patients with a diagnosis of primary tongue cancer and corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples, treated at the Instituto Oncológico Nacional de la Sociedad de Lucha Contra el Cancer (ION-SOLCA), Guayaquil, Ecuador, between January 1, 2006 and December 31, 2011, were identified. Clinical characteristics of the patients , and alcohol use (yes or no)) were recorded after review of the patient medical records. Tumor size was classified following the American Joint Committee on Cancer (AJCC) staging manual, eighth edition (2017). Institutional Review Board approval (including the ethics committee) was obtained prior to study commencement. All data obtained was treated in accordance with the institution's patient confidentiality rules and regulations, including protection of patient names by coding of the samples.A microtome was used to obtain 5–10-μm thick tissue slices of the FFPE tissue blocks, which were then placed in separate encoded tubes and stored. Contamination was prevented by discarding the first 5–8 slices of each FFPE tissue block, using different blades, and discarding gloves and cleaning the material with 70% ethanol after each case had been sliced. Genetic material was obtained through the QIAmp isolation method, using the protocol described in previous published studies ,16.® HPV genotyping test  according to the recommendations of the manufacturer. This test combines polymerase chain reaction (PCR) amplification of target DNA and reverse line-blot hybridization for the identification of thirty-seven anogenital HPV DNA genotypes, including 14 high-risk (HR) HPV types  and 23 low-risk (LR) HPV types . It contains an internal control (a gene probe corresponding to the human β-globin gene), that determines the presence of sufficient cellular material in the biological samples. A result is considered valid when the bands of the internal β-globin control are positive. All of the cases in this study were valid. Hybridization and HPV genotyping were performed as described by the manufacturer , HPV types were identified by comparing the hybridization signals with the Linear Array HPV reference guide. Both positive and negative controls  were used and adequately typed in order to validate the genotyping test.HPV genetic material and genotypes were identified using the Linear ArrayPrograms Numbers and Excel were used for data entry and management. Comparison between groups was performed using Fisher’s exact test. The level of significance was P<0.05. Continuous data are reported as means±SD. Categorical data are reported as proportions and percentages. IBM SPSS Statistics version 24  was used for statistical analyses.The cohort consisted of 53 patients with tongue cancer. Mean age was 61.8±17.3, 29 patients (55%) were male, 49 patients (92%) had SCC and 22 patients (42%) were positive for HPV. Demographic and clinical characteristics of the cohort are shown in 51= –1.581, P=0.12, 95% CI ). There were no smokers in the HPV-positive patients, compared with 8 smokers in the HPV-negative patients (P=0.029). Four patients reported alcohol intake in the HPV-positive cohort compared with 5 patients in the HPV-negative cohort (P=0.92). Among the HPV-positive cohort, the anterior two-thirds of the tongue was the most common site of cancer , followed by the base of the tongue , with 95% having a histopathological diagnosis of SCC. The most common tumor stages were T2  and T4  (Supplementary Table S1).Of the 22 cases in which HPV genome was detected, 11 were female  while 11 were male ; no statistically significant difference was found between genders (P=0.58). The mean age for the patients that had HPV genome was 57 years with a standard deviation of 16.7; no statistically significant difference was found between the ages of HPV positive and negative cases  and 67 (LR) were the most common, being found in 14% (n=4) each . More thIn a cohort of Ecuadorian patients with tongue cancer, HPV (both HR and LR) was found in almost half of the cancers. There was a higher prevalence of HPV in younger patients compared with older patients. Although this difference was not statistically significant, it may have approached significance with a larger sample size. Tobacco consumption was significantly higher in HPV-negative patients, consistent with prior studies where an inverse correlation between smoking and HPV has been established . There wA number of Ecuadorian studies identifying HPV types have been performed in anogenital cancers, but not in HNC. The majority of these studies show HPV 16 as the most prevalent type –21. I® (HPV 16 and 18), which is recommended at age 9 years and has been available since 2014 in the national immunization program . These vaccines prevent HPV infection and associated anogenital cancer (both) and anogenital warts (Gardasil®)  in theory may be more effective in reducing tongue cancer (and other HPV-associated HNC) rates given the prevalence of HPV 33, 45, 52, and 58 in this cohort, however, it is cost-prohibitive for the vast majority of the Ecuadorian population.Two HPV vaccines are currently commercially available in Ecuador - bivalent Cervarix program ,25, and rdasil®) . It has rdasil®) ,28, whicrdasil®) ,30. The This study had limitations, including its retrospective nature, which resulted in some missing data regarding alcohol and tobacco use, and precise cancer location. This may have introduced bias to the results; however, the number of missing data was low and thus may not have affected the overall results. The sample size was low despite involving all patients with a diagnosis of tongue cancer with available cancer tissue for 5 consecutive years. Given the time elapsed between initial specimen acquisition and the HPV analyses in FFPE material, there may have been partially degraded DNA at the time of testing. Moreover, while HPV presence was determined in the samples, their biological activity  was not assessed in this sample.In conclusion, this pilot study identified HPV genome in a high percentage of tongue cancer tumors, further supporting the hypothesis that HPV has an important role in this disease. In this Ecuadorian tongue cancer cohort, HPV 16 and 18 were not present, which may have implications for the current HPV vaccination program should HNC be considered in the future. Larger studies are required to confirm if this HPV type pattern is representative of the Ecuadorian population. Furthermore, identification of the presence of p16 would be of particular interest when identifying the prevalence of HPV infection.pdf].Click here to view [


EPB41L3 and the viral regions of HPV16‐L1/L2, HPV18‐L2, HPV31‐L1, and HPV33‐L2 were determined by a highly accurate quantitative pyrosequencing of bisulfite converted DNA. There was a significant trend of increased methylation with disease grade comparing normal to CIN1 and CIN3 (p < 0.0001). Adjacent‐CIN1 predominantly shared the same HPV types as the CIN3, however, methylation differed substantially between adjacent‐CIN1 and CIN3 (p = 0.008). In contrast diagnostically principal‐CIN1 had an indistinguishable methylation distribution compared to adjacent‐CIN1 . Our results suggest that progression from normal epithelium to CIN1 or CIN3 is usually promoted by the same HPV type but occurs via distinct DNA epigenotypes, thus favoring the “molecular switch” model.The evolution of precancerous cervical lesions is poorly understood. A widely held model of cervical intraepithelial neoplasia grade 3 (CIN3) development is sequential progression from normal through CIN1 and CIN2 to CIN3. Another hypothesis, the “molecular switch” model, postulates that CIN3 can evolve directly from human papillomavirus (HPV)‐infected normal epithelium without progressing through CIN1 and CIN2. To shed light on this process, we compared DNA methylation of selected human biomarkers and HPV types in two groups of CIN1: CIN1 that were near or adjacent to CIN3 (adjacent‐CIN1) and CIN1 that were the principal lesions with no CIN3 detected . 354 CIN (CIN1 and CIN3) and normal tissue areas were dissected and typed for HPV from 127 women who underwent loop electrosurgical excision procedures (LEEP). Methylation of genes  What's new?Do cervical intraepithelial neoplasias always start at CIN1 and progress to CIN3? Or can CIN3 arise directly from normal epithelium, after HPV infection? The authors investigated by testing DNA methylation in different CINs. They compared CIN1 with CIN3 within the same cervix, and also compared CIN1 adjacent to CIN3 with CIN1 from patients without detectable CIN3. CIN1 and their neighboring CIN3, they found, each had distinct patterns of methylation, despite carrying the same HPV infection. All the CIN1 samples, however, exhibited similar methylation, regardless of the presence of CIN3. This suggests methylation can switch cells directly into a CIN3 state. AdjadjacentAVERaverageCINcervical intraepithelial neoplasiaCpG5'‐cytosine‐phosphate‐guanine‐3'2CTT χCuzick test for trend statisticDNAdeoxyribonucleic acidhrhigh‐riskE2BSE2 binding siteEDTAethylenediaminetetraacetic acidFFPEformalin‐fixed paraffin‐embeddedHClhydrochloric acidHPVhuman papillomavirusHPV16human papillomavirus type 16HPV18human papillomavirus type 18HPV31human papillomavirus type 31HPV33human papillomavirus type 33HPVmeHPV methylationIMPdata imputation2KWT χKruskal‐Wallis test statisticLEEPloop electrosurgical excision procedurennumber of samplesNormnormalqPCRquantitative polymerase chain reactionPrinprincipalSDSsodium dodecyl sulphateTSStranscription start siteURRupstream regulatory region.Human papillomavirus (HPV) infections account for an estimated 530,000 new cervical cancers and 270,000 deaths annually, most of which occur in developing countries.Looking at the methylation level of certain genes is a way to shed light on the molecular progression of precancerous cervical lesions. DNA methylation is a reproducible physical epigenetic change involved in a variety of cellular processes that plays an important role in cancer progression.i) adjacent‐CIN1 versus CIN3 from the same women and (ii) adjacent‐CIN1 versus principal‐CIN1 lesion from different women. We hypothesised that if methylation levels were different between adjacent‐CIN1 and CIN3 on the same cervix but similar between adjeacent‐CIN1 and principal‐CIN1, this would add support to the “molecular switch” model. On the other hand, if the methylation levels overlapped substantially between grades and adjacent‐CIN1 exhibited higher methylation levels than principal‐CIN1 then; “the sequential progression” model would be favored. A secondary aim was to determine whether adjacent lesions were usually infected by the same or different HPV types. The study was made possible by our collection of carefully annotated Loop Electrosurgical Excision Procedure (LEEP) cervical tissues  collected from 2005 to 2009 was used.For the biopsies, twelve 5 μm FFPE sections of normal, CIN1 and CIN3 were cut on a microtome using a new blade for each block. Sections were stored at −70°C until DNA extraction. Entire sections were scraped from the slides using a scalpel and deparaffinised using three washes in xylene and one wash with 100% ethanol. DNA was extracted using QIAamp DNA FFPE Tissue Kit  according to manufacturer's instructions with an initial incubation step at 56°C for 16–18 hr with proteinase K. The DNA concentration was measured using a NanoDrop 1000 Spectrophotometer .For the LEEP blocks, sections were cut using a sandwich cutting procedureThe 354 dissected areas from LEEP samples were tested using the PapType High Risk HPV Detection and Genotyping kit  according to the manufacturer's instructions Fig. . The kitDNA methylation was measured by a highly accurate pyrosequencing assay as previously described.EPB41L3  and MAL .The punch biopsies were used to determine the most informative HPV regions for the main study using the LEEP sections Fig. . AnalyseEPB41L3, the L1 and L2 regions of HPV16 and the L2 region of HPV18 , and HPV18‐L2 is presented in Supporting Information Figure S2 and Table S1. MAL and the HPV16‐E2 binding sites were dropped from further study due to inadequate effect size. Although the L2 region of HPV18 was not significant in the pilot study due to small sample size , methylation of this region was assayed in the main study because of the importance of HPV18 as a carcinogenic HPV. To these assays we added biomarkers in the L1 region of HPV31 and the L2 region of HPV33, which were validated as informative in different studies.A pilot study was performed on 104 punch biopsies to select a set of informative biomarkers for the main study. The methylation of candidate biomarkers n = 53) of the CIN3 cases were classed as multifocal because they presented adjacent lesions of different grades (adjacent‐CIN1 or adjacent‐CIN3). The process gave us a total of 354 dissected CIN or normal areas  and HPVme‐All . The average methylation of EPB41L3 increased with the disease progression. In the multifocal CIN3 cases, the trend was significant . Another way to view the data is to test the differences in methylation levels between the paired histopathological groups on a per‐cervix basis. In Figure EPB41L3 and HPVme‐All markers. A large majority of methylation differences were positive, suggesting an increase of methylation as diagnoses changed from CIN1 to CIN3 in each woman.A paired analysis on tissue isolated from the same cervices indicated that methylation of the normal tissue and CIN1 adjacent to CIN3 were significantly different from the CIN3 lesions. This was true for both EPB41L3 (p = 0.21) and HPVme‐All  nor HPVme‐All  lower than for primary‐CIN1 and supports a model where EPB41L3 DNA methylation levels may exist as discrete haplotypes, characterizing and possibly controlling the morphological appearance of pre‐cancerous lesions.Methylation levels of normal tissues located near CIN1 or CIN3 were similar for both 354 dissected areas from LEEP samples were HPV typed Fig. . A majorIn multifocal CIN3, a majority of adjacent‐CIN1 were infected by the same hrHPV as the CIN3 . HPV type results were classed as compatible when some HPV types matched but one or the other paired sample also presented additional types. Normal tissues exhibited a somewhat lower similarity to their adjacent‐CIN1  and adjacent CIN3 (51%). Furthermore, we noticed that when multiple lesions of the same grade were present in a multifocal CIN3, they were generally infected by the same HPV type (13/16 = 81% in CIN3 and 11/12 = 91% in CIN1), which is compatible with a single infection event from which multiple lesions arose. However, in principal‐CIN1 only 56% (9/16) of adjacent CIN1 were infected by the same HPV type, consistent with greater diversity of low‐grade lesions, perhaps due to multiple infection events combined with both transient and short‐term persistent HPV infections.HPV types in macro‐dissected LEEP samples were also compared to their corresponding exfoliated cells from cervical scrapes at colposcopy and a good agreement was found between the two (Supporting Information Table S2). The HPV types found in 99% (77 out of 78) of the CIN3 and 88% (43 out of 49) of CIN1 lesions matched or were compatible  with the corresponding exfoliated cell genotyping results.The main goal of our study was to characterize DNA methylation events associated with progression to CIN3. We show in our detailed methylation and HPV typing analyses that the topography of cervical HPV infection, the resultant intraepithelial neoplasia and the molecular patterns are heterogeneous and quite complex. We report here for the first time that, although adjacent CIN of different grades usually contain the same hrHPV type(s), methylation patterns are significantly different and characteristic of the lesion grade.EPB41L3 methylation, may occur before hrHPV infection and facilitate the virus genome amplification and genetic instability phase, allowing in some women relatively rapid progression to CIN3. Furthermore, although most normal tissues have close to 0% methylation for EPB41L3, a sizeable proportion have modest elevation of EPB41L3 methylation of up to 10%.Previously published evidence of genetic alterations that accumulate during cervical tumorigenesis indicate a common cellular origin for multifocal lesions, suggesting that different intraepithelial lesions arise from the same or clonally related progenitor cells.et al. have suggested that multiple high‐grade lesions on the cervix are often caused by a single carcinogenic genotype while other carcinogenic HPV types detected in cervical smears of the same patients are related to independent transient infections.As regards to HPV types, we found that most CIN1 and CIN3 lesions  were infected by a single hrHPV type. Our data partly confirm a similar study by QuintWe also showed that when multiple lesions of the same grade were present in a multifocal CIN3, they were very often infected by the same HPV type (81% in CIN3 and 91% in CIN1). However, in principal‐CIN1 only 56% of the multiple CIN1 dissected were infected by the same HPV type. This indicates that when lower‐grade lesions are the primary diagnostic manifestation of HPV infection the distinct lesions are often infected by different and presumed transient HPV types.EPB41L3 and HPVme‐All, and were very similar to results we obtained earlier on exfoliated cervical specimens. The correlation between biopsies and scrapes allows a generalization of our findings to specimens obtained by cervical scraping and vaginal self‐sampling.There were consecutively large differences and an overall significant Cuzick test trend of increased methylation with increasingly severe intraepithelial histopathology, going from normal to CIN1 to CIN3. These effects were seen for both in vivo mapping would no doubt quickly run into ethical concerns. We need to bear in mind that the two models are compatible and possibly both are correct in certain situations. Although our data support the idea that most CIN3 arise via a haplotype epigenetic switch some CIN3 may develop via a sequential progression.Our study has some limitations. While the new model can underpin added insights for understanding epidemiologic and molecular distinctions between disease transitions in cervical pathogenesis a limitation is our inability to formally prove the methylation haplotype switch model. However, the same caveat applies to the sequential model, conception of which developed over decades, predominantly on the basis of logical but unproven assumptions. A definitive study would require lengthy (possibly decades long) follow‐up of large groups of women with HPV infection. The cervical lesions would require detailed topological mapping with frequent sampling of changing tissues areas by biopsies (which may affect the course of disease) and carefully directed scrapes, coupled to extensive molecular investigations. Such an A strength of our study is the use of an expertly rendered consensus CIN3 histopathological diagnosis as the primary endpoint rather than CIN2/3 or routine practice CIN3. Previous studies have shown that CIN2 is an equivocal diagnosis of precancer and more importantly in this context CIN2 has very poor inter‐rater agreementWhile our data suggest at least two discrete epigenetic states (CIN1 and CIN3), we cannot say whether there are more epigenotypes. It is unlikely that our results were biased by the contamination of dissected lesions with other CIN or surrounding normal cells. All lesions were expertly marked by our team histopathologist (JC) and then dissected, carefully avoiding areas of other CIN and normal tissue. While the isolated CIN DNA probably had a low level of contamination from normal DNA the latter tissues were predominantly either not methylated or were methylated at lower levels than the CIN. Furthermore normal contamination on average would have been similar for principal‐CIN1 and adjacent‐CIN1, which were our most important comparison groups.There are many remaining questions and it is clear that relatively little is known about specific genetic and epigenetic mutations that underpin the evolution of CIN3, nor exactly which steps have a required sequence that then further facilitate the transformation of CIN3 to malignancy. A lot is known about molecular and mechanistic aspects of oncogenes, tumor suppressor genes, regulatory RNA pathways, multi‐protein pathways etc. and also that many such pathways may become abnormal during carcinogenesis. However, for any given woman it remains not possible to predict accurately whether she will develop cervical cancer, what specific constellation of pathway abnormalities would lead to the cancer and over what timeframe this will occur.In conclusion, we show for the first time that, although adjacent CIN of different grades usually contain the same hrHPV type(s), methylation patterns are significantly different and characteristic of the lesion grade, supporting a “molecular switch” model of progression to CIN3 that may be characterizable by distinct methylation haplotype patterns. Methylation testing may be an effective triage tool to detect and characterize women at high risk of developing CIN3 and cancer. Our results may influence the screening process in many ways, for example by providing an objective method to reach more accurate prognoses or by helping to avoid overtreatment of women with non‐progressive lesions. Reducing the numbers of younger women treated by surgical excisions would have an important effect in preserving the ability of women to have uncomplicated pregnancies and to overall improve childbearing at a later date.The authors declare no conflict of interest.Study conception and design: C.R., A.L., and B.N.Acquisition of data: C.R., S.B., J.C., R.B., and R.W.Analysis and interpretation of data: B.N., C.R., A.L., A.A., and J.C.K.Drafting of article: B.N., C.R., and A.L.Critical revision: B.N., C.R., A.L., A.A., and J.C.K.Supporting Information LegendsClick here for additional data file.Supporting Information Figure 1Click here for additional data file.Supporting Information Figure 2Click here for additional data file.Supporting Information Figure 3Click here for additional data file.Supporting Information TablesClick here for additional data file.


Penile carcinoma (PC) is a rare, highly mutilating disease, common in developing countries. The evolution of penile cancer includes at least two independent carcinogenic pathways, related or unrelated to HPV infection.To estimate the prevalence, identify HPV genotypes, and correlate with clinicopathological data on penile cancer.A retrospective cohort study involving 183 patients with PC undergoing treatment in a referral hospital in Goiânia, Goiás, in Midwestern Brazil, from 2003 to 2015. Samples containing paraffin embedded tumor fragments were subjected to detection and genotyping by INNO-LiPA HPV. The clinicopathological variables were subjected to analysis with respect to HPV positivity and used prevalence ratio (PR), adjusted prevalence ratio (PRa) and 95% confidence interval (CI) as statistical measures.The prevalence of HPV DNA in PC was 30.6% (95% CI: 24.4 to 37.6), high-risk HPV 24.9% (95% CI: 18.9 to 31.3), and 62.5% were HPV 16. There was a statistical association between the endpoints HPV infection and HPV high risk, and the variable tumor grade II-III (p = 0.025) (p = 0.040), respectively. There was no statistical difference in disease specific survival at 10 years between the HPV positive and negative patients (p = 0.143), and high and low risk HPV (p = 0.325).The prevalence of HPV infection was 30.6%, and 80.3% of the genotypes were identified as preventable by anti-HPV quadrivalent or nonavalent vaccine. HPV infections and high-risk HPV were not associated with penile carcinoma prognosis in this study. Penile carcinoma (PC) is a rare and aggressive disease with high mutilating potential. The incidence in the United States and Western Europe is estimated at 0.4%, however, in Africa, Asia and South America the incidence is about 6.0% –3.Brazil is a country with a high incidence of PC, accounting for about 2% of neoplasias that affect males, its frequency is associated with the studied region and socio-economic conditions of individuals –7.The origin of penile carcinoma is multifactorial, and the incidence is mainly related to poor personal hygiene, high number of sexual partners, phimosis in adulthood and infections by bacteria and viruses, such as human papillomavirus (HPV) , 8–11. PHuman papillomavirus (HPV) is the most common cause of sexually transmitted infection (STI) ,13 and iThe variations in the prevalence of HPV in PC, according to the literature, are due to differences in sampling, molecular testing, and study population . The oveIn Brazil, studies carried out in patients with PC found HPV prevalence ranging between 30.5% and 63.1% in the states of São Paulo and Maranhão, respectively ,26. In 2Squamous carcinoma (SCC) is the most common histologic type of PC, representing about 95% of cases of this neoplasm. The HPV prevalence and penile carcinoma may differ between histologic types of squamous carcinoma ,28,29. GThe clinicopathological characteristics of penile tumors are factors that predict disease progression, the need for surgery, and death . TherefoThis is a retrospective cohort study in patients with penile carcinoma treated in the Uro-Oncology service in a referral hospital in Goiânia, Goiás, Brazil, from January 2003 to November 2015. For this study, 225 patients received treatment during the defined period and were included in the study; of these, 42 were excluded, resulting in a total of 183 cases.Inclusion criteria of patients in the study were: diagnosis with penile carcinoma and treatment in a referral hospital; biopsy or amputation of the penis in the institution; paraffin block with PC fragment and records located.Cases whose paraffin blocks containing the fragment of the primary tumor were not found, and those submitted to neoadjuvant chemotherapy or penis surgery at another institution were excluded .This study was approved by the Ethics Committee of the Association to Combat Cancer in Goiás (CEP / AACG) under a consolidated number CEP: 901.094.Slides containing paraffin processed tumor tissue fragments were stained with hematoxylin and eosin and evaluated by two pathologists independently to confirm the diagnosis of PC. After the selection of the cases, the blocks were cut into slices and stored in sterile 2 mL microtubes and identified by block number.The viral DNA extraction was performed with the following reagents: Xylol PA for removal of paraffin; Proteinase-K for cellular digestion; a commercial kit (Wizard Genomic DNA Purifications Kit—Promega) to precipitate protein: isopropanol for DNA precipitation and 70% ethanol for DNA purification.The paraffin removal process results in loss of tissue, and therefore degradation of the DNA contained in the sample , so the Detection and genotyping the HPV DNA was accomplished using commercial kit HPV INNO-LiPA HPV genotyping extra  that amplifies the L1 viral region (65pb) using primer SPF 10. This method uses a primer which amplifies the human gene HLA-DPB1, used to monitor the quality of extraction of DNA from the sample. All reactions included negative control (without HPV-DNA) and positive control.Genotyping was performed by reverse hybridization following amplification of the HPV L1 region, biotinylated amplicons were denatured and hybridized with specific probes fixed in parallel lines in strips. This method detected 28 genotypes, 15 genotypes of high-risk HPV, three probable high-risk, seven low risk, and three that were not classified according to risk. The tests were performed according to the manufacturer's instructions. To avoid cross-contamination between samples, specific laboratory work areas were designated for the handling of reagents and samples and for the manipulation of amplified products. Positive and negative controls were included in all DNA extractions and PCR amplification reactions. The protocols used for extraction, detection and genotyping of HPV DNA can be found in The dependent variables were: (i) HPV infection (no or yes) and (ii) High risk HPV infection (no or yes), and the following independent variables were analyzed: (i) age, categorized as < 60 years and ≥ 60 years; (ii) phimosis (no or yes); (iii) Jackson stage (0/II or III/IV); (iv) tumor grade (I or II/III); (v) tumor invasion ; (vi) inguinal metastasis (no or yes); (vii) inguinal lymphadenectomy (no or yes); (viii) inguinal recurrence (no or yes); (ix) lymphovascular invasion (absent or present) and (x) death (live or dead).The variable phimosis was not included in regression analysis due to the large number of cases missing these variables in this study ,33.Data were analyzed in STATA, version 14.0. Initially, descriptive analysis was performed on all variables investigated. Quantitative variables were presented as mean and standard deviation (SD) and the qualitative variables as absolute and relative frequency. Factors associated with infection with HPV were made by Poisson regression with robust variance ,35. VariA total of 183 individuals with penile carcinomas were included in the study. Most patients (51.4%) were 60 years or older at the time of diagnosis of PC. Phimosis was reported by (90.1%) of the participants. Tumor grade II-III was observed in 50.2% individuals, Jackson stage III-IV in 23.6% of cases, deep tumor invasion (51.9%), inguinal metastasis (36.1%), inguinal lymphadenectomy (38.8%) and post-surgical inguinal recurrence in 8.7% of patients. Regarding the primary treatment of lesions: partial penectomy 72.7%, total penectomy in 14.2% and emasculation in 4.4% of cases. The occurrence of death due to PC happened in 18.6% of the participants, according to records . The stuThe prevalence of HPV DNA was 30.6% in paraffin embedded tissue samples of PC. The high oncogenic risk genotypes were found in 24.9% of cases and low risk HPV in 3.8% . Simple Among HPV DNA-positive samples, the most frequent HPV type was HPV 16 (62.5%) and HPV 18 (5.4%). HPV 6 and HPV 11 in total accounted for approximately 12.4% of penile carcinomas .The clinical and pathological variables were submitted to bivariate analysis, with the outcome being the prevalence of HPV-DNA, only the variable tumor grade (II/III) remained associated after multivariate analysis .The same variables were analyzed with the outcome as positive for high-risk HPV, age and tumor grade [II and III] included in the multivariate analysis model, remained statistically associated with the outcome the variable tumor grade II/III .2: 2.14, p = 0.143)  was found  . Regardias found .To our knowledge this is the largest number of cases of PC collected with the goal of estimating the prevalence, genotypic characterization, and HPV association with clinical and pathological characteristics of this tumor in Brazil.Penile cancer occurs more frequently in men aged 50–70 years . In factIn this study, presence of phimosis was observed in 90.1% of PC cases, corroborating literature data ,41. The The pathogenesis of penile cancer is not well understood. A substantial percentage of penile carcinomas are associated with HPV while the remaining tumors rely on molecular mechanisms other than HPV ,19.The prevalence of HPV-DNA in penile carcinoma in this study was 30.6% 95% CI: 24.4–37.6). In a multicenter study conducted in 25 countries, the prevalence of HPV in PC was 33.1% (95% CI: 30.2–36.1) .4–37.6. ,43,44. OIn Brazil, the prevalence of HPV in penile carcinoma ranges from 30.5 to 63.1% in paraffin-embedded samples ,46,47,48INK4a protein and detection of HPV E6*I mRNA [In this study, the prevalence of HPV and HPV types was estimated in 183 cases of PC, however, the research does not allow for inferences regarding viral activity, whether this viral infection is transcriptionally active or not. Other active infection markers would need to be investigated, for example, to evaluate expression of the p166*I mRNA ,24. HoweThe prevalence of high-risk oncogenic HPV genotypes in this study was 24.9% 45/183) and HPV-16 was identified in 62.5% of DNA-HPV positive samples, corroborating national and international data 3 and HPV,47,50. IHPV 6 and 11 are responsible for the development of 90% of genital warts  and haveThe role of HPV as a prognostic factor in penile cancer remains unclear . It is uIn this study, infection by HPV and high-risk HPV was associated with tumor grade II / III (p = 0.025 / p = 0.040) in multivariate analysis, as well as in other studies ,46,52. T2: 2.14, p = 0.143) and high-risk and low-risk HPV  and disease-specific survival rate at 10 years. It is possible that these conflicting results may be due to different study designs, sample sizes and sampling methods for DNA-HPV .A recent Netherlands study reported that High-risk HPV positive tumors appear to providence a significant survival benefit over High-risk HPV negative tumors on multivariable analsysis  . HoweverThis study has some limitations, by its own design. Because it was a retrospective investigation, there was no possibility of retrieving clinical and histopathological data not recorded in medical records. In addition, it was not possible to identify the histological subtypes of the cases of penile carcinoma and to correlate them with the prevalence of HPV, since the majority of anatomopathological reports of the tumors did not include the histological subtype. The study also did not investigate the transcriptional activity of HPV in PC, however, the study design was to estimate the prevalence of HPV and to correlate positivity with clinicopathological factors of PC cases.The results of the study showed that 80.3% of the types of HPV identified  in individuals with PC are immunopreventable types using the quadrivalent or nonavalent anti-HPV vaccine. This information emphasizes the importance of HPV vaccination in males, especially in developing countries, with a high incidence of penile carcinoma.S1 Database(XLSX)Click here for additional data file.S1 Protocols(PDF)Click here for additional data file.


Anal squamous cell carcinoma (ASCC) is a rare, potentially fatal malignancy primarily caused by high-risk human papillomaviruses (HPV). The prognostic implication of programmed death-ligand 1 (PD-L1) expression remains controversial, and glucose transporter 1 (GLUT1) expression has never been examined in ASCC. Covalently closed circular RNAs have recently been shown to be widespread in cancers and are proposed to be biomarkers. We discovered HPV16 expresses a circular E7 RNA (circE7) which has not been assessed as a potential biomarker. A retrospective, translational case series at UT Southwestern was conducted to analyze PD-L1, GLUT1, HPV-ISH, and HPV circE7 in relation to the clinical features and overall survival of patients with ASCC. Twenty-two (22) subjects were included in the study. Improved overall survival was predicted by basaloid histology (p= 0.013), PD-L1 expression (p= 0.08), and HPV-ISH positivity (p& 0.001), but not GLUT1 expression. High levels of circE7 by quantitative RT-PCR predicted improved overall survival in ASCC (p= 0.023) and analysis of The Cancer Genome Atlas sequencing from HPV-positive head and neck cancer and cervical cancer suggested high circE7 marked improved survival in 875 subjects (p= 0.074). While our study suggests that circE7 levels correlate with improved survival in ASCC, larger, prospective studies are necessary to confirm the potential role of circE7 as a biomarker. Anal Squamous Cell Carcinoma (ASCC) has 30,000 new cases diagnosed annually worldwide and the incidence is rising by 2% each year . A majorInvestigations for both novel therapies and biomarkers in ASCC are necessary. Past studies have shown that epidermal growth factor receptor (EGFR) overexpression occurs in almost all cases of ASCC . ClinicaThe facilitative glucose transporter 1 (GLUT1) regulates glucose transport in the skin, especially keratinocytes. The transporter may play an important role in sustaining carcinogenesis by fueling anaerobic glycolysis in the hypoxic environment of the tumor . HPV-driHigh-risk human papillomaviruses (HPVs), especially HPV16, is implicated in 70-90% of cases of ASCC and is a primary driver of oncogenesis . Recent In this report, we examine both established  and novel (i.e. HPV16 circE7) biomarkers and their role in prognosis in HPV-driven ASCC. We also correlate these findings to demographic, clinical, and prognostic factors in these patients. We identify high levels of circE7 as a positive prognostic marker in ASCC, and potentially other HPV-driven cancers.in-situ hybridization. A schematic of the study design is included in the Supplementary Figures. The demographic, clinical, and histopathologic characteristics of patients who underwent molecular studies are listed in p = 0.034), higher T stage , and higher N stage . Decreased overall survival could also be predicted by greater smoking pack-years  and greater size of tumor (>5 cm dimension) upon presentation  (p = .0375), independent of other factors listed above. Age, ethnicity, medical comorbidities, chemotherapeutic agent, and use of radiotherapy did not correlate to prognosis.Of the 22 patients with ASCC included in the study for clinical analysis, 18 cases had tissue available for molecular studies and 16 had available tissue for immunohistochemistry and  = 0.03) . Patientp = 0.013)  of the biopsies as the sole architecture . Three . Notably= 0.013) . Architep& 0.001), high circE7 levels (p = 0.038), and smaller tumor size (p = 0.020) (p = 0.0837) (p& 0.001)  . PD-L1 p 0.0837) . All 16  0.0837) . GLUT1 w 0.0837) . HPV-ISH 0.0837) –2F; thes& 0.001) .p = 0.023) (p& 0.001), positive PD-L1 expression (p = 0.038), lower T-stage (p = 0.038) and towards basaloid architecture. In contrast, linear E6*I levels did not have prognostic significance (p = 0.26).Endpoint RT-PCR confirmed that inverse primers specific to circE7 amplified products of the expected size . Some sa= 0.023) . The higp = &0.0001)  and head and neck squamous cell carcinoma (HNSC) from the Cancer Genome Atlas . Consist&0.0001) . Of note&0.0001) . In contThe aim of this research was to analyze a novel (HPV16 circE7) and established biomarkers  and correlate these markers to clinical outcomes in patients with ASCC. We also demonstrate for the first time that circE7 is stable enough to be detected in formalin fixed paraffin embedded (FFPE) tumor samples. Although this study was not powered to identify the clinical characteristics that may predict poorer prognosis, we found that male sex, smoking, greater size of tumor upon presentation, and higher TNM stage all correlated with poorer prognosis. Although small in size, our retrospective cohort appears to possess clinical prognostic factors consistent with several larger studies in patients with ASCC , 22.Also, consistent with previous reports, we found that the keratinizing subtype was associated with worse prognosis, and the basaloid subtype with better prognosis , 24. KerPD-L1, an immune-checkpoint inhibitor found on some cancer cells, immune cells, and other normal tissues, suppresses T cell function by interacting with the lymphocyte PD-1 receptor. While expression of immunosuppressive cell surface receptors might be assumed to portend a poorer prognosis in cancer patients, the opposite has been shown in some cancers, including ASCC , 27. TumTumor cells may exhibit an altered metabolism to support their unregulated proliferation and sustain their greater energy demands for proliferation and metastasis. In fact, the GLUT1 transporter has been shown to be strikingly upregulated in several cancers . The incIn general, studies on HPV-associated malignancies reveal a favorable prognosis in HPV-positive carcinomas compared to the HPV-negative counterparts , 32, 33.in vitro and in tumor xenograft studies [Analysis of RNA-Seq from HPV+ head and neck cancer and cervical cancers from TGCA data showed a similar trend for improved survival in high circE7 tumors. We have found that circE7 can be translated to the E7 oncoprotein and that its circE7 is essential to the transformed behavior of CaSki cervical carcinoma cells  studies . While i studies . Additio studies , 36. Fin studies , levels  studies .CircRNAs have been shown to be more stable than linear mRNAs in multiple studies , 21, 37.Limitations of this study include its small sample size and the retrospective nature of clinical data collection. This cohort also contained a heterogenous sample of ASCC severity ranging from locoregional to metastatic and the data was collected at a large, tertiary care center, and thus may have a selection bias for more severe cases of ASCC. Additionally, the nature of examination of biomarkers from histology may lead to sampling error of the tumor and thus exact measurements of biomarkers present in the sample may differ in regions of the patient’s tumor.In summary, this retrospective, translational pilot study examined a novel biomarker, HPV16 circE7, as a potential prognostic indicator given the unique properties of circRNA and our ability to extract it from FFPE tissue. Our study also contributes to the literature on the role of existing clinical, histologic, and immunohistochemical markers for ASCC. Larger, prospective studies are recommended to analyze the role of circE7 roles in HPV-driven cancers.Patients over the age of 18 with a diagnosis of ASCC established by UT Southwestern Medical Center in Dallas, Texas between 01/01/2005 to 3/01/2019 were recruited in a retrospective, translational case series. The UT Southwestern Institutional Review Board approved this retrospective study and the need for consent was exempted. Inclusion criteria consisted of patients with a biopsy-proven diagnosis of ASCC that had follow-up at University of Texas Southwestern Medical Center. Demographic and clinical characteristics of the study cohort, including age, sex, ethnicity, comorbidities, smoking history, initial presenting symptoms, were collected through the electronic medical record. Cancer stage was based on the American Joint Committee on Cancer (AJCC) TNM classification (8th edition) . Treatmehttps://www.ensembl.org/) as well as high-risk HPV genomes and then the reads with the alignment scores on HPV genomes greater than on human transcripts were kept for the following steps for detecting circular RNA. HPV-positive samples were defined by the number of reads mapped on an HPV genome ≥10,000 and the mapping rate on the genome ≥0.00001. Based on these definitions, 125 CESC cases and 51 HNSC cases were HPV16+. The survival analysis was performed using R packages, survival  and survminer (https://cran.r-project.org/web/packages/survminer/). Kaplan–Meier survival curve of comparing high circE7 (≥30 read count and back-splice junction ratio ≥0.2) with non-circE7-high HPV+ cancers from TCGA. The GLUT1 (SLC2A1) and PD-L1 (CD274) gene expression levels were calculated by DESeq2 rlog (regularized log) after read mapping by STAR and read counting by HTSeq-count. The read ratio of E6*I (spliced isoform I) was calculated by the number of E6*I splicing reads divided by all reads covering 5’ splicing junction site.We downloaded raw sequencing data of cervical squamous cell carcinoma and endocervical adenocarcinoma  and head and neck squamous cell carcinoma  from The Cancer Genome Atlas (TCGA). As the read lengths were 50-51bp, to avoid false mapping of human reads onto HPV genomes, the reads were aligned to human transcript sequences downloaded from Ensembl   buffer, primary antibody incubation was performed for 50 minutes in a 25°C incubation oven, using gentle orbital rotation at 40 rpm. Following another rinse in PBS, incubation with the appropriate anti-rabbit (for GLUT1 and PD-L1) horseradish peroxidase-conjugated polymer  was performed for 45 minutes at 25°C, using gentle orbital rotation at 40 rpm as before. Finally, the slides were immersed for 8 minutes in 25°C diaminobenzidine (DAB) , enhanced with 0.5% copper sulfate in phosphate buffered saline (PBS) for 3 minutes at 25°C, counterstained in hematoxylin, dehydrated in graded alcohols, cleared in xylene, and coverslipped. In all cases, IHC was simultaneously performed on an in-house (ProPath) tumor tissue microarray to ensure the expected staining pattern for GLUT1 and PD-L1 stains. All controls performed appropriately.FFPE were sectioned at 4 μm and mounted on adhesive slides, along with multi-tumor sandwich block sections containing normal and tumor tissues for external positive control and external negative control purposes. After drying the slides for 1 minute in an 1100W microwave oven set on high, the slides were transferred to a 65°C oven and dried for an additional 20 minutes. The slides were then deparaffinized in xylene and rehydrated in graded alcohols to distilled water. Endogenous peroxidase activity was quenched for 10 minutes at room temperature, using 0.3% Hin-situ hybridization (HPV-ISH) shows the highest sensitivity (88%) and specificity (97%) for detecting HPV+ head and neck squamous cell carcinomas as a stand-alone assay [in situ hybridization (HPV-ISH) probe  was obtained from Advanced Cell Diagnostics (https://acdbio.com) and was used in conjunction with their RNAscope 2.5 HD assay-brown detection kit  in accordance with the manufacturer’s instructions. Known external positive control and external negative control tissue were mounted on the same slides as the patient tissue to ensure appropriate performance of the stains. HPV-ISH screened for high-risk subtypes including 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, and 68.HPV ne assay . The higTwo board-certified pathologists (S.H. and G.H.) were blinded to clinical information and analyzed all histologic specimens. On H&E, the tumor architecture was recorded as “keratinizing,” “basaloid,” or “mixed.” Basaloid architecture is characterized by a solid and lobular growth pattern composed of basaloid cells with minimal cytoplasm, increased nuclear to cytoplasmic ratio, and the formation of tumor nests showing peripheral nuclear palisading with surrounding stromal fibroplasia. Keratinizing tumors consisted of tumor cells with abundant eosinophilic cytoplasm, keratinization, and the presence of prominent intercellular bridges. If discrepancies in the tumor architecture were noted between the pathologists, the sample was scored as “mixed.” The presence of perineural invasion, lymphovascular invasion, necrosis, the inflammatory response, and the tumor grade were all recorded. GLUT1 staining was measured by a weighted score, measured by the product of the percentage of stained tumor cells  and the intensity of signal . High and low GLUT1 were differentiated by the median of the weighted scores. PD-L1 was measured as positive or negative with positive if >1% staining of the tumor . HPV-ISH was measured as positive or negative. The percentage of positive tumor cells was also recorded, as was the nature of the staining pattern .Total RNA was extracted from FFPE slides using the Qiagen mRNAeasy kit. The extracted RNA was converted to cDNA with SuperScript IV First-Strand cDNA Synthesis Reaction using random hexamer priming. RNA levels were normalized against β-actin mRNA using back-splice specific primers for circularized E7 . Subjects were separated into high circE7 if the quantified mRNA was above the median value of all sample mRNA levels and low/absent circE7 if the levels were at or below the median. High and low/absent linear E6*I values were obtained similarly using the linear splice primers .p-value of &0.05 was considered significant.Continuous data is presented as means with standard deviations and categorical data as counts with percentages. Demographic and histologic differences between biomarker levels and clinical values were compared using Pearson chi-squared for categorical data and ANOVA for continuous data. The Kaplan–Meier curves were utilized to construct the visual differences for overall survival and the biomarkers of GLUT1, PD-L1, and HPV-ISH. Univariable with the log-rank test was used to analyze factors which affect overall survival. A


A small but significant proportion of military veterans become involved in the criminal justice system (CJS) after leaving service. Liaison and Diversion (L&D) services aim to identify vulnerable offenders in order to provide them with the health/welfare support they need, and (where possible) divert them away from custody. An administrative database of L&D service-users was utilised to compare the needs of veterans with those of non-veteran L&D service-users.National data collected from 29 L&D services in 2015–2016 was utilised. Of the 62,397 cases, 1,067 (2%) reported previous service in the Armed Forces, and 48,578 had no previous service history. The associations between veteran status and socio-demographic characteristics, offending behaviour, health- and mental health-problems were explored. The associations between specific types of offending and mental health problems within the veterans in the sample were also investigated.Veterans tended to be older, and less likely to be unemployed than non-veterans, but just as likely to have unstable living arrangements (including homelessness). Veteran status was associated with increased levels of interpersonal violence, motoring offences, anxiety disorders and hazardous drinking patterns. Veteran status was associated with decreased levels of acquisitive offending, schizophrenia, ADHD, and substance misuse. Among veterans, the presence of an anxiety disorder  was associated with increased interpersonal violence, alcohol misuse was associated with increased motoring offences, and substance use was associated with increased acquisitive offending.Our study indicates that among offenders in the CJS who have been identified as needing health or welfare support, veterans differ from non-veterans in terms of their health and welfare needs and offending behaviour. These differences may be influenced by the impact of military service and the transition into civilian life. Our findings support the identification of military personnel within the CJS to provide appropriate interventions and support to improve outcomes and reduce offending. The majority of service leavers make successful transitions back into civilian life . HoweverGovernment statistics show that veterans form the largest single occupational group within the prison and probation services, and that they are more likely to have committed a violent or sexual offence than offenders who have not served in the military ,6. HowevExisting research also suggests that socioeconomic needs, such as relationship problems, financial instability, unemployment and lack of stable accommodation, are strong risk factors for offending among veterans and that stability in these areas can be protective against the risk of offending in the presence of mental health problems ,15,16. TThe purpose of Liaison and Diversion (L&D) services is to provide an assessment of individuals within the CJS who have been identified as having a psychosocial need , to ensure that they receive the appropriate support, and to (where possible/appropriate) divert them out of the CJS and into health, social care or other services. Referral to L&D services usually occurs at the earliest opportunity, but may take place at any stage of the CJS. Furthermore, referrals can be made by a wide range of agencies, including: police, Crown Prosecution Service, youth offending teams, social workers, drugs/alcohol services, defence lawyers, and parents/guardians/family members. This broad referral process maximises the reach of the L&D service to the most vulnerable of individuals at the earliest opportunity. Individuals referred to L&D services are offered a screening appointment with a mental health practitioner, during which they are asked whether they have ever served in the UK Armed Forces. To date, there has been no formal comparison of L&D service offenders with offenders who have not been referred to these services. However, it is likely that those offenders referred to L&D services represent a particularly vulnerable group of offenders with a range of psychosocial vulnerabilities.The research to date has focused either on aspects of military service that are risk factors for offending, or comparisons of offending between veterans and non-veterans within the CJS without considering other factors such as health, mental health and welfare needs. Our study aims to address this gap in the literature. We utilised the national L&D database of all offenders, both Armed Forces veterans and non-veterans, who were assessed by the 29 services across England during 2015–2016. Our primary aim was to identify differences between veterans and non-veterans within L&D services, in terms of: socioeconomic and welfare needs; offending behaviour; and health and mental health problems. In addition, we examined factors associated with specific types of offending  among veterans referred to L&D services.This study employed routinely-collected data from 29 separate L&D services from April 2015 until April 2016. Data were gathered by L&D service practitioners, and included information pertaining to the individual’s: military status; socio-demographics; current offence; mental health needs; alcohol/substance use; and other vulnerabilities . Data were entered onto the database on a case-by-case basis: each referral was treated as a separate case. Thus, in the absence of an individual identifier, the same individual may have multiple entries relating to different offences. As shown in Permission to access the L&D database was sought from and approved by NHS Digital following ethical approval (reference: LRS15/162992) to ensure all information governance policies and procedures were met. The dataset was fully anonymised before it was accessed.In accordance with the UK Government’s criteria, a military veteran was classified by L&D services as anyone who had served for at least one day in the UK Armed Forces, and had left service at the time of contact with L&D services. All other non-military cases were classified as non-veterans. We note that our data are cross-sectional, thus our analyses reflect associations with veteran status, rather than its role as a risk factor for offending or physical/mental health problems.Socio-demographic factors included: age, gender, ethnicity , employment status , and accommodation status . Age was recorded in 5-year increments, and reflected the age-group of the individual at their first meeting with the L&D service. For the purposes of the statistical analyses, age was recoded in to six categories: 25 years and under, 26–35 years, 36 to 45 years, 46 to 55 years, 56 to 65 years, and over 65 years.This was recorded as the most serious offence that the individual was charged with, or suspected of having committed, at the time of their referral to the L&D service. Offences were classified as: violence against the person ; sex offence; non-interpersonal violence ; acquisitive offence ; drug offence; public order offence; motoring offence; breach of court order; and other. For the purposes of statistical analyses, this was recoded into nine separate binary variables, each indicating the presence or absence of each offence type, in line with previous research . HoweverBoth alcohol and substance misuse were assessed using standardised instruments: the Alcohol Use Disorders Identification Test  for alcoThe presence of learning difficulties (where suspected) was established using standard cut-off scores on the Learning Disability Screening Questionnaire  or the HIndividuals were screened for, or diagnosed with, the following: schizophrenia, bipolar affective disorder, depression, anxiety disorders , adjustment disorder, eating disorders, dementia, attention deficit/hyperactivity disorder (ADHD), and personality disorder. Up to three mental health problems could be recorded for each case. Whilst all mental disorders were assessed using standardised screening tools and information from medical records where available, the screening and diagnostic methods varied among the different sites. As a consequence, some recorded mental disorders reflect actual diagnoses, whereas others reflect elevated scores on screening questionnaires.We conducted the analyses using a series of univariate and multivariate logistic regression models in Stata 14 . StandarWe then examined the factors associated with particular offence types in the veteran sample . In these analyses, we only examined offences that were independently associated with veteran status in the preceding analyses: violence against the person ; acquisitive offences ; and motoring offences . As in the preceding analyses, we first identified any socio-demographic variables that were independently associated with each offence type using separate logistic regression analyses. We then conducted a series of logistic regressions to examine the univariate and adjusted associations between each offence type and each of the mental health variables. In these analyses we did not adjust the standard errors based on L&D site due to the small number of veterans per site . First, As we were unable to identify which cases belonged to separate individuals in the database, and thus unable to assess the extent of clustering in data, we performed a crude sensitivity analysis. We matched the cases on a series of “static” variables , removed the duplicates (by selecting the first case in a cluster), and repeated our analyses. Our main results remained unchanged, therefore we report the results of the analysis of the full dataset.The veterans in the sample were predominantly male, aged 26–35, and of white ethnicity. These characteristics were significantly associated with veteran status see . VeteranOffences classed as violence against the person were the most common type of offending among the whole sample (32%), followed by acquisitive (16%), public order (11%), and non-interpersonal violence offences (10%). The remaining offence types were relatively uncommon. As shown in The health needs of veterans and non-veterans are shown in Veterans were more likely than non-veterans to be recorded as having an anxiety disorder , depression , and dementia . In addition, veterans were more likely to have multiple mental health problems . Veterans were less likely than non-veterans to be recorded as having personality disorder , schizophrenia , and ADHD . All of these mental health problems remained independently associated with veteran status, after adjusting for age, gender, ethnicity and employment status, except for personality disorder and depression see .Results of the multivariate logistic regression analyses for factors associated with each offence type among veterans are presented in the supporting information see  Tables.Age and employment status were both independently associated with violence against the person  with PTSD –42. It iVeteran status was associated with hazardous (but not dependent) drinking and physical health problems. Research shows that rates of hazardous and dependent drinking among UK Armed Forces personnel are higher than among those the general population, irrespective of gender . Some phWithin the veteran sample, factors associated with violence against the person, acquisitive, and motoring offending were also examined. Violence against the person offences were associated with a recording of anxiety disorders . There is a well-established link between anxiety and violence/aggression ,49, whicAcquisitive and motoring offences were associated with substance misuse and alcohol misuse, respectively . There is a well-established link between substance misuse and crime in general, and this is consistent across different types of substances and different types of offending . AlthougA major strength of our study was the large sample size, especially of the non-veteran group, which was likely to be representative of L&D service-users. This allowed us to account for the effects of potentially confounding socio-demographic factors, which would not have been possible using a smaller sample. We were also able to directly compare veterans and non-veterans that were members of the same population–i.e. vulnerable individuals in the CJS. This is a major advantage, as any differences between them are unlikely to be biased due to different sampling methods. Furthermore, given that individuals are referred to L&D services from a range of settings, our data include individuals who had committed (or were suspected of having committed) a range of offences: from summary offences to murder and manslaughter. This increases the generalisability of our findings.Despite these strengths, the results of our study should be interpreted in the light of a number of limitations. First, there was a significant amount of missing data. For example, 20% of cases had no veteran status, suggesting that the question is not always asked by L&D service practitioners. However, this level of missingness is not unusual among administrative datasets . FurtherThis study has highlighted the utility of using secondary routinely collected data from services engaged with offenders early in their CJS journey in order to identify those with specific needs and allow early intervention. We have identified that, among L&D service users, those who have served in the Armed Forces have offending, welfare, physical health, mental health and substance misuse needs that differ from general population offenders. In some jurisdictions in the United States of America such is the recognition of these differences in need that special veteran courts operate to divert some veterans into justice reform projects for veterans –61. The In other jurisdictions and countries, such as the UK, where veteran courts are not viable, the differences observed between veterans and non-veterans highlight the need for workforce training across the CJS to improve the identification of veterans and understanding of their needs within the CJS. The different pattern of mental health needs among veterans, such as the higher rate of anxiety disorders, and the links between mental disorders and offending behaviour, especially the link between anxiety disorder and interpersonal violence, emphasises the need for improved availability of psychological therapies in general in prison and the need for staff with an understanding of the impact of military service and life after the military on mental health and risk of offending. This may be especially important, given research showing that veterans tend to improve less from standard PTSD treatment approaches than non-veterans . Thus, iS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Young women (20-35 years) are at high risk of HPV infection, although the majority of the infections are asymptomatic and are cleared spontaneously by the host immune system. These are also the group of women who are sexually active and are in the population of pregnant women. During pregnancy, the changes in the hormonal milieu and immune response may favor persistence of HPV infection and may aid in transgenerational transmission thereby furthering the cancer risk. In the present study, we determined the prevalence of vaginal HPV infection in early pregnancy and attempted to relate with pregnancy outcome. Vaginal cytology samples were collected from the condoms used to cover the vaginal sonography probe during a routine first trimester visit to the hospital. All women were followed up throughout pregnancy and childbirth. Maternal and neonatal outcomes were recorded. We found a prevalence of HPV infection around 39.4% in our population. Interestingly all HPV positive women were infected with one or more high risk HPV viruses with an overlap of intermediate and low risk in 43% and 7.3%, respectively. Women with preterm prelabor rupture of membranes (PPROM) showed a statistically higher incidence in HPV positive (7.3%) group as compared to the HPV negative (3.2%) group. The prevalence of genital HPV infection is high during pregnancy (around 40%) and was associated with higher incidence of PPROM. Human Papillomavirus (HPV) is the most common sexually transmitted viral infection. More than 100 human types of HPV are identified which are known to infect epithelial cells including skin, respiratory mucosa, or the genital tract. HPV types specific to genital tract infections are classified into three risk categories; low risk , intermediate , and high-risk  types based on their relative malignant potential [Young women (20-35 years) are at maximum risk of HPV infection, although majority of these are asymptomatic and get cleared spontaneously because of strong immune system. This is the age when women are more sexually active. In the developing nations this age group forms a major cohort among the pregnant population. During pregnancy the changed hormonal milieu and immune response might favor presence or persistence of HPV infection.A systematic review of literature demonstrated a wide variation in the prevalence of HPV in pregnant women from 5.5 to 65% . The higIn the present study, we determined prevalence of vaginal HPV infection in early pregnancy and its correlation with the pregnancy outcome.This prospective study was conducted in a University Teaching Hospital over a twenty-month period (November 2015–June 2016). The study protocol was approved by institutional review board (IEC127/2016). The study cohort included random obstetric population who presented to us in first trimester (up to 14 weeks) with singleton pregnancy and were planning to continue in one centre throughout pregnancy and delivery. Written informed consent was obtained from all participants. Those women who were previously diagnosed with HPV infection or were detected to have abnormal cervical cytology in Pap smear tests were excluded from the study.Vaginal cytology samples were collected from the condoms used to cover the transvaginal sonography (TVS) probe during a routine first trimester scan. Condoms used to cover the TVS probe were used, as regular speculum examination in asymptomatic pregnant women is not a part of routine antenatal care in our set-up and it is not acceptable to many pregnant women. However every patient undergoes a TVS in first trimester either for confirmation or dating of first trimester early anomaly screening. Condom thus obtained after TVS was immediately put in a 50 cc sterile plastic container with phosphate buffer saline solution (PBS), vigorously shaken, and condom was discarded. The solution was kept at 4°C and processed within one hour. All women were followed up throughout pregnancy and childbirth and maternal and neonatal outcomes were recorded.Tubes were centrifuged at 2000rpm for 10min and the pelleted cells were digested with DNA extraction buffer and proteinase k followed by standard phenol chloroform and ethanol precipitation method. The quality and quantity of the DNA were assessed by agarose gel electrophoresis and spectrophotometrically and stored at -20°C until used. On an average we could extract 200-250 ng of DNA from the condom which was used to cover the TVS probe during first trimester ultrasonography.HPV DNA testing was performed as published earlier. In brief, 100-200 ng of DNA was amplified by nested PCR using PGMY09/11 and GP5+/GP6+ primers with beta globin as internal control. PCR product was gel eluted, purified, and sequenced using Big Dye terminator kit  in Genetic Analyzer 3130XL . The HPV types were identified by NCBI BLAST search. The PCR was performed using appropriate positive and negative controls. The samples were scored as negative after two rounds of independent testing.HPV positivity and type were then correlated with various maternal and fetal variables and pregnancy outcomes.The data on categorical variables is shown as n (% of cases) and the data on continuous variables is presented as Mean and Standard deviation (SD) across two study groups. The intergroup comparison of categorical variables is performed using Chi-square test/Fisher's exact probability test. The statistical significance of intergroup difference of mean of continuous variables is tested using independent sample ‘t' test or unpaired ‘t' test. The underlying normality assumption was tested before subjecting the study variables to ‘t' test. The entire data is entered and cleaned in MS Excel before its statistical analysis.The p-values less than 0.05 are considered to be statistically significant. All hypotheses were formulated using two tailed alternatives against each null hypothesis (hypothesis of no difference). The entire data is statistically analyzed using Statistical Package for Social Sciences  for MS Windows.A total of 115 samples were collected; however 104 samples completed the study. HPV prevalence: in our study cohort out 41/104 (39.4 %) were HPV positive, while 63/104 (60.6%) were found to be negative from HPV vaginal infection. Interestingly, typing of the HPV positive samples indicated that all these 41 samples were positive for high-risk HPV types . Eighteen of them showed evidence for coinfection with intermediate risk HPV types such as HPV 87, HPV35, HPV51, HPV52, and HPV82, while only 3 samples were coinfected with low risk HPV . Most frequent HPV types in our cohort were HPV45 (60%), followed by HPV18 (48.8%) and HPV16 (43.9%) .HPV status and pregnancy outcome: in order to compare the pregnancy outcome in HPV positive versus HPV negative women, we analyzed the basic demography in the two groups. The mean age  and mean number of pregnancies  were comparable in both of the groups, while the years of married life in HPV positive pregnant women were 3.6±2.7 years and they were 2.8±2.7 years in HPV negative women (p<0.05) .On comparing the pregnancy complications  between We found HPV prevalence of around 39.4% in pregnant women in our population. Interestingly all HPV positive women were infected with one or more high risk HPV viruses with an overlap of intermediate and low risk in 43 % and 7.3%, respectively. HPV positive status was seen more frequently in women with longer married life  and higher BMI.HPV infection is common among sexually active young adults, with an estimated prevalence between 20.0% and 46.0% . During In African pregnant women prevalence of HPV DNA was 33.3%. Around 62% of HPV-positive women were infected with high risk and/or possible or probable high risk (pHR) also known as intermediate risk HPV types. The five most prevalent HPV types in this study were HPV-52 and HPV-67, HPV-53, HPV-45, and HPV-18. HPV-16 was rare (1.2%) .HPV in pregnancy is interesting and important to study for two reasons. First vaginal infections are known to adversely affect pregnancy outcome. Secondly HPV infection of the placenta is possible as trophoblast cells appear to have the machinery for HPV replication , 12 and In our cohort only the incidence of PPROM was found to be more among HPV positive pregnant women as compared to those who were HPV negative. There was no difference in risk of abortions or preterm births (without PPROM). In other studies too there is equivocal evidence for placental infection with HPV causing placental dysfunction leading to spontaneous abortions and preterm births , 13. A rAs per our knowledge, this is the first prospective study from Indian subcontinent to find out the prevalence of HPV in our population during pregnancy and its effect on pregnancy and childbirth. The factors affecting HPV infection in our sociocultural environment might be different. Furthermore, though cervical cancer screening with cytology is available all over India, it has not yet become practical for population screening in our country. Thus this kind of simple effortless collection of samples for screening, in the target population during pregnancy, is an interesting area to study further. However, the cost of HPV testing needs to be justified if it is done beyond the periphery of research. HPV screening during pregnancy might also help to optimise our goals towards a better reproductive and child health care, as we found it to be associated with poor pregnancy outcomes. Another novelty of this study was the technique with which we collected samples from the condoms which were used to cover the TVS probe. No other study has mentioned this technique of sample collection and its feasibility until date, in scientific English literature.Small size of study population is the limiting factor here. Other limitations include only one time testing in first trimester for HPV status. New HPV infection towards term or clearance of HPV before delivery would be interesting to study and its correlate with pregnancy outcomes might be more meaningful, as would be the study of the vertical transmission rate by studying the placental affliction and HPV status of the newborn.The prevalence of genital HPV infection is high (40%) during pregnancy and was associated with higher incidence of PPROM in our study. Condom used to cover TVS probe during sonographic evaluation of pregnancy can be used to collect samples for HPV detection where sociocultural belief prohibits direct speculum examination during routine antenatal care in asymptomatic women.


Objective. The study aimed to assess the impact of HPV immunization campaigns organizational aspects, the characteristics of immunization program (vaccination targets and type of offer), and communicative strategies adopted by four Italian administrative regions on vaccination coverage observed. Methods. From November 2017 to March 2018, regional and Local Health Units (LHUs) representatives were invited to complete an online survey including 54 questions evaluating vaccination invite systems, access systems to vaccination centres, reminder and recall systems, and adverse events surveillance. An overall descriptive analysis was conducted. Since observed vaccine coverage (VC) obtained in females (2002-2004 birth cohorts) was lower than objectives fixed by the Italian Ministry of Health, variables were assessed using the national VC mean obtained in the 2003 girls birth cohort as outcome. Results. Twenty-six LHUs belonging to 4 Northern and Southern Italian regions participated in the study. Organizational aspects significantly related to VC lower than the national mean were access to vaccine centres without appointment and parents' reservation as appointment planning system. Recall systems for both the first and the second dose, including the appointment in the invitation letter, the availability of regional immunization registry, and education of healthcare workers on universal HPV immunization strategies, instead, were related to higher VC. As regards preadolescent immunization strategies, both VC obtained in girls and boys were far from the Ministerial goals. Only 20% of LHUs introduced multicohort female strategies while all LHUs adopted copayment targeting both men and women. Immunizations strategies targeting subjects at risk were implemented only in half of participating LHUs. Conclusions. VC observed in participating LHUs are largely lower than the national objectives in all anti-HPV vaccine targets. Both organizational and educational strategies have to be implemented to improve the VC goals. Human papillomavirus (HPV) is considered one of the most common sexually transmitted infections in the world, affecting people especially in developed countries. About 70-80% of the sexually active women and men will acquire an HPV infection during their life . Since HInitially, HPV vaccination has been recommended for adolescent women prior to sexual debut, and it has already been showed to be highly effective in reducing HPV-related lesions, such as genital warts as well as Cervical Intraepithelial Neoplasia (CIN) . IncreasNowadays USA, Canada, Australia, and over 37 out of 53 European (EU) countries have introduced the vaccine into their national routine immunization schedules and several of these both for males and females .In 2014, HPV vaccination coverage in the EU Region accounted for about 50% in the primary target, while African countries reported a mean 88% HPV vaccination coverage (VC) . In ItalIn Europe, several studies have previously indicated that common reasons for not receiving or completing the HPV vaccine were the perception of low risk or not needing the vaccine, low perception of HPV vaccination benefits, doubt about the safety and efficacy of the vaccines, fear of side effects, low participation at school seminar on HPV for school-based immunization campaign, lack of physician recommendations, and cost of the vaccine –17.Organizational aspects to improve HPV vaccine uptake were also investigated, in particular the implementation of immunization services' accessibility, the role of reminder systems, and communication technologies. Nevertheless, previous evaluations were rarely comprehensive or specific, thus compromising the estimate of the effect of interventions' interaction and the consideration of HPV vaccine peculiarities  –29.Aim of the present study was assessing the different HPV immunization campaigns carried out in four Italian administrative regions, evaluating the impact of organizational aspects, the characteristics of immunization program , and communicative strategies adopted by the regions on vaccination coverage observed.An online questionnaire was administered through Google Drive platform to 26 Local Health Units (LHUs), belonging to 4 Northern and Southern Italian regions, where the universal preadolescents HPV vaccine recommendation was introduced before the PNPV 2017-2019, covering 25.7% of the national population.From November 2017 to March 2018, regional and LHUs representatives were invited to complete an online survey including 54 questions evaluating HPV immunization policies targeting preadolescents, adults and subjects at risk, obtained results in different targets, communication and education strategies, and organizational characteristics of vaccination centres. In particular, vaccination invite systems, subjects who invites preadolescents, sending information with invitation letter, subjects who administers the vaccine, access system to vaccination centres, appointment planning system, reminder of the first dose appointment, specific HPV vaccine sessions, recall of subjects who missed the first and the second dose, second dose appointment planning, and AE surveillance within 30 minutes and since the day after were investigated. An overall descriptive analysis was conducted.Since observed VC obtained in females 2002-2004 birth cohorts were lower than the objectives fixed by the Ministry of Health, VC national mean was used as outcome. In particular, the possible association of organizational aspects with VC obtained in the female 2003 birth cohort higher than the national mean (64.7%)  was testVC obtained in boys were not considered as outcome since the objectives fixed by the Ministry of Health in PNPV 2017-2019 refer to the 2006 birth cohort for which data were not available.After the collinearity assessment, factors resulting statistically significant in univariable comparisons (p < 0.05) were included in a multivariable model, by means of a stepwise backward procedure.Statistical analyses were conducted by the JMP software, version 13.The study protocol was approved by the Regional Ethic Committee of the Liguria region, Italy (P.R. 162REG2017).The main results concerning universal preadolescents HPV vaccination organizational aspects are shown in As regards preadolescent immunization strategies, both VC obtained in boys and girls are largely suboptimal in participating LHUs and very far from the goals set by the Ministry of Health in the PNPV 2012-2014 and 2017-2019 Figures . In partFurthermore, only about 20% of LHUs introduced multicohort female strategies while all adopted copayment targeting both men and women.Immunizations strategies targeting subjects at risk are implemented only in half of participating LHUs and a multidisciplinary network to identify them is active in 27% of cases. The healthcare workers who recommend the HPV vaccine are various but low in numbers , while the administration of HPV vaccine is centralized in vaccine centres.As regards vaccine registries, they are digitized in all participating LHUs but only about the half of them are present on a regional level.Communication tools were also investigated . In partRecall systems for both the first and the second dose, including the appointment in the invitation letter, the availability of regional immunization registry, and education of healthcare workers on universal HPV immunization strategies, instead, were related to higher VC.After multivariable analysis, one variable resulted statistically significant: access without appointment to vaccination centres (p=0.038); recall systems resulted borderline, with p=0.063 . In partThis survey allowed us to obtain a detailed picture of a wide range of HPV vaccine offer and promotion strategies and to identify actions adopted in Italian LHUs that are associated with VC higher than the national mean.In our knowledge, the most studied strategies aimed at gaining adequate HPV VC deal with the evaluation of knowledge, attitudes, and determinants of acceptance and refusal, and they mainly focus on preadolescents girls and their parents –41.Nevertheless, immunization strategies and organizational aspects of vaccine centres also have a relevant role in determining the vaccine compliance.Some studies investigated interventions to improve HPV vaccine uptake but each of them was evaluated separately –23. OtheAmong the abovementioned interventions, the active invitation of eligible subjects represents one of the more effective interventions to increase the VC. In this context, almost all participating LHUs invite adolescents' parents by letter and adopt the appointment as access system to vaccination centres, limiting the free access to 19.2% of cases. In particular, the appointment is included in the invitation letter in 76% of cases.On the contrary, the access to vaccine centres without appointment and parents' reservation as appointment planning system are significantly related to VC lower than the national mean. Further, recall systems for both the first and the second dose is adopted only in 69.2% and 61.5% of cases, even if recommended with strong evidence by scientific authorities  and resuAmong recommended with strong evidence immunization information systems, the digitized vaccine registries could be considered. Even if they are active in all participating LHUs, only about the half reported regional immunization strategies, that resulted significantly related to higher VC. This limits the sharing of immunization data between LHUs and from LHUs to regional authorities, compromising the governance of immunization policies.Even though adverse events surveillance could not be strictly considered an organizational aspect, we investigated routine system used by LHUs to monitor adverse events as relevant quality and safety standard of immunization policies. Surprisingly, 30 minutes surveillance and regional vaccine-vigilance systems are not conducted in all participating LHUs and thus they should be extended and homogenised.As regards communication strategies, almost all participating LHUs reported education activities on universal HPV immunization strategy targeting health care workers. Education targets the main professionals involved in HPV vaccine counselling but the proportions are low and the interventions are conducted periodically only in the half of LHUs, highlighting the need of promoting further activities. This is of particular relevance in the field of HPV primary and secondary prevention since a variety of professionals operating in different healthcare settings are involved along a decision path where the immunization centre often represent the point of arrival. Furthermore, available evidences demonstrate the importance of the role of trusted healthcare provider in taking decisions in the field of immunization , 40–42. Furthermore, sharing a unique and coherent message among different stakeholders using available communication tools is crucial to obtain the best results in terms of adherence to HPV vaccine. As healthcare planning activities should include the phase of feedback and report, the discussion of obtained VC and the analysis of vaccine hesitancy determinants among healthcare workers were also investigated, showing a prevalence as low as 57.7% and 34.6%, respectively.The studied communications strategies directed to target subjects included the availability of informative material at immunization centres that was prepared by LHUs and regions in 50% and 42.3% of cases, respectively, and translated in other languages only in 19.2% of cases. Even if these factors are not significantly related to VC, they could contribute to the circulation of scientifically correct information and compensate the spread of misleading messages by “no-vax”. Furthermore, call centres coordinated by immunization centres to obtain information and address doubt about vaccines are available in about the half of participating LHUs. The involvement of media and the conduction of focus group in particular in the school setting could increase the vaccine demand by target when combined with other activities; nevertheless, they are reported in less than half of participating LHUs. This could be due to the suboptimal economic and human resources currently available for prevention in the majority of Italian regions.Our findings are in line with the most recent evidences on interventions aimed at improving HPV vaccine uptake –46. In pOf concerns, no participating LHUs gained VC objectives fixed by the Italian Ministry of Health, not only for the recent target represented by preadolescent boys but also for the more consolidated girls target. Furthermore, even if the PNPV 2017-2019 recommend the immunization of male who have sex with men and some regions correctly included among subjects at risk the HIV positive, the implementation of specific strategies is largely suboptimal. These observations are particularly serious from a public health perspective, given the high prevalence of HPV infection and the variety and burden of HPV-related diseases . Istituto Superiore di Sanità in order to improve the compliance to HPV vaccine and provide the regional and LHUs authorities the operational tools to increase VC [The strengths of our study are represented by the wide spectrum of organizational aspects and communication tools evaluated and the estimate of their role in determining the best outcome in terms of VC. In Italy, from 2010 to 2013 the study “local and evaluation of HPV immunization campaigns against HPV, VALORE” was coordinated by the Ministry of Health and by therease VC . VariousThe main limitations of the study are the difficulty in quantifying and exhaustively evaluating all organizational aspects of immunization centres and communication strategies and the number of participating LHUs that could compromise the representativeness of the national picture. Nevertheless, participating LHUs belong to four among the most populous Italian regions, with heterogeneous sociodemographic characteristics and healthcare systems. In particular, school-based programs were not investigating, even if robust evidences demonstrate their role in increasing the compliance to HPV vaccine. Nevertheless, the paucity of human resources in immunization centres reported in a wide proportion of participating LHUs limits the feasibility of this strategy.In conclusion, our study demonstrated that the majority of Italian LHUs implemented proved actions  concurrently with some HPV vaccine promotion and communication strategies directed to target subjects and healthcare workers. Nevertheless, VC observed in participating LHUs are largely lower than the national objectives in all HPV vaccine targets and organizational strategies to reach subjects at risk are suboptimal.Since multicomponent interventions have a synergistic effect, both organizational and educational strategies have to be implemented to improve HPV VC.