HPV-16 genotyping was performed in HPV-positive tissues and the physical status of the HPV-16 genome was determined by E2 detection. HPV was detected in 19 of 45 (42.2%) CRC cases  and in 1 of 36 (2.8%) controls  with an OR = 25.58 (95% CI 3.21 to 203.49). HPV-16 was detected in 63.2% of the HPV-positive colorectal tumors; genome integration was observed in all HPV-16 positive cases. This is the first report showing the high prevalence of HPV infections in Caribbean Hispanic colorectal tumors. Despite evidence of HPV integration into the host genome, further mechanistic analysis examining HPV oncoprotein expression and the putative role of these oncoproteins in colorectal carcinogenesis is warranted.The role of Human Papillomavirus (HPV) in colorectal carcinogenesis remains elusive. Based on the high incidence of HPV-associated malignancies among Puerto Rican Hispanics, this study aimed to assess the prevalence of HPV infection and viral integration in colorectal tissues in order to evaluate its putative role in colorectal cancer (CRC). In this case-control study, the prevalence of HPV infection in CRC (cases  HPVs are epitheliotropic, double-stranded DNA viruses that infect the squamous epithelia of mucosal cells in the skin. Viral multiplication occurs in the cell nuclei and is tightly linked to the cell's differentiation state. There are well over 100 genotypes of HPV. Types 6, 11, 40, and 42 are commonly associated with benign lesions and are classified as low-risk types. HPV-16, HPV-18, HPV-31, and HPV-45 are considered to possess high oncogenic potential and are referred to as high-risk types [Human Papillomavirus (HPV) infections are the most common sexually transmitted infections in the United States (US) . All procedures were in accordance with the ethical standards of the IRB.Cases and controls were recruited consecutively using convenience sampling and were frequency-matched by gender and age. Study participants aged 21 years or older were recruited when they visited the Puerto Rico Medical Center facilities for colonoscopies due to routine screening, symptoms, and/or referrals by gastroenterologists and colorectal surgeons. The purpose of this study was explained and all study participants gave informed consent prior to inclusion. All of the participants included in this analysis were Hispanics. Being Hispanic was defined by the participant's self-reported heritage or place of birth. All of the control subjects included in this study had normal results in their colonoscopy. Individuals with CRC had a diagnosis of adenocarcinoma confirmed by histopathology. Subjects with hereditary cancer syndromes and inflammatory bowel disease were excluded. proximal , distal (from the splenic flexure to the sigmoid colon), or rectum (last 20 cm of the colon). Control colorectal tissue biopsies were obtained from the distal colon during routine colonoscopies.Fresh frozen tissue was obtained from 45 nonfamilial, sporadic CRC patients (cases) and 36 cancer-free individuals (controls). Tissues were harvested from CRC patients during tumor resection surgery. Anal or perianal tumors and tissue samples from individuals who reported to be HIV-positive were excluded. Tumor location was classified ashttp://coloncfr.org/), clinical and sociodemographic data was collected from each patient including gender, lifestyle, medical history, and family history of cancer. The sociodemographic and clinical characteristics analyzed in the study were gender , median age (<61 years versus ≥61 years), type 2 DM diagnosis (yes versus no), family history of any cancer (yes versus no), and family history of CRC (yes versus no). Family history is defined as having a first-, second-, or third-degree relative with cancer. The lifestyle characteristics that were analyzed in this study were alcohol consumption (ever versus never) and smoking status (ever versus never).Using the Collaborative Family Registries' Colorectal Cancer Risk Factor Questionnaire  was extracted from ≈100 mg of tissue using the DNA Isolation Kit for Cells and Tissues  following the manufacturer's instructions. For quality control purposes, The following standardized precautions were taken to minimize sample-to-sample cross-contamination: all instruments and work benches were wiped down with DNAZap , followed by 10% bleach and 70% ETOH prior to sample manipulation. New, sterile blades were used for each sample. Tissue processing and nucleotide extraction were limited to a maximum of 10 samples per day. Approximately 100 mg of each specimen was randomly coded in a blinded manner and used for further analyses.μL containing 200 ng of gDNA, 12.5 μL of Bullseye HS-Taq 2x Master Mix , and 100 nmol/L of pooled PGMY09/11 primers. The following conditions were used: 15 minutes at 95°C, followed by 35 cycles of 60 seconds each at 94°C, 60°C, and 72°C, with a final extension of 10 minutes at 72°C. Two microliters of the first PCR reaction was used as the template for the nested PCR. The conditions for the nested PCR were identical to the first run PCR with the exception of the annealing temperature which was 52°C. Amplified products were electrophoresed and analyzed using a ChemiDoc . Detection of pHPV-16 purified plasmid DNA (ATCC 45113D) was used as a positive control and water as a negative control.Human HPV DNA detection was performed via a nested PCR strategy using PGMY09/PGMY11 as outer primers and GP5+/GP6+ as inner primers , 28. TheHPV-16 DNA genotyping was carried out with the type-specific primers targeting HPV-16 E6 . HPV-16En = 12). Nested PCR analysis was performed using 2 sets of specific HPV-16 primers which included 2 forward primers, Pr7581 (5′-CACTGCTTGCCAACCATTCC-3′) and Pr7677 (5′-GCC AAC GCC TTA CAT ACC G-3′), and 2 reverse primers, Pr128 (5′-GTCGCTCCTGTGGGTCCTG-3′) and Pr223 (5′-ACGTCGCAGTAACTGTTGC-3′). Both PCRs were performed under the same conditions previously described with the exception of the annealing temperature which was 60°C. Amplicons were analyzed as described. DNA extracted from CaSki (CRL-1550) and SiHa (HTB-35) human cervical cell lines were used as positive controls. The absence of PCR product was interpreted as E2 sequence disruption and integration of viral DNA into the host genome. The presence of E2 amplicons indicates the presence of episomal HPV-16 genomes.The physical status of the HPV-16 genome was determined by examining E2 sequence integrity using a previously described nested PCR strategy . All tumt-test or Mann-Whitney test to compare quantitative variables. Unconditional logistic regression models were used to estimate the OR with 95% confidence of CRC in relation to HPV status and other variables. Statistical analyses were performed using SPSS 17.0 (SPSS Inc.) and Epi InfoTM 7 (CDC).Sociodemographic, clinical, and lifestyle characteristics in cases and controls were described using frequency distributions for categorical variables and summary measures for quantitative variables. Two-sided tests were used to compare study groups: the chi-square test or Fisher's exact test was used for categorical variables and Student's n = 81; 45 CRC cases and 36 controls) are presented in The demographic and clinical characteristics of the study population  CRC samples and in 1 of the 36 (2.8%) control samples studied . HPV inf < 0.001 . The assThe mean age of the HPV-positive CRC cases was 60.3 years . No significant associations were found between HPV-positive status and the following: gender, age, use of tobacco or alcohol, history of diabetes, family history of any cancer, or family history of CRC . No signHPV-16 was detected in 12 of the 19 HPV-positive CRC samples (63.2%); the remaining 7 (36.8%) corresponded to other HPV genotypes (nontyped). HPV-16 was not detected in the HPV-positive control sample. The distributions of HPV-16 positive tumors regarding tumor location were as follows: 33.3% (4 of 12) of the samples were found in the proximal colon , 50.0% (6 of 12) were found in the distal colon , and 16.7% (2 of 12) were detected in the rectum. For non-HPV-16 tumors, 2 out of 7 (28.6%) were located in the distal colon and out 5 of 7 (71.43%) were found in the rectum. The physical status of HPV-16 DNA in CRC tissues was determined by detection of HPV-16 E2. The amplicon corresponding to the intact E2 was not detected in any of the HPV-16 positive cases indicating that the HPV-16 genome was integrated.HPV has been detected in colorectal tumors , 30–33. n = 103; 30 controls, 30 adenomas, and 43 CRC) detected HPV DNA in 27% and 69% of the adenomas and CRC, respectively [n = 37) and in 53% of the CRC (n = 70) [n = 55) and not in control samples (n = 10) from individuals in the USA [n = 75) [n = 30) [A high prevalence of HPV infection (42.2%) was detected in the tumor tissues analyzed. Only 1 of the 36 (2.8%) control samples was positive for a non-HPV-16 type. The prevalence of HPV infections in colorectal tumors found in our cohort is comparable to percentages previously reported in studies from different countries using different experimental approaches . Immunohectively . HPV typ the USA . Other s[n = 75) . However[n = 30) –41.n = 107) [HPV-16 is the most common high-risk type in the Americas , 42. HPVn = 107) . Using nn = 107) , 45, 46. L1 (450 bp). This primer combination has been found to be more type sensitive than the MY/GP+ primer sets, detecting a wider range of HPV types [The discrepancies in the literature reporting the detection of HPV and HPV-16 in colorectal tumors can be attributed to methodological differences including study design, type of tissue sample, sample size, sample collection, and differences in the sensitivity of the HPV detection or genotyping technique used. Frozen tissue samples were used because the DNA extracted is of superior quality compared to DNA from FFPE, which is degraded , 48. The L1 4 bp. ThisPV types , 34. ForPV types .HPV was detected in CRC tissues in multiple tumor samples obtained throughout the colon. HPV was detected in 21.05% of proximal tumors, 42.11% of distal tumors, and 36.84% of the tumors in the rectum. The wide anatomical distribution of HPV infections in CRC tumor tissue implies that HPV infection in CRC is not a result of a retrograde viral transmission from the anogenital area. Given these results, we cannot discard the possibility of hematologic spread, as suggested by Bodaghi et al. . There iVirus integration into the host genome is a critical step in cervical carcinogenesis. Integration of HPV in the host genome has been reported in approximately 90% of cervical carcinomas. These cases show expression of the E6 and E7 viral oncogenes . The HPVOur study has a relatively small sample size, which can be a limitation for stratified and restricted analyses. These could result in some imprecise estimates . Nevertheless, the magnitude of the observed odds ratios for the association between HPV and CRC is very high and unlikely to result from a type I statistical error. The data generated in this study in combination with reports in the literature are not yet sufficient to conclude that HPV is a causative agent of CRC according to Bradford Hill's Criteria of Causation . Five of


Hospital Emergency Codes are used in hospitals worldwide to alert staff to various emergencies. The use of codes is intended to convey essential information quickly and with minimal misunderstanding to staff, while preventing stress and panic among visitors to the hospital. A Code Blue is the term used to alert the Code Blue team (Resuscitation team) to an area where a person has had a cardiac/respiratory arrest. We in our hospital have intensive care unit personnel  assigned to attend the code blue calls.The purpose of this study is to evaluate the policy, standardize and analyze results of code blue over last 3 years of its implication in our hospital setting.A retrospective chart analysis of all code blue cases done, noting down the cause for code blue, time to reach the spot, resuscitation measures& outcome over last 3 years.We had 192 code blue calls in the study period over 3 years, with a monthly average of 4-5 calls. Nephrology patients had maximum number of calls (33.33%) followed by cardiology patients (14.35%). Most of the code blue calls (72.46%) were during off times (8PM-8AM). Bradycardia is the commonest cause (44%) of alert. Our code blue response time was 3 minutes. Return of spontaneous circulation achieved in 54.25% cases, of which 22.80% patients discharged home in conscious state.Code blue team has made a definite positive impact after its implementation in our hospital with improvement in patient outcome with passing time.


P<0.001). The positive rate of HPV 16 was parallel with the chronic inflammation degrees from mild to severe inflammation (P<0.05). The positive rate of HPV 16 was progressively improved with the malignant progression of oral mucous (P<0.05). In addition, CD11b+ LIN- HLA-DR-CD33+ MDSCs were a critical cell population that mediates inflammation response and immune suppression in HPV-positive OPSCC. These indicated that persistent chronic inflammation-related HPV infection might drive oropharyngeal carcinogenesis and MDSCs might pay an important role during this process. Thus, a combination of HPV infection and inflammation expression might become a helpful biomedical marker to predict oropharyngeal carcinogenesis.Oropharyngeal squamous cell carcinoma (OPSCC) has been known to be a highly aggressive disease associated with human papilloma virus (HPV) infection. To investigate the relationship between HPV and chronic inflammation in oropharyngeal carcinogenesis, we collected 140 oral mucous fresh specimens including 50 OPSCC patients, 50 cancer in situ, 30 precancerous lesions, and 10 normal oral mucous. Our data demonstrated that there was a significantly higher proportion of severe chronic inflammation in dysplastic epithelia in comparison with that in normal tissues ( Head and neck squamous cell carcinoma (HNSCC) includes cancers arising from the lining epithelium of the oral cavity, larynx, pharynx, and nasopharynx. High-risk human papilloma virus (HPV) has been recognized as an important risk factor in a subset of oropharyngeal squamous cell carcinomas (OPSCC) and HPV-positive OPSCC has a more favorable prognosis compared to HPV-negative OPSCC–4. The iChronic inflammation is a well-documented risk factor for carcinogenesis. The link between cancer and inflammation was first discovered by Rudolf Virchow nearly 150 years ago, on the basis of observations that a large number of leukocytes infiltrated in tumor tissues . Later oIn this present study, we investigated the relationship between HPV status and chronic inflammation in normal oral mucous, dysplasia, as well as cancer in situ and OPSCC. The results demonstrated that HPV status in normal and non- normal tissues correlated with enhanced chronic inflammation and was also associated with progression of precancerous lesions, suggesting a pivotal role of chronic inflammation in HPV-positive oropharyngeal carcinogenesis.This study was approved by the Institutional Ethics Committee of the West China College of Stomatology, Sichuan University, China.A total of 140 tissue samples and blood including 50 OPSCC patients, 50 cancer in situ, 30 precancerous lesions, and 10 normal oral mucous  were obtained from the Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University between 2012 and 2015 . Part of fresh samples with cancer, cancer in situ, precancerous lesions, and normal oral mucous were fixed, dehydrated and paraffin embedded, and their pathological features and diagnoses were verified by two pathologists with hematoxylin and eosin staining. Part of fresh samples and blood of cancer, cancer in situ, precancerous lesions, and normal oral mucous was examined by flow cytometry. Every patient signed separate informed consent forms for sampling and molecular analysis. This study was approved by the Institutional Ethics Committee of the West China College of Stomatology, Sichuan University, China.Oral mucous inflammation was assessed by counting the number of neutrophils and lymphocytes observed in microscope fields on Pap slides from each study subject. Slides were observed at × 1000 to identify neutrophils and lymphocytes by their nuclei morphology. The numbers of neutrophils and lymphocytes were counted in five nonadjacent fields, which were not contaminated by squamous epithelial cells. Importantly, to minimize bias, readings and countings of Pap slides for cytopathology were independently done (Tang YL and Jiang YP).2O) and positive control (DNA from HeLa cell line). The integrity of the PCR products was visualized by agarose gel electrophoresis. Samples found to be positive for HPV by PCR were confirmed using p16 staining by immunohistochemistry (IHC). PCR primer sequence for HPV 16 is Forward 5- CAC AGT TAT GCA CAG AGC TGC-3; Reverse 5-CATATATTCATGCAATGTAGGTGTA-3[Frozen biopsies were cut in half for parallel RNA and genomic DNA extraction with the TRIzol reagent (Invitrogen) and the QIAamp Mini Kit (Qiagen), respectively. For PCR amplification of the HPV 16, 100 ng of DNA was amplified using consensus primers HPV 16 (457-bp product). All samples were run with a negative  and treated with RNase-free DNase I (Takara) to avoid genomic DNA contamination. The complementary (c)DNA was stored at −80°C until used for PCR. PCR amplification of the cDNA template was done using Thunderbird SYBR qPCR mix (TOYOBO) on ABI PRISM 7300 sequence detection system (Applied Biosystems). The primers of IFN-γ, IL-10, IL-12 and IL-2 were used as the previous publication.Paraffin-embedded samples were serially sectioned at 4 μm and IHC of P16 and MPO was performed on 4-mm-cut representative sections by the streptavidin- peroxidase method followed as previously described . PBS was3 tissue block and put into burnisher. Then cell suspension was collected and centrifugated. To blood specimen, heparin sodium and PBS/Hanks was added into blood of individuals. Ficol was used to separate lymphocyte. Serum free medium containing 1% BSA was added into cell suspension, and then incubated on ice for 10 minutes. PE-Cy Mouse Anti-Human CD11b, FITC- Mouse Anti-Human LIN, APC Mouse Anti-Human HLA-DR, PE Mouse Anti-Human CD33 (BD Biosciences) were added to cell suspension and incubated on ice for 30 minutes. Cells were washed and resuspended in 500 mL buffer and analyzed using Flow Cytometer .Tissues were trim into 1-2mmP<0.05 was considered statistically significant.All statistical analyses were performed using SPSS 13.0 . One-way analysis of variance was used for labeling index analysis in multiple groups. The difference of labeling index between two groups was evaluated with t-test. P<0.001, P>0.05, To clarify the possible role of chronic inflammation in oropharyngeal carcinogenesis, we observed the morphology of neutrophils and lymphocytes and counted their number in microscope fields on smear slides from 140 samples including the specimens of 10 normal oral mucous, 30 dysplasia, 50 caner in situ and 50 oropharyngeal cancer . The numP<0.001, Then, to confirm the results of the cytological examination in oral mucous smear, we analyzed inflammation status in tissues with different pathological types in microscope fields. The results showed that severe chronic inflammation was more frequently observed in dysplastic tissues (75.90%), such as lichen planus and leukoplakia, compared to normal oral mucous .We have now showed the close relationship between HPV infection and chronic inflammation, as well as the relevance of inflammation and oropharyngeal carcinogenesis. Then we hypothesized that HPV infection is supposed to be evident in serous inflammation precancerous lesions and cancer tissue. To test our hypothesis, we statistically analyzed the association of HPV 16 infection with normal oral mucous specimens, dysplasia, caner in situ and oropharyngeal cancer. The result showed that the positive rate of HPV 16 progressively increased in the sequential stages from histology-normal epithelia to cancer P<0.05, , indicatP <0.05). These data confirmed that MDSCs are a critical cell population that mediates inflammation response and immune suppression in HPV-positive OPSCC patients.Myeloid-derived suppressor cells (MDSCs), previously called immature myeloid cells  or myeloP = 0.001). This indicated that HPV infection combined with inflammation might become a helpful biomedical marker for forecasting malignant transformation of oropharyngeal mucous.To investigate the accumulative effects of HPV-positive, HPV-negative, and mild inflammation, severe inflammation expression on the OPSCC progression, we divided these 50 OPSCC patients into three groups according to the number of positive markers from HPV and inflammation. The patients were scored according to the number of positive markers: 1 (one positive), 2 (two positive) and 3 (no expression of the two markers). The results of regression analysis showed that patients with over expression of HPV and inflammation (score 2) had a highest risk to suffer from OPSCC (HNSCC has continued to have poor prognosis despite the improvement of treatment with surgery, chemotherapy and radiation therapy . HoweverCarcinogenesis including OPSCC is a multi-step process involving progression from precancerous cells to cancer cells that undergo a succession of intermediate stages in order to uncontrollably proliferate, metastasize, and reach a fully malignant functional phenotype . During During many microorganisms studied, viral infections are suggested to involve in cancer development especially that was caused by HPV, which combined various inflammation cells and cytokines in tumor microenvironment to contribute to carcinogenesis, and tumors have been called as “wounds that do not heal” . The incIn addition, we also detected that there was the higher level of MDSCs in tissues with different degrees of dysplasia compared to normal mucous, as well as the higher level of MDSCs in HPV-positive OPSCC compared to normal mucous, suggesting that the aggregation of MDSCs facilitated HPV-positive oropharyngeal carcinogenesis. MDSCs are known to inhibit the process that cell-mediated immunity protects from tumor occurrence and progression , 43. TheChlamydia trachomatis could enhance the persistence of cervical HPV infection and the risk of cervical cancer. Yilmaz et al [The observed association between chronic inflammation and HPV infection may be explained through the direct effects of some microorganisms, such as bacteria. Samoff et al  have shoaz et al reported az et al  reportedaz et al , 54. Theaz et al . In addiaz et al –58.In conclusion, our present results plus the previous observations support the hypothesis that chronic inflammation and HPV infection contribute to oropharyngeal tumorigenesis and MDSCs play an important role during this process, thereby establishing a favorable microenvironment for tumor progression. Thus, HPV infection may be selected as a promising biomedical marker for indentifying potential oropharyngeal carcinogenesis in individuals with chronic inflammation.


To evaluate the HPV vaccination coverage in Boa Vista, Roraima (Brazil) and to identify personal and socioeconomic factors related to non-compliance to HPV vaccination.A school-based, cross-sectional study was conducted by distributing a self-administered questionnaire to the parents or guardians of pre-adolescent girls. The questionnaire addressed compliance to, knowledge about and perception of HPV and the HPV vaccine. Between July and November 2015, 13 private and public schools were visited based on a random cluster sampling method.p = 0.003). Most parents (60 %) incorrectly answered more than half of the questions related to HPV knowledge, and limited knowledge about HPV and the HPV vaccine correlated with lower compliance to vaccination . In the perception analysis, the belief that the HPV vaccine is important for the daughter was an important protective factor , and concern about adverse effects of the HPV vaccine was a risk factor for non-compliance . Family income, religion and education level of the parents or guardians did not correlate with compliance to vaccination.A total of 1337 questionnaires were distributed to all female students in the target age group, and 797 were completed and returned (the participation rate was 59.6 %). The vaccination coverage rate was 82.7 % and was higher among public school students than among private school students  contains supplementary material, which is available to authorized users. Cervical cancer (CC) is the second most common type of cancer among women in Brazil, following nonmelanoma skin cancer . In partThe main risk factor for the development of cervical cancer is infection with human papillomavirus (HPV), and the predominant route of transmission is sex. Approximately 12 HPV types are considered oncogenic, and types 16 and 18 are responsible for nearly 70 % of cervical cancer cases . To estaEvidence shows that the HPV vaccine is safe and effective for the prevention of HPV infection . HoweverAttaining satisfactory vaccination coverage is the greatest challenge of HPV vaccination in Brazil. According to official data, the state of Roraima has achieved a participation rate of 85.7 % for the first dose and only 49.5 % for the second dose of the HPV vaccine . The decThis was a school-based cross-sectional study that used quantitative analysis to evaluate vaccination coverage and its determinants in Boa Vista between July and November 2015.Boa Vista is the capital of the state of Roraima and is located in the Brazilian Amazonian region. Boa Vista has 265,000 inhabitants, a concentrated population representing 65 % of all inhabitants of the state. In 2014, the city had 44 public schools and 12 private schools, attended by 9,974 and 2,300 female students within the target age range (6th to 9th grade), respectively. The study population was the parents or guardians of pre-adolescent girls (between 12 and 14 years of age in 2015) who were students of middle schools in the capital city Boa Vista.http://www.random.org) to determine the order in which the schools were visited. The random sequence generation was weighted by the number of students (in the target age group) enrolled in each school.Every middle school in Boa Vista was registered and numbered. The sampling process consisted of random cluster sampling, assuming each school was a sample cluster. A sequence of random numbers was generated .The schools were visited after receiving approval by the board of each school. The researchers visited the classrooms of pre-adolescent girls who were within the target vaccination age range in 2014. After explaining the procedure, the students were asked to take a sealed envelope to their homes and to deliver it to their parents or guardians. The envelope contained a consent form and a self-administered questionnaire. The semistructured questionnaire addressed socioeconomic and demographic data in the first section . The questionnaire had been previously tested and validated with persons of different levels of education. The second section contained 6 questions that addressed the main outcome, the compliance to HPV vaccination and its circumstances. The third section consisted of 10 questions that evaluated the knowledge of the parents / guardians about HPV, cervical cancer and the HPV vaccine. The fourth and final section was composed of 8 assertive statements that assessed the parents’ perception of the HPV vaccine. The questionnaire, adapted from Ragin et al.[The primary outcome variable was non-compliance to vaccination, defined as the rate of girls between the ages of 11 and 13 in 2014 who either received at least 2 doses of the HPV vaccine (in 2014 or 2015) or received their first dose of the vaccine in early 2015. Personal and demographic data of the participating families were considered to be explanatory/descriptive variables.t test for normally distributed variables when homogeneity of the sample variance was observed. Otherwise, the Mann–Whitney test was used for this purpose. We used the chi-square test to compare differences in the proportions of categorical variables. Odds ratios (ORs) and 95 % CIs were calculated for the univariate analysis, and adjusted odds ratios (aORs) were calculated for the multivariate logistic regression analysis. The criterion used to select explanatory variables for entry in the multivariate analysis model was the threshold value of p < 0.15 in the univariate analysis. The data were tabulated and analyzed using MedCalc® software .Descriptive analysis was performed and included a frequency distribution for categorical variables and means (with standard deviation) for continuous variables. The prevalence of the outcome variable and the 95 % confidence intervals (95 % CIs) were estimated based on a binomial distribution. To compare the sample means, we used Student’s This study was approved by the Research Ethics Committee of the Universidade Federal de Roraima. None of the invited school boards refused to participate. All participants signed the informed consent term.Thirteen schools, 11 public and 2 private, were included. In these schools, all classrooms with girls within the target age range were visited and invited to participate. A total of 1,337 questionnaires were distributed, and 933 students returned the envelopes. Of these, 136 were discarded because they contained either a blank questionnaire or a blank consent form. The final sample consisted of 797 respondents (59.6 % participation rate).n = 101). The most common questionnaire respondent was the mother of the student , followed by the father . The mean age of the respondents was 39.1 years (±8.0), and most respondents had completed high school , followed by those who did not complete high school . It is noteworthy that only 24 respondents (3.1 %) declared themselves illiterate. The majority of respondents were Catholic , followed by Protestant  (Table n = 353) reported a monthly household income below I$400 , which was the minimum wage at the time of the study.The mean age of the students was 13.0 (±0.8) years. Only 12.6 % were studying in a private school  had received at least 1 dose of the HPV vaccine. Of these, 659 were considered to be adherent to vaccination . Therefore, the rate of non-compliance to HPV vaccination was 17.3 % (95 % CI: 16.8 to 17.8 %), and this group was composed of 39 never-vaccinated girls and 99 girls in the waiting period to receive the second dose of the HPV vaccine. Considering only the cases with some type of non-compliance, 50 parents (36.2 %) reported that they intended to vaccinate their daughters to rectify the delay in vaccination during the year of the study. When asked about the cause of non-vaccination, the main reasons reported were concern about the side effects (14.8 %), forgetfulness (11.8 %), lack of time due to a busy life (7.4 %), and lack of knowledge about the vaccine (7.4 %). There was no significant difference in these reasons between public and private schools.On average, the respondents correctly answered 4.5 (±2.3) out of 10 questions about the HPV vaccine and cervical cancer. The questions that were most commonly answered correctly involved the causal relationship between HPV and cervical cancer (80.9 %) and the purpose of the vaccine, which was to prevent cervical cancer (80.9 %). Few respondents knew that HPV infection is usually asymptomatic (23.5 %), the recommended age for vaccination (12.2 %) and that there is no curative treatment for HPV infection (8.3 %) . Other sociodemographic variables, such as religion, respondent education level and family income, did not correlate with the outcome variables studied. However, knowledge about the HPV vaccine and HPV correlated with compliance to HPV vaccination. Correctly answering less than 50 % of the knowledge-related questions increased the likelihood of non-compliance to vaccination by 60 % . When each knowledge-related question was analyzed individually, the following items increased non-compliance: not knowing that HPV is common , not knowing that the vaccine was effective in preventing HPV , not knowing that the purpose of the HPV vaccine is to prevent cervical cancer  and not knowing the recommended age for HPV vaccination . Table Private school students had a significantly increased rate of non-compliance to HPV vaccination compared to public school students  than those who were not concerned about adverse events . Those concerned with the quality of the vaccine freely offered by the Brazilian Unified Health System similarly showed an 80 % higher likelihood of non-vaccination . The perception that the HPV vaccine is important for the health of the student was a protective factor. Parents who agreed with the importance of the vaccine had a significantly lower rate of non-compliance than those who disagreed , reducing the likelihood of non-vaccination by approximately one-third. The other questions on perception did not correlate with the outcome variables. These analyses are presented in Table Based on analysis of the correlation between the perception of the HPV vaccine and compliance to vaccination, the respondents who were concerned about the adverse effects of the vaccine showed a 60 % higher rate of non-vaccination , followed by concern about the adverse effects of the vaccine  and having little knowledge about HPV and the HPV vaccine . In contrast, the knowledge that HPV infection is common remained as a protective factor  and the belief that the vaccine is important for the health of the student reduced the chance of non-compliance by approximately 40 % . The other selected variables were not confirmed to be factors related to vaccination  in our sample was relatively high, at 17,3 %. A similar study conducted in Japan (Fukuoka) in 2015 also presented a self-administered questionnaire to parents and guardians of 2,097 students and reported a similar rate of non-compliance to free HPV vaccination   conducteMultiple factors, such as issues related to the availability and distribution system for vaccines, cultural factors, family influences and socioeconomic factors, may influence compliance to vaccination . HoweverThis relationship between knowledge about and compliance to vaccination was also demonstrated in our study. Parents and guardians with higher performance on the knowledge questions had an increased likelihood of vaccination compliance. One aim of this study was to identify which aspects of knowledge related to HPV had a significant influence on the decision to vaccinate. In our sample, the facts with the greatest influence were knowing that HPV infection is not rare, that the HPV vaccine is effective and that its purpose is to prevent cervical cancer. In addition, the perception of parents that the vaccine is important for the health of their daughter was a protective factor for vaccination. Corroborating these results, Osis et al.  reportedIt was evident that attitudinal questions also substantially influenced this process. A portion of the respondents did not vaccinate their daughters due to forgetfulness or lack of time, which were the second and third leading causes of vaccine evasion in our study, respectively. Regarding the causes of forgetfulness, Ramos et al.  reportedStokley et al.  analyzedCompliance to vaccination was more frequent among public school students than among private school students in our sample. There may be socioeconomic differences between the parents of students from these two types of schools, and these factors may influence the acceptance of the vaccine. One study by Osis et al.  in CampiThis study has limitations. First, the cross-sectional design did not permit the use of temporality as a criterion of causality because risk factors and outcome variables were measured synchronously and because the bias of reverse-causality could not be eliminated. Second, the difference in vaccine coverage between private and public schools is difficult to explain, especially because there was no evident difference in questionnaire responses regarding the causes of non-compliance between these groups. Finally, the use of questionnaires for data collection related to risk factors may be inaccurate due to concealment of the truth and answer masking. Moreover, there is the possibility of selection bias inherent in the return of the questionnaires, which may have encouraged more of those who were vaccinated to respond. However, the achieved sample target and the random selection of schools strengthened the credibility of the results.We conclude that the rate of compliance to HPV vaccination was high in Boa Vista, Brazil. Knowledge about the HPV vaccine and cervical cancer was deficient among parents and guardians of pre-teen girls, and this deficiency negatively influenced compliance to vaccination. Attitudinal deficits and concerns about the adverse effects of the HPV vaccine were also substantial barriers to vaccination. HPV vaccination was less frequent in private schools than in public schools, and further analysis is needed to confirm and understand this relationship. Our data suggest that programs aimed at informing the poorest and richest segments of the population about HPV vaccination, including its risks and benefits, are needed to fill knowledge gaps, to eliminate distortions of perception and to attain high HPV vaccination coverage to control cervical cancer.


To examine parents’ preferences and willingness-to-pay (WTP) for HPV vaccines.A discrete choice experiment (DCE) was used. Parents with at least one daughter aged 9–13 years residing in Songkhla province were asked to choose one alternative from each DCE choice set describing HPV vaccines by four attributes, including cervical cancer risk reduction, genital warts risk reduction, common side effects, and cost. Multinomial logit model was used for data analyses.Parents preferred higher risk reductions for cervical cancer and genital warts, and lower common side effects. They valued the quadrivalent and bivalent HPV vaccines at 21,189.9 and 10,479.9 Baht, respectively. Results also showed that mothers valued both vaccines more than fathers did.Parents valued net benefits for both quadrivalent and bivalent HPV vaccines, but they were willing to pay for the quadrivalent vaccine more than for the bivalent vaccine. Genital human papillomavirus (HPV) is a sexually transmitted infection. Even though most HPV infections are transient and spontaneously eliminated by human immune system, it is a leading cause for the development of cervical cancer with high mortality in women –3. EspecRecently, WHO revealed the core principle of a comprehensive approach for cervical cancer prevention and control . Its recFor the primary prevention, two HPV vaccines, including Gardasil® and Cervarix®, have been available in Thailand since 2009. Even though WHO recommends pre-adolescent HPV vaccination and those two existing vaccines are for HPV genotype 16 and 18, which are responsible for 73.8 % of invasive cervical cancers in the country –12. All Several countries cannot afford HPV vaccines as well, but some of them receive assistance. In 2000, the Global Alliance for Vaccines and Immunization (GAVI) was formally established to help children across the globe to access vaccines. GAVI has aimed to support the vaccinations for 30 million girls in 40 countries by 2020 . After GDuring this long haul, these two HPV vaccines have been available in private insurance and out-of-pocket markets. The manufacturers of these vaccines have made some revenues from those markets, which reflected that some parents were willing to pay for the vaccines. This wiliness-to-pay (WTP) could be an explanation for the long haul. However, it has never been examined rigorously. Also, if the country understands how parents, as taxpayers, view or are willing to pay for the HPV vaccines, policy makers then can consolidate parents’ views into their own views to design better policy. Previously, there was a study that showed 41 % of parents would like their children to be vaccinated . Those pIn general, a DCE describes various choice sets of a study intervention by its attributes, e.g. efficacy, side effects, and costs. Each choice set contains various hypothetical alternatives with different attributes and levels, randomly combined by a rigorous method of DCE study design. Respondents are asked to choose one alternative that they prefer from each choice set. Finally, statistical analysis based on Random Utility Theory is used to determine the influences of attributes on respondent preference. This study followed a user’s guide published by Lancsar and Louviere .We reviewed the clinical literatures of HPV vaccines to develop a list of attributes for this study , 4, 7, 9From all possible combinations of selected attributes and levels (3x3x4X3), it was not feasible to present them to an individual respondent. An orthogonal and level balance design was used to randomly draw a subset of all combinations by using Ngene® software version 1.1.1). In this study, 36 choice sets were generated and divided into six blocks. A self-administered questionnaire was developed in Thai language. Each questionnaire contained six choice sets from each block. Therefore, we had six different questionnaires. Each choice set consisted of two alternatives describing hypothetical vaccines and a reference alternative explaining the effects of no vaccination. Figure .1.1. In In January 2014, parents with at least one daughter aged 9–13 years residing in Songkhla province were conveniently sampled from public areas where they needed to wait for some purposes, e.g. bus stations, schools, etc. This specific age range was chosen because WHO Guidance note recommended HPV vaccination for girls at these ages . PreviouIn the questionnaire, respondents received a brief description of HPV infection and its consequences. Descriptions of the choice task, attributes and levels, and an example of a completed choice set were included. Each respondent received only one questionnaire, which contained a total of seven choice sets.ij, was estimated:All data analyses were conducted for father, mother, and overall groups. Based on Random Utility Theory, respondents’ responses for each choice set were observed and analyzed in DCE . The fol0 is the constant reflecting respondents’ preference for selecting vaccination relative to no vaccination, β1, β2, β3 are the coefficients or the mean attribute weights of cervical cancer risk (Cervic), genital warts risk (Warts), side effect (SE), and cost (Cost), respectively, εij is error term. Multinomial logit model by using Nlogit® version 4 was used to estimate the utility model. The value of each coefficient indicated the relative importance of each attribute, while the sign of the coefficient reflected whether the attribute had a positive or a negative effect on utility or preference, as compared with the base level. The level of statistical significance was set at .05.where βMarginal WTPs of the attributes were calculated by taking the ratio of the mean attribute coefficient to the mean coefficient of cost attribute. Each of them represented how much one was willing to pay for a one-unit change in the attribute . KrinskyA total of 400 questionnaires were distributed to parents; 150 and 164 were returned with complete responses, including correct responses on the choice set for validity test, from fathers and mothers, respectively. All 314 responses (78.5 %) were included for data analyses. Table The results of the multinomial logit model are presented in Table p < 0.1. The positive signs of the cervical cancer and genital warts risk parameters indicated that the parents preferred vaccines with higher reduction of these risks. The negative signs of the common side effects and cost parameters reflected that they preferred lower side effects and paying less money for vaccines.All estimated coefficients had expected signs and were statistically significant in every model. The positive constant in the parent’s model implied that they generally preferred getting their children vaccinations, although fathers’ model showed the constant was significant only at only The coefficient strength of each attribute can be interpreted as its preference weight on parents’ utility when other attributes are assumed constant. According to the coefficient strengths on the same scales of the cervical cancer risk and genital warts risk, parents weighted on reducing cervical cancer risk more than reducing genital warts risk, as indicated by the higher coefficient value (0.298 VS 0.013). The results from fathers’ and mothers’ models were relatively similar with a note that fathers put slightly higher weight on the cervical cancer risk than mothers did.Table Table This study suggested that parents with daughters aged 9–13 years had different preferences for vaccines’ attributes and were willing to trade among these attributes when they chose vaccines for their daughters. The results were intuitive that parents preferred higher vaccine efficacy, lower side effect, and lower cost. Parents highly weighted on more serious efficacy, which was cervical cancer risk in this case, than less serious efficacy, which was genital warts risk. Results showed that fathers put lower weight on vaccination, as compared to no vaccination, and slightly higher weight on the cervical cancer risk, as compared to mothers did. One of the reasons could be that the proportion of fathers in this study, who knew someone who had either cervical cancer or genital warts and knew about HPV vaccine, was less than the proportion of mothers; therefore they might not value the vaccination as much as mothers did. The study result about the knowledge of fathers was consistent with the results from various studies indicating that fathers might have poor knowledge or awareness of HPV vaccine –31. AlsoThe parents’ average WTP for the quadrivalent HPV vaccine was extremely high, as compared to its current price  in Thailand. It was much higher than parents’ WTP for the HPV vaccine in two previous studies (about 500–3000 Baht for three doses) , 17. TheCompared to other vaccines and even the prices of other new and expensive vaccines, e.g. invasive pneumococcal disease vaccine , the parents’ WTP for the quadrivalent HPV vaccine was still higher. Their WTP for the bivalent HPV vaccine was also relatively high, but closer to the vaccine’s current price in the country. This might be partially accounted for by the fact that cervical cancer in general is a dreaded and fatal disease . Also, pCompared to Vietnamese mothers’ WTP  for the vaccine concerning only cervical cancer risk reduction and efficacy period , which wIn Thailand, there has been a substantial effort and debate to include the HPV vaccines in the EPI. Even though currently the vaccine prices are approximately the cost-effective prices from the economic evaluation studies, they still have large impact on health care budget. Also, all screenings are still effective alternatives for cervical cancer prevention. In 2012, when the Ministry of Public Health proposed to pay for 500 Baht per dose, HITAP suggested that the cost-effective price of HPV vaccine should not exceed 190 Baht per dose . CertainOnes must “not” interpret the WTP calculated in this study as an appropriate or reasonable market price. Instead, using the same explanation as discussed in a previous study , as a siThis study suffered from some limitations. First, even though DCE used in this study is a state-of-the-art stated preference method, ones may always argue that it does not reveal or reflect true preference or value since all decisions are not really made. However, this study already tried to minimize validity threats as many as possible, e.g. providing an opt-out alternative in each choice set. Second, all choice sets comprised only limited number of vaccine attributes from literatures. There were other attributes that could also affect parents’ preferences. Further research should adopt qualitative interviews to focus on parents’ views on the vaccine attributes. Third, the inclusion of the low risk reduction of cervical cancer might be too difficult for parents to understand its real meaning. Finally, this study examined preferences among parents in a Southern province in Thailand, the results could not be directly generalized to parents in the whole country since they might have different socioeconomic characteristics, especially income and education. For instance, the country’s average household income was approximately 25,000 Baht , which wThis study revealed that parents weighted on the risk reduction of cervical cancer and common side effects more than they did on the risk reduction of genital warts when they chose their preferred HPV vaccine. They valued net benefits for both quadrivalent and bivalent vaccines, but they were willing to pay for the quadrivalent vaccine more than for the bivalent HPV vaccine due to the additional benefit against genital warts.


A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied. Recurrent Respiratory Papillomatosis (RRP) is characterized by the proliferation of multiple papillomas within the respiratory tract affecting especially the larynx ,2. RRP cL1 gene [The prototype HPV-6b clone was originally isolated from a condyloma acuminatum specimen . BecauseL1 gene . RecentlL1 gene . While tL1 gene .The viral long control region (LCR) comprises approximately 10% of the viral genome and encloses cis-regulatory elements for cellular and viral transcription factors (TFs) that modulate early gene expression and replication . The repHPVs 16 and 18 intratype variability has been epidemiologically correlated to viral persistence and development of clinically relevant lesions –18. ConcWe analyzed 23 laryngeal papillomas biopsy specimens from 13 patients diagnosed with RRP from 2005 to 2010, and treated at the Laryngology Clinic of the School of Medicine, University of São Paulo, Ribeirão Preto, Brazil. Disease severity was accessed using the Derkay system at each surgical intervention . This sthttp://trap.molgen.mpg.de/cgi-bin/home.cgi) using the TRANSFAC database [DNA was extracted using the QIAamp DNA Micro kit (Qiagen Inc.). The complete HPV-6 LCR (nt 7292 to 101) was amplified in two independent reactions. Amplicons were cloned using the pCR XL-TOPO Vector (Invitrogen), and clones were purified using the GeneJET Plasmid Miniprep Kit (Fermentas Life Sciences). Sequencing reactions were performed in an ABI 3130XL sequencer (Applied Biosystems) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Variants were identified by alignment of sequences with the references of HPV-6b (GenBank n° X00203), HPV-6a (GenBank n° L41216) and HPV-6vc (GenBank n° AF092932) using CLUSTALW [database .E6/E7 or pLXSN-HPV-16-E6/E7 were maintained in keratinocyte serum free medium (KSFM), supplemented with 5ng/mL epidermal growth factor and 50μg/mL bovine pituitary extract (Invitrogen).C33A (ATCC HTB-31) and C33A cells permanently transfected with pGL3-HPV-6-LCR were grown in Dulbecco’s Modified Eagle medium supplemented with 10% fetal calf serum and antibiotics. Primary human foreskin keratinocytes (PHFK) (Clonetics), and PHFK infected with pLXSN-HPV-6b-6 C33A cells were plated in 10cm diameter dishes. Cells were co-transfected using Lipofectamine (Invitrogen) with 4μg of pGL3-LCR-Luc plasmids and 1μg of pCMV-βGal vector for internal control of transfection efficiency [The complete LCR from the different HPV-6 variants was cloned upstream the luciferase gene in the pGL3-Basic vector (Promega). Accurate construction of all plasmids was confirmed by sequencing. Approximately 24 hours before transfection, 4x10ficiency . ExtractC33A cells were co-transfected in 96-well plates using Fugene (Promega) with 100ng of pGL3-LCR-Luc plasmids of different variants of HPV-6 together with increasing amounts of selected TFs expression plasmids , and 50ng of pCMV-β-Gal vector. Cells were harvested 48 hours after transfection by addition of 100μL of 1X Reporter Lysis Buffer (Promega). Relative luciferase activity was normalized to β-galactosidase measurements. Transcriptional activity was compared to basal activity of the same variant with no TFs superexpressed. Averages and standard deviations are based on the mean of triplicates of at least twelve independent experiments.Fisher´s exact test was used to compare HPV-6 variant distribution between JORRP and AORRP cases. We used T-test to compare Derkay mean values with 95% confidence interval (CI) between HPV-6a and -6vc-related RRP cases. These analyses were conducted using the GraphPad Prism statistical package (version 5.01). Kruskal-Wallis and Tukey’s tests were employed to compare LCR activity among HPV-6 variants. For TFs validation assays, one-way analysis of variance and Tukey’s multiple comparison tests were performed using the SPSS statistical package version 20.0. P< 0.05 was considered significant for all tests.5 cells were incubated with 20μg of anti-GATA1 (Abcam), anti-ELF1 (Santa Cruz Biotechnology), or anti-FOXA1 (Santa Cruz Biotechnology) overnight at 4°C with moderate agitation. Recovered DNA was subjected to PCR using specific primers surrounding putative TFs binding sites.ChIP assays were perfomed using the MAGnify Chromatin Immunoprecipitation System (Invitrogen). Briefly, C33A cells permanently transfected with pGL3-HPV-6-LCR from different variants were cross-linked with methanol-free formaldehyde (Polyscience) and further sonicated using a Sonic Dismembrator Model 100 (Fisher Scientific). Production of DNA fragments of 100-500bp was checked by gel electrophoresis in agarose 2%. Approximately 1x10Cells were washed with ice-cold phosphate-buffered saline (PBS), scraped, and centrifuged. Radio immunoprecipitation assay (RIPA) buffer  containing complete protease inhibitor cocktail (Roche) was used for protein extraction. Eighty micrograms of each lysate were submitted to 12% SDS polyacrylamide gel electrophoresis, and further transferred to nylon membranes (Hybond). Incubations with anti-GATA1 (1:500), anti-ELF1 (1:200), anti-FOXA1 (1:500), or anti-tubulin (1:1.000) (Sigma) were conducted overnight in PBS-T  at 4°C with 5% milk. We followed incubation with anti-HRP-conjugated antirabbit secondary antibody (1:5.000)  for 1 hour at room temperature. Proteins were visualized using the Amersham ECL Western Blotting Detection Reagents  in a ImageQuant LAS4.000 equipment , and quantified using the ImageQuant TL software .We sequenced the LCR of 23 HPV-6 RRP specimens obtained from 13 patients. Individuals with more than one biopsy harbored identical HPV genomes. We detected 7 different HPV-6 variants .Maximum genomic distance was 7 mutations between HPV-6a-ref and -6vc-ref (2 insertions and 5 substitutions). HPV-6a-var1, -6a-var2, -6vc-var1, -6vc-var2 and -6vc-var3 have unique substitutions at nucleotide positions 16 (G→A), 7320 (A→G), 7626 (T→G), 7978 (C→T) and 7681 (A→G), respectively. We detected HPV-6a-related sequences  in 32.5% (4/13) patients. Additionally, 67.5% (9/13) individuals contained HPV-6vc-related sequences , of which HPV-6vc-ref was the most prevalent.Age at RRP diagnosis ranged from newborn to 51 years old. All JORRP cases harbored HPV-6vc-related variants, whereas most AORRP specimens had HPV-6a-related variants  (p = 0.02) . AverageLuciferase assays were used to access the impact of LCR nucleotide sequence heterogeneity on early viral transcription. Unfortunately, we were unable to construct a recombinant plasmid containing the HPV-6a-var2 LCR. We observed an 11.5 fold enhanced promoter activity for HPV-6vc-ref variant as compared to HPV-6a-ref. A transition at nucleotide position 16 detected solely in HPV-6a-var1 led to an increase in transcriptional activity similar to that of the HPV-6vc-ref, whereas the transversion at position 7630 inherent of the HPV-6vc-var1 abolished the increased activity observed for HPV-6-vc-ref. HPV-6vc-var2 and -6vc-var3 showed promoter activities similar to HPV-6vc-ref Search for putative TFs binding sites was restricted to nucleotide positions divergent among HPV-6a-ref, -6a-var1, -6vc-ref, -6vc-var1 because these isolates showed differences in transcription. This approach revealed that nucleotide heterogeneity of HPV-6 variants may impact on binding of different TFs including FOXA1, GATA1 and ELF1 .The substitution in nucleotide position 16 (G→A) detected in HPV-6a-var1 leads to the elimination of putative binding sites for GATA1 and ELF1 as compared to the HPV-6a-ref . We obseDivergence in nucleotide position 7626 (T→G) between HPV-6vc-ref and HPV-6vc-var1 results in the replacement of a putative ELF1 for a FOXA1 binding site as suggested by computational analysis . ELF1 inE6/E7 from both HPV-16 and HPV-6b  ELF1, (B) GATA1 and (C) ELF1 levels in primary human foreskin keratinocytes (PHFK), and PHFK infected with pLXSN-HPV-6b-E6/E7 or pLXSN-HPV-16-E6/E7. One representative experiment from two is shown.(TIFF)Click here for additional data file.


INK4a expression as a marker of viral infection is controversial in HNSCC. We evaluated HPV types and HPV-16 variants prevalence, and p16INK4a expression in HNSCC specimens provided by two different Institutions in São Paulo.Human papillomavirus (HPV) prevalence in head and neck squamous cell carcinomas (HNSCC) diverges geographically. The reliability of using p16INK4a protein levels by immunohistochemistry.HPV DNA from formalin-fixed specimens was accessed by Inno-LiPA, HPV-16 variants by PCR-sequencing, and p16INK4a was more common among HPV positive tumors.Overall, HPV DNA was detected among 19.4 % of the specimens (36/186). Viral prevalence was higher in the oral cavity  then in other anatomical sites  when samples from both Institutions were analyzed together. HPV prevalence was also higher in the oral cavity when samples from both Institutions were analyzed separately. HPV-16 was the most prevalent type identified in 69.5 % of the HPV positive smaples and specimens were assigned into Asian-American (57.2 %) or European (42.8 %) phylogenetic branches. High expression of p16Our results support a role for HPV-16 in a subset of HNSCC. Worldwide head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer with an estimated annual incidence of approximately 600,000 cases . HNSCC cAlthough alcohol and tobacco consumption is the leading predisposing risk factor for HNSCC development , there aL1 gene, HPV-16 molecular variants are clustered in five branches of phylogenetic and geographical relatedness: European (E), Asian-American (AA), Asian (As), African-1 (Af-1) and African-2 (Af-2) [HPV-16 intratype nucleotide heterogeneity has been extensively studied . Based o2 (Af-2) , 10. The2 (Af-2) –13.INK4 overexpression. In the cervix, p16INK4a upregulation is being used as an indirect measure of oncogenically active HPV infection [INK4a has also been used as surrogate marker of biologically relevant viral infections in the oropharynx [INK4a protein levels in these samples.The detection of HPV DNA in HNSCC is not a sufficient proof for causal viral association. High-risk HPV E7 inactivates the retinoblastoma protein, thus promoting p16nfection . Althougopharynx , for othInclusion criteria consisted of histological confirmation of HNSCC, information about the year of diagnosis and no treatment prior to surgical procedure. Formalin-fixed Paraffin Embedded (FFPE) tumor specimens from 96 HNSCC patients diagnosed between 1991 and 2010 were retrieved from the pathology archives of the Santa Casa de Sao Paulo, School of Medicine (FCMSCSP). Further, 109 FFPE HNSCC specimens from patients treated at the Cancer Institute of São Paulo (ICESP) from 2009 to 2012 were included in this study. Samples were selected from the pathology services of both institutions following a chronological sequence, availability and quality of specimen . All sections were reviewed by a pathologist of each participant Institution. Based on anatomical subsite localization of the tumor, specimens were categorized as oral cavity , oropharynx , and larynx, according to The International Classification of Diseases criteria of the World Health Organization. The ethics committees of both institutions approved all study procedures.About ten 6 μm paraffin sections were obtained; the first and last sections were submitted to histological analysis, while the remaining was submitted to DNA isolation. After removing paraffin with xylene, tissues were digested with proteinase K 1 mg/mL-SDS 0.1 % for 24 h. DNA was obtained by organic extraction: after addition of Phenol/Chloroform/Isoamyl Alcohol at 25:24:1 followed by vigorous homogenization and centrifugation, DNA precipation was conducted with ethanol 100 %. The DNA pellet was dried and was dissolved in 100 μL of TE. DNA concentration was determined with a Nanodrop 2000 spectrophotometer . Samples were diluted to 50 ng/uL and 2 μl were used in each PCR. DNA quality was assessed by amplification of a 110 bp fragment of the human β-globin gene using PCO3/PCO4 primers followed by analysis in 8 % acrylamide gel electrophoresis . The InnE6 gene (nt 266–374)  were amplified using AmpliTaq Gold polymerase . PCR products were cloned with TOPO TA Cloning® kit for Sequencing . Sequencing was conducted in a PRISM® 3100 Genetic Analyser  using the BigDye Terminator v3.1 Cycle Sequencing kit . Sequences alignment was achieved using Bioedit 7.2.5, and HPV-16 variants were defined as previously described [A 193 bp segment of the LCR (nt 7744–33)  and/or a 108 bp fragment of the INK4a was performed using the CINtec® P16INK4a Histology Detection kit  in a Ventana Benchmark GX equipment  according to the manufacturer's instructions. Staining of the samples from both institutions (FCMSCSP and ICESP) were analyzed by the same pathologist at ICESP (S.F.F). Expression of p16INK4a was scored as high (≥75 % of cells stained), low (<75 % of cells stained) or negative (no staining). Diverse studies use this scoring system for defining p16INK4a as positive in oropharyngeal SCC staining [Immunohistochemistry for p16 staining .P ≤ 0.05) and SPSS® version 17.0  was used.Qualitative data were expressed as absolute numbers and relative rates with 95 % confidence interval (95 % CI), and quantitative data as means, medians and range. The Chi-square test was used to compare phenomena between qualitative variables. For all analysis the probability of a making an α or type I error was equal to or less than 5 % (n = 17); remaining 94 patients from each institution (FCMSCSP and ICESP). The median age of patients from FCMSCSP was 58 years, ranging from 39 to 87 years, and most individuals were male (86 %). Among specimens from ICESP, median age was 61.5 years with a range of 43 to 91 years , followed by the oral cavity  and the oropharynx . For two samples (2.1 %) the anatomic site could not be defined due to the extension of the tumor, and both specimens were also excluded from analysis. Most cases from ICESP were derived from the oral cavity , followed by the oropharynx  and the larynx .Concerning samples obtained from FCMSCSP, most tumors were from the larynx (P > 0.05). For both series of cases, HPV-16 was the most commonly detected viral type, identified in 69.5 % (25/36) of the HPV positive specimens . When the two cohorts were analyzed together, overall HPV DNA prevalence was 19.4 % (36/186); HPV prevalence was higher in the oral cavity , and similar viral DNA rates were observed in the oropharynx  and the larynx . HPV prevalence was also the highest in the oral cavity when samples from both institutions were analyzed separately: 18.2 % and 27.2 % of the samples from FCMSCSP and ICESP were PCR positive, respectively  and 9 (42.8 %) of the specimens, respectively  was more common among HPV positive specimens.It was possible to evaluate p16Since the early 1980s morphological and immunohistochemical evidence points towards the involvement of HPV in oral SCC , 21. ActReported detection rates of HPV DNA in HNSCC worldwide vary considerably, ranging from 0 % to 100 % in the oropharynx . NeverthConsistent with all studies conducted worldwide HPV-16 was the most prevalent viral type detected independently of the HNSCC anatomical subsite, and was identified in 69.5 % 25/36) of the HPV positive tumors. We and others have reported that in the uterine cervix non-European variants are linked to a higher oncogenic potential , 34, and of the HINK4a may also derive from non-viral alterations [INK4a high expression (>75 % cells stained) was more commonly observed in HPV positive tumors, especially among oropharyngeal samples for which 75 % of HPV positive samples overexpressed this protein.Only the detection of HPV DNA in HNSCC biopsies is not enough evidence of tumor causation and other parameters should be analyzed to prove the pathogenic role of viral infection. Even though overexpression of p16erations , it has erations . The useerations ; howevererations . We obseIn summary, we observe that in our population overall HPV prevalence is lower than reported in developed countries, even though HPV-16 was the most prevalent viral type as observed in all studies conducted worldwide. HPV-16 variant distribution detected in our samples seemed to replicate data of the uterine cervix in our population. However, the role of HPV-16 intratypic variability in HNSCC development deserves further analysis. Nevertheless, the fact that HPV16 accounted for almost all HPV positive HNSCC samples indicates that developed prophylactic vaccines for cervical cancer could also be relevant for HNSCC prevention in our population.


Underground pipelines are widely applied in the so-called lifeline engineerings. It shows according to seismic surveys that the damage from soil liquefaction to underground pipelines was the most serious, whose failures were mainly in the form of pipeline uplifting. In the present study, dynamic centrifuge model tests were conducted to study the uplifting behaviors of shallow-buried pipeline subjected to seismic vibration in liquefied sites. The uplifting mechanism was discussed through the responses of the pore water pressure and earth pressure around the pipeline. Additionally, the analysis of force, which the pipeline was subjected to before and during vibration, was introduced and proved to be reasonable by the comparison of the measured and the calculated results. The uplifting behavior of pipe is the combination effects of multiple forces, and is highly dependent on the excess pore pressure. Pipelines are the artery of modern industries and urban life, widely used in lifeline engineerings such as water supply, electricity supply, natural gas transportation line, and communication cables. According to a large number of seismic disaster surveys , 2, the There have been quite a lot of studies concentrating on the “uplifting” phenomenon of pipelines during soil liquefaction processes. The factors that affect the stress and deformation of pipelines during floating; for example, the buried depth, diameter, thickness, stiffness of the pipelines, the type, liquefied area, stiffness, and strength of soil, have been studied , 8. It iFBuoyancy = ρsatgV, where the saturated soil is considered as fluidized material having a unit weight equivalent to its saturated unit weight [Despite that the above mentioned studies have investigated the pipeline uplifting behaviors in liquefied fields with respect to influencing factors involving the pipe itself and the soil properties, through a comprehensive means of field investigations, numerical simulations, and model tests, there still exists disagreement on the understanding of the mechanism of pipeline uplifting. Some , 14 belit weight –18. It it weight .It is significantly favorable that researches are made on the stress conditions of the pipelines during uplifting for an in-depth understanding of the mechanism of pipe uplifting behaviors. In the present study, dynamic centrifuge model tests were conducted to investigate the mechanism of pipeline uplifting phenomenon during soil liquefaction. Based on the measurements of acceleration, excess pore pressure and earth pressure around the pipelines, the forces on the pipelines before and during soil liquefaction were estimated, and the mechanism and the main influencing factors of pipeline uplifting were analyzed.The tests are conducted on the ZJU400 centrifuge with a shaking table, shown in A Rigid container was used to prepare the model, whose inner dimension is 0.6 m (length) × 0.4 m (width) × 0.5 m (height), and its front perspex made window is convenient for direct observation of the experimental phenomena. A 25 mm thick piece of mouldable Duxseal was placed on each side of the container to reduce reflecting incident stress waves by at least 65% .3, a length of 390 mm, and an inner and outer diameter of 36 mm and 40 mm, respectively. And they are used to simulate large diameter pipes like oil or gas pipelines. Each end of the pipes was sealed by a perspex disc with PTFE, and petroleum jelly was also used to reduce end friction. Microearth pressure transducers were installed on the bottom, side and crown of pipes to measure earth pressures .The model pipes are made of aluminum tube, with a density of 2.7 g/cmTwo model pipes were buried in the ground. One was used for measuring the uplift displacement, named pipe 1#; the other was used for measuring the stabilizing force during soil liquefaction, named pipe 2#. Pipe 1# could move freely during tests. Pipe 2# was installed to the rigid container through a connecting rod. The force, provided by the connecting rod to keep pipe 2# stable in the vertical direction, was defined as stabilizing force. In order to acquire the stabilizing force of pipe in the centrifuge, a load cell was installed on the connecting rod.In order to measure vertical displacements of underground structures in the centrifuge, draw-wire displacement potentiometers are commonly used . HoweverA simple device of a connecting rod with ball joint was developed, as shown in D50) 0.16 mm; the uneven coefficient (Cu) and the curvature coefficient (Cc) are 1.6 and 0.95, respectively. The maximum and the minimum void ratios are 0.96 and 0.61, respectively. The model foundation was prepared by pluviation method. The sand was rained from a sieve in a hopper into the container, where the falling height of the hopper and the shape of the sieve were kept unchanged based on the precalibrated results to obtain a constant relative density. The designed relative density of the two tests was 60%. The heights of the model foundation were 29 cm and 32 cm in test 1 and test 2, respectively.Fujian standard sand, which is widely used in China for geotechnical physical modeling tests , 24, wasn, and the latter is 1/n2. To solve the problem, viscous fluid was introduced to reduce the permeability of soil. Methyl cellulose fluid, which is commonly used in geotechnical centrifuge modeling tests, has similar compressibility and density to water [There is a conflict between dynamic and permeability time scale, for the former is 1/to water . Further2 and 153 cm/s2, respectively.Three types of excitation waves were adopted, that is, EL-Centro wave, Taft wave, and Zhejiang seism wave. El-Centro wave was recorded in the Imperial Valley earthquake of California in 1940, with a primary period of 0.5 s, belonging to near earthquake. Taft wave was recorded in the earthquake happened in Kern of California in 1952, with a primary period of 0.5 s, belonging to distant earthquake. These two waves are commonly used. Zhejiang seism wave is an artificial seismic wave suited the seismic zoning type of Zhejiang Province in China, with a 10 s duration. Assuming the exceeding probability of Zhejiang seism wave to be 10% and 2%, the maximum acceleration is 63.1 cm/sD) for test 1 and 80 mm  for test 2. Accelerometers, pore pressure transducers, earth pressure transducers, potentiometers, and load cell were used in the tests [In the present study, two centrifuge tests were conducted on pipes of different buried depths under the same ground conditions. The centrifuge accelerations were both 30 g. The buried depths, measured from the top of pipes to the ground surface, were 20 mm  when pipe 1# began to move up were shown in Actually, the maximum uplift displacement was not present right after the vibration stopped each time. It can be seen in The stabilizing force, which kept the pipe 2# stable in the vertical direction, was measured by a load cell fixed on the device. The variation of stabilizing force in test 1 under El-Centro wave with different amplitudes is shown in The force components acting on pipes were investigated with the results obtained from centrifuge tests in this section. These components were adapted from static analysis to a dynamic condition where soil liquefaction occurs. Furthermore, the force analysis was validated by the comparison between the measured and calculated data.L and Wp are the length and weight of the pipe, respectively, which can be obtained according to scaling principle from the length and density of pipe in 1 g condition. T is the stabilizing force of pipe 2# which can be measured by load cells (T of pipe 1# is 0). Ws is the effective weight of overlying soil; N is the support force from the soil underlying the pipe; u1 and u2 are pore pressures around the pipe. The total force of these three parts can be calculated by the integral of the earth pressure difference between the upper and the bottom of pipe. It can be written asP denoted the integral of earth pressure difference, which can be obtained by the interpolation according to the earth pressure transducers distributed on the pipes, defined as dD · L is the integral of excess pore pressure during soil liquefaction, labelled as ΔU. ΔWs and ΔN are the increments of support force of soil underlying the pipe and the effective weight of soil overlying the pipe, respectively, due to the flowing deformation of soil around the pipe during vibration [P.As pipe 2# was fixed to the rigid container, no displacement in the vertical direction occurred during vibration. And consequently the shear strength of soil could not excite. The force state of pipe 2# during vibration is shown in ∫−D/2D/2u′dD·L+Wp T′+Ws′+∫−ibration . The thrracting  · Vpipe, which is commonly adopted by other researchers to calculate the buoyancy force, is given in u1′ = γsatf(h1), and the value on the bottom of the pipe is u2′ = γsatf(h2)). Integrating the difference between u1′and u2′ givesThe value of  · Vpipe for fully liquefied ground. The value of dynamic buoyancy force will be overestimated for incompletely liquefied soil by  represents the corresponding forces or stresses during vibration of pipe 1#. Subtracting dD · L is dynamic buoyancy force during soil liquefaction, labelled as Δ*U. Δ*Ws and Δ*N are the increments of support force of soil underlying pipe 1# and the effective weight of soil overlying pipe 1#, respectively, due to the excess pore pressure variation induced flowing deformation of soil around the pipe during vibration. As the different motion patterns of pipe 1# and pipe 2#, the stresses measured by the transducers around them are different. The total force of the three parts can also be calculated by the integral of earth pressure around pipe 1#. All the forces in (Fs.The force state of pipe 1# during uplifting is shown in Fs+Ws′′+∫racting (mpa=Fs+Δ∗Fs from the shear plane is estimated using the equation introduced by DNV (2007) [σH′ refers to the effective vertical stress of the overlying soil. Before vibration, σH0′ = γ′H. fp is a parameter related to the soil property ranging from 0.4 to 0.6 for medium dense sand.The frictional force V (2007)  as folloa) can be obtained. And acceleration time-history is shown in Given the relationship between the degree of liquefaction and the frictional contact between the soil grains, vertical effective stress declines linearly with the increase of excess pore pressure. Considertituting  and 11)(11)σH′=σtituting , the uplaeff). And effective vertical acceleration time-history is illustrated in As the behavior of pipe in liquefied soil is extremely complicated, a few assumptions are made in the following to calculate the displacement of pipe. The uplifting of the pipe 1# occurs as soon as the value of the uplift acceleration is greater than zero. Zero is deemed as the yield acceleration. The movement of pipe occurs when its acceleration exceeds the yield acceleration, which is in accordance with Newmark's analysis. The excess in acceleration above yield acceleration is termed as effective vertical acceleration (aeff twice. The uplifting displacement time history of the pipe 1# calculated under 0.4 g Taft wave in test 1 is shown in Based on the assumptions above, the accumulated uplifting displacement can be obtained by integrating the effective vertical acceleration Although the uplifting phenomenon of pipelines in the liquefied soils always happens during the seismic vibration, the observation in our tests shows the begin and end time point of uplifting is not directly related to the seismic motion. The uplifting is highly dependent on the buildup of the excess pore pressure. Moreover, the quantitative relationship between the uplifting behavior and the generation of the excess pore pressure needs further studies.The uplifting movement of pipe is the combination effects of multiple forces. During seismic vibration, excess pore pressure generates and soil around the pipeline gradually flow in an oval-like trace, which causes both the variation of effective weight of overlying soil and supporting force of soil underlying the pipeline, as well as the shear resistance from shear planes that varies with the degree of liquefaction. As a result, the equilibrium of pipeline during shaking is broken and the pipe consequently uplifts. However, in most existing research, the variations of the overlying soil weight and the supporting force of the underlying soil are ignored.For incompletely liquefied field, the buoyancy force is overestimated by multiplying the saturated unit weight of soil and pipeline volume.The uplifting behavior of shallow buried pipe in liquefied field was investigated through dynamic centrifuge model tests in the present study, and the main conclusions of the research are summarized as follows.


P < 0.001). In conclusion, HPV infections may contribute to the risk of prostate cancer.Despite the increasing number of studies conducted recently to evaluate the association between HPV infections and the risk of prostate cancer, the results remain inconclusive. Furthermore, the prevalence and distribution of overall and individual HPV types worldwide in prostate cancer has not been reported until now. Therefore, we estimated the prevalence of HPV in prostate cancer by pooling data of 46 studies with 4919 prostate cancer cases, taking into account the heterogeneity of major related parameters, including study region, specimen type, HPV DNA source, detection method, publication calendar period and Gleason score. Moreover, we tested the association of HPV infections with prostate cancer risks by a meta-analysis of 26 tissue-based case-control studies. We found that the prevalence of HPV infection was 18.93% (95% CI = 17.84–20.05%) in prostate cancer cases, and most of which were high-risk HPV types . The prevalence varied by region, PCR primers used, publication calendar period and Gleason score. Our study also showed a significantly increased risk of prostate cancer with the positivity of overall HPV detected in prostate tissues  and revealed the geographic variation of association strength ( Human papillomavirus (HPV) is most commonly transmitted through sexual activity. Carcinogenetic types of HPV, such as HPV 16 and 18, have been proved to be a necessary cause of invasive cervical canceret al. reported men with anal cancer, a disease that has been associated with HPV, had an increased risk for developing subsequent prostate cancerStudies have found positive associations of prostate cancer with sexual activities and sexually transmitted diseasesWe retrieved published data on HPV prevalence and combined individual studies on the association between HPV infections and prostate cancer risks through a meta-analysis. We further evaluated how influential parameters  affected the results from the meta-analysis.Overall, 46578910111213141516171819202122232425262728293031323334353637383940414243444546474849508910111215161720222324252627282930313334353637384446474849505714181921323941421340571314A9/A7 = 2.94, 95% CI = 2.55–3.38 and OR A9/A10 = 5.83, 95% CI = 3.78–9.43, respectively). The 5 most common high-risk HPV types identified, in order of decreasing prevalence, were HPV16 , HPV31 , HPV33 , HPV18  and HPV58 . HPV11 and HPV6 were also common in prostate cancer with the prevalence of 2.34% (95% CI = 1.29–3.90%) and 1.02% (95% CI = 0.28–2.60%)  . Further8–2.60%) .After stratified by study region, we found that overall HPV prevalence in prostate cancer cases was highest in Africa  with only one study, followed by Asia  and Latin America  . The HPVin situ hybridization (ISH), immunohistochemistry and HC2, were used. HPV prevalence was 17.72% (95% CI = 15.84–19.72%) when PCR-based method was used but only 5.36% (95% CI = 1.12–14.87%) when non-PCR-based method was used. Further analysis showed that the type-specific PCR primers were generally demonstrated to be more efficient with a detection rate of 27.34% (95% CI = 22.28–32.86%)  and fixed tissues   . With re–32.86%) . Meanwhi–32.86%) .Thirty eight case-control studies891012131617181920212223242526272829303235363738394041424344454647484950181921323941428910121316172022232425262728293035363738444647484950401625408910121317181920212223242627282930323536373839414243444546474849181921323941428910121317202223242627282930353637384449Q = 73.87, P<0.001). However, the statistically significant association was absent for studies based on sera  while persisted in studies based on tissues . Hence, the following analyses focused on 26 studies8910121317202223242627282930353637384449Q = 56.29, P < 0.001), the random-effect model was chosen to evaluate the pooled OR . In subgroup analysis, we found the geographic heterogeneity of the magnitude of association estimates between overall HPV infections and prostate cancer risks (P for between-strata heterogeneity < 0.001) and revealed that the increased risk of prostate cancer with HPV infections was evident in Europe , Asia  and Latin America . However, there was no significant heterogeneity for OR estimates across the other subgroups.Overall, infection of any type of HPV was significantly associated with an increased risk of prostate cancer  with significant between-studies heterogeneity  in further analyses that have never been shown in the previous studies.In general, the prevalence of overall and individual HPV types was higher when detected in sera than in tissues except HPV 16. The distribution of individual types also varied despite bio-samples used in HPV detection . This suL1 gene is adopted, HPV type-specific primers are usually designed to amplify shorter sequences of HPV DNA and might be more sensitive to detect HPV DNA sequencesFurther analysis focused on cancer tissues showed that PCR-based method with the type-specific primer was more suitable in HPV DNA detection in prostate cancer cases. Comparing with the low detection rate of HPV DNA when PCR amplification of long DNA fragments in the Our study also suggested a moderate geographical variation in HPV prevalence and association strengths with prostate cancer. Meta-analyses on the prevalence of cervical HPV DNA worldwide showed a higher HPV detection rate in Africa, and a lower prevalence in North America and EuropeThe prevalence of HPV was higher  in studies published in 2000–2015  than those in 1990–1999 . The increased prevalence is expected and it is primarily due to improved HPV detection protocols. Since the majority of histological type of prostate cancer is adenocarcinoma, it has been hypothesized that HPV 18 to be more predominant than HPV 16 as that in cervical adenocarcinoma1313In addition to the prostate, HPV are also discussed in the pathogenesis of anogenital and urinary cancers, including penis, anus, bladder, testis and renal cancer. HPV appears not to play a major causative role in renal60Very limited studies exploring the association between HPV infections and main clinical features of prostate cancer, e.g., cancer types and prostate-specific antigen (PSA) levels, made it difficult to do further sub-analysis by these features in the present meta-analysis. Most of the cancer cases were adenocarcinoma. Only one study24The mechanism of HPV infections and prostate cancer development is far from clear. It has been proposed that exposure to environmental factors such as infectious agents and dietary carcinogens, and hormonal imbalances lead to injury of the prostate gland and to the development of chronic inflammation and regenerative ‘risk factor’ lesions, referred to as proliferative inflammatory atrophy (PIA)66Until today, prostate cancer is not clearly linked to any preventable risk factorsGenerally, the present study suggested the link between HPV infections and prostate cancer, though the risk estimates varied by study region. Furthermore, as we are aware, it is the first study to summarize the HPV prevalence and the distribution of individual oncogenic types worldwide. Our study highlighted the importance of detection methods in studies of HPV infections and prostate cancer. Considering the great variation in HPV prevalence and the risk estimates by the influential parameters, multi-center large-scale prospective studies are needed. In addition, the etiological and biology functional researches on HPV infections and prostate cancer are necessary.We used Medline to search for relevant articles published from January 1989 to May 2015 using the MeSH terms “Papillomavirus”, “Human”, and “Prostate cancer”. We also evaluated citations in retrieved articles. The work flow is shown in the 26a priori for this study. Disagreement was resolved by consensus. For each study included, the following information was extracted: first author, year of publication, country of origin, specimens type (tissue or serum), HPV DNA source (fixed or fresh), detection method , sample size, HPV prevalence overall by disease status (case or control) and type-specific: HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 58, 59 and 68), and matching criteria if controls were present. When HPV was assessed in both prostate tissues and sera, only the results obtained from tissues was used1340Two reviewers (Lin Yang and Shuanghua Xie) independently extracted data from selected articles according to a standard form created Gleason score of prostate cancer cases was also extracted. The majority of studies classified prostate cancers into two subgroups as high (≥7) and low (<7) Gleason scores. However, 2 studies32We described characteristics of included studies, and calculated prevalence and its 95% confidence intervals of overall, clade-specific  and type–specific HPV prevalence  in total prostate cancers and by detection method. An unconditional logistic regression model was used to adjust and compare the HPV prevalence by the influential parameters including study region, specimen type and published year.χ2-based Q test, and the heterogeneity was considered significant if P < 0.05. Publication bias was evaluated using Egger’s linear regression asymmetry testWe also pooled risk estimates for HPV infections and prostate cancer risk using included case-control studies. A fix-effect or random-effect model was used to pool the data, based on the Mantel-Haenszel method and the DerSimonian and Laird method, respectively. These two models provide similar results when between-studies heterogeneity is absent; otherwise, random-effect model is more appropriate. Between-studies heterogeneity test was performed by using the How to cite this article: Yang, L. et al. Worldwide Prevalence of Human Papillomavirus and Relative Risk of Prostate Cancer: A Meta-analysis. Sci. Rep.5, 14667; doi: 10.1038/srep14667 (2015).


Background. Although alcohol and tobacco use are known risk factors for development of squamous cell carcinoma in the head and neck, human papillomavirus (HPV) has been increasingly associated with this group of cancers. We describe the case of a married couple who presented with HPV-positive oropharynx squamous cell carcinoma within two months of each other. Methods. Tumor biopsies were positive for p16 and high-risk HPV in both patients. Sanger sequencing showed a nearly identical HPV16 strain in both patients. Both patients received chemoradiation, and one patient also underwent transoral robotic tongue base resection with bilateral neck dissection. Results. Both patients showed no evidence of recurrent disease on follow-up PET imaging. Conclusions. New head and neck symptoms should be promptly evaluated in the partner of a patient with known HPV-positive oropharynx cancer. This case expands the limited current literature on concurrent presentation of HPV-positive oropharynx squamous cell carcinoma in couples. Human papillomavirus (HPV), a sexually transmitted virus, is well known for its role in the development of cervical cancer in women. However, high-risk HPV—especially HPV16—has been increasingly associated with oropharynx squamous cell carcinoma (OPSCC) , and oroHPV appears to be more transmittable than other sexually transmitted viruses, and HPV infection is nearly ubiquitous among sexually active adults . Althoug60-year-old woman presented to otorhinolaryngology with one month of progressive otalgia, nasal congestion, odynophagia, and hemoptysis. Her medical history was significant for diabetes mellitus type II, hyperlipidemia, depression, gastroesophageal reflux disease, and obstructive sleep apnea. She had a tonsillectomy as a child and a significant smoking history of 25 pack-years. Examination revealed cervical lymphadenopathy and an ulcerated right tonsillar lesion extending across the palate and into the nasopharynx. A right tonsil biopsy revealed invasive squamous cell carcinoma that was positive for p16 by immunohistochemistry. In situ hybridization (ISH) showed the presence of diffuse high-risk HPV RNA. Positron emission tomography (PET) revealed prominent uptake in the right tonsil, with extension across the midline into the left tonsil and into the lateral nasopharynx, and involvement of the bilateral cervical lymph nodes without evidence of distant metastases (T4bN2cM0). Due to the extent of the primary tumor, she was not deemed a surgical candidate. She was treated with definitive concurrent chemoradiation therapy over 7 weeks in 35 fractions to 7,000 cGy of intensity modulated radiation therapy with weekly cisplatin. A PET scan performed 13 weeks after treatment showed no evidence of residual or recurrent squamous cell carcinoma, and physical examination corroborated a complete response. There was no evidence of recurrent disease at a 1-year follow-up visit.60-year-old man, Patient A's husband of 31 years, presented to family medicine with concern of a left supraclavicular mass that he had noticed two weeks before. His medical history was significant for hypertension, hyperlipidemia, hypothyroidism, coronary artery disease with drug-eluting stent placement, and chronic low back pain. He had a tonsillectomy as a child and had no smoking history but was exposed to second-hand smoke from his wife. Ultrasound imaging revealed the presence of multiple enlarged left supraclavicular lymph nodes. An excisional biopsy was performed, revealing metastatic squamous cell carcinoma that was positive for p16 by immunohistochemistry. In situ hybridization was positive for high-risk HPV RNA. This diagnosis was made approximately two months after his wife's diagnosis of HPV-positive squamous cell carcinoma. He was referred to otorhinolaryngology, where a smooth exophytic lesion was discovered at the left base of the tongue. Subsequent PET imaging suggested malignancy in the left base of tongue with metastases to the bilateral cervical lymph nodes without evidence of distant metastases (T2N2cM0). The patient underwent transoral robotic tongue base resection and bilateral neck dissections. Negative margins were achieved, and 9 of 76 lymph nodes were positive, the largest greater than 5 cm with gross extracapsular extension. He received a six-week course of adjuvant concurrent chemoradiation with 6,000 cGy in 30 fractions with weekly cisplatin. A PET scan performed 20 weeks after treatment showed no evidence of residual or recurrent squamous cell carcinoma. There was no evidence of recurrent disease at a 1-year follow-up visit.Surgical pathology archival material was histopathologically reviewed using hematoxylin and eosin-stained slides. Immunohistochemistry for p16INK4a was performed on formalin-fixed, paraffin-embedded tissue sections with the CINtec Histology Kit  using the automated Ventana BenchMark XT (Roche Diagnostics). RNA ISH for high-risk HPV E6/E7 mRNA was manually performed using the RNAscope HPV kit .DNA was extracted using the QIAamp DNA FFPE Tissue Kit  following the manufacturer's protocol. End point PCR was performed using AmpliTaq Gold 360 master mix  and HPV16 L1 primers. The HPV16 L1 primer sequence is as follows: forward: 5′-CACAGTTATTCAGGATGGTGATATGG-3′, reverse: 5′-GAAGTAGATATGGCAGCACATAAT-3′. The PCR product was purified using the QIAquick PCR Purification kit prior to Sanger sequencing. Sanger sequencing was performed in both directions using both forward and reverse primers.This case presents the rare scenario of a married couple receiving a diagnosis of HPV-positive oropharynx squamous cell carcinoma within two months of each other. Given HPV's ubiquitous nature, it can often be difficult to say whether HPV was transmitted from one spouse to the other or whether each partner carried the infection before their relationship. In this case, genome analysis of tumor biopsies revealed that the couple was infected with a nearly identical strain of HPV16. We performed PCR amplification and Sanger sequencing to identify the presence of HPV sequence in both husband and wife. A specific ~500 bp HPV16 L1 PCR product was successfully amplified from both husband and wife genomic DNA . We thenHPV-induced genital warts exhibit a 60% risk of passing the infection to one's sexual partner, suggesting that HPV is highly transmittable. HPV appears to be more transmittable than other sexually transmitted viruses but about the same as sexually transmitted bacterial infections . The synThe susceptibility of the oropharynx to HPV infection has not been fully explained. The invagination of the tonsillar mucosal surface may favor the retention of viral particles and their access to basal mucosal cells . The metAlthough a rare occurrence, HPV-positive oropharynx squamous cell carcinoma can occur concurrently in couples. As shown in this case, the same HPV strain can be transmitted between a couple and lead to cancer in both partners. It is important to recognize that synchronous presentation of HPV-positive OPSCC in a couple does not necessarily imply that both partners carry the same viral strain or that one partner gave the infection to the other; indeed, it is possible that both partners carried the HPV infection before even meeting and then had similar time courses of cancer development. As demonstrated in this case, HPV genome amplification and DNA analysis may suggest transmission of the same viral strain between partners depending on genome similarity. HPV is a highly transmittable infection and is common among sexually active adults; the growing use of HPV vaccination will play an important role in combating HPV-positive oropharynx cancers. Prompt evaluation of new head and neck symptoms in the partner or spouse of a patient with a known HPV-positive oropharynx squamous cell carcinoma is warranted.