The incidence of oropharyngeal cancer and oral cancer is growing worldwide, both in young non-smokers and in young non-drinkers (smoking and drinking are considered the main risk factors). Epidemiologic studies suggest a strong association between the infection by human papillomavirus (HPV), especially types 16 and 18  which have already demonstrated their etiological role in anal tumours as well as in cervix cancer. There is clear epidemiologic evidence that both types of tumours relate to changes in sexual behaviour and that both are linked to sexual transmission of HPV. The number of oral and oropharyngeal cancer cases is rising nowadays, especially among young individuals with no typical toxic habits, such as tobacco and/or alcohol.In this review we set out to update the aspects related to the onset of oral cancer, its relationship with HPV infection and whether this association may be due to the sexual transmission of the virus. Key words:Human papillomavirus, oral sex, head and neck cancer, oral cancer, squamous cell carcinoma, oropharyngeal cancer. Head and neck carcinomas (HNCs) stem from the mucosa lining the oral cavity, the oropharynx, the hypopharynx, the larynx, the nasopharynx and the sino-nasal tract. The most common histological type is squamous cell carcinoma (SCC) ,2.Over 650,000 patients are diagnosed with head and neck squamous cell carcinoma (HNSCC) worldwide every year and over 350,000 die of it ; furtherThe similarities in the clinical aspects between the oral and genital injuries associated with HPV led clinicians in the eighties to suggest that HPV might be involved in oral carcinogenesis . Some ofRecently, data from case-control and meta-analytic studies show that HPV would be, indeed, an independent risk factor for the development of oropharyngeal and oral carcinomas ,6-8. HNSMoreover, a strong association seems to exist between patient age and HPV-16’s infection prevalence . These cThe association between these oncogenic HPV types (16 and 18) in cervix cancer as well as with anal cancer has been clearly demonstrated, just like the role of certain sexual conducts in their transmission . In this“head and neck squamous cell cancer”; “HPV” or “oral cancer”. Articles related to HPV and larynx cancer have been excluded, as they exceed this field. For our purposes, data bases such as Medline, Pubmed, Current Contents Connect, Cochrane plus and Catálogo Cisne have been used.In order to carry out this review, a search has been conducted for articles from 2000 up to 2010 related to HNSCC, especially at orpharyngeal and OSCC levels, using keywords such as -Relationship among HPV infection, oral cancer and oropharyngeal cancer. The role of sexual conducts.Tobacco, alcohol, poor oral hygiene and genetics remain important risk factors in head and neck tumour’s global figures , although HPV is currently acknowledged as one of the primary causes behind the rise in oropharyngeal cancer cases. In the USA, between 40% and 80% of oropharyngeal cancers are caused by HPV, while in Europe figures vary from Sweden’s 90% to less than 20% found in other communities with a higher prevalence of tobacco consumption .HNC’s total incidence in the USA has diminished over the last years due to a lower consumption of tobacco. Yet the incidence of HPV related oropharyngeal cancer seems to be rising, as shown by SEER (Surveillance Epidemiology and End Results), not only in the USA but also in Sweden, highlighting the relevance of this causal association . SEER’s As for OSCC, Miller et al.  performeTermine et al. , in a meEven though it has been known for a long time that HPV is a key etiological factor in anogenital cancer, it is only recently that its etiological relationship with an HNSCC subtype has been proved. There are over 100 different types of HPVs, and at least 15 of these have oncogenic potential. Notwithstanding, most HNSCCs (>90%) are caused by the HPV-16 type, the same type involved in anogenital cancers associated with HPV  these days, with an incidence of about 5.5 million worldwide. About 75% of sexually active men and women have been exposed to HPV at some point in their lives . HPV typThe likely ethiological role of HPV in cancer has been put forward after finding out that the sexual partner of patients diagnosed with an HPV infection presented a higher risk of developing a head and neck cancer . HoweverAs has already been said, HPV infection is a STI and the consulted studies suggest that the number of sexual partners in the lifetime would be an important risk factor, not only for anal and cervix cancer, but also for the development of HNSCC . In caseA recent study suggests a rise in HPV infection incidence in young adults, mainly due to an earlier initiation of sex and to a higher number of sex partners . In the Yet it is also important to add that HPV-positive HNSCC cases were present in individuals who had had less sexual partners. Thus, over 50% of patients with HPV-positive tumours reported having had five or less sexual partners while between 8% and 40% reported having never had oral sex. Therefore, even though sexual conducts seem to be a key risk factor for HPV-positive HNSCC, the absence of a high number of sexual partners does not prevent the development of an HPV-positive HNSCC .Both men and women can be asymptomatic HPV carriers and the virus transmission to the oral cavity would mainly happen through unprotected sex practices. Research carried out by D’Souza et al.  shows anA thorough study published in 2010 about sexual behaviour and HNC risk carried out by INHANCE , shows that sexual behaviour is linked to the risk of developing an HPV associated cancer, depending on its localization. Thus, the study found out that oropharyngeal cancer was significantly associated with having had six or more sex partners and four or more oral sex partners .As for amygdala cancer, it was associated with having had four or more oral sex partners; and among men, to having ever had oral sex and to a younger age at the time of their first sexual relationship. As far as tongue base cancer is concerned, it was linked, among women to having had oral sex at least with two partners; among men, to having had sex with other men. Little evidence was found, though, of association between the analysed sexual behaviours and their relationship with larynx and oral cavity cancer .D’Souza et al.  found thIt has also been proved that the prevalence of oral infection caused by high oncological risk HPV is higher in seropositive individuals and that it increases with age, male gender and infection by VHS-2 .HIV-positive individuals also have a higher likelihood of developing an oral infection caused by HPV, of being infected by more than one type of HPV and of HPV being of high oncogenic risk . The risLastly, some authors disagree and consider the possibility of HPV infection in HSNCC patients being just a temporal non-causative infection. Acha-Sagredo et al.  did not In this sense, Herrero et al.  suggeste-Pathogenic mechanism of oral/oropharyngeal cancer onset in relation with HPVPapillomaviridae family. The most important ones (of high oncogenic risk) are types 16 and 18 (Human Papillomavirus (HPV) belongs to the 6 and 18 . Infecti6 and 18 ,26. The 6 and 18 ,16. All 6 and 18 ,13,16,27When an HPV infection happens, antibodies such as HPV-16 L1 as well as HPV-16 E6 and E7 viral proteins show up in serum. These viral proteins are capable of producing a malignant transformation of upper respiratory, anal and genital tracts cells. The risk of developing OSCC when having antibodies against these proteins would be between 1.5 and 3.4 times higher, for the oral cavity, and between 3.5 and 19 times higher, for the oropharynx, than otherwise ,26.P16 is another protein involved in cell cycle used by many authors as a likely marker for oral cancer associated with an HPV infection. It has been found out that an over-expression of P16 would be linked to HPV-positive oropharyngeal carcinomas, poorly differentiated and in locally more advanced stages T4, N2-3) . On the , N2-3 . All this would support the existing evidence of oral/oropharyngeal cancer etiologically associated with HPV having an increased survival and a better prognostic, as both an over-expression of P16 and very low P53 mutation rates would happen, and all this might be linked to infected cells being more sensitive to current radiotherapy and chemotherapy ,29.  Tab-Treatment of oral/oropharyngeal cancer associated with HPV. The odontologist’s role in its early prevention and detection.The average time span from the moment in which HPV infection occurs to the onset of cancer has been estimated as around twelve years, so according to some authors the number of young adults who will develop oropharyngeal cancer is expected to increase over the coming years ,31.In line with what has been expressed in the previous section, several authors agree in pointing out that patients who display small size tumours, over-expression of P16 and high HPV viral charge in the tumour sample, have an increased survival in spite of the presence of lymphatic nodules ,16,28.Nguyen et al.  suggest This modality should be considered experimental, since surgery plus post-operative chemotherapy or radiotherapy remains the treatment of choice in patients with locally advanced HNC .HPV vaccine is the first explicitly designed to prevent virus induced cancer ,31. HPVsThere are two commercially available prophylactic vaccines against HPV today: the bivalent (VPHs 16 and 18) Cervarix® (GSK) and the tetravalent  Gardasil® (Merck). Theoretically, there is no reason for these vaccines to fail to work against these same viruses in different localizations . Proving that the vaccine also prevents oropharyngeal cancer would mean not only a landmark in the prevention of these diseases, but it would also provide the missing link in the chain of evidences with the ultimate proof of HPV induced viral etiology of these tumours ,33.Vaccination is approved in females aged 9 to 26. The vaccination primary target population should be females aged 11 and 12. However the vaccine can also be administered to females up to 9 years old and to those aged between 13 and 26 who have been sexually active. There are no available data about the vaccine’s efficacy in individuals aged over 26 ,17,34.Clinicians, including professionals involved in oral health should pay attention to the risk of oropharyngeal cancer in young individuals, who neither smoke nor drink, to avoid an unnecessary diagnostic and therapeutic delay. There seems justified, considering all that has been said so far in this paper, the inclusion of questions over sexual practices in the anamnesis of young patients presenting enlarged lymphatic nodules and/or masses in amygdalae and/or sore throat, to rule out a possible underlying HPV associated neoplasia. According to some authors, an early referral to the specialist would reduce morbidity as well as the cost of the treatment . RoutineSo far, recommendations on early detection of oral cancer focused on patient groups who traditionally had a higher risk of developing it, i.e. individuals aged over 60, tobacco and/or alcohol consumers as well as individuals with certain hereditary diseases, such as Fanconi’s anaemia, or with a past history of oral cancer .Brondani, in his editorial published in an Odontology reference review, also wonders what our patients know about HPV, about oral sex and about oral cancer. Even more, he acknowledges that dentists have not been properly educated in these aspects, so he encourages a more detailed study of these subjects, their registration in the patient’s clinic history and to supply information about them to patients .Scientific evidence in this point seems to indicate that we must be educated, and educate our children, on other potential risk factors in the onset of oral cancer, such as non-protected oral sex, and not only about tobacco and/or alcohol consumption, especially to early detect this injuries in adolescents and young adults or, better yet, to try to prevent them .Patients with HPV associated HNSCC are younger and present reduced tobacco and/or alcohol intakes. There are certain molecular profiles of HPV-positive HNSCCs that differentiate them from HPV-negative cancers, as a result they could be considered a biological entity separated from the rest of HSNCC, and of likely sexual transmission. According to the current literature revised, risk factors associated with this HNSCC variant are similar to those of cervix cancer, including number of sexual partners, younger age at their first sexual intercourse, oral sex practices, a history of genital warts and younger age. Dentists, as health professionals, ought to get more heavily involved in the detection of these factors implied in the etiology and pathogenesis of these injuries in order to contribute to the early detection and prevention of this kind of injuries, especially in young patients with no risky habits traditionally linked to oral cancer, such as tobacco and/or alcohol consumption.


A study including 166 subjects was performed to investigate the frequency and persistence over a 6-month interval of concurrent oral and anal Human Papillomavirus (HPV) infections in Human Immunodeficiency Virus (HIV)-infected men who have sex with men (MSM).Patients with no previously documented HPV-related anogenital lesion/disease were recruited to participate in a longitudinal study. Polymerase chain reaction (PCR) was performed to detect HPV from oral and anal swabs and to detect Human Herpes Virus 8 (HHV-8) DNA in saliva on 2 separate specimen series, one collected at baseline and the other collected 6 months later. A multivariate logistic analysis was performed using anal HPV infection as the dependent variable versus a set of covariates: age, HIV plasma viral load, CD4+ count, hepatitis B virus (HBV) serology, hepatitis C virus (HCV) serology, syphilis serology and HHV-8 viral shedding. A stepwise elimination of covariates with a p-value > 0.1 was performed.The overall prevalence of HPV did not vary significantly between the baseline and the follow-up, either in the oral  or the anal specimens (88.6 and 86.3%). The prevalence of high-risk (HR) genotypes among the HPV-positive specimens was similar in the oral and anal infections . Among 68 patients with either a HR, low-risk (LR) or undetermined genotype at baseline, 75% had persistent HPV and the persistence rates were 71.4% in HR infections and 76.7% in LR infections. There was a lack of genotype concordance between oral and anal HPV samples. The prevalence of HR HPV in anus appeared to be higher in the younger patients, peaking (> 25%) in the 43-50 years age group. A decrease of the high level of anal prevalence of all genotypes of HPV in the patients > 50 years was evident. HHV-8 oral shedding was positively related to HPV anal infection (p = 0.0046). A significant correlation was found between the persistence of HHV-8 shedding and HIV viral load by logistic bivariate analysis .A high prevalence of HPV infection was found in our cohort of HIV-infected MSM, with a negative correlation between anal HPV infection and CD4 cell count. Human papillomavirus infections are among the most common sexually transmitted infections worldwide, representing a significant health problem due to their high prevalence and transmissibility. HPV is a leading cause of anogenital malignancies. The incidence of anal cancer is particularly high among women with a history of cervical dysplasia and cervical cancer, HIV-positive individuals, Men who have sex with Men (MSM) and transplant recipients . Anal caSeveral studies have found high rates of anal HPV infections in HIV-infected patients -9. PrevaRecent molecular and epidemiological studies have demonstrated a role for HPV in the aetiology of oropharyngeal cancers ,11. HighHPV infection is associated with oral squamous cell carcinoma (OSCC). Although the incidence of HPV-unrelated OSCC associated with tobacco and alcohol use has declined over recent decades, OSCC related to HPV has significantly increased, particularly among white men and younger individuals . This maThe few available studies analysing concurrent or sequential oral and anogenital HPV infections have used the similar study populations: high-risk sex workers, women with a history of cervical HPV infection, and HIV-positive women -18. SeveThis study was designed to investigate the frequency and persistence over a six-month interval of concurrent oral and anal HPV infection in a cohort of HIV-infected MSM and to investigate correlations between these infections and virological and immunological status related to HIV and other infections, including HHV-8, HBV, HCV, and syphilis.From December 2007 to June 2009, HIV-positive MSM attending the Infectious Disease Unit of the Padua University Hospital as outpatients were recruited to participate in a longitudinal study of HPV infection. Eligibility criteria included being > 18 years of age, able to provide informed consent and having no history of or current symptomatic HPV-related anogenital lesions/diseases. Each subject underwent clinical evaluation and sample collection on two different occasions separated by 6 months. HPV was assayed from oral and anal swabs.All virologic and serologic tests were performed as a part of the diagnostic procedures performed periodically on these HIV patients. All subjects enrolled gave their informed consent to all procedures and to the use of their data for scientific evaluation and publication in a blinded form. The local government in the form of the Veneto Regional Health Authority approved this study and provided funds. This study was conducted in accordance with the Helsinki Declaration and with local legislation.g for 15 min at 22°C, the supernatant was discarded, the cell pellet was dried and resuspended in 300 ul of 20 mmol/L Tris buffer (pH 8.3) and then frozen at -80°C. Total DNA was extracted using the QIAamp DNA Mini Kit , and DNA extracted from the samples was first tested with primers PC04 and GH20, targeting a 268 bp fragment of beta-globin. Samples negative for beta-globin were not considered for further analysis. DNA was purified using ExoSAP-IT , and the presence of HPV DNA was investigated by PCR using MY09/MY11 primers and, if HPV DNA was not detected, GP5+/GP6+ primers .The prevalence of HPV anal infection and the HR genotype prevalence in various age groups are reported in Figure A bivariate pairwise correlation analysis was performed to explore relationships between the main variables. A positive correlation between positive results from any syphilis diagnostic test and the presence of HR HPV genotypes in anal swabs was found, and this correlation was statistically significant (p = 0.0346). Positive syphilis tests were positively related to age (p = 0.0169) and positive HCV serology (p = 0.016). Anal swab HPV detection at either time-point was positively related to higher HIV plasma viral load (p = 0.0184) and negatively to antiretroviral therapy (p = 0.0419).When the logistic multivariate analysis was performed, after stepwise elimination of the covariates with a p-value > 0.1, only two significant covariates, "age" and "CD4", were retained in the final model Table . The rel2 (1 d.o.f.) = 8.03, p-value = 0.0046). Moreover, a significant correlation was found between the persistence of HHV-8 shedding and HIV log10  by logistic bivariate analysis .HHV-8 oral shedding was positively related to HPV infection in anal area  . Oral seOur findings show that the prevalence of HR HPV genotypes was highest in the 43-50 year old age group, where it was over 25%. The persistence of HR genotypes (71.4%) did not exceed the persistence of low-risk genotypes (76.7%), although further surveillance of this cohort will be necessary to understand of the determinants of HPV persistence. The presence of an HPV-positive anal swab was negatively related to age, CD4 count and antiretroviral treatment, and positively related to HIV viral load. The negative correlation between anal HPV infection and CD4 count, albeit expected, warrants further study and corresponds with the positive correlation with HIV plasma viral load. These observations support a role for HIV viral replication and immunodeficiency in affecting susceptibility to infection with all genotypes of HPV infection in the anal area or the ability to clear HPV.The relationship between anal HPV infection and age was negative (decreasing) both by linear regression (using age as a dependent variable) and by logistic regression. However, we interpret this finding to be somewhat artifactual, as the age-related prevalence of all HPV genotypes shows a plateau followed by a decline in the group older than 50 years, whereas the high-risk HPV genotype infections show a peak in the 43-50 years age group, in partial agreement with Nyitray . MoreoveT. pallidum infection was close to the significance level and became significant when the possible high-risk genotypes 53 and 66 were included in the analysis. Syphilis  was positively related to age, HCV infection, and, unexpectedly, lower HIV plasma viral load and higher CD4 count. The latter two correlations are probably explained by the characteristics of the older HIV patients, as the older patients were more often receiving ART and as a consequence, they had lower viral loads and higher CD4 cell counts. The association between syphilis and HCV infection is somewhat unexpected, since recent data suggests that permucosal HCV transmission might result from high-risk sexual and non-injecting drug use behaviours among MSM, raising the question of the importance of sexual transmission of HCV [The correlation between HPV infection and n of HCV .HHV-8 oral shedding was positively related to HPV infection in anal area: HPV infection rates were 63.6% in the HHV-8-positive and 30.0% in HHV-8-negative group. It has been demonstrated that the risk of HHV-8 infection among men in the United States and Western Europe is closely related to the number of male sexual partners, and in these populations sexual intercourse is the major mode of HHV-8 transmission ,47. A siSome limitations of our study should be addressed. Our data lack information regarding a number of subjects and the robustness of our correlations should be confirmed with further observations. HPV genotyping was performed using a population sequencing method that does not allow us to fully characterise co-infections and to correctly trace all genotypes found during the double sampling. Nevertheless, this method allowed us to demonstrate a high prevalence of infection by undetermined-risk HPV types.In conclusion, the negative relation between anal HPV infection and CD4 count detected by this multivariate logistic procedure, albeit expected, deserves interest and is in agreement with the positive correlation with HIV plasma viral load found using a bivariate analysis.These observations support a role for the HIV viral replication rate and immunodeficiency in affecting patient susceptibility to infection with all genotypes of HPV infection in the anal area and with HPV clearance. Moreover, two subpopulations were detected: a larger and younger subpopulation with a lower antiretroviral treatment rate, less control of HIV replication, lower CD4 counts, and higher prevalence of anal HPV infection including high-risk genotypes, and a smaller, older subpopulation with opposite features. The increasing incidence of anal cancer in the HIV-positive population make new strategies addressing HPV infection monitoring, control and prevention necessary; further studies addressing vaccine efficacy, HPV viral load and the role of undetermined risk variants are needed.No conflictsSGP designed and coordinated the study, supervised laboratory experiments, collected the data, interpreted the findings and wrote the paper; MC helped design the study, interpret the data and write the paper; RS helped design the study, managed the patients and collected the samples; CB performed laboratory experiments; SA performed laboratory experiments; FDB performed HPV sequencing; SP contributed to HPV genotyping; AB managed patients and collected the samples; AS managed patients and collected the samples; CM helped design the study and interpret the data and performed statistical analysis; GP designed and coordinated the study and interpreted the data.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/150/prepub


In France, it is recommended that girls and women aged 14–23 are vaccinated against the human papillomavirus (HPV). However, French women’s knowledge of and attitude towards the vaccine has been little studied.Thirty-nine general practitioners, representative of those working in the large Rhône-Alpes region, offered a self-administered questionnaire on cervical cancer (CC) prevention to all 18–65 year-old women who came for consultation during June and July 2008. In addition, semi-structured interviews were undertaken with a sample of those who had daughters aged 14–18.pneumococcus (OR=3.28 [1.32-8.11]) and knowing the target population for HPV vaccination (OR=2.12 [1.15-3.90]). Knowing the recommended frequency of Papanicolaou smear testing (Pap test) screening was associated with lower acceptance (OR=0.32 [0.13-0.82]).Of the 1,478 women who completed the questionnaire, only 16.9% mentioned HPV as the cause of CC, even though 76.2% knew of the vaccine. 210 women had daughters aged 14–18, and 32 were interviewed. Compared with the wider group, more of these women were aware of the HPV vaccine (91.4%). 44.8% knew the target population and 17.1% the recommended ages for vaccination. 54.3% favoured HPV vaccination; 37.2% were undecided and only 0.9% were opposed. The main barrier to acceptance was the recency of the vaccine’s introduction and concern about possible side effects (54.9%); 14.1% preferred to rely on their GP’s decision. Factors associated with acceptance of the HPV vaccine were having previously vaccinated a child against Few mothers are opposed to HPV vaccination. Factors associated with acceptability were knowledge about the vaccine, acceptance of other vaccines and, unexpectedly, lack of knowledge about the recommended frequency of Pap testing. On multivariate analysis, compliance with recommendations for Pap test screening and socioeconomic factors had no effect on views about HPV vaccination. Given that concern about possible side effects is the major barrier to wider acceptance of the HPV vaccine in France, GPs have a key role in providing information. Cervical cancer (CC) is the tenth most common cancer among French women. In 2011More recently, a new primary prevention tool has become available with the development of two vaccines 5;6] targeting the high-risk HPV 16 and 18 genotypes responsible for 70% of CC. These v;6 targetAttitudes towards this new vaccine seem to be positive, with most studies on parental attitudes reporting acceptance rates above 60%-11. NeveMost previously published studies on CC and HPV awareness have been from North America: with the exception of the United Kingdom, little such research has been carried out in Europe. SeveralThe first objective of this study, which combined quantitative and qualitative techniques, was to assess knowledge about CC, the Pap test and HPV vaccination in 18–65 year-old French women one year after the introduction of the vaccine. The second objective was to assess mothers’ acceptance of HPV vaccination for their 14–18 year old daughters and determinants of that acceptability. The age range 14–18 was chosen to encompass girls above the age at which vaccination is recommended in France and below the age at which they can themselves legally assent to the procedure. This study was part of the REMPAR  programme aimed at evaluating means of preventing HPV-mediated disease.This cross-sectional study, conducted in the Rhône-Alpes region of France in June and July 2008, used both quantitative (self-administered questionnaire) and qualitative methods (semi-structured interviews).The study population was women aged 18–65 living in the Rhône-Alpes region who visited a participating general practitioner (GP) within the two months specified above, without exclusion criteria. Participants were consecutively recruited by GPs during a consultation (which can have been for any reason). The 39 physicians who agreed to participate were volunteers from a sample of 279 GPs who took part in an earlier study. They weGPs offered the self-administered questionnaire to all women who met the inclusion criteria (There were no exclusion criteria.). The anonymous questionnaire had five parts: 1) socio-demographic data , 2) practices related to disease prevention , 3) gynaecological history , 4) knowledge about CC including its cause, the role of HPV, CC prevention (the role of the Pap test and HPV immunization) and 5) the acceptability of HPV vaccination.Questions on socio-demographic variables, preventive health practices and gynaecological history were multiple-choice. Questions on knowledge were multiple-choice in relation to the Pap test and open-ended with regard to the cause of CC and understanding of HPV vaccination. Answers to open-ended questions were recoded according to predefined categories. The acceptability of HPV vaccination was assessed by asking respondents to choose a single response from six options Table. Based oWomen completing the questionnaire who had daughters aged 14–18 were also asked if they would volunteer to take part in a semi-structured, face to face interview conducted in their own home by a sociologist. It was thought particularly important to understand the opinions, and the reasons underlying them, of mothers from an underprivileged, lower socioeconomic background, and of women whose questionnaire responses showed them to be opposed to HPV vaccination. Among the volunteers, selection for interview was designed to include a high proportion of mothers from both groups. Interviews explored in greater depth topics covered in the questionnaire, notably gynaecological, history, practices related to disease prevention, women’s understanding of HPV vaccination, and factors related to its acceptability. There was particular emphasis on the latter, given our concern to better understand the drivers of and barriers to acceptance. Interviews lasted 30 to 60 min, were audio-taped and were transcribed verbatim. A content analysis was carried out using an analysis grid designed to build on the topics addressed in the quantitative part of the study and explore them in greater depth. The results were then compared with analysis using specific software, NVivo  according to the methodology proposed by Miles & Huberman.versus undecided or opposed) and potential predictive factors was studied using Chi-square or Fisher's exact test for qualitative variables and using Student’s t or Mann–Whitney U test for quantitative variables. A stepwise backward logistic regression was used to determine the most suitable model for multivariate analysis. Variables with a p value ≤ 0.20 in univariate analysis were entered in the model and a p value ≤ 0.05 was considered statistically significant. Data analysis was performed using the SAS 9.1 software .Descriptive data were calculated for the survey population as a whole and for the subgroup composed of mothers of one or more 14–18 year old girls. The relationship between mothers’ views  were 18–39 years old and 776 (52.5%) aged 40–65 years. This age distribution is similar to that for the Rhône-Alpes region as a whole, for which the corresponding figures are 47.2% and 52.8% (p=0.81). Among these women, 210 (14.2%) had one or more daughters aged between 14 and 18 at the time of the study. Of these mothers, 32 were interviewed.The socio-demographic characteristics, immunization status and gynaecological history of the study population as a whole and of the subgroup with daughters of vaccination age are given in TableAmong the 1,478 respondents as a whole, knowledge about the Pap test was quite good (61% knew its role), and better than that about the causes of CC: only 16.9% of women mentioned HPV in this context. However, the question on the causes of CC was open-ended while that on the Pap test was multiple-choice. Awareness of the HPV vaccine was high (76.2%) but the recommended target population and age for vaccination were not precisely known Table. The medIt is for prevention” without specifying the disease that was being prevented. The mothers who knew about HPV in the interview were mainly those who had already vaccinated their daughters. These women had also had the situation explained by their GPs before vaccination. Even so, many were not clear about the link between HPV infection and CC. All but one of the 32 mothers interviewed (91.4%) knew about the HPV vaccine. As in the wider population of women, mothers had heard of this vaccine mainly through television (56.2%), but a greater proportion (39.1% vs 16.0% for respondents as a whole) had had information from their physicians.Knowledge about the Pap test among mothers of 14–18 year old daughters was not different from that of the wider population of women surveyed (65.2% vs 61.0% knew its role). As with the wider sample, relatively few mothers of teenage girls mentioned HPV as a cause of CC (20.9% vs 16.9%). However, during the interview, without multiple-choice options, mothers had more difficulties than the wider group in explaining the role of Pap testing: many said “It is for teenagers”, without mentioning ages. They knew from their physicians who should be vaccinated “My physician told me that this concerned my daughter”, because of her age and sexual history. However, few mothers had a detailed recollection of the information provided.Despite this, more than a half of the mothers did not know the target population or the precise recommended ages for vaccination . In interviews, they explained “Among the 210 mothers who completed the questionnaire, 194 responded to the question regarding HPV vaccine acceptability: 2 were opposed (1.0%), 78 were undecided (40.2%) and 114 were favourable (58.8%). A total of 46.4% had already vaccinated their daughters (Tablefear of cancer” and their wish to “protect (their) child”. One explained: “I don’t want my daughter to tell me ‘I have a cervical cancer’ while a vaccine exists”. The second most frequent reason for vaccination (reported by 10% in questionnaires and 40% in interviews) was the favourable opinion of the physician. Those interviewed explained that physicians reassured them if they had any questions: “She recommends it. I think I can trust her. She told me if she had a daughter of this age, she would vaccinate her against HPV.” Nevertheless, in the questionnaires, 14.4% of favourably disposed mothers mentioned incorrect expectations of the vaccine such that it would eradicate risk of all gynaecological cancers or prevent all sexually transmitted diseases (STDs).The major reason given in interviews by favourably disposed mothers (and cited by 65.8%) was that vaccination offered the opportunity of preventing a severe and potentially fatal disease, namely CC, in their daughters. The perceived danger of CC was frequently mentioned as driving the decision to vaccinate. Mothers mentioned the “He told me not to vaccinate my daughter”. Mothers from low socio-economic background who were interviewed seemed to adhere totally to their physicians’ opinion while those in higher professional categories wanted to know his opinion but had a more critical point of view.Undecided and opposed mothers justified their position by the fact that we have little experience of the vaccine to look back on and by fear of side effects with what is a new vaccine (such reasons were cited by 54.9% in questionnaires). Some mothers interviewed had in mind the controversy over a possible link between hepatitis B vaccination and multiple sclerosis which affected France after a mass hepatitis B immunization campaign among adolescents in 1994. Another 14.1% preferred to rely on their physician’s decision and waited to know his opinion, and 7.0% preferred to let their daughters decide by themselves. The key role played by physicians is illustrated by a mother opposed to the vaccine who explained in an interview that she did not want to vaccinate her daughter because her GP was against it: “In questionnaires, other concerns mentioned related to sexual issues: 5.6% found it difficult and too early to have a discussion about sexuality with their adolescent daughters, fearing that this might encourage sexual activity. Others preferred good gynaecological follow-up with regular Pap tests (4.5%). The cost of the vaccine was not mentioned by mothers.versus undecided/opposed opinion are presented in Tablepneumococcus. They were more aware of the target population for HPV vaccination but less knowledgeable about how frequently women should have Pap test screening. Educational level, tobacco consumption and history of STDs were excluded from the final model.Analysis of factors associated with a favourable One year after the licensing of the HPV vaccine in France, knowledge about the role of the Pap test in CC prevention is quite good. However, when asked an open-ended question about the cause of CC, the link between HPV and CC was not widely made. Even so, among mothers of 14–18 year old girls, the majority accepted the value of HPV vaccination -- although 37% were still undecided.Responding to multiple choice questions, 93.7% of women knew that the Pap test should be conducted yearly or every 2–3 years and 82.0% answered correctly that screening should continue throughout adult life. However, only 61% answered that the purpose of Pap testing is to prevent CC. It seems that women passively accept the Pap test without really knowing why: 80% of French women are reported to have Pap tests on the advice of their physician and not on their own initiative . Our datIn contrast, the HPV vaccine itself is now well known: 76% of our overall sample and 91% of mothers had heard of it. However, they did not know precisely who should be vaccinated or at which age. This may be explained by the fact that the predominant source of information on the vaccine is the media (mentioned by 54.7%). Few women had been individually informed by their physicians.The majority of mothers with a 14–18 year old daughter were supportive of HPV vaccination. A substantial proportion (38% in our sample) is still undecided, but very few (< 1%) are clearly opposed. Even if French physicians have also been shown to be supportive of HPV vaccination, the reaPhysicians represent a key influence, and thepneumococcus was associated with a positive attitude towards HPV vaccination. This may reflect a wider attitude towards vaccination. Mothers who believe vaccines in general are safe are more willing to vaccinate their daughters against HPV[We also identified other determinants. Having already vaccinated their children against inst HPV,33-35. MHowever, on multivariate analysis, Pap test adherence itself was not associated with less acceptance of the vaccine.Socio-economic characteristics were not associated with HPV vaccine acceptance. Unfavourably disposed mothers did not mention cost as a barrier to vaccination in either the questionnaire or interviews. This may be due to the partial reimbursement (65%) by French public health insurance.Our results do not support the concern raised previously that underprivileged populations or populations with low adherence to Pap test screening would be less accepting of HPV vaccination. With regard to educational level, the results tended to be the reverse, with a lower acceptance of HPV vaccination among better educated mothers, as has previously been found,11,17,27Possible limitations in our findings should be mentioned. Certain factors previously shown to be associated with HPV vaccine acceptance, such as the younger age of mothers, STD history, tobacco consumption and educational level, were significant in univariate analysis but not after multivariate adjustment. A lack of power due to the relatively small sample of mothers could explain these discrepancies. Another limitation is that since recruitment took place during a GP consultation our respondents may have been more attuned than the wider population to their medical needs. Women included had a history of good gynaecological follow-up and a high rate of Pap test screening: 82.9% of the women sampled (and 87.0% of mothers) had had their most recent Pap testing within the past three years. Data from the French public health insurance system estimate that Pap test coverage nationally is only 56.6%. HoweverOur choice of question to assess the acceptability of HPV vaccination, and the multiple choice responses offered, could also be debated. Our intention was to cover the range of mothers’ attitudes and behaviour. Focus groups prior to the study showed a good understanding of this question and of the options for response. Moreover, all respondents were able to justify their opinion and give further detail in an open ended question; and those interviewed had additional opportunities to discuss why they were favourable towards vaccination, or otherwise.Finally, we used a cross-sectional design. This did not allow us to determine whether the associated factors identified are the cause or the consequence of HPV vaccine acceptance: greater knowledge about the vaccine could be the result of information given during its administration or, alternatively, might have driven the decision to seek vaccination.One year after French national health authorities recommended HPV vaccination, knowledge and awareness about HPV as the cause of CC is still poor a mothers of girls in the targeted age range are globally favourable towards the HPV vaccine but are seeking additional information. The results of this study suggest the type of information that could be disseminated to improve vaccine uptake. This relates principally to the record of vaccine safety. However, women do not fully understand the place of HPV vaccination alongside Pap testing in CC prevention because they are not fully aware of HPV as the cause. The need to complete this link in the information chain could also be addressed. Appropriate information on these topics may improve mothers’ acceptance of vaccination for their daughters. Physicians seem best placed to answer their questions.Women know that the aim of Pap testing is to prevent CC; and they know that there is an HPV vaccine. But they do not know that HPV causes CC. The majority of mothers of 14–18 year old daughters were favourable towards HPV vaccination. Those who were unfavourable justified their opinion mainly by the fear of side effects. Factors associated with acceptability were knowledge about the HPV vaccine, acceptance of other vaccines and lack of knowledge about the recommended frequency of Pap testing. Compliance with recommendations for Pap test screening and socioeconomic factors did not significantly affect views on HPV vaccination. GPs seem to have a key role in providing further information about HPV vaccination (particularly in relation to the virus as the cause of CC) and in reassuring women of its safety.CC: Cervical cancer; GP: General practitioner; HPV: Human Papillomavirus; OR: Odds ratio; STD: Sexually transmitted disease.Authors declared no competing interests expect Yann Leocmach and Anne-Carole Jacquard who are employees of Sanofi Pasteur MSD, Lyon, France.JH: performed the statistical analysis and interpretation of data, wrote the manuscript. DL-M: participated in the design of the study, helped to draft the manuscript and revised it critically for important intellectual content. JK: conducted and interpreted the interviews, wrote the qualitative part of the manuscript. GB: participated in the design of the study and coordinated the data collection. A-CJ: has given final approval of the version to be published. VR: participated in the design of the study. YL: helped in the interpretation of data and has given final approval of the version to be published. PV: participated in the design of the study. FC: participated in the design of the study and helped in the interpretation of data. CL: participated in the design of the study and its coordination and helped in the interpretation of data; revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/1034/prepub


P = 0.002). The infection of HPV has increased the risk of breast cancer . The high-risk HPV genotypes (types 16 and 18) in samples of breast cancer patients were the predominant types (53.34%). Other genotypes detected in breast cancer were HPV-6, HPV-11, HPV-15, HPV-23, and HPV-124, and one isolate could not be genotyped compared to HPV reference sequences. While the sole detected HPV in control specimens was HPV-124. Our study reveals that HPV infection and age are the risk factors in breast cancer development in the north part of Iran.We have analyzed the possible relevance of HPV infection for breast cancer risk among Iranian women from north part of Iran. Among women with breast cancer, 25.9% had positive test results for HPV DNA in breast tumor samples in contrast to 2.4% of women with noncancer status ( Breast cancer is the most common cancer in women worldwide, and there were representing 22.9% of all new cancers in 2008 (an estimated 1.378 million new cases) and ranking second overall when both sexes are considered together . The GenBank accession numbers for the reference HPV nucleotide sequences are as follows:HPV-1 [V01116], HPV-2 [X55964], HPV-3 [X74462], HPV-4 [X70827], HPV-5 [M17463], HPV-6 [X00203], HPV-7 [X74463], HPV-8 [M12737], HPV-9 [X74464], HPV-10 [X74465], HPV-11 [M14119], HPV-12 [X74466], HPV-13 [X62843], HPV-14 [X74467], HPV-15 [X74468], HPV-16 [K02718], HPV-17 [X74469], HPV-18 [X05015], HPV-19 [X74470], HPV-20 [U31778], HPV-21 [U31779], HPV-22 [U31780], HPV-23 [U31781], HPV-24 [U31782], HPV-25 [X74471], HPV-26 [X74472], HPV-27 [AB211993], HPV-28 [U31783], HPV-29 [U31784], HPV-30 [X74474], HPV-31 [J04353], HPV-32 [X74475], HPV-33 [M12732], HPV-34 [X74476], HPV-36 [U31785], HPV-37 [U31786], HPV-38 [U31787], HPV-39 [M62849], HPV-40 [X74478], HPV-41 [X56147], HPV-42 [M73236], HPV-43 [AJ620205], HPV-44 [U31788], HPV-45 [X74479], HPV-47 [M32305], HPV-48 [U31789], HPV-49 [X74480], HPV-50 [U31790], HPV-51 [M62877], HPV-52 [X74481], HPV-53 [X74482], HPV-54 [U37488], HPV-55 [U31791], HPV-56 [X74483], HPV-57 [X55965], HPV-58 [D90400], HPV-59 [X77858], HPV-60 [U31792], HPV-61 [U31793], HPV-62 [AY395706], HPV-63 [X70828], HPV-64 [U12495], HPV-65 [X70829], HPV-66 [U31794], HPV-67 [D21208], HPV-68 [X67161], HPV-69 [AB027020], HPV-70 [U21941], HPV-71 [AB040456], HPV-72 [X94164], HPV-73 [X94165], HPV-74 [U40822], HPV-75 [Y15173], HPV-76 [Y15174], HPV-80 [Y15176], HPV-81 [AJ620209], HPV-82 [AB027021], HPV-83 [AF151983], HPV-84 [AF293960], HPV-85 [AF131950], HPV-86 [AF349909], HPV-87 [AJ400628], HPV-88 [EF467176], HPV-89 [AF436128], HPV-90 [AY057438], HPV-91 [AF419318], HPV-92 [AF531420], HPV-93 [AY382778], HPV-94 [AJ620211], HPV-95 [AJ620210], HPV-96 [AY382779], HPV-97 [DQ080080], HPV-98 [NC_012744], HPV-99 [NC_012745], HPV-100 [NC_012746], HPV-101 [NC_008189], HPV-102 [DQ080083], HPV-103 [NC_012750], HPV-104 [NC_012750], HPV-105 [NC_012747], HPV-106 [DQ080082], HPV-107 [EF422221], HPV-108 [NC_012213], HPV-109 [NC_012485], HPV-110 [EU410348], HPV-111 [EU410349], HPV-112 [NC_012486], HPV-113 [NC_012748], HPV-114 [NC_013931], HPV-115 [NC_013591], HPV-116 [NC_013035], HPV-117 [GQ246950], HPV-119 [GQ845441], HPV-120 [GQ845442], HPV-121 [NC_014185], HPV-122 [GQ845444], HPV-123 [GQ845445], HPV-124 [GQ845446], PcPV [X62844], RhPV1 [M60184].χ2 (Pearson chi-square) and Fisher's exact test. Odds ratios and logistic regression were also calculated. Statistical significance was set as a P-value less than 0.05.Data were processed by SPSS statistical software program version 16.0. The correlations were subjected to The characteristics of study subjects including age, HPV DNA, HPV genotype, and breast cancer histopathologic types are shown in P ≤ 0.0001, The mean ages were 47.77 ± 12.552 (S.D.) and 34.20 ± 9.704 (S.D.) years in breast cancer and control groups, respectively (P = 0.002) and HPV genotype  between these two groups  were more prevalent than other HPV types in the cases  and HPV infection  are the significant risk factors in breast carcinogenesis in the studied women in north part of Iran, Mazandaran province . We suggest that HPV infection is significantly related to breast cancer in women live in Mazandaran province (OR 14.247, 95% CI 1.558–130.284; P = 0.019, In our previous studies , 21, we High-risk HPV types, such as HPV-16 and HPV-18, were detected in our study, and they comprised the majority of isolates . AlthougThe pathogenesis of breast cancer is complex. This study demonstrates the presence of HPV genome in tumor tissues in women with breast cancer in north part of Iran. HPV infection is associated with the development of breast cancer in women live in Mazandaran province. Further studies are needed to clarify the role and the risk assessment of HPV in human breast cancer. Confirming an etiologic role for HPV in breast cancer in Iranian females may help develop vaccine strategies for combating this increasingly common cancer.


Production of the chemokine CCL2 by cells of the neurovascular unit (NVU) drives critical aspects of neuroinflammation. Suppression of CCL2 therefore holds promise in treating neuroinflammatory disease. Accordingly, we sought to determine if the compound bindarit, which inhibits CCL2 synthesis, could repress the three NVU sources of CCL2 most commonly reported in neuroinflammation – astrocytes, microglia and brain microvascular endothelial cells (BMEC) – as well as modify the clinical course of neuroinflammatory disease.in vivo was similarly investigated by qRT-PCR following LPS injection in mice. And to further gauge the potential remedial effects of bindarit on neuroinflammatory disease, its impact on the clinical course of experimental autoimmune encephalomyelitis (EAE) in mice was also explored.The effect of bindarit on CCL2 expression by cultured murine astrocytes, microglia and BMEC was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bindarit action on mouse brain and spinal cord in vivo following LPS administration. Bindarit also significantly modified the course and severity of clinical EAE, diminished the incidence and onset of disease, and evidenced signs of disease reversal.Bindarit repressed CCL2 expression by all three cultured cells, and antagonized upregulated expression of CCL2 in both brain and spinal cord in vivo. It further modulated the course of clinical EAE in both preventative and therapeutic ways. Collectively, these results suggest that bindarit might prove an effective treatment for neuroinflammatory disease.Bindarit was effective in suppressing CCL2 expression by cultured NVU cells as well as brain and spinal cord tissue  The chemokine CCL2  is a critical mediator of neuroinflammation in a myriad of diseases states, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) , AlzheimThese actions and pathogenic role, along with the fact that constitutive expression of CCL2 in the healthy central nervous system is severely limited , render While highly effective in moderating neuroinflammation experimentally, many pharmacological agents that abrogate CCL2 expression and/or activity have nevertheless failed clinically. This disappointing performance in clinical trials might stem, in part, from overly broad suppression of microglia and astrocytes, a potential caveat that could curtail beneficial action of these cells in resolving neuroinflammation ,29, as wAn attractive candidate in this regard is the well-characterized compound 2-((1-benzyl-indazol-3-yl) methoxy)-2-methyl propionic acid (bindarit) . A smallin vivo was well determined. And to gauge bindarit’s potential clinical efficacy, its effect on EAE, a prototypical neuroinflammatory disease [Given this clinical history of bindarit suppressing various examples of peripheral inflammation, we investigated its effect on expression of CCL2 in culture by the three cell types that represent the most frequently reported CNS sources of this chemokine during neuroinflammation: astrocytes, microglia and brain microvascular endothelial cells (BMEC). These cells serve as integral components of the neurovascular unit (NVU)  and, via disease ,45, was 35-55 was synthesized by the WM Keck Biotechnology Resource Center at Yale University, New Haven, CT, USA.All reagents and antibodies were purchased from Sigma-Aldrich , unless specified otherwise. Bindarit was synthesized by and obtained from Angelini . MOG peptidein vivo experiments, bindarit was prepared as a suspension in 0.5% methylcellulose (MTC) at a concentration of 20 mg/ml as previously described [For experiments with cultured cells, a stock solution of 100 mM bindarit was prepared in dimethyl sulfoxide (DMSO), and dilutions  of the DMSO stock were made in culture medium. For escribed .2-mediated euthanasia carried-out, according to the Animal Care and Guidelines of the University of Connecticut Health Center .C57BL/6 mice were obtained from the Charles River Laboratories, Inc. . All animal studies were performed, and COd-glucose, 4 mM sodium bicarbonate, 100 μg/ml penicillin and 100 μg/ml streptomycin. The tissue was mildly triturated to produce a single cell suspension, and the dissociated cells plated onto tissue culture flasks (T-75 cm2) coated with poly-lysine . Cultures were maintained up to 1 week in plating medium in a humidified atmosphere (5% CO2) at 37 °C. After this time, cultures were shaken at 200 rpm for 2 hr at 4 °C, and supernatants containing dislodged microglia collected. Supernatant material was then centrifuged at 1000 × g for 5 minutes to pellet microglia. Microglia were then resuspended in microglia culture medium  supplemented with 10% heat-inactivated fetal calf serum, 100 μg/ml penicillin and 100 μg/ml streptomycin) and cultured in a 24-well plate. Following removal of microglia from the initial mixed glial cultures, the latter were shaken for an additional 18 hr at 37 °C to remove residual neurons. The enriched astrocyte population that remained was further depopulated of remaining microglia by treatment with L-leucine methyl ester (LME) [Brain tissue obtained from mice at postnatal days 2 to 3 was used as the source of astrocytes and microglia. After decapitation, brains were removed immediately and separate astrocyte and microglial cultures prepared following a modified version of the protocol described by Ge and Pachter . Cerebraer (LME) . LME wasBMEC were isolated as previously detailed by this laboratory ,48. PrimEscherichia coli Serotype 026:B6) for 4 or 20 hr in the continued presence of bindarit. After treatments, cells were extracted for RNA purification.To examine effects of bindarit on basal CCL2 expression, cultured microglia and BMEC were incubated with different concentrations of bindarit for 4 hr or exposed to 300 μM bindarit for different time. To gauge effects of bindarit on LPS-stimulated CCL2 expression, astrocytes and microglia were pretreated with 300 μM bindarit for 1 hr; then cells were incubated with ± 100 ng/ml LPS . Then, 4 hr after LPS injection, mice were euthanized and brain and spinal cord dissected for CCL2 mRNA and protein analysis.To determine effects of bindarit on LPS-stimulated CCL2 expression in brain and spinal cord, C57BL/6 female mice were given intraperitoneal (i.p.) injection of bindarit (200 mg/kg) or methylcellulose vehicle, once a day, for 4 consecutive days. At 30 minutes following the last bindarit injection, mice were given i.p. injection of LPS  of rat origin, by a modification of the method previously described [Mycobacterium tuberculosis  in complete Freund’s adjuvant (CFA) (DIFCO) into the right and left flank, 100 μl per site. Mice were also injected i.p. with 200 ng pertussis toxin  in PBS on days 0 and 2 following the first immunization. Animals were monitored and scored daily for clinical disease severity according to the following scale: 0 = normal; 1 = tail limpness; 2 = limp tail and weakness of hind legs; 3 = limp tail and complete paralysis of hind legs; 4 = limp tail, complete hind leg and partial front leg paralysis; and 5 = death. Several disease parameters were calculated as described [For active induction of EAE, C57BL/6 female mice were immunized with MOGescribed . Brieflyescribed . The meaTo investigate the effects of bindarit on both the clinical course of EAE and CCL2 level during disease, mice were given daily i.p. injection of bindarit (or vehicle MTC) at 200 mg/kg for three consecutive days, beginning the day before MOG immunization (day −1), then injections every other day till day 20. This schedule was designed to mitigate, as much as possible, trauma associated with daily injections at times of peak neurologic disease and physical compromise.Total RNA was extracted from cell cultures using the RNeasy kit according to the manufacturer’s instructions. RNA was treated with Turbo DNAse  according to the protocol provided by the manufacturer. RNA yield and purity were determined by spectrophotometry absorption at 260 and 280 nm.RNA and protein were differentially extracted from the same mouse brain and spinal cord samples using the AllPrep RNA/Protein kit  following the manufacturer’s instructions. RNA was treated with Turbo DNAse  according to the protocol provided by the manufacturer. RNA yield and purity were determined by spectrophotometry absorption at 260 and 280 nm. Protein level was determined using the Micro BCA protein assay kit , using bovine serum albumin as a standard.cDNA was synthesized from the total RNA using a SuperScript III  First-strand synthesis system for RT-PCR with a standard protocol. The resulting cDNA was stored at −80 °C until used for further analysis.Measurements of cDNA levels were performed by quantitative (q) RT-PCR using an ABI PRISM 7500 Sequence Detection System Version 1.3, and SYBR green  fluorescence was used to quantify relative amplicon amount. Separate controls included a no template-control and no reverse transcriptase-control, and standard curves were constructed for all primers used. Cycle time (Ct) values for all samples were normalized to RPL-19, the housekeeping gene encoding the 60 S ribosmal protein L19. Specifically, relative amplicon quantification was performed using the formula: in vivo LPS studies, relative CCL2 gene expression values  were expressed as percentage of control. For EAE studies, relative CCL2 gene expression values were designated as percentage of RPL-19 expression, as control CCL2 level (time-point 0) was undetectable. The primer sequences used in this study were as following: for mouse CCL2, 5′- GGC TCA GCC AGA TGC AGT TAA-3′ (forward) and 5′- CCA GCC TAC TCA TTG GGA TCA −3′ (reverse); for RPL-19, 5′- CGC TGC GGG AAA AAG AAG-3′ (forward) and 5′- CTG ATC TGC TGA CGG GAG TTG −3′ (reverse).For all cell culture studies and The level of CCL2 was measured with mouse JE/CCL2 commercial enzyme-linked immunoassay kit  according to the manufacturer’s instructions.t-test, while comparisons of bindarit treatment on LPS-treated mice were performed using a two-tailed t-test for independent samples. For analysis of bindarit effects on clinical EAE, a chi-squared (χ2) test was used for comparisons of disease incidence; a Mann–Whitney U-test was used for comparisons of disease index; and a two-tailed t-test for independent samples was used for comparison of disease onset. A P-value < 0.05 was considered significant in all cases.Statistical significance of differences between mean values of bindarit-treated cultures and control cultures was analyzed using a paired two-tailed The effects of bindarit on cultured glial and BMEC were investigated first Figures , 2, 3. FBMEC demonstrated a similar qualitative response in basal CCL2 mRNA to increasing bindarit concentration, but suppression was not as severe as seen with microglia  it blocked LPS induction of CCL2 in both brain and spinal cord; and 2) it therapeutically modified the course of EAE while suppressing CCL2 expression in both brain microvascular and parenchymal compartments.Given the success of the CCL2 synthesis inhibitor bindarit in ameliorating several animal disease models and human clinical conditions associated with peripheral inflammatory disease, initial studies were conducted to examine its effects on elements critical to neuroinflammatory disease. Focusing on the three main cell types responsible for CCL2 expression during neuroinflammation, experiments revealed bindarit significantly suppressed CCL2 in cultured BMEC, microglia and astrocytes. Bindarit was further shown to be effective As to the effects on the seminal sources of CCL2, it was critical to determine whether each was susceptible to bindarit, as the specific cellular pool(s) responsible for CCL2’s pathogenic actions during neuroninflammatory disease remain unclear . While ain vivo in mice suffering acute EAE [That bindarit also suppressed CCL2 mRNA in BMEC is noteworthy. As these cells form the first line of defense in the BBB , their ecute EAE , as wellcute EAE , possiblcute EAE , and hascute EAE ,76. Bindcute EAE , possiblin situ in the naïve state [in vivo might well exert profound influence on pathologic events.Bindarit action on CCL2 expression by cultured astrocytes had to be studied in the context of LPS stimulation, as these cells exhibit barely detectable CCL2 mRNA in culture or ve state ,51. Yet ve state , suppresin vivo to effectively suppress neuroinflammation was evident in both the LPS and EAE paradigms. Injection of bindarit dramatically reduced LPS-stimulated expression of CCL2 in both brain and spinal cord, dropping mRNA levels to near 10% of vehicle-injected control values, while cutting protein levels approximately by half. In this case, the efficacy of bindarit in suppressing brain CCL2 may have been aided by the fact that LPS can severely disrupt the BBB [That bindarit could indeed act  the BBB ,80, and  the BBB , CCL2 geet al.[The effects of bindarit on clinical EAE suggest that bindarit exerted both preventative and therapeutic actions. Preventative action is indicated by the considerable delay in disease onset in the bindarit-treated group, as well as the reduced incidence and severity of disease displayed by these mice. Possible therapeutic action is conveyed by the steady decline in disease severity following diminished peak clinical score. Such decline was in marked contrast to the typical plateau in clinical score exhibited by EAE mice given vehicle. These results are qualitatively similar to those recently reported by Laborde et al. who, empThe effect of bindarit on clinical EAE was accompanied by significant reduction of CCL2 mRNA in both brain microvessel and parenchymal fractions, consistent with bindarit’s mechanism of action being inhibition of CCL2 transcription . Reinforin situ. Bindarit was further effective in delaying, preventing and attenuating clinical EAE, and evidenced signs of possibly reversing disease course while also suppressing elevation of CCL2 in brain microvascular and parenchymal compartments. Collectively, these data are consistent with the widely proposed critical role for CCL2 in neuroinflammation [In summary, the CCL2 synthesis inhibitor bindarit, previously shown to be highly effective in myriad experimental disease models as well as human conditions having inflammatory involvement -40, was ammation -20, and ammation , might hBBB, Blood–brain barrier; BMEC, Brain microvascular endothelial cells; CFA, Complete Freund’s adjuvant; CNS, Central nervous system; Ct, Cycle time; DMSO, Dimethyl sulfoxide; EAE, Experimental autoimmune encephalomyelitis; GAG, Glycosaminoglycan; GFAP, Glial fibrillary acid protein; Hepes, Hydroxyethyl piperazineethanesulfonic acid; i.p., Intraperitoneal; LME, L-leucine methyl ester; LPS, Lipopolysaccharide; MTC, Methylcellulose; MS, Multiple sclerosis; NVU, Neurovascular unit; PBS, Phosphate buffered saline; PCR, Polymerase chain reaction; RT, Reverse transcription.The author(s) declare that they have no competing interests.SG, BS, DP and CK performed all the experiments. SG assisted with design of the experiments and data analysis, prepared the figures, and participated in drafting the manuscript. AG and RC assisted with the data analysis. JP designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.


Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV) is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA) to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology.Liquid-based (PreservCyt) cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA . Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center.Nine of 25 (36%) women in the ASCUS group, and 64 of 180 (36%) women in the LSIL group developed cervical intraepithelial neoplasia (CIN) grade 2 or worse during 4 years of follow-up. 162 (74%) women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100%) and LSIL (78.1 and 75.8%) groups, although specificity was insufficient (<50%). HPV16 DNA testing and repeat cytology were more specific than APTIMA.The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL. Cytology-based cervical cancer screening programs have significantly reduced the incidence and mortality of cervical cancer Persistent infection with high-risk human papillomavirus (HR-HPV) is a prerequisite for developing precancerous cervical lesions and invasive cervical carcinoma (ICC) In Sweden today, approximately 8% of all cytological samples show some kind of abnormality, 80% of which are minor  or low-grade squamous intraepithelial lesions (LSIL)) Due to the high sensitivity (>90%) and negative predictive value (NPV) of HR-HPV DNA testing to predict CIN, HPV triage has become an attractive approach for the management of women with ASCUS HR-HPV infections results in progression to cervical cancer in only a small percentage of infected women, after a long period of latency. Thus, detection of mRNA transcripts of HPV genes known to be involved in oncogenesis may be more useful for detecting active and potentially persistent infection than HPV DNA tests. The expression of viral E6/E7 oncogenes of HR-HPV has been proposed as a marker of a transforming HPV infection and relevant clinical progression of cervical disease Studies of mRNA testing have shown consistently high sensitivity and a higher specificity than HR-HPV DNA testing both in primary screening and in ASCUS and LSIL triage The present longitudinal study aims to compare, for the first time, the triage efficacy and usefulness of HR-HPV E6/E7 mRNA testing of APTIMA HPV Assay (APTIMA) to that of HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology in the population-based cervical cancer screening program.The study population was composed of 219 HR-HPV-positive women diagnosed with ASCUS or LSIL within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Details on recruitment have been described elsewhere The mean age of the study participants was 32.0 (range: 23–60 years (standard deviation (SD) 8.5 years)). Half of the women were aged 30 years or younger and there was no statistically significant difference in age between the ASCUS and LSIL groups (p = 0.60). The mean age was 32.8 years (SD 9.0) and 31.9 years (SD 8.4) in the ASCUS and LSIL groups, respectively. The age distribution of the study participants was as follows: 25.1% were 23–24, 23.7% were 25–29, 15.1% were 30–34, 16.0% were 35–39, 10.5% were 40–44, and 9.6% were 45–60 years of age. Women were followed for 4 years after the index ASCUS/LSIL cytology, during which time all histological and cytological results were obtained through medical and laboratory records, and through the Stockholm Oncology Center in cases where the information were insufficient. Yearly follow-up of low-grade disease was performed according to local clinical recommendations. Treatment by conization was performed if low-grade disease persisted after two years or immediately if high-grade disease was diagnosed.HPV DNA testing had been previously performed on the baseline ASCUS/LSIL samples from this study population. HPV DNA was extracted  from a lysed cell pellet of 1 milliliter of the PreservCyt sample. The DNA of 37 HPV types In the present study, PreservCyt samples used for HPV DNA testing were retrieved from the archives and tested by APTIMA . Samples were transferred to 2.9 ml of buffered detergent solution and a 400 µl aliquot of the mixture was then tested according to the manufacturer’s instructions. APTIMA is a qualitative nucleic acid amplification test that detects the E6/E7 mRNA of 14 HR-HPV types , and has been validated for cervical specimens in PreservCyt medium The most severe histological or cytological diagnosis recorded during the 4-year follow-up was considered as the outcome. Accuracy parameters for the prediction of CIN2+ and CIN3+ were computed for APTIMA, HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. The parameters calculated included: sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), diagnostic odds ratio and likelihood ratios (LR), stratified by ASCUS and LSIL diagnosis at baseline. Relative sensitivities, specificities, PPV, and NPV with 95% confidence intervals (CIs) were computed for APTIMA compared to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology at three different cut-off levels : ASCUS+, LSIL+ and high-grade squamous intraepithelial lesions or worse (HSIL+).Analyses were performed using Stata 13 .This study was approved by the Regional Ethical Review Board in Stockholm, Sweden (No. 04-679/3 and No. 2010/944-32) and written informed consent for all women were obtained before inclusion.With regard to the distribution of the index smears, LSIL dominated (87.0%). All 219 women were HR-HPV DNA-positive and HPV16 was the most frequently detected HPV type in the ASCUS (20.7%) and LSIL groups (31.1%). The second most common HPV type in the ASCUS group was HPV53 (17.2%), while HPV51 and HPV52 were prevalent in the LSIL group (15.3%). HPV18 was found in 13.8% of the ASCUS and 11.6% of the LSIL group. In the ASCUS group, 31.0% were HPV16/18-positive as compared to 41.1% of the LSIL group. In total, 162 women (74.0%) were mRNA positive with the APTIMA-test, 17 (58.6%) from the ASCUS and 145 (76.3%) from the LSIL group . The majResults for repeat cytology within 12 months were the following: normal cytology was recorded in 113 (57.0%), ASCUS in 16 (8.1%), LSIL in 53 (26.3%), and atypical squamous cells-cannot rule out high-grade lesions (ASC-H) in 17 (8.6%). Histopathological results for 209 women during follow-up were missing in 8 (3.8%) women, not representative in 3 (1.4%), no CIN in 56 (26.8%), CIN1 in 69 (33.0%), CIN2 in 37 (17.7%), and CIN3+ in 36 (17.2%). Nine of 25 women in the ASCUS group (36.0%) and 64 of 180 (35.6%) women in the LSIL group developed CIN2+ during 4 years of follow-up.Altogether, 205 women came for a follow-up visit within 12 months from the index cytology, whereof 198 had a repeat cytology and, if indicated by colposcopy, a biopsy was taken. Seven women had only a biopsy test result available. Four women came for follow-up visits later than 12 months and therefore their cytological test result could not be categorized as a repeat cytology. Ten women were lost to follow-up for unknown reasons. The characteristics of those that were lost to follow-up did not differ largely from the women that were followed, although the mean age was 30.3 years (slightly younger).The sensitivity of APTIMA to predict CIN2+ and CIN3+ was 77.8% (95% CI 40.0–90.0) and 100.0% (95% CI 40.0–100), respectively in the ASCUS group. Specificity to predict the absence of CIN2+ or CIN3+ was 50.0% (CI 30.0–70.0) and 45.5% (CI 30.0–60.0), respectively , Table 2APTIMA was the most sensitive test for triage in the ASCUS group, but the difference reached statistical significance only when compared with repeat cytology using a cut-off of HSIL+ to predict CIN2+ . APTIMA was significantly less specific than HPV16 DNA testing to predict CIN2+ and CIN3+ (relative specificity 0.6 (CI 0.3–0.9) and 0.6 (CI 0.3–0.9), respectively) and significantly less specific than repeat cytology using a cut-off of LSIL+ and HSIL+ to predict CIN3+ (0.6 (CI 0.3–0.9) and 0.4 (CI 0.3–0.7)) .The PPV for all test options ranged from 29.4–100.0% for CIN2+ and from 12.5–100.0% for CIN3+. The PPV of APTIMA (46.7% (95% CI 24.8–69.9) for CIN2+ and 20.0% (95% CI 7.0–45.2) for CIN3+ was lower than that of the other tests, with the exception of repeat cytology using a cut-off of ASCUS+ to predict CIN2+ , and HPV 16/18 DNA testing to predict CIN3+ , but the differences were not significant. The relative PPV of APTIMA compared to repeat cytology using a cut-off of HSIL+ was 0.5 (CI 0.3–0.8) to predict CIN2+ and 0.2 (CI 0.1–0.6) to predict CIN3+  which weThe risk of disease following a negative triage test result  ranged from 5.3–11.8% for the outcome CIN3+, with the exception of APTIMA, for which no risk was detected. A negative APTIMA test resulted in a lower risk of disease compared to the other tests, but the difference was only significant when compared to repeat cytology using a cut-off of LSIL+ and HSIL+ .The sensitivity of APTIMA was 78.1% (95% CI 70.0–90.0 and 75.8% (95% CI 60.0–90.0) for predicting CIN2+ and CIN3+, respectively, in the LSIL group. The specificity for predicting the absence of CIN2+ and CIN3+ was 25.0% (95% CI 20.0–30.0) and 23.8% (95% CI 20.0–30.0), respectively . APTIMA PPVs ranged from 36.5–76.9% for CIN2+ and from 18.2–46.2% for CIN3+. The PPV of APTIMA was lower than the other tests, but this was only significant when compared with repeat cytology using a cut-off of HSIL+, where the relative PPV was 0.5 (CI 0.3–0.7) for CIN2+ and 0.4 (CI 0.2–0.8) for CIN3+.The risk of disease was still high when triage tests were negative (cNPV ranged from 28.0–32.7% for CIN2+ and from 14.0–18.6% for CIN3+.). A negative APTIMA test did not result in or predict a decrease in the risk of disease compared to other tests, and the risk of CIN3+ was significantly higher for APTIMA negative women compared to negative HPV16 DNA, HPV16/18 DNA, or repeat cytology result using a cut-off of ASCUS+.Most tests showed accuracy estimates that did not deviate strongly from the neutral diagonal line (LR+ and LR− near 1), indicating poor triage capacity .HPV infection is a necessary factor in the etiology of ICC In the current study, APTIMA detected 100% of CIN3+ and 77.8% of CIN2+ in the ASCUS group. APTIMA was the most sensitive test to predict high-grade CIN compared to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology at three different cut-off levels. The specificity of APTIMA to exclude CIN2+ in the ASCUS group was 50.0%, and the PPV was 46.7%.HPV DNA testing is widely accepted for ASCUS triage due to its higher sensitivity and similar specificity compared to repeat cytology (12), but newer assays like RNA-based APTIMA have also shown good performance in ASCUS triage (due to its higher specificity) APTIMA-negative women in the ASCUS group had the lowest risk of disease, and no risk for CIN3+ was found. In the LSIL group, APTIMA was the most sensitive test, detecting 76% and 78% of all CIN3+ and CIN2+, respectively, but the least specific test (24–25% for detecting CIN3+ and CIN2+). Specificity for CIN3+ might not be considered useful in a clinical setting, as it would consider CIN2 results false-positives, which is not appropriate as most screening programs use CIN2 as the cut-off for treatment Our results are lower than the pooled sensitivity (91.0% and 96.7%) and specificity (42.5% and 38.7%) of APTIMA to detect CIN2+ and CIN3+ among LSILs reported in the aforementioned meta-analysis A limitation of this study could be that specimens were stored for up to 5 years at room temperature in PreservCyt medium before APTIMA testing, as longer storage times might lead to mRNA degradation. However, two other studies have used PreservCyt specimens that had been stored for more than 3 years Strengths of the present study are the case verification and the data quality of the follow-up, based on unique personal identification numbers as part of the organized screening program and register via medical records and in cases with insufficient information data were supplemented with information from the Stockholm Oncology Center. The long observation time covers an entire 3-year screening interval which allows us to comment on the performance of the triage tests within the context of a programmatically relevant follow-up period.Previous studies have compared the APTIMA test with the PreTect HPV-Proofer mRNA test  in women with ASCUS or LSIL cytology. The PreTect test detects mRNA of five HR-HPV types . APTIMA was substantially more sensitive (ratio 1.91 (95% CI: 1.43–2.56) but less specific (ratio: 0.47 (95% CI: 0.34–0.63) for CIN2+) In summary, the tests evaluated showed accuracy estimates that indicated poor LSIL triage capacity, and the risk of disease remained even when triage tests were negative, indicating that these women cannot return to routine screening. In LSIL triage, our results suggested that, using a cut-off of ASCUS+, a negative HPV16 DNA, HPV16/18 DNA, or cytology resulted in lower risks of CIN over follow-up as compared to a negative APTIMA result. APTIMA showed only a limited ability to stratify the LSIL group according to disease risk, and therefore cannot be used to efficiently triage women with LSIL. Additional markers that can effectively triage these women and avoid over-treatment while not overlooking relevant lesions are needed. In the present study, APTIMA predicted 100% of CIN3+ and 77.8% of CIN2+ in the ASCUS group, making it the most sensitive test for detecting underlying high-grade cervical lesions. This corroborates existing data that APTIMA is an excellent test to identify those women with ASCUS who have a higher risk of developing high grade lesions and ICC.


Despite an increase in the number of molecular epidemiological studies conducted in recent years to evaluate the association between human papillomavirus (HPV) and the risk of breast carcinoma, these studies remain inconclusive. Here we aim to detect HPV DNA in various tissues from patients with breast carcinoma using the method of HPV capture combined with massive paralleled sequencing (MPS). To validate the confidence of our methods, 15 cervical cancer samples were tested by PCR and the new method. Results showed that there was 100% consistence between the two methods.DNA from peripheral blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue were collected from seven malignant breast cancer patients, and HPV type 16(HPV16) was detected in 1/7, 1/7, 1/7and 1/7 of patients respectively. Peripheral blood, tumor tissue and adjacent normal tissue were also collected from two patients with benign breast tumor, and 1/2, 2/2 and 2/2 was detected to have HPV16 DNA respectively. MPS metrics including mapping ratio, coverage, depth and SNVs were provided to characterize HPV in samples. The average coverage was 69% and 61.2% for malignant and benign samples respectively. 126 SNVs were identified in all 9 samples. The maximum number of SNVs was located in the gene of E2 and E4 among all samples. Our study not only provided an efficient method to capture HPV DNA, but detected the SNVS, coverage, SNV type and depth. The finding has provided further clue of association between HPV16 and breast cancer. Breast cancer is the second most common type of cancer worldwide, with the highest prevalence rate among women in the world and 1/8 of women suffer from breast cancer during lifetime There were increasing studies reporting on the involvement of HPV DNA in breast cancer in recent years, but the conclusions remained to be highly controversial. Di Lonardo et al. Previous studies were mostly based on PCR based approach, in which unique primers targeting HPV genes were designed to screen the existence of virus DNA. This strategy is easy to access and screen in large number of samples, but limited in sensitivity and specificity, thus probably contribute to so many controversial studies. Here, we adopted the sequence capture and MPS method to detect the signal of HPV DNA fragment which also provided unique opportunity to study HPV variations in its whole genome level. The study validated the efficiency of the novel method in breast cancer and discovered HPV DNA can be detected in blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue of breast cancer patient, furthermore analyze the variations among them.In this study, we not only provided an efficient method to capture HPV DNA in tissues of breast cancer patients, but also found HPV SNVs difference in the samples. This provided new insight in studying the association between HPV infection and breast cancer.9 sets of breast cancer samples were collected respectively from 9 female patients and received surgery at the department of vascular and endocrine surgery, Xijing hospital, the Fourth Military Medical University, China, between October 2010 and October 2011 . ExclusiThe 9 sets of samples were respectively from 7 malignant breast cancer patients whose ages ranged from 37 to 85 and 2 benign breast cancer patients. For malignant breast cancer, each set included 4 types of tissues . For benign patients, each set included 3 types of tissues . The blood samples were collected before surgery and the tissue samples were preserved in liquid nitrogen with programmed freezing in 1 hour after isolation. All samples were used to detect signals of HPV DNA.http://www.ncbi.nlm.nih.gov/Traces/sra/) under the accession number SRA124368 (SRP035570).All 34 read datasets are accessible through the NCBI Short Read Archive were used to design the HPV probes by MyGenostics . Sequencing libraries of 170 bp insert size were constructed following the instruction of Illumina. Genomic DNA was sheared to around 150 bp–200 bp DNA fragments by Covaris E-210 . These fragments were purified, end blunted, “A” tailed, and adaptor ligated. 10 cycles of PCR were performed after size selection in 2% agarose gel. The concentration of libraries was quantified by Bioanalyzer 2100 .The hybridization process was carried according to MyGenostics GenCapTM Target Enrichment Protocol. Libraries were hybridized with HPV probes(Including 17 high-risk types of HPV) at 65°C for 24 hours and then washed to remove un-captured fragments. The eluted fragments were amplified by 18 cycles of PCR to generate libraries for sequencing. Libraries were quantified and preceded to 101 cycles' paired-end index sequencing using the Illumina HiSeq 2000 sequencer according to manufacturer's instructions .The 100 bp paired-end reads were preceded into bioinformatics process. The paired-end reads which contained the signals of HPV were picked out, and then the mapping ratio, depth, coverage and SNVs on HPV genome of these reads would be obtained. The details of the bioinformatics process are presented as follows .Low quality reads  and duplication reads, as well as adaptor contaminated reads, were firstly removed. The remained clean reads were obtained for subsequent analysis.Clean reads were mapped to human genome  and HPV genome  using SOAP2 (-l 40 -v 5 -r 1) If a paired-end reads were not able to map onto human genome but were able to map onto HPV genome, it would be reported as signals of HPV DNA for subsequent analysis.The coverage, depth and mapping ratio on HPV genome were calculated based on these reported reads. A sample was considered HPV positive if the Coverage of the sample on HPV genome was higher than 50%.Covered_region means the size of the covered region on HPV genome by reported reads; genome_size here means the size of the HPV genome. We also calculated average depth and normalized depth.TNBC means the number of bases mapped on HPV genome. Effective reads number means the total number of reads after the process of Raw data filter.For one certain sample, the depth reflects the reads number covered on the HPV16 genome in raw data. Normalized depth was defined to make the depth comparable among samples with different data quantities.The SNV of HPV genome was called based on the alignment results of the reported reads using soapsnp (-r 0.0005 -e 0.001 -t -u -L 100) 1.The Quality score of consensus genotype is not smaller than 20; 2. Count of uniquely mapped second best base/Count of uniquely mapped best base >0.2; 3. Count of uniquely mapped second best base is not less than 2.The criterions of quality control of SNVs calling were: It's well accepted that HPV plays an important role in cervical cancer. We obtained 15 cervical cancer samples which included 5 negative samples and 10 positive samples by PCR diagnosis. We carried on the HPV capture in these 15 samples, and found 100% accuracy in the result with our method. We analyzed 9 sets of samples collected respectively from 7 malignant breast cancer patients and 2 benign breast tumor patients. HPV16 (K02718.1) was the only HPV type detected in all of the samples. For malignant breast cancer, HPV16 was found only in the set of samples from patient C080. We also found HPV16 DNA in 1/2, 2/2, 2/2 of patients with benign breast tumor, in blood, tumor and adjacent normal tissue respectively. MPS metrics and clinical data were reported in Additionally, we tried to detect the HPV integrations using the method of HIVID To further analyze any discrepancies among various tissues, we tried to identify the SNV pattern of HPV16 in all the samples . For theThen we surveyed the SNV number of transition and transversion in different tissue types of all patients. Four tissue types of C080 had the same trend that there were more transversions than transitions. The normal and tumor tissue of T009 had the contrary trend with set samples of C080. Then weWe also performed comparisons between malignant and benign samples for the same tissue type and found that almost every tissue type from malignant sample set C080 and benign sample set T009 shared some common SNVs .In patient C080 with malignant tumor, the numbers of SNV were 24, 9, 20 and 9, respectively in blood, tumor tissue, and adjacent normal tissue and lymph tissues. 3 common SNVs were identified in all tissues which were located in the position 178, 846, 3448 of HPV genome . AccordiTo pinpoint the SNV variations among the different tissue types in C080, we compare the tumor SNVs with other tissues and identify one SNV was tumor specific .There are enough evidences for the relationships between HPV and cervical cancer The PCR based approach has dominated the previous molecular investigations of HPV in breast cancer, and made significant contributions in some discoveries. Although convenient in common molecular laboratories, it lacks enough sensitivity for further sequence analysis and stability which to some extent revealed by so many controversial reports.MPS technology has been widely used to study the origin, selection and evolution of virus in recent years. The novel method we introduced here combined MPS with virus-target probe capture, as well as high-performance bioinformatics algorithm which are capable to detect signals of target virus and analyze its sequences. MPS makes large-scale paralleled detection of virus signal possible and enables us to reconstruct the virus genome in base-pair resolution, and the capture technology greatly enriched the interested region thus reduced the cost. Our method is not only able to detect virus signals in multiple tissues, but also has the ability to detect the signals of multiple viruses at the same time in a single run with high specificity and sensitivity. To avoid the potential false positive signals introduced by contamination reads, we set strict filtering of 50% of HPV genome is covered by at least one read for a sample to be called as HPV positive. This cut off was adopted to judge the existence of HPV in our 15 cervical tumors, and showed a 100% consistency with PCR result.HPV16 DNA was detected in several tissue types of both malignant and benign samples from breast cancer patients using our new method. Analyzing the coverage, depth distribution, mapping ratio and SNVs of virus genome are helpful for us to find some clue about the association between HPV16 and breast cancer.Traditionally HPV was thought to only infect cutaneous and mucosal tissues but not transmissible via blood. Some discoveries of HPV DNA in blood had brought this notion into question. Chen AC. et al. The tissue type which included more SNVS had significant difference between malignant and benign samples. More SNVS were included in the blood and normal tissue of C080, while the tumor and normal tissues of T007 and T009 included more SNVs. Most of SNVS were located in the E2 region. The papillomavirus E2 protein is required for viral replication and regulates both viral transcription and replication, and therefore plays a central role in the viral life cycle. In addition, E2 is also important for repressing oncoprotein transcription. The mutation on E2 region may have an effect on these aspects in order to keep adaptive Furthermore, common SNVs of malignant cancer sample set C080  and that of benign tumor sample set T009  were significantly discrepant. The SNVS of 178,846 had been identified as the most frequent sequence variation site by previous studies and they had been identified as markers of the Asian lineage We carried on the SNVS categorization according to the synonymous and non-synonymous mutations. The total SNVS number was 126. We got more than 126 SNVS when performing the analysis of synonymous and nonsynonymous mutations. Some SNVS located in two genes region was the cause of the phenomenon. For example the SNVS located in the E4 gene could also located in the E2 gene. The synonymous mutations do not change the protein coding and are not affected by natural selection,while nonsynonymous mutations alter the protein sequences and can be affected by natural selection Improvements of awareness in patient and physician about necessity in treatment for HPV infection might be the preferred strategy for clinical prevention. Wang T et al. In our study, we not only found several samples which had high coverage and depth of HPV, but also detected significant SNVs difference in distribution, number and type. The finding implies that a possible causal role of HPV infection in breast cancer carcinogenesis could not be ruled out. We believe the finding has provided further clue of association between HPV16 and breast cancer. Certainly, there is a need of further study to confirm the role of HPV16 in breast cancer.Table S1Summary of clinical data and sequencing analysis.(XLSX)Click here for additional data file.Table S2Supplementary Results and validation of 15 cervical cancer samples.(XLS)Click here for additional data file.Table S3SNVs of all the HPV positive samples.(XLS)Click here for additional data file.Table S4The distribution of SNV in HPV gene.(XLSX)Click here for additional data file.Table S5The information of whether the SNV is synonymous or nonsynonymous.(XLS)Click here for additional data file.Table S6The distribution of SNVS of Nonsynonymous and synonymous in benign and maligant samples.(XLS)Click here for additional data file.Table S7The common SNVs of the same tissue type.(XLS)Click here for additional data file.