This study examined attitudes to human papillomavirus (HPV) testing among a purposively selected sample of women from four ethnic groups: white British, African Caribbean, Pakistani and Indian. The design was qualitative, using focus group discussion to elicit women's attitudes towards HPV testing in the context of cervical cancer prevention. The findings indicate that although some women welcomed the possible introduction of HPV testing, they were not fully aware of the sexually transmitted nature of cervical cancer and expressed anxiety, confusion and stigma about HPV as a sexually transmitted infection. The term ‘wart virus’, often used by medical professionals to describe high-risk HPV to women, appeared to exacerbate stigma and confusion. Testing positive for HPV raised concerns about women's sexual relationships in terms of trust, fidelity, blame and protection, particularly for women in long-term monogamous relationships. Participation in HPV testing also had the potential to communicate messages of distrust, infidelity and promiscuity to women's partners, family and community. Concern about the current lack of available information about HPV was clearly expressed and public education about HPV was seen as necessary for the whole community, not only women. The management of HPV within cervical screening raises important questions about informed participation. Our findings suggest that HPV testing has the potential to cause psychosocial harm to women and their partners and families. Major changes are taking place in cervical screening. The development of sensitive and cost-effective tests for high-risk types of the human papillomavirus (HPV) . The UK Although HPV testing in cervical screening offers potential public health benefits, it also raises some difficult issues for women who undergo screening. As a sexually transmitted infection (STI), HPV is extremely common among the sexually active population, but awareness of the role of the virus may bring stigma to cervical cancer, and resurrect social and psychological barriers to screening that impact negatively on women. Screening for a sexually transmitted agent has important implications  eligible for cervical screening within Greater Manchester were recruited from social and community groups by ethnically matched community researchers, who conducted focus group discussions. Participants from four ethnic groups , varying in age, marital/partner status and socioeconomic position (measured via education), were selected using principles of purposive sampling, a method of nonrandom sampling of participants possessing certain characteristics, to provide a range of demographic backgrounds and experiences of interest to the research area , during July and September 2000, in English, Gujarati or Urdu as appropriate. The group discussions were tape-recorded, transcribed verbatim and translated into English where necessary. To ensure that all participants had the same baseline knowledge, basic information about cervical cancer and screening, and detailed information about HPV testing was provided at the beginning of the discussion session. Information about HPV concerned high-risk types associated with cervical cancer and covered viral transmission, latency, prevalence and association with CIN, and included information about the potential utility of HPV testing within primary cervical screening. All information was approved by clinical specialists (AS and MD). Core questions explored attitudes towards HPV testing covering: reactions to HPV as an STI linked to cervical cancer; anticipated reactions to testing positive for HPV; partner, family and community attitudes to HPV testing, and religious and cultural influences.a priori issues, emergent issues and recurring attitudes or experiences. Once the first themes are identified they are applied to further transcripts where they are refined so that they encapsulate the diversity of participants' experiences.Data were analysed using framework analysis . This isWomen expressed ‘worry’, ‘shock’, ‘surprise’ and even ‘fear’ in response to the information that cervical cancer was linked to a sexually transmitted virus. Responses were set against a background in which cervical cancer itself was perceived as a ‘serious’ and frightening disease. Few women were fully aware of the sexually transmitted nature of cervical cancer. There was confusion between high-risk HPV types  and other HPV types such as genital warts or plantar warts. Women described concern about transmission of infection from warts on hands to the genital and cervical area during sex, and questioned the impact of these and other HPV types  to their risk of CIN.The stigma associated with STIs was implicit in women's dramatic reactions to HPV testing and was similar across all of the ethnic groups studied. Reactions such as ‘I would cry for a month’ and ‘panic’ indicate the strong negative emotional response to HPV as an STI. Stigma was explicitly mentioned by some women, who highlighted the negative connotations of the term ‘wart’, particularly its link with genital warts , which seemed to have a long-standing stigma as an STI, and also with other types of wart. ‘Warts’ in any form were viewed as unpleasant and unwanted, and these perceptions were described as being rooted in early childhood experience that involved others avoiding contact and exclusion from physical education activities carried out barefoot  Blame. Who was to blame for the infection dominated responses to testing HPV positive. Women described anticipating feelings of ‘anger’, ‘hurt’ and ‘suspicion’ towards their current sexual partner and questioned the history of previous sexual partners. Women, particularly those in the Indian and Pakistani groups, also expressed serious concern and, in some cases, fear that their partner might attribute the blame for HPV infection to them, irrespective of their own previous sexual behaviour.(ii) A test of fidelity. It was suggested by women in the Indian and Pakistani groups that the test might be used as a test of faithfulness, ‘we will know if our husbands are being faithful or not’, and an indicator of how much their partners ‘care’.(iii) Protection. Among women in all the ethnic groups studied, the prospect of testing HPV positive raised questions about protection and safe sex behaviour. Women were told in the study information that protection offered by condoms was uncertain, yet they still identified condoms as the means to protect themselves from HPV. The prospect of using condoms raised worries about the complexities involved in suggesting condom use to partners, which could be perceived as indicating ‘mistrust’ and ‘suspicion’, particularly among women with long-term partners/spouses. It was also suggested that becoming HPV positive, after making a deliberate and emotionally costly effort to use condoms, would add to women's feelings of frustration and helplessness. Abstaining from sex completely was suggested by a few women as a possible strategy for preventing HPV: ‘why bother to have sex when you know it may cause you a problem?’(iv) Attending for HPV testing and testing positive for HPV were identified as potentially communicating unwanted messages to one's partner, family and community.Women from all the groups, but notably Indian and Pakistani women, highlighted the messages of distrust or infidelity that may be conveyed to one's partner by simply attending screening, particularly for women in long-term monogamous relationships.Attending screening or testing positive for HPV was also suggested as implying promiscuity and sexual activity to others such as family (particularly parents) and friends, for women in all the groups. Women from the Indian and Pakistani groups suggested that there may be denial that HPV testing is necessary, with their communities viewed as not at risk of sexually transmitted disease. However, such views were not considered to be held universally and were attributed to older family or community members and less relevant for ‘educated’ men and women.For the Indian and Pakistani women, testing was also perceived to reflect nontraditional cultural or religious practices concerning sex and monogamy to their family and community. Muslim beliefs were described by some of the Pakistani women as potentially prohibitive of screening, drawing on examples of the limits imposed on abortion, women visiting male doctors and concerns that cervical screening may affect unmarried women's virginity, with Shari’a lawBox 3There was a universal expression of the need for more information about HPV: ‘many women need to know this information’. The belief that women ‘should’ have information was forcefully expressed with a desire for a better understanding of disease risk and ‘transmission’, to enable women to make ‘choices and changes in their lifestyles’. The recognition that HPV testing had implications beyond the individual led to the request for information to be provided not only for women, but for men and also the wider community: ‘we need to educate the community’. It was recognised, in all groups, that clear and accurate information was critical to everyone's response to HPV and the potential impact of HPV testing. The current lack of accessible information about HPV and its potential to lead to problems was also raised: ‘ignorance causes many problems’.These findings highlight some of the complex issues that may be raised by the introduction of HPV testing in cervical screening. A positive test result was anticipated to lead to considerable anxiety and distress, and HPV was stigmatised as an STI and associated with genital warts, which held their own long-standing stigma. There was confusion between high-risk HPV, genital warts and other low-risk wart types, and concern about cervical cancer risk and transmission. This has practical implications for giving information to women and suggests that the use of the term ‘wart virus’ to describe high-risk HPV, a common practice among health professionals, may encourage confusion and exacerbate stigma.The prospect of testing positive for HPV raised important issues for women's relationships in terms of trust, blame, fidelity and protection through safer sex, and testing had the potential to communicate unwanted messages to one's partner, family and the wider community about trust and sexual behaviour. Concerns were expressed by women from all of the ethnic groups, but appeared to be particularly pertinent for some of the Indian and Pakistani women, for whom sex outside marriage can be strongly proscribed. In such circumstances, HPV infection (or perceived risk of infection) would have more significant consequences since it could not be openly attributed to sexual partners encountered prior to marriage. These findings have serious implications particularly among some South Asian women, whose chosen sexual lifestyle may directly impact on their family and wider community , which compares to 90% within the national population . Although some categories of women were numerically under-represented in the study sample, the full range of women, and their views, were represented.Study participants were purposively selected to represent heterogeneity of attitudes and experiences to HPV and cervical screening relating to their ethnic, socioeconomic and sexual relationship background, and this paper sought to illustrate the key themes that emerged across the sample. As is usual in qualitative research, participants were not selected to be statistically representative of their ethnic and socioeconomic group; rather, they were selected to represent a range of women eligible for cervical screening so that a diversity of experiences and attitudes could be covered. Almost all women had participated in screening previously (93%, Although the study is qualitative and our sample was purposively selected, its findings may be generalised beyond the study population . The fin


To identify risk factors for various cervical abnormalities, 237 women with abnoromal cervical smears and 422 control women were interviewed. Cervical biopsy specimens taken from the patients with abnormal smears were reviewed according to standard criteria by one pathologist and classified as follows: 65 carcinoma in situ, 81 severe dysplasia, 44 mild dysplasia and 47 normal histology. Factors associated with risk of mild dysplasia, severe dysplasia and carcinoma in situ were similar to those previously identified for invasive carcinoma, and included age at first intercourse, multiple sexual partners and pregnancy outside marriage. Analysis to disentangle correlated factors revealed that number of sexual partners exerted effects independently of age at first intercourse, whereas the reverse was not true. This finding fails to support suggestions that adolescence is a period when the cervix is most vulnerable to the effects of sexual behaviour. Other factors relating to risk of cervical abnormalities were smoking and use of oral contraceptives. It was not possible to show that these relationships were incidental, but further investigation is required to establish whether they are causal.


The presence of high-risk human papillomavirus, loss of heterozygosity on chromosome 3p and fragile histidine triad gene expression were assessed as potential markers of cancer and CIN progression in 83 cervical cancers and 74 cervical intraepithelial neoplasia grade 1 lesions. Human papillomavirus type 16 was an indicator of vascular involvement in cancers. Loss of heterozygosity, especially in the fragile histidine triad gene intron 5, was an indicator of high-grade tumours, greater tumour depth and lymph node involvement. Abnormal fragile histidine triad gene expression was more frequent in cervical intraepithelial neoplasia grade 1 lesions with increased risk of disease progression. Cervical cancer is the second most common cancer in women worldwide and is nearly always associated with one or more of the human papillomavirus types which have been shown to carry a high oncogenic risk (HR HPV) (Loss of heterozygosity (LOH) is an early indicator of genomic instability, which increases the risk of malignancy. In many human cancers, LOH is frequently observed in the short arm of chromosome 3 (3p) . This siLoss of heterozygosity in 3p and reduced FHIT expression have been reported in cervical cancer and precancer lesions  indicative of progressive disease. Genetic markers with significant associations were then analysed in 74 low-grade CIN1 lesions to see if these markers could predict the progression of cervical disease.Archival paraffin blocks of cervical biopsies and residual cervical cells after a smear preparation were obtained from 108 unselected women (mean age 50.9±16.3 years) referred to the Queen Elizabeth Hospital, Gateshead, Tyne and Wear for cervical cancer treatment. In all, 25 cancers were excluded from the study. Out of these, 11 were metastases from organs other than the cervix and 14 contained no amplifiable DNA.* in the text. Four CIN1 biopsies contained no amplifiable DNA and were excluded from the study. Data from the remaining 83 cancers and 74 CIN1 lesions are included in this report. All histology was read by a consultant histopathologist.A total of 78 archival paraffin blocks of loop biopsies containing only CIN1 were obtained from women (mean age 33.4±9.5 years) referred for colposcopy. Of these, 24 biopsies were from women who had been referred following a diagnostic punch biopsy showing CIN2/3. In these cases, all of the CIN2/3 lesion was presumably contained and removed in the punch biopsy since only residual CIN1 was found at all levels in the loop biopsy. These lesions are referred to as CIN1μm sections were prepared from each paraffin block. Section 1 was stained with haematoxylin and eosin (H&E), section 2 was stained for proliferating cell nuclear antigen (PCNA) to confirm the proliferative activity of epithelial cells, sections 3 and 6 were stained for FHIT after epitope retrieval, sections 4 and 5 were stained with methyl-green to locate the lesion prior to micro-dissection and section 10 in CIN1 cases was scraped and used for HPV testing. For each case, the sections stained with H&E and PCNA were photographed and stored digitally. The diseased and normal tissues to be dissected were selected after agreement between two observers and dissected from the corresponding methyl-green-stained sections.In all, 10 serial 10 All paraffin-embedded sections were dewaxed with xylene and re-hydrated in graded alcohol and water. For the H&E stain, Gill's haematoxylin and eosin Y were used. FHIT immunostaining and assessment were carried out after epitope retrieval as described previously (μl T/E containing Tween 20 (1%) and proteinase K . Digestion was carried out for 96 h at 55°C with three additions of fresh PK at 24 h intervals. PK was inactivated subsequently at 98°C for 10 min.Sterile double-distilled water was used throughout. Each section was dewaxed three times with DE-WAX (InnoGenex) for 5 min and washed with 70% ethanol. After soaking in water for 5 min, the section was stained with methyl-green (1%) in water for 1 min. Excess stain was removed by washing with water and excess water was removed by gentle shaking. While the section was still moist, normal or diseased tissue was microdissected using a 25 G needle with the help of a dissecting microscope set up inside a tissue culture hood  for 13 HR HPV types were carried out as described previously  or single-nucleotide polymorphism (SNP) markers. The chromosome locations of these markers are taken from the Human Genome Assembly version hg16 (July 2003). A combination of MS and SNP analyses allows LOH to be localised and validated.A total of 13 MS loci were analysed in cancers  at a given marker was calculated as:-Association of molecular and prognostic markers was assessed using Fisher's exact test or HPV16 was preferentially associated with squamous carcinoma and HPV18 with adenocarcinoma, respectively  (Tables 4A total of 29 CIN1 lesions were positive for HR HPV, of which 10 contained HPV16. The incidences of HPV16, HR HPV as a group and LOH in and close to FHIT intron 5 were not significantly different in the two CIN1 categories, but the incidence of abnormal FHIT protein expression was significantly higher in CIN1In all, 93% of cervical cancers contained HR HPV. HPV16 was the most common and was detected in 61% cancers overall and 71% of squamous carcinomas. Although HR HPV is known to be pivotal in the development of cervical cancer, its role in cancer progression as assessed here for HPV16 against histological markers was found to be limited to an association with vascular involvement. HPV16 occurred more frequently in moderately differentiated cancers and did not show any preference for adenosquamous carcinoma, a histological type which is usually poorly differentiated and has a lower 5-year survival rate . Abnorma* lesions should indicate a greater potential for progression because there is good evidence to suggest that co-existing lesions, even when topographically distinct, could have a common origin and could be under the influence of the same ‘field’ effect (* lesions have a similar incidence of HPV16 or other HR HPV (per se may not be sufficient to predict progression of CIN1. Interestingly, the incidence of abnormal FHIT protein expression in CIN1* is similar to that reported for CIN2/3 lesions (It is of current interest to find markers which can identify those CIN1 lesions which are likely to progress to more severe grades, and HR HPV and the FHIT gene are two likely candidates. In this study, we considered that CIN1r HR HPV  so that  lesions . If thes


Human papillomavirus (HPV) causes cervical, vulvar, and vaginal cancers, precancerous dysplasia, and genital warts. We report data for the longest efficacy evaluation to date of a prophylactic HPV vaccine. In total, 552 women (16–23 years) were enrolled in a randomised, placebo-controlled study of a quadrivalent HPV 6/11/16/18 L1 virus-like-particle vaccine with vaccination at months 0, 2, and 6. At regular intervals through 3 years, subjects underwent gynaecologic examination, cervicovaginal sampling for HPV DNA, serum anti-HPV testing, and Pap testing, with follow-up biopsy as indicated. A subset of 241 subjects underwent two further years of follow-up. At 5 years post enrolment, the combined incidence of HPV 6/11/16/18-related persistent infection or disease was reduced in vaccine-recipients by 96% (two cases vaccine versus 46 placebo). There were no cases of HPV 6/11/16/18-related precancerous cervical dysplasia or genital warts in vaccine recipients, and six cases in placebo recipients . Through 5 years, vaccine-induced anti-HPV geometric mean titres remained at or above those following natural infection. In conclusion, a prophylactic quadrivalent HPV vaccine was effective through 5 years for prevention of persistent infection and disease caused by HPV 6/11/16/18. This duration supports vaccination of adolescents and young adults, which is expected to greatly reduce the burden of cervical and genital cancers, precancerous dysplasia, and genital warts. The human papillomavirus (HPV) family is a heterogeneous group of epitheliotropic viruses of which more than 100 genotypes have been fully sequenced to date .HPV infection is common, with a lifetime risk exceeding 50% . The preIn a recent multinational study conducted in 5455 young women who reported a lifetime history of four or fewer sexual partners, nearly one-third had serologic or molecular evidence of infection with high-risk (oncogenic) HPV types 16 or 18, or low-risk (nononcogenic) HPV types 6 or 11 . Owing tThe trial (Merck protocol V501-007) was a Phase II, randomised, multicentre, double-blind, placebo-controlled study of a quadrivalent HPV 6/11/16/18 L1 VLP vaccine. A total of 1158 women aged 16–23 years were recruited in Brazil, Nordic countries , and the USA. The study enrolled nonpregnant, healthy women who had no prior abnormal Pap smears, and reported a lifetime history of four or fewer male sex partners. Among virgins, enrolment was limited to those women who were ⩾18 years of age and seeking contraception. This study did not exclude subjects with prior HPV infection. Participants were asked to use effective contraception during the trial. All subjects or parents/legal guardians signed informed consents following review of the protocol procedures. The study was conducted in conformance with applicable country or local requirements regarding ethical committee review, informed consent, and other statutes or regulations regarding the protection of the rights and welfare of human subjects participating in biomedical research.μg) to provide appropriate safety comparators for the different vaccine formulations. Efficacy, safety, and tolerability through 3 years have been described for the three formulations . The placebo contained the same adjuvant and was visually indistinguishable from vaccine.The quadrivalent vaccine consisted of a mixture of four recombinant HPV type-specific VLPs composed of the L1 major capsid proteins of HPV types 6, 11, 16, and 18 synthesised in During the initial 3-year study, a 0.5-ml dose of quadrivalent vaccine or placebo was administered by intramuscular injections at day 1, month 2, and month 6. A gynaecological examination was conducted at day 1 and at months 7, 12, 24, and 36. A ThinPrep™ Pap test  and external genital, lateral vaginal, and cervical swabs for HPV DNA testing were obtained at day 1 and at months 7, 12, 18, 24, 30, and 36. External anogenital and vaginal lesions noted during the study were biopsied. Serum samples were obtained at day 1 and months 2, 3, 6, 7, 12, 18, 24, 30, and 36. In the extension, 241 subjects from Brazil and the Nordic countries underwent two further years of follow-up. Subjects enrolled in the extension phase had additional follow-up visits at months 54 and 60, which included gynaecologic examination, cervicovaginal sampling for HPV DNA, serum anti-HPV and Pap testing .All Pap Testing and histologic evaluations were performed within the study. Pap tests were read using The Bethesda 2001 System (in situ (AIS), vulvar intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VaIN), external genital warts or cervical, vulvar, or vaginal cancer with vaccine-HPV-type DNA detected in tissue from, or a swab of, the same lesion and in cervicovaginal samples obtained at the visit antecedent to the biopsy visit. The latter condition was not required if vaccine-HPV-type DNA was detected in biopsy thin sections.The study's efficacy objective was to evaluate the impact of the quadrivalent HPV L1 VLP vaccine with respect to the composite end point of persistent HPV 6-, 11-, 16-, or 18-related persistent infection or cervical or external anogenital or vaginal disease, as compared to placebo. The persistent infection component of the primary efficacy end point was defined as follows: the same vaccine-HPV-type DNA detected in cervicovaginal samples collected at two or more consecutive visits  or vaccine-HPV-type DNA detected in a sample collected during the last visit before being lost-to-follow-up. Vaccine-HPV-type-related disease was defined as a tissue sample diagnosed by consensus of the Pathology Panel as cervical intraepithelial neoplasia (CIN), adenocarcinoma The analyses presented here were conducted based on an interim frozen file created using a visit cutoff date of 13 February 2006. The interim frozen file was generated for an FDA Advisory Committee meeting. At the time of the interim frozen file, 226 out of 241 (94%) of subjects had complete data through month 60 that was available for analysis. The primary analysis of efficacy was conducted in type-specific perprotocol populations which consisted of subjects who were PCR and seronegative to HPV 6, 11, 16, or 18 at enrolment, remained PCR-negative to the same vaccine-HPV-type(s) (to which they were naïve at enrolment) through 1 month postdose three, received three doses of vaccine or placebo within 1 year, and did not violate the protocol. For example, to be eligible for the perprotocol population for HPV 16-related end points, a subject had to be both HPV 16 seronegative and HPV 16 DNA-negative by PCR at enrolment, and remain HPV 16 DNA-negative through 1 month postdose three. Prevalent or incident infection with HPV 6, 11, or 18 was not an exclusion criterion for HPV 16-related end points.Follow-up for case ascertainment in the perprotocol population started 1 month postdose three. Vaccine efficacy (VE) was defined as 100% (1−(risk of becoming a case in the vaccine group/risk of becoming a case in the placebo group)). An exact conditional procedure was used to evaluate VE under the assumption that the numbers of cases in the vaccine and placebo groups are independent Poisson random variables. The number of cases in the vaccine group follows a binomial distribution, which is conditional on the total number of cases in the two vaccination groups combined being fixed. This conditional binomial distribution which accounts for any differential follow-up was used as the basis for calculating the point and exact 95% 2-sided confidence interval (CI) estimates of VE . Each suSupportive prespecified analyses were conducted in a modified-intention-to-treat (MITT) population that included all subjects who were naïve to the relevant HPV type(s) at enrolment and had received at least one vaccination. Protocol violators were included. Follow-up for case ascertainment in the modified-intention-to-treat population started 30 days after the first vaccination.Anti-HPV 6, 11, 16, and 18 responses were measured in quadrivalent vaccine recipients who were seronegative to the relevant HPV type(s) at day 1 and remained PCR-negative to the same HPV type(s) through month 60, and who had serology data at month 60. Anti-HPV 6, 11, 16, and 18 responses were also measured in placebo recipients who were seropositive and PCR-negative to the relevant HPV type at day 1 and who had serology data at month 60.The studies were designed by the sponsor  in collaboration with clinical site investigators. The sponsor collected the data, monitored the conduct of the study, performed the statistical analysis and coordinated the writing of the manuscript with all authors. Data were unblinded for statistical analyses after the data were screened for accuracy and completeness, protocol violators were identified, and the databases were locked for the primary analysis at month 36 . Among the 224 extension subjects, there was one case of infection in quadrivalent vaccine recipients  and 22 cases of persistent infection or disease in placebo recipients .High efficacy through 5 years was also observed when considering only study data from those perprotocol subjects who were eventually enrolled in the extension phase  and month 54 (the first visit in the extension phase) there was a small chance that cases of persistent infection may have occurred in this window which were not captured within the study. A sensitivity analysis was thus conducted to estimate the impact of the 18-month gap on VE. The sensitivity analysis involved modification of the definition of persistent infection such that an extension subject with a single time positive infection detected at month 36 or 54 without confirmation of persistence (including samples from any unscheduled biopsies between month 36 and month 54), was additionally counted as a case of persistent infection. The definition of the disease component did not change. Using this modified definition, four placebo and two vaccine recipients were conservatively counted as additional cases of persistent infection due to vaccine-type DNA detected at a single visit at either month 36 or 54, resulting in a total of four cases in the vaccine group and 50 cases in the placebo group. No subject who had an unscheduled biopsy performed between months 36 and 54 had vaccine-type DNA detected in that biopsy sample. No disease cases caused by vaccine HPV types were added in the sensitivity analysis. The VE estimate from the sensitivity analysis was 92% (95% CI: 78–98%).Vaccine-induced anti-HPV GMTs were measured using a competitive assay, which measures the antibody response to known neutralising epitopes. This provides a specific measure of immunogenicity that is clinically related to protection against HPV infection. At month 7 (1 month after dose three) anti-HPV GMTs among quadrivalent vaccine recipients were substantially higher than GMTs among placebo-recipients with a previous history of natural HPV infection . Though The present study demonstrates sustained long-term efficacy and immunogenicity for a multivalent vaccine targeting the HPV types, which are responsible for a significant proportion of anogenital cancers and genital warts. Over 5 years of follow-up, the incidence of HPV 6-, 11-, 16-, or 18-related disease was reduced by 100% among women who received at least one dose (MITT population), compared with placebo recipients. A persistent and measurable immune response equal to or greater than that observed during natural infection was maintained through 5 years. Although the study was not originally powered to conduct analyses of VE for the disease end points, separately phase III studies conducted in >18 000 women have shown this quadrivalent vaccine to be 100% effective against cervical, vaginal, and vulvar precancerous lesions and genital warts through 2 years of follow-up in the perprotocol efficacy population , providiImportantly, there were no breakthrough cases of disease through the entire 5-year study. The design of the study allowed for the full ascertainment of HPV 6-, 11-, 16-, and 18-related genital disease. Both the external genitalia and cervicovaginal tract were sampled. Subjects underwent external genital and cervicovaginal examination at 6-month intervals through 36 months, and then at months 54 and 60. It should be noted that all subjects underwent a mandatory colposcopy at month 36, increasing the likelihood that cervical disease, if present, would be detected. Unscheduled biopsies during the 18-month gap were included in the analysis, and showed that no HPV 6-, 11-, 16-, or 18-related disease was observed among quadrivalent vaccine recipients. In addition, there were no breakthrough cases of infection during the extension phase. All of the 11 cases observed during the extension were in the placebo group . These data support the high observed 5-year duration of efficacy.The WHO defines persistent infection as detection of the same HPV DNA in cervicovaginal specimens obtained in follow-up visits 6–12 months apart in women who are naive for the relevant type at baseline . An addiA potential limitation of the present study is the relatively small number of women who were enrolled in the extension phase. In addition, the study did not follow American women through the extension period due to the predicted difficulties associated with maintaining enrolment for two additional years, as most were recruited from college campuses. However, a previous study of the monovalent HPV 16 component of the quadrivalent vaccine followed 2391 American women through 4 years  and geniIdeally, a prophylactic HPV vaccine would be given to young adolescents before the initiation of sexual activity since HPV is transmitted through genital contact. Our primary analysis considered a susceptible population of women who were naïve to vaccine-HPV-types at baseline. All sexually active individuals are at risk for HPV infection and disease, including those who do not engage in penetrative sexual intercourse. A recent study has shown the 24-month cumulative incidence of HPV infection among virgins before initiation of penetrative sexual intercourse was 15.3% (This study has demonstrated that a prophylactic quadrivalent HPV vaccine induced high-titre anti-HPV serum antibody levels and was highly effective through 5 years for prevention of persistent infection, neoplasia, and genital warts caused by HPV 6, 11, 16, and 18. This duration supports vaccination of adolescents and young adults, which is expected to greatly reduce the burden of cervical and other anogenital cancers, low- and high-grade intraepithelial neoplasias and genital warts.


Not all women infected with HPV-16/18 have detectable levels of HPV-16/18 antibodies, those who seroconvert develop low antibody levels, and seroconversion occurs typically several months post-infection. We evaluated determinants of seropositivity among 646 women infected with HPV-16 and/or HPV-18.10/LiPA25. Odds ratios (OR) and 95% confidence intervals (CI) were computed.Data are from the enrollment visit of the NCI-sponsored Costa Rica HPV Vaccine Trial. Sera were tested for HPV-16/18 antibodies by ELISA; cervical specimens were tested for HPV DNA using HC2 and SPFAmong HPV-16/18 DNA positives, seropositivity was 63.0% and 57.5%, respectively. Among HPV-16 DNA positives, seropositivity increased with lifetime number of sexual partners (p-trend = 0.01). Women with abnormal cytology and/or high viral load had a 1.63-2.79-fold increase in the detection of antibodies compared to women with normal cytology/low viral load. Current users of oral contraceptives had a 1.88-fold  increased detection of antibodies and current users of injectables had a 3.38-fold  increased detection compared to never users. Among HPV-18 DNA positive women, seropositivity was associated with current oral contraceptive use .Factors associated with sustained HPV exposure  were predictive of HPV-16 seropositivity. Hormonal contraceptive use was associated with seropositivity suggesting an effect of hormones on immune responses to HPV. Patterns were less consistent for HPV-18. Follow up of incident HPV infections to evaluate seroconversion and their determinants is needed. Infections with most viruses typically result in rapid generation of antibodies that protect against re-infection. In contrast, not all women infected with human papillomavirus (HPV) 16/18 have detectable levels of anti HPV-16/18 antibodies. Women who seroconvert develop low antibody levels and seroconversion occurs within months and varies among women . The sloSeveral studies have shown that, as expected, sexual behavior is the strongest predictor of HPV-16/18 antibody detection -7. OtherIn contrast to studies that have evaluated the overall determinants of HPV-16/18 seropositivity, which cannot distinguish risk factors for exposure/infection from those associated with seroconversion given infection, little is known about the specific predictors of seropositivity given concomitant infection with cervical HPV-16 or HPV-18. To better understand why some women with cervical HPV-16/18 infection have detectable levels of antibodies while others do not, using data from a community-based HPV-16/18 vaccine trial of 7,466 women aged 18-25 years in Costa Rica, we analyzed the determinants of HPV-16 and18 seropositivity among the subset of 646 women found to be infected with cervical HPV-16 and/or HPV-18 at enrollment.Data are from the enrollment (pre-vaccination) visit of a trial investigating efficacy of an HPV-16/18 vaccine to prevent infections and cervical neoplasia in the provinces of Guanacaste and Puntarenas, Costa Rica. The study design and procedures have been described in detail . BrieflyAt the clinic women gave informed consent and were interviewed. Information regarding sociodemographic factors, reproductive and sexual history, contraceptive use, and smoking were obtained. A physician obtained the medical history and performed a physical exam including a pelvic exam among sexually experienced women. At the pelvic exam, exfoliated cervical cells were collected using a Cervex brush and were rinsed into a vial containing 20 mL of PreservCyt solution. Samples were transported to the laboratory, where two 0.5 ml aliquots were drawn for HPV DNA testing by PCR. ThinPrep slides were prepared for cytology, and the remaining solution was used for HPV DNA detection by Hybrid Capture 2 (HC2). A blood sample was collected from all participants using a 10 ml vacutainer tube without additive. At the local laboratory serum aliquots for the determination of HPV-16 and HPV-18 antibodies by ELISA were obtained and frozen immediately.Protocols were approved by the institutional review boards of INCIENSA, Costa Rica and National Cancer Institute, United States.10 primers followed by DNA enzyme immunoassay detection of amplimers. HPV typing on positive amplimers was performed using line probe assay (LiPA25) , as previously described [HPV DNA detection and genotyping was performed at DDL Diagnostic Laboratory  using PCR amplification with SPFescribed .10 DEIA but negative for HPV-16 or HPV-18 by LiPA25 were tested for the presence of HPV-16 and HPV-18 using type-specific PCR primers set, as previously described [Since the trial uses a bivalent HPV-16/18 vaccine to maximize sensitivity for these types, all specimens that tested positive by SPFescribed ,14.The results of this assay were used to identify the group of women HPV-16 DNA positive or HPV-18 DNA positive at the enrollment visit of the vaccine trial.HC2 is a nucleic acid hybridization assay with signal amplification that combines antibody capture of DNA and RNA probe hybrids and chemiluminescent signal detection. The HPV HC2 test is designed to detect 13 carcinogenic HPV types  without distinguishing the HPV type present. The test was performed according to the manufacturer's instructions in the laboratory at the University of Costa Rica.With each assay a cut-off RLU (relative light units) value is calculated as the mean RLU value of three positive calibrators. Specimens with relative light units/cutoff (RLU/CO) value < 1.00 were considered negative; specimens with RLU/CO value ≥1.00 were considered positive for one or more of the carcinogenic HPV types detected by the test. Given that the magnitude of the RLU value above the cutoff of the assay is indicative of the total amount of high risk HPV type present, the results of this assay were used as a surrogate for HPV viral load.An enzyme-linked immunosorbent assay (ELISA) was used for the detection and quantitative determination of IgG antibodies against HPV-16 or 18 in serum specimens. HPV-16 and HPV-18 antibodies were measured separately; the assay was performed by GlaxoSmithKline Biologicals as described previously . BrieflyOf the 5871 women that at the enrollment visit had a pelvic exam, 8.3% (488) were HPV-16 DNA positive, 3.2% (188) were HPV-18 DNA positive, 0.5% (30) were HPV-16 and 18 positive. We restricted our analysis to the 484 women HPV-16 DNA positive and the 179 women HPV-18 DNA positive with available ELISA results. Additional information regarding the seroprevalence and determinants of HPV16/18 seropositivity for the total population was evaluated in a separate report [Socio-demographic, sexual behavior, contraceptives, smoking and reproductive history characteristics, and cytology and HPV HC2 test results were evaluated as possible determinants of serological response.For each characteristic, we calculated unadjusted odds ratios (ORs) for seropositivity and 95% confidence interval (CIs) using unconditional logistic regression. Of particular interest were variables that could be markers of timing of HPV infection  or of amount/load of exposure . Possible confounding factors were explored and a final model was built for each characteristic of interest adjusting for all other variables that changed the crude OR estimates by 15% or more. The outcome variables were seropositivity for antibodies against HPV-16 and seropositivity for antibodies against HPV-18.We performed a similar set of analyses restricted to women with single HPV-16 and single HPV-18 infections by DNA testing. Results were comparable to those seen overall (data not shown).From the HC2 test we used the ratio of relative light unit values to positive control (RLU/CO) as a surrogate for HPV viral load. Women were classified as having low or high viral load based on the median RLU/CO value observed .We assessed dose response associations (p-trend) by treating ordinal variables as continuous assuming a linear trend in the models. F test for differences in geometric means were calculated using ANOVA. Analyses were performed using Stata 10.0.Among women who were HPV-16 DNA positive, seropositivity for antibodies against HPV-16 was 63.0% (305/484); among HPV-18 DNA positive the seropositivity for antibodies against HPV-18 was 57.5% (103/179).As shown in table Age at sexual debut, years since first sexual intercourse and time with most recent partner were not significantly associated with detection of anti-HPV16 antibodies, although suggestive effect was observed for time with most recent partner . Other factors such as years of education and marital status were not associated with seropositivity (data not shown).Current use of hormonal contraceptives  was associated with seropositivity Table . Women wNumber of pregnancies and smoking were not significantly associated with anti-HPV16 seropositivity Table , althougCytology and HC2 viral load measures were significantly associated with anti-HPV16 seropositivity . Antibody levels were lowest among women with normal cytology and low viral load (GM = 50.0); intermediate among women with either abnormal cytology or high viral load (GM = 71.5); and highest among women with both an abnormal cytology and high viral load .Analyses that parallel those reported above for HPV-16 were performed to evaluate possible determinants of anti-HPV18 seropositivity among HPV-18 DNA positive women. Results are summarized in Tables We assessed the prevalence and determinants of serological response to HPV-16 and HPV-18 infections in a group of 646 women 18-25 years of age with concomitant detection of HPV-16 and/or HPV-18 at the cervix. One strength of this analysis is that unlike previous studies that evaluated determinants of anti-HPV seropositivity irrespective of cervical HPV infection status ,10,20,21The main finding is the observation that factors associated with sustained HPV exposure , were positively associated with anti-HPV16 seropositivity. We also observed that lifetime number of sexual partners and possibly frequency of sexual intercourse were associated with anti-HPV16 seropositivity. To the extent that these factors reflects increased amount of viral exposure among infected women, this finding might also corroborate the association between viral load and seropositivity.We also observed a positive association between current hormonal contraceptive use  and anti-HPV16 seropositivity. The effect of current oral contraceptive use on seropositivity was also observed for anti-HPV18 antibodies. We initially hypothesized that this finding could reflect increased viral load among hormonal contraceptive users given that previous studies suggested that HPV contains hormonal responsive elements and that hormonal exposure may increase viral replication,23. TherThe main limitation of our study is its cross-sectional nature. Since HPV DNA infection and anti-HPV antibodies were assessed at the same timepoint, we cannot determine the amount of time each woman had been infected with HPV-16 and/or HPV-18 at the time of DNA and serum sample collection. Studies that have evaluated time between HPV infection and seroconversion have suggested that, on average, antibody seroconversion occurs 8-12 months after infection, although these studies did not sample frequently enough [Overall, detection of anti-HPV16 and anti-HPV18 antibodies was observed among 63.0% and 57.5% of women infected with HPV-16 or HPV-18, respectively; this seropositivity is consistent with that reported by other investigators that about half of HPV-16 or HPV-18 DNA positive women are seropositive ,24.Despite similar rates of anti-HPV16 and anti-HPV18 seropositivity, our findings for determinants of anti-HPV18 seropositivity were less clear. The only significant predictor of anti-HPV18 seropositivity among HPV-18 infected women was current oral contraceptive use. Possible explanations include differences in the performance of the ELISA assays designed to measure antibodies against HPV-16 and HPV-18, lower power for the HPV-18 analysis since the number of HPV-18 infected women (n=179) was smaller than that of HPV-16 infected women (n=484), or true biological differences resultant from differences in patterns/location of infections caused by these two viruses. The later statement is supported by reports that HPV-18 infections are often under-represented in precancers, and that they are preferentially associated with the development of cervical adenocarcinomas that often arise deep in the endocervical canal ,26.Our evaluation of the prevalence and determinants of anti-HPV16 and anti-HPV18 seropositivity among women with concurrent cervical infection with HPV-16 and/or HPV-18 shows that over half of HPV-16/18 infected women had detectable levels of antibodies to the HPV type with which they were infected. Factors associated with sustained HPV exposure were predictive of seropositivity  as was current hormonal contraceptive use, suggesting a possible effect of hormones on immune responses to HPV. Findings were clearer for HPV-16 than for HPV-18, suggesting the need for additional studies to understand whether these differences are biologically driven or resultant from study design or assay performance. Longitudinal studies that evaluate incidently detected HPV infections and follow these infections to determine whether seroconversion occurs and their determinants are needed.Wim Quint and Leen-Jan van Doorn are employees of DDL Diagnostic Laboratory; Catherine Bougelet is an employee of GSK Biologicals. None of the authors have any potential conflicts of interest to report.All authors contributed to and approved the final version of the manuscript. CP, CB and AH performed the analysis of data and wrote the first draft of the manuscript. AC, RH, SW, DS, MSc and AH designed the study. CP, AC, RH, PG, SJ, AH were responsible for the coordination of the study and recruitment of the participants. LJVD and WQ were responsible for the HPV DNA testing. CB was responsible for the testing of HPV-16/18 antibodies by ELISA. MSa, SC participated in the analysis of data. MH carried out the cytological interpretations.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/238/prepub


Increasing evidence shows that Human Papillomavirus (HPV) is preferentially associated with some head and neck squamous cell carcinomas (HNSCCs), with variable infection rates reported.We assessed HPV involvement in HNSCC using the Roche Linear Array HPV Genotyping Test, which can detect 37 different HPV types. We examined the prevalence of HPV infection in 92 HNSCCs .HPV was frequently detected in oropharyngeal cancers (OPCs) , but was uncommon in oral cavity cancers , and in other HNSCC subsites . HPV positive tumors were associated with patients that were 40-60 years old (p = 0.02), and node positive (p = < 0.0001). HPV 16 was the most prevalent type, but other types detected included 6, 18, 33, 35, 45, and 52/58.Our results show that in contrast to oropharyngeal cancers, oral cancers and other HNSCCs infrequently harbor HPV. In 2009, head and neck squamous cell carcinoma (HNSCC) had an estimated incidence of 48,010 cases  within the U.S. (American Cancer Society), and 4,550 cases  in Canada (Canadian Cancer Society). The most common risk factors for HNSCC development are excessive tobacco and/or alcohol consumption [Most HPV research has primarily focused on cervical cancer, as >99% of cervical cancers harbor HPV . Over 13Data from the literature show widely variable HPV infection rates in HNSCC -10. ThisWe examined the prevalence of HPV in oral squamous cell carcinomas (OSCCs), compared to other head and neck subsites and OPCs. For HPV detection and genotyping, a sensitive and specific PCR-based method, the Roche Linear Array HPV Genotyping test was used for these studies.The University Health Network (UHN) Research Ethics Board approved this study; informed consent was obtained from all patients prior to sample collection. Medical records were examined to obtain detailed clinical and histopathological information, including age, sex, disease site, histopathological diagnosis, disease stage, history of tobacco and alcohol use, nodal metastasis, treatment, and outcome. Subsets of patients were classified as social or rare alcohol drinkers as written in their clinical reports. Patients having no use of tobacco or alcohol within one year prior to surgery were classified as former smokers and drinkers, respectively. Tumors were staged according to the current TNM classification, as recommended by the American Joint Committee on Cancer .Patients over 60 years of age often present with HNSCC, whereas young patients with HNSCC are often characterized as patients under 35-45 years of age . The patThe 92 samples used in this study were retrospectively collected between 1995-2007. 78 HNSCC samples were obtained at the time of surgery from the Toronto General Hospital. Tissues were snap frozen and stored in liquid nitrogen. Also, 14 HNSCC were formalin fixed paraffin embedded samples. H&E stained sections were examined by histopathology (B.P-O.) to confirm >80% tumor in the specimens being tested.DNA was isolated following fresh frozen tissue homogenization in liquid nitrogen using a cold steel mortar and pestle. Homogenized tissue was lysed in SNET buffer , containing 400 μg/mL proteinase K overnight at 55°C. After digestion, 25 mg/mL RNase was added and DNA was extracted by standard techniques using phenol/chloroform and ethanol precipitation. DNA quantity and quality was assessed by spectrophotometry  and electrophoresis on a 0.8% agarose gel. Genomic DNA was isolated from formalin fixed paraffin embedded (FFPE) samples using the DNeasy Blood and Tissue Kit .The Roche Linear Array HPV Genotyping test  was used for the detection of 37 low- and high-risk HPV types, according to manufacturer's instructions. Briefly, it utilizes biotinylated PCR of the HPV L1 region and reverse blotting to multiple HPV genotypes. HPV types were determined by lining up the manufacturer's HPV reference guide with the genotyping strip. A low- and high-copy β-globin internal control is included in each run to assess the quality of DNA sample. All experiments included an HPV positive control, an HPV negative control, and a no-template control. Cases that were HPV positive were repeated, without the presence of a positive control, to verify results and exclude the risk of contamination.Descriptive statistics were examined as median and range for continuous variables, and frequencies and proportions for categorical variables. The Fisher's exact and Pearson's chi-square tests were used for statistical evaluation. Overall survival was calculated using the Kaplan-Meier method. A Cox Proportional Hazard regression model was applied for continuous predictors. Results were considered significant if p ≤ 0.05. Statistical analyses were performed using the SAS 9.1 software package .All 92 HNSCC samples had a positive β-globin internal control on the linear array and were thus suitable for HPV analysis. Of these, 53 were from sites in the oral cavity: tongue, floor of mouth (FOM), palate, buccal mucosa and gingiva; 17 were from other sites ; and 22 were from the oropharynx (base of tongue and tonsil). The oropharyngeal cases were selected for comparison, as these tumors have a higher incidence of HPV infection.HPV positivity was stratified according to distinct HNSCC subsites: 2/53 (4%) OSCCs, 16/22 (73%) OPCs, and 1/17 (6%) tumors from other HNSCC sites were HPV positive. HPV positivity was significantly associated with OPCs (p < 0.0001) (Table vs. node negative patients (p < 0.0001) (Table HPV was significantly associated in node positive patients p < 0.000 (Table 1vs. HPV-negative HNSCCs (p = 0.24)  Table , as non-vs. base of tongue; the latter is associated with a higher HPV prevalence due to the involvement of the oropharynx [vs. HPV-unrelated OPCs from 1973-2004 [et al. used in situ hybridization for HPV detection and showed a higher frequency of HPV in tumors of the oropharynx, low frequency in larynx and oral cavity, and an absence of HPV in the hypopharynx or nasopharynx [In our study, we sought to determine HPV infection rates in OSCC, in comparison to other HNSCC sites such as OPC. We observed significant differences in HPV positivity among different HNSCC sites, with OPCs having the highest infection rate. We also detected a low infection rate within OSCC samples. In the literature, most HPV studies do not provide stratification between oral cavity subsites, e.g. mobile tongue opharynx . Indeed,opharynx . A recenopharynx .In our study, we observed a significant association of HPV positive tumors to be present in HNSCCs from intermediate age patients. It has been suggested that, from the time of sexual transmission of HPV, a clinical lesion can appear within two decades, in contrast to conventional risk factors , which may take several decades .vs. negative tumors, as most OSCCs were HPV negative in our study, and possibly due to the relative small sample size of OPCs analyzed.Interestingly, HPV positive HNSCCS have also been associated with lymph node metastasis. For example, in another study, HPV positive salivary samples were correlated with node-positive patients, suggesting that HPV infection may result from a cellular immunological deficiency that impedes the cancer cell's ability to clear HPV and thus result in cancer development, recurrence and metastasis . Among pet al. detected 7/59 (12%) HPV-18 positivity in oral cancer using quantitative real-time PCR and 0% positivity when using in situ hybridization assays on the same samples [Some studies have shown a higher prevalence of HPV in OSCCs and other head and neck sites outside the oropharynx. For example, examination of HPV status in OSCC from 50 Brazilian patients (excluding OPC), showed 24% HPV positivity, mostly HPV-16/18 . Such di samples . Our dat samples . In our The authors declare that they have no competing interests.JM was responsible for study design and content, literature research, experimental studies, data analysis, and manuscript preparation. PPR was involved in study design, data analysis, manuscript editing and review. TZ performed experimental studies. CS was responsible for data acquistion, and study concepts. WX perfomed the statistical analysis. BPO was involved with the data analysis. DPG, DHB, RWG, PJG, JCI were involved in study concepts, and sample acquistion. SKR was responsible for study content and design, manuscript editing and review.All authors have read and approved the final manuscript.


HPV genotype was determined by the INNO-LiPA HPV genotyping kit. HPV DNA was detected in 17 cases (65%) in Gansu, where ESCC incidence is much higher than in Shandong, where HPV was positive in two samples (6%). HPV genotypes 16 and 18 were detected in 79 and 16% of HPV-positive samples, respectively. Real-time PCR analysis suggested the presence of integrated form of HPV DNA in all the HPV-16-positive samples, but its viral load was estimated to be only <1–2 copies cell−1. We could not detect HPV 16/18 E6 protein expression by immunostaining in any of the HPV-16-positive samples. Neither p16INK4a nor p53 expression was related to HPV presence in ESCCs. Further studies seem warranted to examine the possible aetiological roles of HPV in ESCC.To examine the potential roles of human papillomavirus (HPV) in oesophageal squamous cell carcinoma (ESCC) development, we examined the presence of HPV DNA in paraffin-embedded ESCC tissues collected from two areas with different ESCC incidence rates in China, that is, Gansu ( In malignant transformation of uterine cervical epithelia, integration of high-risk HPV DNA into the host genome is considered an important step  tumour suppressor proteins, respectively, and downregulates their antitumour functions (Human papillomavirus (HPV) is a nonenveloped, double-stranded DNA virus with more than 90 genotypes. To date, molecular and epidemiological studies have convincingly demonstrated that HPV infection with certain genotypes, that is high-risk HPV, plays an essential role in the development of uterine cervical cancer . An extensive review by Syrjanen published in 2002 showed that HPV was positive in 22.9% of 1485 ESCCs analysed by in situ hybridisation (ISH) and in 15.2% of 2020 ESCCs analysed by PCR .HPV DNA detection rates in ESCC samples appear to be different from area to area. An explanation, offered by Syrjanen in his review, is the hypothesis that the contribution of oncogenic HPVs to ESCC risk is higher in the areas with high ESCC risks . Indeed,Taylor INK4a in order to shed light on the aetiological roles of HPV in ESCC development.In the present study, we compared HPV DNA detection rates in Gansu and Shandong, China, where age-adjusted mortality rates of oesophageal cancer were 29 out of 100 000 in 1996–2000  at 55°C overnight. The presence of DNA was confirmed by PCR with β-globin (110 bp) using PCO3 primer 5′-ACACAACTGTGTTCACTAGC-3′ and PCO4 primer 5′-CAACTTCATCCACGTTCACC-3′.Formalin-fixed, paraffin-embedded samples were cut into 10 L1 gene , 50 mM of each primer, 0.2 mM of each dNTP and 10 × HotStar Taq PCR buffer as supplied by the enzyme manufacturer (QIAGEN) 2SO4 pH 8.7). Different amounts of template for each sample were used . The amplification was carried out with initial enzyme activation at 95°C for 15 min, followed by 45 cycles that included a 1 min denaturation step at 94°C, a 2 min annealing step at 40°C and a 1.5 min chain elongation step at 72°C; and a final elongation at 72°C for 5 min. As positive control for amplification, full genomes of HPV 6 and 18  were used, and water as template was used as negative control. PCR products were visualised on 2% agarose gel with ethidium bromide staining by electrophoresis.The presence of HPV DNA was evaluated by PCR using GP5+/GP6+ primers (150 bp), consensus primers for the HPV M NaOH buffer. Hybridisation and detection of HPV DNA was carried out using the ECL Nucleic Acid labelling and detection kit . The probe used to detect HPV DNA was obtained by purifying from agarose gel the GP5+/GP6+ PCR products from cloned HPV 6 and 18 using QIAEX II Extraction kit .DNA from agarose gel was transferred by upward capillary blotting to a Hybond N+ nylon membrane  using 0.4 L1 gene of the HPV genome was amplified by PCR using SPF10 biotinylated primers 5′-GCiCAGGGiCACAATAATGG-3′ and 5′-GTiGTATCiACAACAGTAACAAA-3′, where ‘i’ indicates inosine (μl of this PCR product was denaturated and hybridised with specific oligonucleotide probes (25 HPV type-specific probes) immobilised as parallel lines on a nitrocellulose membrane strips, following the manufacturer's instructions . The 28 probes for 25 different HPV genotypes in each INNO-LiPA strip are described elsewhere . The amplification conditions were 10 min at 95°C and a two-step cycle of 95°C for 15 s and 60°C for 60 s for a total of 45 cycles. The used primer sets were as follows: E6 (81 bp) and E2 (76 bp) ORFs, respectively . There was a linear relationship between the threshold cycle values plotted against the log of the copy number over the entire range of dilutions. All the experiments were made in duplicate. The specificity of amplification was confirmed using dissociation analysis starting at 60°C and agarose gel electrophoresis of amplified products.Those primers amplify a fragment of μm were placed on silane-coated glass slides, and deparaffinised by passage through xylene. After the endogenous peroxidase activity was blocked with 0.3% H2O2/methanol, the slides were rehydrated with distilled water. For antigen retrieval, the slides were heated in 0.01 mol l−1 citrate buffer, pH 6.0, at 95°C for 5 min (at 121°C for p53 and E6) and then left to cool at room temperature (RT). After rinsing in 0.01 mol l−1 phosphate-buffered saline (PBS), pH 7.4, nonspecific antibody binding was reduced by incubating the sections with 1% foetal bovine serum in PBS at RT for 30 min. Then, the sections were incubated overnight at RT with a mouse monoclonal antibody against p16INK4a protein , p53 protein  or HPV 16 E6/18 E6 . After washing thoroughly with PBS, the slides were incubated with biotinylated horse anti-mouse IgG (1 : 200 dilution) for 30 min, washed with PBS followed by a 1 : 50 dilution (1 : 100 dilution for p53 and E6) of the avidin–biotin–peroxidase complex  for an additional 30 min and washed with PBS. The peroxidase signal was visualised by treatment with DAB substrate-chromogen system  for 10 min. Finally, the sections were stained lightly with haematoxylin. In statistical analysis, the cases with less than 10% cells stained positive were classified as negative cases, and the other cases were regarded as positive cases . In the following, we present the results using SPF10 primers.First, we examined all the 59 samples using GP5+/GP6+ primers for PCR and visualised only five HPV-positive cases in agarose gel. When agarose-gel electrophoresis was replaced with Southern blot hybridisation, HPV DNA was detected in an additional 11 cases . As an alternative approach, we examined cancer specimens using SPF10 primers, and amplicons were visualised on 4% agarose gel. In this method, HPV genome was confirmed to be present in all the 16 HPV-positive samples identified by Southern blot hybridisation. In addition, three cases became HPV positive in this new approach, and, in total, we detected HPV DNA in 19 specimens, 17 samples from Gansu (65%) and only two samples from Shandong (6%). The difference in detection rates between the two regions was highly significant . The presence of HPV 16 genome was more frequent in moderately and poorly differentiated carcinomas. The observed association was statistically significant , but was not significant in a multivariate analysis adjusting for area (P=0.813).Since the majority of HPV genotype detected was HPV 16, we compared HPV-16-positive and HPV-negative tumours with respect to clinicopathological features (data not shown). The area difference was statistically significant  cases, E2 DNA was not detected. In the rest of the cases, E2 DNA was detected, but the E2/E6 ratio was less than unity.In the present study, using PCR with SPF10 primers or PCR with GP5+/GP6+ primers combined with Southern blot hybridisation, we detected HPV DNA in 6% of Shandong samples while HPV DNA was positive in 65% of samples from Gansu, where ESCC incidence is much higher than in Shandong. Our finding suggests possibility of a strong geographical difference in the proportion of HPV-associated ESCCs. Note, however, that our ESCC cases were convenient samples and, therefore, may not represent the ESCC cases in the study areas. All HPV strains that could be genotyped were found to be high-risk types. Prevalent HPV genotypes were HPV 16 and 18, which were found in 79 and 16% of ESCC samples, respectively.Detection rate varied upon the method used, with a high sensitivity when using Southern blot hybridisation, and it was even higher when using SPF10 primers PCR. However, even if such a sensitive detection method was used, HPV genome was detectable only in a small portion of cancer specimens collected from Shandong (6%).E2 gene , we determined E2/E6 ratio and found that 92% of the samples had a mixture of episomal and integrated forms of HPV genome, and 8% of the samples had HPV integrated into the host genome. The method, similar to ours, showed that 65–97% of the cervical cancer cases had HPV 16 integration, which was frequently accompanied by episomal-form HPV (Arias-Pulido ).Using real-time PCR, we confirmed the presence of HPV 16 in 15 ESCC samples. Since HPV integration is considered to result in deletion of the E2 gene , we dete−1 in order to make comparisons with the results of other studies  (−1) (Koskinen ). Considering the presence of relatively low viral loads of HPV DNA in some cervical carcinoma cell lines, for example SiHa (1–2 copies of HPV 16 cell−1) and HeLa (10–50 copies of HPV 18 cell−1), the low viral load in ESCC may be sufficient to promote carcinogenesis, especially if viral DNA is integrated into the cell . In our study, we found a downregulation of p16INK4a expression in 68% of ESCC cases regardless of HPV presence. The failure to detect p16INK4a overexpression in HPV-positive ESCC may therefore be explained by the fact that p16 is downregulated through hypermethylation.HPV E6 and E7 are considered to be the major oncoprotein interfering with cell cycle regulation. The E6 protein of high-risk HPV binds to p53, leading to rapid degradation of p53 through the ubiquitin pathway . In the present study, we could not detect viral E6 protein expression by IHC in any of the HPV-16-positive samples.It should be noted that the cancer specimens used in the present study contain noncancerous tissues, and that our findings do not necessarily indicate the presence of HPV in carcinoma cells. Studies showed the presence of HPV 16 E6 protein in the cytoplasm and nucleus of ESCCs, which showed the presence of HPV 16 in PCR analysis as well (Yao ). In the present study, we observed multiple infection of HPV with different genotypes in 16% of the genotyped samples, using INNO-LiPA genotyping assay. Double infections in ESCC have also been reported by a study in China (−1). It is of note that Studies on the presence of HPV DNA in cervical samples showed that 10% or more of clinical lesions contain at least two different HPV genotypes (Schellekens in China . InteresINK4a or p53 protein. Further studies seem warranted to examine the possible aetiological roles of HPV in ESCC.In conclusion, the present study showed that a large proportion of ESCC specimens harbour HPV 16 genome in the integrated form in a certain area with a high ESCC incidence. Detection rate varied upon the method used, with a higher sensitivity when using Southern blot hybridisation or SPF10 primers PCR. Real-time PCR analysis suggested the presence of only a small number of HPV 16 copies in carcinoma cells. There was no HPV 16/18 E6 protein expression, and on top of that, HPV 16 presence was not related to the expression of p16


To provide guidelines for health-care providers on strategies for cervical cancer prevention based on HPV testing and anti-HPV vaccination.Overall efficacy of different preventive strategies, assessing reduction in the incidence of invasive cervical cancer and precancerous lesions.Medline and the Cochrane Database were searched for articles in English on subjects related to HPVs, HPV diagnosis, HPV anogenital lesions, cervical cancer, HPV testing, and HPV vaccines, in order to elaborate an up-dated document. Relevant Italian Government publications and position papers from appropriate health and family planning organizations were also reviewed.The quality of the evidence and ranking of recommendations for practice were rated using criteria defined by SIV, which were adapted from the Canadian Task Force on Preventive Health Care. This report summarizes recommendations on cervical cancer screening and prevention strategies proposed by experts of the Italian Society for Virology  on the basis of systematic review of evidence from the scientific literature and current guidelines produced by International Societies, in view of an European consensus strategy for cervical cancer screening, human papillomavirus (HPV) testing, and anti-HPV vaccination programs. The guidelines were discussed with experts in the field (see acknowledgment section) and with SIV members, and reviewed and approved by SIV Scientific Committee, during the annual SIV Conference of 2008, following a specific Round table on the subject. These recommendations recognize the diagnostic value of HPV testing and genotyping in the management of women with abnormal PAP test and in screening programs for prevention of cervical carcinoma and recommend HPV vaccination for primary prevention of cervical cancer. The searching strategies, the study selection criteria and other methodologies adopted for the literature review can be asked to the correspondig author.The demonstration that human papillomavirus (HPV) infection has a pathogenetic role on cervical cancer development and the understanding of the epidemiology of HPV infection and its relationship with the natural history of precancerous lesions and cervical cancer have been crucial in the development of diagnostic and vaccination strategies for prevention of one of the most common and lethal cancers, especially in developing countries.Epidemiological data on HPV infection and associated disease have been reviewed in a recently published IARC Monograph . Cervicahigh-risk or low-risk types, based on their association with cervical cancer . SSeveral studies demonstrated that HPV testing, performed at 4–6 months intervals following ablative therapy for high-grade lesions (CIN2–3), has a higher sensitivity and specificity than cytology in detecting residual disease or recurrence -75. In aHPV testing at 6–12 months for post treatment management of CIN2,3 is recommended by current guidelines of the American Society for Colposcopy and Cervical Pathology , whereasThe potential role of HPV testing as a stand-alone test or in conjunction with cytology in cervical cancer screening has been investigated in several studies . A meta-analysis including 25 non-randomized clinical trials, which compared the performance of cytology vs. HPV test in screening programs for prediction of CIN2+ , showed et al. [vs. 76.6%, 95% CI 65.1–85.1%) but less specific  for detecting CIN2+ [Likewise, the meta-analysis by Cuzick et al. , which iet al. . The HARng CIN2+ . Comparing CIN2+ . Interesng CIN2+ . Based ong CIN2+ .Randomized longitudinal studies , which hResults from the second recruitment phase of the NTCC study showed that, in women aged 35–60 years, HPV testing alone with a cut-off of 2 pg/mL achieved a substantial gain in sensitivity over conventional cytology with only a small reduction in PPV  , confirmPersistence of the additional lesions detected at baseline by HPV testing in older women was also suggested by the recently published results from two longitudinal randomized controlled trials, that included only women at least 30  or 32 8 years ofA direct comparison between Pap test and HPV test (by using the HC2 assay) as stand alone tests for cervical cancer screening was evaluated in a recent randomized trial in Canada, involving 10,154 women aged 30–69 years . Both teRecommendations on the use of HPV testing in cervical cancer screening programs (grading of recommendations is reported in Table • HPV testing is recommended for ASC-US triage since HPV test is more sensitive than repeat cytology for detection of CIN2+ (IA).• HPV testing is, in general, not a management option in case of LSIL (ID), even if it may be cost effective in older women with LSIL (IB). Local assess is needed to identify a good triage test for women with LSIL (research need).• HPV testing, alone or in combination with cytology, is recommended for follow-up after treatment of high-grade cervical lesions, since it is more accurate than repeat cytology in diagnosing residual disease or relapse (IA).• HPV testing, both based on hybridization (HC2) and PCR amplification with detection of high-risk HPVs, has been widely used in women aged >30 years in addition to Pap cytology because of the high NPV of a combined negative test (I). Women with normal Pap test result and negative HPV test have a very low risk to develop cervical cancer and should not perform the subsequent follow-up visit earlier than 5 years (IA). However, combination of HPV testing and Pap cytology is not a cost-effective strategy (I), so, strategies based on HPV testing as a stand alone test should be preferred in women aged >30 years (IA).• Follow-up with HPV testing or repeat cytology at 12 months is recommended for cytology-negative, HPV-positive women aged ≥ 30 years (IA). Women who are persistently HPV-positive on repeat testing should undergo colposcopy, whereas those who are negative on both tests may be rescreened in 5 years (IA).• Using the HPV test in screening programs leads to earlier detection of CIN2+ and CIN3+ and reduction of the number of high-grade lesions detected at subsequent follow-up in women aged ≥ 30 years (I), thus allowing longer screening intervals. The extent of long-term protection against high-grade lesions after a negative HPV DNA test has been estimated to be longer than 5 years, but further research is need to establish the most appropriate screening interval (research need).vs. HPV testing in screening programs, confirm the higher efficacy of HPV screening that Pap screening (I). Local assessment is needed before screening policies based on primary HPV testing can be recommended (research need).• HPV testing as a stand alone test is more effective than cytology alone in identification of CIN2+ lesions in women aged ≥ 35 years (I) and might be used as a primary screening test in this age category. First follow-up results from ongoing controlled randomized trials, which compare longitudinal performance of Pap test • In women aged < 35 years, HPV testing with immediate referral to colposcopy leads to over diagnosis of CIN2+ as compared with more conservative management strategies (I). Thus, in women younger than 35 years of age with a positive HPV test, cytology triage or repeat HPV testing at 12 months could be appropriate management strategies to avoid over diagnosis and over treatment of CIN2+, but this hypothesis needs confirmation in longitudinal studies (research need).Due to the relatively low specificity of HPV-DNA testing and the high rate of false negative results at Pap test, new molecular tests have been proposed in order to identify women with a higher risk of progression to invasive cancer. These tests include (i) detection of type-specific HPV persistence; (ii) detection of high-risk HPV E6/E7 mRNAs; (iii) HPV genotyping to detect HPV-16 and HPV-18, which have been associated with a higher risk of CIN2,3 or invasive cancer; (iv) measurement of high-risk HPV load; (v) demonstration of high-risk HPV DNA integration; (vi) HPV subtyping by E6, L1, and LCR sequencing; (vii) analysis of p16-INK4A expression. The implementation of these new molecular tests in cervical cancer screening could be useful to identify the subpopulation of high-risk HPV-positive women who deserve a closer follow-up and appropriate treatment.In women younger than 25 years of age, 20% of high-risk HPV infections persist, whereas the risk of persistent infection is over 50% in women older than 55 years . Only a From a practical point of view, persistence can be defined as the detection of the same HPV type  two or more times over a certain period. There is no consensus as to the length of time that implies persistence, but at least 6 months to 1 year is the time frame that is usually chosen . Evaluat® HPV by bioMérieux SA, Marcy l'Etoile, France). Analysis of E6/E7 mRNA expression with the PreTect™ HPV Proofer kit in 204 histologically confirmed invasive cervical squamous cell carcinomas from Norwegian women demonstrated the presence of oncogenic transcripts of HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45 in 97% of HPV-positive cases, thus confirming the expression of oncogenic E6/E7 in most invasive cancers and the involvement of the above HPV types in most cases [® Assay, developed by Gen-Probe Incorporation , employs transcription-mediated amplification and target capture to detect HPV E6/E7 mRNA from 14 high-risk HPV types, but it does not differentiate among the 14 high-risk types. Preliminary results obtained with this assay suggest it has higher sensitivity and specificity than HPV testing for identification of CIN2+ lesions [Cervical cancer is characterized by overexpression of E6/E7 mRNA of high-risk HPVs. A multiplex nucleic acid sequence based amplification system (NASBA) assay was developed for the identification of E6/E7 mRNA from HPV types 16, 18, 31, 33, and 45  is mainly directed against type-specific immunodominant conformational epitopes, although cross-reactivity has been documented, such as between HPV-6 and HPV-11, HPV-31 and HPV-33, HPV-18 and HPV-45. Serum levels of anti-HPV antibodies are stable, even after virus clearance, so HPV serological assays simply demonstrate previous exposure to the virus. Sensitivity of HPV serology is low (50–60%) while specificity is high (about 90%). The low sensitivity of the test is in part explained by lack of seroconversion in some individuals with documented HPV infection [Both antibody-mediated and cell-mediated immune responses are essential for clearance of HPV and HPV-related cervical lesions. The immune response against HPV ; persistent high-risk HPV infection and high-risk HPV E6/E7 expression are necessary events for cervical cancer development and represent risk factors for cervical cancer (I).• HPV typing and subtyping provides epidemiological data on HPV distribution and diffusion (II).• In epidemiological surveys, type-specific HPV detection could contribute to the evaluation of anti-HPV vaccine efficacy (III).• High HPV DNA load is a risk factor for cervical cancer (II).• HPV testing with p16-INK4A triage improves test specificity for CIN2+ (II).• Detection of anti-HPV antibodies demonstrates previous HPV exposure, but this test has low sensitivity (II); a high antibody titre might indicate persistent infection (II).• Patients with invasive cervical carcinoma have a deficient HPV-specific cell-mediated immune response (II).• In order to demonstrate HPV persistence, repeat HPV testing should be done at intervals of at least 12 months (IA).• HPV typing and subtyping in women with a normal or dubious Pap test is useful since it allows to document persistent infection by the same oncogenic HPV type (II) and to demonstrate the presence of HPV-16 and HPV-18 DNA which are markers of increased risk for cervical cancer (II). Women with persistent high-risk HPV infection or with the presence of HPV-16 and HPV-18 DNA should be referred to colposcopy (B).• Measurement of HPV-16 and other high-risk HPV DNA load could be employed for triage of women with dubious Pap test or with a positive HPV test (B), but further research is needed to identify appropriate management strategies for women with high HPV load (research need).• Detection of high-risk HPV E6/E7 mRNA could be employed for triage of women with dubious Pap test or with a positive HPV DNA test (IIB). Due to its high sensitivity and specificity (II) HPV E6/E7 mRNA testing should be investigated as a stand alone test in primary screening for cervical cancer (research need).• HPV typing and subtyping is useful for epidemiological investigation of HPV distribution and diffusion (B).• Detection of p16-INK4A expression could be used as triage of HPV positive cases (B) and abnormal Pap smears (B) and to confirm histological analysis of cervical biopsies (B).• Further research is needed to develop standardized methods to measure anti-HPV immune response (research need).• In epidemiological surveys, evaluation of antibody-mediated and cell-mediated anti-HPV immune response can be used to assess immune response to anti-HPV vaccination and to monitor duration of vaccine protection (B).Two vaccines targeting HPV-16 and HPV-18, the types that are responsible for 70% of invasive cervical cancers ,16, have®, produced by GlaxoSmithKline), which is administered i.m. in three doses , and a quadrivalent alum adjuvanted L1 VLP vaccine against HPV-16, HPV-18, HPV-6, and HPV-11 , which is administered i.m. in three doses . The bivalent vaccine is advised for reduction of precancerous cervical lesions and cancer incidence. The quadrivalent vaccine, besides cervical lesions, is advised for reduction of vulvar precancerous lesions and genital condylomas.Two HPV VLP vaccines have been developed and are available for primary vaccination in the European Union: an AS04 adjuvanted L1 VLP vaccine against HPV-16 and HPV-18  and aged 10–14 years . These studies demonstrated that both vaccines are safe and immunogenic, and induce antibody titre which is more than 50 times the titres induced by natural infection ,150-156.Evaluation of vaccine efficacy in phase II-III clinical trials was based on the demonstration of reduction of the incidence of persistent HPV infection  and on reduction of the incidence of precancerous lesions (CIN2 and CIN3) caused by HPV vaccine types. International standards for treatment of CIN2/3 are ablative therapy. Moreover, in prospective studies, it is not ethical to allow a woman to develop invasive disease in order to demonstrate efficacy of prevention strategies. In this regard, WHO and other international agencies indicated CIN2/3 as clinical endpoint surrogate to demonstrate efficacy of cancer prevention.per protocol analysis, which demonstrates the pure efficacy of the vaccine, obtained by analysis, in the experimentation context, of clinical data of women who did not violate the protocol (woman who received three doses of vaccine or placebo as expected), who resulted negative for all HPV genotypes included in the vaccine both at enrolment or during the administration of the vaccine. (ii) modified intention to treat analysis, where evaluation of efficacy is more similar to the clinical setting in the general population, since it includes all enrolled women, as long as they receive at least the first dose of vaccine or placebo and who resulted negative for all HPV genotypes of the vaccine merely at the time of administration of first vaccine dose.Randomised clinical trials demonstrated that three doses vaccination is effective for the prevention of persistent infection and for precancerous lesions caused by HPV genotypes included in the vaccine. Results on efficacy reported in published studies are mainly two types: (i) Per protocol efficacy analysis demonstrated that Gardasil® reduced by 90%  the incidence of persistent infection and genital lesion associated with the HPV genotypes included in the vaccine [Modified intention to treat analysis demonstrated that vaccination reduced incident infections and cervical lesions by 94%  [A phase II randomised trial was carried out on 552 women (277 randomised in the vaccine group and 275 in the placebo group).  vaccine . A subse vaccine . Modifie3–98.3%) .® were conducted in the context of the two protocols Females United to Unilaterally Reduce Endo/Ectocervical Disease (FUTURE) I and FUTURE II:Phase III efficacy studies of the quadrivalent vaccine GardasilPer protocol analysis in over 10,000 women  aged 15–26 years demonstrated that, a after a mean 3 year follow-up period, vaccine efficacy was 98%  for prevention of CIN2,3 and in situ adenocarcimoma associated with HPV-16 and HPV-18 [Modified intention to treat analysis  in randomised women (95% of the study population), who were negative for the vaccine types at the first dose administration, and who did not strictly adhere to the study protocol, demonstrated vaccine efficacy was 95% . Intention to treat analysis including all randomised subjects, without any pre-vaccine screening, was used to predict efficacy of vaccination on incident/prevalent HPV infections and related lesions in the general population, who demonstrated an impact of 44% (95% CI 26–58%) at 3 years follow-up.- FUTURE II trial. d HPV-18 . It is tPer protocol analysis on over 5,000 women  aged between 16 and 24 years showed that after an average period of 3 years since vaccine administration, the vaccine had 100% efficacy (95% CI 94–100%) for prevention of condylomas, vaginal and vulvar intraepithelial neoplasia related to HPV-6/11/16/18 and 100% efficacy (95% CI 94–100%) for prevention of CIN1–3 or in situ adenocarcinoma associated with vaccine HPV genotypes [unrestricted susceptible population) showed 95% efficacy (95% CI 87–99%) regarding precancerous genital lesions, vulvar or vaginal, or condylomas and 98% efficacy (95% CI 92–100%) for CIN1+ or in situ adenocarcinoma caused by vaccine HPV types. The intention-to-treat analysis showed a 73% efficacy  in preventing external anogenital or vaginal lesions of any degree and a 55% efficacy  in preventing incident and prevalent cervical lesions of any grade associated with vaccine HPV genotypes, at 3 years follow up. In both FUTURE I and II studies, vaccination did not appear to significantly alter the course of infection or lesions due to HPV which were already present before the first dose of vaccine.- FUTURE I trial. enotypes . ModifiePer-protocol efficacy analysis of the results achieved by the combination of three randomized clinical trials of vaccination  versus placebo  showed that vaccination, after an average follow-up period of 3 years, has an efficacy of 100% (95% CI 72–100%) in preventing vulvar and vaginal lesions grade 2 and 3 associated with HPV-16 and HPV-18. Modified intention-to-treat analysis demonstrated an efficacy of 97% (95% CI 79–100%) and intention-to-treat analysis, on a total of 18,174 randomised women, showed an efficacy of the vaccine at three years of 71%  in the prevention of incident/prevalent HPV-associated lesions [-  lesions .Per-protocol efficacy analysis of the results achieved by the combination of four randomized clinical trials of vaccination with the quadrivalent vaccine  and its HPV-16 L1 component  vs. placebo  showed that, after a follow-up of 3 years, in HPV-16 and HPV-18 negative women, vaccine efficacy was 99% (95% CI 93–100) in preventing development of CIN2/3, in situ adenocarcinoma or invasive cervical cancer associated with HPV-16 and HPV-18. Efficacy was 98%, (95% CI 93–100) in the population of modified intention-to-treat analysis. The modified intention-to-treat analysis demonstrated an impact of the vaccine of 44%  in reducing the incidence of cervical lesions associated with HPV-16 and HPV-18, in the case of HPV type vaccine diseases already prevalent/incident in the population [- pulation .per protocol analysis, vaccine efficacy was 91.6%  against incident infection and 100% against persistent infection  with HPV-16/18 at 18 months post-vaccination. In the intention-to-treat analyses, vaccine efficacy was 95.1%  against persistent cervical infection with HPV-16/18 and 92.9%  against cytological abnormalities associated with HPV-16/18 infection. In a subsequent report from the same study, after a follow-up of 4–5 years, per protocol analysis on the results obtained from a group of 732 women (367 in the vaccination group and 365 in the placebo group) who received all three doses of vaccine, demonstrated vaccine efficacy against incident infection was 96.9%  and 100% (33.6%–100%) against persistent infection for 12 months [Efficacy the AS04 adjuvanted bivalent VLP vaccine has been assessed in phase II-III studies. A pilot randomized phase II study on safety, immunogenicity, and efficacy of the vaccine for prevention of incident and persistent HPV-16 and HPV-18 infection enrolled and randomized 1,113 women aged 15–25 years to receive either the vaccine or placebo . Study p2 months . Efficac2 months . In thisIn women positive for HPV DNA, HPV VLP vaccination does not accelerate clearance of the virus nor prevents incident CIN2+ caused by HPV vaccine types. This finding has been demonstrated in a randomized phase III trial in 2189 women aged 18–25 years, who received the AS04 adjuvanted bivalent VLP vaccine  and in t® HPV quadrivalent vaccine on the rates of infection and disease associated with 10 HPV types not included in the vaccine was estimated in a population of women that was, pre-vaccination, seronegative and PCR-negative to all HPV types for which testing was available and had a normal Pap test [Two clinical trials have evaluated the efficacy of Cervarix™ on other oncogenic types -146. ThePap test . Within Pap test .Data on safety of the quadrivalent HPV VLP vaccine reported a higher rate of side effects in the vaccination group than in the placebo group. Side effects included local reaction at the site of injection   and systemic side effects, such as fever  . Also dain situ adenocarcinoma, invasive cervical cancer, high-grade vulvar and vaginal lesions, and genital warts correlated with HPV-16, HPV -18, HPV -6, and HPV -11 (I).• HPV vaccination with the quadrivalent vaccine is safe and efficacious for the prevention of incident high-grade precancerous lesions, • HPV vaccination with the AS04 adjuvanted bivalent vaccine is safe and efficacious for the prevention of incident and persistent HPV-16 e HPV-18 infection and CIN1+ correlated with HPV vaccine types (I)• Long-term efficacy (> 5 years) of HPV vaccination is currently under investigation (research need).• Efficacy of HPV vaccination has been demonstrated in women aged 15–26 years, who had not been previously exposed to HPV vaccine types (I); results on efficacy in women older than 26 years are not available yet (research need).• Results on immunogenicity and safety (but not efficacy) justify vaccination in girls aged 9–14 years (II).• Efficacy of HPV vaccination in males is currently under investigation (research need).• HPV vaccination in males is not considered to be cost-effective (III).• HPV vaccination should be done before exposure to genital HPV, since it lacks efficacy in women who have already been infected (IA), and it does not enhance virus clearance (IA).• Screening for cervical cancer should be recommended both in vaccinated and non-vaccinated women according to current guidelines (II). Screening protocols could be modified only after appropriate clinical trials (research need).• Further research is needed to assess the duration of vaccine protection, variations in the prevalence of HPV types in vaccinated women and in the general population, and surveillance of side-effects of vaccination (research need).• in ASCUS triage (IA).• in the follow-up after treatment of CIN2/3 (IA).• as a screening test for cervical cancer, as an alternative to cytology, in women older than 30 years, with cytology or repeat HPV testing as triage tests (IA).Efficacy in cervical cancer screening has been proven for both PCR-based and signal amplification-based HPV tests (I).• Evaluation of HPV persistence may be useful to identify patients at risk for cervical cancer, who should be referred for colposcopy (A).• HPV typing and subtyping can be used for epidemiological evaluation of HPV distribution and diffusion (B), for identification of persistent infection (B), for identification of HPV-16 and HPV-18, which are associated with a higher risk of precancerous lesions (B) or invasive cervical cancer (B), and for monitoring the efficacy of vaccination (A). But, there is insufficient evidence to define the best protocol to manage women with positive tests (I).• Detection of high-risk HPV E6/E7 mRNA may be useful to identify women at risk for cervical cancer and with worse prognosis (IIB). Further research is needed to define the best protocol to manage women with a positive test and to evaluate test efficacy in primary screening programs.• Measurement of HPV-16 DNA load in women with normal cytology may be useful to identify women at risk for cervical cancer (IIB). But, there is insufficient evidence to define the best protocol to manage women with positive tests (I).• In epidemiological surveys, HPV serological testing and measurement of anti-HPV cellular immune response could be useful to monitor the duration of vaccine protection (B).in situ adenocarcinoma, invasive cervical cancer and genital warts associated with vaccine HPV types in women aged 15–26 yrs (I).• Quadrivalent HPV VLP vaccine is safe and effective in the prevention of cervical and vulvar precancerous lesions, • HPV vaccination with the AS04 adjuvanted bivalent vaccine is safe and efficacious for the prevention of incident and persistent HPV-16 e HPV-18 infection and CIN1+ correlated with HPV vaccine types in women aged 15–26 yrs (I)• HPV vaccine should be administered before genital HPV exposure, since the benefit of vaccination declines in women already positive for HPV DNA (IA).• In epidemiological surveys, HPV serological testing and HPV genotyping could be useful to monitor vaccine efficacy (research need).• Cervical cancer screening is recommended both in vaccinated and non-vaccinated women, in agreement with current guidelines for cervical cancer prevention (III).• Further research is needed to assess the duration of vaccine protection, variations in the prevalence of HPV types in vaccinated women and in the general population, and surveillance on side-effects of vaccination (research need).The authors declare that they have no competing interests.LB drafted the manuscript. CG, FMB, and GP contributed to the preparation of guidelines. All authors read and approved the final manuscript.


These numbers include multiple infection. These findings indicate that the induction of tetrasomy is a property restricted to high and intermediate-risk HPV types but that it is not type-specific. The factors governing which lesions will develop this abnormality are as yet unclear. © 2000 Cancer Research CampaignHuman papillomavirus (HPV) infection appears to be an early event in cervical carcinogenesis with additional abnormalities being required for biological transformation. We have analysed 179 low-grade cervical squamous intra-epithelial lesions (SILs) and 15 normal cervices for the presence of HPV using both in situ hybridization and polymerase chain reaction (PCR). PCR was performed with GP5+/GP6+ primers followed by hybridization using probes for low , intermediate  and high-risk HPVs . Interphase cytogenetic analysis using pericentromeric probes for chromosomes 1, 3, 4, 6, 10, 11, 17, 18 and X was also performed to identify numerical chromosomal abnormalities. Tetrasomy of all nine chromosomes was identified within basal keratinocytes, was restricted to epithelia infected with high risk (17 of 46) or intermediate risk (23 of 83) HPVs but was not HPV type-specific. Tetrasomy was not identified in any of the epithelia infected with low risk HPVs (


Hybrid Capture II (HC-II) is a commercial human papillomavirus (HPV) detection test designed to detect 18 HPV types divided into high-risk and low-risk groups. We have tested 1647 scrapes from 1518 unselected women attending routine cytological screening by this assay for the detection of histologically proven high-grade lesions. The reliability of this test was also evaluated on 117 fresh cone biopsy samples. HPV DNA has been detected in 400 scrapes (24.3%), 296 containing a high-risk HPV (18.0%). All the smears evocative of high-grade lesions were positive for high-risk HPV, and high-risk HPV were detected in all the 34 cases presenting a histologically proven high-grade lesion and in 68 (97.1%) of the 70 cone biopsy samples showing a high-grade lesion or an invasive carcinoma. Thus, the sensitivity was superior to the sensitivity of cytology (85.3%). Nevertheless, the quantitative approach provided by the HC-II assay for the assessment of the viral load could not clearly distinguish among cases with or without high-grade lesions. Thus this assay is recommended for the screening of high-grade lesions on a large scale, in association with classic cytology. © 1999 Cancer Research Campaign