This study assesses knowledge, attitudes, and behavioural intention towards human papillomavirus (HPV) infection and vaccination in a random sample of 1348 adolescents and young women aged 14–24 years in Italy. A self-administered anonymous questionnaire covered demographics; knowledge about HPV infection, cervical cancer, and HPV vaccine; the perceived risk for contracting HPV infection and/or for developing cervical cancer, the perceived benefits of a vaccination to prevent cervical cancer, and willingness to receive an HPV vaccine. Only 23.3% have heard that HPV is an infection of the genital mucosa and about cervical cancer. Those older, with at least one parent who is a health care professional, with personal, familiar, or friendly history of cervical cancer, and having underwent a health checkup in the last year with information about HPV vaccination were significantly more knowledgeable. Risk perception scores (range: 1–10) of contracting HPV infection and of developing cervical cancer were 5.8 and 6.5. Older age, not having a parent who is a health care professional, having had a personal, familiar, or friendly history of cervical cancer, and need of additional information were predictors of the perceived susceptibility of developing cervical cancer. The vast majority professed intent to receive an HPV vaccine and the significant predictors were having at least one parent who is a health care professional, a high perceived risk of contracting HPV infection and of developing cervical cancer, and a high belief towards the utility of a vaccination for preventing cervical cancer. Knowledge about HPV infection and cervical cancer should be improved with more attention to the benefit of HPV vaccination. Human papillomavirus (HPV) is one of the most common sexually transmitted infections in sexually active adolescent girls and young women of several economically developed countries  and the American Academy of Pediatrics, have recommended routine vaccination for girls aged 11 or 12 years . In ItalWidespread acceptance of HPV vaccine is likely to provide enormous public health benefits  in two Universities and six public secondary schools in the geographic area of the Campania region, in the South of Italy, were randomly chosen. A simple random technique was adopted in selecting students from each faculty and school.Before the study, a meeting with the Head and the Dean of each randomly selected school and faculty was arranged to present the project and the permission and collaboration were obtained. A questionnaire was pilot tested on 25 students before initiation of the survey, the objective of which was to identify the basic questions most valid for testing the comprehensiveness and relevance of our terms. As part of the process of informed consent, all potential candidates were invited to participate through a letter containing information about purpose and objectives, also indicating that participation in the survey was voluntary, and that privacy and confidentiality would be strictly protected as no personal identifiers were included in the questionnaire and data would be presented only in an aggregated manner. For those under 18 years of age, a separate information letter was addressed to the parents. These policies were also printed explicitly on the front page of the questionnaire. Provided that respondent and parental consent were obtained, data were collected by self-administered anonymous structured questionnaire administered with a reply envelope. The questionnaires were delivered to students in their classroom one time and were completed at the time of distribution. A respondent's consent was taken into account while filling the questionnaire. The questionnaire was used to explore several topics, including: (1) student characteristics, included age, marital status, employment status, whether they were living alone or not, and whether they have had a personal, familiar, or friendly history of cervical cancer; (2) knowledge about HPV infection (definition and modes of transmission), cervical cancer (definition and preventive measures), and availability of an HPV vaccine; (3) attitudes pertaining to assess whether or not the respondent perceived a risk for contracting HPV infection and/or for developing cervical cancer, opinions about the perceived benefits of a vaccination to prevent cervical cancer, and their willingness to receive an HPV vaccine; (4) behaviour, included questions whether or not respondents have ever had sex, age at first sex, number of acts of intercourse and of partners in the last year, and how frequently they were protected by condoms during intercourse with the partner(s) in the last year; health-promoting behaviour was assessed with questions about how often the respondent underwent a health checkup in the last year, whether respondents' health care provider informed them about HPV vaccine, and whether they have received the HPV vaccine; (5) sources of and needs of information regarding HPV vaccine and cervical cancer.For items regarding their knowledge, participants were asked to provide answers with options for ‘no’, ‘do not know’, and ‘yes’. All attitudes were measured on a 10-point Likert scale with a score ranging from 1 to 10. The responses for the two questions about perceived risk were recorded as 1 for no risk at all and 10 for very high perceived risk; for the perceived benefits of a vaccination to prevent cervical cancer, 1 for not at all and 10 for high utility; and for their willingness to receive HPV vaccine, 1 for unwillingness and 10 for willingness. The respondents were also asked to report directly reason(s) for willingness or unwillingness to receive HPV vaccine. Questions pertaining to behaviours were close ended with nominal or categorical (yes or no) responses; the frequency of the use of condoms in the last year was measured on a five-point Likert scale ranging from ‘never’ to ‘every time’. Ethics committee approval was obtained.P-values were assessed using two-sided tests and statistical significance was taken as a cutoff of P⩽0.05 throughout. The statistical package Stata (Version 8.1) was used for the analysis ; perception of risk of developing cervical cancer (Model 2); willingness to receive an HPV vaccine (Model 3). The following explanatory variables were included in all models: age (continuous), at least one parent is a health care professional , personal, familiar, or friendly history of cervical cancer , at least a health checkup in the last year and the physician informed them about HPV vaccination , number of sexual partners in the last year , and need of information about HPV vaccination and cervical cancer . The following variables were also included: have already heard about HPV infection and cervical cancer  in Models 2 and 3; perception of risk of contracting HPV infection (continuous), perception of risk of developing cervical cancer (continuous), and perceived benefits of a vaccination to prevent cervical cancer (continuous) in Model 3. The significance level for variables entering the logistic and linear regression models was set at 0.2 and for removing at 0.4. Odds ratios (ORs) with 95% confidence intervals (CIs) were used in the logistic regression analyses. All reported analysis .Subjects who participated in the survey were 1341 and a response rate of 99.5% was achieved. The mean age of participants was 19 years, almost all were single, the majority was full-time students, 8.9% had at least one parent who is a health care professional, and 16.3% has had a personal, familiar, or friendly history of cervical cancer.The distribution of respondents who answered correctly to each of the different items of the knowledge about HPV infection and cervical cancer are presented in Risk perception of contracting HPV infection and of developing cervical cancer was similar with the respondents inferring a level of perception with scores of 5.8 and 6.5, respectively and only 12.7 and 19.9% perceived themselves at a high risk, respectively by responding ‘10’. For those who stated that they would consider the HPV vaccination in future, the main reasons were the belief that the vaccination reduces the risk of contracting HPV infection (73.8%) and of developing cervical cancer (59.7%), and felling at risk (10%). By contrast, for those who stated that they would not consider HPV vaccine, the most common reasons were the belief that the vaccine was dangerous (59.6%) and that they feel to be not at risk (42%).According to sexual behaviour pattern, 45.4% reported current or previous sexual activity, and the mean age of first sexual intercourse of the sexually experienced was 17 years. Overall, only 27.1% of those sexually experienced reported that they have always used a condom during the intercourse in the last year. Approximately, half had at least one health checkup over the last year (48%) and for only 3%, the physician informed them about HPV vaccination.When asked about their main source of information of HPV infection and cervical cancer, only 6% claimed health professionals. Among those who knew that a vaccine is available in Italy, 62.7 and 17.4% had received such information from television/mass media and from health professionals, respectively. Almost all respondents wanted more information about HPV vaccination (94.4%) and cervical cancer (95.7%).This large study expands the previous reports and findings on knowledge, attitudes, and behaviour towards HPV infection, vaccination, and cervical cancer patterns among young women are of particular interest, because the responses reflect real issues in this population setting. This study included only women, mainly because when it has been conducted, the available HPV vaccine was approved for use in female subjects only and, as already indicated, international guidelines recommend vaccination of girls aged 11–12 years and in our country, it was free of charge only for girls of 12 years of age. Moreover, this approach is supported by cost–effectiveness analyses that have indicated that the additional vaccination of boys has a limited effect compared to girls-only vaccination  professed intent to receive the HPV vaccination. This finding is consistent with the already mentioned study in Finland where 86% declaring they would accept the vaccination (Study participants showed that 12.7 and 19.9%, respectively, perceived themselves likely to acquire the infection and the disease by responding ‘10’. In the already mentioned studies of Brazil and United States, 68.1% perceived themselves at moderate-to-high risk (This study has some potential limitations. First, the research questions were investigated by a cross-sectional study design. Such design precludes determination of causal relationships between different factors and outcomes. Second, information was gathered by self-reported questionnaire and so the possibility exists that some responses may have reported incorrect information. Notwithstanding this criticism, the anonymity of the study may have reduced the variation in the responses. This study, however, displays important strengths: first, it provides information from a large number of participants and this allows exploration of very weak associations between variables; second, the data were essentially complete; third, the participation rate was very high, probably reflecting heightened interest for this survey.In conclusion, our results suggest that the continuing efforts should be directed at focusing attention towards the benefits of following HPV vaccination recommendations emphasising those factors positively associated. There is also an urgent need for designing educational interventions to improve the level of knowledge about HPV infection and cervical cancer.


Although cervical cancer is an AIDS-defining condition, infection with human immunodeficiency virus (HIV) may only modestly increase the risk of cervical cancer. There is a paucity of information regarding factors that influence the natural history of human papillomavirus (HPV) in HIV-infected women. We examined factors associated with cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) in Rwandan women infected with both HIV and HPV (HIV+/HPV+).In 2005, 710 HIV+ Rwandan women ≥25 years enrolled in an observational cohort study; 476 (67%) tested HPV+. Each woman provided sociodemographic data, CD4 count, a cervical cytology specimen and cervicovaginal lavage (CVL), which was tested for >40 HPV genotypes by MY09/MY11 PCR assay. Logistic regression models calculated odds ratios (OR) and 95% confidence intervals (CI) of associations of potential risk factors for CIN3+ among HIV+/HPV+ women.3, CI = 0.40-0.97).Of the 476 HIV+/HPV+ women 42 (8.8%) were diagnosed with CIN3+. Factors associated with CIN3+ included ≥7 (vs. 0-2) pregnancies, malarial infection in the previous six months (vs. never), and ≥7 (vs. 0-2) lifetime sexual partners. Compared to women infected by non-HPV16 carcinogenic HPV genotypes, HPV16 infection was positively associated and non-carcinogenic HPV infection was inversely associated with CIN3+. CD4 count was significantly associated with CIN3+ only in analyses of women with non-HPV16 carcinogenic HPV (OR = 0.62 per 100 cells/mmIn this HIV+/HPV+ population, lower CD4 was significantly associated with CIN3+ only in women infected with carcinogenic non-HPV16. We found a trend for higher risk of CIN3+ in HIV+ women reporting recent malarial infection; this association should be investigated in a larger group of HIV+/HPV+ women. Cervical infections by carcinogenic human papillomavirus (HPV) genotypes cause virtually all cervical cancer and its immediate precursor lesion, cervical intraepithelial neoplasia grade 3 (CIN3) Evidence suggests that secondary factors, or HPV cofactors, influence the progression of HR-HPV infections to CIN3 and cancer (CIN3+), including exogenous factors such as parity, smoking tobacco and possibly oral contraceptive use We conducted a cross-sectional analysis of risk factors associated with CIN3 and cancer in a population of HIV-infected Rwandan women. We previously found in this population that HPV prevalence and presence of cytologic abnormalities were inversely associated with CD4 cell counts Each participant provided written informed consent after viewing a video demonstrating study procedures. The Rwanda National Ethics Committee and the Institutional Review Board of Montefiore Medical Center, Bronx, NY approved the study protocol and the consent process.The Rwanda Women's Interassociation Study and Assessment (RWISA) is an observational cohort study of 710 HIV-infected and 226 HIV-uninfected Rwandan women enrolled May-November, 2005. Methods have been previously described in detail Participants underwent pelvic examinations. To minimize contamination of gynecologic specimens by blood, exfoliated cervical cells (used for HPV DNA testing) were obtained by cervicovaginal lavage (CVL) prior to collection of a cervical cytology specimen, as previously described CD4 counts were measured at the National Reference Laboratory of Rwanda by FACS Count .HPV testing was performed on CVL specimens taken at the enrollment visit.In brief, 100 µl of each CVL specimen was mixed (1∶1) with a 2× solution of K buffer  and incubated at 55°C for 2 hours followed by a further incubation at 95°C for 10 minutes. After the CVL specimens were digested with Proteinase K, 10 µL of each cell digest was taken to detect HPV DNA using the L1 MY09/MY11 modified PCR system with AmpliTaq Gold polymerase as described elsewhere Biopsies and excised tissue were read independently and blindly by pathologists at Montefiore Medical Center in the Bronx, New York or Evanston Hospital in Chicago Illinois, and were categorized as Negative, CIN1, CIN2, CIN3, and cancer. We used the most severe histopathologic diagnosis as the final participant diagnosis. Among the 202 biopsies, 3 were diagnosed as cancer, 39 CIN3, 21 CIN2, and 76 CIN1.other than HPV16.Of the 710 HIV-infected women 476 (67.0%) tested positive for any HPV DNA. Although non-carcinogenic HPV has not been shown to be a cause of cervical cancer, we included those women positive for non-carcinogenic HPV because some HPV genotypes categorized as non-carcinogenic HPV may be borderline carcinogenic Standard contingency table methods, with exact tests and when appropriate Mantel extension trend tests assessed univariate associations of categorical variables with CIN3 and cancer versus <CIN3. Two-sided P values <0.05 were considered statistically significant. The associations of ordinally categorized continuous variables with grade of neoplasia were assessed with logistic regression and trend tests. Odds ratios (OR) and 95% confidence intervals (CI) adjusted for variables identified in univariate analyses were calculated using logistic regression. Multivariate logistic regression models were fit using variables that had statistically significant (P≤0.05) unadjusted associations with CIN3+ and women that had complete information on these variables: 454 of 476 (95.4%) women positive for any HPV; 303 of 316 (95.9%) positive for carcinogenic HPV; and 241 of 251 (96.0%) women with non-16 carcinogenic HPV. Because of the relationship of CD4 counts with immunosuppression, all models included CD4 counts. We repeated these analyses with the outcome defined as CIN2 or more severe (CIN2+) (n = 63), including 21 women with CIN2, an equivocal precancer diagnosis.. Of the 476 HPV+ women, 223 had a colposcopy and 202 had a cervical biopsy. Among women diagnosed with CIN3+  the most common HPV types detected were HVP16 (33%), HPV58 (26%), HPV31 and 33 (21% each) and HPV 35 and 51 (19% each). HPV52 was harbored by 4.8% and 64.3% were infected with multiple HPV types, compared to 11.1% and 49.8% of women with <CIN3, respectively. Compared to women with a less severe histologic diagnosis, women diagnosed with CIN3+ reported more pregnancies (ptrend <0.001) and lifetime sexual partners (ptrend  = 0.03), shared their residence with more people (ptrend  = 0.01), had a higher income (ptrend  = 0.02), were more likely to have had a recent malarial infection (ptrend = 0.047) and to harbor carcinogenic HPV (ptrend <0.001). CD4 counts were not related to cervical histologic findings among all HIV+/HPV+ women.Demographic and clinical characteristics of the included women are shown in The results of the three logistic regression models are shown in Analyses restricted to  carcinogenic HPV genotypes showed significant associations of CIN3+ with ≥7 pregnancies (vs. 0-2) and HPV16 (vs. non HPV16 carcinogenic genotypes). In analyses restricted to women with non-HPV16 carcinogenic HPV, ≥7 pregnancies (vs. 0-2) were associated with CIN3+. In both restricted analyses the magnitude of the associations of CIN3+ with 7 or more lifetime sexual partners and recent malarial infection were similar to that found in all HPV+ women, but were not statistically significant, perhaps because of the smaller sample sizes.Both the magnitude (OR) and strength  of the association of CD4 count with CIN3+ was greatest in the women with non-HPV16 carcinogenic HPV : per 100As in multivariate analysis using CIN3+ as the endpoint, CIN2+ was independently and positively associated with number of pregnancies , number of sexual partners , and inversely associated with non-carcinogenic HPV . Unlike in women with CIN3+, CIN2+ was not significantly associated with harboring HPV16  or with recent malarial infection   and the strength of association and magnitude of the odds ratio for the association of HPV16 with CIN3+ were smaller than anticipated. This is further albeit indirect evidence that the relative effects of HPV16 in the development of cervical cancer may be less enhanced in HIV-infected compared to uninfected women, consistent with our prior report We found that recent malarial infections had a significant positive association with CIN3+ in HIV+/HPV+ women. To our knowledge, this is the first report of any evidence that malarial infections might contribute to the risk of cervical precancer and cancer. The mechanisms by which malarial infections could increase the risk of CIN3+ are unknown, but might include additional challenges to the already compromised immune system of HIV+ women. HIV, HPV and malarial infections are highly prevalent in much of Africa, and any increased risk of CIN3+ associated with malaria could result in a substantial attributable risk. However, given the limited sample size in this analysis, this observation needs to be confirmed in other populations.Several factors previously identified as associated with cervical precancer and cancer in general populations of women were also identified here in HIV+/HPV+ Rwandan women. We found that multiple pregnancies (7 or more) were associated with increased risk of CIN3+, independent of number of reported sexual partners. High parity has been reported as a risk factor for cancer in populations of unknown HIV serostatus Our study has several limitations. It is a cross-sectional analysis and temporal or causal relationships cannot be inferred. The lack of follow-up does not allow observation of progression with specific HPV types. An additional limitation of this study is that HPV+ women without clinical indication of abnormal cytology or visual lesions were not colposcoped and therefore not biopsied. Thus visually unapparent, HPV+ high-grade lesions could have been missed In conclusion, we found that CIN3+ was inversely associated with CD4 cell count in HIV+ women co-infected with non-HPV16 carcinogenic HPV. The implication of these data is that a greater proportion of cervical precancer and cancer may be attributable to carcinogenic HPV genotypes other than HPV16 in HIV+ women compared to HIV-negative women but larger studies are needed to evaluate this directly. We also found some evidence that malarial infections may increase the risk of CIN3+; this warrants confirmation in larger population-based studies.Table S1Demographic and clinical characteristics in human immunodeficiency virus-infected, human papillomavirus (HPV)-infected women with and without cervical intraepithelial neoplasia grade 2 or more severe (CIN2+)(0.11 MB DOC)Click here for additional data file.Table S2Multivariate associations of clinical and demographic characteristics with cervical intraepithelial neoplasia grade 2 or more severe versus less severe than CIN2 among 454 human immunodeficiency virus-infected, human papillomavirus (HPV)-infected women with complete data.(0.07 MB DOC)Click here for additional data file.


While 18 of 20 (90%) specimens of normal epithelia stained with methylene blue, 11 of 16 (69%) specimens of metaplastic epithelia were stained, with no staining obtained in cancerousepithelia. The cancerous epithelia stained significantly less often than either the normal (p = 0.000005) or the metaplastic (p = 0.001) epithelia. Evaluation of methylene blue staining during PTCS revealed that this stain was absorbed by the cholangial epithelia, not superficially stuck to it. The difference in methylene blue staining properties between the cancerous and normal epithelia could be helpful to clarify the boundary of superficial lateral spread of bile duct cancer.Longitudinal cancer spread is very important for staging of resectability in bile duct cancer. We verified the difference in methylene blue staining properties between cancerous and noncancerous epithelia that are usually observed by cholangioscopy. We obtained 45 biopsy specimens from the common bile duct of 20 patients with bile duct disease using percutaneous transhepatic cholangioscopy (PTCS) after staining with 0.05% methylene blue. We compared the microscopic staining properties with the gross endoscopic observations and evaluated the characteristics of methylene blue staining on frozen sections of each type of cholangial epithelium. Microscopic staining properties were significantly associated with endoscopic observations (


P<0.001). Among the patients with penile squamous cell carcinoma seroprevalence was 38% (P<0.001), among patients with oropharyngeal carcinoma 33% (P=0.04) and among patients with oesophageal squamous cell carcinoma 14% (P=0.7). The serological evidence for association between HPV 16 infection and both oropharyngeal carcinoma and penile carcinoma was established. The conclusion that no association was found between the presence of antibody against HPV 16 L1 capsids and oesophageal squamous cell carcinoma was in accordance with results of other studies carried out in the Netherlands using HPV DNA technology. In the subjects with HPV 16 L1 capsid antibody, no association was found between the antibody against HPV 16 E7 and clinical outcome.A retrospective seroepidemiologic study was performed to examine the association between human papillomaviruses (HPV) 16 infection and carcinomas of the oropharynx, the oesophagus, penis and vagina. Sera were selected from the serum bank from the Antoni van Leeuwenhoek Hospital (Netherlands Cancer Institute) and the Slotervaart Hospital in Amsterdam, the Netherlands. Presence of HPV 16 specific antibody was assessed using HPV 16 L1 capsids. Sera positive for HPV 16 capsid antibody were further tested for antibody against HPV 16 E7 peptides. Prevalence of antibody against HPV 16 L1 capsids among both the negative control group without cancer and the negative control group with gastric cancer was 18%, while seroprevalence among the control group of patients with HPV-associated cervical squamous cell carcinoma was 47% ( The causative association between infection with oncogenic human papillomaviruses (HPV) and cervical squamous cell carcinoma has been established by molecular and epidemiological studies . CurrentResults of some studies suggest that HPV infection may be a risk factor for squamous cell carcinoma of the head and neck or encompasses a distinct entity with an increased sensitivity to radiotherapy . With rePresence of HPV DNA in carcinomas of the penis is observed in a proportion of the specimens analysed  containing L1 proteins among Dutch patients with carcinomas in the oropharyngeal region, tongue and larynx, patients with oesophageal carcinoma, women with vaginal carcinoma and men with penile carcinoma. Sera positive for antibody against HPV 16 L1 were tested for the presence of antibody to E7.Serum specimens were taken from the serum bank at the Antoni van Leeuwenhoek (AvL) Hospital and Netherlands Cancer Institute in Amsterdam, the Netherlands. Cases were selected over a period of 10 years, 1989–1999, based on the diagnosis as stated in the anonymised medical records. The inclusion criterion was the site of the cancer: penis, cervix, vagina, the upper aerodigestive tract , oesophagus and stomach. The anatomical sites were defined according to the codes of the International Classification of Diseases . Sera frThe sera from the AvL patients were obtained at the first visit to the hospital during the period wherein the diagnosis was made. The sera from the SL hospital patients were also taken at the first visit to the gastroenterology clinic. The specimens were stored at −20°C and transported on dry ice to the Karolinska Institute, where the assays were performed by CM Korse and JCGM Buning-Kager.The pathological diagnosis was made at the Department of Pathology of the AvL hospital and the SL hospital, respectively, using standard methods, and coded according to international standards .First, all sera were screened on the presence of IgG antibody against HPV 16 capsids containing L1 proteins. The positive sera were further tested on the presence of IgG antibody against HPV 16 E7 peptides.μg ml−1, coated overnight at 4°C onto microtiter plates and subsequently blocked with 10% horse serum in PBS (HS-PBS). Sera were diluted 1 : 30 with HS-PBS and added to the plates for 2 h at room temperature (RT). A mouse monoclonal antibody to human IgG, diluted 1 : 800, was added and incubated for 90 min at room temperature, followed by a horseradish peroxidase-conjugated antibody to mouse IgG. Before every addition, the plates had been washed with PBS-0.1% Tween. For each sample the absorbency of the same sample obtained from a well coated with disrupted bovine papilloma virus (BPV) was subtracted from the absorbency obtained for the corresponding well coated with HPV 16 capsids.Baculovirus-expressed HPV16 capsids containing both the L1 proteins were diluted in cold phosphate-buffered solution (PBS) to 1 The assays included two internal positive and one negative control specimens on each ELISA plate. Three pools of sera from patients with cervical carcinoma, from patients with cervical intraepithelial neoplasia (CIN) and from healthy blood donors were used for this purpose. The absorbencies read from each plate were normalised relative to the results of the internal standard. The internal standard was measured in a plate with negative control sera obtained from sexually inexperienced women. Cutoff level was defined as the mean value of the absorbencies of the plate with the negative controls plus three standard deviations.−1 in Tris buffer and coated overnight at room temperature. As negative control another plate was coated with Tris buffer only. The E7 peptide has the designation E7-2 and has the amino-acid sequence PETTDLYCYEQLNDSSEEED  and Epi Info Version 6.0 (P<0.001). A total of 46 of the 122 (38%) individuals with penile cancer were positive for HPV 16 L1 antibody (P<0.001). The proportion of individuals with oesophageal squamous cell carcinoma with antibody against HPV 16 L1 capsids was only 14% (8/56), whereas 11 of the 48 (23%) sera from patients with oesophageal adenocarcinoma were positive for HPV 16 L1 antibody. In the group of patients with oropharyngeal carcinoma, 16 out of 48 (33%) individuals were positive for antibody against HPV 16 L1 antibody (P=0.04). The seroprevalence found in the patients with tongue carcinoma or laryngeal cancer was 12 out of 56 (21%) and 25 out of 127 (20%), respectively. A total of 8 out of 31 (26%) of the patients with vaginal carcinoma were positive (P=0.47). In the negative control sera that were positive for HPV 16 L1 antibody, two of 27 (7%) sera were also positive for antibody against HPV 16 E7 peptides, and seven of the 24 (29%) of the sera from patients with cervical squamous cell carcinoma, who were considered as positive control group . No statistically significant difference was observed for the presence of HPV 16 E7 antibody in any of the other groups studied.The characteristics such as gender and age of the patients from whom the serum specimens were obtained are presented according to pathological diagnosis in Table 1P=0.21, Fisher's exact test, two-sided). The same analysis was executed for the subjects with penile carcinoma, again no differences could be found .In the group of patients with cervical carcinoma and penile carcinoma, the possible association between clinical outcome and presence of antibody both against L1 capsids and E7 peptide was analysed. As clinical outcome the following categories were chosen: alive with or without tumour and died with or without tumour. For 21 of the 24 subjects with cervical carcinoma, who were positive for HPV 16 L1 antibodies and in whose serum samples antibody against E7 was determined, the follow-up data were available. There were no statistical differences between the subjects with or without antibodies against E7 with regard to survival  among control subjects reported in a recently published nested case–control study among patients with head and neck cancers in the Nordic countries  in the present study is comparable with the prevalence of 38% (10 out of 26) vs 7%  type 4. BPV 4 DNA and antigens can be found in high copy numbers in the premalignant oesophageal papillomas, but not in the carcinomas. Cattle infected with BPV 4, which feed on bracken fern, have a high risk of oesophageal cancer, but the BPV 4 viral genome is not present in frank cancers or fully transformed cells . If thisin situ penile cancer of 20.3% was found, and 28.5% for invasive penile cancer (Only a few studies are reported on penile cancer and the role of HPV; a review is given by in situ and the invasive case subjects had antibodies to HPV 16 L1 compared to 15% of the control women.The elevated HPV 16 seroprevalence among patients with vaginal cancer was not statistically significant compared to the control women. This is in contrast to the results of a recently published report that antibodies to HPV 16 L1 were strongly related to the risk of vaginal cancer (Presence of antibodies against papillomavirus might be used as prognostic marker in cervical cancer patients (In conclusion, we found evidence for a strong association between the presence of HPV 16 L1 antibody and both cervical squamous cell carcinoma and penile squamous cell carcinoma. A relation between the presence of HPV 16 antibody and oropharyngeal cancer was also found. No serological evidence was demonstrated for an association between HPV 16 antibody and oesophageal, tongue, laryngeal and vaginal carcinoma.


The role of human papillomavirus (HPV) detection in the management of patients with persistent low-grade (mild dyskaryosis or less) cervical cytological abnormalities is unclear. We have analysed cytological material from 167 such patients both cytologically and by non-isotopic in situ hybridisation (NISH) for HPV 16, 18, 31 and 33 and consensus primer polymerase chain reaction (PCR) amplification followed by both generic and specific typing for these HPV types. Cervical intraepithelial neoplasia (CIN) 2 or 3 was present in 40 of 167 patients (23.9%), and the positive predictive values (PPVs) for the presence of CIN 2 or 3, of moderate or severe dyskaryosis at repeat cytology and an HPV-positive NISH and generic PCR signal were 100%, 66% and 42% respectively. The corresponding sensitivities were 48%, 68% and 87%. Addition of cytology to molecular analysis improved both PPV and sensitivity, the best combination being NISH and cytopathology . These data demonstrate that the presence of CIN 2 or 3 in patients with mild cytological abnormalities can be predicted by molecular detection of HPV in some cases, particularly when combined with cytological analysis. However, the magnitude of this prediction is dependent on the population of patients studied, and the clinical role of this approach therefore remains to be defined.


According to the Dutch guidelines follow-up after treatment consists of cervical cytology at 6, 12 and 24 months. Colposcopy is only performed in case of abnormal cervical cytology. In this observational study 184 women treated for CIN 2 or 3 were prospectively monitored by cervical cytology and high-risk HPV testing 3, 6, 9, 12 and 24 months after treatment. Post-treatment CIN 2/3 was present in 29 women (15.8%). A positive high-risk HPV test 6 months after treatment was more predictive for post-treatment CIN 2/3 than abnormal cervical cytology . At 6 months the negative predictive value of a high-risk HPV negative, normal smear, was 99%. Largely overlapping, partly different groups of women with post-treatment CIN 2/3 were identified by HPV testing and cervical cytology. Based on these results we advocate to include high-risk HPV testing in monitoring women initially treated for CIN 2/3. In case of a high-risk HPV positive test or abnormal cervical cytology, colposcopy is indicated. All women should be tested at 6 and 24 months after treatment and only referred to the population-based cervical cancer screening programme when the tests are negative on both visits. © 2001 Cancer Research Campaign


Particular types of human papillomavirus (HPV) infection may preferentially progress from high-grade squamous intraepithelial lesions (HSIL) to squamous cell carcinoma of the cervix (SCC). We performed a meta-analysis of published data to compare HPV type distribution in HSIL and SCC. HPV16, 18 and 45 were each more prevalent in SCC than HSIL, whereas the reverse was true for other oncogenic types including HPV31, 33, 52 and 58. These data suggest that HSILs infected with HPV16, 18 and 45 preferentially progress to SCC. This may have implications for follow-up protocols of future HPV-based cervical cancer screening programmes and for HPV vaccine trials. Epidemiological studies have established human papillomavirus (HPV) infection as the central cause of invasive cervical cancer (ICC) and its precursor lesions  to ICC has implications not only for follow-up protocols in ICC screening programmes, but also for prophylactic type-specific HPV vaccine trials. For ethical reasons, final outcome measures in such trials will be the prevention of HSIL. However, it is important to know whether the HPV type distribution in HSIL is representative of those that go on to cause cancer.Medline. Studies had to include at least 20 cases of squamous cell or histologically unspecified cervical cancer (detailed information on the HSIL studies is reported in the This report includes 8594 squamous cell carcinoma of the cervix (SCC) cases (including 2725 of unspecified histology), as previously reported in Type-specific prevalence is presented for the 14 most common high-risk (HR) types identified in SCC  than HSIL (84.2%)  . HPV16 wThe SCC : HSIL ratios for the eight most common HPV types were additionally calculated within more homogeneous study subgroups: (i) studies that did not report any multiple infections , (ii) studies testing for HPV from biopsies  and (iii) studies using ‘broad’-spectrum PCR primers . The SCC : HSIL ratios were also calculated separately for HSILs classified by the Bethesda system and for CIN3 only. Across all these subanalyses, SCC : HSIL ratios remained consistent for HPV16 (range: 1.04–1.25), HPV18 (1.46–1.93) and HPV45 (1.20–4.61). HPV31, 33, 35, 52 and 58 were consistently associated with ratios of 0.3–0.9, with the exception of HPV58 for biopsy studies .Where sample size permitted, subanalyses were also stratified by region. When estimated from studies within Asia, Europe and South/Central America, respectively, there was no material difference in SCC : HSIL ratios for HPV16 , HPV18 , HPV45 , HPV33 , HPV52  or HPV58 . However, notably high ratios were observed for HPV31 in South/Central America  in comparison to Europe  and Asia , and for HPV58 in China (including Taiwan and Hong Kong)  in comparison to non-Chinese Asian countries , raising the possibility of localised variation in the malignant potential of particular HPV types (Our findings suggest that worldwide, HSIL infected with HPV16, 18 or 45 are more likely to progress to SCC than HSIL infected with other HR types. This could be interpreted in two ways: either these types have a greater potential to induce fully malignant transformation, and/or these infections somehow preferentially evade the host immune system. Compared to other HPV types, HPV18 has been associated with increased oncogenic potential in cell culture, screening failures and poorer cancer prognosis (Even in countries with established screening programmes, women still die from rapidly progressing cancers that escape periodic examination. Given that HPV16, 18 and 45 appear to have greater progressive potential, and in the event that future cervical screening programmes include HPV typing, women infected with HPV16, 18 and 45 may require closer surveillance than women infected with other HR HPV types.The demonstration that the HPV type distribution in HSIL is not entirely representative of those that go on to cause cancer also has important implications for prophylactic type-specific HPV vaccine evaluation. This is because any beneficial effect identified by randomised trials from the proportion of HSIL preventable by HPV16 or HPV16/18 vaccines may be an underestimate of the beneficial effect on the prevention of ICC.


Sir,We reported the unambiguous presence of human papilloma virus (HPV) type 18 and HPV-associated koilocytes in breast cancers that had occurred in Australian women  in human breast milk from normal unselected women. We used standard and The implication of these various observations, including the identification of high-risk HPV in the Australian milk samples, is suggestive of a possible causal role of high-risk HPV in some breast cancers.


INK4A as a marker for the presence of an active HPV oncoprotein E7. These findings were correlated with clinical and pathological prognostic factors of CRC.Based on the well-recognized etiological role of human papillomavirus (HPV) in cervical, anogenital and oropharyngeal carcinogenesis, a potential role of HPV in colorectal carcinogenesis has been suggested. For that reason, the aim of the present study was to investigate the presence of HPV DNA in colorectal carcinomas (CRC) and to study overexpression of p16The presence of HPV was assessed using a multiplex PCR system of 10 non-biotinylated primers. The amplified fragments of HPV positive samples were further analyzed by a highly sensitive, broad spectrum SPF10 PCR and subsequently genotyped using reverse hybridization in a line probe assay.INK4A protein expression was investigated in a subset of 90 (30 HPV positive and 60 HPV negative) CRC samples by immunohistochemistry.P16INK4A (p = 0.325).HPV DNA was found in 14.2% of the CRC samples with HPV16 as the most prevalent type. No significant differences in clinical and pathological variables were found between HPV positive and negative CRCs, except for age. HPV positive patients were significantly younger (p = 0.05). There was no significant correlation between the presence of HPV and overexpression of p16INK4A does not seem to be a surrogate marker for an active HPV infection in CRC. Therefore, further functional analyses are necessary to elucidate the role of HPV in CRC.In conclusion, the presence of oncogenic HPV DNA in a small cohort of CRC samples may suggest that HPV may be involved in the carcinogenesis of some CRC. However, contrary to what has been observed in head and neck squamous cell cancer and cancer of the uterine cervix, p16 Colorectal cancer (CRC) is one of the most common malignancies throughout the Western World. Surgery is the cornerstone in the treatment of patients with CRC and is followed by adjuvant chemotherapy and radiotherapy for specific subgroups of patients . AlthougINK4A. P16INK4A is a tumor suppressor protein that inhibits cyclin dependant kinases (CDK)-4 or -6 binding to cyclin D which regulates the G1 cell cycle checkpoints [INK4A is considered to be strong and consistent in HPV-induced cancers [INK4A, as detected by immunohistochemistry, has shown to be a useful adjunct to cytology in cervical cancer screening [INK4A and the presence of high-risk HPV [The HPV viral oncogenes E6 and E7 have shown to be the main contributors to the development of HPV induced cancers. These oncogenes have the ability to bind host cell regulatory proteins, especially tumor suppressor gene products . The HPVckpoints ,20. Over cancers . Therefocreening , a reliacreening , and a ucreening . Furtherrisk HPV . HoweverINK4A was investigated as a marker for the presence of an active HPV oncoprotein E7 in a subset of the above mentioned series of colorectal cancers. Subsequently, the results were analyzed for correlation with prognostic clinical features for disease outcome and pathological variables.The aim of the present study was to investigate the presence of HPV DNA in a series of colorectal carcinomas. In a second part of the study, overexpression of p16Material from a previous study of patients with CRC treated at the Antwerp University Hospital in Edegem or the St. Augustinus Hospital in Wilrijk  was usedINK4A immunohistochemistry. Three HPV positive samples could not be investigated by immunohistochemistry since the paraffin blocks were no longer available. The study was approved by the local Ethics Committee of the University of Antwerp and was conducted in accordance with the ethical principles stated in the most recent version of the Declaration of Helsinki.All HPV positive tumors, except three (n = 30), plus two randomly chosen HPV negative tumors per HPV positive tumor (n = 60) were used for p16Tumor DNA was obtained from formalin-fixed, paraffin embedded tissue blocks. After manual microdissection to enrich for tumor cells, DNA was isolated as described previously . After D2, 200 μM deoxynucleotide triphosphate, 1× iTaq buffer, 15 pmol of each of the forward and reverse primers and 10 μl of isolated DNA. The PCR reactions were carried out using the iCycler (BioRad) as previously described [10 primers .Since formalin-fixed paraffin embedded materials often yield poorly amplifiable DNA, the efficacy of the primer pair is inversely correlated with the length of the amplimers and the primers should be designed to amplify a relatively short PCR fragment . DNA samescribed , except 10 LiPA contains probes for high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70, and low risk HPV genotypes 6, 11, 34, 40, 42-44, 53, 54 and 74. The hybridization steps were carried out following the INNO-LiPA kit's instruction. Briefly, oligonucleotide probes (containing a poly d(T) tail) were immobilized in parallel lines on nitrocellulose membrane strips by the supplier; 10 μl of the PCR product, containing biotin at the 5' end of the primers, was denaturated by adding 10 μl of NaOH solution. After hybridization of the PCR product to the probes on the strip under stringent conditions, followed by stringent washing, the hybrids were detected by alkaline phosphatase/streptavidin conjugate and subtrate (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium), resulting in a purple precipitate at the positive probe lines. After drying, the strips were interpreted visually by using the INNO-LiPA HPV genotyping v2 interpretation chart [HPV genotyping was performed using reverse hybridization by the INNO-line probe assay  as described earlier . The curFive μm-thick sections were prepared from formalin-fixed paraffin-embedded tissue for IHC. Sections were deparaffinized in toluene, dehydrated and subjected to heat antigen retrieval in Epitope retrieval solution  in a heating bath for 30 min. at 95 (± 1)°C.INK4A monoclonal antibody (diluted 1:100) was performed for 30 minutes at room temperature. Sites of binding were detected using 3,3'-diaminobenzidine (DAB+) as chromogen according to the manufacturers instructions. The sections were counterstained with haematoxylin, dehydrated, cleared and mounted.Sections were subsequently stained using the CINtec Histology Kit (mtm laboratories) on a Dako Autostainer Plus system . Endogenous peroxidase activity was quenched by incubating the slides in peroxidase blocking reagent for 10 minutes. Incubation with mouse anti-human p164 (pH 8.9), 18 mM (NH4)SO4 and 2 mM MgSO4) and 1 unit Discoverase dHPLC DNA polymerase . Fluorescent PCR products were analyzed by capillary electrophoresis using an ABI 3100 Genetic Analyzer  and Genemapper Software 3.7.All cases had been previously analyzed for MSI status . After mINK4A IHC.Prognostic relevance of HPV was assessed by survival analysis. Survival probability was estimated using the Kaplan and Meier method. Differences were tested using the log rank statistic. The median follow up for OS and DFS was 4.5 and 3.7 years respectively for the entire study population and 5.8 and 4.8 years respectively for the subpopulation of 90 colorectal tumors used for p162-test or Fisher's exact test (when appropriate) for categorical variables and using Student t-test or Mann-Whitney U test (when appropriate) for continuous variables. In order to assess the independent prognostic contribution of HPV, a multiple Cox regression analysis was conducted. All analyses were conducted using SPSS (version 16.0). Significance for all statistics was two-tailed and recorded if p < 0.05.Possible associations between the presence of HPV-DNA and clinicopathological parameters of colorectal cancers were investigated using the χ10 PCR.All tissue samples were positive for the β-globin gene, indicating that DNA was available for molecular analysis. HPV DNA was detected in colorectal tissue in 33 out of 232 patients (14.2%) using SPF10 LiPA by reverse hybridization (Innogenetics). In about half of the samples a single HPV infection was identified (54.5%) whereas the other HPV-DNA positive samples contained multiple HPV infections (45.5%). A relative broad spectrum of HPV genotypes was found, HPV 16 (57.6%) being the most prevalent type, followed by HPV 18 (45.5%)  than HPV negative patients (median age: 60 years) but the difference was of borderline significance (p = 0.05). Anatomic location of the tumor had no correlation with the presence of HPV infection. HPV prevalence was similar in proximal colon, distal colon, and rectum (p = 0.565). The location of the tumors throughout the colon in correlation to the presence of HPV is shown in Table Clinical and pathological features were studied between HPV positive and negative carcinomas. The results are shown in Table INK4A stained slides (n = 90) were scored on two separate aspects: the number of P16INK4A positive tumor cells present , and the intensity of p16INK4A staining . Some examples of the different scores are shown in Figure INK4A IHC in relation to presence or absence of HPV are given in Table INK4A (p = 0.325) in the colorectal cancer tissues examined.P16INK4A. Tissues obtained from the proximal colon showed significantly less expression of p16INK4A compared to tissues taken from the distal colon and the rectum (p = 0.002). The location of the tumors throughout the colon in correlation to the p16INK4A expression is shown in Table INK4A expression level and stage (p = 0.066). Clinical and pathological features were studied between carcinomas with and without expression of p16INK4A. The results are shown in Table Anatomic location of the tumor showed a significant correlation with the overexpression of p16INK4A expression.In addition, follow-up for overall survival (OS) and disease free survival (DFS) was available for 88 and 86 colon cancer patients, respectively. At the end of the observation period, 37 (42%) patients had died while 31 (36%) had experienced a recurrence of the tumor. All deaths were tumor related. No statistically significant difference in OS  and DFS  was found between CRC patients with and without p16Oncogenic papillomaviruses have shown to be involved in benign and malignant lesions of the cervix and other anogenital sites . AlthougBased on its well-known role in cervical and anogenital carcinogenesis, some studies proposed an association between HPV and CRC -12. DNA 10 PCR is proven to be a very sensitive HPV detection technique [The presence of HPV DNA in colonic neoplasms is a conflicting issue. Although earlier studies have failed to detect HPV DNA in colon biopsy samples ,14, moreechnique ,31. Modeechnique  and Damiechnique , there wechnique . One posechnique . Likewisechnique ,43. Tranechnique .As seen in most other studies, high-risk HPV type 16 was the most prevalent type in colorectal tissues in this study, followed by high-risk HPV type 18. These types have been reported to suppress tumor suppressor proteins functions and play an important part in carcinogenesis . Low risNo significant differences in clinical and pathological variables were found between HPV positive and negative colorectal carcinomas. HPV positive patients showed a trend to be younger than HPV negative patients. A similar observation has been made in HPV positive oral squamous cell cancer, a disease in which HPV appears to play an etiologic role -46. OtheINK4A was investigated as a marker for the presence of an active HPV oncoprotein E7 in a subpopulation of colorectal carcinomas. It has been demonstrated that p16INK4A overexpression might specifically identify HPV infections that are biologically relevant in the carcinogenesis of head and neck squamous cell carcinomas and cervical carcinomas. However, it should be noted that expression of p16INK4A is not limited to HPV-positive tumors, and the use of this marker alone as an indicator of biologically relevant HPV infections inevitably entails the risk of including some HPV negative p16INK4A positive results [INK4A expression and the presence of an HPV infection. This might be explained by the fact that p16INK4A is aberrantly methylated in about 40% of sporadic CRC and is significantly correlated with loss of tumor suppressor function [INK4A expression, which is in agreement with the literature. Few studies have investigated the relationship between p16INK4A expression and colorectal adenocarcinoma. The reported frequency of p16INK4A protein expression in CRC varies from 17 to 99%, with the majority of investigations showing p16INK4A expression in more than two third of CRC [INK4A protein was expressed in 74% of the colorectal adenocarcinomas and more than half (n = 38) showed a high level of p16INK4A expression. P16 is a nucleoprotein; the presence of staining in both the nuclei and the cytoplasm supports the finding that p16 gene is overexpressed. The change in subcellular location of the overexpressed nucleoprotein might account for its role in CRC carcinogenesis. The mechanism inducing the p16INK4A overexpression is probably different from promoter methylation and could presumably result from a compensatory response to cell cycle deregulation [INK4A and to a break in the G1-S transition through pRb phosphorylation [In the second part of the study, overexpression of p16 results . In the function . In the d of CRC . In thisgulation . CDK4 ovrylation .INK4A and those not expressing p16INK4A, except for location. In accordance with others [INK4A protein expression was more often seen in the distal colon and the rectum. In addition, there was a trend towards an association between strong p16INK4A expression and stage II and III tumors.No significant differences in clinical and pathological variables were found between CRC samples expressing p16h others ,48 p16ININK4A protein has been investigated in five studies. Three studies noted that p16INK4A expression was associated with poorer survival [INK4A expression and patient survival.The prognostic role of p16survival -50. In asurvival  and Tadasurvival , we founIn conclusion, the presence of oncogenic HPV DNA in a small cohort of CRC samples, with high risk HPV 16 as the most prevalent type, was confirmed in the present study. However, in order to suggest that HPV might be involved in colon cancer, viral DNA incorporation into the host genome needs to be demonstrated by in situ hybridization. Additionally, the presence or absence of HPV in a non-malignant control group and tumor adjacent tissue needs to be investigated in order to determine whether HPV is merely an epiphenomenon in CRC or rather a potential cofactor in the development of the disease.INK4A does not seem to be a surrogate marker for an active HPV infection in CRC. Therefore, further functional analyses are necessary to elucidate the significance of the presence of HPV in CRC.In addition, contrary to what has been observed in head and neck squamous cell cancer and cancer of the uterine cervix, p16The authors declare that they have no competing interests.VD designed the study, carried out the PCR and genotyping analysis, the IHC scoring, the statistical analysis and the interpretation of the results and drafted the manuscript. MB participated in the study design, set up the IHC scoring system, carried out the IHC scoring and helped to draft the manuscript. VMV participated in setting up the IHC scoring system carried out the IHC scoring and revised the manuscript. EVM participated in the IHC and supervised the scoring and revised the manuscript. CW participated in the PCR and genotyping analysis and VF supervised these analyses and revised the manuscript. PV provided tissue samples and coordinated sifting through the medical files. FL and JBV participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/117/prepub


Aims. We investigated the effect of HR-HPV infection on the capacity of the cytokine network in whole blood cultures during carcinogenesis of cervical carcinoma. Methods. Thirty-nine women with moderate dysplasia, severe dysplasia, cervical carcinoma, or without dysplasia formed the study group. The control group consisted of 10 HR-HPV-negative women without CIN. Whole blood cultures were stimulated with phytohemagglutinin (PHA) and concentrations of tumour necrosis factor Results. A significant increase in cytokine release was detected in HR-HPV-positive women without dysplasia. In women with cervical cancer, release of IFNConclusions. (1) Infection with HR-HPV without expression of cervical dysplasia induces activation of the cytokine network. (2) Increases in ratios of Th1-type to Th2-type cytokines at the stage of cervical carcinoma were found by comparison with stage CIN III. (3) Significant changes in the kinetics of cytokine release to a Th2-type immune response in blood of women withcervical dysplasia occurred progressively from CIN II to CIN III. It is well established that high-risk (HR) human papillomavirus (HPV) types are causativefor the development of cervical cancer –3. The mimmunity . Qualitaimmunity –16 and oimmunity  afterstunity [γ –18, IL-2unity [γ ; and occunity [γ  or TNFα ty [γ [α  were meaty [γ [α . Generalty [γ [α , 17, 19.Wepreviously observed manifestation of a Th2-type cytokine pattern in plasma ofHR-HPV-positive women during carcinogenesis of cervical cancer at the stage ofCIN III . Recent In thepresent study we used whole blood cultures from HR-HPV-negative controls,HR-HPV-positive women without cervical dysplasia and HR-HPV positive patientswith different grades of CIN and cervical cancer to investigate changes inimmunocompetence expressed in the capacity of circulating leukocytes to releasecytokines in response to a mitogenic challenge. Of interest were the effect ofHR-HPV infection without clinical manifestations, the special position of CINIII with a Th2-type cytokine response, and a possible revival of inflammatorycytokine activity in cervical carcinoma.Inclusion tookplace at the outpatient clinic of the Obstetrics and Gynaecology Department of the ErasmusUniversity Medical Center  between July 2000 and August 2002. Our selection of patients for this study wasbased on the presence of HR-HPV and the grade of cervical intraepithelialneoplasia. HPV sampling and a cervical biopsy were carried out on allparticipating women. Histology results were defined as no dysplasia, milddysplasia (CIN I), moderate dysplasia (CIN II), severe dysplasia (CIN III), or(micro-) invasive cancer. An experienced pathologist revised all histologicalsamples. Women with CIN I lesions (mild dysplasia) were excluded since morethan fifty percent of our patients with CIN I turned out to be HR-HPV-negative.Healthy women who attended the outpatient clinic for a regular sterilisationprocedure were recruited as HR-HPV-negative controls after sampling forhistology and HPV. Exclusion criteria for all participants were (anamnesticrequired): postmenopausal state, pregnancy at time of sampling, chronicdiseases , presence of sexually transmitteddiseases (STDs) and infection with human immunodeficiency virus (HIV), signs ofacute infection at time of sampling, and an immune-compromised state. With theexception of oral contraceptives, no participant used medication on a regularbase. No participant had used pain-medication (including NSAIDs) for at leasttwo weeks prior to sampling in order to avoid the well-known influence ofNSAIDs on cytokine release from PBMCs. The study protocol was approved by theEthics Committee of the Erasmus Medical Center and all women voluntarily gavesigned informed consent.Cervical scrapesfor HPV detection and typing were taken using a cervical bio-sampler . HPV testing was performed with the consensusGP5+/GP6+ PCR enzyme immunoassay (EIA) using a cocktail probe covering 37(sub- )types, including all (probably) HR-HPV types, as previouslydescribed . This teFor the preparation of whole blood cultures,peripheral venous blood samples were collected between 8–12 amin sterile endotoxin-free vacutainers coated with Na-heparin as anticoagulant, and immediately processed.For a leukocyte count peripheral venous bloodsamples, collected between 8 and 12 am, were drawn into endotoxin-freevacutainers  with ethylene-diaminetetra-acetic acid (EDTA)as anticoagulant and leukocyte counts performed with a Sysmex XE-2100.2. Blood cultureswithout PHA were run as controls. All cultures were sampled at 0,24, 48, 72, and 96 hours, centrifuged for 10 minutes at 4°C and 1500 g, and culture supernatants kept at −80°Cuntil analysis.For preparation of whole blood cultures, bloodwas diluted <6 and <10%; IFN<5 and <10%; IL-2:MDC, 7 pg/ml; VCs, <6 and <10%; IL-4: MDC, 2 pg/ml; CVs, <5 and <7%; IL-10: MDC, 1 pg/ml; CVs, <5 and <10%; IL-12 + p40: MDC, 1.5 pg/ml,CVs, <10 and <10%.All samples wereanalysed by commercially available enzyme-linked immunoassays  for the cytokines TNFnd IL-12 . The detPreliminaryKomolgoroff-Smirnov tests showed an abnor mal distribution of cytokine valuesin PHA-stimulated whole blood cultures. Accordingly, cytokine data arepresented as medians with ranges unless stated otherwise. The nonparametricKruskal-Wallis test (K. W. test) and Mann-Whitney's U-test were used asappropriate to assess differences in cytokine levels between groups. Levels of statistical significance were adjustedfor the number of comparisons according to Bonferroni's method, as indicated inthe graphics. Differences in patient characteristics between groups wereevaluated by one-way ANOVA and unpaired two-tailed T-tests. Spearman's correlations were used toinvestigate possible relations between age at time of sampling and releasedcytokines.Thirty-five patients with different grades ofCIN were selected. Five of them were excluded because of diabetes . The changes in immune-competence in ourstudy are not related to age.6 leukocytes, in order to stratify forpossible different numbers of cytokine-producing leukocytes between studysubjects [The results of cytokineassays were calculated per 10subjects , 24. PreIngeneral our data of maximum cytokine release are in accordance with kineticstudies of PBMC's . On the A significant differencein cytokine release was observed between the two groups of women withoutdysplasia: with the exception of IL-12 all investigated cytokines weresignificantly increased in HR-HPV-positive women. The results are summarized inIn HR-HPV-infected women,release of Th1-type cytokines IFNIn order to characterize a possible Th1-type/Th2-type shift we calculated the ratios of Th1-type cytokines IL-12, IFNIn order to characterize a possible Th-1 typecytokine pattern after establishment of an invasive carcinoma we comparedcytokine levels in PHA-stimulated blood cultures of patients with invasivecarcinoma with levels in HR-HPV-positive women without dysplasia. There was nodifference between levels of IL-12 + p40 and IFNThe significantincrease in Th1-type as well as Th2–type cytokines inour HR-HPV-positive women with normal histology suggests viral activation ofthe systemic cytokine network and induction of cell-mediated immunity afterinitial HR-HPV infection . To our Cytokine releasechanged to an antiinflammatory, tumour-promoting pattern by increase in IL-4and IL-10 expression at the stage of CIN III. This result confirms and extendsour earlier observations of a change to a Th2-type cytokine pattern in thecirculation of patients with CIN III  and is is et al. describei et al. where PHA shift to aTh2-type cytokine pattern in CIN III was more obvious when the ratios betweenTh1-type and Th2-type cytokines  are evalThe course ofIL-12 secretion in our study groups merits consideration. IL-12 is one of thefirst cytokines released during an innate immune reaction and stimulates a Th-1type cytokine response in cell-mediated immunity. Our HR-HPV-positive womenwith normal histology demonstrated significantly increased Th1- and Th2-type cytokine release, with theexception of IL-12 which was low. Our observation of high secretion of IL-12 inCIN II might be explained by an observation made by Moscicki et al. . TheseaThe significantincrease of the four cytokine ratios between CIN III and carcinoma may indicatethat the presence of a tumour with an inflammatory reaction and exposure ofviral antigens  eventually induces a certain T-cell response.This response remains incomplete as shown in our cytokine data presented inThe increase in the IFNg et al.  whenT-cstigated .It was our goalto study changes in the cytokine network in blood of HR-HPV-infected women atvarious stages of CIN upto onset of cervical carcinoma. The cytokine networkis probably best represented in a whole blood culture. The use of whole bloodcultures for determination of mitogen-stimulated cytokine release byimmunocompetent leukocytes has distinct advantages over cultures of isolatedleukocytes or lymphocytes. It permits interaction between different leukocytes,preserves concentrations of stimulatory and inhibitory mediators, and avoidsactivation and changes in cell ratios associated with procedures of isolationand purification . For stiMost studies dealing with cytokine patterns in HR-HPV-relatedcervical neoplasia and cancer concentrate on infections with HPV 16 (the mostfrequently observed oncogenic HPV type in Caucasian population) . In cont1. Our study suggests that infection with HR-HPV in women withoutcervical dysplasia induces activation of the cytokine network.2. Manifestation of a tumour induces a second deregulated andincompetent immune response.3. Ourresults confirm and expand our earlier observations on circulating cytokines:significant changes in the kinetics of cytokine release to a Th2-type immuneresponse in blood of women with cervical dysplasia occur progressively from CINII to CIN III.Theseimmunological findings are supported by clinical observations: many CIN I or IIlesions usually regress without treatment, whereas CIN III lesions mostly willdevelop into invasive cancer if not properly treated .


We evaluated the performance of primary high-risk human papillomavirus (hrHPV) testing by hybrid capture 2 (HC2) with different thresholds for positivity, in comparison with conventional cytology.We used data of 25 871 women (aged 30–60 years) from the intervention group of the VUSA-Screen study , who were screened by cytology and hrHPV. Primary outcome measure was the number of cervical intraepithelial neoplasia grade 3 or higher (CIN3+), detected within 3 years. We compared baseline cytology testing with three possible hrHPV screening strategies at different relative light unit/cutoff (RLU/CO) thresholds.Compared with baseline cytology testing, hrHPV DNA testing as a sole primary screening instrument did not yield a superior sensitivity, as well as lower colposcopy referral rate and lower false positivity rate at any RLU/CO threshold. The hrHPV screening at 1 RLU/CO threshold with cytology triage at baseline and at 12 months revealed the highest sensitivity for CIN3+ (relative sensitivity of 1.32), although still displaying a lower colposcopy referral rate than cytology testing (relative colposcopy rate of 0.94). Higher thresholds (>1RLU/CO) yielded lower colposcopy rates, but resulted in substantial loss in sensitivity.The hrHPV testing at the commonly used threshold of 1 RLU/CO with cytology triage at baseline and at 12 months showed a much higher sensitivity with a lower colposcopy referral rate compared with cytology testing. New promising methods of cervical cancer prevention have been introduced since the recognition that infection with high-risk human papillomavirus (hrHPV) is the necessary cause of cervical cancer  in the generally healthy population. Cytological triage of hrHPV DNA-positive women has been found to improve the specificity of the screening test , a study performed within the routine cervical programme of the Netherlands. Women participating in this cohort received combined hrHPV testing and cytology. The primary end points were cervical intraepithelial neoplasia grade 3 or higher (CIN3+), detected within 3 years.The VUSA-Screen is a population-based study designed to evaluate the effectiveness of combined cervical cytology screening with hrHPV testing by HC2 hybridisation assay . The study was carried out in the Utrecht province of the Netherlands in the setting of the regular screening programme that invites women, aged between 30 and 60 years of age, to be screened every 5 years. The design of the study has also been described elsewhere . After preparation of a conventional smear on a glass slide, the brush was placed in a vial containing 1 ml UCM  for hrHPV testing. Cervical cytology results were reported, blinded to the hrHPV testing results, according to the CISOE-A classification, which is routinely used in The Netherlands and can be converted into the 2001 Bethesda system  hrHPV-positive women as well as a subset of hrHPV-negative cytologically normal women (n=3063) were included. To select the hrHPV-negative women, each hrHPV-positive woman was matched to three randomly chosen hrHPV-negative women of the same age. Women with normal cytology were not informed about the hrHPV test result. The hrHPV-positive women with normal cytology were offered cytology and a blinded hrHPV test at 12 months, and combined hrHPV testing and cytology at 24 months. A woman was referred at 12 months if cytology was abnormal and at 24 months if the hrHPV test was positive and/or cytology was abnormal. The hrHPV-negative, cytologically normal women in the sub-study were invited for combined testing at 24 months, and referred if cytology was abnormal and/or the hrHPV test was positive. If a woman with normal cytology and a negative hrHPV test was not invited for repeat testing after 24 months, cytological and/or histological follow-up results was not included.In the women with normal cytology at baseline, a sub-study was carried out. In this sub-study, all  and registered in the trial register .Of the women who were referred to a gynaecologist for colposcopy, colposcopy-directed biopsies were taken from suspicious areas of the cervix, according to standard procedures in the Netherlands . Cytolog−1 of cloned HPV-16 DNA and negative controls (provided by the manufacturer) were included in each assay (Qiagen). The results of the HC2 assay were expressed as RLU/CO ratio, representing the ratio between the emission from a sample to the average emission of three positive controls. Initially, the threshold of 1 RLU/CO, as proposed by the manufacturer, was used to classify a specimen as positive or negative.The hrHPV testing was performed by the HC2 high-risk HPV DNA test in an automated format on a rapid capture system according to the manufacturer's instructions (Qiagen). This test uses a cocktail probe to detect 13 high-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Positive controls containing 1 pg mlin situ were also included.The primary outcome measure of the study was histologically confirmed CIN3+, detected cumulatively within 3 years after baseline. A secondary outcome was cumulatively detected CIN2+. In the calculations of the number of CIN3+ and CIN2+ lesions, cases of cervical adenocarcinoma and cervical adenocarcinoma The absolute specificity of hrHPV testing with RLU/CO thresholds between 1 and 100 and the absolute specificity of cytology were computed as follows. Specificities were adjusted for non-attendance at repeat testing by applying Bayes's rule, which means that the specificities were computed from the positive and negative predictive values for CIN3+ (and CIN2+), as well as the baseline prevalences of HC2 and cytology test outcome strata  with three possible hrHPV screening strategies at RLU/CO thresholds between 1 and 100. The following hrHPV screening strategies were used: (1) baseline hrHPV testing only; (2) cytology triage of hrHPV-positive women at baseline and one repeat cytological test for cytologically normal women; and (3) cytology triage of hrHPV-positive women at baseline and one repeat combined cytology and hrHPV HC2 test  for cytologically normal women. For each comparison, we computed the relative sensitivity for CIN3+ (and CIN2+), relative false positivity rate and relative colposcopy referral rate. Analogous to the calculation of the specificity, the relative rates were calculated by combining positive and negative predictive values  and baseline test outcomes. Because double-negative women cancel out when calculating relative rates , we onlyThe 95% confidence intervals (CIs) were calculated for absolute specificity using the Wilson Score method , and Excel .Of the 25 871 women from the intervention group of VUSA-Screen study, 25 658 (99.2%) had an adequate baseline Pap smear. Among women with adequate Pap smears, 25 196 had normal cytology of whom 1021 (4.1%) tested hrHPV-positive, 337 women had a BMD result of whom 167 (49.6%) tested hrHPV-positive and 125 women had a >BMD result of whom 115 (92.0%) tested hrHPV-positive. The median age of participating women was 44.0 years . The hrHPV results of positive RLU/CO  showed a mean of 224.5 .The number of test positives and negatives, CIN3+ and CIN2+ detected, stratified for cytology and HC2 thresholds are presented in vs 98.7%) and CIN2+ (95.9 vs 98.9%). The specificity for CIN3+ and CIN2+ increased with increasing RLU/CO thresholds. Only at a RLU/CO threshold of 100, hrHPV testing reached the same specificity for CIN2+  and CIN3+  as cytology.vs baseline cytology testing are presented in The relative colposcopy referral rate, relative sensitivity and relative false positivity rate of primary HC2 testing at different RLU/CO thresholds At the standard test positivity threshold for HC2 , the relative sensitivity of hrHPV was superior to that of cytology, both for CIN3+ (relative sensitivity of 1.36) and CIN2+ (relative sensitivity of 1.50). With increasing HC2 threshold values, the relative sensitivity for CIN3+ decreased, resulting in a relative sensitivity of 1.22 at 10 RLU/CO and 0.80 at 100 RLU/CO. Results were comparable using CIN2+ as outcome measure.There was no HC2 threshold that resulted in an improved false positivity rate and concomitant colposcopy referral rate, without compromising its sensitivity. In fact, the HC2 threshold  at which a lower colposcopy referral rate was reached compared with cytology, also revealed lower sensitivities for CIN3+ and CIN2+ . As no sFor the strategy with cytology triage at baseline and combined cytology and HC2 testing (with the same threshold as used at baseline) at 12 months, only at threshold 30, a lower false positivity and colposcopy rate in combination with higher sensitivities for CIN3+ and CIN2+ than baseline cytology testing was found. However, at RLU/CO 30, the gain in sensitivity compared with cytology was only marginal.vs relative false positivity rate of the three investigated screening strategies, compared with baseline cytology testing, is graphically shown in The relative sensitivity In this study, we evaluated three possible cervical screening strategies that are based on hrHPV testing with different HC2 thresholds and compared them with baseline cytology testing (threshold BMD). We aimed to improve the specificity of hrHPV screening by increasing the RLU/CO threshold, while maintaining a higher sensitivity than baseline cytological testing. The results are based on data from the VUSA-Screen study, a population-based cohort study carried out in the Utrecht province of the Netherlands. We found that compared with baseline cytology testing, there was no HC2 RLU/CO threshold for which a screening strategy of hrHPV testing as a sole primary screening instrument resulted in both superior sensitivity as well as similar (or lower) colposcopy rate and equal (or higher) specificity. As baseline hrHPV testing cannot improve baseline cytology testing, even at increased RLU/CO thresholds, we conclude that some form of triage and follow-up is required in hrHPV screening.Given that follow-up testing is required in hrHPV-positive women, we searched for a strategy that did not increase colposcopy referral rate compared with cytological testing. A screening strategy that was clearly superior to baseline cytological testing was primary hrHPV screening, with RLU/CO thresholds between 1 and 30 and cytology triage at baseline and repeated cytology testing at 12 months. This strategy was not only more sensitive than baseline cytology testing but also resulted in lower false positivity rates and in fewer colposcopy referrals. Using HC2 RLU/CO thresholds between 1 and 5 results in higher sensitivity  and a reduced colposcopy referral rate  compared with baseline cytology testing. The current threshold of 1 RLU/CO makes optimal use of the superior sensitivity of the hrHPV test for CIN3+/CIN2+, without actually increasing the colposcopy referral rate compared with baseline cytology. The colposcopy referral rate of this strategy is therefore substantially lower than a screening scenario with baseline hrHPV testing only. In addition, a screening strategy with a high sensitivity may allow for extension of the screening interval, which in turn reduces colposcopy referral rates , which is the age for which hrHPV testing is most widely advocated (To conclude, no RLU/CO threshold was found for which HC2 testing at baseline resulted in a similar or lower colposcopy referral rate than baseline cytology, while maintaining a higher sensitivity.Superior combinations of sensitivity and colposcopy rate are possible for HC2 testing with cytology triage at baseline and repeated cytology testing after 1 year. As increasing the RLU/CO threshold only marginally decreases colposcopy referral rate while substantially reducing sensitivity, we suggest maintaining the currently used RLU/CO threshold of 1. This results in more than 30% higher sensitivities for CIN3+ than cytology testing at a 20% lower false positivity rate and 6% lower colposcopy referral rate.


Correlation between cytology, age and prevalence for the 14 different high-risk HPV types is given. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology. Coinfection with multiple HPV types also increased with cytological abnormalities, and was highest in HSIL (16.7%). In Flanders, HSIL was most often associated with HPV types 16, 33, 35, 31, 18 and 51. Using thin-layer liquid-based cytology and PCR to detect HPV, it is feasible to screen large numbers of women.The objective of this study was to document the occurrence and to correlate the prevalence of different human papillomavirus (HPV) types with the cytological results on simultaneously performed thin-layer preparations in a large population of Flemish women. During 1 year, 69 290 thin-layer preparations were interpreted using the Bethesda classification system. Using an algorithm for HPV testing based on consensus primers and type-specific PCRs in combination with liquid-based cytology, we determined the occurrence and distribution of 14 different oncogenic HPV types . Reflex HPV testing was performed on cytologically abnormal samples and on an age matched randomly selected control group with normal cervical cytology ( During recent years, there has been increasing evidence implicating infection with high-risk human papillomavirus (HPV) as a causal factor in the development of cervical intraepithelial neoplasia and invasive carcinoma .More than 40 genital HPV types have been shown to infect the genital mucosa and have been characterised . Recent We used the MY09/MY11 L1 consensus primer-based PCR . The aimFor thin-layer liquid-based cytology preparations (LBP), cervical samples were collected using the Cervex-Brush® . After collection, brush heads were transferred directly into alcohol-based preservative , and the vials were transported to the laboratory. The LBP were made with the fully robotic AutoCyte® PREP System  , using tBetween July 2000 and June 2001, the Laboratory for Clinical Pathology  received 69 290 cervical samples for cytological evaluation from women that were taken by general practitioners and gynaecologists in Flanders (Belgium).HPV DNA reflex testing was performed on all cytologically abnormal samples. For cytological normal samples, HPV DNA testing was performed only on explicit request of the clinician (WNLr), and on a randomly selected representative control group of 100 samples with normal cervical cytology (WNLc) of women with the same age distribution as the total group.Study-specific patient identification codes were assigned and transmitted in such a manner that patient confidentiality was preserved.μl of the remaining cells suspension was transferred to an eppendorf tube and cells were pelleted by centrifugation. The supernatant was discarded and the pellet resuspended in 50 μl digestion solution  and digested for 3 h at 55°C. The digestion was followed by a 10 min incubation at 95°C to inactivate the proteinase K. The DNA extracts were stored at −20°C until PCR was performed.After thin-layers were made, 400 Several precautions were taken to prevent false positive results. Different steps such as DNA extraction, sample preparation, amplification and post-PCR were performed in strictly separated rooms. Aerosol-resistant pipette tips were used for all handling of liquids. In all runs, distilled water samples were included as negative PCR controls. As positive control samples, HPV-containing cell lines SiHa , containing 1 copy of HPV-16/cell, and Hela  containing 10–50 HPV-18 copies/cell were used.β-globin with GH20 and PC04 primers  according to the degree of cytological abnormality. Statistical tests were considered significant at P<0.05. Statistical analysis was performed using the MedCalc® program  , followed by Student–Newman–Keuls test for all pairwise comparisons. The P<0.05) compared to all other groups.From a total of 69 290 samples, 19 (0.03%) were inadequate, 67 474 (97.38%) had normal cytology, 992 (1.43%) were classified as ASCUS, 641 (0.93%) as LSIL and 164 (0.24%) as HSIL. In total, 1797 (2.59%) samples were classified as abnormal  compared to all other groups. There was no significant difference in age between the cytologic groups tested for HPV and cytologic groups of the whole group (P>0.05).From the total group of abnormal samples, 129 samples were not tested because the clinician did not want HPV reflex testing on abnormal samples, and 610 samples of women who were tested shortly before were excluded from the study population. Ultimately 1058 abnormal samples were included, 552 ASCUS, 369 LSIL and 137 HSIL cases. Also, 193 samples with normal cytology were tested for HPV DNA on explicit demand of the clinician . Also foβ-globin could be amplified, and 57 tested positive for HPV types 18, 35, 39, 51 or 66. HPV51 was the most frequent type that was not detected by the consensus PCR (71.9%), followed by HPV39 (12.3%), HPV18 and 35 (7.0%), and HPV66 (3.5%). When we consider the number of samples tested in each cytological diagnosis group, the number of consensus PCR-negative samples, but containing HPV types 18, 35, 39, 51, 66, was most frequent in the LSIL (8.9%) and HSIL (4.5%) groups  were positive for one of these HPV types. Subsequent typing of the remaining 389 samples with multiplex PCRs for types 18, 39, 51 and 66 showed that 52.2% (203 out of 389) of samples contained these oncogenic HPV types. From the remaining 186 untyped samples, only 45 were positive when tested with type-specific PCRs for HPV types 45, 52, 56, 58, 59 and 68. Ultimately, 141 samples were negative for all type-specific PCRs and were considered to contain presently unidentified HPV genotypes of unknown malignant potential (HPV X).β-globin DNA were excluded from further analysis. Using HPV consensus L1 and E7 type-specific PCRs, the total number of HPV-positive samples was 875 out of 1339 (65.4%). Overall, HPV was detected in 21% of the samples of the WNLc group, in 25.7% of the WNLr group, in 58.7% of the ASCUS group, in 95.4% of the LSIL group and in 98.5% of the samples in the HSIL group . The percentage of HPV-positive samples infected with only one HPV type in the WNLc and the WNLr group was 19.0 and 25.1%, respectively, and was not significantly different (P>0.2). There was a significant increase in samples with single HPV infection with increasing abnormal cytology . The percentage of samples infected with multiple HPV types was low in the control group (2.0%) and did not differ significantly (P>0.9) from the percentage found in the WNLr group (0.5%). There was a significant increase in samples infected with multiple HPV types with increasing abnormal cytology as compared to the WNLr group . A linear trend was also found in samples infected with high-risk HPV types with increasing abnormal cytology .The overall HPV prevalence and type distribution according to cytological diagnosis is given in In the WNLc control group only a limited number of oncogenic HPV types were detected, namely HPV 16, 18, 31, 35, 39 and 51 . The HPVThe prevalence for each individual HPV type as a function of increasing abnormal cytology is shown in Figure 2P<0.05). Whereas in the WNLc, LSIL and HSIL groups no significant difference in age between HPV-positive and HPV-negative patients was observed. Patients with LSIL smears were always the youngest irrespective of their HPV status.The mean age of the different cytologic groups according to HPV DNA positivity is given in Each year approximately 1 200 000 cervical smears are taken in Belgium, of which 660 000 come from women living in Flanders . BetweenUsing an algorithm for HPV testing based on consensus primers and type-specific PCRs in combination with liquid-based cytology, we determined the occurrence and distribution of 14 different oncogenic HPV types in a population of Flemish women.β-globin DNA could not be detected. Therefore, the CytoRich fixative provides an excellent preservation of the samples making a simple and cheap DNA extraction possible. Liquid-based cytology improves the overall adequacy of the cell sampling, smearing and staining, as seen by the low number of samples judged as inadequate (0.03%) or interpreted as ASCUS (1.43%). Overall, 2.6% of the smears were classified as abnormal. This is within the range observed in other Flemish cytological laboratories  is high, not all samples were positive. Previous studies on HPV-type prevalence in Belgium were carried out only in the female population of Antwerp on DNA extracted from paraffin-embedded tissue  in ASCUS cases. This is in agreement with previous studies .In this study, the prevalence of women with a normal cytology to be HPV positive was 21%, and is in agreement with other studies in which the same MY09/MY11 PCR technique was used (26.0%, We showed that in the Flemish population HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52 and 66 are present in high-grade lesions, contrary to a previous study conducted in Antwerp that detected HPV39 and 51 only in low-grade lesions (Screening in Belgium is performed essentially at opportunistic basis, characterised by overscreening in coexistence with underscreening in indigent groups, heterogeneous quality, lack of quality control and impossibility to document effectiveness of screening by lack of adequate monitoring .Previous studies in the Netherlands have shown that there is a clear relation between HPV positivity and severity of disease, and that severe dyskaryosis is associated with persistent HPV infections, whereas minor abnormalities are more often associated with fluctuating or transient infections (HPV DNA detection allows a more effective triage of women showing equivocal cytological results than repeat cytology . NeverthOur study confirms that polyvalent vaccines including the main cancer-associated HPV types would be needed to prevent most cases of cervical disease in the screened, high-risk population (Finally, with this study we have proven that HPV typing is feasible in a high-throughput or routine setting.


P< 0.001, whereas the difference between HCIN and invasive adenocarcinomas was not statistically significant, P = 0.604. Prototype HPV16 and its E6 variant L83V are both prevalent in preinvasive and invasive cervical lesions in Swedish women. However, the obvious predominance of HPV16 variant in squamous carcinomas was not seen in adenocarcinomas. A single amino-acid shift in the HPV16 E6 gene appears to result in a different transforming potential in squamous and glandular cervical lesions. © 2000 Cancer Research CampaignA previous Swedish study revealed that both prototype and variant HPV16 E6 oncoprotein, occur in about equal numbers in high-grade cervical intraepithelial neoplasia (HCIN), whereas variant HPV16 predominates in invasive cervical squamous carcinoma. Most of the malignant HPV16 variants contain a common mutation, L83V, in the E6 oncoprotein. In the present investigation, 28 HPV16 positive, invasive cervical adenocarcinomas were collected from a total number of 131 adenocarcinomas. These HPV16-positive cases were evaluated with analysis of the E6 gene, using a recently described PCR-SSCP method for identification of the specific mutation (L83V) in the E6 gene. The results obtained were correlated to findings in 103 preinvasive, HCIN, and 31 invasive cervical squamous carcinomas also infected with HPV16. The HPV16 E6 variant L83V was present in 40% of the HCIN lesions, in 54% of the invasive adenocarcinomas, in comparison to 81% of the invasive squamous carcinomas. The difference between HCIN and squamous carcinomas was statistically significant,


However, irregular bleeding is quite common in initial Mirena® users and may thereby mask underlying cervical pathology.The levonorgestrel-releasing intra-uterine device (Mirena® initially presented with irregular bleeding, posing a diagnostic pitfall which resulted in doctor and patient delay.Two women with cervical cancer and a Mirena® and must be repeated in case of persisting symptoms.Proper evaluation of irregular vaginal bleeding, including cervical cytology, should be a prerequisite among all women opting for a Mirena It is tof women . However® was inserted. She returned to our clinic 2 weeks later due to abdominal cramping. The Mirena® was in place. She was treated with antibiotics and painkillers on a presumptive diagnosis of pelvic inflammatory disease. Two months later at follow-up, the Mirena® was still in place and irregular bleeding continued. As this is a common symptom in the first 3 months of Mirena® use, she was reassured and instructed to make a new appointment if the bleeding were to persist.A 40-year old nulligravida was referred to our outpatient clinic because of menorrhagia and irregular bleeding since 2 years, unresponsive to oral contraceptives. Her general practitioner obtained a Papanicolaou (Pap) smear at initial presentation of symptoms, which revealed no abnormalities. Inspection of the cervix showed no lesions, polyps or unusual bleeding. By vaginal ultrasound an intramural fibroid was seen in the fundus, measuring 4,5 cm × 4,5 cm and no further uterine anomalies. The next day, a MirenaSeven months after initial referral, she was seen for a cyst of Bartholin. During follow-up of the cyst, inspection by speculum revealed inflammation of the cervix. A Pap smear was performed, and during colposcopic examination, a lesion suspicious for neoplasia with abnormal vessels was seen. Two biopsies were taken and revealed a moderately differentiated squamous cell carcinoma. The Pap smear was reported as squamous cell carcinoma. Revision of the initial smear showed that due to blood staining it was difficult to interpret, but in retrospect it was suspect of malignancy. Clinical staging allocated our patient to cervical cancer stage IB. She was referred to a centre for Oncology for a radical hysterectomy with pelvic node dissection.®. Her medical history revealed no prior gynaecological problems and she had no history of abnormal cervical cytology. Examination by speculum and ultrasound prior to insertion of the Mirena® revealed no abnormalities. Due to ongoing irregular bleeding and abdominal pains since insertion, the Mirena® was removed several months later and oral contraception was prescribed. The irregular bleeding was attributed to the Mirena® and no Pap smear was obtained in this period. Nine months after initial referral, she was referred again by her general practitioner due to persisting irregular bleeding and rebound tenderness during bimanual palpation. An ulcerative lesion on the cervix was seen by speculum, suspect for malignancy. Cervical cytology revealed moderate dysplasia, biopsies a micro-invasive squamous cell carcinoma of the cervix. Clinical staging allocated her to stage IIA cervical cancer. Assessment of new biopsies revealed adenosquamous carcinoma. She was also referred to a centre for Oncology for a radical hysterectomy with pelvic node dissection. During surgery, however, the procedure was aborted due to tumour extension into the vesicouterine fold. Lymph nodes were negative and she was treated by radiotherapy with brachytherapy.A 29-year-old woman was referred by her general practitioner 2 months after delivery of her first child for insertion of a Mirena® without proper evaluation of its cause. Persistence of symptoms was attributed to the Mirena®. Results of prior long-term studies on cervical cytology in women using a Mirena® have shown that there is no increased incidence of cervical intraepithelial neoplasia [® may be more common than presumed, considering the fact that irregular bleeding is almost inherent to initial Mirena® use.The cases presented above both illustrate the doctor's delay caused by treating young women presenting with vaginal bleeding with a Mirenaeoplasia ,4. NeverThe second pitfall is the limited sensitivity of a Pap smear, varying from 50–80% depending on the study and type of cytology assessment used . Among t®, and must be repeated in case of persisting symptoms.In conclusion, we suggest that proper evaluation of irregular bleeding including cervical cytology should be performed among all women opting for a MirenaWritten informed consent was obtained from the patients for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.SdW was responsible for interpretation of data and drafting of the manuscript, PJW analyzed the Pap smears of the cases presented and revised and interpreted the Pap smears of the 80 women diagnosed with cervical cancer at our clinic between 1999 and 2004 as described in the discussion, and GSK was responsible for acquisition of data and conception of the manuscript.