We followed a population-based cohort of 5696 women, 32–38 years of age, by registry linkage with cytology and pathology registries during a mean follow-up time of 4.1 years to assess the importance for CIN2+ development of type-specific HPV DNA positivity at baseline. HPV 16, 31 and 33 conveyed the highest risks and were responsible for 33.1, 18.3 and 7.7% of CIN2+ cases, respectively. Women infected with HPV 18, 35, 39, 45, 51, 52, 56, 58, 59 and 66 had significantly lower risks of CIN2+ than women infected with HPV 16. After adjustment for infection with other HPV types, HPV types 35, 45, 59 and 66 had no detectable association with CIN2+. In summary, the different HPV types found in cervical cancer show distinctly different CIN2+ risks, with high risks being restricted to HPV 16 and its close relatives HPV 31 and HPV 33. Infection with ‘high-risk’ types of HPV is the major cause of cervical cancer, and the distribution of different HPV types in cancer tissue has been extensively analysed  associated with infections with 14 different so-called ‘high-risk’ HPV types.A population-based multicentre study was started in Sweden in May 1997 with the main purpose to evaluate the effect of HPV testing in primary cervical cancer screening. Women aged between 32 and 38 years (mean age: 35.1 years) in five regions in Sweden  who took part in organised cervical screening were invited to take part in the study. Following informed consent, 12 527 women were enrolled and randomised either to action on HPV tests (6257 women) or to no action on HPV tests (6270 women), as described in detail elsewhere (β-globin-negative) HPV test at baseline (172 women) or who had no cytological or histological samples registered during follow-up (796 women) were excluded from the analysis. The final population-based cohort thus consisted of 5696 women with a mean follow-up time of 4.1 years.All women were followed by registry linkages using unique personal identification numbers with both the regional cytology and pathology registries in the enrolling regions, as well as with the national cervical screening registry, to detect development of CIN2+. All women with an abnormal histopathological diagnosis as well as all women invited for colposcopy within the study protocol had their specimens re-evaluated by a single expert pathologist (WR) who was masked to the HPV status of the women. For 22 specimens that could not be located in the pathology archives, the original diagnosis was retained. Overall, 126/148 (85%) of the CIN2+ diagnoses in the present study have been confirmed by expert pathologist review. Women who had an inadequate  using HPV type-specific plasmids corresponding to the different HPV types tested for in the PCR-EIA   and thus we adjusted for time of diagnosis by introducing a term in the regression model representing follow-up of more or less than 6 months. Because the risk associated with one HPV type can be confounded by co-infection with other HPV types, we adjusted for infection with other HPV types by including type-specific HPV data as single variables in a multivariate regression model. Population attributable proportions were calculated as pc*(RR-1)/RR where pc is the proportion exposed to an HPV type among cases and RR is the type-specific relative rate adjusted for infection with other HPV types and censoring before or after 6 months of follow-up  were calculated for each HPV type. Relative rates for CIN2+ and their 95% CI were calculated using Poisson regression. Women were censored at their last testing date, except for women who developed CIN2+ who were censored at the date when the diagnostic biopsy was taken. Since lesions that were detected early during follow-up might have been present already at baseline we split the data according to length of follow-up of more or less than 6 months. A likelihood ratio test was then performed to assess if there was interaction between HPV infection and time of CIN2+ diagnosis. There was weak evidence for interaction  of these had a positive HPV test at baseline. The HPV type with highest population-based prevalence of infection was HPV 16 (2.5%), followed by type 31 (1.4%), 45 (0.9%) and 18 (0.7%). A single HPV type was detected in 401 (87.0%) of the HPV-positive women, while 60 (13.0%) women had more than one virus type detected in the same sample. Most multiple HPV infections were infections with two HPV types, but up to four different HPV types could be detected in the same sample. The absolute risk of CIN2+ among HPV-positive women ranged from 0% for HPV 59 to 48.0% (95% CI: 27.8–68.7) for HPV 33 . HPV 16 The relative rates of CIN2+ for each HPV type were assessed using women negative for the corresponding HPV type as the reference group . It shouAfter adjustment for concomitant infections with other HPV types, the type-specific relative rates segregated into three groups. HPV 16, 31 and 33 had very high relative rates (exceeding 10) , with alPopulation attributable proportions were based on the type-specific relative rates adjusted for all other HPV types. HPV 16 attributed to 33.1% (95% CI: 24.7–40.6) followed by HPV 31 (18.3% (95% CI: 11.5–24.5)), HPV 33 (7.7% (95% CI: 3.1–12–0)) and HPV 18 (5.7% (95% CI: 1.5–9–7)) . HPV 35,This prospective large-scale study provides individual estimates of the risk for future histopathologically confirmed high-grade CIN for each one of 14 genital HPV types, commonly included in ‘high-risk’ HPV tests. The large size and prospective study design, nested within the national screening programme and our comprehensive follow-up argue in favour of reliability of estimates.We report that several HPV types previously classified as ‘high-risk’ types . The population-attributable proportions were based on HPV type-specific relative rates, that is the rates for a specific HPV type compared to the rate among women negative for that HPV type . This choice of reference group is more appropriate for studying attributable proportions since the elimination of one HPV type does not imply that the population would become negative for all HPV types. It should be noted that even though our type-specific relative rates as well as our classification of which types are oncogenic differs from the above-mentioned studies  jointly attributed to almost 65% of all CIN2+, and prevention of infection with these four types would therefore be most important. By contrast, HPV types 35, 39, 45, 56, 59 and 66 each contributed to 2.0% or less of CIN2+ in the population and inclusion of these types in HPV vaccines therefore seems less important, as the theoretical possibility exists that inclusion of too many HPV types in second generation HPV vaccines might impair the response against the most important HPV type . In a cohort of 10 000 women in Costa Rica, HPV 16 was the type that contributed most to prevalent cases of CIN3+ followed by HPV 58, 18 and 31 . Also, detection of CIN2+ is exceedingly rare when HPV-negative, cytology-negative women are referred to colposcopy suggesting that verification bias has not materially affected our estimates (It has been suggested that CIN 3+ is a more appropriate proxy for the risk of future cervical cancer than CIN2+ since the regression rate is higher for CIN2 than for CIN 3 . AlthougIn conclusion, we found that different so-called ‘high-risk’ HPV types convey very different risks for future CIN2+, with HPV 16 and its close relatives HPV 31 and 33 consistently conferring the highest risks. Appreciation of the HPV-type-specific risks for CIN2+ can be helpful for interpretation and design of HPV tests as well as for second-generation vaccines against HPV.


The cellular transactivator Brn-3a has previously been shown to be expressed at elevated levels in the cervix of women with squamous cell carcinoma of the cervix (SCC) and to activate the expression of HPV E6 mRNA. In this study, we show that common and rare cervical precancer lesions, including those of adenocarcinoma (AC), which are usually difficult to diagnose using classical procedures, also expressed high levels of Brn-3a and can be diagnosed by measuring the levels of Brn-3a and E6 mRNAs. Cervical cancer is the second most common gynaecological cancer worldwide, with developing countries having the highest incidence . Of the Unfortunately, in developing countries such as Brazil, cervical cancer screening programmes are too costly. As a result, unsuitable less costly cervical programmes are often put into place, thus mortality rates due to AC and other associated cervical ailments have been on the increase –14, and It is therefore imperative for appropriate and cost-effective diagnostic programmes which could detect the early onset of AC, SCC, and SSCC to be made available in both the developed and developing worlds –7, 15–17Interestingly, several studies have portrayed HPV as a pivotal aetiological agent in AC , 19, 20,Although studies have suggested that HPV could be the main aetiological determinant for AC and SCC –7, otherMany studies have shown that HPV depends on its EORF E6/E7 oncoproteins –24 to trIn addition, several of our studies have shown not only that Brn-3a mRNA is elevated uniformly in the cervix of women with cervical ailments , but alsImportantly, the level of E6 mRNA, which parallels that of Brn-3a in cervical scrapes, has previously been shown to have considerable potential in screening and diagnosis of cervical abnormalities , 30–32. Here we report that the cellular transcription factor Brn-3a measured from cervical scrapes of women with AC and SCC precancer lesions can potentially be used for the screening and diagnosing of AC and SCC. This is the first time in which the activity of the primary aetiological agent of two different types of cervical cancer has been evaluated on the basis that the cellular factor, which regulates production of its oncogenic transforming proteins , 35, is The present study comprised two different groups of patients. Group one is made up of patients in Recife-Brazil a developing country, and Group two comprises of patients in London-England in the United Kingdom, a developed country. The study group in Recife-Brazil, consisted of 85 women with cervical abnormalities attending one of the following gynaecological units: (1) Instituto Materno Infantil De Pernambuco (IMIP) Recife-Pernambuco Brazil, (2) The Gynaecological Oncology Research Unit Bar de Lucena Hospital-Recife, and (3) The Cancer Hospital Recife. The mean age for this group was 44.8 years (the median: 42 years and the range: 21–81 years). The study group in London-England consisted of 51 women attending either the colposcopy or gynaecological clinics at the Whittington Hospital London. The mean age for this group was 34.7 years (median: 30.5 years and the range: 19–61). In both groups, ethical permission was obtained from the respective hospital's ethics committee after review of the study protocol. All the women in both groups provided informed consent to obtain and use their cervical material for the purpose of the study. Most of the women acknowledged the desire to participate for a long-term follow-up programme. This study focuses on women with High-grade Cervical Glandular Intraepithelial Neoplasia (HG CGIN) (adenocarcinoma (AC) precancer lesion, FIGO stages; adenocarcinoma in situ stages 1a1 and 1a2), women with High-grade Cervical Intraepithelial Neoplasia (HG CIN) (squamous cell carcinoma (SCC) precancer lesion; CIN2 and CIN3), and as our control group, women with cervicitis, which is an inflammation of the uterine cervix usually attributed to infection caused by sexually transmitted diseases . ConversCervical cells were obtained and processed using our previously described routine methodology but with some slight modifications to reduce cost, so that it could be conveniently adapted in developing countries . In brieImportantly, the specimens used in this Material section were not preselected according to their pathological classifications. Indeed, the specimens were obtained by the Clinicians and processed blindly by the Researchers.t-test. All other statistical analyses were performed using Microsoft Excel. At all instances, P values of less than  .05 (P < .05) with 95% Confidence Interval (CI) was used as the cut-off for statistical significance.We used the linear regression model (Microsoft Excel) to derive the coefficient of regression between Brn-3a and E6, and the correlation coefficient between Brn-3a and E6 mRNA levels was derived using the Pearson Correlation coefficient (“PEARSON” in Microsoft Excel). The differences of the mean between variables were analysed using Microsoft Excel Student's R = 0.6), , are presented in  = 0.6), . P = .000052) and 33/49 (67.3%) high E6 (P = .00046) (Tables P = .000026) and (19/21) of them high E6 (P = .0001)  of patients have SCC precancer of which 48/49 (97.9%) registered high Brn-3a  Tables and 2. Fu of patie) Tables and 2. TwAs expected, given that AC and SSCC constitute rare forms of cervical cancer, 33.3% (17/51), or 1 in 3, of the specimens in Brazil were AC precancer, but less than 20% (4/21) of the specimens in England were AC precancer . InteresSimilar prevalence was also observed with the cases in England , where STaken together, we have demonstrated that Brn-3a and HPV E6 mRNA were detected in the cervical scrapes of women with different forms of cervical precancer in both developing and developed countries. This is in accordance with many other studies that have suggested HPV DNA as the primary aetiological agent for these ailments worldwide –11, but r = 0.85 was obtained. Moreover, the coefficient of regression between Brn-3a and E6 for SCC and AC precancers was respectively deduced to be R2 = 0.72 and R2 = 0.74  and for London (P = .0003), which is also true for the E6 mean in Recife (P = .000002) and for London (P = .01). Interestingly, a similar significant difference was observed between the cervicitis control and the AC precancer specimens in Recife (P = .00033) for Brn-3a and E6 (P = .001). In addition, the mean values of Brn-3a and E6 in the cervical scrapes of women with SCC and AC precancers, SSCC and cervicitis as a control group were also determined . The resIn both Brazil and England, the mean value of Brn-3a and E6 in the AC precancer specimens was greater than that of the control group as depicted in Taken together, this result could be of particular importance for the early diagnosing of rare and common forms of cervical cancers, since the elevated level of Brn-3a occurs uniformly in the cervix, and it is not confined only to the transformation zone, so a high level of Brn-3a in the cervix predicts rapid disease progression upon viral infection. In agreement with this, it could therefore be employed for predicting the early stages of AC in countries where cost is hampering cervical screening programmes, or where classical procedures are inadequate in diagnosing the early stages of SCC and other related cervical diseases. This is because, as we have shown in several studies, the rate of transactivation of the oncogenic EORF gene product E6 depends on the level of Brn-3a. Thus, the level of the cellular factor Brn-3a predicts the rate of viral activity, and hence the rate of the accelerated neoplastic transformation. Indeed, Brn-3a measurements have been used successfully in the past in our clinics to recall women for further gynaecological examinations , 32. P = .001) as compared to women in Brazil a developing country with SCC precancer  [P = .00033) as compared to that observed in London, a city in a developed country  [However, further studies are needed to ascertain why young women in a developed country, such as in England, do have high prevalence of SCC precancer  , 31, 32..000001) –16, the 3a 0.46) –16. However, no correlation was observed between age and Brn-3a level in the cervix, suggesting that the high levels of Brn-3a in the cervix of women with cancer in both the developing and developed countries could be due to genetic or other environmental factors rather than hormones , 36, whiThus, the expression of the oncogenic HPV cellular transactivator Brn-3a observed in cervical material in this study suggests that it could be used to predict the early onset of cervical adenocarcinoma and squamous cell carcinoma. The few Sarcomatoid squamous cell carcinoma samples available for this study registered the presence of low but detectable levels of Brn-3a and HPV E6 mRNA, and so further work still needs to be done using material obtain from SSCC biopsies, to investigate what role Brn-3a and the transactivated viral oncogenic products play in the natural history of this rare cervical cancer. Thus, studies have shown that, AC, SCC, and even SSCC share a similar aetiological profile that hinges on the expressed oncogenic products of HPV –10. ThisIn conclusion, this study suggests that in both in the developed and developing worlds, the cellular factor Brn-3a could be used alongside the oncogenic E6 mRNA in the screening and early diagnosing of both the common and rare types of cervical cancer.


Among dietary habits, the highest vs lowest intake tertile for vegetables and fruit was associated with an OR of 0.5 (95% CI 0.2–1.0). Low education level and low body weight were also risk factors for ICC, but they did not account for the associations of paan chewing and low vegetable and fruit intake. In the analyses restricted to HPV-positive cases and controls, the inverse association with vegetable and fruit intake was confirmed. Conversely, the adverse influence of paan chewing on ICC risk seemed to be attributable to a higher prevalence of cervical HPV infection in women who chewed.Non-viral factors contribute to human papillomavirus (HPV)-related cervical carcinogenesis. We investigated the role of paan chewing and dietary habits among 205 women with invasive cervical cancer (ICC) and 213 age-matched control women in Chennai, India. Odds ratios (OR) and 95% confidence intervals (CI) were computed by means of unconditional multiple regression, taking into account major correlates of ICC risk. Paan chewing showed a dose-dependent direct association with ICC (OR for ≥5 paan day Infection with oncogenic types of human papillomavirus (HPV) has been established as the central cause of invasive cervical cancer (ICC) .Several studies have shown that low vegetable and fruit intake , Southern India, one of the areas with the highest ICC incidence rates   a diagnosis of anogenital tract cancers , cancer of the breast, endometrium, ovary or colon, benign genital tumours and tobacco-related diseases ; (2) a history of hysterectomy or cervical conisation; and, as for ICC cases, (3) physical or mental problems. A total of 179 visitors and 37 in-patients were thus contacted. Of the eligible 216 controls, three declined to participate in the study and three refused to have a gynaecological examination. Thus, a sample of exfoliated cells from the cervix was available for 210 control women.Each participant was administered a standardised questionnaire on socioeconomic status, sexual behaviour, reproductive history, contraceptive practices, smoking habits, genital hygiene, history of sexually transmitted infections and cervical cytological screening. Questions on weekly consumption of 21 foods or food groups, and summary questions on overall consumption of all vegetables and fruits were included. Information on height (cm) at time of interview and weight (kg) 2 years before interview was self-reported. Two trained female interviewers administered the questionnaire to all case and control women in the hospital.After the interview, each consenting woman had a pelvic examination performed by a gynaecologist, who took two cervical scrapes with two Ayre spatulas and one endocervical brush. A Papanicolaou smear was prepared, and the remaining cells were eluted in phosphate-buffered saline (PBS), pelleted in PBS (2000 r.p.m. for 20 min at room temperature), and then stored at −70°C. A punch biopsy sample from the tumour (cases only) and a 10-ml sample of peripheral blood were also collected and stored at −70°C.The study was approved by the ethical review committees of the IARC in Lyon and the Cancer Institute in Chennai.β)-globin gene-specific primers were used. β-Globin-negative and HPV-negative samples (i.e. two ICC cases and 26 control women) were considered inadequate and excluded from HPV-related analyses, thus leaving 191 invasive cancer cases (179 squamous-cell carcinoma cases and 12 adeno/adenosquamous carcinoma cases) and 184 control women with adequate HPV-DNA results.To analyse the quality of target DNA, beta  using two HPV oligoprobe cocktails that together detect the following 36 HPV types: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, 11, 26, 34, 40, 42, 43, 44, 53, 54, 55, 57, 61, 70, 71 , 72, 73, 81 , 82 (IS39 and MM4 subtypes), 83 , 84  and CP 6108. Probes and procedures used for EIA detection are described elsewhere  (2), and intake of foods or food groups were computed based on the combined distribution of cases and controls. Tests for linear trend were performed assigning an increasing score for each level of categorical variables. Scores were then fitted into the model as a continuous variable. In order to elucidate the influence of HPV infection, major analyses were repeated: (1) among all cases and controls, (2) among HPV-positive vs HPV-negative control women, and (3) among HPV-positive cases vs HPV-positive control women only.To estimate the risk of cervical cancer associated with various HPV types and the other risk factors, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) by unconditional logistic regression. The ORs were adjusted for age and area of residence (Chennai and out of Chennai), education level, occupation, marital status, age at first marriage , number of pregnancies and husband's extramarital affairs, as indicated in the Tables. Only 4.9% of cases and 1.4% of control women reported more than one lifetime sexual partner and zero cases and 1.4% of control women reported oral contraceptive use. Therefore, these two variables were not included in the models. Approximate tertiles of weight, height, body mass index . Approximately half of the cases and controls combined reported to work in agriculture, and farmers showed an OR of 4.3 compared with housewives. Being a widow or divorced rather than currently married (OR=8.1), having had ⩾5 vs 1–2 pregnancies (OR=5.0), and reporting that one's husband had had (or probably had) extramarital affairs (OR=7.0) were associated with significantly increased risk of cervical cancer . Two cases and no controls reported to have ever smoked  . A nonsignificant inverse association was found also for height .The mean weight was 52 and 54 kg among cases and controls, respectively. Women who weighted less than 50 kg had an OR of 1.9 compared with women who weighted 55 kg or more (vs lowest intake tertile=0.4)and bananas (OR=0.3). The inverse associations with intake of fresh tomatoes, carrots, cruciferae and other citrus fruit were not statistically significant. There was no indication that the consumption of green vegetables, apples or pears, rice, milk, yogurt, egg, fish, meat, cakes or desserts and various oils affected ICC risk. A direct association was found for the intake of bread (OR=2.0).vs never OR=2.4) and vegetable and fruit intake (summary question) (highest vs lowest intake tertile OR=0.5) were associated with ICC risk. In the analyses restricted to HPV-positive women, however, only the association with low vegetable and fruit intake (OR=0.4) was confirmed, even if it became of borderline statistical significance. Conversely, paan chewing was not related to ICC risk among HPV-positive women but showed a hint of direct association with HPV infection among control women.This is the first report of an association between paan chewing and cervical cancer. The topic is of interest in the light of the well-established association between ICC and tobacco smoking . The mecIn addition to paan chewing, low education, occupation in farming, low weight and low intake of vegetables and fruit were risk factors for cervical cancer in our study. An excess of cervical cancer among low socioeconomic class women is one of the most consistent findings of epidemiological studies on this tumour , the apparent adverse effect of weight and BMI must be interpreted with caution, as it may be a consequence rather than a risk factor for cervical cancer. The findings on height, although not statistically significant, point to a possible influence of nutritional deficiencies in childhood and adolescence that might have prevented some women from attaining their full height. Unfortunately, the reliability of self-reported weight and height in India is unknown.Our present evaluation of dietary habits also has limitations, since we have been using a short frequency questionnaire that included 21 indicator foods or food groups only and we did not explore serving size. Furthermore, the questionnaire had not been validated in India, and it was especially difficult to capture any association with staple foods like rice, which are eaten daily by the majority of the population, and with food groups which are eaten rarely  or as ingredients of complex recipes . No reliable translation into specific macro- and micronutrients has been possible, but in the summary questions on overall intake of all vegetables and fruit, ICC cases did report a significantly lower intake than control women, in agreement with findings of some previous reports  and for cervical cancer among HPV-positive cases and HPV-positive controls can help to elucidate whether these influence chiefly the acquisition and persistence of HPV infection or the progression from HPV infection into malignant cervical lesions (Unfortunately, the subgroup analyses in our study are based on small numbers of women and have very broad confidence intervals. However, the analyses restricted to HPV-positive women support the possibility that low intake of vegetables and fruit contributes to the progression from HPV infection to cervical cancer. With respect to paan chewing, either its adverse effect on cervical cancer is real and, possibly, mediated by a decreased ability of women who chew to clear HPV infection, or the habit of chewing is a surrogate marker for increased HPV exposure. Future prospective studies could evaluate the influence of paan chewing in the natural history of HPV infection and the development of preinvasive cervical lesions.


We conducted studies in Vanuatu to evaluate potential screening and treatment strategies to assist with control of cervical cancer. In a pilot study of 496 women, visual inspection and cytology were evaluated as screening tests for detection of CIN 2 or worse (CIN2+), observed in 21 of 206 subjects biopsied on the basis of abnormal visual inspection or cytology. Sensitivity of visual inspection with Lugol's Iodine for detection of CIN2+ on biopsy was 0.63, specificity was 0.32, and the positive predictive value was 0.09. For HSIL cytology, sensitivity was 0.99, specificity was 0.77, and the positive predictive value was 0.88. HSIL cytology was significantly more sensitive and had a significantly higher PPV for CIN 2+ than visual inspection (p<0.01). In a further study of 514 women, we compared testing for HR HPV and cytology as predictors of biopsy proven CIN 2+. Sensitivity of HSIL cytology for CIN2+ as established by loop excision of the cervix was 0.81, specificity was 0.94, and positive predictive value was 0.48. Sensitivity of a positive test for HR HPV for detection of CIN2+ was non-significantly different from cytology at 0.81, specificity was 0.94, and positive predictive value was 0.42. Combining the two tests gave a significantly lower sensitivity of 0.63, a specificity of 0.98, and a positive predictive value of 0.68. For women over 30 in a low resource setting without access to cytology, a single locally conduced test for high risk HPV with effective intervention could reduce cervical cancer risk as effectively as intervention based on cytology conducted in an accredited laboratory. Cervical cancer is a disease of the developing world, and is responsible for over 250,000 deaths annually. Cytological screening programs can be effective for prevention of cervical cancer in the developed world, but rely heavily on effective cytopathology services and call/recall systems to ensure compliance with regular screening assessments amongst at risk women To overcome this problem, alternate strategies have been proposed to detect women in low resource settings requiring intervention to prevent cervical cancer. These include visual inspection of the cervix Vanuatu is a Pacific island nation with limited health care facilities and a poorly documented incidence of cervical cancer, believed similar to the high rates reported for the neighbouring country of Fiji A pilot study recruited female subjects aged between 30 and 50 in 2006 by poster and flier advertisement, radio publicity, and nurse “awareness” visits to villages round Port Vila, Efate Island, Vanuatu. Women with a history of gynaecological surgery were excluded. Informed consent was obtained from all participants. A follow on study used a similar recruitment strategy in 2008 amongst non-participants in the first study, and 519 further subjects were recruited. The studies were approved by the Human Experimentation Ethics Committee of the University of Queensland and by the Parliament of the Nation of Vanuatu, and conducted in accordance with the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants. Where necessary this was supplemented by verbal consent in an appropriate language for the participant. The studies are registered in the Australian and New Zealand Clinical Trials Registry (Registration No ACTRN12610000702011).For the pilot study , a briefCervical cytology samples were taken using a cytobrush and a Cervex sampler, and were processed for both slide and liquid based cytology and for HR HPV DNA testing (Digene HC2 High- Risk HPV DNA Test from QIAGEN). For the first study, samples were also tested for Chlamydia , for gonorrhoea  and subjects were also tested for HIV-1 serology (no confirmed positives). Biopsies and LLETZ specimens were fixed in neutral buffered formalin for transportation to Australia. All cytology and histology examinations were conducted by accredited Australian diagnostic pathology providers participating in National Association of Testing Laboratories (NATA) approved quality control and quality assurance schemes for cervical cytology and pathology, and blinded to the clinical and HPV findings. For the pilot study, HPV DNA testing was performed according to the manufacturer's instructions in Australia by a NATA approved laboratory, and for the subsequent study, locally by a trained nurse technician.Statistical analysis was performed using appropriate modules of Statistica V9 .Apparently healthy Ni-Vanuatu women age 30–50 were recruited to a study to evaluate possible strategies for cervical cancer screening. Amongst a study cohort of 499 women (mean age 39.3y ±5.9y), clinically evident cervical cancer was confirmed on biopsy in five subjects who were not included further in the analysis. The remaining women were examined for visual abnormalities of the cervix, by VIA and VILI without magnification, independently by a gynaecologist and a trained nurse, and subsequently colposcopically by a gynaecologist. They were also tested for cytological abnormalities of the cervix using liquid based cytology. If any test were abnormal, subjects underwent colposcopically directed biopsy of the cervix, or LLETZ excision if cancer was suspected clinically. Of 494 subjects evaluated, 206 were biopsied, and 21 were found at biopsy to have CIN 2+.Correlation between VIA and VILI findings, and between visual inspection findings by doctor and by nurse, was only moderate . AmongstMost subjects in this study were also tested for high risk (HR) HPV using the liquid based cytology specimen. Of 488 subjects tested for high risk HPV, 44 were positive. A similar rate of positive HPV tests was observed in each 5 year age cohort (30-35y (n = 156), 10.2%; 36-40y (n = 139), 7.9%; 41-45y (n = 99); 12.1%; 46-50y (n = 95), 8.4%). Of the 44 subjects with a positive HPV test, 9 had normal visual inspection and cytology: two of these subjected to biopsy had normal histology. Of the 36 subjects with a positive HPV test and either abnormal cytology or abnormal visual inspection, and therefore subjected to biopsy, 14 had biopsy proven CIN 2+. Normal cytology, if used to triage those with positive HPV tests, would have excluded 12 subjects without CIN 2+ from biopsy, and none with CIN 2+. Of 163 women with normal cytology and a negative HPV test who were subject to biopsy because of abnormal visual inspection, 5 had CIN 2+ on directed biopsy. However, on subsequent LLETZ excision, none had CIN 2+ identified within the pathology specimen.On specific questioning, a history of post coital or inter-menstrual bleeding was elicited from one of 5 subjects with invasive cancer, none of 21 with biopsy proven CIN 2+, 13 of 182 with normal or low grade pathology on biopsy, and 18 of 291 subjects for whom no biopsy was taken. Thus, elicited symptoms were of no predictive value for selecting women for further screening.Of 21 subjects from this study with CIN 2+ on biopsy, 15 were predicted by HSIL cytology, of whom 14 also had HR HPV. No subject had CIN2+ predicted only by HR HPV, and the 5 who were detected only by abnormal VIA/VILI were not confirmed on LLETZ excision. Overall, 6 of 21 biopsy identified CIN2+ lesions would have been missed in this study if possible HSIL or worse cytology or a positive test for HR HPV had been used as the screening tests without regard to cervical appearance.We were aware of the possible ascertainment bias inherent in the first study, in which cytology results, but not HR HPV test results, were used to determine collection of a biopsy in the absence of visual abnormality. The number of women with HR HPV but with normal inspection and cytology, and therefore not subjected to biopsy, and therefore the maximum error in true case ascertainment, was 7, or 25% of subjects with actual or potential CIN2+. We therefore undertook a further study in a new cohort of women drawn from the same population. Women between 30 and 50  were recruited using the same strategy as for the first study, and tested for abnormal cytology, defined as possible HSIL or worse, and for HR HPV on the same specimen. If either test were abnormal (n = 77), LLETZ excision was offered . Of 74 s5 women. Cervical pre-cancer is also common in this population with a point prevalence of about 500/105 women. Biopsy proven CIN 2+ in over 30 year old women is a reliable predictor of lifetime risk of development of cervical cancer In this study we show that cervical cancer is common amongst unscreened apparently healthy Ni-Vanuatu women over 30, with a point prevalence estimate of approximately 100/10To reduce the cervical cancer burden in a low resource setting, a single screening test for women over 30 years of age has been proposed. A single assessment of cervical cytology has a 30%–50% false negative rate for significant cervical pathology, as subsequently detected by colposcopically directed biopsy, as was demonstrated in the current study. Colposcopically directed biopsy can be supplemented by multiple biopsies A study of 500 women, even in a country with a high prevalence of disease, has limited power to compare sensitivity and specificity of the various tests. However, visual inspection of the cervix, assisted by acetic acid or Lugols iodine staining, identified 43% of all women in the current study as having significant abnormalities in need of further investigation, a figure significantly higher than the prevalence of disease requiring treatment. The high prevalence of visual abnormalities that would then require treatment, and their relatively poor sensitivity and specificity for high grade premalignant lesions would make visual inspection a poor choice as a feasible single test approach to cervical cancer screening in this population. Thus this study lends support in another geographical setting to the conclusion from a recent report that in India treatment of abnormalities identified through visual inspection is not, in contrast to HPV testing, a practical means of reducing the risk of death from cervical cancerThe high frequency of observed visual abnormality in the current study contrasts with the findings of a meta-analysis of over 52,000 subjects participating in similar studies in which 17% of subjects had abnormal visual inspection. In the meta-analysis, about half the rate of CIN 2+ observed in the current study was reported (2.3% cf 4.2%) The better sensitivity and specificity of cytology for detecting CIN2+ in the current study when compared with the meta-analysis might reasonably be attributed to the use of highly experienced cytology laboratory Our second study was performed to define the relative utility of abnormal cytology and/or HPV test findings as a screen for CIN 2+, and to establish more precisely the nature of cervical pathology present than was possible by directed biopsy. Relative sensitivity and specificity of HC2 HPV detection for incident CIN 2+ was similar in our two studies, and, consistent with removal of possible ascertainment bias inherent in the first study design, the sensitivity of cytology was somewhat lower.www.hpv2010.org) that where routine colposcopy and biopsy services are available, screening findings with a >40% probability of an underlying high grade cervical lesion should be treated definitively, and those with less should be further assessed. Cryotherapy of visual abnormalities of the cervix has been proposed as a strategy for prevention of progression of CIN2+ to cancer but is not recommended by WHO for large lesions, or lesions within the cervical canal, which are more likely to persist and to progress to cancer HC2 HPV detection is somewhat less specific and considerably more sensitive than cytology for detection of CIN2+ lesions in populations preselected by a prior abnormal pap smearWe propose from our findings that a single low cost HPV test, as currently being developed by PATH in cooperation with QIAGEN and the Gates Foundation, with loop excision of the cervix for those with a positive test who have completed their reproductive plan, would be the most appropriate approach to cervical cancer reduction in resource poor countries with high disease prevalence.


Seven (12.7%) samples of apparently normal cervical scrapes were weakly positive for telomerase activity. We observed a good correlation (P<0.001) between telomerase activity and HR-HPV 16/18 positivity with a sensitivity of 88.1% for HPV and 100% for telomerase activity. It is suggested that telomerase activity may be used as an adjunct to cytology and HPV DNA testing in triaging women with cervical lesions.Telomerase is a ribonucleoprotein comprising an RNA template, the telomerase-associated protein and its catalytic subunit, human telomerase reverse transcriptase (hTERT). Telomerase activation is a critical step in cellular immortalisation and development of cancer. Enhanced telomerase activity has been demonstrated in cervical cancer. In the present study telomerase activity and hTERT mRNA expression were evaluated and correlated with the presence of human papillomavirus (HPV) infection and cytological changes in the cervical lesions. Telomerase activity was assayed by telomeric repeat amplification protocol, hTERT mRNA expression by reverse transcriptase polymerase chain reaction and presence of high risk HPV (HR-HPV) infection by polymerase chain reaction. Out of 154 cervical samples of different cytology, 90 (58.44%) were positive for HR-HPV types 16/18, while among 55 normal cervical scrapes, 10 (18.18%) were HPV DNA positive. All 59 invasive cancer samples showed a very high telomerase activity. Among dysplasia, seven (63.6%) mild dysplasia, 18 (100%) of moderate, 20 (100%) of severe dysplasia and 6 (100%) carcinoma  Cervical cancer is one of the most common causes of mortality among women worldwide with an annual incidence rate of approximately 400 000 cases and 200 000 deaths per year  are the primary causal agents in the development of cervical carcinoma .Studies on the oncogenic potential of these HPV types have clearly demonstrated that HR-HPVs are a necessary cause for the development of cervical cancer , a catalytic protein subunit, human telomerase reverse transcriptase (hTERT) and other associated proteins namely hTP1 etc. Most adult somatic cells exhibit low or no telomerase activity and thus experience progressive telomere attrition with each round of cell cycle. In contrast, germline cells, differentiating cells and tumour cells have elevated or detectable levels of telomerase activity and have long or stable telomeres. Consequently, telomerase may be required for the long-term proliferation of tumours. Stabilisation of telomere lengths by activation of telomerase is thought to be the key mechanism of indefinite cell proliferation and immortalisation. Telomerase activity has been reported in different tumour types The main transforming genes of HPV 16 and other HR-HPV types are E6 and E7. HPV E6 protein generally target and degrade tumour-suppressor protein, p53, through ubiquitin pathway . SpragueIn the present study, we investigated whether the status of HPV infection, telomerase activity, hTERT, hTR and hTP1 mRNA expressions in cervical tissues and/or cervical scrapes have clinical value in the triage of women or useful as an adjunct to cytology, particularly undefined or atypical squamous cells of undetermined significance (ASC-US) and mild, moderate or severe dyskaryosis.n=11), moderate (n=18), severe dysplasias (n=20) and CIS (n=6) cases and 40 normal cervical tissue controls. The study also included 55 cervical scrapes from asymptomatic, normal healthy women. All the biopsies were colposcopy-directed, and were either histopathologically or cytologically confirmed by the pathologists. Before taking punch biopsy, cervical scrapes were taken from all invasive cancer patients.This study was conducted in 154 tissue biopsies from patients in the age group 25–60 years and included 59 invasive cervical carcinomas, 55 dysplastic tissues belonging to various grades namely, mild  of Lok Nayak Hospital, New Delhi. The clinical staging of the tumours ranged from stage I to stage IV and their histological tumour grading was either of poor, moderate or well-differentiated squamous cell carcinomas and adenocarcinomas and subtypes thereof. Control biopsy specimens were collected from asymptomatic, apparently normal women visiting OPD and undergoing hysterectomy for gynaecological reasons other than cervical cancer such as fibroids, dysfunctional uterine bleeding etc. Cervical scrapes were also collected from asymptomatic normal women coming for checkups other than gynaecological complaints. Informed consent was obtained from each patient before recruitment for the study.Biopsies and the scraped cervical cells were collected in chilled PBS and transported from cancer clinic to the laboratory on ice. Each biopsy specimen was bisected after grossing by pathologists; one-half was subjected to histopathological examination after fixing the sample in 10% formaldehyde. The remaining half was employed for DNA and RNA extraction. The whole procedure was completed within 3 h from the time of collection.β-globin was used as an internal control to check the integrity of the specimens. Further HPV typing was carried out using type-specific primers for HPV types 16, 18, 6 and 11 , using the consensus primers MY09 and MY11, with an expected product size of about 450 base pairs (bp), for the amplification of the most conserved LI region of the HPV genome. A gene fragment of the μl ice-cold CHAPS lysis buffer . After 30 min of incubation on ice, lysates were centrifuged at 10 000 r.p.m. for 20 min at 4°C, the supernatant was aliquoted and stored at −70°C until used for assay.A portion of the tissue grinded in liquid nitrogen (40–100 mg) was suspended in 200 μl reaction mixture containing (20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20, 1 mM EGTA, 50 μM each of dNTPs, 1 μg of T4 gene 32 protein , bovine serum albumin (1 mg ml−1), 0.1 μg of TS primer (5′-AATCCGTCGAGCAGAGTT-3′) and [α-P32]dCTP10Ci/μl (specific activity 3000 Ci mmole−1) with 5 μg of protein. After telomerase mediated strand formation 0.1 μg of CX primer was added at 72°C and PCR amplification was performed. Lysate from HeLa cells were used as positive control. Sample treated with RNAse A was treated as negative control. Lysis buffer was used as reagent blank to monitor reagent contamination.Telomeric repeat amplification protocol (TRAP) assay was carried out as described by The PCR products were then analysed by electrophoresis on 12% polyacrylamide nondenaturing gel and autoradiographed. The level of telomerase activity was quantified by comparing the density of the ladder signals with the positive control (HeLa cells) and expressed in relative units (RU). Samples which did not give ladder were comparable to negative control and were graded as 0, the ladder which gave stronger signal compared to positive signal as 4+(++++), equal signal as 3+, moderate as 2+ and mild in 1+ RU.μg μl−1 of RNA. The reaction was carried out in 20 μl containing 50 ng of random hexamer primers (N6) , 250 μmol of each of nucleotides , 0.1 M of dithiothreitol, 40 U μl−1 of RNAse inhibitor  and MMLV reverse transcriptase . The cDNAs were stored at −70°C until PCR amplification.RNA was extracted from the tissue biopsies using TRI reagent according to the manufacturer's specifications. The cDNA was synthesised for each of the sample using 5 μl reaction mixture, containing 10 mM dNTPs, 10 μM of each set of primers, 5 U of Taq DNA polymerase, by using gene-specific primers for hTERT, hTR and hTP1 along with the primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The cDNA was amplified for 36, 32 and 30 cycles, respectively, for hTERT, hTR, hTP1 along with house keeping gene G3PDH. The amplified PCR products were resolved by electrophoresis on ethidium bromide stained 1.5% agarose gel and visualised in BIO-RAD gel documentation system.Two microlitre of cDNA was taken in a total volume of 50 In all, 52 out of 59 invasive cervical carcinomas (88.1%), 38 of 55 (69.1%) dysplasias, two (5%) out of 40 normal control tissues and 13 of 55 (23.6%) normal cervical scrapes were positive for HPV by LI consensus primers. Further typing of HR-HPV 16 and 18 was carried out by PCR amplification of most conserved upstream regulatory region (URR) sequence of HPV 16, which results in a PCR product of 217, and 100 bp band HPV 18 E6 region .All 59 (100%) invasive cervical tumours showed a very high telomerase activity when compared to that of controls (5%). Cervical smears from the same patients also showed similar increased activity. Of the 55 dysplasia samples, seven (63.6%) of 11 mild dysplasia, samples revealed low telomerase activity. While all 18 (100%) moderate, 20 (100%) of severe dysplasia and six (100%) of CIS cases were positive for moderate to high to very high telomerase activity respectively. Out of 40 normal control tissues only two (5%) were positive for telomerase activity but seven cases (12.7%) of apparently normal cervical scrapes showed weak telomerase activity. Telomerase activity in different cervical lesions is shown in P<0.001) showed 88.1% sensitivity in invasive carcinomas compared to 100% sensitivity of telomerase activity. It is further observed that as the positivity of oncogenic HPV types 16/18 increased telomerase activity also increased with the increased severity of the disease. Severe dysplasia cases showed 100% sensitivity for telomerase activity compared to 76.9% for HPV. Telomerase activity thus appears to be a better molecular marker than HR-HPV 16/18 in detecting lesions even in mild dysplasia cases. RT–PCR experiments for hTERT, hTR and hTP1 mRNA expressions revealed that both hTR and hTP1 were constitutively expressed in all invasive carcinomas, dysplastic lesions and normal controls of the uterine cervix. However, the expression of hTERT levels was greater in invasive cervical cancer samples and showed an increasing trend with the increasing grade of dysplasia/cervical cancer .P<0.001).As regards to hTERT, a very high expression levels was observed in all 59 invasive cervical carcinomas, which also well correlated with their high telomerase activity. Human telomerase reverse transcriptase expression in eight mild, 18 moderate, 20 severe dysplastic lesions and six CIS cases ranged from mild to moderate to high and very high levels, respectively. While seven samples of normal cervical scrapes revealed a weak hTERT mRNA expression, none of the normal control tissue samples had any detectable levels of hTERT expression. A specific PCR amplimer of 145 bp for hTERT and a 103 bp for hTR are shown in P<0.001). Human telomerase RNA and hTP1 expressions were found even in HPV negative samples, although not as high as in HPV-positive samples. However, their expression levels increased with increasing severity of the disease.When HPV infection in the different categories of cervical lesions was correlated with the expression of telomerase components hTERT, hTR and hTP1 as determined by RT–PCR, a good correlation with the presence of oncogenic HPV type 16 was observed. Human telomerase reverse transcriptase expression was detected in as many as 88 of 90 (97.8%) specimens which were positive for HR-HPV type 16, with the exception of only one (1.11%) of HPV-positive samples not expressing hTERT  software.We studied the relationship between HPV status, telomerase activity, hTERT, hTR, hTP1 mRNA expression during the development of cervical cancer through different grades of cervical dysplasia to invasive cervical cancer.n=40) compared to 18% of (n=10) normal cervical scrapes were positive for HR-HPV types 16/18 infection. This observation in the present study is interesting. It is possible that these women who were attending OPD with symptomatic complaints, may not really be normal women in strict sense, thus they may be having latent or an occult or commensal papillomavirus infection, that is, the presence of HPV DNA in the absence of a visible, histologic or cytologic abnormality. Cytology is considered as the gold standard for the diagnosis of dysplastic and invasive uterine cervical lesions. Pap smear test routinely employed for the diagnosis of premalignant cervical lesions is, however, found to have variable sensitivity and specificity. Testing for oncogenic type of HPVs is another viable method to screen women with cytological abnormalities since HR-HPV infections are considered as the most common causal factor and are reported in more than 90% of cervical cancers . We havein-situ was 69.1%. The results were comparable to earlier published reports  which is in agreement with other studies, while the frequency of HPV type 18 (3.38) is very low when compared to other ethnic population . AbsenceEx vivo studies showed that transfection of normal epithelial cervical keratinocytes with the HPV E6 gene resulted in telomerase activation even before the occurrence of ‘crisis’  of 11 mild dysplasia samples. All 18 moderate, 26 severe dysplasias which included six CIS samples were positive for moderate to high to very high telomerase activity, respectively. High telomerase activity in all samples of CIS, at par with invasive carcinomas, is justified since this stage is a pre-invasive stage. Similar results were reported by several other authors (n=59) also revealed similar results suggesting that the telomerase activity of exfoliated cells reflects that of the lesions or tumour tissues. This is a very interesting finding which indicates that cervical exfoliated cells can be used for the detection of telomerase activity. It will help in easy screening of women for telomerase activity along with Pap test in the same scraped cervical cells. Possible inhibition of the TRAP assay by the presence of normal cervical cells is not going to interfere with sensitivity of the assay since Cervical scrapes collected from the same cervical cancer patients (We observed a positive association between telomerase activity and infection with HR-HPV type 16/18. These findings are in accordance with earlier reports (n=2) and ASC-US (n=1) and the colposcopic examination revealed cervical erosion in four cases. This is in agreement with earlier studies that showed presence of mild telomerase activity even in ASC-US, inflammation and cervical erosions  using PCR based clonality assay, It has been established that there is variation in the interpretation of ASC-US Pap smears even among expert cytopathologists . It may The levels of hTERT mRNA expression complemented well with the telomerase activity levels observed in different grades of cervical lesions to invasive cervical cancer samples. One mild dysplasia sample, which was negative for telomerase activity, expressed hTERT. This may be explained due to the upregulation of hTERT before the reactivation of telomerase activity. Similar observations were made in other cancers including cervical cancer by several authors (It is remarkable that high rates of cervical cancer deaths still occur despite the fact that cervical cancer is an excellent model for early detection due to long interval between the time of infection or initial lesions and development of invasive cancer with a well-known natural history. Early identification and intervention should have a significant impact on the reduction of cervical cancer morbidity and mortality. Identification of women at risk for cervical cancer would not only minimise the unnecessary follow-up visits a woman has to undergo, but would also avoid invasive procedures without compromising on the disease detection. We suggest that telomerase activation is a relatively early-stage event in cervical carcinogenesis, and this activation is associated with the initiation and progression of cervical lesions. Detection of telomerase activity may serve as a tool for reliable diagnosis and prognosis of cervical neoplasias along with cytology and HPV testing.


We have investigated the use of the antibody MIB1 as a proliferative and prognostic marker in breast cancer and whether changes in proliferative activity could account for differences in prognosis of premenopausal women operated on during different phases of the menstrual cycle. MIB1 expression was strongly correlated with S-phase fraction and histological grade. There was no difference in MIB1 scores between different phases of the menstrual cycle. Both MIB1 score and timing of surgery correlated significantly with duration of survival, while the two together were even stronger predictors of overall survival. Women with slowly proliferating tumours surgically removed in the luteal phase had a very good prognosis, whereas women with rapidly proliferating tumours excised at other times of the cycle had a worse prognosis.


High loads of human papillomavirus (HPV) 16 and HPV 18/45 increase the risk of developing invasive cervical carcinoma, revealing higher risk in percentiles of highest viral loads for HPV 16 (odds ratio (OR) 58.7, 95% confidence interval (CI) 21.9–151.4) compared to HPV 18/45 . Thus, HPV load is a type-dependent risk marker for invasive carcinoma. Controls were selected from the same organised screening programme and have been described in detail elsewhere  and 548 controls (1049 smears) . In all,Our results indicate that increasing viral load of HPV 16 in cervical smears increases the risk of future invasive cervical cancer. For HPV 18/45, a significant OR was only seen for the highest viral-load percentile. The relationship between OR of invasive cancer and viral load of HPV 16 is similar to that previously described for CIS ; consist


Published reports have demonstrated that introduction of the ThinPrep Imaging System (Imager) to the cytology screening services has increased the detection rate of high-grade squamous intraepithelial lesions (HSILs). In accordance with recent clinical treatment guidelines, patients with atypical squamous or glandular cells of undetermined significance (ASC-US or AGUS) are often tested for high-risk HPV infection using the Hybrid Capture HPV DNA test. We took the opportunity to investigate whether the Imager had resulted in any significant differences in our diagnostic categories, as well as whether the Imager increased the detection of high-risk HPV-DNA-positive (HRHPV+) ASC-US or AGUS.Cytology cases with the diagnosis of ASC-US and AGUS were retrieved from the archival files of our institution during periods of 11 months prior to and 11 months after the introduction of the Imager. The total number of cases in each category was correlated with results of reflex high-risk HPV DNA testing when the latter were available. All AGUS diagnoses were correlated with subsequent biopsy follow-up. Statistical analyses were performed using the chi-Square test with Yate's Correction and Fisher's Exact test.P>0.124). Similarly, 0.14% and 0.12% AGUS were found in the pre- and post-Imager period. The positive HPV reflex test was 14% versus 23% (P = 0.1690). The abnormal biopsy follow-up rate in the AGUS category was increased from 20.9% in the pre-Imager period to 31% in the post-Imager period (P = 0.1471). The ASCUS/SIL ratios were 1.9 and 1.6 respectively.A total of 108,371 and 104,555 of ThinPrep® Pap Test (TPPT) cases were reviewed during 11 months pre- and post-imager introduction. The ASC-US rate was 5.4% in the pre-Imager and 5.3% in the post-Imager period. The HPV reflex test was 38% and 34% positive respectively in the pre- and post-Imager period (The ASC-US and AGUS rates did not change statistically before and after the introduction of the Imager in our cytology laboratory. Although use of the Imager did not increase detection of HPV+ ASC-US, it did appear to increase the detection rate of HPV+ AGUS and subsequent abnormal biopsy follow-up rates in all categories. However, the increase in the detection rate did not reach the point of statistical significance Recently published reports have demonstrated that the introduction of the ThinPrep Imaging System (Imager) to the cytology screening services has increased the detection rate of high-grade squamous intraepithelial lesions (HSILs) and low-grade lesions (LSILs).–5 Lozanop Imager.7 Howeverp Imager. while anp Imager. Report op Imager. ThinPrepp Imager. The TIS p Imager. Our highThe Cytology Laboratory of Women and Infants Hospital processes over 100,000 cases a year. Our laboratory introduced ThinPrep® Pap Test (TPPT) in late 90s, and since then almost 100% of our Pap tests are ThinPrep. Only a rare conventional smear is occasionally encountered here. The ThinPrep 3000 Processor (HOLOGIC) instrument is used to prepare ThinPrep Slides following manufacturer's instructions. TIS was introduced in our Laboratory on May 01, 2005 and since then 100% cases are screened by TIS.The cytotechnologists, cytopathologists, and the patient population did not change during the study period. We did not open up any new account with any large physician group, and no previous large physician group disaffiliated its practice with our laboratory during the entire time period of study.Cytology cases with the diagnosis of ASC (ASC-US) and AGUS were retrieved from the archival files of our institution during periods of 11 months prior to and 11 months after the introduction of the Imager. All AGUS diagnoses were correlated with the subsequent follow-up. All cytology materials were examined by the same staff of cytopathologists and cytotechnologists during the study period. The total number of cases in each category was correlated with results of reflex high-risk HPV DNA testing when the latter were available. Reflex HPV testing was performed by using Hybrid Capture 2 method  using a cocktail of probes for all 13 high-risk HPV viruses. The test was done by the Microbiology Department following the manufacturer's guidelines. Statistical analyses were performed using the chi-square test with Yate's Correction and Fisher's Exact test.A total of 108,371 cases were reviewed during the 11-month period of manual screening of ThinPrep Pap smear prior to the introduction of TIS. A total of 104,555 cases were screened in the following 11 months after implementation of the TIS.Of these cases, during the manual screening period of 11 months, 5,884 (5.4%) cases were reported as ASC-US. HPV DNA test was performed on 5,536 cases (94%). In comparison, during the 11 - month period after the TIS introduction, 5,559 (5.3%) cases were reported as ASC-US, and HPV DNA test was done on 5,515 (99%) cases.P = 0.124).The rate of high-risk HPV positivity detected in ASC-US was 38% versus 34% pre- and post-TIS introduction in our laboratory. The detection rate of high-risk HPV pre- and post-TIS was not significantly different (P = 0.1690).In contrast, 158 (0.14%) cases of AGUS were reported during the pre-TIS and 126 (0.12%) during the post-TIS period. Of these, HPV DNA test was performed on 116 (73%) during the pre-TIS and on 102 (81%) during the post-TIS period. The high-risk detection rate pre- and post-TIS was 14% versus 23% respectively. The rate of increased detection of HPV-positive AGUS during post-TIS period was higher in our laboratory, although not statistically significant (P>0.05). The ASCUS/SIL ratio was 1.9 and 1.6 respectively during the pre- and post-TIS period. Our rate of HSILs during same pre- and post-TIS periods were 249 (0.23%) versus 260 (0.25%). The difference was statistically not significant In the current study, the findings on AGUS appear similar to what Friedlander reported. TIS did The abnormal biopsy follow-up rate in the AGUS category was increased from 20.9% in the pre-Imager period to 31% in the post-Imager period. Although a percentage-wise increase in detection is seen in all categories, a significant increase is seen in the LSIL category (from 7.6 to 14.3%). As expected, the benign follow-up percentage, in the AGUS diagnosis category, is decreased (60.3 from 72.1%) in the post-Imager period compared to the pre-Imager period respectively.Since our laboratory serves a relatively stable population and the laboratory personnel (cytotechnologists and pathologists) were the same during the study period, we can conclude that the increased detection rate of high-risk HPV-positive AGUS is authentic and can be attributed to TIS.P>0.05).It is often stated that comparison of cervical cancer screening methodologies should include the detection rate of HSILs. Our rate of HSILs during same pre- and post-TIS periods were 249 (0.23%) versus 260 (0.25%). The difference was statistically not significant   of all the institutions associated with this study. Authors take responsibility to maintain relevant documentation in this respect.


Although human papillomavirus (HPV) genome has been detected in lung cancer, its prevalence is highly variable around the world. Higher frequencies have been reported in far-east Asian countries, when compared with European countries. The present study analysed the HPV-16 presence in 60 lung carcinomas from the Asian countries China, Pakistan and Papua New Guinea.HPV-16 was present in 8/59 (13%) samples. According to histological type, HPV-16 was detected in 8/18 (44%) squamous cell carcinomas (SQCs), which were mainly from Pakistan; 0/38 (0%) adenocarcinomas (ACs), which were mainly from China; and in 0/4 (0%) small cell carcinomas (SCLCs). The observed histological difference was statistically significant (p < 0.001). HPV-16 viral load was also determined using real-time polymerase chain reaction (qRT-PCR); it ranged between 411 to 2345 copies/100 ng of genomic DNA. HPV-16 genome was found integrated into the host genome in every HPV-16 positive carcinoma.These results support the notion that HPV-16 infection is highly associated with SQCs in Pakistan. Our results show a frequent HPV-16 integration in SQCs, although the low viral load casts doubt respect a direct etiological role of HPV in lung carcinomas from Asia. Additional HPV-16 characterization is necessary to establish a direct or indirect etiological role of HPV in this malignancy. Epidemiological, clinical and basic research has allowed to conclude that HPV infection is the necessary cause of cervix uterine cancer [Human papillomaviruses (HPVs) are epitheliotropic double-stranded DNA viruses that belong to the e cancer . In addie cancer . The pree cancer . In addie cancer . Relative cancer  and stude cancer . Howevere cancer : HPV wase cancer . A Koreae cancer . On the e cancer . Interese cancer . Note the cancer .In general, high risk-HPVs are frequently integrated in cervical cancer, and low risk-HPVs are frequently found to be episomal . MoreoveEpidemiological evidence suggests that pathways involved in HPV-induced oncogenesis may be influenced by ethnic factors and anatomical location as well as exposures to risk factors such as smoking and alcohol . Thus, iP = 0.295, 0.133 and 0.179, respectively). All ACs analysed in this study were obtained from China and all SQCs were obtained from Pakistan and Papua New Guinea. Thus, in this study there was a statistically significant difference between analysed histological type of lung cancer and countries involved (P < 0.05).The present study analysed the HPV-16 presence in lung carcinomas from Pakistan, China and Papua New Guinea. The clinicopathological features of analysed specimens are shown in Table P= 0.091, 0.669 and 0.131, respectively). In addition, we used real-time PCR to determine viral load and physical status of HPV. All the HPV-16 positive specimens were successfully amplified for an 81 bp fragment of E6 gene but only 3 specimens were successfully amplified for an 82 bp fragment of E2 gene  and its concentration was ranged between 50 to 100 ng/μL. In addition, to check the DNA fragmentation and presence of amplifiable DNA for PCR, we successfully amplified a 110 betaglobin DNA fragment in all of specimens. PCR with GP5+/6+ primers revealed that 8/60 13%) cases were HPV positive. In order to determine the HPV genotype, we used PCR for amplification of a HPV-16 E6 specific DNA fragment. All the cases that were HPV GP5+/6+ positive were typed as HPV-16. We confirmed these results using PCR for amplification of another 65 bp DNA fragment of HPV-16 E6  Figure  y 1B. No Figures  and 2C.  Figures  and 2D. % cases wIn this study HPV-16 was found in 13% of the lung carcinomas. This finding is consistent with other reports where HPV-16 has been the most common HPV genotype detected in lung cancer ,11. SincEpidemiological evidence suggests that pathways involved in HPV-mediated oncogenesis are influenced by ethnic factors and anatomic location as well as exposures to risk factors such as smoking or alcohol . The mosThe variable frequencies of HPV-positive lung carcinomas between countries and continents may be explained by differences in sensitivity of the molecular methods used for HPV detection. PCR has been the most popular method used to detect HPV infection ; howeverThe present study found HPV-16 integration in cellular genome in every HPV-16 positive carcinoma. Even so, the low HPV load observed in the present study and in our previous study in Chile makes it difficult to interpret its etiological significance. It should be pointed out however that human cancer can be considered as a stem cell disease originated from a small fraction of cancer cells that show self-renewal and pluripotency and are capable of initiating and sustaining tumor growth . HPV may"in vivo". Second, the amplification through PCR is not able to detect HPV directly in tumoral cells, so in the future a very sensitive "in situ" method to detect HPV or an alternative method to obtain only tumoral cells from clinical specimens, as tissue microdissection are warranted.This report has some methodological biases that need to be commented: First, DNA specimens from paraffin embedded tissues are known to be degraded because of heat during embedding and formalin fixation. Therefore, the present study might have underestimated the viral load The results obtained in this study support the notion that HPV-16 infection is more strongly associated with SQCs than with ACs in the areas other than far-east Asian countries. Even though HPV-16 was frequently integrated in lung carcinomas, its low viral load casts doubt on a direct etiological role, which has been well established in the case of uterine cervical cancer. However, whether HPV-16 has a direct or indirect impact in the tumour development or modulation remains to be investigated.We examined 60 bronchoscopic biopsy specimens of paraffin-embedded tissue of SQCs (N = 30) and ACs (N = 32) of the lung from Pakistan, China and Papua New Guinea (PNG). The cases from Pakistan (N = 22) were diagnosed in Pathology Department of King Edward Medical University, Mayo Hospital, Lahore, Pakistan from 1996 to 2002. Cases from PNG were diagnosed in the period 1986-88 at Pathology Department of Port Moresby General Hospital, the major teaching center of School of Medicine & Health Sciences, University of Papua New Guinea. Cases from China were diagnosed during the period between 1989 and 2002 in hospitals in eastern China. Based on 1982 WHO classifications of lung tumors, lung carcinomas were classified into four broad categories: SQCs, ACs, small cell carcinomas and large cell carcinomas . In the Ten-μm-thick sections of each formalin-fixed paraffin-embedded sample were prepared. The samples were added with 1 mL of xylene, and then with 1 mL of ethanol. After centrifugation, the pellet was resuspended in digestion buffer  containing 200 μg of Proteinase K  and incubated during 24 hours at 56°C. The solution was heated at 100°C for 10 min and centrifuged at 10,000 rpm for 10 min. After phenol/Chloroform extraction, the obtained DNA was quantified using the Nanodrop 1000 spectrophotometer. HPV amplification with GP5+/GP6+ primers  was made7 and 1 × 101 copies of HPV-16 cloned in pUC plasmid . The specificity of amplification was confirmed using dissociation analysis starting at 60°C and agarose gel electrophoresis of the amplified products. In order to find out the physical status of HPV-16, the ratio of E2 to E6 copy numbers was calculated.Real-time PCR was performed using the ABI Prism 7000 Sequence Detection System and SYBR-Green PCR master mix . The amplification conditions were 10 min at 95°C and a two-step cycle of 95°C for 15 sec and 60°C for 60 sec for a total of 45 cycles. The primers used were: E6F: gagaaactgcaatgtttcaggacc and E6R: tgtatagttgtttgcagctctgtgc, E2F: aacgaagtatcctctcctgaaattattag and E2R: ccaaggcgacggctttg that amplify a fragment of 81 and 76 bp of E6 and E2 ORFs, respectively . The finFisher's exact test was used to examine the statistical significance of the results. A p-value <0.05 was considered statistically significant. P values presented are two-sided.The authors declare that they have no competing interests.FA, SA and CK conceived of the study, analysed the data and participated in the redaction of the manuscript. FA and AC made all the analysis of the clinical specimens. MA, QS and JM gave clinical information and got specimens for analysis. YE and CK gave analytical support for all the analysis. All authors read and approved the final manuscript.


Cytology performed very well in this older cohort of women. The newer Hybrid Capture II microtitre test may be a useful adjunct, especially if the results reported here are reproducible in other studies. A combined screening test offers the possibility of greater protection and/or longer screening intervals, which could reduce the overall cost of the screening programme. © 1999 Cancer Research CampaignCertain types of the human papilloma virus (HPV) are well established as the primary cause of cervical cancer. Several studies have shown that HPV testing can improve the detection rate of high-grade cervical intraepithelial neoplasia (CIN), but these have been carried out primarily in younger women. In this study we evaluated the role of HPV testing as an adjunct to cytology in women aged 35 or over. An additional aim was to evaluate commercially available kits for HPV testing. A total of 2988 eligible women aged 34 or more attending for a routine smear in 40 general practitioner practices received HPV testing in addition to routine cytology, after having given written informed consent. Samples were assayed by polymerase chain reaction (PCR) and two versions of the Hybrid Capture test for HPV, and women were invited for colposcopy if there was any cytological abnormality (including borderline smears) or the PCR test was positive. Any apparent abnormality was biopsied and loop-excision was performed as necessary. CIN was judged by histology; 42 women had high-grade CIN, of which six were cytology negative (86% sensitivity for borderline or worse) and three had a borderline smear (79% sensitivity for mild dyskaryosis or worse). The positive predictive value of a borderline smear was only 3.1%. Eleven high-grade lesions were negative by the PCR HPV test (sensitivity 74%). The first generation Hybrid Capture II test had a similar sensitivity but an unacceptably high false positive rate (18.3%), while the newer Hybrid Capture II microtitre kit had a 95% sensitivity and a 2.3% positivity rate in normal women when used at a 2 pg ml


Uptake of HPV-5 PsV was not inhibited by heparin in C33A cells and only minor inhibition was detected in HaCaT cells. HPV-16 PsV uptake was significantly more inhibited by heparin in both cells and completely blocked in C33A cells.Papillomaviruses can roughly be divided into two tropism groups, those infecting the skin, including the genus beta PVs, and those infecting the mucosa, predominantly genus alpha PVs. The L1 capsid protein determines the phylogenetic separation between beta types and alpha types and the L1 protein is most probably responsible for the first interaction with the cell surface. Virus entry is a known determinant for tissue tropism and to study if interactions of the viral capsid with the cell surface could affect HPV tropism, the net surface charge of the HPV L1 capsid proteins was analyzed and HPV-16  and HPV-5 (beta) with a mucosal and cutaneous tropism respectively were used to study heparin inhibition of uptake. The negatively charged L1 proteins were all found among HPVs with cutaneous tropism from the beta- and gamma-PV genus, while all alpha HPVs were positively charged at pH 7.4. The linear sequence of the HPV-5 L1 capsid protein had a predicted isoelectric point (pI) of 6.59 and a charge of -2.74 at pH 7.4, while HPV-16 had a pI of 7.95 with a charge of +2.98, suggesting no interaction between HPV-5 and the highly negative charged heparin. Furthermore, 3D-modelling indicated that HPV-5 L1 exposed more negatively charged amino acids than HPV-16. Uptake of HPV-5 (beta) and HPV-16  was studied  Papillomaviridae family and consists of a large family of non-enveloped double stranded DNA viruses that infect the basal layer of cutaneous or mucosal epithelia of a dozen vertebrate species with a strict species tropism [Papillomavirus (PV) belongs to the  tropism . Over 10 tropism . HPVs cain vitro of several HPV types from the alpha genus [The first step of PV infection is binding of the major capsid protein L1 to the cell surface. The cell surface glycosaminoglycan (GAG) heparan sulfate (HS) is important for HPV infection ha genus -7. HS coha genus . Furtherha genus -11. Sincha genus  and the The predicted isoelectric point (pI) of the receptor binding L1 capsid for all available HPV types was examined and found to correlate with the cutaneous or mucosal tropism . Plasmids and 293TT cells used for pseudovirus production were a kind gift from Chris Buck  and Martin Müller . Detailed protocols are available at the website . The human epithelial cell line HaCaT, from adult trunk skin [4 cells/well in 24-well plates. PsV doses were calibrated according to [50 = 15.5 μg/ml) and it was also dose-dependent (IC50 = 24.5 μg/ml) with a maximum inhibition level of 72% in HaCaT cells. The difference in inhibition between HPV-5 and HPV-16 was significant (using a 2-tailed student T-test) both in C33A cells (P < 0.0004) and in HaCaT cells (P < 0.03).Heparin is highly negatively charged and was used for inhibition studies of alpha HPV-16 and beta HPV-5 pseudovirus (PsV) uptake, analyzed by expression of the GFP marker gene ,15. GFP-unk skin  and the unk skin  were plarding to  and befording to . There wIn summary, beta HPV-5 PsV uptake was not inhibited in C33A cells and there was only a minor inhibition detected in HaCaT cells similar to previous results where HPV-5 pseudovirus infection of HeLa cells was not inhibited by heparin . Alpha Hin vivo [In a study comparing HS expression in skin and mucosa, more GAGs were found in vaginal tissue than in perianal skin, although this difference could possibly be explained by differential expression of chondroitin sulfates and dermatan sulfate and not HS . Both thin vivo  and a dyin vivo . This sein vivo  and is min vivo . Tropismin vivo . In HaCain vivo .To conclude, the first step of a virus infection is attachment to the cell surface and net charge is important for determining virus binding to cell receptors, as has been shown for certain adenoviruses . The obsThe authors declare that they have no competing interests.NM participated in the design of the study, carried out virus infection experiments, 3D modelling and drafted the manuscript. CW carried out calculation of theoretical pI and 3D modelling experiments. ME conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


In a study involving 13 842 women and 113 gynaecologists, liquid-based cytology and HPV testing for detecting cervical cancer were compared. A total of 1334 women were found to be positive for one or both tests and were invited for colposcopy with biopsy. A total of 1031 satisfactory biopsies on 1031 women were thereafter collected using a systematic biopsy protocol, which was random in the colposcopically normal-appearing cervix or directed in the abnormal one. In all, 502 women with negative tests were also biopsied. A total of 82 histologic high-grade squamous intraepithelial lesion (HSIL) were reported in biopsies, all from the group with one or both tests positive. Sensitivity and specificity to detect histologic HSIL were 59 and 97% for cytology, and 97 and 92% for HPV. In total, 14% of reviewed negative cytological preparations associated with histologic HSIL contained no morphologically abnormal cells despite a positive HPV test. This suggested a theoretical limit for cytology sensitivity. HPV viral load analysis of the 1143 HPV-positive samples showed a direct relationship between abnormal Pap test frequency and HPV viral load. Thus, not only does the HPV testing have a greater sensitivity than cytology but the probability of the latter being positive can also be defined as a function of the associated HPV viral load. The Pap test is currently central to the detection of cervical cancer precursors. Conventional smears, whose utilisation started in the early 1950s, have been progressively replaced by liquid-based methods over the past 10 years. Discovery of the role of HPV in the natural history of cervical cancer and the ability to detect it in liquid cytology medium are rapidly modifying the standard in cervical cancer detection. HPV detection is nowadays mainly used in atypical squamous cells of undetermined significance (ASC-US) triage. The next step will be the utilisation of combined HPV testing and cytology to detect cervical cancer and its precursors. After 50 years of cytology as the sole test in cervical cancer prevention, new methods are appearing to palliate the Achilles’ heel of cytology, which is sensitivity. Indeed, many reports  and a test for high-risk HPV DNA, Hybrid Capture® II . The investigation was conducted in the laboratory Cytopath based in Geneva, Switzerland, a private laboratory member of the Unilabs Group. Cytopath has used liquid-based cytology since 1996, analysing approximately 100 000 slides per year.A target of 15 000 cytologic samples was established in the hope of collecting approximately 250 histologic HSIL, providing adequate power to estimate the differences, if any, in performance characteristics between the tests. Women 30 or more years of age had been favoured for study participation in order to optimise HSIL collection. Most study participants had been screened yearly with liquid-based cytology starting at least 5 years before this study. They were thereby considered to be at low risk for cervical cancer. Recruitment was not from sexually transmitted disease clinics or other high-risk populations. Study participants were recruited by 113 gynaecologists from six Swiss cantons , most of whom are in private practice. Participants were given the option of study participation following a discussion with their gynaecologists of the risks and benefits of enrolment, and the completion of the informed consent document. Prior to initiation, the study was reviewed and approved by the Ethics Committee of the ‘Association des Médecins du canton de Genève’.μl distilled water for further HPV testing. In the text, ‘CYTO’ refers to the Surepath® liquid-based cytology test.All cervical samples were taken using the Surepath®  liquid-based preparation. In agreement with the manufacturer, the gynaecologists put the collecting device in the Surepath vial after having performed a cervical scrape. Cervex Brush®  was the only provided collecting device. All vials were sent to the Cytopath Laboratory and prepared according to the protocol recommended by the manufacturer. A homogenised fraction of the sample was used to prepare the cytological preparation with the AutocytePrep® processor. The residual cellular material underwent centrifugation and was resuspended in 500 −1 HPV 16 DNA) tested in triplicate. Specimen RLU values were then converted into a ratio to the cutoff value. Specimens with a ratio <1.00 were considered negative. Specimens with ratios between 1.00 and 5.00 were systematically retested in duplicate and the mean of the three values was calculated. If discrepant RLU values were observed in the two tests, samples were tested in duplicate again (maximum three thawing cycles). Specimens with a final ratio ⩾1.00 were considered positive (HPV+). In the text, HPV always refers to the Hybrid Capture® II test. Therefore, an HPV− might refer to the absence of HPV virus or to an undetected type by the Hybrid Capture® II test or detection of the virus at an RLU ratio <1.00. All positive RLU values were collected and inserted into the database in order to compare the viral load expressed in RLU with the cytologic and biopsy results.HPV testing was performed at the Bioanalytique-Riotton Laboratory (another member of the Unilabs group). Cell suspensions were stored at 2–8°C for up to 1 week before HPV testing. The HPV DNA assay was performed blind to cytologic and histologic diagnoses. Specimens were tested for high- and intermediate-risk HPV types  using the Digene High-Risk HPV DNA Test Hybrid Capture® II . The DNA assay was processed according to the manufacturer's instructions, using microtubes and a water bath for hybridisation and manual washing. In agreement with the kit manufacturer, clinical specimens were denatured for 90 min in order to reduce borderline results. Relative light units (RLU) were measured on a DML 2000 luminometer . Quality control was assessed using the manufacturer's assay calibration criteria and by the regular use of HC2 HPV DNA Test Panel . The cutoff value was calculated as the mean RLU value of the calibrator  before the cytologic report was sent to the gynaecologist. The cytologic interpretation was done according to the Bethesda 2001 recommendations , or random by strongly brushing the proximal endocervical canal if no lesion was visualised. In the latter case, the collecting device (a brush) was sent to the laboratory in formalin. At the laboratory, the brush was scraped with a scalpel and all material was paraffin blocked in order to obtain a histological preparation.t-test was used to compare mean values with a threshold of 5% for P-value to be significant. In order to correctly estimate the sensitivity and specificity of both tests, a method for correcting biopsy verification bias was used. Verification bias is caused by the fact that the percentages of biopsy verification vary considerably among HPV/CYTO subsets because of practical limitations. Indeed, only 4% (502 out of 12 508) of women HPV−/CYTO− and 77% (1031 out of 1334) of women positive for at least one test, HPV+/CYTO−, HPV−/CYTO+ or HPV+/CYTO+, underwent colposcopy with biopsy. Given the low prevalence of disease in this population, the bias causes a falsely decreased specificity for both tests. The correction relies on the estimation of lesion frequency found in each subset HPV/CYTO if all patients had been colposcoped and biopsied. The adjusted value (AHCL) was derived from the formula AHCL=FHCL+UHC*+FHC(LSIL)+FHC(HSIL)))  were positive for one or both tests (CYTO or HPV), 8.2% of women (1143 out of 13 842) were positive for HPV and 6.1% (841 out of 13 842) were positive for HPV alone (HPV+CYTO−). In all, 96.4% (13345 out of 13 842) of women were 30 years old or more, with a percentage of HPV infection of 7.9%. This percentage rose to 17.4% in the 495 women younger than 30 years. For two women, the age was not available. Mean age of the 13 840 women was 44.4 years (range 17–93). The higher frequency of HPV positivity in younger women is portrayed in Of the 1334 women with abnormal tests, 1086 (81.4%) underwent colposcopy with biopsy. Out of 1086, 1031 (95%) biopsies were satisfactory for evaluation. A total of 18.5% women (248/ out of 1334) refused to be colposcoped, missed appointments or underwent the follow-up in other laboratories. In order to verify the negative group (HPV−CYTO−), random invitations for colposcopy were regularly sent to women with both tests negative during the study. Since some women declined to participate, up to 700 invitations were sent before being able to gather the target number of 500. Finally, 502 women with normal tests were colposcoped and biopsied. These biopsy results were divided into three categories: normal, LSIL (CIN 1) and HSIL (CIN2/3). No invasive lesion was found on cytology and biopsy in any group. The details of the results are presented in The unbiased estimation of frequencies of lesions  per HPV/CYTO subset for the entire study population  permits The 1143 HPV-positive results have RLU values ranging from 1 to 3180. The distribution of RLU values is nonlinear. Most RLU values are small: cumulatively, 22% of all the HPV-positive results are less than 2.2 RLU, 38% are less than 5.0 RLU and 51% are less than 11.2 RLU. In order to compare the HPV distribution values more easily against frequency, the RLU values were transformed using the natural logarithm function. Therefore, the RLU interval (1 : 3200) becomes the lnRLU interval (0 : 8). The latter was subdivided in 10 subintervals as illustrated in The black line with small white squares in The black line with small white circles in n=34) and 23.1 for HPV+/CYTO+ cytological preparations . A total of 14% (11 out of 79) of cytological preparations HPV+/CYTO− did not show any positive spots. The mean lnRLU values were 2.6 for cytological preparations HPV+/CYTO−  and 4.8 for HPV+/CYTO+ cytological preparations . Both differences (number of positive spots and lnRLU) were found significant with t-test (P<0.05). The two cytological preparations HPV−/CYTO+ associated to biopsy-proven HSIL had six and 10 positive spots (not shown in It is important to note that the methodology of the present study required that the collecting device be kept in the vial so that 100% of the cervical scrape was sent to the laboratory. The conventional smear, by selecting a random fraction of the cervical scrape, involves an important loss of material (The relationship between the percentage of HPV infection and the age shown in At the sample level, the study of HPV viral load explains the reason that CYTO has inferior sensitivity. As shown in While some false negative cytological preparations can be explained by human misreading, most are not. False negative preparations would contain few abnormal cells and relatively low HPV viral load. Inspection of At the population level, the study of HPV viral load explains why cytology sensitivity varies considerably among reports. The relationship between the probability that a Pap test is abnormal and the viral load is thought to be independent from intrinsic factor related to this study. Conversely, the viral load distribution (Derived from This study found a higher sensitivity for HPV than liquid-based cytology testing in the detection of cervical cancer precursors. This adds to previous similar reports with the distinction that our study involved a nonacademic setting, thereby demonstrating that the method is robust and readily applicable as a routine test. By collecting all RLU values, a clearcut correlation between the probability for a Pap test to be positive and the associated HPV viral load was found. The findings can also explain how cytology sensitivity can vary among different studies since different populations will differ in their viral load distributions: at one extreme, cytology performs badly when the screened population is at low –risk, while at the other extreme, cytology performance would be much closer to that of HPV testing in a high-risk population. Rare HSIL are not detected by HC2, these cases being presumably related to HPV types that are not detected by the cocktail of probes used in HC2. The combination of Pap and HPV testing identifies all of the women harbouring HSIL that can be detected by the best colposcopic methods currently available, and thereby permits investigators and clinicians to focus their efforts on the population at risk, while reassuring the majority of women who are negative on both tests.


P< 0.0001). Prolonged oral contraceptive use and sexual history were associated with CIN 2–3 in univariate analysis, but these associations lost significance after taking HPV into account. Smoking was associated with CIN 2–3 (odds ratio (OR) 2.6, 95% confidence interval (CI) 1.7–4.0), the effect was dose-dependent (P = 0.002) and the smoking-associated risk was not affected by adjusting for HPV, neither when adjusting for HPV DNA  nor when adjusting for HPV seropositivity . In conclusion, after taking HPV into account, smoking appeared to be the most significant environmental risk factor for cervical neoplasia. © 2000 Cancer Research CampaignSmoking, nutrition, parity and oral contraceptive use have been reported as major environmental risk factors for cervical cancer. After the discovery of the very strong link between human papillomavirus (HPV) infection and cervical cancer, it is unclear whether the association of these environmental factors with cervical cancer reflect secondary associations attributable to confounding by HPV, if they are independent risk factors or whether they may act as cofactors to HPV infection in cervical carcinogenesis. To investigate this issue, we performed a population-based case–control study in the Västerbotten county of Northern Sweden of 137 women with high-grade cervical intra-epithelial neoplasia (CIN 2–3) and 253 healthy age-matched women. The women answered a 94-item questionnaire on diet, smoking, oral contraceptive use and sexual history and donated specimens for diagnosis of present HPV infection  and for past or present HPV infections (HPV seropositivity). The previously described protective effects of dietary micronutrients were not detected. Pregnancy appeared to be a risk factor in the multivariate analysis (