This multicenter study describes the type-specific prevalence of HPV infection in the general population from central and southern Italy, comparing the data with previously published Italian studies.Women aged from 25 to 65 who attended cervical cancer screening in five different Italian regions were tested for HPV infection with Hybrid Capture II (HCII) low and high risk probes. Women repeating Pap-test upon unsatisfactory or positive results, or as a post-treatment and post-colposcopy follow-up analysis, were excluded from our study. High risk (HR) HPV positive samples were typed using GP5+/GP6+ primed PCR, followed by Reverse Line Blot for 18 high/intermediate risk HPV types, while low risk (LR) HPV positive samples were tested with type specific primers for HPV6 and HPV11.3817 women had a valid HCII test: 350 of them (9.2%) were positive for HR probes, 160 (4.2%) for LR probes, while 57 women were positive for both. Multiple infections were detected in 97 HR HPV positive women. The most common types were HPV 16 (3%), 31 (1.2%), 51 (1%). HPV6 ranked fifth (0.6%), HPV18 ranked tenth (0.5%) and HPV11 sixteenth (0.3%).In Sardinia the prevalence of high-risk infection was 13%, significantly higher than the mean value (p < 0.00005).The distribution of the most frequent types did not significantly differ by centre (p = 0.187) and age (p = 0.085).Because cervical cancer incidence and Pap test coverage is lower in southern than in northern Italy, a lower prevalence of high-risk infections in the general population was expected in the south. However, prevalence detected in this study for the south of the country is slightly but significantly higher than the rest of Italy. The consequence may be an epidemic of cervical cancer in the next decades if adequate screening programs are not implemented there. The identification of HPV as a necessary cause of cervical cancer  has beenIn Italy the vaccine has been recommended, free and offered to 11-year old girls since the beginning of 2008 . In. In10]. The aim of the present study was to measure the prevalence of different HPV types in the general population attending population-based screening programmes in Rome and southern Italy and to compare the data with previously published Italian studies.The study was conducted within cervical cancer screening programmes that actively invite the entire target population (age ranging from 25 to 64) in Rome and southern Italy. A convenience sample of the existing screening programmes was selected: in Sicily and Sardinia only two programmes issued invitations regularly and were therefore selected, in Molise, Apulia and Calabria there was no consolidated programme when the study started and no data were collected from these regions, in Abruzzo it was possible to collect samples from the whole region, while in Rome metropolitan area and Campania, the most populated areas, we selected two programmes each.In 2007, the target population for cervical cancer screening, i.e. women aged 25-64, included 5.71 million units in southern Italy and 1.18 million units in the metropolitan area of Rome. The active screening programmes in these areas actually covered 4.75 million women (69%) , while tDuring the study period the programme invited the target population, without any selection criteria (nor age neither previous screening participation). We later sampled about 800 women in each region . 25 to 65 year old women were included in our study. Those repeating Pap-test upon unsatisfactory or positive results, or as a post-treatment and post-colposcopy follow-up, were excluded.Sample size (4000 women), was chosen in order to achieve the two following objectives:1.+/-3% precision in the estimate of the proportion of infections of a type responsible for at least 10% of infections;2. 95% power to see any type responsible for at least 1% of infections.Considering overall HPV prevalence to be 10%, we expected 400 samples would be HPV positive and therefore available for typing.Cervical scrape samples were collected in ThinPrep vials containing PreservCyt  transport medium or in Specimens Transport Medium (STM) . Before testing, 400 μl of STM samples, vortexed on a shaking platform at 1000 rpm for at least 10 minutes, were biobanked at -80°C.® (HCII)  [® (no more than 20 samples at a time) were processed with Digene Sample Conversion kit , according to the manufacturer's instructions. HCII assay followed. HPV DNA testing was done locally in 3 sites (one in Abruzzo and two in Rome) while samples collected in Cagliari and Catania were analysed in Florence (ISPO). Results of HCII tests for HPV DNA were expressed as relative light units (RLU): specifically, the ratio of the specimen light emission to the average emission of three concurrently tested positive controls (1 pg/ml HPV DNA). All samples with a ratio value over 1 were considered HPV positive. HCII quality assurance procedures were performed as previously described [The presence of high risk (HR) and low risk (LR) HPVs in cervical specimens was evaluated by Hybrid Capture IIrg, USA) -17 usingescribed .An additional internal control was then used in each HCII test plate. At the beginning of the study, each laboratory denatured a quality control LR HPV sample (HPV type 6 DNA) and a quality control HR HPV sample (HPV type 16 DNA) provided with the kit. 100 μl of denaturated control sample were aliquoted in 15 tubes and stored at -20°C. Single aliquots were then used in every HCII plate. Quality controls for both LR and HR HPV had a specific concentration of virus DNA (5 pg/ml) and therefore an expected ratio value ranging from 2 to 8.® medium, sent 15 additional ThinPrep samples (4 ml) to the same facility, where the whole HCII procedure was repeated.For external quality assurance, 22 STM samples (100 μl) were sent to one of the laboratories involved in the study  and samples had to be tested twice. Laboratories that used PreservCytResults were expressed as specimens in RLU/CO; a modal value  was defined for each case in the external control set and considered as the reference result. The result provided from each single laboratory was compared with the modal value.Typing procedures were centralized in Florence at the ISPO laboratory and all the centres involved in the study sent HR or LR HPV positive samples there.® solution and 200 μl of STM samples were used to collect DNA with a QIAamp DNAMini Kit. Samples were eluted in 100 μl AE buffer preheated at 70°C. An HPV negative sample was included in each batch of extraction to exclude any contamination during this phase.For DNA extraction, 1.5 ml of samples in PreservCytHCII HR HPV positive specimens were amplified and typed with "consensus High Risk HPV genotyping kit" (Qiagen); this system uses PCR with biotinilated GP5+/GP6+ primers, followed by reverse Line Blot for 18 high risk HPV types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.2 , 10 μl 1 mM dNTPs, 0,3 μl AmpliTaqGold (Applied Biosystem) DNA polymerase (5 U/μl) and 2 μl of Qiagen Primer set, as recommended by the manufacturer. The thermal cycler program contained ramping times between the temperature used for denaturation, annealing and elongation that appeared essential for optimal performance [PCR was performed in 50 μl final volume with 10 μl DNA, 5 μl 10× buffer II, 7 μl 25 mM MgClformance . Activatformance . AmplifiAll samples, independently from gel results, underwent reverse line blot analysis. Biotinylated PCR products were hybridized with specific oligonucleotide probes bound to membrane strips. After stringent washing, streptavidin-conjugated alkaline phosphatase was added and a non-radioactive reaction performed using a chromogenic substrate. Strips containing the resulting purple precipitates were analysed visually from an interpretation grid supplied with the kit. A biotinylated poly(dT) control for conjugate reaction is present in each strip to ensure good test performance and a proper alignment of the strips on the interpretation sheet. Samples were considered positive for a specific HPV type if the precipitate was viewed in the specific position of the strip. In each run we included two negative controls  and two positive controls (HPV16 plasmid purchased with the kit and a known non-16 HR HPV DNA).GP5+/6+PCR-negative and reverse Line Blot-negative samples were amplified for the β-globin gene using primers GH20-PC04 (268 bp amplicon lenght) to assess the integrity of DNA [β-globin positive samples were re-typed with "INNO-LiPA HPV genotyping Extra Amp"  following the manufacturer's instructions, because this system can detect 28 HPV HR or LR types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74 and 82. The β-globin negative samples were extracted and typed again.The remaining un-typed samples  were considered HPV X type.HCII LR HPV positive specimens  were typed using HPV 6 and HPV 11 specific primers and resulting negative samples were considered positive for the other LR types included in probemix A . No LR type, other than 6 and 11, was searched for.Primer sequences for HPV 6 were 5'-TAGTGGGCCTATGGCTCGTC-3' (sense) and 5'-TCCATTAGCCTCCACGGGTG-3' (antisense) which amplified a 289 bp product.Primer sequences for HPV 11 were 5'-GGAATACATGCGCCATGTGG-3' (sense) and 5'-CGAGCAGACGTCCGTCCTCG-3' (antisense) which amplified a 360 bp fragment .2, 2 μl 2,5 mM dNTPs, 1,25 μl 10 μM primers, 0,2 μl Taq Gold (Applied Biosystems). Hot-starting at 94°C for 6 min was followed by 40 cycles as follows: 95°C for 30 sec, 58°C for 30 sec, 72°c for 30 sec and an hold at 72°C for 5 min. Amplified products were run on 2% agarose gels containing ethidium bromide and visualized by ultraviolet light.PCR reactions were performed in 25 μl reaction volume using 5 μl DNA, 2,5 μl 10× buffer II, 1,5 μl 25 mM MgClWe searched the Medline using the terms ("HPV" OR ("human" AND "Papillomavirus")) AND ("type" OR "Typing") AND  AND  AND "prevalence". The search was updated on 15/12/2009. We excluded all studies that selected the pathological population, women in follow-up or with a positive Pap test. We included only studies reporting prevalence in the general healthy population. We extracted the total HPV positivity, the positivity to high risk HPV, the proportion of HPV16 among isolated HPV, the proportion of HPV 18 and the proportion of cases with HPV16 or 18, the age of the population, the geographical area.The following analyses were planned a priori in the study design: prevalence of HPV positivity to high and low risk probes by age, by centre; prevalence of HPV 16-18 by age, by centre; proportion of HPV types by age, by centre. We calculated 95% confidence intervals taking into account the two stages sample using STATA 8 survey module .The study has been approved by the Ethics Committee of the Istituto Superiore di Sanità . Women were informed of an additional HPV test in the screening procedure and asked to sign a consent form.In this study, 3817 women had one valid sample for HPV testing, with both HR and LR probes. The overall positivity was 9.2% (350/3817) (95% CI 6.2-12.2) for high and 4.2% (160/3817) (95% CI 2.3-6.1) for low risk types.The prevalence of cytological modification was 3% of ASCUS or more severe, in the centres using the Pap test as primary screening.Figure The overall prevalence varies significantly by centre (p < 0.00005 for HR and p < 0.00005 for LR types). The observed variability is mostly due to a higher prevalence of HR HPVs in Cagliari (Sardinia) and to a lower prevalence of LR types in Rome Figure .The age-specific prevalence curves do not show heterogeneity except in Cagliari (Sardinia) that has a steeper negative slope of the curve  Figure .The HCII quality control included co-testing of synthetic samples with known HPV DNA concentration and the circulation of clinical samples between laboratories to assess reproducibility, which was found to be very high (kappa coefficient = 0.93 for positive vs. negative).Samples that showed high and/or low risk HPV with HCII were 449: 293 among them displayed infection by HR HPVs only, 99 by LR HPVs only, while 57 by both HR and LR HPVs. In women younger than 25, we detected 12 infections: 6 HR HPVs only, 4 LR HPVs only and 2 HR and LR HPVs. These cases were excluded from the analyses.Out of 350 HR HPV positive samples, 300 (85.7%) were immediately typed using the "Consensus High Risk genotyping HPV" (Qiagen), 15 (4.3%) were typed with INNO-LiPA "HPV Genotyping Extra Amp" (Innogenetics), 12 (3.4%) samples resulted PCR positive but remained un-typed with reverse line blot Digene and INNO-LiPA (10 HR only and 2 HR + LR) and the remaining 23 (6.6%) beta-globin positive samples were classified as HPV negative samples. Co-infections were present in 97 HR positive women .Out of 160 LR HPV identified by probemix A HCII , four samples were not available for typing, 24 (15.4%) samples resulted HPV 6, 10 samples (6.4%) were positive for HPV 11 (4 of them were HPV 6 and 11 co-infections) and 126 samples (80.7%) were classified positive for other LR HPV types. No other specific LR type was searched for.The most common types were 16 (2.8%), 31 (1.1%), 51 (0.8%), 66 (0.7%) and 6 and 56 (0.6% both); HPV18 ranked tenth (0.5%) and HPV11 ranked sixteenth (0.3%). Other low and undertermined risk types were found for a total of 31 different types, some of these were not included in the HCII probemixes. This fact is due to cross-hybridization. Previous studies, in fact, suggested that HCII using probemix B may cross-react with HPV types either not associated or with undetermined associations with cancer (non-oncogenic HPV) .Table Table Out of 97 women with HR HPV co-infections, 43 (44.3%) included HPV 16 and/or HPV 18. Table We have information on 17 histologically-confirmed CIN2 or more severe lesions found among HPV positive women. Including three co-infections, in these samples we detected 21 types: the most frequent types were 16 (12 cases) followed by HPV types 31, 35, 66 (2 cases each) and HPV types 33, 58, 73 (1 case each).We found 72 papers in the PubMed search. Eleven of them fulfilled the inclusion criteria, two more papers were later excluded thorough analysis because prevalence data on HPV types were available only for a subset of the healthy population selected according to Pap test finding. Further literature was found in the IARC meta-analysis: none of these papers were eligible because they investigated only HR HPV prevalence and did not report any typing.Table HPV 16 prevalence ranges from 2.8% to 9.6%; again the lowest prevalence is registered in population-based studies. HPV 18 prevalence ranges from 0 to 6%.Among included and excluded studies, only 4 were large enough to give age-specific prevalence of HR-HPV. Figure The overall prevalence observed in our study is lower than what observed in most of the previous Italian studies even if we consider the same age classes table . All preHPV 16 prevalence has a similar variability than the overall HPV prevalence, reflecting the same difference due to the study-base. The variability in HPV 18 prevalence is higher; this difference may reflect the stronger influence of random fluctuations for this quite rare type. Not surprisingly the two studies on migrant population show a higher prevalence and a wider spectrum of types ,29. In fIn our study, we observed a strong variability among centres in the circulating types, with the exception of HPV 16. Most of the differences are due to Cagliari (Sardinia). This area is relatively isolated, compared with other Italian regions, as supported by observations on genetic background .The ratio between high and low risk infection types decreases with age. This finding has been observed in other studies . There aThe incidence of invasive cancer in Italy is slightly lower in the South, 6.2/100.000, than in the North and Centre, 7.1/100.000 and 7.3/100.000 respectively : 83%, 78In our opinion, lower prevalence of HPV infection in the previous decades is still the only plausible explanation for lower incidence of invasive cancer in the South. In fact the incidence currently observed by cancer registries is the consequence of infections acquired several years ago. In this scenario, the slightly higher prevalence of infection observed now in southern compared to in northern Italy, particularly in younger cohorts, may indicate that the South is in transition from low to medium prevalence of HPV. As a result, we may expect an increase in invasive cervical cancer incidence in the next decades if a better control by screening programme is not implemented. This interpretation is coherent with the cervical cancer incidence trends estimated by De Angelis and coll. , predictActually, the implementation of cervical cancer screening in southern Italy is going on, even if with great difficulties, and the effect on Pap test coverage in some areas is now detectable .We tested women who were invited and participated to cervical cancer screening programmes in Rome and southern Italy. Response to organised screening is quite low, ranging from 25 to 50% in the selected areas; the low participation may introduce a self-selection bias. Nevertheless, active invitation was the only means we had of reducing self-selection. Previous studies found few socio-economic differences between participants and not participants ,37.A second limit, due to the nesting of this study into organised screening programmes, is that we could not include some regions, such as Calabria and Apulia, because at the time the study started there were no active programmes that contacted the whole target population.We decided not to gather information on the cytology results in recruited women, since one centre used HPV as the primary screening test. This choice limited the opportunity of analyses.We typed only women positive to the HR and LR HCII probes; according to this protocol we may have missed women who are positive to other HPV types not included in the probes or samples with very low number of virus DNA copies.Pap-test coverage in southern Italy is lower than in central and northern Italy. Nevertheless cancer registries until 2003 showed a similar or even lower cervical cancer incidence in southern Italy than the rest of the country. In this situation, a lower prevalence of HPV infection in southern Italy was expected. Actually, the observed HPV prevalence in the general female population is slightly higher, in particular in younger cohorts.We interpret this finding as indicative of a transition in southern Italy, from a low to medium-high prevalence area.The final effect of this transition may be an epidemic of cancer in the next decades, if appropriate screening programmes are not implemented to control the disease.®; RLU/CO: Relative light units/cut-off; STM: standard transport medium; NTCC: New Technologies in Cervical Cancer; ASCUS+: Atypical Squamous Cells of Undetermined Significance or more severe cytologyHPV: human papillomavirus; HR HPV: high risk human papillomavirus types; LR HPV: low risk human papillomavirus types; PCR: polymerase chain reaction; HCII: Hybrid Capture II Financial: PGR received travel reimbursement for two international conferences by Sanofi Pasteur MSD; FMC is occasional advisor to Gen-Probe, Abbot, Sanofi and GlaxoSmithKline.Non financial: PGR is employed in the HTA Unit of the Agency for Public Health of the Lazio Region, this Agency contributes in establishing public screening programs.PGR designed and conducted the study and drafted the paper; FC designed the laboratory procedures, coordinated the laboratory quality control and contributed in drafting the paper; SB and IP performed the typing test; FC designed the data flow, managed the data and performed the statistical analyses; AS, CA, RM, MTP, ADI coordinated the study in the recruiting centres. The Working Group contributed in recruiting the women, performing the HPV tests, collecting the data.All the authors approved the final version of the paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/214/prepub


We report results on risk factors for invasive squamous cell and adenocarcinomas of the cervix in women aged 20–44 years from the UK National Case–Control Study of Cervical Cancer, including 180 women with adenocarcinoma, 391 women with squamous cell carcinoma and 923 population controls. The risk of both squamous cell and adenocarcinoma was strongly related to the lifetime number of sexual partners, and, independently, to age at first intercourse. The risk of both types of cervical cancer increased with increasing duration of use of oral contraceptives, and this effect was most marked in current and recent users of oral contraceptives. The risk of squamous cell carcinoma was associated with high parity and the risk of both squamous cell and adenocarcinoma increased with early age at first birth. Long duration smoking (20 or more years) was associated with a two-fold increase in the risk of squamous cell carcinoma, but smoking was not associated with the risk of adenocarcinoma. Further studies are needed to confirm the suggestion from this and other studies of differences in risk related to smoking between squamous cell and adenocarcinomas of the cervix. Among cancers of the uterine cervix, adenocarcinomas are much less common than squamous cell carcinomas. As for squamous cell carcinoma, human papillomavirus (HPV) infection appears to be a major risk factor for adenocarcinoma of the cervix , with additional cases from hospital treatment centres. Squamous cell and adenocarcinoma cases were frequency matched by age group; the mean (and median) age of squamous cell carcinoma cases was 33 years and of adenocarcinoma cases was 35 years. For each woman with cervical cancer, between one and three control women matched for age (within 12 months) were selected from the patient lists of the case's general practitioner. Women were excluded from the study if they had prior malignancy , severe mental handicap or mental illness, insufficient knowledge of the English language, were a member of the Armed Services or were born outside Europe, Africa, Australasia or North America.Cases and controls were interviewed at home by a trained nurse interviewer. A structured questionnaire was used to obtain information on occupation, past medical history, smoking and sexual and reproductive factors, including use of barrier and oral contraceptives and history of screening for cervical cancer. Information on obstetric history and prescriptions for oral contraceptives was obtained from the notes of the general practitioner and, if relevant, the family planning clinic. Results of cervical smear tests were confirmed through the cervical screening service. A total of 181 women with adenocarcinoma or adenosquamous carcinoma and three matched controls per case , and 397 women with squamous cell carcinoma, each with one matched control, were recruited. For analysis, subjects who were virgins and those with unknown values for any of the adjustment variables were excluded; final analyses were based on 180 cases of adenocarcinoma and their 532 matched controls, and 391 matched pairs of squamous cell carcinoma cases and controls.In order to increase the stability of risk estimates, and to provide a better comparison between the two histological types, the matching was broken and analyses were performed using a single, pooled, group of controls. Odds ratios (ORs) and 95% confidence intervals (CIs) for the variables of interest were calculated separately for adenocarcinoma and for squamous cell carcinoma compared with controls, and for squamous cell carcinoma cases compared with adenocarcinoma cases, using unconditional multiple logistic regression. Tests for linear trends were obtained by using the midpoint values of each category and treating this scored variable as continuous. Potential confounders were investigated and the following were adjusted for, as appropriate, in all models: age , recruitment centre, age at first intercourse, duration of oral contraceptive use, level of education, number of negative screening results, smoking status and total number of sexual partners.P=<0.0001). The equivalent odds ratio for adenocarcinoma was 1.98 (1.21–3.26), and there was no significant trend (P=0.1). For both types of cervical cancer, the association with sexual partners was seen only for regular (relationship lasting more than 3 months) and not for non-regular partners. Odd ratios for cervical cancer in women with three or more regular partners compared with women with one or no regular partners were 3.40 (2.38–4.86) for squamous cell carcinoma and 2.18 (1.36–3.47) for adenocarcinoma. Direct comparison showed a higher risk of marginal statistical significance for squamous cell compared with adenocarcinoma for five or more total partners (OR=1.78 (1.00–3.18)) and for three or more regular partners (OR=1.63 (0.97–2.71)). Owing to the small numbers of adenocarcinoma cases in the upper categories for number of partners, these results are given for three categories only; finer stratification by number of partners within the upper category for lifetime or regular partners did not substantially alter the results.For both squamous cell carcinoma and adenocarcinoma, the risk of cervical cancer increased with increasing lifetime number of sexual partners (Table 1P for trend ⩽0.0001) and adenocarcinoma (P=0.004). Women with an age at first intercourse of 17 years or less had a 2–3-fold higher risk of cancer than those with first intercourse at 20 years or older. Direct comparison showed no significant difference between squamous cell and adenocarcinoma cases.The risk of cervical cancer was higher for earlier age at first intercourse for both squamous cell (No relationship was seen between either self-reported weight or body mass index (1 year before diagnosis or pseudodiagnosis) and the risk of squamous cell or adenocarcinoma of the cervix .P trend=0.001). For adenocarcinoma, the odds ratio for three or more live births was 1.44 (0.76–2.73), with no significant trend (P=0.3). Both squamous cell and adenocarcinoma showed an increased risk with early age at first birth, with women with age at first birth of 15–19 years having a risk of cervical cancer about twice that of women whose first birth was at 25 years or older. On direct comparison, there were no statistically significant differences between squamous cell carcinoma and adenocarcinoma cases in relation to reproductive factors.Of 391 women with squamous cell carcinoma, 350 had been pregnant, of whom 331 reported one or more live births, six reported one or two stillbirths and 152 reported one or more interrupted pregnancies (termination of pregnancy or spontaneous miscarriage). For 180 women with adenocarcinoma, the equivalent numbers were 163 ever pregnant, 159 reporting one or more live births, two reporting stillbirth and 54 reporting interrupted pregnancy. Table 2P trend=0.001 for adenocarcinoma and <0.0001 for squamous cell carcinoma). Compared with never use (including use for less than a year), odds ratios for 10 or more years of use were 1.89 (1.22–2.93) for squamous cell carcinoma and 2.06 (1.19–3.57) for adenocarcinoma. Risk was also related to time since last use of oral contraceptives, with higher risk for more recent use. When duration of use was considered in relation to time since last use, a significant increase in cancer risk with increased duration of use of oral contraceptives was seen only in recent users. Over 97% of oral contraceptive users among controls had used combined oral contraceptives, and analysis restricted to use of combined oral contraceptives showed an effect for duration of use very similar to that for all oral contraceptives. There was no association between ever use of progestagen-only oral contraceptives and risk of squamous cell or adenocarcinoma in analyses adjusted for the duration of use of combined oral contraceptives; these analyses are based on limited numbers. Direct comparison showed no significant differences between squamous cell and adenocarcinoma in relation to oral contraceptive use.Use of oral contraceptives was associated with an increased risk of cervical cancer  for squamous cell carcinoma and 0.87 (0.61–1.24) for adenocarcinoma). Twelve women in the study reported using barrier methods always with all regular sexual partners; all of these women were controls.P trend=0.05). For adenocarcinoma, no association was seen between cancer risk and duration of smoking. No association was found between either type of cervical cancer and intensity of smoking or the age at which smoking started. Direct comparison between squamous cell carcinoma and adenocarcinoma cases showed a consistently higher risk associated with smoking for squamous cell carcinoma. The difference between cancer types was statistically significant for current smoking (OR for squamous cell compared with adenocarcinoma=1.74 (1.11–2.71) and for duration of smoking (OR=2.44 (1.26–4.72) for smoking duration of 20+ years, with a statistically significant trend in relation to duration of smoking (P trend=0.04)). This difference is due both to increased risk for squamous cell carcinoma and to decreased risk (with odds ratios of around 0.8) for adenocarcinoma  or of adenocarcinoma (OR=0.80 (0.56–1.13)). For squamous cell carcinoma, the results suggest that the risk of cancer was higher for current smokers (OR=1.26 (0.93–1.71)) than for ex-smokers (OR=0.70 (0.47–1.03)). The risk of squamous cell carcinoma was significantly increased in long-term (20 or more years) smokers, with an odds ratio of 2.05 (1.29–3.26) and a trend for duration of smoking of borderline significance (This study was designed to provide a direct comparison of risk factors for invasive adenocarcinoma (including adenosquamous carcinoma) and squamous cell carcinoma of the cervix in premenopausal women. For squamous cell carcinoma, increased risk was associated with increased number of regular sexual partners, early age at first intercourse, use of oral contraceptives, high parity, early age at first birth and long-term smoking. Adenocarcinoma risk was associated with the number of regular sexual partners, early age at first intercourse, oral contraceptive use and early age at first birth. No significant associations were seen between parity or smoking and the risk of adenocarcinoma. The difference between squamous cell and adenocarcinoma in relation to smoking was statistically significant.Adenocarcinomas and adenosquamous carcinomas of the cervix account for about 15% of invasive cervical cancers, and both absolute and relative numbers of adenocarcinomas in screened populations have increased in recent years. This may reflect a cohort effect similar to that seen for squamous cell carcinomas and related to increased exposure to HPV infection in women born since 1960 , and theWhile there is strong evidence that HPV infection is the major factor in the development of adenocarcinomas and adenosquamous carcinomas as well as of squamous cell carcinomas of the cervix, adenocarcinomas have been associated particularly with HPV type 18 and related virus types, unlike squamous cell carcinomas in which in most populations HPV 16 and related types predominate  cervical cancers , and thiOral contraceptive use is a well-established risk factor for cervical cancer; recent analyses of case–control and cohort studies have confirmed that risk for both squamous cell and adenocarcinoma is directly related to the duration of use of oral contraceptives in all and in HPV-positive women, .et al, the results suggested that smoking may increase the risk of squamous cell carcinoma, but decrease the risk of adenocarcinoma (with odds ratios for ever smoking of 1.6 (0.9–2.9) for squamous cell carcinoma and 0.6 (0.3–1.1) for adenocarcinoma). In our analysis, smoking was also consistently associated with an increased risk of squamous cell carcinoma and a decreased risk of adenocarcinoma. Further evidence is needed to clarify the relationship between smoking and different histological types of cervical cancer, but it appears that smoking may be a risk factor for squamous cell and not for adenocarcinoma. A greater effect of smoking on the risk of squamous cell carcinoma would be consistent with the pattern of risk identified for some other types of epithelial cancer, for example, cancers of the oesophagus, of the nasal cavity and possibly of the lung  relationship with smoking. More recent evidence discussed here does not on the whole support this view. There is clear evidence that strong risk factors such as HPV infection, sexual activity and oral contraceptive use are common to both histological types of cervical cancer ; and no good evidence that squamous cell and adenocarcinomas of the cervix differ in relation to body weight, socioeconomic status, diabetes or hypertension. Unlike endometrial cancer, neither type of cervical cancer is related to age at menarche or age at menopause. The relationship between parity and adenocarcinoma of the cervix is less clear than that seen for squamous cell carcinoma, but the suggestion that adenocarcinomas resemble endometrial carcinomas (which show an inverse relationship with parity) in this respect has not been confirmed. The only consistent difference between cervical squamous cell and adenocarcinomas appears to be in relation to smoking.Our study has a number of limitations, many of which apply also to other published studies. We were unable to take HPV status into account directly in the present analyses, although adjustment for number of sexual partners should to some extent act as a surrogate for adjustment for HPV. We combined cases of adenocarcinoma and of adenosquamous carcinoma in order to obtain larger numbers of these relatively rare cancers. However, risk factors for adenocarcinoma and adenosquamous carcinoma may differ , and the fact that we were able to adjust for major potential confounding factors in the analyses. This study provides information on risk factors for cervical cancer in young women, a relatively little studied group in whom the incidence of adenocarcinoma of the cervix has increased in recent years. Our findings are generally consistent with those from a previous study of squamous cell carcinoma of the cervix in young women in the UK  related to the risk of adenocarcinoma. This finding is consistent with those from other studies. Further information is needed to confirm these results.


Chlamydia trachomatis infection were revealed as significant risk factors for CIN after adjustment for HPV antibodies.To identify the risk factors for cervical intraepithelial neoplasia (CIN), we reanalysed the data from our previous case–control study by adjusting for human papillomavirus (HPV) antibodies. Unlike our previous study based only on HPV DNA, smoking and  Infections by oncogenic human papillomaviruses (HPVs) are established as a major risk factor for cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC). However, only a fraction of infected women develop cervical cancer, suggesting the involvement of additional cofactors in cervical carcinogenesis. In numerous case–control studies various epidemiological factors have been suggested as relevant to CIN and ICC, although the results from these studies have not been entirely consistent . In epidTo address this issue, we reanalysed the data from our previous case–control study of CIN by adjusting for HPV DNA and antibodies.Chlamydia trachomatis was determined by using an enzyme immunoassay (EIA) kit  that does not detect antibody to Chlamydia pneumoniae. The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by a logistic regression analysis. The analysis was carried out using JMP 4.0J statistics package . The P-values obtained in all tests were considered significant at <0.05.The present study was conducted on women who previously participated in a Japanese case–control study of CIN. The details about this case–control study have been provided elsewhere , although the relative risk for CIN was considerably different between HPV DNA positive and seropositive women   and CIN development because of the low prevalence of OC users among study subjects. The effect of smoking on CIN development did not appear to vary between HPV-positive and HPV-negative subjects, while the effect of C. trachomatis infection was stronger among the former.Human papillomavirus status among cases and controls was determined by using HPV DNA testing and HPV capsid serology . Since HPV DNA testing at a single time point cannot identify past infections, the analysis based on HPV DNA may miss cofactors determining whether HPV infection is cleared or becomes persistent. In fact, a very recent study reported that smoking is associated with a reduced probability of clearing oncogenic HPV infections (C. trachomatis infection with persistent HPV infections may be supported by several studies suggesting the modulation of host immunity by smoking (With regard to smoking and Although the DNA adjustment enhanced the CIN risk among current smokers as well as the antibody adjustment, the association of smoking with an increased risk of CIN was not statistically significant in the DNA-based analysis. This may be due to the small numbers in the statistical analysis.C. trachomatis infection are significant cofactors for CIN development after adjustment for HPV antibodies. Simi-larly, associations of smoking and C. trachomatis infection with cervical neoplasia have been consistent in case–control studies employing HPV capsid serology (Unlike our previous study based on HPV DNA (


No data exist on the population prevalence of, or risk factors for, human papillomavirus (HPV) infection in predominantly Muslim countries in Asia.Cervical specimens were obtained from 899 married women aged 15–59 years from the general population of Karachi, Pakistan and from 91 locally diagnosed invasive cervical cancers (ICCs). HPV was detected using a GP5+/6+ PCR-based assay.The prevalence of HPV in the general population was 2.8%, with no evidence of higher HPV prevalence in young women. The positivity of HPV was associated with women's lifetime number of sexual partners, but particularly with the age difference between spouses and other husbands’ characteristics, such as extramarital sexual relationships and regular absence from home. The HPV16/18 accounted for 24 and 88% of HPV-positive women in the general population and ICC, respectively.Cervical cancer prevention policies should take into account the low HPV prevalence and low acceptability of gynaecological examination in this population. To date, there are no data on the population prevalence of, or risk factors for, human papillomavirus (HPV) infection in predominantly Muslim countries in Asia, where sexual mores differ from many other world populations  for HPV testing and liquid-based cytology.β-globin negative and/or without histological evidence of tumour, 91 ICCs remained .In parallel, formalin-fixed tumour biopsies were retrieved from women presenting with histologically confirmed ICC between 2004 and 2008 to the Ziauddin and Aga Khan University Hospitals, Karachi. After exclusion of 40 biopsies that were Liquid-based cytology and HPV testing were carried out at the Vrije University, Amsterdam, the Netherlands. A general primer GP5+/6+-mediated PCR was used for the detection of 44 genital HPV types : 1.08–10.41), as was being a working woman  . In addiThe positivity of HPV did not vary significantly by age . NeitherThis study disclosed a very low burden of HPV infection in the general female population of Karachi, considerably lower than that found using similar protocols in nearby India (17%) , highligThe HPV16 accounted for three-quarters of ICC in Pakistan, confirming the high prevalence of HPV16 observed in ICC from the Indian subcontinent compared with other world regions  ) is high


To evaluate alternative cervical cancer screening methods, digital colposcopy and collection of cervical exfoliated cells for liquid-based cytology (LBC) and hybrid capture 2 (HC2) testing were performed among 2562 women aged 15–59 years in three study sites in the People's Republic of China . Visual inspection with acetic acid (VIA) was also evaluated independently from colposcopy. A total of 74 cases of histologically confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) were identified, and 16 CIN2+ cases were imputed among unbiopsied women to correct for verification bias. Corrected sensitivity for CIN2+ was 37% for VIA, 54% for colposcopy, 87% for LBC with a threshold of atypical cells of undetermined significance (LBC⩾ASCUS), 90% for HC2, 84% for LBC using HC2 to triage ASCUS and 96% for positivity to LBC⩾ASCUS or HC2. For VIA, sensitivity was much lower among women ⩾40 years (12%) than those aged ⩽39 years (50%). Specificity varied from 77% for positivity to LBC⩾ASCUS or HC2, up to 94% for LBC using HC2 to triage ASCUS. In conclusion, LBC, HC2 and their combinations performed well, whereas VIA missed a majority of CIN2+, particularly in older women. Digital colposcopy performed better than VIA, but still missed nearly half of CIN2+ in this study. Important reductions in cervical cancer incidence have been seen in developed countries; thanks to cytological screening .During the last decade, alternatives to cytological screening have been evaluated in low-resource settings  HPV Prevalence Surveys.Between March 2004 and July 2005, the Cancer Institute of the Chinese Academy of Medical Sciences (CI-CAMS) and the IARC carried out three population-based surveys of HPV prevalence among women aged 15–59 years in China, namely in rural Shanxi Province  for the present analysis.All participants in our study underwent a gynaecological examination performed by a gynaecologist. A cytobrush was inserted into the endocervical canal and rotated gently to collect cells from the endo- and ectocervix. The brush containing cellular material was then placed in a vial containing CytoRich transport media , to be used to perform both liquid-based cytology (LBC) and HPV testing.Human papillomavirus genotyping has been reported previously . Visual inspection with acetic acid was also performed everywhere, but only in Liaoning and Guangdong was it performed by a different gynaecologist than digital colposcopy. In Shanxi, it was performed by the same gynaecologist as colposcopy, and hence was not evaluated .ad hoc evaluation of an optical device employing fluorescence spectroscopy technology. It was used to take readings in each quadrant of the cervix of all women before VIA, with positive diagnoses determined automatically by the device's algorithm. Fluorescence spectroscopy showed very poor sensitivity (18.3%) and specificity , and is not formally evaluated in this report. However, findings from biopsies prompted by fluorescence spectroscopy-positive examinations were used to reduce verification bias.In Shanxi and Liaoning, there was also an The cervix was inspected with the naked eye using a speculum and a bright halogen focus lamp 1 min after application of 5% acetic acid. The VIA result was reported by quadrant, as per the criteria in the IARC manual . Visual All women underwent digital colposcopic assessment  as a primary screening test, performed by assessing abnormal areas after application of acetic acid . ObservaLiquid-based cytology was performed either at CI-CAMS, Beijing, China (for Shanxi and Liaoning), or the Shenzhen Hospital of Beijing University, Shenzhen, China (for Guangdong). Slides were prepared from the CytoRich-preserved specimen according to the manufacturer's standard protocol, and results were classified according to the Bethesda System  was performed in CI-CAMS, using the same samples as for LBC, according to the manufacturer's instructions. The test uses an RNA probe mixture of 13 high-risk HPV types. The manufacturer-recommended cutoff of 1.0 pg mlColposcopy was used during the first screening visit to obtain a biopsy from the suspicious area of all women with abnormal findings by VIA, colposcopy and/or fluorescence spectroscopy.Colposcopy was also subsequently used to obtain biopsies from all women with low-grade squamous intraepithelial lesions (LSILs) or worse, or those with HC2-positive atypical squamous cells of undetermined significance (ASCUS), but without biopsy in the first visit, when these results became available (approximately one month later). In the absence of a specific suspicious area, biopsies were taken from each quadrant of the transformation zone. Women whose entire squamocolumnar junction could not be seen upon colposcopy underwent endocervical curettage.Cervical biopsies were prepared and read by a pathologist at CI-CAMS, Beijing, China. In this study, cervical abnormalities were defined as the presence of histologically confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+). Treatment of colposcopy-detected lesions was performed according to local protocols, primarily using loop electrosurgical excision procedures.A total of 653 biopsies were obtained, 195 of which were triggered exclusively by positive findings at fluorescence spectroscopy and 458 of which were indicated by positive findings at one or more of the four screening approaches assessed in this study. Among these colposcopy-directed biopsies, 74 cases of CIN2+ (11.3%) were diagnosed .χ2-test and corresponding P-values were used to calculate eventual heterogeneity of test performance by age and centre.Conventional screening indices, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and their 95% confidence intervals (CIs) were calculated in two ways: first, ‘crude’ indices using only histologically confirmed CIN2+ and assuming that all 1909 women without a biopsy were histolgically negative; and second, ‘corrected’ indices after imputation of the missing data . Robust Selected characteristics of the three study populations are described in Screening test positivity is also shown in With respect to the 22 CIN3 cases, all were HC2-positive, 21 were LBC⩾ASCUS-positive and 10 were colposcopy-positive. Of the nine CIN3 cases arising in study sites where VIA was evaluated, four were VIA-positive. The only case of invasive cancer was an HC2-positive, but colposcopy-negative, high-grade squamous intraepithelial neoplasia.χ12=7.51; P=0.006). Also colposcopy was slightly more sensitive, whereas LBC⩾ASCUS, HC2 and their combinations were slightly less sensitive in younger than older women, although none of these differences were statistically significant.The sensitivity and specificity of different screening tests varied somewhat across study sites, but no significant heterogeneity emerged (data not shown). Furthermore, the performance of the screening tests relative to each other, most notably the relatively lower sensitivity of visual methods in comparison with LBC, HC2 and their combinations were consistent across the three study sites (data not shown).In our present evaluation of different cervical cancer screening approaches in China, LBC, HC2 and their combinations performed well, whereas VIA missed a majority of CIN2+, particularly in older women.Most cervical screening studies suffer, to a varying extent, from verification bias, especially due to the reliance on a visual method (colposcopy) as a gold standard, without the possibility for a positive result using morphological (cytology) and/or virological (HPV) methods to trigger disease evaluation in colposcopy-negative women . This isVisual inspection with acetic acid is an inexpensive and simple screening approach that has been widely evaluated, although often with substantial verification bias . In thisAn important factor in the relatively poor performance of VIA in our study was a strong decline in the sensitivity of the test in women aged 40 years or older. Some earlier studies have reported VIA sensitivity to be stable with age .Colposcopy performed only moderately well as a primary screening test in our study, in agreement with suggestions from recent work (Using ASCUS as a cutoff, LBC showed a sensitivity of 87% and a specificity of 85%, which is consistent with that seen in earlier evaluations (Hybrid capture 2 sensitivity and specificity estimates were within the range of those from earlier studies (Strengths of our study included the large number and broad age range of participants who were drawn from three geographically and socioeconomically diverse areas in China. Screening tests were performed independently of each other and according to similar protocols in the study sites, and an invitation procedure based on population lists ensured the representativeness of the study populations. A limitation of this study was the number of women who were lost to follow-up before a histological confirmation of abnormal screening findings could be made. As a remedy, we envisaged a model that corrected for the lack of certain biopsies, but it is of note that these imputed biopsies were few and had relatively little impact on the conclusions of this study, which would not have differed substantially if crude estimates of sensitivity and specificity had been used.Given their requirement for specialised infrastructure and relatively high cost, the potential application of LBC and/or HC2 to low-resource settings remains unclear. Given the robustness of LBC and HPV samples to both time and temperature, however, our study suggests the feasibility of sending specimens to centralised laboratories, which can bring down testing costs through economies of scale. Both tests can be automated and show good interlaboratory agreement. However, such an approach would still require additional visits for diagnostic work-up and treatment of women with positive tests, which is known to result in considerable losses to follow-up. The potential advent of a new rapid and inexpensive HPV DNA test requiring minimal laboratory infrastructure  would al


Our nested case–control study of EH progression included 138 cases, who were diagnosed with EH and then with carcinoma (1970–2003) at least 1 year  later, and 241 controls, who were individually matched on age, date, and follow-up duration and counter-matched on EH classification. After centralised pathology panel and medical record review, we generated rate ratios (RRs) and 95% confidence intervals (CIs), adjusted for treatment and repeat biopsies. With disordered proliferative endometrium (DPEM) as the referent, AH significantly increased carcinoma risk . Risk was highest 1–5 years after AH , but remained elevated 5 or more years after AH . Progression risks for SH  and CH  were substantially lower and only slightly higher than the progression risk for DPEM. The higher progression risks for AH could foster management guidelines based on markedly different progression risks for atypical vs non-atypical EH.Classifying endometrial hyperplasia (EH) according to the severity of glandular crowding (simple hyperplasia (SH)  Abnormal vaginal bleeding often prompts endometrial biopsy or curettage to exclude endometrial carcinoma and endometrial hyperplasia (EH), its precursor . To descDistinguishing low-risk EH lesions that can be conservatively managed with progestogen-based treatment plus surveillance from higher-risk EH lesions that require immediate surgical treatment has clinical implications . There iThe Kaiser Permanente Center for Health Research (KPCHR) is the research arm of the Kaiser Permanente Northwest (KPNW) pre-paid health plan  who received a diagnosis of CAH at hysterectomy at least 1 year later. We henceforth refer to biopsy or curettage as biopsy  and to tWe retrieved all pathology slides and reports for all index biopsies, follow-up biopsies, and hysterectomy procedures. One pathologist (MES) initially reviewed all slides, assigned a WHO classification, and selected one representative slide for each specimen. Two gynaecologic pathologists (BMR and OBI), who were masked to all specimen data except patient age and accession date, then independently classified the selected slides according to agreed-upon WHO nomenclature .N=6) or cancer (N=2) and seven cases whose panel diagnosis did not confirm their original end point diagnosis of CAH or carcinoma at hysterectomy. Of the remaining 214 potential cases, 138 (65.4%) with index biopsy panel diagnoses of DPEM (N=33), SH (N=42), CH (N=21), or AH (N=42) were eligible. This included 28 cases whose original index biopsy was downgraded by the panel but who had a subsequent biopsy that met all eligibility criteria. The 76 ineligible cases had index biopsies with panel diagnoses of inactive (N=14), atrophy (N=8), polyp (N=4), SEM (N=8), PEM (N=22), or carcinoma (N=13); or their slides were unsatisfactory (N=7) or unavailable (N=1) for review. Of the eligible cases, 127 clinically progressed from EH to carcinoma and 11 clinically progressed to AH at hysterectomy  for at least as long as the progression interval of the case to whom they were matched. Each control within each risk set was matched to that risk set's case on age at (±1 year) and date of (±1 year) index biopsy and assigned a matched censoring date based on the case's progression interval. When necessary, we relaxed the date- and age-matching criteria in successive 1-year intervals, up to 5 years, to populate risk sets that were initially empty. To avoid selecting controls who were diagnosed with endometrial carcinoma shortly after their matched censoring date, we required each potential control to remain at risk for one additional year after her matched censoring date. Therefore, the controls who were eligible to be selected had all been diagnosed with EH at the same age and date as the cases and had the same duration of at-risk follow-up as the cases.Almost three-quarters of all EH diagnoses at the KPNW are SH . Random selection of controls would have led to underrepresentation of patients who had CH or AH, especially in risk sets for older cases or cases with longer progression intervals. To avoid this potential limitation, we selected controls via counter-matching, a form of weighted random sampling . Counter-matching  oversampThe 138 risk sets (one per eligible case) had 5891 total potential controls  who represented 2946 unique patients because controls could appear in multiple risk sets. To counter-match, we needed to know the original EH classifications for all 5891 potential controls. The KPNW pathology database recorded EH diagnoses, but EH classifications – SH, CH, or AH – were only available in the pathology reports. Reviewing 5891 pathology reports was impractical, so we used batch-quota sampling  to revieWe selected three counter-matched controls from each eligible case's risk set. First, we chose two controls whose index biopsy original EH classification differed from the case's index biopsy original EH classification . Then, regardless of the case's original EH classification, we chose a third control who had AH. We intentionally oversampled controls with AH because we expected our pathology panel to downgrade many of those original AH diagnoses. If a risk set did not contain the desired AH control(s), we substituted a control originally classified as CH for the desired AH control. We selected 413 potential controls (one case had only two controls): 129 SH (31%), 153 CH (37%), and 131 AH (32%).N=44; atrophic, N=7; polyp, N=2; SEM, N=24; PEM, N=83) or carcinoma (N=3). The 241 controls (58%) with index biopsy final diagnoses of DPEM , SH , CH , or AH  were eligible (Our pathology panel review of controls’ slides employed the same protocol that was used for cases. Nine (2%) index biopsy slides were unavailable or unsatisfactory. Of the 404 (98%) controls whose slides were reviewed, 160 (39%) were ineligible because their index biopsies had final pathology panel diagnoses of negative  and 95% confidence intervals (CIs) to estimate the relative risk of being diagnosed with carcinoma after a diagnosis of EH. Risk estimates were based on the panel diagnoses of EH type, with DPEM as the reference group.−2) and repeat biopsies  were statistically significantly associated with both EH type and case–control status. Because of its clinical importance, we also adjusted for treatment , even though treatment was similar for cases and controls  were endometrioid adenocarcinomas, five were clear cell carcinomas (4%), and one was a mucinous carcinoma (1%). Eighty-two percent were well differentiated and 11% had spread beyond the uterus.Age, date, and length of progression interval were similar among eligible cases and controls, as expected from our matched design . The oveThe median interval between index biopsy and diagnosis of carcinoma was 6.7 years . Cases’ intervals were longer for DPEM (10.4 years) and SH (8.6 years) than for CH and AH. For each EH classification, some cases had progression intervals longer than 10 years.Three-fourths of cases and a slightly higher percentage of controls had at least one follow-up biopsy after the index biopsy but before the biopsy that prompted hysterectomy. Controls were twice as likely as cases to have had follow-up biopsies within 6 months of the index biopsy. However, total mean (2.2) and median (2) numbers of biopsies were similar. Over 80% of both groups received progestogen-based treatment during follow-up, including similar proportions of oral and injectable progestogens.−2) was present in 21% of cases and 15% of controls. Oral contraceptive use and menopausal hormone therapy use were less common in cases than in controls. Approximately 60% of both cases and controls were pre- or perimenopausal at the time of their index biopsy.Age at menarche, parity, menopausal status, age at menopause, and smoking were similar between cases and controls . Cases wWomen diagnosed with EH were significantly more likely  than women diagnosed with DPEM to be subsequently diagnosed with carcinoma . IncreasTo improve the statistical precision of risk estimates by time interval since index biopsy, we expanded the reference group to include DPEM or SH . ProgresResults did not change after also adjusting for the number of progestogen prescriptions; restricting analyses to women with documented pharmacologic treatment or women who were postmenopausal or over 50 years of age at the time of index biopsy; or excluding the eight cases and one control who used tamoxifen before or during follow-up or the 11 cases (and their 18 matched controls) whose clinical end point was AH at hysterectomy.Analyses that successively excluded risk sets with short progression intervals revealed the same pattern of risks. The RRs for SH, CH, and AH were 1.9, 2.8, and 14.6, respectively, after excluding progression intervals less than 2 years; 1.8, 1.9, and 9.8, respectively, after excluding progression intervals less than 3 years; and 1.6, 1.8, and 6.6, respectively, after excluding progression intervals less than 4 years. None of these RRs for SH or CH were statistically significant, whereas all of these RRs for AH were statistically significant (data not shown).Progression risks were somewhat higher among women who had one or more follow-up biopsies. Many cases and controls had repeat biopsies within 3 or 6 months of their index biopsy; when risk was based on the most severe panel diagnosis for any biopsy within the first 3 or 6 months of follow-up, results did not materially change (data not shown).Rigorous estimates of progression risks for EH could foster improved clinical management of abnormal vaginal bleeding. Numerous methodologic deficiencies limit the available data on progression. In our analysis, biopsy diagnoses of SH and CH did not substantially increase the risk of progression to carcinoma compared with DPEM. Thus, DPEM, SH, and CH seem to be low-risk lesions that may be amenable to attempts at conservative management with close surveillance. Progression risks for AH were substantially elevated and remained increased for years. Our findings support the development of a simplified, potentially dichotomous, pathologic classification that would differentiate non-atypical EH from AH.Risk of carcinoma after AH was markedly higher 1–5 years after index biopsy and remained significantly elevated well after 5 years. Most of the women who received a panel diagnosis of AH in our study were later diagnosed with carcinoma. In previous studies, up to 50% of women who received biopsy diagnoses of AH had occult endometrial carcinoma when hysterectomy was performed soon thereafter . NonetheOf the 25 potential cases whose index biopsies were originally diagnosed as AH, our pathology panel reclassified 28% as carcinoma, confirmed 24% as AH, and downgraded the severity of 44% to SH, CH, DPEM, or negative. These results resemble findings from a recent Gynecologic Oncology Group (GOG) study of 289 patients who underwent hysterectomy within 12 weeks of receiving a community-based biopsy diagnosis of AH , samplinAll patients in our study had at least one endometrial biopsy that was originally classified as EH or DPEM. Most of them received repeat biopsies and progestogen therapy. Approximately 75% of the endometrial carcinomas diagnosed at KPNW between 1970 and 2003 occurred among women who either had no earlier EH or underwent hysterectomy within weeks or months after an EH diagnosis. The well-differentiated stage 1 endometrioid carcinomas  that forHigh BMI, a history of menstrual irregularities, and less exogenous hormone use were more common in cases than in controls. Statistical adjustment for treatment and minor differences in the repeat biopsies did not substantially affect progression risks, but it remains possible that residual differences contributed to our observations. Varied approaches to clinical follow-up of EH contribute to the inherent difficulties of assessing progression risk . Our resOur analysis could theoretically underestimate true progression risk for non-atypical EH if the EH lesions that were treated with hysterectomy within 1 year (whom we excluded) were markedly more aggressive than those that were included in our analysis, but this seems unlikely. The nearly identical progression intervals across categories of original EH diagnoses suggest that those classifications did not predict subsequent clinical behaviour of non-atypical EH. There were no substantial differences in clinical or patient characteristics at the time of index biopsy that could have been used to reliably identify high-risk SH or CH patients. Cases and controls came from one health plan and likely received similar clinical care after their EH diagnoses. Therefore, the experience of women diagnosed with SH or CH in our study is likely representative of the general population of women with non-atypical EH.Our study design represents an improvement over previous studies that lacked control groups  or 29% oOur study used one large, essentially population-based health plan with linked data covering 34 years. We reviewed all available pathology from patients with a full range of EH and DPEM and accounted for clinical follow-up, repeat biopsies, and specific treatments. However, few women with panel-confirmed AH had extended follow-up. Our analysis captured clinical progression in the context of contemporary clinical management , rather vs atypical EH, along with refined detection and classification of endometrial carcinoma precursors, could improve clinical management of abnormal vaginal bleeding.In conclusion, the risk that a woman who receives a biopsy diagnosis of AH will progress to carcinoma is high during early and long-term follow-up. The overall progression risks for SH and CH are lower than previously reported, but a small percentage of these patients progress to carcinoma despite conventional clinical follow-up. On the basis of these findings, a dichotomous classification of non-atypical EH


Cytology and histology records and cervical samples for HPV assay were obtained from a prospective cohort of 49 655 women attending clinics for routine cervical cytology in or near Manchester between 1988 and 1993. The women were followed up for cytological abnormality and neoplasia through the cytology laboratory's records. HPV at entry was assayed in an age- and period-stratified random sample of 7278 women and in prevalent and incident CIN3 cases. The prevalence of newly diagnosed CIN3 increased with time since last normal smear, indicating that most cases persist for several years. CIN3 prevalence did not increase further for screening intervals exceeding 5 years, however, suggesting that CIN3 eventually regresses cytologically. CIN2 prevalence increased less steeply with screening interval, while the prevalence of lesser abnormality was almost independent of screening interval. The prevalence of oncogenic HPV at entry declined from 19% among women aged under 25 to less than 3% at age 40 or above. Oncogenic HPV infection was strongly predictive of subsequent CIN3 , but only weakly related to CIN2  and lesser abnormality . At current incidence rates, the lifetime risk of developing CIN3 will be 9% in this population. The cumulative risk of CIN3 diagnosis among cytologically normal women with oncogenic HPV detected at entry was 28% (CI 18–43%) after 14 years. Persistence of oncogenic HPV may be more sensitive and specific than cytology for early detection of CIN3 and invasive cancer. Population-based prospective data on cervical neoplasia rates in relation to age, screening interval and history of type-specific HPV infection are still limited. Few cohorts are representative of regularly screened women in the general population, and those using the Bethesda system under which the high-grade category (HSIL) includes both CIN2 and CIN3 . HPV tesThis report describes the relationship between HPV detection at entry and cytological and histological follow-up based on routine laboratory records in a prospective population-based cohort of 61 564 women recruited between 1987 and 1993. Each woman contributed at least one cervical sample for HPV analysis at a routine cervical smear test. Since the national cervical screening programme was launched in 1988, all British women aged 20–64 have been invited for cervical screening every 3–5 years, according to regional policy. Many women were therefore being screened for the first time. The cohort, in which 86% of screening intervals were over 2 years, is contemporary with the Portland cohort of over 20 000 American women in a health care plan with a policy of annual screening , 78 062 cervical cell samples were collected from 61 564 women attending for routine screening. There was no age restriction. Participating practices and clinics covered a wide area in and around the city of Manchester, and offered screening either in the context of well-woman clinics or in association with family-planning services.The study was approved by the local ethics committee. Verbal informed consent obtained when the smear was taken was deemed appropriate, as the study database was anonymised, and the clinical significance of HPV infection was not then known. HPV assays were performed after recruitment had ended, and no HPV results were reported either to the cytology laboratory or to the women.At recruitment, a cervical smear was obtained from each woman, usually using an Ayre spatula. A second scrape was then taken using the same spatula and the tip, together with adherent cervical cells, was broken off into a sterile container containing 10 ml of storage buffer (0.9% PBS (pH 7.2) +0.1% SDS). Spatula samples were sent together with the corresponding smears and pathology request forms to the Christie Hospital Cytology Department. Smears that were accompanied by a spatula were flagged on receipt on the laboratory database. The name, address and date of birth are routinely used by the cytology laboratory to match new smears against a woman's previous screening record. Spatulas were vortexed, and the cell suspensions transferred to freezer tubes, which were stored at −30°C. Samples taken before July 1988 were centrifuged and only the pellet was stored. These pelleted samples were deemed unsatisfactory for HPV assay, so all analyses are restricted to the 54 060 women who provided a sample on or after 1st July 1988.As a sampling frame for studies nested within the cohort, the 78 062 spatulas were stratified according to the 12-month period in which they were taken and 5-year age groups. To avoid the cost of testing all spatulas for HPV DNA, we used this sampling frame to select an age- and period-stratified random sample for HPV assay. Entry spatulas for CIN3 diagnoses notified up to June 1998 were also assayed.Smear and histology results in the study database were updated at 6-monthly intervals during recruitment from laboratory records. Matching was on laboratory number, so named data were not required. The database was updated in the same way after recruitment had ended, and the present report includes cytology results for all smears recorded up to the end of January 1996 and histology results for biopsies taken up to the end of December 1998.  In addition, screening histories of 232 women in the HPV-tested random sample who were cytologically normal but were infected with an oncogenic HPV type at entry were updated by matching against the laboratory's current database to extend their follow-up to the end of June 2002 for cytology and to the end of March 2004 for histology.μl suspension were washed in normal saline to remove the collection buffer, then resuspended and digested with 200 μg ml−1 proteinase K by incubation at 55°C for 1 h in 25 μl 50 mM Tris-HCl buffer (pH 8.5) containing 1 mM EDTA (TE buffer), 1% (v v−1) Tween-20. The protease was inactivated by incubation at 95°C for 10 min and the crude digests were made up to 250 μl in TE buffer. Aliquots, 5 μl, of the digests were then used for HPV L1 consensus PCR amplification in 100 μl reaction volume using the MY09/MY11 primers  and negative controls were run in each experiment. Overall, 89% of all samples assayed gave a satisfactory (i.e. β-globin positive) HPV result. Aliquots, 10 μl, of PCR product were run on agarose gel, vacuum blotted onto nylon membranes and immobilised by UV crosslinking. Membranes were hybridised with a β-globin oligonucleotide probe, then with a generic HPV probe in order to determine HPV positivity. Biotin-labelled probes were used and positive hybridisation was detected using enhanced chemoluminescence. Positive samples were dot blotted onto new membranes and hybridised with a series of biotinylated type-specific probes including 6/11/42 (mixed), 16, 18, 26, 31, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 66, 68, 73, 82, 83 and 84. Samples positive with the generic probe but negative on all dot blots were considered positive but untyped.PCR analysis followed recommended anticontamination procedures , they had a record of an abnormal smear within the previous year (n=509), or they had ever had a diagnosis of CIN3 (n=505). All analyses are based on the remaining 49 655 women from whom a sample for HPV assay was collected at a routine smear test that gave an adequate cytological result.The present report is based on 54 060 women who entered the study on or after 1 July 1988  result was obtained for 6462 (89%). These 6462 women are referred to as the random cohort.The data were analysed as two cohort studies . The retThe entry smear in the retrospective cohort and the next smear in the prospective cohort were categorised by 5-year age strata and time since the preceding normal smear for analysis by unconditional logistic regression of the odds ratios for CIN3, CIN2 and lesser abnormality. The underlying incidence of CIN3, estimated by dividing the number of cases by the woman-years of follow-up since the last smear within each age and interval stratum, was analysed by Poisson regression. HPV analysis was performed systematically on all entry spatulas (i) for the 7278 eligible women in the age-stratified random sample (ii) for all CIN3 cases diagnosed at entry and (iii) for subsequent CIN3 cases notified within the study period (i.e. with histology before the end of 1993). HPV status at entry was determined for 87% (314 out of 361) of prevalent CIN3 cases diagnosed at entry and for 41% (84 of 204) of incident cases diagnosed at the next routine smear. Logistic regression analysis was used within this nested case–control study to determine the effect of HPV infection in a normal smear on the risk of cytological abnormality at the following smear. Confidence intervals (95%) are shown for parameter estimates and significance levels are two-sided. The cumulative risk for diagnosis of CIN3 or cancer was calculated by the Kaplan–Meier method, defining diagnosis date as described above and censoring at the latest recorded smear date.Prevalences of CIN3+, CIN2 and lesser abnormality at entry to the study and concurrent HR-HPV infection rates are summarised in n=1611) or after an interval of 10 or more years (n=1821) are excluded in Prevalence rates at the next smear for CIN3+, CIN2 and lesser abnormality in the combined prospective and retrospective cohorts are shown in P<0.001) for intervals of under 5 years, reaching a maximum of 1.0%  between 4 and 5 years. CIN3+ prevalence was 0.8%  for intervals between 5 and 10 years, showing no further increase. CIN2 was more common than CIN3+ initially and increased by a factor of 1.22  per year up to 5 years. The ratio of CIN2 to CIN3+ showed a significant (P<0.005) initial decline, from 1.5 (19 : 13) in smears taken within 18 months of a normal smear to 0.5 (211 : 415) thereafter. The OR for lesser cytological abnormality increased by only 1.05  per year since the last normal smear, from 1.03 (CI 0.82–1.29) below a year to 1.30 (CI 1.13–1.49) at 4–5 years and 1.18 (CI 1.03–1.35) beyond 5 years. The multivariate analysis shown in P=0.07).All three grades of neoplasia showed a statistically significant trend in OR with increasing screening interval, but the rate of increase was greater for CIN3+ than for CIN2, and the prevalence of lesser abnormality was almost constant  suggests that most new cases persist for at least 5 years. We therefore fitted the same multivariate model by Poisson regression, including the variables shown in P<0.02). The underlying age-specific incidence of CIN3 in a screened population can thus be estimated from data on screening intervals not exceeding 5 years. In The linear increase in CIN3+ prevalence with increasing screening interval up to 5 years , giving a cumulative actuarial CIN3+ diagnosis rate of 16% (CI 11–23%) after 10 years and 28% (CI 18–43%) after 14 years . Analysis restricted to the 40 incident CIN3+ cases  HPVs can be classified phylogenetically into groups . All eight women who were initially HR-HPV positive and were diagnosed with CIN3 at their following smear had the same HR-HPV type in both samples . The same HR-HPV type was detected at entry and at the next smear in 21 (32%) of the 65 women whose next smear was taken after an interval of more than 2 years. The next smear was abnormal in 13 (62%) of these 21 women with persistent HR-HPV, including eight with CIN3+, two with CIN2 and three with lesser abnormality. HPV16 was more likely to persist than other HR-HPV types. The proportion of women with HR-HPV at entry who still had the same HPV type at their next smear was 53% (18 out of 34) for HPV16, 26% (nine out of 35) for HPV18, HPV31 or HPV33 and none of 27 for all other HR-HPV types.The relationship between HR-HPV infection at entry and low-grade abnormality at the next smear was weaker in this study  than in several other cohorts . Any relβ-Globin could be amplified in only 89% of samples, due probably to the inhibitory effect of the wooden spatula tip, which remained in the sample at room temperature for several weeks before decanting and freezing. Among those that were β-globin positive, however, these sampling and assay methods detected HR-HPV in 82% (257 out of 314) of the CIN3+ cases diagnosed at an abnormal entry smear  is also consistent with a continuing increase in HPV prevalence during the 1990s.The relationship between HPV prevalence based on a single sample and lifelong risk of infection is not known. Persistent or recurrent infection is more likely to be detected than a transient episode due to the length-biased sampling effect. The cumulative HPV rate was higher than the cross-sectional rate by a factor of 1.6 among American students sampled weekly on 10 occasions . At currP=0.03). This reduction in risk supports the long-standing belief that even biopsy of HPV lesions may reduce the subsequent risk of CIN3 , but a much weaker and nonsignificant relationship for abnormalities other than CIN3+ . Third, the much lower incidence of CIN3 in women aged under 25 despite their higher HPV prevalence compared with those aged 25–34 . CIN2 is also commoner in young women. The CIN2:CIN3+ ratio fell from 1.1 (58 : 53) below age 25 to 0.5 (142 : 308) at older ages (P<0.001). Persistently detectable HPV preceding cytological abnormality is common in the development of CIN3 and cancer but not of CIN2. In a prospective cytological and colposcopic follow-up of women in the Netherlands with abnormal smears (P<0.01). Despite disagreement between pathologists over the classification of individual cases, these objective differences between CIN2 and CIN3 support the view that most CIN2s are biologically and clinically indistinguishable from the lower grade HPV lesions that usually regress (in situ or because they are precursors in its progression. For clinical purposes, the inclusion of CIN2 with CIN3 in the HSIL category of the Bethesda classification may therefore be justified, although a repeat smear would avoid colposcopy and biopsy in the transient majority of CIN2s (in situ were superseded (An increase in prevalence of CIN2 and CIN3 but not of lesser abnormality with increasing interval since the last normal smear was reported in Icelandic women, but data for CIN2 were not presented separately . There ader ages ; P<0.001 regress  confirmsof CIN2s , and resperseded .The age-specific CIN3 incidence pattern shown in If most CIN3s eventually regress cytologically, an increasing proportion will be missed as the screening interval increases and many may not reappear until they become malignant. CIN3 regression is also relevant in choosing the age at which screening should begin. The age at which routine screening begins has recently been raised from 20 to 25 years in the UK, and it has been suggested that this should be further increased to 30 years because invasive cancer is so rare in young women . RegisteCIN3 in an older woman who has not been screened regularly may often be a recurrence of an earlier undiagnosed lesion. The CIN3 rate in England was increasing at all ages before 1988, but the rate among women aged over 45 has fallen progressively since the introduction of national screening. Women whose CIN3 has already regressed before their first smear and does not recur may suffer an increased risk of invasive cancer for the rest of their lives, and this long-term risk could be an important disadvantage of delaying the age at which routine screening begins (Our results support the conclusion that the screening interval should be less than 5 years, except perhaps for older women who are HPV negative, whose risk of CIN3 is very low . 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Rochdale: Whitworth Medical Centre. Salford: Bevendon Square Clinic, Higher Broughton Health Centre, Hope Hospital, Lanceburn Health Centre, Liverpool Street Clinic, Lower Broughton Clinic, Ordsall Health Centre, Langworthy Clinic. Stockport: Adswood Clinic, Bramhall Clinic, Brinnington Clinic, Burley House Clinic, Cheadle Heath Clinic, Hazel Grove Clinic, Heaton Norris Clinic, Maple Clinic, Marple Cottage Surgery, North Reddish Clinic, Offerton Clinic, Offerton Green Clinic, Robins Lane Medical Centre, Romiley Clinic, Romiley Health Centre, Shaw Heath Clinic, Stepping Hill Clinic, Wellwoman Clinic, Woodley Clinic, Bredbury Clinic, Brookfield Clinic, Heaton Norris Clinic. Warrington: Derby Road Clinic. Wigan: Beech Hill Health Centre, College Road Clinic, Grasmere Street Health Centre, Haig Road Clinic, Liverpool Road Health Centre, Longshoot Lane Health Centre, Orrell Road Clinic, Pemberton Clinic, Platt Bridge Clinic, Queens Road Clinic, Standish Clinic


Virtually all cases of cervical cancer are caused by persistent infections with a restricted set of human papillomaviruses (HPV). Some HPV types, like HPV16 and HPV18, are clear and powerful carcinogens. However, the categorization of the most weakly carcinogenic HPV types is extremely challenging. The decisions are important for screening test and vaccine development. This article describes for open discussion an approach recently taken by a World Health Organization International Agency for Research on Cancer (IARC) Monographs Working Group to re-assess the carcinogenicity of different HPV types. As a group, human papillomaviruses (HPV) are proven human carcinogens. But there are >> 100 HPV genotypes and only a small fraction have any known carcinogenic potential , it is iIARC formally considers the carcinogenicity of exposures to humans. Whereas human carcinogenicity might best be considered for some agents like HPV as a continuum of probabilities without a clear breaking point, IARC classifies carcinogens categorically as carcinogenic (Group 1), probably carcinogenic (Group 2a), possibly carcinogenic (Group 2b), not classifiable (Group 3), or probably not carcinogenic Group 4). There has been very little experimental work on the carcinogenicity of HPV types except for HPV16 and HPV18; thus, epidemiologic evidence has been unusually important. Epidemiologic evidence for the carcinogenicity of HPV was originally presented in IARC Monograph Volume 64 . There h. In FebrHPV carcinogenicity has been established most convincingly for cervical cancer, and this discussion will be limited to cervical carcinogenicity. To date, no HPV type has been proven to be carcinogenic only at sites other than the cervix.HPV behaviour at the cervix is strongly correlated with phylogenetic (i.e. evolutionary) categories . All HPVIn Monograph 64 in 1995, HPV 16  and HPV 18  were classified as cervical carcinogens. HPV 31 and HPV 33 in alpha-9 were categorized as probably carcinogenic  When detected, even without detection of a more likely causal type, the Working Group judged that misclassification of some kind was a more likely explanation than causality. As given in Table By this logic, four more types were categorized as having sufficient epidemiologic evidence leading to their classification as definite (Group 1) carcinogens: alpha-5 type 51 , alpha-6 type 56 (0.78%), and alpha-7 HPV types 39 (1.29%) and 59 (1.28%).The remaining types in the high-risk alpha clade were considered, as a group, to have limited evidence to support carcinogenicity. If phylogeny can be taken to predict behaviour, it is possible that most of these types can very rarely cause cancer. Indeed, many of the types have been detected, albeit uncommonly (not significantly greater than HPV 6), in cancers. There are not enough data, even after testing of many thousands of specimens, to be sure which ones are carcinogenic or not. Furthermore, some of these types, namely 30, 34, 67, 85 and 97 have not yet been studied in the IARC cervical cancer meta-analysis approach. Nevertheless, within this group, there are two types, alpha-7 type HPV 68 and alpha-11 type HPV 73, for which the data are slightly stronger than for the others despite methodologic challenges. One of the major PCR-based testing methods (SPF10) cannot distinguish these two types because their amplicons using those primers are identical . NeitherOverall, the Working Group approach led to the re-classification from Monograph 90 of HPV 66 to possible (Group 2B), although the epidemiologic evidence of carcinogenicity was previously judged sufficient (Group 1). In the assembly of much more testing data from cancer cases, HPV 66 has been found so rarely that its percentage of detection is less than the relative percentage of detection among the general population. In the Working Group review of each individual article, HPV 66 was found as a single infection in cancers with extreme rarity, well below the threshold of possible confounding and misclassification.There is no perfect way to categorize a continuum of carcinogenic potential. The Working Group arrived at the following conclusions, with a healthy scepticism concerning the process:a) Persistent HPV 16 infection is a uniquely powerful human carcinogen.b) HPV 18 is also important, particularly for adenocarcinoma.c) Six additional types in alpha-7 (HPV 45) and alpha-9  comprise the remainder of the eight types that are the most important globally, with some regional variation in the etiologic fractions of cancers due to each type.d) There are small and less certain, incremental etiologic contributions of another group of carcinogenic types from alpha-5 (HPV 51), alpha-6 (HPV 56), and alpha-7 (HPV 39 and HPV 59). Each causes a few percent at most of cervical cancer cases worldwide. There has not been any large-scale study of regional variability for these uncommon typese) There is an unresolved dividing line between the HPV types with the weakest evidence judged to be sufficient and those judged to have highly suggestive yet limited data .f) The expanded data for HPV66 were re-evaluated and the evidence was judged to be very limited now that more cases have been studied showing that it is very rarely found in cancers despite being relatively common in the newly collated data on women with normal cytology. HPV 53, also in alpha species 6, shows the same pattern of relative common population prevalence with extremely rare cases of occurrence alone in cancer. The Working Group noted that for these types in particular, there could be harm to public health if the types are viewed with excessive concern; including these types as carcinogenic in screening assays would decrease the specificity and positive predictive value of the assays with virtually no gain in sensitivity and negative predictive value .g) There are several types within the high-risk clade that have extremely sparse or no evidence of carcinogenicity. For some types there are anecdotal but very interesting cases that merit pursuit of additional carcinogenic types. There have been potentially underappreciated reports of alpha-9 type HPV 70 found as single infections in cancer, but the supportive data are sparse. There are only a few reports of HPV 67 in cancer -24, which) The possible role of immunosuppression in HPV carcinogenicity was not emphasized in the Working Group discussions. To cause cancer, an HPV infection must persist and it is possible that some HPV types are only weak carcinogens because they persist poorly. For example, the carcinogenicity of alpha-5 type HPV 26 has been supported by a recent report of multiple peri-ungual cancers in an immunosuppressed individual, containing high viral loads and active transcription of HPV 26 alone . HPV 26 When epidemiology serves as the science informing public health policy, its limitations must be acknowledged. Weak causal associations with one HPV type are extremely hard to prove in the presence of powerful confounding by strong carcinogens like HPV16. A coming generation of intensive molecular studies of microdissected cancers, with examination of transcriptional activity, may clarify the borderline between carcinogenic and non-carcinogenic HPV types. For public health purposes, the main agents that merit consideration in screening tests and vaccines have already been identified.