Rates of surgically treated carpal tunnel syndrome (CTS) among blue- and white-collar workers and housewives in the general population were compared.Surgically treated cases of idiopathic CTS were investigated among 25–59-year-old residents of Tuscany, Italy, during 1997–2000, based on obligatory discharge records from all Italian public/private hospitals, archived according to residence on Tuscany’s regional database. Population data were extracted from the 2001 census.After excluding repeat admissions, 8801 eligible cases were identified. Age-standardised rates (per 100 000 person-years) of surgical CTS were: “blue-collar women”, 367.8; “white-collar women”, 88.1; “housewives”, 334.5; “blue-collar men”, 73.5; and “white-collar men”, 15.3. Compared with reference categories (same-sex white-collar workers): female blue-collar workers experienced a 4.2-fold higher standardised rate; housewives, a 3.8-fold excess; and male blue-collar workers, a 4.8-fold excess . Male and female blue-collar workers showed approximately three to sevenfold higher age-specific rates compared to their white-collar counterparts . Housewives’ rates were similar to those of blue-collar female workers up to 40–44 years of age, after which they were significantly lower (p<0.002). At all ages, housewives’ rates were much higher (p<0.001) than those of white-collar women.Surgically treated CTS was three to seven times more common (depending on age/gender) in blue-collar than in white-collar workers, which is difficult to explain by differences in body weight or other individual factors. Thus, occupational risk factors seem relevant throughout working life. The high rates for full-time housewives suggest that domestic chores should be investigated as a possible risk factor for CTS. Carpal tunnel syndrome (CTS) is a disabling condition6A survey of the general population in southern Sweden found that blue-collar workers had approximately double the risk of symptomatic CTS  compared to white-collar workers.Surgery is the treatment of choice for cases of severe chronic CTS refractory to conservative approaches.Using hospital discharge records and census data, we evaluated age- and sex-specific incidence rates of surgically treated CTS among blue- and white-collar workers and full-time housewives in the general population of Tuscany (3.5 million inhabitants), Italy in 1997–2000. In Italy during this period, diagnosis of CTS supported by median nerve conduction studies was considered a prerequisite for surgical treatment, and out-of-hospital CTS surgery was almost completely absent due to reimbursement regulations. In Italy, all public/private hospitals are obliged to provide individual, codified discharge records, even after day treatment. These discharge records are stored in databases of the patients’ region of residence, irrespective of hospital location. The discharge records of hospitals within the administrative Region of Tuscany (Regione Toscana) from the period also contain codified information on the generic occupational categories listed in –To restrict the study to idiopathic CTS, we excluded cases with secondary  diagnoses of conditions thought to be associated with a increased risk of CTS,16Population data for the age groups of interest in the study area were extracted from the most recent national census, which was conducted in 2001; of note, we are not aware of any major workforce changes in Tuscany between 1997 and 2001. To facilitate white-/blue-collar classification we chose to use census data that had been tabulated based on the ISTAT-2001 Classification of Occupations.19We calculated age-sex specific incidence rates (per 100 000 person-years) and standardised rates (age adjusted by the Standard European Population proposed by WHO)The process used to identify surgically treated cases of CTS meeting the study eligibility criteria is described in Overall age-standardised incidence rates of surgically treated CTS (per 100 000 person-years) were 255.4 (95% CI 249.6 to 261.2) for women and 46.4 (95% CI 43.8 to 49.0) for men. Among women, age-standardised rates were 367.8 (95% CI 355.1 to 380.5) for blue-collar workers, 334.4 (95% CI 322.0 to 346.9) for housewives, and 88.1 (95% CI 81.9 to 94.2) for white-collar workers. Thus, with respect to their white-collar counterparts, female blue-collar workers had a 4.2-fold higher rate of surgically treated CTS, and housewives had a 3.8-fold excess. Among men, the age-standardised rates were 73.5 (95% CI 68.9 to 78.0) for blue-collar workers and 15.3 (95% CI 13.1 to 17.5) for white-collar workers. Thus, male blue-collar workers experienced a 4.8-fold higher rate of surgically treated CTS with respect to their white-collar counterparts. This study indicates that surgically treated idiopathic CTS may be as much as three to seven times more common (depending on age and gender) in blue-collar workers than in their white-collar counterparts. It must be stressed that we were unable to adjust for factors other than age and sex. Nevertheless, we believe that such high excess risks among male and female blue-collar workers of various ages would be difficult to attribute to only moderate differencesUnsurprisingly,29The primary explanation for the greatly increased risk of surgically treated CTS recorded among male and female blue-collar workers at all ages is likely to be exposure to manual work which involves taxing hand-wrist activities, such as prolonged, highly repetitive wrist flexion/extension, forceful grip in awkward postures, and use of hand-held vibratory tools.It might be argued that blue-collar workers are more likely to access surgical treatment due to their greater need to conserve manual fitness. However, in a comparison of surgically treated and electromyographically diagnosed CTS patients in the Tuscan town of Siena,The characteristics of the Tuscan coding system also allowed us to evaluate the risks associated with unemployed housewife status. The remarkably similar patterns of surgically treated CTS among Tuscan housewives and female blue-collar workers highlights the possibly aetiological relevance of domestic work. In a case–control study of determinants of surgically treated CTS, we found that full-time housewives again appeared to have an approximately fourfold excess risk in comparison with white-collar workers (unpublished data). Furthermore, in an evaluation of associations between marital status and surgically treated CTS in several regions of Italy,33The study design allowed estimation of age-related risks associated with blue-collar employment and housewife status in the general population. We are confident that use of the hospital discharge records which every Italian public/private hospital is obliged to supply to local administrations allowed us to identify the vast majority of surgically treated cases of idiopathic CTS which occurred among residents of Tuscany between 1997 and 2000, when out-of-hospital CTS surgery was almost completely absent in Italy and going abroad for treatment was presumably not a widely considered option for CTS. Nevertheless, subsequent exclusion of retired subjects from the main analysis due to lack of information on occupational history limits the validity of the results around retirement age. Furthermore, our attempt to restrict the numerators to cases of “idiopathic” CTS may have been affected by under-reporting of concomitant conditions in the discharge records, perhaps especially in the context of patients with lower educational status. This factor might have led to slight overestimates of (i) the overall rates of surgically treated idiopathic CTS, and (ii) the rate ratios for blue-collar workers and housewives. The routine data on which the analysis was based did not permit adjustment for likely confounding factors other than age and gender. The results regarding housewives may have been particularly vulnerable to unadjusted confounding from factors such as BMI, parity and past working history, which could have contributed to an overestimate of risk. Thus, we think that our findings regarding housewives should primarily be considered a stimulus for further study. Clearly, investigation of possible aetiological relationships between manual work and body weightPossible discrepancies in blue-/white-collar/housewife classification between cases and the general population must be considered. After exclusion of military personnel and subjects with unknown active occupational status, the occupational groupings used for the census data and the hospital discharge records were readily classifiable into blue-/mixed collar and white-collar status. However, it is likely that both the hospital discharge records and census data contain some incorrectly coded information . Of note, we do not think that differential classification of housewives should be a major concern since both the hospital discharge records and the census data provide specific categories for full-time housewives.Manual work-related risk factors for surgical treatment of idiopathic carpal tunnel syndrome (CTS) appear to be relevant throughout working life for both men and women.As an occupational category, full-time housewives seem to have a raised risk of surgically treated CTS, possibly related to domestic work.In aetiological studies of carpal tunnel syndrome, attention should be paid to domestic chores as a potentially relevant risk factor.Although we were able to exclude repeated admissions with a principal diagnosis of CTS during the study period, we had no reliable way of identifying previous admissions before the study period (an issue likely to mainly effect the early part of the 4-year study period): thus the rates recorded are likely to be a slight overestimate. Of note, a sensitivity analysis based on the last 2 years of the study period did not show signs of distortion due to the inevitable inclusion of some prevalent cases. As regards the external validity of the findings, it is noteworthy that the overall rates of surgical treatment were broadly in line with those reported in other population-based surveys.In conclusion, blue-collar workers appear to have much higher rates of surgically treated idiopathic CTS than white-collar workers, irrespective of gender and age group. It is not easy to attribute such discrepancies exclusively to confounding factors such as differential body weight. Thus, the present work seems to underline the relevance of occupational risk factors for surgically treated CTS. Moreover, the remarkably similar patterns recorded for full-time housewives suggest that the role of domestic chores deserves more investigation as a possible risk factor for CTS (not only in full-time housewives). In conjunction with analysis of the possible risks associated with domestic duties, study of the biomechanical profiles of manual domestic chores could also be informative.


Garcinia mangostana Linn, is a tree found in South East Asia, and its pericarps have been used as traditional medicine. Phytochemical studies have shown that they contain a variety of secondary metabolites, such as oxygenated and prenylated xanthones. Recent studies revealed that these xanthones exhibited a variety of biological activities containing anti-inflammatory, anti-bacterial, and anti-cancer effects. We previously investigated the anti-proliferative effects of four prenylated xanthones from the pericarps; α-mangostin, β-mangostin, γ-mangostin, and methoxy-β-mangostin in various human cancer cells. These xanthones are different in the number of hydroxyl and methoxy groups. Except for methoxy-β-mangostin, the other three xanthones strongly inhibited cell growth at low concentrations from 5 to 20 μM in human colon cancer DLD-1 cells. Our recent study focused on the mechanism of α-mangostin-induced growth inhibition in DLD-1 cells. It was shown that the anti-proliferative effects of the xanthones were associated with cell-cycle arrest by affecting the expression of cyclins, cdc2, and p27; G1 arrest by α-mangostin and β-Mangostin, and S arrest by γ-mangostin. α-Mangostin found to induce apoptosis through the activation of intrinsic pathway following the down-regulation of signaling cascades involving MAP kinases and the serine/threonine kinase Akt. Synergistic effects by the combined treatment of α-mangostin and anti-cancer drug 5-FU was to be noted. α-Mangostin was found to have a cancer preventive effect in rat carcinogenesis bioassay and the extract from pericarps, which contains mainly α-mangostin and γ-mangostin, exhibited an enhancement of NK cell activity in a mouse model. These findings could provide a relevant basis for the development of xanthones as an agent for cancer prevention and the combination therapy with anti-cancer drugs.Mangosteen,  Garcinia mangostana. The entire fruit is typically 2.5–7.5 cm in diameter, roughly the size of a tangerine  from the pericarps of mangosteen against human leukemia HL60 cells. Interestingly, α-mangostin was observed to induce mitochondrial dysfunction . MoreoveThe subsurface chemistry of the mangosteen pericarp comprises an array of polyphenolic acids including xanthones and tannins that assure astringency to discourage infestation by insects, fungi, plant viruses, bacteria and animal predation while the fruit is immature. Color changes and softening of the pericarp are natural processes of ripening, which indicates that the fruit can be eaten and the seeds finish developing. Among the constituents of the pericarps, xanthones are biologically active phenols that naturally occur in a restricted group of plants –17. Over50, the inhibitory activity was estimated; βM-ME<βM<αM<γM , β-mangostin (βM), γ-mangostin (γM), and methoxy-β-mangostin (βM–ME)] from the pericarps of mangosteen wereexamined for the effect on the growth of human colon cancer DLD-1 cells . Except βM<αM<γM . The HoeβM<αM<γM .50 of α-mangostin was close to that of 5-FU  and Akt kinase are key regulatory proteins in cells. MAPKs are a widely conserved family of serine/threonine protein kinases involved in many cellular processes such as cell proliferation, differentiation, motility, and death . Akt, anThe levels of phosphorylation of p38 and p-JNK appeared to change within 24 h after the treatment with α-mangostin, but their changes could not be properly explained . The levERK5 by introducing miRNA-143 into DLD-1 cells . miR. miR13].-1 cells ,26. α-MaIn view of recent phytochemical studies, it has been pointed out that such substances included in vegetables and fruits could affect the efficacy of anti-cancer drugs and their metabolism , becauseWe demonstrated the synergistic effect on cell growth when 5-FU was used with α-mangostin  . The groIt is possible that the mechanism of growth suppression by α-mangostin is different from that of 5-FU at more than 15 μM, because the growth inhibition obtained by a single treatment with α-mangostin was greater compared with that by the combined treatment. It is possible that the more potent apoptosis-inducing activity of α-mangostin which was observed at more than 15 μM, was not induced by the combination with 5-FU at both 7.5 μM and 10 μM. In this context, the activation of MAPKs and Akt signal pathways, which were changed by the treatment with 20 μM α-mangostin alone, could be reduced in the single treatment of α-mangostin or 5-FU. Thus, phytochemicals are conceivable to exert a considerable effect on the efficacy of anti-cancer agents, depending on their concentrations, by modulating the intracellular signaling pathways . The enhWe examined whether α-mangostin has short-term chemopreventive effects on putative preneoplastic lesions involved in rat colon carcinogenesis . Rats inNatural killer (NK) cell works as the main immune cells of the innate immunity, and it is especially important in the eradication of the tumor cells and the virus infected cells. However, the activity of the NK cell decreases with aging after the peak at about 15 years old. The morbidity rate of people with cancer is rising by aging, which may be related to the decline of the activity of the NK cell activity.We investigated the effect of α-mangostin on NK cell activity using a mouse model. We administrated the α-mangostin-enriched extract from pericarps named Panaxanthone  to mice everyday by gavage with different doses for 30 days. The NK cell activity was determined by measuring LDH after the incubating of YAC-1 cells (target cells) and splenocytes (effector cells) at the ratio of 1:50. The activities of 20 and 40 mg/kg groups were significantly elevated compared with that in control group 0 mg/kg . A signi50 of α-mangostin is almost same as that of 5-FU in DLD-1 cells. Its activity is mainly due to apoptosis. The apoptotic observations such as morphological changes and DNA ladder formation emerged at 24 h-treatment with α-mangostin, while the decrease in the mitochondrial membrane potential and the release of Endo-G observed at 6 h-treatment. These events were preceded by the inactivation of the signaling cascades involving Erk1/2 and Akt at 3 h-treatment. The cell cycle regulatory proteins cyclin D1 and cdc2 were also down-regulated at 3 h-treatment. Since the swelling of mitochondria was observed at 1 h-treatment, α-mangostin most likely attacks the proteins involved in permeability transition of mitochondria. This event could trigger the cell cycle arrest and apoptosis . α-Mangostin increased the expression of glutathione S-transferase (GST) at 0.5–5.0 μM for 12 h-treatment in human hepatocellular carcinoma HuH-7 cells. Furthermore, recent study has revealed that the suppression of inflammatory reaction by phytochemicals leads to cancer prevention. The xanthones, which have anti-oxidant activity, have been reported to reduce the expression of cyclooxygenase-2 (COX-2) [Considering the chemopreventive effects of phytochemicals, they may depend on three main activities: anti-oxidant, apoptosis inducing, and phase II enzymes inducing. In our studies, we have verified such activities in α-mangostin. It has been already known that the ORAC  value of mangosteen is makedly high (www.natu (COX-2) –39 and t (COX-2) ,40.Recently, we succeeded in crystallizing the extract from the pericarps and found that the substance contains more than 90% xanthones . This was named Panaxanthone, and assessed to be safe by a conventional safety test using mouse model. The safety was further confirmed by the fact that these xanthones have been used as a folk medicine for many years and more than 160 kinds of fruits juice containing whole extract of mangosteen are distributed worldwide. The safety clinical trial of Panaxanthone is under progress, leading to development for a cancer preventive and therapeutic agent.


To define the spectrum of human papillomavirus (HPV) types and establish an age limit for triage HPV testing in atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL).343 liquid-based cytological samples from the population-based screening programme with minor abnormalities were subjected to HPV genotyping .P<0.001). High-risk human papillomavirus prevalence was age-dependent in LSIL (P=0.01), with decreasing prevalence until the age of 50 years, followed by a slight increase. Human papillomavirus type 16 was the most common HR-HPV, found in 23% of HPV-positive women. Human papillomavirus type 18 was the sixth most common, found in 9.9% (P<0.001). An age-dependent quadratic trend was observed for multiple infections (P=0.01) with a trough at about 42 years. The most common HR-HPV types to show a coinfection with HPV16 (clade 9) were HPV39 (28%), 45 (38%), and 59 (46%), belonging to HPV18 clade 7. The frequency of low-risk (LR) vs probable HR and HR-HPV also followed an age-dependent quadratic trend.High-risk human papillomavirus (HR-HPV) was found in 71% of LSIL and 49% of ASCUS cases (After the age of 25 years, HR-HPV prevalence is similar in LSIL and ASCUS cases, motivating a low age limit for triage HPV testing. Multiple infections and LR/HR-HPV dominance are age-dependent. Genotyping in longitudinal design is needed to elucidate the importance of multiple infections in cancer progression and in cross-protection from vaccination. Persistent infection with high-risk human papillomavirus (HR-HPV) types has a critical aetiological function in the development of cervical intraepithelial neoplasia (CIN) and cervical cancer  and to describe the prevalence of multiple infections in relation to age before the implementation of vaccination programmes.From September 2005 through to September 2006, 4204 LBC samples from a population-based screening programme were obtained from seven maternity health centres in Southern Stockholm as previously reported , according to the manufacturer's instructions . The method includes a PCR amplification of extracted HPV DNA using a pool of biotinylated primers that hybridise in the L1 region of the HPV genome, chemical denaturation of HPV DNA amplicons to single-stranded DNA, followed by hybridisation with matching type-specific DNA probes immobilised on nylon strips, and detection by colorimetric determination. The result is a pattern of blue lines on a nylon strip, which is visually read by comparing the pattern with a reference guide. The LA test covers 37 HPV types including 12 HR-HPV types , 6 probable high-risk (pHR-) HPV types , and 19 low-risk or undetermined-risk (LR-) HPV types  (χ2 and Yates corrected χ2 (for n<5) tests were used to compare proportions, and Student's t-test was used to compare continuous variables between two groups. After plotting proportions of HR-HPV- and HPV-infected women and multiple infections, respectively, in each age group, a linear or polynomial fit was visually assessed to be the best model for logistic regression. We tested whether the proportion in each age group decreased linearly with age (log(p/1−p)=b0−b1x) and whether this was significantly different from null. Then we added a second degree (log(p/1−p)=b0−b1x+b2x2) to test whether the increase in women over a certain age was significantly different from null. We controlled for covariation between age and age2 using centred age and centred age2 in the model. The larger model with a second degree provided a better explanation as tested with Nagelkerke R2. Therefore, we concluded that the quadratic function, log(p/1−p)=b0−b1x+b2x2, could best describe the relationship between prevalence of HR-HPV in LSIL cases and age, multiple infections and age, and HR/LR-HPV dominance and age. Logistic regressions were also carried out using age group and cytological diagnosis as categorical predictors, as well as HPV prevalence as the dependent variable. The null hypothesis of no difference was rejected at a significance level of P<0.05.Data were analysed with the software STATISTICA 6.1 . Pearson's P=0.002). In all, 73% of the women were younger than 40 years. Women with ASCUS were older than women with LSIL; 38% of women with ASCUS were 40 years or older, compared with 21% of women with LSIL .There were 223 cases of LSIL and 120 cases of ASCUS, including three cases of atypical glandular cells of undetermined significance (AGUS). Age was illegible on one LSIL sample and on one ASCUS sample; therefore age was recorded in 341 of 343 women. The mean age was 33.6 years . Women were grouped according to age at 5-year intervals from age 20–49 years, whereas the oldest women, aged 50–60 years, were placed into a single comparably sized group. β-globin gene from all 343 samples was successfully amplified as shown by the presence of both low- and high-concentration bands on the array. A total of 282 (82%) women tested positive for one or more of the 37 included HPV types. Human papillomavirus prevalence ranged from 92% in the 20–29-year age group to 62% in women aged 40–45 years. Prevalence was 67% in women over 50 years. Age-specific prevalence for all HPV types and HR-HPV types (12 or 13 types) was higher in younger age groups and there was a linear decline with age . Age-specific prevalence of all HPV types in women with ASCUS compared with women with LSIL is shown in P=0.03), but did not reach a statistically significant difference between ASCUS and LSIL within each age group using logistic regression (P=0.098). However, there is an apparent tendency for increasing discrepancy with lower and especially higher age. χ2-tests to compare proportions gave significant differences in all age groups, except in the 30–39 year age group.Human papillomavirus was more common in women with LSIL  than in women with ASCUS . High-risk human papillomavirus (12 types) was found in 59 (49%) cases among women with ASCUS, compared with 158 (71%) in women with LSIL . Considering 13 types (HR-HPV and pHR-HPV 68), these were found in 60 (50%) ASCUS cases and in 159 (71%) LSIL cases . P=0.001, logistic regression), with a decreasing prevalence until the age of 50 years. A small increase was found in older women, but this was not statistically significant (P=0.19 for age2). A linear correlation between HR-HPV prevalence and age (but not age group) was also found in ASCUS cases (P=0.15). χ2-tests revealed a significant difference in HR-HPV prevalence between ASCUS and LSIL in the youngest age group (High-risk or pHR-HPV (18 types) was found in 68 (57%) ASCUS cases, compared with 181 (81%) LSIL cases . Remarkably, HPV18 was only found in 28 (9.9%) of all HPV-positive women . Human papillomavirus types 51, 52, 56, and 31 were also more common than HPV18. Human papillomavirus type 51 was equally or more common than HPV16 among women over 45 years of age. Among women with ASCUS, the most prevalent HR-HPV genotype was HPV16 (16%), followed by HPV51 and 52 (10%). Among women with LSIL, HPV16 was detected in 21%, followed by HPV56 in 11% of women .of women . Table 2P<0.001, χ2). Taking only HPV-positive samples into account, multiple infections were equally common in women with ASCUS and LSIL . The age-specific prevalence of multiple infections followed a quadratic function in LSIL cases . Among HR-HPV-positive samples, 39% (85 out of 217) were infected by more than one HR-HPV type and similar results were found for LSIL and ASCUS samples .Multiple infections were common in our material . On averP=0.21, χ2). Individuals infected with HR-HPV genotypes were somewhat less likely to be coinfected with a second HPV type . The high-risk types that were most commonly seen in coinfection with HPV16 (clade 9) were HPV39 (28%), 45 (38%), and 59 (46%), belonging to HPV18 clade 7. Interestingly, HPV18 did not occur in either HPV39 or HPV45 , but was found in 13% of HPV59 infections . The high-risk types most commonly seen in coinfection with HPV18 were HPV56 (17%), 33 (14%), and 51 (14%), which did not differ from frequencies of HPV16 coinfection . P=0.06, χ2). Human papillomavirus type 18-infected women carried genotypes from clade 9 in 43% of cases, compared with similar clade 7 genotypes in 11% .In all, there were 710 individual HPV infections. Coinfection was equally common among LR-HPV genotypes  and pHR-HPV genotypes . These findings suggest that age 30, or even younger, is a suitable cutoff point for HR-HPV triage in LSIL, when consideration is given only to the prevalence argument. This is supported by a histological follow-up study of 112 of these cases by our group, in which 48% of women over 30 years with LSIL and 60% of women with ASCUS were HR-HPV negative and none of these had a histology result of CIN2+  than in ASCUS cases (49%), which is consistent with a previous meta-analysis . The ALTViral persistence is believed to be the most important risk factor for cervical cancer. Yet, this is true only for certain oncogenic types , as postulated by As far as we know, our study is the only one to describe the prevalence of HPV genotypes in ASCUS and LSIL cases among women of all ages in Sweden. The relative frequencies of individual HR-HPV types were similar to those found during primary screening in a Swedish multicentre study comprising 5696 women aged 32–38 years (Swedescreen) (Minor cytological abnormalities associated with certain HPV genotypes may progress to cervical cancer. High-risk human papillomavirus prevalence is similar among women with ASCUS and LSIL after the age of 30 years, supporting an age limit of 30 years or even younger for triage testing in LSIL cases and no age limit in ASCUS cases, in order to improve the effectiveness of the screening programme. Human papillomavirus genotyping in longitudinal studies is needed to elucidate the importance of multiple infections for cancer progression, as well as cross-protection from vaccination.


Objective. When pathologic discrepancy arises between high-grade cytology on Papanicolaou (Pap) smear and low-grade histology on cervical biopsy, Loop Electrosurgical Excisional Procedure (LEEP) is one management alternative. Our objective was to determine whether the time from initial HGSIL Pap to LEEP affects the pathologic grade of the LEEP specimen. Study Design. We performed a retrospective case-control study identifying LEEPs performed for discrepancy over a 10-year period (1997–2007). 121 subjects were separated into two groups based on LEEP pathology  and compared using χ2. Results. Of the 121 LEEP specimens, 67 (55.4%) had CIN 2,3. CIN 2,3 was more often discovered when LEEP was performed within 3 months of the HGSIL Pap smear versus after 5 months . Conclusion. Women undergoing LEEP for discrepancy >5 months from their HGSIL Pap demonstrated a trend toward less CIN 2,3 on LEEP pathology. Women with high-grade Papanicolaou (Pap) smears have a 43%–66% risk of having moderate- to high-grade intraepithelial neoplasia CIN 2 or CIN 3) on subsequent biopsy and a 2% risk of having invasive cancer . However or CIN 3In nonadolescent patients, a loop electrosurgical excisional procedure (LEEP) is a reasonable management option for discrepancy, as there is a concern that the high-grade lesion found on Pap smear was missed on the colposcopically directed biopsy. However, we know that as many as 35% of women with HGSIL will regress spontaneously, making the excisional procedure unnecessary . Data evThis was a retrospective case-control study of all LEEP procedures done for discrepancy at the University of Washington Medical Center and Harborview Medical Center in Seattle, Washington. We identified potential cases by performing a search in the pathology database to identify all LEEP specimens between January 1, 1997 and August 31, 2007. Of note, the Dysplasia Clinics at these two medical centers serve as referral centers for women diagnosed with abnormal cytology at local clinics. Thus, not all initial Pap smears originated from these two medical centers. In the two Dysplasia Clinics, a standardized colposcopy form is used to record impression and results of colposcopy, but the Pap smear is not routinely repeated, nor do we have a complete history of prior Pap smears. Based on colposcopic findings, biopsies were performed at the discretion of the attending physician. To determine which LEEPs were done for discrepancy, we linked all LEEPs with their respective preceding Pap smear, colposcopy report, and cervical biopsy result. We included all women, 18–49 years old, who had pathologic discrepancy as defined by HGSIL Pap smear and a subsequent colposcopy with cervical biopsies of CIN 1 or less. We excluded those who had an unsatisfactory colposcopy exam, previous excisional procedure, and positive endocervical curettage and those who were pregnant or HIV positive. Once discrepant cases were identified, we performed a chart review and used a standardized data collection instrument to gather general demographic information and risk factors associated with progression of cervical neoplasia . On this form we also documented cytologic and histologic results of the Pap smear, cervical biopsy, and LEEP procedure, and recorded the length of time between the Pap smear and the LEEP. α = 0.05 to detect this difference in pathologic result, demonstrating that the study was appropriately powered to test our hypothesis.A power calculation was based on the assumption that, in LEEPs done for discrepancy, the prevalence of CIN 2 or 3 found in the LEEP specimen will be higher when the LEEP was performed closer to the time of the HGSIL Pap. We divided the time intervals from Pap smear to LEEP into three intervals: less than three months, between three and five months, and greater than five months. As this was a retrospective study, we used our data of 55% prevalence of CIN 2,3 in LEEP specimens when the time interval from the Pap smear to the LEEP is less then 3 months and 16% when the interval is greater than five months. From this, the calculated sample size was 23 women to give 80% power and t-test and χ². Univariate and multivariate logistic regressions were used to calculate odds ratios and 95% confidence intervals for the association between time since HGSIL and histologic grade of LEEP. Based on their LEEP histology, subjects were separated into two groups: CIN 1 or less and CIN 2,3. The “CIN 1 or less” group included normal, cervicitis, and CIN 1. The “CIN 2,3” group included CIN 2 and CIN 3. We chose these two groups based on clinical application, as this is the general division that determines treatment management. Statistical analysis was executed with SPSS v.16. Differences between the two groups were examined using Student's This study was approved by the International Review Board at the University of Washington, IRB Application Number 07-8885-E/A 01.Of the 1,356 patients who underwent a LEEP during this time period, 157 were performed for discrepancy. Of these, 36 women were excluded: 24 had unsatisfactory colposcopy, six had endocervical curettings positive for neoplasia, four were HIV positive, and two were older than 49. The 121 remaining patients were divided into two groups based on the pathologic grade of CIN in their LEEP specimen . As demonstrated by P = .096). Though not statistically significant, this suggests a trend toward decreased prevalence of CIN 2,3 in the LEEP specimen as the time from HGSIL Pap smear increased. In order to explore this further, a univariate logistic regression was performed to test for trend of LEEP histology over time using CIN 2,3 as the outcome  had CIN 2,3 on their LEEP pathology specimen and 54 (44.6%) had CIN 1 or less. We examined the time interval from the initial HGSIL Pap smear to the LEEP procedure in the CIN 2,3 group. Of the 67 who had CIN 2,3 on LEEP histology, 37 (55.2%) had their LEEP within three months of the initial Pap versus 11 (16.4%) who had their LEEP greater than five months after the Pap smear . Althoug outcome . In thisP = .02).Although it was not our initial intent to examine the effect of birth control method on the histologic grade of the LEEP specimens, there was a significant difference in the use of Depo-Provera between women with low-grade and moderate- or high-grade CIN . We therefore performed a multivariate regression using the covariates of time interval and birth control method to analyze the effect of birth control method on dysplasia . Birth cP = .07).Because of this significant relationship, we performed the multivariate regression in the CIN 2,3 group controlling for contraceptive method. After controlling for the birth control variable, we demonstrated a trend toward fewer CIN 2,3 LEEP findings when the procedure was performed more than five months from the HGSIL Pap, a trend that neared but did not achieve significance (OR 0.39, This study was initiated prior to the 2006 ASCCP Consensus Guidelines, which were released in October 2007. New changes announced in the guidelines included those that direct the management of abnormal cytology in adolescents of 20 years and younger. For adolescents in the discrepant situation, it is now recommended that they be observed and monitored with Pap smears and colposcopy examinations every 6 months for 24 months . ConservThe role of exogenous estradiol and progesterone in the development of cervical cancer has been discussed extensively. There is still insufficient data as to whether oral contraceptive pills are associated with the development of cervical cancer in HPV-infected women . Thomas One limitation of our study is that the University of Washington and Harborview Medical Centers are referral centers for cervical neoplasia. As many patients are referred from providers outside the system, original Pap smears are not always available to us to confirm the HGSIL nor is the Pap smear routinely repeated at the time of colposcopy. Studies demonstrate that pathologists vary in their determination of cytologic grading of Pap smears . If therAnother limitation of this study is the inherent disadvantage of inter observer variation on colposcopy examination because, in our clinics, multiple providers perform colposcopy. We know that colposcopy is imperfect and up to 33% of CIN 2,3 can be missed on a single colposcopy examination . MissingIn addition, we used medical records of LEEP pathology to identify cases of discrepancy. This may open the potential of selection bias as some cases could be missed, or inadvertently miscoded. These LEEPs were identified, though, as a pathology database, and Finally, our examination of birth control method as it affected the LEEP pathology was not adequately investigated. There are many subtle effects of birth control on cervical dysplasia. We grouped non-hormonal methods together, when in fact this may represent a heterogeneous group of women. Women who are using no method may or may not be abstaining from intercourse. Those that are abstaining may have less exposure to HPV, which could lead to a higher rate of HPV regression. In the same way, we would expect condom users to have less exposure to HPV as Winer et al. have shown that condoms prevent HPV transmission . A separTo our knowledge, this is the first study that examines the effect of time on histologic grade of CIN in the LEEP specimen when LEEP is performed for discrepancy. Research demonstrates that the neoplasia caused by HPV may regress spontaneously with time , 5. HencMany have explored the option of “See-and-Treat” for HGSIL management with immediate LEEP in recent years. The 2006 ASCCP Guidelines advocate caution and observation rather than an immediate excisional procedure in women who are still considering childbearing due to the increased pregnancy complications . We cann


TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors. Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) and in RCC in the Eker rat. Tuberin downregulates the DNA repair enzyme 8-oxoguanine DNA-glycosylase (OGG1) with important functional consequences, compromising the ability of cells to repair damaged DNA resulting in the accumulation of the mutagenic oxidized DNA, 8-oxo-dG. Loss of function mutations of OGG1 also occurs in human kidney clear cell carcinoma and may contribute to tumorgenesis. We investigated the distribution of protein expression and the activity of OGG1 and 8-oxo-dG and correlated it with the expression of tuberin in kidneys of wild type and Eker rats and tumor from Eker rat.Tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, Tuberin expression, OGG1 protein expression and activity were higher in kidney cortex than in medulla or papilla in both wild type and Eker rats. On the other hand, 8-oxo-dG levels were highest in the medulla, which expressed the lowest levels of OGG1. The basal levels of 8-oxo-dG were also higher in both cortex and medulla of Eker rats compared to wild type rats.TSC2 allele is associated with loss of OGG1 expression. Immunostaining of kidney tissue shows localization of tuberin and OGG1 mainly in the cortex.In kidney tumors from Eker rats, the loss of the second These results demonstrate that OGG1 localizes with tuberin preferentially in kidney cortex. Loss of tuberin is accompanied by the loss of OGG1 contributing to tumorgenesis. In addition, the predominant expression of OGG1 in the cortex and its decreased expression and activity in the Eker rat may account for the predominant cortical localization of renal cell carcinoma. OGG1-/- mice compared to wild-type animals [OGG1 gene is somatically mutated in some cancer cells and is highly polymorphic among humans [OGG1 allele, located on chromosome 3p25, was found in 85% of 99 human kidney clear cell carcinoma samples, identifying the loss of OGG1 function as a possible consequence of multistep carcinogenesis in the kidney [Oxidative DNA damage is one of the most common threats to genomic stability; DNA repair enzymes provide protection from the effects of oxidized DNA bases. Mutations that influence the repair of oxidized DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation -3. Many  animals . The OGGg humans ,14. Losse kidney .TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) [+/-) kidneys is lower than in wild-type rats [Tuberous sclerosis complex (TSC) is a genetic disorder associated with tumors in many organs, including renal cell carcinoma. It affects about 1 million individuals worldwide, with an estimated prevalence of up to one in 6,000 newborns . Loss ofma (RCC) ,18. The ma (RCC) ,20. The ype rats  suggestiKidney homogenates from cortex, inner medulla and papilla were subjected to western blot analysis. As expected, tuberin expression in kidney of wild type animals was higher than in Eker rats. Kidney cortex including the outer medulla shows higher tuberin and OGG1 expression compared to inner medulla and papilla. Deficiency of tuberin in Eker rats was associated with decrease in OGG1 expression compared to wild type rat in all three regions of the kidney Fig. .32P-labeled 21 oligonuleotide. OGG1 activity was higher in cortex compared to the medulla  kidneys is lower than in wild-type rats [This study is the first report that the loss of tuberin is associated with loss of OGG1 suggesting that both proteins may play a major role in development of kidney tumor in Eker rat. Our data demonstrate that tuberin deficiency is associated with deficiency of protein as well as with marked inhibition of OGG1 activity indicating that tuberin is upstream of OGG1. In addition, our data show that inhibition of OGG1 activity is associated with increased accumulation of 8-oxodG in kidneys of Eker compared to wild type rats. We have show that the constitutive expression of OGG1 in TSC2 heterozygous Eker rat , suggesting that loss of tuberin was biologically significant in affecting OGG1. Treatment of Eker rats with an oxidative DNA damaging agent increases 8-oxodG formation [In mammalian cells, the base excision pathway initiated by OGG1 represents the main defense against the mutagenic effects of 8-oxodG. However, the decrease in OGG1 expression in ormation , but theormation .OGG1 gene in mammalian cells causes a mutator phenotype, it can be expected that cells lacking OGG1 activity could have an enhanced probability of undergoing malignant transformation [OGG1 gene are nonfunctional. The human OGG1 gene is located on chromosome 3p25. Human chromosome 3p cytogenetic abnormalities and LOH have been observed at high frequency in sporadic forms of RCC. The 3p21.2-p21.3 locus is frequently deleted in RCC and also in other cancers, but to date, no candidate gene has been identified.The OGG1 protein initiates the base excision repair process by recognizing and excising the modified base. Consistent with the yeast findings, mice lacking a functional OGG1 protein accumulate abnormal levels of 8-oxoG in their genomes and display a moderately elevated spontaneous mutation rate in nonproliferative tissues . Becauseormation . The valIn summary, deficiency of tuberin is associated with loss of function of OGG1 in tumors of Eker rat. Impaired OGG1 repair enzyme activity results in the accumulation of 8-oxodG, suggesting that tuberin plays a significant role in protecting the cells from oxidative DNA damage. We propose that loss of OGG1 expression and function in tuberin-deficient tumor tissue predisposes to further genetic alterations as a result accumulation of mismatched DNA base lesions, a form of genomic instability that if left unrepaired promotes additional genetic alterations leading to the full blown tumor phenotype in kidney patients with TSC.The protein and activity levels of OGG1 were higher in cortical region than in the medullary region of wild type rat. However, OGG1 protein and activity were lower in kidney cortices and medullae of Eker compared to wild type rats. 8-Oxo-dG levels were highest in the medulla, which expressed the lowest levels of OGG1. The basal levels of 8-oxo-dG were also higher than in both cortex and medulla of Eker rats compared to wild type rats. OGG1 expression was low in kidney of Eker rat and that associated with high amount of 8-oxodG compared to wild rat suggesting that Eker rats are more susceptible to oxidative DNA damage. In addition, tumor kidney of Eker rat show null tuberin and OGG1 expression indicating that tuberin is an upstream regulator of OGG1 protein and gene expression.TSC-2+/+) and Eker male rats (mutant TSC-2+/-) were purchased from a breeding colony maintained at the University of Texas MD Anderson Cancer Center, Smithville, TX. The animals were allowed food and water ad libitum throughout the experiments. Animals were euthanized at 2 and 12 months for nephrectomy. The kidneys were dissected longitudinally, half preserved in 10% formalin in PBS, pH 7.4 for immunostaining and the remaining tissue was used for biochemical assays. Cortex, outer stripe of the outer medulla (OSOM) and papilla of the kidney were excised and frozen immediately in liquid nitrogen for biochemical analysis.Wild type  containing phenylmethylsulfonyl fluoride (10 mg/ml), leupeptin (10 mg/ml), and aprotinin (20 mg/ml). Tissue homogenates was centrifuged at 14,000 × rd assay  using bord assay  and antiLocalization of OGG1 and tuberin was assessed by immunofluorescence histochemistry as previously described . AcetoneKidney tissue was homogenated in 0.25 ml lysis buffer . The homogenate was sonicated for 8 sec at 4°C with pulses of 1 sec each between 10 sec intervals. After centrifugation  at 4°C for 15 min, the supernatant was recovered and protein content was determined by Bradford method (242). A 24-mer oligonucleotide containing the oxidized G base  , was labDNA was isolated from rat kidney tissue and detection of dG and 8-oxodG was performed on DNA hydrolyzed with nuclease P1 and alkaline phosphatase as previously described and validated . AliquotP values less than 0.05 and 0.01 were considered statistically significant.Data are presented as mean ± standard error. Statistical differences were determined using ANOVA followed by Student Dunnett's (Exp. vs. Control) test using 1 trial analysis. TSC2, tuberous sclerosis complex-2; 8-oxodG, 8-oxodeoxyguanine; OGG1, 8-oxoG-DNA glycosylase, RCC, renal cell carcinoma.The author(s) declare that they have no competing interests.SLH and HEA conceived the concept, designed the study, and prepared the manuscript. SS performed the western blotting. JB performed the immunohistochemistry and the pathological analyses of the animal tissue samples. All authors have read and approved the manuscript.


P=0.55) were not significantly different. Among the cervical intraepithelial neoplasia 3 and squamous cell carcinoma specimens, HPV 16 and HPV 52 were predominantly detected using the PCR method. Although some DNA samples extracted from the fluid-based specimens were degradaded, PCR and direct sequencing could be performed without difficulty even after 1 year of specimen storage. We conclude that fluid-based Papanicolaou specimens can be applied to investigate HPV infection.A fluid-based Papanicolaou test has been established to improve sample collection and preparation. This study was the first large-scale investigation in Japan to examine the feasibility of using fluid-based Papanicolaou specimens to detect human papillomavirus (HPV) using Hybrid Capture II and polymerase chain reaction (PCR). Three thousand patients who visited Keio University Hospital between October 2000 and February 2001 were enrolled in the study. The results of the fluid-based Papanicolaou tests corresponded well with those of conventional Papanicolaou smears (96.8% concordance). The sensitivities of cervical neoplasia detection using the fluid-based Papanicolaou test (73.9%) and Hybrid Capture II (76.3%,  The fluid-based Papanicolaou specimen collection method is widely used for primary cervical cancer screening , largelyHPV testing with PCR using fluid-based specimens, in conjunction with cytologic and biopsy follow-up, has been reported to be useful for estimating the significance of atypical squamous cells of undetermined significance , according to the manufacturer's directions. Out of the 3000 cases, colposcopy and biopsy studies were performed in 557 patients. The ThinPrep sample vials were stored at ambient temperature and used for the HPV-DNA analysis within 12 months of collection. Specimens from 477 random patients in whom biopsy studies had also been performed were subjected to HPV-DNA detection using HC II , while specimens from 146 random cases diagnosed as having cervical neoplasia on the basis of biopsy studies were subjected to HPV-DNA detection using PCR. The HC II and PCR test results were compared in 133 cases. HPV typing and sequencing analysis were performed in all the PCR-positive cases. Informed consent was obtained from each biopsy study participant after an oral explanation of the study.Thin-layer slides were prepared using the ThinPrep 2000 Automated Slide Processor , according to the manufacturer's instructions. Briefly, the vial containing the cells was placed in the processor, and a dispersion cycle homogenised the cell suspension. The cells were then automatically collected on a polycarbonate filter membrane. A thin, evenly dispersed monolayer of cells was deposited from the filter onto the slide in a circle with a diameter of 20 mm. Extraneous mucus and blood were removed in the process. The slides were then manually removed from the processor and stained using the Papanicolaou method. All slides, both conventional and ThinPrep, were screened by three cytotechnologists . The diagnostic adequacy of the fluid-based Papanicolaou specimens was compared with those of both conventional Papanicolaou smear and biopsy specimens.HPV-DNA detection was performed using the commercially available HC II hybrid capture technique. Fluid-based specimens were analysed for the presence of low-risk HPV types 6, 11, 42, 43, and 44 using a Probe A cocktail, and for high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 using a Probe B cocktail. The sensitivity of the HC II method for detecting cervical neoplasia was then compared with that of the ThinPrep slides. The enzyme-linked immunosorbent assay was based on a sandwich hybridisation followed by a nonradioactive alkaline phosphatase reaction with chemiluminescence on microplates.Approximately 10 ml of preserved fluid was centrifuged at 3000 r.p.m. for 30 min. The pellet was washed once in phosphate-buffered saline, followed by genomic DNA extraction using proteinase K and phenol–chloroform treatment. The quality and quantity of the extracted genomic DNA were monitored using ethidium bromide-stained agarose gel electrophoresis.μl volume containing 20 mM Tris HCl buffer (pH 8.0), 50 mM KCl, 0.2 mM dNTP mix, 2 mM MgCl2, 0.5 μM of each forward and reverse primer, and 0.25 U of Taq polymerase  was used for each reaction. After an initial period of denaturation at 95°C for 10 min, 43 cycles of reactions were performed, each consisting of denaturation at 95°C for 1.5 min, annealing at 48°C for 1.5 min, and extension at 70°C for 2 min. PK114/K, a variant HPV16 clone provided by Dr Mattias Durst, was used as a positive control.The presence of intracellular HPV DNA was determined using PCR analysis with consensus primer pairs  (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) using Sequencing Analysis 3.3 software .A partial L1 sequence was amplified by the primer L1C1. The amplified PCR products were purified, and automated sequencing was performed using an ABI Prism 3100 Genetic Analyser . The HPV type was determined based on an approximately 200 bases of L1 sequence and a search of the NCBI database .Cytologic studies were performed in all 3000 cases. Twelve cases of glandular dysplasia, 11 cases of adenocarcinoma, five cases of adenocarcinoma in situ, three cases of condyloma acuminatum, two cases of adenosquamous cell carcinoma, and one case of small cell carcinoma were excluded from the 557 cases. A comparison between the cytological and histological results for the remaining 541 cases is shown in P=0.12). The detection sensitivities for cervical intraepithelial neoplasia1 (CIN1), CIN2, CIN3, and SCC were 53.2, 81.5, 94.3, and 100.0%, respectively, for the ThinPrep slides, and 55.3, 83.3, 96.2, and 100.0%, respectively, for the conventional Papanicolaou smear specimens. These differences were not significant for any histological category (P>0.4). In the same manner, the CIN and SCC detection sensitivities also did not differ between the ThinPrep slides  and the conventional Papanicolaou smear specimens .Biopsy studies were performed in 557 of the 3000 cases. Based on the histological diagnosis, five cases of adenocarcinoma, five cases of adenocarcinoma P=0.56) test results.An HC II analysis was randomly performed in 477 of the 541 cases who underwent biopsy studies. Only cases positive for high-risk HPV were regarded as positive in this study; cases positive for only low-risk HPV were regarded as negative. The sensitivity of HC II for detecting CIN1, CIN2, CIN3, and SCC was 59.1% (55 out of 93 cases), 84.9% (45 out of 53 cases), 90.6% (48 out of 53 cases), and 100.0% (16 out of 16 cases), respectively. The overall CIN and SCC detection sensitivities did not differ significantly between the HC II  and the ThinPrep slide . We also found that the frequency of cases with adenocarcionoma or adenocarcinoma in situ, compared to those with CIN or SCC, in the HPV 18-positive cases  was higher than that of the other HPV types . HPV 18 was also detected in one case of the adenosquamous cell carcinoma.P<0.01).The concordance rate between the HC II and PCR test results was 68.1% (32 out of 47 cases) in cases with CIN1, 88.4% (76 out of 86 cases) in cases with CIN2, CIN3, or SCC, and 81.2% (108 out of 133 cases) overall  was lower than that in more progressive lesions (88.4%). The cause of the lower concordance rate among CIN1 cases is probably related to a low copy number of the HPV genome and a higher PCR sensitivity for the detection of HPV. In addition, HPV types that were not included in Probe B cocktail of HC II or unknown types of HPV can be detected using the PCR method.The concordance rate between HC II and PCR test results has been reported to be high (In conclusion, the fluid-based Papanicolaou test produces specimens of the same quality as those produced using conventional Papanicolaou smears. ThinPrep sample vials can be stored at ambient temperature for periods of more than 1 year; furthermore, the detection of HPV DNA can be performed using residual specimens after cytological or biopsy studies have been completed. Moreover, a follow-up protocol consisting of concurrent cytology and HPV-DNA detection can be performed in patients with a high risk of CIN or SCC. This collection system should enable significant advances in molecular epidemiology and is useful for identifying high-risk cases of cervical cancer.


Also, normal cervical smears of 114 age-matched control women were analysed. High-risk HPV DNA was detected in 37 of the 57 (65%) baseline smears of the case women, and 7 (6%) of 114 smears of the control women . The HPV positive subsequent smears and cervical cancer biopsies of the case women contained the same HPV type as was detected in the baseline smear. After cytological revision, the baseline smears of 48 case women (84%) were reclassified as abnormal, 33 (69%) of which scored high-risk HPV DNA positive. Ultimately, an undisputable normal baseline smear was found in only 10 case women. In 7 (70%) of them this smear was HPV positive, whereas only 7 (7%) of 104 revised, undisputable normal smears of control women were high-risk HPV positive . The results showed that (1) high-risk HPV presence precedes abnormal cytology in women who develop cervical cancer, and (2) high-risk HPV testing signals false-negative smears of women at risk of cervical cancer. © 2001 Cancer Research Campaign


Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at −70°C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast. It is accepted that human papillomavirus (HPV) types 16 and 18 are carcinogenic, and that probably HPV types 31 and 33 are also carcinogenic in human cervical and anogenital cancers . The susHuman papilloma virus 16 has been identified in breast tumours in Italian women and breast tumours in Norwegian women who had previous cervical neoplasia (The aims of our investigation are to determine: (1) if HPV types 16, 18 and 33 gene sequences  are present in breast cancers that have occurred in predominantly Caucasian Australian women, and (2) if there are any correlations between the presence of HPV sequences and grade of tumour, patient mortality, hormone receptor status, abnormal p53 protein expression, the presence of p53 mutations and ERB-2 expression.OC from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. The DNA samples were amplified twice per sample using GenomiPhi™ DNA Amplification Kit (Amersham Biosciences). The DNA quality was confirmed by PCR, amplifying 268 bp of the β-globin gene. These samples were then screened for the presence of HPV by PCR using primers that could detect 140 bp in the E6 region of HPV 16, 18 and 33  of the 50 samples were HPV positive. Sequencing of 18 unselected samples showed them to be variants of HPV 18 . In each separate PCR the percentage of breast cancer DNA samples that was HPV positive ranged from 2 to 32%. An added amplification of DNA before PCR and use of SYBR Green I for detection improved viral detection. As shown in As shown in These results confirm the presence of HPV 18 gene sequences in human breast tumours. The identification of such sequences proved to be difficult and required additional amplification of DNA before PCR and use of SYBR Green I to optimise the detection. These technical difficulties may account for the negative results obtained in several other studies (reviewed in three).We did not identify HPV types 16 and 33 in these breast tumours. A plausible explanation is that HPV 18 may be present in breast tumours in predominantly Caucasian populations and HPV 33 in breast tumours that occur in predominantly Chinese and Japanese populations (Five variants of HPV 18 were identified. Variations in the gene sequences indicate that these were not contaminants. Sequence variations were observed in the Sp1-binding site, E2 protein-binding site number 2, between E2 protein-binding site number 1 and 2, a region 12 bp upstream of the first starting codon, and within the translated E6 sequence. As far as we know, these variations are novel and no one has yet studied the effect of these variations on the expression of HPV genes. However, it is anticipated that these variations would not have an impact on the HPV gene expression. This is because those variations do not alter the SP1 and E2 protein-binding motifs, and the base pair change does not alter the E6 amino-acid sequence.The role of HPV in breast cancer development is not elucidated. As in other studies, the presence of HPV sequences in breast tumour samples is not associated with tumour grade, patient mortality, expression of ER, PR, ERB-2, p53 expression and mutation. Hence, it is not clear if HPV promotes cancer development or the presence of HPV sequences represents a parasitic infection independent of cancer. However, given that HPVs are proven human oncoviruses in cervical cancer, it is unlikely that they act as parasitic infections in human breast tumours.Human papilloma viruses are mainly transmitted by cell surface contact. Therefore, how are HPVs transmitted to the breast? A suggestive clue comes from the observation by In conclusion, our studies demonstrate the presence of HPV 18 variants in Australian breast cancer specimens. Further studies are required to elucidate the role and pathogenesis of HPV in breast cancer.


Ninety-four per cent of the scrapes had one or more of these virus types and 66% were HPV16-positive. HPV18 was more frequent in adenocarcinoma. No evidence was found for an increased cancer risk associated with the HPV16 E6 350G variant. The HLA DRB1* and DQB1* alleles in these women and in 155 women with normal cytology and negative for HPVhrDNA were compared. DQB1*0301 alone (2P = 0.02) and in combination with DRB1*0401 (2P = 0.02) was found to be associated with cervical cancer. This was more marked in cancers positive for HPV types other than HPV16. In contrast, DRB1*1501 alone and in combination with DQB1*0602 was not significantly elevated in cancers overall, but did show some excess in HPV16-positive cancers (2P = 0.05), associated with HPV16-positive cervical cancers. Taking all cancers together, a marginally significant protective effect was found for DQB1*0501 (2P = 0.03) but no protective effect could be seen for DRB1*1301. © 2000 Cancer Research CampaignCervical scrapes from 116 British women referred with cervical cancer were tested for the presence of high oncogenic risk human papillomavirus (HPV) genotypes (HPV


Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have potentially important implications for HPV control. Human papillomaviruses (HPV), which comprise more than 100 genotypes, are the most prevalent sexually transmitted infection and are associated with multiple human cancers including all cervical cancers, many other anogenital cancers, and 25% of head and neck cancers. The HPV life cycle is closely linked to epithelial differentiation of skin keratinocytes, with initial infection occurring only in the undifferentiated proliferating basal compartment of the epithelium and progeny virus production only in the terminally differentiated suprabasal compartment. So far, little is known about how host cells restrict the HPV life cycle to specific stages of skin cell development. Here, by identifying small molecule inhibitors of HPV infection, we discovered that cell cycle progression through mitosis is critical for the establishment of HPV infection. In addition, our further chemical genetic dissection of this process showed that early steps of mitosis are required for HPV infection and early gene expression. Our findings provide one reason why HPV only infects undifferentiated proliferating cells and provide new leads for the development of preventive and therapeutic strategies against HPV infection. Papillomaviruses (HPV) are small, non-enveloped, double stranded DNA viruses that infect the cutaneous and/or mucosal epithelium in many vertebrates The HPV life cycle is tightly linked to the differentiation of its host epithelial cells The tight link between keratinocyte differentiation and the HPV life cycle has made large scale production of mature infectious HPV particles difficult, greatly restricting studies of the mechanisms of natural HPV infection To define the pathways and mechanisms involved in these early steps of HPV infection, we tested approximately 5,000 bioactive compounds with known mechanisms of action for effects on the entry of HPV capsids containing reporter genes or the full HPV genome, and identified a subset of cell cycle inhibitors that completely blocked wild type HPV infection. Our further studies showed that cell cycle progression through early stages of mitosis is critical for successful HPV infection. These findings reveal new insights into the mechanism by which HPV infects cells and provide one reason why HPV infects only undifferentiated, proliferating cells. These results also provide new leads for developing preventive and therapeutic strategies against HPV infection.hpvSEAP). High activity effectors identified in this assay were retested in a second infectivity assay using HPV pseudovirions containing renilla luciferase driven by the natural HPV16 promoter (hpv16wpA-RL) to exclude false positive compounds that may selectively affect the SV40 promoter or alkaline phosphatase (data not shown). This screening assay was performed in human kidney 293T cells owing to the high signal to noise ration, and resulting high z scores obtained in this cell line. Secondary screens performed in HaCaT cells, an immortalized line of human keratinocytes, which are natural host cells for HPV infection, confirmed the validity of the hits, as indicated below.To identify mechanistic pathways and informative modulators of HPV infection, we tested nearly 5,000 compounds in known bioactive molecule libraries from Prestwick  and LOPAC , using HPV pseudovirions containing an SV40 promoter-driven secreted alkaline phosphatase (SEAP) reporter gene  is critical for early HPV infection, we synchronized HaCaT cells with aphidicolin or etoposide and then tightly controlled subsequent cell cycle progression. In the first set of experiments  breakdown, which is triggered by a signal cascade that involves polo I like kinase 1 (plk1) and cyclin B1-associated CDK1 Another notable cellular event in early mitosis is the reorganization of cytoplasmic microtubules to support mitotic spindle formation, chromosome segregation, and cell division In addition, restructuring of PML oncogenic bodies (PODs) and chromatin in early mitosis could be necessary for establishing HPV infection in the nucleus. PODs, also known as ND10, are subnuclear bodies implicated in multiple cellular functions including transcription, DNA repair, viral defense, stress, cell cycle regulation, proteolysis and apoptosis Many genes such as polo-like kinases and aurora kinases are exclusively expressed in early mitosis to certify completion of DNA replication, overcome mitotic checkpoint, and initiate mitosis in vivo models such as mouse and non-human primate. In addition, real-time particle imaging studies could be employed to identify what specific steps in infection are blocked upon cell cycle arrest; however, the ability to interpret accurately such imaging studies rests on the ability to distinguish particles leading to infectious events from the vast majority of particles that give rise to abortive or non-infections events. To date no imaging method exists able to make such a distinction at the individual particle level.HPV is thought to be able to infect the proliferating basal layer of stratified epithelium only when wounds allow HPV to penetrate the physical barrier of upper skin layers. Wounding also can provide HPV particles access to laminin 5 or heparin sulfate moieties, both components of the extra cellular matrix (ECM) component of the epithelial basement membrane for which HPV particles have high affinity, thereby localizing the virus particles to basal surface of epithelial cells it needs to infect in vivo mouse model of HPV infection in mouse reproductive organs The recent development of HPV vaccines offers an extremely important avenue for control of HPV infections and their associated cancers pEF399, containing the complete W12E HPV16 genome Human embryonic kidney cell line 293T from ATCC, and its enhanced SV40 T antigen-expressing daughter cell line 293TT HPV virions and pseudovirions were prepared as described previously Known Bioactive Library (KBA01) consists of 3 commercially available collections totaling 4,160 compounds. KBA01 consists of 880 high purity compounds of known safety and bioavailability in humans of which over 85% are marketed drugs from Prestwick Chemical. The Prestwick compounds cover several therapeutic areas including neuropsychiatry, cardiology, immunology, inflammation, analgesia, etc. Also included in the KBA01 library is 2000 diverse FDA approved drugs and natural products from Spectrum Chemical Collection From Microscource Discovery Systems, Inc. and 1280 compounds from the LOPAC of Sigma representing marketed drugs, failed development candidates and “gold standards” that have well-characterized activities. These compounds are the results of lead optimization efforts and have been rationally designed by structure-activity relationship studies. The NCI03 library consists of 235 natural products that were selected from the NCI open repository on the basis of structural diversity and availability of compound. Etoposide, Aphidicolin, Monastrol, 5-Fluorouracil, CGP-74514A were purchased from Sigma.hpvSEAP at 50 vge /cell for 48 hrs. Culture supernatant 2.5 µl was used for virus infectivity assay using a Phospha-Light a chemiluminescent alkaline phosphatase assay (Applied Biosystems) and cell lysate was used for cell viability assay using CellTiter-Glo Luminescent cell viability assay (Promega). Biomek FX (Beckman Coulter) was used for automated liquid handling and a Victor 3-V plate reader (Perkin Elmer) was used for measuring luminescence. Screening was performed in the Small Molecule Screening Facility at the University of Wisconsin Comprehensive Cancer Center.293TT cells in 384-well plates were treated with 3.3 µM of library compounds for 4 hrs and inoculated with hpvSEAP) was measured using a Phospha-Light a chemiluminescent alkaline phosphatase assay (Applied Biosystems) and the activity of pHPV16wpA-RL-encapsidating pseudovirions (hpv16wpA-RL) was assayed using Renilla Luciferase Assay System (Promega). Infectivity of wild type HPV16 virions was examined by quantitative reverse transcriptase PCR (qRT-PCR) by amplifying viral mRNA signals from HPV16-treated 293TT cells. 293TT cells were inoculated with 50–100 vge/cell of packaged virus and incubated for 48 h at 37°C. Total RNA was isolated using the RNeasy total RNA purification kit (Qiagen), treated with RQ DNaseI (Promega) to remove possible DNA contaminants, purified again on RNeasy columns to remove DNaseI, and quantified by spectrophotometer. cDNA was synthesized from 20 µg of total RNA with oligo (dT) using a SuperScript cDNA synthesis kit (Invitrogen), and qPCR was performed with QuantiTect SYBR Green PCR Kit (Qiagen). Oligonucleotide primers  incorporation method. Briefly, HaCaT cells were harvest, homogenized, fixed with cold 70% ethanol for 30 min at −20°C, and incubated with PI staining solution  for 30 min at room temperature. Stained cells were filtered through 35 µm-pore nylon mesh cell filtering caps  and analyzed by flow cytometry using Becton Dickinson FACSCalibur (488 nm laser excitation).Figure S1Selected cell cycle inhibitors abrogate HPV infection. HPV16 early gene (A) and β-actin expression (B) was measured by quantitative RT-PCR. 293T cells were inoculated with wild type HPV16 after 4 hr pre-treatment with 3 µM of each compound or with 1∶100 dilution of neutralizing antibody (H16.7E). Total RNA was extracted after 48 hrs, and expression levels of HPV16 RNA transcripts were measured by qRT-PCR using E7, E2, and E5 sequence specific primers . The exp(0.12 MB PDF)Click here for additional data file.Figure S2Cell cycle progression through M phase is critical for HPV infection. 293T cells were synchronized with aphidicolin  and etoposide (set 3) 4 hrs before wild type HPV16 inoculation. Cell cycle progression was released in set 1 and kept blocked in sets 2 and 3 using aphidicolin and etoposide, respectively. In set 4, cell cycle progressed through S phase but arrested at G2 phase by switching the compounds in medium from aphidicolin to etoposide. In set 5, cell cycle was released for 20 hrs to progress through M phase and arrest at G2 phase by etoposide addition. Wild type HPV16 gene expression was measured from total RNA extracts as indicated in (0.06 MB PDF)Click here for additional data file.Figure S3Quantitative PCR primers of HPV16 early genes. (A) Oligonucleotide primers were designed using the Primer3 primer design program. (B) The HPV genomic position of each primer is indicated by an arrow.(0.01 MB PDF)Click here for additional data file.


Human papillomavirus (HPV) causes cervical cancer and external genital warts. The purpose of this study is to document the genotype distribution of HPV in females aged between 18 and 34 who self-referred to an STI clinic with visible external genital warts (EGW). Scrapings were taken from visible external genital warts (EGW). These scrapings were analysed by PCR for the presence of HPV DNA. Positive samples were then genotyped by means of a commercially available assay (LiPA). A comparison of genotyping results determined by the LiPA assay and direct amplicon DNA sequencing was also performed.Ninety-two patients out of 105 samples (88%) had detectable levels of HPV DNA. The majority of individuals with EGW (66%) showed the presence of two or more genotypes. The most common HPV genotypes present in the study population were HPV-6, HPV-11, HPV-16, HPV-18, HPV-33 and HPV-53. Potential effects of vaccination on HPV molecular epidemiology indicate that 40% of the patients could have been protected from the high risk genotypes HPV-16 and HPV-18.This is the first report of the molecular epidemiology of external genital warts in women aged between 18 and 34 from Ireland based on results from a LiPA assay. The study shows that most individuals are infected with multiple genotypes including those with high oncogenic potential and that the newly available HPV vaccines could have a significant impact on prevalence of the most common HPV genotypes in this study population. Human papillomavirus (HPV) is a group of non-enveloped DNA viruses, which cause benign and malignant epithelial lesions . Over a An exciting advancement in cervical cancer prevention is the recent development of two HPV vaccines. A quadrivalent vaccine  against the four most medically significant HPV genotypes (6/11 and 16/18), which cause 90% of external genital warts and 70% of cervical cancers respectively was approved by the Food and Drug Administration (FDA) in June 2006 . As thesThe molecular epidemiology of external genital warts [EGWs] in Irish females has not been studied but likely reflects that of other European countries. The prevalence and nature of the mixed genotype infections in this population is also unknown. PCR combined with reverse line probe hybridisation (RLPH) is a fast and sensitive method of genotyping viral infections. The routine use of these assays allows a more thorough analysis of HPV molecular epidemiology and assessment of likely cervical cancer risk. The study group presented here was collated through a voluntary screening program for females, aged between 18 and 34, who self presented to an STI clinic in Southern Ireland. Scrapings from EGW were taken and tested by PCR for HPV DNA; positive samples were genotyped using a commercially available RLPH assay. The DNA sequence of the preliminary amplicon was also determined for the first 52 patients. The results of this screening programme provide information on the frequency of sexually transmitted HPV strains circulating in a sample of the Irish population.To determine the presence of HPV, DNA from 114 consecutive samples was applied to a SYBR Green I assay utilising MY09-MY-11 degenerate primers. To ascertain the sensitivity of the assay a serial dilution of genomic DNA known to have integrated HPV-16 viral DNA was performed (CaSki HTC98018). The assay was capable of detecting 30 copies of HPV DNA per amplification. 92 samples were found to be positive by HPV PCR using this method. Nine of the 114 samples were blinded negative controls. Therefore, taking the blinded controls into account, 92 out of 105 samples were HPV positive.In total, 21 different genotypes were recorded; 6, 11, 16, 18, 31, 33, 35, 39, 40, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 70 and 74. These genotypes account for approximately 90% of the 17 genotypes identified as high risk or probable high risk for cervical cancer according [52 of the 92 HPV positive samples were genotyped by direct DNA sequencing of the positive PCR product. 72% of samples were identified as having either genotypes 6 or 11; 29 samples were positive for HPV-6, 13 samples were positive for HPV-11. Only 2 samples were positive by DNA sequence analysis for HPV-16. An additional 2 samples were positive for genotype 18. Other genotypes detected were 33, 39, and 53 at a frequency of 3/52, 1/52 and 2/52, respectively.The primary aim of this study was to ascertain for the first time the molecular epidemiology of sexually transmitted HPV in EGW. This genotype information should assist in estimating the potential benefits of the newly available HPV vaccines and highlight the importance of multiple infections.et al only two relatively uncommon genotypes, i.e., 82 and 26, were not identified in the study population [DNA was successfully extracted from a limited scraping of externally visible EGW using an automated DNA extraction system. The potential falsenegative rate for this study was 12% 13/105). A possible explanation is that insufficient DNA was present to generate an amplicon or the MY09-11 primer set, used in the initial screening for HPV positivity, may be sub-optimal for specific genotypes. With regard to this latter argument, it should be noted that a total of 15 out of a possible 17 high or probably high risk genotypes were detected with this methodological approach. Of the panel of high or probably high risk genotypes proposed by Meyer . A possiet al., 2008 questioned the validity of using PCR and sequencing to determine HPV genotypes especially those associated with cervical cancer [The combination of the real-time and automated DNA extraction allowed for a fast sample throughput (t = 5 hours). In 100% of cases (n = 52) DNA sequencing revealed an equivalent genotype when compared to the LiPA assay. Nevertheless DNA sequencing using consensus primers doesn't readily enable the detection of multiple infections . DNA seql cancer . Our resThe combination of real-time PCR and LiPA assay identified low risk genotypes 6 and 11 or both in 88% of EGWs tested. This trend is evident in HPV infections worldwide as 90% of all genital warts are related to types 6 and 11 . In spitHPV-16, HPV-18 and HPV-31 which account for 75% of cervical cancers . The curThe optimum time for vaccination with the new HPV vaccine (subject to the duration of immune protection) is recognised to be at the onset of puberty . Both thThis study identifies for the first time the molecular epidemiology of HPV in the EGWs of young Irish women. The results of the study found that the majority of EGWs contained the low risk genotypes 6 and 11. Despite this, a large number 70% of the patients were co-infected with one or more high risk genotypes. The new HPV vaccines would have prevented a significant number of these patients from contracting HPV or provided protection against the most oncogenic HPV genotypes 16, 18 and 31. It is to be presumed that with sufficient usage of these vaccines that genotype replacement will become a feature of the epidemiology of HPV infection.The study outlined here was approved by the Clinical Research Ethics Committees of the Cork teaching Hospitals and University College Cork, Ireland. Participation within the study was voluntary and patients gave informed consent before entry into the study.Between October 2006 and October 2008 samples were taken from 114 females who self referred to an STI clinic in Cork, Ireland. The age of the patients ranged from 19–34 years of age with an average age of 24.Scrapings were taken from visible external genital warts of the patients using small brushes. The brushes were stored at room temperature in 500 μl of Lysis Binding buffer (LBB) from the ROCHE™ MagNa Pure LC Total Nucleic Acid isolation Kit. Samples were received from the clinician in a blinded manner. 9 random negative samples were included and the knowledge of these and individual patient status was kept hidden from the diagnostic laboratory for the duration of the investigation. Subsequent to study completion sample identities were revealed.300 μl of sample HPV-LBB was added to 200 μl of nuclease free water and applied to the MagNa Pure LC Total Nucleic acid isolation Kit using the Roche Magna Pure Robotic extraction machine, yielding 50 μl of extracted DNA. A negative control consisting of water was included with every extraction.HPV positive samples were detected by real time PCR using My09-MY-11 generic primers  and SYBRTo ascertain the sensitivity of the assay serial dilutions of commercially available genomic DNA from the CaSki cell line (HTC98018) containing 600 copies of HPV16/genome were generated. A standard curve was created where DNA from the CaSki cell line with a range of 300,000, serially diluted by a factor of 10, to a final concentration of 3 copies per PCR. The dilutions are then applied in 5 μl volumes to the Real-time assay described above to create a standard curve.Positive samples were amplified using Biotin labelled primers provided in the INNOGENTICS HPV genotyping assay . A no template control was performed with every biotinylated PCR to ensure no cross contamination was occurring during pre-amplification stage or during the automated process. 15 μl of each biotin labelled PCR product including the negative control was then applied to a disposable line probe strip provided and placed in an AuoLipA 30 and Reverse Line Probe Hybridisation was performed at an annealing temperature of 49°C using the HCVV3 programme. Interpretation of the genotype of the positive samples was performed using the key provided with the kit. The kit can detect the following genotypes 6, 11, 16, 18, 31, 33, 40, 45, 51, 53, 54, 58, 59, 66, 68, 70 (n = 16). In cases where the CE marked LiPA assay could not resolve the exact genotype of HPV present, the sample was applied to a second LiPA assay (version 2). The HPV LiPA version 2 assay can detect the following genotypes 6, 11, 16, 18, 31/54, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 56/74, 58, 59, 66, 68/73, 70 (n = 24). The HPV LiPA Extra assay became available towards the end of the study. This assay covers all currently known high-risk HPV genotypes and probable high-risk HPV genotypes  as well as a number of low-risk HPV genotypes  and some additional types .All these assays carry a CE mark which means the assays meet European union stringent quality controls in terms of sensitivity and reproducibility. Where confounding genotype heterozygosity was observed the potential genotypes were excluded from the analysis for this individual. Where the CE LiPA assay could not resolve the genotype present the sample was applied to the LiPA V2 assay or the LiPA extra assay.. All sequences were submitted to the Genbank database and accession numbers were acquired for this study (EU056593–EU056644).The PCR amplicons of the first 60 positive samples were purified using the High Pure PCR product purification kit (Roche) and air dried PCR products were outsourced to MWG  for DNA sequencing. Sequencing was performed using the MY-11 primer. Fifty-two samples were successfully sequenced and these were analysed by submitting the obtained DNA sequence to the BLASTn programme available at Genbank. HPV: Human papillomavirus; EGW: external genital wart; PCR: Polymerase chain reaction; RLPH: Reverse Line Probe Hybridisation.The authors declare that they have no competing interests.JFM performed DNA extractions, PCR testing, genotype determination, DNA sequence analysis and manuscript preparation. SC collected samples and patient details. LC performed some PCR testing and genotype determination. MH was involved with project design and manuscript preparation. LJF was involved in project design, project management and manuscript preparation.


Following the demonstration of the superior validity of human papillomavirus (HPV) tests in screening for cervical cancer and the arrival of highly efficacious HPV 16 and 18 vaccines, cervical cancer prevention enters a time of sustainable introduction in developing countries. Multidisciplinary efforts and novel protocols are being developed, and challenging situations are being faced to make cervical cancer, still the number two cancer in women worldwide, an eradicable condition. British Journal of Cancer could have at first sight, a quick and intuitive answer: we will need both. We need human papillomavirus (HPV) vaccines to significantly reduce the health care burden currently required for cervical cancer prevention, and we need screening because of the limitations of current HPV vaccines both in their lack of therapeutic effect (thus not protecting women with an ongoing neoplastic processes) and in their limited number of HPV types . However, the answer only applies in scenarios in which screening is already developed and reasonably efficient. In populations without adequate screening, one could equally argue that HPV vaccines at affordable prices are the only realistic option. While these arrive, more efficient screening schemes, for example with low-cost HPV tests, requiring fewer visits or strategies involving rapid intervention like ‘screen and treat’ protocols, remain the only option for currently living adult women. Moreover, should polyvalent vaccines (including some five–eight HPV types) result in extending protection against more than 90%+ of the oncogenic HPV types, vaccination alone would be the answer for both scenarios. Thus, a complex answer to an apparent straightforward question.The provocative title proposed by the editors of the With the publication of the key short-term results of the two major Phase III trials of HPV vaccines, the perspective of tackling cervical cancer prevention with vaccination has been unambiguously open. While recognising the limitations of the still moderate (5–6 years) follow-up in a few tens of thousand young women, two vaccines to date have shown high efficacy, safety, immunogenicity, long-term duration of protection and a strong suggestions of induction of immune memory .A number of clinically relevant issues remain to be fully described, including the magnitude and the HPV spectrum included in the cross protection effect, and the long-term effects of each of the HPV vaccines on cancer protection and safety. However, to solve these questions, it is unavoidable to continue the studies for additional follow-up time and the organisation of large Phase IV studies, some of which are already in place.The currently available vaccines offer full protection to HPV 16- and 18-naïve women for these two HPV types that cause an estimated 70% of cervical cancer and a slightly lower fraction of its precursors. Once the cross protection impact is fully described and the geographical variation of the HPV types in cervical cancer is better known, these estimates will likely increase in some areas to perhaps 75–80%. Human papillomavirus 16 and 18 account for a higher proportion of cervical adenocarcinomas, in the range of 80–85%  at recruitment and a follow-up time of 15 months.http://www.path.org/news/).On a worldwide scale, the centre of gravity of cervical cancer prevention has shifted towards a more established mindset in the field of vaccinology such as ensuring production and access to HPV vaccines and devising the logistics to ensure wide coverage of the target populations. The Expanded Program of Immunization (EPI) is probably one of the most successful public health efforts in place. vs oral), number of doses required (as well as antigen amount per dose), immune response and duration of protection when administered in these age groups, and tolerability and safety when administered with other EPI vaccines. The need and the opportunity of a booster dose latter in life  should also be evaluated.Public health researchers thus have an important task ahead, namely to organise the trials that will guide how to best evaluate and, if appropriate, to integrate HPV vaccines within the structure of the EPI. These will certainly take time and should address issues such as routes of administration  could be considered in some countries with adequate infrastructure.The population of women in the world continues to increase. The number of women in any 1-year age cohort aged between 10 and 14 years has been estimated to be close to 60 million . Of thesHuman papillomavirus vaccines have a cost in 2007 that exceeds the current possibilities of many countries. It is thus anticipated that for some time after introduction, access to vaccination will also reflect the different opportunities related to socioeconomic status. Previous experience with the introduction of the hepatitis B virus vaccine in developing countries has documented that vaccine cost is an essential component of a successful introduction and a determinant of the time to introduction in may parts of the world  repeated at regular intervals has not been sustainable, and active research is in place to evaluate screening alternatives  at the time of screening. Under this concept, a woman aged 30–35 years or above found positive for a high-risk HPV DNA and normal in the cytological examination would be labelled as a  false positive, weighing against specificity. Longitudinal studies have shown, however, that these cases (might be as high as 10% of screening participants) particularly if HPV DNA persists are indeed at higher risk of progression to CIN 2+ within the next decade as compared to women who show normal cytology and are HPV negative , self-sampling strategies for HPV DNA, reduction of screening protocols to two-step (screen and treat) rather than three-step , and development of lower cost, lower technology screening tests for HPV is ongoing, and results have already been reported. In Peru, comparisons were made between conventional cytology, liquid-based cytology, VIA and HPV testing (laboratories for HPV tests were out of the country). The study concluded that a strategy involving HPV testing as the primary screening test was the best option, provided that local infrastructure is developed and new HPV testing methods that result in lower cost are used  and of women exposed to both 16 and 18 is less that 1%. The lifetime cumulative exposure to any of these two viral types is in the order of 20% in high-risk populations. Therefore, the vast majority of women remain susceptible to new infections throughout their sexually active life. Vaccines including HPV 6 and 11 will further increase their attraction for the prevention of genital warts (GWs) in sexually active women and possibly among men as well , and, therefore, one could assume that protection is consistent in the 15–26 years range . The FutThe vaccines are immunogenic in a wide age range. Bridging studies have shown that vaccinating women in the age groups 26 years and above induces universal antibody response and, although the antibody titres are significantly lower as women age, they remain at a significantly higher level than the titres induced by natural infection; moreover, more indication of immune memory in the quadrivalent vaccine trial has been reported  of HPV DNA. The interest of these new protocols, in which the advantages of an HPV test over cytology in terms of sensitivity and positive predictive value are fully recognised, is further enhanced by the understanding that after eliminating HPV 16 and 18 from the spectrum of HPV infections and related lesions in vaccinated adolescents, the performance of the cytology as screening test will be further reduced , primary prevention of cervical lesions has significant clinical and human advantages over secondary prevention, because it avoids most of the uncertainties and unwanted side effects of cytology-based screening programmes. These include the requirement of frequent and repeated visits of millions of women with a normal cervix, the 4–5% uncertain cytological results (ASCUS and others) requiring additional diagnostic protocols, the often required biopsies and the eventual surgical treatments for conditions that otherwise would regress spontaneously in a sizeable proportion . The latter, a direct consequence of some of the most recent classifications of cervical lesions , has repeatedly been linked to unwanted obstetrics consequences (One of the two vaccines available includes HPV 6 and 11 antigens and has shown high efficacy against external GWs (The tools and the momentum seem now ready to redesign novel strategies for cervical cancer prevention in developed and developing countries and populations. The axes of action seem to call for efforts in at least four directions:Both professional and public need to renew and increase awareness on the burden of disease and the new opportunities for prevention. Educational messages need to understand the complexities of a vaccine against a sexually transmitted infection, and the cultural specificities that have to be considered to ensure acceptability and high uptake.Continue the work initiated by a number of institutions and funding agencies to ensure that support is being directed to make full use of these new opportunities. Cervical cancer might not be regarded as a social priority in some countries if compared to other vaccines or therapies for the prevention of more prevalent conditions such as malaria or AIDS. However, excellent HPV vaccines are the feasible option now and all efforts have to be made to integrate them into the vaccine delivery schemes.The arrival of a new vaccine is a complex interdisciplinary exercise requiring different social abilities and expertise. Multiple partial initiatives are under way, and coherence in the messaging and programme development should be viewed as an advantage. This refers to the management of information, addressing research issues and deploying the network and the logistics to reach women in the world.The qualitative new protocols for cervical cancer prevention after the seminal work of Papanicolaou are now being written, taking full advantage of the HPV-based technology for screening and vaccination. The deployment of such protocols has the potential to complete Papanicolaou's goal of cervical cancer eradication by extending the benefits of prevention to the developing populations of the world.