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Gudlaugsson E, Skaland I, Janssen E A M, Smaaland R, Shao Z, Malpica A, Voorhorst F & Baak J P A (2012) Histopathology Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer Aims: The proliferation factor Ki67 is prognostic in breast cancer and included in international therapy guidelines, but measurement procedures differ between laboratories. We compared the reproducibility and prognostic value of different Ki67 sampling and measurement methods. Methods and results: In 237 T1,2N0M0 breast cancers without adjuvant systemic treatment, strictly standardized section thickness, automated antigen retrieval and immunohistochemistry were used. The percentages of Ki67-positive nuclei were assessed using (i) a ‘quick-scan rapid estimate’, (ii) ocular-square-guided counts by independent pathologists, (iii) computerized point-grid-sampling interactive morphometry (CIM) and (iv) automated digital image analysis (DIA). Quick-scan rapid estimates were poorly reproducible. The optimal prognostic thresholds of Ki67 counts by two pathologists differed greatly (4%, 14%; kappa: 0.36), with many therapeutic differences. CIM-Ki67 and DIA-Ki67 were strongly prognostic (P

Purpose In breast cancer, different tools are used for prognostication and adjuvant systemic therapy selection. We compared the accuracy of the online program Adjuvant!, the Norwegian Breast Cancer Group (NBCG) guidelines, and the proliferation factor mitotic activity index (MAI) in patients with lymph node (LN) –negative disease (pN0). Patients and Methods Adjuvant! and MAI thresholds were set to 90% to 95% breast cancer–specific survival (BCSS) rates. These thresholds were 95% for Adjuvant!, 3 for MAI, and as follows for NBCG: pT1 grade 1 + pT1a-b grade 2 to 3; all pN0M0 and estrogen receptor/progesterone receptor positive versus all others. In 516 patients younger than age 55 years (T1-3N0M0) without adjuvant systemic therapy, univariable and multivariable 10-year BCSS rates were estimated. Results Median follow-up time was 118 months. The concordance between MAI and Adjuvant! or NBCG was fair (κ = 0.35 and κ = 0.29, respectively). Adjuvant!, NBCG, and MAI were all prognostically significant (P ≤ .001). In the univariable analysis, the 10-year BCSS of MAI less than 3 versus ≥ 3 was 95% v 71%, respectively, with a hazard ratio of 7.0. In multivariable analysis, MAI was superior to Adjuvant! and NBCG. The 10-year survival of Adjuvant! ≥ 95% versus less than 95% was 91% v 74%, respectively, but stratification by MAI identified subgroups with different prognosis. Similar results occurred for NBCG and MAI. Adjuvant! and NBCG were not prognostic to each other. Conclusion MAI is superior to Adjuvant! and NBCG in prognostication of patients with LN-negative breast cancer younger than age 55 years.

Introduction: High-risk human papillomavirus (hrHPV) DNA testing is an increasingly used instrument in cervical cancer prevention along cervical cytology. The inclusion of hrHPV testing in cervical screening requires efficient management as many hrHPV infections are transient. We investigated the potential value of hrHPV genotyping in normal and borderline/mildly dyskaryotic (BMD) smears. Materials and Methods: From a screening population of 44,102 women in the Netherlands, we included hrHPV-positive women with a normal or BMD smear. We assessed the type-specific 18-month risk of high-grade cervical intraepithelial neoplasia (CIN). Results: In hrHPV-positive women, 18-month risk of CIN grade 3 or invasive cancer (≥CIN3) was 6% [95% confidence interval (95% CI), 4-9] after normal cytology and 20% (95% CI, 16-25) after BMD. If positive for HPV16, ≥CIN3 risks were 14% (95% CI, 9-21) and 37% (95% CI, 28-48), respectively. In the subset of hrHPV-positive women without HPV16, HPV18 was associated with an increased risk of high-grade CIN after normal cytology and HPV31 and HPV33 were associated with an increased risk, particularly after BMD. HPV16 and HPV18 were also associated with an increased risk of high-grade CIN in women with an hrHPV-positive normal baseline smear and a repeat normal smear at 6 months. Discussion: HrHPV-positive women without type 16, 18, 31, or 33 had a relatively low risk of high-grade CIN. Among women with baseline normal cytology and among women with a baseline and repeat normal smear, HPV16/18–positive women showed an increased risk of high-grade CIN. This warrants more aggressive management of HPV16/18–positive women compared with other hrHPV-positive women. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1268–73)

Background: Adjuvant systemic chemotherapy (ASCT) in lymph node-negative breast (LN−) cancers improves survival. The majority of (LN−) patients receive ASCT when the St. Gallen criteria or its modifications are used, as accurate identifiers which patients benefit from ASCT are lacking. This may imply over-treatment in many patients. Aim: To evaluate which patients or primary tumor factors predict ASCT success. Material and method: Retrospective analysis by single and multivariate survival analysis of clinical and tumor characteristics in (LN−) breast cancers n = 125) or-not (n = 516). Results: The two patient groups did not differ in age, tumor diameter, grade, type, number of mitoses and other factors. Fourteen-year survival for the ASCT and non-ASCT patients was 83% and 74% (Hazard Ratio = HR = 0.33; p < 0.0001, 9% absolute = 12% relative difference). Subgroup analysis showed that the recurrence-free survival = RFS of ASCT treated vs. non-treated patients differed in patients with grade 1 cancers (p = 0.008), grade 2 cancers (p = 0.004), grades 3 (p = 0.02), tumors under and ≧2 cm (p = 0.001 and 0.0002), oestrogen receptor-positive or -negative tumors (p = 0.003, 0.04), MAI < 10 and ≧10 (p = 0.005, 0.003) and fibrotic focus absent (p = 0.002). With multivariate analysis the most important predictor of ASCT effect was the MAI. In patients with slowly proliferating tumors (MAI < 3) no advantage was found between patients treated-or-not with adjuvant chemotherapy (RFS = 92% and 91%, p = 0.13, p = 0.63 for overall survival), contrasting those with MAI ≧ 3 (p = 0.0001; HR = 0.32, 95% CI 0.18–0.58). Conclusion: MAI is the strongest predictor of adjuvant systemic chemotherapy success. In patients with MAI < 3 (31% of all patients), ASCT does not improve survival.

BACKGROUND: Genital human papillomavirus (HPV) infections are generally accepted to be sexually transmitted, but studies of HPV infections in sex partners are limited. We investigated HPV type-specific concordance and viral load in 238 heterosexual couples. Women with cervical intraepithelial neoplasia were the index patients in these couples.METHODS: GP5+/6+ polymerase chain reaction (PCR), followed by reverse-line blot analysis, was used for the detection of 45 HPV types in cervical and penile scrape samples. Viral loads were subsequently determined in scrape samples positive for HPV types 16, 18, 31, and 33 by LightCycler-based real-time PCR assays.RESULTS: A total of 89.9% of the women and 72.9% of their male partners were HPV positive. Predominantly high-risk HPV types were found in persons of both sexes, but infections with multiple and non-high-risk HPV types were more common in men. Of the HPV-positive couples, 57.8% of the men had the same HPV type as their partners; this rate was significantly higher than that expected by chance (P < .001). Moreover, these HPV-concordant men had higher penile scrape viral loads than did the non-HPV-concordant men. For HPV type 16-positive women, higher cervical viral loads were predictive of presence of HPV type 16 in their sex partners.CONCLUSIONS: In sexually active couples, HPV type concordance was more prevalent than expected by chance and was associated with increased viral loads. These data provide biological support for HPV transmission between sex partners.

Human papillomavirus (HPV) infections and HPV-associated penile lesions are frequently found in male sexual partners of women with cervical intraepithelial neoplasia (CIN). To determine the significance of these findings, we studied the prevalence of HPV and HPV associated penile lesions in a male hospital population with non-STD complaints. Penoscopy was performed after application of acetic acid to identify flat lesions, papular lesions, condylomata acuminata and pearly penile papules (PPPs). Presence of HPV DNA in penile scrapes was tested by GP5+6+ PCR. In case of HPV 16 positivity, viral loads were quantified using a LightCycler based real-time PCR method. Comparing the non-STD male hospital population (n = 118) with the male sexual partners of women with CIN (n = 238), flat penile lesions were found in 14% vs. 60% and penile HPV in 25% vs. 59% of the men, respectively. We found that the presence of penile HPV and, in case of HPV 16 positivity, higher viral loads were associated with the presence of flat penile lesions. Amongst the HPV-positive men, flat penile lesions were more common and larger in size in male sexual partners of women with CIN than in the non-STD hospital population. HPV infections and HPV-associated flat penile lesions are commonly found in the non-STD male population. However, these lesions are less frequently present and smaller in size than in male sexual partners of women with CIN. Higher viral loads in penile scrapes of male sexual partners of women with CIN are reflected by a higher prevalence of flat penile lesions and a larger size of these lesions.

In the Netherlands 2% of cervical smears in the cervical cancer screening program are read as borderline or mildly dyskaryotic cytology (BMD smear). Only in about 10% of these women a high-grade CIN lesion (CIN II-III) is present; therefore referral is for the majority unnecessary. In our study triage with high-risk HPV (hrHPV) testing was used to identify women at risk for development of high-grade CIN lesions after a repeat BMD smear. A "wait-and-see" period was incorporated allowing clearance of HPV and regression of the lesion. Women with a low-grade lesion, irrespective of their HPV status, were monitored at 12 months; women with a high-grade lesion were monitored at 6 and 12 months. Fifty-one of the 105 women (49%) were hrHPV negative at baseline; none of them showed progression of the lesion within the first year of follow-up (NPV 100%). High-grade CIN was present in 1 patient who was HPV negative at baseline (2%); she demonstrated regression after 12 months. Nineteen of the hrHPV positive women (35%) demonstrated a high-grade CIN lesion at baseline and 3 cleared hrHPV after 6 months, with a subsequent regression of CIN. Ten women remained hrHPV positive with persistence of high-grade CIN and were eventually treated. At baseline, 35 hrHPV positive women demonstrated a low-grade lesion, 19 remained hrHPV positive after 12 months and 5 developed high-grade CIN. Sixteen out of the 35 cleared the hrHPV infection without progression of the lesion. In conclusion, triage, using hrHPV testing for women with persistent BMD cytology, can select women who are not at risk for development of high-grade CIN. We recommend return to the screening program without referral for colposcopic examination if hrHPV is absent. For hrHPV positive women, a repeat hrHPV test after another 6 months is suggested. Referral is only required if persistence of hrHPV is established.

BACKGROUND: On the question of how to counsel adolescents with irregular menstrual cycles or oligomenorrhoea, no clear answer has been given. Adolescents with oligomenorrhoea especially show endocrine abnormalities and may be at risk for ovulatory dysfunction and the polycystic ovary syndrome in adulthood. METHODS: We followed a cohort of adolescents to document changes in menstrual cycle pattern between ages 15 and 18 years in the general population. RESULTS: Two per cent (2/128) of adolescents with regular menstrual cycles developed oligomenorrhoea, and 12% (17/148) of those with irregular menstrual cycles did so. Fifty-one per cent (34/67) of the oligomenorrhoeic adolescents remained oligomenorrhoeic. Increase in body mass index (BMI), concentration of LH, androstenedione or testosterone, and polycystic ovaries (PCO) were associated with persistence of oligomenorrhoea. In multivariate analysis only a normal to high BMI (>19.6 kg/m 2 ) consistently contributed significantly to predict persistent oligomenorrhoea. Glucose:insulin ratio as a marker for insulin resistance was not associated with an increased risk for oligomenorrhoea. CONCLUSIONS: Oligomenorrhoea at age 18 years is better predicted by menstrual cycle pattern at age 15 years than by LH or androgen concentrations or PCO at this age. Not only obese, but also normal weight oligomenorrhoeic, adolescents have a high risk of remaining oligomenorrhoeic.

Cytological cervical screening is rather inefficient because of relatively high proportions of false negative and false positive smears. To evaluate the efficiency of high-risk human papillomavirus (hrHPV) testing, by GP5+/6+ PCR-enzyme immunoassay (EIA), in conjunction with cytology (Intervention Group) to that of the classical cytology (Control Group), we initiated the Population Based Screening Study Amsterdam (POBASCAM). POBASCAM is a population-based randomized controlled trial for implementation of hrHPV testing in cervical screening. The outcome measure is the proportion of histologically confirmed ≥CIN3 lesions in each study arm up to and including the next screening round after 5 years. We present the design, methods and baseline data of POBASCAM. When, in the next 5 years, the follow-up will be completed, the data obtained will be used in model studies, including a cost-effectiveness study, to advise the Dutch Ministry of Public Health in deciding whether cervical screening should be based on combined hrHPV and cytology testing instead of cytology alone. Between January 1999 and September 2002, 44,102 women (mean age = 42.8 years; range = 29-61) that participated in the regular Dutch screening program were included in our study. In the Intervention Group the distribution of cytology and hrHPV by cytology class was as follows: normal cytology 96.6% (3. 6% hrHPV positive); borderline and mild dyskaryosis (BMD) 2.5% (34.6% hrHPV positive); and moderate dyskaryosis or worse (>BMD) 0.8% (88.3% hrHPV positive), i.e., 0.4% moderate dyskaryosis (82.9% hrHPV positive), 0.3% severe dyskaryosis (92.5% hrHPV positive), 0.1% carcinoma in situ (95.2% hrHPV positive), BMD, i.e., 0.4% moderate dyskaryosis, 0.3% severe dyskaryosis, 0.1% carcinoma in situ, BMD of 13.7% among women with a positive hrHPV test was not age-dependent. Our study indicates that large-scale hrHPV testing by GP5+/6+ PCR-EIA in the setting of population-based cervical screening is practically feasible, is accepted by both participating women and general practitioners and yields highly reproducible results.

Penile HPV-associated lesions are frequently seen in male sexual partners of women with CIN. The natural course and clinical significance of these lesions are unclear. Women with CIN and their male sexual partners were randomized for condom use (condom group n = 68, noncondom group n = 68). Males were screened for the presence of penile lesions, i.e., flat lesions, papular lesions and condylomata acuminata, and of HPV in their penile swabs by PCR testing. Median follow-up time was 13.1 months (range 2.9-57.4). The outcome of our study was clinical regression of penile lesions defined as disappearance of lesions at penoscopy. Potentially prognostic factors, i.e., HPV status, lesion type and age, were studied as well. Outcomes were assessed in 57 men of the condom group and in 43 men of the noncondom group. Condom use shortened the median time to regression of flat penile lesions (7.4 months condom group vs. 13.9 months noncondom group; HR = 2.1, 95% CI 1.2-3.7). This effect was not found for papular lesions (HR = 0.5, 95% CI 0.1-2.8). HPV-negative men showed a significantly shorter median time to regression of flat lesions (3.8 months) compared to men with either HPV-positive status (8.5 months; HR = 0.4, 95% CI 0.2-0.9) or inconsistent HPV status (13.1 months; HR = 0.2, 95% CI 0.1-0.6). Regression of flat penile lesions is HPV-dependent and accelerated by condom use. This effect is probably the result of blocking viral transmission between sexual partners.

Objectives To analyze the predictive power of Ki67 area% (Ki67), mitotic activity index (MAI), p53 area% (p53), and the mean area of the 10 largest nuclei (MNA10) for progression of stage in 195 primary consecutive TaT1 urothelial cell carcinomas of the urinary bladder. Methods Ki67- and p53-positive versus negative nuclei, MAI, and MNA10 using motorized systematic random sampling morphometry were determined. Kaplan-Meier curves and multivariate survival analysis (Cox model) were used to assess the prognostic value of the quantitative and classic clinicopathologic risk factors (age, sex, stage, grade, carcinoma in situ, multicentricity). Results Thirteen (6.7%) of the 195 patients had progression (0 [0%] of 36 low-risk, 1 [1.1%] of 85 intermediate-risk, and 12 [16.2%] of 74 high-risk patients). In univariate analysis (all variables), the strongest predictors with the highest hazard ratios were Ki67 (threshold 25.0%), MAI (threshold 30), and MNA10 (threshold 170 μm2). In multivariate analysis, the strongest independent combinations for progression—MNA10 (170 μm2) plus MAI (threshold 30) and MNA10 (threshold 170 μm2) plus Ki67 (threshold 25.0%)—overshadowed all other features. p53 was weaker but, combined with Ki67, still predicted progression fairly well. In the total group, the sensitivity, specificity, and positive and negative predictive values of MNA10-MAI and MNA10-Ki67 at the thresholds mentioned were 100%, 89%, 38%, and 100%, respectively. These feature combinations were also strongest prognostically in the high-risk treatment group. Conclusions The combined biomarkers MNA10-MAI or MNA10-Ki67 are accurate, well reproducible, and easy to assess progression predictors in all patients with TaT1 urothelial cell carcinomas, as well as in high-risk (bacille Calmette-Guerin-treated) patients.

We followed 353 women referred with abnormal cervical cytology in a non-intervention cohort study. In 91 pregnant women we compared high-risk human papilloma virus rates in the subsequent trimesters and postpartum in comparison to 262 non-pregnant women. High-risk human papilloma virus clearance was compared with 179 high-risk human papilloma virus positive non-pregnant women. Our main questions were: (1) do high-risk human papilloma virus rates change during pregnancy?; and (2) is there any difference between high-risk human papilloma virus clearance in pregnant and non-pregnant women? Women were monitored 3–4 monthly by cytology, colposcopy, and high-risk human papilloma virus testing. The median follow-up time was 33 months (range 3–74). Non-pregnant women showed prevalence rates of high-risk human papilloma virus of 64, 57, 53, and 50%, respectively, in four subsequent 3-months periods since the start of the study. These high-risk human papilloma virus rates were higher than in the three trimesters of pregnancy, and during the first 3 months postpartum, i.e. 50, 44, 45, and 31%, respectively. Postpartum only, this difference was statistically significant (P=0.004). Paired comparisons of high-risk human papilloma virus prevalence rates of the different trimesters with the postpartum rate showed (McNemar test) decreased rates: first trimester: 18% (P=0.02), second trimester: 13% (P=0.02) and third trimester: 23% (P

The relation between human papillomavirus type 16 (HPV 16) viral load in cervical scrapes and development of high-grade cervical intraepithelial neoplasia (CIN II or III) was studied in a nested case-control study of women with normal cytology (group A) and in a cohort of women with abnormal cytology (group B). HPV 16 DNA load was determined using a quantitative real-time PCR assay. In group A, case women (women with CIN II/III, n = 12) had a significantly higher viral load than control women (women with CIN ≤ I, n = 47). This resulted in an increased relative risk of women with the 50% highest viral load for development of CIN II/III (OR 7.7; CI 1.6–33). In group B, women with CIN II/III (n = 38) had a significantly higher viral load than women with CIN ≤ I (n = 25). Women with the 50% highest viral load had an increased relative risk of CIN II/III (OR 3.2; CI 1.1–9.3) and a decreased chance of both viral clearance and cytologic regression. Our data suggest that in women with normal cytology an increased HPV 16 load confers an increased risk of developing a CIN lesion. A sustained high viral load is subsequently informative for progression to a high-grade CIN lesion. © 2002 Wiley-Liss, Inc.

In a retrospective case–control study, we investigated high-risk HPV DNA presence by general primer GP5+/6+ PCR in the last normal cervical smear in the patient archives (i.e. baseline smear) of 57 women who later developed 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 (OR 28, 95% Cl 11–72). 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 (OR 32, 95% Cl 6.8–153). 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 http://www.bjcancer.com

We assessed a possible role for high-risk human papillomavirus (HPV) testing in the policy after treatment for cervical intraepithelial neoplasia (CIN) 2 or 3 (moderate to severe dysplasia). According to the Dutch guidelines follow-up after treatment consists of cervical cytology at 6, 12 and 24 months. Colposcopy is only performed in case of abnormal cervical cytology. In this observational study 184 women treated for CIN 2 or 3 were prospectively monitored by cervical cytology and high-risk HPV testing 3, 6, 9, 12 and 24 months after treatment. Post-treatment CIN 2/3 was present in 29 women (15.8%). A positive high-risk HPV test 6 months after treatment was more predictive for post-treatment CIN 2/3 than abnormal cervical cytology (sensitivity 90% and 62% respectively, with similar specificity). At 6 months the negative predictive value of a high-risk HPV negative, normal smear, was 99%. Largely overlapping, partly different groups of women with post-treatment CIN 2/3 were identified by HPV testing and cervical cytology. Based on these results we advocate to include high-risk HPV testing in monitoring women initially treated for CIN 2/3. In case of a high-risk HPV positive test or abnormal cervical cytology, colposcopy is indicated. All women should be tested at 6 and 24 months after treatment and only referred to the population-based cervical cancer screening programme when the tests are negative on both visits. © 2001 Cancer Research Campaign http://www.bjcancer.com

Study Objective: To evaluate the possible role of inappropriate LH secretion, hyperandrogenism, and hyperinsulinemia in the development of polycystic ovaries (PCO) and the polycystic ovary syndrome. Design: Observational. Setting: General population samples. Participants: 58 adolescents with regular menstrual cycles, 50 with irregular menstrual cycles, and 29 with oligomenorrhea (age 16.7 ± 0.9 years). Interventions: Transabdominal pelvic ultrasonography and vena puncture. Main Outcome Measures: PCO; LH, androstenedione, and testosterone levels; overnight fasting insulin concentrations; and oligomenorrhea. Results: The prevalence of PCO increased significantly with the irregularity of the menstrual cycle pattern, as illustrated by the study, finding PCO in 9% of the girls with regular menstrual cycles, 28% of those with irregular menstrual cycles, and 45% of oligomenorrheic girls. The LH and androgen concentrations were significantly higher in girls with PCO; the insulin levels and the glucose–insulin ratio did not differ when the girls with PCO were compared with girls with normal ovaries. Oligomenorrheic girls with PCO had the highest androgen and LH concentrations; their insulin concentrations and glucose–insulin ratio were in the same range as girls with regular menstrual cycles and normal ovaries; and both their hip and waist girths were wider, although their waist–hip ratio was normal. Conclusions: PCO in adolescents is associated with irregular menstrual cycles, oligomenorrhea, and/or high androgen and LH levels; but no relationship was found with the insulin level or glucose–insulin ratio. Thus, it is doubtful that hyperinsulinemia is an important factor in the development of PCO or polycystic ovary syndrome.

Objective To study the significance of the presence of high risk human papillomavirus (HPV) in women with initially normal cervical cytology for the development of abnormal cytology and an abnormal colposcopic impression. Design Prospective, observational study Participants and methods Sixty-eight women with cytomorphologically normal smears and at least one positive HPV test result were evaluated every six months by cytology, colposcopy and HPV testing. The endpoint of the study was abnormal cervical cytology. Results The median time of follow up from the first positive HPV test was 34 months. A total of 17 women developed abnormal cytology, of whom 16 (94%) had persistence of a high risk HPV infection. Women with persistent high risk HPV were more likely to develop abnormal cervical cytology than women without high risk HPV (hazard ratio 28.2, 95% CI 3.72–215.2); they also had an increased risk of developing an abnormal colposcopic impression (hazard ratio 4.4, 95% CI 1.69–11.7). Among the 17 women with abnormal cytology, high grade dysplasia was histopathologically demonstrated in eight women. Conclusion Persistent presence of high risk HPV in normal cervical smears is associated with a significantly increased risk of developing abnormal cytology and to a lesser degree with developing an abnormal colposcopic impression.

The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the β-globin gene appeared to be superior for the GTC-based assays. Using competitive β-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 × 105to 3 × 105genome equivalents in smears containing 5 × 105to 20 × 105nucleated cells, indicating a mean efficiency of 26% (range of 15–44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3–6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 ± 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears. © 2000 Cancer Research Campaign

Data on changes in hormone concentrations during the first years after menarche are scarce. We studied the relation between gynecological age (age minus age at menarche), hormone concentrations, and body measurements from the 1st to the 6th yr after menarche in 229 observations of girls with regular menstrual cycles, 157 observations of girls with irregular menstrual cycles, and 104 observations of girls with oligomenorrhea. Body Mass Index, waist circumference, hip circumference, LH, androstenedione, testosterone, and dehydro-epiandrosterone sulphate increased significantly (linear regression, P < 0.05) by gynecological age in all menstrual cycle pattern groups. For PRL and estradiol a significant increase with gynecological age was only doc umented in the regular menstrual cycle group and for waist to hip ratio only in the irregular menstrual cycle group. No significant correlation could be documented between gynecological age and overnight fasting insulin concentrations or glucose to insulin ratio. We found no significant correlation between insulin concentrations or glucose to insulin ratio and androgen concentrations. Significant positive correlations were found between LH and androgens. LH and androgen levels increase during the first years after menarche, and reference values should be adjusted for gynecological age. In these years, no significant correlation between hyperinsulinemia and hyperandrogenemia could be documented. Chemicals/CAS: Androgens; Androstenedione, 63-05-8; Dehydroepiandrosterone Sulfate, 651-48-9; Estradiol, 50-28-2; Gonadotropins, Pituitary; Insulin, 11061-68-0; Luteinizing Hormone, 9002-67-9; Testosterone, 58-22-0

This study aimed to assess the role of specific human papillomavirus type 16 (HPV-16) variants, in combination with p53 codon 72 polymorphism genotypes, in cervical carcinogenesis. An initial sequence analysis of HPV-16 long control, E6 and E7 regions of 53 well-defined cervical samples containing HPV-16 revealed that a T to G transition at nucleotide position 350 within the E6 open reading frame was the most common variation, the frequency of which seemed to decrease with increasing severity of the lesion. Therefore, a total of 246 cervical samples of residents of The Netherlands was specifically analysed for HPV-16 350G/T variants and/or p53 codon 72 genotypes. These comprised HPV-negative normal cervical scrapes (n=40), normal cervical scrapes containing HPV-16 (n=46), scrapes containing HPV-16 from women with abnormal cervical cytology participating in a non-intervention follow-up study without (n=38) and with (n=51) a histologically proven cervical intraepithelial neoplasia (CIN) III lesion at the end of the study, and cervical squamous cell carcinomas (n=71). Neither specific HPV-16 350G/T variants nor specific p53 genotypes were associated with a higher risk of developing CIN III or cervical cancer. However, HPV-16 350T variants were significantly over-represented in p53 Arg homozygous women with cervical cancer. This suggests that, in p53 Arg/Arg women, infection with HPV-16 350T variants confers a higher risk of cervical cancer.

Before guidelines can be set for the use of high-risk human papillomavirus (HR HPV) testing in cervical cancer screening and vaccine preparation, age-related prevalence of HR HPV types in cytologically normal smears has to be known. Therefore, in a cross-sectional study the prevalence of 37 different HPV genotypes and putatively unidentified HPV types was determined in 3,305 cytologically normal cervical smears from the general female population (15-69 years of age) using an HPV general primer GP5+/bioGP6+ mediated PCR assay. Subsequently, HPV-positive cervical smears were typed for 19 HR and 18 low-risk (LR) HPVs with an enzyme immunoassay using HPV type-specific oligoprobes in cocktails and individually, respectively. Overall, -HR and -LR HPV prevalences appeared to be of 4.6%, 3.3%, and 1.0%, respectively. Twenty-six different HPV types were detected in the 152 HPV-positive samples, the most prevalent types being HPV 16, 31, and 18. With regard to age, a peak prevalence of 19.6% for all HPVs was found in women 25-29 years of age, which declined to a mean of 4.3% in women over 30 years. With regard cytologically normal cervical smears (n = 3, 011) of women participating in the population-based screening program in the Netherlands (30 to 60 years), all HR HPVs showed decreased occurrence with increasing age, whereas the prevalence of LR HPV types remained constant. We suggest that screening for abnormal cytology implies screening for HR HPV infections and the subsequent treatment results in a decline of HR HPV prevalence in contrast to LR HPV prevalence during the years of screening.

BACKGROUND: The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for triaging women with mild to moderate dysplasia. AIMS: To determine the intermethod agreement between different GP5+/6+ and MY09/11 PCR based protocols for the detection and typing of high risk (HR) HPV DNA in cervical smears and to assess the intramethod reproducibility of the GP5+/6+ PCR enzyme immunoassay (EIA) for HR-HPV detection. METHODS: For the intermethod comparison, crude aliquots of 20 well characterised cervical smears comprising five HPV negative samples, and six and nine samples containing single and multiple HPV infections, respectively, were coded and sent from reference laboratory (A) to three other laboratories. One of these (laboratory B) used the GP5+/6+ PCR-EIA and was provided with standard protocols. Another laboratory (C) used GP5+/6+ PCR combined with sequence analysis and type specific PCR, whereas two laboratories (D and E) used MY09/11 PCR followed by restriction fragment length polymorphism (RFLP) analysis for the detection and typing of HR-HPV. The intramethod agreement of GP5+/6+ PCR-EIA was analysed in a subsequent study with four other laboratories (F to I) on crude aliquots of 50 well characterised cervical smears, consisting of 32 HR-HPV positive and 18 HPV negative samples. Standardised protocols, primers, and probes were also provided by the reference laboratory for HR-HPV detection. RESULTS: In the intermethod comparison, pairwise agreement of the different laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% (kappa values: 0.5 to 1). Typing data revealed a broader range in pairwise agreement rates between 32% and 100%. The highest agreement was found between laboratories A and B using standardised protocols and validated reagents. In the intramethod evaluation, pairwise comparison of the laboratories F to I with reference laboratory A revealed excellent agreement rates from 92% to 100% (kappa values: 0.88 to 1.0) with an overall sensitivity of 97.5% (195/200) and specificity of 99.5% (199/200). CONCLUSIONS: The detection of HR-HPV as a group is highly reproducible with GP5+/6+ PCR-EIA provided that standardised protocols and validated reagents are used.

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and β-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach. © 1999 Cancer Research Campaign

We prospectively evaluated the 5-year predictive values of adding high-risk human papillomavirus (hrHPV) testing to cytology for the detection of > or = cervical intraepithelial neoplasia (CIN)3 lesions in a population-based cohort of 2810 women. At baseline, nine (0.3%) women had prevalent lesions > or = CIN3, all being hrHPV positive. After 5 years of follow-up, four (6.5%) of the 62 hrHPV-positive women with normal cytology developed lesions > or = CIN3, vs only one (0.05%) of the 2175 hrHPV-negative women with normal cytology. High-risk human papillomavirus testing or combined screening revealed a much higher sensitivity, at the cost of a small decrease in specificity, and a higher negative predictive value for the detection of lesions > or = CIN3 till the next screening round (5 years) than cytology alone.

Cervical-cancer screening programmes using cytomorphological criteria could be more efficient if the screening included objective individual risk factors for women with normal cytology, such as a test for high-risk human papillomavirus (HPV). The value of a PCR-based test for high-risk HPV types was studied in a cohort of 1622 women presenting in a routine triannual population-based screening programme. Women were included in the study when they had no previous history of cervical dysplasia; and their initial Pap smear was read as normal (Pap 1 or 2). The mean age of the women was 42 years (range 34-54 years) and mean follow-up time was 40 months (range 5-73 months). Women were referred for colposcopically directed biopsies if they had had 2 successive cervical smears read as Pap 3a (mild to moderate dyskaryosis) or one read as > or = Pap 3b (severe dyskaryosis). Women with histologically confirmed cervical intraepithelial neoplasia grade III (CIN III) were considered positive cases. All women were tested for 14 high-risk HPV genotypes. Of the 86 high-risk HPV-positive women, 6 developed CIN III, whereas only 1 of the 1536 HPV-negative women did. The women with normal Pap smears containing high-risk HPV genotypes were 116 times (95% CI, 13-990) more at risk of developing CIN III, in contrast to women without high-risk HPV. These results support the view that the interval between successive smears in cervical-cancer screening can be increased considerably for women with cytomorphologically normal and high-risk HPV-negative cervical smears as determined by PCR.

The purpose of this work was to study intraobserver and interobserver variation in the interpretation of colposcopic images of cervical intraepithelial neoplasia (CIN). Twenty-three experienced colposcopists were asked to assess colposcopic images presented on slides and to select the biopsy site. Eleven cases were independently interpreted twice with an interval of 2-3 months by all observers. No information about the cytological classification was available. In each case the "majority assessment" was considered as the standard, being "no CIN" in 2 cases, CIN I in 4 cases, CIN II in 3 cases, and CIN III in 2 cases. Intraobserver concordance was 66.7%, the kappa value was 0.54. Interobserver agreement was found to be 52.4 and 51.0% in the first and second sessions, respectively, while the mean kappa values were 0.41 and 0.33, respectively. In selecting the site for biopsy, 77.4% of all observers agreed while the same site was selected in 85.3% of cases by the individual colposcopist in the two sessions. Overall, CIN I and II interpretations revealed lower levels of agreement than no CIN or CIN III interpretations. It is concluded that observer variability in interpreting colposcopic images and selecting the site for biopsy is in the same range as observer variation in other subjective diagnostic tests such as cytology and histopathology. This variation should be taken into account in the colposcopical management of patients with abnormal cytology.

ObjectiveCervical intraepithelial neoplasia grades 2-3 (CIN2-3) are usually treated by cone excision, although only 30% progress to cancer and 6-50% regress spontaneously. The aim of this study was to examine the influence of clinical factors like smoking habits, number of lifetime sexual partners, age at first sexual intercourse, sexual activity span and hormonal versus non-hormonal contraception type on the regression rate of CIN2-3.MethodsIn this prospective population-based cohort study 170 women aged 25-40 with abnormal cytology and colposcopy-directed biopsies showing first time onset CIN2-3 were consecutively included. The interval between biopsy and cone excision was standardized to minimum 12 weeks. Regression was defined as ≤ CIN1 in the cone biopsy.ResultsThe regression rate was 22%. Consistent condom use, defined as those women whose partners used condoms for all instances of sexual intercourse, was infrequent (n=20, 12%). In univariate analysis consistent condom use, hormonal contraception and age at first sexual intercourse significantly predicted regression. In a multivariate analysis only consistent condom use remained as an independent predictor of regression (regression rate 55%, p=0.001, hazard ratio=4.4).ConclusionConsistent condom use between punch biopsy and cone excision in first-time onset CIN2-3 patients significantly increases the regression rate.

A retrospective study of 227 patients presenting with abnormal cervical cytology was conducted to investigate the relationship between human papillomavirus (HPV) and progression of untreated cervical intraepithelial neoplasia (CIN) lesions. All patients had colposcopically directed biopsies for histologic diagnosis. The patients were followed cytologically and colposcopically for a mean of 19 months (range 6–42 months). Progression of a cervical lesion was defined as progression to a higher CIN grade confirmed histologically by directed biopsy. HPV DNA detection was done on material remaining from the cervical swabs by the general primer polymerase chain reaction (PCR) and type-specific PCR method, which made the detection of HPV types 6, 11, 16, 18, 31, 33 and not yet sequenced DNA types (X) possible. The presence of HPV DNA increased with the severity of the lesion (P< 0.001). In CIN III, a 100% HPV DNA prevalence was found, with HPV type 16 being the most prevalent type in 75%. Progression was significantly related to the presence of HPV DNA, in particular HPV type 16. The percentage of progressive disease was 21% in the case of HPV DNA positive lesions (n= 130) and 29% in the presence of HPV type 16, whereas HPV DNA negative lesions (n= 97) showed no progression. The detection of HPV DNA and HPV genotype can be used to identify patients with high-risk cervical lesions, since the presence of HPV DNA and genotype 16 in particular are closely related to CIN progression.

Objective Different Human Papilloma Virus (HPV) tests are currently used. An integrated comparison of the Amplicor, Cobas4800, PreTect HPV-Proofer and APTIMA HPV tests has not been done. Methods We compared the high-risk HPV detection power of these HPV tests in 528 consecutive population-based follow-up Liquid-Based Cytology samples (LBC) after ASCUS/LSIL index cytology. Their sensitivity and specificity to detect HPV in LBC, their predictive values of histopathologic CIN2–3 in follow-up punch biopsies and CIN2–3 regression in the subsequent cones was assessed. The HPV subtypes detected by the Linear Array genotyping-test (LA), PreTect HPV-Proofer and Cobas4800 were also compared. The follow-up histopathology was consensus expert-reviewed and Ki67/p16-supported. The predictive values of the HPV results in LBC by the different tests for presence of CIN2–3 in follow-up biopsies, and regression in subsequent cones, was assessed. Results Amplicor, Cobas4800 and APTIMA show good agreement for HPV-positivity/negativity. PreTect HPV-Proofer has many discrepancies versus any of the other methods. The sensitivities for Amplicor, Cobas4800 and APTIMA to detect CIN2–3 were very high (96–100%), but rather low for PreTect HPV-Proofer (53%). Specificity in case of CIN1 or less in follow-up biopsies of Amplicor and Cobas4800 is lower than APTIMA and highest for PreTect HPV-Proofer. HPV subtyping by LA agreed in 90% with Cobas4800 but 70% with PreTect HPV-Proofer. Conclusions The Amplicor, Cobas4800 and APTIMA give comparable results but PreTect HPV-Proofer differs from the other tests, with low sensitivity but higher specificity. None of the methods predicted regression of CIN2–3.

Fetal growth charts currently used aggregate birth weights of infants with various natural histories from 1931 until 1967. In order to modernize these charts, avoiding deviation from the natural history of fetal development, we report data from infants born after spontaneous onset of labour in 'normal' pregnancy from a gestational age of 267 to 295 days between 1972 and 1982 (n = 14,113). The relationship between birth weight and gestational age in days was studied by multiple regression analysis, containing dummy variables for parity and gender. The estimated proportion of the variance in the model, attributed to these characteristics, was 15%. This could be improved to 22% by supplementing the model with maternal characteristics such as age, height, mid-pregnancy weight and ethnic origin. According to this extended model, in the Dutch section of the population 511 (4.6%) babies had a birth weight below the 5th percentile, whereas 412 (3.7%) babies would be labeled as such according to the conventional birth weight tables. Moreover, 93 babies would be wrongly considered too small, corresponding with a sensitivity of 62.4%, and 192 babies would be wrongly considered normal, corresponding with a specificity of 99.3%. Integration of the four currently used tables into one, and adjustment for easily available maternal characteristics, could substantially improve classification methods.

Serum CA 125 regression after cytoreductive surgery and during the first three courses of chemotherapy was studied in 60 ovarian cancer patients and compared to known prognostic factors. Various methods reported in the literature to calculate a CA 125 half-live value were compared. Using two exponential regression models (Van der Burg et al., 1988; Buller et al., 1991), mean half-lives in stage I-II patients after complete cytoreductive surgery were respectively 10.7 days (range: 5-23) and 9.8 days (range: 7-15). Within stage III-IV patients, a significant positive correlation was seen between survival and (a) stage III (P = 0.002), (b) residual tumour < or = 1 cm (P = 0.02), (c) CA 125 normalisation after three courses (P = 0.003) and (d) CA 125 half-life < or = 20 days (P = 0.02-0.004, depending on the method used for half-life calculation). The median survival times of patients with and without a CA 125 normalisation after three courses were 27 and 14 months respectively (P = 0.003). When using the model of Buller et al. patients with a CA 125 half-life < or = 20 days had a median survival of 28 months compared to a median survival of 19 months for patients with CA 125 half-lives > 20 days (P = 0.004). Half-life calculations only showed a significant correlation with survival, if pre-surgery CA 125 levels were used as a baseline. In a survival analysis using the Cox proportional hazards model, stage of disease was the most predictive variable for survival (P = 0.006). The only additional independent prognostic factor for survival was the CA 125 half-life calculated according to Buller [derived from the formula: CA 125 = exp. [i-s x (days after surgery)], in which i is the y-axis intercept and s is the slope of the CA 125 regression curve]. A CA 125 half-life < or = 20 days vs > 20 days calculated using this formula, provides an independent prognostic factor for survival in stage III-IV patients early in the course of therapy (P = 0.04).

Evaluation of prognostic factors in lymph node negative (LNneg) invasive lobular cancers (ILCs). Pro- spective analysis of proliferation and other prognosticators in 121 LNneg ILCs (119 months median follow-up, range 19-181), without adjuvant chemotherapy. ILC subtype was assessed in accordance with WHO-2003 criteria. Immu- nohistochemical E-cadherin and estrogen receptor were used. With a median follow up time of 83 months (range 19-181), 30 of the 121 (25%) ILC patients developed distant metastases and 27 (22%) died. None of the cases classified as solid/pleomorphic lobular were E-cadherin or estrogen receptor positive, contrasting the other ILCs. The solid/alveolar ILCs (n = 17) had a worse survival (50%) than the other ILCs (n = 104; 83%, P \ 0.0001). Mitotic activity index (MAI) (but not nuclear grade or tubule formation) was prognostic with a threshold 0-5 versus (5 (=MAI-5) (contrasting MAI \ 10 vs. C10 in breast cancers in general; 85 and 54% survival, P \ 0.0001). In multi- variate analysis only subtype and MAI but none of the other characteristics had independent prognostic value. Histologic subtype and MAI have independent prognostic value in node negative invasive lobular cancers.

The prevalence of urinary and faecal incontinence was investigated in a sample of 1049 women aged 60 years and over in the municipality of Amstelveen, the Netherlands; 719 postal histories were completed. The overall prevalence of urinary incontinence was 23.5%. Daily urine loss was reported by 14.0% of all women. In women aged 60 to 84 years and 85 years and over 4.2% and 16.9% were faecally incontinent, respectively. In all age groups poor mobility and frequency were associated with urinary incontinence. Urgency was independently associated in women aged 60-85 years as was nocturia in women aged 85 years and over.

A rapid urinary luteinizing hormone (LH) test was used to plan a late luteal phase endometrial biopsy from 20 women undergoing an infertility evaluation. Histologic dating was correlated with the day of urinary LH surge detection, the day of the basal body temperature (BBT) nadir, and the onset of the next menstrual period (NMP). From 17 interpretable specimens, histologic dating correlated well with the day of the biopsy as determined following a positive LH test detection ( P = 0.079). No correlation was found following the BBT shift ( P = 0.65), and it was significantly correlated with the NMP ( P = 0.016). Moreover, the urinary LH test showed to be the best method to predict the onset of the NMP. These findings confirm urinary LH testing as a valuable adjunct in the investigation of luteal phase disorders.

Two recently developed monoclonal antibody (MAb)-based anti-mucin assays, CA M26 and CA M29, were studied in 250 cancer patients and compared to 3 well-established marker tests, viz., CA 125, CA 15.3 and SCC, in order to assess their clinical usefulness as serum tumor markers. Pre-treatment sera were obtained from patients with predominantly low-stage epithelial malignancies comprising 200 adenocarcinomas (of the ovary, endometrium, breast and large intestine) and 50 squamous-cell carcinomas (of the uterine cervix). Pretreatment sera of 50 patients with benign ovarian tumors were included to evaluate levels in benign disease. CA M26 and CA M29 cut-off levels were established in 89 healthy controls. In patients with adenocarcinomas, overall positivity for CA M29 was 24%, ranging from 10% in breast cancer to 60% in ovarian cancer. Overall positivity was highest for CA 125 (30%) and lowest for CA M26 (18%) with CA M29 (24%) being similar to CA 15.3 (25%). In adenocarcinomas the combined CA M26-CA M29 assays equalled results obtained with the CA 125-CA 15.3 combination (33% vs. 36%). Elevation of 2 or more markers was highly indicative of advanced disease (p

Background: The proliferation factor mitotic activity index (MAI) is the strongest prognosticator in lymph nodenegative invasive breast cancer patients under age 71. The question remains, whether this also holds for ‘favourable prognosis’ subgroups. Patients and methods: The study was a multicentre prospective analysis of the MAI for recurrence-free survival and overall cancer-related survival of grade, MAI, and other prognosticators in 853 long-term follow-up, T1–3N0M0 breast cancer patients under 71 years. Results: In all tumours together (N = 853), in grade 3 (n = 269), in tumours

Summary Background Tests for the DNA of high-risk types of human papillomavirus (HPV) have a higher sensitivity for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) than does cytological testing, but the necessity of such testing in cervical screening has been debated. Our aim was to determine whether the effectiveness of cervical screening improves when HPV DNA testing is implemented. Methods Women aged 29–56 years who were participating in the regular cervical screening programme in the Netherlands were randomly assigned to combined cytological and HPV DNA testing or to conventional cytological testing only. After 5 years, combined cytological and HPV DNA testing were done in both groups. The primary outcome measure was the number of CIN3+ lesions detected. Analyses were done by intention to treat. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN20781131. Findings 8575 women in the intervention group and 8580 in the control group were recruited, followed up for sufficient time (≥6·5 years), and met eligibility criteria for our analyses. More CIN3+ lesions were detected at baseline in the intervention group than in the control group (68/8575 vs 40/8580, 70% increase, 95% CI 15–151; p=0·007). The number of CIN3+ lesions detected in the subsequent round was lower in the intervention group than in the control group (24/8413 vs 54/8456, 55% decrease, 95% CI 28–72; p=0·001). The number of CIN3+ lesions over the two rounds did not differ between groups. Interpretation The implementation of HPV DNA testing in cervical screening leads to earlier detection of CIN3+ lesions. Earlier detection of such lesions could permit an extension of the screening interval.

Women not attending cervical screening programs are at increased risk of cervical cancer. We investigated in these nonresponders to what extent offering self-sampling devices for cervicovaginal brushes for high-risk human papillomavirus (hrHPV) testing would induce participation and, if so, what the yield of precursor (i.e. CIN2 or worse) lesions following self-sampling would be. In addition, we assessed screening history of participants and costs per detected high-grade CIN2 or worse (‘‘CIN21’’) lesion in comparison to the regular program in the Netherlands. Nonresponders received a device for hrHPV testing (self-sampling group, n 5 2,546) or an extra recall for conventional cytology (control group, n 5 284). The percentage of self-sampling responders were compared with responders in the recall group. hrHPV positive selfsampling responders were invited for cytology and colposcopy. CIN21 yield and costs per detected CIN21 were evaluated. Active response was higher in the self-sampling than in the control group (34.2 vs. 17.6%; p < 0.001). hrHPV positive self-sampling responders were less likely to have a prior screening history than screening participants (p < 0.001), indicating that they are regular nonresponders. hrHPV prevalence was similar (8.0 vs. 6.8%; p 5 0.11), but CIN21 yield was higher in self-sampling responders compared to screening participants (1.67 vs. 0.97%; OR 5 2.93, 95% CI 1.48–5.80; p 5 0.0013). Costs per CIN21 lesion detected via self-sampling were in the same range as those calculated for conventional cytological screening (€8,836 vs. €7,599). Offering self-sampling for hrHPV testing in nonresponders is an attractive adjunct to effectively increase population coverage of screening without the adverse effect of markedly increased costs per detected CIN21 lesion. ' 2006 Wiley-Liss, Inc.

Although it has been widely accepted that high-risk human papillomavirus (hrHPV) is sexually transmitted, limited insight is available about the clinical manifestations of hrHPV infection in men and their contribution in the viral spread. Here, we reviewed the literature on the relationship between hrHPV and the presence of penile lesions. Flat penile lesions have similar predilection sites as HPV, often contain hrHPV as identified by DNA in situ hybridization in biopsy specimens, show a high association with hrHPV as identified by PCR in penile scrapes of lesional sites and are associated with high viral copy numbers. Absence of flat lesions is generally associated with very low HPV copy numbers or absence of HPV. Therefore, we argue that these lesions form the reservoir of hrHPV in men and contribute to the viral spread. Their bare visibility with the naked eye and their high degree of spontaneous healing explain why flat penile lesions have slipped the attention of the clinician. Combining an HPV DNA test with a visual inspection after acetic acid application offers a more reliable interpretation of a positive HPV test in men, as it helps to distinguish positivity that is very likely to reflect a productive HPV infection from potentially HPV infections with very low copy numbers or HPV contamination by the sex partner. Future trials of HPV vaccines in men should take into account not only the presence of penile HPV but also the presence of flat penile lesions as an outcome measure for the efficacy of a vaccine.

An increased high-risk human papillomavirus (hrHPV) viral load in cervical scrapings has been proposed as a determinant for high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer (> or =CIN 2), but data so far for HPV types different from HPV 16 are limited and inconsistent. In addition, a viral load threshold to distinguish hrHPV positive women without > or =CIN 2 still has not been defined. Here, we used baseline cervical scrapings of women with normal cytology participating in a large population-based cervical screening trial (i.e. POBASCAM) who were GP5+/6+-PCR positive for 4 common hrHPV types, i.e. HPV 16, 18, 31 or 33, as a reference to arbitrarily define various viral load thresholds (i.e. 25th, 33rd, 50th, 67th and 75th percentiles of the lowest viral load values) for distinguishing women having single infections with these types without high-grade CIN. Viral load assessment was performed by real time type-specific PCR. The viral load threshold values were subsequently validated on abnormal cervical scrapes of 162 women with underlying, histologically confirmed CIN lesions containing 1 of these 4 hrHPV types. All 59 women with CIN 3 had viral load levels that were higher than those of 33% of the women with normal cytology containing the respective hrHPV type detectable by GP5+/6+-PCR (i.e. higher than the 33rd percentile of viral load). By using this 33rd percentile viral load cut-off, sensitivity for CIN 3 of 100% (95% CI 93.9-100) was obtained. Hence, application of this viral load threshold would increase the specificity of HPV testing for HPV 16, 18, 31 and 33-associated prevalent CIN 3 without the cost of a marked reduction in sensitivity. In practice, on the basis of viral load analysis, a less aggressive management can be foreseen for 33% of the women with normal cytology participating in a population-based screening program who are GP5+/6+-PCR positive for HPV 16, 18, 31 or 33.

High-risk human papillomavirus (hrHPV) types are causally related to cervical cancer and its high-grade precursor lesions. The risk posed by the different hrHPV types for the development of cervical intraepithelial neoplasia grade 2 or worse (> or =CIN2) needs to be established. Here, we present the hrHPV type-distribution in relation to cytology and histology for women participating in a cervical screening program. From 44,102 women who participated in a population-based cervical screening program in the Netherlands, 2,154 hrHPV GP5+/6+ PCR positive women were recruited to determine the distribution of 14 hrHPV types by reverse line blotting of GP5+/6+ PCR products. For each HPV type, associations with cytology and histologically confirmed > or =CIN2 were measured by odds ratios. HPV types 16 and 33 were more prevalent in women, amongst those containing a single hrHPV type, with moderate dyskaryosis or worse (>BMD) than in women with normal cytology, but only in case of underlying > or =CIN2 (OR 4.10, 95%CI 2.98-5.64 and OR 2.68, 95%CI 1.39-5.15, respectively). Similar results were obtained for women with double infections (OR 3.29, 95% CI 1.61-6.75 and OR 4.37, 95% CI 1.17-16.34). Coexisting types did not influence the prevalence of > or =CIN2 in HPV 16 or 33 positive women. The increased prevalence of type 16 and 33 in hrHPV positive women with > or =CIN2, compared to women with normal cytology, suggests that infection with these types confers an increased risk for development of > or =CIN2. Distinguishing these types may therefore have implications for future cervical screening strategies.

BACKGROUND. The objective of this study was to compare the accuracy of disease progression prediction of the molecular genetics and morphometry-based Endometrial Intraepithelial Neoplasia (EIN) and World Health Organization 1994 (WHO94) classification systems in patients with endometrial hyperplasias. METHODS. A multicenter, multivariate analysis was conducted on 477 patients with endometrial hyperplasia who were required to have a 1-year minimum disease-free interval from the time of the index biopsy (1-18 years of follow-up). The results from that analysis were compared with the results from 197 patients who had < 1 year of follow-up. RESULTS. Twenty-four of 477 hyperplasias (5.0%) progressed to malignant disease over an average of 4 years (maximum, 10 years). According to the WHO94 classification, 16 of 123 atypical hyperplasias (13%) and 8 of 354 nonatypical hyperplasias (2.3%) progressed (hazard ratio [HR] = 7). Twenty-two of 118 EINs (19%) and 2 of 359 non-EINs (0.6%) progressed (HR = 45). EIN was prognostic within each WHO94 subcategory. Progression rates were 3% in simple hyperplasias, 22% in complex hyperplasias, 17% in simple atypical hyperplasias, and 38% in complex atypical hyperplasias with EIN, compared with progression rates of 0.0-2.0% in all hyperplasias if EIN was absent. EIN detected precancerous lesions (sensitivity, 92%) better than WHO94 atypical hyperplasias collectively (67%) or complex atypical hyperplasias alone (46%). In a Cox regression analysis, EIN was the strongest prognostic index of future endometrial carcinoma. The same was true for patients with < 1 year of follow-up (HR for EIN, atypical hyperplasia, and complex atypical hyperplasia: 58, 7, and 8, respectively). CONCLUSIONS. The EIN classification system predicted disease progression more accurately than the WHO94 classification and identified many women with benign changes that would have been regarded as high risk according to the WHO94 classification system.

We earlier demonstrated, in a randomised clinical trial, that the regression time of flat penile lesions in male sexual partners of women with cervical intraepithelial neoplasia (CIN) was shorter in men who used condoms compared to those who did not. To further evaluate this finding, we examined whether the effect of condom use on the regression of flat penile lesions depends on the presence of human papillomavirus (HPV) type concordance in sexual couples, as determined in cervical and penile scrapes by GP5+/6+ PCR testing. A Cox model with time-dependent covariates showed a beneficial effect of condoms on regression of flat penile lesions in concordant couples (hazard ratio 2.63, 95% CI 1.07-6.48) but not in those who were nonconcordant. When both partners harboured different HPV types, no effect of condoms was found (hazard ratio 0.90, 95% CI 0.27-2.96). Delayed regression of flat penile lesions was associated with either stable lesions or with new penile lesions developing at sites surrounding pre-existing lesions suggesting reinfection of the penile epithelium. We conclude that condom use blocks sexual HPV transmission by preventing reinfection and development of new penile lesions in men who are susceptible to the same type as present in the female partner.

Purpose To validate the independent strong prognostic value of mitotic activity index (MAI) in lymph node (LN) –negative invasive breast cancer patients younger than 55 years in a nationwide multicenter prospective study. Patients and Methods Analysis of routinely assessed MAI and other prognosticators in 516 patients (median follow-up, 118 months; range, 8 to 185 months), without systemic adjuvant therapy or previous malignancies. Results Distant metastases occurred in 127 patients (24.6%); 90 (17.4%) died as a result of metastases. MAI (< 10, ≥ 10) showed strong association with recurrence (hazard ratio [HR], 3.12; 95% CI, 2.17 to 4.50; P ≤ .0001) and mortality (HR, 4.42; 95% CI, 2.79 to 7.01; P < .0001). The absolute difference in 10-year Kaplan-Meier estimates of time to distant recurrence as well as survival was 22% between MAI less than 10 versus ≥ 10. This effect was independent of age, estrogen receptor (ER) status, and tumor diameter (which were significant prognosticators). In multivariate analysis with regard to patient age, tumor diameter, grade, ER status, and the St Gallen criterion, MAI proved to be an independent and the strongest prognosticator. Tubular formation (TF) and nuclear atypia (NA), as constituents of (expert revised) grade, had no (for TF) or limited (for NA, P = .048) additional prognostic value to the MAI. In the group with MAI less than 10, MAI less than 3 versus more than 3 had additional value but the classical threshold of 0 to 5 v 6 to 10 did not. With this additional subdivision of MAI as less than 3, 3 to 9, and more than 9, NA lost its additive prognostic value. Conclusion The MAI is the strongest, most widely available, easily assessable, inexpensive, well-reproducible prognosticator and is well suited to routinely differentiate between high- and low-risk LN-negative breast cancer patients younger than 55 years.

Expression levels of hTERT mRNA were investigated by RT-PCR in tissue specimens of patients with (Group A) and without (Group B) clinically overt bronchial carcinoma, respectively. Bronchial carcinoma (n = 9) and distant normal (n = 9) specimens were analyzed in Group A. The chance of carcinoma seemed to increase with increasing hTERT mRNA levels (OR = 6.04, 95% CI = 1.02–37). Group B was comprised of 21 patients who underwent autofluorescence bronchoscopy. After analysis of 66 bronchial biopsies the chance of prevalent carcinoma in situ or carcinoma increased with increasing hTERT mRNA levels (OR = 6.19, 95% CI = 1.55–25). Variables like age, gender, smoking history, history of cancer within the airways or the degree of lymphocyte infiltrate in the specimens did not modify this relation. In 7 Group B patients in whom bronchial cancer was diagnosed during follow-up, biopsies taken before cancer diagnosis from both the area of the newly developed tumor and distantly from this area had been analyzed for hTERT expression. The median hTERT mRNA level in the biopsies from the area of future cancer was significantly higher than in biopsies taken from distant sites (p < 0.03). These data indicate that elevated hTERT mRNA is associated with an increased relative risk of prevalent and incident bronchial squamous cell carcinoma (in situ). © 2004 Wiley-Liss, Inc.

According to the current guidelines in most western countries, women treated for cervical intraepithelial neoplasia grade 3 (CIN 3) are followed for at least 2 years after treatment by cytology. High-risk human papillomavirus (hrHPV) infections are necessary for the development and maintenance of CIN 3. HrHPV testing could be used to improve monitoring of women treated for CIN 3. This has prompted numerous studies for the implementation of hrHPV testing in monitoring of women treated for CIN 3. Included in this review are 20 studies, published between 1996 and 2003, comparing hrHPV testing with either resection margins or cervical cytology to predict recurrent/residual disease, and 11 of them could be used in a meta-analysis. In the meta-analysis of the 11 studies, the negative predictive value (NPV) for recurrent/residual disease of hrHPV testing was 98% (95% CI 97-99%), that of resection margins 91% (95% CI 87-94%), and that of cervical cytology 93% (95% CI 90-95%). When hrHPV testing was performed in conjunction with cytology, the sensitivity was 96% (95% CI 89-99%), specificity was 81% (95% CI 77-84%), the associated positive predictive value (PPV) was 46% (95% CI 38-54%), and the NPV was 99% (95% CI 98-100%). Combined hrHPV and cytology testing yielded the best test characteristics. We propose to include hrHPV testing an conjunction with cytology for monitoring women treated for CIN 3. Some follow-up visits for women testing negative for both hrHPV and cytology can be skipped. In western countries, this could mean that for women double negative at 6 months, retesting at 12 months should be skipped while keeping the 24-month follow-up visit.

Women with persistent HPV infections have increased risk of progressive CIN lesions. Transmission of HPV between sexual partners might maintain viral infection and, consequently, may influence the clinical course of CIN. We investigated the effect of condom use on regression of CIN lesions and on clearance of HPV. Women with CIN and their male sexual partners were randomized for condom use (condom group n = 72 and noncondom group n = 76). They were conservatively managed and followed every 3-6 months by colposcopy, cytology and HPV testing by GP5+/6+ PCR. Baseline cervical biopsy specimens were taken. Median follow-up time for women was 15.2 months (range 3.0-85.4). Outcomes of interest were clinical regression of CIN at colposcopy and clearance of HPV. Outcomes were assessed in 64 women of the condom group and 61 women of the noncondom group. Women in the condom group showed a 2-year cumulative regression rate of 53% vs. 35% in the noncondom group (p = 0.03). The 2-year cumulative rates of HPV clearance were 23% vs. 4%, respectively (p = 0.02). Although lower regression rates were found if women were HPV-positive and had > or =CIN2 lesions at baseline, effects of condom use were found both in women with CIN1 and in women with > or =CIN2 lesions. Condom use promotes regression of CIN lesions and clearance of HPV.