Risk of cervical precancer among HPV-negative women in the Netherlands and its association with previous HPV and cytology results: A follow-up analysis of a randomized screening study.

BACKGROUND: Human papillomavirus (HPV)-based screening programs still use one-size-fits-all protocols but efficiency and efficacy of programs may be improved by stratifying women based on previous screening results. METHODS AND FINDINGS: We studied the association between cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) and previous screening results in the Population-Based Screening Study Amsterdam (POBASCAM) trial, performed in the Netherlands in the setting of regular screening, where women aged from 29 to 61 years old were invited to cytology and HPV co-testing at enrolment in year 1999/2002 and at the next round in 2003/2007. We selected 18,448 women (9,293 from the intervention group and 9,155 from the control group) who tested HPV-negative in 2003/2007 and did not have cervical intraepithelial neoplasia grade 2 or worse (CIN2+) or hysterectomy after enrolment. Follow-up was collected until 14 years after the 2003/2007 screen, covering 4 rounds of screening. Risk of CIN3+ and CIN2+ among women with an HPV-negative test, irrespective of previous round results and stratified according to previous round HPV and cytology results, were calculated by the Kaplan-Meier method. During 14 years of follow-up, 62 CIN3+ cases (24 in the intervention group and 38 in the control group) were detected. HPV-negative women had a 14-year CIN3+ risk of 0.48% (95% confidence interval 0.37 to 0.62) and CIN2+ risk of 1.17% (0.99 to 1.38). The CIN3+ risk among HPV-negative women was increased in women with a previous positive HPV test (2.36%, 1.20 to 4.63; p < 0.001) or co-test (1.68%, 0.87 to 3.20; p < 0.001) and, equivalently, decreased in women with a previous negative HPV test (0.43%, 0.33 to 0.57) or a negative co-test (0.43%, 0.33 to 0.57). The CIN3+ risk was not influenced by the previous cytology result. The CIN3+ risk among HPV-negative women was increased after both a previous HPV16-positive test (3.90%, 1.47 to 10.12; p < 0.001) and a previous HPV16-negative/HPVother-positive test (1.91%, 0.76 to 4.74; p = 0.002). For endpoint CIN2+ (147 cases), findings were similar except that the CIN2+ risk was increased after previous abnormal cytology (4.06%, 2.30 to 7.12; p < 0.001). The presented risk estimates were calculated by tracking histological results through the Dutch nationwide pathology archive (PALGA) and were not adjusted for non-compliance with the colposcopy referral advice. CONCLUSIONS: HPV-negative women had an increased long-term risk of CIN3+ when the HPV test in the previous screening round was positive. This supports the implementation of risk-based intervals that depend on HPV results in the current and previous screening round. TRIAL REGISTRATION: POBASCAM trial, trial registration number ISRCTN20781131.

Characterization of cervical biopsies of women with HIV and HPV co-infection using p16ink4a, ki-67 and HPV E4 immunohistochemistry and DNA methylation.

This study aims to characterize cervical intraepithelial neoplasia (CIN) in women living with HIV using biomarkers. Immunohistochemical (IHC) staining for human papillomavirus (HPV) E4 protein indicates CIN with productive HPV infection, whereas Ki-67 and p16ink4a indicate CIN with transforming characteristics, which may be further characterized using DNA hypermethylation, indicative for advanced transforming CIN. Cervical biopsies (n = 175) from 102 HPV positive women living with HIV were independently reviewed by three expert pathologists. The consensus CIN grade was used as reference standard. IHC staining patterns were scored for Ki-67 (0-3), p16ink4a (0-3), and E4 (0-2) and correlated to methylation levels of four cellular genes in corresponding cervical scrapes. Reference standards and immunoscores were obtained from 165 biopsies:15 no dysplasia, 91 CIN1, 31 CIN2, and 28 CIN3. Ki-67 and p16ink4a scores increased with increasing CIN grade, while E4 positivity was highest in CIN1 and CIN2 lesions. E4 positive CIN1 lesions had higher Ki-67 and p16ink4a scores and higher methylation levels compared with E4 negative CIN1 lesions. E4 positive biopsies with low cumulative Ki-67/p16 ink4a immunoscores (0-3) had significantly higher methylation levels compared with E4 negative biopsies. No significant differences in Ki-67 and p16ink4a scores and methylation levels were observed between E4 negative and positive CIN2 or CIN3 lesions. The presence of high methylation levels in scrapes of CIN lesions with IHC characteristics of both productive (E4 positive) and transforming infections (increased Ki-67/p16ink4a expression) in women living with HIV might indicate a rapid aggressive course of HPV infections towards cancer in these women.

Comparing the performance of FAM19A4 methylation analysis, cytology and HPV16/18 genotyping for the detection of cervical (pre)cancer in high-risk HPV-positive women of a gynecologic outpatient population (COMETH study).

Recently, DNA methylation analysis of FAM19A4 in cervical scrapes has been shown to adequately detect high-grade cervical intraepithelial neoplasia and cervical cancer (≥ CIN3) in high-risk HPV (hrHPV)-positive women. Here, we compared the clinical performance of FAM19A4 methylation analysis to cytology and HPV16/18 genotyping, separately and in combination, for ≥ CIN3 detection in hrHPV-positive women participating in a prospective observational multi-center cohort study. The study population comprised hrHPV-positive women aged 18-66 years, visiting a gynecological outpatient clinic. From these women, cervical scrapes and colposcopy-directed biopsies (for histological confirmation) were obtained. Cervical scrapes were analyzed for FAM19A4 gene promoter methylation, cytology and HPV16/18 genotyping. Methylation analysis was performed by quantitative methylation-specific PCR (qMSP). Sensitivities and specificities for ≥ CIN3 were compared between tests. Stratified analyses were performed for variables that potentially influence marker performance. Of all 508 hrHPV-positive women, the sensitivities for ≥ CIN3 of cytology, FAM19A4 methylation analysis, and cytology combined with HPV16/18 genotyping were 85.6, 75.6 and 92.2%, respectively, with corresponding specificities of 49.8, 71.1 and 29.4%, respectively. Both sensitivity and specificity of FAM19A4 methylation analysis were associated with age (p ≤ 0.001 each). In women ≥ 30 years (n = 287), ≥ CIN3 sensitivity of FAM19A4 methylation analysis was 88.3% (95%CI: 80.2-96.5) which was noninferior to that of cytology [85.5% (95%CI: 76.0-94.0)], at a significantly higher specificity [62.1% (95%CI: 55.8-68.4) compared to 47.6% (95%CI: 41.1-54.1)]. In conclusion, among hrHPV-positive women from an outpatient population aged ≥ 30 years, methylation analysis of FAM19A4 is an attractive marker for the identification of women with ≥ CIN3.

FAM19A4 methylation analysis in self-samples compared with cervical scrapes for detecting cervical (pre)cancer in HPV-positive women.

BACKGROUND: High-risk human papillomavirus (hrHPV)-positive women require triage to identify those with cervical high-grade intraepithelial neoplasia and cancer (⩾CIN3 (cervical intraepithelial neoplasia grade 3)). FAM19A4 methylation analysis, which detects advanced CIN and cancer, is applicable to different sample types. However, studies comparing the performance of FAM19A4 methylation analysis in hrHPV-positive self-samples and paired physician-taken scrapes are lacking. METHODS: We compared the performance of FAM19A4 methylation analysis (and/or HPV16/18 genotyping) in self-samples and paired physician-taken scrapes for ⩾CIN3 detection in hrHPV-positive women (n=450,18-66 years). RESULTS: Overall FAM19A4 methylation levels between sample types were significantly correlated, with strongest correlation in women with ⩾CIN3 (Spearman's ρ 0.697, P<0.001). The performance of FAM19A4 methylation analysis and/or HPV16/18 genotyping did not differ significantly between sample types. In women ⩾30 years, ⩾CIN3 sensitivity of FAM19A4 methylation analysis was 78.4% in self-samples and 88.2% in scrapes (ratio 0.89; CI: 0.75-1.05). In women <30 years, ⩾CIN3 sensitivities were 37.5% and 45.8%, respectively (ratio 0.82; CI: 0.55-1.21). In both groups, ⩾CIN3 specificity of FAM19A4 methylation analysis was significantly higher in self-samples compared with scrapes. CONCLUSIONS: FAM19A4 methylation analysis in hrHPV-positive self-samples had a slightly lower sensitivity and a higher specificity for ⩾CIN3 compared with paired physician-taken scrapes. With a similarly good clinical performance in both sample types, combined FAM19A4 methylation analysis and HPV16/18 genotyping provides a feasible triage strategy for hrHPV-positive women, with direct applicability on self-samples.

p16/Ki-67 dual-stained cytology for detecting cervical (pre)cancer in a HPV-positive gynecologic outpatient population.

Women who test positive for a high-risk type of the human papillomavirus (HPV) require triage testing to identify those women with cervical intraepithelial neoplasia grade 3 or cancer (≥CIN3). Although Pap cytology is considered an attractive triage test, its applicability is hampered by its subjective nature. This study prospectively compared the clinical performance of p16/Ki-67 dual-stained cytology to that of Pap cytology, with or without HPV16/18 genotyping, in high-risk HPV-positive women visiting gynecologic outpatient clinics (n=446 and age 18-66 years). From all women, cervical scrapes (for Pap cytology, HPV16/18 genotyping, and p16/Ki-67 dual-stained cytology) and colposcopy-directed biopsies were obtained. The sensitivity of p16/Ki-67 dual-stained cytology for ≥CIN3 (93.8%) did neither differ significantly from that of Pap cytology (87.7%; ratio 1.07 and 95% confidence interval (CI): 0.97-1.18) nor from that of Pap cytology combined with HPV16/18 genotyping (95.1%; ratio 0.99 and 95% CI: 0.91-1.07). However, the specificity of p16/Ki-67 dual-stained cytology for ≥CIN3 (51.2%) was significantly higher than that of Pap cytology (44.9%; ratio 1.14 and 95% CI: 1.01-1.29) and Pap cytology combined with HPV16/18 genotyping (25.8%; ratio 1.99 and 95% CI: 1.68-2.35). After exclusion of women who had been referred because of abnormal Pap cytology, the specificity of p16/Ki-67 dual-stained cytology for ≥CIN3 (56.7%) remained the same, whereas that of Pap cytology (60.3%) increased substantially, resulting in a similar specificity of both assays (ratio 0.94 and 95% CI: 0.83-1.07) in this sub-cohort. In summary, p16/Ki-67 dual-stained cytology has a good clinical performance and is an interesting objective microscopy-based triage tool for high-risk HPV-positive women.

Follow-up of high-risk HPV positive women by combined cytology and bi-marker CADM1/MAL methylation analysis on cervical scrapes.

OBJECTIVES: Triage of HPV screen-positive women is needed to identify those with underlying cervical intraepithelial neoplasia grade 2/3 or worse (CIN2/3+). Presently, cytology on a physician-taken cervical scrape is mostly accepted as triage test, but needs follow-up testing in order not to miss severe disease. Here, we evaluated the performance of combined cytology and bi-marker CADM1/MAL-methylation analysis as triage test on physician-taken cervical scrapes of HPV positive women. METHODS: In this post-hoc analysis, we used 364 left-over HPV positive cytology triage samples of participants of a randomized controlled trial (PROHTECT-3: n=46,001) performed in population-based cervical screening. Study endpoints were CIN2+ and CIN3+ detection. Cytology testing with and without methylation marker analysis was evaluated with regard to sensitivity, specificity, positive and negative predictive value, and referral rate. RESULTS: Bi-marker CADM1/MAL-methylation positivity increased proportionally with severity of underlying lesions. Overall, cytology and bi-marker CADM1/MAL-methylation analysis yielded similar performances with regard to CIN3+ detection, yet in combination a significantly higher sensitivity for CIN3+ (88.7%) was obtained at a specificity of 53.6% and a colposcopy referral rate of 53.6%. The combined strategy detected all six cervical cancers, whereas triage by cytology alone failed to detect two of them. CONCLUSIONS: Cytology and bi-marker CADM1/MAL-methylation analysis perform complementary for CIN2+/CIN3+ detection when used as triage tool on cervical scrapes of HPV positive women. This approach not only results in a higher CIN3+ sensitivity than cytology triage with an acceptable referral rate, but also seems to reduce the risk of missing cervical cancers and advanced high-grade lesions.

Triage by methylation-marker testing versus cytology in women who test HPV-positive on self-collected cervicovaginal specimens (PROHTECT-3): a randomised controlled non-inferiority trial.

BACKGROUND: Cytology is a widely used method of triaging women who test positive for human papillomavirus (HPV). However, self-sampled specimens, which can substantially increase participation in screening programmes, are not suitable for accurate cytological assessment. We investigated whether direct DNA methylation-based molecular triage on self-sampled cervicovaginal specimens was non-inferior to cytology triage on additional physician-collected cervical samples in the detection of cervical intraepithelial neoplasia grade 2 (CIN2) or worse in women who did not attend cervical screening programmes. METHODS: In this randomised controlled non-inferiority trial, we invited women (aged 33-63 years) registered as non-attendees of cervical screening in the Netherlands in 2007 to submit a self-collected cervicovaginal sample for HPV testing. Using a computer-generated sequence, we randomly allocated women who tested positive for high-risk hrHPV on a self-sample to either triage by cytology on an additional physician-taken smear or direct triage on the self-sample by methylation analysis of MAL and miR-124-2 genes (1:1; stratified by age and region, with block sizes by age group). Triage-positive women in either group were referred for colposcopy. The primary endpoint was detection of CIN2 or worse, analysed by intention to treat. The non-inferiority margin was 0·80. This study is registered in the Primary Trial Register of the Netherlands, number NTR6026. FINDINGS: We invited 46,001 women to participate, 12,819 of whom returned self-sampled material; 1038 samples tested positive for high-risk HPV. Between Nov 1, 2010, and Dec 31, 2011, after exclusion of women who were ineligible, we enrolled and randomly allocated 515 women to methylation triage and 509 to cytology triage. The detection of CIN2 or worse with methylation triage was non-inferior to that with cytology triage (90 [17%] of 515 women vs 75 [15%] of 509 women; relative risk 1·19, 95% CI 0·90-1·57). Referral for colposcopy was more common in the molecular group (284 [55%] women) than in the cytology group (149 [29%] women; p<0·0001). Mean time to CIN2 or worse diagnosis was shorter in the molecular triage group (96 days, range 44-101) than in the cytology triage group (158 days, 71-222; p=0·00084). INTERPRETATION: DNA methylation analysis of MAL and miR-124-2 genes on HPV-test-positive self-samples is non-inferior to cytology triage in the detection of CIN2 or worse, opening the way to full molecular screening. FUNDING: Midden-West and Oost Screening Organisations and Stichting Achmea Gezondheidszorg.

Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer.

OBJECTIVES: Methylation marker analysis using bi-marker panel MAL/miR-124-2 is a promising triage test for identifying cervical (pre)cancer in high-risk human papillomavirus (hrHPV) positive women. Bi-marker panel MAL/miR-124-2 can be applied directly on self-sampled cervico-vaginal material and its sensitivity is non-inferior to that of cytology, yet at the cost of more colposcopy referrals. Our objective was to increase specificity of MAL/miR-124-2 methylation analysis by varying the assay thresholds and adding HPV16/18 genotyping. METHODS: 1019 hrHPV-positive women were selected from a randomized controlled self-sampling trial (PROHTECT-3; 33-63 years, n=46,001) and nine triage strategies with methylation testing of MAL/miR-124-2 and HPV16/18 genotyping were evaluated. The methylation assay threshold was set at four different predefined levels which correspond with clinical specificities for end-point cervical intra-epithelial grade 3 or worse (CIN3+) of 50%, 60%, 70%, and 80%. RESULTS: The CIN3+ sensitivity of methylation analysis decreased (73.5 to 44.9%) while specificity increased (47.2 to 83.4%) when increasing the assay threshold. CIN3+ sensitivity and specificity of HPV16/18 genotyping were 68.0% and 65.6%, respectively. Combined methylation analysis at threshold-80 and HPV16/18 genotyping yielded similar CIN3+ sensitivity as that of methylation only at threshold-50 (77.6%) with an increased specificity (54.8%). CONCLUSIONS: Combined triage by MAL/miR-124-2 methylation analysis with threshold-80 and HPV16/18 genotyping reaches high CIN3+ sensitivity with increased specificity to identify women with cervical (pre)cancer among HPV self-sample positive women. The combined strategy is attractive as it is fully molecular and identifies women at the highest risk of cervical (pre)cancer because of strongly elevated methylation levels and/or HPV16/18 positivity.

Serum testosterone plays an important role in the metastatic ability of castration resistant prostate cancer.

PURPOSE: Prostate cells are dependent on androgens for growth and proliferation. Androgen deprivation therapy is the recommended treatment for advanced/metastatic prostate cancer. Under this therapy, prostate cancer will inevitably progress to castration resistant prostate cancer (CRPC). Despite putative castration resistance, testosterone might still play a crucial role in the progression of CRPC. The goal of this study was to determine the role of testosterone in the formation of metastases of CRPC in both in vitro and in vivo settings. METHODS: In vitro, the effect of testosterone and the non-aromatizable androgen methyltrienolone on migration, invasion and proliferation of a castration-resistant prostate cancer rat cell line (Dunning R3327-MATLyLu) was assessed using a transwell assay and a sulforhodamine B assay and immunohistochemical detection of ki67. Androgen receptor status was determined using Western blot. In vivo, Copenhagen rats were divided in four groups (males, females, castrated males and females with testosterone suppletion) and inoculated with MATLyLu cells. Tumor size was assessed daily. RESULTS: Testosterone increased cell migration and invasion in a concentration-dependent manner in vitro. Testosterone did not affect in vitro cell proliferation. No difference was shown between the effect of testosterone and methyltrienolone. In vivo, in groups with higher levels of circulating testosterone, more rats had (micro)metastases compared with groups with low levels of testosterone. No effect was observed on primary tumor size/growth. CONCLUSIONS: Despite assumed castration resistance, progression of prostate cancer is still influenced by androgens. Therefore, continuous suppression of serum testosterone in patients who show disease progression during castration therapy is still warranted.

Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial.

BACKGROUND: Human papillomavirus (HPV) testing is more sensitive for the detection of high-grade cervical lesions than is cytology, but detection of HPV by DNA screening in two screening rounds 5 years apart has not been assessed. The aim of this study was to assess whether HPV DNA testing in the first screen decreases detection of cervical intraepithelial neoplasia (CIN) grade 3 or worse, CIN grade 2 or worse, and cervical cancer in the second screening. METHODS: In this randomised trial, women aged 29-56 years participating in the cervical screening programme in the Netherlands were randomly assigned to receive HPV DNA (GP5+/6+-PCR method) and cytology co-testing or cytology testing alone, from January, 1999, to September, 2002. Randomisation (in a 1:1 ratio) was done with computer-generated random numbers after the cervical specimen had been taken. At the second screening 5 years later, HPV DNA and cytology co-testing was done in both groups; researchers were masked to the patient's assignment. The primary endpoint was the number of CIN grade 3 or worse detected. Analysis was done by intention to screen. The trial is now finished and is registered, number ISRCTN20781131. FINDINGS: 22,420 women were randomly assigned to the intervention group and 22 518 to the control group; 19 999 in the intervention group and 20,106 in the control group were eligible for analysis at the first screen. At the second screen, 19 579 women in the intervention group and 19,731 in the control group were eligible, of whom 16,750 and 16,743, respectively, attended the second screen. In the second round, CIN grade 3 or worse was less common in the intervention group than in the control group (88 of 19 579 in the intervention group vs 122 of 19,731 in the control group; relative risk 0·73, 95% CI 0·55-0·96; p=0·023). Cervical cancer was also less common in the intervention group than in the control group (four of 19 579 in the intervention group vs 14 of 19,731; 0·29, 0·10-0·87; p=0·031). In the baseline round, detection of CIN grade 3 or worse did not differ significantly between groups (171 of 19 999 vs 150 of 20,106; 1·15, 0·92-1·43; p=0·239) but was significantly more common in women with normal cytology (34 of 19,286 vs 12 of 19,373; 2·85, 1·47-5·49; p=0·001). Furthermore, significantly more cases of CIN grade 2 or worse were detected in the intervention group than in the control group (267 of 19 999 vs 215 of 20,106; 1·25, 1·05-1·50; p=0·015). In the second screen, fewer HPV16-positive CIN grade 3 or worse were detected in the intervention group than in the control group (17 of 9481 vs 35 of 9354; 0·48, 0·27-0·85; p=0·012); detection of non-HPV16-positive CIN grade 3 or worse did not differ between groups (25 of 9481 vs 25 of 9354; 0·99, 0·57-1·72; p=1·00). The cumulative detection of CIN grade 3 or worse and CIN grade 2 or worse did not differ significantly between study arms, neither for the whole study group (CIN grade 3 or worse: 259 of 19 999 vs 272 of 20,106; 0·96, 0·81-1·14, p=0·631; CIN grade 2 or worse: 427 of 19 999 vs 399 of 20,106; 1·08, 0·94-1·24; p=0·292), nor for subgroups of women invited for the first time (CIN grade 3 or worse in women aged 29-33 years: 102 of 3139 vs 105 of 3128; 0·97, 0·74-1·27; CIN grade 2 or worse in women aged 29-33 years: 153 of 3139 vs 151 of 3128; 1·01, 0·81-1·26; CIN grade 3 or worse in women aged 34-56 years: 157 of 16,860 vs 167 of 16 978; 0·95, 0·76-1·18; CIN grade 2 or worse in women aged 34-56 years: 274 of 16,860 vs 248 of 16 978; 1·11, 0·94-1·32). INTERPRETATION: Implementation of HPV DNA testing in cervical screening leads to earlier detection of clinically relevant CIN grade 2 or worse, which when adequately treated, improves protection against CIN grade 3 or worse and cervical cancer. Early detection of high-grade cervical legions caused by HPV16 was a major component of this benefit. Our results lend support to the use of HPV DNA testing for all women aged 29 years and older. FUNDING: Zorg Onderzoek Nederland (Netherlands Organisation for Health Research and Development).

Experience with high-risk human papillomavirus testing on vaginal brush-based self-samples of non-attendees of the cervical screening program.

We evaluated the effect of offering brush-based vaginal self-sampling for high-risk human papillomavirus (hrHPV) testing to non-attendees of the cervical screening program on response rate, compliance to follow-up and cervical intraepithelial neoplasia grade 2 or 3 (CIN2+/CIN3+) yield. In addition, concordance of hrHPV test results between physician-taken cervical scrapes and vaginal self-samples was determined. A total of 26,409 nonattending women were randomly assigned to receive a vaginal brush device for hrHPV testing by Hybrid Capture-2 method (i.e., self-sampling group, n = 26,145) or a reinvitation for regular cytology-based screening (i.e., recall control group, n = 264). hrHPV-positive self-sampling responders were invited for a physician-taken scrape for cytology and blinded hrHPV testing. If cytology was abnormal, women were referred for colposcopy. Response rate in the self-sampling group was significantly increased compared to the recall control group (30.8% versus 6.5%; p < 0.001). The concordance rate between hrHPV detection in self-samples and corresponding physician-taken cervical scrape samples was 68.8%. Amongst women with CIN3+ and CIN2+, the concordance rates in hrHPV positivity between both samples were 95.5% and 93.8%, respectively. Adherence at baseline to cytology triage of hrHPV-positive self-sampling women (89.1%) and colposcopy referral of those with abnormal cytology (95.8%) was high. The CIN2+/CIN3+/carcinoma yields were 1.5%, 1.0% and 0.1%, respectively, in self-sampling responders. In conclusion, offering hrHPV testing on self-sampled vaginal material with a brush device to non-attendees significantly increases the attendance to the regular screening program, yields hrHPV test results that are in very good concordance with those of physician-taken scrapes in women with CIN2+/CIN3+, and is effective in detecting CIN2+/CIN3+.

Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening.

High-risk human papillomavirus (hrHPV) testing has a higher sensitivity but lower specificity than cytology for detection of high-grade intraepithelial neoplasia (CIN). To avoid over-referral to colposcopy and overtreatment, hrHPV-positive women require triage testing and/or followup. A total of 25,658 women (30-60 years) enrolled in a population-based cohort study had an adequate baseline Pap smear and hrHPV test. The end-point was cumulative two-year risk of CIN grade 3 or worse (CIN3+). In a post-hoc analysis, fourteen triage/followup strategies for hrHPV-positive women (n = 1,303) were evaluated for colposcopy referral rate, positive (PPV) and negative predictive value (NPV). Five strategies involved triage testing without a repeat test and nine strategies involved triage testing followed by one repeat testing. The tests were cytology, hrHPV, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping. Results were adjusted for women in the cohort study who did not attend repeat testing. Of the strategies without repeat testing, combined cytology and HPV16/18/31/33/45 genotyping gave the highest NPV of 98.9% (95%CI 97.6-99.5%). The corresponding colposcopy referral rate was 58.1% (95%CI 55.4-60.8%). Eight of the nine strategies with retesting had an estimated NPV of at least 98%. Of those, cytology triage followed by cytology at 12 months had a markedly lower colposcopy referral rate of 33.4% (95%CI 30.2-36.7%) than the other strategies. The NPV of the latter strategy was 99.3% (95%CI 98.1-99.8%). Triage hrHPV-positive women with cytology, followed by repeat cytology testing yielded a high NPV and modest colposcopy referral rate and appear to be the most feasible management strategy.

Comparison of HPV and cytology triage algorithms for women with borderline or mild dyskaryosis in population-based cervical screening (VUSA-screen study).

We studied the effectiveness of high-risk human papillomavirus (hrHPV) triage for immediate colposcopy in women with borderline or mild dyskaryosis (BMD). In the Utrecht province of the Netherlands, women aged 30-60 years who participated in the regular cervical screening programme were offered hrHPV testing and cytology (intervention group) or cytology only (control group). In the intervention group (n = 337), women with BMD were immediately referred for colposcopy only if the sample was hrHPV positive. Women with a hrHPV negative test were advised to repeat cytology at 6 and 18 months and were referred for colposcopy if and when the repeat test result was positive (BMD or worse). In the control group (n = 329), referral of women with BMD was delayed until cytology was repeatedly positive at 6 or 18 months. The CIN3 detection rates were 10.7% (36/337) in the intervention group and 6.4% (21/329) in the control group (p = 0.047). Moreover, hrHPV triaging resulted in shorter time to diagnosis (154 vs. 381 days). Although the number of colposcopy referrals was 51.5% higher in the intervention group than in the control group, the medical costs per detected CIN3 were slightly lower ([euro] 4781 vs. [euro] 6235). If, in addition, hrHPV negative women had been referred back to routine screening at baseline, the CIN3 rate would have been 10.1% (34/337) and colposcopy rate would only have been 30.4% higher than in the control group. This study shows that hrHPV triaging of women with BMD is at least as effective for detecting CIN3 as repeat cytology, also when hrHPV negative women are referred back to routine screening.

Renal imaging in 199 Dutch patients with Birt-Hogg-Dubé syndrome: Screening compliance and outcome.

Birt-Hogg-Dubé syndrome is associated with an increased risk for renal cell carcinoma. Surveillance is recommended, but the optimal imaging method and screening interval remain to be defined. The main aim of our study was to evaluate the outcomes of RCC surveillance to get insight in the safety of annual US in these patients. Surveillance data and medical records of 199 patients with Birt-Hogg-Dubé syndrome were collected retrospectively using medical files and a questionnaire. These patients were diagnosed in two Dutch hospitals and data were collected until June 2014. A first screening for renal cell carcinoma was performed in 172/199 patients (86%). Follow-up data were available from 121 patients. The mean follow-up period per patient was 4.2 years. Of the patients known to be under surveillance, 83% was screened at least annually and 94% at least every two years. Thirty-eight renal cell carcinomas had occurred in 23 patients. The mean age at diagnosis of the first tumour was 51. Eighteen tumours were visualized by ultrasound. Nine small tumours (7-27 mm) were visible on MRI or CT and not detected using ultrasound. Our data indicate that compliance to renal screening is relatively high. Furthermore, ultrasound might be a sensitive, cheap and widely available alternative for MRI or part of the MRIs for detecting clinically relevant renal tumours in BHD patients,but the limitations should be considered carefully. Data from larger cohorts are necessary to confirm these observations.

Reproducibility and Prognostic Performance of the 1973 and 2004 World Health Organization Classifications for Grade in Non-muscle-invasive Bladder Cancer: A Multicenter Study in 328 Bladder Tumors.

BACKGROUND: Histologic grade is an important prognosticator in patients with non-muscle-invasive bladder cancer (NMIBC). Currently, 2 classifications for grade are widely used; the World Health Organization (WHO) 1973 and the WHO 2004. We compare inter-observer variability of both classifications and investigate which histologic criteria cause this variability. Furthermore, the prognostic value of both classifications was assessed. PATIENTS AND METHODS: Three pathologists reviewed 328 bladder tissue samples of 232 patients with NMIBC in a blinded manner. WHO 1973 grade, WHO 2004 grade, histologic criteria of both classifications, and T-category were evaluated. Reproducibility was analyzed using the weighted Fleiss κ, association between criteria scores and grade with the χ2 test, and time-to-recurrence and time-to-progression with the log-rank test and Cox regression. RESULTS: Reproducibility of both classifications was poor. The WHO 2004 showed better reproducibility (κ = 0.35; 95% confidence interval (CI), 0.29-0.42) compared with the WHO 1973 as a 3-tiered (κ = 0.24; 95% CI, 0.19-0.28), but not as a 2-tiered (G1 + G2 vs. G3) classification (κ = 0.36; 95% CI, 0.29-0.42). Reproducibility of individual criteria was poor (κ range, -0.05 to 0.25). All criteria were associated with grade (P < .05). After a median follow-up of 60 months, 33 of 232 and 112 of 232 patients developed progression and recurrence, respectively. In 1 out of the 3 pathologists, progression was predicted by both the WHO 1973 grade and the WHO 2004 grade in multivariable analysis. Recurrence was not predicted by grade (multivariable). CONCLUSIONS: Reproducibility of the WHO 2004 and WHO 1973 classification for grade are poor. Scoring of individual criteria is poorly reproducible, suggesting that descriptions of these criteria for grade are not specific. The prognostic value of both the WHO 1973 and the WHO 2004 differ per pathologist.

Selection of women at risk for cervical cancer in an HIV-infected South African population.

OBJECTIVE: Cervical cancer is the leading cause of cancer-related death in women in South Africa. This study evaluates DNA methylation levels in cervical (pre)cancer and aims to assess the value of high-risk human papillomavirus (hrHPV) testing and methylation analysis, alone or in combination, on physician-taken cervical scrapes to detect cervical cancer, and cervical intraepithelial neoplasia grade 3 (CIN3) in an HIV-infected South African population. DESIGN: Prospective observational multicentre cohort study. METHODS: Women from a cohort of women living with HIV (n = 355) and a referral cohort (n = 109, 60% HIV seropositive) were included. Cervical scrapes were collected for hrHPV testing and methylation analysis of cell adhesion molecule 1, T-lymphocyte maturation-associated protein, and microRNA124-2 genes. Histologic endpoints were available for all participants. Performance for detection of CIN3 or worse (CIN3+) was determined in the cohort of women living with HIV and different testing strategies were compared. RESULTS: HrHPV and methylation positivity rates increased with severity of cervical disease in the two study cohorts, each reaching 100% in samples of women with carcinoma. HrHPV testing showed a sensitivity for CIN3+ of 83.6%, at a specificity of 67.7%. Methylation analysis showed a comparable CIN3+ sensitivity of 85.2%, but a significantly lower specificity of 49.6%. HrHPV testing with reflex methylation analysis showed a CIN3+ sensitivity of 73.8%, at a specificity of 81.5%. CONCLUSION: In this HIV-infected South African population, stratifying hrHPV-positive women with reflex methylation analysis detects all cervical carcinomas and yields an acceptable sensitivity and specificity for CIN3+.

Safety of extending screening intervals beyond five years in cervical screening programmes with testing for high risk human papillomavirus: 14 year follow-up of population based randomised cohort in the Netherlands.

OBJECTIVES:  To provide an early risk assessment of extending screening intervals beyond five years for a human papillomavirus (HPV) based cervical screening programme in the Netherlands. DESIGN:  14 year follow-up of a population based randomised cohort from the POBASCAM randomised trial. SETTING:  Organised cervical screening in the Netherlands, based on a programme of three screening rounds (each round done every five years). PARTICIPANTS:  43 339 women aged 29-61 years with a negative HPV and/or negative cytology test participating in the POBASCAM trial. INTERVENTIONS:  Women randomly assigned to HPV and cytology co-testing (intervention) or cytology testing only (control), and managed accordingly. MAIN OUTCOME MEASURES:  Cumulative incidence of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+). Associations with age were expressed as incidence rate ratios. In HPV positive women, reductions in CIN3+ incidence after negative cytology, HPV type 16/18 genotyping, and/or repeat cytology were estimated. RESULTS:  The cumulative incidence of cervical cancer (0.09%) and CIN3+ (0.56%) among HPV negative women in the intervention group after three rounds of screening were similar to the cumulative among women with negative cytology in the control group after two rounds (0.09% and 0.69%, respectively). Cervical cancer and CIN3+ risk ratios were 0.97 (95% confidence interval 0.41 to 2.31, P=0.95) and 0.82 (0.62 to 1.09, P=0.17), respectively. CIN3+ incidence was 72.2% (95% confidence interval 61.6% to 79.9%, P<0.001) lower among HPV negative women aged at least 40 years than among younger women. No significant association between cervical cancer incidence and age could be demonstrated. CIN3+ incidence among HPV positive women with negative cytology, HPV 16/18 genotyping, and/or repeat cytology was 10.4 (95% confidence interval 5.9 to 18.4) times higher than among HPV negative women. CONCLUSIONS:  Long term incidences of cervical cancer and CIN3+ were low among HPV negative women in this study cohort, and supports an extension of the cervical screening interval beyond five years for women aged 40 years and older. HPV positive women with subsequent negative cytology, HPV16/18 genotyping, and/or repeat cytology have at least a fivefold higher risk of CIN3+ than HPV negative women, indicating that HPV based programmes with long intervals (>five years) should be implemented with risk stratification.Trial registration POBASCAM trial number ISRCTN20781131.

miR-129-3p controls centrosome number in metastatic prostate cancer cells by repressing CP110.

The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.

Ventricular myocarditis coincides with atrial myocarditis in patients.

INTRODUCTION: Atrial fibrillation (AF) is a common complication in myocarditis. Atrial inflammation has been suggested to play an important role in the pathophysiology of AF. However, little is known about the occurrence of atrial inflammation in myocarditis patients. Here, we analyzed inflammatory cell numbers in the atria of myocarditis patients without symptomatic AF. METHODS: Cardiac tissue was obtained postmortem from lymphocytic myocarditis patients (n=6), catecholamine-induced myocarditis patients (n=5), and control patients without pathological evidence of heart disease (n=5). Tissue sections of left and right ventricle and left and right atrium were stained for myeloperoxidase (neutrophilic granulocytes), CD45 (lymphocytes), and CD68 (macrophages). These cells were subsequently quantified in atrial and ventricular myocardium and atrial adipose tissue. RESULTS: In lymphocytic myocarditis patients, a significant increase was observed for lymphocytes in the left atrial adipose tissue. In catecholamine-induced myocarditis patients, significant increases were found in the atria for all three inflammatory cell types. Infiltrating inflammatory cell numbers in the atrial myocardium correlated positively with those in the ventricles, especially in catecholamine-induced myocarditis patients. CONCLUSIONS: To a varying extent, atrial myocarditis occurs concurrently with ventricular myocarditis in patients diagnosed with myocarditis of different etiology. This provides a substrate that potentially predisposes myocarditis patients to the development of AF and subsequent complications such as sudden cardiac death and heart failure.

Somatic mutation in PIK3CA is a late event in cervical carcinogenesis.

Somatic mutations in cervical intraepithelial neoplasia (CIN) are largely unknown. Here, we profiled 35 cervical carcinomas and 23 CIN grade 2/3 (CIN2/3) for mutations in 48 cancer-related genes using a Next Generation Sequencing-based cancer panel. PIK3CA exon 9 was the most frequently mutated locus in cervical carcinoma and the only mutated locus detected in CIN2/3. These PIK3CA exon 9 mutation findings were verified in a large, independent series (n = 647) covering all stages of cervical carcinogenesis using high resolution melting-guided Sanger sequencing. PIK3CA exon 9 mutation frequency was 37.1% (13/35; 95%CI 21.2-54.0%) in cervical carcinoma, and 2.4% (5/209; 95%CI 0.5-4.7%) in CIN3. No PIK3CA exon 9 mutations were detected in CIN2 (0/144), CIN1 (0/154) and normal cervix (0/105). In a third series of 46 CIN2/3 lesions from women with a known 5-year history of preceding high-risk human papillomavirus (hrHPV) infection, detection of PIK3CA exon 9 mutation was confined to 2 (5.4%; 95%CI 0.0-13.2%) CIN3 lesions with preceding hrHPV infection ≥5 years, and was absent in those with a short duration (<5 years) of preceding hrHPV infection. In conclusion, somatic mutation in PIK3CA represents a late event during cervical carcinogenesis, detected in a substantial subset of cervical carcinoma, but only in a minority of CIN3.