Accurate detection of copy number aberrations in FFPE samples using the mFAST-SeqS approach.

BACKGROUND: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. METHODS: Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. RESULTS: Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. CONCLUSION: mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.

Analytical validation and diagnostic performance of the ASCL1/ZNF582 methylation test for detection of high-grade anal intraepithelial neoplasia and anal cancer.

DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN). For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10-8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity. In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.

Exposure Opportunity: The Advantages of Including Men in Analyses of Female-Related Risk Factors.

Intuitively, researchers do not include subjects who do not have the opportunity to be exposed, such as men in studies on oral contraceptives (OCs). We aimed to explore in which situations it is nevertheless beneficial to do so. We considered the effect of including men in case-control analyses of 8 different hypothetical data sets on the effect of OC use and venous thrombosis. In all scenarios, OC use was the exposure of interest, sex the factor that determined exposure opportunity, and air travel another risk factor. In some of these scenarios, sex and air travel were included as confounders or effect modifiers. Logistic regression was used to estimate odds ratios. Standard errors of the estimated log odds ratios, including and excluding men, were compared. We also studied the effect of including men using data from 1999-2004 from a case-control study on risk factors for venous thrombosis, conducted in the Netherlands. In all hypothetical examples, and in the real-data study, addition of men to the analysis yielded the same odds ratios when correctly adjusting for confounding. Moreover, use of additional subjects often led to more precise estimates. We suggest that subjects who do not have the opportunity to be exposed should not routinely be excluded from epidemiologic studies.

Cervical cancer incidence after normal cytological sample in routine screening using SurePath, ThinPrep, and conventional cytology: population based study.

Objective To compare the cumulative incidence of cervical cancer diagnosed within 72 months after a normal screening sample between conventional cytology and liquid based cytology tests SurePath and ThinPrep.Design Retrospective population based cohort study.Setting Nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA), January 2000 to March 2013.Population Women with 5 924 474 normal screening samples (23 833 123 person years).Exposure Use of SurePath or ThinPrep versus conventional cytology as screening test.Main outcome measure 72 month cumulative incidence of invasive cervical cancer after a normal screening sample for each screening test. Cox regression analyses assessed the hazard ratios, adjusted for calendar time, age, screening history, and socioeconomic status and including laboratories as random effects.Results The 72 month cumulative cancer incidence was 58.5 (95% confidence interval 54.6 to 62.7) per 100 000 normal conventional cytology samples, compared with 66.8 (56.7 to 78.7) for ThinPrep and 44.6 (37.8 to 52.6) for SurePath. Compared with conventional cytology, the hazard of invasive cancer was 19% lower (hazard ratio 0.81, 95% confidence interval 0.66 to 0.99) for SurePath, mainly caused by a 27% lower hazard (0.73, 0.57 to 0.93) of a clinically detected cancer. For ThinPrep, the hazard was on average 15% higher (hazard ratio 1.15, 0.95 to 1.38), mainly caused by a 56% higher hazard of a screen detected cancer (1.56, 1.17 to 2.08).Conclusions These findings should provoke reconsideration of the assumed similarity in sensitivity to detect progressive cervical intraepithelial neoplasia between different types of liquid based cytology and conventional cytology.

Public Health Benefits of Routine Human Papillomavirus Vaccination for Adults in the Netherlands: A Mathematical Modeling Study.

BACKGROUND: Expanding routine human papillomavirus (HPV) vaccination to adults could be an effective strategy to improve prevention of HPV infection and cervical cancer. METHODS: We evaluated the following adult vaccination strategies for women only and for both women and men in addition to the current girls-only vaccination program in the Netherlands, using the established STDSIM microsimulation model: one-time mass campaign, vaccination at the first cervical cancer screening visit, vaccination at sexual health clinics, and combinations of these strategies. RESULTS: The estimated impact of expanding routine vaccination to adult women is modest, with the largest incremental reductions in the incidence of HPV infection occurring when offering vaccination both at the cervical cancer screening visit and during sexually transmitted infection (STI) consultations (about 20% lower after 50 years for both HPV-16 and HPV-18). Adding male vaccination during STI consultations leads to more-substantial incidence reductions: 63% for HPV-16 and 84% for HPV-18. The incremental number needed to vaccinate among women is 5.48, compared with 0.90 for the current vaccination program. CONCLUSIONS: Offering vaccination to adults, especially at cervical cancer screening visits (for women) and during STI consultations (for both sexes), would substantially reduce HPV incidence and would be an efficient policy option to improve HPV prevention and subsequently avert cervical and possibly male HPV-related cancers.

The role of pre-invasive disease in overdiagnosis: A microsimulation study comparing mass screening for breast cancer and cervical cancer.

OBJECTIVE: Although early detection of cancer through screening can prevent cancer deaths, a drawback of screening is overdiagnosis. Overdiagnosis has been much debated in breast cancer screening, but less so in cervical cancer screening. We examined the impact of overdiagnosis by comparing two screening programmes in the Netherlands. METHODS: We estimated overdiagnosis rates by microsimulation for breast cancer screening and cervical cancer screening, using a cohort of women born in 1982 with lifelong follow-up. Overdiagnosis estimates were made analogous to two definitions formed by the UK 2012 breast screening review. Pre-invasive disease was included in both definitions. RESULTS: Screening prevented 921 cervical cancers (-55%) and 378 cervical cancer deaths (-59%), and 169 (-1.3%) breast cancer cases and 970 breast cancer deaths (-21%). The cervical cancer overdiagnosis rate was 74.8% (including pre-invasive disease). Breast cancer overdiagnosis was estimated at 2.5% (including pre-invasive disease). For women of all ages in breast cancer screening, an excess of 207 diagnoses/100,000 women was found, compared with an excess of 3999 diagnoses/100,000 women in cervical cancer screening. CONCLUSIONS: For breast cancer, the frequency of overdiagnosis in screening is relatively low, but consequences are evident. For cervical cancer, the frequency of overdiagnosis in screening is high, because of detection of pre-invasive disease, but the consequences per case are relatively small due to less invasive treatment. This illustrates that it is necessary to present overdiagnosis in relation to disease stage and consequences.

Cervical Cancer Screening in Partly HPV Vaccinated Cohorts - A Cost-Effectiveness Analysis.

BACKGROUND: Vaccination against the oncogenic human papillomavirus (HPV) types 16 and 18 will reduce the prevalence of these types, thereby also reducing cervical cancer risk in unvaccinated women. This (measurable) herd effect will be limited at first, but is expected to increase over time. At a certain herd immunity level, tailoring screening to vaccination status may no longer be worth the additional effort. Moreover, uniform screening may be the only viable option. We therefore investigated at what level of herd immunity it is cost-effective to also reduce screening intensity in unvaccinated women. METHODS: We used the MISCAN-Cervix model to determine the optimal screening strategy for a pre-vaccination population and for vaccinated women (~80% decreased risk), assuming a willingness-to-pay of €50,000 per quality-adjusted life year gained. We considered HPV testing, cytology testing and co-testing and varied the start age of screening, the screening interval and the number of lifetime screens. We then calculated the incremental cost-effectiveness ratio (ICER) of screening unvaccinated women with the strategy optimized to the pre-vaccination population as compared to with the strategy optimized to vaccinated women, assuming different herd immunity levels. RESULTS: Primary HPV screening with cytology triage was the optimal strategy, with 8 lifetime screens for the pre-vaccination population and 3 for vaccinated women. The ICER of screening unvaccinated women 8 times instead of 3 was €28,085 in the absence of herd immunity. At around 50% herd immunity, the ICER reached €50,000. CONCLUSION: From a herd immunity level of 50% onwards, screening intensity based on the pre-vaccination risk level becomes cost-ineffective for unvaccinated women. Reducing the screening intensity of uniform screening may then be considered.

Comparing SurePath, ThinPrep, and conventional cytology as primary test method: SurePath is associated with increased CIN II+ detection rates.

PURPOSE: Within the last decade, SurePath and ThinPrep [both liquid-based cytology (LBC) tests] have replaced conventional cytology (CC) as primary test method in cervical cancer screening programs of multiple countries. The aim of our study was to examine the effect in the Dutch screening program. METHODS: All primary smears taken within this program from 2000 to 2011 were analyzed using the nationwide registry of histo- and cytopathology (PALGA) with a follow-up until March 2013. The percentage of smears classified as borderline/mildly dyskaryotic (BMD) and >BMD as well as CIN and cervical cancer detection rates were compared between SurePath and ThinPrep versus CC by logistic regression analyses (adjusted for age, screen region, socioeconomic status, and calendar time). RESULTS: We included 3,118,685 CC, 1,313,731 SurePath, and 1,584,587 ThinPrep smears. Using SurePath resulted in an increased rate of primary smears classified as >BMD [odds ratio (OR) = 1.12 (95% confidence interval (CI) 1.09-1.16)]. CIN I and II(+) detection rates increased by 14 % [OR = 1.14 (95% CI 1.08-1.20)] and 8 % [OR = 1.08 (95% CI 1.05-1.12)]. Cervical cancer detection rates were unaffected. Implementing ThinPrep did not result in major alterations of the cytological classification of smears, and it did not affect CIN detection rates. While not significant, cervical cancer detection rates were lower [OR = 0.87 (95% CI 0.75-1.01)]. CONCLUSIONS: The impact of replacing CC by LBC as primary test method depends on the type of LBC test used. Only the use of SurePath was associated with increased CIN II(+) detection, although it simultaneously increased the detection of CIN I.

Cervical histology after routine ThinPrep or SurePath liquid-based cytology and computer-assisted reading in Denmark.

BACKGROUND: We compared the sensitivity and specificity of liquid-based cytology (LBC) and computer-assisted reading for SurePath/FocalPoint and ThinPrep with those of manually read conventional cytology in routine cervical screening in four Danish laboratories. METHODS: Using data from five nationwide registers, technological phases were identified by slide preparation, reading technique, and triage of borderline cytology. Trends in the detection of cervical intraepithelial neoplasia (CIN) were an indicator of the technology's relative sensitivity, and trends in false-positive tests an indicator of relative specificity. RESULTS: At 23-29 years, SurePath/FocalPoint statistically significantly increased the detection of ⩾CIN3 by 85% compared with manually read conventional cytology. The 11% increase with ThinPrep was not significant. At 30-44 years, the increase with SurePath/FocalPoint was 58%; the 16% increase with ThinPrep was not significant. At 45-59 years, both technologies led to nonsignificant decreases in the detection. SurePath/FocalPoint doubled the frequency of false-positive tests at any age. With ThinPrep, these proportions remained the same at 23-29 years, but decreased by two-thirds at 45-59 years. In a fourth laboratory with continuous use of manually read conventional cytology, no such trends were seen. CONCLUSIONS: The sensitivity and specificity of modern LBC and computer-assisted reading technologies may be brand- and age-dependent.

The estimated impact of natural immunity on the effectiveness of human papillomavirus vaccination.

BACKGROUND: Mathematical modelling is used to estimate the effectiveness of HPV vaccination. These estimates depend strongly on herd immunity and thus on naturally acquired immunity, a mechanism of which little is known. We estimated the impact of different vaccination strategies on HPV-16 and HPV-18 transmission and cervical cancer incidence in the Netherlands, considering different acquired immunity mechanisms. METHODS: We used the STDSIM microsimulation model, and considered two mechanisms for acquired immunity after infection: (I) full immunity with variable duration; (II) cumulatively decreasing susceptibility to reinfection. Girls aged 13-16 years received vaccination (94.7% efficacy for HPV-16 and 92.3% for HPV-18) during a once-off catch-up campaign with 50% coverage, followed by annual vaccination of 12-year-old girls (60% coverage). Alternative vaccination scenarios included increased coverage, including boys, and lower vaccine efficacy. RESULTS: HPV-16 incidence reduced by 64% under mechanism I and 75% under mechanism II; HPV-18 incidence reduced by 58% and 73%, respectively, and these reductions lead to 48-56% fewer cervical cancer cases. Increasing coverage can lead to over 96% reduction in HPV incidence. Vaccinating boys reduced incidence by 79-89% for HPV-16 and 83-98% for HPV-18 in women. CONCLUSIONS: Effectiveness estimates of HPV vaccination differ slightly between different acquired immunity mechanisms, yet these differences are unlikely to affect policy decisions. Offering vaccination to boys as well may be considered to further reduce cancer incidence.

Exploring the trend of increased cervical intraepithelial neoplasia detection rates in the Netherlands.

OBJECTIVE: Over the last decade, cervical intraepithelial neoplasia (CIN) detection has increased in the Netherlands. We investigated the underlying mechanism by quantifying the increase, and analyzing patterns of CIN and cervical cancer detection over time. METHODS: We observed annual CIN and cervical cancer detection rates (DRs) per 10,000 primary smears within the Dutch screening programme for 2000-2011. Joinpoint analyses were performed to determine changes in time trends, logistic regression analyses to assess the relative risk of calendar time on histological outcomes, adjusted for demographic factors and type of primary cytology test used. RESULTS: Trends of increased detection occurred for all CIN grades (ie. DRs increased from 17.8 to 36.1, from 21.0 to 35.5, and from 43.4 to 64.6 for CIN I, II, and III from 2003 to 2009). After adjusting for demographic factors, DRs were still 2.11 (95% confidence interval (CI): 1.95, 2.29), 1.79 (95% CI: 1.66, 1.92) and 1.59 (95% CI: 1.50, 1.67) times larger in 2009. When also adjusting for the type of cytology test, DRs were 1.90 (95% CI: 1.62, 2.22), 1.48 (95% CI: 1.22, 1.79) and 1.55 (95% CI: 1.39, 1.73) times larger. No trends in cervical cancer DRs were found. CONCLUSIONS: The implementation of liquid-based cytology contributed to the CIN increase. If some of these extra detected CIN are regressive this leads to overdiagnosis. Other factors, such as an increased cervical cancer risk, and implementation of imaging-assisted reading, could also have contributed.

Offering Self-Sampling to Non-Attendees of Organized Primary HPV Screening: When Do Harms Outweigh the Benefits?

BACKGROUND: Human papillomavirus (HPV) self-sampling might be a promising tool to increase effectiveness of primary HPV screening programs when offered to non-attendees. However, effectiveness could decrease if regular attendees "switch" to self-sampling, because self-sampling test characteristics may be inferior. We examined under which conditions the harms would outweigh the benefits. METHODS: The MISCAN-cervix model was used to estimate quality-adjusted life years (QALY) gained and costs of offering HPV self-sampling to non-attendees. We varied the relative CIN2(+) sensitivity and specificity (self-sampling vs. regular sampling), extra attendance, risk of extra attendees, and the switching percentage. RESULTS: Without switching, offering self-sampling is (cost-)effective under every studied condition. If the attendance due to self-sampling increases by ≥6 percentage points, higher primary background risk women (unscreened women who will never attend regular screening) attend and the relative CIN2(+) sensitivity and specificity are ≥0.95; it is (cost-)effective to offer self-sampling to non-attendees, even if all regular attendees switch. If the relative sensitivity decreases to 0.90 combined with either a 3 percentage points extra attendance or the absence of higher primary background risk women, QALYs are lost when more than 30% to 20% of the regular attendees switch. CONCLUSIONS: Offering self-sampling will gain health effects if the relative CIN2(+) sensitivity is ≥0.95, unscreened attendees are recruited, and the total attendance increases by ≥6 percentage points. Otherwise, switching of regular attendees may decrease the total effectiveness of the program. IMPACT: Self-sampling needs to be implemented with great care and advantages of office-based sampling need to be emphasized to prevent switching.

How many cervical cancer cases can potentially be prevented using a more sensitive screening test at young age?

The human papilloma virus (HPV) DNA test has higher sensitivity than cytology for cervical cancer screening. Therefore, cervical cancer cases that are missed by cytology could potentially be identified if we use primary HPV testing. Studies showed that HPV screening is the preferred primary test at age 35 and over. Given the high prevalence of harmless HPV infections, the use of HPV testing at younger age is less obvious. The number of cancers in young age is often mentioned to indicate the possible benefits of a more sensitive test. We actually estimated the proportion of those cases that is potentially preventable in The Netherlands by the use of a more sensitive screen-test at the first screening age 30, given that the more sensitive test is used at age 35 and over. We analysed the screening history of women diagnosed with cervical cancer in the period 2004 to March 2009, using data from the Dutch National Pathology Registry. Only 15-30% (two to four cases per 100,000 women) of the cases was preceded by negative cytology under age 35 and therefore could have been prevented by a more sensitive test at age 30. The lower the screening coverage and the shorter the screening interval in those screened at young age, the smaller the gain of a more sensitive test. So, as long as the current screening pattern is not changed, the majority of the cervical cancer cases at young age would still occur even when applying a more sensitive test at the younger ages.