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This review is based on the recent EUROGIN scientific session: “Assessing risk of cervical cancer in the post-vaccination era,” which addressed the demands of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesion (SIL) triage now that the prevalence of vaccine-targeted oncogenic high-risk (hr) human papillomaviruses (HPVs) is decreasing. Change in the prevalence distribution of oncogenic HPV types that follows national HPV vaccination programs is setting the stage for loss of positive predictive value of conventional but possibly also new triage modalities. Understanding the contribution of the latter, most notably hypermethylation of cellular and viral genes in a new setting where most oncogenic HPV types are no longer present, requires studies on their performance in vaccinated women with CIN/SIL that are associated with nonvaccine HPV types. Lessons learned from this research may highlight the potential of cervical cells for risk prediction of all women's cancers.

Endometrial cancer incidence is rising and current diagnostics often require invasive biopsy procedures. DNA methylation marker analysis of minimally- and non-invasive sample types could provide an easy-to-apply and patient-friendly alternative to determine cancer risk. Here, we compared the performance of DNA methylation markers to detect endometrial cancer in urine, cervicovaginal self-samples and clinician-taken cervical scrapes. Paired samples were collected from 103 patients diagnosed with stage I to IV endometrial cancer. Urine and self-samples were collected at home. All samples were tested for nine DNA methylation markers using quantitative methylation-specific PCR. Methylation levels measured in endometrial cancer patients were compared to unpaired samples of 317 healthy controls. Diagnostic performances were evaluated by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Each methylation marker showed significantly higher methylation levels in all sample types of endometrial cancer patients compared to healthy controls (P

Background High-grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasia (CIN) grade 2/3 lesions in human papillomavirus (HPV)–positive women Methods A European multicenter retrospective study was designed evaluating the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in cervical scrapes of 1061 HPV-positive women aged 15–29 years (690 ≤CIN1, 166 CIN2, and 205 CIN3+). A subset of 62 CIN2 and 103 CIN3 were immunohistochemically characterized by HPV E4 expression, a marker for a productive HPV infection, and p16ink4a and Ki-67, markers indicative for a transforming infection. CIN2/3 lesions with low HPV E4 expression and high p16ink4a/Ki-67 expression were considered as nonproductive, transforming CIN, compatible with advanced CIN2/3 lesions in need of treatment. Results FAM19A4/miR124-2 methylation positivity increased significantly with CIN grade and age groups ( Conclusions Compared with HPV16/18 genotyping, the FAM19A4/miR124-2 methylation test detects nonproductive, transforming CIN2/3 lesions with high specificity in women aged

PDF file - 46K, Metabolite analysis. Mean fractions of parent 11Cerlotinib, nonpolar metabolites and polar metabolites for groups 1 (filled symbols) and 2 (open symbols). Vertical bars indicate standard deviations.

PDF file - 43K, Synthesis of 11Cerlotinib. Reaction conditions: 1.0 mg (2.6 μmole) of (1) 6-O-desmethyl-erlotinib, 11CCH3I, 250 μL of acetonitrile, 4.6 μmole of tetra-n-butylammonium hydroxide (2.0 μL of a 60% solution in water), 80 {degree sign}C, 5 min.

Purpose: To evaluate whether, in patients with non–small cell lung carcinoma (NSCLC), tumor uptake of [11C]erlotinib can be quantified and imaged using positron emission tomography and to assess whether the level of tracer uptake corresponds with the presence of activating tumor EGF receptor (EGFR) mutations.Experimental Design: Ten patients with NSCLCs, five with an EGFR exon 19 deletion, and five without were scanned twice (test retest) on the same day with an interval of at least 4 hours. Each scanning procedure included a low-dose computed tomographic scan, a 10-minute dynamic [15O]H2O scan, and a 1-hour dynamic [11C]erlotinib scan. Data were analyzed using full tracer kinetic modeling. EGFR expression was evaluated using immunohistochemistry.Results: The quantitative measure of [11C]erlotinib uptake, that is, volume of distribution (VT), was significantly higher in tumors with activating mutations, that is, all with exon 19 deletions (median VT, 1.76; range, 1.25–2.93), than in those without activating mutations (median VT, 1.06; range, 0.67–1.22) for both test and retest data (P = 0.014 and P = 0.009, respectively). Good reproducibility of [11C]erlotinib VT was seen (intraclass correlation coefficient = 0.88). Intergroup differences in [11C]erlotinib uptake were not correlated with EGFR expression levels, nor tumor blood flow.Conclusion: [11C]erlotinib VT was significantly higher in NSCLCs tumors with EGFR exon 19 deletions. Clin Cancer Res; 19(1); 183–93. ©2012 AACR.

Background Epstein–Barr virus positivity (EBV+) and microsatellite instability (MSI-high) are positive prognostic factors for survival in resectable gastric cancer (GC). However, benefit of perioperative treatment in patients with MSI-high tumors remains topic of discussion. Here, we present the clinicopathological outcomes of patients with EBV+, MSI-high, and EBV−/MSS GCs who received either surgery only or perioperative treatment. Methods EBV and MSI status were determined on tumor samples collected from 447 patients treated with surgery only in the D1/D2 trial, and from 451 patients treated perioperatively in the CRITICS trial. Results were correlated to histopathological response, morphological tumor characteristics, and survival. Results In the D1/D2 trial, 5-year cancer-related survival was 65.2% in 47 patients with EBV+, 56.7% in 47 patients with MSI-high, and 47.6% in 353 patients with EBV−/MSS tumors. In the CRITICS trial, 5-year cancer-related survival was 69.8% in 25 patients with EBV+, 51.7% in 27 patients with MSI-high, and 38.6% in 402 patients with EBV−/MSS tumors. Interestingly, all three MSI-high tumors with moderate to complete histopathological response (3/27, 11.1%) had substantial mucinous differentiation. No EBV+ tumors had a mucinous phenotype. 115/402 (28.6%) of EBV−/MSS tumors had moderate to complete histopathological response, of which 23/115 (20.0%) had a mucinous phenotype. Conclusions In resectable GC, MSI-high had favorable outcome compared to EBV−/MSS, both in patients treated with surgery only, and in those treated with perioperative chemo(radio)therapy. Substantial histopathological response was restricted to mucinous MSI-high tumors. The mucinous phenotype might be a relevant parameter in future clinical trials for MSI-high patients.

Methylation of host-cell deoxyribonucleic acid (DNA) has been proposed as a promising biomarker for triage of high-risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host-cell DNA methylation markers for triage in an hrHPV-positive cohort derived from primary HPV-based cervical screening in The Netherlands. Methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST were evaluated relative to the ACTB reference gene by multiplex quantitative methylation-specific PCR (qMSP) in clinician-collected cervical samples (n = 715) from hrHPV-positive women (age 29-60 years), who were enrolled in the Dutch IMPROVE screening trial (NTR5078). Primary clinical end-point was cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3+). The single-marker and bi-marker methylation classifiers developed for CIN3 detection in a previous series of hrHPV-positive clinician-collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) curves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined threshold calibrated at 70% clinical specificity. Individual methylation makers showed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi-marker panel with predefined threshold, ASCL1/LHX8 yielded a CIN3+ sensitivity of 76.9% (70/91; 95% CI 68.3-85.6%) at a specificity of 74.5% (465/624; 95% CI 71.1-77.9%). In conclusion, our study shows that the individual host-cell DNA methylation classifiers and the bi-marker panel ASCL1/LHX8 have clinical utility for the detection of CIN3+ in hrHPV-positive women invited for routine screening.

Background Host-cell DNA methylation analysis can be used to triage women with high-risk human papillomavirus (HPV)-positive self-collected cervicovaginal samples, but current data are restricted to under-/never-screened women and referral populations. This study evaluated triage performance in women who were offered primary HPV self-sampling for cervical cancer screening. Methods Self-collected samples from 593 HPV-positive women who participated in a primary HPV self-sampling trial (IMPROVE study; NTR5078), were tested for the DNA methylation markers ASCL1 and LHX8 using quantitative multiplex methylation-specific PCR (qMSP). The diagnostic performance for CIN3 and cervical cancer (CIN3 + ) was evaluated and compared with that of paired HPV-positive clinician-collected cervical samples. Results Significantly higher methylation levels were found in HPV-positive self-collected samples of women with CIN3 + than control women with no evidence of disease (P values ASCL1/LHX8 yielded a sensitivity for CIN3 + detection of 73.3% (63/86; 95% CI 63.9–82.6%), with a corresponding specificity of 61.1% (310/507; 95% CI 56.9–65.4%). The relative sensitivity for detecting CIN3+ was 0.95 (95% CI 0.82–1.10) for self-collection versus clinician-collection, and the relative specificity was 0.82 (95% CI 0.75–0.90). Conclusions The ASCL1/LHX8 methylation marker panel constitutes a feasible direct triage method for the detection of CIN3 + in HPV-positive women participating in routine screening by self-sampling.

Anal cancer has increasing incidence and is preceded by high‐grade anal intraepithelial neoplasia (HGAIN; AIN2–3). Previously, we identified and validated several methylation markers for accurate detection of anal cancer and HGAIN with cancer risk in HIV‐positive (HIV+) men who have sex with men (MSM). This study aimed to evaluate these markers in HIV‐negative risk groups. A cross‐sectional series of 176 tissue samples of anal cancer, AIN3, AIN2, AIN1 and control biopsies obtained in HIV‐negative women and men was tested for six methylation markers (ASCL1, LHX8, SST, WDR17, ZIC1 and ZNF582). Accuracy for detection of AIN3 and cancer (AIN3+) was determined by univariable and multivariable mixed‐effect ordinal logistic regression. Methylation levels of all markers increased with increasing severity of disease (P, Host cell DNA methylation plays a role in anal carcinogenesis in HIV‐negative (HIV‐neg) and HIV‐positive (HIV+) individuals, including men who have sex with men (MSM). Methylation levels are increased in high‐grade anal intraepithelial neoplasia at risk of progression towards anal cancer, making methylation markers promising prognostic biomarkers.

Methylation of host-cell deoxyribonucleic acid (DNA) has been proposed as a promising biomarker for triage of high-risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host-cell DNA methylation markers for triage in an hrHPV-positive cohort derived from primary HPV-based cervical screening in The Netherlands. Methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST were evaluated relative to the ACTB reference gene by multiplex quantitative methylation-specific PCR (qMSP) in clinician-collected cervical samples (n = 715) from hrHPV-positive women (age 29-60 years), who were enrolled in the Dutch IMPROVE screening trial (NTR5078). Primary clinical end-point was cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3+). The single-marker and bi-marker methylation classifiers developed for CIN3 detection in a previous series of hrHPV-positive clinician-collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) curves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined threshold calibrated at 70% clinical specificity. Individual methylation makers showed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi-marker panel with predefined threshold, ASCL1/LHX8 yielded a CIN3+ sensitivity of 76.9% (70/91; 95% CI 68.3-85.6%) at a specificity of 74.5% (465/624; 95% CI 71.1-77.9%). In conclusion, our study shows that the individual host-cell DNA methylation classifiers and the bi-marker panel ASCL1/LHX8 have clinical utility for the detection of CIN3+ in hrHPV-positive women invited for routine screening.

High-grade cervical intraepithelial neoplasia (CIN2 and CIN3) represent a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a , Ki-67 and host-cell DNA methylation), could provide guidance for clinical management in women with high-grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (score 0 - 3) and Ki-67 (score 0 - 3), referred to as the 'immunoscore' (IS), in 262 CIN2 and 235 CIN3 lesions derived from 5 European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124-2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0 - 2 (6.0%), 151 lesions within IS group 3 - 4 (30.4%) and 316 lesions within IS group 5 - 6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (p

Background: In human papillomavirus (HPV)-based cervical screening programs, management of HPV-positive women with normal cytology is debated. Longitudinal information on HPV type persistence may be employed for risk stratification. Methods: We assessed the risk of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) after repeatedly testing positive for the same HPV type(s) in the randomized population-based screening study Amsterdam (POBASCAM). We compared 18-month CIN3+ risks in HPV-positive women (intervention, n = 1,066) to those in HPV-positive/cytology-negative women who tested HPV-positive in the next screening round (control, n = 111) five years later, stratified for HPV type concordance. Results: The 18-month CIN3+ risk was 15% in HPV-positive women in the intervention group, 40% in the control group after two-round type concordance (relative risk 2.6, 95% confidence interval 1.9–3.4), and 20% in the control group after a type switch (1.3, 0.5–3.2). The relative increase in CIN3+ risk after two-round type concordance was similar in Conclusions: Five-year HPV type concordance signals high CIN3+ risk and warrants referral for colposcopy without additional cytology triage. Impact: HPV screening programs become highly efficient when HPV-positive women with negative triage testing at baseline are offered repeat HPV genotyping after five years.

BACKGROUND: Bhutan implemented a national program for human papillomavirus (HPV) vaccination in 2010 involving girls aged 12 to 18 years and achieving nearly 90% coverage.OBJECTIVE: To estimate HPV vaccine effectiveness in a city in Bhutan.DESIGN: 2 cross-sectional surveys, 2011-2012 and 2018.SETTING: 2 hospitals in Thimphu, capital of Bhutan.PARTICIPANTS: Sexually active women aged 17 to 29 years: 1445 participants from the baseline survey and 1595 from the repeated survey.INTERVENTION: National HPV vaccination program.MEASUREMENTS: HPV was assessed in cervical cell samples by using general primer GP5+/GP6+-mediated polymerase chain reaction. Human papillomavirus types were stratified as vaccine types (HPV6/11/16/18) and nonvaccine types. Age- and sexual behavior-adjusted overall, total, and indirect (herd immunity) vaccine effectiveness (VE) was computed as (1 - HPV prevalence ratio) for HPV among all women and among unvaccinated women.RESULTS: Between the 2 surveys, the prevalence of HPV vaccine types decreased from 8.3% to 1.4%, whereas the prevalence of nonvaccine types increased from 25.8% to 31.4%. The overall and indirect adjusted VE against vaccine-targeted HPV types was 88% (95% CI, 80% to 92%) and 78% (CI, 61% to 88%), respectively. Among women younger than 27 years, who were targeted by the vaccination program, the overall and indirect adjusted VE was 93% (CI, 87% to 97%) and 88% (CI, 69% to 95%), respectively. No impact on nonvaccine HPV types was detectable.LIMITATION: Hospital-based recruitment; self-reported vaccination status.CONCLUSION: In Bhutan, the prevalence of vaccine-targeted HPV types has decreased sharply, providing the first evidence of the effectiveness of a high-coverage national HPV vaccination program in a lower-middle-income country.PRIMARY FUNDING SOURCE: Bill & Melinda Gates Foundation.

Cervical screening can prevent cancer by detection and treatment of cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3). Screening also results in considerable overtreatment because many CIN2/3 lesions show spontaneous regression when left untreated. In this multicenter longitudinal cohort study of women with untreated CIN2/3, the prognostic value ofWomen with CIN2/3 were prospectively followed for 24 months. Surgical excision was replaced by a wait-and-see policy.One hundred fourteen women (median age, 30 years; range, 20-53 years) were included, 80 of whom were diagnosed with CIN2 and 34 with CIN3. During the study, 65.8% of women (75/114) did not receive surgical treatment. Women with aMost women with untreated CIN2/3 and a negative baseline

Background Molecular tumor boards (MTBs) provide rational, genomics‐driven, patient‐tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. Materials and Methods MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision‐making method, reporting, and registration of the MTBs was completed through on‐site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. Results Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type–specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). Conclusion MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a “Dutch MTB model” for an optimal, collaborative, and nationally aligned MTB workflow. Implications for Practice Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing., Worldwide, molecular tumor boards (MTBs) differ in terms of scope, composition, methods, and recommendations. This article assesses differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands.

HSIL or CIN2/3 lesions in HPV-positive women30 years have high spontaneous regression rates. To reduce overtreatment, biomarkers are needed to delineate advanced CIN lesions that require treatment. We analysed the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in HPV-positive women30 years aiming to identify CIN2/3 lesions in need of treatment.A European multicentre retrospective study was designed evaluating the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in cervical scrapes of 1,061 HPV-positive women aged 15-29 years (690 ≤CIN1, 166 CIN2 and 205 CIN3+). A subset of 62 CIN2 and 103 CIN3 were immunohistochemically characterized by HPV E4 expression, a marker for a productive HPV infection and p16ink4a and Ki-67, markers indicative for a transforming infection. CIN2/3 lesions with low HPV E4 expression and high p16ink4a/Ki-67 expression were considered as non-productive, transforming CIN, compatible with advanced CIN2/3 lesions in need of treatment.FAM19A4/miR124-2 methylation positivity increased significantly with CIN grade and age groups (25, 25-29 and ≥30 years), while HPV16/18 positivity was comparable across age groups. FAM19A4/miR124-2 methylation positivity was HPV type independent. Methylation-positive CIN2/3 lesions had higher p16ink4a/Ki-67-immunoscores (p = 0.003) and expressed less HPV E4 (p = 0.033) compared with methylation-negative CIN2/3 lesions. These differences in HPV E4 and p16ink4a/Ki-67 expression were not found between HPV16/18-positive and non-16/18 HPV-positive lesions.Compared with HPV16/18 genotyping, the FAM19A4/miR124-2 methylation test detects non-productive, transforming CIN2/3 lesions with high specificity in women30 years, providing clinicians supportive information about the need for treatment of CIN2/3 in young HPV-positive women.

Contains fulltext : 288072.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Immunostaining with p16(INK4a) (p16), a tumor-suppressor surrogate protein biomarker for high-risk human papillomavirus (hrHPV) oncogenic activity, may complement standard hematoxylin and eosin (H&E) histology review, and provide more objective criteria to support the cervical intraepithelial neoplasia (CIN) diagnosis. With this study we assessed the impact of p16 immunohistochemistry on CIN grading in an hrHPV-based screening setting. MATERIAL AND METHODS: In this post-hoc analysis, 326 histology follow-up samples from a group of hrHPV-positive women were stained with p16 immunohistochemistry. All H&E samples were centrally revised. The pathologists reported their level of confidence in classifying the CIN lesion. RESULTS: Combining H&E and p16 staining resulted in a change of diagnosis in 27.3% (n = 89) of cases compared with the revised H&E samples, with a decrease of 34.5% (n = 18) in CIN1 and 22.7% (n = 15) in CIN2 classifications, and an increase of 18.3% (n = 19) in no CIN and 20.7% (n = 19) in CIN3 diagnoses. The level of confidence in CIN grading by the pathologist increased with adjunctive use of p16 immunohistochemistry to standard H&E. CONCLUSIONS: This study shows that adjunctive use of p16 immunohistochemistry to H&E morphology reduces the number of CIN1 and CIN2 classifications with a proportional increase in no CIN and CIN3 diagnoses, compared with standard H&E-based CIN diagnosis alone. The pathologists felt more confident in classifying the material with H&E and p16 immunohistochemistry than by using H&E alone, particularly during assessment of small biopsies. Adjunctive use of p16 immunohistochemistry to standard H&E assessment of CIN would be valuable for the diagnostic accuracy, thereby optimizing CIN management and possibly decreasing overtreatment.

Sodium bisulphite conversion of DNA to separate methylated from unmethylated cytosines is a standard for methylation analysis. This study evaluated a direct cell conversion protocol on cervical samples as alternative to isolated genomic DNA as input. Clinician-collected cervical samples (n = 120) were subjected to a direct conversion protocol, or genomic DNA was isolated with a fixed amount used for subsequent bisulphite conversion. Converted samples were compared for ACTB control gene and methylation of FAM19A4 and miR124-2 genes using quantitative methylation-specific PCR (QIAsure Methylation Test). Direct conversion resulted in a high success rate, i.e., 119/120 (99.2%) samples reported a valid test result. ΔΔCq values of FAM19A4 and miR124-2 were significantly correlated between both protocols (Spearman Rho 0.708 and 0.763, respectively, all p-values = 0.000). Agreement between both the bisulphite protocols was demonstrated by Bland–Altman plots. A direct cell conversion protocol shows good technical and analytical performance and offers a streamlined workflow for methylation analysis.

We studied whether triage of human papillomavirus (HPV)-positive women participating in an HPV-based screening programme can be improved by including the HPV result at the previous screen in the triage algorithm. We analyzed data of a subgroup of 366 women from the POBASCAM trial, screened by cytology and HPV cotesting. Women were included if they tested HPV-positive in the second HPV-based screening round. We evaluated the clinical performance of 16 strategies, consisting of cytology, HPV genotyping, and/or previous screen HPV result. The clinical endpoint was cervical precancer or cancer (CIN3+). The current Dutch triage testing policy for HPV-positive women is to refer women for colposcopy if they have abnormal cytology at baseline or after 6-18 months. In the second HPV-based screening round, this strategy yielded a negative predictive value (NPV) of 95.8% (95% confidence interval: 91.9-98.2) and colposcopy referral rate of 37.6% (32.3-43.2%). Replacing repeat cytology by the previous screen HPV result yielded a similar NPV (96.9%, 93.3-98.9) and colposcopy referral rate (38.8%, 33.4-44.4). A higher NPV (99.2%, 96.3-100%) at the cost of a higher colposcopy referral rate (49.2%, 43.6-54.8) was achieved when cytology was combined with HPV16/18 genotyping. The other 13 triage strategies yielded a lower NPV, a higher colposcopy referral rate or performed similarly but required additional testing. HPV-positive women in the second HPV-based screening round can be suitably managed by cytology, HPV16/18 genotyping and the HPV result at the previous screen, obviating the need for repeat testing of HPV-positive, cytology negative women.

Several countries offer HPV self-sampling for screening non-attendees. It is assumed that screening attendees also prefer self-sampling to clinician-based sampling, however, little research has been conducted with respect to this. Women participating in the IMPROVE-study were randomised (1:1) to self- or clinician-collected HPV testing, and HPV-positive women were retested using the other collection method. Three different questionnaires were sent out among a subset of participating women: Q1) HPV-positive women from both study groups were asked about their experiences with self-sampling and clinician-based sampling (n = 497); Q2) HPV-negative women from the self-sampling group were asked about their experiences with self-sampling (n = 2366); and Q3) HPV-negative women in the clinician-collection group were asked about their experiences with clinician-based sampling (n = 2092). Response rates ranged from 71.6 to 79.4%. Women reported significantly lower levels of shame, nervousness, discomfort and pain during self-sampling compared to clinician-based sampling. However, trust in correct sampling was significantly higher during clinician-based sampling. The majority of women in group Q1 preferred self-sampling (76.5%) to clinician-based sampling (11.9%) in future screening, while 11.6% of women reported to have no preference for either method. To conclude, women from a regular screening population have a positive attitude towards self-sampling but express some concerns with respect to accuracy. The majority prefers self-sampling to clinician-based sampling in future screening. Based on these results, a screening approach where women can choose for either self-sampling or clinician-based sampling seems highly justifiable.

Current clinical and histological classifications are unable to determine the risk of vulvar squamous cell carcinoma (VSCC) in high‐grade vulvar intraepithelial neoplasia (VIN), making prognostic biomarkers highly needed. We studied host‐cell DNA methylation markers in high‐grade squamous intraepithelial lesion (HSIL) and differentiated VIN (dVIN) without VSCC, in HSIL and dVIN adjacent to VSCC and in human papillomavirus (HPV) positive and negative VSCC, relative to control vulvar tissues. A series of 192 formalin‐fixed paraffin‐embedded vulvar samples, including VSCC (n = 58), VIN adjacent to VSCC (n = 30), VIN without VSCC during follow‐up (n = 41) and normal vulvar tissues (n = 63), were tested for 12 DNA methylation markers with quantitative multiplex methylation‐specific PCR (qMSP). HPV status was determined by p16INK4A immunohistochemistry and high‐risk HPV PCR analysis. Logistic regression analyses were used to determine methylation patterns and methylation marker performance for VIN and VSCC detection. Methylation markers showed significantly higher methylation levels with increasing severity of disease. VIN adjacent to VSCC showed a similar methylation‐high pattern as VSCC, while VIN without VSCC displayed a heterogeneous methylation pattern. Vulvar carcinogenesis is associated with increased DNA methylation. Higher DNA methylation levels in VIN seem to reflect higher cancer risk, emphasizing the high potential of DNA methylation biomarkers in the diagnostic workup of VIN. As a next step, longitudinal studies are needed to verify the prognostic value of methylation biomarkers as a clinical tool for stratification of cancer risk in women with VIN., What's new? In high‐grade vulvar intra‐epithelial neoplasia (VIN), existing clinicopathological classifications lack accuracy in predicting cancer risk, resulting in a need for objective prognostic biomarkers. Here, 12 DNA methylation markers were investigated for prognostic capability in a series of high‐grade squamous intraepithelial lesion, differentiated VIN, and vulvar carcinomas. For all 12 methylation markers, as disease severity increased, methylation levels also increased. Methylation patterns were more heterogeneous for VIN without vulvar squamous cell carcinoma. The results link elevated DNA methylation levels with increased VIN cancer risk and indicate that methylation biomarkers are promising diagnostic tools for cancer risk stratification in VIN.

This paper briefly reviews the role of hypermethylation of host cell genes in cervical carcinogenesis and discusses potential clinical applications of methylation analysis in the management of high‐risk HPV (hrHPV) ‐positive women. We argue that methylation assays can be used: 1. for primary triage of hrHPV‐positive women to detect cervical cancer and advanced cervical intraepithelial neoplasia (CIN); 2. as secondary triage for women with minor cytological abnormalities to identify those with the highest risk of CIN3 or worse; 3. as exit test for women leaving the screening programme to identify cervical cancer and advanced CIN; and 4. to support management of CIN. Tweetable abstract This paper discusses potential clinical applications of DNA methylation analysis in the management of women with a high‐risk HPV infection., Tweetable abstract This paper discusses potential clinical applications of DNA methylation analysis in the management of women with a high‐risk HPV infection.

Aim: To evaluate the triage performance of six host-cell DNA methylation markers derived from two genome-wide discovery screens for detection of cervical precancer (cervical intraepithelial neoplasia 3 [CIN]) and cancer. Materials & methods: Human papillomavirus-positive cervical scrapes of controls (≤CIN1; n = 352) and women diagnosed with CIN3 (n = 175) or cervical cancer (n = 50) were analyzed for methylation of ASCL1, LHX8, ST6GALNAC5, GHSR, SST and ZIC1. Results: Methylation levels increased significantly with disease severity (all markers p 0.800 after leave-one-out cross-validation. Bi-marker panel ASCL1/LHX8 had highest area under the curve (0.882), and detected 83.4% of CIN3 and all cervical cancers at specificity of 82.4%. Conclusion: All six methylation markers showed an equivalent, high performance for the triage of human papillomavirus-positive women using cervical scrapes with complementarity between markers.

Widespread adoption of primary human papillomavirus (HPV)‐based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124‐2 genes has shown promise for the triage of high‐risk (hr) HPV‐positive women. In our study, we assessed the consistency of FAM19A4/miR124‐2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation‐specific PCR (qMSP)‐based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7–99.2) tested FAM19A4/miR124‐2 methylation‐positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124‐2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV‐negative carcinomas. These results indicate that a negative FAM19A4/miR124‐2 methylation assay result is likely to rule out the presence of cervical cancer., What's new? Methylation analysis of host cell genes is a promising strategy for the triage of women who test positive for high‐risk human papillomavirus (hrHPV). Its ability to consistently detect cervical cancer, however, warrants further evaluation. In this retrospective cross‐sectional study of more than 500 cervical cancer cases worldwide, methylation analysis using FAM19A4 and miR124‐2 genes successfully detected the vast majority of cervical carcinomas. Detection by FAM19A4/miR124‐2 methylation analysis was consistent regardless of multiple factors, including hrHPV status and genotype, cancer histotype, sample type, and geographical region. The findings suggest that a negative FAM19A4/miR124‐2 methylation test result is likely to rule out cervical cancer.

OBJECTIVES: Epidermal growth factor receptor (EGFR) exon 20 insertions comprise 4-10 % of EGFR mutations in non-small cell lung cancer (NSCLC) and are associated with primary resistance to first and second generation EGFR tyrosine kinase inhibitors (TKIs). In vitro and preclinical animal studies have shown that osimertinib exerts antitumor activity against EGFR exon 20 mutation positive NSCLC. We report on a cohort of advanced stage NSCLC patients who harbor an EGFR exon 20 mutation and received osimertinib treatment.MATERIAL AND METHODS: Twenty-one patients were treated with osimertinib 80 or 160 mg once daily from April 2016 to June 2018, in four institutions in the Netherlands. Data were obtained retrospectively. Progression free survival (PFS), disease control rate (DCR), overall survival (OS) and objective response rate (ORR) were assessed using RECIST v1.1.RESULTS: Thirteen patients received prior platinum-based chemotherapy, and three patients a first - or second generation EGFR TKI. We observed 1 partial response, 17 patients with stable disease and 3 with progressive disease as best response to osimertinib (ORR 5 %). Median PFS was 3.6 (95 % CI, 2.6-4.5) months. PFS did not differ for patients with co-occurring TP53 mutations (p = 0.937). The DCR at three months was 71 %. Median OS was 8.7 (95 % CI, 1.1-16.4) months.CONCLUSION: Osimertinib has limited antitumor activity in patients with EGFR exon 20 mutated NSCLC, with an ORR of 5 %.

The most widely applied algorithm for human papillomavirus (HPV) detection in formalin-fixed, paraffin-embedded (FFPE) specimens of oropharyngeal head and neck squamous cell carcinoma (HNSCC) consists of p16INK4A immunostaining followed by PCR-based detection of high-risk HPV DNA on p16INK4A-immunopositive samples. However, in nonoropharyngeal HNSCC this algorithm fails, hampering correct interpretation of the prevalence and prognosis of HPV in these cases. In this study, we developed and validated a molecular HPV detection method for FFPE specimens of oropharyngeal and nonoropharyngeal HNSCC. Sectioning of FFPE blocks was circumvented by using punch biopsies from tumor-enriched regions of FFPE tissue blocks, and combined extraction was applied to obtain high-quality DNA and RNA from the punch biopsy. Next, PCR-based detection of HPV DNA was performed for 15 high-risk HPV types with subsequent detection of E6 mRNA for validation. The combined DNA/RNA FFPE test of tissue cores was assessed in well-characterized cohorts with known HPV status based on earlier work, that is, a cohort of oropharyngeal HNSCC (n = 80) and oral cavity HNSCC (n = 25), and reached an accuracy of 97% and 100%, respectively. In conclusion, our method is rapid, simple, and shows an excellent diagnostic performance for detection of HPV type 16. Ultimately, it can be applied for large cohort studies to determine the etiologic fraction and prognostic implication of HPV in nonoropharyngeal HNSCC.

Offering self-sampling for HPV testing improves the effectiveness of current cervical screening programs by increasing population coverage. Molecular markers directly applicable on self-samples are needed to stratify HPV-positive women at risk of cervical cancer (so-called triage) and to avoid over-referral and overtreatment. Deregulated microRNAs (miRNAs) have been implicated in the development of cervical cancer, and represent potential triage markers. However, it is unknown whether deregulated miRNA expression is reflected in self-samples. This study is the first to establish genome-wide miRNA profiles in HPV-positive self-samples to identify miRNAs that can predict the presence of CIN3 and cervical cancer in self-samples. Small RNA sequencing (sRNA-Seq) was conducted to determine genome-wide miRNA expression profiles in 74 HPV-positive self-samples of women with and without cervical precancer (CIN3). The optimal miRNA marker panel for CIN3 detection was determined by GRidge, a penalized method on logistic regression. Six miRNAs were validated by qPCR in 191 independent HPV-positive self-samples. Classification of sRNA-Seq data yielded a 9-miRNA marker panel with a combined Area Under the Curve (AUC) of 0.89 for CIN3 detection. Validation by qPCR resulted in a combined AUC of 0.78 for CIN3+ detection. This study shows that deregulated miRNA expression associated with CIN3 and cervical cancer development can be detected by sRNA-Seq in HPV-positive self-samples. Validation by qPCR indicates that miRNA expression analysis offers a promising novel molecular triage strategy for CIN3 and cervical cancer detection applicable to self-samples. This article is protected by copyright. All rights reserved.

Urine samples provide a potential alternative to physician-taken or self-collected cervical samples for cervical screening. Screening by primary hrHPV testing requires additional risk assessment (so-called triage) of hrHPV-positive women. Molecular markers, such as DNA methylation, have proven most valuable for triage when applied to cervical specimens. This study was set out to compare hrHPV and DNA methylation results in paired urine and cervical scrapes, and to evaluate the feasibility of DNA methylation analysis in urine to detect cervical cancer. Urine samples (n = 41; native and sediment) and paired cervical scrapes (n = 38) from cervical cancer patients, and urine from 44 female controls, were tested for hrHPV and 6 methylation markers. Results on native urine and sediment were highly comparable. A strong agreement was found between hrHPV testing on urine and scrapes (kappa = 0.79). Also, methylation levels in urine were moderately to strongly correlated to those detected in scrapes (r = 0.508–0.717). All markers were significantly increased in urine from cervical cancer patients compared to controls and showed a good discriminatory power for cervical cancer (AUC = 0.744–0.887). Our results show a good agreement of urine-based molecular analysis with reference cervical samples, and suggest that urine-based DNA methylation testing may provide a promising strategy for cervical cancer detection.

Cervical cancer development following a persistent infection with high-risk human papillomavirus (hrHPV) is driven by additional host-cell changes, such as altered DNA methylation. In previous studies, we have identified 12 methylated host genes associated with cervical cancer and pre-cancer (CIN2/3). This study systematically analyzed the onset and DNA methylation pattern of these genes during hrHPV-induced carcinogenesis using a longitudinal in vitro model of hrHPV-transformed cell lines (n = 14) and hrHPV-positive cervical scrapings (n = 113) covering various stages of cervical carcinogenesis. DNA methylation analysis was performed by quantitative methylation-specific PCR (qMSP) and relative qMSP values were used to analyze the data. The majority of genes displayed a comparable DNA methylation pattern in both cell lines and clinical specimens. DNA methylation onset occurred at early or late immortal passage, and DNA methylation levels gradually increased towards tumorigenic cells. Subsequently, we defined a so-called cancer-like methylation-high pattern based on the DNA methylation levels observed in cervical scrapings from women with cervical cancer. This cancer-like methylation-high pattern was observed in 72% (38/53) of CIN3 and 55% (11/20) of CIN2, whereas it was virtually absent in hrHPV-positive controls (1/26). In conclusion, hrHPV-induced carcinogenesis is characterized by early onset of DNA methylation, typically occurring at the pre-tumorigenic stage and with highest DNA methylation levels at the cancer stage. Host-cell DNA methylation patterns in cervical scrapings from women with CIN2 and CIN3 are heterogeneous, with a subset displaying a cancer-like methylation-high pattern, suggestive for a higher cancer risk.

Differentiation between multiple primary lung cancers and pulmonary metastases (PM) has important implications in staging, prognosis, and treatment strategies. Clinical and immunohistopathologic criteria have been standardized; however, a substantial number of cases remain difficult to classify. Using next-generation sequencing, it is now possible to improve the classification of multiple lung cancer lesions. This study systematically investigated the value of routine morphologic and IHC characteristics, p53 protein expression, TP53 mutation analysis, and 50-gene panel sequencing (GPS) in 111 lesions from 50 patients with multiple lung lesions. Based on immunohistopathologic criteria, 32 paired lesions were classified as multiple primary lung cancer (MPLC) and 21 as PM. TP53 mutation analysis indicated MPLC in 23 and PM in 6 pairs, but in the majority of cases (n = 28, 49%) no mutation was observed and no conclusion could be drawn. In contrast, only 2 pairs were not conclusive using GPS. In a significant number of matching tumor samples (n = 19, 39%), sequencing results were contradictory to the initial immunohistopathology diagnosis. No separation in overall survival for classifications based on immunohistopathology was observed, while a clear but nonsignificant trend was observed concerning survival in MPLC patients (hazard ratio=3.98) using 50-gene GPS. In about one-third of the patients, GPS provided additional information to improve the differentiation between MPLC and PM.

DNA methylation analysis of cervical scrapes using FAM19A4 and mir124‐2 genes has shown a good clinical performance in detecting cervical cancer and advanced CIN lesions in need of treatment in HPV‐positive women. To date, longitudinal data on the cancer risk of methylation test‐negative women are lacking. In our study, we assessed the longitudinal outcome of FAM19A4/mir124‐2 methylation analysis in an HPV‐positive screening cohort with 14 years of follow‐up. Archived HPV‐positive cervical scrapes of 1,040 women (age 29–61 years), who were enrolled in the POBASCAM screening trial (ISRCTN20781131) were tested for FAM19A4/mir124‐2 methylation. By linkage with the nationwide network and registry of histo‐ and cytopathology in the Netherlands (PALGA), 35 cervical cancers were identified during 14 years of follow‐up comprising three screens (baseline, and after 5 and 10 years). The baseline scrape of 36.1% (n = 375) women tested positive for FAM19A4/mir124‐2 methylation, including 24 women with cervical cancer in follow‐up, and 30.6% (n = 318) had abnormal cytology (threshold borderline dyskaryosis or ASCUS), including 14 women with cervical cancer in follow‐up. Within screening round capability of FAM19A4/mir124‐2 methylation to detect cervical cancer was 100% (11/11, 95% CI: 71.5–100). Kaplan–Meier estimate of 14‐year cumulative cervical cancer incidence was 1.7% (95% CI: 0.66–3.0) among baseline methylation‐negative and 2.4% (95% CI: 1.4–3.6) among baseline cytology‐negative women (risk difference: 0.71% [95% CI: 0.16–1.4]). In conclusion, a negative FAM19A4/mir124‐2 methylation test provides a low cervical cancer risk in HPV‐positive women of 30 years and older. FAM19A4/mir124‐2 methylation testing merits consideration as an objective triage test in HPV‐based cervical screening programs., What's new? While HPV testing is increasingly being used for cervical‐cancer screening, there is a problem with this approach: Most HPV infections won't progress to (pre)malignant disease, which results in a significant number of unnecessary colposcopy referrals and over‐diagnoses. A better triage test is needed to discern which HPV+ women have clinically relevant disease. In this longitudinal study, the authors found that a methylation test may provide adequate predictive power. Low cervical‐cancer incidence after a negative FAM19A4/mir124‐2 methylation test among HPV+ women supports use of this methylation assay as safe, objective triage tool.

Background:In human papillomavirus (HPV)-based screening, a repeat HPV test is often recommended for HPV-positive women with normal cytology (HPV-pos/cyt-neg), but its absolute risk of cervical precancer (CIN3+) over two screening rounds needs to be assessed.Methods:We compared the 5-year risk of HPV infection and CIN3+ in HPV-pos/cyt-neg women with a negative repeat HPV test to the risk in HPV-negative women with normal cytology (double negatives) in the POBASCAM cohort. We obtained histology data from the Dutch pathology registry (PALGA).Results:Human papillomavirus infection risk was 20.4% (19 of 93) in HPV-pos/cyt-neg, repeat HPV-negative women and 3.2% (294 of 9186; P

OBJECTIVE: This study evaluates the long-term risk for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) among HPV positive women triaged with FAM19A4/miR124-2 methylation analysis.METHODS: In a post hoc analysis, data on FAM19A4/miR124-2 methylation, cytology, and HPV16/18 genotyping of HPV positive women (n = 1025) from a large population-based screening cohort with 14-year follow-up were evaluated. Cumulative CIN3+ incidences over 3 screening rounds (5-year intervals) of 4 triage strategies were compared: FAM19A4/miR124-2 methylation analysis, cytology, HPV16/18 genotyping with FAM19A4/miR124-2 methylation, and HPV16/18 genotyping with cytology.RESULTS: Kaplan-Meier estimates of 14-year cumulative CIN3+ incidence of HPV positive women with a negative methylation and a negative cytology triage test were comparable (16.3% and 15.6%, respectively). The cumulative CIN3+ incidence of methylation positive and cytology positive women were 39.8% and 46.5%, respectively. HPV16/18 genotyping with methylation and HPV16/18 genotyping with cytology resulted in the lowest 14-year cumulative CIN3+ incidence among triage negative women (10.7% and 10.0%, respectively), but cumulative CIN3+ incidence among triage positive women was lower (33.4% and 35.7%, respectively) compared with triage by methylation alone and cytology alone.CONCLUSIONS: Among HPV positive women of 30 years and older, a negative FAM19A4/miR124-2 methylation triage test provides a similar long-term CIN3+ risk compared with a negative cytology triage test. Because of their high CIN3+ risk, women with a positive methylation triage test could be referred for colposcopy. Therefore, FAM19A4/miR124-2 methylation analysis is a promising alternative to cytology for triage of HPV positive women.

Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2–3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4. Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening.

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity. Electronic supplementary material The online version of this article (10.1007/s00428-019-02555-3) contains supplementary material, which is available to authorized users.