Colposcopy referral and CIN3+ risk of human papillomavirus genotyping strategies in cervical cancer screening.

BACKGROUND: High-risk human papillomavirus (hrHPV)-based cervical cancer screening in the Netherlands led to a substantial increase in number of colposcopy referrals and low-grade lesions detected. Genotyping strategies may be employed to lower the screening-related burden. METHODS: We evaluated fourteen triage strategies with genotyping (HPV16/18 or HPV16/18/31/33/45/52/58) for hrHPV-positive borderline or mild dyskaryosis (BMD) or normal cytology, using data from a population-based hrHPV-based screening trial with 5-year interval (POBASCAM). We considered colposcopy referral at baseline, after 6-month repeat cytology and after 5-year hrHPV testing. Performance was evaluated by one-round positive and negative predictive value (PPV and NPV) for CIN3+ and by two-round colposcopy referral rate. To identify efficient strategies, they were ordered by the one-round colposcopy referral rate. Adjacent strategies were compared by the marginal PPV for detecting one additional CIN3+ (mPPV). RESULTS: The most conservative strategy (repeat cytology after BMD and HPV16/18/31/33/45/52/58-positive normal cytology, next round otherwise) yielded an mPPV of 28%, NPV of 98.2%, and colposcopy rate of 47.2%. Adding direct referral after BMD or genotype-positive BMD yielded an mPPV≤8.2%, NPV≥98.5% and an increase in colposcopy rate of 1.9-6.5%. Adding direct referral after HPV16/18-positive normal cytology yielded an mPPV≤3.5%, NPV≥99.5% and an increase in colposcopy rate of 13.9%. CONCLUSIONS: Direct colposcopy referral of women with BMD or normal cytology is unlikely to be efficient, but genotype-guided direct referral after BMD may be considered because the increase in colposcopies is limited. IMPACT: HrHPV screening programs can become very efficient when immediate colposcopy referral is limited to women at highest CIN3+ risk.

Evaluation of six methylation markers derived from genome-wide screens for detection of cervical precancer and cancer.

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 < 0.001). Three markers (ASCL1, LHX8, ZIC1) showed receiver operating characteristic curves with area under the curve >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.

Role of FAM19A4/miR124-2 methylation analysis in predicting regression or non-regression of CIN2/3 lesions: a protocol of an observational longitudinal cohort study.

INTRODUCTION: The clinical course of high-grade cervical intraepithelial neoplasia (CIN2/3) is characterised by a high spontaneous regression rate. Histological assessment is unable to differentiate between CIN2/3 lesions likely to regress and those likely to persist or progress. Most CIN2/3 lesions are treated by surgical excision, leading to overtreatment of a substantial proportion. In this prospective study, we evaluate the value of DNA methylation of host cell genes, which has shown to be particularly sensitive for the detection of advanced CIN2/3 and cervical cancer, in the prediction of regression or non-regression of CIN2/3 lesions. METHODS AND ANALYSIS: This is a multicentre observational longitudinal study with 24-month follow-up. Women referred for colposcopy with an abnormal cervical scrape, who have been diagnosed with CIN2/3 and a small cervical lesion (≤50% of cervix) will be asked to participate. Participants will be monitored by 6-monthly cytological and colposcopic examination. In case of clinical progression, participants will receive treatment and exit the study protocol. At baseline and during follow-up, self-sampled cervicovaginal brushes and cervical scrapes will be collected for high-risk human papillomavirus (HPV) testing and FAM19A4/miR124-2 methylation analysis. A colposcopy-directed biopsy will be taken from all participants at the last follow-up visit. The primary study endpoint is regression or non-regression at the end of the study based on the histological diagnosis. Regression is defined as CIN1 or less. Non-regression is defined as CIN2 or worse. The secondary study endpoint is defined as HPV clearance (double-negative HPV test at two consecutive time-points). The association between methylation status and regression probability will be evaluated by means of χ2 testing. ETHICS AND DISSEMINATION: Ethics approval was obtained in all participating clinics. Results of the main study will be submitted for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NTR6069; Pre-results.

Detection of hypermethylated genes as markers for cervical screening in women living with HIV.

INTRODUCTION: To evaluate the performance of hypermethylation analysis of ASCL1, LHX8 and ST6GALNAC5 in physician-taken cervical scrapes for detection of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 in women living with HIV (WLHIV) in South Africa. METHODS: Samples from a prospective observational cohort study were used for these analyses. Two cohorts were included: a cohort of WLHIV who were invited for cervical screening (n = 321) and a gynaecologic outpatient cohort of women referred for evaluation of abnormal cytology or biopsy proven cervical cancer (n = 108, 60% HIV seropositive). Cervical scrapes collected from all subjects were analysed for hypermethylation of ASCL1, LHX8 and ST6GALNAC5 by multiplex quantitative methylation specific PCR (qMSP). Histology endpoints were available for all study subjects. RESULTS: Hypermethylation levels of ASCL1, LHX8 and ST6GALNAC5 increased with severity of cervical disease. The performance for detection of CIN3 or worse (CIN3+ ) as assessed by the area under the receiver operating characteristic (ROC) curves (AUC) was good for ASCL1 and LHX8 (AUC 0.79 and 0.81 respectively), and moderate for ST6GALNAC5 (AUC 0.71). At a threshold corresponding to 75% specificity, CIN3+ sensitivity was 72.1% for ASCL1 and 73.8% for LHX8 and all samples from women with cervical cancer scored positive for these two markers. CONCLUSIONS: Hypermethylation analysis of ASCL1 or LHX8 in cervical scrape material of WLHIV detects all cervical carcinomas with an acceptable sensitivity and good specificity for CIN3+ , warranting further exploration of these methylation markers as a stand-alone test for cervical screening in low-resource settings.

Evaluation of p16/Ki-67 dual-stained cytology as triage test for high-risk human papillomavirus-positive women.

The aim of this study was to evaluate the clinical utility of p16/Ki-67 dual staining, for the identification of CIN in high-risk HPV-positive women from a non-responder screening cohort. P16/Ki-67 dual staining, Pap cytology, and HPV16/18 genotyping were performed on physician-taken liquid-based samples from 495 women who tested high-risk HPV positive on self-sampled material (PROHTECT-3B study). Different triage strategies involving p16/Ki-67 dual staining were evaluated for sensitivity, specificity, and predictive value for ≥CIN2 and ≥CIN3, and compared to Pap cytology with a threshold of atypical cells of undetermined significance. Centrally revised histology or an adjusted endpoint with combined high-risk HPV negative and cytology negative follow-up at 6 months was used as gold standard. Pap cytology (threshold atypical cells of undetermined significance) triage of high-risk HPV-positive samples showed a sensitivity of 93% (95% confidence interval: 85-98) with a specificity of 49% (95% confidence interval: 41-56) for ≥CIN3. Three triage strategies with p16/Ki-67 showed a significantly increased specificity with similar sensitivity. P16/Ki-67 triage of all high-risk HPV-positive samples had a sensitivity of 92% (95% confidence interval: 84-97) and a specificity of 61% (95% confidence interval: 54-69) for ≥CIN3. Applying p16/Ki-67 triage to only high-risk HPV-positive women with low-grade Pap cytology showed a similar sensitivity of 92% (95% confidence interval: 84-97), with a specificity for ≥CIN3 of 64% (95% confidence interval: 56-71). For high-risk HPV-positive women with low-grade and normal Pap cytology, triage with p16/Ki-67 showed a sensitivity of 96% (95% confidence interval: 89-99), and a specificity of 58% (95% confidence interval: 50-65). HPV16/18 genotyping combined with Pap cytology showed a sensitivity and specificity for ≥CIN3 similar to Pap cytology with an atypical cells of undetermined significance threshold. Because the quality of Pap cytology worldwide varies, and differences in sensitivity and specificity are limited between the three selected strategies, p16/Ki-67 triage of all high-risk HPV-positive samples would be the most reliable strategy in triage of high-risk HPV-positive women with an increased specificity and similar sensitivity compared with Pap cytology triage.

Detecting resistance in EGFR-mutated non-small-cell lung cancer after clonal selection through targeted therapy.

Tumor heterogeneity plays an important role in the development of treatment-resistance, especially in the current era of targeted therapies. Although tumor heterogeneity is a widely recognized phenomenon, it is at present unclear how this knowledge should be incorporated into daily clinical practice. In this report, we describe an innovative nuclear imaging method that may play a role in detecting tumor heterogeneity in the future.

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.