Primary testing for human papillomavirus (HPV) in cervical screening requires triage to differentiate women with transient infection from those with persistent infection who require more intensive management given their risk for cervical (pre)cancer. In this study, the clinical performance of a novel methylation marker FAM19A4 for the triage of high-risk (hr)HPV-positive women was evaluated. Using a trainingvalidation set approach, we analyzed a FAM19A4 quantitative methylation-specific PCR (qMSP). The training set comprised hrHPV-positive cervical scrapes of 43 women with cervical intraepithelial neoplasia grade 3 or worse (CIN3þ) and 135 women with CIN1. The validation set comprised hrHPV-positive cervical scrapes of 52 women with CIN2þ, including 33 CIN3þ, 19 CIN2, and 166 women with CIN1. The methylation threshold of FAM19A4 qMSP that gave rise to CIN3þ specificity of 70% in the training set was applied in the validation set. This resulted in CIN3þ sensitivity of 75.8% [95% confidence interval (CI), 61.1-90.4] at 67.0% (95% CI, 60.3-73.8) specificity. Next, the validated qMSP was applied to an independent series of hrHPV-positive cervical scrapes of 22 women with cervical cancer, 29 with advanced CIN2/3 [i.e., women with a known preceding hrHPV infection (PHI) lasting !5 years as proxy of longer duration of lesion existence], and 19 with early CIN2/3 (i.e., PHI <5 years). All carcinomas (22/22) and advanced CIN2/3 lesions (29/29) were FAM19A4 methylation-positive, compared with 42.1% (8/19; 95% CI, 19.9-64.3) of early CIN2/3 lesions. In conclusion, FAM19A4 is an attractive triage marker for hrHPVpositive women, with a high reassurance for the detection of cervical carcinoma and advanced CIN2/3 lesions. Cancer Prev Res; 7(12); 1251-7. Ó2014 AACR.
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.
Aims Gene promoter hypermethylation is recognized as an essential early step in carcinogenesis, indicating important application areas for DNA methylation analysis in early cancer detection. The current study was set out to assess the performance of CADM1, MAL and miR124-2 methylation analysis in cervical scrapes for detection of cervical and endometrial cancer.Methods A series of cervical scrapes of women with cervical (n=79) or endometrial (n=21) cancer, CIN3 (n=16) or CIN2 (n= 32), and women without evidence of CIN2 or worse (n=120), was assessed for methylation of CADM1, MAL and miR124-2. Methylation analysis was done by the PreCursor-M assay, a multiplex quantitative methylation-specific PCR.Results All samples of women with cervical cancer (79/79, 100%), independent of the histotype, and 76% (16/21; 95% CI:58.0-94.4) of women with endometrial cancer scored positive for DNA methylation of at least one of the three genes. In women without cancer, methylation frequencies increased significantly to severity of disease, from 19.2% (23/120; 95% CI:12.1-26.2) in women without CIN2 or worse to 37.5% (12/32; 95% CI: 20.7-54.3) and 68.8% (11/16; 95% CI:46.0-91.5) in women with CIN2 and CIN3, respectively. Not only overall methylation positivity, but also the number of methylated genes increased proportionally to the lesion severity.Conclusions DNA methylation analysis of CADM1, MAL and miR124-2 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 lesions, and has the capacity to broaden its use on cervical scrapes through the detection of a substantial subset of endometrial carcinomas.
ObjectivesDNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples.MethodsWe determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined.ResultsTraining set analysis revealed similar FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4–80.6) at a specificity of 67.8% (95%CI: 62.7–73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8–80.1) at a 76.4% (95%CI: 70.2–82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4–95.6) and 46.0% (95%CI: 40.4–51.5) for lavage self-samples, and 84.7% (95%CI: 76.4–93.0) and 54.9% (95%CI: 47.7–62.2) for brush self-samples.ConclusionsFAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity.
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