Methylation Analysis of the FAM19A4 Gene in Cervical Scrapes Is Highly Efficient in Detecting Cervical Carcinomas and Advanced CIN2/3 Lesions

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 training-validation 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 (CIN3+) and 135 women with ≤CIN1. The validation set comprised hrHPV-positive cervical scrapes of 52 women with CIN2+, including 33 CIN3+, 19 CIN2, and 166 women with ≤CIN1. The methylation threshold of FAM19A4 qMSP that gave rise to CIN3+ specificity of 70% in the training set was applied in the validation set. This resulted in CIN3+ 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 hrHPV-positive women, with a high reassurance for the detection of cervical carcinoma and advanced CIN2/3 lesions

Validation of the FAM19A4/mir124-2 DNA methylation test for both lavage- and brush-based self-samples to detect cervical (pre)cancer in HPV-positive women

Objectives DNA 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. Methods We 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. Results Training 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. Conclusions FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive la

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

Background:High-risk human papillomavirus (hrHPV)-positive women require triage to identify those with cervical high-grade intraepithelial neoplasia and cancer (≥CIN3 (cervical intraepithelial neoplasia grade 3)). FAM19A4 methylation analysis, which detects advanced CIN and cancer, is applicable to different sample types. However, studies comparing the performance of FAM19A4 methylation analysis in hrHPV-positive self-samples and paired physician-taken scrapes are lacking.Methods:We compared the performance of FAM19A4 methylation analysis (and/or HPV16/18 genotyping) in self-samples and paired physician-taken scrapes for ≥CIN3 detection in hrHPV-positive women (n=450,18-66 years).Results:Overall FAM19A4 methylation levels betw

Triage of HPV-positive women by methylation marker analysis

Snijders, P.J.F. [Promotor]Meijer, C.J.L.M. [Promotor]Heideman, D.A.M. [Copromotor]Steenbergen, R.D.M. [Copromotor

Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

BACKGROUND: Quantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design. METHODS: Primers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings. RESULTS: Upon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R(2)=0.985), MAL (R(2)=0.986) and hsa-miR-124-2 (R(2)=0.944). CONCLUSION: Multiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions

La ricostruzione dell'immaginario violato in tre scrittrici italofone del Corno D'Africa : aspetti teorici, pedagogici e percorsi di lettura

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