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

Abstract

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.

Management of high-risk HPV-positive women for detection of cervical (pre)cancer

“…Furthermore, host cell methylation analysis of CADM1, MAL, mir124-2, and FAM19A4 in cervical scrapes allows the discrimination of high-grade CIN with a long-term preceding hrHPV-infection from those with a short-term hrHPV infection [95,96]. Concordantly, in both cervical scrapes and selfsampled cervicovaginal lavages, methylation levels of FAM19A4 have been shown to correspond with the estimated CIN lesion volume [100].…”

“…Concordantly, in both cervical scrapes and selfsampled cervicovaginal lavages, methylation levels of FAM19A4 have been shown to correspond with the estimated CIN lesion volume [100]. Methylation levels of CADM1, MAL, mir124-2, and FAM19A4 are extraordinarily high in cervical scrapes of women with carcinomas [96,101], resulting in a 100% sensitivity for carcinomas of methylation assays targeting these genes, even when very high thresholds are used to define methylation positivity. As previously described, all these findings underline that -in contrast to cytology -host cell promoter methylation analysis specifically detects so-called advanced CIN lesions, which harbor a cancer-like methylation profile and have a high short-term risk of progression to cancer [32,33].…”

“…Methylation levels of certain host cell genes (such as CADM1, MAL, mir124-2, JAM3, TERT, C13ORF, EPB41L3, ANKRD18CP, GFRA1, CDH6, LHX8, GATA4, PRDM14, and FAM19A4) in cervical scrapes have been shown to increase with the severity of underlying cervical (pre)cancer [51,[95][96][97][98][99]. Furthermore, host cell methylation analysis of CADM1, MAL, mir124-2, and FAM19A4 in cervical scrapes allows the discrimination of high-grade CIN with a long-term preceding hrHPV-infection from those with a short-term hrHPV infection [95,96].…”

“…Therefore, host cell DNA methylation analysis might be of value as a marker for the detection of cervical (pre)cancer among hrHPV-positive women [32,92]. A variety of methylation markers has been studied in clinical cohorts of hrHPVpositive women [51,80,81,96,97,99,[102][103][104][105][106]. In general, these studies were cross-sectional or had a short-term follow-up and the majority of studies were performed in nonscreening cohorts.…”

“…[98] has been evaluated on cervical scrapes from a screening cohort (n = 234 [96]) and a gynecologic outpatient population (n = 508 [51]). FAM19A4 methylation analysis has been shown to be dependent on age, with a substantially higher sensitivity (75.8-88.3%) and lower specificity (62.1-67.0%) for ≥CIN3 in women aged ≥30 years [51,96] compared to younger women (≥CIN3 sensitivity 50%, specificity 81.7%; Table 1 [51]). Regardless of age, the NPV of FAM19A4 methylation analysis for ≥CIN3 was relatively consistent, ranging between 91.2% and 95.3% [51].…”