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

Abstract

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.

Cervical cancer risk in HPV‐positive women after a negative FAM19A4/mir124‐2 methylation test: A post hoc analysis in the POBASCAM trial with 14 year follow‐up

“…Cervical cancer development after a persistent infection with high‐risk HPV is driven by additional host cell changes such as altered DNA methylation 12, 17, 20, 21. In earlier work, we have shown that methylation assays targeting FAM19A4 and/or mir124‐2 genes have competitive performance against other triage options 22, 23, 24, 25, 26. In cross‐sectional and short‐term clinical follow‐up studies among both cervical screening and gynecologic outpatient populations, FAM19A4 methylation analysis has shown a similar sensitivity for cervical intraepithelial neoplasia lesions or worse (CIN3+) as compared to cytology 22, 24.…”

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

“…The performance of HPV16/18 genotyping has been evaluated on both lavage-and brush-based selfsamples of hrHPV-positive non-attendees, revealing an accuracy which appears to be comparable to cervical scrapes (≥CIN3 sensitivity 65.4-69.4%, specificity 65.0-70.9%, PPV 25.0-48.5%, NPV 85.4-92.4% [80,81]). It is obvious thatregardless of sample type -HPV16/18 genotyping does not suffice as a direct stand-alone triage strategy for hrHPV-positive women, but the combination of HPV16/18 genotyping with other tests has been frequently suggested.…”

“…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.…”

“…In a large randomized controlled trial among hrHPV-positive screening non-attendees (n = 1019) [103], combined methylation analysis of MAL/mir124-2 on self-sampled cervicovaginal lavage material yielded a ≥CIN3 sensitivity of 73.5% and a specificity of 47.2% (Table 2) [80,103]. Combined methylation analysis of FAM19A4 and mir124-2 was evaluated in a subset of these hrHPV-positive non-attendees using self-collected lavages (n = 389), as well as hrHPV-positive non-attendees from another cohort who submitted self-collected brushes (n = 254) [81]. This marker panel yielded a similar clinical performance for both devices (≥CIN3 sensitivity 69.4-70.5%, specificity 67.8-76.4%), resulting in a similar sensitivity and higher specificity compared to the previously described MAL/ mir124-2 marker panel (Table 2) [81].…”

“…Combined methylation analysis of FAM19A4 and mir124-2 was evaluated in a subset of these hrHPV-positive non-attendees using self-collected lavages (n = 389), as well as hrHPV-positive non-attendees from another cohort who submitted self-collected brushes (n = 254) [81]. This marker panel yielded a similar clinical performance for both devices (≥CIN3 sensitivity 69.4-70.5%, specificity 67.8-76.4%), resulting in a similar sensitivity and higher specificity compared to the previously described MAL/ mir124-2 marker panel (Table 2) [81]. When comparing the different host cell DNA methylation markers (in both cervical scrapes and self-collected samples), several technical aspects should be considered.…”