Frederique J. Vink

Methylation markers FAM19A4 and miR124‐2 as triage strategy for primary human papillomavirus screen positive women: A large European multicenter study

AbstractIn human papillomavirus (HPV) cervical cancer screening, cytology is used as triage to counter the low specificity of HPV testing. VALID‐SCREEN is a EU‐multicenter, retrospective study conducted to evaluate the clinical performance of the FAM19A4/miR124‐2 methylation‐based molecular triage test as a substitute or addition to cytology as reflex testing of HPV screen positive women. FAM19A4/miR124‐2 methylation test (QIAsure Methylation Test) was evaluated in 2384 HPV‐positive cervical screening samples, from women 29‐76 years of age, derived from four EU countries. Specimens were collected in ThinPrep or SurePath media, HPV‐status, concurrent cytology, and histology diagnosis were provided by the parent institutes. The control population consisted of women with no evidence of disease within 2 years of follow‐up. A total of 899 histologies were retrieved; 527 showed no disease, 124 CIN2 (5.2%), 228 CIN3 (9.6%) and 20 cervical cancers (0.8%); 19 of 20 screen‐detected cervical cancers were found methylation‐positive (sensitivity 95%). Overall specificity of FAM19A4/miR124‐2 methylation test was 78.3% (n = 2013; 95%CI: 76‐80). The negative predictive value of hrHPV positive, methylation‐negative outcomes were 99.9% for cervical cancer (N = 1694; 95%CI: 99.6‐99.99), 96.9% for ≥CIN3 (95%CI: 96‐98), and 93.0% for ≥CIN2 (95%CI: 92‐94). Overall sensitivity for CIN3 using FAM19A4/miR124‐2 methylation test was 77% (n = 228; 95%CI: 71‐82). CIN3 sensitivity was uniform between centers independent of sample collection medias, DNA extraction methods and HPV screening tests. Being objectively reported compared to the subjectivity of cytology, equally performing across settings and screening methods, the FAM19A4/miR124‐2 methylation constitute an alternative/supplement to cytology as triage method to be investigated in real‐life pilot implementation.

FAM19A4/miR124‐2 methylation in invasive cervical cancer: A retrospective cross‐sectional worldwide study

Widespread adoption of primary human papillomavirus (HPV)‐based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124‐2 genes has shown promise for the triage of high‐risk (hr) HPV‐positive women. In our study, we assessed the consistency of FAM19A4/miR124‐2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation‐specific PCR (qMSP)‐based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7–99.2) tested FAM19A4/miR124‐2 methylation‐positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124‐2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV‐negative carcinomas. These results indicate that a negative FAM19A4/miR124‐2 methylation assay result is likely to rule out the presence of cervical cancer.

Classification of high‐grade cervical intraepithelial neoplasia by p16ink4a, Ki‐67, HPV E4 and FAM19A4/miR124‐2 methylation status demonstrates considerable heterogeneity with potential consequences for management

AbstractHigh‐grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a, Ki‐67 and host‐cell DNA methylation) could provide guidance for clinical management in women with high‐grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0‐3) and Ki‐67 (Scores 0‐3), referred to as the “immunoscore” (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124‐2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0‐2 (6.0%), 151 lesions within IS group 3‐4 (30.4%) and 316 lesions within IS group 5‐6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). Methylation positivity increased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5‐6 (38/316). Notably, in a minority (43/497, 8.7%) of high‐grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high‐grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a “wait and see” policy to reduce overtreatment of high‐grade CIN lesions.

Risk-stratification of HPV-positive women with low-grade cytology by FAM19A4/miR124-2 methylation and HPV genotyping

Abstract Background The introduction of primary HPV screening has doubled the number of colposcopy referrals because of the direct referral of HPV-positive women with a borderline or mild dyskaryosis (BMD) cytology (ASC-US/LSIL) triage test. Further risk-stratification is warranted to improve the efficiency of HPV-based screening. Methods This study evaluated the discriminative power of FAM19A4/miR124-2 methylation, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping in HPV-positive women with BMD (n = 294) in two Dutch screening trials. Absolute CIN3+ risks and colposcopy referrals within one screening round were calculated. Results Methylation analysis discriminated well, yielding a CIN3+ risk of 33.1% after a positive result and a CIN3+ risk of 9.8% after a negative result. HPV16/18 and HPV16/18/31/33/45 genotyping resulted in a 27.6% and 24.6% CIN3+ risk after a positive result, and a 13.2% and 9.1% CIN3+ risk after a negative result. Colposcopy referral percentages were 41.2%, 43.2%, and 66.3% for FAM19A4/miR124-2 methylation, HPV16/18 and HPV16/18/31/33/45 genotyping, respectively. The CIN3+ risk after a negative result could be lowered to 2.8% by combining methylation and extended genotyping, at the expense of a higher referral percentage of 75.5%. Conclusion The use of FAM19A4/miR124-2 methylation and/or HPV genotyping in HPV-positive women with BMD can lead to a substantial reduction in the number of direct colposcopy referrals.