Analytical validation and diagnostic performance of the ASCL1/ZNF582 methylation test for detection of high-grade anal intraepithelial neoplasia and anal cancer.

DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN). For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10-8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity. In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.

DNA methylation markers have universal prognostic value for anal cancer risk in HIV-negative and HIV-positive individuals.

Anal cancer has increasing incidence and is preceded by high-grade anal intraepithelial neoplasia (HGAIN; AIN2-3). Previously, we identified and validated several methylation markers for accurate detection of anal cancer and HGAIN with cancer risk in HIV-positive (HIV+) men who have sex with men (MSM). This study aimed to evaluate these markers in HIV-negative risk groups. A cross-sectional series of 176 tissue samples of anal cancer, AIN3, AIN2, AIN1 and control biopsies obtained in HIV-negative women and men was tested for six methylation markers (ASCL1, LHX8, SST, WDR17, ZIC1 and ZNF582). Accuracy for detection of AIN3 and cancer (AIN3+) was determined by univariable and multivariable mixed-effect ordinal logistic regression. Methylation levels of all markers increased with increasing severity of disease (P < 0.0001) and were comparable to results in HIV+ MSM. All markers showed high accuracy for AIN3+ detection [area under the curve (AUC): 0.83-0.86]. The optimal marker panel (ASCL1 and ZIC1; AUC = 0.85 for AIN3+) detected 98% of cancers at 79% specificity. In conclusion, DNA methylation markers show a high diagnostic performance for AIN3+ detection in HIV+ and HIV-negative risk groups, justifying broad application of methylation analysis for anal cancer prevention programmes.

Cancer Risk Stratification of Anal Intraepithelial Neoplasia in Human Immunodeficiency Virus-Positive Men by Validated Methylation Markers Associated With Progression to Cancer.

BACKGROUND: High-grade anal intraepithelial neoplasia (HGAIN; AIN2-3) is highly prevalent in HIV+ men, but only a minority of these lesions progress towards cancer. Currently, cancer progression risk cannot be established; therefore, no consensus exists on whether HGAIN should be treated. This study aimed to validate previously identified host cell DNA methylation markers for detection and cancer risk stratification of HGAIN. METHODS: A large independent cross-sectional series of 345 anal cancer, AIN3, AIN2, AIN1, and normal control biopsies of HIV+ men was tested for DNA methylation of 6 genes using quantitative methylation-specific PCR. We determined accuracy for detection of AIN3 and cancer (AIN3+) by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Methylation levels were assessed in a series of 10 anal cancer cases with preceding HGAIN at similar anatomic locations, and compared with the cross-sectional series. RESULTS: Methylation levels of all genes increased with increasing severity of disease (P < .05). HGAIN revealed a heterogeneous methylation pattern, with a subset resembling cancer. ZNF582 showed highest accuracy (AUC = 0.88) for AIN3+ detection, slightly improved by addition of ASCL1 and SST (AUC = 0.89), forming a marker panel. In the longitudinal series, HGAIN preceding cancer displayed high methylation levels similar to cancers. CONCLUSIONS: We validated the accuracy of 5 methylation markers for the detection of anal (pre-) cancer. High methylation levels in HGAIN were associated with progression to cancer. These markers provide a promising tool to identify HGAIN in need of treatment, preventing overtreatment of HGAIN with a low cancer progression risk.