Plasma cell free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study

Jörn Lewin, Denise Kottwitz, Johanna Aoyama, Theo deVos, Jorge Garces, Oliver Hasinger, Stefanie Kasielke, Florian Knaust, Preeti Rathi, Sebastian Rausch, Gunter Weiss, Alexander Zipprich, Edward Mena, Tse-Ling Fong, Jörn Lewin, Denise Kottwitz, Johanna Aoyama, Theo deVos, Jorge Garces, Oliver Hasinger, Stefanie Kasielke, Florian Knaust, Preeti Rathi, Sebastian Rausch, Gunter Weiss, Alexander Zipprich, Edward Mena, Tse-Ling Fong

Abstract

Background: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis, primarily due to failed early detection. HCC screening is recommended among individuals with cirrhosis using biannual abdominal ultrasound, for earlier tumor detection, administration of curative treatment, and improved survival. Surveillance by imaging with or without biomarkers such as alpha-fetoprotein (AFP) remains suboptimal for early stage HCC detection. Here we report on the development and assessment of methylation biomarkers from liquid biopsies for HCC surveillance in cirrhotic patients.

Methods: DNA methylation markers including the HCCBloodTest (Epigenomics AG) and a DNA-methylation panel established by next generation sequencing (NGS) were assessed using a training/testing design. The NGS panel algorithm was established in a training study (41 HCC patients; 46 cirrhotic non-HCC controls). For testing, plasma samples were obtained from cirrhotic patients (Child class A or B) with (60) or without (103) early stage HCC (BCLC stage 0, A, B). The assays were then tested using blinded sample sets and analyzed by preset algorithms.

Results: The HCCBloodTest and the NGS panel exhibited 76.7% and 57% sensitivities at 64.1% and 97% specificity, respectively. In a post-hoc analysis, a combination of the NGS panel with AFP (20 ng/mL) achieved 68% sensitivity at 97% specificity (AUC = 0.9).

Conclusions: Methylation biomarkers in cell free plasma DNA provide a new alternative for HCC surveillance. Multiomic panels comprising DNA methylation markers with other biological markers, such as AFP, provide an option to further increase the overall clinical performance of surveillance via minimally invasive blood samples.

Trial registration: Test set study-ClinicalTrials.gov (NCT03804593) January 11, 2019, retrospectively registered.

Keywords: Biomarker; Carcinoma; Cell-free nucleic acids; DNA methylation; Early detection of cancer; Hepatocellular; Liver cirrhosis.

Conflict of interest statement

Authors indicated by 1,2 are full time employees of Epigenomics AG, the sponsor of the study.

Figures

Fig. 1
Fig. 1
Receiver operating characteristic (ROC) analysis of the NGS Panel, AFP and AFP + NGS Panel. ROC curves for the NGS panel (black), AFP (red) and combination of NGS panel with AFP (blue) using logistic regression for 60 HCC versus 102 controls in the testing set. Areas under the curve (AUC) are written at the bottom right using the corresponding colors. Selected sensitivity/specificity pairs indicated on the curves are reported AFP ≥ 20 ng/mL, the NGS panel using the trained algorithm, and the post hoc combination of the NGS panel and AFP

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