Exploring the prognostic value in methylation markers in early detection of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has an unfavourable prognosis with little difference between incidence and mortality due to late detection. Unfortunately, despite an established surveillance method early detection of liver cancer remains elusive. Focusing on detecting earlier signals of the cancer development means catching earlier stages of the disease. Here epigenetic alterations come into play as they occur early in the cancer development. We focused on discovering epigenetic alterations such as differentially methylated regions of HCC patients and establishing their biological relevance in the context of patient prognosis/survival and early detection.

Differential methylation analysis pipeline was used to identify significant CpGs between 66 paired HCC tumour and adjacent non-tumour tissue samples (GSE54503). Those regions of interest were further validated in the second dataset of 30 HCC tumour and adjacent non-tumour tissue samples from an independent cohort (GSE113017). Gene ontology (GO) and Biological Pathway analysis was performed on Differentially Methylated Regions (DMRs) of interest. Additionally, 8 of these DMRs, reported to be relevant in HCC context, were evaluated in plasma cell-free DNA (cfDNA) from 10 HCC patients (7 stage I-III) and 20 matching control patients (including patients with cirrhosis and steatosis), using univariate statistics. Finally, 5-year survival analysis with Kaplan-Meier was done to assess the capability of methylation markers to predict patient prognosis.

Initial differentially methylated CpG analysis resulted in 5904 potential markers. Validation on a second ethnically distinct dataset showed 4068 of them remained relevant. This indicates that cancer- related methylation signals are robust regardless of the patient cohort. GO pathway analysis of the validated markers (n=4068) revealed that top pathways were involved in cell adhesion processes, which have implications in cancer initiation, progression, and treatment response. Other top pathways were involved in regulation of transcription and RNA metabolic processes. Whereas pathway analysis of non-overlapping markers (n=1836) showed that they were interestingly enriched for embryonic morphogenesis pathways which might indicate ethnicity-related developmental differences in the two cohorts. Targeted hybrid-capture based methylation sequencing of 8 significant DMRs, in plasma cfDNA of patients with HCC (stage I=3, stage II=2, stage III=2, non-metastatic [without further stage classification] =2 and unknown=1) and 20 age and gender-matching controls demonstrated early-detection potential, with univariate statistics on individual marker regions showing high accuracy with AUC= 0.97 for DLX1, AUC= 0.98 for UCHL1 and AUC= 0.89 for GSTP1. All 3 genes have been previously linked to HCC initiation, progression and/or prognosis. Kaplan-Meier correlation analysis showed DLX1 having significant 5-year survival prediction at p < 0.0162.

Methylation markers carry distinct biological signals in HCC, regardless of ethnical background of the patients. High individual accuracy for cancer detection was observed for early-stage cancers when compared to cfDNA from matching controls. These markers extend their relevance beyond early cancer detection, exhibiting strong potential also in HCC prognostication and patients’ stratification, and are potentially leading to better outcomes and improved survival of the patients. Further large-scale confirmation studies are underway.

The authors.

Funded by Universal Diagnostics S.A.

A. Furman: Full / Part-time employment: Universal Dx, d.o.o Ljubljana, Slovenia. J. Higareda-Almaraz: Full / Part-time employment: Universal Diagnostics SL. U. Kunej: Full / Part-time employment: Universal DX, d.o.o., Ljubljana, Slovenia . M. Chersicola: Full / Part-time employment: UniveralDX d.o.o. K. Kruusmaa: Shareholder / Stockholder / Stock options: Universal Dx d.o.o.; Full / Part-time employment: Universal Dx d.o.o.; Officer / Board of Directors: Universal Dx d.o.o..