Abstract 1507: Regional DNA methylation profiling reveals novel epigenetic intra-tumoral heterogeneity signatures and aberrant molecular clocks in hepatocellular carcinoma
Authors: Bubie Adrian , Restrepo Paula , Craig Amanda , Labgaa Ismail , Schwartz Myron , Thung Swan , Stolovitzky Gustavo , Losic Bojan , Villanueva Augusto 
Published: September 2020

Abstract
Introduction: There is limited understanding of epigenetic intra-tumoral heterogeneity (ITH) in hepatocellular carcinoma (HCC) and its potential role in driving cancer evolution. We aimed at deciphering the contribution of DNA methylation to molecular heterogeneity in HCC and identifying novel epi-drivers of cancer evolution.
Methods: We conducted reduced-representation bisulfite sequencing (RRBS) in 35 HCC and 12 adjacent non-tumoral regional samples across 9 patients with treatment-naive, early stage tumors. We computed regional differential DNA methylation ITH signatures across 17,276 well-covered sites within (hg19) promoter regions in CpG islands. We integrate these data with RNAseq, CNV, tumor-infiltrating lymphocyte (TIL) and hepatitis-B viral (HBV) expression on the same regional samples. We used linear models to regress out regional methylation signatures of TIL expression and clonality and HBV expression. Since RRBS significantly overlaps Horvath's clock CpG islands1 (237/354), we trained an expanded, aberrant HCC molecular clock function which quantifies clonal evolution in terms of a regionally varying apparent tumor molecular age and provides potential proxies for estimating tumor growth rates and transverse velocities.
Results: There was significant epigenetic ITH in 4/9 patients as determined by tumor regions for one patient clustering closer with other regions from a different patients than to the other tumor regions of the same patient. These epigenetic outliers were enriched in regions with differential TIL burdens (FDR&lt;0.05). We found ITH signatures that highlighted regional demethylation of MGAT1 (FDR &lt; 1e-3), a novel transcriptional target for the beta catenin signaling pathway, and tumor suppressive cyclin dependent kinase CDK3 (FDR &lt; 1e-2). We also found that while epigenetic tumoral age exceeds patient age (p &lt; 0.001), there exist well-defined conditions for highly regionally varying tumor age that implicate punctuated clonal evolution. Finally, using our regionally trained HCC clock, we leveraged the TCGA LIHC (HCC) cohort of 377 HCC single-biopsy patients profiled with Illumina 450k methylation array to predict the distribution of effective tumor ages. We found that our ITH methylation signatures and effective tumor age are significant predictors of survival under bootstrap validation, controlling for tumor stage.
Conclusions: Novel epigenetic ITH signatures in a subset of HCC tumors which further implicate beta catenin signaling and upregulation of key cyclin dependent kinases predict patient survival in the TCGA, and allow for a quantification of an accelerated aberrant HCC molecular time which estimates tumor age (tumor age ~ [1.5 - 10] x patient age) and growth history.
References:
1. Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, 1-20 (2013).
Citation Format: Adrian Bubie, Paula Restrepo, Amanda Craig, Ismail Labgaa, Myron Schwartz, Swan Thung, Gustavo Stolovitzky, Bojan Losic, Augusto Villanueva. Regional DNA methylation profiling reveals novel epigenetic intra-tumoral heterogeneity signatures and aberrant molecular clocks in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1507.