Abstract 1273: RASSF1 promoter hypermethylation is associated with activated CTNNB1 pathways in hepatocellular carcinoma
Authors: Kwee Sandi Alexander , Peplowska Karolina , Farrar Christine , Wong Linda , Song Min-ae , Tiirikainen Maarit 
Published: September 2020

Abstract
BACKGROUND: The promoter-region of the Ras association domain family member 1 (RASSF1) gene is frequently hypermethylated in several solid tumors, including hepatocellular carcinoma (HCC). Wnt-pathway deregulation and increased accumulation of nuclear catenin-beta-1 (CTNNB1) has been associated with RASSF1 inactivation, and more recently, with diminished response to immune checkpoint blockade in HCC. There is limited data from clinical tumor and liquid biopsy samples on the relationship between RASSF1 promoter methylation and Wnt beta-catenin pathway activation status in HCC.
METHODS: Pyrosequencing was performed to assess RASSF1 promoter methylation in DNA from paired HCC tumor/adjacent-liver tissues as well as cell-free DNA (cfDNA) and buffy coat DNA from blood samples collected in Streck cfDNA tubes from 40 patients with surgically-resectable HCC participating in an IRB-approved diagnostic clinical trial (NCT01395030). All blood samples were collected pre-operatively while all tissue samples were collected upon surgical tumor resection. The DNA methylation analysis results from tumors were compared to a CTNNB1 pathway activation gene expression signature obtained from the same tumor samples. Targeted next-generation DNA sequencing (NGS) was also performed on a limited number of cfDNA samples using a 56 gene Oncology Panel (56G v2, Swift Bioscience) to obtain mutational profiles (including CTNNB1 mutation status) from each HCC case. Allele variant detection was performed using the ERASE-Seq algorithm (Fluxion Biosciences).
RESULTS: RASSF1 promoter hypermethylation was detected in 35/40 tumor samples. Levels of RASSF1 promoter methylation were significantly higher in tumor as compared to liver tissue DNA (55.2% vs. 23.5%, p &lt; 0.0001). Methylation levels in the RASSF1 promoter were also significantly higher in cfDNA as compared to buffy-coat DNA (7.5% vs. 0.4%, p &lt; 0.0001). Tumors enriched for genes associated with Wnt beta-catenin pathway activation demonstrated significantly higher RASSF1 promoter methylation as compared to tumors enriched for immune/interferon-related genes (63% vs. 20%, Tukey p = 0.0004). Pilot liquid biopsy studies involving DNA NGS and methylation analysis in a subset of pre-operative blood samples also revealed higher RASSF1 promoter methylation among the cell-free DNA samples carrying activating CTNNB1 exon 3 mutations as compared to p53 mutations (23.4% vs. 1.4%, Tukey p = 0.009) and PIK3 mutations (23.4% vs. 1.5%, Tukey p = 0.031). RASSF1 promoter methylation in tumor tissue DNA was also significantly higher in patients with liquid biopsy detected CTNNB1-mutations as compared to p53-mutations (65.5% versus 37%, p = 0.030).
CONCLUSION: CTNNB1 mutations and related gene expression patterns may be associated with RASSF1 promoter hypermethylation in HCC. The clinical implications for immunotherapy and the mechanisms underlying these associations deserve further investigation.
Citation Format: Sandi Alexander Kwee, Karolina Peplowska, Christine Farrar, Linda Wong, Min-ae Song, Maarit Tiirikainen. RASSF1 promoter hypermethylation is associated with activated CTNNB1 pathways 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 1273.