Abstract 582: Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma
Authors: Kahana-Edwin Smadar , Minoche Andre E. , Cain Lucy E. , McCowage Geoffrey , Woodfield Sarah E. , Vasudevan Sanjeev A. , Kummerfeld Sarah , Karpelowsky Jonathan 
Published: July 2021

Abstract
Driver mutations in CTNNB1 are a hallmark of hepatoblastoma (HB) and offer a common biomarker for a liquid biopsy approach that is based on the presence of CTNNB1 circulating tumor DNA (ctDNA). Initial results 1 showed an association of CTNNB1 ctDNA variant allele frequency (VAF) identified through digital droplet PCR (ddPCR) and the levels of serum Alpha-fetoprotein (AFP, current HB biomarker) throughout the course of treatment of three patients with HB. However, applying custom probe-designed ddPCR assays may pose challenges for real-time data at diagnosis. Our primary objective is to investigate the utility of a universal next-generation sequencing (NGS) assay to detect CTNNB1 ctDNA in patients with HB. Our secondary objective is to compare the levels of: (1) NGS ctDNA, (2) ddPCR ctDNA, (3) AFP, and verify if they correlate with tumor burden and treatment response. We developed and tested a custom NGS assay covering exons 2 to 4 of CTNNB1 for detection of somatic mutations in plasma. We used the QIASeq platform which: (1) is based on a single primer extension and, thus, captures both single nucleotide variants (SNV) and structural variants (SV), and (2) integrates unique molecular indexing (UMI), thus allowing determination of ctDNA levels from VAF. Our cohort included 18 patients, 13 with Sanger sequencing confirmed CTNNB1 somatic mutations in the matched tumors: 6 missense SNVs and 7 deletions ranging from 96 to 348 base pairs. Our NGS assay was able to accurately detect mutations in 9/13 (69%, 7 SV and 2 SNV) of the Sanger confirmed cases, and SV and missense SNV in 2/5 (40%) cases pending orthogonal Sanger confirmation. Available ddPCR data for 3 patients (8 samples) 1 and corresponding NGS data showed similar ctDNA levels (deviation ranging between 1.2-8.5%). NGS CTNNB1 variants were detected in samples taken at initial diagnosis in 8/9 (89%), following neoadjuvant chemotherapy in 4/9 (44%), post-operatively for fully resected localized disease in 0/4 (0%), and metastatic recurrence in 1/5 (20%), suggesting a positive correlation between CTNNB1 variants and tumor burden. AFP levels were available for 24/27 samples, with abnormal levels detected in 23 samples. The ctDNA levels determined from NGS did not correlate with AFP levels; however, longitudinal plasma samples (n=9) showed similar dynamics of both ctDNA and AFP, reflecting the response to treatment. To conclude, we show that ctDNA detectable with our universal HB NGS assay is a good surrogate marker of tumor burden and shows correlation with treatment response. SV, prevalent in approximately 50% of cases of HB, may be inadequately captured and thereby misrepresented in probe-capture NGS platforms but were highly detectable in our assay. Further work is needed to understand the interplay of both AFP and ctDNA in HB monitoring. Reference: 1. Kahana-Edwin S, McCowage G, Cain L, et al. Exploration of CTNNB1 ctDNA as a putative biomarker for hepatoblastoma. Pediatr Blood Cancer. 2020. doi:10.1002/pbc.28594
Citation Format: Smadar Kahana-Edwin, Andre E. Minoche, Lucy E. Cain, Geoffrey McCowage, Sarah E. Woodfield, Sanjeev A. Vasudevan, Sarah Kummerfeld, Jonathan Karpelowsky. Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 582.