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Driver mutations in CTNNB1 are a hallmark of hepatoblastoma and offer a common biomarker for a liquid biopsy approach based on the presence of CTNNB1 circulating tumor DNA (ctDNA). We developed and investigated the utility of a quantitative universal next-generation sequencing (NGS) ctDNA assay for hepatoblastoma (QUENCH) to detect CTNNB1 ctDNA and assessed the links between ctDNA and current clinical indicators/biomarkers in hepatoblastoma. Applied to patients with hepatoblastoma, we demonstrate quantitation of various variants including single base substitutions and deletions down to 0.3% variant allele frequency, with 65% sensitivity and 100% specificity at the patient level, to allow biopsy-free tumor genotyping and sensitive ctDNA quantitation. CtDNA positivity correlates with tumor burden and ctDNA levels correlate with macroscopic residual disease and treatment response, thus providing promising evidence for the utility of quantitative ctDNA detection in hepatoblastoma.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB.Summary StatementIn this work, we developed and thoroughly characterized several unique models of hepatoblastoma derived from the HepT1 cell line that show high-risk features.