Abstract B008: The role of circulating tumor DNA (ctDNA) in profiling hepatoblastoma tumor heterogeneity

Abstract Purpose: Hepatoblastoma (HB) is the most common pediatric liver tumor. Prior studies of HB have associated some somatic variants with poor outcomes, but the intratumoral heterogeneity in HB makes it difficult to capture all genomic features with tissue biopsy. Circulating tumor DNA (ctDNA) has been used to detect heterogeneous somatic features in pediatric solid tumors. While ctDNA has been detected in HB using tumor-informed PCR, ctDNA profiling with next-generation sequencing (NGS) has not been used to study HB heterogeneity. Further, while studies show that ctDNA has tumor-specific fragment features compared to background cell-free DNA, these have not been used to improve ctDNA detection sensitivity in HB. Here, we aimed to detect and quantify ctDNA and to profile heterogeneity in HB patient blood samples by combining NGS and ctDNA fragment features. Methods: Plasma and tumor tissue were collected for 18 patients with HB through an institutional banking protocol. All collected plasma (n=77) and matched FFPE tumor tissue (n=14) underwent ultra-low pass whole-genome sequencing (ULP-WGS) at 0.2X target depth across the genome. The validated ichorCNA algorithm was used to detect and quantify ctDNA based on aneuploidy. Fragment length and end motifs were compared between plasma from HB patients and healthy controls. A subset of plasma with detectable ctDNA and matched tumor tissue underwent panel sequencing targeting 400 genes at 25000X target depth. Results: Of 18 patients who had plasma collected prior to or early on therapy, 8 (44.4%) had at least one plasma sample with detectable ctDNA by ULP-WGS. Panel sequencing was performed on 8 plasma samples from 6 patients. We detected mutations or deletions in CTNNB1 in 5/8 samples with ctDNA>3%. For a patient with multifocal HB, one of two liver lesions was biopsied and showed a c.104T>G CTNNB1 variant and copy-neutrality for 1q. While the biopsied lesion regressed during therapy, the second tumor progressed. Plasma samples collected after 2, 4, and 20 cycles of palliative chemotherapy, while the second tumor was progressing, demonstrated a 1q gain and a c.101G>A CTNNB1 variant with increasing variant allelic fraction (5.3%, 17.0%, 22.8%), suggesting that the second tumor was a unique lesion. To further improve sensitivity for ctDNA detection, fragment features were studied and showed different end motifs (p<0.01) and shorter fragments in HB than healthy plasma (p<0.01). Both unsupervised clustering based on end motifs and restricting ichorCNA analyses to short fragments improved sensitivity for detecting ctDNA in multiple samples where ctDNA was not detected based on aneuploidy (in 9/18 and 5/18 patients, respectively). Conclusions: ctDNA is detectable and can be profiled in patients with HB by NGS to identify variants not anticipated by the patient's diagnostic biopsy and to study tumor evolution and heterogeneity. Furthermore, cell-free DNA fragment features appear to differentiate HB ctDNA from healthy donor samples and may be useful for improving sensitivity of non-invasive assays to profile HB. Citation Format: Nensi M. Ruzgar, Kelly Klega, Cora Ricker, Josephine H. Wilson, Mohammad Tanhaemami, Natalie Greenwood, Catherine Clinton, Sydney O'Donnell, Joann Cassama, Sam Pollock, Carrie Cibulskis, Allison F. O'Neill, Brian Crompton. The role of circulating tumor DNA (ctDNA) in profiling hepatoblastoma tumor heterogeneity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B008.