Abstract 1977: Implementation of a comprehensive clinical cfDNA analysis platform for aqueous humor liquid biopsy in retinoblastoma
Authors: Laura A. Kagami, Eirini Christodoulou, Venkata Yellapantula, Dong Xu, Cindy Fong, Dejerianne Ostrow, Liya Xu, Jesse Berry, Jaclyn A. Biegel
Journal: Cancer Research (2025)

Introduction: The development of liquid biopsy testing based on sequencing analysis of cell-free DNA (cfDNA) has the potential to transform cancer care in pediatric tumors, which are characterized by a wide array of genomic alterations including copy number alterations (CNAs), sequence variants, epigenetic alterations, and structural rearrangements in tumor-associated genes. In patients with retinoblastoma where direct tumor biopsy is not possible due to the risk of tumor spread, aqueous humor (AH) is a rich source of cfDNA. Comprehensive cfDNA testing, combining a low pass whole genome sequencing (LP-WGS) assay to detect CNAs and a custom targeted sequencing panel (TSP) to detect mutations, can provide a highly effective platform to inform diagnosis, risk stratification, response to therapy, and surveillance in patients with retinoblastoma. LP-WGS Results: We validated a liquid biopsy LP-WGS assay, LBSeq4Kids, to detect CNAs in cfDNA from plasma, cerebrospinal fluid, or AH. Clinical testing has been performed for the past two years for diagnostic evaluation and monitoring response to therapy using AH from retinoblastoma patients. Thirty-six children with unilateral or bilateral retinoblastoma (44 eyes) underwent LBSeq4Kids LP-WGS analysis. CNAs were detected in 30/32 (94%) samples at diagnosis or recurrence. Serial sampling of 22/44 (50%) eyes was performed using a range of 1-10 samples per patient. Seventeen of those 22 (77%) eyes demonstrated clearance of CNAs with treatment. Five eyes showed persistence of abnormal profiles, necessitating enucleation of 2 eyes. Treatment and evaluation are ongoing for the remaining three eyes. Targeted Sequencing Panel Results: We are validating a custom cfDNA TSP to detect single nucleotide variants and small indels in the coding sequence of 136 genes and fusions involving EWSR1, FOXO1, and BRAF for patients with ocular disease, solid tumors and brain tumors. To date, 17 children (21 eyes) diagnosed with retinoblastoma have had TSP testing using AH. All eleven (100%) patients with germline variants demonstrated the same RB1 alterations in the AH using the panel. Six of these patients (55%) had a second somatic alteration in RB1 detected with the panel. Two of 11 (18%) with known germline RB1 mutations also demonstrated mutations in BCOR, ARID1A, or MSH6. Somatic variants in RB1, ARID1A and BCOR were detected in the remaining six patients with sporadic retinoblastoma. Targeted sequencing of the AH surveillance samples from these patients is in progress. Conclusion: Here we demonstrate clinical utility for aqueous humor liquid biopsy molecular testing as a complementary means of diagnosis and monitoring treatment response in patients with retinoblastoma. Continued surveillance of these patients is ongoing to monitor disease status and rule out recurrence, as well as identify novel prognostic biomarkers. Citation Format: Laura A. Kagami, Eirini Christodoulou, Venkata Yellapantula, Dong Xu, Cindy Fong, Dejerianne Ostrow, Liya Xu, Jesse Berry, Jaclyn A. Biegel. Implementation of a comprehensive clinical cfDNA analysis platform for aqueous humor liquid biopsy in retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1977.