Abstract PR018: Tumor-Free RB1 Molecular Diagnosis: Aqueous Humor Liquid Biopsy Enables Comprehensive In Vivo Characterization of Biallelic RB1 Inactivation in Retinoblastoma

Abstract

Retinoblastoma is one of the few pediatric solid tumors in which diagnostic tumor biopsy is contraindicated, making molecular characterization during eye-sparing treatment uniquely challenging. Our group previously established aqueous humor (AH) cell-free DNA (cfDNA) as a tumor-derived liquid biopsy for retinoblastoma, demonstrating robust detection of somatic copy number alterations. However, comprehensive RB1 molecular diagnosis, including identification of both RB1 inactivating events, has remained technically challenging because of the limited quantity of cfDNA recovered from AH. We therefore evaluated whether a clinically validated targeted sequencing assay optimized for the AH matrix could enable comprehensive in vivo RB1 molecular diagnosis without requiring tumor tissue. Diagnostic AH samples from 65 eyes of 53 patients with retinoblastoma underwent targeted sequencing using the LBSeq4Kids assay, a CLIA laboratory-developed test (LDT) analytically validated for AH specimens, to identify RB1 sequence variants, deletions, promoter alterations, and loss of heterozygosity (LOH). Germline RB1 status was determined by blood testing when available. RB1 detection rates, biallelic RB1 inactivation patterns, and RB1 alteration spectra were compared with published retinoblastoma tumor cohorts. AH cfDNA identified at least one RB1 alteration in 57 of 65 eyes (87.7%), while complete biallelic RB1 molecular diagnosis was achieved in 53 eyes (81.5%). Complete identification of both RB1 inactivating events was achieved in 86.1% of germline-associated tumors and 75.9% of non-heritable tumors. AH detected the complete spectrum of clinically relevant RB1 alterations, including nonsense, frameshift, splice-site, missense, promoter variants, deletions, and LOH. Remarkably, the frequencies and architectures of RB1 alterations closely recapitulated those reported in published tumor cohorts, demonstrating that AH faithfully reproduces tumor-derived RB1 molecular findings in living eyes. In addition, pathogenic alterations in BCOR, ARID1A, MSH6 and FANCL were identified in a subset of samples, supporting the broader capability of AH liquid biopsy for molecular profiling beyond RB1. These findings demonstrate that comprehensive in vivo RB1 molecular diagnosis, including accurate resolution of biallelic RB1 inactivation, can now be achieved without tumor tissue. Building upon our previous development of AH liquid biopsy for retinoblastoma, this work extends the platform from copy number profiling to comprehensive RB1 molecular diagnosis, overcoming a major technical barrier created by the limited DNA input of AH specimens. The high concordance between AH and published tumor cohorts supports the clinical implementation of AH liquid biopsy for precision molecular diagnosis in retinoblastoma. Disclosure: Generative artificial intelligence was used only for language editing and grammar refinement. The scientific content was developed by the authors. All authors reviewed, verified, and take full responsibility for the accuracy and integrity of the abstract.