Establishing the Clinical Utility of ctDNA Analysis for Diagnosis, Prognosis, and Treatment Monitoring of Retinoblastoma: The Aqueous Humor Liquid Biopsy

Abstract

Simple Summary Due to prohibition of direct tumor biopsy for patients with retinoblastoma, the prospect of a liquid biopsy for the identification of tumor derived biomarkers for this cancer is enticing. The aqueous humor (AH) is a rich source of eye-specific tumoral genomic information. This is the first prospective study wherein we demonstrate that molecular profiling of the AH at diagnosis and longitudinally throughout therapy has clinical utility for diagnosis, prognosis, and monitoring of treatment response. Tumoral genomic information was detected in 100% of diagnostic aqueous humor samples, including single nucleotide variants in the RB1 tumor suppressor gene and large-scale somatic chromosomal alterations. All eyes that failed therapy and required enucleation had poor prognostic biomarkers for ocular salvage present in the aqueous humor at time of diagnosis. This highlights the potential of the AH liquid biopsy for direct clinical applications to precision oncology to direct genome-specific, personalized treatment for retinoblastoma patients. Abstract Because direct tumor biopsy is prohibited for retinoblastoma (RB), eye-specific molecular biomarkers are not used in clinical practice for RB. Recently, we demonstrated that the aqueous humor (AH) is a rich liquid biopsy source of cell-free tumor DNA. Herein, we detail clinically-relevant molecular biomarkers from the first year of prospective validation data. Seven eyes from 6 RB patients who had AH sampled at diagnosis and throughout therapy with ≥12 months of follow-up were included. Cell-free DNA (cfDNA) from each sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing for pathogenic variants using a RB1 and MYCN custom hybridization panel. Tumoral genomic information was detected in 100% of diagnostic AH samples. Of the seven diagnostic AH samples, 5/7 were positive for RB SCNAs. Mutational analysis identified RB1 variants in 5/7 AH samples, including the 2 samples in which no SCNAs were detected. Two eyes failed therapy and required enucleation; both had poor prognostic biomarkers (chromosome 6p gain or MYCN amplification) present in the AH at the time of diagnosis. In the context of previously established pre-analytical, analytical, and clinical validity, this provides evidence for larger, prospective studies to further establish the clinical utility of the AH liquid biopsy and its applications to precision oncology for RB.