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

Abstract

Purpose : There are currently no eye-specific molecular biomarkers in clinical practice for retinoblastoma (RB), a rare, pediatric intraocular cancer, because direct tumor biopsy is prohibited. We overcome this problem by using the aqueous humor (AH) as a liquid biopsy source of cell-free tumor DNA (ctDNA).This study is aim to detail the first prospective data using the AH liquid biopsy to identify diagnostic and prognostic molecular biomarkers present at diagnosis and discuss the validity and clinical utility of the platform.

Methods : Subjects included 7 eyes of 6 RB patients with at least 12 months of follow-up. The AH liquid biopsy was used at diagnosis and prospectively throughout therapy until the eye was cured or required enucleation due to persistent disease. Cell-free DNA (cfDNA) was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs) and mutation in RB1 gene. Results were compared to peripheral blood RB1 testing. Tumor fraction (TFx) was calculated using ichorCNA. Primary clinical endpoint was ocular salvage versus enucleation.

Results : Tumoral genomic information was detected in 100% of diagnostic AH samples. Of the 7 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 secondary enucleation; both had poor prognostic biomarkers (chromosome 6p gain and MYCN amplification) present in the AH at the time of diagnosis. No patients had any complications from AH sampling or demonstrated extraocular spread of disease.

Conclusions : This initial prognostic study demonstrates feasibility, safety, and utility of an AH liquid biopsy at diagnosis. Molecular profiling of AH provides impactful tumor-derived diagnostic and prognostic information from a single 100 µL AH sample. 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.

This is a 2021 ARVO Annual Meeting abstract.