Aqueous Humor as a Surrogate Liquid Tumor Biopsy in Retinoblastoma

Abstract

Purpose : The aim of this study is to determine whether tumor-derived nucleic acids and tumor DNA copy number alterations can be detected in the aqueous humor (AH) of retinoblastoma (RB) eyes.

Methods : AH was analyzed for DNA, RNA and miRNA using Qubit HS kits. Circulating cell-free DNA (cfDNA) isolation and sequencing library protocols were optimized to retain cfDNAs from the AH and these optimized methods were applied to AH samples from RB patients. Shallow whole genome sequencing was performed on Illumina platform followed by genome-wide copy number variation (CNV) profiling to assess the presence of tumor DNA fractions in AH cfDNA.

Results : Eighteen AH samples from 8 patients (6 patients pre-intravitreal injection and 2 post-enucleation) were examined. All had measurable DNA, RNA and miRNA, with miRNA having the highest concentrations. Whole genome sequencing of AH cfDNA from 2 primarily enucleated eyes and sequential AH cfDNAs obtained from 2 eyes undergoing intravitreal melphalan injections for treatment of tumor seeding revealed tumor-derived cfDNA based on CNV profiles that demonstrated tumor copy number alterations.

Conclusions : This is the first study to evaluate AH from RB eyes undergoing salvage therapy with intravitreal injection of melphalan. We were able to assay quantifiable levels of nucleic acids in treated and untreated eyes and found that AH cfDNA had retinoblastoma-related DNA copy number alterations in all four tested RB patients. This suggests that AH can serve as a ‘surrogate tumor biopsy’ when tumor tissue is not available.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.