Chromosome 6p Amplification in Aqueous Humor Cell-Free DNA Is a Prognostic Biomarker for Retinoblastoma Ocular Survival

Abstract

Aqueous humor contains tumor-derived cell-free DNA (cfDNA) and can serve as a liquid biopsy for retinoblastoma. We previously associated somatic copy-number alteration (SCNA) 6p gain with a 10-fold increased risk of enucleation. Here we provide a 2-year update to further explore 6p gain as a prognostic biomarker for ocular survival. Patients diagnosed with retinoblastoma from December 2014 to July 2019 from whom aqueous humor was sampled were included. cfDNA was extracted and shallow whole-genome sequencing performed to identify highly recurrent retinoblastoma SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). 116 aqueous humor samples from 50 eyes of 46 patients were included: 27 eyes were salvaged, 23 were enucleated. Highly recurrent retinoblastoma SCNAs were found in 66% eyes. 6p gain was the most prevalent SCNA (50% eyes). It was particularly more prevalent in enucleated eyes (73.9%) than in salvaged eyes (29.6%; P = 0.004). 6p gain in aqueous humor cfDNA portended nearly 10-fold increased odds of enucleation (OR = 9.87; 95% confidence interval = 1.75-55.65; P = 0.009). In the enucleated eyes, 6p gain was associated with aggressive histopathologic features, including necrosis, higher degrees of anaplasia, and focal invasion of ocular structures. With extended follow-up and nearly double the aqueous humor samples, we continue to demonstrate 6p gain as a potential prognostic biomarker for retinoblastoma. IMPLICATIONS: Aqueous humor is a high-yield source of tumor-derived DNA in retinoblastoma eyes. Detection of 6p gain in the aqueous humor allows for targeted, patient-centered therapies based on this molecular prognostic marker. Prospective, multicenter studies with aqueous humor sampled from all eyes at diagnosis are warranted to validate these findings.

Conflict of interest statement

Disclosures: Drs. Berry, Xu, and Hicks have filed a provisional patent application entitled, Aqueous Humor Cell Free DNA for Diagnostic and Prognostic Evaluation of Ophthalmic Disease. Otherwise the authors declare no potential conflicts of interest.

©2020 American Association for Cancer Research.

Figures

Figure 1.

Flow diagram summarizing the treatments given and the presence or absence of 6p gain in each treatment group.

Figure 2.

Kaplan-Meier analysis demonstrating a 3.8-times greater risk of enucleation in eyes with 6p gain compared to eyes without a 6p gain after 1 year.

Figure 3.

A, Composite somatic copy number alteration (SCNA) profile of cell-free DNA (cfDNA) sampled from the aqueous humor (AH) of enucleated (Enuc., red) and salvaged (Salv., blue) eyes. B, Box plots demonstrating the range of ratio to median amplitudes for SCNAs in enucleated (Enuc.) and salvaged (Salv.) eyes (median, black bar; mean, green bar). The median of the ratio to the median amplitude of chromosome 6p gain is significantly greater in enucleated eyes compared to salvaged eyes (P = 0.005).

Figure 4.

Chromosome 6 heatmap showing the lengths of 6p gain (red) in 25 eyes with retinoblastoma. Most eyes demonstrated a gain of the entire 6p arm. The minimum region of gain (MRG) was localized to a 19 Mbp region that included a gain of DEK (Case 11).