Abstract 2877: Genomic cfDNA analysis of aqueous humor in retinoblastoma (RB) predicts eye salvage
Authors: Xu Liya , Berry Jesse L. , Kooi Irsan , Murphree A Linn , Prabakar Rishvanth K. , Reid Mark W. , Stachelek Kevin , Le Bao Han A. , Welter Lisa , Jubran Rima , Lee Thomas C. , Kim Jonathan W. , Kuhn Peter , Cobrinik David , Hicks James B. 
Published: August 2019

Abstract
Background: Retinoblastoma (RB) was one of the first tumors to demonstrate a genetic basis to the development of cancer. However, unlike other cancers, RB cannot be directly biopsied due to the high risk of extraocular cancer spread. Therefore, unless the eye is enucleated, tumor tissue is not evaluated for genetic and genomic changes and these alterations are not used to inform diagnosis or prognosis for this disease. However, in a 2017 publication in JAMA Ophthalmology, we demonstrated that tumor-derived cell-free DNA can be extracted from the aqueous humor (AH) of RB eyes, which is safe to remove even with active intraocular disease. The purpose of this current study (Berry JL, Xu L et al. Molec Canc Res 2018) was to identify somatic chromosomal copy number alterations (SCNA) in tumor-derived cell-free DNA in the AH of RB eyes and to correlate with clinical outcomes particularly tumor relapse requiring enucleation.
Methods: AH was extracted via paracentesis from RB eyes during intravitreal injection of chemotherapy or post enucleation. Shallow whole genome sequencing was performed to assess for cell-free tumor DNA fractions and highly-recurrent SCNAs in RB which include gain of 1q, 2p, 6p and loss of 13q and 16q. Globe salvage was recorded.
Results: 26 patients were included; 3 patients had both eyes included for 29 eyes. From these, 63 samples of AH were analyzed; 5 post-enucleations and 58 during intravitreal chemotherapy injection. Ultimately 13 eyes required enucleation and 16 eyes were salvaged. Follow-up ranged from 8-43 months (median 17 months).
The presence of any detectable SCNA was 92% in enucleated eyes versus 38% in salvaged eyes (p=0.006). 6p gain was the most common SCNA found in 77% of enucleated eyes versus 25% of salvaged eyes (p=0.0092). 6p gain was associated with a ten-fold increased odds of enucleation (OR=10, 95% CI:1.8-55.6). The mean amplitude of 6p gain was 1.47 in enucleated eyes versus 1.07 in salvaged eyes (p=0.001). The probability of ocular survival was higher in eyes without detectable SCNAs in the AH (p=0.0028).
Serum testing was positive for a germline mutation in 17 eyes of 14 patients with the following incidence of RB SCNAs 1q (35%); 2p (18%); 6p (47%); 13q (18%) and 16q (35%) versus 12 eyes of 12 patients without germline disease with 1q (33%); 2p (8%); 6p (50%); 13q (17%) and 16q (33%) (p=0.71).
Conclusions: This is the first study to show that clinical outcomes correlate with highly-recurrent SCNAs in the AH from RB eyes. This study suggests that the AH can reliably serve as a surrogate to tumor biopsy and improves upon current clinical staging to predict tumor response to therapy and the ability to salvage the eye. Unlike previous studies that suggest a greater incidence of RB SCNAs in patients with non-germline disease, this was not seen in this evaluation which may be because all previous work was done on tumor from enucleated eyes instead of salvaged eyes.
Citation Format: Liya Xu, Jesse L. Berry, Irsan Kooi, A Linn Murphree, Rishvanth K. Prabakar, Mark W. Reid, Kevin Stachelek, Bao Han A. Le, Lisa Welter, Rima Jubran, Thomas C. Lee, Jonathan W. Kim, Peter Kuhn, David Cobrinik, James B. Hicks. Genomic cfDNA analysis of aqueous humor in retinoblastoma (RB) predicts eye salvage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2877.