Abstract 452: Simultaneous identification of clinically relevant RB1 Mutations and somatic copy number alterations in the Aqueous Humor of retinoblastoma eyes
Authors: Xu Liya , Shen Lishuang , Polski Ashley , Prabakar Rishvanth K. , Reid Mark , Kuhn Peter , Cobrinik David E. , Hicks James , Gai Xiaowu , Berry Jesse 
Published: September 2020

Abstract
Purpose: Retinoblastoma (RB) tumorigenesis is associated with mutations in the RB1 tumour suppressor gene and somatic copy number alterations (SCNAs). Both alleles of the RB1 gene must be mutated for tumour development. The initial RB1 mutation may be germline or somatic. However, the somatic RB1 mutation was only detectable in enucleated RB eyes, when the tumour samples were available. The aqueous humor (AH) can serve as a surrogate to direct tumour biopsy and can be done in vivo. In this study, we demonstrate that both whole genome SCNAs and RB1 mutations can be simultaneously assayed in the AH liquid biopsy.
Methods: AH from 7 eyes of 6 RB patients and 4 matched tumor samples were included. AH was extracted via clear corneal paracentesis. Cell-free DNA (cfDNA) was isolated and constructed into whole genome libraries. Low pass whole genome sequencing was performed for copy number alteration analysis. The same libraries were further used for capture-based targeted NGS for mutation detection. Custom hybridization capture panel manufactured from Agilent includes the whole gene region of RB1 gene, MCYN gene, and all exons of BCOR and CREBBP genes (Agilent SureSelect Tier1). Captured libraries were sequenced on Illumina platform with paired-end 150 cycles mode. Bioinformatics analysis was performed for single-nucleotide variant (SNV) and loss of heterozygosity (LOH) detection using an in-house pipeline (information available on request). Comparison was also made to clinically available peripheral blood RB1 mutation screening (CHLA, CPM).
Results: Somatic whole genome SCNAs were detected in all 4 enucleated tumours. Among 7 evaluated AH samples, 5 contained SCNAs and the remaining 2 were flat without alterations under current analysis setting. Mutational analysis of tumour DNA from 4 enucleated eyes identified 6 RB1 SNVs. Mutation results from AH cfDNA were concordant with results obtained from corresponding tumour tissue and peripheral blood screening, when available. In the 3 AH samples without paired tumours, both RB1 hits were identified with high variant allele frequency (VAF), indicating high tumour fraction in AH of RB eyes.
Conclusions: Use of a customized hybridization capture approach facilitated identification of RB1 mutation(s), together with low pass whole genome analysis for SCNA profiling in the same AH liquid biopsy provides the opportunity to identify the genomic and genetic alterations simultaneously for one diseased eye. This refined approach provides the potential to further investigate the spectrum of RB1 mutations and SCNA subtypes in both enucleated and salvaged RB eyes. It is crucial to distinguish between these alternative mechanisms, for the wider implications for management of patient and family members.
Citation Format: Liya Xu, Lishuang Shen, Ashley Polski, Rishvanth K. Prabakar, Mark Reid, Peter Kuhn, David E. Cobrinik, James Hicks, Xiaowu Gai, Jesse Berry. Simultaneous identification of clinically relevant RB1 Mutations and somatic copy number alterations in the Aqueous Humor of retinoblastoma eyes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 452.