Abstract 6052: Single cell profiling of vitreous humor seeds in retinoblastoma
Authors: Sirivolu Shreya , Xu Liya , Kuhn Peter , Hicks James , Berry Jesse 
Published: June 2022

Abstract
Purpose: Previous studies of retinoblastoma (RB) aqueous humor (AH) analysis for tumor-derived cell-free DNA (cfDNA) have shown that the somatic copy number alteration (SCNA) profiles derived from the AH are highly concordant to profiles derived from the tumor in most but not all cases. As one of the reasons for the discordance in certain cases, it was suggested that these patients had multiple retinal tumors that had developed different subsets of SCNAs. The AH was therefore speculated to be a heterogenous mixture of cfDNA from each tumor clone, a heterogeneity that could not be captured in a single tumor biopsy after enucleation. In such cases that do not show high concordance, analyzing the genomic profile of tumor seeds in the VH can help us determine the degree of heterogeneity between the different subclones in the tumor. As single tumor cells from the vitreous humor (VH) have not been studied in RB before, this study demonstrates a technique that could be used to investigate the SCNA profiles of these cells with the aim to further understand the possible origins of AH cfDNA.
Methods: The subject of this study is a 24-month-old male diagnosed with advanced unilateral RB (Group D/Stage CT2B) in the left eye and treated with primary enucleation after seven days. VH was collected from the enucleated eye, cfDNA was isolated from the AH and VH, and tumor DNA was extracted from tumor biopsy. Thirty live VH single cells were isolated using fluorescence-activated cell sorting using DAPI staining. The cells were then amplified using GenomePlex® Single Cell Whole Genome Amplification Kit (WGA4). Tumor DNA and single-cell WGA amplicons were constructed using NEBNext Ultra II FS Kit, and whole genome libraries of cfDNA from AH and VH were constructed using QIAseq Ultralow Input Library Kit. Shallow WGS of these libraries were sequenced using the IIlumina paired-end (2 X 150bp) platform to assess genome-wide SCNAs. Heatmap with hierarchical clustering was generated in R using the heatmap.2 function in the ggplots package. Cells were clustered by Ward’s method with Manhattan distance by their median centered data. Cutoffs for gains and losses were 1.2 and 0.8 over the median respectively.
Results: There was high concordance in SCNA profiles across AH cfDNA, VH cfDNA, and tumor DNA, showing highly recurrent RB SCNAs such as gains on 1q, 2p, and 6p, in addition to gains on 2p, 5p, 7q, 9q, 10q, 13q, 15q, 17q and loss on 3q. The SCNA profiles of the 30 VH seeds demonstrated the highly recurrent RB SCNAs (gains on 1q, 2p, and 6p) that were consistent with the tumor SCNA signature. The heatmap of the VH single cell SCNA profiles demonstrated low levels of heterogeneity between the seeds.
Conclusions: Our results suggest that VH seeds are tumor cells. Analyzing genomic material of the VH seeds using single cell SCNA profiling provides a valuable platform to study tumor heterogeneity in RB.
Citation Format: Shreya Sirivolu, Liya Xu, Peter Kuhn, James Hicks, Jesse Berry. Single cell profiling of vitreous humor seeds in retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6052.