Simultaneous Copy Number Alteration and Single-Nucleotide Variation Analysis in Matched Aqueous Humor and Tumor Samples in Children with Retinoblastoma

Abstract

Retinoblastoma (RB) is a childhood cancer that forms in the developing retina of young children; this tumor cannot be biopsied due to the risk of provoking extraocular tumor spread, which dramatically alters the treatment and survival of the patient. Recently, aqueous humor (AH), the clear fluid in the anterior chamber of the eye, has been developed as an organ-specific liquid biopsy for investigation of in vivo tumor-derived information found in the cell-free DNA (cfDNA) of the biofluid. However, identifying somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) of the RB1 gene, typically requires either: (1) two distinct experimental protocols-low-pass whole genome sequencing for SCNAs and targeted sequencing for SNVs-or (2) expensive deep whole genome or exome sequencing. To save time and cost, we applied a one-step targeted sequencing method to identify both SCNAs and RB1 SNVs in children with RB. High concordance (median = 96.2%) was observed in comparing SCNA calls derived from targeted sequencing to the traditional low-pass whole genome sequencing method. We further applied this method to investigate the degree of concordance of genomic alterations between paired tumor and AH samples from 11 RB eyes. We found 11/11 AH samples (100%) had SCNAs, and 10 of them (90.1%) with recurrent RB-SCNAs, while only nine out of 11 tumor samples (81.8%) had positive RB-SCNA signatures in both low-pass and targeted methods. Eight out of the nine (88.9%) detected SNVs were shared between AH and tumor samples. Ultimately, 11/11 cases have somatic alterations identified, including nine RB1 SNVs and 10 recurrent RB-SCNAs with four focal RB1 deletions and one MYCN gain. The results presented show the feasibility of utilizing one sequencing approach to obtain SCNA and targeted SNV data to capture a broad genomic scope of RB disease, which may ultimately expedite clinical intervention and be less expensive than other methods.

Keywords: RB1 gene; aqueous humor; liquid biopsy; retinoblastoma; somatic copy number alterations; targeted sequencing; variant calling.

Figure 1

Schematic of study design. (A) Eleven patients were present in the cohort. ((A) Top) Sample counts for each biospecimen collected that was processed through the dual-targeted SNV and SCNA methods are displayed. Eleven patients contained matched AH and tumor-targeted sequencing samples. ((A) Bottom) Sample count of low-pass WGS sequencing samples. (B) SCNAs from the SureSelect Targeted Sequencing approach were compared to the gold standard low-pass WGS, and concordance was calculated. The targeted reads were then used to call SNVs, and the 11 matched AH and tumor-targeted reads were directly compared for concordance. This figure was generated with BioRender.

Figure 2

Copy number alteration analysis shows high concordance between targeted AH and tumor samples. (A) Concordance of derived tumor SCNAs between targeted sequencing reads and their matched low-pass WGS reads. (B) Concordance of SCNAs between targeted AH sequencing samples and their respective targeted tumor sequencing samples. (C) Copy number representation for case 48’s tumor sample overlaying targeted sequencing reads (teal) to low-pass WGS reads (black). A focal MYCN gain was detected on chromosome 2 with both methods. (D) Copy number plot for case 50’s tumor sample overlaying targeted sequencing reads (teal) to low-pass WGS reads (black) showing focal RB1 gene deletion on chromosome 13 and CREBBP loss on chromosome 16.

Figure 3

SNV landscape of RB patients: (A) Allele frequency plot for deleterious SNVs detected to the RB1 gene. Case 33 had two AH samples (pt33-dx and pt33-es) but had a single tumor sample that was taken at enucleation. Cases that are bold and italicized are germline variants. Cases with asterisk (*) indicate nonsense variants that result in truncated protein. Dark grey boxes indicate the variant was not found in that sample. (B) Read depth for each genomic site in (A). Depth accounts for total reads at each specified site, including both variant reads and normal allele reads. X-axis for depth was cut off at 500 bases for legibility.

Figure 4

Summary of variants observed in the Cohort. Germline and somatic RB1 SCNAs and SNVs, and other RB-signature genetic variants are displayed. Germline variants detected in blood (separate clinical diagnostic test), AH, and tumor for cases 1, 9, and 13. Cases 33-dx and 33-es were merged; they have the same RB1 somatic SNV detected at diagnosis and enucleation. Variants depicted are found in both targeted and tumor samples.

Figure 5

Mutational landscape to CREBBP and BCOR: (A) Allele frequency plot for deleterious mutations to BCOR and CREBBP detected in the cohort. Cases with asterisk (*) indicate nonsense variants that result in truncated protein. (B) Read depth for each genomic site in (A). Depth accounts for total reads at each specified site, including both variant reads and normal allele reads. X-axis for depth was cut off at 500 bases for legibility. (C) SCNA plot for case 8 displaying a loss to chromosome 16 impacting CREBBP.