Abstract 6941: Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA)
Authors: Stachelek Kevin , Cobrinik David E. 
Published: March 2024

Abstract
Retinoblastoma is a pediatric retinal cancer that initiates in response to biallelic RB1 loss or MYCN amplification in a cone precursor cell of origin. After initial emergence, RB1-/- retinoblastomas progress to more aggressive forms by accumulation of single nucleotide variants (SNVs) and highly recurrent somatic copy number alterations including 1q+, 2p+, 6p+ and 16q- (RB SCNAs), resulting in heterogeneous mixtures of subclonal cell populations. SCNAs are enriched in highly evolved and aggressive tumor subtypes and show prognostic value in retinoblastoma liquid biopsies. However, the transcriptomic effects of retinoblastoma SCNAs are poorly understood. Single cell methods may enable comparison of tumor subclones within emerging retinoblastoma tumors and thereby provide insight into tumor progression.
We retrieved single cell RNA sequencing data for 25 retinoblastoma tumors and inferred SCNAs and subclone phylogenies from scRNAseq information using a recently published tool, Numbat. We identified tumors with clonal variation in RB SCNAs and excluded samples with poor transcriptome quality or excessive additional SCNAs. We proceeded with analysis of 11 tumors with subclonal heterogeneity for an SCNA of interest, including 10 with 1q+, eight with 2p+, four with 6p+, and six with 16q-. After removal of nontumor cells, we defined cell clusters, cluster marker genes, and cell cycle phase gene signatures, and we compared cell state distributions for subclones with an acquired SCNA relative to their immediate antecedents.
These analyses revealed recurrently discriminated transcriptomic clusters corresponding to six discrete cell cycle states (post-mitotic G1, G1, G1/S, S, S/G2, G2/M) and variable cell stress states, including a cluster with hypoxia and heat shock related marker gene expression in all samples. In each tumor, SCNA-defined tumor subclones had a distinct cell state distribution. Tumor subclones with acquired 1q+ were enriched in G2 states and depleted in G1 states. Subclones with 2p+ were enriched in G2 states. Subclones with 6p+ were enriched in S phase states. Subclones with 16q- were diminished in post-mitotic cell states. Tumor clones with 1q+ and 16q- were most strongly enriched for G2 phase cells. Such 1q+/16q- tumor cells were significantly differentially distributed in three tumor replicates. Tumor clones with other RB SCNAs were enriched over diploid clones in proliferating G2 cells without SCNA specific effects.
To identify candidate drivers of SCNA-specific transcriptomic effects we measured RB SCNA clonal variation in gene expression within and across transcriptomic states. We identified recurrently differentially expressed in-segment genes in 1q+, 2p+, 6p+, and 16q-, as well as out-of-segment genes. Finally, we propose molecular mechanisms underlying SCNA recurrence that may enable tumor evolution in retinoblastoma.
Citation Format: Kevin Stachelek, David E. Cobrinik. Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6941.