Abstract B074: Clonal decomposition and DNA replication states defined by scaled single-cell DNA and RNA sequencing suggest clone-specific therapeutic vulnerabilities in neuroblastoma
Authors: Bhalshankar Jaydutt , Bellini Angela , Jimenez Irene , Thirant Cécile , Saberi-Ansari Elnaz , Iddir Yasmine , Charles Alexandra Saint , Butterworth Charlotte , Kramdi Amira , Raynal Virginie , Baulande Sylvain , Surdez Didier , Zaidi Sakina , Pierron Gaelle , Carcaboso Angel Montero , Geoerger Birgit , Zinovyev Andrei , Delattre Olivier , Janoueix-lerosey Isabelle , Schleiermacher Gudrun 
Published: September 2024

Background: In neuroblastoma (NB), intratumor heterogeneity (ITH) is frequently observed, but the role of cell-to-cell allele-specific copy number alterations in phenotypic variation, clonal evolution and treatment response remains to be determined. Here we investigate ITH, timing of specific genomic aberrations, single-cell replication timing and the co-evolution of the genome and transcriptome in NB tumors at single-cell resolution, with an aim to analyse subclonal dynamics and clone-specific response or resistance under targeted therapeutic pressure. Methods: In addition to germline/tumor bulk whole exome sequencing (WES), ultra-low depth (0.25x) single-cell whole-genome DNA sequencing (scDNAseq) was performed using 10x genomics Chromium single-cell CNV (scCNV) kit and 9410 tumor cells were characterized from 14 patient-derived xenografts (PDX) NB-models and 4 tumor biopsies from NB-patients, either at diagnosis (n=7), progression (n=3) or relapse (n=8). Single-cell RNA sequencing (scRNAseq) data was obtained from the same PDX and patient tumor samples (Thirant et al, 2023). 6/14 PDX models were subjected to different treatment combinations (targeted treatment with/without chemotherapy) and bulk WES was performed at two time-points, pre- and post-treatment. Results: Both monoclonal (n=7) and polyclonal (n=11) genomes were determined by allele and haplotype specific copy number (CN) alteration using both scDNAseq and scRNAseq data analysis, with 2 to 11 clones observed per polyclonal NB tumor. Whole genome duplication events (n=7) were observed in both polyclonal and monoclonal genomes. Known driver CN (segmental loss in chr1p and chr11q and gain at chr17q, or MYCN/ALK amplification) or somatic mutations (ALK/ATRX/TP53/NF1) were early clonal events.Study of replication timing (RT) based on scDNAseq revealed significant differences in RT between the MYCN amplified (n=7) and non-amplified groups (n=5), with NB tumors without MYCN amplification (no MNA) characterized by a predominance of late replicating domains, in contrast to MYCN amplified (MNA) tumors, which are enriched in early replicating domains In a PDX model of interest, scDNAseq analysis showed parallel copy number evolution of two distinct clones, subclone s1/s2. Data integration of clonal mutational profiles with pre- and post-targeted therapy (Lorlatinib) revealed clone-specific treatment response. Subclone s2 was partially responding with extinction of a sub-set of somatic alterations, whereas no change was observed in subclone s1. The replication timing (RT) profile of these two clones, subclone s1 (early-RT) and s2 (late-RT) were mutually exclusive. Genotype to phenotype analysis revealed subclone s1 genotype was preferentially expressed at transcriptomic level. Conclusion: Together, these results determine the evolutionary trajectories of NB tumors, linked to distinct replication timing and highlight opportunities for targetable early clonal alteration detection.

Citation Format: Jaydutt Bhalshankar, Angela Bellini, Irene Jimenez, Cécile Thirant, Elnaz Saberi-Ansari, Yasmine Iddir, Alexandra Saint Charles, Charlotte Butterworth, Amira Kramdi, Virginie Raynal, Sylvain Baulande, Didier Surdez, Sakina Zaidi, Gaelle Pierron, Angel Montero Carcaboso, Birgit Geoerger, Andrei Zinovyev, Olivier Delattre, Isabelle Janoueix-lerosey, Gudrun Schleiermacher. Clonal decomposition and DNA replication states defined by scaled single-cell DNA and RNA sequencing suggest clone-specific therapeutic vulnerabilities in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B074.