Fig. 5. Impact of temporal and spatial intratumour heterogeneity on gene expression, ultra-high-risk stratification and putative therapy options.

a RNA sequencing was used to analyse gene expression in 48 spatially and temporally distinct samples from a cohort of 10 patients. Principal component analysis (PCA) was conducted for risk-associated gene expression in the 48 samples. Legend describes patient number and sampling time. Arrows within the plot indicate samples from patients CB1003 and CB1008 collected at different time points during the disease. Arrows adjacent to the axis annotate high-risk and low-risk/intermediate-risk neuroblastomas. b Molecular classification of ultra-high risk (UHR) for the patient (termed UHR diagnosis here) was performed for each tumour sample separately using TERT mRNA expression, MYCN copy number and mutations (WES) in the TP53 and RAS/MAPK pathways. Variant allele frequency (VAF) is given for all mutations as % of reads that support the mutation. Mutations in the TP53/RAS/MAPK pathways with a VAF > 5% were defined as UHR mutations. Other druggable mutations based on the INFORM criteria⁸ are shown for each sample. TERT expression was defined as “high” according to Ackermann et al.³.