Abstract
For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our “neuroblastoma hybrid capture sequencing panel” (NB-HCSP) assay employs a technology for the high-coverage sequencing (>1000×) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection.
Highlights
Neuroblastoma is the most common extracranial childhood tumor and is characterized by a broad clinical heterogeneity [1,2]
We selected hybrid-capture probes to analyze copy number variations to detect amplifications of MYCN, chromosome 17q gain, and 1p36 and 11q loss, which are routinely analyzed at diagnosis to allow risk stratification according to current protocols using fluorescence in situ hybridization (FISH) or other appropriate methods [44]
To detect alterations associated with telomere maintenance, a crucial mechanism of neuroblastoma tumor biology, we selected probes to detect ATRX
Summary
Neuroblastoma is the most common extracranial childhood tumor and is characterized by a broad clinical heterogeneity [1,2]. The risk-based therapy stratification of neuroblastoma patients has been the foundation of successful treatment, and it relies on clinical and molecular covariates [1,2,4,5,6,7]. Activating mutations of anaplastic lymphoma kinase (ALK) or amplifications of the ALK gene, found in >10–15% of high-risk neuroblastomas, are correlated with adverse outcomes, and define a potential target for a molecular-targeted therapy with ALK inhibitors [19,20,21,22,23,24,25,26]
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