Background: Relapsed neuroblastoma tumors harbor increased mutational burdens and we recently reported that relapse-specific somatic mutations are enriched in ALK and/or activation of the RAS-MAPK pathway. Importantly, many of these mutations were subclonal or not detectable in the matched primary tumor sample. We therefore hypothesized that stringent design and optimization of a custom amplicon sequencing panel will allow prospective detection of prevalent relapse-specific subclonal mutations in primary tumors. This could allow for the integration of targeted therapy earlier, preventing the emergence of resistant subclones.
Methods: We used our previously published relapse sequencing data and analyzed available FoundationOne targeted sequencing data from primary (N=78) and relapse (N=67) samples to design a 28-gene panel. The most frequently mutated genes were ALK, PTPN11, ATRX, HRAS, KRAS, NRAS, TP53 and MYCN. Our panel contains roughly 200 amplicons covering 55 unique mutations, plus the coding regions of TP53 and NF1. The panel was designed with Illumina’s dual-strand design in order to detect and correct for DNA deamination events occurring during formalin fixation. High-risk primary neuroblastoma tumors (N=299) were sequenced across a total of ~12,000bp. To increase sensitivity and likelihood of detection, we used 100 ng of genomic DNA as input for TruSeq Low Input library construction, added unique molecular identifiers to identify PCR duplicates, and sequenced to an average depth of coverage of 100,000X using NextSeq500 high output flowcells.
Results: Control blood samples (N=3) were used to determine the background variability and normalize base calling prior to variant detection. Relapse tumors (N=3) harboring known mutations were used as positive controls and a Horizon Tru-Q standard, genomic DNA containing verified low-level variants (1.3%) in ALK, IDH2, KRAS, and NRAS, was used to determine the limit of detection and assess variant calling accuracy at subclonal levels.
Conclusions: Data are currently being generated and will be presented at the meeting. We plan to use this panel to improve patient outcomes prospectively by coupling targeted therapy to chemotherapy.