Abstract 4831: Analysis of genetic variations in neuroblastoma using deep targeted sequencing
Authors: Javanmardi Niloufar , Fransson Susanne , Ostensson Malin , Sjoberg Rose-marie , Kogner Per , Martinsson Tommy 
Published: August 2015

Abstract
Neuroblastoma tumors (NBs) show complex patterns of genetic abnormalities, which may include amplification or oncogenic mutations of ALK kinase in sporadic and familial cases.
In analyzing genomic deviations it is sometimes highly efficient to focus on deep sequencing of specific parts of the genome with high clinical relevance to NB prognosis. This can be achieved by studying specific regions using targeted or amplicon sequencing, and thereby reducing the complexity of the analysis. The main benefits of this approach are reduced costs and shorter analysis time, due to the sequencing of fewer bases and the associated reduction in the volume of data generated. Additional benefit comes from achieving higher coverage that provides means for detection tumor specific variants present at subclonal level.
The HaloPlex method, the custom target enrichment kit from Agilent, uses a different approach among enrichment kits, namely the fragmentation of DNA by restriction enzymes in conjunction with biotinylated probes designed to hybridize to both ends of a targeted DNA restriction fragment. This will form circular DNA strands that will be then closed by ligation (http://www.genomics.agilent.com) and it requires only 250 ng of input material. We targeted 35kb of genomic sequence including exons of related genes, as well as 50 bp of flanking exon regions. Samples were sequenced in a single MiSeq run (Illumina) using 2×150 paired-end sequencing.
We compiled a gene panel for Next Generation Sequencing (NGS) comprising of three genes, ALK, ATRX and PPM1D, where the two first are associated with different subgroups of NBs. The Haloplex panel was evaluated on a pilot cohort of 40 patients including four controls with known and defined mutations (missense and indels), which produced a total of 4 Gb of data. Data were analyzed using an “in-house” bioinformatics pipeline based on mapping reads against hg19 using bwa, with realignment and recalibration using GATK haplotype caller, and variant annotation using ANNOVAR. For each position, the frequency for each base was calculated. The analysis revealed that we were able to generate sufficient sequencing data with very low background variability for mutation as well as deletion analysis using our diagnostic panels. We identified all expected mutations in our control samples. Sanger sequencing were used to validate the results and for analyzing sensitivity and specificity of the detected variants.
Overall, the panel fulfills diagnostic criteria and will offer a fast and reliable procedure that will be useful in clarifying molecular genetic defects underlying NBs.
Citation Format: Niloufar Javanmardi, Susanne Fransson, Malin Ostensson, Rose-marie Sjoberg, Per Kogner, Tommy Martinsson. Analysis of genetic variations in neuroblastoma using deep targeted sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4831. doi:10.1158/1538-7445.AM2015-4831