Abstract B41: Whole genome sequencing identifies tumor evolution events in relapse neuroblastoma
Authors: Eleveld Thomas F. , Schild Linda , Koster Jan , Zwijnenburg Danny , Ebus Marli E. , van Peter Sluis , Schulte Johannes H. , Caron Huib N. , Versteeg Rogier , Molenaar Jan J. 
Published: May 2015

Abstract
Treatment of primary neuroblastoma tumors tends to initially cause tumor regression, but relapse occurs in 50-60% of patients with high-risk tumors. These relapses are generally very aggressive and to date no curative regimen is known for these refractory tumors. We performed whole genome sequencing for six triplets of primary NB tumors, corresponding relapses and normal tissue from the same patient to determine if tumor evolution events on DNA level occurred.
We first analyzed the number of mutations and structural variations affecting the coding sequence in the primary tumors and relapses. The relapses consistently show more aberrations than the primary tumors, although this varies between pairs. Some mutations are conserved from primary tumor to relapse, while others that are observed in the primary tumor are not present (at detectable levels) in the relapse. The majority of events however appear de novo in the relapses which indicates that clonal selection occurs and that secondary mutations take place in the formation of neuroblastoma relapses.
We next focused on the mutations that are detected in the relapse tumor and not in the primary tumor. Analysis revealed several tumor evolution events in known oncogenes which have also been reported in primary neuroblastoma tumors in low frequency. Two relapses show strong enrichment for point mutations in ALK and PTPN11. Tree other tumors show de novo structural aberrations in NF1 (homozygous deletion), ALK (amplification) and BRAF (truncated protein). All events affect the RAS-MAPK signaling pathway which suggests involvement of this pathway in neuroblastoma tumor evolution. To further investigate the high frequency of events in this pathway in relapsed tumors we performed WGS on a panel of 25 neuroblastoma cell lines which were mainly derived from pretreated or relapsed neuroblastoma tumors. The frequency of genomic aberrations in NF1, NRAS, KRAS, BRAF, PTPN11 and ALK was much higher compared to primary tumor samples.
Currently we are validating the effect of the tumor evolution events in non-malignant neuroblasts and neuroblastoma cell lines. The effect on RAS-MAPK pathway activity and phenotypic affects are studied. For the ALK mutation and the NF1 deletion we could indeed show activation of the RAS-MAPK pathway in vitro which is accompanied by an increase in colony forming capacity.
Our findings show that tumor evolution events do occur in neuroblastoma mainly in genes which are also mutated in primary tumors. We found a high frequency of mutations that affect the RAS-MAPK pathway which suggests involvement of this pathway in NB tumor evolution. This warrants further validation of the RAS-MAPK pathway as a potential therapeutic target in relapsed refractory neuroblastoma.
Citation Format: Thomas F. Eleveld, Linda Schild, Jan Koster, Danny Zwijnenburg, Marli E. Ebus, Peter Sluis van, Johannes H. Schulte, Huib N. Caron, Rogier Versteeg, Jan J. Molenaar. Whole genome sequencing identifies tumor evolution events in relapse neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B41.