Abstract 4952: Whole exome sequencing of circulating tumor DNA highlights spatial and temporal tumor heterogeneity in neuroblastoma
Authors: Chicard Mathieu , Daage Leo Colmet , Clement Nathalie , Bellini Angela , Baulande Sylvain , Bernard Virginie , Pierron Gaelle , Lapouble Eve , Janoueix-Lerosey Isabelle , Michon Jean , Combaret Valérie , Delattre Olivier , Schleiermacher Gudrun 
Published: July 2017

Abstract
Background : Liquid biopsies are revolutionary tools to monitor tumour-specific genetic alterations in sequential samples. In neuroblastoma (NB), significant levels of circulating tumor DNA (ctDNA) in the bloodstream enable the detection of tumour cell-specific markers including MYCN amplification or activating ALK mutations. As clonal evolution plays a role in NB progression, analysis of a single genetic marker will be insufficient for ctDNA-based disease follow-up. Methods : To gain further insights into mechanisms of clonal evolution in NB, we isolated ctDNA from plasma at diagnosis (n=19) and during follow-up (final time-point: partial or complete remission (PR/CR), n=7; progressive disease (PD), n=9) for 19 NB patients for whom primary NB and matched germline DNA whole exome/whole genome sequencing data (WES/WGS) was available. CtDNA (7-100ng) was subjected to Illumina 100PE WES following modified library construction and capture approaches to account for small ctDNA molecules (target depth 100x). SNVs/mutations were called using GATK-UnifiedGenotyper, GATK-HaplotypeCaller and Samtools. Copy-number profiles were generated using Varscan and DNAcopy. Results : CtDNA WES yielded satisfactory depth in all cases. At diagnosis, a majority of observed SNVs were common to the primary NB and corresponding diagnostic ctDNA of a given patient (mean number of SNVs: 19; range 9-69) with MAF (mutated allele fractions) corresponding to the estimated ctDNA content in the total cell free DNA. At diagnosis, few SNVs specific to the NB (mean: 6; range 0-18) or specific to ctDNA (mean:22; range 9-69) were observed, suggesting spatial heterogeneity with different ctDNA amounts released by different clones. In PR or CR ctDNA samples, lower numbers of SNVs with lower MAFs were detected (mean: 11, range 0-12). Interestingly, PD ctDNA samples harboured an increase in MAFs and a higher numbers of SNVs, with additional relapse-specific SNVs (mean: 22; range 0-55) targeting, amongst others, the protein kinase A signaling pathway. Analysis of additional ctDNA samples obtained between diagnosis and relapse (2-6 samples/patient) using deep sequencing techniques demonstrated a disappearance of SNVs coinciding with response to therapy, and re-appearance of SNVs at the time of tumor progression. Discussion and Conclusion: CtDNA WES proves to be an extremely powerful tool to study spatial and temporal heterogeneity in NB, providing further proof of the importance of clonal evolution in NB progression. Full characterization of ctDNA, which might represent more aggressive clones, might orient targeted treatment approaches.
Citation Format: Mathieu Chicard, Leo Colmet Daage, Nathalie Clement, Angela Bellini, Sylvain Baulande, Virginie Bernard, Gaelle Pierron, Eve Lapouble, Isabelle Janoueix-Lerosey, Jean Michon, Valérie Combaret, Olivier Delattre, Gudrun Schleiermacher. Whole exome sequencing of circulating tumor DNA highlights spatial and temporal tumor heterogeneity in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4952. doi:10.1158/1538-7445.AM2017-4952