Oral Presentation Advances in Neuroblastoma Research Congress 2016

Whole exome sequencing of circulating free tumour DNA for study of spatial and temporal tumor heterogeneity: accumulation of new mutations at tumor progression of neuroblastoma (#10)

Mathieu Chicard 1 , Leo Colmet Daage 1 , Nathalie Clement 2 , Angela Bellini 1 , Sylvain Baulande 3 , Virginie Bernard 4 , Gaelle Pierron 5 , Eve Lapouble 5 , Isabelle Janoueix-Lerosey 6 , Jean Michon 7 , Olivier Delattre 6 , Valérie Combaret 8 , Gudrun Schleiermacher 9
  1. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique" and INSERM U830, Institut Curie, Paris, France
  2. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique", Institut Curie, Paris, France
  3. Plateforme de Séquençage ICGEX, Institut Curie, Paris, France
  4. Plateforme ICGEX and U900 INSERM, Institut Curie, Paris, France
  5. Unité de Génétique Somatique, Institut Curie, Paris, France
  6. U830 INSERM, Institut Curie, Paris, France
  7. Department of Pediatric Oncology, Institut Curie, Paris, France
  8. Centre Léon Bérard, Lyon, France
  9. Laboratoire RTOP "Recherche Translationelle en Oncologie Pédiatrique", INSERM U830 and Department of Pediatric Oncology, Institut Curie, Paris, France

Liquid biopsies are revolutionary tools to monitor tumour-specific genetic alterations. In neuroblastoma (NB), significant levels of circulating free tumor DNA (ctDNA) in the bloodstream enable the detection of tumour-specific markers including MYCN amplification or mutations. As clonal evolution plays a role in NB progression, monitoring of a single genetic marker will be insufficient for ctDNA-based disease follow-up.

To study NB clonal evolution, 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.

CtDNA WES yielded satisfactory depth in all cases. At diagnosis, SNVs common to the NB and corresponding diagnostic ctDNA of a given patient were observed (mean number of SNVs: 19; range 9-69), with few SNVs specific to the NB (mean: 6; range 0-18), and others specific to ctDNA (mean:22; range 9-69), suggesting spatial heterogeneity with different ctDNA amounts released by different clones. In PR or CR ctDNA, lower numbers of SNVs were detected (mean: 11, range 0-12). Interestingly, PD ctDNA samples harboured 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 will determine the time of appearance of new driver clones.

In 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.