Whole-exome Sequencing Cell Free DNA Analysis Documents New Tumor Specific Alterations at Relapse of High-Risk Pediatric Cancers

Background : Pediatric cancers are characterized by few recurrent genetic alterations, but genetic heterogeneity and clonal evolution can play a role in tumor progression. Liquid biopsies now enable monitoring of tumor-specific genetic alterations in sequential samples by analysis of cell free DNA (cfDNA). Methods : We have enrolled 28 consecutive patients with newly diagnosed high risk pediatric cancer (neuroblastoma n=8; rhabdomyosarcoma n=7; Ewing sarcoma n=3; other cerebral or extracerebral high risk cancers n=10) in a prospective clinical study NGSkids (clingov trial : NCT02546453) with an aim to study clonal evolution based on sequential cfDNA analysis. With a median follow up of 22 months, 7 patients have experienced relapse. Molecular analysis consisted of standardized Illumina© 100PE 100x whole exome sequencing (WES) of tumor tissue and paired germline material , and WES of cfDNA extracted from plasma samples at diagnosis, during treatment and follow up, with 2-9 sequential samples available per patient. cfDNA analysis was performed using an in-house procedure, with 20-200 ng of cfDNA subjected to WES following modified library construction and capture approaches to account for cfDNA molecule size (target depth 100x). Following filtering on germline to focus on tumor cell specific alterations, SNVs were called using GATK-UnifiedGenotyper, GATK-HaplotypeCaller, Samtools and Mutect. Copy-number profiles were generated using Varscan and DNAcopy. Results:: At diagnosis, cfDNA quantities were higher in advanced stages of disease (localized stages - mean 64 ng/ml of plasma (range 24-172); metastatic stages - mean 505 ng/ml of plasma (range 20-2,782)). CfDNA WES analysis yielded satisfactory depth in all cases. At diagnosis, a mean of 9 tumor cell specific SNVs common to both primary tumor and corresponding cfDNA was observed (range 1 - 57), with a mean of 11 and 6 specific to the primary and cfDNA respectively, indicating spatial heterogeneity. Whereas cfDNA samples obtained at follow-up in patients without evidence of disease revealed no or few tumor cell specific SNVs, interestingly, cfDNA samples obtained at relapse harbored additional, new relapse-specific SNVs (mean 10; range 2-42) in all cases with relapse, targeting genes such as MAPK and MLL4. Deep sequencing (10,000X) capture techniques with a panel encompassing all identified SNVs is currently being applied to all cfDNA samples, including 1-8 intermediate samples per patient, with an aim to develop models of clonal evolution. Discussion and Conclusion: CfDNA WES proves to be an extremely powerful tool to study spatial and temporal heterogeneity in pediatric high risk cancers, providing further proof of the importance of clonal evolution in cancer progression. Full characterization of cfDNA at relapse, which might represent more aggressive clones, might orient towards targeted treatment approaches. Citation Format: Mathieu Chicard, Adrien Danzon, Nathalie Clement, Irene Jimenez, Eve Lapouble, Gaelle Pierron, Angela Bellini, Nada Leprovost, Sylvain Baulande, Paul Freneaux, Francois Doz, Daniel Orbach, Isabelle Aerts, Helene Pacquement, Jean Michon, Franck Bourdeaut, Olivier Delattre, Gudrun Schleiermacher. Whole-exome sequencing cell free DNA analysis documents new tumor specific alterations at relapse of high-risk pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2592.