Abstract 2980: Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations
Authors: Oldridge Derek A. , Eleveld Thomas F. , Bernard Virginie , Koster Jan , Daage Leo C. , Diskin Sharon J. , Schild Linda , Bentahar Nadia B. , Bellini Angela , Chicard Mathieu , Lapouble Eve , Combaret Valérie , Legoix-Né Patricia , Michon Jean , Pugh Trevor J. , Hart Lori S. , Rader JulieAnn , Attiyeh Edward F. , Wei Jun S. , Zhang Shile , Naranjo Arlene , Gastier-Foster Julie M. , Hogarty Michael D. , Smith Malcolm A. , Auvil Jaime G. , Watkins Thomas B. K. , Zwijnenburg Danny A. , Ebus Marli E. , van Sluis Peter , Hakkert Anne , van Wezel Esther , van der Schoot C. Ellen , Westerhout Ellen M. , Schulte Johannes H. , Tytgat Godelieve A. , Dolman M. Emmy M. , Janoueix-Lerosey Isabelle , Gerhard Daniela S. , Caron Huib N. , Delattre Olivier , Khan Javed , Versteeg Rogier , Schleiermacher Gudrun , Maris John M. , Molenaar Jan J. 
Published: August 2015

Abstract
Background
The majority of high-risk neuroblastomas initially respond to chemotherapy, but over half of patients experience therapy-resistant relapses. The molecular defects driving relapse and drug resistance are unknown.
Methods
We performed Illumina or Complete Genomics whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas, and corresponding normal lymphocyte DNA, to define genetic alterations associated with relapse. A panel of 18 neuroblastoma cell lines was analyzed for the presence of RAS-MAPK mutations and sensitivity to small molecule inhibitors of this pathway.
Results
Neuroblastomas that relapsed after chemotherapy showed dramatic clonal evolution, with only 33% of primary tumor mutations also detected at relapse. In 21 out of 23 patients, more somatic coding mutations were observed at relapse (median: 29 unique to relapse, range: 4-129). Unbiased pathway analysis of the somatic mutations detected in the relapse tissues identified a strong enrichment in genes associated with RAS-MAPK signaling (p = 6.1×10−7). 18 of the 23 cases (78%) showed somatic mutations (N = 15) or structural alterations (N = 3) predicted to activate the MAPK pathway, and these were mutually exclusive: ALK (N = 10), NRAS (N = 1), KRAS (N = 1), HRAS (N = 1), BRAF (N = 1), PTPN11 (N = 1), FGRF1 (N = 1) and NF1 (N = 2). These RAS-MAPK mutations were clonally enriched at relapse and exist within clonal or major subclonal tumor populations. Seven of these RAS-MAPK mutations were detected only in the relapse tumor by whole genome sequencing (∼50X coverage), and only 2 of these 7 mutations were detectable in the primary tumor with targeted detection methods (104-105X coverage). Similar MAPK pathway mutations were detected in 11 of 18 human neuroblastoma-derived cell lines, and these lesions are predicted to be sensitive to small molecule inhibition of MEK in vitro (p&lt;0.001) and in vivo (p&lt;0.05).
Conclusions
In this study of 23 neuroblastoma cases selected based solely on having diagnostic-relapse specimens available for analysis, MAPK pathway mutations were highly enriched in the relapsed genomes, providing a potential biomarker for new therapeutic approaches to chemotherapy refractory disease. The fact that several ALK-RAS-MAPK mutations were found in the relapse but not in the corresponding primary tumors favors a model in which rare subclones with secondary driver mutations expand over time. However, it remains to be determined whether these mutations occurred de novo after treatment, were present in rare subclones below detection limits, or were undetectable due to spatial heterogeneity of the primary tumor, which will impact the clinical utility of targeted sequencing at diagnosis. Our study provides strong rationale for performing biopsies on relapse neuroblastoma tumors in order to comprehensively characterize the molecular lesions that underlie treatment-refractory disease, determine their prognostic relevance, and guide treatment decisions for patients.
Citation Format: Derek A. Oldridge, Thomas F. Eleveld, Virginie Bernard, Jan Koster, Leo C. Daage, Sharon J. Diskin, Linda Schild, Nadia B. Bentahar, Angela Bellini, Mathieu Chicard, Eve Lapouble, Valérie Combaret, Patricia Legoix-Né, Jean Michon, Trevor J. Pugh, Lori S. Hart, JulieAnn Rader, Edward F. Attiyeh, Jun S. Wei, Shile Zhang, Arlene Naranjo, Julie M. Gastier-Foster, Michael D. Hogarty, Malcolm A. Smith, Jaime G. Auvil, Thomas B. K. Watkins, Danny A. Zwijnenburg, Marli E. Ebus, Peter van Sluis, Anne Hakkert, Esther van Wezel, C. Ellen van der Schoot, Ellen M. Westerhout, Johannes H. Schulte, Godelieve A. Tytgat, M. Emmy M. Dolman, Isabelle Janoueix-Lerosey, Daniela S. Gerhard, Huib N. Caron, Olivier Delattre, Javed Khan, Rogier Versteeg, Gudrun Schleiermacher, John M. Maris, Jan J. Molenaar. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations. [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 2980. doi:10.1158/1538-7445.AM2015-2980