EGAGenomicsIllumina HiSeq 2500;https://ega-archive.org/datasets/EGAD0000100135625EGA dataset EGAD00001001356EGAThe majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations.Eleveld Thomas F TF, Oldridge Derek A DA, Bernard Virginie V, Koster Jan J, Colmet Daage Léo L, Diskin Sharon J SJ, Schild Linda L, Bentahar Nadia Bessoltane NB, Bellini Angela A, Chicard Mathieu M, Lapouble Eve E, Combaret Valérie V, Legoix-Né Patricia P, Michon Jean J, Pugh Trevor J TJ, Hart Lori S LS, Rader JulieAnn J, Attiyeh Edward F EF, Wei Jun S JS, Zhang Shile S, Naranjo Arlene A, Gastier-Foster Julie M JM, Hogarty Michael D MD, Asgharzadeh Shahab S, Smith Malcolm A MA, Guidry Auvil Jaime M JM, Watkins Thomas B K TB, Zwijnenburg Danny A DA, Ebus Marli E ME, van Sluis Peter P, Hakkert Anne A, van Wezel Esther E, van der Schoot C Ellen CE, Westerhout Ellen M EM, Schulte Johannes H JH, Tytgat Godelieve A GA, Dolman M Emmy M ME, Janoueix-Lerosey Isabelle I, Gerhard Daniela S DS, Caron Huib N HN, Delattre Olivier O, Khan Javed J, Versteeg Rogier R, Schleiermacher Gudrun G, Molenaar Jan J JJ, Maris John M JMglycogen synthase kinase activity, Ras1/RAs85D, RAS, Ras, DmErk, extracellular signal-regulated kinase activity, l(1)G0098, pp44mapk, protein kinase A activity, Mpk2, c-rasHa, p42mapk, E(sev)3C, Ras-1, CG11485, Prp4 protein kinase activity, SR2-1, ras, c-ras2, LeMPK3, ATP-protein transphosphorylase activity, p44mpk, ATP:protein phosphotransferase (non-specific) activity, kinase-related transforming protein, Dp38, mitogen-activated protein kinase activity, Mutations., SAPK2, mpk1, SEM, Sem, D-Ras1, MAP-k, Erk/Map kinase, RTK, pp42, l(1)G0436, DERK-A, Dsor2, ras85B, ras85D, E(sina)7, sem, Rl, neuroblastoma (morphologic abnormality), Raf kinase activity, [M]Neuroblastoma NOS (morphologic abnormality), MP kinase activity, serine kinase activity, high frequency, DERK, D-ras-2, atypical protein kinase C activity, MBP kinase II activity, mitogen-activated S6 kinase activity, protein-aspartyl kinase activity, dERK, NB - Neuroblastoma, CG1799, ERK-A, p82 kinase activity, Neuroblastoma (Schwannian Stroma-Poor), l(3)s1747, MAP kinase 2 activity, dpERK, dpErk, Ras2, RAS2, Ras1, RAS1, Mapk, Ki-ras, betaIIPKC, Erk1, ERK1, ERK2, mapk1a, DmMAPK, PMK-2, dp-ERK, PMK-1, Sympathicoblastoma, PMK-3, M phase-specific cdc2 kinase activity, Wee-kinase activity, mapk1b, MapK, MAPK, dRas85D, pMAPK, pMapK, NOS, p21[Ras1], dRas1, dRAS1, ras1, serine protein kinase activity, Dras85D, ras2, erk2, mapk, ras-2, BcDNA:RE36103, ERKa, phosphorylase B kinase kinase activity, l(1)G0351, DmERKA, LD02673, rl/MAPK, BcDNA:RE08694, protein glutamyl kinase activity, Neuroblastomas, DmelCG1799, c-Ha-ras, l(2R)EMS45-39, D-ras1, l(1)G0238, D-ras2, Hras-1, DmelCG12559, C-ras1, non-specific serine/threonine protein kinase activity, p38, Dm Ras1, l(2)41Ac, Dras64B, C-ras2, l(3)06677, DpErk, DpERK, l(1)G0002, ErkA, RasI, ERKA, l(1)G0482, hydroxyalkyl-protein kinase activity, CT34260, WEE1Hu, dpERk, c-H-ras, serine-specific protein kinase activity, epsilon PKC, l(1)G0127, SAPK, HIPK2, cytidine 3', Wee 1-like kinase activity, Harvey-ras, pERK, Hras1, ribosomal S6 protein kinase activity, calcium/phospholipid-dependent protein kinase activity, neuroblastoma, glycogen synthase A kinase activity, RasV12, GroupII, dRas, Central neuroblastoma, Kras2, glycogen synthase kinase 3 activity, CG1167, 12559, H-ras, DRAS1, DRas2, DmelCG9375, IMPDH, LD06825, phosphorylase b kinase kinase activity, EK2-1, Dras2, Dras1, l(1)G0388, D-Ras, Kras-2, serine(threonine) protein kinase activity, IMPdH, ribosomal protein S6 kinase II activity, Ha-ras, l(1)G0380, Erk, ERK, T-antigen kinase activity, Dras, p21B, PRKM1, ras 1, galactosyltransferase-associated kinase activity, PRKM2, protein-serine kinase activity, dras1, Dm-ras-64B, raspberry/impd, xp42, STK32, Ras[V12], l(1)G0391, EK3-4, p42 mitogen-activated protein kinase activity, CG18732, ATP:protein phosphotransferase (MAPKK-activated) activity, erk, DRas, DRas85D/Ras, Ras 85D, Dmras64B, protein kinase (phosphorylating) activity, protein-cysteine kinase activity, Raf-1, [M]Neuroblastoma NOS, Hpr kinase activity, rll, DmERK-A, STK26, ras-l, p38-2, casein kinase (phosphorylating) activity, myelin basic protein kinase activity, l(1)G0056, ert1, CG9375, prkm2, stress-activated kinase activity, prkm1, CT39192, MAP-2 kinase activity, K-ras, D-ras-1, protein serine kinase activity, protein phosphokinase activity, E(faf), p38delta, DmelCG1167, fs(3)05703, twitchin kinase activity, PKA, PKC, EP(X)1093, stress-activated protein kinase activity, mapk2, AP50 kinase activity, mapk1, S35097, MBP kinase I activity, NB, AI929937, protein kinase p58 activity, Xp42, serine/threonine protein kinase activity, threonine-specific protein kinase activity, RAS85D, Dmras85D, CG12559, frequent, l(1)9Eb, Neuroblastoma, mitogen activated kinase activity, Su(Raf)2B, dpERK1, EY2-2, dsk1, Su(tor)3-2, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, neuroblastoma NOS (morphologic abnormality), D-ERK, (Neuroblastoma NOS) or (sympathicoblastoma), dpMAPK, protein serine-threonine kinase activity, SAP kinase activity, MAP kinase 1 activity, A-kinase activityVasp, VASP, DmelCG15112, ENHANCER OF ATNSI ACTIVITY, CURIE, Ci, Curie., Data Set, MENA, NDPP1, l(2)02029, Enb, enb, ENA, Ena, CG15112, ENA/VASPKinase Kinases, RAS, Ras, DmErk, extracellular signal-regulated kinase activity, l(1)G0098, pp44mapk, DSORT, Surrogate Endpoints, Laboratory, acetylglucosaminyltransferase-like protein, MEK1/2, Tumor, Ras-1, Prp4 protein kinase activity, Mutations, ras, c-ras2, LeMPK3, ATP-protein transphosphorylase activity, Biological, p44mpk, ATP:protein phosphotransferase (non-specific) activity, Line, symptoms, Dp38, Kinase, myd, SAPK2, SEM, Sem, treatment, MAP-k, like-acetylglucosaminyltransferase, Genomes, pp42, MAPK-ERK Kinases, proportionality to, Mbp-1, Dsor2, Evolution, sem, DSor1, DSOR1, allergic reaction, Map Kinase, Map Kinase Kinase, high frequency, sample, Homo sapiens disease, CG1799, Tumors, ERK-A, p82 kinase activity, close to, screening, Neuroblastoma (Schwannian Stroma-Poor), dpERK, dpErk, Viral, Ras2, RAS2, Ras1, RAS1, betaIIPKC, familial, DmMAPK, PMK-2, dp-ERK, PMK-1, Sympathicoblastoma, predicted, PMK-3, Wee-kinase activity, Benign, Recrudescence, Marker, mKIAA0609, dRas85D, MAP Kinase Kinases, pMAPK, pMapK, Chemotherapy, DMEK-1, ras1, ras2, BcDNA:RE36103, EK1-1, fg, DmERKA, rl/MAPK, End Points, proportionality, Hras-1, non-specific serine/threonine protein kinase activity, signs, Benign Neoplasms, rate, Dm Ras1, l(2)41Ac, whole genome, Immunologic, Laboratory Marker, Malignant Neoplasms, Phenotypes, MDC1D, RasI, CT34260, dpERk, enr, MAPK-ERK, serine-specific protein kinase activity, SAPK, HIPK2, cytidine 3', other neoplasm, Wee 1-like kinase activity, Harvey-ras, Hras1, MAPK Kinases, MEKs, glycogen synthase A kinase activity, chemotherapy, Clinical Marker, Neoplasms, dRas, MAPK-ERK Kinase, Central neuroblastoma, glycogen synthase kinase 3 activity, CG1167, 12559, H-ras, DRAS1, DRas2, IMPDH, LARGE1, LD06825, phosphorylase b kinase kinase activity, EK2-1, Dras2, Dras1, D-Ras, sensitive, Kras-2, disease or disorder, IMPdH, ribosomal protein S6 kinase II activity, Ha-ras, Drug Therapies, MAP, Map, Erk, ERK, Dras, PRKM1, ras 1, PRKM2, protein-serine kinase activity, proportion, Clinical, Recrudescences, MDDGB6, raspberry/impd, STK32, MEK 7, Ras[V12], EK3-4, erk, DRas, Neoplasias, DRas85D/Ras, Dmras64B, protein-cysteine kinase activity, Kinase Kinase, Hpr kinase activity, rll, STK26, SOR, Sor, p38-2, constitutitional genetic, myelin basic protein kinase activity, Biologic, Cancer, findings, Malignant Neoplasm, MAP-ERK, Serum Markers, stress-activated kinase activity, disorders, MAP-2 kinase activity, sor, function, D-ras-1, protein serine kinase activity, Cell, MK, protein phosphokinase activity, Immune Marker, E(faf), near to, DmelCG1167, PKA, PKC, MAPK ERK Kinases, Surrogate End Point, stress-activated protein kinase activity, mapk2, mapk1, Neoplasm, S35097, MEK, Mek, condition, NB, MAP Kinase, protein kinase p58 activity, Biologic Markers, serine/threonine protein kinase activity, RAS85D, Dmras85D, CG12559, l(1)9Eb, Mitogen Activated Protein Kinase Kinases, Surrogate, dpERK1, Endpoints, Specificity, mek, Cancers, dsk1, 5'-cyclic monophosphate-responsive protein kinase activity, atypical PKC activity, neuroblastoma NOS (morphologic abnormality), like-glycosyltransferase, (Neuroblastoma NOS) or (sympathicoblastoma), dpMAPK, vicinity of, quotient, hereditary, protein serine-threonine kinase activity, Neoplasia, SAP kinase activity, Immune Markers, glycogen synthase kinase activity, Ras1/RAs85D, Biological Markers, Viral Marker, insensitive, protein kinase A activity, dsor1, MAP-ERK Kinase, Mpk2, Mbp1, Biochemical, D-MEK/Dsor, c-rasHa, p42mapk, Endpoint, E(sev)3C, Serum, PRKMK7, CG11485, SR2-1, Laboratory Markers, relapse, Clonal, diseases, kinase-related transforming protein, Su(Raf)34B, diseases and disorders, mitogen-activated protein kinase activity, Relapses, mpk1, D-Ras1, human disease, Erk/Map kinase, RTK, Recurrences, l(1)G0436, DERK-A, ras85B, ras85D, E(sina)7, Rl, neuroblastoma (morphologic abnormality), Raf kinase activity, [M]Neuroblastoma NOS (morphologic abnormality), genetic, MP kinase activity, serine kinase activity, DERK, D-ras-2, Immune, Markers, atypical protein kinase C activity, Viral Markers, disease management, Therapies, MBP kinase II activity, mitogen-activated S6 kinase activity, protein-aspartyl kinase activity, Malignancies, dERK, NB - Neuroblastoma, dSor1, Therapy, Relapse, l(3)s1747, MAP kinase 2 activity, SAPKK4, Surrogate Endpoint, sor/MEK1, gyltl1b-b, Mapk, D-sor-1, Ki-ras, Erk1, DSor, ERK1, ERK2, mapk1a, Biochemical Markers, Biologic Marker, DRODSOR1, M phase-specific cdc2 kinase activity, mapk1b, MapK, MDDGA6, MAPK, Diseases, NOS, p21[Ras1], median, dRas1, dRAS1, KIAA0609, serine protein kinase activity, Dras85D, erk2, Mitogen Activated Protein Kinase Kinase, acetylglucosaminyltransferase-like 1A, mapk, dSor, ras-2, Lines, ERKa, phosphorylase B kinase kinase activity, l(1)G0351, gyltl1b, LD02673, Pharmacotherapy, BcDNA:RE08694, MAP ERK Kinase, resistant, protein glutamyl kinase activity, Neuroblastomas, DmelCG1799, c-Ha-ras, l(2R)EMS45-39, D-ras1, l(1)G0238, D-ras2, mdc1d, DmelCG12559, C-ras1, p38, Dras64B, LARGE_HUMAN, Treatments, C-ras2, l(3)06677, DpErk, DpERK, l(1)G0002, dMEK, ErkA, ERKA, disease, l(1)G0482, hydroxyalkyl-protein kinase activity, WEE1Hu, Patient, Specificity and Sensitivity, c-H-ras, Biochemical Marker, epsilon PKC, l(1)G0127, Dsor, medical condition., inherited genetic, MAPK Kinase, pERK, ribosomal S6 protein kinase activity, other disease, calcium/phospholipid-dependent protein kinase activity, MAPKK7, neuroblastoma, RasV12, GroupII, Clinical Markers, Kras2, Benign Neoplasm, pharmacotherapy, Pharmacotherapies, DmelCG9375, Malignant, froggy, D-mek, Chemotherapies, Gyltl1a, Surrogate End Points, l(1)G0388, Surrogate Markers, Dmek, serine(threonine) protein kinase activity, JNKK2, l(1)G0380, sensitivity, CG15793, T-antigen kinase activity, p21B, galactosyltransferase-associated kinase activity, Somatic, Biomarker, MAPK ERK Kinase, dras1, Dm-ras-64B, MAPKKs, Malignancy, xp42, MKK7, Biological Marker, LARGE, D-sor, l(1)G0391, p42 mitogen-activated protein kinase activity, CG18732, ATP:protein phosphotransferase (MAPKK-activated) activity, non-neoplastic, BPFD#36, Ras 85D, protein kinase (phosphorylating) activity, Immunologic Markers, Raf-1, Clients, [M]Neuroblastoma NOS, Sensitivity, disorder, DmERK-A, ras-l, casein kinase (phosphorylating) activity, D-Mek, D-MEK, Immunologic Marker, l(1)G0056, ert1, Mitogen-Activated Protein Kinase Kinase, CG9375, prkm2, prkm1, CT39192, Cell Lines, End Point, K-ras, Client, Somatic Evolution, p38delta, MEK/Dsor1, fs(3)05703, twitchin kinase activity, EP(X)1093, AP50 kinase activity, MBP kinase I activity, AI929937, D-SOR, D-Sor, Xp42, threonine-specific protein kinase activity, DmelCG15793, Serum Marker, MAPKK, frequent, Neuroblastoma, mitogen activated kinase activity, Kinases, Su(Raf)2B, SAPKK-4, EY2-2, Su(tor)3-2, sample population, Surrogate Marker, Drug, D-ERK, Therapeutic, approaches, Treatment, pharmacologic therapy, glycosyltransferase-like protein LARGE1, MAP kinase 1 activity, A-kinase activityAU043776, HB-6, Materials, degree (angle), biml, Laboratory, gdf3-a, bam, der, Elp-B1, Elp-1, NINA C, Mini Exon, Tumor, Diagnosis, prevention, Mutations, 5730420M11Rik, symptoms, Whole Transcriptome, Transcriptome Sequencing, Software Engineering, Research Activity, notch, Laboratory Research, Virion-associated nuclear shuttling protein, Priorities, average, SET, thymus nucleic acid, vgr2, Genomes, Basen, Plant Embryo, Ax, ABIN1, Evolution, CDLS3, D17Mit170, flb, Fgfr7, DmelCG4299, GPR14, set, CG10728, Bek, co, sample, Gpr14, Nucleotide, T7H20_80, NECD, Diagnose, l(1)N, Tumors, NK/GPI, Gpi, screening, DMT1, preventive therapy, Elp, Chp, Co, DEgfr, T7H20.80, Exome, Research Priorities, Tl3, Tl2, Diagnoses, Software Tools, DmelCG5125, Computer Applications, torpedo/egfr, Postmortem, Benign, Recrudescence, shd, Base2, Computer Applications Software, Base1, NK|GPI, desoxyribose nucleic acid, CG32315, fa, nucleotides, DRONINAC, Research and Development, egfr, A20-binding inhibitor of NF-kappa-B activation 1, Complete Transcriptome, Software Applications, HD-33, El, HLA-DR-associated protein II, DI-2, Dlt, Source Software, DER/EGFR, EGFR, EGfr, EgfR, I-2Dm, signs, Benign Neoplasms, DER flb, Plant Zygote, whole genome, alpha, MISS, UTR2, Torpedo/Egfr, CG107278, Malignant Neoplasms, Degfr, Activities, I-2PP1, Applications, BIM, TAF-IBETA, nip40-1, clone 1.12, Material, DEGFR, ds DNA, l(2)05351, Whole Exome Sequencing, TAF-Ibeta, School-Age Population, DNA, other neoplasm, Bglap-rs1, Computer Software Applications, bim, DNS, D-Egf, (Deoxyribonucleotide)n, BELLRINGER, Neoplasms, number, ABIN, EK2-6, fs(3)neo61, CG10422, Deoxyribonucleic Acid, Core Genome, EGFr, Mini-Exon, Screening, torpedo/Egfr, bim-beta7, Pgi, TOP, Antemortem, NINAC, CG54125, Application, l(1)Ax, CG13969, School-Age Populations, Open Source Softwares, Complete, Whole Genome, Exome Sequencings, Genetic Heterogeneities, BLR, NinaC, Recrudescences, bim-beta6, Gpi-1r, Software Application, Gpi-1s, Accessory Genome, Phi, Complete Genome Sequencing, Gpi-1t, Open Source Software, Double Stranded, top, 2pp2a, dre1, Zygote, Deoxyribonucleic acid, Diagnoses and Examinations, Population, BMH, Nramp2, Sequencing, CG10574, Computer Software Application, Neoplasias, Whole Exome, 2PP2A, Tools, Whole, Nef, dSET, dSet, (Deoxyribonucleotide)m, Development and Research, 2.2, CSPG6, l(3)dre1, CT13012, DmelCG10728, Cancer, Elp-B1RB1, 16-178, Antemortem Diagnoses, Elaiosomes, 16-55, findings, Malignant Neoplasm, Complete Exome Sequencings, cou, dEGFR1, DNAn+1, 38B.5., C14orf32, UTR, 2310009E07Rik, BOD, anon-EST:Liang-1.12, MF, derriere, Amf, Pangenome, mk, Tool, Exome Sequencing, Lr, I-2PP2A, hVAN, chemical analysis, spl, Dm I-2, Neoplasm, BEL1-LIKE HOMEODOMAIN 9, NB, 1.1, bod, nd, background, DmelCG32315, DER/top, Naf1, NAF1, bimel, NK, VAAMANA, ensemble, prophylaxis, Heterogeneities, Exon, Cancers, Understanding, School Age Populations, Computer Programs, Pan-genome, Applications Softwares, control, Bam-C, Desoxyribonukleinsaeure, Bra, nucleobases, Neoplasia, DER/faint little ball, IPP2A2, Antemortem Diagnosis, BamF, Activity, BamC, determination, Dm NinaC, CG5125, PNY, School-Age, Readability, l(3)04276, relapse, Clonal, Relapses, DmelCG10422, d-egf-r, Elaiosome, Gpi-1, prevention and control, Computer Program, me75, reference sample, PENNYWISE, HCAP, TAF-I, c14_5346, Nlk, Complete Exome Sequencing, long, Recurrences, Whole Transcriptome Sequencing, Open, bases, DmelCG13969, Computer Programs and Programming, T1, preventive measures, IGAAD, Nina C, DmelCG10574, malignant neoplasm, Diaspores, svs, D-EGFR, Double-Stranded DNA, Malignancies, deoxyribonucleic acids, Research Priority, DNAn, TL, REPLUMLESS, EG:140G11.1, CT16120, DER1, swb, phapii, Relapse, AU018810, l(2)09261, Nucleobase, Genome Sequencing, StF-IT-1, ham, Double-Stranded, Fgfr-7, Fgfr-2, CG10079, Source Softwares, Programs, (Deoxyribonucleotide)n+m, Program, BLH9, Screenings, Complete Genome, mOC-X, untranslated region, School Age, Examinations and Diagnoses, Computer Applications Softwares, Genetic Materials, Seed, Softwares, Postmortem Diagnosis, Genetic Material, Diagnoses and Examination, Populations, Postmortem Diagnoses, Diaspore, Org, ORG, Neuroblastomas, dEgfr, Heterogeneity, Plant, Gpi1-r, Gpi1-s, CG4299, Gpi1-t, Patient, l(2)57DEFa, Base, SMC3L1, dEGFR, Cistron, DmHD-33, n[fah], Transcriptome Sequencings, i2pp2a, Gpi1s, base, Nip40-1, VAL, Van, VAN, AU043015, DCT1, nuclear mRNA cis splicing, Complete Exome, KGFR, AI461847, C-erb, Benign Neoplasm, EG:163A10.2, mor1, Gene, Computer, Malignant, aligned, presence, dacer, Deoxyribonucleic acids, PHAPII, val, van, Genetic heterogeneity, c-erbB, Mass, tumor sample, Egf-r, Low, l(1)3Cb, CG1977, study, Somatic, mVAN, EGF-R, WES, FOCUS, Genetic, Malignancy, ninac, Research, AW556123, DmelCG3936, ipp2a2, Torpedo/DER, dNotch, CG 5125, top/flb, Table, via U2-type spliceosome, l(2)57EFa, taf-ibeta, Clients, Zygotes, l(2)57Ea, School Age Population, NICD, Controlled, HIV-1 Nef-interacting protein, Applications Software, WGS, Open Source, Controlling, ABIN-1, Computer Software, igaad, aligned to, Cistrons, Somatic Evolution, Client, Examination and Diagnoses, group, count in organism, Software Tool, LSN, Priority, DmelCG10079, Mass Screenings, Embryos, Research Activities, I2PP2A, CT42491, ds-DNA, Software, LARSON, Embryo, KGFRTr, Complete Transcriptome Sequencing, distinct, top/DER, Neuroblastoma, Mini-Exons, Nef-associated factor 1, Engineering, arc degree, Plant Embryos, sample population, introduction, UR-2-R, dSET/TAF-Ibeta, Egf, 2610030F17Rik, EFG-R, vgr-2, Plant Zygotes, 38B.6, BAM, cardinality, Bam, Der, DER, assay, l(3)62Ba, CG3936, AA407739, DER/torpedofalseena-DATASET-CURIE-LC-04-05-2015-16:47:47:270-89 - samplesNeuroblastoma, a clinically heterogeneous pediatric cancer, is characterized by distinct genomic profiles but few recurrent mutations. As neuroblastoma is expected to have high degree of genetic heterogeneity, study of neuroblastoma's clonal evolution with deep coverage whole-genome sequencing of diagnosis and relapse samples will lead to a better understanding of the molecular events associated with relapse. Samples were included in this study if sufficient DNA from constitutional, diagnosis and relapse tumors was available for WGS. Whole genome sequencing was performed on trios (constitutional, diagnose and relapse DNA) from eight patients using Illumina Hi-seq2500 leading to paired-ends (PE) 90x90 for 6 of them and 100x100 for two. Expected coverage for sample NB0175 100x100bp was 30X for tumor and constitutional samples. For the seven other patients expected coverage was 80X for tumor samples with PE 100x100, 100X in the other tumor samples and 50X for all constitutional samples (see table 1). Following alignment with BWA (Li et al., Oxford J, 2009 Jul) allowing up to 4% of mismatches, bam files were cleaned up according to the Genome Analysis Toolkit (GATK) recommendations (Van der Auwera et al., Current Protocols in Bioinformatics, 2013, picard-1.45, GenomeAnalysisTK-2.2-16). Variant calling was performed in parallel using 3 variant callers: GenomeAnalysisTK-2.2-16, Samtools-0.1.18 and MuTect-1.1.4 (McKenna et al., Genome Res, 2010; Li et al., Oxford J, 2009 Aug; Cibulskis et al., Nature, 2013). Annovar-v2012-10-23 with cosmic-v64 and dbsnp-v137 were used for the annotation and RefSeq for the structural annotation. For GATK and Samtools, single nucleotide variants (SNVs) with a quality under 30, a depth of coverage under 6 or with less than 2 reads supporting the variant were filter out. MuTect with parameters following GATK and Samtools thresholds have been used to filter our irrelevant variants. .SNVs within and around exons of coding genes overlapping splice sites.. Then,variants reported in more than 1% of the population in the 1000 genomes (1000gAprl_2012) or Exome Sequencing Project (ESP6500) have been discarded in order to filter polymorphisms. Finally, synonymous variants were filtered out. MuTect focuses on somatic by filtering with constitutional sample. Mpileup comparison between constitutional and somatic DNAs allowed us to focus also on tumor specific SNVs with GATK and Samtools. Finally, every SNV called by our pipeline and also supported in any constitutional samples were filtered our in order to prevent putative constitutional DNA coverage deficiency. Then we analyzed CNVs (copy number variants) with HMMcopy-v0.1.1 (Gavin et al., Genome Res, 2012) and control-FREEC-v6.7 (Boeva et al., Bioinformatics 2011) with a respective window of 2000bp and 1000 bp, and auto-correction of normal contamination of tumor samples for Control-FREEC. Finally we explored Structural variants (SVs) including deletions, inversions, tandem duplications and translocations using DELLY-v0.5.5 with standard parameters (Rausch et al., Oxford J, 2012). In tumors, at least 10 supporting reads were required to make a call and 5 supporting reads for the sample NB0175 with a coverage of only 40X (see table 2). To predict SVs in constitutional samples for subsequent somatic filtering, only 2 supporting reads were required in order not to miss one. To identify somatic events, all the SVs in each normal sample were first flanked by 500 bp in both directions and any SVs called in a tumor sample which was in the combined flanked regions of respective normal sample was removed (see graph 1). Deletions with more than 5 genes impacted or larger than 1Mb and inversions or tandem duplications covering more than 4 genes, were removed. We focused on exonic and splicing events for deletions, inversions, and tandem duplications. For translocation, we keep all SVs that occurred in intronic, exonic, 5'UTR, upstream or splicing regions. Bioinformatics detection of variations with Deep sequencing approach Once PE reads merged and adaptors trimmed by SeqPrep with default parameters, merged reads were aligned via the BWA (Li H. and Durbin R. 2009 PMID 19451168) allowing up to 1 differences in the 22-base-long seeds and reporting only unique alignments. Only reads having a mapping quality 20 or more have been further analysed. Variant calling software was not used, since we aimed to predict variations at low frequencies, observed in less than 1% of reads. Such variants require a custom approach. Using DepthOfCoverage functions of the Genome Analysis Toolkit (GATK) v2.13.2 (McKenna A, et al., 2010 Genome Research PMID: 20644199), we focused on high quality coverage of bases A, C, G and T at the targeted variant position. Depth of coverage of each base following a mapping quality higher than 20 and a base quality higher than 10 have been taken into account in order to focus only on high quality data. Aiming to determine the background level of variability at the studied regions, 10 control samples were included in the analysis. The same approach and filtering criteria have been applied as introduced above over the entire amplicons. In order to highlight variants, for each sample the frequencies of each bases at each amplicon position were then compared to those observed in the set of controls. Statistical analyses were performed with the R statistical software (http://www.R-project.org). Fisher’s exact two-sided tests with a Bonferroni correction were performed to compare percentages of bases between the data sets, i.e. for a given base between a case and the controls. Finally, significant variations were filtered-in once (i) a significant increase in the percentage of avariant base and (ii) a significant decrease in the percentage of it's reference base following our p.values criteria was observed (p.val < 0.05).2020-07-16 15:33:08EGAD00001001356960626121087EGAC00001000319EGAS00001001184