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Relapsed precursor T-cell acute lymphoblastic leukemia is characterized by resistance against chemotherapy and is frequently fatal. We aimed at understanding the molecular mechanisms resulting in relapse of T-cell acute lymphoblastic leukemia and analyzed 13 patients at first diagnosis, remission and relapse by whole exome sequencing, targeted ultra-deep sequencing, multiplex ligation dependent probe amplification and DNA methylation array. Compared to primary T-cell acute lymphoblastic leukemia, in relapse the number of single nucleotide variants and small insertions and deletions approximately doubled from 11.5 to 26. Targeted ultra-deep sequencing sensitively detected subclones that were selected for in relapse. The mutational pattern defined two types of relapses. While both are characterized by selection of subclones and acquisition of novel mutations, 'type 1' relapse derives from the primary leukemia whereas 'type 2' relapse originates from a common pre-leukemic ancestor. Relapse-specific changes included activation of the nucleotidase NT5C2 resulting in resistance to chemotherapy and mutations of epigenetic modulators, exemplified by SUZ12, WHSC1 and SMARCA4. While mutations present in primary leukemia and in relapse were enriched for known drivers of leukemia, relapse-specific changes revealed an association with general cancer-promoting mechanisms. This study thus identifies mechanisms that drive progression of pediatric T-cell acute lymphoblastic leukemia to relapse and may explain the characteristic treatment resistance of this condition. ALL-BFM 86/90, 1989-1998 INS 98 protocol based on ALL-BFM 95 40 , 1998-2003 and ALL Intercontinental (IC) -BFM 200315, 2003-2005 analyzed at the time of primary diagnosis, during remission and at relapse. Pediatric T-cell lymphoblastic leukemia evolves into relapse by clonal selection, acquisition of mutations and promoter hypomethylation ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n Methods Patients' clinical characteristicsPatients were treated according to ALL-BFM 2000 or related frontline protocols 14 IC 15 ). One patient was aged 18 at diagnosis, all others were children or adolescents. The 13 patients (Table 1) were recruited between 1993 and 2007 from the ALL-REZ BFM 2002 trials (patients T-ALL-H-A61, -E114, -F110, -KI17, -MD40, -T92, -T128) or from Schneider Children's Medical Center of Israel, Petah Tikva, Israel (patients T-ALL-H-S00169, -S00207, -S00285, -S00438, -S00456, -S00472) and selected on the basis of sufficient material being available from the time points of first diagnosis, remission and relapse. Minimal residual disease (MRD) response was assessed as described previously 2,16 (Online Supplementary Table S3).This study was approved by the institutional review boards of the Charité Universitätsmedizin Berlin and the Medical Faculty Heidelberg. Informed consent was obtained in accordance with the Declaration of Helsinki. Exome capture, target capture and Illumina sequencingThe Agilent SureSelect Target Enrichment Kit (Agilent...