Abstract: Background: Approximately 25% of pediatric B-cell precursor ALL are characterized by the ETV6-RUNX1 fusion and are associated with a favorable prognosis. Monozygotic twin studies with concordant ALL and ‘backtracking’ studies using archived neonatal blood spots established that ETV6-RUNX1 is a likely initiating event arising prenatally in a committed B-cell progenitor. However, the fusion gene is not sufficient on its own to cause overt leukemia and a number of subsequent studies have now provided strong evidence that additional mutations, arising post-natally, are essential for the clinical development of ALL. Aims: To obtain a detailed portrait of the composite genetic events that, in concert with the ETV6-RUNX1 fusion gene, drive this subtype of ALL, we have carried out extensive genomic analysis in patients with ETV6-RUNX1 ALL. Methods: A total of 57 cases with diagnostic (leukemic) cell DNA paired with matched, remission samples as a source of constitutive DNA were used for exome sequencing (n=56) and low-depth whole genome sequencing for structural variation analysis (n=51). Integrative analysis of exome and whole genome data was performed and data were evaluated for recurrent gene mutations, copy number alterations and genomic rearrangements. Signatures of somatic mutation and structural variation were studied for insights into operative mutational processes and exposures that may drive ETV6-RUNX1 pathogenesis. Single cell analysis in two patients was performed using a PCR-based, microfluidic platform, to study timing of mutational processes and how these affect the patterns of subclonal segregation of mutations and clonal phylogeny. Results: We confirmed 775 somatic substitutions and 16 indels across 715 protein coding genes and 3 micro RNAs. Each patient had on average 14 gene coding mutations consistent with the low number of acquired somatic mutations identified by systematic sequencing screens of haematological malignancies and other childhood cancer. Whole genome profilings identified 524 SVs (average:11, range 0-49) including 34 tandem duplications, 66 inversions, 106 intrachromosomal translocations and 317 deletions. Oncogenic mutations in KRAS, NRAS, CTCF, DAXX, EZH2, and KDM6A are described as well as patterns of complex rearrangement including the balanced chain of chromosomal rearrangements seen in prostate cancer. Most mutations are subclonal to ETV6-RUNX1. Integrative analysis of whole-genome and exome data identify genes with deletions and inactivating mutations including MGA and ZMYM2 that would have not been identified by either dataset alone. Sequence context analyses on the observed mutations identifies two distinct mutational signatures that are operative in ETV6-RUNX1 ALL, and collectively account for the majority of the acquired mutations observed. Summary and Conclusions: We report the identification of a novel spectrum of somatic mutations in ETV6-RUNX1 ALL and present the first detailed characterization of the genomic landscape of this common ALL subtype. We provide new insights into the molecular pathogenesis of ETV6-RUNX1 ALL and discuss the potential biological and clinical implications.