Using next generation sequencing to characterise in detail the biological mechanisms that underpin genomic instability in ETV6-RUNX1 ALL and identify recurrent drivers associated with leukemic transformation


Session type:

Elli Papaemmanuil1
1Wellcome Trust Sanger Institute, Cambridge, UK


Current sequencing technologies have delivered the ability to profile cancer genomes with great precision allowing for the characterisation of the complete spectrum of genetic events (single base pair events, small insertions or deletions as well as structural variations) with a base pair resolution.

The ETV6-RUNX1 fusion characterises 25% of B-cell precursor ALL but on its own is not sufficient for overt leukemia. We surveyed the genomes of 56 leukemic samples of ETV6-RUNX1 ALL by low-depth genome and high-depth exome sequencing to: 1. Identify critical secondary events to the ETV6-RUNX1 fusion gene necessary for leukemic transformation; 2. Perform detailed analysis of the composite genomic architecture of ETV6-RUNX1 ALL; 3. Study patterns of mutations (composition, location, chromatin landscape, timing) for insights into potential biological mechanisms of genomic instability.

We confirmed 523 structural variations (average of 11 per case; range of 0-49) in 44 of the samples in the study. We identified 779 somatic substitutions and 16 indels across 715 protein-coding genes and 3. Each sample had on average 14 coding point mutations (range of 1-95), consistent with the low number of acquired somatic mutations reported in hematological cancers and childhood malignancies. A remarkable paucity of recurrent gene mutations was observed. However, 76% of intrachromosomal rearrangements were deletions, and accounted for many of the frequent secondary events in ETV6-RUNX1 ALL.

A striking enrichment of deletion breakpoints adjacent to recombination signal sequence (RSS) sites was observed. RSS sites are conserved sequence motifs recognized by the recombination activating gene (RAG) endonucleases that mediate V(D)J recombination during antibody diversification. Clusters of recurrent gene deletions targeting the same or adjacent nucleotides were observed across samples with evidence of re-iterated events within the same sample. RAG-mediated deletions emerge as the dominant mutational process, characterised by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localised clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumour-suppressor genes in ALL.

Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1-positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation is characterised. Taken together our data present the first detailed evaluation of the entire genomic alterations that come together in the wiring of ETV6-RUNX1 ALL and deliver a clear understanding of the mechanisms that initiate genomic instability and result in ALL.