The Genomic Landscape of TEL-AML1+ (ETV6-RUNX1) Acute Lymphoblastic Leukaemia
Authors: Papaemmanuil Elli , Rapado Inmaculada , Ford Anthony M , Raine Keiran , Hinton Jonathan , Jones David , Marshall John , Mudie Laura J , Zuna Jan , Cazzaniga Giovanni , Kempski Helena , de Oliveira Maria S Pombo , Stratton Mike R , Futreal P Andrew , Campbell Peter , Greaves Mel 
Published: October 2019

Abstract
Abstract 403
TEL-AML1 (ETV6-RUNX1) fusions are common (∼25%) in paediatric B cell precursor acute lymphoblastic leukaemia (ALL) and associated with a very favourable prognosis. Our prior studies on monozygotic twins with concordant ALL and ‘backtracking' studies using archived neonatal blood spots established that ETV6-RUNX1 fusion is usually an early or initiating mutational event arising pre-natally. Subsequent studies, have provided strong evidence that additional genetic changes are essential for the clinical development of ALL. Twin studies suggest that these additional events are most likely to be post-natal and secondary to the ETV6-RUNX1 fusion (Bateman C et al, Blood 2010).
To obtain a more in depth portrait of the composite genetic events that drive this subtype of ALL, we have carried out an extensive genomic analysis of 58 cases with diagnostic (leukaemic) cell DNA paired with matched, remission sample as a source of constitutive DNA. For all 58 cases, both leukemic and remission samples were subjected to massively parallel sequencing across all protein coding exons and a subset of selected non-coding RNAs. All cases were sequenced to at least 35x sequence coverage. Likely acquired mutations were called using algorithms developed in house for single base pair substitutions, small insertions and deletions. All putative somatic mutations were validated using conventional Sanger sequencing and to allow for estimation of allelic burden we further validated all somatic calls by massively parallel pyrosequencing.
More than 700 mutations were confirmed to be somatically acquired. The majority of these mutations (>500) represent non-synonymous coding changes or stop-codons. Similar to other paediatric neoplasms, each patient harboured a modest number of total mutations (n=13.5, 95% CI: 9.6–17.4). Interestingly, recurrent mutations were present in 42 genes, however the majority (n=37) were only present in two cases each. Allelic burden estimates across all confirmed somatic variant positions in a patient sample, demonstrated evidence of sub-clonal events at presentation. This is being validated by quantitative PCR and single cell analysis. Mutation signatures were profiled in respect to occurrence, frequency and sequence context. To implement a detailed characterisation of these patterns, further sequencing of the entire genome is currently under way in selected samples.
To further characterise the genomes of ETV6-RUNX1+ ALL and to identify novel cryptic rearrangements, whole genome paired-end sequencing at a physical depth of at least 16x was performed on 49 cases. Paired reads were mapped to build 37 of the human reference genome. Mapping of the breakpoints to base-pair resolution demonstrated that the ETV6-RUNX1 fusion is the result of complex rearrangements involving several chromosomal events. A further series of genomic rearrangements were defined and selected for validation by conventional PCR of the breakpoint junctions in the matched sample pairs.
In summary, we report the identification of a novel spectrum of somatic mutations in ETV6-RUNX1+ ALL and present the first detailed characterisation of the genomic landscape of this ALL subtype. We provide new insights into the molecular pathology of ETV6-RUNX1+ ALL and discuss the potential implications of our findings both in understanding the underlying molecular mechanisms and also its clinical management.

Disclosures:
No relevant conflicts of interest to declare.