Focal BTG1 Deletions Occur in Specific Precursor B-Cell Acute Lymphoblastic Leukemia Subtypes At Defined Hotspots Due to Aberrant V(D)J Recombination
Authors: Scheijen Blanca , Waanders Esmé , van Reijmersdal Simon V. , van der Meer Laurens T. , van Emst Liesbeth , Verwiel Eugene , Kroeze Yvet , Sonneveld Edwin , Hoogerbrugge Peter M. , van Kessel Ad Geurts , van Leeuwen Frank N. , Kuiper Roland P. 
Published: October 2019

Abstract
Abstract 399
Recurrent deletions in genes affecting key cellular pathways are a hallmark of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B cell translocation gene 1 (BTG1), in a large cohort of pediatric BCP- and T-lineage ALL cases.
The BTG1 gene was found to be exclusively affected by genomic microdeletions, which were detected in 65 out of 722 BCP-ALL patient samples (9%), but not in 109 T-ALL cases. Genomic sequencing of the entire BTG1 open reading frame (n=135) and the main coding second exon (n=158), as well as bisulphite sequencing of its promoter (n=25) provided no evidence for the presence of BTG1 point mutations or promoter methylation, respectively. Eight distinct deletion sizes (ranging from 101 to 557 kb) were identified, which all clustered at the telomeric site within a 30 bp stretch in the second and last exon of the BTG1 gene. The deletions resulted in almost identical truncations of the open reading frame and a concomitant loss of two conserved C-terminal protein interaction domains. Truncated BTG1 fusion transcripts specific for each type of deletion could be detected exclusively in the deletion-positive BCP-ALL cell lines and primary BCP-ALL samples, whereas wild-type BTG1 mRNA was expressed in both deletion-positive and -negative cases. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests RAG1/RAG2-mediated recombination as the responsible mechanism. In line with these observations, BCP-ALL, but not T-ALL, cell lines showed increased levels of histone H3 trimethylation at lysine 4 (H3K4me3) at the BTG1 gene locus. This epigenetic mark is associated with actively transcribed loci and acts as a docking site for RAG2 binding, thereby facilitating V(D)J recombination.
BTG1 deletions were found to be unevenly distributed between the different cytogenetic subgroups, being present in 19% (n=27/142) of the ETV6-RUNX1 (TEL-AML) and 26% (n=6/23) of the BCR-ABL1 positive cases, and in only 3% (n=5/160) of the hyperdiploid cases (P<0.001, P=0.003 and P=0.002, respectively). In addition, targeted copy number analysis of recurrently affected genes in ALL revealed that cases with BTG1 deletions more frequently harbor deletions of ETV6, RB1 and EBF1 (P=0.007, P<0.001 and P<0.001, respectively). Using a sensitive PCR-based screening assay, we identified (multiple) additional BTG1 deletions at the subclonal level in 19 of the 65 deletion-positive BCP-ALL cases (29%), and in 21 of the 89 deletion-negative BCP-ALL cases (24%), but not in the T-ALL cases (n=77) or bone-marrow samples from healthy donors (n=26). Similar to the clonal BTG1 deletions, these subclonal events were enriched in the ETV6-RUNX1 subgroup and absent in the hyperdiploid cases.
In conclusion, our results indicate that BTG1 deletions act as ‘driver' mutations in specific BCP-ALL subtypes, in which they can arise independently in multiple subclones in a locus that appears to be prone to aberrant RAG1/RAG2-mediated recombination events.

Disclosures:
No relevant conflicts of interest to declare.