Deregulated expression of the type I cytokine receptor,
CRLF2
, is observed in 5-15% of precursor B-cell acute lymphoblastic leukaemia (B-ALL). We have previously reported the genomic landscape of patients with
CRLF2
rearrangements (
CRLF2
-r) using both whole genome and exome sequencing, which identified a number of potential clonal and sub-clonal genomic alterations. In this study, we aimed to assess when the
CRLF2
-r;
IGH-CRLF2
or
P2RY8-CRLF2
, arose during the evolution of both Down syndrome-ALL (DS-ALL) and non-DS-ALL. Using fluorescence
in situ
hybridisation, we were able to track up to four structural variants in single cells from 47
CRLF2
-r B-ALL patients, which in association with our multiplex single cell analysis of a further four patients, permitted simultaneous tracking of copy number alterations, structural and single nucleotide variants within individual cells. We observed
CRLF2-
r arising as both early and late events in DS and non-DS-ALL patients. Parallel evolution of discrete clones was observed in the development of
CRLF2
-r B-ALL, either involving the
CRLF2
-r or one of the other tracked abnormalities. In depth single cell analysis identified both linear and branching evolution with early clones harbouring a multitude of abnormalities, including the
CRLF2
-r in DS-ALL patients.