FISH evidence that P2RY8-CRLF2, IKZF1, and PAX5 are secondary events to iAMP21 and a putative model showing the clonal architecture of sample 7219. (A) Five interphase nuclei from patient 7219 hybridized with a probe targeting RUNX1 (red), which detects the multiple copies of this gene that defines iAMP21, and a probe targeting CSF2RA and IL3RA, centromeric to CRLF2, which when deleted indicates the presence of P2RY8-CRLF2 (green). The example shows evidence of clonal heterogeneity; populations of iAMP21-positive cells both with and without P2RY8-CRLF2 are indicated. (B) Three interphase nuclei from patient 7219 hybridized using the same RUNX1 probe (red) and a probe for IKZF1 (green) showing iAMP21-positive cells both with and without the IKZF1 deletion. (C) Examples showing data indicating subclonal architecture in 3 patients: 20684, 7219, and 4444. In patients 7219 and 20684, 9% and 6% of cells, respectively, had amplification of RUNX1 with no deletion of IKZF1, whereas 79% and 89%, respectively, showed an IKZF1 deletion. In patient 4444, 83% of cells had amplification of RUNX1 with no PAX5 deletion, whereas 10% of cells showed the deletion. The major clone (53%) detected in patient 7219 showed iAMP21 without P2RY8-CRLF2, whereas 39% showed the P2RY8-CRLF2 fusion. These observations provide evidence that these events are secondary to iAMP21. (C) Putative model predicting the temporal order of events in the diagnostic sample from patient 7219; apparent linear architecture with 3 populations indicating that iAMP21 is the primary event, followed by deletion of IKZF1 and the presence of P2RY8-CRLF2, is shown.