Interphase FISH analysis of 9p21 (CDKN2A) lesions reveals the existence of genetically distinct subclones. The clonal composition of ALL samples was analyzed using dual-color FISH probes against 9p21 (CDKN2A, red) and the centromer of chromosome 9 (CEN9, green) as control in samples from diagnosis and corresponding xenografts. (A) Representative images are shown, indicating wild-type cells with no deletion in the 9p21 locus (left), a heterozygous deletion (middle), and a homozygous deletion (right). (B) The clonal distribution was analyzed in samples from up to 6 rounds of serial transplantation. A total of 200 cells were counted in each sample. The original data are reported in supplemental Table 7. ALL cells with germline, heterozygous deletion, and homozygous deletions are represented by colored dots of size proportional to the percentage of cells in each category in relation to the total cell number as indicated in the legend. One case, VHR-01, shows clonal selection toward additional deletions at this locus, whereas the 4 other cases analyzed are more stable with respect to the clonal composition based on this genetic feature. Original magnifications ×40 (Leica LX Widefield Microscope DMI6000B with the DFC 350 FX R2 camera).