The KMT2A::AFF1 circRNAome is highly dysregulated. (A) Principal component (PC) analysis and (B) hierarchical clustering using Euclidean distance of samples from patients with KMT2A::AFF1 ALL and CD34⁺ cells and B-cell populations from HDs, based on circRNA expression profiles. (C) Heat map of standardized expression of the 86 circRNAs differentially expressed when comparing KMT2A::AFF1 ALL vs B cells and CD34⁺ cells from HDs; (left) the log₂ fold change (LFC) in the 2 comparisons and the average AE in all samples are shown. (D) CircRNA expression quantification by RQ-PCR in an extended patient cohort (relative expression calculated as 2^-ΔΔCT values using glyceraldehyde-3-phosphate dehydrogenase as reference and bone marrow samples from at least 3 HDs as calibrator; Mann-Whitney U test P values: ∗P < .05 and ∗∗ P < .01); N = 10 MLL:AF4 BCP-ALL for circPROM1; N = 17 KMT2A::AFF1 BCP-ALL for circFKBP5, circKLHL2, circNR3C1, circHIPK3, and circPAN3; and N = 27 MLL:AF4 BCP-ALL for circFLT3_2. (E) Relation between dysregulation of circRNA expression and proportion in KMT2A::AFF1 ALL for the same 86 DE circRNAs in panel C. The dot size represents the CLP value in patients with KMT2A::AFF1, the dot color indicates DP variation categories in the 2 comparisons; names of circRNAs DP in at least 1 comparison (KMT2A::AFF1 vs B cells and/or KMT2A::AFF1 vs CD34⁺ cells) are shown, and the number of circRNAs in each quadrant is indicated. UP, upregulated; NS, unvaried; DW, decreased.