Intraclonally stable methylation patterns in CLL reflect signatures of the potential normal B-cell of origin. (A) Unsupervised HC of the fractions by Manhattan distance measurement of DNA methylation (unscaled β-values). Colored horizontal labels indicate IGHV mutation status (U- or M-CLL) and BR (low or high BR, at the threshold of 0.35% cells/day). (B) PCA of DNA methylation data from all fractions, colored by either IGHV mutational status or BR. (C) Scatter plots for pairwise comparisons of DNA methylation between U-CLL and M-CLL (top) or between low BR and high BR (bottom) patients by CXCR4/CD5 intraclonal fractions. Sites were highlighted at false discovery rate (FDR) <0.05 (black dots), and further considered significant at FDR <0.01 and absolute mean methylation difference of ≥10% (red dots). Colors in the cartoon representation of cell fractions are maintained throughout the panels. (D) Venn diagram showing relations between common overlap (c-DMC) and fraction-specific differentially methylated CpGs (f-DMC), which were identified as red dots in panel C after pairwise comparison of U- and M-CLL patients. (E) Heatmap of c-DMCs and f-DMCs between U- and M-CLL, ordered by IGHV status and by fractions. Colored horizontal labels indicate other established predictors of CLL progression: CD38⁺ (>30% positive), ZAP70⁺ (>20% positive). Framed quadrants in the heatmap highlights f-DMCs initially identified in the corresponding fraction, despite largely comparable methylation profiles. IF, intermediate fraction; PC, principal component; PF, proliferative fraction; RF, resting fraction.