ABSTRACT: Genetic and epigenetic changes have provided essential insights into the biology and clinical behavior of chronic lymphocytic leukemia (CLL). Overall, increasing number of chromosomal aberrations are related to disease evolution. Complex karyotype (CK) is defined as presence of at least 3 numerical and/or structural aberrations detected by chromosome banding analysis. CK frequently occur in the context of CLLs with unmutated IGHV (U-CLL) and are associated with mutations in ATM or TP53. In this context, the relationship of karyotype complexity and DNA methylation aberrations in CLL remains largely unexplored. In the present study, we selected a highly curated cohort of 52 U-CLL cases with known karyotypes and ATM and TP53 aberration status. An unsupervised Principal Component Analysis using all the CLL samples showed that there is no specific clustering according to karyotype complexity. Although CK was not a major driver of epigenetic variability, a differential methylation (DM) analysis between CK vs non-CK samples led to a small signature of 59 CpGs, suggesting only a subtle relationship between karyotypic complexity and DNA methylation. We noticed that cases with deletions in ATM and TP53 tend to cluster differently in the first two principal components independently of karyotype complexity. A consensus clustering analysis using the 5,000 most variable CpGs confirmed the two clusters, which were enriched in ATMdel (U-CLL C1, P=0.015) and TP53del/mut (U-CLL C2, P=0.004), respectively. U-CLL C1 was also enriched in ATM mutations (P=0.017) and del(13q) (P=0.007), while U-CLL C2 was also related to trisomy 12 (tri12) (P=0.004). Both clusters showed a similarly unfavorable clinical behavior, both in terms of time to first treatment and overall survival. A DM analysis between them resulted in 4,333 differential CpGs. Surprisingly, despite the fact that all the cases are U-CLLs, and therefore germinal center-inexperienced, 2,150 CpGs were modulated in normal B-cell differentiation. Remarkably, 87% of those CpGs showed that cases in the U-CLL C1 cluster are associated with a DNA methylome of less mature B cells (more similar to NBC) whereas cases in the U-CLL C2 were related to more mature B cells (more similar to MBC). A phylogenetic analysis using previously reported CpGs significant for normal B-cell differentiation confirmed this observation. The remaining DM CpGs between the two clusters were largely related to a de novo DNA hypomethylation signature (n=1,679 CpGs) in the U-CLL C2 cluster. In summary, despite not finding a major relationship between complex karyotype and DNA methylation, we discovered the presence of two epitypes in the U-CLL dataset with distinct genetic profiles and methylation signatures related to different levels of epigenetic imprinting, a finding reinforcing the perception that CLL rises from a continuum of B-cell maturation stages.
