(Peri)Centromeric Rearrangements Are Recurrent Recombination Events in Complex-Karyotype Chronic Lymphocytic Leukemia

Background: A retrospective cytogenetic study encompassing 5290 patients with chronic lymphocytic leukemia (CLL) identified complex karyotype (CK) as adverse prognostic factor. In cases with ≥5 chromosomal aberrations (CAs), this finding was independent of TP53 alterations (Baliakas et al., 2019) Which chromosomal rearrangements dominate CK-CLL is insufficiently defined impeding the identification of genomic events responsible for poor prognosis. To delineate cytogenetic aberrations in CK-CLL, we analysed conventional karyotypes from 125 patients classified as high-risk during the era of chemoimmunotherapy.

Methods: Chromosome banding (CBA, CpG oligonucleotide and IL-2 stimulation, N=131), SNP-array analysis (Affymetrix 6.0, N=109), DNA methylation analysis (Illumina Infinium HumanMethylation450 BeadChips, N=45) and telomere length measurement (N=122) was conducted on samples taken at enrolment on the CLL2O trial of the GCLLSG/FCLLSG (NCT01392079). This trial encompassed CLL patients (pts) with TP53 alteration (deletion/mutation, TP53^alt; treatment-naïve or purine-analogue relapsed/refractory) and TP53^intact purine-analogue refractory disease. CBA and TP53 mutation data from the CLL11 trial of the GCLLSG (NCT01010061, N=152) was integrated for multiple linear regression.

Results: An informative karyotype was obtained in 125/131 pts (for clinical/genetic features see Table). The total number of CBA-detected CAs was 618 with an average of 4.9 CAs per case (range 0-21). Pts with TP53^alt had a significantly higher karyotype complexity than pts without (5.3 vs 2.7 CAs mean, p=0.02). A normal karyotype was found in 2 pts, a non-CK in 32 pts (≤2 CAs; 20 pts TP53^alt), a low- or intermediate-CK in 34 pts (3 or 4 CAs, 30 pts TP53^alt), and a high-CK in57 pts (≥5CAs, 54 pts TP53^alt).

Cytogenetic aberrations comprised 341 derivative chromosomes, 131 interstitial/terminal losses, 65 monosomies, 53 balanced translocations, 21 trisomies, 5 inversions, and 2 interstitial gains. Remarkably, 308/618 CAs had at least one breakpoint in the centromere (p10/q10; N=70) or in p11/q11 (N=238) directing our interest towards (peri-)centromeric breakage. Chr17 was most frequently affected linking (peri-)centromeric breakage to TP53 loss. Whole arm translocations (WAT) constituted the most common cause for TP53 loss (N=47 in 39 pts) with chr18 (N=9), chr17 (N=6), chr8 (N=6) and chr15 (N=6) being the most frequent translocation partners. Other cytogenetic events underlying TP53 loss were complex unbalanced rearrangements (N=40 in 39 pts), simple interstitial/terminal deletion (N=22 in 22 pts) and dicentric chromosomes (DIC, N=8 in 8 pts, 6 with breakpoints in p11/q11). In total, 98/117 aberrations causing TP53 loss had breakpoints in (peri-)centromeric cytobands. Mapping chr17 breakpoints based on SNP-array results revealed that more than half (58%) clustered within a 1 Mb region on both sides of the centromere. In contrast, chr17 breakpoints in CK-AML are random suggesting that chr17 (peri-)centromeric instability is specific for high-risk CLL.

(Peri-)centromeric rearrangements in form of WAT and DIC were also found to occur without chr17 involvement (N=24 in 20 pts and N=12 in 8 pts, respectively). Chromosome involvement was random pointing towards general (peri-)centromeric instability. Multiple linear regression revealed only karyotype complexity but not TP53^alt or prior treatment as significant risk factor for WAT/DIC substantiating the notion that (peri-)centromeric instability is an intrinsic hallmark of high-risk CLL.

DNA methylation patterns in regions adjacent to pericentromeric breakpoint clusters located on chr17p and chr18p showed no abnormalities when compared to cases without chr17/chr18 aberrations. Telomere length was significantly shorter in cases harboring at least one (peri-)centromeric rearrangement (p<0.001), but this association could not be confirmed when restricting the analysis to non-CK cases (N=20 vs 13, p=0.08).

Conclusions: Karyotype complexity was confirmed as typical feature of high-risk CLL. (Peri-)centromeric aberrations showed up as new hallmark characteristic of CK-CLL and WAT/DIC as otherwise rare events in hematologic malignancies were frequently observed. The underlying reasons for (peri-)centromeric breakage and its prognostic impact beyond karyotype complexity and TP53 alterations require further investigation.

No relevant conflicts of interest to declare.