A Breakage-Fusion-Bridge Cycle Generated by Telomeric Loss May Underlie the dup(21q) of Acute Lymphoblastic Leukemia.

Abstract

A highly complex, rearranged chromosome 21, arising from duplication of 21q and associated with amplification of RUNX1 [dup(21q)], is linked to a poor prognosis in acute lymphoblastic leukemia (ALL). Using BAC array-based CGH (Spectral Genomics 1Mb, USA) (aCGH), we originally identified a characteristic pattern of imbalance, with common regions of amplification (CRA) and deletion (CRD) along 21q, in a series of 10 patients. The extent of these regions was refined to 6.6Mb (between 33.192 and 39.796Mb) and 3.3Mb (between 43.7 and 47Mb, which included sub-telomeric sequences), respectively, by tiling-path oligonucleotide-based aCGH (NimbleGen Inc., USA) (n=15). Six BAC clones (including a sub-telomeric one) from the 1Mb arrays, corresponding to the variable regions of amplification along 21q were used as probes for interphase FISH (iFISH) in 48 patients. The same CRA was confirmed in all, while the CRD was observed in 34/46 (77%) of them. iFISH showed that the degree of amplification corresponding to each chromosomal region differed between patients. Although these techniques, which measure genomic copy number changes, showed consistent patterns of amplification and deletion along 21q, it was intriguing as to why, at the cytogenetic level, the abnormal chromosome 21 had many different forms. The same locus-specific probes, applied to metaphases from 10 patients, revealed complex signal patterns, unique to each patient, in which the signals from each probe were distributed along 21q in unexpected positions relative to each other. Often signals from the same probe were located to more than one region of the abnormal chromosome 21. These findings were indicative of duplications of the chromosomal regions to which the probes were located, in combination with intricate intrachromosomal rearrangements, including inversions. We were able to illustrate these unique rearrangements by multicolor FISH banding (XCyte21, Metasystems, Germany) in two cases. Supervised gene expression analysis (HG-U133A arrays, Affymetrix, USA) showed a distinct signature for eight patients with dup(21q). Genomic copy number correlated with overall gene expression levels within areas of gain or loss. However, there was considerable inter-genic differences and variation between individuals. Of the 40 genes contained within the amplicon, no promising targets were upregulated when compared to patients with high hyperdiploidy, comprising at least one additional copy of chromosome 21. Collectively, our results have provided evidence for extensive intrachromosomal rearrangements and instabilityn of 21q in these patients. Although there was no evidence of other established chromosomal changes, the clonal heterogeneity we observed in the karyotypes was indicative of genome-wide chromosomal instability. These types of genomic alterations, arising from a series of chromatid breaks and reunions, which lead to intrachromosomal amplifications and deletions, are the hallmark of the breakage-fusion-bridge (BFB) cycle in solid tumors. This is the first time that cytogenetic features linked to the BFB mechanism have been described in ALL. We hypothesize that the loss or abnormal functioning of telomeric sequences may be the causal event behind this poor-risk 21q abnormality.