Sequential Evolution Versus a Single Catastrophic Event (Chromothripsis) in the Pathogenesis of AML with Complex Aberrant Karyotype

In AML, the concept of gradual evolution through a sequence of genetic alterations and clonal expansion was favored thus far, but has been recently challenged by a hypothesis that one catastrophic event generates multiple lesions across the genome in a single step. The term “chromothripsis” was introduced for a single catastophic event leading to the shattering of a single chromosome followed by rejoining and thereby resulting in a highly recombined chromosome (Stephens PJ et al., Cell 2011).

We addressed the question whether AML with complex karyotype - defined as 4 or more abnormalities - evolves by sequential gradual acquisition of chromosome abnormalities or by a single catastrophic event.

We selected 889 AML cases (de novo: n=634, secondary AML: n=164, therapy-related AML: n=91) presenting a complex karyotype at diagnosis. These were analyzed by chromosome banding analysis, 24-color-FISH, interphase-FISH, array CGH (n=78) and TP53 mutation analysis (n=195).

In 518/889 (58.3%) cases at least one subclone was observed that showed extra chromosome aberrations, thus demonstrating clonal evolution already at the timepoint of AML diagnosis. Within these, 77/518 (14.9%) cases showed a primary clone with only one cytogenetic abnormality. Two of these were recurrent single abnormalities: del(5q) (n=62), +8 (n=4). Clonal evolution was more frequent in cases with del(5q) as compared to those without (404/666 (60.7%) vs 117/223 (52.5%); p=0.034) while no association with loss of 7q, loss of 17p, TP53 mutation or type of AML (de novo vs secondary vs therapy-related AML) was observed. In 46 cases which evolved from MDS (n=43) or MPN (n=3) chromosome banding analysis had been performed prior to the diagnosis of AML. In 21/46 (45.7%) cases karyotype had not changed while in 25/46 (54.3%) cases clonal evolution had occurred. 57 cases were analyzed at relapse of AML; in 28 (49.1%) cases clonal evolution was detected.

Additionally, 78/889 cases were evaluated by array CGH. The occurrence of chromothripsis was analyzed following the definition by Rausch et al. (Cell 2012) with at least 10 segmental copy-number changes involving two or three distinct copy-number states on a single chromosome. Evidence of at least one “shattered” chromosome was found in 24/78 (30.8%) cases. In 21 cases only one chromosome fulfilled these criteria, while in 3 cases chromothripsis affected two or more chromosomes. The chromosome most frequently affected by “shattering” was chromosome 11, observed in 18 (85.7%) cases, followed by chromosomes 2 and 21, which were affected in 2 cases each. Chromosomes 1, 5, 7, 13, 15, 16 and 20 showed signs of chromothripsis in single cases only. In 19/24 (79.2%) cases showing evidence of chromothripsis a high level amplification was observed for the MLL gene (11q23) in 17 cases and for the ERG gene (21q22) in 2 cases. Thus, amplifications were more frequent than in cases without chromothripsis (21/54, 38.89%; p=0.001), while no association was observed between chromothripsis and deletions of 5q, 7q or 17p or TP53 mut, presence of clonal evolution or type of AML (de novo vs secondary vs therapy-related AML). With respect to outcome within the subgroup of AML with complex karyotype only TP53 mutations and the presence of 5q deletions were significantly associated with overall survival (relative risk (RR) for shorter OS in TP53 mut cases: 3.19, p<0.0001, and in del(5q) cases: 1.61, p=0.006; median OS in TP53mut vs TP53wt cases: 4.6 vs 22.0 months, p<0.0001; median OS in del(5q) vs non-del(5q) cases: 5.7 vs 14.4 months, p=0.006), while presence of deletions of 7q, or 17p, chromothripsis and clonal evolution showed no impact on outcome. In multivariable Cox regression analysis only TP53mut had an independent association with shorter OS (RR: 3.12, p=0.001).

1. In AML with complex karyotype harboring a 5q deletion the acquisition of additional abnormalities is more frequent than in cases without del(5q). 2. Chromothripsis does occur in AML with complex karyotype. However, the “shattering” of one chromosome was never observed as the sole abnormality, indicating that chromothripsis and sequential genetic evolution are not alternative but more likely combined mechanisms in AML. 3. Our data demonstrates that stepwise clonal evolution is more frequent than chromothripsis and thus likely to be more important in the pathogenesis of AML with complex karyotype.

Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.