The single catastrophic event chromothripsis, initially uncovered in a case of chronic lymphocytic leukemia by whole genome sequencing,1 is a novel phenomenon of genomic instability. It is characterized by extensive genomic rearrangements reflected by an oscillating pattern of DNA copy-number levels in one or few chromosomes as assessed by e.g., single-nucleotide polymorphism (SNP) microarray analysis or next-generation sequencing (NGS) techniques.21 The chromosome shattering and subsequent erroneous repair mechanisms lead to the formation of highly derivative chromosomes with losses of tumor suppressor genes, activation of oncogenes, and/or generation of novel fusion genes; thus, chromothripsis is thought to promote and perhaps even to cause cancer development. The prevalence of chromothripsis is very heterogeneous across various tumor entities, but on average it can be found in approximately 2–3% of all cancers with up to 25% in bone cancers.1 While the underlying mechanisms resulting in chromothripsis are still largely unknown, previous reports have associated chromothripsis with TP53 mutations in subsets of medulloblastoma and acute myeloid leukemia (AML), however, several additional mechanisms have been proposed, including DNA damage in micronuclei, premature chromosome condensation, breakage-fusion-bridge cycles, and telomere dysfunction.42 In AML, TP53 alterations (loss and/or mutation) are almost exclusively found in cases with complex karyotype (CK) AML and define a subset of patients with distinct genomic alterations and dismal outcome.65
To gain further insight into the role of chromothripsis in AML, we performed an integrative analysis consisting of genomic profiling (Affymetrix GeneChip Human Mapping 250K Array (n=61), Genome-Wide Human SNP Array 6.0 profiling (n=51) [GEO accession number GSE 34542]) and TP53 mutation screening in 112 CK-AML patients.75 The findings were correlated with clinical and genetic data of all CK-AML, whereas outcome analysis was restricted to 62 patients treated with intensive chemotherapy. In addition, global gene expression profiling (GEP) was performed in a subset of cases (Affymetrix GeneChip Human Exon 1.0 ST Array (n=27) [GEO accession number GSE 21337]).
SNP profiling identified a total of 67 rearrangement patterns in 39 out of 112 (35%) CK-AML patients, consistent with chromothripsis defined by the presence of at least ten switches between two or three copy-number states on an individual chromosome.2 In comparison to recent reports,8 the incidence of 35% rearrangement patterns in CK-AML is higher than in other tumor entities. Fifteen of the 39 CK-AML patients exhibited patterns of chromothripsis on up to six different chromosomes (chr; on 2 chr (n=8), on 3 chr (n=4), on 4 to 6 chr [n=1 each]). While chromothripsis was seen in almost all chromosomes, it most frequently affected chr 7 (n=7; 10%) followed by chr 3 and 12 (n=6; 9% each), chr 5, 16, and 17 (n=5; 7% each), chr 6 and 21 (n=4; 6% each), chr 4, 8, 11, and 18 (n=3; 4% each), chr 1, 10, 15, and 19 (n=2; 3% each), and chr 2, 9, 14, 20, 22 (n=1; 2% each) (Figure 1A). CK-AMLs with chromothripsis were characterized by a higher total number of losses (mean±SD; 12.3±8.1 versus 7.0±7.9, P<0.001), gains (5.6±4.0 versus 3.2±4.0, P=0.004), high-level DNA amplifications (1.1±1.7 versus 0.2±0.6, P<0.001), and by a higher general degree of genomic complexity, as measured by total number of copy-number alterations per case (24.0±10.1 versus 10.5±9.5, P<0.001) (Table 1 and Figure 1B). Moreover, cases with chromothripsis were associated with specific genomic alterations, i.e., monosomy 5 or losses of 5q (-5/5q-) (P<0.001), -7/7q- (P=0.02), -12/12p- (P=0.01), - 16/16q- (P=0.01), -17/17p- (P=0.02), and “monosomal karyotype” (P<0.001). As previously reported, chromothripsis was also correlated with the presence of TP53 alteration (33/39 (85%) chromothripsis-positive CK-AML versus 39/73 (53%) chromothripsis-negative CK-AML, P=0.001; Table 1).
Clinically, chromothripsis-positive CK-AML patients were older (median age 61 (range: 19–82) versus 56 (range: 21–76) years, P=0.009) and showed lower bone marrow blast counts (median 40% versus 80%, P=0.01; Table 1). Chromothripsis was associated in trend with a lower CR rate (4/16 (25%) chromothripsis-positive versus 21/45 (47%) chromothripsis-negative CK-AML, P=0.15), but predicted for inferior survival. The 2-year estimated survival rates for chromothripsis-positive versus chromothripsis-negative patients were as follows: event-free survival (EFS), 0% versus 13% (log-rank; P<0.001), relapse-free survival (RFS), 0% versus 34% (P<0.008; see Table 1), and overall survival (OS), 0% versus 28% (P<0.001), respectively (Table 1 and Figure 1C). Other variables predicting for inferior OS and EFS in univariable analysis were age >60 years (P=0.001 and P=0.01, respectively), “monosomal karyotype” (P=0.004 and P=0.03, respectively), and TP53 alteration (P<0.001 and P=0.001, respectively). Within chromothripsis-positive CK-AML there was no correlation regarding clinical and genetic characteristics or outcome for any specific chromosome affected by chromothripsis.
Albeit chromothripsis was associated with TP53 alteration, which has been demonstrated to be the most important prognostic factor in CK-AML thus far, outweighing all other known prognostic variables in multivariable analysis,5 chromothripsis provides additional independent prognostic information. Multivariable analysis stratified anew for age >60 years revealed chromothripsis as the exclusive significant variable for OS and EFS (OS: hazard ratio (HR), 2.47; 95%- confidence interval (CI), 1.14 to 5.34, P=0.02; EFS: HR, 2.20; 95%-CI, 1.04 to 4.64, P=0.04). Age retained its significance for OS alone (HR, 3.01; 95%-CI, 1.49 to 6.11, P=0.002), whereas TP53 alteration and “monosomal karyotype” were not significant in the multivariable model in our study (Table 1).
To gain further molecular insight into mechanisms underlying chromothripsis, we performed GEP and analyzed data using iPathwayGuide. Comparing chromothripsis-positive (n=13) and –negative (n=14) CK-AML, GEP disclosed a distinct gene signature of 421 significantly differentially expressed genes at a nominal P-value <0.05 (the top 200 differentially expressed genes are listed in Online Supplementary Table S1). Among the top most deregulated genes in chromothripsis-positive CK-AML were CCNA1 (−2.9 log fold change, P<0.009; see Online Supplementary Table S1) involved in cell cycle control and proliferation9 and reflecting our observation of lower bone marrow blast counts in chromothripsis-positive CK-AML, PSMB10 (−1.7 log fold change, P<0.001), known to be critical for immune surveillance,10 and PIM1 (1.9 log fold change, P<0.002; see Online Supplementary Table S1), encoding a serine threonine kinase involved in FLT3-mediated cell survival,11 respectively (Figure 1D).
Pathway analysis using “Impact Analysis” revealed ten pathways significantly impacted in chromothripsis-positive CK-AML (Online Supplementary Table S2). These pathways are implicated in tumorigenesis, transformation, progression, and/or proliferation (e.g., “transcriptional misregulation in cancer”, “RNA degradation”, or “Notch signaling”) and have been linked to “acute myeloid leukemia”. In accordance, a gene set enrichment analysis (GSEA) using the Molecular Signatures Database (MSigDB)12 revealed similar results, in particular with regard to genes involved in RNA degradation or proliferation. Beyond that, GSEA disclosed significantly deregulated genes involved in genomic stability (e.g., “chromatin remodeling complex” (ARID1A, ASF1A), “cell cycle” (CCNA1, RAD52, AURKA), “negative regulation of cell cycle”, or “cell cycle arrest” [ZAK, HPGD, KAT25]) in chromothripsis-positive CK-AML (Online Supplementary Table S3).
As chromothripsis was significantly correlated with TP53 alteration, but not all TP53 CK-AML exhibited excessive genomic rearrangements, we performed an explorative subset analysis for chromothripsis associated genes within TP53 CK-AML to gain additional insight into the causal processes of chromothripsis. Comparing chromothripsis-positive (n=10) versus –negative (n=7) TP53 CK-AML, GEP disclosed a distinct chromothripsis-associated gene signature of 337 significantly differentially expressed genes at a nominal P-value <0.05 (the top 200 differentially expressed genes are listed in Online Supplementary Table S4). The top most deregulated genes in chromothripsis-positive TP53 CK-AML were, once more, PSMB10 (−1.8 log fold change, P<0.001) and BCL9 (1.4 log fold change, P<0.001), respectively (Figure 1D). BCL9 encodes the B-cell CLL/lymphoma 9 protein that is involved in signal transduction through the Wnt pathway and plays a role in B-cell malignancies and tumor progression of several human cancers, but has not been associated with myeloid malignancies as of yet. Pathway analysis pointed again to “transcriptional misregulation in cancer” and “RNA degradation” pathways. Moreover, chromothripsis-positive TP53 CK-AML also showed a significant deregulation of genes associated with genomic instability (e.g., “Fanconi anemia pathway genes” [ERCC1, FANCA, MLH1, and EME1]) and transcriptional regulation (e.g., CEBPA) (Figure 1D, Online Supplementary Table S5). Since some of the impacted pathways are linked to telomere dysfunction (e.g., “Fanconi anemia pathway”, “cell cycle”, or “RNA metabolic process”; Online Supplementary Table S3) which may (i) result in chromothripsis by telomere fusions leading to chromatin bridges13 and (ii) drive aberrant hematopoietic differentiation and myelodysplastic syndrome (MDS),14 our data support a role for telomere dysfunction as one of the molecular mechanisms underlying chromothripsis in CK-AML.
Next, the hypothesis that chromothripsis might promote and perhaps even cause oncogenesis was strengthened by sequential genomic profiling of four cases with antecedent MDS. SNP analysis revealed that the patterns of chromothripsis delineated in AML were already present at diagnosis of MDS, thereby supporting chromothripsis as an early event in myeloid leukemogenesis.
In summary, chromothripsis is a frequent genomic alteration in CK-AML and correlates with TP53 alteration, genomic instability, specific copy-number alterations, and “monosomal karyotype”. While further studies confirming these findings with state-of-the-art NGS technology are warranted,15 our SNP-based analysis nevertheless clearly shows that chromothripsis defines a subset of CK-AML cases with very dismal outcomes, and indeed outweighs TP53 alteration in multivariable analysis. Detailed molecular profiling links chromothripsis to altered TP53 in CK-AML and to an additional pathogenic role as, for instance, the deregulation of genes associated with telomere dysfunction, genomic instability (e.g., Fanconi anemia pathway genes), mitotic regulation (e.g., CCNA1), or transcriptional regulation (e.g., CEBPA). These findings also point to novel potential treatment targets such as PIM1 in this acutely high-risk AML.
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