Gene Amplifications In 84 Patients With Acute Myeloid Leukemia and 31 Patients With Myelodysplastic Syndrome Investigated By Array CGH

Gains and losses of chromosomal material are frequent in AML and MDS and usually lead to loss or gain of a single copy of a whole chromosome, a chromosome arm or small stretches of the chromosome that may be microscopically invisible. More rarely, amplifications of chromosomal regions (defined as the presence of more than 6 copies of a region per cell) are observed. These supernumerary copies are located either extrachromosomally as small acentric chromosomal structures - so called double-minutes (dmin) - or intrachromosomally as large contiguous stretches of amplified DNA, so called homogeneously staining regions (HSR).

Characterize AML and MDS cases with gene amplifications with respect to size, affected genes and accompanying chromosomal abnormalities as well as TP53 status.

84 AML and 31 MDS cases with cytogenetically visible amplifications were selected for this study. All cases were analyzed by array CGH, chromosome banding analysis, sequencing for TP53 mutations as well as FISH for TP53 deletions.

The cohort comprised 55 (47.8%) males and 60 (52.2%) females with a median age of 72.0 years (range 38.0 - 90.3 years). A complex karyotype (≥4 aberrations) was present in 92/115 (80.0%) cases (AML=65/84 (77.4%); MDS=27/31 (87.1%)). In total, 385 amplified regions were identified by array CGH. In more detail: 3q26 (AML: n=6; MDS: n=3), 8q24 (AML: n=15; MDS: n=1), 11q21-25 (AML: n=42; MDS: n=13), 13q12 (AML: n=3; MDS: n=1), 13q31 (AML: n=3; MDS: n=2), 19p13 (AML: n=2; MDS: n=4), and 21q21-q22 (AML: n=24; MDS: n=5). The median number of amplified regions was 3 (range 1-18). In 14/115 (12.2%) cases, the amplification was located in dmins (AML: n=11; MDS: n=3) and in 101/115 (87.8%) patients in HSR (AML: n=73; MDS: n=28). In 40 of the latter 101 cases (39.6%) (AML: n=24; MDS: n=16) the amplification was located on a ring chromosome (rc). In patients with complex karyotypes we detected a significantly higher number of amplified regions as compared to non-complex karyotypes (3.5 vs. 2.8; p=0.015). No association between the complexity of the karyotype and the structural type of the amplification (dmin vs rc) was observed. Cases with non-complex karyotypes frequently harbored a 5q deletion (6/23; 26.1%) or chromosome 8 abnormalities (3/23; 13.0%). Within the subgroup of non-complex karyotypes del(5q) cases showed a tendency to a higher number of amplified regions (3.6 vs. 1.9; p=0.140). Further, amplifications of 11q genes were more frequent in complex karyotypes (54.4% vs. 21.7%; p=0.005), whereas 8q amplifications were more frequent in non-complex karyotypes (43.5% vs. 4.4%; p<0.001). We detected a large region on band 11q24, which was amplified in 41/53 (77.4%) cases. This commonly amplified region contains 1,575 genes including the MLL gene. Cases harboring dmins had shorter amplified regions compared to cases with rc (4,428,112.5 bp vs. 18,265,496.9 bp; p=0.028). Moreover, we detected a positive correlation of patients having a rc and gene amplification on chromosome 11q23-25 (p<0.05). On chromosome 3q, 8/9 (88.9%) cases shared a minimal amplified region covering the EVI1 gene. In comparison to samples obtained from healthy donors (n=47), the EVI1 expression was significantly higher in cases with EVI1 amplification (87.4 vs. 0.5; p=0.048). On chromosome 21q the regions of amplifications were heterogeneous. However, we detected a minimal region containing 11 genes including ERG which was amplified in 26/29 (89.7%) patients. ERG expression data was available in 8 cases and was significantly higher compared to a control cohort of AML with normal karyotype (n=331) (729.2 vs. 229.0; p=0.05). On chromosome 8 an amplified region was identified in 15/16 cases. In 14 of these cases (87.5%) the region included MYC. TP53mut were present in 93/115 (80.9%) patients, accompanied by a TP53del in 28/93 (30.1%) cases. Interestingly, cases harboring a TP53mut had more amplified regions compared to TP53wt (3.4 vs. 1.7; p<0.001).

1. MLL is the most frequently amplified gene in AML and MDS. 2. Patients with complex karyotypes or TP53mut harbored more amplified regions compared to patients with non-complex karyotypes and TP53wt. 3. Amplifications on 11q were more frequent in complex karyotype whereas gene amplifications on 8q were predominantly observed in non-complex karyotypes. 4. EVI1 and ERG gene amplifications lead to a higher expression of the respective genes.

Roller:MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.