Gene Amplifications Are Rare Events in AML and MDS and Are Associated with Complex Karyotype, TP53 Deletions and Very Poor Survival

Gene amplifications are usually defined as the presence of more than 6 copies of a gene per cell. These supernumerary copies are located either extrachromosomally in double minutes (small acentric chromosome structures) or intrachromosomally in homogeneously staining regions. Such gene amplifications are rare but recurrent phenomenons in AML and MDS. So far, only small case studies have been reported. Aims: 1) to determine the frequency of gene amplifications in a large AML and MDS cohort, 2) to characterize the amplified regions and accompanying abnormalities, 3) to analyze the impact of specific amplifications on outcome. Patients and Methods: Out of 4,248 AML and 3,689 MDS studied by chromosome banding analysis (CBA) we identified 105 AML patients (2.5%) with gene amplifications (80/3,478 (2.3%) de novo AML, 7/478 (1.5%) s-AML, 18/292 (6.2%) t-AML) and 46 (1.2%) MDS. All cases with gene amplification were studied by 24-color FISH in addition to CBA in order to characterize the amplified regions and the accompanying abnormalities in detail. Further, interphase (IP)-FISH was performed with probes for TP73, HOXD cluster, EVI1, CMYC, JAK2, NUP214, MLL, ZNF4, GLTSCR1, ERG, RUNX1, BCR and CLRF, if 24-color FISH suggested amplification of these genes. In a subcohort of 12 patients genomic arrays (Human CGH Whole-Genome Array, NimbleGen, Madison, WI; Genome-Wide Human SNP Array 6.0, Affymetrix, Santa Clara, CA) were performed to characterize the amplified region in more detail. Results: In 28/151 pts (18.5%) the amplification was located in double minutes and in the remaining 123 cases intrachromosomally (81.5%). The following regions were found to be amplified: 1p (n=1, containing TP73), 2q (n=1, containing HOXD cluster), 3q (n=1, containing EVI1), 7p (n=1), 8q (n=29, containing CMYC in 28/29 pts), 9p (n=2, containing JAK2), 9q (n=1, containing NUP214), 11q (n=81, containing MLL in 80/81 cases), 13q (n=2), chromosome 19 (n=10, containing ZNF4 in 5 cases and GLTSCR1 in 2 cases), 21q (n=19, containing ERG in 16 and RUNX1 in 6 cases), 22q (n=2, containing BCR) and Xp (n=1, containing CLRF). In median, 8 accompanying chromosomal aberrations per cases were observed (range 0–21). 124/151 (82.1%) cases had a complex aberrant karyotype, defined as 4 or more abnormalities. However, in 2 cases the double minutes were the sole abnormalities. Gene amplifications were not observed in patients with disease defining aberration like t(8;21), inv(16), t(15;17) or those carrying NPM1 or CEPBA mutations (mutation status available in 89 and 37 patients, respectively). However, 2 cases with t(6;11)(q27;q23)/MLL-AF6 harbored an amplification of CMYC. In 88 cases the copy number status of TP53 was determined by IP-FISH. A TP53 deletion was detected in 49 (55.7%) pts. Interestingly, 14/16 (87.5%) cases with double minutes compared to 35/72 (48.6%) patients with intrachromosomal gene amplifications showed a TP53 deletion (p=0.004). Only 3 chromosomal regions were amplified in double minutes: 8q24/CMYC (n=14), 11q23/MLL (n=12) and 13q (n=2). In 6 cases with 8q amplification, 2 cases with 11q amplification and 4 cases with 21q amplification genomic arrays were performed. While the amplified region was quite homogeneous in cases with 8q amplification and contained in all cases the CMYC gene, amplified regions on 11q and 21q were heterogeneous and amplified regions were interspersed with regions of deletions. Interestingly, MLL and CBL were amplified in all analyzed cases with 11q23 amplification. In all analyzed cases with 21q22 amplification ERG was located within the amplified region while RUNX1 was amplified in 3/4 cases and deleted in the remaining case. In AML, overall survival was short in cases with gene amplification (median OS 11.3 months) and was particularly short in cases accompanied by complex karyotype (6.3 mo vs 18.6 mo in cases with non-complex karyotype, p=0.049). Conclusions: 1) MLL is the most frequently amplified gene in AML and MDS. 2) Gene amplifications occur predominantly in complex aberrant karyotypes. 3) Prognosis is poor in this subset of cases, and even more dismal if these amplifications are accompanied by complex karyotype. 4) The association of gene amplifications, complex karyotypes and TP53 deletions suggests that the unfavorable prognosis is due to chromosome instability facilitating the occurrence of additional genetic aberrations triggering resistance to chemotherapy.

Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann: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.