High-Resolution Genome-Wide Profiling of DNA Copy Number Abnormalities and Gene Resequencing in Pediatric Acute Myeloid Leukemia (AML).
Authors: Radtke Ina , Mullighan Charles G. , Ishii Masami , Ma Jing , Su Xiaoping , Goorha Salil , Ganti Ramapriya , Cai Zhongling , Phillips Letha A. , Pounds Stanley B. , Dalton James , Raimondi Susana C. , Rubnitz Jeffrey E. , Ribeiro Raul C. , Shurtleff Sheila A. , Downing James R. 
Published: October 2019

Abstract
To define the complement of genetic lesions in pediatric AML, we performed high resolution genome-wide analysis on bone marrow blasts from 111 cases using Affymetrix 100K and 500K SNP microarrays and genomic resequencing. Cases included t(8;21) (N=20), inv(16) (N=16), t(15;17) (N=7), MLL rearranged (N=16), FAB-M7 (N=9), miscellaneous cytogenetic abnormalities (N=24) and normal karyotype (N=19). Germline DNA was available for 67 of the cases. The mean number of somatic copy number abnormalities (CNA) was 2.38 (range 0–45) with 1.32 gains (range 0–41) and 1.06 losses (range 0–12). Focal CNAs were detected at the breakpoints of known chromosomal translocations, most commonly in inv(16) cases (7/16) and t(8;21) cases (4/20). Based on this observation, we examined focal CNAs that affected the 5’ or 3’ regions of genes for their possible involvement in cryptic translocations. CNAs involving NUP98 and MLL led to the identification of two cases with t(5;11)[NUP98-NSD1], and two with t(6;11)[MLL-MLLT4] not evident on cytogenetic analysis. RT-PCR for these fusions identified two additional NUP98-NSD1 cases. Identification of focal CNAs are thus useful in identifying clinically significant translocations in AML. Other recurring somatic CNAs were uncommon. Twelve regions containing a single gene were identified, including amplification of EBF, PRDM5, CCDC26*, GDF10, ABCC4*, and deletion of FAM20C*, TUSC1, GPC5, A2BP1, INSR, BCOR* (* involved in N>2 cases). The most frequent abnormality was amplification of CCDC26 at 8q24.21 (N=15; focal in 4 cases, broad in 11) which encodes a putative mediator of retinoic acid receptor signaling. The focal amplifications are predicted to abolish expression of normal transcripts. Seventeen additional recurring CNAs involving 2 to 25 genes were identified, many of which contain known or putative tumor suppressor genes or oncogenes, including deletions of regions at 7q36, 9p21 (CDKN2A/B), 11p14-p12 (WT1, WIT1), and amplification at 19p13 (JUNB, LYL1). These regions showed significant correlation between copy number and local gene expression in an integrated analysis of CNA and Affymetrix U133A gene expression data. Copy-neutral LOH was uncommon and recurrent only with LOH of chromosome 13 in 3 cases. Sequence analysis has been completed for several genes involved in recurring CNAs (CCDC26, TUSC1, FBXW7) and for genes known to be mutated in AML (N/KRAS, PTPN11, BRAF, SOS1, FLT3, CEPBA, NPM1, AML1, CKIT, GATA1). No mutations were identified in CCDC26, TUSC1, FBXW7, BRAF, or SOS1. Marked differences in the combination of CNA and sequence mutation were identified across the different genetic subtypes of AML. FAB M7 cases had the highest frequency of CNA (mean 9.3 lesions/case) but had sequence mutations limited to GATA1. Mutations of FLT3, CEPBA and NPM1 were most frequent in cases with no or miscellaneous cytogenetic abnormalities, where as RAS mutations were most frequent in t(8;21) and inv(16) cases. Importantly, approximately 30% of the cases with recurring translocations had no other sequence or numerical abnormalities. These data demonstrated that, in contrast to pediatric ALL, AML is characterized by relatively few recurring CNAs, and that spectrum of CNA and sequence mutation is significantly associated with disease subtype.