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Acute myeloid leukemia (AML) is a heterogeneous disease. Risk factors such as karyotype, FAB subtype, FLT3 status and response to induction therapy are determinants of outcome with current therapies. We hypothesize that array comparative genomic hybridization (CGH) will identify gene copy number changes that are determinants of outcome. Array CGH was performed on diagnostic bone marrow samples from patients on the COG study POG #9421. In order to determine regions of altered gene copy number, labeled genomic DNA samples were hybridized together with sex-matching normal human reference DNA to cDNA microarrays with 41,751 features (corresponding to 24,473 unique Unigene cluster IDs), arrays were obtained from the Stanford University Microarray Core Facility. Control hybridizations were performed to assess intra- and inter-experimental variability. We studied 70 samples with adequate high-quality DNA. Circular binary segmentation was used to distinguish discrete gene copy number transition points from chance noise events and to transform primary clone-by-clone data into genomic regions of equal copy number. Using gain/loss threshold, based on two-standard deviation range of control self-to-self distribution, novel gene amplifications and deletions were found in profiled samples. The highest alteration recurrence was observed for gains of chromosome 8 (21%) and losses of chromosome 6 (29%). The area of chromosome 8 which was found to be gained is notable for the presence of potential oncogenes such as ERK8. The deleted area of chromosome 6 is notable for the presence of potential regulators of oncogenesis: MDC1, DDR1, NFKBIL1, TNF, and BRD2. In summary, array CGH has identified novel areas of gene copy number gain and loss in this population of pediatric de novoAML patients. Further studies are needed to assess whether these genes are associated with outcome, known risk factors and whether they will provide insight into the heterogeneity of de novoAML.

The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;21), inv(16) and t(15;17) are currently used to make therapeutic decisions. Additionally, specific mutations such as those involving the FLT3 gene are of prognostic importance as they indicate patients likely to relapse early or fail initial induction therapy. Approximately 20% of children with AML have normal karyotypes at diagnosis and no identifiable chromosomal abnormality using standard methods of analysis. In this subgroup there is an increased incidence of FLT3 mutations (internal tandem duplications or point mutations). We observed that patients with normal karyotypes who were enrolled in the Pediatric Oncology Group (POG) study #9421 had two significantly different clinical outcomes that were associated with the expression of FLT3 mutations. We hypothesized that gene expression profiles would identify genes that cooperate with FLT3 mutations in conferring poor clinical outcome.

Daunorubicin and cytarabine are the most effective chemotherapeutic agents for AML. Using MTT (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays as an in vitrotest for sensitivity to these agents, investigators have found that leukemia cells from pediatric AML patients with Down Syndrome, which are more sensitive to cytarabine than blasts from non-Down Syndrome AML patients, have been associated with an increased ratio of deoxycytidine kinase to cytidine deaminase and an increased level of cystathionine-beta-synthetase [Ge, Jensen, Stout et al. Cancer Res 2004; 64, 728–35]. Looking for related findings in non-Down syndrome AML, leukemic blasts from non-Down syndrome children with AML were examined utilizing gene expression profiling and MTT assays. Expression signatures from cDNA arrays were related to the drug sensitivity results to daunorubicin and cytarabine by supervised clustering. These results will then be analyzed to look for relationships to the patient's clinical outcomes and identify genes that may be potential therapeutic targets for drug resistance reversal.