Abstract

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 vitro test 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.

Bone marrow or blood specimens from 103 patients registered on the Pediatric Oncology Group (POG) protocol 9421 were studied. The diagnostic specimens were tested using MTT assays to measure the IC50 (drug concentration that causes 50% of the cells to die) for both daunorubicin and cytarabine. Samples from ninety-three de novo childhood AML patients were analyzed with a 43,760 element spotted array (containing 41,751 unique genes and expression sequence tags [ESTs]) from the Stanford University Microarray Core Facility. We selected the gene expression profile from 10 samples with the highest and lowest IC50’s for each drug. Then, we used Significance Analysis of Microarrays (SAM) to find significant differences in the gene expression between samples sensitive and resistant to each drug. A cluster of 118 overexpressed genes was associated with resistance to daunorubicin (e.g., BCL6, BAPX1, BCL2A1, MBD2, RAB31 and CDKN1A). A cluster of 24 overexpressed genes was associated with resistance to cytarabine (e.g., RIT1, SH3BP2, BCL2A1, NFKBIA, and PTPRE).

In summary, MTT assays and gene expression profiling have identified genes that correlate with daunorubicin and cytarabine resistance. Study of the relationship among drug resistance patterns, gene expression profiles and clinical outcome may allow us to better predict patients’ prognoses at diagnosis and also can be used to identify novel targets for drug resistance reversal.

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