Expression of IDO (indoleamine 2,3-dioxygenase) In Blasts of Acute Myeloid Leukemia Patients Predicts Poor Clinical Outcome

Indoleamine 2,3-dioxygenase (IDO) is endowed with intense immunomodulatory effects due to its enzymatic activities that catalyse the breakdown of the essential amino acid L-tryptophan. Recently, blasts of patients with acute myeloid leukemia were shown to express IDO. We determined IDO mRNA expression in leukemic blasts of patients with acute myeloid leukemia (AML) by reverse transcriptase PCR.

After informed consent and according to the recommendations as defined in the declaration of Helsinki, bone marrow derived samples were collected from patients with AML. Patients were classified according to the French-American-British (FAB) classification. We investigated 37 patients between December 2000 and March 2007 who were diagnosed with AML. All follow-up data were updated on July 1, 2010. Bone marrow derived mononuclear cell fractions from patients with AML were obtained by Ficoll centrifugation. For reverse transcriptase PCR, RNA was isolated using RNA-Bee solution (Tel-Test Inc, Friendswood, TX, USA). Total RNA was stored at -80°C. cDNA synthesis was performed. PCR amplification was performed with a LightCycler real-time PCR machine (Roche Diagnostics, Almere, the Netherlands). Reaction volumes were 20 μ L, consisting of 2 μ L cDNA, 2 μ L of LightCycler Fast Start DNA SYBR Green Mastermix (Roche) and 0.5 μ M reverse and forward primers. MgCl2 was added to a final concentration of 3.5 μ M. qPCR conditions consisted of an initial denaturation step at 95°C for 10 min, followed by 45 cycles, each for 15 sec at 95°C, 10 sec at 58°C and 10 sec at 72°C. Primer sequences were: IDO forward: 5’-GTGTTTCACCAAATCCACGA-3’, reverse: 5’-CTGATAGCTGGGGGTTGC-3’; (Nijmegen, the Netherlands). To determine associations between variables, Spearman's correlation coefficient was used. Differences between patients’ characteristics were analyzed with the Mann-Whitney U test. The effects of several pretreatment characteristics including IDO expression upon survival were examined by univariate analyses using the Kaplan-Meier method and the log-rank test. P values of <0.05 indicated significance.

Thirty seven patients were analyzed for the expression of IDO by reverse transcriptase PCR. We confirmed that 17 patients with IDO mRNA expression and 19 patients were without IDO mRNA expression by reverse transcriptase PCR. No significant differences were found between IDO mRNA expression of different FAB subtypes and cytogenetic risk profiles. We found no significant correlation between IDO mRNA expression and age, gender, or white blood cell counts. The 5-year OS rates for patients with IDO mRNA expression and without IDO mRNA expression were 64% and 41%, respectively (P <0.05). Other significantly worse factors were not found.

IDO mRNA expression was correlated to significantly shortened overall survival. Inhibition of IDO expressed by AML blasts may result in breaking immune tolerance and offers new therapeutic options for patients with acute myeloid leukemia. IDO mRNA expression might be a significant prognostic factor and a useful tool for selecting appropriate therapeutic strategies for patients with AML.

No relevant conflicts of interest to declare.