Tryptophan Catabolism Is Associated with the Prognosis of Patients with De Novo Acute Myeloid Leukemia,

Indoleamine 2,3-dioxygenase (IDO) exerts immunomodulatory effects due to enzymatic activities catalyzing the essential amino acid L-tryptophan. IDO activity might play an important role in regulating immune responses exerted by antigen-presenting cells. We have recently been able to clarify the utility of either serum L-kynurenine or tissue IDO expression as prognostic factors for DLBCL patients treated using R-CHOP. In addition, blasts of patients with acute myeloid leukemia (AML) were shown to express IDO. In the present study, we examined serum l-kynurenine levels and IDO mRNA expression in leukemic blasts of patients with AML by rtPCR.

The study protocol used a retrospective case series design that was approved by our institutional review board. We investigated data from 35 patients between December 2000 and March 2011 who were histologically diagnosed with AML according to the WHO classification. All follow-up data were updated on June 1, 2011. After informed consent and according to the recommendations as defined in the declaration of Helsinki, serum and bone marrow derived samples were collected from patients with AML. All serum samples were obtained at admission, separated by low-speed centrifugation (800 g, 15 min) at 4°C and stored at -20°C until analysis. We measured serum L-kynurenine concentrations by high-performance liquid chromatography (HPLC) using a spectrophotometric as described. Bone marrow was obtained from every patient by marrow aspiration before initiation of therapy. Bone marrow derived mononuclear cell fractions were obtained by Ficoll centrifugation and were stored. 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 μL, consisting of 2 μL cDNA, 2 μL of LightCycler Fast Start DNA SYBR Green Mastermix (Roche) and 0.5 μM reverse and forward primers. MgCl2 was added to a final concentration of 3.5 μM. 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)

According to WHO classification, the subtype of underlying comprised AML with recurrent genetic abnormalities (n = 13), AML with myeloidysplasia-related changes (n = 3), and AML, not otherwise specified (n = 19). The median serum L-kynurenine level was 1.79 μM (range 0.78 – 5.19). According to FAB classification, the subtype of underlying comprised M0 (n = 1), M1 (n = 2), M2 (n = 9), M3 (n = 10), M4 (n = 8), M5 (n = 2), M6 (n = 3), and M7 (n = 0). The median serum L-kynurenine level was 1.79 μM (range 0.78 – 5.19).The 5-year overall survival (OS) rates for patients with L-kynurenine <2.0 and ≥2.0 μM were 68% and 11%, respectively (P < 0.05). XX patients were analyzed for the expression of IDO by reverse transcriptase PCR. We confirmed that xx patients with IDO mRNA expression and 19 patients were without IDO mRNA expression. The 5-year OS rates for patients with IDO mRNA expression and without IDO mRNA expression were 25% and 56%, respectively (P <0.05).

Serum L-kynurenine and IDO mRNA expression might be novel prognostic factors to determine the treatment outcome of AML. Inhibition of IDO expressed by AML blasts may result in breaking immune tolerance and offers new therapeutic options for patients with AML. IDO might thus represent a candidate therapeutic target for AML patients who show resistance to chemotherapy. Since these results are based on a small sized retrospective analysis, further investigation is required.

No relevant conflicts of interest to declare.