Indoleamine 2,3-dioxygenase expression and serum kynurenine concentrations in patients with diffuse large B-cell lymphoma

We have recently been able to clarify the utility of either serum l -kynurenine or tissue indoleamine 2,3-dioxygenase (IDO) expression as a prognostic factor for patients with diffuse large B-cell lymphoma (DLBCL) treated with a combination of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) and rituximab (R-CHOP) [1,2]. Here, we examined the relationship between the serum level of l -kynurenine and tissue IDO expression in patients with DLBCL. Th e study protocol used a retrospective case series design that was approved by the institutional review board. We investigated 65 patients who had been histologically diagnosed with DLBCL according to the World Health Organization (WHO) classifi cation of hematopoietic tumors between December 2003 and November 2006. Patients in this study were required to have both serum and specimens available. All patients in this study had enrolled in both our kynurenine study [1] and IDO study [2]. Th e following information at the time of diagnosis was collected: age; sex; performance status (PS); clinical stage (CS); number of extranodal sites involved; serum lactate dehydrogenase (LDH) levels; presence or absence of B symptoms; serum soluble interleukin 2 receptor (sIL-2R); International Prognosis Index (IPI) score [3]; and revised IPI (R-IPI) score [4]. Staging was performed for all patients according to Ann Arbor criteria. No patients were infected with human immunodefi ciency virus or human T-cell lymphotrophic virus I. All follow-up data were updated as of 31 May 2011. Serum samples and tumor tissues were obtained from every patient by lymph node biopsy before the initiation of therapy. All patients received either R-CHOP or R-THP (tetrahydropyranyl adriamycin)-COP therapy as the initial therapy. Th e R-THP-COP regimen included THP, an anthracycline derivative of doxorubicin that reportedly off ers lower cardiotoxicity than doxorubicin. We have recently reported the results of phase II studies of R-THP-COP therapy for elderly patients [5] and patients below 70 years old [6]. Th e R-THP-COP regimen showed similar remission and survival rates to the R-CHOP regimen. Tumors that had progressed at any of these time points were considered to represent treatment failure. Responses to treatment were categorized based on the Cheson criteria [7]. We measured serum concentrations of l -kynurenine by high-performance liquid chromatography using a spectrophotometric detector (Tosoh UV-8000; Tokyo, Japan) or a fl uorescence spectrometric detector (Hitachi, Tokyo, Japan). Briefl y, l -kynurenine was separated by reverse-phase column chromatography (column: Brave ODS 3 μ m, 150 4.6 mm; Alltech, IL) and a mobile phase at a fl ow rate of 0.75 mL/min comprising 0.1 M sodium acetate, 0.1 M acetic acid and 1% acetonitrile. Fluorescence excitation and emission wavelengths were set at 270 and 360 nm, respectively. Ultraviolet signals were monitored at 355 nm for l -kynurenine. Tissue samples were fi xed in 10% formalin-acetone and embedded in paraffi n, then cut at a thickness of 2 μ m. Slides were deparaffi nized in xylene and rehydrated. Each slide was heated in 0.1 M citrate buff er (pH 6.0) using the Pascal heat-induced target retrieval system (Dako, Carpinteria, CA). Endogenous peroxidase activity was blocked by incubation with 0.3% H 2 O 2 in methanol for 30 min at room temperature. Th e primary antibody was mouse anti-human IDO monoclonal antibody, kindly provided by O. Takikawa [8], used at a dilution of 1:100. Th e slide was incubated with the antibody at 4 ° C overnight. After washing in 50 mM Tris-buff ered saline, a refi ned avidin-biotin technique in which biotinylated secondary antibody reacted with several peroxidaseconjugated streptavidin molecules was employed for amplifi cation using a labeled streptavidin-biotin/ horseradish peroxidase (LSAB /HRP) kit (Dako, Tokyo, Japan). Diaminobenzidine tetrahydrochloride was used to visualize immunoreactive cells. Finally, nuclei were counterstained using Mayer ’ s hematoxylin. Cases with IDOpositive cells constituting more than 30% of tumor cells were considered positive. L eu k L ym ph om a D ow nl oa de d fr om in fo rm ah ea lth ca re . c om b y M cM as te r U ni ve rs ity o n 01 /0 5/ 15