CLB-08707; No. of pages: 2; 4C: Clinical Biochemistry xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem

Letter to the Editor

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Serum kynurenine/tryptophan ratio is not a potential marker for detecting prostate cancer

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Keywords: Prostate cancer Biomarker IDO Tryptophan Kynurenine

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Tryptophan (Trp) is an essential amino acid in humans that plays an important role in protein synthesis and it is a precursor of many biologically active metabolites, including kynurenine (Kyn) [1]. The enzyme that converts tryptophan into kynurenine is indoleamine 2,3-dioxygenase (IDO), which has been associated to immune escape of tumor cells [2]. Kynurenine was part of a subset of six metabolites found to be associated to prostate cancer (PCa) progression as described by Srekumar et al. [3] in plasma, urine and tissue. More recently, Kyn was proposed by McDunn et al. [4], as a metabolite that strongly correlates with aggressiveness of this malignancy as measured by Gleason scores. We determined concentrations of Kyn and Trp by fluorescence detection in serum samples from patients diagnosed with PCa, and classified in two groups: Gleason score 6 (n = 15) and Gleason score 7 or higher (n = 28). We compared these concentrations with the ones in a group of male subjects with low PSA levels and without PCa, denominated as PCa Controls (n = 20). Power analysis indicated that these number of samples resulted in 70% of power with 95% confidence and with effect size of 0.8 according to Cohen's suggestions [5]. Serum samples from patients diagnosed with colorectal liver metastases (n = 10), liver adenoma (n = 8), and hepatocellular carcinoma (n = 6), as well as serum samples from healthy kidney donors (n = 12), were also included as extra groups to generate information about tumor specificity. The use of serum samples for research purposes was approved by the Erasmus MC Medical Ethics Committee according to the Medical Research Involving Human subjects Act (MEC-ERSPC). Clinical-pathological characteristics of the patients involved in this study are presented in Table 1. Serum samples were obtained according to the protocols reported by the Rotterdam Centre for the European Randomized Study of Screening for Prostate Centre (ERSPC) [6], and were stored at −80 °C until further processing. Sample preparation included thawing of the samples on ice for 1 h, protein precipitation/dilution with trichloroacetic acid at room temperature, and centrifugation at 14,000 g at 4 °C for 15 min. Kyn and Trp in the supernatant were separated by liquid chromatography (LC) and detected by fluorescence (FLD) using the standard addition method. This method is used to avoid matrix interferences by means of the addition of known quantities of the analyte of interest, with a concomitant increment in signal response of the instrument. We used zinc-mediated fluorescence for the accurate determination of Kyn [7,8], and endogenous concentrations of Trp and Kyn were calculated

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by extrapolation using at least four points in a calibration curve [9]. In all cases, the percentage of error was less than 15%, indicating that the small variances in the measurements allow differentiation between cases and controls. Stability was evaluated by consecutive measurements of pooled serum obtained from ten healthy individuals and also of the same pooled serum spiked with known amounts of Kyn and Trp to produce high, medium and low concentrations. Measurements were performed within a period of 20 h, which is the time the samples are stored in the autosampler before analysis. In all cases, relative standard deviations were lower than 5%. Mean serum concentrations of Kyn for PCa patients were found at 2.1 ± 0.7 μM (mean ± SD) in Gleason 6, and 1.91 ± 1.2 μM in Gleason 7; these concentrations were not statistically different compared to the Control group (2.1 ± 0.7 μM). These values are consistent with reported data using different analytical approaches in healthy individuals [10]. Trp concentrations for the PCa patients were also not significantly different (73.0 ± 13.8 μM for Gleason 6 and 70.7 ± 16.8 μM for Gleason 7) compared to those for the Control group (77.7 ± 21.7 μM). Mean concentrations of Trp and Kyn, as well as the calculated Kyn/Trp ratio are shown in Fig. 1a–c. Differences among groups were not significant (p N 0.05). Possible associations between metabolites concentrations in the serum of the PCa patients and clinical information available such as pathological stage and Gleason Score indicated no statistical significance (p N 0.05). Our data show that there are no major alterations in extracellular concentrations of Trp and Kyn. Therefore, these parameters or the calculated Kyn/Trp ratio, have no potential as biomarker for the diagnosis of prostate cancer in serum. Metabolic signatures between PCa tissue and benign prostate tissue have suggested that an increment exists in the concentration of Kyn in PCa tissue [3,4]. Although the number of serum samples used

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Table 1 Clinical and pathological characteristics of cases and controls. PCa controls, and PCa patients.

PSA (ng/mL)

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t1:1 t1:2 t1:3

Controls/ERSPC

PCa patients

t1:4

(n = 20)

(n = 43)

t1:5

Median Range Median Range

68.2 58.2–69.4 0.2 0.1–4.4

65.3 56.2–69.1 5.4 2.0–15.1

pT1 pT2 pT3 pT4 Unknown

n (%) – – – – –

n (%) 5 (11.6) 27 (62.8) 6 (13.9) 4 (9.3) 1 (2.3)

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– – – – –

15 (34.9) 21 (48.8) 1 (2.3) 4 (9.3) 2 (4.6)

t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22

Characteristic

Age (years)

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Pathological state

Gleason score + + + + +

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http://dx.doi.org/10.1016/j.clinbiochem.2014.05.001 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Please cite this article as: Rodríguez-Blanco G, et al, Serum kynurenine/tryptophan ratio is not a potential marker for detecting prostate cancer, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.05.001

Letter to the Editor

Acknowledgments

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This work is financially supported by the Prostate Research Organizations-Network of Early Stage Training (PRO-NEST) — FP7 Marie Curie initial training network (grant agreement no. 238278), and by the framework of CTMM, the Center for Translational Molecular Medicine, PCMM (grant 03O-203).

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References

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[1] Chen Y, Guillemin GJ. Kynurenine pathway metabolites in humans: disease and healthy states. Int J Tryptophan Res 2009;2:1–19. [2] Mellor AL, Munn DH. Tryptophan catabolism and T-cell tolerance: immunosuppression by starvation? Immunol Today 1999;20(10):469–73. [3] Sreekumar A, Poisson LM, Rajendiran TM, Khan AP, Cao Q, Yu J, et al. Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 2009;457(7231):910–4. [4] McDunn JE, Li Z, Adam K-P, Neri BP, Wolfert RL, Milburn MV, et al. Metabolomic signatures of aggressive prostate cancer. Prostate 2013;73(14):1547–60. [5] Cohen J. Statistical power analysis. Curr Dir Psychol Sci 1992;1(3):98–101. [6] Roobol MJ, Kirkels WJ, Schröder FH. Features and preliminary results of the Dutch centre of the ERSPC (Rotterdam, the Netherlands). BJU Int 2003;92:48–54. [7] Luo X, Tang A, Pi L, Xiao L, Ao X, Pu Y, et al. Determination of kynurenine in serum by high-performance liquid chromatography with on-column fluorescence derivatization. Clin Chim Acta 2008;389(1–2):186–8. [8] Xiang ZY, Tang AG, Ren YP, Zhou QX, Luo XB. Simultaneous determination of serum tryptophan metabolites in patients with systemic lupus erythematosus by high performance liquid chromatography with fluorescence detection. Clin Chem Lab Med Apr 2010;48(4):513–7. [9] Bruce GR, Gill PS. Estimates of precision in a standard additions analysis. J Chem Educ 1999;76(6):805. [10] Zhen Q, Xu B, Ma L, Tian G, Tang X, Ding M. Simultaneous determination of tryptophan, kynurenine and 5-hydroxytryptamine by HPLC: application in uremic patients undergoing hemodialysis. Clin Biochem 2011;44(2–3):226–30.

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Fig. 1. Serum concentrations of a) tryptophan, b) kynurenine and c) Kyn/Trp ratio in PCa controls, PCa patients Gleason 6 (n = 15) and Gleason 7&8 (n = 28), HKD: healthy kidney donors (n = 12, median age: 57.2 years), LA: liver adenoma (n = 8, median age: 33.8), HCC: hepatocarcinoma (n = 6, median age: 56.3), and LM: liver metastasis (n = 10, median age: 64.2).

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in this study was relatively small, the results presented here indicate that the reported metabolic signatures do not translate to changes in serum. Therefore, although metabolic alterations in kynurenine pathway might be occurring at the level of the prostate, this pathway cannot be considered as a target for serum biomarkers and of clinical applicability.

Giovanny Rodríguez-Blanco Peter C. Burgers Lennard J.M. Dekker Department of Neurology, Erasmus Medical Centre, Rotterdam, The Netherlands

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Mirella S. Vredenbregt-van den Berg 128 Department of Urology, Erasmus Medical Centre, 129 Rotterdam, The Netherlands 130 131

Jan N.M. Ijzermans 132 Department of Surgery, Erasmus Medical Centre, 133 Rotterdam, The Netherlands 134 Ellen A.M. Schenk-Braat Guido Jenster Department of Urology, Erasmus Medical Centre, Rotterdam, The Netherlands Theo M. Luider Department of Neurology, Erasmus Medical Centre, Rotterdam, The Netherlands Corresponding author at: Laboratories of Neuro-Oncology/Clinical and Cancer Proteomics, Department of Neurology, Erasmus Medical Centre, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. Fax: +31 10 704 4661. E-mail address: [email protected].

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25 November 2013 149 Available online xxxx 150

Please cite this article as: Rodríguez-Blanco G, et al, Serum kynurenine/tryptophan ratio is not a potential marker for detecting prostate cancer, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.05.001

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