INDOLEAMINE-2,3-DIOXYGENASE ASSAY FOR PROSTATE CANCER DIAGNOSIS AND PROGNOSIS

Abstract

The invention relates to a method to predict a patient's risk of developing prostate cancer, or to distinguish indolent vs. aggressive cancers among patients suspected of bearing PCa and therefore selected for biopsy, and/or predict the risk of progression of prostate cancer in a patient having undergone prostate cancer treatment, particularly by radical prostatectomy, brachytherapy, and/or external beam radiation therapy, wherein said method comprises detecting/quantifying the level of indoleamine-2,3-dioxygenase (IDO) mRNA or protein in a urine sample obtained from said patient.

Claims

1. A method to predict a patient's risk of having or developing prostate cancer, assign a patient to prostate biopsy, predict a patient's risk of having clinically relevant PCa, predict a patient's risk of having indolent PCa, distinguish indolent vs. aggressive cancers, predict a patient's risk of relapse or biochemical recurrence of prostate cancer, and/or predict a patient's risk of progression of an indolent prostate cancer to clinically relevant prostate cancer; wherein said method comprises detecting/quantifying the level of indoleamine-2,3-dioxygenase (IDO) mRNA or protein in a urine sample obtained from said patient.

2. The method according to claim 1, wherein no DRE is performed prior to obtaining said urine sample.

3. The method according to claim 1, wherein a risk of having prostate cancer is excluded for said patient if said level of IDO mRNA is below a predetermined mRNA threshold 1.

4. The method according to claim 3, wherein a risk of having prostate cancer is assigned to said patient if said level of IDO mRNA is below a predetermined mRNA threshold 2, but above said predetermined mRNA threshold 1.

5. The method according to claim 1, wherein said patient is assigned to prostate biopsy if said level of IDO mRNA is above a predetermined mRNA threshold 3.

6. The method according to claim 1, wherein a high risk of having a prostate cancer with a Gleason score7 is assigned to said patient if said level of IDO mRNA is above a predetermined threshold 3.

7. The method according to claim 1, wherein a high risk of biochemical recurrence of prostate cancer within 5 years is assigned to a patient having undergone prostate cancer treatment if said level of IDO mRNA is above said predetermined threshold 3.

8. The method according to claim 1, wherein a risk of relapse is assigned to said patient if said level of IDO mRNA is above a predetermined mRNA threshold 4.

9. The method according to claim 1, wherein quantifying IDO mRNA is performed by polymerase chain reaction (PCR), in particular quantitative real-time PCR (qPCR).

10. The method according to claim 1, wherein a low risk of having a prostate cancer is assigned to said patient if the level of IDO protein is below a predetermined protein threshold 1.

11. The method according to claim 1, wherein a definite risk of clinically relevant prostate cancer is assigned to a patient if the level of IDO protein is above a predetermined protein threshold 2.

12. The method according to claim 1, wherein quantifying IDO protein is effected by enzyme-linked immunosorbent assay (ELISA).

13. The method according to claim 1, wherein said urine sample is an unprocessed volume of urine.

14. The method according to claim 1, wherein said urine sample is a pellet obtained by centrifuging a volume of urine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0127] FIG. 1A shows the definition of the thresholds for IDO mRNA and the feasibility of the test without performing DRE (prostate massage). Patients with GS7 and GS6 have PCa and the GS was evaluated from the biopsy or after prostatectomy, when available. GS7 means clinically relevant PCa and GS6 means indolent PCa. Cut-off 1 (a0.0015, assignment to biopsy or risk of having clinically relevant PCa: NPV 100%, PPV 47%; risk of having PCa: NPV=100%, PPV=82%), cut-off 2 (b0.0075, assignment to biopsy or risk of having clinically relevant PCa: NPV 81%, PPV 72%; risk of having PCa: NPV=47%, PPV=100%), cut-off 3 (c0.0096, assignment to biopsy or risk of having clinically relevant PCa: NPV 83%, PPV 88%) and cut-off 4 (d0.0479, risk of having BR within 5 years from treatment or treatment resistant-PCa: NPV 100%, PPV 40%). IDO relative gene expression was analysed in urine of patients collected without execution of a DRE.

[0128] The IDO test reduces the number of unnecessary biopsies by: 14.8% with cut-off 1, or 51.8% with cut-off 2, or 66.6% with cut-off 3.

[0129] FIG. 1B shows the thresholds for IDO protein and the feasibility of the test without performing DRE (prostate massage). Protein cut-off 1 (a42 pg/ml, assignment to biopsy or risk of having clinically relevant PCa: PPV 83%, NPV 63%, risk of having PCa: PPV 90%, NPV 83%,), protein cut-off 2 (b200 pg/ml, assignment to biopsy or risk of having clinically relevant PCa: PPV 69%, NPV 100%; risk of having PCa PPV 100%, NPV 85%) and the feasibility of the test without performing DRE (prostate massage). IDO relative gene expression was analysed in urine of patients collected without execution of a DRE. GS7, GS6 and no PCa are the results from the biopsy or prostatectomy. The IDO test reduces the number of unnecessary biopsies by 29.4% with protein cut-off 2.

[0130] FIG. 2 shows the feasibility of the test in urine without centrifugation (free-RNA vs pellet).

[0131] FIG. 3: Prostatic massage increases the amount of mRNA PSA in urine by a factor of 50; (test performed in five patients comparing gene expression before and after DRE). Normally, DRE increases PSA protein by a factor of 3 in serum. Differently from PSA, the prostatic massage slightly increased the amount of IDO by 2 to 3-fold.

MATERIALS AND METHODS

[0132] Collection of Urines

[0133] Patient must restrain from any sexual activity in the previous 24 hours before collection. First urines of the morning are collected in a sterile container.

[0134] Pellet: 20 ml are centrifuged at 2000 rpm for 10 minutes, the supernatant is discarded and the pellet is used for RNA extraction.

[0135] Cell-free RNA and protein: 500 ul of urines are used for RNA extraction.

[0136] RNA Extraction

[0137] RNA extraction is performed according to the RNA Aqueous Kit protocol (Ambion). 700 ul of lysis solution is added either to the pellet or to 500 ul of urines. The elution step is performed twice in the same tube with a volume of 50 ul each time (final volume is 100 ul). Samples are stored at 80 C.

[0138] Retro transcription 30 ul of RNA are added to 30 ul of mix prepared with the High Capacity cDNA Reverse Transcription Kits (Applied Biosystems).

[0139] 10 ul of mix contains 2.0 l of 10RT Buffer, 0.8 ul of dNTPs, 2.0 ul of random primers, 1.0 ul of multiscribe reverse transcription enzyme and 4.2 ul of water. The reaction is performed in a thermocycler in 4 steps: 10 minutes at 25 C., 120 minutes at 37 C., 5 minutes at 85 C. and 5 minutes at 4 C. Samples are stored at 80 C.

[0140] Real-Time PCR

[0141] The 001 assay is designed covering the exon-exon junction between exon 9 and 10. The primers are designed in order to have a melting temperature between 58 C. and 61 C., with an optimal length of 20 bp and CG content between 30% and 80%. The probe is designed in order to have a melting temperature 10 C. higher than the one of the primers and does not start with a G. Primers and probe are used at a final concentration of 400 nM and 200 nM, respectively.

[0142] An exemplary TaqMan assay is represented by the sequences:

TABLE-US-00002 (SEQIDNO01) PrimerF 5-GAAGACCCAAAGGAGTTTGC-3 (SEQIDNO02) PrimerR 5-TGGAGGAACTGAGCAGCAT-3 (SEQIDNO03) Probe: 5-TGGGCATCCAGCAGACT-3.

[0143] The probe is modified with FAM (6-carboxy fluorescein) on the 5 prime and a MGB (minor grove binder, non-fluorescent quencher fitting the FAM spectral qualities, purchased from Applied Biosystems) on the 3 prime terminus. The mix for the quantitative PCR is prepared using the TaqMan Gene Expression Master Mix (Applied Biosystems): it contains (per well) 10 ul of master mix 2, 7 ul of water, 0.8 ul of primer F (10 uM), 0.8 ul of primer R (10 uM) and 0.4 ul of probe (10 uM). 1 ul of cDNA is tested per well. Samples are run in triplicate. For each run, a negative control is also tested in triplicate.

[0144] The PCR program is as follows: UNG incubation (2 minutes at 50 C.); Polymerase activation (10 minutes at 95 C.); PCR cycles (40): denaturation (15 seconds at 95 C.) and annealing (1 minute at 60 C.).

[0145] IDO's Protein Quantification

[0146] 125 ul of urines were used for the quantification of IDO's protein by ELISA. The assay utilizes the two-site sandwich technique with two selected polyclonal antibodies that bind to human indoleamine 2,3-deoxygenase 1 (IDO1) (IDKR IDO ELISA kit K7727 by Immundiagnostik AG, Stubenwald-Allee 8a, 64625 Bensheim, Germany). The urine samples are added to the wells of a microplate coated with a high affine polyclonal anti-human IDO1 antibody. During the first incubation step IDO1 in the samples is captured by the immobilized antibody. Then, the biotinylated detection antibody, a polyclonal anti-human IDO1 antibody, is added. Peroxidase labeled streptavidin is added to the reaction and Tetramethylbenzidine (TMB) is used as a substrate for the peroxidase. An acidic stop solution is added to stop the reaction. A plate reader is used to measure the absorbance at 450 nm and the concentration of IDO1 is calculated, using the values obtained from the standard curve.

[0147] Data Analysis

[0148] Normalization of gene expression was performed using either GAPDH or 18S. A 2.sup.Ct method was used to compute fold change of genes of interest (PSA and IDO) before and after DRE. A 2.sup.Ct method was also used to compute fold decrease of IDO as compared to GAPDH gene expression as 1. All value10.sup.7 were considered undetectable.

[0149] Selection of Specific Cut-Offs

[0150] By comparing patients diagnosed with clinically relevant PCa (GS7), with indolent PCa (GS6) and patients with PCa-free biopsies, specific cut-offs were selected.

[0151] FIG. 1 illustrates the definition of A) cut-off 1 (0.0015), cut-off 2 (0.0075), cut-off 3 (0.0096), cut-off 4 (0.0479), B) cut-off 5 (42 pg/ml), cut-off 6 (200 pg/ml).

[0152] In order to identify patients undergoing biopsy with negative results (NPV 100%; 0% false positive), a first cut-off (cut-off 1) of 0.0015 was generated.

[0153] In order to gather low risk PCa, a second cut-off (cut-off 2) of 0.0075 was generated. 82% of patients with PCa GS6 and BR or no PCa expressed IDO<0.0075. For patients below this cut-off 2 a biopsy might not be recommended.

[0154] In order to gather intermediate/high risk PCa, a third cut-off (cut-off 3) of 0.0096 was generated. 93.7% of patients with PCa GS6 and BR or no PCa expressed IDO<0.0096. For patients above this cut-off 3 a biopsy is always recommended.

[0155] In order to diagnose patients with a high probability to progress, a forth cut-off (cut-off 4) of 0.0479 was generated. Patients above this cut-off 4 should undergo immediate biopsy and probably treatment.

[0156] In order to identify patients undergoing biopsy with negative results (risk of PCa: PPV=90%; NPV=83%), a first protein cut-off (protein cut-off 1) of 42 pg/ml was generated.

[0157] In order to gather intermediate/high risk PCa, a second protein cut-off (protein cut-off 2) of 200 pg/ml was generated (PPV=100%, NPV=69%). For patients above this protein cut-off 2 a biopsy is always recommended.