Method of detecting proteins in human samples and uses of such methods

Abstract

A method for collecting information about the health status of a subject is proposed involving the quantitative detection, in serum, plasma or blood of the subject, of the concentration of THBS1, the proportion of free PSA (% fPSA), preferably including the concentration of at least one protein selected from the group consisting of CTSD, OLFM4, ICAM1.

Claims

1. A method comprising quantitative measurement in serum, plasma, or blood of a concentration of THBS1 and CTSD in a subject, as well as a proportion of free PSA (% fPSA), wherein for the measurement of the concentration of THBS1 and CTSD, the subject's serum, plasma, or blood is diluted using a buffer having a pH value in a range of 7-7.4 and comprising an agent controlling the pH value selected from at least one of the following systems: Tris (Tris(hydroxymethyl)-aminomethane), Pipes (Piperazine-1,4-bis-2-ethane sulfonic acid), Mes (4-Morpholino ethane sulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid), phosphate buffered saline (PBS), and wherein the buffer has an ionic strength in a range of 50-850 mM, wherein the method includes a first step being performed by contacting the subject's serum, plasma, or blood, with at least one affinity reagent for each protein and detecting whether binding occurs between the respective protein and the at least one affinity reagent and using quantitative readout of the respective protein's concentration, allowing a calculation of the respective concentration in the original serum, plasma, or blood, or in case of free PSA its proportion, wherein either a sandwich enzyme linked immunosorbent assay specific to the respective protein is used, or a sandwich bead based antibody assay to the respective protein is used, and wherein in said sandwich enzyme linked immunosorbent assay or said sandwich bead based antibody assay, mouse monoclonal antibodies that bind the respective protein are used, said mouse monoclonal antibodies having been generated through immunization of mice with the respective recombinant human THBS1 or CTSD protein; a second step of calculating, based on all the protein concentrations as well as the free PSA proportion determined in the first step, a combined score value, further taking account of an age of the subject, a third step of determining a risk of a positive biopsy and/or prostate cancer of the subject to discriminate between benign and malignant prostatic conditions based on the combined score value as determined in the second step, wherein surpassing a corresponding threshold value of the combined score value is taken as positive prostate cancer information and/or as necessity of biopsy, wherein said combined score value is calculated using the following formula (1): $\begin{array}{cc}\mathrm{Combined}\mathrm{Score}=\frac{1}{1+e^{-\left({\beta }_0+{\beta }_1.Math.x_1+\mathrm{.Math.}+{\beta }_k.Math.x_k\right)}}& \left(1\right)\end{array}$ wherein, in the formula (1), β.sub.i are regression coefficients as determined before with an optimization using experimental data, β.sub.0 being an intercept, and wherein x.sub.i is the measured concentration of the respective protein in the original serum, plasma, or blood and in case of free PSA (% fPSA) the proportion in the original serum, plasma, or blood, wherein, for the calculation of the combined score value, the regression coefficients are chosen as follows: β.sub.0 in the range of 3-3.8; β.sub.CTSD in the range of 0.005-0.05; β.sub.THBS1 in the range of (−0.00009)-(−0.00003); β.sub.% fPSA in the range of (−7.5)-(−2.5) wherein the concentration of THBS1, expressed in ng/ml, the proportion, expressed in the numerical range of 0-1, of free PSA (% fPSA), as well as the concentration of CTSD, in each case expressed in ng/ml, are measured in the first step, and wherein, for a 90% sensitivity, a threshold value of a combined score value is 0.3-0.35.

2. The method according to claim 1, involving the quantitative detection, in serum, plasma or blood of the subject, of the concentration of TEMS1 and CTSD, the proportion of free PSA (% fPSA), as well as the concentration of OLFM4, and ICAM1.

3. The method according to claim 2, wherein the concentration of THBS1, expressed in ng/ml, the proportion, expressed in the numerical range of 0-1, of free PSA (% fPSA), as well as the concentration of OLFM4, ICAM1 as well as CTSD, in each case expressed in ng/ml, are measured in the first step, and wherein, for a 90% sensitivity, a threshold value of a combined score value is 0.3-0.35.

4. The method according to claim 2, wherein for the dilution to measure THBS1 a dilution factor in the range of 1:1,000-1:20,000 is chosen for an enzyme linked immunosorbent assay and in the range of 1:5,000-1:15,000 for a bead-based assay, and/or wherein for the dilution to measure the protein CTSD a dilution factor in the range of 1:5-1:70 is chosen, for an enzyme linked immunosorbent assay and in the range of 1:10-1:20 for a bead-based assay is chosen, wherein for the dilution to measure ICAM1 a dilution factor in the range of 1:50-1:200 is chosen or wherein for the dilution to measure OLFM4 a dilution factor in the range of 1:5-1:30 is chosen.

5. The method according to claim 2, wherein at least one of OLFM4 and ICAM1 as measured comprises at least one post-translational modification selected from the group consisting of glycosylation, phosphorylation, and lipidation.

6. The method according to claim 1, wherein at least one of THBS1 and CTSD as measured comprises at least one post-translational modification selected from the group consisting of glycosylation, phosphorylation, and lipidation.

7. The method according to claim 1, wherein for the dilution to measure THBS1 a dilution factor in the range of 1:1,000-1:20,000 is chosen for an enzyme linked immunosorbent assay and in the range of 1:5,000-1:15,000 for a bead-based assay, and/or wherein for the dilution to measure the protein CTSD a dilution factor in the range of 1:5-1:70 is chosen, for an enzyme linked immunosorbent assay and in the range of 1:10-1:20 for a bead-based assay is chosen.

8. The method according to claim 1, wherein the quantitative detection of the respective concentration involves the determination of the concentration of such biomarkers relative to an external protein standard, involving the preparation of a reference standard curve by measuring defined concentrations of several protein standards diluted in the same buffer as for the protein dilution to be measured in the same set of measurements of the samples.

9. The method according to claim 1, further comprising at least one of monitoring, diagnosis, predicting prostate biopsy outcome, prognosis, risk assessment, therapy selection, therapy monitoring of cancer.

10. The method according to claim 1, wherein the concentration of THBS1, expressed in ng/ml, as well as the proportion, expressed in the numerical range of 0-1, of free PSA (% fPSA), and further the concentration of CTSD, expressed in ng/ml, is measured in the first step, and wherein for the calculation of the combined score value in of measuring THBS1, the proportion of free PSA (% fPSA) as well as CTSD, the regression coefficients are chosen as follows: β.sub.0 in the range of 3.1-3.3; β.sub.CTSD in the range of 0.008-0.012; β.sub.THBS1 in the range of (−0.00007)-(−0.00006); β.sub.% fPSA in the range of (−5.2)-(−2.5).

11. The method according to claim 1, wherein the concentration of THBS1, expressed in ng/ml, as well as the proportion, expressed in the numerical range of 0-1, of free PSA (% fPSA), and further the concentration of CTSD, expressed in ng/ml, is measured in the first step, and wherein, for the calculation of the combined score value of measuring THBS1, the proportion of free PSA (% fPSA) as well as CTSD, the regression coefficients are chosen as follows: β.sub.0 in the range of 3.1-3.3; β.sub.CTSD in the range of 0.008-0.012; β.sub.THBS1 in the range of (−0.00007)-(−0.00006); β.sub.% fPSA in the range of (−5.2)-(−2.5), and wherein for a 90% sensitivity a threshold value of the combined score value of 0.33-0.34 is selected.

12. The method according to claim 2, wherein the concentration of THBS1, expressed in ng/ml, the proportion, expressed in the numerical range of 0-1, of free PSA (% fPSA), as well as the concentration of OLFM4, ICAM1 as well as CTSD, in each case expressed in ng/ml, are measured in the first step, and wherein, for the calculation of the combined score value, the regression coefficients are chosen as follows: β.sub.0 in the range of 3-3.8; β.sub.OLFM4 in the range of 0.001-0.003; β.sub.ICAM1 in the range of (−0.004)-(−0.002); β.sub.CTSD in the range of 0.005-0.05; β.sub.THBS1 in the range of (−0.00009)-(−0.00003); β.sub.% fPSA in the range of (−7.5)-(−2.5) and wherein for a 90% sensitivity a threshold value of the combined score value of 0.3-0.35 is selected.

13. The method according to claim 2, wherein the concentration of THBS1, expressed in ng/ml, the proportion, expressed in the numerical range of 0-1, of free PSA (% fPSA), as well as the concentration of OLFM4, ICAM1 as well as CTSD, in each case expressed in ng/ml, are measured in the first step, and wherein for the calculation of the combined score value the regression coefficients are chosen as follows: β.sub.0 in the range of 3.5-3.6; β.sub.OLFM4 in the range of 0.0015-0.0025; β.sub.ICAM1 in the range of (−0.0035)-(−0.00025); β.sub.CTSD in the range of 0.008-0.012; β.sub.THBS1 in the range of (−0.00007)-(−0.00006); β.sub.% fPSA in the range of (−5.2)-(−4.8), and wherein for a 90% sensitivity a threshold value of the combined score value of 0.32-0.335 is selected.

14. The method according to claim 2, wherein for the measurement of the concentration of at least one of THBS1, CTSD, OLFM4, and ICAM1, the subject's serum, plasma or blood is diluted using a buffer having a pH value in the range of 7-7.4 and comprising an agent controlling the pH value, with additional components selected from at least one system as follows: non-ionic detergent, in a concentration of 0.01 to 0.1% (v/v), and selected from at least one of the group consisting of: Dodecylpoly(ethyleneglycolether)m, wherein m is an integer of 5 to 40; 1-O-n-Octyl-β-D-glucopyranoside (n-Octylglucoside); Alkylphenolpoly(ethyleneglycol-ether)m, wherein m is an integer of 5 to 40; 1-O-n-Dodecyl-β-D-glucopyranosyl (1-4)alpha-D-glucopyranoside; Dodecylpoly-(ethyleneglycolether)m, wherein m is an integer of 5 to 40; Poly(oxyethylene)(20)-sorbitane mono fatty acid ester; Octylphenolpoly(ethyleneglycoiether)m, wherein m is an integer of 5 to 40; bovine serum albumin; mouse IgG; bovine gamma globulins; fetal bovine serum; horse serum.

15. The method according to claim 2, wherein for the measurement of the concentration of at least one of THBS1, CTSD, OLFM4, and ICAM1, the subject's serum, plasma or blood is diluted using a buffer having a pH value in the range of 7-7.4 and comprising an agent controlling the pH value, selected from at least one of the following systems: Tris (Tris(hydroxymethyl)-aminomethane), Pipes (Piperazine-1,4-bis-2-ethane sulfonic acid), Mes (4-Morpholino ethane sulfonic acid), Hepes (4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid), phosphate buffered saline (PBS), with or without additional components selected from at least one system as follows: non-ionic detergent, in a concentration of 0.025-0.05% (v/v), and selected from at least one the group consisting of: Dodecylpoly(ethyleneglycolether)m, wherein m is an integer of 5 to 40; 1-O-n-Octyl-β-D-glucopyranoside (n-Octylglucoside); Alkylphenolpoly(ethyleneglycol-ether)m, wherein m=11; 1-O-n-Dodecyl-β-D-glucopyranosyl (1-4)alpha-D-glucopyranoside; Dodecylpoly-(ethyleneglycolether)m, wherein m=23; Poly(oxyethylene)(20)-sorbitane mono fatty acid ester, selected from Poly(oxyethylene)(20)-sorbitane monooleate, Poly(oxyethylene)(20)-sorbitane monolaurate, Poly(oxyethylene)(20)-sorbitane monopalmitat, Poly(oxyethylene)(20)-sorbitane monostearate); Octylphenolpoly(ethyleneglycoiether)m, wherein m=10; bovine serum albumin; mouse IgG; bovine gamma globulins; fetal bovine serum; horse serum; wherein the buffer has a ionic strength in the range of 250-370 mM.

16. The method according to claim 2, wherein, for the dilution to measure THBS1, a dilution factor is in the range of 1:2,000-1:3,000 for an enzyme linked immunosorbent assay and in the range of 1:5,000-1:15,000 for a bead-based assay, or wherein, for the dilution to measure the protein CTSD, a dilution factor is in the range of 1:10-1:30 for an enzyme linked immunosorbent assay and in the range of 1:10-1:20 for a bead-based assay, and wherein the buffer used is further supplemented with a non-ionic detergent selected as Poly(oxyethylene)(20)-sorbitane monolaurate to lead to an additional concentration thereof of 0.05% (v/v), or wherein, for the dilution to measure ICAM1, a dilution factor is in the range of 1:80-1:150, and wherein the buffer used is further supplemented with sodium chloride to an additional sodium chloride content of 250 mM, or wherein, for the dilution to measure OLFM4, a dilution factor is in the range of 1:5-1:20, and wherein the buffer used is further supplemented with sodium chloride to an additional sodium chloride content of 250 mM as well as a reduction agent, selected as dithiothreitol to lead to a concentration of 5 mM thereof.

17. The method according to claim 1, wherein the method includes: a first step being performed by contacting the subject's serum, plasma or blood, after dilution thereof, with at least one affinity reagent for each protein and detecting whether binding occurs between the respective protein and the at least one affinity reagent and using quantitative readout of the respective protein's concentration or in case of free PSA its proportion, allowing the calculation of the respective concentration in the original serum, plasma or blood, and wherein in this first step either a sandwich enzyme linked immunosorbent assay specific to the respective protein with visible readout is used, or a sandwich bead based antibody assay to the respective protein with fluorescent readout is used.

18. The method according to claim 1, wherein the quantitative detection of the respective concentration involves the determination of the concentration of such biomarkers relative to an external protein standard, involving the preparation of a reference standard curve by measuring defined concentrations of 5-7 protein standards diluted in the same buffer as for the protein dilution to be measured in the same set of measurements of the samples.

19. The method according to claim 1, further including at least one of monitoring, diagnosis, predicting prostate biopsy outcome, prognosis, risk assessment, therapy selection, therapy monitoring of localized or non-localized prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

(2) FIG. 1 shows a schematic presentation of a two-step sandwich immunoassay, wherein for the Luminex system the detection is realized using fluorescence with fluorescent Streptavidin-phycoerythrin conjugate (Strep-PE) attaching to the Biotin (B) of the detection antibody, while for ELISA this is realized using color change in the visible range obtained with Streptavidin-enzyme conjugation and a chromogenic substrate for such enzyme, preferably Streptavidin-horseradish peroxidase (HRP) conjugate instead of Strep-PE and the HRP chromogenic substrate TMB;

(3) FIG. 2 shows the optimized receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa, for the case when THBS1 is measured alone, when total PSA is measured alone, when % fPSA is measured alone, and when the concentration of THBS1 is used together with the proportion of % fPSA for the evaluation of the combined score value;

(4) FIG. 3 shows the optimized ROC curves when CTSD is measured alone, when % fPSA is measured alone, and when the concentration of CTSD is used together with the proportion of % fPSA for the evaluation of the combined score value;

(5) FIG. 4 shows the optimized ROC curves when ICAM1 is measured alone, when % fPSA is measured alone, and when the concentration of ICAM1 is used together with the proportion of % fPSA for the evaluation of the combined score value;

(6) FIG. 5 shows the optimized ROC curves when OLFM4 is measured alone, when % fPSA is measured alone, and when the concentration of OLFM4 is used together with the proportion of % fPSA for the evaluation of the combined score value;

(7) FIG. 6 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, CTSD and THBS1, and the combination of % fPSA, CTSD and THBS1; ROC curves of the optimized models are shown;

(8) FIG. 7 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, ICAM1 and THBS1, and the combination of % fPSA, ICAM1 and THBS1; ROC curves of the optimized models are shown;

(9) FIG. 8 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, OLFM4 and THBS1, and the combination of % fPSA, OLFM4 and THBS1; ROC curves of the optimized models are shown;

(10) FIG. 9 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, ICAM1 and CTSD, and the combination of % fPSA, ICAM1 and CTSD; ROC curves of the optimized models are shown;

(11) FIG. 10 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, OLFM4 and CTSD, and the combination of % fPSA, OLFM4 and CTSD; ROC curves of the optimized models are shown;

(12) FIG. 11 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, ICAM1 and OLFM4, and the combination of % fPSA, ICAM1 and OLFM4; ROC curves of the optimized models are shown;

(13) FIG. 12 shows the reduction of negative biopsies at 90% sensitivity; percentages given for % fPSA alone, CTSD and THBS1, and the combination of % fPSA, CTSD and THBS1;

(14) FIG. 13 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, CTSD, THBS1, OLFM4, ICAM1, and the combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1 ROC curves of the optimized models are shown; and

(15) FIG. 14 shows the reduction of negative biopsies at 90% sensitivity; percentages given for % fPSA alone, CTSD, THBS1, OLFM4 and ICAM1, and the combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1;

(16) FIG. 15 shows the optimized receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa, for the case when THBS1 is measured using ELISA alone, when total PSA is measured alone, when % fPSA is measured alone, and when the concentration of THBS1 measured using ELISA is used together with the proportion of % fPSA for the evaluation of the combined score value;

(17) FIG. 16 shows the optimized ROC curves when CTSD is measured using ELISA alone, when % fPSA is measured alone, and when the concentration of CTSD measured using ELISA is used together with the proportion of % fPSA for the evaluation of the combined score value;

(18) FIG. 17 shows receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa; % fPSA alone, CTSD and THBS1 both measured using ELISA, and the combination of % fPSA, CTSD and THBS1 both measured using ELISA; ROC curves of the optimized models are shown;

(19) FIG. 18 shows the reduction of negative biopsies at 90% sensitivity; percentages given for % fPSA alone, CTSD and THBS1 both measured using ELISA, and the combination of % fPSA, CTSD and THBS1 both measured using ELISA.

DESCRIPTION OF PREFERRED EMBODIMENTS

(20) We have previously identified numerous protein biomarkers for PCa diagnosis using a two staged genetics-guided discovery approach focusing on the PI3K/PTEN cancer pathway. In the first stage, glycoproteins were identified from sera and prostate tissue of wild-type and Pten-null cancer mouse models. Following prioritization of the proteins, proteomic profiles were identified in the sera of PCa patients and control individuals in a second stage validation step. The objective was to identify and develop and validate highly sensitive and highly specific immunoassays for the individual protein biomarkers. The transition from mass spectrometry to immunoassay technology represents an important step in order to enable high-throughput clinical validation in large sample cohorts. Furthermore, it facilitates clinical application of the test in routine diagnostic laboratories. The microparticle-based Luminex platform was initially chosen due to the improved kinetics resulting from a higher surface area of micro particles when compared to microtiter plates or tubes. In addition, magnetic microparticle-based assays are more amenable to automation than tube- or microtiter plate-based assays and thus widely distributed in random-access immunoassay systems. Finally, the multiplexing capabilities of the Luminex system facilitated the antibody selection process for immunoassay development, specifically for the subsequent development of the more common and simple and more user-friendly ELISAs.

(21) In this section, the identification, the development and technical validation according to the guidelines from the Center for Drug Evaluation and Research (CDER) is described for four individual immunoassays for human cathepsin D (CTSD), human intercellular adhesion molecule 1 (ICAM1), human olfactomedin 4 (OLFM4), and human thrombospondin 1 (THBS1). The mouse homologues of these human glycoproteins were previously identified by mass spectrometry using a Pten conditional knock-out mouse model. The human homologues were measured in clinical serum samples for testing the capability of discriminating benign prostatic conditions from PCa. As a result it was identified that the claimed combination is the optimum to overcome the above-mentioned defects of prior art approaches and which shows the highest sensitivity and highest specificity.

(22) The development yielded several individual immunoassays with inter and intra-variability (CV) <15% and linearity on dilution of the analytes. In serum, ex vivo protein stability (<15% loss of analyte) was achieved for a duration of at least 24 hours at room temperature and 2 days at 4° C.

(23) Materials and Methods

(24) Gene names, Entry names, Protein names (shortened) and Accession numbers as generally used in this specification are as defined according to the UniProt Consortium (www.uniprot.org), which is comprised of the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB), and the Protein Information Resource (PIR). The annotated or predicted cellular localization is according to Emanuelsson O, Brunak S, von Heijne G, Nielsen H. (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc. 2, 953-71.

(25) TABLE-US-00001 annotated or predicted Gene Accession cellular name Entry name Protein name number localization THBS1 TSP1_HUMAN Thrombospondin 1 P07996 secreted OLFM4 OLFM4_HUMAN Olfactomedin-4 Q6UX06 secreted CTSD CATD_HUMAN Cathepsin D P07339 lysosomal ICAM1 ICAM1_HUMAN Intercellular adhesion P05362 plasma molecule 1 membrane KLK3 KLK3_HUMAN Prostate-specific antigen P07288 secreted (PSA)

(26) Protein Standard/Calibrator

(27) Expression

(28) Recombinant human CTSD (residues 1-412, followed by 6His tag), ICAM1 (residues 1-480, followed by 8His tag), THBS1 (residues 19 to 1170, preceded by a signal peptide and followed by 11His tag) and OLFM4 (residues 1-510) were each expressed and purified from cell culture supernatants of transfected HEK293 cells. CTSD, ICAM1, THBS1 was purified using a His Trap column (GE Healthcare).

(29) Purification

(30) For ICAM1 ammonium sulfate precipitation was first carried out. Proteins precipitating at 35% were eliminated by centrifugation. The supernatant was transferred to clean tubes and ammonium sulfate was gradually added to reach 75% saturation. After centrifugation, the supernatant was eliminated and the pellet dissolved in buffer for purification on His Trap column.

(31) OLFM4 was purified by ammonium sulfate precipitation at 30%.

(32) Native THBS1 purified from human platelets was purchased from Creative Biomart and used for immunization. Recombinant THBS1 protein was initially purchased from R&D Systems and later expressed and purified from cell culture supernatants of transfected HEK293 cells in house.

(33) Antibodies

(34) The capture and detection antibodies were mouse monoclonals generated through immunization of BALB/c mice with the recombinant proteins of human CTSD, ICAM1, OLFM4 and the native THBS1, respectively. A large number of antibodies recognizing different epitopes of the respective protein was isolated and antibody pairs were selected for subsequent development and optimization of the sandwich bead based immunoassays and the ELISA immunoassays, respectively.

(35) The microparticle-based Luminex technology used provided a versatile platform to select antibodies for the newly developed immunoassays. The multiplexing technology facilitated the identification of antibody sandwich pairs by coupling several candidate capture antibodies to different microsphere sets. When mixed together, these sets allowed for the simultaneous testing of separate detection antibodies saving reagents, sample and time (see e.g. Baker H N, Murphy R, Lopez E, Garcia C. Conversion of a capture ELISA to a Luminex xMAP assay using a multiplex antibody screening method. J Vis Exp. 2012). Once suitable antibody pairs were selected, buffer compositions and antibody concentrations were optimized accordingly in order to yield the most optimal conditions (signal to background ratio, dilutional linearity, dynamic range of standard curve, assay sensitivity) for the individual immunoassays.

(36) Bead Based Immunoassays

(37) Bead-based sandwich immunoassays were established on the Luminex system as follows. Capture antibodies were covalently conjugated to carboxylated Luminex microparticles and the detection antibodies were labeled with biotin (B) according to standard procedures. 96-well half area microtiter plates (Corning Inc.) were blocked for a minimum of 15 min with 1× Blocking Reagent for ELISA (Roche Diagnostics). A mix of the capture antibody-coated microparticle and the biotinylated detection antibody at the appropriate concentrations was prepared and added to the protein (sample or standard) diluted in assay buffer within the 96-well plate. Following a 60 or 120 min incubation at 21° C. or 37° C. depending on the assay and shaking at 650 rpm in an Eppendorf ThermoMixer C, the plates were washed with PBS+0.05% Tween20 using a magnetic plate separator (Luminex Corporation). Streptavidin-phycoerythrin conjugate (Strep-PE, Moss Inc.) diluted in assay buffer was added for 30 min, incubated with 650 rpm at 21° C. in an Eppendorf ThermoMixer C. After washing, the bead conjugates were resuspended in Blocking Reagent. Read-out was performed with either a Luminex FlexMap3D or Luminex MAGPIX instrument operated with xPONENT 4.1 or 4.2 software which was also used to calculate the concentrations using 4-parameter curve fit. All samples were measured in independent duplicates on the same plate. Quality control samples with defined protein concentrations were included on each plate.

(38) For the various epitopes of the antibodies, respective capture antibody beads and biotinylated detection antibodies were generated and tested, and the optimum pair of capture and detection antibodies was empirically determined based on optimum readout intensity (see also FIG. 1).

(39) Enzyme-Linked Immunosorbent Assay

(40) Sandwich ELISAs were established as follows. Capture antibodies as determined and selected using the bead-based immunoassays described in the previous chapter were diluted in 50 mM sodium phosphate buffer, pH 8.0 (alternative: 50 mM carbonate buffer, pH 9.6) and coated to 96-well Maxisorp plates (Nunc) overnight at 4° C. (alternative: at 30° C. for 75 min or at room temperature for 1 to 6 hours). After removal of the solution and wash once with PBS/0.05% Tween20, plates were blocked with BSA-block (Candor Bioscience) (alternative: PBS with 1% BSA) for 1.5 hours. Plates were then washed 3 times. The standard or serum sample were diluted in Low Cross Buffer (LCB; Candor Bioscience) (alternative: PBS-based or 10 mM Tris, 0.9% sodium chloride-based buffers, both supplemented with 1% BSA, 0.1% bovine gamma globulins, 0.1% mouse IgG) and mixed in the wells with an equal volume the biotinylated detection antibody as determined and selected using the bead-based immunoassays described in the previous chapter diluted in LCB buffer. Following a 60 min incubation at 37° C. and shaking at 650 rpm in an Eppendorf ThermoMixer C, the plates were washed 3 times with PBS/0.05% Tween20. Streptavidin-HRP conjugate (Jackson ImmuneResearch) was diluted in LCB (alternative: BSA-block diluted with an equal volume of PBS) and added for 30 min, incubating at 37° C. and shaking at 650 rpm in an Eppendorf ThermoMixer C. After washing three times with PBS/0.05% Tween20, TMB substrate (Sigma) solution (diluted in 30 mM Citric acid, pH 4.1 with H.sub.2O.sub.2) (alternative: TMB, Enhanced K-Blue TMB Substrate ready to use, Neogen) was added for 30 min, incubating at 37° C. and shaking at 650 rpm in an Eppendorf ThermoMixer C. The reaction was blocked by addition of an equal volume of 0.25 M H.sub.2SO.sub.4 (alternative: 1.0 M HCl). Absorbance was measured on FLUOStart Optima ELISA reader (BMG LabTech) reading at 450 nm and subtracting reading at 620 nm. Concentrations were calculated using 5-parameter curve fit (alternative: use 4-parameter curve fit) with the FLUOstar OPTIMA software or with Magellan from Tecan.

(41) PSA Measurements

(42) Serum total PSA (tPSA) and free PSA (fPSA) were analyzed using the ADVIA Centaur immunoassay system (Siemens Healthcare). Percent free PSA (% fPSA) was calculated using the measured values of tPSA and fPSA with the following formula % fPSA=fPSA/tPSA. Alternatively the total PSA (tPSA) and complexed PSA (cPSA) can be measured and the proportion of free PSA can be calculated as follows: % fPSA=(tPSA−cPSA)/tPSA. The proportion fPSA/tPSA was used for the evaluations.

(43) Specific Bead Based Assays

(44) For the THBS1 assay, the sample serum (at final dilution of 1:10,000) or recombinant standard was incubated with the capture antibody-coated microparticle and the biotinylated detection antibody in Low Cross Buffer (Candor Bioscience) (LCB, pH 7.2) for 60 min at 37° C. All samples were quantified in the linear range of detection of the assay and the determined concentration (CV<20% for each sample, average CV for samples on one plate of 5.1%) ranged between 14.2 and 209 μg/ml.

(45) For the dilution of the THBS1 assay the following system was used: LCB, pH 7.2.

(46) For the CTSD assay, the sample serum (at final dilution of 1:15) or recombinant standard was incubated with the capture antibody-coated microparticle and the biotinylated detection antibody in LCB (pH 7.2)+0.05% Tween for 120 min at 37° C. All samples were quantified in the linear range of detection of the assay and the determined concentration (CV<20% for each sample, average CV for samples on one plate of 2.8%) ranged between 40 and 453 ng/ml.

(47) For the dilution of the CTSD assay the following system was used: LCB (pH 7.2)+0.05% Tween

(48) For the ICAM1 assay, the sample serum (at final dilution of 1:100) or recombinant standard was incubated with the capture antibody-coated microparticle and the biotinylated detection antibody in LCB (pH 7.2)+250 mM NaCl for 60 min at 37° C. All samples were quantified in the linear range of detection of the assay and the determined concentration (CV<15% for each sample, average CV for samples on one plate of 2.2%) ranged between 44 and 287 ng/ml.

(49) For the dilution of the ICAM1 assay the following system was used: LCB (pH 7.2)+250 mM NaCl.

(50) For the OLFM4 assay, the sample serum (at final dilution of 1:10) or recombinant standard was incubated with the capture antibody-coated microparticle and the biotinylated detection antibody in LCB (pH 7.2)+250 mM NaCl+5 mM DTT for 60 min at 37° C. All samples except three were quantified in the linear range of detection of the assay and the determined concentration (CV<15% for each sample, average CV for samples on one plate of 5.5%) ranged between 1 and 291 ng/ml.

(51) For the dilution of the OLFM4 assay the following system was used: LCB (pH 7.2)+250 mM NaCl+5 mM DTT.

(52) For the calibration of the concentrations to allow for quantitative readout standard solutions of the respective proteins were used using the following procedure:

(53) Measurement of protein biomarkers means the quantification of the concentration of such biomarkers relative to an external protein standard. Namely, a reference standard curve is prepared by measuring defined concentrations of 5-7 protein standards diluted in assay buffer in the same set of measurements of the samples.

(54) The following concentrations of the individual standards were used for the bead-based assays: THBS1 (ng/ml): 50.00, 20.00, 8.00, 3.20, 1.28, 0.51, 0.20 CTSD (ng/ml): 75.0, 2.0, 8.33, 2.78, 0.93, 0.31, 0.10 ICAM1 (ng/ml): 12.6, 5.04, 2.02, 0.81, 0.32, 0.13, 0.05 OLFM4 (ng/ml): 120, 34.29, 9.79, 2.79, 0.80, 0.23, 0.07

(55) The following concentrations of the individual standards were used for the ELISA assays: THBS1 (ng/ml): 200 or 250, 100, 50 or 40, 25 or 16, 12.50 or 6.4, 6.25 or 2.6, 3.125 or 1.0 CTSD (ng/ml): 15.00, 10.00, 6.25 or 6.7, 3.91 or 4.4, 2.44 or 3.0, 1.53 or 2.0, 0.95 or 1.3

(56) The standard curve was calculated using a computer program that allows a higher-grade polynomial curve fit. The serum samples were diluted in assay buffer so that their measurements fell within the range covered by the standards. Their concentration was calculated based on the curve of the standards, multiplying with the dilution factor to determine the concentration in the serum samples.

(57) Specific Enzyme-Linked Immunosorbent Assays

(58) The sample serum was measured at final dilution of 1:2,500 for the THBS1 assay and 1:20 for the CTSD assay. Also possible is measurement with 1:50 dilution for CTSD.

(59) Results

(60) The proposed test measures multiple cancer-specific proteins including cathepsin D (CTSD), thrombospondin 1 (THBS1) and olfactomedin 4 (OLFM4) and intercellular adhesion molecule 1 (ICAM1) individually and in various combinations in order to determine the optimum measurement strategy allowing for the highest sensitivity and specificity at the lowest measurement expenses. The test result additionally includes the % fPSA proportion (ratio of fPSA to tPSA). A combined score is calculated using the above-mentioned formula (1) resulting from a mathematical combination of % fPSA, with each of the systems CTSD, THBS1, OLFM4 and ICAM1, as well as all possible combinations of % fPSA, with a pair of the systems CTSD, THBS1, OLFM4 and ICAM1. Also % fPSA is looked at with CTSD and THBS1 and optionally OLFM4 and ICAM1. The tests were evaluated in a validation study to show its added value to established clinical parameters such as % fPSA in improving PCa diagnosis.

(61) Objective: The objective of the study was to test which combination of the proposed protein biomarkers is able to reduce the number of negative biopsies in men with elevated tPSA (2-10 ng/ml) most efficiently and most reliably. In addition, men included in the study had negative (lack of suspicion for the presence of cancer) digital rectal examination (DRE) and enlarged prostates. This patient subgroup is considered as the most difficult to decide if a prostate biopsy should be performed, because many men have benign conditions, resulting in increased tPSA values in that range, resulting in large numbers of false positive cases. The goal of the study was to reduce the number of negative biopsies by at least 50% while having at least 90% sensitivity for PCa and a high (90-95%) negative predictive value (NPV) for high-grade PCa (Gleason score ≥7). The NPV is an important measure for the urologists indicating the probability that a negative result is truly negative.

(62) Design, setting and participants: A retrospective study of men with a tPSA 2-10 ng/ml cancer negative DRE and enlarged prostate (volume ≥35 ml) who underwent prostate biopsy was conducted. Whole blood samples of prostate cancer-positive and negative men were collected before undergoing prostate biopsy at a leading cancer center in Northern Europe. All samples were taken between 2011-2016 following written patient consent.

(63) Measurements: Serum tPSA and fPSA were analyzed for all samples using the ADVIA Centaur immunoassay system (Siemens Healthcare). Concentration levels of CTSD, THBS1, OLFM4 and ICAM1 were measured using the developed bead-based immunoassays as detailed above. In addition, concentration levels of CTSD and THBS1 were measured using the developed ELISA immunoassays.

(64) Results: Of the 474 men included in this study, 236 men had a negative biopsy and 238 were diagnosed with PCa based on biopsy. Of these, 130 had low-grade (Gleason score ≤6) and 106 had high-grade PCa (Gleason score ≥7) according to prostate biopsy. Optimally correlating the measured concentration/proportion values of the samples with the actual biopsy results of the men included in this study using the above-mentioned formula for the combined score value optimizing the logistic regression formula for a maximum area under the curve was used to determine optimum sensitivity and specificity.

(65) The ROC curves illustrated in FIGS. 2-11,13, and 15-17 are training curves, i.e. the curves obtained after optimization of the model correlating in an optimized manner the measured proportion of % fPSA, and the respective concentrations of the proteins with the true diagnosis based on the prostate biopsies.

(66) Models for CTSD, THBS1, OLFM4 and ICAM1 Individually

(67) In a first step, the four proteins to be analyzed were looked at individually and the optimized combined score value was determined for the respective protein alone as well as in combination with % fPSA.

(68) FIGS. 2-5 show the optimized receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa, for the case when each protein of the group THBS1, CTSD, ICAM1 and OLFM4 is measured alone, when % fPSA is measured alone, and when the concentration of the respective protein is used together with the proportion of % fPSA for the evaluation of the combined score value.

(69) FIGS. 15 and 16 show the optimized ROC training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa, for the case when THBS1 and CTSD are measured alone using ELISA, when % fPSA is measured alone, and when the concentration of the respective protein measured by ELISA is used together with the proportion of % fPSA for the evaluation of the combined score value.

(70) As one can see from the ROC curves, by far the best sensitivity and specificity is made available by THBS1, which so far was known to have some indicative value, but which so far has never been identified to be such a strong and reliable correlator. What one can also recognize is that CTSD, ICAM1 and OLFM4 looked at individually hardly give any benefit when comparing with % fPSA alone. In particular one notices that CTSD is essentially uncorrelated and seems to have no diagnostic value when looked at individually or in combination with % fPSA.

(71) Total PSA concentration values are conventionally used for negative prediction of PCa because of the high sensitivity of total PSA. But as one can see from FIG. 2, the total PSA concentration values are not helpful at all for a positive prediction because of the low specificity of total PSA. As a matter of fact, only if the % fPSA value is used, reasonable AUC values can be obtained, and further only if the % fPSA values are used combined with THBS1 a high-sensitivity can be obtained. Also, if the total PSA values are evaluated in combination with THBS1, only a specificity at 90% sensitivity which is significantly below 50% is obtained and an AUC value significantly below 0.83.

(72) Models for Pairs Selected from CTSD, THBS1, OLFM4 and ICAM1 in Combination with % fPSA

(73) In a second step, the four proteins to be analyzed were looked at in pairs and the optimized combined score value was determined for the respective protein pair alone as well as in combination with % fPSA.

(74) FIGS. 6-11 show the optimized receiver operating characteristic (ROC) training curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa, for the case when all possible parings of the proteins of the group THBS1, CTSD, ICAM1 and OLFM4 are measured alone, when % fPSA is measured alone, and when the concentration of the respective protein pairings is used together with the proportion of % fPSA for the evaluation of the combined score value.

(75) As one can see from the ROC curves, by far the best sensitivity and specificity is made available by THBS1 in combination with CTSD, which is very surprising, since CTSD alone is not giving any discrimination (see FIG. 3). This was confirmed when measuring THBS1 and CTSD using ELISA (see FIG. 17). Also THBS1 with ICAM1 and OLFM4 lead to some additional benefit, but not to the same extent as THBS1 combined with CTSD, which is completely unexpected considering the superior behavior of ICAM1 and OLFM4 taken alone (see FIGS. 4 and 5) compared with CTSD alone (see FIG. 3). There seems to be a high degree of correlation between THBS1 and CTSD levels in blood, serum and plasma hitherto unknown.

(76) By contrast, the parings not involving THBS1 do not give any additional benefit, as can be seen from FIGS. 9-11. This is also unexpected, since also in these cases there could have been a high correlation leading to an additional benefit in the ROC analysis. However, no such benefit could be identified on the tested cohort.

(77) Specific Model: % fPSA, CTSD, THBS1

(78) FIG. 6 shows Receiver Operating Characteristic (ROC) curves for this approach. % fPSA resulted in an AUC=0.6498 (P<0.001; 95% CI=0.6004-0.6992). CTSD and THBS1 together discriminated between men with negative and positive prostate biopsies with an AUC=0.8343 (P<0.001; 95% CI=0.7974-0.8712). The combination of % fPSA, CTSD THBS1 resulted in an AUC=0.8448 (P<0.001; 95% CI=0.8097-0.8798). These results were confirmed when measuring THBS1 and CTSD using ELISA (see FIG. 17). CTSD and THBS1 together discriminated between men with negative and positive prostate biopsies with an AUC=0.8376 (P<0.001; 95% CI=0.8010-0.8742). The combination of % fPSA, CTSD THBS1 resulted in an AUC=0.8508 (P<0.001; 95% CI=0.8161-0.8855). At ≥90% sensitivity for positive prostate biopsy and thus PCa, the specificity of the combination of % fPSA, CTSD and THBS1 was 60%. In comparison, the % fPSA test commonly used in clinical practice had a specificity of 21% at the same sensitivity. This shows that CTSD and THBS1 combined with % fPSA would have avoided 141 (60%) of 236 negative biopsies and delayed the diagnosis of 10% of PCa (FIG. 12). In addition, a high NPV of 94% for high-grade PCa (Gleason score ≥7) was achieved. These results indicate that the combination of % fPSA, CTSD and THBS1 results in a significant improvement.

(79) FIG. 12 illustrates the reduction of negative biopsies at 90% sensitivity for positive prostate biopsy and thus PCa. Percentages are given for % fPSA alone, CTSD and THBS1, and the combination of % fPSA, CTSD and THBS1. These results were confirmed when measuring THBS1 and CTSD using ELISA (see FIG. 18).

(80) In this model like in the other cases, based on the measured concentrations of % fPSA alone, CTSD and THBS1, and the combination of % fPSA, CTSD and THBS1, respectively, in each case a combined score value is calculated using the above-mentioned formula (1) with the constants obtained after optimization:

(81) Logistic Regression Formula:

(82) The logistic regression model used in all these results provides an estimate of the coefficients to be used in the equation (1):

(83) $\begin{array}{cc}\frac{1}{1+e^{-\left({\beta }_0+{\beta }_1.Math.x_1+...+{\beta }_k.Math.x_k\right)}}& \left(1\right)\end{array}$
Where the βs are the regression coefficients, β.sub.0 being the intercept, and the x.sub.i are the values of the matching independent variables. The result (p.sub.i) is the probability for an observation with the given pattern of values of the independent variables to have the event.
These p.sub.i are the scores that are used to build the ROC curve.

(84) Variables in this equation for the situation of the specific Model: % fPSA, THBS1 is as follows:

(85) TABLE-US-00002 Standard Wald Parameter DF B Error Chi-Square Pr > ChiSq Exp(B) Intercept 1 4.663205 0.486095 92.029501 8.54E−22 105.975153 THBS1 1 −0.000053 0.000005 95.644963 1.37E−22 0.999947 % fPSA 1 −5.381332 1.372877 15.364416 0.000089 0.004602
For the situation where the concentration of THBS1 as well as the % fPSA proportion is measured in the first step, for the calculation of the combined score value the regression coefficients are as follows:

(86) β.sub.0=4.663; β.sub.THBS1=−0.000053; β.sub.% fPSA=−5.381.

(87) For a 90% sensitivity a threshold value of the combined score value of larger than 0.316 is given.

(88) Variables in this equation for the situation of the specific Model: % fPSA, THBS1, where THBS1 is measured using ELISA, is as follows:

(89) TABLE-US-00003 Standard Wald Parameter DF B Error Chi-Square Pr > ChiSq Exp(B) Intercept 1 4.902422 0.504822 94.307202 2.70E−22 134.615457 THBS1 1 −0.000086 0.000009 96.648592 8.28E−23 0.999914 % fPSA 1 −5.145655 1.410257 13.313279 0.000264 0.005825

(90) For the situation where the concentration of THBS1 (using ELISA) as well as the % fPSA proportion is measured in the first step, for the calculation of the combined score value the regression coefficients are as follows:

(91) β.sub.0=4.902; β.sub.THBS1=−0.000086; β.sub.% fPSA=−5.146.

(92) For a 90% sensitivity a threshold value of the combined score value of larger than 0.335 is given.

(93) Variables in this equation for the situation of the specific Model: % fPSA, CTSD, THBS1 is as follows:

(94) TABLE-US-00004 Wald Standard Chi- Parameter DF B Error Square Pr > ChiSq Exp(B) Intercept 1 3.233 0.561 33.248 8.113E−9 25.367 CTSD 1 0.010 0.002 19.340 0.000011 1.010 THBS1 1 −0.000063 0.000006 102.843 3.6272E−24 1.000 % fPSA 1 −4.946 1.393 12.612 0.00383  0.007

(95) For a 90% sensitivity a threshold value of the combined score value of larger than 0.330 is given.

(96) Thus, for example when the following concentrations/proportions are measured/determined in a specific sample:

(97) TABLE-US-00005 UPN CTSD (ng/ml) THBS1 (ng/ml) % fPSA (0-1) HH0106 227 78501 0.27

(98) The following is to be evaluated:

(99) $p_i=\frac{1}{1+e^{-\left({\beta }_0+{\beta }_1.Math.x_{1,i}+...+{\beta }_k.Math.x_{k,i}\right)}}=p_{\mathrm{HH}0106}=\frac{1}{1+e^{-\left(\mathrm{Intercept}+\mathrm{CTSD}+\mathrm{THBS}1+\%\mathrm{fPSA}\right)}}=P_{\mathrm{HH}0106}=\frac{1}{1+e^{-\left(3.233+0.010*227-0.000063*78501-4.946*0.27\right)}}=p_{\mathrm{HH}0106}=0.31644$
For a 90% sensitivity for positive prostate biopsy and thus PCa, the threshold of the combined score value p.sub.i is 0.330, so for the sample HH0106 no prostate biopsy would be advised since the corresponding concentration values did not indicate a positive prostate biopsy and thus the presence of PCa.

(100) Variables in this equation for the situation of the specific Model: % fPSA, CTSD, THBS1, where CTSD and THBS1 are measured using ELISA, is as follows:

(101) TABLE-US-00006 Wald Standard Chi- Parameter DF B Error Square Pr > ChiSq Exp (B) Intercept 1 3.892 .567 47.116 6.69E−12 49.022 CTSD 1 0.004 0.001 11.997 .000533 1.004 THBS1 1 −0.000100 0.000010 99.352 2.11E−23 1.000 % fPSA 1 −4.843 1.435 11.391 .000738 0.008
For a 90% sensitivity a threshold value of the combined score value of larger than 0.362 is given.

(102) For the situation where the concentration of THBS1, the proportion of free PSA (% fPSA), as well as the concentration of OLFM4 is measured in the first step, for the calculation of the combined score value the regression coefficients are as follows:

(103) β.sub.0=4.606; β.sub.OLFM4=0.00271; β.sub.THBS1=−0.000054; β.sub.% fPSA=−5.423.

(104) For a 90% sensitivity a threshold value of the combined score value of larger than 0.323 is given.

(105) For the situation where the concentration of THBS1, the proportion of free PSA (% fPSA), as well as the concentration of ICAM1 is measured in the first step, for the calculation of the combined score value the regression coefficients are as follows:

(106) β.sub.0=4.769; β.sub.ICAM1=−0.00084; β.sub.THBS1=−0.000053; β.sub.% fPSA=−5.395.

(107) For a 90% sensitivity a threshold value of the combined score value of larger than 0.318 is given.

(108) Model: % fPSA, CTSD, THBS1, OLFM4, ICAM1

(109) FIG. 13 shows Receiver Operating Characteristic (ROC) curves for this approach. % fPSA resulted in an AUC=0.6498 (P<0.001; 95% CI=0.6004-0.6992). CTSD, THBS1, OLFM4 and ICAM1 together discriminated between men with negative and positive prostate biopsies with an AUC=0.8345 (P<0.001; 95% CI=0.7978-0.8713). The combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1 resulted in an AUC=0.8463 (P<0.001; 95% CI=0.8122-0.8817).

(110) FIG. 13 shows receiver operating characteristic (ROC) curves depicting the accuracy of individual predictors of positive prostate biopsy and thus PCa. % fPSA alone, CTSD, THBS1, OLFM4, ICAM1, and the combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1 ROC curves are shown.

(111) At ≥90% sensitivity for positive prostate biopsy and thus PCa, the specificity of the combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1 was 58%. In comparison, the % fPSA test commonly used in clinical practice had a specificity of 21% at the same sensitivity. This shows that the proposed set of markers combined with % fPSA would have avoided 136 (58%) of 236 negative biopsies and delayed the diagnosis of 10% of PCa (FIG. 13). In addition, a high NPV of 93% for high-grade PCa (Gleason score ≥7) was achieved. These results indicate that the combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1 results in a significant improvement over % fPSA alone, but no significant further improvement over % fPSA, CTSD, THBS1.

(112) FIG. 14 shows the reduction of negative biopsies at 90% sensitivity. Percentages given for % fPSA, CTSD, THBS1, OLFM4 and ICAM1, and the combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1.

(113) Also here, the ROC curves illustrated in FIG. 13 are training curves, i.e. the curves obtained after optimization of the model correlating in an optimized manner the measured concentrations with the true diagnosis based on the biopsies.

(114) Also in this model, based on the measured concentrations of % fPSA alone, CTSD, THBS1, OLFM4 and ICAM1, and the combination of % fPSA, CTSD, THBS1, OLFM4 and ICAM1, respectively, in each case a discriminator is calculated using the following formula with the constants obtained after optimization:

(115) β.sub.0=3.567; β.sub.OLFM4=0.002; β.sub.THBS1=−0.000063; β.sub.CTSD=0.01; β.sub.ICAM1=−0.003; β.sub.% fPSA=−5.033.

(116) For a 90% sensitivity a threshold value of the combined score value of larger than 0.329 is given.

(117) In terms of the formulae for p this means the following:

(118) $p_i=\frac{\left(1\right)}{1+e^{-\left(3.567+0.01\left[\mathrm{CTSD}\right]-0.000063\left[\mathrm{THBS}1\right]+0.002\left[\mathrm{OLFM}4\right]-0.003\left[\mathrm{ICAM}1\right]-5.033*\left(\frac{\mathrm{fPSA}}{\mathrm{tPSA}}\right)\right)}}$$\mathrm{cut}\text{-}\mathrm{off}{p}_{i=90\%\mathrm{sensitivity}}=0.329\left(\text{>}0.329=\mathrm{positive}\right).$

(119) Conclusions:

(120) Unexpectedly, the combination of THBS1 and % fPSA, and even more so the combination of CTSD, THBS1 and % fPSA is significantly more accurate than tPSA or % fPSA alone in determining the absence of PCa in men with elevated tPSA (2-10 ng/ml), enlarged prostate (≥35 ml) and negative DRE. The implementation of a test comprising these three parameters in clinical practice has the potential to significantly lower the rate of unnecessary biopsies by up to 60%.