Use of a marker set for prostate carcinoma diagnostics

Abstract

The invention relates to the use of a marker set comprising at least four substances in an in- vitro method for determining whether a patient suffers from a prostate carcinoma. The at least four substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, and v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol such that substances of at least four of subgroups i) to v) are present in the marker set.

Description

[0001] Use of a marker set for prostate carcinoma diagnostics

[0002] Description

[0003] The present invention relates to the in-vitro use of a marker set for determining whether a patient suffers from a prostate carcinoma according to the preamble of claim 1 , to the further medical use of such a marker set according to the preamble of claim 7 as well as to an analysis method for determining whether a patient suffers from a prostate carcinoma according to the preamble of claim 8.

[0004] Prostate cancer (PCa) affects 1 out of 6 to 8 men. 15 to 35 % of newly diagnosed prostate cancers are locally advanced and / or metastatic. The currently applied standard for diagnosing prostate cancer is determining the concentration of prostate-specific antigen (PSA). However, PSA has low specificity so that there is a high number of patients who obtain a misdiagnosis and an overtreatment.

[0005] The assessment of PCa and its distinction from other prostate conditions is typically based on prostate biopsy results. This assessment has low detection sensitivity and is therefore prone to misclassify the cancer. The invasive nature of the biopsy is associated with a risk of infection.

[0006] Consequently, there exists a need for novel biomarkers to improve clinical decision-making and management of PCa.

[0007] It is an object of the present invention to provide novel methods and biomarkers for determining whether a patient suffers from a prostate carcinoma.

[0008] This object is achieved with the in-vitro use of a marker set having the claim elements of claim 1. Such a marker set comprises at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14) substances chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, and v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol. The substances are chosen such that substances of at least four of subgroups i) to v) are present in the marker set (e.g., substances belonging to subgroups i), ii), iii), and iv)). I.e., if exactly four substances are chosen, each substance belongs to a different subgroup. If more than four substances are chosen, they can all belong to different subgroups (in case that exactly five substances are chosen). Alternatively, some of the substances may belong to the same subgroup, as long as substances of at least four subgroups are present in the marker set. According to an aspect of the present invention, this marker set is used for determining whether a patient suffers from a prostate carcinoma (regardless of being aggressive or indolent), i.e., for distinguishing between PCa-positive patients and healthy individuals.

[0009] For this purpose, the concentration of the substances contained in the marker set is determined in a body fluid obtained from a patient. This concentration determination can be carried out by any appropriate measuring or analysis method, such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and infrared spectroscopy such as Fourier-transform infrared (FT-IR) spectroscopy.

[0010] An alteration of concentration of at least four substances of the marker set with respect to the concentration in a control group or an alteration of a concentration ratio between at least four substances with respect to the concentration ratio of the same substances in a control group was correlated in a statistically significant way with the presence of prostate cancer at the time of analysis (i.e., enabling a distinction between prostate cancer and healthy individuals).

[0011] For identifying the marker set and the substances that can make up the marker set, urine (as representative body fluid) of patients suffering from prostate cancer was used. These patients showed

[0012] • i) a PSA concentration in blood of more than 4 ng / ml and ii) a tumor detection by needle biopsy or an extension of the tumor beyond the prostate capsule and showing no signs of metastasis; or

[0013] • a newly diagnosed prostate cancer with high tumor burden, confirmed by i) an advanced local tumor, and ii) a PSA concentration in blood of more than 10 ng / ml, and iii) a Gleason score of at least 7 and / or bone metastases.

[0014] The Gleason scoring system is the most common grading system used to assess the aggressiveness of prostate cancer (i.e., for grading prostate cancer). It is based on the histological evaluation of the prostate tissue obtained after prostatectomy. The score ranges from 1 to 5 according to the pattern of cell growth of the tumor. The total score (Gleason sum) is the sum of two grades: A primary grade given to the predominant (most extensive) cell morphology and a secondary grade describing the cells of the next largest area of the tumor. The Gleason sum can range from 2 (non-aggressive cancer) to 10 (very aggressive cancer). The higher the score, the more aggressive is the cancer. The Gleason scoring system is described, e.g., in detail by Humphrey (Humphrey, P. A. (2004). Gleason grading and prognostic factors in carcinoma of the prostate. Modem Pathology, 17(3), 292-306) and by Shah and Zhou (Shah, R. B., & Zhou, M. (2016). Recent advances in prostate cancer pathology: Gleason grading and beyond. Pathology international, 66(5), 260-272).

[0015] The control group consisted of healthy individuals. These individuals showed no sign of a prostatic disease. In particular, they i) showed no palpable abnormalities in a rectal examination, and ii) had a prostate volume of not more than 25 ml, and iii) had a PSA concentration in blood of less than 1.5 ng / ml.

[0016] When testing individual biomarkers of the substances contained in the marker set, two biomarkers of this marker set at the same time, or even three biomarkers of this marker set at the same time, no significant results could be obtained for distinguishing the two groups (prostate cancer versus healthy individuals). Rather, the area under the curve (AUC) values of receiver operating characteristic (ROC) plots showed values mainly lying in a range of from 0.5 to 0.65. The AUC value of ROC plots is an aggregated metric that evaluates how well a logistic regression model classifies positive and negative outcomes at all possible cut-offs. It can range from 0 to 1 .0. An AUC value of 0 represents a prediction of the opposite of the trained correlation. An AUC value of 0.5 represents a random prediction. An AUC value of higher than 0.5 represents a classification of an event as fulfilling the trained correlation wherein higher values represent better classification.

[0017] The marker sets were tested against training datasets and test datasets and iteratively crossvalidated. Cross-validation was performed by splitting the training dataset into, e.g., five parts and by using four parts for training (training subset) and the fifth part for testing (testing subset). The individual parts were iteratively removed from and returned to the training set so that each of the five parts belonged - in different training rounds - to the training subset and to the testing subset.

[0018] After all test and validation processes, scyllo-inositol; at least one substance chosen from L- threonine, galactaric acid, and D-galactonic acid; at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate; trigonelline; and at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol turned out to be valid biomarkers for the underlying question (i.e., distinguishing between prostate cancer patients and healthy individuals). However, a valid distinction could only be made if the concentration of at least four of these biomarkers was determined at the same time (i.e., in one or more body fluid samples from the same patient obtained at the same time point). Furthermore, it is necessary that these at least four biomarkers belong to at least four different subgroups. The first subgroup consists of scyllo- inositol. The second subgroup consists of L-threonine, galactaric acid, and D-galactonic acid. The third subgroup consists of cotinine, 2-ethylpyrazine, inosine triphosphate, and formate. The fourth subgroup consists of trigonelline. The fifth subgroup consists of 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

[0019] The concentration determination can be made with a method being able to determine the concentration of the substances by a single measurement or by a method requiring more than one measurement for such determination. NMR spectroscopy is particularly appropriate for such a concentration determination since it enables a highly accurate concentration determination in a body fluid by a single measurement in a very short measuring time.

[0020] In an embodiment, the concentration of the substances is standardized to the concentration of creatinine in the same sample or, alternatively, to the concentration of another substance that is naturally present in the sample.

[0021] In an embodiment, the marker set comprises or consists of i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) trigonelline, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, and iv) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol. Such a marker set showed an AUC value of 0.758 in the test dataset and an AUC value of 0.681 in the training dataset (cf. Figure

[0022] 1)-

[0023] In an embodiment, the marker set comprises or consists of i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) scyllo-inositol, iii) trigonelline, and iv) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol. Such a marker set showed an AUC value of 0.736 in the test dataset and an AUC value of 0.696 in the training dataset (cf. Figure

[0024] 2).

[0025] In an embodiment, the at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate is or comprises formate. In an embodiment, the at least one substance chosen from 4-aminohippuric acid, 4- aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol is or comprises 4- aminohippuric acid.

[0026] In an embodiment, the marker set comprises trigonelline.

[0027] In an embodiment, the marker set comprises formate.

[0028] In an embodiment, the marker set comprises 4-aminohippuric acid.

[0029] In an embodiment, the marker set comprises at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid.

[0030] In an embodiment, the marker set comprises at least one substance chosen from 4- aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p- Cresol.

[0031] In an embodiment, the marker set comprises scyllo-inositol .

[0032] Upon analyzing a plurality of NMR spectra for identifying appropriate biomarkers for the underlying question, the inventors were able to identify NMR signals in bin A232 that appeared to be highly appropriate for determining whether a patient suffers from prostate cancer. This bin comprises a doublet signal around 5.60 ppm, wherein a first line of the doublet lies under exemplary measuring conditions at approximately 5.620 ppm and a second line of the doublet lies under the same measuring conditions at approximately 5.607 ppm. So far, the inventors were not yet successful in assigning a specific metabolite to these signals observed in bin A232. Therefore, the metabolite being responsible for the signals in bin A232 will be referred to in the following as substance Y.

[0033] In an independently claimed aspect, the present invention relates to uses of a marker set comprising the substances listed above and additionally comprising substance Y, as well as to related methods. It turned out that it is fully sufficient to use three substances as biomarkers for determining whether a patient has prostate cancer, as long as substance Y is one of these three substances.

[0034] Therefore, in an independently claimed aspect, the present invention relates to the in-vitro use of a marker set comprising at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15) substances chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol, and vi) substance Y. The substances are chosen such that the marker set comprises substance Y and at least two substances of two different subgroups i) to v) (e.g., substance Y and substances belonging to subgroups i) and ii); or substance Y and substances belonging to subgroups iii) and iv)).

[0035] In an embodiment, the marker set comprises or consists of i) substance Y, ii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iii) trigonelline, and iv) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol. Such a marker set showed an AUC value of 0.739 in the test dataset and an AUC value of 0.695 in the training dataset (cf. Figure 3).

[0036] In an embodiment, the marker set comprises or consists of i) substance Y, ii) scyllo-inositol, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, and iv) trigonelline. Such a marker set showed an AUC value of 0.674 in the test dataset and an AUC value of 0.695 in the training dataset (cf. Figure 4).

[0037] In an embodiment, the marker set comprises or consists of i) substance Y, ii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, and iii) trigonelline. Such a marker set showed an AUC value of 0.674 in the test dataset and an AUC value of 0.679 in the training dataset (cf. Figure 5). Thus, the performance of this marker set was only slightly less good than the performance of the marker set additionally comprising at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol. While any of these substances slightly increases the performance of the marker set, very good result can already be obtained with the marker set comprising or consisting of i) substance Y, ii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, and iii) trigonelline.

[0038] In an embodiment, the marker set comprises or consists of i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) substance Y, iii) trigonelline, and iv) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate. Such a marker set showed an AUC value of 0.658 in the test dataset and an AUC value of 0.698 in the training dataset (cf. Figure 6). In an embodiment, the marker set comprises or consists i) substance Y, ii) scyllo-inositol, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, and iv) trigonelline. Such a marker set showed an AUC value of 0.655 in the test dataset and an AUC value of 0.690 in the training dataset (cf. Figure 7).

[0039] In an embodiment, the marker set comprises substance Y and trigonelline.

[0040] In an embodiment, the marker set comprises substance Y and at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate.

[0041] In an embodiment, the marker set comprises substance Y and formate.

[0042] In an embodiment, the marker set comprises substance Y and scyllo-inositol.

[0043] In an embodiment, the marker set comprises substance Y and at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

[0044] Upon analyzing a plurality of NMR spectra, the inventors found out that, under certain preconditions, a marker set comprising at least three biomarkers is also able to distinguish patients having prostate cancer from healthy individuals, even if substance Y does not form part of the marker set.

[0045] Therefore, in an independently claimed aspect, the present invention relates to the in-vitro use of a marker set comprising at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15) substances chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol, and optionally vi) substance Y. The substances are chosen such that the marker set necessarily comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) scyllo-inositol, and iii) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol. In an aspect, the present invention relates to the further medical use of a marker set comprising at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14) substances chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, and v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p- Cresol for in-vivo diagnostics of prostate cancer, in particular for in-vivo determining whether a patient has prostate cancer or is healthy. The substances are chosen such that substances of at least four of subgroups i) to v) are present in the marker set (e.g., substances belonging to subgroups i), ii), iii), and iv)). I.e., if exactly four substances are chosen, each substance belongs to a different subgroup. If more than four substances are chosen, they can all belong to different subgroups (in case that exactly five substances are chosen). Alternatively, some of the substances may belong to the same subgroup, as long as substances of at least four subgroups are present in the marker set.

[0046] In an aspect, the present invention relates to the further medical use of a marker set comprising at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15) substances chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2- ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol, and vi) substance Y for in-vivo diagnostics of prostate cancer, in particular for in-vivo determining whether a patient has prostate cancer or is healthy. The substances are chosen such that the marker set comprises substance Y and at least two substances of two different subgroups i) to v) (e.g., substance Y and substances belonging to subgroups i) and ii); or substance Y and substances belonging to subgroups iii) and iv)).

[0047] In an aspect, the present invention relates to the further medical use of a marker set comprising at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15) substances chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2- ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol, and optionally vi) substance Y for in-vivo diagnostics of prostate cancer, in particular for in-vivo determining whether a patient has prostate cancer or is healthy. The substances are chosen such that the marker set necessarily comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) scyllo-inositol, and iii) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol.

[0048] In an aspect, the present invention relates to a method for analyzing an isolated body fluid sample in vitro, comprising the steps explained in the following. This method is carried out on an isolated body fluid sample originating from an individual.

[0049] In a first step, the concentration of at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14) substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least four substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D- galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, and v) at least one substance chosen from 4- aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p- Cresol. A very well suited measuring technique for determining the concentration of the individual substances is nuclear magnetic resonance spectroscopy (NMR spectroscopy). The substances are chosen such that substances of at least four of subgroups i) to v) are present in the marker set (e.g., substances belonging to subgroups i), ii), iii), and iv)).

[0050] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for determining whether a patient has prostate cancer or is considered healthy.

[0051] The score can be calculated by taking into consideration the concentrations measured or expected in a body fluid sample from a control group. To give a simple example, the score can be the median of the concentration ratios of the at least four substances between the body fluid test sample of the patient and corresponding control values of a body fluid control sample that have been measured the past. If the score is above a predetermined threshold value, a significant increase of the marker substances is present in the body fluid test sample that is indicative for the presence of prostate cancer. It should be noted that other calculation methods as well as a weighting of individual marker concentrations with respect to other marker concentrations can also be performed in an embodiment.

[0052] A suited way to calculate the score is disclosed on pages 25 to 27 of WO 2012 / 045773 A9. Another suited way to calculate the score is the following: wherein n at = a + > bx• Ix—L

[0053] X=1 a = const. bx= substance specific coefficient

[0054] I = parameter being indicative for the concentration of substance x

[0055] Thereby, the individual factors a, b need to be adjusted according to the underlying model and can vary in dependence on the specific substances considered in the marker set. Parameter “I” can be, e.g., the signal intensity or signal integral of an according signal observed in the evaluated measuring result. To give an example, “I” can be the signal intensity or signal integral of an NMR signal in an NMR spectrum if NMR spectroscopy is used as measuring technique.

[0056] In an embodiment, “I” is a ratio between two signal intensities or two signal integrals. In such a case, it is, e.g., possible to standardize the concentration of a first substance (or a plurality of substances) by the concentration of a second substance such as, e.g., creatinine.

[0057] The score is a (semi-)quantitative measure for distinguishing patients suffering from a prostate carcinoma (either aggressive or indolent) from healthy individuals. Thus, the score serves for (semi-)quantitatively distinguishing for distinguishing patients suffering from a prostate carcinoma (either aggressive or indolent) from healthy individuals.

[0058] Calculating the score comprises multiplying each of the concentrations of the substances by a substance-specific weighting factor to provide a plurality of weighted values and combining the weighted values into a risk equation. Afterwards, an output of the risk equation is compared to a predefined threshold. If the score is above the threshold, there is a likelihood that a patient suffers from prostate carcinoma. In an embodiment, the likelihood is higher, the higher the score is (i.e., the likelihood increases with increasing distance of the score from the threshold).

[0059] In an embodiment, the calculated score is output and presented to the individual and / or to a third person such as a physician or medical staff. The output can be performed on a display (i.e., in an electronic way) or in printed form. Thereby, it is also possible to generate a report indicating the score, optionally in combination with a comparative scale of possible scores and their meaning with respect to distinguishing prostate cancer patients from healthy individuals.

[0060] In an embodiment, the method is a computer-implemented method. In particular, all steps of spectral analysis and concentration determination as well as of score calculation are performed on a computer. Such steps are far too complex to be done in a manual way. The computer- implemented concentration determination is, in an embodiment, based on a spectral analysis, such as an analysis of NMR spectra. The spectral analysis and the further required steps until the score can be output can be done on the same computer that is used for controlling a spectrometer performing the spectral analysis or on a different computer.

[0061] In an embodiment, the body fluid sample is a urine sample or a blood sample. In an embodiment, the blood sample is a whole blood sample, a blood serum sample, a blood plasma sample, or any other blood preparation derivable from whole blood or from other blood preparations.

[0062] In an embodiment, the body fluid sample (and therewith the patient from whom the body fluid sample originates) is grouped into one of at least two predefined groups based on the calculated score. Typically, one group encompasses patients suffering from prostate cancer, wherein the other group encompasses healthy individuals. The resulting grouping can also be indicated on an according report.

[0063] In an aspect, the present invention relates to another method for analyzing an isolated body fluid sample in vitro, comprising the steps explained in the following. This method is done on an isolated a body fluid sample originating from an individual.

[0064] In a first step, the concentration of at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15) substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least three substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4- aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p- Cresol, and vi) substance Y. The substances are chosen such that the marker set comprises substance Y and at least two substances of two different subgroups i) to v).

[0065] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for determining whether a patient has prostate cancer or is considered healthy.

[0066] In an aspect, the present invention relates to another method for analyzing an isolated body fluid sample in vitro, comprising the steps explained in the following. This method is done on an isolated a body fluid sample originating from an individual. In a first step, the concentration of at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15) substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least three substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4- aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p- Cresol, and optionally vi) substance Y. The substances are chosen such that the marker set necessarily comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) scyllo-inositol, and iii) at least one substance chosen from 4- aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p- Cresol.

[0067] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for determining whether a patient has prostate cancer or is considered healthy.

[0068] In an aspect, the present invention relates to a medical method for diagnosing prostate cancer (and thus distinguishing prostate cancer patients from healthy individuals). This method comprises the steps explained in the following.

[0069] In a first step, a body fluid sample is gathered from a patient. In a second step, the concentration of at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14) substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least four substances are chosen from the group consisting of i) scyllo- inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, and v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol. The substances are chosen such that substances of at least four of subgroups i) to v) are present in the marker set (e.g., substances belonging to subgroups i), ii), iii), and iv)).

[0070] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for determining whether the patient has prostate cancer. In a further method step, a diagnosis on the presence of prostate cancer is made. The respective result is then output to the patient or to a third person like a physician or medical staff.

[0071] In a further aspect, the present invention relates to another medical method for diagnosing prostate cancer (and thus distinguishing prostate cancer patients from healthy individuals). This method comprises the steps explained in the following.

[0072] In a first step, a body fluid sample is gathered from a patient. In a second step, the concentration of at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15) substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least three substances are chosen from the group consisting of i) scyllo- inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4- aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol, and vi) substance Y. The substances are chosen such that the marker set comprises substance Y and at least two substances of two different subgroups i) to v).

[0073] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for determining whether the patient has prostate cancer.

[0074] In a further method step, a diagnosis on the presence of prostate cancer is made. The respective result is then output to the patient or to a third person like a physician or medical staff.

[0075] In a further aspect, the present invention relates to another medical method for diagnosing prostate cancer (and thus distinguishing prostate cancer patients from healthy individuals). This method comprises the steps explained in the following.

[0076] In a first step, a body fluid sample is gathered from a patient. In a second step, the concentration of at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15) substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least three substances are chosen from the group consisting of i) scyllo- inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4- aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol, and optionally vi) substance Y. The substances are chosen such that the marker set necessarily comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) scyllo- inositol, and iii) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

[0077] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for determining whether the patient has prostate cancer.

[0078] In a further method step, a diagnosis on the presence of prostate cancer is made. The respective result is then output to the patient or to a third person like a physician or medical staff.

[0079] In a further aspect, the present invention relates to a decision support system for analyzing an isolated body fluid sample in vitro, the decision support system comprising: a) a unit for providing a body fluid sample from an individual; b) a unit for determining the concentration of at least four substances in the body fluid sample from the individual by analyzing the body fluid sample with a suited measuring technique, wherein the at least four substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D- galactonic acid, iii) formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol such that substances of at least four of subgroups i) to v) are present in the marker set; and c) a unit for calculating a score from the determined concentrations, the score being indicative for a likelihood that the body fluid originates from a patient suffering from a prostate carcinoma. In an embodiment, the unit for determining the concentration of the at least four substances is configured to determine the concentration of any substance combination of the embodiments explained above.

[0080] While some of the explained uses and methods are described as in vitro uses and methods and some of the explained uses and methods are described as in vivo uses and methods, it should be noted that each in vitro use or method can also be carried out as in vivo use or method, and vice versa.

[0081] All embodiments of the use of the marker set can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the further medical use of the marker set as well as to the different methods and to the decision support system. Likewise, all embodiments of the further medical use of the marker set can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the use of the marker set, to the different methods, and to the decision support system. Finally, all embodiments of the different methods can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the use of the marker set, to the further medical use of the marker set, to any other of the described methods, and to the decision support system.

[0082] Further details of aspects of the present invention will be explained in the following making reference to exemplary embodiments and accompanying Figures. In the Figures:

[0083] Figure 1 shows an ROC plot of the ability of a first marker set for identifying prostate cancer;

[0084] Figure 2 shows an ROC plot of the ability of a second marker set for identifying prostate cancer;

[0085] Figure 3 shows an ROC plot of the ability of a third marker set for identifying prostate cancer;

[0086] Figure 4 shows an ROC plot of the ability of a fourth marker set for identifying prostate cancer;

[0087] Figure 5 shows an ROC plot of the ability of a fifth marker set for identifying prostate cancer;

[0088] Figure 6 shows an ROC plot of the ability of a sixth marker set for identifying prostate cancer; and Figure 7 shows an ROC plot of the ability of a seventh marker set for identifying prostate cancer.

[0089] All ROC plots shown in Figures 1 to 7 were obtained by analyzing urine samples of patients suffering from a prostate tumor (i.e., having prostate cancer). These patients showed

[0090] • i) a PSA concentration in blood of more than 4 ng / ml and ii) a tumor detection by needle biopsy or an extension of the tumor beyond the prostate capsule and showing no signs of metastasis; or

[0091] • a newly diagnosed prostate cancer with high tumor burden, confirmed by i) an advanced local tumor, and ii) a PSA concentration in blood of more than 10 ng / ml, and iii) a Gleason score of at least 7 and / or bone metastases.

[0092] The control group consisted of healthy individuals. These individuals showed no sign of a prostatic disease. In particular, they i) showed no palpable abnormalities in a rectal examination, and ii) had a prostate volume of not more than 25 ml, and iii) had a PSA concentration in blood of less than 1.5 ng / ml.

[0093] NMR measurements

[0094] All measurements were carried out on a Broker Avance II+ 600MHz or a Broker Avance III HD 600MHz NMR spectrometer, each using a PATXI 1 H / D-13C / 15N Z-GRD probe. All samples were kept at 5-7°C in the SampleJet and brought to the target temperature in the integrated preheating block before measurement. A standard pulse program with 30-degree excitation pulse and pre-saturation for water suppression was used (zgpr30).

[0095] Samples were measured in batches of up to 93 samples per run. In addition to the analytical samples, each run included one Axinon® urine calibrator sample and two Axinon® urine control samples (before and after the analytical urine samples, respectively) in order to assure ideal measurement conditions throughout the run.

[0096] Signal analysis

[0097] NMR spectra underwent automatic referencing, phase correction and baseline correction before further analysis.

[0098] Subsequently, the NMR spectra underwent an automatic standardization and calibration procedure to minimize between-device, between-day and between-run effects. The quality of each of these spectra was assessed by a custom spectrum qualification algorithm that analyzes general spectral properties, e.g., offset and tilt of the baseline in selected spectral regions, and properties of selected indicator signals, e.g., signal position, shape and width. Spectra that did not meet the predefined quality criteria were excluded from further analysis.

[0099] Successfully qualified spectra (typically covering a chemical shift from -5 to 14 ppm) were subjected to further modifications. In particular, broad background signals were separated with a suitable algorithm, e.g., background intensities (such as generated from proteins) were subtracted from the spectra, resulting in spectral intensities devoid of such background signals.

[0100] The cohort (i.e., the plurality) of modified spectra was checked for regions in which the cohort does not show a significant number of signals. These regions - like the region of the water signal and the regions in which additive signals are located - were ignored in the steps explained in the following.

[0101] The remaining spectral regions were subject to an adaptive binning, which divides the spectrum in bins of differing size or extent (typically covering 0.01 to 0.05 ppm, but in extreme cases also covering 0.005 to 0.5 ppm).

[0102] For quantification of individual substances, quantification models making use of the previously assigned bins were applied. After substance identification, substance labels have been assigned to these bins. The quantification was then determined by the bin value, which calculates as [(sum of intensities in bin) / (number of data points in bin)]. The standardization by data points is used to compensate for a varying number of data points in the bins. The number of data points in a bin may vary by one data point due to shifts of the applied discretization grid. A true numerical integration would use a multiplication by the bin extent. However, this is a constant factor and can be omitted.

[0103] Test of identified marker substances

[0104] The identified marker substances were tested in different combinations to assess their suitability for distinguishing an aggressive prostate tumor from an indolent prostate tumor. In doing so, the result of the determination based on the marker substances (probability of prostate tumor aggressiveness) has been checked against clinical signs of an aggressive prostate tumor in the patient who donated the urine sample, as already explained above.

[0105] The results are summarized in receiver operating characteristic (ROC) plots. In these plots, the area under the curve (AUC) indicates the fitness of the prediction. If the AUC is 0.5, the prediction is to be considered random and thus not well suited. The higher the AUC, the better is the prediction model.

[0106] All marker sets, the ROC plots of which are shown in Figures 1 to 7, were able to distinguish between the patient group and the control group in a statistically significant way. The results shown in Figures 1 to 7 are summarized in the following Table 1 .

[0107] Table 1 : Summary of results depicted in Figures 1 to 7.

[0108] Summarizing, the presented marker sets comprise highly appropriate biomarkers for distinguishing between prostate cancer patients and healthy individuals.

Claims

Claims1. Use of a marker set comprising at least four substances in an in-vitro method for determining whether a patient suffers from a prostate carcinoma, wherein the at least four substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, and v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol such that substances of at least four of subgroups i) to v) are present in the marker set.

2. Use according to claim 1 , characterized in that marker set comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) trigonelline, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, and iv) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

3. Use according to claim 1 or 2, characterized in that marker set comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) trigonelline, iii) formate, and iv) at least one substance chosen from 4-aminohippuric acid, 4- aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

4. Use according to any of the preceding claims, characterized in that marker set comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) scyllo-inositol, iii) trigonelline, and iv) at least one substance chosen from 4- aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

5. Use according to any of the preceding claims, characterized in that marker set comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iii) trigonelline, and iv) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

6. Use according to any of the preceding claims, characterized in that marker set comprises i) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, ii) formate, iii) trigonelline, and iv) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol.

7. Marker set comprising at least four substances for use in in-vivo diagnostics of a prostate carcinoma, wherein the at least four substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D-galactonic acid, iii) at least one substance chosen from cotinine, 2-ethylpyrazine, inosine triphosphate, and formate, iv) trigonelline, and v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3- hydroxymandelic acid, acetaminophen, and p-Cresol such that substances of at least four of subgroups i) to v) are present in the marker set.

8. Method for analyzing an isolated body fluid sample in vitro, comprising the following steps: a) determining the concentration of at least four substances in an isolated body fluid sample from an individual by analyzing the body fluid sample with a suited measuring technique, wherein the at least four substances are chosen from the group consisting of i) scyllo-inositol, ii) at least one substance chosen from L-threonine, galactaric acid, and D- galactonic acid, iii) formate, iv) trigonelline, v) at least one substance chosen from 4-aminohippuric acid, 4-aminobenzoic acid, 3-hydroxymandelic acid, acetaminophen, and p-Cresol such that substances of at least four of subgroups i) to v) are present in the marker set, and b) calculating a score from the determined concentrations, the score being indicative for a likelihood that the body fluid originates from a patient suffering from a prostate carcinoma.

9. Method according to claim 8, characterized in that the body fluid sample is a urine sample or a blood sample.

10. Method according to claim 8 or 9, characterized in that that the sample is grouped into one of at least two predefined groups on the basis of the calculated score.

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