Use of a marker set for determining the risk of having or developing bladder cancer

Abstract

The present invention relates to the use of a marker set comprising at least two substances chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid in an in vitro method for determining the risk of an individual to have or to develop bladder cancer.

Description

[0001] Use of a marker set for determining the risk of having or developing bladder cancer

[0002] Description

[0003] The present invention relates to the in-vitro use of a marker set for determining the risk of an individual to have or to develop bladder cancer according to the preamble of claim 1 , to the further medical use of such a marker set according to the preamble of claim 12 as well as to an analysis method for determining the risk of an individual to have or to develop bladder cancer according to the preamble of claim 13.

[0004] Bladder cancer (BCa) is a malignant cancer arising from the epithelial lining (i.e., the urothelium) of the urinary bladder. In case the tumor progresses, the cancer cells may invade underlying layers of the bladder wall as well as other parts of the urinary tract. Bladder cancer grows both flat and papillary. In early stages the carcinoma symptoms are rather unspecific, i.e. none or only few complaints are reported such as hematuria, frequent (painful) urination, increased urge to urinate or pain in the lower abdomen and in the lower back. However, micro hematuria is the most important early symptom. Micro-hematuria (or non-visible hematuria) is a microscopically traceable hematuria.

[0005] Besides age and genetic disposition several lifestyle-associated risk factors were identified for BCa. Tobacco consumption is an essential risk factor; about 50 % of the diagnoses can be traced back to smoking. Furthermore, the exposition to specific chemical substances (in particular aromatic amines, benzene, and colorants), chlorinated drinking water, pain killer abuse (in particular high doses of phenacetin), chemotherapy with cytostatics (in particular on the basis of cyclophosphamide), as well as chronic cystitis and urogenital schistosomiasis, increase the risk of BCa.

[0006] In 2010, the incidence for BCa in the German general population was approximately 15,500

[0001] , With respect to incidences, BCa ranks on positions four and fourteen for men and women, respectively [1]. Worldwide, BCa ranks on position nine with an incidence of approximately 350,000 cases per year [2], The average age at time of diagnosis is 73 years for men and 77 years for women [3]. Men are 3.8-times more likely to be affected by BCa compared to women [4]- The 5-year survival rate is 59 % for men and 50 % for women, respectively [1]. However, survival is highly dependent on tumor stage at time of diagnosis. At initial diagnosis, about 75 % of the patients show a superficial, non-muscle invasive BCa with a 5-year survival rate of more than 90 % [5]. In the remaining 25 % of patients, the tumor already affects deeper layers of the bladder, which significantly increases the cancer-related mortality [4], Depending on stage and grade of the tumor, 5-year survival rates decreases to as low as 65 to 15 %.

[0007] It is thus evident that reliable early detection is essential for curative therapy and a positive prognosis.

[0008] Micro-hematuria represents one of the earliest symptoms of BCa [6]. However, only in 2 to 5 % of the cases, micro-hematuria is caused by a malignant bladder tumor [7], The large majority is traced back to benign urological diseases. Therefore, a key problem in BCa diagnosis is the detection of the relatively rare BCa cases among micro-hematuria patients. [8]. According to the DEGAM Guidelines [9], persistent micro-hematuria in patients > 40 years in presence of an additional risk factor warrants further urologic diagnostics.

[0009] The first diagnostic step is an investigation of potential renal change via ultrasound followed by a cystoscopy. During cystoscopy, a cystoscope - a thin tube with a camera and light on the end - is inserted through the urethra into the bladder for visual examination. In the course of the cystoscopy, samples of suspicious areas of the urethra and urinary bladder can be taken via biopsy. Cystoscopy is considered as “gold standard” for BCa diagnosis. The AUC of narrow band imaging (NBI) cystoscopy was 0.98 and 0.93 for white light imaging (WLI) cystoscopy in a recent meta-analysis

[0010] . However, cystoscopy is invasive, expensive and for the majority of patients with micro-hematuria unnecessary

[0011] , Therefore, according to the DEGAM Guidelines, cystoscopy for patients under age of 40 years - provided that no other BCa risk factors are present - is not recommended as the likelihood of bladder cancer is less than 1 % [9]-

[0010] To avoid unnecessary, expensive and invasive cystoscopies, a wide range of molecular markers for detecting BCa in patients with persistent micro-hematuria were proposed over the last 20 years [8]. Today, there exist several molecular protein-based urine-biomarkers, the following two tests (both FDA approved and CE marked) being the most widely used systems:

[0011] - BTA stat

[0012] with an AUC of 0.75 (95 % confidence interval (Cl): 0.70-0.81 ), and a sensitivity of 67 % (95 % Cl: 0.64-0.69) at a specificity of 75 % (95 % Cl: 73-77 %).

[0012] NMP22 Bladder Chek

[0013] with an AUC of 0.78 (95 % Cl: 0.74-0.83), and a sensitivity of 71 % (95 % Cl: 69-72%) at a specificity of 80 % (95 % Cl: 79-81 %). However, both tests are rarely used in routine diagnostics. The reason for this seems to be twofold.

[0013] First, the published studies report a large variance in sensitivity and specificity. Hence, urologists are potentially confused about the performance of these tests.

[0014] Second, studies reporting a high diagnostic accuracy are often derived from studies comparing healthy individuals to bladder cancer patients, which results in a more difficult assessment of the clinical benefit for patients with urologic comorbidities such as kidney stone or cystitis, which may be associated with micro-hematuria. A main reason might be that these biomarkers are “single biomarkers” focusing on one single aspect of the disease. As a result, it seems that in order to get an acceptable performance, it is necessary to carefully select the population to which these tests can be applied. For example, the overall recommendation is that BTA stat should not be applied in case of micro-hematuria or macro-hematuria as these conditions significantly influence the test results [14, 15]. This limits clinical applicability as hematuria is one of the cardinal symptoms of BCa.

[0015] There are more novel biomarkers in discussions, but none is available for clinical routine application [16, 17], Currently, none of these biomarkers are sufficiently well established in clinical routine to replace cystoscopy or at least reduce the number of unnecessary cystoscopies [9].

[0016] It is an object of the present invention to provide a less invasive method and appropriate biomarkers for determining the risk of an individual to have or to develop bladder cancer.

[0017] 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 two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20) substances chosen from the group consisting of methylpyridinium, 2- furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3- hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid. According to an aspect of the present invention, this marker set is used for determining the risk of an individual to have or to develop bladder cancer. A marker set comprising as few as 2, 3, or 4 of these substances is already very appropriate for determining this risk with a high accuracy (i.e., a high sensitivity and a high specificity) and is thus a particular appropriate embodiment. The term “proteinogenic branched chain amino acid” comprises valine, leucine and isoleucine (being the only proteinogenic branched chain amino acids).

[0018] For the purpose of determining the risk of an individual to have or to develop bladder cancer, 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. These analysis methods are carried out with a measuring device such as an NMR spectrometer, a mass spectrometer, or an infrared spectrometer. Furthermore, it is necessary to carry out these measuring techniques with the help of a computer, since neither the measuring itself nor the evaluation of the measured data represent mental processes that could be done by a human being without assistance of a computer.

[0019] An alteration of concentration of at least two substances of the marker set with respect to the concentration in a control group or an alteration of a concentration ratio between at least two 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 bladder cancer at the time of analysis (i.e., enabling a determination of the risk of an individual to have bladder cancer) or the onset of bladder cancer at a time point after analysis (i.e., enabling a determination of the risk of an individual to develop bladder cancer).

[0020] For identifying the marker set and the substances that can make up the marker set, urine (as representative body fluid) of patients having bladder cancer was used. The control group consisted of i) patients that were subjected to cystoscopy due a suspicion of bladder cancer (e.g., because of having observed a hematuria) but that did not have bladder cancer based on the cystoscopy result and ii) patients not having bladder cancer but an infect of the urogenital tract. Thus, the individuals of the control group can be denoted as patients having a non- cancerous urogenital impairment.

[0021] When testing individual substances identified in the urine, two substances identified in the urine at the same time, or even three or four substances identified in the urine at the same time, no significant results could be obtained for most of the identified substances when trying to distinguish the two groups (bladder cancer patients versus patients having a non-cancerous urogenital impairment). 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.

[0022] However, surprisingly some of the identified substances showed much higher AUC values (above 0.7). This was not the case if the substances were used alone, but only when at least two of these substances were analyzed together.

[0023] 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.

[0024] After all test and validation processes, methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid turned out to be valid biomarkers for the underlying question (i.e., determining the risk of having or developing bladder cancer), provided that the concentration of at least two 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). 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.

[0025] 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.

[0026] In an embodiment, the group of substances comprises at least one of succinate and pyroglutamic acid. Both substances show NMR signals in a very similar spectral region so that their NMR signals cannot always be easily distinguished from each other. In an embodiment, the group of substances comprises at least one of citrate and pyroglutamic acid. These two substances show NMR signals in a very similar spectral region so that their NMR signals cannot always be easily distinguished from each other, too.

[0027] In an embodiment, the group of substances comprises at least one of uridine and pseudouridine or pyroglutamic acid. These substances also show NMR signals in a very similar spectral region so that their NMR signals cannot always be easily distinguished from each other.

[0028] In an embodiment, the group of substances comprises at least one of valine, leucine, and isoleucine as proteinogenic branched chain amino acid, since all of these branched chain amino acids show NMR signals in a very similar spectral region so that their NMR signals cannot always be easily distinguished from each other.

[0029] In an embodiment, the group of substances comprises at least one of valine, leucine, isoleucine, and 3-hydroxybutyrate, since each of these branched chain amino acids and 3- hydroxybutyrate have NMR signals in a very similar spectral region so that their NMR signals cannot always be easily distinguished from each other.

[0030] In an embodiment, the group of substances comprises at least one of isobutyrate and 2- ketobutyrate, since also these substances have NMR signals in a very similar spectral region so that their NMR signals cannot always be easily distinguished from each other.

[0031] In an embodiment, succinate and pyroglutamic acid are not the only substances in the marker set.

[0032] In an embodiment, pyroglutamic acid and citrate are not the only substances in the marker set.

[0033] In an embodiment, the marker set does not comprise more than one proteinogenic branched chain amino acid as the only substances.

[0034] In an embodiment, the marker set does not comprise more than one proteinogenic branched chain amino acid and 3-hydroxybutyrate as the only substances.

[0035] In an embodiment, isobutyrate and 2-ketobutyrate are not the only substances in the marker set. In an embodiment, the marker set necessarily comprises methylpyridinium. Methylpyridinium turned out to be a particular appropriate biomarker for determining the risk of having or developing bladder cancer when used together with least one further substance of the above group of substance.

[0036] In an embodiment, the marker set necessarily comprises 2-furoylglycine. 2-Furoylglycine likewise turned out to be a particular appropriate biomarker for determining the risk of having or developing bladder cancer when used together with least one further substance of the above group of substance.

[0037] A particular appropriate combination of biomarkers consists of or comprises methylpyridinium and 2-furoylglycine.

[0038] In an embodiment, the marker set necessarily comprises succinate and / or pyroglutamic acid. Each of these substances can be very well combined with methylpyridinium, 2-furoylglycine, or a combination of methylpyridinium and 2-furoylglycine. However, as explained above, succinate and pyroglutamic should not be used as the only substances in the marker set to achieve a particularly high sensitivity and specificity of the marker set for the underlying question.

[0039] In an embodiment, the marker set necessarily comprises trans-aconitate, trans- Aconitate can be very well combined with methylpyridinium, 2-furoylglycine, or a combination of methylpyridinium and 2-furoylglycine.

[0040] In an embodiment, the marker set comprises methylpyridinium and at least one second substance that is chosen from the group consisting of 2-furoylglycine, trans-aconitate, succinate, and pyroglutamic acid.

[0041] In an embodiment, the marker set comprises an additional substance chosen from the group consisting of 2‘-deoxyguanosine, 2‘-deoxyinosine, 2‘-deoxyuridine, 2-hydroxybutyrate, 2- hydroxyglutarate, 2-hydroxyvalerate, 2-ketobutyrate, 3,4-dihydroxybenzenacetate, 3- hydroxyisobutyrate, 3-hydroxyisocaleric acid, 3-hydroxykynurenine, 3-hydroxyphenylacetate, 3-hydroxyvalerate, 3-methyladipate, 3-methylxanthine, 4-hydroxyphenylacetate, 4-pyridoxate, 5-hydroxy-3-acetate, 5-hydroxytryptophan, acetoin, acetylcarnitine, allothreonine, alphahydroxyisobutyrate, chlorogenate, cytidine, dimethylurate, dimethylxanthine, fructose, fucose, galactose, glucose, glucuronate, glutamine, glycerate, glycocholate, guanosine, histidine, homovalinate, hypoxanthine, inosine, isobutyrate, isocitrate, kynurenine, L-carnitine, L-cystine, L-malate, L-proline, L-tryptophan, methylsuccinate, myo-inositol, N-acetylaspartate, N-acetyl- L-leucine, N-carbamoylaspartate, N-isovaleroglycine, O-acetylcarnitine, pantothenate, phenylalanine, pyridinedicarboxylate, pyridoxine, pyroglutamate, pyruvate, quinolinic acid, serotonin, S-threo-isocitrate, succinate, succinylacetone, sucrose, threonine, thymidine, tropate, UDP-glucuronate, valerianate, valproate, and valproinic acid. None of the beforementioned substances is appropriate to determine the risk of having or developing bladder cancer when using these substances alone or in combinations of two or more substances. However, these substances are very well appropriate to increase the sensitivity and / or specificity of a marker set that comprises at least two substances of the above-mentioned substance group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid.

[0042] In an embodiment, the additional substance is chosen from the group consisting of sucrose, L-proline, kynurenine, cytidine, and 3-hydroxykynurenine.

[0043] In an embodiment, the additional substance is chosen from the group consisting of L-cystine, hypoxanthine, glucuronate, L-tryptophan, myo-inositol, sucrose, 2‘-deoxyguanosine, 2‘- deoxyinosine, 2‘-deoxyuridine, 2-hydroxybutyrate, 2-hydroxyglutarate, 2-hydroxyvalerate, 4- hydroxyphenyllacetate, 5-hydroxytryptophan, chlorogenate, fucose, galactose, glycerate, guanosine, histidine, inosine, phenylalanine, proline, pyroglutamate, thymidine, tropate, fructose, glucuronate, isocitrate, sucrose, and UDP-glucuronate.

[0044] In an embodiment, the additional substance is chosen from the group consisting of dimethylurate, dimethylxanthine, 3,4-dihydroxybenzenacetate, 3-hydroxyphenylacetate, 3- methylxanthine, 4-hydroxyphenylacetate, and homovalinate.

[0045] In an embodiment, the additional substance is chosen from the group consisting of threonine, alpha-hydroxyisobutyrate, acetoin, 3-hydroxyisocaleric acid, 2-hydroxybutyrate, pyruvate, and valproinic acid.

[0046] In an embodiment, the additional substance is chosen from the group consisting of 5-hydroxy- 3-acetate and serotonin. In an embodiment, the additional substance is chosen from the group consisting of N-acetyl- L-leucine, glycocholate, 3-methyladipate, N-isovaleroglycine, pantothenate, valproate, valerianate, 2-hydroxybutyrate, and 2-ketobutyrate.

[0047] In an embodiment, the additional substance is chosen from the group consisting of L-malate, L-carnitine, isobutyrate, S-threo-isocitrate, 3-hydroxyisobutyrate, glutamine, N- acetylaspartate, O-acetylcarnitine, N-carbamoylaspartate, succinate, 3-hydroxyvalerate, 4- pyridoxate, pyridoxine, pyruvate, methylsuccinate, and succinylacetone.

[0048] In an embodiment, the additional substance is chosen from the group consisting of glucose, allothreonine, acetylcarnitine, quinolinic acid, and sucrose.

[0049] In an embodiment, the additional substance is pyridinedicarboxylate.

[0050] In an embodiment, the marker set comprises or consists of methylpyridinium, 2-furoylglycine, and a third substance chosen from the group consisting of trans-aconitate, succinate, pyroglutamic acid, citrate, uridine, and pseudouridine.

[0051] A combination of methylpyridinium, 2-furoylglycine, and trans-aconitate turned out to be particularly appropriate. In an embodiment, the marker set comprises or consists of methylpyridinium, 2-furoylglycine, and trans-aconitate. Such a marker set showed an AUC value of 0.794 when used for distinguishing between patients suffering from bladder cancer and healthy individuals in an independent test dataset (not used for training) (cf. Figure 1 ).

[0052] A combination of methylpyridinium, 2-furoylglycine, and at least one of succinate and pyroglutamic acid turned out to be particularly appropriate, too. In an embodiment, the marker set comprises or consists of methylpyridinium, 2-furoylglycine, and at least one of succinate and pyroglutamic acid. Such a marker set showed an AUC value of 0.791 when used for distinguishing between patients suffering from bladder cancer and healthy individuals in an independent test dataset (not used for training).

[0053] A combination of methylpyridinium, 2-furoylglycine, and at least one of uridine, pseudouridine, and pyroglutamic acid turned out to be particularly appropriate, too. In an embodiment, the marker set comprises or consists of methylpyridinium, 2-furoylglycine, and at least one of uridine, pseudouridine, and pyroglutamic acid. Such a marker set showed an AUC value of 0.767 when used for distinguishing between patients suffering from bladder cancer and healthy individuals in an independent test dataset (not used for training). In an embodiment, the marker set comprises a fourth substance chosen from the group consisting of 3-hydroxybutyrate, carnitine, hydroxyindoleacetate, isobutyrate, 2-ketobutyrate, lactate, mannitol, pyroglutamic acid, citrate, succinate, taurine, valine, leucine, isoleucine. In this context, the fourth substance is a different substance than the third substance. Thus, even though some of the individual substances can be used as third substance or as fourth substance, it is necessary that the third substance and the fourth substance differ from each other.

[0054] A combination of methylpyridinium, 2-furoylglycine, at least one of succinate and pyroglutamic acid, and at least one of pyroglutamic acid and citrate, turned out to be particularly appropriate, too. In an embodiment, the marker set comprises or consists of methylpyridinium, 2- furoylglycine, at least one of succinate and pyroglutamic acid, and at least one of pyroglutamic acid or citrate (e.g., i) methylpyridinium, 2-furoylglycine, succinate, and citrate; or ii) methylpyridinium, 2-furoylglycine, and pyroglutamic acid; or ill) methylpyridinium, 2- furoylglycine, succinate, and pyroglutamic acid; or iv) methylpyridinium, 2-furoylglycine, pyroglutamic acid, and citrate). Such a marker set showed an AUC value of 0.840 when used for distinguishing between patients suffering from bladder cancer and healthy individuals in an independent test dataset (not used for training) (cf. Figure 2).

[0055] A combination of methylpyridinium, 2-furoylglycine, trans-aconitate, and at least one of isobutyrate and 2-ketobutyrate turned out to be particularly appropriate, too. In an embodiment, the marker set comprises or consists of methylpyridinium, 2-furoylglycine, trans-aconitate, and at least one of isobutyrate and 2-ketobutyrate. Such a marker set showed an AUC value of 0.790 when used for distinguishing between patients suffering from bladder cancer and healthy individuals in an independent test dataset (not used for training) (cf. Figure 3).

[0056] In an aspect, the present invention relates to the further medical use of a marker set comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20) substances chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid for in in-vivo diagnostics of bladder cancer and / or in the prediction of the risk of an individual to develop bladder cancer 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.

[0057] In a first step, the concentration of at least two substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least two substances are chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3- hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid. A very well suited measuring technique for determining the concentration of the individual substances is nuclear magnetic resonance spectroscopy (NMR spectroscopy).

[0058] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for the risk of the individual to have or to develop bladder cancer.

[0059] 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 two 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 bladder 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.

[0060] 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 a) = a + > bx• IxX=1 a = const. bx= substance specific coefficient

[0061] I = parameter being indicative for the concentration of substance x 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 integral of an according signal observed in the evaluated measuring result. To give an example, “I” can be the signal intensity or integral of an NMR signal in an NMR spectrum if NMR spectroscopy is used as measuring technique.

[0062] 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.

[0063] More generally, calculating the risk or 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.

[0064] 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 determining the risk of an individual to have or develop bladder cancer.

[0065] 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.

[0066] 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 bladder cancer, wherein the other group encompasses healthy individuals. The resulting grouping can also be indicated on an according report.

[0067] In an aspect, the present invention relates to a medical method for making a diagnosis of bladder cancer in a patient and / or for determining the risk of an individual to develop bladder cancer. This method comprises the steps explained in the following. In a first step, a body fluid sample is gathered from a patient. In a second step, the concentration of at least two substances in the body fluid sample is determined by analyzing the body fluid sample with a suited measuring technique. The at least two substances are chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid.

[0068] Afterwards, a score is calculated from the determined concentrations, wherein the score is indicative for the risk of the individual to have or to develop bladder cancer.

[0069] In a further method step, a diagnosis of bladder cancer is made on the basis of the previously calculated score. Alternatively or additionally, the risk of the patient to develop bladder cancer is determined on the basis of the previously calculated score. The respective result is then output to the patient or to a third person like a physician or medical staff.

[0070] 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 two substances chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid in the body fluid sample by analyzing the body fluid sample with a suited measuring technique; and c) a unit for calculating a score from the determined concentrations, the score being indicative for the risk of the individual to have or to develop bladder cancer.

[0071] In an embodiment, the unit for determining the concentration of the at least two substances is configured to determine the concentration of any substance combination of the embodiments explained above.

[0072] 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.

[0073] 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.

[0074] 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:

[0075] Figure 1 shows an ROC plot of the ability of a first marker set for determining the risk of an individual to have or to develop bladder cancer;

[0076] Figure 2 shows an ROC plot of the ability of a second marker set for determining the risk of an individual to have or to develop bladder cancer; and

[0077] Figure 3 shows an ROC plot of the ability of a third marker set for determining the risk of an individual to have or to develop bladder cancer.

[0078] All ROC plots shown in Figures 1 to 3 were obtained by analyzing urine samples of patients having bladder cancer. The control group consisted of patients having a non-cancerous urogenital impairment, as explained above.

[0079] Patient characteristics

[0080] For the training dataset, approximately 100 patient samples (“case”) and approximately 200 control samples (“control”) have been used according to the following distribution:

[0081] For the test dataset, approximately 50 patient samples (“case”) and approximately 100 control samples (“control”) have been used according to the following distribution:

[0082] NMR measurements

[0083] 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).

[0084] 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.

[0085] Signal analysis

[0086] NMR spectra underwent automatic referencing, phase correction and baseline correction before further analysis.

[0087] 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.

[0088] 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. 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 with signals arising from substances, e.g. buffer substances, added during sample preparation - were ignored in the steps explained in the following.

[0089] 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).

[0090] Depending on the cohort of modified spectra, the size and thus the number of bins varies. Typical numbers of bins lie in a range of from 350 to 450. In the present case, 374 bins resulted from the precedingly explained analytic steps.

[0091] Signals were not fitted. Rather, 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.

[0092] Test of identified marker substances

[0093] The identified marker substances were tested in different combinations to assess their suitability for distinguishing castration-resistant prostate cancer from non-castration resistant prostate cancer and / or for determining the risk of an individual to develop castration-resistant prostate cancer. In doing so, the result of the determination based on the marker substances (predicted risk) has been checked against clinical signs of castration-resistant prostate cancer in the patient who donated the urine sample, as already explained above.

[0094] 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. All marker sets, the test dataset ROC plots of which are shown in Figures 1 to 3, were able to distinguish between the patient group and the control group in a statistically significant way. The results shown in Figures 1 to 3 are summarized in the following Table 1 together with additional data on the obtained AUC values in the training dataset and after crossvalidation.

[0095] Table 1 : Summary of results depicted in Figures 1 to 3.

[0096] Furthermore, additional marker sets were tested in an identical way, wherein no ROC plots are represented here. However, the respective AUC values of such ROC plots are listed in the following Table 2. These marker sets were able to distinguish between the patient group (patients having bladder cancer) and the control group (patients having a non-cancerous urogenital impairment, as explained above) in a statistically significant way, too.

[0097] Table 2: Summary of AUC values of further marker sets.

[0098] Summarizing, the presented marker sets comprise or consist of highly appropriate biomarkers for determining the risk of an individual to have or to develop bladder cancer. List of references cited in the preceding sections

[0099] 1. Peter, K., et al., Krebs in Deutschland 2009 / 2010, in Beitrage zur Gesundheitsberichterstattung des Bundes. 2013, Robert Koch-lnstitut: Berlin.

[0100] 2. Parkin, D.M., The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl, 2008(218): p. 12-20. 3. H., R., D. J., and K. Th., Blasenkrebs. 2007, Deutsche Krebshilfe e.V.: 2007.

[0101] 4. Witjes, J.A., et al., EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol, 2014. 65(4): p. 778-92.

[0102] 5. Urquidi, V., C.J. Rosser, and S. Goodison, Multiplex Urinary Tests for Bladder Cancer Diagnosis. Eur Med J Urol, 2013. 1 : p. 70-73. 6. Familienmedizin, D.G.f.A.u., DEGAM S1: Nicht-sichtbare Hamaturie 2013.

[0103] 7. Elias, K., et al., High-risk patients with hematuria are not evaluated according to guideline recommendations. Cancer, 2010. 116(12): p. 2954-9.

[0104] 8. Schmitz-Drager, B.J., et al., Immunocytology in the assessment of patients with asymptomatic hematuria. World J Urol, 2008. 26(1): p. 31 -7. 9. Armin, M., Nicht-sichtbare Hamaturie in DEGAM S1-Handlungsempfehlung. 2013, DEGAM - Deutsche Gesellschaft fur Allgemeinmedizin und Familienmedizin.

[0105] 10. Zheng, C., et al., Narrow band imaging diagnosis of bladder cancer: systematic review and meta-analysis. BJU Int, 2012. 110(11 Pt B): p. E680-7.

[0106] 11 . Schwartz, G.L., Proper evaluation of asymptomatic microscopic hematuria in the era of evidence-based medicine-progress is being made. Mayo Clin Proc, 2013. 88(2): p. 123-5.

[0107] 12. Guo, A., et al., Bladder tumour antigen (BTA stat) test compared to the urine cytology in the diagnosis of bladder cancer: A meta-analysis. Can Urol Assoc J, 2014. 8(5-6): p. E347-52.

[0108] 13. Fu , L. , et al . , Nuclear matrix protein 22 ( NMP22) test in the diagnosis of bladder cancer: a meta-analysis. Int J Clin Exp Med, 2016. 9(5): p. 7965-7975.

[0109] 14. Hennenlotter, J., et al., Point-of-Care Tests for Bladder Cancer: The Influencing Role of Hematuria. Adv Urol, 2011. 2011 : p. 937561 .

[0110] 15. Ludecke, G., et al., Comparative analysis of sensitivity to blood in the urine for urinebased point-of-care assays (UBC rapid, NMP22 BladderChek and BTA-stat) in primary diagnosis of bladder carcinoma. Interference of blood on the results of urine-based POC tests. Anticancer Res, 2012. 32(5): p. 2015-8.

[0111] 16. Ghafouri-Fard, S., L. Nekoohesh, and E. Motevaseli, Bladder cancer biomarkers: review and update. Asian Pac J Cancer Prev, 2014. 15(6): p. 2395-403.

[0112] 17. Herman, M.P., et al., Urine-based biomarkers for the early detection and surveillance of non-muscle invasive bladder cancer. Minerva Urol Nefrol, 2008. 60(4): p. 217-35.

Claims

Claims1 . Use of a marker set comprising at least two substances chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid in an in vitro method for determining the risk of an individual to have or to develop bladder cancer.

2. Use according to claim 1 , characterized in that i) succinate and pyroglutamic acid, and / or ii) pyroglutamic acid and citrate, and / or ill) uridine, pseudouridine and pyroglutamic acid, and / or more than one proteinogenic branched chain amino acid, and / or v) one or more proteinogenic branched chain amino acids and 3-hydroxybutyrate, and / or vi) isobutyrate and 2-ketobutyrate are not the only substances in the marker set.

3. Use according to claim 1 or 2, characterized in that one of the at least two substances is methylpyridinium.

4. Use according to any of the preceding claims, characterized in that one of the at least two substances is 2-furoylglycine.

5. Use according to any of the preceding claims, characterized in that the marker set comprises at least one of succinate and pyroglutamic acid.

6. Use according to any of the preceding claims, characterized in that one of the at least two substances is trans-aconitate.

7. Use according to any of the preceding claims, characterized in that the marker set comprises methylpyridinium and at least one second substance that is chosen from the group consisting of 2-furoylglycine, trans-aconitate, succinate, and pyroglutamic acid.

8. Use according to any of the preceding claims, characterized in that the marker set comprises an additional substance chosen from the group consisting of 2‘- deoxyguanosine, 2‘-deoxyinosine, 2‘-deoxyuridine, 2-hydroxybutyrate, 2- hydroxyglutarate, 2-hydroxyvalerate, 2-ketobutyrate, 3,4-dihydroxybenzenacetate, 3- hydroxyisobutyrate, 3-hydroxyisocaleric acid, 3-hydroxykynurenine, 3- hydroxyphenylacetate, 3-hydroxyvalerate, 3-methyladipate, 3-methylxanthine, 4-hydroxyphenylacetate, 4-pyridoxate, 5-hydroxy-3-acetate, 5-hydroxytryptophan, acetoin, acetylcarnitine, allothreonine, alpha-hydroxyisobutyrate, chlorogenate, cytidine, dimethylurate, dimethylxanthine, fructose, fucose, galactose, glucose, glucuronate, glutamine, glycerate, glycocholate, guanosine, histidine, homovalinate, hypoxanthine, inosine, isobutyrate, isocitrate, kynurenine, L-carnitine, L-cystine, L-malate, L-proline, L- tryptophan, methylsuccinate, myo-inositol, N-acetylaspartate, N-acetyl-L-leucine, N- carbamoylaspartate, N-isovaleroglycine, O-acetylcarnitine, pantothenate, phenylalanine, pyridinedicarboxylate, pyridoxine, pyroglutamate, pyruvate, quinolinic acid, serotonin, S- threo-isocitrate, succinate, succinylacetone, sucrose, threonine, thymidine, tropate, UDP- glucuronate, valerianate, valproate, and valproinic acid.

9. Use according to any of the preceding claims, characterized in that the marker set comprises methylpyridinium, 2-furoylglycine, and a third substance chosen from the group consisting of trans-aconitate, succinate, pyroglutamic acid, citrate, uridine, and pseudouridine.

10. Use according to claim 9, characterized in that the third substance is chosen from the group consisting of trans-aconitate, succinate, and pyroglutamic acid.11 . Use according to claim 9 or 10, characterized in that the marker set comprises a fourth substance chosen from the group consisting of 3-hydroxybutyrate, carnitine, hydroxyindoleacetate, isobutyrate, 2-ketobutyrate, lactate, mannitol, pyroglutamic acid, citrate, succinate, taurine, valine, leucine, isoleucine, wherein the fourth substance is a different substance than the third substance.

12. Marker set comprising at least two substances chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid for use in in-vivo diagnostics of bladder cancer and / or in the prediction of the risk of an individual to develop bladder cancer.

13. Method for analyzing an isolated body fluid sample in vitro, comprising the following steps: a) determining the concentration of at least two substances chosen from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindoleacetate, 3-hydroxybutyrate, carnitine,mannitol, taurine, a proteinogenic branched chain amino acid, isobutyrate, 2- ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid in an isolated body fluid sample from an individual by analyzing the body fluid sample with a suited measuring technique, and b) calculating a score from the determined concentrations, the score being indicative for the risk of the individual to have or to develop bladder cancer.

14. Method according to claim 13, characterized in that the body fluid sample is a urine sample or a blood sample.

15. Method according to claim 13 or 14, characterized in that that the body fluid sample is grouped into one of at least two predefined groups on the basis of the calculated score.

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