Use of a marker set to determine 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 selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 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 a person having or developing bladder cancer.

Description

[0001] The present invention relates to the in vitro use of a marker set for determining a person's risk of having or developing bladder cancer according to the preamble of claim 1, the further medical use of such a marker set according to the preamble of claim 12, and an analytical method for determining a person's risk of having or developing bladder cancer according to the preamble of claim 13.

[0002] Bladder cancer (BCa) is a malignant cancer that originates in the epithelial lining (i.e., the urothelium) of the urinary bladder. As the tumor progresses, the cancer cells can invade the underlying layers of the bladder wall, as well as other parts of the urinary tract. Bladder cancer grows in both flat and papillary forms. In early stages, the symptoms of cancer tend to be nonspecific, meaning there are few or no complaints, such as hematuria, frequent (painful) urination, increased urgency, or pain in the lower abdomen and lower back. However, microscopic hematuria is the most important early symptom. Microscopic hematuria (or non-visible hematuria) is hematuria that can be detected microscopically.

[0003] In addition to age and genetic predisposition, several lifestyle-related risk factors for BCa have been identified. A significant risk factor is tobacco use; approximately 50% of diagnoses can be attributed to smoking. Furthermore, exposure to certain chemicals (particularly aromatic amines, benzene, and dyes), chlorinated drinking water, painkiller abuse (particularly high doses of phenacetin), chemotherapy with cytotoxic drugs (particularly cyclophosphamide-based drugs), as well as chronic cystitis and urogenital schistosomiasis, increase the risk of BCa.

[0004] In 2010, the incidence of BCa in the German general population was approximately 15,500 [1]. In terms of incidence, BCa ranks fourth and fourteenth in men and women, respectively [1]. Worldwide, BCa ranks ninth, with an incidence of approximately 350,000 cases per year [2]. The average age at diagnosis is 73 years for men and 77 years for women [3]. Men are 3.8 times more likely to develop BCa than women [4].

[0005] The 5-year survival rate is 59% for men and 50% for women [1]. However, the survival rate depends heavily on the tumor stage at diagnosis. At initial diagnosis, approximately 75% of patients have superficial, non-muscle-invasive BCa, with a 5-year survival rate of over 90% [5]. In the remaining 25% of patients, the tumor has already invaded deeper layers of the bladder, which significantly increases cancer-related mortality [4]. Depending on the stage and grade of the tumor, the 5-year survival rate drops to only 65% ​​to 15%.

[0006] It is therefore obvious that reliable early detection is essential for curative therapy and a positive prognosis.

[0007] Microhematuria is one of the earliest symptoms of BCa [6]. However, only in 2 to 5% of cases is microhematuria caused by a malignant bladder tumor [7]. The vast majority are attributed to benign urological diseases. A central challenge in BCa diagnosis is therefore the detection of the relatively rare BCa cases among patients with microhematuria. [8]. According to the DEGAM guidelines [9], persistent microhematuria in patients ≥ 40 years of age justifies further urological diagnostics if an additional risk factor is present.

[0008] The first diagnostic step is an ultrasound examination for possible kidney abnormalities, followed by cystoscopy. During cystoscopy, a cystoscope—a thin tube with a camera and a light at the end—is inserted through the urethra into the bladder for visual examination. During cystoscopy, samples can be obtained from suspicious areas of the urethra and bladder via biopsy. Cystoscopy is considered the "gold standard" for BCa diagnosis. The AUC for cystoscopy with narrow band imaging (NBI) was 0.98 and 0.93 for cystoscopy with white light imaging (WLI) in a recent meta-analysis

[10] . However, cystoscopy is invasive, expensive, and unnecessary for the majority of patients with microscopic hematuria

[11] .Therefore, according to the DEGAM guidelines, cystoscopy is not recommended in patients under 40 years of age - unless other BCa risk factors are present - because the probability of bladder cancer is less than 1% [9].

[0009] To avoid unnecessary, expensive, and invasive cystoscopies, a wide range of molecular markers have been proposed over the past 20 years for the detection of BCa in patients with persistent microhematuria [8]. Today, several molecular protein-based urinary biomarkers exist, with the following two tests (both FDA-approved and CE-marked) being the most commonly used systems: - BTA statistic

[12] with an AUC of 0.75 (95% confidence interval (CI): 0.70-0.81) and a sensitivity of 67% (95% CI: 0.64-0.69) with a specificity of 75% (95% CI: 73-77%). - NMP22 Bladder Check

[13] with an AUC of 0.78 (95% CI: 0.74-0.83) and a sensitivity of 71% (95% CI: 69-72%) with a specificity of 80% (95% CI: 79-81%).

[0010] However, both tests are rarely used in routine diagnostics. The reason for this appears to be twofold.

[0011] First, published studies show wide variance in sensitivity and specificity, so urologists may be confused about the performance of these tests.

[0012] Second, studies reporting high diagnostic accuracy often come from studies comparing healthy individuals with bladder cancer patients, making clinical utility more difficult to assess for patients with urological comorbidities such as kidney stones or cystitis, which may be associated with microscopic hematuria. A major reason for this may be that these biomarkers are “single-biomarkers” that focus on a single aspect of the disease. Therefore, to achieve acceptable performance, it seems necessary to carefully select the population to which these tests can be applied. For example, the general recommendation is that BTA stat should not be used in cases of microscopic or macroscopic hematuria, as these conditions significantly influence test results [14, 15]. This limits its clinical applicability, as hematuria is one of the cardinal symptoms of BCa.

[0013] Other new biomarkers are under discussion, but none are available for routine clinical use [16, 17]. Currently, none of these biomarkers is sufficiently well established in clinical routine to replace cystoscopy or at least reduce the number of unnecessary cystoscopies [9].

[0014] An object of the present invention is to provide a less invasive method and suitable biomarkers for determining a person's risk of having or developing bladder cancer.

[0015] This object is achieved by the in vitro use of a marker set comprising the claim elements of claim 1. Such a marker set comprises at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) substances selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched-chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid. According to one aspect of the present invention, this marker set is used to determine a person's risk of having or developing bladder cancer. A marker set containing only 2, 3 or 4 of these substances is already very well suited to determine this risk with high accuracy (i.e. high sensitivity and high specificity) and thus represents a particularly suitable embodiment.

[0016] The term “proteinogenic branched-chain amino acid” includes valine, leucine, and isoleucine (the only proteinogenic branched-chain amino acids).

[0017] To determine a person's risk of developing 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 performed using any suitable measurement or analysis method, such as nuclear magnetic resonance spectroscopy (NMR), mass spectrometry, and infrared spectroscopy, such as Fourier transform infrared spectroscopy (FT-IR). These analysis methods are carried out using a measuring device such as an NMR spectrometer, a mass spectrometer, or an infrared spectrometer. Furthermore, these measurement procedures must be performed with the aid of a computer, since neither the measurement itself nor the evaluation of the measurement data represent mental processes that could be performed by a human without the aid of a computer.

[0018] A change in the concentration of at least two substances of the marker set compared to the concentration in a control group or a change in the concentration ratio between at least two substances compared to the concentration ratio of the same substances in a control group was statistically significantly correlated with the presence of bladder cancer at the time of the analysis (ie, with the presence of bladder cancer at the time of the analysis (ie, the determination of the risk of a person to develop bladder cancer is possible) or the occurrence of bladder cancer at a time after the analysis (ie, the determination of the risk of a person to develop bladder cancer is possible).

[0019] To identify the marker set and the substances that can comprise it, urine (as a representative body fluid) from patients with bladder cancer was used. The control group consisted of i) patients who underwent cystoscopy due to suspected bladder cancer (e.g., due to observed hematuria), but who did not have bladder cancer based on the cystoscopy results, and ii) patients who did not have bladder cancer but had a urogenital tract infection. Thus, the control group can be described as patients with non-cancer-related impairment of the urogenital tract.

[0020] When examining single substances identified in the urine, two substances identified simultaneously in the urine, or even three or four substances identified simultaneously in the urine, no significant results could be obtained for most of the identified substances when attempting to distinguish the two groups (bladder cancer patients versus patients with non-cancer urogenital impairment). Rather, the area under the curve (AUC) values ​​of the receiver operating characteristic (ROC) plots showed values ​​that mainly ranged from 0.5 to 0.65. The AUC value of ROC plots is an aggregate metric that evaluates how well a logistic regression model classifies positive and negative outcomes at all possible cutoffs. 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 greater than 0.5 represents a classification of an event that satisfies the trained correlation, with higher values ​​representing a better classification.

[0021] Surprisingly, however, some of the identified substances exhibited significantly higher AUC values ​​(over 0.7). This was not the case when the substances were used alone, but only when at least two of these substances were analyzed together.

[0022] The marker sets were tested on training and test datasets and iteratively cross-validated. Cross-validation was performed by dividing the training dataset into, for example, five parts, with four parts used for training (the training dataset) and the fifth part for testing (the test dataset). The individual parts were iteratively removed and added back to the training dataset, so that each of the five parts belonged to both the training dataset and the test dataset in different training rounds.

[0023] After all testing and validation procedures, methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched-chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid were found to be valid biomarkers for the underlying research question (i.e., determining the risk of developing bladder cancer) provided that the concentration of at least two of these biomarkers was determined simultaneously (i.e., in one or more body fluid samples from the same patient obtained at the same time point). The concentration determination can be performed using a method capable of determining the concentration of the substances using a single measurement or using a method requiring more than one measurement for this determination.NMR spectroscopy is particularly suitable for such concentration determination because it enables highly accurate concentration determination in a body fluid through a single measurement in a very short measurement time.

[0024] In one embodiment, the concentration of the substances is normalized to the concentration of creatinine in the same sample or, alternatively, to the concentration of another substance naturally present in the sample.

[0025] In one embodiment, the group of substances comprises at least one of the two substances succinate and pyroglutamic acid. Both substances exhibit NMR signals in a very similar spectral range, so their NMR signals are not always easily distinguishable from each other.

[0026] In one embodiment, the group of substances includes citrate and / or pyroglutamic acid. These two substances exhibit NMR signals in a very similar spectral range, so their NMR signals are not always easily distinguishable from each other.

[0027] In one embodiment, the group of substances includes uridine and / or pseudouridine and / or pyroglutamic acid. These substances also exhibit NMR signals in a very similar spectral range, so their NMR signals are not always easily distinguishable from each other.

[0028] In one embodiment, the group of substances comprises valine and / or leucine and / or isoleucine as branched-chain proteinogenic amino acids, since all of these branched-chain amino acids show NMR signals in a very similar spectral range, so that their NMR signals are not always easily distinguishable from each other.

[0029] In one embodiment, the group of substances comprises valine and / or leucine and / or isoleucine and / or 3-hydroxybutyrate, since each of these branched-chain amino acids and 3-hydroxybutyrate have NMR signals in a very similar spectral range, so that their NMR signals are not always easily distinguishable from each other.

[0030] In one embodiment, the group of substances comprises isobutyrate and / or 2-ketobutyrate, since these substances also have NMR signals in a very similar spectral range, so that their NMR signals are not always easily distinguishable from each other.

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

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

[0033] In one embodiment, the marker set contains no more than one proteinogenic branched-chain amino acid as the only substance.

[0034] In one embodiment, the marker set contains no more than one proteinogenic branched-chain amino acid and 3-hydroxybutyrate as the only substances.

[0035] In one embodiment, isobutyrate and 2-ketobutyrate are not the only substances in the marker set.

[0036] In one embodiment, the marker set necessarily includes methylpyridinium. Methylpyridinium has proven to be a particularly suitable biomarker for determining the risk of developing bladder cancer when used together with at least one other substance from the above-mentioned group of substances.

[0037] In one embodiment, the marker set necessarily includes 2-furoylglycine. 2-furoylglycine has also proven to be a particularly suitable biomarker for determining the risk of developing bladder cancer when used together with at least one other substance from the above-mentioned substance group.

[0038] A particularly suitable combination of biomarkers consists of or contains methylpyridinium and 2-furoylglycine.

[0039] In one embodiment, the marker set necessarily includes succinate and / or pyroglutamic acid. Each of these substances can be combined very well with methylpyridinium, 2-furoylglycine, or a combination of methylpyridinium and 2-furoylglycine. However, as explained above, succinate and pyroglutamic acid should not be used as the only substances in the marker set in order to achieve a particularly high sensitivity and specificity of the marker set for the underlying research question.

[0040] In one embodiment, the marker set necessarily includes trans-aconitate. Trans-aconitate can be combined very well with methylpyridinium, 2-furoylglycine, or a combination of methylpyridinium and 2-furoylglycine.

[0041] In one embodiment, the marker set comprises methylpyridinium and at least one second substance selected from the group consisting of 2-furoylglycine, trans-aconitate, succinate, and pyroglutamic acid.

[0042] In one embodiment, the marker set comprises an additional substance selected from the group consisting of 2'-deoxyguanosine, 2'-deoxyinosine, 2'-deoxyuridine, 2-hydroxybutyrate, 2-hydroxyglutarate, 2-hydroxyvalerate, 2-ketobutyrate, 3,4-dihydroxybenzeneacetate, 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, methyl succinate, myo-inositol, N-acetylaspartate, N-Acetyl-L-Leucine, N-Carbamoylaspartate, N-Isovaleroglycine, O-Acetylcarnitine, Pantothenate, Phenylalanine,Pyridine dicarboxylate, pyridoxine, pyroglutamate, pyruvate, quinolinic acid, serotonin, S-threoisocitrate, succinate, succinylacetone, sucrose, threonine, thymidine, tropate, UDP-glucuronate, valerianate, valproate, and valproic acid. None of the aforementioned substances is suitable for determining the risk of developing bladder cancer when these substances are used alone or in combinations of two or more substances. However, these substances are very well suited to increase the sensitivity and / or specificity of a marker set that contains at least two substances from the above-mentioned substance group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched-chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid.

[0043] In one embodiment, the additional substance is selected from the group consisting of sucrose, L-proline, kynurenine, cytidine and 3-hydroxykynurenine.

[0044] In one embodiment, the additional substance is selected 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-hydroxyphenyl acetate, 5-hydroxytryptophan, chlorogenate, fucose, galactose, glycerate, guanosine, histidine, inosine, phenylalanine, proline, pyroglutamate, thymidine, tropate, fructose, glucuronate, isocitrate, sucrose and UDP-glucuronate.

[0045] In one embodiment, the additional substance is selected from the group consisting of dimethylurate, dimethylxanthine, 3,4-dihydroxybenzeneacetate, 3-hydroxyphenylacetate, 3-methylxanthine, 4-hydroxyphenylacetate and homovalinate.

[0046] In one embodiment, the additional substance is selected from the group consisting of threonine, alpha-hydroxyisobutyrate, acetoin, 3-hydroxyisocaleric acid, 2-hydroxybutyrate, pyruvate and valproic acid.

[0047] In one embodiment, the additional substance is selected from the group consisting of 5-hydroxy-3-acetate and serotonin.

[0048] In one embodiment, the additional substance is selected from the group consisting of N-acetyl-L-leucine, glycocholate, 3-methyladipate, N-isovaleroglycine, pantothenate, valproate, valerianate, 2-hydroxybutyrate and 2-ketobutyrate.

[0049] In one embodiment, the additional substance is selected 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.

[0050] In one embodiment, the additional substance is selected from the group consisting of glucose, allothreonine, acetylcarnitine, quinolinic acid, and sucrose.

[0051] In one embodiment, the additional substance is pyridine dicarboxylate.

[0052] In one embodiment, the marker set consists of or comprises methylpyridinium, 2-furoylglycine and a third substance selected from the group consisting of trans-aconitate, succinate, pyroglutamic acid, citrate, uridine and pseudouridine.

[0053] A combination of methylpyridinium, 2-furoylglycine, and trans-aconitate proved particularly suitable. In one embodiment, the marker set consists of or comprises methylpyridinium, 2-furoylglycine, and trans-aconitate. Such a marker set showed an AUC value of 0.794 when distinguishing between bladder cancer patients and healthy individuals in an independent test dataset (not used for training) (see [Figure 1]. Fig. 1).

[0054] A combination of methylpyridinium, 2-furoylglycine, and at least one of the components succinate and pyroglutamic acid also proved particularly suitable. In one embodiment, the marker set consists of or comprises 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 to distinguish between patients with bladder cancer and healthy individuals in an independent test dataset (not used for training).

[0055] A combination of methylpyridinium, 2-furoylglycine, and uridine and / or pseudouridine and / or pyroglutamic acid also proved particularly suitable. In one embodiment, the marker set consists of or comprises methylpyridinium, 2-furoylglycine, and uridine and / or pseudouridine and / or pyroglutamic acid. Such a marker set demonstrated an AUC value of 0.767 when used to distinguish between patients with bladder cancer and healthy individuals in an independent test dataset (not used for training).

[0056] In one embodiment, the marker set comprises a fourth substance selected from the group consisting of 3-hydroxybutyrate, carnitine, hydroxyindole acetate, isobutyrate, 2-ketobutyrate, lactate, mannitol, pyroglutamic acid, citrate, succinate, taurine, valine, leucine, and isoleucine. In this context, the fourth substance is a different substance than the third substance. Therefore, while some of the individual substances can be used as the third or fourth substance, it is necessary that the third and fourth substances be different from each other.

[0057] A combination of methylpyridinium, 2-furoylglycine, and succinate and / or pyroglutamic acid, as well as pyroglutamic acid and / or citrate, has also proven particularly suitable. In one embodiment, the marker set consists of or comprises methylpyridinium, 2-furoylglycine, and succinate and / or pyroglutamic acid, as well as pyroglutamic acid and / or citrate (for example, i) methylpyridinium, 2-furoylglycine, succinate, and citrate; or ii) methylpyridinium, 2-furoylglycine, and pyroglutamic acid; or iii) methylpyridinium, 2-furoylglycine, succinate, and pyroglutamic acid; or iv) methylpyridinium, 2-furoylglycine, pyroglutamic acid, and citrate). One such marker set showed an AUC value of 0.840 when used to distinguish between patients with bladder cancer and healthy individuals in an independent test dataset (not used for training) (cf. Fig. 2).

[0058] A combination of methylpyridinium, 2-furoylglycine, trans-aconitate, and at least one of isobutyrate and 2-ketobutyrate also proved particularly suitable. In one 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 to distinguish between patients with bladder cancer and healthy individuals in an independent test dataset (not used for training) (see Fig. 3).

[0059] In one aspect, the present invention relates to the further medical use of a marker set comprising at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) substances selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched-chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate and quinolinic acid for the in vivo diagnosis of bladder cancer and / or for predicting the risk of a person developing bladder cancer.

[0060] In one aspect, the present invention relates to a method for the in vitro analysis of an isolated body fluid sample, comprising the steps explained below. The method is performed on an isolated body fluid sample derived from a person.

[0061] In a first step, the concentration of at least two substances in the body fluid sample is determined by analyzing the body fluid sample using a suitable measurement technique. The at least two substances are selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched-chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid. A highly suitable measurement technique for determining the concentration of the individual substances is nuclear magnetic resonance spectroscopy (NMR spectroscopy).

[0062] A score is then calculated from the determined concentrations, with the score indicating the person's risk of having or developing bladder cancer.

[0063] The score can be calculated taking into account the measured or expected concentrations in a body fluid sample from a control group. A simple example: The score can be the median of the concentration ratios of the at least two substances between the patient's body fluid sample and the corresponding control values ​​of a body fluid control sample measured in the past. If the score is above a predetermined threshold, there is a significant increase in the marker substances in the body fluid sample, which indicates bladder cancer. It is noted that in one embodiment, other calculation methods, as well as a weighting of individual marker concentrations relative to other marker concentrations, can also be used.

[0064] A suitable method for calculating the score is disclosed on pages 25 to 27 of patent document WO 2012 / 045773 A9. Another suitable method for calculating the score is: Score=11+e−ω where ω a+∑x=1nbx⋅Ix a const. b x substance-specific coefficient I Parameter that indicates the concentration of substance x

[0065] The individual factors a and b must be adjusted depending on the underlying model and can vary depending on the specific substances considered in the marker set. The parameter "I" can, for example, be the signal intensity or the integral of a corresponding signal observed in the evaluated measurement result. For example, "I" can be the signal intensity or the integral of an NMR signal in an NMR spectrum if NMR spectroscopy is used as the measurement technique.

[0066] In one embodiment, "I" is a ratio between two signal intensities or two signal integrals. In such a case, it is possible, for example, to standardize the concentration of a first substance (or several substances) by the concentration of a second substance, such as creatinine.

[0067] More generally, the risk or score calculation involves multiplying each of the substance concentrations by a substance-specific weighting factor to obtain a plurality of weighted values ​​and combining the weighted values ​​into a risk equation.

[0068] In one embodiment, the calculated score is output and presented to the individual and / or a third party, for example, a physician or medical personnel. The output may be on a display (i.e., electronically) or in printed form. It is also possible to generate a report indicating the score, optionally in combination with a comparison scale of possible scores and their significance in determining a person's risk of having or developing bladder cancer.

[0069] In one embodiment, the body fluid sample is a urine sample or a blood sample. In one embodiment, the blood sample is a whole blood sample, a blood serum sample, a blood plasma sample, or another blood preparation that can be obtained from whole blood or other blood preparations.

[0070] In one embodiment, the body fluid sample (and thus the patient from whom the body fluid sample was obtained) is classified into one of at least two predefined groups based on the calculated score. Typically, one group includes patients suffering from bladder cancer, while the other group includes healthy individuals. The resulting grouping can also be specified in a corresponding report.

[0071] One aspect of the present invention relates to a medical method for diagnosing bladder cancer in a patient and / or for determining a person's risk of developing bladder cancer. This method comprises the steps explained below.

[0072] In a first step, a body fluid sample is taken 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 using a suitable measuring technique. The at least two substances are selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched-chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate, and quinolinic acid.

[0073] A score is then calculated from the determined concentrations, with the score indicating the person's risk of having or developing bladder cancer.

[0074] In a further step, a diagnosis of bladder cancer is made based on the previously calculated score. Alternatively or additionally, the patient's risk of developing bladder cancer is determined based on the previously calculated score. The respective result is then provided to the patient or to a third party such as a physician or medical staff.

[0075] In a further aspect, the present invention relates to a decision support system for in vitro analysis of an isolated body fluid sample, the decision support system comprising: (a) a unit for providing a body fluid sample from a person; b) a unit for determining the concentration of at least two substances selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 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 using a suitable measuring technique; and (c) a unit for calculating a score from the determined concentrations, the score indicating the person's risk of having or developing bladder cancer.

[0076] In one embodiment, the unit for determining the concentration of the at least two substances is configured to determine the concentration of any combination of substances of the embodiments explained above.

[0077] While some of the uses and methods illustrated are described as in vitro uses and methods and some of the uses and methods illustrated are described as in vivo uses and methods, it is noted that any in vitro use or method may also be performed as an in vivo use or method, and vice versa.

[0078] All embodiments of the use of the marker set can be combined as desired and can be applied, either individually or in any combination, to further medical use of the marker set, as well as to the various methods and the decision support system. Likewise, all embodiments of the further medical use of the marker set can be combined as desired and can be applied, either individually or in any combination, to the use of the marker set, to the various methods, and to the decision support system. Finally, all embodiments of the various methods can be combined as desired and can be applied, either individually or in any combination, to the use of the marker set, to further medical use of the marker set, to any of the other described methods, and to the decision support system.

[0079] Further details of aspects of the present invention are described below with reference to exemplary embodiments and the accompanying figures. In the figures: Fig. 1 an ROC diagram of the ability of an initial set of markers to determine a person's risk of having or developing bladder cancer; Fig. Figure 2 shows an ROC diagram of the ability of a second set of markers to determine a person's risk of having or developing bladder cancer; and Fig. Figure 3 shows an ROC plot of the ability of a third set of markers to determine a person's risk of having or developing bladder cancer.

[0080] All in the Fig. The ROC diagrams shown in Figures 1 to 3 were generated by analyzing urine samples from patients with bladder cancer. The control group consisted of patients with non-cancer-related urogenital impairment, as explained above. Patient characteristics

[0081] For the training dataset, approximately 100 patient samples (“case”) and approximately 200 control samples (“control”) were used according to the following distribution: Female Masculine sum control 33 162 195 case 22 84 106 sum 55 246 301

[0082] For the test dataset, approximately 50 patient samples (“case”) and approximately 100 control samples (“control”) were used according to the following distribution: Female Masculine sum control 15 80 95 case 12 41 53 sum 27 121 148 NMR measurements

[0083] All measurements were performed using a Bruker Avance II+ 600 MHz or a Bruker Avance III HD 600 MHz 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 preheater block prior to measurement. A standard pulse program with a 30-degree excitation pulse and presaturation for water suppression (zgpr30) was used.

[0084] The samples were measured in batches of up to 93 samples per run. In addition to the analytical samples, each run contained an Axinon ® Urine calibration sample and two Axinon ® Urine control samples (before or after the analytical urine samples) to ensure ideal measurement conditions throughout the run. Signal analysis

[0085] The NMR spectra were subjected to automatic referencing, phase correction, and baseline correction before further analysis.

[0086] The NMR spectra were then subjected to an automatic standardization and calibration procedure to minimize inter-instrument, inter-day, and inter-run effects. The quality of each of these spectra was assessed using a custom spectral qualification algorithm that analyzed general spectral properties, such as baseline offset and slope in selected spectral regions, and properties of selected indicator signals, such as signal position, shape, and width. Spectra that did not meet the predefined quality criteria were excluded from further analysis.

[0087] Successfully qualified spectra (typically with a chemical shift of -5 to 14 ppm) were subjected to further modifications. In particular, broad background signals were removed using a suitable algorithm. For example, background intensities (e.g., from proteins) were subtracted from the spectra, so that the spectral intensities were free of such background signals.

[0088] The cohort (i.e., the multitude) of modified spectra was examined for regions where the cohort did not exhibit a significant number of signals. These regions—such as the water signal region and the regions with signals originating from substances, such as buffer substances, added during sample preparation—were ignored in the steps explained below.

[0089] The remaining spectral ranges were subjected to adaptive binning, in which the spectrum is divided into bins of different sizes or extents (usually for 0.01 to 0.05 ppm, but in extreme cases also for 0.005 to 0.5 ppm).

[0090] Depending on the cohort of modified spectra, the size and thus the number of bins varies. Typically, the number of bins ranges from 350 to 450. In this case, the analysis steps described above resulted in 374 bins.

[0091] The signals were not fitted. Instead, quantification models were applied that utilize the previously assigned bins. After identifying the substances, substance names were assigned to these bins. Quantification was then determined by the bin value, which is calculated as [(sum of intensities in the bin) / (number of data points in the bin)]. Standardization by data points is used to compensate for a different number of data points in the bins. The number of data points in a bin can vary by one data point due to shifts in the discretization grid used. A true numerical integration would use multiplication by the bin extent. However, this is a constant factor and can be omitted. Test of the identified marker substances

[0092] The identified marker substances were tested in various combinations for their ability to distinguish castration-resistant prostate cancer from non-castration-resistant prostate cancer and / or to determine an individual's risk of developing castration-resistant prostate cancer. The marker substance determination result (risk prediction) was compared with clinical signs of castration-resistant prostate cancer in the patient who donated the urine sample, as explained above.

[0093] The results are summarized in receiver operating characteristic (ROC) plots. In these plots, the area under the curve (AUC) indicates the adequacy of the prediction. If the AUC is 0.5, the prediction is considered random and therefore not a good fit. The higher the AUC, the better the prediction model.

[0094] All marker sets whose ROC plots for the test data set are in the Fig. 1 to 3 were able to distinguish statistically significantly between the patient group and the control group. Fig. The results presented in Figures 1 to 3 are summarized in Table 1 below, along with additional data on the AUC values ​​achieved in the training dataset and after cross-validation. Table 1: Summary of the results presented in Figures 1 to 3. figure Composition of the marker set AUC value Training dataset Cross-validation Test data set 1 • trans-aconitate 0,775 0,770 0,794 • 2-Furoylglycin • Methylpyridinium 2 • Succinate and / or 0,847 0,838 0,840 Pyroglutamic acid • 2-Furoylglycin • Methylpyridinium • Pyroglutamic acid and / or citrate 3 • trans-aconitate 0,797 0,780 0,790 • Isobutyrate and / or 2-ketobutyrate • 2-Furoylglycin • Methylpyridinium

[0095] In addition, other marker sets were tested in an identical manner; ROC plots are not shown here. However, the respective AUC values ​​of such ROC plots are listed in Table 2 below. These marker sets were also able to statistically significantly distinguish between the patient group (patients with bladder cancer) and the control group (patients with non-cancer-related urogenital impairment, as explained above). Table 2: Summary of AUC values ​​of the other marker sets. Sample No. Composition of the marker set AUC value Training dataset Cross-validation Test data set • trans-aconitate 0,795 0,781 0,775 4 • 2-Furoylglycin • Methylpyridinium • trans-aconitate 0,802 0,785 0,753 • 2-Furoylglycin 5 • Methylpyridinium • Taurine 6 • 2-Furoylglycin 0,786 0,786 0,791 • Methylpyridinium • Succinate and / or Pyroglutamic acid 7 • 2-Furoylglycin 0,843 0,832 0,759 • Methylpyridinium • Succinate and / or pyroglutamic acid • Valine and / or Leucine and / or Isoleucine 8 • 2-Furoylglycin 0,832 0,819 0,757 • Methylpyridinium • Succinate and / or pyroglutamic acid • Taurine 9 • 2-Furoylglycin 0,826 0,814 0,752 • Methylpyridinium • Succinate and / or pyroglutamic acid • Isobutyrate and / or 2-ketobutyrate 10 • 2-Furoylglycin 0,731 0,711 0,749 • Methylpyridinium 11 • Methylpyridinium 0,718 0,680 0,707 • trans-aconitate 12 • at least two of succinate, pyroglutamic acid and citrate 0,740 0,741 0,705

[0096] In summary, the presented marker sets contain or consist of highly suitable biomarkers for determining an individual's risk of developing or developing bladder cancer. List of references cited in the previous sections 1. Peter, K., et al., Cancer in Germany 2009 / 2010, in Contributions to the Federal Health Report. 2013, Robert Koch Institute: Berlin. 2. Parkin, DM, The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl, 2008(218): pp. 12-20. 3. H., R., DJ, and K. Th., Bladder Cancer. 2007, German Cancer Aid: 2007. 4. Witjes, JA, et al., EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol, 2014. 65(4): pp. 778-92. 5. Urquidi, V., CJ Rosser, and S. Goodison, Multiplex Urinary Tests for Bladder Cancer Diagnosis. Eur Med J Urol, 2013. 1: pp. 70-73. 6. Family Medicine, DGfAu, DEGAM S1: Non-visible Hematuria 2013. 7. Elias, K., et al., High-risk patients with hematuria are not evaluated according to guideline recommendations. Cancer, 2010. 116(12): pp. 2954-9. 8. Schmitz-Drager, B.J., et al., Immunocytology in the assessment of patients with asymptomatic hematuria. World J Urol, 2008. 26(1): S. 31-7. 9. Armin, M., Nicht-sichtbare Hämaturie in DEGAM S1-Handlungsempfehlung. 2013, DEGAM - Deutsche Gesellschaft für Allgemeinmedizin und Familienmedizin. 10. Zheng, C., et al., Narrow band imaging diagnosis of bladder cancer: systematic review and meta-analysis. BJU Int, 2012. 110(11 Pt B): S. E680-7. 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): S. 123-5. 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): S. E347-52. 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): S. 7965-7975. 14. Hennenlotter, J., et al., Point-of-Care-Tests für Harnblasenkrebs: The Influencing Role of Hematuria. Adv Urol, 2011. 2011: S. 937561. 15. Ludecke, G., et al., Comparative analysis of sensitivity to blood in the urine for urine-based point-of-care assays (UBC rapid, NMP22 BladderChek and BTAstat) in primary diagnosis of bladder carcinoma. Beeinflussung der Ergebnisse von urinbasierten POC-Tests durch Blut. Anticancer Res, 2012. 32(5): S. 2015-8. 16. Ghafouri-Fard, S., L. Nekoohesh, and E. Motevaseli, Bladder cancer biomarkers: review and update. Asian Pac J Cancer Prev, 2014. 15(6): S. 2395-403. 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): S 217-35. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] WO 2012 / 045773 A9

[0064] Cited non-patent literature

[0000] Peter, K., et al., Cancer in Germany 2009 / 2010, in Contributions to Federal Health Reporting. 2013, Robert Koch Institute: Berlin

[0096] Parkin, DM, The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl, 2008(218): pp. 12-20

[0096] H., R., DJ, and K. Th., Bladder Cancer. 2007, German Cancer Aid: 2007

[0096] Witjes, JA, et al., EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol, 2014. 65(4): pp. 778-92

[0096] Urquidi, V., CJ Rosser, and S. Goodison, Multiplex Urinary Tests for Bladder Cancer Diagnosis. Eur Med J Urol, 2013. 1: pp. 70-73

[0096] Family Medicine, DGfAu, DEGAM S1: Non-visible Hematuria 2013

[0096] Elias, K., et al., High-risk patients with hematuria are not evaluated according to guideline recommendations. Cancer, 2010. 116(12): pp. 2954-9

[0096] Schmitz-Drager, BJ, et al., Immunocytology in the assessment of patients with asymptomatic hematuria. World J Urol, 2008. 26(1): pp. 31-7

[0096] Armin, M., Non-visible hematuria in DEGAM S1 action recommendation. 2013, DEGAM - German Society for General Practice and Family Medicine

[0096] Zheng, C., et al., Narrow band imaging diagnosis of bladder cancer: systematic review and meta-analysis. BJU Int, 2012. 110(11 Pt B): S. E680-7

[0096] 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): S. 123-5

[0096] 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): S. E347-52

[0096] 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): S. 7965-7975

[0096] Hennenlotter, J., et al., Point-of-Care-Tests für Harnblasenkrebs: The Influencing Role of Hematuria. Adv Urol, 2011. 2011: S. 937561

[0096] Ludecke, G., et al., Comparative analysis of sensitivity to blood in the urine for urine-based point-of-care assays (UBC rapid, NMP22 BladderChek and BTAstat) in primary diagnosis of bladder carcinoma. Beeinflussung der Ergebnisse von urinbasierten POC-Tests durch Blut. Anticancer Res, 2012. 32(5): S. 2015-8

[0096] Ghafouri-Fard, S., L. Nekoohesh, and E. Motevaseli, Bladder cancer biomarkers: review and update. Asian Pac J Cancer Prev, 2014. 15(6): S. 2395-403

[0096] 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): S 217-35

[0096]

Claims

[1] Use of a marker set comprising at least two substances selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 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 a person having or developing bladder cancer. [2] Use according to claim 1, characterized by that i) succinate and pyroglutamic acid and / or ii) pyroglutamic acid and citrate and / or iii) 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 by that one of the at least two substances is methylpyridinium. [4] Use according to any one of the preceding claims, characterized by that one of at least two substances is 2-furoylglycine. [5] Use according to any one of the preceding claims, characterized by that the marker set contains succinate and / or pyroglutamic acid. [6] Use according to any one of the preceding claims, characterized by that one of at least two substances is trans-aconitate. [7] Use according to any one of the preceding claims, characterized by that the marker set comprises methylpyridinium and at least one second substance selected from the group consisting of 2-furoylglycine, trans-aconitate, succinate and pyroglutamic acid. [8] Use according to any one of the preceding claims, characterized bythat the marker set comprises an additional substance selected from the group consisting of 2'-deoxyguanosine, 2'-deoxyinosine, 2'-deoxyuridine, 2-hydroxybutyrate, 2-hydroxyglutarate, 2-hydroxyvalerate, 2-ketobutyrate, 3,4-dihydroxybenzene acetate, 3-hydroxyisobutyrate, 3-hydroxyisocaleric acid, 3-hydroxykynurenine, 3-hydroxyphenyl acetate, 3-hydroxyvalerate, 3-methyladipate, 3-methylxanthine, 4-hydroxyphenyl acetate, 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, methyl succinate, myo-inositol, N-acetylaspartate, N-acetyl-L-leucine, N-carbamoyaspartate, N-isovaleroglycine, O-acetylcarnitine, pantothenate, phenylalanine, pyridine dicarboxylate, pyridoxine,Pyroglutamate, pyruvate, quinolinic acid, serotonin, S-threo-isocitrate, succinate, succinylacetone, sucrose, threonine, thymidine, tropate, UDP-glucuronate, valerianate, valproate and valproic acid. [9] Use according to any one of the preceding claims, characterized by that the marker set comprises methylpyridinium, 2-furoylglycine and a third substance selected from the group consisting of trans-aconitate, succinate, pyroglutamic acid, citrate, uridine and pseudouridine. [10] Use according to claim 9, characterized by that the third substance is selected from the group consisting of trans-aconitate, succinate and pyroglutamic acid. [11] Use according to claim 9 or 10, characterized bythat the marker set comprises a fourth substance selected from the group consisting of 3-hydroxybutyrate, carnitine, hydroxyindole acetate, 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 selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 3-hydroxybutyrate, carnitine, mannitol, taurine, a proteinogenic branched-chain amino acid, isobutyrate, 2-ketobutyrate, uridine, pseudouridine, lactate and quinolinic acid for use in the in vivo diagnosis of bladder cancer and / or in predicting the risk of an individual to develop bladder cancer. [13] A method for the in vitro analysis of an isolated body fluid sample, comprising the following steps: a) determining the concentration of at least two substances selected from the group consisting of methylpyridinium, 2-furoylglycine, trans-aconitate, succinate, pyroglutamic acid, citrate, hydroxyindole acetate, 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 of a person by analyzing the body fluid sample using a suitable measuring technique, and b) Calculating a score from the determined concentrations, the score indicating the person's risk of having or developing bladder cancer. [14] Method according to claim 13, characterized by that the body fluid sample is a urine sample or a blood sample. [15] Method according to claim 13 or 14, characterized bythat the body fluid sample is divided into one of at least two predefined groups based on the calculated score.

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