In vitro diagnosis of prostate cancer by means of urine biomarkers

Abstract

The present invention relates to a method for in vitro / ex vivo diagnosis of prostate cancer and / or its aggressiveness by means of urine biomarkers.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims the priority of Italian Patent Application No. 102018000007264, filed on Jul. 17, 2018, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a method for in vitro / ex vivo diagnosis of prostate cancer and / or its aggressiveness by means of urine biomarkers. Background Art

[0004] Prostate cancer (PCa) is the most common disease and the second leading cause of cancer-related death in men worldwide.

[0005] Currently, the diagnosis of prostate cancer is made only after histological evaluation of the tumor on various prostate tissue samples obtained by biopsy. Prostate biopsy is performed after evaluation of circulating levels of prostate specific antigen (PSA) and digital rectal examination (DRE).

[0006] PSA and DRE are initial tests designed to identify patients most likely to have a prostate tumor. In the presence of an abnormality, a prostate biopsy is performed to confirm or deny the clinical evidence. However, the accuracy of these tests is limited, leading to a large amount of overdiagnosis and, in turn, overtreatment.

[0007] In fact, on average, only one in four men who undergo a prostate biopsy due to suspicious PSA levels will be affected by prostate cancer following the biopsy. Furthermore, many of the prostate cancers that are diagnosed progress slowly and may not show symptoms throughout the patient's lifetime. Because it is impossible to distinguish overdiagnosed tumors from other tumors, the majority of patients who screen positive are offered surgery, which is often accompanied by adverse effects that have a significant impact on quality of life (e.g., erectile dysfunction, urinary incontinence, infection).

[0008] In addition to the inconvenience to patients, the economic impact of such a high rate of unnecessary testing on health system resources should also be considered. One way to assess whether resources are well used (value for money) is to express the clinical benefit in relation to economic aspects. The two existing economic studies of PSA screening agree that the procedure is not cost-effective.

[0009] In short, this means that once a PSA test has been performed (due to biopsy, treatment, possible damage), the costs associated with the performed procedure, quantified from an economic point of view, for the entire population, exceed the benefits.

[0010] In view of the above, it is clear that there is an urgent need to identify specific biomarkers for the diagnosis of prostate cancer to pinpoint men at risk of developing prostate cancer and predict the natural progression of the tumor.

[0011] Some recent studies suggest that male infertility may be a risk signal for developing aggressive prostate cancer. In particular, patients diagnosed with infertility have been found to be three times more likely to develop aggressive prostate tumors than fertile men (Walsh TJ et al., Cancer. 2010 May 1; 116(9):2140-7).

[0012] The prostate is a gland whose main function is to produce semen, which contains elements essential for sperm survival, motility and quality. Chronic inflammatory diseases have been found to affect fertility by causing changes in the physico-chemical properties of the ejaculatory fluid fraction (changes in viscosity and fluidity, changes in pH, the presence of white globules, changes in PSA, PAP, zinc, fructose and citric acid levels).

[0013] During neoplastic transformation, cells may undergo profound phenotypic and functional changes. As most prostate tumors are classified as adenocarcinomas, i.e., tumors involving portions of the gland, it is plausible that malignant transformation within the prostate may impair the secretory function of the gland, including the composition of prostate secretions.

[0014] It has been found from some studies that advanced prostate tumors may lose the expression of PSA (Aihara M et al., J Urol. 1994; 151: 1558-1564; Weir EG et al., J Urol. 2000; 163: 1739-1742; Augustin H et al., J Cancer Res Clin Oncol. 2003; 129: 662-668). More detailed studies using tissue microarray technology have confirmed that the expression of PSA in tissues can be used as both a diagnostic parameter and a prognostic parameter for prostate cancer (Erbersdobler A et al., Urology. 2009 Nov; 74 (5): 1169-73).

[0015] Since the urinary tract is in close contact with the prostate, factors produced by prostate tissue can be transferred and therefore detected in urine, which represents biomarkers for diagnosis and prognosis of prostate cancer.

[0016] On this basis, measurement of urinary PSA should provide useful information about the physiological and pathological status of the prostate, since PSA is a normal product of the epithelial cells surrounding the prostatic acini and prostatic tubules (Irani J et al., Eur Urol 1996; 29: 407-12).

[0017] EP2515115 describes the use of urine PSA as a prostate tumor marker. However, in EP2515115, consistent prostate secretions cannot be obtained without prostate massage before collecting urine. For healthy subjects, the average value identified is about 50ng / ml, and for subjects affected by tumors, the average value is about 5ng / ml.

[0018] In a paper published in 2007, it was observed that the ratio between urine PSA and plasma PSA was different in patients with prostate tumors, patients with benign organ hyperplasia and healthy subjects (Bolduc S et al., Can Urol Assoc J. 2007 Nov; 1(4): 377-81).

[0019] Recently, it has been found that circulating PSA exists in free form and in complex with different molecules of the protease inhibitor family. PSA is often complexed with alpha1-antichymotripsin (ACT), and it has been observed that patients with prostate tumors have a higher percentage of PSA bound to ACT than healthy donors, who have a higher percentage of free PSA (Christensson A et al., J Urol. 1993; 150: 100–5).

[0020] Various studies have shown that ACT expression at the tissue level is higher in prostate tumors as mRNA and protein compared to benign hyperplasia, demonstrating higher content of composite PSA (Zhu L et al., Prostate. 2013 Jan;73(2):219-26).

[0021] WO0002052 describes the use of complexed and free, cleaved and uncleaved PSA as biomarkers in plasma / serum to distinguish healthy subjects from subjects with prostate cancer. Summary of the invention

[0022] The object of the present invention is to provide a method for in vitro / ex vivo diagnosis of prostate cancer by means of new biomarkers present in urine, which method can provide reliable and accurate results quickly and inexpensively.

[0023] According to the invention, this object is achieved by a method according to claims 1 and 4 .

[0024] According to the present invention, a kit for use in the above method is also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] For a better understanding of the present invention, reference will now be made to the accompanying drawings, which illustrate the following:

[0026] - Figure 1 A graph showing absolute values ​​of urine biomarkers zinc (Zn), total PSA (utPSA), free PSA (ufPSA), spermine, spermine oxidase (SMOX) in individuals affected by prostate cancer and healthy individuals;

[0027] - Figure 2 A graph showing the values ​​of urine biomarkers Zn, utPSA, ufPSA, spermine (Spm), spermine oxidase (SMOX) normalized to creatinine in individuals affected by prostate cancer and healthy individuals;

[0028] - Figure 3 The receiver operating characteristic (ROC) curves of Zn, spermine (Spm), utPSA, ufPSA, and spermine oxidase (SMOX) are shown;

[0029] - Figure 4a ROC curves for the combinations of Zn-Spm, Zn-utPSA, Zn-ufPSA, ufPSA-Spm, utPSA-ufPSA, and utPSA-Spm are shown;

[0030] - Figure 4b ROC curves for the combinations of Zn-Spm-utPSA, Zn-Spm-ufPSA, Zn-utPSA-ufPSA, Spm-utPSA-ufPSA, and Spm-utPSA-ufPSA-Zn are shown;

[0031] - Figure 4c ROC curves of the combinations of Zn-Spm-SMOX, Zn-utPSA-SMOX, Zn-ufPSA-SMOX, ufPSA-Spm-SMOX, utPSA-Spm-SMOX, and Zn-utPSA-SMOX-Spm are shown;

[0032] - Figure 5 Shown are graphs of absolute values ​​of urine biomarkers zinc (Zn), total PSA (utPSA), free PSA (ufPSA), spermine, spermine oxidase (SMOX) in individuals affected by prostate cancer divided into low, intermediate and high risk based on the aggressiveness of the tumor and in healthy individuals. DETAILED DESCRIPTION

[0033] According to the present invention, a method for in vitro / ex vivo diagnosis of prostate cancer in an individual comprises the following steps.

[0034] In the first step, a urine sample is provided from the individual. The urine sample is preferably obtained after a prostate massage is performed on the patient.

[0035] In the second step, the zinc in the urine sample is quantified, preferably by colorimetry.

[0036] In a third step, the zinc value is compared with a reference value. The reference value corresponds to a value that is characteristic for individuals not affected by prostate cancer.

[0037] The method according to the invention can not only diagnose prostate cancer, but also assess its aggressiveness. The marker according to the invention is expressed differently in the urine of patients with low, medium and high risk of disease progression. "Aggressiveness of a tumor" refers to the risk level of disease progression.

[0038] Preferably, in the second step of the above method, total PSA (prostate specific antigen) (utPSA) is also quantified, and in the third step, the quantified value of total PSA (utPSA) is compared with a reference value.

[0039] Even more preferably, in the second step, free PSA (prostate specific antigen) (ufPSA) is quantified, and in the third step, the quantified value of free PSA (ufPSA) is compared with a reference value.

[0040] Preferably, quantification of total PSA (utPSA) and / or free PSA (ufPSA) is performed by immunoassay, spectroscopy, spectrophotometry, colorimetry, electrophoresis, complexometry, amperometry, even more preferably, by immunoassay.The PSA sequence is provided as SEQ ID NO:1.

[0041] Even more preferably, in the second step, the prostate cancer biomarker is further quantified, and in the third step, the quantitative value of the biomarker is compared with a reference value. The biomarker is selected from prostatic acid phosphatase (PAP), fibrinolysin, profibrinolysin, citrate, fructose, putrescine, spermidine, spermine, spermine oxidase, prostaglandin, calcium, copper. The biomarker is preferably spermine (C 10 H 26N4) or spermine oxidase (SEQ ID NO: 2). Even more preferably, it is spermine oxidase. Preferably, the quantification of spermine or spermine oxidase is performed by immunoassay, spectroscopy, spectrophotometry, colorimetry, electrophoresis, complexometry, amperometry, even more preferably by immunoassay.

[0042] Preferably, in the method according to the present invention, the values ​​of biomarkers (zinc, total PSA (utPSA), free PSA (ufPSA), spermine, spermine oxidase, etc.) are normalized relative to the values ​​of creatinine to improve the accuracy of the test (to be further explained in detail in the examples).

[0043] Therefore, in the preferred embodiment, the method according to the present invention comprises the following steps.

[0044] In the first step, a urine sample is provided from an individual.

[0045] In the second step, zinc is quantified in urine samples.

[0046] In the third step, the molecule with the molecular formula C4H7N3O (creatinine) in the urine sample is quantified. Preferably, the quantification is performed by immunoassay, enzymatic method or Jaffe method.

[0047] In the fourth step, the quantitative value of zinc is normalized with the quantitative value of a molecule having the molecular formula C4H7N3O (creatinine).

[0048] In a fifth step, the normalized zinc values ​​are compared to reference values. The reference values ​​correspond to values ​​that are characteristic of individuals not affected by prostate cancer.

[0049] Also in this embodiment, preferably the total PSA (prostate specific antigen) value (utPSA) is quantified and normalized, and even more preferably the free PSA (prostate specific antigen) value (ufPSA) is quantified and normalized.

[0050] Therefore, preferably, total PSA (prostate-specific antigen) (utPSA) is also quantified in the second step, in the fourth step, the quantitative value of total PSA (utPSA) is normalized by the quantitative value of a molecule with the molecular formula C4H7N3O (creatinine), and then in the fifth step, the normalized value of total PSA (utPSA) is compared with the reference value.

[0051] Even more preferably, free PSA (prostate-specific antigen) (ufPSA) is also quantified in the second step, in the fourth step, the quantitative value of free PSA (ufPSA) is normalized by the quantitative value of a molecule with the molecular formula C4H7N3O (creatinine), and in the fifth step, the normalized value of free PSA (ufPSA) is compared with a reference value.

[0052] Even more preferably, in the second step, the prostate cancer biomarker is further quantified, in the fourth step, the quantitative value of the biomarker is normalized with the quantitative value of a molecule with the molecular formula C4H7N3O (creatinine), and in the fifth step, the normalized value of the biomarker is compared with a reference value. The biomarker is selected from prostatic acid phosphatase (PAP), plasmin, plasminogen, citrate, fructose, putrescine, spermidine, spermine, prostaglandin, calcium, copper. The biomarker is preferably spermine or spermine oxidase.

[0053] The kit for use in the method according to the invention comprises

[0054] - Devices for collecting and preparing urine samples from individuals;

[0055] - Device for quantifying zinc.

[0056] Preferably, the kit further comprises:

[0057] - means for quantifying total PSA (utPSA); and / or

[0058] - a device for quantifying free PSA (ufPSA); and / or

[0059] - means for quantifying other prostate cancer biomarkers, preferably spermine or spermine oxidase; and / or

[0060] -A device for quantifying the molecule with the molecular formula C4H7N3O (creatinine).

[0061] Example

[0062] Urine samples were collected from 110 patients aged 51 to 86 years with an indication for prostate biopsy.

[0063] Urine samples were collected after a digital rectal examination and prostate massage, starting from the base, moving toward the middle and apex, with three digital compressions per lobe for 180 seconds. Within 30 minutes of collection, samples were agitated and frozen in Falcon tubes at -80°C.

[0064] Twelve biopsy samples were also taken from each patient for normal diagnostic procedures in the anatomic pathology department. Histological evaluation determined the presence of tumor cells (PCa) in 53 patients. In the remaining patients, 29 were free of disease, benign pathological conditions were found in 16, and possible precancerous conditions were found in 12. This suggests that approximately 55% of patients with suspected PSA / DRE / ECO underwent unnecessary biopsies.

[0065] Urine samples were thawed and assessed for the presence of utPSA and ufPSA biomarkers by ELISA assay (Sigma Aldrich), zinc was quantified by colorimetry (Zinc assay, Sigma Aldrich), spermine was quantified by ELISA assay (MyBiosource), and spermine oxidase was quantified by ELISA assay (MyBiosource).

[0066] Because the amount of biomarkers in urine depends on the amount of urine collected and its concentration, a calculation, called "creatinine normalization," is performed to improve the accuracy of the test. Creatinine is a substance produced by the body that is excreted through urine, and its level is an indicator of urine concentration. The higher the creatinine value, the more concentrated the urine. The lower the creatinine value, the more dilute the urine.

[0067] To eliminate possible confounding factors, cases with precancerous conditions and benign lesions were excluded.

[0068] Based on the absolute values ​​of the biomarkers, statistically significant differences between healthy and diseased patients can be determined (see Figure 1 ).

[0069] After creatinine normalization, even more pronounced differences were observed between diseased and healthy patients for all five markers studied (see Figure 2 ).

[0070] The values ​​of Zn, spermine, utPSA, ufPSA, and spermine oxidase were correlated with the presence of tumors, and their AUCs were 0.656, 0.628, 0.644, 0.617, and 0.684, respectively (see Figure 3 ).

[0071] By performing multivariate analysis, it was observed that the combination of multiple factors increased the power of the test in correlating biomarker levels and prostate tumors (see Figure 4a , 4b and 4c).

[0072] Analyzing the same values ​​after dividing the patients according to the aggressiveness of their tumors (low, intermediate, and high risk), it is important to emphasize that in patients with more advanced tumors, all five markers analyzed were reduced to a greater extent (see Figure 5 ).

[0073] From the results of the embodiments, the advantages of the present invention are apparent.

[0074] Analysis of the absolute values ​​of the individual biomarkers zinc, total PSA, free PSA, spermine, spermine oxidase in urine samples has been indicative of prostate cancer. Analysis of normalized values ​​relative to creatinine allows even more accurate information to be obtained. Simultaneous analysis of several biomarkers allows for more reliable identification of prostate cancer patients. In particular, the combination of Zn, utPSA and spermine oxidase improves the discrimination between healthy patients and prostate cancer patients (AUC = 0.711).

[0075] For each analyte, a value of 80% sensitivity was determined as the optimal threshold.

[0076] Samples that present a biomarker in an amount above the threshold are considered positive.

[0077] In this way, based on the number of positive biomarkers for each sample, a scale from 0 to 3 (score) was established and the probability of having prostate cancer (risk) was defined.

[0078] Table 1

[0079] Points risk 0 0 1 14 2 43 3 66

[0080] As shown in Table 1, for individuals without positive biomarkers, the probability of having prostate cancer is zero (0%).

[0081] In individuals with 1, 2, and 3 positive biomarkers, the probability increases to 14%, 43%, and 66%, respectively. These results show that based on the use of biomarkers according to the present invention, it is possible to identify individuals with a very low probability of prostate cancer, thus avoiding them from undergoing invasive clinical examinations.

[0082] The method according to the invention represents a rapid and inexpensive way of obtaining a reliable diagnosis, avoiding invasive surgery. Sequence Listing <110> NIB Biotech Co., Ltd. <120> In vitro diagnosis of prostate cancer by means of urine biomarkers <130> 235-18 <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <220> <223> Prostate-specific antigen <400> 1 Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu 65 70 75 80 Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe 85 90 95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg 100 105 110 Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu 115 120 125 Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln 130 135 140 Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu 165 170 175 His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val 180 185 190 Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr 195 200 205 Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln 210 215 220 Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro 225 230 235 240 Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr 245 250 255 Ile Val Ala Asn Pro 260 <210> 2 <211> 555 <212> PRT <213> Homo sapiens <220> <223> Spermine oxidase <400> 2 Met Gln Ser Cys Glu Ser Ser Gly Asp Ser Ala Asp Asp Pro Leu Ser 1 5 10 15 Arg Gly Leu Arg Arg Arg Gly Gln Pro Arg Val Val Val Ile Gly Ala 20 25 30 Gly Leu Ala Gly Leu Ala Ala Ala Lys Ala Leu Leu Glu Gln Gly Phe 35 40 45 Thr Asp Val Thr Val Leu Glu Ala Ser Ser His Ile Gly Gly Arg Val 50 55 60 Gln Ser Val Lys Leu Gly His Ala Thr Phe Glu Leu Gly Ala Thr Trp 65 70 75 80 Ile His Gly Ser His Gly Asn Pro Ile Tyr His Leu Ala Glu Ala Asn 85 90 95 Gly Leu Leu Glu Glu Thr Thr Asp Gly Glu Arg Ser Val Gly Arg Ile 100 105 110 Ser Leu Tyr Ser Lys Asn Gly Val Ala Cys Tyr Leu Thr Asn His Gly 115 120 125 Arg Arg Ile Pro Lys Asp Val Val Glu Glu Phe Ser Asp Leu Tyr Asn 130 135 140 Glu Val Tyr Asn Leu Thr Gln Glu Phe Phe Arg His Asp Lys Pro Val 145 150 155 160 Asn Ala Glu Ser Gln Asn Ser Val Gly Val Phe Thr Arg Glu Glu Val 165 170 175 Arg Asn Arg Ile Arg Asn Asp Pro Asp Asp Pro Glu Ala Thr Lys Arg 180 185 190 Leu Lys Leu Ala Met Ile Gln Gln Tyr Leu Lys Val Glu Ser Cys Glu 195 200 205 Ser Ser Ser His Ser Met Asp Glu Val Ser Leu Ser Ala Phe Gly Glu 210 215 220 Trp Thr Glu Ile Pro Gly Ala His His Ile Ile Pro Ser Gly Phe Met 225 230 235 240 Arg Val Val Glu Leu Leu Ala Glu Gly Ile Pro Ala His Val Ile Gln 245 250 255 Leu Gly Lys Pro Val Arg Cys Ile His Trp Asp Gln Ala Ser Ala Arg 260 265 270 Pro Arg Gly Pro Glu Ile Glu Pro Arg Gly Glu Gly Asp His Asn His 275 280 285 Asp Thr Gly Glu Gly Gly Gln Gly Gly Glu Glu Pro Arg Gly Gly Arg 290 295 300 Trp Asp Glu Asp Glu Gln Trp Ser Val Val Val Glu Cys Glu Asp Cys 305 310 315 320 Glu Leu Ile Pro Ala Asp His Val Ile Val Thr Val Ser Leu Gly Val 325 330 335 Leu Lys Arg Gln Tyr Thr Ser Phe Phe Arg Pro Gly Leu Pro Thr Glu 340 345 350 Lys Val Ala Ala Ile His Arg Leu Gly Ile Gly Thr Thr Asp Lys Ile 355 360 365 Phe Leu Glu Phe Glu Glu Pro Phe Trp Gly Pro Glu Cys Asn Ser Leu 370 375 380 Gln Phe Val Trp Glu Asp Glu Ala Glu Ser His Thr Leu Thr Tyr Pro 385 390 395 400 Pro Glu Leu Trp Tyr Arg Lys Ile Cys Gly Phe Asp Val Leu Tyr Pro 405 410 415 Pro Glu Arg Tyr Gly His Val Leu Ser Gly Trp Ile Cys Gly Glu Glu 420 425 430 Ala Leu Val Met Glu Lys Cys Asp Asp Glu Ala Val Ala Glu Ile Cys 435 440 445 Thr Glu Met Leu Arg Gln Phe Thr Gly Asn Pro Asn Ile Pro Lys Pro 450 455 460 Arg Arg Ile Leu Arg Ser Ala Trp Gly Ser Asn Pro Tyr Phe Arg Gly 465 470 475 480 Ser Tyr Ser Tyr Thr Gln Val Gly Ser Ser Gly Ala Asp Val Glu Lys 485 490 495 Leu Ala Lys Pro Leu Pro Tyr Thr Glu Ser Ser Lys Thr Ala Pro Met 500 505 510 Gln Val Leu Phe Ser Gly Glu Ala Thr His Arg Lys Tyr Tyr Ser Thr 515 520 525 Thr His Gly Ala Leu Leu Ser Gly Gln Arg Glu Ala Ala Arg Leu Ile 530 535 540 Glu Met Tyr Arg Asp Leu Phe Gln Gln Gly Thr 545 550 555

Claims

1. A kit for use in a method for in vitro / ex vivo diagnosis of prostate cancer and / or its aggressiveness in an individual, comprising: - Devices for collecting and preparing urine samples from individuals; - Device for dosing zinc; - a device for quantifying total prostate specific antigen and / or a device for quantifying free prostate specific antigen; - a device for quantifying spermine oxidase; Wherein, the method comprises the following steps: a) providing a urine sample from the individual; b) quantifying zinc and total prostate specific antigen and / or free prostate specific antigen in the urine sample; c) comparing the quantified value of zinc and the quantified value of total prostate-specific antigen and / or the quantified value of free prostate-specific antigen with reference values, respectively; In step b), spermine oxidase is also quantified, and in step c), the quantified value of spermine oxidase is compared with a reference value.

2. The kit according to claim 1, further comprising a device for quantifying spermine, wherein: Spermine is also quantified in step b) and the quantified value of spermine is compared with a reference value in step c).

3. The kit according to claim 1, wherein the quantification of zinc is performed by a colorimetric test.

4. The kit according to claim 1, wherein the quantification of total prostate specific antigen and / or free prostate specific antigen is performed by immunoassay, spectroscopy, spectrophotometry, colorimetry, electrophoresis, complexation, amperometry.

5. A kit for use in a method for in vitro / ex vivo diagnosis of prostate cancer and / or its aggressiveness in an individual, comprising: - Devices for collecting and preparing urine samples from individuals; - Device for dosing zinc; - a device for quantifying total prostate specific antigen and / or a device for quantifying free prostate specific antigen; - A device for quantifying creatinine molecules; - a device for quantifying spermine oxidase, Wherein, the method comprises the following steps: a') providing a urine sample from the individual; b') quantifying zinc in said urine sample; c') quantifying creatinine molecules in said urine sample; d') Normalization of the quantitative value of zinc with the quantitative value of creatinine molecules; e') Comparison of normalized values ​​of zinc with reference values; Wherein, in step b'), total prostate specific antigen and / or free prostate specific antigen are also quantified, In step b'), spermine oxidase is quantified, in step d'), the quantified value of spermine oxidase is normalized with the quantified value of creatinine molecules, and in step e'), the normalized value of spermine oxidase is compared with a reference value.

6. The kit according to claim 5, wherein In step d'), the quantitative value of total prostate specific antigen is normalized with the quantitative value of creatinine molecules and / or the quantitative value of free prostate specific antigen is normalized with the quantitative value of creatinine molecules, and in step e'), the normalized value of total prostate specific antigen is compared with a reference value and / or the normalized value of free prostate specific antigen is compared with a reference value.

7. The kit according to claim 5, further comprising a device for quantifying spermine, wherein: In step b'), spermine is quantified, in step d'), the quantified value of spermine is normalized with the quantified value of creatinine molecules, and in step e'), the normalized value of spermine is compared with a reference value.

8. The kit according to claim 5, wherein In step b'), total prostate specific antigen, zinc and spermine oxidase are quantified, in step d'), the quantified values ​​of total prostate specific antigen, zinc and spermine oxidase are normalized with the quantified values ​​of creatinine molecules, and in step e'), the normalized values ​​of total prostate specific antigen, zinc and spermine oxidase are compared with reference values.

9. The kit according to claim 5, wherein the quantification of total prostate specific antigen and / or free prostate specific antigen is performed by immunoassay, spectroscopy, spectrophotometry, colorimetry, electrophoresis, complexometry, amperometry.

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