In vitro method for diagnosing prostate cancer using urinary biomarkers

Abstract

The present invention relates to a method for in vitro / ex vivo diagnosis of prostate cancer and / or its malignancy using urinary biomarkers. [Selected Figure] Figure 1

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This patent application claims priority to Italian Patent Application No. 102018000007264, filed July 17, 2018, the entire disclosure of which is hereby incorporated by reference.

[0002] The present invention relates to an in vitro / ex vivo method for diagnosing prostate cancer and / or its malignancy using urinary biomarkers. [Background technology]

[0003] Prostate cancer (PCa) is the most common pathology and the second leading cause of cancer-related death in men worldwide.

[0004] Currently, the diagnosis of prostate cancer is made only after histological evaluation of the tumor on various prostate tissue samples taken by biopsy. A prostate biopsy is indicated after evaluation of circulating levels of prostate-specific antigen (PSA) along with a digital rectal examination (DRE).

[0005] PSA and DRE are preliminary tests aimed at identifying patients who are likely to develop prostate tumors. If abnormal, a prostate biopsy can be performed to confirm or refute the clinical evidence. However, the accuracy of these tests is limited, leading to a large number of overdiagnoses and, consequently, overtreatment.

[0006] In fact, for every four men who undergo a prostate biopsy due to a suspicious PSA level, on average only one individual is affected by prostate cancer after the biopsy. Furthermore, many of the diagnosed prostate cancers are very slow-growing and may not show symptoms throughout the patient's life. Since it is impossible to distinguish overdiagnosed tumors from other tumors, the majority of patients found to be screen-positive are often offered surgery with adverse effects (erectile dysfunction, urinary incontinence, infections, etc.) that greatly impact their quality of life.

[0007] In addition to the inconvenience to patients, one should consider the economic impact of such a high rate of unnecessary testing on the resources of the health care system. One way to evaluate whether resources are being well utilized (value for money) is to express the clinical benefit in economic terms. The two economic studies that exist for PSA-based screening are unanimous in determining that the procedure is not cost-effective.

[0008] This means that across the population, the costs associated with the processes that take place after a PSA test is performed (due to biopsies, treatments, and potential damage) outweigh the quantified benefits in economic terms.

[0009] In view of the above, it is clear that there is an urgent need to identify specific biomarkers for the diagnosis of prostate cancer in order to accurately identify men at risk of developing prostate cancer and to predict the natural progression of the tumor.

[0010] Some recent studies suggest that male infertility may signal the risk of developing malignant forms of prostate cancer. In particular, it has been found that patients diagnosed with infertility are three times more likely to develop malignant prostate tumors than the fertile male population (Non-Patent Document 1).

[0011] The prostate is a gland whose main function is to produce semen, which contains the factors underlying sperm survival, motility and quality. Chronic inflammatory conditions have been shown to cause changes in the physicochemical properties of the liquid portion of the ejaculate (variations in viscosity and fluidification, pH, presence of white globules, changes in the levels of PSA, PAP, zinc, fructose and citric acid) that affect fertility.

[0012] During neoplastic transformation, cells can undergo profound phenotypic and functional changes. Since the majority of prostate tumors are classified as adenocarcinomas, i.e., tumors involving the glandular portion, it is not surprising that neoplastic transformation within the prostate can threaten the secretory function of the gland and compromise the composition of prostatic secretions.

[0013] Several studies have revealed that PSA expression can be lost in advanced prostate tumors (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). More detailed studies using tissue microarray technology have confirmed that PSA expression in tissues can be used as both a diagnostic and prognostic parameter for prostate cancer (Non-Patent Document 5).

[0014] Because the urinary tract is in close contact with the prostate, factors produced by prostate tissue are transported and, as a result, can be detected in the urine, providing biological markers for the diagnosis and prognosis of prostate cancer.

[0015] On this basis, since PSA is a normal product of epithelial cells surrounding the prostatic chambers and ducts, measurement of urinary PSA should provide useful information regarding the physiology and pathological status of the prostate (Non-Patent Document 6).

[0016] US Patent No. 5,399,663 describes the use of urinary PSA as a prostate tumor marker. However, in US Patent No. 5,399,663, consistent prostate secretions cannot be obtained without prostate massage before urine collection. The mean values ​​identified are about 50 ng / ml in healthy subjects and 5 ng / ml in tumor-affected subjects.

[0017] A study published in 2007 observed differences in the ratio between urinary PSA and plasma PSA between patients with prostate tumors, patients with benign hyperplasia of the organ, and healthy subjects (Non-Patent Document 7).

[0018] Recently, it has been found that circulating PSA exists in a free form and in a form complexed with various molecules of the protease inhibitor family. More frequently, PSA is complexed with α1-antichymotrypsin (ACT), and it has been observed that patients with prostate tumors have a higher percentage of PSA bound to ACT than healthy donors, while healthy donors show a higher percentage of free PSA (Non-Patent Document 8).

[0019] The higher amount of complexed PSA makes sense, since various studies have shown that the expression of ACT as mRNA and protein is higher at the tissue level in prostate tumors compared to benign hyperplasia (Non-Patent Document 9).

[0020] US Patent No. 5,399,633 describes the use of complexed and free, cleaved and uncleaved PSA as biomarkers in plasma / serum to distinguish between healthy subjects and those with prostate cancer. [Prior art documents] [Patent documents]

[0021] [Patent Document 1] European Patent Application Publication No. 2515115 [Patent Document 2] International Publication No. 0002052 [Non-patent literature]

[0022] [Non-Patent Document 1] Walsh TJ et al., Cancer. 2010 May 1; 116(9):2140-7 [Non-Patent Document 2] Aihara M et al., J Urol. 1994; 151:1558-1564 [Non-Patent Document 3] Weir EG et al., J Urol. 2000; 163:1739-1742 [Non-Patent Document 4] Augustin H et al., J Cancer Res Clin Oncol. 2003; 129:662-668 [Non-Patent Document 5] Erbersdobler A et al., Urology. 2009 Nov; 74(5):1169-73 [Non-Patent Document 6] Irani J et al., Eur Urol 1996; 29:407-12 [Non-Patent Document 7] Bolduc S et al., Can Urol Assoc J. 2007 Nov; 1(4):377-81 [Non-Patent Document 8] Christensson A et al., J Urol. 1993; 150:100-5 [Non-Patent Document 9] Zhu L et al., Prostate. 2013 Jan; 73(2):219-26 Summary of the Invention [Problem to be solved by the invention]

[0023] The aim of the present invention is to provide an in vitro / ex vivo method for diagnosing prostate cancer using new biomarkers present in urine, which is rapid, inexpensive, reliable and accurate. [Means for solving the problem]

[0024] According to the invention, this object is achieved by the methods according to claims 1 and 4.

[0025] The present invention also provides kits for use in the above-described methods.

[0026] For a better understanding of the invention, it will now also be described with reference to the accompanying drawings. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 shows graphs of absolute values ​​of urinary biomarkers zinc (Zn), total PSA (utPSA), free PSA (ufPSA), spermine, and spermine oxidase (SMOX) in individuals affected by prostate cancer and healthy individuals. [Diagram 2] FIG. 1 shows graphs of the values ​​of the urinary biomarkers Zn, utPSA, ufPSA, spermine (Spm), spermine oxidase (SMOX) normalized to creatinine in individuals affected by prostate cancer and healthy individuals. [Diagram 3] FIG. 1 shows ROC (Receiver Operating Characteristic) curves for Zn, spermine (Spm), utPSA, ufPSA, and spermine oxidase (SMOX). [Figure 4a] FIG. 1 shows ROC curves for the combinations of Zn-Spm, Zn-utPSA, Zn-ufPSA, ufPSA-Spm, utPSA-ufPSA, and utPSA-Spm. [Figure 4b] FIG. 1 shows ROC curves for the combinations Zn-Spm-utPSA, Zn-Spm-ufPSA, Zn-utPSA-ufPSA, Spm-utPSA-ufPSA, and Spm-utPSA-ufPSA-Zn. [Figure 4c] FIG. 1 shows ROC curves for the combinations Zn-Spm-SMOX, Zn-utPSA-SMOX, Zn-ufPSA-SMOX, ufPSA-Spm-SMOX, utPSA-Spm-SMOX, and Zn-utPSA-SMOX-Spm. [Diagram 5] FIG. 1 shows graphs of absolute values ​​of urinary biomarkers zinc (Zn), total PSA (utPSA), free PSA (ufPSA), spermine, and spermine oxidase (SMOX) in individuals with prostate cancer divided into low, intermediate, and high risk based on tumor grade and in healthy individuals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] According to the present invention, a method for diagnosing prostate cancer in an individual in vitro / ex vivo comprises the steps of:

[0029] In a first step, a urine sample from an individual is prepared, preferably obtained after the patient has undergone a prostate massage.

[0030] In a second step, zinc is quantified in the urine sample, preferably by a colorimetric test.

[0031] In a third step, the zinc level is compared to a reference value, which corresponds to a value typical of individuals not affected by prostate cancer.

[0032] The method according to the invention not only allows the diagnosis of prostate cancer, but also allows the assessment of its aggressiveness. The markers according to the invention are differentially expressed in the urine of patients with low, medium and high risk of disease progression. By "tumor aggressiveness" is meant the level of risk of disease progression.

[0033] Preferably, in a second step of the above-mentioned method, total PSA (prostate specific antigen) (utPSA) is also quantified, and in a third step, the quantified value of total PSA (utPSA) is compared with a reference value.

[0034] Even more preferably, in a second step, free PSA (prostate specific antigen) (ufPSA) is quantified and in a third step the quantified value of free PSA (ufPSA) is compared with a reference value.

[0035] Preferably, quantification of total PSA (utPSA) and / or free PSA (ufPSA) is performed by immunoassay, spectroscopy, spectrophotometry, colorimetry, electrophoresis, complexometry, amperometry, and even more preferably by immunoassay. The PSA sequence is provided as SEQ ID NO:1.

[0036] Even more preferably, in a second step, further prostate cancer biomarkers are quantified and in a third step, the quantified values ​​of said biomarkers are compared to reference values. The biomarkers are selected from the group consisting of prostatic acid phosphatase (PAP), fibrinolysin, profibrinollysin, citrate, fructose, putrescine, spermidine, spermine, spermine oxidase, prostaglandins, calcium, copper. The biomarkers are preferably spermine (C 10 H 26 N4) or spermine oxidase (SEQ ID NO: 2). Even more preferably, said biomarker 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.

[0037] Preferably, in the method according to the invention, the values ​​of the biomarkers (zinc, total PSA (utPSA), free PSA (ufPSA), spermine, spermine oxidase, etc.) are normalised to the value of creatinine to increase the accuracy of the test (as explained in further detail in the Examples).

[0038] Thus, in this preferred embodiment, the method according to the invention comprises the following steps:

[0039] In a first step, a urine sample from an individual is prepared.

[0040] In the second step, zinc is quantified in the urine sample.

[0041] In a third step, the molecule with the molecular formula C4H7N3O (creatinine) is quantified in the urine sample. Preferably, the quantification is performed by immunoassay, enzymatic method or Jaffe method.

[0042] In the fourth step, the quantified value of zinc is normalized with the quantified value of a molecule having the molecular formula C4H7N3O (creatinine).

[0043] In a fifth step, the normalized zinc value is compared to a reference value, which corresponds to a typical value for individuals not affected by prostate cancer.

[0044] Also in this embodiment, total PSA (prostate specific antigen) values ​​(utPSA) are preferably quantified and normalized, and even more preferably, free PSA (prostate specific antigen) values ​​(ufPSA) are quantified and normalized.

[0045] Thus, preferably, in a second step, total PSA (prostate specific antigen) (utPSA) is also quantified, in a fourth step the quantified value of total PSA (utPSA) is normalized with the quantified value of a molecule having the molecular formula C4H7N3O (creatinine), and in a fifth step the normalized value of total PSA (utPSA) is compared with a reference value.

[0046] Even more preferably, in a second step, free PSA (prostate specific antigen) (ufPSA) is also quantified, in a fourth step the quantified value of free PSA (ufPSA) is normalized with the quantified value of a molecule having the molecular formula C4H7N3O (creatinine), and in a fifth step the normalized value of free PSA (ufPSA) is compared with a reference value.

[0047] Even more preferably, in a second step, a further prostate cancer biomarker is quantified, in a fourth step, the quantified value of said biomarker is normalized with the quantified value of a molecule having the molecular formula C4H7N3O (creatinine), and in a fifth step, the normalized value of said biomarker is compared with a reference value. The biomarker is selected from the group consisting of prostatic acid phosphatase (PAP), fibrinolysin, profibrinollysin, citrate, fructose, putrescine, spermidine, spermine, prostaglandins, calcium, copper. The biomarker is preferably spermine or spermine oxidase.

[0048] The kit for use in the method according to the invention comprises: a means for collecting and preparing a urine sample from an individual; A means for quantifying zinc; Includes.

[0049] Preferably, the kit also comprises: a means to quantify total PSA (utPSA), and / or A means to quantify free PSA (ufPSA), and / or means to quantify other prostate cancer biomarkers, preferably spermine or spermine oxidase; and / or A means for quantifying a molecule having the molecular formula C4H7N3O (creatinine); Includes. EXAMPLES

[0050] Urine samples were collected from 110 patients between the ages of 51 and 86 years with indications for prostate biopsy.

[0051] Urine samples were collected after digital rectal examination and prostate massage consisting of three digital pressures on each lobule starting from the base and moving to the middle and apex over a period of 180 seconds. Samples were vortexed in Falcon tubes and frozen at -80°C within 30 minutes of collection.

[0052] Additionally, 12 biopsy samples were taken from each patient and used for routine diagnostic procedures by the anatomic pathology department. Histological evaluation confirmed the presence of tumor cells in 53 patients (PCa). Of the remaining patients, 29 were disease-free, 16 had benign pathological conditions, and 12 had potential precancerous conditions. This indicates that approximately 55% of PSA / DRE / ECO suspicious patients underwent unnecessary biopsies.

[0053] Urine samples were thawed and the presence of utPSA and ufPSA biomarkers was assessed by ELISA assay (Sigma Aldrich), zinc was quantified by a colorimetric test (Zinc Assay Test, Sigma Aldrich), spermine was quantified by ELISA assay (MyBiosource), and spermine oxidase was quantified by ELISA assay (MyBiosource).

[0054] Because the amount of biomarker in urine depends on the volume of urine collected and its concentration, a calculation called "normalization to creatinine" was performed to improve the accuracy of the test. Creatinine is a substance produced in the body that is excreted via urine, and its level is an indicator of urine concentration. The higher the creatinine level, the more concentrated the urine. The lower the creatinine level, the more diluted the urine.

[0055] To eliminate possible confounding factors, cases with premalignant conditions and benign pathology were excluded.

[0056] The absolute values ​​of the biomarkers allowed us to identify statistically significant differences between healthy and diseased patients (see Figure 1).

[0057] After normalization to creatinine, even greater significant differences were observed between diseased and healthy patients for all five markers studied (see Figure 2).

[0058] The values ​​of Zn, spermine, utPSA, ufPSA, and spermine oxidase correlated with the presence of tumor, with AUCs of 0.656, 0.628, 0.644, 0.617, and 0.684, respectively (see Figure 3).

[0059] By performing multivariate analysis, it is observed that combining several factors increases the statistical power of the correlation between biomarker levels and prostate tumors (see Figures 4a, 4b, and 4c).

[0060] Analysis of the equivalents after dividing patients based on tumor grade (low, intermediate, high risk) highlights that each of the five markers analyzed is reduced to a greater extent in patients with more advanced stage tumors (see Figure 5).

[0061] From the results of the examples, the advantages of the present invention are clear.

[0062] Analysis of the absolute values ​​of the single biomarkers zinc, total PSA, free PSA, spermine, and spermine oxidase in urine samples is already indicative of prostate cancer. Analysis of values ​​normalized to creatinine can provide even more accurate information. Simultaneous analysis of several biomarkers allows even more reliable identification of patients with prostate cancer. In particular, the combination of Zn, utPSA, and spermine oxidase allows improved discrimination between healthy patients and patients with prostate cancer (AUC=0.711).

[0063] For each of these analytes, a value at 80% sensitivity was identified as the optimal threshold.

[0064] Samples in which the biomarker was present in amounts higher than the threshold were considered positive.

[0065] In this way, a scale (score) from 0 to 3 was established based on the number of positive biomarkers for each sample, defining the probability (risk) of having prostate cancer.

[0066] [Table 1]

[0067] As can be seen from Table 1, individuals without positive biomarkers have zero (0%) chance of having prostate cancer.

[0068] For individuals with one, two and three positive biomarkers the probability increases to 14%, 43% and 66%, respectively. These results show that based on the use of the biomarkers according to the invention it is possible to identify individuals with a very low probability of prostate cancer and therefore to avoid them undergoing invasive clinical testing.

[0069] The method according to the invention represents a rapid and inexpensive method resulting in a reliable diagnosis, avoiding invasive procedures.

Claims

1. 1. A method for aiding in the in vitro / ex vivo determination of prostate cancer risk and / or aggressiveness in an individual, comprising: a) providing a urine sample from said individual; b) quantifying zinc and total (utPSA) and / or free (ufPSA) PSA in said urine sample; c) comparing the value of zinc quantified in step b) and the value of total PSA (utPSA) quantified in step b) and / or the value of free PSA (ufPSA) quantified in step b) with corresponding reference values; A method comprising:

2. 2. The method of claim 1, wherein in step b) spermine is also quantified and in step c) the quantified value of spermine is also compared to a reference value.

3. 3. The method according to claim 1, wherein in step b) spermine oxidase (SMOX) is also quantified and in step c) the quantified value of spermine oxidase is also compared to a reference value.

4. 4. The method according to claim 3, wherein in step b) total PSA (prostate specific antigen) (utPSA), zinc and spermine oxidase (SMOX) are quantified, and in step c) the quantified values ​​of total PSA (prostate specific antigen) (utPSA), zinc and spermine oxidase (SMOX) are compared with reference values.

5. 1. A method for aiding in the in vitro / ex vivo determination of prostate cancer risk and / or aggressiveness in an individual, comprising: a') providing a urine sample from said individual; b') quantifying zinc and total (utPSA) and / or free (ufPSA) PSA in said urine sample; c') quantifying creatinine in said urine sample; d') normalizing the value of zinc quantified in step b') and the value of total PSA (utPSA) quantified in step b') and / or the value of free PSA (ufPSA) quantified in step b') with the quantified value of creatinine; e') comparing the normalized value of zinc from step d') and the normalized value of total PSA (utPSA) with corresponding reference values ​​and / or comparing the normalized value of free PSA (ufPSA) from step d') with a reference value; A method comprising:

6. The method of claim 5, wherein in step b'), spermine is also quantified, in step d'), the quantified value of spermine is also normalized with the quantified value of creatinine, and in step e'), the normalized value of spermine is also compared to a reference value.

7. 6. The method according to claim 5, wherein in step b') spermine oxidase (SMOX) is also quantified, in step d') the quantified value of spermine oxidase (SMOX) is also normalized with the quantified value of creatinine, and in step e') the normalized value of spermine oxidase (SMOX) is also compared to a reference value.

8. 8. The method according to claim 7, wherein in step b') total PSA (prostate specific antigen) (utPSA), zinc and spermine oxidase (SMOX) are quantified, in step d') the quantified values ​​of total PSA (prostate specific antigen) (utPSA), zinc and spermine oxidase (SMOX) are normalized with the quantified value of creatinine, and in step e') the normalized values ​​of total PSA (prostate specific antigen) (utPSA), zinc and spermine oxidase (SMOX) are compared with reference values.

9. The method according to any one of claims 1 to 8, wherein the quantification of zinc is carried out by a colorimetric test.

10. The method according to any one of claims 1 to 9, wherein the quantification of the total PSA (utPSA) and / or the free PSA (ufPSA) is performed by immunoassay, spectroscopy, spectrophotometry, colorimetry, electrophoresis, complexometry, amperometry.

11. a means for collecting and preparing a urine sample from an individual; A means for quantifying zinc; a means to quantify total PSA (utPSA), and / or a means for quantifying spermine oxidase (SMOX); and / or and a means for quantifying creatinine.

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