WO2022043890A2

WO2022043890A2 - Method for the diagnosis of prostate cancer based on the psa marker and zinc values in urine

Info

Publication number: WO2022043890A2
Application number: PCT/IB2021/057787
Authority: WO
Inventors: Sergio OCCHIPINTI; Fabio DI NARDO
Original assignee: Nib Biotec S.R.L.
Priority date: 2020-08-25
Filing date: 2021-08-25
Publication date: 2022-03-03
Also published as: WO2022043890A3

Abstract

The present invention relates to a method for the diagnosis of prostate cancer which takes into account determined PSA marker and zinc values in a urine sample.

Description

“Method for the diagnosis of prostate cancer based on the PSA marker and zinc values in urine”

DESCRIPTION

Technical field of the invention

The present invention finds application in the medical field and, in particular, in the field of prostate cancer diagnosis.

Prior art

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

Currently, a diagnosis of prostate cancer is declared after histological analysis of prostate tissue taken by means of biopsy.

In turn, the prostate biopsy is performed after evaluating prostate specific antigen (PSA) levels together with digital rectal examination (DRE).

PSA and DRE are the preliminary tests conducted in order to identify those subjects who are more likely to have developed prostate cancer.

Prostate biopsy is performed to confirm or deny the clinical evidence; however, due to the limited accuracy of these tests, there is a high number of overdiagnoses and consequently over-treatments.

In fact, for every four patients who undergo a prostate biopsy, after appropriate selection by suspected PSA levels, on average only one individual is actually affected by prostate cancer; furthermore, many of the diagnosed prostate cancers have a very slow course and may not show symptoms for the patient’s entire life.

Since it is impossible to distinguish the over-diagnosed cancer from the others, surgical treatment is still offered to most patients with a positive biopsy. However, it is known that such treatment often causes adverse effects, such as: erectile dysfunction, urinary incontinence, infections and therefore has a strong impact on the quality of life.

In addition to the discomfort for patients, the economic impact on the healthcare system caused by such a high percentage of investigations must be considered which, in the end, turn out to have been conducted in vain.

Economic and statistical analyses have concluded that, for the entire population, the costs related to the process which starts once the PSA test has been carried out (due to biopsies, treatments, any consequential damage) outweigh the benefits, quantified economically.

In light of the above, there is a clear need to develop alternative and more reliable methods for the diagnosis of prostate cancer and to be able to identify patients at risk of developing prostate cancer and predict the progression thereof.

It is now known that the growth of prostate cancer is in many cases influenced by testosterone, the male sex hormone produced mainly by the testicles.

By decreasing the level of testosterone in the blood or reducing the possibility of this hormone binding to the cells of the prostate, it is possible to slow down, or even block, the growth of the cancer cells, reduce the size of the tumor and control the symptoms thereof.

The level of testosterone or the binding capacity thereof can be reduced with drug therapy.

The drugs used today in the treatment of prostate cancer are divided according to the mechanism of action thereof.

Drugs which block the production of male hormones comprise:

- LHRH agonists, which block the production of luteinizing hormone (LH) with which the pituitary stimulates the activity of the ovaries and testes, and, in man, inhibit the production of testosterone by obtaining so- called chemical or pharmacological castration;

- LHRH antagonists, which directly inhibit LHRH at the pituitary level through a competitive mechanism and block the secretion of LH and FSH without causing agonist effects.

Other drugs instead bind to the proteins on the surface of cancer cells, preventing testosterone from entering.

In some cases, drug therapy includes the use of both types of drugs in combination in order to enhance the effect thereof, obtaining what is called total androgenic block.

The administration of hormone therapy can also occur before or after radiotherapy or surgery.

Before radiotherapy or surgery, it aims at reducing the size of the tumor and increase the chances of success of subsequent treatments; such an approach is followed in the case of tumors confined to the prostate gland, but potentially at high risk, or in those which have extended beyond the prostate capsule, but which have not yet spread remotely. The treatment generally lasts 3-6 months.

Hormone therapy is instead used after radiotherapy or surgery in order to reduce the risk of relapses and metastases; such an approach is followed when tests indicate the presence of a high-risk cancer. The treatment lasts approximately 3 to 36 months, although there are patients who continue to take it for life.

Studies have shown that approximately 90% of all patients with metastatic prostate cancer initially respond to hormone therapy; however, some cells, particularly in the advanced stages of the disease, proliferate independently of androgenic stimulation and no longer respond to hormone therapy, as they may have, for example, mutations in the genes for androgen hormone receptors, such as to proliferate even in the presence of very low concentrations of male hormones.

The amount of resistant cells can increase over time, making the disease “resistant to castration” and therefore requiring other forms of treatment.

The side effects of hormone therapy are obviously particularly linked to the suspension of the production of male hormones, whose effects are lacking, including the reduction of sexual desire, erectile dysfunction, hot flashes and sweats, reduction of muscle mass, osteoporosis, swelling and breast pain.

To date, the response to hormone therapy is assessed a posteriori by following the PSA and testosterone levels in the blood during or at the end of treatment.

It is also clear that being able to predict a patient’s response to hormone therapy or androgen deprivation avoids treating those patients who will not benefit from the therapeutic effects, but will bear the side effects, important not only from a physiological point of view but also psychological.

Summary of the invention

The inventors of the present patent application have surprisingly found that the severity of prostate cancer is correlated with the PSA marker and zinc values in urine.

Appropriate values of the two markers allow to identify, with greater probability, patients with prostate cancer to be subjected to biopsy, excluding healthy patients.

The same inventors have also surprisingly found that the evaluation of two urinary markers, PSA and zinc values, of prostate cancer, individually or in combination with each other, allows identifying the subjects suffering from prostate cancer among those already previously subjected to a negative prostate biopsy with surprising precision.

In an aspect of the invention, such subjects are therefore those who preferably must undergo prostate biopsy for confirmation of the disease.

The same inventors of the present patent application have surprisingly found that it is also possible to diagnose prostate cancer with particular precision according to the age group of the subject by analyzing the presence of PSA and zinc markers present in the urine.

The same inventors of the present patent application have also found that it is possible to identify individuals who, after a prostate cancer diagnosis, will benefit from hormone therapy by analyzing the presence of PSA and zinc markers present in the urine.

Brief description of the figures

Figure 1 shows Table 1, which reports data related to the percentage of subjects for each category below different uPSA concentration values; Figure 2 shows Table 2, which reports data related to the percentage of subjects for each category below different absolute uPSA values; Figure 3 shows Table 3, which reports data related to the percentage of subjects for each category below different urinary Zinc concentration values; Figure 4 shows Table 4, which reports data related to the percentage of subjects for each category below different urinary Zinc absolute values; Figure 5 indicates the percentage of healthy subjects or subjects with irrelevant tumor (Healthy), patients with tumor with Gleason Score (GS) equal to 7 (3+4), patients with tumor with GS equal to 7 (4+3), patients with tumor with GS equal to or greater than 8, with uPSA concentration values below 2500 ng/ml and Zinc below 4 ng/ml; Figure 6 indicates the percentage of healthy subjects, patients with tumor with GS equal to 7 (3+4), patients with tumor with GS equal to 7 (4+3), patients with tumor with GS equal to or greater than 8, with the absolute uPSA values in urine below 100 pg and of Zinc below 200 ng, Figure 7 presents the graphs of the urinary biomarker values PSA (uPSA) and zinc in individuals with prostate cancer (PCa) and healthy individuals (Sani); Figure 8 depicts the ROC curve (Receiver Operating Characteristic) for uPSA and Zinc; Figure 9 depicts the ROC curve for uPSA-Zinc combinations (Urine) ;Figure 10 depicts the ROC curve (receiver operating characteristic') for the uPSA concentration value at different age groups; Figure 11 depicts the ROC curve for the urinary zinc concentration value at different age groups; Figure 12 depicts the ROC curve for the uPSA and urinary zinc concentration value at different age groups; Figure 13 presents the graphs of the urinary biomarker values of PSA (uPSA) and zinc in subjects subsequently subjected to hormone therapy or androgen deprivation; Figure 14 depicts the ROC curve for the concentration value of uPSA and respectively urinary zinc in subjects subsequently subjected to hormone therapy or androgen deprivation; Figure 15 depicts the ROC curve for the concentration value of uPSA and urinary zinc, in combination, in subjects subsequently subjected to hormone therapy or androgen deprivation.

Object of the invention

A first object of the present invention is represented by a method for the diagnosis of prostate cancer in a subject, which comprises the determination of the PSA marker values and the zinc value in a urine sample from said subject.

A second object of the present invention is represented by a method for the diagnosis of the tumor grade comprising the determination of the PSA marker and zinc values in a urine sample from said subject.

A first further object of the present invention is represented by a method for the diagnosis of prostate cancer in a subject who has previously undergone a prostate biopsy which resulted negative for cancer, said method comprising the analysis of the PSA marker values or the zinc value, or both, in a urine sample from said subject.

In a second further object of the present invention, a method is described for the selection of a subject to undergo a second prostate biopsy, who has previously undergone a first negative prostate biopsy, said method comprising the analysis of the PSA marker value or the zinc value, or both, in a urine sample from said subject.

A still further object of the present invention is represented by a method for the prognosis of prostate cancer in a subject who has previously undergone examination and has received an indication for prostate biopsy.

Another object of the present invention describes a method for identifying subjects who, after a diagnosis of prostate cancer, can benefit from hormone therapy or hormone deprivation therapy.

Detailed description of the invention

Prostate cancer classification

The grade of advancement of prostate cancer is determined following prostate biopsy and is indicated by the Gleason score.

The Gleason score takes into account the type of cells which are most numerous in the sample and adds them up.

The score varies between 6 and 10, based on the aggressiveness of the cancer, where level 10 indicates the most aggressive form; therefore:

When expressing the Gleason score, the type of cells most present in the biopsy sample can be indicated individually in parentheses.

For the purposes of the present invention, “PSA marker value in a urine sample” (uPSA) means the concentration or absolute amount of such a marker in a urine sample.

For the purposes of the present invention, “zinc value in a urine sample” (uZn) means the concentration or absolute amount of zinc in a urine sample.

According to a first object of the present invention, an in vitro method for the diagnosis of prostate cancer in a subject is described.

In an aspect of the present invention, the subject who can be subjected to the in vitro method of the present invention is a subject who has previously undergone examination and who has received a prostate biopsy indication for suspected prostate cancer.

According to an aspect of the present invention, in the in vitro method described, the urine sample is obtained after a prostatic stimulation lasting about 10- 120 seconds.

In particular, such prostatic stimulation can be conducted internally or externally.

In an aspect of the invention, the prostatic stimulation can be conducted by means of prostatic massage.

In particular, such a method comprises the determination of the PSA marker value and/or of the zinc value in a urine sample from said patient.

More specifically, said PSA marker value in the urine sample is a concentration value less than about 500 ng/ml, while said zinc value in the urine sample is a concentration value less than about 3 ng/ml.

For the purposes of the present invention, the concentration value of the PSA marker in the sample can be gradually lower, preferably about 1000 ng/ml, about 1500 ng/ml, about 2000 ng/ml, about 2500 ng/ml, about 3000 ng/ml and about 5000 ng/ml.

By normalizing the PSA concentration value on the collected urine volume, the absolute value thereof is obtained.

The PSA concentration value on the collected urine volume was then normalized to determine the absolute value thereof.

For the purposes of the present invention, the value of the PSA marker content in the urine sample can be gradually lower, preferably about 40 pg, about 60 pg, about 85 pg, about 100 pg, about 120 pg and about 150 pg.

For the purposes of the present invention, the zinc concentration value in the urine sample can be gradually lower, preferably about 3.3 ng/ml, preferably less than about 3.5 ng/ml, more preferably less than about 4 ng/ml and even more preferably less than about 5 ng/ml.

By normalizing the zinc concentration value on the collected urine volume, the absolute value thereof is obtained.

The zinc concentration value on the collected urine volume was then normalized to determine the absolute value thereof.

For the purposes of the present invention, the Zn content value in the urine sample can be gradually lower, preferably about 110 ng, about 150 ng, about 200 ng, about 250 ng.

In a preferred aspect of the present invention, the patient is diagnosed with prostate cancer when both of the following conditions are met:

In accordance with a second object, the present invention describes an in vitro method for the diagnosis of the grade of prostate cancer in a subject.

In particular, such a method comprising the determination of the PSA marker and zinc concentrations in a urine sample from said subject according to the description provided above.

More specifically, a uPSA marker concentration of about <2,500 ng/ml and a uZn concentration of about <4 ng/ml corresponds to a prostate cancer with Gleason score >7 and, more in detail, a Gleason score 7(3+4).

According to a preferred aspect, said concentrations of the uPSA marker and uZn corresponds to a prostate cancer having a Gleason score >7 and, more in detail, a Gleason score (4+3).

According to an even more preferred aspect, said concentrations of the uPSA marker and uZn corresponds to a prostate cancer having a Gleason score >8.

According to an alternative embodiment of the invention, the in vitro diagnostic method described allows to identify patients who, with the methods known in the art, would have been indicated as healthy, when, on the contrary, they were suffering from prostate cancer.

In particular, according to such an alternative aspect, in the in vitro method of the invention, the PSA marker concentration in the urine sample is less than about 5000 ng/ml, further less than about 3000 ng/ml, preferably less than about 2500 ng/ml, more preferably less than about 2000 ng/ml, even more preferably less than about 1500 ng/ml and most preferably less than about 1000 ng/ml.

Consequently, the PSA marker content in the urine sample is less than about 150 pg, further less than about 120 pg, preferably less than about 100 pg, more preferably less than about 85 pg, even more preferably less than about 60 pg and most preferably less than about 40 pg.

Still according to the alternative aspect of the present invention, the zinc concentration in the urine sample is less than about 5 ng/ml, further less than about 4 ng/ml, preferably less than about 3.5 ng/ml, more preferably less than about 3.3 ng/ml and even more preferably less than about 3 ng/ml.

Accordingly, the zinc content in the urine sample is less than about 250 ng, further less than about 200 ng, preferably less than about 150 ng, more preferably less than about 110 ng.

For the purposes of the present invention, the methods described comprise the determination of the PSA marker value in said urine sample by means of an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method; and preferably, by means of ELISA.

Again for the purposes of the present invention, the described method comprises the determination of the zinc value in said urine sample with an assay selected from: colorimetric assay, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy and, preferably, by means of colorimetric assay. For the purposes of the present invention, “PSA marker value in a urine sample” means the concentration or amount of the PSA marker measured in a urine sample collected from a subject.

For the purposes of the present invention, “zinc value in a urine sample” means the concentration or amount of the zinc marker measured in a urine sample collected from a subject.

A “negative prostate biopsy” means a biopsy of the prostate, in which the histopathological analysis did not reveal the presence of prostate cancer or, preferably, the presence of clinically significant prostate cancer.

According to a first further object of the present invention, a method is described for the diagnosis of prostate cancer in a subject who has previously undergone a prostate biopsy which resulted negative for cancer.

For the purposes of the present invention, said method comprising the analysis of the PSA marker values or the zinc value, or both, in a urine sample from said subject.

According to a preferred aspect of the invention, the urine sample is obtained after a prostatic massage lasting about 10-120 seconds and, preferably, about 30 seconds.

According to another preferred aspect of the present invention, the urine sample from said subject is obtained on the same day in which the biopsy which resulted negative for prostate cancer is conducted.

In an aspect of the present invention, said prostate biopsy which resulted negative for cancer was carried out at an earlier time, which can be from a few days to even a few years earlier.

For example, such a period can be starting from 7 days earlier. In another example, such a period can be up to 3 or 4 years.

In a further example, said biopsy is conducted approximately 15 to 900 days before said urine analysis.

Once the PSA or zinc marker analyses, or of both markers, have been performed, a diagnosis of positive prostate cancer is declared where the AUC values are as follows:

For the purposes of the present invention, the method described comprises the determination of the PSA marker value in said urine sample by means of an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method.

Again for the purposes of the present invention, the described method comprises the determination of the zinc value in said urine sample with an assay selected from: colorimetric assay, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy and, preferably, by means of colorimetric assay.

In accordance with a second further object of the present invention, an in vitro method is described for the selection of a subject to undergo a second prostate biopsy, among subjects who have previously undergone a first prostate biopsy which resulted negative for cancer, said method comprising the analysis of the PSA marker values or the zinc value, or both markers in an isolated urine sample from said subject.

“Selection of a subject to undergo a second prostate biopsy” means that the method described provides an indication of the need to subject said subject to a second prostate biopsy or not.

It is clear that such an indication must subsequently be confirmed by a doctor.

For the purposes of the present invention, such a method comprises the features described above in relation to the diagnosis method.

If the analysis of the PSA or zinc value, or both, in the urine sample gives a negative result, an indication will be given not to subject said subject to a second biopsy.

In accordance with a still further object of the present invention, an in vitro method is described for the prognosis of prostate cancer in a subject who has previously undergone examination and received an indication for prostate biopsy.

Once the determination of the PSA marker or zinc concentration, or both, in the isolated urine sample from the subject has been carried out, a prostate cancer positivity diagnosis is declared, as a function of the subject’s age, where the AUC values are the following:

As indicated above, the diagnosis is made based on only one of the values of uPSA or uZn or both.

For the purposes of the present invention, the method described comprises the determination of the PSA marker value in said isolated urine sample from the subject by means of an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method; and preferably, by means of ELISA.

Again for the purposes of the present invention, the described method comprises the determination of the zinc value in said isolated urine sample from the subject with an assay selected from: colorimetric assay, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy and, preferably, by means of colorimetric assay.

In accordance with another object of the present invention, there is described a method for identifying subjects who, after a diagnosis of prostate cancer, can benefit or not benefit from hormone therapy or hormone deprivation therapy.

The term “benefit” means that, at the end of the hormone therapy or hormone deprivation therapy, the subject has obtained partial or complete regression of the prostate cancer. Therefore, the method described allows to predict the good outcome and the success of a hormone therapy or androgen deprivation in a subject with a prostate cancer diagnosis.

The terms “hormone therapy” and “androgen deprivation” mean those pharmacological treatments aimed at decreasing testosterone levels in the blood or diminishing the effects thereof in the patient.

For the purposes of the present invention, said method comprising the analysis and determination of the PSA marker values or the zinc value, or both, in a urine sample from said subject, preferably after diagnosis.

Once the PSA or zinc marker analyses, or both markers, have been performed, the likelihood of benefiting from hormone therapy is stated, where the AUC values are as follows:

For the purposes of the present invention, the method described comprises the determination of the PSA marker value in said isolated urine sample from the subject by means of an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method.

Again for the purposes of the present invention, the described method comprises the determination of the zinc value in said isolated urine sample from the subject with an assay selected from: colorimetric assay, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy..

Methods

Urinary PSA determination by means of ELISA (R&D System) according to the manufacturer’s instructions.

The urine PSA value is obtained by comparing the absorbance in the urine sample with the curve prepared using recombinant PSA molecule at known scalar concentrations.

Urinary zinc determination by means of colorimetric assay (Sigma Aldrich) according to the manufacturer’s instructions.

The urinary zinc value is obtained by comparing the absorbance in the urine sample with the curve prepared using the Zinc molecule at known scalar concentrations.

The invention will be further described with reference to the following non-limiting examples of the present invention.

Example 1

Urine samples were collected from 313 subjects with indication of prostate biopsy for suspected prostate cancer.

The urine samples were collected after prostatic massage for a duration of 30 seconds. The sample was shaken and frozen in Falcon tubes at -80°C within 30 minutes of collection.

Furthermore, 12 biopsy samples were taken from each subject, used for the normal diagnostic procedure by pathological anatomy section. The histological evaluation revealed the presence of neoplastic cells in 168 subjects (PCa), while 144 subjects were free from cancer.

Of the 168 PCa, 26 had a low-grade tumor, 62 a favorable intermediate -grade tumor, 43 an unfavorable intermediate-grade tumor, 37 a high-grade tumor.

The urine was thawed and the presence of urinary PSA (uPSA) and zinc biomarkers was quantified by means of ELISA and colorimetric tests, respectively.

We observed that 68% of the PCa had a uPSA with concentrations below 500 ng/mL versus 29% of the healthy subjects. 77% of the PCa had a uPSA with concentrations below 1000 ng/ml versus 44% of the healthy subjects. 84% of the PCa had a uPSA with concentrations below 1500 ng/ml versus 54% of the healthy subjects. 92% of the PCa had a uPSA with concentrations below 2000 ng/ml versus 59% of the healthy subjects. 95% of the PCa had a uPSA with concentrations below 2500 ng/ml versus 65% of the healthy subjects. 96% of the PCa had a uPSA with concentrations below 3000 ng/ml versus 71% of the healthy subjects. 99% of the PCa had a uPSA with concentrations below 5000 ng/mL with respect to 80% of the healthy subjects. (Figure 5).

We normalized the uPSA concentration values on the collected urine volume to determine an absolute value.

We observed that 80% of the PCa had a uPSA amount below 40 pg versus 44% of the healthy subjects. 87% of the PCa had a uPSA amount below 60 pg versus 51% of the healthy subjects. 90% of the PCa had a uPSA amount below 85 pg versus 59% of the healthy subjects. 91% of the PCa had a uPSA amount below 100 pg versus 63% of the healthy subjects. 94% of the PCa had a uPSA amount below 120 pg versus 65% of the healthy subjects. 96% of the PCa had a uPSA amount below 150 pg versus 71% of the healthy subjects. (Figure 6). We observed that 78% of the PCa had a Zinc with concentrations below 3 ng/ml versus 63% of the healthy subjects. 86% of the PCa had a Zinc with concentrations below 3.3 ng/ml versus 69% of the healthy subjects. 90% of the PCa had a Zinc with concentrations below 3.5 ng/ml versus 71% of the healthy subjects. 91% of the PCa had a Zinc with concentrations below 4 ng/ml versus 76% of the healthy subjects. 97% of the PCa had a Zinc with concentrations below 5 ng/ml with respect to 85% of the healthy subjects (Figure 3).

We normalized the Zinc concentration values on the collected urine volume to determine an absolute value.

We observed that 80% of the PCa had a zinc amount below 110 ng versus 53% of the healthy subjects. 87% of the PCa had a zinc amount below 150 ng versus to 71% of the healthy subjects. 94% of the PCa had a zinc amount below 200 ng with respect to 85% of the healthy subjects. 99% of the PCa had a Zinc amount below 250 ng versus 90% of the healthy subjects. (Figure 4).

In the case of the concentrations, considering as the optimal threshold value 2500 ng/ml for uPSA and 4 ng/ml for Zinc, it can be seen that in the urine of 92% of patients with advanced cancer (GS > 8), 88% of the patients with GS 7 (4+3) and 85% of the patients with cancer GS 7 (3+4) both biomarkers are detectable beyond the threshold, versus 59% of the healthy subjects or subjects with irrelevant disease (Figure 5).

In the case of the absolute values, considering as the optimal threshold value 100 pg for uPSA and 200 ng for Zinc, it can be seen that in the urine of 95% of patients with advanced cancer (GS > 8), 89% of the patients with GS 7 (4+3) and 81% of patients with cancer GS 7 (3+4) both biomarkers are detectable beyond the threshold, versus 59% of the healthy subjects or subjects with irrelevant disease (Figure 6).

Example 2

Urine samples were collected from 71 subjects with indication of prostate rebiopsy, after a period of time between 21 and 883 days passed since a previous negative biopsy.

Furthermore, 12 biopsy samples were taken from each subject, used for the normal diagnostic procedure by pathological anatomy section. Histological evaluation revealed the presence of neoplastic cells in 36 subjects (PCa). The remaining 35 subjects were free from cancer (absence of diseases, benign hyperplasia, high grade intraepithelial prostatic neoplasia).

This indicates that 49% of subjects, considered suspicious based on standard parameters (serum PSA, digital rectal examination, multiparametric magnetic resonance imaging) underwent an unnecessary biopsy examination twice.

The urine was thawed and the presence of urinary PSA (uPSA) and urinary zinc biomarkers was quantified by means of EEISA and colorimetric tests, respectively.

A statistically significant difference was observed between sick (PCa) and healthy (Sani) subjects for each of the markers studied (Figure 7).

The uPSA and zinc values correlate with the presence of cancer, with an AUC of 0.77 and 0.73, respectively (see Figure 8).

Performing a multivariate analysis, it is observed that the combination of the two factors increases the power of the test in correlating the biomarker level and prostate cancer with AUC of 0.8 (see Figure 9).

Example 3

Urine samples were collected from 186 subjects between the ages of 50 and 87 with indication of prostate biopsy for suspected prostate cancer.

Furthermore, 12 biopsy samples were taken from each subject, used for the normal diagnostic procedure by pathological anatomy section. The histological evaluation revealed the presence of neoplastic cells in 98 subjects (PCa), while 92 subjects were free from cancer.

Of the 98, 18 had a low-grade tumor, 28 a favorable intermediate -grade tumor, 24 an unfavorable intermediate-grade tumor, 24 a high-grade tumor.

On the total population, the uPSA values correlate with the presence of cancer with an AUC of 0.803.

Limiting the analysis to the subjects aged less than 60 years, over 60 years and over 70 years, the uPSA values correlate with the presence of cancer with an AUC of 0.741, 0.801, 0.876, respectively (see Figure 10).

The urinary zinc values correlate with the presence of cancer with an AUC of 0.689.

Limiting the analysis to subjects aged less than 60 years, over 60 years and over 70 years, the uPSA values correlate with the presence of cancer with an AUC value of 0.790, 0.670, 0.739, respectively (see Figure 11).

The combined values of uPSA and urinary zinc correlated with the presence of cancer with an AUC of 0.797.

Limiting the analysis to subjects aged less than 60 years, over 60 years and over 70 years, the uPSA and urinary zinc values correlate with the presence of cancer with an AUC of 0.790, 0.792, 0.875, respectively (see Figure 12).

Example 4

Urine samples were collected from 44 subjects diagnosed with prostate cancer and indicated for treatment with hormone therapy or androgen deprivation.

The urine was thawed and the presence of urinary PSA (uPSA) and urinary zinc biomarkers was quantified by means of ELISA and colorimetric tests, respectively (Figure 13).

A statistically significant difference was observed between subjects who benefited (Responder) and subjects who on the contrary are resistant to hormone therapy (No responder) for each of the markers studied.

The uPSA and zinc values correlate with the presence of cancer, with an AUC of 0.77 and 0.71, respectively (see Figure 14) and 0.73 in combination (Figure 15).

From the description provided above and from the experimental evidence gathered, and reported above, the numerous advantages offered by the present invention will be apparent to the person skilled in the art.

The analysis conducted according to the present invention in urine samples of the uPSA and zinc biomarker values, both as concentration and as absolute value, allows to discriminate healthy subjects from patients with prostate cancer with greater precision and more efficiently than the standard parameters.

Therefore, the method of the present invention represents a quick and economical manner to obtain a reliable diagnosis, while avoiding unnecessary and invasive procedures for the patient or which can have significant side effects without any therapeutic benefit.

From an economic point of view, the method of the present invention allows a better management of the economic resources available to the healthcare service and above all to choose the best therapeutic path for the patient.

Claims

1. An in vitro method for the diagnosis of prostate cancer in a subject, said method comprising the analysis of the PSA marker values and/or the zinc value, both in a urine sample from said subject, wherein:

- said PSA marker value in a urine sample is less than about 500 ng/ml; and/or wherein

- said zinc value in a urine sample is less than about 3 ng/ml.

2. The in vitro method for the diagnosis of prostate cancer in a subject, said method comprising the analysis of the PSA marker values and the zinc value, both in a urine sample from said subject, wherein:

- said PSA marker value in a urine sample is less than about 500 ng/ml; and wherein

- said zinc value in a urine sample is less than about 3 ng/ml.

3. The in vitro method according to the preceding claim, wherein said subject has previously been subjected to examination and has received a prostate biopsy indication for suspected prostate cancer.

4. The in vitro method according to any one of the preceding claims, wherein said urine sample is obtained after prostatic stimulation lasting about 10-120 seconds, and preferably 30 seconds.

5. The in vitro method according to any one of the preceding claims, wherein the concentration of said PSA marker in said urine sample is less than about 1000 ng/ml, further less than about 1500 ng/ml, preferably less than about 2000 ng/ml, more preferably less than about 2500 ng/ml, even more preferably less than about 3000 ng/ml, and most preferably less than about 5000 ng/ml.

6. The in vitro method according to any one of the preceding claims,

24 wherein the content of said PSA marker in said urine sample is less than about 40 pg, further less than about 60 pg, preferably less than about 85 pg, more preferably less than about 100 pg, even more preferably less than about 120 pg, and most preferably less than about 150 pg.

7. The in vitro method according to any one of the preceding claims, wherein the zinc concentration in said urine sample is less than about 3 ng/ml, further less than about 3.3 ng/ml, preferably less than about 3.5 ng/ml, more preferably less than about 4 ng/ml, and even more preferably less than about 5 ng/ml.

8. The in vitro method according to any one of the preceding claims, wherein the zinc content in said urine sample is less than about 110 ng, further less than about 150 ng, preferably less than about 200 ng, more preferably less than about 250 ng.

9. The in vitro method for the diagnosis of the grade of prostate cancer in a subject, comprising the analysis of the concentrations of PSA marker and zinc in a urine sample from said subject, wherein when said concentration of PSA marker is about <2,500 ng/ml and said zinc concentration is about <4 ng/ml, it corresponds to a prostate cancer with Gleason score >7 (3+4), preferably >7 (4+3), and more preferably >8.

10. The in vitro method for the diagnosis of the grade of prostate cancer in a subject according to the preceding claim, wherein when said amount of PSA marker is about <100 pg and said amount of zinc is about <200 ng, it corresponds to a prostate cancer with Gleason score >7 (3+4), preferably >7 (4+3), and more preferably >8.

11. The in vitro method according to any one of the preceding claims 1 to 4, wherein the concentration of said PSA marker in said urine sample is less than about 5000 ng/ml, further less than about 3000 ng/ml, preferably less than about 2500 ng/ml, more preferably less than about 2000 ng/ml, even more preferably less than about 1500 ng/ml, and most preferably less than about 1000 ng/ml.

12. The in vitro method according to any one of the preceding claims 1 to 4, wherein the content of said PSA marker in said urine sample is less than about 150 pg, further less than about 120 pg, preferably less than about 100 pg, more preferably less than about 85 pg, even more preferably less than about 60 pg, and most preferably less than about 40 pg.

13. The in vitro method according to any one of the preceding claims 1 to 4 and 11 to 12, wherein the zinc concentration in said urine sample is less than about 5 ng/ml, further less than about 4 ng/ml, preferably less than about 3.5 ng/ml, more preferably less than about 3.3 ng/ml, and even more preferably less than about 3 ng/ml.

14. The in vitro method according to any one of the preceding claims 1 to 4 and 11 to 13, wherein the zinc content in said urine sample is less than about 250 ng, further less than about 200 ng, preferably less than about 150 ng, more preferably less than about 110 ng.

15. The method according to any one of the preceding claims, wherein the value of the PSA marker in said sample is analyzed by an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method.

16. The method according to any one of the preceding claims 1 to 15, wherein the zinc value in said sample is analyzed with an assay selected from: colorimetric, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy.

17. A method for the diagnosis of prostate cancer in a subject who has previously undergone a prostate biopsy which resulted negative for cancer, said method comprising the analysis of the PSA marker values or the zinc value, or both, in a urine sample from said subject.

18. The method according to the preceding claim, wherein said urine sample is obtained after a prostatic massage lasting about 10-120 seconds and, preferably, 30 seconds.

19. The method according to claim 17 or 18, wherein said PSA marker value, zinc value, or PSA and zinc value, in said urine sample are correlated with a positive prostate cancer diagnosis with an AUC value of 0.77, 0.73 and 0.8, respectively.

20. The method according to any one of the preceding claims 17 to 19, wherein said prostate biopsy which resulted negative for cancer was carried out from about 15 to 900 days before said urine analysis.

21. The method according to any one of the preceding claims 17 to 20, wherein said urine sample is obtained on the same day in which said biopsy which resulted negative for cancer is conducted.

22. The method according to any one of the preceding claims 17 to 21, wherein the PSA marker value in said urine sample is analyzed by means of an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method.

23. The method according to any one of the preceding claims 17 to 22,

27 wherein the zinc value in said urine sample is analyzed with an assay selected from: colorimetric, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy.

24. A method for selecting a subject to undergo a second prostate biopsy, who has previously undergone a first prostate biopsy which resulted negative for cancer, said method comprising the analysis of the PSA marker values or the zinc value, or both, in a urine sample from said subject, wherein said urine sample is obtained on the same day in which said first prostate biopsy which resulted negative for cancer is conducted.

25. A method for the diagnosis of prostate cancer in a subject who has previously undergone examination and received an indication for prostate biopsy, said method comprising the analysis of the PSA marker values or the zinc value, or both, in a biological fluid sample from said subject.

26. The method according to claim 25, wherein said urine sample is obtained after a prostatic massage lasting about 10-120 seconds and, preferably, 30 seconds.

27. The method according to claim 25 or 26, wherein said subject is under 60 years old or preferably over 60 years old or even more preferably over 70 years old and the value of said PSA marker in said sample is correlated with a prostate cancer positivity diagnosis with an AUC value of 0.537-0.889, 0.730-0.861, 0.777-0.942, respectively.

28. The method according to claim 25 or 26, wherein said subject is over 60 years old or preferably over 70 years old or even more preferably under 60 years old and the value of said zinc in said sample is correlated with a prostate cancer positivity diagnosis with an AUC value of 0.591-0.922, 0.590-0.744, 0.620-0.838,

28 respectively.

29. The method according to claim 27 and 28, wherein said subject is under 60 years old or preferably over 60 years old or even more preferably over 70 years old and the combined value of said zinc and PSA in said sample is correlated with a prostate cancer positivity diagnosis with an AUC value of 0.590-0.923, 0.721-0.855 and 0.620-0.838, respectively.

30. The method according to any one of the preceding claims 24 to 29, wherein the PSA marker value in said urine sample is analyzed by means of an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method.

31. The method according to any one of the preceding claims 24 to 30, wherein the zinc value in said sample is analyzed with an assay selected from: colorimetric, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy.

32. The method according to any one of the preceding claims 24 to 31, wherein said urine sample can be replaced with an isolated biological sample of sperm, pro static secretion or urine.

33. An in vitro method to predict the success or benefit of hormone therapy or androgen deprivation in a subject diagnosed with prostate cancer comprising the step of determining the PSA and/or zinc values in an isolated urine sample, where such success or benefit of the therapy is correlated with the AUC values of said PSA and/or zinc markers in the sample of 0.77 and 0.71, respectively, and of 0.73 for both.

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34. An in vitro method to predict the success or benefit of hormone therapy or androgen deprivation in a subject diagnosed with prostate cancer according to the preceding claim, comprising the step of determining the PSA marker values in an isolated urine sample, where such success or benefit of the therapy is correlated with the AUC values of said PSA marker in the sample of 0.77.

35. The in vitro method according to the preceding claim 33 or 34, wherein said urine sample is obtained after prostatic stimulation lasting about 10-120 seconds, and preferably 30 seconds.

36. The method according to any one of the preceding claims 33 to 35, wherein the PSA marker value in said sample is analyzed by means of an assay selected from: ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, colorimetric, electrophoretic, complexometric, amperometric method.

37. The method according to any one of the preceding claims 33 to 36, wherein the zinc value in said sample is analyzed with an assay selected from: colorimetric, ELISA, immunoassay, immunochemical, spectrometric, spectrophotometric, electrophoretic, complexometric, amperometric method, atomic absorption spectroscopy, atomic emission spectroscopy.

30