TEST FOR DETECTING MALIGNANT KIDNEY CANCER

Abstract

The present invention relates to a method for detecting malignant kidney cancer in an individual, said method conducted in vitro comprising the steps of providing a body fluid sample, in particular a urine sample, obtained from the individual, and determining one or more biomarker levels selected from the group consisting of the Mxi-2 level, the Vim3 level, the MAPKp38 level and the Atg7 level in the sample. Further, the present invention refers to a kit and a dipstick for use in such method.

Claims

1. A method for detecting malignant kidney cancer in an individual, said method conducted in vitro, comprising determining an Mxi-2 level in a urine sample U from the individual.

2. The method of claim 1, comprising the following steps: (i) providing a urine sample obtained from the individual; and (ii) determining the Mxi-2 level in the urine sample.

3. The method of claim 1, wherein the malignant kidney cancer is renal cell carcinoma.

4. The method of claim 1, wherein the step of determining the Mxi-2 level is determining a level of Mxi-2 polypeptide.

5. The method of claim 1, wherein the step of determining the Mxi-2 level is determining a level of Mxi-2 messenger RNA.

6. The method of claim 1, wherein an increased Mxi-2 level indicates a presence of malignant kidney cancer in the individual.

7. The method of claim 2, wherein the method further comprises the step of: (iii) comparing the Mxi-2 level determined in step (ii) with (a) a predetermined reference value (R1) indicating a borderline between a sample indicating a presence of malignant kidney cancer and a sample indicating an absence of malignant kidney cancer; (b) an Mxi-2 level determined in a control sample obtained from a control individual of the same species known to be free of malignant cancer cells; or (c) a combination of (a) and (b), wherein an Mxi-2 level determined in the urine sample that is higher than R1, at least 20% higher than the Mxi-2 level of the control sample, or both indicates the presence of malignant kidney cancer in the individual, wherein the Mxi-2 level in each case is related to a total polypeptide content comprised in the respective sample.

8. The method of claim 1, wherein the method further comprises the step of determining a Vimentin variant 3 (Vim3) level in the urine sample.

9. The method of claim 8, wherein the method further comprises the step of (v) comparing the Vim3 level determined in step (iv) with (a) a predetermined reference value (R2) indicating a borderline between a sample indicating a presence of malignant kidney cancer and a sample indicating an absence of malignant kidney cancer; (b) Vim3 level determined in the control sample C; or (c) a combination of (a) and (b), herein an Vim3 level determined in the urine sample that is lower than R2, that is at least 50% lower than the Vim3 level of the control sample, or that is lower than R2 and at least 50% lower than the Vim3 level of the control sample indicates the presence of malignant kidney cancer in the individual, wherein the Vim3 level in each case is related to a total polypeptide content comprised in the respective sample.

10. The method of 8, wherein an increase of a ratio of Mxi-2:Vim3 levels in comparison to a predetermined reference value R3 indicating a borderline between a sample indicating a presence of malignant kidney cancer and a sample indicating an absence of malignant kidney cancer indicates the presence of malignant kidney cancer in the individual.

11. The method of claim 2, wherein step (ii) and if present step (iv) comprises at least one of a) determining a respective polypeptide level of Mxi-2, Vim3, or both by means of conducting at least one step selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), immuno-electrophoresis, immuno-blotting, Western blot, SDS-PAGE, capillary electrophoresis (CE), spectrophotometry or enzyme assay for example, dipsticks (lateral flow), and combinations of two or more thereof; b) determining a respective messenger RNA level of Mxi-2, Vim3, or both by means of conducting at least one step selected from the group consisting of polymerase chain reaction (PCR), real time PCR (RT-PCR), by in situ hybridization, gel electrophoresis, Northern Blot, Southern Blot, and combinations of two or more thereof; or c) determining the respective polypeptide level and the respective messenger RNA level of Mxi-2, Vim3, or both according to a) and b).

12. The method of claim 2, wherein step (ii) and if present step (iv) comprises staining of the respective polypeptide Mxi-2, Vim3, or both.

13. A kit for use in a method according to claim 8, comprising: (A) means for determining the Mxi-2 level in an urine sample; (B) means for determining the Vim3 level in an urine sample; and (C) instructions for carrying out the method according to claim 8.

14. A dipstick usable for the method of claim 1 comprising, placed in the direction of flow of the urine sample, on a carrier that is suitable for soaking the urine sample, the following: (0) an edge or segment suitable for receiving the urine sample; (1) optionally a stripe (1) comprising labeled Mxi-2-specific antibodies or antibody fragments which are not immobilized and freely movable when the urine sample passes through this stripe (1); (2) a stripe (2) comprising immobilized unlabeled MAPK p38 or Mxi-2-specific antibodies or antibody fragments; and (3) optionally a stripe (3) of immobilized unlabeled antibodies or antibody fragments specifically binding the labeled Mxi-2-specific antibodies or antibody fragments of stripe (1).

15. A dipstick usable for the method of claim 8 comprising, placed in the direction of flow of the urine sample, on a carrier that is suitable for soaking the urine sample, the following: (0) an edge or segment suitable for soaking the urine sample U; (1) optionally a stripe (1) comprising labeled Mxi-2-specific antibodies or antibody fragments which are not immobilized and freely movable when the urine sample passes through this stripe (1); (1′) optionally a stripe (1′) comprising labeled Vim3-specific antibodies or antibody fragments which are not immobilized and freely movable when the urine sample U passes through the one or more stripe(s) (1); (2) a stripe (2) comprising immobilized unlabeled MAPK p38-specific or Mxi-2-specific antibodies or antibody fragments; (2′) a stripe (2′) comprising immobilized unlabeled vimentin-specific or Vim3-specific; antibodies or antibody fragments; (3) optionally a stripe (3) of immobilized unlabeled antibodies or antibody fragments specifically binding the labeled Mxi-2-specific antibodies or antibody fragments of stripe (1); and (3′) optionally a stripe (3′) of immobilized unlabeled antibodies or antibody fragments specifically binding the labeled Vim3-specific antibodies or antibody fragments of stripe (1).

16. An antineoplastic agent for use in a method for treating an individual bearing malignant kidney cancer, wherein the malignant kidney cancer has previously been detected in the individual by means of a method of claim 1.

17. An antibody or fragment or variant thereof specific for Mxi-2 polypeptide.

18. A cell capable of producing the antibody or fragment or variant thereof according to claim 17.

19. A method for detecting an oncocytoma in an individual, said method conducted in vitro, comprising determining a Vim3 level in a urine sample from the individual.

20. The method of claim 19, comprising the following steps: (i) providing a urine sample obtained from the individual; and (ii) determining Vim3 level in the urine sample.

21. The method of claim 20, wherein the method further comprises the step of (iii) comparing the Vim3 level determined in step (ii) with (a) a predetermined reference value R1 indicating a borderline between a sample indicating a presence of a oncocytoma and a sample indicating an absence of malignant kidney cancer; (b) Vim3 level determined in a control sample C-obtained from a control individual of the same species free of an oncocytoma; or (c) a combination of (a) and (b), wherein an Vim3 level determined in the urine sample that is higher than R1, at least 20% higher than the Vim3 level of, or both indicates the presence of an oncocytoma in the individual, wherein the Vim3 level in each case is related to the total polypeptide content comprised in the respective sample.

22. The method of claim 1, wherein the method further comprises the step of (iv) determining a Vimentin variant 3 (Vim3) level in the urine sample, wherein determining the Vim3 level comprises determining the level of Vim3 polypeptide, the level of Vim3 messenger RNA, or both.

23. The method of claim 1, wherein the method further comprises the step of (iv) determining a Vimentin variant 3 (Vim3) level in the urine sample, wherein a decreased Vim3 level or the absence of Vim3 indicates the presence of malignant kidney cancer in the individual.

24. The method of 10, wherein the individual is known to comprise either (a) malignant renal cell carcinoma, or (b) benign oncocytoma, and the method is conducted to differentiate between (a) and (b), wherein (a) is characterized by an increase of a ratio Mxi-2:Vim3 levels.

25. The method of claim 2, wherein step (ii) and if present step (iv) comprises staining of the respective polypeptide Mxi-2, Vim3, or both by: (iia) direct immunodetection comprising providing at least one labeled antibody or antibody fragment (AB1-L) specific for the respective polypeptide, and enabling the binding of said AB1-L to the respective polypeptide; or (iib) indirect immunodetection comprising providing at least one unlabeled antibody or antibody fragment (AB1-ul) specific for the respective polypeptide and at least one labeled antibody or antibody fragment (AB2-L) specifically binding to AB1-ul, enabling the binding of AB1-ul to the respective polypeptide, and enabling the binding of AB2-L to AB1-ul.

26. The antibody or fragment or variant thereof of claim 17, wherein said antibody or fragment or variant thereof binds to the Mxi-2 polypeptide with a dissociation constant of not more than 20 nM and to full length MAPK p38 with a dissociation constant of more than 20 nM.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0388] FIG. 1 shows the comparison between renal cell carcinoma (RCC), oncocytoma (OC) and healthy control (Crt) urine samples. FIG. 1A shows the Mxi-2 level in RCC and OC urines in comparison to the controls (Ctr): Western blot analysis (above) and densitometric analysis of Western blots averaged from 10 independent experiments (below). The Mxi-2 level is particularly increased in RCC urine samples (***p<0.001). FIG. 1B shows the Vim3 level in RCC and OC urines in comparison to the controls (Ctr): Western blot analysis (above) and densitometric analysis of Western blots averaged from 10 independent experiments (below). The Vim3 level is particularly increased in OC urine samples (***p<0.001). All samples were neutralized to the housekeeping gene (β-actin).

[0389] FIG. 2 shows an exemplary setup of a dipstick usable for the method of the present invention. Such dipstick is usable for detecting and quantifying Mxi-2 or Vim3. FIG. 2A shows the dipstick before use. Herein, the urine sample U may be premixed with a labeled Mxi-2-specific (or Vim3-specific) antibodies (depicted as stars). S indicates the urine sample U to be added to the dipstick. (2) indicates a stripe comprising immobilized unlabeled Mxi-2-specific (or Vim3-specific, alternatively 38- or vimentin-specific) antibodies. (3) indicates a stripe of immobilized unlabeled antibodies specifically binding the labeled Mxi-2-specific (or Vim3-specific) antibodies (depicted as stars). (4) indicates the flow direction of the moisten urine sample U. FIG. 2b shows the results when the respective Mxi-2-containing (or Vim3-containing) sample (S+) premixed with a labeled Vim3-specific antibodies (depicted as stars) is added to the dipstick. Then, the labeled Mxi-2-specific (or Vim3-specific) antibodies (depicted as stars) and the Mxi-2 (or Vim3) in the urine sample U form a Mxi-2:Mxi-2-specific antibody (or Vim3:Vim3-specific antibody) conjugate. After adding this urine sample U to the dipstick, it flows through the dipstick (4). The antibody conjugate is then binding to the unlabeled Mxi-2-specific (or Vim3-specific) antibodies of stripe (2). FIG. 2c shows the results when a sample lacking Mxi-2 (or Vim3) (S-) premixed with a labeled Mxi-2-specific (or Vim3-specific) antibodies (depicted as stars) is added to the dipstick. Then, the labeled Mxi-2-specific (or Vim3-specific) antibodies (depicted as stars) are not binding to its target. Thus, upon flowing through the dipstick (4), the labeled Mxi-2-specific (or Vim3-specific) antibodies are not bound until the stripe (3). Thus, the ratio between signal intensity of the label in stripe (2) and (3) indicates the presence of Mxi-2 (or Vim3). A higher (2):(3) ratio indicates higher level of Mxi-2 (or Vim3), whereas a lower (2):(3) ratio indicates lower level.

[0390] FIG. 3 shows another setup of a dipstick usable for the method of the present invention as an alternative to the one shown in FIG. 2. FIG. 3A shows the dipstick before use. Herein, (1) indicates a stripe comprising labeled Mxi-2-specific (or Vim3-specific) (depicted as stars), which are not immobilized and freely movable when the urine sample U passes through this stripe. (2) indicates a stripe comprising immobilized unlabeled Mxi-2-specific (or Vim3-specific) antibodies. (3) indicates a stripe of immobilized unlabeled antibodies specifically binding the labeled Mxi-2-specific (or Vim3-specific) antibodies (depicted as stars). S indicates the urine sample U to be added to the dipstick. (4) indicates the flow direction of the moisten urine sample U. FIG. 3b shows the results when a Mxi-2-containing (or Vim3-containing) sample (S+) is added to the dipstick. Then, upon flowing through the dipstick (4), the labeled Mxi-2-specific (or Vim3-specific) antibodies are binding to Mxi-2 (or Vim3) in the urine sample U and form a Mxi-2:Mxi-2-specific antibody (or Vim3:Vim3-specific antibody) conjugate. This conjugate is then binding to the unlabeled Mxi-2-specific (or Vim3-specific) antibodies of stripe (2). FIG. 3c shows the results when a sample lacking Mxi-2 (or Vim3) (S-) is added to the dipstick. Then, upon flowing through the dipstick (4), the labeled Mxi-2-specific (or Vim3-specific) antibodies are not bound until the stripe (3). Thus, the ratio between signal intensity of the label in stripe (2) and (3) indicates the presence of Mxi-2 (or Vim3). A higher (2):(3) ratio indicates higher level of Mxi-2 (or Vim3), whereas a lower (2):(3) ratio indicates lower level.

[0391] FIG. 4 shows an example of a dipstick usable for the method of the present invention for concomitantly detecting Mxi-2 and Vim3. This reflects a dipstick as shown in FIG. 3, wherein each of stripes (1), (2) and (3) are each present twice, each for detecting Mxi-2 (indicated by stars) and for detecting Vim3 (indicated by x). The definitions are corresponding to those indicated in the context of FIGS. 2 and 3 above. It will be understood that stripe (3) can alternatively also be represented by a single stripe of immobilized unlabeled antibodies concomitantly binding the labeled Mxi-2-specific and Vim3-specific antibodies (depicted as stars and x) when these have the same or a comparable Fc fragment. It will be understood that the stripes (1) and (1′) may alternatively also form a single stripe comprising a mixture of Mxi-2-specific antibodies and Vim3-specific antibodies.

[0392] FIG. 5 shows the comparison of Mxi-2 levels in different urine samples by means of an ELISA assay. Urine samples from healthy individuals (control, average of samples from ten individuals) were compared to urine samples from individuals having chromophobe RCC (chromo RCC, average of samples from eight individuals), eosinophilic RCC (eosino RCC, average of samples from four individuals), papillary RCC (pap RCC, average of samples from eight individuals), oncocytoma (average of samples from eight individuals), and RCC (average of samples from ten individuals). The Mxi-2 level is increased in RCC urine samples in comparison to samples from healthy controls (**p<0.001) and samples from individuals having an oncocytoma (**p<0.001), but is not significantly altered in a sample from an individual having an oncocytoma. The dashed line indicates the average of the control sample.

[0393] FIG. 6 shows the comparison of MAPK p38 levels in different urine samples by means of an ELISA assay. Urine samples from healthy individuals (control, average of samples from ten individuals) were compared to urine samples from individuals having chromophobe RCC (chromo RCC, average of samples from eight individuals), eosinophilic RCC (eosino RCC, average of samples from four individuals), papillary RCC (pap RCC, average of samples from eight individuals), oncocytoma (average of samples from eight individuals), and RCC (average of samples from ten individuals). The MAPK p38 level is increased in RCC urine samples in comparison to samples from healthy controls (*p<0.001), but is not significantly altered in a sample from an individual having an oncocytoma. Increase is comparably lower in papillary RCC. The dashed line indicates the average of the control sample.

[0394] FIG. 7 shows the comparison of Vim3 levels in different urine samples by means of an ELISA assay. Urine samples from healthy individuals (control, average of samples from ten individuals) were compared to urine samples from individuals having chromophobe RCC (chromo RCC, average of samples from eight individuals), eosinophilic RCC (eosino RCC, average of samples from four individuals), papillary RCC (pap RCC, average of samples from eight individuals), oncocytoma (average of samples from eight individuals), and RCC (average of samples from ten individuals). The Vim3 level is increased in oncocytoma samples in comparison to samples from healthy controls and various RCC samples (*p, **p and ***p<0.001), but is not significantly altered in a sample from an individual having an RCC of any type. The dashed line indicates the average of the control sample.

[0395] FIG. 8 shows the comparison of Atg7 levels in different urine samples by means of an ELISA assay. Urine samples from healthy individuals (control, average of samples from ten individuals) were compared to urine samples from individuals having chromophobe RCC (chromo RCC, average of samples from eight individuals), eosinophilic RCC (eosino RCC, average of samples from four individuals), papillary RCC (pap RCC, average of samples from eight individuals), oncocytoma (average of samples from eight individuals), and RCC (average of samples from ten individuals). The Atg7 is increased in RCC urine samples in comparison to samples from healthy controls (*p and **p<0.001), but is not significantly altered in a sample from an individual having an oncocytoma. Increase is comparably low in papillary RCC. The dashed line indicates the average of the control sample.

EXAMPLES

Example I—Western Blot Analysis

[0396] Western blot analysis of urine samples from patients with either RCC or oncocytoma (OC) compared with “healthy” control urine samples (Crt). All samples were neutralized to beta-actin (β-actin) used as housekeeping gene. For densitometry (FIGS. 1A and 1B), each 10 patient samples were measured. The background of the blot was always measured separately and calculated against the densitometry of the protein lane. Afterwards, the results from the housekeeping gene were determined as 100% and either the Mxi-2 or Vim3 signal was calculated in regard to the 100% of the housekeeping. Calculation was performed by 1st way ANOVA.

[0397] Western blot was performed as followed: Proteins were separated according to their molecular weight. This was done by SDS polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous gel system to enhance the sharpness of the bands within the gel. The discontinuous gel system is composed of a stacking and a separating gel which differs in salt concentration, pH and acrylamide concentration. A 10% separating gel was used and performed as followed: the separating gel contains 0.4 M Tris-HCl pH 8.8, 0.1% SDS, 10-12% acrylamide/bisacrylamide (29:1), 0.5% ammonium persulfate and 0.06% TEMED. The stacking gel contains 0.125 M Tris pH 6.8, 0.1% SDS, 3% acrylamide/bisacrylamide (29:1), 0.5% ammonium persulfate and 0.12% TEMED.

[0398] To load the gel, 500 to 1000 μl urine were centrifuged at full speed and resuspended with PBS and “5× Western loading dye” and heated for 5 min at 99° C. The gel run was performed in “Laemmlis running” buffer. The running time was between 1.5 h to 2 h (15 mA until dye front reached separating gel, then 30 mA) depending on the protein size. After separation in the SDS-PAGE the proteins were transferred onto PVDF membrane using a semidry blotting system in Towbin buffer. 9 layers of 3 MM Whatman paper were placed in the semidry chamber with the gel on top. The gel was covered by an activated PDVF membrane. Finally 9 further layers of Whatman paper were placed on top. The 3 MM Whatman paper was previously moistened in “Towbin buffer”. The PDVF membrane was activated according to the manufacturing protocol. The transfer proceeded at 1.2 mA/cm2 at 4° C. for exactly 70 min.

[0399] After the transfer the membrane was blocked by putting it in blocking solution (5% milk in TBST) for 1 hour, shaking at room temperature, to avoid unspecific binding of the primary antibody. Following that, the membrane was incubated with the first antibody in blocking solution (5% milk in TBST) over night at 4° C., shaking. After this time and after a 30 min washing step in TBST stock solution, the membrane was incubated with the secondary antibody in blocking solution (5% milk in TBST) for 1 hour, shaking at room temperature and then washed again for 30 min in TBST stock solution. Finally the membrane was incubated 1.5 min with ECL reagent and developed in a chemiluminescence reader (ChemiDoc, Biorad).

[0400] The antibodies usable in the context of the method of the present invention are exemplarily specifically binding to the following epitopes:

[0401] epitope present at a region of Mxi-2:

[0402] GKLTIYPHLMDIELVMI (SEQ ID NO: 4)

[0403] epitope present at the C-terminal region of Vim3:

[0404] NLRGKHFISL (SEQ ID NO: 5)

[0405] It has been found surprisingly that individuals suffering from renal cell carcinoma (RCC) show increased levels of Mxi-2, whereas individuals bearing oncocytoma show increased levels of Vim3.

Example II—Analysis Via Dipstick

[0406] For dipstick analysis the same procedure can be used as for the sample analysis, at least when a single dipstick test is performed (Mxi-2 or Vim3). This is further exemplified in FIGS. 2 and 3. Also a combinational (i.e., combined) dipstick for the concomitant/parallel analysis of Mxi-2 and Vim3 in one urine sample U is usable. This is further exemplified in FIG. 4. This analysis indicates the level of Mxi-2 and Vim3, respectively, and optionally the ratio of Mxi-2:Vim3. The levels found in a urine sample U of interest are comparable with predetermined threshold level(s) and/or with those levels determined in one or more control samples.

Example III—Analysis Via Enzyme-Linked Immunosorbent Assay (ELISA)

[0407] Furthermore, also an ELISA is usable in the context of the present invention. For this purpose, an ELISA plate was labelled with an antibody which detects both variants, the full length and the truncated. ELISA plates (Corning Costar®96-Well EIA/RIA Stripwell™ Plates) were washed before start 2× with. 100 μl of the urine sample was added to the plate and incubated for 1 hour. After incubation plates were washed 2× with PBS and incubated for 1 h at room temperature with the primary antibody (1:1000) □ Atg7 (Santa Cruz, H-300; Vim3 monoclonal, clon 51, Davids Lab; Mxi-2, Nanotools, clon 2F2; MAPK p38, Santa Cruz, C-20). The samples were washed again with PBS 2× (alternatively 3×) and incubated with the corresponding, labelled secondary antibody. The signal detection depends on the used secondary antibody used. More in detail, after incubation plates were washed 2× with PBS and incubated for 1 h at room temperature with the primary antibody (1:1000) Atg7 (Santa Cruz, H-300; Vim3 monoclonal, clone 51, Davids Lab; Mxi-2, Nanotools, clone 2F2; MAPK p38, Santa Cruz, C-20). After incubation plates were washed again with PBS 2× and incubated for 1 h at room temperature with secondary goat-anti mouse antibody (1:50000) (Columbia Biosciences, HRP112). After final incubation step ELISAs were washed 3× with PBS and TMB solution was add for 10 min. afterwards the reaction was stopped with ELISA stopping solution and plates were analysed at 450 nm. The levels found in a urine sample U of interest were comparable with predetermined threshold level(s) and/or with those levels determined in one or more control samples.

[0408] Urine samples from healthy individuals (control, samples from ten individuals) were compared to urine samples from individuals having chromophobe RCC (samples from eight individuals), eosinophilic RCC (samples from four individuals), papillary RCC (samples from eight individuals), oncocytoma (samples from eight individuals), and RCC (samples from ten individuals).

[0409] ELISA assays were performed to determine the levels of Mxi-2, MAPK p38, Atg7 and Vim3 in the urine samples.

[0410] The results obtained by ELISA analysis confirmed the results performed by Western Blot analysis above. It was found that the Mxi-2 level, the MAPK p38 level and the Atg7 level in urine samples obtained from individuals suffering from RCC is statistically significantly increased in comparison to the levels found in urine samples from healthy control individuals and individuals having an oncocytoma.

[0411] Remarkably, the Mxi-2 level, the MAPK p38 level and the Atg7 level determined in urine samples obtained from individuals having chromophobe RCC or eosinophilic RCC were similar to those determined in urine samples obtained from individuals having RCC. The MAPK p38 level and the Atg7 level determined in urine samples obtained from individuals having papillary RCC was somewhat lower. Papillary RCC is typically considered as being averagely somewhat less malignant and having a better prognosis than chromophobe RCC, eosinophilic RCC or common RCC. This indicates that the presently claimed assay indicates the malignancy of a malignant renal carcinoma.

[0412] It was found that the Vim3 level in urine samples obtained from individuals having an oncocytoma is statistically significantly increased in comparison to the levels found in urine samples from healthy control individuals and individuals having an RCC.

[0413] The comparison between the levels of Mxi-2 in urine (see FIG. 5) and MAPK p38 (see FIG. 6) further shows that the Mxi-2 antibody appears to be even more specific. The statistic deviations between control samples are smaller for the Mxi-2 antibody.

Example IV—Analysis Via Polymerase Chain Reaction (PCR)

[0414] Furthermore, also PCR is usable in the context of the present invention. For PCR analysis the following primers are exemplarily usable:

TABLE-US-00006 Mxi-2: Forward: (SEQ ID NO: 6) 5′-GACTCAGATGCCGAAGAT-3′ Reverse: (SEQ ID NO: 7) 5′-TCAACTAATGGTACTTTATTTGG-3′ Vim3: Forward: (SEQ ID NO: 8) 5′-GAGAACTTTGCCGTTGAAGC-3′ Reverse: (SEQ ID NO: 9) 5′-GAAATAAAATGCTTACCCCTCAG-3′

[0415] The levels found in a urine sample U of interest are comparable with predetermined threshold level(s) and/or with those levels determined in one or more control samples.