WO2015199529A1

WO2015199529A1 - Method of diagnosis and/or discrimination of prostate cancer

Info

Publication number: WO2015199529A1
Application number: PCT/MY2015/000021
Authority: WO
Inventors: HASHIM.Onn HAJI; Jaime Jacqueline JAYAPALAN; Azad Hassan ABDUL RAZACK
Original assignee: Universiti Malaya
Current assignee: Universiti Malaya
Priority date: 2014-06-27
Filing date: 2015-03-27
Publication date: 2015-12-30
Anticipated expiration: 2016-12-27
Also published as: MY170330A

Abstract

The present invention provides a method of diagnosis and/or discrimination of prostate cancer in a patient, comprising the steps of: -determining the content of ITIH4 fragment peptide in urine taken from the body of the patient, and -determining the content of ITIH4 fragment peptide in serum taken from the body of the patient, and/or determining the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine taken from the body of the patient, -calculating at least one of: -serum : urinary ITIH4 peptide fragment index, and -urinary ITIH4:ITIL peptide fragment index, -comparing said one or both peptide fragment indexes with the indexes of healthy controls and/or individuals suffering from benign prostatic hyperplasia.

Description

Method of diagnosis and/or discrimination of prostate cancer Field of Art The present invention relates to a method of diagnosis and/or discrimination of prostate cancer using peptide biomarkers.

Background Art Prostate cancer (PCa) is an aging-associated cancer of enlarged prostate and voiding dysfunction. However, a similar clinical presentation is also observed among patients with early benign prostatic hyperplasia (BPH). The comprehensive approach to patients presenting with lower urinary tract symptoms attributed to the prostate gland involves digital rectal examination, uroflowmetry, post void residual urine, urinalysis, assessment of validated International Prostate Symptom Score questionnaire, and the measurement of serum prostate specific antigen (PSA).

In patients with a suspicious digital rectal examination of hard nodular prostate gland or a raised PSA, a transrectal ultrasound (TRUS) guided biopsy of the prostate gland is required so as to rule out any malignancy on histopathology. Currently, PSA is the most widely used biomarker for PCa but it lacks specificity and sensitivity. Many benign pathologies like BPH, inflammation and infection can often cause raised PSA, whilst on the other hand, patients whose tissue sections demonstrated belligerent PCa features had been shown to have normal PSA levels. If specific biomarkers are made available, it is possible to limit the number of TRUS guided prostate biopsies to be carried out. This will reduce the diagnosis of low grade and insignificant cancers. It is then possible to reduce or overcome the problem of overdiagnosis and overtreatment in the management of PCa.

Disclosure of the Invention

The present invention provides a method of diagnosis and/or discrimination of prostate cancer in a patient, comprising the steps of: - determining the content of ITIH4 fragment peptide in urine taken from the body of the patient, and

- determining the content of ITIH4 fragment peptide in serum taken from the body of the patient, and/or determining the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine taken from the body of the patient,

- calculating at least one of:

- serum:urinary ITIH4 peptide fragment index, being the ratio of the content of ΠΤΗ4 fragment peptide in serum and the content of ITIH4 in urine, and

- urinary ΠΊΗ4:ΠΊί peptide fragment index, being the ratio of the content of ITIH4 in urine and the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine,

- comparing said one or both peptide fragment indexes with the indexes of healthy controls and/or individuals suffering from benign prostatic hyperplasia (BPH), wherein:

- when the ratio of the content of ITIH4 fragment peptide in serum to the content of ΓΤΓΗ4 in urine is lower than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient, and/or

- when the ratio of the content of ΓΤΙΗ4 in urine to the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine is higher than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient.

It was shown that the following trends apply for prostate cancer patients vs. benign prostatic hyperplasia patients:

- relative lower abundance of ITIH4 fragment peptide in sera of patients with prostate cancer compared to those with BPH and healthy control subjects,

- relative higher abundance of ITIH4 fragment peptide in urine of patients with prostate cancer compared to those with BPH and healthy control subjects,

- relative lower abundance of inter-alpha trypsin inhibitor light chain (ITIL) fragment peptide 1 (ITILfl) in urine of patients with prostate cancer and those with BPH compared to healthy control subjects,

- relative lower abundance of ITIL fragment peptide 2 (ITILf2) in urine of patients with prostate cancer compared to those with BPH and healthy control subjects. Thus, these biomarkers in principle allow to discriminate between the patients with prostate cancer versus patients suffering from BPH.

In order to make the diagnosis and/or discrimination more reliable and robust, the present invention uses ratios of these biomarkers. The use of the two indexes instead of individual levels of serum or urinary biomarkers would enhance efficacy of the assay since the relative abundances of the peptide fragments have been shown to be reciprocally altered in prostate cancer patients compared to those with other cancers or benign tumours, including BPH.

In one embodiment, the content of ITIH4 fragment peptide in urine taken from the body of the patient, and the content of ΓΓΓΗ4 fragment peptide in serum taken from the body of the patient are determined, then serum:urinary ITIH4 peptide fragment index, being the ratio of the content of ITIH4 fragment peptide in serum and the content of ITIH4 in urine, is calculated, and when the ratio of the content of ΙΤΓΗ4 fragment peptide in serum to the content of ΓΓΙΗ4 in urine is lower than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient.

In another embodiment, the content of ITIH4 fragment peptide in urine taken from the body of the patient, and the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine taken from the body of the patient are determined, then urinary ITIH4:ITIL peptide fragment index, being the ratio of the content of ITIH4 in urine and the content of ITIL fragment peptide 1 and/or ITiL fragment peptide 2 in urine, is calculated, and when the ratio of the content of ITIH4 in urine to the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine is higher than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient.

It is preferred that the contents of ΓΓΙΗ4 fragment peptide in urine taken from the body of the patient, and of ΠΤΗ4 fragment peptide in serum taken from the body of the patient, and of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine taken from the body of the patient are determined, and then both the serum:urinary ITIH4 peptide fragment index, being the ratio of the content of ITIH4 fragment peptide in serum and the content of ITIH4 in urine, and the urinary ITIH4:ITIL peptide fragment index, being the ratio of the content of ITIH4 in urine and the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine, are calculated, and the conclusions as to the diagnosing of prostate cancer in said patient are drawn from both indexes.

ITIH4 refers to inter-alpha-trypsin inhibitor heavy chain H4 (Swiss-Prot accession number: Q14624).

ITIL refers to inter-alpha trypsin inhibitor light chain (Swiss-Prot accession number: P02760). ITIL fragment peptides 1 (ITILfl) and 2 (ITILf2) were differentiated when they resolved at different pis as well as molecular weights (M_r) possibly due to different post- translational modifications (Jayapalan et al., 2013). The ITILfl resolved at an acidic region, marking a M_r of -24 kDa whilst the mLf2, was seen to resolve at relatively an alkaline zone with a M_r of -18 kDa. Identities of ITILfl and ITILf2 were confirmed by mass spectrometry and database search, in which their trypsin digested peptides were shown to match amino acids 206-352 of ITIL.

The content of the peptide fragments can be determined by any suitable method known in the art, such as 2-dimensional electrophoresis (2-DE), Western blot (WB) (preferably using champedak galactose binding (CGB) lectin and apposite antibodies for detection, ELISA, Bio-Plex assay or any other known method.

Brief description of Figures Figures 1 to 3 correspond to Table (A) 2-DE, CGB-lectin and/or silver staining.

Figure 1 shows serum ΠΊΗ4, typical O-glycosylated serum proteins profiles of (A) BPH patients, and (B) PCa patients using enzyme-conjugated CGB lectin.

Figure 2 shows urinary ITIH4, typical O-glycosylated urinary proteins profiles of (A) Healthy controls, (B) BPH patients, and (C) PCa patients using enzyme-conjugated CGB lectin.

Figure 3 shows urinary ITIL, typical acetone precipitated urinary proteins 2DE and silver stain profiles of (A) healthy controls, (B) BPH patients, and (C) PCa patients. N.B.: Protein AMBPfl and AMBPf2 are also known as ITILfl and ITILf2, respectively, as described in the text.

Figures 4 to 6 correspond to Table (B) Western blot, CGB -lectin and/or antibody detection.

Figure 4 shows serum ΙΤΓΗ4, typical SDS-PAGE serum proteins profile using enzyme- conjugated CGB lectin. Key: Lane 1 - Marker, Lane 2 - pooled serum of healthy controls subjects, Lane 3 - pooled serum of subjects with BPH, Lane 3 - pooled serum of subjects with PCa.

Figure 5 shows urinary ITIH4, typical SDS-PAGE urinary proteins profile using enzyme- conjugated CGB lectin. Key: Lane 1 - Marker, Lane 2 - pooled urine of healthy controls subjects, Lane 3 - pooled urine of subjects with BPH, Lane 3 - pooled urine of subjects with PCa.

Figure 6 shows urinary ITIL/bikunin, Lane 1 - Marker, Lane 2 - pooled urine of healthy controls subjects, Lane 3 - pooled urine of subjects with BPH, Lane 3 - pooled urine of subjects with PCa.

Figures 7 to 9 correspond to Table (C) Western blot, immunodetection.

Figure 7 shows serum ITIH4, Lane 1 - Marker, Lane 2 - pooled urine of healthy controls subjects, Lane 3 - pooled urine of subjects with BPH, Lane 3 - pooled urine of subjects with PCa.

Figure 8 shows urinary ITIH4, Lane 1 - Marker, Lane 2 - pooled urine of healthy controls subjects, Lane 3 - pooled urine of subjects with BPH, Lane 3 - pooled urine of subjects with PCa.

Figure 9 shows urinary ITIL/bikunin, Lane 1 - Marker, Lane 2 - pooled urine of healthy controls subjects, Lane 3 - pooled urine of subjects with BPH, Lane 3 - pooled urine of subjects with PCa.

Examples of carrying out the Invention

2-DE and Silver staining

2-DE was performed using 800 μg and 100 μg of serum and urinary proteins, respectively in a method that was previously described (Jayapalan et al., 2012; Jayapalan et al., 2013). In brief, serum samples were initially incubated in sample buffer containing 2% v/v IPG sample buffer pH 4-7, 9 M urea, 60 mM DTT, and 0.5% v/v Triton X-100 at room temperature (RT) for 30 min followed by a similar incubation in a rehydration solution buffer containing 8 M urea, 0.5% v/v IPG buffer, 0.5% v/v Triton X-100. IPG Immobiline Drystrips pH 4-7, 11 cm (GE Healthcare,Uppsala, Sweden) were rehydrated in the mix, overnight. The strips were then subjected to isoelectric focusing (IEF) using the Multiphor Flatbed electrophoresis system (GE Healthcare) for a total duration of 15 kV/h. Focused strips were equilibrated in 1.5M Tris-HCl (pH 8.8) solution containing 6M urea, 2% w/v SDS, 30% v/v glycerol, and 0.06M DTT for 15 min on gentle agitation, and further incubated in a similar equilibration solution but containing 4.5% v/v iodoacetamide instead of DTT for another 15 min. The equilibrated strips were overlaid onto 8-18% gradient polyacrylamide gels and electrophoresis was performed following an optimized protocol using the SE 600 Ruby Electrophoresis System and Power Supply-EPS601 (GE Healthcare).

On the other hand, lyophilized urine samples were first precipitated using acetone. The samples were incubated in a rehydration buffer containing 0.5% v/v Pharmalyte pH 3- 10, 8M urea, and 0.5% v/v NP-40 at RT for one hour. The strips were subjected to IEF but, for a total duration of 12 kV/h and similarly equilibrated. The second dimensional electrophoresis was performed on a 12.5% homogenous polyacrylamide gels following an optimized protocol. The electrophoresed 2-DE gels were developed by silver staining according to the method described by Heukeshoven and Dernick (1988).

SDS-PAGE

SDS-PAGE was performed using 30 μg each of serum proteins, urinary proteins, standard human ITIH4 (USCN, Houston, USA) and human bikunin+trypstatin full length protein (Abeam, Cambridge, UK) in 10% resolving gel. In brief, the samples were mixed at a ratio of 1 : 1 with 4x SDS-PAGE sample buffer containing 62.5 raM Tris-HCl pH 6.8, 10% (w/v) glycerol, 2% (w/v) SDS and 1 % (w/v) DTT and boiled at 80 °C for 5 minutes. The samples were then loaded into the wells alongside molecular weight standards and electrophoresis was performed using the Mini-PROTEAN® Tetra Cell and PowerPac™ HC Power Supply (BioRad Laboratories, Hercules, USA).

Western blotting (WB) and detection using CGB-lectin and antibody

The 2-DE and 1-D SDS-PAGE resolved proteins were electrophoretically transferred onto (0.45 mM) nitrocellulose membranes (Whatman, Dassel, Germany) using the NovaBlot Kit of the Multiphor Flatbed System for 2 h at a constant current of 0.8 mA/cm² gel. Detection of transferred (9-glycosylated serum and urinary proteins from 2- DE (biological replicates) and 1-D SDS-PAGE gels (pooled samples; 2-4 technical replicates) was performed using the champedak galactose binding (CGB) lectin conjugated to horseradish peroxidase (CGB-HRP). Membranes were blocked with 3% gelatin in tris- buffered saline (TBS) containing 0.1 % v/v Tween 20 (TBS-T), washed and incubated overnight at 4°C in TBS-T containing 1 μg/ml of CGB-HRP. The membranes were washed thrice with TBS-T for 5 min each and developed using freshly prepared diamino- benzoic acid chromogen solution (Sigma Aldrich, St Louis, USA). The reaction was finally stopped by washing with distilled water.

Immunodetection was performed using appropriate antibody-protein of interest pairs on ID SDS-PAGE electrotransfered NC membranes in 2-4 technical replicates. For pooled serum proteins (30 μg), the membranes were blocked with 5% skim milk in TBS-T for 1 hour at RT and incubated overnight at 4°C in 1 % blocking solution containing goat polyclonal antiserum to human ITIH4 (1 : 10,000 dilution; Biorbyt, San Francisco, USA). Later, it was incubated with peroxidase-conjugated donkey anti-goat antibody (1 : 10,000 dilution; SantaCruz, Heidelberg, Germany) for 1 hour at RT. The membrane was similarly washed, developed and the reaction stopped as iterated above.

For pooled urinary proteins (30 μg), the membrane was similarly blocked as above but incubated overnight at 4°C in 1 % blocking solution containing goat polyclonal antiserum to human ΠΤΗ4 (1 : 1 ,000 dilution; Biorbyt) or rabbit polyclonal antiserum to human ITIL (bikunin) (1 : 10,000 dilution, Abeam). It was then incubated with peroxidase- conjugated donkey anti-goat (1:700 dilution) and goat anti-rabbit secondary antibodies (1 : 10,000 dilution; Santa Cruz), respectively for 1 hour at RT. The membrane was similarly washed, developed and the reaction stopped as iterated above.

Image analysis

Silver stained 2-DE gels, lectin and antibody-probed NC membranes were scanned using the Imaging Densitometer GS690 (Bio-Rad Laboratories, Hercules, USA). Expression of proteins was analyzed in terms of the percentage of volume contribution using the Image Master 2D Platinum software, version 7.0 (GE Healthcare Biosciences, Uppsala, Sweden). Relative quantitation of peptides of interest

Human recombinant ITIH4 (USCN, Houston, USA) and human bikunin+trypstatin full length protein (Abeam) were used as standard for quantitative determination of the ΓΓΓΗ4 and ITILfs protein fragments in the samples. The mean percentage volume contribution of the standard at 30 Mg/ml concentration was used to relatively quantify concentration of the respective protein bands of interest.

Reference:

1. Jayapalan, J.J.; Ng, K. L; Shuib, A. S.; Razack, A. H.; Hashim, O. H., Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter- alpha-trypsin inhibitor heavy chain 4 fragment. Electrophoresis 2013, 34 (11), 1663- 9.

2. Jayapalan, J. J.; Ng, K. L.; Razack, A. H.; Hashim, O. H., Identification of potential complementary serum biomarkers to differentiate prostate cancer from benign prostatic hyperplasia using gel- and lectin-based proteomics analyses. Electrophoresis 2012, 33 (12), 1855-62.

3. Heukeshoven J, Dernick R. Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis. 1988;9(1 ):28-32.

Results:

A) 2-DE experiments - CGB-Lectin &/or Silver stain detection.

Mean % volume*

Parameter

Healthy controls BPH PCa

Serum ITIH4f ^¥ 1.81 ± 0.23 6.87 + 0.33^a 2.05 + 0.20

Urine ITHM 0.16 + 0.04 0.31 + 0.06 0.91 + 0.21^b

Mean ratio¹ 10.97 22.13 2.26

Range of ratio 4.72 - 31.89 7.23 - 51.69 0.83 - 33.36 Urine ITIH4f* 0.16 + 0.04 0.31 + 0.06 0.91 + 0.21"

Urine ITILf 3.78 + 0.63° 1.93 + 0.29 1.62 + 0.36

Mean ratio² 0.04 0.16 0.56

Range of ratio 0.02 - 0.16 0.07 - 1.73 0.04 - 2.33

Urine ITIH4f* 0.20 + 0.04 0.30 + 0.06 0.9 + 0.21

Urine ITrLf2^∞ 1.92 + 0.28" 1.91 + 0.29⁶ 0.88 + 0.15

Mean ratio³ 0.09 0.16 1.03

Range of ratio 0.05 - 0.27 0.07 - 0.83 0.07 - 2.79

*data presented in mean + SEM of the mean % volume contribution of the protein bands, method applied: 2-DE and subsequently by CGB-Lectin detection,

^method applied: 2-DE and subsequently Silver stain detection.

'ratio of mean % volume of serum ITIH4f to urine ITIH4f.

ratio of mean % volume of urine ITIH4f to urine ITILf 1.

3ratio of mean % volume of urine ITIH4f to urine ITILf2. Significance (p < 0.05):

a, BPH vs Healthy and PCa; b, Healthy vs PCa; c, Healthy vs PCa; d, Healthy vs PCa; e, BPH vs PCa

B) Western Blotting - CGB-Lectin &/or Antibody detection.

Mean % volume*

Parameter^*

Healthy controls BPH PCa

Serum ΠΊΗ4ι* 0.49 + 0.07 0.29 + 0.03 0.22 + 0.07

Urine ITIH4f* 0.60 + 0.05 0.65 + 0.01 3.66 + 0.24^a

Mean ratio¹ 0.82 0.45 0.06

Range of ratio 0.69 - 0.94 0.41 - 0.50 0.04 - 0.08

Urine ΠΊΗ4ί* 0.60 ± 0.05 0.65 + 0.01 3.66 + 0.24^a

Urine ITILfr 0.74 + 0.07 0.52 + 0.07 0.39 + 0.01"

Mean ratio² 0.81 1.25 9.31

Range of ratio 0.74 - 0.94 1.10 - 1.46 8.71 - 9.50 Urine ITIH4f^¥ 0.60 ± 0.05 0.65 + 0.01 3.66 + 0.24³

Urine ITILf2^x 0.38 ± 0.09 0.40 ± 0.04 0.12 + 0.04^c

Mean ratio 1.57 1.63 31.12

Range of ratio 1.28-2.02 1.45-2.01 23.14-47.45

*data presented in mean + SEM of the mean % volume of the protein bands.

¥method applied: SDS-PAGE and subsequently WB - CGB lectin detection,

^method applied: SDS-PAGE and subsequently WB - antibody detection.

'ratio of mean % volume of serum ITIH4f to urine ITIH4f.

ratio of mean % volume of urine ITIH4f to urine ITILf 1.

3ratio of mean % volume of urine ITIH4f to urine ITILf2.

Significance (p < 0.05):

a, BPH vs Healthy controls & PCa; b, PCa vs Healthy controls; c, PCa vs BPH.

C) Western blot - immunodetection.

Concentration in g/ml

Parameter ⁰⁰

Healthy controls BPH PCa

Serum ITIH4f 10.21 ± 1.40 9.00 + 0.88 11.93 + 0.63

Urine ITIH4f 3.13 + 1.04 1.45 + 0.16 7.14 + 0.96"

Mean ratio¹ 2.39 6.22 1.68

Range of ratio 1.96-5.11 4.88-7.75 1.18-2.18

Urine ITIH4f 3.13 + 1.04 1.45 + 0.16 7.14 + 0.96³

Urine ITILf 1 19.66+1.96 13.69+1.89 10.45+0.17"

Mean ratio² 0.16 0.11 0.68

Range of ratio 0.10-0.27 0.08-0.13 0.52 - 0.70

Urine ITIH4f 3.13 + 1.04 1.45 + 0.16 7.14 + 0.96³

Urine ITILf2 10.17 ±2.29 10.53 ± 1.00 3.13 + 1.08^c

Mean ratio 0.31 0.14 2.28

Range of ratio 0.20-0.51 0.11-0.18 1.70-3.48 *data presented in mean + SD of the concentration of ITIH4, ITILf 1 or ITIlf2 in μ^πιΐ, respectively. The concentration was derived using the mean % volume contribution of the known standard concentration (30 μg/ml) of the purified recombinant proteins ΓΓΓΗ4 and niL, respectively. ^xmethod applied: SDS-PAGE and subsequently WB - antibody detection.

'ratio of concentration of serum ITIH4f to urine ITIH4f.

2ratio of concentration of urine ITIH4f to urine ITILf 1.

" ratio of concentration of urine ITIH4f to urine ITILf 2.

Significance (p < 0.05):

a, PCa vs Healthy controls & BPH; b, PCa vs Healthy controls; c, PCa vs BPH.

Claims

1. A method of diagnosis and/or discrimination of prostate cancer in a patient, comprising the steps of:

- determining the content of ITIH4 fragment peptide in urine taken from the body of the patient, and

- calculating at least one of:

- serum:urinary ITIH4 peptide fragment index, being the ratio of the content of ITIH4 fragment peptide in serum and the content of ITIH4 in urine, and

- urinary ITIH4:ITIL peptide fragment index, being the ratio of the content of ITIH4 in urine and the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine,

- when the ratio of the content of ITIH4 fragment peptide in serum to the content of ITIH4 in urine is lower than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient, and/or

- when the ratio of the content of ITIH4 in urine to the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine is higher than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient.

2. The method according to claim 1 , wherein the content of ITIH4 fragment peptide in urine taken from the body of the patient, and the content of ITIH4 fragment peptide in serum taken from the body of the patient are determined, then serum:urinary ITIH4 peptide fragment index, being the ratio of the content of ITIH4 fragment peptide in serum and the content of ITIH4 in urine, is calculated, and when the ratio of the content of ITIH4 fragment peptide in serum to the content of ITIH4 in urine is lower than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient.

3. The method according to claim 1, wherein the content of ITIH4 fragment peptide in urine taken from the body of the patient, and the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine taken from the body of the patient are determined, then urinary ITIH4:ITIL peptide fragment index, being the ratio of the content of ITIH4 in urine and the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine, is calculated, and when the ratio of the content of ITIH4 in urine to the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine is higher than said ratio of the healthy control and/or individuals suffering from BPH, then prostate cancer is diagnosed in said patient.

4. The method according to claim 1, wherein the contents of ITIH4 fragment peptide in urine taken from the body of the patient, and of ITIH4 fragment peptide in serum taken from the body of the patient, and of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine taken from the body of the patient are determined, and then both the serum:urinary ITIH4 peptide fragment index, being the ratio of the content of ITIH4 fragment peptide in serum and the content of ITIH4 in urine, and the urinary ITIH4:ITIL peptide fragment index, being the ratio of the content of ITIH4 in urine and the content of ITIL fragment peptide 1 and/or ITIL fragment peptide 2 in urine, are calculated.