JP2002506210A - How to identify prostate cancer - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for identifying prostate cancer patients from patients with benign prostatic hyperplasia (BPH) and healthy men without prostate cancer (PCa). In this method, the concentration of human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) in an individual body fluid is determined. In the present invention, a combination of hK2 and PSA is used as a marker for distinguishing PCa patients from BPH patients and non-PCa individuals. However, PSA means free PSA, complexed PSA or total PSA. The invention further relates to a method for distinguishing painless or low-progression prostate cancer patients from aggressive advanced prostate cancer patients.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to a diagnostic method for distinguishing prostate cancer from healthy controls or benign prostate conditions, comprising an analytical level of sensitivity and specificity for human glandular kallikrein 2 (hK2). By combining the results of immunoassays with sexuality with the results of immunoassays that specifically detect various types of prostate-specific antigen (PSA or hK3), most notably, free PSA. The present invention relates to the diagnosis of prostate cancer in screening asymptomatic individuals and in distinguishing cancer from benign symptoms in humans with clinical symptoms.

[0002] The diagnostic method of the present invention compares the conventional method of quantifying total PSA and the recently introduced method of determining the ratio of complexed or free PSA to total PSA, It has the advantage of higher clinical sensitivity and / or specificity. Biological samples suitable for quantification in this immunoassay are serum, plasma or whole blood.

BACKGROUND OF THE INVENTION The literature and other materials mentioned for the purpose of describing the background of the invention are incorporated herein by reference. Human linear kallikrein 2 (hK2) and prostate-specific antigen (PSA; designated as hK3) are very closely related serine proteases that are expressed in large amounts mainly in prostate tissue [Wang et al. , Invest Urol 1979, 17: 159-163; Chapdelaine et al.,
FEBS Lett 1988, 236: 205-208]. They are very homologous (79
%), But their expression ratios are quite different (hK2 mRNA levels are about 10-20% of PSA mRNA levels) [Schedlich et al., DNA 1987, 6: 429-437].

[0004] Enzymatically active PSA is secreted into semen at high concentrations (0.2-5 mg / ml) [Christens
son et al., Eur J Biochem 1990, 194: 755-763; Ahlgren et al., 1995, J An
drol 16: 491-498]. In semen, PSA breaks down gel-forming proteins from the seminal vesicles, resulting in semen liquefaction and release of sperm with forward motility [Lilja,
J Clin Iavest 1985, 76: 1899-1903]. Recently, recombinant hK2 has been shown to convert inactive recombinant pro-PSA to active mature PSA in vitro [Loevgren et al.
l, Biochem Biophys Res Commun 1997, 238: 549-555; Takayama et al. J Biol
Chem 1997, 272: 21582-21588; Kumar et al. Cancer Res 1997, 57: 3111-311
Four].

In the last decade, the incidence of prostate cancer has increased, mainly due to longer lifespans and increased screening. Thus, there is a need for improved diagnostic methods and new treatments. Analysis of PSA in serum has been well established in the diagnosis and testing of prostate cancer (PCa) patients [Oesterling, J Urol 1991, 145: 907-923]. However, increased serum PSA levels are associated with other prostate disorders, such as benign prostatic hyperplasia (BPH)
[Hudson et al., J Urol 1989, 142: 1011-1]
017].

[0006] PSA has been shown to have chymotrypsin-like protease activity [Lilja e
t al., J Biol Chem 1989, 264: 1894-1900; Christensson et al., Eur J Bioc
hem 1990, 194: 755-763). The active single-chain form of PSA has been identified by several extracellular protease inhibitors, such as α ₁ -antichymotrypsin (ACT), α ₂ -macroglobulin (A
MG), forms a stable covalent complex with maternal band protein (PZP), protein C inhibitor (PCI), and α ₁ -antitrypsin [Christensson et al., Eur J Bi
ochem 1990, 194: 755-763; Stenman et al., Cancer Res 1991, 51: 222-226;
Espana et al., Thromb Res 1991, 64: 309-320; Christensson and Lilja, Eur
J Biochem 1994, 220: 45-53; Zhang et al., Prostate 1997, 33: 87-96].

[0007] The discovery of several molecular forms of PSA in serum has led to some improvements in the diagnostic specificity of prostate cancer. The major form of PSA that is immunologically detectable in blood is ACT
And free form (PSA-F) is only present in small amounts [Stenman et al.,
Cancer Res 1991, 51: 222-226; Lilja et al., Clin Chem 1991, 37: 1618-16
twenty five] .

[0008] In general, the proportion of PSA-F in serum is higher in BPH patients than in prostate cancer patients. This suggests that PSA-T (PSA-T = PSA-F + PSA-ACT + and other quantitatively insignificant) can be used to distinguish BPH from prostate cancer at moderately elevated PSA levels. PSA-
Ratio (PSA-F to serpin complex) has been used [Stenman et al.,
Cancer Res 1991, 51: 222-226; Christensson et al., J Urol 1993, 150: 10
0-105]. As a result, the diagnostic specificity of prostate cancer has improved, but there is still a strong need for markers that further improve the discrimination between cancer patients and normal and benign patients.

HK2 is also expressed at fairly high levels in normal prostate epithelium [Chapd
elaine et al., FEBS Lett 1988, 236: 205-208]. In prostate adenocarcinoma, hK2 is expressed at even higher levels [Darson et al., Urology 1997
, 49: 857-862]. hK2 can be detected in semen plasma (Deperthes et al., Bioch.
im Biophys Acta 1995, 1245: 311-316], not purified (in large quantities) and not fully characterized. Although the physiological function of hK2 has not yet been elucidated, hK2
The primary structure of 2 suggests that it may have trypsin-like catalytic activity [Schedlich et al., DNA 1987, 6: 429-437].

[0010] Recombinant production has allowed further studies of the properties of hK2 [Eerola et a
l., Prostate 1997, 31: 84-90]. Since hK2 is very homologous to PSA in amino acid sequence, there is a possibility of cross-reactivity in PSA immunoassay based on anti-PSA antibody. The present inventors have previously shown that 23 anti-PSA monoclonal antibodies and recombinant hK2
Examination of the cross-reactivity with, found that only 5 of them cross-reacted with hK2 with an affinity similar to that of PSA. Recently, the present inventors have confirmed this fact through detailed epitope determination studies [Piironen et al., Protein
Science 1998, 7: 259-269].

An immunoassay for hK2 utilizing blocking with an anti-PSA monoclonal antibody that does not cross-react with hK2, followed by detection with an anti-PSA monoclonal antibody that fully cross-reacts with hK2, has been constructed, and hK2 in patient serum samples has been constructed. Specificity and sensitivity to the drug (detection limit 0.1 μg / L and cross-reactivity with PSA 0.7%)
[Pillronen et al., Clin Chem 1996, 42: 1034-1041].

From examination of a group of patient samples that have not been clinically defined, the following has been concluded.
1) Serum hK2 and total PSA correlate with each other over the entire PSA range (1-34).
Within the PSA range of 00 μg / L, r = 0.84), and within a more limited range, the correlation was low (r = 0.32 within the PSA range of 1-20 μg / L). 2) The concentration of hK2 was generally lower than that of PSA (hK2 exceeded 5% of total PSA only in 7% of the cases). Based on these results, there are two analytical criteria for any hK2 test applied to clinical samples,
That is, the critical importance of the cross-reactivity with PSA and the lower limit of detection becomes clear.

When a serum sample containing hK2 at a high concentration was subjected to gel filtration, immunodetectable hK2 was eluted as a size of about 30 kDa. This means that the immunodetectable hK2 is free. In addition, the inventor has applied this method to the sera of a closely characterized group of patients to identify and improve the clinical efficacy characteristics of this hK2 test and to determine its clinical utility in urology. Applied. We report a comparison of PSA-T, PSA-F and PSA-ACT tests performed simultaneously on three well-defined clinical sample groups.

SUMMARY OF THE INVENTION and Preferred Embodiments The present invention provides a sensitive and specific human glandular kallikrein hK2 for differentially diagnosing prostate cancer, distinguishing it from control healthy subjects or patients with benign prostate conditions. Related to using immunoassays. This hK2 immunoassay can be performed on a body fluid, eg, serum, plasma or whole blood sample, obtained from the test subject.

According to a central finding of the present invention, the use of hK2 as an effective tumor marker
By combining the concentrations of various forms of prostate-specific antigen (total PSA, complexed PSA, free PSA), most notably, the measured free PSA concentration with the measured hK2 concentration, Best achieved.

Thus, according to one aspect, the present invention distinguishes patients with prostate cancer (PCa) from patients with benign prostatic hyperplasia (BPH) and healthy males without prostate cancer. The method comprises the steps of: human glandular kallikrein 2 (h) in a body fluid of an individual;
K2) and by determining the concentration of prostate specific antigen. This method is characterized in that a combination of hK2 and PSA is used as a marker for distinguishing prostate cancer patients from BPH patients and individuals without prostate cancer. In this case, PSA means free PSA, complex PSA or total PSA.

According to one preferred embodiment, the marker has a ratio hK2 / F or F / hK2,
Where F represents the concentration of free PSA. According to another preferred embodiment, the marker is the ratio hK2 / F or F / hK2 multiplied by a quantity representing the PSA concentration. The term "amount representing PSA concentration" refers to free PSA, total
PSA or complexed PSA (i.e., PSA complexed with ACT), or the PSA (free
PSA, total PSA or complexed PSA), and can be any amount calculated from two or three (all) components, such as “total PSA / free PSA”.

Thus, according to a particularly preferred embodiment, the marker is the product of hK2 / F and T ((hK2 / F
2 × T) / F), where T is the total PSA concentration. According to another particularly preferred embodiment, the marker is the product of hK2 / F and T / F ((hK2 / F) × T / F), where T is the total
PSA concentration. According to a particularly preferred embodiment, the cut-off limit for said ratio is selected by different methods depending on whether high detection sensitivity or high detection specificity is desired.

According to a third preferred embodiment, the combination of hK2 and PSA is performed by a logical regression analysis. However, PSA means free PSA, complexed PSA or total PSA. According to a particularly preferred embodiment, the combination by logical regression analysis is c [hK2, F], where F is free PSA. According to another particularly preferred embodiment, the combination by logical regression analysis is c [hK2, F, T], where F is free PSA and T is total PSA
It is.

According to a preferred embodiment of the present invention, the total PSA concentration is limited by the intermediate PSA concentration, ie, the PSA concentration at which the diagnostic discrimination between cancer and non-cancerous symptoms by total PSA alone is more uncertain, and When present, it is most effectively applied to samples of a subpopulation of patients that are organ-restricted and would be suitable for treatment. Such an area is often defined as a range of 1-20 μg / L, preferably 3 or 4-10 μg / L total PSA.

In accordance with another aspect of the present invention, to further identify the subpopulations in identified prostate cancer patients, more particularly, the use of prostate cancer (Gleason
HK2 was used to identify patients with organ-restricted or localized cancers with low scores from those with more aggressive and progressive cancers (organ-restricted or localized cancers with high Gleason scores). Or a combination by logical regression analysis of hK2 and other measured parameters.

Thus, in accordance with this aspect, the present invention is directed to a method for distinguishing a patient having indolent or low-progression prostate cancer from a patient having aggressive, prostate cancer.
In this method, the concentration of human glandular kallikrein 2 (hK2) and prostate specific antigen (PSA) in an individual body fluid is determined. This method is characterized by using a combination of hK2 and PSA as a marker for distinguishing painless or low-progressive prostate cancer patients from aggressive prostate cancer patients. However, PSA is free PSA, complexed
PSA or total PSA. This expression "combination of hK2 and PSA, where PSA is
"Free PSA, complexed PSA, or total PSA" is as discussed above.

The marker is preferably a ratio hK2 / F or a ratio F / hK2, where F is free
It is the concentration of PSA. Alternatively, the marker is preferably the ratio hK2 / F or the ratio
F / hK2 multiplied by the amount representing the PSA concentration, where "quantity representing the PSA concentration"
Has the previously described meaning. Most preferably, the markers are hK2 / F and T / F
((HK2 / F) × T / F), where T is the concentration of total PSA. According to a particularly preferred embodiment, the cut-off limit for the ratio is selected by different methods depending on whether a high detection sensitivity or a high detection specificity is desired.

Alternatively, the marker is a combination of hK2 and PSA, provided that PSA is free
PSA, complexed PSA or total PSA, the combination being by logical regression analysis. For example, the combination by this logical regression analysis is c [hK2, F], where F is free PSA, and c [hK2, F, T], where F is free PSA;
T is the total PSA.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a sensitive immunoassay specific for human glandular kallikrein 2 (hK2) to differentiate and diagnose prostate cancer patients from control healthy subjects or patients with benign prostate disease. Related to using.

According to the present invention, the ratio of hK2 to free PSA (or conversely, free PS to hK2
HK2 specific immunoassay applied to the biological sample of the subject patient to screen or test for the presence of malignant or benign prostate disease, with the results obtained from the free PSA assay Combine. Identify cut-off limits or decision points to best or as desired discriminate individuals who are likely to have prostate cancer, benign prostatic hyperplasia, or no prostate injury I do. The cutoff value selected will vary depending on whether one wants high sensitivity or high specificity in detecting prostate disease (PCa or BPH).

The above ratio was determined by examining total PSA (= free and complexed PSA) specific, total PSA and hK2
A test to determine the sum of and PSA complexed with ACT and / or other serpins (
Or by the concentration provided by the test for PSA + hK2), or by the ratio of PSA-F to PSA-T (PSA-T can also be replaced by PSA-T + hK2). Such a ratio is, for example, hK2 × PSA-T / PSA-F or (h
K2 / PSA-F) × (PSA-T / PSA-F) or design many other modified ratios that have the ability to better differentiate prostate cancer from benign prostate and healthy conditions Can be.

The combination of hK2 with PSA-F (and / or with other types of PSA or with measurements of PSA + hK2) can be carried out by another method (method not related to the formation of ratios or products), but This can be achieved through combination by regression analysis. Combination by logical regression analysis,
For example, (hK2, PSA-F) or (hK2, PSA-F, PSA-T) is often compared to the ratio calculated from individual measurements for each patient, as described in the ROC analysis described in the experimental section. To provide better identification.

Logical regression analysis is a means of providing evidence for various “risk analysis systems that support medical decisions”. Other examples of such data processing systems include artificial neural networks (ANNs), neuro-fuzzy networks (NFNs), multilayer perceptrons (MLPs).
) And learning vector quantization (LVG) [Freeman et al. In “Neural Networks: A
lgorithms, Applications and Programming Techniques by Addison-Wesley Pub
lishing Company "1991, Zadeh Information and Control 1965, 8: 338-353; Z
adeh “IEEE Trans. on Systems, Man and Cybernetics” 1973, 3: 28-44; Gersh
o et al. In “Vector Quantization and Signal Compression by Kluywer Acad
emic Publishers, Boston, Dordrecht, London "1992, Hassoun“ Fundamentals
of Artificial Neural Networks by The MIT Press, Cambridge, Massachusett
s, London "1995].

According to another aspect of the present invention, the total PSA concentration is limited by an intermediate concentration, that is, a PSA concentration at which the diagnostic discrimination between cancer and non-cancer symptoms by total PSA alone is less certain.
Moreover, if cancer is present, it is most effectively applied to a sample of a subpopulation of patients that is organ-restricted and would be suitable for treatment. Such areas are often defined as a range of 3 or 4 to 10 μg / L total PSA, but differ in both the lower range (which can be lower) or the upper range (which can be higher). Can be specified.

According to yet another aspect of the present invention, to identify a subpopulation from among the identified prostate cancer patients, more particularly, from a group of cancer patients that would be more aggressive,
Ratios containing hK2 or a combination of hK2 and other measured parameters by logical regression analysis can also be used to identify a group of prostate cancer patients that may be painless or less advanced.

The Gleason score is currently the best established factor (predictive marker) to predict the pathological potential of a tumor. Nearly undifferentiated tumors (high Gleason score) are aggressive diseases, while well differentiated tumors (low Gleason score) have an indolent course (Cookson et al., J Urol 1997, 157: 559).

Experiment 1 Study Population and Sample Processing Study Population 1 (SP1) SP1 was from February 1995 to September 1997, the Clinic of Urology, Kantonsspit.
It consisted of 393 consecutive male patients with lower urinary tract symptoms hospitalized in al Aaran, Switzerland. The mean total IPSS symptom score was 20.4 ± 7.3 (obstructive 11.8 ± 4.9 and inflammatory 8.6 ± 3.2). Criteria added to the population were no prostate treatment within three weeks, no signs of infection, and no long-term catheterization prior to hospitalization. These patients were divided into two groups. Group 1: Prostate cancer (PCa
), N = 104; Group 2: Patients with benign prostatic hyperplasia (BPH), n = 289. The median age (and upper and lower quartiles) was 72 (64, 77) in the PCa group and 72 in the BPH group.
(66, 78) years old.

All patients underwent digital rectal examination (DRE). Usually a laboratory test,
All patients up to 70 years of age or with a minimum life expectancy of 10 years and> 4 ng / mL total PSA underwent transrectal ultrasound (TRUS) guided 6-section biopsy of the prostate. The same test was performed on patients suspected of having DRE. Biopsy revealed PCa in 76 cases. Of these patients, 29 underwent radiation prostate removal (16 had pT2, 12 had pT3, and 1 had pT4) and 34 had transurethral prostatectomy (TURP) (12 cT
2, 15 cT3, 7 cT4), 8 received antiandrogen treatment (7 were castrated by orchiectomy (5 cT4, 2 cT3), 1 had pharmacological androgen deprivation) (CT4)).

Five had conservative treatment (intrinsic Foley catheter or suprapubic catheter) due to surgery contraindications or were readjusted for signs of infection at admission. After TUR-P, histological examination of resected specimens revealed PCa in 28 cases (
12 pT1a, 16 pT1b). Twenty-five of the 104 patients had metastatic PCa. BPH
Of the patients, 270 received TUR-P, three underwent transvesical prostatectomy, and 16
Conservative treatment (intrinsic Foley catheter or suprapubic catheter) was given due to contraindications to surgery or readjusted for signs of infection at admission.

Blood samples were obtained before any treatment of the prostate. After coagulation, the samples were centrifuged, the serum was collected and frozen at -70 ° C. The sample was thawed immediately before the measurement.

Study Population 2 (SP2) SP2 was published in the Clinic of Urology, Kantonsspit from February 1995 to September 1997.
Consisting of 115 consecutive male patients with lower urinary tract symptoms admitted to al Aaran, Switzerland (mean of total IPSS symptom score 18.9 ± 8.0; obstructive 10.7 ± 5.3 and inflammatory 8.2 ± 3.2). Criteria to be added to the population include PSA-T at admission of 3-10 μg / L, no prior treatment for prostate disease, no prostate treatment within 3 weeks, and no signs of infection And no long-term catheterization prior to admission. These patients were divided into two groups. Group 1: PCa patients, n = 25; Group 2
: Patients with BPH, n = 90. The median age (and upper and lower quartiles) was 70 (6
He was 2,72) and 71 (64,76) in the BPH group.

[0038] All patients received DRE. As a routine clinical examination, all patients up to 70 years of age or with a minimum life expectancy of 10 years underwent a TRUS-guided 6-section biopsy of the prostate. The same test was performed on patients suspected of having DRE. Biopsy revealed PCa in 15 cases. Of these patients, 6 underwent radiation prostate removal (5 had pT3 and 1 had pT2c) and 6 had TUR-P (1 cT2, 2 cT3, 1 Man
cT4), and three received conservative treatment. After TUR-P, histological examination of resected specimens revealed PCa in 10 cases (6 pT1a, 4 pT1b). TUR- for all BPH patients
P received.

Blood samples were obtained before any treatment of the prostate. After coagulation, the samples were centrifuged, the serum was collected and frozen at -70 ° C. The sample was thawed immediately before the measurement.

Study Population 3 (SP3) SP3 was 51-66 years old and participated in a mass screening for prostate cancer in Sweden, Gothenburg area, and initially had a total PSA concentration> 3 μg / L 360 (Hugosson et al. Submitted). Additional serum samples were obtained prior to DRE, TRUS and TRUS-guided sextant biopsy of the prostate. After blood clotting and centrifugation within 3 hours, the samples were frozen at -70 ° C and thawed immediately before performing the immunoassay. A sextant biopsy revealed prostate cancer in 80 of the 360 tested. 44 of them are clinical grade
T1c, 13 are T2a, 12 are T2b, 4 are T2c, 4 are T3a, 1 is T3b,
And two were T4. Gleason scores were determined from histological examination of needle biopsy samples.

[0041] 2. Purified proteins, monoclonal antibodies, reagents and instruments Recombinant hK2 (rec hK2), recombinant PSA (rec PSA) and
And purified semen PSA [Eerola et al., Prostate 1997, 31: 84-90; Christ
ensson et al., Eur J Biochem 1990, 194: 755-763]. Anti-PSA monoclonal anti
Antibodies H117 and H50 (which fully cross-react with rec hK2) were obtained from Abbot (USA) using anti-PSA
Clonal antibody 2H11 (which does not cross-react with rec hK2) with Wallac Oy (Turku, Finland)
From and anti-PSA monoclonal antibodies 36 and 10 (does not cross-react with rec hK2)
Was obtained from Olle Nilsson, CanAg (Gothenburg, Sweden). These monoc
The binding specificity of antibodies has been well documented [Piironen et al., Protein
Sciense 1998, 7: 259-269]. Microphone coated with streptavidin
Rotita Well, DELFIA^TM 1234 Plate fluorimeter, DELFIA PSA test buffer, DE
LFIA cleaning solution, DELFIA enhancement solution, and DELFIA Prostatus for PSA-T and PSA-F ^TM Tests were obtained from Wallac Oy (Turku, Finland).

[0042] 3. Methods for immunofluorescence testing of hK2 and various types of PSA.The hK2 test was based on previously reported serum hK2 assays [Piironen et al.
, Clin Chem 1996, 42: 1034-1041], using anti-PSA monoclonal antibodies H50 and H117 with similar affinities for PSA and hK2, and monoclonal antibody 2H11 as a blocker sufficiently specific for PSA [Loevgren et al. Biochem Bio
phys Res Commun 1995; 213: 888-895, Piironen et al. Protein Science 1998
7: 259-269], but with significant changes. The new test procedure implements a method of blocking PSA by adding two more PSA-specific anti-PSA monoclonal antibodies (Mab 36 and 10). The incubation time was also changed.

The procedure of this new inspection is as follows. 25 μl or 50 μl of the sample or reference solution was added to 50 μl of DELFIA PSA containing Mab 2H11, 36 and 10 (2000 ng / well each).
Along with the test buffer, add to the wells coated with streptavidin,
Incubated at room temperature for 1 hour. Add biotinylated Mab H50 (200n
g / well), and add 50 μl of PSA test buffer containing
Incubated for hours. After the washing step, the Eu-labeled tracer Mab H117 (100
ng / well) were added and the wells were incubated for 1 hour at room temperature. Finally, after the second washing step, 200 μl / well of enhancement solution was added and the fluorescence was measured for 1 second on a plate fluorimeter.

In vitro produced recombinant hK2 with a 100-fold molar excess of ACT and protein C
Using the complex with the inhibitor (PCI), the property that the reactivity of the complexed hK2 with the free hK2 in the hK2 test was sufficiently equal (equimolar) was examined.

The hK2 test was calibrated using the calibration PSA (range 0.2-500 μg / L) in the DELFIA Prostatus EQM commercial kit, but without the blocking monoclonal antibody. This kit utilizes a sandwich method using a monoclonal antibody similar to the hK2 test. As a control sample, a hand-held control based on rec PSA and serum was used.

The cross-reactivity with PSA as determined by recombinantly produced PSA was less than 0.1%. The analytical limit of detection was 0.01 μg / L hK2. Functional sensitivity (i.e., the concentration at which the test CV: s repeat measurement was less than 20%) was measured with a sample volume of 25 μl (S
P1) is 0.05 μg / L, and sample volume 50 μl (SP2 and SP3) is 0.03 μg / L.
Met.

DELFIA Prostat using monoclonal antibody combinations H117 / H50 and H117 / 5A10
A us PSA F / T double test was performed to detect PSA-T and PSA-F, respectively. The monoclonal antibody combination H117 / H50 determined free PSA and PSA complexed in an equimolar manner with α-1-antichymotrypsin and other serpins [Mitrune
n et al., Clin Chem 1995, 41: 1115-1120], and hK2 is also sufficiently simultaneously detected [Loevgren et al., Biochem Biophys Res Commun 1995, 213: 888-895]. The ratio of hK2 to PSA in serum is generally very low [Piironen et al., Clin C
hem 1996, 42: 1034-1041], the concentration measured by the combination H117 / H50 (this is the DELF
The IA Prostatus PSA F / T (equivalent to the T portion of the duplicate test) is generally referred to below as the PSA-T concentration. In the PSA-F test (H117 / 5A10), hK2 is not detected simultaneously.

The specific measurement of PSA (and hK2) complexed with ACT was performed by a research two-site immunofluorescence assay based on the monoclonal antibody combination H117 / 241 [Oesterlin
g et al., J Urol 1995, 154: 1090-1095].

[0049] 4. Statistical analysis To compare PSA-T, PSA-F and PSA-ACT with hK2, regression analysis was performed. Descriptive statistics were given as the median and upper and lower quartiles (25 and 75% values). PSA-T, PSA-F, hK2, PSA-ACT, PSA-F / PSA-T, hK2 / PSA
-T, hK2 / PSA-F, PSA-F / PSA-ACT and hK2 × PSA-T / PSA-F and (hK2 / PSA-F) × (PSA
-T / PSA-F) to statistically test for differences between Kruskal Wallis and Mann Whitney.
Was subjected to a non-parametric test. PSA-T, PSA-F, hK2,
Specificity of PSA-F / PSA-T, hK2 / PSA-F and (hK2 / PSA-F) × (PSA-T / PSA-F), and logical regression analysis (Bangma et al., Br J Urol 1997 , 79: 756-762] by PSA-F + PS
Receiver operating characteristics (ROC) plots were used to compare the specificity of the AT, PSA-F + hK2 and PSA-F + PSA-T + hK2 combinations. And the area under the curve (AUC) is calculated using the Graph ROC program, version 2.0
[Kairisto and Poola Scand, J Clin Lab Invest Suppl 1995, 2
22: 43-60].

Results Equimolar response of free and complexed hK2 in hK2 studies From studies of equimolar reactivity of hK2, compared to free hK2, complexed hK2 with ACT or PCI was almost equally well was detected. In the case of hK2-ACT, the response had a negative bias of 6%, whereas in the case of the complex of hK2 and PCI, there was no recognizable response effect compared to the case of the same amount of free hK2 alone. Was.

Results from SP1 Table 1 shows the median of each, as well as the lower and upper quartiles. All single parameters measured distinguished the BPH and PCa groups with high statistical significance. All medians in the PCa group were increased compared to those in the BPH group: 156% for PSA-T, PS
75% for AF and 192% for hK2. The six calculated ratios were also examined for their discriminating ability. Among them, PSA-F / PSA-T, PSA-F / PSA-ACT, hK2 / PSA-F, and combinations of these ratios (hK2 / F) × (T / F) and hK2 × PSA-T / PSA- F distinguished the two groups with high statistical significance. The median percentage of free PSA in the BPH group was
82% higher than in the PCa group. The ratio of hK2 to PSA-F in the PCa group was BP
It was 92% higher than in the H group. On the other hand, the combination of the two ratios in the PCa group was 234% higher than in the BPH group. In contrast, the ratio of hK2 to PSA-T was not significantly different between the BPH and PCa groups.

In SP1 (patient group without any restriction by total PSA), hK2 was significantly different (p <0.000
Correlation with PSA-T, PSA-F and PSA-ACT in 1). In the BPH group, the corresponding correlation coefficients (r) were 0.63, 0.77 and 0.57, respectively, and in the PCa group they were 0.73, 0.79 and 0.66.

Results from SP2 Table 2 shows the variables PSA-T, PSA-F, PSA-ACT and hK measured in the PCa and BPH groups.
2, and the ratio PSA-F / PSA-T, hK2 / PSA-T, hK2 / PSA-F, PSA-F / PSA-ACT, hK2 × PS
Median values for AT / PSA-F and (hK2 / PSA-F) x (PSA-T / PSA-F), and lower and upper quartiles are shown. In calculating these ratios, hK2 below the analytical detection limit
Patients who showed concentrations (PCa group n = 1 and BPH group n = 1) had a concentration (0.0
1 μg / L). The PSA-T values in the PCa group and the BPH group were similar,
And 5.6 μg / L. Statistical comparison between the two groups (Mann-Whitney) showed that only two single parameters, PSA-F and hK2, were significantly different. All of the tested ratios except for hK2 / PSA-T distinguished the two groups with high significance.

FIGS. 1 a-c and Tables 3 and 4 show single parameters and various calculated ratios,
And (PSA-F, PSA-T), (hK2, PSA-F) and (hK2, PSA-F, PSA
-T) shows the results from the receiver operating characteristic (ROC) analysis performed on the combination (expressed as c []). Table 3 shows the area under the curve (AUC) and plotted them as PSA-T
It was compared with AUC alone or in the ratio PSF-F, PSA-T. All AUCs of said ratio,
And all AUC of the combination by the logical regression analysis improved significantly with respect to the AUC of PSA-T. When those AUCs were compared with those of PSA-F / PSA-T, only the AUC of the combination of (hK2, PSA-F) and (hK2, PSA-F, PSA-T) by logical regression analysis was significantly significant. Improved.

Table 4 compares the performance of each parameter analyzed with the performance of PSA-T and PSA-F / PSA-T at a given sensitivity and specificity level. At a sensitivity level of 90%, all ratios analyzed and combinations by logical regression analysis significantly improved the specificity level compared to that obtained from PSA-T. At that sensitivity level, the specificity (7%) obtained from PSA-T was increased to 41% for hK2 / PSA-F and 63% for c [hK2, PSA-F]. The values at sensitivity level 80% are 23% (PSA-T), 63% (hK2 / PSA-F) and 74%
(c [hK2, PSA-F]). (hK2 / PSA-F) × (PSA-T / PSA-F) and c (hK2, PSA-F,
PSA-T] performed somewhat better and the results were further accentuated at the 70% sensitivity level. On the other hand, comparing the specificity obtained with that of the ratio PSA-F / PSA-T,
The parameters tested for which the specificity was significantly improved were those containing hK2 (two ratios containing hK2, as well as 2 by logical regression analysis containing hK2).
Combinations). c [hK2, F] showed the following improvements over PSA-F / PSA-T: sensitivity from 90% to 27% to 63%, 80% from 52% to 74%, and 70% From 67% to 80%
%.

Analyzing the ROC curve from the other side, ie, regarding the increase in sensitivity at high specificity levels, the analyzed parameters containing hK2 and PSA-F generally show that
A significant improvement was observed compared to PSA-T. For example, at a specificity level of 90%, PSA-T
Was 16%, but for hK2 / PSA-F it was 56%, (hK2 / PSA-F) × (PSA-T / PSA-F
) Increased to 64%, and c [hK2, F, T] increased to 64%. At a specificity level of 80%, the corresponding percentage was increased from 36% to 60%, to 76%, and to 76%, respectively. Compared to the sensitivity obtained with PSA-F / PSA-T, the ratio (hK2 / PSA-F) × (PSA-T / PSA-F)
, And c [hK2, PSA-F, PSA-T] were the only parameters that were significantly improved (at specificity levels of 90% and 95%).

In ROC analysis based on the use of combinations by ratio or logical regression analysis, PSA-
When T was replaced with PSA-ACT, its performance was virtually unchanged (data not shown). Adding PSA-ACT to c [hK2, PSA-F, PSA-T] did not improve its performance (data not shown). This clearly shows that replacing PSA-T with PSA-ACT is sufficiently feasible to maintain, but does not significantly improve differential diagnosis. In addition, PSA-T by the combination of H117 / H50 monoclonal antibody is PSA and
As we detect the total amount of hK2, we obtained a "true" PSA-T concentration by subtracting the hK2 concentration from that concentration and examined whether this would have a significant effect on the ROC analysis. This did not show a significant result.

In SP2, which includes only patients with a total PSA range of 3-10 μg / L, hK2 correlates with high significance (p <0.0001) with PSA-T, PSA-F and PSA-ACT in the BPH group However, their correlation coefficients (r) were not significant, being 0.43, 0.56 and 0.29, respectively. The corresponding correlation coefficients in the PCa group were 0.22, 0.66 and 0.05, respectively, and only the correlation coefficient between hK2 and PSA-F was statistically significant (p = 0.0003).

SP3 Results Of the single parameters measured in SP3, the screening substances PSA-T and hK2 were
High significance distinguished cancer from non-cancer (Table 5). The ratio PSA-F / PSA-T, which also contains both PSA-F and PSA-T as components, as well as the ratio of the two containing hK, was identified. However, no discrimination was made between the ratios hK2 / PSA-T (p = 0.12) and hK2 / PSA-F (p = 0.056). As shown in Table 6, limiting the analysis to the PSA-T range of 3-10 μg / L, ie, the intermediate region in the diagnosis of PSa for PCa, the following parameters will distinguish between PCa and benign cases: PSA- F, the ratio PSA-F / PSA-T, and all those containing PSA-F as an ingredient
hK2 ratio. PSA-T, hK2 and ratio hK2 / PSA-F could not distinguish between cancer and non-cancer cases.

If the analysis included all benign cases (r = 0.336) or cancer cases (r = 0.402),
The measured hK2 concentration was significantly correlated with the PSA-T concentration, but the correlation coefficient was not significant. No significant relationship was found between hK2 concentration and PSA-F concentration. In the diagnostic intermediate region, PSA-T 3 -10 μg / L, the significant correlation between hK2 and PSA-T is
Lost in both cancer and benign cases.

FIGS. 2 a-c show the following ROCs for subjects in the range of PSA-T 3 -10 μg / L in SP3
AUCs by curves were shown and compared: three single parameters, three ratios, two of which were related to hK2, and by logical regression analysis (PSA-T, PSA-F,
hK2), (PSA-F, hK2) and (PSA-F, PSA-T). A significant improvement in AUC over PSA-T AUC was observed in PSA-F / PSA-T, c [T, F] and c [hK2, F, T]. The ratio (hK2 / PSA-F) × (PSA-T / PSA-F) showed a p-value just above the 0.05 limit. PSA-F / PSA-T showed no improvement compared to AUC.

Table 8 compares the diagnostic specificity and sensitivity from the ROC analysis (FIG. 2) for the different parameters. Statistically significant increase in specificity at sensitivity 90, 80 and 70% for the ratio PSA-F / PSA-T (and c [T, F]) (range + 4% to + 25%)
Was observed in all parameters containing at least hK2 and PSA-F compared to that obtained with total PSA alone. A similar statistically significant increase in sensitivity was observed at particularly high levels of specificity. Particularly powerful here is the formula (h
K2 / PSA-F) x (PSA-T / PSA-F), which is a PSA-T
Showed an increase in specificity (range + 19% to + 29%). These are PSA-F / PS
The sensitivity was much higher than that obtained with AT (but this material did not show statistical significance).

Table 9 shows the results from a statistical comparison of the two groups of cancers based on pathological examination of biopsied tissue specimens. The Gleason score is the best established factor (predictive marker) to predict the pathological potential of a tumor. Nearly undifferentiated tumors (high Gleason score) are aggressive diseases, while well differentiated tumors (low Gleason score) have an indolent course (Cookson et al., J Urol
1997, 157: 559). In comparisons involving all 80 cancers, many of the parameters analyzed significantly distinguished the Gleason 4-6 and Gleason 7-8 groups (most notably PSA-T, PSA-F / PSA -T, hK2 and hK2 x PSA-T / PSA-F). In the clinically interesting PSA-T range 3-10 μg / L (where the probability of organ-restricted benign tumors is still high), the ratios hK2 / PSA-F and (hK2 / PSA-F) × (PSA-T / Only PSA-F) is a factor that statistically significantly distinguishes the two groups, and hK2 and hK2 × PSA-T / PSA-F showed a positive tendency. However, in this PSA-T range, both PSA-T and PSA-F / PSA-T clearly lost discrimination ability.

Discussion and Conclusion In this research study, the present inventors have established a methodological strategy for the sensitive and specific immunological quantification of hK2 in the blood circulation. The lower detection limit of this test is achieved in a carefully optimized test design by a sandwich reagent with excellent technical performance (especially high affinity constant). The good specificity of the test, especially with regard to cross-reactivity with PSA, is mainly due to the scavenger step, ie, the presence of three excess PSA-specific monoclonal antibodies in the PSA or PSA complex in a sandwich (capture / detection) reaction. Achieved due to reduced involvement. The present inventors have also established that this hK2-specific immunoassay behaves in a near equimolar fashion for the two most prominent serpin inhibitors, ACT and the hK2 complex with PCT.

From this study, it was established that hK2 concentration in the population studied correlated with PSA-T and PSA-F, but that this correlation was in a general fashion and had a non-significant correlation coefficient. . Limiting the analysis to a range of clinically interesting PSAs below 10 (where it is still likely to find prostate cancer restricted to the prostate), the correlation will be weaker or completely abolished. These observations suggest that hK2
Support the notion that it is an independent analytical variable that may provide different clinical information compared to PSA-F and PSA-T.

Although hK2 itself, like PSA, could be argued to be an independent marker of prostate cancer, the main conclusion of this study is that the most useful clinical information by specific quantification of hK2 Is found when the specific quantification of hK2 is combined with the measurement of certain forms of PSA, especially the free form of PSA. This is largely the result of limiting the analysis to patients who still have moderately elevated PSA-T concentrations, in which case the tumor is organ-restricted or localized and treatment for curative purposes is Would be eligible for

In a study population 2 (SP2) consisting of men with a median age of 70 to 71 years, presenting with clinical symptoms (due to PCa or BPH) and having a total PSA of 3-10 μg / L, the ROC analysis Thus, it can be seen that PSA-T and hK2 as single parameters hardly discriminate between PCa and BPH. However, various ratios, especially for example the ratio hK2 / PSA-F or its combination with PSA-T (multiply) or PSA-F / PSA-T (divide), are diagnostically specific at high sensitivity levels. In both gender and diagnostic sensitivity at high specificity levels
More often than not significantly more recently introduced ratio PSA-F / PSA-T compared to AT alone
Showed a significant improvement as compared to. Interestingly, the pre-calculated hK2,
By investigating those same parameters in the combined ROC analysis by logical regression analysis instead of the ROC analysis based on the ratio of PSA-F (and PSA-T),
Diagnostic performance has been substantially improved.

Similar results were obtained in Study Population 3 (SP3), a selected population of younger asymptomatic men. The important difference was that PSA-F / PSA-T did not significantly improve the results.

Significant observations on the cancers identified in the selection process were noted by comparison of the biopsy samples to the Gleason score. When the total PSA range was 3-10 μg / L, PSA-T, PSA-F and PSA-F / PSA-T could not discriminate between Gleason score groups 4-6 and 7-8, but hK2 / PSA-F and (hK2 / PSA-F) x (PSA-T / PSA-F) significantly distinguished those groups (p <0.05). This observation supports that measurement of hK2 may provide a predictive indicator of the potential for aggressive tumors in the future.

The method of the present invention can be performed in various embodiments, a few examples of which are disclosed herein. Other aspects will occur to those skilled in the art without departing from the spirit of the invention.
It will be clear that it exists. The described embodiments are exemplary and not limiting.

Table 1. Study population 1: PSA-T, PSA-F, hK2, PSA-ACT, PSA-F / PSA-T, hK2 / PSA- in patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) T, hK2 / PSA-F,
PSA-F / PSA-ACT, median serum levels of hK2 x PSA-T / PSA-F and (hK2 / PSA-F) x (PSA-T / PSA-F), around 25% and 75% Order. Non-parametric Mann Whitney
Statistical significance between groups is tested by -U test (* indicates statistical significance, i.e. p-value <0.05).

[Table 1]

Table 2. Study population 2: Patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa)
PSA-T, PSA-F, hK2, PSA-ACT, PSA-F / in patients with a PSA range of 3-10 μg / L
PSA-T, hK2 / PSA-T, hK2 / PSA-F, PSA-F / PSA-ACT, hK2 × PSA-T / PSA-F and (hK2 / P
Median serum levels of (SA-F) x (PSA-T / PSA-F), 25% and 75%. A non-parametric Mann Whitney-U test is used to test for statistically significant differences between groups (
* Indicates statistical significance, i.e., p-value <0.05).

[Table 2]

Table 3. Study population 2: Area under the ROC curve (AUC). PSA-T range 3-10 μg / L, n (PCa) = 2
5, n (BPH) = 90. The AUC that was statistically significantly improved compared to T or F / T is indicated by * (p <0.05).

[Table 3]

Table 4. Study Population 2: Comparison of ROC curves at certain sensitivity or specificity levels. PSA-
T range 3-10 μg / L, n (PCa) = 25, n (BPH) = 90. Sensitivity or specificity with statistically significant improvement over T or F / T are indicated by * and +, respectively (p <0.05).

[Table 4]

Table 5. Study Group 3: Prostate cancer participants in the Gothenburg screening study (cancer)
, And PSA-T, PSA-F, PSA-F / PSA-T, hK2, hK2 / in patients without (benign)
Median serum levels of PSA-T, hK2 / PSA-F, hK2 × PSA-T / PSA-F and (hK2 / PSA-F) × (PSA-T / PSA-F), around 25% and About 75%. Non-parametric Mann Whitn
Statistical significance between groups is tested by ey-U test (* indicates statistical significance, ie p-value <0.05).

[Table 5]

Table 6. Study Group 3: Prostate cancer participants in the Gothenburg screening study (cancer)
And in patients without (benign) total PSA range of 3-10 μg / L
T, PSA-F, PSA-F / PSA-T, hK2, hK2 / PSA-T, hK2 / PSA-F, hK2 × PSA-T / PSA-F and
Median serum levels of (hK2 / PSA-F) x (PSA-T / PSA-F), 25% and 75%. Statistical significance between groups is tested by non-parametric Mann Whitney-U test (* indicates statistical significance, ie p-value <0.05).

[Table 6]

Table 7. Study population 3: Area under the ROC curve (AUC). PSA-T range 3-10 μg / L, n (PCa) = 4
1, n (non-cancer) = 204. The AUC that was statistically significantly improved compared to T or F / T is indicated by * (p <0.05).

[Table 7]

Table 8. Study Population 3: Comparison of ROC curves at certain sensitivity or specificity levels. PSA-
T range 3-10 μg / L, n (PCa) = 41, n (non-cancer) = 204. Sensitivity or specificity with statistically significant improvement over T or F / T are indicated by * and +, respectively (p <0.05).

[Table 8]

Table 9. Study Population 3: Prostate cancer with low (4-6) and high (7-8) Gleason grade (P
Ca) patient comparison. Low and high non-parametric Mann Whitney-U test
Test for statistical significance between patients with Gleason score (* indicates statistical significance, ie p-value <0.05).

[Table 9]

[Brief description of the drawings]

FIG. 1A-1C shows PSA-T, PSA-F, hK2, PSA-F / PSA-T, and hK in Study Group 2 (SP2).
2 / PSA-F, (hK2 / PSA-F) × (PSA-T / PSA-F), c [PSA-T, PSA-F], c [hK2, PSA-F], and c [hK2, PSA -F, PSA-T] shows the ROC curve.

FIG. 2A-2C shows PSA-T, PSA-F, hK2, PSA-F / PSA-T, and hK in Study Group 3 (SP3).
2 / PSA-F, (hK2 / PSA-F) × (PSA-T / PSA-F), c [PSA-T, PSA-F], c [hK2, PSA-F], and c [hK2, PSA -F, PSA-T] shows the ROC curve.

[Procedure amendment]

[Submission date] September 5, 2000 (2009.5)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventors Lilyya, Hans Sweden, S-239 35 Scansel, Gesellensgatan 17 (72) Inventors Loebgren, Timo Finland, Efien-20100 Turku, Irio Piston Katsu 2 Ho 155 (72) Inventor Petterson, Kim Finland, EFIEN-20810 Turku, Thirentekyankatsu 14 A 20 (72) Inventor Pironen, Timo Finland, EFIEN-20540 TURKU, Frant Sinkatu 4 Aes. 8 (72) Inventor Wrecker, Franz Zeher-5016, Switzerland Oberel Rinsbach, Elzbergbeek 14

Claims (19)

[Claims] Claims 1. Patients with prostate cancer (PCa) are treated with benign prostatic hyperplasia (B
A method for distinguishing from patients with PH) and healthy males without PCa, comprising human glandular kallikrein 2 (hK2) and prostate specific antigen (PSA) in the body fluid of an individual.
Using a combination of hK2 and PSA as a marker to distinguish PCa patients from BPH patients and other non-PCa patients, where PSA is free PSA, complex The above method, wherein the term means generalized PSA or total PSA. 2. The marker according to claim 1, wherein said marker is a ratio hK2 / F or a ratio F / hK2, wherein F is the concentration of free PSA or a ratio hK2 / F or a ratio F / hK2 and a quantity representing the PSA concentration. The method of claim 1, wherein the product is: 3. The method of claim 2, wherein the marker is the product of hK2 / F and T ((hK2 × T) / F), where T is the concentration of total PSA. Method. 4. The method according to claim 1, wherein the marker is the product of hK2 / F and T / F ((hK2 / F) × T / F), wherein T is the concentration of total PSA. The method of claim 2. 5. The method according to claim 2, wherein the cut-off limit for the ratio is selected in different ways depending on whether high detection sensitivity or high detection specificity is desired. the method of. 6. The method according to claim 1, wherein the combination of hK2 and PSA is a combination by logical regression analysis, wherein PSA means free PSA, complexed PSA or total PSA. the method of. 7. The method of claim 6, wherein the combination by logical regression analysis is c [hK2, F], wherein F is free PSA. 8. The combination according to the logical regression analysis is c [hK2, F, T].
7. The method of claim 6, wherein F is free PSA and T is total PSA. 9. The method according to claim 1, wherein the body fluid of the individual is serum, plasma or whole blood. 10. The method according to claim 1, wherein the total PSA of the individual ranges from 1 to 20 μg / L. 11. The method of claim 10, wherein said individual has a total PSA in the range of 3-10 μg / L. 12. A method for distinguishing a patient having painless or low-progression prostate cancer from a patient having aggressive progression prostate cancer, comprising the steps of: human glandular kallikrein 2 (hK2) in a body fluid of an individual HK2 and PSA as markers to distinguish patients with painless or low-progression prostate cancer from patients with aggressive prostate cancer, including quantifying prostate cancer-specific antigen (PSA) levels. Wherein said PSA means free PSA, complexed PSA or total PSA. 13. The marker according to claim 1, wherein said marker is a ratio hK2 / F or a ratio F / hK2, wherein F is the concentration of free PSA, or a ratio hK2 / F or a ratio F / hK2 and a quantity representing the PSA concentration. 13. The method of claim 12, wherein the product is: 14. The marker is a product of hK2 / F and T / F ((hK2 / F) × T / F), wherein T is the concentration of total PSA. 14. The method of claim 13. 15. The method according to claim 13, wherein the cut-off limit for the ratio is selected in different ways depending on whether high detection sensitivity or high detection specificity is desired. . 16. The method according to claim 12, wherein the combination of hK2 and PSA is a combination by logical regression analysis, wherein PSA means free PSA, complexed PSA or total PSA. the method of. 17. The combination according to the logical regression analysis is c [hK2, F].
17. The method of claim 16, wherein F is free PSA. 18. The method according to claim 16, wherein the combination by the logical regression analysis is c [hK2, F, T], wherein F is free PSA and T is total PSA.
the method of. 19. The method according to claim 12, wherein the total PSA of the PCa patient ranges from 1 to 20 μg / L, preferably from 3 to 10 μg / L.