RU2740382C1 - Method for detecting patients with high risk of developing prostate cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to medical biochemistry and oncology, and aims at detecting patients with a high risk of developing prostate cancer. Method comprises producing a sample of tissue (biopsy material) of prostate, preparing homogenate from biopsy material with subsequent determination of such parameters, as 1) ACE activity in interaction with two artificial substrates of ACE - ZPHL and HHL with calculation of catalytic characteristic of ACE, characterized by ratio of hydrolysis rates of these two substrates ZPHL and HHL; and 2) the ACE immunoreactivity coefficient, characterized by the ratio of the amount of ACE which has attached 9B9 antibody, to the activity of the given ACE when reacting with the ZPHL substrate. If observing an increase in the values of each of the obtained parameters by 1.5 times or more as compared to control values characteristic of normal prostate tissue, a high risk of developing prostate cancer is stated.

EFFECT: use of the invention allows to identify patients with a high risk of developing prostate cancer on the basis of phenotyping ACE of prostate tissues obtained in transrectal biopsy under the control of ultrasonic examination, requiring more close observation of the attending physician with the probability of prescribing additional studies to exclude the early stage of prostate cancer.

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Description

The technical field to which the invention relates

The present invention relates to the field of medical biochemistry and can be used to identify patients at increased risk of developing prostate cancer.

State of the art

Prostate cancer (PCa) is the second most common cause of death from malignant neoplasms in men in the world - each year, prostate cancer is diagnosed in more than one million men, about a third of whom are expected to die from this disease [Ferlay J, Soerjomataram I, Ervik M, et al. GLOBOSCAN 2013 v.1.0. Cancer Incidence and mortality worldwide. IARCCancerBase no. eleven.]. Determination of an elevated level of prostate-specific antigen (PSA) in serum is the most commonly used marker for identifying patients with prostate cancer. However, the analysis for PSA (including its modifications - free PSA, the ratio of free PSA to total PSA) has low specificity (a significant percentage of false-positive results), since an increase in this parameter in the blood can occur in prostatic hyperplasia and acute prostatitis [Chatterjee SK and Zetter BR (2005) Cancer biomarkers: knowing the present and predicting the future. Future Oncology 1: 37-50; Ahmed HU (2014) Melbourne consensus - noble but misguided. Nature Reviews (Urology) 11: 250-251) and even when manipulating the prostate (prostate massage); Park SC, Shin YS, Zhang LT et al. (2015) Prospective investigation of change in the prostate-specific antigens after various urological procedures. Clin Intervent Aging 10: 1213-1218.].

As a result of the low specificity of the serum PSA test, only one third of patients referred for a prostate biopsy with a PSA level of 2 to 10 ng / ml (the so-called "gray" zone) histologically confirmed the presence of prostate cancer [Catalona WJ, Partin AW, Slawin KM, et al. (1998) Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter clinical trial. JAMA 279: 1542-1547.].

Diagnosis of prostate cancer using markers such as pro-PSA or PSA3 is based on different identification principles and has greater diagnostic accuracy than PSA, but due to the higher cost (and low prevalence), these methods are used only in a limited number of laboratories [Fillela X and Foj L ( 2015) Emerging biomarkers in the detection and prognosis of prostate cancer. Clin Chem Lab Med 53: 963-973.].

The histochemical study of prostate biopsies is currently the gold standard for confirming the diagnosis, showing morphological changes in prostate tissue. Diagnosis of suspected prostate cancer is carried out using multifocal puncture biopsy under the control of transrectal ultrasound (TRUS). A significant disadvantage of this method is a rather high percentage of false negative conclusions (up to 20-30%). In particular, patients were identified in whom the initial biopsy did not reveal morphological signs of prostate cancer (with the initial conclusions: ASAP or atypical small acinar proliferation, HPIN or high-grade prostatic intraepithelial neoplasia), but repeated biopsies confirmed the presence of a malignant tumor.

Thus, it seems extremely necessary to identify new markers of prostate cancer with higher sensitivity and specificity. A technical problem is the identification of patients with an increased risk of developing prostate cancer in the early stages of the disease, characterized by the absence of clinical manifestations of symptoms of the disease.

Disclosure of invention

The technical result of the claimed invention is to develop a method for identifying patients at risk of developing prostate cancer in the early stages of the disease, which, while maintaining a high degree of sensitivity (comparable to the PSA sensitivity level), has a higher degree of specificity. This method allows diagnosing biochemical changes in the prostate tissue before the appearance of morphological changes, which makes it possible to make a diagnosis at an earlier stage and increases the accuracy of the diagnosis.

The technical result is achieved by implementing a method for identifying patients with an increased risk of developing prostate cancer, including obtaining a tissue sample (biopsy) of the prostate, preparing a homogenate from a biopsy with the subsequent determination of the following parameters characterizing the ACE phenotype of the prostate:

- ACE activity when interacting with two artificial ACE substrates - ZPHL and HHL with the calculation of the catalytic characteristics of ACE, characterized by the ratio of the rates of hydrolysis of these two substrates ZPHL and HHL;

- coefficient of ACE immunoreactivity, characterized by the ratio of the amount of ACE that has attached the 9B9 antibody to the activity of this ACE when interacting with the ZPHL substrate;

and with an increase in the values of each of the obtained parameters by 1.5 times or more, compared with the control values typical for normal prostate tissue (i.e., an increase in the ratio of the rates of hydrolysis of two substrates by 50% or more (Fig.1B), and an increase in the ACE immunoreactivity coefficient by 50% or more (Fig. 2B)), the patient is referred to a group with an increased risk of developing prostate cancer.

Thus, the technical result is achieved through the implementation of a method that allows to identify patients with biochemical characteristics of the angiotensin-converting enzyme (ACE), characteristic of prostate cancer, based on the results of ACE testing in homogenates of prostate tissue biopsies. The method is based on the simultaneous determination of two parameters: 1) the activity of ACE when interacting with two artificial ACE substrates - ZPHL and HHL with the calculation of the catalytic characteristics of ACE, namely, the ratio of the rates of hydrolysis of these two substrates ZPHL and HHL; 2) the coefficient of ACE immunoreactivity, which is determined by the ratio of the amount of ACE that has attached the 9B9 antibody to the activity of this ACE when interacting with the ZPHL substrate. These parameters may indicate a high risk of developing prostate cancer. At the same time, the accuracy of determining a high risk of developing prostate cancer increases if the ACE activity is reduced by more than 2 times (Fig. 1A) in combination with a 50% or more increase in each of the parameters - the ratio of the rates of hydrolysis of two substrates (Fig. 1B); 2) and the coefficient of ACE immunoreactivity (Fig. 2B).

It is known that glandular epithelial cells of the prostate express significant amounts of angiotensin-converting enzyme (ACE), therefore, the concentration of ACE in seminal fluid is 50 times greater than in blood plasma [Krassnigg F, Niederhauser H, Fink E, Frick J, Schill WB (1989 ) Angiotensin converting enzyme inhuman seminal plasma is synthesized by the testis, epididymis and prostate. Int J Androl 12: 22-28; Nikolaeva MA, Balyasnikova IV, Alexinskaya MA, et al. (2006) Testicular isoform of angiotensin I-converting enzyme (ACE, CD143) on the surface of human spermatozoa: Testicular isoform of angiotensin I-converting enzyme (ACE, CD 143) on the surface of human spermatozoa: revelation and quantification using monoclonal antibodies ... Am J Reprod Immunol 55: 54-68.]. Replacement of highly specialized cells of the epithelium of the prostate with tumor cells leads to a significant decrease in ACE activity in the prostate tissue [Yokoyama M, Hiwada K, Kokubu T, Takaha M, Takeuchi M (1980) Angiotensin-converting enzyme in human prostate. Clin Chim Acta 100: 253-258.]. Therefore, phenotyping of ACE in biopsy specimens of prostate tissue may have important clinical implications for identifying patients at increased risk of developing prostate cancer.

Our earlier studies of the binding of a panel of monoclonal antibodies (mAbs) to various conformational epitopes on the surface of human ACE with ACEs from different human tissues (ACE conformational fingerprint) showed that binding is tissue-specific, mainly due to tissue-specific glycosylation of ACE [Danilov SM, Balyasnikova IB, Danilova AS, et al. (2010) Conformational fingerprinting of the angiotensin-converting enzyme (ACE): Application in sarcoidosis. J Proteome Res 9: 5782-5793; Danilov SM, Tikhomirova VE, Metzger R, et al. (2018a) ACE phenotyping in Gaucher disease. Mol Genet Metab 123: 501-510.].

Moreover, the results were obtained showing significant differences in the conformational fingerprint of the ACE of the prostate from the ACE of the lung [Kryukova OV, Tikhomirova VE, Golukhova EZ, et al. (2015) Tissue Specificity of human angiotensin I-converting enzyme. PLoS One 10: e0143455], which opens up the theoretical possibility of using the appearance of prostate ACE in the blood as an early marker of prostate cancer. Due to the fact that the concentration of ACE in semen exceeds the concentration of ACE in plasma by only 50 times, compared with the excess of PSA in semen over plasma by 1,000,000 times [Lovgren J, Valtonen-Andre C, Marsal K, Lilja H, Lundwall A (1999) Measurement of prostate-specific antigen and human glandular kallikrein 2 in different body fluids. J Androl 20: 348-355], it can be assumed that the frequency of false-positive tests in the detection of ACE in the prostate in the blood will be significantly less than in the determination of PSA in the blood.

At the first stage of our studies (phenotyping of ACE in early diagnosis of prostate cancer), ACE analysis was carried out in prostate tissue samples obtained by biopsy from patients with prostate hyperplasia, in patients with prostate cancer, as well as in autopsy material of prostate tissue, as a sample tissues from the normal prostate gland.

The results of the studies showed that: firstly, the catalytic characteristic of ACE (the ratio of the relative rate of hydrolysis of one artificial substrate of ACE (ZPHL) to that of another artificial substrate (HHL) - ZPHL / HHL ratio was significantly increased in patients with prostate cancer. Second, the conformational fingerprint of ACE from the tissue of a patient with prostate cancer was significantly different from that for patients with prostatic hyperplasia or ACE of the prostate tissue in apparently healthy individuals.

The study identified a group of patients in whom the initial biopsy did not reveal morphological signs of prostate cancer, but repeated biopsies confirmed the presence of a malignant tumor. The initial histological findings included: ASAP or atypical small acinar proliferation (31-40% risk of PCa with repeat biopsy), HPIN or high-grade prostatic intraepithelial neoplasia detected in more than three biopsies (30% risk of PCa with repeat biopsy). These conclusions are indications for a second biopsy.

Taking into account the revealed features of ACE in the prostate tissue in the presence of PCa, it was proposed to analyze the prostate tissue for ACE to reduce the number of false-negative primary systemic and targeted biopsies. Thus, even with a morphological conclusion about the absence of PCa in a patient, but with significant signs of PCa according to the immunochemical analysis of prostate tissue for ACE, the appointment of a repeated targeted biopsy from an area with a pathological ACE phenotype can improve the primary diagnosis of PCa at earlier stages. This opportunity is important for improving the clinical prognosis and survival of this group of patients.

Thus, phenotyping of ACE in biopsy specimens of prostate tissue promises to be an effective approach for identifying patients at increased risk of developing prostate cancer.

Brief Description of Drawings

The invention is illustrated by drawings, which show the results of the studies performed: Fig. 1 shows a scheme for determining the activity of ACE in homogenates of prostate tissue obtained as a result of transrectal biopsy of the prostate. A) ACE activity with ZPHL substrate; B) ACE activity with the HHL substrate: C) The ratio of the rates of hydrolysis of the ZPHL substrate to that for the HHL substrate (ZPHL / HHL ratio); D) Determination of the concentration of PSA (Prostate-Specific Antigen) in the blood plasma of the tested patients.

Quantitatively, data are presented as% of values for homogenates from tissues of control prostates (2 to 6 controls for each determination).

FIG. 2. A) demonstrated the activity of ACE in the tissues of the prostate with the substrate ZPHL; B) ACE precipitation by 9B9 antibody; C) The ratio of the immunoreactive protein ACE (with the antibody 9B9) to the activity of ACE in the same homogenate (that is, the ratio B \ A); D) Determination of PSA concentration. Quantitative data are presented as% of values for homogenates from control prostates. Column coloration as in FIG. 1. Bars that are less than 50% of normal prostate tissue values are shown in black.

Implementation of the invention

The inventive method, based on the study of the ACE phenotype in homogenates of prostate tissue, includes the following steps: a) obtaining samples of prostate tissue using transrectal biopsy under the control of ultrasound; b) preparation of a homogenate from a biopsy; c) determination of the following parameters characterizing the ACE phenotype of the prostate of a given patient: 1) determination of ACE activity in interaction with two artificial ACE substrates - ZPHL and HHL with the calculation of the catalytic characteristics of ACE (the ratio of the rates of hydrolysis of these two substrates ZPHL and HHL) and 2) calculation of the coefficient of immunoreactivity ACE (the ratio of the amount of ACE that has attached the 9B9 antibody to the activity of this ACE when interacting with the ZPHL substrate).

A decrease in ACE activity in prostate tissue in patients with prostate cancer is known from the prior art. However, the conducted studies have shown the possibility of using a marker characterizing the nativeness of the catalytic characteristics of ACE using artificial substrates ZPHL and HHL (ZPHL / HHL ratio) for early diagnosis of prostate cancer.

It was found that the ratio of the amount of immunoreactive protein ACE in the homogenate of prostate tissue (determined using the monoclonal antibody 9B9) to the activity of ACE sharply decreases precisely in homogenates of biopsies from prostate cancer tissue, but not from prostate tissue with signs of benign hyperplasia.

Phenotyping of ACE on 35 prostate tissue samples obtained from 18 patients with prostate hyperplasia, 6 patients with prostate cancer and 11 control prostate tissues (autopsy material from apparently healthy individuals) showed that:

a) A sharp drop in ACE activity (more than 3 times) was observed in all 6 cases of prostate cancer (Fig. 1A and B). At the same time, a significant (more than 50%) increase in the ratio of the rates of hydrolysis of the two substrates (ZPHL / HHL ratio) was observed in 4 out of 6 patients (Fig. 1B). At the same time, a positive test result for blood PSA was observed only in two patients of this group (out of 6 patients with histologically verified prostate cancer) (Fig. 1D).

b) A significant (more than 50%) increase in the ratio of immunoreactive protein ACE (determined using mAb 9B9) to ACE activity in the same homogenate (Fig. 2B) was observed in all 6 cases of prostate cancer (Fig. 2B). In this case, a positive test for blood PSA was observed only in two patients of this group out of 6 (Fig. 2D).

c) It should be noted that some patients with prostatic hyperplasia (verified histologically) had the same quantitative characteristics of the ACE phenotype as in patients with prostate cancer (an increase in the ratio of the rates of hydrolysis of two substrates (ZPHL / HHL ratio) - Fig. 1B, and the ratio of the immunoreactive ACE protein to ACE activity in the same homogenate (Fig. 2B).

Studies have shown that phenotyping of ACE makes it possible to identify patients in whom, along with hyperplasia, degeneration of the prostate tissue begins (most likely, as clonal growth of tumor cells), which does not yet change the prostate tissue histologically, but biochemical changes in ACE (introduced by tumor cells) are already beginning manifest.

Thus, the task of developing an easy-to-use and relatively inexpensive method for identifying patients with an increased risk of developing prostate cancer was solved by using a new technology - phenotyping of ACE on homogenates of prostate tissue obtained as a result of transrectal biopsy under ultrasound guidance.

Below is a more detailed description of the research and implementation of the invention, demonstrating the achievement of the technical result.

1. Criteria for selecting patients with prostate pathology for analysis.

We examined 24 patients who were followed up in the urology department of the Moscow State University Medical Center. Lomonosov in the period from January 2017 to October 2018. The inclusion criteria were suspicion of PCa and a signed informed consent to participate in the study. The selection was carried out according to the recommendations of the European Society of Urology, according to which, in case of suspected prostate cancer, the indications for prostate biopsy are: elevated PSA levels (more than 4 ng / ml) and / or suspicious results of digital rectal examination and / or suspicious results of transrectal ultrasound [Mottet N, Bellmunt J, Bolla M. et al. (2017) EAU-ESTRO-SIOG Guidelines on prostate cancer. Part 1: Screening, diagnosis, and local treatment with curative intent. European Urology 71 (2017) 618-629.].

The diagnosis of hyperplasia or prostate cancer was established histologically according to generally accepted criteria.

Based on the results of histological examination of biopsies, 2 groups of patients were formed: with identified benign prostatic hyperplasia (N = 18) and with prostate cancer (N = 6). In addition, a separate group consisted of control samples of prostate tissue (N = 11) obtained by autopsy of patients with fatal outcomes as a result of accidents, without signs of morphological changes in the prostate.

2. Conducting a biopsy of the prostate.

24 patients were recruited for prostate biopsy under the guidance of transrectal ultrasound and shear wave ultrasound elastography.

All patients underwent transrectal ultrasound examination (TRUS) of the prostate and seminal vesicles using an Aixplorer apparatus (Supersonic Imagine, France) using an intracavitary transducer operating in the 3-12 MHz frequency range. TRUS of the prostate was performed using a gray-scale mode, an energy Doppler mode, and a shear wave elastography mode, which allows assessing the stiffness of the peripheral zone of the prostate. The prostate gland was divided into 12 sectors for ultrasound assessment and subsequent systemic biopsy of the prostate (each of the base, middle and apex sextants on the right and left of the gland was subdivided into lateral and medial subregions - a total of 12 sectors).

After TRUS and ultrasound elastography, all patients underwent transrectal multifocal systemic puncture biopsy of the prostate from 12 points under ultrasound guidance using a Bard Magnum biopsy gun and disposable 18G needles. In addition to systemic biopsy, targeted biopsy was performed from lesions suspected of malignancy on the basis of ultrasound and shear wave elastography. The ultrasound criteria, on the basis of which the targeted biopsy was performed, were considered: hypoechoic foci of the peripheral zone (gray-scale mode); zones of local increased blood flow with possible deformation of the vascular pattern (power Doppler mapping); rigid lesions on shear wave elastography. The foci were considered rigid, the Young's modulus of which was more than 35 kPa [Barr R.G., Cosgrove D., Brock M., Cantisani V., Correas J.M., Postema A.W., Salomon G., Tsutsumi M., Xu H.X., Dietrich C.F. WFUMB Guidelines and Recommendations on the Clinical Use of Ultrasound Elastography: Part 5. Prostate // Ultrasound Med. Biol. 2017. V. 43. No. 1.P. 27-48. Doi: 10.1016 / j.ultrasmedbio.2016.06.020.]. In the biopsy protocol, the exact localization of the suspicious lesion was noted on a diagram of the prostate, divided into sectors according to the above technique. Then, three additional columns of tissue were taken from each suspicious lesion for subsequent measurements of the enzymatic activity and immunochemical characteristics of the prostate tissue in this area.

Histological examination of biopsies was carried out according to the standard technique. The samples were fixed in 4% neutral buffered formalin for 48 h, followed by immersion in paraffin. Then hematoxylin and eosin staining was performed and the sections were examined under a microscope.

Additional columns of prostate tissue obtained for the analysis of the immunochemical characteristics of ACE were cooled to 4 ° C without immersion in formalin.

According to the results of histological examination, the morphological characteristics of all suspicious foci were analyzed and compared with the activity and immunochemical characteristics of ACE in the prostate tissue obtained from these foci.

3. Preparation of homogenates from prostate tissue samples.

All operations for the preparation of prostate tissue homogenates were performed at 4 ° C. For this, 2-3 biopsies (weighing about 3-5 mg each) or tissue of the control prostate obtained at dissection (no more than 10 mg) were homogenized in 1 ml of 0.05 M phosphate buffer, pH 7.5 containing 0.15 M NaCl , 1 μM ZnCl ₂ , (buffer A) using a Speed Mill tissue homogenizer (Analytik Jena, Jena, Germany) with a steel ball (3 = minute cycle). Then Triton X-100 was added to the homogenate to 0.025%, stirred by inversion for 1 hour and centrifuged in a microfuge for 5 min at 13000 rpm. The sediment was discarded, and the supernatant was used for further work. Protein concentration in homogenates using Bradford's reagent.

4. Determination of ACE activity

ACE activity in the supernatants of homogenates of prostate tissue biopsies was determined by the initial rates of hydrolysis of substrates Z-Phe-His-Leu (ZPHL) and Hip-His-Leu (HHL), measuring the accumulation of the reaction product His- on a Hitachi multifunctional microplate reader (Japan) Leu with ortho-phthalic aldehyde. To do this, 100 μl of 2.4 mM ZPHL and 6 mM HHL in 0.1 M phosphate buffer pH 8.3 containing 0.3 M NaCl and 1 μM ZnCl ₂ were placed in the wells of 96-well plates, 20 μl of supernatant was added homogenate in Buffer A and incubated the mixture for 60 minutes at 37 ° C in a thermostat. The final concentration of substrates in the reaction mixture is 5 mM and 2 mM, respectively. The reaction time was selected so that the conversion of the substrate did not exceed 5%. The reaction was stopped by the addition of 25 μL of 1.4 N. NaOH, then 25 μl of a solution of ortho-phthalic aldehyde (3.3 mg / ml) in methanol was added, the mixture was incubated for 10 minutes, then 25 μl of 2.1 N HCI was added. HCl. The fluorescence of the formed adduct was measured at an excitation wavelength of 370 nm and an emission of 500 nm. Background fluorescence was measured under the same conditions, but the enzyme (prostate homogenate supernatate) was added after addition of alkali. Standard fluorescence was measured in the same way as background fluorescence, but His-Leu solution was used instead of the substrate. The ratio of the rates of hydrolysis of the two substrates was calculated by dividing the average fluorescence in three wells of the plate with the ZPHL substrate by the average fluorescence in three wells with the HHL substrate.

5. Determination of the immunoreactive protein ACE (using antietl 9B9)

The amount of immunoreactive protein ACE - that is, the relative efficiency of binding of mAbs with ACE (in this case, mAb 9B9), was determined by the method of immunosorption [Kryukova OV, Tikhomirova VE, Golukhova EZ, et al. (2015) Tissue Specificity of human angiotensin I-converting enzyme. PLoS One 10: e0143455.]. For this purpose, polyclonal goat antibodies against mouse immunoglobulins (50 μl, 10 μg / ml) in 0.05 M phosphate buffer with pH 7.5 and 0.15 M NaCl were applied to a Corning plate with a high sorption capacity. Then the plate was left in a thermostat at 37 ° C for 1 hour. Then the solution was removed and the wells were washed with buffer A containing 0.05% Tween-20. The wells were supplemented with 50 μl of a solution of monoclonal antibodies (mAb) 9B9 to human ACE with a concentration of 3 μg / ml in buffer A containing 100 μg / ml BSA (in triplicates). After incubation for an hour at 37 ° C, the wells were washed again with buffer A containing 0.05% Tween-20, and 50 μl of ACE solution (in the supernatant of prostate tissue homogenate) was added and left overnight at 4 ° C. The plate was washed first 5 times with 0.05 M phosphate buffer pH 7.5 and 0.15 M NaCl containing 0.05% Tween-20. The activity of the precipitated ACE of the prostate with the 9B9 antibody was determined by the enzymatic activity of the ACE bound to the plate surface. A plate was loaded with 100 μl of a ZPHL substrate solution (2 mM) in buffer A. The reaction was carried out for one hour at 37 ° С and was stopped by adding 25 μl of 1.4 N. NaOH, then 25 μl of a solution of ortho-phthalic aldehyde (3.3 mg / ml) in methanol was added, the mixture was incubated for 10 minutes, then 25 μl of 2.1 N HCI was added. HCl. The fluorescence of the formed adduct was measured at an excitation wavelength of 370 nm and an emission wavelength of 500 nm. Background fluorescence was measured under the same conditions, but in cells where, instead of mAb 9B9, 0.05 M phosphate buffer with pH 7.5 and 0.15 M NaCl was added.

6. Statistical processing of results

Statistical processing of the data was carried out using the Microsoft Office Excel 2007 and Statistica 10.0 packages using Fisher's two-sided exact test and Pearson's% 2 test (for qualitative features), Mannie-Whitney and Kruskal-Wallis test (for quantitative features with distribution, different from normal). Differences were considered significant at p <0.05 and 95% confidence interval (CI) for OR, not including "1".

A 50% (i.e., 1.5-fold) increase in the ratio of the rates of hydrolysis of the ZPHL substrate to that for the HHL substrate (ZPHL / HHL ratio, since this parameter differs among individuals with a standard deviation of no more than 5%) was taken as prognostically significant. For the ratio of the ACE immunoreactive protein (according to 9B9) to the prognostically significant activity, we took a 50 percent (that is, one and a half-fold) increase in this parameter.

7. Evaluation of the results obtained.

Catalytic characterization of ACE of prostate tissue - specific for ACE from prostate cancer tissue

Out of 24 biopsies of prostate tissue, a sharp drop in ACE activity (more than 3 times) was observed in all 6 cases of prostate cancer (Fig. 1A and B). At the same time, a significant increase (more than 50%) in the ratio of the rates of hydrolysis of the two substrates (ZPHL / HHL ratio) was observed in 4 out of 6 patients (Fig. 1B). At the same time, a positive test for blood PSA was observed in only two patients (out of 6 patients) with histologically verified prostate cancer (Fig. 1D). From which it follows that the new catalytic characteristic of ACE of prostate tissue is a more sensitive marker of prostate cancer than a blood PSA test. It is especially interesting that among 18 patients with prostatic hyperplasia (verified histologically) in two patients (3-6 and 3-8 Fig. 1) the ACE activity in the prostate tissue for both substrates was sharply reduced (less than 50% of the control - black bars) , but at the same time the ZPHL / HHL ratio was sharply increased (more than 200% of the control) only in patient 3-8. Only a sharp decrease in ACE activity in a separate biopsy is not a prognostic sign (since the biopsy can pass not through the prostate tissue, but through, for example, the connective tissue), but a combination of a decrease in activity and an increase in the ZPHL / HHL ratio (Fig. 1B, patient 3-8, highlighted by a rectangle), signals a sharp change in the ACE microenvironment in the prostate tissue. For example, increased expression of some tumor protein, which, moreover, has the ability to bind to ACE and change its catalytic properties.

In addition to patient 3-8, an increase in the ZPHL / HHL ratio was noted in patients 4-6 and 5-1 (Fig. 1B). At the same time, patient 5-1 also showed an increase in PSA in the blood (Fig. 1B and C- marked with a rectangle). This fact adds confidence that patients with an increase in the ZPHL / HHL ratio for ACE from the prostate are a risk group (for the development of prostate cancer) and should be under the special supervision of doctors with the likelihood of additional tests (for example, immunohistochemistry for the expression of specific tumor markers).

The ratio of ACE immunoreactive protein to activity in prostate tissue - specific for ACE from prostate cancer tissue

Out of 24 prostate biopsies, a significant increase in the ratio of immunoreactive protein ACE to ACE activity was observed in all 6 cases of prostate cancer (Fig. 2B). Whereas a positive blood PSA test was noted only in two patients (out of 6 patients with histologically verified prostate cancer (Fig.2D). It follows from this that the new conformational characteristic of ACE of prostate tissue is a more sensitive marker of prostate cancer than the PSA blood test It is especially interesting that among 18 patients with prostatic hyperplasia (verified histologically) in 3 patients (3-3, 3-4 and 3-6 - Fig. 2) this ratio was sharply increased. (with an increased ratio of ACE immunoreactive protein to activity) blood PSA was also increased.

The data obtained indicate that the joint determination of two new parameters of the ACE phenotype in prostate tissues 1) the ratio of the rates of hydrolysis of the two substrates (ZPHL / HHL ratio) and 2) the ratio of the immunoreactive ACE protein to activity makes it possible to identify patients (with histologically verified prostatic hyperplasia) who represent a risk group for developing prostate cancer.

Example 1.

Patient R., 76 years old, during an outpatient examination, the PSA level was revealed in the blood. 5.6 ng / ml (norm 0-4 ng / ml), the volume of the prostate gland was 65 cm3. When performing TRUS in both lobes of the prostate gland, calcifications up to 3 mm were determined. According to the results of a transrectal biopsy of the prostate with IHC study, the patient was diagnosed with BPH. The conclusion of the pathomorphological study: “Pieces of prostate tissue with glands of various sizes and shapes with preserved basement membrane, in places - with signs of proliferation, convoluted glands, with epithelial atrophy. Stroma with areas of sclerosis, hemorrhages and signs of chronic inflammation. " However, according to the measurement of ACE activity, the patient was suspected of prostate cancer, since a decrease in ACE activity was detected by more than 2 times compared with the norm and the ratio of the rates of hydrolysis of two substrates increased by 50%, and the coefficient of ACE immunoreactivity was increased by more than 50%. ... Subsequently, due to severe symptoms of the lower genital tract (18 points on the IPSS scale), 3 months after transrectal biopsy of the prostate, the patient underwent a transurethral biopsy of the prostate gland. According to the data of the pathomorphological examination of the resected prostate tissue, the diagnosis of prostate cancer was confirmed in the patient. The patient underwent surgical treatment.

Claims (4)

A method for identifying patients with an increased risk of developing prostate cancer, including obtaining a tissue sample (biopsy) of the prostate, preparing a homogenate from a biopsy, followed by determining the following parameters characterizing the ACE phenotype of the prostate: - ACE activity when interacting with two artificial ACE substrates - ZPHL and HHL with the calculation of the catalytic characteristics of ACE, characterized by the ratio of the rates of hydrolysis of these two substrates ZPHL and HHL; - coefficient of ACE immunoreactivity, characterized by the ratio of the amount of ACE that has attached the 9B9 antibody to the activity of this ACE when interacting with the ZPHL substrate; and when detecting an increase in the values of each of the obtained parameters by 1.5 times or more in comparison with the control values typical for normal prostate tissue, it is concluded that there is an increased risk of developing prostate cancer.

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