RU2430371C1 - Method of differential diagnostics and prognosis of endometrium myoma and cancer - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: claimed is method of differential diagnostics of malignant tumour and myoma of endometrium. In material of patient's uterus tumour, obtained during operation, content of zinc and level of metalloprotease (MMP) expression are determined. If amount of zinc in tumour tissue increases from 105 to 160 ppm and MMP expression 3.9 is present, prognosis about presence of malignant tumour with tendency to metastases is made, if zinc amount decreases from 153 to 75 ppm in tumour tissue in comparison with non-transformed and MMP expression 2 is present, conclusion about uterus myoma is made. ^ EFFECT: increase of prediction accuracy. ^ 1 ex, 1 tbl

Description

The invention relates to medicine, in particular to oncology. The method provides an increase in the degree of objectivity of the differential diagnosis of uterine fibroids and endometrial cancer. Material is taken from uterine tumors obtained after surgery.

Description of sample preparation and measurement

As a result of the development of the sampling preparation procedure, tissue samples that were previously frozen were crushed in liquid nitrogen and then dried at a temperature of 45-60 ° C. Nitrogen allows the tissue to remain frozen throughout the entire grinding process, which contributes to finer tissue grinding. This method turned out to be very effective for preparing dry fine material for the formation of tablets of equal thickness with a smooth surface, which would eliminate roughness and protrusions during the passage of synchrotron radiation through the sample. The results for samples prepared in this way gave the best reproducibility and made it possible to use the external standard method for quantifying the concentration of elements. The experiments were carried out at the X-ray fluorescence analysis station using synchrotron radiation (RFA SI) of the Institute of Nuclear Physics SB RAS, Novosibirsk. The set of elements that was identified in the spectra of the samples taken was as follows: K, Ca, Cr, Fe, Ni, Cu, Zn, As, Br, Rb, Cd, Sn, Sb. We studied healthy and tumor (malignant and benign) tissues of one patient. For biological tissues, the excitation energy was chosen at 30 keV, and the exposure time was about 600 s. The choice of exposure time was determined by the set of defined elements and reproducibility of the results, which depends on the time of collection of statistics. The measurement errors of elements such as Fe, Cu, Zn, Br, with an excitation energy E = 43 keV less than or about 1-2%.

In the same samples, the level of gene expression of matrix metalloproteinases MMP 2, 3, 9 is determined by the multiplex RT-PCR method. From an analysis of the distribution of malignant uterine tumors by MMP expression and Zn ion content, it can be concluded that in most cases, an increase in MMP 3 and 9 expression coincides with an increased Zn content compared to untransformed tissue. This may be due to an unfavorable prognosis of endometrial cancer. A feature of uterine fibroids tumors is an increased content of Zn in normal tissue compared to tumor tissue, which correlates with increased expression of MMP-2, whereas for MMP-3 and MMP-9 such patterns were not detected. Then, the determination of the concentration of Zn in endometrial tissues is one of the strategies in determining the degree of malignancy of a tumor and its ability to metastasize. This parameter can serve as the basis for the diagnosis and prognosis of the disease.

DESCRIPTION OF THE INVENTION

The invention relates to medicine, in particular to oncology, and can be used for the differential diagnosis of benign and malignant uterine tumors.

A known method for the differential diagnosis of benign and malignant tumors of the uterus, including taking the tumor material with subsequent histological analysis

(1. Hecht J.L. and Mutter G.L. (2006) Molecular and Pathologic Aspects of Endometrial Carcinogenesis. // J Clin Oncol 24: 4783-4791).

This method allows us to distinguish between benign and malignant tumors only as a first approximation without involving molecular markers for fine diagnosis and prognosis. Therefore, objective criteria are needed to diagnose and evaluate disease prognosis factors. It is important to evaluate the stage of cancer, the degree of invasion of the myometrium, the type of tumor. None of these factors is determined accurately using histological analysis and it all depends on a subjective assessment. Molecular biological and elemental analysis of tissues allows a deeper insight into the process of tissue transformation into tumor, as well as understanding which molecular processes underlie the redistribution of zinc and the activity of MMPs that are zinc-dependent (2. Oehler MK, Brand A ., Wain GV (2003) Molecular genetics and endometrial cancer. // J. Br. Menopause Soc, 9: 27-31.

3. Shamamian, P., Schwartz, JD, Pocock, BJ, Monea, S., Whiting, D., Marcus, SG, and Mignatti, P. (2001) Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis. J. Cell Physiol. 189, 197-206).

This serves practical purposes and the basis for developing a method for the diagnosis and prognosis of diseases of organs of the reproductive sphere of women. The proposed method allows with a high degree of accuracy to quantify the content of zinc and the activity of metalloproteinases in uterine tumors

(4. Braundmeier AG, Fazleabas AT., Lessey BA, Guo H., Toole BP, Nowak RA (2006) Extracellular Matrix Metalloproteinase Inducer Regulates Metalloproteinases in Human Uterine Endometrium. // The Journal of Clinical Endocrinology & Metabolism, 91: 2358- 2365.

5. Visse R., Nagase H. (2003) Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases: Structure, Function, and Biochemistry. // Circ. Res., 92: 827-839. Malemud C.J. (2006) Matrix metalloproteinases (MMPs) in health and disease: an overview. // Front Biosci., 11: 696-1701.

6. Curry T.E., Jr., Osteen K.G. (2003) The Matrix Metalloproteinase System: Changes, Regulation, and Impact throughout the Ovarian and Uterine Reproductive Cycle. // Endocr. Rev.24: P.428-465)

and correlate them with the degree of malignancy of the uterine tumor. A feature of the method is its high sensitivity and good reproducibility. Closest to the claimed is a method for determining the level of matrix metalloproteinases 2, 9 in uterine washes

(7. Lopata A., (AU), Salamonsen L.A. (AU), Quinn M A (AU) Matrix metalloproteinase levels as indicators of endometrial cancer, US 6607894),

which can be both qualitative and quantitative and adapted to screening in clinics. The authors used the accumulated scientific knowledge on enhancing the activity of certain MMPs during tissue transformation. On this basis, the authors developed a non-invasive screening method to assess the risk of endometrial cancer.

The disadvantage of this method is the dependence of MMP activity on the phase of the menstrual cycle, which may complicate the diagnosis. In addition, this method requires additional monitoring of the presence of endometrial cells in the swabs, which complicates screening diagnostics. In our case, activity is determined directly in the tissue when transformation processes are already present. Such information is essential for developing a strategy for controlling the biological behavior of the endometrium and other tissues.

The problem to which the invention is directed, is to increase the degree of objectivity in the diagnosis of uterine tumors and differential recognition of fibroids and endometrial cancer. The solution to this problem is achieved by evaluating the zinc content and expression level of MMP2.3.9 in the tumor and adjacent untransformed uterine tissue.

Description of the invention

A method for differential diagnosis and prognosis of endometrial fibroids and cancer according to the zinc content and activity of matrix metalloproteinases according to the claimed invention includes:

1) taking the surgical material of the uterus from the area affected by the tumor and adjacent to it without a tumor. Namely, 41 pairs of tissue samples of benign tumors and non-transformed adjacent to them, and 37 pairs of tissue samples of malignant tumors and surrounding non-transformed were taken;

2) preparation of 2 samples from the taken tissue - one for X-ray fluorescence elemental analysis and determination of the content of elements in the tissues; the second - to determine the expression of metalloproteinases MMP, which is placed in liquid nitrogen;

3) X-ray fluorescence elemental analysis using synchrotron radiation to determine the zinc content in the studied tissues by comparing the spectrograms of these tissues with the spectrograms of the external standard, which is taken as the standard “Bull liver” NIES 1577, the matrix of elements of which is closest to the matrix of the studied tissues;

4) isolation of total RNA from a piece of tissue frozen in liquid nitrogen, followed by measurement of the expression of MMP genes by multiplex RT-PCR;

5) assessment of quantitative characteristics: a change in the zinc content, an increase in the activity of MMP2, 3, 9;

6) determination for each quantitative sign of the correlation between the zinc content, the activity of MMP and the degree of tumor malignancy, as determined in the clinic;

7) analysis of the obtained quantitative characteristics of patients, namely, the zinc content in tumor and non-transformed tissues, on the significance level of concentration differences using a program such as SPSS 13;

8) conducting a correlation analysis of the obtained values of zinc concentrations and the level of expression of metalloproteinases in tissues.

Case Studies

Zinc was evaluated in 41 samples of fibroids and 37 samples of endometrial cancer. Patients were divided into 2 groups on the basis of changes in the concentration of Zn in normal and tumor tissues. One part of the patients (large) had a lower concentration of zinc in normal tissues (75-105 ppm) than in malignant (130-160 ppm), and the other part was vice versa. The same applies to tissue samples from patients with endometrial myoma. Elevated zinc levels in non-transformed tissue adjacent to a benign tumor were observed in most patients. A statistical analysis of the data obtained using the SPSS13 program confirmed the need for dividing the tissue samples of various patients according to the type of zinc change in the studied tissues. So, in untransformed tissues adjacent to the tissue of the endometrial myoma, in most patients (> 60%), the increase in zinc was about 1.5 with a significance level of p <0.0003 and amounted to 153 ± 12 ppm. In the malignant tissues of uterine cancer in most patients (> 65%), the increase in zinc was about 1.4 with a significance level of p <0.008 and amounted to 120 ± 12 ppm.

Before starting to measure the relative level of mRNA of the MMP2, 3, and 9 genes, as well as ERα and ERβ in tumor and normal tissues of the endometrium and myometrium, optimal conditions for conducting multiplex PCR were selected. Multiplex PCR was carried out in a reaction volume of 20 μl. As an endogenous internal control, relative to which the amplification products of the studied genes were aligned, the “household” genes, β-actin, were used to normalize MMP2, 3, and 9; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to normalize ERα and ERβ. Each PCR sample contained 2 μl of cDNA, 1-fold PCR buffer, 0.2 mM deoxyribonucleotide triphosphates (each), 20 pcM of each primer and 2 units. ac. Taq DNA polymerase. For each pair of primers, the optimal concentrations of MgCl ₂ were selected: MMP2, MMP3, MMP9 and ERβ - 2.0 mM, ERα - 1.8 mM. Primers for determining the expression of MMP-2 genes: direct: TCG CCC ATC ATS AAG TTC and reverse: GTG ATC TGG TTC TTG TCC, MMP-3: direct: TGC TTT GTC CTT TGA TGC TG and reverse: GGG AAA CCT AGG GTG TGG AT , MMP-9: direct: CCC TTC STT ATC GCC GAC AAG and reverse: AAC CGG TCC ACC TCG C. For all primer pairs, annealing temperatures of 59 ° C were selected for the MMP2, MMP3, MMP9 genes; 63 ° C for the ERα gene and 65 ° C for the ERβ gene. Optimum conditions were considered for PCR under which there was a lack of nonspecific amplification products, and the intensity (brightness) of the band of the PCR product was sufficient for densitometry.

Next, the optimal number of amplification cycles was determined. The optimal number of cycles was considered such that a linear relationship was maintained between the number of PCR product and the number of cycles. In our conditions, the most suitable number of cycles for MMP2 is 32, for MMP3 - 38, for MMP9 - 38, for ERα - 38, for ERβ - 38, for GAPDH - 32 and β-actin - 32.

Multiplex PCR products were separated by horizontal electrophoresis in a 2% agarose gel. Electrophoresis was carried out in 1 × TBE (0.018 M Tris-borate, 0.018 M boric acid, 0.4 mm EDTA) at a voltage of 8 V / cm. Gel scanning was carried out in ultraviolet light using a DNA Analyzer video system. Densitometry was performed using the Total Lab computer program. The relative mRNA content of the studied genes was evaluated in relative units as the ratio of the staining intensity of a specific gene band to the intensity of the “housekeeping” gene band. When analyzing the expression level of metalloproteinases, it was found that the expression profile depends on both the type of tumor and the class of MMP. So, MMP-2 is equally effectively expressed in malignant tissues of the endometrium and in fibroids. And only in 2 patients its expression is not registered. Another enzyme, MMP-3, on the contrary, is expressed mainly in the cancerous tissues of the endometrium, while its expression is weaker in myoma, and in some patients it is completely absent. In the malignant tissues of many patients, a sufficiently high expression of MMP-3 is observed, while MMP-9 is also expressed in endometrial cancer tissues, but not in myoma. Thus, MMP-3 and MMP-9 are expressed mainly in malignant tissues of the endometrium, but not in benign. Based on this, these metalloproteinases may be suitable candidates for characterizing the malignancy of these tumors.

The following analysis of the distribution of uterine tumors by hormonal status, MMP expression and zinc ion content led to the following conclusion: in most cases (from 60 to 90%), increased expression of MMP-3 is observed in endometrial malignant tissues (from 0.4 to 1.5 p.u. .) and MMP-9 (from 0.01 to 0.8 pu), which coincides with an increased content of Zn (130-160 ppm) compared with the non-transformed tissue adjacent to the tumor. A feature of uterine fibroids tumors is an increased content of Zn in normal tissue (153 ± 12 ppm) compared with the tumor, which correlates with increased expression of MMP-2 (from 1 to 3 p.u.), whereas for MMP-3 and MMP- 9 such patterns were not identified. Thus, determining the concentration of Zn in endometrial tissues may be one of the strategies in determining the degree of malignancy of a tumor and its ability to metastasize. This indicator can be used in the differential diagnosis of fibroids and endometrial cancer. The following analysis of the distribution of uterine tumors by hormonal status, MMP expression and zinc ion content led to the following conclusion: in most cases (from 60 to 90%), increased expression of MMP-3 is observed in endometrial cancer (from 0.4 to 1.5 p.u. .) and MMP-9 (from 0.01 to 0.8 pu), which coincides with an increased content of Zn (130-160 ppm) compared with the non-transformed tissue adjacent to the tumor. A feature of uterine fibroids tumors is an increased content of Zn in normal tissue (153 ± 12 ppm) compared with the tumor, which correlates with increased expression of MMP-2 (from 1 to 3 p.u.), whereas for MMP-3 and MMP- 9 such patterns were not identified. Thus, determining the concentration of Zn in endometrial tissues may be one of the strategies in determining the degree of malignancy of a tumor and its ability to metastasize. This indicator can be used in the differential diagnosis of fibroids and endometrial cancer.

Examples of differential diagnosis of gynecological tumors in patients 1 and 2 according to the claimed invention Patient 1 Patient 2 Zn concentration [N]> [T] [N] <[T] ERα expression level 0 + ERβ expression level 0 - The expression level of MMP 2 ++ +++ The expression level of MMP 3 + ++ MMP expression level 9 0 +++ Conclusion Uterine fibroids Endometrial cancer Note: N is normal non-transformed tissue, T - tumor tissue, 0 - gene expression does not change, + - gene expression is increased by 1.5-2 times (in tumor tissue compared with untransformed), ++ - gene expression is increased by 2.2-5 times (in tumor tissue compared to untransformed), ++ - gene expression is increased by 2.2-5 times (in tumor tissue compared to untransformed), +++ - gene expression is increased by 5.1 or more times (in tumor tissue compared with untransformed), - - gene expression decreases (in tumor tissue compared to untransformed).

Claims (1)

A method for differential diagnosis of benign and malignant uterine tumors, including taking the material of a uterine tumor of a patient removed during surgery, for analysis, which consists in determining the expression level of MMP 2, 3, 9 metalloproteinases in samples taken, characterized in that the activity of metalloproteinases is determined directly in the tissue , part of which is used to prepare tissue samples by grinding it in liquid nitrogen, then drying and forming a sample tablet for X-ray fluorescence analysis lysis using synchrotron radiation of the elemental composition of the tissue, obtaining the spectrum of the sample matrix followed by computer recognition of the spectrograms of the elements and calculating the quantitative concentration values, analyzing the changes in the zinc content in the tumor and non-transformed adjacent to the tumor tissue, determining the group to which the patient belongs by the type of zinc change in tissues, correlation of data on zinc content with the level of expression and type of metalloproteinases, with an increase in zinc in tumor tissue from 105 to 160 ppm and the presence of MMP 3, 9 expression make a prediction about the presence of a malignant tumor with a tendency to metastasis, with a decrease in the zinc content from 153 to 75 ppm in the tumor tissue compared with untransformed and the presence of MMP 2 expression, we conclude the presence of endometrial fibroids .