RU2691606C1 - Method for detecting the presence of estrogen and progesterone receptors in tissue of her2-negative breast cancer - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention refers to medicine and biotechnology, particularly to determination of estrogen and progesterone expression by HER-negative breast cancer cells. Method involves preparation of paraffin blocks from tumor tissue and histological sections, their dewaxing in hexane. Then, non-dyed dewaxed histological sections of tumor tissue are subjected to fluorimetric analysis, fluorescence excitation spectra are measured from 230 to 360 nm at detection wavelength 410 nm, and ratio between intensities of curves with maxima within 262 and 303 nm is determined (I/I) and presence of maximum at 340 nm; at I/Iless than 1.0 and absence of peak on spectrum of fluorescence excitation at 340 nm conclude presence of receptors to estrogen and progesterone; at I/I, greater than and equal to 1.0, and the presence of a peak on the fluorescence excitation spectrum at 340 nm, the absence of estrogen and progesterone receptors is stated.EFFECT: invention increases objectivity of obtained results.1 cl, 3 dwg, 2 ex

Description

The invention relates to medicine and can be used in pathological anatomy, medical diagnostics and for medical research, in particular to determine the presence of estrogen and progesterone receptors in breast cancer tissue, which is necessary for determining the tactics of treatment of a patient.

Currently, the expression of estrogen receptors in> 1% of tumor cells is an indication for the appointment of hormone therapy and significantly changes the prognosis of patients with breast cancer. It is important that co-expression with estrogen receptors is characteristic of progesterone receptors (Clinical guidelines for the diagnosis and treatment of patients with breast cancer. - M., 2014. - 43 p.).

There is a way to determine the presence of estrogen and progesterone receptors in breast cancer tissue in histological sections prepared by standard paraffin wiring followed by immunohistochemistry with mouse monoclonal antibodies to estrogen receptors and progesterone. A breast cancer tissue fragment, obtained by biopsy or during surgery, is subjected to standard paraffin wiring followed by immunohistochemistry on paraffin sections by peroxidase-antiperoxidase method with unmasking antigens in a PT module (DAKO) using demasking buffer (Tris / EDTA pH 9 ) (DAKO). As a visualization system, a universal set with EnVision Flex Polymer (DAKO) is used. The nuclei are stained with Mayer's hematoxylin. Mouse monoclonal antibodies to estrogen receptors and progesterone receptors are used (Petrov S.V., Raikhlin N.T. Guidelines for the immunohistochemical diagnosis of human tumors. - Kazan, 2004. - 452c). Then, the intensity of the immunohistochemical expression of estrogen and progesterone receptors in epithelial cell nuclei is estimated by the Allred method (estimation of the percentage of stained cell nuclei (grades from 0 to 5) and the intensity of nuclear staining (grading from 0 to 3), the final value is obtained by summing the values. Value up to 2 is considered negative expression, from 3 to 8 is positive). (Quresh A., Pervez S. ): 350-353).

The disadvantages of this method:

1) The need for expensive equipment for automatic standardized painting and special expensive reagents.

2) The need for special preparation of the material before paraffin wiring.

3) The complexity and low objectivity in the processing of results.

4) The results of the method are largely subjective and depend on the qualifications of the specialist conducting the assessment of expression.

5) High probability of distortion of the result during the study without expensive automated equipment due to the high sensitivity of the method to the conditions of its implementation.

The technical result of the proposed method is to simplify the method and increase the objectivity of the results obtained for determining the expression of receptors for estrogen and progesterone by HER-2 negative breast cancer cells.

The essence of the invention is that unstained dewaxed histological sections of breast cancer tissue prepared by standard paraffin wiring, after confirming the absence of HER-2 / neu receptors by immunohistochemistry, are subjected to a fluorimetric study by placing them on quartz glass. Removal of paraffin from the histological section is carried out by washing it in a solution of hexane. After that, the fluorescence excitation spectrum of the tumor tissue is measured in the wavelength range from 230 to 360 nm at a registration wavelength of 410 nm and the ratio between the intensities of the curves with maxima in the 262 and 303 nm regions (I ₂₆₂ / I ₃₀₃ ) is determined, and the maximum is 340 nm; at I ₂₆₂ / I ₃₀₃ less than 1.0 and the absence of a maximum in the fluorescence excitation spectrum at 340 nm, it is concluded that estrogen and progesterone receptors are present; when I ₂₆₂ / I _{303 is} more than and equal to 1.0 and there is a maximum in the fluorescence excitation spectrum at 340 nm, it is concluded that there are no receptors for estrogen and progesterone.

The method is as follows.

A piece of breast cancer tissue obtained by biopsy or during surgery, is subjected to standard paraffin wiring. After confirming the absence of HER-2 / neu receptors in breast cancer tissue by immunohistochemistry, a parallel section 6–7 µm thick is prepared from the paraffin block without staining with hematoxylin and eosin, it is dewaxed in hexane for 3 minutes, placed on quartz glass, without staining, and measure the fluorescence excitation spectra from 230 to 360 nm at a recording wavelength of 410 nm and fluorescence spectra at an excitation wavelength of 300 nm and recording wavelengths from 330 to 450 nm using a SOLAR SM-2203 spectrofluorometer. The graphs of figure 1 depicts typical fluorescence excitation spectra of breast cancer samples that do not have estrogen and progesterone receptors. The fluorescence excitation spectrum has a complex structure consisting of three distinct maxima: the first at 268 nm, the second 303 nm, and the third at 340 nm. In the presence of HER-2 tissue, negative estrogen and progesterone receptors for breast cancer with a maximum of 340 nm are absent, and a maximum of 239 nm appears (figure 2).

To analyze the spectra, they are decomposed into components using the Gaussian – Lorentz curve method. Figure 3 shows an example of such a decomposition of a typical spectrum. Curve 1 represents the intensity of light, after absorption of which ionization of the fluorophore molecules of the tissue under study will occur. Curves 2 and 3 characterize the luminescence of the endogenous fluorophores of the tissue studied. Curve 4 characterizes diffused light. Curve 5 characterizes the measured spectrum, which is decomposed into components. After the decomposition of the spectrum into components, the ratio of the maxima of curves 2 and 3, which characterizes the presence or absence of receptors to estrogen and progesterone, is calculated.

The method is based on changes in the spectral properties of the tissue due to changes in its biochemical composition in the presence of estrogen and progesterone receptors. These receptors have a protein nature, they are composed of amino acids that selectively absorb in the studied wavelength range.

Example 1. Patient A., 45 years old. Performed right-sided mastectomy for ductal breast cancer, verified by preoperative trepanobiospia. Histological examination of the surgical material confirmed the preoperative diagnosis. An immunohistochemical study revealed HER2-negative breast cancer, the use of monoclonal antibodies to estrogen and progesterone revealed pronounced expression (8 points according to Allred).

The results of the fluorimetric study: the ratio of I ₂₆₅ / I ₃₀₅ is 0.75, on the fluorescence spectrum is missing a maximum of 340 nm., Concludes that there are receptors for estrogen and progesterone in breast cancer tissue and the need for endocrinotherapy.

Example 2. Patient O., 61 years old. A left-sided mastectomy for ductal breast cancer, verified by preoperative trepanobiospia, was performed. Histological examination of the surgical material confirmed the preoperative diagnosis. An immunohistochemical study revealed HER2-negative breast cancer, the use of monoclonal antibodies to estrogen and progesterone revealed no expression (0 Allred points).

The results of the fluorimetric study: the ratio of I ₂₆₅ / I ₃₀₅ is 1.25, the fluorescence excitation spectrum is present at a maximum of 340 nm, it is concluded that there are no receptors for estrogen and progesterone in the breast cancer tissue and no need for endocrinotherapy.

In this way, 33 cases of HER2-negative breast cancer were investigated, in 100% of cases the results of a fluorimetric study corresponded to the results of determining the presence of estrogen and progesterone receptors in the breast cancer tissue by immunohistochemistry.

The advantages of this method are low cost, no need to purchase expensive equipment and reagents that require certain temperature storage conditions, simplify the method of determining the hormonal status of breast cancer, eliminate the influence of subjectivity on the processing of results, no need for special preparation of the material for the study and the possibility of distorting the results under the influence of external conditions, the speed of obtaining a result, which is especially important for cancer nyh for an early decision on the appointment of a specific therapy.

Claims (1)

A method for determining the expression of estrogen and progesterone receptors by HER-negative breast cancer cells, comprising preparing paraffin blocks from tumor tissue and histological sections of them with verification of the absence of expression of HER-2 / neu receptors by immunohistochemistry, characterized in that unpainted dewaxed histological sections tumor tissue is subjected to fluorimetric study, fluorescence excitation spectra are measured from 230 to 360 nm at a registration wavelength of 410 nm, and the ratio of shenie curves between intensities of peaks in the region of 262 and 303 nm (I ₂₆₂ / I ₃₀₃₎ and having a maximum at 340 nm; at I ₂₆₂ / I ₃₀₃ less than 1.0 and the absence of a peak at 340 nm concludes that there are receptors for estrogen and progesterone; at I ₂₆₂ / I ₃₀₃ , more than and equal to 1.0, and the presence of a peak on the fluorescence excitation spectrum at 340 nm, it is concluded that there are no receptors for estrogen and progesterone.

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