RU2692561C1 - Method for determining proliferative activity of triple-negative breast cancer tumor cells - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention refers to medicine and biotechnology, particularly to determining the proliferative activity of tumor cells of triple-negative breast cancer. Method involves preparation of paraffin blocks from tumor tissue and histological sections, their dewaxing in hexane. Uncoloured dewaxed histological sections of tumor tissue are subjected to fluorimetric analysis and fluorescence excitation spectra are measured from 230 to 360 nm at a detection wavelength of 410 nm and fluorescence spectra at excitation wavelength of 300 nm and detection wavelength from 330 to 450 nm, determining the ratio between intensities of curves with maxima in region of 265 and 305 nm (I/I), and if I/I= 1.0, the proliferative activity is low, the ratio I/Iindicates high proliferative activity.EFFECT: invention increases efficiency of obtained results.1 cl, 3 dwg, 1 tbl, 4 ex

Description

The invention relates to medicine, can be used in pathological anatomy, medical diagnostics and for medical research, in particular to determine the proliferative activity of tumor cells of triple-negative breast cancer with the subsequent decision on the appointment of therapy and prognosis of the patient.

Currently, triple-negative breast cancer is considered to be a heterogeneous disease and is divided into basal-like and non-basal-like subtypes, characterized by different projections and responses to chemotherapy. The index of proliferative activity, determined by expression of Ki-67 protein, is a predictor of the effectiveness of neoadjuvant chemotherapy in patients with operable triple-negative cancer. A high level of proliferative activity, determined by the Ki-67 index (more than 10%), is reliably associated with high efficacy of neoadjuvant chemotherapy and the achievement of complete morphological remission, but low survival, a low level of proliferative activity (Ki-67 index less than 10%) has normal clinical significance (Fasching PA, Heusinger K, Haeberle L., et al. Ki-67; Breast Cancer Research. Vol. 11 (486). - P. 2-13. Doi: 10.1186 / 1471-2407-11-486).

There is a method for determining the proliferative activity of breast cancer cells on histological sections prepared by standard paraffin wiring using immunohistochemistry with monoclonal antibodies to Ki-67 protein. Fragment tissue triple-negative breast cancers obtained by biopsy or during surgery, subjected to a standard paraffin wiring, followed by immunohistochemical study on paraffin sections peroxidase-antiperoksidaznym method to unmask antigens PT module (DAKO) using a buffer for unmasking (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 proliferation factor Ki-67 protein (clone MIB 1, RTU) are used. The proliferative activity is estimated on the basis of the calculation of the proliferative activity index in percent based on the counting of 400 cells with a high magnification of the microscope in the zones with the maximum concentration of positively stained nuclei (hot spot) (Petrov SV, Raikhlin NT. Guidelines for the immunohistochemical diagnosis of tumors person - Kazan, 2004. - 452 c). The disadvantages of this method:

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

2) Ki-67 immunoreactivity largely depends on a number of methodological parameters, such as the timing of fixation of a tissue sample, its processing conditions and antigen unmasking method, and conditions for performing the method.

3) The complexity of processing the results.

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

There are ways to determine the proliferative activity: radioautography with ³ H-thymidine, detection of cells labeled with BrdU; immunohisto-and cytochemical detection of proteins regulating the cell cycle, such as PNCA, caps H3, cyclin D1 (Kirsch OV, GV Beznin, DE Korzhevsky Proliferation markers used in histological studies // Morphology. - No. 6, T136. - S. 95-100). The disadvantages of these methods are the high cost, the need for preliminary preparation of the material, the expression of markers only in certain phases of the cell cycle, as well as the toxicity of the first method.

The technical result of the proposed method is to simplify the methodology and increase the objectivity of the obtained results to determine the proliferative activity in patients with triple-negative breast cancer.

The essence of the invention is that unstained dewaxed histological sections of breast cancer tissue after determining its triple-negative status, prepared by standard paraffin wiring, subjected to fluorimetric study, placing them on quartz glass, previously dewaxed in a solution of hexane. After that, a measurement of the fluorescence excitation spectrum of the tumor tissue is carried out in the wavelength range from 230 to 360 nm at a registration wavelength of 410 nm. Additionally, fluorescence spectra of the tumor tissue are measured at an excitation wavelength of 300 nm and recording wavelengths from 330 to 450 nm and determine the ratio between the intensities of the curves with maxima in the 265 and 305 nm (I ₂₆₅ / I ₃₀₅ ) and if I ₂₆₅ / I ₃₀₅ = 1.0, then the proliferative activity of triple-negative cancer is low, an increase in the value of the ratio I ₂₆₅ / I ₃₀₅ indicates a high proliferative activity.

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 triple-negative status of breast cancer (no expression of estrogen, progesterone and HER-2 / neu receptors) by immunohistochemistry, a parallel section 6-7 µm thick without staining with hematoxylin and eosin is prepared from the paraffin block, it is dewaxed in hexane in for 3 min, placed on a quartz glass, without staining, and measure the fluorescence excitation spectra from 230 to 360 nm at a recording wavelength of 410 nm and the fluorescence spectra at an excitation wavelength of 300 nm and lengths register n from 330 to 450 nm using a spectrofluorometer SOLAR CM-2203. The graphs of figure 1 shows typical fluorescence excitation spectra of samples of triple-negative breast cancer. All fluorescence excitation spectra have a complex structure consisting of three distinct maxima: the first at 260–265 nm, the second at 305–310 nm, and the third at 335–340 nm. Curves 1, 2, 3, 4 represent the fluorescence excitation spectrum of the tissue studied with a proliferative activity index of 10%, 70%, 80% and 90%, respectively (the results were compared with the results of immunohistochemistry with mouse monoclonal antibodies to Ki-67 protein (clone MIB 1, RTU) with the subsequent calculation of the index of proliferative activity by the method described above). With the smallest index of proliferative activity, two peaks are poorly resolved and therefore form one common peak with a maximum at 287 nm, the other peak is clearly pronounced with a maximum at 334 nm. The remaining curves have three distinct well-resolved peaks in the 265, 305, and 335 nm regions. As the proliferative activity index increases, the ratio between the first two maxima changes.

To analyze the spectra, they are decomposed into components using the Gaussian – Lorentz curve method. Figure 2 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 molecules of the fluorophores of the tissue studied 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 decomposition of the spectrum into components, it is necessary to calculate the ratio of the maxima of curves 2 and 3, corresponding to a specific index of proliferative activity.

Table 1 shows the ratios between the intensities of the curves with maxima at 265 and 305 nm (I ₂₆₅ / I ₃₀₅ ) obtained after decomposition of the curves of figure 1. The highest value of the ratio I ₂₆₅ / I ₃₀₅ , where I ₂₆₅ is the fluorescence intensity of the curve with a maximum of around 265 nm, i ₃₀₅ is the fluorescence intensity of a curve with a maximum around 305 nm, observed in a sample with an index of proliferative activity equal to 90%. With an increase in the proliferative activity index the contribution of I ₂₆₅ increases.

The graphs of figure 3 shows typical fluorescence spectra of the same tumor tissue samples as in figure 1. Curve 1 is the fluorescence spectrum of triple-negative breast cancer tissue with a proliferative activity index of 10%. Curves 2, 3, 4 represent the fluorescence excitation spectrum of the studied tumor tissue of triple-negative breast cancer with a proliferative activity index of 70%, 80% and 90%, respectively. Curve 1, as well as curves 2,3 and 4, has two peaks. However, the second peak is very small with a maximum at 428 nm. On the axes of coordinates, it is depicted separately by a dashed line. With an index of proliferative activity from 70 to 90%, the peak with a maximum at 440 nm is well pronounced and gradually decreases. Such a difference in the spectra of samples with an index of proliferative activity of less than and more than 10%, which is the threshold value for the appointment of neoadjuvant chemotherapy, allows you to replace immunohistochemistry with more accessible fluorimetry.

The method is based on the identification of biochemical atypism, which is an early manifestation of the malignant transformation of cells at the molecular level. Due to the selective luminescence of fluorophores (nicotinamide dinucleotide, collagen, elastin and vitamin B6) at a wavelength of 410 nm and energy transfer to them from tryptophan and tyrosine-containing whites, radical differences in the fluorescence excitation spectra of tissues with different proliferative activity indexes can be identified.

Example 1. Patient A., 45 years old. Performed right-sided mastectomy for nonspecific breast cancer, verified by preoperative trepanobiopsy. Histological examination of the surgical material confirmed the preoperative diagnosis. An immunohistochemical study revealed a triple-negative breast cancer, the use of monoclonal antibodies to the Ki-67 protein determined an index of proliferative activity of 10%.

The results of the fluorimetric study: the value of the ratio I ₂₆₅ / I ₃₀₅ is 1.0, which corresponds to an index of proliferative activity equal to 10%.

Example 2. Patient K., 54 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 triple-negative breast cancer, the use of monoclonal antibodies to Ki-67 protein revealed an index of proliferative activity of 80%.

The results of the fluorimetric study: the value of the ratio I ₂₆₅ / I ₃₀₅ is 1.22, which corresponds to an index of proliferative activity equal to 80%.

Example 3. Patient C, 76 years old, underwent left-sided mastectomy for unspecified breast cancer, verified by preoperative trepanobiospia. Histological examination of the surgical material confirmed the preoperative diagnosis. An immunohistochemical study revealed a triple-negative breast cancer, the use of monoclonal antibodies to the Ki-67 protein revealed a proliferative activity index of 90%.

Results of the fluorimetric study: the ratio of I ₂₆₅ / I ₃₀₅ is 1.31, which corresponds to a proliferative activity index of 90%.

Example 4. Patient O., 62 years old, performed trepanobiospia of a tumor of the left mammary gland. The histological conclusion: unspecified breast cancer. An immunohistochemical study revealed triple-negative breast cancer, the use of monoclonal antibodies to Ki-67 protein revealed a proliferative activity index of 70%.

The results of the fluorimetric study: the ratio of I ₂₆₅ / I ₃₀₅ is 1.15, which corresponds to the proliferative activity index of 70%.

This method investigated 28 fragments of triple-negative breast cancer, in 100% of cases the proposed method differentiated high (more than 10%) and low (less than 10%) proliferative activity in the tissue of triple-negative breast cancer, in 6 cases with high proliferative activity results of immunohistochemical studies differed by 10% with the data of fluorimetric studies, which can be neglected, given the presence of subjectivity in the analysis of the proliferative activity index by determining the Ki-67 protein, and also lack of value for prescribing therapy.

The advantages of the proposed method are low cost, no need to purchase expensive equipment and reagents that require certain temperature storage conditions, simplify the method for determining proliferative activity of cells of triple-negative 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 distortion results under the influence of external conditions, the speed of obtaining a result that It is especially important for cancer patients to quickly resolve the issue of chemotherapy.

Claims (1)

The method of determining the index of proliferative activity of tumor cells of triple-negative breast cancer, including the preparation of paraffin blocks from tumor tissue and histological sections of them, characterized in that the unstained dewaxed histological sections of the tumor tissue are subjected to fluorimetric examination and fluorescence excitation spectra are measured from 230 to 360 nm at a registration wavelength of 410 nm and fluorescence spectra at an excitation wavelength of 300 nm and recording wavelengths from 330 to 450 nm, o limit the ratio between the intensities of the curves with maxima in the region of 265 and 305 nm (I ₂₆₅ / I ₃₀₅ ), and if I ₂₆₅ / I ₃₀₅ = 1.0, then the proliferative activity is low, an increase in the value of the ratio I ₂₆₅ / I ₃₀₅ indicates a high proliferative activity.

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