RU2730993C2 - Method for differential diagnostics of fibroadenoma and breast cancer - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, particularly to oncology. Disclosed is a method for differential diagnosis of fibroadenoma (FA) and breast cancer (BC). Contents of mammary nodular aspirate are placed in microtube containing 800 mcl of Hanks medium, cell suspensions are distributed 50–100 mcl into centrifuge containers and centrifuged for 5 min in 1,000 rpm. Two liquid smears are prepared. Obtained cytospin preparations are dried at room temperature and atomic-force microscopy is performed. If the ratio of the height of the nucleus and cytoplasm is 2.5 or more, the BC is differentiated, if 1.7–2.4, then the FA is differentiated.EFFECT: invention provides a method which enables to differentiate BC from FA with high objectivity and reliability to determine further therapeutic approach to the patients.1 cl, 2 dwg, 1 tbl, 2 ex

Description

The invention relates to medicine, namely to oncology, and discloses a method for differential diagnosis between fibroadenoma and breast cancer.

The incidence of breast cancer (BC) increases with age, starting at 40 and peaking at 60-65 years. The decline in mortality from breast cancer in a number of developed countries is associated with early diagnosis. For this reason, one of the topical issues is the timely differential diagnosis of breast cancer. This is important for the choice of programs for further treatment of the patient, which vary enormously in volume and consequences, depending on the pathology identified.

In the mammary glands, in addition to various forms of mastopathy, fibroadenomas and cancer, there are lipomas of various sizes, lipogranulomas that occur after trauma, galactocele in lactating women, angiomatous tumors, sarcomas, the differential diagnosis of which is carried out by clinical, mammographic, ultrasound and cytological studies.

Significant difficulties arise in the differential diagnosis of breast cancer and fibroadenoma (FA).

Fibroadenoma is a benign tumor of fibroepithelial structure. This is the most common form of benign breast tumors. It is mainly found in the hormonally active age between 20-40 years. Only one fourth of FA reaches such a size when they can be detected macroscopically or detected on mammograms, the rest remain at the microscopic level. Having reached a certain stage, the PA stops growing and can remain stable for years. The dependence of the size and histological structure of the tumor with cyclic hormonal processes in the body has been established: during the menstrual cycle, the tumor grows, and after menstruation it returns to its original state. During pregnancy and lactation, fibroadenomas can reach gigantic proportions.

Fibroadenomas calcify with the same frequency as cancer (about 30%), but the character of calcifications is different: they are few in number, larger, shapeless, lumpy.

Timely differential diagnosis of FA and breast cancer is of great importance.

From the prior art, there is a method for the differential diagnosis of fibroadenoma and breast cancer in which the number of parenchymal cells, fibroblasts and fibrocytes localized periarteriolar is determined morphometrically in histological sections of the tumor, and when the number of parenchymal cells is equal to or less than 18.67 ± 1.96, the number fibroblasts equal to or more than 11.91 ± 0.81, the number of fibrocytes equal to or more than 14.00 ± 1.07, fibroadenoma is diagnosed, and if the number of parenchymal cells is equal to or more than 44.62 ± 6.56, the number of fibroblasts, equal or less than 8.32 ± 0.61, the number of fibrocytes equal or less than 8.65 ± 0.72, breast cancer is diagnosed (RU 2092843, 10.10.1997).

However, this method does not take into account the objective parameters of parenchymal cells, but only their number and ratio with fibrocytes and fibroblasts, which excludes the diagnosis of initial breast cancer on the background of fibroadenoma. In addition, the method is designed for histological diagnosis and cannot be used in cytology.

The known method of morphometric research, which increases the information content of the pathological examination. For example, it is proposed to pay attention to the DNA content of cell nuclei as a criterion for differential diagnosis of tumors. The presence of epithelial cells with an increased more than 4 times the DNA content against the norm increases the likelihood of developing breast cancer by 9.1 times (Neishtadt E.L. Possibilities of using morphological criteria in determining the likelihood of breast cancer. Abstract of the thesis. . St. Petersburg, 1991, p. 44).

However, the method is not designed for cytological research, it takes into account only one parameter, namely, the DNA content in the nuclei of breast cells, which allows only in a tentative form to express the likelihood of contracting breast cancer.

A known method for the differential diagnosis of benign tumors and breast cancer in histological sections stained with hematoxylin and eosin. The method is based on microscopic detection of nodular formation in the mammary gland, in which, from the size and shape of the glandular tubes and the nature of the location of connective tissue, in some cases, pericanalicular, in others intracanalicular FA can be distinguished (Laskina A. V. Breast tumors. In the book. Pathological diagnosis of human tumors. 2 ed. / Ed. by N. A. Kraevsky, A. V. Smolyannikov. M. Medicine, 1976, pp. 191-211).

However, the disadvantage of this method is the lack of unambiguous objective pathohistological criteria distinguishing benign from the initial forms of malignant fibroadenomas, i.e. from minimal breast cancer, into which in 5% of the total number of breast fibroadenomas are transformed (Ermilova V.D. Breast tumors. In the book. Guidelines for the pathological diagnosis of human tumors / Ed. by N.A. Kraevsky, A.V. . Smolyannikova, DS Sarkisova. 3rd ed. M. Medicine, 1982. p. 210 232). The method is used for histology and depends on the experience and qualifications of the histologist, does not imply a cytological examination.

The known method of research using microscopy, the conduct of which allows you to confirm or refute the diagnosis of malignant neoplasm. This method is a cytological examination of a biopsy specimen or scraping from a breast tumor (Guidelines for the cytological diagnosis of human tumors. Edited by Petrova AS, Ptokhova MP M. "Medicine", 1975).

However, this study at some of its stages is subjective (assessment of the relative and absolute sizes of cell elements, difficulties in taking into account the inevitable variability of the conditions for material preparation, which leads to variability in both the color and size characteristics of the preparation cells).

There is an approach to recognition in the process of diagnostic search for the three-dimensional structure of the tissue under study based on computer analysis of images of successive histological sections (Rene Albert, Tomas Schinderwolf, Irith Baumann, Harry Harms., Three-Dimensional Image Processing for Morphometric Analysis of Epithelium Sections. Cytometry 13: 759 -765 (1992)).

However, the method contains two possible sources of errors that may turn out to be critical when studying the details of the cell: firstly, the volumetric structure of the cell is not measured directly, but is built by the program, and secondly, the accuracy of construction is limited from below by the thickness of the sections that the microtome prepares. Attempts to improve accuracy through the use of ultramicrotomes lead to the need to use these expensive devices, as well as to a rapid increase in labor intensity at the histological stage and to an increase in the computational complexity of software algorithms. As an example, we point out that to build a three-dimensional image of a cell with a size of 10 micrometers (a fairly common and not the largest size) with a resolution of 10 nanometers (a typical resolution achieved by the proposed research method), it is necessary to prepare, microscopize and jointly process with a high accuracy (10 nanometers) software algorithm of at least 1000 histological preparations.

The known method of atomic force microscopy (AFM) is one of the types of scanning probe microscopy, based on van der Waals interactions of the probe with the sample surface (Mironov V.L. Fundamentals of scanning probe microscopy M. Technosphere, 2004). The principle of operation of AFM is based on the use of atomic bond forces acting between the atoms of a substance. At small distances between two atoms, repulsive forces act, and at large distances, attractive forces. Using AFM, images are obtained of both physical objects (surfaces of solids) and biological and chemical objects (viruses and bacteria, atoms and molecules). The resolution of such microscopes reaches a fraction of nanometers, which makes it possible to observe atoms. As a result of AFM, it is possible to build a volumetric relief of the sample surface in real time. The resolution of this method is approximately 0.1-1 nm horizontally and 0.01 nm vertically. The use of AFM opens up new possibilities in cytological diagnostics, since the method allows for a short time (minutes) to obtain an image of the cell surface with a resolution of the order of several nanometers. The physical basis for AFM operation is the interaction between the tip of the probe scanning the sample under study and the surface. In the process of scanning, the probe moves line by line along the studied sample. In this case, the tip of the probe rises and falls, outlining the microrelief of the sample surface.

The proposed method is aimed at overcoming the disadvantages of previously known research methods and is aimed at developing the most accurate differential diagnosis of FA and BC.

The technical result of the invention is the creation of a method that allows with high objectivity and reliability to differentiate breast cancer from FA to determine the further tactics of treatment of such patients.

The specified technical result is solved by the fact that, as in the known method, cytospin preparations are prepared using liquid cytology and AFM is carried out, then on the basis of computer analysis of the microrelief of cell surfaces and measurement of cell parameters, such as the height of the cytoplasm above the substrate, the height of the nucleus above the substrate and the ratio the heights of the nucleus and cytoplasm determine the objective morphometric data of the studied cells.

The peculiarity of the proposed method lies in the fact that the contents of the aspirate of the nodules of the mammary gland are placed in a microtube containing 800 μl of Hanks medium, the suspension of cells is distributed in 50-100 μl in centrifuge containers and centrifuged for 5 minutes at 1000 rpm, two liquid cytological smears, then the resulting cytospin preparations are dried at room temperature and atomic force microscopy is performed, and if the ratio of the height of the nucleus and cytoplasm:

- 2.5 or more, then differentiate breast cancer;

- 1.7-2.4, then fibroadenoma is differentiated.

The invention is illustrated by a detailed description, table, clinical examples of execution and illustrations, which depict:

FIG. 1 - AFM image of a mammary gland epithelium cell: the average ratio of the height of the nucleus to the height of the cytoplasm corresponded to 1.8 - the diagnosis of fibroadenoma.

FIG. 2 - AFM image of invasive breast cancer cells of a nonspecific type: the average ratio of the height of the nucleus to the height of the cytoplasm was 2.7.

The method is carried out as follows.

The patient is examined using ultrasound, and if there are nodules in the mammary gland, a fine-needle puncture biopsy is performed under ultrasound control. Thin strokes are prepared from punctates. If a liquid is obtained as a result of puncture, it is pre-centrifuged and several thin smears are prepared from the sediment. The swabs are air-dried and then stained with azure-eosin mixtures according to standard techniques. Thus, routine cytological preparations are prepared.

For AFM, we used the method of preparing cytospin preparations using liquid cytology. The contents of the aspirate of the nodules of the mammary gland are placed in Hanks medium, in a microtube containing 800 μl of Hanks medium. The cell suspension is dispensed in 50-100 μl into containers of a Cytospin system centrifuge (Shandon, UK) and centrifuged at 1000 rpm for 5 min. Two liquid smears are prepared. The obtained cytospin preparations are dried at room temperature and then used for AFM, having previously carried out quality control of the obtained monolayer smears, for which one cytopreparation is stained using the routine Leishman method (azure eosin mixtures).

Received routine cytological preparations of the mammary gland, stained with azure eosin mixtures, are analyzed for diagnosis in two stages: first by means of conventional light microscopy, and then by AFM.

For AFM, an atomic force microscope is used (for example, the Integra complex produced by ZAO NT NDT, Zelenograd, Russia). Scanning is carried out in a semi-contact mode.

The obtained data of the microrelief of cell surfaces and its local properties are processed by standard software that is part of the software of the Integra research complex.

Statistical processing of the results of measurements of the morphological parameters of cells (determination of average values of parameters, standard deviations, reliability of differences in statistical parameters for cells of various types) is carried out by conventional statistical methods using the Statistica 6.0 software package.

We examined 51 cytological preparations of breast tumors obtained by fine-needle aspiration biopsy under the control of ultrasound and with the help of scrapings from the surgical material. AFM investigated both routine cytological smears and liquid monolayer preparations stained with azure eosin mixtures. Each study took 20 minutes.

When AFM is performed in BC cells, a rough microrelief of the nuclear surface is observed, the nucleoli are clearly visible in the form of local elevations, the nuclear-cytoplasmic ratio is shifted towards the nucleus. The contours of the nucleus and cytoplasm are uneven. In FA, the cells are usually located in the form of complexes, cell size enlargement and rough nuclear chromatin are observed. At the same time, the cells are located approximately at the same distance and are oriented in one direction, the contours of the nuclei are even, the chromatin is evenly distributed, and the smoothness of the microrelief of the nuclei is clearly visible (see table).

Clinical example 1.

Patient M., 42 years old. History of breast cancer on the right six years ago, now there is a nodule in the left breast. According to ultrasound data, fibroadenoma, according to mammography, suspicion of cancer. During cytological examination by light microscopy of fine-needle aspiration biopsy material taken from the nodule of the mammary gland, it turned out to be impossible to exclude breast cancer in the presence of fibroadenoma. The material was analyzed using AFM.

With AFM, along with the absence of nucleoli, the average ratio of the height of the nucleus to the height of the cytoplasm corresponded to 1.8 (Fig. 1). The diagnosis was FA, which was confirmed by subsequent histological examination.

Clinical example 2.

Patient J., 51 years old. She has a history of fibrocystic mastopathy in both mammary glands for 25 years; currently, a nodular formation is revealed in the right mammary gland. According to ultrasound, the diagnosis of fibroadenoma is questionable, according to mammography, cancer is suspected. Cytological examination by light microscopy of fine-needle aspiration biopsy material taken from the nodule of the mammary gland proved impossible to exclude breast cancer. The material was analyzed using AFM. With AFM, the average ratio of the height of the nucleus to the height of the cytoplasm was 2.7 (Fig. 2). The diagnosis was breast cancer, which was subsequently confirmed by subsequent histological examination.

The proposed AFM method allows:

- to study the structure and various properties of surfaces in various media with high spatial resolution, of the order of several nanometers (7-8 nm), in a short time (20 minutes);

- to obtain a three-dimensional image of cells and macromolecules on their surface;

- to study the behavior of macromolecules and living cellular systems under conditions close to physiological.

- to obtain information on the chemical composition of the investigated object, as well as to detect the physical properties of the investigated surfaces;

- to obtain differences for FA cells and BC cells.

The claimed method can provide the cytologist with additional invaluable assistance in the pathology of the mammary glands by searching for the most accurate, objective and reliable criteria that allow for differential diagnosis between normal cells and BC cells.

Claims (3)

A method for the differential diagnosis of fibroadenoma and breast cancer, including the preparation of cytospin preparations using liquid cytology and atomic force microscopy with measurement of cell parameters: the height of the cytoplasm above the substrate, the height of the nucleus above the substrate and the ratio of the height of the nucleus and cytoplasm, characterized in that the content of the aspirate nodules of the mammary gland are placed in a microtube containing 800 μl of Hanks medium, the cell suspension is distributed in 50-100 μl in centrifuge containers and centrifuged for 5 minutes at 1000 rpm, two liquid smears are prepared, then the resulting cytospin preparations are dried at room temperature and carry out atomic force microscopy, and if the ratio of the height of the nucleus and cytoplasm: - 2.5 or more, then differentiate breast cancer; - 1.7-2.4, then fibroadenoma is differentiated.

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