RU2437096C1 - Method of formation of risk groups of neoplastic disorders in cervical epithelium - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: formation of a risk group of neoplastic disorders in a cervical epithelium is carried out by IF staining of a cervical epithelium smear with using a monoclonal antibody reacting with a sialylated conformational glycopeptide antigen determinant as a part of MUC1 followed by with cell finish staining with chromogenic nuclear stain. The sample is examined by alternated fluorescent light and transmission light. Each cell with a depolarised stained surface membrane detected by fluorescent light is examined in transmission light. If the cells with the depolarised stained surface membrane detected by fluorescent light show cytological symptoms of dyscariosis, and/or proliferative parabasal cell complexes with no symptoms of dyscariosis show depolarised stained surface membranes or granular stained cytoplasm, a patient is subsumed to the risk group of the presence of neoplastic disorders in the cervical epithelium. ^ EFFECT: higher analysis accuracy. ^ 5 dwg

Description

The invention relates to medicine, in particular to methods of physical analysis of biological materials in vitro. The invention can be used for cytological diagnosis of dysplasia of cervical epithelium and cervical cancer (cervical cancer).

The development of cervical cancer passes through the stages of dysplasia and intraepithelial cancer. The difficulty in diagnosing the disease in the early stages lies in the fact that the initial forms of neoplasia can be focal in nature, can be clinically asymptomatic, masked by background diseases.

The “gold standard” in the primary diagnosis of cervical disease (CMM) is a cytological study. In cytological studies, the determination of the nature of the disorders and the severity of pathological changes is based on the characteristics of the nuclei and the degree of cell maturity. In the vast majority of cases, the limitations of the method are related to the quality of the sampling and preparation of the material, low information content of the material with small sizes of pathological foci. In particular, as a separate problem, experts identify difficulties in identifying and interpreting the cytological picture in cases where dysplastic disorders are predominantly represented by a population of small diskariotic cells. Thus, the search for additional methods that would improve the efficiency of the cytological diagnosis of precancerous lesions and malignant pathology in cervical epithelium is relevant.

Currently, immunocytochemical (ICC) staining of cervical epithelial cells is used as additional methods. Monoclonal antibodies to various biological markers, which are produced by epithelial cells, the expression of which depends on the differentiation and proliferative activity of cells and is increased by neoplastic changes, are used to identify cells.

A known method for the diagnosis of dysplasia and cervical cancer in cervical mucosa (US 7.455.973), based on the ICC determination of several markers in the cervical epithelium sample that are expressed in the cell nucleus: pl4ARF protein, p21.sup.waf1 protein, Toro2A topoisomerase, cyclin E, proteins belonging to the MSM group. The expression of these markers is increased in severe pathological cervical disorders. Due to the variability of their expression, the best indicators of the specificity and sensitivity of the test for the diagnosis of intraepithelial disorders, as the authors indicate, can be obtained using a combination of antibodies to three different markers. The main criteria for cytological diagnosis are the features of the structure of the nucleus and the degree of maturity of the cells. Since ICC staining of nuclear proteins screens the structure of the nucleus, the method is also used in combination with cytological examination of smears stained with traditional methods to verify the existing pathological disorders.

There is also a method of determining the presence or absence of cervical cancer using an antibody kit (EP 1586903), comprising at least one antibody from each of the following groups:

- 1 - antibodies that interact with glandular epithelial cells: anti-MUC1, anti-cytokeratin 7, anti-cytokeratin 18;

- 2 - antibodies interacting with adenocarcinoma cells: anti-cytokeratin 8 and anti-HIC1083;

- 3 - antibodies interacting with atypical squamous cells: anti-NMP179, anti-p16 ^INK4A , anti-Ki-67, anti-p53, anti-p21, anti-EMA, anti-CEA and anti-MIB-1.

Antibodies interact with nuclear, membrane and cytoplasmic proteins. Antibodies of each group are conjugated with different fluorescent labels, which allows us to differentiate the binding of an object to an antibody of the corresponding group. The main task that the known method solves is the differential diagnosis of pathological disorders in the glandular epithelium from those in the squamous epithelium. However, the known method does not make it possible to determine the nature of disorders in squamous cells. Antibodies to MUCl-anti-MUCl and anti-EMA - are used as markers of different types of cells. Perhaps the authors suggest the use of different antibody clones that differ in reactivity, but their specificity for the antigenic determinants of MUCl is not specified. The method is intended for automated screening, and first of all, for selecting patients at risk. The method does not exclude the need for subsequent verification of pathological disorders by traditional cytological methods. In addition, the method requires the use of special equipment and software and makes it possible to carry out research only in large medical centers.

As a prototype of the claimed invention, according to a combination of features, a method for assessing the state of cervical epithelium in relation to the presence of precancerous and tumor diseases of CM is adopted, which uses a panel of two or more antibodies interacting with membrane and cytoplasmic proteins of cervical epithelial cells (US 6.869.801). In the prototype method, a panel of two or more antibodies is used that interact with membrane and cytoplasmic proteins of cervical epithelial cells. This panel includes at least one antibody that binds to cells of the cylindrical epithelium and at least one antibody that binds to cells of the squamous epithelium. Antibodies that bind to squamous cells differ in interaction with cells of varying degrees of maturity. The following antibodies are used:

- antibody 6B5, which detects a surface glycoprotein, a dimeric protein with a molecular weight (MB) of about 180 kDa and interacts with basal and parabasal cells and also cross-reacts with cylindrical cells and stromal elements;

- antibody 2C7, which detects a high molecular weight glycoprotein with an MB of more than 500 kDa (unidentified mucin) and interacts with cylindrical cells;

- 9G5 antibody, which detects a protein with MB 40 kDa and interacts with surface and intermediate squamous cells;

- an HG3 antibody that detects a protein with MB 180 kDa and interacts with surface and intermediate squamous cells;

- BC4 antibody, which detects a protein with MB 200-210 kDa and interacts with parabasal and intermediate squamous cells.

The method is based on registration in cervical smears of the ratio of cells stained with antibodies of various specificity. An increase in the relative number of mature and undifferentiated, immature cells of the parabasal layers of squamous epithelium is regarded as a sign of possible pathological disorders in the mucosa of the cervix. The assessment of the characteristics of the cellular composition of the smear is carried out either qualitatively, based on a visual assessment of the relative number of cells stained with antibodies of various specificity, or quantitatively, by counting the corresponding cells. The disadvantage of this method is the need for analysis of several samples stained with various antibodies. A quantitative assessment can lead to a false negative result with a small number of atypical cells in the sample. In addition, the appearance of cells with insufficient differentiation in squamous epithelium can occur with atrophic and reactive changes not associated with neoplasia, which may be the reason for the low specificity of the test. The test result allows you to identify cases with a potentially high risk of pathological disorders, but it does not make it possible to verify their nature.

The claimed invention is directed to solving the problem of increasing the efficiency of cytological diagnosis of precancerous disorders and cervical cancer.

Using in clinical practice the proposed method allows to achieve the following technical and economic results:

- to simplify the technical procedure of the study by combining in one study the effective detection of atypical cells and verification of the nature of the violations according to the characteristics of ICC staining;

- reduce the cost of the technical research procedure;

- reduce the number of false-negative results of a cytological study, due to the lack of informativeness of the smears, the complexity of the interpretation of the cytological picture in the small-cell variant of severe diskariosis or inferiority of the material;

- effectively visualize cells with insufficient differentiation during ICC staining and conduct a targeted assessment of the presence of pathological disorders in accordance with morphological criteria;

- increase the efficiency of detection of neoplastic disorders with small sizes of the pathological focus;

- provides the opportunity to refine the diagnosis of latent forms of severe cervical neoplasia by the anomalous nature of ICC staining in proliferating complexes of parabasal cells that do not have sufficient morphological signs of atypia;

- available for research in laboratories equipped with a fluorescence microscope.

These technical results in the implementation of the invention are achieved due to the fact that, as in the known method, the formation of a risk group for neoplastic disorders in CM epithelium is carried out by immunofluorescence staining of a smear of cervical epithelium using a monoclonal antibody that interacts with a high molecular weight glycoprotein MUCl followed by staining of the nuclei with chromogenic cells .

A feature of the proposed method is that as a monoclonal antibody interacting with a high molecular weight glycoprotein MUCl, a monoclonal antibody interacting with a sialylated conformational glycopeptide antigenic determinant in the composition of MUCl is used. The sample is examined by alternating fluorescent lighting and transmitted light. Each cell with depolarized staining of the surface membrane detected by fluorescent lighting is examined in transmitted light. If cytological signs of discariosis are detected in transmitted light in cells with depolarized staining of the surface membrane and / or if proliferating parabasal cells in the complexes without signs of dicariosis exhibit depolarized staining of the surface membrane or granular staining of cytoplasm, the patient is at risk of neoplastic disorders in the cervical epithelium.

The invention consists in the following.

Mucous CMM is represented by two types of epithelium: flat epithelium covering the ectocervix, and cylindrical lining the inner surface of the canal. Under the influence of damaging factors, the cylindrical epithelium can undergo metaplasia - transform into a flat one.

Dysplasia is a pathological process in which cell proliferation and differentiation are impaired. Morphologically, this is expressed in an increase in the thickness of the parabasal layer of the epithelium and the appearance of atypical cells. Dysplastic disorders can progress to intraepithelial cancer and are often associated with invasive cancer. In the cervical mucosa, dysplasia in the overwhelming majority of cases is found in stratified squamous and metaplasized epithelium in the transformation zone, in areas of squamous metaplasia in the cervical canal.

With dysplasia and initial cervical cancer, an increase in the number of atypical cells with dyskariosis is observed in the scraping material from the cervical mucosa. Signs of diskariosis are variability in the size and shape of the nuclei, a high nuclear-cytoplasmic ratio, a violation of the chromatin structure, hyperchromia, multinucleation, an abnormal number of nucleoli. With an increase in the severity of disorders, the relative number of parabasal type cells and the severity of discariosis in them increase.

In normal epithelium, CMM is known to be produced by cells of the cylindrical epithelium and not produced by mature squamous cells. Antigen expression is retained in metaplastic squamous epithelium.

In case of pathological abnormalities in the squamous CM epithelium, atypical cells in cervical smears are almost always stained by ICH reaction with anti-MUCl antibodies. However, due to the fact that the expression of MUC1 can be observed in both normal and atypical cells, the use of anti-MUCl antibodies for the diagnosis of pathological abnormalities in the mucosa of CM has been considered to be impractical to date.

The MUCl molecule is highly polymorphic. Due to the long polypeptide chain polymorphic in size and the variable composition of the side carbohydrate chains, the structure of the MUCl molecule forms many potential antigenic determinants. As a result, most monoclonal antibodies produced against MUCl are unique in their ability to interact with carbohydrate and peptide epitopes in the composition of its molecule. The structure of MUCl, which is produced by normal epithelial cells of various organs, is different. The experience of numerous studies and a comparative analysis of published data shows that the results of the determination of MUCl in tissues significantly depend on the specificity of the monoclonal antibodies that are used for analysis.

The inventors found that determining the expression of MUCl in cervical epithelial cells using a monoclonal antibody, which is produced by a strain of hybrid cultured Mus.Musculus L cells No. VSCK / P / 456D (ICO25) and interacts with a sialylated glycopeptide conformational antigenic determinant in MUCl, can be used to detect pathological abnormalities in the mucosa of CMM.

So, normally surface and intermediate squamous cells in most cases are not stained by reaction with IKO25, or relatively weak uniform staining of the entire surface membrane is observed in them. With atrophy or reparative changes in parabasal cells of squamous epithelium, a polarized homogeneous staining of the cytoplasm or a weak uniform staining of the entire cytoplasm is observed.

In undifferentiated cells of the parabasal layer of squamous and metaplasized epithelium, the expression of antigen recognized by ICO25 (MUC1 / HKO25) is increased compared with cells of the overlying layers. In immature squamous metaplasia cells, staining of the surface membrane is polarized. Cells of mature and ripening squamous metaplasia lose their polarization; they exhibit variable in intensity uniform staining of the entire surface membrane and / or cytoplasm.

In differentiated cells of the cylindrical epithelium, the apical surface of cells is normally stained. In contrast, undifferentiated proliferating reserve cells located in layers and groups either do not stain, or relatively weak staining of the cytoplasm is observed in them, uniformly present in all cells. With inflammatory changes, the nature of staining of the cylindrical epithelium does not fundamentally differ from the norm.

In neoplastic disorders, high expression of MUCl / IKO25 is observed in basal and parabasal cells of squamous and metaplasized epithelium. Intensive depolarized staining of the surface membrane distinguishes insufficiently differentiated cells from more differentiated cellular elements.

In mild dysplasia, proliferating parabasal cells occupy no more than a third of the epithelial layer and rarely enter the smear when scraping from the mucosa. The picture of ICC staining with ICA IKO25 with mild dysplasia practically does not differ from that normal. Signs of mild dyskariosis are detected predominantly in unstained, ICC-negative, intermediate cells.

In moderate and severe dysplasia, intraepithelial cancer (pronounced cervical intraepithelial disturbances, CIN) or microinvasive cervical cancer among stained cell elements in the overwhelming majority of cases, atypical cells stained with a reaction with ICA ICO25 with varying degrees of severity are detected. The exception is cases in which dystrophic changes are expressed in most cells and bare atypical nuclei predominate in the cytogram. Indirect signs of the presence of atypia when examining in fluorescent illumination a smear stained with an ICC reaction with ICA ICO25 are the high intensity of staining of cells of variable size and shape, the uneven distribution of staining on the surface membrane, and the presence of stained intracellular inclusions.

In severe dysplasia and initial cervical cancer in the cervical epithelium, along with atypical cells, in some cases, smears contained massive complexes of proliferating parabasal type cells stained by ICX reaction with ICO25, sometimes enlarged, sometimes with hyperchromic nuclei, with a weak polymorphism. Intensive depolarized staining of the surface membrane or granular staining of the cell cytoplasm in these complexes distinguished them from proliferating reserve cells, proliferating glandular epithelium, and cell strata of immature squamous metaplasia. The presence of groups of cells with similar staining is closely associated with pathological disorders, since they were not observed normally and with reactive changes in the epithelium.

The inventive method is based on ICC staining of cervical smears with anti-MUCl monoclonal antibody, the product of a strain of hybrid cultured Mus.Musculus L cells No. VSCK / P / 456D (ICO25), analysis of the fluorescence staining pattern and the search for cells with depolarized intracellular staining distribution, morphology analysis cells by alternating fluorescent lighting and lighting in transmitted light.

Features of ICC staining of cervical epithelial cells with ICA ICO25 serve to identify a pool of undifferentiated cells with pathological changes. Using a fluorescent staining option allows you to alternate fluorescent lighting with light in transmitted light and analyze the morphology of the cell nucleus. This approach allows us to determine the presence of diskariosis in cells, morphological changes in which are the main criterion for assessing the presence and severity of pathological disorders in the cervical epithelium.

Intensive immunofluorescence staining effectively visualizes even single parabasal cells among the unpainted or homogeneously stained component of the smears - blood cells, mucus, debris, in massive layers of proliferating epithelium. An aimed assessment of the morphology of the nucleus in cell elements with depolarized staining of the surface membrane significantly increases the likelihood of detecting cells with diskariosis, and also makes it possible to verify the degree of violations in accordance with generally accepted cytological criteria.

The use of this approach is especially relevant in cases where smears are not informative for cytological analysis due to the small number of atypical cells, the illegibility of the material due to pronounced inflammatory changes, a large number of blood elements, as well as in cases where the pathological changes are mainly represented by a population of small discarotic cells.

Thus, the use of immunofluorescence staining technique in the case of the use of anti-MUCl monoclonal antibody IKO25 allows combining the high sensitivity of the ICC detection and the high specificity of the cytological analysis method.

The method is as follows.

Scraping of the cervical epithelium is obtained in the same way as for routine cytological examination with a spatula with a mucosa of the external pharynx of the cervix in the area of its transition into the cervical canal, with a spatula or brush from the cervical canal. A smear is prepared on a glass slide and fixed in 95% ethanol or another alcohol cytological fixative.

ICH staining was performed using the immunofluorescence assay technique. The smears are kept in pH7.6 buffered physiological saline (PBS) for 5 minutes, then 200 μl of a solution of monoclonal antibody IKO25 at a concentration of 10 μg / ml prepared in a 1% solution of bovine serum albumin (BSA) in PBS is applied to the glass. Glasses are incubated in a humid chamber for 1 hour at room temperature (20-24 ° C). At the end of the incubation, the glasses are washed three times in PBS and 200 μl of a solution of antispecies antibodies labeled with a fluorescent label prepared in a 1% solution of BSA in PBS are applied to them. Incubated in a humid chamber for 1 hour, washed three times in PBS.

After completion of the ICC staining procedure, the drug is stained with hematoxylin, enclosed in a mixture of glycerol and PBS (pH 7.4, v / v 1: 1) and analyzed using a fluorescence microscope with an integrated daylight illuminator.

Sequentially examine the entire area of the smear.

1) In transmitted light at a low magnification (objective × 10), the presence and general nature of the morphology of cellular elements in the smear are evaluated, including obvious signs of atypia, which may be sufficient to assess the nature of pathological changes. In the absence of obvious signs of atypia, they go on to study the smear under fluorescent lighting.

2) Under fluorescence illumination at a higher magnification (lens × 20), the presence of cells stained with immunofluorescence reaction, their shape and size, the nature of intracellular staining are assessed.

3) The criteria for assessing the nature of immunofluorescence staining of cells are the distribution of staining relative to the borders and nucleus of the cell. Differentiate staining:

- polarized - staining of a limited area of the surface membrane and / or cytoplasm;

- depolarized - uniform staining of the entire surface membrane and / or cytoplasm;

- membrane - the intensity of staining the borders of the cell is higher than the intensity of staining of its inner part; fluorescent staining completely or partially screens the core;

- cytoplasmic - the intensity of staining of the cell border is not higher than the intensity of staining of its inner part; a dark area in the cytoplasm is determined, corresponding to the shape and location of the nucleus.

4) Detect separately lying and grouped cells with depolarized staining of the surface membrane. Alternating luminescent lighting and light in transmitted light, the morphology of the nuclei of cells at a higher magnification is accurately assessed (lens × 40).

5) In accordance with generally accepted cytological criteria, a conclusion is made about the presence or absence of pathological disorders. The degree of existing pathological disorders is established in accordance with cytological criteria.

In cases where atypical cells with unambiguously interpreted signs of discariosis were not found during the study, the presence of a parabasal type of proliferating cells in the cytogram with intensive granular staining of the cytoplasm or intense depolarized staining of the membrane indicates a high probability of latent or small focal pathological disorders. In such cases, re-examination and regular monitoring are advisable.

After fluorescence analysis, smears can be stained with hematoxylin and eosin and subjected to cytological examination.

Examples of the proposed method are demonstrated in the attached micrographs of preparations processed in accordance with the described methodology.

Figure 1 Patient X., the clinical diagnosis of severe dysplasia of the epithelium CM. Intensive depolarized staining of the surface membrane with ICA ICO25 in parabasal cells with severe discariosis, normal squamous cells are not stained. A - image in fluorescent lighting, B - image of the same area in transmitted light, hematoxylin staining (UV. × 400).

Figure 2. Patient S., the clinical diagnosis is severe dysplasia of the epithelium of CM. Intensive depolarized staining of the surface membrane and cytoplasm with ICA IKO25 in parabasal cells with severe discariosis, red blood cells are not stained. A - image in fluorescent lighting, B - image of the same area in transmitted light, hematoxylin staining (UV. × 400).

Figure 3. Patient M., the clinical diagnosis is microinvasive cervical cancer. Intensive depolarized staining with ICA IKO25 of the surface membrane of small discarotic cells. A - image in fluorescent lighting; B - image of the same area in transmitted light, hematoxylin staining (UV. × 400).

Figure 4. Patient S., the clinical diagnosis is microinvasive cervical cancer. Intensive granular cytoplasmic staining with ICA ICO25 of proliferating parabasal cells. A - image in fluorescent lighting; B - image of the same area in transmitted light, hematoxylin staining (UV. × 400).

Figure 5. Patient K., the clinical diagnosis is microinvasive cervical cancer. Examples of visualization of single cells with severe diskariosis in uninformative and illegible smears of cervical epithelium ICC staining with ICA ICO25. A, B - image in transmitted light, hematoxylin staining; B, D - image of the same area in fluorescent lighting (UV. × 200).

Thus, the use of the proposed method for the formation of a risk group for neoplasia of CMM allows to identify pathological disorders in the epithelium of CMM in the early stages of their development. Timely treatment of precancerous disorders makes it possible to prevent the development of malignant neoplasms. Early detection of cervical cancer will allow for more effective, safe and organ-preserving treatment, which is especially important for patients of a young, reproductive age.

Claims (1)

A method of forming a risk group for neoplastic disorders in the cervical epithelium, including immunofluorescence staining of a smear of a cervical epithelium using a monoclonal antibody, which interacts with a high molecular weight glycoprotein MUC1, followed by staining the cells with a chromogenic nuclear dye, characterized in that it is a high molecular weight monoclonal antibody MUC1, use monoclonal antibody IK025, product of a hybrid cultured cell strain Mus.Musculus L No. VSCK / P / 456D, the sample is examined by alternating fluorescent lighting and transmission light, each cell with depolarized staining of the surface membrane detected by fluorescent lighting is examined in transmitted light; when detecting in transmitted light in cells with depolarized staining of the surface membrane cytological signs of discariosis and / or detecting proliferating parabasal cells in complexes without signs of discariosis, depolarized staining of the surface membrane or granular staining of cytoplasm, the patient is at risk of neoplastic disorders in the cervical epithelium.

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