RU2322676C1 - Cytometric diagnosis method for predicting prostate tumor diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves fixing prostate bioptate cell imprints with alcohol and stained with 50% AgNO₃ solution. Nucleus-nucleolus computer morphometry is carried out and 6 morphological nucleus types are selected with the following mean area values. Nuclei of 34.43 mcm², nucleoli of 1.43 mcm² belong to the first morphological type. Nuclei of 45.71 mcm², nucleoli of 2.78 mcm² belong to the second morphological type. Nuclei of 51.64 mcm², nucleoli of 4.60 mcm² belong to the third morphological type. Nuclei of 62.70 mcm², nucleoli of 6.96 mcm² belong to the fourth morphological type. Nuclei of 94.91 mcm², nucleoli of 11.07 mcm² belong to the fifth morphological type. Nuclei of 111.46 mcm², nucleoli of 16.35 mcm² belong to the sixth morphological type. Cells being of 1-3 morphological types, benign hyperplasia is to be diagnosed. Cells being of 1-4 morphological types, intraepithelial neoplasia is to be diagnosed. Cells being of 1-6 morphological type, prostate carcinoma is to be diagnosed.

EFFECT: high accuracy of diagnosis.

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Description

The invention relates to histology, histopathology, urology and oncology.

The main method for diagnosing prostate cancer is a polyfocal biopsy followed by histological analysis. Noting its high information content, it should also indicate the complexity and the fact that it does not always provide 100% reliability. This may be due to both the small size of the tumor, and the availability of appropriate equipment and the qualifications of the medical staff.

And in this regard, cytological examination of prostate juice is of particular interest. Since intraepithelial neoplasia and prostate cancer in the initial stages are localized processes, it can be assumed that the epithelial component of the prostate juice in these cases will be presented as unchanged cells, and with signs of neoplasia and malignancy.

Bearing in mind certain restrictions imposed on prostate massage when cancer is suspected, it must be pointed out that in practice there are cases when, during cytological examination of prostate juice, for example, regarding prostatitis, a differential diagnosis is necessary.

Known methods for the cytological diagnosis of diseases are based on the classical panchromatic staining of cells and their visual analysis. A definite drawback of these approaches is that their resolution does not allow in some cases to differentiate severe dysplastic changes in cells with benign diseases and highly differentiated forms of cancer. In these cases, new methods of cell staining and quantitative, morphometric analysis come to the rescue [G. Avtandilov "Medical morphometry. Guide. - M .: Medicine 1990 - 384 p .; Avtandilov G.G., Glukhov Yu.K. and Shabalova I.P. Ploidometric diagnosis of precancerous processes and cervical cancer by cytological preparations // Clinical Laboratory Diagnostics . - 2004. - No. 3. - P.45-47, Novik V.I., Agroskin L.S., Papayan G.V. et al. Biometric studies in the diagnosis of precancerous conditions and endocervical cancer. // Archive pathology. 1989, No. 12, pp. 45-49; Pogorelov V.M., Baidurin S.A., Kaplanskaya I.B. et al. Morphometric parameters of malignant cells in case of non-leukemic hemoblastosis with primary damage to the lymph nodes. // Hematology and Transfusiology, 1992, No. 3, pp. 5-10; Bittinger A, von Keitz A., Ruschoff J., Melekos MD Silver staining nucleolar organizer region in prostate cytology. // Zentralbl Pathol. 1994. Mar. 140 (1): 103-6].

However, it should be mentioned that in these works, as a rule, averaged group metric characteristics of cell populations as a whole are given, which makes identification of single malignant cells very difficult. In this regard, it becomes relevant to develop methods for the morphometric identification of such cells in prostate secretion preparations, which will help to improve the quality of the diagnosis of prostate cancer.

The closest analogue of our proposal is the work of V.I. Tsygankov, N.P. Melnikova N.P. and GL Mogileva [Morphotypes of glandular epithelium in benign and malignant diseases of the mammary glands. // Clinical laboratory diagnostics. 2004, No. 1. - S.43-45], in which the authors, analyzing the cytological material of the mammary gland stained with a 50% silver nitrate solution, were able to divide the population of nuclei into 6 morphotypes, linking them to certain clinical and morphological diagnoses. It has been shown that breast cancer is characterized by nuclear morphotypes that are not found in dysplasia, and on this basis it is believed that their detection may indicate the presence of cancer. This approach is of interest for diagnosis when single malignant cells are present among an array of benign cells. An undoubted advantage of this approach is the fact that it is difficult to apply visual diagnostic criteria to cells stained with a 50% silver nitrate solution, which are used for cells stained with classical cytological dyes.

The disadvantage of this prototype is that morphometric parameters are given for punctate cells of the mammary gland, which excludes their use for analysis of cytological preparations of the prostate gland.

The objective of the invention is the development of cytometric approaches to the differential diagnosis of benign hyperplasia, prostatic intraepithelial neoplasia (IDU), cancer, as well as cytometric identification of rare atypical cells in cytological preparations of the prostate gland.

The specified problem is solved as follows. Cells of prostate biopsy samples from 27 patients were fixed with alcohol and stained to identify nucleolar organizers with 50% AgNO ₃ solution. Measurements were carried out using an Mekos-C image analyzer. The grouping of drugs (benign hyperplasia, intraepithelial neoplasia and prostate cancer) was carried out on the basis of histopathological data. The data of the measurement results were statistically processed in the program "Statistica 5".

A morphometric study showed that with benign prostatic hyperplasia (BPH), the area of the nuclei is 35.74 ± 7.52 μm ² , and the nucleoli are 1.88 ± 0.53 μm ² . With prostatic intraepithelial neoplasia (IDU), the area of the nuclei of epithelial cells increases to 46.88 ± 3.55, and the nucleoli to 3.94 ± 0.39 μm ² . The average number of nucleoli and the values of the nucleolar – nucleus ratio in IDUs also statistically significantly exceed the corresponding indicators of BPH (p <0.01). In prostate cancer, the average values of all the studied morphometric parameters are statistically significantly higher than the similar indicators not only for BPH, but also for IDUs (Table 1).

The indicator of the correct classification of all cases based on a discriminant analysis of the values of the four morphometric indicators was 100%.

All 1122 nuclei of cytological preparations studied in 27 patients were divided into 6 groups using cluster analysis procedures. The number of groups was selected empirically, when choosing which two constraints were adhered to: the first is that their number be quite large, and the second is that the separation of the values of the nucleolar nucleolar parameters of the cells should not be too fractional, i.e. so that the values of neighboring groups differ significantly enough from each other. The primary cell separation was carried out on the basis of cluster analysis (K-means) of the values of the total area of the nucleoli, the results of which were subsequently used for discriminant cell separation according to four morphometric indicators. Each group is a collection of cells of a certain morphotype with close values of morphometric parameters (Table 2). The indicator of the general correct classification amounted to 98.31%.

Although the average values of the nuclei of each morphotype are statistically significantly different from each other, the extreme values of both min and max and 25 and 75 %% quartiles overlap the values of neighboring morphotypes, which makes it difficult to identify them based only on an analysis of the areas of the nuclei or nucleoli (table 3).

A multi-parameter description of the nucleolar nucleolus apparatus and separation based on it using discriminant analysis allows us to identify the qualitative uniqueness of the representation of the nuclei of various morphotypes in benign hyperplasia (BPH), intraepithelial neoplasia (IDU) and prostate cancer. So, with BPH, nuclei of 1-3 morphotypes are found, with IDU nuclei of 4 morphotypes appear, and with cancer - 5 and 6 morphotypes (Table 4).

The advantages of the method according to the invention:

- improving the quality of diagnosis of tumor diseases of the prostate based on computer morphometry of the nucleolar nucleolar apparatus of cells in cytological preparations of the gland secretion;

- the ability to identify rare, single malignant cells based on the cytometric classification of nuclear morphotypes;

- this method does not require expensive reagents, it is technically simple and its implementation with the issuance of results is possible within one to two days.

DIAGNOSTIC EXAMPLES:

Example 1. Observation No. 165. Morphological diagnosis: benign prostatic hyperplasia. Morphometric measurements: core area 39.68 ± 1.31 μm ² ; the area of the nucleoli 1.68 ± 0.09 μm ² . Nuclear morphotypes: I - 73.2%; II - 26.8%.

Example 2. Observation No. 184. Morphological diagnosis: prostatic intraepithelial neoplasia.

Morphometric measurements: core area 45.41 ± 1.28 μm ² ; the area of the nucleoli is 3.40 ± 0.19 μm ² . Nuclear morphotypes: I - 26.3; II - 39.4; III - 20, 2%; IV-14, 1%.

Example 3. Observation No. 229. Morphological diagnosis: prostate cancer. Morphometric measurements: core area 84.07 ± 3.46 μm ² ; the area of the nucleoli is 9.49 ± 0.63 μm ² . Nuclear morphotypes: I - 7.0%; II - 2.3%; III - 9.3%; IV - 13.9%; V - 53.5%; VI - 14.0%.

Table 1 Morphometric parameters (M ± cc) of the nucleolar nucleolar apparatus of epithelial cells in benign hyperplasia (BPH), intraepithelial neoplasia (IDU) and prostate cancer Diagnosis n Morphometric indicators The number of nucleoli The area of nuclei, microns ² The area of the nucleoli, μm ² Nucleolus-nuclear ratio BPH 8 1.52 ± 0.47 35.74 ± 7.52 1.88 ± 0.53 0,054 ± 0,008 PIN 9 2.00 ± 0.21 * 46.88 ± 3.55 * 3.94 ± 0.33 * 0.0841 ± 0.009 * Cancer 10 3.57 ± 0.81 * / ** 87.47 ± 13.66 * / ** 10.03 ± 3.19 * / ** 0.109 ± 0.022 * / ** Note: * - p <0.01 in comparison with BPH;
** - p <0.01 in comparison with IDUs.

table 2 Average values of morphometric parameters of the nucleolar nucleolar apparatus of cells of various morphotypes in cytological preparations of the prostate gland Morphotypes The number of nucleoli The area of nuclei, microns ² The area of the nucleoli, μm ² Nucleolus-nuclear ratio Number of cores one 1.29 34.43 1.43 0,043 284 2 1.73 45.71 2.78 0,064 357 3 2,36 51.64 4.60 0,093 239 four 3.15 62.70 6.96 0.116 85 5 4.26 94.91 11.07 0.118 101 6 4.17 111.46 16.35 0.149 56

Table 3 Statistical parameters of the areas of nuclei and nucleoli, various morphotypes of nuclei Morphotypes Morphometric indicators Average value Min Max 25% Quartile 75% Quartile one The area of nuclei, microns ² 34.43 13.76 57.77 30.06 40.09 The area of the nucleoli, μm ² 1.43 0.47 2.22 1,08 1.73 2 The area of nuclei, microns ² 45.71 20.76 79.91 37.69 50.67 The area of the nucleoli, μm ² 2.78 1.28 4.27 2,34 3.08 3 The area of nuclei, microns ² 51.64 21.43 95.40 42.54 59.32 The area of the nucleoli, μm ² 4.60 1.31 6.31 4.14 5.11 four The area of nuclei, microns ² 62.70 39.44 90.51 52.86 75.08 The area of the nucleoli, μm ² 6.96 5.69 8.91 6.43 7.30 5 The area of nuclei, microns ² 94.91 69.71 150.82 81.45 108.1 The area of the nucleoli, μm ² 11.07 8.56 14.60 9.59 12.37 6 The area of nuclei, microns ² 111.46 80.00 150.82 98.92 122.71 The area of the nucleoli, μm ² 16.35 13.74 20.14 14.59 18.09

Table 4 Representation of various nuclear morphotypes (%) in cytological preparations for BPH, IDU and prostate cancer Diagnosis Morphotypes one 2 3 four 5 6 BPH 55,99 38.16 5.85 PIN 12.55 40.21 36.81 10.43 Cancer 8.19 10.58 15.36 12.29 34.47 19.11

Claims (1)

A method for the cytometric diagnosis of tumor diseases of the prostate gland, which consists in staining cytological preparations with a 50% solution of silver nitric acid, computer morphometry of the nucleolar nucleolus apparatus, characterized in that 6 morphotypes of nuclei are distinguished having the following characteristics (average area values): 1- morphotype - nuclei 34.43 μm ² , nucleoli 1.43 μm ² ; 2nd morphotype - nuclei 45.71 μm ² , nucleoli 2.78 μm ² ; 3rd morphotype - nuclei 51.64 μm ² , nucleoli 4.60 μm ² ; 4th morphotype - nuclei 62.70 μm ² , nucleoli 6.96 μm ² ; The 5th morphotype — nuclei 94.91 microns ² , nucleoli 11.07 microns ² , the 6th morphotype — nuclei 111.46 microns ² , nucleoli 16.35 microns ² , and with the detection of 1-3 morphotypes, benign hyperplasia is diagnosed, 1-4 morphotypes - intraepithelial neoplasia, 1-6 morphotypes - prostate cancer.