INVENTION
Russian Federation Patent RU2127429

A method for diagnosing hyperplasia and endometrial cancer

A method for diagnosing hyperplasia and endometrial cancer

Name of the inventor: Kogan Emmanuel Markovic; Saveliev Galina; Umahanova Madina Musaevna; Zhukotsky Alexander; Victor F. Kopylov
The name of the patentee: Kogan Emmanuel Markovic; Saveliev Galina; Umahanova Madina Musaevna; Zhukotsky Alexander; Victor F. Kopylov
Address for correspondence: 119828, Moscow G-435, ul.M.Pirogovskaya 1a NIIFHM patent department
Starting date of the patent: 1995.06.06

The invention relates to medicine and can be used for differential diagnosis of hyperplastic processes and endometrial cancer. The method is based on a quantitative study of changes morfodensitometricheskom interphase chromatin nuclei of glandular cells of endometrial hyperplastic processes and endometrial cancer. A method of diagnosis of hyperplastic processes and endometrial cancer by cytology taken from a patient of the biological material is characterized in that the biological material taking fingerprints endometrial obtained in diagnostic curettage endometrium, fixed with a mixture of Nikiforov carried ribonuclease treatment, stained with gallocyanin, chrome alum, enclosed in Canada balsam, mikrodensitometriruyut interphase chromatin glandular cells of the endometrium and on the basis of these parameters (Comp IOD 1, C Std OD2, IOD, PERIMETR) calculates: DF = 0,00013 Comp IOD 1 + 3,98725 C Std OD2 + 0,00001 IOD + 0,01213 PERIMETR - 13,4409 and the positive value of the indicator was diagnosed with cancer of the endometrium, with a negative - benign endometrial processes. The invention allows for the diagnosis with greater specificity and sensitivity.

DESCRIPTION OF THE INVENTION

The proposed method relates to the field of medicine and can be used for differential diagnosis of hyperplastic processes and endometrial cancer.

For endometrial hyperplastic processes according to the histological WHO classification in 1980 are background processes hyperplasia (glandular, glandular-cystic, glandular polyp) and precancerous conditions - atypical glandular hyperplasia and glandular polyp with the phenomena of proliferation and atypia; Endometrial cancer - adenocarcinoma of varying degrees of maturity. Differential diagnosis of background and precancerous processes of endometrial cancer is extremely important practical significance, since it is carried out based on the selection of adequate treatment (1,2). So, for glandular endometrial polyps are acceptable methods of removing the polyp under the control of the hysteroscope and subsequent hormonal treatment; in the presence of glandular polyp with the phenomena of proliferation and atypia resort, depending on the age of the patient or to the long-term hormone replacement therapy or for surgical treatment. It is found that in 72% of cases of endometrial cancer is preceded or atypical glandular hyperplasia, and the interval between the development of cancer and endometrial hyperplasia or endometrial cancer, and polyps are respectively 8, 4, 4, 6 years. The complexity of the diagnosis of hyperplastic (GGE) and cancerous processes (AC) of the endometrium is the similarity of the clinical picture, that is the main symptom of the disease - uterine bleeding. Existing clinical diagnostic methods (ultrasound, radiometry, hysterosalpingography, the study of hormonal status, determination of steroid hormone receptors in endometrial tissue, immunological methods, gisterokopiya, scraping the endometrium, uterine probe) can not fully meet the clinicians due to insufficient information content, lack of opportunity to reflect nosological specificity pathological process, time-consuming and cumbersome to perform and almost all methods are invasive (2).

The primary diagnostic method in detecting endometrial pathology is currently a histological examination of scrapings of the endometrium obtained by dilatation and curettage (3). However, a prerequisite is pridelnyh biopsy and removal of the entire uterus mucosa. Leaving the changed portions of the endometrium in the future may lead to unnecessary diagnosis of recurrent disease. Besides the morphological structure of the tumor is not always fully correspond to the biological course of tumor process, does not allow to evaluate the structural and functional changes in the nuclear chromatin, which are a key element in the process of ontogeny and cellular adaptation and during the early stages of the pathogenesis of many diseases, including malignant tumors (4). The most common worldwide screening method for diagnosis of endometrial cancer is the cytological study, which the diagnostic accuracy is 60 - 84% (2.5).

When cytology (light microscopy) analysis is performed visually and the result is a high quality morphological description, which does not objectify and quantify the results, interpretation of cytological research can be difficult due to poor quality products, but also factors that can cause changes in the endometrium especially inflammation.

Electron microscopic study allows you to get a general idea about the distribution of chromatin in the cell nucleus, but the method does not allow to evaluate the functional state of the genome, rather laborious, especially in the preparation of material for the study (4).

Cytochemical and immunochemical studies of endometrial material can be used as an auxiliary for the formation of test groups risk of endometrial cancer, since most of them are detected as a high frequency in cancer, and endometrial hyperplasia.

Currently oncomorphology used cytogenetic methods to understand the nature of genetic changes in cells during the development of cancer and its progression. Since the core is the carrier of genetic information, and the structural condition of the chromosomes in interphase nuclei corresponds to the activity of genes, as of heterochromatin can be seen on the functioning of the genome (6).

On the basis of scanning tsitospektrofotometrii cell nuclei, followed by mathematical processing of scans using different statistical indicators (7) showed an objective increase in the heterogeneity of the structure of chromatin during interphase nuclei of atypical hyperplasia and endometrial cancer compared with those with glandular hyperplasia of the endometrium and unchanged.

Morphometric and light microscopic study of the nuclei of endometrial epithelial precancerous lesions and cancer of the endometrium in the diagnosis of endometrial lesions cytomorphological VN been taken Novick et al. (1990). Studied material smears obtained from 57 women with uterine cavity via syringe Browne (aspirates). Cytological preparations 96-degree fixed with alcohol and dyed with hematoxylin-eosin and destained Feulgen overcoated with cold 5N hydrolysis. HCl. Morphometric parameters were investigated nuclei endometrial epithelial cells (area, perimeter, maximum and minimum diameter) and the average optical density and dispersion and integrated optical density. Biometric data confirmed Cytomorphological diagnosis and 85.7% of cases, contributed to the division of normal and hyperplastic epithelium of precancer and cancer cells of highly differentiated cells (8).

This method can be considered as a prototype because it is the most informative for the differential diagnosis of hyperplastic processes and endometrial cancer. However, unlike the proposed herein a method in the prototype method as object aspirates uterus were used, which involves getting less rich and high quality cellular material for research, less gentle way fence preparation and fixation of drugs that can have damaging effect on the structural elements of the kernel. As evidenced by the results obtained in the present process (compared to the prior art) was used a dye which is more sensitive and specific for the detection of structural changes in chromatin of interphase cell nucleus.

The present invention aims to provide a process more sensitive and specific for the differential diagnosis of hyperplastic processes and endometrial cancer.

The method is as follows:

Scraping the endometrium, resulting in diagnostic curettage of the endometrium, washed free of erythrocytes Hank's solution and make prints on a glass slide. Then a mixture of Nikiforov record mark (ethanol and ether in a ratio of 1: 1) for 20 minutes and air-dried. Processing carried ribonuclease, ribonuclease (Reanal, Hungary) diluted in osmolar sucrose solution at a concentration of 200 ME / 100ml. Treatment lasts 60 minutes at a temperature of 37 o C, and then washed with the imprint in 3 changes of distilled water. Produce color in the solution for 3 hours gallocyanin-chrome alum (MCC), prepared according to standard procedures (9) at 37 o C. Preparation of the supernatant were washed 5 min with tap water. dyeing process takes about 5 hours.

After painting, the drug is in Canada balsam and mikrodensitometricheskoe conducted research on image analysis system. microdensitometers result is optical, topological and geometric characteristics of the interphase chromatin component (euchromatin and heterochromatin) and the nucleus as a whole.

Study Procedures one print takes 30 - 40 minutes.

Previously on training samples for different groups of hyperplastic processes (74 patients) and endometrial cancer (12 patients) were installed the most informative indicators of conventional structure of interphase chromatin:

CompIOD1 - integrated optical density of heterochromatin;

CStdOD2 - medium deviation of the optical density of heterochromatin;

IOD - integrated optical density of the chromatin of the nucleus;

PERIMETR - the perimeter of the core.

For a comprehensive assessment of these indicators is calculated DF parameter for each group

DF = 0,00013 × CompIOD1 + 3,98725 × CStdOD2 + 0,00001 × IOD + 0,01213 × PERIMETR-13,4409.

The formula for calculating the DF parameters obtained using a linear discriminant analysis (10) using a standard statistical software package. With a positive value index DF diagnosed with cancer of the endometrium (AC), with a negative - hyperplastic processes endometpiya (GGE) - glandular hyperplasia of the endometrium (ZHGE), endometrial polyps (PE), endometrial atrophy (AE).

A similar connection between the numerical values ​​established when examining groups of patients pre- and postmenopausal, are examined and treated in the gynecological department of the Moscow City Clinical Hospital 31 suspected endometrial pathology. The analysis showed the most significant indicators of the structure of interphase chromatin endometrial glandular cells derived parameter DF calculated to identify the type of endometrial pathology.

The proposed method demonstrates the following clinical examples:

Example 1.

Patient M., 54 years old N history - 940, 31 GKB.

He admitted to the hospital with complaints of bleeding in postmenopausal (PMP) for the first time, RAP-5 years.

Clinical diagnosis: fibrous glandular endometrial polyp. During examination and treatment in the clinic of the proposed method was investigated by chromatin of interphase nuclei of glandular endometrial cells obtained by diagnostic curettage of the endometrium. The study was laundered endometrial tissue from red blood cells in Hanks' solution and prints are made on the slide. The resulting print was air-dried, fixed with Nikiforov. Further, eliminating the effects of ribonucleic acid scan results ribonuclease treatment was carried out. Ribonuclease diluted osmolar sucrose solution in a concentration of 2000 E per 100 ml of solution. Preparations treated in this solution for 1 hour at 37 o C, after which they were washed in 3 changes of distilled water. Then the staining agents in MCC solution. MCC solution was prepared by the standard method (9). Staining was carried out for 3 hours in a thermostat at 37 o C, after which the preparations were washed for 5 minutes with tap water supernatant.

Drugs frequently embedded in Canada balsam, after drying, were examined for the image analysis system.

chromatin structure indicators to examine the patient:

CompIOD1 - 4932,3; CStdOD2 - 0,9779; IOD - 129 710;

PERIMETR - 469,76

Further integrated assessment indicator was calculated DF = - 1.90529.

The value of the DF <0, therefore, the patient revealed changes in the structure of interphase chromatin corresponding benign processes (PE) of the endometrium, which is fully consistent with the data of clinical and laboratory research.

Example 2.

Patient G., 61 years, N history - 1843, 31 GKB.

He received gynecology department GKB 31 with complaints of smearing blood from the genital tract.

Clinical diagnosis: endometrial polyps?

With the proposed method is studied endometrial glandular cells chromatin structure of the patient (see. Example 1). chromatin structure indicators to examine the patient:

CompIOD1 - 4020,12; CStdOD2 - 0,698019; IOD - 108 930; PERIMETR - 528,769

For a comprehensive evaluation index was calculated DF = - 2.63184.

The value of the DF <0, therefore, identified chromatin structure changes correspond to changes in benign process (PE) of the endometrium.

Example 3.

Patient S., 51 years old, N history - 2640, 31 GKB. Admitted to the hospital with complaints of violations of menstrual function (irregular menstruation delayed up to 2 months).

In history - twice performed diagnostic curettage of the uterus on the bleeding, previously diagnosed with a polyp and glandular hyperplasia of the endometrium.

In addition to the clinical examination with diagnostic curettage produced prints and studied changes in the structure of interphase chromatin endometrial glandular cells (see. Example 1).

chromatin structure indicators to examine the patient:

CompIOD1 - 7228,66; CStdOD2 - 0,870088; IOD - 115035; PERIMETR - 493,088

Further integrated assessment indicator DF = -1,90041 was calculated.

The value of the DF <0, therefore, identified chromatin structure changes correspond to changes in benign process (ZHGE) of the endometrium, which is confirmed by clinical and laboratory investigations.

Example 4.

Patient K., 47 years old, N history - in 2823, 31 GKB.

I received complaints of excessive bleeding from the genital tract for the first time.

Clinical diagnosis: Dysfunctional uterine bleeding premenopausal period, adenomyosis.

Using the proposed method identified the following indicators of the chromatin structure of glandular endometrial cells (see Example 1).:

CompIOD1 - 11069,9; CStdOD2 - 0,823681; IOD - 149 059; PERIMETR - 565,161

The calculated ratio is equal to DF - 0.3716.

The value of the DF <0, therefore, found the interphase chromatin structure changes are characteristic of benign processes (ZHGE) of the endometrium, which is confirmed by histological examination of the endometrium.

Example 5.

Patient F., 64 years old, N history - 6274, 31 GKB.

Complaints on admission. Postmenopause, 10 years. Heredity is burdened: my grandmother and mother - uterine cancer. From history: twice performed scraping the endometrium and endometrial polyps removal. The hospital examination revealed thickening of the endometrium. Produced hysteroscopy and separate diagnostic curettage of the endometrium, thus found a thin endometrium and mucus. Histologically: small pieces of hypoplastic endometrium.

With the help of the method studied the chromatin structure of glandular cells of the endometrium (see. Example 1).

chromatin structure indicators to examine the patient:

SompIOD1 - 5154.77; CStdOD2 - 0,847275; IOD - 102923; PERIMETR - 574,834

Further integrated assessment indicator DF = -1,39052 was calculated.

The value of the DF <0, therefore, identified chromatin structure changes correspond to changes in benign changes (AE) of the endometrium.

Example 6.

Patient D., 51 years, N history - 6888, 31 GKB.

Received complaints of bleeding from the genital tract on the background of 2 years post-menopause.

Clinical diagnosis: Dysfunctional uterine bleeding postmenopausal period.

In history - makes diagnostic curettage of the uterus on the bleeding.

In addition to the clinical examination and studied prints restructuring of interphase chromatin endometrial glandular cells produced during dilatation and curettage (see Example 1).:

SomrIOD1 - 5202.47; CStdOD2 - 0,542494; IOD - 113 359; PERIMETR - 459,794

The calculated ratio is equal to DF - 3.89064.

The value of the DF <0, therefore, found the interphase chromatin structure changes characteristic for the processes of benign endometrium.

Clinical and laboratory studies confirmed the presence of the patient bleeding to endometrial atrophy background.

Example 7.

Patient G., 69 years, N history - 3942, 31 GKB.

Received complaints of bleeding from the genital tract on the background of 19 years of menopause.

From history - since 1988 after a bilateral mastectomy received radiation therapy and long-term tamoxifen. Double-produced diagnostic curettage of the endometrium on the uterine bleeding.

The diagnosis on admission: endometrial polyps, uterine fibroids.

The hospital performed a hysteroscopy and diagnostic curettage, in which the endometrial polyp is found.

With the help of the method studied the chromatin structure of glandular cells of the endometrium (see. Example 1).

chromatin structure indicators to examine the patient:

CompIOD1-17148,5; CStdOD2 - 1,04596; IOD - 160650;

PERIMETR - 574,917

Further integrated assessment indicator was calculated DF = 1.53916.

The value of the DF> 0, therefore, identified chromatin structure changes correspond to changes in endometrial cancer.

Histological study of the removal of polyps detected sites of malignancy by type highly differentiated adenocarcinoma. The patient is made operative treatment.

Example 8.

Patient G., 54 years, N history - 5582, 31 GKB.

He admitted to the hospital with complaints of bleeding from the genital tract, premenopausal 6 months.

With the help of the method studied the structure of interphase chromatin endometrial glandular cells (see. Example 1).

chromatin structure indicators to examine the patient:

CompIOD1 - 14652,5; CStdOD2 - 0,918475: IOD - 177132; PERIMETR - 6 06.035

Further integrated assessment indicator was calculated DF = 1.24863

The value of DF> 0, therefore, identified chromatin structure changes correspond to changes in endometrial cancer.

Clinically with hysteroscopy and dilatation and curettage revealed glandular tissue, decaying under the current injected into the cavity of the uterus fluid. Histological examination revealed well-differentiated endometrial adenocarcinoma.

Thus, the clinical examination confirmed the presence of the patient, the patient endometrial cancer.

The coincidence of the results of tests using the proposed method, and clinical and laboratory research suggests the specificity of the method of diagnosis of hyperplastic processes and endometrial cancer.

Thus, compared with the prototype, the method of diagnosis of hyperplastic processes and endometrial cancer allows:

1. significantly speed up the diagnostic process;

2. The method is widely used in medical practice when examining large populations, providing good reproducibility tsitodiagnosticheskogo analysis, by improving the quality of the drug;

3. To improve the accuracy of diagnosis of hyperplastic processes and endometrial cancer.

USED ​​BOOKS

1. YV Bohman Guide onkoginekologii.- Health Leningrad.-1989. from. 68-75, 275-336.

2. VG Breusenko The pathology of the endometrium in postmenopausal (pathogenesis, clinical picture, diagnosis, therapy) Diss. DOCG. honey. .: Science, Moscow, 1989.

3. Hoffken H., Hemmersbach E., Hebeirling D., Leppien G.- Moglichkeiten und Grenzen der konventionellen Differentiol -. Diagnostik am Endometrium.- Zentralbl Gynakol, 1984 106.7, 421-430.

4. KP Ganina Cytogenetic diagnostics Tissue Diagnostics. Naukova Dumka. 1980.-176 with.

5. Ferenczy A. Cytodynamics of endometrial hyperplasia and neoplasia.- Part 2: in vitro DNA histodutoradiography.- Hum. Pathol., 1983, 14, 1, 77-82.

6. Zhukotsky AV, Kogan EM: Automated analysis of chromatin structure in interphase cell nuclei. Sov. Med. Rev. B. Physicochem. Asp. Med. Vol.2, 25 - 77, 1989.

7. Y. Boyko Morphological and functional features of the chromatin of the interphase nuclei of epithelial cell hyperplasia and endometrial cancer. Kand. diss. Kiev. 1990.

8. Novik VI, Agroskin LS Papayan GV, Soloviev LV Biometric studies cytomorphological diagnosis of endometrial pathology. The experimental. Oncology. Volume 12, N. 2, 30 - 33, 1990.

9. E. Pierce Gistohimiya.-M., 1962, p. 190 - 192, 746.

10. A. Afifi, S. Eisen Statistical analysis: an approach using a computer. Trans. from English. - M .: Mir, 1982 - 488 p.

CLAIM

A method of diagnosis of hyperplastic processes and endometrial cancer by cytology taken from patients with endometrial and research heterophasic chromatin cell nuclei of cells, characterized in that they take fingerprints of endometrial cells is performed ribonuclease treatment, stained gallocyanin-chrome alum, enclosed in Canada balsam, mikrodensitometriruyut interphase chromatin glandular endometrium and on the basis of these parameters cells - Comp10D1 - integrated optical density of heterochromatin, CStd OD2 - the standard deviation of the optical density of the heterochromatin, 10 - the integral optical density of nuclear chromatin, PERIMETR - core perimeter calculated diskrimenantnuyu function DF

DF = 0,00013 × Comp10D1 + 3,98725 × CStdOD2 + 0,00001 × 10D + 0,01213 × PERIMETR - 13,4409,

and if positive indicator DF diagnose endometrial cancer, with a negative - benign endometrial processes.

print version
Publication date 27.03.2007gg