4 курс / Акушерство и гинекология / Клинико_морфологические_особенности_злокачественной

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Chapter 2

MATERIAL AND METHODS

2.1 Characteristics of the research

The work consisted of two parts: the first part was an epidemiological, and its purpose was to determine the frequency of EH occurrence (according to the WHO 2014 criteria).

The study was conducted on the basis of the Clinical Hospital No. 122 named after. L.G. Sokolov, V.A. Almazov Clinical Hospital and Medical Research Center on the basis of a retrospective analysis of the journals of histological conclusions of the pathoanatomical departments. The sample included patients who underwent hysteroscopy with the collection of material for morphological studies. Analysis of the histological assessment of the endometrium was carried out in 818 women: 675 patients underwent an invasive examination of the endometrium over 4 years from 2016-2019. in 122 clinical hospital and 143 patients over 3 years from 20172019. in Almazov centre, in which the clinical characteristics were also analyzed and published in the article [26].

Indications for surgical treatment were AUB, infertility, suspicion of endometrial pathology according to ultrasound examination of the pelvic organs.

The structure and prevalence of endometrial diseases in the studied group was revealed by the results of histological examination.

The second stage was the recruitment of patients who met the inclusion/exclusion criteria and whose histological material was available for revision and immunohistochemical staining.

Characteristics of the investigated material

The material for the analysis of the second stage of the study was clinical and anamnestic data and histological preparations (glasses and paraffin blocks) of

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2.2 Method of morphological research

All collected histological material was reviewed on the basis of the Russian Scientific Center for Radiology and Surgical Technologies named after Academician

A.M. Granov” of the Ministry of Health of Russia, St. Petersburg using the 2014

WHO morphological classification criteria.

Of the 152 samples, 10 cases were excluded from the study: 2 cases were serous endometrial cancer, which is not the object of study in this work, 4 cases were represented by atypical mucinous proliferation, 2 were combinations of serous cancer with atypical endometrial hyperplasia, 2 cases were a combination of clear cell carcinoma and endometrioid endometrial cancer, 3 cases – endometrial polyp without endometrial hyperplasia, 2 cases – proliferative endometrium. In 35 patients, it was not possible to adequately assess the histological material due to its quality or quantity.

The remaining 107 cases after revision according to the 2014 WHO morphological criteria were divided into three groups: 35 patients diagnosed with benign endometrial hyperplasia, 36 with endometrioid intraepithelial neoplasia, 36 with endometrioid adenocarcinoma of the endometrium.

At the same time, in 23 cases (22%) there was a discrepancy in the morphological diagnosis: 18 cases after revision by different pathologists, in 5 cases after the analysis of biopsy and surgical material.

Morphological criteria WHO 2014:

1. Endometrial hyperplasia without atypia – increased proliferation of glands with a change in their shape and size, accompanied by an increase in the gland / stroma ratio compared to proliferative endometrium, but without significant signs of cellular atypia.

Synonyms: benign EH, simple non-atypical EH, complex non-atypical EH, simple EH without atypia, complex EH without atypia.

Histological picture: The spectrum of changes is typical. Glands vary in size and shape and can be separated from each other by varying amounts of stroma,

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The cytoplasm of neoplastic cells is eosinophilic or granular. Nuclear atypia is usually mild to moderate with inconspicuous nucleoli, except in poorly differentiated adenocarcinoma. The mitotic index is highly variable. In contrast to EIN, stromal invasion is present, there is no intermediate stroma (confluent or cribriform glands), there is damaged endometrial stroma (desmoplastic reactions) or papillary structures.

2.3 Method of immunohistochemical research

Immunohistochemical examination was performed according to a standard protocol (Table 4) with determination of the tumor receptor status (estrogen and progesterone receptors), expression of BAF250a (ARID1A), PTEN, CTNNB1(β- catenin), MSH6, MSH2, PMS2, MLH1, PAX2, PD-L1, proliferation index (Ki-67) (Table 3).

Table 3 – Specification of antibodies used for differential diagnosis of endometrial hyperplasia without atypia, with atypia and endometrial adenocarcinoma

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Next, sections were applied to glasses with poly-L-lysine coating by Menzel and dried at a temperature of 35-37 °C for 60 minutes. After that, the material was dewaxed in two o-xylenes, two minutes each, washing and dehydration in two 96% alcohols for five minutes each and 70% alcohol for ten minutes. In the next step, the glasses were washed in distilled water and exposed to antigen unmasking in a DAKO citrate buffer (Target Retrieval Solution pH 6.0, code S169984-2) in a water bath at a temperature of 95 ° with a duration of 40 minutes. After that, they were cooled to room temperature together with the buffer in which the unmasking was carried out, and washed in a Tris buffer for 10 minutes. Then the slices were outlined with a paraffin pencil (DakoCytomation Pin, code S200230-2) and re-lowered into the Tris buffer (TBS pH 7.4) for 10 minutes. After that, it is treated with 3% hydrogen peroxide for 5 minutes to suppress endogenous peroxidase.

To dilute the first antibodies, a buffer (Antibody Diluent with Background Reducing Components by DakoCytomation, code S3022) was used to dilute antibodies with a component that prevents their nonspecific binding. The first antibodies were kept for 30 minutes at a constant temperature of 30 °C, which was maintained using a LEICA HI1220 heating plate (Histoplat). Then the glasses were washed in a Tris buffer for 10 minutes.

To determine the binding of the first antibodies to tumor cells, the DAKO EnVision Flex polymer imaging system with diaminobenzidine as a chromogen was used. Then the sections were washed in distilled water and additionally stained with Mayer hematoxylin for 1-2 minutes. Then they were washed in water for 15 minutes, dehydrated in 96% alcohol for 10 minutes and clarified with carbol xylene or xylene for 5 minutes.

The slices were enclosed in special liquids Ultramounts, Paramount, Aquatic Mounting Medium, Ready-to-Use by DAKO code S302580-2.

Evaluation and interpretation of the results was performed using light microscopy (Olympus CX41, DP72 camera) at magnifications of 200 and 400.

The PD-L1 staining was carried out on a Ventana BenchMark Ultra closedtype autostainer using ready-to-use PD-L1 clone SP 263 antibodies and a two-stage

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Table 4 continuation

The evaluation of immunohistochemical staining was carried out for each indicator in accordance with standard and generally accepted methods of determination. Some of the indicators have a qualitative reaction and were determined by the presence or absence of staining of the cell cytoplasm and nuclei. The other part was quantified. The methodology for evaluating each indicator is presented below.

Quality indicators:

1.PAX2 – the absence of nuclear staining was assessed as the absence of expression of this gene. The presence of colored and unpainted nuclei in the preparation was assessed as a partial loss of expression of this gene.

2.PTEN – the absence of cytoplasmic and nuclear staining was assessed as a complete loss of PTEN expression. The presence of stained and unpainted cells in the preparation was assessed as a partial loss of expression of this gene.