[Exosomes' role in intercellular interactions in different variants of lung injury in fatal cases of COVID-19]. / Rol' ekzosom v mezhkletochnykh vzaimodeistviyakh pri razlichnykh variantakh porazheniya legkikh v letal'nykh sluchayakh COVID-19.

BACKGROUND: Extracellular vesicles are surrounded by a phospholipid bilayer, carrying various active biomolecules and participating in many physiological and pathological processes, including infectious ones. OBJECTIVE: To research the role of exosomes in intercellular interactions in the pathogenesis of various types of lung damage in fatal cases of COVID-19. MATERIAL AND METHODS: We conducted a clinical and morphological analysis of 118 fatal cases caused by coronavirus infection in Moscow. We selected 32 cases with morphological signs of various types of lung lesions for immunohistochemical reaction (IHC) with antibodies against tetraspanin proteins (CD63, CD81), which are involved in the assembly of exosomes, as well as with antibodies against viral proteins: nucleocapsid and spike protein. We determined the main producing cells of extracellular vesicles and cells containing viral proteins, carried out their comparison and quantitative analysis. RESULTS: IHC reaction with antibodies against CD63 showed cytoplasmic granular uniform and subapical staining of cells, as well as granular extracellular staining. We determined similar staining using antibodies against viral proteins. Extracellular vesicles were found in the same cells as viral proteins. The main producing cells of vesicles and cells containing viral proteins were found to be macrophages, type II pneumocytes, and endothelial cells. CONCLUSION: Taking into account the results of the literature, the localization of viral proteins and extracellular vesicles in the same cells indicates the key role of vesicles in the pathogenesis of various forms of lung damage by the SARS-CoV-2 virus, in the dissemination of the pathogen in the organism, which leads to interaction with the adaptive immune system and the formation of immunity.

[Features of the cell composition of inflammatory infiltrate in different phases of diffuse alveolar lung damage with COVID-19]. / Osobennosti kletochnogo sostava vospalitel'nogo infil'trata v raznye fazy diffuznogo al'veolyarnogo povrezhdeniya legkikh pri COVID-19.

BACKGROUND: Acute respiratory distress syndrome (ARDS) with COVID-19 has a worse prognosis than ARDS with other diseases. Mortality from ARDS with COVID-19 is 26.0 - 61.5%, and due to other causes - 35.3-37.2%. OBJECTIVE: To find of the correlation between polymorphonuclear leukocytes (PMNs), lymphocytes, and macrophages in the cellular composition of the inflammatory infiltrate at different stages and phases of diffuse alveolar damage (DAD) with COVID-19, analyzing the autopsy material. MATERIAL AND METHODS: The lung tissue of 25 patients who died from ARDS with COVID-19 without a secondary bacterial or mycotic infection, another thanatologically significant pathology of the lungs, was studied. To study the cellular composition of the inflammatory infiltrate and the dynamics of its changes a double immunohistochemical analysis of the expression of antibodies to CD15, CD3, and CD68 was used. RESULTS: The inflammatory infiltrate and intraalveolar exudate in the exudative phase of DAD was represented by 56.8% of PMNs (CD15-positive cells; hereinafter - the average value of the percentage of positive cells to the total number of cells of the inflammatory infiltrate), 6.9% - lymphocytes (CD3-positive cells) and 19.5% macrophages (CD68-positive cells). In the early stage of the proliferative phase: 14.1% PMNs, 38.7% lymphocytes and 13.5% macrophages. In the late stage of the proliferative phase: 11.3% PMNs, 14.5% lymphocytes and 39.3% macrophages. CONCLUSIONS: In the exudative phase of DAD a statistically significant predominance of PMN was revealed, which could determine the main volume of lung damage and the severity of ARDS with COVID-19. In the early stage of the proliferative phase of DAD, a statistically significant change in the composition of the inflammatory infiltrate was revealed to compare with the exudative phase: a significant decrease in the content of PMNs relative to the total number of cells in the inflammatory infiltrate; an increase in the number of lymphocytes, which is probably associated with the start of organization and repair processes. In the late stage of the proliferative phase of DAD, compared with its early stage, was revealed a statistically significant increase in the number of macrophages in ratio.

The Relationship of Immunoexpression of Ki-67 and Hsp70 with Clinical and Morphological Parameters and Prognosis of Papillary Thyroid Cancer.

We studied the correlations of the expression of Ki-67 proliferation marker and localization of heat shock protein Hsp70 in tumor cells of papillary thyroid cancer with some clinical and morphological parameters and a 10-year survival prognosis. High variability of the proliferative activity (by Ki-67 index) of tumor cells was revealed. It was found that Hsp70 protein has different localization in tumor cells: cytoplasmic, nuclear, nucleolar, and mixed. Nuclear translocation of the Hsp70 protein correlated with the stage of the process and the prognosis of the disease, but did not correlate with sex, tumor size, capsule invasion, and lymph node metastasis. Ki-67 index of tumor cells correlated with sex, stage of the process, size of tumor formation and prognosis, but did not correlated with age, invasion into the capsule and metastasis to the lymph nodes. Depending on the variants of Hsp70 and Ki-67 co-expression, three molecular types of papillary thyroid cancer can be distinguished: favorable, intermediate, and poor prognosis.

Loss of Heterozygosity in BRCA1 and BRCA2 Genes in Patients with Ovarian Cancer and Probability of Its Use for Clinical Classification of Variations.

Changes (or variants) in BRCA1 and BRCA2 gene sequences can have different lengths and clinical significance: from single nucleotide variants (SNV) and short insertions/deletions (<50 bp) to extended deletions and duplications (so-called copy number variations, or CNV). According to their clinical significance, all variants can be divided into pathogenic, likely pathogenic, variants of uncertain significance, likely benign, and benign. Moreover, variants can be germinal (i.e. inherited from parents) and somatic (arising in the process of development of the organism). A specific somatic event is loss of heterozygosity (LOH), i.e. transition of one or many point and short variants from heterozygous to homozygous state. Such an event can be the key to the development of carcinogenesis for cells carrying a pathogenic variant, if we consider it within the framework of the Knudson's two-hit carcinogenesis theory. We studied the prevalence and nature of LOH in of ovarian cancer samples carrying or not carrying a pathogenic variant. To this end, a full coding sequence of BRCA1/2 genes was determined in 30 pairs of DNA samples isolated from blood cells and paraffinized histological blocks of patients on a MiSeq Illumina instrument. Analyss of the obtained reads revealed 9 pathogenic point and short variants (30% patients): 6 germinal (20%) and 3 somatic (10%), and 8 somatic CNV (3 deletions and 5 duplications of several or all exons of the BRCA1 gene). LOH was detected in 70% patients; among the carriers of pathogenic variants - in 83%. For pathogenic variants, the percentage of reads with the alternative allele increased more often than for benign variants located in another gene, or detected in other patients (67% vs. 44%). However, the difference was statistically insignificant, which can be due to insufficient number of patients. Only in 3 of 21 cases of LOH (14%), it can be attributed to CNV. In other cases, LOH is most likely determined by gene conversion, but further research is needed.

Mast Cells in Renal Cancer: Clinical Morphological Correlations and Prognosis.

Morphofunctional activity of mast cells in renal cancer is studied on specimens from the center of the tumor, peritumorous zone (with an obligatory fragment of the pseudocapsule), and from renal tissues maximally distant from the tumor. The counts and areas of tumor cells were maximum in the peritumorous zone in comparison with intact kidney tissue and central zone of the tumor. Analysis of correlations revealed a direct correlation between the tumor cell count in the peritumorous zone and some clinical morphological parameters of the tumor (anaplasia degree, size, presence of invasion and metastases, and angiogenesis). The count of mast cells in the renal peritumorous zone inversely correlated with 5-year survival: 100% cumulative 5-year survival in patients with mast cell count <6.7±1.6 per field of view.

[Morphometric and molecular biological features of femoral head tissue in different nosological entities of coxarthrosis]. / Morfometricheskie i molekulyarno-biologicheskie osobennosti tkani golovki bedra pri razlichnykh nozologicheskikh formakh koksartroza.

AIM: to comparatively analyze the morphometric and molecular biological characteristics of femoral head tissue in different nosological entities of coxarthrosis. MATERIAL AND METHODS: A total of 95 samples of femoral head tissue extirpated during hip endoprosthesis in patients with coxarthrosis were investigated. Clinical findings were used to identify the following nosological entities of coxarthrosis: dysplastic, postischemic and posttraumatic. Histological, immunohistochemical and morphometric studies were used. Osteoclast resorptive activity was assessed by determining the cytoplasmic expression of TRAcP (9C5, «Cell Marque¼). Vasculogenesis was evaluated by estimating the mean area of vessels with CD34 (QBEnd/10, «Ventana¼) from the positive stained endothelium and by determining the cytoplasmic expression level of VEGF (SP28, «Spring Bio¼) in osteoblasts and osteoclasts. RESULTS: Specific histopathological signs were described for each nosological entity of coxarthrosis. Morphometric analysis could reveal a number of additional characteristics of the magnitude of fibrous changes and the thickness of the articular surface and bone rods. Immunohistochemical assessment of molecular biological parameters, such as the expression level of VEGF and TRAcP, also pointed to the characteristic features of bony tissue in the above-mentioned nosological entities of coxarthrosis. In dysplastic coxarthrosis, the maximal expression level of VEGF was recorded in osteoblasts and the expression of VEGF and TRAcP in osteoclasts remained at the minimum level. The lowest expression of VEGF in osteoblasts was found in posttraumatic coxarthrosis. In postischemic coxarthrosis, the highest expression of VEGF and TRAcP was recorded in osteoclasts. CONCLUSION: The comparative analysis of the morphometric and molecular biological characteristics of femoral head tissue in different nosological entities of coxarthrosis indicated a number of peculiar features. The most specific manifestations of certain morphological and molecular biological signs were identified for each nosological entity of coxarthrosis.

Expression of B23/Nucleophosphamine Nonribosomal Nucleolar Protein in Smooth Muscle Tumors of the Corpus Uteri.

Immunohistochemical study of B23/nucleophosmine (NPM), the key argyrophilic nonribosomal protein associated with the nucleolus-producing regions, in smooth muscle formations of the body of the uterus and myometrium was carried out. The uterus index, number of positively stained nucleoli in the nucleus, integral optical density, and area of positively stained nucleoli were studied by computer-aided image analysis. The results indicated significant differences in the integral optical density and less so in the areas of NPM+ nucleoli in leiomyosarcomas, but not in intact myometrium and leiomyomas. Coefficients of variations in the integral optical density and NPM area proved to be significant parameters for differentiating leiomyosarcomas by size (≤7 cm or larger), evaluating the malignancy grade and stage of the process. NPM parameters in leiomyomas and leiomyosarcomas differed significantly, depending on the levels of Ki-67 and p53 expression and the receptor status of leiomyosarcoma. The index of area and the integral optical density of NPM could serve a accessory objective differential diagnostic criteria in cellular and mitotically active liomyomas and well-differentiated leiomyosarcoma of the corpus uteri.

[THE PROCESSES OF CELL PROLIFERATION, APOPTOSIS AND ANGIOGENESIS IN PATHOLOGICALLY UNCHANGED LUNG AND IN NON-SMALL CELL LUNG CANCER].

The objective of this study was to examine cell proliferation, apoptosis and angiogenesis in pathologically unchanged lung and in non-small cell lung cancer with the use of appropriate markers. The material studied included samples of pathologically unchanged lung (n=80) and those obtained at operations in 237 cases of non-small cell lung cancer. Immunohistochemical methods were used to demonstrate Ki-67, topoisomerase IIalpha (TopoIIalpha), p53, bcl-2, bax, CD34 and podoplanin. Argyrophilic proteins associated with nucleolar organizer regions (Ag-NOR proteins), were detected by impregnation with silver nitrate. The quantitative data were obtained and the peculiarities of the expression of markers associated with proliferation, apoptosis and angiogenesis in pathologically unchanged lung were determined. In the alveoli, the labeling index of Ki-67 and Topolla was less than 1%, AgNOR area index was equal to 1.31 +/- 0.20; p53, bcl-2, bax expression was absent, density of blood vessels was equal to 86 (73-102), while lymphatic vessels were absent. In the bronchus, the labeling index of Ki-67 and TopoIIalpha were respectively 4 (1-8) and less than 1%, AgNOR area index--1.85 +/- 0.24, bax expression--100%, density of blood and lymph vessels--22 (17-31) and 4 (2-7) respectively; p53 and bcl-2 expression were absent. The results were compared with the expression of markers in non-small cell lung cancer. This comparison has fundamental and differential diagnostic value in the study of histopathological lung material. The expression of markers associated with proliferation, apoptosis and angiogenesis changes from pathologically unchanged lung to non-small cell lung cancer.

[Non-small cell lung cancer: HER2 oncogene status].

OBJECTIVE: to study HER2 protein and HER2 gene, their heterogeneity in non-small cell lung cancer. MATERIAL AND METHODS: 218 intraoperative non-small cell lung samples were examined using tissue matrix methods. HER2 protein was determined by immunohistochemistry (clone 4B5, <> and HER2 gene and CEP1 7 were evaluated by in situ hybridization (SISH, <>). RESULTS: Positive and indefinite statuses were found in 59 (27%) and 47 (22%) cases, respectively; intratumor heterogeneity was detected in 32 (30%) cases. Amplification of the HER-2 gene was found in 12 (6%) cases; that of the HER2 gene along with an increase in CEPI 7 was observed in 7 (3%) cases; elevated CEP1 7 levels were seen in 19 (9%) cases. Intratumor heterogeneity of HER2 gene amplification was not found; however, one case of adenocarcinoma showed high-level HER2 gene amplification in the gland-like areas and low-level HER2 gene amplification in the solid areas. HER2-positive status and amplification were more common in adenocarcinoma than in squamous cell carcinoma (p<0.001). There was a moderate correlation between HER2 immunohistochemical status and amplification (r=0.38; p<0.001). CONCLUSION: Thus, in non-small cell lung cancer, there is an elevated HER2 protein level and, well less frequently, altered activity in the HER2 gene (amplification) as a cause of enhanced protein synthesis.

Relationship of argyrophilic proteins of nucleolal organizer regions in Ki-67+ cells with clinical and morphological parameters in lung adenocarcinoma.

Ninety-four lung adenocarcinoma samples obtained during surgeries were examined using a combination of immunohistochemical staining for Ki-67 antigen and silver nitrate staining for argyrophilic nucleolar organizer (Ag-NOR) proteins. In lung adenocarcinoma, we studied the correlation between the area of Ag-NOR proteins in Ki-67(+) cells and clinical and morphological TNM parameters: maximum tumor diameter (T) and stage of the disease and tumor differentiation degree (N). Survival of patients with small area of Ag-NOR proteins in Ki-67(+) cells was higher that in patients with great area of these proteins. The area of Ag-NOR proteins in Ki-67(+) cells is an independent prognostic factor in lung adenocarcinoma. The area of Ag-NOR proteins in Ki-67(+) cells correlates with clinical and morphological TNM parameters and survival of patients with lung adenocarcinoma.

Correlation of HER-2/Neu protein expression and HER2 gene amplification with clinical morphological parameters of nonsmall cell lung cancer.

A total of 218 operation specimens from patients with nonsmall cell lung cancer were examined. The expression of HER-2 protein was studied by the immunohistochemical method, amplification of HER2 gene and centromere part of chromosome 17 (CEP17) - by the in situ chromogenic hybridization method. Moderate correlation (r=0.38, p<0.001) between the immunohistochemical status and HER2 gene amplification was detected. A relationship between TNM clinical morphological parameters and HER-2 protein content and chromosome 17 polysomia (for N parameter), HER-2 protein content, and HER2 gene amplification (for tumor histogenesis) was detected. The content of HER-2 protein and amplification of HER2 and CEP17 correlated with the clinical morphological parameters of patients with nonsmall cell lung cancer.

Argyrophilic proteins of nucleolar organizer regions and proliferative activity of cells in squamous cell carcinoma of the lung.

Argyrophilic proteins associated with nucleolar organizer regions (Ag-NOR proteins) and Ki-67 antigen were analyzed in 118 samples of squamous cell carcinoma of the lungs. Tumors with low and high content of Ag-NOR proteins and Ki-67 index were selected. It was found that the content of Ag-NOR proteins correlated with some clinical and morphological parameters (indexes T and N, tumor size less and more that 3 cm, stage of the disease, and tumor differentiation degree) and survival rate. High survival was associated with low content of Ag-NOR proteins and Ki-67 index and low survival correlated with high content of Ag-NOR proteins and Ki-67 index, while intermediate survival was associated with opposite values of Ag-NOR protein content and Ki-67 index. The tumor size, parameter N, and the content of Ag-NOR proteins had independent effects on patient's survival. In patients with squamous cell carcinoma of the lung without metastases in lymph nodes, survival correlated with the content of Ag-NOR proteins; in patients without metastases, survival correlated with tumor size.

Relationship between the argyrophilic proteins of the nucleolar organizer region and stage of squamous-cell carcinoma of the lung.

Argyrophilic proteins associated with chromosome nucleolar organizer regions (Ag-NOR proteins) were studied in lung squamous-cell carcinoma cells. After routine staining with silver nitrate, the area index and coefficient of variations of Ag-NOR protein were estimated by computer-aided image analysis. The Ag-NOR protein area indexes of tumor cells differed in clinical morphological variants of squamous-cell cancer (differing by T and N parameters, size of the primary node <3 cm or larger, by disease stage, and differentiation degree). Ag-NOR protein variation coefficient can be used for discriminating the tumors by size (<3 cm and larger), presence/absence of metastases, and evaluation of squamous-cell cancer differentiation degree. Ag-NOR protein area index and variation coefficient can be used as additional objective differential diagnostic criteria of clinical and morphological forms of squamous-cell carcinoma of the lung.

Spectrum of TP53 Mutations in BRCA1/2 Associated High-Grade Serous Ovarian Cancer.

Objective: Mutations in TP53 lead to loss of function (LOF) or gain of function (GOF) of the corresponding protein p53 and produce a different effect on the tumor. Our goal was to determine the spectrum of somatic TP53 variants in BRCA1/2 associated high-grade serous ovarian cancer (HGSOC). Methods: The population under study comprised of HGSOCs with pathogenic variants in BRCA1 (n = 78) or BRCA2 (n = 21). Only chemo-naive and platinum-sensitive patients were included in this study. The case group of the IARC database (n = 1249) with HGSOC not stratified by BRCA status was used as a reference. A custom NGS panel was used for sequencing TP53 and mutational hot-spots of other genes, and p53 expression was evaluated by immunohistochemistry for 68 cases of HGSOCs. Results: Somatic TP53 variants (95) or inhibition of wild-type p53 expression (3) were observed in 98 cases. The sample with normal p53 had CDKNA1 variants. The frequency of truncating variants was significantly higher than in the reference cohort (30.3 vs. 21.0%, p = 0.01). Most of the samples (41/68) demonstrated low (or absent) expression of p53, and 17 samples overexpressed p53. LOH was typical for TP53 nonsense variants (14/15). In total, 68/95 samples were LOH positive and showed LOH in all tumorous cells, thus indicating the driver effect of TP53 mutations. Three specimens had KRAS, BAX, APC, and CTNNB1 subclones variants. Conclusion: High frequency of TP53 truncating variants, the low expression of mutant p53, and low incidence of oncogene mutations show potential GOF properties of p53 to be poorly represented in BRCA1/2 associated HGSOC.