ROLE OF STROMAL MICROENVIRONMENT IN THE FORMATION OF INVASIVE, ANGIOGENIC, AND METASTATIC POTENTIAL OF ENDOMETRIOID CARCINOMA OF ENDOMETRIUM.

The aim of the study was to determine the association of indicators of the progression of endometrioid carcinoma of the endometrium (ECE) with the type of stromal microenvironment, the counts of CXCL12+ fibroblasts and CD163+ macrophages, and the expression of the chemokine CXCL12 and its receptor CXCR4 in tumor cells. MATERIALS AND METHODS: Histological preparations of ECE samples (n = 51) were analyzed. Expression of CXCL2 and CXCR4 antigens in tumor cells, the content of CXCL12+ fibroblasts and CD163+ macrophages, and the density of microvessels were determined by the immunohistochemical method. RESULTS: Groups of ECE with desmoplastic and inflammatory stromal reactions were delineated. The majority (80.0%) of tumors with desmoplasia were of low differentiation grade, deeply invading the myometrium; 65.0% of patients with these tumors were at stage III of the disease. In ECE cases of stages I-II, 77.4% of ECE showed an inflammatory type of stroma. The high angiogenic and invasive potential of EC of stages I-II was associated with an inflammatory stromal type, high counts of CD163+ macrophages and CXCL12+ fibroblasts in the tumor microenvironment, high expression of the chemokine receptor CXCR4, and reduced expression of its ligand CXCL12 in tumor cells. In the majority of EC of stage III, the increase in angiogenic, invasive, and metastatic potential was accompanied by the presence of desmoplastic stroma, increased expression of CXCR4 in tumor cells, and a high count of CXCL12+ fibroblasts. CONCLUSIONS: The obtained results showed that the morphological architecture of the stromal ECE component is related to the molecular features of its constituents and tumor cells. Their interaction modulates the phenotypic characteristics of ECE associated with the degree of malignancy.

VALIDATION OF A PANEL OF BIOMARKERS ASSOCIATED WITH AGGRESSIVE PHENOTYPE OF ENDOMETRIOID CARCINOMA OF ENDOMETRIUM.

AIM: To evaluate the prognostic significance of a panel of biomarkers for the identification of a highly malignant molecular subtype of endometrioid carcinoma of the endometrium (ECE). MATERIALS AND METHODS: The expression of a number of markers (CD24, CD44, E2F1, FOXP3, Her2/neu, p21WAF1/CIP1, p53, ß-catenin, vimentin, Е-cadherin, с-Myc, cyclins D1 and Е1) was determined by the immunohistochemical method in the samples of resected tumors of 127 patients with ECE of I-II stage. The Kullback method and the PanelomiX web tool were used to assess the informativeness and identify the aggressive subtype of ECE. The associative relationships of the studied markers were determined using the STRING v11 database. RESULTS: The study of the prognostic significance of a number of biomarkers in ECE has revealed the informativeness, high specificity and sensitivity (> 95%) of the Ñ€53+FOXP3-c-Myc+ phenotype, which is associated with a more aggressive tumor process. Bioinformatics analysis confirmed the correlative relationships between p53, FOXP3 and c-Myc, which are significant prognostic markers associated with cancer progression in ECE patients. CONCLUSIONS: The identified molecular phenotype of ECE (р53+FOXP3-c-Myc+) has differential and prognostic significance and objectively reflects a highly malignant subtype of this form of cancer.

Expression of microRNA in tumor cells of endmetrioid carcinoma of endometrium.

BACKGROUND: It is known that more than half of the genes encoding human proteins are regulated by various microRNAs (miRNAs, miR), the expression of which may be associated with various pathological conditions. At the same time, the question of assessing the relationship between the expression of particular miRNAs and the aggressive molecular subtype of endometrial cancer remains open. Aim of the study was to determine the relationship between the expression of miR-34a, miR-125b, miR-142 and miR-101 in endometrioid carcinomas of the endometrium (ECE) and the features of the disease course. MATERIALS AND METHODS: The samples of surgical material of 51 patients with ECE (mean age 59.8 ± 7.1 years), I-III stage were investigated using morphological, immunohistochemical methods, real time polymerase chain reaction (PCR), cytofluorometry. RESULTS: In endometrial tumors with high proliferation index (< Me), the expression of miR-34a, miR-142 and miR-125b significantly decreased (1.8, 2.7 and 1.5 times, respectively) compared with those in ECE with low proliferation index (> Me). The expression of all studied miRNAs was lower in G3 tumors and those that deeply invaded the myometrium compared to G2 carcinomas and tumors with an invasion of > 1/2 myometrium and significantly decreased in tumors of patients with low stage III compared with stage I-II. The high (< Me) microvessel density in ECE was associated with a significant decrease of miR-125b and miR-101 expression, and the presence of signs of epithelial-mesenchymal transition - with a decreased expression of miR-34a and miR-101. CONCLUSIONS: The study revealed a significant heterogeneity of expression of miR-34a, miR-125b, miR-142 and miR-101 in ECE, which is associated with changes in morphofunctional characteristics of endometrial carcinoma.

Molecular phenotype of high-grade endometrioid carcinoma of the endometrium.

BACKGROUND: Prognosis of the course of tumor progression is one of urgent problems of clinical oncology. A relevant specificity of endometrial cancer is its clinical polymorphism within the same histological type of the disease. The search for molecular-biological features associated with the aggressive phenotype of endometrioid carcinomas is indisputably urgent. AIM: To study molecular-biological features of endometrioid carcinoma of the endometrium (ECE) and to identify the molecular subtype of tumors with high potential of malignancy. MATERIALS AND METHODS: Surgical specimens of 127 patients with EC, stages I-II, aged 36-72 (the average age - 59.3 ± 3.2) were studied using morphological and immunohistochemical methods. The multivariant analysis with the Kullback's informative measure and PanelomiX were used to estimate the significance of the expression of specific biomarkers. RESULTS: The expression of a complex of multifunctional markers was evaluated in ECE cells of different malignancy stage: p53, FOXP3, p21WAF1/CIP1, р16INK4a, E2F1, cyclins Е and D1, Her2/neu, с-Myc, Е-cadherin, ß-catenin, vimentin, CD44, CD24. A triad of biomarkers with threshold expression levels was determined (р53 < 45%; FOXP3 > 14%; с-Myc < 10%). The high expression of oncogene c-Myc and oncosuppressor p53 along with the low level of FOXP3 in tumor cells of ECE was associated with high proliferative potential, low differentiation grade, and deep invasion of a tumor into the myometrium. CONCLUSIONS: The molecular phenotype of ECE, most informative in terms of specificity and sensitivity (95%) - р5highFOXP3lowc-Mychigh, was first characterized, which would help identify a high-grade subtype of this cancer form.

Morphological characteristics and expression of adhesion markers in cells of low differentiated endometrial carcinoma.

The aim of the study was to evaluate the morphological features of endometrioid carcinoma of the endometrium (ECE) of low differentiation grade with different invasive potential and to characterize their molecular phenotype by the expression of a number of adhesion markers. MATERIALS AND METHODS: We have studied the samples of operation material of 37 patients with ECE of low differentiation grade with deep invasion (> ½ myometrium), n = 26, and with invasion < ½ myometrium, n = 11, with the use of morphological and immunohistochemical methods, and flow cytometry. RESULTS: In the morphological study of tumors with deep invasion in the myometrium, we have detected pronounced structural heterogeneity, which became the basis for the discretion of two groups of tumors with different characteristics of morphological phenotypes. In the majority of cases, solid layers and glandular-like structures are detected, and the similarity of the tumor epithelium with the elements of the endometrium is completely lost. In such tumors high expression of adhesion molecules - E-cadherin, CD44, CD24, and ß-catenin and low expression of the marker of mesenchymal tissues - vimentin were determined. Other tumors were characterized by morphological features of the epithelial-mesenchymal transition (EMT), with the decrease of the expression of E-cadherin, ß-catenin, CD24, CD44, and a significant increase in vimentin expression in comparison with these indices in tumors without signs of EMT. In ECEs that invade < ½ myometrium, the morphological indices of malignancy were less pronounced, which was associated by the changes in the expression of the molecular markers. CONCLUSION: This comprehensive study has established associations between the morphological heterogeneity of ECE and the expression of adhesion markers and vimentin, which is important for understanding the mechanisms of tumor cell migration.

Markers of the epithelial-mesenchymal transition in cells of endometrial carcinoma.

AIM: To study the expression of adhesion markers (E-cadherin, ß-catenin and vimentin) associated with epithelial-mesenchymal transition (EMT) and their role in progression of endometrial carcinoma (EC). MATERIALS AND METHODS: Expression of E-cadherin, ß-catenin and vimentin was studied immunohistochemically in the samples of surgical material of 55 EC patients stage I-III. The proliferation index was determined by flow cytometry. RESULTS: In the group of vimentin-negative EC, tumors of low differentiation grade and deep invasion in myometrium as well as high expression of E-cadherin and ß-catenin prevailed compared with the cases with high expression of vimentin. In addition, in EC with high expression of vimentin, an increase in the number of cells with expression of E-cadherin in the cytoplasm (78.9 ± 3.6%) and ß-catenin with cytoplasmic-nuclear localization (73.7 ± 3.2%) was observed compared with these indices in vimentin-negative tumors (45.4 ± 4.2%, p < 0.001 and 54.5 ± 2.6%, respectively, p < 0.005), which may indicate EMT-associated changes in EC with high expression of vimentin. CONCLUSIONS: The progression of the endometrioid carcinoma may occur in the setting of various molecular changes, in particular, with decreased expression of E-cadherin and ß-catenin and high expression of vimentin, or in the absence of vimentin, utilizing other mechanisms of regulation of proliferative and metastatic potential.

Phenotypic features of endometrial tumors in patients with family history of cancer.

AIM: To determine the peculiarities of expression of a number of proteins-regulators of the cell cycle in endometrial cancer (EC) cells in patients with a family history of oncological pathologies. PATIENTS AND METHODS: 95 EC patients (stage І-ІІ) were included into the study. Clinical-genealogical analysis was performed. 54 patients (group I) had healthy relatives, and in families of 41 patients (group II) an aggregation of malignant tumors of different genesis (mainly tumors of the gastrointestinal tract and the female reproductive system) was recorded. p53, p21WAF1/CIP1, p16INK4a, and Ki-67 were assessed immunohistochemically in the surgical samples. RESULTS: In the majority of patients, both from group I and II, moderately differentiated tumors were observed (in 38.9 and 46.3% of cases, respectively), mainly with deep myometrium invasion (64.8 and 58.5% of cases, respectively). In EC patients from group II, a significantly higher number of p16INK4a-positive cells (17.7 ± 1.7%; p = 0.001) and lower number of p53-positive (30.9 ± 3.2%; p = 0.05) and Ki-67-positive (26.9 ± 2.7%; p = 0.048) cells was observed compared to those in tumors of patients from group I (12.0 ± 1.6; 37.7 ± 2.8 and 36.7 ± 3.4%, respectively). CONCLUSION: Phenotypic features of the EC in the patients with family history of cancer differ from those in tumors of patients without such aggregation. The biological heterogeneity of EC seems to relate to the oncogenealogical history of patients. Also this biological heterogeneity is linked to the molecular features of EC cells, which affects cancer aggressiveness and the course of the disease.

The study of chromosomal instability in patients with endometrial cancer.

AIM: Study is devoted to evaluation of sensitivity of peripheral blood T-lymphocytes (PBL) of patients with endometrial cancer (EC) to genotoxic effect of bleomycin and detection of patients with hidden chromosomal instability. METHODS: PBL of 24 EC patients (mean age 58.9 ± 2.9) and 10 healthy women-volunteers (mean age 55.7 ± 2.3) were subjected to cytogenetic analysis. RESULTS: Mean spontaneous level of chromosomal aberrations (CA) per 100 analyzed lymphocytes (CA/100) of healthy women has equaled 2.7 ± 0.6, i.e. has not exceeded maximal values of healthy population and was significantly lower (p < 0.05), than in PBL of EC patients (6.9 ± 0.6). After incubation of PBL with bleomycin, number of CA/100 significantly was increased both in control (11.5 ± 1.3) and in EC patients (21.9 ± 1.0). Spontaneous chromosomal instability has been observed in 41.7%, increased sensitivity to bleomycin - in 54.2% and hidden chromosomal instability in 37.5% of patients with EC. It has been shown that level of specific damage of genome in EC patients has constituted 2·10(-5), and after exposure with bleomycin, it was increased 4.5 times (9·10(-5)) that was significantly higher (p < 0.05) compared to control (8·10(-6) and 1.0·10(-5), respectively). CONCLUSION: These results have demonstrated that PBL of most patients with EC are characterized by apparent genome alterations, which are manifested by increased number of spontaneous and induced chromosomal damages, hypersensitivity to mutagens and hidden chromosomal instability.

Risk assessment of cancer of the female reproductive system.

AIM: To create an information resource concerning multifactorial oncological diseases of the female reproductive system. MATERIALS AND METHODS: A comprehensive search of the literature in the PubMed and Ukrainian scientific sources published from 1995 to 2014 and the results of researches performed in R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine. Development environment of information resource "Multifactorial oncological disease" was Borland Delphi. RESULTS: The information content of web page concerning cancers of the female reproductive system was posted in the information resource "Multifactorial oncological disease". The assessment algorithm of genetic contribution to cancers of the female reproductive system and recurrent risk of cancer development in families have been described. These algorithms can be used in assessment of contribution of genetic and environmental factors in the development of malignant tumors.

Expression of the estrogen and progesterone receptors as prognostic factor in serous ovarian cancers.

AIM: To study the expression of estrogen receptors (ER) and progesterone receptors (PR) and proliferation marker Ki-67 in ovarian tumors using immunohistochemistry, and evaluate possible prognostic significance of these markers. METHODS: Immunohistochemical evaluation of Ki-67, ER and PR expression was performed on serous ovarian cancer (OC) tissue samples from 81 OC patients. RESULTS: Serous OC is characterised by high proliferative activity and increased expression of steroid hormone receptors compared to nontransfomed ovarian surface epithelium. It has been shown that ER and PR expression levels depend on tumor histologic grade and the stage of the disease, and are variable between tumors of the same grade. The ER and PR expression levels correlate with OC patients' survival. CONCLUSION: Proliferative activity and steroid hormone receptor status along with clinical and morphological characteristics of serous OC possess prognostic significance and may be used for evaluation of the disease course.

Expression of p53, p21WAF1/CIP1, p16INK4A and Ki-67 proteins in serous ovarian tumors.

AIM: The analysis of p53, p21(WAF1/CIP1), p16(INK4A) and Ki-67 expression in serous ovarian carcinomas of different grade. MATERIALS AND METHODS: In total, 43 ovarian adenocarcinomas and 8 non-altered ovarian epithelial tissues were immunohistochemically investigated for expression of Ki-67, p53, p21(WAF1/CIP1) and p16(INK4A). RESULTS: It has been shown that expression of Ki-67, p53, p21(WAF1/CIP1) and p16(INK4A) in non-altered ovarian epithelial tissue is absent. Serous ovarian carcinomas are characterized by high proliferative activity (PI Ki-67 = 30.0 +/- 0.3%), p53 and p16(INK4A) overexpression (LI is 40.3 +/- 0.3% and 31.1 +/- 0.6% respectively) and low expression of p21(WAF1/CIP1) (LI = 6.8 +/- 0.3%). The association between expression of these markers and ovarian tumor grade was defined: the maximal level of Ki-67, p53 and p16/(INK4A) and minimal of p21(WAF1/CIP1) expression were observed in G3 tumors. So, low p21(WAF1/CIP1) expression (LI < 7.0%) combined with p16(INK4A) overexpression is considered to be the factor for a poor prognosis in serous ovarian cancer. CONCLUSIONS: The present study has indicated that biomolecular markers of cell proliferation along with traditional clinical and morphologic characteristics can be used for differential diagnostics of ovarian tumors.

Different trends of p53, MDM2 and p14 ARF expression patterns in endometrial adenocarcinomas versus hyperplasia.

AIM: To study the expression of p53, MDM2, and p14 ARF in the highly, moderately and low differentiated endometrial adenocarcinomas, compared to hyperplasia. MATERIAL AND METHODS: Surgical material and the scrapes of endometrial cancer, glandular and atypical hyperplasia patients. Expression of 53, MDM2 and p14 ARF was evaluated by immunohistochemical method using respective monoclonal antibodies. RESULTS: High p53 expression level is accompanied by decreased level of MDM2 expression in endometrial cancers. On contrary, in endometrial hyperplasia, there was clear connection between the expression levels of p53 and MDM2. We hypothesize that the high p53 and low MDM2 levels in endometrial cancers could arise due to the inhibition of transcriptional activity of p53 by its binding to estrogen receptors. CONCLUSION: High p53 expression level with low MDM2 and p14 ARF levels may be the characteristic features of low differentiated endometrial carcinoma.

Expression of the cell cycle regulators p53, p21(WAF1/CIP1) and p16(INK4a) in human endometrial adenocarcinoma.

AIM: To study correlation links between expression level of proteins p53, p21(WAF1/CIP), p16(INK4) and proliferative potential in human endometrial adenocarcinoma (EC). MATERIAL AND METHODS: The immunohistochemical analysis of expression level of Ki-67, p53, p21(WAF1/CIP) and p16(INK4) was carried out on surgically resected endometrial cancer samples (n = 74). Scrapes of normal endometrium from 10 patients with polyps of cervical canal of the uterus served as the control. RESULTS: The data showed that endometrial malignant tumors possess high proliferative activity (proliferation index was 37.3 +/- 0.2%), overexpression of p53 (labeling index (LI) = 46.1 +/- 0.5%) and high expression of p21(WAF1/CIP) (LI = 11.2 +/- 0.4%) and p16(INK4) (LI = 12.0 +/- 0.2%). In low differentiated endometrial adenocarcinomas the highest level of Ki-67, p53 and p21(WAF1/CIP) expression and lowest content of p16(INK4) protein were observed. CONCLUSION: The indicated markers may be used along with traditional morphological and clinical characteristics for diagnosis of endometrial neoplasia.

[Cytogenetic changes in the Namalwa cell line of human malignant lymphoma induced by inhibitors of DNA replication and synthesis]. / Tsitogeneticheskie izmeneniia v kletkakh perevivaemoi linii Namalwa zlokachestvennoi limfomy cheloveka, indutsirovannye ingibitorami replikatsii i sinteza DNK.

Namalwa cells originating from the malignant human lymphoma have been analyzed cytogenetically upon short-time exposure to subtoxic doses of inhibitors of DNA replication and synthesis, either etoposide or fludarabine. The intact cells were characterized by the modal class of the chromosomes within the diploid range with the proportion of the aberrant cells amounting to 16.0 +/- 0.5%. Upon exposure to etoposide the percentage of the aberrant cells increased amounting to 26.1 +/- 2.9 through 39.8 +/- 1.7% depending on the duration of the exposure and the dose of the drug. At the same time the number of the polyploid cells increased but the modal class retained within the diploid range. Upon exposure to fludarabine the percentage of the cells with the aberrant chromosomes increased to 57.1 +/- 2.9%. Two modal classes appeared--the first approaching the diploid number and the second being polyploid. The exposure to either etoposide or fludarabine resulted in increasing number of the chromatide aberrations with more frequent involvement of #1, #2, #5, #6, #7, #11, #13, #14, #16 and #17 chromosomes. The data obtained have shown the susceptibility of Namalwa cells to the subtoxic concentrations of the inhibitors of DNA synthesis and replication used in the study resulting in the survival of the novel clones resistant to the drugs.

Effects of etoposide and fludarabine in subtoxic doses on karyotype of human malignant lymphoid Namalwa cells.

We studied changes in the karyotype of transplanted Namalwa cells induced by DNA-damaging antitumor preparations etoposide and fludarabine in subtoxic doses. The relative number of cells containing increased number of chromosomes and the incidence of chromatid aberrations with primary damage to chromosomes 2, 5, 11, 16, and 17 increased. Cytogenetic changes developed even after short-term incubation of cells with antitumor preparations and were observed during further culturing in a medium not containing etoposide or fludarabine.

[The determination of chromosome fragile sites in the peripheral blood lymphocytes of patients with colorectal cancer taking into account the cancer pathology predisposition in the familial anamnesis]. / Opredelenie khromosomnoi sait-lomkosti v limfotsitakh perifericheskoi krovi bol'nykh kolorektal'nym rakom s uchetom otiagoshchennosti semeinogo anamneza po onkopatologii.

Results of comparative study of spontaneous and 5-bromdeoxyuridine-induced fragility of peripheral blood lymphocytes chromosomes in 9 patients with colorectal adenocarcinoma were presented. It was shown the increase of average spontaneous level of chromosomal fragility in patients with tumor aggregation in family as well as without it to 4.5 +/- 1.0 and 5.3 +/- 1.1 per 100 tested cells, accordingly. The increase of average level of damaged chromosomes in spectrum of rare sites to 12.5 +/- 2.6 in the patients with tumor aggregation in pedigree comparing to the patients without oncopathology in family 8.0 +/- 1.7 was observed. The most number of rare fragile sites was observed in 1q21 site of the chromosome 1. Possible connection between fragile sites of chromosomes in normal cells and malignant processes in the patients with colorectal cancer is discussed.

[Structural chromosome aberrations in the peripheral blood lymphocytes in patients with precancer and endometrial cancer]. / Strukturnye aberratsii khromosom v limfotsitakh perifericheskoi krovi u bol'nykh predrakom i rakom éndometriia.

The level of chromosomal aberrations in cultured blood lymphocytes from patients with hyperplasia, atypical hyperplasia of endometrium and endometrial cancer was studied. Chromosomal aberration frequency significantly increased up to 5.000 +/- 00.47% in patients with atypical and endometrial hyperplasia and to 5.21 +/- 00.53% in patients with endometrial cancer in comparison to 0.67 +/- 0.21% in the control. All indices were more pronounced in endometrial cancer patients with pathology in pedigree.

[A cytogenetic examination of persons subjected to radiation exposure in certain regions of Ukraine]. / Tsitogeneticheskoe obsledovanie lits, podvergshikhsia radiatsionnomu vozdeistviiu v nekotorykh regionakh Ukrainy.

We have summarized the results of cytogenetic studies of peripheral blood lymphocytes conducted in the institutions of Kiev and Kharkov in persons irradiated after the accident of the Chernobyl NPP. The average level of chromosomal aberrations and the appearance of cytogenetic markers characteristic of their irradiation effect was higher in the examinees in comparison with the control. The increase in the number of aberrations in certain Kiev residents as well as the development of lymphogranulomatosis in some liquidators of the accident were detected. The increase in the frequency of chromosomal aberrations in liquidators of the accident was accompanied by the reduction in the level of reparative DNA synthesis of blood lymphocytes. The necessity to expand cytogenetic examination in persons that participated in the liquidation of the accident on the ChNPP and Kiev inhabitants is discussed.

[Polymorphism of the C-band segments of chromosomes 1, 9 and 16 in the peripheral blood lymphocytes of patients with endometrial cancer]. / Polimofizm C-segmentov 1, 9 i 16 khromosom v limfotsitakh perifericheskoi krovi bol'nykh rakom éndometriia.

The paper presents results of investigation of constitutive heterochromatin of chromosomes 1, 9 and 16 in 23 patients with endometrial cancer and 5 women of the control group. The analysis was carried out on slides obtained by the routine method of the peripheral blood cell culture. The C-band segment variability was studied by the C-binding method. The investigation established an increase of polymorphism of constitutive heterochromatin in cancer patients, extreme variants and heteromorphism of homologues of chromosomes 1, 9 and 16. It is shown that polymorphism of the C-band on chromosomes 1, 9 and 16 in patients with cancer in their families increases as compared to the patients without cancer pathology in pedigrees.

[Familial-populational studies of endometrial cancer: segregation and genetic dispersion analyses]. / Semeino-populiatsionnye issledovaniia raka éndometriia: segregatsionnyi i genetiko-dispersionnyi analiz.

A clinico-genealogic investigation was carried out in 216 patients with endometrial cancer. Familial accumulation of endometrial and other cancer incidence was established. The segregation rates appeared to be lower than those expected from simple Mendelian models (2-11%). A multifactorial nature of endometrial cancer in overall susceptibility to the disease was found to be at 61%. A genetic correlation analysis showed endometrial cancer to share common genes with breast and gastric cancer in females. Tables of recurrent risk of the disease for relatives were prepared to be used in medico-genetic counseling.