e15530 Background: Circulating microRNAs (miRNAs) are promising non-invasive biomarkers for colorectal cancer (CRC), but their diagnostic performance has not been systematically compared across a broad spectrum of miRNAs. This network meta-analysis evaluated the diagnostic potential of circulating miRNAs and their panels for early CRC detection. Methods: A systematic review and network meta-analysis were conducted (PROSPERO CRD420251062003). PubMed and Embase were searched, including studies published from 2000–2025. Histologically confirmed CRC patients and healthy controls were eligible. The primary outcome was AUC. Risk of bias was assessed using QUADAS-2. Statistical analyses were performed in R. Results: Forty-eight studies (69 miRNAs/panels) were included. MiR-155 and MiR-182 served as reference markers. Funnel plots indicated substantial publication bias. Heterogeneity was significant (I²=74.8% [53.0–86.5]; Q=35.7, p<0.001). No miRNA showed statistically significant benefit over references. SUCRA ranking identified three miRNA-21-based panels and two standalone candidates among the top five tools. Conclusions: This network meta-analysis provides the most comprehensive comparative evaluation of circulating miRNAs for early CRC detection. The combination miRNA-21 + miRNA-25 + miRNA-18a + miRNA-22 demonstrated the highest diagnostic potential and represents a promising candidate for non-invasive clinical implementation. Further clinical applicability will be clarified by pooled sensitivity and specificity analyses. Top five circulating miRNAs/miRNA panels by SUCRA ranking. miRNA Pooled AUC SUCRA score 21 + 25 + 18a + 22 0.93 [0.88; 0.96] 0.889 21 + 92a + 221 0.89 [0.78; 0.95] 0.799 21 + 221 + 150 0.82 [0.74; 0.88] 0.797 760 0.92 [0.83; 0.97] 0.774 221 0.81 [0.67; 0.90] 0.754

Myogenic differentiation is a powerful mechanism for generating diverse cell types from fibroblasts. Here, we show that targeted suppression of β-actin by RNA interference in human fibroblasts triggers coordinated molecular and structural changes consistent with trans-differentiation toward SMC-like phenotype. This conversion is marked by upregulation of smooth muscle differentiation markers (α- and γ-smooth muscle actins, SM22, smooth muscle myosin, desmin, vinculin) at mRNA and protein levels, together with distinct morphological alterations: increased cell area, loss of polarity, and reorganization of the actin cytoskeleton. Notably, β-actin-downregulated fibroblasts exhibited a focal adhesion architecture that differed from parental fibroblasts. These findings indicate that β-actin downregulation may provide a novel in vitro method to induce SMC-like differentiation, with potential implications for vascular biology and tissue engineering.

Background. Cervical cancer (CC) is the fourth leading cause of cancer-related morbidity and mortality among women. Despite the established etiologic factor of cervical cancer, especially high-risk human papillomavirus (HPV, human papillomavirus) and available vaccine prophylaxis, the issue persists both in understanding the mechanism of HPV-associated carcinogenesis and in developing new approaches for diagnosis and treatment of cervical cancer. Epigenetic regulation of gene expression by means of microRNAs plays an important role in the pathogenesis of cervical cancer. Therefore, the search for new differentially expressed microRNAs in order to reveal new mechanisms of HPV-associated transformation of tumor cells is of high priority. AIM: To explore microRNAs with differentiated expression in the cervical cancer tissue and to assess the functional potential of their detection in silico. MATERIALS AND METHODS: The spectrum of microRNAs characterizing by modified expression in the tumor tissue of HPV16-positive squamous cell carcinomas of the cervix was determined using NGS sequencing (Next Generation Sequencing) of tumor tissue and of apparently unchanged adjacent epithelium obtained with the use of microdissection. Potential target genes of the investigated microRNAs were identified using MirTargetLink, Tarbase v9.0, and LinkedOmics services. Gene Set Enrichment Analysis was performed using Metascape. The relations between the level of microRNA expression and clinical SCC features (according to TCGA data, CESC sample) were searched using USCS Xena service. RESULTS: NGS-sequencing of paired HPV16-positive specimens of tumor tissue and normal cervical tissue resulted in the identification of 42 differentially expressed microRNAs. Specifically, the levels of 22 microRNAs in the tumor tissue were higher than in the apparently normal adjacent epithelium, while 20 microRNAs demonstrated lower levels in the tumor specimens. Analysis of the potential targets of significant microRNAs revealed multiple functional gene categories, potentially involved in carcinogenesis as well as an association with clinical features. We showed that increased expression levels of microRNA-20b in the tumor tissue correlated with the risk of distant metastases, whereas the lower levels of microRNA-218-1 and microRNA -218-2 were associated with unfavorable prognosis of disease. With regard to microRNA-363, -615, and -769, their increase in cervical cancer was described for the first time, and the potential targets and signaling pathways associated with THEIR EXPRESSION LEVELS WERE IDENTIFIED. CONCLUSION: The search for differentially expressed microRNAs in cervical cancer has revealed a spectrum of microRNAs with potentially important role in the process of malignant transformation and persistence of tumor phenotype. In addition to microRNAs demonstrating functional characteristics described in the world literature, we found microRNAs that play an unknown role in cervical cancer. In this relation, the potentially relevant targets were identified that could be helpful in understanding the mechanisms of carcinogenesis. The data obtained may form the basis for the development of new approaches to the diagnosis and therapy of SCC.

BACKGROUND: Cervical cancer (CC) is the fourth most common cancer among women worldwide in terms of incidence and mortality. High-risk human papillomaviruses (HPVs) are etiologic factor of CC in more than 90% of cases, with type 16 HPV (HPV16) revealed in 50% of cancers. Dysregulation of expression of viral oncogenes E6 and E7 is the main cause of malignant transformation in infected cervical epithelial cells. The mechanisms of impaired expression of these genes are still underexplored. The dysfunction of viral microRNAs may be among the underlying factors. AIM: To analyze the expression of HPV16-associated microRNA-H1 and microRNA-H2 in cervical cancer specimens, evaluate the correlation of their expression to viral load and overall patient survival, and analyze in silico their potential viral and cellular targets. MATERIALS AND METHODS: The expression of HPV16 microRNA-H1 and HPV16 microRNA-H2 was evaluated in the real-time polymerase chain reaction. With this purpose, small RNAs were isolated from 36 specimens of HPV16-positive squamous cell carcinomas of the cervix. Further, the viral load was assessed after calculating the value of HPV16 DNA copies per cell. The association between microRNA expression and the viral load was evaluated using the nonparametric Spearman’s correlation coefficient. Kaplan-Meier curves were plotted to analyze the dependence of 5-year overall survival on the level of viral microRNA expression. The miRanda algorithm and online services mirDB, MR-microT and TargetScan Custom 5.2 were used for in silico search of theoretical microRNA targets. RESULTS: MicroRNA-H1 expression was revealed in 33 of 38 specimens (86.8%), microRNA-H2 was detected in 37 of 38 specimens (97.4%) of HPV16-positive cervical cancer. There was a positive correlation between both microRNA-H1 (r=0.36, p=0.042) and microRNA-H2 (r=0.51, p=0.001) expression and HPV16 viral load. Higher level of expression of viral microRNA-H1 and microRNA-H2 tended to correlate with better overall patient survival. The theoretical microRNA-H1 (E7, E2, E5, L2 and URR) and microRNA-H2 (E1, E2, E5, L2, L1, URR) targets in the HPV16 genome were identified in silico, as well as theoretical cellular targets indicating possible regulation of cellular signaling pathways by means of viral microRNAs, both controlling normal viral cycle and promoting tumor transformation. CONCLUSION: The results of this study demonstrate promising further investigation of the functions of viral microRNAs in relation with the infectious process and virus-induced malignant transformation, and their potential importance in the diagnosis of HPV16-associated cancers.

The widespread occurrence of breast cancer and its propensity to develop drug resistance highlight the need for a comprehensive understanding of the molecular mechanisms involved. This study investigates the intricate pathways associated with secondary resistance to taxol in triple-negative breast cancer (TNBC) cells, with a particular focus on the changes observed in the cytoplasmic actin isoforms. By studying a taxol-resistant TNBC cell line, we revealed a shift between actin isoforms towards γ-actin predominance, accompanied by increased motility and invasive properties. This was associated with altered tubulin isotype expression and reorganisation of the microtubule system. In addition, we have shown that taxol-resistant TNBC cells underwent epithelial-to-mesenchymal transition (EMT), as evidenced by Twist1-mediated downregulation of E-cadherin expression and increased nuclear translocation of β-catenin. The RNA profiling analysis revealed that taxol-resistant cells exhibited significantly increased positive regulation of cell migration, hormone response, cell–substrate adhesion, and actin filament-based processes compared with naïve TNBC cells. Notably, taxol-resistant cells exhibited a reduced proliferation rate, which was associated with an increased invasiveness in vitro and in vivo, revealing a complex interplay between proliferative and metastatic potential. This study suggests that prolonged exposure to taxol and acquisition of taxol resistance may lead to pro-metastatic changes in the TNBC cell line.

Introduction. Human papillomavirus (HPV) infection with high-risk HPVs is an etiological factor in the development of cervical cancer, with HPV type 16 (HPV16) being the most common. The mechanisms leading to disruption of viral oncogene expression and initiation of epithelial cell transformation are poorly understood. Epigenetic regulatory factors, including cellular miRNAs, may play an important role in HPV-induced carcinogenesis, and aberrantly expressed miRNAs may be promising markers for the diagnosis of HPV-associated lesions.Aim. To search for miRNAs involved in the pathogenesis of HPV16-associated cervical cancer and to evaluate their diagnostic potential for the detection of cervical cancer and precancerous lesions.Materials and methods. MiRNA expression in clinical samples was assessed by both next generation sequencing and quantitative stem-loop polymerase chain reaction (sl-qPCR). Plasma miRNAs from patients with precancerous and cancerous lesions and healthy donors were analyzed using sl-qPCR. Loss of heterozygosity in cervical cancer samples was assessed by copy number ratio of MIR135A1 and ACTB genes. A total of 67 patients with cervical cancer, 21 with precancerous cervical lesions and 24 healthy donors were included in the study. The effect of DNA methylation on miRNA-135A1 expression was evaluated after treatment with a demethylating agent of the cervical HPV16-positive SiHa cell line. Changes in the expression of the HPV16 E6 oncogene were analyzed after transfection with synthetic analogues of the mature forms of miRNA-135А1 (miRNA-135a-3p and miRNA-135a-5p).Results. A significant decrease in the expression of miRNA-135A1 and miRNA-135A2 was detected in tumor tissue samples from HPV16-positive cervical cancer, which was confirmed by sl-qPCR in an independent panel of tumor samples. A decrease in miRNA-135A1 expression was shown to result from both loss of heterozygosity of the gene and aberrant DNA methylation. Transfection of mature forms of miRNA-135A1 into SiHa cells resulted in decreased expression of the E6 oncogene of HPV16. Blood plasma samples from patients with cervical cancer and precancerous lesions showed lower levels of miRNA-135a-3p than healthy donors, and ROC analysis indicated its high diagnostic potential.Conclusion. Levels of miRNA-135A1 are significantly reduced in cervical lesions, both in tumor tissue and plasma, and the ability of this miRNA to suppress the expression of the HPV16 E6 oncogene suggests its oncosuppressive properties. Thus, miRNA-135A1 can be used as a promising new marker for the diagnosis of HPV-associated lesions.

Aim: Resistance to hormonal and targeted therapies in breast cancer limits treatment efficacy. Epigenetic alterations, including changes mediated by DNA methyltransferases, play a key role in this process. Previously, we identified that resistance to tamoxifen and rapamycin is associated with the suppression of DNMT3A. This study aims to further explore the mechanisms underlying this suppression, with a focus on identifying NR6A1 as a novel regulatory factor. Methods: Acquisition of resistant breast cancer cell sublines, MTT-test, immunoblotting, transient transfection and reporter analysis, lentiviral infection, qRT-PCR, and analysis of methylation using bisulfite pyrosequencing. Results: Our findings indicate that the development of cross-resistance in breast cancer cells to hormonal and targeted therapies involves a shift in cell signaling to alternative AKT pathways, marked by a localized suppression of the NR6A1/DNMT3A axis and associated DNA methylation changes. We demonstrated the critical role of NR6A1 downregulation in resistance development. Additionally, we observed activation of Snail - a key regulator in the epithelial-mesenchymal transition - as a mediator of the effects of NR6A1 depletion, establishing a direct link between Snail expression and resistance formation. Conclusion: The coordinated suppression of NR6A1 and DNMT3A may contribute to sustaining the resistant phenotype in breast cancer cells. This pathway could serve as a predictive marker, helping guide the selection of optimal therapeutic strategies for breast cancer treatment in the future.

The rearrangement of molecular pathways and the activation of bypass signaling determine the progression of tumor cell resistance to various drugs that specifically block target signaling proteins. The present work was performed on the MCF-7 breast cancer cells and established sublines, resistant to mTOR inhibitor rapamycin or antiestrogen tamoxifen, developed under prolonged cell treatment with rapamycin or tamoxifen, respectively. We have shown that both resistant sublines demonstrate the cross-resistance to rapamycin and tamoxifen and are characterized with the common signaling changes, namely—blocking of the estrogen receptor α (ERα) transcriptional activity and constitutive activation of Akt signaling. Analysis of the epigenetic machinery revealed the drastic suppression of the level of DNA methyltransferase 3A (DNMT3A) in both the resistant sublines that were correlated with the demethylation of the LINE-1 repeats. Knockdown of the DNMT3A via siRNA results in the progression of partial resistance of MCF-7 cells to both tamoxifen and rapamycin, supporting the important role of DNA methylation in the formation of the resistant phenotype. Totally, the results obtained highlight the possible mechanism of the tumor cell resistance to targeting/hormonal drugs based on the rearrangement of DNA methylation profile and activation of the bypass signaling pathways.

Human papillomaviruses (HPV) are epitheliotropic viruses that affect the cutaneous and mucosal epithelium of the genitals and oral cavity. High-risk HPV (HPV type 16, 18, etc.) infection induces the development of cervical cancer, anogenital carcinomas, and a large proportion of head and neck squamous cell carcinomas. Low-risk HPVs, mainly HPV types 6 and 11, cause benign lesions, such as exophytic condyloma of the anogenital tract, as well as inverted papilloma of the nasal cavity and paranasal sinuses, laryngeal papillomatosis, which also have the potential for malignant transformation in a long-term recurrent course. Head and neck squamous cell carcinoma (HSCC) is the main histological subtype of head and neck cancer and one of the most common types of cancer worldwide. The main risk factors for the development of head and neck cancer are alcohol and tobacco consumption, as well as high-risk HPV infection. In recent decades, there has been a downward trend in the incidence of the HNSCC associated with smoking, but an increase in HPV-induced tumors in younger adults. Despite significant advances in diagnostics and modern clinical oncology, head and neck cancer mortality has not declined for several decades. One of the effective methods of combating viral infection is vaccine prevention. Currently available vaccines against HPV 6, 11, 16, 18, and other high-risk HPVs are widely used to prevent HPV-associated malignant and benign lesions according to the registered indications for use in both males and females. Вирусы папилломы человека (ВПЧ) относятся к эпителиотропным вирусам, которые поражают кожный и слизистый эпителий половых органов и ротовой полости. ВПЧ высокого канцерогенного риска (ВПЧ типа 16, 18 и др.) являются этиологическим фактором развития злокачественных опухолей шейки матки, карцином аногенитального тракта и значительной части плоскоклеточных карцином различных отделов головы и шеи. ВПЧ низкого онкогенного риска, главным образом, ВПЧ 6 и 11 типов, вызывают доброкачественные поражения, такие как экзофитные кондиломы аногенитального тракта, а также инвертированные папилломы полости носа и околоносовых пазух, папилломатоз гортани, которые при длительном рецидивирующем течении также обладают потенциалом злокачественной трансформации. Плоскоклеточные карциномы головы и шеи (ПКГШ) являются основным гистологическим подтипом опухолей головы и шеи и одним из самых распространенных форм рака во всем мире. Основными факторами риска развития опухолей головы и шеи являются употребление алкоголя и табака, а также инфекция ВПЧ высокого канцерогенного риска. В последние десятилетия отмечается тенденция снижения заболеваемости ПКГШ, связанных с курением, но при этом возрастает число ВПЧ-индуцированных опухолей у людей более молодого возраста. Несмотря на значительные достижения в диагностике и современной клинической онкологии, смертность от злокачественных новообразований головы и шеи не снижается уже на протяжении нескольких десятилетий. Одним из эффективных методов борьбы с вирусной инфекцией является вакцинопрофилактика. Существующие на сегодняшний день вакцины против ВПЧ 6, 11, 16, 18 и других высокоонкогенных ВПЧ получили широкое распространение в качестве средства профилактики ВПЧ-ассоциированных злокачественных и доброкачественных поражений в соответствии с зарегистрированными показаниями к применению, как у женщин, так и у мужчин.

Introduction . Rearrangement of molecular pathways and activation of bypass signaling determine the progression of tumor cell resistance to various drugs. Study of the common features of resistant formation mechanisms is essential for breast and other cancer beneficial treatments. Materials and methods . The present work was performed on estrogen receptor α ERα-positive (ERα – estrogen receptor α) McF-7 breast cancer cells, established sublines resistant to the mTOR inhibitor rapamycin or antiestrogen tamoxifen, and ERα-negative MDA-MB-231 breast cancer cells. Methods used include MTT test, transient transfection, immunoblotting, real-time polymerase chain reaction and methylation analysis by bisulfite pyrosequencing. Results . We have shown that the resistance of breast cancer cells to targeted and hormonal drugs is associated with the suppression of DNA methyltransferase 3A (DNMT3A) and respective changes in DNA methylation; DNMT3A knockdown results in the partial resistance to both drugs demonstrating the pivotal role of DNMT3A suppression in the progression of cell resistance. Conclusion . Totally, the results obtained highlight the possible mechanism of tumor cell resistance to targeting/hormonal drugs based on the deregulation of DNMTs expression and demonstrate direct connection between DNMT3A suppression and resistance progression.

Extracellular vesicles (EVs), including exosomes, are key factors of intercellular communication, performing both local and distant transfers of bioactive molecules. The increasingly obvious role of EVs in carcinogenesis, similarity of molecular signatures with parental cells, precise selection and high stability of cargo molecules make exosomes a promising source of liquid biopsy markers for cancer diagnosis. The uterine cavity fluid, unlike blood, urine and other body fluids commonly used to study EVs, is of local origin and therefore enriched in EVs secreted by cells of the female reproductive tract. Here, we show that EVs, including those corresponding to exosomes, could be isolated from individual samples of uterine aspirates (UA) obtained from epithelial ovarian cancer (EOC) patients and healthy donors using the ultracentrifugation technique. First, the conducted profiling of small RNAs (small RNA-seq) from UA-derived EVs demonstrated the presence of non-coding RNA molecules belonging to various classes. The analysis of the miRNA content in EVs from UA performed on a pilot sample revealed significant differences in the expression levels of a number of miRNAs in EVs obtained from EOC patients compared to healthy individuals. The results open up prospects for using UA-derived EVs as a source of markers for the diagnostics of gynecological cancers, including EOC.

INTRODUCTION:High carcinogenic-risk human papillomaviruses (hrHPVs) are recognized as etiological agents of cervical cancer. Constant expression of the viral oncoproteins, E6 and E7, is required for maintenance of the malignant phenotype of tumor cells. The exact mechanism of regulation of viral oncogenes expression in tumor cells is not fully elucidated.THE PURPOSE:identification of viral noncoding RNAs (ncRNAs) in HPV16-positve cervical cancer.MATERIALS AND METHODS:The reverse transcription polymerase chain reactions were used to detect viral ncRNAs in HPV16-positve primary cervical squamous cell carcinomas and SiHa and CasKi cell lines. The knockdown technique with oligonucleotides complementary to ncRNAs was used to elucidate their functions.RESULTS:We have identified ncRNAs transcribed in the upstream regulatory region of HPV16 in the cervical carcinoma cell lines and in 32 out 32 cervical squamous cell carcinomas with episomal or integrated forms of HPV16 DNA. Knockdown of sense or antisense strains of ncRNAs by oligonucleotides results in a decrease or increase of the E6 and E7 oncogenes mRNA levels in cells, respectively. These changes of oncogenes mRNA levels are accompanied by the modulation of the levels of the p53 protein, the main target of the E6 oncoprotein.CONCLUSION:The presence of regulatory ncRNAs in all examined tumors and cell lines revealed for the first time indicates their necessity for maintenance of constant expression of E6 and E7 oncogenes in them. The findings can be useful for understanding of the fundamental aspects of the viral expression regulation in HPV16-positive tumors.

EVs are involved in local and distant intercellular communication and play a vital role in cancer development. Since EVs have been found in almost all body fluids, there are currently active attempts for their application in liquid diagnostics. Blood is the most commonly used source of EVs for the screening of cancer markers, although the percentage of tumor-derived EVs in the blood is extremely low. In contrast, GJ, as a local biofluid, is expected to be enriched with GC-associated EVs. However, EVs from GJ have never been applied for the screening and are underinvestigated overall. Here we show that EVs can be isolated from GJ by ultracentrifugation. TEM analysis showed high heterogeneity of GJ-derived EVs, including those with exosome-like size and morphology. In addition to morphological diversity, EVs from individual GJ samples differed in the composition of exosomal markers. We also show the presence of stomatin within GJ-derived EVs for the first time. The first conducted comparison of miRNA content in EVs from GC patients and healthy donors performed using a pilot sampling revealed the significant differences in several miRNAs (-135b-3p, -199a-3p, -451a). These results demonstrate the feasibility of the application of GJ-derived EVs for screening for miRNA GC markers.

Secreted extracellular vesicles (EVs) contain active biomolecules, including miRNAs, composition of which reflects epigenetic changes occurring in cells during pathological processes, in particular, malignant transformation. The accumulated pool of data on the role of EVs in carcinogenesis has stimulated investigations of the EV-derived cancer markers. The most important factor limiting development of this scientific direction is lack of "gold standards" both for methods of EV isolation from biological fluids and for analyzing their molecular content, including composition of miRNAs. Here we first examined efficacy of various methods for small RNA isolation from EVs contained in ascitic fluid for subsequent miRNA analysis. Comparison of different commercial kits showed advantages of the methods based on phenol-chloroform extraction: Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit. Analysis of the small RNA transcriptome showed presence of various classes of molecules in the EVs, among which proportion of miRNAs averaged 6% and reaching 10% with the Total Exosome RNA & Protein Isolation Kit. The PureLink miRNA Isolation Kit demonstrated the lowest efficiency. The miRNeasy Advanced Serum/Plasma Kit showed the highest concentration of the small RNA fraction, miRNA proportion of which, however, did not exceed that obtained with the miRNeasy Serum/Plasma Kit and Total Exosome RNA & Protein Isolation Kit. Moreover, RT-PCR analysis of the individual molecules showed lower levels of each of investigated miRNAs (miR-1246, miR-200b-5p, miR-200c-3p, and miR-23a-3p) when using the miRNeasy Advanced Serum/Plasma Kit. In conclusion, Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit can be considered as optimal kits in terms of performance based on combination of the studied characteristics, including small RNA concentration, percentage of microRNA according to bioanalyzer and sequencing results, and levels of individual miRNAs detected by RT-PCR.

The recombinant proteins E6-CBD and E7-CBD, which are antigens E6 and E7 of human papillomavirus type 16 (HPV16), were connected by a glycine-serine spacer with the cellulose-binding domain (CBD) of Anaerocellum thermophilum in preparations obtained via synthesis in a heterologous expression system in Escherichia coli. A simple, effective, one-step technology for the isolation and purification of recombinant proteins E6-CBD and E7-CBD has been developed. The presence of antigenic properties in the obtained protein preparations was confirmed via Western blot. Three variants of immunogenic compositions with the recombinant proteins E6-CBD and E7-CBD and a three-dose immunization scheme have been developed. The immunogenicity was assessed in the BALB/c mice, and the antitumor effect of immunogenic compositions was studied on an experimental model of BALB/c mice with HPV16-associated tumors. It is shown that E6-CBD and E7-CBD are highly immunogenic and have antitumor effects in vivo. Both proteins, E6-CBD and E7-CBD, must be present to achieve the highest efficiency as part of the immunogenic composition. The obtained HPV16 recombinant antigens E6-CBD and E7-CBD can be used in two ways: (a) in clinical practice as the main specific components of therapeutic vaccines for the treatment of HPV16-associated neoplastic lesions of various localizations (cervical cancer, carcinoma of the anogenital tract, oropharynx) and (b) in diagnostics for the development of diagnostic kits to detect antibodies to E6 and E7 oncoproteins in patients with HPV16-associated malignant neoplasms.

Background. Ovarian cancer is a complex and poorly studied disease that kills nearly 70–80 % of patients. Therefore, practitioners are interested in any opportunity of improving survival of these patients. From this point of view, investigation of genetic and epigenetic functions associated with this pathology is quite promising. Objective: to assess clinical and morphological characteristics of tumors in ovarian cancer patients, considering the presence of mutations and methylation in the BRCA1/2 gene. Materials and methods. This study included 180 ovarian cancer patients (FIGO stage I–IV) treated in the N. N. Blokhin Russian Cancer Research Center between 2008 and 2019. Study participants were divided into 3 groups according to their BRCA status and the number of primary tumors. We collected and analyzed venous blood, biopsy samples of ovarian cancer, archived histological sections, and paraffin-embedded tissue blocks. DNA isolated from venous blood was used to identify the following germline mutation by pyrosequencing: BRCA1 5382insC, BRCA1 4153delA, BRCA1 185delAG, and BRCA26174delT. DNA isolated from biopsy specimens and paraffin-embedded tissue specimens was used to analyze methylation in the promoter regions of the BRCA1 and BRCA2 genes by bisulfite sequencing (PyroMark Q24 DNA Sequencer; Qiagen, USA) with specific primers targeting promoter regions of the BRCA1 and BRCA2 genes. Results. Molecular testing demonstrated that the frequency of BRCA1 gene mutations was 21.1 % (38/148) in patients with solitary ovarian cancer and 40.6 % (13/32) in patients with multiple primary ovarian cancers. The frequency of methylation of the BRCA1 gene promoter was 2.2 % (18/148) in patients with solitary ovarian cancer and 3.1 % (1 case) in patients with multiple primary ovarian cancers. All BRCA1 methylated ovarian tumors were serous adenocarcinomas, including high grade tumors in 15 patients (78.9 %) and low-grade tumors in 4 patients (21.1 %). Conclusion. Hypermethylation of the BRCA1 gene promoter was observed only in individuals with sporadic serous ovarian cancer. No methylation was detected in patients with non-serous ovarian cancer, as well as in patients carrying BRCA1 gene mutations (both with solitary ovarian cancer and with primary multiple ovarian tumors).

Objective of the study: To increase the efficiency of the diagnosis of serous ovarian cancer by the method of immunocytochemical (ICC) analysis of the expression of p53, p16, wt1 markers in cells obtained in aspiration material from the uterine cavity. Materials and methods: The cellular expression of p53, p16 and wt1 markers was determined in aspiration material from uterine cavity of 71 patients with a morphologically confirmed diagnosis of serous ovarian cancer III-IV stages (51 patients with high grade serous carcinoma and 20 patients with low grade). The results were compare with cellular expression of the same markers in groups of patients with benign ovarian tumors (n = 50), patients with secondary (metastatic) ovarian lesions (n = 50) and healthy women (n = 50). The study did not include patients with early stages of ovarian cancer because of too small number of similar cases . For cytological studies, cytopin multilayer preparations of the cytospin system were obtained. An ICC study was conducted with monoclonal antibodies to p53, p16, wt1. Results: p53 demonstrated the greatest diagnostic significance in group of patients with high grade serous carcinomas: expression was traced in 31 of 51 observations of serous carcinomas of the ovaries, which amounted to 61%. A positive reaction of wt1 was observed in 24 of 51 cases (47%), p16 expression was observed in 25 of 51 cases (49%). In the group of patients with low grade serous carcinomas (n = 20), in contrast to the group of patients with high grade serous ovarian cancer, a positive reaction of p53 was observed in 3 out of 20 cases (15%), p16 in 2 (10%), but wt1 expressed in samples of 12 (60%) patients. By ICC examination of samples collected from uterine cavity of patients with benign ovarian tumors we identify focal moderate expression of p53 in separate endometrial epithelial calls in 2 cases. Histologically, serous cystadenoma of the ovaries was detected in these patients, and in another 48 samples no positive reactions with antibodies to p53,p16,wt1 were observed. In group of patients with secondary lesion of the ovaries, as well as in the control group, positive marker reactions were not observed. Conclusions: The increased expression of p53, p16, wt1 markers in cells obtained by aspiration from the uterine cavity during an ICC study can be used as a diagnostic test for high grade serous carcinoma.