Цель. Изучение связи полиморфных маркеров генов репарации ДНК ERCC2 (rs13181), ERCC5 (rs17655), транспортного белка ABCB1 (rs1045642, rs2032582) с клиническим ответом и степенью патоморфологической регрессии (ПР) опухоли у беременных женщин с онкологическими заболеваниями после ХТ препаратами платины.

Hereditary form of prostate cancer is often caused by pathogenic variants in genes associated with the DNA repair system. Identification of genetic aberrations allows to stratify patients into groups for personalization and improvement of therapy effectiveness. With this approach, it is important to take into account that the frequency of pathogenic variants can vary significantly in different ethnic populations. The article presents a case of metastatic castration-resistant prostate cancer in a carrier of hereditary pathogenic variant in the BRCA2 gene c.6341del (р.Pro2114fs). The results support the need for genetic testing using up-to-date methods capable of detecting rare genetic variants.

The distribution of allele frequencies of polymorphic markers of the genes of the apoptosis and cell cycle control system TP53 (rs1042522), MDM2 (rs2279744), and CDKN1A (rs1801270) was obtained. The relationship of these markers with the risk of ovarian cancer was analyzed in women of the Moscow region. The study included 70 healthy female donors and 66 patients with ovarian cancer. DNA isolated from the blood of healthy donors and tissue and blood of patients with ovarian cancer was studied. The polymorphic markers of genes were analyzed by real-time PCR with fluorescent allele-specific probes. In the work, the odds ratios (OR) of ovarian cancer risk were obtained: OR = 0.94, 95% CI = 0.57–1.65, p = 1.0 for the Pro allele of the Arg72Pro of the TP53 gene; OR = 0.96, 95% CI = 0.49–1.89, p = 0.90 for the G allele of the T(–410)G of the MDM2 gene; OR = 1.53, 95% CI = 0.76–3.09, p = 0.29 for the Arg allele of the Ser31Arg of the CDKN1A gene. Obtained results show no association with ovarian cancer for studied markers. It was revealed that the carriage of minor alleles of the Arg72Pro of the TP53 gene, T(–410)G of the MDM2 gene, and Ser31Arg of the CDKN1A gene did not affect the risk of development of ovarian cancer in women of the Moscow region.

Background. Renal cell carcinoma (RCC) is asymptomatic up to severe stages and is characterized by a high mortality rate, reaching 90 % with the development of a metastatic process.Objective: to determine the group of microRNA genes, the methylation of which is associated with the progression of the disease, in particular, with metastasis.Materials and methods. Methylation-specific polymerase chain reaction in a representative sample of RCC patients (98 cases) showed an increase in the methylation status of 6 microRNA genes (MIR9-1, MIR9-3, MIR34b/c, MIR130b, MIR1258, MIR107) in tumor DNA samples relative to matched samples of histologically unchanged tissue.Results. For 4 genes (MIR9-1, MIR107, MIR130b, MIR1258), a significant association of methylation with late (III-IV) stages, tumor size, loss of differentiation, and metastasis to lymph nodes or distant organs was shown. These 4 genes were used to compose a potential metastatic prognosis marker system with a clinical sensitivity of 68 % and a specificity of 84 % (area under curve 0.83), which will be applied in the final development of a system for personalized therapy of RCC patients.Conclusion. The association of methylation of the MIR1258 with RCC metastasis has been shown for the first time and is of independent interest as a new promising marker for the prognosis of metastatic relapses.

Systemic analysis of the relationship between the levels of methylation of 21 microRNA genes and the parameters of breast cancer progression was performed on a representative sample of 91 paired specimens of breast cancer and histologically normal tissues and a system of markers for prediction of metastasis was proposed. A significant association of hypermethylation of 11 genes with late (III-IV) clinical stages was found, and for 6 genes (MIR124-1, MIR127, MIR34B/C, MIR9-3, MIR1258, and MIR339) this association was highly significant (p≤0.001, FDR=0.01). For MIR9-3 and MIR339, an association with tumor size was demonstrated (p<0.001, FDR=0.01). No association of the levels of methylation of the analyzed microRNA genes with the degree of differentiation were found. An association with lymph node metastasis was established for 9 microRNA genes; the most significant association was shown for 6 genes MIR125B-1, MIR127, MIR9-3, MIR339, MIR124-3, and MIR1258 (p<0.005, FDR=0.05). Based on these 6 genes, a marker system for predicting breast cancer metastasis was developed by ROC analysis. This system is characterized by 87% sensitivity and 77% specificity (AUC=0.894). The proposed system may have clinical application in the personalized treatment of breast cancer patients.

Changes in the methylation levels of 21 microRNA genes in 91 breast cancer samples in comparison with paired samples of histologically unchanged tissue were studied by quantitative methylation-specific PCR. For 19 microRNA genes, a significant increase in the methylation level in tumors in comparison with normal tissues was shown (Mann—Whitney test). When considering the data for breast cancer samples only from patients with clinical stages I and II (59samples), 17 genes with a significantly increased level of methylation were identified. Increased methylation level for 11 genes (MIR124-1, MIR124-3, MIR125B-1, MIR127, MIR129-2, MIR132, MIR137, MIR193a, MIR34B/C, MIR375, and MIR9-1) compared to the paired norm was highly significant (p<0.001, FDR=0.01). The ROC analysis was used to optimize a set of markers for diagnosing breast cancer at the early stages consisting of 4 microRNA genes: MIR125B1, MIR127, MIR1258, and MIR132; the system is characterized by 100% specificity, 85% sensitivity, and AUC=0.924. Importantly, 100% specificity eliminates false positive results. Detection of methylation of at least one of the 4 genes of this set is sufficient to classify the patient’s sample as breast cancer.

Background. The development of hereditary cancer syndromes is caused by genetic disorders in the DNA repair system, which consists of more than 100 genes. However, at present, in the majority of medical centers in Russia, the diagnosis of hereditary OC and BC is limited to the determination of the most frequent mutations (8 points) in BRCA1 and BRCA2 genes using PCR methods. However, the given mutations are frequent for Slavic population while in other populations of Russia they occur less often or do not occur at all. Aim. To reveal the landscape of hereditary pathogenic variants in the genes of the reparation system and develop methods for a fast analysis of the NGS data. Methods. A panel of 34 genes of the reparation system was analyzed by next generation sequencing (NGS) in 1644 samples of patients with inherited cancer syndromes. Results. The NGS analysis revealed 119 pathogenic mutations of BRCA1/BRCA2 genes in 374 patients, with a marked difference in mutation frequencies between different ethnic groups Slavs, Tatars, Bashkirs and Chuvash. A public database of the frequencies of mutations and polymorphisms of the genes of the DNA reparation system in different ethnoses was created at https://oncobrca.ru. Methods were created for automatic parallel processing of any number of samples from the raw data to the final report. Conclusions. The NGS method has created a possibility to identify rare mutations characteristic of different ethnic groups, which makes it possible to prescribe optimal chemotherapy and develop diagnostic methods of population screening for carrying gene mutations of the DNA repair system. The modern approaches to the analysis of genome sequencing data allowed reducing the time of obtaining the result to several hours after the analysis.

We studied the association of polymorphic markers of cell cycle control genes (Arg72Pro of the TP53 gene, T(-410)G of the MDM2 gene, and Ser31Arg of the CDKN1A gene) in ovarian cancer and progression-free survival following platinum-based chemotherapy. Tumor tissue samples obtained from 49 patients who had undergone chemotherapy were examined. Patients received standard platinum-based chemotherapy and were observed until disease progression. Polymorphic markers of genes were evaluated by PCR-RFLP and real-time PCR. In patients carrying the G allele of the T(-410)G marker of the MDM2 gene, a decreasing trend was observed in median progression-free survival. An increase in the median progression-free survival was observed in carriers of the Pro allele of the TP53 gene (p=0.045). Furthermore, a stronger association was noted with carriers of the minor Pro/Pro homozygous genotype relative to the Arg/Arg genotype (p=0.007). In the subgroup of patients who underwent optimal or complete cytoreductive surgery, carriage of the minor Arg allele of the Ser31Arg marker (CDN1A gene) was associated with a decrease in the median progression-free survival time (p=0.004).

Обоснование . Развитие наследственных онкологических синдромов обусловлено генетическими нарушениями в системе репарации ДНК, состоящей более чем из 100 генов. Однако в настоящее время в большинстве медицинских центров России диагностика наследственных заболеваний раком яичника и раком молочной железы сводится к определению наиболее частых мутаций (8 точек) в генах BRCA1 и BRCA2 с помощью методов полимеразной цепной реакции. При этом данные мутации являются частыми для славянской популяции, в то время как в остальных популяциях России они встречаются реже или не встречаются вообще. Цель исследования — получить представление о ландшафте наследственных патогенных вариантов в генах системы репарации; разработать методы быстрого анализа данных полногеномного секвенирования. Методы. Методом секвенирования нового поколения (Next Generation Sequencing, NGS) была проанализирована панель из 34 генов системы репарации в 1644 образцах биоматериала пациентов с наследственными онкологическими синдромами. Результаты. Выявлены 119 патогенных мутаций генов BRCA1/BRCA2 у 374 пациентов, при этом обнаружена выраженная разница в частоте встречаемости мутаций между различными этносами — славянами, татарами, башкирами и чувашами. Созданы публичная база данных частоты мутаций и полиморфизмов генов системы репарации ДНК в различных этносах ( https://oncobrca.ru ), а также методы для автоматической параллельной обработки любого количества образцов от сырых данных до готового отчета. Заключение. С помощью метода NGS появилась возможность выявления редких мутаций, характерных для различных этносов, что дает возможность назначать оптимальную химиотерапию и разрабатывать диагностические методы популяционного скрининга носительства мутаций генов системы репарации ДНК. Современные подходы к анализу данных полногеномного секвенирования позволили сократить время получения результата до нескольких часов после проведения анализа.

Systems of markers for the diagnosis of breast cancer based on DNA methylation of a group of suppressor protein-coding genes, hypermethylated microRNA genes, and their combinations were compiled. On a representative sample of 70 paired breast cancer specimens (tumor/normal), MS-PCR analysis revealed a significant increase in the methylation frequency of 5 protein-coding genes: RASSF1A suppressor and apoptosis genes APAF1, BAX, BIM/BCL2L11, and DAPK1 (34-61% vs. 4-24%) and 6 microRNA genes: MIRG124G1, MIRG125bG1, MIRG129G2, MIRG148a, MIRG34b/c, and MIRG9G3 (36-76% vs. 6-27%). ROC-analysis showed that a combination of 4 genes (APAF1, BAX, BIM/BCL2L11, and DAPK1) and MIRG125bG1 gene constitute a highly efficient 5-marker system with 100% specificity and sensitivity of 94-96% at AUC=0.98-0.97, suitable also for patients with stage I and II breast cancer. Detection of methylation of at least one gene in this system in biopsy or postoperative material is sufficient to refer the sample to breast cancer.

We identified a group of miRNA genes whose methylation is associated with ovarian cancer metastasis. Based on these data, new markers and the systems of markers predicting tumor dissemination were selected. Using methylation-specific PCR and a representative set of 54 ovarian cancer samples, we identified 10 microRNA genes (MIR-124a-2, MIR-127, MIR-125b-1, MIR-129-2, MIR-137, MIR-193a, MIR-203a, MIR-34b/c, MIR-130b, and MIR-1258) whose methylation is associated with tumor metastasis. The greatest association was established for 4 genes: MIR-137, MIR-193a, MIR-34b/c, and MIR-130b (p<0.01). ROC analysis revealed 3 most optimal marker systems including 4-5 miRNA genes and characterized by high sensitivity (82-94%) and specificity (76-86%) at AUC=0.89-0.92. Methylation of any three genes from these systems is sufficient to predict metastasis with the specified accuracy. Detection of the group of hypermethylated miRNA genes with predictive value for ovarian cancer metastasis is of great importance for personalized treatment of the patients.

Obesity is a high-risk factor for such comorbidities as cardiovascular disease, several types of cancer, and type 2 diabetes; however not all individuals with obesity have such complications. Approximately 20% of individuals with obesity are metabolically healthy. This study focused on differences between obese individuals with and without type 2 diabetes (T2D+ and T2D–, respectively) on the transcriptome level. Subjects included were 35 T2D– patients with obesity and 35 T2D+ patients with obesity with the same body mass index (BMI). The study was based on the transcription analysis of mRNA and microRNAs (miRs) by RNAseq. In the first step, we performed RNAseq of miRs, in the second step, we analyzed only those mRNA, which appeared targets for significant miRs from the first step. All RNAseq results were validated by qPCR. There were seven miRs differently expressed with adjusted p-value <0.1, which were confirmed by qPCR. Five among them: miR-204-5p, miR125b-5p, miR-125a-5p, miR320a, miR-99b—were upregulated in T2D+ patients with obesity, while only two miRs, miR-23b-3p, and miR197-3p, were increased in T2D– patients with obesity. These seven miRs target two groups of genes: matrix metalloproteinases and TGFβ signal pathway genes. According to the results of transcriptome analysis, the main difference between T2D+ and T2D– patients with obesity was in adipogenesis and fibrosis regulation by matrix metalloproteinases and SMAD4-RUNX2 signal cascade. Based on the data about transcription profiles of both groups, we suggested that the process of fibrosis in T2D+ patients with obesity is more pronounced than in T2D– patients with obesity.

miRNA genes play an important role in cancer pathogenesis, while they may be suppressed by hypermethylation. Here, we assess the diagnostic potential of a group of hypermethylated miRNA genes (MIR-124-1, MIR-124-3, MIR-125B-1, MIR-127, MIR-132, MIR-193a, and MIR-34b/c) in a representative set of 70 breast cancer samples and 17 breast tissue samples from deceased donors with no malignancies. For these seven genes, the methylation status is determined using the methylation-specific PCR. Methylation reached 26-76% in tumor specimens, 1-27% in paired considered normal breast tissues, and 0-18% in breast tissue from deceased donors. By quantitative RT-PCR, reduced expression levels of the investigated miRNAs are detected, with a negative correlation of expression levels with gene hypermethylation. Combinations of three or four hypermethylation biomarkers, namely, MIR-124-1, MIR-125B-1, MIR-127, and MIR-34b/c are found suitable for breast cancer diagnostics; with sensitivity (76-93%), specificity (88-100%), and AUC (0.88-0.94). Notably, the MIR-127 gene was hypermethylated only in the tumor samples of patients with metastases, and, therefore, should be tested as a marker of breast cancer dissemination. These findings may lead to improvement in the management of breast cancer.

Groups of microRNA genes, methylation of which is associated with the initial (I-II) stages of breast cancer, are determined, and new markers and marker systems for the disease diagnosis were created on the basis of these data. A total of 14 genes in which methylation was associated with breast cancer were identified with the use of methyl-specific PCR on a representative sample of 70 tumor specimens. Analysis of 46 specimens from patients with clinical stages I and II detected 9 genes (MIR-124-1, MIR-124-3, MIR-125b-1, MIR-129-2, MIR-132, MIR-148a, MIR-193a, MIR-34b/c, and MIR-9-3), in which methylation was associated with the initial stages of the disease. Using ROC analysis, we formed two systems including 6 markers each and detecting breast cancer at stages I-II with high sensitivity (89 and 91%) and specificity (88%) at AUC=0.92-0.93. These sets were validated on the total sample of 70 specimens including all disease stages; they showed 93 and 94% sensitivities, 88% specificity, and AUC=0.95. Highly sensitive systems of markers, based on microRNA gene methylation, were created for the diagnosis of breast cancer at stages I-II.

The study was designed to determine genes of microRNAs hypermethylated in malignant ovarian tumors and to select new diagnostic and prognostic markers of the disease and effective system of markers. Using methyl-specific PCR and a representative sample of 54 ovarian cancer specimens, we determined 5 microRNA genes ( MIR-34b/c , MIR-9-1 , MIR-124-3 , MIR-129-2 , and MIR-107 ) hypermethylated in the majority of tumor samples in comparison with paired samples of histologically unchanged tissue (48-57% vs . 4-19%, p <0.001). Using ROC-analysis, we selected an effective system of 4 markers for diagnosis of ovarian cancer ( MIR-9-1 , MIR-124-3 , MIR-129-2 , and MIR-107 ) characterized by high sensitivity and specificity (up to 87-94% at AUC=0.92) relative to the conventional norm (54 paired samples of histologically unchanged tissue) and absolute norm (18 ovarian tissue samples from subjects who died from non-tumor diseases). It was also shown that methylation of MIR-129-2 , MIR-9-1 , and MIR-34b/c genes is significantly ( p <0.01) correlated with the clinical stage or the presence of metastases. The results indicate that epigenetic modifications of the studied microRNA genes are involved in the pathogenesis and progression of ovarian cancer and attest to their diagnostic and prognostic potential.

The high mortality in non-small cell lung cancer (NSCLC) and the lack of effective diagnostics indicate a need for new NSCLC biomarkers. Epigenomic studies have shown that the proportion of hypermethylated miRNA genes is several times higher than of protein-coding genes, which makes them promising markers. The aim of the study was to evaluate diagnostic characteristics of a group of hypermethylated miRNA genes in NSCLC and its subtypes, squamous cell lung cancer (SLC), and adenocarcinoma (AC). On a set of 39 NSCLC samples and 20 samples of lung tissue from «donors» (dead from non-oncological disease), methylation-specific PCR was used to perform a comparative analysis of methylation of a group of genes, MIR-125B-1, MIR-129-2, MIR-137, and MIR -375. The ROC-analysis identified 6 potential diagnostic systems of markers for NSCLC, SLC, and AC, which had high sensitivity (85—92%) and specificity (90—95%) and the AUC value of 0.89—0.94. The diagnostic systems for SLC and NSCLC showed the best values.