[Epigenetic Regulation Disturbances on Gene Expression in Imprinting Diseases].

Epigenetic regulation is hereditary and non-hereditary changes in the expression of a particular gene without any corresponding structural changes in its nucleotide sequence. Genomic imprinting is an epigenetic mechanism for regulating the expression of homologous genes depending on parental origin, i.e., they are expressed monoallelically in the mammalian diploid cell. Being genetically imprinted, only the maternal or only the paternal genome is unable to ensure normal embryonic development. The most studied epigenetic modification, which plays one of the main roles in the maintenance of imprinting processes, is the specific methylation of cytosine in CpG-dinucleotides. All known imprinted genes contain differential DNA methylation regions on homologous parent chromosomes, which are necessary for their monoallelic expression. However, it is now known that not only DNA methylation, but chromatin remodeling, histone modifications, and non-coding RNAs also ensure the proper functioning of imprinted genes in the human body. Structural and functional disturbances of epigenetic mechanisms lead to imprinting diseases.

[Current possibilities of the differential diagnosis of plaque parapsoriasis and the early stages of mycosis fungoides]. / Sovremennye vozmozhnosti differentsial'noi diagnostiki bliashechnogo parapsoriaza i rannikh stadii gribovidnogo mikoza.

Mycosis fungoides (MF) is the most common primary cutaneous epidermotropic T-cell lymphoma (80%). The accurate diagnosis of MF confirmed only by clinical, histological and immunohistochemical signs amounts to 50-75%. OBJECTIVE: To investigate genetic markers (FOXP3, STAT4, IL-12B) for the early diagnosis of MF, to estimate the informative value of used diagnostic techniques (histology, immunophenotyping), and to determine clonality by the T-cell receptor γ-chain genes. MATERIAL AND METHODS: Fifty patients with MF and plaque parapsoriasis (PP) who had been treated at the V.A. Rakhmanov Clinic of Skin and Venereal Diseases and at the National Medical Research Center for Hematology were followed up. A MF group consisted of 27 patients; a PP group included 23 patients, and a control group comprised 10 healthy individuals. The expression of the FOXP3, STAT4, and IL-12B genes was analyzed by TaqMan real time-PCR. The objectives of the study were affected skin portions from patients with MF or PP and healthy individuals. RESULTS: The investigation revealed a increase in the expression level of STAT4 mRNA transcripts by 9 times in patients with MF compared with those with PP and by 553 times in healthy individuals. There was also a statistically significant predominance of the expression level of STAT4 mRNA transcripts in patients with spotted and plaque stages of MF (180; 318) compared with those with PP and healthy individuals, as well as a sharp decrease in those with erythrodermic MF, which was statistically significant. CONCLUSION: MF cannot be diagnosed without comprehensively assessing the clinical, anamnestic, histological, immunophenotypic, and molecular genetic data. The expression level of STAT4 mRNA transcripts is of great importance for the early diagnosis of MF. Inclusion of the level of STAT4 expression in the list of diagnostic signs increases the accuracy of differential diagnosis of MF and PP from 59.1 to 81.8%, respectively.

Structural Alterations in Human Fibroblast Growth Factor Receptors in Carcinogenesis.

Fibroblast growth factor (FGF) plays an important role in human embryogenesis, angiogenesis, cell proliferation, and differentiation. Carcinogenesis is accompanied by aberrant constitutive activation of FGF receptors (FGFRs) resulting from missense mutation in the FGFR1-4 genes, generation of chimeric oncogenes, FGFR1-4 gene amplification, alternative splicing shift toward formation of mesenchymal FGFR isoforms, and FGFR overexpression. Altogether, these alterations contribute to auto- and paracrine stimulation of cancer cells and neoangiogenesis. Certain missense mutations are found at a high rate in urinary bladder cancer and can be used for non-invasive cancer recurrence diagnostics by analyzing urine cell pellet DNA. Chimeric FGFR1/3 and amplified FGFR1/2 genes can predict cell response to the targeted therapy in various oncological diseases. In recent years, high-throughput sequencing has been used to analyze exomes of virtually all human tumors, which allowed to construct phylogenetic trees of clonal cancer evolution with special emphasis on driver mutations in FGFR1-4 genes. At present, FGFR blockers, such as multi-kinase inhibitors, specific FGFR inhibitors, and FGF ligand traps are being tested in clinical trials. In this review, we discuss current data on the functioning of the FGFR family proteins in both normal and cancer cells, mutations in the FGFR1-4 genes, and mechanisms underlying their oncogenic potential, which might be interesting to a broad range of scientists searching for specific tumor markers and targeted anti-cancer drugs.

[Long Noncoding RNAs and Their Role in Oncogenesis].

The noncoding part of the human genome, which was previously considered nonfunctional or junk DNA, has been the subject of extensive research this decade. Nevertheless, long noncoding RNAs still represent one of the least investigated fields because of their complexity, multiplicity, and diversity. While some long noncoding RNAs have been characterized fairly well, the functions of many others remain poorly understood. Long noncoding RNAs play an essential role in the regulation of gene expression in all tissues and on all developmental stages. They are involved in a number of signaling pathways, and their aberrant functioning can be pathogenic. This review aims to summarize current state-of-the-art structures of these transcripts in this research field, their genomic localization, their functions, and underlying mechanisms. It also focuses on cancer-associated aberrations of long noncoding RNAs, as well as on prospects of their application in tumor diagnostics and therapy. Examples of decreasing the levels of oncogenic long noncoding RNAs via silencing with short interfering RNAs, antisense oligonucleotides, or low molecular-weight inhibitors are also described.

[Hormone Resistance and Neuroendocrine Differentiation Due to Accumulation of Genetic Lesions during Clonal Evolution of Prostate Cancer].

Progression of malignant tumors is largely due to clonal evolution of the primary tumor, clones acquiring different sets of molecular genetic lesions. Lesions can confer a selective advantage in proliferation rate or metastasis on the tumor cell population, especially if developing resistance to anticancer therapy. Prostate cancer (PCa) provides an illustrative example of clinically significant clonal evolution. The review considers the genetic alterations that occur in primary PCa and the mechanism whereby hormone-refractory PCa develops on hormone therapy, including mutations and alternative splicing of the androgen receptor gene (AR) and intratumoral androgen synthesis. Certain molecular genetic lesions determine resistance to new generation inhibitors (AR mutations that block the antagonist effect or allow other hormones to activate the receptor) or lead to neuroendocrine differentiation (repression of the AR signaling pathway, TP53 mutations, and amplification of the AURKA or MYCN oncogene). Multistep therapy based on the data about somatic mutations associated with progression and metastasis of the primary tumor can be expected to significantly improve the survival of patients with advanced PCa in the nearest future.

[Expression of microRNA let-7a, miR-155, and miR-205 in tumor and tumor-adjacent histologically normal tissue in patients with non-small cell lung cancer]. / Ekspressiya mikroRNK let-7a, miR-155, miR-205 v opukholevoi i smezhnoi morfologicheski normal'noi tkani u patsientov nemelkokletochnym rakom legkogo.

UNLABELLED: Non-small cell lung cancer (NSCLC) is a main group of lung malignancies. Epigenetic changes are as important as genome structural changes in carcinogenesis. MicroRNA (miRNA) is a class of non-coding single-stranded RNAs that play an important role in the regulation of matrix RNA (mRNA) translation and degradation. MicroRNA expression changes occur in many cancers. According to the field cancerization theory, tumor-adjacent histologically normal tissue takes part in tumor progression by triggering cell transformation. The important clinical implication is that the fields may serve as the basis for a recurrence after surgery. Thus, the aim of our study was to determine the expression levels of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues to evaluate these changes as potential prognostic markers in NSCLC patients. METHODS: The expression of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues at 2 and 5 cm was determined by real-time PCR with subsequent quantification using a 2-ΔΔСt method. The findings were then analyzed to reveal possible associations with clinical and morphological parameters, such as age, cancer stage, and tumor grade. RESULTS: The expression of miRNA let-7a was found to be significantly lower in tumor than that in tumor-adjacent apparently normal tissue at 2 and 5 cm. In groups of patients older than 63 years with Stage III-IV NSCLC, the expressions of microRNA let-7a and miR-155 in tumor tissue were substantially lower than that in the adjacent normal tissue. Beyond that point, patients with high-grade tumors had also a significantly lower expression of miRNA let-7a in relatively adjacent apparently normal tissue. CONCLUSION: The findings suggest that miRNA let-7a and miR-155 may be used as poor prognostic markers for patients with NSCLC.

[. Role of molecular and genetic factors in survival from uveal melanoma]. / Vyzhivaemost' pri uveal'noi melanome: rol' molekulyarno-geneticheskikh faktorov.

AIM: to analyze survival rates in uveal melanoma (UM) patients and establish correlations with chromosome 3 monosomy, chromosome 1p deletion, and RASSF1A methylation. MATERIAL AND METHODS: Methylation-specific PCR analysis was performed in 104 patients with histologically verified UM. RESULTS: A statistically significant correlation has been found between chromosome 3 monosomy, on the one hand, and mixed/epithelioid cell melanomas and ciliary body involvement, on the other. As for chromosome 1p deletion, it has demonstrated association with extrabulbar tumor growth. We have also calculated 5-year survival and mortality rates in «large¼ UMs and their relationship with chromosome 3 monosomy, chromosome 1p deletion, and RASSF1A methylation. CONCLUSION: Chromosome 3 monosomy is associated with lower survival rates, while RASSF1A methylation - with a better prognosis. A combination of molecular and genetic changes (particularly, chromosome 3 monosomy and chromosome 1p deletion) also leads to reduced survival in UM patients.

[The molecular genetic alterations in mucosa of intestines as markers of oncologic progression and estimate of effectiveness of anti-reflux operations in patients with Barrett's esophagus].

The development of disease of Barrett's esophagus is based on processes of metaplasia of epithelium of esophagus when as a result of reflux of gastric juice and bile acids the normal planocellular epithelium of esophagus is replaced by cylindrical epithelium of intestinal type. Thereupon, Barrett's esophagus is progressing up to dysplasia and adenocarcinoma of esophagus. The progression from precancerous states up to tumor is related to development of genome disorders in cells associated with malignant transformation. The genetic and epigenetic alterations conditioning tumor growth can be used as markers of prognosis of clinical course of disease. To receive possible markers of progression of Barrett's esophagus the study was organized concerning methylation of such genes-suppressors of tumor growth as MGMT, CDH1, p16/CDKN2A, DAPK, RAR-ß and RUNX3 in patients with Barrett's esophagus and adenocarcinoma of esophagus. The effectiveness of applied anti-reflux surgical treatment was evaluated too. The abnormal methylation of studied genetic panel in patients with Barrett's esophagus prior to surgical treatment was observed reliably more frequently in altered epithelium as compared with unaltered epithelium (p<0.0001), under dysplasia as compared with metaplasia (p<0.0358) and in the presence of long (>3 cm) segments of altered epithelium as compared with short (<3 cm) segments (p=0.0068). In normal epithelium, prior to operation, abnormal methylation of panel of genes was detected in 7/60 (12%) of patients. Against the background of surgical treatment number of long and short segments of altered epithelium of esophagus reliably decreased (p<0.05). At that, in short segments after operation rate of methylation increased significantly (p=0.0068). Though after operation number of patients with Barrett's esophagus and dysplasia and metaplasia decreased, the rate of abnormal methylation in the other patients increased. It is demonstrated that anti-reflux operation ameliorates condition of mucous membrane of esophagus under Barrett's esophagus. However, in cases without regression significant increasing of rate of abnormal methylation of studied panel of genes is occurred. This is a proof that abnormal methylation of system of genes is related to worse response to application of anti-reflux surgical treatment.

[Allelic imbalance of loci 17p13.1 (TP53), 1p36.1 (RUNX3), 16p22 (CDH1) and microsatellite instability in gastric cancer].

We examined allelic imbalance (AI) on loci 17p13.1 (TP53), 1p36.1 (RUNX) and 16p22 (CDHI) and microsatellite instability (MI) with BAT26 in 78 patients with gastric cancer. We have shown a significant difference in the frequency of allelic imbalance of the studied loci among different types of gastric cancer. Frequency of AI in 16p22.1 (CDH1) (p = 0.023), 17p13.1 (TP53) (p = 0.038), microsatellite instability (p = 0.047) and AD two and more loci in a single sample (p = 0.0176) was significantly higher in the intestinal type of gastric cancer than in the diffuse type. We have shown, that, frequency of AI in 16p22.1 (CDH1), and AD two and more loci in a single sample, was higher in thetumors with high or moderate type of tumor cells differentiation (p = 0.0414, p = 0.0057 respectively). We found no significant differences in the groups with metastases in regional lymph nodes, different tumor stage, localization of tumors and the generalization process.

[Structural and functional analysis of tumor genomes and the development of test systems for early diagnosis, prognosis and cancer therapy optimization].

The article discusses results of the structural and functional analysis of molecular genetic abnormalities in various malignant tumors. Investigations have discovered more than 20 new markers for sporadic breast cancer. Several of them formed the test system, allowing the diagnosis with a specificity of 100%. Appearance of TMPRSS2/ERG4 chimeric gene is a frequent tumor-specific event, its expression is correlated with more aggressive forms of prostate cancer, may serve as a molecular marker for tumor cells and androgen assessment of tumor response to hormonal therapy. The effective systems for the early diagnosis of cervix and endometrium cancer were developed as well. Mutations in the VHL, deletions of chromosome 3 and methylation of several genes can predict the course and selection of effective therapy of clear cell kidney cancer, a number of molecular markers were identified for early diagnosis and prognosis of recurrence of bladder cancer. For diagnosis, prognosis and treatment of brain tumors we developed an effective complex system of markers. Protocol of molecular genetics investigation reveals the cause of the disease by more than 90% of patients with retinoblastoma. In order to study abnormal methylation in tumor genomes an innovative technology AFLOAT has been developed that allows to efficiently identify new markers with diagnostic value. Test systems of molecular genetic and epigenetic markers for early diagnosis and prognosis as well as for cancer therapy optimization have shown to be effective, have been approved for use in clinical practice and are being introduced into practical healthcare.

[The systems of molecular genetic markers under cancer of stomach].

The study was organized to investigate the anomalous methylation of genes NA?1, RASSF1A, MLH1, N33, DAPK, the expression of genes hTERT. metalloproteinase MMP7, MMP9, survivin. COX-2, p53. The activity of telomerase in 106 samples of stomach tumors taken through intra-operation way and 53 samples of stomach tumors taken through endoscopic way and 50 samples of biopsy taken from patients with chronic calculous cholecystitis (comparison group) was analyzed too. These changes can be used as additional markers both in diagnostic of cancer of stomach and dynamic monitoring of operated patients.

[Localization of point mutations in the coding part of the VHL gene in clear cell renal cancer].

VHL gene is often inactivated in sporadic clear cell renal cancer (CCRC) due to somatic mutations, and it's germline mutations cause hereditary CCRC--von Hippel-Lindau syndrome. Localization of mutations in VHL, identification of new mutations and their influence on CCRC progression and sensitivity to targeted therapy are actual problems in modern oncogenetics. We have provided search and characterization of mutations in 248 primary CCRC using SSCP-analysis and sequencing. Somatic mutations were detected in 37.5% of samples, 72% of mutations were identified for the first time. New missense-mutations were analyzed by alignment programs and three-dimensional structure modeling. Mutation frequency was compared in different groups of patients in respect to stage, grade, and metastases. It was demonstrated that 39.1% samples with stage I harbor somatic mutations, however, no association with progression or metastases was found. We also have investigated localization of mutations in the VHL coding part and positions of missense-mutations and inframe deletions/insertions focusing on VHL critical sequences. VHL mutation analysis performed in this study improve the possibilities of laboratory diagnostics of familial and sporadic CCRC.

[Clonal origin of multiple foci of urinary bladder cancer].

To analyze the pattern of molecular damages in urinary bladder cancer (UBC), the authors studied allelic imbalance of chromosome loci 9p21 and 17p13 in 22 patients diagnosed as having multiple primary UBC (2 to 5 foci in each patient). The state of markers has no informative value in 3 (13.6%) cases; in 9 (47.4%) of 19 informative cases, deletion of the same allele was determined in at least one of the loci in question in all tumor nodules, which may point to the monoclonal origin of multiple tumors in these patients. Five (26.3%) of the 19 patients exhibited deletion of the same allele in different tumor nodules, which is suggestive of the active process of clonal evolution and the feasibility of tumor subcloning, which does not preclude the possibility of monoclonal origin. Five (26.3%) of the 19 patients had an imbalance of different alleles in varying nodules, which may show the oligoclonal origin of the tumor nodules concerned. The concordant and discordant patterns of molecular damages are encountered virtually with the same frequency in the tumors of multiple primary UBC, which supports the view that its synchronous tumors can develop both monoclonally through intraluminal dissemination of tumor cells and when there are cancerization fields that determine the occurrence of oligoclonal tumors.

[Allelic disbalance in 1q32 area and microsatellite instability renal papillary adenocarcinoma].

Papillary adenocarcinoma is an abundant form of renal cell carcinoma. At present any diagnostic and prognostic molecular markers of papillary adenocarcinoma are absent, however some cytogenetic and molecular-genetic features of disease are known. According to literary data, the 1q32 duplication is associated with progressive deterioration of primary tumor. We have done a genetic typing (D1S2142 and D1S3465 locus) of 39 papillary adenocarcinoma cases, used PCR and fragment analyses of the 1q32 area. Frequency of the allelic disbalance was 36.8%; the microsatellite instability was found out in 48.7% of cases. The association of genetic disturbances with clinic-morphological features of papillary adenocarcinoma wasn't revealed. In some cases genetic heterogeneity of tumor-adjacent renal parenchyma and primary tumors was found out at multifocal renal carcinoma. For the first time we ve demonstrated that the allelic disbalance in 1q32 area and the microsatellite instability are frequent molecular-genetic disturbances in sporadic papillary carcinomas at all stages of the disease. Probably, the microsatellite instability is connected with progressive deterioration of primary tumor at renal papillary adenocarcinoma.

[Some molecular-genetic markers, defining the pathogenesis of superficial and invasive bladder cancer].

We have investigated deletions of 3p14, 9p21, 9q34, 17p13 (TP53) loci, activating FGFR3 mutations in exon 9 and aberrant methylation of RASSF1, RARbeta, P16, P14, CDH1 genes with the aim of the molecular pathogenesis pathways analysis of bladder cancer. FGFR3 activating mutations and 9p21 deletions were observed significantly more frequent in the group of non-invasive bladder cancer pTa than in minimally-invasive cancers pT1 (p = 0.004 and 0.006 respectively). It was shown that groups of superficial and invasive bladder cancer are significantly differing in the frequency of 17p13 (p = 0.006) and 9q34 (p = 0.04) deletions and in aberrant methylation of the gene P16 (p = 0.02). We have revealed some differing molecular-genetic alterations in groups of superficial and invasive bladder cancers. Therefore we suppose that these two types of bladder cancer might have different pathways of development.

[Fusion genes and transcripts in neoplasia].

Chromosomal rearrangements resulting in the formation of fusion genes are common events in carcinogenesis. There are more than 440 known fusion genes found in both malignant and benign tumors. The mechanism of transcription induced chimerism (TIC) contributes to fusion transcripts in normal human tissues. However, there is no clarity about the role of TIC in carcinogenesis. Hybrid proteins resulting from chimeric genes regarded as ideal markers which are specific for disease entities can be potential targets for the treatment due to their key roles in malignant transformation. In some tumors fusion genes may play primary role, and in the others may represent an additional mechanism during subclonal selection. The aim is to briefly review and discuss the occurrence and biologic relevance of chimeric genes in hematologic malignant diseases, sarcomas and epithelial neoplasms.

[Analysis of SYT/SSX1 and SYT/SSX2 fusion genes from synovial sarcoma].

The t(X;18)(p11;q11) translocation has been shown to be the specific alteration for synovial sarcomas. The translocation leads to production of chimeric protein SYT/SSX by fusion of SYT and SSX genes involved. The expression analysis of SYT/SSX1 and SYT/SSX2 chimeric transcripts was performed in formalin-fixed soft tissue tumour specimens and the diagnostic validity of immunohistochemistry, FISH and RT-PCR methods was compared. The chimeric transcripts were detected in 12 from 16 synovial sarcomas: 7 SYT/SSX1 and 5 SYT/SSX2 fusion variants; by fluorescence hybridization in situ (FISH) the translocation was found in 13 from 16 sarcoma samples. As synovial sarcoma represents a diagnostically challenging group, genetic analysis of translocations and chimeric transcripts is an extremely useful confirmatory diagnostic tool providing higher sensitivity than immunohistochemistry markers do.

[Amplification of intermethylated sites experimental design and results analysis with aims in silico computer software].

Amplification of intermethylated sites (AIMS) is a powerful tool for differential methylation screening of genomes. Its applications have nevertheless been limited until recently for the absence of systemic approach to AIMS experimental design and of appropriate computer software for the analysis of AIMS results. We have developed AIMS in silico computer suggestion tool capable of predicting possible experimental outcomes, which assists in designing AIMS experiments depending on the research aims and available instrumentation, and in analyzing experimental results from the point of view of genomic locations of the DNA fragments under study. With AIMS in silico we have characterized qualitatively and quantitatively AIMS products obtainable under different conditions; to ease experimental design we demonstrate AIMS products hierarchical structure. We discuss examples of designing AIMS experiments and results analysis as well as possible relative to AIMS alternative approaches to differential methylation screening. AIMS in silico computer software is intended to standardize AIMS applications and to turn it into one of the principal approaches towards cancer epigenomes studies as well as towards diagnostics in oncology, including early screening.

[Molecular diagnosis of liposarcomas: identification of the chimeric genes FUS/CHOP and EWS/CHOP].

This paper presents the results of an analysis the chimeric genes FUS/CHOP and EWS/CHOP in patients diagnosed as having liposarcoma in order to make a differential diagnosis in both soft tissue tumors and various variants of liposarcoma. Liposarcomas were found in 5 of 7 cases of primary tumors: 4 chimeric transcripts of the FUS/CHOP type (5-2), a variant of alternative splicing of the FUS/CHOP type (5-2) with depletion in 14 p.n. anda rare variant of the EWS/CHOP type (7-2). Fluorescence in situ hybridization (FISH) confirmed translocations in the tumor samples with the chimeric genes being detected. Reverse transcription-polymerase chain reaction and FISH revealed no chimeric genes specific to myxoid sarcoma in a group of patients with other variants of liposarcoma. Thus, the findings support the strict specificity of the chimeric genes FUS/CHOP and EWS/CHOP for myxoid liposarcoma and the expression of these genes in most tumors of this type.

[Inactivation of the VHL gene in sporadic clear cell renal cancer].

Renal cell carcinoma is the most common variant of the kidney cancer, which accounts approximately 75% patients with this disease. The majority of those tumors are characterized by inactivation of the VHL gene suppressor as a result of mutations, allelic deletions and/or methylation. We have conducted the complex molecular-genetic analysis of 64 samples obtained from patients with the clear cell renal cancer. VHL mutations were detected by single strand conformation polymorphism and subsequent sequencing, loss of heterozygosity was analyzed using two STR-markers, methylation was tested by methylsensitive polymerase chain reaction. All revealed variations were statistically analyzed in respect to the parameters of primary tumors in various groups of patients. Seventeen VHL somatic mutations were detected, 12 from which were described for the first time. Allelic deletions of VHL were found in 31.6%, and methylation--in 7.8% samples of the renal cancer. As a whole, VHL inactivating events were presented in 46.9% cases of disease, in 51.7% -among renal cancer patients with first stage. We have not observed any association of mutations, loss of heterozygosity and methylation with clinical-pathological parameters of disease. Results of this investigation specify for expediency of further studies of molecular genetics aberrations in the VHL gene. Perhaps, it would promote renal cancer molecular markers evaluation, for example, a determination of suppressor genes methylated in renal cancer.