[Determination of the Subclonal Tumor Structure in Childhood Acute Myeloid Leukemia and Acral Melanoma by Next-Generation Sequencing].

Intratumoral heterogeneity and clonal variability are among the central problems of clinical oncology, leading to resistance to therapy, relapse, and metastasis. High-throughput sequencing of the tumor exome makes it possible to investigate the subclonal tumor organization. Target panel, clinical exome, and complete exome sequencing data were compared in tumors with different mutational burden, acute myeloid leukemia (AML) in children and acral melanoma. Targeted sequencing of AML samples detected more than one potential driver mutation in the signaling pathway genes KIT, NRAS, KRAS, CBL, and FLT3 in one patient, reflecting the complex clonal structure of the tumor substrate. Clusters of mutant alleles corresponding to different populations of leukemic cells in a sample were isolated based on exome sequencing data from the same AML patients. A comparison of the mutation profile for a primary AML sample and samples obtained in remission and relapse made it possible to trace the dynamic changes in the clonal composition of the tumor. The subclonal tumor structure was investigated in an acral melanoma case as an example. Mutant alleles present in the sample with close frequencies were clustered using the SciClone and ClonEvol packages. The results were used to predict the intratumoral clonal composition and to assume a clonal evolution model, which described the changes in the clonal composition of the tumor during metastasis, including the appearance of new mutations that might be associated with further disease progression. The approach used in the work is suitable for identifying the mutations that cause the formation of new tumor clones, which may have a proliferative advantage, in particular, in conditions of antitumor therapy.

[Germline and Somatic Mutations in Archived Breast Cancer Specimens of Different Subtypes].

Molecular profiling of tumors may provide promising options for personalized treatment. We have examined the spectrum of germline and somatic mutations in 23 breast cancers (ВС) of various molecular subtypes, including tumors 1) with expression of estrogen, progesterone and/or epidermal growth factor receptor HER2/neu, and 2) with a triple negative phenotype. Genomic DNA specimens were isolated from archived tumor and normal tissue samples and subjected to targeted sequencing of the coding regions of 25 cancer-associated genes with a mean coverage of x 1000. In the triple negative subtype of ВС, the pathogenic germline mutations BRCA1 c.66_67delAG (185delAG) and BRCA1 c.3226_3227AG (3347delAG) were detected, while the germline mutation BRCA2 658_659del (886delGT) was found in patients with positive receptor staining. Mutations in BRCAl/2 were overrepresented by frequency (80%), pointing at common loss of heterozygosity affecting the normal allele. Somatic mutations in the TP53 gene were found in 7/10 (70%) patients with the triple negative subtype of ВС and in 3/13 (23%) in the group with positive receptor staining. Additionally, in both groups of patients, somatic mutations of the PTEN, MSH2, MSH6, and MUTYH genes were detected.

[Driver Mutations in Acute Myeloid Leukemia with Inversion of Chromosome 16].

Certain subtypes of acute myeloid leukemia occur as a result of the cooperation of several events these are, the formation of fusion genes as a result of chromosomal rearrangements, which leads to the disruption of cell differentiation, and the emergence of mutations that enhance cellular proliferation by activating intracellular signaling pathways. High-throughput sequencing methods reveal characteristic mutation spectra in leukemia associated with different chromosomal disorders. However, the role of mutation events in malignant cell transformation processes remains obscure. We searched for driver mutation events in leukemic cells containing the chimeric CBFB-MYH11 gene, which results from inversion of chromosome 16. Using target enrichment, the coding regions of 84 genes in genomes of 12 children with acute myeloid leukemia with inv(16) were investigated. Somatic mutations have been found in the genes of the proteins of intracellular signaling cascades mediated by receptor tyrosine kinases, such as KIT (41%), NRAS (25%), KRAS (17%), and FLT3 (8.3%). Comparative analysis of samples at the time of diagnosis and during remission was used to assess the role of mutations in the pathogenesis of the disease. Previously undescribed mutations in the KDM6A, NOTCH1, and IDH1 genes, which may be involved in leukemogenesis processes have been identified.

H2S counteracts proinflammatory effects of LPS through modulation of multiple pathways in human cells.

BACKGROUND: Hydrogen sulfide donors reduce inflammatory signaling in vitro and in vivo. The biological effect mediated by H2S donors depends on the kinetics of the gas release from the donor molecule. However, the molecular mechanisms of H2S-induced immunomodulation were poorly addressed. Here, we studied the effect of two different hydrogen sulfide (H2S)-producing agents on the generation of the LPS-induced inflammatory mediators. Importantly, we investigated the transcriptomic changes that take place in human cells after the LPS challenge, combined with the pretreatment with a slow-releasing H2S donor-GYY4137. METHODS: We investigated the effects of GYY4137 and sodium hydrosulfide on the release of proinflammatory molecules such as ROS, NO and TNF-α from LPS-treated human SH-SY5Y neuroblastoma and the THP-1 promonocytic cell lines. Transcriptomic and RT-qPCR studies using THP-1 cells were performed to monitor the effects of the GYY4137 on multiple signaling pathways, including various immune-related and proinflammatory genes after combined action of LPS and GYY4137. RESULTS: The GYY4137 and sodium hydrosulfide differed in the ability to reduce the production of the LPS-evoked proinflammatory mediators. The pre-treatment with GYY4137 resulted in a drastic down-regulation of many TNF-α effectors that are induced by LPS treatment in THP-1 cells. Furthermore, GYY4137 pretreatment of LPS-exposed cells ameliorates the LPS-mediated induction of multiple pro-inflammatory genes and decreases expression of immunoproteasome genes. Besides, in these experiments we detected the up-regulation of several important pathways that are inhibited by LPS. CONCLUSION: Based on the obtained results we believe that our transcriptomic analysis significantly contributes to the understanding of the molecular mechanisms of anti-inflammatory and cytoprotective activity of hydrogen sulfide donors, and highlights their potential against LPS challenges and other forms of inflammation.

[Somatic Mutations Associated with Metastasis in Acral Melanoma].

Acral melanoma is one of the most aggressive and fast-growing forms of cutaneous melanoma and is characterized by a predominant location on the palms and feet. Primary tumors, metastases, and normal tissue samples from five acral melanoma patients were examined by massive parallel sequencing, focusing on the coding regions of 4100 genes involved in the origin and progression of hereditary and oncology diseases. Somatic mutations were found in genes related to cell division, proliferation, and apoptosis (BRAF, NRAS, VAV1, GATA1, and GCM2); cell adhesion (CTNND2 and ITGB4); angiogenesis (VEGFA); and the regulation of energy metabolism (BCS1L). Comparisons of target DNA sequences between morphologically normal and primary tumor tissues and between normal and metastatic tissues identified the candidate genes responsible for rapid metastasis in acral melanoma.

[Evidence database for deprescribing of antipsychotic drugs in elderly and senile patients]. / Dokazatel'naia baza snizheniia kolichestva i doz antipsikhotikov u patsientov pozhilogo i starcheskogo vozrasta.

The review deals with the problem of polypragmasia and associated adverse drug reactions, which is very relevant for the elderly and senile age. Based on the frequent unjustified prescription of antipsychotic drugs in clinical practice and the serious consequences associated with it, especially in elderly people with cognitive impairment, the aim of this review was to analyze the current literature and an evidence base for antipsychotic therapy optimization in elderly. One of the most effective way to decrease drug-associated harm is deprescribing, the planned process of decreasing dose, discontinuation of drug or switching to another one aimed to improve quality of life of the patient. The article describes different types of deprescribing, presents the results of the analysis of literature on deprescribing of antipsychotics in long-term use in elderly patients with dementia. Central to this is the analysis of a systematic review of Cochrane E. Van Leeuwen and co-authors (2018), the leading research in the evidence base of deprescribing. Based on the available literature, the authors make the conclusion about the safety of deprescribing of antipsychotic drugs. The effect of abrupt discontinuation of treatment with antipsychotic drugs was evaluated in available literature. Most of the evidence relates only to residents of nursing homes or to patients in long-term psychogeriatric or geriatric wards (in-patient treatment). However, the evidence base of deprescribing of antipsychotic drugs is small, many studies have methodological limitations, the initial characteristics of the patients included in the study are extremely heterogeneous, methodologies for diagnosing and determining the severity of dementia, types and dosages of antipsychotic drugs, duration of observation periods differed greatly. Attention is drawn to the short duration of observation periods. All of the above dictates the need for specially planned randomized clinical trials, the results of which will develop detailed algorithms for deprescribing antipsychotics.

[Mutational Profiling of Pediatric Myeloid Leukemia Subtypes without Clinically Significant Chromosomal Aberrations].

The discovery of novel significant molecular and genetic markers is important for the diagnostics, prognosis, and therapy selection in hematological malignancies. Distinct cytogenetic aberrations leading to the formation of fusion genes are found in more than 40% of pediactric cases of acute myeloid leukemia (AML); however, the tumor cells in approximately 20% of these patients display cytogenetically normal karyotype (NK-AML). Here we present the analysis of the mutational profiles of leukemic cells collected from pediatric AML cases without known clinically significant chromosomal aberrations aimed at identifying AML specific markers. In 34 pediatric cases of different AML types, the coding regions of 26 genes involved in the AML pathogenesis were analyzed by massive parallel sequencing. Sequencing revealed the somatic mutations in genes that are involved in various intracellular signaling pathways, including the CEBPA, ETV, IDH1, JAK2, and NRAS genes. In addition, rare genetic variants were found in CUX1, FLT3, TET2, PTPN11, and NUP98 genes. This data may contribute to the understanding of the mechanisms of malignant cell transformation in the case of leukemogenesis.

[CAX3 Gene is Involved in Flax Response to High Soil Acidity and Aluminum Exposure].

Understanding the molecular mechanisms of plant response to unfavorable conditions is necessary for the effective selection of tolerant genotypes. Earlier, using high-throughput transcriptome sequencing of flax plants after exposure to aluminum ions (Al^(3+)) and high soil acidity, we detected stress-induced alteration in the expression of several genes, including CAX3, which encodes Ca^(2+)/H^(+)-exchanger involved in calcium ion transport. Here we describe CAX3 mRNA levels in flax cultivars either tolerant (Hermes and TMP1919) or sensitive (Lira and Orshanskiy) to Al^(3+). Stress-induced increased expression of CAX3 was detected only in aluminum-tolerant flax cultivars. The product of CAX3 gene may participate in flax response to high soil acidity and high Al^(3+) concentration through Ca^(2+)-mediated intracellular regulation.

[Identification of Fusion Transcripts in Leukеmic Cells by Whole-Transcriptome Sequencing].

Genetic aberrations in leukemia often lead to the formation of expressed chimeric genes, which should be assessed for proper diagnosis and therapy. Modern methods of molecular diagnostic mainly allow to identify already known fusion genes. RNAseq is an efficient tool for identification of rare and novel chimeric transcripts. Here we present the results of the whole transcriptome analysis of bone marrow samples from five patients with acute myeloblastic leukemia and one, with myelodysplastic syndrome. The whole-transcriptome analysis was performed using Illumina/Solexa approach. We found rare or unknown chimeric transcripts including ETV6-MDS1, MN1-ETV6, OAZ1-PTMA, and MLLT10-GRIA4. Each of these transcripts was confirmed by RT-PCR and Sanger sequencing.

[Overexpression of microRNAs miR-9, -98, and -199 Correlates with the Downregulation of HK2 Expression in Colorectal Cancer].

Glycolysis activation is one of the main features of energy metabolism in cancer cells that is associated with the increase in glycolytic enzyme synthesis, primarily, hexokinases (HKs), in many types of tumors. Conversely, in colorectal cancer (CRC) the decrease in the expression of HK2 gene, which encodes one of the key rate-limiting enzyme of glycolysis, was revealed, thus, the study of the mechanisms of its inhibition in CRC is of particular interest. To search for potential microRNAs, inhibiting the expression of HK2 in CRC, we have performed the analysis of data from "The Cancer Genome Atlas" (TCGA) and five microRNA-mRNA target interaction databases (TargetScan, DIANA microT, mirSVR (miRanda), PicTar, and miRTarBase) using original CrossHub software. Seven microRNAs containing binding site on mRNA HK2, which expression is negatively correlated with HK2 expression, were selected for further analysis. The expression levels of these microRNAs and mRNA HK2 were estimated by quantitative PCR on a set of CRC samples. It has been shown, that the expression of three microRNAs (miR-9-5p, -98-5p, and -199-5p) was increased and correlated negatively with mRNA level of HK2 gene. Thus, downregulation of HK2 gene may be caused by its negative regulation through microRNAs miR-9-5p, -98-5p, and -199-5p.

[The Effect of Beta-Amyloid Peptides and Main Stress Protein HSP70 on Human SH-SY5Y Neuroblastoma Proteome].

The accumulation and aggregation of ß-amyloids are major molecular events underlying the progression of Alzheimer's disease. In neural cells, recombinant HSP70 reduces the toxic effect of Aß and its isomeric forms. Here we describe the proteome of the neuroblastoma cell line after incubation with amyloid peptides Aß42 and isomerized Asp7 (isoAß42) without and with human recombinant heat shock protein 70 (HSP70). Incubation of SH-SY5Y cell culture with the synthetic Aß-peptides leads to a decrease in the levels of several cytoskeleton proteins (e.g., ACTN1, VIME, TPM3) and several chaperonines involved in the folding of actin and tubulin (TCPQ, TCPG, TCPE, TCPB). These changes are accompanied by an increase in the expression of calmodulin and the proteins involved in folding in the endoplasmic reticulum and endoplasmic cell stress response. The presence of exogenous HSP70 has led to an increase in expression of several chaperones and a few other proteins including endogenous HSP70. A combined effect of recombinant HSP70 with Aß peptides reduced cell apoptosis and significantly decreased the level of tubulin phosphorylation caused by the addition of Aß peptides.

[Treatment with anti-cancer agents results in profound changes in lncRNA expression in colon cancer cells].

Using real-time RT-PCR in combination with bioinformatics, we have shown for the first time that the treatment of HCT-116 and HT-29 colon cancer cells with two anti-cancer agents (doxycycline or 3,3'-diindolylmethane) results in profound changes in the intracellular content of several lncRNAs (by up to 100 times). Since many of these RNAs are secreted by tumors into the bloodstream, the obtained results provide a basis for developing more sensitive protocols for serological monitoring of tumor relapse and metastasis, as well as for search of new anti-cancer drugs.

[Differential expression of an ensemble of the key genes involved in cell-cycle regulation in lung cancer].

Targeted cancer therapy directed at individual targets is often accompanied by the rapid development of drug resistance. The development of a new generation of antitumor drugs involves the search for many targets simultaneously to block or, conversely, restore their activity. In this regard, simultaneous analysis of gene expression in a complex network of interactions, primarily cell cycle control elements, is relevant for the search of specific molecular markers for the differential diagnosis of adenocarcinoma (ADC) and squamous cell lung cancer (SCC), as well as new targets for therapy. In this paper we performed an extended quantitative analysis of the expression of two suppressor genes, CTDSPL and its target RB1, as well as 84 genes of the main participants of the p16^(INK4A)-Cdk/cyclin D1-Rb and p53/p21^(Waf1) signaling pathways in the histological types of non-small-cell lung cancer (NSCLC), i.e., ADC and SCC, using the special panel of the Human Cell Cycle Regulation Panel. The expression profile of some genes shows the specificity to the histological type of NSCLC and the presence of metastases. The genes with a significantly increased expression that affect the activity of Rb (cyclins, cyclin-dependent kinases, their activators, inhibitors, etc.) can serve as potential targets for combined therapy of both ADC and SCC.

[Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation.

[Molecular genetic mechanisms of drug resistance in prostate cancer].

The major problem in prostate cancer treatment is the development of drug resistance and especially important, cross-resistance. The mechanisms of drug resistance, which are divided into ligand-dependent (requiring the presence of androgens in the cell) and independent (not requiring the presence of androgens) are reviewed. The mechanisms are mainly represented with mutations of the androgen receptor and expression of aberrant constitutively active splice variants, as well as up-regulation of genes involved in androgens synthesis.

[Downregulation of OGDHL expression is associated with promoter hypermethylation in colorectal cancer].

Cell metabolic reprogramming is one of the cancer hallmarks. Glycolysis activation, along with suppression of oxidative phosphorylation and, to a lower extent, the TCA cycle, occurs in the majority of malignant tumors. A bioinformatics search for the glucose metabolism genes that are differentially expressed in colorectal cancer (CC) was performed using the data of The Cancer Genome Atlas (TCGA) Project. OGDHL for an oxoglutarate dehydrogenase complex subunit, which is involved in the TCA cycle and is indirectly responsible for the induction of apoptosis, was identified as one of the most promising candidates. A quantitative PCR analysis showed, on average, an eightfold downregulation of OGDHL in 50% (15/30) of CC samples. Based on the TCGA data, promoter hypermethylation was assumed to be a major mechanism of OGDHL inactivation. Bisulfite sequencing identified the OGDHL promoter region (+327 ... +767 relative to the transcription start site) that is often methylated in CC samples with downregulated ODGHL expression (80%, 8/10) and is possibly crucial for gene inactivation. Thus, frequent and significant OGDHL downregulation due to hypermethylation of a specific promoter region was demonstrated for CC. The OGDHL promoter methylation pattern was assumed to provide a marker for differential diagnosis of CIMP+ (CpG island methylator phenotype) tumors, which display dense hypermethylation of the promoter region in many genes.

[Evaluation of Gene Expression of Hexokinases in Colorectal Cancer with the Use of Bioinformatics Methods].

One of the hallmarks of cancer is the change of energy metabolism, mainly activation of glycolysis that occurs even at early stages of tumorigenesis. The glycolysis activation can be caused by overexpression of hexokinases, primarily HK1 and HK2. Colorectal cancer, which takes the third place in the cancer morbidity and mortality rates worldwide, is believed to be accompanied with overexpression of HK2, which is .considered a marker of poor prognosis. With the use of the developed CrossHub tool, we performed the analysis of the Cancer Genome Atlas RNA-Sequencing data, which, on the contrary, revealed the prevalence of the down-regulation of HK2 gene and only slight expression alterations in HK1 gene. The Cancer Genome Atlas is the largest resource in the field of molecular oncology that accumulated genomic, transcriptomic and methylomic data for thousands of sample of more than 20 cancers. The transcriptome analysis data for colorectal cancer (283 tumor samples and 41 matched normal samples) were in accord with the results of further qPCR expression level evaluation. Up-regulation of HK1 and HK2 genes was observed only in a part of samples: 12% for HK1 and 30% for HK2. At the same time, the HK2 mRNA level decrease was shown in 50% of cases. Correlation analysis revealed the consistency in HK1 and HK2 expression alterations (Spearman's rank correlation coefficient r(s) = 0.43, p < 0.01), that could be explained by common deregulation mechanisms of these genes in colorectal tumors. The HK3 expression level was significantly increased in 60% of samples. Most likely, just hexokinase 3 contributes significantly to the activation of glycolysis in colorectal cancer.

[Cell analogs of viral proteins].

Horizontal transfer of genes between viruses and their hosts played an important role in the evolution of various eukaryotes including contemporary mammals as well as the pathogens themselves. Elements of viruses of various types can be found in the genome of animals. Endogenous retroviral elements composing up to 8% of human genome length not only determine its high flexibility and rapid adaptation potential. Many of virus genes such as Fv1, Lv1, Lv2 being analogues of capsid and other proteins determine effective suppression of viral replication after cell penetration by the causative agent. Introduction of these elements into genome of a wide variety of animals from fish to primates could have taken place against the background of global natural cataclysms of viral origin. Integration of retrovirus genes coding surface glycoproteins with immunosuppressing domains into genetic apparatus of animals served as an impetus to the development of viviparity and spread ofplacental mammals. Their cell analogs syncytins perform a dual function: take direct part in the formation of syncytiotrophoblast layer of placenta and ensure tolerance of immune system of mother to embryo. The acquisition of cell genes by viruses also played an important role in their evolution: various interleukins and other modulators of immune response introduced into viral genome from cell genetic apparatus became one of the most important factors of pathogenicity of a wide variety of causative agents including poxviruses, cytomegalovirus, Epstein-Barr virus and many others. Evolutionary pathways of the virus and host are thus inseparable from each other, and character of one of these directions is largely dictated by the vector of another.

[Serological diagnosis of tumors: methods of marker's search].

This review describes the most popular methods of search for serological markers of tumors that are used in clinical setting, provided with comparison of their efficiency.

[Mechanisms of retroviral immunosuppressive domain-induced immune modulation].

Immunosuppressive domains (ISD) of viral envelope glycoproteins provide highly pathogenic phenotypes of various retroviruses. ISD interaction with immune cells leads to an inhibition of a response. In the 1980s it was shown that the fragment of ISD comprising of 17 amino acids (named CKS-17) is carrying out such immune modulation. However the underlying mechanisms were not known. The years of thorough research allowed to identify the regulation of Ras-Raf-MEK-MAPK and PI3K-AKT-mTOR cellular pathways as a result of ISD interaction with immune cells. By the way, this leads to decrease of secretion of stimulatory cytokines (e.g., IL-12) and increase of inhibitory, anti-inflammatory ones (e.g., IL-10). One of the receptor tyrosine kinases inducing signal in these pathways acts as the primary target of ISD while other key regulators--cAMP and diacylglycerol (DAG), act as secondary messengers of signal transduction. Immunosuppressive-like domains can be found not only in retroviruses; the presence of ISD within Ebola viral envelope glycoproteins caused extremely hard clinical course of virus-induced hemorrhagic fever. A number of retroviral-origin fragments encoding ISD can be found in the human genome. These regions are expressed in the placenta within genes of syncytins providing a tolerance of mother's immune system to an embryo. The present review is devoted to molecular aspects of retroviral ISD-induced modulation of host immune system.