[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.

Investigation of ion acceleration effect influence on formation of ambipolar potential profile in the expander region.

This paper presents a study of plasma flux characteristics flowing out from the gas-dynamic mirror trap along the magnetic field lines to a metal wall. The main feature of the current work is that the effect of ion acceleration by ambipolar potential is considered in the expander region. The developed model also takes into account a possibility of transition from the collisional expansion of electron flow in the vicinity of the magnetic plug to the collisionless regime. The developed model allows calculation of the ambipolar potential profile and plasma characteristics in the expander region.

[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.

[Transcription Factor SAP30 Is Involved in the Activation of NETO2 Gene Expression in Clear Cell Renal Cell Carcinoma].

Clear cell renal cell carcinoma (ccRCC) is a common oncourological disease with a high mortality level. The incidence of this type of cancer is constantly increasing, while molecular mechanisms involved in the disease initiation and progression remain far from being fully understood. A problem of the search for novel markers is crucial for improvement of diagnosis and therapy of ccRCC. We have previously found that the disease is characterized by increased expression of the NETO2 gene. In the present study, we showed that isoform 1 (NM_018092.4) makes the main contribution to the upregulation of this gene. Using original CrossHub software, "The Cancer Genome Atlas" (TCGA) project data were analyzed to identify possible mechanisms of NETO2 gene activation in ccRCC. The absence of significant contribution of methylation to the increase of mRNA level of the gene was observed. At the same time, a number of genes encoding transcription factors, which could potentially regulate the expression of NETO2 in ccRCC, were identified. Three such genes (MYCBP, JMY, and SAP30) were selected for the further analysis of their mRNA levels in a set of ccRCC samples with quantitative PCR. We showed a significant increase in mRNA level of one of the examined genes, SAP30, and revealed its positive correlation with NETO2 gene expression. Thus, upregulation of NETO2 gene is first stipulated by the isoform 1 (NM_018092.4), and the probable mechanism of its activation is associated with the increased expression of SAP30 transcription factor.

[Multiplex Assay to Evaluate the Genetic Risk of Developing Human Melanoma].

A genotyping procedure based on single-step PCR and subsequent allele-specific hybridization on a hydrogel biochip was developed to address the polymorphisms of HERC2, OCA2, SLC24A4, SLC45A2, TYR, IRF4, MC1R,MITF, PIGU, MYH7B, NCOA6, and CDK10. Amplified gene fragments were fluorescently labeled in PCR, and fluorescent signals from biochip cells were detected to evaluate how efficiently the PCR product formed a perfect duplex with an immobilized probe. The analytical characteristics of hybridization analysis were estimated for several fluorophores with different optical spectra. Cyanine dyes fluorescing in the range of Cy5 and Cy7 were synthesized for the purpose and used as 5'-tags of universal primers in single-step PCR. A Cy7 analog fluorescing in the near infrared range was found to increase the sensitivity of hybridization analysis by producing a lower background signal in the cases where target gene amplification was low.

[Allelic variants of immune response genes in children with infectious complications during the treatment of acute leukemia].

Infectious complications that arise during the treatment of children with acute leukemia with chemotherapeutic agents at high doses represent a serious problem in oncohematology. To find genetic conditions that may lead to the development of postchemotherapy infections, the genomes of 12 children with acute leukemia who had severe infectious complications during therapy were examined. At the same time, the coding regions of 17 genes involved in the regulation of the immune response were determined by massive parallel sequencing. The analysis revealed 39 nonsynonymous SNPs that lead to amino acid substitutions, including the following informative genetic markers: PTPN22 c.1858C>T (rs2476601), TLR4 c.896A>G (rs4986790) and TLR4 c.1196C>T (rs4986791), IL7R c.197T>C (rs1494555) and IL7R c.412G>A (rs1494558). The results of massive parallel sequencing were validated by Sanger sequencing. The identification of genetic markers associated with the predisposition to infectious complications may allow one to assess the individual risk of the severe infection development in children with acute leukemia during the treatment with chemotherapeutic agents and to begin the development of personalized approaches to anticancer therapy.

[Detection of BCR-ABL gene mutations in chronic myeloid leukemia using biochips].

A biochip-based method was developed to identify the BCR-ABL mutations that affect the thyrosine kinase domain and determine resistance to targeted therapy with thyrosine kinase inhibitors. The method is based on RT-PCR followed by allele-specific hybridization on a biochip with immobilized oligonucleotide probes. The biochip addresses 11 mutations, which are responsible for up to 85% of imatinib resistance cases. A method to decect the clinically significant mutation T315I was designed on the basis of LNA-clamped PCR and proved highly sensitive, detecting the mutation in clinical samples with a leukemic cell content of 5% or higher. The method was validated using clinical samples from chronic myeloid leukemia (CML) patients with acquired resistance to imatinib. The results of hybridization on biochip were verified by Sanger sequencing.

[Infrared fluorescent markers for microarray DNA analysis on biological microchip].

To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

[Remote results of semiclosed endarterectomy with a loop from the superficial femoral artery and femoropopliteal bypass grafting].

AIM: To compare the results of semiclosed retrograde loop endarterectomy from the superficial femoral artery (SFA) and proximal femoropopliteal bypass grafting (FPBG) with a synthetic prosthesis. MATERIAL AND METHODS: From 2007 to 2011, at the Department of Vascular Surgery of Municipal Clinical Hospital No 15 named after O.M. Filatov we operated on a total of 97 patients with occlusive and stenotic lesions of the femoropopliteal segment, with 83 (86%) of them being men. The mean age of the patients was 62.7 ± 8.9 years. By the degree of chronic ischaemia the patients were subdivided according to the Fontain-Pokrovsky classification as follows: 52 (53.6%) patients had stage 2B ischaemia, 22 (22.7%) patients presented with stage 3, and 14 (14.4%) patients had stage 4. Nine (9.3%) patients were diagnosed with symptoms of acute ischaemia. Depending on the operative treatment performed, the patients were subdivided into two groups: Group I (n=56) comprising patients subjected to FPBG above the knee-joint fissure and Group II (n=41) consisting of those who underwent loop endarterectomy from the SFA. RESULTS: Technical success was obtained in 97 (100%) patients. After the operation the average ankle-brachial index (ABI) amounted to 0.87 ± 0.22 in Group I and 0.83 ± 0.22 in Group II. One (1.8%) patient from Group I developed shunt thrombosis successfully treated by thrombectomy. In Group II after surgery we also observed 1 thrombosis (2.4%) of the desobliterated superficial femoral artery, treated by thrombectomy from the SFA. The remote results were followed up during 4-7 years. Primary patency in the group of patients undergoing FPBG amounted after 1 and 2 years to 73.1% and 63.2% (p<0.005), and after 3 and 4 years to 34.1% and 30.7% (p>0.05). In the group of patients after loop EAE from the SFA 1-, 2-, 3- and 4-year primary patency amounted to 92.1%, 74.6% (p<0.05) and 63.1%, 58.9% (p>0.05), respectively. CONCLUSION: Semiclosed retrograde loop endarterectomy (EAE) demonstrated better results both in the immediate and remote postoperative periods as compared with femoropopliteal proximal bypass grafting using a synthetic prosthesis.

[Hybrid operations in multilevel lesions of lower-limb arteries].

Reflected in the article is experience in treating a total of 24 patients suffering from lower limb chronic ischaemia and diagnosed as having concomitant lesions of the aortofemoral and femoropopliteal segments, with a total of 24 hybrid operations performed. Of these, nineteen interventions consisted of implanting a stent graft into iliac arteries and revascularization of the femoropopliteal segment, whereas the remaining five cases included balloon angioplasty of the iliac artery with revascularization of the femoropopliteal segment. Good immediate outcomes were obtained in 23 patients. The ankle-brachial index increased averagely from 0.38 to 0.88. The remote outcomes (from 12 months to 5 years) were followed up in fifteen patients. Cumulative patency of the bypass grafts after 1, 3 and 5 years amounted to 78%, 41.5% and 41.5%, respectively. The cumulative survival rates after 1, 3 and 5 years equalled 90.8%, 76.2% and 76.2%, respectively. All extremities were eventually spared, with the long-term limb salvage rate thus amounting to 100%.

[Semi-closed endarterectomy from the aortic-iliac-femoral segment].

Presented herein is a review of the literature dedicated to extended semi-closed endarterectomy from the aortoiliac segment. Considered in the article are historical data concerning the development of semi-closed loop endarterectomy, also showing present-day approaches to revascularization of the arteries of the aortofemoral zone. Also given are indications/or and contraindications to surgical management and the data on immediate and remote outcomes of semi-closed endarterectomy, which are not inferior to the classical aortofemoral reconstructions. The advantages of this method are as follows: a minimally traumatic nature, a possibility to be performed in patients presenting with severe concomitant pathology, and no use of synthetic materials.

[Role and place of ophthalmological methods in the diagnosis of lesions of the main vessels of the brain]. / Rol' i mesto oftal'mologicheskikh metodov v diagnostike porazhenii magistral'nykh sosudov golovnogo mozga.

The study was aimed at detecting changes in visual functions and organ of vision in patients with involvement of the main vessels of the head and injuries to this vessels in patients with expressed ischemic diseases of the organ of vision. Twenty-four patients with occlusive disorders of the carotid arteries and 22 with retinal and ocular nerve diseases with the ischemic component were examined. The share of carotid artery abnormalities in the second group was 27.3%. Total efficacy of ophthalmological methods of diagnosis in the group with carotid artery involvement was 89.4%, this demonstrating high efficacy of ophthalmological methods for the diagnosis of involvement of the main cerebral vessels. Static quantitative perimetry proved to be the most effective method. It permits reliable detection of the slightest changes and assess the efficacy of surgical treatment.

[Indications for repairing the internal carotid artery after carotid endarterectomy]. / O pokazaniiakh k primeneniiu plastiki vnutrennei sonnoi arterii posle karotidnoi éndarteréktomii.

From 1983 to 1989, a total of 182 patients with atherosclerotic stenosis of the internal carotid artery (ICA) were examined and operated on. They underwent 198 carotid endarterectomies (EAE). In the long-term postoperative period, Doppler sonography and spectral analysis of an ultra Doppler signal were used to study 143 patients. Depending on the type of endarterectomy, 2 groups of patients were identified (1) 61 who had after EAE a plastic bifurcation operation with transition to the ostium of the ICA by means of a flap from a fragment of a synthetic prosthesis, autovein or xeno-pericardium; (2) 82 underwent EAE without dilatation of ICA. The analysis of the results of the studies revealed that after carotid EAE, the incidence of restenoses was 18.2%, that of thromboses was 8.4%, the incidence of atherosclerotic restenoses depended on the method to close the arteriotomic ostium: when the dilating flap was used, it was 1.5%, in isolated carotid EAE without its use, it was 11.03%. The incidence of myointimal restenoses in patients undergone plasty or no plasty of ICA was virtually equal. In atherosclerotic restenosis, 18.2% developed a negative dynamics of cerebrovascular insufficiency, whereas in thromboses 25% did, myointimal restenoses ran without clinical signs. The low incidence of atherosclerotic restenosis in plasty of carotid bifurcation made it the method of choice. Taking into account the nature of myointimal hyperplasia and the absence of clinical signs it should be noted that repeated reconstruction in this restenosis is not indicated, while in atherosclerotic restenosis, reconstruction is indicated in the presence of clinical manifestations.

[Tactics and indications for surgical treatment of patients with combined extra- and intracranial lesions of the carotid arteries]. / Taktika i pokazaniia k khirurgicheskomu lecheniiu bol'nykh s sochetannymi ékstra- i intrakranial'nymi porazheniiami sonnykh arterii.

The authors present the results of surgical treatment of patients with concomitant extra-intracranial and bilateral lesions of the carotid arteries. The patients underwent 163 operations for establishment of extra-intracranial microanastomosis (EICMA) including reconstruction of the extracranial segments with EICMA establishment (75 cases). In 66 cases, EICMA was performed for contralateral stenosis of the internal carotid artery (ICA). Of these, 22 patients later underwent carotid endarterectomy. In 14 patients, EICMA was established as the first stage before reconstruction of the iliofemoral segment. The operation was also performed in 8 patients with occlusion of the median cerebral artery or with the crimp of the carotid arteries. Examinations that included ultrasound dopplerography, transcranial dopplerography, studies of the cerebral blood flow according to 133Xe clearance and angiography were carried out. A study was made of the long-term results of the surgical treatment (from 0.5 to 5 years). It has been discovered that to estimate indications for surgical treatment, it is necessary that the data on the regional blood flow (RBF) be taken into account. In patients with stable cerebral hemodynamics according to the RBF that characterizes an uneventful course of ICA occlusion and in the decompensated type most typical of patients with pronounced postinsult neurological dificit, the establishment of EICMA is not indicated. Surgical treatment is most desirable in ICA occlusion (with preliminary reconstruction of the extracranial segments provided they are impaired) and in patients with unstable cerebral hemodynamics most typical of patients with transitory ischemic attacks, dyscirculatory encephalopathy, and minor brain stroke with chiefly mild residual phenomena of ischemic brain stroke.

[Tactics and indications in the surgical treatment of patients with combined extra- and intracranial lesions of the carotid arteries]. / Taktika i pokazaniia k khirurgicheskomu lecheniiu bol'nykh s sochetannymi ékstra-intrakranial'nymi porazheniiami sonnykh arterii.

The article deals with the results of surgical treatment of 159 patients with concomitant extra-intracranial and bilateral lesions of the carotid arteries; 163 operations were conducted for the formation of an extra-intracranial microanastomosis (EICMA) among which 75 operations comprised reconstruction of extracranial segments with creation of EICMA. In 66 cases EICMA was performed in contralateral stenosis of the internal carotid artery (in 22 with subsequent carotid endarterectomy), in 14 cases as the first stage before reconstruction of the iliofemoral segment, and in 8 cases in occlusion of the middle cerebral artery or in tortuous carotid arteries. Complex examination was conducted, including ultrasonic dopplerography, transcranial dopplerography, study of cerebral blood flow according to Xe-133 clearance, and angiography. The late-term results of surgery were studied in follow-up periods of 6 months to 5 years. It was found that to appraise the indications for surgical treatment it is necessary to take into account the data obtained in studying the regional cerebral blood flow. Surgical treatment is indicated in occlusion of the internal carotid artery (with preliminary reconstruction of extracranial segments when they are involved) in patients with unstable type of cerebral hemodynamics, which is most characteristic in patients with transitory ischemic episodes, dyscirculatory encephalopathy, small apoplexy and predominantly with mild sequelae of ischemic apoplexy.