Practical application of massively parallel reporter assay in biotechnology and medicine

The development and viability of an organism relies on tissue-specific gene programs. The genome regulatory elements play a key role in the regulation of such programs, whereas its disfunction can lead to the establishment of various pathologies, including cancer, congenital disorders, and autoimmune diseases. The development of high-throughput approaches in genomics has led to the emergence of massively parallel reporter assays (MPRA), which enable genome-wide screening and functional verification of regulatory elements. Although MPRA was originally used for investigation of fundamental aspects of epigenetics, it also has a great potential for clinical and practical biotechnology. Currently, MPRA is used for validation of clinically significant mutations, identification of tissue-specific regulatory elements, identification of the favorable loci for transgene integration, as well as represents an essential tool for creating highly efficient expression systems, with a wide range of applications from protein production and design of novel therapeutic antibody super-producers to gene therapy. In this review, the basic principles and areas of practical application of high-throughput reporter assays will be discussed

Methods of massive parallel reporter assays for investigation of enhancers

The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation

IMMUNOPHENOTYPIC CHARACTERISTICS OF INFLAMMATORY BREAST CANCER

The investigation enrolled 31 patients with inflammatory breast cancer (IBC) treated at the N. N. Blokhin Cancer Research Center from 2006 to 2008. IBC is diagnosed on the basis of signs of rapid progression, such as localized or generalized breast induration, red- ness and edema. IBC accounts for less than 5% of all diagnosed breast cancers and is the most lethal form of primary breast cancer. We studied tumor markers of the immunophenotype of IBC and levels and subpopulations of immunocompetent tumor-infiltrating cells. We found that expression of HLA-DR is in negative correlation with MUC-1 expression and lymphoid cells tumor infiltration is asso- ciated with the increase in T-cell subpopulations

Circulating microRNAs in lung cancer: prospects for diagnosis, prognosis, and prediction of antitumor treatment efficacy

The review considers the main techniques to extract microRNA (miRNA) from various biological fluids (in particular, the serum and plasma), approaches to the analysis of miRNA concentration and composition, and methods to normalize the results in data analyses. Advantages and drawbacks of the methods are described. Special attention is given to circulating miRNAs, which can be used as markers for minimally invasive diagnosis, prediction of antitumor treatment efficacy, and disease prognosis in lung cancer. The review discusses the prospects and limitations that arise as the clinical significance is evaluated for miRNAs as potential tumor markers and a better understanding is gained for the roles various miRNAs play in the pathogenesis of lung cancer

Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation

Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA. Concentration of cirDNA and methylation of RARβ2, RASSF1A and HIC-1 gene promoters were investigated in cell-free and cell-surface-bound fractions from healthy donors, patients with breast cancer, and patients with breast fibroadenoma. Tumour development was shown to lead to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions. Analysis of RARβ2 and RASSF1A methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women. Results of the study indicate that methylation-specific PCR of RARβ2 and RASSF1A genes based on the total cirDNA combined with the quantitative analysis of cirDNA distribution between cell-bound and cell-free fractions in blood provide the sensitive and accurate detection and discrimination of malignant and benign breast tumours

CLINICAL-MORPHOLOGIC AND MOLECULAR-BIOLOGIC PROGNOSTIC FACTORS FOR RARE FORMS OF UTERINE CANCER

This review of literature is devoted to rare forms of uterine cancer. Comparative characteristics of each histological type are given. Clinical-morphologic and molecular-biologic prognostic factors are analyzed in details

The clinical and morphological features of ovarian steroid cell tumors

Ovarian steroid cell tumors are rare, unusual neoplasms. There is now no unified management tactics for patients with this pathology. The paper reviews the literature on the clinical and morphological characteristics, prognostic factors, diagnosis, and treatme nt in patients with ovarian steroid cell tumors

Circulating DNA-markers in lung cancer: changes in retrotrasposons methylation status in response therapy and during the post-treatment follow-up

Malignant cell transformation is accompanied by two processes of DNA methylation changes

ГЕПАТОЦЕЛЛЮЛЯРНЫЙ РАК ПРОМЕЖУТОЧНОЙ СТАДИИ. BCLC B – ОФИЦИАЛЬНЫЕ РЕКОМЕНДАЦИИ, КАК СТРАТЕГИЯ БАЗИСНОГО ЛЕЧЕНИЯ И ТОЧКА ОТСЧЕТА В ОЦЕНКЕ ЭФФЕКТИВНОСТИ НОВЫХ ПОДХОДОВ

Представлен анализ Барселонской клинической классификации (BCLC) гепатоцеллюлярного рака (ГЦР). Подробно освещены лечебные рекомендации, предлагаемые BCLC для промежуточной стадии, рассмотрены современные мировые тенденции по внедрению эффективных  лечебных стратегий. Проведен анализ клинической гетерогенности промежуточной стадии (BCLC В), проблем диагностики и лечения. Подчеркнута важность стандартизованного алгоритма обследования для точной диагностики  промежуточной  стадии ГЦР. Рассмотрены перспективы оптимизации лечебных стратегий при BCLC В: поиск  и создание возможностей для радикального оперативного лечения, а также рациональное использование консервативных методов – химиоэмболизации и системной терапии – в лечении неоперабельных больных ГЦР