The paper discusses the possible mechanisms of antitumor action of indolocarbazole derivatives. Here we present a data that show interaction drugs based on indolocarbazole derivatives with several intracellular targets and consequently activation different pathways of cell death. Also we present results of our studies on the mechanisms of antitumor action of compounds LCS-1006 and LCS-1208 synthesized in the N.N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia.

The review presents a discussion on articles and patents, describing new in vitro and in vivo models of pigmented or non-pigmented human cutaneous melanoma, received in NMRCO from the patients» metastases. Molecular genetic characteristics of the new models is supported by the arguments in addition to the given data and visual materials. The subjects of the discussed publications are 3 polyclonal cell lines, 2 subclones and 4 subcutaneous (s/c) xenografts in immunodeficient mice Balb/c nude. All the models are stored in Cryo Collection with xenografts at N.N. Blokhin NMRCO as well as in the Russian Collection of Cell Cultures of Vertebrae (RCCCV, St. Petersburg). This mini-collection is recommended for use in basic research of cutaneous melanoma and pre-clinical studies of anti-melanoma agents. The basis for these studies are the appropriate characteristics of the models, including cytological, immunologic, transplantation and molecular-genetic ones, as well as in vivo drug sensitivity to the corresponding target therapy.

Adjuvants are important components of cancer vaccines because they enhance immune responses to vaccination. However, adjuvants licensed for clinical use, e. g. aluminum salts, fail to stimulate an effective immune response. Research and development of new adjuvants with combined functions, including immune stimulation and antigen delivery, are a vital task for antitumor immunotherapy. Clinical trials of immune stimulating compounds, in particular Toll-like receptor (TLR) ligands, reveal their therapeutic potential as both antitumor agents and vaccine adjuvants.

Renal cell carcinoma (RCC) ranks first in mortality among urogenital tumors and is the most common disease after prostate and bladder cancer. Early detection of RCC allows immediately undertaking appropriate treatment, which significantly increases the survival of patients. In the case of the asymptomatic RCC, timely diagnosis in the early stages is usually difficult. To date, the problem of searching for molecular markers of clear cell RCC, which allows to determine the stage, metastatic potential and prognosis of disease, or select a treatment regimen remains topical. Of particular interest are early-stage biomarkers of RCC and its metastatic potential, as well as markers that can be obtained by non-invasive or minimally invasive methods. This review presents modern methods for diagnosing RCC using biomarkers.

Introduction. Previously we»ve shown that there are typical changes in haematopoiesis in diffuse large B-cell lymphoma patients (DLBCL). Attention was paid to the elevation of oxyphylic erythroblasts, as this sign was of prognostic significance. The aim of this work was to see
if there is possible to identify bone marrow erythrokaryocyte maturation stages using flow cytometry method.

Materials and methods. Study was done in 60 DLBCL patients. Elevation of oxyphilic erythrobast levels was noted in 50 patients (83,3 %). Flow cytometric study of bone marrow were done in 30 patients. We used monoclonal antibodies to Glycophorin A, CD45, CD36, CD117, CD71, CD105, CD34, HLA-DR, as well as nuclear staining for SYTO 16, SYTO 41.

Results. Use of Glycophorin A together with of SYTO 16 or SYTO 41 is effective for identification of erythrocaryocytes, main proportion of which is represented by CD71+ and Glycophorin A+ cells, which does not express any precursor markers. In CD45-negative cellular elements there are as well CD71- glycophorin А– cells, which represents mainly precursors – CD34+ CD117+ HLA-DR+/–. Erythro caryocytes with low levels of glycophorin A expression and prominent expression of CD71 (committed to erythroid differentiation) show slight expresiion of CD117 and HLA-DR, but CD34 is completely absent. The informative method for characterization of most mature erythrocaryocyes was the approach based on identification of erythrocaryocytes according to CD36 expression on cels with low SSC (in that case usage of nuclear stain, CD45 and glycophorin A may be omitted as CD36 does not stain erythrocytes). After peak of CD36, CD71, CD105 expression levels of these antigens are declining. Namely that fraction represents most mature erythrocaryocytes. Cor relation of the percentages of that cells with oxyphilic normoblasts (identified morphologically) was near to significant value.

Introduction. Increasing of blast cell percentage in bone marrow of diffuse large B-cell lymphoma patients is a sign of unfavourable prognosis. We estimated the frequency of this phenomenon and made attempts to idenify minimal bone marrow involvement in DLBCL by flow cytometry.

Materials and methods. Study has been done in 60 DLBCL patients. Diagnosis in all cases was done according to WHO (2008) criteria. Bone marrow study was done on smears (myelogram). Immunophenotypic study was done with a large panel of B-lineage monoclonal antibodies as well as kappa and lambda light chains to membrane immunoglobulins.

Results. Increase in blast cell content in bone marrow was noted in 73.3 % of DLBCL patients. That group of patients had lower surviv al. We used the following methods for identification of monoclonal B-lymphocytes: monoclonality according to membrane light immu noglobuline chains within mature B-lymphocytes and aberrancy of immunophenotype. Levels of different B-cell antigen expression – CD20, CD21, CD22, CD24. It was identified B-lymphocytes with aberrant expression of СD21.

Conclusion. The most informative methods of identification of minimal bone marrow involvement in DLBCL are estimation of clonality according to light chain expression of mature B-lymphocytes (CD45++CD20+CD5–), as well as assessement of aberrancy of CD21 expression of mature B-lymphocytes.

Introduction. B1-lymphocytes are a subpopulation of B-cells, the proportion of which in the spleen accounts for 5 % of the total number of B-cells. B1-lymphocytes predominantly secrete IgM, which plays an important role in the induction of tumor cell apoptosis. Splenec tomy for the purpose of adequate lymph node dissection in gastric cancer causes marked and prolonged immunological disorders. It primarily affects the B1a-lymphocyte subpopulation, which provides a thymus-independent immune response.

Objective. To study the features of the B-cell component of immunity in patients with gastric cancer.

Materials and methods. The subpopulation composition of B-lymphocytes of peripheral blood of gastric cancer patients undergoing sur gical treatment using flow cytometry (Facs Can, Lysys II and FacsCanto II programs, Facs Diva program) was analyzed. Cells were stained simultaneously with three monoclonal antibodies labeled with different fluorochromes.

Results. The data obtained demonstrate a violation of the composition of B-cell subpopulations. In the group of patients with standard D2 lymph node dissection and splenectomy at the preoperative stage and three months after surgery, a significant correlation was found between the relative number of B lymphocytes (p = 0.018), CD5 + B lymphocytes (p = 0.012) and the number of CD19 + CD38 + cells (p = 0.035). In addition, after surgical treatment, the percentage of CD5 + B-lymphocytes increased significantly from 12.9 to 21.8 %, while the total number of CD19 + lymphocytes and CD19 + CD21 + cells decreased.

Introduction. Currently, the following approaches are used for cancer treatment: surgical tumor removal, chemotherapy, targeted therapy and immunotherapy. The combination of different drugs may have additional advantages due to cumulative effect. Moreover, some additional effects like changes in PD–L1 and PD–L2 expression levels may be observed.

Aim. The aim of this study was to determinate the influence of aranose, cisplatin or paclitaxel and their combination on the expression of mRNA level and proteins PD–L1 and PD–L2 in melanoma cell lines and to compare the results with the differentiation status and with the appearance of mutations in melanoma cells.

Materials and methods. Melanoma cell lines used in this study were derived from surgical species of patients with disseminated melanoma. The mRNA expression level of PD–L1 and PD–L2 genes was measured by RQ-PCR. The expression of PD–L1 and PD–L2 proteins was measured by flow cytometry. The Pearson’s correlation and median test were used for statistical analysis.

Results. The expression level of PD–L2 gene was correlated with melanomas cell’s differentiation status (Pearson’s coefficient 0.937, p <0.15). The expression levels of PD–L1 gene and PD–L1 and PD–L2 proteins were not correlated with differentiation status of melanoma cells as well as TP53 mutations. In case of BRAF mutations the expression of PD–L2 was low detectable (p = 0.0117). It is worth noting that the TP53 mutations were associated with BRAF mutations (Pearson’s coefficient 1, p <0.15). The exposure of cells to aranose led to increased PD–L1 gene expression (p = 0,23). Incubation with cisplatin in combination with paclitaxel also resulted in an increase in PD–L1 protein expression (p = 0.037). Cisplatin or paclitaxel had no effect on the expression of PD–L1 protein. The expression level of PD–L2 gene and protein decreased under the action of any of these two drugs: these data are statistically (p = 0.6).

Introduction. The section «Pharmaceutical development» is an integral part of the registration dossier when developing a dosage form, however, this process is of particular importance for lyophilized dosage forms (LF), due to the sensitivity of lyophilization to the slightest changes in temperature, pressure and other factors. The purpose of this work is to demonstrate the capabilities of the methods shown in ICHQ8 Pharmaceutical Development, ICH Q9 Quality Risk Management and Guidelines for the Development and Production of a Drug Product (Pharmaceutical Development) (EurAsEC).

Materials and methods. These methods are used in the development of a LF for parenteral use based on the original pharmaceutical substance hexamethylene amide bis- (N-monosuccinyl-L-glutamyl-L-lysine) GK-2, which has neuroprotective activity. The studies were carried out using the substance GK-2, and as excipients: lyoprotectant – sucrose, cryoprotectants – medium chain polyethylene glycols 1500, 4000, 6000.

Results. The risks arising during the production of LF of a lyophilisate for parenteral use are estimated on the basis of a cause and effect diagram (Ishikawa diagram). Based on the chart, identified the main factors affecting the quality of the final product. Also, the critical
parameters of the processes, critical control points and interrelated critical parameters of quality, model compositions of the lyophilisate GK-2 for parenteral use were analyzed.

Introduction. Antibiotics of aureolic acid group are highly effective anticancer drugs. New data about their mode of action caused the need for creation of analogs with improved pharmacological properties. The methods of selective chemical modification of aureolic acid antibiotic olivomycin A were developed at the Gause Institute of New Antibiotics. Some semisynthetic derivatives have been prepared. The most active compound – N, N-dimethylaminoethylamide 1’-des-(2,3-dihydroxybutteroil)-1’-carboxy-olivomycin A (olivamide) – was selected for advanced preclinical testing.

Objective. The aim of the study was to investigate the toxicological safety of olivamide drug formulation in chronic experiment on rabbits.

Materials and methods. The study was performed in male and female “Soviet chinchilla” rabbits. Drug formulation was administrated intravenously at the total doses of maximum tolerated dose and 50 % lethal dose (15 × 0.02 mg/kg or 15 × 0.04 mg/kg with 24-h inter
val). During the experiment body weight, hematological parameters, blood biochemical parameters, electrocardiography and urinalysis were performed. Animals were sacrificed 1 and 30 days post treatment. The internal organs were subjected to histological evaluation.

Results. It has been shown that the treatment with total dose of olivamide maximum tolerated dose produces an increase of aspartate aminotransferase, urea and creatinine level in serum. Urinalysis revealed the elevation of protein, urobilinogen and urine specific grav
ity. Administration of high dose of olivamide in addition to the above-mentioned laboratory parameter caused the raising of alkaline phosphatase and total bilirubin level in serum. Microscopic pathology observation showed structure abnormalities of varying severity in liver and kidneys.

Introduction. In N.N. Blokhin National Medical Research Center of Oncology Ministry of Health of Russia were made preclinical tox icological studies the lyophilized dosage form of ormustine (Ormustine), a new antitumor drug from the class of nitrosoureas. The paper presents some results of study the subchronic toxicity of Ormustine on rats.

Objective. The objective of present study was to investigate the subchronic toxicity of Ormustine on rats with a daily 3 times intravenous administration.

Materials and methods. The study was performed on 40 outbred male rats. Ormustine was administered intravenously daily 3 times in total doses of 300, 200 and 100 mg/kg. The observation period was 45 days. During the observation period, the necessary clinical and laboratory tests were performed. To study the damaging effect of Ormustine to organs and tissues, a pathomorphological examination was carried out at the 3rd and 45th day of observation.

Results. It has been established that Ormustine has hemato-, nephro-, cardio- and gastrointestinal toxicity when administered repeat edly to rats in the three doses studied. The depth and extent of damage, as well as their reversibility, depend on the magnitude of the applied dose of Ormustine.

Objective of the study. Identification of genes, the expression of which is associated with the metastasis of gastric cancer tumor.

Materials and methods. Quantitative real-time PCR on paired tumor – normal samples.

Results. An association with the metastasis of the VEGFR1, FGFR2 and NRP-1 gene expression is shown. The odds ratio (OR) was the highest for the FGFR2 gene: OR 175.00; 95 % CI 8.972–3413.271; for the VEGFR1 gene, OR 4.622, 95 % CI 1.240–17.227, for NRP1 – OR 2.667; 95 % CI 0.597–11.915. When assessing the jointly elevated expression of VEGFR1 and NRP1 genes, OR 5,778; 95 % CI 1.393–23.909; p = 0.0147. The frequency of increased expression of FGFR2 was 5 % in case of metastasis, while in the case of localized GC it reached 53 %.

Objectives. Quantitative assessment of the expression and co-expression levels of estrogen receptors of different types (ERα and ERβ) in malignant tissues of large cohort of patients with non-small cell lung cancer (NSCLC).

Materials and methods. Measurement of ERα and ERβ expression levels was carried out on 167 malignant tissue samples by immunofluorescence flow cytometric method. Level of the ERα and ERβ expression was calculated as a ratio of specifically fluorescent cells (%) compared to the control (incubated with the secondary antibody only).

Results. The expression of ERα and ERβ was detected in all the NSCLC samples investigated. High variability in the level of the marker expression was revealed and it was shown 1,5–2,0-fold higher ERβ expression in the most tumors than that for ERα. The Spearman rank correlation coefficient of 0,364 (p <0,001) and the Pearson correlation coefficient of 0,401 (p <0,001) indicate statistically significant weak positive correlation between the levels of expression of ERβ and ERα.