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Cancer-testis antigens (CTA) are antigens expressed by tumor cells of various histological types, but practically absent in cells of normal tissues, with the exception of germ cells. CTA includes more than 100 proteins, most of which are grouped into large families. Currently, the use of CTA for immunotherapy in the treatment of oncological diseases has been tested in many studies, and an increase in survival time has been achieved for many cases. Therefore, they can be promising targets for the creation of antitumor drugs, targeted therapy of tumors and as diagnostic biomarkers.The purpose of this review was to study the GAGE family of antigens, one of the CTA groups recognized by T cells. Proteins of this family, expressed in tumor cells, stimulate the development of a humoral and cellular immune response against them. It follows from this that they fully meet the requirements for targets for tumor immunotherapy. The review provides information about the structure and sequence of genes encoding proteins of the GAGE family. The question of the role of GAGE in apoptosis is considered in detail and the results of studies proving that GAGE-7C makes cells resistant to apoptosis mediated by interferon γ or Fas are presented. The results of clinical studies of the expression of GAGE group genes and proteins in various types of tumor diseases are considered and examples of the reported correlation between GAGE expression and poor prognosis in some types of cancer are given.Thus, the proteins of the GAGE group, with a detailed study, can become a possible diagnostic and prognostic marker of cancer diseases, and in the future be used to assess malignancy and monitor tumors for the selection of treatment tactics.

Determination of the expression level of tumor marker PRAME protein is important both for predicting the course of the disease and for monitoring the effectiveness of anticancer therapy. A fluorescently labeled monoclonal antibody to the PRAME protein was obtained by periodate oxidation of glycans followed by modification with a bifunctional azido-oxyamine reagent and a “click” reaction with alkyne-modified sulfonated cyanine dye Cy3. A new approach to the synthesis of a bifunctional azido-oxyamine reagent using the ethoxyethylidene protecting group for oxyamine is proposed. The labeled antibodies were characterized with UV/Vis absorption spectra and the stoichiometry of the modification was determined. It has been demonstrated that the fluorescent antibodies retain affinity and can be used as a diagnostic tool for detecting the residual marker (the PRAME protein) after anticancer therapy.

Two monoclonal antibodies recognizing non-overlapping epitopes of the PRAME protein were injected into immunocompetent mice to study their influence on the growth of subcutaneous tumor nodes. The B16F10 murine melanoma line, either expressing human PRAME protein or bearing only a vector without PRAME gene, were used as transplants. Each of the antibodies showed the ability to suppress tumor growth of a PRAME-expressing tumour, but not a tumor without PRAME.

Molecular genetic detection of CALR gene somatic mutations is required for myeloproliferative neoplasms diagnosis and treatment according to the novel WHO clinical recommendations. CALR mutations are found in approximately 25–35 % cases of essential thrombocythemia and primary myelofibrosis and they are associated with benign clinical outcome. In this study we have compared sensitivity and selectivity of seve ral different options of CALR mutation molecular genetic detection in blood samples of 379 CMD patients and 17 healthy donors. Among methods compared in our study there have been conventional polymerase chain reaction with electrophoretic detection, real-time quantitative polymerase chain reaction, direct Sanger sequencing of polymerase chain reaction fragments and polymerase chain reaction high resolution melting curve analysis. By means of melting curve analysis CALR mutations have been found in 97 (25.5 %) patients, whereas in the cases of Sanger sequencing and polymerase chain reaction there have been 87 (23.0 %) and 84 (22.1 %) CALR mutation positive patients respectively.

Background. TP53 gene mutations impede cell apoptosis and lead to additional oncogenic events contributing to tumor progression. Aim. To assess TP53 gene mutation rate in patients with high-grade B-cell lymphoma double-hit (HGBCL DH) and not otherwise specified (HGBL NOS); to analyse its relationship with disease prognosis. Materials & Methods. Retrospective materials from medical data of 10 HGBL DH and 26 HGBL NOS patients were analyzed. Median follow-up was 26.5 months (range 0.6-160.9 months). Selection was based on the presence of available biological materials (paraffin blocks) for Sanger sequencing of TP53 gene from exon 5 to exon 8 (encoding DNA-binding domain of TP53 gene). FISH analysis of the tumor was performed in all patients to identify translocations involving c-MYC/8q24, BCL2/18q21, and BCL6/3q27 gene locus. To analyze differences between groups χ2 and Mann-Whitney tests were applied. Univariate event analysis (Kaplan-Meier and log-rank tests) and Cox regression analysis were used to assess the influence of molecular markers on the disease prognosis. Results. TP53 gene mutations in lymphoma cells were found in 13 (36 %) out of 36 patients, 10 (77 %) out of 13 mutations were pathogenic. In 8 out of 10 patients with TP53 mutations c-MYC/8q24 gene translocation was identified. Groups with wild (TP53-WT) and mutant (TP53-MUT) types of TP53 gene were similar in terms of main clinical characteristics. Patients with TP53-MUT in tumor cells showed worse 3-year overall survival (OS) compared with the group without TP53-MUT (30 % vs. 73 %; p = 0.026) as well as higher probability of disease progression in the period of 3 years (66 % vs. 15 %; p = 0.004). In multivariate analysis significant OS predictor was the presence of TP53 mutation (p = 0.006). Relapse/ progression probability was higher in combined cases of TP53 mutation and translocation involving c-MYC gene locus (p = 0.0003). Conclusion. Translocation involving c-MYC gene along with TP53 gene mutation in tumor cells can serve as a criterion for dividing HGBL DH and HGBL NOS patients into different lymphoma relapse/progression risk groups.

Aim. To set survival parameters for follicular lymphoma (FL) patients with different PRAME expression levels in tumor cells. Materials & Methods. The study was conducted on samples of lymph nodes, blood, and bone marrow of 34 patients with newly diagnosed FL. PRAME expression levels were measured in tumor cells (centrocytes and centroblasts) by quantitative real-time PCR. The impact of different PRAME expression levels on survival parameters was studied with median follow-up of 29 months. Clinical and laboratory characteristics used for FLIPI-1 and FLIPI-2 calculations in different patient groups were compared. Results. A high (> 5 % against ABL control gene) PRAME expression level correlates with higher Ki-67 activity (p = 0.043) and larger tumor mass (p = 0.04). Survival parameters were worse with high PRAME expression level in FL cells. Combination of both high FLIPI-1/FLIPI-2 risk and high PRAME expression level determines extremely unfavorable prognosis and may result in death. Conclusion. In FL patients high PRAME expression level in tumor cells has negative prognostic value, but only in the presence of parameters determining high FLIPI-1 and FLIPI-2 risk. Juxtaposition of PRAME expression level and FLIPI-1/ FLIPI-2 values enables most reliable prediction of early mortality in FL patients.

Two monoclonal antibodies recognizing non-overlapping epitopes of the PRAME protein were injected into immunocompetent mice to study their influence on the growth of subcutaneous tumor nodes. The B16F10 murine melanoma line, either expressing human PRAME protein or bearing only a vector without PRAME gene, were used as transplants. Each of the antibodies showed the ability to suppress tumor growth of a PRAME-expressing tumour, but not a tumor without PRAME.

Background. T-cell lymphomas (T-CL) represent a heterogeneous group of malignant lymphoproliferative disorders characterized by unfavorable prognosis. The cancer-testis PRAME gene is notable for its spontaneous expression in transformed cells as observed in solid tumors, B-cell lymphoproliferative and chronic myeloproliferative diseases. Activity and clinical significance of PRAME in T-CL was not studied before, which determines the relevance and provides ground for the present trial. Aim. To assess the clinical significance of the PRAME gene expression in T-CL. Materials & Methods. PRAME gene expression level was measured in samples of lymph nodes, blood, and bone marrow from 35 T-CL patients. Among them 3 patients received allogeneic hematopoietic stem cell transplantation, and 6 patients received autologous hematopoietic stem cell transplantation. A correlation was established between the PRAME expression in bone marrow and peripheral blood with morphological markers of disseminated disease with bone marrow lesions and leukemic blood. PRAME expression level was correlated with survival parameters and tumor proliferative activity (Ki-67). Results. PRAME activity was observed in 21 (60 %) patients. PRAME hyperexpression is associated with advanced stages of disease (p = 0.0734), bone marrow lesions (p = 0.0289), leukemic blood (p = 0.0187), worsening of the overall survival (OS) (p = 0.0787) and event-free survival (EFS) (p = 0.7185), also after hematopoietic stem cell transplantation (p = 0.2661 for OS and p = 0.0452 for EFS), and with a high Ki-67 expression level (p = 0.0155). Conclusion. PRAME expression in T-CL is often observed and related with unfavorable clinical prognosis.

Background: We have previously shown that the FAS, TNFR2, TRAIL, DR3, DR4/5 gene expression in patients with newly diagnosed chronic lymphoblastic leukemia (CLL) correlates with clinical manifestations of the disease: they are minimal in patients with high activity of the proapoptotic genes and low activity of the apoptosisinhibiting genes, and advanced in patients with high expression of the anti-apoptotic and low expression of the pro-apoptotic genes.Aim: To compare the levels of expression of the external apoptosis pathway genes in patients with newly diagnosed CLL before and after chemotherapy with fludarabine, cyclophosphamide and rituximab (FCR), taking into account baseline clinical data and the response to treatment.Materials and methods: This prospective one-center cohort study included 23 patients with newly diagnosed CLL, who underwent clinical and diagnostic assessments and treatment from November 2014 to December 2017. Immunophenotyping of peripheral blood lymphocytes for CLL diagnosis was done by fourcolor flow cytometry. Expression of the external apoptosis pathway genes was assessed by realtime reverse transcriptase polymerase chain reaction. All patients were treated with a standard FCR regimen with subsequent maintenance treatment with rituximab.Results: There were more men (n = 16) than women among our 23 CLL patients. Median age was 64 years (range, from 47 to 77 years). Sixteen (16) patients had CLL Rai Grade I and II, and 7 patients had CLL Grades III and IV. For convenience of analysis, all patients were divided into two groups depending on the FAS gene expression. At baseline, the patients with high FAS expression had higher TNFR2 (p < 0.0015) and TRAIL (p < 0.0053) expression levels. Before FCR therapy, the patients with low FAS expression had higher lymphocyte counts (р = 0.0016) and lower erythrocyte counts (р = 0.0159). At baseline, there were more Grade I and II patients in the group with higher FAS expression (р = 0.0205). At day 3 after the end of a four day FCR cycle, there was an increase only of the FAS (p = 0.0025) and TRAIL (p = 0.0045) expression. After the completion of the first FCR cycle, lymphocyte counts in the patients with low FAS expression decreased earlier than those in the patients with high FAS expression (p = 0.0019). After six FCR cycles, complete or partial remission was obtained in 82% (19/23) of the patients. The patients with high FAS expression had higher complete remission rate (р = 0.026). No adverse events related to FCR were registered.Conclusion: The external apoptosis pathway genes are one of the key factors of the tumor progression in CLL. Our data on the effect of FCR therapy on the FAS and TRAIL gene expression make it possible to consider them as a target for this combination regimen and may become the rationale to develop new pharmaceutical molecules.

Aranosa, nitrozourea derivative is a DNA-methylating agent that has been approved for treatment of patients with disseminated melanoma. Aranoza effect is based on DNA damage and then as a result apoptosis mechanisms start launching. The important role in this process must be played by p53 protein, and its different dysfunctions can result in drug resistance. Objective. The purpose is to study p53 mutational status in cell lines of human skin metastatic melanoma and to estimate its connection with сell lines resistance to aranoza. Materials and methods. The research was conducted on 14 cell lines of human skin metastatic melanoma. Aranoza IC50 for cell lines was determined by MTT-test. The 17р chromosome’s condition was estimated by fluorescence in situ hybridization. The presence of point mutations in DNA-binding domain of human p53 was researched by Sanger sequencing. Results. Skin metastatic melanoma cell lines had different sensitivity to aranoza. Almost all cell lines were heterogeneous in the condition of 17 th chromosome. P53 point mutations were found in 2 cell lines. But one part of resistant cell lines almost didn’t have any mutational disorders of p53, another part of resistant lines on the contrary had plenty of p53 mutational disorders. Conclusion. The correlation of resistance and p53 mutations can be established for one part of human skin metastatic melanoma cell lines. But for another part of human skin metastatic melanoma cell lines resistance most likely are driven by other mechanisms.

Objective:to study the prognostic significance of the expression of cancer-testis (CT) genes PRAME, NY-ESO1, GAGE1, MAGE A3, MAGE A6, MAGE A12, SSX1, SLLP1, PASD1 in patients with multiple myeloma (MM) and their influence on overall survival and relapse rate. To determine their effect on suсh clinical parameters as levels of lactate dehydrogenase, leucocytes, hemoglobin, calcium, albumen, creatinine, beta-2-microglobulin.Materials and methods.Real-time polymerase chain reaction was performed on complementary DNA obtained from bone marrow of 77 patients with MM. The statistical analysis was performed using the Statistica 10.0 software package. To estimate prognostic values of the CT gene expression data were analyzed by the Kaplan – Meier method.Results.The study was conducted to determine the level of expression of CT genes PRAME, NY-ESO1, GAGE1, MAGE A3, MAGE A6, MAGE A12, SSX1, SLLP1, PASD1 in a group of patients with MM. The group included primary and receiving cancer treatment in MM patients. According to the log-rank criterion expression of any of the CT genes PRAME, NY-ESO1, GAGE1, MAGE A3, MAGE A6, MAGE A12, SSX1, SLLP1, PASD1 exerts a significant influence on overall survival and progression-free survival/relapse. It was also determined that providing expression of some CT genes, the levels of creatinine, calcium, beta-2-microglobulin were much higher to compare with patients without expression.

Background. Kinase domain mutations of BCR-ABL gene is the most common cause of tyrosine kinase inhibitor resistance. Aim. To present the data on prognostic value of BCR-ABL mutation burden in Russian patients over the last 10 years. Materials & Methods. The study included 1885 chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitor resistance who were followed up from 2006 to 2016. BCR-ABL point mutations in mRNA samples were analyzed by means of polymerase chain reaction and subsequent Sanger sequencing. Results. In 1257 CML patients with signs of tyrosine kinase inhibitor resistance BCR-ABL expression level was > 1 %. BCR-ABL mutations were detected in 31.8 % of patients. Total mutation count was 467 (70 mutation types). Total count of patients with mutation-associated tyrosine kinase inhibitor resistance decreased from 36.6 % (2006-2008) to 24.95 % (2013-2016) and to marked decrease of 23.12 % in 2014. Detection rate of imatinib-resistant mutations and F359V mutation was shown to decrease within the period from 2010-2011 to 2014-2015. F317L level, which is responsible for dasatinib resistance, considerably increased in 2015. T315I frequency was the highest in 2014, afterwards it was gradually decreasing. Mutation-associated resistance rates varied by region of the Russian Federation. Conclusion. The analysis of trends of mutation incidence in patients with CML can be of extreme significance in longterm prognosis of resistance development and in improvement of treatment planning.

Background. The V617F mutation of JAK2 is known to manifest in Ph-negative chronic myeloproliferative diseases (cMPD), such as polycythemia vera, thrombocythemia, and myelofibrosis. These diseases not infrequently advance into more aggressive forms up to acute leukemia. As the progression mechanism is still unknown, its study retains a high priority. JAK2 carrying the V617F mutation is believed to cause constant activation of V(D)J recombinase in myeloid tumor cells in cMPD patients. Aberrant activation of V(D)J recombinase in tumor cells in cMPD patients can lead to t(9;22)(q34;q11) chromosomal rearrangement. Aim. To study the expression of BCR-ABL1 resulting from translocation t(9;22)(q34;q11) in cMPD patients at the progression stage in order to test the suggested hypothesis. Materials & Methods. The BCR–ABL1 expression was assessed in peripheral blood granulocytes in cMPD patients by real-time PCR. The JAK2 V617F mutation was identified by quantitative allele-specific PCR. The JAK2 exon 12 mutations were determined using Sanger direct sequencing of PCR products. Results. The BCR-ABL1 expression was discovered in 29 % of patients with cMPD progression. The BCR-ABL1 expression in these patients correlated with hepatosplenomegaly and hyperleukocytosis. Conclusion. In a significant proportion of cMPD patients the disease progression can be associated with activation of the BCR-ABL expression.

In this review we discuss some biological activities of quinazolinon derivatives. The derivatives of quinazolinon possess their activity by binding to some proteins important for the survival, proliferation and metastasis of tumor cells. There are grounds to initiate clinical trials of these substances to create new treatment protocols for a wide range of cancer diseases, several parasitic diseases, and certain neurodegenerative diseases.

Introduction. Chemotherapy is an extremely ineffective and unsatisfactory means of treating malignant melanoma due to drug resistance, which is characteristic of this disease. A number of studies have shown that liposomal forms of anticancer drugs are able to overcome the multidrug resistance, but the mechanism by which this occurs is still remained to be elucidated. Aranoza (DNA-alkylating agent, a derivative of nitrosourea) has been approved for the treatment of patients with metastatic melanoma. Objective: to examine the influence of liposomal aranoza as well as the empty liposomes and “liophilisate for the preparation of solution for injections” (aranoza-lio) on the expression of mRNA of p53, MDM2, NFkB1, NFkB2, MyD88. Materials and methods. The study was performed with 10 melanoma cell lines, 4 of which carried the BRAF mutation. The level of p53, MDM2, NFkB1, NFkB2, MyD88 mRNA was investigated by quantitative polymerase chain reaction in real time. Results. Aranoza-lio increased slightly the expression of p53 mRNA in BRAF-mutated cells. We have observed also the increased expression of MDM2 mRNA (p = 0.0013). The expression of NFkB2, MyD88 mRNA did not change significantly as compared to control. Liposomal aranoza increased the expression of NFkB1 mRNA. Conclusion. Based on the data obtained we conclude that the liposomal aranoza triggers the mechanisms that contribute to sensitivity of cells toward anticancer drugs while aranoza-lio favored the enhancing of the expression of MDM2 mRNA and increase the resistance to chemotherapy.

We modified the preparative-scale method for the synthesis of 2-aryl 1,2-dihydro-4(3H)-quinazolinone derivatives obtained in high yields by the reaction of new and commercially available aromatic aldehydes with anthranilic acid amides. A series of quinazolinone derivatives possessing anticancer and antiparasitic activities, as well as capable of preventing the progress of neurodegenerative diseases were characterized. There are grounds for clinical trials of these substances in order to select compounds being promising for clinical application.

We investigated the epitope specificity of different monoclonal antibodies recognizing the cancer testis antigen PRAME. Antibody 5D3 binds to the fragment of PRAME corresponding to 160-180 amino acid residues. Antibodies 6H8 and F11 bind to the fragment corresponding to 180-200 amino acid residues of PRAME. These antibodies retained the ability to recognize these PRAME fragments after chimerization.

Background Autologous dendritic cells (DC) loaded with tumor-associated antigens (TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient’s dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are minimally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells. Patients and methods We compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation. Results The majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME’s was significantly different (adj. p SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpression of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p Conclusion Our results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials.

Mediastinal gray-zone lymphoma (MGZL, lymphoma with features intermediate between classical Hodgkin lymphoma and diffuse large B-cell lymphoma) was declared as a separate entity in WHO classification of Tumors of Haematopoetic and Lymphoid Tissues in 2008 and 2017 years. Despite of similar pathomorphological characteristics between primary mediastinal B-cell lymphoma and Hodgkin lymphoma, clinical features and optimal therapeutic approach to MGZL are not clearly defined. Usually MGZL manifests with mediastinal lymphadenopathy, although extranodal lesions often occur (grey-zone lymphoma, GZL). Patients with MGZL have unfavorable prognosis, taking into account high rate of relapse. This article describes two cases of MGZL. First case manifested by arrhythmias due to primary heart involvement. In spite of cardiac failure antracycline-containing chemotherapy (6 courses of R-DA-EPOCH) it allowed to achieve a complete remission and resolving of arrhythmias. Second case was represented by metachronous tumors: primary mediastinal B-cell lymphoma at the time of disease onset and classical Hodgkin lymphoma, NS II, diagnosed after disease progression. Thus, we demonstrated two examples of MGZL that differ by clinical manifestation, response to chemotherapy, which emphasizes an importance of pathogenesis studying, and using of new therapeutic approaches.

Introduction. PRAME protein is a promising target for cancer immunotherapy. PRAME is not expressed in normal tissues, but active in number of the tumor types. We have developed the mouse monoclonal antibodies 5D3F2 and 6H8F12 against PRAME epitopes. Aim. To determine the effects provided by the monoclonal antibodies 5D3F2 and 6H8F12 against the cells with different levels of PRAME gene expression. Materials and methods. We used different cell lines: NOMO-1 and WI-38 with low levels of expression PRAME; THP-1 with intermediate level of PRAME expression; K562 and WI-38-PRAME with high level of PRAME expression. We incubated these cell lines in the presence of monoclonal antibodies 5D3F2 and 6H8F12. The final concentration of monoclonal antibodies in culture varied from 6 pg/ml to 120 mcg/ml. The live cells were counted at the 24, 48 and 72 hours after incubation. The number of dead cells was evaluated by the MTT-test after 24 hours. Results. Cell growth rate is significanely decreased during incubation with monoclonal antibodies. This effect is correlated with increase of monoclonal antibody concentrations (Pearson coefficient 0,67;p = 0,0219). K562 growth rate was much less compared to the THP-1’s rate (p = 0,0061), NOMO-1 (p = 0,0005) and WI-38 (p = 0,0002) in the presence of the same amount of monoclonal antibody 6H8F12. K562 cell growth rate was lower than the WI-38-PRAME’s rate (p = 0,0027), despite the comparable level of PRAME expression. Effects of monoclonal antibody 5D3F2 and 6H8F12 were similar (p = 0,3946). According to the MTT-test, the comparable number of death cells in K562 and WI-38-PRAME was observed (p = 0,8405). Under the same conditions the amount of death cells in THP-1 was smaller than K562 (p = 0,6335). To compare with K562, fewer cells died in NOMO-1 and WI-38 (p = 0,0026 and p = 0,0005, respectively). Conclusion. It was shown that monoclonal antibody 5D3F2 and 6H8F12 exhibit a significant cytotoxic effect against PRAME-express-ing cells. In case of higher levels of PRAME expression the cytotoxic effect was stronger.

Background. Aranose is alkylating antineoplastic agent with cytotoxic and cytostatic properties. Liposomal form of aranose has advantages compared with traditional dosage form. Another alkylating agent, cisplatin, promotes mRNA increase expression level of genes, which control external apoptosis way. Objective. We assume that liposomal form of aranose can change the CD95/Fas, TNFR1, TNFR2, TRAIL, DR3 and DR4/5 expression level in melanoma cell lines mel Kor, mel Z, mel Mtp, mel Mtp-X, mel Ibr, mel Ch, mel Is and mel R. Materials and methods. There are two forms of aranose were used in this work: liposomal form and traditional diluted form for injection. Melanoma cell lines were incubated with both forms. We used RQ PCR to evaluate mRNA expression mRNA expression of DR3, DR4/5, CD95/Fas, TNFR1, TNFR2 and TRAIL relatively gene ABL. Results. CD95/FAS mRNA expression level was most changeable. Liposomal aranose increase CD95/FAS expression level in mel Ibr and mel Mtp-X cell lines and reduced CD95/FAS expression level in mel Ch and mel Is. Conclusion. Thus, liposomal aranose can change mRNA CD95/FAS expression level in melanoma cell lines. Using this process, liposomal form can affect the apoptotic cell death of melanoma.

Tumor antigens recognized by CTLs have been identified several years ago and are major targets for creating anticancer vaccines. PRAME is an antigen which is highly expressed in various malignant tumors including melanomas and hematopoietic malignancies such as acute and chronic leukemias (AML, CML). Technology for producing recombinant antigen PRAME is based on creating a bacterial producer strain containing cDNA of human PRAME gene. We have obtained two producers of recombinant PRAME protein and its N-half, the synthesis of the target protein in the producers occurs in the inclusion bodies. The schemes of isolation and purification of soluble proteins have been developed. The protein purity was approximately 95-96%. The monoclonal antibodies raised against truncated recombinant PRAME were used for PRAME protein analysis by Western blot on the various tumor cells. Specific monoclonal antibodies recognized the native PRAME protein in tumor cell lines as well as in tumor samples from patients. Our findings support the suggestion that this recombinant antigen may be further used as a target for diagnostic and therapeutic approaches. The monoclonal antibodies can be used for immunoassays of tumor samples from patients with hematologic malignancies to reveal clinical features and to monitor tumor progression.

Apoptosis can be triggered from external signals. Several homologous receptors transmit apoptotic signals from outside into the cell. For successful activation of apoptosis receptors should interact with their ligands. For example, FAS receptor must bind with FAS-ligand, TNFR1 with TNFα, TRAIL-R1 and TRAIL-R2 with TRAIL, DR3 - with TL1A, respectively. In majority of cases ligands should be anchoring in the cell membrane to perform their functions. FAS and TNFR1 receptors trigger apoptosis only when they are internalized into the cell’s cytoplasm. If FAS and TNFR1 are not internalized, then anti-apoptotic program won’t start. In contrast, TRAIL-R1, TRAIL-R2 and DR3 aren’t internalized during apoptotic signal transduction. Other receptors, TNFR2, TRAIL-R3 and TRAIL-R4 start an anti-apoptotic program. The apoptotic signal starts when DISC complex is formed on the inner side of the cell membrane. FADD, procaspase-8 and intracellular domain of receptor form together DISC complex. If the DISC complex wasn’t formed, signal is transmitted by the NFкB-way via MAP-kinase cascade. In such conditions anti-apoptotic program starts. A variety of receptors and ligands provides for multiple biological functions. For example, receptor-mediated apoptosis takes a part in elimination of infected or transformed cells, regulation of inflammation, modulation of ontogenesis, hematopoiesis and antibody production.

A growing number of evidence indicates that cancer-testis antigens (CTA) can be used as specific targets for immune therapy of malignant melanoma. The aim of this study was to provide a basis for selecting the most suitable CTA by analyzing the mRNA expression profile of genes encoding CTA in melanoma cell lines. We used a real-time quantitative PCR to measure the expression level for the following genes: GAGE1, NY-ESO-1, MAGEA1, PASD, SCP1, SEMG1, SPANXA, SSX1, and PRAME. The objects of study were cell lines mel P, mel Si, mel Mtp, mel Il, mel Hn, mel Ibr, and mel Kor obtained from patients diagnosed with disseminated melanoma. We established that the highest frequency of occurrence and the highest expression level had the following genes: GAGE1, NY-ESO-1, MAGEA1, SCP1, SPANXA, SSX1, and PRAME. Their mRNA translation products can be promising candidates for immunotherapy.

Introduction. MYC increases proliferative capacity of malignant B-cells, independently from mechanisms led to increased protein expression. Tumors with solely MYC expression are highly effective curable with doses intensification. Ability of cells to escape apoptosis by several mechanisms like BCL2 or P53 соехpression promotes malignant B-cells growth and survival and represents a big therapeutic problem. Aim. To investigate mechanism of MYC hyperexpression in absence of c-MYC -rearrangement in DLBCL (diffuse large B-cell lymphoma). To analyze efficacy of intensive chemotherapy in pts with DLBCL who underwent modified NHL-BFM-90 (m-NHL-BFM-90) plus rituximab (R) in correspondence with MYC and BCL2 protein expression. Patients and methods. Data of 62 DLBCL pts (35 males and 27 females) who underwent m-NHL-BFM-90+R in National Research Center for Hematology (Moscow) between 2004 and 2013 years were analyzed. Tumor samples were stained with antibody to BCL2 (clone 124, Dako) and MYC (clone Y69, Epitomics). We used a previously reported cut off for MYC expression ≥40% and BCL2 ≥50% (N. Johnson et al., 2012). G-banding data were available in 19 pts. In one case was revealed c-MYC rearrangement into heavy chain locus t(8;14)(q24;q32). In all other cases FISH didn't reveal c-MYC orBCL2 rearrangements with DNA probes Vysis LSI MYC Dual color, Break Apart Rearrangement Probe, Vysis LSI BCL2 Dual color, Break Apart Rearrangement Probe. RQ-PCR was performed in 17 cases to estimate mRNA level of c-MYC expression relatively to ABL. To assess relation between mRNA of c-MYC and MYC protein expression was used correlation analysis (R²). To estimate treatment results were performed Kaplan-Meyer and Cox regression analyses (SAS 9.3). Results: Majority of pts - 48/62 (78%) attended to a high-risk group according IPI (3-5). 9/62 (14,5%) patients had MYC+/BCL2- tumors, 15 (24%) - "double-expressor" (DE) MYC+/BCL2+, 21 (34%) - MYC-/BCL2+, 17 (27,5%) - MYC-/BCL2-. Median age of DE pts was statistically higher than others (61 (25-73) vs 47 (15-73) years old, P Conclusion. We illustrated that MYC protein expression level correlates with c-MYC transcriptional activity in absence of c-MYC rearrangement. MYC hyperexpression alone didn't influence on prognosis, only coexpression of MYC and BCL2 had a crucial role increasing probability of relapse or progression in DLBCL patients. Disclosures No relevant conflicts of interest to declare.

B-cell lymphoma that is unclassifiable and intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma (BL)--(UBL) is an aggressive lymphoma that has simultaneously morphological, immunophenotypical, and cytogenetic signs of both DLBCL and LB. At the same time due to its biological characteristics and clinical manifestations, UBL cannot be referred to as one of the above nosological entities. UBL has been first identified as an individual nosological entity in the 2008 World Health Organization classification. The review details the epidemiology, clinical manifestations, morphological pattern, immunophenotypic and karyotypic features, as well as therapeutic approaches and prognostic factors.В-клеточная лимфома неклассифицируемая, занимающая промежуточное положение между диффузной В-крупноклеточной лимфомой (ДВККЛ) и лимфомой Беркитта (ЛБ) - ВНЛ, является агрессивной лимфомой, имеющей одновременно морфологические, иммунофенотипические и цитогенетические признаки как ДВККЛ, так и ЛБ. В то же время по биологическим характеристикам и клиническим проявлениям ВНЛ не представляется возможным отнести ни к одной из указанных нозологий. ВНЛ впервые выделена как отдельная нозологическая форма в классификации Всемирной организации здравоохранения 2008 г. В обзоре подробно рассмотрены эпидемиология, клинические проявления, морфологическая картина, особенности иммунофенотипа и кариотипа, а также подходы к терапии и прогностические факторы.

Background. TP53 mutations were described to have a negative impact on prognosis of patients with B-chronic lymphoid leukemia and diffuse large cell lymphoma. However, a role of TP53 mutations in high-grade B-cell lymphomas (HGL) is not well defined in context of other genetic aberrations. Materials and methods. 23 patients (7 males and 16 females) had diagnosis of HGL were treated in National Research Center for hematology, Moscow, Russia. Median age was 49 years old (30-76). 5 patients had HGL with c-MYC (MYC-R) and BCL2 genes rearrangements and 2 - HGB with MYC-R and BCL6 rearrangements. 17 (74%) patients had MYC-R, 11/23 (48%) had double expresser lymphoma (MYC≥40%, BCL2≥50%) (DE). Median of observation time was 29,1 months (6,3-99,8). 19 (82%) of patients had IPI score 3-5 points. 16 patients underwent LM-B-04 with rituximab (Table 1), 5 - R-(DA)-EPOCH, 2 - R-CHOP-21. In 5 cases autologous stem cell transplantation was performed. Sanger sequencing was performed to identify mutations in exons 5-8 of TP53 gene using DNA extracted from formalin fixed paraffin embedded tissue («Extra-DNA» kit, «Genetechnology» LLC). Primers to TP53 gene were synthesized based on nucleotide sequences data available online on website ncbi.com by «Evrogene». To evaluate an influence of such factors as TP53 mutation (TP53mut), MYC-R, DHL, DE, gender, therapy on overall survival (OS) and time to progression (TTP) were performed multivariate dispersion analysis and Cox regression analysis (STATISTICA 10). Results: 8 (35%) cases with TP53mut were identified: c.535C>T 45,6% p.H179Y, c.524G>C 15,6% p.R175P, c.743G>A 75,6% p.R247Q, c.487T>A 25,2% p.Y163N, c.824G>A 75% p.C275Y, c.713G>A 87,7% p.C238Y, c.745A>G 31,9% p.R249G, c.639A>G 41,8% p.R213R. 7/8 of them harbored MYC-R, 2/8 had DHL. In univariate (Picture 1, 2) and multivariate analysis pts harboring TP53mut had worse OS (median OS was 6,2 (0,7-9,5) vs 25,5 (0,7-99,8) months, p=0,004) and shorter TTP (median TTP 3,5 (0,7-9,5) vs 23,1 (0,7-99,8) months, p=0,027) than patients without TP53mut. DHL status had also an adverse effect on OS with lower significance than in pts with TP53mut (p=0,022). Adverse effect of TP53mut trends to play a role in combination with c-MYC gene rearrangement, thus5 pts TP53mut/MYC-R had shorter TTP than 5 DHL pts (3,4 (1,1-9,5) vs 7,3 (0,6-67,1) months, p=0,07). Conclusion: High-grade lymphoma has a more powerful and independent prognostic factor than double-hit status - TP53 mutation that contribute inferior prognosis. This factor shouldn't be underestimated in routine diagnostics because of its frequency and requirement of a different therapeutic approach. Disclosures No relevant conflicts of interest to declare.