Background. Diffuse large B-cell lymphoma (DLBCL) is a potentially curable biologically heterogeneous lymphatic tumor. Standard R-CHOP therapy shows disappointing results, both immediate and longterm. To improve efficacy without additional toxicity, it is worth considering the possibility of using biologically oriented therapy.Aim. To evaluate the clinical efficacy and toxicity of the genotypedirected R-CHOP-X in patients with newly diagnosed DLBCL in real clinical practice.Materials and methods. A single-center prospective interventional clinical study included 30 patients with newly diagnosed DLBCL between September 2023 and September 2024. The median age was 60 (38–78) years. According to the international prognostic index, 23 (77 %) patients were classified as having a high risk of progression. Genotype incidence in the study cohort: MCD – 7 %, N1 – 20 %, BN2 – 7 %, EZB– 16 %, ST2 – 7 %, NOS – 43 %.Results. 30 patients received personalized genotype-directed therapy. Of these, 21 (70 %) patients completed treatment: the overall response rate was 100 % (complete metabolic response – 100 %). 9 (30 %) patients continue therapy: the overall response rate is 100 %. At 12 months, overall survival and progression-free survival were 100 % (95 % confidence interval 100 %). Hematological toxicity was assessed depending on the number of cycles (n = 144): grade III–I neutropenia was detected in 7 % of cycles, grade III–I anemia and grade III–I thrombocytopenia in 1.4 and 3.5 % of cycles, respectively. Non-hematological toxicity was generally grade ≤I–II.Conclusion. The results of this clinical trial are promising and provide preliminary evidence for the benefit of personalized genotype-directed antitumor therapy in newly diagnosed DLBCL. This therapeutic strategy demonstrates high clinical efficacy, particularly in the main target group – DLBCL with a high risk of progression with low toxicity. Further randomized studies are needed to confirm the effectiveness and implement the new approach in routine clinical practice.

Primary or secondary resistance is an important problem when treating any type of tumor. It is often associated with changes in target genes’ functioning. This raises the question of understanding functional intracellular interactions of genes and proteins in oncological processes and therapeutic resistance occurring. When searching target proteins of targeted therapy, it is necessary to identify biomolecules, participating in cell signaling life, which differ significantly in normal and oncological processes and interact with a large number of pathways. It is also important that these biomolecules are not an artifact of tumor therapy or cell line cultivation, and that it is possible to influence them directly, obtaining complex effect. In addition, it is important to study changes occurring during therapy with the biomolecules, which include proto-oncogene of SRC family kinase LYN and gene of the estrogen receptor α ESR1. All these factors may help to overcome the emerging resistance.Objective – to study the way genes of SRC kinase LYN and estrogen receptor α ESR1 influence oncological processes and occurrence of therapeutic resistance.

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.

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. PRAME gene spontaneous expression is frequently observed in a cancer cell. The protein encoded by this gene increases the viability of tumour cell. NF-κB signalling pathway takes part in PRAME upregulation. It proposes, that stress conditions may increase the expression level of PRAME in the tumour cell and increase cell’s viability after it. We hypothesized that this phenomenon determines chemoresistance of PRAME-expressing cell, which can be overcome by NF-κB inhibitors, such as bortezomib.Materials and methods. We incubated A875 melanoma cells with cisplatin, bortezomib and dexamethasone, as well as with a mixture of cisplatin with bortezomib and cisplatin with dexamethasone within 24 hours. To assess the cytotoxicity of these combinations MTT-test was used. For evaluation of PRAME expression level, real-time polymerase chain reaction was used. All data were analyzed with Wilcoxon test for coupled samples.Results. It was found that cisplatin and dexamethasone increased an expression level of PRAME compared to control (p <0.03). The addition of dexamethasone to cisplatin reduced cytotoxic effect of cisplatin. Bortezomib has a cytotoxic effect, but it did not increase the activity of PRAME gene (p = 0.12). PRAME gene activity in cells incubated with a mixture of cisplatin and bortezomib was observed at a lower level in comparison with cells incubated with cisplatin (p = 0.0277).Conclusion. The results of experiments show that an increase of PRAME expression level reduces the sensitivity of melanoma cells to the cytotoxic effect of cisplatin. PRAME activity increases under stress conditions. Using of bortezomib can inhibit the growth of PRAME expression and makes the tumour cell more vulnerable to cytotoxic agents. On the other hand, dexamethasone may increase a resistance of PRAME-expressing cell to cytotoxic effects.

Background . Multiple myeloma (MM) is a malignant lymphoproliferative B-cell disease characterization by clonal proliferation of plasma cells in the bone marrow and beyond its borders. Currently, a wide range of cytogenetic anomalies and molecular-biological parameters are studied as prognostic factors. Objective: a comparative study of the frequency, features and clinical significance of chromosomal abnormalities in MM by conventional cytogenetic and fluorescent in situ hybridization (FISH) methods. Materials and methods . 77 patients with MM, which admitted in N.N. Blokhin National Medical Research Center of Oncology, were included in the study from 2016 to 2017. Results . Chromosomal alterations were detected only in one case (1/77) by conventional cytogenetic method G-banding. However cytogenetic aberrations were revealed in 26 % of cases (20/77) using FISH. Deletions of different regions of chromosomes, indicating the possible presence of a hypodiploid clone or loss of some regions, were found in one patient in the second FISH analysis after 6 months. In the cohort of patients with chromosomal abnormalities (n = 20) a partial trisomy 11q, a deletion of the region q32 of the chromosome 14, a translocation t(4;14)(p16;q32) and IGHV gene rearrangement were determined in 30 % (6/20) as sole anomalies. Two or more cytogenetic aberrations were identified in the remaining 14 patients. Our study confirms that chromosomal abnormalities are more likely detected at later stages of MM (IA и IIA – 0 %, IIIA и IIIВ – 27 and 47 % respectively). Conclusion . FISH allows to detect chromosomal changes in tumor plasma cells regardless of the mitosis phase. In MM, it becomes particularly important in connection with low proliferative activity of plasma cells. Additionally, in the fourth of MM patients in the study submicroscopic chromosomal aberrations were discovered using FISH. The improvement of the probe panel and the widespread use of locus specific FISH don’t replace G-banding that allows to see damages of all chromosomes at once.

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 . Multiple myeloma (MM) is a malignant lymphoproliferative B-cell disease characterization by clonal proliferation of plasma cells in the bone marrow and beyond its borders. Currently, a wide range of cytogenetic anomalies and molecular-biological parameters are studied as prognostic factors.Objective: a comparative study of the frequency, features and clinical significance of chromosomal abnormalities in MM by conventional cytogenetic and fluorescent in situ hybridization (FISH) methods.Materials and methods . 77 patients with MM, which admitted in N.N. Blokhin National Medical Research Center of Oncology, were included in the study from 2016 to 2017.Results . Chromosomal alterations were detected only in one case (1/77) by conventional cytogenetic method G-banding. However cytogenetic aberrations were revealed in 26 % of cases (20/77) using FISH. Deletions of different regions of chromosomes, indicating the possible presence of a hypodiploid clone or loss of some regions, were found in one patient in the second FISH analysis after 6 months. In the cohort of patients with chromosomal abnormalities (n = 20) a partial trisomy 11q, a deletion of the region q32 of the chromosome 14, a translocation t(4;14)(p16;q32) and IGHV gene rearrangement were determined in 30 % (6/20) as sole anomalies. Two or more cytogenetic aberrations were identified in the remaining 14 patients. Our study confirms that chromosomal abnormalities are more likely detected at later stages of MM (IA и IIA – 0 %, IIIA и IIIВ – 27 and 47 % respectively).Conclusion . FISH allows to detect chromosomal changes in tumor plasma cells regardless of the mitosis phase. In MM, it becomes particularly important in connection with low proliferative activity of plasma cells. Additionally, in the fourth of MM patients in the study submicroscopic chromosomal aberrations were discovered using FISH. The improvement of the probe panel and the widespread use of locus specific FISH don’t replace G-banding that allows to see damages of all chromosomes at once.

Intrоduction.Human antigen PRAME is preferentially expressed in a number of different tumor types and may be a potent target for anti-tumor immunotherapy.Purpose.To study anti-tumor action of immunogenic mix recombinant PRAME protein and adjuvant in mice with innate immunity.Materials and methods.C57BL/6 female mice were used for immunization with purified human recombinant protein PRAME. Human PRAME gene coding sequence was cloned in mammalian expressing vector pCEP4 and resulting plasmid was introduced in mouse melanoma B16F10 cells by transfection followed by RQ-PCR, Western blot and flow-cytometry analysis. Then stably PRAME-transfected melanoma cells were injected in mice.Results.The mouse melanoma B16F10 cells stably expressing human PRAME protein were obtained. We demonstrate the 10-fold decreased tumor volume in mice with melanoma B16F10 expressing human PRAME after preventive immunization series with recombinant PRAME protein. The tumor volume reducing was correlated with high titer (6.14 × 10 5) of anti-PRAME antibodies in mice sera.Conclusion.These data indicate that recombinant protein PRAME is immunogenic and may be a potent antigen for immunotherapuetics studies.

Background. BCR-ABL gene mutations are the main course of tyrosine kinase 1 st generation inhibitor (TKI-1) imatinib resistance in chronic myelogenous (CML) patients. Some of them are resistant both for imatinib and TKI-2. The nilotinib and dasatinib mutation spectra are not the same. To ensure a correct choice of TKI-2 for imatinib CML resistant patient treatment it is necessary to investigate frequency of BCR-ABL gene mutations. Aim. To evaluate BCR-ABL gene mutation frequency in imatinib resistant CML patients. Materials and methods. Peripheral blood of imatinib resistant CML patients were studied by means of direct PCR product Sanger sequencing in order to reveal BCR-ABL gene mutations. Results. BCR-ABL gene mutations were found in 31 % (n=262/846) imatinib resistant patients. Proportion of resistant mutations were 40,3 % for nilotinib and 21 % for dasatinib. Conclusion. More than a halve of BCR-ABL mutations resistant for imatinib were resistant either against nilotinib or dasatinib. Consequently, prior to replace imatinib for nilotinib or dasatinib it is necessary to perform BCR-ABL mutational analysis. If to prescribe TKI-2 blindly there may be high risk that nilotinib or dasatinib would be ineffective due to improper mutation in BCR-ABL gene.

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.

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.