Background. The V617F mutation of JAK2 is known to manifest in Ph-negative chronic myeloproliferative diseases (cMPD), such as polycythemia vera, thrombo-cythemia, 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.

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.

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.

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.

Все больше данных говорит в пользу того, что раково-тестикулярные антигены (РТА) могут служить специфической мишенью при проведении иммунотерапии диссеминированной меланомы. Цель данного исследования – обеспечить основу для выбора наиболее подходящего РТА путем анализа экспрессии мРНК генов, кодирующих РТА, в клетках линий меланомы методом количественной ПЦР в реальном времени. Экспрессию генов GAGE1, NY-ESO-1, MAGEA1, PASD, SCP1, SEMG1, SPANXA, SSX1 и PRAME определяли в клеточных линиях mel P, mel Si, mel Mtp, mel Il, mel Hn, mel Ibr и mel Kor, полученных из образцов опухолевого материала пациентов с метастатической меланомой. Наиболее высокий уровень экспрессии в большинстве рассмотренных клеточных линий обнаружен у генов GAGE1, NY-ESO-1, MAGEA1, SCP1, SPANXA, SSX1 и PRAME. Таким образом, продукты трансляции их мРНК можно рассматривать в качестве перспективных кандидатов на роль мишени для иммунотерапии.

Real Time PCR enables an adequate molecular monitoring of the chronic myelogenous leukemia (CML) without which in the case of this disease it is impossible to thoroughly evaluate therapy response especially when modern target inhibitory molecules involved. Unfortunately, discrepancy and methodological deviations of the results obtained by different researches hinder a proper involvement of different labs and clinics into international research protocols and makes it impossible to compare the data obtained. In this paper we discuss the different approaches to standardize Real Time PCR technique and stress the significance of the international effort to incorporate International Scale (IS) into BCR / ABL gene expression monitoring of CML as well as a great importance of the regular sample exchange between participating labs and necessity of the calculation of individual conversion factor (CF) for each lab involved. Nowadays CF is the only way to turn the data collected from different labs into the universal and comprehensive IS scale. The Lab for Genetic Engineering of Research Center for Hematology (Moscow) is the only one in Russia that successfully fulfilled all necessary steps of standardization procedure and obtained a position of the national reference lab for Russia. We report here the preliminary results of the Russian program of BCR / ABL Real Time PCR standardization that has been started under our supervision.

SUMMARY Real Time PCR enables an adequate molecular monitoring of the chronic myelogenous leukemia (CML) without which in the case of this disease it is impossible to thoroughly evaluate therapy response especially when modern target inhibitory molecules involved. Unfortunately, discrepancy and methodological deviations of the results obtained by different researches hinder a proper involvement of different labs and clinics into international research protocols and makes it impossible to compare the data obtained. In this paper we discuss the different approaches to standardize Real Time PCR technique and stress the significance of the international effort to incorporate International Scale (IS) into BCR / ABL gene expression monitoring of CML as well as a great importance of the regular sample exchange between participating labs and necessity of the calculation of individual conversion factor (CF) for each lab involved. Nowadays CF is the only way to turn the data collected from different labs into the universal and comprehensive IS scale. The Lab for Genetic Engineering of Research Center for Hematology (Moscow) is the only one in Russia that successfully fulfilled all necessary steps of standardization procedure and obtained a position of the national reference lab for Russia. We report here the preliminary results of the Russian program of BCR / ABL Real Time PCR standardization that has been started under our supervision.

Modern treatment options for the chronic myelogenous leukemia require the regular performance of molecular monitoring of the BCR / ABL expression level by means of the Real Time PCR. Achievement of the major molecular response (MMR) is considered to be a prominent hallmark of the successful therapy with the inhibitors of tyrosine kinase activity and a very important prognostic factor. Our aim was to report the data of molecular monitoring of BCR / ABL gene expression in CML patients treated with imatinib during the 5 years experience of this work in the real clinical conditions of the Research Center for Hematology (Moscow, Russia). The strict correlation between cytogenetic and molecular data was shown and unevenness of CML patients in complete cytogenetic response (CCR) when they were analyzed in the terms of molecular response. We found it out that 1 % IS was a threshold that shouldn't be exceeded to keep cytogenetic response for a long time of observation. The level of BCR / ABL expression at the start of imatinib treatment had a great prognostic significance. All changes in cytogenetic and molecular data showed the same trend and their dynamics adequately reflected evolution of disease.

Molecular diagnosis of chronic myeloid leukemia. A.V.Misyurin, E.V.Aksyonova, A.A.Krutov, A.V.Lukyanenko, Yu.P.Finashutina, M.V.Suchkova, V.V.Tikhonova, E.Yu.Chelysheva, I.N.Soldatova, A.G.Turkina, N.D.Khoroshko. Hematology Research Center, Moscow. Philadelphian (Ph) chromosome is the specific cytogenetic marker of chronic myeloid leukemia (CML). It emerges as a result of t(9-,22)(q34;q11) reciprocal translocation. Ph-chromosome is a genetic abnormality, most fully analyzed at a molecular level. Southern blot hybridization is the first method which was effectively used for the molecular diagnosis of CML. This method was replaced by the polymerase chain reaction (PCR) and real time PCR, due to which the molecular diagnosis of CML transformed from an almost artistic manipulation into a routine method of laboratory analysis. Due to this the routine protocols for examination of CML patients in complete remission were appreciably simplified. CML patients can develop resistance to gleevek during therapy with this drug; one of the causes of this resistance is emergence of tumor cell clones carrying mutations at the BCR/ABL gene site determining the tyrosine kinase activity. Molecular methods detect these mutations.

New systems are proposed for the PCR analysis of HindIII polymorphic sites in the gamma A and gamma G globin genes and of TaqI polymorphic site in the human factor IX gene of blood population. DNA fragments amplified according to the systems described contain constant restriction site of the appropriate endonuclease, in addition to the polymorphic one, which significantly improves the reliability of the RELP analysis. The systems proposed are highly specific and may be used for DNA diagnosis of beta-thalassemia and haemophilia B.

Using oligonucleotide probes, sequences containing the Mbcr locus involved in chromosome translocation t(9:22) were cloned form the library of human genes in the Charon 4A vector. The recombinant clone lambda BCR 1.1 obtained contained Mbcr sequences, but the 3' region of the Mbcr locus in lambda BCR 1.1 clone was strongly altered. Subcloning of a fragment of the altered region and blot hybridization analysis using it as a DNA probe revealed recombination in the 3' region of the Mbcr locus in clone lambda BCR 1.1 which resulted in insertion of unknown sequences into the region. A modified system is suggested for chromosome 22 breakpoint identification using restriction analysis of genome DNA with four restriction endonucleases and one 5'-DNA probe.