Search

A universal approach to the construction of antibody–drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug–antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

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.

Aim. To compare NHL BFM-90 and CHOEP efficacy in adult patients with ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL). Materials & Methods. Within the period from June 2014 to December 2019 the prospective randomized comparative study at the National Research Center for Hematology in Moscow included 23 ALK+ ALCL patients. In one study arm (n = 11) CHOEP was administered, whereas the other one (n = 12) received high-dose chemotherapy (CT) according to NHL BFM-90 protocol. The median age of patients in both arms was 33 and 40 years, respectively. Results. Overall survival (OS) and event-free survival (EFS) within 3 years were 91 % in the arm receiving CHOEP (this protocol was administered to all 11 patients), and 100 % in the arm receiving NHL BFM-90 (complete remission was achieved in all patients). Due to its toxicity NHL BFM-90 was fully implemented in 9 out of 12 patients. The 3-year OS and EFS in the CHOEP and NHL BFM-90 arms are comparable, and the difference between them is not significant. Conclusion. In ALK+ ALCL treatment high-dose CT according to NHL BFM-90 protocol has no advantage in terms of the 3-year OS and EFS compared to less toxic regimen CHOEP. A larger sample of patients is required to achieve significant results, which will further lead to a final judgement on feasibility of high-dose regimens in the treatment of adult patients with ALK+ ALCL.

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.

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 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.

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: Anaplastic large cell ALK-positive lymphoma (ALCL ALK+) is a lymphoproliferative disease of the “young” age. ALCL ALK+ has a favourable prognosis. A failure in therapy (relapse) is diagnosed within the first 1.5-2 years of the follow up period. Aim: The aim of the present study was to compare the effectiveness of the NHL BFM-90 and CHOEP protocols intended for the treatment of adult patients presenting with the ALCL ALK+. Materials and methods: This prospective comparative randomized study was carried out based at the National Medical Research Centre of Hematology, Moscow, during the period from June 2014 to December 2019. It involved 23 patients with the ALCL ALK+ disease randomly allocated to two groups. One of them included 11 patients given a CHOEP, the other consisted of 12 patients treated using a high-dose CT in accordance with the NHL BFM-90 protocol (age medians 33 and 40 years respectively). Results: The 3-year OS and EFS rates among the patients receiving CHOEP was 91%, with all the members of this study group having completed the treatment regimen. All patients comprising the latter (NHL BFM-90) group achieved complete remission. However, only 9 of the 12 patients fully complied with the treatment protocol because of its toxic effects. It can be inferred that the two study groups are comparable in terms of the 3-year OS and EFS rates among the patients treated in accordance with the CHOEP and NHL BFM-90 programs, the differences between them being insignificant. Conclusion: High-dose CT based on the NHL BFM-90 protocol has no advantages over the less toxic CHOEP regimen as far as the treatment of the ALCL ALK+ is concerned (if the efficacy of both modalities is estimated in terms of the 3-year OS and EFS rates). However, additional studies with larger patient samples are needed to obtain statistically significant results before a definitive conclusion can be made as regards clinical feasibility of the use of high-dose therapeutic regimens for the treatment of adult patients presenting with the ALCL ALK+ disease.

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.

Porphobilinogen deaminase (PBGD) is a key enzyme of the heme biosynthetic pathway. Defects in the PBGD gene lead to an autosomal dominant disease, acute intermittent porphyria (AIP). Almost all AIP patients with rare exceptions are heterozygous for the defective gene. To date, at least 160 different mutations causing AIP are identified. Extensive investigations along this line are conducted in many countries of the world. In Russia these studies had not been hitherto performed. Here we report the results of molecular genetic examination of four Russian patients with AIP diagnosed from clinical symptoms. By direct sequencing of the PBGD gene or the corresponding cDNA, we have detected four mutations, three of which were not previously encountered in the world population. These are TAAG deletion in intron 7 between positions +2 and +5 (IVS7 2–5 delTAAG); T deletion in the initiation codon ATG of exon 3, and the G for C replacement at position –1 of intron 5 (IVS5 as –1 G–C), which disrupts splicing. In addition, in one female patient, a known deletion CT in codon 68 was revealed. In two patients, expression of PBGD gene alleles was significantly disproportional, so that normal mRNA prevailed in one case and mutant mRNA of nonerythroid type in the other. Deletion in intron 7 was easily detectable due to the formation of a heteroduplex fragment with abnormal electrophoretic mobility directly in PCR. This simple heteroduplex analysis allowed us to exclude AIP carriage in son and daughter of a female patient with the genetic defect.

Abstract 4420 In the period from 18.01.06 to 25.05.11 we have performed mutation analysis in CML pts with the obvious manifestation of resistance against imatinib. Chronic phase CML patients have been treated by imatinib and monitored by means of RQ-PCR using IS units. At first signs of the evident BCR-ABL level increase (more than 5–10% IS), the mutation analysis has been performed by means of direct PCR fragment sequencing. We have used original primers that enable to search for mutations in the area spanning from a3 to a11 ABL exons of BCR-ABL. Sequencing has been performed for 274 CML pts that were suspected to acquire imatinib resistance. Among them there were 47,45% (130/274) of females (median age 51 years, from 15 to 73), 52,55% (144/274) of males (median age 50 years, from 15 to 74). In the group of mutation negative pts (80/274 – 29,2%) the BCR-ABL level was more than 9% (IS). Interestingly, in this group there were a little bit more females than in the total cohort of pts (58,7% against 47,45%, p=0,034). The median age of the mutation negative females (51 years) was quite the same as in the total cohort, whereas the median age of mutation negative males significantly differed from the one in the total cohort (58 vs. 51 year, p=0,041). 70,8% (194/274) pts tested occurred to be mutation positive (42,8% females (83/194), 57,2% males (111/194)). 214 point mutations of 38 different types have been detected, among them T315I (26/194 – 12%), G250E (26/194 – 12%), T317L (16/194 – 7,4%), F359V (15/194 – 7,0%), H396R (15/194 – 7,0%), M244V (15/194 – 7,0%), E255K (12/194 – 5,6%), Y253H (12/194 – 5,6%), E255V (9/194 – 4,2%), L248V (8/194 – 3,7%), E355G (6/194 – 2,8%), M315T (6/194 – 2,8%), Q252H (5/194 – 2,3%), L387F (4/194 – 1,8%), S348L (4/194 – 1,8%), F311I (3/194 – 1,4%), F359C (3/194 – 1,4%), E255D (2/194 – 0,9%), E275K (2/194 – 0,9%), E279A (2/194 – 0,9%), K247R (2/194 – 0,9%), E292V (1/194 – 0,46%), E334G (1/194 – 0,46%), E450K (1/194 – 0,46%), E459A (1/194 – 0,46%), E459K (1/194 – 0,46%), F359I (1/194 – 0,46%), F486S (1/194 – 0,46%), L383F (1/194 – 0,46%), P441L (1/194 – 0,46%), Q252M (1/194 – 0,46%), Q491L (1/194 – 0,46%), T305I (1/194 – 0,46%), T345I (1/194 – 0,46%), V299A (1/194 – 0,46%), Y312C (1/194 – 0,46%), T520S (1/194 – 0,46%). As rare events we have observed some other types of mutations. In one CML patient with primary imatinib resistance there was a G425Stop (1/194 – 0,46%) mutation which caused a significant truncation of the C-end of the BCR-ABL protein. There were also 2 highly imatinib resistant pts with deletions of exon a7 ABL (2/194 – 0,9%). One more unusual mutation was an insertion of 72 bp between the a8 and a9 exons of ABL. The origin of this insertion was a middle part of the 8th intron of the ABL gene. This sequence is arranged as a typical human exon because it is flanked by donor and acceptor splicing signals. We found this mutation in 3 imatinib resistant CML pts (3/194 – 1,4%). This mutation gives rise to the abnormal stop-codon within the exon a9 due to a CD’s frameshift. In all pts with point L248V mutation (8/194 – 3,7%) we have also observed additional abnormal variant of BCR-ABL gene as a splicing isoform lacking 81 bp of exon a4. The point mutation L248V (742C>G) exchange the motif CAAGCT for CAAGGT and the latter is a strong splice acceptor site. It seems that this cryptic splice acceptor site in the middle of 4a exon carrying 742C>G point mutation may compete with natural splice acceptor site from 4th intron. It gives rise to two BCR-ABL transcripts: one carrying a point mutation and the other one with 81 bp’s deletion of the 3’end of exon 4a, without frameshift. These findings suggest that in the case of mutational analysis the considerable attention should be paid not only to BCR-ABL point mutations but also to the possible emergence of the other changes of the BCR-ABL mRNA structure which may occur due to arbitrary activity of splicing machinery. Our findings also suggest that among imatinib resistant CML pts some of them already acquired mutations that prevented adequate response for the 2nd generation of TKI. In our study 12% of BCR-ABL mutation positive CML pts resistant to imatinib (n=26/194) had T315I which also render nilotinib and dasatinib resistance. 23,8% (n=51/194) were positive for mutations that decreases nilotinib response (E255K/V, F359C/V, Y253H) and 7,5% (n=16/194) were positive for T317L that hinders dasatinib response. Disclosures: No relevant conflicts of interest to declare.

Abstract 893 Introduction: A European collaborative harmonization study involving 61 laboratories is being conducted under the auspices of the European Treatment and Outcome Study (EUTOS) for CML that aims to facilitate reporting of molecular BCR-ABL quantification results according to the International Scale (IS). The aim of this analysis was to investigate the effectiveness of this process and specifically the stability of conversion factors (CF) over time. Methods: The currently accepted way of adopting the IS is to establish and validate a laboratory-specific CF which is then used to convert local results to the IS. For round 1, preliminary CFs were calculated by centrally distributing standard samples containing 10–20 million WBC approximating to 10%, 1%, 0.1%, and 0.01% BCR-ABL IS. Rounds 2 and 3 were employed to refine the CF calculations using 25–30 CML patient samples from each participating laboratories covering a range of BCR-ABL levels between 0.01% and 10%. Log BCR-ABL values for the same samples were compared between reference and local laboratories applying the Bland-Altman bias plot. In order to judge the stability of each laboratory`s methodology, a CF index (ratio of round 3 CF divided by round 2 CF) was calculated and evaluated according to its capability to achieve optimum concordance of results. Results: Of the 61 laboratories participating in round 1, evaluable patient samples have been provided to date by 56 and 30 laboratories in rounds 2 and 3, respectively. Of the 30 laboratories with complete data, 12 had stable CFs (defined as a CF index within 0.75–1.33) whereas 18 laboratories were outside this range. Comparison of the CFs derived from round 2 with those derived from round 3 revealed better and more consistent concordance between laboratories with stable CFs compared to those with unstable CFs. For the 12 stable laboratories, 79% (round 3 CF) vs 79% (round 2 CF) of the samples were within a 2-fold range (0.5–2.0) and 93% vs 89% were within a 3-fold range (0.33–3.0). For the 18 unstable laboratories, 74% vs 55% of the samples were within a 2-fold range (0.5–2.0), p=0.0005 and 92% vs 77% were within a 3-fold range (0.33–3.0), p=0.0005. 2 of 12 laboratories with stable CFs and 8 of 18 laboratories with unstable CFs indicated changes in either one or more components of their procedures (cDNA synthesis, PCR platform, RQ-PCR protocol) that may have impacted on their CFs. Conclusion: These data indicate that CFs may be unstable in some laboratories even in the absence of significant changes to laboratory protocols. Further, it supports the need for continuous revalidation of CFs. In laboratories with unstable CFs we suggest revalidation within 3 to 6 months whereas those with stable CFs should be assessed on a yearly basis. We also suggest that laboratories with unstable CFs need to rigorously examine their internal processes to identify potential sources of variation. Disclosures: Müller: Novartis: Honoraria, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Molecular monitoring of PML/RARA chimeric gene expression is stated to be necessary for diagnosis of acute promyelocytic leukemia (APL), as well as for monitoring of minimal residual disease (MRD). In our study we aimed to modify leukemia treatment according to MRD results. PML/RARA expression was measured by RT-PCR. Synthesis of primers and RT-PCR were performed using recommendations of BIOMED-1 Concerted Action (1999). Sensitivity of method was estimated as 10−4 – 10−6. Fresh bone marrow (BM) aspirates were investigated before treatment, during induction and consolidation, every 2–3 months during maintenance therapy, and after completion of treatment. A total of 73 morphologically suspected APL patients were included in the study; expression of PML/RARA was found in 69 patients (94.5%), bcr1/2 type in 31 patients (45%), and bcr3 type in 38 patients(55%). MRD screening during maintenance therapy was performed in 47 PML/RARA-positive APL patients. Median follow-up was 4 years (2 mo – 8 years). If chimerical transcript was revealed in a BM sample once, this was considered probable molecular relapse, and when PML/RARA was find twice in 2–3 weeks, we considered this proven molecular relapse. Molecular relapses were revealed in 19 of 47 (40.4 %) APL patients. In 6 of 19 patients (31.5 %) relapses were proven, and in 13 (68.5%) relapses were probable. When the molecular relapses were detected, maintenance chemotherapy was changed in 3 of 13 (23%) patients with probable relapses and in 5 of 6 (83%) patients with proven relapses. None of them developed hematological relapse. Therapy was not changed in 11 patients, and 3 of 10 (30%) patients with probable molecular relapse and 1 pt with proven relapse demonstrated recurrence of the disease. Thus, 4 of 11 (36%) patients with unchanged therapy developed hematological relapse. These results are significantly different from patients with modified maintenance treatment (P=0.01). Interesting data were obtained while investigating BM samples obtained from 5 patients in whom BM sampling were performed at 2 different sites (sternal and iliac). Simultaneously performed RT-PCR of those samples showed different results in 3 of 5 (60%) patients. Though we have a small cohort of patients, we may conclude that it is advisable to change chemotherapy when proved or probable molecular relapse is revealed., When the single positive result of chimerical transcript expression is obtained, it is important to repeat RT-PCR analysis in 2–4 weeks in order to prove the molecular relapse, and then to adjust the maintenance program. However, even when it is impossible to perform the second analysis, changing the therapy may be advisable.

Monitoring of chimerism levels after hemopoietic stem cell transplantation (HSCT) has become a standard test for documentation of important clinical events such as engraftment, graft rejection or leukemic relapse. Sensitive and informative molecular techniques (PCR for STR/VNTR markers) are preferable methods for many laboratories for assessment of donor- or recipient-derived hemopoiesis. However, the sensitivity and specificity of this method may be improved using lineage-specific analysis. Separation of cell population allows not only to detect the kinetics of lineage engraftment but, in some cases, to reveal residual potentially malignant cells. We performed chimerism analysis in 10 pts with high risk haematological malignancies underwent alloHSCT in 2002–2004 ( 4pts - ALL, 3 of them - in 2-nd CR, 2pts - acute non-differentiated leukemia in 2-nd CR, 2 - AML in 2-nd CR, 1 pt with MDS and 1 pt - CML). 6 pts were transplanted after conventional conditioning (Busulfan 16 mg/kg and Cytoxan 120 mg/kg) and 4 - after reduced-intensity conditioning (Busulfan 8 mg/kg, Fludarabin 150 mg/m2 and ATG 40 mg/kg or Cytoxan 900 mg/m2). GVHD was prophylacted by Cyclosporin A (CSA) and Methotrexate(MTX) in 5 pts and Cy +MTX +Prednisone in 5 pts. The median follow-up was 339 days (90–600), median age was 32 yrs. The evaluation of chimerism was performed on bone marrow (BM) and following subpopulations of peripheral blood (PB): CD3+, CD19+ and granulocytes (GR) on +30, +60,+90, +180, +270, +360 days after HSCT using PCR-based method for VNTR/STR analysis. The analysed subpopulations have been obtained using immunomagnetic separation technique (Dynal). We also investigated the mononuclear fraction (MN) left after immunomagnetic separation for revealing of possible residual malignant cells. We revealed differencies in chimerism pattern in BM and PB cell populations. Only 4 pts showed complete chimerism (CC) in PB cell subsets while 7 patients had CC in BM on day+30. Four pts showed stable CC in all cell populations during all time of observation. Six pts demonstrated mixed chimerism (MC) in PB subpopulations (CD3+ - 6 pts, CD19+ - 4 pts, MN - 4 pts, GR - 2pts). Four of them showed stable or increasing donor signal (> 80%) and 2 pts expressed decreased donor chimerism (< 80%). In 4 pts with stable or decreasing MC CSA was cesated and 3 of them became CC to +180 d. 3 pts relapsed, 2 of them - with decreased donor chimerism in MN and CD3 subsets and one patient relapsed on day +90 despite CC in all cell populations. GVHD developed in 6 pts and always was predicted by CC in CD3+ subset. Pts with MC never demonstrated any GVHD. Conditioning regimen had no impact on chimerism status. In conclusion, investigation of chimerism in PB cell subsets permitted detection of minimal persistent recipient cell populations with high sensitivity than in BM. Close monitoring of chimerism allowed to identificate the patients with risk of relapse and convert them to CC using cessation of immunosupression.