[CASC5 Gene Expression Changes Correlate with Targeted Mutations in Leukemia].

Dysfunction of genes that control mitosis and are responsible for the correct segregation of sister chromatids in anaphase is often accompanied by aneuploidy, which is frequently detected in leukemia. One of the components of the kinetochore complex, namely, the AF15q14/KNL1/CASC5 protein, is an important factor ensuring the correct binding of the pericentromeric region of chromosomes with the spindle microtubules. As shown recently, in some leukemias, the gene of this protein can be involved in the generation of the chromosomal translocation t(11;15)(q23;q14) or a variant of the chimeric MLL-AF15Q14 oncogene, which serves as a biomarker of poor prognosis. Despite the implication of mRNA of the CASC5 gene in oncogenesis of solid tumors, expression of this gene in hematopoietic neoplasms has not been studied. We analyzed expression levels of the CASC5 gene and the nearest regulatory genes, including WT1, APOBEC3A (A3A), and N-MYC. A pronounced decrease in CASC5 expression in bone marrow cells of primary leukemia patients compared with healthy donors was found. It was also shown that reduced expression of the CASC5 gene correlates with the detection of targeted mutations in patients composed two prognostic subgroups (favorable, unfavorable) with a significance level (p <0.05). It was noted that the change in the expression level of the CASC5 gene in acute myeloid leukemia is associated with overexpression of the genes WT1, A3A, and in some cases N-MYC and SPT16, which is consistent with the resistance to chemotherapy and leukemia progression. However, the question of which regulatory gene initiates leukemogenesis remains open.

European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia.

The therapeutic landscape of chronic myeloid leukemia (CML) has profoundly changed over the past 7 years. Most patients with chronic phase (CP) now have a normal life expectancy. Another goal is achieving a stable deep molecular response (DMR) and discontinuing medication for treatment-free remission (TFR). The European LeukemiaNet convened an expert panel to critically evaluate and update the evidence to achieve these goals since its previous recommendations. First-line treatment is a tyrosine kinase inhibitor (TKI; imatinib brand or generic, dasatinib, nilotinib, and bosutinib are available first-line). Generic imatinib is the cost-effective initial treatment in CP. Various contraindications and side-effects of all TKIs should be considered. Patient risk status at diagnosis should be assessed with the new EUTOS long-term survival (ELTS)-score. Monitoring of response should be done by quantitative polymerase chain reaction whenever possible. A change of treatment is recommended when intolerance cannot be ameliorated or when molecular milestones are not reached. Greater than 10% BCR-ABL1 at 3 months indicates treatment failure when confirmed. Allogeneic transplantation continues to be a therapeutic option particularly for advanced phase CML. TKI treatment should be withheld during pregnancy. Treatment discontinuation may be considered in patients with durable DMR with the goal of achieving TFR.

Cepeginterferon alfa-2b in the treatment of chronic myeloproliferative diseases.

AIM: A comparative evaluation of the effectiveness of different therapeutic strategies in patients with polycythemia vera (PV) and essential thrombocythemia (ET). MATERIALS AND METHODS: Patients with PV or ET, diagnosed according to the criteria WHO 2016 were included in the study. The primary endpoint - 6 months of therapy (clinical-hematological and molecular responses). The secondary endpoint - 12 months of therapy (clinico-hematologic, molecular, histological responses). Sixty three patients were included in the analysis: the first group consisted of 33 patients who received the therapy with ce-pegiterferone alpha-2b (ce-pegalpha-INF-α-2b), 10 of them received previous treatment; the second group - 23 patients btained hydroxycarbamide; the third group - 7 patients were treated with recombinant interferon alpha therapy (rINFα). In comparison groups, differences in age were revealed: patients receiving hydroxycarbamide therapy were older. Phlebotomy occurred in 36% of patients in the first group, 9% in the second group, and 14% in the third group. RESULTS: By the 6th month of therapy, 43% of the patients receiving the ce-pegalpha-INF-α-2b had complete clinical-hematologic response, 36% had partial clinical-hematologic remission and stabilization of the disease was established in 21% cases. No disease progression occured. By the 12th month of therapy, statistically significant differences in terms of efficacy between the different therapeutic groups (p = 0.2462, Fisher's exact test). In all three groups, the allelic load of JAK2V617F decreased: from 50 to 19%, from 22.3 to 15.8%, from 50 to 7.19%, respectively. The lower the allele load positively correlated with better response to therapy, which was observed in all analyzed groups. Hematologic adverse events (AEs) were more frequently observed in patients receiving ce-pegalpha-INF-α-2b therapy. Local reactions developed on 3-7 days of therapy as a hyperemic macula at the injection site. Both these reactions and hair loss did not influence on patient's condition. In the second group (patients with hydroxycarbamide therapy) there were changes in the skin and mucous membranes: dry skin, stomatitis, and in older patients new keratomas appeared. The flu-like syndrome was the most common adverse event associated with the therapy of ce-pegalpha-INF-α-2b, which fully relived during the first month of therapy. There was only one case with the flu-like syndrome we observed at the 11th month of therapy. As a rule, the biochemical blood test changes did not influence on patient's condition, were mostly associated with dietary violations, had a tendency to self-resolution and did not require medical interventions. Serious AEs were reported in one case - pulmonary embolism in a patient treated with rINFα. The reasons for the therapy discontinue in group 1: toxic hepatitis, intolerance, by the request of the patient, inadequate efficacy of therapy; in group 2: skin toxicity, in group 3: thromboses. CONCLUSION: Treatment of ce-pegalpha-INF-α-2b in patients with PV and ET is highly effective - the most patients pbtained clinical and hematological responses. There were no statistically significant differences in these parameters in comparison with hydroxycarbamide and rINFα. The use of the ce-pegalpha-INF-α-2b had an acceptable safety profile. The estimated therapeutic dose should be calculated according to body weight. To reduce the frequency of hematologic AE, titration of the drug dose is required.

Treatment and outcome of 2904 CML patients from the EUTOS population-based registry.

The European Treatment and Outcome Study (EUTOS) population-based registry includes data of all adult patients newly diagnosed with Philadelphia chromosome-positive and/or BCR-ABL1+ chronic myeloid leukemia (CML) in 20 predefined countries and regions of Europe. Registration time ranged from 12 to 60 months between January 2008 and December 2013. Median age was 55 years and median observation time was 29 months. Eighty percent of patients were treated first line with imatinib, and 17% with a second-generation tyrosine kinase inhibitor, mostly according to European LeukemiaNet recommendations. After 12 months, complete cytogenetic remission (CCyR) and major molecular response (MMR) were achieved in 57% and 41% of patients, respectively. Patients with high EUTOS risk scores achieved CCyR and MMR significantly later than patients with low EUTOS risk. Probabilities of overall survival (OS) and progression-free survival for all patients at 12, 24 and 30 months was 97%, 94% and 92%, and 95%, 92% and 90%, respectively. The new EUTOS long-term survival score was validated: the OS of patients differed significantly between the three risk groups. The probability of dying in remission was 1% after 24 months. The current management of patients with tyrosine kinase inhibitors resulted in responses and outcomes in the range reported from clinical trials. These data from a large population-based, patient sample provide a solid benchmark for the evaluation of new treatment policies.

The EUTOS population-based registry: incidence and clinical characteristics of 2904 CML patients in 20 European Countries.

This population-based registry was designed to provide robust and updated information on the characteristics and the epidemiology of chronic myeloid leukemia (CML). All cases of newly diagnosed Philadelphia positive, BCR-ABL1+ CML that occurred in a sample of 92.5 million adults living in 20 European countries, were registered over a median period of 39 months. 94.3% of the 2904 CML patients were diagnosed in chronic phase (CP). Median age was 56 years. 55.5% of patients had comorbidities, mainly cardiovascular (41.9%). High-risk patients were 24.7% by Sokal, 10.8% by EURO, and 11.8% by EUTOS risk scores. The raw incidence increased with age from 0.39/100,000/year in people 20-29 years old to 1.52 in those >70 years old, and showed a maximum of 1.39 in Italy and a minimum of 0.69 in Poland (all countries together: 0.99). The proportion of Sokal and Euro score high-risk patients seen in many countries indicates that trial patients were not a positive selection. Thus from a clinical point of view the results of most trials can be generalized to most countries. The incidences observed among European countries did not differ substantially. The estimated number of new CML cases per year in Europe is about 6370.

Children and adults with acute lymphoblastic leukaemia have similar gene expression profiles.

OBJECTIVES: To compare the gene expression pattern in children and adults with acute lymphoblastic leukaemia (ALL) in order to improve our understanding of the difference in disease biology and prognosis. METHODS: The gene expression profiles in diagnostic samples from 29 children and 15 adults with ALL were analysed using the oligonucleotide chip Hu95ver2a, produced by Affymetrix. RESULTS: Unsupervised hierarchical cluster analysis revealed that, in spite of differences in outcome, patients clustered irrespective of age, first by T-cell or B-precursor immunophenotype, and second by cytogenetic changes within the B-precursor group. The expression pattern analysis allowed the reclassification of some samples into the proper cytogenetic group. We also showed that separate clustering of samples with the BCR/ABL translocation could be explained by different breakpoint regions in the BCR. No significant difference in gene expression was observed between samples with and without CDKN2A deletion within the B-precursor group. Analysis of different age groups revealed a similarity in expression profiles when infants with the MLL translocation and adults over 40 yr of age were compared irrespective of karyotype. CONCLUSIONS: In spite of the difference in clinical outcome, the gene expression pattern in children and adults with ALL is very similar and is primarily dependent on immunophenotype and cytogenetic aberrations. However, when age groups are compared, the expression patterns of infants and adults over 40 show a remarkable similarity.

Stromal cells prevent apoptosis of AML cells by up-regulation of anti-apoptotic proteins.

The aim of this study was to study interactions between stromal bone marrow microenvironment and leukemic cells. We tested the hypothesis that stromal cells prevent apoptosis of AML cells by up-regulating anti-apoptotic proteins in leukemic blasts. In HL-60 and NB-4 cells, serum deprivation- and ara-C-induced apoptosis was diminished when cells were cocultured with murine MS-5 stromal cells (P < 0.02). This effect was reproduced with conditioned medium from MS-5 cells. Cocultivation with stromal cells induced Bcl-2 expression levels, both by PCR analysis and flow cytometry. In primary AML (n = 14), ara-C-induced apoptosis was significantly lower in cells cocultured with MS-5 cells than in controls (P < 0.001). This effect was partially preserved when leukemic cells were separated from stromal cells by a microporous insert (in 5/9 samples, P = 0.04). In addition, Bcl-2 levels were significantly higher in stroma-supported than in control CD34(+) AML cells (P < 0.01). Bcl-X(L) levels were higher in 5/7 samples grown on stromal layers. Of note, in AML patients resistant to induction chemotherapy (n = 6), Bcl-2 increased significantly after cultivation with stromal cells, but no such increase was noted in cells from chemotherapy-sensitive patients. In conclusion, MS-5 stromal cells prevented apoptosis in HL-60 cells and in primary AML blasts via modulation of Bcl-2 family proteins. The observed association of high Bcl-2 expression in stroma-supported AML blasts in vitro with resistance to chemotherapy in vivo suggests that the same mechanisms may be operational in vivo.

Ultraviolet irradiation induces multiple DNA double-strand breaks and apoptosis in normal granulocytes and chronic myeloid leukaemia blasts.

Major leucocyte subpopulations were isolated from peripheral blood of healthy donors, and patients with chronic myeloid leukaemia (CML) and chronic lymphoid leukaemia (CLL). In vitro UV irradiation was performed at the wavelength of 257nm (UVC band). DNA double-stranded breaks (DNAdsbs) were detected immediately after UV-irradiation, by means of agarose gel electrophoresis. Cell viability was estimated after 18h in culture, as relative numbers of residual non-apoptotic cells. Evaluation of the dose-response curves revealed that normal CLL lymphoid cells showed only moderate damage after UV-irradiation, as assessed by DNAdsbs and cell viability criteria. However, normal granulocytes and myeloid blasts from CML patients expressed a sharp increase in DNAdsbs, even at lower doses of UV-radiation. UV-induced amplification of endogenous oxidative systems (e.g. NADPH-dependent oxidase) is suggested as a probable reason for enhanced DNA breakage and apoptosis in cells of the granulocytic lineage.