Filip Rázga

Mechanism of impaired glucose metabolism during nilotinib therapy in patients with chronic myelogenous leukemia

Hyperglycemia represents frequent adverse event reported in chronic myelogenous leukemia (CML) patients treated with nilotinib. In order to determine the major mechanism of glucose metabolism impairment, we performed a metabolic analysis using an oral glucose tolerance test as well as assessment of incretins and adipokines at baseline and after 3 months of nilotinib treatment in patients with CML. We proved that rapid insulin resistance, compensatory hyperinsulinaemia, and hypoadiponectinaemia develop after initiation of nilotinib therapy, which clarifies not only the mechanism of impaired glucose metabolism, but also explains the fast development of dyslipidaemia and peripheral artery occlusion in nilotinib-treated CML patients.

The role of reactive oxygen species in the genotoxicity of surface-modified magnetite nanoparticles

The generation of reactive oxygen species (ROS) has been proposed as the underlying mechanism involved in the genotoxicity of iron oxide nanoparticles. The data published to date are, however, inconsistent, and the mechanism underlying ROS formation has not been completely elucidated. Here, we investigated the capacity of several surface-modified magnetite nanoparticles (MNPs) to generate ROS in A549 human lung adenocarcinoma epithelial cells and HEL 12469 human embryonic lung fibroblasts. All MNPs, regardless of the coating, induced significant levels of DNA breakage in A549 cells but not in HEL 12469 cells. Under the same treatment conditions, variable low levels of intracellular ROS were detected in both A549 and HEL 12469 cells, but compared with control treatment, none of the coated MNPs produced any significant increase in oxidative damage to DNA in either of these cell lines. Indeed, no significant changes in the total antioxidant capacity and intracellular glutathione levels were observed in MNPs-treated human lung cell lines regardless of surface coating. In line with these results, none of the surface-modified MNPs increased significantly the GPx activity in A549 cells and the SOD activity in HEL 12469 cells. The GPx activity was significantly increased only in SO-Fe3O4-treated HEL 12469 cells. The SOD activity was significantly increased in SO-PEG-PLGA-Fe3O4-treated A549 cells but significantly decreased in SO-Fe3O4-treated A549 cells. Our data indicate that oxidative stress plays, at most, only a marginal role in the genotoxicity of surface-modified MNPs considered in this study in human lung cells.

Monitoring of minimal residual disease in acute myeloid leukemia with frequent and rare patient‐specific NPM1 mutations

Nucleophosmin (NPM1) mutations in exon 12 are the most common genetic alternation in cytogenetically normal AML (CN‐AML). Although mutation types A, B, and D represent the majority of cases, rare mutation variants of the NPM1 gene in individual patients do occur. In this study, we have evaluated a novel, DNA‐based real‐time quantitative polymerase chain reaction (RQ‐PCR) for the detection of three of the most commonly occurring mutations and for six rare patient‐specific mutation types, which represent 28% of all of the NPM1 mutations in our group of 25 CN‐AML patients. Furthermore, the prognostic relevance of NPM1‐based monitoring of minimal residual disease (MRD) in peripheral blood (PB), bone marrow (BM), and in specific cell subsets (CD34+, CD34−, CD34dim) of BM were evaluated. In 80% of the evaluable patients, a molecular relapse preceded a hematological relapse. Moreover, in this subset of patients, the molecular relapse occurred at a median of 97 days before the hematological relapse. Our compartment analysis showed a strong correlation between BM and PB (r = 0.907, P < 0.001) as well as a high copy number of mutated NPM1 in CD34+ BM cells. In conclusion, we have demonstrated applicability of our presented RQ‐PCR method for a large percentage of mutated NPM1 patients with CN‐AML as well as the usefulness for long‐term follow‐up monitoring of MRD and the prediction of hematological relapse. Am. J. Hematol., 2010.

The assessment of human organic cation transporter 1 (hOCT1) mRNA expression in patients with chronic myelogenous leukemia is affected by the proportion of different cells types in the analyzed cell population

The monitoring of hOCT1 mRNA expression in patients with chronic myelogenous leukemia (CML) was used for predicting the response to imatinib treatment. However, different cell populations from patients who received various degrees of pretreatment were used for this analysis. Therefore, several biases in the results and their interpretation may arise. We investigated hOCT1 mRNA expression in different cell populations of peripheral blood (PB) from healthy volunteers and in imatinib nave de novo CML patients by analyzing changes in hOCT1 mRNA expression during the first 6 months of imatinib therapy. The hOCT1 mRNA expression was significantly higher in PB polymorphonuclears compared to mononuclears. The hOCT1 mRNA expression in total PB leukocytes is, therefore, preferentially determined by the percentage of polymorphonuclears. Expression in each analyzed group of cells was always significantly lower in imatinib nave de novo CML patients compared to healthy volunteers. This difference disappeared after the initiation of imatinib therapy, suggesting that CML tumor burden and the degree of pretreatment at the time of monitoring were both influencing factors.

Increasing hematopoietic microchimerism is a reliable indicator of incipient AML relapse

Introduction:  The reoccurrence or increase in autologous hematopoiesis after allogeneic transplantation has been linked to incipient leukemia relapse. However, the importance of such an emergency regarding microchimerism (i.e. mixed chimerism below 1% of autologous cells) still remains controversial, as fluctuating microchimerism can be observed for a very long time after transplantation.

Imatinib as the first-line treatment of patients with chronic myeloid leukemia diagnosed in the chronic phase: Can we compare real life data to the results from clinical trials?†

Imatinib (IM) dramatically improved the prognosis of chronic myeloid leukemia (CML), particularly with newly diagnosed patients in a chronic phase (CP) [1]. The most robust source of data about IM efficacy in this setting is the IRIS trial. However, every day clinical practice data are still scarce. We analyzed IM efficacy and safety in the first-line therapy of 152 consecutive adult CP-CML patients from a defined region. The estimated 4-year cumulative incidences of complete hematologic, complete cytogenetic, major, and complete molecular responses were 95.3%, 80.6%, 65.4%, and 39.2%, respectively. The 4-year probability of overall and progression-free survival (PFS) defined as with the IRIS [2] was 91.5% and 78.1%, respectively. We thus confirmed very good IM efficacy also in patients not participating in clinical trials. However, the estimated 4-year event-free survival (EFS), which also counted failure events according to valid recommendations [3] or IM discontinuation due to intolerance, was only 60.7%. The 4-year probability of an alternative treatment-free survival, our newly defined parameter, which better reflects the proportion of patients remaining on IM despite an event, was 67.6%. Therefore, more appropriate selection and unification of survival analyses end-points is desirable to describe and compare IM real efficacy.

Assessment of adenosine triphosphate-binding cassette subfamily B member 1 (ABCB1) mRNA expression in patients with de novo chronic myelogenous leukemia: the role of different cell types

In this study, we investigated differences in ABCB1 mRNA expression measured in peripheral blood (PB) leukocytes (LEU) as well as polymorphonuclear (PMNCs) and mononuclear cells (MNCs) of PB LEU obtained from healthy volunteers and patients with de novo CML. In addition, we analyzed the relationship between the percentage of individual cells that comprise the total LEU count and ABCB1 mRNA expression assessed from the total LEU. We have shown that ABCB1 mRNA transcript levels are significantly higher in PB MNCs than in PB PMNCs and are lower in patients with de novo CML compared to healthy individuals. Moreover, we have demonstrated the importance of the cell composition of analyzed samples in ABCB1 mRNA expression analysis.

Quantitative assessment of the CD26+ leukemic stem cell compartment in chronic myeloid leukemia: patient-subgroups, prognostic impact, and technical aspects

Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26− HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors.

Vibration Technology for Microencapsulation: The Restrictive Role of Viscosity

Microencapsulation employed in a broad range of applications, including those with a strict demand on standardization, requires to understand the limitations of respective encapsulation technologies. Among the most frequently used technologies, vibration technology has gained a significant interest due to high capacity and capability to produce uniform and monodisperse microspheres. In this contribution, the restrictive role of viscosity and practical consequences regarding the production of microspheres using vibration technology are reported and discussed.

The predictive value of human organic cation transporter 1 and ABCB1 expression levels in different cell populations of patients with de novo chronic myelogenous leukemia

In this study, we investigated the predictive value of pretreatment mRNA expression levels of hOCT-1 and ABCB1 in different cell populations with regard to the response to therapy at 6 and 12 months of IMA therapy. Expression levels were assessed in peripheral blood (PB) leukocytes (LEU, n = 30), polymorphonuclear cells (PMNC, n = 23), and mononuclear cells (MNC, n = 21) of PB LEU obtained from 30 patients with de novo chronic myelogenous leukemia (CML). Moreover, the available bone marrow cells (BM) were also included and analyzed (BM, n = 11). The PB and BM samples were obtained, processed, and analyzed as previously described. Responses to therapy were classified according the European LeukemiaNet 2009 (ELN) criteria: responders show an optimal response at 6 months and 12 months, while non-responders reflect a suboptimal response or therapy failure. The assessed pretreatment expression levels were stratified into two groups according to the median-a low-mRNA expression group below the median and a high-mRNA expression group equal to or above the median. The statistical evaluation of the data obtained was performed using the Fisher’s exact tests and summarized in Table 2.