Daniela Zackova

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 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.

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.

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.

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.

Successful treatment of steroid-refractory hepatitic variant of liver graft-vs-host disease with pulse cyclophosphamide

OBJECTIVE Corticosteroid-resistant graft-vs-host disease (GVHD) is difficult to manage and is associated with high morbidity and mortality. No standard treatment exists. We have previously seen good results with pulse cyclophosphamide (Cy) in the treatment of liver GVHD in contrast to gastrointestinal GVHD, and here we report results of pulse Cy protocol in the treatment of steroid-refractory hepatitic variant of liver GVHD, with no association to the gut. MATERIALS AND METHODS Cy was infused at a dose of 1,000 mg/m(2). Twenty-nine cyclophosphamide administrations were given to 21 patients. Median time of GVHD onset and Cy administration after transplantation, or donor lymphocyte infusion, were 58 and 69 days, respectively. RESULTS Eleven patients (52%) achieved complete remission and 6 patients (29%) achieved partial remission. Four patients (19%) did not respond, however, their condition stabilized and, upon additional therapy, three achieved partial remission and one complete remission. Overall survival of all 21 patients is 86%, with median and maximal follow-up of 33 and 81 months, respectively. Toxicity was mild and easily manageable without influencing chimerism or disease status. CONCLUSIONS Pulse Cy seems to be an effective treatment for steroid-refractory hepatitic variant of liver GVHD with a good toxicity profile, which may favor its use instead of drugs with more pronounced immunosuppressive effects.

Current survival measures reliably reflect modern sequential treatment in CML: Correlation with prognostic stratifications

Using the data of 723 chronic myeloid leukemia (CML) patients in the chronic phase, we analyzed the prognostic value of the Sokal, Euro, and EUTOS scores as well as the level of BCR‐ABL1 and the achievement of complete cytogenetic response (CCgR) at 3 months of imatinib therapy in relation to the so‐called current survival measures: the current cumulative incidence (CCI) reflecting the probability of being alive and in CCgR after starting imatinib therapy; the current leukemia‐free survival (CLFS) reflecting the probability of being alive and in CCgR after achieving the first CCgR; and the overall survival. The greatest difference between the CCI curves at 5 years after initiating imatinib therapy was observed for the BCR‐ABL1 transcripts at 3 months. The 5‐year CCI was 94.3% in patients with BCR‐ABL1 transcripts ≤ 10% and 57.1% in patients with BCR‐ABL1 transcripts > 10% (P = 0.005). Therefore, the examination of BCR‐ABL1 transcripts at 3 months may help in early identification of patients who are likely to perform poorly with imatinib. On the other hand, CLFS was not significantly affected by the considered stratifications. In conclusion, our results indicate that once the CCgR is achieved, the prognosis is good irrespective of the starting prognostic risks. Am. J. Hematol. 88:790–797, 2013.

No clinical evidence for performing trough plasma and intracellular imatinib concentrations monitoring in patients with chronic myelogenous leukaemia

This multicentre study focused on monitoring imatinib mesylate (IMA) trough plasma (Ctrough) and intracellular (IMA Cintrac) concentrations in 228 chronic myelogenous leukaemia patients. The median of measured IMA Ctrough in our patient group was 905.8 ng ml (range: 27.7–4628.1 ng/ml). We found a correlation between IMA Ctrough and alpha 1‐acid glycoprotein plasma concentrations (rS = 0.42; p < 0.001). All other analysed parameters revealed only weak (gender, dose of IMA per kg) or not significant (age, albumin, creatinine plasma concentration or body mass index) impact on measured IMA Ctrough. The IMA Ctrough decreased during the first 6 months and significantly increased later during treatment. The IMA Ctrough at the first month of therapy did not differ between patients with and without an optimal response at the 12th (p = 0.724) and 18th month (p = 0.135) of therapy. There were no significant differences in medians of IMA Ctrough between both groups measured during the first year of treatment. The IMA Cintrac during the first month were not different between patients with and without an optimal response at the 6th (p = 0.273) and the 12th month (p = 0.193) of therapy. Our data obtained from real life clinical practice did not find a benefit of routine and regular IMA Ctrough nor IMA Cintrac therapeutic drug monitoring in chronic myelogenous leukaemia patients or for subsequent adjustments of the IMA dose based on these results. Moreover, actual alpha 1‐acid glycoprotein plasma concentration should be used for proper interpretation of IMA Ctrough results. Copyright

Role of treatment in the appearance and selection of BCR-ABL1 kinase domain mutations

Background and ObjectiveThe availability of different tyrosine kinase inhibitors (TKIs) with distinct antileukemic potency enables optimization of current therapeutic regimens; however, some patients lose their therapy response and acquire TKI resistance. In this study, we describe a single-center experience of monitoring BCR-ABL1 kinase domain (KD) mutations and discuss the impact of treatment on mutation selection.MethodsChronic myelogenous leukemia (CML) patients treated with TKIs at the Department of Internal Medicine — Hematology and Oncology, Masaryk University and University Hospital Brno during 2003–2011 were included in this study. A total number of 100 patients who did not achieve an optimal therapy response or who lost their therapy response were screened for the presence of BCR-ABL1 KD mutations, using direct sequencing.ResultsOur data show that pretreatment with non-specific non-TKI drugs prior to TKI therapy does not preferentially select for initial BCR-ABL1 KD mutations, in contrast to first-line imatinib therapy, which shows a clear predominance of T315I or P-loop mutations compared with mutations located in other KD regions. In addition, the median time to detection of P-loop mutations was substantially shorter in patients treated with first-line imatinib than in those pretreated with non-TKI drugs. Furthermore, analysis of CML patients who had recurrent resistance to TKI therapy revealed possible therapy-driven selection of BCR-ABL1 KD mutations. Finally, we confirm the previously described poor prognosis of CML patients with mutations in the BCR-ABL1 KD, since 40.0% of our CML patients who harbored a BCR-ABL1 KD mutation died from CML while receiving TKI treatment. Moreover, among the patients who are still on treatment, 27.8% have already progressed. Our data also confirm the unique position of the T315I mutation with respect to its strong resistance to currently approved TKIs.ConclusionOn the basis of the ‘real-life’ data described in this study, it is possible that the therapy itself results in its failure and selects the most resistant mutations under the selective pressure of the applied therapy regimen in some CML patients who harbor BCR-ABL1 KD mutations.

Failure of molecular diagnostics in chronic myeloid leukemia: an aberrant form of e13a2 BCR–ABL transcript causing false-negative results by standard polymerase chain reaction

In 95% of patients suffering from chronic myeloid leukemia (CML), and in 25% of patients suffering from acute lymphoblastic leukemia (ALL), the leukemic cells are characterized by the presence of the Philadelphia chromosome (Phþ), which results from the reciprocal translocation t(9;22)(q34;q11). The resultant BCR–ABL fusion gene encodes oncogenic proteins that vary in size, depending on the breakpoint within the BCR gene. A majority of patients with Phþ CML (*90%) have the most common type of breakpoint, denoted as M-bcr (major), resulting in e13a2 and/or e14a2 fusion transcripts. A minor portion of cases with Phþ CML (*10%) are associated with two other types of breakpoint, termed m-bcr (minor) and m-bcr (micro), involving e1a2 and e19a2 junctions, respectively [1,2]. For the abovementioned fusions (M-bcr, m-bcr, m-bcr), powerful molecular diagnostic tools based on reverse-transcriptase polymerase chain reaction (RT-PCR) have been purposefully used in routine practice. Considering the fact that the majority of patients with Phþ CML and ALL harbor the typical BCR–ABL fusion transcript, many modern molecular diagnostic techniques for the qualitative detection of BCR–ABL fusions are based on a multiplex RT-PCR method reported by Cross et al. in the early 1990s [3]. These authors described a rapid and simple PCR method capable of identifying the common BCR–ABL breakpoints, a technique that was also expected to distinguish all the theoretical in-frame mRNAs, including e19a2, e13a3, and e1a3 transcripts. Unfortunately, to date, atypical types of BCR–ABL fusion genes have also been reported. Involvement of the BCR gene breakpoints outside the cluster regions [4–7], insertion of small sequences [8,9], or genomic breakpoints within individual exons [10,11] represent a risk of misinterpretation during routine investigation at diagnosis. To reduce the possibility of misinterpretation, an improved RT-PCR assay for the simultaneous detection of common (M-bcr, m-bcr, m-bcr) and also rare BCR–ABL fusion transcripts has been recently reported [12]. It is well known that the common strategy for follow-up of patients with Phþ CML on a molecular level is based on characterization of the BCR–ABL fusion transcript at the time of diagnosis (by RT-PCR) and its subsequent quantitative assessment (by real-time quantitative RT-PCR). Therefore, it is evident that exact fusion determination plays a crucial role in the long-term monitoring of minimal residual disease (MRD) in these patients. Here we report on a ‘illustrative’ case where the molecular diagnostics covering mainly common BCR–ABL fusions failed. Moreover, within this case report, we highlight the importance of precise qualitative BCR–ABL fusion detection before quantitative monitoring.