Tomáš Jurček

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.

Constant BCR-ABL transcript level ≥0.1% (IS) in patients with CML responding to imatinib with complete cytogenetic remission may indicate mutation analysis

OBJECTIVE Of 140 chronic myeloid leukemia patients responding to imatinib with complete cytogenetic remission, 32 exhibited a plateau of BCR-ABL values at >or=0.1% level in a minimum of three subsequent samples (minimal duration, 6 - 9 months). Median follow-up of unchanged BCR-ABL transcript level was 12 months (range, 6 - 64). We tested this group of patient for BCR-ABL mutations to reveal resistance development and to evaluate the risk of disease progression. MATERIALS AND METHODS Altogether, 134 samples of peripheral blood of these 32 patients were tested for mutation in BCR-ABL kinase domain. RESULTS Mutation was detected by direct sequencing in 9 of 32 patients (28%). Loss of complete cytogenetic remission or 1 log rise of BCR-ABL was observed in five of nine patients at a median of 5 months (range, 4-17) since first detection of mutation. One patient with no mutation relapsed 12 months after the start of the BCR-ABL plateau. In 5 of 32 patients without mutation (16%), BCR-ABL level significantly decreased after the first plateau to levels that stayed unchanged for a median of 11 months (range, 7-28). CONCLUSION We show here that the BCR-ABL constant levels >or=0.1% (BCR-ABL plateau) in imatinib-responding patients may indicate mutation analysis. This approach highly reduces the number of examinations for mutation in chronic myeloid leukemia responders and may present cost-effective alternative applicable in clinical practice.

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.

A Case of a Novel PML/RARA Short Fusion Transcript with Truncated Transcription Variant 2 of the RARA Gene

Acute promyelocytic leukemia (APL) with atypical breakpoints in the promyelocytic leukemia (PML) and retinoic acid receptor-α (RARA) genes represents a rare leukemic event, which occurs preferentially in patients with variant types of the PML/RARA fusion gene. Here we report on a patient with APL with a unique PML/RARA fusion transcript that harbors a short type of this fusion gene, exhibiting unexpected results of standard PCR diagnostics. The detected transcript originates from fusion of PML exon 4 and a truncated form of transcription variant 2 of the RARA gene, with an additional 9 bp insertion. According to our knowledge, this differs from all previously described fusion transcripts.

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.

CEBPA gene mutational status: a complete screening using high-resolution melt curve analysis.

In recent years, several independent prognostic factors in cytogenetically normal acute myeloid leukemia (CN-AML) have been reported. Mutations or the expression levels of certain genes have been often used as molecular markers for prediction of a patients outcome or for evaluation of treatment outcome. One of them, the gene encoding CCAAT/enhanced binding protein alpha (CEBPA), plays an important role in myeloid differentiation and, when mutated, confers a favorable prognosis for patients with CN-AML. Complete mutation screening of the CEBPA gene is therefore beneficial and requires fast, precise, and sensitive diagnostic tools. Thus, for routine diagnostics, we developed a screening method using high-resolution melt curve analysis prior to direct sequencing, where only positive samples (according to reference) are further sequenced. With this approach, all positive and negative patients were successfully distinguished, and the results obtained were in absolute concordance with the direct sequence analysis.

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.

BCR–ABL activity measured by 50% inhibitory concentration for imatinib, p-CrkL/CrkL ratio or p-CrkL ratio in CD34+ cells of patients with chronic myeloid leukemia does not predict treatment response

In this study, we assessed BCR-ABL kinase activity by measuring the protein levels of CrkL and its phosphorylated form (p-CrkL) in order to predict the clinical outcome of newly diagnosed chronic myeloid leukemia patients. CD34+ cells from these patients were collected before the start of imatinib therapy and treated in vitro with imatinib. The reduction of p-CrkL and CrkL protein levels was then measured by flow cytometry. The data were processed using three independent approaches: an assessment of a) IC50imatinib, b) p-CrkL/CrkL ratio, and c) p-CrkL ratio. The results were subsequently correlated with the clinical response of patients after 3 and 6 months of imatinib therapy. None of the three p-CrkL parameters measured in CD34+ cells was found to be predictive of clinical outcome.

Prospective analysis of low-level BCR - ABL1 T315I mutation in CD34 cells of patients with de novo chronic myeloid leukemia

Th e detection of BCR – ABL1 kinase domain (KD) mutations is frequently associated with resistance to tyrosine kinase inhibitors (TKIs), which results in an impaired prognosis for patients with chronic myeloid leukemia (CML) [1]. Early detection of these mutations can potentially lead to early therapeutic intervention and optimization of an ongoing treatment strategy. Considering the hematopoiesis hierar- chy, it is expected that BCR – ABL1 mutation clones expand directly from hematopoietic stem cells or early progenitor cells [2]. It has already been reported that BCR – ABL1 KD mutations were detected in these cells before they occurred in bone marrow (BM) or peripheral blood (PB) [3]. Particu- lar focus should be given to the T315I mutation, which is resistant to all approved TKIs (imatinib, nilotinib and dasa- tinib) [4,5], meaning that early detection of this key BCR – ABL1 KD mutation in “ source ” cells could have potential clinical benefits.