- MeSH
- bcr-abl fúzové proteiny * MeSH
- lidé MeSH
- myeloproliferativní poruchy diagnóza genetika terapie MeSH
- směrnice pro lékařskou praxi jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- komentáře MeSH
- úvodníky MeSH
- Názvy látek
- bcr-abl fúzové proteiny * MeSH
Monitoring of BCR-ABL transcript level is widely used in chronic myeloid leukemia (CML) to follow up response to therapy. In this study we compare abilities of two quantitative RT-PCR assays to characterize the disease status in CML patients: RT-PCR quantifying the BCR-ABL transcript concentration and RT-PCR determining the BCR-ABL/BCR transcript ratio (R). We demonstrate that in non-responders only R, but not BCR-ABL, unambiguously characterizes the state of disease. Moreover, R values >1 found in all poor responders indicate lower BCR expression compared to BCR- ABL in these patients. Our results demonstrate the importance of BCR-ABL/BCR transcript ratio for the disease status and the disease prognosis characterization and suggest a possible role of the normal BCR gene expression in CML pathogenesis.
- MeSH
- chronická myeloidní leukemie genetika patologie MeSH
- dospělí MeSH
- geny abl * MeSH
- lidé středního věku MeSH
- lidé MeSH
- longitudinální studie MeSH
- nádorová transformace buněk * MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- prognóza MeSH
- progrese nemoci MeSH
- protoonkogenní proteiny c-bcr MeSH
- protoonkogenní proteiny biosyntéza MeSH
- senioři MeSH
- tyrosinkinasy biosyntéza MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- BCR protein, human MeSH Prohlížeč
- protoonkogenní proteiny c-bcr MeSH
- protoonkogenní proteiny MeSH
- tyrosinkinasy MeSH
BACKGROUND: The BCR::ABL1 is a hallmark of chronic myeloid leukemia (CML) and is also found in acute lymphoblastic leukemia (ALL). Most genomic breaks on the BCR side occur in two regions - Major and minor - leading to p210 and p190 fusion proteins, respectively. METHODS: By multiplex long-distance PCR or next-generation sequencing technology we characterized the BCR::ABL1 genomic fusion in 971 patients (adults and children, with CML and ALL: pediatric ALL: n = 353; pediatric CML: n = 197; adult ALL: n = 166; adult CML: n = 255 patients) and designed "Break-App" web tool to allow visualization and various analyses of the breakpoints. Pearson's Chi-Squared test, Kolmogorov-Smirnov test and logistic regression were used for statistical analyses. RESULTS: Detailed analysis showed a non-random distribution of breaks in both BCR regions, whereas ABL1 breaks were distributed more evenly. However, we found a significant difference in the distribution of breaks between CML and ALL. We found no association of breakpoints with any type of interspersed repeats or DNA motifs. With a few exceptions, the primary structure of the fusions suggests non-homologous end joining being responsible for the BCR and ABL1 gene fusions. Analysis of reciprocal ABL1::BCR fusions in 453 patients showed mostly balanced translocations without major deletions or duplications. CONCLUSIONS: Taken together, our data suggest that physical colocalization and chromatin accessibility, which change with the developmental stage of the cell (hence the difference between ALL and CML), are more critical factors influencing breakpoint localization than presence of specific DNA motifs.
- Klíčová slova
- ABL1, Acute lymphoblastic leukemia, BCR, Chronic myeloid leukemia, Genomic breakpoints,
- MeSH
- akutní lymfatická leukemie * genetika patologie MeSH
- bcr-abl fúzové proteiny * genetika MeSH
- body zlomu chromozomu * MeSH
- chronická myeloidní leukemie * genetika patologie MeSH
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- dopisy MeSH
- Názvy látek
- bcr-abl fúzové proteiny * MeSH
Many patients with chronic myeloid leukemia in deep remission experience return of clinical disease after withdrawal of tyrosine kinase inhibitors (TKIs). This suggests signaling of inactive BCR-ABL, which allows the survival of cancer cells, and relapse. We show that TKI treatment inhibits catalytic activity of BCR-ABL, but does not dissolve BCR-ABL core signaling complex, consisting of CRKL, SHC1, GRB2, SOS1, cCBL, p85a-PI3K, STS1 and SHIP2. Peptide microarray and co-immunoprecipitation results demonstrate that CRKL binds to proline-rich regions located in C-terminal, intrinsically disordered region of BCR-ABL, that SHC1 requires pleckstrin homology, src homology and tyrosine kinase domains of BCR-ABL for binding, and that BCR-ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of three core complex members, i.e., GRB2, SOS1, and cCBL. Further, SHIP2 binds to the src homology and tyrosine kinase domains of BCR-ABL and its inositol phosphatase activity contributes to BCR-ABL-mediated phosphorylation of SHC1. Together, this study characterizes protein-protein interactions within the BCR-ABL core complex and determines the contribution of particular BCR-ABL domains to downstream signaling. Understanding the structure and dynamics of BCR-ABL interactome is critical for the development of drugs targeting integrity of the BCR-ABL core complex.
- Klíčová slova
- BCR–ABL, Chronic myeloid leukemia, Protein complex, Signaling,
- MeSH
- adaptorové proteiny signální transdukční metabolismus MeSH
- aminokyselinové motivy MeSH
- bcr-abl fúzové proteiny chemie genetika metabolismus MeSH
- chronická myeloidní leukemie metabolismus patologie MeSH
- čipová analýza proteinů MeSH
- fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy metabolismus MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- pyrimidiny farmakologie MeSH
- signální transdukce * účinky léků MeSH
- src homologní domény MeSH
- transformující protein 1 obsahující src homologní doménu 2 metabolismus MeSH
- vazba proteinů účinky léků MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- bcr-abl fúzové proteiny MeSH
- CRKL protein MeSH Prohlížeč
- fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy MeSH
- inhibitory proteinkinas MeSH
- INPPL1 protein, human MeSH Prohlížeč
- nilotinib MeSH Prohlížeč
- pyrimidiny MeSH
- transformující protein 1 obsahující src homologní doménu 2 MeSH
Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.
- MeSH
- bcr-abl fúzové proteiny analýza normy MeSH
- geny abl MeSH
- kalibrace MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- protoonkogenní proteiny c-bcr genetika MeSH
- referenční standardy MeSH
- Světová zdravotnická organizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- BCR protein, human MeSH Prohlížeč
- bcr-abl fúzové proteiny MeSH
- protoonkogenní proteiny c-bcr MeSH
BACKGROUND: Molecular biology methods based on reverse transcription and polymerase chain reaction (RT-PCR) are able to detect the presence of BCR-ABL transcripts in chronic myeloid leukemia (CML). In this study we present our experience with monitoring of residual disease using real-time PCR with hybridization probes detection in patients treated with imatinib mesylate and in collected peripheral blood progenitor cells (PBPC). METHODS AND RESULTS: We measured the level of BCR-ABL transcripts in peripheral blood cells of 27 subjects before and in the course of the imatinib treatment. The median of relative quantity of BCR-ABL in the blood before imatinib therapy was 2.55%. The number of the transcripts in 23 imatinib-sensitive subjects decreased to 0.02% in 6 months. After 12 months of the treatment the BCR-ABL median was 0.005%. Subsequent levels fluctuated between values below the detection limit (DL, 0.001%) and 0.005%. Three patients were primarily resistant to imatinib with the BCR-ABL range of 0.13%-11.7% during the treatment. One subject showed marks of molecular relapse after 18 months of the treatment. Only two of 16 filgrastim-stimulated patients had BCR-ABL levels in the blood and in collected PBPC below DL. In other subjects BCR-ABL transcripts were determined within the measurable range of RT-PCR. CONCLUSIONS: Taking into account prognostic importance, the measurement of BCR-ABL transcripts is an effective approach to monitoring of residual CML kinetics. Evaluation of BCR-ABL levels in collected PBPC can complete information on quality of the cells in potential autotransplants, and choose subsequent therapeutic protocols and patient prognosis.
- MeSH
- antitumorózní látky terapeutické užití MeSH
- bcr-abl fúzové proteiny analýza genetika MeSH
- chemorezistence genetika MeSH
- chronická myeloidní leukemie diagnóza farmakoterapie genetika MeSH
- dospělí MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery analýza MeSH
- polymerázová řetězová reakce MeSH
- reziduální nádor MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- Názvy látek
- antitumorózní látky MeSH
- bcr-abl fúzové proteiny MeSH
- inhibitory proteinkinas MeSH
- nádorové biomarkery MeSH
Chronic myeloid leukemia (CML) is caused by constituve activity of BCR-ABL tyrosine kinase. Despite of high efficiency of imatinib, selective BCR-ABL inhibitor, about 30% of patients develop resistance. Novel markers and targets for therapy are thus necessary. MicroRNAs are small intereference RNAs whose role in physiological and malignant hematopoiesis has been shown. This study is focused on miR-451 in CML. Following our observation of miR-451 downregulation in CML, we further show its relation to BCR-ABL activity. Our data together with current literature indicate a more complex relationship of miR-451 and BCR-ABL in CML.
- MeSH
- bcr-abl fúzové proteiny antagonisté a inhibitory MeSH
- chronická myeloidní leukemie genetika MeSH
- geny abl fyziologie MeSH
- lidé MeSH
- mikro RNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bcr-abl fúzové proteiny MeSH
- mikro RNA MeSH
- MIRN451 microRNA, human MeSH Prohlížeč
We present two patients with Ph-negative chronic myeloid leukemia (CML) and fusion signal BCR/ABL on both chromosomes 9, located in region 9q34. The first case was a 27 years old man with CML. Molecular studies (RT-PCR) revealed the rearrangement in the major-BCR region and expression of chimeric BCR/ABL mRNA of b3a2 configuration. By classical cytogenetic studies (G-banding) karyotype 46,XY was found in short-term cultivated bone marrow cells and phytohemagglutinin (PHA) stimulated peripheral lymphocytes. FISH studies revealed the BCR/ABL fusion signals on both chromosomes 9 and green BCR signals on both chromosomes 22 in all mitoses studied. Detection of the alleles of ABL1 intragenic STR locus by fluorescence PCR followed by fragmentation analysis in the patient and his parents provided no information about transmission of the ABL gene. Quantitative assessment of BCR/ABL transcript level by RT-PCR showed 60 and 70% BCR/ABL positivity in two peripheral blood samples at 6,5 and 10,5 months after diagnosis, respectively, which does not correspond to the expression from two identical BCR/ABL hybrid genes. Therefore, the possible mechanism of the origin of two BCR/ABL fusion signals present on both chromosomes 9 could not be resolved and remains speculative. The second case was a 53 years old male with diagnosis of chronic phase of CML, with first sign of acceleration one month after diagnosis and death because of sepsis in blastic phase within four months. The cytogenetic findings were identical to those in case No. 1., i.e. karyotype 46, XY by G-banding, two BCR/ABL fusion signals on both chromosomes 9 and RT-PCR molecular studies proved b3a2 breakpoints. It is generally accepted that prognosis of the patients with fused BCR/ABL gene located on chromosome 9 is poor. The presence of two fused genes could be anticipated as two Ph chromosomes in accelerated and blastic phases of the disease. However, in our study, quantitative findings of BCR/ABL transcripts did not corresponded to the expression of two BCR/ABL genes originating from duplication. If this assumption is correct then the expression of both fused genes BCR/ABL was in case No. 1 equally suppressed and total expression reached about the level of one BCR/ABL gene.
- MeSH
- bcr-abl fúzové proteiny genetika MeSH
- chronická myeloidní leukemie genetika MeSH
- dospělí MeSH
- hybridizace in situ fluorescenční MeSH
- leukemie myeloidní chronická atypická BCR-ABL-negativní genetika MeSH
- lidé MeSH
- lidské chromozomy, pár 9 * MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bcr-abl fúzové proteiny MeSH
BACKGROUND AND OBJECTIVE: The availability of different tyrosine kinase inhibitors (TKIs) with distinct anti-leukemic 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. METHODS: Chronic 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. RESULTS: Our 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. CONCLUSION: On 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.
- MeSH
- antitumorózní látky terapeutické užití MeSH
- bcr-abl fúzové proteiny genetika MeSH
- chronická myeloidní leukemie farmakoterapie genetika MeSH
- dospělí MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- mutace MeSH
- senioři MeSH
- tyrosinkinasy genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antitumorózní látky MeSH
- bcr-abl fúzové proteiny MeSH
- inhibitory proteinkinas MeSH
- tyrosinkinasy MeSH
The fact of chromosome 9 and 22 translocation connected with the fusion of BCR and ABL genes occurring in 95% patients with chronic myeloid leukemia (CML) enables us to use molecular biology methods in CML diagnosis. By means of these methods we also can prove the rearrangement of BCR gene in some cytogenetically negative cases that are without so called Philadelphia (Ph) chromosome. 51 patients with myeloproliferative disease have been tested by Southern technique during the last year. The rearrangement of BCR gene was detected in 28 patients, in 13 of 14 patients where the Ph chromosome and also in 3 of 13 patients where the Ph chromosome was not detected. The detection of BCR gene rearrangement helped us to set the diagnosis of CML more precisely.
