Assessment of the Presence and the Level of BCR-ABL Fusion Gene Expression and Mutational Status in ABL Kinase Domain

Abstract Neoplastic cells bearing fusion genes that express an activated tyrosine kinase may set the scene for accumulation of genetic lesions by dysregulating DNA damage repair mechanisms and causing genetic instability. The observation that the BCR-ABL fusion gene alters pre-mRNA splicing in a variety of other genes including Ikaros and PYK2 supports this hypothesis. However, the only current evidence for acquired genetic change in the BCR-ABL gene itself is limited to finding mutations in the BCR-ABL kinase domain in patients treated with imatinib mesylate (IM). Here we report the observation that some patients with CML have abnormally small BCR-ABL transcripts both before and during treatment. Patients with sub-optimal response to IM are screened for mutations specifically within the BCR-ABL kinase domain by performing nested PCR, thereby excluding amplification of the non-translocated ABL allele. In the first round PCR amplification is performed across the fusion and the amplicons generated are subjected to a second round to yield an expected 863 bp (containing ABL exons 4 through 9 and thus the entire BCR-ABL kinase domain) PCR fragment. Smaller amplicons were observed in 49 (9.9%) of the 494 CML patients investigated. There was marked variation in the mRNA species when the abnormally small amplicons were subjected to direct sequencing; we found exon skipping, intra-exon splicing and insertion of intronic sequences. Similarly, in some cases the open reading frame was maintained whilst in others there were frame shifts leading to premature stop codons. The commonest finding, (22 of the 49 patients) was skipping of ABL exon 7 from codons 362 to 424, which includes the activation loop of the kinase domain. The smaller amplicons persisted even after the first round products had been diluted to 1:160. We also noted that the normal 863 bp fragment was present in some cases but was not detectable in others; its absence could reflect preferential amplification of the smaller transcripts. In a number of cases the same abnormally short amplicons were identified in the same patient studied serially on three or more separate occasions. We subsequently performed a second round of nested PCR with primers designed to amplify across the BCR-ABL junction such that the product included sequences from BCR 13 to ABL exon 9. With these new primers the detection frequency of abnormally small transcripts was increased. Furthermore, we observed the smaller transcripts in all of the 12 patients tested prior to beginning IM therapy. We then sought to determine if the normal ABL allele was involved; in order to avoid amplifying the BCR-ABL kinase domain, we performed a single round of PCR and restricted the analysis to patients in complete cytogenetic remission (CCyR). Only the expected 863 bp amplicon was observed in cDNA samples from 19 CML patients in CCyR whose BCR-ABL/ABL ratios ranged from 0.01 to 0.98. Furthermore, the smaller amplicons were not observed in cDNA samples from 20 normal individuals. We conclude that these abnormalities may result either from aberrant alternative splicing or from spontaneous deletions, or from a combination of both mechanisms. They may be a manifestation of the genetic instability believed to be an integral feature of CML.

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Examination of Point Mutations within the ABL Kinase Domain of Bcr-Abl Fusion Gene in Chinese Patients with Chronic Myeloid Leukemia Who Develop Imatinib Resistance.

Blood ◽

10.1182/blood.v110.11.4573.4573 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4573-4573

Author(s):

Dongguang Yang ◽

Ri Zhang ◽

Honglin Gang ◽

Jian Gu ◽

Jian Nong Cen

Keyword(s):

Chronic Myeloid Leukemia ◽

Point Mutation ◽

Mutation Rate ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Point Mutations ◽

Kinase Domain ◽

Chinese Patients ◽

Single Mutation ◽

Abl Kinase

Abstract Objective: To investigate the point mutations within the ABL kinase domain (KD) of Bcr-Abl fusion gene in Chinese patients with chronic myeloid leukemia who develop IM resistance and investigate the mutation. Method: We collect a total of 17 bone marrow samples obtained from 11 patients who showed hematology resistance(n=7) or cytogenetic refractoriness(n=4). A long semi-nest PCR method was used to amplify the ABL KD of the Bcr-Abl fusion gene. After two cycles of PCR reaction, we get a fragment including 863 bases, purified and sequenced it. Result: The ABL point mutation was detected in 45.45% of patients. The mutation rate of hematology resistance is 57.14% and the mutation rate of cytogenetic refractoriness is 25%. In total, we find three point mutation in all of our patients tested, ie, G250E(2), E255K(1) and T315I(2). In patients who develop hematology resistance, the earliest time to detect mutation is 4 months later after the therapy of IM and the longest time to detect mutation is 9 months later after the therapy of IM. In patients who develop cytogenetic refractoriness, the time to detect mutation is 12 months later after the therapy of IM .For the patients whose samples are available, no single mutation happens before IM thearpy. Conclusion: There is high frequency of point mutations clustered within the adenosine triphosphate-binding region of Bcr-Abl fusion gene in Chinese patients with chronic myeloid leukemia, and the mutation is related to IM therapy. It’s good for patients to switch to another therapeutic strategy when the mutations were detected earlier. Figure Figure

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Multilevel Molecular Profiling to Dissect Resistance to Tyrosine Kinase Inhibitors in TEL/ABL Positive Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3637.3637 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3637-3637

Author(s):

Olga Zimmermannova ◽

Veronika Kanderova ◽

Daniela Kuzilkova ◽

Fridtjof Lund-Johansen ◽

Eliska Doktorova ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Kinase Inhibitors ◽

Fusion Gene ◽

Molecular Profiling ◽

Resistant Cell ◽

Resistant Cell Line ◽

Sensitive Cell Line ◽

Abl Kinase ◽

Tki Resistance

Abstract Acute lymphoblastic leukemias (ALLs) bearing TEL/ABL (ETV6/ABL1) fusion gene are generally rare but can be identified within the Ph-like (BCR/ABL-like) ALLs - a poor risk ALL subtype characterized by the presence of various molecular lesions activating kinase signaling. Identification of these druggable lesions brought the possibility to improve the outcome of Ph-like ALL by incorporation of targeted therapeuticals – e.g. tyrosine kinase inhibitors (TKI) – into the current treatment strategies. In vitro studies proved the efficiency of TKI to inhibit TEL/ABL kinase activity justifying TKI use in clinical practice. However, there is a lack of knowledge on potential development of TKI resistance and its mechanism in TEL/ABL-positive ALL. To address this issue we aimed to establish TKI resistant TEL/ABL-positive cell line and study the mechanism of acquired resistance. After a long-term exposure to gradually growing doses of imatinib mesylate we induced a stable pan-TKI resistance not resulting from the most common mechanisms described in BCR/ABL ALL such as ABL kinase domain mutations, fusion gene amplification or enhanced fusion gene expression/activity. To elucidate the molecular basis of the resistance we performed multilevel molecular profiling. Using high density whole genome SNP array we identified a single acquired copy number aberration in the resistant cell line - a 60 kb intragenic deletion in KDM6A gene encoding lysine-specific histone demethylase. However, this deletion did not result in expression of the predicted aberrant protein. Using whole exome sequencing we identified 25 non-synonymous single nucleotide variants (SNVs) within gene coding regions to be gained by the resistant cell line. Only half of these variants (12/25) affected genes transcribed in the studied cell line and were thus subjected to further verification by Sanger sequencing. Finally, 5 of the 12 SNVs were confirmed to be stably present and expressed in the resistant cell line. All the affected genes – AEBP1, GNB1, KMT2C, RREB1, STAM2 – are involved in important biological processes and have not been associated with TKI resistance yet. Gene expression profiling of multiple replicates of parental sensitive and induced resistant cell line identified a gene expression pattern associated with TKI resistance consisting of more than 500 genes with significantly changed expression. Further analysis of this pattern revealed broad changes in many unrelated biological pathways and processes, hence providing a limited potential to specifically focus the study direction. We further employed two independent methods to study the resistant cell line proteome. Using 2-dimensional electrophoresis of the whole cell protein lysates we detected a single differentially expressed protein spot present in the resistant compared to parental sensitive cell line. Strikingly, mass spectrometry identified this protein as a transducin beta chain, product of the aforementioned GNB1 gene, one of the five beta subunits of human G-proteins playing an important role as signal transmitters. Using Size-Exclusion Chromatography-Microsphere-based Affinity Proteomic array (SEC-MAP; Wu, MCP 2009) we analyzed the total expression of more than 300 proteins by flow cytometry. We have found 39 proteins to be differentially expressed in resistant compared to parental sensitive cell line. Interestingly, several distinctive changes indicate enhanced B-cell receptor signaling in the resistant cell line. This mechanism was already implicated as a potential strategy of resistant cells for evading TKI induced apoptosis in BCR/ABL-positive leukemia (Klein, Cell Cycle 2004). In conclusion we have successfully used several high-throughput technologies to study genomic, transcriptomic and proteomic changes associated with acquired TKI resistance in TEL/ABL-positive ALL model and identified dysregulation of G-protein and BCR-signaling as its potential causes. Support: IGA MZ NT/13170-4; GAUK 694414; GAUK 596912; UNCE 204012; RVO-FNM64203; BIOCEV –(CZ.1.05/1.1.00/02.0109) from the ERDF Disclosures No relevant conflicts of interest to declare.

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the Relation of common ABL kinase domain mutations with resistance to Tyrosine Kinase Inhibiters in patients with Chronic Myeloid Leukemia in Middle Euphrates of Iraq

International Journal of Scientific Research and Management ◽

10.18535/ijsrm/v8i02.mp01 ◽

2020 ◽

Vol 8 (02) ◽

pp. 284-292

Author(s):

Mohammed Sadeq Mahdi Al- Musawi ◽

Raheem Mahdy Raheem ◽

Liwaa Hussein Mahdi ◽

Israa Ketab Al-Yasiri

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Significant Risk ◽

Kinase Domain ◽

Control Group ◽

Abl Kinase ◽

Kinase Domain Mutations ◽

Risk Association

Background : Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease, associated with a reciprocal translocation between chromosomes 9 and chromosome 22, lead to the formation of the BCR-ABL fusion gene (Philadelphia chromosome). This fusion gene is believed to play golden role in the initial development of CML with constitutive tyrosine kinase activation. Successful use of tyrosine kinase inhibiters (TKIs) play a role in improve survival and increase prevalence of CML, but un fortunately mutations in the BCR-ABL kinase domain may cause, or contribute to increase, resistance to TKIs in CML patients. . Objective: This study was designed to assess the association of five most common BCR-ABL kinase domain mutations (T315I, M351T, E255K, M244V and E255V) with resistance state of CML patients on TKIs in Iraqi Middle Euphrates region. Patients and methods: A retrospective case-control study in which 85 patients with chronic myeloid leukemia in chronic phase (45 patients as cases group and 40 patient as control group) were selected from three hemato-oncology centers in middle Euphrates in Iraq during the period from January 2016 till October 2016 out of a total of 240 CML patients ( 108 male and 132 female) who were registered during this period in these three centers and all patients on TKI (Imatinib and Nilotinib). Venous blood sampling done for BCR-ABL kinase domain mutations screening. Results: four patients from cases group (4/45) were carriers of one of five selected ABL kinase domain mutations and no one of control group. T315I mutation was detected in 3/45 (6.6 %) of resistant patients, with a significant risk association to develop resistance to TKI therapy (odd ratio and C. I. ) (6.67, 0.3340 -133.2255). E255V was detected in 1/45 ( 2.2 %) and also had significant risk association to develop resistance to TKIs( odd ratio, C.I.) (2.73, 0.1081 -68.9424). No one of these mutation had significance correlation with demographic or hematological features. M351T, E255K and M244V were not detected in any one of our study groups CML patients. Conclusions: T315I and E255V among five ABL kinas domain mutations were detected in our CML patients with resistance to TKIs. All of them may play a role in development variable degree of resistance to first and second generation TKIs weather primary or secondary.T315I mutation is most common mutation within BCR-ABL domain kinase gene.

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Use of Direct Sequence PCR for ABl Kinase Mutations in Patients with CML Blast Crisis, Treated Prior to the Availability of Imatinib Therapy.

Blood ◽

10.1182/blood.v106.11.2002.2002 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2002-2002

Author(s):

Sara Ghorashian ◽

Anna Babb ◽

Jamshid Khorasan ◽

Jaspal Kaeda ◽

David Marin ◽

...

Keyword(s):

Nested Pcr ◽

Mutational Analysis ◽

Fusion Gene ◽

Limit Of Detection ◽

Blast Crisis ◽

Kinase Domain ◽

Imatinib Therapy ◽

Abl Kinase ◽

Major Teaching Hospital ◽

Major Teaching

Abstract We identified all patients who developed blast crisis before the advent of imatinib therapy and in whom stored marrow samples were available from a database of CML patients in a major teaching hospital. mRNA was extracted and cDNA synthesized successfully in 20 of 22 cases. The cDNA was subjected to real time PCR for quantification of BCR-ABL transcripts. The ABL allele not involved in the t(9;22) translocation was excluded from mutational analysis by subjecting the cDNA to nested PCR. This was achieved by amplifying from exons 13 and 10 of the BCR and ABL moieties of the BCR-ABL fusion gene using primers B2A and JAMR, respectively. The resulting amplicon was then subjected to nested PCR using primers, NTPB+ and NTPE-, sited within exons 4 and 10 of the ABL gene. The nested PCR yielded an 863 bp fragment in length containing the entire BCR-ABL kinase domain. To test for successful amplification an aliquot of the PCR products were electrophoresed through 2.0% agarose gel. In all cases, a single amplicon was observed and the PCR reaction subjected to magnetic purification. The purified amplicon were then sequenced by Sanger’s dideoxy chain termination reaction using Big-Dye ABI 310 sequencer (Applied Biosystems, Foster City, USA). In each case, the sequence obtained was compared with the published ABL type 1a sequence, Genbank M14752, using Blast 2 software. No mutations were found in any patient. This suggests the absence of a mutated kinase in a proportion of greater than approximately 30% of the total BCR-ABL kinase, due to the limit of detection of the technique. Our results suggest that the predominant form of BCR-ABL in the setting of blast crisis (in the absence of imatinib) is wild type. This goes against theories that kinase mutations confer an intrinsic ‘gain of function’ or a proliferative advantage to a CML clone. Instead, it is likely that mutant forms of BCR-ABL which are relatively resistant to imatinib binding gain prominence through positive selection with the presence of imatinib within the cell milieu.

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Faculty Opinions recommendation of The kinase domain of titin controls muscle gene expression and protein turnover.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025137.298741 ◽

2005 ◽

Author(s):

Lars-Eric Thornell

Keyword(s):

Gene Expression ◽

Protein Turnover ◽

Kinase Domain ◽

Muscle Gene Expression ◽

Muscle Gene

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Programmed cell death 4 and BCR-ABL fusion gene expression are negatively correlated in chronic myeloid leukemia

Oncology Letters ◽

10.3892/ol.2016.4942 ◽

2016 ◽

Vol 12 (4) ◽

pp. 2976-2981 ◽

Cited By ~ 2

Author(s):

Xia Zhang ◽

Riming Liu ◽

Baohua Huang ◽

Xiaolu Zhang ◽

Weijuan Yu ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

Chronic Myeloid Leukemia ◽

Programmed Cell Death ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Programmed Cell Death 4

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Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia

Journal of Experimental Medicine ◽

10.1084/jem.194.11.1639 ◽

2001 ◽

Vol 194 (11) ◽

pp. 1639-1648 ◽

Cited By ~ 766

Author(s):

Andreas Rosenwald ◽

Ash A. Alizadeh ◽

George Widhopf ◽

Richard Simon ◽

R. Eric Davis ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Germ Line ◽

Gene Expression Signature ◽

Lymphocytic Leukemia ◽

Common Mechanism ◽

Human Leukemia ◽

Mutational Status ◽

Cell Chronic Lymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

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BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet

Blood ◽

10.1182/blood-2010-12-326405 ◽

2011 ◽

Vol 118 (5) ◽

pp. 1208-1215 ◽

Cited By ~ 332

Author(s):

Simona Soverini ◽

Andreas Hochhaus ◽

Franck E. Nicolini ◽

Franz Gruber ◽

Thoralf Lange ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Direct Sequencing ◽

Chronic Phase ◽

Kinase Domain ◽

Abl Kinase

AbstractMutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.

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