Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients

博士 === 慈濟大學 === 醫學研究所 === 98 === The BCR-ABL fusion protein is a constitutively activated tyrosine kinase that plays a central role in the pathogenesis of chronic myeloid leukemia (CML) and Philadelphia (Ph) chromosome-positive acute lymphoid leukemia (ALL). The aberrant tyrosine kinase activity of...

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Main Authors: JU-HUEI CHIEN, 簡如慧
Other Authors: Chan-Ping Lee
Format: Others
Language:en_US
Published: 2010
Online Access:http://ndltd.ncl.edu.tw/handle/11499581818752295819
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spelling ndltd-TW-098TCU055340072016-04-22T04:23:29Z http://ndltd.ncl.edu.tw/handle/11499581818752295819 Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients 慢性骨髓性白血病之分子診斷與Imatinib抗藥性基因突變偵測之研究 JU-HUEI CHIEN 簡如慧 博士 慈濟大學 醫學研究所 98 The BCR-ABL fusion protein is a constitutively activated tyrosine kinase that plays a central role in the pathogenesis of chronic myeloid leukemia (CML) and Philadelphia (Ph) chromosome-positive acute lymphoid leukemia (ALL). The aberrant tyrosine kinase activity of BCR-ABL induces cellular transformation and leukemogenesis. Targeting the tyrosine kinase activity of BCR-ABL is a very promising therapeutic strategy in treating these disorders. Imatinib is a tyrosine kinase inhibitor that has been shown a very effective therapy in patients with chronic phase CML. Although most patients with CML in the blast-crisis phase are initially responsive to Imatinib, majority of the patients relapse and develop resistance to this inhibitor. Studies have shown that many relapsed patients carry mutations in the ABL gene that increase the level of autophosphorylation of BCR-ABL kinase and promote the downstream signal transduction pathway. In order to understand the molecular mechanisms by which ABL gene mutations induce Imatinib resistance, we investigated 19 patients (18 CML patients and 1 Ph (+) ALL patient) who either relapsed or showed no response after Imatinib treatment. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, denaturing high performance liquid chromatography (dHPLC), and direct DNA sequencing to analyze all possible mutations in exons 5 to 9 of the ABL gene. We also used quantitative PCR to monitor minimal residual disease of those patients. Various mutations were found in exons 5, 6, and 7 of the ABL gene in 5 of 19 patients with CML. The patient with Ph (+) ALL had a GAG→AAG (E255K) mutation in exon 5 and a ACT→ATT (T315I) mutation in exon 7, a finding consistent with the literatures reported previously. The E255K substitution had a G to A change, and the T315I substitution had a C to T change in the ABL gene. Other unique mutations found in this study include TAC→CAC (Y253H), ATG→ACG (M351T), CTC→CCC (L213P) and AAG tri-nucleotide insertion. In summary, both quantitative monitoring of residual tumor cells and mutation detection are useful to predict outcome during Imatinib treatment. The different mutations are likely to interfere the Imatinib binding ability, and may alter intrinsic kinase activity by changing the protein structure conformation or phosphorylation status. The alteration of BCR-ABL kinase activity will count for the different transformation potency, which conferred the drug resistant and directly contribute to the relapse and disease progression. It is important to identify the gene mutations, which can provide a new insight into kinase function and better drug treatment strategies to overcome the drug resistance. Chan-Ping Lee 李展平副教授 2010/07/ 學位論文 ; thesis 127 en_US
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description 博士 === 慈濟大學 === 醫學研究所 === 98 === The BCR-ABL fusion protein is a constitutively activated tyrosine kinase that plays a central role in the pathogenesis of chronic myeloid leukemia (CML) and Philadelphia (Ph) chromosome-positive acute lymphoid leukemia (ALL). The aberrant tyrosine kinase activity of BCR-ABL induces cellular transformation and leukemogenesis. Targeting the tyrosine kinase activity of BCR-ABL is a very promising therapeutic strategy in treating these disorders. Imatinib is a tyrosine kinase inhibitor that has been shown a very effective therapy in patients with chronic phase CML. Although most patients with CML in the blast-crisis phase are initially responsive to Imatinib, majority of the patients relapse and develop resistance to this inhibitor. Studies have shown that many relapsed patients carry mutations in the ABL gene that increase the level of autophosphorylation of BCR-ABL kinase and promote the downstream signal transduction pathway. In order to understand the molecular mechanisms by which ABL gene mutations induce Imatinib resistance, we investigated 19 patients (18 CML patients and 1 Ph (+) ALL patient) who either relapsed or showed no response after Imatinib treatment. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, denaturing high performance liquid chromatography (dHPLC), and direct DNA sequencing to analyze all possible mutations in exons 5 to 9 of the ABL gene. We also used quantitative PCR to monitor minimal residual disease of those patients. Various mutations were found in exons 5, 6, and 7 of the ABL gene in 5 of 19 patients with CML. The patient with Ph (+) ALL had a GAG→AAG (E255K) mutation in exon 5 and a ACT→ATT (T315I) mutation in exon 7, a finding consistent with the literatures reported previously. The E255K substitution had a G to A change, and the T315I substitution had a C to T change in the ABL gene. Other unique mutations found in this study include TAC→CAC (Y253H), ATG→ACG (M351T), CTC→CCC (L213P) and AAG tri-nucleotide insertion. In summary, both quantitative monitoring of residual tumor cells and mutation detection are useful to predict outcome during Imatinib treatment. The different mutations are likely to interfere the Imatinib binding ability, and may alter intrinsic kinase activity by changing the protein structure conformation or phosphorylation status. The alteration of BCR-ABL kinase activity will count for the different transformation potency, which conferred the drug resistant and directly contribute to the relapse and disease progression. It is important to identify the gene mutations, which can provide a new insight into kinase function and better drug treatment strategies to overcome the drug resistance.
author2 Chan-Ping Lee
author_facet Chan-Ping Lee
JU-HUEI CHIEN
簡如慧
author JU-HUEI CHIEN
簡如慧
spellingShingle JU-HUEI CHIEN
簡如慧
Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients
author_sort JU-HUEI CHIEN
title Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients
title_short Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients
title_full Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients
title_fullStr Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients
title_full_unstemmed Molecular diagnosis in chronic myeloid leukemia and mutation detection in Imatinib resistant patients
title_sort molecular diagnosis in chronic myeloid leukemia and mutation detection in imatinib resistant patients
publishDate 2010
url http://ndltd.ncl.edu.tw/handle/11499581818752295819
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