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Myeloproliferative neoplasms (MPN) are a group of clonal disorders of hematopoietic stem cells, and JAK2 V617F gene mutation is the main basis for the diagnosis of MPN. Previous studies have shown that BCR-ABL fusion gene and JAK2 V617F gene mutation are mutually exclusive in MPN patients, but in recent years, patients with a double mutation of both genes are often reported. The article synthesizes the relevant domestic and foreign literature in recent years, and reviews the BCR-ABL fusion gene and JAK2 V617F mutation double-positive MPN.
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Introducción: La leucemia mieloide crónica es un desorden clonal maligno de células madres hematopoyéticas pluripotentes que se caracteriza por la presencia del cromosoma Filadelfia, consecuencia de la traslocación cromosómica recíproca entre los brazos largos de los cromosomas 9 y 22. El resultado de esta alteración cromosómica es un gen de fusión que contiene las uniones b2a2 (e13a2) o b3a2 (e14a2). En la mayor parte de los casos, las células de la leucemia mieloide crónica expresan uno de los dos transcritos (b2a2 o b3a2); sin embargo, el 5 por ciento de los pacientes tienen ambos tipos de ARNm como resultado de empalmes alternativos. Se han encontrado otros transcriptos como e19a2, e2a2, e1a3, e6a2, e13a3(b2a3), y e14a3(b3a3), que ocurren con menos frecuencia. Objetivo: Describir el comportamiento de dos pacientes con leucemia mieloide crónica que presentan un trascripto BCR/ABL atípico. Casos clínicos: En el estudio molecular por reacción en cadena de la polimerasa cualitativo realizado a los dos pacientes, se observó un punto de ruptura del gen de fusión BCR/ABL poco frecuente, el cual se correspondía al transcripto e14a3 (b3a3). Estos pacientes iniciaron tratamiento con mesilato de imatinib a dosis de 400 mg diarios. Al primer paciente a los dos meses de tratamiento se le detectó crisis blástica, por lo que se le cambió el tratamiento a nilotinib 400 mg diarios que mantiene hasta la actualidad. La segunda paciente mantuvo igual tratamiento, aunque en ocasiones ha sido necesario incorporar tratamiento citorreductor con hidroxiurea por presentar leucocitosis. Conclusiones: Los pacientes con BCR/ABL a3 presentan un curso más benigno de la enfermedad. Aunque en los pacientes estudiados no se observó una respuesta satisfactoria al tratamiento pues presentaron diversas complicaciones(AU)
Introduction: Chronic myeloid leukemia is a malignant clonal disorder of pluripotent hematopoietic stem cells and characterized by the presence of the Philadelphia chromosome, which is the product of a reciprocal translocation between the long arms of chromosomes 9 and 22. The result of this chromosomal alteration is a fusion gene that contains the e13a2 (b2a2) and e14a2 (b3a2) junctions. In most cases, chronic myeloid leukemia cells express one of the two transcripts (b2a2 or b3a2); however, 5 percent of patients have both types of mRNA, as a result of alternative junctions. Other transcripts have been identified, such as e19a2, e2a2, e1a3, e6a2, e13a3 (b2a3), and e14a3 (b3a3), which occur less frequently. Objective: To describe the behavior of two patients with chronic myeloid leukemia who have an atypical BCR-ABL transcript. Clinical cases: In a qualitative molecular study of polymerase chain reaction carried out with two patients, a BCR-ABL fusion gene breakpoint was observed, which corresponded to the e14a3 (b3a3) transcript. These patients started treatment with imatinib mesylate at a dose of 400mg/d. At two months, the first patient had a diagnose of blast crisis, so the treatment was changed to nilotinib at a dose of 400mg/d, which the patient maintained to date. The second patient maintained the same treatment, although it was sometimes necessary to incorporate cytoreductive treatment with hydroxyurea due to leukocytosis. Conclusions: Patients with BCR-ABL a3 present a more benign evolution of the disease. However, a satisfactory response to treatment was not observed in the patients studied, as long as they presented various complications(AU)
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Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , CubaRESUMO
Objective@#To investigate the clinical features and treatment of child patient with chronic myeloid leukemia (CML) and T315I mutation in the ABL1 kinase domain.@*Methods@#The clinical features, diagnosis and treatment of one child CML patient with T315I mutation in ABL1 kinase domain in Fujian Medical University Union Hospital were retrospectively analyzed, and the literature was reviewed.@*Results@#The patient was treated with imatinib and dasatinib. The BCR-ABLIS value decreased and then increased. The disease progressed to the accelerated phase. At the same time, the T315I mutation was detected in the ABL1 kinase domain, the harringtonine chemotherapy was used, and the condition of patient got better. But eventually the hematopoietic stem cell transplantation could not be performed, the CML progressed to the blast phase and the patient died half a year later.@*Conclusions@#The prognosis of children with CML and T315I mutation in ABL1 kinase domain is poor. In the absence of punatinib treatment, hematopoietic stem cell transplantation should be performed as soon as possible after chemotherapy, which may improve the prognosis.
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Objective To investigate the clinical features and treatment of child patient with chronic myeloid leukemia (CML) and T315I mutation in the ABL1 kinase domain. Methods The clinical features, diagnosis and treatment of one child CML patient with T315I mutation in ABL1 kinase domain in Fujian Medical University Union Hospital were retrospectively analyzed, and the literature was reviewed. Results The patient was treated with imatinib and dasatinib. The BCR-ABLIS value decreased and then increased. The disease progressed to the accelerated phase. At the same time, the T315I mutation was detected in the ABL1 kinase domain, the harringtonine chemotherapy was used, and the condition of patient got better. But eventually the hematopoietic stem cell transplantation could not be performed, the CML progressed to the blast phase and the patient died half a year later. Conclusions The prognosis of children with CML and T315I mutation in ABL1 kinase domain is poor. In the absence of punatinib treatment, hematopoietic stem cell transplantation should be performed as soon as possible after chemotherapy, which may improve the prognosis.
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OBJECTIVE: To provide reference for reasonable selection of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients with BCR-ABL35INS mutation. METHODS: Using “BCR-ABL insertional mutation” “ABL1 35ins mutation” “BCR-ABL c.1423_1424ins35” “ABL1 p.C475Tyrfs*11” as keywords, retrieved from CNKI, Wanfang database, Medline and COSMIC database, BCR-ABL35INS mutation CML patients were summarized and analyzed in respects of general information and treatment (treatment plan, patient compliance and drug withdrawal), therapeutic effect (molecular biological mitigation and disease progress) and safety data (ADR) during 2007-2018. RESULTS: Totally 9 related literatures were included, involving 70 patients with BCR-ABL35INS mutation, all of them were foreign cases. Among them, 39 cases were male and 31 cases were female, with a median age of 49.2 years. The median time from the diagnosis of CML to the detection of BCR-ABL35INS mutation was 19 months. After detecting gene mutation, 39 cases were treated with imatinib (initial dose of 400 mg, po, once a day), and molecular biological remission was achieved in 5 patients (12.9%); 15 cases (38.5%) had molecular biological response but had disease progression; 8 patients (20.5%) had no response. Seventeen patients were treated with dasatinib (100 mg, po, once a day or 2 divided dose), and 8 cases (47.1%) achieved molecular biological response. Twenty-one patients were treated with nilotinib (400 mg, po, 2 divided dose), and 3 patients (14.3%) achieved molecular biological response; 2 patients achieved molecular biological response, but the disease progressed. Seven, three and seven of these patients stopped taking drugs due to adverse reactions, accounting for 17.9%, 17.6% and 33.3% respectively. All the ADRs were classified as grade 3-4 of the National Cancer Institute’s Common Terminology Criteria for Adverse Events, and most of them were hematological toxicity. CONCLUSIONS: CML patients with BCR-ABL35INS mutation are less likely to achieve molecular response on imatinib therapy but are more sensitive to dashatinib. In the course of treatment, we should strengthen the monitoring of blood system and other related indicators to ensure the safety and effectiveness of drug use.
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Platycodin D(PD) has a significantly inhibitory effect on multiple malignant tumors, and can inhibit the proliferation of leukemia cells K562 and induce apoptosis. However, its effect in improving the sensitivity of drug-resistant cells to imatinib and their molecular mechanism remained unclear. To investigate the effect and mechanism of PD alone or combined with imatinib (IM) in inhibiting CML imatinib resistant cell line K562/R, the cell proliferation was examined by CCK8 assay to reveal the effect of PD on the inhibitory function of imatinib. Cell apoptosis was detected by Annexin V-FITC/PI double staining. Protein expressions of cleaved caspase-3, cleaved caspase-9, PARP, cleaved PARP, Bcr/abl, p-AKT and p-mTOR were detected by Western blot. The results showed that the inhibitory effect of PD combined with imatinib on the proliferation and apoptosis of K562/R cells was significantly higher than that of the control group and the single drug group. Protein expressions of cleaved caspase-3, cleaved caspase-9 and cleaved PARP were significantly up-regulated in the combination group, and protein expressions of PARP, Bcr/abl, p-AKT and p-mTOR were down-regulated. The results indicated that PD increased the sensitivity of drug-resistant cells to imatinib, and the inhibitory effect of PD combined with imatinib was significantly better than the single drug on cell proliferation, induction of apoptosis, inhibition of Bcr/abl protein and PI3K/AKT/mTOR signaling pathway.
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Humanos , Antineoplásicos , Farmacologia , Apoptose , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Mesilato de Imatinib , Farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva , Tratamento Farmacológico , Patologia , Saponinas , Farmacologia , Transdução de Sinais , Triterpenos , FarmacologiaRESUMO
The chronic myeloid leukemia( CML) is characterized by a cytogenetic abnormality.The BCR-ABL fusion gene encodes protein 210.With the rapid development of molecular biology and other technologies, the treatment of CML has made great progress.However,patients for TKI resistance,which cannot be tolerated,and TKI will not eliminate CML stem cells.Despite hematopoietic stem cell transplantation( HSCT) is recommended as first-line treatment,it is still faced with many problems.Therefore,to clear CML tiny residual lesions from Ph+malignantly clonal stem cells has become an urgent need,which is expected to be an effective method for CML patients to obtain permanent cure and long-term disease-free survival.In this paper,we review the main advances achieved in the treatment of CML.
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Objective To investigate the detection methods of atypical bcr-abl rearrangement with b3a3 fusion transcript,and to describe the characteristics of this fusion gene.Methods Karyotype analysis,FISH and RT-PCR were applied to detect the break point of bcr-abl fusion gene in a patient who was diagnosed as acute lymphoblastic leukemia.Results The karyotype of the patient was expressed as 45,XY,-7,t(9;22)(q34;q1 1).The translocation event in chromosome 9 and 22 could be successfully detected by FISH,and a rare bcr-abl rearrangement with b3a3 fusion transcript was detected by RT-PCR with specific primers.Conclusions The rare e14a3 (b3a3) fusion of bcr-abl gene is present in this patient.Clinical laboratories using commercial kits that do not cover such rare fusions are likely to generate false result,thereby declaring combination of various methods to detect fusion genes is necessary.More studies are needed to explore the function and significance of rare bcr-abl fusion genes.
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AIM: To observe the expression of β-catenin in patients with chronic myeloid leukemia (CML) at different disease phases, and to analyze the relationship between BCR-ABL and cytogenetic response to imatinib mesylate. METHODS: RT-PCR and Western blotting were used to detect β-catenin mRNA and protein expression in bone marrow mononuclear cells (BMMNCs) from 99 patients with CML. The association with BCR-ABL and BCR-ABL fusion was determined by FISH in 94 patients after one year treatment with imatinib mesylate, and the relationship between β-catenin and cytogenetic response to imatinib mesylate was analyzed. RESULTS: The expression of β-catenin was increased significantly in patients with blast crisis and accelerated phase (P<0.01), while the expression of β-catenin between normal person and chronic phase of CML patients was not statistically different (P>0.05). No significant relation between β-catenin and BCR-ABL expression (r=0.314, P>0.05) was observed. The expression of β-catenin was increased significantly in the patients who did not reach main cytogenetic remission (P<0.01). CONCLUSION: The patients in progression phases of CML over-express β-catenin. The expression of β-catenin is not significantly related to BCR-ABL expression, but related to the therapeutic response of imatinib. Beta-catenin may be involved in the mechanism of CML progression and could be used as a new therapeutic target.
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Objective To increase the sensitivity of residual leukemic cells detectin in chronic myelocytic leukemia(CML) patients with RT-PCR,the optional annealing temperature and PCR cycles were studied to confirm bcr-abl fused gene types,and bcr-abl mRNA transcripts were monitored by RQ-PCR to study the relation with CML at different phases. Methods Through changing the PCR conditions, the annealing temperature was measured from 55℃ to 60℃, and the number of reaction cycles was increased from 30 to 45.All 22 samples were examined, and bcr-abl mRNA transcripts were quantified by RQ-PCR kit. Results Bcr-abl fused gene types were found in 22 samples,of all 9 cases were b_2a_2 type, 13 cases were b_3a_2.When the annealing temperature was set for 58℃ and the number of reation cycles was set for 45,10~3 copies/ul standard samples was detected.18 samples were positive tested by RQ-PCR kit,and the value was between 10~2 to 10~6 copies/g.There were significant differce between the results of chronic phase samples and those of accelerated phase. Conclusions The RT-PCR is a reliable,sensitive and reproducible method of monitoring CML patients.The real-time RT-PCR is useful in evaluating leukemic burden,assessing response to treatment and predicating the prognosis of the disease.
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Blast crisis (BC) transformation in chronic myelogenous leukemia (CML) can involve each differentiation lineage of the hematopoietic system, i.e. granulocyte, monocyte, erythrocyte, megakaryocyte, and lymphocyte lineage. The lymphoid blast crisis (BC) leukemia cells usually belong to B-lineage, commonly having the phenotype of Pre-B stage of the B-lineage, in which cell-surface immunoglobulin (sIg) is not yet expressed. In contrast, T-lineage BC of CML is extremely rare. The objective of this study is to describe the fenotype, fusion transcript of bcr-abl, TdT, and cytoplasmic CD3 in T-lineage BC CML cases. Case report study. This report shows a simple summary of 4 cases of T-lineage BC of CML which have been collected in the phenotypic and genotypic analysis study for 17 years (1987-2004). In all cases, the chromosomal analysis revealed the presence of t(9;22)(q34;q11) at presentation. Cell surface analysis were done at diagnosis. Cases’ mononuclear cells stored as 10% DMSO were retrieved to be performed reverse transcription (RT) PCR BCR-ABL multiplex to demonstrate the presence of the fusion transcript of bcr-abl. RT-PCR was also performed for detecting the expression of cytoplasmic CD3ε and terminal deoxynucleotydil transferase (TdT). The results of cell surface antigen (CSA) at presentation showed that 1 case was CD7+, CD5-, and CD2-; 1 case CD7+, CD5+, and CD2-; and 2 cases CD7+, CD5+ and CD2+ indicating that all these T-lineage BC of CML cells show the phenotype of pre-(pro-) thymic stage phenotype. In the present study, two cases showed b2a2, one e1a2, and one negative bcr-abl transcript. The RT-PCR revealed the presence of CD3ε mRNA in all cases, and TdT mRNA in only one case. These results can constitute a basis for the future analysis of T-lineage BC of CML from now on.
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Leucemia Mielogênica Crônica BCR-ABL PositivaRESUMO
Objective To construct a recombinant eukaryotic expression plasmid of glycosylphosphatidyl inositol(GPI)-anchored bcr/abl and explore its expression at mRNA and protein level.Methods The gene fragment encoding bcr/abl was amplified by PCR using the plasmid containing the cDNA sequence of P210 as template and then inserted into a eukaryotic expression vector pBudCE4.1.The constructed recombinant plasmid pBudCE4.1-bcr/abl was identified by restriction analysis and DNA sequencing.Lymphocytes were isolated from human peripheral blood and their total RNA was extracted.The gene fragment encoding GPI was amplified by RT-PCR using the obtained RNA as template and was inserted into the constructed recombinant plasmid pBudCE4.1-bcr/abl in order to anchor GPI and bcr/abl.The constructed recombinant plasmid pBudCE4.1-bcr/abl-GPI was transfected into COS-7 cells,and the expressions of objective fragment were detected by RT-PCR and Western blotting.Results The results of restriction analysis,PCR and DNA sequencing proved that GPI-anchored bcr/abl fusion fragment was correctly inserted into vector pBudCE4.1.The expression of bcr/abl fusion gene and fusion protein were identified in transfected COS-7 cells and on their membrane.Conclusion The recombinant plasmid pBudCE4.1-bcr/abl-GPI was successfully constructed and expressed on the membrane of COS-7 cells,which found a basis of cell immunity with GPI-anchored bcr/abl fusion gene.
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Objective To construct the transformed mouse BaF3-P210 cell line stably expressing BCR/ABL and to initially investigate the influence of BCR/ABL on the cell biological characteristics of BaF3 cell line. Methods The retroviral vector with bcr/abl gene was transfected into the packaging cell line. The BaF3 cells were infected with the collected viral supernatant. The transgenic BaF3-P210 cell line stably expressing BCR/ABL were screened and subcloned. The integration of the bcr/abl gene in the genome of the target cell was determined by PCR and DNA sequencing,trypan blue staining assay,flow cytometry and MTT assay. Results The bcr/abl gene was integrated into the BaF3 cell genome; RT-PCR and Western blot verified the stable expression of the bcr/abl gene and protein in the screened subclone cell line BaF3-P210. Compared with the control group,the cell proliferation rate was promoted (P
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Objective To establish a quantitative RT-PCR method with self-quenched fluorogenic probe for detection of bcr/abl mRNA in patients with chronic myeloid leukemia for providing a useful tool for diagnosis of CML,evaluation of therapeutic effect and monitoring of minimal residual disease(MRD). Methods bcr/abl gene from cultured K562 cells was amplified by conventional RT-PCR.The standard quantitative plasmid was constructed by A-T clone method.The self-quenched fluorogenic quantitative RT- PCR method(FQ-RT-PCR)for determination of bcr/abl mRNA was established successfully using the ABI PRISM 7000 PCR Detector.The linear range,sensitivity,stability,and repetitiveness of the method were determined.The marrow samples from 25 CML patients and 3 ALL patients were assessed.Results The sensitivity of the FQ-RT-PCR was 10 copies/?l recombined plasmid,and bcr/abl mRNA can be detected from 1 K562 cell in 10~5 normal cells.The linear range was 10~2-10~9 copies/?l recombined plasmid.The coefficient variation(CV)value was 2.1% in intra-assay and 6.1% in inter-assay.The median ber/abl mRNA expression level was 4.50?10~4 copies/?g RNA [(0.45-89.00)?10~4],5.45?10~4 copies/?g RNA [(2.95-19.30)?10~4 ],13.00?10~4 copies/?g RNA [(4.10-89.00)?10~4] and 2.35?10~4 copies/?g RNA [(0.45-5.12)?10~4] in 25 CML patients,11 patients in the incipient chronic phase,6 patients in blastic crisis,8 patients in chronic period after treatment,respectively.The bcr/abl mRNA level in blastic crisis was significantly higher than that in chronic phase(q= 3.41,P
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0^05).In six patients(8 specimens)with complete cytogenetic remission(CCR)and partial cytogenetic remission(PCR),the Ph positive cells diminished remarkably as compared with that of untreated patients(26^3% vs 89^21%,P
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BACKGROUND: Three types of chimeric mRNA can be expressed as a result of fusion of abl gene on chromosome 9 and bcr gene on chromosome 22. These are b3a2, b2a2, and e1a2. Rearranged mRNA of bcr-abl fusion gene is found in majority of chronic myelogenous leukemia (CML) and in 20% of acute lymphoblastic leukemia (ALL) and in other hematologic malignancies. METHODS: We have examined the chimeric mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) since 1994. Positive results for the chimeric mRNA were 167 (118 patients) out of 714 examined cases. 94 patients with CML irrespective of the disease course, 49 with ALL, 120 with acute myelogenous leukemia, 11 with acute mixed leukemia, and 7 with myelodysplastic syndrome were included. RESULTS: 97.9% (92/94) of patients with CML, 40% (20/49) of ALL, 45% (5/11) of acute mixed leukemia, and 14% (1/7) of myelodysplastic syndrome were positive for bcr-abl rearrangement. In CML, positive cases were with b3a2 (64%), b2a2 (32.6%), and b3a2/b2a2 (2.2%). One patient with CML-like feature expressed e1a2 type mRNA. In adult ALL, 62.5% (10/16) expressed minor-bcr (e1a2). CONCLUSIONS: The bcr-abl fusion gene was positive in various hematologic diseases as well as CML and that fact indicates the dysfunction of primitive pleuripotent stem cells. Various expressions and changes of mRNA types in bcr-abl fusion gene might be due to alternative splicing mechanism and detection sensitivity of RT-PCR when two types were intermingled. We should be aware of these facts when interpretating the results.