Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Mutation

OBJECTIVETo revalidate the conversion factor（CF）for the conversion of BCR-ABL （P210）transcript levels to the international scale（BCR- ABLIS）in chronic myeloid leukemia（CML） which validated before.

METHODSPeking University People's Hospital（PKUPH）prepared the exchange samples for revalidation of CFs of 15 laboratories which validated nine or eighteen months ago. The fresh BCR-ABL（P210）（+）bone morrow or peripheral blood nucleated cells were diluted with BCR-ABL （P210）（-）cells to achieve different BCR- ABL levels, totally 16 sets and 24 samples per set were prepared. TRIzol reagent was added in each tube. Each laboratory tested BCR-ABL transcript levels of one set of samples. Agreement between BCR-ABLIS of each laboratory and PKUPH was assessed by the Bland- Altman method. For laboratories which did not meet the criteria of revalidation, linear regression equation was derived after the samples with maximum BCR-ABL deviation were removed until R²>0.98, then new CF was calculated.

RESULTS10 laboratories met the revalidation criteria with both bias within ±1.4 fold and 95% limits of agreement within ±6 folds, and their CFs still could be used for accurately conversion of BCR-ABLIS. New CFs were recalculated as of 1.8-6.3 folds of their previous CFs in 5 laboratories not met the criteria.

CONCLUSIONRevalidation of CF by sample exchange among laboratories was necessary for accurate and continuous application of BCR-ABLIS, which not only tested the validity of CF acquired before but also calculated new available CFs for those with invalid CFs.

OBJECTIVE: To analyze the significance of various abnormal signal patterns appreared in CML and B-ALL patients by using BCR/ABL/ASS1 tricolor dual-fusion probe, and to explore its application value in detecting BCR/ABL fusion gene and ASS1 gene deletion. METHODS: 50 newly diagnosed CML patients and 50 newly diagnosed B-ALL patients were detected by fluorescence in situ hybridization (FISH) with BCR/ABL/ASS1 tricolor dual-fusion probe. Meanwhile, karyotype analysis was performed on all the patients using the 24 hours short-term culture and R-banding. RESULTS: Among the 50 CML patients, Ph was found in 49 cases, 5 normal interphase karyotype was observed in 1 case. FISH detection showed that BCR/ABL fusion gene existed in all patients (100%), while the positive signal pathway showed that 1R1G2B2F was observed in 39 cases (78%), 2R1G2B1F in 2 cases (4%) and 1R1G2B1F in 6 cases (12%), simultaneous existence of 1R1G1B1F and 1R1G2B3F in 1 case (2%), 2R1G1B1F in 1 case (2%) 1R1G3B3F in 1 case (2%). FISH detection also showed that the karyotype of 6 case at ASS1 gene deletion (1R1G1B1F) all were simple t (9; 22) translocation, and other abnormalities not were observed. Among 50 cases of B-ALL, Ph was found in 13 cases, the numerical aberration and structural aberration of non t (9; 22) in 16 cases, normal karyotype in 20 cases, absence of mitotic phase in 1 case. FISH detection showed that 16 cases (32%) had BCR/ABL fusion gene including 13 cases (26%) of 1R1G2B2F, 1 case (2%) of stimultaneous exitance of 1R1G2B2F and 1R1G3B3F 1 case (2%) of 2R1G1B1F, 1 case (2%) of 1R1G3B2F. FISH detection also showed that 3 cases had BCR/ABL fusion gene, including 1 case with ASS1 gene deletion (2R1G1B1F), 1 case with classical t (9; 22) translocation (1R1G2B2F) and 1 case with BCR/ABL fusion gene and increase of ASS1 gene copy (1R1G3B3F). CONCLUSION Tricolor dual-fusion FISH probe for detecting BCR/ABL fusion gene and ASS1 gene deletion is simple, rapid, sensitive and stable. It can detect various forms of molecular fusion and avoid the false positive results due to coincidental overlap of signals generated by D-FISH probe and ES-FISH probe. In addition, this detection method not only can directly observe the presence or absence of ASS1 gene deletion, but also improve the reliability of the positive results of newly diagnosed BCR/ABL fusion gene and accuracy of monitoring results of minimal residual disease for the subsequent visit.
Fusion Proteins, bcr-abl ; genetics ; Gene Deletion ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Reproducibility of Results

Adolescent ; Adult ; Female ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Leukemia, Myeloid ; genetics ; Male ; Oncogene Proteins, Fusion ; genetics ; Retrospective Studies

Transgenic animal model provide a good platform to research the pathogenesis and therapy of chronic myelogenous leukemia (CML). To date, a number of BCR/ABL transgenic animal models have been established using different promoter or tetracycline-controlling system. Some of them appear the characteristics of human CML, which have contributed greatly to research the pathogenesis and therapy of CML. In this article, the researching progress, advantage and drawback, application of BCR/ABL transgenic animal model are reviewed.
Animals ; Animals, Genetically Modified ; Disease Models, Animal ; Fusion Proteins, bcr-abl ; genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive

OBJECTIVE: To investigate the pro-apoptotic effect and mechanism of miR-30a overexpression on chronic myeloid leukemia K562 cells. METHODS: The k562 cells were transfected with the recombinant plasmid pEGFP-pre-miR-30a, the real-time quantitative PCR was used to detect the level of miR-30a and BCR/ABL, and then the cell apoptosis was assessed by flow cytometry with AnnexinV-FITC/PI double staining. Western blot was used to detect the expression of BCR/ABL protein,apoptosis-related protein BCL-2 and BAX, PTEN, AKT and p-AKT. RESULTS: Sequencing and digestion map indicated that the recombinant plasmid was constructed successfully. Compared with 2 control groups, the miR-30a expression in k562 cells transfected with recombinant plasmid pEGFP-pre-miR-30a was obviously up-regulated. The expression of BCR/ABL mRNA and BCR/ABL protein was both significantly down-regulated. Apoptotic rate was significantly enhanced (both P<0.05),and the expression of anti-apoptotic protein BCL-2 was down-regulated while the expression of pro-apoptotic protein BAX was up-regulated. The level of PTEN was significantly up-regulated in omparison with control groups,no variation was found in total AKT, but the expression of p-AKT was down-regulated. CONCLUSION The overexpression of miR-30a is abled to down-regulate the level of BCR/ABL mRNA and BCR/ABL protein, and increase apoptotic rate, its mechanism may be related with inhibition of the activity of BCR/ABL-PTEN/AKT signaling pathway.
Apoptosis ; Cell Proliferation ; Fusion Proteins, bcr-abl ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; MicroRNAs ; genetics

OBJECTIVETo establish a fluorogenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of bcr/abl mRNA fusion gene expression level in leukemia cells, and provide a useful tool for leukaemia diagnosis and minimal residual disease inspectation.

METHODThe conventional RT-PCR was used to amplify bcr/abl gene from cultured K562 cells, the quantitative standard template was constructed with A-T clone method. The fluo-rogenic quantitative RT-PCR method by using Applied Biosystems 7700 Sequence Detector for detecting the expression of bcr/abl fusion gene was successfully. The sensitivity, stability and repetitiveness of this method was determined. The peripheral blood samples from 14 CML patients, one of whom before and one month after bone marrow transplantation (BMT) and 4 cases of ALL in the early stages were detected.

RESULTSThe sensitivity of FQ-RT-PCR for detecting bcr/abl fusion gene was about 10(-5) micro g RNA from K562 cell and 10 copies recombined plasmid. The repetition CT value (cycle threshold) and the coefficient variation (CV) among tubes and batches were 2.0% and 3.7%, respectively. The median bcr/abl fusion gene expression level of 14 CML patients was 5.15 x 10(4) copies/ micro g RNA. The products analyzed by electrophoresis showed that 11 cases were b2a2 and 3 cases b3a2. 1.2 x 10(5) copies/ micro g RNA in one CML patient before BMT was changed to 2.3 x 10(3) copies/ micro g RNA one month after BMT. B2a2 was observed in one of the four (25.0%) patients with ALL, and its level of expression was 8.2 x 10(5) copies/ micro g RNA.

CONCLUSIONThe established FQ-RT-PCR method is sensitive, specific, reliable, accurate and good at repetitiveness. The results expressed in copies were easy for evaluation and comparation. Two different bcr/abl fusion gene form - b2a2, b3a2 can be detected by the method. It can be widely applied to diagnosis and detection of minimal residual disease for CML and some ALL patients.

OBJECTIVE: To explore the expression level of ETV6-ABL fusion gene in different cell populations in patients with myeloproliferative neoplasm (MPN) and therapeutic effect of tyrosine kinase inhibitor (TKI). METHODS: A 42-year-old man who presented with fever, marked leukocytosis and chronic myelogenous leukemia (CML) like MPN was reported. ETV6-ABL fusion gene was detected by real-time PCR and confirmed by direct sequencing. ETV6-ABL mRNA expression in each cell population sorted from peripheral blood by flow cytometry was detected by real-time PCR. RESULTS: ETV6-ABL fusion gene was found out in bone marrow cells and confirmed as type A by direct sequencing. ETV6-ABL fusion gene transcript level in polymorphonuclear cells was nearly 3.6-fold relative to that in total cells, which was significantly higher than that in T cell, B cell and monocyte subsets. The complete blood count (CBC) returned to normal level after treatment with imatinib (400 mg) daily for three months. After TKI treatment for 6 months, the ratio of ETV6-ABL/ABL decreased from 174.1% to 1.9%. CONCLUSION ETV6-ABL fusion gene positive MPN may have a CML clinical presentation and is sensitive to TKI. ETV6-ABL fusion gene is specifically expressed in polymorphonuclear cells.
Adult ; Fusion Proteins, bcr-abl/genetics* ; Genes, abl ; Hematopoietic Stem Cell Transplantation ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics* ; Male ; Myeloproliferative Disorders/genetics*

OBJECTIVETo investigate the inhibitory effect of RNA interference on chronic myeloid leukemia (CML) bcr/abl oncogene expression.

METHODSThe small interference RNAs (siRNAs) were synthesized in vitro. K562 cells stably expressing bcr/abl gene were transfected with the siRNA by electroporation, both the non-transfected cells and non-specific siRNAs transfected cells were taken as controls. The enhanced green fluorescent protein (EGFP) plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay.

RESULTSThe transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. siRNAs could inhibit K562 cell proliferation to 47% and 56% at 24 h and 48 h after transfection, respectively, and induce cell apoptosis from 1.00% in control group to 15.05% and 19.4% at 24 h and 48 h respectively.

CONCLUSIONAt the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study.

Aged ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; RNA Precursors