This study was aimed to investigate the bcr-abl transcription level and its relationship with the clinical status of patients so as to provide some bases for predicting patient status according to absolute value of bcr-abl transcript. The bcr-abl/abl values (%) of bone marrow samples from 30 newly diagnosed CML patients at the baseline bcr-abl/abl value obtained in CML patients with bcr-abl positive were defined, then 161 bone marrow samples from 82 patients were detected at virions time points, and the bcr-abl/abl value of each sample was compared with baseline value and its relationship with clinical status of patient at same time point was investigated. The results showed that bcr-abl/abl values (%) of 30 patients showed positive skew distribution and a large variation with mean 13.5631 (1.0206 - 98.3159) and mathematical mean of 21.1491 (95% CI: 12.3532 - 29.9450). For strict standard, the baseline value of bcr-abl/abl (%) was set as 1, the lower limit of these values. In the detected results of 161 samples, there were 33 samples' values above the baseline value, in which resistance/relapse/progression (R/R/P) 13 (39.4%, 13/33), no remission (NR) 17 (51.5%, 17/33) and complete hematologic remission (CHR) 3 (9.1%, 3/33) were observed. the values of 26 samples decreased by 0 - 1 order of magnitude (0.1 < or = bcr-abl/abl % < 1), in which R/R/P 6 (23.1%, 6/26), NR 7 (26.9%, 7/26), CHR 7 (26.9%, 7/26) and cytogenetic remission (CyR) 6 (23.1%, 6/26) were observed, the values of 19 samples decreased by 1 - 2 order of magnitude (0.01 < or = bcr-abl/abl % < 0.1), in which NR 2 (10.5%, 2/19), CHR 3 (15.8%, 3/19) and CyR 14 (73.7%, 14/19) were determined. 7 samples decreased by 2 - 3 order of magnitude (0.001 < or = bcr-abl/abl % < 0.01) in which major CyR (MCyR) 2 (28.6%, 2/7) and complete CyR (CCyR) 5 (71.4%, 5/7) were determined, the values of 76 samples decreased by 3 or more order of magnitude (bcr-abl/abl % < 0.001), and all these were CCyR. In conclusion, the using decrease degree of one time point-detected value compared to the baseline could well assess the patient clinical status. The bcr-abl/abl % < 0.01 can reliably reflect CyR obtained by patients at the time point, and bcr-abl/abl % < 0.001 can reflect CCyR obtained by patients. However, exact judgments of patient status relies on dynamic and serial monitoring.
Adolescent ; Adult ; Female ; Fusion Proteins, bcr-abl ; analysis ; metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; physiopathology ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Young Adult

OBJECTIVETo investigate the accuracy and consistency of the detection of BCR-ABL tyrosine kinase domain point mutation among different laboratories.

METHODSEvery one of 6 laboratories prepared 10 cDNA samples from tyrosine kinase inhibitors resistant BCR-ABL (P210 or P190) positive patients'bone marrow or peripheral blood. Each cDNA sample was divided into 6 aliquots and delivered to the laboratories. All 6 laboratories tested BCR-ABL point mutations of 60 samples according to their own protocols. Peking University People's Hospital analyzed the comparison results based on both the reports and sequencing chromatogram from all laboratories.

RESULTSAll laboratories reported the same nucleotide and corresponding amino acid mutations in 37 samples (61.7%）. Of 60 samples, 53 had confirmed mutation types, and a total of 23 types were included; 1 had no mutation; mutation types of 6 samples could not be determined because of the big differences among chromatograms from different laboratories. Low percentages of mutants were significantly related to results inconsistency (P=0.008). Inconsistent result of one sample was caused by the unique chromatogram of the mutant L248V, and one by the non-coverage amplification of PCR product from different laboratories. Amplification was failed in 3 samples. Testing or sequencing mistakes occurred in 7 samples. The differences in the mutant percentages among laboratories were less than 20% in the 80.6% of samples with confirmed results. Low internal control gene copies (ABL<10 000) were significantly related to both failed amplification and big differences among chromatograms from different laboratories (P=0.005 and <0.001, respectively).

CONCLUSIONProblems in the clinical routine detection of BCR-ABL point mutation could be exposed and improvement could be achieved by sample exchange and comparison. Low percentage of mutant is the main reason which causes the discrepancy of BCR-ABL point mutation results among different laboratories.

Bone Marrow ; DNA Mutational Analysis ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Point Mutation ; Polymerase Chain Reaction

OBJECTIVETo investigate the unusual bcr/abl fusion gene structures of two Ph chromosome positive chronic myelogenous leukemia (CML) patients in chronic phase (CP).

METHODSBy using general M- and micro -bcr/abl specific primers respectively, bcr/abl fusion transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR). The RT-PCR products sequencing was performed, the DNA sequences were analyzed in Genebank and the bcr and abl sequences at the fusion site were identified. DNA was amplified by PCR using a set of primers designed according to the sequencing result of RT-PCR products.

RESULTSTwo patients showed typical manifestations of CML-CP. Their RT-PCR products were different from usual M- or micro -type; one was longer than M-bcr/abl but shorter than micro -bcr/abl, the other one was shorter than M-bcr/abl. The RT-PCR products sequencing showed that both products contained bcr and abl gene sequences. The first patient's bcr gene was broken within exon 18, and fused to abl gene exon 2(a2), and a 40 bp of partial abl intron 1b fragment was inserted between them, resulting in a novel in-frame bcr/abl fusion transcript-e18-int-a2 which has not been reported in the literature so far. In the second patient, deletion of abl exon2(a2) led to exon 13(b2) of bcr gene fusing with abl exon 3(a3).

CONCLUSIONUncommon bcr/abl fusion gene may occur in typical Ph(+) CML patient.

Adult ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Molecular Sequence Data ; Philadelphia Chromosome ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

To investigate the relationship between the single nucleotide polymorphism (SNPs) of the bcr and abl gene and chronic myelogeous leukemia (CML), the 9 sequence-tagged sites (STS) in bcr and abl gene were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were varified by sequencing. The results showed that the polymorphism sites were detected in 4 out of the 9 STS fragments and there were 3 bases different from the reference sequence found in 3 fragments. In conclusion, the novel SNP in U07000 fragment shows significantly different frequencies between CML and controled people.
Chromatography, High Pressure Liquid ; methods ; Fusion Proteins, bcr-abl ; genetics ; Genes, abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-bcr ; genetics ; Sequence Analysis, DNA ; Sequence Tagged Sites

OBJECTIVETo investigate the apoptotic inducing effect of As(2)S(2) on K562 cells.

METHODSThe apoptotic inducing effect of As(2)S(2) on K562 cells was determined by flow cytometry, DNA fragmentation analysis and morphology observation. Expression of protein was determined by Western-blot. RT-PCR was used to evaluate changes in gene expression.

RESULTSApoptosis of K562 cells was induced by 48 - 72 h exposure to 5 micromol/L As(2)S(2). Apoptosis was induced in (34.4 +/- 3.3)% treated cells by 72 h exposure to 3 micro mol/L As(2)S(2), in (21.8 +/- 3.6)% treated cells by 48 h exposure to 5 micromol/L As(2)S(2) and in (46.0 +/- 5.2)% treated cells by 72 h exposure to As(2)S(2) at the same concentration. With 5 micromol/L As(2)S(2), the protein level of Bcr-Abl and JAK2 decreased, while bax expression was upregulated and c-myc was downregulated both in protein and mRNA level. The activity of caspase 3 in K562 cells was increased by As(2)S(2). As(2)S(2) also induced apoptosis of fresh mononuclear cells derived from chronic myelogenous leukemia (CML) patients.

CONCLUSIONAs(2)S(2) can induce apoptosis of CML cells. The decline of Bcr-Abl may play an important role. The upregulation of bax, increase of the activity of caspase 3, downregulation of c-myc and decrease of JAK2 may also be involved in the mechanism.

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Caspase 3 ; metabolism ; Fusion Proteins, bcr-abl ; analysis ; Humans ; Janus Kinase 2 ; analysis ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; pathology ; Sulfides ; pharmacology ; bcl-2-Associated X Protein ; analysis

OBJECTIVETo explore the specificity of anti-phosphotyrosine monoclonal antibody PY20 in bcr-abl+ cells and its possible clinical applications.

METHODSBcr-abl cell lines( K562, MEG-01) and bcr-abl- cells lines( Jurkat, MCF-7 )were stained with PY20. Phosphotyrosine protein of K562 and MEG-01 cells was detected by flow cytometry before and after treatment with imatinib. Phosphotyrosine protein in bone marrow cells from 49 patients with chronic myeloid leukemia (CML), Ph+ acute lymphoblastic leukemia(Ph(+) -ALL), Ph- ALL, acute myeloid leukemia (AML-M1, M2, M3, M5, FAB classification), chronic lymphocytic leukemia (CML) and 3 normal donor. Positive cells over 5% of total cells was considered positive cases for phosphotyrosine protein. The level of tyrosine phosphorylation was determined by median fluorescence intensity (MFI).

RESULTSBcr-abl cell lines and marrow cells from 10 CML patients and 8 ALL patients were all PY20-positive, while bcr-abl- cell lines and marrow cells from 18 leukemia patients and 3 normal donor were all PY20-negative. MFI decreased remarkably after blocked by imatinib in K562 cells and MEG-01 cells. The positive cell percent of marrow cells from 10 newly diagnosed CML patients and 9 imatinib-sensitive CML patients was (54.20 +/- 19.82)% and (14.84 +/- 6.17)% (P < 0.05), while that of 2 cases of imatinib-resistant was 64.3% and 57.2%. There was significant difference of MFI between imatinib-resistant patients and imatinib-sensitive patients (99.42 +/- 4.87 vs 46.41 +/- 4.67, P < 0.01).

CONCLUSIONPY20 monoclonal antibody is highly specific for bcr-abl+ cells. It might be useful in rapid detection of bcr-abl+ cells and sensitivity to imatinib of CML patients.

Antibodies, Monoclonal ; analysis ; Bone Marrow Cells ; metabolism ; Flow Cytometry ; Fusion Proteins, bcr-abl ; analysis ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; Phosphotyrosine ; analysis ; immunology

No abstract available.
Aged, 80 and over ; Base Sequence ; Bone Marrow/pathology ; DNA/chemistry/metabolism ; Female ; Fusion Proteins, bcr-abl/*genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/*genetics ; Multiplex Polymerase Chain Reaction ; Protein Isoforms/genetics ; Sequence Analysis, DNA

The objective of this study was to investigate the specificity of detecting the phosphotyrosine level with anti-phosphotyrosine monoclonal antibody PY20 for diagnosis and prognosis of patients with chronic myeloid leukemia (CML) and the possibility of its clinical application. The positive rate of PY20 in 28 newly diagnosed CML patients was detected by flow cytometry using anti-PY20 antibody, the bcr-abl fusion gene was detected by nested RT-PCR, the Ph chromosome was measured by R-banding cytogenetic analysis, and the coincidence of PY20 positive rate with results of bcr-abl fusion gene and Ph chromosome detection was compared. In addition, the positive rate of PY20, the changes of bcr-abl fusion gene and Ph chromosome were determined in follow up 7 CML patients after allo-hematopoietic stem cell transplantation. The results indicated that the positive rates of PY20 in 28 newly diagnosed CML patients in groups of chronic phase (CP), accelerated phase (AP), and blast phase (BP) were (40.31% +/- 1.22)%, (77.28 +/- 1.14)% and (78.12 +/- 1.32)% respectively. The positive rate of PY20 in CP was lower than that in AP and BP (p < 0.05). There was no difference in positive rate of PY20 between AP and BP (p > 0.05). PY20 expression level of leukocytes from peripheral blood and bone marrow showed no difference (p < 0.05). The positive rates of PY20 in patients with CR, PR and NR were (15.56% +/- 1.51)%, (38.73% +/- 2.31)% and (60.43% +/- 2.04)% respectively. The positive and negative coincidence between PY20 and RT-PCR was 92.31% and 95.45% respectively. The positive and negative coincidence between PY20 and Ph Chromosome in newly diagnosed patients was 88.46% and 95.46% respectively. Ph chromosome and PY20 were all negative in 7 CML patients after allo-HSCT. Bcr-abl fusion gene was negative persistently in 5 patients, but in the other 2 patients, the fusion gene was persistently positive. In conclusion, the detection of the level of phosphotyrosine in CML cells has high sensitivity and specificity. The results of PY20 cell positive rate combined with detection of bcr/abl fusion gene and Ph chromosome might be useful in diagnosis as a good index of monitoring.
Adolescent ; Adult ; Antibodies, Monoclonal ; chemistry ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Male ; Middle Aged ; Philadelphia Chromosome ; Phosphotyrosine ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Young Adult

The study was purposed to analyze the relationship between the content of T-cell receptor excision DNA circles (TREC) and bcr-abl mRNA levels in CML patients and to evaluate the prognostic significance of recent thymic output function detection in patients with chronic myelogenous leukemia (CML). Quantitative detection of TREC and bcr-abl fusion gene transcripts in peripheral blood from 15 CML patients were preformed by real-time PCR. The change of bcr-abl levels in 6 patients was followed-up for two years. The results showed that there was no significant correlation between TREC and bcr-abl mRNA levels in peripheral blood from CML patients for the first attack. Patients who had higher TREC at diagnosis had a larger reduction of bcr-abl after 2 years of follow-up. While out of 2 patients who underwent haemopoietic stem cell transplantation (HSCT), one patient with higher level of TREC before transplantation was confirmed to express undetectable level of TREC by three consecutive detections after transplantation, other one patient was identified to express low level of bcr-abl. It is concluded that high thymic output function in CML patients can be beneficial for killing the residual CML cells.
Adolescent ; Adult ; Aged ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Gene Rearrangement ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-abl ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcr ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Antigen, T-Cell ; analysis ; immunology ; T-Lymphocytes ; chemistry ; immunology ; Thymus Gland ; immunology

To explore the application value of bcr-abl fusion gene deterction microarray in diagnosis, typing, choosing of treatment variant and prognosis judgment, probe for fusion gene detection was designed, oligonucleotide microarray was prepared; total RNA was extracted, reverse-transcripted and labeled by fluorescence, then cDNA was hybridized with microarray in order to detect bcr-abl fusion gene in leukemia cells. The results showed that better reaction conditions were gained by exploration of hybridizotion temperature and elution conditions, bcr-abl fusion gene in leukemia cells was detected by prepared miccroarray. In conclusion, oligonucleotide microarray is effective in detecting the fusion gene and has some unique advantages and certain clinical application value, but has some deficiency too. If microarray can be improved further, it could be used widely in the field of hematology.
Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia ; genetics ; pathology ; Oligonucleotide Array Sequence Analysis ; methods ; Reproducibility of Results