Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0023418 (leukemia)
93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the sensitivity of a serial dilution of total RNA from a positive cell line (K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ABL ratio in all samples. The two patients treated by IFN showed a progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10(-4). The four patients remained in clinical and cytogenetic CR. The seven other allografted patients were studied immediately after the procedure. Three of them showed a progressive decrease in the BCR-ABL/ABL ratio which reached 10(-4) 7 months after allo-BMT. The three patients remained in hematologic and cytogenetic CR. The remaining four allografted patients had progressive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR in the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round nested PCR. However, evolution changes in the results of real-time quantitative RT-PCR often preceded those of the conventional technique: a decrease of the BCR-ABL/ABL ratio preceded progression from first round to second round positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinical and cytogenetic evolution than conventional qualitative techniques and may help in making early therapeutic decisions in CML, especially after molecular relapse.
Leukemia 1999 Jun
PMID:Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a 'real time' quantitative RT-PCR assay. 1036 Mar 86

Methods of minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) include chromosome analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. We report a novel method to detect intracellular BCR-ABL messenger on single cells using in situ RT-PCR, which can be performed on blood and marrow slides, without extraction of the nucleic acid. After cellular permeabilization and fixation, the mRNA BCR-ABL was reverse transcribed and amplified by PCR using digoxigenin-labelled dUTP. The reaction was revealed with the anti-digoxigenin FITC antibody. On the fluorescent microscope, a strong positive green fluorescence signal was observed in 98-99% cells in Ph1-positive cell lines. A faint signal was detected in 1.5% and 2% of negative cell lines. Likewise, a faint signal was found in 1.6-2.8% of the cells in five normal controls (mean 2.2 +/- 1.1%). The positive threshold for in situ RT-PCR was therefore determined as mean + 2 s.d. = 4.4% cells. We used in situ RT-PCR by comparison to cytogenetics (at least 30 mitoses examined), and two-step RT-PCR (10(-6) sensitivity in our hands) in bone marrow samples from 13 CML patients: two patients at diagnosis and 11 patients in hematological remission after alpha interferon (three patients), hydroxyurea (one patient) autologous bone marrow transplantation (BMT) (one patient) and allogeneic BMT (six patients). In the two diagnostic patients, 90 and 95% cells were respectively strongly positive by in situ RT-PCR. In the six patients treated by allogeneic BMT, the median percentage of positive cells was 2.4% (range 1.8-3.2). All six patients had normal karyotype and negative two-step RT-PCR results. In the five other patients, two were treated by hydroxyurea alone or autologous BMT, and 11 and 13% of the cells were strongly positive; three were treated with interferon and 14-62% of the cells were positive, generally weakly. All five patients had persistence of Ph1 (in 9-56% mitoses), and positive RT-PCR results after one round. In conclusion, in situ RT-PCR can specifically identify cells with BCR-ABL transcript and its results are concordant with those of karyotype and RT-PCR. Because of its limited sensitivity and specificity, however, it appears to have limited value in the analysis of MRD. On the other hand, it can evaluate the presence and intensity of BCR-ABL fusion transcript at the single cell level, and this could be useful in treatment monitoring.
Leukemia 1999 May
PMID:Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay. 1037 89

The expression of the BCR-ABL fusion oncoprotein in primitive hematopoietic cells results in chronic myeloid leukemia. Over the past decade studies of several in vitro and in vivo cell systems revealed multiple signal transduction pathways activated by BCR-ABL. However, the precise function of BCR-ABL in the pathogenesis of CML is still unclear. The goal of this review is to synthesize data on intracellular signaling in the context of the diverse murine assay systems employed. We emphasize the importance of in vivo assays and assays using primary cells in understanding the biology of CML and the molecular mechanisms by which BCR-ABL exerts its effects.
Leukemia 1999 Aug
PMID:Growth factor independence and BCR/ABL transformation: promise and pitfalls of murine model systems and assays. 1045 Jul 47

A novel biphenotypic leukemia cell line, NALM-29, was established from a 46-year-old Japanese male patient with acute lymphoblastic leukemia (ALL). The primary leukemic blasts showed a common ALL phenotype with CD19+, CD10+, CD13-, HLA-DR+ and Igs-. NALM-29 cells display biphenotypic characteristics: expression of the intracellular enzyme myeloperoxidase at the mRNA and protein level and cell surface positivity for CD19, CD10, CD13, CD33 and HLA-DR. NALM-29 fulfills EGIL criteria as B-cell precursor (BCP) leukemia B-II type. NALM-29 cells have a lymphoblastic morphological appearance; the immunoglobulin heavy chain gene is rearranged. NALM-29 cells responded significantly to the proliferative stimuli of FLT-3 ligand and IL-7, but not to GM-CSF, IL-3, IL-6, PIXY-321 or SCF. Proliferation of cells was inhibited significantly by IL-4, TNF-alpha or TNF-beta treatment. Cytogenetic analysis revealed the characteristic t(9;22)(q34;q11); expression of the m-bcr e1-a2 BCR-ABL fusion gene (typically found in ALL) was determined by PCR amplification of cDNA. The immunological, cytogenetic and functional characterization of NALM-29 suggests that this cell line may represent a scientifically significant in vitro model for BCP-type leukemia cells with biphenotypic characteristics.
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PMID:A novel biphenotypic B-cell precursor leukemia cell line (NALM-29) carrying t(9;22)(q34;q11) established from a patient with acute leukemia. 1045 71

We sought to establish a rapid and reliable RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the LightCycler technology. This device combines rapid thermocycling with online detection of PCR product formation and is based on the fluorescence resonance energy transfer (FRET) between two adjacent hybridization probes carrying donor and acceptor fluorophores. A pair of probes was designed that was complementary to ABL exon 3, thus enabling detection of all known BCR-ABL variants and also normal ABL as an internal control. Conditions were established to amplify less than 10 target molecules/reaction and to detect one CML cell in 105 cells from healthy donors. To determine the utility of the assay, we quantified BCR-ABL and ABL transcripts in 254 samples (222 peripheral blood, 32 bone marrow) from 120 patients with CML after therapy with IFN-alpha (n = 219), allogeneic BMT (n = 17), chemotherapy (n = 11), or at diagnosis (n = 7). The level of residual disease in the 245 BCR-ABL positive specimens was expressed as the ratio of BCR-ABL/ABL. This ratio was compared to results obtained by three established methods from contemporaneous specimens. A highly significant correlation was seen between the BCR-ABL/ABL ratios determined by the LightCycler and (1) the BCR-ABL/ABL ratios obtained by nested competitive RT-PCR (n = 201, r = 0.90, P < 0. 0001); (2) the proportion of Philadelphia chromosome positive metaphases determined by cytogenetics (n = 81, P < 0.0001); and (3) the BCR ratio determined by Southern blot analysis (n = 122, P < 0. 0001). We conclude that real-time PCR with hybridization probes is a reliable and sensitive method to monitor CML patients after therapy. The major advantages of the methodology are (1) amplification and product analysis are performed in the same reaction vessel, avoiding the risk of contamination; (2) the results are standardized by the quantification of housekeeping genes; and (3) the complete PCR analysis takes less than 60 min.
Leukemia 1999 Nov
PMID:Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. 1055 58

Childhood leukemia is the commonest form of childhood cancer and represents clonal proliferation of transformed hemopoietic cells as a result of genetic changes. Molecular characterization of these changes, in particular chromosomal translocations, has yielded a wealth of information on the mechanisms of leukemogenesis. These findings have also allowed the development of sensitive assays for the identification of underlying molecular defects, which is applicable to disease diagnosis and to monitor response to treatment. Genetic alterations in childhood leukemia are powerful prognostic indicators. TEL-AML1 fusion and hyperdiploidy >50 chromosomes are associated with a good prognosis in childhood acute lymphoblastic leukemia, whereas BCR-ABL fusion and MLL rearrangements are associated with a poor prognosis. Hence cytogenetic and molecular genetic classification of childhood leukemia will significantly improve the ability of clinicians to predict therapeutic response and prognosis, which paves the way for risk stratification based on clinical and genetic features. Finally, deciphering of genetic lesions in leukemia has allowed elucidation of the molecular basis of current treatment, as typified by the success of all-trans retinoic treatment in acute promyelocytic leukemia, and has identified targets for novel therapeutic approaches. It is envisaged that efforts in characterization of molecular defects in childhood leukemia will ultimately be translated into better clinical outcome for patients.
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PMID:Cytogenetics and molecular genetics of childhood leukemia. 1064 Oct 30

Topological analysis of the three-dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three-dimensionally conserved nuclei with a 'resolution equivalent', many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR-ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state-of-the-art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non-random angle distribution.
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PMID:Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome. 1094 2

Very limited data exists in Thailand regarding the frequency of BCR-ABL leukemic gene and its prognostic implication in Thai CML patients. The objective of this study was to develop a rapid molecular assay for the detection of the two most commonly reported variants of BCR-ABL fusion gene, B2A2 and B3A2 in CML patients. Bone marrow or peripheral blood were used for RNA extraction and reverse-transcribed to cDNA for PCR amplification. 92 per cent of CML patients (91/99) were positive for BCR-ABL gene (61% B3A2 and 31% B2A2). 8/99 CML patients were BCR-ABL-negative. B3A2 and B2A2-positive patients did not have any different clinical and hematological features at presentation although B3A2 patients tended to be slightly older and had higher platelet counts. 71/71 non-CML including other MPD and leukemia cases were all negative for BCR-ABL gene. In conclusion, a rapid RT-PCR assay has now been developed for the detection of this hallmark gene in CML patients. It should be of great value in the differential diagnosis of CML from other diseases. Long-term follow-ups of CML patients with different variants are needed to determine the prognostic importance of each gene variant.
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PMID:Detection of molecular variants of BCR-ABL gene in bone marrow and blood of patients with chronic myeloid leukemia by reverse-transcriptase polymerase chain reaction (RT-PCR). 1099 48

By employing a new semi-quantitative assay system that includes co-culturing leukemia cells with the mouse bone marrow-derived stromal cell line MS-5, we examined the suppressive effect of a selective inhibitor of ABL tyrosine kinase, STI571, on acute lymphoblastic leukemia (ALL) cells with BCR-ABL fusion. Leukemic blast cells from eight patients with B-precursor ALL, including three patients with BCR-ABL-positive ALL, were cultured on monolayers of MS-5 cells for 3 weeks with or without addition of variable amounts of STI571. In all cases, cobblestone areas (CAs) were formed, showing clear linear cell dose-dependent curves, allowing quantitative assessment of blast cell growth. The progenitor frequencies obtained by this direct CA-forming cell (CAFC) assay were equivalent to ALL progenitor frequencies assessed by the standard limiting dilution assay. The number of CAFCs ranged from 12.3 to 140.3/10(4) cells. In BCR-ABL-positive ALL patients, CA-containing cells were examined by FISH, and all contained BCR-ABL fusion genes. STI571 inhibited CA formation of BCR-ABL-positive ALL cells virtually 100% at 0.1-1.0 micromol/l. None of the five BCR-ABL-negative ALL patients showed this growth inhibition by STI571 at 0.1-1.0 micromol/l. Our results indicate that STI571 selectively inhibits in vitro growth of BCR-ABL-positive ALL cells.
Leukemia 2001 Apr
PMID:Effect of a selective Abl tyrosine kinase inhibitor, STI571, on in vitro growth of BCR-ABL-positive acute lymphoblastic leukemia cells. 1136 61

Chronic myeloid leukaemia (CML) is characterised by an indolent, chronic phase (CP) preceding an acute transformation to blast crisis (BC). While the BCR-ABL fusion oncogene is strongly implicated in the CP, the molecular changes underlying BC are largely unknown. The ataxia telangiectasia gene, ATM, is a candidate gene for this transformation because the complex karyotypes associated with BC of CML suggest that DNA double-strand break repair is defective and because the ABL pathway involves the interaction between the Abl and the Atm proteins. We performed a mutational analysis for ATM in CML using genomic DNA from 14 CML cell lines and 59 CML patients in BC. No clearly deleterious nucleotide changes were observed. A new polymorphism C4138T was discovered which results in a non-conservative amino acid substitution (H1380Y). This variant lies in the Atm recognition motif for the Abl protein. While ATM is unlikely to contribute substantially to CML, further investigation of the H1380Y substitution should clarify whether it has any functional effect.
Leukemia 2001 Sep
PMID:Investigation on the role of the ATM gene in chronic myeloid leukaemia. 1151 6


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