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: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytogenetic, interphase fluorescent in situ hybridization (FISH) and RT-PCR methods were used to study minimal residual disease in peripheral blood stem cells collected for autografting in three chronic myeloid leukemia (CML) patients in sustained complete cytogenetic remission after treatment with interferon alpha (IFNalpha). Karyotypic analysis failed to reveal Ph-positive metaphases. FISH detected 9-16% nuclei with a BCR-ABL fusion gene, contrasting with RT-PCR, performed in two cases, which was negative in one case and weakly positive in the other. RT-PCR was also subsequently weakly positive in the third patient. This discrepancy suggests that the BCR-ABL genomic rearrangement persists unexpressed in quiescent cells. These preliminary results, which need to be confirmed in larger series, suggest that monitoring residual disease in CML should be performed both at DNA and RNA levels. Moreover, autografting following IFNalpha therapy should be considered with caution because of the persistence of the BCR-ABL genomic rearrangement in a sizeable proportion of the cells.
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PMID:Does BCR-ABL genomic rearrangement persist in CML patients in complete remission after interferon alpha therapy? 966 93

The number of genetic lesions necessary to generate leukemia in humans is unknown, but it is possible that certain specific abnormalities, eg, fusion genes, known to be associated with acute and chronic leukemia are produced relatively frequently in human cells but require other events to occur before the leukemia becomes manifest. We investigated this possibility by studying peripheral blood leukocytes from normal individuals and various hematopoietic cell lines for the presence and expression of the p210 and the p190 types of the BCR-ABL gene associated with chronic myeloid leukemia (CML) and acute lymphoblastic leukemia. We used two-step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in which batches of 10(8) cells per sample were tested in 40 replicate reactions. We estimate that this assay is 1.5 logs more sensitive than the two-step RT-PCR assays that we use routinely to assess minimal residual disease. BCR-ABL fusion gene transcripts of various configurations were found in circulating leukocytes from 12 of the 16 healthy adults analyzed. Transcripts with an e1a2 junction (p190 BCR-ABL) were present in 11 and p210-type transcripts with b2a2 and/or b3a2 junctions were detected in 4 individuals. The same RT-PCR assays in non-CML cell lines showed the presence of classical or aberrant p210-type mRNA in 3 of 7 lines and of p190-type transcripts in all 7 lines of hematopoietic origin (HL60, KG1, U937, Kasumi, Jurkat, JVM13, and JVM25), whereas the NIH3T3 murine fibroblast line was reproducibly negative for these fusion genes. These findings confirm and extend previous reports on the detection of leukemia-associated genes in normal leukocytes and suggest that certain fusion genes are generated relatively frequently in hematopoietic cells, but only infrequently do the cells acquire the additional changes necessary to produce leukemia in humans. Although there is only a small probability that such innocent BCR-ABL-carrying leukocytes are detected by conventional RT-PCR assays, they may be the source of some sporadically positive tests in leukemia patients in long-term remission.
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PMID:The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease. 978 74

The purpose of this study was to review the progress in clinical and translational research in chronic myelogenous leukemia (CML) over the past 20 years at M.D. Anderson Cancer Center. The CML database updating the clinical and basic research investigations was reviewed as the source of this report. Publications resulting from these investigations were summarized. The long-term results with intensive chemotherapy, IFN-alpha therapy alone or in combination, autologous stem cell transplantation, and new agents such as homoharringtonine and decitabine showed encouraging results. Biological studies related to the BCR-ABL molecular abnormality, other molecular events, and the detection of minimal residual disease were detailed. Future strategies with potential promise in CML were outlined. Significant progress in understanding CML biology and in treating patients afflicted with the disease has occurred. Several therapeutic and research tools are currently investigated, which should hopefully improve further the prognosis of patients with CML.
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PMID:Chronic myelogenous leukemia--progress at the M. D. Anderson Cancer Center over the past two decades and future directions: first Emil J Freireich Award Lecture. 1006 80

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.
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PMID:Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay. 1037 89

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-alpha) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients had < or =34% and seven patients >34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers (r = 0.84, P < 0.0001) or BCR-ABL/ABL ratio (r = 0.86, P < 0.0001) was found. For patients that underwent the procedure in early chronic phase, Ph-negative collections showed different levels of BCR-ABL expression. BCR-ABL transcript numbers varied from a median of 100/microg RNA in the first and second leukaphereses, to 500/microg RNA in the third and fourth leukaphereses, and 1500/microg RNA in the fifth leukapheresis (P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (< or =0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (chi2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.
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PMID:Quantitative competitive reverse transcriptase-polymerase chain reaction for BCR-ABL on Philadelphia-negative leukaphereses allows the selection of low-contaminated peripheral blood progenitor cells for autografting in chronic myelogenous leukemia. 1040 Apr 14

Chronic myelogenous leukemia is characterized by an abnormal 22nd chromosome known as a Philadelphia chromosome, which can be detected in 95% of patients with CML. Molecular equivalent of this aberration is a BCR-ABL translocation resulting in chimeric gene formation. A BCR-ABL chimeric gene plays a key role in the hematopoietic cells proliferation regulation. RT-PCR can be used in diagnosis of CML, to detect chimeric BCR-ABL gene, and to reveal the type of translocation what could have prognostic importance, and in monitoring of minimal residual disease, confirming the eradication of pathological cells clone after treatment and identifying the group of patients with best prognosis and best overall survival. RT-PCR in monitoring of minimal residual disease after allo- or autologous hemopoietic cells transplantation can early reveal relapse of the disease. Detection of molecular relapse is an important prognostic factor and may implicate induction of treatment which should prevent haematological relapse of the disease.
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PMID:[Molecular basis of chronic myelogenous leukemia and significance of diagnostic methods based on BCR-ABL gene amplification]. 1049 84

Because previous PCR-based methodologies for detection of minimal residual disease (MRD) in leukemia patients have been too cumbersome to allow for widespread clinical usefulness, we have employed a real-time quantitative PCR (RQ-PCR) system to develop an MRD assay for t(12;21). We initially determined the expression of the different alternatively spliced TEL-AML1 mRNAs found in t(12;21) breakpoint variants I and II. We then optimized PCR primers for the RQ-PCR system and, using the t(12;21)+ REH cell line in spiking experiments, found a linear detection of TEL-AML1 over at least five logs. Moreover, 1 malignant cell in a background of 1,000,000 normal cells could be detected. The expression of the GAPDH, ABL, and beta(2)-microglobulin (beta2M) housekeeping genes were then compared in normal donors and in leukemic patients, and the very stably expressed beta2M was selected as an internal reference gene, allowing us to compensate for variation in RNA quality and day-to-day variation. In 12 samples from t(12;21)-positive patients at diagnosis, the levels of the TEL-AML1 fusion transcripts were found to vary up to 14-fold after normalization to beta2M. Interestingly, in samples obtained from seven patients at diagnosis, during induction chemotherapy, or relapse, the level of TEL-AML1 in peripheral blood (PB) and bone marrow (BM) was found to differ only by threefold, suggesting that MRD may be evaluated in PB samples in most patients. We conclude that this assay could set new standards for t(12;21) MRD detection with its accuracy, its high throughput, and its short turnover time for samples. Genes Chromosomes Cancer 26:355-365, 1999.
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PMID:Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. 1053 71

We have developed a rapid real-time quantitative PCR method for measuring BCR-ABL mRNA levels in peripheral blood in chronic myeloid leukaemia (CML). The technique was used to monitor minimal residual disease for the early detection of relapse and as an assessment of treatment response. Normal BCR mRNA was quantitated to control for RNA degradation and the results reported as a percentage of BCR-ABL/BCR. Every patient measured at diagnosis (n = 21) had increased expression of BCR-ABL of up to 5-fold above the normal BCR levels. With effective treatment the BCR-ABL levels decreased. The molecular data was correlated with Philadelphia chromosome levels in bone marrow and a good correlation was found when treatment induced a cytogenetic response (Spearman correlation = 0.94, P < 0.0001, n = 67 samples). In patients receiving interferon-alpha therapy we found a significant difference in the BCR-ABL levels between cytogenetic response groups. The method was sensitive, reproducible, and readily detected a change in BCR-ABL transcript levels in serial blood samples. Sample throughput was high because post PCR processing was unnecessary. We conclude that real-time quantitative PCR monitoring of peripheral blood can be used to reliably monitor disease response in CML.
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PMID:Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics. 1058 64

Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A <--> B) and nested PCR (internal primers C <--> D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A <--> B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10-2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10-4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.
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PMID:Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. 1060 11

A substantial minority of patients with chronic myelogenous leukemia (CML) achieve a complete response (CR) to treatment with interferon-alpha (IFN), defined as the disappearance of Philadelphia chromosome-positive metaphases. Currently it is unclear how long IFN treatment should be continued for such patients. We used a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify levels of BCR-ABL transcripts in 297 peripheral blood specimens collected from 54 patients who had achieved CR with IFN. The median duration of observation was 1.9 years (range, 0.3-11.0 years). Total ABL transcripts were quantified as internal control and results were expressed as the ratio BCR-ABL/ABL. All 54 patients had molecular evidence of residual disease, although 3 patients were intermittently PCR negative. The median BCR-ABL/ABL ratio at the time of maximal response for each patient was 0.045% (range, 0%-3. 6%). During the period of observation 14 patients relapsed, 11 cytogenetically to chronic phase disease and 3 directly to blastic phase. The median ratio of BCR-ABL/ABL at maximal response was significantly higher in patients who relapsed than in those who remained in CR (0.49% versus 0.021%, P < 0.0001). Our findings show that the level of residual disease falls with time in complete responders to IFN, but molecular evidence of disease is rarely if ever eliminated. The actual level of minimal residual disease correlates with the probability of relapse. We suggest that for patients who reach CR, IFN should be continued at least until relatively low levels of residual leukemia are achieved. (Blood. 2000;95:62-66)
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PMID:Molecular heterogeneity in complete cytogenetic responders after interferon-alpha therapy for chronic myelogenous leukemia: low levels of minimal residual disease are associated with continuing remission. German CML Study Group and the UK MRC CML Study Group. 1060 85


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