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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neuroblastoma (NB) is characterised by the secretion of catecholamines in approximately 95% of patients. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis pathway. Expression of the tyrosine hydroxylase gene (TH) is regulated in a tissue-specific manner during neonatal development and differentiation, therefore TH mRNA expression is a specific tumour marker for NB. Here we present a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using TaqMan technology for detection and quantification of TH mRNA in bone marrow (BM) NB patients. The degree of TH expression was derived from the ratio of the mRNAs of this gene and the reference gene, beta-actin. A ratio greater than 3x10(-2) was considered as positive for TH mRNA presence. Samples were also examined for TH mRNA by first and nested RT-PCR. Seventeen BM samples from 4 patients with disseminated NB (3 stage IV and 1 stage IVs) were evaluated at diagnosis and during treatment. We found a variable degree of TH expression ranging from 0.0344 to 26.3370 in 12/17 positive samples, while no TH mRNA (value lower than 3x10(-2)) was detected in 5/17 samples obtained after consolidation therapy. Our results show a moderate concordance between different qualitative RT-PCR methods and real-time RT-PCR. The real-time RT-PCR results seem to fit better with the natural short-term clinical follow-up of the evaluated patients, with respect to qualitative methods. Real-time TH RT-PCR could therefore be of clinical value for the assessment of a patient's prognosis by monitoring minimal residual disease (MRD).
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PMID:Real-time RT-PCR of tyrosine hydroxylase to detect bone marrow involvement in advanced neuroblastoma. 1257 72

Acute promyelocytic leukaemia (APL) is characterized by a unique genetic marker in virtually 100% of cases, i.e. the PML/RARalpha fusion gene which is readily amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Several international groups reported the prognostic significance of minimal residual disease (MRD) assessment in APL, indicating that sequential PCR analysis should be used as a guide to therapy. In fact, such evaluation offers the possibility of identifying, after front-line treatment, either patients requiring additional therapy or patients at low risk who are presumably cured and who may be spared unnecessary toxicity. In this view, the terms molecular remission and molecular relapse are now widely employed to define a more advanced therapeutic objective and a condition necessitating anticipated salvage, respectively. The introduction of quantitative PCR through automated technologies is likely to further improve standardization of the method and comparison of results obtained in the context of large clinical trials.
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PMID:The importance of molecular monitoring in acute promyelocytic leukaemia. 1293 66

In the last few years molecular genetic studies of childhood cancer have acquired great importance. Advances in these techniques have increased knowledge of the various genes involved in tumoral development. Genetic alterations can occur in three large groups of genes: oncogenes, tumor suppressor genes, and DNA repair genes. Cytogenetic analyses (karyotyping) are complemented by various molecular techniques, such as fluorescence in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and spectral karyotyping (SKY). These are the most reliable techniques and improve the sensitivity of karyotyping. The present article reviews the most representative and best characterized genes involved in the molecular etiology of childhood cancer, both hematologic malignancies (leukemia and lymphoma) and solid tumors (brain tumors, neuroblastoma, Wilms' tumor, hepatoblastoma, rhabdomyosarcoma, Ewing's sarcoma and retinoblastoma). Molecular techniques have enabled more precise diagnosis as well as identification of new prognostic factors and the development of more effective treatments. These techniques can also be useful in identifying minimal residual disease during and after treatment for leukemias, neuroblastomas and sarcomas, with the aim of predicting recurrence.
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PMID:[The role of molecular genetics in childhood cancer]. 1451 4

We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending on WT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitating WT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-time WT1 assay had high reproducibility. WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed among WT1 transcript values normalized with 3 different control genes (beta-actin, ABL, and glyceraldehyde-3-phosphate dehydrogenase) and between relative WT1 transcript values with WT1 expression in K562 cells as the reference and absolute WT1 transcript copy numbers per microgram RNA. When WT1 expression and minor bcr-abl expression were concurrently monitored in 2 patients with bcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of a WT1 assay.
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PMID:Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1). 1468 94

Detection of the PML/RARalpha fusion gene by RT-PCR in acute promyelocytic leukemia (APL) blasts is not only critical to commence promptly the specific therapy with all-trans retinoic acid (ATRA) or arsenic trioxide (As(2)O(3)), but also essential for the definition of PML breakpoint type and subsequent monitoring of minimal residual disease (MRD). The current PML/RARalpha amplification techniques with conventional nested PCR are laborious and time consuming, which fails to meet the requirements for rapid diagnosis of APL in clinical practice. Therefore, an easily handled RT-PCR methodology for the rapid and accurate amplification of PML/RARalpha fusion transcripts is needed. A modified one round RT-PCR protocol was described with a few variations which includes rapid extraction of high quality cellular total RNA, cDNA synthesis with random hexamer and M-MLV reverse transcriptase, optimal concentrations of MgCl(2) (1 mmol/L), PCR primers (0.4 micro mol/L) and Taq polymerase (0.01 U/ micro l), hot-start procedure, and concomitant amplification of PML/RARalpha fusion gene and RARalpha internal control under the identical thermocycle parameters. The results in 40 patients with newly diagnosed APL showed that the improved RT-PCR protocol allowed the rapid detection of PML/RARalpha fusion gene and the accurate discrimination of its transcript types, and simultaneous amplification of RARalpha internal control under the identical program in less than 6 hours. There were no false positive or negative results found with the assay. In conclusion, the assay reported here is proved to be a simple, easily handled, and highly specific procedure for the diagnosis of APL cases, particularly those requiring such urgent therapeutic intervention as ATRA or As(2)O(3) and meriting its further application in APL management.
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PMID:[Improved RT-PCR for detection of PML/RARalpha fusion gene in rapid diagnosis of acute promyelocytic leukemia]. 1470 40

A quantitative nested reverse transcriptase polymerase chain reaction (QN-RT-PCR) method was developed using a plasmid cDNA containing the AML1/ETO (MTG8) fusion transcript from Kasumi-1 cells, an acute-myelogenous leukemia cell line with the t(8;21) translocation. In this method, the plasmid was detectable at a concentration of 10(-17) m. The fusion transcript in a mixture of 10(7) Rice94 (Burkitt lymphoma cell line) cells containing two Kasumi-1 cells was detectable at 10(-17) m. In a previously published real-time PCR method, the plasmid containing the fusion transcript was detectable at 10(-16) m or higher, and 20 or more Kasumi-1 cells were detectable in 10(7) Rice94 cells. Thus, this QN-RT-PCR method is more sensitive than the real-time PCR. When the same samples were examined by real-time PCR and our QN-RT-PCR method, in one patient in clinical remission after chemotherapy and allogeneic-bone marrow transplantation (BMT), the transcript was detected by QN-RT-PCR 60 days prior to hematological relapse, in contrast to 10 days before hematological relapse by real-time PCR. The transcript level was below 10(-17) m (undetectable) with this QN-RT-PCR in patients in clinical remission after chemotherapy and BMT, while it was 10(-15)-10(-16) m in patients in clinical remission after chemotherapy alone. The quantitative difference of the transcript level in minimal residual disease (MRD) between these two different types of clinical remission was estimated to be at least 10(2)-fold. This QN-RT-PCR method is useful for predicting hematological relapse and for quantitatively estimating MRD in different types of clinical remission.
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PMID:Quantitative nested reverse transcriptase PCR vs. real-time PCR for measuring AML1/ETO (MTG8) transcripts. 1505 4

The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.
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PMID:RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications. 1508 63

Intensive, myeloablative therapy supported by autologous hematopoietic stem-cell transplantation (AHSCT) has improved the outcome for children with high-risk neuroblastoma. However, >50% of patients develop recurrent neuroblastoma, often from minimal residual disease (MRD). Immunocytological and reverse transcriptase polymerase chain reaction (RT-PCR) for genes highly expressed in neuroblastoma both can detect small amounts of MRD in blood and bone marrow, and detection of MRD at certain levels during therapy has prognostic value. Radionucleotide scans using meta-iodobenzaguanidine (MIBG) imaging allows sensitive detection of neuroblastoma in patients, but whether or not all MIBG-positive disease detected after AHSCT will progress remains to be defined and is complicated by use of post-AHSCT therapy. Selective removal of tumor cells from marrow or blood stem cells harvested for AHSCT could decrease recurrence by preventing infusion of tumorigenic cells with AHSCT. Treating MRD after AHSCT with the differentiation-inducing retinoid 13-cis-retinoic acid significantly /improved EFS of high-risk neuroblastoma patients. Randomized clinical trials in the Children's Oncology Group are testing the value of purging blood stem cells and also whether post-AHSCT therapy with an anti-GD2 monoclonal antibody (combined with cytokines) improves outcome over use of 13-cis-retinoic acid alone. New approaches to treating neuroblastoma MRD that are in early clinical trials include the cytotoxic retinoid fenretinide and the hu14.18-IL2 immunocytokine. It is anticipated that testing novel approaches to treating neuroblastoma MRD will be the subject of future phase-III randomized trials.
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PMID:Detection and treatment of minimal residual disease in high-risk neuroblastoma. 1512 7

Wilms' tumor gene WT1 mRNA is a new marker of leukemic blast cells for AML, ALL, and CML. The minimal residual disease(MRD) of leukemia can be detected at frequencies as low as 1 in 10(3) to 10(4) normal bone marrow cells and 1 in 10(5) normal peripheral blood mononuclear cells by means of the quantitation of WT1 mRNA (WT1 assay) using reverse transcriptase-polymerase chain reaction. Thus, the WT1 assay makes it possible to rapidly assess the effectiveness of treatment and to evaluate the degree of eradication of leukemic cells in individual leukemia patients. Furthermore, the WT1 assay can continuously assess the disease progression of myelodysplastic syndrome(MDS) and predict the evolution of MDS to overt AML within 6 months. Moreover, WT1 protein is highly immunogenic, thus, WT1 peptide-based cancer immunotherapy is effective.
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PMID:[Development of a new inspection diagnostic method: genetic screening of cancer]. 1520 29

The WT1 gene is highly expressed in various types of leukemia, particularly in acute type leukemia. The extent of minimal residual disease (MRD) of leukemia can be evaluated by measuring the WT1 gene expression level using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. The WT1 transcript assay can be applied to almost all patients with leukemia regardless of the presence or absence of chimeric DNA markers. Furthermore, because of the fact that WT1 expression levels increase significantly at relapse compared with those at the time of diagnosis, and because of the decrease in background levels of WT1 expression in bone marrow following allogeneic SCT, the WT1 transcript assay possesses a high degree of sensitivity following allogeneic SCT. Frequent monitoring of the WT1 gene expression level predicts the risk of relapse following allogeneic SCT, and furthermore, the kinetics of WT1 transcripts predict the efficacy of immunomodulation therapy such as donor leukocyte infusion.
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PMID:WT1 gene transcript assay for relapse in acute leukemia after transplantation. 1522 32


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