Real-time Quantification Assay to Monitor BCR-ABL1 Transcripts in Chronic Myeloid Leukemia

2014 ◽  
pp. 115-124 ◽  
Author(s):  
Pierre Foskett ◽  
Gareth Gerrard ◽  
Letizia Foroni
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Myeloid Leukemia

scholarly journals Manifold-assisted Reverse Transcription-PCR with Real-Time Detection for Measurement of the BCR-ABL Fusion Transcript in Chronic Myeloid Leukemia Patients

2000 ◽  
Vol 46 (7) ◽  
pp. 913-920 ◽  
Author(s):  
Gisela Barbany ◽  
Anette Hagberg ◽  
Ulla Olsson-Strömberg ◽  
Bengt Simonsson ◽  
Ann-Christine Syvänen ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Minimal Residual Disease ◽  
Reverse Transcription ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Detection System ◽  
Fusion Transcript ◽  
Mrna Quantification ◽  
Minimal Residual

Abstract Background: BCR-ABL fusion mRNA expression in bone marrow or peripheral blood can be used as a measure of minimal residual disease in patients with chronic myeloid leukemia (CML). Methods: We used an oligo(dT)-coated manifold support to capture the mRNA directly from the cell lysate. After reverse transcription, the cDNA was eluted from the manifold support, and BCR-ABL and GAPDH mRNAs were quantified in real time using the TaqMan fluorogenic detection system. Results: The detection limit of the method was one positive K562 cell among 105 negative cells. GAPDH was chosen as a reference gene based on the low variation between samples from different stages of the disease and the low signal in the absence of reverse transcription. The day-to-day variation of the method (CV) was 32%. In 43 blood samples from 13 CML patients, mRNA quantification agreed well with cytogenetic data. Conclusions: The proposed procedure constitutes a reproducible and sensitive BCR-ABL mRNA quantification method and is suitable to monitor minimal residual disease in CML patients.

Imatinib Mesylate Affects the Expression of Lipid Metabolism Genes in K562, a Chronic Myeloid Leukemia Cell Line

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4238-4238
Author(s):  
Osnat Ashur-Fabian ◽  
Adi Har-Zahav ◽  
Aviv Shaish ◽  
Ninette Amariglio ◽  
Dror Harats ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Real Time Pcr ◽  
Apolipoprotein B ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Lipid Lowering ◽  
Pcr Assays ◽  
Lipid Lowering Effect

Abstract Background: Imatinib mesylate (IM) is a tyrosine kinase inhibitor designed to inhibit the BCR-ABL fusion protein, the hallmark of chronic myeloid leukemia (CML). Interestingly, IM treatment possesses a rapid lipid-lowering effect on patients with hyperlipidemia, particularly those receiving 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR) inhibitors. While it is known that IM may competitively inhibit statin metabolism, the lipid-lowering effect of IM is not fully understood. Therefore, we have studied the effects of IM on a family of genes critically involved in lipid metabolism, including low density lipoprotein receptor (LDL-R), HMGCoAR, cholesteryl ester transfer protein (CETP), apolipoprotein B (apoB) and apolipoprotein B editing complex (apobec1), in K562, a CML cell line model. Methods: Cell cultures and treatments: K562 cells were grown in DMEM supplemented with 10% FCS. Cells were treated with IM or DMSO (1mM) and were harvested after 24–96 hours. Untreated cells served as a control as did the DMSO-treated cells. RNA extraction and cDNA synthesis: RNA was extracted using Trizol reagent method and reverse transcribed using random primers and M-MLV. Real-time PCR assays: Real-time PCR assays were conducted using assays on demand for the different genes (Applied Biosystems, Foster City, CA, USA) and performed on Applied Biosystems 7900HT Prism® real-time PCR instrument with a 100ng equivalent of cDNA, in a total volume of 20 ml 1X Taqman master mix (Applied Biosystems). Results were quantified using the delta-delta cycle threshold method, normalized to actin beta and expressed as “fold-change from control”. Statistics: Results were analyzed using unpaired students’ t-test. Results: LDL-R, HMGcoAR, CETP, apobec1 and apoB genes expression were evaluated in untreated K562 cells (control) and following treatment with DMSO (negative control) or IM for 24 to 96 hours. IM treatment of K562 cells significantly affected the expression of lipid-related genes. Namely, a time-dependent increase in LDL-R (3–6 fold), HMGcoAR (2–5 fold) and CETP (4–50 fold) expressions were documented following IM treatment. No increase in expression of these genes was observed in untreated and DMSO treated controls. Basal apobec1 and apoB expression detected in untreated K562 cells was abolished following IM but not DMSO treatment. Discussion: Putative mechanisms for the decrease in serum cholesterol and triglycerides observed in some patients treated with IM include reduced intestinal absorption and inhibition of platelet derived growth factor receptor (PDGFR), resulting in decreased lipoprotein lipase (LPL) synthesis. Our novel observation proposes another explanation, namely a direct effect of IM on central lipid-related genes. Conclusions: The direct effect of IM on lipid-related genes, together with the fact that in cell culture IM inhibits proliferation and induces apoptosis, suggest a possible role of lipids in apoptosis/cell cycle pathways. This possibility is currently the subject of further investigation.

scholarly journals Detection of the BCR-ABL Gene By the Real-Time PCR Method in Patients with Chronic Myeloid Leukemia in Rio Grande Do Norte, Brazil

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5432-5432
Author(s):  
Aldair Sousa Paiva ◽  
Hugo Diogenes De Oliveira Paiva ◽  
Geraldo Barroso Cavalcanti ◽  
Gioconda DR Leão ◽  
Marcos Dias Leão ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Rna Extraction ◽  
Philadelphia Chromosome ◽  
Genetic Material ◽  
Chromosome 9 ◽  
The Real

Abstract Background: The Philadelphia chromosome is a cytogenetic change resulting from a reciprocal translocation of genetic material between ABL genes from chromosome 9 and BCR from chromosome 22 or t(9; 22) (q34; 11), forming the chimeric gene BCR- ABL, being associated with chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). The p190 variant is usually associated with acute forms of leukemia, including AML and ALL, whereas the p210 variant is associated with the chronic phases of CML. Due to the high sensitivity and specificity, nucleic acid amplification techniques by real-time PCR have replaced the conventional cytogenetic techniques for the identification of the Philadelphia chromosome and its p190 and p210 variants. Molecular analysis has been indicated in the initial diagnostic phase and also for the therapeutic monitoring defining the percentage of neoplastic cells present in the patients during the different phases of the treatment (Minimum Residual Disease or MRD).The aim of this study was the transcript BCR-ABL identification in patients with suspected of CML and evaluation of the gene frequency in these patients. Methods: The presence of BCR-ABL gene was investigated in blood samples from 42 patients with suspected CML. The RNA extraction was performed by phenol/chloroform method. The cDNA was submitted to PCR, using specific primers for and BCR-ABL genes by Real time PCR. Results: From all studied patients, 16 (38.10%) were negative, and 26 (59.09%) positive for one of rearrangements: p210 b3a2 and b2a2 in 18 cases (40.91%) and p190 a1a2 in 2 cases (4,76%) and double positive p120/190 in 6 cases (14,28%). We observed that the most common rearrangement was the p210 b3a2, and the molecular results were compatible with clinical and hematologic suspicion. Conclusions: The Real-timePCR, because of its specificity and sensitivity, can be considered the most used technique in routine diagnosis and investigation of MRD of CML patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Frequency of coexistence and kinetics of the BCR-ABL1 transcript level and allele burden of JAK2V617F and CALR Type 1, 2 gene mutations in patients with chronic myeloid leukemia

2020 ◽  
Vol 65 (3) ◽  
pp. 253-280
Author(s):  
A. O. Abdullaev ◽  
E. A. Stepanova ◽  
T. V. Makarik ◽  
E. Y. Nikulina ◽  
S. A. Treglazova ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Transcript Level ◽  
Chimeric Gene ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Kinetics Of

Introduction. The pathogenesis of myeloproliferative neoplasms is associated with the chimeric gene BCR-ABL1 or with one of the driver mutations in the genes JAK2, MPL and CALR (Calreticulin). However, the classifi cation of the World Health Organization lists no myeloid neoplasms with more than one driver genetic abnormality. Aim. To search for mutations in the genes JAK2, MPL and CALR in patients with BCR-ABL1-positive chronic myeloid leukemia (CML), as well as to evaluate the kinetics of the discovered mutations during tyrosine kinase inhibitor (TKI) therapy. Materials and methods. mRNA and DNA samples isolated from blood and bone marrow cells of 567 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2019 period were included in the study The BCR-ABL1 transcript level was determined using a highly sensitive quantitative real-time polymerase chain reaction. The mutations JAK2V617F and MPLW515L/K were detected using real-time quantitative allele-specifi c polymerase chain reaction. Mutations in the CALR gene were investigated using fragment analysis followed by Sanger sequencing. Results. The combination of the BCR-ABL1, JAK2 and CALR gene mutations among CML patients receiving TKIs was 1.23 % (7/567). Out of these, the combination of BCR-ABL1 with JAK2V617F and the combination of BCR-ABL1 with CALR gene mutations were detected in 0.88 % (5/567) and 0.35 % (2/567) of cases, respectively. During TKI therapy, in 5 out of 7 patients, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these patients, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second-generation TKI preparations. Conclusions. The combination of the BCR-ABL1 chimeric gene with gene mutations Jak2 or CALR was a rare event and amounted to 0.88 and 0.35 % of cases, respectively. The combination of BCR-ABL1 with Jak2V617F and CALR mutations does not always impede the achievement of major MR.

FLT3, CD32, PU.1, ERG, uPAR, and TAP2 Are Strongly Associated with the Progression of Chronic Myeloid Leukemia and Combination of Small Interference RNA of FLT3 and STI571 Synergistically Induced Apoptosis of K562 Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4237-4237
Author(s):  
Kyung Im Kim ◽  
Kwang-Sung Ahn ◽  
Juwon Park ◽  
Chansu Lee ◽  
Byoung Kook Kim ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Mtt Assay ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Blast Crisis ◽  
Chronic Phase ◽  
K562 Cells ◽  
Potential Candidate

Abstract To characterize molecular mechanisms by which transition from chronic phase to blast crisis in chronic myeloid leukemia (CML) for developing novel therapeutic targets, we analyzed gene-expression profiles of leukemic cells from 12 patients in chronic phase and 9 patients in blast crisis using a 8.7K cDNA chip. We identified 89 genes that were up-regulated as well as 54 genes that were down-regulated in blast crisis of CML. The expression profile included oncogenes, tumor suppression genes, and human genes encoding proteins involved in transcription, signal transduction, metabolism, cell growth, differentiation, apoptosis and immune functions. 18 genes were selected among the up-regulated group for analysis using real-time PCR. Real-time PCR data indicated that the expression of FLT3 (p < 0.001), CD32 (p < 0.001), ERG (p < 0.001), uPAR (p < 0.001), MAD (p < 0.001) and TAP2 (p < 0.001) showed statistically significant difference between chronic phase and blast crisis. For further analysis, we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human FLT3. These small interfering RNA constructs significantly inhibited FLT3 expression at mRNA and protein levels in K562 cells. After treating both the FLT3 knockdown cells and control cells (FLT3 wild type) with STI571, MTT assay and the expression patterns of apoptosis related genes (PARP, caspase-3, Bax) were examined. MTT assay and caspase-3 activity assay showed that silencing of the gene for FLT3 significantly reduced cell viability and ultimately facilitated the induction of apoptotic cell death by STI571. These findings uncovered evidence of a complex signaling network operating down-stream of FLT3 that actively contributes to tumor progression. Thus, RNA interference-directed targeting of FLT3 can be a potential candidate anticancer agent in association with STI571 against chronic myeloid leukemia.

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