Rapid detection of chimeric bcr/abl mRNAs in acute lymphoblastic and chronic myeloid leukemia by the polymerase chain reaction

1990 ◽  
Vol 61 (6) ◽  
pp. 350-353 ◽  
Author(s):  
Juergen Maurer ◽  
Eckhard Thiel
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Myeloid Leukemia ◽  
Rapid Detection ◽  
Myeloid Leukemia ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Detection of the molecular abnormality in chronic myeloid leukemia by use of the polymerase chain reaction

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 2063-2065
Author(s):  
A Dobrovic ◽  
KJ Trainor ◽  
AA Morley
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Myeloid Leukemia ◽  
Blood Cells ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Great Sensitivity ◽  
Chain Reaction ◽  
Molecular Abnormality ◽  
Polymerase Chain

The bcr-abl translocation characteristic of chronic myeloid leukemia (CML) was detected by the polymerase chain reaction (PCR) modified to use mRNA as the starting material. Amplification of a sequence spanning the bcr-abl junction was obtained by using peripheral blood cells from all of 20 patients with classic CML, one patient with acute lymphoblastic leukemia probably secondary to CML, and two cell lines derived from patients with CML. The presence of bcr exon 3 in the mRNA was determined from the size of the amplified sequence; it was present in 14 and absent in seven patients. One leukemic cell per 1,000 nonleukemic cells could be readily detected, thus indicating the great sensitivity of the method. This technique is of routine value in CML both for diagnosis and for following the course of treatment.

scholarly journals Detection of residual leukemia after bone marrow transplant for chronic myeloid leukemia: role of polymerase chain reaction in predicting relapse

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 874-878 ◽  
Author(s):  
TP Hughes ◽  
GJ Morgan ◽  
P Martiat ◽  
JM Goldman
Keyword(s):  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Chronic Myeloid Leukemia ◽  
Bone Marrow Transplant ◽  
Blood Cells ◽  
Myeloid Leukemia ◽  
Marrow Transplant ◽  
Early Assessment ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract We used the polymerase chain reaction (PCR) to detect residual leukemia- specific mRNA in blood and marrow from 37 patients in complete hematologic and cytogenetic remission after allogeneic bone marrow transplant (BMT) for chronic myeloid leukemia (CML). Our two-step PCR method involved the use of “nested primers” in the second step and could detect one K562 cell diluted into 10(5) normal cells. Elaborate measures were taken to exclude false-positive and false-negative results. In nine patients whose blood and marrow were studied simultaneously the results were concordant (two positive and seven negative). Twenty-three patients transplanted in chronic phase (CP) with unmanipulated donor marrow were studied. Blood cells from nine of these patients were studied 3 to 6 months post-BMT and six were PCR positive; three were negative on subsequent studies. Blood cells from 18 patients studied between 8 months and 8 years post-BMT were all PCR negative. Nine patients transplanted in CP with T-cell-depleted marrow cells were studied. Blood from five was positive 3 to 24 months post- BMT; blood from five was negative 3 to 6 years post-BMT. Four patients no longer in first CP were studied after BMT with unmanipulated donor marrow. Blood from all four was positive 5 to 19 months post-BMT. Based on the known clinical results of transplant in these three cohorts we conclude that PCR may be positive within 6 months of BMT in patients who can expect long-lasting remission, whereas PCR positivity later after BMT may indicate that the probability of cure is reduced. Thus, the technique may prove useful for early assessment of new transplant protocols that might inadvertently increase the risk of relapse.

scholarly journals A new method of "in-cell reverse transcriptase-polymerase chain reaction" for the detection of BCR/ABL transcript in chronic myeloid leukemia patients

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3822-3827 ◽  
Author(s):  
N Testoni ◽  
G Martinelli ◽  
P Farabegoli ◽  
A Zaccaria ◽  
M Amabile ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Single Cells ◽  
Fluorescent Microscopy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Methods of detecting minimal residual disease (MRD) in chronic myeloid leukemia (CML) include chromosome analysis, Southern blotting, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) techniques. We report a novel method to detect intracellular messenger RNA (mRNA) by combining the techniques of reverse transcription (RT) and PCR performed directly inside the cells, without extraction of the nucleic acid. We applied this method, which we call “in-cell RT-PCR”, to detect hybrid BCR/ABL transcript within single cells. After cellular permeabilization and fixation of single cells in suspension, the neoplastic mRNA was reverse transcribed into cDNA, and the cDNA was amplified by PCR with fluorescent primers, specific for bcr/abl. Flow cytometry was used to detect cells positive for the amplified DNA within the cell cytoplasm. After transferring the amplified cells onto slides by cytospin, the positive cells for BCR/ABL cDNA were observed by fluorescent microscopy. The technique was capable of detecting low abundancy signals and distinguishing different levels of gene expression. The amplification products were found in the cells and supernatants. The distribution was critically affected by the protease digestion condition. The specificity of amplification was confirmed by a nested RT-PCR of BCR/ABL performed on extracted mRNA from the same sample, and by reamplification of supernatants. We have used the technique to study 10 Ph+ CML patients and three normal subjects as controls. Four patients were 100% Ph+ at diagnosis time and RT-PCR+ at cytogenetic and molecular analysis, respectively. In-cell RT- PCR showed that the residual non-neoplastic cells could be observed in all cases. In two patients undergoing interferon-alpha (IFN-alpha) therapy and in four bone-marrow transplanted patients, the in-cell RT- PCR was used to compare the level of Ph+ positivity detected by cytogenetic analysis with the number of cells expressing BCR/ABL transcript. In this manner, we could estimate the MRD. Our preliminary application of the technique suggests that it is capable of accurately identifying cells transcribing bcr/abl, and that it may have significant clinical applications in the detection of MRD.

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.

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