Real-time quantitative PCR: a reliable molecular diagnostic and follow-up tool for ‘minimal residual disease’ assessment in chronic myeloid leukemia

Molecular monitoring of BCR-ABL transcript levels by real-time quantitative PCR is increasingly being used to diagnose the disease and assess treatment response in patients with chronic myeloid leukemia (CML). This has become particularly relevant when residual levels of leukemia usually fall below the level of detection by cytogenetic analysis. Forty-two CML patients, including 18 males (42.86%) and 24 females (57.14%) aged 7–75 years, were enlisted for the study and followed-up for the response to imatinib treatment. Patients were subjected to Multiplex RT-PCR (reverse-transcriptase PCR) and were all found to harbor either e13a2 or the e14a2, which could be analyzed by a single Taqman probe based quantitation kit (Geno-Sen’s) to quantitate the BCR-ABL transcript load. The Multiplex RT-PCR and peripheral blood cytogenetics providing specific and sensitive detection of BCR-ABL fusion transcripts and metaphase signal load respectively were used as parallel reference tools to authenticate the q-PCR findings. There was 100% concordance between the multiplex RT-PCR and the q-PCR as every positive RT-PCR assay for a transcript reflected as q-PCR load of above 0% for that transcript. q-PCR also demonstrated a strong Pearson correlation with the cytogenetic response.

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A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib

PLoS ONE ◽

10.1371/journal.pone.0207170 ◽

2019 ◽

Vol 14 (3) ◽

pp. e0207170 ◽

Cited By ~ 3

Author(s):

Hiroaki Kitamura ◽

Yoko Tabe ◽

Tomohiko Ai ◽

Koji Tsuchiya ◽

Maiko Yuri ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Real Time ◽

Minimal Residual Disease ◽

Quantitative Pcr ◽

Myeloid Leukemia ◽

Residual Disease ◽

Real Time Quantitative Pcr ◽

Highly Sensitive ◽

Pcr Method ◽

Minimal Residual

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Sensitive Replicate Real-Time Quantitative PCR of BCR-ABL Shows Deep Molecular Responses in Long-Term Post–Allogeneic Stem Cell Transplantation Chronic Myeloid Leukemia Patients

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2015.06.018 ◽

2015 ◽

Vol 21 (10) ◽

pp. 1852-1855 ◽

Cited By ~ 1

Author(s):

Maya Koren-Michowitz ◽

Avichai Shimoni ◽

Filomena Daraio ◽

Francesca Crasto ◽

Roberta Lorenzatti ◽

...

Keyword(s):

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Stem Cell Transplantation ◽

Real Time ◽

Quantitative Pcr ◽

Allogeneic Stem Cell Transplantation ◽

Myeloid Leukemia ◽

Molecular Responses ◽

Real Time Quantitative Pcr

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Comparison of Real-Time Quantitative PCR and Digital Droplet PCR for BCR-ABL1 Monitoring in Patients with Chronic Myeloid Leukemia

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2019.08.007 ◽

2020 ◽

Vol 22 (1) ◽

pp. 81-89 ◽

Cited By ~ 8

Author(s):

Georg-Nikolaus Franke ◽

Jacqueline Maier ◽

Kathrin Wildenberger ◽

Michael Cross ◽

Francis J. Giles ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Real Time ◽

Quantitative Pcr ◽

Myeloid Leukemia ◽

Real Time Quantitative Pcr ◽

Digital Droplet Pcr ◽

Droplet Pcr

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Increased ABCG2 Expression Could Be Responsible for Resistance to Imatinib Mesylate in Patients with Chronic Myeloid Leukemia Who Do Not Have Mutations in BCR-ABL Kinase Domain

Blood ◽

10.1182/blood.v112.11.5026.5026 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5026-5026

Author(s):

Poonkuzhali Balasubramanian ◽

Preetha Markose ◽

Ezhilarasi Chendamarai ◽

Vikram Mathews ◽

Vivi Srivastava ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Real Time ◽

Imatinib Mesylate ◽

Quantitative Pcr ◽

Myeloid Leukemia ◽

Kinase Domain ◽

Mechanisms Of Resistance ◽

Expression Levels ◽

Real Time Quantitative Pcr ◽

Abl Kinase

Abstract Mutation in the Bcr-Abl kinase domain is one of the most common mechanism of resistance to imatinib mesylate (IM), seen in 30– 90% of patients with chronic myeloid leukemia (CML). A total of 57 patients with CML, 41 in chronic phase, 10 in accelerated phase and 6 in blast crisis, were analyzed for bcr-abl kinase domain (KD) mutations using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the bcr-abl KD followed by direct sequencing. Twelve different mutations including three novel mutations were identified in the bcr-abl KD in 18 patients. Resistance to IM was considered if the patient had rising white blood cell counts or did not achieve hematological remission at 6 months; or had rising bcr-abl levels (as determined by fluorescence in situ hybridization or real-time quantitative PCR) after the start of IM treatment. In order to evaluate other potential mechanisms of resistance in patients without mutations in the bcr-abl kinase domain, we tested the expression levels of transporter genes known to be involved in IM influx and efflux. mRNA expression levels of efflux transporters, ABCG2 and ABCB1, and influx transporter, hOCT1 were measured by real time quantitative PCR using ABL to normalize the expression levels of the same. Serially diluted cDNA made from HepG2 RNA was used to make the standard curve and amplification efficiencies of the target and the house keeping genes were similar. Each sample was analyzed in duplicate and the experiment was repeated twice. In the patients without mutations in the bcr-abl KD, significantly higher ABCG2 mRNA levels were observed compared to patients with mutations (Table). Transcript levels of ABCB1, hOCT1 or bcr-abl were not significantly different between the two groups. This study suggests that over expression of ABCG2 may be one of the mechanisms of resistance to imatinib in patients without mutations in bcr-abl. Future studies should not only compare the expression of these transporters at diagnosis (before the start of IM treatment) but also at the time of clinical resistance. This will help understand the influence of expression levels of these transporters in achieving haematological or molecular response to increased IM doses. Bcr-abl mutation positive (n=18) Bcr-abl mutation negative (n=39) p value ABCG2: median (range) 0.0126 (0.0004–0.45) 0.051 (0.0041–0.51) 0.034 ABCB1: median (range) 0.1842 (0.00349–0.51) 0.1819 (0.0463–0.68) NS hOCT1: median (range) 488 (3.4–3394) 486 (62–3059) NS BCR-ABL: median (range) 53.96 (5.73–179.1) 51.3 (2.36–129) NS

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Kappa deleting element as an alternative molecular target for minimal residual disease assessment by real-time quantitative PCR in patients with multiple myeloma

European Journal Of Haematology ◽

10.1111/ejh.12000 ◽

2012 ◽

Vol 89 (4) ◽

pp. 328-335 ◽

Cited By ~ 13

Author(s):

Noemí Puig ◽

María E. Sarasquete ◽

Miguel Alcoceba ◽

Ana Balanzategui ◽

María C. Chillón ◽

...

Keyword(s):

Multiple Myeloma ◽

Real Time ◽

Minimal Residual Disease ◽

Quantitative Pcr ◽

Residual Disease ◽

Molecular Target ◽

Disease Assessment ◽

Real Time Quantitative Pcr ◽

Minimal Residual

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Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Yeast ◽

10.1002/1097-0061(20000930)17:3<201::aid-yea30>3.0.co;2-r ◽

2000 ◽

Vol 1 (3) ◽

pp. 201-210 ◽

Cited By ~ 40

Author(s):

A. Al-Taher ◽

A. Bashein ◽

T. Nolan ◽

M. Hollingsworth ◽

Brady G.

Keyword(s):

Potassium Channel ◽

Single Cell ◽

Real Time ◽

Quantitative Pcr ◽

Single Cells ◽

Initial Step ◽

Small Samples ◽

Rt Pcr ◽

Real Time Quantitative Pcr ◽

Global Amplification

We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use ofTaqMan™real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst108total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

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