Harmonization of quantitative <scp>BCR</scp>‐<scp>ABL</scp> measurements using the secondary reference material anchored to the <scp>WHO</scp> primary standards

et al. 2018

PLoS ONE

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called “the Eprobe leukemia assay,” for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

Section: Resultsmentioning

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

Tsuchiya

et al. 2018

PLoS ONE

“…Currently, several commercial kits using IS are available (22, 28); however, these are designed to test large-scale samples in commercial laboratories, rather than for hospital laboratories, in which relatively small numbers of samples are examined. Therefore, we developed an in-house RQ-PCR method that is more accurate and flexible than those currently in use (23). In the present study, we evaluated the relevance and accuracy of this in-house RQ-PCR method using samples obtained for the DOMEST trial.…”

Section: Discussionmentioning

confidence: 99%

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

Kitamura

Tsuchiya

et al. 2018

Preprint

21Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the 22 Philadelphia chromosome, have drastically improved the outcomes for patients with 23 chronic myeloid leukemia (CML). Although several real-time quantitative polymerase 24 chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are 25 commercially available, their accuracy and efficiency in laboratory practice require 26 reevaluation. We have developed a new in-house RQ-PCR method to detect minimal 27 residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML 28 from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate 29 discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the 30 international standard (IS) ratio, where IS < 0.01% was defined as a major molecular 31 response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a 32 widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML 33Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in 34 five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, 35BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) 36 that were not positive using the BML method. Nineteen samples were also tested using a 37 commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0007% Page3 38 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels 39 of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) 40 that were positive using the in-house method. These data suggest that our new in-house 41 RQ-PCR method is effective for monitoring MRD in CML. 42 43 44 48 (t(9;22)(q34;q11.2)), referred to as the Philadelphia chromosome, which generates BCR-49 ABL1 fusion transcripts. The BCR-ABL1 protein constitutively activates tyrosine kinase 50

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

Kitamura

et al. 2019

PLoS ONE

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1 / ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.