Quantification of ALL-Type BCR/ABL Fusion Transcripts in Patients with Chronic Myelogenous Leukemia in Chronic Phase Using Real-Time Quantitative Polymerase Chain Reaction

The Philadelphia (Ph′) chromosome in chronic myelogenous leukemia (CML) results in fusion of the bcr gene and c-abl oncogene, which transcribes into two types of chimeric bcr/abl mRNAs: the L-6 junction and the K-28 junction. By means of a highly sensitive assay, combination of reverse transcription and polymerase chain reaction (RT/PCR), we analyzed 38 blood samples obtained from 31 patients with Ph′-positive CML and two patients with Ph′-negative bcr rearranged CML. Among the 21 samples obtained in chronic phase, eight patients had the L-6 mRNA, 11 had the K-28 mRNA, and two had both the L-6 and K-28 mRNAs. Among the nine samples obtained in blast crisis, four contained the L-6 mRNA, two contained the K-28 mRNA, and three contained both the K-28 and L-6 mRNAs. This finding supports the concept of alternative splicing of bcr/abl mRNAs transcribed in Ph′-positive CML. However, it appears to be a rare event. Of the eight samples obtained from eight patients who had achieved complete cytogenetic remission and negativity for bcr region rearrangement for 6 months to 3 years after recombinant alpha interferon (r alpha-IFN) therapy, all of them showed evidence of minimal residual Ph′-positive clones as detected by the RT/PCR assay. This finding suggests that interferon therapy suppresses the proliferation of the Ph′-positive clones, but it does not completely eradicate the Ph′-positive stem cells.

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Monitoring of Residual Disease in Chronic Myelogenous Leukemia by Quantitative Polymerase Chain Reaction and Clinical Decision Making

Blood ◽

10.1182/blood.v94.4.1486.416a37g_1486_1488 ◽

1999 ◽

Vol 94 (4) ◽

pp. 1486-1488 ◽

Cited By ~ 2

Author(s):

Thomas Lion

Keyword(s):

Decision Making ◽

Polymerase Chain Reaction ◽

Chronic Myelogenous Leukemia ◽

Clinical Decision Making ◽

Residual Disease ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Decision ◽

Myelogenous Leukemia ◽

Chain Reaction ◽

Polymerase Chain

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Detection of two alternative bcr/abl mRNA junctions and minimal residual disease in Philadelphia chromosome positive chronic myelogenous leukemia by polymerase chain reaction

Blood ◽

10.1182/blood.v73.8.2165.2165 ◽

1989 ◽

Vol 73 (8) ◽

pp. 2165-2170 ◽

Cited By ~ 84

Author(s):

MS Lee ◽

A LeMaistre ◽

HM Kantarjian ◽

M Talpaz ◽

EJ Freireich ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Chronic Myelogenous Leukemia ◽

Philadelphia Chromosome ◽

Rare Event ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Minimal Residual

Abstract The Philadelphia (Ph′) chromosome in chronic myelogenous leukemia (CML) results in fusion of the bcr gene and c-abl oncogene, which transcribes into two types of chimeric bcr/abl mRNAs: the L-6 junction and the K-28 junction. By means of a highly sensitive assay, combination of reverse transcription and polymerase chain reaction (RT/PCR), we analyzed 38 blood samples obtained from 31 patients with Ph′-positive CML and two patients with Ph′-negative bcr rearranged CML. Among the 21 samples obtained in chronic phase, eight patients had the L-6 mRNA, 11 had the K-28 mRNA, and two had both the L-6 and K-28 mRNAs. Among the nine samples obtained in blast crisis, four contained the L-6 mRNA, two contained the K-28 mRNA, and three contained both the K-28 and L-6 mRNAs. This finding supports the concept of alternative splicing of bcr/abl mRNAs transcribed in Ph′-positive CML. However, it appears to be a rare event. Of the eight samples obtained from eight patients who had achieved complete cytogenetic remission and negativity for bcr region rearrangement for 6 months to 3 years after recombinant alpha interferon (r alpha-IFN) therapy, all of them showed evidence of minimal residual Ph′-positive clones as detected by the RT/PCR assay. This finding suggests that interferon therapy suppresses the proliferation of the Ph′-positive clones, but it does not completely eradicate the Ph′-positive stem cells.

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Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Virology Journal ◽

10.1186/s12985-020-01467-y ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Yang Zhang ◽

Chunyang Dai ◽

Huiyan Wang ◽

Yong Gao ◽

Tuantuan Li ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Samples ◽

Chain Reaction ◽

Pcr Assay ◽

Qrt Pcr ◽

Highly Sensitive ◽

One Step ◽

Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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