Diagnosis and monitoring of PML-RARalpha-positive acute promyelocytic leukemia by quantitative RT-PCR.
The last 15 yr have produced dramatic improvements in the survival rate of patients with acute promyelocytic leukemia (APL). These improvements have been due mainly to the introduction of targeted therapies and improved methods for diagnosing and monitoring this disease. The underlying molecular lesion in APL involves a t(15:17) translocation which leads to the generation of PML-RARalpha fusion transcripts and proteins. The PML-RARalpha fusion transcripts have been shown to be useful markers for establishing the diagnosis and for monitoring the response to treatment. This manuscript describes the application of QZyme reverse-transcription polymerase chain reaction (RT-PCR) to the quantification of PML-RARalpha transcripts as a marker of APL. QZyme is a method for real time detection and quantification of target genes or transcripts. The principle of QZyme analysis is similar to other quantitative PCR systems; however, the mechanism is quite different. QZyme exploits the catalytic activity of DNAzymes (deoxyribozymes), which are oligonucleotides that can bind and cleave nucleic acid substrates. The approach is well suited to monitoring minimal residual disease (MRD) in patients with APL, as a result of its ability to detect low numbers of transcripts and accurately measure differences in concentration over a broad dynamic range. Further, its capacity for duplex analysis has multiple advantages for analysis of clinical specimens. Protocols for duplex, single-tube QZyme RT-PCR assays, which allow simultaneous quantification of PML-RARalpha fusion transcripts (either L-type and V-type, or S-type) and the internal control BCR transcript, are provided. These protocols can be used for analyzing patient RNA specimens and are suitable for clinical trial monitoring. For this type of work, it is recommended that investigators validate the assays to ensure reproducible, accurate, and specific results on the equipment in their own laboratories. Assay validation is critical for real-time quantitative RT-PCR (RQ-PCR) and is often overlooked. A guide to the steps involved in validation and recommendations for acceptance criteria is included in this chapter.
Mokany
Elisa
E
Johnson & Johnson Research Pty Limited, NSW, Australia.
Todd
Alison V
AV
Fuery
Caroline J
CJ
Applegate
Tanya L
TL
D002884:Chromosomes, Human, Pair 15; D002886:Chromosomes, Human, Pair 17; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics*; D008991:Monitoring, Physiologic / Q000379:methods; D009363:Neoplasm Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic; D014178:Translocation, Genetic
D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D012334:RNA, Neoplasm; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein