Diagnosis and molecular monitoring of acute promyelocytic leukemia using DzyNA reverse transcription-PCR to quantify PML/RARalpha fusion transcripts.
BACKGROUND
PML/RARalpha fusion transcripts provide a readily accessible marker for diagnosis of acute promyelocytic leukemia (APL) and for monitoring response to therapy. Survival rates are improved by therapies guided by such monitoring. We assessed the potential of DzyNA reverse transcription-PCR (RT-PCR) for measurement of PML/RARalpha fusion transcripts.


METHODS
Parallel single-tube DzyNA RT-PCR protocols were developed to allow real-time fluorescent quantification of PML/RARalpha fusion transcripts and a low abundance control transcript, normal BCR. Calibration curves, generated using cell line RNA, allowed estimation of these transcripts in RNA from patients with APL at various stages of the disease.


RESULTS
DzyNA RT-PCR calibration curves were linear for both transcripts over a broad range and demonstrated interassay variations of 12% (mean, 658 ng) and 10% (mean, 263 ng), respectively. The protocols detected low concentrations of transcripts and resolved twofold dilutions. PML/RARalpha mRNA was quantified in 10 patients at diagnosis and in 1 patient over a 7-year period. Monitoring of transcript concentrations effectively reflected the disease course in one patient and demonstrated that an increase in PML/RARalpha transcripts can be detected 4-6 months before hematologic relapse, with no false-positive results.


CONCLUSIONS
DzyNA RT-PCR has potential for use in clinical practice as a tool for diagnosis of APL and forsubsequent monitoring of minimal residual disease and detection of molecular relapse.
Applegate
Tanya L
TL
Johnson & Johnson Research Pty Limited, Australian Technology Park, Eveleigh NSW 1430, Australia. tapplega@medau.jnj.com
Iland
Harry J
HJ
Mokany
Elisa
E
Todd
Alison V
AV
D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000175:diagnosis* / Q000235:genetics; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein