Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia.
The characteristic t(15;17) of acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR-alpha) gene on chromosome 17 to a gene on chromosome 15 called PML, a putative transcription factor. This distinct translocation results in a fusion mRNA detected by Northern analysis. Two cDNAs have been isolated that differ in the extent of 3' PML nucleic acid sequence contained. This study describes a reverse transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha fusion transcript, which amplifies PML/RAR-alpha mRNA from APL cells with either reported breakpoint. DNA sequencing of the predominant RT-PCR products from 6 patients showed identical RAR-alpha exonic breakpoints and two PML breakpoints. This RT-PCR assay was positive in leukemic cells from 30/30 APL patients with the molecular rearrangement confirmed by cytogenetics or Northern analysis. In leukemic cells of patients with a morphologic diagnosis of APL lacking the t(15;17) by routine cytogenetics, a positive RT-PCR assay predicted clinical response to all-trans-retinoic acid (RA) therapy. Dilutional studies with leukemic cells that express (NB4) or do not express (HL-60) a PML/RAR-alpha fusion mRNA reveal that this RT-PCR assay detects the transcript from as little as 50 pg of total RNA. In APL cells from 5/6 patients treated with RA alone, a complete response by clinical and cytogenetic criteria accompanied a persistently positive RT-PCR assay. This preceded relapse by 1-6 months. RT-PCR for PML/RAR-alpha mRNA provides a more-sensitive test for the t(15;17) than routine cytogenetics or Northern analysis. This molecular rearrangement detected by RT-PCR best defines this RA-responsive malignancy. The RT-PCR assay for the PML/RAR-alpha transcript yields important diagnostic and prognostic information in the management of APL patients.
Laboratory of Molecular Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
W H
WH
Miller
A
A
Kakizuka
S R
SR
Frankel
R P
RP
Warrell
A
A
DeBlasio
K
K
Levine
R M
RM
Evans
E
E
Dmitrovsky
D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics*; D002869:Chromosome Aberrations / Q000175:diagnosis*; D025063:Chromosome Disorders; D015870:Gene Expression; D015321:Gene Rearrangement; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018168:Receptors, Retinoic Acid; D014178:Translocation, Genetic; D014212:Tretinoin / Q000627:therapeutic use
D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D012194:RNA-Directed DNA Polymerase