A novel, non-nested reverse-transcriptase polymerase chain reaction (RT-PCR) test for the detection of the t(15;17) translocation: a comparative study of RT-PCR cytogenetics, and fluorescence In situ hybridization.
BACKGROUND
The development of a rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) assay is described that identifies the promyelocytic leukemia- retinoic acid receptor alpha (PML-RARa) hybrid messenger RNA (mRNA), a characteristic feature of acute promyelocytic leukemia (APL).


RESULTS
Randomly primed complementary (cDNA) is synthesized from leukocyte RNA and amplified in the presence of Taq Gold in 2 separate reaction tubes containing primer pairs specific for intron 3 (bcr 3, long [L] form mRNA transcript) and intron 6 (bcr 1, short [S] form)/exon 6 (bcr 2, variant [V] form) breakpoints in PML, respectively. The different sized products generated from each RNA transcript (S, L, or V forms) are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. The sensitivity of the assay is 1 in 10,000 to 1 in 100,000. The separate amplification of a b2-microglobulin transcript controls for adequate RNA and cDNA preparation. The newly developed assay was used clinically for the evaluation of 78 patients with APL. It was rapid and more sensitive than cytogenetic karyotyping, both for the diagnosis of APL and the assessment of minimal residual disease (MRD) after therapy. RT-PCR detected PML-RARa mRNA in all cases positive for the t(15;17) translocation by cytogenetics. However, as many as 50% and 80% of the diagnostic specimens and the specimens for MRD assessment, respectively, that were positive by RT-PCR were negative by cytogenetics. The ratio of cases with L-form to S-form PML-RARa fusion transcript was 2:1, whereas 3 cases (10%) had fusion sites in exon 6 of the PML gene (V forms). In addition, approximately 50% of the patients were diagnosed morphologically with microgranular M3V-type leukemia, but no significant correlation with PML breakpoints was found.


CONCLUSIONS
The current assay is rapid, sensitive, and specific without using nested PCR or hybridization.
Rennert
H
H
Department of Pathology and Laboratory Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania, USA.
Golde
T
T
Wilson
R B
RB
Spitalnik
S L
SL
Van Deerlin
V M
VM
Leonard
D G
DG
D014408:Biomarkers, Tumor / Q000235:genetics*; D001856:Bone Marrow Examination; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics / Q000648:ultrastructure*; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics / Q000648:ultrastructure*; D005091:Exons / Q000235:genetics; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D015473:Leukemia, Promyelocytic, Acute / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D008297:Male; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009363:Neoplasm Proteins / Q000235:genetics*; D018365:Neoplasm, Residual; D009360:Neoplastic Cells, Circulating; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012191:Retroviridae Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014178:Translocation, Genetic
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012191:Retroviridae Proteins; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D012194:RNA-Directed DNA Polymerase