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
H. Rennert et al., 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, MOL DIAGN, 4(3), 1999, pp. 195-209
Background: The development of a rapid and simple reverse-transcription pol
ymerase chain reaction (RT-PCR) assay is described that identifies the prom
yelocytic leukemia-retinoic acid receptor alpha (PML-RAR alpha) hybrid mess
enger RNA (mRNA), a characteristic feature of acute promyelocytic leukemia
(APL).
Methods and Results: Randomly primed complementary (cDNA) is synthesized fr
om leukocyte RNA and amplified in the presence of Taq Gold in 2 separate re
action 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 pro
ducts generated from each RNA transcript (S, L, or V forms) are readily and
unambiguously distinguishable after agarose gel electrophoresis without, t
he need for either nested PCR or hybridization. The sensitivity of the assa
y is 1 in 10,000 to 1 in 100,000.The separate amplification of a beta(2)-mi
croglobulin 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, bo
th for the diagnosis of APL and the assessment of minimal residual disease
(MRD) after therapy. RT-PCR detected PML-RAR alpha mRNA in all cases positi
ve 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, r
espectively, that were positive by RT-PCR were negative by cytogenetics. Th
e ratio of cases with L-form to S-form PML-RAR alpha fusion transcript was
2:1, whereas 3 cases (10%) had fusion sites in exon 6 of the PML gene (V fo
rms). In addition, approximately 50% of the patients were diagnosed morphol
ogically with microgranular M3V-type leukemia, but no significant correlati
on with PML breakpoints was found.
Conclusion: The current assay is rapid, sensitive, and specific without usi
ng nested PCR or hybridization.