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

Citation
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
Citations number
34
Categorie Soggetti
Research/Laboratory Medicine & Medical Tecnology
Journal title
MOLECULAR DIAGNOSIS
ISSN journal
10848592 → ACNP
Volume
4
Issue
3
Year of publication
1999
Pages
195 - 209
Database
ISI
SICI code
1084-8592(199909)4:3<195:ANNRPC>2.0.ZU;2-R
Abstract
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.