A rapid practical RT-PCR-based approach for the detection of the PML/RAR alpha fusion transcript in acute promyelocytic leukemia (original) (raw)
Related papers
British Journal of Haematology, 1992
Acute promyelocytic leukaemia (APL; AML M3) is identified by a unique t( 15;17) translocation which fuses the PML gene to the retinoic acid receptor alpha gene (RARA). Keverse transcription coupled with the polymerase chain reaction (RT-PCR) has been used to develop a diagnostic test for APL based on the PML-RARA fusion message. Separate PCR assays were designed to amplify either PML-RARA (1 5q + derived) or RARA-PML (1 7q-derived) chimaeric transcripts. PML-RARA transcripts were detected in every case from a series of 18 APL patients with cytogenetically confirmed t( 1 5; 1 7) translocations, whereas RARA-PML messages were detected in only 67% (12/18) of these
The Hematology Journal, 2001
Introduction: To study the relationship between the expression level of the PML-RARa fusion transcripts and the clinical status and eciency of the therapy in acute promyelocytic leukemia (APL) patients, we applied a very sensitive and speci®c real-time Reverse Transcription Polymerase Chain Reaction (RT ± PCR) system to quantify the dose of PML-RARa fusion transcripts in a series of APL patients at distinct disease stages. Materials and methods: A total of 31 APL patients (19 males and 12 females; aged from 8 to 74 years) from eight hospitals in Shanghai were analysed. Real-time Quantitative RT ± PCR was used to measure the normalized dose (Dose N ) of PML-RARa fusion transcripts. Results: A wide range of PML-RARa Dose N above 1610 3 was noted in 25 newly diagnosed patients. PML-RARa Dose N was signi®cantly decreased after remission induction with ATRA, ATRA/chemotherapy or As 2 O 3 and further reduced after consolidation. The fact that all patients with long disease free survival had a constantly low PML-RARa Dose N below 2610 2 and a higher level predicted impending relapse suggests that this value could serve as à threshold' for molecular remission. PML-RARa Dose N was also of prognostic value in a group of relapsed patients, since good response to As 2 O 3 reinduction was accompanied by a remarkable reduction of fusion transcript level, whereas patients with high PML-RARa Dose N after the second CR tended to relapse again rapidly. Conclusion: These results con®rm that real-time RT ± PCR assay for PML-RARa transcripts in APL patients is useful in re¯ecting leukemic burden, assessing response to treatment and indicating the ultimate clinical outcome or curability of disease.
Vojnosanitetski pregled
Introduction. The accurate diagnosis of acute promyelocytic leukemia (APL), not only on the morphological and clinical, but also on the molecular level, is very important for application of targeted therapies. Case report. A 62- year-old woman presented with APL. By using conventional cytogenetic analysis as well as applying the fluorescence in situ hybridization (FISH) analysis it has not been possible to confirm the presence of t(15;17) in the presented patient. Using reverse transcriptase polymerase chain reaction (RT-PCR) two atypical promyelotic leukemia/ retinoic acid receptor alpha (PML/RAR-?) fusion transcripts were identified. Both detected transcripts were isoforms. The larger transcript was in-frame, coding for functional aberrant PML/RAR-? protein, while the shorter transcript was an out-of-frame. Conclusion. Our study highlights the need for the application of molecular methodology in daily clinical practice. Precise characterization of PML/RAR-? fusion transcript creat...
Blood, 1995
Of 113 acute promyelocytic leukemia cases documented to have diagnostic PML-RAR alpha hybrid mRNA, 10 cases (8.8%) had fusion sites in PML gene exon 6 (V-forms) rather than in the two common hybrid mRNA configurations resulting from breaksites in either PML gene intron 6 (L-forms) or intron 3 (S-forms). In 4 V-form cases, a common break/fusion site was discovered at PML gene nucleotide (nt) 1685, abutting a 3' cryptic splice donor sequence. The fusion site was proximal to the common site in 1 case and more distal in 5 cases. The open reading frame encoding a PML-RAR alpha gene was consistently preserved, either by an in-frame fusion site or by the insertion of 3 to 127 unidentified nts. In 2 V-form cases, hybridization analysis of the reverse transcriptase-polymerase chain reaction products with a PML-RAR alpha juction probe was required for discrimination from L-form cases. Two V-form subgroups were defined by in vitro sensitivity to all-trans retinoic acid (tRA)-induced differ...
…, 1992
The acute promyelocytic leukemia (APL) t(15; 17) translocation generates a myl/retinoic acid receptor-(RAR-) chimeric gene that is transcribed as a fusion myl/RAR-amessenger RNA. Using primer sets derived from RAR-cr and my1 cDNAs, we were able to amplify the breakpoint sites of the fusion transcripts of all 35 APL RNA samples by reverse polymerase chain reaction (PCR) and nested primer approach of two rounds of amplification. DNA fragments of different size were obtained according to the chromosome 15 breakpoints (intron 3-bcr 3; exon &bcr 2; and intron 6-bcr 1). bcr 1 and bcr 3 represent the regions of the my1 locus most frequently involved among APL (48.5 and 34.2 of cases, HE MOLECULAR definition of chromosomal translo-T cation associated with hematopoietic tumors has provided important tools, either at the DNA or RNA level, for the diagnosis and the monitoring of patient response to therapy. Examples are the rearrangements of the bcr and c-ab1 genes in the t(9;22) of chronic myeloid leukemia,' the c-myc and Ig genes in the t(8;14) of Burkitt's lymphoma? and the bcl-2 in the t(14;18) of follicular lymphomas? A consistent translocation t(15;17) is present in the blasts of the majority of acute promyelocytic leukemia (APL) patients? Recently, we and others have identified that the two genes involved in the t(15;17) are my1 (also designated PML) on chromosome 15 and retinoic acid receptor-a (RAR-a) on chromosome 17.5-8 Using DNA probes representative of the chromosome 17 and 15 breakpoint regions, the t(15;17) could be identified in 100% of APL cases? Chimeric RAR-a/myl and myl/RAR-a are generated as a consequence of the reciprocal translocation between the my1 and RAR-a loci.10-12 The chimeric myl/RAR-a and
Expression pattern of the RAR alpha-PML fusion gene in acute promyelocytic leukemia
Proceedings of the National Academy of Sciences, 1992
Two chimeric genes, PML-RARa and RARa-PML, are formed as a consequence of the acute promyelocytic leukemia (APL)-specific reciprocal tranlocation of chromosomes 15 and 17 [t(15;17)]. PML-RARa is expressed as a fusion protein. We investigated the organization and expression pattern of the RARa-PML gene in a series of APL patients representative of the molecular heterogeneity of the t(15;17) and found (i) two types of RARa-PML mRNA junctions (RARa exon 2/PML exon 4 or RARa exon 2/PML exon 7) that maintain the RARa and PML longest open reading frames aligned and are the result of chromosome 15 breaking at two different sites; and (us) 10 different RARe-PML fusion transcripts that differ for the assembly of their PML coding exons.
Clinical Chemistry, 2002
Background: PML/RAR␣ 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/RAR␣ fusion transcripts. Methods: Parallel single-tube DzyNA RT-PCR protocols were developed to allow real-time fluorescent quantification of PML/RAR␣ 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/RAR␣ 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/RAR␣ transcripts can be detected 4 -6 months before hematologic relapse, with no false-positive results. Conclusion: DzyNA RT-PCR has potential for use in clinical practice as a tool for diagnosis of APL and for subsequent monitoring of minimal residual disease and detection of molecular relapse.
Clinical chemistry, 2002
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. 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. 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 quanti...