title: Transcription-mediated amplification and hybridisation protection assay to determine BCR-ABL transcript levels in patients with chronic myeloid leukaemia.
abstract: Detection of BCR-ABL transcripts in chronic myeloid leukaemia (CML) is used to confirm the diagnosis and to monitor residual disease. Quantitative techniques are required to predict response to therapy or early relapse. We have evaluated an assay in which transcription-mediated amplification (TMA) of BCR-ABL and ABL transcripts is achieved using reverse transcriptase and RNA polymerase. The products are quantified in the hybridisation protection assay (HPA) using acridinium ester-labelled DNA probes and chemiluminescence. The method is a single tube procedure which uses small amounts of RNA (<500 ng/triplicate analysis), is technically simple (requiring just two waterbaths and a luminometer), rapid (total assay time <4 h) and sensitive (capable of detecting one BCR-ABL-positive K562 cell in the presence of 10(4)-10(5) BCR-ABL-negative cells). BCR-ABL signals from patient RNA samples were quantified relative to known amounts of K562 RNA and normalised to levels of ABL. BCR-ABL/ABL ratios ranged from 0.15 to 1.59 (median 0.65) in RNA from diagnostic blood or bone marrow of 18 CML patients and were < or =0.0001 in 20 normal controls. Sequential samples analysed from six CML patients post-allogeneic bone marrow transplantation who relapsed and received donor lymphocyte infusions showed BCR-ABL/ABL ratios which reflected patient status or treatment. A BCR-ABL/ABL ratio of 0.01 served as a useful arbitrary indicator value, with results above and below this value generally correlating with relapse or remission, respectively.
authors.affiliation: Department of Haematology, University College London, London, UK.
authors.forename: S E
authors.initials: SE
authors.lastname: Langabeer
authors.forename: R E
authors.initials: RE
authors.lastname: Gale
authors.forename: R C
authors.initials: RC
authors.lastname: Harvey
authors.forename: R W
authors.initials: RW
authors.lastname: Cook
authors.forename: S
authors.initials: S
authors.lastname: Mackinnon
authors.forename: D C
authors.initials: DC
authors.lastname: Linch
mesh_terms: D000166:Acridines; D016026:Bone Marrow Transplantation; D015342:DNA Probes; D016044:Fusion Proteins, bcr-abl / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D006082:Graft Enhancement, Immunologic; D006801:Humans; D006824:Hybridization, Genetic; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D009364:Neoplasm Recurrence, Local / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000032:analysis* / Q000096:biosynthesis; D012307:Risk Factors; D013388:Succinimides; D014158:Transcription, Genetic
chemical_list: D000166:Acridines; D015342:DNA Probes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D013388:Succinimides; C053333:4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium-9-carboxylate; D016044:Fusion Proteins, bcr-abl