title: A novel approach to quantitating leukemia fusion transcripts by qRT-PCR without the need for standard curves.
abstract: Acute myeloid leukemia patients with recurrent cytogenetic abnormalities including inv(16);CBFB-MYH11 and t(15;17);PML-RARA may be assessed by monitoring the levels of the corresponding abnormal fusion transcripts by quantitative reverse transcription-PCR (qRT-PCR). Such testing is important for evaluating the response to therapy and for the detection of early relapse. Existing qRT-PCR methods are well established and in widespread use in clinical laboratories but they are laborious and require the generation of standard curves. Here, we describe a new method to quantitate fusion transcripts in acute myeloid leukemia by qRT-PCR without the need for standard curves. Our approach uses a plasmid calibrator containing both a fusion transcript sequence and a reference gene sequence, representing a perfect normalized copy number (fusion transcript copy number/reference gene transcript copy number; NCN) of 1.0. The NCN of patient specimens can be calculated relative to that of the single plasmid calibrator using experimentally derived PCR efficiency values. We compared the data obtained using the plasmid calibrator method to commercially available assays using standard curves and found that the results obtained by both methods are comparable over a broad range of values with similar sensitivities. Our method has the advantage of simplicity and is therefore lower in cost and may be less subject to errors that may be introduced during the generation of standard curves.
authors.affiliation: ARUP Laboratories, Salt Lake City, UT, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Schumacher
authors.affiliation: ARUP Laboratories, Salt Lake City, UT, USA.
authors.forename: N
authors.initials: N
authors.lastname: Scott Reading
authors.affiliation: ARUP Laboratories, Salt Lake City, UT, USA.
authors.forename: Philippe
authors.initials: P
authors.lastname: Szankasi
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, UT, USA.
authors.forename: Anna P
authors.initials: AP
authors.lastname: Matynia
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, UT, USA. Electronic address: todd.kelley@path.utah.edu.
authors.forename: Todd W
authors.initials: TW
authors.lastname: Kelley
mesh_terms: D002869:Chromosome Aberrations; D050658:Core Binding Factor beta Subunit / Q000235:genetics / Q000378:metabolism; D003362:Cost-Benefit Analysis; D053938:DNA Fragmentation; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D000071156:Promyelocytic Leukemia Protein; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013048:Specimen Handling; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C493692:CBFB protein, human; D050658:Core Binding Factor beta Subunit; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D000071156:Promyelocytic Leukemia Protein; D012333:RNA, Messenger; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human
keywords: Acute myeloid leukemia; Fusion transcripts; Molecular testing; Polymerase chain reaction; inv(16); t(15;17)