title: Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients.
abstract: Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per microg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per microg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested, competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.
authors.affiliation: Department of Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: J Q
authors.initials: JQ
authors.lastname: Guo
authors.forename: H
authors.initials: H
authors.lastname: Lin
authors.forename: H
authors.initials: H
authors.lastname: Kantarjian
authors.forename: M
authors.initials: M
authors.lastname: Talpaz
authors.forename: R
authors.initials: R
authors.lastname: Champlin
authors.forename: M
authors.initials: M
authors.lastname: Andreeff
authors.forename: A
authors.initials: A
authors.lastname: Glassman
authors.forename: R B
authors.initials: RB
authors.lastname: Arlinghaus
mesh_terms: D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis / Q000235:genetics*; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012334:RNA, Neoplasm / Q000032:analysis*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D012334:RNA, Neoplasm; D016044:Fusion Proteins, bcr-abl