title: Reverse transcriptase-polymerase chain reaction for bcr/abl fusion in chronic myelogenous leukemia.
abstract: Reverse transcriptase-polymerase chain reaction (RT-PCR) has shown promise as a means of detecting low levels of cells bearing the Philadelphia chromosome (Ph1) and for detecting cytogenetically inapparent ("masked") Ph1 in patients with chronic myelogenous leukemia (CML). For detection by karyotyping, dividing cells must be used, precluding use of peripheral blood samples in cases with low peripheral blood blast counts. Reverse transcriptase-polymerase chain reaction was performed in 83 bone marrow and 30 peripheral blood samples from patients with CML to compare results with karyotyping and to evaluate utility of this test on peripheral blood samples. Using isolated total cellular RNA and a single primer pair, cDNA was transcribed, amplified, electrophoresed, and probed for bcr/abl fusion involving M-bcr exons 2 and 3 of the bcr gene. Fifty-three samples were from untreated or conventionally treated patients (pre-BMT), and 60 were from patients who had undergone bone marrow transplantation (post-BMT). Fifty of 53 pre-BMT samples were positive by RT-PCR. Two samples, negative by RT-PCR, had complex translocations, t(9;16;22) and t(4;14;22). One case was indeterminate by RT-PCR, but positive on retesting. Forty-five of 53 had Ph1 by karyotyping; 8 were negative, including 5 peripheral blood samples, 2 bone marrow samples with "masked" Ph1, and 1 bone marrow sample with poor growth. Thirty-five of 60 post-BMT samples were positive by RT-PCR. Fourteen of 60 post-BMT samples had Ph1 by karyotyping. Of the RT-PCR+/Ph1- cases, most showed a weak but definite band by RT-PCR, suggesting a low level of the bcr/abl fusion gene. Nineteen patients had concurrent peripheral blood and bone marrow samples analyzed by RT-PCR and karyotyping. Of 16 patients with satisfactory RNA extraction, 15 had concordant results by RT-PCR. Five patients had adequate metaphase cells for karyotypic analysis. All had Ph1 in bone marrow, but were negative in peripheral blood. Our results indicate that RT-PCR for detection of bcr/abl fusion is more sensitive than karyotyping in pre- and post-BMT samples. Furthermore, RT-PCR can be successfully performed on peripheral blood, yielding excellent correlation with bone marrow samples.
authors.affiliation: Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Wells
authors.forename: C N
authors.initials: CN
authors.lastname: Phillips
authors.forename: E F
authors.initials: EF
authors.lastname: Winton
authors.forename: D C
authors.initials: DC
authors.lastname: Farhi
mesh_terms: D001483:Base Sequence; D001853:Bone Marrow / Q000473:pathology; D003001:Cloning, Molecular; D016313:Genes, abl; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase
chemical_list: D012194:RNA-Directed DNA Polymerase