Expression of bcr-abl mRNA in individual chronic myelogenous leukaemia cells as determined by in situ amplification.
We present the results of a novel method developed for evaluation of in situ amplification, a molecular genetic method at the cellular level. Reverse transcription polymerase chain reaction (RT-PCR) was used to study bcr-abl transcript levels in individual cells from patients with chronic myelogenous leukaemia (CML). After hybridizing a fluorochrome-labelled probe to the cell-bound RT-PCR product, bcr-abl mRNA-positive cells were determined using image analysis. A dilution series of bcr-abl-positive BV173 into normal cells showed a good correlation between expected and actual values. In 25 CML samples, the percentage of in situ PCR-positive cells showed an excellent correlation with cytogenetic results (r = 0.94, P < 0.0001), interphase fluorescence in situ hybridization (FISH) (r = 0.95, P = 0.001) and hypermetaphase FISH (r = 0.81, P < 0.001). The fluorescence intensity was higher in residual CML cells after interferon (IFN) treatment than in newly diagnosed patients (P = 0.004), and was highest in late-stage CML resistant to IFN therapy and lowest in CML blast crisis (P = 0.001). Mean fluorescence values correlated with bcr-abl protein levels, as determined by Western blot analysis (r = 0.62). Laser scanning cytometry allowing automated analysis of large numbers of cells confirmed the results. Thus, fluorescence in situ PCR provides a novel and quantitative approach for monitoring tumour load and bcr-abl transcript levels in CML.
The University of Texas M.D. Anderson Cancer Center, Department of Molecular Haematology and Therapy, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
K
K
Pachmann
S
S
Zhao
T
T
Schenk
H
H
Kantarjian
A K
AK
El-Naggar
M J
MJ
Siciliano
J Q
JQ
Guo
R B
RB
Arlinghaus
M
M
Andreeff
D000704:Analysis of Variance; D015153:Blotting, Western; D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics* / Q000628:therapy; D008677:Metaphase; D012333:RNA, Messenger / Q000032:analysis*; D012074:Remission Induction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl