Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.
Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.
Shi
Yan
Y
Department of Hematology, Qilu Hospital, Shandong University, Jinan, 250012, People's Republic of China.
Li
Li-Zhen
LZ
Sun
Jian-Zhi
JZ
Zhang
Ti
T
Peng
Jun
J
Xu
Cong-Gao
CG
D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D016044:Fusion Proteins, bcr-abl / Q000032:analysis* / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008027:Light; D000090063:Proto-Oncogene Mas; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D017931:DNA Primers; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016044:Fusion Proteins, bcr-abl