Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1).
We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending on WT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitating WT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-time WT1 assay had high reproducibility. WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed among WT1 transcript values normalized with 3 different control genes (beta-actin, ABL, and glyceraldehyde-3-phosphate dehydrogenase) and between relative WT1 transcript values with WT1 expression in K562 cells as the reference and absolute WT1 transcript copy numbers per microgram RNA. When WT1 expression and minor bcr-abl expression were concurrently monitored in 2 patients with bcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of a WT1 assay.
Tamaki
Hiroya
H
Department of Molecular Medicine, Osaka University Graduate School of Medicine, Suita City, Osaka, Japan.
Mishima
Machiko
M
Kawakami
Manabu
M
Tsuboi
Akihiro
A
Kim
Eui Ho
EH
Hosen
Naoki
N
Ikegame
Kazuhiro
K
Murakami
Masaki
M
Fujioka
Tatsuya
T
Masuda
Tomoki
T
Taniguchi
Yuki
Y
Nishida
Sumiyuki
S
Osumi
Kazuoki
K
Soma
Toshihiro
T
Oji
Yusuke
Y
Oka
Yoshihiro
Y
Kawase
Ichiro
I
Sugiyama
Haruo
H
Ogawa
Hiroyasu
H
D018628:Gene Dosage; D016162:Genes, Wilms Tumor; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D018365:Neoplasm, Residual / Q000175:diagnosis*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012333:RNA, Messenger