Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML.
The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.
Kim
Yoo-Jin
YJ
Catholic Hemopoietic Stem Cell Transplantation Center, Catholic University of Korea, #62 Youido-dong, Youngdeungpo-gu, Seoul, Korea (South), 150-713.
Kim
Dong-Wook
DW
Lee
Seok
S
Kim
Hee-Je
HJ
Kim
Yoo-Li
YL
Hwang
Ji-Yeon
JY
Oh
Il-Hoan
IH
Park
Yoon-Hee
YH
Lee
You-Kyoung
YK
Min
Chang-Ki
CK
Kim
Tai-Gyu
TG
Han
Tae-Hee
TH
Min
Woo-Sung
WS
Kim
Chun-Choo
CC
D001853:Bone Marrow / Q000473:pathology; D005240:Feasibility Studies; D015321:Gene Rearrangement; D016313:Genes, abl / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000592:standards*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000235:genetics / Q000628:therapy; D008722:Methods; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards
D012333:RNA, Messenger