PMID- 17846772
OWN - NLM
STAT- MEDLINE
DCOM- 20080428
LR  - 20211203
IS  - 1432-0584 (Electronic)
IS  - 0939-5555 (Linking)
VI  - 87
IP  - 1
DP  - 2008 Jan
TI  - Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, 
      in-cell RT-PCR and flow cytometric method.
PG  - 35-41
AB  - 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.
FAU - Shi, Yan
AU  - Shi Y
AD  - Department of Hematology, Qilu Hospital, Shandong University, Jinan, 250012, 
      People's Republic of China.
FAU - Li, Li-Zhen
AU  - Li LZ
FAU - Sun, Jian-Zhi
AU  - Sun JZ
FAU - Zhang, Ti
AU  - Zhang T
FAU - Peng, Jun
AU  - Peng J
FAU - Xu, Cong-Gao
AU  - Xu CG
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070911
PL  - Germany
TA  - Ann Hematol
JT  - Annals of hematology
JID - 9107334
RN  - 0 (DNA Primers)
RN  - 0 (MAS1 protein, human)
RN  - 0 (Proto-Oncogene Mas)
RN  - EC 2.7.10.2 (Fusion Proteins, bcr-abl)
SB  - IM
MH  - Cell Line, Tumor
MH  - DNA Primers/*genetics
MH  - Flow Cytometry/*methods
MH  - Fusion Proteins, bcr-abl/*analysis/*genetics
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - *Light
MH  - Proto-Oncogene Mas
MH  - Reverse Transcriptase Polymerase Chain Reaction/*methods
MH  - Time Factors
EDAT- 2007/09/12 09:00
MHDA- 2008/04/29 09:00
CRDT- 2007/09/12 09:00
PHST- 2007/05/23 00:00 [received]
PHST- 2007/08/02 00:00 [accepted]
PHST- 2007/09/12 09:00 [pubmed]
PHST- 2008/04/29 09:00 [medline]
PHST- 2007/09/12 09:00 [entrez]
AID - 10.1007/s00277-007-0365-8 [doi]
PST - ppublish
SO  - Ann Hematol. 2008 Jan;87(1):35-41. doi: 10.1007/s00277-007-0365-8. Epub 2007 Sep 
      11.