PBPC mobilization with chemotherapy and G-CSF in patients with chronic myeloid leukemia: quantification of bcr/abl-positive cells by interphase fluorescence in situ hybridization and competitive PCR.
BACKGROUND
Autografting of normal stem cells mobilized after chemotherapy is increasingly used in chronic myeloid leukemia (CML). Thus, quantification of possible contamination of progenitor cell apheresis with breakpoint cluster region (bcr)/Abelson murine leukemia (abl)-positive cells is of great clinical interest.


METHODS
Two molecular methods were compared to quantify bcr/abl positivity in leukapheresis components obtained after mobilizing chemotherapy in six patients with CML. To document the efficacy of in vivo purging, the leukapheresis procedures were monitored with interphase fluorescence in situ hybridization (FISH) and quantitative competitive PCR (QC-PCR) as a ratio of bcr/abl:abl.


RESULTS
From the first to the last leukapheresis, bcr/abl positivity in FISH increased from a median of 11 percent to 33 percent. For bcr/abl transcripts, a simultaneous increase in consecutive leukapheresis procedures was seen. The median percentage of bcr cells in a bcr/abl:abl ratio was 3.1 percent in the first apheresis. In the last apheresis after the mobilization with mRNA, the QC-PCR showed a median of 19.5 percent. FISH and QC-PCR showed a statistical significant increase of bcr/abl positivity from the first to the last apheresis.


CONCLUSIONS
Both FISH and QC-PCR were reliable methods of quantifying bcr/abl positivity, and they allowed selection of the optimal apheresis component for autologous transplantation. In both methods, a significant increase in bcr/abl positivity was seen from the first to the last leukapheresis. With FISH, results can be obtained within 24 hours. This method may prevent additional contaminated leukapheresis in case of increasing percentages of bcr/abl-positive cells.
Keil
F
F
Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria. felix.kiel@ akh-wie.ac.at
Mitterbauer
G
G
Chen
X
X
Haas
O A
OA
Kalhs
P
P
Lechner
K
K
Mannhalter
C
C
Moser
K
K
Worel
N
N
Pirc-Danoewinata
H
H
Louda
N
N
Loidolt
H
H
Rabitsch
W
W
Greinix
H T
HT
D000328:Adult; D002467:Cell Nucleus / Q000378:metabolism; D002908:Chronic Disease; D003561:Cytarabine / Q000494:pharmacology; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015255:Idarubicin / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D007937:Leukapheresis; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D015513:Oncogene Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D051562:Proto-Oncogene Proteins c-bcr
D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D003561:Cytarabine; D016179:Granulocyte Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr; D015255:Idarubicin