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Comparison of genomic DNA and cDNA for detection of residual disease after
treatment of chronic myeloid leukemia with allogeneic bone marrow
transplantation
JG Zhang, F Lin, A Chase, JM Goldman and NC Cross
LRF Leukaemia Unit, Royal Postgraduate Medical School, Hammersmith
Hospital, London, UK.
To test whether patients in remission after allogeneic bone marrow
transplantation (BMT) possess a pool of chronic myeloid leukemia (CML)
cells that do not express BCR-ABL mRNA, we have compared the results and
sensitivity of amplification of BCR-ABL from genomic DNA with conventional
reverse transcription-polymerase chain reaction (RT-PCR). Bubble PCR was
used to amplify the genomic BCR-ABL translocation breakpoints from
chronic-phase DNA of 10 patients with CML who subsequently underwent BMT.
After cloning and sequencing of the amplification products,
patient-specific ABL primers were synthesized and tested for both
specificity and sensitivity in nested or heminested combinations with a
variety of primers derived from the major breakpoint cluster region of the
BCR gene. In all cases, combinations of primers were selected that enabled
the detection of chronic-phase DNA from a specific patient at up to a
10(5)x dilution into DNA from a normal individual. Patterns of residual
disease obtained by serial RT- PCR and DNA-PCR analyses of blood and bone
marrow samples obtained after BMT were similar for most patients, including
one treated for relapse by infusion of donor leukocytes. Of the 24 samples
for direct comparison of RT-PCR and DNA-PCR, results were concordant in 19
(79%) cases. Five results were discordant. In two instances, RT-PCR was
positive, while PCR from genomic DNA was negative; this discrepancy might
have arisen due to the slightly greater sensitivity of RT-PCR compared with
DNA-PCR. In three samples from three patients, two of whom had been
transplanted in the accelerated phase, PCR from genomic DNA was positive
while RT-PCR was negative; this could mean that some CML cells in these
samples had a reduced or absent capacity to express BCR-ABL mRNA
post-transplant. Of these three patients, one subsequently relapsed; and
two are in remission at 21 and 24 months after the discordant result. Thus,
the finding of a single DNA-PCR- positive, RT- PCR-negative results does
not necessarily predict relapse. Because the great majority of samples
(79%) gave concordant results with the two assays, we believe that patients
in remission do not generally harbor a substantial pool of CML cells that
do not express BCR-ABL mRNA.
Volume 87,
Issue 6,
pp. 2588-2593,
03/15/1996
Copyright © 1996 by The American Society of Hematology
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