Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 777-778
CORRESPONDENCE
 |
To the Editor: |
Absence of host-derived cells in the blood of patients in
remission after allografting for chronic myeloid leukemia
Chomel and colleagues1 recently reported that
study of blood or marrow cells from patients in complete cytogenetic
remission following treatment with interferon-
or allogeneic bone
marrow transplantation (BMT) for chronic myeloid leukemia (CML) by
fluorescence in situ hybridization (FISH) shows appreciable numbers of
BCR-ABL-positive cells. They examined 10 patients treated by BMT using
RT-PCR and BCR-ABL D-FISH for evidence of the fusion gene
at mRNA and genomic levels, respectively. In all cases the
BCR-ABL transcripts were undetectable (BCR-ABL/ABL
ratio less than 0.002%), whereas FISH studies of
interphase nuclei were positive at levels of 3%-11%. The authors
concluded that these patients must harbor appreciable numbers of
leukemic cells that carried a BCR-ABL fusion gene but did not
express BCR-ABL transcripts. In 2 patients, the donor was of
opposite sex and the level of genomic rearrangement was confirmed by XY
FISH, and in 2 patients the level was confirmed by Southern
hybridization. These results are surprising and are at odds
with our own experience of minimal residual disease in CML after BMT.
We have found BCR-ABL FISH to be technically demanding.
Although in experienced hands with good quality preparations the
technique can give a very low false positive rate of
0.1%-0.2%,2,3 in practice we prefer to use cutoff rate of
1% because accuracy is seriously compromised by several factors,
including slide quality and operator experience. A similar point has
recently been made in a multicenter study of BCR-ABL FISH in
clinical practice.4 In this study, slides scored with
the knowledge that they were normal were given a mean
BCR-ABL score of 0.2% (maximum 0.8%), whereas normal slides
scored blind with other mixed positive slides were given a
mean score of 1.0% (maximum 8.8%). In practice, therefore, the
expectations of the operator can substantially influence the number of
cells scored as positive.
To circumvent the problems associated with interpreting BCR-ABL
FISH data, we decided to study CML patients who had previously received
allogeneic stem cell transplants from opposite sex donors. We made
the assumption that residual leukemic cells in a patient's blood after
allografting would necessarily have the patient's sex
chromosome complement. If so, the proportion of cells with a
BCR-ABL fusion gene could not be greater than the
proportion with a host-type sex pattern. We used a commercial kit
(Vysis, Downer's Grove, IL) for the detection of X and Y
chromosomes in interphase cells. This probe has a false positive rate
less than 0.1% and is less subject to observer bias than are
BCR-ABL probes.5
We have examined 11 consecutive CML patients previously in remission
after BMT with a sex-mismatched donor
(Table).
Residual host cells were detected
using the commercial XY FISH kit, and BCR-ABL mRNA was detected
using RT-PCR as previously described.6 FISH slides were
scored blind with no knowledge of the RT-PCR result. Nine patients were
RT-PCR-negative, and 8 were also negative by FISH; one patient showed
a single poor-quality cell of apparently host sex, which we interpreted
as a false positive cell. Two patients were RT-PCR-positive and showed
very low levels of host cells by FISH. Using these techniques, we
therefore found no evidence of residual leukemic cells in
RT-PCR-negative patients. Three patients were also examined by
BCR-ABL FISH using a commercial kit (BCR-ABL ES probe;
Vysis) with no evidence of BCR-ABL-positive cells in excess of
our false positive rate of 1%.
A previous study by our group has shown similar results using a
different method. Patient-specific PCR primers were designed to amplify
genomic BCR-ABL rearrangements in CML patients at a sensitivity
of one leukemic cell in 105 cells. Ten patients underwent
treatment with BMT, and serial samples were analyzed for levels of
genomic and mRNA BCR-ABL using DNA-PCR with patient-specific
primers and RT-PCR, respectively. Eight patients became
RT-PCR-negative after BMT. All these patients also became
DNA-PCR-negative either concurrently or, in one case, shortly after.
Again, this study was not consistent with the hypothesis of common,
persistent genomic BCR-ABL rearrangement without mRNA expression.
In conclusion, we believe that our data demonstrate concordance of
BCR-ABL mRNA and DNA levels after BMT for CML. But the results
reported by Chomel et al deserve further attention because they would,
if confirmed by others, require radical revision of our concepts of
"minimal residual disease" after allografting for CML.
Andrew Chase
Sally Parker
Jaspal Kaeda
Renuka Sivalingam
Nicholas C. P. Cross
John M. Goldman
Department of Haematology
Imperial College of School of
Medicine
Hammersmith Hospital
London, England
| |
References |
1.
Chomel J-C, Brizard F, Veinstein A, et al.
Persistence of BCR-ABL genomic rearrangement in chronic myeloid leukemia patients in complete and sustained cytogenetic remission after interferon- therapy or allogeneic bone marrow transplantation.
Blood.
2000;95:404-409[Abstract/Free Full Text].
2.
Dewald GW, Wyatt WA, Juneau AL, et al.
Highly sensitive fluorescence in situ hybridization method to detect double BCR/ABL fusion and monitor response to therapy in chronic myeloid leukemia.
Blood.
1998;91:3357-3365[Abstract/Free Full Text].
3.
Grand FH, Chase A, Iqbal S, et al.
A two-color BCR-ABL probe that greatly reduces the false positive and false negative rates for fluorescence in situ hybridization in chronic myeloid leukemia.
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Dewald G, Stallard R, Al Saadi A, et al.
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[Order article via Infotrieve].
6.
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Zheng JG, Lin F, Chase A, Goldman JM, Cross NCP.
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