Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 778-780
CORRESPONDENCE
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To the Editor: |
No evidence for persistence of BCR-ABL-positive cells in patients
in molecular remission after conventional allogeneic transplantation
for chronic myeloid leukemia
Appropriate monitoring of patients after
allogeneic stem cell transplantation (SCT) for chronic myeloid leukemia
(CML) is crucial, because the response to donor lymphocyte infusions is superior in the case of cytogenetic or molecular relapse compared with
hematological relapse.1 Chomel et al recently reported that
fluorescence in situ hybridization (FISH) performed on
interphase nuclei (IP-FISH) detects between 2% and 11%
BCR-ABL-positive cells in the blood or bone marrow of CML patients in
molecular remission (RT-PCR-negative) after allogeneic bone marrow
transplantation.2 The authors concluded that the BCR-ABL
fusion gene was transcriptionally silent in the FISH-positive cells.
We analyzed our database for specimens that fulfilled the
following criteria: (1) classical allogeneic SCT (ie, conventional conditioning) for CML; (2) RT-PCR for BCR-ABL negative; (3) IP-FISH performed with LSI bcr/abl ES kit (Vysis, Stuttgart,
Germany). This probe has a low false-positive rate since it detects an
additional red signal on the derivate chromosome 9. Fourteen patients
after sex-mismatched transplantations and 17 patients after sex-matched transplantations for Ph-positive CML were identified (Table
1). RT-PCR was performed as
described.3 The false-positive rate of the PI-FISH system
was determined in patients with Ph-negative disorders and found to be
in the range of 0.1% (Table 2). In the
patients after allografting, a mean of 360 (range, 135-500) nuclei per
specimen were analyzed. Chimerism was determined by sex chromosome specific probes (SO CEP X /SG CEP Y,
Vysis), with a mean of 496 (range, 100-1000) nuclei per sample
analyzed. If mixed chimerism was detected, cohybridization with BCR-ABL
probes (LSI bcr/abl dual color translocation probe, Vysis)
and sex chromosome probes was carried out; the respective signals,
although of identical colors, could be distinguished based on their
different sizes (Figure).
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Cohybridization of sex-chromosome specific probes and BCR-ABL
fusion probes. (A) BCR-ABL-positive cell male cell. Thin arrows
point to the ABL (red) and BCR (green) signals; the thick arrow points
to the BCR-ABL fusion. The large signals identify the X (red) and Y
(green) chromosomes. (B) BCR-ABL-negative male cell. Thin arrows point
to the 2 separate ABL (red) and BCR (green) signals. The large signals
identify the X (red) and Y (green) chromosomes. Note that the
"conventional" dual-color color FISH probe (without additional
red signal on the derivative chromosome 9) was used in these
experiments.
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Eleven out of 14 patients with sex-mismatched donors were
found to be complete chimeras at a median of 3.5 years after
transplantation. In a total of 24 samples, no BCR-ABL-positive
interphases were detectable in these patients (Table 1).
Three out of 14 patients were mixed chimeras (90%-99.8% donor, total
of 6 samples). Cohybridization revealed that the recipient cells were
BCR-ABL-negative and thus represented residual normal hematopoiesis.
Seventeen patients after sex-matched allografting were followed by FISH
for BCR-ABL only, and a total of 26 samples were analyzed at a mean of
2.1 years after tranplantation. Only one sample was positive (6 of 500 cells).
In our series only a single specimen scored positive, with a
value just above our cutoff (mean + 3 SD = 4.6 for 500 analyzed nuclei). Moreover, complete donor chimerism was present in
approximately 80% of patients after sex-mismatched transplantation,
which rules out the presence of any substantial number of
Ph-positive cells. In the remaining patients recipient cells were
present but they were exclusively BCR-ABL-negative and thus
represented residual Ph-negative hematopoiesis that survived the conditioning.
It is not easy to explain the discordance between the findings of
Chomel et al and our results. Different FISH systems were used in the 2 studies (Chomel et al used one from Oncor, and we used one from Vysis),
but it is not readily obvious why this should result in such a great
discrepancy. False-positive rates appear to be similar in both systems
and are in the range of 0.1%. But Chomel et al studied only 1000 cells
from Ph-negative patients, which might have led to an underestimate of
the false-positive rate in their system. Over 30 000 nuclei were
analyzed to establish the false-positive rate in our laboratory. In
order to resolve this issue it would be crucial to directly compare the
performance of both FISH systems in the same specimens. Although our
results refer exclusively to patients after allogeneic SCT, it
should be noted that Chomel et al also found between 1% and
12% Ph-positive cells in 11 complete cytogenetic responders (CCR) to
therapy with IFN-alpha. Although this is certainly less surprising
than in allografted patients, it is still in contrast to a previous
report that employed the same FISH probe and found a normal
pattern in 4 out of 6 specimens from CCR.4
In conclusion, in our hands RT-PCR reliably predicts negative IP-FISH.
Conversely, reemergence of IP-FISH positive nuclei likely indicates
relapse and warrants close follow-up.
Michael Deininger
Tornalf Lehmann
Rainer Krahl
Eveline Hennig
Christel Müller
Dietger Niederwieser
Department of Hematology/Oncology
University of Leipzig
Leipzig, Germany
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References |
1.
Dazzi F, Szydlo RM, Goldman JM.
Donor lymphocyte infusions for relapse of chronic myeloid leukemia after allogeneic stem cell transplant: where we now stand.
Exp.Hematol.
1999;27:1477-1486[Medline]
[Order article via Infotrieve].
2.
Chomel JC, 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-alpha therapy or allogeneic bone marrow transplantation.
Blood.
2000;95:404-408[Abstract/Free Full Text].
3.
Schulze E, Krahl R, Thalmeier K, Helbig W.
Detection of bcr-abl mRNA in single progenitor colonies from patients with chronic myeloid leukemia by PCR: comparison with cytogenetics and PCR from uncultured cells.
Exp.Hematol.
1995;23:1649-1654[Medline]
[Order article via Infotrieve].
4.
Buno I, Wyatt WA, Zinsmeister AR, Dietz-Band J, Silver RT, Dewald GW.
A special fluorescent in situ hybridization technique to study peripheral blood and assess the effectiveness of interferon therapy in chronic myeloid leukemia.
Blood.
1998;92:2315-2321[Abstract/Free Full Text]
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Response: |
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
We appreciate the letters by M. Deininger et al and by A. Chase et al regarding our report.1 Both letters mainly
address the important question of the residual disease after bone
marrow transplantation (BMT) for chronic myeloid leukemia (CML)
chronic-phase patients. M. Deininger et al reported their own series of
patients undergoing transplantation. The residual disease was assessed on 14 patients after sex-mismatched transplantation and on 17 patients
after sex-matched transplantation. For that purpose, residual disease
was assessed using sex-chromosome-specific probes from Vysis. With
these probes, most of their patients were found to be FISH negative
with complete donor chimerism in 80% of patients. Three out of 14 patients were mixed chimeras, but cohybridization with BCR-ABL probes
was negative. For these 3 cases it is not easy to explain why residual
Ph-negative hematopoiesis survived the conditioning regimen while
leukemic cells, which are likely to be more resistant, were totally
eliminated. In addition we feel that it is not so easy to score nuclei
after cohybridization and to distinguish signals based on their
different sizes. Different delays from the times of BMT and of FISH
analyses could explain the discrepancies between the studies. Hence the
FISH analysis was performed at a median of 3.5 years after
transplantation in the study by Deininger et al whereas it was done at
a median of 2 years afterward in our study. Thus it is possible that
total elimination of the leukemic cells would take a more prolonged period of time. But in our study patient 13 was persistently positive at 56 months after transplantation (with BCR-ABL probes, Y probes, and
Y amplification by RT-PCR). The other 2 studies used Vysis probes, whereas we used the Oncor system. But we agree with Deininger that these 2 systems should not produce substantial differences. Chase
et al have studied the residual disease of 11 consecutive CML patients
who underwent BMT with a sex-mismatched donor. The use of X or Y FISH
probes showed that 8 patients were negative with no host
cells, and 3 were also negative by FISH with the use of BCR-ABL probes
from Vysis. But the absence of host cells does not demonstrate that no
more leukemic cells are present in the bone marrow, although we agree
that residual leukemia blood cells would have the patient's sex
chromosome complement. Chase et al referred also to their previous
study in which they suggested that, at the DNA level, patients were
cured after BMT.2 For that purpose they used patients'
specific PCR primers for genomic DNA amplification, that is, DNA-PCR.
But they also found discrepancies with 3 patients who were
DNA-PCR-positive and BCR-ABL- mRNA-negative by RT-PCR. It is obvious,
however, that their results challenge our data.
We agree with Deininger et al and Chase et al that BCR-ABL FISH is
technically demanding. The analysis of each nucleus requires a
sufficient duration of observation under the microscope in order to
detect the fusion signal indicating a cell possessing the
translocation. We also paid great attention to the
preparation of the slides: only high-quality slides were
scored. To avoid false positive signals, we performed 2 controls: (1) dilutions of Ph+ cells in
Ph
cells analyzed by FISH and Southern-blot that
exhibited a very good concordance between the 3 methods even in
the low percentage of Ph+ cells and (2) the study of 1000 nuclei from patients with hematologic disorders
other than CML showing a false positive rate of 0.1%. This study of 1000 nuclei is sufficient to determine the false positive
rate because we believe that an accurate and sufficient duration of
observation of each nucleus is more important than the observation of a
large number of nuclei. Indeed, the direct observation of fusion
signals is sometimes difficult and can be underestimated. Moreover, to
avoid false negative results, some nuclei were computerized in order to
validate the detection of the fusion signal. The slides were also
scored without knowledge of the RT-PCR results, and this was done for
the IFN-treated patients, as well as for the BMT- treated patients.
An increased risk of leukemia relapse has been associated with
the detection of BCR-ABL mRNA in bone marrow
transplant patients, particularly if such detection has been observed
repeatedly more than 100 days after
transplantation.3 Moreover, an increased relapse rate was
noticed if BCR-ABL transcripts were detected between 6 and 12 months
after transplantation.4 Conversely, a period of sustained
PCR negativity is generally associated with a high probability of cure
or prolonged leukemia-free survival. Hence the risk of relapse must be
evaluated at the RNA levels by quantitative RT-PCR rather than at the
DNA level with FISH.
Françoise Brizard
Andre Brizard
Laboratoire d'Hématologie CHU de
Poitiers
Poitiers, France
Françoise Guilhot
Département d'Hématologie et Oncologie
Médicale
CHU de Poitiers
Poitiers, France
Jean-Claude Chomel
Alain Kitzis
Laboratoire de Génétique Cellulaire et
Moléculaire CHU de Poitiers
Poitiers,
France
| |
References |
1.
Chomel JC, 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.
2.
Guang Zhang JI, Lin F, Chase A, Goldman JM, Cross NCP.
Comparison of genomic DNA and cDNA for detection of residual disease after treatment of chronic myeloid leukemia with allogeneic bone marrow transplantation.
Blood.
1996;87:2588-2593[Abstract/Free Full Text].
3.
Roth MS, Antin JH, Ash R, et al.
Prognostic significance of Philadelphia chromosome-positive cells detected by the polymerase chain reaction after allogeneic bone marrow transplant for chronic myelogenous leukemia.
Blood.
1992;79:276-282[Abstract/Free Full Text].
4.
Radich JP, Gelhy G, Gooley T, et al.
Polymerase chain detection of the BCR/ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients.
Blood.
1995;85:2632-2638[Abstract/Free Full Text]
