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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-06-1831.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Molecular Medicine, Osaka
University Graduate School of Medicine, Japan; Department
of Clinical Laboratory Science, Osaka University Medical School,
Japan; and Department of Medicine, Osaka Minami National
Hospital, Osaka, Japan.
In acute-type leukemia, no method for the prediction of relapse
following allogeneic stem cell transplantation based on minimal residual disease (MRD) levels is established yet. In the present study,
MRD in 72 cases of allogeneic transplantation for acute myeloid
leukemia, acute lymphoid leukemia, and chronic myeloid leukemia
(accelerated phase or blast crisis) was monitored frequently by
quantitating the transcript of WT1 gene, a
"panleukemic MRD marker," using reverse transcriptase-polymerase
chain reaction. Based on the negativity of expression of chimeric
genes, the background level of WT1 transcripts in bone
marrow following allogeneic transplantation was significantly decreased
compared with the level in healthy volunteers. The probability of
relapse occurring within 40 days significantly increased step-by-step
according to the increase in WT1 expression level (100%
for 1.0 × 10 Relapse still remains an obstacle to successful
allogeneic stem cell transplantation (SCT) for patients with acute
leukemia. Early recognition of relapse at the molecular level provides
a window for therapeutic intervention while the burden of disease is
still relatively low.1,2 For chronic myeloid leukemia (CML), several reports have shown that serial quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the chimeric bcr-abl gene after allogeneic SCT can effectively
distinguish those patients who will remain in remission from those
patients who are destined to have a relapse.3,4 However,
for acute types of leukemia, there have been few studies on the
association of minimal residual disease (MRD) levels with the
occurrence of leukemic relapse after allogeneic SCT. In addition to the
heterogeneity of the diseases other than CML, only 40% of patients
with acute leukemia have chimeric tumor markers that make it possible
to design PCR primers for quantitating MRD.5 Furthermore,
the period in which patients can be diagnosed with molecular relapse is
short, because the growth speed of blasts in acute leukemia is usually
much faster than that of leukemia cells in CML in the chronic
phase (CP). To overcome this difficulty, we performed frequent
monitoring of MRD based on the expression levels of WT1, which has been reported as a "panleukemic marker" for
evaluating MRD in leukemia.
WT1 was isolated as a gene responsible for Wilms tumor, a
childhood kidney neoplasm, and categorized as a tumor suppressor gene.6,7 We8,9 and others10-13
have recently shown that the WT1 gene is highly expressed in
various types of leukemia (acute myeloid leukemia [AML], acute
lymphoid leukemia [ALL], and CML) and that the expression of the
WT1 gene is thus a tumor marker for leukemic blast cells of
almost all leukemias. The WT1 expression level, measured by
quantitative RT-PCR, significantly increases at relapse compared with
that at the time of diagnosis.14 For CML, the
WT1 expression level increases by one log as the clinical
stage progresses from CP to the accelerated phase (AP) and also
increases by one log from AP to blast crisis (BC).8 Furthermore, the WT1 expression level in bone marrow (BM) or
peripheral blood (PB) accurately reflects the extent of MRD of 3 types
of leukemia (AML, ALL, and CML).15
In our previous paper, using a quantitative RT-PCR method, we showed
that either a rapid or a gradual increase in the WT1 expression level occurred in BM and PB before the occurrence of hematologic relapse (HR) in all of 3 patients treated by allogeneic transplantation.15 However, the precise relationship
between the WT1 expression level and the risk of relapse
after allogeneic transplantation still remains to be determined. In
addition, one of major problems with the measurement of MRD based on
WT1 transcripts is the background expression of
WT1 in BM that stems from normal hematopoietic stem cells.
In the present study, we first determined the background level
following allogeneic transplantation by quantitating WT1
transcript levels in samples from patients who were diagnosed as
negative for MRD based on the expression of chimeric genes. After
having clearly determined the MRD+ range of WT1
transcripts in BM samples, we analyzed the association of the
WT1 transcript levels obtained by frequent WT1
testing with the probability of relapse occurring within a short period of time (within 40 days), in a retrospective study based on
WT1 data in 72 transplantations performed for acute types of
leukemia (AML, ALL, CML in AP or BC). Furthermore, we studied the
kinetics of the increase in WT1 transcripts in 15 relapsed
cases, and the relationship between the rate of increase of
WT1 expression and the efficacy of immunologic therapeutic
interventions including the discontinuation of immunosuppressive agents
or donor leukocyte infusion (DLI).
Patients
Discontinuation of immunosuppression was performed for patients who had
a relapse while receiving immunosuppressive agents. Infusion of donor
leukocytes containing at least 1.0 × 108 CD3 cells/kg
was performed for patients who had a relapse while off therapy for GVHD
prophylaxis. These immunomodulation therapies were diagnosed as
effective when patients achieved CR or partial remission without the
occurrence of fatal GVHD. Institutional review board approval by the
Osaka University Medical School was obtained for the treatment
protocol, and informed consents were obtained from patients and their families.
Sample preparation and quantitation of transcripts of
WT1, major bcr-abl, minor bcr-abl,
and AML1-MTG8
Mononuclear cells from samples were obtained by Ficoll-Hypaque density
gradient centrifugation and were stored at Until January 1998, quantitative RT-PCR for WT1 transcripts
was performed as previously described.16 Because the
measurement of WT1 transcripts in the exponential
amplification phase was required in this procedure, PCR was performed
for various numbers of cycles according to the WT1
expression levels in samples. Namely, to quantitate WT1
transcript levels that were 10 Since February 1998, we have used a real-time PCR method for
WT1. For real-time PCR, we designed all primers and probe
combinations using Primer-Express software (PE Applied Biosystems,
Foster City, CA). WT1 and For the construction of standard curves of positive control, RNA of
K562 cells was reverse-transcribed into cDNA and serially diluted in 5 log steps. This standard cDNA serial dilution was prepared in large
amounts and stored at
For quantification of WT1 transcripts of patient samples,
samples were always amplified simultaneously with the standard dilution of K562 cDNA. The WT1 transcript level in samples was
determined by reference to the corresponding transcript level of K562
cells on the standard curve. Samples were analyzed in duplicate, and the average value was calculated and adjusted according to the level of
Quantification of transcripts of chimeric genes was performed by
real-time PCR. Real-time PCR for AML1-MTG8 was performed using the primers and protocol described by Marcucci et
al.17 Kasumi-1, a myeloid leukemia cell line containing
t(8;21) was used as a positive control, and the level of
AML1-MTG8 gene transcripts in Kasumi-1 cells was defined as
1.0. Real-time PCR for major bcr-abl was performed using the
primers and protocol described by Mensink et al.18 K562
cells were used as a positive control, and the level of major
bcr-abl transcripts in K562 cells was defined as 1.0. Real-time PCR for minor bcr-abl was performed using the same
protocol and primers as for major bcr-abl except for the use
of the sequence 5'-ACCATCGTGGGCGTCCGCAAGA-3' as the forward primer.
L2,19 a Ph1+ acute lymphoblastic
leukemia-derived cell line, was used as a positive control, and the
level of transcripts in L2 was defined as 1.0. Real-time PCR was
performed using basically the same methods as for WT1. The
sensitivity of real-time PCR for these chimeric genes was at least
10 Analysis of the relationship between the WT1 level and the risk of relapse after transplantation Twenty of 72 transplantations were ultimately followed by an HR. Ten, 8, and 2 patients had relapse within 150 days, between 150 and 400 days, and 400 days or more after transplantation, respectively. Of the patients who received the remaining 52 transplants, 23 had transplantation-related mortality in CR at a median of 286 days after transplantation (range, 52-1244 days), and 29 are alive in CR at a median follow-up of 592 days (range, 106-2755 days).The relationship between the WT1 transcript level in BM after transplantation and the occurrence of relapse until day 400 was analyzed. For patients who had a relapse or developed transplantation-related mortality, WT1 transcript levels were analyzed until these events occurred. A way of standardizing the data was needed because the interval of monitoring of WT1 was different in each patient. The duration was divided into periods of 20 days each: from day 11 to day 30, from day 31 to 50, from day 51 to 70, and so on. When the WT1 test was performed more than once in a given 20-day period, the mean value of the data was used as the representative value of the period. The mean value was calculated after conversion of the WT1 value into a logarithm. Consequently, each transplantation had 5.1 standardized WT1 points on average (range, 1-20). Furthermore, there were 3.35 and 5.50 standardized WT1 points per transplantation on average in the periods until 100 days and until 180 days, respectively. For the analysis of time course of WT1 gene expression in relapsed patients, raw data of WT1 were used. Statistical methods Statistical analyses were performed with SPSS software (Version 7.5; SPSS, Chicago, IL). For the analysis of the background level of transcripts in BM in patients who underwent allogeneic transplantation, when the expression of chimeric genes was less than 10 5
or undetectable (the sensitivity was at least 105), MRD
was defined as negative. The comparison of WT1 transcript levels of healthy volunteer donors and MRD BM
transplantation (BMT) patients was analyzed by the Mann-Whitney U test. The relationship between WT1 transcript
levels and the probability of relapse occurring within 40 days was
analyzed by the Fisher exact test. The relationship between the
doubling time of WT1 transcripts in relapsed patients and
the efficacy of the immunomodulation therapy or the disease status at
transplantation was analyzed by the Mann-Whitney U test.
The data were "locked " for analysis on January 29, 2002.
The background level of WT1 gene expression in patients undergoing BMT We changed the method by which we quantitated the WT1 gene expression from the method we used previously16 to a real-time quantitative PCR method in January 1998, because the real-time PCR method was shown to be simple, rapid, and reliable.17,18,20,21 We compared the 2 quantitative RT-PCR methods by analyzing 50 RNA samples that were obtained before January 1998 and stored at 80°C. WT1 transcript levels obtained
by the previous method and by the real-time PCR method were found to
show a good correlation (r = .998, P < .001).
Therefore, we considered that data obtained by the 2 methods could be
handled equivalently in the subsequent WT1 analysis.
In acute leukemia, the usefulness of high WT1 mRNA
expression during follow-up for detection of MRD is still the subject
of discussion because normal CD34+ progenitors have also
been found to express detectable levels of WT1
mRNA.22 To clarify the usefulness of WT1
expression as an MRD marker in patients undergoing BMT, the background
levels of WT1 transcripts in BM were examined in detail.
First, we examined the WT1 gene expression levels in 34 BM
samples from healthy donors for related transplantation. As shown in
Figure 2, the WT1 gene expression levels in 32 of 34 samples were in the range of
1.0 × 10
Relationship of expression levels of WT1 gene and chimeric genes as determined by quantitative RT-PCR analysis To further investigate the reliability of WT1 expression as an MRD marker, we analyzed the correlation in the expression levels between WT1 and chimeric genes in MRD+ samples from patients who had AML1-MTG8 or minor bcr-abl. MRD in BM samples at different time points after transplantation was analyzed by using the real-time PCR methods for WT1 and these chimeric genes (see "Patients, materials, and methods"). The results showed a strong positive correlation between the levels of gene expression of WT1 and AML1/MTG8 (r = 0.941; Figure 3A), and between those of WT1 and minor bcr/abl (r = 0.924; Figure 3B). The time courses of the gene expression levels of WT1 and AML1-MTG8 in a patient with AML are shown in Figure 3C. The 2 MRD markers generally changed in parallel.
Relationship between WT1 level after transplantation and risk of relapse All of 20 patients who had HR showed WT1 gene expression levels of more than 1.0 × 102 at the time
of relapse. We analyzed whether HR could be predicted based on
WT1 assay. Considering that blasts in acute type of leukemia expand much more rapidly at relapse after transplantation than leukemia
cells in CML-CP, we focused on the occurrence of HR within a short
period of time after sampling for WT1 testing. Because the
interval of monitoring of WT1 was different for each
patient, standardization of the WT1 data was performed (see
"Patients, materials, and methods"). We then analyzed the
association of the standardized WT1 levels with the
occurrence of HR within 40 days, based on 367 WT1 values
obtained from BMT patients with AML, ALL, and CML-AP or CML-BC (Table
1). HR was observed only in patients with
a WT1 expression level over 1.0 × 102. All
patients with WT1 expression levels of
5.0 × 102 and over (level A) had full relapse. Of 13 instances of WT1 expression levels between
1.0 × 102 and 5.0 × 102 (level B), 6 were in patients in HR. However, most BM samples of patients in HR
contained less than 20% blasts, which indicates an early phase of HR.
The remaining 7 instances were in patients in CR, but all of them were
followed by HR within 20 days. Therefore, level B was considered to be
the WT1 expression level at which HR had begun to occur. All
patients with WT1 expression levels less than
1.0 × 102 had morphologic CR. However, the percentage
of patients who had HR within 40 days abruptly increased at
WT1 expression levels over 4.0 × 103. In
fact, of 9 patients with WT1 expression levels between
4.0 × 103 and 1.0 × 102 (level C), 2 and 2 had HR within 20 and 40 days, respectively. The remaining 5 did
not have HR within 40 days, but 3 of them ultimately had HR. Therefore,
level C corresponds to molecular relapse. Of 78 instances of
WT1 levels between 4.0 × 104 and
4.0 × 103 (level D), values still considered
MRD+ (the background level of WT1 after
transplantation was < 4.0 × 104; Figure 2), only 8 instances (10.3%) were followed by HR within 40 days. Of 255 instances
of WT1 levels less than 4.0 × 104 (level
E), values considered MRD, only 2 instances
(0.8%) were followed by relapse within 40 days, suggesting that level
E had almost no risk of relapse occurring within a short time period.
Regarding the incidence of HR within 20 days in cases of CR, there was
a significant difference between level B and level C
(P < .01; Fisher exact test). Furthermore, regarding the
incidence of HR within 40 days in cases of CR, there were significant
differences between level B and level C (P = .03), between
level C and level D (P = .01), and between level D and
level E (P < .001). The ultimate relapse rates of
patients in whom WT1 levels showed level C and level D even
once after transplantation were 77.8% and 29.7%, respectively (Table
1). The ultimate relapse rate of patients in whom WT1 levels
showed level E twice in succession after transplantation was 17.1%.
Furthermore, in patients having a relapse, the median lengths of time
from the first increase of WT1 expression to level B, level
C, and level D until a diagnosis of HR were 11, 43, and 92 days,
respectively (Table 1).
Analysis of time course of WT1 gene expression in patients having a relapse Twenty transplantations ultimately resulted in HR at a median of 139 days after transplantation (range, 14-1386 days). The time course of WT1 gene expression in 16 of 18 patients who had HR within 400 days after transplantation is shown in Figure 4. After rapidly decreasing after transplantation in most patients and reaching trough levels between day 13 and day 104, WT1 levels began to increase exponentially with a constant doubling time that was different in each patient, and the patients went on to HR. In some patients, after reaching trough levels, WT1 levels fluctuated slightly and began to increase exponentially at some points after transplantation, and the patients also went on to HR. The doubling times of WT1 in 15 transplantations in which WT1 levels were monitored at more than 3 points in the increasing phase are listed in Table 2. No association was found between doubling time and type of donor (sibling versus unrelated or HLA match versus HLA mismatch), or presence of GVHD. Similarly, no relationship was found between doubling time and time of relapse after transplantation. Patients who underwent transplantation in CR had a significantly longer doubling time of WT1 compared with those who were in non-CR at transplantation (P = .04; Mann-Whitney U test). Immunomodulation therapy, consisting of the discontinuation of immunosuppressive agents or DLI, was performed for relapsed patients. The doubling time for patients in whom the immunomodulation therapy was effective was significantly longer than that for patients in whom it was not (P = .014; Mann-Whitney U test). No patients with a WT1 doubling time of less than 13 days responded to the immunomodulation therapy. In contrast, 5 of 7 patients with a WT1 doubling time of 13 days and over responded to the therapy.
Because the WT1 gene is expressed at low levels even in
normal hematopoietic stem cells, most studies using qualitative or semiquantitative analyses of WT1 transcripts have produced
negative results with regard to the prediction of
relapse.24,25 To accurately determine the background level
of WT1 gene expression in BM following allogeneic SCT, we
examined WT1 transcript levels in BM samples that could be
considered as MRD By sequential measurement of WT1 transcripts using the
quantitative RT-PCR methods, we attempted to elucidate the kinetics of
MRD following allogeneic SCT in patients with acute-type leukemia. Lin
et al reported that 21 (72%) of 29 patients with CML who had increasing or persistently high bcr-abl expression (> 50
transcripts/µg) following allogeneic transplantation ultimately had
relapse.3 However, for acute leukemia, the length of time
from the initiation of the increase in MRD to a diagnosis of HR must be
short, because the growth speed of leukemic blasts at relapse may be
much more rapid in acute leukemia than in CML-CP. Thus, an early
diagnosis of relapse based on the MRD level is much more difficult in
acute leukemia. In accordance with the kinetics of leukemic blasts, we
analyzed the probability of relapse occurring within a short period of
time (within 40 days) by frequent testing of WT1 expression. The results clearly showed that the probability of relapse was significantly increased according to the increase in the WT1
expression level (Table 1). In fact, the analysis of CR samples showed
that there were significant differences in the relapse rate within 40 days among 4 MRD levels: level B (1.0 × 10 Next, we elucidated the kinetics of WT1 transcripts in patients having a relapse. After rapidly decreasing following transplantation and reaching the trough levels between day 13 and day 104, WT1 levels began to increase exponentially with a constant doubling time that was different for each patient, and eventually the patients underwent HR. Despite the fact that the number of patients who had relapses was relatively small, we found that patients in whom immunomodulation therapy was effective had a significantly longer doubling time of WT1 transcripts (median, 26 days) than patients in whom the therapy was not effective (median, 5.8 days). Lin et al reported similar results for patients with CML, among whom patients with rapidly doubling numbers of bcr-abl transcripts were less likely to respond to DLI than those with a long doubling time.3 We consider that patients with longer doubling times have some residual graft-versus-leukemia (GVL) effect that slows the rate of relapse and that these patients respond to reinforcement of GVL effects by the withdrawal of immunosuppressive agents or DLI. By measuring WT1 expression levels at least once every 2 weeks, we were able to diagnose relapse at the molecular level in patients in whom the WT1 expression levels decreased to as
low as less than 1.0 × 10 In conclusion, molecular monitoring of WT1 transcripts by using quantitative RT-PCR accurately and reproducibly informs us in real time of the kinetics of MRD after allogeneic SCT, regardless of the presence of chimeric DNA markers. As far as we know, this is the first demonstration in acute-type leukemia that the relapse probability following allogeneic SCT significantly increased step-by-step according to the MRD level, and that a rapid doubling time of WT1 transcripts predicted the ineffectiveness of therapeutic interventions including the discontinuation of immunosuppressive agents or DLI. Although our results need to be confirmed in a large-scale, prospective study, we consider that the WT1 expression assay is an essential test for the prevention and management of relapse in allogeneic transplantation. To keep the relapse rate to a minimum, the optimized adjustment of the dose of immunosuppressive agents in real time depending on individual patients' MRD levels may be made possible by use of the WT1 assay in the near future.
We thank Dr Honma and Dr Hayashi for generous gifts of L2 cells, which we used as a positive control in minor bcr-abl PCR analyses.
Submitted June 21, 2002; accepted October 1, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-06-1831.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hiroyasu Ogawa, Department of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2, Yamada-Oka, Suita City, Osaka, 565-0871, Japan; e-mail: ogachan{at}ceres.ocn.ne.jp.
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© 2003 by The American Society of Hematology.
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H. Lapillonne, A. Renneville, A. Auvrignon, C. Flamant, A. Blaise, C. Perot, J.-L. Lai, P. Ballerini, F. Mazingue, S. Fasola, et al. High WT1 Expression After Induction Therapy Predicts High Risk of Relapse and Death in Pediatric Acute Myeloid Leukemia J. Clin. Oncol., April 1, 2006; 24(10): 1507 - 1515. [Abstract] [Full Text] [PDF]
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T. Azuma, T. Otsuki, K. Kuzushima, C. J. Froelich, S. Fujita, and M. Yasukawa Myeloma Cells Are Highly Sensitive to the Granule Exocytosis Pathway Mediated by WT1-Specific Cytotoxic T Lymphocytes Clin. Cancer Res., November 1, 2004; 10(21): 7402 - 7412. [Abstract] [Full Text] [PDF]
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P. Savage, L. Gao, K. Vento, P. Cowburn, S. Man, N. Steven, G. Ogg, A. McMichael, A. Epenetos, E. Goulmy, et al. Use of B cell-bound HLA-A2 class I monomers to generate high-avidity, allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen Blood, June 15, 2004; 103(12): 4613 - 4615. [Abstract] [Full Text] [PDF]
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D. Cilloni, E. Gottardi, M. Fava, F. Messa, S. Carturan, A. Busca, A. Guerrasio, G. Saglio, H. Ogawa, and H. Tamaki Usefulness of quantitative assessment of the WT1 gene transcript as a marker for minimal residual disease detection Blood, July 15, 2003; 102(2): 773 - 774. [Full Text] [PDF]
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| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-actin transcripts.
Rn) increased with the number of amplification cycles in
proportion to the accumulation of PCR products (Figure
and 
indicate WT1 gene expression levels of BM
samples of 34 healthy volunteer donors and of 53 BM samples from 11 BMT
patients, respectively, in the period up to day 400. In patients with
AML, ALL, or CML (BC and AP) who had chimeric DNA markers (major
bcr-abl, minor bcr-abl, and
AML1-MTG8), when the expression of these chimeric genes was
less than 10
WT1 < 5.0 × 10