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Title page
Clinical utility of WT1 monitoring in patients with myeloid malignancy and prior allogenic
hematopoietic stem cell transplantation
Kazuko Ino1,2), Shigeo Fuji1), Kinuko Tajima1), Takashi Tanaka1), Keiji Okinaka1), Yoshihiro
Inamoto1), Saiko Kurosawa1), Sung-Won Kim1), Naoyuki Katayama2), Takahiro Fukuda1)
1) Department of Hematopoietic Stem Cell Transplantation, National Cancer Center
Hospital, Tokyo, Japan.
2) Department of Hematology and Oncology, Mie University Graduate School of Medicine,
Tsu, Japan.
Corresponding author:
Shigeo Fuji, M.D.
Department of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital,
Tokyo, Japan
5-1-1, Tsukiji, Chuo-Ku, Tokyo 104-0045, Japan.
TEL: +81-3-3542-2511
FAX: +81-3-3542-3815
E-mail: [email protected]
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Running title: WT1 as MRD in allo-HSCT
Key words: allogeneic hematopoietic stem cell transplantation, myeloid malignancy,
minimal residual disease.
Word count: abstract 197; main body 24593120
Abstract
Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the
standard treatments for myeloid malignancy, relapse remains a major obstacle to cure.
Early detection of relapse by monitoring of minimal residual disease (MRD) may enable us
to intervene preemptively and potentially prevent overt relapse.
WT1 is well known as a panleukemic marker. We retrospectively examined serially
monitored WT1 levels of peripheral blood in 98 patients (84 with acute myeloid leukemia
and 14 with myelodysplastic syndrome). At the time of allo-HSCT, 49 patients (50%) were
in CR. Patients were divided into three groups according to WT1 levels (<50 copy/gRNA,
50-500 copy/gRNA and >500 copy/gRNA). The cumulative incidence of relapse (CIR)
and overall survival (OS) differed statistically according to the WT1 levels before allo-HSCT
and at days 30 and 60 after allo-HSCT. In multivariate analysis, WT1 >500 copy/gRNA
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before and at day 60 after allo-HSCT, and WT1 >50 copy/gRNA at day 30 were correlated
with CIR. Moreover, WT1 >500 copy/gRNA at day 60 after allo-HSCT was only correlated
with worse OS. Our data suggest that serial monitoring of WT1 levels in peripheral blood
may be useful for MRD monitoring and as a predictor of hematological relapse in allo-
HSCT.
Introduction
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has recently become a
standard treatment for patients with high-risk myeloid malignancies. A number of patients
are cured after allo-HSCT. For other patients and their transplant teams, however, relapse
after allo-HSCT remains a major obstacle to treatment success. Preemptive intervention in
patients with an impending relapse is an attractive option, and identification of such patients
requires monitoring techniques to detect minimal residual disease (MRD).
Some reports have indicated that in patients with acute myeloid leukemia (AML),
detection of MRD after induction chemotherapy and before allo-HSCT correlates with the
risk of relapse after allo-HSCT (ref. 1-9).
In patients with myeloid malignancies characterized by expression of a chimeric fusion
gene such as PML/RAR, AML1/MTG8, or CBF/MYH11, this expression can be used as
the MRD parameter. On the other hand, a significant proportion of patients with myeloid
malignancies do not have such markers, which makes the monitoring of MRD difficult.
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Wilms’ tumor 1 (WT1) was originally identified as a tumor suppressor gene in Wilms’ tumor,
a pediatric renal cancer (ref. 10, 11). WT1 is also overexpressed in many myeloid
malignancies and can be used for monitoring MRD in patients’ peripheral blood (PB) (ref. 2,
4, 9, 12-21). Although the superiority of WT1 level in PB in comparison to that in bone
marrow has not yet been established, the sampling of PB is obviously preferable in clinical
practice. Thus, we assessed the importance of WT1 level in PB only in this study. At our
center, WT1 levels of PB was measured as MRD monitoring routinely. However, clinical
decisions based on the results of WT1 level relies mostly on the decisions of physicians as
the threshold has not been well established. At our institute, patients who underwent allo-
HSCT were followed monthly to monitor WT1 levels in PB. In this report, we retrospectively
assessed the utility of WT1 monitoring and the correlation between WT1 levels and clinical
outcomes.
Patients and methods
Study population and WT1 analysis
Our study included 149 adult patients who had myeloid malignancies and received their
first allo-HSCT at the National Cancer Center Hospital, Tokyo, Japan, from April 2010 to
August 2014. We excluded 47 patients without data of WT1 levels, 3 patients with isolated
myeloid sarcoma, and 1 patient with myeloid NK precursor acute leukemia. We analyzed
the detailed clinical features of the remaining 98 patients. WT1 levels in PB were
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determined before and 30 and 60 days after allo-HSCT, using the WT1 mRNA Assay Kit II
“new kit” (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) as previously reported (ref. 22).
The WT1 mRNA expression levels were calculated by multiplying the value obtained by
dividing the measured WT1 mRNA by the measured value of GAPDH mRNA (number of
WT1 mRNA copies per copy of GAPDH mRNA) with the mean GAPDH mRNA
measurement value per 1 g of RNA (2.7 x 107 copies/g RNA) based on independent
tests in healthy adults. The method to calculate WT1 mRNA expression is shown below. A
unit of WT1 mRNA expression was prescribed as copies/g RNA.
WT1 mRNA expression (copies/g RNA) = [Measured value of WT1 mRNA (copies/mL)
/Measured value of GAPDH mRNA (copies/mL)] x 2.7 x 107 (copies/g RNA)*
*2.7 x 107 (copies/gRNA): mean GAPDH mRNA measurement value per 1 g of RNA in
PB of healthy adult.
The IRB of National Cancer Center approved this study. In this study, complete remission
(CR) and relapse were defined as hematological. These data were analyzed as of March
2015. The median follow-up time of survivors, defined as the time from allo-HSCT to last
observation, was 775 days.
MRD detection
WT1 levels in PB were serially monitored. Patients were divided into three groups
according to WT1 levels, as follows: group 1, <50 copy/gRNA; group 2, 50-500
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copy/gRNA; and group 3, >500 copy/gRNA. The receiver operating characteristic (ROC)
curve for the prediction of hematological relapse indicated that the best cutoff values at
each time point were as follows: WT1 600 copy/gRNA before allo-HSCT, 87 copy/gRNA
at day 30 after allo-HSCT, and 120 copy/gRNA at day 60 after allo-HSCT. We further
analyzed the cutoff value of WT1 500 copy/gRNA. WT1 50 copy/gRNA was the detection
limit in this analysis. The MRD data was obtained before and at approximately days 30 and
60 after allo-HSCT. Patients were classified according to their MRD values.
Statistical methods
To estimate the probabilities of cumulative incidence of relapse (CIR) and overall survival
(OS), the observation time was calculated from the MRD examination to the event date of
the last follow-up. Evaluation of CIR was performed with the Fine and Gray model and
Gray’s test, and non-relapse mortality (NRM) was considered a competing risk for relapse.
NRM was defined as death without prior relapse. The probabilities of OS were estimated
with Kaplan-Meier (KM) statistics. The log-rank test was used for comparisons. Cox
proportional hazards regression model analysis was conducted to identify factors affecting
an adverse subsequent event. Covariates were further investigated in a multivariate Cox
proportional hazard model based on stepwise selection strategy, and the main effect
variable of MRD was held in all steps of model building. The corresponding hazard ratios
(HRs) and their 95% confidence intervals (95%CIs) were calculated.
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All statistical analyses were performed with EZR software (ref. 23), which is a graphical
user interface for R. More precisely, it is a modified version of R commander designed to
add statistical functions frequently used in biostatistics.
Results
Patient characteristics
A total of 98 patients were included in this retrospective study; their detailed characteristics
are shown in Table 1. The median age was 46.5 years (range, 18-68 years). Eighty-four
patients (85.7%) had AML and 14 (14.3%) had myelodysplastic syndrome (MDS). At the
time of allo-HSCT, 49 patients (50%) achieved CR. The median follow-up period among
survivors was 775 days (range, 61-1739 days) after allo-HSCT. Cytogenetic risk was good
in 47 patients (47.9%), intermediate in 27 patients (27.6%), and poor in 20 patients
(20.4%). Of the 47 patients with good cytogenetic risk, 4 were AML1/MTG8 positive, 1 was
CBF/MYH11 positive, and 1 was PML/RAR positive. All patients with good cytogenetic
risk suffered from relapsed disease after first complete remission or primary induction
failure. Thus, it would be reasonable to consider those patients as a candidate for all-HSCT.
This karyotype analysis was based on the International Prognostic Scoring System (ref.
24). Twenty-seven patients (27.5%) received allo-HSCT from related donors and the others
from unrelated donors. In terms of HLA disparity, 23 patients (23.5%) had 1 locus mismatch
and 30 patients (30.6%) had >2-loci mismatches. The conditioning regimens were as
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follows: 28 patients (28.6%) received total body irradiation / cyclophosphamide (CY), 32
patients (32.7%) received busulfan (Bu) / CY, 12 patients (12.2%) received fludarabine
(Flu) / Bu 3.2 mg/kg/day for 4 days as a myeloablative conditioning regimen, and 26
patients (26.5%) received Flu / Bu 3.2 mg/kg/day for 2 days as a reduced-intensity
conditioning regimen. Myeloablative conditioning regimen was used in 72 patients (73.5%).
Monitoring of WT1 levels and the relationship to clinical outcomes
In this study, we retrospectively analyzed the data of WT1 in PB. These results are shown
in Figure 1. Most patients with WT1 levels <500 copy/gRNA before allo-HSCT maintained
WT1 levels <50 copy/gRNA after allo-HSCT. In contrast, of the patients with WT1 levels
>500 copy/gRNA, about one-third demonstrated WT1 levels above 50 copy/gRNA after
allo-HSCT.
CIR and OS differed statistically (P<0.01) according to the WT1 levels before allo-HSCT
(Figure 2a and b). The 2-year relapse rates were 21% (95%CI 9-36%) in the WT1 <50
copy/gRNA group, 23% (95%CI 5-48%) in the WT1 50-500 copy/gRNA group, and 49%
(95%CI 33-64%) in the WT1 >500 copy/gRNA group (Figure 2a). The 2-year OS rates
were 90% (95%CI: 73-96%) in the WT1 <50 copy/gRNA group, 79% (95%CI 36-94%) in
the WT1 50-500 copy/gRNA group, and 52% (95%CI 34-67%) in the WT1 >500
copy/gRNA group (Figure 2b).
CIR and OS differed statistically (P<0.01) according to the WT1 levels at days 30 and
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60 after allo-HSCT (Figure 2c-f). Grouped according to the WT1 level at day 30 after allo-
HSCT, the 2-year relapse rates were 27% (95%CI 18-38%) in the WT1 <50 copy/gRNA
group, 38% (95%CI 13-63%) in the WT1 50-500 copy/gRNA group, and 83% (95%CI 8-
98%) in the WT1 >500 copy/gRNA group (Figure 2c). Grouped according to the WT1
level at day 30 after allo-HSCT, the 2-year OS rates were 76% (95%CI 64-84%) in the WT1
<50 copy/gRNA group, 82% (95%CI 46-95%) in the WT1 50-500 copy/gRNA group, and
42% (95%CI 9-73%) in the WT1 >500 copy/gRNA group (Figure 2d). Grouped according
to the WT1 level at day 60 after allo-HSCT, the 2-year relapse rates were 21% (95%CI 13-
31%) in the WT1 <50 copy/gRNA group, 50% (95%CI 12-79%) in the WT1 50-500
copy/gRNA group, and not available in the WT1 >500 copy/gRNA group (Figure 2e).
Grouped according to the WT1 level at day 60 after allo-HSCT, the 2-year OS rates were
85% (95%CI 74-91%) in the WT1 <50 copy/gRNA group, 55% (95%CI 20-80%) in the
WT1 50-500 copy/gRNA group, and not available in the WT1 >500 copy/gRNA group
(Figure 2f).
Univariate and multivariate analysis for relapse
In univariate analysis, sex (vs. male; female, HR 0.40, 95%CI 0.21-0.78, P<0.01), status of
disease at the time of allo-HSCT (vs. CR; non-CR, HR 5.16, 95%CI 2.46-10.82, P<0.01),
and karyotype (vs. good; poor, HR 2.89, 95%CI 1.53-5.49, P<0.01) were associated with
an increased risk of CIR. In terms of MRD before allo-HSCT, WT1 >500 copy/gRNA was
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associated with an increased risk of CIR (vs. WT1 <50 copy/gRNA; WT1 >500
copy/gRNA, HR 4.22, 95%CI 2.04-8.76, P<0.01). In terms of MRD at day 30, WT1 >500
copy/gRNA was associated with an increased risk of CIR (vs. WT1 <50 copy/gRNA at
day 30; WT1 >500 copy/gRNA at day 30; HR 7.43, 95%CI 2.17-25.43, P<0.01). In terms
of MRD at day 60, WT1 >500 copy/gRNA was associated with an increased risk of CIR
(vs. WT1 <50 copy/gRNA at day 60; WT1 >500 copy/gRNA at day 60; HR 24.73, 95%CI
8.32-73.51, P<0.01, Table 2).
In multivariate analysis, WT1 >500 copy/gRNA before allo-HSCT was associated with
an increased risk of CIR (vs. WT1 <50 copy/gRNA; WT1 >500 copy/gRNA, HR 3.00,
95%CI 1.20-7.49, P=0.02) (Table 3). In terms of MRD at day 30, WT1 50-500 copy/gRNA
and >500 copy/gRNA were associated with an increased risk of CIR (vs. WT1 <50
copy/gRNA at day 30; WT1 50-500 copy/gRNA at day 30, HR 2.98, 95%CI 1.19-7.47,
P=0.02; WT1 >500 copy/gRNA at day 30, HR 7.86, 95%CI 2.28-27.12, P<0.01) (Table 3).
In terms of MRD at day 60, WT1 >500 copy/gRNA was associated with an increased risk
of CIR (vs. WT1 <50 copy/gRNA at day 60; WT1 >500 copy/gRNA at day 60, HR 15.90,
95%CI 4.22-59.92, P<0.01) (Table 3).
Univariate and multivariate analysis of OS
In univariate analysis, the disease status at the time of allo-HSCT (vs. CR; non-CR, HR
6.08, 95%CI 2.28-16.21, P<0.01), and karyotype (vs. good; poor, HR 3.08, 95%CI 1.35-
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7.02, P<0.01) were adverse prognostic factors for OS. In terms of MRD before allo-HSCT,
WT1 >500 copy/gRNA was an adverse prognostic factor for OS (vs. WT1 <50
copy/gRNA; WT1 >500 copy/gRNA, HR 5.41, 95%CI 2.13-13.76, P<0.01). In terms of
MRD at day 30, WT1 >500 copy/gRNA was an adverse prognostic factor for OS (vs. WT1
<50 copy/gRNA at day 30; WT1 >500 copy/gRNA at day 30, HR 5.11, 95%CI 1.70-15.34,
P<0.01). In terms of MRD at day 60, WT1 50-500 copy/gRNA and >500 copy/gRNA were
adverse prognostic factors for OS (vs. WT1 <50 copy/gRNA at day 60; WT1 50-500
copy/gRNA at day 60, HR 3.20, 95%CI 1.04-9.87, P=0.04; WT-1 >500 copy/gRNA at day
60, HR 25.78, 95%CI 6.62-100.40, P<0.01) (Table 4).
In multivariate analysis, WT1 >500 copy/gRNA at day 60 of allo-HSCT was the only
adverse prognostic factor for OS (vs. WT1 <50 copy/gRNA at day 60; HR 13.94, 95%CI
3.59-54.11, P<0.01) (Table 5). In contrast, WT1 levels before and at day 30 after allo-HSCT
were not significant risk factors for OS (Table 5).
Impact of conditioning intensity on the kinetics of WT1 level after allo-HSCT
With regard to the conditioning intensity, a univariate analysis showed a trend toward
higher rate of MRD negativity in patients who received a myeloablative conditioning
regimen (80.5% at day 30, 84.3% at day 60) in comparison to those who received a non-
myeloablative conditioning regimen (76.9% at day 30, 75.0% at day 60) (Supplemental
Table 1). However, conditioning intensity was not a significant variable in multivariate
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analysis.
Subgroup analysis stratified based on the pretransplant disease status
We added the subgroup analysis stratified based on the pretransplant disease status
(Supplemental Figure 1, 2). In terms of patients in hematological CR with WT1 level >500
copy/gRNA, the number of patients was limited (n=6), although the cumulative incidence
of relapse seems to be high. The study population was small, further studies were needed.
Discussion
In this study, we showed that serial monitoring of WT1 in PB before and after allo-HSCT
was a useful method to estimate the risk of hematological relapse in patients with myeloid
malignancies, as elevation of WT1 level was significantly associated with an increased risk
of subsequent hematological relapse.
In multivariate analysis, WT1 >500 copy/gRNA before and at day 60 after allo-HSCT,
and WT1 >50 copy/gRNA at day 30 after allo-HSCT were significantly associated with an
increased risk of CIR. Moreover, WT1 >500 copy/gRNA at day 60 after allo-HSCT was the
only significant prognostic risk factor for OS. The assessment and monitoring of MRD after
allo-HSCT are of crucial importance in patients with myeloid malignancies, as they can
enable us to determine transplantation efficacy and to achieve early diagnosis of relapse.
We had data of WT1 levels at day 90 and day 180 after HSCT (Supplemental table 2).
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At day 90, 74 patients were <50 copy/gRNA, 7 patients were 50-500 copy/gRNA and 2
patients were >500 copy/gRNA. In these 9 patients of MRD positivity, 6 patients (66.7%)
had subsequent hematological relapse. Meanwhile, at day 180, 69 patients were <50
copy/gRNA, 2 patients were 50-500 copy/gRNA and 3 patients were >500 copy/gRNA.
In these 5 patients of MRD positivity, all patients (100%) had subsequent hematological
relapse. Although the number of evaluable patients decreased at later time point, long-term
monitoring of WT1 level in PB might be useful to assess the risk of subsequent
hematological relapse. On the contrary, MRD detection of day 30 may be uninformative
because of recovering hematopoiesis after allo-HSCT. In this study, the possibility of MRD
value of WT1 at day 30 was suggested. Further studies will be needed to detail analysis at
this time point.
WT1 elevation was eventually detected in 40 patients (Figure 3), of whom 27 had
subsequent hematological relapse. Four of these 27 patients were alive after second allo-
HSCT, and only 1 patient was alive after donor lymphocyte infusion (DLI). In total, of the 27
relapsed patients, 5 were still alive, 5 were lost to follow-up, and the remaining 17 died: 6
after second allo-HSCT, 8 after chemotherapy, and 3 after discontinuation of GVHD
prophylaxis. Thus, it is obvious that the outcomes of patients who experienced
hematological relapse after allo-HSCT were dismal. On the other hand, 13 patients who did
not experience hematological relapse but had MRD were all alive. Of these patients, 3 had
WT1 vaccination, 1 received second allo-HSCT for graft failure, and 9 experienced
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spontaneous disappearance of MRD. The level of WT1 elevation in this group was only
between 100 and 200 copy/gRNA. The WT1 threshold to actively consider intervention to
prevent hematological relapse has not yet been established. Another important issue is the
continued lack of clarity regarding the most effective treatment strategy to prevent
hematological relapse at the time of MRD relapse after allo-HSCT. Possible preemptive
treatment interventions in patients with MRD relapse include azacitidine (Aza), FLT3
inhibitors, DLI, and second allo-HSCT as previously reported (ref. 25-31). Recently, Pozzi S
et al reported that preemptive treatment with DLI after allo-HSCT in patients with elevated
WT1 level improved prognosis (ref. 25). In their report, the risk of leukemia relapse was not
significantly reduced, although OS was significantly better in patients who received DLI
than in those who did not. These results may indicate that DLI alone is insufficient to
prevent hematological relapse. Regarding this point, Schroeder T et al showed that the
combination of Aza and DLI was an effective treatment strategy in patients with relapse
after allo-HSCT, in particular those with MDS or with AML characterized by low tumor
burden (ref. 28). In AML patients with high tumor burdens, the effects of these therapies
were limited. Therefore, it is important to detect MRD relapse using serial monitoring of
MRD after allo-HSCT. In addition to these reports, others showed that Aza after allo-HSCT
could induce expansion of immunomodulatory regulatory T cells and enhance the response
of cytotoxic T cells to tumor antigens (ref. 32, 33). Moreover, Aza could induce leukemic cell
differentiation and increase the expression of several tumor-associated antigens (ref. 32,
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33). These effects of Aza after allo-HSCT may increase the graft-versus-leukemia effect
and improve patient outcomes (ref. 29-31). Prospective studies assessing the effectiveness
of preemptive treatment strategies incorporating such drugs are warranted.
There are several limitations to this study. First, we divided patients into three groups
based on their peripheral blood WT1 levels: WT1 <50 copy/gRNA, WT1 50-500
copy/gRNA, and WT1 >500 copy/gRNA. However, the cutoff value for peripheral WT1
level has not yet been well established. In our data-base, ROC curves at each time point to
determine the optimal cutoff values showed different describe each cutoff values: 600
copy/gRNA before allo-HSCT, 87 copy/gRNA at day 30, and 120 copy/gRNA at day 60
each other. However, it is practically complicated if we use different cutoff values at different
time points. Thus, we adopted more simplified cutoff values 50 and 500 copy/gRNA.
However, these values were arbitrary and the importance of these cutoffs should be
reconfirmed in other studies. The second limitation is that we did not use flow cytometry to
monitor MRD level routinely in clinical practice. Thus, to assess the benefit or limitation of
WT1 level in comparison to flow cytometry, further studies which incorporate both
measurements are needed. The third limitation is that this study consisted of a
retrospective analysis at a single center. Prospective studies are needed to establish
appropriate management approaches to elevated WT1 levels after allo-HSCT.
In conclusion, our data suggested that monitoring WT1 levels in PB after allo-HSCT
might be useful to identify patients at high risk of hematological relapse. Further
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prospective studies are necessary to determine how to effectively and efficiently prevent
hematological relapse in patients diagnosed with MRD.
Acknowledgments
The authors thank the inpatient, outpatient, and support staff for their excellent care. This
work was supported by grants from the National Cancer Research and Development Fund
(26-A-26) and the Advanced Clinical Research Organization
Conflict of interest
Authors declare no conflict of interest.
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Table 1. Clinical characteristics of study population (N=98)Characteristic No. %No. of patients 98 100.0Age, years Median 46.5 Range 18 to 68 Sex Male 55 56.1 Female 43 43.9Disease AML, MDS/AML 84 85.7 MDS 14 14.3Status of disease CR 49 50.0 Non-CR 49 50.0Secondary AML No 90 91.8 Yes 8 8.2Cytogenetic risk group Good 47 47.9 Intermediate 27 27.6 Poor 20 20.4 Not available 4 4.1Relation to donor Related donor 27 27.5 Unrelated donor 71 72.5Stem cell source Bone marrow 65 66.3 Peripheral blood 27 27.6 Cord blood 6 6.1HLA compatibility Fully matched 45 45.9 1 locus mismatched 23 23.5 >2-loci mismatched 30 30.6Conditioning regimen TBI/CY 28 28.6 Bu/CY 32 32.7 Flu/Bu4 12 12.2 Flu/Bu2 26 26.5GVHD prophylaxis Tac-based 78 79.6 CyA-based 17 17.4 Post CY 3 3.0ATG No 84 85.7
Yes 14 14.3Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; CR, complete remission; HLA, human leukocyte antigen; TBI, total body irradiation; CY, cyclophosphamide; Bu, busulfan; Flu, fludarabine; GVHD, graft-versus-host-disease; Tac,
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tacrolimus; CyA, cyclosporine A; ATG, anti-thymocyte globulin.
Table 2. Univariate analysis of CIRFactor HR 95% CI PDisease AML, MDS/AML (n=84) 1.00 MDS (n=14) 1.06 0.42 to 2.69 0.89Status of disease CR (n=49) 1.00 Non-CR (n=49) 5.16 2.46 to 10.82 <0.01Sex Male (n=55) 1.00 Female (n=43) 0.40 0.21 to 0.78 <0.01Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.48 0.68 to 3.23 0.32 Poor (n=20) 2.89 1.53 to 5.49 <0.01Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 1.43 0.72 to 2.86 0.31WT1 before allo-HSCT <50 (n=34) 1.00 50-500 (n=13) 1.40 0.35 to 5.58 0.63 >500 (n=41) 4.22 2.04 to 8.76 <0.01WT1 at day 30 after allo-HSCT <50 (n=78) 1.00 50-500 (n=13) 1.60 0.57 to 4.47 0.37 >500 (n=6) 7.43 2.17 to 25.43 <0.01WT1 at day 60 after allo-HSCT <50 (n=77) 1.00 50-500 (n=8) 2.75 0.89 to 8.43 0.08 >500 (n=5) 24.73 8.32 to 73.51 <0.01
Abbreviations: CIR, cumulative incidence of relapse; HR, hazard ratio; CI, confidence interval; allo-HSCT, allogeneic hematopoietic stem cell transplantation.
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Table 3. Multivariate analysis of CIR: before and at days 30 and 60 after allo-HSCTFactor HR 95% CI P
Before allo-HSCTDisease AML, MDS/AML (n=75) 1.00 MDS (n=13) 0.25 0.09 to 0.70 <0.01Status of disease CR (n=44) 1.00 Non-CR (n=44) 4.40 1.56 to 12.42 <0.01Sex Male (n=50) 1.00 Female (n=38) 0.26 0.13 to 0.53 <0.01Cytogenetic risk group Good (n=41) 1.00 Intermediate (n=25) 2.35 0.71 to 7.83 0.16 Poor (n=19) 1.57 0.71 to 3.47 0.27Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 3.03 0.79 to 11.69 0.11WT1 before allo-HSCT <50 (n=34) 1.00 50-500 (n=13) 0.93 0.15 to 5.93 0.94 >500 (n=41) 3.00 1.20 to 7.49 0.02
Day 30 after allo-HSCTDisease AML, MDS/AML (n=83) 1.00 MDS (n=14) 0.58 0.28 to 1.20 0.15Status of disease CR (n=49) 1.00 Non-CR (n=48) 5.04 2.25 to 11.27 <0.01Sex Male (n=54) 1.00 Female (n=43) 0.29 0.13 to 0.65 <0.01Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.73 0.73 to 4.11 0.22 Poor (n=19) 1.79 0.77 to 4.19 0.18Conditioning regimen MAC (n=71) 1.00 RIC (n=26) 1.58 0.81 to 3.10 0.18WT1 at day 30 after allo-HSCT <50 (n=78) 1.00 50-500 (n=13) 2.98 1.19 to 7.47 0.02 >500 (n=6) 7.86 2.28 to 27.12 <0.01
Day 60 after allo-HSCTDisease AML, MDS/AML (n=77) 1.00 MDS (n=13) 0.23 0.05 to 1.05 0.06Status of disease CR (n=49) 1.00 Non CR (n=41) 3.88 1.77 to 8.48 <0.01Sex Male (n=49) 1.00 Female (n=41) 0.54 0.27 to 1.09 0.09Cytogenetic risk group Good (n=45) 1.00 Intermediate (n=25) 1.61 0.59 to 4.35 0.35 Poor (n=16) 1.41 0.64 to 3.11 0.39
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Conditioning regimen MAC (n=60) 1.00 RIC (n=20) 2.39 0.45 to 12.64 0.31WT1 at day 60 after allo-HSCT <50 (n=77) 1.00 50-500 (n=8) 2.94 0.88 to 9.82 0.08 >500 (n=5) 15.90 4.22 to 59.92 <0.01
Table 4. Univariate analysis of OSFactor HR 95% CI PDisease AML, MDS/AML (n=84) 1.00 MDS (n=14) 0.59 0.14 to 2.51 0.47Status of disease CR (n=49) 1.00 Non-CR (n=49) 6.08 2.28 to 16.21 <0.01Sex Male (n=55) 1.00 Female (n=43) 0.58 0.26 to 1.31 0.19Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.03 0.38 to 2.77 0.96 Poor (n=20) 3.08 1.35 to 7.02 <0.01Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 1.38 0.60 to 3.18 0.45WT1 before allo-HSCT <50 (n=34) 1.00 50-500 (n=13) 1.68 0.31 to 9.23 0.55 >500 (n=41) 5.41 2.13 to 13.76 <0.01WT1 at day 30 after allo-HSCT <50 (n=78) 1.00 50-500 (n=13) 0.78 0.18 to 3.34 0.73 >500 (n=7) 5.11 1.70 to 15.34 <0.01WT1 at day 60 after allo-HSCT <50 (n=77) 1.00 50-500 (n=9) 3.20 1.04 to 9.87 0.04 >500 (n=8) 25.78 6.62 to 100.40 <0.01
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Table 5. Multivariate analysis of OS: before and at days 30 and 60 after allo-HSCTFactor HR 95% CI P
Before allo-HSCTDisease AML, MDS/AML (n=75) 1.00 MDS (n=13) 0.12 0.02 to 0.88 0.04Status of disease CR (n=44) 1.00 Non-CR (n=44) 9.00 3.32 to 24.44 <0.01Sex Male (n=50) 1.00 Female (n=38) 0.43 0.18 to 1.00 0.05Cytogenetic risk group Good (n=41) 1.00 Intermediate (n=25) 1.78 0.55 to 5.70 0.33 Poor (n=19) 1.57 0.63 to 3.94 0.34Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 2.29 0.44 to 12.06 0.33WT1 before allo-HSCT <50 (n=34) 1.00 50-500 (n=13) 1.42 0.21 to 9.58 0.72 >500 (n=41) 2.55 0.78 to 8.29 0.12
Day 30 after allo-HSCTDisease AML, MDS/AML (n=84) 1.00 MDS (n=14) 0.29 0.07 to 1.25 0.09Status of disease CR (n=49) 1.00 Non-CR (n=49) 6.09 2.29 to 16.22 <0.01Sex Male (n=55) 1.00 Female (n=43) 0.58 0.26 to 1.33 0.20Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.65 0.57 to 4.76 0.35 Poor (n=20) 2.33 0.99 to 5.48 0.05Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 1.60 0.66 to 3.89 0.30WT1 at day 30 after allo-HSCT <50 (n=78) 1.00 50-500 (n=13) 1.29 0.25 to 6.67 0.76 >500 (n=7) 2.29 0.56 to 9.38 0.25
Day 60 after allo-HSCTDisease AML, MDS/AML (n=80) 1.00 MDS (n=14) 0.25 0.05 to 1.12 0.07Status of disease
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CR (n=49) 1.00 Non-CR (n=45) 4.32 1.55 to 12.04 <0.01Sex Male (n=53) 1.00 Female (n=41) 0.56 0.21 to 1.53 0.26Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.26 0.36 to 4.43 0.72 Poor (n=18) 1.39 0.43 to 4.54 0.58Conditioning regimen MAC (n=70) 1.00 RIC (n=24) 2.64 0.44 to 15.86 0.29WT1 at day 60 after allo-HSCT <50 (n=77) 1.00 50-500 (n=9) 2.90 0.94 to 8.93 0.06 >500 (n=8) 13.94 3.59 to 54.11 <0.01
Figure legends
Figure 1. Monitoring and change of peripheral blood WT1 levels before and after allo-
HSCT.
Data indicate the WT1 levels before and 30 and 60 days after allo-HSCT. The upper row of
text in each box indicates patient group based on WT1 levels (units: copy/gRNA), and the
lower row indicates the number of patients corresponding to each box.
Abbreviations: allo-HSCT, allogeneic hematopoietic stem cell transplantation; NA, not
available.
Figure 2. Cumulative incidence of relapse and overall survival before and after allo-
HSCT according to MRD levels.
Solid line, WT1 <50 copy/gRNA; broken line, WT1 50-500 copy/gRNA; dotted line, WT1
>500 copy/gRNA. a, b; before allo-HSCT, c, d; day 30 after allo-HSCT, e, f; day 60 after
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allo-HSCT. Abbreviations: CI, confidence interval.
Figure 3. The prognosis of cases with elevated WT1.
Of the 27 patients with hematological relapse, only 5 were alive at the end of the study. In
contrast, all 13 patients with MRD but no hematological relapse survived.
Abbreviations: SCT, stem cell transplantation; DLI, donor lymphocyte infusion; GVL, graft
versus leukemia.
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Figure 1.
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Figure 2.
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Supplemental Table 1. Transition of WT1 in PB based on MAC or RIC conditioning
Data of WT1 in PB day 0 (N=72) day 30 (N=72) day 60 (N=70)
MAC N % N % N %<50 27 37.5 58 80.5 59 84.3
50-500 12 16.7 11 15.3 7 10.0>500 26 36.1 3 4.2 4 5.7NA 7 9.7 0 0.0 0 0.0
RIC day 0 (N=26) day 30 (N=26) day 60 (N=24)
<50 7 26.9 20 76.9 18 75.050-500 1 3.9 2 7.7 2 8.3>500 15 57.7 4 15.4 4 16.7NA 3 11.5 0 0.0 0 0.0
With regard to the conditioning intensity, a univariate analysis showed a trend toward
higher rate of proportion of MRD negativity in patients who received a myeloablative
conditioning regimen.
Supplemental Table 2. WT1 data at day 90, 180 of allo-HSCT
Day 90 of allo-HSCT (N=85) Day 180 of allo-HSCT(N=76)
Data of WT1 in PB Total No. of relapse No. of relapse / Total Total No. of relapse No. of relapse / Total
N N % %
<50 74 19 26.7 69 11 15.9
50-500 7 4 57.1 2 2 100
>500 2 2 100 3 3 100
NA 2 2 100 2 1 50
At day 90, in 9 patients of MRD positivity, 6 patients had subsequent hematological relapse,
and at day 180, in 5 patients of MRD positivity, all had hematological relapse.
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Supplemental Figure legends
Supplemental Figure 1. Cumulative incidence of relapse and overall survival before
and after allo-HSCT in hematological CR patients (N=49).
In hematological CR patients, WT1 level of PB was relatively related to CIR. On the other
hand, WT1 level of PB was not related to OS.
Solid line, WT1 <50 copy/gRNA; broken line, WT1 50-500 copy/gRNA; dotted line, WT1
>500 copy/gRNA.
a, b; before allo-HSCT, c, d; day 30 after allo-HSCT, e, f; day 60 after allo-HSCT.
Abbreviations: allo-HSCT, allogeneic hematopoietic stem cell transplantation; CI,
confidence interval; NA, not available.
Supplemental Figure 2. Cumulative incidence of relapse and overall survival before
and after allo-HSCT in hematological non-CR patients (N=49).
In hematological non-CR patients, WT1 level of PB was relatively related to CIR and OS at
the time of day 30 and 60.
Supplemental Figure 3. Cumulative incidence of relapse and overall survival before
and after allo-HSCT in AML patients (N=84).
In AML patients, WT1 level of PB was significantly related to CIR and OS.
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Supplemental Figure 4. Cumulative incidence of relapse and overall survival before
and after allo-HSCT in MDS patients (N=14).
In MDS patients, the number of patients was small, the analysis was not detectable.
Supplemental Figure 5. Cumulative incidence of relapse and overall survival before
and after allo-HSCT according to MRD levels (Cutoff value of WT1 200 copy/gRNA).
In this analysis, WT1 level was divided into 3 groups according to <50 copy/gRNA, 50-200
copy/gRNA and >200 copy/gRNA. The cutoff value was different from the analysis of
above. CIR and OS differed statistically (P<0.05) according to the WT1 levels before and at
day 30 and 60 after allo-HSCT.
Solid line, WT1 <50 copy/gRNA; broken line, WT1 50-200 copy/gRNA; dotted line, WT1
>200 copy/gRNA.
a, b; before allo-HSCT, c, d; day 30 after allo-HSCT, e, f; day 60 after allo-HSCT.
Abbreviations: CI, confidence interval.
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Supplemental Figure 1. CIR and OS before and after allo-HSCT according to MRD
levels in hematological CR patients (N=49).
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Supplemental Figure 2. CIR and OS before and after allo-HSCT according to MRD
levels in hematological non-CR patients (N=49).
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Supplemental Figure 3. CIR and OS before and after allo-HSCT according to MRD
levels in AML patients (N=84).
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Supplemental Figure 4. CIR and OS before and after allo-HSCT according to MRD
levels in MDS patients (N=14).
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Supplemental Figure 5. CIR and OS before and after allo-HSCT according to MRD
levels (WT1 cutoff value 200 copy/gRNA).
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Supplemental Table 3. Multivariate analysis of CIR: before and at days 30 and 60 after allo-HSCT (WT1 cutoff value 200 copy/gRNA)Factor HR 95% CI P
Before allo-HSCTDisease AML, MDS/AML (n=75) 1.00 MDS (n=13) 0.31 0.11 to 0.85 0.02Status of disease CR (n=44) 1.00 Non-CR (n=44) 7.91 3.32 to 18.79 <0.01Sex Male (n=50) 1.00 Female (n=38) 0.33 0.17 to 0.65 <0.01Cytogenetic risk group Good (n=41) 1.00 Intermediate (n=25) 1.68 0.64 to 4.45 0.29 Poor (n=19) 1.86 0.83 to 4.15 0.13Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 2.91 0.78 to 10.78 0.11WT1 before allo-HSCT <50 (n=34) 1.00 50-200 (n=9) 1.45 0.24 to 8.90 0.69 >200 (n=45) 1.71 0.68 to 4.31 0.25
Day 30 after allo-HSCTDisease AML, MDS/AML (n=83) 1.00 MDS (n=14) 0.54 0.22 to 1.33 0.18Status of disease CR (n=49) 1.00 Non-CR (n=48) 5.02 2.17 to 11.60 <0.01Sex Male (n=54) 1.00 Female (n=43) 0.29 0.12 to 0.66 <0.01Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.69 0.80 to 3.61 0.17 Poor (n=19) 1.85 0.77 to 4.45 0.17Conditioning regimen MAC (n=71) 1.00 RIC (n=26) 1.56 0.76 to 3.22 0.22WT1 at day 30 after allo-HSCT <50 (n=78) 1.00 50-200 (n=11) 3.02 1.15 to 7.96 0.03 >200 (n=8) 6.32 2.05 to 19.42 <0.01
Day 60 after allo-HSCTDisease AML, MDS/AML (n=77) 1.00 MDS (n=13) 0.39 0.16 to 0.92 0.03Status of disease CR (n=49) 1.00 Non CR (n=41) 5.82 2.26 to 14.97 <0.01Sex Male (n=49) 1.00 Female (n=41) 0.38 0.17 to 0.86 0.02Cytogenetic risk group Good (n=45) 1.00 Intermediate (n=25) 2.12 0.82 to 5.52 0.12 Poor (n=16) 1.66 0.78 to 3.51 0.19Conditioning regimen MAC (n=60) 1.00
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RIC (n=20) 1.47 0.71 to 3.04 0.30WT1 at day 60 after allo-HSCT <50 (n=77) 1.00 50-200 (n=5) 4.86 1.38 to 17.15 0.01 >200 (n=8) 5.62 1.84 to 17.16 <0.01
Supplemental Table 4. Multivariate analysis of OS: before and at days 30 and 60 after allo-HSCT (WT1 cutoff value 200 copy/gRNA)Factor HR 95% CI P
Before allo-HSCTDisease AML, MDS/AML (n=75) 1.00 MDS (n=13) 0.12 0.02 to 0.88 0.04Status of disease CR (n=44) 1.00 Non-CR (n=44) 9.00 3.32 to 24.44 <0.01Sex Male (n=50) 1.00 Female (n=38) 0.53 0.23 to 1.22 0.14Cytogenetic risk group Good (n=41) 1.00 Intermediate (n=25) 1.75 0.53 to 5.76 0.36 Poor (n=19) 1.88 0.73 to 4.83 0.19Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 3.07 0.60 to 15.71 0.18WT1 before allo-HSCT <50 (n=34) 1.00 50-200 (n=9) 1.58 0.15 to 16.21 0.70 >200 (n=45) 2.60 0.82 to 8.19 0.10
Day 30 after allo-HSCTDisease AML, MDS/AML (n=84) 1.00 MDS (n=14) 0.29 0.07 to 1.25 0.09Status of disease CR (n=49) 1.00 Non-CR (n=49) 6.09 2.29 to 16.22 <0.01Sex Male (n=55) 1.00 Female (n=43) 0.58 0.26 to 1.33 0.20Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.68 0.58 to 4.84 0.34 Poor (n=20) 2.33 0.99 to 5.48 0.05Conditioning regimen MAC (n=72) 1.00 RIC (n=26) 1.60 0.66 to 3.89 0.30WT1 at day 30 after allo-HSCT <50 (n=78) 1.00 50-200 (n=11) 1.70 0.32 to 9.10 0.54 >200 (n=9) 1.86 0.49 to 7.10 0.36
Day 60 after allo-HSCTDisease AML, MDS/AML (n=80) 1.00 MDS (n=14) 0.25 0.05 to 1.22 0.08Status of disease CR (n=49) 1.00 Non-CR (n=45) 4.48 1.62 to 12.39 <0.01Sex Male (n=53) 1.00 Female (n=41) 0.53 0.20 to 1.40 0.20
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Cytogenetic risk group Good (n=47) 1.00 Intermediate (n=27) 1.69 0.51 to 5.55 0.39 Poor (n=18) 1.69 0.45 to 6.45 0.44Conditioning regimen MAC (n=70) 1.00 RIC (n=24) 3.74 0.68 to 20.42 0.13WT1 at day 60 after allo-HSCT <50 (n=77) 1.00 50-200 (n=5) 4.17 0.88 to 19.81 0.07 >200 (n=12) 6.09 2.36 to 15.72 <0.01
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