European Journal of Haematology ISSN 0902-4441

ORIGINAL ARTICLE

Wilms tumour gene 1 overexpression in bone marrow as a

marker for minimal residual disease in acute myeloid
leukaemia
Mauri M. Hämäläinen1,2, Veli Kairisto3, Vesa Juvonen3, Janita Johansson4, Jukka Aurén4,
Kati Kohonen2,5, Kari Remes6, Toivo T. Salmi7, Hans Helenius8, Tarja-Terttu Pelliniemi4
1
Department of Clinical Chemistry, University of Turku, 2Joint Clinical Biochemistry Laboratory of University of Turku, Turku University Central
Hospital and Wallac Oy, 3Laboratory of Molecular Biology, TYKSLAB, 4Laboratory of Haematology, TYKSLAB, 5Turku University of Applied
Sciences, 6Department of Medicine, Turku University Central Hospital, 7Department of Pediatrics, Turku University Central Hospital,
8
Department of Biostatistics, University of Turku, Turku, Finland

Abstract
Objectives: Wilms tumour gene 1 (WT1) is overexpressed in leucocytes of most acute myeloid leukaemia
(AML) patients. However, the clinical relevance of WT1 gene expression as minimal residual disease (MRD)
marker in AML has been questioned. Methods: We determined the expression of WT1 gene in bone mar-
row (BM) mononuclear cells of 100 AML patients at diagnosis and compared it with other MRD markers
during follow up in 16 patients using quantitative reverse transcription-polymerase chain reaction. Results:
The median WT1 gene expression was 9.7% of K562 cell line WT1 expression (lower quartile 1.5%, upper
quartile 29.9%, n = 100) at diagnosis and, 0.053% (lower quartile 0.022%, upper quartile 0.125%, n = 87)
in molecular or immunophenotypic remission. Median WT1 expression in control BM was 0.029% (lower
quartile 0.013%, upper quartile 0.061%, n = 22). The upper 99% percentile of remission samples was
0.3%, which was regarded as the cut-off of increased WT1 gene expression in AML and was exceeded in
87% of all AML patients at diagnosis. WT1 and the other MRD markers showed only minor differences in
profiles during follow-up. WT1 expression at diagnosis with median value 9.7% as the cut-off level or as a
continuous variable had no prognostic significance for 2-yr survival. Conclusions: The sensitivity of WT1 as
a MRD marker was low due to the relatively high background WT1 gene expression in BM cells at remis-
sion and in subjects without haematological malignancies. Therefore, WT1 gene expression analysis would
be beneficial only in those patients who do not have a more specific and sensitive MRD marker.

Key words Wilms tumour gene 1; acute myeloid leukaemia; minimal residual disease

Correspondence Mauri Hämäläinen, PhD, Department of Clinical Chemistry, Turku University Central Hospital, FI-20520 Turku, Fin-
land. Tel: +358 2 3131899; Fax: +358 2 3133924; e-mail: mauham@utu.fi

Accepted for publication 17 November 2007 doi:10.1111/j.1600-0609.2007.01009.x

Wilms tumour gene 1 (WT1) overexpression has been
offered as a panleukaemic molecular marker because it
has been demonstrated in many leukaemia types includ-
ing acute lymphatic and myeloid leukaemias (ALL and
AML), chronic myeloid leukaemia in blast crisis as well
as in myelodysplastic syndromes (1–8). The development
of quantitative reverse transcription-polymerase chain
reaction (RT-PCR) has given a tool for detection of low
levels of WT1 mRNA as a marker of minimal residual
disease (MRD) in AML patients (9–13). WT1 gene is
involved in the embryonic development of kidneys,
spleen and sex organs. It is also expressed in early stages
of leukocyte differentiation (14, 15). Therefore, undiffer-
entiated leucocytes increase the background WT1 gene
expression reducing its usefulness as a MRD marker.
The level of WT1 gene expression has also been offered
as a prognostic marker for survival in AML. Inoue et al.
(3) reported significant correlation between low WT1
expression and higher complete remission rate and longer
survival. Later, both supporting and conflicting data of

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Journal compilation 80 (201–207) ª 2008 Blackwell Munksgaard 201
WT1 gene expression as MRD marker Hämäläinen et al.

the prognostic significance of WT1 expression at diagno- RNA extraction and reverse transcription
sis have been presented (16–23). In the present study we Mononuclear leucocytes were isolated from BM and
determined the upper confidence limit for WT1 gene blood samples by Ficoll-PaqueTM (Amersham Bio-
expression in bone marrow (BM) mononuclear cells and sciences, Uppsala, Sweden) gradient centrifugation. The
evaluated the sensitivity of WT1 gene expression as a cells were frozen in liquid nitrogen in 107 cells aliquots
molecular marker for MRD detection in AML patients. and stored in )80C until used for analyses. Total RNA
We also studied whether the level of WT1 gene expression was extracted form the cells with RNeasy kit and DNA
at diagnosis predicts 2-yr survival in AML patients. were extracted with QiaAmp kit of Qiagen Inc (Valencia,
CA, USA). cDNA synthesis from 1 lg of RNA was car-
Material and methods ried out in 20 lL of buffer with 1 mm nucleotides, 5 mm
MgCl2, 25 lm random hexamers, 20 U RiboLockTM
Subjects RNase inhibitor (Fermentas Inc, Hanover, MD, USA)
One hundred AML patients (57 females, 43 males) and 100 U M-MLV reverse transcriptase (Promega
treated in Turku University Central Hospital for AML Corp., Madison, WI, USA).
participated this study. The numbers of patients in differ-
ent age groups were as follows: 1–16 yr, 8; 17–39 yr, 23; Minimal residual disease analyses
40–65 yr, 45; 66–81, 24. The subtypes of AML according
Multiparameter flow cytometry of BM total leucocytes
to the French-American-British classification were as
was carried out with Becton Dickinson FacsCaliburTM
follows: M0 in 6, M1 in 19, M2 in 18, M3 in 12, M4 in
(4-colour analysis) or FACSCantoTM (6-colour analysis)
12, M5 in 14, M7 in 3, respectively. In addition, 10
flow cytometer (San Jose, CA, USA) based on cross line-
patients with secondary AML, four patients AML with
age antigen expression, antigen overexpression or asyn-
multilineage dysplasia (according to WHO criteria), one
chronous antigen expression depending on the immune
biphenotypic and one triphenotypic AML cases were
phenotype at diagnosis. Sensitivity of the assay was gen-
included in the study. The karyotype of BM cells was
erally around 0.1%.
abnormal in 63 patients and normal in 37 patients.
Fusion gene analyses t(9;22) BCR-ABL, t(15;17)
In addition, 22 BM samples were taken from patients
PML-RARa, t(8;21) AML1-ETO and Inv(16)
suspected to have a haematological disease, but diag-
CBFB-MYH11 were analysed using RT-qPCR accord-
nosed later to suffer either of an infection, iron deficiency
ing to Europe Against Cancer Program protocol (27,
anaemia, mild hypoplacia or trombocytopenia. Blood
28). Nucleophosmin (NPM) mutation analysis was
samples were taken from 12 healthy controls.
carried out by multiplying the gene sequence with
The paediatric patients (1–16 yr) were treated accord-
PCR as described by Falini et al. (29) and sequencing
ing to the common Nordic AML protocol (NOPHO)
the PCR product with Abi Prism 310 genetic ana-
(24). There are two different protocols, NOPHO AML
lyzer (Applied Biosystems, Forster City, CA, USA).
1994 and 2004 with minor modifications. In NOPHO
FMS-like tyrosine kinase 3 (FLT3) point mutations
2004 the intensity of the treatments has increased and
were analysed according to Abu-Duhier et al. (30)
stratification is better defined. Induction consists of
and tandem mutations as described by Kottaridis
6-tioguanin, cytarabin, VP-16, idarubicin and it metho-
et al. (31).
trexate, second part of induction includes cytarabin and
Wilms tumour gene 1 gene expression was analysed
mitoxatrone and it methotrexate. Consolidation protocol
with RT-qPCR method of Ogawa et al. (12). ß-Glucu-
depends on the risk category. The patients aged 17–65 yrs
ronidase gene expression was used for normalizing
were treated according to the protocols of the Finnish
WT1 result with the sample. WT1 gene expression of
Leukaemia Group (25). The patients older than 65 yrs
K562 cells was kept as 100% standard. Accordingly to
were treated with less intensive treatments which typically
the study of Ogawa et al. (12) we found that the stan-
included low-dose cytarabine for 5–7 d and idarubicine
dard curve with K562 cDNA was linear in dilutions
+ ⁄ ) thioguanine. Few patients received per oral induc-
down to 10)5. Determinations were made in duplicates
tion treatment with etoposide + thioguanine + idarubi-
in ABI Prism 5700 Sequence Detector System (Applied
cine (26). After induction the patients received one or two
Biosystems). If the threshold cycle number (Ct) devi-
additional chemotherapy courses with cytarabine plus
ated more than one cycle between the duplicates
idarubicine or mitoxantrone + ⁄ ) thioguanine. Thereafter
the analysis was repeated. WT1 and a molecular or
few patients got also per oral maintenance therapy with
flow cytometric MRD marker were determined simul-
daily mercatopurine + ⁄ )weekly methotrexate. Eighty
taneously from 16 AML patients in the course of
per cent of all patients achieved cytological complete
disease.
remission during induction and consolidation treatments.

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202 Journal compilation 80 (201–207) ª 2008 Blackwell Munksgaard
Hämäläinen et al.
Clinical outcome
Survival of 91 patients was recorded at 2 yrs after diag-
nosis. The patients were divided into two prognostic
groups by the median WT1 value 9.7% or by normal or
pathological karyotype. In addition, the predictive value
of WT1 as continuous variable was studied. The cytoge-
netic prognostic groups in the survival data were based
on Medical Research Council criteria (32, 33) as follows:
favourable group, patients with t(15;17), t(8;21) or
inv(16); adverse group, patients with )5 ⁄ 5q- or )7 or 3q
abnormalities or complex karyotype with five or more
aberrations. All other patients belonged to the intermedi-
ate group.
Statistics
The distribution of WT1 at diagnosis was quite skewed
and the skewness was not eliminated with log-transfor-
mation. That is why the comparisons between karyotype
groups were carried out using non-parametric methods.
First, an over-all comparison of the five karyotype
groups was done with Kruskal–Wallis test. Post-hoc
comparisons between each group pairs were performed
using Mann–Whitney’s U-test with Bonferroni correc-
tion for multiple comparisons. Survival analyses were
performed applying log-rank tests when two or more
groups were compared. The survival curves were esti-
mated with Kaplan–Meier technique. Cox’s regression
analysis was used in situations where continuous explan-
atory variables were studied. Cox’s regression analysis
was also used in multivariate survival analysis. SAS for
Windows 9.1.3 ⁄ 2006 (Cary, NC, USA) and MedCalc
(Mariakerke, Belgium) statistics softwares were used in
analyses.
Figure 1 Wilms tumour gene 1 (WT1) expression of acute myeloid leukaemia patients at diagnosis. Abnormal karyotype group does not include
the patients with translocations t(15;17), t(8,21) or Inv(16). Control bone marrow samples were obtained from haematological patients with non-
malignant disease. Control peripheral blood leucocytes samples were obtained from healthy subjects. Bone marrow WT1 was significantly higher
in patients with t(15;17) than in the normal karyotype, abnormal karyotype or t(8,21) groups (P = 0.02, P = 0.003 or P = 0.016, respectively,
Mann–Whitney’s U-test).
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Journal compilation 80 (201–207) ª 2008 Blackwell Munksgaard
WT1 gene expression as MRD marker
Results
WT1 gene expression at diagnosis
Figure 1 shows the distribution of BM WT1 gene expres-
sion in mononuclear cells of 100 AML patients at diag-
nosis, 22 controls with non-malignant haematological
disease, and 12 control blood samples. Median WT1 at
diagnosis for all patients was 9.7% (lower quartile 1.5%,
upper quartile 29.9%, n = 100) when compared with
K562 cell WT1 gene expression. Median control BM
WT1 was 0.029% (lower quartile 0.013%, upper quartile
0.061%, n = 22) in the BM of patients without malig-
nant disease and 0.00049% in the blood samples of con-
trol subjects. WT1 gene expression of BM samples of
AML patients with either normal karyotype or abnormal
karyotype (excluding the translocation and inversion
groups) did not differ significantly (median values 11.9%
and 4.0%, respectively). Patients with promyelocyte type
leukaemia (FAB M3) carrying 15;17 translocation
showed significantly higher WT1 expression at diagnosis
(median 38.7%) in comparisons with normal karyotype,
abnormal karyotype or t(8;21) groups (P = 0.02,
P = 0.003 or P = 0.016, respectively). WT1 expression
level of AML patients with fusion gene t(8;21) (median
4.8%) did not differ significantly from the other AML
patients except those in the t(15;17) group.
WT1 at remission
Figure 2 shows the distribution of BM WT1 expression
in 87 samples of 21 AML patients at immunophenotypic
or molecular remissions. Median WT1 at remission was
0.053% (lower quartile 0.022%, upper quartile 0.125%)
when compared to the K562 cell WT1 expression. The
203
WT1 gene expression as MRD marker Hämäläinen et al.

0.4
WT1 (per cent of K562 expression)

99% Percentile cut-off 0.3%

0.3

0.2

0.1

0
0 20 40 60 80 100
AML remission samples

Figure 2 Wilms tumour gene 1 (WT1) expression of bone marrow

samples taken from acute myeloid leukaemia patients in remission.
The median value was 0.053%. The 99% percentile value 0.3% was
regarded as cut-off of WT1 gene overexpression in remission.

upper 99% percentile expression level in the remission

samples was 0.3%, which was regarded as the cut-off
value for WT1 gene overexpression in AML at
remission.

Comparison WT1 with other molecular markers

during the disease
Forty-five out of 100 AML patients had a molecular
marker, FLT3 or NPM mutation, or a fusion gene. Both
WT1 gene expression and the other molecular marker
were determined in 12 patients during follow-up. In addi-
tion, WT1 and immunophenotypic data were compared
in four patients. Figure 3 shows the result of four repre-
sentative patient cases during follow-up. Both MRD
methods gave similar results in 10 of 16 patients. In three
patients WT1 decreased faster than the specific molecular
marker at the onset of therapy (one of these cases is
shown in Fig. 3). In three patients WT1 was low,
although a molecular marker indicated leukaemia cell
proliferation (an example in Fig. 3; lowest diagram). The
baseline WT1 expression was detectable in all samples
even if the other molecular marker was below the report-
able range. It is noteworthy that some patients showed Figure 3 Four patient cases of comparisons of Wilms tumour gene 1
low individual baseline WT1 level enabling the 0.1% expression and specific molecular minimal residual disease markers
MRD sensitivity. during the follow-up of acute myeloid leukaemia.

overall survival between the groups (Fig. 4). The patients

Clinical outcome
When the AML patients were divided into two groups
according to the median WT1 value 9.7%, no significant
difference was found in the 2-yr disease free survival or
groups with either abnormal or normal karyotype had
not different survival curves, either (data not shown).
Using WT1 as continuous variable in Cox proportional-
hazards regression model WT1 did not show a significant

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204 Journal compilation 80 (201–207) ª 2008 Blackwell Munksgaard
Hämäläinen et al. WT1 gene expression as MRD marker

levels in AML patients at diagnosis and at remission as

well as in with non-malignant haematological disease
agree with the results in previous studies taking into
account that the standard may be either K562 cell WT1
WT1 > median, mRNA or WT1 plasmid (10, 13). Several research groups
n = 46 have reported 0.1% sensitivity in BM WT1 assays in con-
siderable part of AML patients (11, 13, 22). If the assay
is based on peripheral blood (PB) leucocytes still better
WT1 < median, sensitivity has been obtained because background WT1
n = 45 level in PB leucocytes of healthy control subjects is less
than one tenth of the BM level. The baseline figure for
the determination of WT1 gene overexpression is of great
importance when the sensitivity of the assay is evaluated.
Because patient-specific baseline value is not available
before the achievement of remission, the WT1 gene
expression during treatment has to be compared levels
detected in AML patients in remission or in patients
without haematological malignancies. In our study remis-
sion samples were obtained either after chemotherapy or
after BM transplantation and remission was based on
immunophenotypic or ⁄ and molecular genetic determina-
WT1 > median, tions. As expected, some WT1 gene expression values in
n = 41 remission were higher than those in patients with non-
malignant diseases. Our opinion is that in MRD determi-
nation patient’s WT1 gene expression level should be
WT1 < median, compared to the decision limit calculated from remission
n = 34
values rather than to the control values to avoid false
positive results. If patient’s WT1 at diagnosis is compared
to the highest WT1 remission value seen in this study,
then as high as 340% WT1 gene expression value is
needed to reach 0.1% sensitivity. This level of WT1
expression was not seen in any of the diagnostic samples.
Even if the cut-off value for baseline WT1 expression is
set at the upper 99% percentile (0.30%), the sensitivity of
0.1% in the MRD analysis is not reached. If the highest
WT1 expression value in patients with non-malignant dis-
Figure 4 Kaplan–Meier survival curves of acute myeloid leukaemia eases (0.1%) is accepted as the baseline value, the WT1
patients divided into groups by the median Wilms tumour gene 1 expression level of 100% at diagnosis allows MRD detec-
expression value 9.7% at diagnosis. The curves did not differ signifi- tion with 0.1% sensitivity and even then in our study
cantly (Log rank test). five of 100 AML patients would be suitable for MRD
follow-up by WT1 gene expression analysis. This does
not prevent WT1 assay if a more sensitive analysis is
predictive value (P = 0.39). Accordingly, there was no not available, especially in those patients showing very
significant association of WT1 with the prognostic low individual WT1 expression in remission. However, in
adverse or favourable groups (P-values 0.074 and 0.118, most cases the MRD detection with 0.1% sensitivity can
respectively). be performed by immunophenotypic analyses.
Comparisons of WT1 with other molecular markers in
longitudinal studies showed similar MRD kinetics in
Discussion
most cases. This result is in concordance with other stud-
Wilms tumour gene 1 expression is a tempting method to ies (11–13, 20, 22). However, background WT1 expres-
analyse residual leukaemia cells in AML or ALL patients sion is constantly detectable throughout the remission
who do no have a gene rearrangement, mutation or trans- period whereas a specific molecular marker may fall
locations as a target for PCR-based minimal residual dis- under the detection level in response to therapy. Another
ease determination. Our results in WT1 gene expression noticeable phenomenon is that WT1 may decrease

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Journal compilation 80 (201–207) ª 2008 Blackwell Munksgaard 205
WT1 gene expression as MRD marker
rapidly at the onset of chemotherapy whereas the specific
molecular marker shows the presence of residual leukae-
mic cells for longer time periods. Thus, WT1 may give
too optimistic view of MRD status. On the other hand,
rapid decrease of WT1 gene expression would be
regarded as an indication of effective therapy. The inhib-
itory effect of chemotherapy may also be the reason for
that WT1 did not respond to a rise in leukaemia cells
after the initial decline shown in the lowest diagram of
(Fig. 4). The proposed relapse prediction by elevated
WT1 expression needs more frequent sampling than is
the case in most treatment protocols and is only possible
with blood samples. However, in every case where a rise
in WT1 was found in initially successfully treated patient
it was connected to a relapse. We conclude that, if a spe-
cific molecular marker is available, it cannot be replaced
by the WT1 gene expression analysis as a follow-up mar-
ker for MRD analysis in acute myeloid leukaemia. How-
ever, WT1 may offer additional information of the effect
of therapy thus supporting the specific MRD findings. If
a specific molecular marker is not available, WT1 expres-
sion might be beneficial if the lower sensitivity and the
possible lower values than other molecular markers due
to the therapy are kept in mind.
As the early observations of WTI as a diagnostic tool,
a high level of WT1 expression at diagnosis has been
regarded as a marker of poor prognosis for survival (3,
12, 16, 21). However, contradictory views have been pre-
sented (17, 19). In our study no significant difference
existed in the survival between diagnostic high and low
WT1 groups. WT1 as continuous variable had not a pre-
dictive value, either. The early studies (3, 16, 17) were
based on semiquantitative PCR methods, which prevent
conclusive decisions. In two resent studies with quantita-
tive real-time RT-PCR technology, the cut-off value of
WT1 in outcome studies was stated around median as in
our study but the discrepancy still persists (19, 21). In
the studies of Lapillonne et al. (23) and Weisser et al.
(22) WT1 expression at diagnosis had no predictive value
whereas a high WT1 after induction therapy predicted
poor outcome. Our view is that high WT1 at presenta-
tion has no prognostic significance whereas a rise in
WT1 expression during treatment is a predictor for
relapse.
In this study our aim was to evaluate the usefulness of
WT1 gene expression in BM cells as a universal MRD
marker in 100 AML patients. Although WT1 kinetics
generally was in agreement with other MRD markers,
we found that the baseline WT1 expression in remission
was so high that the sensitivity of MRD analysis based
on WT1 gene expression remained too low. We were not
able to demonstrate a prognostic significance of high
WT1 at presentation. Therefore, we suggest that BM
WT1 gene expression analysis is informative only in
206
Hämäläinen et al.
cases where a more sensitive MRD marker is not
available.
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