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Brief report
Expression of nuclear transcription factor interferon consensus sequence binding
protein in chronic myeloid leukemia correlates with pretreatment risk features
and cytogenetic response to interferon-␣
Manuel Schmidt, Andreas Hochhaus, Andreas Nitsche, Rüdiger Hehlmann, and Andreas Neubauer
Recently, it was shown that interferon
consensus sequence binding protein
(ICSBP), a member of the interferon regulatory factor (IRF) family, has a potential
role in chronic myeloid leukemia (CML).
Deletion of ICSBP gene in mice leads to a
CML-like syndrome and samples from
CML patients exhibited impaired ICSBP
expression. The present study found that
ICSBP expression correlated with risk
features determined by Sokal score in
untreated CML (P ⴝ .007 for high versus
low risk). In addition, analyzing ICSBP
expression during interferon-␣ (IFN-␣)
therapy in “good” (n ⴝ 27) versus “poor”
(n ⴝ 15) cytogenetic responders, high
ICSBP levels were only observed in
“good” responders (P ⴝ .0002). Together,
these data suggest that ICSBP levels are
related to initial presentation of CML and
the therapeutic response of CML to IFN-␣,
indicating an important role of ICSBP in
CML. (Blood. 2001;97:3648-3650)
© 2001 by The American Society of Hematology
Introduction
Chronic myeloid leukemia (CML) is a clonal myeloproliferative
disorder characterized by bcr-abl gene rearrangement and a
typical 3-phase course (chronic, accelerated, and blastic phase)
(see Sawyers1 for a review). Treatment with interferon-␣
(IFN-␣), as monotherapy or in combination with other chemotherapeutic drugs, is frequently used for patients with CML and
has prolonged survival.2-6 IFNs are a family of multifunctional
cytokines known to regulate cell growth, immune response, and
antiviral activity in mammals. They act by activating various
downstream signal cascades, which leads to regulation of
several transcription factors.7-10
One of these factors is the interferon consensus sequence
binding protein (ICSBP), a member of the IFN regulatory factor
(IRF) family. ICSBP is known to be regulated by IFNs and
subsequently binds to IFN-stimulated response elements (ISRE) in
IFN-dependent genes.11,12 ICSBP is preferentially expressed in
cells of hematopoietic origin.12,13 ICSBP has been implicated in
CML, because ICSBP-knockout mice revealed a granulocytic
leukemia similar to CML in humans.14 The deletion of ICSBP gene
led also to a blastic transformation that was more frequent in
ICSBP⫺/⫺ homozygous compared to ICSBP⫹/⫺ heterozygous mice.14
Furthermore, a lack of ICSBP expression in CML patients has been
described, and stimulation experiments suggested a mechanism of
down-regulation.15
To address the question whether ICSBP expression was predictive of the risk profile of untreated patients with CML or later of
their cytogenetic response to IFN-␣, we correlated the transcriptional level of ICSBP in CML with clinical data.
Study design
Patient samples, RNA isolation, and complementary
DNA synthesis
Patient samples were taken from patients enrolled in the ongoing German
CML trials and the Charité-Campus Virchow Klinikum, Humboldt Universität, Berlin, Germany. Heparinized peripheral blood was drawn after
informed consent was obtained. All investigated patients were positive
for bcr-abl.
RNA was extracted from peripheral blood using the commercially
available kits (RNAzol, Paesel, Frankfurt, and RNeasy, QIAGEN, Hilde,
Germany). Total RNA (1 ␮g) was used for complementary DNA (cDNA)
synthesis as described previously.15
ICSBP messenger RNA expression analysis
Because of the restricted availability of RNA amounts from leukemia
patients and the retrospective design of the study, messenger RNA (mRNA)
expression analysis was carried out by reverse transcription-polymerase
chain reaction (RT-PCR). RT-PCR for ICSBP and ␤-actin was performed as
described elsewhere.15 The PCR products were electrophoresed
and the gels were photographed. Integrated optical densities (IntOD) were
calculated using the ONE-Dscan 1.0⬘ software (Scanalytics, Billerica, MA).
Quantitative RT-PCR was performed using a real-time PCR assay
with the ABI PRISM 7700 Sequence Detection System (PE Biosystems,
Foster City, CA) as described elsewhere.16 In addition to the previously
applied primers,15 a labeled ICSBP probe was used (5⬘-6FAMTAAGAGCCCAGATTTTGAGGAAG-(TAMRA)-TGACCGG-3⬘). The
␤-actin was used as reference gene and ⌬CT values were then calculated
(CT ␤-actin ⫺ CT ICSBP).17
From the Klinikum der Philipps-Universität Marburg, Zentrum Innere Medizin,
Abteilung Hämatologie/Onkologie/Immunologie, Marburg, Germany; III.
Medizinische Universitätsklinik, Klinikum Mannheim der Universität
Heidelberg, Germany; and Klinik für Innere Medizin m.S. Hamatologie und
Onkologie, Charité-Campus Virchow Klinikum, Humboldt Universität, Berlin,
Germany.
Kuhlemann-Stiftung (to M.S. and A.N.).
Submitted July 26, 2000; accepted January 24, 2001.
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.
Supported in part by grants from the Deutsche Forschungsgemeinschaft (to
A.N.), the H.W. and J. Hector Stiftung (to M.S. and A.N.), the Deutsche JoseCarreras-Leukämie-Stiftung e.V. (to M.S., A.N., and A.H.) and the A. and U.
3648
Reprints: Andreas Neubauer, Klinikum der Philipps-Universität Marburg,
Zenrum Innere Medizin, Abteilung Hämatologie/Onkologie/Immunologie,
Baldingerstrasse, 35043 Marburg, Germany; e-mail: neubauer@mailer.
uni-marburg.de.
© 2001 by The American Society of Hematology
BLOOD, 1 JUNE 2001 䡠 VOLUME 97, NUMBER 11
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 JUNE 2001 䡠 VOLUME 97, NUMBER 11
Calculation of Sokal score for risk feature discrimination and
determination of cytogenetic response to IFN-␣
The Sokal score at diagnosis was calculated as the percentage of peripheral
blasts, thrombocytes, size of spleen, and age of the patient at diagnosis as
described elsewhere.18 A Sokal score below 0.8 comprises a low risk, from
0.8 to 1.2 an intermediate risk, and above 1.2 a high risk.
Cytogenetic response was assessed analyzing at least 10 metaphases
and was defined as “good” comprising complete responders (CR, 0% Ph⫹
metaphases) and partial responders (PR, 1%-34% Ph⫹ metaphases) or
“poor” including patients with a minor (MinR, 35%-94% Ph⫹ metaphases)
or no response (NR, 95%-100% Ph⫹ metaphases).19 Mean time of IFN-␣
administration was 30.2 months for “good” and 30.1 months for “poor”
responders.
Results and discussion
Pretreatment risk features in CML can be useful for early indication
of allogenic transplantation and are commonly determined by
Sokal score.18 Because ICSBP expression may play a role in CML,
we wanted to study the association of risk profile with ICSBP
levels. Thus, we retrospectively analyzed a total of 72 samples
from patients with CML in chronic phase without IFN-␣ treatment
for their ICSBP expression (median 0.098; range 0.000-0.622). For
33 of these samples the ICSBP level was previously presented.15
Clinical data for calculation of the Sokal score were available
for 24 of the 72 CML samples. Correlating the ICSBP expression
with the Sokal score, we detected a significant difference of ICSBP
Figure 1. Correlation of ICSBP expression with pretreatment risk groups and
cytogenetic response to IFN-␣ in CML patients. (A) The ICSBP level of samples
without IFN treatment was analyzed (total n ⫽ 72). Twenty-four samples were divided
into different risk groups by Sokal score: low risk (n ⫽ 10; below 0.8), intermediate
risk (n ⫽ 4; 0.8-1.2) and high risk (n ⫽ 10; above 1.2). The data revealed a correlation
of low ICSBP level with high risk (low versus high P ⫽ .007; low versus intermediate
P ⫽ .374; intermediate versus high P ⫽ 0.188; Mann-Whitney test). (B) The ICSBP
level of samples under IFN-␣ was analyzed (total n ⫽ 55). Forty-two samples were
divided in groups of “good” (CR/PR; n ⫽ 27) and “poor” (MinR/NR; n ⫽ 15) responders and revealed a significant difference in ICSBP expression (P ⫽ .0002, MannWhitney test).
ICSBP LEVEL CORRELATES WITH CLINICAL DATA IN CML
3649
Table 1. Interferon consensus sequence binding protein levels in chronic
myeloid leukemia samples from different responders to interferon-␣ therapy
Response
N
Median*
Range*
P value†
CR
13
0.657
0.132-0.985
.010‡
PR
14
0.803
0.442-1.072
.001‡
2
0.343
0.258-0.427
ND§
13
0.325
0.072-0.961
—
MinR
NR
N indicates number; CR, complete response; PR, partial response; MinR, minor
response; NR, no response; ND, not determined.
*Relative ICSBP expression is displayed as arbitrary value, calculated as
described.
†Mann-Whitney test calculated against NR.
‡Significant P value (P ⬍ .05).
§Amount of samples of MinR was too low for calculation of P value. The
difference between CR and PR was not significant (P ⫽ .297).
expression in the pretreatment risk groups. Patients from the
high-risk group had lower ICSBP levels (n ⫽ 10; median 0.059;
range 0.000-0.273) than patients from the low-risk group (n ⫽ 10;
median 0.248; range 0.057-0.519) (P ⫽ .007) (Figure 1A). Samples
with intermediate risk (n ⫽ 4) had no significant difference in
ICSBP expression to low (P ⫽ .374) or high risk (P ⫽ .188). Due
to the low amounts of samples and an overlap of ICSBP levels in
the risk groups, the indicative value of ICSBP expression must be
proved with a larger amount of samples. However, we corroborated
our data using a real-time PCR assay. Analysis of each 5 randomly
selected samples from the high- and low-risk groups yielded a
significant difference (data not shown).
We then sought to delineate the implication of ICSBP in CML
with the best known prognostic parameter for survival, cytogenetic
response to IFN-␣ therapy. Because approximately 70% of the
patients exhibit only “poor” cytogenetic response to IFN-␣ therapy,
it is useful to identify these resistant patients as early as possible.5
To study the correlation of ICSBP expression and cytogenetic
response to IFN-␣, we first analyzed a total of 55 patients under
IFN-␣ for ICSBP expression (median 0.556; range 0.072-1.072).
The ICSBP level was significantly higher than in the untreated
samples above (P ⬍ .0001). This is in keeping with data from
sorted B cells, showing that ICSBP expression is impaired in B
cells from CML patients without IFN-␣, whereas B cells from
patients under IFN-␣ exhibit a normal ICSBP level (15, data not
shown). For 42 patients, complete clinical data were available.
They were specified for their cytogenetic response and were
divided into groups of 13 CR, 14 PR, 2 MinR, and 13 NR. The
samples were preselected for their response, in an attempt to obtain
equal amounts of the respective groups. Interestingly, a significant
difference in ICSBP levels between CR versus NR or PR versus
NR was found (P ⫽ .010 or P ⫽ .001, respectively). No significant
difference between CR and PR itself was detected (P ⫽ .297);
however, the ICSBP levels from PR were slightly higher than in CR
(Table 1). By analyzing combined groups of “good” (n ⫽ 27;
CR/PR, 0%-34% Ph⫹) and “poor” responders (n ⫽ 15; MinR/NR,
35%-100% Ph⫹), we detected an even more striking correlation of
“good” response with high ICSBP expression (median 0.715; range
0.132-1.072) and “poor” response with low ICSBP level (median
0.325; range 0.072-0.961) (P ⫽ .0002; Figure 1B). Again, we
corroborated our data using the real-time PCR assay. Analysis of
each 5 randomly selected samples from “good” and “poor”
responders gave also a significant difference (data not shown).
Interestingly, analyzing 15 samples during follow-up, the initial
ICSBP level before IFN-␣ treatment of patients becoming “good”
responders was significantly higher than that of “poor” responders
(data not shown).
Our results suggest that ICSBP expression in untreated CML
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3650
BLOOD, 1 JUNE 2001 䡠 VOLUME 97, NUMBER 11
SCHMIDT et al
patients indicates classification into different risk groups. In
addition, cytogenetic response to IFN-␣ therapy in CML correlates
to ICSBP expression, leading to normal mRNA levels only in
“good” responders. These data suggest a possible usage of ICSBP
levels for deciding therapy strategies in CML, maybe by early
determination of cytogenetic response to IFN-␣, and thus enabling
patients to receive a faster allogeneic stem cell transplantation.
Current studies with mice support the conclusion of the importance
of ICSBP in CML therapy. It was shown that mice transplanted
with bcr-abl–transfected bone marrow cells developed a myeloproliferative disease resembling CML.20,21 Interestingly, mice transplanted with bone marrow cells coexpressing ICSBP and bcr-abl
showed prolonged survival in contrast to the mice transplanted
with cells expressing bcr-abl alone, indicating a protective role of
ICSBP against bcr-abl.21
Taken together, the data presented here strongly suggest that
lack of ICSBP may be involved in chronic myeloid leukemogenesis and an increase of ICSBP may have an important role in the
therapeutic effect of IFN-␣. Further prospective studies are
needed to evaluate the detailed role of ICSBP in CML and a
possible use as marker for prognosis or therapy or seperately as
a target of gene therapy.
Acknowledgments
We thank Prof D. Huhn for his help throughout the study. We are
grateful to the patients, physicians, and cytogeneticists participating in the German CML trials for sending samples and providing
clinical and cytogenetic data.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2001 97: 3648-3650
doi:10.1182/blood.V97.11.3648
Expression of nuclear transcription factor interferon consensus sequence
binding protein in chronic myeloid leukemia correlates with pretreatment risk
features and cytogenetic response to interferon-α
Manuel Schmidt, Andreas Hochhaus, Andreas Nitsche, Rüdiger Hehlmann and Andreas Neubauer
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