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CLINICAL TRIALS AND OBSERVATIONS
Expression of Livin, an antiapoptotic protein, is an independent favorable
prognostic factor in childhood acute lymphoblastic leukemia
Jaewon Choi,1 Yu Kyeong Hwang,2 Ki Woong Sung,1 Soo Hyun Lee,1 Keon Hee Yoo,1 Hye Lim Jung,1 Hong Hoe Koo,1
Hee-Jin Kim,3 Hyong Jin Kang,4 Hee Young Shin,4 and Hyo Seop Ahn4
1Department of Pediatrics, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, South
Korea; 2Division of Immunotherapy, Mogam Biotechnology Research Institute, Yongin, South Korea; 3Department of Laboratory Medicine, Samsung Medical
Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 4Department of Pediatrics, Cancer Research Institute, Seoul National University
College of Medicine, South Korea
Livin, a member of the inhibitor of apoptosis proteins, has been considered to be a
poor prognostic marker in malignancies.
However, little is known about the clinical
relevance of Livin expression in childhood acute lymphoblastic leukemia (ALL).
In this study, the expression of Livin was
analyzed in 222 patients with childhood
ALL using quantitative reverse transcriptase–polymerase chain reaction (RT-PCR)
to investigate a possible association with
the clinical features at diagnosis and treat-
ment outcomes. Both Livin expression
rates and expression levels were higher
in patients with favorable prognostic factors. The expression rate was also higher
in patients with a favorable day 7 bone
marrow response to induction chemotherapy (P < .001). The Livin expression
was related to the absence of relapse
(P < .001). Similarly, the relapse-free survival rate (ⴞ 95% CI) was higher in patients with Livin expression than in patients without Livin expression (97.9% ⴞ
4.0% versus 64.9% ⴞ 11.8%, P < .001).
Multivariate analysis for relapse-free survival demonstrated that Livin expression
was an independent favorable prognostic
factor in childhood ALL (P ⴝ .049). This
study suggests that Livin expression is a
novel prognostic marker in childhood ALL
and thus needs to be incorporated into
the patient stratification and treatment
protocols. (Blood. 2007;109:471-477)
© 2007 by The American Society of Hematology
Introduction
Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. It accounts for one fourth of all childhood
cancers and approximately 75% of all cases of childhood leukemia.
The parameters of initial leukocyte count and age at diagnosis have
traditionally provided the most reliable basis for patient stratification; this is because these parameters are readily available and are
relatively independent predictors of prognosis.1 The immunophenotype and cytogenetic abnormalities of leukemic cells are also
important prognostic factors and are used in the design and analysis
of modern therapeutic trials for childhood ALL.2-4
As a result of the accumulation of knowledge on the molecular
biology of malignancies, new diagnostic modalities are beginning
to be incorporated into diagnostic and therapeutic strategies.5-7 One
of these modalities is the quantitative reverse transcriptasepolymerase chain reaction (RT-PCR) that allows the determination
of messenger RNA expression levels and, therefore, allows researchers to examine the expression patterns of a large number of genes at
the RNA level. If specific patterns of gene expression can be
correlated with clinical features in childhood ALL, a refinement of
current prognosis-based stratification systems would be possible.
Apoptosis is an active biologic mechanism leading to programmed cell death. A tight regulation is required in biologic
systems to ensure a delicate balance between life and death. The
loss of apoptosis might result in the development of a wide variety
of diseases, including cancers. Cellular defects that halt apoptosis
have been shown to be frequently involved in cancer development
and progression.8 The up-regulation of antiapoptotic proteins
would certainly be advantageous for tumor survival.9-12 During the
last decade, a complex network of proapoptotic and antiapoptotic
proteins, which strictly regulate the apoptosis pathways, have been
identified.13-16 Studies investigating the expression of these molecules in acute leukemia have demonstrated that the expression of
proapoptotic or antiapoptotic regulatory molecules varies depending on the types of leukemias and individual patients’ characteristics.9,17-25 These differences can be potentially important for the
prediction of the response to treatment.23-25
A group of proteins known as the inhibitor of apoptosis proteins
(IAP) have been identified. As their name implies, the IAP family
proteins are known to inhibit apoptosis induced by a variety of
stimuli.15,26 They are the only cellular factors that act on both
initiator and effector caspases. Livin (MIM no. 605737; baculoviral
IAP repeat-containing 7, BIRC7; aliases, ML-IAP or KIAP) is a
member of the IAP family proteins; the gene has been localized to
the long arm of chromosome 20 on 20q13.3, a region frequently
amplified in melanomas and other malignancies.27 Two splicing
variants of Livin (␣- and ␤-isoform) have been identified.15,27 Livin
has been shown to antagonize both the death receptor and
mitochondria-based apoptotic pathways through the inhibition of
caspases 3, 7, and 9, as well as the participation of JNK1.27-29
Therefore, Livin expression has been regarded as a poor prognostic
Submitted July 7, 2006; accepted August 30, 2006. Prepublished online as
Blood First Edition Paper, September 21, 2006; DOI 10.1182/blood-2006-07032557.
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 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
© 2007 by The American Society of Hematology
BLOOD, 15 JANUARY 2007 䡠 VOLUME 109, NUMBER 2
471
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472
BLOOD, 15 JANUARY 2007 䡠 VOLUME 109, NUMBER 2
CHOI et al
marker in malignancies. However, only a limited number of studies
are available to date; thus, the clinical relevance of Livin expression is still controversial in different types of malignancies. For
example, Livin expression was associated with poor clinical
outcomes in superficial bladder cancer,30 melanoma,31 and neuroblastoma,32 whereas the Livin expression level was not related to
the survival of patients with nasopharyngeal cancer.33 No study has
been conducted on the clinical relevance of Livin expression in
childhood ALL.
In this study, therefore, we analyzed the expression of Livin in
222 patients with childhood ALL using quantitative RT-PCR to
investigate a possible relation between Livin expression and the
clinical features at diagnosis and treatment outcomes. As a result,
Livin expression in childhood ALL was found to be strongly
associated with both favorable prognostic factors at diagnosis and
better treatment outcomes. This is the first study demonstrating the
prognostic implication of Livin expression in childhood ALL.
The mRNA expression levels of Livin and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were measured by quantitative RT-PCR using an
ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Foster
City, CA). The quantitative RT-PCR amplification was performed using the
predeveloped Assays-on-demand Gene Expression Set for the Livin gene
(Hs00223384_m1; GenBank accession no. NM_139317; Applied Biosystems), and TaqMan GAPDH Control Reagents (Applied Biosystems) for
the GAPDH gene in combination with the TaqMan Universal PCR Master
Mix (Applied Biosystems).
All reactions were performed in triplicate using 20-␮L samples
containing 50 ng cDNA. The reaction protocol used involved heating for 2
minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of
amplification (15 seconds at 95°C and 1 minute at 60°C). Analysis was
performed using ABI PRISM 7000 Sequence Detection software (Applied
Biosystems).
The expression levels of the Livin gene in unknown samples were
normalized and analyzed by the ⌬⌬Ct method [⌬⌬Ct ⫽ (CtLivin ⫺
CtGAPDH )sample ⫺ (CtLivin ⫺ CtGAPDH )calibrator]. After all reactions, the average ⌬⌬Ct (CtLivin ⫺ CtGAPDH ) from all samples combined was defined as
1.0, as calibrator. We previously determined that the amplification efficiencies for Livin and GAPDH showed the same slopes. A negative control
without template was included in each experiment.
Patients, materials, and methods
Patients and treatment protocol
From September 1998 to March 2006, a total of 222 patients with newly
diagnosed childhood ALL who were younger than 15 years at initial
diagnosis were recruited from 2 institutions, the Samsung Medical Center
and the Seoul National University Hospital in Seoul, South Korea. The
diagnosis of ALL was made based on the morphologic findings from
Wright-Giemsa–stained smears of bone marrow aspirates and immunophenotyping analyses of leukemic cells by flow cytometry. Conventional
cytogenetic analyses, including fluorescent in situ hybridization (FISH) for
t(9;22), 11q23 rearrangement, and t(12;21), were also performed as part of
the routine workup. The ploidy was determined directly by the classic
method of counting the modal number of chromosomes on metaphase
karyotype preparations.
Patients were assigned to the standard-risk group if the leukocyte count
was less than 50 ⫻ 109/L and the age was 1 to 9 years at diagnosis.
Otherwise, patients were assigned to a high-risk group. In the standard-risk
patients, the treatment protocols were modified from the Children’s Cancer
Group (CCG)–189134 or CCG-1952 protocols.35 The regimen used was
switched to a modified one from the CCG-1882 protocol36 from consolidation chemotherapy, if the percentage of bone marrow leukemic blasts on
day 7 was greater than 25% during induction chemotherapy. If the
percentage of leukemic blasts on day 14 was still greater than 25%, the
protocol used for the high-risk patients was restarted. In the high-risk
patients, the treatment protocols were modified from the CCG-1882,
CCG-106B,37 or CCG-1901 protocols.38 If a patient had one or more of the
following: a leukocyte count of at least 100 ⫻ 109/L, age younger than
1 year, presence of t(9;22), or the 11q23 rearrangement, the patients
proceeded to undergo hematopoietic stem cell transplantation when an
appropriate donor was available. The protocols used were approved by the
Institutional Review Board of each institution. Informed consent was
obtained from parents or guardians for both laboratory studies and
treatment according to the guidelines of the Korean Food and Drug
Administration.
RNA isolation and real-time quantitative RT-PCR
Mononuclear cells (MNCs) were isolated from 2 mL bone marrow aspirate
at diagnosis by Ficoll density gradient centrifugation. Total RNA was
extracted from MNCs using a QIAamp RNA Blood kit (Qiagen, Hilden,
Germany) according to the manufacturer’s protocol. After treatment with
DNA-free (Ambion, Austin, TX) to remove chromosomal DNA, complementary DNA (cDNA) was synthesized using oligo (dT) 15-mer primer by
SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) and
stored at ⫺20°C until use.
Cytotoxicity assay
Cell proliferation was assessed 24 hours, 48 hours, and 72 hours after the
exposure to methylprednisolone (50 ␮g/mL) by measuring the conversion
of the trazolium salt WST-8 to formazan according to the manufacturer’s
instructions (Cell Counting Kit-8; Dojindo, Kumamoto, Japan).39,40 Briefly,
cells (1.5 ⫻ 105 cells/well) in RPMI-1640 media supplemented with 10%
fetal bovine serum and penicillin were plated onto 96-well plates. The cells
were cultured in triplicate wells with 50 ␮g/mL methylprednisolone for 24,
48, and 72 hours in a humidified atmosphere containing 5% CO2 at 37°C. At
the end of each time point, 10 ␮L WST-8 was added to each well, and the
plates were incubated for an additional 4 hours at 37°C to convert WST-8
into formazan. The absorbance of each plate was measured at 450 nm and
600 nm. The absorbance at 450 nm represents a direct correlation with the
cell number in this analysis. The results were expressed as the percentage of
the absorbance of control (untreated and serial diluted) cells. The control
cell number was assessed by trypan blue exclusion (final concentration,
0.2%) for 5 minutes using a hemocytometer.
Western blotting
MNCs from patients’ bone marrow were obtained by treatment of lysis
buffer (0.8% ammonium chloride solution; StemCell Technology, Vancouver, Canada) to remove red blood cells and were washed 3 times in
phosphate-buffered saline. MNCs were resuspended at a final concentration
of 1 ⫻ 107/mL in RPMI-1640 supplemented with 10% fetal bovine serum
and penicillin. Five milliliters of the suspension was then plated onto a T-25
culture flask (Becton Dickinson, Franklin Lakes, NJ) with methylprednisolone (50 ␮g/mL). A total of 1 ⫻ 107 cells was harvested at 0, 24, 48, and
72 hours. Total proteins were immediately extracted by the Pro-Prep
solution (Intron, Seoul, South Korea).
The proteins extracted were assessed by the Bradford assay (Bio-Rad,
Hercules, CA) according to the manufacturer’s instructions. Briefly, the
samples were resolved on a 10% Bis-Tris precast gel, following the
manufacturer’s instructions (Invitrogen). After transferring of the gel to
polyvinylidene difluoride membrane (Millipore, Bedford, MA), the membrane was blocked with 5% skim milk in Tris-buffered saline containing
0.1% Tween 20 (TBST) for 1 hour at room temperature. Monoclonal
antibodies against Livin (clone 88C570; Abcam, Cambridge, United
Kingdom), caspase 3 (Cell Signaling Technology, Beverly, MA), and poly
(ADP-ribose) polymerase (PARP; Cell Signaling Technology) were diluted
1:1000, and anti–␤-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA)
was diluted 1:5000 in blocking solution for immunoblotting. The membrane was exposed to the primary antibodies in a blocking buffer overnight
at 4°C, followed by three 5-minute washes with TBST. After washing with
TBST 3 times, the blots were incubated with anti–rabbit or anti–mouse IgG
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BLOOD, 15 JANUARY 2007 䡠 VOLUME 109, NUMBER 2
horseradish peroxidase–linked antibody (Cell Signaling Technology) for
1 hour at room temperature. Enhanced chemiluminescence (ECL) reaction
was performed by ECL detection kit (Amersham Bioscience, Piscataway,
NJ) according to the manufacturer’s instructions. The full-length (␣- and
␤-isoform) and cleaved forms of Livin were determined by the Western blot
assay based on the method by Nachmias et al.31 Densitometric quantification of the Livin protein was performed with a GS-800 imaging densitometer (Bio-Rad, Taiwan). The protein levels of Livin and ␤-tubulin from the
same patient were quantified by using Quantity one 4.2.2 software
(Bio-Rad, Hercules, CA), and the ratio of Livin to ␤-tubulin was then
defined as the Livin protein level.
LIVIN EXPRESSION IN CHILDHOOD ALL
Table 1. Patient characteristics and the Livin expression rates
Characteristic
Differences in the expression rate of Livin with respect to common
prognostic factors (ie, sex, age, leukocyte count, risk group, immunophenotype, numerical or structural cytogenetic abnormalities, cerebrospinal fluid
[CSF] involvement, and mediastinal mass) and treatment outcomes (day 7
bone marrow response to induction chemotherapy and occurrence of
relapse) were analyzed using the Pearson chi-square test. Differences in
expression levels of Livin with respect to the prognostic factors were
analyzed using the Mann-Whitney U test. The percentage of survival of
leukemic cells in the cytotoxicity assay was also compared using the
Mann-Whitney U test. The expression rates of Livin were recorded as a
percentage (%) of patients with Livin expression among total patients
regardless of the expression level, and the expression levels were represented as the median values along with the ranges. The patients were
categorized into 2 groups according to the presence or absence of the Livin
expression. Overall survival (OS) rate, relapse-free survival (RFS) rate, and
event-free survival (EFS) rate along with 95% confidence interval (CI) were
estimated using the Kaplan-Meier method. An event was defined as the
occurrence of a relapse or treatment-related death. The differences in
survival rates between the 2 groups were compared using the log-rank test.
Univariate and multivariate analyses comprising common prognostic
factors for relapse-free or event-free survival were performed using the Cox
regression analysis. The correlation between the Livin mRNA expression
level and protein level was estimated using the Spearman test. Statistical
significance was accepted when the P values were less than .05.
No. of Livin expression,
patients
no. (%)
P
Sex
Female
Male
84
28 (33.3)
138
29 (21.0)
161
49 (30.4)
61
8 (13.1)
166
49 (29.5)
56
8 (14.3)
129
42 (32.6)
93
15 (16.1)
171
45 (26.3)
49
12 (24.5)
.042
Age
1 y to less than 10 y
At least 10 y or younger than 1 y
.008
Leukocyte count
Less than 50 ⫻ 109/L
At least 50 ⫻ 109/L
Statistical analysis
473
.024
Risk group
Standard-risk patients
High-risk patients
.006
Immunophenotype
Precursor B cell
Others
.797
Number of chromosomes
50 or more
45-49
44 or fewer
26
1 (3.8)
168
48 (28.6)
9
3 (33.3)
.023
Structural abnormalities of chromosome
t(12;21)
35
29 (82.9)
155
27 (17.4)
32
1 (3.1)
Absent
193
49 (25.4)
Present
12
1 (8.3)
215
57 (26.5)
Absent or others
t(9;22) or 11q23 rearrangement
⬍ .001
CSF involvement
.182
Mediastinal involvement
Absent
Present
7
0 (0)
.114
Day 7 BM response to treatment
Leukemic blasts less than 25%
143
50 (35.0)
69
6 (8.7)
No
185
56 (30.3)
Yes
37
1 (2.7)
Leukemic blasts at least 25%
⬍ .001
Relapse
⬍ .001
Differences in Livin expression rates were analyzed using the Pearson chisquare test. Statistical significance was accepted when P values were less than .05.
Results
Patient characteristics
Clinical characteristics of the patients at diagnosis are presented in
Table 1. The median age at diagnosis among the 222 patients (138
boys and 84 girls) was 65.5 months (range, 3-190 months), and
their median leukocyte count at diagnosis was 9.00 ⫻ 109/L (range,
0.31 ⫻ 109–925.1 ⫻ 109/L). The median proportion of bone marrow leukemic blasts at diagnosis was 92.0% (range, 31%-100%).
The proportion of patients with at least 75% of bone marrow
leukemic blasts was 84.2%. The numbers of standard-risk and
high-risk patients were 129 and 93, respectively, and 171 patients
(77.7%) had the precursor B-cell immunophenotype. The number
of patients with hyperdiploidy (ⱖ 50) and hypodiploidy (ⱕ 44)
were 26 (12.8%) and 9 (4.4%), respectively. Frequent translocations identified by conventional chromosomal analysis, FISH
examination, or both were t(12;21) in 35 patients, t(9;22) in 16
patients, and 11q23 rearrangement in 17 patients. Thirty-seven
(16.7%) patients received allogeneic hematopoietic stem cell
transplant from 11 related and 26 unrelated donors (20 bone
marrow, 15 cord blood, and 2 peripheral blood stem cells).
Twenty-nine patients received a transplant at first CR, 7 at second
CR, and 1 at third CR.
Both expression rates and expression levels of Livin were
higher in patients with favorable prognostic factors
The expression rates of Livin with respect to the common
prognostic factors are presented in Table 1. Livin mRNA was
expressed in 57 (25.7%) of 222 patients. The expression rate of
Livin was higher in female patients (P ⫽ .042), patients with an
age range of 1 to 9 years (P ⫽ .008), patients with a leukocyte
number of less than 50 ⫻ 109/L (P ⫽ .024), standard-risk patients
(P ⫽ .006), and patients with t(12;21) (P ⬍ .001), compared with
male patients, patients at least 10 years old or younger than 1 year,
patients with a leukocyte count of at least 50 ⫻ 109/L, high-risk
patients, and children with structural chromosomal abnormalities
other than t(12;21). Livin expression rates were also higher in
patients without CSF involvement or a mediastinal mass than in
those with CSF involvement or a mediastinal mass, albeit without
statistical significance. There was no difference in the Livin
expression rate with respect to the immunophenotype. The Livin
expression rate was lower in patients with hyperdiploidy (ⱖ 50)
than in patients with hypodiploidy (ⱕ 44) or others (P ⫽ .023).
When the analysis was confined to only the patients who
exhibited Livin expression, the median expression level of Livin
was 3.28 (range, 0.04-382.68). Of note, the expression level of
Livin was higher in patients with favorable prognostic factors than
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474
CHOI et al
BLOOD, 15 JANUARY 2007 䡠 VOLUME 109, NUMBER 2
when the leukemic cells expressed Livin mRNA (Figure 2B).
During the cytotoxicity assay, we observed a persistent presence of
both full-length Livin protein (␣- and ␤-isoform) and a cleaved
form thereof in the leukemic cells with Livin expression.31 The
intensities of caspase 3 and PARP proteins increased as the
apoptotic process continued (Figure 2C).
Livin expression was an independent favorable
prognostic factor
Figure 1. Livin expression level with respect to risk factors. (A) The Livin
expression level in low-risk patients was higher than in high-risk patients.
(B) Similarly, the Livin expression level in patients with t(12;21) was higher than in
patients without t(12;21). The Livin expression levels are presented in box plots
showing the median and 90th percentiles of Livin expression. Maximum and
minimum values are represented by bars. Statistical analysis was performed
using the Mann-Whitney U test.
in patients with unfavorable prognostic factors when the analysis
was confined to only the patients with Livin expression. For
example, the expression level of Livin in the standard-risk patients
(median, 5.01; range, 0.04-382.68) was higher than in the high-risk
patients (median, 0.68; range, 0.04-11.71; P ⫽ .021; Figure 1A).
Similarly, the Livin expression level was also higher in patients
with t(12;21) (median, 7.39; range, 0.86-382.68) than in patients
without t(12;21) (median, 1.26; range, 0.04-37.75; P ⫽ .027;
Figure 1B). The expression level in the only patient with t(9;22)
who exhibited Livin expression was very low (0.18). The Livin
protein level (median, 0.165; range, 0.029-0.819) measured by
Western blot analysis in 7 patients with Livin expression showed a
good correlation with the mRNA level (median, 0.207; range,
0.001-3.784; P ⫽ .044).
Livin expression was associated with a favorable early
response to chemotherapy
Livin expression was associated with a favorable early response to
induction chemotherapy. The Livin expression rate was higher in
patients with a favorable (leukemic blast ⬍ 25%) day 7 bone
marrow response than in patients with an unfavorable (leukemic
blast ⱖ 25%) response (35.0% versus 8.7%, P ⬍ .001; Table 1).
Similarly, the proportion of patients with an unfavorable day 7
bone marrow response was significantly lower in patients with
Livin expression than in patients without Livin expression (10.7%
versus 40.4%, P ⬍ .001; Figure 2A). From the cytotoxicity assay
that was used to evaluate the ex vivo susceptibility of leukemic
blasts to apoptotic stimuli provided by chemotherapeutic agents,
the percentage of surviving leukemic blasts after 24, 48, and 72
hours of cultures with methylprednisolone were significantly lower
The median follow-up duration among 193 live patients was 37
months (range, 1-90 months). The leukemia relapsed in 37 patients
and treatment-related mortality occurred in 7 patients. The 5-year
OS, RFS, and EFS rates (⫾ 95% CI) in 222 patients were
82.2% ⫾ 7.0%, 78.6% ⫾ 6.8%, and 76.0% ⫾ 6.8%, respectively.
Livin expression was significantly associated with the absence of
relapse (P ⬍ .001; Table 1). Similarly, RFS rate was higher in
patients with Livin expression than in patients without Livin
expression (97.9% ⫾ 4.0% versus 64.9% ⫾ 11.8%, P ⬍ .001; Figure 3A). In the only relapsed patient with Livin expression, the
scheduled chemotherapy could not be delivered prior to relapse
because of unexplained severe neurotoxicity during maintenance
chemotherapy. Otherwise, all patients who showed Livin expression, including high-risk patients with unfavorable prognostic
factors at diagnosis, are currently relapse free (Figure 3B). Among
29 patients who underwent allogeneic stem cell transplantation at
first CR, 5 patients with Livin expression are all alive without
relapse, whereas 5-year RFS rate of 24 patients without Livin
expression was 50.0% ⫾ 22.5% (P ⫽ .067).
According to the univariate analyses, age at diagnosis of 1 to 9
years, leukocyte count less than 50 ⫻ 109/L at diagnosis, precursor
B-cell immunophenotype, the presence of t(12;21), and Livin
expression were statistically significant favorable prognostic factors. The presence of t(9;22) or 11q23 rearrangement was also an
unfavorable prognostic factor. According to the multivariate analyses for relapse-free survival, Livin expression and structural
chromosomal abnormalities were independent favorable prognostic factors with a statistical significance in childhood ALL (hazard
ratio of Livin⫺ patients compared with Livin⫹ patients, 8.844; 95%
CI, 1.010-77.479; P ⫽ .049; Table 2). The age at diagnosis also had
a borderline statistical significance as an independent prognostic
factor. Leukocyte count at diagnosis, immunophenotype, and
number of chromosomes were not independent prognostic factors.
During the study period, 14 bone marrow samples were collected
from relapsed patients or from patients who were refractory to chemotherapy after relapse. Only 1 (7.1%) of 14 patients exhibited Livin
Figure 2. Different apoptotic responses to chemotherapeutic agents according to Livin expression. (A) The proportion of patients with unfavorable day 7 bone marrow
response (leukemic blasts ⱖ 25%) was significantly lower in patients with Livin expression compared with patients without Livin expression (P ⬍ .001). (B) In the cytotoxicity
assay to evaluate the ex vivo susceptibility of leukemic blasts to apoptotic stimuli provided by methylprednisolone, the percentages of surviving leukemic blasts after 24, 48, and
72 hours of culture were significantly lower when the leukemic cells expressed Livin mRNA. Graph shows means and SEM. (C) In the cytotoxicity assay, both full-length Livin
protein (␣- and ␤-isoform) and the cleaved form of Livin protein were persistently observed in the leukemic cells with Livin expression. The intensities of caspase 3 and PARP
proteins increased as the apoptotic process continued.
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BLOOD, 15 JANUARY 2007 䡠 VOLUME 109, NUMBER 2
LIVIN EXPRESSION IN CHILDHOOD ALL
Figure 3. Relapse-free survival according to Livin expression. (A) Relapse-free
survival was significantly longer in patients with Livin expression than in patients
without Livin expression (97.9% ⫾ 4.0% versus 64.9% ⫾ 11.8%). (B) All patients in
the high-risk group were relapse free when they exhibited Livin expression (100.0%
versus 48.7% ⫾ 23.7%).
expression. In this patient, who had been transferred from another
hospital after relapse, the day 7 bone marrow response was M1 during
reinduction chemotherapy after relapse, and this patient is currently
disease free at 22 months after relapse.
Discussion
This is the first study evaluating the expression of Livin, a member
of the IAP family proteins, in association with clinical features at
diagnosis and treatment outcomes in childhood ALL. Unexpectedly, this study showed that Livin expression in childhood ALL
was, albeit not always, associated with the presence of favorable
prognostic factors at diagnosis. In addition, Livin expression was
associated with a favorable early response to chemotherapy and
eventually a higher long-term relapse-free survival.
In this study, the Livin expression rate was significantly higher
in patients with favorable prognostic factors. It is particularly
interesting to note that the Livin expression rate was very high in
t(12;21) and was very low in t(9;22)/11q23 rearrangement, because
475
these translocations are known to be strongly associated with the
best41 and worst outcomes,42-44 respectively, in childhood ALL. In
this context, it was also interesting to note that the Livin expression
rate was lower in cases of hyperdiploidy (ⱖ 50) than in cases of
hypodiploidy (ⱕ 44) or others. Although the absolute number of
chromosomes chosen as the “cutoff point” for analysis may vary
slightly between studies, children with hyperdiploidy and hypodiploidy have a better or poorer prognosis, respectively.45-47 The lower
rate of Livin expression in our patients with hyperdiploidy may
explain their relatively poorer RFS rates (65.4% ⫾ 29.0% at 5
years) than have been generally expected. However, it is not clear
why the Livin expression rate was lower in our patients with
hyperdiploidy, whereas it was higher in patients with other
favorable prognostic factors.
It is also interesting to note that not only the Livin expression
rates but also the Livin expression levels were higher in patients
with favorable clinical features compared with those in patients
with unfavorable clinical features. However, a higher Livin expression level was not associated with a better RFS rate compared with
a lower Livin expression level; this was because all but one patient
who exhibited Livin expression are currently relapse free regardless of the Livin expression level. These findings suggest that Livin
expression itself, not the expression level, is an important prognostic determinant in patients with childhood ALL.
Livin expression in this study was strongly associated with
better RFS, EFS, and OS rates in patients with childhood ALL. The
better early bone marrow response to induction chemotherapy and,
similarly, the better ex vivo response to methylprednisolone in the
cytotoxicity assay suggest that Livin expression is associated with
a faster apoptotic response of leukemic cells to apoptotic stimuli
provided by chemotherapeutic agents and eventually associated
with a better relapse-free survival.39 According to the multivariate
analyses, Livin expression was an independent favorable prognostic factor harboring statistical significance.
Table 2. Univariate and multivariate analyses with prognostic factors for relapse-free survival
Variable
No.
Relapse,
no. (%)
5-y RFS,
ⴞ 95% CI
Univariate analysis
HR
95% CI
Multivariate analysis
P
HR
95% CI
P
Sex
Female
Male
84
9 (10.7)
87.4 ⫾ 7.7
—
138
28 (20.3)
73.9 ⫾ 9.3
1.864
—
0.879-3.950
—
—
.104
1.367
0.599-3.120
—
—
⬍ .001
2.095
0.996-4.406
—
—
⬍ .001
1.537
0.617-3.827
—
—
.027
1.186
0.491-2.868
—
—
.458
Age
1 y to less than 10 y
At least 10 y or younger than 1 y
161
20 (12.4)
83.4 ⫾ 7.3
—
61
17 (27.8)
65.3 ⫾ 14.2
3.425
166
20 (12.0)
83.6 ⫾ 8.9
—
56
17 (30.4)
61.7 ⫾ 15.2
3.457
171
24 (14.0)
80.8 ⫾ 7.6
—
49
13 (26.5)
70.5 ⫾ 14.2
2.139
—
1.778-6.598
.051
Leukocyte count
Less than 50 ⫻ 109/L
At least 50 ⫻ 109/L
—
1.806-6.617
.356
Immunophenotype
Precursor B cell
Others
—
1.088-4.204
No. of chromosomes
At least 50
45-49
44 or fewer
.805
Absent or others
t(9;22) or 11g23 rearrangement
.425
26
5 (19.2)
65.4 ⫾ 29.0
—
168
27 (16.1)
81.6 ⫾ 6.6
0.730
0.280-1.899
.518
0.499
0.171-1.455
.203
9
2 (22.2)
75.0 ⫾ 30.0
0.839
0.162-4.352
.834
0.689
0.118-4.038
.680
—
—
—
⬍ .001
Structural abnormality of chromosome
t(12;21)
.705
35
2 (5.7)
93.6 ⫾ 8.6
—
—
.033
155
19 (12.3)
83.1 ⫾ 7.7
2.342
0.545-10.054
.252
0.630
0.131-3.036
.565
32
16 (50.0)
39.7 ⫾ 23.0
12.742
2.923-55.550
.001
1.923
0.344-10.754
.457
—
—
.007
8.844
1.010-77.479
Livin expression
Expressed
Not expressed
57
1 (1.8)
97.9 ⫾ 4.0
—
—
165
36 (21.8)
71.4 ⫾ 9.0
15.098
2.069-110.195
Prognostic effect of each variable was estimated using Cox regression analysis. Statistical significance was accepted when P values were less than .05.
HR indicates hazard ratio; CI, confidence interval; —, not applicable (reference category).
.049
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476
BLOOD, 15 JANUARY 2007 䡠 VOLUME 109, NUMBER 2
CHOI et al
Risk stratification in childhood ALL has been traditionally
based on the initial leukocyte count and age at diagnosis.1 Most
current treatment protocols for childhood ALL differ in their
intensities of treatment according to the risk stratification based on
these 2 factors. The immunophenotype and cytogenetic abnormalities of leukemic cells are also important factors considered for
prognostic stratification and are thus used in the design and
evaluation of modern therapeutic trials in childhood ALL.2-4
However, this study has demonstrated that Livin expression is a
new independent favorable prognostic factor in childhood ALL
and, therefore, suggests that a new risk stratification system is
needed to incorporate this new molecular biologic factor with
the traditional prognostic factors. On the basis of the new
therapeutic algorithm, patients could be offered a less-intensive
therapeutic modality if the leukemic cells exhibit Livin expression. Admitting that the number of cases and the follow-up
period in this study were limited, we hope this study would
prompt other scientists to investigate the implications of Livin
expression in childhood ALL to confirm our observations in a
larger series of patients.
Most ex vivo studies and studies on the clinical relevance of
Livin expression, albeit limited in number, have shown that Livin is
an antiapoptotic regulator; therefore, Livin expression has been
considered to be a poor prognostic factor in malignancies. Therefore, the observations from this study were quite unexpected and
suggest that the role of Livin in the apoptosis system in leukemogenesis or in the maintenance of leukemic cells might be different
from what has been previously recognized. The results from this
study clearly showed that a better RFS in patients with Livin
expression is associated with a faster cell death in response to
apoptotic stimuli. However, the molecular biologic mechanisms to
explain these unexpected observations are elusive.
One possible explanation is that the cleaved form of Livin may
act as a strong proapoptotic regulator in response to apoptotic
stimuli in childhood ALL. Several investigators reported that some
of the IAP family proteins could be changed to isoforms that have
proapoptotic properties.31,48-50 Nachmias et al31 demonstrated that
the caspase-mediated cleavage of Livin converts it from an
antiapoptotic to proapoptotic factor. In this study, we could
demonstrate the presence of the cleaved form of Livin in the
leukemic cells with Livin expression. It seems plausible that this
cleaved form might have had proapoptotic activities and thereby
partly explains our observations. However, the presence of the
cleaved Livin variant has not been investigated in other types of
malignancies. Further studies are needed on the expression and
implications of the Livin variant protein and the regulatory
mechanisms thereof in childhood ALL and other malignant diseases.
Another possible explanation is that the role of Livin expression
might be related to the age of patients. Children, not fully matured,
have a variety of different biologic features distinguishing them
from adults. The regulation of apoptosis, the function of apoptotic
regulators, or both in children appear to be different from that in
adults. For example, Wuchter et al21 reported that the expression
patterns of apoptosis-related molecules were different between
children and adults with de novo acute myeloid leukemia. Another
example is that the overexpression of Bcl-2, a well-known antiapoptotic
protein, was significantly associated with a better prognosis in childhood
ALL,23 whereas it was not associated with distinct clinical or biologic
characteristics in adult ALL.22 These findings suggest that Livin
expression might have a different biologic significance in childhood
ALL compared with adult ALL and may also explain the better
outcomes in the former than in the latter.
In summary, this is the first study that investigated the relation
between Livin expression and the clinical features at diagnosis and
treatment outcomes in childhood ALL. We have shown that Livin
expression is associated not only with the presence of favorable
prognostic factors at diagnosis but also with a significantly better
RFS in childhood ALL. A better RFS appears to be associated with
a faster death of leukemic cells in patients with Livin expression in
response to apoptotic stimuli provided by chemotherapeutic agents.
Therefore, Livin expression can be a potential independent prognostic factor in childhood ALL, and new investigational approaches, as
part of well-controlled trials, would be needed to develop a
modified risk stratification system based on the status of this new
molecular biologic factor.
Acknowledgments
We would like to thank Dr Sun Woo Kim (Samsung Biomedical
Research Institute) for critical reading of this manuscript.
This work was supported by the Samsung Biomedical Research
Institute (grant SBRI C-A6-319-1) and the National R&D Program
of the Ministry of Health and Welfare, Republic of Korea (grant
0520290-1).
Authorship
Contribution: J.C. performed the research and wrote the paper;
Y.K.H. and H.-J.K. performed the research and analyzed the data;
K.W.S. designed the research, analyzed the data, and wrote the
paper; S.H.L. performed the research and collected the data;
K.H.Y., H.L.J., H.H.K., H.J.K., and H.Y.S. designed the research
and collected the data; and H.S.A. designed the research and
analyzed the data.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
K.W.S. and H.H.K. contributed equally to this work.
Correspondence: Ki Woong Sung or Hong Hoe Koo, Department of Pediatrics, Samsung Medical Center, Sungkyunkwan
University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku,
Seoul, South Korea 135-710; e-mail: [email protected]
or [email protected].
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2007 109: 471-477
doi:10.1182/blood-2006-07-032557 originally published
online September 21, 2006
Expression of Livin, an antiapoptotic protein, is an independent
favorable prognostic factor in childhood acute lymphoblastic leukemia
Jaewon Choi, Yu Kyeong Hwang, Ki Woong Sung, Soo Hyun Lee, Keon Hee Yoo, Hye Lim Jung,
Hong Hoe Koo, Hee-Jin Kim, Hyong Jin Kang, Hee Young Shin and Hyo Seop Ahn
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