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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2671-2678
Increased BAX Expression Is Associated With an Increased Risk of
Relapse in Childhood Acute Lymphocytic Leukemia
By
Linda A. Hogarth and
Andrew G. Hall
From The LRF Molecular Pharmacology Specialist Programme, Cancer
Research Unit, Medical School, Newcastle Upon Tyne, UK.
 |
ABSTRACT |
Studies in cell lines have indicated that expression of the BCL-2
family of proteins is an important determinant of chemotherapy-induced apoptosis; however, the level of expression of these proteins in
childhood acute lymphoblastic leukemia (ALL) has not been extensively reported. Using quantitative Western blotting we have determined the
level of expression of BCL-2, BAX, MCL-1, and BCL-X in lymphoblasts from 47 children with ALL (33 at presentation only, 4 at relapse only,
and 10 at both presentation and on relapse). Results were determined as
a ratio to actin as an internal control. BCL-2, BAX, and MCL-1 were
detected in all samples. BCL-XL was only detected in 6 cases (4 at presentation and 2 at relapse) and BCL-XS in none. No correlation was found between expression and white blood cell
count, age at diagnosis, gender, or blast karyotype. BCL-2 levels and
the BCL/BAX and MCL-1/BAX ratios were found to be significantly higher
in B-lineage as compared with T-lineage disease (P < .003, .02, and .02, respectively). No consistent pattern of change in expression was noted in the 10 cases studied at both presentation and
relapse. Kaplan-Meier analysis showed a significant correlation between
high BAX expression and an increased probability of relapse (P < .05 by the log rank test), suggesting that chemosensitivity in
leukemic blasts may be regulated by factors that override the BCL-2 pathway.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CONSIDERABLE evidence now exists that
cytotoxic drugs used in the treatment of malignancies exert their
effects through the triggering of apoptotic pathways.1,2
Preclinical studies have indicated that alterations in the apoptotic
threshold may be a cause of drug resistance, but the direct relevance
of this to clinical practice is, as yet, uncertain. Although apoptosis involves a complex series of intracellular molecular interactions, cell
line studies have indicated that, in most systems, alterations in the
expression of BCL-2 and related proteins can have a marked influence on
chemosensitivity.3 The aim of the study reported here is to
establish if measurement of expression of these proteins could be of
prognostic relevance in childhood acute lymphoblastic leukemia (ALL).
The BCL-2 oncoprotein is a suppressor of apoptosis,3,4
initially described in human B-cell follicular lymphomas with the
t(14;18) chromosomal translocation, where its juxtaposition with the JH
region of the Ig heavy chain results in deregulated overexpression and
elevated levels of a 26-kD protein.5-7 However, BCL-2 has
also been found in normal T- and B-lymphoid cells and in a variety of
lymphoproliferative disorders in which t(14;18) is not
present.5,8 In follicular lymphomas, BCL-2 suppresses apoptosis mediated by several agents9,10 and also
correlates with poor treatment outcome.10 BCL-2 has now
been shown to inhibit apoptosis in many different cell types and under
a variety of conditions, such as treatment with  -irradiation,
chemotherapeutic drugs, glucocorticoids, cytotoxic lymphokines, and
heat shock,11-14 suggesting that it inhibits cell death
triggered by multiple routes and acts at a step common to many
pathways. Protection by BCL-2 against inhibitors of thymidylate
synthase and also DNA-damaging agents, such as nitrogen mustard,
camptothecin, or etoposide, showed that BCL-2 is not involved in the
reduction of drug-induced DNA damage, alterations in the rates of DNA
repair, inhibition of drug-induced alterations in nucleotide pools, or
changes in cell cycle kinetics.15-17 Overall, these
findings suggest that BCL-2 acts downstream of these events.
Since the discovery of BCL-2, a large number of genes have been
identified whose products have homology with BCL-2 and have been termed
the BCL-2 family of proteins. These proteins either suppress or
promote apoptosis. Those, like BCL-2, that suppress apoptosis include
BCL-XL, MCL-1, A1, BCL-W, NR-13, and BFL-1, and those that
promote apoptosis include BAX, BCL-XS (a splice variant of
BCL-XL), BAD, BAK, NBK/BIK, BID, HRK,18,19 and
BOK.20
The BCL-2 family of proteins appears to regulate apoptosis by a process
involving complex protein-protein interactions. They can form
homodimers or heterodimers with each other and in some cases with
structurally unrelated proteins.21 There is evidence that
the relative expression and competitive dimerization between these
proteins may influence response of a cell to apoptotic stimuli. Although a number of studies have been reported in which the expression of BCL-2 has been related to clinical outcome in hematological malignancies, few of these have included measurement of the relative expression of other members of the BCL-2 family. However, recently, Kaufmann et al22 published data describing the expression
of BCL-2, BCL-XL, and MCL-1 in the blasts of adults
presenting with acute myeloid leukemia (AML) or ALL in which they
reported an increase in MCL-1 in the majority of cases at relapse (10 of 19 matched pairs for AML and 3 of 4 matched pairs for
ALL).22 We extend here these observations to childhood ALL
and link measurement of expression in blasts at presentation to
long-term survival.
| |
MATERIALS AND METHODS |
Patients.
Forty-seven children with de novo ALL, referred to the Royal Victoria
Infirmary (RVI; Newcastle upon Tyne, UK) between 1986 and 1997, were
used in this study. Forty-three samples were presentation blasts and 14 samples were obtained at first relapse. Children who failed to achieve
remission were excluded from the study. Samples obtained at relapse
included 10 from children who were also analyzed at presentation. The
diagnosis was established by cytological examination of bone marrow
(BM) smears according to the French-American-British Group
recommendations23 and immunophenotyping of leukemic
cells.24 Immunophenotype was assessed by flow cytometry (FACScan; Becton Dickinson, San Jose, CA) and
immunocytochemistry, using antibodies against terminal deoxynucleotidyl
transferase (TdT), cytoplasmic µ (cyt-µ), surface membrane Ig
(SIg), CD2, CD3, CD7, CD10, CD13, CD19, CD20, CD33, and CD34 by the
Department of Haematology, RVI, Newcastle upon Tyne. Within the cases
diagnosed as ALL, immunologic subgroups were defined as follows: null
ALL (CD19+, CD20±, CD10 ,
cyt-µ, SIg), common ALL (cALL;
CD19+, CD20±, CD10+,
cyt-µ, SIg), pre-B ALL
(CD19+, CD20±, CD10±,
cyt-µ+, SIg), B-ALL
(CD19+, CD20+, CD10±,
cyt-µ+, SIg+), and T-ALL
(CD2±, CD3+, CD7+).
Cytogenetic analysis was performed in the Department of Cytogenetics,
RVI, Newcastle upon Tyne, using standard G-banding techniques to stain
metaphase preparations obtained from unstimulated cultures of BM cells.
A complete karyotype analysis was obtained in 34 presentation samples
and 11 relapse samples, and incomplete karyotype analysis was obtained
in 4 presentation samples and 1 relapse sample.
Of the 43 presentation childhood ALL cases studied, 25 were male and 18 were female. The median age was 5 years (age range, 0.8 to 16 years).
The median presenting white blood cell (WBC) count was 17 × 109/L (range, 0.3 × 109/L to 1,000 × 109/L). There were 30 common ALL, 3 pre-B-cell
ALL, 1 null cell ALL, and 9 T-cell ALL.
Eighteen of the 43 patients studied at diagnosis relapsed. First
relapses included 11 BM, 3 central nervous system (CNS), 1 testicular,
2 with joint BM and CNS relapse, and 1 with joint BM and testicular
relapse. The median follow-up of patients was 36 months (range, 1 to
136 months).
Children in this study had been entered into one of three trials
administered by the UK Medical Research Council (MRC): UKALL X (1995 to
1992, 8 patients), UKALL X1 (1992 to 1997, 33 patients), or ALL97 (1997 to date, 2 patients). Remission induction was achieved with
prednisolone (or dexamethasone in the case of ALL97), vincristine, L-asparaginase, and intrathecal methotrexate. Daunorubicin was also
used in UKALLX. Intensification therapy consisted of prednisolone (or
dexamethosone in ALL97), etoposide, vincristine, cytarabine, thioguanine, intrathecal methotrexate, and daunorubicin. Continuing therapy consisted of prednisolone (or dexamethasone in ALL97), methotrexate, vincristine, and 6-mercaptopurine (or 6-thioguanine in ALL97).
Preparation of samples.
All samples were processed within 5 hours of BM aspiration.
Lymphoblasts and mononuclear cells were separated by centrifugation over Ficoll. The percentage of leukemic lymphoblasts in the Ficoll prepared samples was determined by cytological examination of cells
stained with May Grunwald-Giemsa stain. Cells were cryopreserved at
 135°C before analysis.
Cells were thawed at 37°C and 10 vol of RPMI 1640 (Dutch
modification; GIBCO-BRL Life Technologies Ltd, Paisley, UK) containing 15% heat-inactivated fetal calf serum, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine (RF15) added slowly. Cells were pelleted by centrifugation at 230g for 5 minutes,
resuspended in 5 mL of RF15, and incubated at 37°C in 5%
CO2 for 90 minutes. An aliquot was taken to determine the
percentage of viable and apoptotic cells using Hoescht and trypan blue
staining or acridine orange and ethidium bromide.25 Samples
containing more than 15% of dead or apoptotic cells were excluded from
further study.
The remaining cells were spun at 230g for 5 minutes, and the
pellet was resuspended in 2 mL of 100 µmol/L EDTA, 1 mmol/L
KHCO3, and 0.17 mol/L NH4Cl in deionized water
(pH7.3) for 4 minutes to lyse any contaminating red blood
cells.26 Twenty milliliters of ice-cold phosphate-buffered
saline (PBS) was added; the sample was centrifuged at 230g for
4 minutes; and the pellet was lysed in sodium dodecyl sulfate
(SDS) sample buffer containing 62.5 mmol/L Tris/HCl, pH
6.8, 2% SDS, and 20% glycerol at 100 µL per 5 million cells and was
heated at 100°C for 5 minutes. The sample was sonicated for 5 seconds at 15 µm and microcentrifuged at 14,000 rpm for 10 minutes,
and the pellet was discarded. An aliquot of the supernatant was diluted
1:9 with water and used to estimate the protein level using a
commercially available kit using the bicinchoninic acid (BCA) method
(Pierce, Rockford, IL). Samples were stored at 80°C until
ready for analysis. Immediately before use, 2-mercaptoethanol (5%) and
bromophenol blue (0.005%) were added and the samples were diluted to a
concentration of 0.5 mg/mL in SDS sample buffer containing
2-mercaptoethanol and bromophenol blue before being heated at 50°C
for 5 minutes.
Determination of BCL-2, BAX, MCL-1, and BCL-X by immunoblotting.
The Epstein-Barr virus-immortalized human Philadelphia
chromosome-positive ALL cell line, SD-1 (kindly provided by Dr S. Dhut27 ICRF, Medical Oncology Unit, St Bartholomew's
Hospital, London, UK) was used as a positive control for
BCL-2, BAX, and actin. The erythroleukemia K562 cell line28
was used as a control for MCL-1 and BCL-X. Cells were lysed in SDS
sample buffer as described above for patient lymphoblasts and diluted
to give a five-point standard curve for each blot by loading 20, 15, 10, 5, and 2.5 µg of total cellular protein.
Immunoblotting.
Twenty microliters of sample (equivalent to 10 µg of total protein)
was subjected to SDS-polyacrylamide gel electrophoresis according to
the method of Laemmli29 using minigel equipment supplied by
Bio-Rad Laboratories Ltd (Hertfordshire, UK). Twelve percent acrylamide
gels were used for analysis of BCL-2 and BAX expression, and 10% gels
were used for MCL-1 and BCL-X. For an individual experiment, 5 standards were loaded on the same gel as 4 lymphoblast samples and a
molecular weight (MW) marker (SeeBlue Pre-Stained Standard; Novex
Experimental Technology, San Diego, CA).
After electrophoresis, proteins were transferred to nitrocellulose
membranes (Hybond-C; Amersham, Little Chalfont, UK) using miniblot equipment supplied by Bio-Rad Laboratories Ltd (Hertfordshire, UK) as previously described.30 After transfer, blots for
BCL-2, BAX, and actin analysis were divided between the 30- and 36-kD MW markers. One half was probed for actin (MW 42 kD) and the other for
BCL-2 (MW 26 kD). After developing the BCL-2 blot, it was washed with
two changes of Tris-buffered saline (TBS; 0.01 mol/L Tris, pH 7.5, containing 0.1 mol/L NaCl) and stored at 4°C before reprobing for
BAX within 1 month. One part of the MCL-1/BCL-X blot was probed for
MCL-1 (41 kD) and the other part for BCL-X (BCL-XL 29 kD,
BCL-XS 20 kD).
Immunocomplexes were detected using enhanced chemoluminescence. Blots
were immersed for 1 hour in a blocking buffer of 5% instant dried
skimmed milk (Boots Co PLC, Nottingham, UK) in TBS containing 0.05% tween 20 (TBS-T). Primary antibody was diluted in
blocking buffer and incubated with the blots for 1 hour as follows:
BCL-2 (mouse monoclonal; Dako, Cambridge, UK) at 1/200; BAX (rabbit
polyclonal; Pharmingen, San Diego, CA) at 1/100; BCL-X and MCL-1
(rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) at 1/100;
and actin (mouse monoclonal; Sigma-Aldrich Chemical Co Ltd, Dorset, UK)
at 1/1,000. Blots were washed thoroughly with TBS-T and incubated in
secondary antibody (biotinylated swine antirabbit or rabbit antimouse,
diluted 1 in 1,000 in 0.5% casein in TBS-T) for 30 minutes. After
further washing in TBS-T, blots were incubated for 30 minutes in
streptavidin-conjugated horseradish peroxidase (HRP; Dako)
diluted 1 in 1,000 in 0.5% casein in TBS-T. After a final wash in
TBS-T, HRP was detected using ECL reagent (Amersham Life Science Ltd,
Buckinghamshire, UK) as described in the manufacturer's instructions.
Chemiluminescence was detected by autoradiography using x-ray film
(Fuji, Gateshead, UK). The integrated optical density (IOD) of the
resulting bands was determined by densitometry using a Bio-Image system
(Millipore UK Ltd, Hertfordshire, UK).
Statistical analysis.
Survival analysis was performed using the Kaplan-Meier method and
survival curves were compared using the log-rank test. Protein expression in different prognostic groups was compared using the Mann-Whitney U test. Expression was correlated with WBC count using
Spearman rank analysis. P values of less than .05 were
considered to be statistically significant.
| |
RESULTS |
Percentage of blasts and apoptosis in lymphoblast samples.
The blast count after mononuclear cell separation was greater than 90%
in most cases. No cases were analyzed that contained fewer than 80% blasts.
Reproducibility of immunoblots.
To check intrablot reproducibility, 9 identical samples were run on
10% polyacrylamide gels. As 10 or 20 µL of samples was loaded on
polyacrylamide gel, reproducibility was checked for loading both of
these amounts. On one gel was loaded 20 µL of a 1 µg/µL MCL-1
standard and on another 10 µL of a 2 µg/µL standard. The
coefficient of variation for loading 10 µL samples was 10.8% and for
loading 20 µL samples was 8.7%. To check interblot reproducibility, one sample was loaded on five separate gels with standards, blotted, and probed for BCL-2 and actin. The coefficient of variation for BCL-2
and actin measurements and the BCL-2/actin ratio were 15.7%, 12.3%,
and 12.7%, respectively.
Expression of BCL-2, BAX, MCL-1, and BCL-X in lymphoblasts at
diagnosis and on relapse.
Examples of immunoblots for BCL-2, BAX, MCL-1, and actin are shown in
Fig 1. The identity of the target protein
was determined on the basis of MW where multiple bands were present. A
standard curve was produced for each blot by plotting the IOD of each
standard band against the amount of cell lysate loaded. For the patient samples, these standard curves were used to determine the amount of
cellular protein in the standards required to produce an equivalent IOD. These values were finally expressed as a ratio to actin used as an
internal control for the amount of cell lysate loaded. Because actin
has approximately the same MW as MCL-1, actin and MCL-1 could not be
probed on the same blots. Hence, MCL-1 levels were expressed as a ratio
to actin measured on blots probed for BCL-2 and BAX. Because the ratios
of BCL-2 to BAX and MCL-1 to BAX have been suggested as determinants of
apoptotic threshold, these were determined for each sample.
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| Fig 1.
Immunoblot showing level of expression of BCL-2, BAX,
MCL-1, and BCL-XL with corresponding standards from SD1
cells (in the case of BCL-2 and BAX) and K562 cells (in the case of
MCL-1 and BCL-XL). Actin is included as a loading
control.
|
|
BCL-XL expression was only detected in four lymphoblast
samples at presentation and 2 relapse samples. Levels of expression were less than the 2.5 µg standard except for one patient that was
between the 2.5 and 5 µg standard. BCL-XS was not
detected in any of the samples analyzed.
Relationship between relapse-free survival and expression of BCL-2,
BAX, and MCL-1 at diagnosis.
Relapse-free survival was compared in cases in which expression of
BCL-2 family proteins was above the median for the whole group (high
expressers) with those in which expression was below or equal to the
median value (low expressers). Survival curves were compared using the
log rank test. Relapse-free survival did not differ with expression of
BCL-2, BCL-2/BAX ratio, MCL-1, or MCL-1/BAX ratio. However, there was a
significant correlation between high levels of BAX expression and an
increased probability of relapse (P = .04), as shown in
Fig 2.
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| Fig 2.
Kaplan-Meier analysis of relapse-free survival for
patients with high or low expression of BCL-2 (A), BAX (B), and MCL-1
(C). High expression is defined as greater than the median, and low
expression is defined as less than or equal to the median value
obtained by quantitative Western blotting.
|
|
Expression of BCL-2 family proteins in lymphoblasts at diagnosis and
correlation with presenting WBC count, gender, and age at diagnosis.
There was no significant relationship between expression of BCL-2, BAX,
BCL-2/BAX, MCL-1, and MCL-1/BAX levels with presenting WBC count (data
not shown). Expression of these BCL-2 family proteins was compared
between age groups associated with a poor prognosis (ie, <2 years and
>10 years) and age associated with good prognosis (ie, >2 years and
<10 years). Correlations were also made with gender, because males
with ALL have been reported to have a poorer prognosis than females.
However, there was no correlation between levels of expression of
BCL-2, BAX, BCL-2/BAX, MCL-1, or MCL-1/BAX with age at diagnosis or
gender (data not shown).
Immunophenotype, karyotype, and expression of BCL-2, BAX, MCL-1,
bcl2/BAX, and MCL-1/BAX ratios in lymphoblasts at diagnosis.
Levels of expression of BCL-2, BAX, BCL-2/BAX, MCL-1, and MCL-1/BAX
were compared in B-lineage versus T-lineage ALL. There was no
correlation with BAX or the MCL-1/BAX ratio with the immunophenotype (data not shown). However, BCL-2 and MCL-1 expression and the BCL-2/BAX
ratio were significantly higher in B-lineage ALL compared with
T-lineage ALL cases (P values of .002, .016, and .012, respectively; Fig 3).
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| Fig 3.
Comparison of expression of BCL-2, MCL-1, and the
BCL-2/BAX ratio in B-lineage and T-lineage childhood ALL (*median
value). P values refer to results of analysis by the Mann
Whitney U test.
|
|
Patients were divided into good (hyperdiploid, >50 chromosomes),
standard (diploid), and poor prognosis (pseudodiploid) groups according
to karyotype analysis. Because only 2 patients fell into the poor
prognostic group, as determined by karyotype, the levels of BCL-2, BAX,
BCL-2/BAX, MCL-1, and MCL-1/BAX ratio were correlated only in good
versus standard karyotype prognostic groups. There was no significant
correlation between BCL-2 family protein expression and karyotype (data
not shown).
Expression of BCL-2, BAX, BCL-2/BAX ratio, MCL-1, and MCL-1/BAX in
lymphoblasts at diagnosis and on relapse.
Comparison of expression of BCL-2, BAX, BCL-2/BAX, MCL-1, and MCL-1/BAX
levels in lymphoblasts at diagnosis and on first relapse showed no
significant difference between the two groups (data not shown).
Although there were differences in some cases between expression of
these proteins in paired presentation and first relapse lymphoblasts
from the same patient, there was no consistent pattern of change (data
not shown).
| |
DISCUSSION |
High levels of expression of BCL-2 have been shown to correlate with
poor treatment outcome in some hematological malignancies, including
follicular lymphoma,10 chronic lymphocytic leukemia (CLL),31 and AML.32 Results from the present
study show no correlation between BCL-2 expression in childhood ALL and
known prognostic features, including age at diagnosis, gender,
karyotype, or presenting WBC count. In the present study, no
correlation was found between BCL-2 expression and relapse-free
survival. This is in agreement with several other reports, including a
study of 43 adult ALL cases,33 a study of 40 childhood and
adult ALL cases,34 and studies of childhood ALL involving
5235 and 338 patients.36 Although in the latter
study high BCL-2 levels predicted slow early response in T-lineage but
not B-lineage ALL, as judged by the number of blasts in a BM aspirate
at day 28 of induction therapy, it had no impact on short-term
event-free survival within the first 2 years of diagnosis.
Although BCL-2 is a suppressor of apoptosis and hence may be expected
to promote drug resistance in childhood ALL, increased expression has
been found to be associated with certain favorable prognostic features,
such as non-T-lineage ALL. In this study, BCL-2 was found to be
significantly lower in T-lineage compared with B-lineage cases of ALL,
with a 3.2-fold difference in the median levels of expression. Similar
differences have been reported by others.36-38 High levels
have also been related to expression of the CD10 antigen in B-lineage
ALL (common ALL)38; however, because there were only 3 pre-B ALL cases compared with 30 common ALL cases in our patient
cohort, we could not make this comparison. Increased BCL-2 expression
has been found by others to be associated with a relatively low WBC
count,36,38 another favorable prognostic feature. This
association was not found in our study.
Because the ability of BCL-2 to modulate the apoptotic threshold is
affected by the relative expression of BAX,39-41 several studies have attempted to assess the relationship between the BCL-2/BAX
ratio and clinical outcome. A high BCL-2/BAX ratio has been reported to
correlate with an inability to achieve complete remission in
AML42 and with in vitro resistance to drug-induced apoptosis in CLL.43,44 We could find no correlation between BAX expression or the BCL-2/BAX ratio and age at diagnosis, gender, karyotype, or presenting WBC count. Although there was no significant difference in BAX expression between B-lineage and T-lineage ALL, the
BCL-2/BAX levels were significantly lower in T-lineage compared with
B-lineage ALL, as was found with BCL-2 expression alone. The BCL-2/BAX
ratio did not predict relapse-free survival. However, high BAX
expression alone was shown to be associated with an increased probability of relapse (P = .04). From in vitro data, this
result was unexpected, although a comparable association between high BAX expression and unfavorable prognostic features has been reported for cancers of the breast45 and ovary.46
The unexpected relationship between high BAX expression and poor
prognosis may be explained by the observation that BCL-2 and BAX have a
role in the control of proliferation as well apoptosis. Mature T cells
overexpressing BAX have been shown to have lower levels of
p27Kip1 and enter S phase more rapidly in response to
interleukin-2 stimulation than control T cells. The converse is true
for BCL-2-transfected T cells.47 Transfection of several
mammalian cell lines with BCL-2 has been associated with reduced cell
proliferation and prolongation of the G1 phase of the cell
cycle,48,49 both of which could be abrogated by the
coexpression of BAX.48 A relationship between elevated
BCL-2 expression and a reduced rate of disease progression has been
shown in studies of clinical samples from patients with ovarian,
non-small cell lung cancer, and stage II carcinoma of the
colon.46,50,51 In addition, expression of BAX has been
associated with increased proliferative capacity in ovarian and breast
cancer.45,46 We did not assess cell proliferation in the
current study; however, we did demonstrate that BCL-2 was lower in
T-lineage disease, which has, in turn, been associated with a higher
WBC count than B-cell ALL.52 Although the generalized correlation between high WBC count and T-lineage ALL was not
statistically apparent in the patients analyzed here (data not shown),
Salomons et al,38 who did report a correlation with T-cell
lineage and WBC count, found that, after controlling for WBC count, the
association between low BCL-2 and expression of T-cell markers disappeared.
Because BCL-2 family members other than BCL-2 and BAX may be important
in regulating apoptosis in ALL, levels of expression of MCL-1,
BCL-XL, and BCL-XS were also assessed in the
current study. There was a 14-fold variation in MCL-1 expression and
ninefold variation in MCL-1/BAX expression. MCL-1 and MCL-1/BAX
expression did not correlate with age at diagnosis, gender, karyotype,
presenting WBC count, or event-free survival. BCL-XL was
only detected in 4 cases at diagnosis. Two of these cases have relapsed
(1 after 15 months and the other after 26 months) and 2 remain in
remission (after a follow-up of 96 and 116 months). BCL-XL
was only detected in 2 of 15 cases at relapse, although
BCL-XL was not detected in lymphoblasts from these same
patients at diagnosis. BCL-XS was not detected in any of
the samples analyzed. Hence, this study does not suggest that MCL-1,
BCL-XL, or BCL-XS are of prognostic significance in childhood ALL. However, it is possible that other BCL-2
family members, not assessed in this study, may be important in
regulating apoptosis and determining prognosis in ALL.
There is evidence that posttranscriptional modification regulates the
function of BCL-2 and possibly other family members. Several studies
have suggested that phosphorylation of BCL-2 affects its function,
although there have been conflicting reports that this may
enhance53,54 or reduce55,56 its ability to
suppress apoptosis. It is also possible that mutations may alter the
function of these proteins by, for example, affecting protein
stability. A variety of BAX mutations have been reported in cell lines
derived from hematological malignancies, including
ALL57,58; hence, the high level of BAX in some cases
reported in our series may have been due to the increased transcription
of nonfunctional protein, analogous to the increases seen for some p53 mutations.
In summary, we have shown that, whereas expression of BCL-2 and
determination of the BCL-2/BAX ratio do not appear to have prognostic
significance in childhood ALL, high expression of BAX at diagnosis is
associated with a significant increase in the probability of relapse.
Studies using a larger cohort of uniformly treated patients and
multivariate analysis will need to be performed to determine if this is
an independent prognostic variable that may be used to aid treatment stratification.
| |
ACKNOWLEDGMENT |
The help of Mike Reid, Elizabeth Matheson, and Jill Robson in the
collection and preparation of samples is gratefully acknowledged.
| |
FOOTNOTES |
Submitted July 10, 1998; accepted November 18, 1998.
Supported by grants from the North of England Children's Cancer
Research Fund and the Leukaemia Research Fund.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Andrew G. Hall, PhD, MD,
Cancer Research Unit, Medical School, Framlington Place, Newcastle Upon
Tyne, NE2 4HH, UK.
| |
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ABSTRACT
INTRODUCTION