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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 315-320
Hyperdiploid Acute Lymphoblastic Leukemia With 51 to 65 Chromosomes: A Distinct Biological Entity With a Marked Propensity
to Undergo Apoptosis
By
Chikako Ito,
Masa-aki Kumagai,
Atsushi Manabe,
Elaine Coustan-Smith,
Susana C. Raimondi,
Frederick G. Behm,
K. Gopal Murti,
Jeffrey E. Rubnitz,
Ching-Hon Pui, and
Dario Campana
From the Departments of Hematology-Oncology, Pathology and Laboratory
Medicine, and Virology and Molecular Biology, St Jude Children's
Research Hospital, Memphis; and the University of Tennessee Memphis
College of Medicine, Memphis, TN.
 |
ABSTRACT |
To determine the cellular basis for the excellent clinical outcome
of hyperdiploid acute lymphoblastic leukemia (ALL), defined by a modal
chromosome number of 51 to 65, we assessed the growth potential of
leukemic cells from 129 children with newly diagnosed ALL. Flow
cytometric analysis was used to compare leukemic cell recoveries at the
beginning and at the end of 7-day cultures on allogeneic bone
marrow-derived stromal layers. The median percentage of cell recovery
after culture was 91% (range, <1% to 550%). Among the 25 hyperdiploid cases, only two had cell recoveries above the median
value, compared with 63 of 104 cases with different ploidies (P
< .001); 21 had recoveries within the first quartile, in contrast
to only 12 of the 104 other cases. Cell recoveries in the 16 cases with
duplications of chromosomes 4 and 10, a feature previously associated
with a superior outcome, were all within the first quartile. Flow
cytometric studies indicated that rapid induction of apoptosis was the
underlying cause of low cell recoveries in cases with hyperdiploidy.
The demise of hyperdiploid cells on stroma was not due to failure to
adhere with stromal elements (as shown by electron microscopy) or to
deficiencies of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-6, IL-7,
IL-11, stem-cell factor, interferon- (IFN-), tumor necrosis
factor- (TNF-), or to combinations of these cytokines.
Inactivation of IL-4, IFN- and TNF-, which if secreted by stromal
layers could be toxic to ALL cells, failed to improve the survival of
hyperdiploid blasts. We conclude that leukemic cells bearing 51 to 65 chromosomes have a marked propensity to undergo apoptosis. The
stringent survival requirements of these cells, together with their
potentially higher sensitivity to antileukemic drugs, may well account
for the high cure rates achieved in patients with this form of ALL.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
AMONG THE DISTINGUISHING cellular
features of acute lymphoblastic leukemia (ALL), hyperdiploidy of 51 to
65 chromosomes is one of the most reliable predictors of a favorable
clinical outcome.1-9 Approximately 85% of children with
this form of ALL can be cured with contemporary
treatments.10 Several hypotheses have emerged to account
for this outstanding result. One suggestion is that hyperdiploid cases
have relatively higher percentages of cells in S phase,11
resulting in greater sensitivity to cell cycle-specific drugs. Another
is that hyperdiploid blasts accumulate significantly higher levels of
methotrexate polyglutamates than do cells of lower
ploidy,12,13 rendering them more sensitive to
antimetabolites.14 An intriguing idea is that such cells lack the ability to expand rapidly and cannot grow outside the bone
marrow microenvironment. Support for this hypothesis comes from the
lower leukocyte counts and the general lack of bulky extramedullary
disease typically seen at diagnosis in hyperdiploid cases.1,2,8,15
Underlying our research is the hypothesis that leukemic cell growth
requirements strongly influence treatment outcome. Thus, cells with
less stringent requirements may have an enhanced ability to grow
outside the bone marrow microenvironment, enabling them to infiltrate
pharmacologic sanctuaries with consequent adverse effects on treatment
response. Methods to culture leukemic lymphoblasts on bone
marrow-derived stromal layers provide a unique opportunity to measure
the growth potential of leukemic lymphoblasts. Stromal feeder layers
secrete factors that prevent apoptosis in vitro in most cases of
ALL.16,17 In a previous study of 70 cases of childhood ALL,
we found a strong correlation between in vitro growth potential and
treatment outcome, and observed that hyperdiploid cases were among
those least able to grow in stromal cultures.18 In the
present study, we investigated in greater detail the relation between
ploidy classification and the ability of leukemic cells to survive in
vitro, using bone marrow-derived stromal cells as a source of survival
factors. The findings suggest that hyperdiploid 51-to-65 ALL is unique
among commonly recognized immunophenotypic, karyotypic, and genotypic
subtypes of this disease, by virtue of its exquisitely stringent
survival requirements and its marked propensity to undergo apoptosis.
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MATERIALS AND METHODS |
Characterization of leukemic cells.
Bone marrow samples were obtained at diagnosis from 129 patients with
ALL, aged 3 months to 18 years (median, 5 years). These studies were
approved by the institutional review board, with informed consent
obtained from patients, their parents, or their guardians.
Immunophenotyping was performed by standard methods, as previously
described.19 One hundred fourteen cases were classified as
B-lineage ALL because greater than 80% of the blast cells were CD19+ and CD22+. In the remaining 15 cases, the
blast cells expressed CD7 and cytoplasmic or surface CD3, diagnostic of
T-ALL. Karyotyping was done by standard banding techniques. DNA content
was measured by flow cytometry after propidium iodide
staining.11 Mononuclear cells were collected after
centrifugation on a Lymphoprep density gradient (Nycomed, Oslo, Norway)
and washed three times in phosphate-buffered saline (PBS).
Preparation of stromal layers and cell culture experiments.
To obtain bone marrow stromal cells, we collected mononuclear cells
from healthy bone marrow donors. In previous studies, the source of
stromal layers was not a factor in the survival and proliferation of
leukemic lymphoblasts in vitro.20 The cells were cultured
in RPMI 1640 (Whittaker, Walkersville, MD) with 10% fetal calf serum
(FCS; Whittaker), 10-6 mol/L hydrocortisone (Sigma, St
Louis, MO), 2 mmol/L L-glutamine (Whittaker), and antibiotics in an
incubator set at 33°C and 5% CO2 in 96-well
flat-bottomed microtiter plates (Costar, Cambridge, MA), as
described.18,21-26 Immediately before initiating the cell culture experiments, we removed the media of bone marrow stromal cultures and washed the adherent cells seven times with AIM-V medium
(Gibco, Grand Island, NY). Leukemic cells were resuspended in AIM-V
medium at a final concentration of 1.5 × 106/mL. Two
hundred microliters of the suspension were then placed in
a 96-well tissue culture plate or seeded onto bone marrow stromal cells. In 22 cases (including all T-ALL samples) fresh leukemic cells
were placed in culture within 5 hours of collection; the remaining 107 samples were cryopreserved and used immediately after thawing. In all
samples, cell viability exceeded 80% by Trypan blue dye exclusion. In
preliminary experiments with nine B-lineage ALL cases, in which both
fresh and cryopreserved samples were cultured on stromal layers,
cryopreservation did not significantly affect the survival and growth
of lymphoblasts in culture (not shown). Recombinant human interleukin
(IL)-1, IL-1 , IL-2, IL-3, IL-6, IL-7, IL-11, and stem cell factor
(SCF) were purchased from R & D Systems (Minneapolis, MN); IL-4 and
tumor necrosis factor- (TNF-) were from Genzyme (Cambridge, MA);
and interferon- (IFN-) was from Schering (Kenilworth, NJ).
Cytokines were used at the final concentrations indicated in the
Results, in excess of the active concentrations measured in the
manufacturer's tests. Neutralizing antibodies to IL-4, TNF-, and
IFN- (from R & D) were used at the concentrations recommended by the
manufacturer. All cell cultures were performed at 37°C under 5%
CO2.
At the termination of cultures, cells were obtained by vigorous
pipetting. B-lineage ALL samples were incubated with CD19 monoclonal
antibody conjugated to fluorescein isothiocyanate (FITC); T-ALL samples
were incubated with FITC-conjugated CD7. All antibodies were from
Becton Dickinson (San Jose, CA). Samples were analyzed with a FACScan
flow cytometer with Lysis II or CellQuest softwares (Becton Dickinson),
as previously described.18,21-26 After 7 days of culture
the percentage of cell recovery was calculated as follows: (No. of
CD19+ or CD7+ Lymphoblasts After 7 Days of
Culture) × 100 / (No. of CD19+ or
CD7+ Lymphoblasts After 1 Hour of Culture). All results are
reported as the mean of at least duplicate experiments. Leukemic cells were counted without knowledge of the patient's clinicobiologic features or treatment response.
Determination of apoptosis.
To detect phosphatidylserine residues exposed on the cell surface (a
marker of apoptosis),27 we labeled cells with
FITC-conjugated Annexin-V (Trevigen, Gaithersburg, MD), following the
manufacturer's instructions. In these experiments, cell membrane
permeabilization was revealed by labeling cells with 5 µg/mL of
propidium iodide (Trevigen) for 15 minutes at 20°C. To detect DNA
fragmentation, cells were fixed in 1% wt/vol paraformaldehyde,
permeabilized with 70% vol/vol ice-cold ethanol, and incubated with
bromodeoxyuridin (BrdU)-labeled dUTP plus terminal deoxynucleotidyl
transferase.28 BrdU-dUTP incorporated into fragmented DNA
was visualized with a FITC-labeled anti-BrdU antibody. At the same
time, DNA content was measured after treating the permeabilized cells
with RNAse and propidium iodide (5 µg/mL). All reagents were
purchased from Phoenix (San Diego, CA), and the staining procedures
followed the manufacturer's instructions.
Electron microscopy.
For scanning electron microscopy, stromal layers were prepared on
membrane cell culture inserts (Falcon Cyclopore; Becton Dickinson) as
previously described17 and seeded with leukemic lymphoblasts from four cases of ALL. After 1 to 3 days of culture, the
inserts were washed with PBS, fixed in 2.5% glutaraldehyde in PBS,
rinsed with PBS containing 7% sucrose, postfixed in 2% osmium
tetroxide, rinsed in water, passed through ethanol gradients (30%,
70%, 85%, 95%, and 100%) for 5 minutes each, and placed in fresh
100% ethanol for an additional 5 minutes. The preparations were dried
by the critical point method in an Autosamdri-840 (Tousimis Research,
Rockville, MD), mounted on a specimen holder, and coated with gold in a
sputter-coater (Denton Vacuum, Cherry Hill, NJ). Cells were examined in
the scanning mode of a 1200 EXII TEMSCAN electron microscope (JEOL,
Tokyo, Japan).
Statistical analysis.
Distributions of commonly measured presenting features according to the
percentage of viable leukemic cells (above or below the median value)
recovered after 7 days of culture were compared by Fisher's exact
test.
| |
RESULTS |
Relation of immunophenotypic and genetic features to cell survival on
stromal feeder layers.
Among the 129 diagnostic ALL samples studied, the median percentage of
cell recovery after 7 days of culture on bone marrow-derived stromal
layers was 91% (range, <1% to 550%), compared with 1% (range,
<1% to 151%) among 125 parallel cultures without stroma. Table 1 compares cell recovery on stroma
according to the presenting immunophenotypic features of the cases. The
median percentage of recovered cells did not differ significantly
between B-lineage ALL (n = 114) and T-lineage ALL (n = 15), nor was
there any significant difference in cell recovery between the groups
with an early pre-B (n = 73) or pre-B (cytoplasmic µ+; n = 41) immunophenotype.
We also determined the chromosomal and molecular genetic changes in
these cases and attempted to relate the findings to survival on stromal
layers. Although not statistically significant, there was a clear trend
toward higher cell recoveries among cases with unfavorable genetic
features. Thus, of 25 patients with the Philadelphia (Ph) chromosome (n = 6), MLL gene rearrangements (n = 14), or the t(1;19) (n = 5),
17 had a cell recovery above median value (Table 1). Importantly, in
the 31 cases with a TEL gene rearrangement, an abnormality
generally associated with favorable responses to treatment,29 cell recovery values showed essentially the
same distribution as those in the remaining cases studied (Table 1).
Relation of ploidy to cell survival on stromal layers.
Complete information about ploidy was available for all the 129 cases
studied. In addition to the 25 hyperdiploid cases with modal chromosome
numbers of 51 to 65, 8 cases were classified as hypodiploid, 26 as
having 46 chromosomes or constitutive trisomy 21 with no detectable
abnormalities, and 40 as pseudodiploid. Among the 30 remaining cases,
27 were hyperdiploid with modal numbers of 47 to 50, and 3 were
near-tetraploid. Figure 1 shows the
percentage of cell recovery according to ploidy. The most striking
finding was the significantly decreased survival of the hyperdiploid
51-to-65 cases compared with other ploidy groups. That is, cell
recoveries above the median value of 91% were noted in only two of 25 cases, in contrast to 63 of the remaining 104 cases (P < .001; Table 1). Remarkably, all but four of the recovery values for the
hyperdiploid 51-to-65 group were within the first quartile (<49%),
compared with only 12 of the 104 cases with different ploidy
classification.
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| Fig 1.
Percentage of cell recovery after 7 days of culture on
stroma according to ploidy. Numbers of leukemic lymphoblasts before and
after culture were counted by flow cytometry as described in Materials
and Methods. Horizontal bars indicate the median cell recovery in each
ploidy group. The broken horizontal line indicates the overall median
cell recovery.
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The survival disadvantage of hyperdiploid cells was confirmed by flow
cytometric analysis of cellular DNA content. Of the 128 cases in which
this parameter was studied, 21 had a DNA index (DI) of 1.16 to 1.6, corresponding to a modal chromosome number of 51 to 65. Only 2 of these
cases had cell recoveries above 91%, in contrast to 62 of the 107 cases with a DI less than 1.16 or greater than 1.6 (P < .001).
Within the hyperdiploid group of ALL cases, a duplication of
chromosomes 4 and 10, a modal chromosome number greater than 55, and
the absence of structural abnormalities have been associated with
especially good treatment outcome.30-32 We therefore tested whether these features influenced recovery after culture on stromal feeder layers. As shown in Fig 2, cell
recoveries did not differ significantly among these subsets of
patients, although each of the 16 cases with a +4 and +10 karyotype had
cell recoveries within the first quartile.
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| Fig 2.
Percentage of cell recovery after 7 days of culture on
stroma among different subgroups of hyperdiploid 51-to-65 ALL,
including cases without and with duplications of chromosomes 4 and 10,  or >55 chromosomes, and without and with
structural chromosomal abnormalities. Numbers of leukemic lymphoblasts
before and after culture were counted by flow cytometry as described in
Materials and Methods. Horizontal bars indicate the median cell
recovery in each group.
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Mechanism of cell death of hyperdiploid blasts and effects of
exogenous cytokines.
Hyperdiploid cells died rapidly in stromal cultures, as shown by the
prominent terminal morphologic changes (eg, cell shrinkage, nuclear
fragmentation) observed in situ with an inverted microscope within 48 to 72 hours of culture. In one experiment with a hyperdiploid sample
(55 chromosomes), we counted viable cells after each day of culture for
7 days. After a single day of culture, only 27% of the originally
seeded cells could be recovered. This value decreased progressively to
9% after 7 days of culture (Fig 3). A
mechanism of hyperdiploid cell death was suggested by shifts in
light-scattering properties similar to those of cells undergoing apoptosis after treatment with cytotoxic drugs.28 These
included a reduction in forward scatter (FSC), indicating a reduction
in cell size, and an increase in side scatter (SSC) indicating an increase in cell granularity (Fig 4).
Occurrence of apoptosis was confirmed by binding of Annexin-V, loss of
DNA, and prominent appearance of DNA fragmentation (Fig 4).
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| Fig 3.
Kinetics of cell recovery during culture with and without
stroma in one case of hyperdiploid ALL (55 chromosomes). Numbers of
leukemic lymphoblasts before and after culture were counted by flow
cytometry as described in Materials and Methods. Each point indicate
the means of two measurements.
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| Fig 4.
Hyperdiploid lymphoblasts undergo apoptosis despite the
presence of stromal layers. Flow cytometric dot plots represent
assessment of apoptosis before (top panels) and after 2 days of culture
(bottom panels) in one case of hyperdiploid ALL (55 chromosomes). Left
panels illustrate FSC (a measurement of cell size) versus SSC (a
measurement of cell granularity). Center panels illustrate binding of
Annexin V FITC (an indicator of apoptosis) and staining with propidium
iodide (PI; a sign of membrane permeability). Right panels illustrate
the cells' DNA content, measured by staining with PI after cell
membrane permeabilization, and labeling with dUTP-BrdU, which is
incorporated into cells with fragmented DNA. In all panels, events
outside the gates correspond to cells at various stages of apopotic
death.
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We previously showed an intimate relationship between immature B cells
and stromal elements in which close contact of both normal and leukemic
cells with stromal layers was a prerequisite for continued
survival.17,20 Thus, it seemed reasonable to suspect that
loss of adhesive interactions with stromal layers could account for the
poor viability of hyperdiploid cells in such cultures. This prediction
was tested by scanning electron microscopy studies of two hyperdiploid
cases with low cell recoveries (<50%) after 7-day cultures. There
was no indication that the hyperdiploid cells differed from
nonhyperdiploid cells (from 2 cases studied in parallel) in their
adherence to stromal layers (Fig 5). Thus,
this factor does not appear to be related to the early demise of
hyperdiploid lymphoblasts.
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| Fig 5.
Hyperdiploid lymphoblasts adhere to stroma. Scanning
electron microscopy of hyperdiploid lymphoblasts after 3 days of
culture on bone marrow-derived stroma. Most cells are dying despite
tight adhesion to stromal elements.
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Alternatively, stromal cultures may lack some of the growth factors
essential for the support of hyperdiploid leukemic cells. Therefore, in
experiments representing eight cases of hyperdiploid ALL with 7-day
cell recoveries of less than 1% to 46%, we added a variety of
cytokines to the cultures: IL-1 at 50 to 200 pg/mL (n = 4), IL-1
at 50 to 400 pg/mL (n = 4), IL-2 at 200 ng/mL (n = 4), IL-3 at 2 to 10 ng/mL (n = 5), IL-4 at 100 U/mL (n = 5), IL-6 at 2 to 7.5 ng/mL (n = 6), IL-7 at 25 ng/mL (n = 8), IL-11 at 10 to 50 ng/mL (n = 1), SCF at
10 to 50 ng/mL (n = 6), TNF- at 50 U/mL (n = 5), IFN- at 2,000 U/mL (n = 3), or fetal calf serum 10% (n = 5). None of these exogenous
factors suppressed apoptosis, whether added singly or in combination
(data not shown). In fact, IL-4, IFN-, and TNF- proved toxic to
the cells, as in previous studies.21,33,34
We examined the possibility that low concentrations of toxic cytokines
may have been present in stromal cultures. Thus, neutralizing antibodies to IL-4, IFN-, and TNF- were added either alone or in
combination to cultures supporting one case of hyperdiploid ALL
(chromosome number, 57; cell recovery after stroma-supported culture,
7%). None of these additions significantly affected 7-day cell
recoveries.
Finally, we considered whether allopurinol, which is often administered
to leukemia patients before collection of diagnostic bone marrow, could
have selectively impaired the survival of hyperdiploid cells in
culture. Among the 25 hyperdiploid patients, however, 14 (11 of which
with cell recoveries of <50%) either did not receive allopurinol or
received it after the bone marrow sampling, thus excluding this
possibility.
| |
DISCUSSION |
In this study, we show that hyperdiploid ALL with a modal chromosome
number of 51 to 65 constitutes a biologically distinct subtype of ALL,
characterized by a high rate of spontaneous cell death in vitro. Unlike
results obtained with other ploidy groups, apoptosis in these cases was
not suppressed by culture on bone marrow-derived stromal layers.
Moreover, supplementation of the cultures with exogenous cytokines
known to stimulate hematopoietic cells lacked any discernible effect on
cell recovery, indicating that hyperdiploid lymphoblasts have
exceedingly stringent survival requirements. Finally, it should be
emphasized that extensive apoptosis in culture was not a
feature of hyperdiploidy of less than 51 chromosomes or of other cases
generally acknowledged to have a favorable prognosis, such as those
with TEL gene rearrangement.
The molecular basis underlying the high propensity of hyperdiploid
51-to-65 to undergo apoptosis is still unknown. Tsuchiya et al
postulated that the extra chromosomes of hyperdiploid ALL may control
its tumorigenicity, possibly through increased gene dosage.35 Although plausible, this explanation does not
account for the unfavorable prognostic features of near-triploid and
near-tetraploid ALL, which may represent clinically distinct entities
within the overall category of hyperdiploid ALL.36
Alternatively, the pathogenesis of hyperdiploid ALL could involve
molecular defects leading to both DNA content abnormalities and a
propensity to undergo apoptosis. This idea is suggested by the
observation that ectopic expression of the protein kinase PITSLRE1
induces telophase delay, abnormal chromosome segregation, and an
increase in the rate of apoptosis in mammalian cells.37
Although preliminary studies have failed to detect PITSLRE1
overexpression in hyperdiploid lymphoblasts (J. Lahti, V. Kidd, and D. Campana, unpublished results, March 1996), the possible
involvement of similar mechanisms in the pathogenesis of this leukemia
warrants further careful investigation.
The culture system used in this study simulates the in vivo growth
conditions of leukemic lymphoblasts in that both survival and growth
factors are derived from cells constituting the bone marrow
microenvironment. However, failure of the system to support hyperdiploid cells (which thrive in vivo) clearly shows that the culture conditions are not optimal for all lymphoid progenitors. Nonetheless, our culture system appears to provide a reliable estimate
of leukemic cell growth potential. Indeed, in a previous study with
samples from children enrolled in a single program of chemotherapy,
cell recovery after 7 days of culture on stromal layers emerged as an
independent predictor of treatment outcome.18 Moreover,
among the 25 patients with hyperdiploid ALL included in the present
study, only two have relapsed. Cell recoveries in these cases were
133% and 74%, the first and the third highest values measured in this
group.
In summary, our findings help explain the paradox of higher percentage
of proliferating cells and lower white blood cell counts generally
observed at diagnosis in ALL with 51 to 65 chromosomes.1,2,8,11,15 Hyperdiploid lymphoblasts appear to
be ideal targets for innovative, potentially less toxic therapies
designed to decrease microenvironmental support to leukemic
cells.38,39
| |
ACKNOWLEDGMENT |
We thank Michael Hancock for statistical analysis and John Gilbert for
editorial suggestions.
| |
FOOTNOTES |
Submitted June 11, 1998;
accepted September 4, 1998.
Supported by grants RO1-CA58297, P30-CA21765 (CORE), and CA-20180 from
the National Cancer Institute; and by the American Lebanese Syrian
Associated Charities (ALSAC).
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 Dario Campana, MD, PhD, Department of
Hematology-Oncology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis TN 38105; e-mail:
dario.campana{at}stjude.org.
| |
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Abstract
Introduction