(CANCER RESEARCH 50. 10-14. January I. 1990]
Establishment of Continuous Cultures of T-Cell Acute Lymphoblastic Leukemia
Cells at Diagnosis1
Ruth Gjerset, Alice Yu, and Martin Haas2
Cancer Center [R. 6'.. A. Y., M. //./, Department of Pediatrics [A. Y.J, and Department of Biology [M. H.J, University of California, San Diego, LaJolla, California 92093
ABSTRACT
the samples gave rise to lines and only a small fraction of the
cells grew out (8-10). Recently, Smith et al. (12) have reported
conditions which have enabled them to grow out a cytogenetically abnormal, growth factor-independent population of leu
kemic cells from most patients with T-ALL at diagnosis or in
relapse. The conditions used involved hypoxia and initial sup
plement of insulin-like growth factor I.
Normal T-lymphocytes can be grown for extended periods of
time in the presence of IL-2 (13-15). However, the length of
time is variable (from about 2 weeks to >13 months), and it is
not clear whether all samples are able to give rise to long-term
cultures. One recent study reported that long-surviving T-cell
cultures are rare and that most cultures of human T-lympho
cytes possess a limited in vitro life span (16). It is probable that
long-term cultures of normal T-lymphocytes remain polyclonal
(17).
Here we report new conditions which allow the long-term
culturing of virtually every T-ALL sample taken at diagnosis
with outgrowth of nearly 50% of the cells plated as determined
by trypan blue staining of cells before plating and at daily
intervals after activation. These cultures should enable us to
study the growth properties of T-ALL cells at diagnosis and
possibly identify early events responsible for the development
of the disease.
We have devised methods facilitating the establishment of continuous
cultures of T-cell blasts from patients with acute lymphoblastic leukemia
of T-cell type at diagnosis. The cultured cells closely resemble those of
the patients at the time of diagnosis with respect to surface markers,
karyotype, and T-cell receptor gene rearrangements. Cultured T-cell
acute lymphoblastic leukemia (diagnosis) cells (a) are lymphocytes with
a convoluted nucleus; (b) have doubling times of 24-48 h; (c) are
dependent for growth on interleukin 2; (d) are reverse transcriptase
negative; (<•)
do not form colonies in methyl cellulose; and (/) are clonal
with respect to T-cell receptor 0 chain rearrangements. Three T-cell
acute lymphoblastic leukemia cultures had a normal diploid karyotype,
and one had a 6q- deletion which was also present at the time of
diagnosis.
INTRODUCTION
Childhood T-cell acute lymphoblastic leukemias comprise
some 15% of the heterogeneous group of the acute lympho
blastic leukemias (1). At diagnosis the patients often have a
peripheral WBC of greater than 108/ml, extensive bone marrow
involvement (80-90% blasts), and a rapidly dividing population
of relatively immature, monoclonal or oligoclonal T-lymphoblasts. The patients have a poor prognosis and a short survival
unless treated without delay. Even when treated, relapse is a
common occurrence in T-ALL.1
Little is known about the mechanism underlying the devel
opment of T-ALL. Progress in the cytogenetic characterization
of the disease has been limited, in contrast to the case of the Bcell leukemias and lymphomas where many of the specific
chromosomal changes associated with lymphoid malignancies
have been identified. Recurrent chromosomal abnormalities
involving the a or ßchains of the T-cell receptor or deletions
in the short arm of chromosome 6 are seen in only about onethird of T-ALL cases. However, a surprisingly large percentage
of cases (30-50% depending on stage, Ref. 2; or 3 of 4 cases,
Ref. 3) show apparently normal karyotype. Acquisition of au
tocrine growth properties or activation of specific oncogenes
may play a role in the early phases of the disease (3-7).
However, difficulties in maintaining cultures of leukemic cells
from patients at diagnosis have impeded the study of the
induction of T-ALL.
A number of leukemia or lymphomacell lines of T-cell origin
have been established from patients in relapse (3,8-13). Success
has, until recently, been limited to patient samples provided at
the time of relapse; even in these cases only a small fraction of
MATERIALS
AND METHODS
Source of Leukemic Cells. The cells in this report were derived from
patients (ages 6-16 years) with T-cell acute lymphoblastic leukemia at
diagnosis. The protocol was approved by the Committee on Investiga
tions Involving Human Subjects at the University of California, San
Diego, and informed consent was obtained from the patients or their
parents. Clinical and pathological data on each patient are presented
in Table 1. Bone marrow or peripheral blood lymphocytes were col
lected for the purpose of routine clinical diagnosis, and cells that
remained following the diagnostic procedures were cultured on the
same day they were taken from the patients. Usually this was within 8
h of drawing.
Surface Markers on Cells from Patients and on Cultured Cells. Cells
(IO6) were washed twice in PBS plus 0.2% sodium azide and stained
directly as follows. Monoclonal antibody (50 p\) (anti-T and antiCALLA (Coulter), T101 (Hybritech), anti-Leu M5 (Becton Dickinson
Co.) was added to each tube followed by a 30-min incubation on ice.
Cells were washed twice with PBS plus 0.2% azide and 50 n\ of diluted
(1:20 in PBS) fluorescein isothiocyanate goat anti-mouse IgG (Anti
bodies, Inc.) were added. Following an additional 30 min incubation
on ice, the cells were washed twice in PBS plus 0.2% azide, and fixed
in 75 M!of 1% formalin. The percentage of fluorescent cells was counted
by using a fluorescence microscope and a fluorescence-activated cell
sorter.
Determination of Terminal Deoxynucleotide Transferase. Terminal
deoxynucleotide transferase was determined by standard techniques
(18).
Conditioned Medium from Activated Normal Peripheral Blood Lym
phocytes. Peripheral blood lymphocytes (HPBLs) from normal donors
were separated on Ficoll-Hypaque, washed, activated, and grown as
described below for leukemic cells. For 2 to 3 weeks following activation
these cultures proliferated and secreted factors. HPBL CM was col-
Received 6/26/89; revised 9/17/89; accepted 9/27/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by USPHS Grants CA3415I. CA42432,
and CA40186 awarded by the National Cancer Institute, Department of Health
and Human Services, and by Grant CH46S awarded by the American Cancer
Society.
1To whom requests for reprints should be addressed.
' The abbreviations used are: T-ALL, T-cell acute lymphoblastic leukemia; CI,
calcium ionophore A23187: PMA, phorbol 12-myristate 13-acetate; HPBL CM,
human peripheral blood lymphocyte-conditioned medium; kbp, kilobase pair;
PBS, phosphate-buffered saline; IL-2. interleukin 2.
10
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T-ALL CONTINUOUS
PatientA.
CELL CULTURES
Table 1 Clinical dala
gave rise to continuous cultures which could be maintained for
mpho-blasts
of 1)
essentially unlimited time (more than 6 months).
bonemarrow97939480NDCulturedesignationAndiaTimpaniGemVepaRym
in
Sixty to 90% of T-ALL cells were estimated by visual inspec
PBL*BMPBLrBMPBL'%
+
D.T.
P.G.
M.V.
P.R.
M.*Age12816IS4SexMMMFMSampleculturedBM
°BM, bone marrow; PBL, peripheral blood lymphocytes; ND, not determined.
4 342.000 WBC/VI in peripheral blood, 88% blasts.
' 204,000 WBC/iil in peripheral blood, 83% blasts.
'' Relapse patient.
' 400,000 WBCVul in peripheral blood, 99% blasts.
lected from these cultures every 48 h, filter sterilized, and stored at
4°C.
Activation of Leukemic Cells. Mononuclear cells from bone marrow
or peripheral blood of T-ALL patients at diagnosis were separated on
Ficoll-Hypaque (Pharmacia). Cells were washed in RPMI medium with
10% fetal bovine serum (HyClone), resuspended in the same medium,
and counted. Cells were then resuspended at 4-6 x IO6 cells/ml in
Costar 24-well cluster dishes in medium D (Dulbecco's modified Eagle's
medium, 10% fetal bovine serum (HyClone), 10% human AB serum
(Gibco), 30 MS/"1'human transferrin (Sigma). Cells were activated once
with 2 ¡IM
calcium ionophore (A23187 free acid; Sigma) plus 17 nM
PMA (Sigma). Following an 8- to 10-h treatment with CI plus PMA,
medium was changed by removing about three-fourths of it and replac
ing it with medium D containing 200 units/ml human recombinant IL2 (Genzyme) and 20% HPBL CM (prepared as described above).
Medium was changed every 2-3 days and the cell concentration was
kept high (5-6 x 106/ml).
Karyotype Analysis of Patient Samples and Cultured Cells. Cells were
incubated for 2 h at 37°Cin medium containing 0.1 n%/m\ colcemid.
They were then centrifuged at 1000 rpm in a clinical centrifuge for 6
min. After aspiration of the medium the pelleted cells were gently
resuspended in 5 ml of 0.075 M KC1 for 30 min, during which time the
cell suspension was mixed continually. Five ml of freshly prepared
fixative (methyl alcohohglacial acetic acid, 2:1) were added to the cell
suspension, which was allowed to stand for 5 min at room temperature.
After two changes of fixative, the cells were placed on glass slides for
analysis. G-banding of the chromosomes was accomplished by brief
exposure to a solution of 0.5% trypsin and subsequent staining with
Giemsa.
Analysis of T-Cell /* Chain Rearrangements. High-molecular-weight
DNA was isolated from 1-5 x IO7cells, digested with EcoRl restriction
endonuclease (Promega) at 6 units of enzyme/^g DNA, and subjected
to electrophoresis on an 0.8% agarose gel (Seakem agarose ME, FMC
Corporation), 10 ¿tgDNA/lane. The running buffer was 40 m\i Trisacetate, pH 7.8, 1 min EDTA. The DNA was blotted onto nitrocellulose
(19), and hybridized to a 32P-labeled T-cell receptor ß
chain probe (Q81
plus Cß2;
Oncor) using IO6cpm of probe/ml in 40% formamide, 7 mivi
Tris, pH 7.6, 4x standard saline citrate, 0.8x Denhardt's, 50 jig/ml
herring sperm DNA, 16 h at 45°C.Blots were washed in 0.2x standard
saline citrate (Ix SSC = 0.15 M NaCI, 0.015 M Na, citrate, pH 7) at
55°Cfor 30 min. The hybridized blots were subjected to autoradiography (Kodak X-Omat film, intensifying screens, 48 h).
Reverse Transcriptase Assay. Reverse transcriptase assays were per
formed as described (20).
Cell Cloning in Methyl Cellulose. Cell cloning in methyl cellulose
culture was performed as described (21), using IO4 cells/6-cm tissue
culture dish.
RESULTS
Continuous Cultures of T-ALL Cells. We have found condi
tions facilitating the long-term culture of T-cell blasts from the
peripheral blood and from the bone marrow of T-ALL patients
at diagnosis. Following a single exposure of Ficoll-Hypaque
separated lymphoblasts to CI and PMA, most T-ALL samples
(four of five) grown in medium D with IL-2 and HPBL CM
tion of the cultures to respond to treatment with CI plus PMA
(within 1 to 2 h) by forming large aggregates of blasts. Following
8-10 h of activation some 50% of the cells were viable as
estimated by trypan blue staining. The activated cells responded
to IL-2 plus HPBL CM by dividing continuously (doubling
time, —¿48
h) and their viability remained high (80-90%). In
long-term culture, the cells grew as partially aggregated suspen
sion cells. When human AB serum was omitted from the
medium, the cells appeared to differentiate and form adherent
cell layers with fibroblastoid characteristics, but lacking T-cell
markers (data not shown). T-ALL (diagnosis) culture supernatants were tested for the presence of reverse transcriptase activ
ity and all were found to be negative, indicating lack of retrovirus production. In contrast to established lines of T-ALL
(relapse) cells (e.g., Molt-4, CEM, HSB-2), cultured T-ALL
(diagnosis) cells did not produce colonies in methyl cellulose or
soft agar cultures, suggesting their "nonprogressed" (i.e., nontransplantable) state. Like the cultured T-ALL (diagnosis) cells,
T-ALL cells taken directly from patients at diagnosis do not
grow in methyl cellulose or soft agar cultures nor under the
skin of nude mice (22), indicating that the cells have not yet
undergone "progression" to full transplantability. As we have
shown previously (23) there is a close, though not complete
correlation between the cloning of blood cells in methyl cellu
lose cultures and their transplantation in vivo.
T-Cell Surface Markers. Clinical data on four T-ALL patients
at diagnosis and one patient in relapse from whom successful
continuous cultures have been established are shown in Table
1. Immunophenotypic analysis to identify T-cell surface mark
ers was performed on mononuclear cells separated from periph
eral blood and bone marrow at diagnosis and again following
propagation in culture (Table 2). Also shown in Table 2 are
data on a culture grown from normal peripheral blood mononuclear cells that had been treated identically and propagated
for 13 days. A panel of immunophenotypic markers was re
tained on T-ALL cells through long-term culture in the presence
of IL-2 plus HPBL CM, including Til (CD2, erythrocyte
receptor), T3 (CD3, mitogenic pan-T), T4 (CD4, T-helper),
T101 (CDS, pan-T-lymphocyte), Leu9-3Al (CD7, pan-T-lymphocyte), T8 (CDS, T-cytotoxic), T9 (transferrin receptor), and
TÕO(thymocyte). All cultured T-ALL cells were 100% positive
for terminal nucleotidyl transferase (data not shown). Two
markers that were present on a percentage of the cells of some
of the T-ALL patients at the time of diagnosis (CALLA or
CD 10, and OKT6 or CD1) was lost during long-term culturing
of the cells. This suggests that during culturing, the T-ALL
(diagnosis) cells undergo a degree of differentiation, since the
expression of CD1 is found on cortical thymocytes but not on
medullary thymocytes or normal peripheral blood T-lymphocytes (24), and expression of CD3 [T-cell receptor (25)] is found
on more mature thymocytes. Alternatively, it could be argued
that a subpopulation of mature, polyclonal cells is overgrowing
the population in culture'. This possibility seems unlikely since
analyses of karyotypes and T-cell receptor gene rearrangements
support the argument that a clonal tumor cell population is
growing out (see below). The induction of la seen in all cases is
indicative of activation (26) and is to be expected following
treatment with PMA and calcium ionophore. The absolute
requirement for IL-2 for the proliferation of T-ALL (diagnosis)
cells explains the low incidence of CD25 (Tac, IL-2 receptor)
on the surface of the cultured cells. At the concentrations of
11
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T-ALL CONTINUOUS
CELL CULTURES
Table 2 Comparison ofphenotypes of leukemia cells or normal peripheral blood lymphocytes (PBL) before and after propagation in continuous culture
Table shows percentage of cells positive for a specific cell surface marker. Designations A. D. and T. P. refer to patients' cells at diagnosis. Andia and Timpani
refer, respectively, to cultures established from these samples. Timpani culture established from bone marrow (BM). At time of analysis, Andia cultures had been
propagated 6 weeks. Timpani culture had been propagated 9 weeks, and cultured PBLs had been propagated 13 days.
Surface
antigenCD
antibodyOKT6TilT3T4T101Leu9-3AlT8CALLA-J5TACT9TÕO12LeuMSPatient
A.D.83693279497908633833BM393831479877151635577146AndiaPBL09886258389681162866833Patient
T.P.13948520908821135824Timpani01001007398721000140901768NormalFresh1867359
1CD
2CD
3CD
4CDSCD
7CDSCD10CD
25T9TÕOlaCDllcMonoclonal
IL-2 that were used, most of the receptors for IL-2 can be
expected to be blocked by the ligand and thus be inaccessible
to the Tac antibody. Only growth with a limiting concentration
of IL-2 might enable one to demonstrate the presence of the
IL-2 receptor on the surface of a majority of the cells.
In the case of one patient (A. D.), where continuous cultures
were established both from the bone marrow and from periph
eral blood lymphocytes, we see similarity with respect to cell
surface markers.
Cytogenetic Findings. Three of four T-ALL cultures which
we examined possessed apparently normal karyotypes, as did
the original patient samples from which they were derived. This
result is not surprising since cytogenetics has often failed to
reveal karyotypic abnormalities in T-ALL (2, 3); indeed, cyto
genetics cannot, as a rule, detect mutations or deletions in
single genes. One culture (Timpani) showed a 6q- deletion,
which was also present in the original patient sample (not
shown). This deletion has been correlated with high c-myb
expression in hematopoietic malignancies (27). Since expres
sion of c-myb is directly correlated with proliferation and in
versely correlated with differentiation, it has been suggested
that elevated expression of c-myb could contribute to the pro
liferation of leukemic cells.
The maintenance of the 6q—abnormality during culturing
demonstrates that our culture conditions permit outgrowth of
leukemic cells, and argues that phenotypic changes observed
upon culturing (see above) represent clonal evolution rather
than long-term growth of normal activated T-cells. It is highly
unlikely that a chromosomal deletion such as 6q—would be
present in the unaffected cells of the patient.
Monoclonal Rearrangements of T-Cell Receptor ft Chain (\'ft)
I
m o
CL J3 2
Z
E ^ =5
>s O e
Q_ CC OC <
<
o.
CD
>
23.7-
9.56.7-
i
4.3-
2.252.0Fig. 1. Southern blot analysis of DNA digested with EcoRl restriction enzyme
and hybridized to a T-cell receptor. i chain probe. Molecular-weight-marker bands
are indicated on the ordinate in kbp. Samples are (left to right): normal peripheral
blood lymphocytes (NPBL), placenta, patient sample (RM), 5-month continuous
culture (Arm), patient sample (AD), 6-week continuous culture (Andia), and 7week continuous culture ( Vepa).
Locus in Cultured T-ALL (Diagnosis) Cultures. The available
phenotypic markers of leukemic cells define specific blastogenic
states and are not leukemia cell specific. Similarly, the results
of cytogenetic analysis may fail to provide consistent markers
for leukemic cells. We have therefore relied on Southern blot
analysis of T-cell receptor ßchain gene rearrangements to
demonstrate that our cultures represent the original leukemic
clone. These rearrangements occur during T-cell ontogeny and
are unique for each T-cell. They can therefore be used to identify
clonal leukemic populations and to follow the evolution ofthat
clone in culture.
Fig. 1 shows a Southern analysis of T-cell receptor ß
chain
gene rearrangements in £c0RI-digested DNA isolated from
original patient samples and from cultured T-ALL cells. As a
control we have included human placenta! DNA which yields
two bands at 12 kbp and 4.2 kbp, corresponding to the germ
line configuration of the Cßland C/32 alÃ-eles,respectively (Lane
2). In fcoRI-digested DNA from cultured normal peripheral
blood lymphocytes, the 12-kbp band has disappeared as a result
of polyclonal rearrangements of the C/31 alÃ-ele (C/32 re
arrangements are not revealed by digestion of the DNA with
EcoRl). In contrast, the EcoRI-digested DNA from two T-ALL
(diagnosis) cultures (Andia, Vepa) display clear monoclonal or
oligoclonal rearrangements of the CßlalÃ-ele,visible as distinct
bands between 7 and 12 kbp. DNA obtained from a T-ALL
(relapse) culture (Rym) also shows distinct monoclonal re
arrangements of the C/31 alÃ-ele.DNA from the original patient
sample R. M. shares a common band with the DNA from the
12
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T-ALL CONTINUOUS
CELL CULTURES
is maintained at 4-6 x 106/ml or higher.4 This suggests that
some samples of activated T-ALL (diagnosis) cells secrete an
autocrine factor or factors required for their own proliferation,
possibly by maintaining IL-2 receptor expression.
The nature of the additional factor(s) supplied in HPBL CM
and possibly produced by some T-ALL (diagnosis) cells is not
yet known. Preliminary experiments indicate that this activity
cannot be substituted by recombinant interleukins (IL-1
through IL-6), cerebrospinal fluids, or interferon, nor by a
combination of recombinant factors.
T-ALL (diagnosis) cultures are growth factor dependent (re
quiring IL-2 and an additional factor or factors) and have a
minimum of apparent chromosomal aberrations. They there
fore resemble normal peripheral blood lymphocytes in many
respects. However, they differ in that they contain a dominant
clone (or clones) from the beginning. That they do not grow
autonomously, but require activation and exogenous growth
factor, suggests that the in vivo environment must be playing
an important role in T-ALL. The ability to maintain T-ALL
(diagnosis) cells in culture should facilitate the study of these
cells and their growth requirements. This in turn should lead
to a better understanding of the early events in the disease.
The activated IL-2-dependent continuous cultures of T-ALL
(diagnosis) cells that we describe here differ from the IL-2independent lines recently established under hypoxic conditions
from T-ALL (diagnosis) samples by Smith et al. (12). It is
possible that patient samples contain clonal T-ALL cell blasts
that are heterogeneous with respect to their differentiation state,
as well as their responsiveness to activation. Thus the two
methods might select for two different subpopulations of TALL cells belonging to the same clone. Alternatively, different
culture conditions may induce different changes in the cells.
cell culture grown from it (Rym), and DNA from the original
patient sample A. D. shares a common band with the DNA
from the cell culture grown from it (Andia). This strongly
suggests that the cultured cells derive from the original tumor
cells. The loss or acquisition of bands in the cultured cells
relative to the original patient samples could be due to the
outgrowth of minor leukemic clones that are undetectable in
the original sample, or to subsequent rearrangements arising
during culturing, a phenomenon that has been previously doc
umented (28).
Densitometric scanning of the Southern blot autoradiograms
enabled us to estimate the fraction of clonal leukemic T-cells
present in the original patient samples. Since rearrangements
in the C/32 alÃ-elesare not revealed by digestion with EcoRl, the
intensity of the rearranged Cß\band relative to the Q?2 band
reflects the proportion of the population representing that
particular clonal rearrangement. The relative intensities on the
same blot of the unrearranged C/Õ1and C/32 bands in placental
DNA serve as a control. We thus estimate that the leukemic
clonal population, as defined by TCR gene rearrangement,
represents some 40-50% of the total peripheral blood lympho
cytes in the case of diagnosis patient sample A. D. and nearly
100% in the case of relapse patient sample R. M.
DISCUSSION
This report describes conditions which facilitate the estab
lishment of continuous cultures from T-ALL samples taken
from the peripheral blood or the bone marrow of patients at
the time of diagnosis. Our conditions favor establishment at
high frequency of T-ALL (diagnosis) cultures (4 of 5 samples
gave rise to continuous cultures) with up to 50% of the plated
cells growing out. The cultured cells resembled the original
patient cells with respect to (a) surface phenotype, although
some maturation occurred in culture; (b) karyotype; and (c) Tcell receptor gene rearrangements. Continuous culturing of TALL (diagnosis) cells should enable us to examine the early
events of leukemogenesis, before major secondary events have
occurred, and to compare the diagnosis samples to the relapse
samples as these occur.
The original patient samples are composed of clonal T-ALL
cells and in part of polyclonal normal T-cells. It is unlikely that
outgrowths of minor normal clones contribute to long-term
clonal T-ALL (diagnosis) cultures for the following reasons, (a)
Substantial percentages of the original patient samples are
represented by the leukemic clones and these clones are main
tained in culture; (b) normal polyclonal peripheral blood lym
phocytes that are maintained in culture for up to 90 days with
periodic restimulation remain polyclonal ( 17). These arguments
suggest that the long-term cultures described here represent
outgrowths of the original leukemic clones.
The absolute requirement of T-ALL (diagnosis) cells for
exogenous IL-2 is surprising in light of the published reports
(29, 30) that activation of pre-T-cells with CI plus PMA induces
the secretion of IL-2, in addition to the induction of functional
IL-2 receptors. Apparently the concentration of IL-2 that is
secreted by the activated T-ALL (diagnosis) cells is insufficient
to support long-term cell proliferation in culture.
While HPBL CM has been added routinely to all T-ALL
cells grown in culture to facilitate unlimited proliferation of
virtually all samples at any cell concentration, we have also
seen that some T-ALL (diagnosis) cultures do not require
HPBL CM and can be grown in medium D supplemented only
with human recombinant IL-2 as long as the cell concentration
ACKNOWLEDGMENTS
Timothy Cahill and Roland Jimenez are gratefully acknowledged for
karyotyping and technical assistance. We also thank Susan Wormsley
for phenotyping several of the cultures described and Catherine Stanish
for hybridization of Southern blots with T/J probes. Raymond Taetle,
M.D., was, as always, most helpful with ideas and advice and with
critical reading of the manuscript.
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Establishment of Continuous Cultures of T-Cell Acute
Lymphoblastic Leukemia Cells at Diagnosis
Ruth Gjerset, Alice Yu and Martin Haas
Cancer Res 1990;50:10-14.
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