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[CANCER RESEARCH 47, 1724-1730, March 15, 1987)
Expression of GD3 Ganglioside in Childhood T-Cell Lymphoblastic Malignancies1
William D. Merritt,2 James T. Casper, Stephen J. Lauer, and Gregory H. Reaman
The Departments of Biochemistry [W. D. M.] and Child Health and Development [G. H. R.J, The George Washington University School of Medicine and Health
Sciences, Washington, DC 20037; The Children's Hospital National Medical Center ¡G.H. KJ, Washington, DC 200JO; and The Midwest Children's Cancer Center,
Department of Pediatrics, The Medical College of Wisconsin and Milwaukee Children 's Hospital [J. T. C., S. J. L.¡,Milwaukee, Wisconsin 53233
ABSTRACT
Lymphoblasts from seven children with T-cell lymphoblastic malig
nancies and three children with non-T, non-B acute lymphoblastic leu
kemia (ALL) were analyzed for ganglioside content. Nonmalignant I -
cells from thymus served as controls. Both ganglioside and glycoprotein
sialic acid were increased approximately 3-3.5-fold in T-cell disease
compared to thymic tissue when expressed on a per cell basis, but not on
a per milligram protein basis. Thin-layer chromatography of the isolated
ganglioside fraction from T-cell lymphoblasts revealed two major resor-
cinol-positive bands. One ganglioside comigrated with lI'-a-/V-acetylneu-
raminosyllactosylceramide (GM3), the major ganglioside in normal
lymphoid tissue, and the other ganglioside comigrated with authentic II3-
o-A'-acetylneuraminosyl-a2—»8-jV-acetylneuraminosyllactosylceramide
(GDj) in three different solvent systems. Neuraminidase treatment of the
latter ganglioside yielded (; M <and lactosyl ceramide, hydrolysis products
of (. 1),. Scanning densitometry revealed that whereas thymus cells con
tained 97% CM] and 3% CD*, T-cell lymphoblasts contained from 22 to
86% GD3 and a corresponding decrease in GM3. The shift to increased
GDj was observed in the blasts from all seven T-cell patients, but not in
the blasts from the non-T, non-B patients studied. Only trace quantities
of <.1)i were detected from two continuous T-ALL cell lines, HSB2 and
Kl'MI 8402. The results demonstrate a consistently significant increase
in ganglioside GD3 in uncultured, patient-derived T-cell ALL lympho
blasts when compared to non-T-cell ALL and normal lymphoid tissue.
Therefore, GD3 may represent a tumor-associated antigen for the T-cell
subclass of childhood lymphoblastic malignancy.
INTRODUCTION
Transformation of cells by oncogenic viruses or by chemicals
consistently results in one or more changes in the composition
of neural glycolipids and/or sialylated glycolipids (ganglio-
sides). Altered expression of glycolipids appears to be related
to a block in glycolipid glycosylation and/or a shift in glycolipid
biosynthesis to an inappropriate pathway (for reviews see Refs.
1-4). For instance, during hepatocarcinogenesis of the rat,
synthesis of the disialoganglioside, GD3,3 is blocked, resulting
in lowered expression of complex gangliosides, particularly
trisialoganglioside (5, 6). On the other hand, GD3 is inappro
priately expressed in human melanoma cells (7, 8). In studies
of gangliosides of human leukemias, GD3 is expressed to vary
ing extents in the blasts of patients with chronic lymphocytic
leukemia (9), acute lymphoblastic leukemia (ALL), and acute
myeloblastic leukemia (10,11). We have studied the ganglioside
Received 7/30/86; revised 12/5/86; accepted 12/10/86.
The costs of publication of this article were defrayed in pan 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 study was supported in part by National Cancer Institute Grant
CA33573 and the Milwaukee Athletes Against Childhood Cancer Fund.
' To whom requests for reprints should be addressed, at the Department of
Biochemistry, The George Washington University Medical Center, 2300 Eye
Street, NW, Washington, DC 20037.
3The abbreviations used are: GD3, II3-«-A'-acetylneuraminosyl-a-2—»8-/V-ace-
tylneuraminosyllactosylceramide; GM3, II3-a-/V-acetylneuraminosyllactosyl-
ceramide; GM2, H'-a-W-acetylneuraminosylgangliotriosylceramide; CM,, H3-a-
A'-acetylneuraminosylgangliotetraosylceramide; GD,., II3-a-W-acetylneuramino-
syl-IV3-a-/V-acetylneuraminosylgangliotetraosylceramide; SPG, sialosylparaglo-
boside, 1V3-/V-acetylneuraminosylneolactotetraosylceramide; ALL, acute lympho
blastic leukemia; TLC, thin-layer chromatography; HPTLC, high-performance
thin-layer chromatography; CALLA, common acute lymphoblastic leukemia an
tigen.
composition in malignant blasts of six children with acute T-
cell lymphoblastic leukemia (T-Cell ALL), one with T-cell
lymphoblastic lymphoma, and three with non-T, non-B ALL.
We have found that GD3 was a major ganglioside in each of
the T-cell malignant lymphoblastic samples studied, whereas
this ganglioside was barely detectable or absent in the non-T,
non-B leukemias and normal thymus that we evaluated.
MATERIALS AND METHODS
Isolation and Characterization of Lymphocytes from Human Lymph
oid Tissue and Lymphoblasts from Patients with Acute Lymphoid Ma
lignancies. Fresh tonsils and adenoid tissue or lymphomas were ob
tained immediately after surgical removal; any highly inflammed tissue
was discarded. Thymus tissue was freshly obtained after surgical re
moval from children undergoing cardiac surgery. Tissues were washed
in McCoy's medium (tonsils and adenoids) or RPMI 1640 (thymus)
with penicillin (100 Mg/ml) and streptomycin (100 units/ml), and
trimmed free of hematomas and visible connective/vascular tissue.
Single cell preparations from each tissue were prepared as described
(12). The cells were examined for viability using trypan blue exclusion,
and the final cell pellet was stored at -20°Cfor glycolipid extraction.
Lymphoblasts were separated from whole blood, bone marrow, or
pleural fluid from leukemic patients by either plasmagel sedimentation
in Hanks' balanced salt solution (13), or by Ficoll-Hypaque density
gradient centrifugation, as previously described (14). Cells were washed
two times in PBS, checked for viability by trypan blue exclusion, and
the percentage of lymphoblasts was determined by Wright's staining.
Cells were further characterized with respect to various T-cell and
leukemia-associated (CALLA) cell surface antigens using sheep eryth-
rocyte rosetting assays (14, IS) or immunofluorescent techniques with
heterologous or monoclonal antibodies as previously described (12, 15-
17). Lymphoblasts in all cases were positive for terminal deoxytidyl
transferase (18) and negative for surface immunoglobulin, myeloper-
oxidase, chloroacetate esterase, and a-naphthylbutyrate esterase.
Culture of Human IX ell Leukemia Lines. The cell lines RPMI 8402
(T,o-i) and CCRF-HSB2 HV.) (19, 20) were obtained from the labo
ratory of Dr. J. Minowada, Roswell Park Memorial Institute, Buffalo,
NY. Karyotyping of the cultured cell lines was performed in the
cytogenetics laboratory of Milwaukee Children's Hospital, and the
presence of Epstein-Barr virus was analyzed by immunofluorescence
staining for us by Dr. Warner Henle, Joseph Stokes Jr. Research
Institute, Philadelphia, PA. The cells were cultured in RPMI 1640
media (GIBCO, Grand Island, NY) in the presence of penicillin (100
/jg/ml), streptomycin (100 units/ml), 4 mm 4-(2-hydroxyethyl)-l-pi-
perazineethanesulfonic acid, and 10% heat-inactivated fetal bovine
serum (GIBCO). Cells were cultured in a humidified 5% CO2, 37'C
humidified incubator, and were split two times per week. For analysis
of gangliosides, cells were cultured in 350-ml volumes in closed-roller
bottles.
Isolation and Purification of Gangliosides. Cell pellets were homoge
nized in a small volume of dl I..() and homogenized with a polytron
homogenizer. Aliquots were removed for assay of protein and sialic
acid. Lipids were extracted by overnight stirring of the homogenate in
chloroform:methanol in the ratio, 1:1:0.1 (v/v/v) of chloroform:
methanol: homogenate. The precipitated material was collected by fil
tration through a sintered glass filter and then reextracted twice with
chloroform:methanol:water, 1:1:0.1 (v/v/v). The combined lipid ex
tracts were evaporated by rotary evaporation at 45*C, and gangliosides
and neutral glycolipids were separated by the procedure of Ledeen et
al. (21). Specifically, the residue was dissolved in chloroform:
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CD, IN MALIGNANT T-LYMPHOBLASTS
methanohwater, 60:30:8 (v/v/v), and placed on a DEAE-Sephadex A-
25 column (5 g of resin/2.5 x 10' cells). Nonacidic lipids, including
neutral glycolipids, and acidic lipids, including gangliosides, were eluted
separately. After removal of solvent, lipids in the latter fraction were
saponified with 0. l M methanolic NaOH to destroy phospholipids (22).
The mixture was dialyzed in the presence of EDTA against distilled
H2O for 48 h, and then lyophilized. The lyophilized material was
dissolved in chloroform:methanol, 4:1 (v/v), applied to a 2-g column
of silicic acid (Biosil; Bio-Rad Industries). Sulfatides and other contam
inants were eluted with chloroform:methanol, 80:20 (v/v), and ganglio
sides were eluted with chloroform:methanol, 1:1 (v/v) (21). The gan-
glioside fraction was dried under nitrogen, dissolved in chloro-
form:methanol, 1:1 (v/v) and stored at -20°Cfor further analysis.
Individual gangliosides were purified from T-cell leukemia extracts
by preparative TLC. Chromatography was on 250 n silica gel H 20- x
20-cm plates (Analtech, Newark, DE) with the solvent system chloro-
form:methanol:0.22% CaCl2, 60:40:9 (v/v/v). Silica gel H was used
instead of silica gel G to improve the recovery of the gangliosides away
from the silica gel. Individual gangliosides were located with iodine
vapor, scraped, and then eluted by overnight shaking in chloro-
form: met hanol: water, 1:1:0.1 (v/v/v).
Analytical Procedures. Gangliosides were separated by TLC on
precoated silica gel 60 TLC or HPTLC plates in either «-propanol:
0.22% CaCl2, 8:2 (v/v) (Solvent A), chloroform:methanol:0.22%
CaCl2, 60:40:9 (v/v/v) (Solvent B), or chloroform:methanol:28%
NH4OH:water, 60:40:3:5 (v/v/v/v) (Solvent C). Ganglioside standards
were spotted in lanes alongside experimental lanes. Standards included
GM3, isolated from human liver (23); SPG, isolated from human red
blood cells (24); human brain GD3 and Tay-Sachs brain GM2, gifts
from Dr. Robert Yu, Yale University; and GM, and GDU (Supelco,
Bellefonte, PA). Ganglioside bands were visualized by resorcinol stain
ing (25). The relative proportions of individual gangliosides were as
sessed by scanning densitometry using a Schimadzu model CS-930
TLC scanning densitomer, with drifting baseline and peak integration
capability.
Ganglioside sialic acid was measured by the method of Hammond
and Papermaster (26) at 549 urn, after digestion of gangliosides in 0.1
M H2SO4 for l h at 80'C. Glycoprotein sialic acid was measured from
the chloroform-methanol-insoluble residues collected on sintered glass
filters after lipid extraction of cells (procedure modified from Ref. 27):
Samples (10 mg) were digested for 2.5 h at 80"C to optimize sialic acid
hydrolysis, and digests were placed on a column of Biorad AG 1-X8
acetate ion exchange resin. Sialic acid was eluted with 1.5 ml l N
formate, and 0.5-ml samples were assayed in triplicate as above. Various
concentrations of ALacetylneuraminic acid (Sigma) were processed in
parallel throughout the procedure. Proteins were measured by a linear
transform modification of the Folin-Lowry procedure (28).
Neuraminidase Digestion of GI), Ganglioside. The isolated GD3 (8
nmol sialic acid/treatment) was resuspended in 100 /¿Iof 0.05 M sodium
acetate buffer, pH 5.7, with either 150, 50, or 15 munits neuraminidase
(Clostridium perfringens, Type X; Sigma), (29). Incubations were for
10 min to 24 h at 37*C, and were terminated by addition of 2 ml of
chloroform: met hanol, 2:1 (v/v). The precipitated protein was removed
by centrifugation, and the soluble components were partitioned by
addition of 0.4 ml water, vortexing, and recentrifugation (270 X g, 15
min). The lower phase was dried under N2, and the lipids were resus
pended in a small volume of chloroform:methanol, 2:1 (v/v) and sepa
rated by HPTLC, using the solvent system, chloroform:methanol:water,
50:20:3.3 (v/v/v). Bands were visualized by spraying with orcinol re
agent.
RESULTS
Lymphoblastic Malignancies. Surface marker characteriza
tion of the malignant cells from seven of the ten patients
demonstrated that the cells were of T-cell origin. This was
demonstrated by sheep erythrocyte rosetting and/or reactivity
with either an antithymic antibody or with a monoclonal anti
body against the sheep erythrocyte receptor (OKT11) (Table
1). The remaining ALL patients studied were non-T, non-B
based on absence of T-cell markers and surface membrane
immunoglobulin. These cells were examined for expression of
CALLA, and were either CALLA' (2 cases) or CALLA* (l
case). All samples demonstrated terminal deoxytidyl transferase
activity.
Quantitation of Ganglioside and Glycoprotein Sialic Acid.
Sialic acid was measured colorimetrically in the purified gan-
glioside fractions and lipid-free residue from pooled tin mie
tissue (12 samples), and the lymph olilasts from six separate T-
cell ALL patients. The results showed that both ganglioside
and glycoprotein sialic acid were increased approximately 3-
3.5-fold in leukemia cells compared to thymic cells. Measure
ments of the ganglioside fractions showed that T-cell leukemias
contained 11.80 ±4.3 nmol sialic acid/109 cells, and the thy-
mocytes contained 4.3 ±2.0 nmol/109 cells. Similarly, the
leukemia samples contained 133.0 ±12 nmol glycoprotein
sialic acid/109 cells whereas thymocytes contained 38.0 ±4
nmol/109 cells. On the other hand, T-cell ALL cells and thy
mocytes contained equal amounts of ganglioside and glycopro
tein sialic acid when expressed on a per mg protein basis (0.5
and 3.9 nmol/mg protein respectively).
Ganglioside TLC of Malignant T-Lymphoblasts. The purified
ganglioside fraction of malignant lymphoblasts in each of seven
T-cell patients with malignancies were analyzed by TLC. Fig.
1 shows the ganglioside patterns of three leukemia samples
(lanes 5-7) compared to tonsil and adenoid lymphocytes (lane
3) and thymocytes (lane 4). The patterns show that the ganglio
side profile from T-malignant blasts is more similar to that of
thymus gangliosides than that of tonsil and adenoid lymphocyte
gangliosides. However, the leukemia cells have a lowered
amount of the major doublet ganglioside found in thymus (Fig.
1, single arrow), which cochromatographed with standard GM3,
and a proportionally larger amount of a second but very minor
thymic doublet ganglioside (Fig. 1, double arrow). The latter
ganglioside was also observed in tonsil and adenoid lympho-
Table 1 Characteristics of lymphoblastic malignancies analyzed
PatientDiagnosis1
T-ALL2
T-ALL3
T-ALL4
T-lymphoblasticlymphoma5
T-ALL6
T-ALL7
T-ALL8 non-T, non-BALL9 non-T, non-BALL10 non-T, non-B ALLSexMFMMMMFFMMAge
(yrs)4.51112111635952.5Surface
phenotype"ER*(77%),T-Ag*(70%),CALLA-ER-"(57%),T-Ag*(72%),CALLA-ER*(63%),T-Ag*(70%),CALLA-ER*(85%),T-Ag*(95%),CALLA-ER-(2%),T-Ag*(40%),CALLA-ER*(96%),OKT1
1*(97%),B4-,CALLA-ER*(95%),OKT1
r(97%),B4~,CALLA-ER-.OKT1
l-,B4*,sIg-,CALLA-ER-,T-Ag-,sIg-,CALLA-ER-,T-Ag-,sIg-,CALLA*
" KK. percentage of sheep erythrocyte rosette formation at 4'C; T-Ag, percentage of expression of thymocyte antigen; CALLA, expression of common acute
lymphoblastic leukemia antigen; OKI 11 and B4, expression of Tl 1 and B4 antigens, respectively; slg, expression of surface membrane immunoglobulin. All samples
were positive for terminal deoxytidyl transferase.
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GDj IN MALIGNANT T-LYMPHOBLASTS
Br St Ly Th T-1 T-2 T-3
Fig. 1. Gangliosides of T cd l ALL. Thin-layer chromatogram of gangliosides
from normal human lymphoid tissue and blasts from T-cell acute lymphoblastic
leukemia patients. The separation was on 20- x 20-cm silica gel G TLC plates,
using solvent system C. Br, total bovine brain gangliosides; St, codeveloped
purified ganglioside standards (a, GM3; h, ( ,M.,; c, GM|); Ly. tonsil and adenoid
lymphocytes; Th, thymocytes; T-l-3, cells from three patients with T-cell ALL;
/:';•.human red blood cells. A double-band ganglioside (double arrow) is increased
in patients T-l-3 relative to thymus, while GM3 (single arrow) is decreased. For
tissue samples and standards, 10 nmol and S nmol ganglioside sialic acid was
spotted, respectively.
St T-ALL
Fig. 2. Gangliosides of T-cell ALL. Thin-layer chromatogram of gangliosides
from a T-cell ALL patient. The separation was on 10- x 10-cm silica gel G
HPTLC plates, using solvent system B. ,S"r.codeveloped purified standards (a,
GM3; b, GDj); T-ALL, cells from a patient with T-cell ALL. (Ganglioside sialic
acid, S nmol.)
cytes but not in red blood cells (Fig. 1) and cochromatographed
with a purified GD3 standard (Fig. 2). Further characterization
of the nature of the gangliosides of T-malignant lymphoblasts
followed isolation of the upper and lower doublet gangliosides
from purified total ganglioside by preparative TLC.
TLC and Neuraminidase Treatment of Purified Ganglioside
from Malignant T-Lymphoblasts. Based on the migration of the
two major gangliosides of the mixed ganglioside fractions in
the GM3 and GD3 regions, purified upper and lower doublet
gangliosides were tested for comigration with GM3 and GD3
standards in three different solvent systems. As seen in Fig. 3,
the two major T-lymphoblast gangliosides chromatographed
with identical Rf values to GM3 and GD3, respectively, in each
solvent system. The results also indicate that the doublet gan
gliosides are indeed single ganglioside species, with differences
in the ceramide portion accounting for slight differences in the
migration of each band of the doublets.
Standard GD3 and the lower doublet ganglioside of T-lym-
phoblasts were treated with C. perfringens neuraminidase to
compare the ganglioside and neutral glycosphingolipid neura
minidase products of these gangliosides (Fig. 4). Incubation of
brain GD3 standard with 150 munits neuraminidase for short
times ( 10-40 min) resulted in loss of GD3 and appearance of
both GM3 and lactosyl ceramide. Longer periods of time (2 and
24 h) were required to hydrolyze GM3 completely to lactosyl
ceramide. Neuraminidase treatment of the ganglioside from T-
lymphoblasts also resulted in glycolipids which migrated with
GM3 and lactosyl ceramide, although this ganglioside was more
sensitive to neuraminidase than brain GD3: 150 munits treat
ment for 10 min resulted in complete hydrolysis of the ganglio
side to lactosyl ceramide, 15 munits for 10 min resulted in
appearance of GM3 with no lactosyl ceramide, and 50 munits
resulted in both GM3 and lactosyl ceramide (not shown). These
results are consistent with the preliminary identification of the
lower doublet ganglioside of T-lymphoblasts, based on the
three-solvent TLC, as GD3.
Comparison of Gangliosides of Malignant T-Lymphoblasts to
Gangliosides of Non-T, Non-B Lymphoblasts. Gangliosides were
extracted from cells obtained from three non-T, non-B ALL
patients (two CALLA" and one CALLA*) to determine if high
levels of GD3 were common to childhood lymphoblastic leu-
kemias and lymphomas in general. The results of the TLC
chromatogram of the purified gangliosides showed that the
lower double-band ganglioside observed in malignant T-lym
phoblasts (GD3) was not found in these non-T, non-B leukemic
blasts. Rather, a ganglioside which cochromatographed with
the major ganglioside of human red blood cells, SPG, was the
major ganglioside other than GM3 in these cells (Fig. 5).
Quantitative Scanning Densitometry of Ganglioside Profiles of
Childhood Lymphoblastic Malignancies. Results of scanning
densitometry of the ganglioside profiles of thymocytes, blasts
from six T-ALL patients and one T-cell lymphoma patient, and
blasts from three non-T, non-B ALL patients are shown in Fig.
6. ( ¡I) i was a major ganglioside of cells from all seven patients
with T-cell disease. In contrast to thymus, in which 97% of the
gangliosides was GM3 and 3% was GD3, T-cell lymphoblasts
contained from 2 to 77.7% GM3 and from 22 to 86% GD3,
with the median ganglioside content of 37% GM3 and 63%
GD3. Non-T, non-B ALL, as indicated above, did not contain
detectable GD3, but rather appeared to contain SPG as a major
ganglioside.
Gangliosides of T-Cell Leukemia Cell Lines. Gangliosides
were extracted from two cultured ALL cell lines, CCRF-HSB2
and RPMI 8402. Unlike the results from patient-derived T-
lymphoblasts, these cells did not contain increased levels of
GD3 relative to GM3 (Fig. 7 and Table 2). Rather, GD3 was
only a minor ganglioside in HSB2 and was not detectable in
8402. These cell lines contained high levels of three other
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GD3 IN MALIGNANT T-LYMPHOBLASTS
4A
3A
GD3 T,
3B St
4B
GD3 'u GM-
3C
St 1 2
Fig. 4. Sialidase treatment of GD3 gangliosides. Thin-layer chromatograms of
the products of sialidase hydrolysis of purified brain GD3 (.I ) and purified lower
band ganglioside from T-ALL cells (B). Separations were on silica gel G HPTLC
plates, utilizing the solvent system, CHC13:CH3OH:H:2O, 50:20:3.3 (by volume).
In A, GD3 was treated with 1SOmunits sialidase for 10 min (lane 1), 20 min (lane
2), 40 min (lane 3), 2 h (lane 4), or 24 h (lane 5). In B, T-cell ALL ganglioside
was treated for 10 min with either ISO munits sialidase (lane 1) or IS munits
sialidase (lane 2). Standard lanes include: a, lactosyl ceramide; b, GM3; c. ( .1),.
I u *•*M 3 gangliosides which were not observed in the patients with T-
cell malignancies.
Fig. 3. Purified gangliosides of T-cell ALL. Thin-layer chromatograms of
isolated individual gangliosides of T-cell ALL together with purified standards. DISCUSSION
Separations were on silica gel G HPTLC plates, utilizing solvent system A (A),
solvent system B (B), and solvent system C (Q. Purified ganglioside standards T. . i_-ui j T •
are indicated; Tt, purified lower band ganglioside; r„,purified upper band gan- The results show that childhood T-cell acute lymphoblaStlC
giioside.Gangliosidesialicacid(3nmol)wasspottedineachlane. malignancies consistently expressed ( ìDias the major gangiio-
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GD3 IN MALIGNANT T-LYMPHOBLASTS
a
b
c
d
St N-1 N-2
Fig. 5. Gangliosides of non-T, non-B acute lymphoblastic leukemias. Thin-
layer chromatogram of gangliosides of T-ALL compared to non-T, non-B acute
lymphoblastic (CALLA') leukemias. Gangliosides, separated on HPTLC silica
gel G plates in solvent B, were from T-cell ALL cells (7) and from two patients
with non-T, non-B ALL (N-1. N-2). Standards (St) are: a, GM3; b, GM2; c, SPG;
d, GDj.
side. The purified leukemia ganglioside was identified as GD3
by similar R, values to authentic (il), in three solvent systems,
and by the products of neuraminidasc treatment. GD3 has been
observed in both childhood and adult leukemias in other labo
ratories. In particular, chronic lymphocytic leukemia blasts
were shown by Goff et al. to also contain predominantly GM3
and GD3 (9), unlike chronic myelogenous leukemia cells (30).
In two of three T-cell ALL patients, and one acute myeloblastic
leukemia patient studied by Westrick et al. (IO), a double-band
ganglioside was visualized on TLC plates between IV3 NeuAc-
nLcOse4Cer (SPG) and VI3 NeuAc-nLcOse6Cer, but this gan
glioside was not identified as GD3 in this paper. Among T-cell
malignancies, GD3 may not be restricted to childhood disease:
a ganglioside which migrated in thin-layer chromatograms in
the expected GD3 region compared to a human brain ganglio
side standard was observed in lipid extracts of blasts from two
adult T-cell leukemia patients (31). GD3 has been identified by
anti-GD3 antibody immunostaining in several preparations of
acute and chronic lymphocytic leukemia cells (11), although
the cell surface antigen phenotypes were not further subclassi-
tied immunologically. In our study of the gangliosides of ten
childhood lymphoblastic malignancies, GD3 was expressed in
all seven T-cell malignancies, whereas no GD3 was found in the
three non-T, non-B leukemias which were studied. Although
very low levels of GD3 that are detectable only by sensitive anti-
GD3 but not resorcinol staining may be present in non-T, non-
B ALL, the results suggest that large quantitative differences
in GD3 levels may distinguish these subtypes of childhood
lymphoblastic malignancies.
A major ganglioside of the non-T, non-B (CALLA") blasts,
besides GM3, was a ganglioside tentatively identified as SPG.
SPG was not found exclusively in these non-T blasts, since low
amounts were also observed in two of seven T-ALL cells.
Westrick et al. (10) observed in one non-T, non-B lymphoblastic
leukemia the expression of high SPG by TLC analysis. SPG
was also observed as one of two major gangliosides of acute
myelomonoblastic leukemia (32).
Unlike the results obtained with freshly isolated patient T-
cell leukemic lymphoblasts, two cultured T-ALL cell lines did
not contain a high relative concentration of GD3. Rather, only
trace amounts were observed, in contrast to high levels of three
other gangliosides which were not found in the patient samples.
Several explanations are possible for this observation. One is
that these two leukemias were established from two patients
with rare T-cell leukemias that can be established in culture and
coincidently contained this unique ganglioside pattern. It is well
known that blasts from T-cell ALL are very difficult to establish
in culture (33). Since this ganglioside pattern was observed in
both cultured T-ALL lines, a relationship between establish
ment of these cells in culture and the ganglioside content is
suggested. Secondly, it is possible that the ganglioside compo
sition of T-cell leukemia blasts is altered during establishment
of the cells in culture. In this regard, the ganglioside composi
tion of cells in culture is dependent upon the culture stage (34).
Third, these cultured leukemias may represent a stage of leu-
kemogenesis that is different from any stage of T-cell lymphoid
malignancy in our current patient population, although this is
unlikely due to the relatively large number of T-cell malignan
cies studied. Saito et al. (35), have tested various cultured
Fig. 6. Major gangliosides of T-cell lym
phoblastic malignancies compared to thymus
and non-T, non-B ALL. The gangliosides iso
lated from 10 acute childhood malignancies
and pooled thymus tissue were separated by
TLC or HPTLC. The plates were sprayed with
resorcinol reagent, and the separated ganglio
sides were quantitated as to the relative
amount of GM3, GD,, SPG, and total other
gangliosides using a TLC densitometer. The
T-cell and non-T, non-B malignancies are
numbered according to patient number from
Table 1. m. GM3; •.GD5; B, SPG; M, other
gangliosides.
•OUHo1Z
60
OÃ-oo*
40200.•_
EI 1i
13_¡:;THYMUS
12345T-CELL
MALIGNANCIESi11In10080*seoi>p840
g20n67
8 910NON-T.
NON B-ALL
1728
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CD, IN MALIGNANT T-LYMPHOBLASTS
7A
b
d
an anti-GDj antibody to investigate the surface expression of
GD3 in large numbers of patients with childhood lymphoblastic
malignancies.
We conclude that the immunological subclassification of
childhood lymphoid malignancies appears to correlate with the
ganglioside composition, particularly the expression of ( ¡D,.
Since GD3 is increased in T-cell malignancies relative to normal
thymocytes, GD3 may be a leukemia/lymphoma-specific alter
ation rather than an alteration related to normal T-lymphocyte
ontogeny, although the possibility that GD3 is expressed in very
early stem cells of normal bone marrow and again in these
malignancies cannot be discounted.
HSB2 8402 St St
7B
St HSB2 8402 St
Fig. 7. Gangliosides of IMI cell lines. Thin-layer chromatograms of gan-
gliosides of two T-ALL cell lines compared to primary T cell ALL cells. Ganglio
sides from CCRF-HSB2 cells (USUI) or RPMI 8402 (8402) cells were separated
on silica gel G HPTLC plates, and developed in either solvent system A (A) or
solvent system B (B). T, cells from a T-cell ALL patient; .Vi.standards (a, GM3;
6, GM2; c, SPG; </,GM,; e, GD3;/ CD,.).
Table 2 Gangliosides of T-ALL cell lines
Gangliosides were isolated from the two T-cell ALL-cultured lines HSB2 and
RPMI 8402, and each fraction was separated by HPTLC using solvent system A.
The plates were sprayed with resorcinol reagent, and the relative amount of each
ganglioside was quantitated by TLC densitometry. Stained bands were associated
with a ganglioside region according to migration relative to authentic standards
(see Fig. 7).
Ganglioside
regionGM,
GD,
GMj
GM,
CD,.
OtherPercentage
of totalgangliosideHSB-210.54.5
42.7
18.6
20.3
3.3RPMI
84025.6
ND"
30.6
25.6
38.0
ND
•ND, not detected.
human leukemic cultured cells for ganglioside content. Varied
patterns were observed for the four T-cell lines tested, from
simple to complex, but GD3 does not appear to be present in
these lines according to their densitometric tracings. It is clear
from our results that ganglioside patterns of T-cell lines are
distinctly different from that of freshly obtained, uncultured
cells, and therefore these lines may not necessarily be useful
tools for obtaining information on specific glycolipid markers
in T-cell lymphoblastic malignancies. We are currently utilizing
ACKNOWLEDGMENTS
We would like to thank Dr. Robert Yu, Yale University Medical
School, for providing samples of GD3 and GM? standards, and Dr.
Warner Henle for performing Epstein-Barr virus analysis of our leu
kemia cell lines. We also thank Susan Adams and Phyllis Kirchner
(The Medical College of Wisconsin), and Vicki Cipriano (The George
Washington University Medical Center) for excellent technical assist
ance.
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1987;47:1724-1730. Cancer Res
William D. Merritt, James T. Casper, Stephen J. Lauer, et al.
Lymphoblastic Malignancies
Ganglioside in Childhood T-Cell
3
Expression of GD
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