[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 composition in malignant blasts of six children with acute T- cell lymphoblastic leukemia (T-Cell ALL), one with T-cell Lymphoblasts from seven children with T-cell lymphoblastic malig lymphoblastic lymphoma, and three with non-T, non-B ALL. nancies and three children with non-T, non-B acute lymphoblastic leu We have found that GD3 was a major ganglioside in each of kemia (ALL) were analyzed for ganglioside content. Nonmalignant I - the T-cell malignant lymphoblastic samples studied, whereas cells from thymus served as controls. Both ganglioside and glycoprotein sialic acid were increased approximately 3-3.5-fold in T-cell disease this ganglioside was barely detectable or absent in the non-T, non-B leukemias and normal thymus that we evaluated. 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- MATERIALS AND METHODS raminosyllactosylceramide (GM3), the major ganglioside in normal lymphoid tissue, and the other ganglioside comigrated with authentic II3- Isolation and Characterization of Lymphocytes from Human Lymph o-A'-acetylneuraminosyl-a2—»8-jV-acetylneuraminosyllactosylceramide oid Tissue and Lymphoblasts from Patients with Acute Lymphoid Ma lignancies. Fresh tonsils and adenoid tissue or lymphomas were ob (GDj) in three different solvent systems. Neuraminidase treatment of the tained immediately after surgical removal; any highly inflammed tissue latter ganglioside yielded (;M glycolipid extraction. in ganglioside GD3 in uncultured, patient-derived T-cell ALL lympho blasts when compared to non-T-cell ALL and normal lymphoid tissue. Lymphoblasts were separated from whole blood, bone marrow, or pleural fluid from leukemic patients by either plasmagel sedimentation Therefore, GD3 may represent a tumor-associated antigen for the T-cell in Hanks' balanced salt solution (13), or by Ficoll-Hypaque density subclass of childhood lymphoblastic malignancy. 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. INTRODUCTION Cells were further characterized with respect to various T-cell and Transformation of cells by oncogenic viruses or by chemicals leukemia-associated (CALLA) cell surface antigens using sheep eryth- consistently results in one or more changes in the composition rocyte rosetting assays (14, IS) or immunofluorescent techniques with of neural glycolipids and/or sialylated glycolipids (ganglio- heterologous or monoclonal antibodies as previously described (12, 15- 17). Lymphoblasts in all cases were positive for terminal deoxytidyl sides). Altered expression of glycolipids appears to be related transferase (18) and negative for surface immunoglobulin, myeloper- to a block in glycolipid glycosylation and/or a shift in glycolipid oxidase, chloroacetate esterase, and a-naphthylbutyrate esterase. biosynthesis to an inappropriate pathway (for reviews see Refs. Culture of Human IX ell Leukemia Lines. The cell lines RPMI 8402 1-4). For instance, during hepatocarcinogenesis of the rat, (T,o-i) and CCRF-HSB2 HV.) (19, 20) were obtained from the labo synthesis of the disialoganglioside, GD3,3 is blocked, resulting ratory of Dr. J. Minowada, Roswell Park Memorial Institute, Buffalo, in lowered expression of complex gangliosides, particularly NY. Karyotyping of the cultured cell lines was performed in the cytogenetics laboratory of Milwaukee Children's Hospital, and the trisialoganglioside (5, 6). On the other hand, GD3 is inappro priately expressed in human melanoma cells (7, 8). In studies presence of Epstein-Barr virus was analyzed by immunofluorescence of gangliosides of human leukemias, GD3 is expressed to vary staining for us by Dr. Warner Henle, Joseph Stokes Jr. Research Institute, Philadelphia, PA. The cells were cultured in RPMI 1640 ing extents in the blasts of patients with chronic lymphocytic media (GIBCO, Grand Island, NY) in the presence of penicillin (100 leukemia (9), acute lymphoblastic leukemia (ALL), and acute /jg/ml), streptomycin (100 units/ml), 4 mm 4-(2-hydroxyethyl)-l-pi- myeloblastic leukemia (10,11). We have studied the ganglioside perazineethanesulfonic acid, and 10% heat-inactivated fetal bovine serum (GIBCO). Cells were cultured in a humidified 5% CO2, 37'C 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 humidified incubator, and were split two times per week. For analysis of page charges. This article must therefore be hereby marked advertisement in of gangliosides, cells were cultured in 350-ml volumes in closed-roller accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was supported in part by National Cancer Institute Grant bottles. Isolation and Purification of Gangliosides. Cell pellets were homoge CA33573 and the Milwaukee Athletes Against Childhood Cancer Fund. ' To whom requests for reprints should be addressed, at the Department of nized in a small volume of dl I..() and homogenized with a polytron Biochemistry, The George Washington University Medical Center, 2300 Eye homogenizer. Aliquots were removed for assay of protein and sialic Street, NW, Washington, DC 20037. 3The abbreviations used are: GD3, II3-«-A'-acetylneuraminosyl-a-2—»8-/V-ace-acid. Lipids were extracted by overnight stirring of the homogenate in tylneuraminosyllactosylceramide; GM3, II3-a-/V-acetylneuraminosyllactosyl- chloroform:methanol in the ratio, 1:1:0.1 (v/v/v) of chloroform: ceramide; GM2, H'-a-W-acetylneuraminosylgangliotriosylceramide; CM,, H3-a- methanol: homogenate. The precipitated material was collected by fil A'-acetylneuraminosylgangliotetraosylceramide; GD,., II3-a-W-acetylneuramino- tration through a sintered glass filter and then reextracted twice with syl-IV3-a-/V-acetylneuraminosylgangliotetraosylceramide; SPG, sialosylparaglo- chloroform:methanol:water, 1:1:0.1 (v/v/v). The combined lipid ex boside, 1V3-/V-acetylneuraminosylneolactotetraosylceramide; ALL, acute lympho tracts were evaporated by rotary evaporation at 45*C, and gangliosides blastic leukemia; TLC, thin-layer chromatography; HPTLC, high-performance thin-layer chromatography; CALLA, common acute lymphoblastic leukemia an and neutral glycolipids were separated by the procedure of Ledeen et tigen. al. (21). Specifically, the residue was dissolved in chloroform: 1724 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. CD, IN MALIGNANT T-LYMPHOBLASTS methanohwater, 60:30:8 (v/v/v), and placed on a DEAE-Sephadex A- min). The lower phase was dried under N2, and the lipids were resus 25 column (5 g of resin/2.5 x 10' cells). Nonacidic lipids, including pended in a small volume of chloroform:methanol, 2:1 (v/v) and sepa neutral glycolipids, and acidic lipids, including gangliosides, were eluted rated by HPTLC, using the solvent system, chloroform:methanol:water, separately. After removal of solvent, lipids in the latter fraction were 50:20:3.3 (v/v/v). Bands were visualized by spraying with orcinol re saponified with 0. l M methanolic NaOH to destroy phospholipids (22). agent. 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 RESULTS of silicic acid (Biosil; Bio-Rad Industries). Sulfatides and other contam Lymphoblastic Malignancies. Surface marker characteriza inants were eluted with chloroform:methanol, 80:20 (v/v), and ganglio tion of the malignant cells from seven of the ten patients sides were eluted with chloroform:methanol, 1:1 (v/v) (21). The gan- demonstrated that the cells were of T-cell origin. This was glioside fraction was dried under nitrogen, dissolved in chloro- form:methanol, 1:1 (v/v) and stored at -20°Cfor further analysis. demonstrated by sheep erythrocyte rosetting and/or reactivity Individual gangliosides were purified from T-cell leukemia extracts with either an antithymic antibody or with a monoclonal anti by preparative TLC. Chromatography was on 250 n silica gel H 20- x body against the sheep erythrocyte receptor (OKT11) (Table 20-cm plates (Analtech, Newark, DE) with the solvent system chloro- 1). The remaining ALL patients studied were non-T, non-B form:methanol:0.22% CaCl2, 60:40:9 (v/v/v). Silica gel H was used based on absence of T-cell markers and surface membrane instead of silica gel G to improve the recovery of the gangliosides away immunoglobulin. These cells were examined for expression of from the silica gel. Individual gangliosides were located with iodine CALLA, and were either CALLA' (2 cases) or CALLA* (l vapor, scraped, and then eluted by overnight shaking in chloro- case). All samples demonstrated terminal deoxytidyl transferase form: met hanol: water, 1:1:0.1 (v/v/v). Analytical Procedures. Gangliosides were separated by TLC on activity. precoated silica gel 60 TLC or HPTLC plates in either «-propanol: Quantitation of Ganglioside and Glycoprotein Sialic Acid. 0.22% CaCl2, 8:2 (v/v) (Solvent A), chloroform:methanol:0.22% Sialic acid was measured colorimetrically in the purified gan- CaCl2, 60:40:9 (v/v/v) (Solvent B), or chloroform:methanol:28% glioside fractions and lipid-free residue from pooled tin mie NH4OH:water, 60:40:3:5 (v/v/v/v) (Solvent C). Ganglioside standards tissue (12 samples), and the lymph olilast s from six separate T- were spotted in lanes alongside experimental lanes. Standards included cell ALL patients. The results showed that both ganglioside GM3, isolated from human liver (23); SPG, isolated from human red and glycoprotein sialic acid were increased approximately 3- blood cells (24); human brain GD3 and Tay-Sachs brain GM2, gifts 3.5-fold in leukemia cells compared to thymic cells. Measure from Dr. Robert Yu, Yale University; and GM, and GDU (Supelco, ments of the ganglioside fractions showed that T-cell leukemias Bellefonte, PA). Ganglioside bands were visualized by resorcinol stain contained 11.80 ±4.3 nmol sialic acid/109 cells, and the thy- ing (25). The relative proportions of individual gangliosides were as sessed by scanning densitometry using a Schimadzu model CS-930 mocytes contained 4.3 ±2.0 nmol/109 cells. Similarly, the TLC scanning densitomer, with drifting baseline and peak integration leukemia samples contained 133.0 ±12 nmol glycoprotein capability. sialic acid/109 cells whereas thymocytes contained 38.0 ±4 Ganglioside sialic acid was measured by the method of Hammond nmol/109 cells. On the other hand, T-cell ALL cells and thy 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 mocytes contained equal amounts of ganglioside and glycopro tein sialic acid when expressed on a per mg protein basis (0.5 the chloroform-methanol-insoluble residues collected on sintered glass and 3.9 nmol/mg protein respectively). 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 Ganglioside TLC of Malignant T-Lymphoblasts. The purified hydrolysis, and digests were placed on a column of Biorad AG 1-X8 ganglioside fraction of malignant lymphoblasts in each of seven acetate ion exchange resin. Sialic acid was eluted with 1.5 ml l N T-cell patients with malignancies were analyzed by TLC. Fig. formate, and 0.5-ml samples were assayed in triplicate as above. Various 1 shows the ganglioside patterns of three leukemia samples concentrations of ALacetylneuraminic acid (Sigma) were processed in (lanes 5-7) compared to tonsil and adenoid lymphocytes (lane parallel throughout the procedure. Proteins were measured by a linear 3) and thymocytes (lane 4). The patterns show that the ganglio transform modification of the Folin-Lowry procedure (28). side profile from T-malignant blasts is more similar to that of Neuraminidase Digestion of GI), Ganglioside. The isolated GD3 (8 thymus gangliosides than that of tonsil and adenoid lymphocyte nmol sialic acid/treatment) was resuspended in 100 /¿Iof0.05 Msodium gangliosides. However, the leukemia cells have a lowered acetate buffer, pH 5.7, with either 150, 50, or 15 munits neuraminidase amount of the major doublet ganglioside found in thymus (Fig. (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 1, single arrow), which cochromatographed with standard GM3, chloroform: met hanol, 2:1 (v/v). The precipitated protein was removed and a proportionally larger amount of a second but very minor by centrifugation, and the soluble components were partitioned by thymic doublet ganglioside (Fig. 1, double arrow). The latter addition of 0.4 ml water, vortexing, and recentrifugation (270 X g, 15 ganglioside was also observed in tonsil and adenoid lympho-

Table 1 Characteristics of lymphoblastic malignancies analyzed PatientDiagnosis1 (yrs)4.51112111635952.5Surfacephenotype"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 T-ALL2 T-ALL3 T-ALL4 T-lymphoblasticlymphoma5

T-ALL6 T-ALL7 *(97%),B4-,CALLA-ER*(95%),OKT11 T-ALL8 r(97%),B4~,CALLA-ER-.OKT1 ALL9 non-T, non-B l-,B4*,sIg-,CALLA-ER-,T-Ag-,sIg-,CALLA-ER-,T-Ag-,sIg-,CALLA* ALL10 non-T, non-B non-T, non-B ALLSexMFMMMMFFMMAge " 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. 1725 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. GDj IN MALIGNANT T-LYMPHOBLASTS

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

Br St Ly Th T-1 T-2 T-3 minidase products of these gangliosides (Fig. 4). Incubation of Fig. 1. Gangliosides of T cd l ALL. Thin-layer chromatogram of gangliosides brain GD3 standard with 150 munits neuraminidase for short from normal human lymphoid tissue and blasts from T-cell acute lymphoblastic times (10-40 min) resulted in loss of GD3 and appearance of leukemia patients. The separation was on 20- x 20-cm silica gel G TLC plates, both GM3 and lactosyl ceramide. Longer periods of time (2 and using solvent system C. Br, total bovine brain gangliosides; St, codeveloped purified ganglioside standards (a, GM3; h, ( ,M.,; c, GM|); Ly. tonsil and adenoid 24 h) were required to hydrolyze GM3 completely to lactosyl lymphocytes; Th, thymocytes; T-l-3, cells from three patients with T-cell ALL; ceramide. Neuraminidase treatment of the ganglioside from T- /:';•.humanred blood cells. A double-band ganglioside (double arrow) is increased lymphoblasts also resulted in glycolipids which migrated with 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 GM3 and lactosyl ceramide, although this ganglioside was more spotted, respectively. 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 St T-ALL contained from 2 to 77.7% GM3 and from 22 to 86% GD3, with the median ganglioside content of 37% GM3 and 63% Fig. 2. Gangliosides of T-cell ALL. Thin-layer chromatogram of gangliosides GD3. Non-T, non-B ALL, as indicated above, did not contain 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, detectable GD3, but rather appeared to contain SPG as a major GM3; b, GDj); T-ALL, cells from a patient with T-cell ALL. (Ganglioside sialic ganglioside. acid, S nmol.) Gangliosides of T-Cell Leukemia Cell Lines. Gangliosides were extracted from two cultured ALL cell lines, CCRF-HSB2 cytes but not in red blood cells (Fig. 1) and cochromatographed and RPMI 8402. Unlike the results from patient-derived T- with a purified GD3 standard (Fig. 2). Further characterization lymphoblasts, these cells did not contain increased levels of of the nature of the gangliosides of T-malignant lymphoblasts GD3 relative to GM3 (Fig. 7 and Table 2). Rather, GD3 was followed isolation of the upper and lower doublet gangliosides only a minor ganglioside in HSB2 and was not detectable in from purified total ganglioside by preparative TLC. 8402. These cell lines contained high levels of three other 1726 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. 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 *•*M3 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 ( ìDi as the major gangiio- 1727 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. GD3 IN MALIGNANT T-LYMPHOBLASTS

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 a three non-T, non-B leukemias which were studied. Although very low levels of GD3 that are detectable only by sensitive anti- b GD3 but not resorcinol staining may be present in non-T, non- B ALL, the results suggest that large quantitative differences c in GD3 levels may distinguish these subtypes of childhood lymphoblastic malignancies. d 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). St N-1 N-2 Unlike the results obtained with freshly isolated patient T- cell leukemic lymphoblasts, two cultured T-ALL cell lines did 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 not contain a high relative concentration of GD3. Rather, only lymphoblastic (CALLA') leukemias. Gangliosides, separated on HPTLC silica trace amounts were observed, in contrast to high levels of three 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; other gangliosides which were not found in the patient samples. d, GDj. Several explanations are possible for this observation. One is that these two leukemias were established from two patients side. The purified leukemia ganglioside was identified as GD3 with rare T-cell leukemias that can be established in culture and by similar R, values to authentic (il), in three solvent systems, coincidently contained this unique ganglioside pattern. It is well and by the products of neuraminidasc treatment. GD3 has been known that blasts from T-cell ALL are very difficult to establish observed in both childhood and adult leukemias in other labo in culture (33). Since this ganglioside pattern was observed in ratories. In particular, chronic lymphocytic leukemia blasts both cultured T-ALL lines, a relationship between establish were shown by Goff et al. to also contain predominantly GM3 ment of these cells in culture and the ganglioside content is and GD3 (9), unlike chronic myelogenous leukemia cells (30). suggested. Secondly, it is possible that the ganglioside compo In two of three T-cell ALL patients, and one acute myeloblastic sition of T-cell leukemia blasts is altered during establishment leukemia patient studied by Westrick et al. (IO), a double-band of the cells in culture. In this regard, the ganglioside composi ganglioside was visualized on TLC plates between IV3 NeuAc- tion of cells in culture is dependent upon the culture stage (34). nLcOse4Cer (SPG) and VI3 NeuAc-nLcOse6Cer, but this gan Third, these cultured leukemias may represent a stage of leu- glioside was not identified as GD3 in this paper. Among T-cell kemogenesis that is different from any stage of T-cell lymphoid malignancies, GD3 may not be restricted to childhood disease: malignancy in our current patient population, although this is a ganglioside which migrated in thin-layer chromatograms in unlikely due to the relatively large number of T-cell malignan the expected GD3 region compared to a human brain ganglio cies studied. Saito et al. (35), have tested various cultured

•OUHo1Z

Fig. 6. Major gangliosides of T-cell lym phoblastic malignancies compared to thymus and non-T, non-B ALL. The gangliosides iso 60 lated from 10 acute childhood malignancies Oíoo* 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 40200.•_ g20n67 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.

1i 13_¡:;THYMUSEI 12345T-CELL 8 10NON-T. 9

MALIGNANCIESi11In10080*seoi>p840 NON B-ALL 1728 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. CD, IN MALIGNANT T-LYMPHOBLASTS

an anti-GDj antibody to investigate the surface expression of 7A 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 b thymocytes, GD3 may be a leukemia/lymphoma-specific alter ation rather than an alteration related to normal T-lymphocyte d 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.

ACKNOWLEDGMENTS

We would like to thank Dr. Robert Yu, Yale University Medical HSB2 8402 St St School, for providing samples of GD3 and GM? standards, and Dr. Warner Henle for performing Epstein-Barr virus analysis of our leu 7B 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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1730 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. Expression of GD3 Ganglioside in Childhood T-Cell Lymphoblastic Malignancies

William D. Merritt, James T. Casper, Stephen J. Lauer, et al.

Cancer Res 1987;47:1724-1730.

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