[CANCER RESEARCH 44, 5914-5920, December 1984]

Detection of Ganglioside GD2in Tumor Tissues and Sera of NeuroblastomaPatients1

Gregor Schulz,2 David A. Cheresh,3 Nissi M. Varki, Alice Yu, Lisa K. Staffileno, and Ralph A. Reisfeld4

Department of Immunology, Scripps Clinic and Research Foundation [G. S., D. A. C., N. M. V., L K. S., R. A. R.], and the Department of Pediatrie Oncology, University ofCalifornia at San Diego ¡A.Y.J, La JoHa, California 92037

ABSTRACT

A murine monoclonal antibody (monoclonal antibody 126) produced against cultured human neuroblastoma cells (LAN-1) was

found to be specifically directed to a disialoganglioside (GD2)antigen preferentially expressed on both cell lines and tissuesderived from melanoma and neuroblastoma. In enzyme-linked

immunosorbent assays, monoclonal antibody 126 failed to reactwith leukemic and lymphoblastoid cells as well as with a varietyof carcinoma and sarcoma cell lines. Immunohistological analysisby the immunoperoxidase technique revealed strong reactivityof monoclonal antibody 126 with frozen and formaldehyde-fixed

neuroblastoma and melanoma tissues. Tissues from patientswith glioma or with small cell cancer of the lung showed faintstaining, whereas those from individuals with sarcoma, lym-

phoma, and a variety of other neoplasms proved to be negative.Sera of neuroblastoma patients showed significantly elevatedGo2 levels compared to normal children (p < 0.001) and childrenwith other tumors (p < 0.001 ) as determined by a quantitativecompetitive enzyme-linked immunosorbent assay.

Furthermore, the GD2serum level of one neuroblastoma patient, when followed serially, was found to correlate with progression of disease, suggesting the potential usefulness of thisassay for the diagnosis and monitoring of neuroblastoma.

INTRODUCTION

Several antigenic determinants have recently been detectedon neuroblastoma cells with MAbs5 (4, 10, 13, 17, 22-24, 28,

29). A panel of these antibodies was reported to be helpful inthe differential diagnosis of neuroblastoma and lymphoblasticdisorders (11,12). In these same studies, antibodies were usedeither in immunoperoxidase assays with tumor tissue sectionsor in indirect immunofluorescence assays to detect tumor cellsin bone marrow aspirates (11, 12). However, there have notbeen any reports describing the use of antibodies to neuroblas-toma-associated antigens to detect elevated amounts of these

antigens in the sera of patients and normal children.The effective use of MAbs directed to any tumor-associated

antigens as diagnostic reagents depends on the quantity, expression, and chemical nature of the corresponding antigen. In thisregard, MAbs directed to tumor-associated gangliosides have

been useful in defining antigens associated with melanoma,

' This work was supported by USPHS Grant CA28420. This is Publication 3279-

IMM from the Department of Immunology, Scripps Clinic and Research Foundation,La Jolla, California 92037.

2 Recipient of Deutsche Forschungsgemeinschaft Grant l-3-Schu 512/1-1.3Recipient of NIH Fellowship Award 1F32 CA07544-01.4To whom requests for reprints should be addressed.'The abbreviations used are: MAb, monoclonal antibody; PBS, phosphate-

buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbentassay; TLC, thin-layer chromatography.

Received May 21,1984; accepted September 6,1984.

neuroblastoma, colon carcinoma, and adenocarcinoma (6). Oneof these antibodies was reported to detect a ganglioside antigenshed into the serum of patients with colon carcinomas (15).

In this study, we demonstrate that MAb 126 specifically directed against the disialoganglioside G026(2) detects elevated

amounts of this antigen on tumor tissues and in the sera ofneuroblastoma patients.

MATERIALS AND METHODS

Cell Lines

The following cell lines were used for screening of the antibody.Neuroblastoma: LAN-1, 2, 5 (Dr. R. Seeger, UCLA); SK-N-SH, American

Type Culture Collection (ATCC, Rockville, MD); melanoma; M14, M21(Dr. D. Morton, UCLA); FM3, FM8, F12 (Dr. J. Harper, Scripps Clinic, LaJolla, CA); Melur (Dr. P. Koldovsky, Dusseldorf, W. Germany); Foss (Dr.B. Giovanella, Stehlin Foundation, Houston, TX); glioma: U373 MG,U138MG, U87MG (ATCC); small cell carcinoma of lung: H69 (Dr. J.Minna, National Cancer Institute, Bethesda, MD); B-lymphoblastoid: LG2,L14 (Dr. Gati, UCLA); leukemias: Molt 4, HPB-ALL, Burkitt's lymphoma

Daudi, (ATCC); adenocarcinoma of lung: T291 (Dr. H. Masui and Dr. S.Sato, University of California, San Diego); UCLA-P3 (Dr. D. L. Morton,UCLA); squamous cancer of skin: SCI-1185 (ATCC); anaplastic cancer:CALU-6 (ATCC); adenocarcinoma of adrenal cortex: SW13 (ATCC);Wilms' tumor: WILTU-1 (ATCC); osteosarcoma: U-20S (ATCC); Ewing's

sarcoma:SK-ES-2 (ATCC); rhabdomyosarcoma: A204 (ATCC).

Monoclonal Antibodies

MAb 126 was produced by immunization with the neuroblastoma cellline LAN-1 using standard hybridoma technology (14). Briefly, BALB/c

mice were inoculated i.p. once every week for a total of 4 weeks with 5x 10s LAN-1 cells per injection and their splenocytes fused with the

murine myeloma cell line P3Ag8X63 3 days after the last injection.Hybridoma 126 was selected by growth in Dulbecco's modified Eagle's

medium containing 10% fetal calf serum and hypoxanthine, aminopterin,and thymidine medium, and was subcloned using limiting dilution. Theisotype of the MAb 126 was determined with a Litton Bionetics Kit (LittonBionetics, Kensington, MD) as described in the kit instructions. In addition, MAb MB3.6 (2) which is directed against the ganglioside antigenGDS,was used. Both hybridoma supematants contained approximately10 ng per ml of antibody.

Tissues

Portions of fresh normal and malignant tissues were obtained fromthe Surgical Pathology department of the Ida M. Green Hospital ofScripps Clinic, La Jolla, CA. Other tumor samples were kindly providedby Dr. F. Kung (Department of Pediatrie Oncology, University of Californiaat San Diego) and by Dr. P. Wolf (Department of Pathology, Universityof California at San Diego). Fresh tissue specimens were embedded inTissue Tek-ll O.C.T. (Miles, Napervilte, IL), frozen in blocks in isopentane

•Gangliosides are termed according to the nomenclature as described previously

by Svennerholm (25).

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IMMUNODIAGNOSIS OF NEUROBLASTOMA

at liquid nitrogen temperature, and stored at -70°. Formalin-fixed,

paraffin-embedded blocks were obtained from the files of the Department

of Pathology, University of California at San Diego.

Immunoperoxidase Staining

Frozen Tissues. The reactivity of MAb 126 with different frozentissues was determined by using an indirect immunoperoxidase assayas described earlier (26).

Paraffin-embedded Tissues. The procedure was slightly modifiedfrom that already described (26). Briefly, sections on gelatin-coated slides

were deparaffinized and rehydrated. Endogenous peroxidase was removed with 0.3% H2O2. The following antibodies were applied sequentially to the sections for 30 min with rinses in PBS, pH 7.6, between eachincubation: monoclonal antibody supernatant, 1:50; rabbit anti-mouseantiserum, 1:1000, diluted in 1% BSA in PBS, swine anti-rabbit anti-

serum, 1:100; and rabbit peroxidase antiperoxidase, 1:1000 (AccurateChemicals, Westbury, NY). The color reaction was developed withdiaminobenzidine, and the sections were dehydrated, cleared in xylene,mounted, and viewed.

ELISA for Cultured Cell Lines. The procedure to test the reactivityof MAbs with cell lines in ELISA has been described previously (7).Briefly, target cells were plated in flat-bottomed poly vinyl microtiter plates(Dynatech, Alexandria, VA) at 5 x 104 cells/well. Prior to the ELISA

assay, dried plates were rehydrated by 2 washes of 2 min each with 10mM PBS, pH 7.4, containing 0.1% Tween 20, and 0.02% thimerosal. Inorder'to reduce nonspecific binding, hybridoma supematants were di

luted 1:2 in washing buffer containing 0.1% BSA as diluent. Fifty n\ ofdiluted test supematants were added to each well and incubated for 1hr at 4°on a gyroshaker. Following 2 washes of 2 min each, 50 /¿Iof

peroxidase-linked goat anti-mouse IgG plus IgM (Tago, Buriingame, CA),

diluted 1:2000, were added to each well, and the reaction mixtureincubated at 4°for 1 hr. After 2 final washes, 50 /<!of substrate solution

(o-phenylene diamine, 400 i¿g/m\,in 80 mM citrate-phosphate buffer, pH

6.0, containing 0.12% H2O2)were added to each well. Color developmentwas stopped after 15 min by adding 25 ¡Aof 4 M H2SO4 to each well,and the absorbance at 492 nm was measured with a Multiskan ELISAplate reader.

Serum Samples. Serum samples were provided by Dr. A. Lightsey(Department of Pediatrie Oncology, Navy Hospital, San Diego, CA), Dr.F. Lampert (Department of Pediatrie Oncology, Justus Liebig University,Giessen, West Germany), Dr. R. Castleberry (Department of PediatrieOncology, Birmingham, AL), and the Department of Pediatrie Oncology,University of California, San Diego. All serum samples were drawn beforeintense chemotherapy was started. They were frozen and stored at -20°

until used in the assay. In all cases, the clinical diagnosis was confirmedby histology.

Purification of Gangliosides from Serum Samples. Serum sampleswere dissolved in chloroform:methanol (2:1) at a ratio of 1:20 (v:v) andfiltered through syringes filled with scrubbed nylon fiber (3 denier, 3.81cm, type 200; Fenwal Laboratories, Deerfield, IL) to remove proteinaggregates. The residue was re-extracted with chloroform:rnethanol 1:1

at a ratio of 1:20 (v:v). The combined filtrates were then subjected tonitrogen evaporation. The dried glycolipid extract was dissolved in 5 mlof chloroform :methanol (2:1). Gangliosides were partitioned into anaqueous phase as described by Leeden and Yu (16). This material wasdialyzed exhaustively against cold distilled water and dried down byrotary evaporation.

Ganglioside Standards. Purified GD2was kindly supplied by Dr. R. K.Yu (Yale University, New Haven, CT). Qua and GM2were supplied by Dr.J. Sundsrno from our institution, and GM1,GDI. and GT were purchasedfrom Supleco (Bellefonte, PA).

TLC. Silica gel plates (plastic-backed; E. M. Merck, Darmstadt, WestGermany) were activated by heating at 110° for 1 hr. Chtoro-

form:methanol:0.2% aqueous CaCI2 60:45:10 was used for the development of the chromatograms. Samples were spotted 1.5 cm from the

bottom of the TLC plates which were then placed in a developing tankpresaturated with 100 ml of the above solvent. Chromatograms weredeveloped for 1.5 hr at room temperature, after which the plates wereallowed to dry. Appropriate lanes of chromatograms were cut andsprayed with resorcinol reagent to visualize gangliosides (9).

Immunostaining of Gangliosides Separated by TLC. The reactivityof MAb 126 with gangliosides separated by TLC was determined directlyby using an immunostaining method originally described by Magnani efa/. (18). This procedure was modified by using an ELISA detection system(3).

Lipid ELISA Inhibition Assay. For the preparation of lipid plates, atotal lipid extract was prepared from 2 ml of packed Melur melanomacells, which are known to express the Gm ganglioside. These cells werehomogenized in 40 ml of chlorofomrmethanol (2:1), followed by filtrationthrough a scintered glass filter. The residue was re-extracted with

chlorofomrmethanol (1:1) and refiltered. The combined filtrates werethen dried down. Finally, these glycolipids were resuspended in methanol.Routinely, glycolipids from 50-nl packed-cell volume were plated per wellin flat-bottomed polyvinyl microtiter plates (Dynatech, Alexandria, VA).

Before use in the assay, the plates were preincubated for 2 hr with 5%BSA in PBS.

The assay to detect GM was done by incubating 100 //I of MAb 126supernatant for 1 hr at room temperature with semipurified gangliosidesextracted from different serum samples (300 pi). After centrifugation for5 min in an Eppendorf centrifuge, 75 n\ of this supernatant were diluted1:4, and 50 n\ of the diluents were tested in duplicate for binding to thelipid plates in ELISA. The ELISA assay was similar to that for culturedcell lines described above except that 1% BSA in PBS was used aswashing and diluting buffer. Binding of antibody preincubated with partially purified gangliosides obtained from serum samples was comparedto that achieved with antibody binding alone when incubated with lipidplates.

In addition, the inhibition of antibody binding achieved with differentserum samples was compared to that obtained by preincubation ofantibody with different amounts of purified GD2. By using a log-logitbinding curve, it was possible to determine the amount of GD2in bothpatient and control sera.

The differences of G02 levels in sera from different patient groups andcontrol individuals were evaluated for statistical significance using theRank Testing according to Wilcoxon-Mann-Whitney.

RESULTS

ELISA Reactivity of MAb 126 with Different Tumor CellLines. Table 1 summarizes the reaction pattern obtained whenMAb 126 (IgM) was tested against 30 different cell lines by astandard ELISA technique. It is evident that all 7 melanoma celllines as well as all 4 neuroblastoma cell lines tested were stronglypositive. The glioma cell line U373MG proved negative, whereas2 other glioblastoma cell lines were strongly positive. Five of 7carcinoma cell lines did not react with the antibody. The oat cellcarcinoma cell line H69 and a cell line derived from an adenocar-cinoma of adrenal cortex (SW13) were weakly positive. MAb 126did not bind to 2 B-lymphoblastoid and 2 T-cell leukemia celllines, as well as to cell lines derived from Wilms' tumor, Ewing's

sarcoma, osteogenic sarcoma, and rhabdomyosarcoma. In addition, the antibody failed to react with leukemic cells isolatedfrom peripheral blood from 6 patients with different kinds ofleukemia (acute lymphoblastic, acute myeloblastic, and chronicmyelogenous). These data indicate that the antigen recognizedby MAb 126 is preferentially expressed on cell lines derived fromneuroectodermal tumors with the exception of glioma cell lineU373MG.

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Table 1Reactivity of MAb T26 with different cell lines in an ELISAassay

Table 2Tissuereactivity of MAb 126 by immunoperoxidaseassays

CelllineLAN-1LAN-2LAN-5SK-N-SHM14M21FM3FM8FL2FossMelurU373MGU138MGU87MGTumortypeReactivityNeuroblastoma+++a+++++Melanoma

+++++++++++++++++++++Glioma

-++++++Other

tumorsLG2L14Molt

4HPB-ALLDaudiT291CALU6SCI-1185UCLAP3SW13PanelH69WIL-TU-1SK-ES-2U-20SA204B-lymphoWastoid

cells—T-cell

leukemia-Acutelymphocyte leukemia-Burkitt's

lymphoma-Adenocarcinoma

lung-Anaplasticcarcinoma-Squamousskin carcinoma-Adenocarcinoma

lung-Adenocarcinomaadrenal cortex++Pancreatic

carcinoma-Oatcell carcinoma++Wilm's

tumor-Ewing'ssarcoma-Osteogenic

sarcoma-Rhabdomyosarcoma-*

+++, binding reactivity is more than 4 times higher than nonimmuneimmuno-gtobulin

fromthetimeshigher thanmyeloma

partner, P3Ag8X63; ++, binding reactivity is 3 to4immunogtobulinfrom myeloma P3Ag8X63; +, bindingreactivityis

2 to 3 times higher than immunogobulinfrom myelomaP3Ag8X63.Reactivity

of MAb 126 with Different Normal andMalignantTissues.Whf>n MAb 126 was tested aaainst a relatively laraeine

number ot each type ot tissue testediTumor

tissuesNeuroblastomaMelanomaOat

cell carcinomalungAdenocarcmomasStomachLungBreastColonProstateOvaryFibrosarcomaSeminomaNon-Hodgkin's

lymphomaIslet

cellcarcinomaAstrocytomaGlioblastoma

multiformeLeiomyomauterusPleomorphic

adenomaparotidOsteogenicsarcomaEmbryonalrhabdomyosarcomaWilm's

tumorNormal

tissuesColonSpleenPancreasLiverLungBrain

cortexCerebellumKidneyThyroidSkin

melanocytesBenignnevusFetal

tissuesColon

SpleenAdrenalLiverLungBrain

cortexCerebellumKidneyanged

trom ¿tobFrozen+++*++++_c_c—

c_c_c_c————±

±•f—NDNDND_*___"-±±~r4.+°__—NONPFixed++++++NDNDNDNDNDNDNDNDNDND—NDNDNDND_-—____-4.+-ND+wnnu

NDNDNDND++ND

number of frozen or formalin-fixed normal and malignant tissues

by the immunoperoxidase technique, all 7 neuroblastomas including 2 métastasesto testis and 4 melanomas were found tobe strongly positive (Table 2). Only 50% of the cells were stainedin a tissue derived from small cell carcinoma of lung, and a gliomatissue showed very faint staining. A variety of tumor tissues frompatients with fibrosarcoma, non-Hodgkin's lymphoma, osteo-genic sarcoma, rhabdomyosarcoma, Wilms' tumor, and different

types of carcinoma were negative. Among normal tissues, therewas only very slight reactivity with cerebral and cerebellar graymatter, skin melanocytes, and nevi. MAb 126 also reacted faintlywith smooth muscle of blood vessels, supporting stroma, and abenign leiomyoma. All other organs and tissues were not reactive. Most of the fetal tissues tested were also negative with theexception of those derived from brain and cerebellum.

Fig. 1, A and B illustrates the staining obtained when MAb 126was reacted with a formaldehyde-fixed, paraffin-embedded neu

roblastoma tissue as compared to that observed when the sametissue was reacted with a nonimmune ¡mmunoglobulin, i.e., theP63Ag8X63 supernatant. Staining of this tissue with anotherMAb (KS 1/9) of IgM isotype provided essentially the sameresults as the nonimmune ¡mmunoglobulin (data not shown).These figures clearly depict the very strong staining obtained byMAb 126 with all tumor cells of this tissue section. It is of interest

" +++, strongly positive reaction; -, negative reaction; +, positive reaction; ND,

not determined.6 Smooth muscle around blood vessels is faintly positive.cThe supporting stroma of smooth muscle adjacent to tumor cells is faintly

positive.

that, at the time the test was done, this particular tumor hadbeen fixed and embedded for more than 6 months, indicatingthat the antigen recognized by MAb 126 was not denatured byeither formaldehyde or prolonged storage.

Reactivity of MAb 126 with Gangliosides Extracted fromSerum Samples in an Immunostaining Procedure on TLC. Itwas of interest to determine whether the GD2antigen could bedetected in the circulation, since relatively large amounts of thisantigen are expressed in neuroblastoma tissues. In a first experiment, gangliosides extracted from 750 n\ of serum were separated on a TLC plate and immunostained to assess the bindingof MAb 126 to GD2by an ELISA procedure. Fig. 2 clearly depictsthat GDZpresent in the sera of 4 patients with neuroblastoma(Stage IV and IVs) reacted strongly with MAb 126 (Lanes C toF), whereas control serum samples from 4 normal childrenshowed only faint staining (Lanes G to K). The reactivity of MAb126 with purified GD2is demonstrated in Lane B, while Lane Ashows the migration of different ganglioside standards stainedwith resorcinol. When the same gangliosides were separated onTLC and sprayed with resorcinol instead of using the immuno-

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IMMUNODIAGNOSIS OF NEUROBLASTOMA

A

'

*#•4 .^'

*. a!*fir a

Fig. 1. Staining of a formaldehyde-fixed, paraffin-embedded neuroblastoma tissue by MAb 126 using the ¡m-

munoperoxidase technique (A): staining of the same areaby nonimmune immunoglobulin from the supernatant of themyeloma fusion partner P3Ag8X63 (B), x 1000, oil immer- IHsion. D

V

staining procedure, only the neuroblastoma patients' ganglioside

profile revealed a prominent GD2band (data not shown). Thesedata suggested that neuroblastoma patients may present withrelatively large amounts of circulating GD2in serum. Therefore,we attempted to develop a quantitative assay to determine theexact levels of GDain the sera of patients and control individuals.

G02 Levels in Sera from Neuroblastoma Patients, Childrenwith Other Pediatrie Tumors, and Normal Children. In earlierexperiments, it was shown that the binding of MAb 126 toneuroblastoma or melanoma cells is specifically inhibited bypreincubation with purified GD2 (2). In order to determine theamount of GD2extracted from serum samples, we first calculatedthe inhibition of binding of MAb 126 induced by different amountsof purified GDZ.

Chart 1 depicts the results from one representative experiment. MAb 126 supernatant was preincubated with purified GD2,ranging in concentration from 2.5 to 50 ng. These supematantswere then tested in duplicate in ELISA against glycolipids con

taining a large amount of GD2.The binding was compared to thatobtained with antibody supernatant that was not preincubatedwith Go2, and the data were expressed in a log logit plot. Thecorrelation coefficient of the standard plot was 0.98. Using thesedata, the amount of GD2in semipurified gangliosides extractedfrom serum samples could be determined by the inhibition theyinduced in this competitive ELISA. Whenever the inhibition wasoutside of the range of sensitivity of the standard curve, sampleswere retested at higher dilutions. Because the recovery in ourextraction procedure was >93% in 2 experiments (data notshown), we considered these values to be essentially the sameas the actual amount of GD2in serum.

Chart 2 shows the serum levels of GD2 determined by thecompetitive ELISA in patients with active neuroblastoma (n =23), other pediatrie tumors (n = 23), healthy, age-matched controls (n = 16), and patients with nonactive neuroblastoma (n =

6). The mean age of neuroblastoma patients was 61 ±55 (S.D.)months, compared to 63 ±38 months in children with other

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GM3- •

GM2- »

GM,- «

GD,a- 9

GT-

ABCDEFGHIJKFig. 2. Reactivity of MAb 126 with GM in neuroblastoma sera. Immunostaining

of gangliosidesseparatedon TLC by MAb 126.LanesC to F. gangliosidesextractedfrom sera of patients with neuroblastoma;Lanes G to K, gangliosidesfrom sera ofnormal children; Lane B, purified GM; Lane A, migration of different gangfosktestandards stained with resorcinol.

f

-2

1

LogIngG0¡lChart 1. Inhibition of MAb 126 binding to GD2-containingglycolipids in ELISA

after preincubation with different amounts of purified GD;(2.5 to 50 ng) expressedin a log logit plot.

tumors and 65 ±48 months in patients with nonactive neuroblastoma. Twenty-three patients with active neuroblastoma hadthe following clinical stages at the time of serum collection. Threepresented with Stage IVs, 8 with Stage IV, 6 with Stage III, 3with Stage II, and 3 with Stage I. The patients who after therapyshowed no clinical signs of neuroblastoma were originally classified as: Stage IVs, 2; Stage IV, 2; Stage II, 1; and Stage I, 1.Patients with other tumors presented with the following diagnosis: rhabdomyosarcoma, 4; Ewing's sarcoma, 2, Wilms' tumor,3; osteogenic sarcoma, 2; Hodgkin's disease, 2; mesenchy-

moma, 1; histiocytosis X, 1; retinoblastoma 1; acute lymphoblas-tic leukemia, 4; acute myeloblastic leukemia, 1; non-Hodgkin'slymphoma, 1; and Burkitt's lymphoma, 1.

Data depicted in Chart 2 indicate that 21 of 23 neuroblastomaexhibited GD2levels in serum of more than 26 ng/ml (mean ofcontrols ±3 S.D.) indicating a degree of confidence of >99.8%.The 2 neuroblastoma patients with lower levels had diseaseclassified as Stage I and II. Of 6 patients with very high GD?levels (over 1000 ng/ml), 4 presented with Stage IV and 2 withStage III. One of these patients even presented with a GD2levelof 4300 ng/ml. Because the number of neuroblastoma patientswith Stage I and II was very small, we did not compare GD2levelsof patients with different stages of disease. However, none ofthe neuroblastoma patients with Stage I and II had GD2serumlevels above 100 ng/ml. Among patients with other tumors, only

>10001001

75Ìa1«

50J3

XLÜ25Control

Children•.•;'f"PWJMric

NeuroblastomaTumors E.D.N.E.D.j:!AT••X•.•*l:•

Chart 2. Gm (ng/ml) extracted from sera of patients with neuroblastoma, children with other tumors, and control children. E.D., evidence of disease; N.E.D., noevidenceof disease. Values were determined by competitive ELISA.

2 showed elevated GD2levels in serum. One child had a Wilms'

tumor (69 ng/ml), and the other, an osteogenic sarcoma (33 ng/ml). It is interesting that 2 other patients with Wilm's tumor, as

well as one other child with osteogenic sarcoma, exhibitednormal levels of GD2. Six children, who after treatment wereconsidered to be free of tumor, showed normal levels of GD2intheir serum. The mean GD2serum level of these children (8.8 ±6.8 ng/ml) was similar to that of normal controls (8.8 ±5.8 ng/ml) and children with other tumors (18.2 ±13.6 ng/ml). Whenthe GD2levels were compared in a ranking evaluation test corresponding to Wilcoxon-Mann-Whitney, the difference in serum

Go2 levels of patients with neuroblastoma compared to all othergroups was statistically highly significant (p < 0.001). However,there was no statistically significant difference among the 3 othergroups, i.e., normal individuals, patients without clinical evidenceof neuroblastoma, and patients with other tumors. The reprod-

ucibility of the assay is excellent as illustrated by the fact that,at most, ±10% variability was observed when different individuals tested the same serum sample at various times. For controlpurposes, we preincubated these same neuroblastoma seraextracts with a MAb directed against GD3,i.e., MB3.6 (15) (notreactive with neuroblastoma tissues), and found that its binding

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IMMUNODIAGNOSIS OF NEUROBLASTOMA

was not inhibited in this assay (data not shown). These datasuggest that the inhibition of MAb 126 with these serum extractsis specific. In addition, in one patient, we were able to follow theserum level of GD2during the clinical course of disease. This wasa 16-month-old child who had inoperable neuroblastoma covering the entire abdomen, with infiltration to the kidney, paraaortic,and mesenterio lymph nodes. The patient did not show elevatedurinary levels of catecholamines, vanillylmandelic acid, or homo-

vanillic acid, which are the conventionally used tumor markersfor neuroblastoma. The GD2serum level before treatment was1600 ng/ml. After the first course of chemotherapy with cyclo-

phosphamide and Adriamycin, and 7 days after initiation ofradiotherapy, there was a good clinical response with tumorregression to one-third of the original size. The GD2level corre

spondingly fell to 148 ng/ml. After the second course of chemotherapy and radiotherapy, the patient was still in partial remission. The observed decrease in GD2level remained constant andthus correlated with this patient's response to therapy. After the

completion of 5 courses of chemotherapy, the tumor becameinpalpable, and the G02 level dropped to 16 ng/ml. The patientthen underwent a second surgery in which small clusters ofresidual tumor were removed. The bulk of the residual tumorhad the pathology of ganglioneuroma with very few interspersedneuroblastoma elements. Postoperatively, the patient's GD2level

in the serum decreased further to 3 ng/ml and remained at thislevel thus far. These data suggest that the level of GD2in serummay be a useful indicator for tumor regression and reoccurrencein neuroblastoma.

DISCUSSION

It was demonstrated in this study that MAb 126, recognizinga GD2antigen (2), is strongly reactive with most neuroectoder-

mally derived tumor cell lines and tissues tested thus far. InELISA, this antibody failed to react with nearly all other lympho-mas, sarcomas, and carcinoma cell lines. The screening of fresh-frozen and formaldehyde-fixed normal and malignant tissues by

the immunoperoxidase technique indicated that MAb 126 hadits greatest reactivity with neuroblastoma and melanoma tumortissues. This is the first report that clearly demonstrates elevatedlevels of the ganglioside GD2 in the sera of most patients withneuroblastoma compared to sera of normal children or patientswith other pediatrie tumors. These observations suggest thatthe level of GD2antigen in the serum may be a useful marker fordiagnosis, monitoring, and followup of neuroblastoma patients.We demonstrated in previous studies that the binding of MAb126 to corresponding neuroblastoma target cells was inhibitedby preincubation with purified GD2and that this antibody recognized only GDZin a glycolipid extract isolated from neuroblastomaor melanoma tumors by immunostaining procedures on TLC (2).In this regard, we could now prove that a strong inhibition ofbinding of MAb 126 by preincubation with semipurified ganglio-

sides extracted from the sera of neuroblastoma patients correlates with strong immunostaining of 602 in a TLC overlay assay.Because the recovery of our extraction procedure was nearly100%, it became possible to use the log phase binding inhibitioncurve obtained with purified GD2to calculate the actual amountof GD2in serum samples.

Eighteen of 20 patients with other pediatrie tumors did notpresent with elevated GD2levels. However, it is of some interest

that 1 of 3 serum samples of patients with Wilms' tumor and

another sample from one of 2 osteogenic sarcoma patientsshowed elevated amounts of GD2.This was particularly surprising, since cell lines as well as tissues of these histológica! typesof tumors failed to react with MAb 126. Although we believe thatthese data represent individual abnormalities, further sampleswill have to be tested in order to exclude elevated GD;.levels inthese cancers.

Studies by others suggested that MAbs may be useful in thedetection of tumor-associated antigens in serum of patients with

melanoma and colon carcinoma (5, 15, 19). However, we concentrated our efforts on developing effective methods for diagnosis of neuroblastoma in tissue and serum samples, mainlybecause the differential diagnosis of this tumor is often moredifficult than that for glioma or melanoma (12, 20, 21). In fact,neuroblastomas are at times quite difficult to distinguish histo-

pathologically from other round cell tumors in children, whichinclude lymphoblastic lymphoma, leukemia, rhabdomyosarcoma,or Ewing's sarcoma (12, 20, 21). Consequently, a serum assay

for neuroblastoma requiring only small volumes of blood is indeedmost advantageous, especially since other diagnostic tests forneuroblastoma, I.e., urinary catecholamines and their metabolites, are sometimes negative because some neuroblastomasare cholinergic or inactive rather than adrenergic (22). In addition,specific catecholamines may also be elevated in individuals thatdo not have neuroblastoma, such as patients with pheochro-

mocytoma and patients with iron deficiency.In addition to the potential diagnostic utility of MAb 126, our

data also suggest that it may prove useful for therapeutic monitoring. In this regard, a patient whose serum GD;>level wasfollowed serially showed a remarkable correlation with regressionof disease. Further studies are required to strengthen and corroborate these findings.

To the best of our knowledge, 2 other MAbs were reported tobe directed to neuroblastoma-associated gangliosides. Thus,

Eisenbarth ef al. (4) described a murine MAb directed to the GQganglioside. A monospecific human antibody (anti-OFAI-2), pro

duced in vitro by a lymphoblast cell line derived from a melanomapatient was also shown to detect a GD?ganglioside on humanmelanoma, glioma, and neuroblastoma cells (1, 8). In anotherreport, it was demonstrated that an antibody from a melanomapatient recognized an autoantigenic ganglioside related to GD?(27). Both antibodies proved to react relatively specifically withneuroectodermal-derived tumors when tested by immune adher

ence assays (8, 27). However, both studies using the humanantibodies failed to apply the more discriminating immunoperoxidase technique for staining of frozen and paraffin-embedded

normal and tumor tissues that also provides more useful information for clinical purposes, since it detects even small tumorinfiltrates and métastasesinto normal organs. In addition, thereis thus far no published report providing a complete descriptionof the tissue distribution of the GD2antigen in normal tissues.

In our hands, MAb 126 proved to be most useful in theimmunoperoxidase assay to discriminate between neuroecto-

dermal tumor cells and normal tissues, especially since, in contrast to many other tumor-associated antigens (12), the GDZ

ganglioside antigen recognized by MAb 126 is not denatured bythe routinely used formaldehyde fixation of tissues. This characteristic enhances its potential usefulness for clinical applications.

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IMMUNODIAGNOSIS OF NEUROBLASTOMA

It is quite apparent from our results that additional studies arerequired to optimize the detection of elevated GD2levels in ourserum assay. However, our data obtained thus far suggest thatthe application of MAbs directed against GD2may help to improvethe diagnosis and thereby assist in the treatment of neuroblastoma.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Robert K. Yu, Yate University, for helpfuldiscussions,and Bonnie Pratt Filiault for her expert secretarial assistance.

REFERENCES

1. Cahan, L. D., Irie, R. F., Singh, R. Cassidenti. A., and Paulson, J. C. Identification of a human neuroectodermal tumor antigen (OFA-1-2) as gangliosideG«.Proc. Nati. Acad. Sci.. USA, 79: 7629-7633,1982.

2. Cheresh, D. A., Harper, J. R., Schulz, G., and Reisfeld, R. A. Localization ofthe gangliosidesGM and Gta in adhesionplaquesand on the surfaceof humanmelanomacells. Proc. Nati. Acad. Sci., USA,81: 5767-5771,1984.

3. Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J., andReisfeld, R. A. A monoclonal antibody recognizes an 0-acylated sialic acid ina human melanoma-associatedganglioside. J. Bid. Chem., 259: 7453-7459,1984.

4. Eisenbarth, G. S., Walsh, F. S., and Nierenberg, M. Monoclonal antibody to aplasma membraneantigen of neurons. Proc. Nati. Acad. Sci. USA, 76: 4913-4917, 1979.

5. Gupta, R. K., and Morton, D. L. Studies of a melanoma tumor-associatedantigen detected in the spent culture medium of a human melanoma cell lineby allogeneic antibody. II. Immunobiologic characterization. J. Nati. CancerInst., 72:75-82,1984.

6. Hakomori, S-l . and Kannagi, R. Glycosphmgolipidsas tumor-associated anddifferentiation markers. J. Nati. Cancer Inst., 71, 231-251,1983.

7. Harper, J. R., and Orengo, A. The preparation of an immunoglobulin-amyto-glucosidase conjugate and its quantitation by an enzyme-cyclingassay. Anal.Biochem., 773: 51-57, 1980.

8. Irte, R. F., Sze, L. L., and Saxton, R. E. Human antibody to OFA-1, a tumorantigen,produced in vitro by Epstein-Barrvirus-transformedhumanB lymphoidcell lines. Proc. Nati. Acad. Sci. USA, 79. 5666-5670,1982.

9. Jourdian, G. W., Dean, L., and Roseman, S. The sialic acids XI. A periodate-resorcmol method for the quantitative estimation of free sialic acids and theirglycoskJes.J. Biol. Chem.,246: 430-435,1971.

10. Kemshead, J. T., Bicknell, D., and Greaves, M. F. A monoclonal antibodydetecting an antigen shared by neural and granulocytic cells. Pediatr. Res.,15: 1282-1286,1981.

11. Kemshead, J. T., Goldman, A., Fritschy, J., Malpas, J. S., and Pritchard, J.Use of panels of monoclonal antibodies in the differential diagnosis of neuro

blastoma and lymphoblastic disorders. Lancet 7. 12-15,1983.12. Kemshead, J. T., Walsh, F. S., Pritchard, J., and Greaves, M. Monoclonal

antibody to ganglioside GQ discriminates between hematopotetic cells andinfiltrating neuroblastomatumor cells in bone marrow. Int. J. Cancer,27: 447-452, 1981.

13. Kenne«,R. H., Jonak, Z., and Bechtol, K. B. Characterizationof antigens withmonoclonal antibodies. In: Evans, A. E., (ed.), Advances in NeuroblastomaResearch,pp. 209-217. New York: Raven Press, 1980.

14. Konter, G., and Milstein, C. Continuous cultures of fused cells secretingantibody of pre-definedspecificity. Nature (Lond.),256: 495-497,1975.

15. Koprowski. H., Herlyn, M., and Steplewski, Z. Specific antigen in serum ofpatients with colon carcinoma. Science(Wash. DC),272; 53-55,1981.

16. Ledeen, R. W., and Yu, R. K. Gangliosides:structure, isolation, and analysis.Methods Enzymol.,83:139-191,1982.

17. Uaso, S. K., Clarke, B. J., Kwong, P. C., Brickenden, A., Gallte, B. L., andDent, P. B. Common neuroectodermalantigens on human melanoma,neuroblastoma, retinoblastoma.ghoblastoma.and fetal brain revealedby hybridomaantibodiesraised againstmelanomacell. Eur. J. Immunol., 77:450-454,1981.

18. Magnani, J. L., Smith, D. F., and Ginsburg, V. Detection of gangliosides thatbind cholera toxin: direct binding of 125 l-labeted toxin to thin layer chromat-ograms. Anal. Biochem., 709: 399-402, 1980.

19. Morgan,A. C., Crane,M. M., and Rossen, R. D. Measurementof a monoclonalantibody-defined,melanoma-associatedantigen in human sera: correlation ofcirculating antigen levels with tumor burden. J. Nati. Cancer Inst., 72: 243-249, 1984.

20. Raney, R. B., Lyon, G. M., and Porter, F. S. Neuroblastoma simulating acuteleukemia.J. Ped.89: 433-435,1976.

21. Reynolds, P. C., Smith, G. R., and Frenkel, E. P. The diagnostic dilemma ofthe small round cell neoplasm. Cancer (Phila.),48: 2088-2094,1981.

22. Seeger, R. C., Danon,Y. L., Rayner,S. A., and Hoover, F. Definitionof a Thy-1 determinant on human neuroblastoma,glioma, sarcoma, and teratoma cellswith a monoclonalantibody. J. Immunol., 728: 983-989,1982.

23. Seeger, R. C., Rosenblatt, H. M., Imai, K., and Ferrone, S. Common antigenicdeterminantson human glioma, neuroblastoma,and sarcoma cells defined bymonoclonalantibodies. Cancer Res., 41: 2714-2717,1981.

24. Seeger,R. C., Siegel,S. E., and Side«,N. Neuroblastoma:clinicalperspectives,monoclonal antibodies, and retine«;acid. Ann. Intern. Med., 97: 873-884,1982.

25. Svennerholm,L. Chromatographieseparation of human brain gangliosides. J.Neurochem., 70: 613-623,1963.

26. Taylor, C. R. Immunoperoxidasetechniques. Practicaland theoretical aspects.Pathol. Lab. Med., 702; 113-121,1978.

27. Watanabe, T., Pukel, C., Takeyama, H., Uoyd, K. D., Shiku, H., U, L. T. C.,Travassos, L. R., Oettgen, H. F., and Old, L. J. Human melanomaantigen AHis an autoantigenicgangliosiderelated to Go,. J. Exp. Med., 756:1884-1889,1982.

28. Wikstrand, C. J., and Bigner, D. D. Expression of human fetal brain antigensby human tumors of neuroectodermal origin as defined by monoclonal antibodies. Cancer Res., 42: 267-275,1982.

29. Wikstrand, C. J., Bourdon, M. A., Pegram, C. N., and Bigner, D. D. Humanfetal brain antigen expression common to tumors of neuroectodermal tissueorigin: gliomas.neuroblastomas.and melanomas.J. Neuroimmunol 3: 43-62,1982.

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1984;44:5914-5920. Cancer Res   Gregor Schulz, David A. Cheresh, Nissi M. Varki, et al.   Neuroblastoma Patients

in Tumor Tissues and Sera ofD2Detection of Ganglioside G

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