(CANCER RESEARCH 40, 897-908, March 1980]0008-5472/80/0040-0000$02.00

Adaptation of Mass Spectrometry for the Analysis of Tumor Antigens asApplied to Blood Group Glycolipids of a Human Gastric Carcinoma

MichaelE. Breimer

Department of Medical Biochemistry, University of Göteborg,S-400 33 Goteborg, Sweden

ABSTRACT

Total neutral (nonacidic) glycolipid fractions have been isolated from a gastric adenocarcinoma and the surroundingnormal gastric tissue removed at lapanotomy of a Blood GroupB human individual. Each glycolipid mixture was fractionatedby silicic acid column chromatography into ten different partlypurified glycolipid fractions. These fractions were analyzed bythin-layer chromatography and tested for Blood Group A andB activity. Blood group B activity was found in several fractionsfrom both tumor and normal tissue. Two of the tumor glycolipidfractions reacted with some batches of commercial anti-Aantisera, but other antisera tested did not react. No such BloodGroup A reactivity was found in the fractions from the normalgastric tissue. The two Blood Group A-active tumor glycolipidfractions were methylated and methylated-reduced, and thesetwo derivatives were analyzed by mass spectrometry. It wasshown that these two fractions were mixtures of glycosphingolipids with five to nine sugars. The dominating glycosphingolipids were blood group Leband B-like hexaglycosylceramides, a B-similar heptaglycosylcenamide with an additional fucose, an H-like heptaglycosylcemamide, and a Leb@likeoctaglycosylcemamide. Evidence for small amounts of a BloodGroup A-similar heptaglycosylcenamide with an additional fucose was also found. The finding of a Blood Group A-similarglycolipid in a fraction which reacts with some anti-A antiserais the first chemical evidence for a hetenolog blood groupantigen in human cancer which has previously been found byhistoimmunological techniques. The clinical significance of thisfinding is discussed in relation to diagnostic procedures andimmunotherapy.

INTRODUCTION

The blood group antigens of the ABH, Lewis, and P systemsare known to be of carbohydrate nature (28, 36). They exist in3 different forms, as free oligosacchanides on bound to proteinor lipid. The first 2 are secreted into various body fluids, i.e.,saliva (36), milk (19), urine (26), and ovarian cyst fluid (36). Onthe other hand, most of the membrane-bound blood groupantigens are glycolipids (8, 27, 36) where the immunologicallyactive carbohydrate chain is bound to ceramide, which isburied in the outer part of the plasma membrane bilayer. Thesyntheses of the Blood Group A on B antigens are done byenzymatic addition of galactosamine or galactose, respectively,to the H substance (36). The glycosyltransfenases which catalyze these reactions are genetically controlled, but how this isdone is not yet known.

Several studies on the ABH antigens in different tumors have

I SUppOrted by grants from the Swedish Medical Research Council(No. 3967)

and from the Medical Faculty, university of Goteborg.ReceIved July 26, 1979; accepted December 5, 1979.

been done. Mostly, histoimmunological techniques have beenused, but some attempts to do a chemical characterizationhave also been made. By histoimmunological methods, loss ofA, B, or H blood group antigens from the cell surface ofneoplastic epithelial cells has been reported for several different tumors, e.g. , carcinoma of the stomach (4, 30), lung (3),and transitional cell carcinoma of the urinary bladder (20).Several investigators have found a correlation between the lossof A, B, on H isoantigens and a higher malignancy potential ofthe tumors (4, 20), but contradictory results have also beenreported (5). The finding of noncompatible Blood Group Aactivity in human gastric adenocarcinoma of Blood Group Band 0 individuals (6) is of great interest. This finding was laterconfirmed by Denk et a!. (5), who found Blood Group A- and B-incompatible reactions in adenocancinoma of gastric and coIonic mucosa. They also noted that the blood group substancesfrom poorly differentiated and anaplastic cancers were alcoholsoluble and therefore resembled glycolipids.

The deletion of isolog ABH-active glycolipids in human adenocarcinoma has been reported (8—10, 31). This deletion ofABH antigens was in some cases found to result in a rise of theamount of blood group Lewis-active glycolipid antigens (9). In1967, Hakomoniet a!. (10) isolated a glycosphingolipid fractionfrom a gastric adenocarcinoma of a blood group 0 patient.This fraction had a weak Le0but no ABH activity. Rabbits giveninjections of this preparation produced anti-A antisera.

The deletion of ABH activity in tumors has been found to bedue to a low activity of the enzymes responsible for synthesisof A, B, and H substances instead of a high glycosidase activity(18, 34).

In our laboratory, methods for preparation and structuralcharacterization of blood group active glycosphingolipids on amicroscale (14) have been developed. By a mass spectrometnictechnique, we are able to identify individual glycolipids inmixtures which is impossible by other chemical methods (2,13).

In the present study, this mass spectrometnic technique isused for characterization of 2 glycosphingolipid fractions isolated from a human gastric adenocarcinoma.

MATERIALS AND METHODS

Case HIstory. The patient was an 81-year-old woman whohad had mild diabetes and hypertension for some years. Because of anemia, an X-ray investigation of the stomach wasperformed, and a highly suspected malignant lesion was found.At laparotomy, a gastric carcinoma in fundus ventniculi wasfound; no hepatic metastasis was seen. A total gastric resectionwas carried out, and part of the pancreas and the spleen wasalso removed. The pathological examination revealed a fairlylow-differentiated adenocarcinoma with metastases to the megional lymphatic glands with peniglandular growth. The patient

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Some data of the partly purified neutral glycolipid fractions isolated fromagastricadenocarcinoma (T,@) and normal gastric tissue (N@) from thesamehuman

Blood Group BindividualBlood

group activityGlycolipid Forssman

Glycolipid fraction wt (mg) Ba AbactivityGastric

carcinomaT29.0 NDC NDNDT211.4 ND ND ND

T3 7.9i+dT46.3 2+ ——T5

16.4 4+ ——T611.5 4+ ——T72.5 4+ 1+—T83.1 4+ 3+—T92.1 2+ ——T02.8 2+ ——Normal

gastrictissueN9.3 ND NDNDN24.2 ND NDNDN32.7 1+ ——N40.4 1+ ——N51.1 4+ ——N62.5 4+ ——N7

2+ ——N819 4+ — —

N9 . 3+ ——N04+ — —

M. E. Breimer

remained well for 6 months when 2 cutaneous metastases werefound in the operation scar. She developed clinical symptomsof recurrence of the tumor and died 10 months after theoperation. No autopsy was done. The blood group of thepatient's ABC was B, Rh+.

Tissues. The tumor specimen obtained at lapanotomy wasimmediately dissected macroscopically into cancer tissue andnormal gastric tissue and lyophilized. The dry weight wasdetermined.

Preparation of Glycolipids. The dried tissues were cut intopieces and extracted overnight in a Soxhlet apparatus withchloroform:methanol, first 2:1 and then 1:9 (by volume). Thetotal neutral (nonacidic) glycosphingolipid mixture was obtamed by conventional preparative steps (11)2 including mildalkaline degradation, DEAE-cellulose chromatography, silicicacid chromatography, and by use of native and acetylatedglycolipids. Inasmuch as immunology and mass spectrometryare the major interests in this work, the applied details ofpreparation are omitted.

The total neutral glycosphingolipid mixture obtained from thecancer and the normal gastric tissue were finally separatedinto 10 different, partly purified glycolipid fractions (Fig. 1) bysilicic acid chromatography, using increasing amounts of methanol in chloroform as eluant. Conditions for thin-layer chromatography are given in the legend of Fig. 1.

Immunological Studies. Fonssman hapten activity wastested by inhibition of hemolysis as described (16). Pure Fonssman glycolipid, a pentaglycosylcenamide isolated from horsekidney in our laboratory (16), was used as reference. Thedetection limit was 0.05 @zgof Forssman glycolipid in a glycolipid mixture of 5 @g.

Blood group A, B, Lea, and Leb activity was tested by hemagglutination-inhibition in microscale.3 The glycolipid fractions were tested either in the micelle or liposome form. Themicelles were produced by dissolving the amphipatic glycolipids in 0.9% NaCI solution to a concentration of 0.5 @tg/@zI.Theliposomes were prepared by mixing 50 @zgof the glycolipid

fraction with 50 @sgof hydrated lecithin and 25 @.&gof cholesterol,and the organic solvent was removed. A 0.9% NaCI solution(1 ml) was added, and the mixture was sonicated for 1 hr. Thetest was performed by mixing 2 s1of the antigen preparationwith 1 @lof antiserum under a layer of paraffin oil and incubatingat room temperature for 1 hr. A 5% suspension (1 @tI)of theappropriate human RBC was thereafter added, followed byanother incubation at room temperature for 1 hr. The amountof agglutinates was estimated microscopically. Commercialhuman blood grouping anti-A and anti-B antisera (Gamma Biol.Inc., Houston, Texas) and goat anti-Le6 and anti@Lebantisera(Behningwerke AG, Manburg/Lahn, Federal Republic of Germany) were used. Pure blood group Le8-active pentaglycosylceramide and blood group A-, B-, and Leb@activehexaglycosylceramides were used as positive controls. Pure glycolipidantigen (0.05 @g/@sl)gave a complete inhibition of agglutination(a 4+ reaction) at the antiserum dilution used.

Mass Spectrometry. Five hundred j@gof the 2 Blood Group

2 K-A. Karlsson. An improved and simplified method for the quantitative

isolation of glycosphingolipids, manuscript in preparation.3 K-E. Dahigren, P. Emilsson, G. C. Hansson, and B. E. Samuelsson. Prepa

ration and characterization of liposomes for the immunological study of glycosphingolipids with blood-group activity including a micromethod for haemagglutination-Inhibition, manuscript in preparation.

A-active (see ‘‘results'‘)glycolipid fractions from the gastriccancer were permethylated (7, 12) and analyzed by massspectrometny. The remaining sample was then reduced (12)with LiAIH4and again subjected to mass spectrometry. An MS902 instrument (AEI, Manchester, England) was used. Thesample (about 40 to 60 @g)was introduced through the directinlet system equipped with a separate probe heater. The conditions of analysis are given in the legends for the charts. Fordetails about sample handling, instrumental conditions, andinterpretation of the spectra, see Refs. 13 and 15.

RESULTS

The weight of the total neutral (nonacidic) glycolipid fractionof the adenocarcinoma was 93.0 mg (4.8 mg/g, dry tissueweight), and the weight for the surrounding normal gastrictissue was 22.1 mg (1.2 mg/g, dry tissue weight). The weightof each partly purified fraction obtained after silicic acid chromatognaphy is given in Table 1.

The total neutral glycolipid fractions prepared from the gastnic adenocancinoma 0') and the surrounding normal tissue (N)are shown in the thin-layer chromatogram of Fig. 1 togetherwith the partly purified fractions obtained by silicic acid columnchromatography (T110, N11o). All bands seen were coloredgreen with the anisaldehyde reagent and thus contained cambohydmate(33). The glycolipids separate mainly according tothe number of carbohydrate residues, but the lipophilic partalso influences the mobility giving rise to double bands whichare most easily seen for the glycolipids with 2 carbohydrateresidues. With increasing number of carbohydrate residues,the influence of the ceramide part plays a minor role on themobility of the glycolipids.

When comparing the glycolipid fractions, striking differences

Table 1

a Anti-B antisera diluted 1 :4, glycolipid concentration, 0.5 @g/@d.b Anti-A antisera diluted 1:4, glycolipid concentration, 0.5 @ag/pl.C ND, not determined.d The strength of inhibition of agglutination was graded from 4+ (strong

inhibition) to 1+ (weak inhibition) and —(no inhibition).

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Mass Spectrometry of Tumor G!yco!ipids

T1 T2 T3 T4 T5 T6 T7 T8 Tg ho T N N1 N2 N3 N4 N5 N6 N7 N8 Ng N10

Fig. 1. Thin-layer chromatogram of the total neutral glycolipid fraction prepared from a gastric adanocarcinoma 0') and the surrounding normal gastric tissue (N).The 10 glycollpid fractions obtained by silicic acid chromatography of the total glycolipid mixtures are designated T—T0and N—N0,respectively. The plate wasprecoated HPTLC Fertigplatten, Kiselgal 60, 10 x 20 cm (Merck, Darmstadt, Federal Republic of Germany). The solvent was chloroform:mathanol:watar (60:35:8,by volume),and the glycolipidswere visualizedwiththe anisaldahyde(33) reagent.About40 @sgof the totalneutralglycolipidfractionsand about4 @sgof the purifiedfractions were applied on each lane. The designation to the left indicates the number of carbohydrates in each glycolipid band.

sensitive, was also used but still no activity was found. The Bactivity was unchanged and gave a strong inhibition. The onlychange in the test system was the use of a new batch of anti-Aantisera. Several other batches of anti-A antisera were therefore tested, but no activity was obtained.

When tested as liposomes, Fractions T7 and T8 of the tumorshowed Leb activity but no Le6 activity. The other glycolipidfractions were not tested against these antisera.

No immunological Forssman activity was found in the glycolipid fractions isolated from the tumor or the normal gastricmucosa (Table 1).

Mass Spectrometry. Mass spectrometny of permethylatedand permethylated-reduced glycolipids has proved to be apowerful technique for microscale characterization (less than50 @sg)of pure glycolipids or mixtures of several glycolipids.The conclusion concerning the structures is based on thecombined interpretation of the 2 spectra obtained from themethylated glycolipid fractions (Charts 1 and 5), and methylated-reduced fractions (Charts 2 and 6), respectively. Featuresof primary interest are terminal sugar(s), terminal saccharides,number of sugars, sequence of sugars, and ceramide stnuctunes. In this paper, the evidence for the structures proposedon the base of the reproduced spectra is briefly discussed. Formore details about the interpretation, see Refs. 13 and 15.

Spectra of Tumor Fraction T7. The mass spectrum of themethylated T7 glycolipid fraction is shown in Chart 1, and themethylated-reduced spectrum is shown in Chart 2. Simplifiedformulas of probable structures for the interpretation are givenin Charts 3 and 4, respectively. In the methylated sample (Chart1), terminal fucose is found at m/e 189 and 157 due to loss ofmethanol (32 mass units). Terminal hexose is seen at m/e 219and 187 (219-32). No terminal hexosamine at m/e 260 can beseen. Abundant ions from the terminal saccharides of Leb@6,B-6, and B-7 glycolipids (Chart 3) are observed in the uppermass region. Some of these fragments can be formed from 2different glycolipids, e.g. , m/e 1016 of the Leb@6and B-7

can be seen between the tumor and the normal gastric tissue.Between regions 3 and 4, a new band appears in the tumor.There is an accumulation of the glycolipid with about 5 sugarsin the tumor compared to the normal fraction, and there is alsoan accumulation of slow-moving glycolipids in the tumor. Moreover, one cannot exclude the possibility that each spot in theslow-moving region may contain more than one glycolipid,because it is known that glycolipids with different carbohydratestructures can have the same mobility on thin-layer chromatogmaphy(32).

Immunological Activity. Immunological studies were performed on the partly purified glycolipid fractions from the tumor(T3@1o)and normal tissue (N310). Fractions 1 and 2 were nottested. These small glycolipids are difficult to handle in thewater test system because of their dominating lipophilic properties, and thus fanno blood group activity in the ABH or Lewissystems has been found in glycolipids with less than 5 sugars.

The results of the initial test for A and B activity using themicelle method are shown in Table 1. The strength of inhibitionof agglutination was graded from 4+ (strong inhibition) to +(weak inhibition) and —(no inhibition). As expected for a BloodGroup B individual, Blood Group B activity was found in thenormal gastric tissue. The strongest inhibition at an antiserumdilution of 1:4 was found in Fractions N5, N6, N8, and N10.Bactivity was also found in the tumor with the strongest reactionin Fractions T5 to T8 at an antiserum dilution of 1:4. When aconcentrated anti-B serum was used, the strongest inhibitionwas found in Fraction T6 and Fraction N8. When testing forBlood Group A activity, a 3+ inhibition of agglutination inFraction T8 and a weak reaction of Fraction T7 were found(Table 1). No activity was found in the normal gastric tissueglycolipids. The experiment was repeated, and the findingswere confirmed. Some months later, after the completion ofmass spectrometnic analysis, I tried to reproduce the finding ofBlood Group A activity in the tumor fractions, but no activitywas found at this time. The liposome technique, which is more

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Chart 1. Mass spectrum of the methylated derivative of glycolipid Fraction T7 obtained from a human gastric adenocarcinoma. The conditions of analysis were:electron energy, 65 aV; trap current, 500 @a;acceleration voltage, 6 kV; ion source temperature, 300°;and probe temperature, 2600. Peaks below m/e 80 ware notreproduced. Corresponding formulas for interpretation are shown in Chart 3. REL. INT., relative intensity.

100

80

z

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20

0

Chart 2. Mass spectrum of the mathylatad and reduced derivative of glycolipid Fraction T7 obtained from a human gastric adenocarcinoma. The conditions ofanalysis ware: electron energy, 64 aV; trap current, 500 pta;acceleration voltage, 4 kV; ion source temperature, 3000; and probe temperature, 2800. Peaks belowm/e 80 were not reproduced. Corresponding formulas for interpretation are shown in Chart 4. REL. INT., relative intensity.

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glycolipids, but the reduced spectrum clearly shows the presence of 2 different glycolipids (see below). Fragments oniginating from the Leb@6glycolioid are seen at m/e 780 (812-32)due to the terminal tetrasacchanide, m/e 1016 and 984(1016-32) due to the pentasacchanide. Hexasacchanide peaks arefound at m/e 1220 and 1188 (1220-32). A loss of fucose (189plus a hydrogen) from the hexose of the pentasacchanide givesm/e 826 (1 01 6-1 90), but fucose elimination from the hexosamine also includes the glycosidic oxygen and a hydrogen (m/e 810, 1016-206). The corresponding fragments for the tetrasacchanide of the Leb@6glycolipid are seen at m/e 622 and606. Terminal saccharides from the B-6 glycolipid are found atm/e 842 and 810 (842-32), m/e 1046 due to the tetra- and

pentasacc@anides, respectively. The sacchanide fragmentsformed from the B-7 glycolipid m/e 984, 1016, 1188, 1220,810, and 826 all coincide with the fragments from the Leb@6glycolipid. Earlier mass spectrometnic studies on pure methylated glycolipids have shown that the fragmentation is favoredat the glycosidic linkages of hexosamine residues in the sacchanidechain, e.g. , m/e 812 (Leb@6)and 842 (B-6). Therefore,the fragments formed by the terminal disaccharide (m/e 393)for the Leb@6and terminal tnisacchanide(m/e 597) for the B-6glycolipid are of low intensity (13, 15). The peaks at m/e 1263and 1375 are due to the ceramide part plus the 2 internalhexoses and the hexosamine as shown for the B-6 glycolipidin Chart 3. The 16:0 hydroxy fatty acid gives m/e 1263, and

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Chart 3. SimplIfied formulas of methylated glycolipids to explain the major fragments of Charts 1 and 5. The molecular species shown contains phytosphingosineand hydroxytatracosanoic acid, which was the heaviest species found.

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902

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Mass Spectrometry of Tumor Glycolipids

the 24:0 hydmoxyfatty acid gives m/e 1375. Evidence for 2hexoses nearest to the cenamide comes from m/e 906(905+ 1) explained in Chart 3. The ceramide part of the glycolipids contained predominantly hydroxy fatty acids with 16to 24 carbon atoms together with tnihydroxy base (phytosphingosine, ti 8:0), see formulas, and dihydnoxy base (sphingosine,dl 8:1). The cemamidespecies with ordinary sphingosine andhydroxyhexadecanoic acid is evident at m/e 578 and 562(578-1 6) and the phytosphingosine and hydroxytetnacosanoicacid species at m/e 722 and 706 (722-1 6). Ions specific forsphingosine at m/e 364 and phytosphingosine at m/e 396 areof relatively low abundance.

The mass spectrum of the methylated and reduced T7 glycolipid fraction is shown in Chart 2, and simplified formulas forinterpretation appear in Chart 4. The reduction (LiAIH4) elimmates the amide oxygens (of the ceramide and amino sugars)giving rise to amines (a loss of 14 mass units for each reductionplace). The reduction has a stabilizing effect on the moleculesand gives prominent peaks in the high mass region containingall sugars and the fatty acid. Usually, molecular ions are alsofound. Three prominent series of peaks (m/e 1533—1645,1563—1675, and 1737—1849) are found in the upper massregion. These are due to a loss of part of the long-chain basefrom the Leb@6,B-6, and B-7 glycolipids, respectively, and giveinformation about the number and type of sugars and semiquantitatively the fatty acid composition (i.e. , m/e 1737 for 16:0, 1821 for 22:0, 1835 for 23:0, and 1849 for 24:0 hydroxyfatty acid of the B-7 glycolipid). The peaks at m/e 1577, 1607,and I 781 are due to loss of the fatty acid (see Chart 4) and areevidence for 2-hydmoxygroups of the fatty acids and for phytosphingosine as the dominating long-chain base. Molecularions for the phytosphingosine and hydnoxytetmacosanoicacidspecies of the Leb@6,B-6, and B-7 glycolipids are seen at m/e1929, 1959, and 2133, respectively. Carbohydrate sequenceions are found at m/e 189 and 157, m/e 798, 814, m/e 1002,1018, and m/e 1206 for the Leb@6glycolipid, at m/e 189, 157,m/e 219, 187, m/e 828, 844, m/e 1032, and m/e 1236 forthe B-6 glycolipid and at m/e 189, 157, m/e 219, 187, m/e1002, 1018, m/e 1206, and m/e 1410, 1426, for the B-7glycolipid. The 2 series of peaks at m/e 532-644 and m/e736-848 show the 2 sugars nearest the cemamidepart to be 2hexoses and evidence that these hexoses are not substitutedwith fucose as indicated in Chart 4. The peak at m/e I 253 isthe corresponding peak of m/e 848 with an additional hexosamine and fucose originating from the Leb@6and B-7 glycolipids. The low intensity of this peak compared to m/e 644 and648 explains why the series of peaks due to the fatty acidcomposition (m/e 1141-1253) is overshadowed by the otherfragments in this mass region. The intensity of series of peakscontaining the carbohydrate and fatty acid at m/e 1533—1645,1563—1675, and 1737—1849 roughly reflects the amount ofeach glycolipid species found in the sample mixture. This is inagreement with the thin-layer chmomatogmam(Fig. 1, Lane T7)where it has been shown by references that the upper bandcontains the Leb@6and B-6 glycolipids and the lower bandcontains the B-7 glycolipid.

In addition to these 3 major glycolipids, the T7 fraction alsocontains small amounts amounts of a Blood Group H-likepentaglycosylceramide with one fucose, 3 hexoses, and onehexosamine. The carbohydrate and fatty acid fragments forthis glycolipid are seen in Chart 2 at m/e 1359 for the 16:0

and m/e 1471 for the 24:0 hydroxy fatty acid homologs,respectively.

Spectra of Tumor Fraction T8. The mass spectra of themethylated (Chart 5) and methylated-reduced (Chart 6) T8glycolipid fraction show it to be a mixture of 7 different glycolipids. The simplified formulas for interpretation are given inCharts 3 and 4, respectively. The mass spectrum of the methylated derivative (Chart 5) shows terminal carbohydrate fragments from 3 major glycolipids B-7, H-7, and Leb_8shown inChart 3. The fragments from the B-7 glycolipid at m/e 189,157, 219, 187, 810, 826, 1016, 984, 1220, and 1188 have allbeen discussed above. The terminal tnisacchanide of the H-7glycolipid gives fragments at m/e 638 and 606. The tetrasacchanide is found at m/e 842 and 810, the pentasacchanide isat m/e 1087 and 1055, and the hexasacchanide is at m/e1291 and 1259 (1291-32). The corresponding fragments forthe Leb@8glycolipid are found at m/e 780 (tetrasacchanide),m/e 101 6 and 984 (pentasacchanide), and m/e 1261 and1229 (hexasacchanide). The tetnasacchamidefragment at m/e812 is covered by the isotope peaks of m/e 810. The possibilitythat m/e 780 originates from loss of the terminal hexose of thepentasacchanide of the B-7 glycolipid (1016-21 9-16-1) is unlikely because this fragmentation is not present in the massspectrum of a pure B-7 glycolipid.4 Rearrangement ions due toloss of fucose from the hexose (loss of 190 mass units) or thehexosamine (loss of 206 mass units) of the tetma-, penta-,hexa-, and heptasacchanide give the peaks at m/e 622 and606, m/e 826 and 810, m/e 1055 and 1071 , and m/e 1275and 1259. Carbohydrate peaks originating from a Blood GroupA-similar heptaglycosylcenamide (A-7) are also found and willbe discussed below.

The cenamide composition is identical to that found in Fraction T7 with ordinary sphingosine (m/e 364) and phytosphingosine (m/e 396) in combination with hydnoxy fatty acids with16 to 24 carbon atoms (m/e 578 to 722).

The mass spectrum of the reduced Fraction T8 shows ahighly complex glycolipid mixture (Chart 6). By recording spectra at 2 different probe temperatures (290°and 310°),morestructural information could be obtained because the smallestglycolipid (6 sugars) evaporates at a lower probe temperaturethan the largest one (9 sugars). The 3 major glycolipids in thismixture are B-7, H-7, and Leb@8,which is shown by the intenseseries of peaks at m/e 1737—1849 (B-7), m/e 1794.-i 906 (H-7), and m/e 1968—2080(Leb@8)due to all sugars and the 16:0-24:0 hydnoxy fatty acids. The corresponding fragment, dueto loss of the fatty acid instead of the long-chain base, is foundat m/e 1781 (B-7), 1838 (H-7), and 201 2 (Leb@8).Carbohydrate sequence fragments are seen at m/e 189, 157, m/e219, 187, m/e 1002, 1018, m/e 1206, and m/e 1410, 1426for the B-7 glycolipid, at m/e 189, 157, m/e 624, 640, m/e828, 844, m/e 1059, m/e 1263 and m/e 1467, 1483, for theH-7 glycolipid and at m/e I 89, 157, m/e 798, 814, m/e 1002,1018, m/e 1233, m/e 1437, and m/e 1641 , 1657 for the Leb@8 glycolipid. The series of rearrangement ions (see below,bottom formula of Chart 4) at m/e 532-644 and 736—848areevidence for 2 hexoses next to ceramide. The correspondingfragment with an additional hexosamine for the hydmoxytetra

4 M. E. Brelmer, K-A. Karlsson, and B. E. Samuelsson. Characterization of a

human intestinal difucosyl glycolipid with a blood group B determinant and a type1 carbohydrate chain, submitted for publication.

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88[lolxo.4@—X20 r—@XlOO@—X2S0X07 II

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Chart 5. Mass spectrum of the mathylated derivative of glycolipid Fraction T8obtained from a human gastric adanocarcinoma. The conditions of analysis ware:electron energy, 65 aV; trap currant, 500 ,@a;acceleration voltage, 6 kV; ion source temperature, 300°;and probe temperature, 2800. Peaks below m/e 80 ware notreproduced. Corresponding formulas for interpretation are shown in Chart 3. REL. INT., relative intensity.

100

80

I-

@ 60

-JuJ 40

20

DO

16411611 1 1657

1437 1483t\@@ 467

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00

100

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@ 60

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1400 1SOO •1600@ 1700 •1800@ 1900@ 2000 2100 2200@

1252+1 1849 r—xSOO1206 1253 1426 1467 1737 1781

f@126@ 16@5 @64(@@ ( / ( 2012 1 2142

11876 19683 1 1437)1493 1675 20801906

@@ @A@ ‘@ \@ 2@S4

1@200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

rn/c

Chart 6. Mass spectrum of the methylated-reduced derivative of glycolipid Fraction T8obtained from a human gastric adanocarcinoma. The conditions of analysiswere: electron energy, 64 aV; trap currant, 500 @a:acceleration voltage, 3.5 kV; ion source temperature, 310°;and probe temperature, 290°.The inserted spectrumin the region m/e 1400—2400was recorded at a probe temperature of 310O.Peaks below m/e 80 ware not reproduced. Corresponding formulas for interpretationare shown in Chart 4. REL. INT., ralativa intensity.

cosanoic fatty acid is seen at m/e 1079. The sequence fragment at m/e 1253 has been explained above.

In addition to the major glycolipid species discussed, smallamounts of the Leb@6and B-6 glycolipids are seen at m/e 1645and 1675 for the hydroxytetracosanoic acid species. Theirlower fatty acid homologues are seen in a spectrum recordedat a slightly lower temperature (not reproduced).

Evidence for a previously unknown glycolipid with 9 sugars(3 fucoses, 4 hexoses, 2 hexosamines, phytosphingosine, and16:0-24:0 hydroxy fatty acids) is found by fragments at m/e2142 (16:0) and 2254 (24:0) due to all sugars and the fattyacid. The fragments from loss of the fatty acid instead of thelong-chain base are seen at m/e 2186. No further conclusions

about the structure of this glycolipid can be drawn because ofthe small amount of substance in the sample mixture; i.e. , weakions are overshadowed by the major glycolipid components.The presence of oligosacchanide chains with 3 fucoses hasbeen established in human milk (35).

The finding of a highly complex mixture of glycolipids inFraction T8 is in agreement with the thin-layer chromatogram(Fig. 1, Lane T8)where 3 major bands are seen in the 7- to 8-sugar region together with weak bands in the 6-sugar regioncorresponding to the small amounts of Leb@6and B-6 found inthe spectra (compare Fraction T7).

Identification of a Blood Group A-similar Heptaglycosylceramide. Some years ago, we identified by mass spectrom

904 CANCERRESEARCHVOL. 40

842-32 1016-190 1016-32 1261-2061016- 206________________7-32—810 826 984 ic@0@5 I

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Mass Spectrometry of Tumor G!yco!ipids

etry a novel heptaglycosylceramide from dog intestine, humanpancreas, and small intestine (32). This glycolipid had a stnuctumesimilar to the common Blood Group A-active hexaglycosylceramide but with an additional fucose bound to the internalhexosamine (compare the B-6 and B-7 glycolipids in Chart 3).The schematic structure of this glycolipid is shown in its methylated and methylated-meducedform in Chart 7 together withthe major mass spectmometricfragments. From the immunological studies, one may expect Blood Group A-like carbohydratefragments to be present in the mass spectrum of the methylatedFraction T8 (Chart 5). The problem is that several of the fragments formed by the A-7 glycolipid can also originate fromother glycolipids in the sample mixture, predominantly from theH-7 and Leb@8glycolipids. This is shown in Table 2 where thefragments specific for the A-7 glycolipid have been boxed.Another problem in the identification is that the A-7 specificfragments can be overshadowed by the isotope peaks fromother fragments. For example, the fragment at m/e 1057, dueto the pentasacchanide from the A-7 glycolipid, is covered bythe isotope peak of the prominent peak at m/e 1055 arisingfrom the H-7 and Leb@8glycolipids (Chart 5). Nevertheless,there are fragments which can only be formed by the A-7glycolipid and are not masked by other more intense fragments.In the methylated sample (Chart 5), the peak at m/e 851 isdue to loss of the fucose from the hexosamine of the pentasaccharide (1057—206)and the peak at m/e 1025 is due toloss of methanol from the pentasacchanide (1057—32).These

M —2049 —2161

peaks are prominent in the mass spectrum of a pure A-7glycolipid (32) and cannot originate from the other glycolipidspresent in the sample (Table 2). The fragment originating fromthe terminal hexosamine at m/e 260 is of low intensity, but theions formed by loss of methanol from this terminal sugar arepresent at m/e 228 (260-32) although these ions are notspecific for the A-7 glycolipid (Table 2). In the methylatedreduced derivative (Chart 6), no specific-sequence ions can befound, but the peak at m/e 1764 is due to all the 7 sugars (2fucoses, 3 hexoses, and 2 hexosamines) and the hydroxyhexadecanoic fatty acid. The corresponding fragment for the hydroxytetracosanoic acid homolog is seen at m/e 1876. Theseions can also be formed by the unsaturated 22:1 homolog ofthe H-7 glycolipid (m/e 1906-28-2), but certainly originatepartly from the 24:0 species of the A-7 glycolipid, because it israre that the unsaturated fatty acid species are more prominentthan the saturated ones (see, i.e. , the corresponding fragmentsof the Leb@6,B-6, and B-7 glycolipids in Chart 2).

All glycolipids identified by mass spectrometry in the glycolipid fractions T7 and T8 from a gastric adenocarcinoma of aBlood Group B human individual are summarized in Table 3.

DISCUSSION

In this study, total neutral (nonacidic) glycolipid fractionsfrom a gastric adenocarcinoma and the surrounding normalgastric tissue of a Blood Group B human individual have been

CII I

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Chart 7. SimplIfied formulas of the mathylated (above) and methylated-reduced (below) derivative of the A-7 glycolipid found in the mass spectra (Charts 5 and6) of the T8glycollpld fraction isolated from a gastric adanocarcinoma of a Blood Group B human individual.

M = 2203

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Carbohydrate fragment (m/e)A-7Otherpossible origin of

the carbohydratefragment189,157x8x(B-7)228xx

(H-7)260111606

(638-32)xx(H-7)638xx(H-7)851(1057-206)867(1057-190)1025(1057-32)1055(1261-206)xx(Lab@8)1057E@J1071

(1261—190)xx(Leb@8)1229(1261—32)xx(Leb@8)1259(1465—206)xx(Leb@8)1261xx(Lab@8)1291xx(H-7)1465xx(Leb@8)aFor

abbreviations used, see Charts 3 and 4.

GlycolipidfractionaT7T8(H_5)a.

bB-6(B-6)Leb@6(Le@'-6)B-7B-7

(A-7)H-7Lab@8(Unknown

-9)

M. E. Breimer

Table2

Mass spectrometric fragments formed from the mathylated A-7 glycolipidThe boxed fragments are specific for the A-7 glycolipid. All other fragments

could be formed from other glycolipids present in the sample mixture. CompareCharts 3 and 7.

react with anti-Fonssman antisera which are known to crossreact with ordinary Blood Group A antigen (11). HetemologBlood Group A activity has been found in gastric (5, 6) andcolonic adenocarcinoma (5) by histoimmunological methods,and a glycolipid nature of these antigens has been proposedon the basis of their ethanol solubility (5). The existence of thisBlood Group A-similar glycolipid, having a terminal hexosamineinstead of hexose (B-7) in a tumor from a Blood Group B humanpoints to a changed genetic function in the tumor cells compared to the normal ones. Nothing can yet be postulatedconcerning the level at which this is done.

The first structural identity of a “tumorspecific' ‘antigen wasobtained in 1972 when a pentaglycosylceramide with serological Forssman activity was isolated from a human biliamytractcarcinoma and characterized (17). This finding was recentlyconfirmed for gastric adenocancinoma (11). The Forssmanantigen has a terminal a-N-acetylgalactosamine which probablyexplains the immunological cross-reaction with anti-A antisera.The possibility that the Blood Group A activity found by Häkkinen (6) and Denk et a!. (5) is due to the Forssman antigenhas been proposed by Hakomoni et a!. (11). It is a well-knownstatistical fact that gastric cancer is more common amongBlood Group A individuals compared to Blood Group 0 on Bindividuals (8). This can be explained by a stronger immunological defense reaction among the Blood Group 0 or B mdividuals against these non-self Blood Group A-similar antigens,which are regarded more as self-antigens by the immunesystem of the Blood Group A individuals.

A highly interesting finding of incompatible blood groupantigens in cancer has been reported by Levine (23, 24) forthe blood group P system. He describes the identification ofthe P1 antigen, shown to be a pentaglycosylceramide (28),from a gastric cancer of a woman with the rare genotype ppwho lacks the P1 antigen. An involuntary immunotherapy testwas done by a blood transfusion of 25 ml of incompatibleblood, increasing henanti-P1 titer from 1:4 to 1:512. This hightiter could well be related to the factthat the 66-year-old patientsurvived for another 22 years and died from natural causes,with no clinical evidence of metastases (23). The possibility ofa specific immunotherapy for adenocancinoma with anti-A andanti-P1 for prevention of metastases has been outlined byLevine (25).

The loss of isolog blood group activity in the ABH system indifferent tumors has been reported (3, 4, 20, 30), and this wascorrelated to the grade of malignancy (4, 20) and postulated tobe a help in early diagnosis and prognosis of carcinoma (4). In

Table 3Glycosphingolipids found by mass spectrometry in tumor glycolipid fractions 7'@

and T8isolated from a gastric adenocarcinoma of a Blood Group B humanindividual

isolated. Each glycolipid mixture has been separated into 10partly purified fractions by silicic acid chromatography. Thefractions were analyzed by thin-layer chromatography (Fig. 1)and tested for immunological activity (Table 1). Two of thetumor fractions (T7 and T8) reacted with some commercialhuman anti-A antisera. These fractions were analyzed by massspectrometry as their methylated and methylated-meduceddenivatives. Altogether, 5 major and 3 minor glycolipids wereidentified (Table 3) using less than 500 @gof each glycolipidfraction. The tumor Fraction T8 which had the strongest neaction with anti-A antisera (Table 1) was shown by mass spectrometry to contain small amounts of a Blood Group A-similarheptaglycosylcenamide. When we tried to repeat the immunological reaction with other batches of commercial anti-A antisera, no activity was found.

We have isolated 2 Blood Group A-similar heptaglycosylceramides from dog and human small intestine, and determinedtheir chemical structures.5 The two isomers are shown in Chart8 and are based on a type 1 (human) and a type 2 (dog)sacchanide chain, respectively. They do not react with commercial anti-A or anti@Lebantisera. (Unfortunately, the batch ofanti-A antiserum that reacted with the tumor glycolipid fractionwas not tested on these 2 glycolipids.) The type I isomer is thereceptor for the Siedlen antibody (results obtained in collabonationwith C. A. Tilley and M. C. Crookston), which reacts withhuman A1, Le(a—b+) ABC but not with cells containing the Aon Le'@antigen alone (29). It would be of some interest to testthis antibody on tumor Fraction T8, although we do not knowthe chain type of the proposed glycolipid.

The explanation of the finding that the tumor glycolipidfractions did react with some anti-A antisera is that theseantisera also contained a population of antibodies directedagainst the A-7 antigen by an additional fucose at the glucosamine. This may also explain why the tumor fraction did not

aM. E. Breimer, K-A. Karlsson, J. M. McKibbin, and B. E. Samuelsson.Characterization of intestinal difucosyl glycolipids based on type 1 and 2 carbohydrate chains with blood group A determinants, manuscript in preparation.

6 For abbreviations used, see Fig. 1 and Charts 3 and 4.

b Designations in parentheses, minor components.

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Mass Spectrometry of Tumor G!yco!ipids

GalNAca1-..@3GaII@1-.-3GIcNAcIi1 .@3GalII1 -ø'4GIC@1-'@1CERAMIDE

IFuc

Type 1 Human

1Fuc

GalNAcul .@3GaII@1 .@4GIcNAcfl1 ‘3GalI@1.@4GIcI11 .@1CERAMlDE

Type 2 Dog

‘a

1 1Fuc Fuc

Chart 8. Sfructures of A-similar glycolipids isolated from human and dog small intestine. The A-7 glycolipid (Chart 7) detected in the tumor may be identical withone or both of these glycolipids.

this case, we have shown by chemical and immunologicalmethods that the isolog Blood Group B antigen was present inthe tumor which had a high malignancy potential with a clinically fatal outcome. One major B-similar glycolipid was a difucosyl compound (B-7) not reacting with ordinary anti-B or antiLeb antisera (this has been shown for a pure B-7 glycolipidbased on a type 1 sacchanide chain isolated from human smallintestine4). This is analogous to the A-7 glycolipid discussedabove. Therefore, one should interpret immunological data withcare, especially when using mixtures of antibodies. The availability of well-defined synthetic antigens in the ABH, I, Lewis,and P systems no doubt opens up new possibilities for immunological studies (21 , 22).

Only 2 of all fractions obtained (Fig. 1) were chemicallycharacterized, and these were chosen on the basis of BloodGroup A activity. However, other differences between tumorand normal tissue may be found in the chnomatognam(Fig. 1).As demonstrated for other tumor tissues (8), more complexstructures may be diminished and precursor material be accumulated. In agreement with this, the tumor fraction (T) showsa major band in the 5-sugar region, which may be H-5, aprobable precursor of B-6 and B-7. Furthermore, a narrowband in the 3-sugar region, practically absent in the normaltissue, maybe lacto-N-tniaosylceramide, earlier found in humanmalignant melanoma (14) and in small amounts in humanerythrocytes (1, 14). This glycolipid is a partial structure of allblood group glycolipids and differs from the major glycolipids(double bands) with 3 and 4 sugars (Fig. 1), which probablybelong to the globo series. However, against the rule (8), slowmoving, more complex material (more than 8 sugars) is relatively more abundant in the tumor than in normal tissue (Fig. 1;Table 1).

Although mass spectrometry is not able to discriminate stereoisomers (e.g. , glucose and galactose, a and fi isomers) theexistence of an A-similar heptaglycosylceramide in Fraction T8is probable, due to the specificity of spectral information anduse of 2 derivatives (13, 15). The possibility for the presence

of an alternative glycolipid structure (isomenic) giving rise tothe sequence ions found for the methylated derivative, must ofcourse be kept in mind, but a further characterization of thiscomponent by isolation and degradation is practically impossible on a 3.1-mg fraction where it occupies only a few % ofthe total. This demonstrates the potency of mass spectrometryfor collection of important chemical data in a primary stage ofinvestigation. No other method presently available is capableof this. This kind of fingerprinting of antigenic determinants incombination with immunological analysis will be of help instudies on surface antigens in cancerous diseases.

ACKNOWLEDGMENTS

The author is indebted to I. Pascher for chemical darivatizations and to A.Pakvis for help with illustrations.

REFERENCES

1. Ando, S., Kon, K., Isobe, M., Nagai, Y., and Yamakawa, T. Existence ofglucosaminyl lactosyl ceramide (amino CTH-l) in human arythrocyte mambranas as a possible precursor of blood group-active glycolipids. J. Biochem., 79: 625—632,1976.

2. Breimer, M. E., Hansson, G. C., Karlsson, K. A., Lafflar, H., Pimlott, W., andSamualsson, B. E. Selected ion monitoring of glycosphingolipid mixtures.Identification of several blood group type glycolipids In the small intestine ofan individual rabbit. Biomad. Mass Spectrom., 6: 231—241, 1979.

3. Davidsohn, I., and Ni, L. V. Loss of isoantigans A, B and H in carcinoma ofthe lung. Am. J. Pathol., 57: 307-334, 1969.

4. Davidsohn, I., Ni, L. V., and Stajskal, R. Tissue isoantigens A, B, and H incarcinoma of the stomach. Arch. Pathol.. 92: 456—464,1971.

5. Dank, H., Tappeinar, G., Davidovitz, A., Eckerstorfer, R., and Holznar, J. H.Carcinoembryonic antigen and blood group substances in carcinomas of thestomach and colon. J. Natl. Cancer Inst., 53: 933—942,1974.

6. Häkkinen,I. A-like blood group antigen In gastric cancer calls of patients inBlood Groups 0 or B. J. NatI. Cancer Inst., 44: 1183—1193, 1970.

7. Hakomori, S-I. A rapid permethylation of glycolipid and polysaccharidecatalyzed by mathylsulfinyl carbanion in dimethyl sulfoxide. J. Biochem., 55:205-208, 1964.

8. Hakomori, S-I. Glycolipids of tumor cell membrane. Adv. Cancer Res.. 18:265—315,1973.

9. Hakomori, S-I., and Andraws, H. D. Sphingoglycolipids with Labactivity andthe co-presence of Lee-, Leb@glycolipidsin human tumor tissue. Biochim.Biophys. Acta, 202: 225—228,1970.

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M. E. Breimer

10. Hakomori, S-I., Koscielak, J., Bloch, K. J., and Jaanloz, R.W. Immunologicalrelationship between blood group substances and a fucose-containing glycolipid of human adenocarcinoma. J. Immunol., 98: 31—38,1967.

11. Hakomori, S-I., Wang, S.-M., and Young, W. W. Isoantigenic expression ofForssman glycolipid in human gastric and colonic mucosa: its possibleidentity with ‘‘A-likeantigen' ‘in human cancer. Proc. NatI. Acad. Sci. u. S.A., 74: 3023—3027,1977.

12. Karlsson, K-A. Carbohydrate composition and sequence analysis of aderivative of brain disialogangliosida by mass spactrometry, with molecularweight ions at m/e 2245. Potential use in the specific microanalysis of cellsurface components. Biochemistry, 13: 3643—3647,1974.

13. Karlsson, K-A. Microscale fingerprinting of blood-group fucolipids by massspactromatry. In: L. A. Witting (ad.), Glycolipid Methodology, pp. 97—122.Champaign, Ill.: American Oil Chemists' Society, 1976.

14. Karlsson, K-A. Aspects on structure and function of sphingolipids in cellsurface membranes. In: S. Abrahamsson and I. Pascher (eds.), Structure ofBiological Membranes, pp. 245—274.New York: Plenum Press, 1977.

15. Karlsson, K-A. Mass spactrometric sequence studies of lipid-linked oligosaccharides. Blood group fucolipids, gangliosidas and related call surfacereceptors. Prog. Cham. Fats Other Lipids, 16: 207—230,1977.

16. Karlsson, K-A., Laffler, H., and Samualsson, B. E. Characterization of thaForssman glycolipid hapten of horse kidney by mass spectrometry. J. Biol.Cham., 249: 4819-4823, 1974.

17. Kawanami, J. The appearance of Forssman haptan in human tumor. J.Biocham., 72: 783—785,1972.

18. Kim, Y. S., Isaacs, R., and Pardomo, J. M. Alteration of membrane glycopaptidas in human colonic adenocarcinoma. Proc. NatI. Acad. Sci. U. S. A.,71: 4869-4873, 1974.

19. Kobata, A. Milk glycoprotains and oligosaccharidas. In: M. J. Horowitz andW. Pigman (ads.), The Glycoconjugatas, Vol. 1, pp. 423—440.New York:Academic Press, Inc., 1977.

20. Lange, P. H., Limas, C., and Fraley, E. E. Tissue blood group antigens andprognosis in low stage transitional call carcinoma of the bladder. J. Urol.,119:52—55,1978.

21. Lamiaux, R. Ii. The chemical synthesis of oligosaccharide haptens to achieveimmunoadsorbentsand antibodies possessing humanblood group activities.In: XVIICongress of the International Society of Hematology, Paris, AbstractBook, p. 924. Paris: Librairie Arnatte, 1978.

22. Lemieux, R. U., and Driguez, H. The chemical synthesis of 2-O-(a-D-galactopyranosyl)-D-galactose. The terminal structure of the Blood Group Bantiganic determinant. J. Am. Cham. Soc., 97: 4069—4075,1975.

23. Levine, P. Illegitimate blood group antigens P,, A, and MN (1) in malignancy—apossible therapeutic approach with anti@Tja,Anti-A, and Anti-T.Ann. N. V. Acad. Sci., 277: 428—435,1976.

24. Levine, P. Blood group antigens in adanocarcinoma foreign to the host.Prog. Cancer Res. Ther., 5: 69—73,1977.

25. Levine, P. Specific immunotherapy for adenocarcinoma with anti-A and antiP1for prevention of metastasis. Prog. Cancer Res. Ther. 5: 75—79,1977.

26. Lundblad, A. Urinary glycoprotains, glycopeptidas, and oligosaccharides.In: M. J. Horowitz and W. Pigman (ads.), The Glycoconjugatas, Vol. 1, pp.441—458.New York: Academic Press, Inc., 1977.

27. McKibbin, J. M. Fucolipids. J. Lipid Ras., 19: 131—147,1978.28. Naiki, M., and Marcus, D. M. An immunological study of the human blood

group P,. P. and P1'glycosphingolipid antigens. Biochemistry, 14: 4837—4841, 1975.

29. Seaman, M. J., Chalmers, D. G., and Franks, D. Siedler, an antibody whichreacts with A,Le(a—b+)red cells. Vox Sang., 15: 25—30,1968.

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908 CANCERRESEARCHVOL. 40

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