Proc. Natl. Acad. Sci. USA Vol. 74, No. 7, pp. 3023-3027, July 1977 Medical Sciences

Isoantigenic expression of Forssman in human gastric and colonic mucosa: Its possible identity with "A-like antigen" in human cancer (Forssman-positive and Forssman-negative populations/tumor/globoside/ group) S. HAKOMORI, S.-M. WANG, AND W. W. YOUNG, JR. Division of Biochemical Oncology, Fred Hutchinson Cancer Research Center; and Departments of Pathobiology and Microbiology, University of Washington, Seattle, Washington 98104 Communicated by Philip Levine, May 4, 1977

ABSTRACT The heterogenetic Forssman antigen is a gly- and blood group A antigens (5, 6), the A-like activity observed cosphingolipid, a pentasaccharide with the structure in those studies could have been due to the presence of a GalNAcal-s-3GalNAc,1-a3Galal---4Gal,1--.4Glc-.ceramide. Forssman antigen. In fact, Kawanami (7) described the presence Forssman-positive animals are capable of synthesizing this compound in tissues or in erythrocytes, in contrast to the of Forssman glycolipid in one case of gastric cancer although Forssman-negative species, including humans, which are in- the glycolipid composition of normal mucosa was not examined. capable of adding the last carbohydrate in the sequence of the Consequently, a thorough study has been undertaken using the Forssman antigen, namely aGalNAc. The Forssman glycolipid surgical samples collected by one of the authors (S.M.W.) in and its precursor globoside were examined in twenty-one sam- Taiwan, where there is a high incidence of gastric cancer. ples of surgically extirpated gastrointestinal mucosa and tumors derived therefrom. The results revealed that a few patients had The heterogenetic Forssman antigen (8) is a glycosphingo- chemically and immunologically detectable levels of the (9, 10) whose structure was identified as GalN- Forssman glycolipid as a normal component of their gastroin- Acal1-3GalNAc/ll-3Gala1-'"4Galfll1-'4Glc--ceramide (11, testinal mucosa (F+ population); in contrast, the majority of 12). Forssman-positive animals, irrespective of species, are patients did not contain this glycolipid in their normal mucosa characterized by having a glycolipid of this structure (13-16),* (F- population). Whereas the F- population included blood whereas Forssman-negative animals, including humans,t are groups A, B, and 0, the F+ population did not correspond to bloo group A. The Forssman status in tumors taken from the considered to be unable to complete the carbohydrate structure, F+ or F- population showed the following striking features: (i) although they can synthesize the immediate precursor of the all tumors derived from F- mucosa possessed Forssman gly- Forssman antigen, which has been identified as globoside colipid, whereas (ii) none of the tumors originating in F+ mu- (11). cosa contained Forssman glycolipid. Globoside, the immediate precursor of Forssman antigen, was distributed equally among F+ and F- mucosa and the tumors derived therefrom. Thus, the MATERIALS AND METHODS expression of Forssman antigen in gastrointestinal mucosa ap- Tissues. Normal mucosa and cancer tissues from gastric and pears akin to that of an isoantigen. Furthermore, the Forssman antigen that appears in tumors of the F- population could rep- colonic mucosa were obtained from surgically extirpated resent a human tumor-associated antigen. In view of the strong specimens obtained in the National Taiwan University Hospital crossreactivity of Forssman antigen with blood group A deter- and its associated hospitals. All cases were diagnosed by histo- minants, the appearance of Forssman antigen in human tumors logical examination, and the ABO blood group of patients was could be related to the "A-like antigen" (or "neo-A antigen") of determined by standard procedures. Immediately after the human tumors reported previously [Hakomori, S., Koscielak, specimens were extirpated, the tissues were cleaned by rinsing J., Black, K. J. & Jeanloz, R. W. (1967) J. Immunol. 98, 31-38; with saline and frozen in dry ice and stored at -70°. The Hakkinen, I. (1970) J. Nat]. Cancer Inst. 44, 1183-11931. samples were shipped to this institute in dry ice. After thawing, The appearance of incompatible blood group antigens foreign the tumor tissues and normal mucosa were carefully separated to the host may occur in some human tumors. In 1951 Levine and weighed. et al. (1, 2) reported a case which suggested the synthesis of P Preparation of Glycolipid Fractions, Including Ceramide and PI antigen in gastric cancer tissue of a rare p individual. Tetra- and Pentasaccharide. The total glycolipid fraction was A possible conversion of blood group 0 or B to an A-like antigen prepared from the weighed tissue by the acetylation procedure in human gastrointestinal tumor was suggested by two inde- ("procedure A") (19), and was further separated into neutral pendent studies: (a) rabbit antiserum directed against a "human and by DEAE-Sephadex A-25 ac- tumor glycolipid" fraction agglutinated blood group A eryth- cording to the procedure of Yu and Ledeen (20). The total rocytes more strongly than 0 or B erythrocytes although the glycolipid was prepared from the tumor of a patient of blood * The immunodeterminant of Forssman antigen must reside on the group 0 (ref. 3; see also Discussion in the present paper); and terminal disaccharide GaINAcal -c3GalNAcB1 - R, because a (b) Hakkinen (4) described the presence of A-like antigen in ceramide tetrasaccharide with GalNAcal - 3GaINAcfil - 3Galal stomach cancers from patients whose blood group status was 4Galf,1 - ceramide of hamster fibroblast is equally as active as group 0 or B. The active fraction was described as a the ceramide pentasaccharide (17), and a polysaccharide of Strep- "sulfo- tococcus type C containing the terminal disaccharide is Forssman- glycoprotein." active (18). In view of the extensive crossreaction between the Forssman t In the early literature, reviewed by Buchbinder in 1935 (6), most primates were Forssman-negative in erythrocytes and tissues. Abbreviations: TLC, thin-layer chromatography; CTT, ceramide te- However, humans of blood groups A and AB were found by immu- trasaccharide; CP, ceramide pentasaccharide; F+ and F-, positive and nological means to be Forssman-reactive, but these results could have negative for the presence of Forssman antigen. been due to crossreactions with blood group A structures. 3023 Downloaded by guest on October 1, 2021 3024 Medical Sciences: Hakomori et al. Froc. Natl. Acad. sci. USA 74 (1977) neutral glycolipid fraction was further separated into various from globoside liposomes (both preparations containing 25 MAg classes of glycolipids by a small column (15 X 0.8 cm) of porous of glycolipid pert mol of ). In contrast, rabbit silica gel (latrobeads, latron Co., Tokyo) eluted with a chloro- anti-sheep hemolysin reacts more extensively with globoside form/methanol/water system of increasing polarity (21). liposomes (27). The liposomes prepared for the lysis assay were Ceramide tetrasaccharides were mainly eluted with chloro- used also for inhibition of sheep erythrocyte hemolysis by form/methanol/water 70:30:2.5 (vol/vol) (fraction 5) and the anti-Forssman antibody. Twenty-five microliters of each ceramide pentasaccharides, with chloroform/methanol/water liposome suspension were diluted in a 2-fold series in a micro- 66:34:3.0 (fraction 6). Because these fractions slightly over- titer plate. The liposomes were incubated with 25 Ml of three lapped, aliquots of fraction 5 and 6 were combined for immu- hemolysis doses (about 1:500 dilution) of the anti-Forssman nological assay, but chemical analysis was carried out on sep- antiserum for 1 hr. Then the mixture was incubated with 25 Ml arate fractions. each of complement (1:60 diluted guinea pig serum) and 1% Separation of Forssman Glycolipid and Its Identification. sheep erythrocytes. The extent of hemolysis was determined Because the iorssman glycolipid in the free state was not sep- after 1 hr. arable by thin-layer chromatography from the other ceramide pentasaccharides of fraction 6, the fraction was acetylated in RESULTS pyridine/acetic anhydride. Excellent separation of the acety- lated Forssman glycolipid was accomplished on TLC using Isoantigenic expression of Forssman glycolipid in solvent 1,2-dichloroethane/acetone (55:45, vol/vol). Under gastric and colonic mucosa: Distinction of F+ and F- these conditions the other ceramide pentasaccharides ran much mucosa faster than Forssman glycolipid (see Fig. 1). Following this The results of hemolysis inhibition and liposome lysis assays of purification step, the Forssman glycolipid was deacetylated. the ceramide tetra- and pentasaccharide fractions of 21 samples An aliquot was degraded by a-N-acetylgalactosaminidase of of normal mucosae indicated a clear-cut distinction of two pig liver (22) according to the procedure previously described human populations: one group showed positive Forssman ac- (9). Another portion was methylated (23) and identified by tivity while the other lacked Forssman activity (designated F+ electron impact mass fragmentography (16) using a Finnigan and F-, respectively; Tables 1 and 2). Sixteen of the twenty-one 3300 mass spectrometer. patients were F-; this group encompassed all ABO blood Immunological Analysis. Preliminary analysis of the total groups, while four of the five F+ patients were from blood glycolipid fraction failed to demonstrate any Forssman reac- group 0. Because samples of stomach as well as colonic mucosa tivity. For this reason the immunological assays described below were represented in both F+ and F- populations, no correlation were run on the purified'ceramide tetra- and pentasaccharide could be made between the F- activity and the location of the fractions. Anti-Forssmnan and anti-globoside sera were prepared mucosa. All normal mucosa showed strong reactivity with an- by injecting rabbits with mixtures of the purified glycolipid and tigloboside in the liposome lysis assay. bovine serum albumin emulsified in complete Freund's adju- In agreement with the results of immunological reactivity, vant as previously described (24). Antibodies against the albu- the consistent presence of Forssman glycolipid was chemically min were removed from the sera by passage through a bovine demonstrated in F+ mucosae when the ceramide pentasac- serum albumin-Sepharose column (25). The titer of anti- charide fractions were acetylated-and separated on thin- Forssman serum for hemolysis of sheep erythrocytes was about layer chromatography (TLC) (Fig. 1). In striking contrast, none 1:2500, while the titer of antigloboside serum for hemaggluti- of the F- population yielded a detectable Forssman glycolipid nation of trypsinized human erythrocytes was 1:128 to 1: spot. It should be noted that in the free state Forssman gly- 256. colipid overlapped with the TLC spots of other ceramide The quantity of ceramide tetra- and pentasaccharide frac- pentasaccharides, thus preventing assessment of its presence. tions to be tested was based on the dry weight of the tissue res- The remaining ceramide tetra- and pentasaccharides of frac- idue remaining after extraction of glycolipids with chloro- tions 5 and 6 have not been characterized chemically, but form/methanol. The aliquots of fraction 5 and fraction 6 gly- preliminary results suggest the presence of globoside, para- colipids corresponding to 50 mg of the dry tissue residue were globoside, galactosylparagloboside, and several other complex mixed with 100 Ail of 5 mM sphingomyelin, 10 Ml of 50 mM glycolipids containing fucose. and 15 of 3 mM solutions in chdlesterol, ,l dicetylphosphate Forssman glycolipid antigen in tumors derived from chloroform/methanol (2:1, vol/vol). Multi-compartment li- F- and F+ mucosa posomes were prepared from these lipid mixtures and the final volume of the liposome suspensiot was adjusted to 250ul. These Determination of Forssman activity by both hemolysis inhi- liposomes were tested both for their sensitivity to antibody- bition and liposome lysis assays revealed that all tumors derived complement lysis by a slight modification of the method of Six from F- mucosae contained significant levels of Forssman re- et al. (26), and for hemolysis inhibition. The liposome lysis assay activity (Table '1). In striking contrast, the.ceramide tetra- to detects release of a fluorogenic substrate (4-methylumbellif- pentasaccharide fractions of tumors derived from F+ mucosa erone phosphate) from the liposomal aqueous compartments did not express Forssman activity (Table 2). All tumor fractions and its subsequent conversion by alkaline phosphatase to an from both F+ and F- cases possessed strong globoside reactiv- intense fluorophore. Each assay tube contained 950 Al of buffer ity. (20 mM Tris-HCI, pH 8.0/150 mM NaGl), 5 ul of alkaline The chemical analysis of acetylated glycolipid fractions on phosphatase (2.5 mg/ml), 10 Ml of a 1:10 dilution of either TLC agreed with the immunological data; all the tumors de- anti-Forssman or antigloboside serum, and 25 ,l of guinea pig rived from F- mucosa contained chemically detectable serum as the complement source. The reaction was started by Forssman glycolipid, whereas the tumors derived from F+ the addition of 10 Ml of liposomes (2 mM sphingomyelin). In mucosa did not contain Forssman glycolipid. Some examples control experiments the anti-Forssman serum was found to be are shown in Fig. 1. Several other acetylated glycolipid bands specific for Forssman liposomes, causing the release of 53% of were present in the ceramide tetra- and pentasaccharide the trapped marker from FQrssman liposomes but only 0.4% fractions. When these materials were purified on TLC as Downloaded by guest on October 1, 2021 Medical Sciences: Hakornori et al. Proc. Nati. Acad. Sci. USA 74 (1977) 3025

Table 1. Immunological activities of ceramide tetra- and pentasaccharide fractions prepared from Forssman-negative (F-) mucosae and tum'ors derived therefrom Normal mucosae Tumors PatientPatient lysist Liposome lysist identification Blood Hemolysis ~~~~~~~~~~~~~Liposorfie Hemolysis no., sex, age Tissue* type inhibitions vs anti-F vs anti-globoside inhibitions vs anti-F vs anti-globoside 019 m 54 Stomach 0 0 1.0 66.9 5+ 32.3 38.5 048 f 52 Colon B 0 0.3 44.6 6+ 30.3 42.3 005 m 54 Colon 0 0 1.0 44.7 10+ 57.1 65.4 021 m 62 Colon B 0 5.6 80.6 10+ 84.7 87.9 026 f 52 Colon 0 0 5.6 84.1 11+ 77.6 79.3 039 f 56 Colon 0 0 4.1 81.5 8+ 58.1 90.7 062 m 61 Stomach B 0 0.5 86.5 6+ 48.8 93.3 069 m 48 Stomach B 0 1.6 73.7 6+ 42.4 63.2 077 m 73 Stomach 0 0 0.5 65.0 8+ 61.3 70.0 068 f 51 Colon B 0 2.8 63.6 3+ 23.0 30.1 078 m 28 Stomach cardia B 0 1.0 39.2 3+ 30.0 40.4 082 f 49 Stomach cardia B 0 3.6 45.1 4+ 37.8 52.4 045 m 35 Colon A 0 2.4 57.8 8+ 71.1 76.0 050 f 60 Stomach (cardia) A 0 2.4 61.3 9+ 70.8 81.8 054 m 48 Colon A 0 3.3 81.3 8+ 72.3 82.6 063 m 56 Gastric (cardia) A 0 0.9 46.0 6+ 36.8 50.0 * All tumors were histologically identified as adenocarcinoma. t Numbers for hemolysis inhibition indicate the number of 2-fold dilution wells (in microtiter plate) that show complete inhibition of hemolysis. 0, no inhibition even at the first well. 5+, the 5th well in 2-fold dilution inhibited the hemolysis. Number for liposome lysis indicates the percent of trapped 4-methylumbelliferone phosphate released in 30 min. In the presence of heated (560, 30 min) instead of native guinea pig serum the percent of trapped marker released was less than 4% in all cases.

acetylated derivatives, then deacetylated, and run on TLC in Forssman glycolipid isolated from goat erythrocytes in the free the free state, significant differences in the glycolipid patterns state and as acetate. (ii) The glycolipid of both normal and of normal mucosa and tumor tissue were apparent. Further tumor tissues was converted to globoside by a-N-acetylgalac- extensive study will be necessary to characterize these gly- tosaminidase. (Ni) The permethylated glycolipid showed a mass colipids. fragment pattern identical to that of the reference Forssman compound as to the major mass peaks derived from oligosac- Quantity of Forssman glycolipids in tumors and in charides, namely mass-to-charge ratio, m/e, 228, 260, 505, and normal mucosa, and failure of demonstrating 709 (16). (iv) The liposomes prepared from the purified gly- Forssman antigen activity with crude glycolipid colipid were lysed by anti-Forssman antibody. The concentration of Forssman glycolipid in tumors of F- mucosa was about 8 jsg/100 mg of dry residue following chlo- DISCUSSION roform/methanol extraction (case 019, in Table 1) and that in The expression of Forssman antigen must depend on the action F+ mucosa was about 5 ,g/100 mg of the mucosal tissue (case of a specific gene for synthesis of a-N-acetylgalactosaminyl- 075, in Table 2). All other cases showed approximately a similar transferase, which is necessary to convert globoside to Forssman concentration of Forssman glycolipid. It is worthwhile to em- glycolipid. In F- individuals, the majority of humans,t this phasize that all the immunological data presented in Table 1 enzyme must be absent (or repressed) and is present (or de- are collected from the purified ceramide tetra- to pentasac- repressed) in tumors. Whether this phenomenon is limited to charide fraction. Without this purification step, Forssman an- gastrointestinal tumors or may be found in other malignancies tigen activity could not be demonstrated with either the he- is not known. Although the synthesis of Forssman glycolipid molysis inhibition or liposome lysis assays. The Forssman gly- occurs in mucosal tissue of F+ individuals, it may not take place colipid may have been masked by other lipid components when in erythrocytes. We have no information at present as to what the activity was determined in the presence of a large amount kinds of tissues and cells are involved in synthesis of Forssman of other glycolipids. This situation was encountered previously glycolipid in F+ individuals, but it is clear that the Forssman with H-activity of H-glycolipid (28) and Ii activity of Ii-active antigen is completely absent in tumors derived from F+ mu- glycolipids (unpublished observation). cosa. This phenomenon is similar to that in hamster NIL cells, Characterization of Forssman glycolipid where the synthesis of Forssman glycolipid was completely The Forssman glycolipid was isolated as acetate by preparative suppressed when the cells were transformed by polyoma virus TLC from normal F+ mucosa and from tumor tissue of F- in- (17, 29). Much work is necessary to clearly define Forssman dividuals, and was characterized by the following criteria: (i) * The incidence of 5 cases of F+ against 16 cases of Fi isoantigen may The Forssman glycolipid of both normal and tumor tissues be characteristic of Chinese in Taiwan, and the incidence in other showed a migration rate on TLC identical to that of the racial groups may well be different. Downloaded by guest on October 1, 2021 3026 Medical Sciences: Hakomori et al. Proc. Natl. Acad. Sci. USA 74 (1977) Table 2. Immunological activities of ceramide tetra- and pentasaccharide fractions prepared from Forssman positive (F+) mucosae and tumors derived therefrom Normal mucosae Tumors

Patient L l L identification Blood Hemolysis Liposome lysis Hemolysis Liposome lysis no., sex, age Tissue type inhibition vs anti-F vs anti-globoside inhibition vs anti-F vs anti-globoside 075 m 51 Colon 0 10+ 33.0 44.3 0 0 39.3 017 f 56 Colon 0 10+ 58.3 64.2 1+ 4.1 76.9 036 m 61 Stomach 0 10+ 69.3 71.5 0 4.8 87.2 091 f 51 Colon 0 11+ 84.0 86.5 0 2.9 76.5 061 m 66 Stomach A 8+ 65.0 68.0 0 4.7 17.9 All tumors were histologically identified as adenocarcinoma; numbers for hemolysis inhibition and liposome lysis have the same meaning as in Table 1. expression in human tissue, but it could be possible that ex- man expression may well depend on the cell type even in F- pression of Forssman is essentially of an "oncofetal" nature (30) individuals, because differences of expression in in F- individuals; namely, its synthesis may be repressed during lymphocyte subpopulations are now known (32). development and de-repressed in tumors. The repression of Whatever the mechanism of Forssman expression might be, Forssman synthesis may not take place with development in the presence of Forssman antigen in tumors of F- individuals some tissues, thus yielding F+ individuals. The "incomplete is of great significance because it could be recognized as a synthesis" model (31) can be applied to explain the deletion of tumor-associated antigen. The apparent conversion of 0 or B Forssman glycolipids in tumors derived from F+ tissue. Forss- antigens to an A-like antigen in tumors of 0 or B individuals

i-,_ I ..; y p I I. Fi

1 3 4 5678 911121 131415 , 1 2 345 6 7 8 9

FIG. 1. Thin-layer chromatograms of the acetylated ceramide tetrasaccharide (CTT) and ceramide pentasaccharide (CP) fraction of normal mucosa and cancer tissues derived therefrom. Chromatograms were developed with 1,2-dichloroethane/acetone (55:45) on silica gel G and stained with orcinol. (Left) Examples of blood group A cases. Lanes 1, 2, 7, and 12 are reference acetylated glycolipids. 1, globoside; 2, Forssman glycolipid; 7 and 12, mixture of globoside and Forssman glycolipid. 3 to 6: case 045 (F- colon mucosa and tumor; see Table 1). 3, CTT of normal colon mucosa; 4, CP of normal colon mucosa; 5, CTT of colon cancer; 6, CP of colon cancer. 8 to 11: case 050 (F- cardia mucosa and cardia tumor, Table 1). 8, CTT of normal cardia mucosa; 9, CP of normal cardia mucosa; 10, CTT of cardia cancer; 11, CP of cardia cancer. 13 to 16: case 061 (F+ gastric mucosa and cancer therefrom; see Table 2). 13, CTT of normal stomach mucosa; 14, CP of normal stomach mucosa; 15, CTT of gastric cancer; 16, CP of gastric cancer. (Right) Examples of blood group 0 cases. 1 and 2: case 019 (F- stomach mucosa and cancer therefrom, see Table 1). 1, CP of normal colon mucosa; 2, CP of colon tumor. 8 and 9: case 075 (F+ colon mucosa and cancer therefrom, see Table 2). 8, CP of normal colon mucosa; 9, CP of colon tumor. Lanes 3 to 7 are reference acetylated glycolipid. 3, galactosylparagloboside; 4, Forssman glycolipid; 5, globoside; 6, paragloboside; 7, a mixture of Lex-glycolipid and Lea-glycolipid. Y indicates a yellow spot which is an unknown, nonglycolipid compound probably derived from the acetylation procedure. (This film is sensitive to yellow and records it markedly.) Downloaded by guest on October 1, 2021 Medical Sciences: Hakomori et al. Proc. Natl. Acad. Sci. USA 74 (1977) 3027

as mentioned in the introduction could well be ascribable to the be hereby marked "advertisement" in accordance with 18 U. S. C. this fact. presence of Forssman antigen in tumors frbm 0, F-- D-i, k §1734 solely to indicate individuals. The crossreactivity of blood group A and F6rssinan 1. Levine, P., Bobbitt, 0. B., Waller, R. K. & Kuhmichel, A. (1951) antigen is well known (5, 6); Forssman glycolipid is capable of Proc. Soc. Exp. Biol. Med. 77,403-405. inhibiting A-hemagglutination, and A-antigen inhibits he- 2. Levine, P. (1976) Ann. N.Y. Acad. Sci. 277,428-435. molysis of sheep erythrocytes by anti-Forssman antibody (our 3. Hakomori, S., Koscielak, J., Bloch, K. J. & Jeanloz, R. W. (1967) own experience). However, further extensive study is necessary J. Immunol. 98,31-38. to identify the A-like (or "neo-A") antigen, occurring in clinical 4. Hakkinen, I. (1970) J. Natl. Cancer Inst. 44, 1183-1193. cases as was described by Hakkinen (4). A possible relation of 5. Schiff, F. & Adelsberger, L. (1925) Z. Immunitaetsforsch. Exp. "A-like immunogenicity" displayed by "human tumor gly- Ther. 40, 335-367. colipid" fraction to the Forssman antigen was explored before, 6. Buchbinder, L. (1935) Arch. Pathol. 19,841-880. fraction did not inhibit of 7. Kawanami, J. (1972) J. Biochem. (Tokyo) 72, 783-785. and the tumor glycolipid hemolysis 8. Forssman, J. (1911) Biochem. Z. 37, 78-115. sheep erythrocytes by anti-sheep hemolysin (3). However, this 9. Yamakawa, T., Irie, R. & Iwanaga, M. (1960)J. Biochem. (Tokyo) previous finding is compatible with our present results, namely 48,490-507. Forssman glycolipid is a relatively minor component in tumors 10. Makita, A., Suzuki, C. & Yosizawa, Z. (1966) J. Biochem. (Tokyo) of F- mucosa as compared to coexisting glycolipids and the 60,502-513. Forssman activity must be masked by other glycolipids. Because 11. Siddiqui, B. & Hakomori, S. (1971) J. Biol. Chem. 246,5766- the immunogenicity of Forssman for rabbit is strong as com- 5769. pared to other glycolipids, "A-like immunogenicity" was 12. Stellner, K., Saito, H. & Hakomori, S. (1973) Arch. Biochem. demonstrated with the tumor glycolipid fraction. Forssman Biophys. 155, 464-472. glycolipid might be immunogenic in blood group 0 or B indi- 13. Sung, S. J., Esselman, W. J. & Sweeley, C. C. (1973) J. Biol. Chem. viduals. Previously, Levine suggested that immunotherapy of 248,6528-6533. 14. Taketomi, T., Hara, A., Kawamura, N. & Hayashi, M. (1974) J. human tumors having illegitimate ABO and P (Tja) blood group Biochem. (Tokyo) 75,197-199. antigens could be possible by immunization of patients with 15. Ziolkowski, C. H. J., Fraser, B. A. & Mallette, M. F. (1975) Im- such antigens (1, 2). Immunologic suppression of tumors in 0 munochemistry 12, 297-302. or B individuals by immunization with Forssman glycolipid 16. Karlsson, K.-A., Pascher, I., Pimlott, W. & Samuelsson, B. E. suitably arranged on a carrier macromolecule might be possi- (1974) Biomed. Mass Spectrometry 1, 49-56. ble. 17. Gahmberg, C. G. & Hakomori, S. (1975) J. Biol. Chem. 250, 2438-2446. Note Added in Proof. In contrast to the findings of Levine et al. (1, 18. Coligan, J. E., Fraser, B. A. & Kindt, T. J. (1977) J. Immunol. 118, 2), the studies described above on antigens [either the precursor, glo- 6-11. boside (F-), or the final product, Forssman antigen (F+)] do not directly 19. Saito, T. & Hakomori, S. (1971) J. Lipid Res. 12,257-259. reflect the presence of corresponding human erythrocyte antigens. In 20. Yu, R. K. & Ledeen, R. W. (1972) J. Lipid Res. 13,680-686. an attempt to identify these two classes without resorting to biochemical 21. Ando, S., Isobe, M. & Nagai, Y. (1976) Btochtm. Btophys. Acta procedures, P. Levine, M. Celano, W. W. Young, and S. Hakomori 424,98-105. screened more than 100 normal sera (in a % dilution) for their capacity 22. Weissman, B. & Hinrichsen, E. (1969) 8, 2034- to hemolyze sheep erythrocytes in the presence of complement. In a 2043. separate experiment, 22 normal sera were tested for their capacity to 23. Hakomori, S. (1964) J. Biochem. (Tokyo) 55,205-208. lyse liposomes containing Forssman glycolipid in the presence of 24. Hakomori, S. (1972) in Methods in Enzymology, ed. Ginsburg, complement. Two classes were identified in both tests, i.e., lysers (about V. (Academic Press, New York), Vol. 28, pp. 232-236. 70%) and nonlysers. Double-blind studies are being carrried out to 25. Cuatrecasas, P. & Anfinsen, C. B. (1971) Methods in Enzymol- determine if the lysers and nonlysers correspond with the F- and F+ ogy, ed. Jakoby, W. B. (Academic Press, New York), Vol. 22, pp. populations, respectively. If so, the way will be open to carry out fa- 345-378. milial and racial studies on the two antigens by the immunological 26. Six, H. R., Young, W. W., Jr., Uemura, K. & Kinsky, S. C. (1974) procedures outlined. Buchbinder (6) refers to two studies in 1928 and Biochemistry 13, 4050-4058. 1929 in which the same two classes were found upon screening normal 27. Alving, C. R., Joseph, K. C. & Wistar, R. (1974) Biochemistry 13, human sera for their various capacities to hemolyze sheep erythro- 4818-4824. cytes. 28. Stellner, K., Watanabe, K. & Hakomori, S. (1973) Biochemistry 12,656-661. This investigation was supported by Grant CA 19224 and Contract 29. Sakiyama; H., Gross, S. K. & Robbins, P. W. (1972) Proc. Nat!. NO1-CB-64067 awarded by the National Cancer Institute, Department Acad. Sci. USA 69,872-876. of Health, Education, and Welfare. W.W.Y. is the recipient of National 30. Gold, P. & Freedman, S. 0. (1965) J. Exp. Med. 122, 467- Institutes of Health Postdoctoral Fellowship 1F 32 GM05281. 481. The costs of publication of this article were defrayed in part by the 31. Hakomori, S. (1973) Adv. Cancer Res. 18, 265-315. payment of page charges from funds made available to support the 32. Stein-Douglas, K. E., Schwarting, G. A., Naiki, M. & Marcus, D. research which is the subject of the article. This article must therefore M. (1976) J. Exp. Med. 143,822-832. Downloaded by guest on October 1, 2021