17 Human Carcinoma-Associated Precursors of the Blood
Glycoproteins and Glycolipids in Disease Processes Downloaded from pubs.acs.org by UNIV OF MICHIGAN ANN ARBOR on 11/20/18. For personal use only.
Group MN Antigens G.F.SPRINGER,P.R. DESAI, and M. S. MURTHY Department of Immunochemistry Research, Evanston Hospital, 2650 Ridge Ave., Evanston, IL 60201 E.F.SCANLON Department of Surgery and Microbiology—Immunology, Evanston Hospital, 2650 RidgeAve.,Evanston, IL 60201 M and N are the major antigens of the second human blood group system (1). Glycoproteins are the predominant carriers of the human red blood cell MN specificities which are determined by carbohydrates (cf. 2,3). A look at the structural basis of the interrelation of blood group MN specificities, which also occur on human epithelial tissues (4,5,6), and their carbohydrate precursor specificities T and Tn is important for an understanding of the human carcinoma-associated antigenic specificities T and Tn, as is also the appreciation that all humans possess anti-T and anti-Tn but not anti-M and anti-N antibodies. The biosynthesis of T-, N- and M- specific structures and the surprising significance of the carcinoma-associated precursor antigens T and Tn for the human immune response, humoral as well as cell-mediated, against adenocarcinoma are the main topics of this paper. Materials and Methods Bacteria and Vaccines. Bacteria were obtained from the American Type Culture Collection (Washington, DC); bacterial antigens were prepared in this laboratory or given by Drs. D.A.L. Davies, O. Lüderitz and E. Rietschel. Gram-negative bacteria were grown on fully defined media using procedures described previously (7). Vaccines were from the individuals and Drug Houses listed in Table IV. Carcinomatous and Control Tissue. Mammary gland tissues originated from ductal adenocarcinomata Stage I (International Nomenclature) which had not invaded regional lymph nodes and from Stage II and III ductal carcinomata as well as from nonmalignant breast lesions (fibroadenomatosis and fibrocystic disease) of U.S. women 18 to 86 years old. Glandular tissue of the primary tumor as well as distant metastases were obtained under aseptic conditions during surgery. Specimens left in the Pathology Department for more than a few minutes and autopsy tissues were not used because of autolytic changes. No specimen showed obvious 0-8412-0452-7/78/47-080-311$05.00/0 © 1978 American Chemical Society
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G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
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b a c t e r i a l contamination and none t e s t e d was pyrogenic. The glandular s t r u c t u r e s were s l i c e d and f r e e d of other t i s s u e s . H i s t o l o g i c s e c t i o n s were made to assure that only predominantly malignant regions were used i n case of carcinoma. C e l l membranes were prepared by hypotonic l y s i s as described p r e v i o u s l y f o l l o w i n g procedures used by others ( J 3 , 9 ) . The two human colon carcinoma-derived t i s s u e c u l t u r e c e l l l i n e s were given by Dr. B. Tom (10); c e l l s derived from normal colon e p i t h e l i u m were not a v a i l a b l e . P r e p a r a t i o n o f T antigen from Human Blood Group 0,MN Red Cells. The mode o f p r e p a r a t i o n has been described e a r l i e r (11,12, 13). From r e d blood c e l l stroma the MN antigens were e x t r a c t e d with aqueous 45% phenol p l u s e l e c t r o l y t e a t 23-25°C. A c t i v e m a t e r i a l was i s o l a t e d by f r a c t i o n a l u l t r a c e n t r i f u g a t i o n and ethanol f r a c t i o n a t i o n . The g l y c o p r o t e i n which p r e c i p i t a t e d between 45 and 70% ethanol concentration was exhaustively d i a l y z e d , f r e e z e - d r i e d and used f o r T antigen p r e p a r a t i o n . T a n t i gen was uncovered by the removal o f NAN from the i s o l a t e d 0,MN antigens with V i b r i o cholerae neuraminidase (RDE) a t pH 6.8; RDE was then i n a c t i v a t e d by h e a t i n g at 100°C f o r 5 min. T antigen was prepared a s e p t i c a l l y . Absorption o f A n t i s e r a . Because o f t h e i r s t r i c t i n t r a species s p e c i f i c i t y human a n t i s e r a only were used against blood group antigens M, N, T, and Tn, unless s t a t e d otherwise; a l l s e r a used have been described before (14,37). The a n t i s e r a were absorbed once with the membrane-cytoplasm preparations o r homogenized glandular t i s s u e or colon carcinoma-derived t i s s u e c u l t u r e c e l l s or b a c t e r i a under standard c o n d i t i o n s (7,14,15). C o n t r o l absorptions o f human a n t i - b l o o d group B s e r a with blood group H ( 0 ) - s p e c i f i c human breast glandular t i s s u e , benign o r malignant, r e s u l t e d i n no t i t e r decrease, nor d i d absorption o f human a n t i Rh (D) s e r a with benign o r malignant t i s s u e s from blood groups A l and H(0) persons (see a l s o Table I ) . Q
Blood C o l l e c t i o n and S e r o l o g i c Procedures. Bloods f o r determination of a n t i - T t i t e r s were c o l l e c t e d 24 to 48 h r before o r immediately a f t e r surgery, unless s t a t e d otherwise. The c l o t t e d bloods were coded and recorded by i n d i v i d u a l s who were not i n v o l v e d i n the assays. Sera were prepared and used a t once o r s t o r e d i n a l i q u o t s a t -20°C. For s e r o l o g i c a l assays 0,MM and 0,NN erythrocytes were obtained, s t o r e d , washed, and employed as such o r a f t e r T - a c t i v a t i o n with RDE (2). Tn r e d blood c e l l s were donated by Drs. G. Leonard (Cla.Ric.) and W.D. Bowman (Car.Lip.) ( c f . 16,17). Hemagglutination and hemagglutination i n h i b i t i o n t e s t s i n c l u d i n g c o n t r o l s and standards were performed and i n t e r p r e t e d by r o u t i n e procedures (7,13). Tests were scored ( c f . 18) independently by three i n d i v i d u a l s to whom nature of t i s s u e s used f o r absorption
Anti-T
58 -
0 0
98
10 - 25
100
83
50 -
70 (30 - > 90) 64 (50 - > 80)
53 (33 - 75)
42 (25 - 50)
< 1 ( o -
58
6)
59
< 10 ( o - 25)
49 -
3
OMM erythrocytes
0
0
2
"Buffy coat"
a b c — Number of cases. — No normal e p i t h e l i a l ^ c e l l s a v a i l a b l e . — A r i t h m e t i c averages i f > 2 persons t e s t e d , f i g u r e s i n parentheses = range. — M a c t i v i t i e s were found, with one exception, i f M antigen was expressed on the subject's r e d c e l l s (see t e x t ) . — Membranes from MM i n d i v i d u a l .
Anti-Tn
> 90)
/ m
v
Colon carcinoma . ^ \ (Tissue c u l t u r e ) ' 2-
53 - 100
66 ( 0 * .
15
Stages I I & I I I carcinoma
77 (50 - > 80)
72 (60 - 80)
3
Stage I carcinoma
53 (20 - 70)-
(50 - 66)
Anti-N
(A
5
ign
52^ (30 - 75)
B e n
Anti-M
sera
Human
TT
Breast glandular t i s s u e
Table I . Percent Absorption o f A n t i - B l o o d Group M, N, and Precursor A g g l u t i n i n s with Human Breast Gland Membrane-Cytoplasm P r e p a r a t i o n s , Colon Carcinomata and Controls
314
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
and source o f the sera were unknown; three serum standards were included as c o n t r o l s i n a l l determinations. Specimens were decoded only a f t e r completion o f the t e s t s . B i o s y n t h e t i c Procedures. For enzymatic transformation o f Tn to T group B r e d c e l l s o f C l a . R i c , which are completely Tntransformed, were used (19 ,20). Sera o f blood group B and A]B persons f r e e d o f a n t i - T and -Tn a g g l u t i n i n s served as p-galactos y l t r a n s f e r a s e source, UDP-Gal as Gal donor, and ATP, M n C l and MgCl2 as a c t i v a t o r s . A n a l y t i c a l grade chemicals were used; UDPGal and ATP were from Sigma Chemicals. Tn r e d c e l l s incubated i n absence o f one or more o f the other reactants served as c o n t r o l s ; they d i d not a c q u i r e T s p e c i f i c i t y , nor d i d normal MN r e d blood c e l l s when incubated under experimental c o n d i t i o n s with transf e r a s e - c o n t a i n i n g serum. For b i o s y n t h e s i s o f N- and M- s p e c i f i c determinants, T antigens prepared by d e s i a l a t i o n o f three d i f f e r e n t p r e p a r a t i o n s , each from i s o l a t e d human r e d blood c e l l NN and MM g l y c o p r o t e i n s , were used as s u b s t r a t e s , and f o r M - a c t i v a t i o n o f N, three d i f f e r ent human r e d c e l l NN g l y c o p r o t e i n preparations i s o l a t e d under gentle c o n d i t i o n s (13) were employed. Sera from three to s i x d i f f e r e n t donors o f NN as w e l l as MM blood types served as s i a l y l t r a n s f e r a s e source and CMP-NAN as NAN donor. CMP-NAN was prepared i n Dr. H. Schachter's l a b o r a t o r y as d e s c r i b e d by others (21+ 22) and C-CMP-NAN was from New England Nuclear Corp. In " c o l d experiments, the r e a c t i o n mixtures c o n s i s t e d o f 5 mg (0.5% f i n a l cone.) o f antigen with 2.0 (jmoles CMP-NAN and 500 serum as t r a n s f e r a s e source. In "hot" experiments, a l l r e a c t a n t s were reduced to 12.5% o f those i n the " c o l d " experiment. Incubation and work up were as described p r e v i o u s l y (JL9,_20). C o n t r o l samples were run throughout e x a c t l y as the experiment proper; they c o n s i s t e d o f r e a c t i o n mixtures l a c k i n g i n e i t h e r substrate or CMP-NAN. Substrates incubated with t r a n s f e r a s e i n absence o f CMP-NAN showed no change i n s p e c i f i c i t y . In a l l instances r e covery o f substrate was < 507o a t the end o f the experiment. Controls without added s u b s t r a t e o c c a s i o n a l l y acquired t r a c e s o f activity. 2
14
11
11
In v i v o Measurement o f Delayed-Type " R e c a l l H y p e r s e n s i t i v i ty. T antigen was d i s s o l v e d a t 1% f i n a l c o n c e n t r a t i o n i n buff e r e d s a l i n e c o n t a i n i n g 0.25% phenol; c o n t r o l s were solvent alone and solvent c o n t a i n i n g 1% MN a n t i g e n . A l l reagents were s t e r i l e and free o f pyrogens and Au-antigen. These s o l u t i o n s (0.1 ml each) were i n j e c t e d i . d . i n t o the upper arm. P a t i e n t s with mastectomy were i n j e c t e d i n the c o n t r a l a t e r a l arm. Induration and erythema were measured c a . 24 and 48 h r l a t e r and scored as by H o l l i n s h e a d ejt a l . (23). Reactions, i f any, given by the cont r o l s were s u b t r a c t e d from those due to T antigen. In v i t r o Measurement o f Cell-Mediated Immune Response.
Cell-
17.
SPRINGER E T A L .
Cancer-Associated
Blood Group
MN Precursors
315
u l a r immunity towards T antigen was assessed by the leukocyte migr a t i o n i n h i b i t i o n (LMI) assay i n agarose p l a t e s e s s e n t i a l l y as by Clausen (15,24). As c o n t r o l , leukocytes o f an apparently healthy person were t e s t e d i n p a r a l l e l on the same p l a t e s with those o f the p a t i e n t f o r MI by T antigen (5 o r 10 yg T antigen per 10 u l ) i n each agarose p l a t e . Each t e s t was run i n t r i p l i c a t e , and a r i t h m e t i c averages o f the r e s u l t s were used. C o n t r o l t e s t s with MN i n s t e a d o f T antigen were performed under l i k e c o n d i t i o n s . For healthy i n d i v i d u a l s , the average migration index a t 5 and 10 ug o f T antigen was 1.00; a migration index o f 75% f o r N - a c t i v a t i o n o f T, from four to > 34% f o r M - a c t i v a t i o n o f T and from two to > 75% f o r M - a c t i v a t i o n o f N antigen depending on the human o r animal antiserum i n h i b i t e d . Incorporation of ^C-NAN i n t o T- and Nantigens during incubation with t r a n s f e r a s e - c o n t a i n i n g s e r a was i n keeping with the s e r o l o g i c a l a c t i v i t i e s of the products obtained. S e r o l o g i c a l r e s u l t s were s i m i l a r f o r M - a c t i v a t i o n o f the a-1 glycopeptide i s o l a t e d from NN e r y t h r o c y t e s . The b i o s y n t h e t i c experiments are summarized i n Figure 1. We were s t r u c k by the s t r u c t u r a l s i m i l a r i t y o f the N- and Ms p e c i f i c groups o f the human red c e l l g l y c o p r o t e i n s and glycoprot e i n I ( e p i g l y c a n i n ) (26) of the TA3 mouse mammary adenocarcinoma; we found that t h i s g l y c o p r o t e i n indeed possessed blood group Nl i k e s p e c i f i c i t y (27). TA3 g l y c o p r o t e i n I o f s t r a i n A mice i s carcinoma-associated and does not occur i n normal s t r a i n A mouse t i s sue (28). Thus, a l i n k was e s t a b l i s h e d f o r the f i r s t time between an animal carcinoma-associated antigen and a human blood group substance (N and precursor T ) . The human a n t i - T a g g l u t i n i n of Thomsen and F r i e d e n r e i c h r e a d i l y k i l l e d TA3-St carcinoma c e l l s
316
GLYCOPROTEINS
A N D GLYCOLIPIDS
I N DISEASE
cx-GalNAc-OSer/Thr —
Tn
Figure 1. Biosynthetic pathway of the M- and Nspecific immunodeterminant structures of human blood group M and N glycoproteins
PROCESSES
17.
SPRINGER E T A L .
Cancer-Associated
Blood
Group
MN Precursors
317
i n the presence o f guinea p i g complement (28). We therefore turned our a t t e n t i o n from murine mammary glands and the TA3 carcinoma to human breast t i s s u e and i t s malignancies. Human breast gland c e l l membrane preparations were used to absorb human a n t i s e r a . The r e s u l t s of some o f these s t u d i e s are l i s t e d i n Table I . M- and N- s p e c i f i c s t r u c t u r e s are present i n both benign and malignant human breast glands, T- and Tn- s p e c i f i c i t i e s occurred i n a l l of the human b r e a s t (primary) and colon carcinomata t e s t e d but not i n corresponding preparations from benign o r normal b r e a s t t i s s u e s . T a c t i v i t y was low i n one i n v a s i v e Stage I I I b r e a s t carcinoma. A l l but one gland membrane p r e p a r a t i o n s p e c i f i c a l l y absorbed anti-M and anti-N a n t i b o d i e s i n agreement with the r e p r e s e n t a t i o n o f these antigens on the p a t i e n t ' s r e d cells. There was one exception where N but n o t M was expressed on the p a t i e n t ' s carcinoma c e l l membranes, while both antigens were present on her red c e l l s . We have t e s t e d metastases removed up to 60 months a f t e r surgery f o r primary d u c t a l carcinoma o f the breast from four pat i e n t s . A l l contained T- and the two t e s t e d f o r Tn contained a l s o T n - s p e c i f i c s t r u c t u r e s . This promising observation i n d i c a t e s that T- and Tn- s p e c i f i c i t i e s are not e l i m i n a t e d by e i t h e r somatic mutation or modulation and thus remain a c c e s s i b l e to p o t e n t i a l immunodiagnosis and immunotherapy. The T - s p e c i f i c i t y on adenocarcinomata prompted our determinat i o n of a n t i - T t i t e r scores o f human s e r a , which o r d i n a r i l y range from 22 to 24. They were s e v e r e l y depressed i n a h i g h l y s i g n i f i cant number o f a l a r g e p o p u l a t i o n o f breast carcinoma p a t i e n t s as compared to 270 p a t i e n t s with benign breast d i s o r d e r s and 470 cont r o l s a l l together (Table I I ) . S i m i l a r r e s u l t s were obtained i n p a t i e n t s with lung and g a s t r o i n t e s t i n a l (G.I.) carcinomata (15; to be p u b l i s h e d ) . Of the j u s t described p a t i e n t s with breast disease, 32 with mastectomy f o r carcinoma and 32 with breast biopsy who s u f f e r e d from benign disease were r e b l e d once w i t h i n 14 months of surgery. Of the 32 breast carcinoma p a t i e n t s r e b l e d , 21 (65.6%) showed an i n c r e a s e of > 25 to 90% i n a n t i - T t i t e r , while only one o f 32 (3.1%) benign breast disease p a t i e n t s and none o f 11 p a t i e n t s w i t h major operations f o r non-malignant d i s o r d e r s showed a s i g n i f i c a n t i n c r e a s e i n a n t i - T . This d i f f e r e n c e between the carcinoma pat i e n t s and the c o n t r o l s was s i g n i f i c a n t (JD < 0.001). In some p a t i e n t s , b l e d a t h i r d time, c l i n i c a l recurrence o f the carcinoma was a s s o c i a t e d with renewed lowering o f serum a n t i - T l e v e l s (to be p u b l i s h e d ) . Table I I I summarizes the f i n d i n g s on delayed-type s k i n hypers e n s i t i v i t y (DTH) observed on i . d . i n j e c t i o n of T antigen, and those o f the leukocyte migration i n h i b i t i o n assay (LMI). I t can be c l e a r l y seen that the immune response towards T antigen of breast carcinoma p a t i e n t s extends from humoral to c e l l - m e d i a t e d r e a c t i v i t y and i s demonstrable i n v i v o as w e l l as i n v i t r o . F o r t y four breast carcinoma p a t i e n t s were s k i n t e s t e d (Table I I I ) , 36 o f
3.62
A l l noncarcinomatous persons studied (B) C
17 /470
8.30
8.15
14^/270
£
39 /470
22^/270
61/189
32.28-
40/189
No. o f persons
No. of persons
12 or
: that d i f f e r e n c e between a n t i - T scores of breast carcinoma p a t i e n t s and c o n t r o l group (A) as w e l l as (B) i s due to chance i s < 0.001 throughout (X t e s t ) . £ Two of the 14 p a t i e n t s have s i n c e developed carcinoma.
5.19
Benign breast disease (A)
21.16^
%
10 or
3 weeks a f t e r surgery or chemotherapy and > 12 months a f t e r r a d i a t i o n therapy were s t u d i e d , because of the known immunosuppression due to these i n t e r v e n t i o n s (46). Individuals of Chinese and Japanese descent not i n c l u d e d . — DTH p o s i t i v e = > 4.5 mm i n d u r a t i o n a f t e r 24-48 hr or erythema at 48 hr > 25 mm (average of 2 diameters measured p e r p e n d i c u l a r l y ) (23). — Leukocyte migration i n h i b i t i o n on agarose p l a t e ; 10 (jbg T antigen/1.5 x 10 leukocytes; not t o x i c up to > 45 jig. — Negative was the only i n f i l t r a t i n g l o b u l a r carcinoma. — The two negative persons had i n s i t u l o b u l a r carcinoma. — 2/38 p o s i t i v e . I n t r a d u c t a l papilloma was the most ominous s t r u c t u r e i n both. £ Vietnam war veteran with typhoid, c h o l e r a and tetanus t o x o i d v a c c i n a t i o n s .
"Healthy"
11/84
8/27
14-/16
I 2-/38
17/31
13/13
II
Benign
10/21
8^/9
7/14
Persons p o s i t i v e / P e r s o n s
III
tested
6/6
Persons positive—/Persons
LMI
IV
Disease stage
DTH
Table I I I . Delayed-Type R e c a l l Skin R e a c t i v i t y upon i . d . I n j e c t i o n of T and i n v i t r o Cell-Mediated Immune Response to T Antigen i n P a t i e n t s with Breast Carcinoma, Benign Breast Disease and i n Apparently Healthy Controls-^
320
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE
PROCESSES
these s u f f e r e d from carcinoma of d u c t a l o r i g i n , 2 were noni n v a s i v e . The remaining 8 p a t i e n t s had l o b u l a r carcinoma. A l l p a t i e n t s with d u c t a l carcinoma gave a p o s i t i v e DTH r e a c t i o n regardless of Stage and invasiveness (15,29). F i v e with l o b u l a r carcinomata a l s o gave a p o s i t i v e r e a c t i o n but one of the p a t i e n t s with i n v a s i v e and two with non-invasive l o b u l a r tumors d i d not r e a c t (29). In c o n t r a s t , none of the 23 healthy i n d i v i d u a l s t e s t ed had any delayed h y p e r s e n s i t i v i t y r e a c t i o n toward T antigen, and 36 of the 38 p a t i e n t s diagnosed h i s t o l o g i c a l l y to have f i b r o adenoma-fibrocystic disease had a negative r e a c t i o n . The r i g h t hand s i d e of Table I I I shows that i n LMI 34 (51.5%) of the 66 p a t i e n t s with breast carcinoma Stages I I , I I I and IV had a p o s i t i v e r e a c t i o n , while of 27 p a t i e n t s with Stage I disease, eight (29.6%) were p o s i t i v e . Seven of the e i g h t p a t i e n t s with l o b u l a r carcinoma r e f e r r e d to above were t e s t e d i n LMI. The leukocytes of only two of these p a t i e n t s , both with i n f i l t r a t i n g tumor showed migration i n h i b i t i o n . Of 84 p a t i e n t s with benign breast disease, 11 (13.1%) were p o s i t i v e i n LMI with T antigen. No e f f e c t of T antigen was found on the p e r i p h e r a l leukocytes of a l a r g e number of presumably healthy people. LMI was p o s i t i v e i n only one of 95 healthy i n d i v i d u a l s , a Vietnam war veteran v a c c i n a t e d repeatedly with S.typhi, V. choJLerae and Tetanus t o x o i d a l l of which possess T - s p e c i f i c i t y (see Table IV). T antigen up to 50 yg d i d not a f f e c t v i a b i l i t y (trypan blue) or migration of the white c e l l s of healthy persons, MN antigen at the same high concentration had no e f f e c t on migrat i o n of leukocytes of any of the p a t i e n t s and c o n t r o l s t e s t e d . Discussion In t h i s communication we report that we have reversed the degradative steps l e a d i n g from blood group M - s p e c i f i c s t r u c t u r e s to i t s precursors and synthesized T - s p e c i f i c groups (19,20) on Tn erythrocytes as w e l l as N- and M- s p e c i f i c groups on i s o l a t e d T antigens. The peptide core of T antigen i n M- d i f f e r s i n 2 amino acids from that i n N- i n d i v i d u a l s and M a n t i g e n i c determinants possess more carbohydrate (30,31,32,33,34). Since g l y c o s y l t r a n s ferases g e n e r a l l y recognize only l i m i t e d areas (one to two monosaccharides) u n d e r l y i n g the acceptor molecule, s i a l y l t r a n s f e r a s e s from MM and MN donors produced N and M a c t i v a t i o n , whereas those from NN donors produced only N regardless of the MN type o r i g i n of T antigen. I s o l a t e d N antigen could be transformed to M only with s e r a from MM and MN donors. The g e n e t i c a l sequence of these immunological s p e c i f i c i t i e s i s t h e r e f o r e : Tn-*- T* N->- M (M could conceivably a r i s e d i r e c t l y from T). These s t r u c t u r e s have a l s o been found on l i p i d i c c a r r i e r s , i n c l u d i n g those from a carcinoma (35). The discovery that human adenocarcinoma but not healthy human t i s s u e s and benign s t r u c t u r e s possess T- and Tn- s p e c i f i c i t i e s i n r e a c t i v e , unmasked form e s t a b l i s h e s that these precursors are
P a s t e u r e l l a pseudotuberculosis— Pneumococcus XIV p o l y s a c c h a r i d e — BCG, T i c e s t r a i n (Univ. of Illinois).^ T u b e r c u l i n , PPD ( T u b e r s o l ) - * Influenza v i r u s : A / V i c t o r i a + B/Hong Kong (Parke-Davis )— Pseudomonas—*^
VACCINES:
Aerobacter aerogenes K l e b s i e l l a pneumoniae 3/3 Proteus s t r a i n s
LPS:
BACTERIA:
Absent
(LPS) and
— Non-dialyzable p a r t . SL A c t i v e with some but not a l l human a n t i - T . — Donated by Dr. M. H e i d e l berger. ^ Donated by Dr. R. G. Crispen. — A p o s s i b l e c o n t r i b u t i o n to a c t i v i t i e s by growth^medium has not been excluded. -= From Connaught L a b o r a t o r i e s . Donated by Dr. J . U. Gutterman. — Donated by Wellcome L a b o r a t o r i e s , - i From L e d e r l e , Orimune t r i v a l e n t .
Salmonella t y p h i (Wyeth, E. L i l l y ) Corynebacterium parvum (Coparvax)—>|l C l o s t r i d i u m t e t a n i t o x o i d (Wyeth)—*— V i b r i o cholerae (Wyeth)—>— Poliomyelitis v i r u s ^ ' i
4/8 E. c o l i 6/9 Salmonella s t r a i n s S. typhimurium S. abortus equi S. t y p h i S. minnesota 2/3 S h i g e l l a s t r a i n s S e r r a t i a marcescens Bacterium t u l a r e n s i s 2/3 Chromobacterium violaceum
LPS:
VACCINES:
9/9 E s c h e r i c h i a c o l i 3/3 E. f r e u n d i i Salmonella poona 1/3 Pseudomonas s t r a i n s
T Specificity
T - S p e c i f i c Substances i n Microbes, t h e i r Lipopolysaccharides M i c r o b i a l Vaccines^
Present
IV.
BACTERIA:
Table
322
GLYCOPROTEINS
A N D GLYCOLIPIDS I N DISEASE
PROCESSES
c h a r a c t e r i s t i c o f adenocarcinomata o f human b r e a s t , G.I. t r a c t and probably lung. The occurrence o f f u l l y r e a c t i v e T- and Tna n t i g e n i c s p e c i f i c i t i e s i n these cancers i s probably due to i n complete b i o s y n t h e s i s o f normal c e l l - s u r f a c e components, as i n d i cated by the presence of a l l these s p e c i f i c i t i e s except M, i n the breast carcinoma o f one p a t i e n t whose r e d c e l l s c a r r i e d M and N antigens as w e l l as by the extensive absorption (70%) of anti-N by b r e a s t carcinoma membranes from one p a t i e n t o f e r y t h r o c y t e group MM. T- and Tn- s p e c i f i c a n t i g e n i c determinants have r e c e n t l y been demonstrated d i r e c t l y with immunofluorescent l e c t i n s i n a l l carcinomatous b r e a s t glands t e s t e d but not i n n o n - c a r c i nomatous glandular areas o f the same s e c t i o n s (36; Dr. Cr. McNeil, p e r s o n a l communication). O r d i n a r i l y humans have no anti-N o r anti-M a n t i b o d i e s s i n c e a l l possess b l o o d group N, M, o r NM, and i n d i v i d u a l s homozygous f o r M uniformly have some N as w e l l (1,37). Healthy mammals do not form a n t i b o d i e s against t h e i r own t i s s u e s which are i n contact with t h e i r immune machinery. Furthermore, N and M s p e c i f i c i t i e s are not ubiquitous as are A and B s p e c i f i c i t i e s (3,7,38). On the other hand, T- and Tn- s p e c i f i c s t r u c t u r e s are always masked i n healthy humans, but are widespread among i n t e s t i n a l and a i r b o r n e b a c t e r i a (see below); consequently a n t i b o d i e s against these s t r u c t u r e s are found i n a l l humans (16,39). Levels o f these a n t i bodies are q u i t e constant under o r d i n a r y circumstances (29,39). Anti-T was s e v e r e l y depressed i n p a t i e n t s with carcinoma o f the b r e a s t , lung and G.I. t r a c t much more f r e q u e n t l y when compared to populations o f comparable age and socio-economic s t a t u s who had e i t h e r benign disease o r were apparently h e a l t h y . Of a l l i n d i v i duals with s e v e r e l y depressed a n t i - T , > 80% s u f f e r e d from c a r c i noma. This depression was not due to IgG, IgM, o r IgA decrease. S i m i l a r observations on a n t i - T of p a t i e n t s with b r e a s t cancer have r e c e n t l y been reported by others (40). Anti-T antibody may be depressed because of i t s i n t e r a c t i o n with T - s p e c i f i c substances e i t h e r attached to cancer c e l l - s u r f a c e and/or r e l e a s e d from t h e c e l l - s u r f a c e i n t o the c i r c u l a t i o n . This hypothesis i s supported by the o b s e r v a t i o n of r e l e a s e o f T - s p e c i f i c antigens from human mammary carcinoma-derived t i s s u e c u l t u r e c e l l s i n t o the c u l t u r e f l u i d (41). We d i d not f i n d depressed a n t i - T i n p a t i e n t s with sarcoma, melanoma o r with b r a i n tumors t e s t e d so f a r , n o r Ts p e c i f i c s t r u c t u r e s on these tumors. As mentioned above, there i s evidence that humoral a n t i - T as w e l l as a n t i - T n r e s u l t l a r g e l y from immunization by the host's own i n t e s t i n a l f l o r a (7,14,38,42,43,44,45). We s t u d i e d numerous, predominantly gram-negative b a c t e r i a and/or t h e i r l i p o p o l y s a c c h a r i d e s , and some v a c c i n e s . Table IV shows that many microbes indeed possess T - s p e c i f i c s t r u c t u r e s as determined by hemagglut i n a t i o n i n h i b i t i o n and by absorption assays. Three of the 12 a c t i v e E s c h e r i c h i a c o l i and one o f the two a c t i v e Chromobacterium violaceum s t r a i n s , Salmonella milwaukee and JS. abortus equi i n h i b i t e d human a n t i - T only when t e s t e d a f t e r b o i l i n g . It is
17.
SPRINGER E T A L .
Cancer-Associated
Blood
Group
MN Precursors
323
noteworthy that Corynebacterium parvum v a c c i n e , i n wide use to s t i m u l a t e " u n s p e c i f i c " r e s i s t a n c e against cancer, possessed s i g n i f i c a n t T s p e c i f i c i t y , while the one commercial BCG t e s t e d and the Pseudomonas v a c c i n e (also used as " u n s p e c i f i c " s t i m u l a n t against cancer) and PPD t u b e r c u l i n had no T a c t i v i t y . Tn a c t i v i t y o f b a c t e r i a l p r e p a r a t i o n s was a l s o found i n 25 of 30 Enterobacteariaceae ( i n c l u d i n g S.typhi). Bacterium t u l a r e n s i s , and i n JC. parvum, typhoid, tetanus and p o l i o m y e l i t i s v a c c i n e s . Tn a c t i v i t y was not demonstrable i n BCG, PPD t u b e r c u l i n , three Pseudomonas s t r a i n s and some Enterobacteriaceae. The discovery o f T- and Tn- s p e c i f i c immunologically r e a c t i v e determinants i n the c e l l membranes of breast and colon adenocarcinomata but not i n healthy t i s s u e s e s t a b l i s h e s f o r the f i r s t time a p h y s i c a l l y and chemically w e l l - d e f i n e d immunospecific s t r u c t u r e c h a r a c t e r i s t i c a l l y a s s o c i a t e d with human carcinoma. These precursor s p e c i f i c i t i e s T and Tn i n r e a c t i v e form appear to be due to incomplete b i o s y n t h e s i s (or a c c e l e r a t e d degradation) o f normal c e l l - s u r f a c e components. I t remains to be seen i f T a n t i gen a l s o occurs i n other malignancies and o c c a s i o n a l l y i n some permanently benign human tumors. The T antigen q u i t e l i k e l y can be considered a paradigm f o r other p r e c u r s o r antigens, e.g. Tn, and a l s o f o r the Forssman antigens and r e l a t e d blood group antigens, with s i m i l a r f u n c t i o n s i n malignant processes. T antigen i s a v a i l a b l e i n u n l i m i t e d q u a n t i t y , i s not contaminated with HL-A- and Au- antigens, and i s o b t a i n a b l e pyrogen-free. I t can be r e a d i l y prepared by removal of NAN from i s o l a t e d human r e d c e l l blood group NM antigens. T antigen and the a n t i - T a n t i b o d i e s which are present i n a l l humans (conceivably coupled with r a d i o a c t i v e i s o t o p e s ) may be u s e f u l i n d i a g n o s i s , prognosis, and p o s s i b l y therapy and immunoprophylaxis of some adenocarcinomata. Acknowledgments G. F. S. i s J u l i a S. Michels I n v e s t i g a t o r i n S u r g i c a l Oncology. This work was supported by the N a t i o n a l I n s t i t u t e s o f Health grants CA 19083 and CA 22540. We thank surgeons and patho l o g i s t s of Evanston and St. F r a n c i s H o s p i t a l s , Evanston, IL and the B e r k s h i r e Medical Center, P i t t s f i e l d , MA f o r f r e s h t i s s u e s . We are g r a t e f u l t o Mrs. H. Tegtmeyer, Mrs. T. Mo, Mr. M. Coleman, Mrs. V. Scanlon, Mrs. E. M i t c h e l l and Mrs. J . K e l l o g g f o r e x c e l lent assistance.
324
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Literature Cited 1. Landsteiner, K.; Levine, P. J. Exp. Med. (1928), 47, 757-775. 2. Springer, G. F.; Ansell, N. J. Proc. Natl Acad. Sci. USA (1958), 44, 182-189. 3. Springer, G. F. Naturwissenschaften (1970), 57, 162-171. 4. Kosjakov, P. N.; Tribulev, G. P. J. Immunol. (1939), 37, 283-295. 5. Boorman, K. E.; Dodd, B. E. J. Pathol. Bacteriol. (1943), 55, 329-339. 6. Stalder, K.; Springer, G. F. Fed. Proc. (1960), 19, 70. 7. Springer, G. F.; Horton, R. E. J. Clin. Invest. (1969), 48, 1280-1291. 8. Davies, D. A. L. Immunology (1966), 11, 115-125. 9. Mann, D. L.; Rogentine, G. N., Jr.; Fahey, J. L.; Nathenson, S. G. Nature (1968), 217, 1180-1181. 10. Tom, B. H.; Rutzky, L. P.; Jakstys, M. M.; Oyasu, R.; Kaye, C. I.; Kahan, B. D. In Vitro (1976), 12, 180-191. 11. Springer, G. F.; Desai, P. R.Carbohydr.Res. (1975), 40, 183-192. 12. Springer, G. F.; Desai, P. R.; Scanlon, E. F. Cancer (1976), 37, 169-176. 13. Springer, G. F.; Nagai, Y.; Tegtmeyer, H. Biochemistry (1966), 5, 3254-3272. 14. Springer, G. F.; Desai, P. R.; Banatwala, I. J. Natl. Cancer Inst. (1975), 54, 335-339. 15. Springer, G. F.; Desai, P. R.; Murthy, M. S.; Scanlon, E. F. J. Surg. Oncol. (1978), in press. 16. Moreau, R.; Dausset, J.; Bernard, J.; Moullec, J . Bull. Soc. Méd. Hôp. (1957), 73, 569-587. 17. Sturgeon, P.; McQuiston, D. T.; Taswell, H. F.; Allan, C. J. Vox Sang. (1973), 25, 481-497. 18. Stratton, F.; Renton, P. H. "Practical Blood Grouping", p. 34, Blackwell, Oxford, 1958. 19. Springer, G. F.; Desai, P. R.; Schachter, H.; Narasimhan, S. Naturwissenschaften (1976), 63, 488-489. 20. Desai, P. R.; Springer, G. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. 21. Comb, D. G.; Roseman, S. J. Biol. Chem. (1960), 235, 25292537. 22. Kean, E. L. J. Biol. Chem. (1970), 245, 2301-2308. 23. Hollinshead, A. C.; Stewart, T. H. M.; Herberman, R. B. J. Natl. Cancer Inst. (1974), 52, 327-338. 24. Clausen, J. E. Acta Allergol. (Kbh.) (1971), 26, 56-80. 25. Springer, G. F.; Desai, P. R. Transplant. Proc. (1977), 9, 1105-1111. 26. Codington, J. F.; Sanford, B. H.; Jeanloz, R. W. Biochemistry (1972), 11, 2559-2564. 27. Springer, G. F.; Codington, J. F.; Jeanloz, R. W. J . Natl. Cancer Inst. (1972), 49, 1469-1470.
17.
28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.
SPRINGER ET AL.
Cancer-Associated Blood Group MN Precursors
Springer, G. F.; Desai, P. R. Ann. Clin. Lab. Sci. (1974), 4, 294-298. Springer, G. F.; Desai, P. R.; Murthy, M. S.; Scanlon, E. F. Naturwissenschaften (1978), in press. Springer, G. F.; Yang, H. J.; Huang, I.-Y. Naturwissenschaften (1977), 64, 393. Springer, G. F.; Yang, H. J. Immunochemistry (1977), 14, 497-502. Yang, H. J.; Springer, G. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. Waśniowska, K.; Drzeniek, Z.; Lisowska, E. Biochem. Biophys. Res. Commun. (1977), 76, 385-390. Friedensen, B.; Yang, H. J.; Springer, G. F., in preparation. Cantrell, J. L.; Springer, G. F.; Desai, P. R., in preparation. Fischer, K.; Poschmann, A.; Stegner, H. E. Dtsch. Med. Wochenschr. (1977), 102, 1226-1227. Springer, G. F.; Tegtmeyer, H.; Huprikar, S. V. Vox Sang. (1972), 22, 325-343. Hoskins, L. C.; Boulding, E. T. J. Clin. Invest. (1976), 57, 74-82. Friedenreich, V. "The Thomsen Hemagglutination Phenomenon" Levin and Munksgaard, Copenhagen, 1930. Luner, S. J.; Wile, A. G.; Sparks, F. C. Proc. 68th Annu. Meet. Am. Assoc. Cancer Res. (1977), Abstract 375. Anglin, J. H.; Lerner, M. P.; Nordquist, R. E. Nature (1977), 269, 254-255. Springer, G. F.; Desai, P. R. Annu. Meet. Am. Soc. Microbiol. (1977), Abstracts, E42, p. 88. Springer, G. F.; Desai, P. R.; Murthy, S. M.; Scanlon, E. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. Boccardi, V.; Attinà, D.; Girelli, G. Vox Sang. (1974), 27, 268-272. Springer, G. F.; Horton, R. E.; Forbes, M. J . Exp. Med. (1959), 110, 221-244. Daban, A.; Schneider, M.; Calle, R. Bull, du Cancer (1975), 62, 21-28.
RECEIVED
March 30, 1978.
