Biochimica et Biophysica Acta, 458 (1976) 355-373 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 87030

HETEROGENEITY IMPLICATIONS

OF CARCINOEMBRYONIC ON ITS ROLE

ANTIGEN

AS A TUMOUR

MARKER

SUBSTANCE

G O R D O N T. R O G E R S

Department of Medical Oncology, Charing Cross Hospital, London W.6 8RF (U.K.) (Received April 27th, 1976)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355

1I.

CEA as a glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

356

III.

Origin of heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . .

356

IV.

Definition and detection of CEA . . . . . . . . . . . . . . . . . . . . . . . .

357

V.

Size heterogeneity of CEA . . . . . . . . . . . . . . . . . . . . . . . . . . .

358

VI.

Variation in amino acid and carbohydrate contents . . . . . . . . . . . . . . . .

359

VII.

Non-specific binding of CEA and the effect of perchloric acid . . . . . . . . . . . .

360

VIII. CEA and blood-group activity . . . . . . . . . . . . . . . . . . . . . . . . .

361

IX.

Fractionation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

362

X.

Lectin binding capacity of CEA . . . . . . . . . . . . . . . . . . . . . . . . .

364

XI.

Fractionation studies using concanavalin A affinity chromatography

365

XII.

The antigenic site of CEA . . . . . . . . . . . . . . . . . . . . . . . . . . .

XIII. Clinical implications

........

368

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

369

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

371

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

371

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

371

XIV. Conclusion

I. I N T R O D U C T I O N T h e t e r m c a r c i n o e m b r y o n i c a n t i g e n ( C E A ) w a s first u s e d b y G o l d a n d F r e e d m a n [1,2] t o d e s c r i b e a t u m o u r adenocarcinomas macromolecular

from

associated antigen found in the cellular membrane

entodermally

derived

digestive system epithelia.

of

I t is a

glycoprotein with a molecular weight of approximately 200000 and

Abbreviation: CEA, carcinoembryonic antigen.

356 with beta electrophoretic mobility. It is also present in foetal gastrointestinal tissue and with alpha-fetoprotein has been one of the most widely studied onco-foetal antigens. Extensive studies using radioimmunoassay have now shown that CEA is present in serum and associated with many forms of cancer [3] and also with various non-malignant diseases [3] and it can also be detected in low amounts in various normal tissues [4]. Although ten years have elapsed since the initial work of Gold and Freedman it is only in the last four years that preparations of CEA have been widely examined with regard to their purity, heterogeneity of molecules carrying the antigenic group and relative potencies, despite the fact that such studies promise to provide data which may enable the reproducibility and agreement of CEA radioimmunoassay to be improved and may also provide a way of assessing whether some molecules with CEA activity are more cancer specific than others. In the present article an attempt has been made to discuss recent studies which have a bearing on the heterogeneity of CEA and the implications of these studies to an assessment of the potential role of CEA as a tumour marker substance.

ti. CEA AS A GLYCOPROTEIN That CEA is a glycoprotein was evident from its solubility and apparent stability in perchloric acid [1]. The difficulties encountered in studying CEA are therefore those encountered in general glycoprotein chemistry. Biosynthesis of the carbohydrate moiety of a glycoprotein is believed to be a post-ribosomal event in which the enzymic assembly of the saccharide chains takes place after the protein has been completed [5]. Glycoprotein structure is therefore only partly under genetic control and considerable variation in structure is possible. This variation often takes the form of micro-heterogeneity of the intermediate and peripheral sugar residues whereas the innermost carbohydrate residues are relatively homogeneous in structure. CEA appears to be no exception to this and the physical and chemical properties of a CEA preparation will be the properties of the "average" molecule of CEA found in the mixture. Since the enzymic activity for the biosynthesis and possible breakdown or modification of a glycoprotein may vary in different individuals and in the malignant state [6,7,8] it is not surprising to find variations in the carbohydrate content and composition between different CEA preparations.

!I1. ORIGIN OF HETEROGENEITY Examination of glycoproteins by techniques such as electrophoresis, ionexchange chromatography and isoelectric focussing have demonstrated heterogeneity with respect to charge density and net molecular charge [9]. A major contribution to this is micro-heterogeneity in the form of variation in the number of terminal sialic acid residues and this can be distinguished from charge variation originating in the

357 protein structure. Treatment of a glycoprotein with neuraminidase will remove sialic acid and an increase in its cationic electrophoretic mobility and a considerable simplification in its isoelectric focussing pattern can be expected. Residual heterogeneity observed by the above techniques can be attributed to charge variation on the protein [9]. It is known that heterogeneity in the carbohydrate structure of a glycoprotein can result through variation at the genetic level as well as to variations in post-ribosomal biosynthesis [10, I 1]. It may therefore be important to distinguish between heterogeneity occurring at a single locus due to post-ribosomal events and variation generated in carbohydrate chains as a result of being attached to different positions on the peptide chain depending on variation in the structure of the protein. Aspects of post-ribosomal biosynthesis of glycoproteins which may have a particular bearing on the tumour specificity of a potential marker substance are tissue specificity and developmental differentiation. Identical polypeptide chains have been shown to be glycosylated differently in different tissues. For example, hen serum transferrin synthesised in the liver has an asparagine-linked carbohydrate group of different composition to a similarly linked group in ovo-transferrin synthesised in the hen oviduct [12]. Furthermore developmental changes, possibly associated with changes in glycosyl transferase activity have been detected in other types of glycoproteins [13]. These aspects of heterogeneity are particularly interesting as they provide a biochemical basis for studying components of onco-foetal antigens produced in various malignant and non-malignant conditions of various tissues, in order to find out if these components vary in structure.

IV. DEFINITION AND DETECTION OF CEA In view of the heterogeneous nature of the glycoprotein it is impossible to define CEA precisely in terms of physical and chemical properties. A definition of CEA at the present time has to be based on its specific immunological reaction with a monospecific anti-CEA antiserum which has been shown to be identical in specificity to the original antiserum prepared by Gold and Freedman [1]. This is important since it has been clearly established that CEA possesses at least two different immunogenic groups: a unique group which defines CEA-like molecules and a second determinant which is common to CEA and the glycoprotein N G P which is also found in normal and tumoural tissues [14,15,16]. Double diffusion studies have shown that antibodies to both the groups can easily be obtained by inoculating animals with purified CEA preparations [16]. N G P has a smaller molecular weight than CEA and can be substantially separated from the latter by Sephadex G-200 chromatography [17] but it is still often a contaminant of CEA and antisera raised to conventionally prepared CEA could contain antibodies which react specifically with N G P (Rogers, G. T., unpublished observation). In addition antisera raised to current CEA preparations could also contain other antibodies which react with non-CEA contaminants, even after extensive absorption, indicating the presence of other cancer

358 associated antigens. Immunochemical identity of a particular CEA preparation with authentic CEA from Gold [18] can be obtained by inhibition studies as illustrated by Coligan et al. [19]. However this information does not provide evidence that the antibodies reacting are of the same specificity as those prepared by Gold and which define CEA activity. It is therefore important to check the specificity of antisera by double diffusion using authentic anti-CEA antiserum as a control. The two main methods of detecting CEA, Ouchterlony double diffusion and radioimmunoassay, depending on the specificity of the anti-CEA antiserum will therefore detect the presence of any glycoprotein containing the specific CEA determinant providing that it is not sterically prevented from reacting with the antibody. These methods will therefore enable CEA-like substances to be distinguished from other glycoproteins with similar physical and chemical properties.

v. SIZE HETEROGENEITY OF CEA Variation in the apparent molecular size of CEA can be expected in view of its heterogeneity. Most preparations have been purified using large scale Sepharose 4B and Sephadex G-200 chromatography [18,19] and the CEA usually obtained has a mean molecular weight of 200000 ~ 20000, suggested by the fact that CEA from various sources elutes between IgM and IgG. CEA molecules with molecular weights in this range would not normally be resolved by Sephadex G-200 chromatography. However wider variation has been reported [19,20]. For example Coligan et al. [19] found that immunologically active CEA with a larger molecular size (10.1 S) has sometimes been fractionated on the Sephadex column and also resolved from conventional CEA (6.8 S) by acrylamide gel electrophoresis and ultracentrifugation [19]. This size difference appeared not to result from polymeric modifications of CEA since treatment of the 10.1 S CEA with dithiothreitol failed to cause dissociation. It is interesting however that the 10.1 S CEA had a higher CEA activity/absorbance ratio than conventional CEA indicating that the protein substructure may be linked to additional CEA determinants which could be contained in the carbohydrate part of the molecule. Similar variation in size has been shown to occur with circulating CEA. Using an ~25I-labelled CEA marker which had a molecular weight of 200000 Pletsch and Goldenburg [20] have chromatographed on Sephadex G-200 plasma and perchloric acid extracts of plasma, from patients with certain cancers and assayed the fractions for CEA activity. Whereas the CEA in rectal tumour patients chromatographed with the marker, CEA in the plasma of patients with cancer of the colon, ovary, cervix and bronchus had a molecular size corresponding to 370000 daltons. It is interesting to note that although the main peak of CEA activity chromatographed with a size corresponding to 370000 daltons, variation in molecular size was also evident and this was most marked in the case of CEA in the plasma of patients with bronchogenic cancer. Dithiothreitol, sodium dodecylsulphate and perchloric acid all failed to

359 alter the elution pattern. So far no data on the molecular size of circulating CEA of non-malignant origin has been reported. It is also not known whether the large CEA represents a CEA dimer or m u c i n . CEA complex, or whether it is a different glycoprotein with the CEA specific determinant. If the latter is true it may be pertinent to raise antisera to circulating CEA to search for a different and possibly more specific immunodeterminant so that more discriminating assays might be developed.

VI. VARIATION IN AMINO ACID AND CARBOHYDRATE CONTENTS The most marked variation between different CEA preparations is the content of carbohydrate which can vary between about 80 ~o for purified CEA of gastric origin to about 4 0 ~ for CEA derived from colonic metastases [21]. Unfortunately the content of non-CEA glycoprotein present in most CEA preparations is unknown and so it is difficult to attribute this variation solely to carbohydrate heterogeneity. Furthermore these variations have not been accurately correlated with variation in molecular weight. It is therefore difficult to see if the protein sub-structure varies significantly in size, suggesting variations at the genetic level, or whether a similar protein sub-structure carries different amounts of carbohydrate. The latter case is more likely since the compositions of amino acids for different preparations of CEA prepared from colonic metastases all showed similar features provided that variation in total protein and experimental error were allowed for [21]. Each of the preparations showed high levels of acidic and hydroxy amino avids representing approximately 40 ~ of the protein portion of CEA [22]. There are however marked differences in amino acid composition between CEA of colonic origin and that prepared from gastric tumour metastases [22]. The protein moiety of the latter preparation constituted only 16 ~ of the material and although the acidic and hydroxy amino acids again constituted a large part of the protein, the overall differences in amino acid composition were large enough to suggest that the protein moiety in gastric CEA may be distinctive from that in colonic CEA. The amino acid content and composition of CEA obtained from other tissues has not yet been adequately studied to ascertain whether there are fundamental variations in the protein structure of CEA obtained from tissues other than gastric and colonic tumours. Wide variations in the carbohydrate composition of different CEA preparations have been reported and this is so even between individual CEA preparations isolated from tumours of the same type and tissue origin and within the same laboratory [21]. An obvious source of this variation, heterogeneity in the carbohydrate structure, has been discussed but there are other possible factors. Briefly these are possible autolysis occurring in the autopsy or surgical tissue used as the source of CEA, the chemical effects which may take place on treatment of the tissue with perchloric acid, the degree of resolution achieved in the gel filtration stages employed in the purification and therefore the amount of non-CEA glycoprotein present and the effect of nonspecific binding of CEA to other proteins and glycoproteins which therefore may be

360 TABLE I COMPARISON OF DIFFERENT METHODS OF EXTRACTION OF CEA (SEE TEXT) FROM TUMOUR TISSUE AND SERUM SOURCES Tissue source (CEA ng/g)

Perchloric acid extract

Perchloric acid precipitate

Perchloric acid extract (prior centrifuging)

Ca bronchus (liver 2°) Ca stomach [25] (liver 2°) Ca colon (1° in liver) Serum pool (CEA ng/ml) Non-colonic cancer Colonic cancer Rectal cancer

80 negative 1200

200 positive

140

122 360 140

22 9.5 3.4

2500

purified along with CEA (see below). All these factors could potentially effect the carbohydrate content and composition of the purified material. It is worth pointing out that the method normally employed for preparing CEA is a modified method based on that of Krupey et al [18], but the block electrophoresis step suggested by the latter workers is seldom carried out. The method therefore relies on precipitating out unwanted proteins with perchloric acid and fractionating the remaining soluble glycoproteins by gel filtration. All glycoproteins or polymerically modified material with an apparent molecular weight similar to that of the main CEA fraction will therefore contaminate the product.

vii. NON-SPECIFIC BINDING OF CEA AND THE EFFECT OF PERCHLOR1C ACID Experiments using complement fixation and precipitation assays [23] have shown that the recovery of CEA from colonic tumour homogenates appears to be higher in saline than in perchloric acid extracts and these results have been confirmed using radioimmunoassay where approximately half the activity was recovered in the perchloric acid extract compared with the saline extract (Rogers, G. T. and Searle, F., unpublished result). That the charge variation between the two types of extract is different has been demonstrated by isoelectric focussing [24]. Here certain components present in the saline extract appear to be missing in perchloric acid extracted CEA. Further experiments on the extraction of CEA with perchloric acid (Rogers, G. T. and Searle, F., unpublished data) have shown that a larger amount of CEA activity can be recovered from the tumour homogenate if it is first centrifuged to remove the insoluble material, rather than adding the acid directly to the homogenate (Table I). This suggests that perchloric acid modifies some CEA molecules in such a way that they bind to the insoluble material and are not extracted by the perchloric acid. This has been confirmed by subsequently extracting CEA from the perchloric acid precipitate with 3 M K C I solution. Similar CEA binding has been independently

361 reported to occur in perchloric acid treated homogenates of gastric tumours [25]. The methods of extracting CEA vary. The original CEA [1] was extracted using phosphate buffered saline. However some more recent methods use perchloric acid to extract CEA directly from the homogenate [18,19] whereas others [26] employ a centrifugation step prior to adding perchloric acid. It is therefore important with regard to the assay for CEA to establish whether the bound CEA differs chemically (and may therefore have a different specificity)from non-binding CEA, or whether the effect of perchloric acid is quantitative and merely alters the yield. Similar studies (Rogers, G. T. and Searle, F., unpublished result) on pooled sera from patients with colonic and non-colonic cancers have shown that CEA is also present in perchloric acid precipitates of serum although in contrast to tumour tissue the amount is small relative to that in the supernatant (Table I). These results however differ from those of Sorokin et al. [27] which failed to show the presence of CEA activity in serum precipitates but this discrepancy may be due to an insufficient amount of CEA in the individual sera studied by these workers. Our preliminary studies show that a much larger proportion of the total CEA is obtained from the perchloric acid precipitate of serum from non-colonic cancer patients compared with colonic cancer patients but more studies are required to see if this proves to be general. Although published comparative studies between different CEA assays indicate a 70-85 ~ diagnostic agreement, different absolute values are frequently obtained. For instance the effect of extraction with perchloric acid has been examined in sera which showed positive results for CEA [28]. In 17 patients with ulcerative colitis the CEA activity became undetectable after extraction despite initial levels in excess of 10 ng/ml, whereas the CEA activity remained detectable after extraction in the case of patients with colo-rectal carcinoma. Although this result can be interpreted as a general lowering of a background level of CEA after extraction, it clearly demonstrates that perchloric acid can significantly effect the assay value. Careful comparisons between the direct assays, in which serum or plasma is used, and indirect assays, which employ perchloric acid extracts, are needed to see if discordant values result from different specificities or represent quantitative effects.

Vlll. CEA AND BLOOD-GROUP ACTIVITY Cross-reactivity between CEA and blood group-like antigens has been suggested on the basis of binding of labelled CEA to human sera containing anti-A antibodies [29,30,31] and that the binding can sometimes be inhibited by absorption of the sera with group A erythrocytes. It has been shown [29] that both blood group A substance and CEA could inhibit the binding of anti-A antibodies to 12SI-labelled CEA. On the other hand blood group substances A and B do not inhibit CEA-antibody binding [30]. Other studies illustrate that hyperimmune human antisera against various blood group antigens could bind 30-75 ~ of added labelled CEA [32]. The conclusions drawn from these studies were that the CEA and blood group-like

362 determinants were present on the same molecule. However some of the evidence for cross-reactivity between CEA and blood group antigens provided by the above methods is questionable. First a hyperimmune antiserum may contain other nonspecific binding substances as well as antibodies and secondly the binding of labelled CEA to human blood group A antibodies could be explained if the blood group determinant was present on a separate molecule to which CEA was non-covalently bound [21]. In this case the blood group activity could be purified along with CEA and may account for the small amount of the immunodominant sugar N-acetylgalactosamine detected in some CEA preparations. This explanation would be consistent with the finding that blood group activity detected in CEA was related to the blood group of the donor [32]. However this correlation has not always been checked. Nevertheless the binding studies reported by Gold and Gold [29] were carried out using CEA in which N-acetylgalactosamine was not detected and it is interesting that in this work the group of sera that possessed anti-A activity bound 125I-labelled CEA to a significantly greater extent than the group of sera that did not contain such antibodies. Although blood group A-like activity might be conferred on a certain conformation of heterosaccharides in the absence of N-acetylgalactosamine another explanation is possible, that non-specific CEA binding capacity of human sera might be higher in those sera containing anti-A antibodies. Although the possibility exists that some molecules of CEA might possess blood group activity whereas others may not [33] there is strong evidence which has recently thrown further doubt on the existence of a blood group site on all CEA molecules. Experiments using anti-A and anti-i blood group specific immunoadsorbents for instance have demonstrated that only the corresponding blood group antigen activity was removed from colonic tumour extracts whereas the CEA activity remained essentially unchanged after absorption [34,26]. Again it has been shown that blood group precursors I and i, found in colonic tumour extracts, can be separated from CEA by gel filtration [35]. A more recent study which thows particular doubt on those experiments using antibodies eluted from erythrocytes has been reported [36]. It has been demonstrated that absorption of anti-blood group antisera by erythrocytes, instead of enabling removal of extraneous binding substance from the antisera in fact increases this contamination and therefore may lead to inconclusive results from binding studies using eluted blood group antibodies.

IX. FRACTIONATION STUDIES Various physical methods have been used to fractionate molecules with CEA activity. Many of these depend on variation in net charge on the protein. CEA preparations usually give a single but diffuse band on acrylamide gel electrophoresis as revealed by both staining [22,37] and by CEA activity [19], and an extended and sometimes double-humped arc in the beta region in immunoelectrophoresis at pH 8.6 [15]. These experiments demonstrate charge heterogeneity. The degree of charge

363 variation is extensive as shown by ion exchange chromatography [38,39] and isoelectric focussing studies [22-24,38,40]. Multiple peaks of CEA activity are obtained by each of these methods and there is also a clear correlation between an increased negative charge on the CEA molecule and an increase in the amount of sialic acid [38]. Studies by different groups [38,41,42] have shown that removal of sialic acid from CEA with neuraminidase does not effect its immunological activity. This is consistent with the finding [21] that the specific activity of the isoelectric focussing fractions of CEA does not vary widely except in the case of the most acidic fractions with a high content of N-acetylgalactosamine and therefore probably more non-CEA impurity. Residual heterogeneity in CEA is observed after removal of its sialic acid [38,22] indicating a variation in charge density or net molecular charge in the protein substructure which is either genetically controlled or due to decreased basicity as a result of possible deamination of glutamine or asparagine residues by the perchloric acid extraction. It is interesting that an identical sequence is obtained for the first 20-30 amino acid residues of the protein of CEA from several sources [37] including serum CEA [21], but there is no conclusive evidence ruling out heterogeneity and therefore variation in charge, in other parts of the protein structure. ~he isoelectric focussing fractions of CEA also show other differences. With the limited data available it appears [21 ] that of the neutral sugars, the contents of fucose and galactose decrease and that of mannose increases with increasing electronegativity indicating that the CEA fractions may vary considerably in their intermediate and peripheral carbohydrate structures. However the extent to which the sugar analyses of the fractions is effected by non-CEA impurities is unknown. The above methods have been employed effectively in the isolation of a species of CEA believed to be homogenous, called CEA-S [26]. In these studies hepatic metastases of primary adenocarcinomas of the colon were homogenised and the homogenate clarified by centrifugation and dialysis before extracting CEA with perchloric acid. This procedure therefore differs from the conventional method of Krupey [18] in removing the insoluble CEA-binding material, lsoelectric focussing of the material showed marked heterogeneity and a fraction pI 4.5 was retained for further purification. Subsequent chromatography on Sephadex G-200 however was atypical displaying five immunologically active peaks but this may be attributed to the modified extraction procedure. Further purification of CEA-S was achieved by DEAE cellulose chromatography, immunoabsorbent chromatography using anti-A and anti-B blood group affinity columns and density gradient ultracentrifugation (cf. ref. 43). The product obtained was claimed by double antibody radioimmunoassay employing 125I-labelled CEA-S to be approximately five times the potency of conventional CEA with significant quantitative differences observed between CEA-S and preparation Be from the laboratory of Dr. C. Todd [19] and the MRC reference CEA(73/601) [44]. It is interesting to note that assays employing CEA-S as the radio-labelled ligand were reported to be more specific for colonic cancer than conventional assays [26] suggesting that some molecules with CEA activity may be more specific than others and can be isolated using a combination of physical techniques. An alternative fractiGn-

364 ation of CEA using concanavalin A affinity chromatography is discussed in the next section. X. LECTIN BINDING CAPACITY OF CEA The plant lectins concanavalin A, wheat germ lectin, phytohaemagglutinin and ricin among others have been shown to bind to carbohydrates on radio-labelled CEA [41,45-48]. Wheat germ lectin and concanavalin A in particular have been used in binding studies on both untreated CEA and CEA which has been progressively degraded in order to provide structural information about CEA molecules. In addition affinity chromatography employing concanavalin A-Sepharose, can discriminate differences in the carbohydrate structure of glycoproteins and has proved a valuable tool in studying the heterogeneity of CEA and in providing a means of fractionating CEA into components of differing carbohydrate structure [49-51 ]. Most lectins have been shown to bind to specific groupings on particular carbohydrate residues or heterosaccharide chains [52]. The carbohydrate binding site of concanavalin A for instance appears to be directed primarily towards unmodified hydroxyl groups at the C-3, C-4 and C-6 positions of a-D-mannose, a-D-glucose and a-D-Nacetylglucosamine, whereas wheat germ lectin binds preferentially to N-acetylglucosamine. The binding of concanavalin A to CEA has been studied by many workers [45,46,53]. Chu et al. [45] have shown that the amount of concanavalin A bound to CEA increases proportionally with the amount of lectin and this can be specifically inhibited and reversed by mannose. It is also known that concanavalin A does not inhibit the binding of 125I-labelled CEA to its antibody [45,49] showing that the immunodeterminant group and the concanavalin A binding site are situated on different parts of the CEA molecule. Concauavalin A is able to bind up to 70~o of 12SI-labelled CEA [46] and CEA capable of this binding has been shown to have an increased activity for anti-CEA compared to CEA which does not react with the lectin [45,53,54]. Similarly CEA which binds to WGA is more antigenic than CEA which does not bind [46]. Structural studies [41] on CEA have shown that after three complete Smith's periodate degradations, when all the sialic acid and fucose, 80 ~o of the galactose, 65 ~,, of the mannose and about 40 ~ of the N-acetylglucosamine have been removed, the ability of the degraded CEA to react with anti-CEA antiserum and with wheat germ agglutinin is not impaired. This is clear evidence that the wheat germ lectin binding sites, viz. two or more 1-4 linked N-acetylglucosamine residues, and the antibody combining site are situated in the innermost part of the CEA molecule (Fig. 1). Evidence from binding studies [46] and immunodiffusion analysis [55] has indicated that these sites may have some residues in common. Concanavalin A on the other hand, since it fails to bind with CEA after a single Smith's degradation [41] appears to bind to 1-4 linked mannose residues which are situated in the intermediate or peripheral carbohydrate chains. These structural studies, together with the fact that

365 /NANA 3 Gal

FUCOSE

fL, .~

~ ~ / -

I

'

~

~S~

~

PROTEIN I

~

Limit of SMITH I degradation

/ Poss~le i~ntibody bindi g s t e

] ;i I Intermediate chains = mainly MANNOSE and GALACTOSE = N-ACETYLGLUCOSAMINE

Fig. 1. Schematic model of the general structural features of CEA. The circles represent unknown numbers of sugar residues. It is known however that the carbohydrate moiety of CEA exists as multiple side chains attached through asparagine linkages to glucosamine residues [21]. It is also likely that the side chains are distributed asymmetrically although on average there appears to be about seven residues per side chain and a maximum of about 80 chains [73]. Fucose and sialic acid are present at non-reducingends and the latter residues are attached to the 3-position in galactose [42]. Galactose and mannose constitute the intermediate portion of the carbohydrate moiety where branching occurs [74]. Concanavalin A binding is abolished by a single Smith's degradation [41] indicating that this lectin binds to mannose residues present in the outer chains. The protein moiety of CEA is stabilised by six disulphide bonds [61 ]. The available evidence [41,42] suggests that the antigenic site in CEA is situated in the innermost carbohydrate chains or in the protein and activity is abolished if the three dimensional structure of the protein is destabilised by cleavage of the disulphide bonds.

the lectins c o n c a n a v a l i n A a n d wheat germ lectin do n o t b i n d to all molecules with C E A activity [46,49] establish that there are f u n d a m e n t a l chemical differences in both the outer a n d i n n e r chains of different C E A molecules.

Xl. FRACTIONATION STUDIES USING CONCANAVALIN A AFFINITY CHROMATOGRAPHY The possibility that C E A might be isolated a n d purified by affinity c h r o m a t o graphy using c o n c a n a v a l i n A - S e p h a r o s e was suggested by C h u et al. [45] on the basis o f their finding that C E A can b i n d to c o n c a n a v a l i n A a n d the b i n d i n g reversed by a n o t h e r saccharide. F u r t h e r studies have d e m o n s t r a t e d that c o n v e n t i o n a l C E A o b t a i n e d from metastatic colonic t u r n o u t s can be fractionated by this technique into

366 TABLE 1I ACTIVITY AND CARBOHYDRATE CONTENT OF CEA FRACTIONS CEA activity expressed as per cent activity on a weight to weight basis of assay standard. Neutral sugar including fucose estimated by the orcinol method and expressed as rag/rag protein. The total carbohydrate was deduced from the freeze-dried weight after subtracting protein. (Data from Rogers et al. [54]) Fraction

Activity ~

Neutral h e x o s e

Carbohydrate

Unfractionated

100

-

56

1

2 2A 2B 3

23

1.18

70

170 66 196 110

0.39 0.66 0.31 0.51

42 42 38 44

a non-binding component and either one [46] or two [49,51] concanavalin A binding components depending on the conditions. The concanavalin A bound components can be conveniently separated by eluting the affinity column with increasing concentrations of a-methyl glucoside using either a stepwise or continuous gradient. It has also been shown, using a similar technique, that CEA from rectal, gastric and pancreatic tumours can be fractionated into immunologically active unbound and bound components [56]. Additional studies [54] have shown that Concanavalin A bound CEA can be further fractionated using NaBO2 to elute the column prior to the use of a-methyl glucoside. Borate ions compete in a different way from a-methyl glucoside in that they complex with vicinal cis hydroxyl groups and therefore probably compete with concanavalin A for these groups on non-reducing 0¢-D-mannose residues in CEA. So far CEA has been separated into four components designated fractions 1,2A,2B and 3 by eluting the column sequentially with acetate buffer, borate buffer, 2 ~ methyl glucoside and 10~o methyl glucoside, respectively [54]. All these fractions have been shown to produce a line of identity with anti-CEA antiserum by double diffusion, but although the activity in each fraction was appreciable, they differed in their ability to inhibit the binding of 125I-labelled CEA to its antibody (Table II). Comparative assays have shown [54] parallel inhibition curves between CEA 2B and conventional CEA including the M R C reference preparation 73/601 [44] (Fig. 2). However the potency of CEA 2B is significantly higher than that of conventional preparations of CEA and appears to be comparable with the potency of CEA-S [26] although chemical similarity with the latter preparation has not been established. The carbohydrate contents and compositions of the concanavalin A fractions vary considerably (Tables II and 1II) and, since the proportions of the fractions are different in different preparations (Rogers, G. T., unpublished observation) this may help to explain the wide variation found in the carbohydrate analyses of unfractionated CEA. In the case of CEA 2B in particular the content of neutral sugar is less than that found in the other fractions and in unfractionated CEA but the content of N-acetylglucosamine is comparatively high [54]. It may be significant, in view of the

367

/./V

Z g- 30 20

I0

~..--~'~"J"

~

(labell d M-12 + 'ace' (labelled

l0

antiseru 36antiserum)

100

1000

ng CEA freeze dried wt.

Fig. 2. Comparative inhibition curves of conventional CEA (M-12), fraction 2 B and the MRC reference CEA demonstrating the inhibition of binding of *2SI-labelled CEA to goat anti-CEA. (Data from Rogers et al. [54]).

high potency of CEA 2B, that the latter sugar has been suggested to be an important part of the antigenic site of CEA (see below). The amino acid composition of CEA 2B is very similar to that reported for conventional preparations [54]. Preliminary studies [54] have shown that circulating CEA from patients with colonic cancer can also be fractionated into four components by similar concanavalin A affinitychromatography techniques. A fraction eluted with 2 ~ methyl glucoside contained most of the CEA activity as measured by radioimmunoassay with only minor proportions detected in the fractions eluted with acetate and borate buffers. Different proportions however have been found in the serum of patients with other diseases (Table IV)

TABLE III CARBOHYDRATE ANALYSES OF FRACTION 2B Neutral sugars were determined by gas-liquid chromatography and amino acids estimated on an amino acid analyser after hydrolysis in p-toluenesulphonic acid. The percentage carbohydrate was calculated from the total recovery of monosaccharides per mg of protein. (Data from Rogers'et al [54]).

Fucose Mannose Galactose N-Acetyl glucosamine N-Acetyl galactosamirte Sialic acid Carbohydrate 700

/~mol/100 mg dry weight

mg/mg protein

44.9 42.6 56.4 109.7

0.12 0.13 0.16 0.31 not detected trace 41.00

368 T A B L E IV Distribution o f C E A as m e a s u r e d by r a d i o i m m u n o a s s a y , in fractions obtained by c o n c a n a v a l i n A affinity c h r o m a t o g r a p h y as applied to pools of sera f r o m patients in five different disease groups. All fractions d i a l y z e d / H 2 0 prior to assay. ( D a t a f r o m u n p u b l i s h e d experimental results: Rogers, G. T. a n d Searle, F.) C E A (ng/10 ml patient's serum) Group

Acetate

Borate

2% MG

10% M G

R e c t u m 2° Colonic 2 ° Gastric ca R h e u . arth. Gyn. ca

46 67 -

79 9 -

570 1230 15 15 31

478 720 5

although it is too early to comment on the significance of these studies. (Rogers G. T., and Searle, F., unpublished results).

XII. T H E A N T I G E N I C

SITE O F C E A

Degradation studies [41,42] have shown that 85 ~o of the carbohydrate residues in CEA can be removed without appreciable loss of antigenic activity, showing that the immunodeterminant is present in the innermost carbohydrate residues or in the protein sub-structure. The predominant sugar residue in the degraded material is N-acetylglucosamine and this is consistent with its strong reaction with wheat germ agglutinin. Earlier studies [57] had indicated that carbohydrate is involved in the immunological activity of CEA and that N-acetylglucosamine [57,58], or an asparagine-linked N-acetylglucosamine [59], may play a role in the determinant. These studies involved measuring the activity of CEA fragments obtained either by acid hydrolysis using polystyrene sulphonic acid or proteolysis using the enzyme nagase. Many of the fragments appeared to be predominantly carbohydrate enriched in N-acetylglucosamine relative to neutral sugars, but the activity was always considerably less than that of untreated CEA. More recent studies [60,41,61 ] have shown that integrity of the protein sub-structure of CEA is important for high immunological activity. Thus the activity is abolished by treatment of CEA with 0.5 M NaOH at 20 °C and reduced to 3-5 ~o of the activity of untreated CEA on cleavage of the disulphide bonds. The tentative conclusion reached [41]was that the carbohydrate moeity of CEA does not contain the immunodeterminant. This however may be an oversimplification since particular residues in the innermost part of the carbohydrate may be involved in the antigenic site but require to be held in a particular conformation by the protein sub-structure for full activity as suggested by Westwood and Thomas [61]. This may explain why a variety of N-acetylglucosamine-rich heterosaccharides failed to inhibit ~2SI-labelled CEA-anti-CEA binding [41]. Variation in the affinity of different CEA glycoproteins for anti-CEA antibodies may be accounted for by

369 heterogeneity of carbohydrate residues in or adjacent to the antigenic site or by direct perturbation produced by variation in the protein sub-structure. In view of its association with non-malignant diseases and also its presence in normal tissues (see below), the CEA determinant usually described cannot be strictly termed a tumour specific site (cf. Fuks et al. [48]). However the possibility exists that tumour specificity may be conferred on the structure of intermediate or peripheral sugar residues in some molecules which also possess CEA activity. Similarly there is no evidence ruling out possible cancer specificity residing in the protein structure of some CEA molecules.

XIIl. CLINICAL IMPLICATIONS The original optimism about the specific occurrence of CEA in entodermally derived tumours of the digestive tract [2] and in the serum of patients with such lesions [62] has not been sustained. Further studies [3,4,63,64] have demonstrated that CEA is associated with a variety of other malignancies and also with non-malignant conditions particularly those of an inflammatory nature [65]. CEA, shown to be immunologically indistinguishable from Gold's CEA, has also been found in normal tissues [4,68], in normal serum [67] and in normal saliva [66]. These findings have severely limited the diagnostic value of the CEA assay, but its importance in the follow-up of patients during and after treatment is well documented (see reviews by Terry et al. [21], Fuks et al. [48] and Zamcheck [69]). Obvious areas for improvement in the CEA assay which have a direct bearing on its clinical value are its reproducibility, the agreement with other CEA assays performed in different laboratories, its sensitivity and its specificity. Standardisation of antiserum, CEA label and assay standard are essential if assays using different batches of reagents are to agree. The available MRC reference CEA (73/601), to which the potency of internal laboratory standards can be related is useful and some progress has been made in defining the specificity of antisera used in the assay, and the need to exclude certain cross-reactive antibodies is recognised [16,50]. However differences have emerged between different preparations of CEA actually used for the assay. In view of the foregoing discussion there is clearly a need to standardise the methods of extraction and purification of CEA. The differences between saline and perchloric acid extracts and the binding of CEA to perchloric acid precipitates should be recognised and more studies made in this direction and a comparison made between the direct and indirect assays. It has already been demonstrated that the effect of perchloric acid can significantly effect the assay value. In view of the size heterogeneity of CEA it may be difficult to obtain a standardised CEA sample directly by gel filtration as used in the conventional procedure. However affinity chromatography using mono-specific anti-CEA antisera conjugated to Sepharose, applied to conventional CEA and to a25I-labelled CEA [70] has provided an effective means of separating CEA-like molecules from other impurities and has

370 enabled CEA which has been damaged in labelling to be separated prior to the assay. The results obtained in an assay using these reagents [70] showed the presence of CEA in the sera of patients with a range of cancers in accord with the earlier results of LoGerfo et al. [3]. However further standardisation of CEA particularly with regard to the potency of the molecule is undoubtedly possible using concanavalin A affinity chromatography. The technique is easy to apply in a reproducible manner to the conventional product and enables a product with a greater degree of homogeneity and a significantly higher potency to be obtained [54,46]. The use of concanavalin A Sepharose in the isolation of high activity ~2q-labelled C EA has also been described [71]. Further it has been demonstrated [54] that concanavalin A affinity chromatography can be employed to produce CEA directly from dialysed perchloric acid extracts thus eliminating the need for large scale gel filtration. The possibility that the CEA assay can be made more specific for colonic cancer by using a particular species of CEA as the radio-labelled ligand, has been raised by the work on CEA-S [26] and alternative studies are in progress to establish if fractions obtained by Concanavalin A affinity chromatography can usefully replace conventional CEA in radioimmunoassay (Rogers, G. T., Searle, F. and Bagshawe, K. D., unpublished work). Two points are worth considering in this context. First an improvement in specificity by using a more "specific" radio-labelled ligand is limited in a conventional inhibition assay by its affinity for the antibody compared with the affinity of different competing CEA molecules encountered in the serum being assayed. Secondly, CEA molecules that might have a higher specificity for cancer may not necessarily be the ones with highest immunological potency. To avoid this presupposition studies have recently been implimented (Rogers, G. T., unpublished work), to ascertain whether the proportions of the CEA fractions obtained by concanavalin A affinity chromatography vary with different diseases. If a suitable pattern emerges suggesting that a particular fraction relates to certain diseases it may be possible to design more discriminating assays. One possibility may be to incorporate a separation stage prior to a conventional assay but the ease of handling large numbers of specimens on a routine basis would have to be an important factor in judging its acceptability. A recent study which may have important implications in the CEA assay and emphasise the necessity to standardise reagents has recently been reported [72]. It has been shown that the calculated serum CEA concentration could vary by three orders of magnitude depending on the CEA standard, the amount of sera tested and on the particular anti-CEA antiserum used in the assay. Although different preparations of CEA, including various national standards, could vary in potency due to the presence of impurities, it is evident from this study that serum CEA may be antigenically different from tumour CEA and that the result of the assay may reflect the binding affinity of polyvalent anti-CEA antibodies to a mixture of different CEA iso-antigens. This is consistent with the previous isolation from conventional CEA of molecules with different specific activity by concanavalin A affinity chromatography [54] and of CEA-S [26] which is also immunochemically different from CEA.

371 T h e a b o v e s t u d y [72] also poses the exciting possibility t h a t antisera with high specificity for p a r t i c u l a r C E A iso-antigens m a y offer a new a p p r o a c h for the d e t e c t i o n o f a m o r e cancer-specific f o r m o f C E A a n d a m o r e reliable diagnosis o f malignancy.

x I v . CONCLUSION It has been the a i m o f this article to be selective a n d focus on those studies which d e m o n s t r a t e heterogeneity o f C E A . C E A exhibits m i c r o - h e t e r o g e n e i t y due m a i n l y to v a r i a t i o n in the n u m b e r o f t e r m i n a l sialic acid residues b u t there is also m o r e f u n d a m e n t a l v a r i a t i o n in the inner c a r b o h y d r a t e structure. It is this structure which m a y p l a y an i m p o r t a n t role in characterising C E A molecules a n d its v a r i a b i l i t y m a y be responsible for the discrepancies f o u n d between C E A m e a s u r e m e n t s in different l a b o r a t o r i e s a n d for the difficulty e n c o u n t e r e d in devising a m e t h o d o f p r e p a r a t i o n to o b t a i n C E A o f consistent quality. O f equal i m p o r t a n c e is the exciting possibility t h a t heterogeneity o f the inner a n d i n t e r m e d i a t e c a r b o h y d r a t e residues m a y result in C E A molecules which v a r y in specificity for different diseases. The carlzoh y d r a t e structure o f C E A is c o m p l e x b u t m o r e k n o w l e d g e o f this structure m a y help to solve the p r o b l e m s raised a b o v e p r o v i d i n g t h a t studies in this direction are n o t l o o k e d at w i t h o u t r e g a r d for their possible relevance to the use o f C E A as a t u m o u r m a r k e r substance.

ACKNOWLEDGEMENTS T h e a u t h o r t h a n k s Professor K. D. Bagshawe a n d Drs. F. Searle, M. W a s s a n d P. D. W i l s o n for their helpful suggestions a n d constructive criticism.

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