Transplant. Rev. (1974), Vol.

20
Published by Munksgaard, Copenhagen, Denmark
No part may be reproduced by any process without written permission from the author(s)

Carcinoembryonic Antigen:
Characterization
and Clinical Applications
WILLIAM D . TERRY, PIERRE A. HENKART, JOHN E . COLIGAN
& CHARLES W . TODD

I. INTRODUCTION

Embryonic and fetal tissues appear to contain substances that are absent, or
present in very much diminished concentration, in the mature organism. Some
of these substances can be recognized by immunochemical tests, and the anti-
gens detected are referred to as fetal or embryonic antigens. It is presumed
that at least some of these substances represent 'signals' or 'signal receptors'
that have been hypothesized to be required for differentiation and organo-
genesis of the developing embryo.
It is of great biological interest that malignant tumors in adult animals
frequently contain high concentrations of molecules usually found only in
fetal tissues. This was clearly demonstrated by Abelev et al. (1963), who dis-
covered that mouse liver tumors contained a high concentration of alpha feto-
protein, a substance normally found only in fetal tissue. Gold & Freedman
(1965 a, b) similarly found that tumors of the human gastrointestinal tract
contained substances absent from normal adult gastrointestinal tissues, but
present in the fetal gastrointestinal tract. Immunochemical tests showed that
this material, which was called carcinoembryonic antigen (CEA), could be
detected in the serum of patients with gastrointestinal malignancy, but not in
the serum of normal individuals or of patients with other cancers (Thomson et
al. 1969). The possibility that CEA assays might provide a diagnostic test for

From the Immunology Branch, National Cancer Institute, Bethesda, Maryland 20014,
and the Department of Immunology, City of Hope National Medical Center, Duarte,
California 91010.
This work was supported in part by grant # 12631 and contract # NCI-G-72-3877 from the
National Cancer Institute.
 CARCINOEMBRYONIC ANTIGEN 101

cancer provided impetus for extensive clinical trials, and also for investigations
of the immunochemical, physical and chemical characterization of CEA.
This review attempts to summarize recent publications concerning the
structure of CEA, presents some previously unpublished structural studies,
and briefly summarizes work on the clinical applications of CEA assays.

7.7 Nomenclature
Nomenclature in this field is awkward for historical reasons. Gold & Freedman
(1965 a, b) reported that antisera prepared against human colonic cancer
detected common antigens in extracts of fetal gastrointestinal tissue, but not in
extracts of normal adult colon or other normal adult tissues. These experi-
ments were performed by immunizing rabbits with saline extracts of a primary
colon tumor and then absorbing the antiserum with a similar extract of the
adjacent normal colon tissue from the same donor. The absorbed antiserum
precipitated the immunizing extract, as well as extracts from fetal gastrointes-
tinal tissue, and the reactive materials were therefore designated carcino-
embryonic antigens. Subsequent work has led to partial characterization of a
glycoprotein that carries these antigens, but unfortunately, the carrier has
also been called carcinoembryonic antigen (CEA). Thus, the term CEA has
been used interchangeably to refer to a poorly defined group of antigenic
determinants, and to the glycoprotein (or glycoproteins) upon which these
determinants are detected. While it might be preferable to refer to the carriers
as carcinoembryonic glycoproteins (CEGP) and the antigenic determinants
as CEA, there are sufficient gaps in our information about the carriers of
CEA that a revision of the nomenclature is probably unwise at this time. We
will therefore adhere to present conventions and refer to both the antigenic
determinants and the glycoproteins as CEA.

1.2 Definition and Immunochemistry of CEA

Lack of precision in the general nomenclature of CEA probably reflects the
fact that it is difficult to formulate a precise definition of CEA at the present
time. CEA is operationally defined as a glycoprotein of approximately
200,000 molecular weight, present in low concentration in normal adult tissue,
but in high concentration in fetal gastrointestinal tissue and a variety of malig-
nant tumors (reviewed in Gold 1972, Laurence & Neville 1972, Terry et al.
1973). CEA carries antigenic determinants detected by antisera prepared in
the following manner:
a. glycoproteins are extracted and isolated from primary or metastatic
human gastrointestinal cancers by the- methods described in Section 2.2;
102 TERRY, HENKART, COLIGAN & TODD

b. the purification is monitored using a preexisting antiserum which must

be obtained from one of the other workers in the field (in this sense, all CEA
preparations have at least some relationship to the original CEA of Gold &
Freedman);
c. the purified material is used to immunize any of a variety of species
of animals;
d. the resulting antiserum is shown to precipitate the immunizing material,
as well as a reference preparation of CEA obtained from one of the other
workers in the field, and to give a reaction of complete identity between the
two preparations;
e. the antiserum is absorbed to render it CEA specific (see below for
reasons).
Thus, in the usual immunochemical sense, CEA is circularly defined as the
antigenic determinants detected by the antiserum raised against the glyco-
protein carrying the antigenic determinants. The chemical nature of the anti-
genic determinants (i. e. protein or carbohydrate) is not known at the present
time. Information on this point is summarized in Section 5.3.
It is important to note that much of the work done on CEA has leaned
heavily on immunochemical measurements with much less attention paid to
other molecular parameters. Antisera prepared against complex substances,
such as CEA, may vary in regard to the antigenic determinants they detect,
and improper conclusions will be reached unless the limitations of the method
are kept in mind. The definition of CEA antigenic determinants and carrier
glycoproteins will require considerably more chemical and physical charac-
terization of all substances considered to be CEA by immunochemical tests.

1.3 Distribution and Cellular Localization

CEA is detected in high concentration in tissue extracts of the fetal gastro-
intestinal tract and of most malignant tumors of the gastrointestinal tract. It is
not specific for gastrointestinal tract cancer, however, since CEA can also
be extracted from primary lung tumors (Pusztaszeri & Mach 1973, Sizaret&
Martin 1973) and other non-gastrointestinal malignancies (Pusztaszeri & Mach
1973). Similarly, CEA is present in high concentration not only in the plasma of
patients with gastrointestinal tract malignancies, but also in plasma of
patients with other malignancies and non-malignant diseases (Lo Gerfo et al.
1971, Moore et al. 1971) and, indeed, in normal plasma (Chu et al. 1972)
and normal tissue (Martin & Martin 1970, Burtin et al. 1972 a, Kupchik &
Zamcheck 1972, Lo Gerfo & Herter 1972, Pusztaszeri & Mach 1973). The
CEA detected in malignancies is thus a quantitative rather than a qualitative
alteration from normality. Failure to detect CEA in earlier studies of normal
 CARCINOEMBRYONIC ANTIGEN 103

tissues probably reflects the minute quantities of CEA present in these tissues.
CEA has been referred to as a membrane protein, and it probably should
not be considered as such. It certainly is not an integral membrane protein (Sin-
ger 1971), in that it can be removed from cell membranes by incubation with
saline. CEA can indeed be detected on cell surfaces (Gold et al. 1968, von
Kleist & Burtin 1969, Gold et al. 1970, Denk et al. 1972), but appears to be
loosely adherent and is propably present at the outer surface of the cell mem-
brane following secretion.

1.4 Cross Reactive Tissue Antigens

The question of whether CHA is present in normal tissue has been complicated
by the detection of substances which share antigenic determinants with CEA.
Antisera prepared against purified CEA precipitate these normal tissue com-
ponents which have been called NGP (Mach & Pusztaszeri 1972), NCA (von
Kleist et al. 1972), FSA (Hakkinen 1972), CCEA-2 (Darcy et al. 1973),
BCGP (Kuo et al. 1973) and NCA-2 (Burtin et al. 1973). Preliminary results
suggest that NCA, NGP, CCEA-2 and BCGP may be identical (Mach, per-
sonal communication). Anti-CEA antisera give reactions of partial identity be-
tween CEA and these cross-reacting substances, and can he rendered specific
for CEA by absorption with extracts of normal tissue.
NGP has been partially characterized and shown to be smaller than CEA
and to contain a somewhat lower content of carbohydrate, consisting of the
same monosaccharides as are present in CEA, but in different molar ratios
(Mach & Pusztaszeri 1972).
It is not yet known whether the antigenic determinants shared between
NGP and CEA are located on the protein or carbohydrate portions of these
molecules, and amino acid sequence studies of NGP and related substances
have not yet been reported (see Section 4.3).

2. ISOLATION AND PURIFICATION

2.1 Sources
Various sources have been used to prepare CEA. Studies on the chemistry
of CEA have been carried out on primary colon tumors but, because of the
small size of such tumors at the time of excision, most workers have chosen
to extract the antigen from bowel tumor metastases. These metastases can
become very large (especially in the liver) and, while they have a variable
CEA content (Coligan et al. 1972) many are a convenient source for isola-
tion of large amounts of CEA. In the most favorable cases, as much as one
104 TERRY, HENKART, COLIGAN & TODD

gram of CEA can be obtained from a single metastatic tumor. In comparing

studies on CEA chemistry, it may be important to distinguish between the
tissue sources of antigen used, since CEA may be affected by enzymes in the
environment of the tissues used for isolation. In particular, large metastatic
tumors are usually obtained from cadavers, and it is entirely possible that in
such tissue autolytic degradation of the antigen can take place.
Obtaining large quantities of tumor material for chemical studies can be a
problem. In the future it may be possible to isolate large amounts of CEA,
without danger of extensive autolysis, from colon tumor cell lines which have
been developed by Goldenberg and collaborators and have been carried in vivo
(Goldenberg& Hansen 1972) and in vitro (Goldenberg et al. 1972, Laing et al.
1972, Egan & Todd 1972).

2.2 Purification
Most attempts at purification have started with the initial steps used by Gold
& Freedman (1965 a) who found that CEA was soluble upon treatment of a
tissue homogenate with perchloric acid. This step effects a large purification
since perchloric acid denatures and precipitates almost all proteins other than
those, like CEA, with a high sugar content. The possible physical or chemical
alteration of native CEA by this harsh treatment must be borne in mind, how-
ever.
Subsequent steps in CEA purification by all laboratories invariably include
gel filtration on Sephadex G-200 and/or agarose. The procedure we have used
to isolate CEA from hepatic metastases consists of homogenization and per-
chloric acid extraction followed by two gel filtration columns; the first one
with Sepharose 4B, the second on Sephadex G-200 (Coligan et al. 1972).
Zbne electrophoresis before or after gel filtration is used by some groups
(Krupey et al. 1972, Turner et al. 1972) but not by others (Coligan et al. 1972,
Turberville et al. 1973). Its utility may depend on the resolution in the gel fil-
tration systems previously used in the preparation. Electrofocusing of gel-
filtration purified CEA was reported to result in little or no increase in specific
activity (Coligan et al. 1973). On the other hand. Turner et al. (1972) were
able to eliminate some impurities by electrofocusing their CEA preparation
from primary colon tumors.
An independent approach to CEA isolation not involving perchloric acid
has been taken by Rosai et al. (1972) using primary tumors. They first pre-
pared a crude membrane fraction from the colon tumors, solubilized CEA
with lithium diiodosalicylate, and removed the proteins by a phenol extrac-
tion. The resulting crude glycoprotein was further subjected to chromato-
graphy and electrofocusing to yield purified CEA. The authors claimed a
 CARCINOEMBRYONIC ANTIGEN 105

higher recovery of CEA than was obtained from primary tumors by Krupey
et al. (1968) using a traditional perchloric acid extraction, but it should be
noted that CEA concentration varies markedly in different tumors.

2.3 Homogeneity
Glycoproteins are unlikely to be as homogeneous as proteins because the en-
zymatic synthesis of the sugar moieties is not as faithful as the m-RNA
directed protein synthesis, and well studied purified glycoproteins show heter-
ogeneity of their carbohydrate groups (Montgomery 1972). Thus, sensitive
criteria for purity may demonstrate heterogeneity in a glycoprotein prepara-
tion that contains a single protein backbone with a distribution of different
sugar sidechains.
In the case of CEA, analytical ultracentrifugation has shown a single sym-
metrical peak for preparations derived from various sources (Krupey et al.
1968, Coligan et al. 1972). Other criteria based on electric charge have been
less clear cut. Polyacrylamide gel electrophoresis of CEA under various con-
ditions shows a single broad band when stained for sugar or protein, with no
minor components (Rosai et al. 1972, Terry et al. 1972, Turberville et al. 1973,
Banjo et al. 1974). CEA activity shows the same broad peak with a similar Rf
(Coligan et al. 1972). As discussed below, electrofocusing experiments show
heterogeneity with preparations from liver metastases, while in two cases,
preparations from primary tumors are reported to give a single peak (Rosai
et al. 1972, Turner et al. 1972).
In spite of the heterogeneity demonstrated by electrofocusing, Terry et al.
(1973) report a single ami no-terminal sequence in their CEA preparations.
The finding of an unambiguous sequence is good evidence for the presence
of only a single protein with an unblocked amino terminus, but does not rule
out other proteins with blocked amino termini, or non-protein impurities.
Turner et al. (1972) used centrifugation in a CsCl gradient as a step in puri-
fying their radioiodinated CEA. This technique separates molecules on the
basis of their buoyant density, which in this case is probably largely based on
the ratio of carbohydrate to protein. They found two radiolabeled impurities in
their preparations, a finding which suggests that this technique should be
applied to other CEA preparations as a homogeneity criterion.

3. PHYSICAL PROPERTIES
3.1 Size
No rigorous molecular weight determinations such as by sedimentation equi-
librium have been carried out with CEA. As a rough approximation a value
106 TERRY, HENKART, COLIGAN & TODD

of 200,000 to 300,000 is suggested by the fact that CEA from various sources
normally elutes between IgM and IgG on a Sephadex G-200 column (Coligan
et al. 1972, Puztaszeri & Mach 1973). The sedimentation coefficient is in
reasonable agreement with a value in this range, but without other physical
data not yet available a molecular weight cannot be calculated. This range of
normally elutes between IgM and IgG on a Sephadex G-200 column (Coligan
molecular weight is also compatible with the yield of amino terminal amino acid
recovered during protein sequence studies (Terry et al. 1973).
An unusually large molecular species of CEA has been reported by Coligan
et al. (1972). This large CEA was found along with normal size CEA and was
not convertible to the latter by treatment with dithiothreitol.
It is possible that CEA as it is normally isolated may consist of several
glycoprotein chains held together by disulfide or noncovalent bonds. CEA,
reduced and alkylated with 10 mM dithiothreitol and 20 mM ^*C-iodoacetamide
in 9 M urea, has the same Rf in SDS acrylamide electrophoresis as untreated
CEA (M. Egan, personal communication). In spite of the anomolous behavior
of glycoproteins on SDS gels, the fact that the untreated and the reduced and
alkylated CEA have the same Rf suggests that CEA is composed of only one
polypeptide chain. Amino terminal sequence determinations (Terry et al. 1972)
show only one sequence, and as discussed above, the yield is compatible with
one protein chain per molecule. SDS gel electrophoresis cannot be used to
estimate the molecular weight of the protein chain using standard methods
because of CEA's high carbohydrate content (Weber et al. 1972, Segrest et al.
1971).

3.2 Sedimentation behavior

Purified CEA preparations give a single symmetrical peak in the analytical
ultracentrifuge. Coligan et al. (1972) report a S°2o, w of 6.8 S. Sedimentation
coefficients uncorrected for concentration ranging from 6.9 to 8.5 have been
obtained for CEA from various sources (Krupey et al. 1968, Mach & Pusz-
taszeri 1972).
Turner et al. (1972) obtained 3 peaks upon ultracentrifugation of purified
CEA in a CsCl gradient. The most antigenically active material was found at
a density of 1.42. It would appear to be too low for the buyoant density of a
glycoprotein containing more than 50 per cent carbohydrate.

3.3 Shape
Physical studies capable of giving significant information regarding the shape
of CEA molecules have not yet been performed. The lack of a hypersharp
 CARCINOEMBRYONIC ANTIGEN 107

peak in the analytical ultracentrifuge and the moderate dependence of the

sedimentation coefficient on concentration (Coligan et al. 1972) implies that
CEA is probably not an extremely elongated or fibrous molecule.

3.4 Charge
CEA has been reported to migrate in immunoelectrophoresis as a /^-globulin
(Krupey et al. 1967), although different preparations appear to show differ-
ences in migration (Pusztaszeri & Mach 1973).
Isoelectric focusing, a technique of high resolution which 'focuses' mole-
cules at their isoelectric point in pH gradient, has been applied to CEA by
several laboratories with different results. Rosai et al. (1972) and Turner et al.
(1972) working with extracts of primary colon tumor (prepared by different
methods) report CEA activity in one peak showing little heterogeneity with an
isoelectric point of 4.8 and 4.7, respectively. However, Rule & Goleski-Reilly
(1973 a, b) show multiple CEA activity peaks on their saline extracts of pri-
mary colon carcinomas when electrofused in urea. CEA purified from hepatic
metastases of colon and rectum tumors was found by Coligan et al. (1973)
to show extensive heterogeneity of activity as measured by radioimmunoassay,
with the major peaks at the acidic end near pH 3. Isoelectric heterogeneity
of metastatic CEA has been confirmed by Turberville et al. (1973), who found
the major activity peak at pH 4.4, and by Banjo et al. (1974 b) who found two
major peaks at pH 3.0 and 3.75. Coligan et al. (1973) have shown that the
charge heterogeneity is not entirely due to sialic acid, since after its removal
with neuraminidase some heterogeneity remained. Rule & Goleski-Reilly
(1973 b) recently have shown that perchloric acid treatment of a saline tumor
extract containing CEA causes a marked shift in the complex isoelectric
focusing pattern they obtain. It seems at present difficult to reconcile the
varying results of isoelectric focusing of CEA, but at least three variables may
be important; 1) tissue source of the antigen, 2) prior treatment with perchloric
acid, and 3) individual tumor variation (e. g. Figure 2 in Coligan et al. 1973).

4. PROTHN CHEMISTRY
4.1 Chemical composition
Chemical analyses of purified CEA from hepatic metastases of bowel tumor
indicate that it is a glycoprotein containing approximately 50 per cent sugar
(Tables II and III). Elemental analyses in agreement with this have been
published by Coligan et al. (1972). It is interesting to note that the combined
carbohydrate plus protein yields do not exceed 90 per cent in most of these
108 TERRY, HENKART, COLIGAN & TODD

TABLE I
Amino Acid Composition of CEA Preparations
Moles Amino Acid/10^ g Protein

Amino Acid 1 2 3 4

lys 23.5 25.7 24 32

his 17.2 19.1 12 21
arg 33.9 34.6 32 36
asp 142.0 127.0 150 157
thr 77.6 73.9 88 100
ser 90.2 91.1 108 111
glu 94.5 94.5 104 94
pro 103.8 70.2 76 76
giy 49.0 50.6 52 54
ala 54.1 56.1 56 62
V6 cys 1
val 65.6 68.0 64 nd
met 1.0 2.0 0
ileu 45.6 44.8 48 53
leu 82.3 84.7 88 84
tyr 36.9 49.5 24 38
phe 22.4 nd 20 24
cys+cys—cys^ nd 17.4 nd

a. determined as cysteic acid after performic acid oxidation

b. nd - not determined
1 CEA-Be, Terry et al. (1972).
2 CEA-St, Coligan et al. (1973).
3 Banjo et al. (1972).
4 Turberville et al. (1973).

preparations and in one case the yield does not exceed 50 per cent. Banjo et al.
(1974 b) have reported that CEA does not contain fatty acids. It is unclear
why the combined weight of the analyzed components does not add up to 100
per cent of the dry weight.

4.2 Amino acid analysis

Amino acid analyses of CEA-preparations purified from metastatic bowel
tumors have been performed by several groups, and the results have shown a
general distribution of amino acids which is not unlike that of many proteins.
The comparison of amino acid compositions is rendered more difficult because
the values are usually expressed as moles of amino acid per 10= g of dry
weight. However, since the protein content (per cent of dry weight) of CEA
 CARCINOEMBRYONIC ANTIGEN 109

TABLE II
Carbohydrate composition of various CEA preparations

Micromoles per 100 mg CEA

a b c d e f g
Fucose 37 15 57 54 49 20 55
Mannose 38 49 20 45 38 33 50
Galactose 51 62 72 71 107 26 71
N-Acetyiglucosamine 48 97 118 127 132 62 96
Sialic Acid 34 24 7.5 12 59 7 8.5
N-Acetylgal actosamine NS* NS NS 11 9.8 NS 11
Carbohydrate per cent 42 47 57 58 77 27 52
Protein per cent 40 25 20 34 16 21 38

a. Mach, J. P. & Pusztaszeri, G. (1972), Source of CEA not reported.

b. Banjo et al. (1972), CEA prepared from a liver metastasis of a colon adenocarcinoma.
c. Banjo et al. (1974 a), CEA prepared from hepatic metastases of a colonic adenocar-
cinoma.
d. Banjo et al. (1974 b), CEA prepared from hepatic metastases of colonic adenocarci-
nomas (average of three preparations).
e. Banjo et al. (1974 b), CEA prepared from adsnocarcinoma of the stomach.
f. Values determined by Hansen reported by Kupcbik et al. (1974).
g. Coligan, J. E., Egan, M. L. & Todd, C. W. (unpublished observations), average of
values in Table III.
* NS - Not significant.

seems to be variable, the amino acid levels expressed on a dry weight basis
can vary from preparation to preparation even though the basic protein com-
position remains constant. We therefore have expressed our amino acid com-
positions as moles of amino acid per 10^ g of protein, eliminating the dry
weight measurement as a factor in the value. These data, shown in Table I,
differ somewhat from the original compositions reported (Krupey et al. 1968),
but are similar to more recent reports from the same group (Table I,
column 3).
Table I shows amino acid compositions of 4 different CEA preparations
from three laboratories. All show generally similar features - high levels of
carboxyl and hydroxyl amino acids, rather low levels of basic amino acids,
and very low levels of sulfur-containing amino acids. Some glycoproteins with
a high carbohydrate content, e. g., blood group substances (Watkins 1972),
show an amino acid content which is quite atypical of proteins in general, but
such is not the case with CEA.
no TERRY, HENKART, COUGAN & TODD

TABLE n i
Carbohydrate composition of individual CEA preparation^

Micromoles/lOO mg CEA

Cal-24 OE^70 Rol NeB6 Ro4Bl,3 MiC SiBl

Fucose'' 63 42 46 57 63 57 58
Mannose'' 57 53 61 44 50 44 39
Galactose'' 56 67 81 63 82 70 75
N-Acetyl-
glucosamine'' 77 98 113 102 98 87 99
Sialic Acid^ ND« 6.6 8.8 0.6 0.2 14 21
N-acetyl-
galactosamine*^ 8.3 7.5 7.4 22 8.9 10 13
% carbohydrate 45 49 57 53 53 50 56
% protein 46 45 38 37 35 35 30

Total 91 94 95 90 88 85 86

a Unpublished data, Coligan, Egan & Todd. All CEA preparations were isolated from
individual tumors as described by Coligan et ai. (1972). All the preparations except
Mi and SiBl were isolated from liver metastases. Mi was a primary colon and SiBl
was a primary liver tumor.
I" Neutral sugars were determined by gas chromatography according to the method of
Clamp etal. (1972).
"^ Determined on the Beckman 121 amino acid analyzer using the hydrolysis method of
Liu & Chang (1971).
•J Determined by the thiobarbituric acid assay (Warren 1959) and/or by gas chromato-
graphy (Clamp et al. 1972).
«i Not determined.

4.3 N-terminal amino sequence

The N-terminal amino acid sequence of CEA preparations from liver meta-
stases has been determined (Terry et al. 1973, Figure 1), and a remarkably
'clean' sequence was obtained, with very small amounts of secondary amino
acids appearing at each step. This implies homogeneity of the CEA polypep-
tide homogeneity chain, at least at the N-terminus, despite evidence for hetero-
geneity of the whole molecule. It is possible that the CEA molecule contains
additional polypeptide chains of differing sequence and with blocked termini,
but quantitative considerations make this unlikely (Terry et al. 1972). The
identical sequence has now been found for CEA isolated from five different
metastatic bowel tumors (Terry, Henkart, Coligan & Todd, unpublished ob-
servations) and also for a crude CEA preparation from a human colonic tumor
Une maintained in hamsters (Goldenberg & Hansen 1972). This latter se-
 CARCINOEMBRYONIC ANTIGEN HI

quence shows heterogeneity, consistent with other evidence that purification

of these preparations is incomplete.
The amino acid sequence of CEA isolated from serum has also been deter-
mined. Serum containing several /^g/ml of CEA was obtained from a patient
with advanced metastatic colon cancer. The serum was treated with an equal
volume of 2M perchloric acid and the extract purified with the same proce-
dures used for tissue extracts (Figure 2). The serum CEA activity peak elutes
at the same volume as does CEA from metastatic tumors, indicating that the
two activities are carried on molecules of approximately the same size. The
amino acid sequence of serum CEA is homogeneous and identical to that
shown in Figure 1.

5 10
LYS-LEU-THR-ILE^GLU-SER-THR-PRO-PHE-ASN
15 20
VAl^ALA-GLU-GLY-LYS-GLU-VAL-LEU-LEU-LEU
24
VAU-HIS-ASN-LEU
Figure 1. N-terminal amino acid sequence of CEA. TTie sequence was determined using
an automatic protein sequencer (Beckman 890). From Terry et al. (1972).

As a control, normal serum (containing a low CEA concentration) was

extracted with perchloric acid and the extract filtered through the same
Sepharose and Sephadex columns. Amino acid sequence studies were per-
formed on material eluted in the same vdume as tissue and serum CEA.
Multiple amino acids were detected at each step, and no coherent sequence
was identified. These observations strongly support the conclusion that the
major glycoprotein present in the purified CEA preparations is indeed the
molecule with which CEA activity is associated, since it appears unlikely that
the same contaminant would be isolated from tumor extracts and serum and
that it would be present only when CEA activity is detectable.
Recently Bhargava et al. (1974) have confirmed this N-terminal sequence
in their studies of CEA isolated from hepatic metastases and purified by a
similar procedure. They were able to sequence the first 30 residues from the
N-terminal and found the first 24 to be identical to those shown in Fig. 1,
with the exception that glutamine, rather than glutamic acid, was detected at
positions 5 and 16.

5. CARBOHYDRATE CHEMISTRY

Considerable attention has been focused on the carbohydrate portion of CEA

because it constitutes a large part of the molecule, and because of the suspicion
112 TERRY, HENKART, COLIGAN & TODD

that it might account for some or all of the antigenic sites detected by anti-
bodies to CEA.

5.7 Composition
As can be seen from Tables II and III, the carbohydrate compositions reported
for CEA isolated from different tumors vary widely. Significant differences
are seen even for individual preparations isolated within the same laboratory
(Krupey et al. 1968, Mach & Pusztaszeri 1972, Banjo et al. 1972, 1974 a, b,
Egan et al. 1974 b). Heterogeneity of the carbohydrate portion of CEA
(discussed in Sections 2.1 and 5.2) may account for some of these differences.
Table III gives a sampling of the values obtained from analyses of many
CEA preparations. Values for all the sugars present in CEA can be obtained

500 600 700 800

ELUTION VOLUME, cc

Figure 2. Purification of serum CEA by gel filtration. Upper: Sepbarose 4B column.

The sample was 141 mg of a perchloric acid extract of plasma from a patient with
extensive hepatic metastases from a colon carcinoma. Lower: Sephadex G200 column.
The sample consisted of the pooled effluent of the Sepharose 4B column eluting between
900 cc and 1070 cc which was dialyzed and lyophilyzed before application to the column.
Both columns were 3.5x155 cm, and were eluted with 0.14M NaCl, 0.5M sodium
phosphate, pH 5.5, at lOcc/hr. • • , CEA activity, detected by a double antibody
radioimmunoassay (Egan et al. 1974 b), '-280'
 CARCINOEMBRYONIC ANTIGEN

using the gas chromatographic procedure of Clamp et al. (1972). However, of

the values shown in Table III, only the neutral sugars were obtained using this
method. Glucosamine and galactosamine were determined on the amino acid
analyzer during the amino acid analyses by the method of Liu & Chang (1971).
Amino sugar values obtained by gas chromatography were always 10 to 20
per cent lower. This discrepancy probably can be attributed to the relative
resistance of the hexosaminide bonds if de-acetylation occurs prior to acid
hydrolysis (Rovis et al. 1973) The more severe conditions required for amino
acid analysis probably insure more complete hydrolysis of the hexosaminide
bonds. Although sialic acid values can also be obtained by gas chromato-
graphy, the thiobarbituric assay of Warren (1959) was found to be more
reproducible. The per cent of carbohydrate varies over a narrow range (45 to
57 per cent) with the average being 52 per cent. The per cent of protein shows
much greater variability, ranging from 30 to 46 per cent. Total yields of carbo-
hydrate plus protein range from 85 to 95 per cent. Even though all samples were
dried over P2O5 prior to weighing for analysis, failure to achieve 100 per cent
recovery may possibly be due to tightly bound H2O.
In the CEA preparations studied by Coligan, Egan & Todd, N-acetylglucos-
amine was always the monosaccharide present in highest concentration, but ex-
ceptions have been obtained in other laboratories (Mach & Pusztaszeri 1972,
Burkhard et al. 1973). It has been assumed in these studies that all amino
sugars are in their acetyl form, as is usually the case in glycoproteins. No
quantitative determination of acetyl groups has been performed. Characteriza-
tion by paper chromatography of the sialic acid liberated from CEA by mild
acid hydrolysis indicated it to be N-acetyl-neuraminic acid (Coligan &. Todd,
unpublished observations). Galactose is the predominant neutral sugar except
in one preparation (Cal-24). This agrees with data reported by several labora-
tories (Banjo et al. 1972, 1974 a, b. Mach & Pusztaszeri 1972, Burkhard et al.
1973), but disagrees with data initially reported by Krupey et al. (1968). In
agreement with results initially published by Krupey et al. (1968), the mono-
saccharide showing the greatest variability among different CEA preparations
is sialic acid.
Conversion of the data in Table III to mole per cent of the carbohydrate
(Table IV) illustrates the constancy of the N-acetylglucosamine and galactose
content of CEA. The rather constant values suggest that these sugars may
represent the core of the carbohydrate units in CEA. It is interesting to note
that the decrease in mole per cent of mannose correlates with the decrease in
per cent by weight of protein. Since limited data are available, it may only be
coincidental that the two primary tumors (Mi and SiBl) are lowest in per cent
of protein and mole per cent of mannose.
In accord with observations on other mammalian glycoproteins (Heath 1971),
114 TERRY, HENKART, COLIGAN & TODD

TABLE IV
Carbohydrate composition of individual CEA" preparations

Mole per cent of carbohydrate

Cal-24 OE-70 Rol NeB6 Ro4Bl,3 MiC SiBl

Fucose 24.1 15.3 14.5 19.8 20.9 20.2 19.0
Mannose 21.8 19.4 19.2 15.2 16.6 15.6 12.8
Galactose 21.4 24.4 25.5 21.8 27.1 24.8 24.6
N-acetyl-
glucosamine 29.5 35.8 35.6 35.3 32.4 30.9 32.5
Sialic acid NDb 2.4 2.8 0.2 O.I 5.0 6.9
N-Acetyl-
galactosamine 3.2 2.7 2.3 7.6 2.9 3.5 4.3

* Coligan & Todd, unpublished data.

1" Not determined.

experimental data we have obtained (Coligan et al. 1974, Coligan et al. 1973,
Egan et al. 1974 a) indicate that fucose and sialic acid are expected to be termi-
nal sugars. Due to the heterogeneity commonly experienced in mammalian
glycoproteins (Spiro 1970), these sugars would be expected to vary in different
CEA preparations.
N-acetylgalactosamine has been detected in relatively low amounts in CEA
preparations (Mach & Pusztaszeri 1972, Burkhard et al. 1973, Coligan et al.
1973, Banjo et al. 1974 b, Egan et al. 1974 b. Tables III and IV) and in some
cases is not detectable at all (Krupey et al. 1968, Banjo et al. 1972, 1974 a, b,
Burkhard et al. 1973). The significance of this will be discussed in more detail
later.
Some investigators (Krupey et al. 1967, Haverback & Dyce, 1974) have
noted the presence of glucose in their CHA preparations. In one instance
(Krupey et al. 1967), this was partially attributed to contamination by Sepha-
dex gel. We have observed the presence of trace amounts of glucose in several
of our samples. Whether this glucose is actually present in CEA or represen-
tative of some contamination or impurity is not known. Sephadex and cellulose
dialysis tubing represent likely sources of contamination. In some instances
we have observed that glucose is present in the perchloric acid extract prior
to column chromatography. It is therefore possible that trace amounts of glu-
cose-containing molecules are sometimes isolated along with CEA.

5.2 Relationship to heterogeneity

Fractionation of purified CEA by ECTEOLA-cellulose chromatography and
 CARCINOEMBRYONIC ANTIGEN

TABLE V
Carbohydrate Composition of
Isoelectric Focusing Pools

Micro moles/100 ng

Initial
Fucose 41 52 47 41 40 35
Mannose 48 36 49 50 54 57
Galactose 50 78 56 47 46 29
N-Acetyl-
glucosamine 87 96 83 79 87 84
Sialic Acid 7.5 14 92 7.2 5.5 4.1
N-Acetyl-
galactosamine 3.9 11 5.9 4.4 2.5 3.9

ph Range 1.5 -«—• 3.3 *—> 3.8 -.—.- 4.1 -—• 4.4 *—* 5.2
Carbohydrate per cent<: 43 52 45 41 42 38
Protein per cent 54 44 40 43 41 43

Glucose per cent by wt. 0.86 11.8 12.4 11.0 10.5 17.4
Specific activity^, a 1.23 0.69 1.16 1.54 1.39 1.45

* Coligan & Todd, unpublished data.

'' Ail values were calculated as described in Table II and corrected for glucose contami-
nation (refer to text). Data in a previous study, Coligan et al. (1973), were not cor-
rected for the sucrose contamination, which probably occurred.
" Corrected to eliminate glucose content.
^ radioimmunoassay value
dry weight

by isoelectric focusing indicates that CEA possesses considerable charge

heterogeneity (Coligan et al. 1973, and Section 3.4). The bulk of the material
electrofocuses between pH 2 and pH 4, and fractions vary in their content of
sialic acid, amino sugars, and amino acids. Recent investigation (Coligan et al.
1974) has also shown variability in the neutral sugar content of the fractions
from the electrofocusing column. Table V presents the carbohydrate compo-
sition of the pools from a typical electrofocusing run. The sialic acid and
amino sugar values are similar to those previously reported (Coligan et al.
1973). In both ECTEOLA-cellulose chromatography and isoelectric focusing,
the charge heterogeneity correlates with variable per cent by weight of sialic
acid; the more negative the charge on the CEA molecule, the higher the sialic
acid content. Although removal of sialic acid with neuraminidase renders the
isoelectric focusing patterns more homogeneous and increases the isoelectric
point of the bulk of the material to pH 4.7, some heterogeneity still remains
 TERRY, HENKART, COLIGAN & TODD

(Coligan et al. 1973, Banjo et al. 1974 b). An interesting observation is that
of the neutral sugars, fucose and galactose decrease and mannose increases
with increasing electronegativity. Of the amino sugars, glucosamine remains
relatively constant in all the pools, whereas the majority of the galactosamine
is present in the most electronegative pools. Comparison of the precolumn
sample with the resultant pools indicates that the electrofocused samples
were contaminated with glucose. This can probably be attributed to the sucrose
used to form the electrofocusing column density gradiant. The tenacity of this
binding is remarkable considering that these pools were dialyzed against 3M
urea for 4 days, 2M urea plus 2M NaCI for 2 days, 2M NaQ for 3 days and
deionized water for 1 week.
Despite the chemical differences that exist among the column pools, the
antigenic determinants recognized by goat anti-CEA appear to be similar in all,
as evidenced by the lines of identity obtained in Ouchterlony analysis (Coligan
et al. 1973). The data in Table V show that pool 1 has a specific activity
(radioimmunoassay value divided by dry weight) approximately 50 per cent
less than that of the other pools. This low specific activity correlates with the
high quantity of galactosamine in the most acidic pool. Since previous studies
(Coligan et al. 1973) have shown galactosamine not to be necessary for CEA
activity, and since other authors have reported the absence of galactosamine
in CEA (Krupey et al. 1968, Banjo et al. 1972, Burkhard et al. 1973), this
suggests that the acidic fractions possess impurities containing galactosamine.
Another indication of the contribution of carbohydrate to the heterogeneity
of CEA comes from studies of the interaction of purified CEA with various
lectins (Terry et al. 1974). CEA is precipitated by Conconavalin A, wheat
germ agglutinin, and ricin which specifically bind mannose, N-acetylglucos-
amine and galactose, respectively, as well as by leukoagglutinating phyto-
hemagglutinin, which binds a more complex determinant. The double diffu-
sion precipitin patterns show that the reaction between CEA and anti-CHA
spurs over the reactions between CEA and each of the lectins, which in turn
spur over one another in a manner which varies with the CEA preparation.
Thus, each of these lectins appears to bind a different subpopulation of CEA
molecules, reflecting the heterogeneity of carbohydrate on these molecules.

5.3 Role of carbohydrate in the antigenic site

It has not been determined to what extent the polysaccharide portion of CEA
is involved in the antigenic determinant. Abeyounis & Milgrom (1972) have
boiled specimens containing CEA for 1 hour without loss of antigenic activity.
Stability to boiling has also been reported by Terry et al. (1974). This suggests
that the antigenic determinant(s) may be a polysaccharide or located on a
 CARCINOEMBRYONIC ANTIGEN 117

TABLE VI
Fragments from Nagase Hydrolysis of CEA *

A B C D E
50% Assay % Starting
Inhibition Ratio equivalents Material
Weight activity B/2.7 of CEA 100 D/305
(mg) (ng) (mg)

CEA 2.7 305 100

Nagase — Nondialyzable
>• C E A 34 3.7-ia* 11.9-103 2.86-10-3 0.0009
Dialyzabie

CEA 205 6.5103 2.M03 97.6-10-3 0.032

G-25
.. GP-2 6.6 197 63.1 0.105 0.034
-^ GP-3 24.2 225 72.1 0.336 0.11
-^ GP-4 32.7 175 56.1 0.583 0.191
-^ GP-5 5.1 143 45.8 0.11 0.036
^ GP-6 2.0 AA.A 14.2 0.I4I 0.046
-* GP-7 3.5 105.0 33.7 0.104 0.034
GP-1 75 31.5 10.1 7.43 2.4
Cellulose
powder—
*- OSF" 20
^ GPl-
DW" 40 15.75 5.05 7.92 2.56
AG50W-X4
-^ GPl-A 14 55.0 17.6 0.79 0.26
-^ GPl-B 5.5 71.4 22.9 0.24 0.08
-^ GPl-C 1.0 37.5 12.0 0.08 0.03
-^ G P I - D 1.2 24.4 7.8 0.15 0.05
-^ G P l - E 8.0 35.6 11.4 0.70 0.23
-* G P l - F 1.4 37.5 12.0 0.12 0.04

" Organic soluble fraction

*> Distilled water fraction
* Banjo eL a. (1974 a)

portion of the polypeptide chain that is stabilized by the proximity of poly-

saccharide groups. However, several proteins lacking carbohydrate are known
to be relatively resistant to boiling. The crossreactivity of CEA with blood
group substances also suggests that the antigenic site may be located on the
carbohydrate portion of the molecule.

Transplant. Rev. (1974), Vol. 20

118 TERRY, HENKART. COLIGAN & TODD

Banjo et al. (1972) have isolated four immunologically active heterosaccha-

ride fragments from CEA which are free of amino acids. The only sugar present
in three of the four fragments was N-acetylglucosamine. The most active frag-
ment contained N-acetylglucosamine but was predominantly marmose. How-
ever, these fractions were 5 to 10 thousand times less active in the radioimmuno-
assay than intact CEA molecules. This low activity could possibly be explained
by the expected inability of small molecules containing only a portion of the
antigenic site to compete successfully in the radioimmunoassay with the com-
plete sites present on CEA molecules.
Inhibition studies by Banjo et al. (1973) using synthetic compounds sug-
gested that the linkage between asparagine and N-acetyl-D-glucosaraine was
immunodominant in the antigenic site of CEA. Arguing against this possibility
is the fact that this grouping occurs in a wide variety of other mammalian glyco-
proteins (Spiro 1970), and that concentrations several orders of magnitude
beyond that required for CEA are needed to obtain inhibition in the radio-
immune assay.
Banjo et al. (1974 a) have obtained fragments (molecular weight probably
less than 4,000 daltons and active in the radioimmune assay) by degradation
of neuraminidase-treated CEA with the proteolytic enzyme nagase. The frag-
ments were fractionated as summarized in Table VI. Initially only 0.03 per
cent of the activity of the CEA was recovered. Surprisingly, the total amount
of assay activity increased substantially during the fractionation steps, but
always represented only a small amount of the initial material. In all cases
the fragments were also considerably less active than CEA on a weight basis.
Many appear to be predominantly carbohydrate, enriched in N-acetylglucos-
amine relative to neutral sugars. Considering the difficulties involved in work-
ing with such small quantities of materials, the fact that only 35-50 per cent
of the dry weights of the materials can be accounted for is understandable.
If the antigenic determinants of CEA do indeed involve the carbohydrate
component, it appears clear that sialic acid does not play a major role in these
determinants, since removal of 97 per cent of the sialic acid from CEA by
treatment with neuraniinidase causes no reduction in the antigenic activity
(Coligan et al. 1973, Banjo et al. 1974). More recent studies involving the
treatment of CEA with periodate indicate that the complete destruction of
both sialic acid and fucose causes no concomitant loss in antigenic activity
(Coligan et al. 1974, Egan et al. 1974 a). No destruction of glucosamine was
observed and mannose and galactose were recovered in 70-75 per cent yield.
It is interesting that the observed reductions in the mannose and galactose
content also did not affect the antigenic activity of CEA.
 CARCINOEMBRYONIC ANTIGEN 119

5.4 Arrangement of saccharides in the carbohydrate chains

In mammalian glycoproteins sialic acid and fucose are generally restricted to
the terminal positions of polysaccharide chains (Heath 1971). Studies men-
tioned previously involving neuraminidase (Coligan et al. 1973) and periodate
(Coligan et al. 1974, Egan et al. 1974 a) are consistent with the assumption
that this is probably true for CEA.
Prior to neuraminidase treatment, Banjo et al. (1974) were unable to label
galactose oxidase-treated CEA by reduction with tritiated NaBH^. Removal
of the sialic acid allowed 0.2 per cent of the galactose residues to be labeled.
Depending on the efficiency of labeling, this may suggest that their CEA has
little or no terminal galactose and relatively few galactose residues in penul-
timate positions. Alternatively, it may suggest that sialic acid interferes with
the action of galactose oxidase as it does with proteolytic enzymes.
No studies have been reported on the size of the carbohydrate chains on
CEA, nor on the number of such chains per glycoprotein molecule.

5.5 Linkage of carbohydrate to protein

There are two common ways by which carbohydrates link to peptides in mam-
malian glycoproteins (Spiro 1970). In one instance, the bond involves the C-1
of N-acetylglucosamine and the amide group of asparagine. In the alternative
method, the bond is an O-glycosidic linkage involving C-1 of N-acetylgalac-
tosamine and the hydroxyl of serine or threonine. The low content of N-acetyl-
galactosamine in CEA suggests that the carbohydrate attachment to the pro-
tein chain occurs through the asparagine-N-acetylglucosamine linkage. Exper-
imentally, this has been supported by the finding that the carbohydrate does
not split off from the protein by ^-elimination in the presence of alkali (Egan,
Coligan, Todd & Todd, unpublished observation).

5.6 Relation to carbohydrate in blood group substances

Purified CEA preparations are reported to have antigenic determinants in
common with blood group A (Gold et al. 1972, Turner et al. 1972, Gold &
Gold 1973). Holbum et al. (1974) reported that CEA preparations possessing
blood group A reactivity contain N-acetylgalactosamine, the monosaccharide
associated with blood group A activity, whereas CEA preparations derived
from donors of other blood types do not contain this sugar. However other
investigators (Gold et al. 1972, and Gold & Gold 1973) have not been able
to detect N-acetylgalactosamine in their blood group A reactive CEA pre-
parations Immunochemical evidence of Gold & Gold (1973) and Holbum
120 TERRY, HENKART, COLIGAN & lODD

TAliLE VII
Chemical composition of Be-CEA and blood group substances

Mole per cent carbohydrate

Blood group A substances

Be-CEA
(Hog gastric (Human Ovarian
Mucin)^ Cyst)b
Fucose 19 23 18
Mannose 0 0.3 14
Galactose 37 32 26
N-Acetyl-glucosamine 28 20 36
N-Acetyl-galactosamine 16 23 2.1
Sialic Acid ND= 2.0 4.5
% Carbohydrate 78.4 77.1 56.2
% Amino acid 9.9 18.6 34.1

Kindly supplied by Dr. T. J. Kindt, Rockefeller University, New York.

Kindly donated by Dr. W. T. J. Morgan, Lister Institute, London, England.
Not determined.

et al. (1974) suggests that the CEA and blood group determinants are both
on the same molecule.
Blood group A substance isolated from hog gastric mucin and from human
ovarian cyst fluid has been found to be a relatively inefficient inhibitor when
compared to CEA in the radioimmunoassay described by Egan et al. (1974 b).
Amounts 460,000 and 1,300 times greater than CEA on a weight basis, re-
spectively, for hog gastric mucosa A-substance and human A-substance, were
required to produce equivalent inhibition in the radioimmunoassay (Egan,
personal communication). As can be seen in Table VII, the carbohydrate com-
positions of blood group A substances and CEA are quite different. More-
over, the distribution of the amino acids in the protein has also been found
to be remarkably different in CEA compared to those of the blood group A
substances (Table I and Watkins 1972). Thus, CEA and blood group sub-
stances appear to differ in the protein to which the carbohydrate moiety is
attached, the way it is attached (Watkins 1972), and the monosaccharide con-
tent of the material that is attached. While blood group substances may be
capable of crossreacting in the CEA assay at very low efficiency, clearly they
are not CEA, nor are they closely related chemically.
Holbum et al. (1974) have recently demonstrated that a number of CEA
preparations are reactive with anti-blood group sera (either anti-A, anti-B,
anti-Lewis b or anti-Lewis a). The blood group reactivity of the CEA appears
to correlate with the genetic background of the donor, i. e., CEA from a type
 CARCINOEMBRYONIC ANTIGEN 121

B donor reacts specifically with anti-B sera. Quantitative studies indicate that
the blood group determinants and the CEA determinants are present on the
same molecule, but that the CEA immunodominant antigenic determinants
are distinct from the major blood group antigens. The presence of blood group
specificities can be attributed to the adventitious addition of the characteristic
groupings on the CEA molecule, but other explanations have been proposed
(Holbum etal. 1974).
Considering the distinct chemical differences between CEA and blood group
substances, the relative inefficiency of blood group substances in inhibiting
CEA in the radioimmune assay, and the correlation between CEA blood
group activity and the blood group of the donor, it may be conceivable that
the blood group substances are extracted and purified along with CEA as
noncovalently attached molecules. The stickiness of CEA has been previously
mentioned in relation to the relatively irreversible binding of sucrose to CEA
during isoelectric focusing. It has also been noted that ^^^I tagged bovine
serum albumin in the presence of a relatively large excess of CEA assumes
the isoelectric focusing characteristics of CEA (Coligan & Todd, unpublished
observation). Hughes (1973) has also reported the existence of noncovalently
attached substances being isolated along with CEA. Thus, the possibility
must be considered that blood group substances may be extracted inadvertently
with CEA.

6. CLINICAL IMPLICATIONS

Ever since the demonstration that the serum of some patients with malignancy
contained elevated levels of CEA (Thomson et al. 1969), attempts have been
made to investigate the usefulness of assays for CEA in the detection and diag-
nosis of malignant disease.

6.1 Assays

A number of different assays are presently being used to measure the concen-
tration of CEA in plasma or serum, or to monitor the purification of CEA
(reviewed in Kupchik et al. 1974). Most are radioimmunoassays utilizing the
general principle of inhibition of binding (Egan et al. 1974 b). Antiserum
containing antibodies to CEA is incubated with patient's serum and a known
amount of radiolabelled purified CEA. Any unlabelled CEA in the patient's
senim competes with the labelled CEA for binding to the antibodies. Antibody
and bound CEA are precipitated either chemically (Thomson et al. 1969,
Hansen et al. 1971) or with an anti-immunoglobulin (Egan et al. 1972). The
amount of label left in the supernatant is related to the concentration of CEA
122 TERRY, HENKART, COLIGAN & TODD

TABLE VIII
Occurrence of Elevated CEA Concentration in Various Clinical States

Percent with CEA concentration

above Normal

A - Healthy volunteers 10
Non-smokers 3
Smokers^ ^^
Former smokers ^
B - Non-malignant Disease 34
Pulmonary Emphysema 57
Alcoholic Cirrhosis 70
Ulcerative Colitis 30
Gastric Ulcer 45
Breast Disease 15
C - Endotermal Carcinoma (Colerectal,
Pulmonary, Gastric, Pancreatic) 73
D - Non-entodermal Carcinoma
Breast, Head and Neck, Other) 59
E - Non-Carcinoma
(Leukemias, Lymphomas, Sarcomas) 40
F - Suspected Malignancy, Not confirmed H

1 -Compiled from CEA-Roche: A Clinical Monograph (1974). These data are all based
on the Z-gel assay, with the upper limit of normal taken as 2.5 ng/ml.
2 " Elevations of CEA concentrations in smokers have not been carefully studied with
assays other than the Z-gel assay.

present in the serum. Assays either use serum or plasma, and for some assays,
it is necessary that the test serum be extracted first with perchloric acid (in-
direct assays) rather than utilizing intact serum or plasma (direct assays).
Careful comparative studies between different assays and the reagents used
are needed to determine whether any have special advantages. Published com-
parative studies indicate 70-80 per cent agreement (Kupchik et al. 1973,
Laurence et al. 1972).

6.2 Potential clinical uses, results and implications

The major potential uses for CEA assays are: 1) screening 'normal' popula-
tions for the presence of undetected cancer; 2) aiding in the differential diag-
nosis of patients with suspected cancer; 3) following patients immediately after
surgery or other therapy to determine effectiveness of therapy and 4) follow-
ing patients for a prolonged period of time after apparent curative therapy to
detect recurrent disease before it becomes clinically apparent, so that addi-
tional therapy can be instituted as early as possiWe.
 CARCINOEMBRYONIC ANTIGEN 123

Most of the clinical studies of CEA have been concerned with correlations
between serum CEA concentrations and the clinical status of the patient.
CEA is detected in the serum or plasma of most normal people at a relatively
low concentration. Quantitative studies in normal subjects and patients with
various diseases are summarized in Table VIII. About 10 per cent of normal
people have 'elevated' CEA levels (Table VIII, A), although this percentage
shifts depending on the value chosen as the upper limit of normal. CEA is
elevated in carcinoma of the gastrointestinal tract (Table VIII, C), but is also
elevated in other entodermal (Table VIII, C) and non-entodermal (Table VIII,
D and E) malignancies. In general, relatively large amounts of tumor are
required before CEA values are elevated, and in different series, 50 to 80
per cent of patients with established bowel cancer limited to the bowel wall
had normal CEA values (Dhar et al. 1972, Lo Gerfo et al. 1972, Laurence
et al. 1972, Mach et al. 1974). Similarly, 83 per cent of patients with primary
breast cancer without metastases had normal levels of CEA (Steward et al.
1974). Moreover, 17 per cent of patients suspected of malignancy, but where
no lesion could be detected, had CEA concentrations above normal (Table
vni, F).
These data are important in considering the potential use of CEA assays
mentioned above. It is generally agreed that CEA assays are impractical as a
general population screen. The major reason for this is that about 10 per cent
of the population has elevated CEA serum concentrations. It is not definitely
proven, but it seems reasonable to assume that these are truly false positive
tests, i. e., these individuals do not have cancer or any of the benign conditions
associated with elevated CEA. This 10 per cent of the population would have
to have a total 'cancer' examination to rule out the presence of malignancy
since elevated serum concentrations of CEA are seen with cancers of many
different organs (Lo Gerfo et al. 1971, Moore et al. 1971, Laurence et al.
1972). The resulting load on diagnostic medical facilities would be unaccept-
able. This conclusion may be modified if smoking can be consistently shown
to cause elevated levels, since the per cent of false positive tests is apparently
lower in non-smokers (Table VIII, A). Even if the per cent of false positive
tests can be decreased, the usefulness of this assay as a screen for early cancer
is limited by the fact that 50 to 80 per cent of individuals with small gastro-
intestinal cancers or non-metastatic breast cancers have normal CEA tests;
thus two-thirds to three quarters of those individuals who already have estab-
lished but small cancers would be missed by this assay (Dhar et al. 1972,
Laurence et al. 1972, Lo Gerfo et al. 1972, Steward et al. 1974).
There is no statistically valid proof at the present time that the CEA test
is useful as a diagnostic adjunct when cancer is suspected; that is, no reported
studies have demonstrated that the accuracy of the differential diagnosis of
124 TERRY, HENKART, COLIGAN & TODD

cancer is improved by adding CEA assays to conventional diagnostic proce-

dures. In fact, some of the benign conditions associated with elevated CEA
levels are precisely those (eg., ulcerative colitis, pancreatitis) involved in the
differential diagnosis of various types of malignancies (Laurence et al. 1972,
Holyoke et al. 1972, Zamcheck et al. 1972). It is possible that at some time in
the future, the CEA assay will be shown to be as reliable as one of the con-
ventional diagnostic procedures. If so, CEA assays, which are relatively cheap
and cause no discomfort to the patient, might replace some of the more
expensive or elaborate diagnostic techniques.
Several studies have shown a good correlation between effectiveness of
therapy and CEA serum concentration (Mulcare & Lo Gerfo 1972, Skarin
et al. 1974). Patients having 'curative' surgery show a decrease of CEA serum
concentrations to normal levels (Dhar et al. 1972, Holyoke et al. 1972, Lau-
rence et al. 1972, Lo Gerfo 1972, Mach et al. 1974). Continued elevation of
CEA after therapy is presumptive evidence that therapy was inadequate, but
at this time, no statistically evaluable studies have been reported that establish
this fact or demonstrate a better therapeutic effect if the patient is given addi-
tional therapy on the basis of persistent elevation of CEA. It will be necessary
to demonstrate that second-look surgery, chemotherapy, or some other form
of therapy (initiated because of persistently elevated CEA levels after pre-
sumptive curative surgery) will lead to a better clinical outcome, before it is
possible to conclude that this use of CEA assays is beneficial to patients.
Similarly, there are no statistically evaluable studies showing that in patients
where the CEA level falls to normal after therapy but rises again after an
interval, the CEA assay provides an earlier or more reliable indication of
recurrent disease than presently utilized diagnostic tests. A number of obser-
vations have been reported where rising CEA levels did precede clinical
recurrence (Holyoke et al. 1972, Sorokin et al. 1974) and it is in this area
that the CEA assay may prove most useful.

CONCLUDING COMMENTS

The biologic significance of the substances bearing carcinofetal antigens is

not understood. The functions carried out in the fetus by, for example, the
gjycoproteins bearing CEA determinants are not known, nor is it known why
there is an increased expression of CEA in malignant tissues. It is important
to attain a better understanding of the functional roles of these molecules in
development and malignancy. Progress in this area will be speeded by more
complete immunochemical, physical and chemical characterization of CEA
and related proteins, and this would be a worthwhile research undertaking
even if there were no other considerations.
 CARCINOEMBRYONIC ANTIGEN 125

There are, however, other considerations, and a full exploration of the value
of CEA and related antigens in the detection, diagnosis, and management of
human cancer is of the first importance. At the present time, there appears
to be a relatively small role for CEA in detection and diagnosis. This pessi-
mistic opinion may be altered by future developments, including the develop-
ment of more sensitive assays. For example, it is possible that serial assays on
the same normal individual will be of value in detection or diagnosis, parti-
cularly if the assays can be made 100 times more sensitive. A CEA concen-
tration that rises steadily from 0.01 ngm/ml to 1.0 ngm/ml may be very im-
portant, while the change from 'not detectable' to 1.0 ngm/ml does not help
very much. The observation of a rise over time may distinguish true positive
from false positive results, but to be of real value in detection or diagnosis of
small cancers, it will have to be coupled with the use of more sensitive assays.
More information concerning the chemistry and nature of the antigenic site
of CEA should make it possible to design such assays.
The application of CEA assays to the management of patients with malig-
nancy is a more promising area for the immediate future. Appropriately de-
signed prospective trials will be required to determine a) whether changes in
CEA levels immediately after therapy distinguish reliably between 'cure' and
residual disease, and if so, b) whether additional therapy based on CEA results
improves survival or prolongs remission. Similar prospective trials will be
required to determine a) whether recurrence of an elevation in CEA level after
it had fallen to normal following therapy reliably indicates recurrent disease
and b) whether initiation of therapy based on CEA results improves survival
or prolongs remission. Studies of this type can be carried out now and within
the next year answers to these questions should be available.

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