JOURNAL OF PATHOLOGY, VOL.

179: 358-369 (1996)

REVIEW ARTICLE

CELL SURFACE CARBOHYDRATES AS PROGNOSTIC

MARKERS IN HUMAN CARCINOMAS
ERIK DABELSTEEN

School of’Dentistry, Faculty of Health Sciences, University o j Copenhagen, Deninurk

SUMMARY
Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to
terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes
in the carbohydrate core structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of
carbohydrates in N-linked glycoproteins, and blocked synthesis of carbohydrates in 0-linked mucin-like glycoproteins. In mature
organisms, expression of distinct carbohydrates is restricted to specific cell types; within a given tissue, variation in expression may be
related to cell maturation. Tumour-associated carbohydrate structures often reflect a certain stage of cellular development; most of these
moieties are structures normally found in other adult or embryonic tissues. There is no unique tumour carbohydrate structure, since
certain structures which are tumour-related in one organ may be normal constituents of other tissues. Tumour-associated carbohydrate
changes have been used in the diagnosis of human cancers. Recently, however, it has been demonstrated that the expression of some
carbohydrate structures is associated with prognosis. Tn, sialyl-Tn, and T are cell membrane-bound mucin-like carbohydrate structures
that may be expressed in tumours due to blocked synthesis of the core carbohydrate chain of mucin-like structures. Their expression is
strongly associated with prognosis in certain tumours, but the biological relationship between their expression and tumour progression
is at present unknown. The blood group-related carbohydrate structures Lex, sialyl-Le”, ABH, and Ley are examples of terminal
carbohydrate structures which are related to tumour prognosis. These structures are of increasing interest since they may function as
adhesion molecules; adhesion of tumour cells to endothelial cells of blood vessels may be mediated by an interaction between sialosyl-Le”
and E-selectin and studies indicate that Ley is related to cell motility. These findings are now the basis for tumour therapeutic
experiments.
KEY WORDS-+arbohydrates; prognosis; cancer

INTRODUCTION tumours. Numerous monoclonal antibodies have

In recent years, studies of genetic changes in tumour
cells have advanced to such a stage that correlations
between molecular alterations and tumour behaviour
are becoming clearer. Most often it is the relationship
between changes in cancer-related genes and their
products that has been related to tumour behaviour.
Recently, however, indirect gene products, e.g., cell
surface carbohydrates. have also attracted interest.'^^
Several studies have demonstrated changes in glyco-
sylation of both glycolipids and glycoproteins in
tumours. The changes first demonstrated by Hirtsfeld
(1929) and Thomsen (1930) were later confirmed as
aberrant tumour-associated g l y c o ~ y l a t i o nLater,
. ~ ~ ~ bio-
chemical studies provided further evidence of altered
glycosylation and showed that in many cases these
changes were related to blood group antigens of the
ABO s y ~ t e m . ~ . ~
Monoclonal antibody technology has made it possible
to study in detail the histological distribution of cellular
carbohydrates and the pattern of changes seen in solid
been produced by immunization with tumour cells in the
search for tumour-specific antigens; many of the tumour
antigens detected by these monoclonal antibodies turned
out to be cell surface glycoconjugatesi3
The biosynthesis of carbohydrate chains involves a
stepwise elongation catalysed by specific glycosyltrans-
ferases. l 4 They often have a common precursor structure
and variants are in many cases limited to the terminal
end of this carbohydrate chain (Fig. l).13 Although
carbohydrates are extremely complex structures, their
cellular expression is highly regulated and appears to be
dependent on the pattern of glycosyltransferases found
in the Golgi apparatus.15 l 8 The expression of carbo-
hydrates changes in an orderly fashion during embry-
onic development and a tissue-specific pattern of
expression is seen in the adult.19J0 Within a particular
organ, the carbohydrates may be expressed in a way
that correlates with cell differentiation.*’ In malignant

Fig. ]-Schematic diagram of cell surface carbohydrates. The inner

Addressee for correspondence: Erik Dabelsteen, Department of core may be linked to proteins by an N- or 0-linkage or to lipids (R).
Oral Diagnostics, School of Dentistry, Faculty of Health Sciences, The backbone structure, consisting of a polylactosamine chain, may be
University of Copenhagen, Nsrre Alle 20, DK-2200 Copenhagen N, branched or unbranched. The terminal structures often contain ABH
Denmark. and related blood group antigens

CCC 0022-341 7/96/080358-12 Received 8 August 1995

(C) 1996 by John Wiley & Sons, Ltd Accepted 1I Junuary 1996
 CARBOHYDRATES AND TUMOUR PROGNOSIS 359

+
Gal H GlcNacFR - [ Gal H GlcNAc
R
Lac

LeY

"Hz?(F
GalNAc GlcNAc R

S-LeX LeX
Fig. 2-Biosynthetic pathway of terminal carbohydrate structures with blood group specificity. R represents the backbone structure,
which in most cases is a polylactosamine chain. (a) Lac: N-acetyllactosamine, which by the action of fucosyltransferase may be
transformed into blood group H. The A determinant is formed by the addition of N-acetylgalactosamine to the H structure by the action
of a glycosyltransferase coded for by the A gene. Due to the action of another fucosyltransferase, the H structure may be transformed
into the Ley structure. (b) Sialosyl-Le" (S-Le") may be synthesized by addition of first sialic acid and subsequently fucose to N-
acetyllactosamine (Lac). The synthesis of these structures is dependent on a sequential presence of glycosyltransferases, carbohydrates, and
acceptor molecules. (Gal: galactose; GlcNAc; N-acetylglucosamine; Fuc: fucose; GaINAc: N-acetylgalactosamine; NANA: sialic acid)

tissue, the glycosylation often reflects fetal or immature
differentiation patterns. 1,19,22,23
Tumour-associated aberrant glycosylation is found
both in membrane glycolipids and glycoproteins and
in secreted protein^.^.^+^* The cell surface changes are
classically divided into (i) changes related to terminal
carbohydrate structures, which include incomplete syn-
thesis and modification of normally existing carbo-
hydrates; (ii) changes seen in the backbone structure
(poly-N-acetyllactosamine chain), including chain elon-
gation and decreased branching; and (iii) changes seen in
the inner core structures, which include increased
branching of N-linked glycoproteins and blocked syn-
thesis of 0-linked mucin-like glycoproteins (Fig. 1). The
tumour-associated changes in glycosylation seen in dif-
ferent organs and in serum and the chemical nature
of these changes have been reviewed in detail else-
where.1,3-4,9,13,29,30The present review deals with
carbohydrate changes in carcinomas in relation to
tumour behaviour and in particular tumour spread and
prognosis.
CHANGES IN TERMINAL STRUCTURES
RELATED TO TUMOUR PROGNOSIS
The most detailed information on glycosylation in
tumours comes from immunohistochemical staining
with specific monoclonal antibodies. Different investi-
gators have often used different monoclonal antibodies
for investigating the same carbohydrate structure.
Although the antibodies are specific for the structure in
question, they often bind to related structures, making
comparisons difficult. Furthermore, histological data are
often semi-quantitative, which adds to the problem.
Nevertheless, in recent years the techniques have been
standardized to such an extent that useful information
has been obtained. The monoclonal antibodies are gen-
erally directed towards terminal carbohydrate struc-
tures. The tumour-associated changes detected by these
antibodies are therefore related to the terminal part of
the glycoconjugates, without any indication of the
nature of the core s t r ~ c t u r e . The
~ ~ ~peripheral
' part of
both glycoproteins and glycolipids often carries blood
group antigens related to the ABO and Lewis sys-
~ ~ 2).
t e m ~ (Fig. . ~Such~ antigens are found on erythro-
cytes and other (predominantly epithelial) cells
throughout the body; the term histo-blood group anti-
gens has been used to stress the identity between blood
group antigens in tissues and those found on blood cells,
although the mechanisms that regulate their expression
in different tissues vary. There is correspondence
between erythrocyte blood group and tissue blood
group; consequently, cells from blood group 0 persons
have neither A nor B antigens, but may express the H
antigen, which is their immediate chemical precursor.32
Many of the tumour-associated changes are related to
these antigens or their chemical precursors. Further-
more, these changes are in some cases directly correlated
with p r o g n ~ s i s . ~ , ~ ~ - ~ ~
ABH antigen and prognosis
Several groups have investigated ABH carbohydrate
expression in urothelium in order to study whether
changes can be used as predictive markers for the
behaviour of bladder carcinomas.3740. Mrntoft has
documented the sequence of changes in carbohydrate
antigen expression associated with increasing grade of
dysplasia and the presence of invasion (Table I). A
gradual deletion of ABH antigens is found in the
non-invasive lesions, with an almost total loss of ABH
antigens in invasive tumours. Although the early results
Table I-Changes in ABH histo-blood group antigen expres-
sion associated with increasing grade of dysplasia and invasion
in bladder carcinomass3
Non-invasive carcinomas
1. Disruption of stratification of antigen
2. Change in cellular localization of antigen
3 . Gradual deletion of ABH antigen expression
Invasive carcinomas
4. Almost total loss of ABH antigen
5 . Uniform expression of ABH precursor structures (Leb
and LEY)
360
were somewhat contradictory, most studies have shown
that in non-invasive bladder carcinomas, a benign
course can be predicted if A or B blood group antigens
are expressed in the tumour tissue, the positive predic-
tive value being over 90 per cent. Predicting an un-
favourable course in patients with loss of A and B
antigen expression has, however, been less conclusive,
with predictive values around 30 per cent.37.41-s6
The expression of A and B antigens in bladder carci-
nomas also seems to be related to the outcome of specific
forms of therapy. By combining the expression of A or B
antigens and /32-microglobulin, it has been possible to
predict the response to intravesicd chemotherapy with
the bacillus Calmette-GuCrin (BCG) vaccine. Five
tumours that stained positive for both A or B and
p2-microglobulin responded to BCG chemotherapy,
whereas nine of ten tumours that stained negative for
one or both of the antigens recurred within 2 years.57
Loss of blood group A, B, or H has also been
associated with tumour prognosis in carcinomas of the
head and neck.58 Relapse was found to occur in 78 per
cent (1 8 of 23) of patients whose tumours exhibited loss
of blood group antigen expression, compared with a
relapse rate of 36 per cent (16 of 43) among patients
whose tumours retained blood group antigen expression.
Loss of ABH expression was shown to be an important
independent variable associated with early recurrence.
An increased expression of the integrin a6P4, which is
localized in basal cells of stratified epithelium where it
mediates basal cell adhesion via epiligrin (laminin 5)
present in the basement membrane, was also shown to
be associated with early tumour relapse. The combined
loss of blood group antigen and increased expression of
a6P4 integrin was even more strongly associated with
disease outcome than either variable alone, or tra-
ditional prognostic factors such as tumour site, clinical
stage, and TNM classification. Interestingly, loss of
histo-blood group antigens and increased expression of
a6P4 integrins similar to that seen in invasive cancer
have also been observed during epithelial migration
associated with wound healing in the oral mucosa
(Dabelsteen, unpublished). This relationship between
cell motility and carbohydrate expression has led to the
hypothesis that changes in blood group antigens in
invasive cells are a sign of increased cellular locomotion,
which is a prerequisite for m e t a ~ t a s i sThe
. ~ ~ relationship
between expression of histo-blood group antigen, cell
migration, and prognosis of squamous cell carcinomas
of the oral cavity is further emphasized in the study by
Bryne,60 which showed that loss of expression of histo-
blood group antigen H on infiltrating cells at the deepest
margin of the tumour was associated with metastatic
spread and poor prognosis, whereas assessment of H
antigen in the entire tumour did not correlate with
prognosis.60 Studies of squamous cell carcinomas of the
cervix have also shown a correlation between H-antigen
expression and prognosis; the incidence of death within
2 years after hysterectomy was higher in the H antigen-
negative group than in the H antigen-positive group.61
A relationship between expression of ABH antigens
and survival was also seen in studies of non-small cell
carcinomas of the 1ung.62,63The average survival of
E. DABELSTEEN
those patients with blood group A or AB whose primary
carcinomas were negative for A antigen was significantly
shorter than for those whose tumours showed expres-
sion of this antigen (Fig. 3). However, using a mono-
clonal antibody which defines two AB precursor
antigens (H and Ley, Fig. 2), it was found that patients
who had squamous cell carcinomas of the lung that
expressed these precursor structures had a much worse
survival than patients who had precursor-negative
tumours (Fig. 4).64,6s It is interesting that the same
antibody (anti-H and Ley) has been shown to inhibit the
mobility and metastatic potential of a metastatic mouse
melanoma cell line,66 suggesting again a functional
relationship between carbohydrate expression and cell
motility. The relationship between Ley and cell mi-
gration has recently been further suggested by studies of
oral mucosal wound healing; migrating epithelial cells
show loss of A and B antigens at the same time as they
upregulate the expression of Ley.67
One of the problems inherent in studying ABH blood
group antigens in tissues is that expression in a particu-
lar tissue may vary between individuals of the same
ABO blood group.18,41,68This variation is to some
degree regulated by the secretor genes (Se/se) which
regulate expression of A and B antigens by controlling
the presence of their chemical precursor, the H anti-
gen.41-68,69
As non-secretors (se/se) constitute 20 per cent
of the population, a detailed knowledge of the patient's
blood group and secretor status is needed before any
conclusions on changes of blood group antigens in a
tumour can be r e a ~ h e d . ' . ' ~ . ~ ~
LeX and sialosyl-Le" and prognosis
In some tumours, incomplete synthesis of cell surface
carbohydrates results in an increased expression of ABH
precursor structures such as Le" and sialosyl-Le" and
their isomers, Lea and sialosyl-Lea (Fig. 2).1,5,9,33 As
synthesis of Le" and sialosyl-Le" (in contrast to A, B,
and H antigens) is independent of the patient's ABO
and secretor status, they are more suitable for clinical
studies. Sialosyl-Le" and related carbohydrate structures
have recently been shown to function as adhesion
molecules and they may thus theoretically be involved
in tumour metastasis. It is therefore interesting that
their expression in certain tumours is correlated to
prognosi~.~~,~~-~~
Dimeric sialosyl-Le" was found in extracts of most
colon carcinomas but not in normal colonic m u c o ~ a . ~ ~ . ~
Metastases showed a stronger expression than primary
tuniours and when the tumours were divided according
to stage, sialosyl-Le" showed a lower expression in
Dukes' A and B tumours than in more advanced
stages.74 One of the problems with studies of tissue
extracts is that the sialosyl-Le" detected may not solely
reflect tumour-derived carbohydrates, but may also
come from other cells such as granulocytes, which
may be present in tumours with necrosis. However,
immunohistochemical studies have also revealed a
higher proportion of sialosyl-Lex-positive tumour cells
in metastatic lesions than in primary turn our^.^^-^^ In
two clinical studies, each involving more than 100
 CARBOHYDRATES AND TUMOUR PROGNOSIS 36 1

1.0

'1 -*a*. f
i,:. b
0.8 . ....
I *:
-1 'a.

0.6
1 .....
I ..

1"'
,
l-15 m o *.
-L - - - -?& - - -- a
- -- -------
c, .a.
....
0.4 1
I .............
L-,
7 .............
0.2

I I 1 I I I I 1 -

0 12 24 36 48 60 72 84 96 108 120
Months of Survival
Fig. 3-The survival of patients with non-small cell lung cancer. Blood group A patients who had a primary
tumour negative for histo-blood group antigen for A, n=28, had a significantly shorter survival than patients
who had a primary tumour positive for A, n=43, and a shorter survival than the patients with blood group B
or 0, n = 9 P 3

n
8
Y

Q)
c,
Q
a
-
Q
.->
L
3
cn
100

80

60

40
\ ga
-7-5 MIA-negative (n = 29)

P<O. 001

20
MIA-positive (n = 38)
b
0
1 2 3 4 5 6 7 8 9 10

Years of Survival
Fig. +Survival of patients with squamous cell carcinoma of the lung. Correlation between expression of blood
group antigen A precursor structures and survival. MIA reacts with the carbohydrate structures H, Ley, and the Ley
isomer Leb. The MIA-negative patient has a better survival than the MIA-positive one; the MIA-negative tumours
may be the tumours which have retained histo-blood A or B antigens65
362
100
-
-
-
50 41
1 I
1000
Time after operation (days)
Fig. S--Overall survival of patients with colorectal cancer in relation to sialosyl-Le" expression. Patients with sialosyl-Le"-
negative tumours have a better survival than patients who have tumours which express sialosyl-Le" (S-Le": ~ialosyl-Le")~'
patients increased expression of sialosyl-LeX in colo-
rectal carcinomas was correlated with overall 5-year
survival (Fig. 5). Although immunohistochemical
expression of sialosyl-Le" in the two studies was detected
by two different monoclonal antibodies with slightly
different specificities, the results were almost identical.
In both studies, sialosyl-Le" expression proved to be
an important discriminant for predicting disease-free
~ u r v i v a l , and
~ ~ .expression
~~ of sialosyl-Le" added pre-
dictive value to the tumour TNM stage, which is often
used as a prognostic factor. In patients with stage 11 and
I11 disease, disease-free survival was significantly greater
in the sialosyl-Lex-negative group than in the sialosyl-
Lex-positive group. However, in patients with stage I
disease, there was no difference in disease-free survival
between the two groups. From a practical point of view,
it is important to note that sialosyl-Le" epitopes may be
detected in small amounts in normal colonic mucosa
when antibodies are used that recognize sialosyl-Le"
epitopes irrespective of neighbouring sugars. However,
when more specific antibodies are used that recog-
nize only the dimeric version of sialosyl-Le", reactivity
is not seen in normal colonic mucosa, but only in
carcinoma^.^^.^^
A study of 300 patients with breast cancer showed
an increased 2-year survival in the group with sialosyl-
Lex-negative tumours compared with the group with
sialosyl-Le"-positive tumours.80 Furthermore, in a
recent study of metastatic prostate cancer it was demon-
strated that increased expression of sialosyl-Le" in
the invasive parts of the tumours was significantly
associated with a poor prognosis.8'
E. DABELSTEEN
S-LeX negative (n = 33)
60 6'0 >
S-LeX positive (n = 87)
P = 0 0263
I I 1 1
3000
The fact that sialosyl-Le" structures play a role in
tumour prognosis has been supported by experimental
animal studies, using various human cancer cell lines
showing different degrees of colonization in organs of
nude mice. By investigating different subclones of a
human lung adenocarcinoma cell line, it was shown that
those characterized by the highest lung colonization
potential expressed high amounts of sialosyl-Le",
whereas those with moderate lung colonization potential
expressed low amounts.82 Studies of highly and poorly
metastatic sublines derived from human colonic carcino-
mas have also demonstrated that 'metastatic' cell lines
express more sialylated Le" than 'non-metastatic' ones.83
It has further been shown that cells producing high levels
of sialosyl-Le" are more invasive in vitro than those
producing low levels of this structure.84 It has been
pointed out that although highly metastatic tumour cells
seem to express increased amounts of sialosyl-Le" deter-
minants, chemical analysis may show that in some cases
there is little difference between highly and poorly meta-
static cells, as they express the same number of sialosyl-
Lex molecules.85 This apparent disparity has been
explained by the fact that sialosyl-Lex is often carried on
polylactosamine structures, which may be extended on
the metastatic cells leaving the sialosyl-Le" residues more
exposed, or that sialosyl-Le" is exposed due to organiz-
ational changes in the cell membrane. It is likely that it is
the degree of expression and not the actual amount of
sialosyl-Lex which plays a role in the interaction between
cells and subsequent m e t a s t a s e ~ . ~ . ' ~ , ~ ~
Although sialosyl-Lea is expressed on many types of
tumour, the relationship between the expression of
 CARBOHYDRATES AND TUMOUR PROGNOSIS
sialosyl-Lea and metastatic behaviour remains
~ ~ , ~ et~ a1.88reported that the presence of
u n ~ l e a r .Arends
sialosyl-Lea in gastrointestinal cancers as not correlated
with parameters known to be of prognostic significance.
Walker and Days9 found no correlation between
sialosyl-Lea in breast carcinomas and lymph node status,
while Nakajoe et ~ 2 1 found . ~ ~ that sialosyl-Lea was less
frequently expressed in metastatic lesions of colorectal
carcinomas than in primary carcinomas without lymph
node metastasis.
Sialosyl- Lex, sialosyl- Lea, and cellulur adhesion
The significance of increased expression of sialosyl-
Lex on certain tumour types may relate to the presence
of receptors for sialosyl-Lex-carrying ligands on acti-
vated endothelial ~ e l l s . ~ O When
- ~ ~ endothelial cells are
treated with interleukin-1 or TNFa, they express
endothelial leukocyte adhesion molecule 1 (ELAM- l),
which is an adhesion molecule belonging to the selectin
family. The selectins are transmembrane molecules
containing a terminal carbohydrate binding domain
(a lectin) followed by an epidermal growth factor-like
domain and a variable number of complement-like
consensus repeats.94 Soluble mediators released from
inflammatory cells or tumour cells may induce expres-
sion of ELAM-1 on endothelial cells. ELAM-1 was
initially identified as an inducible endothelial cell surface
adhesion molecule for neutrophils and is believed to play
a key role in the extravasation of these cells at the site of
acute i n f l a m m a t i ~ n ELAM-1
.~~ is now known to func-
tion as a lectin, recognizing sialylated forms of Le" and
related structures in genera1.94-96
In many respects, tumour metastasis is a similar
phenomenon to leukocyte trafficking. Tumour cells and
leukocytes circulating in the blood are able to extrava-
sate and populate new tissues using the same types of
adhesion molecule^.^^^^^ Several studies have indicated
that tumour cells expressing sialosyl-Le" and its
positional isomer sialosyl-Lea do adhere to cultured
endothelial cells activated by interleukin.86~s7~yy~100
Takada et investigated 18 cancer cell lines express- not detected by monoclonal antibodies) also shows
ing sialosyl-Lex and/or sialosyl-Lea and found that the
adhesion of tumour cells to endothelium correlated with
the degree of sialosyl-Le" and/or sialosyl-Lea expression.
This adhesion could be significantly inhibited by pre-
treatment with antibodies to either ELAM-1, sialosyl-
Lea, or sialosyl-Le". They also showed that the binding
of tumour cells could be inhibited by addition of
sialosyl-Lex liposomes prior to the addition of cancer
cells. Inhibition of tumour cell adhesion to activated
endothelial cells was also achieved by blocking
glycosylation of cell surface proteins. l o ] Rais and
Bevilacqualo2 suggested that tumour cells producing
cytokines may promote expression of ELAM-1 and
thereby facilitate adhesion of malignant cells arrested
within the vessels. Yet another possibility for adhesion
between sialosyl-Lex and other selectins has been sug-
gested, involving the ability of tumour cells to activate
platelets. Activated platelets also express P-selectin,
which interacts with sialosyl-Le" and sialosyl-Lea.lo3,104
In this way, P-selectin-dependent adhesion of tumour
363
cells to platelets may be an important step leading to
tumour cell aggregation and microembolism. lo5
Sialosyl-Lea and sialosyl-Le" are released to the blood
from tumour cells and are present at increased levels in
the blood of many cancer patients. Measurement of
sialosyl-Lea carbohydrate epitope is the basis for the
Ca19-9 immunoassay for pancreatic cancer. lo6 Sialosyl-
Lea and sialosyl-Le" in serum may theoretically modify
the binding of cancer cells to endothelial cells by block-
ing the ELAM-1 receptors. Sawada et al.lo7 showed that
adhesion of human pancreatic adenocarcinoma cell lines
which express high amounts of sialosyl-Le" and -LeX
could be inhibited by sera from patients with pancreatic
cancer, but not by sera from normal individuals. More-
over, the extent of this inhibition was related to the
sialosyl-Le" antigen content. These findings indicate that
high blood levels of sialosyl-Lex antigen may reduce
adhesion of tumour cells to endothelial cells by the
E-selectin mechanism. Conversely, successful interaction
of tumour cells with endothelial cells would appear more
likely when the blood levels of the ligands are low, as is
the case during the early course of malignancy, or after
surgical resections. However, Sawada et al. also point
out that leukocytes which normally use these ligands
might be adversely affected by high blood levels, thereby
contributing to an impairment of anti-tumoural immune
responses. Administration of sialosyl-Le" or sialosyl-
Lea, for example by use of sialosyl-Le" and -Lea oligo-
saccharides in the form of liposomes, has been suggested
in the therapy of cancer.lo3 Anti-cancer treatment by
blocking sialosyl-Le" or -Lea expression of the target cell
s ~ r f a c e l may
~ , ~ be
~ especially useful when serum levels
of tumour-derived sialosyl-Le" are low.
CHANGES IN CORE STRUCTURES RELATED
TO TUMOUR PROGNOSIS
Studies based on the use of lectins and chemical
analysis of carbohydrates have demonstrated that the
carbohydrate core structure of glycoproteins (which is
marked changes during tumourigenesis. 3,108,109
Glycoproteins can be classified into two groups,
N-glycans and O-glycans. In O-glycans, saccharides are
attached to the hydroxyl group of the amino acids serine
or threonine, whereas in N-acetylglycosamine they are
linked to the amino group of asparagine, forming an
asparatylglucoamine linkage. Both types of carbo-
hydrate may contain repeating units of N -
acetyllactosamine that may serve as a backbone for
ABH and Lewis-related structures.I3
O-Glycuns and prognosis
Mucins are high molecular weight O-glycans synthe-
sized by a variety of epithelial cells. Mucin consists of
approximately 20 amino acid residues repeated in tan-
dem. These repeats may vary from organ to organ but
are always heavily glycosylated. Mucin molecules con-
tain 50-85 per cent O-linked oligosaccharides by weight.
These oligosaccharides contain from one to more than
364 E. DABELSTEEN
I
, I
Thri
Term P-Lac Gal GalNAc -- Se r
I
Fig. (C~Schematicdiagram of a mucin-type glycosylation. The initial
glycosylatIon of serine o r threonine, amino acids o f proteins, with
N-acetylgalactosainine constitutes the T n antigen. This structure may
be replaced with sialic acid (NANA), resulting in the sialosyl-Tn
antigen (STN). Another possibility is that the T n may be extended
with galactose to form the T antigen, which may be sialylated to form
sialyl-T or may be extended by a polylactosamine branched or
unbranched ohgosaccharide structure (P-Lac) and terminated by
ABH, Le', and Le' antigens (Term)
20 sugars per chain. The term mucin is used primarily to
refer to glycoproteins found in secretions: very similar
glycoproteins, however, are found to be present at the
cell surface in a number of different cell types."O Studies
of mucins in malignant tissue have indicated both
increased level of mucin mRNA and an aberrant glyco-
sylation of the tandem repeats. These changes lead to
unusual expression of the core proteins, partly due to
demasking caused by blocked synthesis of carbohydrates
and partly due to an increased amount of the mucin
protein.
Altered glycosylation of tumour-associated mucins
may result in shorter carbohydrate side-chains, which on
the other hand may show increased, or altered,
sialylation.'"j,'''-' l 5 Of special interest for tumour prog-
nosis is the expression of T antigen and its precursor Tn
and sialylated forms of these antigens, although an
explanation for the observed correlation between prog-
nosis and expression of these structures is still obscure
(Fig. 6).
Springer et were the first to draw attention to
Tn and T antigens as cancer markers. These antigens are
mono- and di-saccharides respectively, attached to
mucin-type peptide structures, and can be detected by
immunohistochemical techniques in most adenocarcino-
mas of the colon, breast, lung, bladder, endometrium,
and ovary, and during e m b r y o g e n e s i ~ . ~It~is~ ,gener-
'~~
ally believed that Tn and T antigens in normal adult
tissues are masked by ABH determinants, sialylation, or
further chain branching and elongation of the sacchar-
ide chaini2 (Fig. 6). The altered glycosylation of mucins
leading to exposure of T and Tn antigens may also
unmask other epitopes that may elicit a potent immune
response which may influence tuniour development.
That this may be the case has been suggested by recent
studies showing that vaccination of patients with breast
carcinomas using Tn carbohydrate structures may
improve prognosis.
Siulosyl- Tn antigen and prognosis
Immunohistochemical studies of colonic mucosa have
shown that Tn, sialosyl-Tn, and T antigens are expressed
in fetal colonic mucosa and in carcinoma of the colon
but not in normal adult colonic inucosa.115,122
Follow-up studies indicate that sialosyl-Tn in particular
is an important prognostic marker. Multivariate analysis
showed that sialosyl-Tn was an independent predictor of
disease-free and overall 5-year survival in 128 patients
who underwent curative surgical resection for colorectal
carcinoma. Sialosyl-Tn antigen expression occurred in
87 per cent of the tumours and was independent of
factors such as tumour location, Dukes' stage, depth of
invasion, degree of differentiation, and ploidy status.
Although more advanced Dukes' stages are associated
with poor overall 5-year survival, antigen-negative
tumours within each Dukes' stage had a much better
prognosis than antigen-positive tumours (Fig. 7). One
hundred and eighteen patients had colonic tumours
classified as Dukes' B or C with an overall survival of 82
and 60 per cent, respectively. In this group, all 13
patients with sialosyl-Tn-negative tumours survived. It
has traditionally been considered that colorectal carci-
nomas of the mucinous type are more prone to have
a poor clinical outcome. However, in the study by
Itzkowitz et al. it was shown that there was no statisti-
cally significant association between the presence of
sialosyl-Tn antigen in a tumour and the histological
type, although it was found that mucinous carcinomas
and poorly differentiated carcinomas were nearly all
sialosyl-Tn-positive. 22
Studies of gastric carcinoma have shown expression of
sialosyl-Tn in 20 per cent of tumours, independent of the
histological type.123,'24Death from disease or disease
recurrence occurred in 17 out of 21 patients with
sialosyl-Tn-positive tumours, compared with only 2 out
of 10 patients with sialosyl-Tn-negative tumours. The
study highlights the fact that sialosyl-Tn expression
does not have to be correlated with histopathological
features in order to serve as a useful marker for tumour
prognosis. 123.125
The relationship between expression of sialosyl-Tn
antigen and prognosis in 86 oesophageal carcinomas
was studied by Ikeda et al. 126 Sialosyl-Tn was expressed
in almost 50 per cent of the lesions. The 3-year survival
rate for patients with sialosyl-Tn-positive tumours was
29 per cent, compared with 42 per cent for patients with
sialosyl-Tn-negative tumours. Although these results
show the same trend observed in patients with colonic
and gastric carcinomas, they are not statistically signifi-
cant. Other investigators found that sialosyl-Tn in
oesophagus and oral mucosa could only be detected
in apparently normal mucosa adjacent to carcinomas
and in non-malignant hyperplastic lesions, suggesting
a relationship between increased proliferation and
expression of this structure. 127,128
Sialosyl-Tn expression has been noted in under 50 per
cent of human breast carcinomas. Although patients
with sialosyl-Tn-positive tumours had a lower survival,
this finding was not statistically significant.31"29.'30
However, in subgroups defined by nodal status,
sialosyl-Tn expression was significantly associated with a
poorer prognosis in the node-positive group (Fig. 8).13"
Interestingly, it was also noted that patients with
sialosyl-Tn-negative tumours who received adjuvant
chemotherapy had improved survival compared
with those who were not thus treated. By contrast,
 CARBOHYDRATES AND TUMOUR PROGNOSIS 365

I
Dukes AIS Tn in=3)
Dukes AIS T r i + (n=7)
1.0 ’==- Dukes BiS T n in=9)
.- -I&--
1 Dukes ZfSTn in=4j
.-> “q-- I--
L3 -- 7
1- 1-
1- - ---1
-1
.80 -
I
a I
I
l-7
t -1 L-, 3ukes BiS Tn + in=721
.-0
c,
I
--I
1

L I----,
I
0
.60 -
b
Q L __----
------- Ddkes CIS Tn + in-33,
0
L
P
Q)
.-> .40 -
-
cI
(P
i-
P<0.02

12 24 36 48 60 72

Survival (months)
Fig. 7-Overall survival of patients with colorectal cancer according to Dukes’ stage and sialosyl-Tn status. Although Dukes’
B and C patients in general have a poorer survival than patients with Dukes’ A turnours, it is seen that patients
with sialosyl-Tn-negative turnours in general have a much better prognosis than those with positive tumours (S-Tn:
’’
sialosyl-Tn)

100

80
STn.

Lr
60

N t . STn-
40

20 N t . STn+
I S-Tn
I P<O.OOl
L I I I 1 I I

3 6 9 12 15 18
Time (years)
Fig. 8-Overall survival of patient with breast carcinoma in relation to nodal status and sialosyl-Tn
expression. In the node-positive group, survival was better for patients with sialosyl-Tn-negative tumours
than for those with sialosyl-Tn-positive tumours (S-Tn: sialosyl-Tn; N: nodal status)”’
366
Term P-Lac Gal
Term P-Lac Gal
Term P-Lac Gal GlcNAc
NANA Gal GlcNAc
Fig. 9 - Schcrnatic drawing of a complex N-linked oligosaccharide. Increased p1-6 branching (printed in bold type) at
the trimannosyl core, has been correlated with invasiveness and metastatic potential (Term: terminal carbohydrate
structures may carry ABO and precursor structures; P-Lac: polylactosamine chains; Gal: galactose: GlcNAc:
M-acetylglucosamine NANA; Man: mannosc: Asn: asparagine)
adininistration of adjuvant chemotherapy to those
patients whose tumours expressed sialosyl-Tn did not
influence survival. It was therefore concluded that
sialosyl-Tn positivity, whether acting through direct
or indirect mechanisms, appeared to be a marker of
resistance to adjuvant chemotherapy.I3O
Tn antigen and prognosis
A study of Tn antigen expression in squamous cell
carcinomas from the uterine cervix showed that the
degree of expression was higher in metastases than in the
primary lesions.1'' I n a 5-year follow-up study, it was
demonstrated that T n antigen expression is a useful
indicator for metastatic potential of cervical carcinoma,
although this was only apparent when assessment of
expression of Tn antigen was combined with the degree
of invasion in the cervical stroma. Tn antigen-positive
tumours with deep invasion had a 5-year survival rate of
43.3 per cent, whereas tumours with deep invasion but
which were Tn antigen-negative had a survival rate of
92.9 per cent.'?'
T antigen und prognosis
Expression of T antigen has been associated with the
invasiveness of gastric carcinoma. In a study of 87
tumours, T antigen was seen in 18 cases; these were all
advanced carcinomas with metastases. However, as the
authors pointed out, a high percentage of the advanced
tuinours did not express T antigen, and the predictive
value of expression of T antigen or the absence of this
antigen remains to be determined.124
There have been various studies of the relationship
between T antigen expression, recurrence, and progres-
sion of bladder tumours.13' 139 In normal urothelium,
the T antigen is present in a cryptic form masked by
sialylation. In low-grade tumours, T antigen is expressed
due to loss of sialic acid, whereas the T antigen is
successively deleted with decreasing differentiation and
is absent in most invasive tuniours.i38Although most of
the invasive carcinomas are negative for T antigen, the
presence of T antigen in the initial non-invasive stage
seems to be a better predictor of subsequent tumour
invasion than the loss of this antigen.'38 In a study of 34
non-invasive (Ta) transitional cell carcinomas, it was
E. DABELSTEEN
I
I
Man I
I
I
Man H GICNACHGICNAC+- Asn
I
I
I
I
Man I
found that 7 out of 13 T antigen-positive tumours
subsequently progressed to invasion, whereas only 3 of
21 T antigen-negative tumours became invasive.
N-Glycans and prognosis
N-Linked oligosaccharides can be classified into com-
plex oligosaccharides, high-mannose oligosaccharides,
and hybrid forms (Fig. 9). N-Glycans have a great
diversity which is generated by a varying number of
side-chains called antennas that attach to the u-mannose
residues, and by variation in branching, length, and
terminal structures of these side-chains.j
Increased branching has been demonstrated as a
tumour-associated change in the N-linked carbohydrate
core structures (Fig. 9). This change has been correlated
with increased invasiveness and metastatic poten-
tia1.3,108,140
Metastatic sublines of the murine lymphoma
RAW117 cell line selected in vitro for enhanced meta-
static potential showed increased branching of N-linked
oligosaccharides, and the binding affinity of cells from a
human urothelial cell line to a lectin suggested that the
cells which were invasive had increased contents of tri-
and tetra-antenna structures. I4l Dennis et ul.Ios were
able to demonstrate an association between pl--6
branched complex type oligosaccharides and increased
metastatic potential in three independent tumour cell
lines. They found that tumour cells lacking the enzyme
required for branching vl-6 GlcNAc-transferase) did
not metastasize. It was further demonstrated that induc-
tion of increased PI-6 branching in a non-metastatic
murine mammary carcinoma cell line correlated strongly
with the acquisition of metastatic potential (Fig. 9).
An increased PI-6 branching of N-linked oligosac-
charides was also found in human colonic carcinomas
where regional nodal metastases had been detected.
Unfortunately, no follow-up studies have yet been per-
formed, but the authors suggested that increased
branching and subsequent elongation of the carbo-
hydrates by polylactosamine structures may be
associated with increased expression of terminal carbo-
hydrates, such as Le" and sialosyl-Le", and in this way
are responsible for the expression of the extended-chain
Lex, sialosyl-Le", and Ley antigens which appear to be
more cancer-specific than the shorter versions as
described above."
 CARBOHYDRATES AND TUMOUR PROGNOSIS
CONCLUSIONS
The field of tumour glycobiology evolved from devel-
opments in immunopathology demonstrating that aber-
rant expression of blood group antigens occurred in
tumours and parallel developments in carbohydrate
chemistry showing that blood group ABH antigens were
carbohydrates and that many tumour-related glycolipids
were structurally related to ABH antigens. Later devel-
opments have demonstrated that certain cell surface
carbohydrates are involved in cell-cell interactions and
that expression of these carbohydrates in tumours is
directly correlated to a poor prognosis. Ongoing studies
are expected to give detailed information concerning
carbohydrate-protein interaction and its possible role
in cellkell adhesion, cell homing, and the formation of
metastases. Such studies will eventually open up new
pathways for therapeutic intervention targeted at these
molecules. Synthetic carbohydrates, glycosphingolipids,
and clonal antibodies to carbohydrates have been
proposed as possible anti-adhesion agents for cancer
therapy.Io5 There is also the possibility of using well-
defined carbohydrate antigens for active, specific im-
munotherapy of cancer; recent clinical trials in this area
herald a growing potential for tumour glycobiology in
cancer treatment.
ACKNOWLEDGEMENTS
We are grateful for permission from Cancer to publish
Figures 5 and 7, from The New England Journal o j
Medicine to publish Figures 3 and 4 (1991; 324; 1084-90
and 1992; 327: 14-18. Massachusetts Medical Society),
from the British Journal of Cancer to publish Figure 8,
and from the authors T. Nakagoe, S. H. Itzkowitz, J. S.
Lee, M. Miyake and D. W. Miles.
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