Full text of DOI:10.1016/S0194-5998(00)70010-6

Correlation of galectin-3/galectin-3-binding sites with
low differentiation status in head and neck squamous
cell carcinomas
SOPHIE DELORGE, MD, SVEN SAUSSEZ, MD, PATRICIA PELC, MD, BENOÎT DEVROEDE, MD, HADELIN MARCHANT, MD,
MARIA BURCHERT, PhD, FU-YUE ZENG, PhD, ANDRÉ DANGUY, PhD, ISABELLE SALMON, MD, PhD, HANS-JOACHIM GABIUS, PhD,
ROBERT KISS, PhD, and SERGIO HASSID, MD, PhD, Brussels, Belgium, and Munich, Germany
The accurate determination of levels of differentia-
tion is of prognostic value in human head and neck
squamous cell carcinomas (HNSCCs). Because the
deliberate selection of biochemical determinants
accompanying certain stages of differentiation can
refine the predictive power of histochemical
assessments, the application of the quantitative
evaluation of staining distribution and intensity by
computer-assisted microscopy is one prerequisite
to potential improvements. We used 2 innovative
approaches with peanut agglutinin based on
encouraging results with respect to common lectin-
histochemistry. First, we used a custom-made neo-
glycoprotein to monitor the presence of Thomsen-
Friedenreich (T) antigen-binding sites. Second, we
measured the presence of 2 galectins immunohis-
tochemically and, at the same time, measured
lectin-histochemically the presence of accessible
ligands for the endogenous lectins. We also moni-
tored the presence of calcyclin, a protein with rele-
vance to cell cycle progression or exocytosis. With
61 cases of HNSCC as their basis, including 31 oral,
20 laryngeal, and 10 hypopharyngeal lesions, the
data show that the main modifications observed in
connection with a loss of differentiation are related
to a modification in the levels of both galectin-3/
galectin-3-binding site and T-antigen/T-antigen-
binding site expressions. The data obtained also
suggest that galectin-3 could act as an acceptor
site for the T antigen. Because the level of differen-
tiation is known to be indicative of the recurrence
rate in HNSCCs and our data clearly indicate that
galectin-3 and the T antigen (and their respective
binding sites) are involved in dedifferentiation
processes, further investigation is warranted into the
roles of galectins in HNSCC tumor progression and
recurrence analysis. (Otolaryngol Head Neck Surg
2000;122:834-41.)
A
s recently emphasized by Khuri et al,1 head and neck
cancer (HNC) is a notable health problem worldwide,
with an estimated 900,000 cases in 1995. Pathologists
have a number of tools for the evaluation of malignancy
levels in HNCs. Between 85% and 90% of epithelial
HNCs are squamous cell carcinomas2 (HNSCC). The
level of malignancy can be assessed by means of both
the clinical TNM staging system and histopathologic
grading. As stated by Urist et al,3 although the TNM
system4 is the accepted standard for HNSCC classifica-
tion, there are often discrepancies between TNM-pre-
dicted prognostic values and actual patient survival.
These discrepancies occur with respect to both the T3,5
and the N6,7 variables.
From the Departments of Otolaryngology–Head and Neck Surgery (Drs
Delorge, Saussez, and Hassid) and Pathology (Dr Salmon), Cliniques
Universitaires de Bruxelles, Hôpital Erasme; the Department of
Otolaryngology–Head and Neck Surgery, Hôpital des Enfants Reine
Fabiola (Dr Pelc); Laboratory of Histopathology, Faculty of
Medicine, Université Libre de Bruxelles (Drs Devroede, Danguy, and
Kiss); Department of Otolaryngology–Head and Neck Surgery,
Hôpital Brugmann (Dr Marchant); Institute of Physiological
Chemistry, Ludwig-Maximilians University (Drs Burchert, Zeng, and
Gabius); and Fonds National de la Recherche Scientifique (Dr Kiss).
Supported by grants from the Fonds de la Recherche Scientifique
Médicale, Belgium, and the Dr.M.-Scheel-Stiftung für Krebsfor-
schung, Germany.
Histopathologic grading relates to the determination
of HNSCC differentiation levels with their 3 categories
of good, moderate, and poor differentiation in accor-
dance with various criteria, including the progressive
loss of keratinization.8 The determination of the level
of differentiation is of proven prognostic value in
HNSCCs.7,9 In this respect, correlations have been
described between differentiation level, tumor vascular-
ization,10 and the cumulative incidence of metas-
tases.7,11 According to various studies published in the
literature, the differentiation level may10,11 or may not5
mirror the values provided by the TNM staging system.
However, there is agreement on the notion that the pro-
gressive loss of HNSCC differentiation is accompanied
Reprint requests: Robert Kiss, PhD, Laboratory of Histology, Faculty
of Medicine, Free University of Brussels, 808 route de Lennik,
1070 Brussels, Belgium.
Copyright © 2000 by the American Academy of Otolaryngology–
Head and Neck Surgery Foundation, Inc.
0194-5998/2000/$12.00 + 0 23/77/100451
doi:10.1067/mhn.2000.100451
834

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DELORGE et al 835
by an increase in biologic, and therefore clinical,
aggressiveness. This alteration of the level of differenti-
ation not only is visible through an examination of the
tissue architecture but also is apparent when distinct
biochemical determinants are monitored histochemically.
Such changes may account for the altered cell behavior.
Because of the enormous extent of biologic information
that can be stored in oligosaccharide structures, these
glycan chains have gained increasing attention as a
research focus to decipher the glycocode system in bio-
logic information transfers.12-14
sites), and calcyclin, and their levels of differentiation.
We also used peanut agglutinin (PNA) to locate the T
antigen because it continues to be of supposed impor-
tance in the field of tumor biology. A tailor-made neo-
glycoprotein with the synthetic T-antigen structure
enables endogenous sites with binding capacity to this
disaccharide to be visualized, thereby detecting both
components of an assumed interaction.31 Quantitative
measurements of the histochemical expression of the
selected biologic markers were carried out by means of
computer-assisted microscopy.32-34
In normal oral epithelia, cell surface carbohydrates
are predominantly expressed in basal cells as part of
mucins, and these carbohydrate chains are elongated
parallel to terminal differentiation.15,16 The loss of cer-
tain membrane-associated carbohydrate epitopes, which
belong to the blood group antigen system, is attributable
to the HNSCC stage and invasion level17 as well as to
early recurrence.18 Certain blood group antigen-consti-
tuting oligosaccharides, including the sialosyl-T
(Thomsen-Friedenreich) or the sialosyl-Tn antigens, are
expressed in HNSCCs, but not in normal epithelia.19
Several families of mammalian lectins have already
been defined14 in connection with the receptor sites of
recognitive protein-carbohydrate interactions. Because
of the widespread occurrence of β-galactoside-contain-
ing glycoconjugates, the galectin group deserves special
attention as mediators.20 Galectin-1 and/or galectin-3
having initiated immunohistochemical and biochemical
studies on rodent tumors and human breast cancer,21,22
their expression patterns have been examined in tumors
of the endometrium,23 colon,24 and thyroid.25 Gillen-
water et al26 recently reported that galectin-1 and galectin-
3 occur in HNSCCs and are located on the cell surface,
where they may participate in cell interactions. These
authors had previously observed that the expression pat-
tern of galectins appeared to be associated with the
degree of squamous differentiation; this suggests a
potential role for galectins as biologic and differentia-
tion markers in the case of HNSCCs.
METHODS AND MATERIAL
Clinical and Histopathologic Data
This series included 31 cancers from the oral cavity, 20
from the larynx, and 10 from the hypopharynx. The 31 can-
cers from the oral cavity included 8 from the tongue, 8 from
the tonsils, 7 from between the tongue and the tonsils, and 8
from the buccal mucosa. All the patients (52 men and 9
women, age range 45-79 years) had undergone partial or rad-
ical surgery. The diagnoses were established on the basis of
the histologic criteria described by Hyams et al.35 Clinical
staging was carried out by means of the TNM classification.4
There were 5 T-I, 17 T-II, 14 T-III, and 25 T-IV cases. Of these
61 HNSCCs, 44 were node negative, and 17 were node posi-
tive. All 61 patients had stage M0 tumors.
The 61 HNSCCs were categorized as well (n = 24), mod-
erately (n = 25), or poorly (n = 12) differentiated on the basis
of the following criteria. The well-differentiated cancers were
characterized by the presence of intercellular bridges and
numerous keratinization foci, whereas the poorly differentiated
ones were characterized by sparse (if not a total lack of) kera-
tinization foci and an absence of intercellular bridges.
Individual cell keratinization and the presence of cell junc-
tions are consistent with the moderately differentiated pheno-
type.
Ligand Histochemistry
HNSCC tissue specimens were fixed in 4% formaldehyde
and embedded in paraffin, and 5-µm-thick sections were
processed with the various histochemical probes and kit re-
agents under study. As described elsewhere,36-38 the markers
were prepared and biotinylated under activity-preserving con-
ditions, and specific polyclonal antibodies to calcyclin were
raised. The p-aminophenyl derivative of the T antigen was
synthesized and conjugated to bovine serum albumin, as pre-
viously described.10 Incubation with labeled probes was car-
ried out at room temperature for 60 minutes at a concentration
of 10 µg/mL.
Proteins associated with cell proliferation were
investigated principally in the search for markers asso-
ciated with predictive values. This group of proteins in
HNSCCs includes members of the large S100-related
protein family27; one of these proteins is the S100A6
member, denoted as calcyclin,28 which has the ability to
interact with negatively charged sugars and peptide
motifs of annexin II, annexin VI, and glyceraldehyde-3-
phosphate dehydrogenase.29,30
The aim of this study has been to investigate a series
of 61 patients with HNSCCs to determine whether there
is a significant correlation between the expressions of
galectin-1, galectin-3 (and their respective binding
The extent of specifically bound markers (the biotinylated
probes) was revealed by means of avidin-biotin-peroxidase
complex (ABC) kit reagents (Vector Labs, Burlingame, CA),
with diaminobenzidine and H2O2 as chromogenic substrates;

Otolaryngology–
Head and Neck Surgery
June 2000
836 DELORGE et al
A
B
C
D
E
F
G
Fig 1. Development of the mean (open squares), SE (solid rectangles), and SD (bars) values quanti-
tatively describing (by means of the LI variable) the percentage of HNSCC tissue area specifically
stained by 7 distinct biotinylated probes or target-specific antibodies (carrier-immobilized T antigen
[A], PNA [B], anticalcyclin [C], antigalectin-1 antibody [D], galectin-1 [E], antigalectin-3 antibody
[F], and galectin-3 [G]) as a function of the level of tumor differentiation. The data analyzed includ-
ed 24 well, 25 moderately, and 12 poorly differentiated cancers.

Otolaryngology–
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DELORGE et al 837
Table 1. Coexpression of the various determinants in HNSCCs
Tag
PNA
Calc
Gal-1
aGal-1
Gal-3
At LI-dependent value level
PNA
Calc
cGal-1
aGal-1
Gal-3
aGal-3
NS
NS
NS
NS
NS
—
0.011
0.012
0.006
0.002
0.001
—
0.009
NS
—
NS
—
0.0005
0.031
0.0012
0.0003
0.0012
0.0011
—
0.0001
0.0001
At MOD-dependent value level
PNA
Calc
Gal-1
aGal-1
Gal-3
aGal-3
0.0006
<0.00001
NS
NS
0.003
NS
—
0.00006
NS
NS
NS
—
0.023
NS
0.00001
NS
—
0.0001
0.021
NS
—
NS
0.013
—
NS
NS
Values represent the level of statistical significance obtained by means of the Kendall rank correlation test. Whereas the LI relates to the percentage of tissue area
specifically stained by a glycohistochemical probe, the MOD relates to the staining intensity (ie, the histochemically revealed probe concentration in the tissue area
analyzed).
Tag, T antigen as a ligand part of a biotinylated neoglycoprotein; PNA, biotinylated PNA; Calc, anticalcyclin antibody; Gal-1, biotinylated galectin-1; aGal-1, anti-
galectin-1 antibody; Gal-3, biotinylated galectin-3; aGal-3, antigalectin-3 antibody; NS, not significant.
this is detailed elsewhere.34,37 Control reactions included
competitive inhibitions to ascertain sugar specificity, and the
omission of the incubation step with a labeled marker served
to exclude any staining by the binding of kit reagents, such as
the mannose-rich glycoproteins horseradish peroxidase and
avidin. Counterstaining with toluidine blue concluded the pro-
cessing.
µm2, were scanned on each histologic slide. The 2 quantitative
variables computed for each of the 4 histochemical probes
included: the labeling index (LI), which corresponds to the
percentage of tissue area specifically stained by a glycohisto-
chemical probe; and the mean optical density (MOD), which
corresponds to the staining intensity (ie, the histochemically
revealed probe concentration in the tissue area analyzed).
Computer-assisted Microscopy
Statistical Analysis
The expression of calcyclin, galectin-1, and galectin-3 by
the tumor cells of the 61 HNSCCs under study was immuno-
histochemically shown by the binding of specific anticalcy-
clin, antigalectin-1, and antigalectin-3 antibodies. The expres-
sion of the galectin-1- and galectin-3-binding sites was
demonstrated glycohistochemically by means of biotinylated
galectin-1 and galectin-3. The expression of the T antigen was
glycohistochemically demonstrated by means of biotinylated
PNA, whereas the expression of the T-antigen-binding sites
(which correspond to endogenous PNA-related lectins) was
shown glycohistochemically by means of the biotinylated car-
rier-immobilized T antigen (ie, with the T antigen used as a
ligand part of a biotinylated neoglycoprotein).39 Seven bio-
logic markers were thus available for each of the 61 HNSCCs.
Two quantitative variables were computed for each of the 7
markers by means of a SAMBA 2005 computer-assisted
microscope system (Alcatel-TITN, Grenoble, France) with a
20× (aperture 0.50) magnification lens. The way in which we
used this system to quantify the histochemical staining fol-
lowed an optimized protocol, as did the ways in which we
dealt with the problems relating to quantitative histochem-
istry.32-34 Fifteen fields, each of between 60,000 and 120,000
Some of the results are given as the mean ± SEM. In such
cases the statistical comparisons of the data were made by a 1-
way analysis of variance after a check on the equality of vari-
ance by means of the Levene test and the normal distribution
fitting of the data by means of a χ2 test. The nonparametric
Mann-Whitney U test was applied if the variances were not
equal or if the value distribution was not normal. Correlations
between the expression levels of the various histochemical
probes were evidenced by means of the Kendall rank correla-
tion test. All the statistical analyses were performed with
Statistica/Windows software (StatSoft, Tulsa, OK).
RESULTS
Development of the Biologic Marker-related LI
and Mod Indexes in Relation to the HNSCC
Differentiation Levels
Fig 1 shows that for 5 of the 7 markers the mean LI
values (describing the percentage of HNSCC tissue area
labeled by each histochemical probe) decreased signifi-
cantly in the monitoring of the well, moderately, and
poorly differentiated lesions. These 5 markers were
biotinylated PNA, indicating the presence of the T anti-

Otolaryngology–
Head and Neck Surgery
June 2000
838 DELORGE et al
A
B
C
D
E
F
G
Fig 2. Development of the mean (open squares), SE (solid rectangles), and SD (bars) values quanti-
tatively describing (by means of the MOD variable) the concentration of the target epitopes for each
of the 7 probes (carrier-immobilized T antigen [A], PNA [B], anticalcyclin [C], antigalectin-1 antibody
[D], galectin-1 [E], antigalectin-3 antibody [F], and galectin-3 [G]) as a function of the HNSCC dif-
ferentiation levels. The data analyzed included 24 well, 25 moderately, and 12 poorly differentiated
cancers.

Otolaryngology–
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DELORGE et al 839
DISCUSSION
gen in the tumor cells (Fig 1B; P = 0.0009), anticalcy-
clin antibody (Fig 1C; P = 0.003), antigalectin-1 anti-
body (Fig 1D; P = 0.005), antigalectin-3 antibody (Fig
1F; P = 0.01), and biotinylated galectin-3, which visu-
alized accessible binding sites for galectin-3 (Fig 1G; P
= 0.004). In contrast, the mean LI values obtained after
the application of the carrier-immobilized T antigen as
a ligand part of a biotinylated neoglycoprotein (Fig 1A)
and biotinylated galectin-1 (Fig 1E) did not significant-
ly (P > 0.05) follow an unambiguous pattern in line with
the differentiation levels.
In comparison with Fig 1, Fig 2 shows that the mean
MOD values developed less significantly than the LI
ones in relation to the HNSCC differentiation level.
Indeed, only the mean MOD values obtained by the
immunohistochemical detection of galectin-3 (Fig 2F;
P = 0.006), as well as by the lectin-histochemical detec-
tion of galectin-3 ligands (Fig 2G; P = 0.002), signifi-
cantly decreased in parallel to the loss of extent of dif-
ferentiation.
The search for refinements in the set of biologic
markers is fueled by the suggested ensuing clinical con-
sequences. Wolf et al18 stated that the clinical use of
biologic staging parameters in the initial assessment of
HNSCC patients should enable more aggressive and
successful primary treatment strategies to be selected
for individual patients. This point of view is motivated,
at least in part, our present study, which aims to assess
whether the expression of calcyclin, galectin-1, and
galectin-3 relates to HNSCC differentiation levels. Two
biologic markers were monitored whose intrinsic diag-
nostic and/or prognostic values in HNSCCs, and par-
ticularly in oral types, have already been under-
scored.15-19,34 These two markers are the T antigen and
the T-antigen-binding sites, both of which were visual-
ized by the innovative combination of chemical syn-
thesis and histochemistry. The presence of T-antigen-
binding sites (which, in fact, can be considered as an
equivalent of a PNA-type endolectin in terms of sugar
affinity) was detected in the 61 HNSCCs under study by
means of the carrier-immobilized T antigen, whereas
biotinylated PNA was used to reveal the presence of the
T antigen in the HNSCC tumor cells. Our results on
PNA binding dovetail with those previously reported by
others. A similar observation was particularly made for
the neoglycoprotein, which has so far only been tested
in a few cases. Indeed, T-antigen-binding site expres-
sion was reduced in parallel with the decrease in differ-
entiation level (ie, from a well to a moderately to a
poorly differentiated status). Considering the complete
panel of probes selected, this study intimates clearly
that, in addition to these two markers, the loss of differ-
entiation in HNSCCs can also affect calcyclin and
galectin expression. Furthermore, because the expres-
sion level of these various markers was quantified by
means of computer-assisted microscopy, we were able
to unravel various previously undetected coexpressions
of targets for certain probes.
Coexpression of Various Carbohydrate-binding
Proteins in HNSCCs
The histologic specimens that were used to carry out
the histochemical staining came from serial sections of
each of the individual tumors analyzed. This means that
contiguous areas in each tumor were monitored by each
of the markers. This prompted us to investigate whether
the pattern of expression of certain target molecules in
any given section in fact correlated with the expression
of other markers in the contiguous areas. When the dis-
tributions of the 61 LI values that characterized the
expression pattern of the different determinants were
compared, a remarkable correlation surfaced between
the T-antigen-dependent (showing PNA-like endolectin)
and antigalectin-3 antibody-dependent LI value distrib-
utions (Table 1). The PNA-related LI value distribution
correlated significantly with each of the other LI value
distributions (Table 1). Whereas the calcyclin-related LI
value distribution correlated significantly with the
galectin-1-, galectin-3-, and antigalectin-3-dependent
LI value distributions, it failed to do so with the anti-
galectin-1-dependent distribution (Table 1). The same
feature was observed in the case of the galectin-1-
dependent LI value distribution. The antigalectin-1-
related LI value distribution correlated significantly
with both the galectin-3- and antigalectin-3-dependent
patterns, as did galectin-3 with antigalectin-3 (Table 1).
Table 1 clearly shows that very high levels of statis-
tical significance were obtained from distinct MOD dis-
tribution values. This was the case for the Tag/Calc,
PNA/Calc, Calc/Gal-3, and Gal-1/aGal-1 paired rank
correlations (Table 1).
The data in Fig 1 indicate that the loss of HNSCC
differentiation from a well to a moderately to a poorly
differentiated status is reflected biologically, not only in
a decrease in the proportion of cells and/or extracellular
matrix tissue presenting the T antigen (but not the PNA-
like endolectin; Fig 1A) but also in a decrease in those
exhibiting calcyclin, galectin-1 (but not the galectin-1-
binding site; Fig 1E), and both galectin-3 and galectin-
3-binding site positivity. As indicated in Fig 2, in some
cases it was not only the proportion of cells harboring
binding sites for a given marker that decreased but also
the concentration of this marker per cell. This holds true
for galectin-3 and its binding sites, for example.
Because no direct measurement of T antigen as a li-

Otolaryngology–
Head and Neck Surgery
June 2000
840 DELORGE et al
gand for galectins has been carried out so far, common-
ly assaying panels of carbohydrates in inhibition assays,
a discussion is precluded on the actual potency of the T
antigen as a binding partner for galectins. However, the
LI parameter correlations suggest an interaction at the
galectin-3 level.
Checkerboard correlations enable us to discuss fur-
ther relationships. Although no T-antigen-binding site-
positive cells and only a limited percentage of T-anti-
gen-positive ones were correlated with the percentage
of calcyclin-positive cells, both the T-antigen and T-
antigen-binding site concentrations correlated markedly
(P < 0.00001 and P = 0.00006, respectively) and posi-
tively with the calcyclin concentration. Unless this cor-
relation is fortuitous, regulatory mechanisms linking the
two marker classes can be tentatively suggested on the
basis of this result.
intriguing implication to link the detection of the T anti-
gen functionally to that of certain galectins, at least in
part. This result has relevance for lectin-histochemical
studies on this new class.
It should be emphasized that although our data clearly
demonstrate the intergroup correlation between the
molecular markers that we used and the tumor differen-
tiation status, there is a substantial overlap between
individual patients. Additional markers are therefore
needed to allow pathologists to use these markers to
provide prognostic information on an individual basis.
The characterization of the levels of expression of new
galectins (including galectin-9) and S-100 proteins
(including S-100A1, S-100A2, S-100A3, S-100A4, S-
100A5, and S-100B) is under way in our laboratory.
CONCLUSION
Although initially detected as a growth factor–
inducible gene product of quiescent fibroblasts, calcy-
clin can evidently exert other functions besides playing
a part in cell cycle progression.30,40,41 It has recently
been reported that its RNA gene expression can be evi-
denced in HNSCCs, but not in benign lesions.42 These
data clearly reveal the expression of this protein.
However, they show that the loss in HNSCC differenti-
ation level is paralleled by a significant decrease in cal-
cyclin expression.
The data from this study reveal that modifications in
the level of HNSCC differentiation—a prognosis-relat-
ed feature—are accompanied by changes in the extent
of galectin expression, of their accessible binding sites,
and of calcyclin.
We thank Dr J. L. Wang for his kind gift of the expression
vector for murine galectin-3.
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With respect to galectins, the loss of HNSCC differ-
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galectin-3 expression. Although malignant thyroid tis-
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normal and even benign tissue does not express these
markers.25,43 Schoeppner et al24 observed that galectin-
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to Dukes’ stage, thereby indicating a linear relationship
with advancing stage. Enhanced galectin-3 expression
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Indeed, colon cancer mucins have been evidenced as
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