EXPRESSION OF HYALURONAN IN NORMAL AND DYSPLASTIC BRONCHIAL
EPITHELIUM AND IN SQUAMOUS CELL CARCINOMA OF THE LUNG
Risto T. PIRINEN1,2*, Raija H. TAMMI3, Markku I. TAMMI3, Paavo K. PA ¨A ¨KKO ¨5, Jyrki J. PARKKINEN1,4, Ulla M. ÅGREN3,
Risto T. JOHANSSON2, Markku M.T. VIREN2, Ulla TO ¨RMA ¨NEN5, Ylermi M.J. SOINI5, and Veli-Matti KOSMA1,4
1Department of Pathology and Forensic Medicine, University of Kuopio, Kuopio, Finland
2Department of Oncology, Kuopio University Hospital, Kuopio, Finland
3Department of Anatomy, University of Kuopio, Kuopio, Finland
4Department of Pathology, Kuopio University Hospital, Kuopio, Finland
5Department of Pathology, University of Oulu and Oulu University Hospital, Oulu, Finland
A series of 85 lung/bronchial tissue samples from 76 pa-
tientsconsisting ofnormal, metaplastic and dysplastic epithe-
lium and different types of lung carcinomas were analyzed for
the distribution of hyaluronan (HA), using a biotinylated
hyaluronan binding complex as an HA-specific probe.
The normal pseudo-stratified columnar bronchial epithe-
lium was either negative for HA or displayed a weak staining
around the basal cells. The epithelia of serous and mucous
bronchial glands were HA negative whereas the submucosal
connectivetissuewasstrongly positive.Inmetaplastic, dysplas-
tic and carcinoma in situ lesions the whole epithelium from
basal to uppermost cells expressed HA on plasma mem-
branes. Epithelial HA wasalso foundinsquamouscell carcino-
mas, but not in adenocarcinomas, carcinoid tumors or small
cell carcinomas of the lung. W hereas epithelial HA was
present in all lesions of the squamous cell type, the staining
intensity displayed great local variability in 50% of the cases
with severe dysplasia, carcinoma in situ and squamous cell
carcinomas. In squamous cell carcinomas, such an irregular
staining pattern was significantly associated with poor differ-
entiation. Our results indicate that the expression of HA in
different bronchial lesions and lung tumors is restricted to
those showing squamous cell differentiation, being absent
from other types of lung carcinomas. The increase of HA-
depleted areasin poorly differentiated squamouscell carcino-
mas emphasizes the important role of HA in tumor differen-
tiation. HA on carcinoma cell surface may influence tumor
growth and metastatic behavior. Int. J. Cancer (Pred. Oncol.)
?1998 Wiley-Liss, Inc.
Lung cancer has become one of the leading causes of death
worldwide. At the time of the diagnosis, lung cancer is usually
widespread, and, in spite of improvements in therapy, prognosis is
often poor (Carney, 1995).To understand the biological behavior of
lung cancer better and to define new strategies for improving
prognosis, attempts have been made to find new prognostic and
predictive markers (Carney, 1995; Szabo and Mulshine, 1993).
These include the p53 protein, which is overexpressed more
frequently in severe bronchial dysplasia and carcinoma in situ than
in metaplasia but never in the normal bronchial epithelium (Nuorva
et al., 1993). Increased epidermal growth factor receptor (EGFR)
expression, K-ras mutations and loss of nuclear heterozygosity
have also been associated with lung cancer (Graziano, 1997). Many
cell adhesion molecules act as both positive and negative modula-
tors during the metastatic process (Freemont, 1995; Zetter, 1993).
The role of these adhesion factors in the proliferation and
development of lung cancer has been studied less extensively.
Hyaluronan (HA) is a glycosaminoglycan ubiquitous in all
connective tissue and is also present in several types of epithelia
(Tammi et al., 1988, 1994a,b). HAis synthesized at the cell surface
by the membrane-bound enzyme hyaluronate synthase (Prehm,
1984) and contributes to control of cell migration, differentiation
and proliferation (Toole, 1981), thereby influencing tissue morpho-
genesis, wound healing and tumor growth (Mast et al., 1992;
Zhang et al., 1995). Increased HA content has been reported in
several tumors (Auvinen et al., 1997;Wang et al., 1996), and it may
influence tumor growth and invasiveness by providing an ex-
panded, hydrated loose matrix for carcinoma cells. HA may also
facilitate tumor growth by protecting the tumor from cytotoxic
cells (McBride and Bard, 1979). Fragments of HA created by the
enzyme hyaluronidase stimulate angiogenesis and probably malig-
nant neovascularization, supporting tumor growth (Deed et al.,
1997; West and Kumar, 1991).
In normal bronchial tissue, most of the HA exists in the
connective tissue stroma, although weak staining of HA and its
receptor, CD44, is present around the basal cells of normal
pseudo-stratified bronchial and bronchiolar epithelium (Green et
al., 1988). Elevated HA concentration in the tissues and broncho-
alveolar fluid occurs in many benign diseases with lung injury,
probably reflecting tissue repair (Juul et al., 1993, 1995). Elevated
HAexpression is valuable in the differential diagnosis of mesothe-
lioma (Azumi et al., 1992). Concentrations of HA in serum and
broncho-alveolar lavage fluid are also higher in patients with
bronchogenic carcinoma, especially in small cell lung carcinoma,
than in those with benign disease (Hernandez-Hernandez et al.,
1995). Whether the HA originates from the tumor parenchyma,
stroma or adjacent tissues is unclear.
The expression of epithelial HA is modulated in premalignant
and malignant esophageal and breast tumors (Auvinen et al., 1997;
Wang et al., 1996). We hypothesized that HA expression may also
be changed in bronchial metaplasia and dysplasia, which are
presumably stages in the evolution of squamous cell lung carci-
noma (Pendleton et al., 1993, 1996). To test this hypothesis, we
collected samples of metaplastic and dysplastic lesions and malig-
nant lung tumors and stained them for HA using a specific probe
(Wang et al., 1992).
MATERIAL AND METHODS
Our tissue material consisted of 85 lung/bronchial samples from
76 patients treated in the Kuopio and Oulu University Hospitals,
Finland, by lobectomy or pulmectomy. Paraffin-embedded samples
collected from the archives of the Departments of Pathology were
sectioned at 5 µm. Hematoxylin and eosin-stained sections were
used to type and grade the lesions histologically according to the
WHO (1981) criteria by 2 observers (RP, V-MK). Normal epithe-
lium was present in 15 samples and metaplastic epithelium without
cellular atypia in 2 samples. Dysplastic epithelia were found in 19
samples; 3 of these were classified as mild, 2 as moderate and the
rest as severe or carcinoma in situ lesions.The invasive carcinomas
consisted of 30 squamous cell carcinomas, 6 adenocarcinomas, 11
small cell carcinomas and 2 carcinoid tumours (Table I).
Grant sponsors: Kuopio University Hospital (EVO); Research Founda-
tion, ORION Corporation; North-Savo CancerAssociation, Finland.
*Correspondence to: Department of Pathology and Forensic Medicine,
University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Fax:
?358-17-162753. E-mail: email@example.com
Received 20 November 1997
Int. J. Cancer (Pred. Oncol.): 79, 251–255 (1998)
?1998 Wiley-Liss, Inc.
Publication of the International UnionAgainst Cancer
Publication de l’Union Internationale Contre le Cancer
Preparation of the biotinylated HA probe
The biotinylated hyaluronan binding complex (bHABC) used as
a specific probe was prepared from bovine articular cartilage as
described previously (Tammi et al., 1994a; Wang et al., 1992). In
brief, proteoglycans containing mostly aggregans were extracted
from cartilage with 4 M guanidium chloride in 50 mM sodium
acetate, pH 5.8. The extract was dialyzed against distilled water in
the presence of high m.w. HA (Healon, Pharmacia, Uppsala,
Sweden). The complex of HA binding region and link protein
(HABC) and HA was isolated from the rest of the proteoglycan
molecule using trypsin treatment (type XI; Sigma, St. Louis, MO).
The complex between the HAbinding region of aggregan (HABR),
link protein and HA was purified by hydroxyapatite chromatogra-
phy (DNAgrade, Bio-Rad, Richmond, CA), and Sephacryl S-1000
gel filtration (Pharmacia). The proteins in the complex were then
biotinylated (Wang et al., 1992), and HA was separated from the
probe by dissociative gel filtration on Sephacryl S-400 (Pharma-
cia). The purity of the preparation was confirmed by polyacryl-
amide gel electrophoresis and Western blotting using monoclonal
antibodies (MAbs) against the HA binding region of aggregan and
link protein as described by Caterson et al. (1985).
Staining of HA
The slides were deparaffinized in xylene and rehydrated in
graded alcohols followed by washing in 0.1 M phosphate buffer,
pH 7.4 (PB). To block endogenous peroxidase activity, the slides
were washed with 10% H2O2for 3 min and then incubated for 30
min with 1% BSA in PB to block non-specific binding. The slides
were incubated overnight with bHABC (protein concentration 5
µg/ml, diluted in 1% BSAin PB) at 4°C, washed throughly with PB
and treated with avidin-biotin-peroxidase (ABC; Vector, Burlin-
game, CA; dilution 1:200) for 1 hr at room temperature. Following
washes in PB, the slides were incubated in DAB (0.05% 3.3?
diaminobenzidine; Sigma) containing 0.03% H2O2for 5 min. The
slides were dehydrated in graded alcohols and mounted in DPX
without counterstaining. The specificity of the staining was tested
by digesting the sections with Streptomyces hyaluronidase (Seik-
agagu,Tokyo, Japan; 100TRU/ml in 50 mM sodium acetate buffer,
pH 5.0, for 3 hr) in the presence of protease inhibitors prior to
staining (Tammi et al., 1994a) or preincubating the bHABC probe
with hyaluronan oligosaccharides (Ripellino et al., 1985).
Evaluation of staining
The bHABC staining was simultaneously evaluated by 2 observ-
ers (RP, V-MK) who were unaware of the clinical data. The
bronchial epithelium and the tumoral areas were analyzed sepa-
rately. The distribution of HAwas classified as homogeneous when
all parts of the epithelium or tumor tissues showed the same
intensity of staining reaction. The staining was classified as
irregular when the staining intensity varied throughout the tumor
area, showing some negative areas, but being mostly HA positive.
The intensity of homogeneous HA staining was classified as
negative (0), weak (1), moderate (2) or intense (3). In irregularly
stained lesions, all intensities (0–3) existed in the same slide, and so
specific classification was not possible (Table I).
In statistical calculations, the SPSS for Windows program was
used. Fischer’s exact probability test was used to calculate the
possible significant associations between the groups.
Normal bronchial and bronchiolar epithelium
The normal pseudostratified columnar epithelium was either
totally HA negative (6 cases) or displayed a weakly positive HA
staining in the basement membrane zone and around the basal cells
(9 cases); the superficial ciliated and goblet cells of the pseudo-
stratified epithelium were HA negative in all cases (Fig. 1a, Table
I). In smaller bronchioles (5 cases, in which the epithelium was
more cuboidal), a weak basal staining for HA extended to about
halfway up the whole epithelium; the rest of the epithelium was
negative. The epithelial cells in bronchial serous and mucous
glands were always totally HA negative, in clear contrast to the
normal mucosal and submucosal connective tissues of the bron-
chial wall, which displayed intense HA staining (Fig. 1a,b). The
bronchial cartilage was weakly stained for HA.
Bronchial squamous cell metaplasia and dysplasia
In 2 cases of squamous metaplasia, the epithelium displayed
moderate to intense HAstaining throughout all cell layers (Fig. 1b),
and the staining was localized on the plasma membranes. The
adjacent normal columnar bronchial epithelium was HA negative.
All dysplastic epithelia displayed HA throughout the epithelium
(Fig. 1c). In contrast to the metaplasias, in about half of the cases
dysplasias showed irregular HA staining (Table I), varying from
virtually negative to intensely stained areas. The dysplastic lesions
with homogeneous staining exhibited moderate (43%) or intensive
Squamous cell carcinomas
The epithelium was HApositive in all squamous cell carcinomas
of the lung (Fig. 1d,e, Table I). While an irregular staining pattern
was seen in all histological grades (I–III), the proportion of
irregularly stained specimens was higher in grade III (79%), than in
TABLE I – DISTRIBUTIONAND STAINING INTENSITY OF HYALURONAN IN NORMAL, METAPLASTICAND DYSPLASTIC
EPITHELIUMAND IN LUNG CARCINOMAS
Severe/carcinoma in situ
Squamous cell carcinoma
Small cell carcinoma
1Different staining intensities (0–3) were seen in the same slide.–2Homogeneously stained epithelia/
tumors: 0, negative; 1, weak; 2, moderate; 3, intense HAstaining.–3Weak staining reaction around the basal
cells was seen in 9 cases.–4Grade III squamous cell carcinomas showed an irregular HA staining pattern
significantly more often than grade I–II tumors (p ? 0.012, Fisher’s exact test).
PIRINEN ET AL.
FIGURE 1 – (a)The normal columnar bronchial epithelium is mostly hyaluronan (HA) negative; note weak staining around the basal cells (star).
The adjacent sub-epithelial stroma was always intensely stained. Scale bar ? 35 µm. (b) Metaplastic bronchial epithelium stained intensely and
homogeneously with HA. Scale bar ? 35 µm. (c) Severe dysplasia/carcinoma in situ of the bronchial epithelium shows homogeneous and intense
HA staining pattern. Scale bar ? 35 µm. (d) Typical homogeneous HA staining pattern noted in grade I squamous cell carcinoma. Scale bar ?
20 µm. (e) HA staining pattern is irregular in grade III squamous cell carcinoma. Arrow indicates the location of basement membrane and
arrowhead the stromal staining pattern. Scale bar ? 35 µm.
HYALURONAN IN BRONCHIAL LESIONS
grade I (25%) or grade II tumors (33%) (Fig. 1e,Table I). Hence the
irregular staining pattern showed a significant association with
tumor grade (Fisher’s test, p ? 0.012). The staining intensity in
homogeneously stained specimens was either moderate or intense
in 13 of the 14 samples (Fig. 1d), and only one tumor was weakly
Adenocarcinoma of the lung, small cell lung carcinoma and
bronchial carcinoid tumor
The epithelial components in the well-differentiated adenocarci-
nomas of the lung, including 2 bronchiolo-alveolar subtypes, were
HA negative (Fig. 2a, Table I). The epithelial tissue in small cell
lung carcinomas and bronchial carcinoid tumors (Fig. 2b) was also
devoid of HA.
Tumor stroma was HA positive in all carcinoma types. In small
cell carcinomas, adenocarcinomas and bronchial carcinoid tumors,
the amount of stromal tissue was scant, but the intensity of HA
staining in the stroma was comparable to that in squamous cell
carcinomas and in the mucosal stroma of the normal bronchial wall
(Fig. 1a,b). The stromal HA staining intensities of grades I, II and
III squamous cell tumors were equal.
We have tested here the hypothesis that HA expression pattern
might change in parallel with the structural change of normal
lium into squamous metaplasia, dysplasia or carcinoma was
associated with increased expression of HA. The strong signal for
HAcorresponded to the pattern of normal stratified epithelia found
earlier in organs such as skin (Tammi et al., 1988), gingiva (Tammi
et al., 1990) and esophagus (Wang et al., 1996). The expression of
HAwas specific for the bronchial squamous cell carcinomas, since
adenocarcinomas, small cell lung carcinomas and bronchial carci-
noid tumors expressed no epithelial HA.Also, poorly differentiated
squamous cell carcinomas showed irregular staining, with a
considerable proportion of tumor parenchyma negative for HA.
Our present findings suggest that HA expression is an integral part
of the squamous differentiation pattern, even in abnormal ectopic
In a contrast to normal squamous epithelia, where HAdisappears
when the superficial cells differentiate terminally (Tammi et al.,
1988; Wang et al., 1996), the metaplastic and neoplastic bronchial
cells continued to express HAup to the surface.The lack of HAloss
in the superficial cells indicates that the metaplastic cells are
partially defective in dedifferentiation and resemble in this respect
the carcinoma in situ lesions of the esophagus (Wang et al., 1996).
The pseudo-stratified columnar epithelium of normal bronchus,
like that in the male reproductive tract (Tammi et al., 1994b)
contained only traces of HAclose to the basal area. The basal cells
in bronchial epithelium represent progenitor cells responsible for
the renewal of the epithelium. The presence of HA around them is
consistent with the hypothesis that HA is needed for cell prolifera-
tion (Inoue and Katakami, 1993).
The well-differentiated bronchial adenocarcinomas in the pres-
ent study were HA negative, like normal bronchial glands, their
proposed tissue of origin (Noguchi et al., 1995). This finding is
consistent with a previous report on lung adenocarcinomas, of
which only 8% were positive for HA (Azumi et al., 1992).
Similarly, most well-differentiated adenocarcinomas of the gastric
tract (Wang et al., 1996) and breast (Auvinen et al., 1997) are also
devoid of HA. Interestingly, in a large study of colorectal carcino-
mas, there was a strong association with the intensity of HA
staining on tumor cells and poor prognosis (Ropponen et al., 1998).
The malignant cells in the small cell carcinoma were totally devoid
of HA, although the concentration of HA in serum and bronchial
lavage fluid increase in these patients (Hernandez-Hernandez et al.,
1995). This is obviously due to the inflammatory tissue reactions
and continuous stromal remodeling that occurs around malignant
tumors and that increases HArelease.
The significant association between irregular distribution of HA
and tumor grade suggests that in squamous cell carcinoma, there
may be a situation opposite to that in adenocarcinoma, with the
partial loss of HA now indicating increased malignancy and
unfavorable outcome. The partial loss of HA in poorly differenti-
ated squamous cell carcinomas may reflect cessation of HA
synthesis, loss of HA receptors or local expression of HA-
degrading factors like hyaluronidase (Thet et al., 1983) or oxygen-
free radicals (Ågren et al., 1997). The degradation products may be
important signals for the alveolar macrophages (McKee et al.,
1996). Local changes in HAsynthesis in squamous cell carcinomas
has not been studied, but expression of the HA receptor, CD44,
occurs in all squamous cell carcinomas of the lung (Ariza et al.,
1995; Jackson et al., 1994; Penno et al., 1994).
By analogy to the proteolytic enzymes known to facilitate tumor
invasion (McCormick and Zetter, 1992), a hyaluronidase has been
described in colon carcinomas, presumably active in the extracellu-
lar milieu (Liu et al., 1996). There is a possibility that the patchy
disappearance of epithelial HA in the grade III squamous carcino-
mas is due to local expression of a hyaluronidase. While the
presence of hyaluronidase is completely speculative at present, the
consequences of such an activity could support tumor growth, since
FIGURE 2 – (a) Adenocarcinoma cells are HA negative; only scant stroma is HA positive. Scale bar ? 50 µm. (b) The stroma of the bronchial
carcinoid tumor stains positively with HA, whereas the tumor cells are HAnegative. Scale bar ? 35 µm.
PIRINEN ET AL.
esis and migration of endothelial cells (Deed et al., 1997; West and Download full-text
In summary, we have shown that in normal, metaplastic, and
dysplastic bronchial epithelia, and in different types of lung
carcinomas, the expression of HA is restricted to the lesions
exhibiting squamous cell differentiation. The cell surfaces of
squamous cell metaplastic and dysplastic epithelium revealed a
consistent HA signal. In the direction of poor differentiation, the
squamo-cellular carcinomas started to display areas lacking HA, a
finding having the potential to identify cases with an unfavorable
The technical assistance of Mrs.A. Parkkinen, Mrs. S. Haatanen
and S. Eskelinen is gratefully acknowledged.
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