Prognostic significance of syndecan-1 expression in human endometrial cancer

Hasengaowa, J. Kodama*, T. Kusumoto, Y. Shinyo, N. Seki and Y. Hiramatsu

Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

* Correspondence to: Dr J. Kodama, Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: +81-862357320; Fax: +81-862259570; Email: kodama{at}cc.okayama-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:: Syndecan-1 binds to various extracellular matrix components via its heparan sulfate glycosaminoglycans. The aim of this study was to investigate syndecan-1 expression in endometrial cancers.

Patients and methods:: We investigated the expression of the syndecan-1 core protein by immunohistochemistry in 109 endometrial cancers, and analyzed correlation with various clinicopathological features, including patient outcome.

Results:: Epithelial syndecan-1 expression was significantly lower in advanced stage, high grade, deep myometrial invasion, cervical involvement, lymph node metastasis, lymph vascular space involvement and positive peritoneal cytology. Stromal syndecan-1 expression was significantly higher in high-grade tumors. The disease-free and overall survival rates of patients exhibiting both low epithelial and high stromal syndecan-1 expression was poor. Multivariate analysis showed that high stromal syndecan-1 expression was an independent prognostic factor for both disease-free and overall survival. Low epithelial syndecan-1 expression was a prognostic factor only in the univariate analysis.

Conclusions:: Loss of epithelial syndecan-1 and induction of stromal syndecan-1 expression may be associated with tumor progression. Stromal syndecan-1 expression can serve as an indicator of poor prognosis in patients with endometrial cancer.

Key words: endometrial cancer, prognosis, syndecan-1


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Endometrial cancer is the most common disease among gynecological malignancies and remains a major health concern worldwide. Tumor cell invasion and subsequent metastasis via blood and lymph vessels are critical steps in malignant tumor progression, including endometrial cancer.

Heparan sulfate proteoglycans (HSPGs) consist of a core protein to which heparan sulfate glycosaminoglycan (HS-GAG) chains are covalently attached. These molecules are classified into several families according to the amino acid sequence of the core protein, such as syndecans and perlecan [1Go, 2Go]. Syndecans, cell-surface HSPGs, participate in cell–cell and cell–extracellular matrix (ECM) interactions [1Go, 3Go]. To date, four members of the syndecan family have been identified, all having homologous transmembrane and cytoplasmic domains, but different extracellular domains [4Go]. Syndecan-1 (CD138) is the best characterized member of the family and its expression is localized entirely in epithelial cells, with stratified squamous epithelia showing the most abundant expression [5Go]. Decreased expression of syndecan-1 has previously been reported to correlate with increased tumorigenicity and with tumor invasion [6Go–8Go]. Syndecan-1 binds to various ECM components, such as collagen, fibronectin, thrombospondin and tenascin, via its HS-GAG, and most of its biological functions are considered to be associated with this process [1Go–3Go]. Syndecan-1 also binds to the heparin-binding growth factor family, of which basic fibroblast growth factor is the best-studied example, but also includes hepatocyte growth factor (HGF), a splice variant of platelet-derived growth factor, heparin-binding epidermal growth factor (EGF), vascular endothelial growth factor, neuregulins and others [9Go]. The loss of epithelial syndecan-1 expression has been associated with poor prognosis in some forms of cancer, although this has not been previously assessed in endometrial cancer [10Go–16Go]. In contrast to the results of studies on a variety of other cancer forms, it has been reported that epithelial syndecan-1 expression may be an indicator of poor prognosis in breast cancer [17Go, 18Go]. Recently, the appearance of stromal expression of syndecan-1 has been correlated with a devastating clinical course in gastric, ovarian and breast cancers [11Go, 18Go, 19Go].

Here, we investigated the expression of syndecan-1 core protein in 109 endometrial cancers. We then analyzed correlation with various clinicopathological features, including patient outcome.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and tissue samples
The patient population consisted of 109 individuals presenting with endometrial cancer. All patients underwent hysterectomy, bilateral salpigo-oophorectomy, pelvic and/or para-aortic lymphadenectomy and partial omentectomy at the Department of Obstetrics and Gynecology of Okayama University Graduate School of Medicine and Dentistry, Japan between January 1997 and August 2002. Patients with distant metastasis were excluded from this study. Tumor specimens were obtained at the time of surgery and immediately fixed in 10% neural-buffered formalin and embedded in paraffin. Informed consent was obtained from each patient before sample collection. Histological cell types were assigned according to the WHO classifications: 104 were classified as endometrioid adenocarcinomas, four as adenosquamous and one as serous carcinomas. Histological grades according to the International Federation of Gynecology and Obstetrics (FIGO) staging classification were as follows: 37 were grade 1, 59 were grade 2 and 13 were grade 3.

Surgical staging was reviewed based on the FIGO staging system: 58 were allocated to stage I, 10 to stage II, 35 to stage III and six to stage IV. The median age at the time of surgery was 58 years (range 28–85). Patients with grade 3 tumor, non-endometrioid histological subtype, deep myometrial invasion or extrauterine disease were treated with adjuvant combination chemotherapy consisting of etoposide, epirubicin and cisplatin or paclitaxel, pirarubicin and carboplatin. Disease-free and overall survival rates were defined as the interval between the initial operation to either clinically or radiologically proven recurrence and death, respectively. The end date of the follow-up study for analysis was 30 September 2004 and the median duration of the follow-up was 45 months (range 2–85).

Immunohistochemistry
Sections 4 µm thick from several representative areas of the tumor specimens were mounted onto glass slides and immunostained according to the labeled streptavidin biotin procedure of the Dako LSAB kit (Dako, CA, USA). Briefly, after the slides were dewaxed in xylene and rehydrated in an alcohol series, and antigen retrieval was performed in a microwave oven in 10 mM citric acid buffer (pH 6.0) for 3x 10 min. The sections were incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity followed by incubation with normal horse serum for 5 min at room temperature. Immunostaining was then performed by incubation with a 1:100 dilution of mouse monoclonal anti-human syndecan-1 core protein (clone B-B4; Immunotech, Marseille, France) for 2 h at room temperature. The sections were next incubated for 20 min with biotinylated goat anti-mouse immunoglobulin followed by peroxidase-conjugated streptavidin for 20 min, and 10 min with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Wako Pure Chemical Industries, Osaka, Japan) containing hydrogen peroxide. Finally, the slides were counterstained with Mayer's hematoxylin and mounted in aqueous mounting medium. At each step the slides were washed carefully in phosphate-buffered saline (pH 7.4). As the negative control, the sections were incubated with normal mouse serum (Dako, Copenhagen, Denmark) at a concentration of 10 µg/ml. As the positive control, normal cervical squamous epithelia were available for analysis.

Staining evaluation
The level of syndecan-1 immunoreactivity in epithelial and stromal cells was expressed by scoring the percentage of syndecan-1 positive cells into three groups: strong (2), >50% of the cells stained; moderate (1); 10–50% of cells stained; and weak (0), <10% of cells stained. Microscopic analyses were evaluated independently by two of the authors with no prior knowledge of the clinical data. Final decisions in controversial cases were made using a conference microscope.

Statistical analyses
The Mann–Whitney U-test was used to examine the association between clinicopathological factors and syndecan-1 expression. Survival rates were calculated by the Kaplan–Meier method and differences were examined by the log-rank test. Factors found to be significant were then chosen for stepwise Cox's multivariate proportional hazard model to determine their prognostic values. These analyses were performed utilizing StatView 5.0 software (Abacus Concepts, Berkeley, CA, USA). P values <0.05 were considered statistically significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Epithelial syndecan-1 expression
Figure 1 illustrates representative immunostaining patterns of syndecan-1 in endometrial cancers. Strong epithelial staining was seen in 63 tumors (58%), moderate staining in 32 tumors (29%) and weak staining in 14 (13%) tumors. The mean score of epithelial staining according to clinicopathological factors is shown in Table 1. The score of epithelial syndecan-1 expression was significantly lower in patients with advanced FIGO stage (P=0.007), high FIGO grade (P=0.02), deep myometrial invasion (P=0.01), cervical involvement (P<0.0001), lymph node metastasis (P=0.0002), lymph vascular space involvement (P=0.0002) and positive peritoneal cytology (P=0.04). There was no significant association between epithelial syndecan-1 immunoreactivity and age or ovarian metastasis.



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Figure 1. Immunohistochemical staining of syndecan-1 in endometrial cancers using the anti-human syndecan-1 core protein B-B4. (A) Weak epithelial cell and moderate stromal cell staining. (B) Strong epithelial cell and weak stromal cell staining. (C) Weak epithelial cell and weak stromal cell staining. (D) Moderate epithelial cell and strong stromal cell staining.

 

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Table 1. Association between epithelial syndecan-1 expression and clinicopathological factors in endometrial cancers

 
Stromal syndecan-1 expression
Strong stromal staining was seen in 17 tumors (16%), moderate staining in 42 tumors (38%) and weak staining in 50 (46%) tumors. The mean score of stromal staining according to clinicopathological factors is shown in Table 2. The score of stromal syndecan-1 expression was significantly higher in high FIGO grade (P=0.05) tumors. There was no significant association between stromal syndecan-1 immunoreactivity nd any other clinicopathological factors. Furthermore, no significant association was noted between epithelial syndecan-1 expression and stromal syndecan-1 expression (data not shown).


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Table 2. Association between stromal syndecan-1 expression and clinicopathological factors in endometrial cancers

 
Univariate survival analysis
Figure 2 shows the disease-free and overall survival curves of 109 patients displaying endometrial cancer, according to epithelial syndecan-1 expression status. The disease-free and overall survival rates of patients exhibiting low epithelial syndecan-1 expression (score 0–1) were significantly lower than those of patients exhibiting high epithelial syndecan-1 expression (score 2) (P=0.004 and 0.005, respectively). Figure 3 shows the disease-free and overall survival curves, according to stromal syndecan-1 expression status. The disease-free and overall survival rates of patients exhibiting high stromal syndecan-1 expression (score 1–2) was significantly lower than those of patients exhibiting low stromal syndecan-1 expression (score 0) (P=0.004 and 0.02, respectively). Furthermore, the disease-free and overall survival rates of patients exhibiting both low epithelial and high stromal syndecan-1 expression were poor (P <0.0001) (Figure 4). The results of the univariate survival analyses of other variables are shown in Table 3.



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Figure 2. (A) Disease-free and (B) overall survival curves of the 109 patients displaying endometrial cancer according to their epithelial (E) syndecan-1 expression status. Low epithelial expression, score 0–1; high epithelial expression, score 2.

 


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Figure 3. (A) Disease-free and (B) overall survival curves of the 109 patients displaying endometrial cancer according to their stromal (S) syndecan-1 expression status. Low stromal expression, score 0; high stromal expression, score 1–2.

 


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Figure 4. (A) Disease-free and (B) overall survival curves of the 109 patients displaying endometrial cancer according to their epithelial (E) and stromal (S) syndecan-1 expression status. Low epithelial expression, score 0–1; high epithelial expression, score 2; low stromal expression, score 0; high stromal expression, score 1–2.

 

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Table 3. Disease-free and overall survival analyses of prognostic factors using the log-rank test

 
Multivariate survival analysis
Multivariate analysis showed that lymph node metastasis was the strongest independent prognostic factor for disease-free survival, followed by ovarian metastasis, deep myometrial invasion, high grade and high stromal syndecan-1 expression (Table 4). In addition, lymph node metastasis was the strongest independent prognostic factor for overall survival, followed by deep myometrial invasion, high stromal syndecan-1 expression and high-grade tumor (Table 4). Low epithelial syndecan-1 expression was not an independent prognostic factor for either disease-free or overall survival.


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Table 4. Prognostic factors for disease-free and overall survival selected by Cox's multivariate proportional hazard model analysis

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Invasion and metastasis are characteristics of malignant solid tumors. Many mechanisms are involved in these processes and cell adhesion molecules, such as integrins, cadherins and cell-surface HSPGs, are particularly important in the regulation of cell differentiation, morphology and migration [20Go–22Go]. Syndecans, which are cell-surface HSPGs, participate in cell–cell and cell–ECM interactions. In our current study, syndecan-1 core protein expression in endometrial cancer specimens was investigated.

Epithelial syndecan-1 expression was reduced with advanced stage, high grade, deep myometrial invasion, cervical involvement, lymph node metastasis, lymph vascular space involvement and positive peritoneal cytology. Miturski et al. [23Go] reported in their small series that the immunoreactivity of anti-syndecan-1 antibody correlates statistically with histological differentiation of endometrial cancers. They also reported that sections of proliferating endometrium were positively and regularly stained. Reduced expression of epithelial syndecan-1 in colorectal, gastric and laryngeal cancers, hepatocellular carcinoma, head and neck carcinoma, cholangiocarcinoma, and malignant mesothelioma has been reported to be associated with de-differentiating cancer cells or increasing metastatic potential [10Go–13Go, 15Go, 16Go, 24Go]. These results suggest that epithelial syndecan-1 is an important molecule in the regulation of cell proliferation and differentiation in a wide range of tumors. Indeed, it has been shown that the suppression of endogenous syndecan-1 expression in epithelial cells by transfection with syndecan-1 antisense cDNA causes loss of the epithelial characteristics of their parental cells, and acquiring of an elongate fusiform morphology, the ability to invade and migrate within collagen gels and anchorage-independent growth. The fusiform antisense transfectants show rearrangement ß1-integrins, markedly reduced E-cadherin expression, and altered disposition of the actin cytoskeleton [25Go]. Contradictory findings have also been reported, and increased epithelial syndecan-1 expression was related to an aggressive phenotype and poor prognosis in patients with breast cancer [17Go, 18Go]. The effects of epithelial syndecan-1 in tumor progression may be dependent on organ and tumor type examined.

Stromal syndecan-1 expression was first demonstrated by Stanley et al. [26Go] in infiltrating breast carcinoma. They speculated that because syndecan-1 interacts with heparin-binding growth factors such as FGF-2, accumulation of syndecan-1 within the tumor stroma may contribute to the extensive angiogenesis and stromal proliferation characteristic of infiltrating breast carcinoma. Mukunyadzi et al. [27Go] showed that stromal expression of syndecan-1 is present in high-grade head and neck squamous cell carcinoma. This is consistent with our finding that stromal syndecan-1 expression was significantly higher in high-grade endometrial cancer. In general, high-grade tumors lose epithelial syndecan-1 expression. This shift in syndecan-1 expression from epithelial cells to stromal cells is interesting, although the mechanism of syndecan-1 expression in stroma remains to be elucidated. Maeda et al. [28Go] recently reported that syndecan-1 expression in stromal cells promotes carcinoma cell growth. They speculated that syndecan-1 expression in stromal fibroblasts creates a favorable microenvironment for accelerated tumor cell growth by storing and presenting growth factors to the carcinoma cells, since many epithelial mitogens, including FGFs, HGF and heparin-binding EGF, bind to syndecan-1 HS-GAG chains.

Our study is the first to demonstrate that both loss of epithelial syndecan-1 expression and induction of stromal syndecan-1 expression are associated with reduced survival outcomes in patients with endometrial cancer. Decreased expression of epithelial syndecan-1 has been previously reported to correlate with a poor prognosis in colorectal, gastric, head and neck, and laryngeal cancers, non-small-cell lung carcinoma, cholangiocarcinoma and malignant methothelioma [10Go–16Go]. Furthermore, the multivariate analysis in the present study showed that stromal syndecan-1 expression is an independent prognostic factor for both disease-free and overall survival. Wiksten et al. [11Go] reported that stromal syndecan-1 immunoreactivity was a prognostic factor independent of stage, surgery for cure and tumor size in gastric cancer. Davies et al. [19Go] reported that stromal syndecan-1 expression was a poor prognostic factor for survival in ovarian cancer. These studies indicate that syndecan-1 may be a promising prognostic factor for these kinds of cancer.

In conclusion, loss of epithelial syndecan-1 and induction of stromal syndecan-1 expression may be associated with tumor progression in endometrial cancer. Stromal syndecan-1 expression can serve as an indicator of poor prognosis in patients with endometrial cancer.


    Acknowledgements
 
We thank Ms Chisae Kodera for her help with histochemistry and section cutting.

Received for publication December 8, 2004. Revision received February 19, 2005. Accepted for publication February 21, 2005.


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