Annals of Oncology Advance Access originally published online on October 25, 2006
Annals of Oncology 2007 18(2):269-274; doi:10.1093/annonc/mdl370
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© 2006 European Society for Medical Oncology
gynecologic tumors |
Prognostic significance of stromal versican expression in human endometrial cancer
1 Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2 Department of Obstetrics and Gynecology, Inner Mongolia Agriculture University Hospital, Huhehaote, China
* Correspondence to: Dr J. Kodama, Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: +81-862357320; Fax: +81-862259570; E-mail: kodama{at}cc.okayama-u.ac.jp
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Abstract |
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Background: Versican expression may enhance tumor invasion and metastasis. However, the expression of versican in human endometrial cancer has seldom been characterized. The aim of this study was to investigate versican expression in endometrial cancers.
Patients and methods: We immunohistochemically investigated the expression of versican protein in 167 endometrial cancers and analyzed the correlation with various observed clinicopathological features, including patient outcome.
Results: Stromal versican expression was significantly higher in the advanced-stage (P = 0.010) and high-grade (P = 0.049) cancers, lymph node metastasis (P = 0.012), and ovarian metastasis (P = 0.024). Epithelial versican expression was significantly higher in patients with lymph node metastasis (P = 0.014) and lymph-vascular space involvement (P = 0.014). The disease-free survival (DFS) and overall survival (OS) rates of patients exhibiting high stromal versican expression were significantly lower than those of patients exhibiting low stromal versican expression (P < 0.0001). Multivariate analysis showed that high stromal versican expression was an independent prognostic factor for DFS and OS.
Conclusions: Versican enrichment of the stroma may be associated with tumor progression in endometrial cancer. Stromal versican expression can serve as an indicator of poor prognosis for patients with endometrial cancer.
Key words: endometrial cancer, prognosis, tumor progression, versican
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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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 the progression of malignant tumor, including endometrial cancer. It is well known that the tumor environment is one of the major factors that determine the behavior of malignant cells. Remodeling of the extracellular matrix (ECM) through altered expression of molecules integrated in the functional network of cell-to-cell and cell-to-matrix interactions is essential for local tumor cell invasion and metastasis [1].
Proteoglycan is one of the components of the ECM that has the ability to alter cell function. Versican is a member of the large aggregating chondroitin sulfate proteoglycan (CSPG) family [2]. Structurally, versican is composed of an N-terminal G1 domain, a glycosaminoglycan (GAG) attachment region, and a C-terminal G3 domain. Alternative splicing generates at least four isoforms of versican, named V0, V1, V2, and V3 [3–5]. V0, the largest versican isoform, contains two GAG-binding regions called the CS
and CSß domains. The V1 isoform contains a CSß domain, and the V2 isoform contains a CS domain. Versican V3 is solely composed of the G1 and G3 domains, lacking all potential GAG attachment sites. Versican is highly expressed in the early stages of tissue development, and its expression decreases after tissue maturation. Its expression is also elevated during wound repair and tumor growth [6–8]. An increase in versican expression in the ECM facilitates local tumor invasion and metastasis by decreasing the cell–ECM adhesion [9]. In fact, it has been demonstrated that versican expression is related to tumor progression in some types of malignant tumors [10–13].
Thus, versican expression may enhance tumor cell invasion and metastasis, although its expression in human endometrial cancer has seldom been characterized. Therefore, we investigated the expression of the versican protein in 167 endometrial cancers. We then analyzed its correlation with various observed clinicopathological features, including patient outcome.
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patients and methods |
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patients and tissue samples
In this study, we examined 167 patients with endometrial cancer. Between January 1997 and November 2004, each of these patients underwent a hysterectomy, bilateral salpingo-oophorectomy, pelvic and/or para-aortic lymphadenectomy, and partial omentectomy at the Department of Obstetrics and Gynecology of Okayama University Medical School. Patients with distant metastasis were excluded from this study. Tumor specimens were obtained at the time of surgery and were immediately fixed in 10% neutral buffered formalin and embedded in paraffin. Informed consent was obtained from each patient before sample collection. Histological cell types of the specimens were determined according to the World Health Organisation classifications. Consequently, 159 specimens were classified as endometrioid adenocarcinomas, five as adenosquamous carcinomas, and three as serous carcinomas. Histological grades were assigned according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Sixty were classified as grade 1; 81, as grade 2; and 26, as grade 3. The surgical staging status was determined based on the FIGO staging system. Thus, 96 specimens were allocated to stage I, 15 to stage II, 46 to stage III, and 10 to stage IV. The median age of patients at the time of surgery was 58 years (range, 27–85 years). Patients with a grade 3 tumor, a nonendometrioid histological subtype, a deep myometrial invasion, or an extrauterine disease were treated with three to six courses of adjuvant combination chemotherapy. Disease-free survival (DFS) and overall survival (OS) times were defined as the interval between the initial surgery and either clinically or radiologically proven recurrence and death, respectively. The end date of the follow-up study for analysis was March 31, 2006, and the median duration of the follow-ups was 45 months (range, 1–108 months). In all, 22 of 167 patients developed recurrent disease, and 17 patients died during the follow-up time.
immunohistochemistry and staining evaluation
Four-micrometer-thick sections from several representative areas of the tumor specimens were placed onto glass slides and immunostained according to the labelled streptavidin–biotin procedure of the Dako LSAB kit (Dako North America Inc., Carpinteria, CA). Briefly, after the slides were dewaxed in xylene and rehydrated in an alcohol series, antigen retrieval was performed in a microwave oven in 10 mM citric acid buffer (pH 6.0) for 3 x 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 : 500 dilution of mouse monoclonal anti-human versican (clone: 2B1; Seikagaku Corporation Ltd, Tokyo, Japan) for 2 h at room temperature. The sections were next incubated for 20 min with biotinylated goat anti-mouse immunoglobulin followed by incubation with peroxidase-conjugated streptavidin for 20 min and with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Wako Pure Chemical Industries Ltd, Osaka, Japan) containing hydrogen peroxide for 10 min. 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). The blood vessel walls in the myometrium were taken as a positive control for the analysis.
staining evaluation
The level of versican immunoreactivity in the stroma was expressed by classifying the area of peri- and intratumoral versican-positive stroma into four groups: strong, >50% of the stroma stained; moderate, 10%–50% of the stroma stained; weak, <10% of the stroma stained; and negative, no staining. We also evaluated versican-positive cancer cells. Any case with cancer cell-associated versican staining was considered as positive. Microscopic analyses were evaluated independently by two of the authors who had no prior knowledge of the clinical data. Final decisions in questionable cases were made using a conference microscope.
statistical analyses
The chi-square test was used to examine the association between clinicopathological factors and versican expression. The survival rates were calculated by the Kaplan–Meier method, and the differences between the survival curves were examined by using the log-rank test. Factors found to be significant were then analyzed by a stepwise Cox's multivariate proportional hazard model to decide their prognostic values. These analyses were performed by utilizing the Stat-View 5.0 software (Abacus Concepts Inc., Berkley, CA). P values <0.05 were considered to be statistically significant.
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stromal versican expression
In all cases, versican staining was observed in the walls of the relatively large blood vessels of the myometrium (Figure 1A). Figure 1B–D illustrate the representative immunostaining of stromal versican in the endometrial cancers. Strong stromal staining was observed in 12 tumors (7%), moderate staining in 57 tumors (34%), weak staining in 39 (23%) tumors, and no staining in 59 tumors (35%). The association between stromal versican staining and clinicopathological factors is shown in Table 1. Stromal versican expression was significantly higher in cases assigned a high-FIGO stage (P = 0.010) and a high-FIGO grade (P = 0.014) and in lymph node metastasis (P = 0.012) and in ovarian metastasis (P = 0.024). There was no significant association between stromal versican immunoreactivity and other clinicopathological factors.
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epithelial versican expression
Figure 1C illustrates the characteristic immunostaining of epithelial versican in an endometrial cancer. Epithelial versican staining was observed in 36 tumors (22%) and no staining in 131 tumors (78%). The frequency of epithelial versican staining was significantly higher in cases with stromal versican staining (P = 0.006). The association between epithelial versican staining and clinicopathological factors is shown in Table 2. Epithelial versican expression was significantly higher in patients with lymph node metastasis (P = 0.049) and lymph-vascular space involvement (P = 0.014). There was no significant association between epithelial versican immunoreactivity and any other clinicopathological factor.
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univariate survival analysis
Figure 2 presents both the DFS and OS curves for the 167 patients displaying endometrial cancer, according to the stromal versican expression status. The DFS and OS rates of patients exhibiting high stromal versican expression (strong and moderate staining) were significantly lower than those of patients exhibiting low stromal versican expression (weak and no staining) (P <0.0001). Figure 3 presents both DFS and OS curves, according to the epithelial versican expression status. The DFS and OS rates of patients exhibiting epithelial versican expression were lower than those of patients exhibiting negative epithelial versican expression, although the difference was not statistically significant. The results of the univariate survival analyses of other variables are shown in Table 3.
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multivariate survival analysis
Multivariate analysis showed that FIGO stage IV was the strongest independent prognostic factor for DFS; this was followed by high stromal versican expression, ovarian metastasis, and the FIGO grade (Table 4). In addition, lymph node metastasis was the strongest independent prognostic factor for OS, followed by the FIGO grade, FIGO stage IV, and high stromal versican expression (Table 4). In a cohort of 159 endometrioid adenocarcinomas, FIGO stage IV was the strongest independent prognostic factor for DFS; this was followed by high stromal versican expression. FIGO stage IV was the strongest independent prognostic factor for OS followed by lymph node metastasis and the FIGO grade (data not shown).
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discussion |
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This is the first study to analyze versican protein expressions in a large series of human endometrial cancer specimens. In all the specimens, versican was enriched in the relatively large vascular structures. This observation is consistent with a previous report that demonstrated the expression of versican in various adult tissues, including blood vessels [14]. Versican, which is abundantly produced by vascular smooth muscle cells, may be implicated in the normal hemostasis of blood vessels and may also be involved in some of the pathological conditions that affect these tissue structures [15]. Furthermore, normal endometria express small to moderate amounts of stromal versican throughout each reproductive cycle, although there is no expression in atrophic endometria (data not shown). We speculate that dynamic changes in the endometrium may be regulated, in part, by stromal versican expression.
We demonstrated that the overexpression of stromal versican was associated with a high-FIGO stage, a high-FIGO grade, lymph node metastasis, and ovarian metastasis. Hanekamp et al. [16] reported that in their small series, CSPG/versican is preferentially expressed in poorly differentiated endometrial cancers. The overexpression of stromal versican in epithelial ovarian cancer, breast cancer, non-small-cell lung cancer (NSCLC), and prostate cancer has been reported to be associated with tumor progression [10–13]. These results suggest that stromal versican is an important molecule in the progression of several malignant tumors. Indeed, it has been shown that the versican G1 domain can enhance cell proliferation and reduce cell adhesion in different cell types [17, 18], and the versican G3 domain enhances tumor growth and angiogenesis [19]. However, a contradictory observation has also been reported; increased stromal versican expression was found to be related to less advanced tumors in patients with pharyngeal squamous cell carcinoma [20]. The effects of stromal versican expression on tumor progression may therefore be dependent on the organ and tumor type examined.
In the present study, versican was mainly present in the peritumoral stroma; however, tumor cell-associated versican was also observed. The occurrence of versican-positive tumor cells was significantly higher in cases with lymph node metastasis and lymph-vascular space involvement. Voutilainen et al. [10] reported that epithelial versican expression is significantly higher in early-stage epithelial ovarian cancer. Pirinen et al. [12] reported that tumor cell-associated versican is not significantly associated with clinicopathological factors in NSCLC, although the frequency of the expression was insufficient to allow statistical analysis. Touab et al. [21] reported that cell-associated versican is involved in the progression of melanomas. Although malignant cells can synthesize versican, the mechanism and functional role of epithelial versican expression remains to be elucidated [21–23]. Further studies are required to clarify this issue.
Our study is the first to demonstrate that high stromal versican expression is associated with the reduced survival outcomes in patients with endometrial cancer. An increased level in the expression of stromal versican has been previously reported to correlate with a poor prognosis in some types of cancers [10–13]. Furthermore, the multivariate analysis in the present study showed that stromal versican expression is an independent prognostic factor for both DFS and OS. Suwiwat et al. [11] have also reported that stromal versican immunoreactivity is an independent prognostic factor in node-negative breast cancer. These studies indicate that stromal versican expression may be a promising prognostic factor for these kinds of cancers.
In conclusion, versican enrichment of the stroma may be associated with tumor progression in endometrial cancer. Our findings also provide evidence that stromal versican expression can serve as an indicator of poor prognosis in patients with endometrial cancer.
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We thank Ms Chisae Kodera for her help with histochemistry and section cutting.
Received for publication May 12, 2006. Revision received August 23, 2006. Accepted for publication September 6, 2006.
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