Annals of Oncology Advance Access originally published online on October 8, 2007
Annals of Oncology 2008 19(1):81-85; doi:10.1093/annonc/mdm344
gynecologic tumors |
Twist expression in patients with cervical cancer is associated with poor disease outcome
Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan
* Correspondence to: Dr K. Shibata, Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan. Tel: +81-52-744-2263; Fax: +81-52-744-2268; E-mail: shiba{at}med.nagoya-u.ac.jp
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Abstract |
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Background: Twist, a basic helix–-loop–helix transcription factor, has been reported to be associated with the development and progression of human cancer. We examined the distribution and expression of Twist in cervical cancer to examine its clinical significance.
Patients and methods: We examined the distribution and expression of Twist in 101 cervical cancer specimens and determined the association between their expression and the clinico-pathological features observed, including patient outcome.
Results: Of the 101 specimens, 55 cases were negative for Twist immuno-expression, whereas 46 were positive. When categorized into negative versus positive expression, Twist was not associated with any of the clinico-pathological parameters examined. Positive Twist expression significantly predicted poorer overall survival (OS) and progression-free survival (PFS) when compared with negative expression (P < 0.01). In the multivariate analyses, positive Twist expression was an independent prognostic factor for OS (P < 0.05).
Conclusions: Our data imply that positive Twist expression seems to be a useful marker in patients with cervical cancer likely to have an unfavorable clinical outcome.
Key words: cervical cancer, epithelial mesenchymal transition (EMT), prognosis, Twist
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introduction |
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Cervical cancer is the second most frequently diagnosed malignancy in women worldwide, and it is the only major gynecologic malignancy that is staged clinically according to the International Federation of Obstetrics and Gynecology (FIGO) recommendation [1]. Despite the generally good prognosis for early-stage cervical cancer, approximately one-third of these patients are expected to eventually die from the disease. To improve the assessment of an individual patient's risk of disease progression, several clinical parameters, including lymph node status, tumor size, depth of stromal invasion, and lymphovascular space invasion (LVSI) are employed. The invasion of lymphatic or blood vessels by carcinoma cells is considered a critical step in the formation of lymphatic or distant metastasis. Involvement of lymphatic vessels clearly is an indicator of an unfavorable prognosis, even in early-stage cervical cancer. The identification and functional characterization of molecules involved in lymphovascular space invasion may, therefore, reveal targets for diagnostic and therapeutic techniques.
Epithelial-mesenchymal transition (EMT) is a recently identified novel concept implicated in tumor metastasis. This process was first recognized as a feature of embryogenesis in the early 1980s [2]. The resulting cells lose their basal/apical polarity and phenotype, become elongated, are mobile, and are capable of migrating through the extracellular matrix [3, 4]. The basic helix–loop–helix transcription factor Twist is a major regulator of mesenchymal phenotypes. It has been shown that loss of appropriate levels of expression or mutations of normal human Twist results in developmental defects [5, 6]. It has recently been proposed to be relevant in cancer as well. Suppression of Twist by small interfering RNA in highly metastatic mammary or prostate cancer cells, induced the expression of epithelial components and specifically inhibited their capacity for invasion and metastasis [7]. In addition, increased expression of Twist in four tumor cell lines was found to be associated with resistance to paclitaxel as well as other drugs that similarly disrupt microtubles [8]. Furthermore, MCF-7 cells overexpressing Twist exhibited a deregulated p53 response to 
-radiation, including cell cycle progression and down-regulation of downstream target genes like p21Waf1/Chip1 and MDM-2 [9].
These facts prompted us to hypothesize that Twist acts as a tumor-progression factor for cervical cancer, and that Twist may have clinical usefulness as a novel molecular target or as a prognostic indicator in the treatment of cervical cancer. Based on this hypothesis, the present study examined the immunohistochemical expression of Twist in cervical cancer tissues to determine whether Twist expression is correlated with clinico-pathological factors or the prognosis of cervical cancer patients.
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materials and methods |
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tissues
Tissue specimens were obtained from 101 patients who were surgically treated at Nagoya University Hospital. Patients who underwent pre-operative treatment, such as radiotherapy and chemotherapy, were excluded. Informed consent was obtained from each patient for sample use. All tissue samples were fixed in 10% formalin, embedded in paraffin, and routinely stained with hematoxylin and eosin for histological examination.
immunohistochemistry
Formalin-fixed, paraffin-embedded tissue sections were cut at a thickness of 4 µm. For heat-induced epitope retrieval, deparaffinized sections in 0.01M citrate buffer (Target Retrieval Solution, pH 6.1, Dako) were heated three times for 5 min each at 90°C and 750 W using an H2500 microwave oven. Immunohistochemical staining was performed employing the avidin-biotin immunoperoxidase technique using the Histofine SAB-PO kit (Nichirei, Tokyo, Japan) according to the manufacturer's protocol. Briefly, endogenous peroxidase activity was blocked by incubation with 0.3% H2O2 in methanol for 15 min, and non-specific immunoglobulin binding was blocked by incubation with 10% normal goat serum for 10 min. Sections were incubated at 4°C for 12 h with anti-Twist antibody (anti-rabbit-incubated at 1 : 100 dilution, Santa Cruz Biotech, CA, USA). The sections were rinsed and incubated for 30 min with biotinylated secondary antibody. After washing, the sections were incubated for 30 min with horseradish peroxidase-conjugated streptavidin, and finally treated with 3-amino-9-ethylcarbazole in 0.01% H2O2 for 10 min. The slides were counterstained with Meyer's hematoxylin. As a negative control, the primary antibody was replaced with normal rabbit IgG at an appropriate dilution. Immunostaining intensity was scored based on the percent positivity of stained cells as negative (0–10%), positive (10–50%), and strongly positive (<51%). Each case was scored independently by two different investigators.
statistical analysis
The association between negative vs positive Twist expression and clinico-pathological parameters was evaluated using 
2-tests. The univariate survival analysis was based on the Kaplan–Meier method. Comparison between the survival curves was analyzed using the log-rank test. The OS was defined as the time between the date of surgery and the last date of follow-up or date of death owing to cervical cancer. The PFS was defined as the time interval between the date of surgery and the date of progression/recurrence or date of last follow-up. The prognostic significance of Twist expression concerning other pathological variables was assessed using the multivariate Cox's proportional hazard's analysis. Stat View software ver.5.0 (SAS, Institution Inc., Cary, NC, USA) was used for all statistical analyses and a P-value of <0.05 was considered significant.
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results |
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immunohistochemical detection of Twist in cervical cancer tissues
Diffuse staining for Twist was detected both in the cytoplasm and on the cell membrane of cervical cancer cells (Figure 1). There was little immunoreactivity of Twist in the tumor stroma. We found cases in which cancer involving vascular spaces exhibited strong Twist staining (Figure 1E). Of the 101 cervical cancer specimens examined in this study, 46 (45.5%) cases were positive for Twist immuno-reactivity, of which eight cases were strongly positive. The strongly positive cases were included in positive cases in the statistical analysis. The association between Twist expression and clinicopathologic variables is shown in Table 1. There was no significant correlation between the expression of Twist and patient age, FIGO stage, histological type, or LVSI.
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There was a tendency whereby high Twist expression is associated with pelvic lymph node metastasis, but the association was not significant (P = 0.093).
association of twist expression with survival of cervical cancer patients
The median follow-up for all patients was 63 months (range: 33–118). Among 101 patients, 23 patients (22.8%) had relapses of cervical cancer at the time of the last follow-up, and 20 patients (19.8%) died. The 5-year OS rates of patients who were negative (n = 55) and positive (n = 46) for expression of Twist were 92.7 and 70.2%, respectively (Table 2). Figure 2 shows the OS and PFS curves with respect to Twist expression. Both OS and PFS in patients positive for expression of Twist were significantly lower than those in patients who were negative for Twist expression (OS: P = 0.0006; PFS: P = 0.0068). In univariate analyses, FIGO stage, histological type, positive for LVSI, positive for lymph node metastasis, and positive expression of Twist were significant predictors of poor OS (Table 2). In addition, for PFS, four prognostic factors (age, FIGO stage, positive for lymph node metastasis, and positive expression of Twist) were significant (Table 2). We further divided all cases into two groups according to the FIGO stage (early stage: stage I–IIA; advanced stage: stage IIB–IV). In either group, Twist expression was significantly associated with the prognosis (stage I–IIA: P = 0.0153, stage IIB–IV: P = 0.0239) (Figure 3A, B). Similarly, all cases were stratified according to histological subtype (squamous and adenocarcinoma). The expression of Twist was a significant prognostic factor in the squamous type, but not in the adenocarcinoma type (squamous: P = 0.0091; adenocarcinoma: P = 0.104) (Figure 3C, D). However, the small number of specimens employed in the adenocarcinoma group may account for the lack of significance.
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multivariate analysis of prognostic variables in cervical cancer patients
In multivariate OS analyses, FIGO stage, histological type, status of LVSI and lymph node metastasis, and Twist expression were entered into Cox proportional hazard analysis. Twist expression (relative risk, 4.167; P = 0.0165), along with FIGO stage, were identified as independent predictive factors. Furthermore, multivariate analysis for PFS, including age, FIGO stage, lymph node matastasis and Twist expression, revealed that only the FIGO stage was a significant, independent prognostic factor (relative risk, 3.391, P = 0.0164). Twist expression could not be a significant independent prognostic factor by a narrow margin (relative risk, 2.378: P = 0.0502) (Table 3).
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discussion |
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Various studies have demonstrated the involvement of Twist as a crucial regulator of the metastatic process in cancer [10–12]. In the present study of 101 cervical patients with long-term follow-up, the Twist expression level was not associated with any of several clinico-pathological parameters, The Twist expression level tended to correlate with lymph node metastasis, although not significantly. High Twist expression was associated with poor overall survival in both univariate and multivariate analyses of all factors that influenced survival. There have been a few other reports showing a correlation between Twist expression and clinical significance in patients with malignancies [13–15]. There are several possible reasons for the association of Twist with a poor prognosis in various cancers. In a previous report, Kyo et al. demonstrated that Twist expression in endometrial cancers was significantly associated with the depth of myometrial invasion and reduced survival [13]. In this study, too, Twist expression tended to correlate with lymph node metastasis, although not significantly. Regarding the mechanism, one possible explanation of our current finding that Twist expression was linked to a poor prognosis may be an increased metastatic potential. Recently, a novel function of Twist has been reported in the development of acquired chemoresistance in human cancer cells. Up-regulation of Twist was associated with cellular resistance to microtube-targeting anticancer drugs in various types of cancers [8]. It was also shown that its mechanism involves the Akt-dependent anti-apoptotic pathway [16]. Other studies demonstrated that down-regulation of Twist by antisense in mouse fibroblasts increased sensitivity to etoposide-induced apoptosis [17]. Thus, Twist may be involved in a drug-induced apoptosis pathway. In addition, a recent study by Stasinopoulos et al. has shown that Twist expression influences the expression of p53-regulated genes in breast cancer cells, thereby controlling their radiation response [9]. In this study, we did not examine the function of Twist in a cervical cancer cell line; however, in our center, we have been performing post-operative radiotherapy and/or chemotherapy for advanced cervical cancers, and the results to date suggest that Twist expression influenced the radiosensitivity or chemosensitivity, thereby being involved in the prognosis. In this study, the analysis of the histological type-stratified data showed that Twist expression was a significant prognostic factor in squamous cell carcinomas with a reportedly high radiosensitivity, but not in adenocarcinomas with a reportedly low radiosensitivity, suggesting that Twist expression influenced the radiosensitivity. Green et al. have reported that Bid, an anti-apoptotic Bcl-2 family member, serves as a prognostic factor in cervical cancer after radiotherapy [18]. Twist may also have anti-apoptotic activity against radiation, although functional experiments on it are needed.
In conclusion, in this study, we present evidence for a role of Twist in cervical cancer patients as an independent prognostic predictor of clinical outcome. Further functional experiments will allow us to elucidate the molecular mechanism by which Twist influences the prognosis of cervical cancer. Moreover, Twist may become not only a prognostic factor for cervical cancer, but also a target for molecular-targeted therapy.
Received for publication March 6, 2007. Revision received May 17, 2007. Accepted for publication June 4, 2007.
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