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ANTICANCER RESEARCH 28: 1905-1910 (2008)

Prognostic Significance of Fascin Expression in Advanced Poorly Differentiated Serous Ovarian Cancer ALEXANDROS DAPONTE1, EVA KOSTOPOULOU2, MARCO MINAS3, GEORGE KOUKOULIS2 and IOANNIS E. MESSINIS1

Departments of 1Obstetrics and Gynaecology, 2Pathology, and and Epidemiology, University Hospital of Larissa, Larissa, Greece

3Hygiene

Abstract. Background: High expression of the actin-

bundling protein fascin correlates well with histological grade and clinical stage of ovarian carcinoma. This study addresses fascin expression in advanced poorly differentiated serous ovarian cancer with respect to progression free interval (PFI) and overall survival. Patients and Methods: Fascin and Ki-67 expression were analysed in paraffin blocks tissue sections of 56 stage III, poorly differentiated (G3) serous adenocarcinoma patients by immunohistochemistry. Fascin expression was tested for correlation with PFI and overall survival. Results: Fascin expression inversely correlated with Ki-67 expression (p=0.016). Strong fascin immunoreactivity was associated with poor prognosis; patients with low fascin expression had a median survival of 36.5 months versus 32 months for high fascin expression (p=0.041), and the median PFl was 24 versus 17.5 months (p=0.034). Conclusion: Fascin expression is an independent prognostic factor for survival of advanced ovarian serous carcinoma, and may represent a novel therapeutic target for patients with aggressive forms of ovarian cancer.

A better understanding of ovarian cancer is urgently needed because two thirds of patients currently present with already advanced disease requiring major surgery and adjuvant chemotherapy, and the great majority experience recurrence. Despite many therapeutic improvements, the development of secondary metastatic tumours that are resistant to conventional treatment remains a major cause of morbidity and mortality (1). Tumour invasion and metastasis are the result of highly coordinated processes involving multiple intracellular and extracellular factors. Understanding the early events that Correspondence to: A. Daponte, MD, FCOG, M. Skyllakou 10, 41334 Larissa, Greece. Tel: +30 2410 672505, Fax: +30 2410 672506, e-mail: [email protected]

Key Words: Fascin, Ki-67, immunohistochemistry, serous ovarian cancer, prognosis.

0250-7005/2008 $2.00+.40

enable carcinoma cell migration and invasion is an important research goal that has the potential to improve early diagnosis of aggressive tumours and stimulate new approaches towards molecular adjuvant therapies. Carcinoma cell migration is facilitated by the altered differentiation status of the epithelial cells, including changes in cell cell and cell matrix adhesion properties and in the organization of the actin cytoskeleton. The cytoskeleton is a complex network that includes three types of protein: actin filaments, microtubules and intermediate filaments. Changes in cytoskeletal components or associated binding proteins may be implicated in the progression and metastasis of tumours. Fascin is a highly conserved actin-bundling protein, expression of which induces membrane protrusions and cell motility. Fascin has emerged as a very interesting candidate biomarker because its expression is low or absent in the majority of normal adult epithelia, yet up-regulation of the protein has been reported in many forms of human carcinoma. Irrespective of the tissue source of the tumor, high levels of fascin expression in primary carcinomas consistently correlate with a clinically aggressive phenotype and poor prognosis (2-10). To date, the only reports concerning fascin expression in ovarian carcinoma are from two teams of investigators who showed that fascin immunoreactivity is increased in ovarian carcinomas of advanced stage (11-13), and a report by our group demonstrating that fascin expression is associated with tumour aggressiveness and histological type and is further increased in samples from metastatic sites (14). No data exist on the clinical relevance of fascin expression in serous ovarian cancer and, more specifically, in the most common subgroup of poorly differentiated serous advanced ovarian cancer. Thus, the primary aim of our study was the assessment of any correlation of fascin expression with the progression free interval (PFI) and overall survival in this subgroups. Fascin is a critical cytoskeletal component required for actin organization and cell migration responses (15). It is recognized that malignant progression is caused by progressive mutations in cancer-related genes that result in 1905


protein productions that have been categorized as “early”, such as fascin, and “late”, such as tumour growth or proliferative fraction, assessed by Ki-67 antigen (16, 17). A second aim of our study was to evaluate the correlation between the expression of these two factors associated with tumour growth; fascin as a marker of “early” alterations associated with tumour cell motility and Ki-67 antigen as a “late” gene product associated with proliferative fraction of cells, in poorly differentiated serous ovarian adenocarcinoma tissues.

Patients and Methods

Patients and samples. Fifty-six patients with a diagnosis of serous ovarian carcinoma of stage III, G3 who received six cycles of postoperative chemotherapy (TC: 175-180 mg/m2 paclitaxel and carboplatin after calculating the area under the concentration curve) with complete medical records were selected for further analysis. Patients had an average age of 62.5 years. Pathological samples from paraffin-embedded tissue from each of the above patients were included in this study. Clinicopathologic information was obtained from medical records. Institute Review Board (IRB) approval was obtained. Cancer patients were classified after a staging laparotomy; the most common initial surgical procedure consisted of abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy and lymph node sampling. The surgery was classified as complete when 4/≤4, because a fascin expression immunohistochemistry score of 4 was the calculated median value for these patients. These two groups (high vs. low expression) contained patients with stage III, G3 serous adenocarcinomas with no statistically significant differences in age, family history or level of cytoreduction (complete vs. incomplete). The mean follow-up was 3 years. At the time of analysis, 13/56 patients were deceased due to their disease. Different regimens were given as second- and third-line therapy, after further classification according to second-line chemotherapy, the groups contained too few patients to be analyzed separately. Therefore, although both PFI and overall survival were calculated, we report PFI after first-line chemotherapy (TC) as indicative of the response to the first-line chemotherapy.

Immunohistochemical staining of ovarian tumour tissue (paraffin blocks). Immunohistochemical staining of ovarian tissue was performed in a commercially available automated immunostainer (Bond Max, Vision Biosystems, Australia). The samples were fixed in 10% buffered formalin solution, embedded in paraffin blocks and cut at 4 μm sections. For fascin expression, slides were incubated with clone IM20 (Novocastra, Newcastle upon Tyne, UK) diluted 1:300 for 20 min at room temperature (RT). Bond Epitope Retrieval Solution 2 (Vision BioSystems, Mount Waverley, Australia; 30min) was used for antigen retrieval. Binding of the primary antibody was assessed by Bond Polymer Refine Detection (Vision Biosystems, Newcastle upon Tyne, UK). For double immunohistochemical staining of fascin and Ki-67 the slides were pretreated in target retrieval solution at high pH (DakoCytomation, Glostrup, Denmark)

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and stained manually with the double Envision Detection System (Dako, Denmark). The first antibody was anti-Ki-67 MIB-1 (DakoCytomation, dilution 1:80, 25 min RT) stained with Envision horse radish peroxidase (HRP) with diaminobenzidine DAB as a chromogen (nuclear stain), and the second antibody was anti-fascin (clone IM20, dilution 1:300, 20 min RT) stained with Envision/AP with Fast Red as a chromogen (cytoplasmic stain).

Evaluation and scoring of fascin immunohistochemical staining. All slides were initially evaluated blindly and independently by two pathologists (G.K., E.K.). During a subsequent joint evaluation, a final consensus immunoreactivity score was obtained and used for statistical analysis. Cytoplasmic immunoreactivity of tumour cells was assessed in comparison with endothelial cells, which were used as internal positive controls. Two aspects of immunoreactivity were semiquantitatively evaluated: the extent and the intensity of staining. Intensity was considered as “weak to moderate” when it was less than that of endothelial cells and “intense” when it was similar to that of the endothelial cells. After preliminary analysis, the pathologists involved in the evaluation of immunohistochemical staining realized that the observed differences in immunoreactivity were best represented by counting only the cellular subpopulation showing intense immunohistochemical staining and expressing this as the HIES (highest immunohistochemical expression score). To calculate HIES, a value from 0 to 4 was assigned according to the percentage of cells showing intense staining (0: 0% , 1: 75% ). Statistical analysis. Group comparisons were based on Spearman’s test for nominal variables (age, CA125) and on the linear χ2 test for ordinal variables (complete/incomplete cytoreduction) (data not shown). The fascin and Ki-67 expression scores of the different patient groups were compared using the non-parametric test for multiple comparisons (Kruskal Wallis-Mann Whitney) followed by Dunn’s test, which generalizes the Bonferroni adjustment. Factors possibly influencing the PFI (fascin, Ki-67, age) were determined by binary logistic regression analysis using the forward likelihood ratio method. Univariate and multivariate analyses were performed. The covariates fascin, Ki- 67 and age were used for multivarite analysis; all patients had serous, stage III, G3 carcinomas. Progression-free survival and overall survival were estimated by Kaplan and Meier’s method. The log-rank test was used to compare differences between survival curves and the Cox regression to calculate hazard ratios (18, 19). P2) and 35 with low (immunohistochemistry score 0,1,2), of which five (9% ) and showed no staining. Representative examples of immunostaining are shown in Figure 1.

Relationship between fascin and Ki-67. Statistical analysis confirmed an inverse relationship between fascin and Ki-67 immunostaining in the overall tumour cell population (Figure

Daponte et al: Fascin Expression in Advanced Serous Ovarian Cancer

2).There was no association of fascin and Ki-67 in the multivariate analysis for survival (fascin independent prognostic factor), but the two variables were not independent in the multivariable analysis for PFI, which is indicative of the primary chemotherapy response (Table I).

Fascin expression correlates with a poor prognosis in serous stage III poorly differentiated adenocarcinoma. Patients with low fascin expression had a median PFI of 24 months compared with 17.5 months for patients with high fascin expression (p=0.034). Furthermore, Kaplan-Meier survival curves showed that stage III, G3 patients with high fascin expression had significantly reduced overall PFI compared with patients with low fascin expression (p=0.031) Cox regression analysis demonstrated an increased hazard ratio (HR 2.549 (95% CI 1.039 to 6.252) (Figure 3a) for high fascin expression. This correlation was not an independent prognostic factor (p=0.230) (see multivariate analysis, Table I). In the 56 patients with stage III G3 serous carcinoma, the overall survival of patients with tumours that stained strongly for fascin was significantly worse than that of patients with tumours that stained weakly or were negative for fascin (p=0.041). Patients with low fascin expression displayed a median survival of 36.5 months compared with 32 months for patients with high fascin expression. In addition, Kaplan Meier survival curves also showed that high fascin expression reduced overall survival compared with low fascin expression (p=0.032) (Figure 3b). Cox regression analysis demonstrated demonstrated a decreased hazard ratio (HR)=0.4269 (0.1751 to 1.041) for low fascin expression. Increased fascin expression was an independent adverse prognostic factor for survival (p=0.025; see multivariate analysis, Table I).

Discussion

Ovarian cancer is the most common cause of gynaecological cancer-related mortality. Patients with this disease generally undergo surgery followed by platinum-taxane chemotherapy (TC), with additional chemotherapy at relapse. Although the prognosis for patients with advanced cancer is poor, with a five-year survival of only 30-40% , there is a wide range of outcomes for individual patients. Clinicopathological variables such as age, stage, grade, histology, debulking status and response to chemotherapy continue to provide the basis on which treatment decisions are made for individual patients. Additional biomarkers need to be extensively evaluated in cancer tissues of patients of the same histology and no different in age, debulking status and chemotherapy to individualize future experimental treatment approaches. For example, the most common ovarian cancer histological subtype, high-grade serous carcinoma, was previously characterized by different researchers by p53 mutations,

Table I. Fascin expression correlations in univariate and multivariate analysis. Univariate analysis

Fascin vs. Ki-67

Multivariate analysis

Independent variables

Fascin Age Ki-67 PFI Fascin Ki-67 Survival Fascin Ki-67

R2

–0.441

95% CI

–0.688 - –0.103

P-value 0.0156

p-value 0.1107 0.0210

0.2308 0.2979

0.0257 0.1722

identifying a subgroup with an adverse impact on prognosis, and numerous therapeutic approaches based on this finding are being investigated (20). The actin-bundling protein fascin is expressed in mature dendritic cells, mesenchymal cells endothelial cells, and neurons during development and in the adult. It is absent from most normal epithelia, but is expressed in multiple epithelial neoplasms, including carcinomas of the pancreas, lung, esophagus, stomach and breast. Immunohistochemical analyses of human carcinomas have consistently correlated up-regulation of fascin with a clinically aggressive phenotype and poor prognosis (2-10). In node-negative, invasive hereditary breast carcinomas, fascin is frequently expressed by tumours associated with the tumour suppressor BRCA1 (21). Previous studies of fascin expression in ovarian cancer suggested that up-regulation of fascin in tumour tissue may promote invasion of ovarian carcinoma (11). Fascin immunoreactivity was elevated in established ovarian tumour cell cultures from advanced stage carcinoma (13). Our group recently reported the first demonstration of increased fascin expression in surgical material from advanced stage primary ovarian carcinomas of different histologies and in samples obtained from metastatic sites (14). The data presented here represent the largest reported study on surgical material to date, both confirming our previous findings and providing several novel insights into the clinical relevance of fascin in the most common subtype of ovarian cancer, high-grade serous carcinoma. Fascin was expressed in 9/10 of these aggressive tumours and high expression of fascin was shown to be an independent adverse prognostic factor of survival. Similarly, strong and diffuse 1907


Figure 1. A. Serous tumor of low malignant potential. Immunostaining for fascin. Note weak to moderate cytoplasmic staining. B. Serous carcinoma of high malignant potential. Immunostaining for fascin. Note moderate to strong cytoplasmic staining.

fascin expression was seen in a subset of advanced colorectal adenocarcinomas and correlated with shorter survival in stage III and IV patients (6), and recent reports implicated fascin as an independent prognostic factor (22-24). These findings indicate the potential clinical significance of fascin as a marker or prospective therapeutic target for advanced aggressive adenocarcinomas. Most significantly, fascin expression correlated significantly with reduced PFI, suggesting a role in the response to the standard first-line chemotherapy regime of TC. Nonetheless, fascin could have value as a prognostic marker to identify individuals who should receive additional monitoring and a larger study is needed to confirm this. 1908

In general, the relationship between fascin expression and cell proliferation in carcinomas is not entirely clear and may be tumour specific. In non-small cell lung carcinoma, highly fascinpositive tumours tended to be highly proliferative, as established by Ki-67 antibody staining; however, it was also noted that individual Ki-67-positive cells stained less strongly for fascin than surrounding tumour cells (24). In colonic adenomas, fascin and Ki-67 tended to be inversely correlated at the cellular level; this trend was less apparent in adenocarcinomas. Our study demonstrated an inverse relationship between fascin and Ki-67 immunostaining that could be explained by the finding that cycling cells (Ki-67 positive) express little, if any, fascin. This observation, which was made by our group before (14), was

Daponte et al: Fascin Expression in Advanced Serous Ovarian Cancer

Figure 2. a-b. Double immunohistochemical staining for Ki-67 and fascin often revealed an inverse association.

Figure 3. High fascin expression correlates with a clinically aggressive tumor phenotype. The Kaplan-Meier survival curves show that stage III patients with high fascin expression [fascin (+)] ] had a reduced progression free interval (A) and overall survival (B) compared to patients with low fascin expression [fascin (–)] tumors.

confirmed by immunostaining a greater number of samples of a specific histology (serous adenocarcinomas). Findings to support this notion include: i) a statistically verified inverse relationship between fascin and Ki-67 staining; ii) double immunostaining showing that this inverse relationship could be attributed at a cellular level to low or no fascin expression in proliferating cells. Additionaly in serous ovarian carcinomas, fascin expression was not only a negative prognostic factor for survival, but its increased expression was also independent of Ki-67

immunoreactivity and therefore of tumour cell proliferative activity. Further studies are underway to investigate a possible mechanism associated with these findings.

Conclusion

In conclusion, we provide the first demonstration that increased expression of the actin-bundling protein fascin correlates with poor clinical outcome in a subset of patients 1909


with poorly differentiated serous ovarian carcinoma. Our findings not only implicate fascin as a potential biomarker for closer monitoring and for prediction of tumour recurrence and clinical outcome of ovarian cancer, but also provide impetus for research into fascin-related therapeutic approaches and mechanisms of metastasis.

References

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Received December 18, 2007 Revised March 17, 2008 Accepted March 24, 2008