Clinicopathological Significance of Fatty Acid Synthase Expression in Carcinoma of the Ampulla of Vater

Objective: Increased fatty acid synthase (FAS) expression is detected in various cancers. It has been demonstrated that the intensity of FAS expression in cancer correlates with clinical outcome. Here, we investigated whether FAS expression in carcinoma of the ampulla of Vater has any relationship to clinical or pathological features. Methods: We studied 32 patients surgically treated for carcinoma of the ampulla of Vater in our hospital. We compared FAS immunohistochemical staining with other pathological features, including Ki-67, p53, and MUC1, and 2 staining, and with clinical findings. Results: Of these 32 cases, 16 were considered FAS-positive under our criteria. Pathologically, FAS positivity was correlated to Ki-67 positivity (p: 0.0045), but there was no significant relationship between FAS expression and p53 staining(p: 0.6715). FAS-positive cases showed a higher rate of lymph node metastases (p: 0.0373). However, FAS expression was not related to tumor size, differentiation of adenocarcinoma, duodenal invasion, pancreatic invasion and MUC1 or MUC2 positivity. Clinically, the disease survival rate and the overall survival rate of FASpositive cases was lower than that of FAS-negative ones (p: 0.0365, 0.0007). Conclusion: Histological FAS expression is related to aggressiveness of cancer of the ampulla of Vater and FAS expression can be considered to be a prognostic marker in cancer of the ampulla of Vater.

Biliary tract cancer is regarded as one of the cancers with poor prognoses in clinical practice [17]. Carcinoma of the ampulla of Vater is one of the carcinomas that occur in the biliary tract. The prognosis of carcinoma of the ampulla of Vater is considered to be better than those of carcinomas derived from other parts of the biliary tract or pancreatic head [17][18][19]. However, recurrence of carcinoma of the ampulla of Vater occurs even though the curative resection has been performed [17][18][19].
However, FAS expression in cancer of the ampulla of Vater has not been reported. It is unclear whether FAS is also expressed in carcinoma of the ampulla of Vater, and whether FAS expression has any relationship to the clinical outcome of this type of carcinoma. The aim of this study is thus to investigate the relationship between the expression of FAS and clinicopathological features using immunohistochemistry in carcinoma of the ampulla of Vater. Then, we considered whether FAS could be regarded as a prognostic marker in this type of carcinoma.

Samples
We studied 32 patients who underwent resection for carcinoma of the ampulla of Vater between January 2002 and January 2014 in our hospital. Pathological diagnoses were performed by hematoxylin and eosin staining. We investigated the expression of FAS, Ki-67, p53, and MUC1, and MUC2 on tissue sections cut from carcinoma specimens formalin-fixed and paraffin-embedded by immunohistochemical staining. Pathological aspects, tumor size, node metastases, tumor differentiation, duodenal invasion, pancreatic invasion, Ki-67, p53 staining, and MUC1, and MUC2 expression were also compared with FAS expression. We compared FAS expression with clinical information. Clinical data such as gender, age, preoperative serum CA19-9 level, diabetes mellitus complication and prognoses were obtained from the clinical records. The study protocol conformed to

Journal of Gastrointestinal & Digestive System
the ethical guidelines of the World Medical Association Declaration of Helsinki, and has been approved by the ethical committee of our hospital.

Immunohistochemical studies
We used 4-m tissue sections cut from specimens that had been formalin-fixed and paraffin-embedded. Immunohistochemical staining was performed using an automated slide stainer (Bench-Mark XT; Ventana Medical Systems Inc., Tucson, AZ) [20,21]. Anti-human FAS rabbit IgG was obtained from Immuno-Biological Laboratories Co., Ltd. (Fujioka, Gunma, Japan). Monoclonal mouse anti-human Ki-67 and p53 antigens were obtained from Dako (Carpinteria, CA, USA). Monoclonal mouse anti-human MUC1 and MUC2 antigens were obtained from Dako (Carpinteria, CA, USA).
Staining intensity was measured according to the classification previously described [22,23], as 0, negative staining; 1+, low staining for up to 1/3 of cells stained; 2+, moderate staining for up to 2/3 of cells stained; and 3+, intense staining for more than 2/3 of cells stained. For statistical analysis we used a method that the results were dichotomized as high (3+ and 2+) or low (1+ and 0) expression ( Figures 1-5).
Cancer of the ampulla of Vater expressed MUC1 or MUC2. MUC1positive cases were determined as those predominantly stained with MUC1 antibody. MUC2-positive cases were determined in the same fashion.

Statistical analysis
Relationships of the scores of tissue FAS expression and those of tissue Ki-67 expression or those of tissue p53 expression were analyzed using Spearman's correlation coefficient by rank test. Cut-off endpoints were determined according to positive and negative immunohistochemical tissue FAS expressions observed. FAS positivity was tested for association with clinicopathological features using Fisher's exact test or Mann-Whitney's test. Overall survival rate and disease-free survival rate were plotted using the Kaplan-Meier method. These survival rates were analyzed using log-rank test. All analyses were conducted using the GraphPad Prism5® statistical software package (GraphPad Software Inc., La Jolla, CA, USA).
We estimated survival based on FAS expression patterns using the Kaplan-Meier analysis and compared survival between groups using log-rank tests. A p value less than 0.05 was considered significant.

Results
The clinical features of the 32 cases were listed (Table 1). All 32 cases had adenocarcinoma and recurrences were noted in 10 patients, and they died due to carcinoma of the ampulla of Vater. A total of 16 cases were positive for FAS staining and 16 were negative.  The correlation between scores of tissue FAS expression and scores of tissue Ki-67 expression was analyzed (Figures 6 and 7). FAS positivity was correlated with Ki-67 positivity (p: 0.0045). Next, we analyzed the correlation between scores of tissue FAS expression and scores of tissue p53 expression. There was no relationship between these two variables (p: 0.6715).   Table 2. FAS-positive cases showed higher rates of lymph node metastases (p: 0.0373). However, FAS expression was not related to tumor size, differentiation of adenocarcinoma, duodenal invasion, pancreatic invasion.
There were no significant differences between the two groups in terms of Ki-67 positivity, p53 positivity. MUC1-positive cases were seventeen and MUC2-positive cases were fifteen. Ten cases (58.8 %) were FAS-positive in MUC1-positive cases.
6 cases (40%) were FAS-positive in MUC2-positive cases. There was no significant difference between FAS positivity and MUC1 or MUC2 positivity.
The clinical features of the two groups were also analyzed. There were no significant differences between the two groups in terms of gender, age, and preoperative level of CA19-9, diabetes mellitus complication. Recurrences were seen in both FAS-positive and FASnegative groups.
Overall survival rate and disease-free survival are shown using the Kaplan-Meier method in Figures 8 and 9. The overall survival rate and the disease survival rate of FAS-positive cases were lower than that of FAS-negative ones (p: 0.0007, 0.0365).
Furthermore, immunohistochemical researches to detect the prognostic factors of this type of cancer have been studied and reported. MIB-1 labeling index and p53 were investigated, same as in this study. Shyr et al. reported that MIB-1 index had prognostic value in resectable carcinoma of the ampulla of Vater, although they did not show the relationship between MIB-1 index and clinical stage and lymph node metastases [30]. The prognostic value of p53 expression has also been reported in several manuscripts. However, it was not clear whether p53 had a possible role in predicting survival [31][32][33][34][35].
Immunohistochemical analyses on the expression of keratin, apomucin subtypes for detecting prognostic factors of carcinoma of the ampulla of Vater have been performed, and those immunohistochemical features were related to the prognoses of carcinoma of the ampulla of Vater [28,29,36,37]. Kimura et al. demonstrated that analysis using keratin7 and MUC2 was useful for the prediction of prognoses in cases of carcinoma of the ampulla of Vater [36,37]. They concluded that prognoses of keratin7-positive cases showed worse than those of MUC2-positive cases. That meant that pancreatobiliary-type adenocarcinoma was highly aggressive compared with the intestinal-type. They also reported pancreatobiliary-type carcinomas showed more frequent involvement of lymph nodes that was responsible for the shorter survival time. However, Catrer et al. reported that there was no significant difference in node metastases between pancreatobiliary-type and intestinal-type [28]. Relation node metastasis and pancreatobiliary-or intestinalsubtypes classification is considered to be still controversial.
In this study, we found that FAS expression is associated with tumor aggressiveness in carcinoma of the ampulla of Vater and had relation to lymph node metastases and their prognoses. That is, FAS expression in carcinoma of the ampulla of Vater is also useful for predicting prognosis, the same as in MIB-1 labeling index and keratin, apomucin subtypes [28,30,36,37]. Our result showed FAS expression had a significant relation to Ki-67 expression and node metastases. However, we could not find any significant relationship between MUC1 or MUC2 positivity and FAS positivity. Our study contains a small number of samples, so we need to accumulate more cases for further consideration in order to determine the relationship between pancreatobiliary-or intestinal-subtypes classification and FAS expression.
Recently, FAS has been said to play an important role not only in tumor growth, but also in drug resistance [38,39]. Both cellar proliferation and drug resistance can contribute cancer survival. Theses abilities are based of elevated FAS activity [40]. From previous reports and our results, it is suspected that FAS expression is related to cancer aggressiveness. FAS-positive expression can be considered to be a prognostic marker of cancer.

Conclusion
FAS expression may be related to clinical behavior in carcinoma of the ampulla of Vater. Histologically, positive FAS expression related to high incidence of lymph node metastases and poor prognoses. Histological FAS expression can be a prognostic factor in carcinoma of the ampulla of Vater same as other types of carcinoma.