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Journal of Gastrointestinal & Digestive System
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Differences in Expression of Fatty Acid Synthase among Histological Subtypes of Intraductal Papillary Mucinous Neoplasm of the Pancreas – An Experience of Immune Histochemical Analysis

Hiroshi Maekawa1*, Hajime Orita1, Tomoaki Ito1, Mutsumi Sakurada1, Tomoyuki Kushida1, Koichi Sato1 and Ryo Wada2

1Department of Surgery, Juntendo University School of Medicine, Shizuoka Hospital, Shizuoka, Japan

2Department of Pathology, Juntendo University School of Medicine, Shizuoka hospital, Shizuoka, Japan

*Corresponding Author:
Hiroshi Maekawa
Department of Surgery
Juntendo University School of Medicine
Shizuoka Hospital, Shizuoka, Japan
Tel: +81 55.948.3111
Fax: +81 55.946.0514
E-mail: [email protected]

Received date: June 14, 2013; Accepted date: September 30, 2013; Published date: October 10, 2013

Citation: Maekawa H, Orita H, Ito T, Sakurada M, Kushida T, et al. (2013) Differences in Expression of Fatty Acid Synthase among Histological Subtypes of Intraductal Papillary Mucinous Neoplasm of the Pancreas – An Experience of Immune Histochemical Analysis. J Gastroint Dig Syst 3:144. doi:10.4172/2161-069X.1000144

Copyright: © 2013 Maekawa H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Background: Fatty acid synthase plays an important role in tumorigenesis. We investigated the expression of fattyacid synthase in cases of intraductal papillary mucinous neoplasm of the pancreas, thought to be one of the precancerous lesions of pancreatic cancer, using immunohistochemistry. We also examined whether there are any differences between histological subtypes in intraductal papillary mucinous neoplasm of the pancreas. Methods: Eight cases of intraductal papillary mucinous neoplasm were examined using immunohistochemistry. Fatty acid synthase and Ki-67 expressions were investigated, and positive cellular rates were compared with histological subtypes of intra-ductal papillary mucinous neoplasm. Results: Pathologically, cases of the gastrictype were represented as low-grade dysplasia; however, cases of the intestinal or pancreatobiliary type were classified as intermediate- or high-grade dysplasia. Concerning fatty acid synthase expression, positive cellular rates of the gastric type were lower than those in cases of the intestinal or pancreatobiliary type. The positive cellular rates of Ki-67 expression were similar to those of fatty acid synthase expression. Conclusion: Although this study included only a small sample number, fatty acid synthase expression differed between the gastric and other subtypes in intraductal papillary mucinous neoplasm of the pancreas.

Keywords

Fatty acid Synthase; Immunohistochemistry; IPMN

Introduction

Fatty Acid Synthase (FAS) is usually expressed in hormonesensitive cells and cells showing high-level lipid metabolism in animals [1]. FAS increases the de novo biosynthesis of fatty acids, which contributes to cellular activities [2] including energy metabolism, building cellular membranes, and intracellular second messengers [3,4]. FAS expression is suppressed in normal cells because serum fatty acids are usually rich in the diet. In cancer cells and preneoplastic lesions, FAS expression has been found to be up-regulated. It has been suggested that FAS contributes to tumorigenesis [5,6] because it’s over expression has been demonstrated in precancerous lesions and early cancers using immunohistochemical staining [6-8]. IPMN is considered to be one of the precancerous lesions of pancreatic ductal adenocarcinoma. In the 2010 WHO classification, IPMNs are classified as showing low-, intermediate-, or high-grade dysplasia on the basis of the degree of architectural and cytological atypia identified [9]. Previously, Walter et al. [10] reported that FAS tissue expression in 30 cases of IPMN was correlated with the histologic grade. However, differences in FAS expression among histological subtypes of IPMN, such as the gastric, intestinal, and pancreatobiliary types have not been fully discussed. In this report, we examined the differences of FAS expression among subtypes of IPMN resected in our department using immunohistochemistry.

Methods

Eight cases of IPMN resected in our department between 2001 and 2012 were examined. Clinical data such as the gender, age, recurrence, and prognosis were obtained from clinical records. Pathological diagnoses were performed under H.E. staining and immunohistochemistry according to the 2010 WHO classification [9]. The subtypes of IPMN were determined according to immunohistochemical staining using MUC1, MUC2, and 45M1 mouse monoclonal antibody obtained from LEICA MICROSYSTEMSNEWCASTLE Ltd. (Newcastle, UK).

We investigated the expressions of FAS and Ki-67 in tissue sections cut from resected specimens formalin-fixed and paraffin-embedded with immunohistochemical staining.

Immunohistochemical procedure for FAS expression

We used 4-μm-thick tissue sections cut from specimens formalinfixed and paraffin-embedded. Sections were deparaffinized by treatment with xylene for 3 min twice and were rehydrated by passage through 100% ethanol for 30 sec (x3). Antigen retrieval was performed by microwaving slides in 10 mM sodium citrate buffer (T.R.S.) obtained from DAKO (Carpinteria, CA, USA) for 8 min. Endogenous peroxidase activity was quenched by incubating slides for 5 min in 3% H2O2. Slides were then washed in tris-buffered saline -0.05% Tween 20 (TBST) for 5 min twice and blocked with a solution containing 5% goat serum for 15 min at room temperature. Samples were incubated with the primary antibody for 60 min at room temperature. Anti-human FAS rabbit IgG was obtained from IMMUNO-BIOCHEMICAL LABORATORIES Co., Ltd. (Fujioka, Gunma, Japan). Slides were washed with TBST for 5 min (x3) and incubated with the secondary antibody, anti-rabbit IgG (Envison+/HRP from DAKO), for 60 min at room temperature. Slides were washed in TBST for 5 min (x3) and incubated with diaminobenzidine solution containing 20 mg/ml DAB-4HCl in 0.01M PB and 30% H2O2 until colorarion developed. Slides were then stained with hematoxylin, dehydrated, and mounted with cover slips.

Immunohistochemical procedure for Ki-67 expression

An immunoperoxidase assay for Ki-67 analysis was performed using a commercially available kit obtained from ROCHE DIAGNOSTICS Co., Ltd. (Tokyo, Japan) in the same manner as that described in the immunohistochemical procedure for FAS analysis. Monoclonal mouse anti-human Ki-67 antigens were obtained from DAKO (Carpinteria, CA, USA).

Scoring of immune reactivity

Cellular FAS expression was classified into positive and negative. We classified FAS staining of IPMN cells relative to the staining of adipose cells using the following criteria. Scoring of cellular FASpositivity was determined as follows: negative for not stained or stained less than adipose cells, positive for stained the same as or more than adipose cells.

Percentages of FAS-positive cells were evaluated in high power magnification chosen at random. Data show the average of 10 series of these counts. Quantification of Ki-67-positive cells was performed in the same manner.

Ethics

Informed consent was obtained from all patients included in this study. The study protocol conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki. This study was approved by the Ethical Committee of our hospital.

Results

Clinical features of IPMN cases

Clinical features of the 8 cases are presented in Table 1. Five were male and 3 were female. The average age was 70 years old. Six cases were treated with pancreatoduodenectomy, and one case underwent total pancreatectomy. The remaining case underwent spleen-preserving distal pancreatectomy. No patient showed recurrence of the disease or pancreatic ductal adenocarcinoma during the postoperative follow-up.

  Gender Age (years old) Serum CA19-9 level (IU/l) Diabetes mellitus Surgical procedure
Case 1 F 67 12.7 + PD
Case 2 M 70 27.4 - PD
Case 3 M 62 2 - TP
Case 4 F 70 73 + PD
Case 5 F 71 31 + PD
Case 6 M 68 7 - PD
Case 7 M 83 2 - PD
Case 8 M 69 14 - SPDP

Table 1: Clinical features of the 8 IPMN cases.

Pathological features of IPMN cases

Histopathological features of the 8 cases are shown in Table 2. Five cases had the main duct type, two had the mixed type, and the last one had the branch duct type. The cases are shown with the histological subtypes and dominant type of dysplasia based on the WHO classification. Five were classified as the gastric type, and all corresponded to low-grade dysplasia. Two were the pancreatobiliary type, which corresponded to high-grade dysplasia, and the last one was the intestinal type, corresponding to intermediate-grade dysplasia. We encountered no cases of the oncocytic type of IPMN. FAS expression was detected in all cases.

  Gross appearance Histological subtypes Degree of dysplasia FAS- positivity (%) Ki-67- positivity (%)
Case 1 MD Gastric IPMN-low 5.3 7.2
Case 2 BD Gastric IPMN-low 7.6 0.9
Case 3 MD Pancreatobiliary IPMN-high 59 12.6
Case 4 MD Intestinal IPMN-intermediate 18.7 23.4
Case 5 MD Gastric IPMN-low 10 2.4
Case 6 Mixed Gastric IPMN-low 5 0.8
Case 7 Mixed Pancreatobiliary IPMN-high 60 40.9
Case 8 MD Gastric IPMN-low 6.4 0.8

Table 2: Histopathological features of the 6 IPMN cases.

Difference in FAS or Ki-67 expression in the subtypes of IPMN

In the gastric-type, FAS expression in tumor tissue was similar to that of normal duodenal mucosa. In duodenal mucosa, FAS-positive cells were found in the proliferative cell zone (Figure 1A). In the gastric type, FAS-positive cells were more often seen at the base than top of the papillae (Figure 1B). In FAS expression, positive cellular rates of the gastric type were lower than those of the intestinal or pancreatobiliary type (Table 2). In Ki-67 expression, positive cellular rates of the gastric type were also lower than those of other sub types, similar to FAS expression (Table 2) (Figures 2-4). We divided these eight cases into two groups: the gastric type and others (non-gastric type). Then, we compared FAS- and Ki-67-positivity of the gastric type and the others (non-gastric types) statistically. The positivity of the gastric type was lower than that of the non-gastric type (Table 3).

  Gastric type Non-gastric type p-value
FAS-positivity 6.9 45.9 0.0357
Ki-67-positivity 2.4 25.6 0.0358

Table 3: Comparison of gastric with non-gastric type for FAS- and Ki-67-positivity.

gastrointestinal-digestive-normal-duodenal-mucosa

FAS-positive cells were found in the proliferative cell zone of the duodenal mucosa.
Figure 1A: FAS expression in normal duodenal mucosa (Case 5).

gastrointestinal-digestive-FAS-expression-IPMN

In the IPMN gastric type, FAS positive cells were more often seen at the base than top of the papillae.
Figure 1B: FAS expression in the IPMN gastric type (Case 6).

gastrointestinal-digestive-IPMN-gastric-type

In the IPMN gastric type, tumor papillae were relatively low and dilatation of the pancreatic duct was marked.
Figure 2A: Histological findings in the IPMN gastric type (Case 6: H.E.staining, low-power magnification).

gastrointestinal-digestive-high-power-magnification

The epithelium of the tumor was composed of columnar cells. The nuclei two groups: the gastric type and others (non-gastric type). Then, we were small and located basally. Architectural and cytological atypia were not marked.
Figure 2B: Histological findings in the IPMN gastric type (Case 6: H.E.staining, high-power magnification).

gastrointestinal-digestive-stained-FAS-staining

FAS-positive cells were mainly located at the bases of papillae, and less than 10% of tumor cells were positively stained in FAS staining.
Figure 2C: Immunohistochemical findings of FAS staining in gastric-type IPMN (Case 6).

gastrointestinal-digestive-were-seldom-seen

In Ki-67 staining, positively stained cells were seldom seen. The positive cellular rate was less than 10%.
Figure 2D: Immunohistochemical findings of Ki-67 staining for gastric-type IPMN (Case 6).

gastrointestinal-digestive-papillary-villous-manner

In intestinal-type IPMN, the papillae were tall and proliferated in an intraluminal, papillary, or villous manner. The epithelium was similar to colonic villous adenoma.
Figure 3A: Histological findings in the IPMN intestinal type (Case 4: H.E. staining, low-power magnification).

gastrointestinal-digestive-goblet-like-cells

Tall columnar cells were packed tightly and contained hyperchromatic nuclei. However, cytological atypia was not evident. In this case, goblet-like cells were not marked.
Figure 3B: Histological findings in the IPMN intestinal type (Case 4: H.E. staining, high-power magnification).

gastrointestinal-digestive-positively-visualized-FAS

Some tumor cells were positively visualized on FAS staining. FAS-positive cells were more frequent than in the gastric type.
Figure 3C: Immunohistochemical findings of FAS staining in intestinal-type IPMN (Case 4).

gastrointestinal-digestive-Ki-67-staining

Positive cells were more often seen than in the gastric type.
Figure 3D: Immunohistochemical findings of Ki-67 staining in intestinal-type IPMN (Case 4).

gastrointestinal-digestive-low-power-magnification

The tumor cells proliferated intraluminally and occupied the main pancreatic duct.
Figure 4A: Histological findings in the IPMN pancreatobiliary type (Case 3: H.E. staining, low-power magnification).

gastrointestinal-digestive-Tumor-cells-cuboidal

Tumor cells were cuboidal and had hyperchromatic nuclei. The tumor cells showed prominent nucleoli.
Figure 4B: Histological findings in the IPMN pancreatobiliary type (Case 3: H.E. staining, high-power magnification).

gastrointestinal-digestive-half-tumor-cells

More than half of the tumor cells were positively stained on FAS staining.
Figure 4C: Immunohistochemical findings of FAS staining in pancreatobiliarytype IPMN (Case 3).

gastrointestinal-digestive-findings-Ki-67-staining

Some of the tumor cells showed positive staining for Ki-67. The positive cellular rate of the pancreatobiliary type was higher than that of the gastric type.
Figure 4D: Immunohistochemical findings of Ki-67 staining in pancreatobiliarytype IPMN (Case 3).

Discussion

Recently, it was suggested that FAS plays an important role in tumor growth, and FAS expression has been associated with cancer progression, a high risk of recurrence, and shorter survival in several kinds of cancer [11,12]. This means that FAS expression is an indicator of a poor prognosis in several kinds of cancer [13,14].

Some previous studies reported that FAS over expression is also detected in precancerous lesions or carcinoma in situ [5-8]. These results show that FAS over expression is an early event in cancer development.

IPMN has been considered to be one of the precancerous lesions of pancreatic ductal adenocarcinoma [15]. In cases of IPMN, concomitant pancreatic ductal adenocarcinoma is detected synchronously or metachronously at a relatively high incidence [16-18]. Approximately 30% of resected IPMNs have an associated invasive carcinoma, which can be uni- or multifocal. Most invasive carcinomas arise in association with main-duct type IPMNs with high- grade dysplasia [9].

Adsay et al. proposed that IPMNs could be sub classified according to the direction of differentiation using immunohistochemistry in 2004 [19]. In the 2010 WHO classification, four subtypes of IPMN were also defined [9]. The gastric typeis flat or has thick, finger-like papillae lined with epithelia resembling gastric foveolar epithelia with abundant apical mucin and basally located nuclei [20]. The gastric type is considered to be a common precursor of the other types of IPMN [19]. The intestinal type is characterized by long villous papillae resembling the epithelium of colorectal villous adenomas. The intestinal type is usually classified with intermediate- or high-grade dysplasia [9,20]. The pancreatobiliary type is characterized as forming thin, branching papillae with high-grade dysplasia. The neoplastic cells are cuboidal, with round, hyperchromatic nuclei, prominent nucleoli, moderately amphophilic cytoplasm, and have a less mucinous appearance [9].

In this investigation, regarding FAS and Ki-67 expression, the gastric type showed a lower positivity than the other subtypes. These results suggest that the gastric type grew slowly. The gastric type is not considered to be a candidate for pancreatic ductal carcinoma. However, the intestinal or pancreatobiliary type might be regarded as a candidate for pancreatic ductal adenocarcinoma because they showed higher rates of FAS or Ki-67 expression.

We need to accumulate more reports on FAS expression in IPMN in order to clarify the exact significance of FAS expression.

Conclusion

Although this study included only a small sample number, fatty acid synthase expressions were different among the IPMN subtypes. FAS expression showed a difference based on tumor aggressiveness between the gastric type and other subtypes.

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