Ampullary carcinomas (ACs), although uncommon, have a better prognosis than other periampullary tumors such as pancreatic and bile duct carcinomas.

The ampulla of Vater consists of 4 minor anatomic regions: the ampulloduodenum (Ad), the ampullopancreatobiliary common duct (Ac), the ampullopancreatic duct (Ap), and the ampullobiliary duct (Ab) 1 2 . The ampulla is formed by the union of 2 distinct types of mucosa. The Ad is covered by intestinal mucosa, while the other parts of the ampulla of Vater (the Ap, Ab, and Ac) are lined with pancreatobiliary-type ductal mucosa 2 3 . Therefore, ACs may arise from the intestinal-type mucosa as well as from the pancreatobiliary-type mucosa; this may explain the broad histomorphologic spectrum of these tumors 1 . Tumor progression and prognosis are affected by the primary AC tumor sites 1 3 4 .

Kimura et al. classified ACs into 2 histological subtypes: intestinal and pancreatobiliary 3 . Albores-Saavedra et al. further defined the characteristics of these 2 types and also described unusual types such as signet-ring cell carcinoma and undifferentiated carcinoma 4 .

While histopathological typing is a useful method for classifying ACs, some cases cannot be easily classified by using histomorphology 1 . Determination of the cytokeratin (CK) and apomucin (MUC) immunophenotypes of an AC can facilitate identification of the primary tumor site 1 2 5 6 7 . Most pancreatobiliary adenocarcinomas express CK7 and low levels of CK20 6 8 9 . Among ACs, the pancreatobiliary type expresses CK7 but does not express CK20, while the intestinal type expresses CK20 but does not express CK7 5 .

The pancreatobiliary type of ACs usually express MUC1 but do not express MUC2 6 7 9 10 . Most intestinal-type ACs express MUC2 1 2 5 6 .

In the present study, we analyzed the spectrum of CK and MUC expression in 43 patients with ACs. We then evaluated the immunohistochemical subtypes of ACs by analyzing the expressions of CK7, CK20, MUC1, MUC2, MUC5AC, and MUC6 in these tumors. Further, we assessed the correlations between the histomorphological findings and the defined immunohistochemical subtypes and evaluated the clinical significance of these immunohistochemical AC subtypes; the classification of ACs on the basis of their immunohistochemical characteristics may be useful to predict the clinical outcome.

Clinical data were obtained retrospectively from ACs that were surgically resected from 43 patients (22 men and 21 women) with an average age of 66.4 years (range, 44-82 years). All resected specimens had been obtained between 1983 and 2007 and were maintained at the Department of Digestive and General Surgery, Faculty of Medicine, Shimane University. All but 5 patients underwent pancreatoduodenectomy. The other 5 underwent pancreas-sparing duodenectomy. All tumors included in this study histologically showed surgically negative margins.

The study was approved by the hospital's ethics committee. Informed consent was obtained from all patients for the subsequent use of resected tissues.

Histopathological examinations were performed according to the guidelines of the Japanese Society of Biliary Surgery 11 . The Tumor, Node, Metastasis (TNM) Staging System put forth by the International Union Against Cancer was used for tumor classification 12 . All tumors were classified histologically according to the criteria published by Albores-Saavedra et al 4 . Intestinal-type carcinomas are composed of well-formed tubular to elongated glands, complex cribriform areas, and solid nests indistinguishable from those found in colorectal adenocarcinoma, whereas pancreatobiliary-type carcinomas mostly consist of simple or branching glands and small solid nests of cells surrounded by a strikingly desmoplastic stroma (Figure 1). Mixed-pattern tumors were classified into the intestinal- or pancreatobiliary-type groups on the basis of their predominant component. Carcinomas of the unusual types included undifferentiated, mucinous, signet-ring cell, and solid carcinomas.

Albores-Saavedra et al. defined 3 types of ACs--the intestinal type, the pancreatobiliary type, and the unusual type 4 .

In the present study on ACs, we histologically classified these tumors into these 3 types, and 37% were of the intestinal type, 42% were of the pancreatobiliary type, and 21% were of the unusual type 4 . According to the histological criteria, Zhou et al. reported the rates of the histological intestinal, pancreatobiliary, and unusual types to be 27%, 44%, and 29%, respectively 5 . According to their histological criteria, the unusual type included mucinous, signet-ring cell, solid, or undifferentiated carcinomas 4 5 . However, Kimura et al. simply classified ACs into 2 types, intestinal and pancreatobiliary 3 .

Because these systems of histological classification use different sets of criteria, 3 4 we tried to create a simple AC classification system based on immunohistochemical staining of CKs and MUCs. Goldstein et al. reported positive CK7 expression in 100% and positive CK20 expression in 43% of ACs, but it was difficult to distinguish between pancreatic carcinomas and ACs by the coordinate staining patterns of CK7 and CK20 9 . Very few reports have examined the expression patterns of CK and MUC in the AC subtypes 1 5 6 .

In a previous study, the CK7+/CK20-/MUC2- pattern in the histological intestinal-type carcinoma and the CK7-/CK20+/MUC2+ pattern in the histological pancreatobiliary-type carcinoma indicated that these different types of ACs had developed from 2 different types of mucosa in the ampulla of Vater 1 . Chu et al. reported positive expression of CK7, CK20, and MUC2 in the histological intestinal-type and positive expression of CK7 and MUC1 in the histological pancreatobiliary-type and that ACs of pancreatobiliary origin showed immunophenotypes similar to that of pancreatic ductal carcinoma 6 . Zhou et al. were the first to show agreement between the histological classification and the immunohistochemical characterization based on cytokeratins 5 . However, their immunohistochemical classification did not correlate with tumor progression and prognosis.

Although most other studies have described immunohistochemical classification systems based on the expression of either MUC 2 or CK 5 , we analyzed the expression of both in ACs.

In the present study, significant differences were noted in the expression levels of the histological intestinal type and the histological pancreatobiliary type, with the sensitivity being 100% for CK20 and 94% for MUC1 expression, respectively. These results indicate that the CK20+/MUC1- pattern fully corresponds to the immunohistochemical I-type and that the CK20-/MUC1+ pattern fully corresponds to the immunohistochemical PB-type.

With regard to immunohistochemical classification systems, Zhou et al classified ACs on the basis of the combined expression of CK7 and CK20, while Chu et al classified ACs on the basis of the combined expression of CDX2, CK17, MUC1, and MUC2.

Little is known, however, about the combined expression of CK20 and MUC1 in ACs. The possibility of identifying the primary AC site is increased when the combined expression of CK and MUC, rather the expression of either one of them, is taken into account.

We found that the classification of the immunohistochemical subtypes based on the expression of both CK20 and MUC1 correlated well with histological typing (κ-coefficient = 0.5184). Using this immunohistochemical classification system based on the coordinated expression of CK20 and MUC1, we were able to determined that 2 of 9 tumors classified as the histological unusual type expressed the pancreatobiliary pattern in immunohistochemical analysis, while 1 of these 9 tumors expressed the intestinal pattern.

Previous studies have shown that the prognosis of AC patients depends on the pT stage, nodal metastasis, and histological type 1 4 13 14 15 16 17 . Similarly, our results indicated that the pT stage, nodal metastasis, and histological subtype correlated significantly with cumulative survival. According to our histological classification method, a large number of intestinal type ACs were at stage pT1 (60%) and node-negative (81%). These results indicate that progression of the intestinal-type tumor is slower and the risks of nodal metastasis lower than those in the case of the pancreatobiliary- and unusual-type tumors.

In our immunohistochemical classification, the pT stage correlated significantly with the immunohistochemical subtypes. Although the number of immunohistochemical I-type tumors in the early pT stages was significantly greater than the number of immunohistochemical PB-type and O-type tumors in the same stages, there were no significant differences in the cumulative survival. This result may be attributable to the lack of differences in the nodal metastasis risks associated with the immunohistochemical I-type and PB-type. This may indicate that the risks of nodal metastasis are similar among the intestinal, pancreatobiliary, and other types. Therefore, pancreatoduodenectomy with lymph node dissection should be performed for adequate surgical resection in AC 18 . If function-preserving surgery has been selected for AC, the histological classification system is currently more useful than our immunohistochemical classification method.

Gürbüz et al. reported that MUC5AC- and MUC6-positive expression patterns were regarded as representing gastric differentiation and that negative expression of both indicated the intestinal type of AC 7 . Zhou et al. showed that gastric MUC5AC expression correlated well with PB-type carcinomas 5 . Interestingly, we found that the frequency of the coexpression of gastric MUC5AC and MUC6 in the immunohistochemical O-type was significantly higher than those in the immunohistochemical I-type or PB-type. These results provide evidence that both the immunohistochemical I-type and immunohistochemical PB-type have low-grade expression of gastric mucins. Expression of these gastric MUCs in the immunohistochemical O-type indicates that both gastric foveolar and pyloric gland metaplasia occur in the immunohistochemical O-type 19 20 21 . In addition, patients with tumors of the immunohistochemical O-type and MUC5AC and MUC6 coexpression had a significantly longer survival than those with tumors that did not show this coexpression. Among the tumors of the immunohistochemical O-type, those negative for the coexpression were at a more advanced pT stage than those positive for the coexpression. The prognosis of tumors of the immunohistochemical O-type that were positive or negative for the coexpression may depend on the tumor stage. Thus, gastric differentiation of the immunohistochemical O-type is associated with good prognosis. Interestingly, our observations of the histological unusual type were similar.

In summary, we studied the expression of CK7, CK20, MUC1, MUC2, MUC5AC, and MUC6 in ACs. On the basis of the histological classification of ACs, we found that CK20 had a high sensitivity for the histological intestinal type and MUC1 had high sensitivity for the histological pancreatobiliary type carcinoma. On the basis of the observed differences in the expression patterns of both CK and MUC, we defined immunohistochemical subtypes. These immunohistochemical subtypes correlated well with the conventional histomorphological classification but did not correlate with prognosis. However, the coexpression of gastric MUC5AC and MUC6 correlates with the prognosis of patients with the immunohistochemical O-type of AC.

The authors declare that they have no competing interests.

YK participated in the design of the study, histological diagnoses, data processing, and drafting of the manuscript. TT participated in the design of the study and reviewed the manuscript. TN established histological diagnoses and reviewed the manuscript. TI, TN, and SY participated in the design of the study, established histological diagnoses, and reviewed the manuscript. All authors read and approved the final manuscript.