Invasive micropapillary carcinoma of the extrahepatic bile duct and its malignant potential

Invasive micropapillary carcinoma (IMPC) was originally described as a distinctive type of invasive carcinoma in the breast, but it has not been recognized as a histological type of the extrahepatic bile duct cancer. The present study demonstrated clinicopathological features and patient prognosis of IMPC. We examined histological reviews of 93 consecutive cases of the extrahepatic bile duct cancer and identified 13 cases which included IMPC component. The component of IMPC ranged from 5 to 60% of the primary tumor tissue, which was mainly detected at the invasive front of the tumor. Of the 13 cases, 12 (92.3%) carcinomas with IMPC showed lymph node metastasis more frequently compared to conventional adenocarcinoma (39.2%, P<0.001). Presence of IMPC component was significantly associated with poor overall survival (P=0.003). In conclusion, extrahepatic bile duct carcinoma with IMPC component showed significant lymphatic invasion, lymph node metastasis, and resulted in poor prognosis.


Introduction
Extrahepatic bile duct carcinoma is an epithelial cancer originating from the bile ducts with features of cholangiocytic differentiation. There is no significant geographical variation in the incidence of extrahepatic bile duct carcinoma. In the USA, extrahepatic bile duct carcinoma accounts for 0.16% of all invasive cancers in males and 0.15% in females in the general population (1). Surgical treatment is the only curative therapy for extrahepatic bile duct carcinoma and is therefore the treatment of choice if feasible. The spreading cancer cells via the lymphatic to regional lymph nodes are an important factor for tumor progression. In a recent study, the median disease-specific survival rate after surgery in patients with lymph node metastasis was lower than that of patients without lymph node metastasis (19.3 vs. 53.5 months, P<0.0001) (2).

Materials and methods
Patients. We investigated consecutive bile duct carcinoma surgical cases treated between January 2007 and December 2012, after obtaining each patient's informed consent for use of their clinical records and pathological specimens at Hirosaki University Hospital. The series consisted of 69 men and 24 women with a median age of 70 years (range, 31-83 years). The carcinomas were located in the perihilar (34 cases) and distal bile duct (59 cases), according to the anatomic location (24). Curative resection and regional lymph node dissection were dependent on the location of primary tumors: pancreaticoduodenectomy or pylorus-preserving pancreaticoduodenectomy was performed in 56 patients, bile duct resection in 1 patient and combined hepatectomy with bile duct resection in 29 cases and combined hepatectomy and pancreaticoduodenectomy in 7 patients. Survival data were obtained from hospital medical charts and the median observation period was 25.6 months (79 cases).  (24). We also investigated phenotypes of IMPC components using the immunohistochemical procedure, as described below.
Immunohistochemistry. For histological examination, extrahepatic bile duct carcinoma specimens were routinely fixed with formalin, embedded in paraffin and thin-sectioned. Sections 4-µm-thick were mounted on saline-coated glass slides. Immunohistochemical examination was performed on deparaffinized sections using the standard avidin-biotin- Evaluation of immunohistochemistry. Luminal membranous immunoreactivities of the tumor were judged as positive for MUC1, and cytoplasmic immunoreactivities as positive for MUC2, MUC5AC and MUC6. The results were classified into two groups based on the percentage of positive cells with each staining as follows: negative group in which <5% of cancer cells were stained, and positive group in which ≥5% was stained.
Statistical analysis. Statistical comparisons between two groups were analyzed using the Pearson's Chi-square test for categorical data and the Mann-Whitney test for continuous data. Survival curves were constructed using the kaplan-Meier method and differences in survival were evaluated using the log-rank test. The relative prognostic factors were analyzed with the Cox proportional hazards regression model. Differences were considered to be statistically significant if the P-value was <0.05. All statistical evaluations were performed using R (http://www.r-project.org) and PASW statistics software (version 18.0; SPSS, Inc., Chicago, IL, USA).

Results
Histological and immunohistochemical findings of IMPC.
We reviewed 93 cases of the extrahepatic bile duct cancer  (Fig. 1A). The tumor was characterized by small round to ovoid micropapillary tumor cell clusters with no fibrovascular cores, lying within clear stromal spaces (Fig. 1b). The clear stromal spaces resembled lymphatic vessels, but were immunohistochemically negative for D2-40, a marker of lymphatic vessel (Fig. 1C). Metastatic carcinomas of lymph nodes also had IMPC component (Fig. 1D). The carcinoma cells characteristically displayed a reverse polarity, known as an ̔inside-out' growth pattern, mimicking extensive lymphatic invasion ( Fig. 2A and b).  The results of immunohistochemistry are summarized in Table II. Nine (69.2%) of the 13 cases of IMPC were positive for MUC1. MUC1 immunoreactivity was predominantly detected at the surface of the cell cluster and clearly exhibited the ̔inside-out' growth pattern (Figs. 2C and 3A). MUC5AC was focally found in the cytoplasm, as well as at the cell surface, in 4 of the 13 cases (Fig. 3C). MUC2 and MUC6 staining was negative in all cases of IMPC ( Fig. 3b and D).
Clinicopathological findings of IMPC. The clinicopathological findings of extrahepatic bile duct carcinoma with and without IMPC component are summarized in Table III. The presence of IMPC component was significantly correlated with lymph node metastasis, lymphatic invasion and the mode of infiltration pattern (P<0.001, P=0.016 and P=0.027, respectively). In addition, the extrahepatic bile duct cancer with IMPC compo-nent frequently showed lymph node metastasis with IMPC component (Table Iv,  Univariate and multivariate analysis. Survival curves based on univariate survival analysis demonstrated that the patients with IMPC were associated with poor prognosis (Fig. 4, P=0.003) and poor disease-free survival (Fig. 5, P<0.001). To clarify potential prognostic indicators, we analyzed various pathological factors investigated (    and lymph node metastasis (RR 3.868, 95% CI 1.946-7.684, P<0.001).

Discussion
In the present study, we clarified clinicopathological characteristics of IMPC of the extrahepatic bile duct. We clarified that IMPC frequently showed aggressive tumor growth with lymphatic invasion and lymph node metastasis, resulting in short overall/disease-free survival of the patients. This is the first report describing clinicopathological malignant potential of IMPC of the extrahepatic bile duct.
The molecular mechanisms of the malignant potential of IMPC have not yet been fully elucidated, while IMPC is histologically characterized by the ̔inside-out' pattern. A previous study proposed that extracellular matrix (ECM) contributed to the IMPC structure (28). Madin-Darby canine kidney (MDCK) cells exhibited ̔inside-out' structure without type Ⅰ collagen, but were able to reorient their cell polarity under the presence of type Ⅰ collagen in ECM. Here, the reorientation of cell polarity was shown to be related to RAC1, PI3-kinase and a PkC (28). Cancer cells of IMPC may have abnormalities of RAC1 suppression cascade and show the characteristic ̔inside-out' structures (29). We also revealed that MUC1 expression was predominantly at the surface of tumor clusters. The MUC1 expression was similar to the other organs (30). Human MUC1 is a high molecular weight transmembrane glycoprotein, which is apically expressed in the majority of glandular epithelia (31). Increased MUC1 expression has been shown to inhibit integrin-mediated cell adhesion between   cancer cells and ECM (32) and to decrease adhesion to type Ⅰ collagen (33). based on this evidence, IMPC expressing MUC1 may reduce the cell adhesion from ECM and result in forming the characteristic ̔inside-out' structures.

Mucin phenotype
Our study revealed that IMPC structures were found not only in the primary carcinoma lesions, but also in the foci of lymphatic vessels and metastatic lymph nodes. The ̔inside-out' IMPC structures were thought to play an important role in  the lymph node metastasis. Of note, IMPC exhibited stromal desmoplastic reactions around the ̔inside-out' cancer cell clusters. The desmoplastic changes consisted of proliferation of fibroblasts and collagen fibers and were found not only in the primary lesion, but also in the parts of lymph node metastasis. The desmoplastic changes are thought to be associated with epithelial-mesenchymal transition, which may contribute to an aggressive growth of the invasive cancer.
The results of univariate analysis revealed that IMPC was significantly correlated with poor patient prognosis, but the multivariate analysis using Cox proportional hazards model showed that IMPC was not an independent prognostic factor for overall survival. We suspected the reason why it was not an independent factor was that IMPC may be strongly associated with lymphatic invasion and lymph node metastasis. An important clinical issue is that the presence of IMPC indicates malignant potential, even if the component is small. Therefore, pathologists should describe presence of IMPC component in the diagnostic report, even if the component is a small part of the extrahepatic bile duct carcinoma.