Lymphoepithelioma-like cholangiocarcinoma in middle-aged woman with Epstein-Barr Virus infection: a case report and review of the literature

Lymphoepithelioma-like carcinoma(LELCC) is a rare variant of intrahepatic cholangiocarcinoma (ICC), LELCC is composed of prominently reactive lymphoid infiltration and scattered malignant epithelial cells, which may lead to misdiagnosis of pathologist, especially on rapid freezing during operation. Recognizing the tumor is important to avoid misdiagnosis. We report a case of LELCC in a 53-year-old Chinese female patient with EBV infection, who was found to have a nodules in the junction of the left and right lobes of the liver on abdominal Computed tomography(CT) and magnetic resonance imaging(MRI). Microscopically, the boundary of the tumor was clear with incomplete fibrous capsule around the tumor, the tumor was arranged in a nest or tubular gland, tumor cells were cubic, with vesicular nuclei, prominent nucleoli, rare mitotic figures, and abundant lymphoid infiltration and lymphofollicular formation. Immunohistochemically, tumor cells were diffusely positive for biliary-type cytokeratins (CK7, CK19), CK8,CK18,Ki-67 proliferation index (about 20%) and negative for CK20, polyclonal carcinoembryonic antigen(CEA) and CD34, hepatic marker (HepPar-1), The tumor-infifiltrating lymphocytes are predominant for CD3 and CD45RO-positive T cells, CD20 and CD79α are positive scattered in the germinal center and surrounding areas. In situ hybridization, EBV-encoded RNA (EBER) was strongly positive in tumor cells. The


Background
Lymphoepithelioma-like carcinoma (LELC) is a malignant tumor resembling undifferentiated nasopharyngeal carcinoma (NPC), which is histologically composed of undifferentiated epithelial cells with prominent lymphoid infiltration [1]. LELC have been reported in varios organs including salivary gland, skin, lung,the stomach, and liver and so on [2][3][4][5][6][7]. Liver is a rare site of occurrence. In recent years, more and more cases have been reported with the further understanding of LELC [8]. LELC in the liver can be divided into lymphoepithelioma-like hepatocellular carcinoma (LELHCC), lymphoepithelioma-like cholangiocarcinoma (LELCC) and mixed LELHCC and LELCC [8][9]. LELCC, which is defined as tumors arisen in the hepatic tract and composed of undifferentiated epithelial cells arranged in gland forming, sheets, or cords with a dense lymphoid infiltration, is a rare variant of intrahepatic cholangiocarcinoma (ICC) [1,10]. Ling et al [10] retrospectively analyzed 40 cases of LELCC and found 80.0% of these cases were related to EBV. To date, only 29 cases of LELCC with Epstein-Barr Virus(EBV) Infection have been reported in the English literature [11], in addition, the diagnosis and differential diagnosis of LELCC are rarely reported [1,11]. The diagnosis and differential diagnosis of this extremely rare tumor are important for its identification. This paper reports a case of LELCC with EBV infection with the review of the literature, and explores its clinicopathological characteristics, immunophenotype, molecular genetic features and differential diagnosis, in order to improve the understanding of pathologists on this tumor.

Case Presentation
A 53-year-old female patient was admitted to the hospital for discovering liver mass  In situ hybridization for EBV-encoded RNA (EBER) showed strongly positive nuclear signal in tumor cells (Fig. 1j),but not in the surrounding non-neoplastic liver tissue.

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Based on these histopathological, immunohistochemical and in situ hybridization findings, a diagnosis of EBV-associated LELCC was made.
The patients were followed up for 12 months without any radiotherapy or chemotherapy.
The liver, gallbladder, pancreas, and bilateral renal enhanced CT were reviewed every three months within one year after the operation. The fourth review was liver enhanced MRI. No recurrence or metastasis was found. At present, the follow-up is continuing.

Discussion And Conclusions
Epidemiology and clinical characteristics: EBV-associated ICC is very rare and can be divided into 3 types: LELC, conventional-type ICC and LELCC on the basis of the tumor cellular differentiation, and host cellular immune responses in the tumors [12].Indicating that LELCC, which was first reported by Jeng et al [13] in 2001, is a rare variant of ICC.
Literature reported, EBER in situ hybridisation was positive in 70% of LELCC cases, while conventional cholangiocarcinomas are not associated with EBV infection [13] According to previous studies, 26 cases of LELCC was analyzed, found that 73.1% (19 of 26) patients were EBV-positive [1,8].These results suggested that LELCC is highly associated with Epstein-Barr virus (EBV) infection [14] and EBV infection might promote the development of LELCC. Hepatitis B virus (HBV) and hepatitis C virus (HCV) has been confirmed as one of the etiologic factors for ICC [15]. However,only 26.9% (7 of 26) and 7.7% (2 of 26) LELCC patients were HBV and HCV positive, respectively. This low incidence of HBV and HCV infection suggested that these two viruses might not be significant factors in the pathogenesis of LELCC [8].Despite these findings, the molecular mechanism of EBV, HBV and HCV remain unknown in the tumorigenesis and development of LELCC. The published literatures reported that majority of LELCC patients were discovered in the Asian countries [11,13,16]. LELCC with EBV infection tends to occur in middle-aged women with a female:male ratio of more than 3:1, the range of age was 19 to 71 years (mean age, 7 53.39 years) [8][9][10][11][12]. No preference in the tumor location was found between the left and the right hepatic lobe [17]. The size of LELCC tumors ranged from 1.2 to 7.3 cm in diameter (mean, 3. 3 cm) [12,18].Clinical symptoms were nonspecifific, usually found incidentally or abdominal fullness and abdominal pain [10]. Literature reported that gene hypermethylation was more frequent in LELCC than IHCC. No LELCC harboured any EGFR or KRAS mutation [25].Molecular analysis of the original periportal mass showed single-nucleotide variations in the tumour suppressor gene TP53 as well as single-nucleotide variations of uncertain significance in MLH1, a gene involved in DNA mismatch repair. The tumour was microsatellite stable by PCR [11]. Chiang et al [14]reported that 4 LELCCs with 0 to 5 mutations per sample. proliferative. There is no bile in the lumen, no atypia and mitosis, and there is chronic inflammatory cell infiltration. There were no dilated bile ducts in the tumor, In advanced stage, there were more collagen, fewer small bile ducts and inflammatory cells. CD56 was positive for bile duct adenoma. However, the epithelial cells of LELCC are atypical and CD56 was negative for epithelial cells. 6,Other less likely neoplasms were also ruled out using immunohistochemistry, including inflammatory myofibroblastic tumour (Anaplastic lymphoma kinase (ALK)), malignant melanoma (HMB45 and S100) and histiocytic sarcoma (CD163). These tumours can have large cells with a lymphocyte-rich tumour stroma, but EBV were negative in situ hybridization [11] .
Treatment and prognosis: LELCC might be associated with EBV infection, but whether EBV infection is associated with a better outcome or poorer prognosis, which is controversial.
Compared to patients with EBV-negative ICCs, after surgery, patients with EBV-associated LELCCs usually have favorable outcomes [25]. However,Wang et al [18]reported that EBV infection leads to a poorer prognosis in LELCC. The role of EB infection in LELCC prognosis is still unclear, and more clinical studies are needed for further analysis. Zhang et al [26]found that Only 8 cases had recurrence in 52 cases of LELCC reported by searching English literature. Gearty et al [11] retrospectively analyzed the 29 LELCC cases with EBV infection, found that 8 patients died of the disease. Most cases were early stage tumours treated with surgical resection, including one successfully resected lymph node metastasis. These findings indicate that LELCC patients have a better prognosis. The prognosis of LELCC patients seems to be better than that of conventional-type cholangiocarcinoma at 5-year survival rate(100% and 13.2%) [25].
In all cases reported, surgical resection was the the most effective choice of treatment, but the necessity of lymph node dissection had not been reported. However, postoperative radiotherapy, postoperative chemotherapy, or targeted therapy was rarely adopted. Some reports had suggested that postoperative radiation therapy and chemotherapy may be benefificial [24]. However, Zhang et al [26]reported that that surgery could be an effective way of treating lymph node metastasis of LELCC. In recent years, immunotherapy has become a new treatment option for many tumors. Of the one LELCC patient who had anti-PD-1 treatment after gemcitabine/ cisplatin therapy failure got a durable partial response to the anti-PD-1 immunotherapy [14].Wang et al [18] found that PD-L1 levels were higher in LELCC than in ICC.Therefore, Immunotherapy of targeted PD-L1/PD-1 may be effective therapeutic strategy in EBV-associated LELCC. Because of the limited number of reported cases, no consensus on standardized treatment strategy for LELCC had been reported, treatment methods and prognosis need further observation and exploration. In our study,the patient, which did not receive any radiation and chemotherapy, was followed up for 12 months without recurrence or metastasis after hepatectomy. We will Currently continue to follow up.
In summary,EBV-associated LELCC which have a better prognosis than conventional-type CC, is a rare variant of ICC, The diagnosis depends on histopathology, immunohistochemistry and EBER detection, and the recognition and correct diagnosis of