Mucoepidermoid carcinoma arising in Warthin’s tumor of the parotid gland: Clinicopathological characteristics and immunophenotypes

Mucoepidermoid carcinoma (MEC), an extremely rare tumor, arises from the epithelial component of preexisting parotid Warthin tumors (WT). Among the 309 cases of surgically resected WTs in Chinese PLA General Hospital and Beijing Shijitan Hospital of Capital Medical University, 5 cases (1.6%) fulfilled the criteria for MECs transformed from WTs. Clinicopathological characteristics of MECs was demonstrated in order to avoid misdiagnosis of this rare type of tumor. All the 5 patients, 3 males and 2 females, presented painless masses in the parotid gland. MECs were located inside or at the edge of WTs, with an obvious transitional zone between WT and MEC. Basal cells of WTs and epidermoid cells of MECs were strongly positive for cytokeratin CK5/6, CK34βE12, and P63; whereas negative for CK7, CK20, and CEA. Mucous cells of MECs were positive for CK7, CEA, as well as periodic acid-Schiff (PAS), whereas negative for CK5/6, CK34βE12, CK20, and P63. MECs patients were followed up for 25–69 months after surgery, presenting no evidence of recurrence or metastasis. Collectively, MECs arising from WT is very rare. The pathological diagnosis was based on histological morphology, especially the transitional zone between WT and MEC.

Only 19 cases of WT co-existing with MEC have been reported worldwide 7 . Here we reported 5 new cases of MECs arising in Warthin's tumor of the parotid gland. The purpose of this study was to improve the understanding of clinical and pathological characteristics of MECs, and thus to avoid misdiagnosis of this very rare type of tumor.

Results
Clinicopathological characteristics. The demographic, clinical and pathological information of MECs patients were summarized in Table 1. Among these 5 cases, 3 males and 2 females; the median age was 43 years (range 26-63 years). All patients presented painless masses in the parotid gland region, except for Case 5, whose first symptom was progressive facial paralysis. Four cases developed tumors in the superficial lobe of the parotid gland, and 1 case in the deep lobe. In Case #1, CT scanning displayed circular nodule shadow with slightly high density in the right parotid gland, with clear boundary and multiple nodules surrounding the shadow (Fig. 1A). Enhanced CT scanning described lesions in moderate strengthening, with lower density inside an oval shaped area, which was considered as internal cystic or necrosis of the tumor (Fig. 1B). In Case #5, MRI imaging displayed multiple long T-1 and short T-2 signals varying in sizes in the right parotid gland globe with irregular shapes (Fig. 1C). MRI enhanced scanning could reveal moderate enhancement of MECs tumors (Fig. 1D). Three cases received single surgery treatment, and 2 cases received surgery combined with radioactive particle implantation.
Gross appearance. Each mass exhibited a nodular or oval shape. There were multiple tumors (ranged from 0.5-2.5 cm in diameter) in Case #5. The tumor size of another 4 cases ranged from 2 cm to 4.2 cm in the maximum diameter ( Table 1). The masses of Cases #2 and #3 were completely encapsulated with clear boundaries. Masses of the other cases were mostly encapsulated with ambiguous boundary to the surrounding parotid gland tissues. Cross sections of the masses in Cases #1, 3, and 4 were solid, presenting grayish-yellow or grayish-brown color. Cross section of the mass in Case #2 was cystic-solid, with mucous fluid in the cystic areas. In Case #5, the mass had invaded the outside border of the capsule (Fig. 2).
Microscopic features. Warthin's tumor was composed of large gland and microcystic lumen. The gland and the inside of the cystic walls were covered with two layers of epithelia, with columnar oncocytic cells in the inner layer and smaller basal cells in the outer layer. Oncocytic secretion could be observed in the glands and cystic lumens. MECs were identified within WTs in all five cases. MECs had abundant fibrous stroma with focal hyalinization and scarce lymphoid elements. MECs exhibited invasive growth into the lymphoid tissues of WTs, with fibrous stroma. In Cases #2 and 4, the invasion of nerve fibers by MECs was evident. In Cases #1, 4, and 5, the invasion of capsules by MECs was observed. In all five cases, transitional zones between WTs and MECs elements were distinguished (Fig. 3).
Periodic acid-Schiff (PAS) staining was positive in mucous cells of MECs. Mucus secretion from the cystic lumen was observed in WTs, while exudative mucus component was present in the stroma of MECs (Fig. 10).
Follow up. MECs patients were followed up for 25-69 months. Currently, there is no local recurrence or distant metastasis.   Local chronic inflammation, ischemia or hypoxic microenvironment may play important roles in mediating epithelial metaplasia to atypical hyperplasia 9 . Warthin's tumor occurs mostly in older males. In the reported cases of MEC arising from WT, the youngest one was a 35-year-old female 10 and the oldest one was a 64-year-old male 11 . Age of onset of most patients was between 50-65 years, and the ratio between males and females was almost 1:1. There were 3 males and 2 females in the present study. The average age was 45.6 years, and the youngest was a 26-year-old female.
Warthin's tumor occurs frequently in parotid glands and their lymph nodes, occasionally in minor salivary glands. WT is located in the tail lobe or deep lobe of parotid gland 9 . Classic clinical presentation is a slow-growing painless mass, although a few cases can cause pain or exhibit rapid growth 21 . If the mass increases suddenly, the possibility of malignancy or inflammation should be considered. Clinical manifestations of 5 patients in our study were similar to those reported in the literature. All patients presented painless masses in the parotid glands. Notably, in one case, the first symptom was facial paralysis. Facial paralysis is rare in WT, attributed to secondary inflammation, fibrosis, metaplasia 22 , or malignancy. MEC arising from WT was mostly a single nodular or oval mass located in lateral parotid gland 11 , rarely multiple nodules or located in bilateral parotid glands 5 . When MEC occurred in the interior part of WT, the capsule was clearly observed macroscopically. If MEC was located at the margin or invades the capsule, the boundary became ambiguous.
Cross sections of the masses were solid or cystic-solid, grayish-brown in color, and mucous. Cancerous region was usually desmoplastic, grayish-white in color. WT and MEC can co-exist as MEC is located in the interior of or adjacent to WT. Transitional zone between the epithelial component of WT and MEC confirms the diagnosis. It is difficult to distinguish metaplastic goblet cells of WT from mucous cells of a low-grade MEC 11 . In these 5 cases, oncocytic epithelium was present in mucous and squamous metaplasia of WT, while metaplastic goblet cells and epidermoid cells exhibited as a patchy distribution in MEC and mucous lake. Direct evidence of malignant transformation was infiltration of lymphoid tissues and encapsulation of WT by MEC 13         MEC arising from WT should be differentiated from the following lesions. The first is WT accompanied by metaplasia of squamous cells and mucous cells. Similar to normal parotid gland, WT accompanied by focal squamous and mucous cells is not uncommon. Approximately 7.5% of WT has extensive squamous cell metaplasia 13 . Normal epithelial tissues in WT exhibit a double-layer structure; while abnormal structure and atypical cells appear with the metaplasia of squamous and mucous cells, characterized by an increase in epithelial layers, and the formation of cystic structures lined by mucous and intermediate cells. Therefore, it is difficult to determine the presence of malignant transformation. The key point of identification is that metaplasia lacks atypical cells and infiltrative growth.
Second, hyperplasia of stromal fibrous tissue and hyaline degeneration can be observed in MEC 12 . It is difficult to distinguish WT's metaplastic mucous epithelium from MEC's mucous epithelium based on dysplasia. In these 5 cases, mucous and oncocytic cells were mixed and distributed in mucous metaplasia of WT. Patchy distribution of mucous glands, irregular gland structure, mucous lakes, mucus spillover, and cancer stroma suggested the possibility of MEC. In future, more samples will be collected to identify evidence of malignant transformation.  Third, WT and MEC can develop independently and co-exist 9 . MEC arising from WT should be differentiated from another pattern of malignant transformation of WT, i.e., squamous cell carcinoma arising from WT. The key point of differential diagnosis is the presence of mucous cells, represented by positive CEA and AB/PAS staining. In addition, the epithelioid cells of MECs are rarely keratinized 8 .
Fourth, an extremely rare condition "tumor-to-tumor metastasis" should be considered. WT may act as a recipient of the malignancy that metastasizes from distant sites. It has been reported that lung carcinoma and renal carcinoma metastasize to WT 23 . The diagnosis is mainly based on the fact that other tumor is identified according to clinical manifestations and imaging results; and there is no transitional zone between the two tumors. The patients in this study had received comprehensive examinations. There was no tumor at other sites, while there was a transitional zone between the two tumors. The differential diagnosis between the malignant transformation of WT's epithelial component and metastatic tumor is very important for the treatment and prognosis of the patients. If WT is combined with a metastatic tumor, local surgical resection alone is not adequate to achieve a satisfactory outcome.
The treatment of MEC arising in WT is mainly surgical operation, including resection of the primary tumor combined with neck lymph node dissection. MEC arising in WT is susceptible to recurrence or metastasis, which requires a long-term follow-up. In this study, 3 cases received surgery only, and 2 cases received surgery combined with radioactive particle implantation. So far, the reported cases of MEC arising from WT have been very limited, and most cases have no recurrence or distant metastases 11,13 . The 5 cases in the present study were followed up for 25-69 months. Currently, serum CEA, CA125, AFP and CA-199 levels are all in normal range. No recurrence or distant metastasis of the tumor has been found by imaging examination. A long-term prognosis still awaits further observations.

Materials and Methods
Patients. This study has been approved by Institutional Review Board (IRB) committees at Chinese PLA General Hospital and Beijing Shijitan Hospital. Informed consent was acquired from each participate before the operation. All procedures were conducted according to the guidelines approved by the ethics committees at Chinese PLA General Hospital and Beijing Shijitan Hospital. Among 309 cases of consecutively, surgically resected Warthin tumors at The Chinese People's Liberation Army (PLA) General Hospital (n = 286, recruited from 2009 to 2014) and Beijing Shijitan Hospital of Capital Medical University (n = 23, recruited from 2010 to 2014), 5 cases fulfilled the criteria for MECs arising from WTs. None of the cases had MECs at any other sites at the time of diagnosis or during the follow-up after surgery.