Primary Small Cell Neuroendocrine Carcinoma of the Endometrium: A Cytologically Diagnosed Case with Immunocytochemical, Immunohistochemical, and Electron Microscopic Features

Endometrial neuroendocrine tumors are rare, accounting less than 1% of endometrial cancers. They include small cell neuroendocrine carcinoma (NEC) and large cell NEC and usually occur in postmenopausal patients. Although common symptoms include postmenopausal bleeding, most patients are diagnosed at an advanced stage. We report an extremely rare case of small cell NEC developed in the endometrium of a 75-year-old Japanese woman. This case was initially suspected based on the findings of endometrial cytology: a number of small round malignant cells were present on the endometrial smear specimens. The immunocytochemical and immunohistochemical examinations revealed diffusely cytoplasmic positive reaction of neuron eight months after the first visit without any viability of lung metastases.


Introduction
Neuroendocrine tumors could occur in the female genital organs, but endometrial neuroendocrine tumors are extremely rare. According to the recent WHO classification, they are categorized as low-grade and high-grade neuroendocrine neuroendocrine carcinomas [1]. They are further sub-classified into small-cell and large-cell types [1] [2]. Approximate 100 cases of small-cell neuroendocrine carcinoma (SCNEC) of the endometrium have been reported in the English literatures [3] [4] [5] [6]. SCNEC of the endometrium demonstrates aggressive clinical behavior [1] [4] [5] [7]. Endometrial SCNEC is known to be highly aggressive, because this malignancy is usually diagnosed at advanced stages of the diseases [8]. Furthermore, the prognosis of a pure type of SCNEC is worse, when comparing to that of a mixed type of SCNEC [9].
While most of the SCNECs of the uterine cervix possess mutations in PIK3CA, K-ras, and p53, molecular pathology of the endometrial SCNECs is unknown [10]. Abnormal mismatch repair protein expression was described in a few cases of endometrial SCNECs, but the majority of reported cases have not been included mismatch repair testing [5].
Endometrial cytological examination is a useful and minimally invasive tool for detecting endometrial malignancies, premalignancies, and benign lesions [11]. However, studies on the cytological features of SCNECs of the endometrium are scare [3] [4]. In the present report, we describe cytological, immunocytochemical, immunohistochemical, and ultrastructural features of endometrial SCNEC developed in an old Japanese woman, her clinical course as well.

Case Presentation
A 75-year-old female, gravida four para four, presented to our hospital with vaginal bleeding for more than three months in 20xy. She had a slight dementia due to previous cerebral bleeding, but did not have chronic diseases, such as hypertension, diabetes. The patient had inserted a soft pessary due to complete prolapse uteri three years ago. She did not visit the gynecologic clinic until then.
When the pessary in the vagina was removed, we found that the bleeding came from the uterine cavity. The uterine corpus was enlarged as over new born head size. The endometrial cytology and curettage were performed. On the endometrial cytology specimens, we found atypical cells suggestive of SCNEC [ Figure   K

Discussion
The primary SCNEC of the endometrium, which was reported to be approximately 100 cases in the literature is an extremely rare neoplasm [3] [4] [5] [6].
Mean age at the diagnosis is over 60, our case being 75-year-old. Abnormal or postmenopausal bleeding is the most frequent symptom, as experienced in this case [15]. The cytological and clinicopathological characteristics, including electron microscopic findings and a variety of immunohistochemical features of primary endometrial SCNEC are described. In addition, the management and clinical outcomes of our patient are also presented. We also determined DNA repair function of cancer cells using the MSI-IVD Kit and immunohistochemistry with primary antibodies, such as MSH2, MLH1, MSH6, and PMS2. However, cancer cells did not have DNA repair dysfunction.
The endometrial SCNEC is reported to have aggressive clinical behavior [1,7].
Five-year survival rate of stage IVB in ENCs was found to be 12.0% (0.7% -40.8%), while that of endometrioid adenocarcinoma was 27.7% (25.2% -30.3%) [6]. The aggressive surgical resection and adjuvant chemotherapy with or without radiotherapy appeared to be responsible for the favorable outcomes. Our case was histopathologically a pure type of endometrial SCNEC. When compared to a mixed type of endometrial SCNEC, prognosis of a pure type was reported to be worse [9]. Treatment strategies for endometrial NEC are not standardized [6], and most cases of endometrial SCNEC include platinum-based chemotherapy with etoposide or paclitaxel [6] [15], as performed in the small cell lung cancers. In our case, reductive surgery and following EP chemotherapy suppressed recurrence and lung metastases.
There are a few reports on the cytological features of endometrial SCNEC [3] [4]. The characteristics of cytological include the presence of single or small nests of neoplastic cells with a high nuclear/cytoplasmic ratio, round to oval nuclei containing coarse chromatin in necrotic backgrounds [3], corresponding to the typical cytological features of lung SCNEC [16]. As reported [17], immu-  [20].
Although a transcription factor, PAX-8, in the regulation of organogenesis of the thyroid gland, kidney, and Müllerian system, is commonly expressed in the nuclei of most endometrial adenocarcinoma cells [21], approximate 30% of endometrial NECs were immunohistochemically positive for PAX-8 [5]. Pocrnich et al. reported that the NECs tend to be PAX-8 negative and may be associated with microsatellite instability [5]. However, their cases were large cell NECs. In our case, PAX-8 was positive and p53 was focally positive in the cancer cell nuclei. It is known that nuclear expression of PAX-8 and p53 is well-correlated in the endometrial carcinomas regardless histopathological types such as endometrioid and non-endometrioid types. Pax-8 positivity correlating with p53 expression might be one of useful prognostic parameters [22]. Most commonly observed mutations were reported to be PIK3CA, K-ras, and p53 in the SCNEC of the uterine cervix [23] [24] [25], the molecular alterations in the endometrial SCNEC were not well-understood because of its rarity [10]. Nuclear p53 was focally positive in the tumor cells, suggesting the importance of p53 mutation for the development of SCNEC in our case.

Conclusion
We report here an extremely rare case of primary endometrial SCNEC initially