Diagnosis and Clinical Management of Neuroendocrine Tumor of the Breast: Report of Six Cases and Systematic Review of Existing Literature

guidelines. Materials and Methods: We presented our experience of six cases of bNENs. Moreover, we conducted a systematic review of published data on diagnosis, treatment and outcome of this kind of tumors. Results: bNENS usually presented as palpable breast masses, classically appearing as irregular hypoechoic lesions at US examination and as hyperdense masses at mammography. Usually pre-operative tumor biopsy is not able to recognize the neuroendocrine components and the final diagnosis is performed only on definitive histopathological assessment. The most frequent subtype seems to be neuroendocrine carcinoma and synaptophysin is positive in most specimens. Treatment strategies, including surgical treatment, radiotherapy and medical treatment are nowadays based on current non-endocrine breast cancer guidelines, independently from neuroendocrine components, even if some studies have proposed the use of somatostatin analogues for bNET and cisplatin-etoposide for NEC. Prognosis of all bNENs, especially of poorly differentiated neoplasia, seems worse compared to non-neuroendocrine breast cancer and stage and morphology seem the best predictor of tumor outcome. Conclusions: We provide an algorithm for clinical management of bNETs, basing on available data. More studies are necessary for confirming the best treatment strategy for these patients, in order to improve clinical outcome . year of publication, type of study, number of patients included, age at diagnosis, sex, familiarity for breast tumors, other known risk factors for breast cancer, clinical presentation, palpability, diagnostic procedures (ultrasound, mammography, MRI, CT, PET, fine needle aspiration and biopsy), treatment strategy (surgery, medical treatment, radiotherapy), histopathological examination including immunohistochemistry, stadiation and outcomes.


Introduction
The first description of a neuroendocrine tumor (NET) of the breast dates back to 1963: an invasive breast cancer morphologically similar to intestinal carcinoids [1]. World Health Organization (WHO) recognized neuroendocrine tumors of the breast as a separate entity of breast cancer only in 2003, defining them as primary neuroendocrine carcinomas exhibiting morphological features of gastrointestinal and pulmonary NETs in which more than 50% of the cells expresses neuroendocrine markers (chromogranin A and synaptophysin) [2]. In 2012 the cut-off of 50% of the cells expressing neuroendocrine markers was eliminated and bNENs were divided in groups according to morphology: welldifferentiated (carcinoid-like) neuroendocrine tumor (bNET), poorly differentiated neuroendocrine carcinoma (NEC) small-cell neuroendocrine carcinoma (SCNC) and invasive carcinoma with neuroendocrine differentiation (ICNE) [3]. According to WHO data, bNENs represent about 2-5 % of all breast cancer [4]. In data from SEER database bNENs represent less than 0.1% of total invasive carcinomas of the breast [5]. Probably these frequencies may underestimate the real incidence of bNENs: retrospective studies on breast tumor specimens showed high incidence of neuroendocrine cells with positive neuroendocrine markers [6,7]. Nowadays, the impact of neuroendocrine differentiation of breast cancer on diagnosis, treatment and outcome is still unclear. Because of the low incidence of this kind of neoplasia, no clinical trials or guidelines are available on this topic. The aim of this systematic review is to summarize clinical presentation, diagnosis, treatment and outcomes of all available cases in Literature, adding our personal experience of six cases.

I Article Identification
We searched PubMed, Embase, Google Scholar and Cochrane databases for English language studies on neudoendocrine tumor of the breast. Search terms used were: "neuroendocrine tumor" AND breast, "neuroendocrine tumour" AND breast; "neuroendocrine cancer" AND breast; "neuroendocrine carcinoma" AND breast.

II Eligibility Criteria
We included English-language studies on humans with any of the following design: randomized clinical trials, prospective nonrandomized trials, retrospective studies, case reports and case series. We selected cases classified by the pathologist as neuroendocrine breast tumor, according to WHO classification used at time of publication (2003 or 2012). For article published before 2003, we included cases defined as breast neuroendocrine tumors or carcinoids by the Authors. We included in the systematic reviews only articles with data on at least one of the following topics: clinical presentation, treatments and outcomes of neuroendocrine tumors of the breast. Last search date was February 2019.

III Article Selection
Each study was screened by abstract and title and potentially eligible studies were further assessed in detail by retrieving full-length articles. Each full-length article was independently reviewed by two separate Authors following inclusion criteria. Two authors independently extracted data from the articles that met the inclusion criteria. A standardized form was used to extract the following information: year of publication, type of study, number of patients included, age at diagnosis, sex, familiarity for breast tumors, other known risk factors for breast cancer, clinical presentation, palpability, diagnostic procedures (ultrasound, mammography, MRI, CT, PET, fine needle aspiration and biopsy), treatment strategy (surgery, medical treatment, radiotherapy), histopathological examination including immunohistochemistry, stadiation and outcomes.

I Case Series
We present six cases of bNENs diagnosed in Humanitas Research Hospital of Milan from 2012 to 2018. All patients provided written informed consent to case publication. All cases were females, mean age 64.2 ± 13.7. All patients presented with breast lumps (in one case painful). When performed, breast ultrasound (US) always showed a mass (in three cases hypoechoic mass) and mammography showed 4 spiculated and 1 regular margin hyperdense lesion of 0.8-2.7 cm of maximum diameter. Breast magnetic resonance imaging (MRI) was not performed in all cases due to lack of indication. Biopsy showed in all cases infiltrating breast carcinoma but only in one case succeeded in identifying neuroendocrine differentiation. All patients underwent surgical intervention. Surgery on tumor mass was in 4 cases breast conservative surgery (BCS) and in 2 cases total mastectomy; axillary surgery consisted of 3 lymphadenectomies and 3 sentinel lymph node biopsies. Radiotherapy was performed in the 4 cases of BCS. Definitive histopathological evaluation confirmed in all cases the neuroendocrine differentiation: 2 bNET, 2 breast NEC and 2 ICNE. In our case series synaptophysin has been the most important neuroendocrine marker, been positive in 6/6 cases. Chromogranin was positive in 1/3 cases while NSE was never evaluated. 5/6 (83,3%) cases showed positivity for both oestrogen and progesterone receptors. Ki67 ranged from 10 to 90%.
After definitive diagnosis, all patients performed a total body scans ( 18 FDG PET/CT and contrast-enhanced total body CT scans) for excluding neuroendocrine neoplasm of other origin. After surgical removal, patients underwent chemotherapy or hormonal therapy according to associated non-endocrine breast tumor histotype guidelines (2 only hormone therapy, 1 chemotherapy, 3 hormone therapy associated to chemotherapy). One patient with NEC developed liver and bone metastasis after 6 months and is now alive with metastatic disease after one year of follow-up. Medium follow-up of other cases was 65  months: 3 patients are today alive and disease free, two are alive with local recurrence. All data are summarized in Table 1A.

II Systematic Review
From the initial search we retrieved 445 articles. After screening for title and abstract we identified 140 potentially eligible articles. After full text examination a total of 117 articles were included in this systematic review (Figure 1). 102 articles were case reports on a total of 113 bNENs. Available data are summarized in Table 1B. 15 articles were retrospective studies or case series on a total number of 731 patients: data are summarized in Table 2.

Clinical Characteristics
From the analysis of 113 reported cases (Table 1), the most frequent clinical presentation was breast mass, which was present in 37 cases (in 7 cases also associated to axillary adenopathy and in 5 cases painful) followed by breast lump in 22 patients (of which 3 associated to axillary adenopathy, 2 to bloody nipple discharge, 1 to nipple retraction) and symptoms due to metastatic diffusion (1 jaundice, 1 haematuria, 1 bone pain, 1 respiratory symptoms, 1 perianal pain, 2 neuralgia). Less frequent clinical presentations were: isolated bloody nipple discharge (3 cases), only skin retraction (2 cases), anorexia (2 cases), locally advance disease in 3 cases (2 ulcerated breast masses, 1 carcinomatous mastitis, 1 Paget like mass). In 33 cases clinical presentation was not reported. Tumor was palpable in 58/77 cases (75%).

Radiological Findings
Radiological findings of bNENs were often similar to other breast cancer histotype, like ductal or lobular breast tumors. From available data, sonography was performed in 61 cases. In 11 cases US failed to detect breast lesions. In the other cases tumor appeared as irregular hypoechoic lesion. Data on mammographic finding was present in 61 cases and tumor always appeared as hyperdense mass. Notably, tumor was detected in all cases in which US and mammography were both performed. Only in 14 cases reported data on breast MRI: tumors appeared as irregular mass, hyperintense in T2-weighted sequences.

Discussion
bNENs are rare entities and no guidelines are available for the management of this kind of neoplasia. According to our systematic review, the most frequent clinical presentation is palpable breast mass, sometimes associated to axillary adenopathy or bloody nipple discharge. bNENs appears as hypervascular and irregular hypoechoic lesions at US examination and as hyperdense masses at mammography [8]. The detection rate of these two instrumental evaluations is high, even if is not possible to clearly differentiate this kind of tumor from another breast cancer histotype [9]. When performed, breast MRI shows irregular masses, usually hyperintense in T2-weighted sequences [8]. Before establishing treatment strategies, as recommended in all suspicious breast lesions, tumor biopsy should be performed, even if it is not always able to recognize a breast neuroendocrine tumor, which is often detected only by definitive histopathological assessment [10].
The contemporary presence of neuroendocrine cells with ductal carcinoma is usually considered a sign of the breast origin of the neuroendocrine lesions, even if a total body examination is mandatory for excluding neuroendocrine neoplasm of other origin [11]. Recommended imaging techniques are total body CT or PET/CT scan: 68 Gallium PET/CT in case of well-differentiated neuroendocrine tumors or 18 FDG PET/CT in case of poorly differentiated NEN (NEC, ICNE, SCNC) as commonly performed in other neuroendocrine neoplasia [12]. Considering available data, the most frequent subtype is NEC. Most all cases were positive for synaptophysin staining, followed by chromogranin; hormone receptors and Her2 expressions were heterogeneous but luminal type (estrogen and progesterone receptors positive and HER2 negative) was the most common, as previously published [13]. This finding is in accordance with the hypothesis that bNENs develop from breast stem cells which divides into neuroendocrine and epithelial cells [14].
Surgical treatment strategies are nowadays based on tumor size and lymph node status basing on current breast cancer guidelines, independently of neuroendocrine component. Likewise, radiotherapy is usually performed after BCS [10,15]. Medical therapy depends on immunohistochemical analysis: in case of strong hormone receptors positivity, hormonal therapy is usually indicated [10]. In hormonenegative tumors, chemotherapy regimens, based on anthracyclines and or taxanes, is often used [10]. The possibility of using a cisplatin and etoposide regimen in breast NEC, as indicated for small cells carcinomas of other origin, has been evaluated only in small studies [16]. somatostatin receptors have been found in non-neuroendocrine breast tumors with high estrogen and progesterone receptor expression and low HER2 [17][18][19][20]. Moreover, somatostatin analogues are able to reduce breast cancer cells proliferation especially in case of low estrogen levels, providing the rationale for contemporary administration of hormonal therapy and somatostatin analogue therapy [21,22]. In Figure 2, we propose a diagnostic and therapeutic algorithm for bNENs. Finally, if the prognosis of all bNENs is different compared to nonneuroendocrine breast cancer is still debated. From the published cases, only 8 patients on a total of 91 deceased for the disease. When available, histotype of these neoplasms was NEC/SCNC. In the other 4 cases, tumor histology was not reported but tumor stage was advanced, implying that tumor stage and histology could be the main predictors of poor outcome. Data from the SEER database, comparing 142 breast NEC and non-neuroendocrine breast tumors, demonstrated a shorter overall survival and disease-specific survival of breast NEC and in a multivariate analysis neuroendocrine differentiation was an independent determinant of poorer prognosis [5]. Similarly, Bogina et al. have demonstrated a worse prognosis in 55 breast NEC patients compared to 115 matched non-neuroendocrine breast tumors patients [7].

Conclusions
bNENs are rare tumors, usually identified only during definitive histopathological examinations of surgical specimen. bNENs are nowadays treated similarly to non-neuroendocrine breast cancer, but they are very heterogeneous and not well understood. Similarly, to NEN of other origin, we should probably distinguish between well differentiated tumors, NET, and poorly differentiated tumors, NEC/small cells carcinomas regarding treatment and prognosis. Specific trials on adjuvant therapy, for example with somatostatin analogues for well differentiated form, bNET, or classical chemotherapy with cisplatin and etoposide in NEC and SCNC are necessary for establishing the best treatment strategy for these patients and improving clinical outcome.

Consent for Publication
All patients provided written informed consent to case publication.

Conflicts of Interests
The Authors have no conflicts of interest for this Paper. All authors disclose any financial and personal relationships with other people or organizations in the writing of this Paper.

Funding
None.

Author Contributions
Dr. Federico Frusone and Dr. Giulia Puliani cowrote this paper. Dr. Federico Frusone collected information of the case series from the database of Humanitas Research Hospital of Milan. Dr. Andrea Sagona,