Neuroendocrine Neoplasms of Breast:A Rare Breast Tumor Worthy of Attention


 Background: Neuroendocrine neoplasms of breast(NENB) is a rare and underrecognized subtype of breast neoplasms.The clinical significance, prognostic risk factors and optimal treatment modalities are limited. This study was focused on clinical and pathological features of 27 NENB cases to improve the understanding of these diseases and to further investigate the behavior of these neoplasms and to provide more factual evidence.Methods: We retrospectively analyzed the clinicopathological features and follow-up data of 27 patients diagnosed with NENB at the First Affiliated Hospital of Bengbu Medical College between February 2003 and February 2015.Results: The proportion of NENB from all invasive breast carcinomas(BC) in our hospital was 0.24% (27/11352). The expression of specific immunohistochemical markers was different: 48.1% cases showed the expression of chromogranin A (CgA)(13/27), CD56 was positive in 77.8%cases (21/27), the positive rate of INSM1 and synaptophysin (Syn) were 85.2%（23/27）and 100.0% (27/27). NENB occurred in older patients (median age,64 ).11cases (40.7%) were well-differentiated NETs and 16 cases (59.3%) were poorly differentiated NEC. In NETs, The positive rate of ER was 10/11 (90.9%) , while in NECs, The positive rate of ER was 9/16(56.3%) , On the basis of immunophenotypes, most of NENBs were of the luminal molecular subtype ,6 cases were luminal A and 15cases were luminal B ,6 cases were triple negative breast cancer(TNBC) and had no HER-2 overexpression subtypes. Conclusions: NENB more likely occures in elderly patients.Well-differentiated NETs are more often positive for hormone receptors than poorly differentiated NEC. NENBs are almost negative for HER-2. The combination of INSM1 is an effective supplement and improvement for traditional neuroendocrine markers.


Introduction
Neuroendocrine neoplasms of breast(NENB) is one of the rarest subtypes of breast tumor .Since neuroendocrine differentiation in breast carcinomas(BC) was rst described by Feyrter and Hartmann in 1963 1 ,various de nition and criteria for what constitutes breast neuroendocrine carcinomas have been used.The newest 5th Edition of World Health Organization (WHO) classi cation of Breast Tumors has moved to a dichotomous classi cation of neuroendocrine neoplasms in breast in order to become standardized with classi cations of other organ system 2 .The most signi cant feature of this Edition is that it divides NENB into two categories:well-differentiated neuroendocrine tumor(NET) and poorly differentiated neuroendocrine carcinoma (NEC).The latter including highly aggressive small cell carcinomas and large cell neuroendocrine carcinoma and exclusion of special histologic types (solid papillary carcinoma and hypercellular variant of mucinous carcinoma) 3 .
Because the incidence rate of NENB is low and the pathological character is variable,The clinical signi cance, prognostic risk factors and optimal treatment modalities are limited. This study collected clinical and pathological data of 27 NENB cases in the First A liated Hospital of Bengbu Medical College from February 2003 to February 2015 and investigated the clinicopathological features and the prognosis data to improve the understanding of these diseases.

Study Design and Patients
This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Tt was approved by institutional ethics board of The First A liated Hospital of Bengbu Medical College (No. BBMEC-2021088) and informed consent was taken from all the patients. Specimens were double-blind pathological reviewed by two pathologists. Histological evaluation standards refer to the 5th Edition WHO and TNM staging of breast cancer developed by the American Joint Committee on Cancer (AJCC, 7th edition) . All patients were followed up telephonically using outpatient records.

Immunohistochemistry
Formalin-xed para n embedded tissue sections were employed in each case using a standard protocol.
HE-stained sections (4 µm thickness) were re-examined to evaluate the tumor's histological features and immunohistochemistry were performed with Elivision technique. Antibody details are given in Table 1.
The threshold used for positive ER and PR expression was any nuclear labeling 1% or higher 4 . HER-2 immunoreactivity was evaluated on a standardized scale from 0-3 based on the intensity of staining of the cell membrane and the proportion of invasive tumor cells stained. Strong complete staining of the membrane in >10% of tumor cells (score, 3+) was considered positive. Intensity patterns with scores 0-1+ were considered negative, and samples scored as 2+ were further assessed by FISH test. Fluorescence in situ hybridization ratio of 2.0 or more was considered positive for HER-2 gene ampli cation 5 .

Clinicopathological characteristics
The clinicopathological characteristics of 27 NENB are summarized in Table 2. All were female patients,the median age was 64 years.All patients were presented with a palpable, painless lump in the breast.The tumors were located in the left breast in 18 cases(66.7 %) and in the right breast in 9 cases(33.3%). Tumor sizes were determined by gross pathological examination and ranged from 1.  In general observation, 15 cases of tumors were well-de ned, expansive growing.The cut surface was gray and white, in which 7 cases were partially or mostly gray and red. The other 12 cases, the boundary was unclear and gray-yellow necrotic areas were visible.
Under microscope,the tumor cells of well-differentiated NET were arranged in solid island, trabecular shape or ribbon shape with low to intermediate nuclear grade. Blood sinusoids can be seen between the tumor cell nests. The tumor cells were of medium size and relatively uniform in shape. The nucleus were single, round and had smooth or irregular borders. The cytoplasm were rich and some were more transparent. The mitotic gures were rare.
Poorly differentiated NEC were in ltrative with densely packed hyperchromatic cells with scant cytoplasm. the tumor cells were ovoid to spindled and showed dense chromatin with crush artifact.
Mitotic gures were abundant. Primary small cell carcinoma of the breast was histologically similar to lung counterpart, characterized by densely packed hyperchromatic cells with scant cytoplasm (Figure 1).  Figure  2).
11cases (40.7%) were well-differentiated NETs and 16 cases (59.3%) were poorly differentiated NEC.In NETs the positive rate of ER was 10/11(90.9%) ,while in NEC the positive rate of ER was 9/16(56.3%) . On the basis of immunophenotypes,most of NENBs were of the luminal molecular subtype ,6 cases were luminalA and 15cases were luminal B ,6 cases were TNBC and had no HER-2 overexpression subtypes.

Treatment modalities
Currently there is no standard therapy for NENB,most treatments are similar to the treatment of invasive carcinomas, no special type(NST). Surgery was the rst-line therapy,followed by chemotherapy and endocrine therapy.In our group all patients received surgical treatment. The most common surgical procedure was modi ed radical mastectomy (MRM), which was performed in 21 patients,4 cases received simple resection and sentinel lymph node biopsy.2 cases underwent puncture diagnosis and neoadjuvant chemotherapy plus radical mastectomy. All cases had received chemotherapy, 14 cases had received radiotherapy and 19 cases with positive ER expression had received adjuvant hormonal therapy.
Outcome, recurrence and prognosis All patients were followed up until February2020, the median follow-up time was 76 months (range, 39-87 months).In well diferentiated NET group(H), 4 cases developed disease progression, of them one patient had local recurrence and survived long-term after treatment, 3 cases had distant metastasis(1, lung metastasis; 1, liver metastasis; and 1, multiple metastasis with lung, liver ,brain).In poorly diferentiated NEC group(L), 12 patients had disease progression, of them 4 patients had local recurrence:2 cases survived long-term after treatment and 2 cases had progressed to distant metastasis after treatment.8 patients had distant metastasis (1, lung metastasis; 2, liver metastasis; and 5, multiple metastasis with lung, liver ,brain,bone).For 5-year DFS, the well diferentiated NET group was 63.6% and poorly diferentiated NEC group was 25%. There was a statistical difference between the two groups (P=0.033).For 5-year OS the well diferentiated NET group(72.7%) was also signi cantly better than poorly diferentiated NEC group (37.5%) (P=0.046). (Figure 3)

Discussion
Neuroendocrine differentiation in BC has been a matter of discussion since it was rst described almost 60 years ago. Indeed NENB is a less well-de ned group than analogous entity in other organ system, such as lung and gastroenteropancreatic tract.Various de nitions and criteria on NENB have been used 6-8 . So the reported frequency of NENB and BC with neuroendocrine differentiation ranges from approximately 0.3%-30%.
In 2019, The 5th Edition WHO updated neuroendocrine neoplasms of breast again.Key change was classi cation of neuroendocrine neoplasms into NET and NEC and exclusion of special histologic types (solid papillary carcinoma and hypercellular variant of mucinous carcinoma) and the inclusion of large cell neuroendocrine carcinoma.The 5th edition classi cation was based on an expert consensus from the International Agency for Research on Cancer (IARC) and WHO, whose aim was to adapt a "common classi cation framework" to standardize the categorization of neuroendocrine tumors across all anatomic sites 9 .
Neuroendocrine expression is common in invasive breast carcinoma NST.With typical morphological characteristics and expression of neuroendocrine markers can the BC be classi ed as NENBs.The 5th Edition WHO recommends classifying invasive carcinomas with <10% neuroendocrine morphology as invasive carcinoma NST and those with >90% as NET or NEC.
Our cohort strictly followed 2019 WHO criteria and each case was reviewed by two senior pathologists. NET diagnosis was referred to neoplasms with typical solid nests or trabeculae of spindle or polygonal cells, separated by fbrovascular stroma. NEC was composed either of small cells with extensive necrosis, uniform small hyperchromatic cells with high nuclear or cytoplasm ratio or large cells with evident cytoplasm and highly pleomorphic nuclei ,morphologically resembling pulmonary high-grade NEC.At least one extensive positivity of NE marker was required to con rm the morphological diagnosis.In our actual case selection process, we found that the classi cation standard was not very clear in some respect, especially for highly differentiated NETs. The grading system used for NENB was the Nottingham system and not the proliferative grade proposed for GEP and pulmonary NETs (neither with Ki67 proliferative index nor with mitotic index).At this point, breast NET cannot t well with gastrointestinal NET.
Immunohistochemical markers most commonly used to demonstrate neuroendocrine expression include Syn, CgA and CD56. Syn and CgA are speci c markers, while CD56 is sensitive and less speci c.A novel marker called insulinoma-associated protein 1 (INSM1) has been identifed in insulinoma tissue 10

and subsequently detected in different NE human cells and tumors. INSM1 is strongly expressed in most
NETs with a specifc nuclear staining 11 . In our group,INSM1was positively expressed in 23 of the 27 cases,Both sensitivity and speci city are relatively strong.Practice proved that the combination of INSM1 is an effective supplement and improvement for the traditional neuroendocrine markers.
90.9% (10/11) NETs show positive expression of estrogen receptor (ER) with variable rates of progesterone receptor (PR) and 56.3% (9/16)NEC show ER expression .All the cases are typically HER2negative.Ki-67 labeling index is high (>90%) in small cell and large cell neuroendocrine carcinoma. Molecular classi cation is the basis for breast cancer clinical treatment.In our cohort, most of NENBs were of the luminal molecular subtype ,In 11 NETs,6 cases were luminalA and 5cases were luminalB ,In 16 NECs, 10cases were luminal B and 6 cases were TNBC and had no HER-2 overexpression subtype.
Research has shown that NENB is genetically heterogenous and harbor molecular alterations that differ from invasive carcinoma NST 12 . Targeted sequencing analysis of NENBs revealed FOXA1, TBX3, GATA3, and ARID1A as the most frequently mutated genes 13  anatomic locations 20 . The genetic differences observed between breast NETs and NECs suggest that these entities might not be part of the same spectrum, but rather that they might arise through different molecular mechanisms 21 .
The most important differential diagnosis for neuroendocrine tumors in breast is metastatic tumor. There is considerable morphologic overlap between primary breast neuroendocrine tumors and those from other sites.we should have detailed medical history and comprehensive clinical information in our diagnosis and choose a series of immunohistochemical antibodies to help diagnosis and differential diagnosis.The positive expression of ER,PR,mamaglobin,GCDFP-15 and GATA3 and lack of expression speci c to other sites of origin such as CDX2 and TTF-1 support primary breast NENB .We must remind us that neuroendocrine morphology and immunophenotype in the absence of ER expression and of in situ or invasive non-neuroendocrine carcinoma of the breast should drive the attention to the possibility of a metastasis and prompt the search for the primary site.
There is no standard therapy for NENB yet.In practical work,molecular typing,clinical staging and histological grading remain the main prognostic factors for NENB. Most treatments of NENB are similar to the treatment of invasive carcinoma NST, with surgery as the rst-line therapy, followed by chemotherapy and endocrine therapy 22 .From our data, it can be seen that the prognosis of poorly differentiated NEC was worse than that of well differentiated NET. In terms of both overall and diseasefree survival, the poorly differentiated neuroendocrine nature of the neoplasm is an independent prognostic factor. As for advanced disease, some cases have been treated with platinum and etoposidebased regimens, in analogy with lung NEC, whereas in others a taxanes and anthracycline-based chemotherapy therapy has been attempted, but no consensus on a standard treatment has been reached, due to the small number of cases 23 .

Conclusion
Considering the low frequency of NENB, there is limited knowledge on the clinical presentation and management of this neoplasm. Further understanding of the molecular mechanism of breast neuroendocrine neoplasms will aid in the treatment and prognostication of these tumors.  Disease-free survival curves(A) and Overall survival curves (B) between well diferentiated NET group(H) and poorly diferentiated NEC group(L).