Clinical Utility of Endoscopic Ultrasound-Guided Fine-Needle Aspiration Biopsy In Mixed Ductal-Neuroendocrine Neoplasms of The Pancreas

Background: Endoscopic ultrasound-guided ne needle aspiration (EUS-FNA) biopsy is the rst line diagnostic tool used in the evaluation of solid pancreatic masses with high sensitivity and specicity prior to the surgery. Mixed ductal-neuroendocrine neoplasms of the pancreas are rare entity consisting of both ductal adenocarcinoma and neuroendocrine neoplasms, with each component representing at least 30%. Methods: We performed a retrospective analysis of patients with a diagnosis of pancreatic mixed ductal adenocarcinoma and neuroendocrine neoplasms by EUS-FNA specimens at Peking Union Medical College Hospital between 2010-2019 and presented a review of the literature. Results: A total of 3 cases (1 male and 2 females) were evaluated, aged 46, 38 and 62 years, and presented with 3-cm, 2.3-cm and 1.8-cm pancreatic masses, respectively. All the patients presented with abdominal pain and underwent EUS-FNA biopsy. FNA smears revealed a mixture of ductal adenocarcinoma and neuroendocrine neoplasms, identied by immunohistochemistry. According to Ki-67 proliferation index and mitotic count, the tumor grades of the neuroendocrine component were classied as NET G2 in 2 cases (cases 2 and 3) and NEC in 1 (case 1). All of the patients received adjuvant therapy. The diagnosis of patient 1 was conrmed on surgical excision, and she died 5 months after surgery due to hepatic metastases. The other two patients did not undergo surgery, but achieved long – term survival of 3 and 5 years, respectively. To date, 34 cases of pancreatic mixed ductal adenocarcinoma and neuroendocrine neoplasms have been reported in the English language literature including our present three cases. Among them, 5 of 12 patients who underwent preoperative biopsy were diagnosed as combined ductal adenocarcinoma and neuroendocrine neoplasms, of which 3 surgical resection specimens available were identied as mixed ductal-neuroendocrine neoplasms. Conclusion: We suggest that EUS-FNA biopsy of two in (1) dispersed single plasmacytoid cells with eosinophilic cytoplasm, with round to oval nuclei, prominent nuclear molding, ‘salt-and-pepper’ chromatin, and nely granular cytoplasm. Immunohistochemistry staining revealed diffuse positivity for synaptophysin and chromogranin A. Focal weak cytokeratin positivity was seen. (2) A minor component abortive glandular structures of cells with round to oval nuclei, coarse chromatin, nucleoli, and relatively rich cytoplasm with scant intracytoplasmic mucin, which were immunohistochemically positive for cytokeratin but for of primary ductal mitotic Ki-67 There male (21/34, 61.8 %) and patient age ranged from 21 to 76 years (median age 63 years). The tumors were mostly located in the head of the pancreas (18/33, 54.5 %), with a mean size of 4.1cm in greatest dimension (range, 1.0–19.0 cm). The most common symptom was abdominal pain (13/33, 39.4%) and jaundice (13/33, 39.4 %), followed by weight loss (8/33, 24.2 %). Most of tumors were non-functional (26/33, 78.8%) according to the symptoms and blood test results. Preoperative cytological or biopsy examination of ERCP (Endoscopic retrograde cholangiopancreatography)/EUS-FNA was performed in 12 patients, among which 5 exhibited mixed morphologic features, 6 with a pure adenocarcinoma appearance, and 1 with a pure neuroendocrine appearance. Ten surgical resection specimens available showed 30% (3/10) correlation between preoperative cytology/histology and postoperative pathology. Furthermore, a review of ERCP/EUS-FNA smear of cases 26 and 29 showed a mixture of the two components.


Background
Mixed tumors exhibiting both exocrine and neuroendocrine features can originate in all organs. The mixed adeno-neuroendocrine carcinoma (MANEC) was rst introduced in the World Health Organization (WHO) 2010 classi cation of digestive system and has been renamed as mixed neuroendocrine non-neuroendocrine neoplasms (MiNENs) in the WHO 2017 classi cation of pancreatic neuroendocrine neoplasms (NENs) [1].
MiNENs are composite neoplasms containing both neuroendocrine and non-neuroendocrine component, each comprising at least 30% of the tumor volume [1,2].
The pancreas is an organ composed of endocrine and exocrine (acinar and ductal) components [3]. Pancreatic MiNENs are extremely rare, which comprise 0.2% of all pancreatic neoplasms and 3.0% of pancreatic NENs [4]. The most common subtype is mixed acinar-neuroendocrine carcinoma [5]. To our knowledge, only 31 cases of pancreatic mixed ductal-neuroendocrine neoplasms have been published in the English literature [3,.
The cases were mostly identi ed by surgical specimens, with 3 of them detected incidentally during autopsy [10,13,17]. Furthermore, twelve cases were performed by preoperative biopsy, only 2 cases were accurately diagnosed both ductal and neuroendocrine components [21,33].
Endoscopic ultrasound guided ne needle aspiration (EUS-FNA) biopsy has been widely used and become the most effective technique for preoperative evaluation of pancreatic tumors. However, the use of EUS-FNA to help the diagnosis of pancreatic MiNENs is limited, since such condition is extremely unusual [5,34]. The rarity of these neoplasms and the limitation of biopsy samples are obstacles to diagnosis and treatment.
As the treatment depends on morphological diagnosis, cytological and biopsy analysis via EUS-FNA are particularly important [29,[35][36][37]. Here, we report three cases of pancreatic mixed ductal adenocarcinoma and neuroendocrine neoplasms diagnosed by preoperative EUS-FNA biopsy, one of which was veri ed as mixed ductal-neuroendocrine carcinoma by operation. We also presented a literature review and focused on the correlation between preoperative cytology and subsequent surgical pathology.

Methods
The pathology records over a 10-year period (2010-2019) were retrieved from the archive les of the Department of Pathology, Peking Union Medical College Hospital. Three patients with mixed pancreatic ductal adenocarcinoma and neuroendocrine neoplasms diagnosed by preoperative EUS-FNA were selected. Hematoxylin and eosin (H&E) slides and immunohistochemistry (IHC) results were reviewed by two expert pathologists. The clinical and follow-up data were collected from the medical records or telephone inquiry. Patient data was analyzed to the last follow-up before February 15, 2020. Patients were enrolled after providing oral consent, and this study was approved by the Institutional Review Board of Peking Union Medical College Hospital.
All EUS-FNAs were performed using 22-gauge needles (Echotip, Wilson-Cook, and Winston-Salem, NC). The aspirated material was smeared onto 6-10 glass slides and immediately xed in 95% ethanol for Hematoxylin and eosin (H&E) staining. Additionally, the subsequent materials from cases 1 and 2 were formalin-xed and para n-embedded for cell blocks. Patient 1 underwent surgical resection after 6 cycles of neoadjuvant chemotherapy.
The specimens of the surgery were xed in 10% neutral formalin and processed by routine histology techniques.

Clinicopathological Features
The clinical and pathological data of the 3 cases are summarized in Table 1. 1 male and 2 females were evaluated, aged 46, 38 and 62 years, respectively. All of the patients presented with abdominal pain at admission. Ultrasonography of the abdomen, contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) showed the pancreatic solid mass in the head in 2 cases and in the body in 1, with a diameter of 2.3-cm, 2.5-cm and 3-cm, respectively (Fig. 1). The image also demonstrated the presence of multiple hepatic metastases in 2 cases (cases 1 and 2) and extensive vascular vascular invasion in 1 (case 3).  The clinical features were shown in Table 1. All of the three patients were not candidates for surgery, so they received systemic treatment targeting the neuroendocrine component. Patient 2 received chemotherapy with octreotide acetate and molecular -targeted drug (everolimus) with inhibitory effect on tumor growth and had no sign of disease progression 3 years later after diagnosis. Patient 3 received chemotherapy treatment with gemcitabine hydrochloride (1000mg/m2) and tegafur (60mg D1-D14) followed by local radiation and was still alive with no disease progression 5 years later at the last follow-up.
After six cycles of chemotherapy using VP-16 (140mg D1-D3) and cisplatin (40mg D1, 30mg D2-D3), the patient of case 1 underwent pancreatoduodenectomy with regional lymph node dissection and partial hepatectomy. Grossly, the pancreas revealed an ill-de ned, hard and grey mass within the head invading duodenal wall. Microscopically, the tumor showed a moderate differentiated adenocarcinoma and a poorly differentiated neuroendocrine carcinoma (NEC) intermingling together (Fig. 4a). The adenocarcinoma exhibited tubular growth pattern with positive expression of epithelial markers (cytokeratin, CK7 and CK19), while negative expression of neuroendocrine markers (synaptophysin and chromogranin A). The NEC cells were arranged in irregular sheets with amphophilic cytoplasm, prominent nucleoli and a high mitotic rate (30/10 HPFs). Comedo-like necrosis and geographic necrosis were seen in NEC component. The "organoid" structure and pseudorosette pattern was rarely seen. NEC component occupied approximately 60% of the lesion, which immunoreactive to synaptophysin, chromogranin A (Fig. 4b) and partly immunoreactive to cytokeratin but negative to CK7 or CK19. Ki-67 index was about 85%. Four of 24 dissected regional lymph nodes and the liver were involved. The liver and 2 involved lymph nodes were positive for metastatic neuroendocrine carcinoma only (Fig. 4c). The other 2 lymph nodes were in ltrated by a mixture of adenocarcinoma and neuroendocrine carcinoma (Fig. 4d). The patient received six cycles of adjuvant gemcitabine hydrochloride (1200mg D1-D8) and cisplatin (40mg D1-D8) therapy and died due to diffuse hepatic metastases 5 months after operation.
Total analysis of thirty-four cases reported in the literature (including our present three) Thirty-four cases of mixed ductal neuroendocrine neoplasms of the pancreas have been identi ed, including three cases reported herein ( showed a mixture of the two components.

Discussion
Mixed ductal-neuroendocrine neoplasms of pancreas are de ned as tumors composing of ductal adenocarcinomas and neuroendocrine neoplasms, with each part accounting for at least 30%. To date, the nal pathological diagnosis relies mainly on postoperative specimen. To our knowledge, this was the largest cohort worldwide of pancreatic mixed ductal -neuroendocrine neoplasms diagnosed by EUS-FNA specimen.
EUS-FNA is often the rst-line diagnostic tool used in the evaluation of solid pancreatic masses with a reported sensitivity of 85%-95%, speci city of 95%-98%, and diagnostic accuracy of 78%-95% [38,39]. In general, the cytomorphologic feature of adenocarcinomas and NENs is easy to distinguish, however, the accurate cytological diagnosis of mixed tumors is limited by a potential sampling bias and overlapping the cytological characteristics, that should be taken a particular caution [5,[40][41][42]. Immunohistochemical stain should be considered when encountering a tumor showing mixed morphological features, particularly when the two components are poorly differentiated [40,43]. The immunohistochemical markers of ductal differentiation include CK-7, CK-8, CK-18, CK-19, CA19-9, CEA, CA125, B72.3, MUC1 and MUC5AC, whereas neuroendocrine differentiation is identi ed by expression of synaptophysin, chromogranin A, CD56 and hormone production [2]. Tian et al. [44] reviewed 10 cases of pancreatic MiNENs in which an EUS-FNA and surgical treatment were performed, only 3 (30%) were accurately diagnosed by biopsy. Similarly, Sullivan et al. [5] revealed that only 2 out of 9 patients (22.2%) of mixed acinar-endocrine carcinoma were accurately diagnosed by EUS-FNA specimens before surgical resection. Of the previous reported cases of mixed ductal -neuroendocrine neoplasms, only 2 cases had two components observed in preoperative biopsy specimens [21,33] [46,47]. As most of the cases with a high grade neuroendocrine component, it is usually treated as pure NEC [47]. Kaji et al. [37] expressed that the NEC-targeted chemotherapy for MiNENs had been effective. EP (etoposide and cisplatin) therapy has been the most widely used in GEP NEC, which is typically followed by the treatment of the small-cell lung cancer [48]. Other drugs including oxaliplatin, temozolomide, and gemcitabine can also be assessed in mixed neuroendocrine with non-neuroendocrine component [49]. It has been reported that the molecular targeted drugs such as everolimus and sunitinib showing signi cantly prolonged progression -free survival among patients with advanced PanNETs [50]. In our cases, all of three patients targeted the predominant NENs component for treatment. Patients 2 and 3 achieved long-term survival with unresectable pancreatic tumor, which are far longer than the median overall survival of the current cohort. It suggests that our treatment strategy has been effective. hence, if the neuroendocrine component of these tumors was classi ed as NET G2, we propose appropriate adjuvant therapy rather than surgery.
The pathogenesis of MiNENs remains controversial and current molecular/genetic studies showed that the two components may have a common monoclonal origin [51][52][53]. In addition, the neuroendocrine components usually have a higher number of aberrations and a higher allele imbalance than non-neuroendocrine components, suggesting a more aggressive tumor biology [45]. The prognosis of MiNENs is not well known and the median overall survival (OS) ranging between 10.5 and 78 months [45]. Some scholars believed that the survival outcome of MiNENs was close to that of pure GEP-NEC, and it was more obvious in the advanced stage [54][55][56]. Similarly, Harada et al. [57] reported that the NEC component de ned the prognosis, which was involved in most of the lymph node metastasis and vascular as well as perineural invasion in patients with MiNENs. In our study, the median OS of the reported cases of pancreatic mixed ductal -neuroendocrine neoplasms was 10 months, similar to that of panNEC [58].
In summary, we suggest that EUS-FNA is the most effective tool for diagnosis and subsequent guidance of therapy. It is di cult to diagnose mixed ductal-neuroendocrine neoplasms by EUS-FNA biopsy, however, it can identify mixed morphology and has high sensitivity. If the tumor grades of the neuroendocrine component were classi ed as NET G2 by EUS-FNA biopsy, we propose appropriate adjuvant therapy rather than surgery.