In all localized MiNENs, curative-intent surgery is recommended as the first treatment of choice, if available. Even in high-grade MiNENs, because of their less aggressive behavior than pure NECs, tumors with an acinar component of the pancreas belonging to this category have benefited from such treatment[48-50]. In the same group, although combinations of etoposide (VP16) and platinum salt or a combination of 5-fluorouracil (5FU) with irinotecan (IRI)- or oxaliplatin (OX)-based preoperative and postoperative chemotherapy have been used in tumor management similar to PDNEC therapy, the role of adjuvant therapy is still not completely defined[44,51-53]. Moreover, in the intermediate group, recent studies suggest replacing this therapy with a combination of 5FU and IRI and/or OX or gemcitabine (GEM) and/or OX for treatment parallel to the chemotherapy applied to adenocarcinomas, and in some cases, radiotherapy has been used as a treatment option in addition to chemotherapy[3,54-57].

Unfortunately, extensive surgery does not seem to be an option in this group because the risk outweighs the benefits, especially in high-grade MiNENs. Therefore, systemic chemotherapy is generally performed according to the type present at metastatic sites. In patients with both components diagnosed either at the metastatic site or the primary tumor, therapy is based on the most aggressive component[3,54-57]. Since some intermediate-grade MiNENs with predominant NET components frequently express type 2 somatostatin receptors, such cases may also benefit from long-acting somatostatin analogs and peptide irradiation nucleotide therapy[58-60]. Although MANET is not categorized as MiNEN, its neuroendocrine component can metastasize. Therefore, pure NET-based chemotherapy and peptide irradiation nucleotide therapy are recommended for their treatment. At present, Akt/mTOR mutations have not been investigated in MiNENs. However, some patients who benefit from everolimus have been described[61].

Despite all these findings, the fact that further studies in large series are needed to define the treatment of patients more precisely with MiNEN should not be overlooked. Future studies aiming to determine the molecular vulnerability of both components in MiNEN cases in the GIS diagnosed based on criteria recommended for the diagnosis of these tumors may allow for the development of targeted therapies against both components and improve their treatment.

The treatment approach in MiNENs in both groups is briefly presented in Tables 3 and 4.

Recently, a comprehensive systematic review and a multicenter study performed by Frizziero et al[3,15] showed that esophageal MiNENs accounted for between 5.9% and 15.9% of all GIS MiNENs. These tumors, which account for approximately one-quarter of the NENs observed in this localization, show an apparent male predominance and are observed at advanced ages (6th decade) in the distal third of the esophagus[2,19,62]. Although the tumor consists of NEC and squamous cell carcinoma (SCC) in most cases, there are rare adenocarcinoma cases, particularly adenocarcinoma, in the background of Barrett's esophagus[40,63,64]. Molecular data indicate their monoclonal origin, as demonstrated by LOH, RB1, TP53, and alterations in TP63, SOX2, DVL3, PTEN, PIK3A, and KRAS[40]. However, the monoclonal origin of esophageal MiNEN with a nonendocrine component composed of adenocarcinoma on the background of Barrett’s metaplasia remains to be elucidated, and some authors postulate that this group reflects a true collision tumor rather than MiNEN[64].

In the esophagus, because the discrimination of basaloid SCC and small-cell NEC (SNEC) has paramount importance and may pose a diagnostic pitfall in routine microscopic evaluation, immunohistochemical staining for high molecular weight cytokeratins, p63, and p40, is highly recommended to discriminate basaloid SCC from SCNEC[17].

Unfortunately, their lower metastatic capacity (25% vs 54%) and longer survival time (28 vs 15 mo) relative to pure PDNEC do not change their poor prognosis[65]. Another important finding is the predictive role of the Ki-67 proliferation index of NEC for prognosis[15,66]. More recently, any statistically significant difference in OS between gastroesophageal GEP MiNEN vs colorectal MiNEN was detected[66].

These tumors constitute 6%–20% of MiNENs located in the GIS, and 7% of NENs are located in the stomach[14,15,67]. Similar to those in the esophagus, stomach tumors are observed in elderly patients (5^th-6^th decades) and show a male predominance. Based on the macroscopic appearance of these tumors, which are observed equally in the corpus and antrum of the stomach, they are not different from adenocarcinomas, and specific diagnostic findings on endoscopy have not been obtained[19]. Most of these aggressive tumors consist of well-differentiated adenocarcinomas, and the PDNEC component is mainly located deeper in the organ[68,69]. Although cases composed of adenocarcinoma and NETs have been recorded, it is suggested that the term MANEC can be retained for MiNEN at this location[19]. MiNENs composed of gastric NEN and adenocarcinoma have been described in the setting of chronic atrophic gastritis, etiological factors have not yet been entirely identified[70]. Recent molecular studies indicated a monoclonal origin[37,44,71]. Ishida et al[72] compared the molecular pathology of poorly differentiated NEC and MiNEN of the stomach by whole-exome sequencing. The analysis revealed recurrent mutations in 62% of TP53 cases, and they were more frequent in MiNENs than in NECs. Frameshift mutations of APC were observed in two MiNEN cases. In cases of MiNEN, two histological components shared mutations in TP53, APC, and ZNF521, whereas alterations in CTNNB1, KMT2C, PTEN, and SPEN were observed in neuroendocrine components only. They concluded that TP53 is a single, frequently mutated gene in gastric NEC and MiNEN, and alterations in other genes are less common, thus resembling the mutation profiles of gastric adenocarcinomas. Another interesting previous finding is the presence of ATRX gene mutations (primary partial loss) in 37% of cases involving a substantial proportion of gastric MiNEN[73]. However, these findings should be investigated in further studies. Gastric MiNENs are tumors with a poor prognosis that show lymph node and distant metastases at the time of initial diagnosis, and the prognosis is slightly better than that of pure PDNEC[15,74,75]. Similar to these findings, in a recent study including 401 patients, the 5-year disease-free survival was 51.1%, which was significantly better than that of NEC (47,6%) and worse than that of adenocarcinoma (57,8%). Furthermore, in the same series, advanced stages and lymph node metastasis were independent risk factors related to distant recurrence[76].

MiNENs of the small intestines frequently in the duodenum, where they are mainly located in the ampulla[77-81]. MiNENs of the jejunum and ileum are exceedingly rare, similar to PDNECs encountered in these regions[19]. They are equally observed in both sexes and older patients. While adenocarcinoma constitutes the nonneuroendocrine component of tumors in many cases, rare cases of SCC have also been recorded[77,78]. Since the neuroendocrine component is frequently located in the deeper part of the intestinal wall, they are frequently diagnosed as adenocarcinoma from biopsies[81]. The histological subtype of adenocarcinoma forming these tumors was also found to be associated with tumor behavior. MiNENs with intestinal-type adenocarcinoma have a better prognosis than those with the pancreaticobiliary subtype[20,25]. However, ampullary MiNENs are aggressive tumors with a poor prognosis and generally present at advanced stages[15,17]. This finding contrasts with MiNEN in the other part of the duodenum, which combine intestinal-phenotype adenocarcinoma and a well-differentiated somatostatin-secreting NET[20]. They are mostly superficial and not highly aggressive, and distant metastasis is rare.

The last WHO classification of MiNEN indicated that these tumors were composed of two morphologically recognizable components generally represented by adenocarcinoma and NEC, and this classification has provided knowledge about mixed tumors in the appendix[18]. The exclusion of goblet cell carcinoids, which are currently determined to be amphicrine tumors, from the MiNEN group has led to the limited applicability of past clinicopathological data on these tumors located in the appendix, thus necessitating further evaluation of the findings related to this group[29]. A few studies conducted in the recent past indicate that MiNENs constitute 10% of malignancies at this location. On the other hand, a systematic review showed that among the lower gastrointestinal tract organs, these tumors were most frequently (60.3%) localized in the appendix[82]. An interesting finding is that the age-adjusted incidence for MiNENs increased from 0.01/100000 person-years to 0.07/100000 person-years (range 2004-2016), with an annual percentage change (APC) of 13.8%[83]. This finding can be attributed to the increase in clinical recognition and better diagnostic technologies over the years. They are observed in advanced age (between 58-60 years) mostly encountered incidentally and discovered at advanced stages[56,83,84]. Although recent studies indicate that these tumors do not show sex predilection, new findings that APC shows significant differences according to sex (13.81% in females vs 12.24% for males) need to be clarified[83].

Their overall survival rate is 6.5 years, which is better than that of signet ring cell carcinomas (2.1 years) and worse than that of goblet cell adenocarcinomas (13.8 years) and pure NETs (39.4 years)[56,84]. In a more recent study, the prognosis of 315 patients with MiNEN was compared with that of other histological subtypes in the appendix, including NETs, NECs, goblet cell carcinoma, signet ring cell carcinoma, mucinous adenocarcinoma and nonmucinous adenocarcinoma, based on the surveillance, epidemiology and end results program 18 registries. The overall 5-year survival rate was 57.4%, and the level of invasion was the only independent factor influencing tumor behavior. In addition, multivariate analysis demonstrated that the prognosis of MiNENs was worse than that of NETs, NECs, goblet cell carcinoma, and mucinous adenocarcinoma but better than that of nonmucinous adenocarcinoma and signet ring cell carcinoma[83].

Their pathological differential diagnosis should include goblet cell carcinomas, tubular-type carcinoids and pure NETs with glandular configurations.

Tumors in this region constitute more than half of all MiNENs of the GIS[67,85]. In particular, evidence has shown that they are observed more frequently than pure NETs in resections performed in this region[85]. Their distribution among NENs is 14-20% and 1%-3% in the colon and rectum, respectively. They are more common in men than women and observed at an advanced age (6^th decade)[25,67,85]. Although they do not have specific clinical findings, they have been defined in the background of inflammatory bowel diseases[86-89]. As a result of their more frequent detection in these regions, the number of molecular studies performed in MiNEN exceeds that of other localizations, and they have provided important data regarding their pathogenesis. The same genetic alterations in both components strongly support their monoclonal origin from a common precursor progenitor cell[33,35,37,38,42]. Parallel to these findings, a recent case showed that in addition to microsatellite instability due to MLH1 promoter methylation, the same mutations affecting the ARID1A, ASXL1, BLM, and RNF43 genes occur in both components, as determined by a multigene next-generation sequencing panel. On the other hand, BRCA2 has been explicitly altered in the neuroendocrine area. Although the latter observation suggested that BRCA2 could be a potential new target for MiNEN, the lack of this alteration in the nonneuroendocrine part of the tumor requires further consideration concerning intratumor heterogeneity[90].

Macroscopically, these tumors form masses (average 5 cm) without distinguishing features from adenocarcinomas. Histologically, most cases are composed of adenocarcinoma and NEC. Rare cases in which the nonneuroendocrine component consisted of squamous cell carcinoma have also been noted[43,91]. If the neuroendocrine component consists of PDNEC, it is consistently observed in metastases. In comparison, adenocarcinomas are observed in one-third of cases[47]. Such tumors show aggressive behavior and have poor prognosis (overall survival: 12.2 mo) according to the Ki67 proliferative index of the NEC component as well as the MSI status and stage[42,43,67]. Recently, a systematic review demonstrated that if the neuroendocrine component consists of NETs, it is unknown which component will be encountered in metastatic sites because of a higher grade, and the predominance of one component does not warrant their presence in metastatic sites[47,92,93]. This finding emphasizes the complexity of MiNENs and the need for an accurate morphological description of all components. A recent systematic review also demonstrated that in MiNENs of the lower gastrointestinal tract, the site of origin in those with metastatic disease at diagnosis appeared to influence prognosis. The median survival time was 12.3 mo for those with primary colonic tumors vs 11.7 mo for those with primary anorectal tumors, with hazard ratios of 1.13 vs 0.80, respectively[82].

Pancreatic MiNENs are rare tumors in which the nonneuroendocrine component can be formed by ductal or acinar carcinoma[18]. In addition, tumors with a nonneuroendocrine part consisting of ductal and acinar carcinomas are extremely rare and defined as mixed ductal-acinar-neuroendocrine carcinomas[94]. Tumors with mixed ductal–neuroendocrine carcinoma are rare and account for approximately 0.5%–2% of all ductal adenocarcinomas and 5% of all NENs arising from this organ[27]. There is no sex predilection. Although the average age of onset is 68, they are observed in a wide age range, from 21 to 68 years old. They usually consist of NEC accompanying ductal adenocarcinoma[95-97]. They can be located anywhere in the organ and produce clinical symptoms similar to ductal adenocarcinoma without specific clinical findings. Because of the rare nature of these tumors, molecular data are scarce and limited[46,67,98]. Therefore, the diagnosis should be performed using adenocarcinoma and neuroendocrine tumor-specific markers separately and morphologically because they should be clearly distinguished from ductal adenocarcinomas with entrapped islets and NETs with entrapped ductules in the differential diagnosis[17,27]. In the former, the islands present an ovoid shape and have regular contours constituted by endocrine cells without atypia, and express all hormones, which is inconsistent with a predominant cell line of tumor cells; in the latter, however, the absence of atypia of ductal cells , the lack of aberrant P53 staining and the low proliferative index are essential clues in the differential diagnosis (Figure 2). An increase in the number and size of Langerhans islands that accompany chronic obstructive pancreatitis is another diagnostic pitfall in the differential diagnosis. Previous reports indicated that mixed ductal–neuroendocrine carcinomas are aggressive tumors with poor prognoses (5-year survival is 0%)[18,98]. More recently, lymph node metastasis was indicated as an adverse prognostic factor of disease-specific survival in 7 patients with mixed ductal–neuroendocrine carcinomas[99]. Similar findings were also observed by Zhang et al[92] in a larger number of patients. Although data for surgically resected cases are very limited in the literature, a cohort study reported that the median survival was 15.3 mo and all cases died due to disease[100]. However, in a recent study evaluating 8 cases with a median follow-up of 21 mo, the overall survival was 88 mo and the 5-year OS was 58%. In addition, the survival of these tumors was better than that of pancreatic ductal adenocarcinomas; thus, further investigation is warranted[101].

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Figure 2 Histopathological pitfalls in the diagnosis of mixed neuroendocrine-nonneuroendocrine neoplasms of the pancreas. A: A ductal adenocarcinoma of the pancreas surrounding and invading an islet in the background of chronic pancreatitis (x 200). The islet has regular contours despite an invasion; B: A neuroendocrine tumor of the pancreas with entrapped two ductulus without atypia. Such areas should be evaluated carefully to avoid a misdiagnosis of mixed neuroendocrine-nonneuroendocrine neoplasms (x 200).

Cases in which the neuroendocrine component consists of NETs have been reported, although their morphology resembles true collision tumors and their monoclonal origin remains to be proven[95,96].

Mixed acinar–neuroendocrine carcinomas are rare and account for nearly one-fifth of all pancreatic ACCs[49,94]. The features of these tumors do not differ from the macroscopic features of ACC, and the tumors are quite large. In the differential diagnosis, morphological and IHC findings should be evaluated together, such as in ductal carcinoma. It is worth noting that 20%-30% of ACCs show a small neuroendocrine cell population, and this morphologically undetected component should not lead to the diagnosis of MiNEN[49]. Immunohistochemical staining with trypsin and bcl-10 [monoclonal antibody directed against the C-terminal portion of bcl-10 (clone 331.3)] is very useful for identifying the acinar component[102] (Figure 3). However, it should be kept in mind that a significant portion of the ACC is stained with synaptophysin.

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Figure 3 Acinar carcinoma of the pancreas. A: The tumor is composed of cells that demonstrate the presence of monomorphic nuclei, sometimes forming minute lumens. Tumor cells are in a monolayer with basally located nuclei and have a granular eosinophilic cytoplasm (x 400); B: Bcl-10 expression with higher staining in the apical portion of tumor cells (x 400).

Moreover, since pure NETs of the pancreas have a better prognosis than these tumors, advanced immunohistochemical evaluations with bcl-10 and trypsin are recommended for all tumors with neuroendocrine-appearing pancreatic neoplasms that show a high mitotic index, abundant necrosis, and evident nucleoli[17]. Although these tumors seem to share the genetic changes observed in pure ACCs, they do not show characteristic mutations that can be found in pancreatic NETs[103,104].

A recent study suggested that c-MYC alterations are involved in mechanisms leading to the neuroendocrine differentiation of ACCs[46]. Surgical resection and tumor stage are the most important prognostic factors, and the reported 5-year survival rate is 30%–50% for patients who undergo surgery[49,105].

The nonneuroendocrine component of MiNENs is mostly hepatocellular carcinoma (HCC) and less frequently cholangiocellular carcinoma, and they are rare liver tumors encountered at advanced ages (43-84 years) predominantly in men[2,106,107]. The neuroendocrine components of these tumors are predominantly NECs, and they have a dismal prognosis, with many cases presenting distant metastasis at the time of diagnosis[2,108,109]. More recently, the 1-year cumulative survival rate of patients was reported to be 53%[107].Although the pathogenesis has not been fully elucidated; neuroendocrine differentiation from existing HCC has been suggested[108,109].

MiNENs of the gallbladder and biliary tract account for 10% of all biliary carcinomas and 2% of all hepatobiliary carcinomas[110,111]. Although considered rare, adenocarcinoma is detected in approximately 30% of NENs, particularly in the gallbladder, and these tumors constitute 35% of NENs in this region, thus indicating that they are more frequent than previously described[79,110]. While the age range is relatively wider than that of many MiNENs, these tumors are observed at an advanced age (mean: 65 years), which is similar to those in other regions of the GIS. However, compared with the male dominance observed in other MiNENs, these tumors are more common in females[18]. The close relationships between MiNENs and inflammatory diseases in these locations suggest that inflammation plays a role in pathogenesis. Recent findings indicate that the NEC component of the tumor is composed of large cell NECs in a great majority of cases (59%), and NECs are incidentally discovered during imaging studies without any specific clinical findings[112]. In parallel, specific findings that differ from the findings for adenocarcinomas on macroscopic examination have not been observed. Although a considerable portion of the tumors are confined to the gallbladder wall at the time of diagnosis, one-fifth of the cases have serosa and one-third have adjacent organ invasion[16]. Therefore, the rarity of distant metastases in these tumors does not exclude the possibility that half of them metastasize to neighboring organs (liver, peritoneum, lymph nodes) at the time of diagnosis. On histopathologic evaluation, the nonneuroendocrine component often consists of adenocarcinoma and rarely consists of squamous cell carcinoma or carcinoma with sarcomatous or osteosarcomatous differentiation[112,113]. Few cases with intracystic papillary neoplasms (IPNs) have also been reported[16]. The NEN component, which is usually more deeply located, often consists of NEC. Molecular studies support that MiNENs in this region also consist of a common precursor[16,114]. Although studies on IPN have indicated that the endocrine component originates from these areas, further studies are needed to support this finding. The one-year survival was four times higher in patients with organ-confined tumors than in those with distant metastases, revealing that distant metastasis is the most effective predictive parameter for the course of the disease, thus emphasizing the importance of staging[79,110]. A recent systemic review based on 53 studies to predict the clinicopathological features and prognosis of biliary MiNENs, including gallbladder MiNENs, showed a median overall survival time of 21 mo. In addition, radical resection and small morphological subtype were independent prognostic factors associated with higher overall survival, and radical resection (R0) and younger age (< 65 years) were associated with higher recurrence-free survival time[115].