Langenbecks Arch Surg (2011) 396:273–298 DOI 10.1007/s00423-011-0739-1 REVIEW ARTICLE The diversity and commonalities of gastroenteropancreatic neuroendocrine tumors Simon Schimmack & Bernhard Svejda & Benjamin Lawrence & Mark Kidd & Irvin M. Modlin Received: 3 January 2011 / Accepted: 7 January 2011 / Published online: 28 January 2011 # Springer-Verlag 2011 Abstract Background Recent data demonstrate that the incidence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has increased exponentially (overall ~500%) over the last three decades, thus refuting the erroneous concept of rarity. GEP-NETs comprise 2% of all malignancies and in terms of prevalence, are the second commonest gastrointestinal malignancy after colorectal cancer. Diagnosis is usually late since there is no biochemical screening test and symptoms are protean and overlooked. As a consequence, 60–80% exhibit metastases with a consequent suboptimal outcome. Discussion The gastrointestinal tract and pancreas exhibit ~17 different neuroendocrine cell types, but neither the cell of origin nor the biological basis of GEP-NETs is understood. This review examines GEP-NETs from the cellular and molecular perspective and addresses the distinct patterns of functional tumor biology pertinent to clinicians. Although grouped as a neoplastic entity (NETs), each lesion is derived from distinct cell precursors, produces specific bioactive products, exhibits distinct chromosomal abnormalities and somatic mutation events and has uniquely dissimilar clinical presentations. GEP-NETs demonstrate very different survival rates reflecting the intrinsic differences in malignant potential and variations in proliferative regulation. Apart from the Supported by NIH: DK080871 S. Schimmack : B. Svejda : B. Lawrence : M. Kidd : I. M. Modlin (*) Gastrointestinal Pathobiology Research Group, Department of Gastroenterological Surgery, Yale University School of Medicine, PO Box 208602, New Haven, CT, USA e-mail: imodlin@optonline.net S. Schimmack Visceral- and Transplantation-Surgery of Heidelberg, University Hospital of General-, Heidelberg, Germany identification of the inhibitory role of the somatostatin receptors, there is limited biological knowledge of the key regulators of proliferation and hence a paucity of successful targeted therapeutic agents. IGF-I, TGFβ and a variety of tyrosine kinases have been postulated as key regulatory elements; rigorous data is still required to define predictably effective and rational therapeutic strategy in an individual tumor. A critical issue in the clinical management of GEPNETs is the need to appreciate both the neuroendocrine commonalities of the disease as well as the unique characteristics of each tumor. The further acquisition of a detailed biological and molecular appreciation of GEP-NETs is vital to the development of effective management strategy. Keywords Chromogranin . Enterochromaffin . GEP-NET . Neuroendocrine . Serotonin . Somatostatin Introduction Although initially considered rare tumors, recent data indicate that the incidence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) (Fig. 1) has increased exponentially over the last three decades, and they are as common as myeloma, testicular cancer, and Hodgkin's lymphoma [1]. As such, they comprise 2% of all malignancies and in terms of prevalence, GEP-NETs represent the second commonest gastrointestinal malignancy after colorectal cancer [1]. The increase in incidence and prevalence most likely reflects improvement in disease awareness and diagnostic techniques [1]. GEP-NETs present a considerable diagnostic and therapeutic challenge since their clinical presentation is nonspecific. Diagnosis is usually therefore late in the natural history of the disease with metastases evident at presentation in 60–80% [2]. Most GEP-NETs are sporadic lesions, 274 Langenbecks Arch Surg (2011) 396:273–298 Fig. 1 Neuroendocrine cell types and tumors. Individual neuroendocrine cells (circle) and their associated tumors (rectangles) represent the most commonly clinically encountered gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Chromogranin A (CgA) positive staining (central immunohistochemical image) in a NET (red = Cy-5 labeled CgA, blue = DAPI (nuclear stain)) is the common denominator histopathology biomarker for NETs. The majority (>90%) of tumors express CgA but poorly differentiated lesions (NEC) may lose their neuroendocrine phenotype and be CgA-negative although some, especially pancreatic neuroendocrine tumors (pNETs), may occur as part of familial tumor syndromes such as multiple endocrine neoplasia type 1 (MEN1 syndrome), von Hippel-Lindau disease (VHL), neurofibromatosis type 1 (NF-1), and tuberous sclerosis (TSC) [3]. The origin of the cells from which GEP-NETs arise is not well understood. Overall, the gastrointestinal tract including the pancreas has at least 17 different neuroendocrine cell types (Fig. 2). The term “neuroendocrine” is a composite description of a cell type that exhibits mixed morphological and physiological attributes of both the neural and endocrine regulatory systems. The bicameral cell embraces the phenotypic relationship of tumor precursor cells to neural cells in the expression of certain proteins, such as synaptophysin, neuron-specific enolase, and chromogranin A (CgA) and the physiological secretory/regulatory role classically ascribed to endocrine cells [4]. Individual GEP-NETs usually originate from a neuroendocrine cell that is specific to a particular part of the gut or pancreas. In some circumstances, neuroendocrine cells may be part of complex lesions that have adenocarcinomatous elements and the precise lineage of such cells is unclear. This review addresses differences and distinct patterns of each GEP-NET, and focuses on the cell and organ of origin as well as the functionality of the tumor. ganglia, and the adrenal medulla) and diffusely distributed dispersed cells that constitute a disseminated system (diffuse neuroendocrine system (DNES)) and comprise at least 17 different cells. These cells, either individually or in aggregations, populate the skin, thyroid, lung, thymus, pancreas or gastrointestinal tract (Table 1), biliary tract, and urogenital tract; and are the largest group of hormone-producing cells in the body. In some organs (e.g., stomach, pancreas) the presence of a distinct neuroendocrine system provides a functional duality whereby an endocrine and an exocrine function are biologically assimilated. Thus, the acid pepsin digestive function of the oxyntic part of the stomach is intrinsically dependent upon the endocrine role of the gastrin-secreting antrum. Similarly, the digestive role of the pancreas is enmeshed with the glucose homeostatic role of the pancreatic islets embedded within the exocrine parenchyma of the pancreas. Presumably, such specialized regulatory cell collections represent biology in the process of transformation much as diffuse collections of sympathetic neurons evolved into para-renal endocrine organs (adrenals). There has been considerable and prolonged controversy and debate in respect of the developmental origin of gut neuroendocrine cells [5–7]. In the past, it was considered that the neuroendocrine cell system was based upon migration from the primitive neural crest to specific anatomical sites [8]. Currently the most accepted proposal is the “Unitarian Theory” of intestinal cytogenesis, which opines that gastrointestinal cell lineages are derived from a common stem-cell precursor, located in the base of intestinal crypts or in the neck region of gastric glands (Fig. 3) [9]. Recent studies of gastric and intestinal epithelia have identified that neuroendocrine cells are among the progeny of Neuroendocrine cell phenotypes: development and embryology Broadly speaking, the neuroendocrine cell system can be divided into two systems, namely aggregations of cells that constitute glands (the pituitary, the parathyroids, the para- Langenbecks Arch Surg (2011) 396:273–298 275 Fig. 2 Gut neuroendocrine cell morphology. Electron micrograph of an isolated EC cell demonstrates the typical admixture of electron dense and electron-luscent granules (inset) that characterize neuroendocrine secretory cells (top left). Neuroendocrine cells are largely distributed at the base of the gland (bottom left—yellow arrows: immunofluorescent CgA stain (FITC green), nuclei are blue (DAPI)). A higher magnification of the mucosa (demonstrates EC cells predominantly located at the gland periphery (brown DAB staining of CgA; gland cross section—top right). DAB staining of isolated, fixed EC cells exhibits long, axonal-like structures (bottom right) that “synapse” with either adjacent mucosal cells or neurons within the gut mucosa providing the neural phenotype component such multipotential stem cell [10–13]. It therefore seems likely that gastrointestinal neuroendocrine cells are derived from local tissue-specific stem cells, probably through a committed precursor cell. In the pancreas, it is thought that the common precursor cell resides within the ductal epithelium and that this structure provides the basis for the genesis of the pancreatic islets [14]. A second hypothesis suggests that islet neogenesis or development of an islet-precursor cell may occur from an already differentiated pancreatic cell (i.e., transdifferentiation of an acinar cell) [15]. Within the gastrointestinal tract and pancreas, at least 17 individual neuroendocrine cell types have been identified as derived from local multipotent gastrointestinal stem cells. The precise mechanism of differentiation of cells of the DNES is still poorly understood although individual transcription factors including protein atonal homolog 1 (PATCH1), neurogenin-3 (NGN3) and neuroD have been identified as regulatory components responsible for lineage transformation. An example of this cell specification role is the effect of loss-of-function mutations in NGN3 evident in individuals with congenital malabsorptive diarrhea. This is associated with failure to promote neuroD transcription, resulting in a specific loss of intestinal enteroendocrine cell populations [16]. In general, neuroendocrine cells are terminally differentiated and considered non-proliferating as demonstrated by the absence of proliferation makers for example Ki67 in CgA-expressing cells [17]. An alternative proliferative mechanism, however, probably exists since neuroendocrine cells are able to adapt to pathological and physiological stimuli within their environment [18]. Evidence for this phenomenon is provided in the instance of gastric enterochromaffin-like (ECL) cells [19]. The ECL cells, located in the oxyntic mucosa, interact with antral G-cells, which secrete gastrin and activate ECL-cell histamine production which, in turn, drives acid secretion from parietal cells. Loss of parietal cells (e.g., in atrophic gastritis) or acid suppression results in increased gastric pH, increased gastrin secretion, and culminates in increased ECL-cell hyperplasia and even neoplasia. If ECL cells are terminally differentiated, this suggests that the mechanism of proliferation likely resides within the ECL-cell stem cell or progenitor. Since a similar phenomenon occurs as a component of hypergastrinemiaassociated MEN1 syndrome, it is plausible that an intrinsic gastrin-activated genetic event may also be implicated [20]. Neuroendocrine cell phenotypes: secretory function A characteristic of neuroendocrine cells is the production of a variety of bioactive peptides and amines (Table 1). Secretory products are stored in large dense-core vesicles 276 Langenbecks Arch Surg (2011) 396:273–298 Table 1 Gastroenteropancreatic neuroendocrine (GEP-NET) cell types: distribution and bioactive products Cell type STOM Oxy A B D EC ECL G Gr GIP n I L M N P/D1 PP S VIP X +++ +++ +++ +++ + + STOM Ant DUOD PANC +++ +++ +++ +++ +++ +++ +++ + +++ +++ + +++ + + + +++ + +++ + +++ n +++ + JEJ ILEUM APPX COLON +++ +++ + +++ + +++ + +++ + +++ + + + + +++ + +++ +++ +++ + + + +++ +++ RECTUM + ++ n +, n +++ ++ +++ + + + +++ + + + Bioactive product Glugagon Insulin Somatostatin 5-HT Histamine Gastrin Ghrelin GIP/Xenin Cholecystokinin GLI/PYY Motilin Neurotensin Pancreatic Polypeptide Secretin/5-HT VIP Amylin Although neuroendocrine cells are distributed throughout the gut, there is evidence of some spatial restriction, for example ECL cells—oxyntic gastric mucosa, G cells to the antrum, and duodenum. Neuroendocrine cells of the pancreas are, however, tightly spatially aggregated into islet architecture. The majority of gut neuroendocrine cells are scattered throughout the gastrointestinal tract, for example somatostatin (D) and serotonin (EC) cells. This suggests a more ubiquitous role for the products of these cells. Despite a similarity in distribution, EC cell-derived tumors are ~30× more common than somatostatinomas (yellow rows) STOM = stomach, APPX = appendix, DUOD = duodenum, JEJ = jejunum, PANC = pancreas, Ant = antrum, Oxy = oxyntic mucosa, n = neonatal and fetal period, + = few cells, ++ = cells present, +++, major site (LDCV) and in small synaptic-like vesicles (SSV), and some proteins associated with these vesicles (e.g., CgA or synaptophysin) have been utilized as specific markers of NECs [21]. Peptide hormones for regulated secretion are packaged into secretory granules (LDCV) that bud from the trans-Golgi network where prohormones and proneuropeptides are stored and processed (Fig. 4). The size, shape, and electron density of the secretory granules have been used with a varying degree of efficacy to characterize individual NEC types. Different granules store individual peptide hormones; however, in some neuroendocrine cells, several different peptides or amines may co-localize in the same granule [22]. A key protein in the genesis of vesicles is CgA which, among other functions, regulates the biogenesis of dense-core secretory granules. Other granins (e.g., chromogranin B (CgB)) adjust proteolytic processing of peptide precursors and promote aggregation-mediated sorting into mature secretory granules, enabling granules to mature into regulatable exocytotic carriers. In certain neuroendocrine cell types, for example ECL cell—histamine, or enterochromomaffin (EC) cell— serotonin (5-HT), amines are co-packaged with chromogranins in secretory vesicles. This process is energy dependent, driven by proton gradients and involves vesicular monoamine transporters (VMAT) [23]; ECL cells are identified by VMAT2 and EC cells by VMAT1 [24, 25]. Neuroendocrine cell secretion is regulated by a complex variety of G-protein-coupled receptors, ion-gated receptors, and receptors with tyrosine-kinase activity [1]. Secretagogue-evoked stimulation (via cAMP/PKA signaling, through MAPK or via ion channel-mediated depolarization [26]) induces actin re-organization through sequential ordering of carrier proteins at the interface between granules and the plasma membrane. This calcium-dependent step is a prerequisite for regulated exocytosis, and it allows granule membrane trafficking and release of neuroendocrine contents. Regulators include neural, for example α- or β-adrenergic, muscarinic (both stimulatory and inhibitory), and VPAC/PAC1 receptors; hormonal, for example gastrin/CCK2, histamine H1–4, or 5HT1–7 receptors and somatostatin (usually types 2 and 5) which are invariably inhibitory (Fig. 5) [26]. Exocytosis, the mechanistic process by which bioactive products are delivered from the cell into the adjacent milieu, comprises a series of sequential intracellular events. In general the exocytotic process involves three steps: (1) Langenbecks Arch Surg (2011) 396:273–298 277 Fig. 3 Neuroendocrine cell differentiation in the gastrointestinal tract. Basal crypt stem (totipotential and pluripotential) cells give rise to a variety of mucosal cell types: Math1 expression directs cells to the secretory lineage and NGN3 to the neuroendocrine lineage. Specific hormone transcription is regulated by several transcription factors such as Pax4, Pax6, and BETA2 (from [9], with permission) the transport of dense core vesicles to the plasma membrane (recruitment step), (2) their initial interaction with the plasma membrane (docking step), and (3) their subsequent fusion with the plasma membrane (fusion step). Molecular mechanisms leading to regulated exocytosis have been Fig. 4 Calcium-dependent exocytosis. CgA is a key protein in the genesis of vesicles and regulates the biogenesis of dense-core secretory granules. Secretory products are stored in large dense-core vesicles (LDCV) and in small synaptic-like vesicles (SSV). Proteins associated with these vesicles (e.g., CgA or synaptophysin) have been summarized in the SNARE (synaptosomal-associated protein receptor) hypothesis [27]. These include the following. 1. Docking of the vesicle to the plasma membrane. SNAREs are present on both the vesicle membrane utilized as biomarkers of neuroendocrine cells [21]. Prohormones and proneuropeptides are stored and processed in the trans-Golgi network prior to packaging into secretory granules (LDCV) as bioactive peptides for regulated secretion (from [2], with permission) 278 Langenbecks Arch Surg (2011) 396:273–298 Fig. 5 Regulation of serotonin release from enterochromomaffin (EC) cells. Tryptophan is transported into the cell from the apical luminal compartment and converted to serotonin (5-HT) by tryptophan hydroxylase. Serotonin accumulates in secretory vesicles which undergo exocytosis in response to cell activation. Positive regulators (green) include noradrenaline, dopamine, serotonin itself and pituitary adenylate cyclase-activating peptide (PACAP), which excite secretion through receptor activation and either cAMP or calcium ([Ca2+]) signaling pathways. Inhibitors (red) of secretion include serotonin, dopamine, acetylcholine, glutamic acid, and somatostatin. Therapeutic activation of somatostatin receptors by somatostatin analogs have proved effective in the inhibition of excess serotonin secretion in “carcinoid” syndrome (v-SNAREs) and on target membranes (t-SNAREs). The docking site is formed by the tight binding of one vSNARE, synaptobrevin or vesicle-associated membrane protein (VAMP), with two t-SNAREs, syntaxin, and synaptosome-associated protein (SNAP-25), resulting in a stable trimeric core complex (Fig. 6). 2. Priming of the exocytotic machinery. After docking, vesicles are not immediately competent to fuse with their target membrane. The trimeric core complex serves as a binding site for N-ethylmaleimide-sensitive fusion protein (NSF) and thereafter NSF crosslinks multiple core complexes leading to hemifusion of vesicle and target membrane. 3. Triggering of exocytosis by calcium. Synaptotagmins are v-SNAREs that comprise a component of a clamping apparatus that prevents spontaneous fusion of vesicles with their target membrane. Synaptotagmins most likely function as calcium sensors and after a sub-plasmalemmal Fig. 6 Mechanism of vesicle docking and exocytosis at the plasma membrane. SNAREs have been identified both on the vesicle membrane (v-SNAREs) and on target membranes (t-SNAREs). The docking site is formed by the tight binding of one v-SNARE, synaptobrevin or vesicle-associated membrane protein (VAMP), with two t-SNAREs, syntaxin and synaptosome-associated protein (SNAP-25), resulting in a stable trimeric core complex. At the completion of docking, vesicle content is released into the paracellular space Langenbecks Arch Surg (2011) 396:273–298 279 rise in intracellular calcium, interact with syntaxins (t-SNAREs) as well as membrane phospholipids and other yet-unidentified target proteins to allow. 4. Fusion of the vesicular membrane with the plasmalemma. This final step results in exocytosis [28]. Neuroendocrine tumors: etiology and pathogenesis NETs are generally thought to represent malignant transformations of either terminally differentiated neuroendocrine cells or a precursor/stem cell. The mechanism of these events is largely unknown. It is postulated that damage to early, neuroendocrine precursor cell types leads to the development of high grade or poorly differentiated neuroendocrine carcinomas (NECs). G1-NETs (previously called NETs) and G2-NETs (previously called WDNEC (well differentiated neuroendocrine carcinoma)) develop from later stage or partially differentiated cells (Fig. 7). The mechanisms for “damage” are not known but the sequelae are largely considered to be either epigenetic modifications, for example differences in histone acetylation or chromosomal methylation, or spontaneous mutations in critical genes, for example MEN1 [29]. Although an attractive pathological concept, there is little evidence to support the proposal of progression from a GEP-NET-G1 to NET-G2 and finally to a high-grade NEC (the so-called “NET–NEC sequence”) [30]. While the majority (>95%) of GEP-NETs are sporadic [1], a small percentage are either familial or associated with five independent autosomal dominant inherited syndromes. Evidence for the familial basis of gastrointestinal NETs has been assessed in large cancer databases, for example Swedish Family Cancer database [31] which indicated that the risk of developing NETs was significantly higher among individuals with a parental history of NETs (relative risk (RR): 4.33) and in individuals with a sibling history of NETs (RR 2.88). Parental NETs were strongly associated with the development of small intestinal (RR 11.80) and colon NETs (RR 2.78) in the offspring. Although this type of analysis cannot identify candidate genes, it indicates that there exists an individual predisposition to GEP-NET development. A separate defined group of NET genetic disorders includes MEN types 1 and 2, which are the most common forms, VHL disease, von Recklinghausen disease or neurofibromatosis (NF1), tuberous sclerosis (TSC), and Carney complex (CNC). MEN1 is an inherited disease classically constituted by parathyroid hyperplasia/adenoma, pancreatic endocrine tumors, and pituitary tumors. Variations of MENI include in addition adrenocortical secreting or nonfunctional Fig. 7 Transformative events (putative) in the development of GEPNETs. NETs develop in inherited/familial tumors of the stomach (gastric type II) and pancreas (pNETs) as a consequence of either a second hit or LOH. Somatic mutations, the most common event, perhaps due to environmental damage at a committed neuroendocrine precursor stage, lead to well-differentiated NETs (NET-G1). If damage occurs early in stem cell progress (e.g., stem cell 1), poorly differentiated neuroendocrine carcinomas develop. If damage occurs at a later stage, for example to a pluripotent cell (stem cell 2), then a well-differentiated NEC (G2-NET) is the consequence. There is little evidence for evolution from a NET to a NEC in this schema [29] tumors, thymic NETs, and bronchial NETs [3, 32]. The diversity of MEN1-related lesions and the divergent embryonic origins of affected tissues implicate the MEN1 gene as exhibiting a critical role in early embryogenesis. The MEN1 gene is located on the long arm of chromosome 11, band q13 [33], and comparative genomic analysis of tumoral and constitutional genotypes has identified evidence of somatic loss of heterozygosity (LOH). This is consistent with the likelihood that development of MEN1associated tumors is a two-step process, a germline mutation affecting the first MEN1 allele, and a second somatic inactivation of the unaffected allele (LOH). Tumorigenesis in MEN1 likely involves loss of function of the growth-suppressor gene MEN1 [34]. Menin is a 610amino acid nuclear protein encoded by the MEN1 gene and interacts with Jun D and the AP1 transcription factors to modify growth-regulatory signaling. It also interacts with a putative tumor metastasis suppressor nm23H1/nucleoside 280 diphosphate kinase (nm23), and exerts GTPase activity [35]. Truncation or instability of MEN1 gene products has been proposed to culminate in loss of transcriptional regulation and/or GTP hydrolysis, thereby providing a possible mechanism of MEN1-driven tumor formation [34]. Germline mutations of the RET proto-oncogene encoding a transmembrane tyrosine-kinase-receptor, confer predisposition to clinical variants of MEN2, which can be subdivided into 2A (Sipple’s syndrome), 2B (Gorlin’s syndrome), and Familial Medullary Thyroid Cancer [36, 37]. In MEN2A, medullary thyroid carcinoma is associated with pheochromocytoma (30–50%) and primary hyperparathyroidism (10– 20%). In MEN2B, the major clinical features are medullary thyroid carcinoma, pheochromocytoma, mucosal neuromas, and cranio-skeletal abnormalities sometimes associated with a marfanoid habitus. Additionally, angioneuromatosis of the gastrointestinal tract may also occur [38]. It is noteworthy that the majority of lesions associated with the MEN2/RET abnormality are NETs outside of the gastrointestinal system. VHL disease is an autosomal dominant syndrome whose cardinal features include a predisposition to renal cancers, retinal and/or cerebellar hemangioblastoma, pheochromocytoma, and cystic and/or pancreatic endocrine tumors [39]. Ten percent to fifteen per cent of patients with VHL develop pancreatic islet or ductal endocrine cell tumors [40] and more than 50% exhibit multiple tumors. The VHL gene is located on chromosome 3p35-26 [41], and its product interacts with the elongin family of proteins to regulate transcriptional elongation [42]. Other functions involving the VHL protein are hypoxia-induced cell regulation and extracellular matrix fibronectin expression and localization [43]. NF1- and TSC -related GEP-NETs include multiple tumors in the pancreas and/or duodenum with psammomatous glandular histological features and immunohistochemical expression of somatostatin and/or insulin [3]. The NF gene, located on chromosome 17q11.2, acts as a tumor suppressor. Mutated (non-functional) neurofibromin, the NF-1 gene product, results in a loss of normal function, downregulation of the P21ras signaling pathway; this loss leads to a constitutively activated GTP which results in abnormal cell proliferation [44]. TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The protein products of the tuberous sclerosis complex genes, hamartin (TSC1), and tuberin (TSC2), have important cellular regulatory functions. These include a role in cell signaling in growth and translation regulation via the PI3K/AKT pathway, in cell adhesion via the glycogen synthase kinase 3 pathway, and in proliferation via the mitogen-activated protein kinase (MAPK) pathway [45]. The CNC, described in 1985 [46], is an autosomal dominant disease comprising skin pigmentation, myxomas, Langenbecks Arch Surg (2011) 396:273–298 melanotic Schwannomas and endocrine tumors of the adrenal glands, Sertoli cells, somatotrophs, thyroid, and ovary [47]. The CNC gene, located on chromosome 17q22q23, encodes PRKARIA, the protein kinase A (PKA) regulatory subunit 1α (R1α), and is a tumor suppressor gene. The role of this gene in GEP-NETs is unclear. Neuroendocrine tumors: “functional” versus “non-functional” Some NETs are associated with specific symptomatology consequent upon the release of bioactive peptides and amines, for example insulin and 5-HT into the systemic circulation. Such tumors have in the past been designated as “functioning” tumors and recognized as the cause of a variety of syndromes, for example hypoglycemia related to insulinoma (insulin), peptic ulceration and gastrinoma (gastrin), and the diarrhea, abdominal pain, sweating, flushing, bronchospasm, tachycardia, and fibrotic heart disease of "carcinoid syndrome" (5-HT). Other functioning tumor syndromes reflect pancreatic primary lesions associated with either excessive secretion of glucagon (glucagonomas) or vasoactive intestinal polypeptide (VIPomas). In contrast, many NETs (~50%) are not associated with a clinically defined “hypersecretory” symptom complex (syndrome) and were previously termed “non-functioning”. The wide variation previously reported (~10–85%) reflects the limitations in current reporting systems and the difference between older series and more recent ones where more sophisticated biological tools are available. This clinical distinction has artificially led to the conclusion that there are two separate types of NETs. In general, however, NETs are indistinguishable at pathological, immunohistochemical, and transcriptomic level, while therapeutically they appear to respond in an almost indistinguishable fashion. There currently appears to be little scientific information to support the concept that functional NETs are in any biological fashion different to non-functional NETs. Gastric nets—predominantly ECL-cell tumors The stomach contains at least five types of endocrine cells which collectively comprise ~2% of the cells in the gastric mucosa [48]. Each endocrine cell secretes a “dominant” chemical messenger: ECL cells secrete histamine, G cells secrete gastrin, while EC, D and P cells secrete 5-HT, somatostatin, and ghrelin, respectively [49]. The histaminesecreting ECL cells are the most common gastric neuroendocrine cell type, constitute up to 80% of oxyntic mucosal neuroendocrine cells, and constitute the predominant neuroendocrine tumor type in the stomach. Gastric (ECL Langenbecks Arch Surg (2011) 396:273–298 281 cell) NETs are classified into three subgroups based upon their responsiveness to gastrin: Type I and type II tumors occur in the setting of hypergastrinemia (Table 2). Type I lesions represent a physiological response to low acid states such as chronic atrophic gastritis (CAG) and pernicious anemia. Type II tumors are driven by autonomous gastrin secretion from a gastrinoma, almost always in the setting of MEN1. These ECL tumors are similar to type I in respect of their gastrin-responsiveness but the MEN1 genetic abnormality renders them more susceptible to malignant transformation. Type III tumors occur in the absence of hypergastrinemia, are not always of ECL origin, and include a more malignant subtype described as “atypical”. These three types of tumors are usually considered distinct from a poorly differentiated subtype (type IV in some classifications), which had been previously been regarded as anaplastic, high-grade NET, or small cell carcinoma of the stomach [50]. Etiology/pathogenesis Gastrin is the most important growth factor in type I and II gastric NETs. In type I tumors, a loss of parietal cells is associated with a diminution of acid production and an elevation in luminal pH with consequent loss of negative Table 2 Gastroenteropancreatic neuroendocrine (GEP-NET) tumor type, distribution and 5-year survival Cell Type Tumor Incidence (%) Five-year survival (%) EC 30 68 L β ECL α G δ or D I PP Intestinal NET (Carcinoid) NET Insulinoma Gastric NET Glugagonoma Gastrinoma Somatostatinoma CCKoma Ppoma 16 7 6 3 2.5 1 <1 <1 72–92 97 65–91 50-60 30–100 40 ND 30–50 VIP ? VIPoma Non-funtional* <1 10–85* 60–95 31–59* GEP-NETs exhibit a wide range in incidence and outcome. Survival for the individual tumors is linked to the cell type of origin. For example, β cells that give rise to insulinomas are only rarely malignant as are gastric ECL cell tumors (type I). In other cases, for example EC cell NETs “carcinoids”, survival is predicated on the proliferative index of the tumor as well as the presence of metastasis. The precursor cells of non-functional tumors (~50% of pNETs) are unknown and may be different in individual non-functional lesions. The variable survival, which ranges from 31–59%, may reflect different cells of origin ND = no data; ? = cell type unknown; * refers to pNETs feedback on G cells culminating in increased gastrin production and thereafter G-cell hyperplasia (Fig. 8). The destruction of parietal cells is most commonly associated with CAG, pernicious anemia [51, 52], and a variety of autoimmune diseases [53]. Sustained elevation of gastrin secretion results sequentially in ECL cell hyperplasia, with progression to dysplasia, and culminates in neoplastic transformation to a gastric tumor [49]. There has been some debate in relation to the causative role of potent acid suppressive medications (proton pump inhibitors (PPIs)) in the etiology of type I NETs; little evidence or support, however, exists for this assertion [54, 55]. Animal studies have identified that Helicobacter pylori may have a causative role in type I gastric NETs and studies in a Mongolian Gerbil model infected with H. pylori (toxigenic cagPAI + strains) noted elevation in serum gastrin and the development of ECL hyperplasia and neoplasia [56]. H. pylori eradication was followed by diminution in gastrin levels and a decrease in neoplasia in these animals. To date, there is little evidence to support a role for H. pylori in human gastric neuroendocrine tumorigenesis. The trophic effect of gastrin is also responsible for neoplastic transformation in type II tumors in which the source of hypergastrinemia is autonomous secretion from a gastrinoma; approximately 20–40% of gastrinomas occur in patients with MEN1 [57]. Gastrinomas in MEN1 occur predominantly in the duodenum (70–100%) with the remainder in the pancreas; for reasons that are not understood, they very rarely occur in antral G cells. The subsequent gastrin-driven type II tumors occur exclusively in the ECL cells of the gastric oxyntic mucosa. In contrast to the type I gastric carcinoids, parietal cells remain functional, and type II lesions are associated with excessive histamine production, excessive parietal cell acid production, reduction in luminal pH, and the characteristic gastrinoma-associated peptic ulceration (Fig. 8) [52]. Gastrinomas may also occur sporadically, however, the frequency of associated gastric NETs are much lower (2%) than in MEN1 related gastrinoma (~40%) [57]. This difference is consistent with the MEN1 gene mutation being permissive in gastric carcinoid type II pathogenesis [58]. Neoplastic transformation in type III tumors and NECs is independent of gastrin and is not associated with a known familial predisposition to malignancy. The absence of hyperplastic and dysplastic ECL cells (commonly identified in type I and II lesions) suggests a different mechanism of tumorigenesis, and type III tumors are substantially more aggressive exhibiting early gastric mural invasion [59], and frequent penetration into lymph and blood vessels [52]. Type III lesions and NECs often contain cells other than ECL cells [49]; these can be of endocrine (e.g., EC cells, G cells, and pancreatic polypeptide positive-cells) or of nonneuroendocrine origin [54]. Concurrent gastric adenocarci- 282 Langenbecks Arch Surg (2011) 396:273–298 Fig. 8 Gastric NET (ECL cell) pathogenesis. The physiological regulation of acid secretion (top) involves luminal amino acid activation of G-cell gastrin secretion to provoke fundic ECLcell histamine release, which stimulates parietal cell acid secretion. Luminal acid increase counter-regulates gastrin via a somatostatin-modulated negative feedback loop. In Type I gastric tumors (left), diminution of parietal cell function (e.g., pernicious anemia or atrophic gastritis) increases luminal pH which stimulates gastrin secretion and sustained hypergastrinemia, culminating in ECL cell proliferation. This growth proceeds through phases of hyperplasia, dysplasia, and neoplasia. In Type II lesions (right), hypergastrinemia originates from autonomous secretion by a gastrinoma (G-cell neoplasia). In type III tumors (bottom), neoplastic transformation occurs independently of gastrin levels noma can be present in 5–10% of type III gastric NETs [60], but is more common (34%) in NECs [60]. The coexistence of endocrine and epithelial cells in both NETs and adenocarcinomas has led to several etiological hypotheses: that both types of cancer derive from a multidirectional gastric stem cell; that secretory products of neoplastic endocrine cells create a transforming milieu that encourages epithelial carcinogenesis [6]; or that poorly differentiated NECs may represent transformation from a well-differentiated carcinoid tumor, although this appears unlikely. These proposals all remain unproven. Growth factors implicated in ECL cell proliferation include connective tissue growth factor (CTGF) [61, 62], insulin-like growth factor I (IGF-I) [61], pituitary adenylate cyclase activating polypeptide (PACAP) [63], and transforming growth factor-alpha (TGFα) [64]. Epidemiology and survival Type I gastric lesions are the most common gastric NET comprising 74–78% of gastric NETs; in two series of gastric NETs, the frequency of type II, III, and NEC was 2– 6%, 13%, and 6%, respectively [50, 51]. There is a female preponderance for type I gastric NETs (as might be predicted given the female preponderance of atrophic gastritis), and a male predominance for type III gastric tumors and gastric NECs [50, 52]. The incidence of gastric NETs is steadily increasing and is currently 6% of all NETs reported between 2000 and 2007 (Surveillance, Epidemiology, and End Results (SEER)) [65]. The 5-year-survival of patients with gastric NETs has improved from 51% in the 1970s to 71% [66]. This largely reflects inclusion of more benign (type I and II lesions) in datasets and earlier identification based on increasing availability of upper GI endoscopy. Molecular markers Gastric NETs are classically identifiable by immunohistochemical labeling for the neuroendocrine markers synaptophysin and CgA, although synaptophysin, cytosolic neuron specific enolase or PGP9.5 may be the only indicators of neuroendocrine origin in poorly differentiated gastric NETs [52, 67]. An ECL cell origin can be confirmed by staining for histamine and VMAT2. Gastric NETs that contain other non-ECL NEC types or exhibit an adenocarcinoma pheno- Langenbecks Arch Surg (2011) 396:273–298 type can be identified based on properties of the other cell types (e.g., immunostaining of a specific peptide). At a transcript level, CgA discriminates gastric NETs from other gastric neoplasms (including GISTs) [68]. Over expression of MAGE-D2 and MTA1 differentiate type III/IV from type I/II GCs and MTA1 appears to be a marker of tumor invasion [68]. 283 Duodenal tumors comprise 50–88% of gastrinomas in sporadic ZES patients and 70–100% of gastrinomas in MEN1/ZES patients. In rare cases, non-pancreaticoduodenal gastrinomas have been described in the stomach, liver, bile duct, ovary (5–15%), and extra-abdominal (heart, lung) locations [69, 76]. Overall, more than 50% of duodenal gastrinomas have liver metastases at the time of diagnosis. Epidemiology and survival Duodenal GEP-NETs—G- and D-cell tumors These are usually small, non-functioning tumors, which do not cause any symptoms, and are often diagnosed by a gastroduodenoscopy performed with a different indication. Most duodenal NETs are discovered at an early treatable stage (tumor diameter ≤10 mm) [69, 70]. In the instances when tumors are associated with a syndrome, for example ZES, lesions are often metastatic [71]. G cell—gastrinomas Gastrinomas arise in the duodenum and in the pancreas. Up to 70% occur in the duodenum, arise directly from G cells, and tend to be small, multiple, and exhibit a less malignant clinical course than those arising in the pancreas [72]. Since G cells are not identifiable in the adult pancreas it is assumed that pancreatic gastrinomas arise from a different neuroendocrine cell or precursor. The more aggressive nature of most pancreatic gastrinomas would support the concept of a different model of tumorigenesis compared to duodenal gastrinomas [73]. Clinically, gastrin secreting tumors, irrespective of their origin, are associated with ZES. This syndrome comprises gastric acid hypersecretion, recurrent, multiple, and intractable peptic ulceration (duodenum and proximal jejunum) and is often accompanied by a secretory diarrhea. Etiology/pathogenesis Sporadic duodenal gastrinomas constitute 80% of all gastrinomas, and their etiology and pathogenesis is unknown. The residual 20% of duodenal gastrinomas are part of the MEN1 syndrome. Duodenal gastrinomas are located in the first and second part of the duodenum (90%) [69] and are limited to the submucosa in ~50% of patients [69]. The anatomical area comprising the head of the pancreas, the superior, and descending portion of the duodenum, and the relevant lymph nodes have been entitled the “gastrinoma triangle”, since it harbors the vast majority of these tumors [74]. In some instances, gastrinomas in peri-pancreatic and peri-duodenal lymph-nodes have been considered to represent a primary tumor rather than metastases from an occult lesion in the duodenum, and reports exist of occasional individuals having been cured after resection of the lymph nodes [75, 76]. The reported incidence of gastrinomas is between 0.5 and 4 per million of the population per year [69]. They are the most common functional duodenal tumor. Approximately 0.1% of patients with duodenal ulcers have evidence of ZES. In general, the progression of gastrinomas is relatively slow with a 5-year survival rate of 65% and 10-year survival rate of 51% [72]. The most significant predictor of survival is the presence and extent of liver metastases at diagnosis [76, 77]. The 10-year survival of patients with local tumors is 96% and only 30% in those with metastatic tumors [77]. Molecular markers There is no established marker to predict the biological behavior of a gastrinoma, although LOH at 11q13 occurs in 44–48% of sporadic duodenal gastrinomas [78, 79]. Hypermethylation (with subsequent gene silencing) of the promoter region of p16INK4A occurs in ~50% of gastrinomas [80]; the relevance of this is unknown. Some investigators have suggested that HER-2/neu amplification, over expression of epidermal growth factor (EGF), and hepatocyte growth factor (HGF) may be associated with an aggressive growth phenotype [76, 81, 82]. D cell—somatostatinomas Somatostatinomas secrete the bioactive product, somatostatin. This tetradecapeptide is a ubiquitous neurotransmitter, paracrine agent that in general exerts a widespread inhibitory effect on exocrine and endocrine secretion and bowel motility. These tumors usually produce local symptoms secondary to local mass effects, for example bile duct obstruction, jaundice, abdominal pain, and gastrointestinal bleeding [83, 84]. The somatostatinoma syndrome (steatorrhea, cholelithiasis, diabetes mellitus-like symptoms) is very rare, and a small proportion are associated with the MEN1 or NF1 disorders. The majority of somatostatinomas are solitary, sporadic, and malignant. Etiology/pathogenesis Approximately 14–43% of somatostatinomas develop in NF1 patients. The lesions occur predominantly in the duodenum, in 284 the vicinity of the ampulla of Vater but rarely in the pancreas [85, 86]. Tumors are often small, well differentiated, and low grade. Nevertheless, lymph node and liver metastases occur in ~35%, and the risk of metastases significantly increases when tumors are >20 mm [87]. Tumors that occur in the setting of NF1 are usually identified earlier due to increased surveillance [84]. Langenbecks Arch Surg (2011) 396:273–298 the liver, invasion of adjacent organs, tumor size more than 2 cm, angioinvasion, and elevated proliferative activity (more than 2% of cells positive for Ki67) [92]. Recent reports suggest that angioinvasion may represent a more critical role than previously assumed [93]. It is probable that a tumor be considered malignant if angioinvasion is evident even if no other criteria of malignancy are demonstrable. Epidemiology and survival Embryology, development, and phenotype To date, more than 100 cases of duodenal somatostatinomas have been reported [88], and the relative frequency of duodenal primaries is ~6 times higher than pancreatic primaries [85]. There is no statistically significant difference in the rate of metastases and malignancy between pancreatic and extra pancreatic tumors [89]. The overall 5year survival rate is 75% when the tumor is localized but decreases to 60% when metastases are present [90]. Molecular markers Somatostatinomas exhibit positivity for endocrine markers, especially synaptophysin and CgA, and most cells express somatostatin receptors [85]. Expression of the latter indicates tumors may be amenable to somatostatin receptor targeting. Human islets consist of approximately 3,000 cells producing insulin (β cells, 54%), glucagon (α cells, 34%), somatostatin (δ cells, 10%), VIP (δ 2 cells), pancreatic polypeptide (PP) (PP cells), and substance P/5-HT (EC cells). Gastrin-producing G cells are present in fetal but not normal adult pancreatic islets [94, 95]. The quotient of all pancreatic endocrine cells is 1–2% of the entire pancreatic cell mass [96]. Both, alpha and beta cells appear to arise from the same precursor cell, namely the ductal epithelial cell. By the fifth month of fetal life, islet cells produce insulin and glucagon [96, 97]. Cytokeratin 20, a marker for adult rat pancreatic ductal cells [98, 99] which is not found in normal adult islet cells, is co-expressed with insulin or glucagon during islet cell neogenesis in the fetal [14] and neonatal [99] pancreas. Pathogenesis and pathology Pancreatic endocrine tumors Pancreatic neuroendocrine tumors (pNETs) represent 1–2% of all pancreatic neoplasia [91].The majority of pNETs are G1 or G2 neuroendocrine tumors in the WHO classification of 2010, and approximately half secrete measurable levels of site-specific bioactive peptides (insulin, gastrin, VIP, glucagon), or other non-pancreatic hormones, for example adrenocorticotropic hormone (ACTH) or growth hormone (GH). These peptides are associated with characteristic syndromes (ZES, Verner-Morrison syndrome, glucagonoma syndrome, Cushing’s syndrome, and acromegaly) and lessspecific symptoms (hypoglycemia, hyperglycemia) [4]. Depending on the predominant bioactive agent secreted, individual tumors are identified as insulinomas, gastrinomas, VIPomas, glucagonomas, etc. (Table 2). However, approximately half of pNETs (~10–85%) are not associated with symptoms from a clinically defined “hypersecretory” syndrome and are termed “non-functioning”. Irrespective of the secretory status, pNETs are usually well demarcated, often solitary, ovoid tumors that can occur in all parts of the pancreas. Although pNETs are histologically “well differentiated”, they are frequently malignant, with the exception of insulinomas. Tumor characteristics associated with poor prognosis include metastases to regional lymph nodes and The pathogenesis of pNETs is not clear. However, since ductal cells may be a precursor cell type, at least for normal pancreatic endocrine cells, these are also considered precursors for pNETs themselves. The mechanism of malignant transformation is unknown but for nonhereditable tumors, is considered similar to other GEPNETs, that is, reflects an environmental damage-driven somatic mutation event. The etiology of inherited tumors, for example MEN1, has been discussed but usually is based on the general schema of an overactive/unregulated growth phenomenon. Histological examination is not able to define whether a lesion is functional or identify the type of hormone production. There are two exceptions to this rule: amyloid deposits are indicative of insulinomas, and glandular structures containing psammoma bodies are commonly observed in somatostatin-producing tumors [100–102]. Poorly differentiated endocrine carcinomas can be misdiagnosed as pancreatic adenocarcinoma unless appropriate immunohistochemical is undertaken to define their neuroendocrine phenotype. Such tumors exhibit pleomorphic cells with high mitotic index (≥10/10 HPF) and often angioinvasion. PNETs can be identified using antibodies to markers common to all or most NECs: that is, CgA, Langenbecks Arch Surg (2011) 396:273–298 synaptophysin, NSE, and protein gene product 9.5 (PGP 9.5) but can also express cytokeratin 8, 18, and 19 [103] and sometimes either VMAT1 or VMAT2. Molecular markers Under some conditions, the molecular basis of familial pNETs has been identified and comprises inherited mutations and a second-hit somatic mutation of MEN1 and VHL genes. However, in contrast to other tumors, for example pancreatic adenocarcinoma, activation of classical oncogene-mediated pathways does not seem a common event in pNETs. Little is known in respect of pancreatic neuroendocrine oncogenesis and the molecular basis of the progression of sporadic NETs [103]. Thus, mutations in k-ras, P53, myc, fos, jun, src, and the Rb gene have not been specifically implicated [104, 105]. In contrast, copy number alterations, for example in proteins that regulate some of these pathways, MDM2 and P53, have been noted [106]. Transcription factors (TFs) regulate organogenesis and PAX8 (paired box gene 8) is a part of a group of TFs that are cell-lineage specific in multiple organ systems [107]. TF analysis has identified that PAX8 is expressed in normal adult pancreatic islet cells, and is also expressed in a significant proportion of primary and metastatic welldifferentiated PETs [108]. It has been proposed that loss of PAX8 may have prognostic significance since PAX8negative tumors are significantly larger, associated with malignant behavior, and are associated with an increased incidence of liver metastases [108]. At a chromosomal level, molecular and cytogenetic analyses have identified a number of chromosomal alterations in pNETs. Comparative genomic hybridization studies indicate that chromosomal losses have occurred slightly more frequently than gains, while amplifications are uncommon (Fig. 9 top) [109, 110]. Furthermore, the total number of genomic changes per tumor appears to be associated with both the tumor volume (size) and disease stage, indicating that genetic alterations accumulate during the natural history of the lesion. Thus, large tumors with increased malignant potential—and especially metastases—tend to harbor more genetic alterations than small and clinically benign neoplasms. This suggests the loss of tumor suppressor pathway(s) and genomic instability as important mechanisms associated with progression but not initiation of a pNET. However, losses of chromosomes 1 and 11q as wells as gains on 9q appear to be early events in the development of pNETs, since they may already be present in small tumors. Prevalent chromosomal alterations common in metastases include gains of both chromosome 4 and 7 and losses of 21q, implying that these chromosome imbalances may contribute to tumor metastasis (Fig. 9 top) [111, 112]. 285 Deletions of 9p which occur in ~30% of pNETs, contain the location of the p16INK4A and p14ARF genes, both of which encode tumor suppressors; loss of this gene locus may lead to tumorigenesis due to deregulation of the p53 and Cyclin D1/Rb pathways. Alterations in the cyclin D1 pathway in pNETs indicate over expression of this proto-oncogene in 43% of tumors [113]. Chromosome 16p, which contains TSC2 (a tumor suppressor of the AKT/mTOR pathway with GTPase activating function), is lost in ~40% of PETs [111, 114], while PTEN a second tumor suppressor at this locus, is lost in 10–29% of lesions [111, 114, 115]. Low expression of either TSC2 or PTEN correlates with pNET aggressiveness, a “non-functional” status, proliferation index, presence of liver metastasis at diagnosis or followup, and with time to progression [116]. This suggests the involvement of the AKT/mTOR pathway in pNET tumorigenesis and progression. PNETs also over-express MDM2, MDM4, and WIP1, all of which may attenuate the function of p53. Since p53 is critical in maintaining genomic stability, alterations in regulators of p53 are therefore considered potentially permissive for pNET pathogenesis [106]. Fibroblast growth factor 13 (FGF13) is upregulated in metastatic compared to non-metastatic pNETs [116], and is an independent predictor for shorter progression free survival [116]. Little, however, is known about the mechanisms by which FGF13 regulates pNET proliferation and metastasis. Deletions on the X-chromosome were associated in one study with 100% of pNETs [117]. Two studies of pNETs found no evidence of microsatellite instability [118, 119]. Glucagonomas (α-cell tumors) Glucagonomas represent about 5% of pNETs and 8–13% of functional tumors. Etiology/pathogenesis Although the majority of α-cell-derived tumors are large and malignant only a minority (8–13% of functioning tumors) are associated with the glucagonoma syndrome [120]. Nonsyndromic glucagon-producing tumors have been described under four different conditions: (1) as solitary tumors that become symptomatic because of their size and/or malignant growth; (2) as micro tumors (≤0.5 cm) found incidentally; (3) as multiple microadenomas and macro tumors in patients with MEN1 [121]; and (4) so-called glucagon cell adenomatosis. The latter constitutes multiple pancreatic neoplasms exclusively producing glucagon, associated with glucagon cell hyperplasia of the islets and unrelated to MEN1, VHL, or the recently identified [122] p27 MEN syndrome [123, 124]. Glucagonomas commonly occur in the tail of the 286 Langenbecks Arch Surg (2011) 396:273–298 Fig. 9 Chromosomal abnormalities identified in gastroenteropancreatic neuroendocrine tumors (GEP-NETs). In pNETs (top), the commonest losses occur on 6, 11, X, and Y. Common gains include Chr 9, 12, and 17. In small intestinal NETs (bottom), the commonest loss is on 18, while 17 and 19 exhibit the most common gains. It is evident that pancreatic and small intestinal NETs express significantly different patterns of chromosomal rearrangements, however, the precise implications of the alterations are as yet unresolved pancreas. Extra pancreatic glucagonomas are extremely rare [125, 126]. One example is a kidney enteroglucagonoma (described in 1971 [127, 128]), which was associated with a massive hypertrophy of the small bowel villi and slow bowel transit times. Markers Markers include CgA, CgB, and the glucagon peptide. Insulinomas (β cell tumors) Epidemiology and survival Insulinomas are the most frequent of all functioning pNETs. The estimated incidence of the glucagonoma syndrome is 1 per 20 million of the population per year [103]. Approximately 60–70% of glucagonomas are metastatic at the time of diagnosis, and only 50–60% of patient survive 5 years [129]. Even small glucagonomas are considered tumors of uncertain behavior since in some instances the neoplasm may grow slowly, and patients may survive for many years whereas other glucagon-secreting lesions are locally invasive, metastasize early and follow an accelerated course. Occasionally, in multihormonal tumors, the glucagonoma syndrome may be associated with or followed by another syndrome, such as hypoglycemia syndrome or ZES [130]. Etiology/pathogenesis The etiology and pathogenesis of insulinomas is unknown, and no known risk factors have been identified. Clonality studies on pNETs suggest that insulinomas may be primarily a polyclonal or oligoclonal neoplasm which is eventually overgrown by a more aggressive cell clone that may give rise to invasive growth and metastasis [131]. The majority of insulinomas are located in the pancreas or are directly attached to it. Ectopic (extrapancreatic) insulinomas with symptoms of hypoglycemia are extremely rare (<2%) Langenbecks Arch Surg (2011) 396:273–298 and are most commonly found in the duodenal wall. Tumors are equally distributed between the head, body, and tail of the pancreas. Approximately 85% of insulinomas are single lesions, 6–13% are multiple, and 4–6% are associated with MEN1 [103]. 287 Epidemiology and survival The number of patients undergoing successful complete resection ranges from 60% to 80% [140]. The overall 5year survival rate of all somatostatinomas is 75%, but decreases to to 60% when metastases are present. Epidemiology and survival Molecular markers The incidence of insulinoma is reported to be one to four patients per million of the population per year, and 5–10% are malignant [132]. The molecular basis for the latter is not known. It is difficult to predict the malignant nature of an insulinoma on the basis of histology alone [133]. Criteria which are associated with poor prognosis are the presence of metastases, gross invasion, larger tumor size, higher percentage of mitoses and proliferative index, and vascular invasion [134]. The distinction between malignant and benign insulinomas is often difficult and is generally based on intraoperative evidence (metastases in the liver, regional nodes or local invasion). In some circumstances, the tumor may be the identified at the time of a recurrent hypoglycemic episode [134]. Overall, the 5-year survival is ~97% [135, 136]. Molecular markers Islet 1 (Isl1, 349 amino acids, 39 kd) is a transcription factor that binds to a β-cell-specific enhancer element in the insulin gene, and is required for the development of the dorsal pancreas mesenchyme and for differentiation of islet cells [137]. It is expressed in the majority of β-cell-derived pNETs and their metastases [138]. Little is known regarding molecular markers for these lesions. Rare pancreatic endocrine tumors VIPomas—Verner–Morrison syndrome Tumors secreting VIP are associated with the watery diarrhea syndrome (WDS), eponymously referred to as the Verner–Morrison syndrome, pancreatic cholera and WDHA (watery diarrhea/hypokalaemia/achlorhydria) syndrome. Pancreatic VIPomas constitute about 80% of diarrheogenic neoplasms and 3–8% of all endocrine pancreatic tumors in the pancreas. The majority (80%) of VIPomas are located in the pancreas and most of these occur in the tail [103]. The prognosis of VIPomas in childhood is more favorable than in adults, because~two thirds of VIPomas are of neurogenic origin and benign. A small percentage of VIPomas (5%) are associated with MEN1. ACTHomas, GRFomas, and other -omas Somatostatinomas (δ cell tumors) Somatostatinomas represent <5% of pNETs. Based on its secretion of somatostatin, the lesion has been proposed to generate a so-called “inhibitory syndrome”. Etiology/pathogenesis Little is known regarding the etiology and pathogenesis of δcell tumors. They are occasionally associated with NF1, although rarely with MEN1 [86]. Somatostatinomas usually present in the 5th decade of life, are usually large (average diameter 5.1 cm), have a predilection for the pancreatic head, and are associated with local symptoms and/or symptoms of excessive somatostatin secretion (steatorrhoea, cholelithiasis, diabetes mellitus-like symptoms) [86]. Between 70% and 92% demonstrate metastatic spread at diagnosis or operation [139]. A third of somatostatinomas may, in addition to somatostatin, produce multiple peptides which is usually an indication of a more malignant phenotype. Other rare pNETs have been reported, including ACTHomas (adrenocorticotropic hormone, 110 cases), GRFomas (growth hormone releasing factor, 50 cases), neurotensinomas (50 cases), and parathyrinomas (35 cases) [141]. In rare instances, pNETs can secrete enteroglucagon, cholecystokinin (CCK), gastric inhibitory peptide (GIP), gastrin-releasing peptide (GRP/bombesin), and ghrelin. The occurrence of Cushing’s syndrome as the only manifestation of a pNET occurs in 37–60% of ACTHomas and may precede any other hormonal syndrome. Ectopic ACTH secretion originating from a pancreatic tumor is responsible for 4–16% of Cushing’s syndrome [142] and ~5% of sporadic gastrinomas may also secrete ACTH. ACTH secreting gastrinomas generally exhibit metastatic disease, aggressive behavior, a poor response to chemotherapy and a poor prognosis [142]. Acromegaly due to pancreatic GRFoma can be cured by surgical resection in the absence of metastases [143] while individuals with unresectable disease may respond to somatostatin analog therapy [103]. 288 Small bowel NETs (EC Cell “Carcinoids”) The EC cell is the predominant neuroendocrine cell of the gastrointestinal tract and plays a key role in the physiological regulation of secretion, motility, blood flow and visceral pain. Intestinal EC cells synthesize, store, and release this amine and contain the majority (95%) of 5-HT in the body [144–149] (Fig. 10). Abnormalities in 5-HT release and availability (reuptake and catabolism) are associated with altered gastrointestinal secretion and motility culminating in diarrhea, constipation, and pain, as in the carcinoid syndrome [150, 151]. Pathogenesis and pathology The biological basis of small intestinal neuroendocrine pathogenesis, malignancy, and metastasis is unknown. Although EC cells are considered terminally differentiated, they express proliferation-associated transcripts, for example Ki67 [90]. Despite this, EC cell tumors are considered to arise from abnormal mucosal precursor cells. It is likely that the cell which accumulates the mutations necessary for development of NETs is a committed neuroendocrine progenitor, a cell not as yet defined in the human gastrointestinal tract. The precise mechanisms underlying the lineage pathways of neuroendocrine cells and their precursors remains poorly defined but the Notch signaling pathway is implicated in regulation of cell differentiation from stem cells [152]. Basic helix-loophelix transcription factors Math1, NGN3, and beta2/ Fig. 10 The distribution and role of serotonin secreting EC cells within the gastrointestinal tract. EC cells are ubiquitous throughout the gut and represent 0.25–0.5% of the total mucosal volume (left). They are chemomechanosensory cells (center) and respond both to luminal products and mechanical activity of the bowel by serotonin (5-HT) secretion. Locally produced serotonin regulates mucosal secretion and Langenbecks Arch Surg (2011) 396:273–298 NeuroD are expressed in neuroendocrine precursors although Notch is inactive. Precursor cells induce Notch in adjacent cells, switching off neuroendocrine differentiation. Math1 commits cells to one of three secretory lineages: goblet, paneth, and neuroendocrine while NGN3 appears to be essential for neuroendocrine cell differentiation [9]. The regulatory role of these transcription factors is considered essential for final entero-endocrine cell specification [16]. Typical small bowel EC cell tumors display an insular growth pattern (type I), which consists of solid nests or cords of cells with clearly defined boundaries [153]. A trabecular pattern (type II) consists of narrow cell bands forming ribbons, regularly anastomosing along a highly differentiated vascular network. Type III has a glandular pattern, consisting of cells arranged in alveolar, acinar, or rosette patterns with glandular cavities or pseudo-cavities. Type IV and V NETs consist of undifferentiated and mixed cells, respectively. Multifocal lesions are evident in ~30% of small bowel EC cell tumors [153]. Most of tumors develop as independent primary lesions, and only a minority are due to metastasis from a single primary suggesting a polyclonal series of synchronous neoplastic events [154]. In general, hyperplasia of neuroendocrine cells in the associated mucosa is also evident [155, 156]. Transmural invasion and an extensive local desmoplastic response are common features contributing to the aggressive local behavior of the neoplasm [153]. Local spread into the adjacent mesentery and peritoneum are common as are regional lymph node and distant metastases. The latter are predominantly hepatic but also may involve lung, bone, and absorption as well as peristalsis and secretory reflexes (right). Abnormalities, for example increased EC cell numbers (e.g., small intestinal NETs), result in excess 5-HT production with accentuation of normal physiological events, for example increased mucus secretion, secretory diarrhea, and excessive peristalsis Langenbecks Arch Surg (2011) 396:273–298 brain [153]. The tumor cells are characteristically argyrophil and argentaffin [153] and over 85% of the tumors exhibit positive immunohistochemistry for CgA, Leu-7, NSE, and 5-HT [153]. The vast majority of these lesions are “classical” ileal carcinoids with production of 5-HT and substance P, but rare tumors producing enteroglucagon, PP, or peptide YY may occur. EC cell NETs exhibit the highest frequency of non-NET tumor association, for example colorectal cancers (39%) [157, 158]. Other associated non-endocrine tumors include adenocarcinomas of the small bowel, stomach, lung, prostate, and cervix uteri [159]. Several factors appear to be determinants of malignancy, including lesion size, local spread and extent of metastases at the time of diagnosis, mitotic rate, multiplicity, female gender, depth of invasion, and the presence of carcinoid syndrome [153]. Epidemiology and survival Small intestinal EC cell tumors are the second most frequent type of NET (17.3% in the 2007 SEER analysis) [160]. The highest frequency of small intestinal NETs is in the ileum, and is ~7 times more frequent than in the duodenum and the jejunum [158, 161]. Small intestinal NETs exhibit an overall higher frequency of metastases at the time of diagnosis (~60% of staged tumors, SEER) compared to all GEP-NETs (26% of staged tumors SEER) [1]. The overall 5-year-survival rate is 68.1% [1]. The 5year-survival rate of patients with hepatic tumor spread is 18–32% [1]. An increased median survival (4.4 years) is evident in patients with jejuno-ileal carcinoids which exhibit a mixed insular/glandular pattern [162]. In contrast, patients with an undifferentiated pattern have a median survival of only 6 months. In those lesions with a pure insular and trabecular pattern, an intermediate prognosis is evident with a median survival time of 2.9 years and 2.5 years, respectively [162]. The relatively poor prognosis of small intestinal NETs reflects the inherent clinical difficulty in identifying small bowel malignancies (Table 3), as well as the intrinsically malignant nature of the tumor with dissemination to both the lymph nodes and the liver. Molecular markers Comparative genomic hybridization strategies have identified gains in chromosomes 17q and 19p (57%) and in 19q and 4q (50%) in EC cell tumors [163]. Gains were also found in 4p (43%), 5 (36%), and 20q (36%) and losses in 18q or 18p (43%), while 21% had full or partial loss of 9p [163]. Of 14 tumors, six had full gain of chromosome 4 of which four samples also had gain of chromosome 5. There 289 were four tumors with a gain of chromosome 4 along with a partial or full trisomy of chromosome 14 [163]. In a separate CGH study, losses in 18q22-qter (terminal end of chromosom18q) (67%) and 11q22-23 (33%) were the most common genetic defects although losses of 16q (22%) and gains of 4p (22%) were also identified [164]. Of note, since the 18q and 11q chromosomal losses occurred more frequently this suggests that they are early events in EC cell tumorigenesis while a loss on chromosome 16 and some gain-of-function on chromosome 4 are later events in tumor/carcinoid development (Fig. 9 bottom). This proposal is supported by a report that aberrations in 16q and 4p tend to occur in metastases [165]. Lollgen et al. reiterated the notion that 18q deletions were characteristic of midgut NETs by finding losses in 88% of tumors [166]. These findings, particularly losses and gains in chromosomes 18 and 14 have been confirmed by more recent reports [167, 168]. One of the genes encoded on Chr18 (18q21) is the tumor suppressor gene DCC (deleted in colorectal carcinoma). Loss of this gene, which has been linked to the tumor suppressor NCAM (neural cell adhesion molecule) on 11q [169], is thought to play a role in carcinoid genesis [170]. A 40 kb heterozygous deletion in Chr18q22.1 has been suggested as a potential inherited factor, but the low occurrence of this (only ~6% of cases) make it difficult to appreciate the real significance [171]. A gain of chromosome 14 has been identified as a marker of poor prognosis [167], while the anti apoptotic protein DAD1 has been identified in one of the chromosome 14 foci, and confirmed to be overexpressed at an immunohistochemical level [168]. A separate CGH study identified that ~20% of EC cell tumors exhibited alterations in the distal part of 11q (location of succinate ubiquinone oxidoreductase subunit D gene—SDHD) [165]. Furthermore, two of five EC cell tumors exhibited a missense mutation in the SDHD gene in association with LOH of the other allele, suggesting that alterations of the SDHD gene might be implicated in the tumorigenesis of these lesions [165]. An analysis of microsatellite instability in well-differentiated EC cell tumors or their metastases using an analysis of the BAT26 microsatellite locus in intron 5 of hMSH2 and the BATII microsatellite region of TGFβRII [172] identified no MSI. In contrast, carcinomas of the small intestine exhibit MSI in approximately 20% of cases [173, 174], suggesting that neuroendocrine cell tumors probably evolve differently to epithelial tumors in this organ [172]. Affymetrix transcriptional profiling has identified >1,500 over-expressed and ~400 transcripts that are decreased in expression in a large group (~30 samples) of EC cell tumors [175]. Further analysis of this data identified three potentially useful malignancy-marker genes. Specifically, over expression of NAP1L1, MAGE-D2, and MTA1 mRNA and MTA1 290 Table 3 Gastroenteropancreatic neuroendocrine (GEP-NET) classification 2010 (based on [201]) Langenbecks Arch Surg (2011) 396:273–298 Nomenclature Type NET G1 NET G2 Stomach Neuroendocrine tumor G1 (carcinoid)* Neuroendocrine tumor G2** Gastric NET (ECL cell)† Gastrin-producing NET (G cell) Serotonin-producing NET (EC cell) ACTH-producing NET Serotonin-producing NET (EC cell) Somatostatin-producing NET Gangliocytic paraganglioma (periampullary) Non-functional pNET Insulinoma Glucagonoma Gastrinoma Somatostatinoma VIPoma, PPoma Duodenum Pancreas Appendix *G1: <2 mitoses per 10 highpower field (HPF) and/or ≤2% Ki67-index **G2: 2–20 mitoses/10 HPF and/or 3–20% Ki67-index ***G3: >20 mitoses/HPF and/or >20% Ki67-index Bowel NEC G3 † Associated with autoimmune chronic atrophic gastritis (ACAG) or MEN1-ZES Appendix protein in tumor and metastatic EC cell NETs was confirmed suggesting these genes may be markers for identifying metastatic tumors while NAP1L1 may be a neuroendocrine tumor-specific marker [175]. Expression of these markers as well as CgA has been demonstrated as effective in the prediction of EC cell tumor grade and stage [176]. Other candidate marker genes have been identified in EC cell carcinomas [177], the utility of which are still being examined. Over-expression of MTA1 has been confirmed [178]. Analysis of microRNAs in EC cell tumors has identified the cardiac-specific miRNA-133a to be down regulated in metastases [179]; the relevance of this observation remains to be determined. The Swedish Family Cancer database study identified an increased risk of EC cell NETs among the offspring of patients with squamous cell skin cancer (RR 1.79) and nonHodgkin’s lymphoma (RR 2.06), while the relative risk of this tumor was 2.21 for individuals whose mother had endometrial cancer [31]. The offspring of patients diagnosed with EC cell NETs had an increased risk of cancer of the breast (RR 1.39), kidney (RR 2.08), and brain (RR 1.65). This and other epidemiological-based studies [31, 180] suggest that an increased risk of developing EC cell Serotonin-producing NET (carcinoid) Serotonin-producing NET (EC cell) Tubular carcinoid L-cell, glucagon-like peptide and PP/PPY-producing NETs Serotonin-producing tumor (EC cell) L-cell, glucagon-like peptide and PP/PPY-producing NETs Neuroendocrine carcinoma*** Small cell NEC Large cell NEC Goblet cell carcinoid (mixed adenoneuroendocrine carcinoma (MANEC)) NETs occurs in individuals with a parental history of the disease while a family history of any cancer, that is, not only NETs, is a risk factor for the development of these tumors [181]. This study could not identify predisposing genetic factors but suggests that gene mutations common to these different tumors may play a role in triggering EC cell NET development. Genetic analyses in a family with three consecutive first-degree relatives [182] could not identify that inheritance of EC cell NETs was linked to MEN1. This, and other studies [3, 183], provide further support that inheritance of the tumor in this location (small intestine) is not linked to the MEN1 syndrome. At a growth-regulatory level, EC cell NETs, which do not express mutations of the menin gene are, however, characterized by a loss-of-responsiveness to TGFβ1mediated growth inhibition that characterizes normal small intestinal EC cell proliferation (Fig. 11) [184]. Colorectal NETS Colorectal “carcinoid” tumors comprise ~35% of all gastrointestinal NETs and 25% of all NETs [65]. The Langenbecks Arch Surg (2011) 396:273–298 Fig. 11 Proliferative regulation of EC cells. Proliferation is differentially regulated by a variety of growth factors through activation of a number of signal transduction pathways such as AKT/ERK/SMAD and mTOR pathways. The somatostatin receptor system is the best characterized negative regulator and signal transduction is predominantly via the p38/cGMP pathway. It is likely that the majority of neuroendocrine cells exhibit similar signal transduction circuitry distribution is cecum (~11%), ascending colon and appendix (~22%), transverse and descending (~3%), sigmoid colon with recto sigmoid junction (~10.5%), and 51% rectum. The increased distribution in the right colon may be explained by a higher density of neuroendocrine cells in this region or reflect inability to clearly differentiate terminal ileal lesions that have invaded the cecum [185]. 291 increase) [189]. The true incidence of colorectal NETs, however, is likely higher since these tumors are considered benign by some pathologists who do not therefore register the tumor for the SEER database. Colon NETs proximal to the rectum appear more aggressive, with a 5-year survival of ~62% across all stages [190]. Data for the 5-year-survival rates of colonic NETs, however, are inconsistent. Some data report 80% [191], and others 6% [192] which are worse than colonic adenocarcinoma (~43%) [191]. The heterogeneity is likely due to a recruiting bias of NETs and NECs in different series or inclusion of lesions of different as yet unclassified neuroendocrine cell types. An analysis of ~8,000 colorectal NETs demonstrated a 5-year-survival rate of 41.6% compared to 60.9% in colonic adenocarcinomas [185]. Rectal NETs are associated with the highest 5-yearsurvival rate of 88% in comparison to other NET primaries [190]. This finding reflects that most of rectal carcinoid tumors (82%) [190] are localized at diagnosis, with a median size of only 0.6 cm. Tumor size, depth of invasion, and lymph node involvement significantly predict malignant behavior in localized rectal NETs. A literature analysis reported that metastases were observed in 2% of patients with rectal NETs measuring less than 1 cm, 10–15% of tumors measuring 1–2 cm, and 60–80% in patients with tumors measuring greater than 2 cm [193]. Another study has shown that metastases occurred in only 2% of tumors smaller than 2 cm, which had not invaded the muscularis propria, compared to 48% in tumors with muscularis invasion [194]. Five-year-survival rate in rectal NET is 72–92% depending on selection bias for small tumors. Overall, the prognosis is substantially better than for rectal adenocarcinoma which exhibits an overall 5-year-survival rate of 46–59% [191, 195]. Pathogenesis and pathology Molecular markers Little is known about these tumors in terms of pathogenesis. Due to the embryonic development of the mid- and hindgut, two types of common well-differentiated endocrine NETs have been identified in the colon and rectum: Lcell tumors and EC cell tumors [186]. Rectal tumors are usually L-cell tumors, producing glicentin-related products and PP-PYY peptides. EC tumors with typical 5-HT production are rare in the colon and rectum [187, 188]. Specific markers that identify rectal NETs include those that identify L cells, such as glucagon- 29, glucagon-37, glicentin, PYY, and PP and their precursors. Epidemiology and survival The incidence of colorectal NETs has increased from approximately 0.2 per 100,000 in 1973 to 0.86 per 100,000 per year in the 2004 SEER database (430% Neuroendocrine marker molecules differentially expressed in large bowel NETs include α/β-SNAP and synaptophysin as well as SNAP25. In contrast, CgA and VAMP2 are less frequently expressed [196]. Proximal colonic tumors are usually EC cell tumors with similar markers to small intestinal NETs. Metastatic colonic disease is only rarely associated with the carcinoid syndrome and only a small fraction of hindgut NETs (<1%) produce and secrete 5-HT or other bioactive hormones [193]. Therefore, measurement of serum 5-HT or urine 5-hydroxyindoleaceticacid (5-HIAA) is not recommended. Poorly differentiated small cell carcinomas of the large bowel usually have extensive expression of synaptophysin and cytosolic markers of neuroendocrine differentiation like PGP9.5 and NSE. Prostate-specific acid phosphatase is expressed in 80–100% of rectal carcinoids [187], and may be useful clinically. Serum CgA can be of 292 some utility for monitoring patients with metastatic disease [197, 198] or for surveillance in patients with resected stage II or III tumors. False positive elevations in CgA are frequently associated with the use of proton-pump inhibitors. Elevated levels of CgA can also occur in patients with chronic gastritis or other inflammatory diseases. P53 may be useful as a marker of poorly differentiated tumors. Immunohistochemistry for somatostatin receptor-2A may be performed in specialized laboratories. βHCG may be expressed, and is associated with greater malignancy of the lesions [199]. Identification of lesions with high malignant potential can be determined by mitotic indexing and Ki67 percentage staining to determine the tumor proliferative index [200]. Conclusion In this review, we examined GEP-NETs, focusing on the distinct patterns of each type, by examining both the cell type and organ of origin. GEP-NETs are derived from distinct cell precursors, undergo specific chromosomal abnormalities/ somatic mutation events, have different clinical presentations (are either syndromic or not), and are associated with widely differing survival rates reflecting the different proliferative regulation as well as intrinsic difference in malignant potential of different neuroendocrine cell types. In the past, these tumors were considered under a common rubric “carcinoid” as indolent and uncommon. Overall NETs occur far more frequently than previously considered and exhibit distinct cellular and clinical behaviors; each cell-specific lesion should therefore be considered and examined as a separate entity. In general the tumors exhibit both commonalities as well as significant diversity. The former include common secretory machinery and physiological responses (neural/hormonal regulators), a relative high proportion of sporadic tumors and development from precursor cell types. Overall there is a paucity of information regarding transformation from the naïve to the neoplastic cell state except that microsatellite instability does not occur often and adenocarcinomaassociated oncogenic events, for example K-RAS mutations are very uncommon. Differences include restrictions in locoregional distribution (e.g., gastric ECL cell in the oxyntic mucosa compared to extensive distribution of EC cells), significantly different growth patterns such as the relative benign nature (high long-term survival) of some organrestricted cell specific tumors, particularly gastric Type I gastric NETs and pancreatic β-cell NETs (insulinomas) compared to others, for example colonic or non-functioning pNETs; the significant difference in chromosomal abnormalities (and potentially etiopathogenesis) between pNETs and small intestinal NETs; and the relative lack of information regarding growth regulation (except for gastric ECL cells). Langenbecks Arch Surg (2011) 396:273–298 Conflicts of interest None. References 1. Modlin IM, Oberg K, Chung DC, Jensen RT, de Herder WW, Thakker RV, Caplin M, Delle Fave G, Kaltsas GA, Krenning EP, Moss SF, Nilsson O, Rindi G, Salazar R, Ruszniewski P, Sundin A (2008) Gastroenteropancreatic neuroendocrine tumours. 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