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Pancreatic Neuroendocrine Tumors: Pathologic and Molecular Characteristics Chanjuan Shi MD, PhD, David S. Klimstra MD

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To appear in: Seminars in Diagnostic Pathology

Cite this article as: Chanjuan Shi MD, PhD, David S. Klimstra MD, Pancreatic Neuroendocrine Tumors: Pathologic and Molecular Characteristics, Seminars in Diagnostic Pathology, http://dx.doi.org/10.1053/j. semdp.2014.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pancreatic Neuroendocrine Tumors: Pathologic and Molecular Characteristics 1

Chanjuan Shi, MD, PhD and 2David S. Klimstra, MD

1

Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN; 2Department of Pathology, Memorial Sloan Kettering Cancer Center, New York City, NY

Corresponding Author: David S. Klimstra, MD Department of Pathology Memorial Sloan Kettering Cancer Center 1275 York Avenue New York, NY 10065

ABSTRACT Pancreatic neuroendocrine neoplasms include mainly well-differentiated neuroendocrine tumors, but also rare poorly-differentiated neuroendocrine carcinomas. Molecular mechanisms underlying pancreatic neuroendocrine tumorigenesis have recently been elucidated. While alterations in the chromatin remodeling and PI3K/Akt/mTOR pathways are present in most well-differentiated pancreatic neuroendocrine tumors, mutations in TP53 and RB may contribute to the development of pancreatic poorly-differentiated neuroendocrine carcinomas. With these discoveries, new molecular targeted therapies have become available and show promise in some patients with pancreatic well-differentiated neuroendocrine tumor.

INTRODUCTION Pancreatic neuroendocrine neoplasms, including well-differentiated neuroendocrine tumors (PanNETs) and poorly-differentiated neuroendocrine carcinomas (NECs), are malignant neoplasms with significant neuroendocrine differentiation. Although rare, they are the second most common neoplasm of the pancreas, accounting for approximately 5% of all pancreatic malignancies. The annual incidence is less than 1 per 100,000 persons per year in the general population;1-4 however, autopsy studies have shown a much higher prevalence.4 In addition, the incidence of PanNETs has steadily increased over the last 40 years,1, 3 partly due to increased awareness of these tumors among clinicians and widespread use of modern imaging techniques.

According to the World Health Organization (WHO) 2010 classification of digestive neuroendocrine tumors, neuroendocrine neoplasms of the pancreas are divided into 3 grades: well-differentiated PanNET WHO grade 1, well-differentiated PanNET WHO grade 2, and poorly-differentiated pancreatic NEC, WHO grade 3.5 The vast majority of pancreatic neuroendocrine neoplasms are well-differentiated PanNETs. PanNETs can be classified based on clinical symptoms into functional and nonfunctional tumors, with nonfunctional tumors accounting for more than 50% of the total cases in contemporary studies. While most PanNETs occur sporadically, some are associated with a genetic syndrome, such as the multiple endocrine neoplasia type 1 (MEN1) and von HippelLindau (VHL) syndromes.6

Surgery is the only curative treatment for PanNETs and is recommended only when complete resection is possible. However, more than half of patients present with distant metastatic disease at initial diagnosis,3 and most PanNETs are incurable once they have metastasized. Although PanNETs are associated with a much better prognosis compared to pancreatic ductal adenocarcinoma, the 5-year survival rate is still less than 40%.1, 3 With recent discoveries in PanNETs, new molecular targeted therapies have become available and show promise in some patients with PanNETs. In this review, we discuss clinical, pathologic and molecular characteristics of PanNETs.

CLINICAL FEATURES Most patients with PanNETs are in the sixth and seventh decade of life, with a slight male predominance.1, 2 Nonfunctional PanNETs are not associated with a hormone hypersecretion syndrome, nonetheless these tumors secrete a number of substances, such as pancreatic polypeptide and chromogranin, and may secrete hormones at levels insufficient to cause symptoms.7 Due to lack of specific symptoms, nonfuctional PanNETs tend to be diagnosed at more advanced stages, with a large primary tumor, invasion to adjacent organs, or distant metastases, compared with functional PanNETs such as insulinomas.3 However, with increasing numbers of PanNETs being discovered incidentally, more patients with nonfunctional PanNETs are being diagnosed at an early stage.

Nine well-established syndromes have been described to be associated with functional PanNETs. Insulinoma is considered the most common functional tumor, followed by gastrinoma, VIPoma and glucagonoma (Table 1). Other rare functional PanNETs with a

well-defined syndrome include somatostatinoma, GRHoma, ACTHoma, PanNET causing carcinoid syndrome, and PTHrp-oma.8, 9 These tumors secrete a specific hormone that causes a characteristic clinical syndrome. While most insulinomas are associated with an indolent clinical course, other functional tumors, such as the glucagonoma, are mostly aggressive.

GENETIC SYNDROMES While most PanNETs are sporadic, some of them occur in patients with an underlying genetic syndrome, including the MEN-1 syndrome, VHL syndrome, neurofibromatosis type 1 (NF-1), and tuberous sclerosis complex (TSC). Table 2 outlines their inheritance pattern, the specific gene involved, and clinical features of these syndromes. The occurrence of PanNETs in patients with these genetic disorders raises the potential that these same genes may be involved in the pathogenesis of sporadic PanNETs as well (see below).

MEN1 Syndrome Up to 100% of patients with MEN1 syndrome have PanNETs, which are usually multiple and occur in younger patients than sporadic PanNETs. While most PanNETs seen in MEN1 patients are nonfunctional, some are functional tumors.6 Of note, duodenal NETs are also very common in MEN1 patients, and most are gastrinomas. These small duodenal gastrinomas can metastasize to peripancreatic lymph nodes and mimic a pancreatic primary. The pancreas in MEN1 patients usually demonstrates numerous microadenomas and enlarged or architecturally abnormal islets (hyperplasia and dysplasia, respectively) in addition to the frank PanNETs. Loss of heterozygosity at

11q13 (containing the MEN1 gene) is always detected when islets transform into microadenomas in the MEN-1 patients.10, 11

VHL Syndrome PanNETs occur in 11-17% of VHL patients, with a median age of 37 years.12 VHLassociated PanNETs are often small and multifocal, and they are almost always nonfunctional.13 VHL-associated PanNETs can have clear cell features, and there is a propensity for mixed serous-neuroendocrine tumors. In addition, VHL-associated PanNETs are associated with a better prognosis compared to sporadic PanNETs. Patients with the VHL syndrome usually do not have numerous microadenomas and abnormal islets, at least based on evaluation of routinely stained sections, but abnormal islets can be detected by immunohistochemistry based on aberrant expression of hypoxia induced factor 1 (HIF-1

and Carbonic Anhydrase IX.14

NF-1 Up to 10% of NF-1 patients develop NETs, most of which are non-functional glandular NETs that express somatostatin and arise in the periampullary duodenum (so-called “ampullary somatostatinoma” or “psammomatous somatostatinoma”).6 Rare functional and nonfunctional PanNETs have also been reported in these patients. 6

TSC Both functional and nonfunctional PanNETs have been reported in a small percentage of patients with TSC.15 It appears that PanNETs predominantly occur in patients with a germline TSC2 mutation.

PATHOLOGY PanNETs are well-differentiated tumors by definition. Only a small group of pancreatic neuroendocrine neoplasms are poorly-differentiated; these are termed poorlydifferentiated NECs.

WHO Classification The 2010 WHO classification uses both mitotic count and Ki67 index to assign pancreatic neuroendocrine neoplasms into grade 1 PanNETs (mitoses20%, Figure 2). In addition, nonfunctional tumors measuring less than 0.5 cm are designated neuroendocrine microadenomas. The Ki67 index is to be based on counting 500-2000 cells in the highest labeling regions within the tumor (“hot spots”) identified by scanning at low power.16, 17 Mitoses should be counted in 50 high power fields to ensure accuracy. Similar to Ki67, different parts of the tumor may have different mitotic rates, so pathologists should carefully evaluate all tumor sections and should perform the count on the section with the highest mitotic rate. Discordance between Ki67 index and

mitotic count occurs in about one third of the cases.18 In such cases, the higher grade should be assigned.

Poorly-differentiated NECs, by definition, have a mitotic rate > 20/10 HPF and a Ki67 index >20%, but the latter is usually >50%. Occasionally, neuroendocrine tumor with relatively uniform nuclei and other features more typically seen in well-differentiated PanNETs have a Ki67>20% (Figure 3). Based on the current WHO criteria, these tumors are classified as WHO grade 3, NEC. However, they have a typical morphology of well-differentiated tumors, a mitotic rate usually 20% or a mitotic rate >20/10 HPF. NECs include small cell carcinoma and large cell neuroendocrine carcinomas. They are rare in the pancreas, and are associated with early metastasis and poor outcomes.28 Most pancreatic poorly-differentiated NECs are large cell NEC. Approximately 70% are located in the head of the pancreas, with a median tumor size of 4 cm. Grossly they are described as a tan-red or yellowish, solid masses that are relatively circumscribed, frequently hemorrhagic, and occasionally necrotic. 28 Microscopically, large cell NECs are composed of round to polygonal cells with moderate amounts of cytoplasm and round nuclei with vesicular chromatin or prominent nucleoli, forming organoid or nested structures, trabeculae, or rosettes (Figure 2). Apoptotic cells and mitoses are abundant. Expression of at least one neuroendocrine marker (chromogranin or synaptophysin) is required to diagnose large cell NEC.

Small cell carcinoma of the pancreas shares similar morphology to its lung counterpart, and metastases to the pancreas always have to be ruled out before establishing the

diagnosis. Microscopically, the carcinoma present with sheets or nests of small cells with a high nucleus-to-cytoplasm ratio, hyperchromatic and finely granular chromatin, inconspicuous nucleoli, and nuclear molding. There are confluent areas of necrosis, extensive apoptotic cells, and abundant mitoses (Figure 2). The morphology itself is diagnostic of small cell carcinoma, and expression of neuroendocrine markers is not required for the diagnosis. However, given the rarity of both types of poorlydifferentiated NEC in the pancreas, other diagnostic possibilities (acinar cell carcinoma, mixed acinar carcinomas, primitive neuroectodermal tumor, etc.) should be excluded before rendering this diagnosis.

Immunohistochemical Studies Although immunohistochemistry may not be needed to diagnose some primary PanNETs, immunolabeling for chromogranin A and synaptophysin, the two most sensitive and specific general neuroendocrine markers, can be used to confirm the diagnosis. Most PanNETs express synaptophysin and chromogranin A with synaptophysin expression being more diffuse and intense than chromogranin. A number of markers have adverse prognostic significance in PanNETs, including CK19, CD117, CD99, CD44, p27, c-Met, progesterone receptor (loss), and phosphatase and tensin homolog (PTEN, loss); however, these biomarkers have not been validated in a large cohort and are not in general use. Ki67 is the only well-established biomarker that shows independent prognostic significance18, 29, 30 and is in widespread use for grading, as discussed above.

PanNETs and other primary NETs share similar morphology; however, they have a different genetic basis and biologic behavior, and are therefore sometimes treated differently. Discrimination of metastatic PanNETs from an extra-pancreatic primary is therefore of clinical importance. NETs primary to the lung frequently express TTF-1, which is not expressed by PanNETs; however, TTF-1 can be expressed by small cell carcinoma of non-pulmonary sites. The vast majority of intestinal NETs express CDX2; however, CDX2 expression is seen in approximately 10% of PanNETs. Islet 1 gene product (ISL1), pancreatico-duodenal homeobox 1 gene product (PDX1), and PAX8 were thought to be specific markers for NETs of pancreatic origin. However, recently studies have demonstrated that these markers can also be expressed by NETs of other origins.31-35 More sensitive imaging techniques using somatostatin receptor (SSTR) imaging can also be helpful to identify an occult primary NET.

A number of other neoplasms primary to the pancreas can mimic PanNETs. Mixed acinar neuroendocrine carcinoma can mimic pancreatic poorly-differentiated NECs. Immunohistochemical studies for neuroendocrine and acinar cell markers aid the diagnosis of mixed tumors. Solid-pseudopapillary neoplasms (SPNs) are another hypercellular pancreatic neoplasm that shares with PanNETs features such as solid and nesting growth patterns, stromal sclerosis, eosinophilic hyaline globules, and expression of synaptophysin and CD56. A number of immunohistochemical stains can differentiate SPN from PanNET; SPN shows abnormal β-catenin nuclear accumulation, loss of cell surface E-cadherin expression, CD10 expression, and does not express chromogranin.

Activation of SSTRs (inhibitory G-protein coupled receptors) by somatostatin and its analogs leads to inhibition of hormone secretion and cell proliferation. Five subtypes, including SSTR1, SSTR2A, SSTR3, SSTR4, and SSTR5, have been characterized, all of which have been detected in PanNETs.36-38 SSTR2A is expressed by most PanNETs and shows high affinity for somatostatin analogs (SSAs). It has been used as a target for molecular imaging and treatment of PanNETs.39-44 Immunohistochemical staining for SSTR2A is more widely performed in Europe.

MOLECULAR GENETICS AND SIGNALING PATHWAYS Recent advances in next generation sequencing technologies have greatly facilitated the discovery of the molecular basis of PanNETs. Unlike pancreatic ductal adenocarcinoma, well-differentiated PanNETs harbor no mutations in KRAS, CDKN2A, and SMAD4, and only 4% have TP53 mutations. Instead, the most frequently mutated genes in PanNETs are those implicated in chromatin remodeling, including MEN1 (consistent with the occurrence of PanNETs in patients with MEN1 syndrome), death domain- associated protein gene (DAXX), and alpha thalassemia/mental retardation Xlinked gene (ATRX).45 In addition, approximately 15% of PanNETs harbor a mutation in the genes in the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway (which relates to their occurrence in VHL syndrome and tuberous sclerosis). Dysregulation of the PI3K/Akt/mTOR signaling pathway has also been demonstrated by altered expression of proteins in the pathway. In addition, the TP53/RB signaling pathways have been implicated in the tumorigenesis of NECs, as mutations in these genes are present in pancreatic poorly-differentiated NECs (Table 3).

Chromatin Remolding Pathway Approximately 45% of sporadic PanNETs harbor mutually exclusive mutations in either DAXX or ATRX45. DAXX and ATRX encode nuclear proteins which form a chromatin remolding complex and are involved in chromatin remolding at telomeric and pericentromeric regions. Mutations of these genes are associated with loss of DAXX/ATRX protein expression. The loss of protein expression shows a near 100% correlation with the alternative lengthening of telomere (ALT) phenotype, a telomerase-independent pathway for telomere maintenance.46 Comparative genomic hybridization array studies have demonstrated chromosome instability (CIN) in PanNETs with loss of DAXX/ATRX and ALT. DAXX/ATRX loss followed by ALT may lead to chromosomal instability, resulting in a large spectrum of mutations and chromosomal abnormalities,47 and eventually promote tumor progression. In addition, studies have shown that loss of DAXX/ATRX proteins and the ALT phenotype are late events in the development of PanNETs.47, 48 The relationship between mutations in DAXX/ATRX and prognosis is complicated with conflicting reports in the literature depending on whether primary or metastatic lesions are studied.45, 47

MEN1 is mutated in PanNETs arising from patients with MEN1 syndrome and approximately 50% of sporadic PanNETs.45 MEN1 encodes menin, which serves as both an activator and a repressor of gene transcription via interaction of different chromatin modification complexes. Menin interacts with MLL-HMT complex, a histone methyltransferase (HMT) complex that mediates histone H3 K4 methylation, and

activates transcription of antiproliferative genes such as cyclin-dependent kinase inhibitors, consequently resulting in inhibition of cell proliferation.49, 50 In addition, menin participates in histone deacetylation through interactions with complexes of the general transcription repressor, histone deacetylases-mSin3A complexes, leading to repression of transcription of genes targeted by the transcription factor JunD.51 JunD is a growth suppressor when it is bound with menin, and a growth promoter when it is free from menin. Disruption of the JunD-menin interaction due to loss of menin may be one of the tumorigenetic mechanisms underlying both sporadic and hereditary PanNETs with MEN1 mutations.

PI3K/Akt/mTOR Signaling Pathway Genetic and expression studies have revealed that both aberrant expression and genetic alterations of the PI3K/AKT/mTOR signaling pathway are frequently present in PanNETs (Figure 12). In normal cells, the activation of growth factor receptors, including receptors for vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor (PDGFR), triggers the PI3K/Akt/mTOR signaling pathway. These receptors and/or their factors are frequently overexpressed by PanNETs, 52 and have been implicated in the carcinogenesis of PanNETs. In addition, overexpression of VEGF by PanNETs may contribute the rich vascular networks of the tumor.

Activation of the PI3K/Akt/mTOR signaling pathway can lead to cell proliferation/survival, invasion, and angiogenesis through activation of mTOR downstream targets, including p70S6 kinase (S6K), eukaryotic initiation factor 4E-

binding protein (4E-BP1) and HIF-1α (Figure 12). The tumor suppressor proteins TSC1/2 and PTEN are the two key negative regulators of the pathway. In addition, S6K negatively regulates the signaling pathway through suppression of insulin receptor substrate-1, the adaptor protein linking IGF-1 to PI3K. Altered activation of Akt, mTOR and S6K has been reported in PanNETs and is correlated with tumor invasion, proliferation, and tumor progression.53, 54 On the other hand, TSC2 and PTEN are commonly downregulated in PanNETs, and their low expression is significantly associated with shorter disease-free and overall survival.55

Whole-exome sequencing studies have demonstrated that genes in the mTOR pathway are frequently mutated in PanNETs. In the first report of whole exome sequencing of PanNETs, 10 of 68 (15%) sporadic PanNETs harbored mutations in one or more genes in the pathway, including three with a PTEN mutation only, five with a TSC2 mutation only, one with mutations in both PIK3CA and PTEN, and one with mutations in both PTEN and TSC2.45 Additionally, a missense single-nucleotide polymorphism (SNP) of the FGFR4 (fibroblast growth factor receptor 4) gene may be involved in PanNET progression.56

The mTOR pathway also plays an important role in the development of hereditary PanNETs. pVHL inhibits angiogenesis through inhibition of HIF-1 .57 PanNETs in VHL patients have VHL dysfunction, which may lead to enhanced HIF-1 activity and consequently promote tumor angiogenesis. TSC1 or TSC2, mutated in patients with TSC and in sporadic PanNETs, are also key molecules in the PI3K/Akt/mTOR signaling

pathway. In addition, studies have shown that menin is involved in repression of the PI3K signaling pathway.58 Enhancement of the PI3K signaling pathway due to loss of menin function may be one of the mechanisms underlying the development of PanNETs in patients with MEN1 syndrome.

TP53/RB Signaling Pathways Mutations in TP53 and RB are rare in well-differentiated PanNETs, with only 4% with a mutation in the TP53 gene and none in the RB gene.45 In addition, immunohistochemical studies only detect rare p53 abnormalities in PanNETs. 59, 60 However, dysregulation of the TP53 pathway may still be involved in the initiation or progression of many PanNETs, as gene amplification and protein overexpression of three p53 negative regulators, MDM2, MDM4, and WIP1, are detected in some PanNETs.60 Aberrant activation of these negative regulators may down-regulate p53 function, which could be an important mechanism underlying PanNET tumorigenesis.

Dysregulation of the RB pathway may also contribute to pancreatic neuroendocrine tumorigenesis. In mice, genetic inactivation of the RB pathway leads to PanNETs.61, 62 In addition, gene amplification and overexpression of two Rb negative regulators, Cdk4 and Cdk6, are detected in a majority of PanNETs, which correlates increased expression of phosphorylated Rb-1.63

Although they are rare in well-differentiated PanNETs, mutations of TP53 and RB are more common in pancreatic poorly-differentiated NECs. A recent study has shown that

most pancreatic NECs abnormally immunolabel for p53 (nuclear expression) and Rb (loss of expression).59 In this study, the abnormal expression of these proteins correlated with intragenic mutations in the genes coding for these proteins. By contrast, DAXX and ATRX expression, which is frequently lost in well-differentiated PanNETs, was intact in these NECs. These findings suggest that poorly-differentiated NECs of the pancreas are genetically distinct from well-differentiated PanNETs.

MANAGEMENT Surgery is the treatment of choice for most PanNETs. When patients with unresectable PanNET are symptomatic, or have progressive disease, chemotherapy, biotherapy and molecular targeted therapies can be used. Debulking surgery or hepatic regional therapy may be considered in those with severe hormone-related symptoms insufficiently controlled by medical therapy.

Chemotherapy Streptozotocin-based chemotherapy is often used for patients with inoperable progressive liver metastasis from PanNETs.64-66 In addition, drug regimens including temozolomide and capecitabine are promising.67 The efficacy of temozolomide may rely on O(6) methylguanine DNA methyl transferase (MGMT) expression; lack of MGMT expression due to promoter methylation confers sensitivity to temozolomide in PanNETs.68 Immunohistochemistry for MGMT expression can be used to identify this subgroup of patients.

Somatostatin Analogs SSAs have been used to treat PanNETs and have effects limiting hormonal symptoms but also can prolong survival.40, 42-44, 69-75 In addition, peptide-receptor radionucleatide therapy with radiolabeled SSAs may be a promising new tool in the management of patients with inoperable or metastasized SSTR-expressing PanNETs.41, 76 Immunohistochemical assessment of SSTR2A expression can be used to identify patients who will benefit from these therapies.

Molecular Targeted Therapies Recently, two small-molecular inhibitors, everolimus and sunitinib, that interfere the PI3k/Akt/mTOR signaling pathway, have shown anti-tumor activity in patients with advanced PanNETs, and were approved by the Federal Drug Administration in 2011. The identification of molecular alterations in the mTOR pathway may explain the efficacy of this therapeutic approach. Everolimus, a mammalian target of rapamycin complex 1 (mTORC1) inhibitor, has been shown to achieve an objective response rate of 10% and a stable disease rate of 68%, and to significantly prolong progression-free survival among patients with progressive advanced PanNETs as compared with placebo.77, 78 Sunitinib, a tyrosine kinase inhibitor for several growth factor receptors, including VEGFR and PDGFR, has demonstrated clinically meaningful improvements in progression-free survival in patients with advanced PanNETs.79, 80

Although both inhibitors achieve significant rates of stable disease, the rate of tumor remission is low. This might be related to escape mechanisms along with primary and

secondary resistance to PI3K/Akt/mTOR inhibition. For example, inhibition of mTORC1 by everolimus leads to decreased activation of S6K. Decreased S6K activation may diminish the inhibitory effect of S6K on IRS-1, eventually activating mTORC2-Akt through activation of PI3K. Various studies and clinical trials are therefore exploring the efficacy of combined approaches.

In addition, different molecular mechanisms underlying PanNET pathogenesis may confer different responses to the inhibitors. mTOR inhibitors would be effective in PanNETs with aberrant PI3K/Akt/mTOR signaling pathway, either due to gene mutations or altered protein expressions. PanNETs with overexpression of receptor tyrosine kinases, especially VEGFR and PDGFR, would be predicted to respond to tyrosine kinase inhibitors such as sunitinib. Gene mutational analysis and immunohistochemical studies of the signal pathway genes/molecules may help stratify patients with PanNETs that are sensitive to these therapies.

SUMMARY Recent advance in understanding the molecular mechanisms underlying carcinogenesis and tumor progression has led to development of specific targeted therapies that demonstrate significant efficacy in patients with PanNETs. Although a high rate of stable disease is achieved with these targeted therapies, the cure rate remains very low. Better strategies to overcome resistance and increase efficacy are being explored. Combined target therapies, identification of predictive biomarkers, and defining patient

subgroups that may benefit from new treatment options may improve the outcome in patients with PanNETs.

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FIGURE LEGENDS

Figure 1. Examples of well-differentiated pancreatic neuroendocrine tumors (original magnification 200X). A and B: WHO grade 1 with Ki67 50% (200X); C and D: large cell neuroendocrine carcinoma with Ki67>50% (400X).

Figure 3. An otherwise well-dfferentiated pancreatic neuroendocrine tumor (A, 200X) with Ki67>20%. Because of the high Ki-67 rate this neoplasm would be classified as a NEC (B, 200X).

Figure 4. A representative gross picture of well-differentiated pancreatic neuroendocrine tumor.

Figure 5. A well-differentiated pancreatic neuroendocrine tumor showing markedly degenerative nuclei with no increase in mitosis (200X).

Figure 6. A well-differentiated pancreatic neuroendocrine tumor with abundant hyalinized stroma (100X).

Figure 7. An insulinoma with amyloid deposition (200X).

Figure 8. A well-differentiated pancreatic neuroendocrine tumor from a patient with VHL disease showing clear cytoplasm (clear cell variant, 200X).

Figure 9. A well-differentiated pancreatic neuroendocrine tumor with abundant eosinophilic cytoplasm (oncocytic variant, 200X).

Figure 10. A well-differentiated pancreatic neuroendocrine tumor arising adjacent to the pancreatic duct (A, 40X) showing dense stromal fibrosis (B, 200X).

Figure 11. Two examples of cystic pancreatic neuroendocrine tumor showing multiple cysts lined with multilayer of well-differentiated neuroendocrine tumor cells (A and B, 100X).

Figure 12. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. Activation of receptor tyrosine kinase by growth factors leads to activation of PI3K. Activated catalytic subunit of PI3K (PIK3CA) converts phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)trisphosphate (PIP3). PIP3 activates downsteam molecules Akt/mTOR. mTOR is present in two complexes: mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. mTORC2 phosphorylates and further activates Akt, whereas mTORC1 results in protein synthesis, cell growth, cell survival, and angiogenesis through activation of p70S6 kinase (S6K) and hypoxia-induced factor 1 (HIF-1 ), and inhibition

of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Akt, itself, plays an important role in tumor invasion through snail. Phosphatase and tensin homolog (PTEP) inactivates the signaling pathway through dephosphorylation of PIP3 into PIP2. TSC1/2 negatively regulates mTOR. In addition, activated S6K inhibits the signaling pathway through suppression of insulin receptor substrate 1 (IRS-1), playing a negative feedback role. The Von-Hippel-Lindau tumor suppressor (pVHL) suppresses angiogenesis through inhibition of HIF-1 , whereas menin reduces the activation of the PI3K/Akt/mTOR signaling pathway through suppression of insulin-like growth factor binding protein 2 (IGFBP2). Sunitibin inhibits activation of receptor tyrosine kinases by multiple growth factors. Everolimus is an mTORC1 inhibitor. Its effect is solely on the mTORC1 protein complex and not on the mTORC2 complex. Inactivated mutations in PTEN and TSC2 and activated mutations in PIK3CA are detected in some welldifferentiated pancreatic neuroendocrine tumor. In addition, severe growth factors (GFs) and multiple receptor tyrosine kinases (RTKs) are overexpressed in the tumors. VEGF, vascular endothelial growth factor; elF-4E, eukaryotic initiation factor 4E.

Table 1. Incidence, hormone secretion and clinical features of the four most common functional pancreatic neuroendocrine tumors

Incidence

Insulinoma

Association Hormone Syndrome with MEN(million/year) 1

Clinical behavior

1-3

5%

Insulin

Whipple triad: neuroglycopenic symptoms, low blood sugar, and reversal of symptoms by glucose administration

Indolent (only 10% demonstrating a aggressive clinical course)

Gastrinoma 0.1

20%

Gastrin

Zollinger-Ellison syndrome: complicated and uncomplicated ulcers and/or gastroesophageal reflux disease

Liver metastasis seen in 22-35% of cases at the initial diagnosis

VIPoma

0.05-0.2

6%

VIP

large-volume secretory diarrhea, dehydration, and electrolyte disturbances

Aggressive clinical course seen in 50% of case

Glucagoma

0.01-0.1

20%

glucagon

Glucose intolerance, weight loss, and necrolytic migratory erythema

Aggressive clinical course in seen in >50% of cases

Abbreviations: MEN-1, multiple endocrine neoplasia type 1; VIP, vasoactive intestinal polypeptide

Table 2. Genetic Syndromes Associated with Well-Differentiated Pancreatic Neuroendocrine Tumors (PanNETs) syndrome

Multiple Endocrine Neoplasia, Type 1

von Hippel Lindau

Inherita nce pattern AD

Gene invol ved MEN -1

locu Clinical presentations s

Frequency of PanNET

Type of PanNET

11q 13

Up to 100%

AD

VHL

3q2 526

Nonfunct ional gastrino ma insulino ma glucagon oma VIPoma Nonfunct ional only

Hyperplasia/neoplasms in multiple endocrine tissues, including parathyroid glands, pituitary, pancreatic islets, and duodenum

Pheochromocytoma/par aganglioma, hemangioblastoma, clear cell renal cell carcinoma, PanNETs, serous cystadenoma of the pancreas, and tumors of the middle ear and epididymis NF-1 17q Café-au-lait macules, Neurofibrom AD atosis type 11 neurofibromas (usually 1 plexiform), skin fold freckling, iris Lisch nodules and bony dysplasia TSC 3q3 Hamartomas in almost Tuberous AD 1 Sclerosis 4 every organ, disabling TSC 16p neurologic disorders, 2 13 and dermatologic findings Abbreviation: AD, autosomal-dominant.

11-17%

Rare (mostly ampullary somastosti noma)

Nonfunct ional Function al

2%

Nonfunct ional Function al

Table 3. Altered signaling pathways in neuroendocrine neoplasms of the pancreas Signaling pathway

Genes or molecules

Frequency

Chromatin DAXX/ARTX 45% remodeling

PI3K/Akt/ mTOR

MEN1

50%

PIK3CA, PTEN, TSC2

15%

Growth factors

majority

Growth factor receptors

TP53/Rb

Possible mechanisms

Clinical significance

Loss of DAXX/ARTX ALT CIN

prognosis

1) Loss of menin decreased histone H3 K4 methylation increased expression of CDKIs increased cell proliferation 2) Loss of menin decreased histone deacetylation decreased interaction with JunD cell proliferation 1) Mutation in PIK3CA activation of downstream Akt/mTOR 2) Mutations in PTEN/TSC2 loss negative regulation of the PIK3/Akt/mTOR pathway 3) Overexpression of growth factors/growth factor receptors increased activation of the pathway

Prognosis

MEN1

50%

Loss of menin loss inhibitory effect on IGFBP2 activation of IGF-associated PI3K/Akt/mTOR activation

VHL

Small subset

Loss of VHL increase HIF1α (downstream of mTOR) activity angiogenesis

TP53

Mutation: common in PD-NECs

1) Loss of TP53 entry into cell cycle (cell proliferation) and decreased cell apoptosis Overexpression of 2) Loss of Rb entry into cell MDM2/MDM4/WIP1: cycle cell proliferation common in

Therapeutic targets

Potential therapeutic targets

PanNETs Rb

Mutation: common in PD-NECs Overexpression of Cdk4/Cdk6: common in PanNETs

Abbreviations: DAXX, death domain- associated protein gene; ARTX, alpha thalassemia/mental retardation X-linked gene; ALT, alternative lengthening of telomere; CIN, chromosome instability; CDKIs, cyclin-dependent kinase inhibitor Proteins; MEN1, multiple endocrine neoplasia type 1 gene, PIK3CA, phosphatidylinositol-4,5bisphosphate 3-kinase, catalytic subunit alpha gene; PTEN, phosphatase and tensin homolog gene; TSC2, tuberous sclerosis complex 2 gene; mTOR, mammalian target of rapamycin; IGFBP2, Insulin-like growth factor-binding protein 2; IGF, insulin-like growth factor; VHL, Von-Hippel-Lindau tumor suppressor gene; HIF-1 , hypoxia-induced factor 1 ; PD-NECs, poorly-differentiated neuroendocrine carcinoma; PanNET, pancreatic well-differentiated neuroendocrine tumor.