The genetic basis of sporadic PNETs has yet to be fully elucidated; however, varied observationsmay contribute to a greater understanding regarding the molecular development and progression of these neoplasms. Traditionally, malignant neoplasms have been associated with multiple genetic mutations. Paradoxically, PNETs are overall regarded as being relatively mutational inert neoplasms.

Genetic instability has been implicated in the development and progression of PNETs, with genetic losses occurring more commonly than gains. Four chromosomal abnormalities have been detected using single nucleotide polymorphism studies in 60% of PNETs. Such losses accumulate as the tumour progresses, and are associated with tumour volume and stage [29] . Malignant behaviour is linked with loss of chromosomes 3q, 6pq, and 10 pq, and gains of 5q, 12a, 18q, and 20q [12] . These tumors contain microsatellite losses, with a median fractional loss of 0.37, significantly different from the median fractional loss of 0 amongst benign tumors. This is significant, as a fractional allelic loss of greater than 0.2 is associated with increased progression and mortality [30] . Chromosome 1 and 11q loss with gain of 9q are thought to occur early in development as they have been observed in tumours <2 cm, suggesting genetic instability may be associated with development of malignancy [5] [29] .

Several genetic mutations have been described in association with PNETs. A study investigating the genetic basis of PNETs identified 157 somatic mutations in 149 genes with a mean of 16 mutations per tumor, and the most common somatic mutations were MEN1 (44.1%), DAXX (25%), ATRX (17.6%), PTEN (7.3%), TS47 (8.8%) and PIK3CA (1.4%) [31] . The most frequent mutation is MEN1, which occurs in the syndromic PNETs. Other specific genes involved in PNETs include B1N1, Serpine 10, BST2, IGFBP3, LCK, MET, fibronectin, PDGF, IGF-1, fibroblast growth factor, TGF-alpha and -beta, EGFR, and stem cell factor receptor [32] [33] . Markers that may predict a more aggressive malignancy include cytokeratin 19, E-cadherin, and CEACAM1 [5] . Many PNETs are positive for somatostatin receptor which is involved in hormone secretion, apoptosis and endocrine proliferation [34] [35] .

Impaired G1/S checkpoint is reported in PNETs. It is hypothesized that mutations of PTEN in PI3K/mTOR signaling and p53 expression lead to tumor progression [9] . Overexpression of cyclin D1 is common in PNETs, reported in 43% [29] , and PTEN mutations are identified in 7.4% of sporadic PNETs all of which had concurrent mutations of either MEN1 or DAXX. Increased activity of protein kinase B (AKT) is reported in 61 to 76% of gastroenteropancreatic neuroendocrine tumors, with inhibition of AKT signaling reducing PNET proliferation. Abnormal activation of mTOR, a component of the PI3K pathway, is common in PNETs and therapeutic targeting of this pathway with sirolimus decreases relapse rates [9] . The DAXX/p53 pathway is also implicated in PNETs. While a mutation in TP53 is rare in well-differentiated PNETs and is possibly a late event for poorly-differentiated tumours, mutation of the DAXX adaptor protein causes disassociation of MDM2/USP7, degradation of MDM2, and subsequent p53 stabilization, causing arrest of the G2/S checkpoint [9] [29] . DAXX normally functions with ATRX; mutation of either of these proteins is mutually exclusive and correlated with alternative lengthening of telomeres. These mutations are present in 45% of PNETs and are not identified in pancreatic neuroendocrine carcinoma. Patients with mutation of MEN1 and DAXX/ATRX have double the median survival compared with other mutations [9] .

The Ras-Raf-MEK-ERK pathway is implicated in the pathogenesis of PNETs. While KRAS mutations are not present in these tumors, low levels of Raf1 are reported in PNETs which is hypothesized to contribute to invasion and migration via activation of focal adhesion kinase (FAK). The PNET molecular profile is thus opposite of pancreatic neuroendocrine carcinoma (PNEC), where this pathway is activated and Raf1 inhibition causes antitumor activity [9] . PNECs have 60% more mutations than PNETs and have a higher likelihood of TGF-β, CDKN2A and TP53 mutations compared with PNETs and they are thus proposed to arise through different independent pathways [31] .

In gastrinomas, deletion or hypermethylation of p16/MTS1 and/or deletion of p16INK4a has been reported; however, this mutation is not recognized in insulinomas. By contrast, 93% of both benign and malignant insulinomas have loss of heterozygosity (LOH) on 22q [29] . PNET metastases often have gain of function in chromosomes 4 and 7, with loss of 21q [5] . Specific factors involved in this metastatic process include VEGF-C, MAGE-1, p27, thrombomodulin, and SRC kinases [32] .

Despite an improved understanding of the genetic basis of these tumours, the clinical implications remainunclear, as traditionally genetic testing could only be done on tissue samples post-resection. Recently, it has been shown that an analysis of microsatellite loss could be performed on EUS-FNA samples in which a mean fractional allelic loss with > 0.2, was associated with disease progression [12] , thus providing an insight into future tumor behavior. Further investigation is warranted to determine the reliability of this technique in terms oftumorrelated clinical outcomes. Figure 2 summarizes the molecular profile of PNET’s as discussed above.

Though they are often spontaneous, PNETs may be associated with one of several underlying genetic conditions. Patients diagnosed with a PNET secondary to any of these conditions follow a more indolent course, and tumours are often multifocal [8] . These include von Hippel Lindau (vHL), Multiple Endocrine Neoplasia-1 (MEN-1), Tuberous sclerosis (TS), and Neurofibromatosis-1 (NF- 1).

Von Hippel Lindau (vHL) is caused by an autosomal dominant mutation of the vHL tumor suppressor gene on chromosome 3p25 that increases the patient’s susceptibility to neoplasms in the central nervous system and visceral

organs, including neuroendocrine tumours [5] [36] . Loss of heterozygosity of this chromosome has been demonstrated in 30% of sporadic PNETs though it is suggested the VHL gene doesn’t play a direct role in these sporadic lesions [29] . The VHL gene is responsible for the regulation of ubiquitination of hypoxia- inducible factors (HIF) 1 and 2, which causes upregulation of VEGF, PDGFR- beta, TGF-alpha, and erythropoietin. Mutation of this protein in vHL syndrome results in the development of hemangioblastomas, renal and hepatic cysts, adrenal/pulmonary/hepatic hemangiomas, renal cell carcinomas, and pheochromocytomas [5] . 10% - 17% of patients with vHL will develop a PNET, and these tumours are non-functional and often asymptomatic [29] [36] [37] [38] . The average age at diagnosis is 29 - 38 years, and 67% - 70% haveunifocal tumours [38] . It is suggested pancreatic screening should begin at age 15, when screening begins for renal cell carcinoma with follow-up every 2 - 3 years. These PNETs are often locally invasive; however, they have a lower rate of metastases, at 11% - 20%, potentially due to constant screening resulting in earlier diagnosis than sporadic non-functional PNETs. The decision of when to operate can be a challenge due to the presence of multiple tumours and recurrences. Criteria for predicting metastases includes: tumour size >3 cm, exon mutations, and a tumour doubling time <500 d. The presence of these factors favors early surgical intervention. However, these PNETs are slow-growing which contributes to their overall good prognosis; the death rate of PNET with vHL is 0.3% [37] .

Multiple Endocrine Neoplasia type 1 (MEN-1) results when the tumour suppressor gene on chromosome 11q13 is inactivated and is characterized by primary hyperparathyroidism in >95% secondary to parathyroid hyperplasia or adenoma, pituitary tumours in 20% - 40%, and PNETs [29] . Over 1,300 mutations of the MEN1 gene have been identified, leading to dysfunction of the Menin protein [9] . This gene is mutated in one-third of sporadic non-function- ing PNETs, insulinomas, and gastrinomas and does not affect tumour size of metastases. It is additionally hypothesized that additional oncogenes and tumour suppressor genes may be at 11q, downstream from MEN-1, that play a role in PNET development [29] . The Menin gene interacts with a number of downstream proteins, regulating transcription, genomic stability, cell division and the cell cycle [38] [39] . It is not yet understood from which cells MEN-1-associated PNETs arise, though it is thought they arise from the acinar and ductal cells rather than the islets themselves [5] [38] .

The lifetime risk of developing a PNET in the context of MEN-1 is nearly 100% [40] , though 54% - 93% of MEN-1 patients are asymptomatic [41] . Patients more often present with hyperparathyroidism or hyperpituitarism, with the PNET being diagnosed incidentally after confirmatory diagnosis of MEN-1. As such, continued screening should be offered to these patients. The most common type of PNET in patients with MEN-1 is non-functioning, gastrinoma, insulinoma, and VIPoma, which occur in 55%, 50% - 60%, 20%, and 3% - 5% respectively [8] . These tumours occur at a younger age, usually 30 - 50 years [4] . Up to 50% of these patients will develop metastases [42] , and 30% - 50% of patients will already have liver metastases at the time of symptomatic presentation [43] .

PNETs associated with MEN-1 have a higher rate of recurrence than sporadic PNETs and consequently are the most significant cause of death in patients with MEN-1. There remains no reliable clinical, histologic, or molecular marker to predict the aggressiveness of a MEN-1-associated PNET. Early and aggressive treatment is suggested to prevent metastases; however, most will develop recurrences [42] . PNETs are often multiple with tiny lesions that may only be detected with EUS/intraductal ultrasonography. Unresectable advanced PNET burden are controlled through hormone regulation, hepatic metastases management, and systemic chemotherapy [40] . There remains no consensus on the best treatment guidelines for non-functional PNETs associated with MEN-1. If the lesion is >3 cm in diameter it should be resected due to the increased risk of liver metastases; however, this has not improved survival [38] . Additionally, no best-practice guidelines have been established for the management of smaller tumours. The timing and extent of surgical intervention for gastrinoma is also debated [42] . Some authors argue aggressive interventions such as Whipple’s procedure are not indicated due to high postoperative morbidity with unclear benefits; additionally, chemoembolization cannot be used to treat liver metastases after this procedure due to the risk of an ascending infection. This procedure therefore should be reserved for healthy patients with large tumours in the pancreatic head with positive nodes not amenable to simple resection [38] . These lesions are often multicentric, suggesting pharmacological interventions such as H2-blockers or protein pump inhibitors rather than surgical resection are indicated [31] [42] . In contrast, insulinomas in patients with MEN-1 should undergo surgical resection including subtotal pancreatic resection with splenic preservation combined with enucleation providing there are no metastases, as cure rates of 83% - 100% can be achieved [42] [44] . A distal pancreatectomy is often required to decrease recurrence risk [40] . In these patients, enucleation alone is less effective as tumours are often small and multifocal. It is proposed laparoscopic surgery may be an option for insulinomas, with intraoperative laparoscopic ultrasound to identify pre-operatively unidentified tumours [44] .

Metastases are less common in PNETs associated with MEN-1 than their sporadic counterparts. The risk of hepatic metastases is decreased by 62% for gastrinomas, 50% for VIPomas and glucagonomas, 30% for somatostatinomas, and 80% for non-functional PNETs. The only exception is insulinomas, in which hepatic metastases occur more frequently. Patients diagnosed with MEN-1 generally have a shortened lifespan, with a mean survival of 46 - 54 years. The 5- and 10-year survival in patients with MEN-1 with PNETs is 82% and 75% respectively. This can be further broken down based on hormonal activity, with 10- year survival in insulinomas/gastrinomas of 82% - 91%, non-functional PNETs of 62% and glucagonomas, VIPomas, and somatostatinomas of 54% [38] .

Tuberous Sclerosis (TSc) is the result of a mutation of either TSC-1, which codes for hamartin, or TSC-2, which codes for tuberin, and is characterized by hamartoma development resulting in neurological and dermatologic disorders. Both functional and non-functioning PNETs have been reported with tuberous sclerosis, particularly in patients with TSC-2 mutations; however, it is not clear whether this relationship is a causal or a casual association [5] [38] .

Neurofibromatosis type-1 (NF-1) is an autosomal dominant genetic disorder caused by alterations, including nonsense, frameshift, splice mutations, translocations, and partial or complete deletions, of the NF1 gene on chromosome 17q11.2 that encodes expression of neurofibromin [29] . Neurofibromin regulates p21 activation to affect cell proliferation, growth and signalling [38] . The most common PNET in NF-1 patients are somatostatinomas, but these tumours are usually functionally silent and present due to mass effect. PNETs in NF-1 are overall rare (0% - 10%) [38] .

Chromogranin-A, a glycoprotein in the secretory granules of neuroendocrine cells, is expressed in the majority of PNETs. The sensitivity depends on tumor burden, ranging from 50% - 100% and the majority of studies show a relationship between the level of chromogranin-A and patient prognosis (Table 3). False positives may be due to Parkinson’s disease, hypertension, glucocorticoids, chronic atrophic gastritis, renal/liver failure, or pregnancy. An exception is insulinomas, in which elevated chromogranin-A is rare. Other serologic markers include neuronal serum enolase, human chorionic gonadotropin, and pancreatic polypeptide, which are elevated in 20% - 40% of PNETs [45] .

Specific elevated hormone levels (insulin, gastrin, glucagon, VIP) can be assessed in the serum of symptomatic patients with functional PNETs. Diagnostic criteria are explored in their respective sections under the heading “Functional PNETs”.

Several radiological imaging techniques can be used for PNET detection, characterization, and staging (Table 3). These radiological interventions can be divided by their use into the “anatomic techniques” that determine the location and extent of the tumour, which include CT, MRI and EUS, and the “functional techniques” that define metastatic spread and biological behaviour, which includes scintigraphy (Octreoscan) and PET [46] . Functional PNETs are more difficult to diagnose radiologically than non-functional, as these tumors are small and seldom alter the pancreatic contour [47] .

The most common radiological tools used in the work-up of PNETs are computed tomography (CT) and magnetic resonance imaging (MRI), and all patients should have at least one of these to localize the tumour, determine its resectability and assess metastatic spread [48] . The sensitivity of these modalities has been reported to range from 14% - 77% [40] . On such imaging, PNETs are

*Elevated in 20% - 40% of PNETs**elevated in functional PNETs.

solid, hypervascular lesions, differing from the hypovascular appearance of adenocarcinoma. Features suggestive of PNET include calcification, cystic degeneration and the absence of ductal obstruction, vascular encasement or desmoplastic reaction. Findings suggestive of malignancy in PNET include necrosis, calcification, and retroperitoneal invasion [48] . By contrast, pancreatic adenocarcinomas are hypovascular; only 2% are calcified, and often have ductal obstruction. CT scanning can be combined with Single-Photon Emission Computed Tomography (SPECT) to improve attenuation correction, increase specificity, improve tumour localization, and assess for and detect invasion [14] . Identification of regions of uptake using both CT/SPECT aids in the differentiation between true- and false-negatives [49] .

On magnetic resonance imaging (MRI), PNETs exhibit low signal intensity on T1 weighted imaging (T1WI) with intermediate-to-high intensity on T2 weighted imaging (T2WI). Because of the rich vasculature, contrast-enhanced imaging shows enhancement on arterial phase, either homogenously for tumours <2 cm or heterogeneously in a ring-like appearance for larger tumours [14] . CT scans are optimized with angiography and images should be obtained 25 - 30 s following contrast administration for the arterial phase, and 60 seconds post-administration for the portal venous phase [8] . MRI sensitivity is reported between 74% - 100% [46] . The sensitivity of CT and MRI for detecting metastases is up to 94% [8] . These metastases often have low-density T1WI though patient-to-patient variability exists [49] . MRI imaging can be combined with diffusion-weighted imaging (DWI) to improve visualization of small lesions. A recent study that investigated the fusion of DWI with T2 imaging found significant improvements in interpretation, strong inter-interpreter reliability, and an increased overall confidence [50] .

Endoscopic ultrasonography (EUS) is an investigational technique that can be used in the evaluation of small <1cm cystic pancreatic lesions [8] . When combined with biphasic thin-section helical CT, diagnostic sensitivity approaches 100% [44] . EUS allows for anatomic identification of the PNET in relation to the pancreatic duct for pre-operative planning. EUS is better for visualization of intra-pancreatic lesions compared with extra-pancreatic lesions. Unlike other imaging modalities, EUS identifies lesions as small as 2 - 3 mm, and is the preferred diagnostic modality of choice for the detection of small insulinomas [45] . EUS evaluation may be combined with fine needle aspiration biopsy (FNAB), allowing the retrieval of a specimen for pathological examination. It is a technique often used for screening patients with MEN-1 or VHL who are prone to pancreatic lesions. It has a reported sensitivity of 82% and specificity of 95% [11] ; however, it is unable to evaluate metastatic spread and its accuracy is highly operator- dependent. Differentiation between pancreatic nodules and peripancreatic lymph nodes is often difficult using this technique [11] .

Somatostatin receptors are expressed on 80% - 90% of PNETs; therefore, somatostatin receptor scintigraphy has become a standard technique for detection and staging [51] [52] . As somatostatin has a short half-life, the analog octreotide labelled with indium-111 (111-DTPA octreotide) is the most common technique for detecting all gastropancreatic neuroendocrine tumours, with sensitivity of 67% - 100% [48] . Though rare, octreotide has also been labelled with Gallium-67 citrate to improve detection of PNETs [53] . Insulinomas are an exception as they are poorly detected by this technique [11] . Scintigraphy scans the entire body; therefore, identification of distant metastases can be identified with this one technique. Scintigraphy combined with CT imaging has a higher sensitivity than CT and MRI for detecting metastatic disease, with a sensitivity of 90% [8] . OctreoScan can be also combined with positron emissions tomography (PET) scans to visualize lesions not seen by CT or MRI and to differentiate malignant from benign based on uptake functional images [48] . PET (FDG-PET) alone may play a role in assessing poorly differentiated neuroendocrine tumours, which are often negative on OctreoScan [14] . Radiopharmaceuticals used with PET to detect PNETs include fluorodeoxyglucose, Gallium-68, fluorodehydrox- yphenylalanine, and 5-hydroxytryptophan [49] [52] .

The selective arterial secretagogue injection (SASI) test has two parts: first, percutaneous transhepatic portal venous sampling followed by arterial calcium stimulation with hepatic venous sampling. Percutaneous transhepatic portal venous sampling involves the placement of a venous catheter through the liver to the portal vein to permit hormone sampling from the splenic vein, SMV, and portal vein. This technique has a sensitivity of 70% - 95%. Arterial calcium stimulation with hepatic venous sampling, also called an Imamura test, is conducted by serial injection of calcium into the splenic, gastroduodenal, and inferior pancreaticoduodenal arteries, with samples taken from the hepatic vein pre- and post-each injection [46] . SASI with calcium is a preoperative tool to locate MEN-1 associated insulinomas [43] .

On gross examination, PNETs are most commonly tan to pink, well-demarcated soft tumours; however they have been reported to be hard and white/gray when associated with fibrosis or amyloid. Less common appearances include papillary, angiomatous, and cystic [5] . Rarely, PNETs with attendant tubules or ductules are recognized [54] . Larger tumors may reveal areas of haemorrhage and/or necrosis [19] . Insulinomas are generally <2 cm in diameter, the smallest of all PNETs while non-functional PNETS usually present as a larger tumour.

Cytological features of PNETs include round-to-ovoid cells, an eosinophilic/ granular cytoplasm, dispersed chromatin in the nucleus and nucleoli [5] . On microscopic examination, well-differentiated tumours may demonstrate a number of architectural patterns including solid, nesting, trabecular, gyriform, glandular, tubular/acinar, pseudorosettes and mixed [5] [19] . Well-differentiated tumours exhibit tumour cell monomorphism with little to no cytologic atypia and a low mitotic and proliferative index. Occasionally clear cells, vacuolated cells, and oncocytes may be seen [19] . The histological architecture is not indicative of the functional state except for the presence of amyloid, which is more typical in insulinomas, and psammoma bodies indicating somatostatinoma [19] . Malignancy is evidenced by the presence of local spread, vascular invasion, nodal involvement and/or organ metastases rather than specific microscopic histopathological features.

As PNETs are epithelial in origin, they are often immunoreactive to keratin 8% and 18%, and 50% are immunoreactive to vimentin. As with other neuroendocrine tumours, PNETs usually stain positive for neuroendocrine markers such as chromogranin A and synaptophysin, thus differentiating them from pancreatic adenocarcinomas. One of both of these markers is positive in approximately 90% of PNETs [55] . Well-differentiated PNETs are positive for both chromogranin and synaptophysin; however, poorly differentiated PNETs may be only positive for synaptophysin [56] . As discussed previously, chromogranin A is the most frequently secreted and measured hormone in PNETs. Levels are correlated with the tumour burden, with 60% - 100% sensitivity in metastatic disease yet less than 50% in localized disease [8] . Insulinomas and somatostatinomas may be negative for chromogranin A, though are often reactive to chromogranin B or C; however, antibodies to B and C are usually commercially unavailable [55] . False positives can result from renal insufficiency, Parkinson’s disease, untreated hypertension, pregnancy, steroid use, and achlorhydria [8] .

Other markers that have been described to define neuroendocrine differentiation include protein gene product 9.5, CD57 and neuron specific enolase [55] . PNETs have been shown to stain strongly to PDGFRA (33%), CK19 (26%), CD56 (25%), CD20 (5%), S100 (6%), and CK7 (2%) [3] . Pancreatic polypeptide has a sensitivity of 63% and specificity of 81% [8] .

A recent PNET study investigated the prognostic role of geminin, a negative regulator of DNA proliferation that has been reported to confer a negative prognosis in a variety of malignancies including breast cancer, renal cell carcinoma, prostatic adenocarcinoma, salivary gland carcinoma, and lung cancer. The study suggested geminin is a greater predictor of disease-free survival than Ki67 or ENETS staging [6] .

Surgical resection remains the only curative treatment for PNETs and alleviates symptoms associated with hormone secretion and mass effect. Surgical options include radical excision with a curative intent, palliative excision aimed at symptomatic relief, and surgical treatment of complications [23] [25] [47] . The 5-year overall survival rate of resected PNETs is significantly greater than unresected ones, from 77% to 46% [57] . Complete excision with a curative intent plays a central role for patients with localized tumours at presentation. Major aggressive resection, including either pancreaticoduodenectomy or distal pancreatectomy, may effectively treat tumour-related endocrinopathies and local symptoms due to mass effect [57] . These surgical procedures, however, are associated with a high incidence of exocrine or endocrine pancreatic insufficiency. Some authors suggest that sporadic malignant tumours, or tumors over 3cm, are best managed with Whipple’s resection or distal pancreatectomy, with resection of adjacent organs and vasculature as indicated in relation to tumor size and its localization [44] . One notable exception is patients with MEN-1 with non-functional PNETs, in whom multiple microadenomas are often found throughout the pancreas; thus restricting the effectiveness of limited tumour resection [46] . Among these patients, subtotal (80%) distal pancreatectomy with enucleation of any pancreatic head tumor is often recommended [43] .

Laparoscopic surgery of the pancreas was first introduced in 1994, since which sufficient evidence in the international literature has proven its suitability in the management of low-risk PNETs [44] [47] . The feasibility of a complete resection with optimal oncologic outcome using laparoscopic techniques remains debateable. Laparoscopic surgery, without proper pre-operative imaging, has a localization failure rate of 19% [48] . Laparoscopic distal pancreatectomy is recommended for tumors of the pancreatic body or tail, with five surgical variations: 1) spleen and splenic vessel preserving distal pancreatectomy, 2) spleen-preserving distal pancreatectomy, 3) distal pancreatectomy with splenectomy, 4) central pancreatectomy, and 5) laparoscopic pancreaticoduodenectomy [47] . With proper preparation, laparoscopic resection success rates reach 60% - 100% [44] . The morbidity rate of laparoscopic surgery is between 8% - 50% and improves with technology and surgical skill [48] . No significant difference in mortality, morbidity, reoperation, or readmission has been found when a laparoscopic vs. an open resection was used [1] . Overall, minimally invasive treatment of PNETs is associated with lower complication rates, shorter length of stay, with comparable rates of fistula formation, postoperative complications and patient mortality when compared with open resection. Besides laparoscopic resections, robotic assisted surgery and single port surgery are new upcoming promising approaches [68] .

A specific laparoscopic technique for PNET management is enucleation, a technique that is reported to decrease the risk of postoperative complications such as diabetes, as a great amount of pancreatic parenchyma is spared. This technique, however, increases the risk of causing a pancreatic fistula, most commonly when the tumour is in the pancreatic duct [47] . The decision to choose enucleation vs. resection additionally depends on tumour location, focality, and intraoperative ultrasound findings [44] . Large or fast-growing tumours are not suitable for enucleation and require open excision. Similarly, enucleation is contraindicated if the surgical plane between pancreatic parenchyma and tumor capsule is not identified [68] . Ideal tumors for laparoscopic enucleation are well-circumscribed lesions <3 cm with noninvasive features located along the pancreatic periphery [69] . Prediction of which tumors will have a benign natural history is challenging. Enucleation can be an open or laparoscopic procedure. It is suggested subtotal (80%) distal pancreatectomy should be performed in conjunction with enucleation for syndromic non-functional PNETS >2 cm or for functional PNETs of any size [43] . The postoperative morbidityrate of laparoscopic enucleation is 20% - 30% [44] . A second minimally invasive technique, central pancreatectomy, has also been reported that, with enucleation, has a lower morbidity and shorter hospital stay than a standard pancreatectomy [66] .

Robotic-assisted surgery has been used to perform pancreaticoduodenectomy, central pancreatectomy, and distal pancreatectomies [47] [68] . This technique offers several advantages, most notably a three-dimensional and significantly magnified view of the surgical field and a 540 degree range of motion. Aside from the expense and technical expertise required, other limitations of robotic surgery include technical issues such as collisions between the robotic arms and the inability of changing table position after robot docking [47] .

Unresectable primary tumors may be treated with radiofrequency ablation performed percutaneously with ultrasonographic guidance, or intraoperatively [47] . Solitary small tumors (<3 cm), ideally located distal from peripancreatic structures are the best candidates for this technique.Thermal-induced pancreatitis and injury to peri-tumoral structures (duodenum, pancreatic duct, and blood vessels) are potential adverse effects [47] . Another technique, ethanol ablation, causes dehydration, protein denaturation and vascular occlusion of the tumor resulting in coagulation necrosis; however, the technique poses the risk of late recurrence, incomplete ablation, and progression [47] .

Rates of metastases among small tumors < 1.5 cm and < 3 cm are 8% and 31% respectively. The significance of lymph node metastases on patient outcomes remains uncertain; therefore, guidelines for the management of lymph nodes in PNETs remain controversial. A small (n = 136) retrospective study showed higher rates of lymph node metastases among large tumors (>1.5 cm), tumors involving the pancreatic head, tumors with a high (>20%) Ki67 index, and tumors with vascular invasion; lymph node metastases was associated with a shorter disease free survival (4.5 years vs. 14.6 years without nodal involvement). These authors concluded that lymph node metastases are predictive of poor outcome [66] . While some authors have also reported a relationship between tumor size and lymphadenopathy, others have failed to show this correlation. Recommendations from the National Comprehensive Cancer Network suggest lymph node resection is indicated for tumors between 1 - 2 cm in size [41] . The exact extent of lymph node resection, whether regional, radical, extended or lymph node “picking” is still unclear.

The presence of metastases is the most definitive indicator of a malignant PNET. The risk of metastatic spread depends on the functional status of the tumour and the hormone being expressed. Complete resection and/or hepatic debulking for metastases is associated with improved quality of life and survival [70] . Among all PNETs, 60% - 80% will present with metastatic spread [71] . The most common site of metastases is the liver; however, hepatic dysfunction is rare despite the large tumour mass [72] . Optimal treatment of liver metastases remains controversial, with options including cytoreduction, debulking surgeries, transplantation, or observation with or without pharmaceutical interventions. Cytoreductive surgery is indicated if metastases are localized or if >90% of the tumor burden is resectable [10] . This number is recently contested, with Maxwell et al suggesting that a target threshold of 70% may increase patient eligibility for cytoreduction and increase patient survival [73] . If left untreated, the median survival of a patient with hepatic metastases from a PNET is 2 - 4 years; this number is improved when the patient is aggressively treated, whether through resection or debulking. Resection of a metastasis should be considered if less than 50% of the liver is involved and a minimum of 90% of the tumour burden can be safely resected. Such conditions account for approximately 10% of cases. The rate of hepatic recurrence is up to 76% within 2 years. The risk/benefit ratio for aggressive resection must be evaluated in relation to its morbidity and mortality, versus pharmacotherapy or locoregional techniques [8] .

Hepatectomy with transplantation may be considered for patients younger than 55 - 60 years of age [25] . This option should be reserved for patients with no extrahepatic disease, who are unresponsive to other therapies, and the tumour should be well-differentiated with a low Ki67 index [8] . Few orthotopic liver transplantations have been attempted which have incurred high rates of mortality and recurrence. The perioperative mortality rate, which ranges from 11% - 28%, may be reduced by choosing a staged procedure [72] . Post-trans- plant, the rate of recurrence is up to 63% within months [8] .

If the hepatic metastasis is unresectable, patients may benefit from surgical excision of the primary lesion to decrease the risk of biliary obstruction, gastric outlet obstruction, or haemorrhage. This may, however, depend on the extent of metastases, as one study found no significant difference in the survival of patients with more than 50% liver involvement treated surgically or nonsurgically [74] . Data from the Surveillance, Epidemiology, and End Results from 882 patients with non-functional PNETs indicates that resection of the primary tumor improves patient survival from 0.83 years to 5.42 years [75] . These results, however, may be confounded given its retrospective study design, as patients selected for resection may have been healthier with less tumor burden. Recent study by Morgan recommends expanding the criteria for liver debulking in PNET to include a new threshold of >70% debulking, intermediate grade tumors, positive margins, and extrahepatic metastases, thus increasing the operative eligibility with associated higher survival rates [76] .

Ablation techniques to the liver include radiofrequency ablation, cryoablation, microwave ablation, and alcohol ablation. Radiofrequency ablation can be used as a monotherapy or in combination with other therapies including surgery. This technique is indicated when less than 10 lesions are present, with none over 4 cm. [8] . These techniques are used to treat metastases less than 5 cm [46] . Hepatic arterial embolization is based on the observation 75% - 80% of blood supporting PNETs is derived from the hepatic artery [8] [46] . This staged procedure involves sequential catheterization of hepatic arterial branches to one liver lobe then 6 - 8 weeks later, cannulation and embolization of the branches to the other lobe [8] . This procedure is contraindicated in patients with portal vein thrombosis, cirrhosis, or a history of biliary reconstruction [8] . Side effects secondary to ischemic hepatitis include nausea, abdominal pain, fever, and fatigue [46] . Symptomatic response rates in non-functional PNETs with liver metastases are 50% - 100%, with tumour volume response rates from 25% - 86%. This response may last from 6 - 42 months. A longer response is associated with isolated hepatic metastatic disease, prior resection of the primary tumour, involvement of less than 75% of the liver, and a tumour size <5 cm [8] . Hepatic artery embolization can be combined with intra-arterial chemotherapeutics like doxorubicin or cisplatin. Radioembolization using yttrium-90 radiolabeled microspheres may be distributed by arterial injection, delivering direct intratumoral radiation. This technique has a response rate of 12% - 18% with a median survival of 22 - 36 months [77] .

Aggressive surgical management with improved survival of patients with advanced PNETs include pancreatectomy, splenectomy and SMV reconstruction. In some patients with symptomatic, low-volume advanced disease, careful observation without treatment may be sufficient until symptoms of progression present; however, the most recent review of the Surveillance Epidemiology and End Results (SEER) database reported improved cancer-specific and overall survival in patients treated with surgical resection among metastatic PNETs with improved Cancer specific survival [p = 0.028] and overall survival benefit [p = 0.025] [78] . In patients with symptomatic disease, a number of palliative measures may be used to improve the patient’s quality of life. The 5-year survival for patients presenting with advanced PNETs ranges from 25% - 75% [57] . Surgical debulking may help to relieve symptoms of mass effect or hormone excess and is an appropriate choice if a minimum of 90% of the tumour is resectable [41] [46] . For patients who do not fit this criterion, or who are not good surgical candidates, a number of non-operative techniques exist including radiofrequency ablation, cryotherapy, hepatic artery embolization, and/or chemoembolization. Chemoembolization involves direct injection of chemotherapeutics including doxorubicin or cisplatin directly into the liver. This technique has the best results when combined with systemic chemotherapy, with a mean survival of 3.5 years [25] .

Whereas systemic cytotoxic agents remain the primary treatment for poorly- differentiated and/or rapidly progressing PNETs, for most patients with advanced unresectable well or moderately-well differentiated PNET treatment options include observation, nonsurgical liver-directed therapy, and systemic therapy to control tumor growth and symptoms related to the disease. The systemic options include somatostatin analogs, peptide receptor radionuclide therapy, interferon-alpha and systemic chemotherapy and targeted therapy [71] . These pharmacological interventions may be used for non-resectable PNETs, in patients unsuitable for surgery, or for patient’s symptomatic post-surgical resection.

Octreotide and lanreotide are somatostatin analogs that bind to somatostatin receptors that are expressed in most neuroendocrine tumors and inhibit the secretion of multiple hormones. Somatostatin analogs have not only shown efficacy in the management of symptoms associated with hormone hypersecretion but are also effective in controlling the tumor growth. Recent reports suggest that somatostatin analogs help to stabilize the disease and prolong the progression free interval [79] [80] . For example, the PROMID study group conducted a randomized placebo-controlled, double-blind study to assess the ability of octreotide long-acting repeatable (LAR), to control the growth of well-differentiated metastatic neuroendocrine tumors [79] . The results showed that median time to tumor progression was significantly longer in the group treated with octreotide LAR compared with the placebo group (14.3 versus 6 months with HR of 0.34; 95% CI, 0.20 to 0.59). After 6 months of treatment, stable disease was observed in 66.7% of patients in the octreotide LAR group compared with 37.2% in the placebo group. The findings of the PROMID study was subsequently confirmed by the CLARINET investigators in a placebo-controlled randomized phase involving patients with grade 1 or 2 nonfunctioning, somatostatin receptor-posi- tive neuroendocrine tumors of pancreas and gastrointestinal tract [80] . This study randomly assigned 204 patients to receive either 120 mg lanreotide or placebo. The estimated rate of progression-free survival at 2 years was 65.1% in the lanreotide group compared with 3.0% in the placebo group (HR for progression or death, 0.47; 95% CI: 0.30 to 0.73). Side effects include mild nausea, abdominal discomfort, bloating, loose stools, and fat malabsorption.

A variant of somatostatin treatment is peptide receptor radioligand therapy or targeted radiotherapy using radiolabeled somatostatin analogs [70] . Yttrium and lutetium are the most commonly used radionuclides for targeted radiotherapy [80] [82] . These therapies are most useful in patients with somatostatin-receptor positive tumors. In a large observational study 1109 patients were treated with median two cycles of ⁹⁰Y-DOTA-TOC [82] . The objective radiographic response rate was 34.1%, biochemical response rate was 15.5%, and clinical response rate was 29.7%. The median survival was 94.6 months and survival was correlated with any type of response. Transient bone marrow suppression and renal dysfunction were major toxicities. Tumoral uptake in the initial imaging study was predictive for overall survival (HR, 0.45; 95% CI, 0.29 to 0.69).

Another study evaluated the efficacy and toxicity of ¹⁷⁷Lu-DOTATATE in over 500 patients. Complete and partial tumor remissions occurred in 2% and 28% of 310 patients with gastroenteropancreatic NET, respectively. Minor tumor response noted in 16% patients. Median time to progression was 40 months. Median overall survival from start of treatment was 46 months, and from diagnosis was 128 months [81] . In addition to radiolabeled somatostatin analogs, 131I- metaiodobenzylguanidine (¹³¹I-MIBG) therapy in MIBG positive metastatic neuroendocrine tumorshas demonstrated activity [83] .

Streptozocin with either 5-fluorouracil or doxorubicin is one of the standard chemotherapy regimens in the management of advanced low-grade PNET [25] . In a randomized trial that compared two streptozocin-based regimen, combination of streptozocin and doxorubicin was associated with a significantly better combined biochemical and radiographic response rate (69% vs. 45%) and median overall survival (2.2 vs 1.4 years) compared with streptozocin and 5-FU [70] [84] . Nevertheless, due to major toxicities such as severe myelosuppresion and renal dysfunction, currently the use of streptozocin-based regimens has declined.

Similar to streptozocin, decarbazine (DTIC) and its orally active analog Temozolomide are alkylating agents that have shown activity in PNET. For example, a phase II trial assessed decarbazine (DTIC) in 50 patients with advanced symptomatic or progressive PNET and showed a response rate of 33% in 42 patients with measurable disease and median overall survival of 19.3 months [85] . However, due to better side effect profile and dosing convenience, temozolomide-based regimens have replaced the use of DTIC in PNET. The optimal temozolomide-based regimen and dosing schedule is not known. It has been used as a monotherapy or in combination with: 1) capecitabine, with an overall response rate of 70% and median progression-free survival of 18 months, and 2) thalidomide with an overall response rate of 45% [77] . Temozolomide has also been evaluated in combination with everolimus and bevacizumab in advanced PNET with response rate of 40% and 33% and median progression free survival of 15.4 months and 14.3 months, respectively [86] [87] . Long-term use of temozolomide has been associated with lymphopenia and pneumocystis pneumonia, therefore prophylaxis is recommended inthese patients. Other common non- hematological toxicities include fatigue, nausea, vomiting, anorexia, constipation, and diarrhea.

Oxaliplatin is a third generation platinum compound that in combination with fluoropyrimidine and bevacizumab has shown efficacy in selected patients with well differentiated and poorly differentiated PNET [88] . Oxaliplatin has also been combined with gemcitabine, with a response rate of 27% [77] . Other chemotherapeutics that have been failed to affect the course of PNETs include endostatin, irinotecan & cisplatin and capecitabine & rofecoxib [48] .

Sunitinib and everolimus are approved for the treatment of unresectable PNETS. Sunitinib is a potent inhibitor of VEGFR-1, VEGFR-2, FL26, KIT, PDGFRα, and PDGFRβ [36] [77] [89] . Of note, the mechanism of angiogenesis in neuroendocrine tumors is not well understood as these malignancies do not have the same dense vascularizationas other solid organ cancers [90] . A phase II trial that evaluated activity of sunitinib in patients with advanced neuroendocrine tumors found an overall response rate of 16.7%, with 62.1% demonstrating some response and the time to progression while was 7.7 months [33] . Subsequently, a randomized, double-blind, placebo-controlled phase 3 trial involving 171 patients with advanced, well-differentiated pancreatic neuroendocrine tumors demonstrated superiority of sunitinib over placebo [91] . The response rate was 9.3% in the sunitinib group compared with no response in the placebo group. The median progression-free survival of the group treated with sunitinib was 11.4 months compared with 5.5 months in the placebo group (HR, 0.42; 95% CI, 0.26 to 0.66; P < 0.001). Although follow-up period was short, mortality rate was also lower in the group treated with sunitinib compared with the placebo group (10% versus 25%) [HR, 0.41; 95% CI, 0.19 to 0.89; P = 0.02]. The most frequent adverse events in the sunitinib group were diarrhea, nausea, vomiting, asthenia, and fatigue. Nevertheless, there were no differences in the quality-of-life index between the two groups.

Two other oral multi-targeted tyrosine kinase inhibitors: sorafenib and pazopanib, have also demonstrated efficacy in well-differentiated PNET [92] . In addition, the VEGF inhibitor bevacizumab has been used to treat advanced PNETs, with 18% response rate. When combined with oxaliplatin and fluorouracil, the response rate increases to 19% in one study, and 50% in another [77] .

The mTOR pathway is involved in a number of the genetic syndromes associated with PNETs including von Hippel Lindau, tuberous sclerosis, neurofibromatosis-1 and MEN-1; sporadic PNETs express IGF-1, which stimulates mTOR pathway and thereby tumor growth and proliferation [93] . Everolimus is an oral inhibitor of mTOR. A phase II trial found a 22% response rate, with 70% stable disease when combined with octreotide. Later a phase 3 trial showed that everolimus in patients with PNET was associated with 65% reduction in the estimated risk of progression or death [94] . In RADIANT-3 trial 410 patients who had advanced, low-grade or intermediate-grade pancreatic neuroendocrine tumors with radiologic progression within the previous 12 months were randomized to everolimus, or placebo. The median progression-free survival was 11.0 months with everolimus compared with 4.6 months with placebo (HR, 0.35; 95% CI, 0.27 to 0.45; P < 0.001). Most drug-related adverse events were mostly mild to moderate and included stomatitis, rash, diarrhea, fatigue, and hyperglycemia.

Everolimus has been combined with bevacizumab for low- to intermediate- grade PNETs, octreotide, temozolomide, and pasireotide [70] [95] . Given the fact that both VEGF pathway and mTOR inhibitors are active in pancreatic NET, a phase 2 trial evaluated the benefit of adding bevacizumab to everolimus in 150 patients with advanced PNET [96] . Although combination therapy was associated with higher response rate of 31% compared with 12% and superior progression free survival of 16.7 compared with 14 months, it was associated with higher rates of severe adverse effects including diarrhea (14% vs. 3%), hyponatremia (12% vs. 3%), hypophosphatemia (11% vs. 3%), proteinuria (16% vs. 1%), and hypertension (41% vs. 12%). Contrary to everolimus, temsirolimus appears to have little activity as single agent treatment in PNET [97] .

Interferon-alpha-2b is used in the treatment of PNETs because of its role in anti-proliferation, anti-angiogenesis, apoptosis and differentiation in both functional and non-functional tumours [52] . Interferon can stabilize tumour growth in 10% - 15% of patients [33] [52] . Interferon therapy can be used as a monotherapy or, for better effects, in combination with octreotide [52] . Adverse effects may include fatigue, myelosuppression, or depression and currently its use has declined [33] .

Somatostatin analogs including octreotide (Sandostatin), lanreotide, and pasireotide are useful treatments of the symptoms of PNETs, particularly VIPomas and glucagonomas [70] [77] . Somatostatin itself cannot be used due to its short half-life [5] . In addition to blocking the release of hormones thereby decreasing symptoms such as diarrhea, flushing, or acromegaly, octreotide and somatostatin analogs also stabilize the disease and lengthen the time to progression [70] [98] [99] . It has been suggested that in asymptomatic patients these drugs should be started after a 12-month observation period in which growth pattern can be established; however, clinical trials are needed to support or refute this hypothesis [77] . Adverse effects include biliary disorders (62%), gastrointestinal disorders (14% - 38%), injection site pain (20% - 50%), hypoglycaemia (4%), hyperglycemia (27%), and bradycardia (19%) [98] . In patients with insulinomas, somatostatin analogs may cause a transient worsening of hypoglycaemia as half of these tumours don’t express somatostatin receptor II and the octreotide blunts the glucagon response [77] .

The other systemic treatments including peptide receptor radioligand therapy, cytotoxic agents, and targeted therapy that as discussed above are also effective in symptomatic management of PNET.