Neuroendocrine tumors (NETs) are tumors arising from the neuroendocrine tissues of the upper respiratory tract or gastrointestinal (GI) tract. When originating from the islets of the Langerhans in the pancreas, they are called pancreatic NETs (panNETs). Of all newly diagnosed pancreatic cancers every year, panNETs comprise approximately 1% to 3%, with an incidence of 3.2 cases per 100,000.1 The number of new cases has increased recently owing to advances in imaging, increased diagnostic procedures, and increased awareness in the medical field.2–4
The clinical presentation varies widely among patients. Approximately 85% of the cases do not secrete clinically significant hormones and are designated as nonfunctional tumors, whereas those that secrete hormones leading to clinical symptoms are known as functional panNET (15% of cases).5,6 The more common peptides secreted include insulin or gastrin leading to insulinoma or gastrinoma, respectively. A small subset of these tumors may secrete somatostatin, glucagon, and vasoactive intestinal peptide leading to the clinical syndromes of somatotastinomas, glucagonomas, and VIPomas, respectively. Most panNETs are nonfunctional panNETs, and these patients usually present with more advanced disease stage because of the lack of the symptoms to seek early medical attention. Treatment modalities as surgical removal become less feasible during advanced stages of the tumor.1,5 Nonfunctional panNETs are postulated to have worse prognosis compared with hormone-secreting functional panNETs.5,7 According to American Cancer Society, the 5-year survival rate for patients who had surgical interventions for panNETs from 1985 to 2004 ranges from 61% in stage 1 to 16% in stage 4 where surgical removal was not feasible.8
Pancreatic NETs mostly occur sporadically and occasionally in association with other genetic syndromes such as multiple endocrine neoplasm (MEN) syndrome, Von Hipple-Lindau syndrome, tuberous sclerosis (complexes 1 [TSC1] or 2 [TSC2]), and neurofibromatosis type-1.9–11 Familial syndromes account for less than 10% of all the cases and are characterized by an inherited deleterious germline mutation in a tumor suppressor gene that leads to increased tumor susceptibility in the pancreas and in other neuroendocrine organs, leading to the development multiple tumors.
With the advances in molecular biology and information gathered through sequencing studies, there has been a recent surge to investigate biomolecular pathways to improve understanding of the pathophysiology of these tumors and help develop targeted therapies as potential treatment modalities. In this review, we will provide a comprehensive summary of the current knowledge of signaling pathways involved in the tumorigenesis of panNETs. We will describe studies targeting these pathways at preclinical and clinical level. This will help to foster and develop further treatment strategies.
Classification of PanNETs
The classification of NETs has evolved considerably. The newest version of the World Health Organization classification system established in 2017 includes a classification specifically for panNETs. These are classified as well-differentiated tumors or poorly differentiated tumors, with grading based on the proliferation rate of the neoplastic cells, as determined by the mitotic count and/or the Ki-67 labeling index. Well-differentiated panNETs tumors include those of grade 1 (G1) and grade 2 (G2) as well as a new subset of grade 3 (G3) well-differentiated tumor. Poorly differentiated pancreatic neuroendocrine carcinomas are those with G3 histology12 and are subclassified into small cell or large cell type. Low-grade (G1) tumors have a mitotic count of less than 2 per 10 high-power fields (HPFs) or a nuclear Ki-67 labeling index of less than 3%. Intermediate-grade (G2) panNETs are those with 2 to 20 mitoses per 10 HPFs or a nuclear Ki-67 labeling index of 3% to 20%. Both the G3 NETs and neuroendocrine cancers (NECs) have mitotic counts of more than 20 per 10 HPFs or more than 20% nuclear Ki-67 labeling index.
This updated 2017 World Health Organization classification formally recognizes the heterogeneity of G3 tumors.13
This change was prompted after multiple studies noticed heterogeneity in prognosis and response to treatments among the G3 group. Sorbye at al14 first reported the heterogeneity among the G3 patients and proposed further classifying the group based on the Ki-67.
Another study revealed that approximately 50% of G3 neuroendocrine neoplasms (NENs) had well-differentiated morphology similar to the G2-NET and had longer median survival compared with the poorly differentiated G3 NEC.15 A study by Basturk et al16 described that G3 NEN, which showed discordant between Ki-67 and mitotic rate, had longer overall survival similar to G2 NET, when compared with grade-concordant G3 NEN.
A large European study evaluating G3 gastroenteropancreatic NEN, including 37 G3 NETs and 167 G3 NECs, showed that G3 NET had relatively higher overall survival at 99 months versus 17 months in G3 NEC (P < 0.001).17 However, they also noted that the G3 NET group had poorer response rate to treatment with platinum-based chemotherapy regimens compared with the G3 NEC.17
This grading system is considered useful for diagnostic purposes, for treatment decisions, and to guide discussions of prognosis.18–20 There still remains a degree of heterogeneity among the low- and intermediate-grade tumors21 highlighting the need of biomarkers and molecular fingerprints to further stratify the NENs and optimize treatment outcomes.
Genetic Differences Between G3 NET and G3 NEC and Their Implications
In addition to the histological differences, recent advances have demonstrated genetic differences for G3 NET and G3 NEC, increasing our understanding of the underlying biology and aggressiveness of these tumors and thus changes in clinical treatments.
Sequencing of the G3-NET has revealed mutations in DAXX, ATRX, and MEN1 genes.22 RB1 gene mutation with Rb loss or abnormal p53 expression was not seen in the G3 NET,23–25 hence making them genetically more similar to G1 and G2 NET. On the other hand, RB1 and TP53 gene mutations are commonly found in G3 NEC. A study by Hijioka et al24 found that 54.5% of G3 NECs had negative Rb expression, whereas 48.7% of G3 NECs had KRAS gene mutation. A study evaluating the molecular expression in 19 poorly differentiated pancreatic NEC revealed that 95% of them had abnormal expression of the p53 protein and 74% had abnormal Rb protein.26 In addition, 74% of the NEC had overexpression of bcl-2 protein.26
The treatment landscape for pancreatic NEN is also changing based on new genotyping data, and treatment recommendations for G3 NET are still evolving given that it is a new subtype classification. The European Neuroendocrine Tumor Society has recommended treatment regimens containing alkylating agents such as temozolamide and streptozotocin.27
Studies are also ongoing to evaluate the role of everolimus in G3 NET with the knowledge that the mTOR is overexpressed in a large number of G3 NECs.28 Furthermore, immune checkpoint inhibitors are also being evaluated in G3 NET, as recent data have shown evidence of immune dysregulation in NET.29
On the other hand, platinum-based chemotherapy regimens have been recommended by the European Neuroendocrine Tumor Society27 and the National Comprehensive Cancer Network30 for G3 NEC, as they are thought to share similar features to the poorly differentiated NEC in other organs such as the lungs.31 Most common regimens used are cisplatin and etoposide or cisplatin and irinotecan. There are no prospective evidence for the efficacies of these treatment, and most of the data are based on prior retrospective studies.32 A multicenter Japanese study on panNEN had revealed that the response rate among G3 NEC to platinum-based regimen was 61.3%.24 To investigate the predictive factors for response to platinum-based treatment, Hijioka et al32 had analyzed immunolabeling in 49 patients with G3 NEC. They found that the group with loss of Rb protein expression had significantly better response to platinum regimen at 80% compared with 38.4% in the group with retained Rb expression.
The recent changes in the classification, the genetic differences, and the variable response to treatments between G3 NET and G3 NEC highlight the need for further research for these entities, which will continue to impact treatment recommendations.
Genetics of Sporadic PanNETs
Most of our knowledge of pathways involved in panNET tumorigenesis was gained from genomic exome sequencing of patients with panNETs. In 2011, whole-exome sequencing of a 10 sporadic metastatic panNETs identified a set of commonly mutated genes. These were subsequently explored in 58 additional panNETs.7 The most commonly somatically mutated genes encoded for proteins implicated in chromatin remodeling: 44% of the tumors had somatic inactivating mutations in MEN1, and 43% had mutations in genes encoding for a transcription/chromatin remodeling complex composed of death domain–associated protein (DAXX) and a thalassemia/mental retardation syndrome X-linked (ATRX).7 In addition. 14% of the patients had mutations in genes from the mammalian target of rapamycin (mTOR) pathway and mutations in the catalytic subunit of phosphatidylinositol 3-kinase (PI3K). Table 1 summarizes the most common mutations identified by Jiao et al,7 its prevalence and pathway involved. We will describe in detail the knowledge up to date for each of these pathways at the preclinical and clinical levels.
Chromosomal Aberrations and MicroRNA in PanNET
In the last few years, there is a shift from classifying tumor primarily based on histopathological data to including genetic and molecular markers. Studies have identified various biomarkers that not only affect the prognosis of the tumor but also affect tumor management. In panNET, various genomic differences and chromosomal aberrations have been identified among primary and metastatic panNETs.
A study evaluating 37 primary panNET and 11 metastatic panNET revealed that there were significant genomic differences among the primary tumor and the metastatic tumors.33 They found that genomic aberrations involving 6p22.2 to 6p22.1, 8q24.3, 9q34.11, and 17p13.1 were associated with a poorer prognosis.33 Another study also noted that aberrations at 5p12 to 5p13, 4q13 to 4q24, 5p15, 5q11 to 5q31, 9q21 to 9q22, 11p11, 11p14 to 11p15, 11q23, 11p12 to 11p13, and 11q22. Tumors with lower Ki-67, smaller size, and no distant metastasis were found to have less aberrations.34 Genomic gains were found to be more common than genomic losses in both studies.33,34
MicroRNAs, which are noncoding RNA composed of 22 nucleotides, have also been implicated to play an important role in pathogenesis of panNET. A Korean study on 37 surgically resected panNET revealed that miR-196a was associated with poor prognosis and shorter disease-free survival.35 Pancreatic NET with higher expression of miR-196a had higher pathological stage, mitotic rate, and Ki-67.35
Another study revealed that there was increased expression of miR-155, miR-146a, miR-142-3p, and miR-142-5p in panNET.36 MiR-155 is also expressed in pancreatic intraepithelial neoplasms and has been associated with progression of the disease to pancreatic adenocarcinoma.37 Its role in panNET is not clear at this point.
Although several genomic aberrations and overexpression of microRNA have been associated with panNET, more research is needed to further establish its prognostic role and usefulness in individualizing treatment of panNET.
PATHWAYS OF RELEVANCE FOR PANNETS
The PI3K/AKT/mTOR pathway is involved in regulation of cell proliferation, growth, survival, autophagy, and other vital functions.38 This pathway is initiated when PI3K is activated by the receptor tyrosine kinase, which in turn catalyzes conversion of the phosphatidylinositol-4,5-diphosphate to phosphatidylinositol-3,4,5-triphosphate, initiating the activation of protein kinase B (AKT). Through an unknown mechanism, mTOR complex 2 (mTORC2) gets activated causing cascade activation of AKT, mTOR complex 1 (mTORC1), and other effectors to initiate multiple vital cellular processes. Phosphatase and tensin homolog (PTEN) is a negative regulator of the pathway that acts as a tumor suppressor by converting phosphatidylinositol-3,4,5-triphosphate back to phosphatidylinositol-4,5-diphosphate, hence inhibiting tumorigenesis and angiogenesis.38,39 PTEN, TSC1, and TSC2 also inhibit phosphorylation of mTOR, hence suppressing tumor progression.40
Exome sequencing has shown that approximately 14% of well-differentiated panNETs have somatic mutations that occur in the PIK3/AKT/mTOR pathway. These include mutations in PTEN (7%), TSC2 (9%), and PIK3CA (1%).7 Similarly, in a gene expression profile study on a large cohort of patients, proteins encoded by TSC2 and PTEN, 2 key inhibitors of the mTOR pathway, were underexpressed in most primary panNETs.41 Taken together, these findings suggest a role of the mTOR pathway in tumorigenesis of panNETs (Fig. 1). Additional mechanisms of pathway activation is through aberrant tyrosine kinase mutations, such as overexpression of insulin-like growth factor 1 (IGF-1) receptor and fibroblast growth factor receptor 3, resulting in downstream activation of PI3K and RAS signaling pathways.38,39 The effects of these mutations are far more extensive as this signaling pathway interacts with numerous other signaling loops.7,39,42
PI3K is the upstream component in the mTOR pathway and is composed of regulatory subunit (PIK3R1 p85α) and catalytic subunit (PIK3CA p110α). Its activation results in phosphorylation of 3-phosphoinositide–dependent protein kinase-1, which in turn is a downstream regulator of mTOR.40 PI3K mutation has been reported in 1.5% of panNETs as a single mutation, although more frequently, it is associated with other regulator mutations such as PTEN or TSC.7
Preclinical studies using BON cell lines, derived from metastatic lymph nodes of patients with panNET, have shown treatment with LY294002 blocks the downstream activation of PI3K in panNET cells.1 BON cell secretes IGF-1 and activates IGF-1 receptor, which in turn activates PI3K.43 PI3K is involved in regulation of expression of cyclin D1 (Fig. 2) and p27kip144 as well as inhibition of p110α, the catalytic subunit of class 1 PI3K involved in tumorigenesis in vitro.45 Loss of function of p110α causes unregulated activity of AKT through inactivation of PTEN.
AKT is a serine/threonine kinase with 3 isoforms and a key regulator of PI3K and mTOR pathway. AKT plays a major role in cell migration, proliferation, and apoptosis and hence plays a crucial role in the regulation of tumorigenesis. AKT phosphorylates the TSC2 resulting in the activation of the PI3K pathway, hence promoting tumor progression and angiogenesis.46 Approximately 61% to 76% of panNETs have increased AKT expression.47,48
Preclinical studies examining the effects of perifosine, a pan-AKT inhibitor on BON1 cells, revealed that AKT 1 and AKT3 are of particular importance in panNET cell survival, as their inhibition using perifosine resulted in decreased phosphorylation of ERK1/2 (extracellular signal regulated kinases) and subsequent induction of apoptosis in the BON cells.49
Mammalian Target of Rapamycin
mTOR is an important target of the PI3K/AKT/mTOR pathway. It is a serine/threonine kinase encoded by the FRAP-1 gene. It is made of 2 proteins, namely, mTORC1 sensitive to rapamycin and mTORC2 that is resistant to rapamycin.50 mTOR undergoes activation by phosphorylation of the serine moiety on the molecule through AKT in the PI3K/AKT pathway.51 mTOR is involved in many vital cellular processes including G1/S phase cell-cycle transition,52 and its protein mTORC1 interacts with many other pathways by controlling other modulators such as ornithine decarboxylase, glycogen synthase, and hypoxia-inducible factor 1α (HIF-1α).53,54 The activation of mTOR pathway is associated with elevated MKi-67 and higher proliferative index and is usually associated with more aggressive malignancy and adverse outcomes. As such, MKi-67 has been used as a prognostic marker of NETs.55,56
Earlier preclinical studies in carcinoid cell lines BON-1 and NCI-H727, rapamycin (prototype mTOR inhibitor) showed a significant reduction in cell proliferation in vivo and in vitro,57 although it induced AKT phosphorylation leading to upstream signaling. Subsequently, a preclinical study by Chiu et al58 examined dual inhibition targeting the endothelial growth factor receptor (EGFR) and mTOR pathway with erlotinib and rapamycin in a Rip-Tag2 transgenic mouse model of panNET. The study revealed that that dual therapy was more effective in increasing overall survival and reducing tumor burden in the animal model. Interestingly, dual therapy also prevented the development of resistance to mTOR inhibitors that occurred with the monotherapy.
Tuberous Sclerosis Complex 2
TSC2 protein is a regulatory protein of the mTOR pathway and its expression is down-regulated in panNETs. It is associated with worse prognosis and increased aggressiveness of the tumor.41 There is higher incidence of panNETs in patients with tuberous sclerosis at 1.8% compared with general population, with incidence of 0.002%. Taken together, these findings suggest that TSC1 and TSC2 proteins have an important role in the development and progression of panNETs.59,60
Phosphatase and Tensin Homolog
PTEN is a tumor suppressor gene that negatively regulates mTOR in the PI3K/AKT/mTOR pathway. Loss of PTEN function results in overexpression of mTOR pathway with subsequent tumor progression. PTEN expression is lost in approximately 7.4%,5 although higher incidence of 10% to 29% has been described in some studies.61 Loss of PTEN expression correlates with more advanced stages of cancer.5 In a study by Missiaglia and colleagues,41 they reported that low expression or loss of PTEN and TSC2 was associated with increased risk of liver metastasis. Approximately 85% of primary panNETs have altered levels of PTEN and TSC2, although the rate of actual somatic mutations was lower at 10%.7,41
Clinical Studies Targeting the mTOR Pathway
Everolimus has been tested on panNETs and other NETs in 4 large multinational phase II and III trials. RADIANT-1 was a phase II trial that examined the effects of everolimus versus everolimus and octreotide in patients with chemotherapy-refractory panNETs. It showed a statistically significant relationship between the reduction in the initially elevated chromogranin A levels and the increase in progression-free survival (PFS) in panNET patients.60 RADIANT-2 was a phase III trial conducted on patients with advanced NETs and carcinoids. Although RADIANT-2 did not meet its desired end point, the trial showed increased PFS in patients treated with everolimus after adjusting for all the confounding factors.39,62,63 The median PFS was greater for patients receiving everolimus and octreotide compared with patients in the placebo arm. RADIANT-3 was the trial that led to the Food and Drug Administration approval of the use of everolimus in panNETs in 2011. The trial examined 410 patients with unresectable or metastatic low- or intermediate-grade panNETs. Everolimus, as compared with placebo, was associated with a significant prolongation of the median PFS (11.0 months vs 4.6 months). Although the response rate by conventional Response Evaluation Criteria in Solid Tumors (RECIST) was low at 5% in everolimus arm versus 2% in the placebo arm, the rate of patients experiencing stable disease was increased to 73% versus 51% in the placebo arm.39,63,64 The RADIANT-4 trial examined the use of everolimus in treatment of NET, mainly lung and GI NETs and showed significant improvement in the median PFS from 3.9 months in the placebo arm versus 11.0 months in the everolimus arm.65
Although an approved regimen, everolimus is only effective in delaying tumor progression without significant effect on the response rate or the patterns of tumor progression.64,66 The limited effectiveness is partly because everolimus is only active against mTORC1. Inhibition of mTORC1 releases the negative feedback on mTORC2 resulting in mTORC2-mediated activation of AKT.67 Another therapeutic limitation with everolimus as monotherapy is the reactivation of different arms along the PI3K/AKT/mTOR pathway. As a result, research was steered to the direction of alternative inhibitors of the PI3K/AKT/mTOR pathway either targeting both mTORC1 and mTORC2 or testing everolimus combined with other agents.
Challenges With mTOR Monotherapy and Resistance
Although mTOR inhibitors are approved in panNET, the efficacy of these molecules has been far less than anticipated with nondurable responses. Both primary and secondary resistance to mTOR inhibitors has been described in panNET patients and represents a major challenge in patient care.68 The first-generation mTOR inhibitors (rapamycin and its analogs) act by binding to the FRB domain with the FKBP12, hence blocking the access of substrate to the active kinase site.69,70 This only blocks the mTORC1 partially leading to the limited clinical efficacy.71 As such, second-generation mTOR inhibitors, named as ATP-competitive inhibitors of mTOR, have been developed. They act on the active kinase domain directly to block both the mTORC1 and mTORC2 pathway.72,73 These drugs are being tested in clinical trials and have not been approved for clinical use. Recent studies have reported the development of a third-generation mTOR inhibitor, Rapalink, that will be able to overcome the existing resistance to the first- and second-generation mTOR inhibitors.74 It has been proposed that the Rapalink will act on both the FRB and the kinase domain to exert its effects.74
Other mechanisms of secondary or acquired resistance to mTOR inhibitors have been described, such as appearance of mTOR mutations, loss of function of PP2A (a phosphatase involved in dephosphorylation of AKT), activation of a feedback loop, or alternative pathways.
Similarly, activation of other pathways to bypass inhibition of mTORC1 may shift the balance to increased mTORC2 activity, leading to AKT phosphorylation and activation as a contributor to diminished rapalogs anticancer activity.75 In a preclinical study on various cancer cell lines, it was revealed that inhibition of mTOR resulted in up-regulation of AKT kinase activity through IGF-I signaling.76 Investigations to use combination regimens of mTOR inhibitors with PI3K inhibitors have been suggested to possibly increase the efficacy of these drugs.
mTOR Inhibitors Combined With Other Agents
mTOR Inhibitors Combined With Antiangiogenesis Drugs
Temsirolimus, another mTOR inhibitor, was studied in phase II trial in patients with progressive panNETs; although it only reported 9% objective response rate, 67% of the patient had disease control.77 The promising results prompted next-level trial with combination of temsirolimus with bevacizumab, which will be discussed in the section on angiogenesis/HIF-1α pathway. Recent trials involving combination temsirolimus and bevacizumab (phase II) have demonstrated increased response rate and PFS, with response rate of 44% with 6-month PFS of 79% by RECIST criteria within 7 months of study entry in the latter.78
mTOR Inhibitors Combined With AKT Inhibitors
The AKT inhibitor MK-2206 was recently tested in panNET BON cell lines to study its effects on cell proliferation and bioactive hormone production in vitro.79 The study revealed significant suppression NET markers such as ASCL1, CgA, and NSE expression as well as antiproliferative effect through apoptosis induction.80 A phase I clinical trial of MK-2206 on 33 patients with various solid tumor types revealed partial responses in 2 patients with advanced panNETs with tumor shrinkages of 13.1% and 17.5%, respectively.81 Both patients had remained on the trial for 32 weeks, with the latter who was on a lower-dose regime experienced marked reduction in the ascites and peripheral edema, as well as computer tomography–confirmed central tumor necrosis.81 With promising results in both preclinical and clinical trials,79 various combination trials have been initiated with erlotinib, AZD6244, lapatinib, and chemotherapy on various malignancies, hoping to maximize therapeutic potential in panNETs.81
Blocking the PI3K/AKT/mTOR pathway and TORC2 may theoretically limit challenges for mechanisms of resistance to everolimus. In this context, BEZ-235, a potent oral dual PI3K/mTOR inhibitor was investigated in clinical trials. In preclinical studies with BON-1 and QGP-1 cell lines, it had proven to be more efficient in inhibiting proliferation of the panNET cell. BEZ235 in combination with everolimus exhibited synergistic effect in the inhibition of panNET cells proliferation and possible reversal of the everolimus-acquired resistance among the BON cells.82,83 Multiple phase 1/1b trials were conducted with various different formulations and dosages, as significant variability in the pharmacokinetics among the patients was noticed.84 Two phase II trials with BEZ235 as single agent were designed in patients with panNETs: (1) BEZ235 in patients with panNETs who had failed everolimus and (2) randomized trial with BEZ235 versus everolimus in patients previously not treated with mTOR inhibitors. Both trials were prematurely halted. The former was halted because of unmet statistical end point with early disease progression and high events of adverse effects.85 The latter was halted because of poor tolerability and superiority of everolimus among the randomized patients.86,87
Dual mTORC1/mTORC2 Inhibition
Recently, few mTORC1/mTORC2 inhibitors were developed, namely, INK 128, AZD8055, and OSI-027. INK 128 is used by Nakakura et al141 in an ongoing preclinical study with mouse models of panNETs has shown efficacy in halting tumor growth or shrinking tumor size in everolimus resistant panNETs. This is not tested yet in human trials but may be another mechanism to try to overcome resistance.
Future Directions for mTOR Pathway
There is a need for biomarker-driven trials to stratify the subgroup of patients who will respond to mTOR inhibition and should take in consideration mutations and protein expression. For example, the low prevalence of somatic mutations in mTOR pathway genes does not match the high prevalence of cases with low gene expression. Similarly, it is unclear why a substantial number of patients had disease stability on clinical trials using mTOR inhibition if the prevalence of mutations in this pathway is small. This suggests that, in addition to somatic mutations, alternative mechanisms may cause up-regulation of the mTOR-signaling axis. Future studies seeking to determine if mTOR pathway mutations or protein expression predicts response to these agents would be ideal to appropriately select patients who would benefit from these drugs. Combination strategies of agents targeting multiple pathways to overcome resistance to everolimus will be crucial to advance the field.
Menin (MEN1 Gene)
MEN1 is a tumor suppressor gene located on chromosome 11q13 and is a commonly affected gene in MEN syndrome.89 The rate of somatic mutations of MEN1 in sporadic panNETs has been reported in 25% to 44% of panNETs.7,9,90,91 Previous reports have shown approximately 40% loss of heterozygosity at 11q1389 with abnormally low nuclear staining of Menin, the protein encoded by MEN1 gene.90
Menin is known to have diverse positive or negative regulatory functions including gene transcription.92,93 It causes expression of the antiproliferative genes such as cyclin-dependent kinase inhibitors through the mixed lineage leukemia (MLL) histone methyl transferase complex.94,95 Menin not only mediates the MLL-dependent histone H3 lysine 4 methylation96 to maintain promoter activity of CDKN2C and CDKN1B but also binds directly to the MLL complex.97,98 Menin takes part in the nuclear protein complex that promotes site-specific histone methylation and regulates histone methylation in promoters of specific target genes with a role in growth and differentiation of neuroendocrine cells.
In addition, recent studies have also revealed that there are other possible gene mutations apart from MEN1 that affect the menin expression in the panNET cells. A combined genetic and immunohistochemistry study on MEN1 gene and menin expression revealed that 40% of the panNET had weak or absent nuclear immunostaining for menin, whereas 80% had intensified cytoplasmic immunostaining for menin. Surprisingly, only 25% of these panNET harbored a MEN1 gene mutation. This was compared with normal pancreas islet cell, which showed strong nuclear and very faint cytoplasmic signal.99
Similar results were seen in another study evaluating menin expression in various endocrine tumors of the gastroenteropancreatic system. They reported menin expression in 78% of all tumors and 93% of the panNETs, whereas only 14.3% of the tumors had hypermethylation of the MEN1 gene. These 2 studies suggest that there are other pathways or genes that are involved in the altered expression of menin.100
Taken all together, these findings suggest that MEN1 directly or indirectly plays an important role in islet cell proliferation in panNETs and would be a good target for potential therapeutic approaches. To date, there are no drugs/trials targeting this.
PanNETs are highly vascular tumors that express increased levels of proangiogenic molecules such as HIF-1α and vascular endothelial growth factor (VEGF).101–104 The endocrine and endothelial cells of the vascular system of pancreatic islet cells interact with each other,105 and the high levels of proangiogenic factors are necessary not only for the islet microvasculature but also for the homeostasis of blood glucose levels.106–108 Well-differentiated NETs express higher levels of HIF-1α, VEGF, and microvessel density compared with poorly differentiated NETs.101,102 A denser vascularity in panNETs has been associated with better prognosis.109 Other molecules such as c-kit, mTOR, and PDGF are also involved in angiogenesis in panNETs.110
Hypoxia-Inducible Factor 1α
HIF-1α has a role in tumor progression in sporadic panNETs. Lack of degradation of HIF-1α may lead to enhanced transcription of proangiogenic factors such as VEGF.111,112 Pinato et al112 identified that overexpression of HIF-1α is a predictor of poor prognosis. Prolyl hydroxylase domain proteins (PHDs) regulate the HIF-1 pathway by mediating molecular responses to the changes in tissue oxygenation. Under hypoxic conditions, PHDs are inhibited from mediating HIF-1α ubiquination and proteosomal degradation resulting in stabilization and transcriptional activation of HIF-1α.5 Presence of nuclear expression of HIF-1α and PHDs has been shown to be associated with poorer prognosis, whereas cytoplasmic expression of PHDs was associated with low microvascularity in a retrospective study.102,113 Notably, mTOR also mediates the translation and activity of HIF-1α,38 suggesting that the synergistic targeting of the mTOR pathway with HIF-1α pathway could result in better response rates.
Vascular Endothelial Growth Factor
VEGF is expressed in approximately 78% to 80% of panNETs,101,102 and is generally expressed in well-differentiated tumors. Couvelard et al102 showed that there is loss of VEGF expression with resultant deceased vascular density and activation of hypoxia pathway as the tumor progresses. In NET, one of the most widely used VEGF inhibitors is sunitinib, a potent inhibitor of VEGF, Kit, and PDGF.114 Preclinical studies of sunitinib in RIP1-Tag2 transgenic mouse model of panNETs revealed reduction in tumor burden and better prognosis with increased survival in the animal model.115 Sunitinib caused 75% reduction in the endothelial cells by inhibition of VEGF and 63% reduction of the pericyte coverage of tumor microvasculature by inhibition of PDGF.102,115
Clinical Trials Using Antiangiogenesis Strategy
Given the role of angiogenesis in panNET tumorigenesis, clinical trials using antiangiogenic strategies with VEGF inhibitors (bevacizumab) or the VEGF-receptor targeted tyrosine kinase inhibitor (sunitinib, pazopanib, cabozantinib) were initiated (Fig. 3).
A phase II trial tested sunitinib in patients with metastatic panNETs and showed a promising response rate of 16.7%.116 Subsequent phase III study evaluated the effect of sunitinib versus placebo in patients with advanced well-differentiated panNETs. The study was halted at interim analysis because of significantly more adverse events in the placebo arm and improved PFS in the treatment arm.117 Investigators noted a PFS of 5.5 months in placebo arm compared with 11.4 months on the treatment arm.117,118 The findings lead to sunitinib approval by the Food and Drug Administration in 2011 for the management of nonresectable locally advanced or metastatic panNETs.119
With promising results from phase III trials with everolimus and sunitinib and preclinical data suggestive of better result with combination target therapy, single-arm 2-stage phase II clinical trials with temsirolimus with bevacizumab were designed.78 The response rate was notably 41%, whereas the 6-month PFS was 79% by RECIST criteria within 7 months of study entry. Median PFS was also significantly prolonged at 13.2 months with overall survival of 34 months.78
Subsequently, a multicenter phase II trial with single-agent bevacizumab was conducted in patients with well- to moderately differentiated panNETs with no prior therapy with mTOR inhibitor to evaluate the efficacy of bevacizumab as a single agent. The response rate was reduced at 9%, with 6- and 12-month PFS being 95% and 54%, respectively.120
A few other tyrosine kinase inhibitors with VEGF inhibition activities worth noting are sorafenib and pazopanib. Phase II trial with sorafenib revealed a response rate of 9% among 41 patients with panNETs.121 Pazopanib, on the other hand, was examined in 29 patients with panNETs on stable doses of octreotide long-acting release (LAR) and demonstrated a response rate of 22%.122
Cabozantinib is a tyrosine kinase inhibitor that targets the VEGF and MET pathways. A recent phase II trial tested cabozantinib in carcinoid and panNETs,123 after preclinical studies showed decreased cell stability and reduced metastatic potential in NET models.124 This study involved 20 patients with metastatic or unresectable well-differentiated G1 to G2 panNETs, and response was observed in approximately 15% of patients with a median PFS of 21.8 months.123 The findings on initial studies with this drugs were very encouraging, and larger phase III trials are planned in the short-term future after these results.
Death Domain–Associated Protein/Thalassemia/Mental Retardation Syndrome X-Linked
DAXX and ATRX are proteins involved in chromatin remodeling and stabilization,125 especially in the telomeric areas.126 Mutations in DAXX/ATRX are mutually exclusive and can promote tumorigenesis.5 Both copies of DAXX are usually inactivated, one by mutation and the other by loss or epigenetic silencing. Both copies of ATRX are also inactivated, one through mutation and other through chromosome X inactivation.7 The presence of homozygous mutations or the possibility of inactivating nonsense mutations in both genes defines their role as tumor suppressor genes and in panNET tumorigenesis.
DAXX and ATRX are H3.3 histone chaperone required for incorporation of H3.3 at the telomeres.126 DAXX is involved in the regulation of ubiquination mediated by ligase MDM2, whereby DNA damage results in degradation of MDM2 leading to p53 stabilization and subsequent cell-cycle arrest.5 The loss of up-regulation of p53 with DAXX mutation in panNETs leads to tumor progression.127 ATRX, on the other hand, is involved in the suppression of telomeric repeats with RNA expression128,129 as well as of the G-rich tandem repeats near the telomeric regions.130 Studies with mouse models have been proposed to study the pathway as well as for potential development of target therapies focusing on DAXX/ATRX. These will be crucial for future therapeutic strategies.
Mutations in the DAXX/ATRX were initially associated with longer overall survival.7,131
Because the proteins encoded by ATRX and DAXX participate in chromatin remodeling at telomeres and other genomic sites, subsequent studies examined the telomere status of panNETs and found that 61% of panNETs displayed abnormal telomeres that are characteristic of a telomerase-independent telomere maintenance mechanism termed alternative lengthening of telomeres (ALT). Pancreatic NETs exhibiting these abnormal telomeres had ATRX or DAXX mutations or loss of nuclear ATRX or DAXX protein.132
De Wilde et al133 reported that ATRX and DAXX defects and ALT phenotype occurred only in panNETs measuring at least 3 cm and their lymph node metastases, suggesting that these changes are late events in panNET development. Larger retrospective studies have shown that loss of DAXX or ATRX protein and ALT are associated with chromosome instability in panNETs and shorter survival times of patients. This supports the hypothesis that DAXX- and ATRX-negative tumors are a more aggressive subtype of panNET.134
Interestingly, disease stage may impact the prognostic value for these markers. Primary ALT-positive and ATRX/DAXX-negative panNETs were independently associated with aggressive clinico pathologic behavior with reduced recurrence-free survival in patients with curative resection135 but seem to confer a better overall survival in patients with metastases.131,135 Studies with mouse models have been proposed to study the pathway as well as for potential development of target therapies focusing on DAXX/ATRX. These will be crucial for future therapeutic strategies.
c-MET is a proto-oncogene expressed in cancers that encodes for a tyrosine kinase expressed on the stem cell and progenitor cells in the embryonic stage, which functions to promote cell regeneration and repair.136 In a study performed on RIP-Tag2 mice with panNET, it was found that c-MET expression was stronger in the tumor cells that were treated with sunitinib (VEGF inhibitor). Subsequent inhibition of c-MET using PF-04217903 further reduced the metastasis of the tumor cells to the liver.124 Another study that looked at the tumors in mouse xenograft models revealed that, although MET was highly expressed in the tumor cells, the tumor cells did not carry the gene for the protein MET ligand, hepatocyte growth factor. Instead, the hepatocyte growth factor was expressed in the neighboring noncancerous cells.136 This is an interesting finding because it reveals that the cancer cells use the c-MET pathway as a rescue mechanism when angiogenesis is inhibited, and they rely on neighboring cells for the activation of this pathway. A recent phase II clinical trial evaluated cabozantinib, which is a TKI that targets MET and VEGF, in panNETs and carcinoid tumors and showed promising results. The details of the study are discussed under the section clinical trials using antiangiogenesis strategy.
Although p53 mutations or dysfunctions are usually associated with tumor progression, these rarely occur in panNETs.137 Instead, panNETs may have high levels of p53 regulators such as MDM2, MDM4, and W1P1,138 which repress p53 activity, hence relieving the cell-cycle arrest and leading to tumor formation. Recent studies revealed that SV40T, which is responsible p53 and Rb inactivation, is associated with more aggressive phenotype of β-cell panNET in mice.137 The data from recent studies are important in realizing that targeting the overexpression of upstream modulators of the p53 pathway may be important in controlling tumorigenesis and increasing panNETs' sensitivity to other therapies, as attenuated p53 function would be restored.137
Previous studies have shown minimal or absent Notch signaling activation in NETs.139–141 The human panNET BON cells lack active Notch-1 protein, despite having expression of all 3 Notch receptors. A study with adenoviral vector caused transient activation of the Notch signaling pathway and confirmed its role as tumor suppressor in panNETs when activated.142 However, another study that examined 120 well-differentiated GI NETs showed that Notch-1 and Hes-1 (a downstream target of Notch pathway) were expressed in approximately a third (34%) of the panNETs.143
In normal conditions, the Notch signaling pathway is not active in panNETs, and controlled activation of the Notch pathway can be used for inhibition of tumorigenesis.142 Given this new information on the preferential expression of Notch in panNETs, more studies are needed to explore the factors surrounding the heterogeneity in the expression of Notch-1. This pathway has not been tested at clinical level.
Scr are nonreceptor tyrosine kinases that play a role in tumorigenesis, specifically cell adhesion and metastasis, in panNETs.144 A study on BON cell line showed that Scr kinases facilitates EGFR transactivation by various G-protein coupled receptors, which are in turn activated by GI hormones or neurotransmitters.145 In the same study, a specific Scr inhibitor PP2 was able to inhibit the transactivation of EGFR in the BON cells. Knowing the involvement of Scr in cancer growth and that anti-EGFR therapies such as sunitinib have shown some promise, it is necessary to examine this pathway further and develop Scr inhibitors that can be used in the clinical setting for panNETs.
With the recent increase of immunotherapy trials in various cancer types, a study looked at the PD-L1 expression in 24 gastroenteropancreatic NETs, of which 14 were panNETs. They found greater expression of PD-L1 in tumors with higher pathologic grade.29 With this important revelation, phase 1b study KEYNOTE-028 (NCT02054806) was designed to evaluate the efficacy of pembrolizumab in patients with PD-L1–positive advanced carcinoid and panNET. Preliminary results were presented at the recent European Society for Medical Oncology meeting and showed that the drug was generally well tolerated and the patients with panNETs had ongoing response of 17.6 months on average.146 These data are certainly promising, although more research is needed to optimize the use of immunotherapy in this area.
Table 2 summarizes all the preclinical and clinical trials included in this review, differentiated by the pathways they act on, whereas Table 3 includes trials and studies that are ongoing or recently completed pending publications.
Pancreatic NETs are unique tumors owing to their heterogeneity in clinical presentation, mutational pattern, and the diversity of activated signaling pathways. Given the multiple pathways involved and the cross-talk between some of these, it is likely that a multiagent therapeutic approach may be needed to improve patient outcomes. Future studies should ideally obtain tissue specimens for correlative studies to better understand the mechanisms of response and mechanisms of resistance to these drugs. This will be crucial to guide subsequent clinical trials and further advance the field.
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Keywords:Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
pancreatic neuroendocrine tumor; panNET; molecular biology; signaling pathways; checkpoints; tumorigenesis