European Neuroendocrine Tumor Society (ENETS) 2023 guidance paper for gastroduodenal neuroendocrine tumours (NETs) G1-G3.

The aim of the present guidance paper was to update the previous ENETS guidelines on well-differentiated gastric and duodenal neuroendocrine tumours (NETs), providing practical guidance for specialists in the diagnosis and management of gastroduodenal NETs. Type II gastric NETs, neuroendocrine carcinomas (NECs), and functioning duodenal NETs are not covered, since they will be discussed in other ENETS guidance papers.

Circulating hsa-miR-5096 predicts 18F-FDG PET/CT positivity and modulates somatostatin receptor 2 expression: a novel miR-based assay for pancreatic neuroendocrine tumors.

Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are rare diseases encompassing pancreatic (PanNETs) and ileal NETs (SINETs), characterized by heterogeneous somatostatin receptors (SSTRs) expression. Treatments for inoperable GEP-NETs are limited, and SSTR-targeted Peptide Receptor Radionuclide Therapy (PRRT) achieves variable responses. Prognostic biomarkers for the management of GEP-NET patients are required. 18F-FDG uptake is a prognostic indicator of aggressiveness in GEP-NETs. This study aims to identify circulating and measurable prognostic miRNAs associated with 18F-FDG-PET/CT status, higher risk and lower response to PRRT. Methods: Whole miRNOme NGS profiling was conducted on plasma samples obtained from well-differentiated advanced, metastatic, inoperable G1, G2 and G3 GEP-NET patients enrolled in the non-randomized LUX (NCT02736500) and LUNET (NCT02489604) clinical trials prior to PRRT (screening set, n= 24). Differential expression analysis was performed between 18F-FDG positive (n=12) and negative (n=12) patients. Validation was conducted by Real Time quantitative PCR in two distinct well-differentiated GEP-NET validation cohorts, considering the primary site of origin (PanNETs n=38 and SINETs n=30). The Cox regression was applied to assess independent clinical parameters and imaging for progression-free survival (PFS) in PanNETs. In situ RNA hybridization combined with immunohistochemistry was performed to simultaneously detect miR and protein expression in the same tissue specimens. This novel semi-automated miR-protein protocol was applied in PanNET FFPE specimens (n=9). In vitro functional experiments were performed in PanNET models. Results: While no miRNAs emerged to be deregulated in SINETs, hsa-miR-5096, hsa-let-7i-3p and hsa-miR-4311 were found to correlate with 18F-FDG-PET/CT in PanNETs (p-value:<0.005). Statistical analysis has shown that, hsa-miR-5096 can predict 6-month PFS (p-value:<0.001) and 12-month Overall Survival upon PRRT treatment (p-value:<0.05), as well as identify 18F-FDG-PET/CT positive PanNETs with worse prognosis after PRRT (p-value:<0.005). In addition, hsa-miR-5096 inversely correlated with both SSTR2 expression in PanNET tissue and with the 68Gallium-DOTATOC captation values (p-value:<0.05), and accordingly it was able to decrease SSTR2 when ectopically expressed in PanNET cells (p-value:<0.01). Conclusions: hsa-miR-5096 well performs as a biomarker for 18F-FDG-PET/CT and as independent predictor of PFS. Moreover, exosome-mediated delivery of hsa-miR-5096 may promote SSTR2 heterogeneity and thus resistance to PRRT.

Liquid biopsy and preclinical tools for advancing diagnosis and treatment of patients with pancreatic neuroendocrine neoplasms.

Pancreatic neuroendocrine neoplasms (pNENs) are rare cancers with broad challenges for their management. The main clinical obstacles are the high rate of patients diagnosed at advanced stages, lack of prognostic markers for early detection of disease recurrence in resected patients, significant limitations in identifying those who will benefit from adjuvant therapy, and timely recognition of treatment response. Therefore, the discovery of new prognostic and predictive markers is necessary for patient stratification and clinical management. Liquid biopsy, which has revolutionized the field of clinical oncology, is extremely under-investigated in pNENs. This review highlights its potential and the recent advances in related technologies, as candidates for the delivery of the new tools that can help to refine pNEN diagnosis and to personalize treatment. In addition, the opportunities and limitations of available preclinical research models with regard to biomarker research are discussed in light of pNEN clinical needs.

Metabolic Syndrome Is Associated with Impaired Survival after Surgery for Pancreatic Neuroendocrine Tumors.

INTRODUCTION: Pancreatic neuroendocrine tumors (pNETs) are a heterogeneous group of neoplasms. Surgery is the only curative treatment option. However, our understanding of predictors of survival after surgery remains incomplete. The aim of the study was to evaluate metabolic syndrome (MetS) as a prognostic factor in pNET. METHODS: In a retrospective single-center cohort study, we examined the influence of MetS in 120 patients with curative intended resection of pNETs on overall survival (OS), recurrence-free survival, and outcome after recurrence. RESULTS: MetS was present in 32 patients (26.6%). Patients with MetS had an impaired OS after curative intended surgery compared to patients without MetS (median OS 72 months [95% CI 13.3-130.7] vs. not reached, p < 0.001). The shortest survival was observed in patients with MetS in the presence of oligometastatic disease at time of surgery. In a multivariable Cox regression analysis, MetS was identified as an independent risk factor for mortality (hazard ratio [HR] = 4.54, 95% CI [1.88-11.00], p = 0.01). In our dataset, MetS was not associated with tumor recurrence or recurrence-free survival. Nevertheless, in patients with recurrence, MetS was associated with shorter time to recurrence (median 3.4 months, 95% CI [2.48-4.24], vs. 20.1 months, 95% CI [10.8-29.49], p < 0.001), and poor outcome (HR = 5.03, 95% CI [1.25-20.20], p = 0.01). CONCLUSIONS: We identified MetS as a negative prognostic factor after curative intended surgery for pNET. In particular, patients with oligometastatic disease might not benefit from extensive surgery in the presence of MetS. Furthermore, MetS had a strong impact on survival after recurrence.

Canine insulinoma as a model for human malignant insulinoma research: Novel perspectives for translational clinical studies.

Insulinomas are considered rare indolent neuroendocrine neoplasms in human medicine, however when metastases occur no curative treatment is available thus, novel therapies are needed. Recently advances have been made in unraveling the pathophysiology of malignant insulinoma still major challenges hinder the development of a functional model to study them. Canine malignant insulinoma have similar recurrence and a poor prognosis as human malignant insulinoma. Additionally, both human and canine patients share extensively the same environment, tend to develop insulinoma seemingly spontaneously with an etiological role for hormones, at a similar incidence and stage of lifespan, with metastasis commonly to liver and regional lymph nodes, which are unresponsive to current therapies. However, the occurrence of metastases in dogs is as high as 95% compared with only 5-16% in human studies. From a comparative oncology perspective, the shared features with human insulinoma but higher incidence of metastasis in canine insulinoma suggests the latter as a model for human malignant insulinomas. With the common purpose of increasing survival rates of human and veterinary patients, in this review we are going to compare and analyze clinical, pathological and molecular aspects of canine and human insulinomas to evaluate the suitability of the canine model for future translational clinical studies.

Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bi-transgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in ß-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. The utility of this model is enhanced by the ability to form tumor-derived allografts. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

Cross-talk among MEN1, p53 and Notch regulates the proliferation of pancreatic neuroendocrine tumor cells by modulating INSM1 expression and subcellular localization.

Genomic analysis of Pancreatic Neuroendocrine Tumors (PanNETs) has revealed that these tumors often lack mutations in typical cancer-related genes such as the tumor suppressor gene p53. Instead, PanNET tumorigenesis usually involves mutations in specific PanNET-related genes, such as tumor suppressor gene MEN1. Using a PanNET mouse model, human tissues and human cell lines, we studied the cross-talk among MEN1, p53 and Notch signaling pathways and their role in PanNETs. Here, we show that reactivation of the early developmental program of islet cells underlies PanNET tumorigenesis by restoring the proliferative capacity of PanNET cells. We investigated the role of INSM1, a transcriptional regulator of islet cells' development, and revealed that its expression and subcellular localization is regulated by MEN1 and p53. Both human and mouse data show that loss of MEN1 in a p53 wild-type genetic background results in increased nuclear INSM1 expression and cell proliferation. Additionally, inhibition of Notch signaling in a p53 wild-type background reduces the proliferation of PanNET cells, due to repression of INSM1 transcription and nuclear localization. Our study elucidates the molecular mechanisms governing the interactions of INSM1 with MEN1, p53 and Notch and their roles in PanNET tumorigenesis, suggesting INSM1 as a key transcriptional regulator of PanNET cell proliferation.

Placenta-Specific 8 Is Overexpressed and Regulates Cell Proliferation in Low-Grade Human Pancreatic Neuroendocrine Tumors.

BACKGROUND/AIMS: Many aspects of the biology of pancreatic neuroendocrine tumors (PanNETs), including determinants of proliferative, invasive, and metastatic potential, remain poorly understood. Placenta-specific 8 (PLAC8), a gene with unknown molecular function, has been reported to have tumor-promoting roles in different human malignancies, including exocrine pancreatic cancer. Since preliminary data suggested deregulation of PLAC8 expression in PanNET, we have performed detailed analyses of PLAC8 expression and function in human PanNET. METHODS: Primary tissue from PanNET patients was immunohistochemically stained for PLAC8, and expression was correlated with clinicopathological data. In vitro, PLAC8 expression was inhibited by siRNA transfection in PanNET cell lines and effects were analyzed by qRT-PCR, Western blot, and proliferation assays. RESULTS: We report that PLAC8 is expressed in the majority of well-differentiated human PanNETs, predominantly in early-stage and low-grade tumors. SiRNA-mediated knockdown of PLAC8 in PanNET cells resulted in decreased proliferation and viability, while apoptosis was not induced. Mechanistically, these effects were mediated by attenuation of cell cycle progression, as Western blot analyses demonstrated upregulation of the tumor suppressor p21/CDKN2A and downregulation of the cell cycle regulator Cyclin D1 as well as reduced levels of phosphorylated ribosomal protein s6 and retinoblastoma protein. CONCLUSION: Our findings establish PLAC8 as a central mediator of cell growth in a subset of human PanNET, providing evidence for the existence of distinct molecular subtypes within this class of tumors.

Extended cycle streptozotocin/5-FU chemotherapy for maintenance therapy in pancreatic neuroendocrine tumors.

PURPOSE: The standard of care treatment for patients with advanced pancreatic neuroendocrine tumors (pNET) is a combination of streptozotocin and 5-FU. Although widely used, little is known about the best long-term strategy with these substances. METHODS: We here report our experience of 28 patients treated with streptozotocin/5-FU for advanced pNET with special consideration for long-term management using an extended cycle protocol. RESULTS: Standard 6-weekly Moertel protocol resulted in a median progression-free survival of 21 months (range 3-128) and a median overall survival of 69 months (range 3-157+) in the whole cohort. Thirteen of the 28 patients were switched to an extended 3-month cycle protocol for maintenance therapy. Of these 13 patients, 2 achieved complete remission, 1 partial remission, and 8 stable disease as best response while 2 showed progressive disease following switch to the extended protocol, resulting in an additional median progression-free survival of 23 months. Median overall survival after the start of chemotherapy in this patient group was 69 months (21-157+). Patients benefitted from extended periods free of chemotherapy-associated side effects after switching to the extended cycle protocol. CONCLUSIONS: Switching to an extended cycle protocol of 3 months for maintenance therapy following initial standard cycles may achieve long-term disease stabilization in selected patients with advanced pNET with good patient acceptance.

Tumour diameter is not reliable for management of non-secreting pancreatic neuroendocrine tumours.

Small non-functioning pancreatic NETs (pNETs) ≤2 cm can pose a management dilemma in terms of surveillance or resection. There is evidence to suggest that a surveillance approach can be considered since there are no significant radiological changes observed in lesions during long-term follow-up. However, other studies have suggested loco-regional spread can be present in ≤2 cm pNETs. The aim of this study was to characterise the prevalence of malignant features and identify any useful predictive variables in a surgically resected cohort of pNETs. 418 patients with pNETs were identified from 5 NET centres. Of these 227 were included for main analysis of tumour characteristics. Mean age of patients was 57 years, 47% were female. The median follow-up was 48.2 months. Malignant features were identified in 38% of ≤2 cm pNETs. ROC analysis showed that the current cut-off of 20 mm had a sensitivity of 84% for malignancy. The rate of malignant features is in keeping with other surgical series and challenges the belief that small pNETs have a low malignant potential. This study does not support a 20 mm size cut-off as being a solitary safe parameter to exclude malignancy in pNETs.

Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo.

UNLABELLED: Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short-term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN-receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting and hepatic stellate cell (HSC)-MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC-MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC-MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC-MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1-dependent manner. CONCLUSION: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status.

Increased angiogenesis protects against adipose hypoxia and fibrosis in metabolic disease-resistant 11ß-hydroxysteroid dehydrogenase type 1 (HSD1)-deficient mice.

In obesity, rapidly expanding adipose tissue becomes hypoxic, precipitating inflammation, fibrosis, and insulin resistance. Compensatory angiogenesis may prevent these events. Mice lacking the intracellular glucocorticoid-amplifying enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1(-/-)) have "healthier" adipose tissue distribution and resist metabolic disease with diet-induced obesity. Here we show that adipose tissues of 11ßHSD1(-/-) mice exhibit attenuated hypoxia, induction of hypoxia-inducible factor (HIF-1α) activation of the TGF-ß/Smad3/α-smooth muscle actin (α-SMA) signaling pathway, and fibrogenesis despite similar fat accretion with diet-induced obesity. Moreover, augmented 11ßHSD1(-/-) adipose tissue angiogenesis is associated with enhanced peroxisome proliferator-activated receptor γ (PPARγ)-inducible expression of the potent angiogenic factors VEGF-A, apelin, and angiopoietin-like protein 4. Improved adipose angiogenesis and reduced fibrosis provide a novel mechanism whereby suppression of intracellular glucocorticoid regeneration promotes safer fat expansion with weight gain.

Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism.

Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of "hepatocytes"; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources.

Persistence of functional hepatocyte-like cells in immune-compromised mice.

BACKGROUND: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver. AIMS: In this study, we assess the therapeutic value of HLCs in long-term cell-based therapies in vivo. METHODS: hESC-derived HLCs were injected into the spleen of acutely injured NODscid(IL-2Rγ) null mice and analysed at various time points post-transplantation up to 3 months. RESULTS: Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct-like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme-linked immunoabsorbant assay. CONCLUSIONS: We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post-translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC-derived HLCs have the potential to play an important role in cell-based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.

Dimethylarginine dimethylaminohydrolase overexpression enhances insulin sensitivity.

OBJECTIVE: Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity. METHODS AND RESULTS: To test this hypothesis we used a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22 to 70 wk) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistance index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2 mumol/L) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle. CONCLUSIONS: These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity.

Human cytomegalovirus-mediated induction of MIF in fibroblasts.

Human cytomegalovirus (HCMV) infection of fibroblasts induces the proinflammatory mediator macrophage migration inhibitory factor (MIF). Our in vitro experiments show that active HCMV infection alone is required to induce an early and sustained induction of MIF mRNA and protein production. Unlike in other infection models, in which MIF has been described to be released from preformed stores, our data conclusively show that HCMV infection triggers de novo synthesis and subsequent secretion of MIF. The kinetics of MIF protein production during HCMV infection points to an efficacious immune modulation, in which lymphocytes and monocytes are initially recruited by the early release of chemokines to sites of infection and locally activated by increasing concentrations of MIF.