Novel diagnostic biomarkers and therapeutic options for neuroendocrine tumors

Neuroendocrine tumors (NETs) represent a heterogeneous group of rare, slow-growing neoplasms, originating from enterochromaffin cells. Their annual incidence has progressively increased, although it is not known whether this is a true increase in NET incidence, the result of increased use of (improved) diagnostic procedures, or a combination of both. While metastatic disease is frequently observed at diagnosis, survival in NET patients has increased, which may be related to the advances in diagnostic and therapeutic procedures. NETs require appropriate standardized diagnostic procedures to assure early diagnosis, monitor disease progression and guide an optimal treatment. Additionally, medical treatment options in NETs have significantly increased and improved in the last years. This thesis evaluates a number of novel diagnostic and therapeutic options for patients with NET. Chapter 1 provides a general overview about NETs, emphasizes the applicability and limitations of the current diagnostic biomarkers and summarizes some putative novel biomarkers in NETs. At the same time, this chapter describes current and novel therapeutic options for functioning a non-functioning NETs. The chapter introduces the main content of this thesis: novel diagnostic and therapeutic options for NETs. Chapter 1 ends with an overview of the main aims of this thesis. Chapter 2 comprehensively evaluates the expression of several components of the somatostatin/cortistatin and ghrelin systems in a large series of well-characterized typical and atypical lung carcinoids. To the best of our knowledge, this study represents the first systematic characterization of the components of these regulatory systems in samples from lung carcinoids (LCs), in which a comparison is made with adjacent non-tumor regions and normal lung tissue. In this study, we observed that the expression of somatostatin, of some receptor subtypes (SSTs), and of the ghrelin receptor GHSR gradually increases from normal to non-tumoral adjacent and tumor tissue. Moreover, the presence of the truncated isoforms SST5TMD4, In1-ghrelin and the ghrelin receptor GHSR1b are first reported in LCs. Higher expression levels of ghrelin-O-acyltransferase (GOAT) were observed in tumors with necrosis, which were tumors with a larger size and higher capacity of peritumoral invasion and distant metastasis. These data suggest that a dysregulation of ghrelin system may be involved in the development/progression of these tumors. Chapter 3 evaluates the expression of the somatostatin/cortistatin system components in gastroenteropancreatic- (GEP-) NETs. The observed overexpression of SST5 in tumor tissue compared to adjacent non-tumor and normal tissue, as well as its clinical relation with vascular and nerve invasion suggests the putative role of SST5 as a target for treating aggressive GEP-NETs. Furthermore, it emphasizes that the evaluation of SSTs profile in GEP-NETs may provide additional information for clinical decision making, including the use of second-generation somatostatin analogs (SSAs). The most novel and relevant finding of chapter 4 is the marked overexpression of GOAT in GEP-NET samples. Whereas the expression of this enzyme is almost absent in the corresponding normal tissues, it is present in adjacent non-tumoral tissue and notably overexpressed tumor tissues. Additionally, its overexpression was demonstrated using immunohistochemistry. Importantly, this enzyme was associated to larger tumors, especially in gastrointestinal-NETs. These findings, in combination with previous publications, suggest a putative role of GOAT as a diagnostic biomarker in GEP-NETs. Chapter 5 evaluates the effect of ketoconazole on proliferation, cell cycle, apoptosis and secretion of adrenocorticotropic hormone (ACTH) or serotonin and chromogranin in an ACTH-producing bronchial NET and a non-ACTH producing pancreatic NET cell line, respectively. Ketoconazole exerted a predominant pro-apoptotic or cytotoxic effect, depending on the cell line. Additionally, ketoconazole induced changes in the cell cycle progression, e.g. it increases G0/G1 phase in both cell lines and an arrest in G2/M phase in the pancreatic NET cell line. Furthermore, a non-previously reported inhibitory effect of the SSA pasireotide on serotonin secretion by pancreatic NET cells was reported, which is probably related to the predominant SST5 expression in this cell line. In chapter 6, a comprehensive characterization of two pancreatic NET models was performed using two- (2D; monolayer) and three- dimensional (3D; spheroids) culture systems in different medium conditions. According to our results, NET 3D spheroid cultures represent a promising method for evaluating cell proliferation and secretion in NET cell lines, even in serum-deprived conditions. A non-previously reported dynamic expression of SSTs and dopamine receptor (D2R) was observed during growth of both 2D and 3D cultures. The effects of the somatostatin analog octreotide, the dopamine agonist cabergoline and of two novel SSTs-D2R multi-receptor targeting drugs (BIM-065, BIM-23A760) were evaluated using both culture systems. Cabergoline and SSTs-D2R multi-receptor targeting drugs, but not octreotide, inhibited CgA and serotonin secretion, but not NET cell growth. This suggests that the effect of the SSTs-D2R multi-receptor targeting drugs on secretion is mediated by D2R, and may indicate a putative role of dopamine agonists for treating D2R expressing functioning NETs, including carcinoid syndrome. Chapter 7 shows the in vitro effects of the novel tryptophan hydoxylase inhibitor telotristat on pancreatic NET cells. Telotristat potently inhibited serotonin release in a dose-dependent manner at a clinically feasible concentration. Its combination with pasireotide, but not with octreotide, had an additive effect on serotonin secretion. In 3D cultured pancreatic NET cells, serotonin secretion was completely blocked by telotristat, whereas this drug did not influence cell growth. These data suggest that serotonin has no autocrine/paracrine effect on cell growth in this 3D PNET model. In chapter 8 decreased expression of SSTs was observed in tumor tissue of GEP-NET patients with type 2 diabetes, compared to non-diabetic GEP-NET patients. This decreased expression was not observed in type 2 diabetic patients that were treated with metformin. The in vitro experiments showed an antiproliferative effect of biguanides and statins in NET cell lines, with consequent increased apoptosis and decreased cell migration. Since metformin and statins are low cost commercially available drugs, with a large experience in their clinical use, they may represent putative options for adjuvant therapy in NETs. In Chapter 9, the results and conclusions of the studies included in this thesis, are discussed in relation to current and possible novel future diagnostic modalities, as well as therapeutic options for NET patients.The general aims of the studies presented in this thesis are: 1. To identify potential novel tissue biomarkers for lung carcinoids and GEP-NETs 2. To evaluate the antitumor effect of registered drugs (for other medical purposes) in NETs 3. To evaluate the effects of novel drugs on NET hormone release and cell proliferation Specifically, we studied the potential applicability of ghrelin and somatostatin systems as biomarkers in tissue samples of lung carcinoids and GEP-NETs. As registered drugs for other medical purposes, we evaluated the antitumor effects of ketoconazole, metformin and statins. Finally, somatostatin-dopamine receptor chimeras and telotristat were studied as novel drugs for hormone release and cell growth control

Effect of the Tryptophan Hydroxylase Inhibitor Telotristat on Growth and Serotonin Secretion in 2D and 3D Cultured Pancreatic Neuroendocrine Tumor Cells

Serotonin, a biologically active amine, is related to carcinoid syndrome in functioning neuroendocrine tumors (NETs). Telotristat ethyl is a novel inhibitor of the tryptophan hydroxylase (TPH), a key enzyme in the production of serotonin. While its use in patients with carcinoid syndrome and uncontrolled diarrhea under somatostatin analogs (SSAs) has been recently approved, in vitro data evaluating its effectiveness are lacking. For this reason, we aimed to evaluate the effect of telotristat as monotherapy, and in combination with SSAs, on proliferation and secretion in a NET cell line model. The human pancreatic NET cell lines BON-1/QGP-1 were used as 2D and 3D cultured models; somatostatin receptor and TPH mRNA expression, as well as the potential autocrine effect of serotonin on tumor cell proliferation using a 3D culture system were evaluated. Telotristat decreased serotonin production in a dose-dependent manner at a clinically feasible concentration, without affecting cell proliferation. Its combination with pasireotide, but not with octreotide, had an additive inhibitory effect on serotonin secretion. The effect of telotristat was slightly less potent, when BON-1 cells were co-treated with octreotide. Octreotide and pasireotide had no effect on the expression of TPH. Telotristat did not have an effect on mRNA expression of somatostatin receptor subtypes. Finally, we showed that serotonin did not have an autocrine effect on NET cell proliferation on the 3D cell model. These results suggest that telotristat is an effective drug for serotonin inhibition, but the effectiveness of its combination with SST2 (somatostatin receptor subtype 2)-preferring SSA should be evaluated in more detail

Effects of Ketoconazole on ACTH-Producing and Non-ACTH-Producing Neuroendocrine Tumor Cells

Prolonged remission of hypercortisolism with steroidogenesis inhibitors has been described in patients with ectopic adrenocorticotropic hormone (ACTH) syndrome. The anti-proliferative and pro-apoptotic effect of ketoconazole in human cancer cells was previously suggested. The aim of this study was to explore the effects of ketoconazole on ACTH-producing and non-ACTH-producing neuroendocrine tumor (NET) cell lines. The effects of ketoconazole alone, and in combination with somatostatin analogs, were evaluated in two human cell lines: DMS-79 (ectopic ACTH-producing small cell lung carcinoma) and BON-1 (human pancreatic NET). Total DNA measurement, apoptosis, cell cycle, chromogranin A (CgA)/proopiomelanocortin (POMC) expression by qRT-PCR, serotonin, CgA, and ACTH secretion assays were performed. In both cell lines, ketoconazole significantly suppressed cell growth and colony formation in a dose and time-dependent manner. The effect in DMS-79 was primarily cytotoxic, while it was more apoptotic in BON-1 cells. Ketoconazole also induced increase in G0/G1 phase in both cell lines and arrest in phase G2/M of BON-1 cells. Ketoconazole did not affect the secretion of serotonin, CgA, ACTH, or the mRNA expression of CgA and POMC. Decreased serotonin secretion was observed after the combination treatment with pasireotide. These results suggest a direct effect of ketoconazole on cell proliferation, apoptosis, and cell cycle in both ACTH- and non-ACTH-producing NET cells

Targeted Systemic Treatment of Neuroendocrine Tumors: Current Options and Future Perspectives

Neuroendocrine tumors (NETs) originate from the neuroendocrine cell system in the bronchial and gastrointestinal tract and can produce hormones leading to distinct clinical syndromes. Systemic treatment of patients with unresectable NETs aims to control symptoms related to hormonal overproduction an

Co-occurrence of mutations in NF1 and other susceptibility genes in pheochromocytoma and paraganglioma

IntroductionThe percentage of patients diagnosed with pheochromocytoma and paraganglioma (altogether PPGL) carrying known germline mutations in one of the over fifteen susceptibility genes identified to date has dramatically increased during the last two decades, accounting for up to 35-40% of PPGL patients. Moreover, the application of NGS to the diagnosis of PPGL detects unexpected co-occurrences of pathogenic allelic variants in different susceptibility genes. MethodsHerein we uncover several cases with dual mutations in NF1 and other PPGL genes by targeted sequencing. We studied the molecular characteristics of the tumours with co-occurrent mutations, using omic tools to gain insight into the role of these events in tumour development. ResultsAmongst 23 patients carrying germline NF1 mutations, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic mutations in H3-3A and PRKAR1A. Three additional patients, with somatic mutations in NF1 were found carrying germline pathogenic mutations in SDHB or DLST, and a somatic truncating mutation in ATRX. Two of the cases with dual germline mutations showed multiple pheochromocytomas or extra-adrenal paragangliomas - an extremely rare clinical finding in NF1 patients. Transcriptional and methylation profiling and metabolite assessment showed an intermediate signature to suggest that both variants had a pathological role in tumour development. DiscussionIn conclusion, mutations affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signalling) co-occurring in the same patient could provide a selective advantage for the development of PPGL, and explain the variable expressivity and incomplete penetrance observed in some patients