Objective: Evidence is limited regarding outcome of patients with ectopic Cushing's syndrome (ECS) due to neuroendocrine tumors (NETs). Design: We assessed the prognostic factors affecting the survival of patients with NETs and ECS. Methods: Retrospective analysis of clinicopathological features, severity of hormonal syndrome, treatments from a large cohort of patients with NETs and ECS collected from 17 Italian centers. Results: Our series included 110 patients, 58.2% female, with mean (±s.d.) age at diagnosis of 49.5 ± 15.9 years. The main sources of ectopic ACTH were bronchial carcinoids (BC) (40.9%), occult tumors (22.7%) and pancreatic (p)NETs (15.5%). Curative surgery was performed in 56.7% (70.2% of BC, 11% of pNETs). Overall survival was significantly higher in BC compared with pNETs and occult tumors (P = 0.033) and in G1-NETs compared with G2 and G3 (P = 0.007). Negative predictive factors for survival were severity of hypercortisolism (P < 0.02), hypokalemia (P = 0.001), diabetes mellitus (P = 0.0146) and distant metastases (P < 0.001). Improved survival was observed in patients who underwent NET removal (P < 0.001). Adrenalectomy improved short-term survival. Clinical Study

Background/Aim: The neuroendocrine tumor (NET) proliferation-based grading system (ENETS/WHO) for gastroenteropancreatic (GEP) tumors has proved reliable for prognostic stratification. To date, concerns exist regarding Ki-67 heterogeneity within the tumor and little is known on whether grade varies between primary and secondary sites. As tumor heterogeneity may have a significant impact on clinical management, our aim was to retrospectively evaluate Ki-67 on a series of GEP NETs in order to establish whether there is variability in different samples of the same lesion or between primary and metastatic disease (local/distant, synchronous/metachronous). Methods: Sixty patients with multiple samples of tumor were accrued from a total of 338 GEP NETs; 44 of them also had tissue from local/distant metastases and a further 5 had multiple metastatic foci from unknown primary tumors. Immunohistochemistry for Ki-67 was performed on all paraffin blocks from both primary and metastatic tumors. Results: Intratumor Ki-67 heterogeneity sufficient to change grade at first diagnosis was seen in 3/60 cases (5%). Out of 49 patients with primary NETs and/or multiple metastases, discrepancy in grade between sites was identified in 19 (39%) cases and in particular in 11/47 (23%) and in 10/12 (83%) patients with synchronous and metachronous metastases, respectively (p = 0.0002). Change in grade was more frequent in distant compared to locoregional metastases (p = 0.024) and in particular in distant sites other than the liver (p = 0.006). Conclusions: NETs show frequent differences in grade between primary sites and their synchronous/metachronous metastases; assessment of Ki-67 at all sites may prove to be significant for patient management.

Preclinical research on neuroendocrine tumors usually involves immortalized cell lines and few animal models. In the present study we described an in vivo model based on patient-derived xenografts of neuroendocrine tumor cells in zebrafish (Danio rerio) embryos, allowing a rapid analysis of the angiogenic and invasive potential. Patient-derived neuroendocrine tumor cells were transplanted in 48 hours post-fertilization Tg(fli1a:EGFP) zebrafish embryos that express enhanced green fluorescent protein in the entire vasculature. Neuroendocrine tumor cells, stained with CM-Dil, were injected into the subperidermal (perivitelline) space, close to the developing subintestinal venous plexus. A proper control group, represented by zebrafish injected with only D-PBS, was included in this study. Angiogenic and invasive potentials of each patient-derived xenograft were evaluated by both epifluorescence and confocal microscopes. Six out of eight neuroendocrine tumor samples were successfully transplanted in zebrafish embryos. Although the implanted tumor mass had a limited size (about 100 cells for embryos), patient-derived xenografts showed pro-angiogenic (5 cases) and invasive (6 cases) behaviors within 48 hours post injection. Patient-derived xenograft in zebrafish embryos appears to be a reliable in vivo preclinical model for neuroendocrine tumors, tumors with often limited cell availability. The rapidity of this procedure makes our model a promising platform to perform preclinical drug screening and opens a new scenario for personalized treatment in patients with neuroendocrine tumors.

Tumor size >15.5 mm, grading G2, and presence of lymphovascular infiltration are factors independently related to nodal metastases in appendiceal NETs. Presence of ≥1 of these features should be considered an indication for oncological radicalization. Although these results represent the largest study currently available, prospective validation is needed.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) raise difficult therapeutic problems despite the emergence of targeted therapies. Somatostatin analogs (SSA) remain pivotal therapeutic drugs. However, the tachyphylaxis and the limited antitumoral effects observed with the classical somatostatin 2 (sst 2 ) agonists (octreotide and lanreotide) led to the development of new SSA, such as the pan sst receptor agonist pasireotide. Our aim was to compare the effects of pasireotide and octreotide on cell survival, chromogranin A (CgA) secretion, and sst 2 phosphorylation/trafficking in pancreatic NET (pNET) primary cells from 15 tumors. We established and characterized the primary cultures of human pancreatic tumors (pNETs) as powerful preclinical models for understanding the biological effects of SSA. At clinically relevant concentrations (1-10 nM), pasireotide was at least as efficient as octreotide in inhibiting CgA secretion and cell viability through caspase-dependent apoptosis during short treatments, irrespective of the expression levels of the different sst receptors or the WHO grade of the parental tumor. Interestingly, unlike octreotide, which induces a rapid and persistent partial internalization of sst 2 associated with its phosphorylation on Ser341/343, pasireotide did not phosphorylate sst 2 and induced a rapid and transient internalization of the receptor followed by a persistent recycling at the cell surface. These results provide the first evidence, to our knowledge, of striking differences in the dynamics of sst 2 trafficking in pNET cells treated with the two SSAs, but with similar efficiency in the control of CgA secretion and cell viability.