Ileal carcinoids are malignant neuroendocrine tumours of the small intestine. The aim of this study was to obtain a high-resolution genomic profile of ileal carcinoids in order to define genetic changes important for tumour initiation, progression and survival. Forty-three patients with ileal carcinoids were investigated by high-resolution array-based comparative genomic hybridization. The average number of copy number alterations (CNAs) per tumour was 7

Neuroendocrine tumours are characterized by their capacity to produce hormones, which are stored in vesicles and secretory granules. Demonstration of granule/vesicle proteins in tumours is taken as evidence of neuroendocrine differentiation. Vesicular monoamine transporters (VMAT1 and VMAT2) mediate the transport of amines into vesicles of neurons and endocrine cells. The expression of VMAT1 and VMAT2 and the usefulness of VMAT1 and VMAT2 in the histopathological diagnosis of gastrointestinal endocrine tumours have not been fully explored. This study therefore investigated the expression of VMAT1 and VMAT2 in 211 human gastrointestinal tumours by immunocytochemistry and western blotting. VMAT1 and/or VMAT2 were demonstrated in the majority of amine-producing endocrine tumours of gastric, ileal, and appendiceal origin. Serotonin-producing endocrine tumours (ileal and appendiceal carcinoids) expressed predominantly VMAT1, while histamine-producing endocrine tumours (gastric carcinoids) expressed VMAT2 almost exclusively. In peptide-producing endocrine tumours such as rectal carcinoids and endocrine pancreatic tumours, only a small number of immunopositive tumour cells were observed. No labelling was found in non-endocrine tumours, including gastric, colorectal and pancreatic adenocarcinomas and gastrointestinal stromal tumours. In conclusion, VMAT1 and VMAT2 are differentially expressed by gastrointestinal endocrine tumours, with a pattern specific for each tumour type, reflecting their neuroendocrine differentiation and origin. VMAT1 and VMAT2 may therefore become valuable markers in the classification of neuroendocrine tumours and may also indicate patients suitable for radioisotope treatment operating via these transporter systems.

The aim of this study was to define the miRNA profile of small intestinal neuroendocrine tumors and to search for novel molecular subgroups and prognostic biomarkers. miRNA profiling was conducted on 42 tumors from 37 patients who underwent surgery for small intestinal neuroendocrine tumors. Unsupervised hierarchical clustering analysis of miRNA profiles identified two groups of tumor metastases, denoted cluster M1 and M2. The smaller cluster M1 was associated with shorter overall survival and contained tumors with higher grade (WHO grade G2/3) and multiple chromosomal gains including gain of chromosome 14. Tumors of cluster M1 had elevated expression of miR-1246 and miR-663a, and reduced levels of miR-488-3p. Pathway analysis predicted Wnt signaling to be the most significantly altered signaling pathway between clusters M1 and M2. Analysis of miRNA expression in relation to tumor proliferation rate showed significant alterations including downregulation of miR-137 and miR-204-5p in tumors with Ki67 index above 3%. Similarly, tumor progression was associated with significant alterations in miRNA expression, e.g. higher expression of miR-95 and miR-210, and lower expression of miR-378a-3p in metastases. Pathway analysis predicted Wnt signaling to be altered during tumor progression, which was supported by decreased nuclear translocation of β-catenin in metastases. Survival analysis revealed that downregulation of miR-375 was associated with shorter overall survival. We performed in situ hybridization on biopsies from an independent cohort of small intestinal neuroendocrine tumors using tissue microarrays. Expression of miR-375 was found in 578/635 (91%) biopsies and survival analysis confirmed that there was a correlation between downregulation of miR-375 in tumor metastases and shorter patient survival. We conclude that miRNA profiling defines novel molecular subgroups of metastatic small intestinal neuroendocrine tumors and identifies miRNAs associated with tumor proliferation rate and progression. miR-375 is highly expressed in small intestinal neuroendocrine tumors and may be used as a prognostic biomarker.

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We have examined the global gene expression profile of small intestinal carcinoids by microarray analysis. High expression of a number of genes was found including amyloid precursor-like protein 1 (APLP1). Quantitative real-time PCR and western blot analysis demonstrated higher expression of APLP1 in carcinoid metastases relative to primary tumours indicating a role of APLP1 in tumour dissemination. Tissue microarray analysis of gastroentero-pancreatic tumours demonstrated a high frequency of APLP1 expression and a low frequency of APLP2 expression in neuroendocrine (NE) tumours when compared with non-NE tumours at the same sites. Meta-analysis of gene expression data from a large number of tumours outside the gastrointestinal tract confirmed a correlation between APLP1 expression and NE phenotype where high expression of APLP1 was accompanied by downregulation of APLP2 in NE tumours. Cellular localization of APLP1, APLP2 and amyloid precursor protein (APP) in carcinoid cells (GOT1) by confocal microscopy demonstrated partial co-localization with synaptophysin. This suggests that the APP family of proteins is transported to the cell membrane by synaptic microvesicles and that they may influence tumour cell adhesion and invasiveness. We conclude that APLP1 is differentially upregulated in gastrointestinal NE tumours and that APLP1 may be important for the dissemination of small intestinal carcinoids. Identification of APLP1 in NE tumours offers a novel target for treatment and may also serve as a tumour-specific marker.