Not only stem cells, but also mature cells, particularly neuroendocrine cells, may develop into tumours: time for a paradigm shift.

Stem cells are considered the origin of neoplasms in general, and malignant tumours in particular, and the stage at which the stem cells stop their differentiation determines the degree of malignancy. However, there is increasing evidence supporting an alternative paradigm. Tumours may develop by dedifferentiation from mature cells able to proliferate. Studies of gastric carcinogenesis demonstrate that mature neuroendocrine (NE) cells upon long-term overstimulation may develop through stages of hyperplasia, dysplasia, and rather benign tumours, into highly malignant carcinomas. Dedifferentiation of cells may change the histological appearance and impede the identification of the cellular origin, as seen with gastric carcinomas, which in many cases are dedifferentiated neuroendocrine tumours. Finding the cell of origin is important to identify risk factors for cancer, prevent tumour development, and tailor treatment. In the present review, we focus not only on gastric tumours, but also evaluate the role of neuroendocrine cells in tumourigenesis in two other foregut-derived organs, the lungs and the pancreas, as well as in the midgut-derived small intestine.

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease.

OBJECTIVE: Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn's disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. MATERIAL AND METHODS: Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3',5'-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. CONCLUSIONS: GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Mucosal toll-like receptor 3-dependent synthesis of complement factor B and systemic complement activation in inflammatory bowel disease.

BACKGROUND: Recent studies link Toll-like receptor 3 (TLR3) to the pathogenesis of inflammatory bowel disease (IBD). Screening TLR3-agonist response in an intestinal epithelial cell line, we found complement factor B mRNA (CFB) potently upregulated and went on to further study localization of complement factor B synthesis and systemic activation of complement in ulcerative colitis and Crohn's disease. METHODS: In a transcriptome analysis of poly (I:C) stimulated HT-29 cells, we found CFB highly upregulated downstream of TLR3. We sought to confirm CFB upregulation in a microarray gene expression analysis on colonic biopsies from an IBD population (n = 133). Immunohistochemical staining and in situ hybridization were done to identify cellular sources of factor B and CFB. Systemic complement activation was assessed in plasma (n = 18) using neoepitope-based enzyme linked immunosorbent assay. RESULTS: CFB mRNA and protein were abundantly expressed in the colonic epithelial cell line, and synthesis enhanced by the poly (I:C) TLR3 ligand. In inflamed versus normal colonic mucosa of ulcerative colitis and Crohn's disease, CFB mRNA was the most significantly overexpressed gene and the mRNA abundance ratio was among the 50 highest. Epithelial cells were the dominating site of factor B expression. Systemic complement activation was significantly higher in active than in nonactive IBD. CONCLUSIONS: This study is the first to link TLR3 to activation of the alternative complement pathway. Complement factor B is potently upregulated locally in IBD in addition to having a possible central role in systemic complement activation. This suggests a prominent role for complement in IBD pathogenesis.

The PAS positive material in gastric cancer cells of signet ring type is not mucin.

PURPOSE: The purpose of this study is to assess the exocrine and neuroendocrine properties of tumour cells in diffuse gastric cancer with signet ring cell differentiation. MATERIAL AND METHODS: Mucin mRNA and protein expressions (MUC1, 2, 3, 4, 5AC, 6 and MUC13) were assessed by immunohistochemistry and in situ hybridization. The neuroendocrine properties were evaluated by protein and mRNA expression of the general neuroendocrine markers chromogranin A and synaptophysin. RESULTS: No MUC expression was observed in signet ring tumour cells including the amorphous substance in any of the nine cases. All cases showed immunoreactivity to synaptophysin, and seven out of nine cases immunoreactivity to chromogranin A in signet ring and non-signet ring tumour cells. Chromogranin A mRNA expression was observed in tumour cells in all samples with retained mRNA. CONCLUSIONS: The lack of MUC protein and mRNA in signet ring tumour cells suggests the amorphous substance is not mucin. The lack of MUC mRNA expression in non-signet ring tumour cells questions exocrine differentiation in this tumour group. The abundant protein expression of the general neuroendocrine markers CgA and synaptophysin, and mRNA expression in tumour cells strengthens the hypothesis that this tumour group may be of neuroendocrine origin.

The stimulatory adenosine receptor ADORA2B regulates serotonin (5-HT) synthesis and release in oxygen-depleted EC cells in inflammatory bowel disease.

OBJECTIVE: We recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC) cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion. DESIGN: The effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2) was compared to NECA (adenosine agonist), MRS1754 (ADORA2B receptor antagonist) and SCH442146 (ADORA2A antagonist) on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo. RESULTS: HIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (~90%) of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE) control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5'-ASA treatment was confirmed in a TNBS-model. CONCLUSION: Hypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD.

REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridisation.

The regenerating islet-derived (REG) gene family encodes a group of proteins highly expressed in several human pathologies, many of which are associated with epithelial inflammation. All human family members, namely REG1A, REG1B, REG3A and REG4, are closely related in genomic sequence and all are part of the c-type lectin superfamily. REGs are highly expressed during inflammatory bowel disease (IBD)-related colonic inflammation and the in vivo expression pattern of REG1A and REG4 has been localised by using immunohistochemistry. However, the function of the encoded proteins is largely unknown and the cellular localisation of REG expression during colonic inflammation has not been described. Therefore, we have used in situ hybridisation to demonstrate the cellular localisation of REG expression in healthy and diseased colonic mucosa. Samples drawn from an IBD cohort including both inflamed and un-inflamed colonic mucosa are described, as are samples from non-IBD inflammation and healthy controls. Immunohistochemistry against known cell-type markers on serial sections has localised the expression of REGs to metaplastic Paneth cells (REG1A, REG1B and REG3A) and enteroendocrine cells (REG4), with a marked expansion of expression during inflammation. The group of REGs can, based on gene expression patterns, be divided into at least two groups; REG1A, REG1B and REG3A with their expression focused in the crypt base spreading from Paneth cells and REG4 being more highly expressed towards the luminal face. This exploration of expression pattern forms provides the background for further exploration of REG function in the intestine.

Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demonstrates lack of major differences between Crohn's disease and ulcerative colitis.

BACKGROUND: In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn's disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns. METHODS: Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores. RESULTS: Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls. CONCLUSIONS: There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.

Relevance of TNBS-colitis in rats: a methodological study with endoscopic, histologic and Transcriptomic [corrected] characterization and correlation to IBD.

BACKGROUND: Rectal instillation of trinitrobenzene sulphonic acid (TNBS) in ethanol is an established model for inflammatory bowel disease (IBD). We aimed to 1) set up a TNBS-colitis protocol resulting in an endoscopic and histologic picture resembling IBD, 2) study the correlation between endoscopic, histologic and gene expression alterations at different time points after colitis induction, and 3) compare rat and human IBD mucosal transcriptomic data to evaluate whether TNBS-colitis is an appropriate model of IBD. METHODOLOGY/PRINCIPAL FINDINGS: Five female Sprague Daley rats received TNBS diluted in 50% ethanol (18 mg/0.6 ml) rectally. The rats underwent colonoscopy with biopsy at different time points. RNA was extracted from rat biopsies and microarray was performed. PCR and in situ hybridization (ISH) were done for validation of microarray results. Rat microarray profiles were compared to human IBD expression profiles (25 ulcerative colitis Endoscopic score demonstrated mild to moderate colitis after three and seven days, but declined after twelve days. Histologic changes corresponded with the endoscopic appearance. Over-represented Gene Ontology Biological Processes included: Cell Adhesion, Immune Response, Lipid Metabolic Process, and Tissue Regeneration. IL-1α, IL-1ß, TLR2, TLR4, PRNP were all significantly up-regulated, while PPARγ was significantly down-regulated. Among genes with highest fold change (FC) were SPINK4, LBP, ADA, RETNLB and IL-1α. The highest concordance in differential expression between TNBS and IBD transcriptomes was three days after colitis induction. ISH and PCR results corresponded with the microarray data. The most concordantly expressed biologically relevant pathways included TNF signaling, Cell junction organization, and Interleukin-1 processing. CONCLUSIONS/SIGNIFICANCE: Endoscopy with biopsies in TNBS-colitis is useful to follow temporal changes of inflammation visually and histologically, and to acquire tissue for gene expression analyses. TNBS-colitis is an appropriate model to study specific biological processes in IBD.

Management of gastric carcinoids (neuroendocrine neoplasms).

Gastric neuroendocrine neoplasms of the stomach can be divided into the usually well-differentiated, hypergastrinemia-dependent type I and II lesions and the more aggressively behaving gastrin-independent type III lesions. Mainly due to better diagnostics and awareness of this tumor, the observed incidence has increased more than tenfold over the last 30 years. Small (<15-20 mm) localized type I and II lesions that are slowly proliferating (Ki67<2%) can usually be managed conservatively with endoscopic surveillance. Reducing hypergastrinemia by surgical removal of an underlying gastrinoma is important in inhibiting growth and induce reduction of type II lesions, while the specific gastrin receptor antagonist YF476 or gastrin antibodies may become useful for both type I and II lesions. Infiltrating and metastasized tumors and type III lesions require a more aggressive approach with surgical resection and consideration of modalities such as somatostatin analogs, cytotoxics, and peptide receptor targeted treatment.

Microencapsulation of small intestinal neuroendocrine neoplasm cells for tumor model studies.

Basic cancer research is dependent on reliable in vitro and in vivo tumor models. The serotonin (5-HT) producing small intestinal neuroendocrine tumor cell line KRJ-1 has been used in in vitro proliferation and secretion studies, but its use in in vivo models has been hampered by problems related to the xeno-barrier and tumor formation. This may be overcome by the encapsulation of tumor cells into alginate microspheres, which can function as bioreactors and protect against the host immune system. We used alginate encapsulation of KRJ-1 cells to achieve long-term functionality, growth and survival. Different conditions, including capsule size, variations in M/G content, gelling ions (Ca(2+) /Ba(2+)) and microcapsule core properties, and variations in KRJ-1 cell condition (single cells/spheroids) were tested. Viability and cell growth was evaluated with MTT, and confocal laser scanner microscopy combined with LIVE/DEAD viability stains. 5-HT secretion was measured to determine functionality. Under all conditions, single cell encapsulation proved unfavorable due to gradual cell death, while encapsulation of aggregates/spheroids resulted in surviving, functional bioreactors. The most ideal spheroids for encapsulation were 200-350 µm. Long-term survival (>30 days) was seen with solid Ca(2+) /Ba(2+) microbeads and hollow microcapsules. Basal 5-HT secretion was increased (sixfold) after hollow microcapsule encapsulation, while Ca(2+) /Ba(2+) microbeads was associated with normal basal secretion and responsiveness to cAMP/PKA activation. In conclusion, encapsulation of KRJ-1 cells into hollow microcapsules produces a bioreactor with a high constitutively activate basal 5-HT secretion, while Ca(2+) /Ba(2+) microbeads provide a more stable bioreactor similar to non-encapsulated cells. Alginate microspheres technology can thus be used to tailor different functional bioreactors for both in vitro and in vivo studies.

Gene network inference and biochemical assessment delineates GPCR pathways and CREB targets in small intestinal neuroendocrine neoplasia.

Small intestinal (SI) neuroendocrine tumors (NET) are increasing in incidence, however little is known about their biology. High throughput techniques such as inference of gene regulatory networks from microarray experiments can objectively define signaling machinery in this disease. Genome-wide co-expression analysis was used to infer gene relevance network in SI-NETs. The network was confirmed to be non-random, scale-free, and highly modular. Functional analysis of gene co-expression modules revealed processes including 'Nervous system development', 'Immune response', and 'Cell-cycle'. Importantly, gene network topology and differential expression analysis identified over-expression of the GPCR signaling regulators, the cAMP synthetase, ADCY2, and the protein kinase A, PRKAR1A. Seven CREB response element (CRE) transcripts associated with proliferation and secretion: BEX1, BICD1, CHGB, CPE, GABRB3, SCG2 and SCG3 as well as ADCY2 and PRKAR1A were measured in an independent SI dataset (n = 10 NETs; n = 8 normal preparations). All were up-regulated (p<0.035) with the exception of SCG3 which was not differently expressed. Forskolin (a direct cAMP activator, 10(-5) M) significantly stimulated transcription of pCREB and 3/7 CREB targets, isoproterenol (a selective ß-adrenergic receptor agonist and cAMP activator, 10(-5) M) stimulated pCREB and 4/7 targets while BIM-53061 (a dopamine D(2) and Serotonin [5-HT(2)] receptor agonist, 10(-6) M) stimulated 100% of targets as well as pCREB; CRE transcription correlated with the levels of cAMP accumulation and PKA activity; BIM-53061 stimulated the highest levels of cAMP and PKA (2.8-fold and 2.5-fold vs. 1.8-2-fold for isoproterenol and forskolin). Gene network inference and graph topology analysis in SI NETs suggests that SI NETs express neural GPCRs that activate different CRE targets associated with proliferation and secretion. In vitro studies, in a model NET cell system, confirmed that transcriptional effects are signaled through the cAMP/PKA/pCREB signaling pathway and that a SI NET cell line was most sensitive to a D(2) and 5-HT(2) receptor agonist BIM-53061.

The archaic distinction between functioning and nonfunctioning neuroendocrine neoplasms is no longer clinically relevant.

BACKGROUND: Neuroendocrine neoplasms (NENs) are increasing in incidence and prevalence. This reflects greater clinical awareness, effective imaging, and increasing pathological diagnostic recognition. Although the identification and treatment of clinical neuroendocrine syndromes are established, there is confusion when a NEN has no discernible clinical symptoms. DISCUSSION: Nonfunctional tumors are usually diagnosed incidentally and at a later stage largely because either they do not secrete a bioactive product or do so, but in a form that is either inactive or in quantities that have no discernible effect. Nevertheless, the histopathology is indistinguishable from functional NENs, and tumors exhibit somatostatin receptor expression, and positive immunohistochemistry for neuroendocrine cell markers (CgA, NSE/synaptophysin). Similarly, their rates of growth and metastatic behavior are, like other NENs, predictably based on staging and grading (mitotic rate and Ki67 expression). Both types are diagnosed biochemically (CgA) and by imaging in an identical fashion with computed tomography, magnetic resonance imaging, somatostatin receptor scintigraphy, and endoscopic ultrasound. NENs, irrespective of function or bioactive secretory profile, respond with equal efficacy to the same regimen of surgery or antitumor drugs (e.g., somatostatin analogs with or without tyrosine kinase inhibitors/antiangiogenics or cytotoxics) depending on grade. Given the efficacy of somatostatin analogs in increasing progression free survival, nonfunctional NENs should be managed identically to symptomatic NENs. The consideration of NENs as functional or nonfunctional is an archaic clinical concept that should be discarded since the tumors are indistinguishable at a cellular, biological, and morphological level. All current evidences indicate that their diagnosis and treatment should follow the same common principles.

The epidemiology of gastroenteropancreatic neuroendocrine tumors.

In this article, updated analyses of the National Cancer Institute Surveillance, Epidemiology and End Results (SEER) registry (1973-2007) are presented and compared with epidemiologic GEP-NET data from Europe and Asia. Several studies have demonstrated a steadily increasing incidence of GEP-NETs, and this escalation is still ongoing (SEER data 2004-2007). The common primary GEP-NET sites exhibit unique epidemiologic profiles with distinct patterns of incidence, age at diagnosis, stage, and survival. Overall, GEP-NET survival has improved over the past 3 decades, although the outcome for poorly differentiated tumors remains dismal.

The clinical relevance of chromogranin A as a biomarker for gastroenteropancreatic neuroendocrine tumors.

Chromogranin A, although it exhibits limitations, is currently the most useful general tumor biomarker available for use in the diagnosis and management of gastroenteropancreatic neuroendocrine tumors (NETs). The value of the chromogranin A lies in its universal cosecretion by the majority of neuroendocrine cells that persists after malignant transformation. Clinicians aware of the physiologic role of chromogranin A and its secretion in a variety of non-NET-related pathologic conditions can use this protein as a moderately effective tumor biomarker in the management of GEP-NETs.

Serotonin and fluoxetine receptors are expressed in enamel organs and LS8 cells and modulate gene expression in LS8 cells.

The selective serotonin re-uptake inhibitor (SSRI) fluoxetine is widely used in the treatment of depression in children and fertile women, but its effect on developing tissues has been sparsely investigated. The aim of this study was to investigate if enamel organs and ameloblast-derived cells express serotonin receptors that are affected by peripherally circulating serotonin or fluoxetine. Using RT-PCR and western blot analysis we found that enamel organs from 3-d-old mice and ameloblast-like cells (LS8 cells) express functional serotonin receptors, the rate-limiting enzyme in serotonin synthesis (Thp1), as well as the serotonin transporter (5HTT), indicating that enamel organs and ameloblasts are able to respond to serotonin and regulate serotonin availability. Fluoxetine and serotonin enhanced the alkaline phosphatase activity in the cell culture medium from cultured LS8 cells, whereas the expression of enamelin (Enam), amelogenin (Amel), and matrix metalloproteinase-20 (MMP-20) were all significantly down-regulated. The secretion of vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) was also reduced compared with controls. In conclusion, enamel organs and ameloblast-like cells express functional serotonin receptors. Reduced transcription of enamel proteins and secretion of vascular factors may indicate possible adverse effects of fluoxetine on amelogenesis.

New pharmacologic therapies for gastroenteropancreatic neuroendocrine tumors.

Successful treatment of unresectable and metastatic gastroenteropancreatic neuroendocrine tumors (GEP-NETs) requires the thoughtful choice of systemic therapy as a component of a multidisciplinary therapeutic approach. The role of somatostatin analogues is established in symptom relief, but the efficacy of interferon and radiopeptide targeted therapy is not clear. The utility of a variety of tyrosine kinase and antiangiogenic agents is variable and under investigation, whereas the role of cytotoxic chemotherapy in poorly differentiated GEP-NETs is accepted. Overall, the ideal treatment of more indolent tumors is less certain. Reassessments of the GEP-NET pathology classification has provided improved logic for the role of a variety of agents, whereas the precise positioning of many new agents that target molecular pathways of angiogenesis and proliferation is under examination. This article describes the current options for systemic therapy for GEP-NETs within the framework of the current World Health Organization classification system.

A nomogram to assess small-intestinal neuroendocrine tumor ('carcinoid') survival.

Neuroendocrine tumors (NETs) are a heterogeneous group of cancers of which the commonest site is the small intestine (SI). Most information available to determine tumor behavior reflects univariate assessment of factors or is anecdotal or experience based. There currently exists no objective multivariate analysis of indices that defines SI NET prognosis. A key unmet need is the lack of a rigorous mathematical-based tool - a nomogram - for the assessment of parameters that define progress, determine prognosis and can guide therapy. Since prediction of NET behavior is a critical criterion in determining clinical strategy, we constructed a NET nomogram (Modlin Score) for prognosis prediction, patient group comparisons and a guide for stratification of treatment and surveillance. We used hazard ratio (HR), Cox analysis and Kaplan-Meier analysis of published data and the current Surveillance, Epidemiology and End Results (SEER) database (approx. 20,000 patients) to develop a nomogram from 15 variables demonstrated to provide significant multivariate HRs. These included age, gender, ethnicity, symptoms, urinary 5-hydroxyindoleacetic acid, plasma chromogranin A, liver function tests, tumor size, invasion, metastasis, histology, Ki-67 index, carcinoid heart disease and therapy (surgery or long-acting somatostatin analogs). Internal validation was assessed using 33 SI NET patients. A NET nomoscore (Modlin Score) was developed by HR weighting and stratification into low (<75), medium (75-95) and high risk (>95). This identified significant differences (p <0.03, Kaplan-Meier) in survival (15.5 ± 4.3, 9.7 ± 2.5 and 6.4 ± 1.1 years, respectively). The Modlin Score was significantly elevated (p <0.01) in deceased compared to alive patients. This nomogram represents an optimized construct based upon currently analyzable data, and application will facilitate accurate stratification for comparison in clinical trials. External validation and amplification by identification of additional indices, e.g. molecular biomarkers, are necessary. The development of a mathematically validated nomogram provides a platform for objective assessment of SI NET disease, a finite basis for precise prognostication and a tool to guide management strategy.

Gastrointestinal neuroendocrine (carcinoid) tumours: current diagnosis and management.

Neuroendocrine tumours (NETs) are increasing in both incidence and prevalence and, as a group, are more prevalent than either gastric, pancreatic, oesophageal or hepatobiliary adenocarcinomas, or any two of these cancers combined. Clinical awareness of the protean and intermittent symptoms of NETs (eg, sweating, flushing, diarrhoea, and bronchospasm) is critical for timely diagnosis; however, the classical carcinoid syndrome is relatively uncommon. The most useful diagnostic test for gastrointestinal NETs is measurement of plasma chromogranin A (CgA) levels. Disease extent is assessed by both anatomical imaging, and nuclear imaging with radiolabelled somatostatin analogues. Pathological evaluation comprises tumour-node-metastasis classification, a minimum pathological dataset, CgA and synaptophysin immunostaining, as well as mitotic count or Ki-67 index (a marker of cell proliferation) to define grading. Resection of the primary lesion and as much metastatic disease as possible increases the efficacy of medical therapy. Other management strategies include hepatic embolisation and peptide receptor radionuclide therapy. Patients with tumours expressing somatostatin receptors should be treated with somatostatin analogues. Depending on the tumour grade, other effective agents include cytotoxics, tyrosine kinase inhibitors, and antiangiogenics. The overarching requirement for best management of patients with NETs is to ensure that they have ready access to experienced multidisciplinary clinician groups located within centres of appropriate subspecialty expertise.

The 5-HT(2B) receptor plays a key regulatory role in both neuroendocrine tumor cell proliferation and the modulation of the fibroblast component of the neoplastic microenvironment.

BACKGROUND: Fibrosis is a cardinal feature of small intestinal neuroendocrine tumors (SI-NETs) both in local peritumoral tissue and systemic sites (cardiac). 5-HT, a commonly secreted NET amine, is a known inducer of fibrosis, although the mechanistic basis for it and growth factors regulating fibrosis and proliferation in the tumor microenvironment are unclear. We hypothesized that targeting 5-HT(2B) receptors on tumor cells would inhibit SI-NET 5-HT release and, thereby, fibroblast activation in the tumor microenvironment. METHODS: We studied the 5-HT(2B) receptor antagonist PRX-08066 in NET cell lines (KRJ-I, H720) and in the coculture system (KRJ-I cells: fibroblastic HEK293 cells) using real time polymerase chain reaction, ELISA, Ki67 immunostaining, and flow cytometry-based caspase 3 assays to assess antiproliferative and profibrotic signaling pathways. RESULTS: In the 5-HT(2B) expressing SI-NET cell line, KRJ-I, PRX-08066 inhibited proliferation (IC(50) 4.6 x 10(-9)M) and 5-HT secretion (6.9 x 10(-9)M) and decreased ERK1/2 phosphorylation and profibrotic growth factor synthesis and secretion (transforming growth factor beta 1 [TGFbeta1], connective tissue growth factor [CTGF] and fibroblast growth factor [FGF2]). In the KRJ-I:HEK293 coculture system, PRX-08066 significantly decreased 5-HT release (>60%), Ki67 (transcript and immunostaining: 20%-80%), TGFbeta1, CTGF, and FGF2 transcription (20%-50%) in the KRJ-I cell line. 5-HT itself stimulated HEK293 proliferation (25%) and synthesis of TGFbeta1, CTGF and FGF2. PRX-08066 inhibition of KRJ-I function reversed these effects in the coculture system. CONCLUSIONS: Targeting the 5-HT(2B) receptor may be an effective antiproliferative and antifibrotic strategy for SI-NETs because it inhibits tumor microenvironment fibroblasts as well as NET cells. Fibrosis and proliferation appear to be biologically interfaced neuroendocrine neoplasia domains.