A multigenomic liquid biopsy biomarker for neuroendocrine tumor disease outperforms CgA and has surgical and clinical utility.

BACKGROUND: Biomarkers are key tools in cancer management. In neuroendocrine tumors (NETs), Chromogranin A (CgA) was considered acceptable as a biomarker. We compared the clinical efficacy of a multigenomic blood biomarker (NETest) to CgA over a 5-year period. PATIENTS AND METHODS: An observational, prospective, cross-sectional, multicenter, multinational, comparative cohort assessment. Cohort 1: NETest evaluation in NETs (n = 1684) and cancers, benign diseases, controls (n = 731). Cohort 2: (n = 1270): matched analysis of NETest/CgA in a sub-cohort of NETs (n = 922) versus other diseases and controls (n = 348). Disease status was assessed by response evaluation criteria in solid tumors (RECIST). NETest measurement: qPCR [upper limit of normal (ULN: 20)], CgA (EuroDiagnostica, ULN: 108 ng/ml). STATISTICS: Mann-Whitney U-test, AUROC, chi-square and McNemar' test. RESULTS: Cohort 1: NETest diagnostic accuracy was 91% (P < 0.0001) and identified pheochromocytomas (98%), small intestine (94%), pancreas (91%), lung (88%), gastric (80%) and appendix (79%). NETest reflected grading: G1: 40 ± 1, G2 (50 ± 1) and G3 (52 ± 1). Locoregional disease levels were lower (38 ± 1) than metastatic (52 ± 1, P < 0.0001). NETest accurately stratified RECIST-assessed disease extent: no disease (21 ± 1), stable (43 ± 2), progressive (62 ± 2) (P < 0.0001). NETest concordance with imaging (CT/MRI/68Ga-SSA-PET) 91%. Presurgery, all NETs (n = 153) were positive (100%). After palliative R1/R2 surgery (n = 51) all (100%) remained elevated. After curative R0-surgery (n = 102), NETest levels were normal in 81 (70%) with no recurrence at 2 years. In the 31 (30%) with elevated levels, 25 (81%) recurred within 2 years. Cohort #2: NETest diagnostic accuracy was 87% and CgA 54% (P < 0.0001). NETest was more accurate than CgA for grading (chi-square = 7.7, OR = 18.5) and metastatic identification (chi-square = 180, OR = 8.4). NETest identified progressive disease (95%) versus CgA (57%, P < 0.0001). Imaging concordance for NETest was 91% versus CgA (46%) (P < 0.0001). Recurrence prediction after surgery was NETest-positive in >94% versus CgA 11%. CONCLUSION: NETest accurately diagnoses NETs and is an effective surrogate marker for imaging, grade, metastases and disease status compared to CgA. A multigenomic liquid biopsy is an accurate biomarker of NET disease.

The clinical utility of circulating neuroendocrine gene transcript analysis in well-differentiated paragangliomas and pheochromocytomas.

CONTEXT: Paragangliomas and pheochromocytomas (PPGLs) exhibit variable malignancy, which is difficult to determine by histopathology, amine measurements or tissue genetic analyses. OBJECTIVE: To evaluate whether a 51-neuroendocrine gene blood analysis has clinical utility as a diagnostic and prognostic marker. DESIGN: Prospective cohort study. Well-differentiated PPGLs (n = 32), metastatic (n = 4); SDHx mutation (n = 25); 12 biochemically active, Lanreotide treated (n = 4). Nine patients had multiple sampling. Age- and gender-matched controls and GEP-NETs (comparators). METHODS: Circulating neuroendocrine tumor mRNA measured (qPCR) with multianalyte algorithmic analysis. Metabolic, epigenomic and proliferative genes as well as somatostatin receptor expression were assessed (averaged, normalized gene expression: mean ± s.e.m.). Amines were measured by HPLC and chromogranin A by ELISA. Analyses (2-tailed): Fisher's test, non-parametric (Mann-Whitney), receiver-operator curve (ROC) and multivariate analysis (MVA). All data are presented as mean ± s.e.m. RESULTS: PPGL were NETest positive (100%). All exhibited higher scores than controls (55 ± 5% vs 8 ± 1%, P = 0.0001), similar to GEP-NETs (47 ± 5%). ROC analysis area under curve was 0.98 for differentiating PPGLs/controls (cut-off for normal: 26.7%). Mutation status was not directly linked to NETest. Genetic and molecular clustering was associated (P < 0.04) with NETest scores. Metastatic (80 ± 9%) and multicentric (64 ± 9%) disease had significantly (P < 0.04) higher scores than localized disease (43 ± 7%). Progressive disease (PD) had the highest scores (86 ± 2%) vs stable (SD, 41 ± 2%) (P < 0.0001). The area under the curve for PD from SD was 0.93 (cut-off for PD: 53%). Proliferation, epigenetic and somatostatin receptor gene expression was elevated (P < 0.03) in PD. Metabolic gene expression was decreased in SDHx mutations. Repeat NETest measurements defined clinical status in the 9 patients (6 SD and 3 PD). Amine measurement was non-informative. Multivariate analysis identified NETest >53% as an independent prognostic factor. CONCLUSION: Circulating NET transcript analysis is positive (100% diagnostic) in well-differentiated PCC/PGL, scores were elevated in progressive disease irrespective of mutation or biochemical activity and elevated levels were prognostic.

Measurement of circulating transcripts and gene cluster analysis predicts and defines therapeutic efficacy of peptide receptor radionuclide therapy (PRRT) in neuroendocrine tumors.

BACKGROUND: Peptide receptor radionuclide therapy (PRRT) is an effective method for treating neuroendocrine tumors (NETs). It is limited, however, in the prediction of individual tumor response and the precise and early identification of changes in tumor size. Currently, response prediction is based on somatostatin receptor expression and efficacy by morphological imaging and/or chromogranin A (CgA) measurement. The aim of this study was to assess the accuracy of circulating NET transcripts as a measure of PRRT efficacy, and moreover to identify prognostic gene clusters in pretreatment blood that could be interpolated with relevant clinical features in order to define a biological index for the tumor and a predictive quotient for PRRT efficacy. METHODS: NET patients (n = 54), M: F 37:17, median age 66, bronchial: n = 13, GEP-NET: n = 35, CUP: n = 6 were treated with (177)Lu-based-PRRT (cumulative activity: 6.5-27.8 GBq, median 18.5). At baseline: 47/54 low-grade (G1/G2; bronchial typical/atypical), 31/49 (18)FDG positive and 39/54 progressive. Disease status was assessed by RECIST1.1. Transcripts were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and multianalyte algorithmic analysis (NETest); CgA by enzyme-linked immunosorbent assay (ELISA). Gene cluster (GC) derivations: regulatory network, protein:protein interactome analyses. STATISTICAL ANALYSES: chi-square, non-parametric measurements, multiple regression, receiver operating characteristic and Kaplan-Meier survival. RESULTS: The disease control rate was 72 %. Median PFS was not achieved (follow-up: 1-33 months, median: 16). Only grading was associated with response (p < 0.01). At baseline, 94 % of patients were NETest-positive, while CgA was elevated in 59 %. NETest accurately (89 %, χ(2) = 27.4; p = 1.2 × 10(-7)) correlated with treatment response, while CgA was 24 % accurate. Gene cluster expression (growth-factor signalome and metabolome) had an AUC of 0.74 ± 0.08 (z-statistic = 2.92, p < 0.004) for predicting response (76 % accuracy). Combination with grading reached an AUC: 0.90 ± 0.07, irrespective of tumor origin. Circulating transcripts correlated accurately (94 %) with PRRT responders (SD+PR+CR; 97 %) vs. non-responders (91 %). CONCLUSIONS: Blood NET transcript levels and the predictive quotient (circulating gene clusters+grading) accurately predicted PRRT efficacy. CgA was non-informative.

The clinical utility of a novel blood-based multi-transcriptome assay for the diagnosis of neuroendocrine tumors of the gastrointestinal tract.

OBJECTIVES: Current monoanalyte blood-based biomarkers for the diagnosis and follow-up of neuroendocrine tumors (NETs) do not achieve satisfactory metrics of sensitivity and specificity. We report the sensitivity and selectivity of the PCR-based test, the NETest, to detect tumors with reference to other benign and malignant gastrointestinal diseases. METHODS: A total of 179 cases (gastrointestinal tumors: n=81; pancreatic disease: n=98) were prospectively collected and assessed using the NETest or chromogranin A (CgA) to determine metrics for detecting small intestinal and pancreatic NETs. RESULTS: For intestinal carcinoids, the accuracy of the NETest was 93% (all NETs positive and 3 (12%) colorectal tumors were positive). CgA was positive in 80%, but 29% (n=7) of colorectal cancers were CgA positive. For pancreatic disease, the NETest accuracy was 94% (96% NETs positive, 2 (6%) of intraductal papillary mucinous neoplasms (IPMNs) were positive). The accuracy of CgA was 56% (29% of pancreatic NETs were CgA positive). Overall, the NETest was significantly more sensitive than CgA for the detection of small intestinal (area under the curve 0.98 vs. 0.75 P<0.0001) and pancreatic NETs (0.94 vs. 0.52, P<0.0001). NETest scores were elevated (P<0.05) in extensive disease and were more accurate (76-80%) than CgA levels (20-32%). The metrics of the multianalyte NETest met the performance criteria proposed by the National Institutes of Health for biomarkers, whereas CgA measurement did not. CONCLUSIONS: This study demonstrates that a blood-based multianalyte NET gene transcript measurement of well-differentiated small intestinal and pancreatic neuroendocrine tumor disease is sensitive and specific and outperforms the current monoanalyte diagnostic strategy of plasma CgA measurement.

Gene transcript analysis blood values correlate with 68Ga-DOTA-somatostatin analog (SSA) PET/CT imaging in neuroendocrine tumors and can define disease status.

PURPOSE: Precise determination of neuroendocrine tumor (NET) disease status and response to therapy remains a rate-limiting concern for disease management. This reflects limitations in biomarker specificity and resolution capacity of imaging. In order to evaluate biomarker precision and identify if combinatorial blood molecular markers and imaging could provide added diagnostic value, we assessed the concordance between (68)Ga-somatostatin analog (SSA) positron emission tomography (PET), circulating NET gene transcripts (NETest), chromogranin A (CgA), and Ki-67 in NETs. METHODS: We utilized two independent patient groups with positive (68)Ga-SSA PET: data set 1 ((68)Ga-SSA PETs undertaken for peptide receptor radionuclide therapy (PRRT), as primary or salvage treatment, n = 27) and data set 2 ((68)Ga-SSA PETs performed in patients referred for initial disease staging or restaging after various therapies, n = 22). We examined the maximum standardized uptake value (SUVmax), circulating gene transcripts, CgA levels, and baseline Ki-67. Regression analyses, generalized linear modeling, and receiver-operating characteristic (ROC) analyses were undertaken to determine the strength of the relationships. RESULTS: SUVmax measured in two centers were mathematically evaluated (regression modeling) and determined to be comparable. Of 49 patients, 47 (96 %) exhibited a positive NETest. Twenty-six (54 %) had elevated CgA (χ(2) = 20.1, p < 2.5×10(-6)). The majority (78 %) had Ki-67 < 20 %. Gene transcript scores were predictive of imaging with >95 % concordance and significantly correlated with SUVmax (R (2) = 0.31, root-mean-square error = 9.4). The genes MORF4L2 and somatostatin receptors SSTR1, 3, and 5 exhibited the highest correlation with SUVmax. Progressive disease was identified by elevated levels of a quotient of MORF4L2 expression and SUVmax [ROC-derived AUC (R (2) = 0.7, p < 0.05)]. No statistical relationship was identified between CgA and Ki-67 and no relationship with imaging parameters was evident. CONCLUSION: (68)Ga-SSA PET imaging parameters (SUVmax) correlated with a circulating NET transcript signature. Disease status could be predicted by an elevated quotient of gene expression (MORF4L2) and SUVmax. These observations provide the basis for further exploration of strategies that combine imaging parameters and disease-specific molecular data for the improvement of NET management.

EGFR/TGFα and TGFß/CTGF Signaling in Neuroendocrine Neoplasia: Theoretical Therapeutic Targets.

Neuroendocrine neoplasms (NENs) are a heterogeneous family of malignancies whose proliferation is partially dependent on growth factors secreted by the microenvironment and the tumor itself. Growth factors which were demonstrated to be important in experimental models of NENs include EGF (epidermal growth factor), TGF (transforming growth factor) α, TGFß and CTGF (connective tissue growth factor). EGF and TGFα bind to the EGF receptor to stimulate an intact RAS/RAF/MAPK pathway, leading to the transcription of genes associated with cell proliferation, invasion and metastasis. Theoretically, TGFα stimulation can be inhibited at several points of the MAPK pathway, but success is limited to NEN models and is not evident in the clinical setting. TGFß1 stimulates TGFß receptors (TGFßRI and TGFßRII) resulting in inhibition of neuroendocrine cell growth through SMAD-mediated activation of the growth inhibitor P21(WAF1/CIP1). Although some NENs are inhibited by TGFß1, paradoxical growth is seen in experimental models of gastric and small intestinal (SI) NENs. Therapeutic targeting of TGFß1 in NENs is therefore complicated by uncertainty of the effect of TGFß1 secretion on the direction of proliferative regulation. CTGF expression is associated with more malignant clinical phenotypes in a variety of cancers, including NENs. CTGF promotes growth in gastric and SI-NEN models, and is implicated as a mediator of local and distant fibrosis caused by NENs of enterochromaffin cell origin. CTGF inhibitors are available, but their anti-proliferative effect has not been tested in NENs. In summary, growth factors are essential for NEN proliferation, and although interventions targeting these proteins are effective in experimental models, only limited clinical efficacy has been identified.

The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion.

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy.

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.

The CCK(2) receptor antagonist, YF476, inhibits Mastomys ECL cell hyperplasia and gastric carcinoid tumor development.

YF476 is a potent and highly selective cholecystokin 2 (CCK(2)) receptor antagonist of the benzodiazepine class. It inhibits gastric neuroendocrine enterochromaffin-like (ECL) cell secretion, proliferation and spontaneous formation of gastric neuroendocrine tumors (carcinoids) in cotton rats. The Mastomys rodent species exhibits a genetic predisposition to gastric ECL neuroendocrine tumor formation which can be accelerated by acid suppression and induction of hypergastrinemia. In this respect, it mimics the human condition of atrophic gastritis, hypergastrinemia and gastric carcinoid development. We investigated whether YF476 could inhibit acid suppression-induced ECL cell hyperplasia and neoplasia in this model. In addition, we examined whether YF476 could reverse established ECL cell hyperplasia and neoplasia. Targeting the CCK(2) receptor during Loxtidine-induced hypergastrinemia resulted in a reduction in ECL cell secretion (plasma and mucosal histamine, and histidine decarboxylase (HDC) transcripts, p<0.05) and proliferation (numbers of HDC-positive cells, connective tissue growth factor (CTGF) and cyclin D1 transcription). This was associated with a decrease in ECL cell hyperplasia and a 60% reduction in gastric ECL cell microcarcinoid (tumors <0.3mm in size) formation. YF476 inhibited ECL cell neoplasia (gastric carcinoid) in animals with hyperplasia, inhibited the formation of ECL cell tumors when co-administered with Loxtidine and reversed the growth and developement of gastric ECL cell carcinoids in long-term acid suppressed Mastomys. Variable importance analysis using a logistic multinomial regression model indicated the effects of YF476 were specific to the ECL cell and alterations in ECL cell function reflected inhibition of transcripts for HDC, Chromogranin A (CgA), CCK(2) and the autocrine growth factor, CTGF. We conclude that specifically targeting the CCK(2) receptor inhibits gastrin-mediated ECL cell secretion and ECL cell proliferation and tumor development in vivo.

Review article: somatostatin analogues in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumours.

BACKGROUND: The discovery of somatostatin (SST) and the synthesis of a variety of analogues constituted a major therapeutic advance in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumours (GEP-NETs). They currently provide the most efficient treatment to achieve symptomatic relief and have recently been demonstrated to inhibit tumour growth. AIM: To review 35 years of experience regarding the clinical application and efficacy of SST analogues. METHODS: The PubMed database (1972-2009) was searched using somatostatin as a search term with combinations of terms including 'treatment'; 'neuroendocrine'; 'carcinoid'; 'tumor'; 'octreotide'; 'lanreotide' and 'pasireotide'. RESULTS: In a review of 15 studies including 481 patients, the slow-release formulations Sandostatin LAR and Somatuline SR/Autogel achieved symptomatic relief in 74.2% (61.9-92.8%) and 67.5% (40.0-100%), biochemical response in 51.4% (31.5-100%) and 39.0% (17.9-58%), and tumour response in 69.8% (47.0-87.5%) and 64.4% (48.0-87.0%) respectively. Novel SST analogues like SOM230 (pasireotide) that exhibit pan SST receptor activity and analogues with high affinity to specific somatostatin receptor (sstr) subtypes may further advance the field, but efficacy studies are lacking. CONCLUSION: As more precise understanding of NET cell biology evolves and molecular biological tools advance, more accurate identification of individual tumours sstr profile will probably facilitate a more precise delineation of SST analogue treatment.

Diagnosis and management of non-erosive reflux disease--the Vevey NERD Consensus Group.

BACKGROUND/AIMS: Although considerable information exists regarding gastroesophageal reflux disease with erosions, much less is known of non-erosive reflux disease (NERD), the dominant form of reflux disease in the developed world. METHODS: An expert international group using the modified Delphi technique examined the quality of evidence and established levels of agreement relating to different aspects of NERD. Discussion focused on clinical presentation, assessment of clinical outcome, pathobiological mechanisms, and clinical strategies for diagnosis and management. RESULTS: Consensus was reached on 85 specific statements. NERD was defined as a condition with reflux symptoms in the absence of mucosal lesions or breaks detected by conventional endoscopy, and without prior effective acid-suppressive therapy. Evidence supporting this diagnosis included: responsiveness to acid suppression therapy, abnormal reflux monitoring or the identification of specific novel endoscopic and histological findings. Functional heartburn was considered a separate entity not related to acid reflux. Proton pump inhibitors are the definitive therapy for NERD, with efficacy best evaluated by validated quality-of-life instruments. Adjunctive antacids or H(2) receptor antagonists are ineffective, surgery seldom indicated. CONCLUSIONS: Little is known of the pathobiology of NERD. Further elucidation of the mechanisms of mucosal and visceral hypersensitivity is required to improve NERD management.

IL1beta- and LPS-induced serotonin secretion is increased in EC cells derived from Crohn's disease.

Gut mucosal enterochromaffin (EC) cells are regarded as key regulators of intestinal motility and fluid secretion via secretion of serotonin (5HT), are increased in numbers in mucosal inflammation and located in close proximity to immune cells. We examined whether interleukin (IL)1beta and Escherichia coli lipopolysaccharide (LPS) induced EC cell 5HT release through Toll-like/IL-1 (TIL) receptor activation, nuclear factor kappa B (NFkappaB) and mitogen-activated protein kinase (MAPK) phosphorylation and evaluated whether somatostatin could inhibit this phenomenon. Pure (>98%) human intestinal EC cells were isolated by fluorescent activated cell sorting from preparations of normal (n = 5) and Crohn's colitis (n = 6) mucosa. 5HT release was measured (ELISA), and NFkappaB and ERK phosphorylation quantitated (ELISA) in response to IL1beta and LPS. 5HT secretion was increased by both E. coli LPS (EC(50) = 5 ng mL(-1)) and IL1beta (EC(50) = 0.05 pmol L(-1)) >2-fold (P < 0.05) in Crohn's EC cells compared with normal EC cells. Secretion was reversible by the TLR4 antagonist, E. coli K12 LPS (IC(50) = 12 ng mL(-1)) and the IL1beta receptor antagonist (ILRA; IC(50) = 3.4 ng mL(-1)). IL1beta caused significant (P < 0.05) NFkappaB and MAPK phosphorylation (40-55%). The somatostatin analogue, lanreotide inhibited IL1beta-stimulated secretion in Crohn's (IC(50) = 0.61 nmol L(-1)) and normal EC cells (IC(50) = 1.8 nmol L(-1)). Interleukins (IL1beta) and bacterial products (E. coli LPS) stimulated 5HT secretion from Crohn's EC cells via TIL receptor activation (TLR4 and IL1beta). Immune-mediated alterations in EC cell secretion of 5HT may represent a component of the pathogenesis of abnormal bowel function in Crohn's disease. Inhibition of EC cell-mediated 5HT secretion may be an alternative therapeutic strategy in the amelioration of inflammatory bowel disease symptomatology.

Pharmacotherapy of neuroendocrine cancers.

BACKGROUND: Neuroendocrine tumors (NETs) of the diffuse neuroendocrine cell system often present a considerable diagnostic and therapeutic challenge. METHODS: We have reviewed the literature on NET treatment between 1979 and 2008 (PubMed search: carcinoid or neuroendocrine tumor/tumour + treatment or management), and summarized current therapeutic options and recommendations. RESULTS: The majority of tumors are diagnosed at a stage that the only curative treatment, radical surgical intervention, is no longer an option. Biotherapy with somatostatin analogs is currently the most efficient treatment to achieve palliation. The interferon class of agents may have a role in selected individuals but substantial adverse events often limit their use. Conventional chemotherapy has minimal efficacy but may have some utility in undifferentiated or highly proliferating neuroendocrine carcinomas and pancreatic NETs. Hepatic metastases, depending on size, location and number, may be amenable to surgical resection, embolization or radio-frequency ablation. Peptide receptor targeted radiotherapy may lead to reduction in tumor size but in most circumstances has a tumor-stabilizing effect. A variety of antiangiogenesis and growth factor-targeted agents have been evaluated but to date the results have failed to meet expectations. Thus, long-acting somatostatin analogs remain the only effective pharmacotherapeutic option that improves symptomatology and quality of life with minimal adverse effects.

[Gastrointestinal neuroendocrine tumors]. / Neuroendokrine Tumoren des Gastrointestinaltraktes.

Two thirds of all neuroendocrine tumors (NET) are located in the gastroentero-pancreatic system. Depending on its localisation, each tumor presents a different histological pattern as well as different clinical symptoms and prognosis. Symptoms are usually due to the mass effects of the local tumor or tumor-related fibrosis. The classical "carcinoid-syndrome" with flush and diarrhea is seen in fewer than 10 %. Tests like chromogranin A (serum) or 5-hydroxyindolacetic acid (24h urine-collection) are indicators for the initial diagnosis of NET. Somatostatin-receptor scintigraphy is the most valuable imaging modality. In addition CT/MRI can be used for further topographical definition. Endoscopic techniques like the use of capsule endoscopy are being evaluated for the diagnosis of small intestinal NETs. The only curative treatment of NET is still complete surgical resection. However, it can only be done in 20 %, depending on localization and local extension of the primary tumor. If the liver is involved local ablation techniques should be considered. The gold standard for medical treatment is the use of somatostatin analogs, although interferons show a comparable therapeutic potential. Traditional cytotoxic agents should only be used for poorly differentiated tumors refractory to other forms of treatment. New compounds that target different pathways at the intra- and intercellular level are under investigation. Supportive therapy should be considered for the control of symptoms.

Carcinoid heart disease.

The carcinoid syndrome is usually evident when enterochromaffin (EC) cell-derived neuroendocrine tumors (carcinoids) metastasize to the liver. In addition to carcinoid symptomatology, about 40% of patients exhibit carcinoid heart disease (CHD) with fibrotic endocardial plaques and associated heart valve dysfunction. The mechanism behind CHD development is not fully understood, but serotonin (5-HT) is considered to be a major initiator of the fibrotic process. Most patients present with right-sided heart valve dysfunction since pulmonary and tricuspid valves lesions are the most common (>95%) cardiac pathology. Left-sided valvular involvement, and angina associated with coronary vasospasm occur in ~10% of subjects with CHD. Pathognomonic echocardiograpic features include immobility of valve leaflets and thickening and retraction of the cusps most commonly resulting in tricuspid valve regurgitation and pulmonary stenosis. Therapeutic options include cardioactive pharmacotherapy for heart failure and, in selected individuals, cardiac valve replacement. Previously valve replacement was reserved for advanced disease due to a perioperative mortality of >20% however in the last decade, technical advances as well as an earlier diagnosis have decreased surgical mortality to <10% and valve replacements are undertaken more frequently. A recent analysis of 200 cases demonstrated an increase in median survival from 1.5 years to 4.4 years in the last two decades. Although the improved prognosis might also reflect the increased use of surgical cytoreduction, hepatic metastatic ablative therapies and somatostatin analogs a robust correlation between diminution of circulating tumor products and an increased long-term survival in CHD has not been rigorously demonstrated.

Molecular mechanisms in therapy of acid-related diseases.

Inhibition of gastric acid secretion is the mainstay of the treatment of gastroesophageal reflux disease and peptic ulceration; therapies to inhibit acid are among the best-selling drugs worldwide. Highly effective agents targeting the histamine H2 receptor were first identified in the 1970s. These were followed by the development of irreversible inhibitors of the parietal cell hydrogen-potassium ATPase (the proton pump inhibitors) that inhibit acid secretion much more effectively. Reviewed here are the chemistry, biological targets and pharmacology of these drugs, with reference to their current and evolving clinical utilities. Future directions in the development of acid inhibitory drugs include modifications of current agents and the emergence of a novel class of agents, the acid pump antagonists.

The elucidation of peptic esophagitis: from Hamperl to heartburn.

Although the esophagus was initially considered as the "humble" organ, it has in recent times evoked both substantial interest and considerable controversy as esophagitis becomes a global pandemic and the debate over the causes of esophagitis and the management of Barrett's has escalated. The esophagus has always defied easy understanding and the origins of its name and its symptomatology continue to confound those who address them. Esophagitis is increasing relentlessly on a global scale and the challenge of defining its symptomatology remains today as great a problem as when initially identified by Herwig Hamperl and Asher Winkelstein. Their identification of the entity they called "peptic esophagitis" in 1934, while initially for the most part overlooked, has today become one of the centers of gastroenterological focus. Although the initial symptomatology of esophagitis was limited to heartburn, it has more recently become apparent that a diverse group of symptoms is produced by the inflammation associated with intermittent reflux of gastric acid. Of interest is the nature of the original observations that led to the identification of the physiopathology of esophagitis and the elucidation of the relationship between symptomatology, acid reflux and inflammation. Although Asher Winkelstein of New York has long been considered the first to define the relationship of acid peptic reflux, esophageal ulceration and reflux symptomatology, it is of note to consider the seminal role of Herwig Hamperl, an Austrian pathologist in the elucidation of acid peptic esophagitis. Indeed, a careful consideration of the relative contributions of these two pioneers suggests that both deserve credit for identifying a disease process that, although initially for the most part ignored by clinicians, has now become one of the most fundamental problems faced by both gastroenterologists and pathologists.

CTGF, intestinal stellate cells and carcinoid fibrogenesis.

AIM: To investigate the role of small intestinal carcinoid tumor-derived fibrotic mediators, TGFbeta1 and CTGF, in the mediation of fibrosis via activation of an "intestinal" stellate cell. METHODS: GI carcinoid tumors were collected for Q RT-PCR analysis of CTGF and TGFbeta1. Markers of stellate cell desmoplasia were identified in peritoneal fibrosis by immunohistochemistry and stellate cells cultured from fresh resected fibrotic tissue. CTGF and TGFbeta1 were evaluated using quantitative tissue array profiling (AQUA analysis) in a GI carcinoid tissue microarray (TMA) with immunostaining and correlated with clinical and histologically documented fibrosis. Serum CTGF was analyzed using a sandwich ELISA assay. RESULTS: Message levels of both CTGF and TGFbeta1 in SI carcinoid tumors were significantly increased (> 2-fold, P < 0.05) versus normal mucosa and gastric (non-fibrotic) carcinoids. Activated stellate cells and markers of stellate cell-mediated fibrosis (vimentin, desmin) were identified in histological fibrosis. An intestinal stellate cell was immunocytochemically and biochemically characterized and its TGFbeta1 (10-7M) initiated CTGF transcription response (> 3-fold, P < 0.05) demonstrated. In SI carcinoid tumor patients with documented fibrosis, TMA analysis demonstrated higher CTGF immunostaining (AQUA Score: 92 +/- 8; P < 0.05), as well as elevated TGFbeta1 (90.6 +/- 4.4, P < 0.05). Plasma CTGF (normal 12.5 +/- 2.6 ng/mL) was increased in SI carcinoid tumor patients (31 +/- 10 ng/mL, P < 0.05) compared to non-fibrotic GI carcinoids (< 15 ng/mL). CONCLUSION: SI carcinoid tumor fibrosis is a CTGF/TGFbeta1-mediated stellate cell-driven fibrotic response. The delineation of the biology of fibrosis will facilitate diagnosis and enable development of agents to obviate its local and systemic complications.

A comparison of the effects of gastrin, somatostatin and dopamine receptor ligands on rat gastric enterochromaffin-like cell secretion and proliferation.

Gastrin regulates ECL cell histamine release and is a critical determinant of acid secretion. ECL cell secretion and proliferation is inhibited by gastrin antagonists and somatostatin but little is known about the role of dopamine agonists in this process. Since the ECL cell exhibits all three classes of receptor we evaluated and compared the effects of the gastrin receptor antagonist, (YF476), lanreotide (SST agonist) and novel dopaminergic agents (BIM53061 and BIM27A760) on ECL cell histamine secretion and proliferation. Highly enriched (>98%) ECL cell preparations prepared from rat gastric mucosa using a FACS approach were studied. Real-time PCR confirmed presence of the CCK2, SS2 and SS5 and D1 receptors on ECL cells. YF476 inhibited histamine secretion and proliferation with IC(50)s of 1.25 nM and 1.3 x 10(-11) M respectively, values 10-1000x more potent than L365,260. Lanreotide inhibited secretion and proliferation (2.2 nM, 1.9 x 10(-10) M) and increased YF476-inhibited proliferation a further 5-fold. The dopamine agonist, BIM53061, inhibited gastrin-mediated ECL cell secretion and proliferation (17 nM, 6 x 10(-10) M) as did the novel dopamine/somatostatin chimera BIM23A760 (22 nM, 4.9 x 10(-10) M). Our studies demonstrate that the gastrin receptor antagonist, YF476, is the most potent inhibitor of ECL cell histamine secretion and proliferation. Lanreotide, a dopamine agonist and a dopamine/somatostatin chimera inhibited ECL cell function but were 10-1000x less potent than YF476. Agents that selectively target the CCK2 receptor may provide alternative therapeutic strategies for gastrin-mediated gastrointestinal cell secretion and proliferation such as evident in the hypergastrinemic gastric carcinoids associated with low acid states.

Role of CCN2/CTGF in the proliferation of Mastomys enterochromaffin-like cells and gastric carcinoid development.

Mastomys enterochromaffin-like (ECL) cell proliferation is initially gastrin driven, but once neoplasia develops, cells become gastrin autonomous. We hypothesized that CCN2 (CTGF), a mitogenic growth factor, may regulate ECL cell proliferation. A Mastomys GeneChip database was examined (dCHIP) to identify CCN2 expression levels. CCN2 in normal and tumor ECL cell preparations obtained using FACS (100 nM acridine orange) was examined by real-time PCR. CCN2 protein was identified in mucosal and ECL cell preparations by immunohistochemistry. Short-term cultured cells were stimulated with either CCN2 or CCN2 + EGF, and proliferation was measured (MTT assay). The ERK1/2 inhibitor PD-98059 (0.1-100 microM) was assessed in terms of CCN2 (1 ng/ml)-mediated proliferation and ERK1/2 phosphorylation. CCN2 transcript and protein was then examined in clinical gastric carcinoids. The ccn2 transcript was upregulated in tumor samples compared with the normal mucosa (+2.36-fold, P < 0.01). PCR demonstrated that ccn2 was not expressed in FACS-prepared (>98% pure) normal ECL cells but was elevated in tumor ECL cell fractions (41.3 +/- 10.7-fold). Immunostaining of the Mastomys gastric mucosa and FACS preparations confirmed that CCN2 protein was present in ECL tumors but not in normal ECL cells. Neither CCN2 nor CCN2 + EGF stimulated normal ECL cell proliferation. CCN2 stimulated tumor proliferation (EC50 approximately 0.01 ng/ml); EGF significantly augmented (P < 0.01) CCN2-induced tumor cell proliferation (EC50 = 20 pg/ml). PD-98059 inhibited CCN2-induced proliferation (-12 +/- 3%, P < 0.05) and ERK1/2 phosphorylation (-34 +/- 5%, P < 0.05) in tumor cells. In clinical samples, both CCN2 transcript and protein were elevated in gastrin-autonomous carcinoids (P < 0.02) compared with the normal mucosa. In conclusion, CCN2 may be a proliferative regulator of Mastomys ECL neoplastic proliferation once these cells become autonomous of gastrin regulation. Identification of CCN2 in gastric carcinoid tissue may be useful both as an indicator of ECL cell transformation and may define gastrin autonomy, a criteria of gastric carcinoid malignancy.