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.

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.

Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.

Mastocytosis is characterized by accumulations of mast cells in various organs (1). Most cases are indolent and confined to the skin, where discrete mast cell infiltrates are associated increased epidermal melanin, a clinical picture known as urticaria pigmentosa (UP). Other forms of mastocytosis combine UP with aggressive involvement of other organs or with haemotologic abnormalities (1-4). It is not known whether all forms of mastocytosis are true neoplasms or whether some might represent reactive hyperplasias (5-7). The c-KIT proto-oncogene encodes a type III receptor tyrosine kinase (KIT) that is critical to the development and survival of mast cells and melanocytes (8-11). The ligand for KIT (KL) can stimulate mast cell development, proliferation, and mediator release (9,12-17), as well as melanocyte proliferation and pigment production (18-20). To determine the role of c-KIT in the pathogenesis of mastocytosis, we examined tissue and cells isolated from a patient with UP and aggressive systemic mastocytosis with massive splenic involvement. We found a mutation that results in constitutive activation and expression of c-KIT in mast cells of both skin and spleen. This is the first in situ demonstration of an activation c-KIT mutation in neoplastic cells. It also demonstrates the clonal and neoplastic nature of this form of mastocytes.

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.

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.

Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor.

The modulation of enterocyte sheet migration was studied using Caco-2 cells, a well-differentiated human colonic cell line. Although Caco-2 cells attached and spread equivalently over collagen types I, III, IV, and V and laminin, migration over laminin was significantly slower than migration over the collagen types. Fibronectin was a poor substrate for attachment, spreading, and migration. Epidermal growth factor (EGF) stimulated migration over laminin but did not alter Caco-2 migration over collagen or fibronectin. This effect was independent of cell proliferation, which was stimulated equivalently on both laminin and collagen I. Expression and organization of cell surface receptors for matrix (integrins) were studied using antibodies specific for beta and alpha integrin subunits. Integrin surface expression was assessed by immunoprecipitation of surface 125iodinated control and EGF-treated cells. Beta 1 surface pools did not change substantially in any condition studied. Alpha 1 subunit pools were decreased after EGF treatment on collagen I but alpha 1 pools increased after EGF treatment on laminin. Surface pools of alpha 2 subunits were increased following EGF treatment whether cells were cultured on laminin or collagen I. However, traditional immunofluorescent and laser confocal imaging demonstrated substantial differences in the character of alpha 2 subunit organization between collagen and laminin in the migrating cell front. Furthermore, a functional antibody to the alpha 2 subunit inhibited EGF stimulation of migration over laminin without substantial effects on basal migration over laminin or collagen I. Thus, EGF appears to exert a matrix-specific effect on enterocyte migration by modulation of integrin expression and organization.

Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high doses of cholecystokinin.

The mechanism by which digestive zymogens become activated during acute pancreatitis remains poorly understood. Given the ability for cholecystokinin (CCK) to induce pancreatitis in vivo, the effects of high dose CCK on preparations of isolated pancreatic acini were examined. Using an immunologic technique for the detection of zymogen activation, CCK was found to stimulate the conversion of procarboxypeptidase A1 to a 35-kD form having the same net charge and electrophoretic mobility as purified recombinant carboxypeptidase A1. This enhanced conversion was proportional to the dose of CCK (maximal at 100 nM), and time dependent. CCK also produced changes in the electrophoretic mobility of procarboxypeptidase B and chymotrypsinogen 2 immunoreactivity, consistent with activation of these zymogens. These events were detectable only within acinar cell pellets and not in the incubation medium, suggesting an intracellular site of conversion. The conversion of procarboxypeptidase A1 to its active form was inhibited by pretreatment with the weak base chloroquine (40 microM) and the protonophore monensin (10 microM). This conversion was also inhibited by pretreatment with the serine protease inhibitor benzamidine (10 mM) but not the cysteine protease inhibitor E64 (100 microM). The results suggest that high dose CCK stimulates the intracellular activation of digestive zymogens within isolated pancreatic acini. This event appears to require an acidic subcellular compartment and serine protease activity.

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.

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.

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.

Review article: from gastrin to gastro-oesophageal reflux disease--a century of acid suppression.

To commemorate Edkins' discovery of gastrin in 1905, we review a century of progress in the physiology and pathobiology of gastrin and acid secretion especially as it pertains to clinical aspects of gastro-oesophageal reflux disease. Although initially ignored, Edkins' observations eventually led to the enthusiastic investigation of gastrin and acid regulation in peptic ulcer disease, culminating in important therapeutic advances in the management of acid peptic disease. Following the improved understanding of gastric secretory physiology, and the development of acid suppressants with increasing efficacy, the use of surgical intervention for peptic ulcer disease was almost eliminated. Surgery became obsolete with the discovery of Helicobacter pylori. Three other advances are also influencing modern practice: the gastrotoxicity of aspirin and non-steroidal anti-inflammatory drugs is now increasingly appreciated, the role of endoscopy in the diagnosis and therapy of upper gastrointestinal bleeding, and the use of intravenous acid-suppressive agents. The major issue for the future resides within the epidemic of gastro-oesophageal reflux disease. How to diagnose, categorize and treat this condition and how to identify and prevent neoplasia, are the challenges of the new century.

Genotoxicity, carcinogenicity and acid-suppressing medications.

With the availability of increasingly potent acid-suppressing medications, questions continue to rise concerning the safety of these compounds in regards to carcinogenetic potential. In this review, we examine current concepts and procedures relating to genotoxicity, the potential for a chemical agent to interact with and alter the genomic information of the cell, and carcinogenesis. A description and discussion of commonly utilized techniques for the determination of (a) in vitro mutagenicity, (b) in vitro and in vivo DNA damage and repair, (c) in vitro and in vivo chromosomal damage and (d) chronically dosed animal tumorigenesis development is presented. Observations from these procedures as they have been applied to a review of the safety of acid-suppressing medications will be discussed. An evaluation of reports relating to potential genotoxic and carcinogenic hazards of therapeutically relevant acid-suppressing medications (cimetidine, ranitidine, omeprazole) is presented. Information related to the effect of prolonged administration of acid-suppressing medications, alterations of serum gastrin levels, and the potential for tumor promotion is discussed.

A new look at an old hormone gastrin.

The origin of gastrin from the G cell has been identified, as have a number of mechanisms regulating both secretion and processing of the peptide. Little, however, is known of the intracellular regulation of processing and secretion. Gastrin receptors have been identified and cloned, although the exact relationship to the cholecystokinin receptor remains to be clarified. The gene for the peptide has been sequenced and various promoters identified. Up- and downstream sequences have been demonstrated to be interrelated with epidermal growth factor and somatostatin, and the existence of a complex gastric-based molecular regulatory system is apparent. The dynamic role of gastrin in the regulation of acid secretion has been delineated by its action on multiple targets in gastric parietal cells and endocrine enterochromaffinlike cells. Similarly, its trophic effects on gastrointestinal tissue have been explored, resulting in its identification as a common denominator of gastric fundic hyperplasia and microcarcinoid evolution in conditions of hypergastrinemia.

Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and confocal microscopic examination.

Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.

Global expression analysis of ECL cells in Mastomys natalensis gastric mucosa identifies alterations in the AP-1 pathway induced by gastrin-mediated transformation.

Enterochromaffin-like (ECL) cell hyperplasia and then irreversible neoplasia can be generated in the African rodent Mastomys natalensis using the H2 receptor blocker, loxtidine, for 8-16 wk. We used a GeneChip approach complemented by standard technologies to identify gene expression alterations in the gastric mucosa during gastrin-mediated ECL cell transformation. Gastric mucosa (mucosal scrapping) and ECL cell-enriched fractions were obtained from untreated Mastomys (controls) and from animals treated with loxtidine for 8 wk (hyperplasia). Tumor ECL cells were obtained by hand-dissection of gastric ECL cell nodules from animals treated with loxtidine for >16 wk and from a spontaneously developed ECL cell tumor. RNA was isolated, examined on rat U34A GeneChips, and comparison analysis was performed to identify altered gene expression. Alterations in gene expressions were examined further by immunohistochemistry, quantitative RT-PCR (Q-RT-PCR), sequencing and Western blot. GeneSpring analysis demonstrated alterations in few genes (<20) in hyperplastic and tumor mucosa. The histamine H1 receptor was consistently increased in proliferating mucosa. This gene change was confirmed by Q-RT-PCR. Other genes showing alterations included neural-(chromogranin A and somatostatin), cell-cycle-, and AP-1-associated genes. Immunostaining confirmed alterations in neural markers. Cluster analysis of ECL cell-enriched samples demonstrated that c-fos and junD were differently regulated. Q-RT-PCR and Western blot in prospectively collected gastric mucosal samples confirmed the differential expression of Fos and Jun. The negative regulators of AP-1, JunD, and Menin were decreased in tumor mucosa. A missense of unknown function was noted in the menin gene. Hypergastrinemia in an animal model of gastric carcinoids differentially altered the histamine type 1 receptor and gene expression and protein composition of AP-1. These results suggest that expression of this receptor and an altered composition of AP-1 with a loss of inhibition play a role in ECL cell transformation.

Histidine decarboxylase expression and histamine metabolism in gastric oxyntic mucosa during hypergastrinemia and carcinoid tumor formation.

Histamine is an important stimulator of gastric acid secretion. In experimental animals, inhibition of acid secretion by long term histamine2 receptor blockade causes hypergastrinemia, proliferation of enterochromaffin-like (ECL) cells, and formation of histamine-producing gastric carcinoids. The aim of this study was to examine the role of gastrin in histamine synthesis and metabolism of the oxyntic mucosa of normal, hyperplastic, and carcinoid-bearing Mastomys natalensis. Administration of exogenous gastrin to normal animals increased histidine decarboxylase (HDC) messenger RNA (mRNA) expression in the oxyntic mucosa within 30 min, indicating that gastrin stimulates histamine synthesis by regulating HDC mRNA abundance. Endogenous hypergastrinemia, induced by short term histamine2 receptor blockade (loxtidine) for 3-29 days, did not induce tumors, but enhanced the expression of HDC mRNA (2- to 4-fold elevated) and histamine contents (2-fold elevated) in the oxyntic mucosa. Long term histamine2 receptor blockade (7-21 months) resulted in sustained hypergastrinemia and ECL tumor formation. Tumor-bearing animals had a 4-fold increase in HDC mRNA expression and histamine contents of the oxyntic mucosa. Urinary excretion of the histamine metabolite methyl-imidazole-acetic acid was 2-fold elevated. Tumor-bearing animals recovering from histamine2 receptor blockade were normogastrinemic and had normal levels of HDC mRNA and histamine in the oxyntic mucosa as well as normal excretion of methyl-imidazole-acetic acid. The results indicate that ECL cell carcinoids developing during hypergastrinemia are well differentiated tumors that respond to high gastrin levels with increased histamine synthesis and secretion.