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Find video protocols related to scientific articles indexed in Pubmed.
A PCR blood test outperforms chromogranin A in carcinoid detection and is unaffected by proton pump inhibitors.
Endocr Connect
PUBLISHED: 10-14-2014
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A critical requirement in neuroendocrine tumor (NET) management is a blood biomarker test that is sensitive, specific and reproducible. We evaluated a PCR-based 51-transcript signature to detect tumors, compared it with chromogranin A (CgA) and examined the confounding effect of proton pump inhibitors (PPIs), which cause falsely elevated CgA levels. The multigene signature was evaluated in two groups. Group 1: 125 prospectively collected NETs: gastroenteropancreatic NETs (n=91, including 42 pancreatic and 40 small intestinal), carcinoids of unknown primary (n=18) and other sites (n=16). Group 2: prospectively collected non-NET patients receiving PPIs (>1 month; dyspepsia, n=19; GERD, n=6; and pancreatitis, n=4) and 50 controls. All samples were analyzed by PCR (marker genes) and ELISA (DAKO-CgA). Sensitivity comparisons included ?(2), non-parametric measurements, and receiver operating characteristic (ROC) curves. Group 1: 123 NETs were PCR-positive (98.4%) compared with 50 (40%) CgA-positive (?(2)=97.3, P<10(-26)). Significant differences (P<0.001) were noted between pancreas: PCR 95% vs CgA 29.2% (P<10(-9)) and small intestine: 100 vs 58% (P<10(-4)). The multigene test was elevated in all grades (G1-G3), in both local and disseminated disease, and was not normalized by somatostatin analog therapy. It was also elevated in 97% of CgA normal NETs. Group 2: PPI administration increased CgA in 83% and CgA was elevated in 26% of controls. PCR values were not elevated in either group. PCR performance metrics were as follows: sensitivity 98.4%, specificity 100%, positive predictive value 100%, negative predictive value 97.8%, and the ROC-derived area under the curve (AUC) was 0.997. These were significantly better than CgA (all metrics <60%; AUC, 0.54; Z-statistic, 10.44, P<0.0001). A 51-panel multigene blood transcript analysis is significantly more sensitive than plasma CgA for NET detection and is unaffected by acid suppression therapy.
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PRRT: Defining the Paradigm Shift to Achieve Standardization and Individualization.
J. Nucl. Med.
PUBLISHED: 09-25-2014
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Peptide receptor radionuclide therapy is a treatment for inoperable or metastatic neuroendocrine tumors. A key issue is the need to standardize the treatment and develop randomized controlled trials. Standardization would help define the characteristics of response, including progression-free survival; provide homogeneous phase II and III studies; delineate the position of peptide receptor radionuclide therapy in the therapeutic algorithm for neuroendocrine tumors; and establish the basis for approval by the regulatory authorities. Standardization of treatments is the starting point to redefine the treatment paradigm from a one-size-fits-all to a personalized treatment. To delineate the treatment paradigm, treatments should be optimized for efficacy and minimization of long-term toxicity, through dosimetry, and adapted to each individual, including relevant patient characteristics. Although differences in therapy outcomes may be explained by the specific absorbed dose (or biologically effective dose), they may also be related to discrete tumor- and patient-specific features. In this respect, a particular area of investigation is the assessment of genetic elements regulating tumor cell proliferation, especially those involved in the response to cytotoxic therapies.
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A historical appreciation of bronchopulmonary neuroendocrine neoplasia: resolution of a carcinoid conundrum.
Thorac Surg Clin
PUBLISHED: 07-29-2014
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In the three-quarters of a century that have elapsed since the first description of a bronchial carcinoid, the field has progressed from serendipitous radiological or bronchoscopic diagnosis to computed tomography, magnetic resonance imaging, and somatostatin receptor imaging identification. Similarly, pathologic techniques have advanced from a naïve assessment of neoplasia to a delineation of several tumor subtypes and an understanding of the neuroendocrine basis of the disease process. A key unresolved question is the identification of the genetic and environmental activators that are responsible for the initiation of pulmonary neuroendocrine cell proliferation and neoplastic transformation.
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Blood transcript analysis and metastatic recurrent small bowel carcinoid management.
BMC Cancer
PUBLISHED: 07-22-2014
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Detection of neuroendocrine tumor (NET) disease progression is a key issue in determining management. Currently, assessment is by imaging (MRI/CT and Octreoscan®) and plasma Chromogranin A (CgA) measurement.
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A multianalyte PCR blood test outperforms single analyte ELISAs (chromogranin A, pancreastatin, neurokinin A) for neuroendocrine tumor detection.
Endocr. Relat. Cancer
PUBLISHED: 07-13-2014
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A critical requirement in neuroendocrine tumor (NET) management is a sensitive, specific and reproducible blood biomarker test. We evaluated a PCR-based 51 transcript signature (NETest) and compared it to chromogranin A (CgA), pancreastatin (PST) and neurokinin A (NKA). The multigene signature was evaluated in two groups: i) a validation set of 40 NETs and controls and ii) a prospectively collected group of NETs (n=41, 61% small intestinal, 50% metastatic, 44% currently treated and 41 age-sex matched controls). Samples were analyzed by a two-step PCR (51 marker genes) protocol and ELISAs for CgA, PST and NKA. Sensitivity comparisons included ?(2), non-parametric measurements, ROC curves and predictive feature importance (PFAI) analyses. NETest identified 38 of 41 NETs. Performance metrics were: sensitivity 92.8%, specificity 92.8%, positive predictive value 92.8% and negative predictive value 92.8%. Single analyte ELISA metrics were: CgA 76, 59, 65, and 71%; PST 63, 56, 59, and 61% and NKA 39, 93, 84, and 60%. The AUCs (ROC analysis) were: NETest: 0.96±0.025, CgA: 0.67±0.06, PST 0.56±0.06, NKA: 0.66±0.06. NETest significantly outperformed single analyte tests (area differences: 0.284-0.403, Z-statistic 4.85-5.9, P<0.0001). PFAI analysis determined NETest had most value (69%) in diagnosis (CgA (13%), PST (9%), and NKA (9%)). Test data were consistent with the validation set (NETest >95% sensitivity and specificity, AUC =0.98 vs single analytes: 59-67% sensitivity, AUCs: 0.58-0.63). The NETest is significantly more sensitive and efficient (>93%) than single analyte assays (CgA, PST or NKA) in NET diagnosis. Blood-based multigene analytic measurement will facilitate early detection of disease recurrence and can predict therapeutic efficacy.
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Neuroendocrine Tumor Biomarkers: Current Status and Perspectives.
Neuroendocrinology
PUBLISHED: 07-08-2014
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The identification of accurate harbingers of disease status and therapeutic efficacy are critical requirements in precise diagnosis and effective management. Initially, tissue analysis was regarded as ideal but invasive strategies represent risk compared to peripheral blood sampling. Thus far, most biomarkers whether in tissue or blood/urine have been single analytes with varying degrees of sensitivity and specificity. Some analytes have not exhibited robust metrics or lacked methodological rigor. Neuroendocrine (NE) disease represents an area of dire biomarker paucity since the individual biomarkers (gastrin, insulin etc.) are not widely applicable to the diverse types of NE neoplasia (NEN). Broad spectrum markers such as CgA have limitations in sensitivity, specificity and reproducibility. Monoanalytes cannot define the multiple variables (proliferation, metabolic activity, invasive potential and metastatic propensity) that constitute tumor growth. The restricted status of the NEN field has resulted in a lack of comprehensive knowledge of the molecular and cellular biology of the disease with tardy application of innovative technology. This overview examines limitations in current practice and describes contemporary viable strategies under evaluation including the identification of novel analytes (gene transcripts, miRNA), circulating tumor cells and metabolic imaging agents that identify disease. Novel requirements are the necessity to develop biomathmatical algorithms for synchronous calibration of multiple molecular markers and predictive nomograms that interface biological variables to delineate disease progress or treatment efficacy. Optimally, application of novel techniques and amalgamations of multianalyte assessment will provide a personalized molecular disease signature extrapolative of NEN status, likelihood of progression and predictive of therapeutic opportunity. © 2014 S. Karger AG, Basel.
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Peptide receptor radionuclide therapy for advanced neuroendocrine tumors.
Thorac Surg Clin
PUBLISHED: 06-09-2014
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Peptide receptor radionuclide therapy (PRRT) consists of the systemic administration of a synthetic peptide, labeled with a suitable ?-emitting radionuclide, able to irradiate tumors and their metastases via internalization through a specific receptor (usually somatostatin S2), over-expressed on the cell membrane. After almost 2 decades of experience, PRRT, with either (90)Y-octreotide or (177)Lu-octreotate, has established itself to be an efficient and effective therapeutic modality. As a treatment, it is relatively safe up to the known thresholds of absorbed and bio-effective isotope dosages and the renal and hematological toxicity profiles are acceptable if adequate protective measures are undertaken.
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Long-term tolerability of PRRT in 807 patients with neuroendocrine tumours: the value and limitations of clinical factors.
Eur. J. Nucl. Med. Mol. Imaging
PUBLISHED: 05-19-2014
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Peptide receptor radionuclide therapy (PRRT) with (90)Y and (177)Lu provides objective responses in neuroendocrine tumours, and is well tolerated with moderate toxicity. We aimed to identify clinical parameters predictive of long-term renal and haematological toxicity (myelodysplastic syndrome and acute leukaemia).
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Stathmin in pancreatic neuroendocrine neoplasms: a marker of proliferation and PI3K signaling.
Tumour Biol.
PUBLISHED: 05-14-2014
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Chromosome 1p35-36, which encodes tumor suppressors and mitotic checkpoint control genes, is commonly altered in human malignancies. One gene at this locus, stathmin 1 (STMN1), is involved in cell cycle progression and metastasis. We hypothesized that increased STMN1 expression may play a role in pancreatic neuroendocrine neoplasm (pNEN) malignancy. We investigated stathmin copy number variation, mRNA, and protein expression using PCR-Taqman Copy Number Assays, Q-PCR, Western blot, and immunohistochemistry. A mechanistic role for stathmin in proliferation was assessed in the BON cell line under growth-restrictive conditions and siRNA silencing. Furthermore, its role in PI3K signaling pathway activation was evaluated using pharmacological inhibitors. mRNA (p?=?0.0001) and protein (p?
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The Status of Neuroendocrine Tumor Imaging: From Darkness to Light?
Neuroendocrinology
PUBLISHED: 04-25-2014
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Diagnostic imaging plays a pivotal role in diagnosis, staging, treatment selection and follow-up for neuroendocrine tumors (NETs). Diagnostic strategies that are available include morphologic imaging with CT, MRI, US techniques, and molecular imaging, with scintigraphy with (111)In-pentetreotide, PET with (68)Ga-DOTA-peptides, (18)F-DOPA and (11)C-5-HTP. A combination of anatomic and functional techniques is routinely performed to optimize sensitivity and specificity. The introduction of diffusion weighted imaging (DWI) MRI and dynamic contrast-enhanced (DCE) techniques represents promising advance in radiologic imaging whereas new receptor binding peptides, including somatostatin agonists and antagonists, represent the recent most favorable innovation in molecular imaging. Future development includes the short-term validation of these techniques but in extension also a more comprehensive multi-level integration of biologic information pertaining to a specific tumor and patient, possibly encompassing genomic considerations, currently evolving as a new entity denoted 'precision medicine'. The ideal is a diagnostic sequence that captures the global status of an individual's tumor and encompasses a multi-dimensional characterization of tumor location, metabolic performance and target identification. To date, advances in imagery have focused on increasing resolution, discrimination and functional characterization. In the future, the fusion of imagery with the parallel analysis of biological and genomic information has the potential to considerably amplify diagnosis. © 2014 S. Karger AG, Basel.
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Gene network-based analysis identifies two potential subtypes of small intestinal neuroendocrine tumors.
BMC Genomics
PUBLISHED: 04-02-2014
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Tumor transcriptomes contain information of critical value to understanding the different capacities of a cell at both a physiological and pathological level. In terms of clinical relevance, they provide information regarding the cellular "toolbox" e.g., pathways associated with malignancy and metastasis or drug dependency. Exploration of this resource can therefore be leveraged as a translational tool to better manage and assess neoplastic behavior. The availability of public genome-wide expression datasets, provide an opportunity to reassess neuroendocrine tumors at a more fundamental level. We hypothesized that stringent analysis of expression profiles as well as regulatory networks of the neoplastic cell would provide novel information that facilitates further delineation of the genomic basis of small intestinal neuroendocrine tumors.
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Recommendations for management of patients with neuroendocrine liver metastases.
Lancet Oncol.
PUBLISHED: 01-04-2014
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Many management strategies exist for neuroendocrine liver metastases. These strategies range from surgery to ablation with various interventional radiology procedures, and include both regional and systemic therapy with diverse biological, cytotoxic, or targeted agents. A paucity of biological, molecular, and genomic information and an absence of data from rigorous trials limit the validity of many publications detailing management. This Review represents the views from an international conference, for which 15 expert working groups prepared evidence-based assessments addressing specific questions, and from which an independent jury derived final recommendations. The aim of the conference was to review the existing approaches to neuroendocrine liver metastases, assess the evidence on which management decisions were based, develop internationally acceptable recommendations for clinical practice (when evidence was available), and make recommendations for clinical and research endeavours. This report represents the final clinical statements and proposals for future research.
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A mechanistic role for the chromatin modulator, NAP1L1, in pancreatic neuroendocrine neoplasm proliferation and metastases.
Epigenetics Chromatin
PUBLISHED: 01-01-2014
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The chromatin remodeler NAP1L1, which is upregulated in small intestinal neuroendocrine neoplasms (NENs), has been implicated in cell cycle progression. As p57(Kip2) (CDKN1C), a negative regulator of proliferation and a tumor suppressor, is controlled by members of the NAP1 family, we tested the hypothesis that NAP1L1 may have a mechanistic role in regulating pancreatic NEN proliferation through regulation of p57(Kip2).
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Biomarkers in neuroendocrine tumors.
JOP
PUBLISHED: 06-19-2013
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Neuroendocrine tumors are a heterogeneous group of tumors with cells of neuroendocrine differentiation that arise from diverse anatomic sites with varying morphologic and clinical features. Since the natural history and prognosis varies widely between individual neuroendocrine tumor types, there is a critical need to identify accurate prognostic and predictive biomarkers and markers predictive of therapeutic efficacy. To date, plasma chromogranin-A levels have generally been accepted as the most useful biomarker, despite the fact that there are substantial concerns in sensitivity and discrepancies in measurement techniques. As a consequence, considerable attention has been focused upon the development of novel biomarkers that can be utilized with more clinical efficacy than chromogranin-A. In addition to amplifying the diagnostic/prognostic landscape, the need to calibrate the efficacy of biological targeted therapy has further accelerated the development of molecular biomarkers. At the 2013 American Society of Clinical Oncology (ASCO) Annual Meeting, Chou et al. (Abstract #e15151) presented data that chromogranin A levels can be monitored during treatment to predict clinical outcome. Modlin et al. (Abstract #4137), demonstrated a promising novel biomarker, serum multi-transcript molecular signature. Grande et al. (Abstract #4140), Heetfield et al. (Abstract #e15071) and Casanovas et al. (Abstract #4139) described sVEGFR2, p-mTOR and IGF1R as molecular markers with potential for use in targeted therapy trials. The authors review and summarize these abstracts in this article.
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Serotonin and the 5-HT7 receptor: the link between hepatocytes, IGF-1 and small intestinal neuroendocrine tumors.
Cancer Sci.
PUBLISHED: 03-26-2013
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Platelet-derived serotonin (5-HT) is involved in liver regeneration. The liver is also the metastatic site for malignant enterochromaffin (EC) cell "carcinoid" (neuroendocrine) neoplasms, the principal cellular source of 5-HT. We hypothesized that 5-HT produced by metastatic EC cells played a role in the hepatic tumor-microenvironment principally via 5-HT? receptor-mediated activation of hepatocyte IGF-1 synthesis and secretion. Using isolated rat hepatocytes, we evaluated 5-HT? receptor expression (using PCR, sequencing and western blot). ELISA, cell transfection and western blots delineated 5-HT-mediated signaling pathways (pCREB, AKT and ERK). IGF-1 synthesis/secretion was evaluated using QPCR and ELISA. IGF-1 was tested on small intestinal neuroendocrine neoplasm proliferation, while IGF-1 production and 5-HT? expression were examined in an in vivo SCID metastasis model. Our results demonstrated evidence for a functional 5-HT? receptor. 5-HT activated cAMP/PKA activity, pCREB (130-205%, P < 0.05) and pERK/pAKT (1.2-1.75, P < 0.05). Signaling was reversed by the 5-HT? receptor antagonist SB269970. IGF-1 significantly stimulated proliferation of two small intestinal neuroendocrine neoplasm cell lines (EC??: 7-70 pg/mL) and could be reversed by the small molecule inhibitor BMS-754807. IGF-1 and 5-HT were elevated (40-300×) in peri-tumoral hepatic tissue in nude mice, while 5-HT? was increased fourfold compared to sham-operated animals. We conclude that hepatocytes express a cAMP-coupled 5-HT? receptor, which, at elevated 5-HT concentrations that occur in liver metastases, signals via CREB/AKT and is linked to IGF-1 synthesis and secretion. Because IGF-1 regulates NEN proliferation, identification of a role for 5-HT? in the hepatic metastatic tumor microenvironment suggests the potential for novel therapeutic strategies for amine-producing mid-gut tumors.
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Gastric carcinoids (neuroendocrine neoplasms).
Gastroenterol. Clin. North Am.
PUBLISHED: 03-01-2013
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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. Studying menin and its complex interrelationship with gastrin may provide insight into tumor biology at the clinical level and in terms of basic cell biology (eg, the role of the epigenome in neuroendocrine cell proliferation), and lead to potential consideration of other targets that are known candidates for molecular-based therapies in other adenocarcinomas.
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Improving the diagnosis and management of neuroendocrine tumors: utilizing new advances in biomarker and molecular imaging science.
Neuroendocrinology
PUBLISHED: 02-11-2013
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Neuroendocrine tumors (NET) are malignant solid tumors that arise in hormone-secreting tissue of the diffuse neuroendocrine system or endocrine glands. Although traditionally understood to be a rare disease, the incidence and prevalence of NET have increased greatly in the past 3 decades. However, during this time, progress in diagnosis and outcome of NET has generally been modest. In order to achieve improved outcome in NET, a better understanding of NET biology combined with more reliable serum markers and better techniques to identify tumor localization and small lesions are needed. Although some NET biomarkers exist, sensitive and specific markers that predict tumor growth and behavior are generally lacking. In addition, the integration of new molecular imaging technologies in patient diagnosis and follow-up has the potential to enhance care. To discuss developments and issues required to improve diagnostics and management of NET patients, with specific focus on the latest advances in molecular imaging and biomarker science, 17 global leaders in the fields of NET, molecular imaging and biomarker technology gathered to participate in a 2-day meeting hosted by Prof. Kjell Öberg at the University of Uppsala in Sweden. During this time, findings were presented regarding methods with potential prognostic and treatment applications in NET or other types of cancers. This paper describes the symposium presentations and resulting discussions.
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Systemic therapeutic options for carcinoid.
Semin. Oncol.
PUBLISHED: 02-09-2013
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"Carcinoids" are mostly slow-growing neuroendocrine neoplasms (NENs) with low proliferative activity. A wide range of therapeutic options with variable efficacy exist, including locoregional ablative strategies. Thereafter, some patients may not require medical therapy for years depending on the rate of progression or recurrence. However, the majority of patients require systemic treatment and therein lies the dilemma, since no antiproliferative agent is currently approved for carcinoids. Somatostatin analogs (SSAs), and to a lesser extent interferon-alpha, are standard therapy for carcinoids associated with the carcinoid syndrome. These drugs have some antiproliferative efficacy. SSAs rarely lead to tumor remission but may modestly prolong time to tumor progression. Chemotherapy is of limited value in carcinoids with low proliferation indices but may be useful in higher grade tumors. Peptide receptor-targeted radionuclide therapy may be of benefit and is mostly used after medical therapies fail. However, it is considered an investigational modality. More recently, targeted drugs such as mammalian target of rapamycin (mTOR) inhibitors and anti-angiogenics have been investigated. Objective remissions are rare. Their value remains to be rigorously elucidated. Increased efficacy requires a better understanding of the underlying tumor biology and identification of molecular pathological criteria to allow appropriate preselection of candidates for targeted therapies.
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Immunohistochemical evidence for an impairment of autophagy in tumorigenesis of gastric carcinoids and adenocarcinomas in rodent models and patients.
Histol. Histopathol.
PUBLISHED: 02-07-2013
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Autophagy has dual roles in tumorigenesis: tumor-promoting or tumor-suppressing. The aim of the present study was to examine autophagy-related markers by immunohistochemistry in gastric carcinoids and adenocarcinomas in rodent models and patients.
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Chromogranin a and its fragments as regulators of small intestinal neuroendocrine neoplasm proliferation.
PLoS ONE
PUBLISHED: 01-01-2013
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Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation.
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The identification of gut neuroendocrine tumor disease by multiple synchronous transcript analysis in blood.
PLoS ONE
PUBLISHED: 01-01-2013
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Gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) are increasing in both incidence and prevalence. A delay in correct diagnosis is common for these lesions. This reflects the absence of specific blood biomarkers to detect NENs. Measurement of the neuroendocrine secretory peptide Chromogranin A (CgA) is used, but is a single value, is non-specific and assay data are highly variable. To facilitate tumor detection, we developed a multi-transcript molecular signature for PCR-based blood analysis. NEN transcripts were identified by computational analysis of 3 microarray datasets: NEN tissue (n?=?15), NEN peripheral blood (n?=?7), and adenocarcinoma (n?=?363 tumors). The candidate gene signature was examined in 130 blood samples (NENs: n?=?63) and validated in two independent sets (Set 1 [n?=?115, NENs: n?=?72]; Set 2 [n?=?120, NENs: n?=?58]). Comparison with CgA (ELISA) was undertaken in 176 samples (NENs: n?=?81). 51 significantly elevated transcript markers were identified. Gene-based classifiers detected NENs in independent sets with high sensitivity (85-98%), specificity (93-97%), PPV (95-96%) and NPV (87-98%). The AUC for the NEN gene-based classifiers was 0.95-0.98 compared to 0.64 for CgA (Z-statistic 6.97-11.42, p<0.0001). Overall, the gene-based classifier was significantly (?(2)?=?12.3, p<0.0005) more accurate than CgA. In a sub-analysis, pancreatic NENs and gastrointestinal NENs could be identified with similar efficacy (79-88% sensitivity, 94% specificity), as could metastases (85%). In patients with low CgA, 91% exhibited elevated transcript markers. A panel of 51 marker genes differentiates NENs from controls with a high PPV and NPV (>90%), identifies pancreatic and gastrointestinal NENs with similar efficacy, and confirms GEP-NENs when CgA levels are low. The panel is significantly more accurate than the CgA assay. This reflects its utility to identify multiple diverse biological components of NENs. Application of this sensitive and specific PCR-based blood test to NENs will allow accurate detection of disease, and potentially define disease progress enabling monitoring of treatment efficacy.
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The clinical implications and biologic relevance of neurofilament expression in gastroenteropancreatic neuroendocrine neoplasms.
Cancer
PUBLISHED: 07-08-2011
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Although gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) exhibit widely divergent behavior, limited biologic information (apart from Ki-67) is available to characterize malignancy. Therefore, the identification of alternative biomarkers is a key unmet need. Given the role of internexin alpha (INA) in neuronal development, the authors assessed its function in neuroendocrine cell systems and the clinical implications of its expression as a GEP-NEN biomarker.
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Small intestinal neuroendocrine cell pathobiology: carcinoid tumors.
Curr Opin Oncol
PUBLISHED: 05-17-2011
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Neuroendocrine tumors, particularly small intestinal tumors, also grouped as carcinoids, are defined by an increasing incidence and prevalence, a poor response to current therapies, and confusion regarding appropriate models for drug development. Despite these issues, approximately 350 studies were published in the last year.
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A clinical perspective on gastric neuroendocrine neoplasia.
Curr Gastroenterol Rep
PUBLISHED: 04-22-2011
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The incidence of gastric neuroendocrine tumors (NETs) has increased exponentially based on widespread use of endoscopy and a greater pathological awareness of the condition. A key concern is the potential association with hypergastrinemia induced by proton pump inhibitor administration. Previous confusion regarding diagnosis and therapy has been diminished by a series of international consensus statements defining the biology and management strategies for the disease. Overall, gastric NETs are categorized as well-differentiated or poorly differentiated neoplasms. Well-differentiated gastric NETs are enterochromaffin-like (ECL) cell tumors subclassified into three types based on their relationship to gastrin, a key regulator of ECL cell neoplastic transformation. The treatment of type 1 and type 2 tumors depends on the size and invasiveness of the tumor, whereas type 3 tumors and poorly differentiated neuroendocrine carcinomas warrant aggressive surgical resection. The disease-specific 5-year survival ranges from about 95% in type 1 gastric carcinoids to about 25% in poorly differentiated gastric NECs. Elucidation of the precise biology of a gastric NET is critical to diagnosis and delineation of a type-specific management strategy.
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Gene network inference and biochemical assessment delineates GPCR pathways and CREB targets in small intestinal neuroendocrine neoplasia.
PLoS ONE
PUBLISHED: 02-28-2011
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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.
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The clinical relevance of chromogranin A as a biomarker for gastroenteropancreatic neuroendocrine tumors.
Endocrinol. Metab. Clin. North Am.
PUBLISHED: 02-26-2011
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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.
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The epidemiology of gastroenteropancreatic neuroendocrine tumors.
Endocrinol. Metab. Clin. North Am.
PUBLISHED: 02-26-2011
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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.
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Parietal cell activation by arborization of ECL cell cytoplasmic projections is likely the mechanism for histamine induced secretion of hydrochloric acid.
Scand. J. Gastroenterol.
PUBLISHED: 02-23-2011
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Enterochromaffin-like (ECL) cells are central in the regulation of acid secretion. G cells release gastrin and activate ECL cell histamine secretion which stimulates parietal cell H(2) receptors initiating acid secretion. It is unclear whether histamine-mediated parietal cell activation is via a vascular or paracrine pathway. To assess this, we utilized immunohistochemistry (IHC) and electron microscopy to examine gastric tissue and used visualization of formalin fixed dispersed gastric cells and glands to investigate and define the anatomical relationship between ECL and parietal cells.
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Limitations in small intestinal neuroendocrine tumor therapy by mTor kinase inhibition reflect growth factor-mediated PI3K feedback loop activation via ERK1/2 and AKT.
Cancer
PUBLISHED: 01-31-2011
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Treatment of small intestinal neuroendocrine tumors (SINETs) with mammalian target of rapamycin (mTOR) inhibitors alone or with somatostatin analogs has been proposed as effective therapy, because both agents have been reported to exhibit antiproliferative activity. Because adenocarcinomas escape mTOR inhibition, we examined whether the escape phenomenon occurred in SINETs and whether usage of somatostatin analogs with mTOR inhibitors surmounted loss of inhibition.
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New pharmacologic therapies for gastroenteropancreatic neuroendocrine tumors.
Gastroenterol. Clin. North Am.
PUBLISHED: 10-19-2010
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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.
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The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems.
Pancreas
PUBLISHED: 07-29-2010
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Neuroendocrine tumors (NETs) arise in most organs of the body and share many common pathologic features. However, a variety of different organ-specific systems have been developed for nomenclature, grading, and staging of NETs, causing much confusion. This review examines issues in the pathologic assessment of NETs that are common among primaries of different sites. The various systems of nomenclature are compared along with new proposal for grading and staging NETs. Although differences persist, there are many common themes, such as the distinction of well-differentiated (low and intermediate-grade) from poorly differentiated (high-grade) NETs and the significance of proliferative rate in prognostic assessment. A recently published minimum pathology data set is presented to help standardize the information in pathology reports. Although an ultimate goal of standardizing the pathologic classification of all NETs, irrespective of primary site, remains elusive, an understanding of the common themes among the different current systems will permit easier translation of information relevant to prognosis and treatment.
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A nomogram to assess small-intestinal neuroendocrine tumor (carcinoid) survival.
Neuroendocrinology
PUBLISHED: 06-29-2010
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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.
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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.
Cancer
PUBLISHED: 06-22-2010
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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.
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Neuroendocrine tumors of the gallbladder: an evaluation and reassessment of management strategy.
J. Clin. Gastroenterol.
PUBLISHED: 04-09-2010
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Gallbladder neuroendocrine tumors (GB-NETs) represent only 0.5% of all NETs, and little is known about their biological behavior. We sought to provide an overview of the current state of knowledge about GB-NETs and provide a recommendation for management.
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Chromogranin A--biological function and clinical utility in neuro endocrine tumor disease.
Ann. Surg. Oncol.
PUBLISHED: 03-09-2010
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Neuroendocrine tumors (NETs) are a form of cancer that differ from other neoplasia in that they synthesize, store, and secrete peptides, e.g., chromogranin A (CgA) and amines. A critical issue is late diagnosis due to failure to identify symptoms or to establish the biochemical diagnosis. We review here the utility of CgA measurement in NETs and describe its biological role and the clinical value of its measurement.
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Gastrointestinal neuroendocrine (carcinoid) tumours: current diagnosis and management.
Med. J. Aust.
PUBLISHED: 02-11-2010
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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.
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Pathology reporting of neuroendocrine tumors: application of the Delphic consensus process to the development of a minimum pathology data set.
Am. J. Surg. Pathol.
PUBLISHED: 02-02-2010
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Epithelial neuroendocrine tumors (NETs) have been the subject of much debate regarding their optimal classification. Although multiple systems of nomenclature, grading, and staging have been proposed, none has achieved universal acceptance. To help define the underlying common features of these classification systems and to identify the minimal pathology data that should be reported to ensure consistent clinical management and reproducibility of data from therapeutic trials, a multidisciplinary team of physicians interested in NETs was assembled. At a group meeting, the participants discussed a series of "yes" or "no" questions related to the pathology of NETs and the minimal data to be included in the reports. After discussion, anonymous votes were taken, using the Delphic principle that 80% agreement on a vote of either yes or no would define a consensus. Questions that failed to achieve a consensus were rephrased once or twice and discussed, and additional votes were taken. Of 108 questions, 91 were answerable either yes or no by more than 80% of the participants. There was agreement about the importance of proliferation rate for tumor grading, the landmarks to use for staging, the prognostic factors assessable by routine histology that should be reported, the potential for tumors to progress biologically with metastasis, and the current status of advanced immunohistochemical and molecular testing for treatment-related biomarkers. The lack of utility of a variety of immunohistochemical stains and pathologic findings was also agreed upon. A consensus could not be reached for the remaining 17 questions, which included both minor points related to extent of disease assessment and some major areas such as terminology, routine immunohistochemical staining for general neuroendocrine markers, use of Ki67 staining to assess proliferation, and the relationship of tumor grade to degree of differentiation. On the basis of the results of the Delphic voting, a minimum pathology data set was developed. Although there remains disagreement among experts about the specific classification system that should be used, there is agreement about the fundamental pathology data that should be reported. Examination of the areas of disagreement reveals significant opportunities for collaborative study to resolve unanswered questions.
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Management of advanced neuroendocrine tumors with hepatic metastasis.
J. Clin. Gastroenterol.
PUBLISHED: 08-06-2009
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Neuroendocrine tumors (NETs) in general and specifically these gastroenteropancreatic-neuroendocrine tumors often present a considerable diagnostic and therapeutic challenge, especially those that behave in an aggressive fashion. The majority of tumors are diagnosed at a stage that the only curative treatment, radical surgical intervention, is no longer an option and thus long-term therapy with somatostatin analogs is focused on symptom amelioration and in the improvement of quality of life. Although biotherapy is currently the most efficient treatment to achieve palliation, conventional chemotherapy 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 or radiofrequency ablation. If surgery is not feasible, embolization either alone (bland), in combination with chemotherapeutic agents, or using radioactive microspheres can be used. Peptide receptor targeted radiotherapy using radiolabeled octapeptide analogs (Yttrium or Lutetium-octreotide) 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 our expectations.
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Autoregulatory effects of serotonin on proliferation and signaling pathways in lung and small intestine neuroendocrine tumor cell lines.
Cancer
PUBLISHED: 07-28-2009
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: Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not altered significantly (5-year survival rate: GI NETs, 64.1%; BP NETs, 87%-89%) in 30 years (from 1973 to 2004). No effective or specific antineoplastic agents are available to date, although somatostatin analogs inhibit NET 5-hydroxytryptophan (5-HT) secretion. Given the expression of 5-HT receptors on NETs, the authors hypothesized that 5-HT autoregulated NET proliferation.
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Principal component analysis, hierarchical clustering, and decision tree assessment of plasma mRNA and hormone levels as an early detection strategy for small intestinal neuroendocrine (carcinoid) tumors.
Ann. Surg. Oncol.
PUBLISHED: 05-01-2009
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Incidence of neuroendocrine tumors (NETs) is increasing (approximately 6%/year), but clinical presentation is nonspecific, resulting in delays in diagnosis (5-7 years; approximately 70% have metastases). This reflects absence of a sensitive plasma marker. The aim of this study is to investigate whether detection of circulating messenger RNA (mRNA) alone or in combination with circulating NET-related hormones and growth factors can detect gastrointestinal NET disease. The small intestinal (SI) NET cell line KRJ-I was used to define the sensitivity of real-time polymerase chain reaction (PCR) for mRNA detection in blood. NSE, Tph-1, and VMAT ( 2 ) transcripts were identified from one KRJ-I cell/ml blood. mRNA from the tissue and plasma of SI-NETs (n = 12) and gastric NETs (n = 7), and plasma from healthy controls (n = 9) was isolated and real-time PCR performed. Tph-1 was a specific marker of SI-NETs (58%, p < 0.03) whereas CgA transcripts did not differentiate tumors from controls. Patients with metastatic disease expressed more marker transcripts than localized tumors (75% versus 18%, p < 0.02). Plasma 5-hydroxytryptamine (5-HT), chromogranin A (CgA), ghrelin, and connective tissue growth factor (CTGF) fragments were measured, combined with mRNA levels, and a predictive mathematical model for NET diagnosis developed using decision trees. The sensitivity and specificity to diagnose SI-NETs and gastric NETs were 81.2% and 100%, and 71.4% and 55.6%, respectively. We conclude that mRNA from one NET cell/ml blood can be detected. Circulating plasma Tph-1 is a promising marker gene for SI-NET disease (specificity 100%) while an increased number of marker transcripts (>2) correlated with disease spread. Including NET-related circulating hormones and growth factors in the algorithm increased the sensitivity of detection of SI-NETs from 58 to 82%.
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Acute pancreatitis: a historical perspective.
Pancreas
PUBLISHED: 04-25-2009
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Among the various pancreatic disorders, the pace of scientific discovery in acute pancreatitis has been particularly slow. The objective of this paper was to briefly review the history of scientific discovery of the clinical features, pathophysiology, and treatment of acute pancreatitis. A clinical description of acute pancreatitis was first presented in 1652 by the Dutch anatomist Nicholas Tulp, and despite the nearly 350 years that have passed, there continue to be many unanswered questions. In the late 19th and early 20th century, Reginald Fitz, Nicholas Senn, Eugene Opie, and others made seminal contributions that continue to influence our present understanding of acute pancreatitis. Despite remarkable progress in the past 6 decades, our ability to accurately diagnose and estimate the severity of acute pancreatitis remains limited. History provides multiple examples of empiric remedies and surgical interventions based on the prevailing theories and opinions of the scientific luminaries du jour, and indeed, after 3 centuries of inquiry, the most effective interventions for acute pancreatitis are purely supportive in nature and not specific to the pancreas. However, the perseverance of successive generations of the finest scientific minds provides hope that we will unravel the many mysteries of this enigmatic gland.
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KRJ-I and BON cell lines: defining an appropriate enterochromaffin cell neuroendocrine tumor model.
Neuroendocrinology
PUBLISHED: 03-18-2009
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Neuroendocrine tumors (NETs) of the gastrointestinal (GI) system are increasing in incidence with minimal improvement in prognosis. Although the cell of origin has been identified as the enterochromaffin (EC) cell, its secretory and proliferative regulation has not been defined at a mechanistic level. To date, the BON cell line has been the most widely used in vitro EC cell model despite its pancreatic origin. Using whole-genome mathematical analysis as well as secretory and proliferative studies, we compared the BON cell line to the small intestine (SI) EC cell-derived NET cell line, KRJ-I, to assess individual cell line validity and applicability for the investigation of GI-NET disease.
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From Leningrad to London: the saga of Kulchitsky and the legacy of the enterochromaffin cell.
Neuroendocrinology
PUBLISHED: 03-17-2009
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By the end of the 19th century, the subject of internal secretion and the consequences of its perturbations had been explored in considerable depth but with little clear understanding. Despite the anatomic delineation of the majority of the glands and tissues that comprised the gross endocrine system, the cellular basis and the interactions between the internal glands and the nervous system had not been clearly delineated. Prominent early investigators in the field included Rudolf Peter Heidenhain (1834-1897), who described a novel class of clear cells (1868), Paul Langerhans (1847-1888), who identified pancreatic islets in 1869, and M.C. Ciacco (1877-1956), who coined the term enterochromaffin (1906). Their contributions facilitated the description of the diffuse neuroendocrine system (DNES) by F. Feyrter (1938) which allowed for the understanding of a syncytial regulatory system that consisted of both endocrine and neural components. This rich developmental history often reveals the name of Kulchitsky, but little recognition has been given to his seminal contributions. Indeed the Russian, Nikolai Konstantinovich Kulchitsky (1856-1925), both due to his modest and unassuming nature and the tragic events of his life, was little recognized and has been relegated to a mere eponymous attribution. In reality, his life bears legacy to rich scientific contributions spanning a great teaching and scientific career at Kharkov University, to responsibilities as the Imperial Minister of Education for all of Russia. He identified the Kulchitsky cell, trained and mentored numerous professors of histopathology, was incarcerated by the Bolsheviks and worked in a soap factory to save his life. He and his family finally fled on a British battleship with the remnants of the Russian Royal family to England where he secured a position with Bayliss and Starling at University College, London (UCL). His mysterious demise in a lift-shaft accident on his 69th birthday tragically terminated a life of great service to science and teaching. He excelled as a histopathologist and was responsible for the early description of tonsillar and gut epithelial leucocytes as well as defining components of the Ascaris life cycle. At UCL, his contributions to the anatomic delineation of muscle nerve endings were highly regarded and widely admired. It is, however, his identification of the enterochromaffin cell in 1897 for which he is most remembered since this observation formed the basis for the subsequent delineation of the DNES and provided the cellular framework on which the discipline of gut neuroendocrinology would be established.
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Predicting neuroendocrine tumor (carcinoid) neoplasia using gene expression profiling and supervised machine learning.
Cancer
PUBLISHED: 02-07-2009
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A more accurate taxonomy of small intestinal (SI) neuroendocrine tumors (NETs) is necessary to accurately predict tumor behavior and prognosis and to define therapeutic strategy. In this study, the authors identified a panel of such markers that have been implicated in tumorigenicity, metastasis, and hormone production and hypothesized that transcript levels of the genes melanoma antigen family D2 (MAGE-D2), metastasis-associated 1 (MTA1), nucleosome assembly protein 1-like (NAP1L1), Ki-67 (a marker of proliferation), survivin, frizzled homolog 7 (FZD7), the Kiss1 metastasis suppressor (Kiss1), neuropilin 2 (NRP2), and chromogranin A (CgA) could be used to define primary SI NETs and to predict the development of metastases.
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Nikolai Konstantinovich Kulchitsky (1856-1925).
J Med Biogr
PUBLISHED: 02-05-2009
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Nikolai Kulchitsky is best remembered for his identification of the Kulchitsky (enterochromaffin) cell. His life spanned a teaching and scientific career at Kharkov University, employment as the Imperial Minister of Education for all Russia, work in a soap factory and flight from the Russian Revolution to London, and a position at the University College with Elliot Smith. His subsequent contributions to the anatomic delineation of dual nerve-endings in the muscle were highly regarded, although his identification of the enterochromaffin cell (1897) remains his enduring scientific legacy. The observation of a cardinal neuroendocrine cell of the gut formed the basis for the subsequent delineation of the diffuse neuroendocrine system and provided the cellular framework on which the discipline of gut neuroendocrinology would be established. Kulchitskys mysterious demise in a bizarre lift-shaft accident at UCL on his 69th birthday tragically terminated a life of service to science.
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Delineation of the chemomechanosensory regulation of gastrin secretion using pure rodent G cells.
Gastroenterology
PUBLISHED: 01-08-2009
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Gastrin is a key regulator of gastric acid secretion. We aimed to isolate pure G cells to identify the mechanistic basis of luminal- and strain-mediated regulation.
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Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods.
Am. J. Surg. Pathol.
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Pathologic grading for prognostic stratification of neuroendocrine tumors (NETs) is critical but presents a challenging interpretive dilemma. Tumor cell proliferative rate is an important factor in the determination of prognosis, and immunohistochemical analysis with Ki67 is becoming more widely used to quantify the proliferative rate. However, Ki67 assessment has limitations due to lack of uniformity and consistency in quantification. These limitations are accentuated in well-differentiated NETs, as differences in the range of 1% to 5% can alter tumor grade, with potential implications for treatment. We therefore performed a concordance study to assess different Ki67 quantification techniques including: (a) digital image analysis (DIA); (b) manual counting (MC) of >2000 cells; and (c) "eyeballed" estimate (EE) of labeling percentage by pathologists (n=18), including individuals experienced in evaluating Ki67 labeling as well as others who had little prior experience assessing Ki67 percentages. Forty-five Ki67 images were selected and analyzed using the 3 methods. On the basis of the recommendations of the World health Organization (WHO) for grading NETs, MC of 2000 cells was used as the "gold standard" reference against which the other techniques were compared. Three images were presented twice, the second being inverted, to assess intraobserver consistency. Statistical analyses were performed to evaluate: (a) the concordance between methods; (b) intraobserver and interobserver consistency; and (c) correlation of NET grades on the basis of Ki67 scores by EE versus the gold standard. Agreement between scores was assessed by intraclass correlation (ICC). DIA and MC were highly concordant (ICC=0.98). The ICC between DIA and the mean EE of all observers was 0.88. However, there was discordance among individual observers on all cases quantified by EE (ICC=0.13). The ICC for intraobserver consistency was 0.39±0.26. With Ki67 in the ranges of <1%, 2% to 3%, and >20%, the mean of Ki67 by EE was, respectively, 93%±2%, 55%±7%, and 55%±15% correct against the gold standard. The ? statistics for EE exhibited low agreement (?=0.24; 95% confidence interval, 0.23-0.25) for all WHO NET grades. Incorrect assessment by EE resulted in upgrading of all WHO G1 group tumors (n=14); in the WHO G2 group, downgrading of 41% cases occurred (n=11) when Ki67 was <5% (by DIA or MC), and upgrading of 59% cases occurred (n=16) when Ki67 was >5%. We conclude that DIA and MC are the acceptable standards for Ki67 assessment. Given the inherent discordance in determining the grade, the use of an approximate EE of the Ki67-labeling index requires critical reevaluation, especially for NETs with a labeling index straddling the cut-points between grades. Consequently, determination of therapeutic strategies should be guided by an amalgamation of clinicopathologic characteristics, including but not limited to the Ki67 index.
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Differential signal pathway activation and 5-HT function: the role of gut enterochromaffin cells as oxygen sensors.
Am. J. Physiol. Gastrointest. Liver Physiol.
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The chemomechanosensory function of the gut enterochromaffin (EC) cell enables it to respond to dietary agents and mechanical stretch. We hypothesized that the EC cell, which also sensed alterations in luminal or mucosal oxygen level, was physiologically sensitive to fluctuations in O(2). Given that low oxygen levels induce 5-HT production and secretion through a hypoxia inducible factor 1? (HIF-1?)-dependent pathway, we also hypothesized that increasing O(2) would reduce 5-HT production and secretion. Isolated normal EC cells as well as the well-characterized EC cell model KRJ-I were used to examine HIF signaling (luciferase-assays), hypoxia transcriptional response element (HRE)-mediated transcription (PCR), signaling pathways (Western blot), and 5-HT release (ELISA) during exposure to different oxygen levels. Normal EC cells and KRJ-I cells express HIF-1?, and transient transfection with Renilla luciferase under HRE control identified a hypoxia-mediated pathway in these cells. PCR confirmed activation of HIF-downstream targets, GLUT1, IGF2, and VEGF under reduced O(2) levels (0.5%). Reducing O(2) also elevated 5-HT secretion (2-3.2-fold) as well as protein levels of HIF-1? (1.7-3-fold). Increasing O(2) to 100% inhibited HRE-mediated signaling, transcription, reduced 5-HT secretion, and significantly lowered HIF-1? levels (?75% of control). NF-?B signaling was also elevated during hypoxia (1.2-1.6-fold), but no significant changes were noted in PKA/cAMP. We concluded that gut EC cells are oxygen responsive, and alterations in O(2) levels differentially activate HIF-1? and tryptophan hydroxylase 1, as well as NF-?B signaling. This results in alterations in 5-HT production and secretion and identifies that the chemomechanosensory role of EC cells extends to oxygen sensing.
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Comparison of PCR-based detection of chromogranin A mRNA with traditional histological lymph node staging of small intestinal neuroendocrine neoplasia.
BMC Res Notes
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Accurate neuroendocrine neoplasia (NEN) staging is vital for determining prognosis and therapeutic strategy. The great majority of NENs express chromogranin A (CgA) which can be detected at a protein or transcript level. The current standards for lymph node metastasis detection are histological examination after Hematoxylin and Eosin (H&E) and CgA immunohistochemical (IHC) staining. We hypothesized that detection of CgA mRNA transcripts would be a more sensitive method of detecting these metastases.
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Ultrastructure of ECL cells in Mastomys after long-term treatment with H2 receptor antagonist loxtidine.
Med Mol Morphol
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Gastric ECL-cell hyperplasia and carcinoids (ECLoma) develop after 1 year in rats treated with omeprazole or 2 months in Mastomys treated with loxtidine. The aim of this study was to examine the ultrastructure of ECL cells in Mastomys after loxtidine treatment with an attempt to evaluate whether an impairment of autophagy was involved in the tumorigenesis. Mastomys were given loxtidine for 8 or 27 weeks. Morphological analysis of ECL cells showed that (1) cell size was not increased after 8 or 27 weeks; (2) secretory vesicles, a hallmark feature of welldifferentiated ECL cells, were unchanged after 8 weeks but reduced after 27 weeks; (3) granules were reduced after 8 or 27 weeks; (4) microvesicles were unchanged after the treatment; and (5) vacuoles and lipofuscin bodies were found occasionally after 8 weeks but not at 27 weeks. In addition, the appearance of ECL-cell ultrastructure differed between loxtidine-treated Mastomys and rats treated with omeprazole or subjected to antrectomy, but was similar between Mastomys treated with loxtidine for 27 weeks and mice deficient in CCK(2) receptor. We suggest that the ultrastructure of ECL cells in Mastomys after long-term treatment with loxtidine displayed an impaired formation of vacuoles and lipofuscin bodies, markers of the autophagic pathway.
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Neuroendocrine tumor disease: an evolving landscape.
Endocr. Relat. Cancer
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Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) represent a heterogenous group of tumors arising from a variety of neuroendocrine cell types. The incidence and prevalence of GEP-NENs have markedly increased over the last three decades. Symptoms are often absent in early disease, or vague and nonspecific even in advanced disease. Delayed diagnosis is thus common. Chromogranin A is the most commonly used biomarker but has limitations as does the proliferative marker Ki-67%, which is often used for tumor grading and determination of therapy. The development of a multidimensional prognostic nomogram may be valuable in predicting tumor behavior and guiding therapy but requires validation. Identification of NENs that express somatostatin receptors (SSTR) allows for SSTR scintigraphy and positron emission tomography imaging using novel radiolabeled compounds. Complete surgical resection of limited disease or endoscopic ablation of small lesions localized in stomach or rectum can provide cure; however, the majority of GEP-NENs are metastatic (most frequently the liver and/or mesenteric lymph nodes) at diagnosis. Selected patients with metastatic disease may benefit from advanced surgical techniques including hepatic resection or liver transplantation. Somatostatin analogs are effective for symptomatic treatment and exhibit some degree of antiproliferative activity in small intestinal NENs. There is a place for streptozotocin, temozolomide, and capecitabine in the management of pancreatic NENs, while new agents targeting either mTOR (everolimus) or angiogenic (sunitinib) pathways have shown efficacy in these lesions.
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Microencapsulation of small intestinal neuroendocrine neoplasm cells for tumor model studies.
Cancer Sci.
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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 ?m. 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.
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Gut neuroendocrine tumor blood qPCR fingerprint assay: characteristics and reproducibility.
Clin. Chem. Lab. Med.
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Abstract Background: We have developed a PCR-based tool that measures a 51-gene panel for identification of gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) in peripheral blood. This manuscript assesses the robustness (performance metrics) of this tool with a specific focus on the effects of individual parameters including collection, storage, acid suppressive medication [proton pump inhibitor (PPI)], age, sex, race and food on accuracy. Methods: Performance metrics were evaluated using a gold standard (mRNA derived from three individual human neuroendocrine tumor cell lines) and clinical samples using qPCR. Results: One hundred percent of the 51 transcripts were amplified in the gold standard (NEN cell line-derived mRNA) (CQ<35, average efficiency 1.94). The inter- and intra-assay variations were 1%-2%. In clinical samples, 50 of 51 targets (98%) were amplified. The inter- and intra-assay reproducibility ranged between 0.4% and 1.2%. The coefficient of variation (CV) was 5.3%. Expression of the reference gene, ALG9, was robust [low variation, low M-value, high (99.5%) PCR efficiency] and unaffected by sample processing. Test meals, long-term PPI use (>1 year), age, sex and ethnicity had no effect on the signature. Expression of two genes, ALP2 and CD59 correlated strongly with RNA integrity (R=0.72, p<0.001) and could be used to assess storage and processing. Conclusions: The 51 marker gene signature was robust and reproducible, exhibiting acceptable inter- and intra-assay metrics (<5%). Feeding, PPI intake, age, sex and ethnicity do not affect the signature. Expression levels of APLP2 and CD59 are effective surrogate markers of proper sample collection and processing.
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