Ignat Drozdov

Neuroendocrine Tumor Epidemiology : Contrasting Norway and North America

The National Cancer Institutes Surveillance, Epidemiology, and End Results (SEER) program has proven to be a significant resource in US neuroendocrine tumor (NET) epidemiology. Norway also holds a robust and detailed cancer registry: the Norwegian Registry of Cancer (NRC).

Long-term tolerability of PRRT in 807 patients with neuroendocrine tumours: the value and limitations of clinical factors

PurposePeptide receptor radionuclide therapy (PRRT) with 90Y and 177Lu 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).MethodsOf 807 patients studied at IEO-Milan (1997–2013), 793 (98xa0%) received 177Lu (278, 34.4xa0%), 90Y (358, 44.4xa0%) or 177Lu and 90Y combined (157. 19.5xa0%), and 14 (2xa0%) received combinations of PRRT and other agents. Follow-up was 30xa0months (1–180xa0months). The parameters evaluated included renal risk factors, bone marrow toxicity and PRRT features. Data analysis included multiple regression, random forest feature selection, and recursive partitioning and regression trees.ResultsTreatment with 90Y and 90Yu2009+u2009177Lu was more likely to result in nephrotoxicity than treatment with 177Lu alone (33.6xa0%, 25.5xa0% and 13.4xa0% of patients, respectively; pu2009<u20090.0001). Nephrotoxicity (any grade), transient and persistent, occurred in 279 patients (34.6xa0%) and was severe (grade 3u2009+u20094) in 12 (1.5xa0%). In only 20–27xa0% of any nephrotoxicity was the disease modelled by risk factors and codependent associations (pu2009<u20090.0001). Hypertension and haemoglobin toxicity were the most relevant factors. Persistent toxicity occurred in 197 patients (24.3xa0%). In only 22–34xa0% of affected patients was the disease modelled by the clinical data (pu2009<u20090.0001). Hypertension (regression coefficient 0.14, pu2009<u20090.0001) and haemoglobin toxicity (regression coefficient 0.21, pu2009<u20090.0001) were pertinent factors. Persistent toxicity was associated with shorter PRRT duration from the first to the last cycle (mean 387 vs. 658xa0days, pu2009<u20090.004). Myelodysplastic syndrome occurred in 2.35xa0% of patients (modelled by the clinical data in 30xa0%, pu2009<u20090.0001). Platelet toxicity grade (2.05u2009±u20091.2 vs. 0.58u2009±u20090.8, pu2009<u20090.0001) and longer PRRT duration (22.6u2009±u200924 vs. 15.5u2009±u20099xa0months, pu2009=u20090.01) were relevant. Acute leukaemia occurred in 1.1xa0% of patients (modelled by the clinical data in 18xa0%, pu2009<u20090.0001).ConclusionIdentified risk factors provide a limited (<30xa0%) risk estimate even with target tissue dosimetry. These data strongly suggest the existence of unidentified individual susceptibilities to radiation-associated disease.

Carcinoid heart disease

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

IL1β‐ and LPS‐induced serotonin secretion is increased in EC cells derived from Crohn’s disease

Abstractu2002 Gut mucosal enterochromaffin (EC) cells are regarded as key regulators of intestinal motility and fluid secretion via secretion of serotonin (5HT), are increased in numbers in mucosal inflammation and located in close proximity to immune cells. We examined whether interleukin (IL)1β and Escherichia coli lipopolysaccharide (LPS) induced EC cell 5HT release through Toll‐like/IL‐1 (TIL) receptor activation, nuclear factor kappa B (NFκB) and mitogen‐activated protein kinase (MAPK) phosphorylation and evaluated whether somatostatin could inhibit this phenomenon. Pure (>98%) human intestinal EC cells were isolated by fluorescent activated cell sorting from preparations of normal (nu2003=u20035) and Crohn’s colitis (nu2003=u20036) mucosa. 5HT release was measured (ELISA), and NFκB and ERK phosphorylation quantitated (ELISA) in response to IL1β and LPS. 5HT secretion was increased by both E. coli LPS (EC50u2003=u20035u2003ngu2003mL−1) and IL1β (EC50u2003=u20030.05u2003pmolu2003L−1) >2‐fold (Pu2003<u20030.05) in Crohn’s EC cells compared with normal EC cells. Secretion was reversible by the TLR4 antagonist, E. coli K12 LPS (IC50u2003=u200312u2003ngu2003mL−1) and the IL1β receptor antagonist (ILRA; IC50u2003=u20033.4u2003ngu2003mL−1). IL1β caused significant (Pu2003<u20030.05) NFκB and MAPK phosphorylation (40–55%). The somatostatin analogue, lanreotide inhibited IL1β‐stimulated secretion in Crohn’s (IC50u2003=u20030.61u2003nmolu2003L−1) and normal EC cells (IC50u2003=u20031.8u2003nmolu2003L−1). Interleukins (IL1β) and bacterial products (E. coli LPS) stimulated 5HT secretion from Crohn’s EC cells via TIL receptor activation (TLR4 and IL1β). Immune‐mediated alterations in EC cell secretion of 5HT may represent a component of the pathogenesis of abnormal bowel function in Crohn’s disease. Inhibition of EC cell‐mediated 5HT secretion may be an alternative therapeutic strategy in the amelioration of inflammatory bowel disease symptomatology.

Differential cytotoxicity of novel somatostatin and dopamine chimeric compounds on bronchopulmonary and small intestinal neuroendocrine tumor cell lines

Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not significantly altered (overall 67%, 5‐year survival) in 30 years (1973‐2004), whereas the incidence has increased (∼ 1000%) in the same time frame. No effective or specific antineoplastic agent is available for treatment, although somatostatin analogs inhibit tumor secretion. Given the coexistence of somatostatin and dopamine regulatory receptors on NET cells, the antiproliferative efficacy as well as the signaling and transcriptional targets of their ligands were evaluated.

Pharmacotherapy of neuroendocrine cancers

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

Predicting neuroendocrine tumor (carcinoid) neoplasia using gene expression profiling and supervised machine learning

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.

The CCK2 receptor antagonist, YF476, inhibits Mastomys ECL cell hyperplasia and gastric carcinoid tumor development

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

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

Incidence of neuroendocrine tumors (NETs) is increasing (approximately 6%/year), but clinical presentation is nonspecific, resulting in delays in diagnosis (5–7xa0years; 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 VMAT2 transcripts were identified from one KRJ-I cell/ml blood. mRNA from the tissue and plasma of SI-NETs (nxa0=xa012) and gastric NETs (nxa0=xa07), and plasma from healthy controls (nxa0=xa09) was isolated and real-time PCR performed. Tph-1 was a specific marker of SI-NETs (58%, pxa0<xa00.03) whereas CgA transcripts did not differentiate tumors from controls. Patients with metastatic disease expressed more marker transcripts than localized tumors (75% versus 18%, pxa0<xa00.02). 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%.

KRJ-I and BON Cell Lines: Defining an Appropriate Enterochromaffin Cell Neuroendocrine Tumor Model

Background: 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. Methods and Results: Principal component analysis and ANOVA of KRJ-I and BON transcriptomes (U133 Plus 2) identified substantially different (<10%) overlap in transcripts with minimal (R2 = 0.24) correlation in gene expression profiles. RT-PCR detected large variability (>12%) in neuroendocrine (NE) marker transcripts in the BON cell line and the absence of Tph-2, DDC, TGFβR2, and M3 transcripts in KRJ-I. The KRJ-I cell line secreted serotonin (5-HT) in response to isoproterenol (EC50 = 100 nM), noradrenaline (EC50 = 1.7 nM), and pituitary adenylate cyclase (PACAP, EC50 = 0.03 nM). Cholecystokinin (IC50 = 430 nM), somatostatin (IC50 = 400 nM), acetylcholine (IC50 = 3.7 nM), and γ-aminobutyric acid A (GABAA, IC50 = 2 nM) all inhibited 5-HT release, while gastrin and bombesin had no effect. 5-HT secretion in the BON cell line was stimulated by isoproterenol (EC50 = 900 nM), noradrenaline (EC50 = 20 nM), cholecystokinin (EC50 = 130 nM), PACAP (EC50 = 0.12 nM), bombesin (EC50 = 15 nM), and acetylcholine (EC50 = 0.2 nM). It was inhibited by somatostatin (IC50 = 300 nM) but not GABAA. KRJ-I responded with proliferation to connective tissue growth factor (CTGF, EC50 = 0.002 ng/ml), transforming growth factor-α (TGFα, EC50 = 0.63 ng/ml) and transforming growth factor-β (TGFβ, EC50 = 0.63 ng/ml). Epidermal growth factor (EGF) and somatostatin had no significant effect. BON cell proliferation was stimulated only by EGF and TGFα (EC50 = 15.8 and 10 ng/ml). TGFβ (IC50 = 0.16 ng/ml), MZ-4-147 (IC50 = 0.5 nM), and BIM23A761 (IC50 = 0.06 nM) all inhibited proliferation. CTGF and somatostatin had no effect. Conclusion: KRJ-I and BON cell lines demonstrate substantial differences in gene level transcripts, inconsistent receptor profile expression, wide variability in NE marker transcript levels, and significantly differential proliferative and secretory responses. Given the EC cell origin of KRJ-I, these results provide evidence that the BON cell line does not represent an EC cell system and is not a valid study model of (carcinoid) EC cell-derived NET.