Resource Library: Neuroendocrine Tumor Clinical Press

Predicting Neuroendocrine Tumor (Carcinoid) Neoplasia Using Gene Expression Profiling and Supervised Machine Learning

Drozdov I, Kidd M, Nadler B, Camp R, Mane M, Gustafsson BI, Hauso O, Modlin IM.

A more accurate taxonomy of small intestinal (SI) neuroendocrine tumors (NETs) is necessary to accurately predict tumor behavior, prognosis and define therapeutic strategy. We identified a panel of such markers implicated in tumorogenicity, metastasis, and hormone production and hypothesized that transcript levels of MAGE-D2, MTA1, NAP1L1, Ki-67, Survivin, FZD7, Kiss1, NRP2, and CgA could be used to define primary SI NETs and predict the development of metastases.

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Gene Network Inference and Biochemical Assessment Delineates GPCR Pathways and CREB Targets in Small Intestinal Neuroendocrine Neoplasia

Drozdov I, Kidd M, Gustafsson BI, Mane S, Hauso O, Pfragner R, Modlin IM.

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. 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 Neuroendocrine Tumor (NET) cell system, confirmed that transcriptional effects are signaled through the cAMP/PKA/pCREB signaling pathway and that a SI Neuroendocrine Tumor (NET) cell line was most sensitive to a D2 and 5-HT2 receptor agonist BIM-53061.

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

Siddique Z-L, Drozdov I, Floch J, Gustafsson BI, Stunes K, Pfragner R, Kidd M, Modlin IM.

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 Neuroendocrine Tumor (NET) cell line, KRJ-I, to assess individual cell line validity and applicability for the investigation of GI-Neuroendocrine Tumor (NET) disease. Our results demonstrate that 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.

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Comparison of PCR-based Detection of Chromogranin A mRNA with Traditional Histological Lymph Node Staging of Small Intestinal Neuroendocrine Neoplasia

Lawrence B, Kenney B, Svejda B, Schimmack S, Alaimo D, Barbieri A, Jedrych J, Kidd M, Modlin IM.

Background: 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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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

Modlin IM, Gustafsson BI, Drozdov I, Kidd M.

We sought to investigate whether detection of circulating messenger RNA (mRNA) alone or in combination with circulating NET-related hormones and growth factors could detect gastrointestinal Neuroendocrine Tumor (NET) disease. The small intestinal (SI) Neuroendocrine Tumor (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. 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 Neuroendocrine Tumor (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 Neuroendocrine Tumor (NET) cell/ ml blood can be detected. Circulating plasma Tph-1 is a promising marker gene for SI-Neuroendocrine Tumor (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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Serotonin and the 5-HT7 Receptor: The Link Between Hepatocytes, IGF-1 and Small Intestinal Neuroendocrine Tumors

Svejda B, Kidd M, Timberlake A, Lawrence B, Harry K, Alaimo D, Kazberouk A, Schimmack S, Modlin IM.

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 tested whether 5-HT produced by metastatic EC cells played a role in the hepatic tumor-microenvironment through 5-HT7 receptor-mediated activation of hepatocyte IGF-1 synthesis and secretion. Our results demonstrated evidence for a functional 5-HT7 receptor in the liver and identified that IGF-1 and 5-HT were elevated in peri-tumoral hepatic tissue in nude mice, while 5-HT7 was increased fourfold compared to sham-operated animals. We conclude that hepatocytes express a cAMP-coupled 5-HT7 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-HT7 in the hepatic metastatic tumor microenvironment suggests the potential for novel therapeutic strategies for amine-producing mid-gut tumors

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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

Svejda B, Kidd M, Kazberouk A, Lawrence B, Pfragner R, Modlin IM.

BACKGROUND: 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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A Principal Component Analysis of gene expression from primary small intestinal NETs and their meetastases compared to normal neuroendocrine cells. This analysis demonstrates that normal, tumor and metastases profiles can be distinguished by the expressions of selected marker genes. This has enabled the development of molecular testing for tumor cells.


The cAMP-signal pathway network in small intestinal NETs (A) and identification of upstream (B) and downstream (C) gene targets. This analysis identifies which genes are activated when a neuroendocrine tumor cell responds to growth signals.


Transcriptome analysis of two common models of NETs - the human small intestinal cell line (KRJ-I-green) and a pancreatic cell line (BON-red). This investigation identified no genes to be commonly unregulated and that the two cell lines were completely different. The BON cell line is therefore not an appropriate experimental NET model.


Comparison of the sensitivity of standard histological approaches (histology - A, stained with chromogranin antibody - B) and PCR for the identification of metastases in lymph nodes. The PCR approach (C) is significantly more sensitive and identifies more positive lymph nodes..


Principal component analysis (A) and hierarchical clustering (B) of blood samples demonstrates that a combination of genes and hormones can be used to differentiate healthy controls from small intestinal NETs. These analyses indicate that the presence or absence of mRNA is adequate to establish distinct regulatory signatures for healthy controls and circulating tumor cells.


The functional regulation of hepatocytes occurs via serotonin (5-HT) activation of 5-HT7 cell surface receptors. This receptor subtype regulates hepatocyte secretion of growth factors. Since serotonin is one of the principal products of intestinal metastases, it is axiomatic that it can regulate hepatocytes and thus self-modify the growth environment of liver metastases.


Schematic of the mTOR pathway in NETs (A). This demonstrates (B) inhibition of the pathway that activates an escape signal (activation of the ERK pathway that results in compensatory growth). These pathways are highly activated in NETs (C, D) providing a basis for understanding the substantial limitations of the mTOR class of drugs.