Caring for Carcinoid Foundation Multi-Institutional Bioconsortium

James C. Yao, MD
Associate Professor and Deputy Chairman
Gastrointestinal Medical Oncology
University of Texas M. D. Anderson Cancer Center

The Caring for Carcinoid Foundation Multi-Institutional Bioconsortium represents an important effort in advancing our understanding of neuroendocrine tumors. Our efforts in recent years have shown that there are significant heterogeneity in neuroendocrine tumors of various sites in terms of epidemiology, genetics, and biologic behavior.1,2 It is likely that the development of neuroendocrine tumors is a result of complex interactions between genetics and environmental factors. While the tools available to scientists have advanced over the past decade, they actually present increasingly complex computational problems in order to understand the data.

There are more then 1.8 million known subtle variations (Single Nucleotide Polymorphisms) in the human genome that makes each of us different but may also predispose some of us to certain diseases. There are often complex interactions between these genetic variations and environmental factors which ultimately determine who will get the disease. For example, not everyone who is exposed to a given carcinogen will develop cancer. Similarly, individuals with similar genetic makeup may not get the same disease if they have different environmental exposures. Today’s high throughput techniques allow us to ask over 1 million questions from a single sample about these genetic variations. Thus, if we looked at these variations in a small number of patients, we would have very little power to detect subtle differences that may predispose patients to develop neuroendocrine tumors.

This means we must work together to have the best chance for success. Member institutions of the bioconsortium will join forces to collect the much needed data and biospecimens to make this meaningful research possible. Our goal would be to work together to collect clinical, epidemiological, and risk factor data that will enable large scale collaborative research.

1. Yao JC, Hassan M, Phan A, et al: One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States. J Clin Oncol 26:3063-72, 2008
2. Kim do H, Nagano Y, Choi IS, et al: Allelic alterations in well-differentiated neuroendocrine tumors (carcinoid tumors) identified by genome-wide single nucleotide polymorphism analysis and comparison with pancreatic endocrine tumors. Genes Chromosomes Cancer 47:84-92, 2008

What’s New in the Treatment of Carcinoid/Neuroendocrine Tumors with Somatostatin Analogs

Edward M. Wolin, M.D.
Co-Director Carcinoid/Neuroendocrine Center
Samuel Oschin Comprehensive Cancer Institute
Cedars-Sinai Medical Center
Los Angeles, CA

Somatostatin is a natural hormone, which works to stop the growth and hormone-secretion of neuroendocrine cells. However, it is destroyed in the body within minutes, making it highly impractical to use it as drug. To overcome this problem, modifications of the somatostatin molecule have been made, allowing it to survive and remain functional for long periods of time. These modifications of somatostatin are called somatostatin analogs. Octreotide (Sandostatin®) and octreotide LAR have long been the only somatostatin analogs available for the therapy of carcinoid syndrome. They are highly effective in the management of diarrhea and flushing associated with metastatic carcinoid tumors.

In an exciting new development, there is now proof that somatostatin analogs can stop the growth of neuroendocrine cells, thus paving the way for their use as anti-cancer drugs (low toxicity “chemotherapy”). In the PROMID study, 85 patients with midgut carcinoids were randomized to octreotide LAR 30mg/month vs. placebo. The average progression-free survival period was 14.3 mo. with octreotide vs. 6 months with placebo. At 6 months, 64% of the octreotide-treated patients remained without cancer progression vs. 37.2% of the placebo-treated patients.(1) This study establishes octreotide as standard therapy for all patients with metastatic carcinoid, whether or not a syndrome of diarrhea and flushing is present.

Another important advance has been the development of new highly effective somatostatin analogs. One, known as lanreotide, can be combined with water to form stable molecular aggregates that dissemble over a period of four weeks, continuously releasing lanreotide into the circulation after a single subcutaneous injection. This product, lanreotide autogel, can thus be administered as a single subcutaneous injection every four weeks. In addition to being less painful than deep IM injection of octreotide LAR, subcutaneous injection of lanreotide autogel allows for the administration of higher and potentially more effective doses in a smaller volume. It provides for reliable blood levels in obese people, when octreotide LAR doses intended for IM use can end up in fat instead of muscle, impairing absorption. Lanreotide autogel can also be easily administered to thin individuals who do not have adequate muscle mass for repeated deep IM injections. Randomized clinical trials exploring lanreotide in carcinoid/neuroendocrine tumors, with or without carcinoid syndrome are ongoing.

The third exciting somatostatin analog being used is called pasireotide (SOM-230). Pasireotide binds to more types of somatostatin receptors on carcinoid/NET cells, and binds more tightly than either octreotide or lanreotide. The long acting formulation which is given every four weeks is called pasireotide LAR. In 27% of patients whose carcinoid syndrome could not be controlled with octreotide or lanreotide, the syndrome could be controlled with pasireotide. Side effects are very similar for all three of these somatostatin analogs, except pasireotide caused elevated blood sugar in 12% of patients and type 2 diabetes mellitus in 7%. (2). Studies are currently under way to compare symptom control and tumor control with pasireotide LAR vs. octreotide LAR.

References:

1) Placebo-Controlled, Double-Blind, Prospective, Randomized Study on the Effect of Octreotide LAR in the Control of Tumor Growth in Patients With Metastatic Neuroendocrine Midgut Tumors: A Report From the PROMID Study Group
Rinke, A et al, JCO Oct 1 2009: 4656-4663.

2) Pasireotide LAR In Patients With Metastatic Carcinoid Tumors: Interim Safety Results From A Randomized, Multicenter Phase I Study. Edward Wolin, Larry Kvols, Shereen Ezzat, Walter Kocha, Eric Van Cutsem, Ulrike Schönherr and Abderrahim Fandi. 2009 Am Soc Clin Oncol Gastrointestinal Cancers Symposium, Abstr. 122

I-131 MIBG Therapy for Carcinoid Tumors

R. Edward Coleman, M.D. is Professor of Radiology and Director of the Division of Nuclear Medicine at Duke University Medical Center (DUMC). After receiving his medical degree from Washington University in St. Louis, he did postgraduate training at Barnes Hospital in St. Louis, the Royal Victoria Hospital in Montreal, and Mallinkrodt Institute of Radiology in St. Louis. After a short stint on the faculty at Washington University, he joined the faculty at the University of Utah for three (3) years before moving to DUMC as Professor of Radiology in 1979. Soon after the introduction of radiolabeled MIBG, Dr. Jerome Feldman, an endocrinologist with special interest and expertise in neuroendocrine tumors, and he began investigating and reporting on the use of MIBG in diagnosing and treating these tumors. He is the principal investigator on a clinical trials contract with Molecular Insight Pharmaceuticals, Inc.

Development of Metaiodobenzylguanidine (MIBG) Imaging

Radiolabeled MIBG was developed in 1980 at the University of Michigan by Wieland and colleagues because of their interest in imaging the adrenal gland and pheochromocytoma, a tumor of the adrenal gland. That group demonstrated prominent uptake of MIBG in the normal adrenal gland as well as in pheochromocytoma.

MIBG Imaging and Therapy at DUMC

MIBG was used in the early 1980’s at Duke for evaluating patients with suspected pheochromocytoma, one of the neuroendocrine tumors that accumulate this agent. Working with Dr. Feldman, we demonstrated that the mechanism of the accumulation of this agent in pheochromocytomas was the same as in platelets, i.e., a neuronal-pump mechanism. We were aware that the same mechanisms were present in carcinoid tumors and this early research resulted in our using it for imaging carcinoid tumors as well as pheochromocytomas. Our initial observation on the use of MIBG of carcinoid tumors in 23 patients was published in 1986 when we demonstrated that 61% of carcinoid tumors were detected by MIBG imaging. Even in this small population, we noted that greater accumulation of MIBG was demonstrated in tumors of midgut origin (ileum, cecum), than in tumors of foregut origin (pancreas, stomach), and it was not significantly concentrated by tumors of other foregut origin (bronchus). In a subsequent publication that reported the results in 82 patients, we confirmed the findings of the earlier study. In addition, we noted that accumulation of MIBG was more likely when the serum serotonin levels were elevated. Shortly after those studies were published, we began treating patients who had metastatic carcinoid tumors with larger doses of I-131 MIBG. In 2004, our results of treating 98 patients with metastatic carcinoid were published. Patients who experienced a symptomatic response to the therapy had improved survival over those who did not have a symptomatic response. Tumor markers such as 5-HIAA levels decreased significantly after the I-131 MIBG treatment. We observed that patients who received an initial high dose of the radioactive material had a better prognosis than those patients who initially received a lower dose. Decreases in blood counts were noted after therapy, but these decreases in counts improved to the baseline level with time.

In addition to studying the role of MIBG in carcinoid tumors, we evaluated its role in diagnosing and treating pheochromocytoma. In a study of 64 patients with suspected pheochromocytoma, the MIBG imaging had a sensitivity and specificity of 88%. In a study that evaluated the impact of MIBG therapy on 33 patients who had metastatic pheochromocytoma or paraganglioma, we noted that a symptomatic response led to an improved survival. Furthermore, patients with a measurable hormonal response demonstrated an increased survival in comparison to those with no response. Patients who received an initial high dose of the therapy had improved survival over those who had a low dose.

New Formulation of MIBG

The method of synthesizing MIBG developed at the University of Michigan and used at several other institutions had non-radioactive MIBG in the formulation. Recently, Molecular Insight Pharmaceuticals, Inc., has begun investigating a no-carrier-added formulation of MIBG (Azedra) for treating neuroendocrine tumors. Preliminary studies in animals and patients have demonstrated greater accumulation of MIBG in the tumors with better results in treating tumors in animal models. Patients with metastatic pheochromocytoma and paraganglioma are being treated with this new formulation. A Phase I study has been completed, and a Phase II study is to begin in early 2009. Although the Phase I trial was primarily designed for determining safety and maximum tolerated dose in patients with metastatic pheochromocytoma and paraganglioma, efficacy was demonstrated in some of the patients. The Phase II study is designed for treating patients with metastatic pheochromocytoma and paraganglioma, but it is anticipated that some patients with carcinoid tumors may be treated with this new formulation.

Future of Radiopharmaceutical Therapy

With the initial demonstration of response to the Azedra therapy in the Phase I trial, a Phase II study is anticipated. The potential for using Azedra therapy in patients with metastatic carcinoid tumor is also an exciting possibility.

In addition to the potential use of Azedra in treating metastatic carcinoid tumors, therapy with radiolabeled somatostatin analog is also promising. The same company that developed Azedra for therapy of neuroendocrine tumors is planning clinical trials of a somatostatin analog labeled with Yttrium-90 for therapy of carcinoid tumors. Previous studies in small numbers of patients have demonstrated therapeutically efficacy, but the agent is not generally available in the U.S. Clinical trials with this agent will be performed in anticipation of leading to approval by the FDA.


Literature cited:

1. Feldman JM, Frankel N, Coleman RE: Platelet uptake of the pheochromocytoma-scanning agent 131-I-meta-iodobenzylguanadine. Metabolism 33:397-399, 1984.

2. Feldman JM, Blinder RA, Lucas KJ, Coleman RE: Iodine-131 metaiodobenzylguanidine scintigraphy of carcinoid tumors. J Nucl Med 27:1691-1696, 1986.

3. Hanson MW, Feldman JM, Blinder RA, Moore JO, Coleman, RE: Carcinoid tumors: Iodine-131 MIBG scintigraphy. Radiology 172:699-703, 1989.

4. Safford SD, Coleman RE, Gockerman JP, Moore J, Feldman J, Leight GS, Tyler DS, Olson JA: Iodine-131 metaiodobenzylguanidine is an effective treatment for malignant pheochromocytoma and paraganglioma. Surgery 134: 956-962, 2003.

5. Safford SD, Coleman RE, Gockerman JP, Moore J, Feldman J, Onaitis MW, Tyler DS, Olson JA Jr: Iodine-131 metaiodobenzylguanidine treatment for metastatic carcinoid. Results in 98 patients. Cancer, 101:1987-93, 2004.

6. Hanson MW, Feldman JM, Beam CA, Leight GS, Coleman RE: Iodine 131-labeled metaiodobenzylguanidine scintigraphy and biochemical analyses of pheochromocytomas. Arch Int Med 151:1397-1402, 1991.

7. DeGrado TR, Zalutsky MR, Coleman RE, Vaidyanathan G: Effects of specific activity on meta-[131I] Iodobenzylguanidine kinetics in isolated rat heart. Nucl Med Biol 25:59-64, 1998.

8. Khan MU, Coleman RE: Diagnosis and therapy of carcinoid tumors – current state of the art and future directions. Nucl Med & Biol, 2008, in press.

9. James O, Coleman RE: Radioiodinated MIBG in paraganglioma and pheochromocytoma: previous results and early experiences using no-carrier-added MIBG. Nucl Med & Biol, 2008, in press.

Identifying Molecular Pathways in Carcinoid

Daniel C. Chung, MD
Harvard Medical School
Boston, MA 02115

The underlying molecular abnormalities in carcinoid tumors are poorly understood. This is an important deficiency in the field of neuroendocrine tumors, and is in sharp contrast to what we know about many other tumors such as colon cancer, breast cancer, or pancreatic cancer. Understanding some of these alterations can provide important insights into why the tumor forms and more importantly, how to develop new therapies. Our laboratory is interested in identifying some of these molecular alterations in carcinoids and in pancreatic neuroendocrine tumors. We have taken a multi-pronged approach to examine changes at the level of DNA, RNA, and protein.

Our strategy was to examine human tumor samples, in contrast to cultured cell lines. While there is some inherent tissue heterogeneity, this approach is appealing in that the changes identified reflect what is going on in actual tumors. We are fortunate to have developed a large collection of tumor samples from our institution to work with. I’d like to share some exciting new data about one gene/protein that we have recently obtained.

This protein is a transcription factor called HoxC6. This Hox gene was of particular interest because it appears to be a target of the Menin gene that is responsible for the Multiple Endocrine Neoplasia-I syndrome. HoxC6 was dramatically upregulated in GI carcinoids. Levels were very low in normal tissues, indicating that this gene is likely to play a role in tumor pathogenesis. To establish a biological role for HoxC6, independent studies were performed in which we overexpressed Hoxc6 and also knocked-down its expression in the Bon carcinoid cell line. Carcinoid cells that expressed high levels of HoxC6 had higher levels of growth and proliferation, and those with lower levels of HoxC6 grew at significantly slower rates. Traditionally, Hox genes are thought to play an important role in embryonic development, so its specific function in tumorigenesis in the adult is uncertain. Independent studies were performed to screen a variety of oncogenic pathways, and we were able to successfully demonstrate that HoxC6 was an important regulator of the AP-1 transforming pathway. This is a novel function for the HoxC6 gene and the first description of the role of the AP-1 pathway in this tumor type. Disrupting this specific interaction between Hoxc6 and AP-1 prevented HoxC6 from activating the AP-1 pathway and stimulating cell growth. This indicates a new and specific target for therapeutic intervention. There are some established inhibitors of AP-1 signaling, and we would like to see if we can identify molecules that may specifically block the protein-protein interaction between HoxC6 and AP-1. These studies are in press in Gastroenterology.

We are grateful for the support of the Caring for Carcinoid Foundation. Through studies such as these, we hope to improve our understanding of the molecular basis of the disease, and then utilize this information to design novel, targeted therapies.

New Cell Lines and Potential Therapy Targets

Lee Ellis, MD
MD Anderson Cancer Center
Houston, TX 77030

Although advances are being made in the therapy of metastatic carcinoid tumors, true responses to biologic or chemotherapy remains less than 20%. In order to develop new therapeutic approaches for patients with metastatic carcinoid tumors, it is essential to understand the molecular alterations that lead to metastatic disease. Unfortunately, there are very few cancer cell lines that have been developed from carcinoid tumors and the few cell lines that are available are not widely used.

The major objective of our research is to develop newly established carcinoid tumor cell lines and determine targets for potential therapy. Working at a cancer center with a large number of patients with carcinoid tumors who come to surgery has allowed us to develop new human cell lines. We can harvest tissues from patients whose tumor has been removed, process it rapidly, and go through an extended period of selection and validation of these newly developed carcinoid cell lines. To date, we have published the results on the establishment and characterization of the first midgut carcinoid cell line reported in many years (Van Buren et al Clinical Cancer Research 2007). At the present time we are establishing and validating other newly developed carcinoid cell lines that can be used in labs throughout the world. Our first cell line has been shared with over twenty different research groups on several continents.

In very preliminary experiments, we have determined that there are several activated signaling pathways that may be good targets for therapy. One such pathway that appears activated in carcinoid tumors in the mTOR pathway. This pathway is currently being targeted in clinical trials and thus we are not pursuing studies regarding inhibition of this pathway in preclinical models. However we have established that the Src tyrosine kinase signaling molecule is activated in nearly all of our carcinoid tumor cell lines. When we use a Src inhibitor in cell culture, we can inhibit proliferation of these cells. In very preliminary studies in mice, when we use a chemical inhibitor of Src activity we are able to markedly inhibit tumor growth. This work requires validation studies that are ongoing.

We are very excited about the above findings, and are now obtaining clinical specimens to determine the frequency of Src activation in liver metastases from carcinoid tumors. Of course a great deal of further study is necessary in order to understand the role of Src in regulating tumor growth and metastases. However, our preliminary results are exciting and we are pursuing this research with a great deal of enthusiasm.

'Modeling' Carcinoid

Seung Kim, MD, PhD
Stanford University School of Medicine
Stanford, CA 94305

What is carcinoid? We know that it constitutes a rare, neuroendocrine tumor, but at the cellular and molecular level, we know very little about what carcinoid is. The past several decades of 'cancer research' has validated repeatedly that a detailed understanding of the basis for how cancers develop, where they come from, and how they progress can lead to development of effective treatments for specific cancers. Moreover, much of the most important work in these areas of cancer research comes from development of specific cancers in animal models, which provide powerful experimental tools for understanding the behavior and origins of cancer. Based on this successful precedent, the Caring for Carcinoid Foundation has made investments in a number of research projects that aim to use experimental animals like mice to investigate the cellular and molecular basis of 'what carcinoid is.'

In my group at Stanford University School of Medicine, we are using CFCF support in attempts to produce a mouse 'model' of carcinoid. This is a somewhat ambitious goal, since no genetic model of carcinoid yet exists. To accomplish this, we are using classical mouse genetic methods to perturb the 'growth control' of cells in the mouse intestine that are thought to be the origins of carcinoid tumors. (The origins and molecular defects leading to carcinoid tumor formation in mice are also being explored by the group led by Dr. Andrew Leiter, another recipient of CFCF support; see Wang et al 2007). In prior studies of endocrine tumors that form in organs like the prostate and pancreas, other groups have previously shown that tumor formation can be 'driven' by production of a foreign viral protein, called 'T-antigen' (Hanahan, 1985; Garabedian et al 1998). These studies led ultimately to new ways of thinking about neuroendocrine tumors in these organs (Hu et al 2004; Karnik et al 2005; Ippolito et al 2005, 2006). Our work in carcinoid, if successful, could lead to better understanding about the molecular and cellular changes that lead to formation of carcinoid tumors, and to better ways of testing and discovering new therapies for carcinoid.


Literature cited:

Garabedian, E.M., et al 1998. A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells. Proc Natl Acad Sci USA 95:15382-7

Hanahan, D. 1985. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315:115-22.

Hu, Y. et al 2004. RNA interference of achaete-scute homolog 1 in mouse prostate neuroendocrine cells reveals its gene targets and DNA binding sites. Proc Natl Acad Sci USA 101:5559-64.

Ippolito, J.E., et al 2006. Linkage between cellular communications, energy utilization, and proliferation in metastatic neuroendocrine cancers. Proc Natl Acad Sci USA 103:12505-10.

Ippolito, J.E., et al 2005. An integrated functional genomics and metabolomics approach for defining poor prognosis in human neuroendocrine cancers. Proc Natl Acad Sci USA 102:9901-6.

Karnik, S.K. et al 2005. Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kip1 and p18INK4c. Proc Natl Acad Sci USA 102:14659-64.

Wang, Y. et al 2007. Enteroendocrine precursors differentiate independently of Wnt and form serotonin expressing adenomas in response to active beta-catenin. Proc Natl Acad Sci USA 104:11328-33.

Genetics of Carcinoid Tumors: Approaches and Progress

Ramesh Shivdasani, MD
Dana-Farber Cancer Institute
Boston, MA 02115

Carcinoid tumors of the small intestine resist conventional chemotherapy. Knowing the underlying genetic changes will inform efforts to develop new treatments, but to date specific genes are not implicated in the disease and critical molecular pathways are largely unknown. My laboratory seeks to define the genetic mutations that drive development of the disease and hence pave the way for new targeted therapies. We are using a variety of genetic approaches to mine one of the largest collections of carcinoid tumors, at the Dana-Farber Cancer Institute.

Genes that are amplified or deleted in tumors provide vital clues about underlying disease mechanisms, which differ from one tumor type to another. One approach we have taken uses single nucleotide polymorphism (SNP) arrays to analyze the genome of carcinoid tumors. This technique identifies genes that have increased or decreased copy numbers in carcinoid tumors compared with normal tissue. To help identify genetic alterations that may be involved in metastatic tumor spread, we also succeeded in some cases in conducting SNP array analysis on both primary tumors and metastases from the same patient. A novel and unexpected result of these studies was that small intestine (ileal) carcinoid tumors seem to belong to two distinct groups: those with simultaneous gain of chromosomes 4, 5, 7 and 14, and those with modest copy number imbalance. One focal region of recurrent gain on chromosome 14q mapped to the locus of the gene encoding the anti-apoptotic protein DAD1. By identifying genes that are amplified or deleted in tumors, we expect to delineate genetic events that might be important in tumor formation and serve as targets for therapy.

A second approach involves using modern techniques to analyze carcinoid tumors simultaneously for dozens of potential gene mutations, particularly those commonly present in a wide range of tumors. My lab is currently performing this analysis on a group of nearly 100 carcinoid tumors, one of the largest collections assembled to date for the purpose of molecular investigation.1

Understanding of tumor biology is considerably advanced by knowledge of the cell of tumor origin, which remains a conspicuous unknown in the case of carcinoid tumors. My laboratory therefore uses a combination of mouse genetics and cell culture techniques to study the origins of normal gastrointestinal endocrine cells and the cell of origin of carcinoid tumors. By tagging specific lineages of enteroendocrine cells in the developing gut, we hope to learn more about how carcinoid tumors arise and hence consider new strategies for tumor prevention or treatment. In particular, definition of neuroendocrine lineage hierarchies will identify molecular pathways that act to generate or maintain discrete endocrine cell lineages; such pathways may be targeted for rational therapy of carcinoid tumors.

Homeodomain transcription factors often function in differentiation of specific cell types. We used targeted gene disruption to generate mice lacking Nkx6.3, a new member of the Nkx6 subfamily. These mice develop and grow normally, with a grossly intact digestive tract, but carry many fewer gastrin-producing (G) cells in the stomach; as a result, blood levels of the hormone gastrin and acid levels in the stomach are significantly reduced. There is also a corresponding increase in somatostatin-producing (D) cells. These studies implicate Nkx6.3 as a novel and selective regulator of G- and D-cell neuroendocrine cell lineages, which are believed to derive from a common progenitor, and they set the stage for further characterization of enteroendocrine differentiation pathways.2

These studies would not be possible without the generous support and far-reaching vision of the Caring for Carcinoid Foundation. For more information please see:

1. Kulke MH, Freed E, Chiang D, Philips J, Zahrieh D, Glickman JN, Shivdasani RA.
High- resolution analysis of genetic alterations in small bowel carcinoid tumors reveals areas of recurrent amplification and loss. Genes Chrom Cancer 2008; 47:591-603.

2. Choi MY, Romer AI, Wang Y, Wu MP, Ito S, Leiter AB, Shivdasani RA. Requirement of the tissue-restricted homeodomain transcription factor Nkx6.3 in differentiation of gastrin- producing G-cells in the stomach antrum. Mol Cell Biol 2008; 28:3208-3218.

You can view these publications on the Caring for Carcinoid Foundation website at: http://www.caringforcarcinoid.org/research/researchprogress.asp