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10.1245/ASO.2004.03.077
Annals of Surgical Oncology 11:413-419 (2004)
© 2004 Society of Surgical Oncology
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ORIGINAL ARTICLES

Identification of Molecular Markers Specific for Pancreatic Neuroendocrine Tumors by Genetic Profiling of Core Biopsies

Mark Bloomston, MD, Alan Durkin, MD, Ivana Yang, PhD, Mumtaz Rojiani, PhD, Alexander S. Rosemurgy, MD, Steven Enkmann, PhD, Timothy J. Yeatman, MD and Emmanuel E. Zervos, MD

From the Department of Surgery (MB, AD, ASR, TJY, EEZ) and Department of Pathology (MR), University of South Florida College of Medicine, and the Department of Interdisciplinary Oncology (SE, TJY), H. Lee Moffitt Cancer Center, Tampa, Florida; and The Institute for Genomic Research (IY), Rockville, Maryland.

Correspondence: Address correspondence and reprint requests to: Emmanuel E. Zervos, MD, Assistant Professor of Surgery, Moffitt Cancer Center, 12902 Magnolia Drive, MRC-30037, Tampa, Florida 33612; e-mail: ezervos{at}hsc.usf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: There is a paucity of known molecular markers that distinguish pancreatic neuroendocrine tumors from other pancreatic tumor types. We hypothesized that novel markers for pancreatic neuroendocrine tumors could be identified with molecular fingerprinting of pooled RNA samples from core biopsies.

Methods: Total RNA was harvested from nine core biopsies of normal pancreas, pancreatitis, pancreatic adenocarcinoma, pancreatic adenocarcinoma metastases, and pancreatic neuroendocrine tumors. RNA from each group of samples was pooled and hybridized to an oligonucleotide-based microarray. Four genes (ANG2, NPDC1, ELOVL4, and CALCR) were selected for further investigation by reverse transcriptase polymerase chain reaction from the top 20 highest expressed genes, on the basis of potential as novel markers.

Results: Neuroendocrine tumors were most unique from normal pancreas. Pancreatitis, pancreatic adenocarcinoma, and metastases are more closely related to each other and to normal pancreas. ANG2 was overexpressed in 89% of neuroendocrine tumors, compared with 22% of normal pancreas, making it the best potential molecular marker or therapeutic target of the four genes selected for analysis.

Conclusion: We have identified a specific set of molecular markers for pancreatic neuroendocrine tumors distinct from pancreatitis and pancreatic adenocarcinoma. These novel markers may prove useful as molecular markers or therapeutic targets unique to pancreatic neuroendocrine tumors.

Key Words: Genetics • Microarray • Neuroendocrine • Pancreas


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuroendocrine tumors of the pancreas are uncommon neoplasms that are estimated to be clinically significant in 3.4 to 4 cases per million population, or approximately 6000 cases per year.1,2 While their clinical features can sometimes distinguish them from ductal adenocarcinomas of the pancreas, as many as 50% may be nonfunctional and thus clinically indistinguishable from adenocarcinoma of the pancreas.3 The distinction is critical because treatment of neuroendocrine tumors is different from that of adenocarcinomas, especially in the presence of locally advanced or metastatic disease, where surgical debulking may be indicated in the more indolent neuroendocrine tumors.4–6

The advent of microarray technology has allowed profiling of tumors by simultaneously probing for thousands of oligonucleotides to generate a genetic fingerprint for a variety of oncologic diseases.7,8 Microarray analysis may prove invaluable in distinguishing various pancreatic tumor types, aiding in early diagnosis and treatment, and guiding therapy. To date, little has been reported about the molecular profile of pancreatic neuroendocrine tumors, primarily because of their relative infrequency. The purpose of this study was to begin to identify molecular markers that may aid in distinguishing pancreatic neuroendocrine tumors from ductal adenocarcinomas and chronic pancreatitis.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Procurement
With approval of the institutional review board, core biopsy specimens of various pancreatic tumors and metastases (when present) were obtained from patients undergoing abdominal exploration with planned resection of pancreatic tumors. In addition, core biopsy specimens were obtained from histologically confirmed adjacent normal pancreas. Biopsy specimens were immediately placed in liquid nitrogen and stored at −70°C until total RNA could be harvested. Final diagnoses were confirmed by routine hematoxylin and eosin staining. None of the neuroendocrine tumors had evidence of functionality according to preoperative serum evaluation or symptomatology.

RNA Extraction and Purification
Within 24 hours of harvest, frozen core biopsy specimens were homogenized in 5 mL of Trizol reagent (Gibco-BRL, Carlsbad, CA) and incubated at room temperature for 5 minutes. One milliliter of chloroform was added, and the samples were shaken vigorously for 15 seconds and then incubated at room temperature for 2 minutes. The samples were then centrifuged at 9000g for 15 minutes at 4°C. The aqueous phase containing RNA was then carefully removed and precipitated with an equal volume of fresh 70% ethanol mixed in RNase-free water. The solution was mixed vigorously and purified with an RNeasy midi column (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Purified RNA was then eluted in 400 µL of RNase-free water, and its concentration was determined by spectrophotometry. RNA was aliquoted to avoid repeated freeze-thaw cycles and was stored at −70°C.

The quality of RNA was determined by running 1 µg of RNA on a 1% agarose degradation gel. Only RNA samples demonstrating distinct 18S and 28S ribosomal RNA bands were used for subsequent experiments.

Microarray Analysis
Five RNA pools were created, representing nine distinct tumors in each pool. Equimolar amounts of RNA were allocated from each sample to comprise 10 µg of pooled RNA for normal pancreas, adenocarcinoma, metastatic adenocarcinoma, chronic pancreatitis, and neuroendocrine tumors. We have previously shown that sample pooling can yield an accurate representation of pool components.9 Pooled RNA was then hybridized to a human genome U133A GeneChip (Affymetrix, Sacramento, CA).

The human genome U133A (HG-U133A) consists of a GeneChip array, which contains probe sets representing more than 13,000 well-substantiated human genes. This chip design uses sequences selected from GenBank, and RefSeq Target synthesis, hybridization, and posthybridization staining were performed according to standard protocols recommended by the manufacturer.

Gene Selection and Reverse Transcriptase-PCR Confirmation
As a point of reference, the 20 most overexpressed genes in neuroendocrine tumors in comparison to normal pancreas were identified (Table 1). Four of these genes were selected for further investigation on the basis of relative expression, potential as novel molecular markers (i.e., not previously described in this tumor type), or potential as prognostic indicators or therapeutic targets (i.e., known antibody or secreted protein) (Table 2).


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  		TABLE 1. Top 20 genes overexpressed in neuroendocrine tumors and degree of increase in expression relative to normal pancreas
 

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  		TABLE 2. Candidate and housekeeping (GPI) genes evaluated by rt-PCR
 
Five hundred nanograms of total RNA were reverse transcribed with AMV Reverse Transcriptase (Roche Applied Science, Indianapolis, IN) and a 24-unit polythymidine primer. PCR with 2 µL of cDNA was carried out in a final volume of 26 µL containing 1.25 U Taq polymerase (Ampli-Taq Gold, Applied Biosystems, Branchburg, NJ), 100 µM dNTPs, 0.4 µM of each primer, 1.5 mM MgCl2, and 5% DMSO (v/v). The primers used and expected product size for each of the four selected genes and the housekeeping gene glucose phosphate isomerase (GPI) are listed in Table 2. The conditions of each PCR reaction are available upon request. PCR products were electrophoresed on 2% agarose gel containing ethidium bromide (0.4 µg/mL) and photographed.

Immunohistochemistry
Angiopoietin-2 protein expression was evaluated in neuroendocrine tumors and normal pancreas samples comprising their respective pools by immunohistochemistry (n = 9 each). In four cases, matched samples of normal pancreas and neuroendocrine tumors were available from the same patients. Immunohistochemistry for CD34 was undertaken in these tissues to compare the degree of new vessel formation. CD34 is a transmembrane glycoprotein expressed by vascular endothelial cells.

Paraffin slides were deparaffinized in xylene, and varying concentrations of ethanol were then quenched with peroxidase and rehydrated. Antigen retrieval was undertaken with 10 mM citrate buffer (pH, 6.0) and heated. After rinsing twice with PBS, anti-CD34 antibody (Pharmingen, San Diego, CA) or anti-angiopoietin-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to tissue at a 1:10 dilution in PBS and allowed to incubate at 4°C overnight. Primary antibody was removed and tissue was rinsed three times with PBS. Secondary antibody removal and immunodetection were undertaken with the Super Sensitive Immunodetection System(Biogenex, San Ramon, CA), in accordance with the manufacturer’s instructions. Slides were then quickly counterstained with hematoxylin, dehydrated, and covered.

Angiopoietin-2 immunohistochemical slides were reviewed by a senior pathologist and assigned an Allred score.10 A score of 6 or higher (corresponding to at least moderate staining intensity over at least 50% of the sample) was considered significant. CD34 slide images were captured electronically, and intraparenchymal vessel counts and area were determined with use of Image-Pro software (Media Cybernetics, Silver Spring, MD).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of Tumor Types to Normal Pancreas
Microarray analysis demonstrated that neuroendocrine tumors were the most dissimilar to normal pancreas. Chronic pancreatitis, pancreatic adenocarcinoma, and metastases from pancreatic adenocarcinoma were more closely related in Euclidean distance to each other and to normal pancreas. In comparison with normal pancreas, 1340 differentially expressed genes were found in neuroendocrine tumors, 783 were found in pancreatic adenocarcinoma metastases, 492 were found in pancreatic adenocarcinoma primaries, and 217 were found in chronic pancreatitis.

Selection of Candidate Genes for Further Study
Table 1 represents the top 20 overexpressed genes in neuroendocrine tumors in comparison with normal pancreas. Four of these top 20 genes were selected for further study on the basis of potential as novel markers (ANG2, NPDC1), prognostic indicators (CALCR, ELOVL4), or therapeutic targets (ANG2, CALCR). The primers used, expected product size, and relative expression by microarray analysis of these genes are listed in Table 2.

Relative Gene Expression in Pooled Samples of Four Candidate Genes by Reverse Transcriptase PCR
ANG2 and NPDC1 showed increased expression by PCR in neuroendocrine tumors, in comparison with normal pancreas (Fig. 1). ANG2 was not expressed in chronic pancreatitis and showed expression in pancreatic adenocarcinoma primaries and metastases that was similar to that in normal pancreas. NPDC1 was similarly overexpressed in neuroendocrine and adenocarcinoma tumors, while showing expression in adenocarcinoma metastases similar to that in normal pancreas and virtually no expression in chronic pancreatitis. ELOVL4 expression was similar between normal pancreas and adenocarcinoma and increased in chronic pancreatitis, neuroendocrine tumors, and adenocarcinoma metastases. Finally, CALCR expression was similar in normal pancreas, chronic pancreatitis, and neuroendocrine tumors but was less in adenocarcinoma primaries and metastases (Fig. 1).



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  		FIG. 1. Gene expression of four selected genes by reverse transcriptase PCR for each of five pooled samples (N = normal pancreas; CA = pancreatic adenocarcinoma; CP = chronic pancreatitis; NE = pancreatic neuroendocrine tumor; M = pancreatic adenocarcinoma metastasis).

 
Relative Gene Expression in Individual Samples of Four Candidate Genes by Reverse Transcriptase PCR
Global gene expression in each of the nine samples comprising pooled RNA for normal pancreas and pancreatic neuroendocrine tumors is shown in Fig. 2. Expression of ANG2 (88% vs. 22%) and NPDC1 (100% vs. 44%) was markedly greater (P < 0.05; Fisher exact test) in neuroendocrine tumors than in normal pancreas. ELOVL4 (55% vs. 11%) and CALCR (55% vs. 22%) was also greatly increased in neuroendocrine tumors, but to a lesser degree (P = NS).



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  		FIG. 2. Global gene expression in nine neuroendocrine (NE) tumors in comparison with nine normal pancreas samples.

 
Immunohistochemical Staining
Angiopoietin 2 was expressed predominantly within the cytoplasm of dysplastic islet cells and was seen in nine (100%) of the pancreatic neuroendocrine tumors, in comparison with two (22%) of the normal pancreas samples (P < 0.05; Fisher exact test). Representative immunohistochemistry slides for angiopoietin-2 are shown in Fig. 3.



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  		FIG. 3. Immunohistochemistry stains for angiopoietin 2 (brown stain) show increased expression in neuroendocrine tumors (top panel) relative to normal pancreas (bottom panel).

 
Neuroendocrine tumors had abundant vascular ingrowth, as demonstrated by immunohistochemistry for CD34 (Fig. 4). Mean intraparenchymal vessel count (124 ± 39 vs. 29 ± 12) was significantly higher in neuroendocrine tumors than in normal pancreas (P < 0.05; Mann-Whitney U test) (Fig. 5).



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  		FIG. 4. Immunohistochemical stains for CD34 show increased angiogenesis in neuroendocrine tumors (top panel) in comparison with normal pancreas (bottom panel). Microvessels stain dark purple.

 


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  		FIG. 5. Intraparenchymal vessel counts in four neuroendocrine tumors and matched normal pancreas by CD34 immunohistochemistry.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although pancreatic neuroendocrine tumors are less common than ductal adenocarcinomas, their diagnosis and their distinction from these more aggressive neoplasms are important in planning therapy and providing meaningful counsel on prognosis. Half of these tumors are nonfunctional, and their distinction from adenocarcinoma can be difficult—if not impossible—without surgical biopsy or radical resection. Microarray technology offers a potential strategy by which core biopsy specimens from undiagnosed pancreatic tumors may be differentiated on the basis of expression (or lack thereof) of specific molecular markers.

In this series of experiments, we have pooled RNA from several patients on the basis of tumor type for microarray analysis. Although detailed analysis of individual tumors is ideal, pooled sample microarray has been validated previously at our institution as a rapid, cost-effective means of delineating a gene expression profile that accurately reflects the profiles of the contributing samples.9 The benefits of sample pooling are multiple.

First, smaller quantities of RNA are needed for analysis. This is particularly helpful for analyzing samples from pancreatic tumors, which are notorious for the difficulty with which quality RNA is obtained from them. Second, the risk of a single specimen significantly altering the genetic profile of a group of tumors is proportionately diluted with increasing representative sample size. Finally, pooling of samples allows for fewer microarray chips to be used, resulting in less cost and fewer computational analyses. We found that neuroendocrine tumors of the pancreas are the most unique type of tumor from a molecular standpoint, in comparison with normal pancreas.

We identified more than 1000 genes differentially expressed in neuroendocrine tumors, some of which were also expressed in chronic pancreatitis, pancreatic adenocarcinoma, and metastases. We selected the 20 most highly overexpressed genes in neuroendocrine tumors only as a basis to focus our experiments. Four of these genes, ANG2, NPDC1, ELOVL4, and CALCR, were selected on the basis of their potential as diagnostic, prognostic, or therapeutic targets as genes of interest.

ANG2 (angiopoietin) is a homologue to Ang1, which codes for ligands of the endothelial cell-specific tyrosine kinase receptor Tie2 to regulate growth and remodeling of blood vessels.11,12 Although ANG2 has been shown to be expressed in the developing mouse pancreas and in human hepatocellular carcinoma and non-small-cell lung cancer, its precise role is not clear.13–15 Neuroendocrine tumors are highly vascular tumors, making ANG2 a logical potential target of investigation. In this series of experiments, we have shown that ANG2 expression is markedly upregulated in pancreatic neuroendocrine tumors and almost universal. This upregulation was associated with increased protein expression by immunohistochemistry and marked increase angiogenesis, an association seen previously in lung cancer.15 The utility of an antiangiogenesis agent targeting this gene is obvious.

The neural proliferation, differentiation, and control gene (NPDC1) is involved in the regulation of cell cycle, survival, and apoptosis in tissues of neural origin.16,17 Although it stands to reason that neuroendocrine tumors could express this gene, it was somewhat surprising to see it expressed, albeit at lower intensity, in normal pancreas as well as adenocarcinoma primaries and metastases. When expressed at normal levels, NPDC-1 allows neuroendocrine cells to moderate their own growth through contact inhibition. Overexpression of this gene, on the other hand, gives the neuroendocrine cell the potential for unchecked cell growth. Further investigation into this gene’s potential as a target for therapy is warranted.

Elongation of very-long-chain fatty acids 4-like protein gene (ELOVL4) is responsible for biosynthesis of fatty acids, and mutations of this gene have been implicated in familial forms of macular degeneration or dystrophy (Stargardt disease).18,19 Although derangements or excessive synthesis of fatty acids are not implicated in the pathophysiology of neuroendocrine tumors of the pancreas, microarray showed that this gene was highly overexpressed in pancreatic neuroendocrine tumors only and not other tumor types. We hypothesized that this may be a novel tumor marker but found in PCR analysis that it was not relatively overexpressed, and its identification is probably more reflective of the oversensitivity of microarray rather than a truly novel finding. Again, this gene was expressed two times more commonly in our neuroendocrine tumors. Utilization of this marker may be helpful in identifying familial forms of neuroendocrine tumors and thus may be useful in genetic counseling.

We chose to further study the gene encoding for the calcitonin receptor (CALCR) because of its promise as a potential therapeutic target. If expression of the calcitonin receptor could be isolated to neuroendocrine tumors, then pharmacologic or genetic blockade at this receptor theoretically may alter growth kinetics of neuroendocrine tumors. Paracrine/neuroendocrine calcitonin-stimulated growth of human prostate cancer cells is well documented.20,21 Calcitonin receptor analogs may offer a potential therapeutic strategy in pancreatic neuroendocrine tumors and warrant further study.

It is widely accepted that neuroendocrine tumors can grow to extraordinary sizes, are exquisitely vascular, and may be stimulated to grow by the products of their differentiation in an autocrine manner. Using oligonucleotide microarrays, we have attempted to identify genes or genetic markers that may be of use in curtailing the growth of these tumors through antagonism of their gene products or aiding in the accurate diagnosis of nonfunctional lesions. Our investigations focused on those genes overexpressed that had not been described in the context of pancreatic neuroendocrine tumors or showed potential as a novel marker or therapeutic target. The next logical step in this project is to apply what we have learned herein and attempt to alter the natural history of these tumors by targeting the gene products or genetic pathways of the genes identified. Angiopoietin makes the most sense to pursue first because of its relative overexpression at the gene and protein levels and functional fit.


   ACKNOWLEDGMENTS
 
Supported by CA85052–01A1 and CA85429–01 (to TJY). The authors thank Amyn Rojiani, MD, PhD, and Enid Gilbert-Barness, MD, for their expert assistance with immunohistochemical staining and interpretation.


   FOOTNOTES
 
Presented at the 56th Annual Society of Surgical Oncology Cancer Symposium.

Microarray analysis was utilized to provide a genetic profile of pancreatic neuroendocrine tumors relative to normal pancreas, pancreatic adenocarcinoma, pancreatic adenocarcinoma metastases, and chronic pancreatitis. With use of microarray technology, more than 1000 genes have been identified in neuroendocrine tumors of the pancreas. Four novel genes were further investigated by reverse transcriptase polymerase chain reaction analysis.

Received for publication March 17, 2003. Accepted for publication November 21, 2003.


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 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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