James IV Article

Molecular strategies and ¹¹¹in-labelled somatostatin analogues in defining the management of neuroendocrine tumour disease: a new paradigm for surgical management

I.M. Modlin, M. Kidd, T. Hinoue and K.D. Lye 
Gastrointestinal Surgical Pathobiology Research Group, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, USA, 06520

J. Murren 
Department of Clinical Oncology, Yale University School of Medicine, New Haven, Connecticut, USA 06520

A. Argiris
Department of Medicine Northwestern University Feinberg School of Medicine and the Robert H. Lurie Comprehensive Cancer Center, Chicago, Illinois, USA, 60611

Correspondence to: I.M. Modlin, Department of Surgery Yale University School of Medicine 333 Cedar Street, P.O. Box 208062 New Haven, Connecticut, USA 06520-8062 Telephone:(203) 937-4785

Introduction The Mastomys model Human tumours

Somatostatin receptor therapy Discussion Conclusion References

Keywords: ECL cell, mastomys, neuroendocrine, tumour, surgery, gene-chip, somatostatin Surg J R Coll Surg Edinb Irel., 1 June 2003, 137-143

This manuscript provides a gene-chip examination of gastric ECL cell proliferation in an animal model of neuroendocrine tumour disease. Data that were used to identify molecular targets were then utilised to develop novel therapeutic strategies as appropriate adjuncts to surgery in human disease. Alterations in growth-mediated cell signaling (the AP-1 pathway) and in the cell cycle were identified in ECL cell tumours in the animal model and confirmed in human tumour tissue. The growth-inhibitory somatostatin receptor subtype 2 was identified as a potential clinical target. An investigation of patients with neuroendocrine tumours treated using SSTR2 targeted radiotherapy [111In]pentetreotide producing encouraging preliminary results. Fifty-six per cent of patients with evaluable hormone markers demonstrated stable levels or a significant decrease in one or more measured markers. This data demonstrate that gene pathways recognised to be altered in an animal model of a human disease can be used to identify therapeutic agents. This approach was successfully used to discover novel strategies that can be both effective and appropriate adjuncts to surgery for patients with neuroendocrine tumour disease

INTRODUCTION
Although the entity of carcinoid tumour was defined almost a century ago by Oberendorfer, the biological behaviour of neuroendocrine tumours (NETs) remains an enigma and their clinical management a source of controversy and concern.¹ As a result, surgical strategy in their management is often based upon relatively arbitrary concepts (e.g. size of primary tumour and numbers of lymph nodes involved).² This situation reflects a lack of understanding of the growth factor regulation of these lesions, an inability to define their rates of cell proliferation, and incapacity to either detect or predict metastatic events.³ The surgeon, thus, is dependent upon clinical judgment and light microscopy (i.e., pattern recognition) to guide his operative strategy. As a result, operations may be either inadequate or, alternatively, very radical and extensive.² Gastric NETs have become relatively common as awareness and recognition, by pathologists and gastroenterologists, has risen. The increase of upper gastrointestinal endoscopy has greatly amplified instances where biopsy reveals enterochromaffinlike (ECL) cell proliferation.² This has resulted in confusion regarding the neoplastic potential and surgical relevance of such an observation in much the same fashion as that engendered by the unexpected identification of an appendiceal carcinoid. The genesis of gastric NETs has not been clearly delineated and the rational basis for surgical management is unclear. Are they “benign,” can they metastasise, or are they harbingers of adenocarcinoma?⁴ The information imparted by light microscopy has failed to enable such determinations; as such, surgical options proposed have run the gamut from total gastrectomy to antrectomy and endoscopic local excision to observation. This broad disparity of therapeutic options reflects an inability to define whether the lesions identified are autonomous neoplastic cells or hyperplastic cell aggregations driven by gastrin, which may simply regress if the trophic agent is withdrawn.

The Mastomys (Praomys natalensis) animal model of gastric NETs, with an advantageously short experimental time frame, has been developed in our laboratory.5 Induction of achlorhydria in these rodents by irreversible histamine² receptor blockade (loxtidine, 1 mg/kg/day) results in ECL cell hyperplasia (100%), dysplasia (80%), and neoplasia (75%) within three months, respectively. ^6,7 Numerous studies have indicated that these gastric carcinoid lesions are similar to those of human disease.² Availability of the Mastomys purified and isolated ECL cell preparations enables identification of the cellular regulatory events involved in ECL cell replication and also the events involved when the normal cell transforms to the neoplastic state.

The purpose of this study was, firstly, to seek to define the growth-regulatory elements of the neuroendocrine ECL cell system--which forms the cellular basis for NETs--using a genechip approach in an animal model of the disease and, secondly, to identify the molecular determinants of cellular proliferation that would enable determination of whether a group of proliferating ECL cells were autonomous in humans. The third aim was to identify molecular targets that could be utilised to develop novel therapeutic strategies as appropriate adjuncts to surgery.

Figure 1: Venn diagram of the distribution of the gene expression in the three Mastomys ECL cell samples, one from an untreated animal (naïve), one from a hypergastrinemic animal treated with loxtidine, and a spontaneous tumour in an aged animal. The numbers quantify the expressed genes which are either unique to a sample (e.g., 226 genes expressed uniquely in the loxtidine-treated animal), shared by two samples (e.g., 118 genes shared by the two tumor types), or shared by all samples (i.e., 801 genes)

To accomplish these aims, growth factors and regulators of cell proliferation were examined in the Mastomys model of gastric NETs. Thereafter, human NETs were studied using reverse transcriptase-polymerase chain reaction (RT-PCR) assays, genomic DNA analyses for gene promoter region methylation status, and protein for western blot analyses, including cyclin D1, cdk4, menin, jun-D, p16, somatostatin receptor type 2 (SSTR₂), and PCNA.

Lastly, 39 patients whose lesions expressed adequate SSTR₂ uptake were treated with high-dose intravenous [¹¹¹In]DTPA⁰-octreotide ([¹¹¹In]pentetreotide), a vehicle for cell-targeted intravenous radiotherapy. ⁸ Somatostatin is an endogenous tetradecapeptide hormone found in neuroendocrine cells of the pancreas, gastrointestinal tract, and nervous system which inhibits cell proliferation, downregulates angiogenesis, and plays a role in immunomodulation and neurotransmission. The effects of somatostatin are mediated through five principal somatostatin receptor subtypes, of which SSTR₂ appears to be over-expressed most commonly in human tumours. ^9,10 In particular, SSTR₂ are expressed in neuroendocrine tumours such as carcinoids, gastrinomas, pituitary tumours, and endocrine pancreatic tumours at sufficiently high quantities to facilitate receptor-based imaging.⁸ These data suggest a biological basis for radioactivity studies with therapeutic intent in patients whose tumours express SSTR₂. We sought, therefore, to establish a therapeutic protocol to identify this known receptor as a target site, based on our ability to identify the presence of the SSTR₂ in the Mastomys model and in human carcinoid tissue.

This article will firstly address the genomic basis of the Mastomys model of gastric NETs. Thereafter, genes identified as altered in this model were examined for expression changes in a databank of human carcinoid tissue. Thirdly, the use of the SSTR2 as a therapeutic target for isotopic delivery was examined in patients with neuroendocrine tumours.

THE MASTOMYS MODEL

Background
A genomic and gastrin-driven animal model (Praomys natalensis) of neuroendocrine ECL cell tumours exists. ¹¹ Irreversible _H2- receptor blockade (loxtidine, 1 mg/kg/day) causes ECL cell hyperplasia, dysplasia, and neoplasia in 100%, 80%, and 75% of animals, respectively, within three months.^12,13 Tumours also arise spontaneously in approximately 20% of normogastrinemic (untreated) animals at the end of their lifespan (around two years of age).⁵ Using immunohistochemistry, isolated pure ECL cells, and bromodeoxyuridine (BrdU) cell proliferation assays, we have previously determined that gastrin, pituitary adenylate cyclase activating peptide (PACAP), and transforming growth factor - a (TGF-a) are the primary determinants of ECL cell proliferation in this model. ^14-16 The administration of the somatostatin analogue octreotide by infusion pump results in a significant inhibition of plasma gastrin levels and is associated with inhibition of ECL cell hyperplasia and tumour development. ¹⁷ The presence of SSTR₂ on both ECL cells and ECL cell tumours has been demonstrated both functionally and by autoradiography. ^18,19 Because the properties of tumour cells vary, it is often not possible to characterise individual tumours by means of a single, or even several, molecular markers. Examination of single-gene pathways, therefore, is unlikely to identify the key regulators of ECL cell proliferation. The isolated cell system, however, allows for a more global approach to gene expression.

Methods
Affymetrix (Santa Clara, California, USA) gene-chip technology was used to examine gene expression (mRNA) in three Mastomys ECL cell samples. The three samples included a naïve Mastomys ECL cell preparation (isolated by counterflow elutriation and Nycodenz gradient centrifugation, resulting in cell preparations of approximately 80% enriched ECL cells) and two Mastomys ECL cell tumours isolated by hand dissection and subsequent tissue mincing; one was from a hypergastrinemic animal treated with the _H2-receptor antagonist loxtidine for 6 months, and the other was from a normogastrinemic high-age animal (2.5 years old) that had spontaneously developed an ECLoma. Total RNA was isolated (RNeasy, Qiagen) from these ECL cells. An Affymetrix rat-specific chip which visualises 8801 genes was then used to examine gene expression using standard technology. ²⁰ Sequence output was statistically analysed using Affymetrix software (MAS 4.0). ²¹

Figure 2. Alterations in three cell cycle regulatory genes in Mastomys samples. The relative increase against gene levels in naive samples is provided (naive= 1)

Results
A range of 1041-1723 probe-pair sets was identified as present in the cell samples (mean ± SEM: 1327 ± 95) using the Affymetrix rat gene-chip. Examination of the three Mastomys ECL cell samples demonstrated that they shared 801 genes with the following distribution: naïve cells 801/1875 (43%), loxtidine tumour 801/1337 (60%) and spontaneous tumour 801/1597 (50%) (Figure 1). The naïve state had 521 genes not present in either of the two tumour states; the expression of these genes is presumed “lost” or downregulated below detectable limits during ECL cell tumourigenesis. The loxtidine-induced tumour had 226 “unique” genes; these are presumed “gained” or substantially upregulated during hypergastrinemia-induced ECL cell proliferation. The spontaneous tumour had 317 genes unique to it; these are presumably gained during normogastrinemic transformation. Both tumour states separately shared 192 genes (loxtidine-induced) and 361 genes (spontaneous) with the naïve state, respectively. The two tumour samples shared 118 genes which are not present in the naïve state and presumably are unique to the transformed ECL cell.

We then examined heterogeneity in gene expression using a cut-off of greater than two-fold differences in gene read-out values between the naïve and tumour ECL cell samples. The same proportion of genes was upregulated in the loxtidine (171/262, 65.3%) and the spontaneous samples (125/190, 65.8%). The loxtidine-induced and spontaneous tumours shared 127 genes that were altered. Expression-change congruency was 99% for those upregulated and 100% for those downregulated. Comparing the ECL cell gene expression in the proliferating samples demonstrated that fos (downregulated 6-12-fold), junD (downregulated 2.4-3.5-fold), junB (downregulated 2.2-4.9-fold) and furin (upregulated 5- 9-fold) were significantly altered during ECL cell transformation. Other genes significantly altered included members of the cyclin D1 pathway; cdk4 and cyclin D1 were upregulated 3.2- and 2.4-fold, respectively, while the cofactor in this pathway, cdc37, was increased 1.5-fold. PCNA was upregulated approximately twofold. These alterations were seen most readily in the hypergastrinemic-tumour animals (Figure 2).

Summary
Using the gene chip approach in the Mastomys demonstrated that 118 genes were specifically upregulated in both the loxtidine-induced and spontaneous ECL cell tumours. An examination of these genes identified alterations in the AP-1 transcription apparatus (fos/junB were downregulated 2.2-12-fold). Also noted was downregulation of junD (2.4-3.5-fold), a transcription factor negatively regulated by menin (the gene product of the MEN-1 locus²²), and upregulation of other cell cycle regulators (cyclin D1 and cdk4, 2.4-3.2-fold). This suggests that these pathways are commonly altered in the Mastomys during ECL cell transformation. We hypothesised that these gene products may also be altered in human tissue.

HUMAN TUMOURS

Background
While the AP-1 apparatus has not been exhaustively investigated in human ECL cells, loss of heterozygosity (LOH) of the MEN-1 locus is a common event; LOH occurs in approximately 17-100% of ECL cell tumours, irrespective of their malignant potential. ^23,24 Over-expression of cyclin D1 occurs in 43% of pancreatic endocrine tumours, ²⁵ while hypermethylation (with subsequent gene silencing) of the promoter region of p16 occurs in about 50% of gastrinomas. ²⁶ The latter is a negative regulator of cyclin D1:cdk4 activity. A role for oncogenes in gastric ECL cell tumours has also been defined. Examination of oncoprotein immunoreactivity in five gastric tumours out of a series of eighty-seven primary carcinoids of the gastroenteropancreatic neuroendocrine system demonstrated that c-Myc was detected in 100% of cases, Bcl-2 (20%), c-Jun (20%), c-ErbB-2 (0%) and c-ErbB-3 (0%). ²⁷ This indicates that oncogenes may play a role in ECL cell pathogenesis. These studies suggest that AP-1-regulated pathways, several oncogenes, and the cyclin D1 regulatory pathway are all altered during gastrointestinal neuroendocrine tumourigenesis. These gene products are also altered in the Mastomys model, and so the expression of genes found to be altered in the Mastomys tumour samples was then examined in a human carcinoid tissue databank.

Methods
Gene (and protein) differences in 13 tissue specimens from normal, precancerous, or neoplastic sites obtained by intraoperative excision or endoscopic gastric biopsy from nine patients (mean age: 57 years, range 38-77 years) were evaluated. mRNA was isolated for RT-PCR, genomic DNA was extracted for gene promoter region methylation status, and protein was isolated for western blot analysis as described. 15 Genes and proteins determined to be regulators of ECL cell proliferation and autonomy, including cyclin D1, cdk4, menin, jun-D, p16, SSTR2, and PCNA were examined.

Results
The data demonstrate that expression of the cyclin D1 gene (mRNA) was correlated with the degree of neoplasia (0% of nonneoplastic tissue vs. 33% of neoplastic specimens) (Figure 3). The cyclin D1 protein, however, was identified in all tested tissue samples; cdk4 mRNA was expressed in 80% of assayed normal and precancerous tissue samples and was present in 83% of tumour samples. The p16 gene, a regulator of cyclin D1/cdk4 activity, was identified in 66% of non-tumour specimens and in less than 30% of all tumour specimens, while the p16 gene promoter was uniformly unmethylated (gene-active) in all normal tissue. The menin gene was expressed in 50% of normal tissues and 57% of tumour tissues while its catalytic partner, junD, was identified in all tested normal and tumour specimens. SSTR2 was identified in all non-neoplastic specimens and in 86% of tumour tissue samples. RTPCR failed to detect the PCNA gene in any non-neoplastic tissue, but did identify it in 60% of tumour specimens. Thereafter, we identified that the human NET itself or the surrounding tissue may produce message for gastrin (Figure 4). This indicates that a gastrin autocrine loop may exist to drive human tumour cell proliferation.

Figure 3: RT-PCR of genes examined in human NET tissue. On the left are the genes involved in the cyclin D1 pathway; on the right are the AP-1 transcription genes and SSTR2. These genes and pathways are indicated to be altered by gene-chip analysis of the Mastomys tissue. Preneoplastic refers to naïve and hyperplastic ECL cell samples

Summary
The results demonstrate that cyclin D1 and PCNA are over-expressed (33-60%) and p16 downregulated in NET tissue. Alterations in certain cell cycle regulators are common to both human NETs and Mastomys ECL cell tumours. These findings highlight the potential for identifying genes in Mastomys ECL tumours that may be markers for human NETs and are indicative of the potential of examining global gene alterations to identify differences in NET gene expression.

SOMATOSTATIN RECEPTOR THERAPY

Background
Radiolabelled somatostatin analogues target neuroendocrine and other tumours that express high levels of SSTR₂ and may be utilised to deliver therapeutic doses of radiation.⁸ Few studies have, however, evaluated the therapeutic safety of the somatostatin analogue [¹¹¹In]pentetreotide in the treatment of gastroenteropancreatic tumours (GEPT).

Methods
In order to determine if a SSTR₂-positive NET could be therapeutically targeted, patients with unresectable advanced malignancy, at least one tumour-bearing site documented by [¹¹¹In]pentetreotide uptake, and no further conventional therapeutic options were included in this trial. Baseline laboratory profiles and computed tomography imaging were acquired prior to the administration of at least four cycles of ¹¹¹In-labeled pentetreotide at one of three radiation dose levels (up to a maximum of 1800 mCi) in a university institutional review board-approved protocol. Patients were scrupulously monitored for changes in haematologic and renal function profile as well as evidence of disease progression with frequent phlebotomy, follow-up octreotide scintigraphy, and biannual computed tomography.

Results
Thirty-nine of 233 patients who were evaluated for screening, post-operative follow-up, and other indications with [¹¹¹In]pentetreotide imaging over the course of a five-year period were recruited into this study. A majority of the 39 patients enrolled in this therapeutic safety trial experienced mild or moderate side-effects, including transient myelosuppression. One patient developed transient acute renal tubular necrosis but successfully underwent six cycles of radiotherapy without developing renal failure. Of twenty-nine evaluable patients with GEPT, 76% demonstrated radiographic stability or modest improvement of disease (Table 1), while 83% had stable or improved symptoms. Twelve of 22 patients (55%) with evaluable hormone markers experienced stability or a notable decrease in at least one measured tumour hormone marker over the course of therapy (Table 2).

Figure 4: RT-PCR of gastrin in human NETs. E, ECL cell tumor; G, gastrinoma; Mr, molecular weight. The two bands represent the gastrin mRNA before splicing (345bp) and after removal of intron 2 (215bp)

Summary
[¹¹¹In]pentetreotide is effective in the identification of patients with NET burden expressing SSTR₂. The haematologic and renal safety as well as the therapeutic efficacy of radiolabelled pentetreotide at divided doses of only 300 mCi suggest that higher doses of this agent, or perhaps other chelating, higher-energy emitters, could be used safely for the treatment of otherwise untreatable neuroendocrine tumours. This study demonstrates the safety and feasibility of intravenously administered ¹¹¹In in the management of neoplastic lesions expressing SSTR₂ and, furthermore, suggests that with increased dosage, this modality of therapy may well be efficacious.

DISCUSSION
The molecular identification of specific gene products in an animal neuroendocrine tumour model using a gene-chip approach has allowed the elucidation of pathways altered during ECL cell tumourigenesis. Specifically, alterations in the AP-1 transcription apparatus (fos/jun and junD) and in the cell cycle regulators (cyclin D1 and cdk4) were identified in both hypergastrinemic and normogastrinemic ECL cell tumours. The alterations in these gene pathways have been confirmed using more traditional methods (RT-PCR, western blot, and immunohistochemistry). ^28-31 These data indicate that such pathways are altered in the Mastomys during ECL cell transformation and also identify a number of candidate gene products that are altered during ECL cell tumourigenesis.

An examination of these gene products in human NET tissue identified that these pathways are similarly altered in the human tumour as in the Mastomys. In particular, p16, a regulator of cyclin D1/cdk4 activity, was downregulated in tumour specimens compared with normal tissue, indicating that the activity of this negative regulator of the cell cycle is altered during ECL cell tumourigenesis in humans. This was supported by the absence of cyclin PCNA (a marker of the cell cycle) in any nonneoplastic tissue, but with the identification of this gene message in tumour specimens. Interestingly, both human NETs and the surrounding tissue may produce message for gastrin, which suggests that a gastrin autocrine/paracrine loop may exist to drive human NET proliferation; this has been previously demonstrated for gastric carcinoids.³² While SSTR₂ was identified in the majority (86%) of tumour tissue samples, it was not present in all samples. This suggests that therapeutically targeting a single somatostatin receptor subtype may not adequately treat all patients with NETs.

A preliminary investigation of carcinoid patients using SSTR₂-targeted [¹¹¹In]pentetreotide radiotherapy generated encouraging results. Specifically, the ease of administration, lack of adverse events associated with treatment, and maintained quality of life are significant factors to be considered in the management of individuals with protracted oncologic disease. Of particular note was the fact that 56% of patients with evaluable hormone markers demonstrated stable levels or a significant decrease in one or more measured markers. This indicates the potential clinical usefulness of this approach in patients with NETs.

CONCLUSION
The growth-regulatory elements of the neuroendocrine ECL cell system, which forms the cellular basis for gastric NETs, have been defined using a gene-chip approach in an animal model of the disease. The definition of ECL cell status and the findings of appropriate molecular targets highlight the potential for identifying genes that may be markers for human gastric carcinoid autonomy. These findings can then be applied to the development of rational and novel therapeutic strategies in patients with ECL cell tumours. One of these molecular targets (SSTR₂) has been successfully used as a novel therapeutic strategy that is an appropriate adjunct to surgery for this disease.

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Copyright: 15 April 2003