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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0986
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 11 4394-4402
Copyright © 2007 by The Endocrine Society

Secretin-Receptor and Secretin-Receptor-Variant Expression in Gastrinomas: Correlation with Clinical and Tumoral Features and Secretin and Calcium Provocative Test Results

Scott H. Long1, Marc J. Berna1, Michelle Thill, Andrea Pace, Tapas K. Pradhan, K. Martin Hoffmann, Jose Serrano and Robert T. Jensen

Digestive Diseases Branch (S.H.L., M.J.B., T.K.P., J.S., R.T.J.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; National Eye Institute (M.T.), National Institutes of Health, Bethesda, Maryland, 20892-1804; Medizinische Klinik I (M.J.B., A.P.), Universitätsklinikum Hamburg-Eppendorf, 20256 Hamburg, Germany; and Department of Paediatrics and Adolescent Medicine (K.M.H.), Medical University of Graz, Auenbruggerplatz 30, A-8036 Graz, Austria

Address all correspondence and requests for reprints to: Dr. R. T. Jensen, National Institutes of Health, Building 10, Room 9C-103, Bethesda, Maryland 20892. E-mail: robertj{at}bdg10.niddk.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context/Objectives: The diagnosis of Zollinger-Ellison syndrome requires secretin testing in 60% of patients. Even with secretin, the diagnosis may be difficult because variable responses occur, and 6–30% have negative testing. The basis for variability or negative responses is unclear. It is unknown whether the tumor density of secretin receptors or the presence of a secretin-receptor-variant, which can act as a dominant negative, is important. The aim of this study was to investigate these possibilities.

Patients/Methods: Secretin-receptor and variant mRNA expression was determined in gastrinomas using real-time PCR from 54 Zollinger-Ellison syndrome patients. Results were correlated with Western blotting, secretin-receptor immunohistochemistry, with gastrin-provocative test results and tumoral/clinical/laboratory features.

Results: Secretin-receptor mRNA was detectible in all gastrinomas but varied 132-fold with a mean of 0.89 ± 0.12 molecules per ß-actin. Secretin-receptor PCR results correlated closely with Western blotting (r = 0.95; P < 0.0001) and receptor immunohistochemistry (P = 0.0015; r = 0.71). The variant was detected in all gastrinomas, but levels varied 102-fold and were 72-fold lower than the total. Secretin-receptor levels correlated with variant levels, {Delta}secretin, but not {Delta}calcium and with tumor location, but not growth, extent, or clinical responses. Variant levels did not correlate with the {Delta}secretin. Detailed analysis provides no evidence that variant expression modified the secretin-receptor response or accounted for negative tests.

Conclusions: Secretin-receptor and secretin-receptor-variant expressions occur in all gastrinomas. Because the expression of the total, but not variant, correlated with the secretin results and no evidence for dominant negative activity of the variant was found, our results suggest that the total secretin-receptor density is an important determinant of the secretin test response.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THE DIAGNOSIS OF Zollinger-Ellison syndrome (ZES) can be difficult in more than 60% of patients because their fasting serum gastrin levels overlap with other conditions causing hyperchlorhydria/hypergastrinemia (1). The secretin test has become a mainstay for ZES diagnosis in this group of patients (2, 3, 4). Secretin test results in more than 800 patients are reported (4) and criteria of positivity extensively studied (2, 3, 4, 5, 6). However, no studies address the variability of up to a 4000-fold difference in gastrin responses to secretin in different ZES patients (4) or why 20–35% have negative responses (3, 4).

A recent case report (7) described one of three patients with ZES whose gastrinoma showed a large amount of a particular secretin-receptor-variant transcript that could function as a dominant negative in transfected cells. It was proposed (7) that this effect might account for the patient’s negative secretin test. At present it is unknown whether the abundance of the secretin-receptor, secretin-receptor-variant, or their ratio could be factors in determining the variability of the secretin-stimulated gastrin response in different ZES patients or the occurrence of a positive test. To address these questions, we assessed the secretin-receptor, secretin-receptor-variant transcript, and their ratio in gastrinomas from 54 ZES patients and correlated these with clinical/laboratory/tumoral features, as well as the secretin and calcium (Ca) test responses.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients, tumors, and clinical variables

A total of 54 ZES patients seen at the National Institutes of Health between 1989 and 2005 were included. The protocol was approved by the Clinical Research Committee-National Institute of Diabetes and Digestive and Kidney Diseases, and all patients gave informed consent. The diagnosis of ZES and multiple endocrine neoplasia type 1 (MEN1) was established as previously reported (8, 9, 10). MEN1 patients had DNA testing for mutations in the MEN1 gene. Seven patients had mutations: E363del, 672del CC/N, and 512delC. Preoperative fasting gastrin, secretin (2 U/kg), and Ca (5 mg/kg·h x 3 h) tests were performed (4). Studies assessing basal acid output (BAO), maximal acid output (MAO), and drug control of acid secretion were performed (11). Conventional imaging (computed tomography, magnetic resonance imaging, and ultrasound), angiography with secretin-stimulation/gastrin sampling, and somatostatin receptor scintigraphy were performed (12, 13) to locate the primary (Prim) and/or extent. Each patient underwent an exploratory laparotomy for attempted cure (14). Patients were reassessed postoperatively (15, 16). Disease-free status and relapse were defined as outlined previously (16, 17). In patients not disease free, annual imaging studies (computed tomography, magnetic resonance imaging, ultrasound, and somatostatin receptor scintigraphy) and, if the results were unclear, angiography provided the basis for assessment of tumor growth/progression (18). For each patient the number and size of each measurable tumor were determined by imaging modalities. Tumor growth rate was calculated as the percent volume increase per month (18). Patients were classified as having nonaggressive ZES if there was no growth or less than 25% increase in volume/month, either with or without liver metastases at all yearly evaluations, and if initially there was not extensive metastatic disease.

Tumor samples and cDNA preparation

Tumors were snap frozen and stored at –70 C. Tumor mRNA was extracted from 5-µm cryosections after analyzing an adjacent slide to determine more than 80% of the section contained tumor (19). Total RNA was extracted using the RNeasy Mini-Kit (QIAGEN Inc., Santa Clarita, CA) from two or three adjacent slides. Random hexamer-primed first strand cDNA was prepared with RT (RNA PCR Kit; Applied Biosystems, Foster City, CA).

Real-time PCR

PCR was done using SYBR Green (Applied Biosystems 2X SYBR GREEN PCR Mater Mix, Warrington, UK) and Stratagene Mx3000P Multiplex Quantitative PCR Systems (La Jolla, CA) Primers for both the total and a secretin-receptor-variant (missing exon 3) were selected through analysis of the secretin-receptor mRNA sequence (GenBank accession no. U28281), and were based on the gene structure described by Ho et al. (20). The total secretin-receptor primer (product 201 bp) was selected from the 3' end of the mRNA sequence. The sense and antisense sequences of the total secretin-receptor primer were as follows: sense, 5'-CTGGCCAGGTCCACTCTC-3' (nucleotides 1126–1143); and antisense, 5'-CCATTGCTGCCACTTCTTCT-3' (nucleotides 1307–1326). The secretin-receptor-variant primers (product 118 bp) were designed so that the sense primer spanned the end of exon 2 and the beginning of exon 4: sense, 5'-CCCGACTATGTGACGTGCTA-3' (nucleotides 194–213); and antisense, 5'-AACAAGGAACCTGGCACTGG-3' (nucleotides 292–419). The abundance of the total secretin-receptor mRNA was determined in all 54 gastrinomas, but because of the low expression of the variant receptor, more concentrated cDNA samples were required in the PCR, and sufficient material was available from 35 gastrinomas. The specificity of all the primers was ensured by their size and their localization in unique sequences across spliced regions of the gene. The PCRs were performed in a final volume of 25 µl. Patient samples were screened for the expression of ß-actin as a reference gene (19). The reaction mixture included the SYBR Green PCR buffer (12.5 µl), H2O (5.5 µl), and 300 nmol/liter specific primer (6 µl), and was run in duplicate. The PCR was conducted with 40 cycles, which were within the linear amplification range. The PCR began with a cycle of 94 C for 10 min, followed by 40 cycles of 94 C for 50 sec (denaturing), 62 C for 50 sec (annealing), and 72 C for 50 sec (elongation.) A final extension period of 94 C for 5 min concluded the amplification. In all experiments, dilutions of standard were included, which was human pancreas (Panc) cDNA (lot no. A901107; BioChain Institute, Inc., Hayward, CA).

Western blot

Pooled tissue from cryosections containing more than 80% tumor was lysed in lysis buffer [50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium azide, 1 mM EGTA, 0.4 mM EDTA, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, and one protease inhibitor tablet per 10 ml]. Equal amounts of total protein were analyzed by SDS-PAGE and Western blotting using rabbit anti-Secretin receptor antibody (AbD Serotec, Oxford, UK) or mouse anti-ß actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The intensity of the protein bands was measured using Kodak ID-Image Analysis (Eastman Kodak Co., Rochester, NY).

Immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded tissue samples from 26 patients were analyzed by IHC for secretin-receptor and gastrin. Slides were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was achieved by boiling slides in 10 mM Tris (pH 10.0) and keeping them at a sub-boiling temperature for 10 min. Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide solution for 10 min. Sections were stained using the RTU Vectastain Universal Quick kit and the DAB Substrate Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s recommendations. Rabbit antigastrin polyclonal antibody (CHEMICON International, Inc.) (1:1000) and rabbit anti-Secretin receptor antibody (1:50) were used. Slides incubated with an isotype-matched Ig were used as a negative control. Sections were counterstained with hematoxylin (Vector Laboratories), mounted, and analyzed by two independent blinded investigators. Staining intensity for secretin-receptor was graded 0–3 as previously described (19), and the percentage of positive cells was graded 1–4 corresponding to less than 25, 25–50, 50–75, and more than 75%, so that the maximal score was 7.

Statistical analysis

Values were expressed as mean ± SEM. Discontinuous variables were compared using the Fisher exact test, and for continuous variables, the Mann-Whitney U test (two variables), Kruskal-Wallis test, or an ANOVA with the Bonferroni Dunn test as a post hoc test. Correlation coefficients were calculated using least-squares analysis. All calculations were performed using the statistical program StatView 5 (SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
The clinical/laboratory characteristics of the 54 ZES patients resemble those in other large series (9, 11, 21) in having almost equal frequency in both sexes, mean age in the fifth decade, long-disease duration, elevated fasting serum gastrin level, and secretin-stimulated gastrin level and preoperative BAOs/MAOs (Table 1Go). Similar to recent series (9), duodenal primaries were more frequent than pancreatic, and Prim tumors in lymph nodes (LNs) as well as other sites were found (9, 17). In approximately one third of patients, the tumor was confined to the Prim site. In contrast, 22% of patients developed liver metastases at some time (Table 1Go). As reported recently, approximately one third had a gastrinoma with an aggressive postoperative course (16, 21), and approximately 45% of the patients had a long-term cure (15, 17).


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  		TABLE 1. Clinical characteristics, laboratory values, and tumor characteristics in the patients studied

 
Total secretin-receptor mRNA abundance varied greatly in different gastrinomas (Fig. 1Go), however, it was detectible in all (Fig. 1AGo). Control studies demonstrated that the PCR products resulted from tumor mRNA because total secretin-receptor primers spanned a long intron and gave no amplification with genomic DNA. The total secretin mRNA levels varied 132-fold, from 0.03–3.95 molecules per ß-actin molecule, with a mean of 0.89 ± 0.12 per ß-actin molecule (Fig. 1AGo).


Figure 1
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  		FIG. 1. Secretin-receptor mRNA expression in gastrinomas. A, Total secretin-receptor mRNA expression. Top, PCR result for total secretin-receptor mRNA expression in gastrinomas of 10 patients showing variable amounts of the 201-bp PCR product. Bottom, Distribution of the total secretin-receptor mRNA expression in gastrinomas from 54 patients with ZES. All values are expressed as the number of total secretin-receptor molecules per ß-actin molecule in the gastrinoma. Each point is the mean value from one patient’s gastrinoma. All patient gastrinoma values are the mean of at least three determinations. The horizontal and vertical lines indicate the mean ± SE for all 54 patients (0.89 ± 0.12). B, Secretin protein expression in gastrinomas. Top, Western blot of secretin receptor protein expression in five gastrinomas. Bottom, Correlation of secretin protein expression and total secretin-receptor mRNA assessed by PCR from 10 gastrinomas. Results are expressed as the amount present compared with ß-actin. C, Secretin-receptor-variant mRNA expression in gastrinomas. Top, PCR result for secretin-receptor-variant mRNA expression in gastrinomas from 10 patients showing the variable amounts of the 118-bp PCR product. Bottom, Distribution of the secretin-receptor-variant mRNA expression in gastrinomas from 35 patients with ZES. All values are expressed as the number of secretin-receptor-variant molecules per ß-actin molecule in the gastrinoma determined, as described in Patients and Methods. Each point is the mean value from one patient’s gastrinoma. All patient values are the mean of at least three PCR determinations. The horizontal and vertical lines indicate the mean ± SE for all patients (0.017 ± 0.003). The different symbols indicate the source of the tumor analyzed in each case: •, Prim-sporadic; {circ}, Prim-MEN1; {blacksquare}, LN-sporadic; {square}, LN-MEN1; and {blacktriangleup}, liver metastases.

 
Because only small amounts of tumor were available from most patients, a PCR analysis was the only method able to assess secretin-receptor abundance in most patients. To be certain the secretin-receptor mRNA expression correlated with secretin-receptor protein expression, the results of PCR and Western blotting were compared in 10 patients with sufficient tissue to perform each (Fig. 1BGo). The results of the two analyses demonstrated a very close correlation (r = 0.95; P < 0.0001). To confirm this association further, IHC for secretin-receptor and gastrin was performed in 26 gastrinomas in which formalin-fixed paraffin-embedded tissue samples were available. All tumors showed positive staining for secretin receptor. The vast majority of cells (>75%) staining positive for gastrin also showed expression of secretin-receptor, with only two tumors showing less than 50% of tumor cells expressing secretin-receptor. The average secretin-receptor score was 5.5 (range 3–7), and we found a highly significant (r = 0.71; P = 0.0015) correlation between the IHC score and the total secretin-receptor mRNA expression.

To assess variant-receptor mRNA expression, specific primers were designed that would only produce a product if exon 3 was deleted. Control studies demonstrated that this primer set produced a single 118-bp amplification product, which was seen only in tissues known to contain the secretin-receptor (three separate pancreatic cDNAs) and not in tissues, which do not contain secretin receptors (placenta, lung cancer). Secretin-receptor-variant expression was detected in all gastrinomas, with its levels varying 102-fold from 0.001–0.102 molecules per ß-actin, with a mean ± SEM level of 0.017 ± 0.003 (Fig. 1CGo). For the different gastrinomas, the total secretin-receptor had a 72 ± 12-fold greater level of expression than the secretin-receptor-variant, with levels ranging from 10.5- to 301-fold. The total secretin-receptor expression level showed a highly significant (r = 0.65; P < 0.0001) direct correlation with the level of the secretin-receptor-variant (Fig. 2Go, D1).


Figure 2
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  		FIG. 2. Correlation of total secretin-receptor and secretin-variant expression, and their ratio with results of secretin and Ca provocative testing (A1–C2) and with each other (D1) or with tumor size (D2). Each dot indicates the results from a gastrinoma from one patient, and each value is the mean of at least three separate PCR determinations. Correlation coefficients and significance were determined by least-squares analysis. The different symbols indicate the source of the tumor analyzed in each case: •, Prim-sporadic; {circ}, Prim-MEN1; {blacksquare}, LN-sporadic; {square}, LN-MEN1; and {blacktriangleup}, liver metastases.

 
Increased age (21), MEN1 (16), short-disease duration (9, 16), female gender (21), or high hormone levels or its effects (i.e. in gastrinomas, BAO and MAO) (21) are associated with a poor prognosis in gastrinomas and/or neuroendocrine tumors. However, none of these characteristics correlated with the magnitude of either the total or variant-secretin-receptor expression, or their ratio (Table 2Go). Similarly, other clinical factors such as race or need for previous gastric-acid reducing surgery, which was frequently performed in patients with more severe disease, did not correlate with the expression levels of the total and secretin-receptor-variant or their ratio. However, a history of ulcer disease correlated with the magnitude of the variant, but not the magnitude of the total secretin-receptor or variant to total ratio (Table 2Go).


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  		TABLE 2. Effect of various demographic, clinical, and laboratory features on total secretin-receptor and variant secretin-receptor mRNA, and their ratio

 
The median {Delta}secretin was 1,566 pg/ml (range 67–158,000), preoperatively. All but four patients (92%) had a positive test using the historical criterion of 200 pg/ml or more (22), or all but two patients (96%) using the recently proposed more sensitive criterion of 120 pg/ml or more (4). The median {Delta}Ca was 1,620 pg/ml (range 9–46,000), with 77% having a positive test using the criterion of a 395 pg/ml or more increase (3, 4). The magnitude of the increase with secretin, but not with Ca, directly correlated with the total secretin-receptor expression (P = 0.0035) (Fig. 2Go, A1 and A2). The mean total secretin-receptor mRNA expression level did not differ in the four patients with a negative secretin test from those with a positive test (1.00 ± 0.53 vs. 0.82 ± 0.11 molecules per ß-actin), suggesting that factors other than total secretin-receptor expression were responsible for these patients’ negative test. The secretin-receptor-variant is proposed to be able to function as a dominant negative (23, 24). To examine this possibility in more detail, we studied the effect of differing amounts of the secretin-receptor-variant on secretin-stimulated gastrin release from different gastrinomas (Fig. 1BGo). No correlation existed between the absolute amount of secretin-receptor-variant or the total to variant ratio and the magnitude of {Delta}secretin or {Delta}Ca (Fig. 2Go, B and C). The magnitude of the {Delta}secretin did not differ for gastrinomas with a high (>70-fold) or low (<30-fold) ratio of total to variant secretin-receptor (Fig. 3AGo), and conversely the total to variant secretin-receptor ratio did not differ in gastrinomas with {Delta}secretin less than 500 pg/ml compared with those with more than 5000 pg/ml {Delta}secretin (Fig. 3CGo). Finally, it was proposed that sufficiently high amounts of secretin-receptor-variant could cause a negative secretin test (7). In our study four patients had a negative secretin test, and we found no significant difference between their total to variant secretin-receptor ratio compared with the patients with positive secretin tests (Fig. 3BGo).


Figure 3
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  		FIG. 3. Comparison of total secretin and variant secretin-receptor expression, or their ratio in gastrinomas with the positivity or magnitude of the secretin response or the tumor location. Panel A compares the median {Delta}secretin in patients with gastrinomas that had less than 30-fold with those that had more than 70-fold greater amounts of total compared with secretin-receptor-variant abundance. Panel B compares the total to variant secretin-receptor ratio in patients with or without a positive secretin test (i.e. >200 pg/ml increase). Panel C compares the total to variant secretin-receptor ratio in gastrinomas from patients who had less than 500 pg/ml increase or more than 5000 pg/ml increase on secretin. D–F, Tumor location was determined at surgery. For the total secretin-receptor expression, results are from 31 duodenal, seven pancreatic, seven Prim LN, and five Prim gastrinomas in other locations. For the variant and ratio, results from 17 duodenal, four pancreatic, seven LN Prim, and four Prim gastrinomas in other locations are shown. Each dot represents the value from one gastrinoma in the indicated location and is a mean of at least three separate PCR determinations. Horizontal and vertical lines show the mean ± SEM for each Prim tumor group. The different symbols indicate the source of the tumor analyzed in each case: •, Prim-sporadic; {circ}, Prim-MEN1; {blacksquare}, LN-sporadic; {square}, LN-MEN1; and {blacktriangleup}, liver metastases. Duo, duodenum; Pan, pancreas.

 
Tumor extent, liver metastases, disease-free status after resection, occurrence of relapse, postoperative tumor growth pattern, or survival did not correlate with total or variant-secretin-receptor expression, or their ratio (Table 3Go and Fig. 2Go, D2). However, both gastrinoma size and location had an effect on at least one of these three variables (Table 3Go and Fig. 3Go, D–F). Specifically, the total secretin-receptor expression was 2.5 times higher for tumors located in the Panc/duodenum (Duod), whereas the secretin-receptor-variant levels were two times higher in duodenal than nonduodenal tumors (Table 3Go and Fig. 3Go, D and E). This resulted in a 2.3-fold higher secretin-receptor-variant to total secretin-receptor ratio in Prim tumors outside the Duod or Panc (Table 3Go and Fig. 3FGo). The secretin-receptor-variant was 2.3 to four times more frequent in small gastrinomas than in large primaries (Table 3Go). The secretin-receptor-variant to total secretin-receptor ratio was significantly higher in gastrinomas outside the Duod or Panc (P = 0.0004; Fig. 3FGo).


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  		TABLE 3. Effect of tumoral characteristics on expression variant and their ratio of total secretin-receptor and secretin-receptor-variant in gastrinomas

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Since the initial description of secretin’s ability to increase gastrin levels in ZES (25), the secretin test was used in ZES diagnosis (2, 4). Although some studies suggest that the increased secretin response in ZES represents an exaggerated normal response (4, 5, 26), recent studies demonstrate that secretin stimulates gastrin release through secretin receptors on gastrinomas. Secretin can stimulate gastrin release from isolated gastrinoma cells (4, 27, 28, 29), stimulate adenylate cyclase activation in these cells (29), and secretin receptors occur on gastrinomas (7, 24).

Many studies have examined the gastrin changes after secretin in ZES patients (2, 4) and proposed criteria diagnostic for ZES (2, 4); however, there is almost no information on the factors that determine the markedly variable secretin-stimulated gastrin change that occurs between patients [i.e. 0- to 4000-fold in one recent review (4)] or explains the nondiagnostic increase in 13% [mean 11 series (range 0–30%)] of ZES patients (3, 4). Recently it was shown in one gastrinoma (7) and a number of pancreatic ductal adenocarcinomas (23) that secretin-receptor-variants occur. In transfected cells one secretin-receptor-variant, which lacks exon 3 encoding amino acids 44–79 in the amino terminus, could function as a dominant negative (7, 23). It was proposed that the dominant-negative action might account for the negative secretin test in the ZES patient studied (7). At present whether the secretin receptor abundance or the proportion of variant actually contributes to the magnitude of the secretin-stimulated gastrin response is unclear. This has occurred because it is difficult to perform pathological-clinical correlations on gastrinomas because of their low incidence and small sizes (1, 16). In the present study, the mean size of the duodenal gastrinomas, which were the majority, was 3 mm, and in most cases, only a few microscopic slides of tissue were available. Therefore, a PCR approach was used for the secretin receptor mRNA. Western blotting in a limited number of tumors with more tissue showed a close correlation (r = 0.95; P < 0.0001) between the expression of the mRNA, with the receptor protein as did secretin-receptor IHC (P = 0.0015; r = 0.71). This result supported the validity of this approach and is similar to recent studies with somatostatin receptor expression in neuroendocrine tumors, which show a close correlation between mRNA expression and receptor expression (30, 31). The present study using this approach was undertaken to address the aforementioned unanswered questions.

A number our results support the conclusion that secretin-receptor is expressed in all gastrinomas, and the expression level is at least one determinant of the secretin-stimulated gastrin release response. First, in all 54 gastrinomas, secretin-receptor mRNA levels were detected and in all 26 gastrinomas by IHC. Second, 15% were pancreatic gastrinomas, with the remaining primarily duodenal, and a lesser number from other locations. These results demonstrate that all gastrinomas express secretin-receptors, even though gastrinomas in different locations are reported to have different origins (32). Third, secretin-receptor level showed a highly significant direct correlation (P = 0.0035) with the maximal secretin-stimulated gastrin release, suggesting secretin-receptor density on gastrinomas was an important variable in determining the maximal gastrin release with secretin. The finding that secretin-receptor expression did not correlate with maximal Ca-stimulated release shows that the association was not just coincidental or due to a nonrelated factor causing increased release. Furthermore, it is unlikely that this difference is due to one stimulant having a direct effect and the other an indirect effect on the gastrinoma because recent studies report that similar to secretin, Ca-stimulated gastrin release from gastrinomas is due to a direct action on the gastrinomas (27, 33, 34, 35). Fourth, in the four patients with negative secretin tests, each had total secretin-receptor mRNA present in their gastrinoma, demonstrating that the presence or absence of secretin-receptor does not determine a positive secretin test result, but other factors such as secretin-receptor amount, coupling to secretory pathways, or gastrin storage could be responsible for the negative secretin test in these patients.

The present study revealed that no clinical/laboratory features of ZES patients correlated with the abundance of total secretion-receptor. This result is consistent with findings in a recent study (4) demonstrating no clinical/laboratory features in 830 ZES patients from the National Institutes of Health and literature, correlated with the secretin test result either with its positivity or magnitude of response. In terms of tumoral features, an association was found between the gastrinoma Prim location and total secretin-receptor mRNA abundance in the gastrinoma, with duodenal/pancreatic tumors having higher levels than nonduodenal-pancreatic tumors. A number of studies report that secretin can have either growth stimulatory (36, 37) or inhibitory effects (23, 38) in pancreatic, gastric, or colonic tumors. Our finding that there is no correlation of total secretin-receptor expression and growth of gastrinomas suggests that it is unlikely that secretin has a potent growth stimulatory or inhibitory effect on the tumor itself.

Alternatively spliced forms of various G protein-coupled receptors as well as other receptors are described that can have important clinical implications (39, 40, 41, 42, 43, 44). In some cases they can alter signaling by the wild-type receptor by functioning as a dominant negative. Such splice-variant behavior has been described for receptors for GHRH (45), calcitonin (46), vasopressin (41), {alpha}-adrenergic agents (44), thyroid hormones (40), and recently for one secretin receptor variant (7, 23). This secretin-receptor-variant (7, 23) lacking expression of the third exon encoding for amino acid residues 44–79 in the amino terminus (7, 23) occurred in one of three gastrinomas (7), in four pancreatic adenocarcinomas cell lines (23), and three Prim pancreatic adenocarcinomas (23). In these studies the secretin-receptor-variant (7, 23) did not alter cell signaling when present alone but functioned as a dominant negative likely by heterodimerizing with the wild-type receptor (23). This dominant-negative ability was proposed as a possible mechanism causing a negative secretin test in one patient who had a high level of expression of the secretin-variant-receptor in his gastrinoma (i.e. 70% of the total secretin-receptor transcript) (7). A number of our results support the conclusion that this secretin-receptor-variant is expressed in all gastrinomas, although its level is low in most tumors, and that its presence neither modifies the ability of the wild-type receptor to stimulate gastrin release, nor is it a cause of the negative secretin test that occurred in a subset of patients. First, secretin-receptor-variant mRNA expression levels could be quantitated in all gastrinomas. Second, the total secretin-receptor transcript had a mean level of expression 72-fold higher than the variant receptor. This means that the variant receptor accounted for a mean of 1.4% of the total secretin transcript, with a range from 0.4–11% in different gastrinomas. This result demonstrates that the occurrence of the variant accounting for 70% of the total secretin-receptor transcript in a gastrinoma, as reported in the original case discussed previously (7), is rare. Third, neither the expression of the secretin-receptor-variant nor the ratio of the variant to the total secretin-receptor transcript correlated with the magnitude of secretin-stimulated gastrin release. Fourth, the ratio of total to variant secretin-receptor expression did not differ between patients whose gastrinomas showed a low or high gastrin release with the secretin test or between patients with a positive or negative secretin test. These results suggest that in most patients with a negative secretin test, some other mechanism than the level of secretin-variant receptor expression accounts for the negative study. It also supports the conclusion that the level of the variant expression in gastrinomas does not account for the marked variability of the secretin test results from one patient to the next. One likely explanation for our failure to see an effect of the variant receptor on secretin-stimulated gastrin release in our patients, in contrast to the previous case report (7), is the marked differences in the level of variant-receptor expression found in the gastrinomas in these two studies. The highest variant receptor level we detected was 6-fold lower than that reported in the case report (11 vs. 70% of total transcript, respectively), and our mean variant receptor level was 50-fold lower than that reported in the case report (7) (i.e. 1.4 vs. 70%).

Not only did the magnitude of the secretin-receptor-variant or its percentage of the total secretin transcript not correlate with secretin-stimulated gastrin release, it also was not influenced by most clinical/laboratory variables. Although no differences were seen between MEN1 and non-MEN1 patients, the number of MEN1 patients was small (n = 9), and further study of this group is warranted. Prim tumors not located in the Duod or Panc had a higher percentage of total but not variant receptor, and small gastrinomas had a 2.3 to four times higher level of the variant receptor. At present the pathogenetic factors responsible for these differences are unknown and could include different origins of the tumors, differences in differentiation, or different influences of local tissue factors. Even though these tumoral differences for the total and variant-receptor expression occurred, they unfortunately did not help to differentiate the 25% of patients with tumors with aggressive growth or the development of liver metastases from those with nonaggressive growth, which is one of the most important clinical differentiations that needs to be made in the long-term management of these patients (16, 21).


   Footnotes
 
This research was partially supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases and National Eye Institute, National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 21, 2007

1 S.H.L. and M.J.B. contributed equally to this work. Back

Abbreviations: BAO, Basal acid output; Ca, calcium; Duod, duodenum; IHC, immunohistochemistry; LN, lymph node; MAO, maximal acid output; MEN1, multiple endocrine neoplasia type 1; Panc, pancreas; Prim, primary; ZES, Zollinger-Ellison syndrome.

Received May 2, 2007.

Accepted August 10, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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