Secretin-receptor and secretin-receptor-variant expression in gastrinomas: correlation with clinical and tumoral features and secretin and calcium provocative test results

Abstract

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 beta-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, Deltasecretin, but not Deltacalcium and with tumor location, but not growth, extent, or clinical responses. Variant levels did not correlate with the Deltasecretin. 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.

Figure 1

Secretin-receptor mRNA expression in gastrinomas. (Left panel). Total secretin-receptor mRNA expression. (Top left panel) PCR result for total-secretin-receptor mRNA expression in gastrinomas of 10 patients showing variable amounts of the 201 bp PCR product. (left bottom panel). 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 ~β-actin molecules 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). (Middle panel). Secretin protein expression in gastrinomas. (Top panel). Western plot of secretin receptor protein expression in 5 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 to β-actin. (Right panel). Secretin-receptor-variant mRNA expression in gastrinomas. (Top panel) PCR result for secretin-receptor-variant mRNA expression in gastrinomas from 10 patients showing the variable amounts of the 118 bp PCR product. (Bottom panel). 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 ~ β-actin molecules in the gastrinoma determined as described in 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. Symbols: ●Primary-sporadic, ○Primary-MEN1, ■LN-sporadic, □LN-MEN1, ▲Liver metastases

Figure 2

Correlation of total-secretin-receptor and secretin-variant expression and their ratio with results of secretin and calcium provocative testing (A, B, C) 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-square analysis.). The different symbols indicate the source of the tumor analyzed in each case. Symbols: ● Primary-sporadic, ○Primary-MEN1, ■LN-sporadic, □LN-MEN1, ▲Liver metastases

Figure 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. (Left Panel). The top panel compares the median Δsecretin in patients with gastrinomas that had <30-fold to those that had >70 fold greater amounts of total compared to secretin-receptor-variant abundance. The middle panel compares the total to variant secretin-receptor ratio in patients with or without a positive secretin test (i.e. >200 pg/ml increase). The lower panel compares the total to variant secretin-receptor ratio in gastrinomas from patients who had <500 pg/ml increase or >5000 pg/ml increase on secretin. (Right panel). Tumor location was determined at surgery. For the total-secretin-receptor expression results are from 31 duodenal, 7 pancreatic, 7 primary lymph node and 5 primary gastrinomas in other locations. For the variant and ratio results from 17 duodenal, 4 pancreatic, 7 lymph node primary and 4 primary 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 3 separate PCR determinations. Horizontal and vertical lines show the mean ± SEM for each primary tumor group.). The different symbols indicate the source of the tumor analyzed in each case. Symbols: ● Primary-sporadic, ○Primary-MEN1, ■LN-sporadic, □LN-MEN1, ▲Liver metastases Abbreviations: Duod, duodenum; Panc, pancreas; Prim, primary; LN, lymph node.